BioMed Research International: Biotechnology The latest articles from Hindawi © 2017 , Hindawi Limited . All rights reserved. Development of Isothermal Recombinase Polymerase Amplification Assay for Rapid Detection of Porcine Circovirus Type 2 Thu, 23 Mar 2017 00:00:00 +0000 Porcine circovirus virus type II (PCV2) is the etiology of postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis, nephropathy syndrome (PDNS), and necrotizing pneumonia. Rapid diagnosis tool for detection of PCV2 plays an important role in the disease control and eradication program. Recombinase polymerase amplification (RPA) assays using a real-time fluorescent detection (PCV2 real-time RPA assay) and RPA combined with lateral flow dipstick (PCV2 RPA LFD assay) were developed targeting the PCV2 ORF2 gene. The results showed that the sensitivity of the PCV2 real-time RPA assay was 102 copies per reaction within 20 min at 37°C and the PCV2 RPA LFD assay had a detection limit of 102 copies per reaction in less than 20 min at 37°C. Both assays were highly specific for PCV2, with no cross-reactions with porcine circovirus virus type 1, foot-and-mouth disease virus, pseudorabies virus, porcine parvovirus, porcine reproductive and respiratory syndrome virus, and classical swine fever virus. Therefore, the RPA assays provide a novel alternative for simple, sensitive, and specific identification of PCV2. Yang Yang, Xiaodong Qin, Yingjun Sun, Guozheng Cong, Yanmin Li, and Zhidong Zhang Copyright © 2017 Yang Yang et al. All rights reserved. Synthesis of L-Ascorbyl Flurbiprofenate by Lipase-Catalyzed Esterification and Transesterification Reactions Tue, 21 Mar 2017 00:00:00 +0000 The synthesis of L-ascorbyl flurbiprofenate was achieved by esterification and transesterification in nonaqueous organic medium with Novozym 435 lipase as biocatalyst. The conversion was greatly influenced by the kinds of organic solvents, speed of agitation, catalyst loading amount, reaction time, and molar ratio of acyl donor to L-ascorbic acid. A series of solvents were investigated, and tert-butanol was found to be the most suitable from the standpoint of the substrate solubility and the conversion for both the esterification and transesterification. When flurbiprofen was used as acyl donor, 61.0% of L-ascorbic acid was converted against 46.4% in the presence of flurbiprofen methyl ester. The optimal conversion of L-ascorbic acid was obtained when the initial molar ratio of acyl donor to ascorbic acid was 5 : 1. kinetics parameters were solved by Lineweaver-Burk equation under nonsubstrate inhibition condition. Since transesterification has lower conversion, from the standpoint of productivity and the amount of steps required, esterification is a better method compared to transesterification. Jia-ying Xin, Li-rui Sun, Shu-ming Chen, Yan Wang, and Chun-gu Xia Copyright © 2017 Jia-ying Xin et al. All rights reserved. Fungal Biomass Protein Production from Trichoderma harzianum Using Rice Polishing Mon, 06 Mar 2017 00:00:00 +0000 Industrially important enzymes and microbial biomass proteins have been produced from fungi for more than 50 years. High levels of crude protein as much as 45% are present in fungal biomass with balanced essential amino acids. The aim of this study was to access the potential of Trichoderma harzianum to produce fungal biomass protein from rice polishings. Maximum biomass yield was obtained at 5% (w/v) rice polishings after 72 h of incubation at 28°C at pH 4. Carbon and nitrogen ratio of 20 : 1 gave significantly higher production of fungal biomass protein. The FBP in the 75 L fermenter contained 49.50% crude protein, 32.00% true protein, 19.45% crude fiber, 9.62% ash, 11.5% cellulose content, and 0.325% RNA content. The profile of amino acids of final FBP exhibited that all essential amino acids were present in great quantities. The FBP produced by this fungus has been shown to be of good nutritional value for supplementation to poultry. The results presented in this study have practical implications in that the fungus T. harzianum could be used successfully to produce fungal biomass protein using rice polishings. Sibtain Ahmed, Ghulam Mustafa, Muhammad Arshad, and Muhammad Ibrahim Rajoka Copyright © 2017 Sibtain Ahmed et al. All rights reserved. Outdoor Growth Characterization of an Unknown Microalga Screened from Contaminated Chlorella Culture Sun, 05 Mar 2017 06:57:29 +0000 Outdoor microalgae cultivation process is threatened by many issues, such as pest pollution and complex, changeable weather. Therefore, it is difficult to have identical growth rate for the microalgae cells and to keep their continuous growth. Outdoor cultivation requires the algae strains not only to have a strong ability to accumulate oil, but also to adapt to the complicated external environment. Using 18S rRNA technology, one wild strain Scenedesmus sp. FS was isolated and identified from the culture of Chlorella zofingiensis. Upon contamination by Scenedesmus sp., the species could quickly replace Chlorella zofingiensis G1 and occupy ecological niche in the outdoor column photobioreactors. The results indicated that Scenedesmus sp. FS showed high alkali resistance. It also showed that even under the condition of a low inoculum rate (OD680, 0.08), Scenedesmus sp. FS could still grow in the outdoor raceway pond under a high alkaline environment. Even under unoptimized conditions, the oil content of Scenedesmus sp. FS could reach more than 22% and C16–C18 content could reach up to 79.68%, showing that this species has the potential for the biodiesel production in the near future. Shuhao Huo, Changhua Shang, Zhongming Wang, Weizheng Zhou, Fengjie Cui, Feifei Zhu, Zhenhong Yuan, and Renjie Dong Copyright © 2017 Shuhao Huo et al. All rights reserved. Selective Removal of Hemoglobin from Blood Using Hierarchical Copper Shells Anchored to Magnetic Nanoparticles Tue, 21 Feb 2017 09:49:27 +0000 Hierarchical copper shells anchored on magnetic nanoparticles were designed and fabricated to selectively deplete hemoglobin from human blood by immobilized metal affinity chromatography. Briefly, CoFe2O4 nanoparticles coated with polyacrylic acid were first synthesized by a one-pot solvothermal method. Hierarchical copper shells were then deposited by immobilizing Cu2+ on nanoparticles and subsequently by reducing between the solid CoFe2O4@COOH and copper solution with NaBH4. The resulting nanoparticles were characterized by scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectrometry, X-ray photoelectron spectroscopy, and vibrating sample magnetometry. The particles were also tested against purified bovine hemoglobin over a range of pH, contact time, and initial protein concentration. Hemoglobin adsorption followed pseudo-second-order kinetics and reached equilibrium in 90 min. Isothermal data also fit the Langmuir model well, with calculated maximum adsorption capacity 666 mg g−1. Due to the high density of Cu2+ on the shell, the nanoparticles efficiently and selectively deplete hemoglobin from human blood. Taken together, the results demonstrate that the particles with hierarchical copper shells effectively remove abundant, histidine-rich proteins, such as hemoglobin from human blood, and thereby minimize interference in diagnostic and other assays. Youxun Liu, Yaokun Wang, Mingyang Yan, and Juan Huang Copyright © 2017 Youxun Liu et al. All rights reserved. Bioengineered Plants Can Be a Useful Source of Omega-3 Fatty Acids Tue, 21 Feb 2017 08:28:21 +0000 Omega-3 fatty acids have proven to be very essential for human health due to their multiple health benefits. These essential fatty acids (EFAs) need to be uptaken through diet because they are unable to be produced by the human body. These are important for skin and hair growth as well as for proper visual, neural, and reproductive functions of the body. These fatty acids are proven to be extremely vital for normal tissue development during pregnancy and infancy. Omega-3 fatty acids can be obtained mainly from two dietary sources: marine and plant oils. Eicosapentaenoic acid (EPA; C20:5 n-3) and docosahexaenoic acid (DHA; C22:6 n-3) are the primary marine-derived omega-3 fatty acids. Marine fishes are high in omega-3 fatty acids, yet high consumption of those fishes will cause a shortage of fish stocks existing naturally in the oceans. An alternative source to achieve the recommended daily intake of EFAs is the demand of today. In this review article, an attempt has, therefore, been made to discuss the importance of omega-3 fatty acids and the recent developments in order to produce these fatty acids by the genetic modifications of the plants. Waleed Amjad Khan, Hu Chun-Mei, Nadeem Khan, Amjad Iqbal, Shan-Wu Lyu, and Farooq Shah Copyright © 2017 Waleed Amjad Khan et al. All rights reserved. Addressing Challenges to Enhance the Bioactives of Withania somnifera through Organ, Tissue, and Cell Culture Based Approaches Thu, 16 Feb 2017 12:52:47 +0000 Withania somnifera is a highly valued medicinal plant in traditional home medicine and is known for a wide range of bioactivities. Its commercial cultivation is adversely affected by poor seed viability and germination. Infestation by various pests and pathogens, survival under unfavourable environmental conditions, narrow genetic base, and meager information regarding biosynthesis of secondary metabolites are some of the other existing challenges in the crop. Biotechnological interventions through organ, tissue, and cell culture provide promising options for addressing some of these issues. In vitro propagation facilitates conservation and sustainable utilization of the existing germplasms and broadening the genetic base. It would also provide means for efficient and rapid mass propagation of elite chemotypes and generating uniform plant material round the year for experimentation and industrial applications. The potential of in vitro cell/organ cultures for the production of therapeutically valuable compounds and their large-scale production in bioreactors has received significant attention in recent years. In vitro culture system further provides distinct advantage for studying various cellular and molecular processes leading to secondary metabolite accumulation and their regulation. Engineering plants through genetic transformation and development of hairy root culture system are powerful strategies for modulation of secondary metabolites. The present review highlights the developments and sketches current scenario in this field. Pritika Singh, Rupam Guleri, Amrita Angurala, Kuldeep Kaur, Kulwinder Kaur, Sunil C. Kaul, Renu Wadhwa, and Pratap Kumar Pati Copyright © 2017 Pritika Singh et al. All rights reserved. Production of Human Cu,Zn SOD with Higher Activity and Lower Toxicity in E. coli via Mutation of Free Cysteine Residues Thu, 16 Feb 2017 00:00:00 +0000 Although, as an antioxidant enzyme, human Cu,Zn superoxide dismutase 1 (hSOD1) can mitigate damage to cell components caused by free radicals generated by aerobic metabolism, large-scale manufacturing and clinical use of hSOD1 are still limited by the challenge of rapid and inexpensive production of high-quality eukaryotic hSOD1 in recombinant forms. We have demonstrated previously that it is a promising strategy to increase the expression levels of soluble hSOD1 so as to increase hSOD1 yields in E. coli. In this study, a wild-type hSOD1 (wtSOD1) and three mutant SOD1s (mhSOD1s), in which free cysteines were substituted with serine, were constructed and their expression in soluble form was measured. Results show that the substitution of Cys111 (mhSOD1/C111S) increased the expression of soluble hSOD1 in E. coli whereas substitution of the internal Cys6 (mhSOD1/C6S) decreased it. Besides, raised levels of soluble expression led to an increase in hSOD1 yields. In addition, mhSOD1/C111S expressed at a higher soluble level showed lower toxicity and stronger whitening and antiradiation activities than those of wtSOD1. Taken together, our data demonstrate that C111S mutation in hSOD1 is an effective strategy to develop new SOD1-associated reagents and that mhSOD1/C111S is a satisfactory candidate for large-scale production. Kun Zhang, Yuejuan Zhang, Jing Zi, Xiaochang Xue, and Yi Wan Copyright © 2017 Kun Zhang et al. All rights reserved. Food Waste to Energy: An Overview of Sustainable Approaches for Food Waste Management and Nutrient Recycling Tue, 14 Feb 2017 10:22:20 +0000 Food wastage and its accumulation are becoming a critical problem around the globe due to continuous increase of the world population. The exponential growth in food waste is imposing serious threats to our society like environmental pollution, health risk, and scarcity of dumping land. There is an urgent need to take appropriate measures to reduce food waste burden by adopting standard management practices. Currently, various kinds of approaches are investigated in waste food processing and management for societal benefits and applications. Anaerobic digestion approach has appeared as one of the most ecofriendly and promising solutions for food wastes management, energy, and nutrient production, which can contribute to world’s ever-increasing energy requirements. Here, we have briefly described and explored the different aspects of anaerobic biodegrading approaches for food waste, effects of cosubstrates, effect of environmental factors, contribution of microbial population, and available computational resources for food waste management researches. Kunwar Paritosh, Sandeep K. Kushwaha, Monika Yadav, Nidhi Pareek, Aakash Chawade, and Vivekanand Vivekanand Copyright © 2017 Kunwar Paritosh et al. All rights reserved. Biotechnological Processes in Microbial Amylase Production Thu, 09 Feb 2017 00:00:00 +0000 Amylase is an important and indispensable enzyme that plays a pivotal role in the field of biotechnology. It is produced mainly from microbial sources and is used in many industries. Industrial sectors with top-down and bottom-up approaches are currently focusing on improving microbial amylase production levels by implementing bioengineering technologies. The further support of energy consumption studies, such as those on thermodynamics, pinch technology, and environment-friendly technologies, has hastened the large-scale production of the enzyme. Herein, the importance of microbial (bacteria and fungi) amylase is discussed along with its production methods from the laboratory to industrial scales. Subash C. B. Gopinath, Periasamy Anbu, M. K. Md Arshad, Thangavel Lakshmipriya, Chun Hong Voon, Uda Hashim, and Suresh V. Chinni Copyright © 2017 Subash C. B. Gopinath et al. All rights reserved. Corrigendum to “Comparison of Different Strategies for Selection/Adaptation of Mixed Microbial Cultures Able to Ferment Crude Glycerol Derived from Second-Generation Biodiesel” Mon, 06 Feb 2017 00:00:00 +0000 C. Varrone, T. M. B. Heggeset, S. B. Le, T. Haugen, S. Markussen, I. V. Skiadas, and H. N. Gavala Copyright © 2017 C. Varrone et al. All rights reserved. Effects of Six Commercial Saccharomyces cerevisiae Strains on Phenolic Attributes, Antioxidant Activity, and Aroma of Kiwifruit (Actinidia deliciosa cv.) Wine Mon, 30 Jan 2017 00:00:00 +0000 “Hayward” kiwifruit (Actinidia deliciosa cv.), widely planted all around the world, were fermented with six different commercial Saccharomyces cerevisiae strains (BM4×4, RA17, RC212, WLP77, JH-2, and CR476) to reveal their influence on the phenolic profiles, antioxidant activity, and aromatic components. Significant differences in the levels of caffeic acid, protocatechuate, and soluble solid content were found among wines with the six fermented strains. Wines fermented with RC212 strain exhibited the highest total phenolic acids as well as DPPH radical scavenging ability and also had the strongest ability to produce volatile esters. Wines made with S. cerevisiae BM 4×4 had the highest content of volatile acids, while the highest alcohol content was presented in CR476 wines. Scoring spots of wines with these strains were separated in different quadrants on the components of phenolics and aromas by principal component analyses. Kiwifruit wines made with S. cerevisiae RC212 were characterized by a rich fruity flavor, while CR476 strain and WLP77 strain produced floral flavors and green aromas, respectively. Altogether, the results indicated that the use of S. cerevisiae RC212 was the most suitable for the fermentation of kiwifruit wine with desirable characteristics. Xingchen Li, Yage Xing, Lin Cao, Qinglian Xu, Shaohua Li, Ranran Wang, Zijing Jiang, Zhenming Che, and Hongbin Lin Copyright © 2017 Xingchen Li et al. All rights reserved. Optimization of Hydrothermal and Diluted Acid Pretreatments of Tunisian Luffa cylindrica (L.) Fibers for 2G Bioethanol Production through the Cubic Central Composite Experimental Design CCD: Response Surface Methodology Tue, 24 Jan 2017 08:28:11 +0000 This paper opens up a new issue dealing with Luffa cylindrica (LC) lignocellulosic biomass recovery in order to produce 2G bioethanol. LC fibers are composed of three principal fractions, namely, α-cellulose (45.80%  ± 1.3), hemicelluloses (20.76%  ± 0.3), and lignins (13.15%  ± 0.6). The optimization of LC fibers hydrothermal and diluted acid pretreatments duration and temperature were achieved through the cubic central composite experimental design CCD. The pretreatments optimization was monitored via the determination of reducing sugars. Then, the 2G bioethanol process feasibility was tested by means of three successive steps, namely, LC fibers hydrothermal pretreatment performed at 96°C during 54 minutes, enzymatic saccharification carried out by means of a commercial enzyme AP2, and the alcoholic fermentation fulfilled with Saccharomyces cerevisiae. LC fibers hydrothermal pretreatment liberated 33.55 g/kg of reducing sugars. Enzymatic hydrolysis allowed achieving 59.4 g/kg of reducing sugars. The conversion yield of reducing sugar to ethanol was 88.66%. After the distillation step, concentration of ethanol was 1.58% with a volumetric yield about 70%. Kaouther Zaafouri, Manel Ziadi, Aida Ben Hassen-Trabelsi, Sabrine Mekni, Balkiss Aïssi, Marwen Alaya, Latifa Bergaoui, and Moktar Hamdi Copyright © 2017 Kaouther Zaafouri et al. All rights reserved. Considering the Specific Impact of Harsh Conditions and Oil Weathering on Diversity, Adaptation, and Activity of Hydrocarbon-Degrading Bacteria in Strategies of Bioremediation of Harsh Oily-Polluted Soils Tue, 24 Jan 2017 06:25:01 +0000 Weathering processes change properties and composition of spilled oil, representing the main reason of failure of bioaugmentation strategies. Our purpose was to investigate the metabolic adaptation of hydrocarbon-degrading bacteria at harsh conditions to be considered to overcome the limitations of bioaugmentation strategies at harsh conditions. Polluted soils, exposed for prolonged periods to weathered oil in harsh soils and weather conditions, were used. Two types of enrichment cultures were employed using 5% and 10% oil or diesel as sole carbon sources with varying the mineral nitrogen sources and C/N ratios. The most effective isolates were obtained based on growth, tolerance to toxicity, and removal efficiency of diesel hydrocarbons. Activities of the newly isolated bacteria, in relation to the microenvironment from where they were isoalted and their interaction with the weathered oil, showed individual specific ability to adapt when exposed to such factors, to acquire metabolic potentialities. Among 39 isolates, ten identified ones by 16S rDNA genes similarities, including special two Pseudomonas isolates and one Citrobacter isolate, showed particularity of shifting hydrocarbon-degrading ability from short chain n-alkanes (n-C12–n-C16) to longer chain n-alkanes (n-C21–n-C25) and vice versa by alternating nitrogen source compositions and C/N ratios. This is shown for the first time. Zulfa Al Disi, Samir Jaoua, Dhabia Al-Thani, Saeed Al-Meer, and Nabil Zouari Copyright © 2017 Zulfa Al Disi et al. All rights reserved. A Mathematical Model of Neutral Lipid Content in terms of Initial Nitrogen Concentration and Validation in Coelastrum sp. HA-1 and Application in Chlorella sorokiniana Wed, 18 Jan 2017 00:00:00 +0000 Microalgae are considered to be a potential major biomass feedstock for biofuel due to their high lipid content. However, no correlation equations as a function of initial nitrogen concentration for lipid accumulation have been developed for simplicity to predict lipid production and optimize the lipid production process. In this study, a lipid accumulation model was developed with simple parameters based on the assumption protein synthesis shift to lipid synthesis by a linear function of nitrogen quota. The model predictions fitted well for the growth, lipid content, and nitrogen consumption of Coelastrum sp. HA-1 under various initial nitrogen concentrations. Then the model was applied successfully in Chlorella sorokiniana to predict the lipid content with different light intensities. The quantitative relationship between initial nitrogen concentrations and the final lipid content with sensitivity analysis of the model were also discussed. Based on the model results, the conversion efficiency from protein synthesis to lipid synthesis is higher and higher in microalgae metabolism process as nitrogen decreases; however, the carbohydrate composition content remains basically unchanged neither in HA-1 nor in C. sorokiniana. Zhenhua Yang, Yue Zhao, Zhiyong Liu, Chenfeng Liu, Zhipeng Hu, and Yuyong Hou Copyright © 2017 Zhenhua Yang et al. All rights reserved. Screening and Expression of a Silicon Transporter Gene (Lsi1) in Wild-Type Indica Rice Cultivars Mon, 16 Jan 2017 09:48:14 +0000 Silicon (Si) is one of the most prevalent elements in the soil. It is beneficial for plant growth and development, and it contributes to plant defense against different stresses. The Lsi1 gene encodes a Si transporter that was identified in a mutant Japonica rice variety. This gene was not identified in fourteen Malaysian rice varieties during screening. Then, a mutant version of Lsi1 was substituted for the native version in the three most common Malaysian rice varieties, MR219, MR220, and MR276, to evaluate the function of the transgene. Real-time PCR was used to explore the differential expression of Lsi1 in the three transgenic rice varieties. Silicon concentrations in the roots and leaves of transgenic plants were significantly higher than in wild-type plants. Transgenic varieties showed significant increases in the activities of the enzymes SOD, POD, APX, and CAT; photosynthesis; and chlorophyll content; however, the highest chlorophyll A and B levels were observed in transgenic MR276. Transgenic varieties have shown a stronger root and leaf structure, as well as hairier roots, compared to the wild-type plants. This suggests that Lsi1 plays a key role in rice, increasing the absorption and accumulation of Si, then alters antioxidant activities, and improves morphological properties. Mahbod Sahebi, Mohamed M. Hanafi, M. Y. Rafii, Parisa Azizi, Rambod Abiri, Nahid Kalhori, and Narges Atabaki Copyright © 2017 Mahbod Sahebi et al. All rights reserved. Effects of Frequency and Acceleration Amplitude on Osteoblast Mechanical Vibration Responses: A Finite Element Study Thu, 15 Dec 2016 12:56:33 +0000 Bone cells are deformed according to mechanical stimulation they receive and their mechanical characteristics. However, how osteoblasts are affected by mechanical vibration frequency and acceleration amplitude remains unclear. By developing 3D osteoblast finite element (FE) models, this study investigated the effect of cell shapes on vibration characteristics and effect of acceleration (vibration intensity) on vibrational responses of cultured osteoblasts. Firstly, the developed FE models predicted natural frequencies of osteoblasts within 6.85–48.69 Hz. Then, three different levels of acceleration of base excitation were selected (0.5, 1, and 2 g) to simulate vibrational responses, and acceleration of base excitation was found to have no influence on natural frequencies of osteoblasts. However, vibration response values of displacement, stress, and strain increased with the increase of acceleration. Finally, stress and stress distributions of osteoblast models under 0.5 g acceleration in Z-direction were investigated further. It was revealed that resonance frequencies can be a monotonic function of cell height or bottom area when cell volumes and material properties were assumed as constants. These findings will be useful in understanding how forces are transferred and influence osteoblast mechanical responses during vibrations and in providing guidance for cell culture and external vibration loading in experimental and clinical osteogenesis studies. Liping Wang, Hung-Yao Hsu, Xu Li, and Cory J. Xian Copyright © 2016 Liping Wang et al. All rights reserved. Corrigendum to “Cytotoxic Effect of Ethanol Extract of Microalga, Chaetoceros calcitrans, and Its Mechanisms in Inducing Apoptosis in Human Breast Cancer Cell Line” Tue, 29 Nov 2016 08:53:03 +0000 Siyamak Ebrahimi Nigjeh, Fatimah Md Yusoff, Noorjahan Banu Mohamed Alitheen, Mehdi R. Pirozyan, Yeap Swee Keong, and Abdul Rahman bin Omar Copyright © 2016 Siyamak Ebrahimi Nigjeh et al. All rights reserved. Expression, Purification, and Characterization of a Sucrose Nonfermenting 1-Related Protein Kinases 2 of Arabidopsis thaliana in E. coli-Based Cell-Free System Thu, 24 Nov 2016 06:15:44 +0000 The plant-specific sucrose nonfermenting 1-related protein kinase 2 (SnRK2) family is considered an important regulator of plant responses to abiotic stresses such as drought, cold, salinity, and nutrition deficiency. However, little information is available on how SnRK2s regulate sulfur deprivation responses in Arabidopsis. Large-scale production of SnRK2 kinases in vitro can help to elucidate the biochemical properties and physiological functions of this protein family. However, heterogenous expression of SnRK2s usually leads to inactive proteins. In this study, we expressed a recombinant Arabidopsis SnRK2.1 in a modified E. coli cell-free system, which combined two kinds of extracts allowing for a convenient and affordable protein preparation. The recombinant SnRK2.1 was produced in large-scale and the autophosphorylation activity of purified SnRK2.1 was characterized, allowing for further biochemical and substrate binding analysis in sulfur signaling. The application of this improved E. coli cell-free system provides us a promising and convenient platform to enhance expression of the target proteins economically. Xu Zhang, Shuangxi Zhang, Jun Wang, Jing Zi, Jianhui Wang, Shaolin Chen, and Yi Wan Copyright © 2016 Xu Zhang et al. All rights reserved. Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from Geobacillus stearothermophilus by Site-Directed Mutagenesis at the Active Site Thu, 17 Nov 2016 08:41:10 +0000 The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1.543 (Bst DNA polymerase) with 5′-3′ DNA polymerase activity while in absence of 5′-3′ exonuclease activity possesses high thermal stability and polymerase activity. Bst DNA polymerase was employed in isothermal multiple self-matching initiated amplification (IMSA) which amplified the interest sequence with high selectivity and was widely applied in the rapid detection of human epidemic diseases. However, the detailed information of commercial Bst DNA polymerase is unpublished and well protected by patents, which makes the high price of commercial kits. In this study, wild-type Bst DNA polymerase (WT) and substitution mutations for improving the efficiency of DNA polymerization were expressed and purified in E. coli. Site-directed substitutions of four conserved residues (Gly310, Arg412, Lys416, and Asp540) in the activity site of Bst DNA polymerase influenced efficiency of polymerizing dNTPs. The substitution of residue Gly310 by alanine or leucine and residue Asp540 by glutamic acid increased the efficiency of polymerase activity. All mutants with higher polymerizing efficiency were employed to complete the rapid detection of EV71-associated hand, foot, and mouth disease (HFMD) by IMSA approach with relatively shorter period which is suitable for the primary diagnostics setting in rural and underdeveloped areas. Yi Ma, Beilei Zhang, Meng Wang, Yanghui Ou, Jufang Wang, and Shan Li Copyright © 2016 Yi Ma et al. All rights reserved. Erratum to “Inhibitory Effect of Long-Chain Fatty Acids on Biogas Production and the Protective Effect of Membrane Bioreactor” Tue, 15 Nov 2016 06:51:26 +0000 Kris Triwulan Dasa, Supansa Y. Westman, Ria Millati, Muhammad Nur Cahyanto, Mohammad J. Taherzadeh, and Claes Niklasson Copyright © 2016 Kris Triwulan Dasa et al. All rights reserved. Studies on the Ecological Adaptability of Growing Rice with Floating Bed on the Dilute Biogas Slurry Tue, 01 Nov 2016 09:06:49 +0000 This study aimed to explore the ecological adaptability and the possibility of growing rice with floating bed on the dilute biogas slurry. The results of the experiments show that the growth stage, rice plant height, and rice yield and quality were significantly affected by multiple dilutions; rice plants cultivated with 45 multiple dilutions had better ecological adaptability than others. In the 45 multiple dilutions’ group, the yield of rice was 13.3 g/bucket (8 rice plants), milled rice rate was 63.1%, and the content of crude protein in the rice was 6.3%. The concentrations of heavy metals in the rice cultivated with 30 multiple dilutions’ slurry, such as total lead, cadmium, mercury, chromium, and arsenic, were all below the national standard. The study shows that it is possible and safe to cultivate rice plants with no soil but diluted biogas slurry. In the experiments, the yield, milled rice rate, and crude protein of the rice cultivated with slurry were not as much as those of rice cultivated with regular way in soil. This study provides the basic theoretical support for the development of biogas projects and the potential achievement of organic farming in special agricultural facilities and circular economy. Qun Kang, Rui Li, Qi Du, Bowen Cheng, Zhiqi Liao, Chengcheng Sun, and Zhaohua Li Copyright © 2016 Qun Kang et al. All rights reserved. Frontiers in the Expansion of Bioproducts Mon, 31 Oct 2016 14:29:36 +0000 Junio Cota, Joachim Venus, Zaira B. Hoffmam, and Lucas F. Ribeiro Copyright © 2016 Junio Cota et al. All rights reserved. Effect of Static Load on the Nucleus Pulposus of Rabbit Intervertebral Disc Motion Segment in an Organ Culture Mon, 31 Oct 2016 08:08:36 +0000 The development of mechanically active culture systems helps in understanding of the role of mechanical stress in intervertebral disc (IVD) degeneration. Motion segment cultures facilitate the application and control of mechanical loads. The purpose of this study was to establish a culturing method for rabbit IVD motion segments to observe the effect of static load on the whole disc organ. Segments were cultured in custom-made apparatuses under a constant, compressive load (3 kg) for 2 weeks. Tissue integrity, matrix synthesis, and matrix gene expression profile were assessed and compared with fresh one. The results showed ex vivo culturing of samples gradually destroyed the morphology. Proteoglycan contents and gene expression were decreased and downregulated obviously. However, immunohistochemical staining intensity and collagen type II gene expression were significantly enhanced and upregulated. In contrast, these trends were reversed under constant compression. These results indicated short-term static load stimulated the synthesis of type II collagen; however, constant compression led to progressive degeneration and specifically to proteoglycan. Through this study a loading and organ-culturing system for ex vivo rabbit IVD motion segments was developed, which can be used to study the effects of mechanical stimulation on the biology of IVDs and the pathomechanics of IVD degeneration. Jia-Wen Zhan, Min-Shan Feng, Li-Guo Zhu, Ping Zhang, and Jie Yu Copyright © 2016 Jia-Wen Zhan et al. All rights reserved. Selection of Lipases for the Synthesis of Biodiesel from Jatropha Oil and the Potential of Microwave Irradiation to Enhance the Reaction Rate Thu, 27 Oct 2016 05:59:25 +0000 The present study deals with the enzymatic synthesis of biodiesel by transesterification of Jatropha oil (Jatropha curcas L.) with ethanol in a solvent-free system. Seven commercial lipase preparations immobilized by covalent attachment on epoxy-polysiloxane-polyvinyl alcohol composite (epoxy-SiO2-PVA) were tested as biocatalysts. Among them, immobilized lipases from Pseudomonas fluorescens (lipase AK) and Burkholderia cepacia (lipase PS) were the most active biocatalysts in biodiesel synthesis, reaching ethyl ester yields (FAEE) of 91.1 and 98.3% at 72 h of reaction, respectively. The latter biocatalyst exhibited similar performance compared to Novozym® 435. Purified biodiesel was characterized by different techniques. Transesterification reaction carried out under microwave irradiation exhibited higher yield and productivity than conventional heating. The operational stability of immobilized lipase PS was determined in repeated batch runs under conventional and microwave heating systems, revealing half-life times of 430.4 h and 23.5 h, respectively. Livia T. A. Souza, Adriano A. Mendes, and Heizir F. de Castro Copyright © 2016 Livia T. A. Souza et al. All rights reserved. Isoprene Production on Enzymatic Hydrolysate of Peanut Hull Using Different Pretreatment Methods Tue, 25 Oct 2016 13:09:50 +0000 The present study is about the use of peanut hull for isoprene production. In this study, two pretreatment methods, hydrogen peroxide-acetic acid (HPAC) and popping, were employed prior to enzymatic hydrolysis, which could destroy the lignocellulosic structure and accordingly improve the efficiency of enzymatic hydrolysis. It is proven that the isoprene production on enzymatic hydrolysate with HPAC pretreatment is about 1.9-fold higher than that of popping pretreatment. Moreover, through High Performance Liquid Chromatography (HPLC) analysis, the amount and category of inhibitors such as formic acid, acetic acid, and HMF were assayed and were varied in different enzymatic hydrolysates, which may be the reason leading to a decrease in isoprene production during fermentation. To further increase the isoprene yield, the enzymatic hydrolysate of HPAC was detoxified by activated carbon. As a result, using the detoxified enzymatic hydrolysate as the carbon source, the engineered strain YJM21 could accumulate 297.5 mg/L isoprene, which accounted for about 90% of isoprene production by YJM21 fermented on pure glucose (338.6 mg/L). This work is thought to be the first attempt on isoprene production by E. coli using peanut hull as the feedstock. More importantly, it also shows the prospect of peanut hull to be considered as an alternative feedstock for bio-based chemicals or biofuels production due to its easy access and high polysaccharide content. Sumeng Wang, Ruichao Li, Xiaohua Yi, Tigao Fang, Jianming Yang, and Hyeun-Jong Bae Copyright © 2016 Sumeng Wang et al. All rights reserved. Comparative Genomic Analysis of Delftia tsuruhatensis MTQ3 and the Identification of Functional NRPS Genes for Siderophore Production Tue, 25 Oct 2016 11:09:56 +0000 Plant growth-promoting rhizobacteria (PGPR) are a group of rhizosphere bacteria that promote plant growth. Delftia tsuruhatensis MTQ3 is a member of PGPR that produces siderophores. The draft genome sequence of MTQ3 has been reported. Here, we analyzed the genome sequence of MTQ3 and performed a comparative genome analysis of four sequenced Delftia strains, revealing genetic relationships among these strains. In addition, genes responsible for bacteriocin and nonribosomal peptide synthesis were detected in the genomes of each strain. To reveal the functions of NRPS genes in siderophore production in D. tsuruhatensis MTQ3, three NRPS genes were knocked out to obtain the three mutants MTQ3-Δ1941, MTQ3-Δ1945, and MTQ3-Δ1946, which were compared with the wild-type strain. In qualitative and quantitative analyses using CAS assay, the mutants failed to produce siderophores. Accordingly, the NRPS genes in MTQ3 were functionally related to siderophore production. These results clarify one mechanism by which plant growth is promoted in MTQ3 and have important applications in agricultural production. Haimeng Guo, Yanan Yang, Kai Liu, Wenfeng Xu, Jianyong Gao, Hairong Duan, Binghai Du, Yanqin Ding, and Chengqiang Wang Copyright © 2016 Haimeng Guo et al. All rights reserved. Isolation and Physicochemical Characterization of Laccase from Ganoderma lucidum-CDBT1 Isolated from Its Native Habitat in Nepal Sun, 16 Oct 2016 13:37:16 +0000 At present, few organisms are known to and capable of naturally producing laccases and white rot fungi are one such group. In the present study, three fungal species, namely, Ganoderma lucidum-CDBT1, Ganoderma japonicum, and Lentinula edodes, isolated from their native habitat in Nepal were screened for laccase production, and G. lucidum-CDBT1 was found to express highest levels of enzyme (day 10 culture media showed 0.92 IU/mg total protein or 92 IU/mL laccase activity with ABTS as substrate). Lignin extracted from rice straw was used in Olga medium for laccase production and isolation from G. lucidum-CDBT1. Presence of lignin (5 g/L) and copper sulfate (30 μM) in the media increased the extracellular laccase content by 111% and 114%, respectively. The laccase enzyme produced by G. lucidum-CDBT1 was fractionated by ammonium sulfate and purified by DEAE Sepharose anion exchange chromatography. The purified enzyme was found to have a molecular mass of 43 kDa and exhibits optimal activity at pH 5.0 and 30°C. The isolated laccase was thermally stable for up to 70°C for 1 h and exhibited broad pH stability. The kinetic constants, , , and , determined using 2,2′-azinobis-(-3-ethylbenzothiazoline-6-sulfonic acid) as substrate were found to be 110 μM, 36 μmol/min/mg, and 246 min−1, respectively. The isolated thermostable laccase will be used in future experiments for delignification process. Prabin Shrestha, Bishnu Joshi, Jarina Joshi, Rajani Malla, and Lakshmaiah Sreerama Copyright © 2016 Prabin Shrestha et al. All rights reserved. Co-Digestion of Sugar Beet Silage Increases Biogas Yield from Fibrous Substrates Tue, 11 Oct 2016 13:31:33 +0000 This study tested the hypothesis that the easily degradable carbohydrates of the sugar beet silage (S) will improve the anaerobic digestion of grass silage (G) more profoundly compared to co-digestion of sugar beet silage with maize silage (M). M : S and G : S mixtures were tested in two continuous laboratory-scale AD experiments at volatile solid ratios of 1 : 0, 6 : 1, 3 : 1, and 1 : 3 at organic loading rates of 1.5 kgVS m−3 day−1. While the sugar beet effects in mixtures with maize silage were negligible, co-digestion with grass silage showed a beneficial performance. There, the specific methane production rate was 0.27  kg−1VS h−1 at G : S ratio of 6 : 1 compared to G : S 1 : 0 with 0.14  kg−1VS h−1. In comparison to G : S 1 : 0, about 44% and 62% higher biogas yields were obtained at G : S 6 : 1 and 3 : 1, respectively. Also, the highest methane concentration was found in G : S at ratio of 1 : 3. Synergistic increase of methane yield was found in co-digestion in both experiments, but higher effect was realized in G : S, independently of the amount of sugar beet silage. The findings of this study emphasize the improvement of AD of grass silage by even low addition of sugar beet silage. Sharif Ahmed, Daniel Einfalt, and Marian Kazda Copyright © 2016 Sharif Ahmed et al. All rights reserved. High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli Wed, 28 Sep 2016 11:44:28 +0000 Human epidermal growth factor (hEGF) is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe) at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO) and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10. The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development. Yi Ma, Jieying Yu, Jinglian Lin, Shaomin Wu, Shan Li, and Jufang Wang Copyright © 2016 Yi Ma et al. All rights reserved. Potential of Polycyclic Aromatic Hydrocarbon-Degrading Bacterial Isolates to Contribute to Soil Fertility Wed, 28 Sep 2016 11:20:18 +0000 Restoration of polycyclic aromatic hydrocarbon- (PAH-) polluted sites is presently a major challenge in agroforestry. Consequently, microorganisms with PAH-degradation ability and soil fertility improvement attributes are sought after in order to achieve sustainable remediation of polluted sites. This study isolated PAH-degrading bacteria from enriched cultures of spent automobile engine-oil polluted soil. Isolates’ partial 16S rRNA genes were sequenced and taxonomically classified. Isolates were further screened for their soil fertility attributes such as phosphate solubilization, atmospheric nitrogen fixation, and indoleacetic acid (IAA) production. A total of 44 isolates were obtained and belong to the genera Acinetobacter, Arthrobacter, Bacillus, Flavobacterium, Microbacterium, Ochrobactrum, Pseudomonas, Pseudoxanthomonas, Rhodococcus, and Stenotrophomonas. Data analysed by principal component analysis showed the Bacillus and Ochrobactrum isolates displayed outstanding IAA production. Generalized linear modelling statistical approaches were applied to evaluate the contribution of the four most represented genera (Pseudomonas, Acinetobacter, Arthrobacter, and Rhodococcus) to soil fertility. The Pseudomonas isolates were the most promising in all three soil fertility enhancement traits evaluated and all isolates showed potential for one or more of the attributes evaluated. These findings demonstrate a clear potential of the isolates to participate in restorative bioremediation of polluted soil, which will enhance sustainable agricultural production and environmental protection. Maryam Bello-Akinosho, Rosina Makofane, Rasheed Adeleke, Mapitsi Thantsha, Michael Pillay, and George Johannes Chirima Copyright © 2016 Maryam Bello-Akinosho et al. All rights reserved. Alkaline Pretreatment of Sugarcane Bagasse and Filter Mud Codigested to Improve Biomethane Production Wed, 21 Sep 2016 08:27:43 +0000 To enhance the codigestion of degradation and improve biomethane production potential, sugarcane bagasse and filter mud were pretreated by sodium hydroxide NaOH 1 N at 100°C for 15, 30, and 45 minutes, respectively. Biomethane generation from 1-liter batch reactor was studied at mesophilic temperature ()°C, solid concentrations of 6%, and five levels of mixing proportion with and without pretreatment. The results demonstrate that codigestion of filter mud with bagasse produces more biomethane than fermentation of filter mud as single substrate; even codigested substrate composition presented a better balance of nutrients (C/N ratio of 24.70) when codigestion ratio between filter mud and bagasse was 25 : 75 in comparison to filter mud as single substrate (C/N ratio 9.68). All the pretreatments tested led to solubilization of the organic matter, with a maximum lignin reduction of 86.27% and cumulative yield of biomethane (195.8 mL·g, digestion of pretreated bagasse as single substrate) obtained after 45 minutes of cooking by NaOH 1 N at 100°C. Under this pretreatment condition, significant increase in cumulative methane yield was observed (126.2 mL·g) at codigestion ratio of 25 : 75 between filter mud and bagasse by increase of 81.20% from untreated composition. Zahir Talha, Weimin Ding, Esmaeil Mehryar, Muhammad Hassan, and Jinhua Bi Copyright © 2016 Zahir Talha et al. All rights reserved. Optimized Collection Protocol for Plasma MicroRNA Measurement in Patients with Cardiovascular Disease Tue, 20 Sep 2016 16:39:51 +0000 Background. Various microRNAs (miRNAs) are used as markers of acute coronary syndrome, in which heparinization is considered mandatory therapy. Nevertheless, a standard method of handling plasma samples has not been proposed, and the effects of heparin treatment on miRNA detection are rarely discussed. Materials and Method. This study used quantitative polymerase chain reaction (qPCR) analysis to investigate how storage temperature, standby time, hemolysis, and heparin treatment affect miRNA measurement in plasma samples from 25 patients undergoing cardiac catheterization. Results. For most miRNAs, the qPCR results remained consistent during the first 2 hours. The miRNA signals did not significantly differ between samples stored at 4°C before processing and samples stored at room temperature (RT) before processing. miR-451a/miR-23a ratio < 60 indicated < 0.12% hemolysis with 100% sensitivity and 100% specificity. Pretreatment with 0.25 U heparinase I recovered qPCR signals that were reduced by in vivo heparinization. Conclusions. For miRNA measurement, blood samples stored at RT should be processed into plasma within 2 hours after withdrawal and should be pretreated with 0.25 U heparinase I to overcome heparin-attenuated miRNA signals. The miR-451a/miR-23a ratio is a reliable indicator of significant hemolysis. Chi-Sheng Wu, Fen-Chiung Lin, Shu-Jen Chen, Yung-Lung Chen, Wen-Jung Chung, and Cheng-I Cheng Copyright © 2016 Chi-Sheng Wu et al. All rights reserved. Strategies for Lipid Production Improvement in Microalgae as a Biodiesel Feedstock Tue, 20 Sep 2016 13:53:27 +0000 In response to the energy crisis, global warming, and climate changes, microalgae have received a great deal of attention as a biofuel feedstock. Due to a high lipid content in microalgal cells, microalgae present as a promising alternative source for the production of biodiesel. Environmental and culturing condition variations can alter lipid production as well as chemical compositions of microalgae. Therefore, application of the strategies to activate lipid accumulation opens the door for lipid overproduction in microalgae. Until now, many original studies regarding the approaches for enhanced microalgal lipid production have been reported in an effort to push forward the production of microalgal biodiesel. However, the current literature demonstrates fragmented information available regarding the strategies for lipid production improvement. From the systematic point of view, the review highlights the main approaches for microalgal lipid accumulation induction to expedite the application of microalgal biodiesel as an alternative to fossil diesel for sustainable environment. Of the several strategies discussed, the one that is most commonly applied is the design of nutrient (e.g., nitrogen, phosphorus, and sulfur) starvation or limitation. Other viable approaches such as light intensity, temperature, carbon dioxide, salinity stress, and metal influence can also achieve enhanced microalgal lipid production. L. D. Zhu, Z. H. Li, and E. Hiltunen Copyright © 2016 L. D. Zhu et al. All rights reserved. Simultaneous Saccharification and Fermentation of Sugar Beet Pulp for Efficient Bioethanol Production Mon, 19 Sep 2016 14:11:02 +0000 Sugar beet pulp, a byproduct of sugar beet processing, can be used as a feedstock in second-generation ethanol production. The objective of this study was to investigate the effects of pretreatment, of the dosage of cellulase and hemicellulase enzyme preparations used, and of aeration on the release of fermentable sugars and ethanol yield during simultaneous saccharification and fermentation (SSF) of sugar beet pulp-based worts. Pressure-thermal pretreatment was applied to sugar beet pulp suspended in 2% w/w sulphuric acid solution at a ratio providing 12% dry matter. Enzymatic hydrolysis was conducted using Viscozyme and Ultraflo Max (Novozymes) enzyme preparations (0.015–0.02 mL/g dry matter). Two yeast strains were used for fermentation: Ethanol Red (S. cerevisiae) (1 g/L) and Pichia stipitis (0.5 g/L), applied sequentially. The results show that efficient simultaneous saccharification and fermentation of sugar beet pulp was achieved. A 6 h interval for enzymatic activation between the application of enzyme preparations and inoculation with Ethanol Red further improved the fermentation performance, with the highest ethanol concentration reaching  g/L and fermentation efficiency relative to the theoretical yield. Joanna Berłowska, Katarzyna Pielech-Przybylska, Maria Balcerek, Urszula Dziekońska-Kubczak, Piotr Patelski, Piotr Dziugan, and Dorota Kręgiel Copyright © 2016 Joanna Berłowska et al. All rights reserved. Copyrolysis of Biomass and Coal: A Review of Effects of Copyrolysis Parameters, Product Properties, and Synergistic Mechanisms Sun, 18 Sep 2016 09:17:42 +0000 Concerns in the last few decades regarding the environmental and socioeconomic impacts of the dependence on fossil fuels have resulted in calls for more renewable and alternative energy sources. This has led to recent interest in copyrolysis of biomass and coal. Numerous reviews have been found related to individual pyrolysis of coal and biomass. This review deals mainly with the copyrolysis of coal and biomass and then compares their results with those obtained using coal and biomass pyrolysis in detail. It is controversial whether there are synergistic or additive behaviours when coal and biomass are blended during copyrolysis. In this review, the effects of reaction parameters such as feedstock types, blending ratio, heating rate, temperature, and reactor types on the occurrence of synergy are discussed. Also, the main properties of the copyrolytic products are pointed out. Some possible synergistic mechanisms are also suggested. Additionally, several outlooks based on studies in the literature are also presented in this paper. Cui Quan and Ningbo Gao Copyright © 2016 Cui Quan and Ningbo Gao. All rights reserved. Corrigendum to “miR-1-Mediated Induction of Cardiogenesis in Mesenchymal Stem Cells via Downregulation of Hes-1” Wed, 14 Sep 2016 09:28:38 +0000 Feng Huang, Liang Tang, Zhen-fei Fang, Xin-qun Hu, Jia-yi Pan, and Sheng-hua Zhou Copyright © 2016 Feng Huang et al. All rights reserved. Inhibitory Effect of Long-Chain Fatty Acids on Biogas Production and the Protective Effect of Membrane Bioreactor Thu, 08 Sep 2016 12:01:06 +0000 Anaerobic digestion of lipid-containing wastes for biogas production is often hampered by the inhibitory effect of long-chain fatty acids (LCFAs). In this study, the inhibitory effects of LCFAs (palmitic, stearic, and oleic acid) on biogas production as well as the protective effect of a membrane bioreactor (MBR) against LCFAs were examined in thermophilic batch digesters. The results showed that palmitic and oleic acid with concentrations of 3.0 and 4.5 g/L resulted in >50% inhibition on the biogas production, while stearic acid had an even stronger inhibitory effect. The encased cells in the MBR system were able to perform better in the presence of LCFAs. This system exhibited a significantly lower percentage of inhibition than the free cell system, not reaching over 50% at any LCFA concentration tested. Kris Triwulan Dasa, Supansa Y. Westman, Ria Millati, Muhammad Nur Cahyanto, Mohammad J. Taherzadeh, and Claes Niklasson Copyright © 2016 Kris Triwulan Dasa et al. All rights reserved. Complex Approach to Conceptual Design of Machine Mechanically Extracting Oil from Jatropha curcas L. Seeds for Biomass-Based Fuel Production Wed, 07 Sep 2016 16:49:03 +0000 One of important sources of biomass-based fuel is Jatropha curcas L. Great attention is paid to the biofuel produced from the oil extracted from the Jatropha curcas L. seeds. A mechanised extraction is the most efficient and feasible method for oil extraction for small-scale farmers but there is a need to extract oil in more efficient manner which would increase the labour productivity, decrease production costs, and increase benefits of small-scale farmers. On the other hand innovators should be aware that further machines development is possible only when applying the systematic approach and design methodology in all stages of engineering design. Systematic approach in this case means that designers and development engineers rigorously apply scientific knowledge, integrate different constraints and user priorities, carefully plan product and activities, and systematically solve technical problems. This paper therefore deals with the complex approach to design specification determining that can bring new innovative concepts to design of mechanical machines for oil extraction. The presented case study as the main part of the paper is focused on new concept of screw of machine mechanically extracting oil from Jatropha curcas L. seeds. Ivan Mašín and Michal Petrů Copyright © 2016 Ivan Mašín and Michal Petrů. All rights reserved. Enzymatic Synthesis of Sorboyl-Polydatin Prodrug in Biomass-Derived 2-Methyltetrahydrofuran and Antiradical Activity of the Unsaturated Acylated Derivatives Wed, 07 Sep 2016 12:02:43 +0000 Efficient and highly regioselective synthesis of the potential 6′′-O-sorboyl-polydatin prodrug in biomass-derived 2-methyltetrahydrofuran (2-MeTHF) was achieved using Candida antarctica lipase B for the first time. Under the optimal conditions, the initial reaction rate, maximum substrate conversion, and 6′′-regioselectivity were as high as 8.65 mM/h, 100%, and 100%, respectively. Kinetic and operational stability investigations evidently demonstrated excellent enzyme compatibility of the 2-MeTHF compared to the traditional organic solvents. With respect to the antioxidant properties, three unsaturated ester derivatives showed slightly lower DPPH radical scavenging activities than the parent agent. Interestingly, further studies also revealed that the antiradical capacities of the acylates decreased with the elongation of the unsaturated aliphatic chain length from C4 to C11. The reason might be attributed to the increased steric hindrance derived from the acyl residues in derivatives. Zhaoyu Wang, Yanhong Bi, Rongling Yang, Xiangjie Zhao, Ling Jiang, Chun Zhu, Yuping Zhao, and Jianbo Jia Copyright © 2016 Zhaoyu Wang et al. All rights reserved. Anaerobic Codigestion of Municipal Wastewater Treatment Plant Sludge with Food Waste: A Case Study Mon, 05 Sep 2016 13:18:00 +0000 The aim of this study was to assess the effects of the codigestion of food manufacturing and processing wastes (FW) with sewage sludge (SS), that is, municipal wastewater treatment plant primary sludge and waste activated sludge. Bench scale mesophilic anaerobic reactors were fed intermittently with varying ratio of SS and FW and operated at a hydraulic retention time of 20 days and organic loading of 2.0 kg TS/m3·d. The specific biogas production (SBP) increased by 25% to 50% with the addition of 1%–5% FW to SS which is significantly higher than the SBP from SS of   VS added. Although the TS, VS, and tCOD removal slightly increased, the biogas yield and methane content improved significantly and no inhibitory effects were observed as indicated by the stable pH throughout the experiment. Metal screening of the digestate suggested the biosolids meet the guidelines for use as a soil conditioner. Batch biochemical methane potential tests at different ratios of SS : FW were used to determine the optimum ratio using surface model analysis. The results showed that up to 47-48% FW can be codigested with SS. Overall these results confirm that codigestion has great potential in improving the methane yield of SS. Zubayeda Zahan, Maazuza Z. Othman, and William Rajendram Copyright © 2016 Zubayeda Zahan et al. All rights reserved. Molecular Characterization of LRB7 Gene and a Water Channel Protein TIP2 in Chorispora bungeana Thu, 01 Sep 2016 08:47:04 +0000 Background. Water channel proteins, also called aquaporins, are integral membrane proteins from major intrinsic protein (MIP) family and involved in several pathways including not only water transport but also cell signaling, reproduction, and photosynthesis. The full cDNA and protein sequences of aquaporin in Chorispora bungeana Fisch. & C.A. Mey (C. bungeana) are still unknown. Results. In this study, PCR and rapid amplification of cDNA ends approaches were used to clone the full cDNA of LRB7 (GenBank accession number: EU636988) of C. bungeana. Sequence analysis indicated that it was 1235 bp, which had two introns and encoded a protein of 250 amino acids. Structure analysis revealed that the protein had two conserved NPA motifs, one of which is MIP signature sequence (SGxHxNPAVT), six membrane helix regions, and additional membrane-embedded domains. Phylogenetic analysis suggested that the protein was from TIP2 subgroup. Surprisingly, semiquantitative RT-PCR experiment and western blot analysis showed that LRB7 and TIP2 were only detectable in roots, unlike Arabidopsis and Raphanus. Connecting with our previous studies, LRB7 was supported to associate with chilling-tolerance in C. bungeana. Conclusion. This is the first time to characterize the full sequences of LRB7 gene and water channel protein in C. bungeana. Our findings contribute to understanding the water transports in plants under low temperatures. Ming Li, Zhaoxu Liang, Cuixia Di, Weikuan Fang, Kaichao Wu, Maoshan Chen, Shanshan He, Yuan Zeng, Yan Jing, Jun Liang, Fang Tan, Song Li, Tuo Chen, Guangxiu Liu, and Lizhe An Copyright © 2016 Ming Li et al. All rights reserved. Extracellular Ribonuclease from Bacillus licheniformis (Balifase), a New Member of the N1/T1 RNase Superfamily Tue, 30 Aug 2016 06:09:26 +0000 The N1/T1 RNase superfamily comprises enzymes with well-established antitumor effects, such as ribotoxins secreted by fungi, primarily by Aspergillus and Penicillium species, and bacterial RNase secreted by B. pumilus (binase) and B. amyloliquefaciens (barnase). RNase is regarded as an alternative to classical chemotherapeutic agents due to its selective cytotoxicity towards tumor cells. New RNase with a high degree of structural similarity with binase (73%) and barnase (74%) was isolated and purified from Bacillus licheniformis (balifase, calculated molecular weight 12421.9 Da, pI 8.91). The protein sample with enzymatic activity of 1.5 × 106 units/A280 was obtained. The physicochemical properties of balifase are similar to those of barnase. However, in terms of its gene organization and promoter activity, balifase is closer to binase. The unique feature of balifase gene organization consists in the fact that genes of RNase and its inhibitor are located in one operon. Similarly to biosynthesis of binase, balifase synthesis is induced under phosphate starvation; however, in contrast to binase, balifase does not form dimers under natural conditions. We propose that the highest stability of balifase among analyzed RNase types allows the protein to retain its structure without oligomerization. Yulia Sokurenko, Alsu Nadyrova, Vera Ulyanova, and Olga Ilinskaya Copyright © 2016 Yulia Sokurenko et al. All rights reserved. Biosynthesis and Characterization of Silver Nanoparticles by Aspergillus Species Mon, 29 Aug 2016 06:08:30 +0000 Currently, researchers turn to natural processes such as using biological microorganisms in order to develop reliable and ecofriendly methods for the synthesis of metallic nanoparticles. In this study, we have investigated extracellular biosynthesis of silver nanoparticles using four Aspergillus species including A. fumigatus, A. clavatus, A. niger, and A. flavus. We have also analyzed nitrate reductase activity in the studied species in order to determine the probable role of this enzyme in the biosynthesis of silver nanoparticles. The formation of silver nanoparticles in the cell filtrates was confirmed by the passage of laser light, change in the color of cell filtrates, absorption peak at 430 nm in UV-Vis spectra, and atomic force microscopy (AFM). There was a logical relationship between the efficiencies of studied Aspergillus species in the production of silver nanoparticles and their nitrate reductase activity. A. fumigatus as the most efficient species showed the highest nitrate reductase activity among the studied species while A. flavus exhibited the lowest capacity in the biosynthesis of silver nanoparticles which was in accord with its low nitrate reductase activity. The present study showed that Aspergillus species had potential for the biosynthesis of silver nanoparticles depending on their nitrate reductase activity. Kamiar Zomorodian, Seyedmohammad Pourshahid, Arman Sadatsharifi, Pouyan Mehryar, Keyvan Pakshir, Mohammad Javad Rahimi, and Ali Arabi Monfared Copyright © 2016 Kamiar Zomorodian et al. All rights reserved. Effect of Different Light Qualities on Growth, Pigment Content, Chlorophyll Fluorescence, and Antioxidant Enzyme Activity in the Red Alga Pyropia haitanensis (Bangiales, Rhodophyta) Tue, 23 Aug 2016 14:41:45 +0000 Spectral light changes evoke different morphogenetic and photosynthetic responses that can vary among different algae species. The aim of this study is to investigate the photosynthetic characteristics of the red macroalgae grown under different spectrum environments. In this study, Pyropia haitanensis were cultured under blue, red, and green LED and fluorescent tubes light. The growth rate, photopigment composition, chlorophyll fluorescence, and antioxidative enzymes activities in different light spectrums were investigated. The results revealed that growth rate was significantly higher in the thalli grown under blue, green, and fluorescent tubes light. Contents of Chl a and phycobiliprotein in red light were lower among all the growth conditions. Furthermore, a striking increase in SOD and CAT activity was observed in red light treatment along with the NPQ increase. The results revealed that the photosynthetic efficiency and increased growth rate of P. haitanensis benefitted from light spectrums such as blue, green, and fluorescent tubes light by pigment composition and photochemical efficiency manipulation, whereas red light has disadvantageous effects. Accordingly, the results for improving quality and the economic yield of algae species in some extent and the combination of different wavelengths could allow better economic resource exploitation. Huanyang Wu Copyright © 2016 Huanyang Wu. All rights reserved. Anaplasma marginale: Diversity, Virulence, and Vaccine Landscape through a Genomics Approach Wed, 17 Aug 2016 16:48:26 +0000 In order to understand the genetic diversity of A. marginale, several efforts have been made around the world. This rickettsia affects a significant number of ruminants, causing bovine anaplasmosis, so the interest in its virulence and how it is transmitted have drawn interest not only from a molecular point of view but also, recently, some genomics research have been performed to elucidate genes and proteins with potential as antigens. Unfortunately, so far, we still do not have a recombinant anaplasmosis vaccine. In this review, we present a landscape of the multiple approaches carried out from the genomic perspective to generate valuable information that could be used in a holistic way to finally develop an anaplasmosis vaccine. These approaches include the analysis of the genetic diversity of A. marginale and how this affects control measures for the disease. Anaplasmosis vaccine development is also reviewed from the conventional vaccinomics to genome-base vaccinology approach based on proteomics, metabolomics, and transcriptomics analyses reported. The use of these new omics approaches will undoubtedly reveal new targets of interest in the near future, comprising information of potential antigens and the immunogenic effect of A. marginale proteins. Rosa Estela Quiroz-Castañeda, Itzel Amaro-Estrada, and Sergio Darío Rodríguez-Camarillo Copyright © 2016 Rosa Estela Quiroz-Castañeda et al. All rights reserved. Corrigendum to “Type 2C Phosphatase 1 of Artemisia annua L. Is a Negative Regulator of ABA Signaling” Wed, 17 Aug 2016 13:37:26 +0000 Fangyuan Zhang, Xueqing Fu, Zongyou Lv, Qian Shen, Tingxian Yan, Weimin Jiang, Guofeng Wang, Xiaofen Sun, and Kexuan Tang Copyright © 2016 Fangyuan Zhang et al. All rights reserved. Expression of Codon-Optimized Plant Glycosyltransferase UGT72B14 in Escherichia coli Enhances Salidroside Production Mon, 15 Aug 2016 13:15:36 +0000 Salidroside, a plant secondary metabolite in Rhodiola, has been demonstrated to have several adaptogenic properties as a medicinal herb. Due to the limitation of plant source, microbial production of salidroside by expression of plant uridine diphosphate glycosyltransferase (UGT) is promising. However, glycoside production usually remains hampered by poor expression of plant UGTs in microorganisms. Herein, we achieved salidroside production by expression of Rhodiola UGT72B14 in Escherichia coli (E. coli) and codon optimization was accordingly applied. UGT72B14 expression was optimized by changing 278 nucleotides and decreasing the G+C content to 51.05% without altering the amino acid sequence. The effect of codon optimization on UGT72B14 catalysis for salidroside production was assessed both in vitro and in vivo. In vitro, salidroside production by codon-optimized UGT72B14 is enhanced because of a significantly improved protein yield (increased by 4.8-fold) and an equivalently high activity as demonstrated by similar kinetic parameters ( and ), compared to that by wild-type protein. In vivo, both batch and fed-batch cultivation using the codon-optimized gene resulted in a significant increase in salidroside production, which was up to 6.7 mg/L increasing 3.2-fold over the wild-type UGT72B14. Feiyan Xue, Huili Guo, Yingying Hu, Ran Liu, Lina Huang, Heshu Lv, Chunmei Liu, Mingfeng Yang, and Lanqing Ma Copyright © 2016 Feiyan Xue et al. All rights reserved. Induction of Osmoregulation and Modulation of Salt Stress in Acacia gerrardii Benth. by Arbuscular Mycorrhizal Fungi and Bacillus subtilis (BERA 71) Mon, 15 Aug 2016 11:08:58 +0000 The role of soil microbiota in plant stress management, though speculated a lot, is still far from being completely understood. We conducted a greenhouse experiment to examine synergistic impact of plant growth promoting rhizobacterium, Bacillus subtilis (BERA 71), and arbuscular mycorrhizal fungi (AMF) (Claroideoglomus etunicatum; Rhizophagus intraradices; and Funneliformis mosseae) to induce acquired systemic resistance in Talh tree (Acacia gerrardii Benth.) against adverse impact of salt stress. Compared to the control, the BERA 71 treatment significantly enhanced root colonization intensity by AMF, in both presence and absence of salt. We also found positive synergistic interaction between B. subtilis and AMF vis-a-vis improvement in the nutritional value in terms of increase in total lipids, phenols, and fiber content. The AMF and BERA 71 inoculated plants showed increased content of osmoprotectants such as glycine, betaine, and proline, though lipid peroxidation was reduced probably as a mechanism of salt tolerance. Furthermore, the application of bioinoculants to Talh tree turned out to be potentially beneficial in ameliorating the deleterious impact of salinity on plant metabolism, probably by modulating the osmoregulatory system (glycine betaine, proline, and phenols) and antioxidant enzymes system (SOD, CAT, POD, GR, APX, DHAR, MDAHR, and GSNOR). Abeer Hashem, E. F. Abd_Allah, A. A. Alqarawi, A. A. Al-Huqail, and M. A. Shah Copyright © 2016 Abeer Hashem et al. All rights reserved. Upstream and Downstream of Recombinants Biomolecules to Health Care Industry Wed, 10 Aug 2016 08:17:31 +0000 Priscila G. Mazzola, Arthur Cavaco-Paulo, Jorge G. Farías, and Jorge F. B. Pereira Copyright © 2016 Priscila G. Mazzola et al. All rights reserved. Cloning, Expression, and Characterization of a Novel Thermophilic Monofunctional Catalase from Geobacillus sp. CHB1 Sun, 07 Aug 2016 09:58:00 +0000 Catalases are widely used in many scientific areas. A catalase gene (Kat) from Geobacillus sp. CHB1 encoding a monofunctional catalase was cloned and recombinant expressed in Escherichia coli (E. coli), which was the first time to clone and express this type of catalase of genus Geobacillus strains as far as we know. This Kat gene was 1,467 bp in length and encoded a catalase with 488 amino acid residuals, which is only 81% similar to the previously studied Bacillus sp. catalase in terms of amino acid sequence. Recombinant catalase was highly soluble in E. coli and made up 30% of the total E. coli protein. Fermentation broth of the recombinant E. coli showed a high catalase activity level up to 35,831 U/mL which was only lower than recombinant Bacillus sp. WSHDZ-01 among the reported catalase production strains. The purified recombinant catalase had a specific activity of 40,526 U/mg and of 51.1 mM. The optimal reaction temperature of this recombinant enzyme was 60°C to 70°C, and it exhibited high activity over a wide range of reaction temperatures, ranging from 10°C to 90°C. The enzyme retained 94.7% of its residual activity after incubation at 60°C for 1 hour. High yield and excellent thermophilic properties are valuable features for this catalase in industrial applications. Xianbo Jia, Jichen Chen, Chenqiang Lin, and Xinjian Lin Copyright © 2016 Xianbo Jia et al. All rights reserved. High Mobility Group Box1 Protein Is Involved in Endoplasmic Reticulum Stress Induced by Clostridium difficile Toxin A Sun, 07 Aug 2016 09:46:45 +0000 High Mobility Group Box (HMGB1), a damage-associated inflammatory factor, plays an important role in the pathogenesis of numerous chronic inflammatory and autoimmune diseases. In this study, the role of the HMGB1 in TcdA-induced ER stress was identified. Clostridium difficile toxin A is one of the major virulence factors of C. difficile infection (CDI) and has been proved to induce apoptotic cell death through ER stress. Our results showed that HMGB1 might play an important role in the TcdA-induced ER stress and unfolded protein response. HMGB1 activated molecular markers and induced the C/EBP homologous protein upregulation (CHOP). This study may provide the essential information for better understanding of the molecular mechanisms involved in CDI. Ji Liu, Yi Ma, Chun-Li Sun, Shan Li, and Ju-Fang Wang Copyright © 2016 Ji Liu et al. All rights reserved. Structural Changes of Lignin after Liquid Hot Water Pretreatment and Its Effect on the Enzymatic Hydrolysis Wed, 03 Aug 2016 12:04:27 +0000 During liquid hot water (LHW) pretreatment, lignin is mostly retained in the pretreated biomass, and the changes in the chemical and structural characteristics of lignin should probably refer to re-/depolymerization, solubilization, or glass transition. The residual lignin could influence the effective enzymatic hydrolysis of cellulose. The pure lignin was used to evaluate the effect of LHW process on its structural and chemical features. The surface morphology of LHW-treated lignin observed with the scanning electron microscopy (SEM) was more porous and irregular than that of untreated lignin. Compared to the untreated lignin, the surface area, total pore volume, and average pore size of LHW-treated lignin tested with the Brunner-Emmet-Teller (BET) measurement were increased. FTIR analysis showed that the chemical structure of lignin was broken down in the LHW process. Additionally, the impact of untreated and treated lignin on the enzymatic hydrolysis of cellulose was also explored. The LHW-treated lignin had little impact on the cellulase adsorption and enzyme activities and somehow could improve the enzymatic hydrolysis of cellulose. Wen Wang, Xinshu Zhuang, Zhenhong Yuan, Wei Qi, Qiang Yu, and Qiong Wang Copyright © 2016 Wen Wang et al. All rights reserved. Microbial Production of Short Chain Fatty Acids from Lignocellulosic Biomass: Current Processes and Market Sun, 31 Jul 2016 13:01:45 +0000 Biological production of organic acids from conversion of biomass derivatives has received increased attention among scientists and engineers and in business because of the attractive properties such as renewability, sustainability, degradability, and versatility. The aim of the present review is to summarize recent research and development of short chain fatty acids production by anaerobic fermentation of nonfood biomass and to evaluate the status and outlook for a sustainable industrial production of such biochemicals. Volatile fatty acids (VFAs) such as acetic acid, propionic acid, and butyric acid have many industrial applications and are currently of global economic interest. The focus is mainly on the utilization of pretreated lignocellulosic plant biomass as substrate (the carbohydrate route) and development of the bacteria and processes that lead to a high and economically feasible production of VFA. The current and developing market for VFA is analyzed focusing on production, prices, and forecasts along with a presentation of the biotechnology companies operating in the market for sustainable biochemicals. Finally, perspectives on taking sustainable product of biochemicals from promise to market introduction are reviewed. Ivan Baumann and Peter Westermann Copyright © 2016 Ivan Baumann and Peter Westermann. All rights reserved. Enhancing the Feasibility of Microcystis aeruginosa as a Feedstock for Bioethanol Production under the Influence of Various Factors Sun, 31 Jul 2016 06:27:44 +0000 Microcystis aeruginosa, a freshwater microalga, is capable of producing and accumulating different types of sugars in its biomass which make it a good feedstock for bioethanol production. Present study aims to investigate the effect of different factors increasing growth rate and carbohydrates productivity of M. aeruginosa. MF media (modified BG11 media) and additional ingredients such as aminolevulinic acid (2 mM), lysine (2.28 mM), alanine (1 mM), and Naphthalene acetic acid (1 mM) as cytokine promoted M. aeruginosa growth and sugar contents. Salmonella showed growth-assisting effect on M. aeruginosa. Enhanced growth rate and carbohydrates contents were observed in M. aeruginosa culture grown at 25°C under red LED light of 90 μmolm−2s−1 intensity. More greenish and carbohydrates rich M. aeruginosa biomass was prepared (final OD660 nm = 2.21 and sugar contents 10.39 mM/mL) as compared to control (maximum OD660 nm = 1.4 and sugar contents 3 mM/mL). The final algae biomass was converted to algae juice through a specific pretreatment method. The resulted algae Juice was used as a substrate in fermentation process. Highest yield of bioethanol (50 mM/mL) was detected when Brettanomyces custersainus, Saccharomyces cerevisiae, and Pichia stipitis were used in combinations for fermentation process as compared to their individual fermentation. The results indicated the influence of different factors on the growth rate and carbohydrates productivity of M. aeruginosa and its feasibility as a feedstock for fermentative ethanol production. Muhammad Imran Khan, Moon Geon Lee, Hyo Jin Seo, Jin Hyuk Shin, Tai Sun Shin, Yang Ho Yoon, Min Yong Kim, Jong Il Choi, and Jong Deog Kim Copyright © 2016 Muhammad Imran Khan et al. All rights reserved. Synthesis of Novel Pyrimethanil Grafted Chitosan Derivatives with Enhanced Antifungal Activity Wed, 27 Jul 2016 06:45:24 +0000 In this study, three pyrimethanil grafted chitosan (PML-g-CS) derivatives were obtained. The structures of the conjugates were confirmed by FT-IR, 1H NMR, and EA. The grafting ratios were measured by HPLC. Antifungal properties of pyrimethanil grafted chitosan (PML-g-CS) derivatives against the plant pathogenic fungi Rhizoctonia solani and Gibberella zeae were investigated at concentrations of 100, 200, and 400 mg/L. The PML-g-CS derivatives showed enhanced antifungal activity in comparison with chitosan. The PML-g-CS-1 showed the best antifungal activity against R. solani, whose antifungal index was 58.32%. The PML-g-CS-2 showed the best antifungal activity against G. zeae, whose antifungal index was 53.48%. The conjugation of chitosan and pyrimethanil showed synergistic effect. The PML-g-CS derivatives we developed showed potential for further study and application in crop protection. Yan Li, Song Liu, Yukun Qin, Ronge Xing, Xiaolin Chen, Kecheng Li, and Pengcheng Li Copyright © 2016 Yan Li et al. All rights reserved. Genotypes-Independent Optimization of Nitrogen Supply for Isolated Microspore Cultures in Barley Mon, 25 Jul 2016 06:48:40 +0000 To establish a high-efficiency system of isolated microspore culture for different barley genotypes, we investigated the effects of nitrogen sources and concentrations on callus induction and plant regeneration in different barley genotypes. The results showed that the organic nitrogen sources greatly increased the callus induction, and the great reduction of total nitrogen sources would significantly decrease the callus induction. And the further optimization experiments revealed that the increasing of organic nitrogen sources was much important in callus induction while it seemed different in plant regeneration. Based on the great effects of organic nitrogen on callus induction, the medium of N6-ANO1/4-2000 might be the best choice for the microspore culture system. In addition, the phylogenetic analysis indicated that there were clear differences of genetic backgrounds among these barley genotypes, and it also suggested that this medium for microspore culture had widespread utilization in different barley genotypes. Ruiju Lu, Zhiwei Chen, Runhong Gao, Ting He, Yifei Wang, Hongwei Xu, Guimei Guo, Yingbo Li, Chenghong Liu, and Jianhua Huang Copyright © 2016 Ruiju Lu et al. All rights reserved. Utilization of Waste Materials for Microbial Carrier in Wastewater Treatment Wed, 20 Jul 2016 11:38:49 +0000 This research focused on the ammonium-nitrogen (NH4-N) removal from the domestic wastewater using the attached growth reactors. Two types of waste material of corncob (biodegradable material) and concrete (nonbiodegradable material) were used as the carrier for microorganisms’ attachment. During operation, both reactors achieved absolutely high performance of ammonium removal (up to 99%) and total nitrogen removal (up to 95%). The significant advantage of corncob carrier was that the corncob was able to be a source of carbon for biological denitrification, leading to no external carbon requirement for operating the system. However, the corncob caused an increasing turbidity of the effluent. On the other hand, the concrete carrier required the minimal external carbon of 3.5 C/N ratio to reach the good performance. Moreover, a longer period for microorganisms’ adaptation was found in the concrete carrier rather than the corncob carrier. Further, the same physiological and biochemical characteristics of active bacteria were found at the two carriers, which were negative gram, cocci shape, and smooth and white-turbid colony. Due to the effluent quality, the concrete was more appropriate carrier than the corncob for wastewater treatment. H. T. Le, N. Jantarat, W. Khanitchaidecha, K. Ratananikom, and A. Nakaruk Copyright © 2016 H. T. Le et al. All rights reserved. Heterologous, Expression, and Characterization of Thermostable Glucoamylase Derived from Aspergillus flavus NSH9 in Pichia pastoris Mon, 18 Jul 2016 13:32:52 +0000 A novel thermostable glucoamylase cDNA without starch binding domain (SBD) of Aspergillus flavus NSH9 was successfully identified, isolated, and overexpressed in Pichia pastoris GS115. The complete open reading frame of glucoamylase from Aspergillus flavus NSH9 was identified by employing PCR that encodes 493 amino acids lacking in the SBD. The first 17 amino acids were presumed to be a signal peptide. The cDNA was cloned into Pichia pastoris and the highest expression of recombinant glucoamylase (rGA) was observed after 8 days of incubation period with 1% methanol. The molecular weight of the purified rGA was about 78 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C. The enzyme was stable at higher temperature with 50% of residual activity observed after 20 min at 90°C and 100°C. Low concentration of metal (Mg++, Fe++, Zn++, Cu++, and Pb++) had positive effect on rGA activity. This rGA has the potential for use and application in the saccharification steps, due to its thermostability, in the starch processing industries. Kazi Muhammad Rezaul Karim, Ahmad Husaini, Md. Anowar Hossain, Ngieng Ngui Sing, Fazia Mohd Sinang, Mohd Hasnain Md. Hussain, and Hairul Azman Roslan Copyright © 2016 Kazi Muhammad Rezaul Karim et al. All rights reserved. A Tobacco-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Wound Healing in Mice Thu, 14 Jul 2016 17:11:35 +0000 Thymosin β4 (Tβ4) is a peptide that is known to play important roles in protection, regeneration, and remodeling of injured tissues in humans, and that shows great promise in a range of clinical applications. However, current strategies to Tβ4 are insufficient to meet growing demand and have a number of limitations. In this current study we investigated whether expression of recombinant Tβ4 in plants, specifically in tobacco (Nicotiana tabacum) leaves, represents an effective approach. To address this question, a 168 bp Tβ4 gene optimized for tobacco codon usage bias was constitutively expressed in tobacco as a 4-unit repeat concatemer, fused to a polyhistidine tag. Quantitative polymerase chain reaction and Western blot analyses were used to verify 4×Tβ4 expression in 14 transgenic tobacco lines and enzyme-linked immunosorbent assay analysis indicated 4×Tβ4 protein concentrations as high as 3 μg/g of fresh weight in the leaves. We observed that direct administration of tobacco-derived Tβ4 was more effective than Tβ4 either obtained commercially or derived from expression in Escherichia coli at promoting splenocyte proliferation in vitro and wound healing in mice through an endothelial migration assay. This study provides new insights into the development of plant-derived therapeutic proteins and their application by direct administration. Rylosona Janarthini, Xiaolei Wang, Lulu Chen, Lei Gao, and Lingxia Zhao Copyright © 2016 Rylosona Janarthini et al. All rights reserved. Laboratory Prototype of Bioreactor for Oxidation of Toxic D-Lactate Using Yeast Cells Overproducing D-Lactate Cytochrome c Oxidoreductase Thu, 30 Jun 2016 09:44:51 +0000 D-lactate is a natural component of many fermented foods like yogurts, sour milk, cheeses, and pickles vegetable products. D-lactate in high concentrations is toxic for children and people with short bowel syndrome and provokes encephalopathy. These facts convincingly demonstrate a need for effective tools for the D-lactate removal from some food products. The main idea of investigation is focused on application of recombinant thermotolerant methylotrophic yeast Hansenula polymorpha “tr6,” overproducing D-lactate: cytochrome c oxidoreductase (EC, D-lactate cytochrome c oxidoreductase, D-lactate dehydrogenase (cytochrome), DLDH). In addition to 6-fold overexpression of DLDH under a strong constitutive promoter (prAOX), the strain of H. polymorpha “tr6” (gcr1 catX/Δcyb2, prAOX_DLDH) is characterized by impairment in glucose repression of AOX promoter, devoid of catalase and L-lactate-cytochrome oxidoreductase activities. Overexpression of DLDH coupling with the deletion of L-lactate-cytochrome oxidoreductase activity opens possibility for usage of the strain as a base for construction of bioreactor for removing D-lactate from fermented products due to oxidation to nontoxic pyruvate. A laboratory prototype of column-type bioreactor for removing a toxic D-lactate from model solution based on permeabilized cells of the H. polymorpha “tr6” and alginate gel was constructed and efficiency of this process was tested. Maria Karkovska, Oleh Smutok, and Mykhailo Gonchar Copyright © 2016 Maria Karkovska et al. All rights reserved. An Experimental Study of Briquetting Process of Torrefied Rubber Seed Kernel and Palm Oil Shell Tue, 21 Jun 2016 14:13:07 +0000 Torrefaction process of biomass material is essential in converting them into biofuel with improved calorific value and physical strength. However, the production of torrefied biomass is loose, powdery, and nonuniform. One method of upgrading this material to improve their handling and combustion properties is by densification into briquettes of higher density than the original bulk density of the material. The effects of critical parameters of briquetting process that includes the type of biomass material used for torrefaction and briquetting, densification temperature, and composition of binder for torrefied biomass are studied and characterized. Starch is used as a binder in the study. The results showed that the briquette of torrefied rubber seed kernel (RSK) is better than torrefied palm oil shell (POS) in both calorific value and compressive strength. The best quality of briquettes is yielded from torrefied RSK at the ambient temperature of briquetting process with the composition of 60% water and 5% binder. The maximum compressive load for the briquettes of torrefied RSK is 141 N and the calorific value is 16 MJ/kg. Based on the economic evaluation analysis, the return of investment (ROI) for the mass production of both RSK and POS briquettes is estimated in 2-year period and the annual profit after payback was approximately 107,428.6 USD. M. Fadzli Hamid, M. Yusof Idroas, M. Zulfikar Ishak, Z. Alimuddin Zainal Alauddin, M. Azman Miskam, and M. Khalil Abdullah Copyright © 2016 M. Fadzli Hamid et al. All rights reserved. Evaluation of Culture Conditions to Obtain Fatty Acids from Saline Microalgae Species: Dunaliella salina, Sinecosyfis sp., and Chroomonas sp. Wed, 08 Jun 2016 11:40:40 +0000 The use of the saline microalgae, Dunaliella salina, Sinecosyfis sp., and Chroomonas sp., was explored as an alternative source for the production of fatty acids using fertilizer and glycerol as culture media. The nutrient medium used contained “Nutrifoliar,” a commercial fertilizer, and/or glycerol, in natural sea water. The microalgae were placed in cultures with different conditions. The parameters that favored the largest production of fatty acids were 24 hours of agitation and illumination, 1620 L/day of air supply, 2.25 L of air/min, and a temperature of 32°C using “Nutrifoliar” as the culture media. Results indicated that, from 3 g of microalgae in wet base of Chroomonas sp., 54.43 mg of oil was produced. The chromatographic characterization of oil obtained revealed the presence of essential fatty acids such as 9,12,15-octadecatrienoic acid (omega-3) and 4,7,10-hexadecatrienoic acid (omega-6) from the species Dunaliella salina. On the other hand, 9,12-octadecadienoic acid (omega-6) and cis-11-eicosenoic acid (omega-9) were identified from the species Chroomonas sp. The temperature variations played an important role in the velocity of growth or the production of the algae biomass, the amount of oil, and the ability to produce fatty acids. D. A. Castilla Casadiego, A. R. Albis Arrieta, E. R. Angulo Mercado, S. J. Cervera Cahuana, K. S. Baquero Noriega, A. F. Suárez Escobar, and E. D. Morales Avendaño Copyright © 2016 D. A. Castilla Casadiego et al. All rights reserved. Microchips in Medicine: Current and Future Applications Tue, 07 Jun 2016 12:39:42 +0000 With the objective of improving efficacy and morbidity, device manufacturers incorporate chemicals or drugs into medical implants. Using multiple reservoirs of discrete drug doses, microchips represent a new technology capable of on-demand release of various drugs over long periods of time. Herein, we review drug delivery systems, how microchips work, recent investigations, and future applications in various fields of medicine. Adam E. M. Eltorai, Henry Fox, Emily McGurrin, and Stephanie Guang Copyright © 2016 Adam E. M. Eltorai et al. All rights reserved. Full-Length cDNA, Prokaryotic Expression, and Antimicrobial Activity of UuHb-F-I from Urechis unicinctus Tue, 07 Jun 2016 10:59:50 +0000 Hemoglobin, which widely exists in all vertebrates and in some invertebrates, is possibly a precursor of antimicrobial peptides (AMPs). However, AMPs in the hemoglobin of invertebrates have been rarely investigated. This study is the first to report the full-length cDNA, prokaryotic expression, and antimicrobial activity of UuHb-F-I from Urechis unicinctus. The full-length cDNA sequence of UuHb-F-I was 780 bp with an open-reading frame of 429 bp encoding 142 amino acids. MALDI-TOF-MS suggested that the recombinant protein of UuHb-F-I (rUuHb-F-I) yielded a molecular weight of 15,168.01 Da, and its N-terminal amino acid sequence was MGLTGAQIDAIK. rUuHb-F-I exhibited different antimicrobial activities against microorganisms. The lowest minimum inhibitory concentration against Micrococcus luteus was 2.78–4.63 M. Our results may help elucidate the immune defense mechanism of U. unicinctus and may provide insights into new AMPs in drug discovery. Rongli Niu and Xiang Chen Copyright © 2016 Rongli Niu and Xiang Chen. All rights reserved. Plant Growth Biostimulants Based on Different Methods of Seaweed Extraction with Water Mon, 06 Jun 2016 11:08:36 +0000 We explored two methods for obtaining aqueous extracts: boiling and soaking of Baltic seaweeds (EB and ES, resp.). Algal extracts were characterized in terms of polyphenols, micro- and macroelements, lipids content, and antibacterial properties. The utilitarian properties were examined in the germination tests on Lepidium sativum for three extract dilutions (0.5, 2.5, and 10%). It was found that the extracts were similar in micro- and macroelement concentrations. Water was proved to be a good solvent to extract phenolic compounds. The algal extract produced by soaking biomass did not show inhibitory effect on Escherichia coli and Staphylococcus aureus. Only the boiled extract had an inhibitory activity against E. coli. Germination tests revealed a positive influence of the bioproducts on the cultivated plants. In the group treated with 10% EB, plants were 13% longer than in the control group; the content of elements B, Mo, Zn, and Na in the group treated with 10% ES was higher by 76%, 48%, 31%, and 59% than in the control group, respectively; the content of chlorophyll was 2.5 times higher in 0.5% ES than in the control group. Extracts showed the slight impact on the morphology of plants. Katarzyna Godlewska, Izabela Michalak, Łukasz Tuhy, and Katarzyna Chojnacka Copyright © 2016 Katarzyna Godlewska et al. All rights reserved. Subcritical Water Technology for Enhanced Extraction of Biochemical Compounds from Chlorella vulgaris Sun, 05 Jun 2016 07:36:07 +0000 Subcritical water extraction (SWE) technology has been used for the extraction of active compounds from different biomass materials with low process cost, mild operating conditions, short process times, and environmental sustainability. With the limited application of the technology to microalgal biomass, this work investigates parametrically the potential of subcritical water for high-yield extraction of biochemicals such as carbohydrates and proteins from microalgal biomass. The SWE process was optimized using central composite design (CCD) under varying process conditions of temperature (180–374°C), extraction time (1–20 min), biomass particulate size (38–250 μm), and microalgal biomass loading (5–40 wt.%). Chlorella vulgaris used in this study shows high volatile matter (83.5 wt.%) and carbon content (47.11 wt.%), giving advantage as a feedstock for biofuel production. The results showed maximum total carbohydrate content and protein yields of 14.2 g/100 g and 31.2 g/100 g, respectively, achieved under the process conditions of 277°C, 5% of microalgal biomass loading, and 5 min extraction time. Statistical analysis revealed that, of all the parameters investigated, temperature is the most critical during SWE of microalgal biomass for protein and carbohydrate production. S. A. Awaluddin, Selvakumar Thiruvenkadam, Shamsul Izhar, Yoshida Hiroyuki, Michael K. Danquah, and Razif Harun Copyright © 2016 S. A. Awaluddin et al. All rights reserved. Biotransformation of Cholesterol and 16α,17α-Epoxypregnenolone and Isolation of Hydroxylase in Burkholderia cepacia SE-1 Thu, 02 Jun 2016 13:28:32 +0000 The metabolism of cholesterol is critical in eukaryotes as a precursor for vitamins, steroid hormones, and bile acids. Some steroid compounds can be transformed into precursors of steroid medicine by some microorganisms. In this study, the biotransformation products of cholesterol and 16α,17α-epoxypregnenolone produced by Burkholderia cepacia SE-1 were investigated, and a correlative enzyme, hydroxylase, was also studied. The biotransformation products, 7β-hydroxycholesterol, 7-oxocholesterol, and 20-droxyl-16α,17α-epoxypregn-1,4-dien-3-one, were purified by silica gel and Sephadex LH-20 column chromatography and identified by nuclear magnetic resonance and mass spectroscopy. The hydroxylase was isolated from the bacterium and the partial sequences of the hydroxylase, which belong to the catalases/peroxidase family, were analyzed using MS/MS analyses. The enzyme showed activity toward cholesterol and had a specific activity of 37.2 U/mg of protein at 30°C and pH 7.0. XiangDong Zhu, CuiPing Pang, Yuting Cao, and Dan Fan Copyright © 2016 XiangDong Zhu et al. All rights reserved. Molecular Identification of Sex in Phoenix dactylifera Using Inter Simple Sequence Repeat Markers Thu, 26 May 2016 09:31:45 +0000 Early sex identification of Date Palm (Phoenix dactylifera L.) at seedling stage is an economically desirable objective, which will significantly increase the profits of seed based cultivation. The utilization of molecular markers at this stage for early and rapid identification of sex is important due to the lack of morphological markers. In this study, a total of two hundred Inter Simple Sequence Repeat (ISSR) primers were screened among male and female Date palm plants to identify putative sex-specific marker, out of which only two primers (IS_A02 and IS_A71) were found to be associated with sex. The primer IS_A02 produced a unique band of size 390 bp and was found clearly in all female plants, while it was absent in all male plants. Contrary to this, the primer IS_A71 produced a unique band of size 380 bp and was clearly found in all male plants, whereas it was absent in all the female plants. Subsequently, these specific fragments were excised, purified, and sequenced for the development of sequence specific markers further in future for the implementation on dioecious Date Palm for sex determination. These markers are efficient, highly reliable, and reproducible for sex identification at the early stage of seedling. Abdulhafed A. Al-Ameri, Fahad Al-Qurainy, Abdel-Rhman Z. Gaafar, Salim Khan, and M. Nadeem Copyright © 2016 Abdulhafed A. Al-Ameri et al. All rights reserved. A Drosophila Model for Screening Antiobesity Agents Mon, 09 May 2016 14:24:29 +0000 Although triacylglycerol, the major component for lipid storage, is essential for normal physiology, its excessive accumulation causes obesity in adipose tissue and is associated with organ dysfunction in nonadipose tissue. Here, we focused on the Drosophila model to develop therapeutics for preventing obesity. The brummer (bmm) gene in Drosophila melanogaster is known to be homologous with human adipocyte triglyceride lipase, which is related to the regulation of lipid storage. We established a Drosophila model for monitoring bmm expression by introducing the green fluorescent protein (GFP) gene as a downstream reporter of the bmm promoter. The third-instar larvae of Drosophila showed the GFP signal in all tissues observed and specifically in the salivary gland nucleus. To confirm the relationship between bmm expression and obesity, the effect of oral administration of glucose diets on bmm promoter activity was analyzed. The Drosophila flies given high-glucose diets showed higher lipid contents, indicating the obesity phenotype; this was suggested by a weaker intensity of the GFP signal as well as reduced bmm mRNA expression. These results demonstrated that the transgenic Drosophila model established in this study is useful for screening antiobesity agents. We also report the effects of oral administration of histone deacetylase inhibitors and some vegetables on the bmm promoter activity. Tran Thanh Men, Duong Ngoc Van Thanh, Masamitsu Yamaguchi, Takayoshi Suzuki, Gen Hattori, Masayuki Arii, Nguyen Tien Huy, and Kaeko Kamei Copyright © 2016 Tran Thanh Men et al. All rights reserved. Production and Partial Characterization of an Alkaline Xylanase from a Novel Fungus Cladosporium oxysporum Tue, 26 Apr 2016 13:53:08 +0000 A new fungus Cladosporium oxysporum GQ-3 producing extracellular xylanase was isolated from decaying agricultural waste and identified based on the morphology and comparison of internal transcribed spacer (ITS) rDNA gene sequence. C. oxysporum produced maximum xylanase activity of 55.92 U/mL with wheat bran as a substrate and NH4Cl as a nitrogen source. Mg2+ improved C. oxysporum xylanase production. Partially purified xylanase exhibited maximum activity at 50°C and pH 8.0, respectively, and showed the stable activity after 2-h treatment in pH 7.0–8.5 or below 55°C. Mg2+ enhanced the xylanase activity by 2% while Cu2+ had the highest inhibition ratio of 57.9%. Furthermore, C. oxysporum xylanase was resistant to most of tested neutral and alkaline proteases. Our findings indicated that Cladosporium oxysporum GQ-3 was a novel xylanase producer, which could be used in the textile processes or paper/feed industries. Guo-Qiang Guan, Peng-Xiang Zhao, Jin Zhao, Mei-Juan Wang, Shu-Hao Huo, Feng-Jie Cui, and Jian-Xin Jiang Copyright © 2016 Guo-Qiang Guan et al. All rights reserved. Biosimilars in Developed and Developing East and Southeast Asian Countries: Japan, South Korea, and Malaysia—Overview, Evolution, and Regulations Assessment Sun, 24 Apr 2016 09:19:04 +0000 The development of biological products has experienced continuous growth over the past three decades. The expiration of patent protection for many biological medicines has led to the development of biosimilars in many countries around the world. This paper reviews the literature on biosimilar drugs and covers their therapeutic status, clinical trials, approved biosimilars, and regulatory guidelines in Japan, South Korea, and Malaysia. The literature suggests that biosimilars are comparable but not identical to the reference product. They are not a generic version of an innovative product and do not ensure therapeutic equivalence. Biosimilars present more challenges than conventional generics and their marketing approval is also much more complicated. Guidelines for biosimilars were published in Japan in July 2009 by the Ministry of Health, Labour and Welfare (MHLW), in South Korea in March 2009 by the Ministry of Food and Drug Safety (MFDS), and in Malaysia in July 2008 by the National Pharmaceutical Control Bureau (NPCB). Tomas Gabriel Bas and Carolina Oliu Castillo Copyright © 2016 Tomas Gabriel Bas and Carolina Oliu Castillo. All rights reserved. Improve the Anaerobic Biodegradability by Copretreatment of Thermal Alkali and Steam Explosion of Lignocellulosic Waste Wed, 20 Apr 2016 13:57:31 +0000 Effective alteration of the recalcitrance properties like crystallization of cellulose, lignin shield, and interlinking of lignocellulosic biomass is an ideal way to utilize the full-scale potential for biofuel production. This study exhibited three different pretreatment effects to enhance the digestibility of corn stover (CS) for methane production. In this context, steam explosion (SE) and thermal potassium hydroxide (KOH-60°C) treated CS produced the maximal methane yield of 217.5 and 243.1 mL/, which were 40.0% and 56.4% more than untreated CS (155.4 mL/), respectively. Copretreatment of thermal potassium hydroxide and steam explosion (CPTPS) treated CS was highly significant among all treatments and improved 88.46% (292.9 mL/) methane yield compared with untreated CS. Besides, CPTPS also achieved the highest biodegradability up to 68.90%. Three kinetic models very well simulated dynamics of methane production yield. Moreover, scanning electron microscopy (SEM), Fourier transform infrared (FTIR), and X-ray diffraction (XRD) analyses declared the most effective changes in physicochemical properties after CPTPS pretreatment. Thus, CPTPS might be a promising approach to deconstructing the recalcitrance of lignocellulosic structure to improve the biodegradability for AD. Muhammad Abdul Hanan Siddhu, Jianghao Li, Jiafu Zhang, Yan Huang, Wen Wang, Chang Chen, and Guangqing Liu Copyright © 2016 Muhammad Abdul Hanan Siddhu et al. All rights reserved. KvLEA, a New Isolated Late Embryogenesis Abundant Protein Gene from Kosteletzkya virginica Responding to Multiabiotic Stresses Wed, 30 Mar 2016 09:06:57 +0000 The LEA proteins are a kind of hydrophilic proteins, playing main functions in desiccation tolerance. However, their importance as a kind of stress proteins in abiotic stress is being clarified little by little. In this study we isolated, cloned, and identified the first KvLEA gene in Kosteletzkya virginica. Bioinformatic analysis showed that the protein encoded by this gene had common properties of LEA proteins and the multiple sequences alignment and phylogenetic analysis further showed that this protein had high homology with two Arabidopsis LEA proteins. Gene expression analysis revealed that this gene had a higher expression in root and it was induced obviously by salt stress. Moreover, the transcripts of KvLEA were also induced by other abiotic stresses including drought, high temperature, chilling, and ABA treatment. Among these abiotic stresses, ABA treatment brought about the biggest changes to this gene. Collectively, our research discovered a novel LEA gene and uncovered its involvement in multiabiotic stresses in K. virginica. This research not only enriched studies on LEA gene in plant but also would accelerate more studies on K. virginica in the future. Xiaoli Tang, Hongyan Wang, Liye Chu, and Hongbo Shao Copyright © 2016 Xiaoli Tang et al. All rights reserved. Next Generation Sequencing Identifies Five Major Classes of Potentially Therapeutic Enzymes Secreted by Lucilia sericata Medical Maggots Mon, 28 Mar 2016 06:33:48 +0000 Lucilia sericata larvae are used as an alternative treatment for recalcitrant and chronic wounds. Their excretions/secretions contain molecules that facilitate tissue debridement, disinfect, or accelerate wound healing and have therefore been recognized as a potential source of novel therapeutic compounds. Among the substances present in excretions/secretions various peptidase activities promoting the wound healing processes have been detected but the peptidases responsible for these activities remain mostly unidentified. To explore these enzymes we applied next generation sequencing to analyze the transcriptomes of different maggot tissues (salivary glands, gut, and crop) associated with the production of excretions/secretions and/or with digestion as well as the rest of the larval body. As a result we obtained more than 123.8 million paired-end reads, which were assembled de novo using Trinity and Oases assemblers, yielding 41,421 contigs with an N50 contig length of 2.22 kb and a total length of 67.79 Mb. BLASTp analysis against the MEROPS database identified 1729 contigs in 577 clusters encoding five peptidase classes (serine, cysteine, aspartic, threonine, and metallopeptidases), which were assigned to 26 clans, 48 families, and 185 peptidase species. The individual enzymes were differentially expressed among maggot tissues and included peptidase activities related to the therapeutic effects of maggot excretions/secretions. Zdeněk Franta, Heiko Vogel, Rüdiger Lehmann, Oliver Rupp, Alexander Goesmann, and Andreas Vilcinskas Copyright © 2016 Zdeněk Franta et al. All rights reserved. Advances in Biotechnology for Sustainable Development Sun, 27 Mar 2016 12:02:27 +0000 Weiqi Fu, Basel Khraiwesh, Hongbing Liu, and Lei Kai Copyright © 2016 Weiqi Fu et al. All rights reserved. Mutation Detection in an Antibody-Producing Chinese Hamster Ovary Cell Line by Targeted RNA Sequencing Sun, 20 Mar 2016 13:47:13 +0000 Chinese hamster ovary (CHO) cells have been used widely in the pharmaceutical industry for production of biological therapeutics including monoclonal antibodies (mAb). The integrity of the gene of interest and the accuracy of the relay of genetic information impact product quality and patient safety. Here we employed next-generation sequencing, particularly RNA-seq, and developed a method to systematically analyze the mutation rate of the mRNA of CHO cell lines producing a mAb. The effect of an extended culturing period to mimic the scale of cell expansion in a manufacturing process and varying selection pressure in the cell culture were also closely examined. Siyan Zhang, Jason D. Hughes, Nicholas Murgolo, Diane Levitan, Janice Chen, Zhong Liu, and Shuangping Shi Copyright © 2016 Siyan Zhang et al. All rights reserved. Silicon Mitigates Salinity Stress by Regulating the Physiology, Antioxidant Enzyme Activities, and Protein Expression in Capsicum annuum ‘Bugwang’ Sun, 20 Mar 2016 10:15:59 +0000 Silicon- (Si-) induced salinity stress resistance was demonstrated at physiological and proteomic levels in Capsicum annuum for the first time. Seedlings of C. annuum were hydroponically treated with NaCl (50 mM) with or without Si (1.8 mM) for 15 days. The results illustrated that saline conditions significantly reduced plant growth and biomass and photosynthetic parameters and increased the electrolyte leakage potential, lipid peroxidation, and hydrogen peroxide level. However, supplementation of Si allowed the plants to recover from salinity stress by improving their physiology and photosynthesis. During salinity stress, Si prevented oxidative damage by increasing the activities of antioxidant enzymes. Furthermore, Si supplementation recovered the nutrient imbalance that had occurred during salinity stress. Additionally, proteomic analysis by two-dimensional gel electrophoresis (2DE) followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed that Si treatment upregulated the accumulation of proteins involved in several metabolic processes, particularly those associated with nucleotide binding and transferase activity. Moreover, Si modulated the expression of vital proteins involved in ubiquitin-mediated nucleosome pathway and carbohydrate metabolism. Overall, the results illustrate that Si application induced resistance against salinity stress in C. annuum by regulating the physiology, antioxidant metabolism, and protein expression. Abinaya Manivannan, Prabhakaran Soundararajan, Sowbiya Muneer, Chung Ho Ko, and Byoung Ryong Jeong Copyright © 2016 Abinaya Manivannan et al. All rights reserved. Cloning and Expression of the γ-Polyglutamic Acid Synthetase Gene pgsBCA in Bacillus subtilis WB600 Thu, 17 Mar 2016 06:46:00 +0000 To clone and express the γ-polyglutamic acid (γ-PGA) synthetase gene pgsBCA in Bacillus subtilis, a pWB980 plasmid was used to construct and transfect the recombinant expression vector pWB980-pgsBCA into Bacillus subtilis WB600. PgsBCA was expressed under the action of a P43 promoter in the pWB980 plasmid. Our results showed that the recombinant bacteria had the capacity to synthesize γ-PGA. The expression product was secreted extracellularly into the fermentation broth, with a product yield of 1.74 g/L or higher. γ-PGA samples from the fermentation broth were purified and characterized. Hydrolysates of γ-PGA presented in single form, constituting simple glutamic acid only, which matched the characteristics of the infrared spectra of the γ-PGA standard, and presented as multimolecular aggregates with a molecular weight within the range of 500–600 kDa. Expressing the γ-PGA synthetase gene pgsBCA in B. subtilis system has potential industrial applications. Biaosheng Lin, Zhijuan Li, Huixia Zhang, Jiangwen Wu, and Maochun Luo Copyright © 2016 Biaosheng Lin et al. All rights reserved. Development of Monoclonal Antibodies against HIV-1 p24 Protein and Its Application in Colloidal Gold Immunochromatographic Assay for HIV-1 Detection Wed, 16 Mar 2016 09:06:38 +0000 Human immunodeficiency virus type 1 (HIV-1) p24 protein is the most abundant viral protein of HIV-1. This protein is secreted in blood serum at high levels during the early stages of HIV-1 infection, making it a biomarker for early diagnosis. In this study, a colloidal gold immunochromatographic assay (GICA) was established for detecting p24 protein using mouse monoclonal antibodies (mAbs). The HIV-1 p24 protein was expressed in E. coli strain BL21 and the purified protein was used to immunize mice. Stable hybridoma cell lines secreting anti-p24 monoclonal antibodies were obtained after ELISA screening and subcloning by limiting dilution. 34 different capture and labeling mAb pairs were selected by a novel antibody-capture indirect sandwich ELISA and then applied in GICA to detect p24 protein. The GICA method has a limit of detection (LOD) of 25 pg/mL and could detect p24 protein in all 10 positive samples obtained from the National Reference of HIV-1 p24 antigen. Out of 153 negative samples tested, 3 false positives results were obtained. The overall specificity of this test was 98.03%. The good sensitivity and specificity of this method make it a suitable alternative to provide a more convenient and efficient tool for early diagnosis of HIV infection. Yi Ma, Chao Ni, Emmanuel E. Dzakah, Haiying Wang, Keren Kang, Shixing Tang, Jihua Wang, and Jufang Wang Copyright © 2016 Yi Ma et al. All rights reserved. Improved Production of Aspergillus usamii endo-β-1,4-Xylanase in Pichia pastoris via Combined Strategies Tue, 15 Mar 2016 13:36:23 +0000 A series of strategies were applied to improve expression level of recombinant endo-β-1,4-xylanase from Aspergillus usamii (A. usamii) in Pichia pastoris (P. pastoris). Firstly, the endo-β-1,4-xylanase (xynB) gene from A. usamii was optimized for P. pastoris and expressed in P. pastoris. The maximum xylanase activity of optimized (xynB-opt) gene was 33500 U/mL after methanol induction for 144 h in 50 L bioreactor, which was 59% higher than that by wild-type (xynB) gene. To further increase the expression of xynB-opt, the Vitreoscilla hemoglobin (VHb) gene was transformed to the recombinant strain containing xynB-opt. The results showed that recombinant strain harboring the xynB-opt and VHb (named X33/xynB-opt-VHb) displayed higher biomass, cell viability, and xylanase activity. The maximum xylanase activity of X33/xynB-opt-VHb in 50 L bioreactor was 45225 U/mL, which was 35% and 115% higher than that by optimized (xynB-opt) gene and wild-type (xynB) gene. Finally, the induction temperature of X33/xynB-opt-VHb was optimized in 50 L bioreactor. The maximum xylanase activity of X33/xynB-opt-VHb reached 58792 U/mL when the induction temperature was 22°C. The results presented here will greatly contribute to improving the production of recombinant proteins in P. pastoris. Jianrong Wang, Yangyuan Li, and Danni Liu Copyright © 2016 Jianrong Wang et al. All rights reserved. Improved Stability of a Model IgG3 by DoE-Based Evaluation of Buffer Formulations Thu, 03 Mar 2016 12:55:18 +0000 Formulating appropriate storage conditions for biopharmaceutical proteins is essential for ensuring their stability and thereby their purity, potency, and safety over their shelf-life. Using a model murine IgG3 produced in a bioreactor system, multiple formulation compositions were systematically explored in a DoE design to optimize the stability of a challenging antibody formulation worst case. The stability of the antibody in each buffer formulation was assessed by UV/VIS absorbance at 280 nm and 410 nm and size exclusion high performance liquid chromatography (SEC) to determine overall solubility, opalescence, and aggregate formation, respectively. Upon preliminary testing, acetate was eliminated as a potential storage buffer due to significant visible precipitate formation. An additional 24 full factorial DoE was performed that combined the stabilizing effect of arginine with the buffering capacity of histidine. From this final DoE, an optimized formulation of 200 mM arginine, 50 mM histidine, and 100 mM NaCl at a pH of 6.5 was identified to substantially improve stability under long-term storage conditions and after multiple freeze/thaw cycles. Thus, our data highlights the power of DoE based formulation screening approaches even for challenging monoclonal antibody molecules. Brittany K. Chavez, Cyrus D. Agarabi, Erik K. Read, Michael T. Boyne II, Mansoor A. Khan, and Kurt A. Brorson Copyright © 2016 Brittany K. Chavez et al. All rights reserved. Biochemical and Metabolic Changes in Arsenic Contaminated Boehmeria nivea L. Sun, 28 Feb 2016 13:51:12 +0000 Arsenic (As) is identified by the EPA as the third highest toxic inorganic contaminant. Almost every 9th or 10th human in more than 70 countries including mainland China is affected by As. Arsenic along with other toxins not only affects human life but also creates alarming situations such as the deterioration of farm lands and desertion of industrial/mining lands. Researchers and administrators have agreed to opt for phytoremediation of As over costly cleanups. Boehmeria nivea L. can soak up various heavy metals, such as Sb, Cd, Pb, and Zn. But the effect of As pollution on the biology and metabolism of B. nivea has been somewhat overlooked. This study attempts to evaluate the extent of As resistance, chlorophyll content, and metabolic changes in As-polluted (5, 10, 15, and 20 mg L−1 As) B. nivea in hydroponics. Toxic effects of As in the form of inhibited growth were apparent at the highest level of added As. The significant changes in the chlorophyll, electrolyte leakage, and H2O2, significant increases in As in plant parts, catalase (CAT), and malondialdehyde (MDA), with applied As revealed the potential of B. nivea for As decontamination. By employing the metabolic machinery of B. nivea, As was sustainably removed from the contaminated areas. Hussani Mubarak, Nosheen Mirza, Li-Yuan Chai, Zhi-Hui Yang, Wang Yong, Chong-Jian Tang, Qaisar Mahmood, Arshid Pervez, Umar Farooq, Shah Fahad, Wajid Nasim, and Kadambot H. M. Siddique Copyright © 2016 Hussani Mubarak et al. All rights reserved. Overexpression of AaWRKY1 Leads to an Enhanced Content of Artemisinin in Artemisia annua Tue, 23 Feb 2016 09:46:59 +0000 Artemisinin is an effective component of drugs against malaria. The regulation of artemisinin biosynthesis is at the forefront of artemisinin research. Previous studies showed that AaWRKY1 can regulate the expression of ADS, which is the first key enzyme in artemisinin biosynthetic pathway. In this study, AaWRKY1 was cloned, and it activated ADSpro and CYPpro in tobacco using dual-LUC assay. To further study the function of AaWRKY1, pCAMBIA2300-AaWRKY1 construct under 35S promoter was generated. Transgenic plants containing AaWRKY1 were obtained, and four independent lines with high expression of AaWRKY1 were analyzed. The expression of ADS and CYP, the key enzymes in artemisinin biosynthetic pathway, was dramatically increased in AaWRKY1-overexpressing A. annua plants. Furthermore, the artemisinin yield increased significantly in AaWRKY1-overexpressing A. annua plants. These results showed that AaWRKY1 increased the content of artemisinin by regulating the expression of both ADS and CYP. It provides a new insight into the mechanism of regulation on artemisinin biosynthesis via transcription factors in the future. Weimin Jiang, Xueqing Fu, Qifang Pan, Yueli Tang, Qian Shen, Zongyou Lv, Tingxiang Yan, Pu Shi, Ling Li, Lida Zhang, Guofeng Wang, Xiaofen Sun, and Kexuan Tang Copyright © 2016 Weimin Jiang et al. All rights reserved. Optimization of Cultural Conditions for Antioxidant Exopolysaccharides from Xerocomus badius Grown in Shrimp Byproduct Mon, 22 Feb 2016 12:49:30 +0000 To optimize the production conditions for exopolysaccharides with higher antioxidant activities from Xerocomus badius cultured in shrimp byproduct medium, Plackett-Burman design, path of steepest ascent, and response surface methodology were explored. Based on the results of Plackett-Burman design and path of steepest ascent, a Box-Behnken design was applied to optimization and the regression models. The optimal cultural condition for high yield and antioxidant activity of the exopolysaccharides was determined to be 10.347% of solid-to-liquid ratio, a 4.322% content of bran powder, and a 1.217% concentration of glacial acetic acid. Culturing with the optimal cultural conditions resulted in an exopolysaccharides yield of  g/L and a total antioxidant activity of  U/mg. These values are consistent with the values predicted by the corresponding regression models (RSD < 5%). Xiujun Gao, Peisheng Yan, Xin Liu, Jianbing Wang, and Jiajia Yu Copyright © 2016 Xiujun Gao et al. All rights reserved. Optimized Biotransformation of Icariin into Icariside II by β-Glucosidase from Trichoderma viride Using Central Composite Design Method Sun, 14 Feb 2016 09:27:33 +0000 A crude -glucosidase has been produced from Trichoderma viride and used to explore a simple method to prepare icariside II from icariin. The crude enzyme has been studied by zymography method and used for hydrolysis of ICA. To achieve a high conversion rate of ICA, various factors have been studied including pH, reaction time, temperature, initial concentration of enzyme, and initial concentration of ICA through central composite design experiments. In the condition of the optimum hydrolysis parameters with pH 4.0, 41°C, 1.0 mg/mL ICA, and 9.8 U/mL crude -glucosidase, the conversion rate of ICA reached 95.03% at 1 h. Moreover, the cytotoxicity test showed that ICA II performed inhibition effects on proliferation of A549 cell, while ICA has no cytotoxicity. It indicated that the hydrolysis transformation study of ICA is valuable for exploration of active new drugs. Tao Cheng, Jun Yang, Tong Zhang, Yi-Shun Yang, and Yue Ding Copyright © 2016 Tao Cheng et al. All rights reserved. Upscaling Arbuscular Mycorrhizal Symbiosis and Related Agroecosystems Services in Smallholder Farming Systems Thu, 28 Jan 2016 12:57:55 +0000 Smallholder farming systems form unique ecosystems that can protect beneficial soil biota and form an important source of useful genetic resources. They are characterized by high level of agricultural diversity mainly focused on meeting farmers’ needs. Unfortunately, these systems often experience poor crop production mainly associated with poor planning and resource scarcity. Soil fertility is among the primary challenges faced by smallholder farmers, which necessitate the need to come up with affordable and innovative ways of replenishing soils. One such way is the use of microbial symbionts such as arbuscular mycorrhizal fungi (AMF), a beneficial group of soil microbiota that form symbiotic associations with majority of cultivated crops and play a vital role in biological soil fertility, plant nutrition, and protection. AMF can be incorporated in smallholder farming systems to help better exploit chemical fertilizers inputs which are often unaffordable to many smallholder farmers. The present review highlights smallholder farming practices that could be innovatively redesigned to increase AMF symbiosis and related agroecosystem services. Indeed, the future of global food security depends on the success of smallholder farming systems, whose crop productivity depends on the services provided by well-functioning ecosystems, including soil fertility. Marjorie Bonareri Oruru and Ezekiel Mugendi Njeru Copyright © 2016 Marjorie Bonareri Oruru and Ezekiel Mugendi Njeru. All rights reserved. Synergy between Rhizobium phaseoli and Acidithiobacillus ferrooxidans in the Bioleaching Process of Copper Thu, 28 Jan 2016 06:39:44 +0000 This study investigates the synergy of Rhizobium phaseoli and Acidithiobacillus ferrooxidans in the bioleaching process of copper. The results showed that additional R. phaseoli could increase leaching rate and cell number of A. ferrooxidans. When the initial cell number ratio between A. ferrooxidans and R. phaseoli was 2 : 1, A. ferrooxidans attained the highest final cell number of approximately 2 × 108 cells/mL and the highest copper leaching rate of 29%, which is 7% higher than that in the group with A. ferrooxidans only. R. phaseoli may use metabolized polysaccharides from A. ferrooxidans, and organic acids could chelate or precipitate harmful heavy metals to reduce their damage on A. ferrooxidans and promote its growth. Organic acids could also damage the mineral lattice to increase the leaching effect. Xuecheng Zheng and Dongwei Li Copyright © 2016 Xuecheng Zheng and Dongwei Li. All rights reserved. Azocasein Substrate for Determination of Proteolytic Activity: Reexamining a Traditional Method Using Bromelain Samples Wed, 27 Jan 2016 10:11:16 +0000 Given the importance of protease’s worldwide market, the determination of optimum conditions and the development of a standard protocol are critical during selection of a reliable method to determine its bioactivity. This paper uses quality control theory to validate a modified version of a method proposed by Charney and Tomarelli in 1947. The results obtained showed that using azocasein substrate bromelain had its optimum at 45°C and pH 9 (Glycine-NaOH 100 mM). We also quantified the limit of detection (LoD) and limit of quantification (LoQ) in the above-mentioned optimum (0.072 and 0.494 mg·mL−1 of azocasein, resp.) and a calibration curve that correlates optical density with the amount of substrate digested. In all analysed samples, we observed a significant decrease in response after storage (around 17%), which suggests its use must be immediately after preparation. Thus, the protocol presented in this paper offers a significant improvement, given that subjective definitions are commonly used in the literature and this simple mathematical approach makes it clear and concise. Diego F. Coêlho, Thais Peron Saturnino, Fernanda Freitas Fernandes, Priscila Gava Mazzola, Edgar Silveira, and Elias Basile Tambourgi Copyright © 2016 Diego F. Coêlho et al. All rights reserved. Treatment of Copper Contaminated Municipal Wastewater by Using UASB Reactor and Sand-Chemically Carbonized Rubber Wood Sawdust Column Wed, 20 Jan 2016 12:13:58 +0000 The performance of a laboratory scale upflow anaerobic sludge blanket (UASB) reactor and its posttreatment unit of sand-chemically carbonized rubber wood sawdust (CCRWSD) column system for the treatment of a metal contaminated municipal wastewater was investigated. Copper ion contaminated municipal wastewater was introduced to a laboratory scale UASB reactor and the effluent from UASB reactor was then followed by treatment with sand-CCRWSD column system. The laboratory scale UASB reactor and column system were observed for a period of 121 days. After the posttreatment column the average removal of monitoring parameters such as copper ion concentration (91.37%), biochemical oxygen demand () (93.98%), chemical oxygen demand (COD) (95.59%), total suspended solid (TSS) (95.98%), ammonia (80.68%), nitrite (79.71%), nitrate (71.16%), phosphorous (44.77%), total coliform (TC) (99.9%), and fecal coliform (FC) (99.9%) was measured. The characterization of the chemically carbonized rubber wood sawdust was done by scanning electron microscope (SEM), X-ray fluorescence spectrum (XRF), and Fourier transforms infrared spectroscopy (FTIR). Overall the system was found to be an efficient and economical process for the treatment of copper contaminated municipal wastewater. Swarup Biswas and Umesh Mishra Copyright © 2016 Swarup Biswas and Umesh Mishra. All rights reserved. Multipurpose, Integrated 2nd Generation Biorefineries Tue, 19 Jan 2016 06:26:32 +0000 Alberto Scoma, Lorenzo Bertin, Maria A. M. Reis, Michael Kornaros, and Marta Coma Copyright © 2016 Alberto Scoma et al. All rights reserved. Organogenesis and Ultrastructural Features of In Vitro Grown Canna indica L. Mon, 18 Jan 2016 16:29:33 +0000 An efficient protocol for micropropagation of Canna indica L., an economically and pharmaceutically important plant, was standardized using rhizome explants, excised from two-month-old aseptic seedlings. Complete plant regeneration was induced on MS medium supplemented with 3.0 mg/L BAP plus 1.5 mg/L NAA, which produced the highest number of shoots (73.3 ± 0.5%) and roots (86.7 ± 0.4%) after 2 weeks. Furthermore, the optimum media for multiple shoots regeneration were recorded on MS enriched with 7.0 mg/L BAP (33.0 ± 0.5%). Plantlets obtained were transplanted to pots after two months and acclimatized in the greenhouse, with 75% survival. In addition, ultrastructural studies showed that rhizomes of in vitro grown specimens were underdeveloped compared to the in vivo specimens, possibly due to the presence of wide spaces. Meanwhile, the leaves of in vivo specimens had more open stomata compared to in vitro specimens, yet their paracytic stomata structures were similar. Hence, there were no abnormalities or major differences between in vitro regenerants and mother plants. Sharifah Nurashikin Wafa, Rosna Mat Taha, Sadegh Mohajer, Noraini Mahmad, and Bakrudeen Ali Ahmed Abdul Copyright © 2016 Sharifah Nurashikin Wafa et al. All rights reserved. Preparation of Microencapsulated Bacillus subtilis SL-13 Seed Coating Agents and Their Effects on the Growth of Cotton Seedlings Thu, 14 Jan 2016 15:56:31 +0000 Inoculation of the bacterial cells of microbial seed coating agents (SCAs) into the environment may result in limited survival and colonization. Therefore, the application efficacy of an encapsulated microbial seed coating agent (ESCA) was investigated on potted cotton plants; the agent was prepared with polyvinyl alcohol, sodium dodecyl sulfate, bentonite, and microencapsulated Bacillus subtilis SL-13. Scanning electron micrography revealed that the microcapsules were attached to ESCA membranes. The ESCA film was uniform, bubble-free, and easy to peel. The bacterial contents of seeds coated with each ESCA treatment reached 106 cfu/seed. Results indicated that the germination rate of cotton seeds treated with ESCA4 (1.0% (w/v) sodium alginate, 4.0% polyvinyl alcohol, 1.0% sodium dodecyl sulfate, 0.6% acacia, 0.5% bentonite, and 10% (v/v) microcapsules) increased by 28.74%. Other growth factors of the cotton seedlings, such as plant height, root length, whole plant fresh weight, and whole plant dry weight, increased by 52.70%, 25.13%, 46.47%, and 33.21%, respectively. Further analysis demonstrated that the peroxidase and superoxide dismutase activities of cotton seedlings improved, whereas their malondialdehyde contents decreased. Therefore, the ESCA can efficiently improve seed germination, root length, and growth. The proposed ESCA exhibits great potential as an alternative to traditional SCA in future agricultural applications. Liang Tu, Yanhui He, Chunhui Shan, and Zhansheng Wu Copyright © 2016 Liang Tu et al. All rights reserved. Extraction of Oleic Acid from Moroccan Olive Mill Wastewater Sun, 10 Jan 2016 07:47:55 +0000 The production of olive oil in Morocco has recently grown considerably for its economic and nutritional importance favored by the country’s climate. After the extraction of olive oil by pressing or centrifuging, the obtained liquid contains oil and vegetation water which is subsequently separated by decanting or centrifugation. Despite its treatment throughout the extraction process, this olive mill wastewater, OMW, still contains a very important oily residue, always regarded as a rejection. The separated oil from OMW can not be intended for food because of its high acidity of 3.397% which exceeds the international standard for human consumption defined by the standard of the Codex Alimentarius, proving its poor quality. This work gives value addition to what would normally be regarded as waste by the extraction of oleic acid as a high value product, using the technique of inclusion with urea for the elimination of saturated and unsaturated fatty acids through four successive crystallizations at 4°C and 20°C to have a final phase with oleic acid purity of 95.49%, as a biodegradable soap and a high quality glycerin will be produced by the reaction of saponification and transesterification. Reda Elkacmi, Noureddine Kamil, Mounir Bennajah, and Said Kitane Copyright © 2016 Reda Elkacmi et al. All rights reserved. Application of Innovative Technologies for Improved Food Quality and Safety Mon, 04 Jan 2016 12:58:15 +0000 Yiannis Kourkoutas, Nikos Chorianopoulos, Aspasia Nisiotou, Vasilis P. Valdramidis, and Kimon A. G. Karatzas Copyright © 2016 Yiannis Kourkoutas et al. All rights reserved. Enhanced and Secretory Expression of Human Granulocyte Colony Stimulating Factor by Bacillus subtilis SCK6 Thu, 31 Dec 2015 13:20:36 +0000 This study describes a simplified approach for enhanced expression and secretion of a pharmaceutically important human cytokine, that is, granulocyte colony stimulating factor (GCSF), in the culture supernatant of Bacillus subtilis SCK6 cells. Codon optimized GCSF and pNWPH vector containing SpymwC signal sequence were amplified by prolonged overlap extension PCR to generate multimeric plasmid DNA, which was used directly to transform B. subtilis SCK6 supercompetent cells. Expression of GCSF was monitored in the culture supernatant for 120 hours. The highest expression, which corresponded to 17% of the total secretory protein, was observed at 72 hours of growth. Following ammonium sulphate precipitation, GCSF was purified to near homogeneity by fast protein liquid chromatography on a QFF anion exchange column. Circular dichroism spectroscopic analysis showed that the secondary structure contents of the purified GCSF are similar to the commercially available GCSF. Biological activity, as revealed by the regeneration of neutrophils in mice treated with ifosfamine, was also similar to the commercial preparation of GCSF. This, to our knowledge, is the first study that reports secretory expression of human GCSF in B. subtilis SCK6 with final recovery of up to 96 mg/L of the culture supernatant, without involvement of any chemical inducer. Shaista Bashir, Saima Sadaf, Sajjad Ahmad, and Muhammad Waheed Akhtar Copyright © 2015 Shaista Bashir et al. All rights reserved. Impact of pH Management Interval on Biohydrogen Production from Organic Fraction of Municipal Solid Wastes by Mesophilic Thermophilic Anaerobic Codigestion Sun, 27 Dec 2015 13:42:19 +0000 The biohydrogen productions from the organic fraction of municipal solid wastes (OFMSW) were studied under pH management intervals of 12 h (PM12) and 24 h (PM24) for temperature of °C and °C. The OFMSW or food waste (FW) along with its two components, noodle waste (NW) and rice waste (RW), was codigested with sludge to estimate the potential of biohydrogen production. The biohydrogen production was higher in all reactors under PM12 as compared to PM24. The drop in pH from 7 to 5.3 was observed to be appropriate for biohydrogen production via mesophilic codigestion of noodle waste with the highest biohydrogen yield of 145.93 mL/g under PM12. When the temperature was increased from 37°C to 55°C and pH management interval was reduced from 24 h to 12 h, the biohydrogen yields were also changed from 39.21 mL/g to 89.67 mL/g , 91.77 mL/g to 145.93 mL/g , and 15.36 mL/g to 117.62 mL/g for FW, NW, and RW, respectively. The drop in pH and VFA production was better controlled under PM12 as compared to PM24. Overall, PM12 was found to be an effective mean for biohydrogen production through anaerobic digestion of food waste. Chaudhry Arslan, Asma Sattar, Ji Changying, Abdul Nasir, Irshad Ali Mari, and Muhammad Zia Bakht Copyright © 2015 Chaudhry Arslan et al. All rights reserved. Production of Monomeric Aromatic Compounds from Oil Palm Empty Fruit Bunch Fiber Lignin by Chemical and Enzymatic Methods Tue, 22 Dec 2015 06:20:05 +0000 In this study, oil palm empty fruit bunch (OPEFBF) was pretreated with alkali, and lignin was extracted for further degradation into lower molecular weight phenolic compounds using enzymes and chemical means. Efficiency of monomeric aromatic compounds production from OPEFBF lignin via chemical (nitrobenzene versus oxygen) and enzymatic [cutinase versus manganese peroxidase (MnP)] approaches was investigated. The effects of sodium hydroxide concentration (2, 5, and 10% wt.) and reaction time (30, 90, and 180 minutes) on the yield of aromatic compounds were studied. The results obtained indicated that nitrobenzene oxidation produced the highest yield ( ppm hydroxybenzoic acid,  ppm p-hydroxybenzaldehyde,  ppm vanillic acid,  ppm vanillin,  ppm syringic acid,  ppm syringaldehyde,  ppm p-coumaric acid, and  ppm ferulic acid), among the tested methods. High sodium hydroxide concentration (10% wt.) was needed to promote efficient nitrobenzene oxidation. However, less severe oxidation condition was preferred to preserve the hydroxycinnamic acids (p-coumaric acid and ferulic acid). Cutinase-catalyzed hydrolysis was found to be more efficient than MnP-catalyzed oxidation in the production of aromatic compounds. By hydrolyzed 8% wt. of lignin with 0.625 mL cutinase g−1 lignin at pH 8 and 55°C for 24 hours, about  ppm hydroxybenzoic acid,  ppm syringaldehyde,  ppm vanillin,  ppm p-coumaric acid, and  ppm ferulic acid were produced. Pei-Ling Tang, Osman Hassan, Mohamad Yusof Maskat, and Khairiah Badri Copyright © 2015 Pei-Ling Tang et al. All rights reserved. Calcium Mitigates Arsenic Toxicity in Rice Seedlings by Reducing Arsenic Uptake and Modulating the Antioxidant Defense and Glyoxalase Systems and Stress Markers Sun, 20 Dec 2015 06:33:38 +0000 The effect of exogenous calcium (Ca) on hydroponically grown rice seedlings was studied under arsenic (As) stress by investigating the antioxidant and glyoxalase systems. Fourteen-day-old rice (Oryza sativa L. cv. BRRI dhan29) seedlings were exposed to 0.5 and 1 mM Na2HAsO4 alone and in combination with 10 mM CaCl2 (Ca) for 5 days. Both levels of As caused growth inhibition, chlorosis, reduced leaf RWC, and increased As accumulation in the rice seedlings. Both doses of As in growth medium induced oxidative stress through overproduction of reactive oxygen species (ROS) by disrupting the antioxidant defense and glyoxalase systems. Exogenous application of Ca along with both levels of As significantly decreased As accumulation and restored plant growth and water loss. Calcium supplementation in the As-exposed rice seedlings reduced ROS production, increased ascorbate (AsA) content, and increased the activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT), glutathione peroxidase (GPX), superoxide dismutase (SOD), and the glyoxalase I (Gly I) and glyoxalase II (Gly II) enzymes compared with seedlings exposed to As only. These results suggest that Ca supplementation improves rice seedlings tolerance to As-induced oxidative stress by reducing As uptake, enhancing their antioxidant defense and glyoxalase systems, and also improving growth and physiological condition. Anisur Rahman, Mohammad Golam Mostofa, Md. Mahabub Alam, Kamrun Nahar, Mirza Hasanuzzaman, and Masayuki Fujita Copyright © 2015 Anisur Rahman et al. All rights reserved. Environmental Biotechnology: Current Advances, New Knowledge Gaps, and Emerging Issues Mon, 14 Dec 2015 09:14:22 +0000 Abd El-Latif Hesham, T. Komang Ralebitso-Senior, Yu Zhang, and Qing X. Li Copyright © 2015 Abd El-Latif Hesham et al. All rights reserved. Towards a Universal Approach Based on Omics Technologies for the Quality Control of Food Sun, 13 Dec 2015 07:21:08 +0000 In the last decades, food science has greatly developed, turning from the consideration of food as mere source of energy to a growing awareness on its importance for health and particularly in reducing the risk of diseases. Such vision led to an increasing attention towards the origin and quality of raw materials as well as their derived food products. The continuous advance in molecular biology allowed setting up efficient and universal omics tools to unequivocally identify the origin of food items and their traceability. In this review, we considered the application of a genomics approach known as DNA barcoding in characterizing the composition of foodstuffs and its traceability along the food supply chain. Moreover, metabolomics analytical strategies based on Nuclear Magnetic Resonance (NMR) and Mass Spectroscopy (MS) were discussed as they also work well in evaluating food quality. The combination of both approaches allows us to define a sort of molecular labelling of food that is easily understandable by the operators involved in the food sector: producers, distributors, and consumers. Current technologies based on digital information systems such as web platforms and smartphone apps can facilitate the adoption of such molecular labelling. Emanuele Ferri, Andrea Galimberti, Maurizio Casiraghi, Cristina Airoldi, Carlotta Ciaramelli, Alessandro Palmioli, Valerio Mezzasalma, Ilaria Bruni, and Massimo Labra Copyright © 2015 Emanuele Ferri et al. All rights reserved. Cadmium Removal from Aqueous Systems Using Opuntia albicarpa L. Scheinvar as Biosorbent Wed, 09 Dec 2015 13:08:16 +0000 The aim of this research was to investigate the use of a natural adsorbent like nopal (Opuntia albicarpa L. Scheinvar) for removing cadmium from aqueous solutions with low concentrations of this metal. Two treatments were applied to the cladodes: a dehydration to get dehydrated nopal (DHN) and heating up to 90°C to obtain a thermally treated nopal (TN). After examining the effect of various pH values (2–7), the capacity of each biosorbent was examined in batch sorption tests at different dosages (0, 500, 1000, 1500, 2000, and 3000 mg L−1). The results indicated that adsorption of cadmium to biomass of DHN and TN was highly dependent on pH and biosorbent dosage. The best removal of cadmium (53.3%, corresponding to of 0.155 mg g−1) was obtained at pH 4.0 by using the TN sorbent. Infrared and Raman spectra confirmed that cadmium removal occurred via adsorption to –OH functional groups. Rosa Icela Beltrán-Hernández, Gabriela Alejandra Vázquez-Rodríguez, Luis Felipe Juárez-Santillán, Ivan Martínez-Ugalde, Claudia Coronel-Olivares, and Carlos Alexander Lucho-Constantino Copyright © 2015 Rosa Icela Beltrán-Hernández et al. All rights reserved. Improvement of D-Ribose Production from Corn Starch Hydrolysate by a Transketolase-Deficient Strain Bacillus subtilis UJS0717 Wed, 02 Dec 2015 13:21:17 +0000 D-Ribose is a five-carbon sugar and generally used as an energy source to improve athletic performance and the ability. The culture conditions for maximum D-ribose production performance from cheap raw material corn starch hydrolysate were improved by using one-factor-at-a-time experiments and a three-level Box-Behnken factorial design. The optimal fermentation parameters were obtained as 36°C culture temperature, 10% inoculum volume, and 7.0 initial pH. The mathematical model was then developed to show the effect of each medium composition and their interactions on the production of D-ribose and estimated that the optimized D-ribose production performance with the concentration of 62.13 g/L, yield of 0.40 g/g, and volumetric productivity of 0.86 g/L·h could be obtained when the medium compositions were set as 157 g/L glucose, 21 g/L corn steep liquor, 3.2 g/L (NH4)2SO4, 1 g/L yeast extract, 0.05 g/L MnSO4·H2O, and 20 g/L CaCO3. These findings indicated the D-ribose production performance was significantly improved compared to that under original conditions. Zhuan Wei, Jue Zhou, WenJing Sun, FengJie Cui, QinHua Xu, and ChangFeng Liu Copyright © 2015 Zhuan Wei et al. All rights reserved. Indirect Manganese Removal by Stenotrophomonas sp. and Lysinibacillus sp. Isolated from Brazilian Mine Water Tue, 01 Dec 2015 12:56:46 +0000 Manganese is a contaminant in the wastewaters produced by Brazilian mining operations, and the removal of the metal is notoriously difficult because of the high stability of the Mn(II) ion in aqueous solutions. To explore a biological approach for removing excessive amounts of aqueous Mn(II), we investigated the potential of Mn(II) oxidation by both consortium and bacterial isolates from a Brazilian manganese mine. A bacterial consortium was able to remove 99.7% of the Mn(II). A phylogenetic analysis of isolates demonstrated that the predominant microorganisms were members of Stenotrophomonas, Bacillus, and Lysinibacillus genera. Mn(II) removal rates between 58.5% and 70.9% were observed for Bacillus sp. and Stenotrophomonas sp. while the Lysinibacillus isolate 13P removes 82.7%. The catalytic oxidation of Mn(II) mediated by multicopper oxidase was not properly detected; however, in all of the experiments, a significant increase in the pH of the culture medium was detected. No aggregates inside the cells grown for a week were found by electronic microscopy. Nevertheless, an energy-dispersive X-ray spectroscopy of the isolates revealed the presence of manganese in Stenotrophomonas sp. and Lysinibacillus sp. grown in K medium. These results suggest that members of Stenotrophomonas and Lysinibacillus genera were able to remove Mn(II) by a nonenzymatic pathway. Natália Rocha Barboza, Soraya Sander Amorim, Pricila Almeida Santos, Flávia Donária Reis, Mônica Mendes Cordeiro, Renata Guerra-Sá, and Versiane Albis Leão Copyright © 2015 Natália Rocha Barboza et al. All rights reserved. Growth Inhibition and Membrane Permeabilization of Candida lusitaniae Using Varied Pulse Shape Electroporation Tue, 01 Dec 2015 12:03:03 +0000 Candida lusitaniae is an opportunistic yeast pathogen, which can readily develop resistance to antifungal compounds and result in a complex long-term treatment. The efficient treatment is difficult since structure and metabolic properties of the fungal cells are similar to those of eukaryotic host. One of the potential methods to improve the inhibition rate or the cell permeability to inhibitors is the application of electroporation. In this work we investigated the dynamics of the growth inhibition and membrane permeabilization of C. lusitaniae by utilizing the various pulse shape and duration electric field pulses. Our results indicated that single electroporation procedure using 8 kV/cm electric field may result in up to % inhibition rate. Also it has been experimentally shown that the electroporation pulse shape may influence the inhibitory effect; however, the amplitude of the electric field and the pulse energy remain the most important parameters for definition of the treatment outcome. The dynamics of the cell membrane permeabilization in the 2–8 kV/cm electric field were overviewed. V. Novickij, A. Grainys, E. Lastauskienė, R. Kananavičiūtė, D. Pamedytytė, A. Zinkevičienė, L. Kalėdienė, J. Novickij, A. Paškevičius, and J. Švedienė Copyright © 2015 V. Novickij et al. All rights reserved. Safety Evaluation of the Coagulase-Negative Staphylococci Microbiota of Salami: Superantigenic Toxin Production and Antimicrobial Resistance Mon, 30 Nov 2015 13:08:47 +0000 The risks of contracting staphylococci food poisoning by the consumption of improperly manufactured salami and the possibility of this food being reservoirs for antibiotic resistance were evaluated. Nineteen coagulase-negative staphylococci (CNS) strains were found in commercial and artisanal salami. The species in commercial salami were S. saprophyticus, S. sciuri, S. xylosus, and S. carnosus. Artisanal salami showed S. succinus, S. epidermidis, and S. hominis but no S. carnosus. Phylogenetic analyses grouped the strains into three major staphylococcal species groups, comprised of 4 refined clusters with similarities superior to 90%. Fifteen strains harbored multiple enterotoxin genes, with high incidence of seb/sec and sea, 57% and 50%, respectively, intermediate incidence of sed/seh/selm and sei/seln/tst-H, 33% and 27%, correspondingly, and low incidence of see/selj/selo and seg, of respectively 13% and 1%. Real time RT-PCR and enzyme-linked-immunosorbent assays confirmed the enterotoxigenicity of the strains, which expressed and produced enterotoxins in vitro. The CNS strains showed multiresistance to several antimicrobials of therapeutic importance in both human and veterinarian medicine, such as β-lactams, vancomycin, and linezolid. The effective control of undue staphylococci in fermented meat products should be adopted to prevent or limit the risk of food poisoning and the spread of antimicrobial-resistant strains. Raquel Soares Casaes Nunes, Eduardo Mere Del Aguila, and Vânia Margaret Flosi Paschoalin Copyright © 2015 Raquel Soares Casaes Nunes et al. All rights reserved. Ethanol and Protein from Ethanol Plant By-Products Using Edible Fungi Neurospora intermedia and Aspergillus oryzae Mon, 23 Nov 2015 12:07:41 +0000 Feasible biorefineries for production of second-generation ethanol are difficult to establish due to the process complexity. An alternative is to partially include the process in the first-generation plants. Whole stillage, a by-product from dry-mill ethanol processes from grains, is mostly composed of undegraded bran and lignocelluloses can be used as a potential substrate for production of ethanol and feed proteins. Ethanol production and the proteins from the stillage were investigated using the edible fungi Neurospora intermedia and Aspergillus oryzae, respectively. N. intermedia produced 4.7 g/L ethanol from the stillage and increased to 8.7 g/L by adding 1 FPU of cellulase/g suspended solids. Saccharomyces cerevisiae produced 0.4 and 5.1 g/L ethanol, respectively. Under a two-stage cultivation with both fungi, up to 7.6 g/L of ethanol and 5.8 g/L of biomass containing 42% (w/w) crude protein were obtained. Both fungi degraded complex substrates including arabinan, glucan, mannan, and xylan where reductions of 91, 73, 38, and 89% (w/v) were achieved, respectively. The inclusion of the current process can lead to the production of 44,000 m3 of ethanol (22% improvement), around 12,000 tons of protein-rich biomass for animal feed, and energy savings considering a typical facility producing 200,000 m3 ethanol/year. Veronika Bátori, Jorge A. Ferreira, Mohammad J. Taherzadeh, and Patrik R. Lennartsson Copyright © 2015 Veronika Bátori et al. All rights reserved. In Vitro Propagation, Phytochemical Analysis, and Evaluation of Free Radical Scavenging Property of Scrophularia kakudensis Franch Tissue Extracts Mon, 16 Nov 2015 08:25:56 +0000 The current study deals with in vitro propagation, antioxidant property estimation, and assessment of acacetin content in Scrophularia kakudensis Franch. Adventitious shoot induction was achieved from the nodal explant with the highest number of adventitious shoots per explant (17.4) on Murashige and Skoog’s (MS) medium fortified with 2.0 mg·L−1 6-benzyladenine (BA) and 0.5 mg L−1 indole-3-acetic acid (IAA). Maximum number of roots per plant (16.5) was noted in half strength MS medium supplemented with 0.5 mg·L−1 IAA. The regenerated plants displayed successful survival ratio (95%) in the greenhouse. The highest content of acacetin, a pharmaceutically important flavonoid, was observed in the shoot extracts (in vitro: 32.83 µg·g−1 FW; in vivo: 30.05 µg·g−1 FW) followed by root extracts. Total phenol and flavonoid contents along with free radical scavenging assays revealed the occurrence of larger amount of antioxidants in shoot extract in comparison with callus and root extracts of S. kakudensis. Thus, the outcome of the present study can be highly beneficial for the germplasm conservation and commercial cultivation of S. kakudensis for therapeutic purposes. Abinaya Manivannan, Prabhakaran Soundararajan, Yoo Gyeong Park, and Byoung Ryong Jeong Copyright © 2015 Abinaya Manivannan et al. All rights reserved. One-Step Recovery of scFv Clones from High-Throughput Sequencing-Based Screening of Phage Display Libraries Challenged to Cells Expressing Native Claudin-1 Sun, 15 Nov 2015 11:35:40 +0000 Expanding the availability of monoclonal antibodies interfering with hepatitis C virus infection of hepatocytes is an active field of investigation within medical biotechnologies, to prevent graft reinfection in patients subjected to liver transplantation and to overcome resistances elicited by novel antiviral drugs. In this paper, we describe a complete pipeline for screening of phage display libraries of human scFvs against native Claudin-1, a tight-junction protein involved in hepatitis C virus infection, expressed on the cell surface of human hepatocytes. To this aim, we implemented a high-throughput sequencing approach for library screening, followed by a simple and effective strategy to recover active binder clones from enriched sublibraries. The recovered clones were successfully converted to active immunoglobulins, thus demonstrating the effectiveness of the whole procedure. This novel approach can guarantee rapid and cheap isolation of antibodies for virtually any native antigen involved in human diseases, for therapeutic and/or diagnostic applications. Emanuele Sasso, Rolando Paciello, Francesco D’Auria, Gennaro Riccio, Guendalina Froechlich, Riccardo Cortese, Alfredo Nicosia, Claudia De Lorenzo, and Nicola Zambrano Copyright © 2015 Emanuele Sasso et al. All rights reserved. Optimization of Extraction Parameters for Enhanced Production of Ovotransferrin from Egg White for Antimicrobial Applications Tue, 10 Nov 2015 13:10:25 +0000 Ovotransferrin is the second most abundant protein (~12-13% of the total egg protein) in egg white after ovalbumin. Ovotransferrin is a potent natural antimicrobial agent as it possesses antibacterial, antifungal, and antiviral properties and is also the major metal binding protein found in egg, which makes it an industrially important protein. Ovotransferrin was extracted from egg white using its metal (iron) binding properties. In the present study, eggs from two different sources were used (fresh local eggs from domestic household source and poultry eggs from shops) to compare the results and Response Surface Methodology was used for the experiment design and data analysis. The following extraction conditions were optimized so as to maximize the yield of ovotransferrin from egg white: ethanol % (v/v) and pH and volume (mL) of 25 mM FeCl3/50 mL of egg white. A maximum yield of ~85 ± 2.5% was obtained near the optimum extraction conditions. The yield was calculated based on the theoretical value (934 mg) of ovotransferrin in 100 mL of 1.5x diluted egg white solution. Our results suggest that efficient downstream processing may reduce the cost of overall production process of this promising enzyme, making it a natural and cost-effective alternative to the existing chemically synthesized antimicrobial agents. Eyad M. A. Alshammari, Saif Khan, Arshad Jawed, Mohd Adnan, Mahvish Khan, Gowher Nabi, Mohtashim Lohani, and Shafiul Haque Copyright © 2015 Eyad M. A. Alshammari et al. All rights reserved. Selection of the Strain Lactobacillus acidophilus ATCC 43121 and Its Application to Brewers’ Spent Grain Conversion into Lactic Acid Tue, 10 Nov 2015 06:02:26 +0000 Six Lactobacillus strains were analyzed to select a bacterium for conversion of brewers’ spent grain (BSG) into lactic acid. Among the investigated strains, L. acidophilus ATCC 43121 showed the highest yield of lactic acid production (16.1 g/L after 48 hours) when grown in a synthetic medium. It was then analyzed for its ability to grow on the hydrolysates obtained from BSG after acid-alkaline (AAT) or aqueous ammonia soaking (AAS) pretreatment. The lactic acid production by L. acidophilus ATCC 43121 through fermentation of the hydrolysate from AAS treated BSG was 96% higher than that from the AAT treated one, although similar yields of lactic acid per consumed glucose were achieved due to a higher (46%) glucose consumption by L. acidophilus ATCC 43121 in the AAS BSG hydrolysate. It is worth noting that adding yeast extract to the BSG hydrolysates increased both the yield of lactic acid per substrate consumed and the volumetric productivity. The best results were obtained by fermentation of AAS BSG hydrolysate supplemented by yeast extract, in which the strain produced 22.16 g/L of lactic acid (yield of 0.61 g/g), 27% higher than the value (17.49 g/L) obtained in the absence of a nitrogen source. Rossana Liguori, Carlos Ricardo Soccol, Luciana Porto de Souza Vandenberghe, Adenise Lorenci Woiciechowski, Elena Ionata, Loredana Marcolongo, and Vincenza Faraco Copyright © 2015 Rossana Liguori et al. All rights reserved. Selection of Single-Stranded DNA Molecular Recognition Elements against Exotoxin A Using a Novel Decoy-SELEX Method and Sensitive Detection of Exotoxin A in Human Serum Mon, 09 Nov 2015 08:32:04 +0000 Exotoxin A is one of the virulence factors of Pseudomonas aeruginosa, a bacterium that can cause infections resulting in adverse health outcomes and increased burden to health care systems. Current methods of diagnosing P. aeruginosa infections are time consuming and can require significant preparation of patient samples. This study utilized a novel variation of the Systematic Evolution of Ligand by Exponential Enrichment, Decoy-SELEX, to identify an Exotoxin A specific single-stranded DNA (ssDNA) molecular recognition element (MRE). Its emphasis is on increasing stringency in directing binding toward free target of interest and at the same time decreasing binding toward negative targets. A ssDNA MRE with specificity and affinity was identified after fourteen rounds of Decoy-SELEX. Utilizing surface plasmon resonance measurements, the determined equilibrium dissociation constant of the MRE is between 4.2 µM and 4.5 µM, and is highly selective for Exotoxin A over negative targets. A ssDNA MRE modified sandwich enzyme-linked immunosorbent assay (ELISA) has been developed and achieved sensitive detection of Exotoxin A at nanomolar concentrations in human serum. This study has demonstrated the proof-of-principle of using a ssDNA MRE as a clinical diagnostic tool. Ka Lok Hong, Kailey Yancey, Luisa Battistella, Ryan M. Williams, Katherine M. Hickey, Chris D. Bostick, Peter M. Gannett, and Letha J. Sooter Copyright © 2015 Ka Lok Hong et al. All rights reserved. Optimization of Arundo donax Saccharification by (Hemi)cellulolytic Enzymes from Pleurotus ostreatus Thu, 05 Nov 2015 13:14:36 +0000 An enzymatic mixture of cellulases and xylanases was produced by Pleurotus ostreatus using microcrystalline cellulose as inducer, partially characterized and tested in the statistical analysis of Arundo donax bioconversion. The Plackett-Burman screening design was applied to identify the most significant parameters for the enzymatic hydrolysis of pretreated A. donax. As the most significant influence during the enzymatic hydrolysis of A. donax was exercised by the temperature (°C), pH, and time, the combined effect of these factors in the bioconversion by P. ostreatus cellulase and xylanase was analyzed by a 33 factorial experimental design. It is worth noting that the best result of 480.10 mg of sugars/gds, obtained at 45°C, pH 3.5, and 96 hours of incubation, was significant also when compared with the results previously reached by process optimization with commercial enzymes. Rossana Liguori, Elena Ionata, Loredana Marcolongo, Luciana Porto de Souza Vandenberghe, Francesco La Cara, and Vincenza Faraco Copyright © 2015 Rossana Liguori et al. All rights reserved. Facets of Nanotechnology as Seen in Food Processing, Packaging, and Preservation Industry Tue, 03 Nov 2015 12:32:25 +0000 Nanotechnology has proven its competence in almost all possible fields we are aware of. However, today nanotechnology has evolved in true sense by contributing to a very large extent to the food industry. With the growing number of mouths to feed, production of food is not adequate. It has to be preserved in order to reach to the masses on a global scale. Nanotechnology made the idea a reality by increasing the shelf life of different kinds of food materials. It is not an entirely full-proof measure; however it has brought down the extent of wastage of food due to microbial infestation. Not only fresh food but also healthier food is being designed with the help of nano-delivery systems which act as a carrier for the food supplements. There are regulations to follow however as several of them pose serious threats to the wellbeing of the population. In coming days, newer modes of safeguarding food are going to be developed with the help of nanotechnology. In this paper, an overview has been given of the different methods of food processing, packaging, and preservation techniques and the role nanotechnology plays in the food processing, packaging, and preservation industry. Neha Pradhan, Surjit Singh, Nupur Ojha, Anamika Shrivastava, Anil Barla, Vivek Rai, and Sutapa Bose Copyright © 2015 Neha Pradhan et al. All rights reserved. Evaluation of Mediterranean Agricultural Residues as a Potential Feedstock for the Production of Biogas via Anaerobic Fermentation Mon, 02 Nov 2015 13:19:46 +0000 Hydrothermal, dilute acid, and steam explosion pretreatment methods, were evaluated for their efficiency to improve the methane production yield of three Mediterranean agricultural lignocellulosic residues such as olive tree pruning, grapevine pruning, and almond shells. Hydrothermal and dilute acid pretreatments provided low to moderate increase in the digestibility of the biomass samples, whereas steam explosion enabled the highest methane yields to be achieved for almond shells at 232.2 ± 13.0 mL CH4/gVS and olive pruning at 315.4 ± 0.0 mL CH4/gVS. Introduction of an enzymatic prehydrolysis step moderately improved methane yields for hydrothermal and dilute acid pretreated samples but not for the steam exploded ones. Christos Nitsos, Leonidas Matsakas, Kostas Triantafyllidis, Ulrika Rova, and Paul Christakopoulos Copyright © 2015 Christos Nitsos et al. All rights reserved. Dual Monitoring of Secretion and ATP Levels during Chondrogenesis Using Perfusion Culture-Combined Bioluminescence Monitoring System Thu, 29 Oct 2015 13:26:12 +0000 Skeletal pattern formation in limb development depends on prechondrogenic condensation which prefigures the cartilage template. However, although morphogens such as TGF-βs and BMPs have been known to play essential roles in skeletal patterning, how the morphogens induce prechondrogenic cells to aggregate and determine patterns of cartilage elements has remained unclear. Our previous study reported that ATP oscillations are induced during chondrogenesis. This result suggests the possibility that ATP oscillations lead to the oscillatory secretion of morphogens, due to the fact that secretion process requires ATP. To examine the correlation between ATP oscillations and secretion levels of morphogens, we have developed perfusion culture-combined bioluminescence monitoring system to simultaneously monitor intracellular ATP levels and secretion levels. Using this system, we found that secretory activity oscillates in phase with ATP oscillations and that secretion levels of TGF-β1 and BMP2 oscillate during chondrogenesis. The oscillatory secretion of the morphogens would contribute to amplifying the fluctuation of the morphogens, underlie the spatial patterning of morphogens, and consequently lead to skeletal pattern formation. Hyuck Joon Kwon and Youngbae Han Copyright © 2015 Hyuck Joon Kwon and Youngbae Han. All rights reserved. Production of Monoclonal Antibodies in Plants for Cancer Immunotherapy Thu, 15 Oct 2015 09:15:33 +0000 Plants are considered as an alternative platform for recombinant monoclonal antibody (mAb) production due to the improvement and diversification of transgenic techniques. The diversity of plant species offers a multitude of possibilities for the valorization of genetic resources. Moreover, plants can be propagated indefinitely, providing cheap biomass production on a large scale in controlled conditions. Thus, recent studies have shown the successful development of plant systems for the production of mAbs for cancer immunotherapy. However, their several limitations have to be resolved for efficient antibody production in plants. Ghislain Moussavou, Kisung Ko, Jeong-Hwan Lee, and Young-Kug Choo Copyright © 2015 Ghislain Moussavou et al. All rights reserved. Current and New Approaches in GMO Detection: Challenges and Solutions Thu, 15 Oct 2015 07:08:43 +0000 In many countries, genetically modified organisms (GMO) legislations have been established in order to guarantee the traceability of food/feed products on the market and to protect the consumer freedom of choice. Therefore, several GMO detection strategies, mainly based on DNA, have been developed to implement these legislations. Due to its numerous advantages, the quantitative PCR (qPCR) is the method of choice for the enforcement laboratories in GMO routine analysis. However, given the increasing number and diversity of GMO developed and put on the market around the world, some technical hurdles could be encountered with the qPCR technology, mainly owing to its inherent properties. To address these challenges, alternative GMO detection methods have been developed, allowing faster detections of single GM target (e.g., loop-mediated isothermal amplification), simultaneous detections of multiple GM targets (e.g., PCR capillary gel electrophoresis, microarray, and Luminex), more accurate quantification of GM targets (e.g., digital PCR), or characterization of partially known (e.g., DNA walking and Next Generation Sequencing (NGS)) or unknown (e.g., NGS) GMO. The benefits and drawbacks of these methods are discussed in this review. Marie-Alice Fraiture, Philippe Herman, Isabel Taverniers, Marc De Loose, Dieter Deforce, and Nancy H. Roosens Copyright © 2015 Marie-Alice Fraiture et al. All rights reserved. Improved Sugar Production by Optimizing Planetary Mill Pretreatment and Enzyme Hydrolysis Process Sun, 11 Oct 2015 12:54:08 +0000 This paper describes an optimization of planetary mill pretreatment and saccharification processes for improving biosugar production. Pitch pine (Pinus rigida) wood sawdust waste was used as biomass feedstock and the process parameters optimized in this study were the buffering media, the milling time, the enzyme quantity, and the incubation time. Glucose yields were improved when acetate buffer was used rather than citrate buffer. Initially, with each process variable tests, the optimal values were 100 minutes of milling, an enzyme concentration of 16 FPU/g-biomass, and a 12-hour enzymatic hydrolysis. Typically, interactions between these experimental conditions and their effects on glucose production were next investigated using RSM. Glucose yields from the Pinus rigida waste exceeded 80% with several of the conditions tested, demonstrating that milling can be used to obtain high levels of glucose bioconversion from woody biomass for biorefinery purposes. Jeong Heo Kwon, Siseon Lee, Jae-Won Lee, Youn-Woo Hong, Jeong Ho Chang, Daekyung Sung, Sung Hyun Kim, Byoung-In Sang, Robert J. Mitchell, and Jin Hyung Lee Copyright © 2015 Jeong Heo Kwon et al. All rights reserved. Biotechnology in Environmental Monitoring and Pollution Abatement 2015 Wed, 07 Oct 2015 09:21:19 +0000 Kannan Pakshirajan, Eldon R. Rene, and Aiyagari Ramesh Copyright © 2015 Kannan Pakshirajan et al. All rights reserved. Enhancing Ecoefficiency in Shrimp Farming through Interconnected Ponds Wed, 07 Oct 2015 07:29:51 +0000 The future development of shrimp farming needs to improve its ecoefficiency. The purpose of this study was to evaluate water quality, flows, and nitrogen balance and production parameters on a farm with interconnected pond design to improve the efficiency of the semi-intensive culture of Litopenaeus vannamei ponds. The study was conducted in 21 commercial culture ponds during 180 days at densities of 30–35 ind m−2 and daily water exchange <2%. Our study provides evidence that by interconnecting ponds nutrient recycling is favored by promoting the growth of primary producers of the pond as chlorophyll a. Based on the mass balance and flow of nutrients this culture system reduces the flow of solid, particulate organic matter, and nitrogen compounds to the environment and significantly increases the efficiency of water (5 to 6.5 m3 kg−1 cycle−1), when compared with traditional culture systems. With this culture system it is possible to recover up to 34% of the total nitrogen entering the system, with production in excess of 4,000 kg ha−1 shrimp. We believe that the production system with interconnected ponds is a technically feasible model to improve ecoefficiency production of shrimp farming. Ramón Héctor Barraza-Guardado, José Alfredo Arreola-Lizárraga, Anselmo Miranda-Baeza, Manuel Juárez-García, Antonio Juvera-Hoyos, and Ramón Casillas-Hernández Copyright © 2015 Ramón Héctor Barraza-Guardado et al. All rights reserved. Fabrication of an Amperometric Flow-Injection Microfluidic Biosensor Based on Laccase for In Situ Determination of Phenolic Compounds Mon, 05 Oct 2015 09:54:11 +0000 We aim to develop an in situ microfluidic biosensor based on laccase from Trametes pubescens with flow-injection and amperometry as the transducer method. The enzyme was directly immobilized by potential step chronoamperometry, and the immobilization was studied using cyclic voltammetry and electrochemical impedance spectroscopy. The electrode response by amperometry was probed using ABTS and syringaldazine. A shift of interfacial electron transfer resistance and the electron transfer rate constant from 18.1 kΩ to 3.9 MΩ and 4.6 × 10−2 cm s−1 to 2.1 × 10−4 cm s−1, respectively, evidenced that laccase was immobilized on the electrode by the proposed method. We established the optimum operating conditions of temperature (55°C), pH (4.5), injection flow rate (200 µL min−1), and applied potential (0.4 V). Finally, the microfluidic biosensor showed better lower limit of detection (0.149 µM) and sensitivity (0.2341 nA µM−1) for ABTS than previous laccase-based biosensors and the in situ operation capacity. Juan C. Gonzalez-Rivera and Johann F. Osma Copyright © 2015 Juan C. Gonzalez-Rivera and Johann F. Osma. All rights reserved. Comparison of Different Strategies for Selection/Adaptation of Mixed Microbial Cultures Able to Ferment Crude Glycerol Derived from Second-Generation Biodiesel Mon, 05 Oct 2015 09:38:56 +0000 Objective of this study was the selection and adaptation of mixed microbial cultures (MMCs), able to ferment crude glycerol generated from animal fat-based biodiesel and produce building-blocks and green chemicals. Various adaptation strategies have been investigated for the enrichment of suitable and stable MMC, trying to overcome inhibition problems and enhance substrate degradation efficiency, as well as generation of soluble fermentation products. Repeated transfers in small batches and fed-batch conditions have been applied, comparing the use of different inoculum, growth media, and Kinetic Control. The adaptation of activated sludge inoculum was performed successfully and continued unhindered for several months. The best results showed a substrate degradation efficiency of almost 100% (about 10 g/L glycerol in 21 h) and different dominant metabolic products were obtained, depending on the selection strategy (mainly 1,3-propanediol, ethanol, or butyrate). On the other hand, anaerobic sludge exhibited inactivation after a few transfers. To circumvent this problem, fed-batch mode was used as an alternative adaptation strategy, which led to effective substrate degradation and high 1,3-propanediol and butyrate production. Changes in microbial composition were monitored by means of Next Generation Sequencing, revealing a dominance of glycerol consuming species, such as Clostridium, Klebsiella, and Escherichia. C. Varrone, T. M. B. Heggeset, S. B. Le, T. Haugen, S. Markussen, I. V. Skiadas, and H. N. Gavala Copyright © 2015 C. Varrone et al. All rights reserved. Perspective for Aquaponic Systems: “Omic” Technologies for Microbial Community Analysis Mon, 05 Oct 2015 08:15:48 +0000 Aquaponics is the combined production of aquaculture and hydroponics, connected by a water recirculation system. In this productive system, the microbial community is responsible for carrying out the nutrient dynamics between the components. The nutrimental transformations mainly consist in the transformation of chemical species from toxic compounds into available nutrients. In this particular field, the microbial research, the “Omic” technologies will allow a broader scope of studies about a current microbial profile inside aquaponics community, even in those species that currently are unculturable. This approach can also be useful to understand complex interactions of living components in the system. Until now, the analog studies were made to set up the microbial characterization on recirculation aquaculture systems (RAS). However, microbial community composition of aquaponics is still unknown. “Omic” technologies like metagenomic can help to reveal taxonomic diversity. The perspectives are also to begin the first attempts to sketch the functional diversity inside aquaponic systems and its ecological relationships. The knowledge of the emergent properties inside the microbial community, as well as the understanding of the biosynthesis pathways, can derive in future biotechnological applications. Thus, the aim of this review is to show potential applications of current “Omic” tools to characterize the microbial community in aquaponic systems. Perla Munguia-Fragozo, Oscar Alatorre-Jacome, Enrique Rico-Garcia, Irineo Torres-Pacheco, Andres Cruz-Hernandez, Rosalia V. Ocampo-Velazquez, Juan F. Garcia-Trejo, and Ramon G. Guevara-Gonzalez Copyright © 2015 Perla Munguia-Fragozo et al. All rights reserved. Magnet-Facilitated Selection of Electrogenic Bacteria from Marine Sediment Sun, 04 Oct 2015 12:18:46 +0000 Some bacteria can carry out anaerobic respiration by depositing electrons on external materials, such as electrodes, thereby creating an electrical current. Into the anode chamber of microbial fuel cells (MFCs) having abiotic air-cathodes we inoculated microorganisms cultured from a magnetic particle-enriched portion of a marine tidal sediment, reasoning that since some external electron acceptors are ferromagnetic, electrogenic bacteria should be found in their vicinity. Two MFCs, one inoculated with a mixed bacterial culture and the other with an axenic culture of a helical bacterium isolated from the magnetic particle enrichment, termed strain HJ, were operated for 65 d. Both MFCs produced power, with production from the mixed culture MFC exceeding that of strain HJ. Strain HJ was identified as a Thalassospira sp. by transmission electron microscopic analysis and 16S rRNA gene comparisons. An MFC inoculated with strain HJ and operated in open circuit produced 47% and 57% of the maximal power produced from MFCs inoculated with the known electrogen Geobacter daltonii and the magnetotactic bacterium Desulfamplus magnetomortis, respectively. Further investigation will be needed to determine whether bacterial populations associated with magnetic particles within marine sediments are enriched for electrogens. Larisa Kiseleva, Justina Briliute, Irina V. Khilyas, David J. W. Simpson, Viacheslav Fedorovich, M. Cohen, and Igor Goryanin Copyright © 2015 Larisa Kiseleva et al. All rights reserved. Fungal Antagonism Assessment of Predatory Species and Producers Metabolites and Their Effectiveness on Haemonchus contortus Infective Larvae Sun, 04 Oct 2015 10:36:57 +0000 The objective of this study was to assess antagonism of nematophagous fungi and species producers metabolites and their effectiveness on Haemonchus contortus infective larvae (L3). Assay A assesses the synergistic, additive, or antagonistic effect on the production of spores of fungal isolates of the species Duddingtonia flagrans, Clonostachys rosea, Trichoderma esau, and Arthrobotrys musiformis; Assay B evaluates in vitro the effect of intercropping of these isolates grown in 2% water-agar (2% WA) on L3 of H. contortus. D. flagrans (Assay A) produced 5.3 × 106 spores and associated with T. esau, A. musiformis, or C. rosea reduced its production by 60.37, 45.28, and 49.05%, respectively. T. esau produced 7.9 × 107 conidia and associated with D. flagrans, A. musiformis, or C. rosea reduced its production by 39.24, 82.27, and 96.96%, respectively. A. musiformis produced 7.3 × 109 spores and associated with D. flagrans, T. esau, or C. rosea reduced its production by 99.98, 99.99, and 99.98%, respectively. C. rosea produced 7.3 × 108 conidia and associated with D. flagrans, T. esau, or A. musiformis reduced its production by 95.20, 96.84, and 93.56%, respectively. These results show evidence of antagonism in the production of spores between predators fungi. Manoel Eduardo Silva, Fabio Ribeiro Braga, Pedro Mendoza de Gives, Jair Millán-Orozco, Miguel Angel Mercado Uriostegui, Liliana Aguilar Marcelino, Filippe Elias de Freitas Soares, Andréia Luiza Araújo, Thainá Souza Vargas, Anderson Rocha Aguiar, Thiago Senna, Maria Gorete Rodrigues, Frederico Vieira Froes, and Jackson Victor de Araújo Copyright © 2015 Manoel Eduardo Silva et al. All rights reserved. Efficacy of Clonostachys rosea and Duddingtonia flagrans in Reducing the Haemonchus contortus Infective Larvae Sun, 04 Oct 2015 10:18:53 +0000 The biocontrol is proven effective in reducing in vitro and in situ free-living stages of major gastrointestinal helminths, allowing progress in reducing losses by parasitism, maximizing production, and productivity. This study aimed at evaluating the predatory activity of fungal isolates of Duddingtonia flagrans and Clonostachys rosea species and its association on infective larvae (L3) of H. contortus in microplots formed by grasses and maintained in a protected environment. All groups were added with 10 mL of an aqueous suspension with 618 H. contortus L3 approximately. Group 1 was used as control and only received the infective larvae. Groups 2 and 3 received D. flagrans chlamydospores and C. rosea conidia at doses of 5 × 106. Group 4 received the combination of 5 × 106 D. flagrans chlamydospores + 5 × 106 C. rosea conidia. D. flagrans and C. rosea showed nematicidal effectiveness reducing by 91.5 and 88.9%, respectively, the population of H. contortus L3. However, when used in combination efficiency decreased to 74.5% predation of H. contortus L3. These results demonstrate the need for further studies to determine the existence of additive effects, synergistic or antagonistic, between these species. Manoel Eduardo da Silva, Fabio Ribeiro Braga, Pedro Mendoza de Gives, Miguel Angel Mercado Uriostegui, Manuela Reyes, Filippe Elias de Freitas Soares, Lorendane Millena de Carvalho, Francielle Bosi Rodrigues, and Jackson Victor de Araújo Copyright © 2015 Manoel Eduardo da Silva et al. All rights reserved. Biosorption of Pb(II) Ions by Klebsiella sp. 3S1 Isolated from a Wastewater Treatment Plant: Kinetics and Mechanisms Studies Sun, 04 Oct 2015 08:57:51 +0000 Lead biosorption by Klebsiella sp. 3S1 isolated from a wastewater treatment plant was investigated through a Rotatable Central Composite Experimental Design. The optimisation study indicated the following optimal values of operating variables: 0.4 g/L of biosorbent dosage, pH 5, and 34°C. According to the results of the kinetic studies, the biosorption process can be described by a two-step process, one rapid, almost instantaneous, and one slower, both contributing significantly to the overall biosorption; the model that best fits the experimental results was pseudo-second order. The equilibrium studies showed a maximum lead uptake value of 140.19 mg/g according to the Langmuir model. The mechanism study revealed that lead ions were bioaccumulated into the cytoplasm and adsorbed on the cell surface. The bacterium  Klebsiella sp. 3S1 has a good potential in the bioremoval of lead in an inexpensive and effective process. Antonio Jesús Muñoz, Francisco Espínola, Manuel Moya, and Encarnación Ruiz Copyright © 2015 Antonio Jesús Muñoz et al. All rights reserved. Structure and Antioxidant Activity of Soy Protein Isolate-Dextran Conjugates Obtained by TiO2 Photocatalysis Thu, 01 Oct 2015 11:12:06 +0000 The aim of this study was to investigate the structural characteristics and antioxidant activities of soy protein isolate- (SPI-) dextran conjugates obtained by TiO2 photocatalysis treatment. Results revealed that the UV-vis absorption and the fluorescence intensity increased as the photocatalytic power increased (). Higher photocatalytic power could promote the extent of glycation and the formation of high molecular weight SPI-dextran conjugates, which were evidenced by free amino group content and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The Fourier transform infrared (FT-IR) spectra suggested that the amide I, II, and III bands of SPI were altered by the glycation induced by TiO2 photocatalysis. Moreover, significant changes of secondary structure occurred in SPI-dextran conjugates. The α-helix, β-sheet, β-turns, and random coil were changed from approximately 10.6%, 37.9%, 12.9%, and 38.6% to 3.8%, 10.4%, 17.7%, and 68.8%, respectively, after treatment at photocatalytic power of 1000 W. In addition, SPI-dextran conjugates obtained by TiO2 photocatalysis treatment exhibited high hydroxyl radical scavenging activity and possessed increased reducing power. All data indicated that TiO2 photocatalysis was an efficient method for promoting protein-polysaccharide copolymerisation. Bei Jin, Xiaosong Zhou, Bing Li, Caiyan Chen, Xiaosa Zhang, and Siqiao Chen Copyright © 2015 Bei Jin et al. All rights reserved. The Regulation by Phenolic Compounds of Soil Organic Matter Dynamics under a Changing Environment Thu, 01 Oct 2015 08:26:04 +0000 Phenolics are the most abundant plant metabolites and are believed to decompose slowly in soils compared to other soil organic matter (SOM). Thus, they have often been considered as a slow carbon (C) pool in soil dynamics models. Here, however, we review changes in our concept about the turnover rate of phenolics and quantification of different types of phenolics in soils. Also, we synthesize current research on the degradation of phenolics and their regulatory effects on decomposition. Environmental changes, such as elevated CO2, warming, nitrogen (N) deposition, and drought, could influence the production and form of phenolics, leading to a change in SOM dynamics, and thus we also review the fate of phenolics under environmental disturbances. Finally, we propose the use of phenolics as a tool to control rates of SOM decomposition to stabilize organic carbon in ecosystems. Further studies to clarify the role of phenolics in SOM dynamics should include improving quantification methods, elucidating the relationship between phenolics and soil microorganisms, and determining the interactive effects of combinations of environmental changes on the phenolics production and degradation and subsequent impact on SOM processing. Kyungjin Min, Chris Freeman, Hojeong Kang, and Sung-Uk Choi Copyright © 2015 Kyungjin Min et al. All rights reserved. A Novel Reference Plasmid for the Qualitative Detection of Genetically Modified Rice in Food and Feed Thu, 01 Oct 2015 07:45:58 +0000 Rice is one of the most important food crops in the world. Genetically modified (GM) technology has been used in rice to confer herbicide tolerance and pathogen or insect resistance. China invests heavily in research on GM rice. By the end of 2014, at least 250 transgenic rice lines had been developed in China. To monitor the presence of GM rice in food and feed, we collected information on foreign elements from 250 transgenic rice lines and found 5 elements, including the Agrobacterium tumefaciens nopaline synthase terminator (T-NOS), the cauliflower mosaic virus 35S promoter (CaMV35S), the ubiquitin gene (Ubi), the bar gene, and the hygromycin phosphotransferase gene (Hpt), that are commonly present in GM rice. Therefore, we constructed a novel plasmid (pBJGMM001) that contains fragments of these elements and two endogenous reference genes (the sucrose phosphate synthase gene, SPS, and the phosphoenolpyruvate carboxylase gene, PEPC). pBJGMM001 can serve as a standard for detecting 96% of GM rice lines in China. The primers, amplicons, reaction mixture, and PCR program were developed based on Chinese National Standards. The protocol was validated and determined to be suitable for practical use in monitoring and identifying GM rice. Liang Li, Mei Dong, Na An, Lixia Liang, Yusong Wan, and Wujun Jin Copyright © 2015 Liang Li et al. All rights reserved. Effect of Chitosan Coating with Cinnamon Oil on the Quality and Physiological Attributes of China Jujube Fruits Thu, 01 Oct 2015 07:41:48 +0000 Effects of chitosan coating with cinnamon oil on the physiological attributes and preservation quality of China jujube fruits during storage at 4°C for 60 days were investigated. Results indicated that weight loss and decay of jujube fruits were significantly reduced by chitosan-oil coating during the period of 60-day storage, which also exhibited a quite beneficial effect on maintaining the sensory quality for jujube fruits. Meanwhile, the contents of vitamin C and titratable acid decreased to 3.08 mg·g−1 and 0.342% for the fruits treated by chitosan-oil coating (1.0% + 0.10%), respectively. Polyphenol oxidase, superoxide dismutase, and peroxidase activities were 13.40 U·g−1, 14.53 U·g−1, and 63.6 U·g−1 at the end of storage, respectively. The contents of total soluble phenolics and MDA were 34.51 mg·g−1 and 19.43 μmol·g−1 for the combined coating treated samples and control fruits, respectively. These results suggested that the chitosan-oil coating might be recognized as one efficiency technology on the preservation quality of jujube fruits during the storage time. Yage Xing, Hongbin Lin, Dong Cao, Qinglian Xu, Wenfeng Han, Ranran Wang, Zhenming Che, and Xihong Li Copyright © 2015 Yage Xing et al. All rights reserved. Integrated Evaluation of Urban Water Bodies for Pollution Abatement Based on Fuzzy Multicriteria Decision Approach Thu, 01 Oct 2015 07:21:58 +0000 Today’s ecology is erected with miscellaneous framework. However, numerous sources deteriorate it, such as urban rivers that directly cause the environmental pollution. For chemical pollution abatement from urban water bodies, many techniques were introduced to rehabilitate the water quality of these water bodies. In this research, Bacterial Technology (BT) was applied to urban rivers escalating the necessity to control the water pollution in different places (Xuxi River (XXU); Gankeng River (GKS); Xia Zhang River (XZY); Fenghu and Song Yang Rivers (FSR); Jiu Haogang River (JHH)) in China. For data analysis, the physiochemical parameters such as temperature, chemical oxygen demand (COD), dissolved oxygen (DO), total phosphorus (TP), and ammonia nitrogen (NH3N) were determined before and after the treatment. Multicriteria Decision Making (MCDM) method was used for relative significance of different water quality on each station, based on fuzzy analytical hierarchy process (FAHP). The overall results revealed that the pollution is exceeding at “JHH” due to the limit of “COD” as critical water quality parameter and after treatment, an abrupt recovery of the rivers compared with the average improved efficiency of nutrients was 79%, 74%, 68%, and 70% of COD, DO, TP, and NH3N, respectively. The color of the river’s water changed to its original form and aquatic living organism appeared with clear effluents from them. Sarfraz Hashim, Xie Yuebo, Muhammad Saifullah, Ramila Nabi Jan, and Adila Muhetaer Copyright © 2015 Sarfraz Hashim et al. All rights reserved. Vulnerability Assessment and Application of Bacterial Technology on Urban Rivers for Pollution Eradication Thu, 01 Oct 2015 06:57:41 +0000 To protect against the environmental pollution, the present research was undertaken to enumerate the Bacterial Technologies (BTs) on the restoration of polluted urban rivers, that is, Fenghu-Song Yang River (FSR) and Xuxi River (XXR). Experimental research accounted for the physiochemical parameters (pH; temperature; dissolved oxygen (DO); chemical oxygen demand (COD); total phosphorus (TP); total nitrogen (TN); and ammonia nitrogen (NH3N)) before and after the BT operation. The results declared that the BT is efficient to restore the polluted rivers up to reliable condition. These results were analyzed by using multivariate statistical techniques (principal component analysis (PCA) and cluster analysis (CA)). These techniques interpreted the complex data sets and expressed the point source information about the water quality of these rivers at SA5, SA6, and SB3 under highly polluted regions. For better understanding, water quality index (WQI) was applied to compute the single numeric value. WQI results are evidence of the above results which prove the water quality of both rivers faced under outrageous condition (below 50 WQI scores) before the BT treatment, but, after the treatment, the rivers were restored from fair to good level (above 50 WQI scores) and overall output of these scores was quite similar to detect the point source of pollution. These results described an abrupt recovery of the urban rivers up to reliable condition for aquatic organism and clear effluents from the rivers. Sarfraz Hashim, Xie Yuebo, Fiaz Ahmad, Chaudhry Arslan, and Muhammad Saifullah Copyright © 2015 Sarfraz Hashim et al. All rights reserved. Evaluation of Natural Materials as Exogenous Carbon Sources for Biological Treatment of Low Carbon-to-Nitrogen Wastewater Thu, 01 Oct 2015 06:54:10 +0000 In the bacterial processes involved in the mitigation of nitrogen pollution, an adequately high carbon-to-nitrogen (C : N) ratio is key to sustain denitrification. We evaluated three natural materials (woodchips, barley grains, and peanut shells) as carbon sources for low C : N wastewater. The amount of organic matter released from these materials to aqueous media was evaluated, as well as their pollution swapping potential by measuring the release of total Kjeldahl nitrogen, N-, , and , and total phosphorous. Barley grains yielded the highest amount of organic matter, which also showed to be the most easily biodegradable. Woodchips and peanut shells released carbon rather steadily and so they would not require frequent replenishment from biological reactors. These materials produced eluates with lower concentrations of nutrients than the leachates from barley grains. However, as woodchips yielded lower amounts of suspended solids, they constitute an adequate exogenous source for the biological treatment of carbon-deficient effluents. Juan Ramírez-Godínez, Icela Beltrán-Hernández, Alejandro Álvarez-Hernández, Claudia Coronel-Olivares, Elizabeth Contreras-López, Maribel Quezada-Cruz, and Gabriela Vázquez-Rodríguez Copyright © 2015 Juan Ramírez-Godínez et al. All rights reserved. Decolorization of Distillery Spent Wash Using Biopolymer Synthesized by Pseudomonas aeruginosa Isolated from Tannery Effluent Thu, 01 Oct 2015 06:31:44 +0000 A bacterial strain was isolated from tannery effluent which can tolerate high concentrations of potassium dichromate up to 1000 ppm. The isolated microorganism was identified as Pseudomonas aeruginosa by performing biochemical tests and molecular characterization. In the presence of excess of carbohydrate source, which is a physiological stress, this strain produces Polyhydroxybutyrate (PHB). This intracellular polymer, which is synthesized, is primarily a product of carbon assimilation and is employed by microorganisms as an energy storage molecule to be metabolized when other common energy sources are limitedly available. Efforts were taken to check whether the PHB has any positive effect on spent wash decolorization. When a combination of PHB and the isolated bacterial culture was added to spent wash, a maximum color removal of 92.77% was found which was comparatively higher than the color removed when the spent wash was treated individually with the PHB and Pseudomonas aeruginosa. PHB behaved as a support material for the bacteria to bind to it and thus develops biofilm, which is one of the natural physiological growth forms of microorganisms. The bacterial growth in the biofilm and the polymer together acted in synergy, adsorbing and coagulating the pollutants in the form of color pigments. Charles David, M. Arivazhagan, M. N. Balamurali, and Dhivya Shanmugarajan Copyright © 2015 Charles David et al. All rights reserved. Antioxidant Potential in Different Parts and Callus of Gynura procumbens and Different Parts of Gynura bicolor Wed, 30 Sep 2015 14:31:43 +0000 Plants from Gynura family was used in this study, namely, Gynura procumbens and Gynura bicolor. Gynura procumbens is well known for its various medicinal properties such as antihyperglycaemic, antihyperlipidaemic, and antiulcerogenic; meanwhile, G. bicolor remains unexploited. Several nonenzymatic antioxidants methods were utilized to study the antioxidant capacity, which include ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, total flavonoid content, total phenolic content, and ascorbic acid content determination. DPPH assay reveals G. procumbens shoot as the lowest (66.885%) and G. procumbens root as the highest (93.499%) DPPH radical inhibitor. In FRAP assay, reducing power was not detected in G. procumbens leaf callus (0.000 TEAC mg/g FW) whereby G. procumbens root exhibits the highest (1.103 TEAC mg/g FW) ferric reducing power. Total phenolic content and total flavonoid content exhibited similar trend for both the intact plants analysed. In all antioxidant assays, G. procumbens callus culture exhibits very low antioxidant activity. However, G. procumbens root exhibited highest phenolic content, flavonoid content, and ascorbic acid content with 4.957 TEAC mg/g FW, 543.529 QE µg/g FW, and 54.723 µg/g FW, respectively. This study reveals that G. procumbens root extract is a good source of natural antioxidant. Vijendren Krishnan, Syahida Ahmad, and Maziah Mahmood Copyright © 2015 Vijendren Krishnan et al. All rights reserved. Characteristics of Biological Nitrogen Removal in a Multiple Anoxic and Aerobic Biological Nutrient Removal Process Wed, 30 Sep 2015 14:09:04 +0000 Two sequencing batch reactors, one with the conventional anoxic and aerobic (AO) process and the other with the multiple AO process, were operated to examine characteristics of biological nitrogen removal, especially of the multiple AO process. The long-term operation showed that the total nitrogen removal percentage of the multiple AO reactor was 38.7% higher than that of the AO reactor. In the multiple AO reactor, at the initial SBR cycle stage, due to the occurrence of simultaneous nitrification and denitrification, no nitrite and/or nitrate were accumulated. In the multiple AO reactor, activities of nitrite oxidizing bacteria were inhibited due to the multiple AO operating mode applied, resulting in the partial nitrification. Denitrifiers in the multiple AO reactor mainly utilized internal organic carbon for denitrification, and their activities were lower than those of denitrifiers in the AO reactor utilizing external organic carbon. Huoqing Wang, Yuntao Guan, Li Li, and Guangxue Wu Copyright © 2015 Huoqing Wang et al. All rights reserved. Antiviral Potential of Algae Polysaccharides Isolated from Marine Sources: A Review Mon, 21 Sep 2015 11:45:49 +0000 From food to fertilizer, algal derived products are largely employed in assorted industries, including agricultural, biomedical, food, and pharmaceutical industries. Among different chemical compositions isolated from algae, polysaccharides are the most well-established compounds, which were subjected to a variety of studies due to extensive bioactivities. Over the past few decades, the promising results for antiviral potential of algae-derived polysaccharides have advocated them as inordinate candidates for pharmaceutical research. Numerous studies have isolated various algal polysaccharides possessing antiviral activities, including carrageenan, alginate, fucan, laminaran, and naviculan. In addition, different mechanisms of action have been reported for these polysaccharides, such as inhibiting the binding or internalization of virus into the host cells or suppressing DNA replication and protein synthesis. This review strives for compiling previous antiviral studies of algae-derived polysaccharides and their mechanism of action towards their development as natural antiviral agents for future investigations. Azin Ahmadi, Soheil Zorofchian Moghadamtousi, Sazaly Abubakar, and Keivan Zandi Copyright © 2015 Azin Ahmadi et al. All rights reserved. Two-Dimensional Crystallization Procedure, from Protein Expression to Sample Preparation Thu, 27 Aug 2015 14:07:58 +0000 Membrane proteins play important roles for living cells. Structural studies of membrane proteins provide deeper understanding of their mechanisms and further aid in drug design. As compared to other methods, electron microscopy is uniquely suitable for analysis of a broad range of specimens, from small proteins to large complexes. Of various electron microscopic methods, electron crystallography is particularly well-suited to study membrane proteins which are reconstituted into two-dimensional crystals in lipid environments. In this review, we discuss the steps and parameters for obtaining large and well-ordered two-dimensional crystals. A general description of the principle in each step is provided since this information can also be applied to other biochemical and biophysical methods. The examples are taken from our own studies and published results with related proteins. Our purpose is to give readers a more general idea of electron crystallography and to share our experiences in obtaining suitable crystals for data collection. Qie Kuang, Pasi Purhonen, and Hans Hebert Copyright © 2015 Qie Kuang et al. All rights reserved. Biological Activities of Tetrodotoxin-Producing Enterococcus faecium AD1 Isolated from Puffer Fishes Mon, 24 Aug 2015 12:24:09 +0000 Puffer fishes were collected from the central sea in Vietnam from spring to summer season. The eggs were incubated in MRS broth that was used to test the toxicity in mice and isolate the lactic acid bacteria community that could produce tetrodotoxin (TTX). Thin layer chromatography (TLC) and high performance lipid chromatography (HPLC) were used to detect and quantify TTX. As a result, Enterococcus faecium AD1 which was identified by biochemical test and 16S rRNA analysis could produce TTX 0.3 mg/mL when cultured in MRS broth. The bacterium was optimized for TTX production and gave 0.18 mg/mL, 0.07 mg/mL, and 0.15 mg/mL in media prepared from the meat-washing water of freshwater fishes (Pangasius bocourti, Oreochromis sp.) and sea fish (Auxis thazard), respectively, that are also hopeful to answer some poisoning cases related to eating fishes. Enterococcus faecium also showed the wide antimicrobial activities on yeast, Gram-negative and -positive bacteria. Extracted exopolysaccharide (EPS) that reacted with 2,2-diphenyl-1-picrylhydrazyl to give IC50 at 5 mg/mL equaled 11 mg/mL ascorbic acid which could show effects on Hela-6 and Hep G2 using sulforhodamine B test. Enterococcus faecium can be claimed as a promising source in tetrodotoxin and biological compounds. Tu Hoang Khue Nguyen, Huu Ngoc Nguyen, Dat Van Nghe, and Kim Hoang Nguyen Copyright © 2015 Tu Hoang Khue Nguyen et al. All rights reserved. Optimization of Fermentation Medium for Extracellular Lipase Production from Aspergillus niger Using Response Surface Methodology Thu, 20 Aug 2015 16:16:13 +0000 Lipase produced by Aspergillus niger is widely used in various industries. In this study, extracellular lipase production from an industrial producing strain of A. niger was improved by medium optimization. The secondary carbon source, nitrogen source, and lipid were found to be the three most influential factors for lipase production by single-factor experiments. According to the statistical approach, the optimum values of three most influential parameters were determined: 10.5 g/L corn starch, 35.4 g/L soybean meal, and 10.9 g/L soybean oil. Using this optimum medium, the best lipase activity was obtained at 2,171 U/mL, which was 16.4% higher than using the initial medium. All these results confirmed the validity of the model. Furthermore, results of the Box-Behnken Design and quadratic models analysis indicated that the carbon to nitrogen (C/N) ratio significantly influenced the enzyme production, which also suggested that more attention should be paid to the C/N ratio for the optimization of enzyme production. Jia Jia, Xiaofeng Yang, Zhiliang Wu, Qian Zhang, Zhi Lin, Hongtao Guo, Carol Sze Ki Lin, Jianying Wang, and Yunshan Wang Copyright © 2015 Jia Jia et al. All rights reserved. Hydrothermal Pretreatment of Date Palm (Phoenix dactylifera L.) Leaflets and Rachis to Enhance Enzymatic Digestibility and Bioethanol Potential Wed, 12 Aug 2015 14:27:47 +0000 Date palm residues are one of the most promising lignocellulosic biomass for bioethanol production in the Middle East. In this study, leaflets and rachis were subjected to hydrothermal pretreatment to overcome the recalcitrance of the biomass for enzymatic conversion. Evident morphological, structural, and chemical changes were observed by scanning electron microscopy, X-ray diffraction, and infrared spectroscopy after pretreatment. High glucan (>90% for both leaflets and rachis) and xylan (>75% for leaflets and >79% for rachis) recovery were achieved. Under the optimal condition of hydrothermal pretreatment (210°C/10 min) highly digestible (glucan convertibility, 100% to leaflets, 78% to rachis) and fermentable (ethanol yield, 96% to leaflets, 80% to rachis) solid fractions were obtained. Fermentability test of the liquid fractions proved that no considerable inhibitors to Saccharomyces cerevisiae were produced in hydrothermal pretreatment. Given the high sugar recovery, enzymatic digestibility, and ethanol yield, production of bioethanol by hydrothermal pretreatment could be a promising way of valorization of date palm residues in this region. Chuanji Fang, Jens Ejbye Schmidt, Iwona Cybulska, Grzegorz P. Brudecki, Christian Grundahl Frankær, and Mette Hedegaard Thomsen Copyright © 2015 Chuanji Fang et al. All rights reserved. Specialized Bioactive Microbial Metabolites: From Gene to Product Wed, 05 Aug 2015 12:07:51 +0000 Flavia Marinelli, Olga Genilloud, Victor Fedorenko, and Eliora Z. Ron Copyright © 2015 Flavia Marinelli et al. All rights reserved. A Novel Microbisporicin Producer Identified by Early Dereplication during Lantibiotic Screening Tue, 04 Aug 2015 11:50:53 +0000 With the increasing need of effective antibiotics against multi-drug resistant pathogens, lantibiotics are an attractive option of a new class of molecules. They are ribosomally synthetized and posttranslationally modified peptides possessing potent antimicrobial activity against aerobic and anaerobic Gram-positive pathogens, including those increasingly resistant to β-lactams and glycopeptides. Some of them (actagardine, mersacidin, planosporicin, and microbisporicin) inhibit cell wall biosynthesis in pathogens and their effect is not antagonized by vancomycin. Hereby, we apply an efficient strategy for lantibiotic screening to 240 members of a newly described genus of filamentous actinomycetes, named Actinoallomurus, that is considered a yet-poorly-exploited promising source for novel bioactive metabolites. By combining antimicrobial differential assay against Staphylococcus aureus and its L-form (also in the presence of a β-lactamase cocktail or Ac-Lys-D-alanyl-D-alanine tripeptide), with LC-UV-MS dereplication coupled with bioautography, a novel producer of the potent microbisporicin complex was rapidly identified. Under the commercial name of NAI-107, it is currently in late preclinical phase for the treatment of multi-drug resistant Gram-positive pathogens. To our knowledge, this is the first report on a lantibiotic produced by an Actinoallomurus sp. and on a microbisporicin producer not belonging to the Microbispora genus. Lucia Carrano, Monica Abbondi, Paola Turconi, Gianpaolo Candiani, and Flavia Marinelli Copyright © 2015 Lucia Carrano et al. All rights reserved. Toxicity of Environmental Contaminants Tue, 04 Aug 2015 11:40:21 +0000 Sunil Kumar, Patrick Hettiaratchi, Nanjappa Ashwath, and Petros Gikas Copyright © 2015 Sunil Kumar et al. All rights reserved. Improved Production of Sublancin 168 Biosynthesized by Bacillus subtilis 168 Using Chemometric Methodology and Statistical Experimental Designs Mon, 03 Aug 2015 12:05:52 +0000 Sublancin 168, as a distinct S-linked antimicrobial glycopeptide produced by Bacillus subtilis 168, is effective in killing specific microorganisms. However, the reported yield of sublancin 168 is at a low level of no more than 60 mg from 1 L fermentation culture of B. subtilis 168 by using the method in the literature. Thus optimization of fermentation condition for efficiently producing sublancin 168 is required. Here, Box-Behnken design was used to determine the optimal combination of three fermentation parameters, namely, corn powder, soybean meal, and temperature that were identified previously by Plackett-Burman design and the steepest ascent experiment. Subsequently, based on the response surface methodology, the quadratic regression model for optimally producing sublancin 168 was developed, and the optimal combination of culture parameters for maximum sublancin 168 production of 129.72 mg/L was determined as corn powder 28.49 g/L, soybean meal 22.99 g/L, and incubation temperature 30.8°C. The results showed that sublancin 168 production obtained experimentally was coincident with predicted value of 125.88 mg/L, and the developed model was proved to be adequate, and the aim of efficiently producing sublancin 168 was achieved. Shengyue Ji, Weili Li, Haiyun Xin, Shan Wang, and Binyun Cao Copyright © 2015 Shengyue Ji et al. All rights reserved. Bioconcentration and Acute Intoxication of Brazilian Freshwater Fishes by the Methyl Parathion Organophosphate Pesticide Mon, 03 Aug 2015 11:49:36 +0000 Three species of freshwater Brazilian fishes (pacu, Piaractus mesopotamicus; piavussu, Leporinus macrocephalus, and curimbatá, Prochilodus lineatus) were exposed to an acute dose of 5 ppm methyl parathion organophosphate pesticide. Three to five individuals per species were exposed, one at a time, to 40 liters tap water spiked with Folidol 600. Pesticide concentrations and cholinesterase (ChE) activities were evaluated in serum, liver, brain, heart, and muscle. The bioconcentration of methyl parathion was similar for all studied fishes. Brain tissue showed the highest pesticide concentration, reaching 80 ppm after exposure for 30 min to methyl parathion. Three to 5 hours of 5 ppm methyl parathion exposure provoked the death of all P. lineatus at 92% brain AChE inhibition, whereas fish from the other two species survived for up to 78 hours with less than 80% brain AChE inhibition. Our results indicate that acute toxic effects of methyl parathion to fish are correlated with brain AChE sensitivity to methyl paraoxon. João Bosco de Salles, Renato Matos Lopes, Cristiane M. C. de Salles, Vicente P. F. Cassano, Manildo Marcião de Oliveira, Vera L. F. Cunha Bastos, and Jayme Cunha Bastos Copyright © 2015 João Bosco de Salles et al. All rights reserved. Considerable Variation of Antibacterial Activity of Cu Nanoparticles Suspensions Depending on the Storage Time, Dispersive Medium, and Particle Sizes Mon, 03 Aug 2015 11:46:27 +0000 Suspensions of Cu nanoparticles are promising for creating the new class of alternative antimicrobial products. In this study we examined copper nanoparticles of various sizes obtained by the method of wire electric explosion: nanopowder average size 50 nm (Cu 50) and 100 nm (Cu 100). The paper presents the complex study of the influence of physicochemical properties such as particle size and concentration of the freshly prepared and 24-hour suspensions of Cu nanoparticles in distilled water and physiological solution upon their toxicity to bacteria E. coli M-17. Ionic solution of Cu2+ and sodium dichloroisocyanurate was used for comparison study. It has been shown that decrease in the nanoparticle size leads to changes in the correlation between toxicity and concentration as toxicity peaks are observed at low concentrations (0.0001⋯0.01 mg/L). It has been observed that antibacterial properties of Cu 50 nanoparticle suspensions are ceased after 24-hour storage, while for Cu 100 suspensions no correlation between antibacterial properties and storage time has been noted. Cu 100 nanoparticle suspensions at 10 mg/L concentration display higher toxicity at substituting physiological solution for water than Cu 50 suspensions. Dependence of the toxicity on the mean particle aggregates size in suspension was not revealed. Olga V. Zakharova, Anna Yu. Godymchuk, Alexander A. Gusev, Svyatoslav I. Gulchenko, Inna A. Vasyukova, and Denis V. Kuznetsov Copyright © 2015 Olga V. Zakharova et al. All rights reserved. Antibacterial Discovery and Development: From Gene to Product and Back Mon, 03 Aug 2015 11:35:01 +0000 Concern over the reports of antibiotic-resistant bacterial infections in hospitals and in the community has been publicized in the media, accompanied by comments on the risk that we may soon run out of antibiotics as a way to control infectious disease. Infections caused by Enterococcus faecium, Staphylococcus aureus, Klebsiella species, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and other Enterobacteriaceae species represent a major public health burden. Despite the pharmaceutical sector’s lack of interest in the topic in the last decade, microbial natural products continue to represent one of the most interesting sources for discovering and developing novel antibacterials. Research in microbial natural product screening and development is currently benefiting from progress that has been made in other related fields (microbial ecology, analytical chemistry, genomics, molecular biology, and synthetic biology). In this paper, we review how novel and classical approaches can be integrated in the current processes for microbial product screening, fermentation, and strain improvement. Victor Fedorenko, Olga Genilloud, Liliya Horbal, Giorgia Letizia Marcone, Flavia Marinelli, Yossi Paitan, and Eliora Z. Ron Copyright © 2015 Victor Fedorenko et al. All rights reserved. Biologically Active Metabolites Synthesized by Microalgae Mon, 03 Aug 2015 11:25:43 +0000 Microalgae are microorganisms that have different morphological, physiological, and genetic traits that confer the ability to produce different biologically active metabolites. Microalgal biotechnology has become a subject of study for various fields, due to the varied bioproducts that can be obtained from these microorganisms. When microalgal cultivation processes are better understood, microalgae can become an environmentally friendly and economically viable source of compounds of interest, because production can be optimized in a controlled culture. The bioactive compounds derived from microalgae have anti-inflammatory, antimicrobial, and antioxidant activities, among others. Furthermore, these microorganisms have the ability to promote health and reduce the risk of the development of degenerative diseases. In this context, the aim of this review is to discuss bioactive metabolites produced by microalgae for possible applications in the life sciences. Michele Greque de Morais, Bruna da Silva Vaz, Etiele Greque de Morais, and Jorge Alberto Vieira Costa Copyright © 2015 Michele Greque de Morais et al. All rights reserved. Violacein: Properties and Production of a Versatile Bacterial Pigment Mon, 03 Aug 2015 11:20:01 +0000 Violacein-producing bacteria, with their striking purple hues, have undoubtedly piqued the curiosity of scientists since their first discovery. The bisindole violacein is formed by the condensation of two tryptophan molecules through the action of five proteins. The genes required for its production, vioABCDE, and the regulatory mechanisms employed have been studied within a small number of violacein-producing strains. As a compound, violacein is known to have diverse biological activities, including being an anticancer agent and being an antibiotic against Staphylococcus aureus and other Gram-positive pathogens. Identifying the biological roles of this pigmented molecule is of particular interest, and understanding violacein’s function and mechanism of action has relevance to those unmasking any of its commercial or therapeutic benefits. Unfortunately, the production of violacein and its related derivatives is not easy and so various groups are also seeking to improve the fermentative yields of violacein through genetic engineering and synthetic biology. This review discusses the recent trends in the research and production of violacein by both natural and genetically modified bacterial strains. Seong Yeol Choi, Kyoung-hye Yoon, Jin Il Lee, and Robert J. Mitchell Copyright © 2015 Seong Yeol Choi et al. All rights reserved. Comparison of Simultaneous Nitrification and Denitrification for Three Different Reactors Mon, 03 Aug 2015 11:18:36 +0000 Discharge of high NH4-N containing wastewater into water bodies has become a critical and serious issue due to its negative impact on water and environmental quality. In this research, the performance of three different reactors was assessed and compared with regard to the removal of NH4-N from wastewater. The highest nitrogen removal efficiency of 98.3% was found when the entrapped sludge reactor (ESR), in which the sludge was entrapped in polyethylene glycol polymer, was used. Under intermittent aeration, nitrification and denitrification occurred simultaneously in the aerobic and anaerobic periods. Moreover, internal carbon was consumed efficiently for denitrification. On the other hand, internal carbon consumption was not found to occur in the suspended sludge reactor (SSR) and the mixed sludge reactor (MSR) and this resulted in nitrogen removal efficiencies of SSR and MSR being 64.7 and 45.1%, respectively. Nitrification and denitrification were the main nitrogen removal processes in the aerobic and anaerobic periods, respectively. However, due to the absence of sufficient organic carbon, denitrification was uncompleted resulting in high NO3-N contents in the effluent. W. Khanitchaidecha, A. Nakaruk, P. Koshy, and K. Futaba Copyright © 2015 W. Khanitchaidecha et al. All rights reserved. Isolation and Characterization of Hydrocarbon-Degrading Yeast Strains from Petroleum Contaminated Industrial Wastewater Mon, 03 Aug 2015 10:49:23 +0000 Two yeast strains are enriched and isolated from industrial refinery wastewater. These strains were observed for their ability to utilize several classes of petroleum hydrocarbons substrates, such as n-alkanes and aromatic hydrocarbons as a sole carbon source. Phylogenetic analysis based on the D1/D2 variable domain and the ITS-region sequences indicated that strains HC1 and HC4 were members of the genera Candida and Trichosporon, respectively. The mechanism of hydrocarbon uptaking by yeast, Candida, and Trichosporon has been studied by means of the kinetic analysis of hydrocarbons-degrading yeasts growth and substrate assimilation. Biodegradation capacity and biomass quantity were daily measured during twelve days by gravimetric analysis and gas chromatography coupled with mass spectrometry techniques. Removal of n-alkanes indicated a strong ability of hydrocarbon biodegradation by the isolated yeast strains. These two strains grew on long-chain n-alkane, diesel oil, and crude oil but failed to grow on short-chain n-alkane and aromatic hydrocarbons. Growth measurement attributes of the isolates, using n-hexadecane, diesel oil, and crude oil as substrates, showed that strain HC1 had better degradation for hydrocarbon substrates than strain HC4. In conclusion, these yeast strains can be useful for the bioremediation process and decreasing petroleum pollution in wastewater contaminated with petroleum hydrocarbons. Boutheina Gargouri, Najla Mhiri, Fatma Karray, Fathi Aloui, and Sami Sayadi Copyright © 2015 Boutheina Gargouri et al. All rights reserved. Prenatal Exposure to Sodium Arsenite Alters Placental Glucose 1, 3, and 4 Transporters in Balb/c Mice Mon, 03 Aug 2015 09:36:50 +0000 Inorganic arsenic (iAs) exposure induces a decrease in glucose type 4 transporter (GLUT4) expression on the adipocyte membrane, which may be related to premature births and low birth weight infants in women exposed to iAs at reproductive age. The aim of this study was to analyze the effect of sodium arsenite (NaAsO2) exposure on GLUT1, GLUT3, and GLUT4 protein expression and on placental morphology. Female Balb/c mice () were exposed to 0, 12, and 20 ppm of NaAsO2 in drinking water from 8th to 18th day of gestation. Morphological changes and GLUT1, GLUT3, and GLUT4 expression were evaluated in placentas by immunohistochemical and image analysis and correlated with iAs and arsenical species concentration, which were quantified by atomic absorption spectroscopy. NaAsO2 exposure induced a significant decrease in fetal and placental weight () and increases in infarctions and vascular congestion. Whereas GLUT1 expression was unchanged in placentas from exposed group, GLUT3 expression was found increased. In contrast, GLUT4 expression was significantly lower () in placentas from females exposed to 12 ppm. The decrease in placental GLUT4 expression might affect the provision of adequate fetal nutrition and explain the low fetal weight observed in the exposed groups. Daniela Sarahí Gutiérrez-Torres, Carmen González-Horta, Luz María Del Razo, Rocío Infante-Ramírez, Ernesto Ramos-Martínez, Margarita Levario-Carrillo, and Blanca Sánchez-Ramírez Copyright © 2015 Daniela Sarahí Gutiérrez-Torres et al. All rights reserved. Gestational Hypertension and Organophosphorus Pesticide Exposure: A Cross-Sectional Study Mon, 03 Aug 2015 09:34:12 +0000 Hypertension is the most common medical problem encountered during pregnancy, complicating 2-3% of pregnancies. High blood pressure (BP) with diastolic BP ≥ 90 mm Hg and/or systolic BP ≥ 140 mm Hg arising after week 22 of pregnancy and resolving after delivery is defined as gestational hypertension (GHY). The aim of this cross-sectional study was to investigate whether occupational and/or environmental exposure to organophosphorus (OP) pesticide affects GHY. Women at approximately 22 weeks of gestation were recruited. OP pesticide exposure in the first trimester of pregnancy was classified into four categories: no exposure, indirect exposure, domestic exposure, and occupational exposure. Application of the exclusion criteria left 2203 participants (mean age 30.4 ± 11.6 years). Data analysis showed that in women with indirect OP pesticide exposure the incidence of GHY was slightly higher than that in the world population, whereas domestic exposure involved a 7% increase and occupational exposure a 12% increase. Analysis of the pesticides used by participants highlighted a possible role for malathion and diazinon (adjusted OR 1.09 and 1.14, resp.). Further investigation of exposed workers and the general population is clearly warranted given the broad diffusion of OP pesticides and their possible public health impact, maybe by including a wider range of health outcomes. Caterina Ledda, Maria Fiore, Lory Santarelli, Massimo Bracci, Giuseppe Mascali, Maria Grazia D’Agati, Alfredo Busà, Margherita Ferrante, and Venerando Rapisarda Copyright © 2015 Caterina Ledda et al. All rights reserved. ZnO Nanoparticles Treatment Induces Apoptosis by Increasing Intracellular ROS Levels in LTEP-a-2 Cells Mon, 03 Aug 2015 09:02:32 +0000 Owing to the wide use of novel nanoparticles (NPs) such as zinc oxide (ZnO) in all aspects of life, toxicological research on ZnO NPs is receiving increasing attention in these days. In this study, the toxicity of ZnO NPs in a human pulmonary adenocarcinoma cell line LTEP-a-2 was tested in vitro. Log-phase cells were exposed to different levels of ZnO NPs for hours, followed by colorimetric cell viability assay using tetrazolium salt and cell survival rate assay using trypan blue dye. Cell morphological changes were observed by Giemsa staining and light microscopy. Apoptosis was detected by using fluorescence microscopy and caspase-3 activity assay. Both intracellular reactive oxygen species (ROS) and reduced glutathione (GSH) were examined by a microplate-reader method. Results showed that ZnO NPs (≥0.01 μg/mL) significantly inhibited proliferation () and induced substantial apoptosis in LTEP-a-2 cells after 4 h of exposure. The intracellular ROS level rose up to 30–40% corresponding to significant depletion (approximately 70–80%) in GSH content in LTEP-a-2 cells (), suggesting that ZnO NPs induced apoptosis mainly through increased ROS production. This study elucidates the toxicological mechanism of ZnO NPs in human pulmonary adenocarcinoma cells and provides reference data for application of nanomaterials in the environment. Caixia Wang, Xiaoke Hu, Yan Gao, and Yinglu Ji Copyright © 2015 Caixia Wang et al. All rights reserved. Study of Cytotoxic Effects of Benzonitrile Pesticides Mon, 03 Aug 2015 06:23:52 +0000 The benzonitrile herbicides bromoxynil, chloroxynil, dichlobenil, and ioxynil have been used actively worldwide to control weeds in agriculture since 1970s. Even though dichlobenil is prohibited in EU since 2008, studies addressing the fate of benzonitrile herbicides in the environment show that some metabolites of these herbicides are very persistent. We tested the cytotoxic effects of benzonitrile herbicides and their microbial metabolites using two human cell lines, Hep G2 and HEK293T, representing liver and kidneys as potential target organs in humans. The cell viability and proliferation were determined by MTT test and RTCA DP Analyzer system, respectively. The latter allows real-time monitoring of the effect of added substances. As the cytotoxic compounds could compromise cell membrane integrity, the lactate dehydrogenase test was performed as well. We observed high toxic effects of bromoxynil, chloroxynil, and ioxynil on both tested cell lines. In contrast, we determined only low inhibition of cell growth in presence of dichlobenil and microbial metabolites originating from the tested herbicides. Petra Lovecka, Marketa Thimova, Petra Grznarova, Jan Lipov, Zdenek Knejzlik, Hana Stiborova, Tjokorda Gde Tirta Nindhia, Katerina Demnerova, and Tomas Ruml Copyright © 2015 Petra Lovecka et al. All rights reserved. Subtoxic Doses of Cadmium Modulate Inflammatory Properties of Murine RAW 264.7 Macrophages Mon, 03 Aug 2015 06:21:31 +0000 Cadmium (Cd) is a toxic heavy metal that exhibits various adverse effects in the human and animal organism. Its resemblance to essential metals such as calcium, iron, and zinc leads to an unintended uptake in cells after intake through inhalation and ingestion. In this study we investigated the toxicity and the immunomodulatory potential of Cd in nonactivated and activated murine macrophages (i.e., cell line RAW 264.7). Cadmium alone caused a dose-dependent decreased viability of exposed cells. Subtoxic Cd concentrations delayed cell death in macrophages, resulting from cytotoxic storm, producing reactive oxygen species (ROS) and nitric oxide (NO), in response to their stimulation by bacterial antigens via pattern-recognition receptors (PRRs). In addition, production of selected pro- and anti-inflammatory cytokines, the chemokine CXCL1 (KC), and NO was determined. We observed that proinflammatory IL-1β and also CXCL1 were highly upregulated whereas anti-inflammatory or regulatory cytokines IL-6 and IL-10 were suppressed by 10 µM Cd. Also production of antibacterial NO was significantly reduced through exposure to 10 µM Cd, maybe explaining better survival of macrophages. Additionally, we could show by analysis via ICP-MS that different effects of Cd in nonactivated and activated macrophages definitely did not result from different Cd uptake rates. Sina Riemschneider, Martin Herzberg, and Jörg Lehmann Copyright © 2015 Sina Riemschneider et al. All rights reserved. Pollution Problem in River Kabul: Accumulation Estimates of Heavy Metals in Native Fish Species Mon, 03 Aug 2015 06:20:28 +0000 The contamination of aquatic systems with heavy metals is affecting the fish population and hence results in a decline of productivity rate. River Kabul is a transcountry river originating at Paghman province in Afghanistan and inters in Khyber Pakhtunkhwa province of Pakistan and it is the major source of irrigation and more than 54 fish species have been reported in the river. Present study aimed at the estimation of heavy metals load in the fish living in River Kabul. Heavy metals including chromium, nickel, copper, zinc, cadmium, and lead were determined through atomic absorption spectrophotometer after tissue digestion by adopting standard procedures. Concentrations of these metals were recorded in muscles and liver of five native fish species, namely, Wallago attu, Aorichthys seenghala, Cyprinus carpio, Labeo dyocheilus, and Ompok bimaculatus. The concentrations of chromium, nickel, copper, zinc, and lead were higher in both of the tissues, whereas the concentration of cadmium was comparatively low. However, the concentration of metals was exceeding the RDA (Recommended Dietary Allowance of USA) limits. Hence, continuous fish consumption may create health problems for the consumers. The results of the present study are alarming and suggest implementing environmental laws and initiation of a biomonitoring program of the river. Habib Ahmad, Ali Muhammad Yousafzai, Muhammad Siraj, Rashid Ahmad, Israr Ahmad, Muhammad Shahid Nadeem, Waqar Ahmad, Nazia Akbar, and Khushi Muhammad Copyright © 2015 Habib Ahmad et al. All rights reserved. Green Tea Increases the Concentration of Total Mercury in the Blood of Rats following an Oral Fish Tissue Bolus Sun, 02 Aug 2015 13:40:25 +0000 Fish has many health benefits but is also the most common source of methylmercury. The bioavailability of methylmercury in fish may be affected by other meal components. In this study, the effect of green tea on the bioavailability of methylmercury from an oral bolus of fish muscle tissue was studied in rats and compared to a water treated control group and a group treated with meso-2,3-dimercaptosuccinic acid (DMSA), a compound used medically to chelate mercury. Rats were given a single oral dose of fish tissue via gavage and one of the treatments. Rats were given access to food for 3 h at 12 h intervals. They were dosed with each of the treatments with each meal. Blood samples were collected for 95 hours. Green tea significantly increased the concentration of total mercury in blood relative to the control, whereas DMSA significantly decreased it. In addition, feeding caused a slight increase in blood mercury for several meals following the initial dose. Elsa M. Janle, Helene Freiser, Christopher Manganais, Tzu-Ying Chen, Bruce A. Craig, and Charles R. Santerre Copyright © 2015 Elsa M. Janle et al. All rights reserved. Evaluation of Ricinus communis L. for the Phytoremediation of Polluted Soil with Organochlorine Pesticides Sun, 02 Aug 2015 13:38:40 +0000 Phytoremediation is an attractive alternative to conventional treatments of soil due to advantages such as low cost, large application areas, and the possibility of in situ treatment. This study presents the assessment of phytoremediation processes conducted under controlled experimental conditions to evaluate the ability of Ricinus communis L., tropical plant species, to promote the degradation of 15 persistent organic pollutants (POPs), in a 66-day period. The contaminants tested were hexachlorocyclohexane (HCH), DDT, heptachlor, aldrin, and others. Measurements made in rhizosphere soil indicate that the roots of the studied species reduce the concentration of pesticides. Results obtained during this study indicated that the higher the hydrophobicity of the organic compound and its molecular interaction with soil or root matrix the greater its tendency to concentrate in root tissues and the research showed the following trend: HCHs < diclofop-methyl < chlorpyrifos < methoxychlor < heptachlor epoxide < endrin < o,p′-DDE < heptachlor < dieldrin < aldrin < o,p′-DDT < p,p′-DDT by increasing order of log values. The experimental results confirm the importance of vegetation in removing pollutants, obtaining remediation from 25% to 70%, and demonstrated that Ricinus communis L. can be used for the phytoremediation of such compounds. Sandra Regina Rissato, Mário Sergio Galhiane, João Roberto Fernandes, Marli Gerenutti, Homero Marques Gomes, Renata Ribeiro, and Marcos Vinícius de Almeida Copyright © 2015 Sandra Regina Rissato et al. All rights reserved. Dichloroacetate Decreases Cell Health and Activates Oxidative Stress Defense Pathways in Rat Alveolar Type II Pneumocytes Sun, 02 Aug 2015 13:31:30 +0000 Dichloroacetate (DCA) is a water purification byproduct that is known to be hepatotoxic and hepatocarcinogenic and to induce peripheral neuropathy and damage macrophages. This study characterizes the effects of the haloacetate on lung cells by exposing rat alveolar type II (L2) cells to 0–24 mM DCA for 6–24 hours. Increasing DCA concentration and the combination of increasing DCA concentration plus longer exposures decrease measures of cellular health. Length of exposure has no effect on oxidative stress biomarkers, glutathione, SOD, or CAT. Increasing DCA concentration alone does not affect total glutathione or its redox ratio but does increase activity in the SOD/CAT oxidative stress defense pathway. These data suggest that alveolar type II cells rely on SOD and CAT more than glutathione to combat DCA-induced stress. Alexis Valauri-Orton, Frizzi Bschorer, and Karen K. Bernd Copyright © 2015 Alexis Valauri-Orton et al. All rights reserved. Air Pollution by Hydrothermal Volcanism and Human Pulmonary Function Sun, 02 Aug 2015 12:59:13 +0000 The aim of this study was to assess whether chronic exposure to volcanogenic air pollution by hydrothermal soil diffuse degassing is associated with respiratory defects in humans. This study was carried in the archipelago of the Azores, an area with active volcanism located in the Atlantic Ocean where Eurasian, African, and American lithospheric plates meet. A cross-sectional study was performed on a study group of 146 individuals inhabiting an area where volcanic activity is marked by active fumarolic fields and soil degassing (hydrothermal area) and a reference group of 359 individuals inhabiting an area without these secondary manifestations of volcanism (nonhydrothermal area). Odds ratio (OR) and 95% confidence intervals (CIs) were adjusted for age, gender, fatigue, asthma, and smoking. The OR for restrictive defects and for exacerbation of obstructive defects (COPD) in the hydrothermal area was 4.4 (95% CI 1.78–10.69) and 3.2 (95% CI 1.82–5.58), respectively. Increased prevalence of restrictions and all COPD severity ranks (mild, moderate, and severe) was observed in the population from the hydrothermal area. These findings may assist health officials in advising and keeping up with these populations to prevent and minimize the risk of respiratory diseases. Diana Linhares, Patrícia Ventura Garcia, Fátima Viveiros, Teresa Ferreira, and Armindo dos Santos Rodrigues Copyright © 2015 Diana Linhares et al. All rights reserved. Three-Dimensional Modelling inside a Differential Pressure Laminar Flow Bioreactor Filled with Porous Media Sun, 02 Aug 2015 11:15:22 +0000 A three-dimensional computational fluid dynamics- (CFD-) model based on a differential pressure laminar flow bioreactor prototype was developed to further examine performance under changing culture conditions. Cell growth inside scaffolds was simulated by decreasing intrinsic permeability values and led to pressure build-up in the upper culture chamber. Pressure release by an integrated bypass system allowed continuation of culture. The specific shape of the bioreactor culture vessel supported a homogenous flow profile and mass flux at the scaffold level at various scaffold permeabilities. Experimental data showed an increase in oxygen concentration measured inside a collagen scaffold seeded with human mesenchymal stem cells when cultured in the perfusion bioreactor after 24 h compared to static culture in a Petri dish (dynamic: 11% O2 versus static: 3% O2). Computational fluid simulation can support design of bioreactor systems for tissue engineering application. Birgit Weyand, Meir Israelowitz, James Kramer, Christian Bodmer, Mariel Noehre, Sarah Strauss, Elmar Schmälzlin, Christoph Gille, Herbert P. von Schroeder, Kerstin Reimers, and Peter M. Vogt Copyright © 2015 Birgit Weyand et al. All rights reserved. Heavy Metal Adsorption onto Kappaphycus sp. from Aqueous Solutions: The Use of Error Functions for Validation of Isotherm and Kinetics Models Thu, 30 Jul 2015 09:44:35 +0000 Biosorption process is a promising technology for the removal of heavy metals from industrial wastes and effluents using low-cost and effective biosorbents. In the present study, adsorption of Pb2+, Cu2+, Fe2+, and Zn2+ onto dried biomass of red seaweed Kappaphycus sp. was investigated as a function of pH, contact time, initial metal ion concentration, and temperature. The experimental data were evaluated by four isotherm models (Langmuir, Freundlich, Temkin, and Dubinin-Radushkevich) and four kinetic models (pseudo-first-order, pseudo-second-order, Elovich, and intraparticle diffusion models). The adsorption process was feasible, spontaneous, and endothermic in nature. Functional groups in the biomass involved in metal adsorption process were revealed as carboxylic and sulfonic acids and sulfonate by Fourier transform infrared analysis. A total of nine error functions were applied to validate the models. We strongly suggest the analysis of error functions for validating adsorption isotherm and kinetic models using linear methods. The present work shows that the red seaweed Kappaphycus sp. can be used as a potentially low-cost biosorbent for the removal of heavy metal ions from aqueous solutions. Further study is warranted to evaluate its feasibility for the removal of heavy metals from the real environment. Md. Sayedur Rahman and Kathiresan V. Sathasivam Copyright © 2015 Md. Sayedur Rahman and Kathiresan V. Sathasivam. All rights reserved. Microbial Diversity for Biotechnology 2014 Tue, 28 Jul 2015 07:50:37 +0000 George Tsiamis, Ameur Cherif, Dimitrios Karpouzas, and Spyridon Ntougias Copyright © 2015 George Tsiamis et al. All rights reserved. Development of Electroactive and Anaerobic Ammonium-Oxidizing (Anammox) Biofilms from Digestate in Microbial Fuel Cells Mon, 27 Jul 2015 08:33:54 +0000 Microbial Fuel cells (MFCs) have been proposed for nutrient removal and energy recovery from different wastes. In this study the anaerobic digestate was used to feed H-type MFC reactors, one with a graphite anode preconditioned with Geobacter sulfurreducens and the other with an unconditioned graphite anode. The data demonstrate that the digestate acts as a carbon source, and even in the absence of anode preconditioning, electroactive bacteria colonise the anodic chamber, producing a maximum power density of 172.2 mW/m2. The carbon content was also reduced by up to 60%, while anaerobic ammonium oxidation (anammox) bacteria, which were found in the anodic compartment of the reactors, contributed to nitrogen removal from the digestate. Overall, these results demonstrate that MFCs can be used to recover anammox bacteria from natural sources, and it may represent a promising bioremediation unit in anaerobic digestor plants for the simultaneous nitrogen removal and electricity generation using digestate as substrate. Enea Gino Di Domenico, Gianluca Petroni, Daniele Mancini, Alberto Geri, Luca Di Palma, and Fiorentina Ascenzioni Copyright © 2015 Enea Gino Di Domenico et al. All rights reserved. Bacterial Diversity and Bioremediation Potential of the Highly Contaminated Marine Sediments at El-Max District (Egypt, Mediterranean Sea) Mon, 27 Jul 2015 08:17:07 +0000 Coastal environments worldwide are threatened by the effects of pollution, a risk particularly high in semienclosed basins like the Mediterranean Sea that is poorly studied from bioremediation potential perspective especially in the Southern coast. Here, we investigated the physical, chemical, and microbiological features of hydrocarbon and heavy metals contaminated sediments collected at El-Max bay (Egypt). Molecular and statistical approaches assessing the structure of the sediment-dwelling bacterial communities showed correlations between the composition of bacterial assemblages and the associated environmental parameters. Fifty strains were isolated on mineral media supplemented by 1% crude oil and identified as a diverse range of hydrocarbon-degrading bacteria involved in different successional stages of biodegradation. We screened the collection for biotechnological potential studying biosurfactant production, biofilm formation, and the capability to utilize different hydrocarbons. Some strains were able to grow on multiple hydrocarbons as unique carbon source and presented biosurfactant-like activities and/or capacity to form biofilm and owned genes involved in different detoxification/degradation processes. El-Max sediments represent a promising reservoir of novel bacterial strains adapted to high hydrocarbon contamination loads. The potential of the strains for exploitation for in situ intervention to combat pollution in coastal areas is discussed. Ranya A. Amer, Francesca Mapelli, Hamada M. El Gendi, Marta Barbato, Doaa A. Goda, Anna Corsini, Lucia Cavalca, Marco Fusi, Sara Borin, Daniele Daffonchio, and Yasser R. Abdel-Fattah Copyright © 2015 Ranya A. Amer et al. All rights reserved. A High Diversity in Chitinolytic and Chitosanolytic Species and Enzymes and Their Oligomeric Products Exist in Soil with a History of Chitin and Chitosan Exposure Sun, 26 Jul 2015 12:45:43 +0000 Chitin is one of the most abundant biomolecules on earth, and its partially de-N-acetylated counterpart, chitosan, is one of the most promising biotechnological resources due to its diversity in structure and function. Recently, chitin and chitosan modifying enzymes (CCMEs) have gained increasing interest as tools to engineer chitosans with specific functions and reliable performance in biotechnological and biomedical applications. In a search for novel CCME, we isolated chitinolytic and chitosanolytic microorganisms from soils with more than ten-years history of chitin and chitosan exposure and screened them for chitinase and chitosanase isoenzymes as well as for their patterns of oligomeric products by incubating their secretomes with chitosan polymers. Of the 60 bacterial strains isolated, only eight were chitinolytic and/or chitosanolytic, while 20 out of 25 fungal isolates were chitinolytic and/or chitosanolytic. The bacterial isolates produced rather similar patterns of chitinolytic and chitosanolytic enzymes, while the fungal isolates produced a much broader range of different isoenzymes. Furthermore, diverse mixtures of oligosaccharides were formed when chitosan polymers were incubated with the secretomes of select fungal species. Our study indicates that soils with a history of chitin and chitosan exposure are a good source of novel CCME for chitosan bioengineering. Malathi Nampally, M. B. Govinda Rajulu, Dominique Gillet, T. S. Suryanarayanan, and Bruno B. Moerschbacher Copyright © 2015 Malathi Nampally et al. All rights reserved. Bacterial Diversity Associated with the Coccolithophorid Algae Emiliania huxleyi and Coccolithus pelagicus f. braarudii Sun, 26 Jul 2015 11:46:22 +0000 Coccolithophores are unicellular calcifying marine phytoplankton that can form large and conspicuous blooms in the oceans and make significant contributions to oceanic carbon cycling and atmospheric CO2 regulation. Despite their importance, the bacterial diversity associated with these algae has not been explored for ecological or biotechnological reasons. Bacterial membership of Emiliania huxleyi and Coccolithus pelagicus f. braarudii cultures was assessed using cultivation and cultivation-independent methods. The communities were species rich compared to other phytoplankton cultures. Community analysis identified specific taxa which cooccur in all cultures (Marinobacter and Marivita). Hydrocarbon-degrading bacteria were found in all cultures. The presence of Acidobacteria, Acidimicrobidae, Schlegelella, and Thermomonas was unprecedented but were potentially explained by calcification associated with coccolith production. One strain of Acidobacteria was cultivated and is closely related to a marine Acidobacteria isolated from a sponge. From this assessment of the bacterial diversity of coccolithophores, a number of biotechnological opportunities are evident, from bioprospecting for novel taxa such as Acidobacteria to helping understand the relationship between obligate hydrocarbonoclastic bacteria occurrence with phytoplankton and to revealing bacterial taxa that have a specific association with algae and may be suitable candidates as a means to improve the efficiency of mass algal cultivation. David H. Green, Virginia Echavarri-Bravo, Debra Brennan, and Mark C. Hart Copyright © 2015 David H. Green et al. All rights reserved. Isolation and Characterization of Phages Infecting Bacillus subtilis Sun, 26 Jul 2015 11:10:47 +0000 Bacteriophages have been suggested as an alternative approach to reduce the amount of pathogens in various applications. Bacteriophages of various specificity and virulence were isolated as a means of controlling food-borne pathogens. We studied the interaction of bacteriophages with Bacillus species, which are very often persistent in industrial applications such as food production due to their antibiotic resistance and spore formation. A comparative study using electron microscopy, PFGE, and SDS-PAGE as well as determination of host range, pH and temperature resistance, adsorption rate, latent time, and phage burst size was performed on three phages of the Myoviridae family and one phage of the Siphoviridae family which infected Bacillus subtilis strains. The phages are morphologically different and characterized by icosahedral heads and contractile (SIOΦ, SUBω, and SPOσ phages) or noncontractile (ARπ phage) tails. The genomes of SIOΦ and SUBω are composed of 154 kb. The capsid of SIOΦ is composed of four proteins. Bacteriophages SPOσ and ARπ have genome sizes of 25 kbp and 40 kbp, respectively. Both phages as well as SUBω phage have 14 proteins in their capsids. Phages SIOΦ and SPOσ are resistant to high temperatures and to the acid (4.0) and alkaline (9.0 and 10.0) pH. Anna Krasowska, Anna Biegalska, Daria Augustyniak, Marcin Łoś, Malwina Richert, and Marcin Łukaszewicz Copyright © 2015 Anna Krasowska et al. All rights reserved. Bioprecipitation of Calcium Carbonate Crystals by Bacteria Isolated from Saline Environments Grown in Culture Media Amended with Seawater and Real Brine Sun, 26 Jul 2015 09:38:01 +0000 The precipitation of calcium carbonate and calcium sulphate by isolated bacteria from seawater and real brine obtained in a desalination plant growth in culture media containing seawater and brine as mineral sources has been studied. However, only bioprecipitation was detected when the bacteria were grown in media with added organic matter. Biomineralization process started rapidly, crystal formation taking place in the beginning a few days after inoculation of media; roughly 90% of total cultivated bacteria showed. Six major colonies with carbonate precipitation capacity dominated bacterial community structure cultivated in heterotrophic platable bacteria medium. Taxonomic identification of these six strains through partial 16S rRNA gene sequences showed their affiliation with Gram-positive Bacillus and Virgibacillus genera. These strains were able to form calcium carbonate minerals, which precipitated as calcite and aragonite crystals and showed bacterial fingerprints or bacteria calcification. Also, carbonic anhydrase activity was observed in three of these isolated bacteria. The results of this research suggest that microbiota isolated from sea water and brine is capable of precipitation of carbonate biominerals, which can occur in situ with mediation of organic matter concentrations. Moreover, calcium carbonate precipitation ability of this microbiota could be of importance in bioremediation of CO2 and calcium in certain environments. G. A. Silva-Castro, I. Uad, A. Gonzalez-Martinez, A. Rivadeneyra, J. Gonzalez-Lopez, and M. A. Rivadeneyra Copyright © 2015 G. A. Silva-Castro et al. All rights reserved. Characterization, Microbial Community Structure, and Pathogen Occurrence in Urban Faucet Biofilms in South China Sun, 26 Jul 2015 08:18:53 +0000 The composition and microbial community structure of the drinking water system biofilms were investigated using microstructure analysis and 454 pyrosequencing technique in Xiamen city, southeast of China. SEM (scanning electron microscope) results showed different features of biofilm morphology in different fields of PVC pipe. Extracellular matrix material and sparse populations of bacteria (mainly rod-shaped and coccoid) were observed. CLSM (confocal laser scanning microscope) revealed different distributions of attached cells, extracellular proteins, α-polysaccharides, and β-polysaccharides. The biofilms had complex bacterial compositions. Differences in bacteria diversity and composition from different tap materials and ages were observed. Proteobacteria was the common and predominant group in all biofilms samples. Some potential pathogens (Legionellales, Enterobacteriales, Chromatiales, and Pseudomonadales) and corrosive microorganisms were also found in the biofilms. This study provides the information of characterization and visualization of the drinking water biofilms matrix, as well as the microbial community structure and opportunistic pathogens occurrence. Huirong Lin, Shuting Zhang, Song Gong, Shenghua Zhang, and Xin Yu Copyright © 2015 Huirong Lin et al. All rights reserved. Diverse Reductive Dehalogenases Are Associated with Clostridiales-Enriched Microcosms Dechlorinating 1,2-Dichloroethane Sun, 26 Jul 2015 07:49:34 +0000 The achievement of successful biostimulation of active microbiomes for the cleanup of a polluted site is strictly dependent on the knowledge of the key microorganisms equipped with the relevant catabolic genes responsible for the degradation process. In this work, we present the characterization of the bacterial community developed in anaerobic microcosms after biostimulation with the electron donor lactate of groundwater polluted with 1,2-dichloroethane (1,2-DCA). Through a multilevel analysis, we have assessed (i) the structural analysis of the bacterial community; (ii) the identification of putative dehalorespiring bacteria; (iii) the characterization of functional genes encoding for putative 1,2-DCA reductive dehalogenases (RDs). Following the biostimulation treatment, the structure of the bacterial community underwent a notable change of the main phylotypes, with the enrichment of representatives of the order Clostridiales. Through PCR targeting conserved regions within known RD genes, four novel variants of RDs previously associated with the reductive dechlorination of 1,2-DCA were identified in the metagenome of the Clostridiales-dominated bacterial community. Giuseppe Merlino, Annalisa Balloi, Massimo Marzorati, Francesca Mapelli, Aurora Rizzi, Davide Lavazza, Francesca de Ferra, Giovanna Carpani, and Daniele Daffonchio Copyright © 2015 Giuseppe Merlino et al. All rights reserved. Expression of Heterologous Cellulases in Thermotoga sp. Strain RQ2 Sun, 26 Jul 2015 07:32:41 +0000 The ability of Thermotoga spp. to degrade cellulose is limited due to a lack of exoglucanases. To address this deficiency, cellulase genes Csac_1076 (celA) and Csac_1078 (celB) from Caldicellulosiruptor saccharolyticus were cloned into T. sp. strain RQ2 for heterologous overexpression. Coding regions of Csac_1076 and Csac_1078 were fused to the signal peptide of TM1840 (amyA) and TM0070 (xynB), resulting in three chimeric enzymes, namely, TM1840-Csac_1078, TM0070-Csac_1078, and TM0070-Csac_1076, which were carried by Thermotoga-E. coli shuttle vectors pHX02, pHX04, and pHX07, respectively. All three recombinant enzymes were successfully expressed in E. coli DH5α and T. sp. strain RQ2, rendering the hosts with increased endo- and/or exoglucanase activities. In E. coli, the recombinant enzymes were mainly bound to the bacterial cells, whereas in T. sp. strain RQ2, about half of the enzyme activities were observed in the culture supernatants. However, the cellulase activities were lost in T. sp. strain RQ2 after three consecutive transfers. Nevertheless, this is the first time heterologous genes bigger than 1 kb (up to 5.3 kb in this study) have ever been expressed in Thermotoga, demonstrating the feasibility of using engineered Thermotoga spp. for efficient cellulose utilization. Hui Xu, Dongmei Han, and Zhaohui Xu Copyright © 2015 Hui Xu et al. All rights reserved. Immobilization of Yarrowia lipolytica Lipase on Macroporous Resin Using Different Methods: Characterization of the Biocatalysts in Hydrolysis Reaction Thu, 09 Jul 2015 09:38:14 +0000 To improve the reusability and organic solvent tolerance of microbial lipase and expand the application of lipase (hydrolysis, esterification, and transesterification), we immobilized marine microbial lipase using different methods and determined the properties of immobilized lipases. Considering the activity and cost of immobilized lipase, the concentration of lipase was fixed at 2 mg/mL. The optimal temperature of immobilized lipases was 40°C and 5°C higher than free lipase. The activities of immobilized lipases were much higher than free lipase at alkaline pH (more than 50% at pH 12). The free lipase lost most activity (35.3%) and immobilized lipases retained more than 46.4% of their initial activity after 3 h heat treatment at 70°C. At alkaline pH, immobilized lipases were more stable than free lipase (more than 60% residue activity at pH 11 for 3 h). Immobilized lipases retained 80% of their activity after 5 cycles and increased enzyme activity (more than 108.7%) after 3 h treatment in tert-butanol. Immobilization of lipase which improved reusability of lipase and provided a chance to expand the application of marine microbial lipase in organic system expanded the application range of lipase to catalyze hydrolysis and esterification in harsh condition. Jingjing Sun, Yiling Chen, Jun Sheng, and Mi Sun Copyright © 2015 Jingjing Sun et al. All rights reserved. Improved Salinity Tolerance in Carrizo Citrange Rootstock through Overexpression of Glyoxalase System Genes Wed, 08 Jul 2015 06:54:54 +0000 Citrus plants are widely cultivated around the world and, however, are one of the most salt stress sensitive crops. To improve salinity tolerance, transgenic Carrizo citrange rootstocks that overexpress glyoxalase I and glyoxalase II genes were obtained and their salt stress tolerance was evaluated. Molecular analysis showed high expression for both glyoxalase genes (BjGlyI and PgGlyII) in 5H03 and 5H04 lines. Under control conditions, transgenic and wild type plants presented normal morphology. In salinity treatments, the transgenic plants showed less yellowing, marginal burn in lower leaves and showed less than 40% of leaf damage compared with wild type plants. The transgenic plants showed a significant increase in the dry weight of shoot but there are no differences in the root and complete plant dry weight. In addition, a higher accumulation of chlorine is observed in the roots in transgenic line 5H03 but in shoot it was lower. Also, the wild type plant accumulated around 20% more chlorine in the shoot compared to roots. These results suggest that heterologous expression of glyoxalase system genes could enhance salt stress tolerance in Carrizo citrange rootstock and could be a good biotechnological approach to improve the abiotic stress tolerance in woody plant species. Ximena Alvarez-Gerding, Rowena Cortés-Bullemore, Consuelo Medina, Jesús L. Romero-Romero, Claudio Inostroza-Blancheteau, Felipe Aquea, and Patricio Arce-Johnson Copyright © 2015 Ximena Alvarez-Gerding et al. All rights reserved. Cyanobacteria: Photoautotrophic Microbial Factories for the Sustainable Synthesis of Industrial Products Thu, 25 Jun 2015 12:16:51 +0000 Cyanobacteria are widely distributed Gram-negative bacteria with a long evolutionary history and the only prokaryotes that perform plant-like oxygenic photosynthesis. Cyanobacteria possess several advantages as hosts for biotechnological applications, including simple growth requirements, ease of genetic manipulation, and attractive platforms for carbon neutral production process. The use of photosynthetic cyanobacteria to directly convert carbon dioxide to biofuels is an emerging area of interest. Equipped with the ability to degrade environmental pollutants and remove heavy metals, cyanobacteria are promising tools for bioremediation and wastewater treatment. Cyanobacteria are characterized by the ability to produce a spectrum of bioactive compounds with antibacterial, antifungal, antiviral, and antialgal properties that are of pharmaceutical and agricultural significance. Several strains of cyanobacteria are also sources of high-value chemicals, for example, pigments, vitamins, and enzymes. Recent advances in biotechnological approaches have facilitated researches directed towards maximizing the production of desired products in cyanobacteria and realizing the potential of these bacteria for various industrial applications. In this review, the potential of cyanobacteria as sources of energy, bioactive compounds, high-value chemicals, and tools for aquatic bioremediation and recent progress in engineering cyanobacteria for these bioindustrial applications are discussed. Nyok-Sean Lau, Minami Matsui, and Amirul Al-Ashraf Abdullah Copyright © 2015 Nyok-Sean Lau et al. All rights reserved. Single-Stranded DNA Aptamers against Pathogens and Toxins: Identification and Biosensing Applications Tue, 23 Jun 2015 11:58:35 +0000 Molecular recognition elements (MREs) can be short sequences of single-stranded DNA, RNA, small peptides, or antibody fragments. They can bind to user-defined targets with high affinity and specificity. There has been an increasing interest in the identification and application of nucleic acid molecular recognition elements, commonly known as aptamers, since they were first described in 1990 by the Gold and Szostak laboratories. A large number of target specific nucleic acids MREs and their applications are currently in the literature. This review first describes the general methodologies used in identifying single-stranded DNA (ssDNA) aptamers. It then summarizes advancements in the identification and biosensing application of ssDNA aptamers specific for bacteria, viruses, their associated molecules, and selected chemical toxins. Lastly, an overview of the basic principles of ssDNA aptamer-based biosensors is discussed. Ka Lok Hong and Letha J. Sooter Copyright © 2015 Ka Lok Hong and Letha J. Sooter. All rights reserved. The 482Ser of PPARGC1A and 12Pro of PPARG2 Alleles Are Associated with Reduction of Metabolic Risk Factors Even Obesity in a Mexican-Mestizo Population Mon, 22 Jun 2015 08:51:13 +0000 The aim of this study was to investigate the relationship between functional polymorphisms Gly482Ser in PPARGC1A and Pro12Ala in PPARG2 with the presence of obesity and metabolic risk factors. We included 375 individuals characterized as Mexican-Mestizos and classified by the body mass index (BMI). Body dimensions and distribution of body fat were measured. The HOMA-IR and adiposity indexes were calculated. Adipokines and metabolic profile quantification were performed by ELISA and routine methods. Genetic polymorphisms were determined by polymerase chain reaction restriction fragment length polymorphism analysis. A difference between obese and nonobese subjects in polymorphism PPARGC1A distribution was observed. Among obese individuals, carriers of genotype 482Gly/Gly were observed to have decreased body fat, BMI, and body fat ratio versus 482Ser/Ser carriers and increased resistin and leptin levels in carriers Gly+ phenotype versus Gly− phenotype. Subjects with PPARG2 Ala− phenotype (genotype 12Pro/Pro) showed a decreased HOMA-IR index versus individuals with Ala+ phenotype (genotypes 12Pro/Ala plus 12Ala/Ala). We propose that, in obese Mexican-Mestizos, the combination of alleles 482Ser in PPARGC1A and 12Pro in PPARG2 represents a reduced metabolic risk profile, even when the adiposity indexes are increased. Mónica Vázquez-Del Mercado, Milton-Omar Guzmán-Ornelas, Fernanda-Isadora Corona Meraz, Clara-Patricia Ríos-Ibarra, Eduardo-Alejandro Reyes-Serratos, Jorge Castro-Albarran, Sandra-Luz Ruíz-Quezada, and Rosa-Elena Navarro-Hernández Copyright © 2015 Mónica Vázquez-Del Mercado et al. All rights reserved. Evaluating Insects as Bioindicators of Heavy Metal Contamination and Accumulation near Industrial Area of Gujrat, Pakistan Thu, 18 Jun 2015 12:19:14 +0000 To study the accumulation and contamination of heavy metals (i.e., Cd, Cr, Cu, Ni, and Zn) in soil, air, and water, few insect species were assayed as ecological indicators. Study area comes under industrial zone of district Gujrat of Punjab, Pakistan. Insects used as bioindicators included a libellulid dragonfly (Crocothemis servilia), an acridid grasshopper (Oxya hyla hyla), and a nymphalid butterfly (Danaus chrysippus) near industrial zone of Gujrat. Accumulation of Cd was highest in insect species followed by Cu, Cr, Zn, and Ni at . Hierarchical cluster analysis (HACA) was carried out to study metal accumulation level in all insects. Correlation and regression analysis confirmed HACA observations and declared concentration of heavy metals above permissible limits. Metal concentrations in insects were significantly higher near industries and nallahs in Gujrat and relatively higher concentrations of metals were found in Orthoptera than Odonata and Lepidoptera. The total metal concentrations in insects were pointed significantly higher at sites S3 (Mid of HalsiNala), S9 (End of HalsiNala), and S1 (Start of HalsiNala), whereas lowest value was detected at site S6 (Kalra Khasa) located far from industrial area. HACA indicates that these insect groups are potential indicators of metal contamination and can be used in biomonitoring. Iqra Azam, Sumera Afsheen, Ahmed Zia, Muqaddas Javed, Rashid Saeed, Muhammad Kaleem Sarwar, and Bushra Munir Copyright © 2015 Iqra Azam et al. All rights reserved. A Mutagenesis Assay for Reporter Gene Screening Using Partially Degenerate Oligonucleotides of the Tandems NNT and NNC Thu, 18 Jun 2015 11:30:26 +0000 Not all proteins are tolerable to mutations. Whether a specific protein can be a mutable target is of importance in the biotechnology and pharmaceutical industry. This study reported a novel mutagenesis assay using tandem NNT and NNC oligonucleotides to test the mutability of a candidate gene. These two tandem oligonucleotides avoid the risk of forming nonsense mutations and render flexibility of truncating or expanding the insertion size. As a reporter gene, ZeoR (zeocin resistance gene) was confirmed to have a high tolerance for mutagenesis by this new assay. Huifen Xu, Cuilan Zhou, Andy K. Zhang, Wen Li, Jia Zhang, and Kai Li Copyright © 2015 Huifen Xu et al. All rights reserved. Anaerobic Treatment of Palm Oil Mill Effluent in Pilot-Scale Anaerobic EGSB Reactor Thu, 18 Jun 2015 08:38:30 +0000 Large volumes of untreated palm oil mill effluent (POME) pose threat to aquatic environment due to the presence of very high organic content. The present investigation involved two pilot-scale anaerobic expanded granular sludge bed (EGSB) reactors, continuously operated for 1 year to treat POME. Setting HRT at 9.8 d, the anaerobic EGSB reactors reduced COD from 71179 mg/L to 12341 mg/L and recycled half of sludge by a dissolved air flotation (DAF). The average effluent COD was 3587 mg/L with the consistent COD removal efficiency of 94.89%. Adding cationic polymer (PAM) dose of 30 mg/L to DAF unit and recycling its half of sludge caused granulation of anaerobic sludge. Bacilli and small coccid bacteria were the dominant microbial species of the reactor. The reactor produced 27.65 m3 of biogas per m3 of POME which was utilized for electricity generation. Jin Wang, Qaisar Mahmood, Jiang-Ping Qiu, Yin-Sheng Li, Yoon-Seong Chang, and Xu-Dong Li Copyright © 2015 Jin Wang et al. All rights reserved. In Vitro Assessment of Cadmium Bioavailability in Chinese Cabbage Grown on Different Soils and Its Toxic Effects on Human Health Thu, 18 Jun 2015 08:31:59 +0000 The minimum concentration of cadmium (Cd), by Chinese cabbage grown on Cd contaminated soils that can initiate toxicity in human liver cells using in vitro digestion coupled with Caco-2/HL-7702 cell models was studied. Cadmium bioaccessibility in the gastric phase for yellow soil (YS) cabbage (40.84%) and calcareous soil (CS) cabbage (21.54%) was significantly higher than small intestinal phase with the corresponding values of 21.2% and 11.11%, respectively. Cadmium bioavailability was higher in YS cabbage (5.27%–14.66%) than in CS cabbage (1.12%–9.64%). Cadmium concentrations (>0.74 μg) transported from YS and CS cabbage were able to induce oxidative (MDA, H2O2) stress by inhibiting antioxidant (SOD, GPx) enzyme activities in human liver cells (HL-7702). Additionally the study revealed that the ingestion of Cd contaminated Chinese cabbage grown in acidic soil (yellow soil) weakened the antioxidant defense system under all levels of contamination (2, 6, and 9 mg·kg−1) which ultimately escalated the oxidative stress in liver cells; however, in case of CS cabbage, a marked oxidative stress was observed only at 9 mg kg−1 Cd level of soil. Therefore, it is necessary to monitor Cd concentrations in leafy vegetables grown on acidic soils to minimize human health risk. Rukhsanda Aziz, Muhammad Tariq Rafiq, Zhenli He, Di Liu, Kewang Sun, and Yang Xiaoe Copyright © 2015 Rukhsanda Aziz et al. All rights reserved. Role of Geitlerinema sp. DE2011 and Scenedesmus sp. DE2009 as Bioindicators and Immobilizers of Chromium in a Contaminated Natural Environment Wed, 17 Jun 2015 08:14:20 +0000 The aim of this work was to study the potential of the two phototrophic microorganisms, both isolated from Ebro Delta microbial mats, to be used as bioindicators and immobilizers of chromium. The results obtained indicated that (i) the Minimum Metal Concentration (MMC) significantly affecting Chlorophyll a intensity in Geitlerinema sp. DE2011 and Scenedesmus sp. DE2009 was 0.25 µM and 0.75 µM, respectively, these values being lower than those established by current legislation, and (ii) Scenedesmus sp. DE2009 was able to immobilize chromium externally in extracellular polymeric substances (EPS) and intracellularly in polyphosphate (PP) inclusions. Additionally, this microorganism maintained high viability, including at 500 µM. Based on these results, we postulate that Geitlerinema sp. DE2011 and Scenedesmus sp. DE2009 are good chromium-indicators of cytotoxicity and, further, that Scenedesmus sp. DE2009 plays an important role in immobilizing this metal in a contaminated natural environment. Laia Millach, Antoni Solé, and Isabel Esteve Copyright © 2015 Laia Millach et al. All rights reserved. Microbial Enzymes and Their Applications in Industries and Medicine 2014 Tue, 16 Jun 2015 07:31:50 +0000 Periasamy Anbu, Subash C. B. Gopinath, Bidur Prasad Chaulagain, Thean-Hock Tang, and Marimuthu Citartan Copyright © 2015 Periasamy Anbu et al. All rights reserved. Bioenergy and Biomass Utilization Thu, 11 Jun 2015 12:02:44 +0000 Guangli Cao, Dexun Zou, Ximing Zhang, Lei Zhao, and Guojun Xie Copyright © 2015 Guangli Cao et al. All rights reserved. Codon Optimization Significantly Improves the Expression Level of α-Amylase Gene from Bacillus licheniformis in Pichia pastoris Wed, 10 Jun 2015 07:07:29 +0000 α-Amylase as an important industrial enzyme has been widely used in starch processing, detergent, and paper industries. To improve expression efficiency of recombinant α-amylase from Bacillus licheniformis (B. licheniformis), the α-amylase gene from B. licheniformis was optimized according to the codon usage of Pichia pastoris (P. pastoris) and expressed in P. pastoris. Totally, the codons encoding 305 amino acids were optimized in which a total of 328 nucleotides were changed and the G+C content was increased from 47.6 to 49.2%. The recombinants were cultured in 96-deep-well microplates and screened by a new plate assay method. Compared with the wild-type gene, the optimized gene is expressed at a significantly higher level in P. pastoris after methanol induction for 168 h in 5- and 50-L bioreactor with the maximum activity of 8100 and 11000 U/mL, which was 2.31- and 2.62-fold higher than that by wild-type gene. The improved expression level makes the enzyme a good candidate for α-amylase production in industrial use. Jian-Rong Wang, Yang-Yuan Li, Dan-Ni Liu, Jing-Shan Liu, Peng Li, Li-Zhi Chen, and Shu-De Xu Copyright © 2015 Jian-Rong Wang et al. All rights reserved. Improvement in Saccharification Yield of Mixed Rumen Enzymes by Identification of Recalcitrant Cell Wall Constituents Using Enzyme Fingerprinting Tue, 09 Jun 2015 08:19:41 +0000 Identification of recalcitrant factors that limit digestion of forages and the development of enzymatic approaches that improve hydrolysis could play a key role in improving the efficiency of meat and milk production in ruminants. Enzyme fingerprinting of barley silage fed to heifers and total tract indigestible fibre residue (TIFR) collected from feces was used to identify cell wall components resistant to total tract digestion. Enzyme fingerprinting results identified acetyl xylan esterases as key to the enhanced ruminal digestion. FTIR analysis also suggested cross-link cell wall polymers as principal components of indigested fiber residues in feces. Based on structural information from enzymatic fingerprinting and FTIR, enzyme pretreatment to enhance glucose yield from barley straw and alfalfa hay upon exposure to mixed rumen-enzymes was developed. Prehydrolysis effects of recombinant fungal fibrolytic hydrolases were analyzed using microassay in combination with statistical experimental design. Recombinant hemicellulases and auxiliary enzymes initiated degradation of plant structural polysaccharides upon application and improved the in vitro saccharification of alfalfa and barley straw by mixed rumen enzymes. The validation results showed that microassay in combination with statistical experimental design can be successfully used to predict effective enzyme pretreatments that can enhance plant cell wall digestion by mixed rumen enzymes. Ajay Badhan, Yu-Xi Wang, Robert Gruninger, Donald Patton, Justin Powlowski, Adrian Tsang, and Tim A. McAllister Copyright © 2015 Ajay Badhan et al. All rights reserved. Molecular Identification of a Newly Isolated Bacillus subtilis BI19 and Optimization of Production Conditions for Enhanced Production of Extracellular Amylase Tue, 09 Jun 2015 08:08:51 +0000 A study was carried out with a newly isolated bacterial strain yielding extracellular amylase. The phylogenetic tree constructed on the basis of 16S rDNA gene sequences revealed this strain as clustered with the closest members of Bacillus sp. and identified as Bacillus subtilis BI19. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Rice flour (1.25%) as a cheap natural carbon source was found to induce amylase production mostly. A combination of peptone and tryptone as organic and ammonium sulfate as inorganic nitrogen sources gave highest yield. Maximum production was obtained after 24 h of incubation at 37°C with an initial medium pH 8.0. Addition of surfactants like Tween 80 (0.25 g/L) and sodium lauryl sulfate (0.2 g/L) resulted in 28% and 15% increase in enzyme production, respectively. Amylase production was 3.06 times higher when optimized production conditions were used. Optimum reaction temperature and pH for crude amylase activity were 50°C and 6.0, respectively. The crude enzyme showed activity and stability over a fair range of temperature and pH. These results suggest that B. subtilis BI19 could be exploited for production of amylase at relatively low cost and time. Biplab Kumar Dash, M. Mizanur Rahman, and Palash Kumar Sarker Copyright © 2015 Biplab Kumar Dash et al. All rights reserved. Production and Characterization of Lipases by Two New Isolates of Aspergillus through Solid-State and Submerged Fermentation Tue, 09 Jun 2015 07:43:00 +0000 Due to the numerous applications of lipases in industry, there is a need to study their characteristics, because lipases obtained from different sources may present different properties. The aim of this work was to accomplish the partial characterization of lipases obtained through submerged fermentation and solid-state fermentation by two species of Aspergillus. Fungal strains were isolated from a diesel-contaminated soil and selected as good lipases producers. Lipases obtained through submerged fermentation presented optimal activities at 37°C and pH 7.2 and those obtained through solid-state fermentation at 35°C and pH 6.0. The enzymes produced by submerged fermentation were more temperature-stable than those obtained by solid-state fermentation, presenting 72% of residual activity after one hour of exposition at 90°C. Lipases obtained through submerged fermentation had 80% of stability in acidic pH and those obtained through solid-state fermentation had stability greater than 60% in alkaline pH. Luciane Maria Colla, Aline M. M. Ficanha, Juliana Rizzardi, Telma Elita Bertolin, Christian Oliveira Reinehr, and Jorge Alberto Vieira Costa Copyright © 2015 Luciane Maria Colla et al. All rights reserved. Statistical Optimization of Laccase Production and Delignification of Sugarcane Bagasse by Pleurotus ostreatus in Solid-State Fermentation Tue, 09 Jun 2015 06:56:06 +0000 Laccases are oxidative enzymes related to the degradation of phenolic compounds, including lignin units, with concomitant reduction of oxygen to water. Delignification is a necessary pretreatment step in the process of converting plant biomass into fermentable sugars. The objective of this work was to optimize the production of laccases and to evaluate the delignification of sugarcane bagasse by Pleurotus ostreatus in solid-state fermentation. Among eight variables (pH, water activity, temperature, and concentrations of CuSO4, (NH4)2SO4, KH2PO4, asparagine, and yeast extract), copper sulfate and ammonium sulfate concentrations were demonstrated to significantly influence laccase production. The replacement of ammonium sulfate by yeast extract and the addition of ferulic acid as inducer provided increases of 5.7- and 2.0-fold, respectively, in laccase activity. Optimization of laccase production as a function of yeast extract, copper sulfate, and ferulic acid concentrations was performed by response surface methodology and optimal concentrations were 6.4 g/L, 172.6 μM, and 1.86 mM, respectively. Experimentally, the maximum laccase activity of 151.6 U/g was produced at the 5th day of solid-state fermentation. Lignin content in sugarcane bagasse was reduced from 31.89% to 26.36% after 5 days and to 20.79% after 15 days by the biological treatment of solid-state fermentation. Susan Grace Karp, Vincenza Faraco, Antonella Amore, Luiz Alberto Junior Letti, Vanete Thomaz Soccol, and Carlos Ricardo Soccol Copyright © 2015 Susan Grace Karp et al. All rights reserved. Biotechnological Aspects and Perspective of Microbial Keratinase Production Tue, 09 Jun 2015 06:52:14 +0000 Keratinases are proteolytic enzymes predominantly active when keratin substrates are available that attack disulfide bridges in the keratin to convert them from complex to simplified forms. Keratinases are essential in preparation of animal nutrients, protein supplements, leather manufacture, textile processing, detergent formulation, feather meal processing for feed and fertilizer, the pharmaceutical and biomedical industries, and waste management. Accordingly, it is necessary to develop a method for continuous production of keratinase from reliable sources that can be easily managed. Microbial keratinase is less expensive than conventionally produced keratinase and can be obtained from fungi, bacteria, and actinomycetes. In this overview, the expansion of information about microbial keratinases and important considerations in keratinase production are discussed. Subash C. B. Gopinath, Periasamy Anbu, Thangavel Lakshmipriya, Thean-Hock Tang, Yeng Chen, Uda Hashim, A. Rahim Ruslinda, and M. K. Md. Arshad Copyright © 2015 Subash C. B. Gopinath et al. All rights reserved. Purification and Characterization of a Polyextremophilic α-Amylase from an Obligate Halophilic Aspergillus penicillioides Isolate and Its Potential for Souse with Detergents Tue, 09 Jun 2015 06:42:45 +0000 An extracellular α-amylase from the obligate halophilic Aspergillus penicillioides TISTR3639 strain was produced and enriched to apparent homogeneity by ammonium sulfate precipitation and Sephadex G100 gel filtration column chromatography. The mass of the purified amylase was estimated to be 42 kDa by SDS-PAGE. With soluble starch as the substrate it had a specific activity of 118.42 Umg−1 and and values of 1.05 µmolmin−1mg−1 and 5.41 mgmL−1, respectively. The enzyme was found to have certain polyextremophilic characteristics, with an optimum activity at pH 9, 80°C, and 300 gL−1 NaCl. The addition of CaCl2 at 2 mM was found to slightly enhance the amylase activity, while ZnCl2, FeCl2, or EDTA at 2 mM was strongly or moderately inhibitory, respectively, suggesting the requirement for a (non-Fe2+ or Zn2+) divalent cation. The enzyme retained more than 80% of its activity when incubated with three different laundry detergents and had a better performance compared to a commercial amylase and three detergents in the presence of increasing NaCl concentrations up to 300 gL−1. Accordingly, it has a good potential for use as an α-amylase in a low water activity (high salt concentration) and at high pH and temperatures. Imran Ali, Ali Akbar, Mohammad Anwar, Sehanat Prasongsuk, Pongtharin Lotrakul, and Hunsa Punnapayak Copyright © 2015 Imran Ali et al. All rights reserved. Proteases of Wood Rot Fungi with Emphasis on the Genus Pleurotus Tue, 09 Jun 2015 06:41:17 +0000 Proteases are present in all living organisms and they play an important role in physiological conditions. Cell growth and death, blood clotting, and immune defense are all examples of the importance of proteases in maintaining homeostasis. There is growing interest in proteases due to their use for industrial purposes. The search for proteases with specific characteristics is designed to reduce production costs and to find suitable properties for certain industrial sectors, as well as good producing organisms. Ninety percent of commercialized proteases are obtained from microbial sources and proteases from macromycetes have recently gained prominence in the search for new enzymes with specific characteristics. The production of proteases from saprophytic basidiomycetes has led to the identification of various classes of proteases. The genus Pleurotus has been extensively studied because of its ligninolytic enzymes. The characteristics of this genus are easy cultivation techniques, high yield, low nutrient requirements, and excellent adaptation. There are few studies in the literature about proteases of Pleurotus spp. This review gathers together information about proteases, especially those derived from basidiomycetes, and aims at stimulating further research about fungal proteases because of their physiological importance and their application in various industries such as biotechnology and medicine. Fabíola Dorneles Inácio, Roselene Oliveira Ferreira, Caroline Aparecida Vaz de Araujo, Tatiane Brugnari, Rafael Castoldi, Rosane Marina Peralta, and Cristina Giatti Marques de Souza Copyright © 2015 Fabíola Dorneles Inácio et al. All rights reserved. Optimization of Culture Conditions for Production of the Anti-Leukemic Glutaminase Free L-Asparaginase by Newly Isolated Streptomyces olivaceus NEAE-119 Using Response Surface Methodology Tue, 09 Jun 2015 06:39:51 +0000 Among the antitumor drugs, bacterial enzyme L-asparaginase has been employed as the most effective chemotherapeutic agent in pediatric oncotherapy especially for acute lymphoblastic leukemia. Glutaminase free L-asparaginase producing actinomycetes were isolated from soil samples collected from Egypt. Among them, a potential culture, strain NEAE-119, was selected and identified on the basis of morphological, cultural, physiological, and biochemical properties together with 16S rRNA sequence as Streptomyces olivaceus NEAE-119 and sequencing product (1509 bp) was deposited in the GenBank database under accession number KJ200342. The optimization of different process parameters for L-asparaginase production by Streptomyces olivaceus NEAE-119 using Plackett-Burman experimental design and response surface methodology was carried out. Fifteen variables (temperature, pH, incubation time, inoculum size, inoculum age, agitation speed, dextrose, starch, L-asparagine, KNO3, yeast extract, K2HPO4, MgSO47H2O, NaCl, and FeSO47H2O) were screened using Plackett-Burman experimental design. The most positive significant independent variables affecting enzyme production (temperature, inoculum age, and agitation speed) were further optimized by the face-centered central composite design-response surface methodology. Noura El-Ahmady El-Naggar, Hassan Moawad, Nancy M. El-Shweihy, and Sara M. El-Ewasy Copyright © 2015 Noura El-Ahmady El-Naggar et al. All rights reserved. Lipase-Secreting Bacillus Species in an Oil-Contaminated Habitat: Promising Strains to Alleviate Oil Pollution Tue, 09 Jun 2015 06:32:07 +0000 Lipases are of great interest for different industrial applications due to their diversity and versatility. Among different lipases, microbial lipases are preferable due to their broad substrate specificity, and higher stability with lower production costs compared to the lipases from plants and animals. In the past, a vast number of bacterial species have been reported as potential lipases producers. In this study, the lipases-producing bacterial species were isolated from an oil spillage area in the conventional night market. Isolated species were identified as Bacillus species by biochemical tests which indicate their predominant establishment, and further screened on the agar solid surfaces using lipid and gelatin as the substrates. Out of the ten strains tested, four potential strains were subjected to comparison analysis of the lipolytic versus proteolytic activities. Strain 10 exhibited the highest lipolytic and proteolytic activity. In all the strains, the proteolytic activity is higher than the lipolytic activity except for strain 8, suggesting the possibility for substrate-based extracellular gene induction. The simultaneous secretion of both the lipase and protease is a mean of survival. The isolated bacterial species which harbour both lipase and protease enzymes could render potential industrial-based applications and solve environmental issues. Li Pin Lee, Hudzaifah Mohamed Karbul, Marimuthu Citartan, Subash C. B. Gopinath, Thangavel Lakshmipriya, and Thean-Hock Tang Copyright © 2015 Li Pin Lee et al. All rights reserved. Response of Arbuscular Mycorrhizal Fungi to Hydrologic Gradients in the Rhizosphere of Phragmites australis (Cav.) Trin ex. Steudel Growing in the Sun Island Wetland Wed, 03 Jun 2015 12:43:58 +0000 Within the rhizosphere, AM fungi are a sensitive variable to changes of botanic and environmental conditions, and they may interact with the biomass of plant and other microbes. During the vegetative period of the Phragmites australis growing in the Sun Island Wetland (SIW), the variations of AM fungi colonization were studied. Root samples of three hydrologic gradients generally showed AM fungi colonization, suggesting that AM fungi have the ability for adaptation to flooded habitats. There were direct and indirect hydrological related effects with respect to AM fungi biomass, which interacted simultaneously in the rhizosphere. Though water content in soil and reed growth parameters were both positively associated with AM fungi colonization, only the positive correlations between reed biomass parameters and the colonization could be expected, or both the host plant biomass and the AM fungi could be beneficial. The variations in response of host plant to the edaphic and hydrologic conditions may influence the effectiveness of the plant-mycorrhizal association. This study included a hydrologic component to better assess the role and distribution of AM fungi in wetland ecosystems. And because of that, the range of AM fungi was extended, since they actually showed a notable adaptability to hydrologic gradients. Li Wang, Jieting Wu, Fang Ma, Jixian Yang, Shiyang Li, Zhe Li, and Xue Zhang Copyright © 2015 Li Wang et al. All rights reserved. Biochemical Modulation of Lipid Pathway in Microalgae Dunaliella sp. for Biodiesel Production Wed, 03 Jun 2015 09:52:51 +0000 Exploitation of renewable sources of energy such as algal biodiesel could turn energy supplies problem around. Studies on a locally isolated strain of Dunaliella sp. showed that the mean lipid content in cultures enriched by 200 mg L−1 myoinositol was raised by around 33% (1.5 times higher than the control). Similarly, higher lipid productivity values were achieved in cultures treated by 100 and 200 mg L−1 myoinositol. Fluorometry analyses (microplate fluorescence and flow cytometry) revealed increased oil accumulation in the Nile red-stained algal samples. Moreover, it was predicted that biodiesel produced from myoinositol-treated cells possessed improved oxidative stability, cetane number, and cloud point values. From the genomic point of view, real-time analyses revealed that myoinositol negatively influenced transcript abundance of AccD gene (one of the key genes involved in lipid production pathway) due to feedback inhibition and that its positive effect must have been exerted through other genes. The findings of the current research are not to interprete that myoinositol supplementation could answer all the challenges faced in microalgal biodiesel production but instead to show that “there is a there there” for biochemical modulation strategies, which we achieved, increased algal oil quantity and enhanced resultant biodiesel quality. Ahmad Farhad Talebi, Masoud Tohidfar, Seyedeh Mahsa Mousavi Derazmahalleh, Alawi Sulaiman, Azhari Samsu Baharuddin, and Meisam Tabatabaei Copyright © 2015 Ahmad Farhad Talebi et al. All rights reserved.