Applied Biochemistry and Biotechnology (v.180, #5)

In situ Removal of Hydrogen Sulfide During Biogas Fermentation at Microaerobic Condition by Mengmeng Wu; Yima Zhang; Yuanyuan Ye; Chunmian Lin (817-825).
In this paper, rice straw was used as a raw material to produce biogas by anaerobic batch fermentation at 35 °C (mesophilic) or 55 °C (thermophilic). The hydrogen sulfide in biogas can be converted to S0 or sulfate and removed in-situ under micro-oxygen environment. Trace oxygen was conducted to the anaerobic fermentation tank in amount of 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, or 10.0 times stoichiometric equivalence, respectively, and the control experiment without oxygen addition was carried out. The results showed that the initial H2S concentrations of biogas are about 3235 ± 185 mg/m3 (mesophilic) or 3394 ± 126 mg/m3 (thermophilic), respectively. The desulfurization efficiency is 72.3 % (mesophilic) or 65.6 % (thermophilic), respectively, with oxygen addition by stoichiometric relation. When the oxygen feeded in amount of 2∼4 times, theoretical quantity demanded the removal efficiency of hydrogen sulfide could be over 92 %, and the oxygen residue in biogas could be maintained less than 0.5 %, which fit the requirement of biogas used as vehicle fuel or combined to the grid. Though further more oxygen addition could promote the removal efficiency of hydrogen sulfide (about 93.6 %), the oxygen residue in biogas would be higher than the application limit concentration (0.5 %). Whether mesophilic or thermophilic fermentation with the extra addition of oxygen, there were no obvious changes in the gas production and methane concentration. In conclusion, in-situ desulfurization can be achieved in the anaerobic methane fermentation system under micro-oxygen environment. In addition, air could be used as a substitute oxygen resource on the situation without strict demand for the methane content of biogas.
Keywords: Biogas; Micro-oxygen; Desulfurization; Rice straw

Candida rugosa lipases were immobilized onto collagen fibers through glutaraldehyde cross-linking method. The immobilization process has been optimized. Under the optimal immobilization conditions, the activity of the collagen-immobilized lipase reached 340 U/g. The activity was recovered of 28.3 % by immobilization. The operational stability of the obtained collagen-immobilized lipase for hydrolysis of olive oil emulsion was determined. The collagen-immobilized lipase showed good tolerance to temperature and pH variations in comparison to free lipase. The collagen-immobilized lipase was also applied as biocatalyst for synthesis of butyl butyrate from butyric acid and 1-butanol in n-hexane. The conversion yield was 94 % at the optimal conditions. Of its initial activity, 64 % was retained after 5 cycles for synthesizing butyl butyrate in n-hexane.
Keywords: Lipase; Immobilization; Collagen; Glutaraldehyde; Esterification; Butyl butyrate

Effect of Vanadyl Rosiglitazone, a New Insulin-Mimetic Vanadium Complexes, on Glucose Homeostasis of Diabetic Mice by Pingzhe Jiang; Zhen Dong; Baicheng Ma; Zaizhong Ni; Huikun Duan; Xiaodan Li; Bin Wang; Xiaofeng Ma; Qian Wei; Xiangzhen Ji; Minggang Li (841-851).
Diabetes has been cited as the most challenging health problem in the twenty-first century. Accordingly, it is urgent to develop a new type of efficient and low-toxic antidiabetic medication. Since vanadium compounds have insulin-mimetic and potential hypoglycemic activities for type 1 and type 2 diabetes, a new trend has been developed using vanadium and organic ligands to form a new compound in order to increase the intestinal absorption and reduce the toxicity of vanadium compound. In the current investigation, a new organic vanadium compounds, vanadyl rosiglitazone, was synthesized and determined by infrared spectra. Vanadyl rosiglitazone and three other organic vanadium compounds were administered to the diabetic mice through oral administration for 5 weeks. The results of mouse model test indicated that vanadyl rosiglitazone could regulate the blood glucose level and relieve the symptoms of polydipsia, polyphagia, polyuria, and weight loss without side effects and was more effective than the other three organic vanadium compounds including vanadyl trehalose, vanadyl metformin, and vanadyl quercetin. The study indicated that vanadyl rosiglitazone presents insulin-mimetic activities, and it will be a good potential candidate for the development of a new type of oral drug for type 2 diabetes.
Keywords: Type 2 diabetes; Vanadium; Insulin-mimetic activities; Vanadyl rosiglitazone; Organic vanadium compounds; Oral drug

The Feasibility of Thermophilic Caldimonas manganoxidans as a Platform for Efficient PHB Production by Li-Jung Hsiao; Ji-Hong Lin; Pantitra Sankatumvong; Tzong-Ming Wu; Si-Yu Li (852-871).
Recently, poly(3-hydroxybutyrate) (PHB) has been found in a few thermophilic strains where several advantages can be gained from running fermentation at high temperatures. Caldimonas manganoxidans, a thermophilic gram-negative bacterium, was investigated for the feasibility as a PHB-producing strain. It is suggested that the best fermentation strategy for achieving the highest PHB concentration of 5.4 ± 1.1 g/L (from 20 g/L glucose) in 24 h is to use the fermentation conditions that are favored for the bacterial growth, yet temperature and pH should be chosen at conditions that are favored for the PHB content. Besides, the above fermentation conditions produce PHB that has a high molecular weight of 1274 kDa with a low polydispersity index (PDI) of 1.45, where the highest Mw of PHB of 1399 kDa (PDI of 1.32) is obtained in this study. To the best knowledge of authors, C. manganoxidans has the best PHB productivity among the thermophiles and is comparable to those common PHB-producing mesophiles.
Keywords: Caldimonas manganoxidans ; Thermophiles; Poly(3-hydroxybutyrate) (PHB); Glucose; Substrate inhibition; Molecular weight

Effect of Plant Growth Promoting Bacteria Associated with Halophytic Weed (Psoralea corylifolia L) on Germination and Seedling Growth of Wheat Under Saline Conditions by Ajay M. Sorty; Kamlesh K. Meena; Khushboo Choudhary; Utkarsh M. Bitla; P. S. Minhas; K. K. Krishnani (872-882).
Halotolerant bacteria associated with Psoralea corylifolia L., a luxuriantly growing annual weed in salinity-affected semi-arid regions of western Maharashtra, India were evaluated for their plant growth-promoting activity in wheat. A total of 79 bacteria associated with different parts viz., root, shoot and nodule endophytes, rhizosphere, rhizoplane, and leaf epiphytes, were isolated and grouped based on their habitat. Twelve bacteria isolated for their potential in plant growth promotion were further selected for in vitro studies. Molecular identification showed the presence of the genera Bacillus, Pantoea, Marinobacterium, Acinetobacter, Enterobacter, Pseudomonas, Rhizobium, and Sinorhizobium (LC027447-53; LC027455; LC027457, LC027459, and LC128410). The phylogenetic studies along with carbon source utilization profiles using the Biolog® indicated the presence of novel species and the in planta studies revealed promising results under salinity stress. Whereas the nodule endophytes had minute plant growth-promoting (PGP) activity, the cell free culture filtrates of these strains enhanced seed germination of wheat (Triticum aestivum L). The maximum vigor index was monitored in isolate Y7 (Enterobacter sp strain NIASMVII). Indole acetic acid (IAA) production by the isolates ranged between 0.22 and 25.58 μg mL−1. This signifies the need of exploration of their individual metabolites for developing next-generation bio-inoculants through co-inoculation with other compatible microbes. This study has potential in utilization of the weed-associated microbiome in terms of alleviation of salinity stress in crop plants.
Keywords: Psoralea corylifolia. halotolerant bacteria; PGP; Biolog; Salinity stress; Wheat

Biological activities of chitosan oligosaccharides (COS) are well documented, and numerous reports of COS production using specific and non-specific enzymes are available. However, strategies for improving the overall yield by making it monomer free need to be developed. Continuous enzymatic production from chitosan derived from marine wastes is desirable and is cost-effective. Isolation of potential microbes showing chitosanase activity from various ecological niches, gene cloning, enzyme immobilization, and fractionation/purification of COS are some areas, where lot of work is in progress. This review covers recent measures to improve monomer-free COS production using chitosanase/non-specific enzymes and purification/fractionation of these molecules using ultrafiltration and column chromatographic techniques. Various bioprocess strategies, gene cloning for enhanced chitosanase enzyme production, and other measures for COS yield improvements have also been covered in this review. COS derivative preparation as well as COS-coated nanoparticles for efficient drug delivery are being focused in recent studies.
Keywords: Chitosanase; Chitooligosaccharides; Immobilization; Gene expression; Fractionation

An Acid-Adapted Endo-α-1,5-l-arabinanase for Pectin Releasing by Chong Lang; Rujian Yang; Ying Yang; Bei Gao; Li Zhao; Wei Wei; Hualei Wang; Shingo Matsukawa; Jingli Xie; Dongzhi Wei (900-916).
An arabinanase gene was cloned by overlap-PCR from Penicillium sp. Y702 and expressed in Pichia pastoris. The recombinant enzyme was named AbnC702 with 20 U/mg of endo-arabinanase activity toward linear α-1,5-l-arabinan. The optimal pH and temperature of AbnC702 were 5.0 and 50 °C, respectively. The recombinant AbnC702 was highly stable at pH 5.0–7.0 and 50 °C. It could retain about 72.3 % of maximum specific activity at pH 5.0 after incubation for 2.5 h, which indicated AbnC702 was an acid-adapted enzyme. The K m and V max values were 24.8 ± 4.7 mg/ml and 88.5 ± 5.6 U/mg, respectively. A three-dimensional structure of AbnC702 was made by homology modeling, and the counting of acidic/basic amino residues within the region of 10 Å around the active site, as well the hydrogen bonds within the area of 5 Å around the active site, might theoretically interpret the acid adaptability of AbnC702. Analysis of hydrolysis products by thin layer chromatography (TLC) combined with high-performance liquid chromatography (HPLC) verified that the recombinant AbnC702 was an endo-1,5-α-l-arabinanase, which yielded arabinobiose and arabinotriose as major products. AbnC702 was applied in pectin extraction from apple pomace with synergistic action of α-L-arabinofuranosidase.
Keywords: Endo-1,5-α-l-arabinanase; GH 43; Acid-adapted; Pectin extraction; Apple pomace

Degraded gelatin (Gel) and oxidized corn starch (OCS) as abundant, recyclable, and biodegradable materials can be applied to agricultural production, which has been investigated in this research. Firstly, the prepared oxidized corn starch-gelatin (OCS-Gel) composite material was characterized through a Fourier transform infrared spectrometer (FT-IR), a scanning electron microscope (SEM) picture, and a thermogravimetric analysis (TGA). The OCS-Gel was then used as a liquid film mulching for agricultural production, and the application performances (hygroscopicity, permeability, water retention, etc.) of the OCS-Gel were measured. Finally, the planting rapeseed experiments were carried out, and the germination and growing state of the rapeseed seeds were observed. The results from the structural analysis indicated that OCS-Gel enriches pore structure and exhibits high thermal stability up to 324.8 °C. In the application experiments, the OCS-Gel showed excellent properties of water-absorbing and water-retention and low permeability. In addition, the germination rate of the rapeseed seed reached 80 %, and the height of rapeseeds obviously increased in pot experiments after adding the liquid film mulching.
Keywords: Agricultural production; Degradable liquid film mulching; Oxidized corn starch; Gelatin; Solid leather waste

Effect of S/N Ratio on the Removal of Hydrogen Sulfide from Biogas in Anoxic Bioreactors by Xi Li; Xia Jiang; Qiying Zhou; Wenju Jiang (930-944).
Both biogas desulfurization and wastewater denitrification can be achieved simultaneously, when nitrate/nitrite is used as the electron acceptor for H2S oxidation. The main objective of this study was to investigate the influence of the molar ratio of sulfide/nitrate (S/N) on biogas desulfurization performance in a biotrickling filter (BTF) and a biobubble column (BBC). The results show that with the decrease of the S/N ratios from 3.6 to 0.7, the removal efficiencies of H2S increased from about 66 to 100 %, while the removal of nitrate decreased from 100 to 70 % in the two bioreactors. The BTF has a better and more stable desulfurization performance than the BBC does, which could be attributed to their different gas-liquid contacting modes. With the increase of the S/N ratios from 1.0 to 2.5 in the BTFs, the removal of H2S in biogas was affected slightly, while the percentages of the produced sulfate decreased evidently. In addition, different supplying methods of nitrate wastewater, i.e., intermittent and continuous, did not affect the removal of H2S significantly, while the intermittent addition of nitrate wastewater increased the percentages of sulfate and denitrification performance.
Keywords: Hydrogen sulfide; Biogas; Desulfurization; S/N ratio; Nitrate wastewater

Levulinic acid production, directly from lignocellulosic biomass, resulted in low yields due to the poor substrate accessibility and occurrence of side reactions. The effects of reaction conditions, enzymatic pretreatment, and inhibitor addition on the conversion of steam-exploded rice straw (SERS) short fiber to levulinic acid catalyzed by solid superacid were investigated systematically. The results indicated that the optimal reaction conditions were temperature, time, and solid superacid concentration combinations of 200 °C, 15 min, and 7.5 %. Enzymatic pretreatment improved the substrate accessibility to solid superacid catalyst, and p-hydroxyanisole inhibitor reduced the side reactions during reaction processes, which helped to increase levulinic acid yield. The levulinic acid yield reached 25.2 % under the optimal conditions, which was 61.5 % higher than that without enzymatic pretreatment and inhibitor addition. Therefore, enzymatic pretreatment coupled with the addition of p-hydroxyanisole increased levulinic acid production effectively, which contributed to the value-added utilization of lignocellulosic biomass.
Keywords: Levulinic acid; Enzymatic pretreatment; Steam-exploded rice straw; Short fiber; Solid superacid

The potential of the modified magnetic nanoparticles for covalent immobilization of porcine pancreatic α-amylase has been investigated. The synthesis and immobilization processes were simple and fast. The co-precipitation method was used for synthesis of magnetic iron oxide (Fe3O4) nanoparticles (NPs) which were subsequently coated with silica through sol–gel reaction. The amino-functionalized NPs were prepared by treating silica-coated NPs with 3-aminopropyltriethoxysilane followed by covalent immobilization of α-amylase by glutaraldehyde. The optimum enzyme concentration and incubation time for immobilization reaction were 150 mg and 4 h, respectively. Upon this immobilization, the α-amylase retained more than 50 % of its initial specific activity. The optimum pH for maximal catalytic activity of the immobilized enzyme was 6.5 at 45 °C. The kinetic studies on the immobilized enzyme and its free counterpart revealed an acceptable change of Km and Vmax. The Km values were found as 4 and 2.5 mM for free and immobilized enzymes, respectively. The Vmax values for the free and immobilized enzymes were calculated as 1.75 and 1.03 μmol mg−1 min−1, in order, when starch was used as the substrate. A quick separation of immobilized amylase from reaction mixture was achieved when a magnetically active support was applied. In comparison to the free enzyme, the immobilized enzyme was thermally stable and was reusable for 9 cycles while retaining 68 % of its initial activity.
Keywords: Magnetic nanoparticles; Covalent Immobilization; Porcine pancrease α-amylase; Enzyme activity; Enzyme stability

Cells of Candida guilliermondii permeabilized with Triton X-100 were able to efficiently produce xylitol from a medium composed only by d-xylose and MgCl2·6H2O in potassium phosphate buffer, at 35 °C and pH 6.5. Under these conditions, the results were similar to those obtained when cofactor and co-substrate or nutrients were added to the medium (about 95 % d-xylose was assimilated producing 42 g/L of xylitol, corresponding to 0.80 g/g yield and 2.65 g/L h volumetric productivity). Furthermore, the permeabilized cells kept the d-xylose assimilation in about 90 % and the xylitol production in approx. 40 g/L during three bioconversion cycles of 16 h each. These values are highly relevant when compared to others reported in the literature using enzyme technology and fermentative process, thereby demonstrating the effectiveness of the proposed method. The present study reveals that the use of permeabilized cells is an interesting alternative to obtain high xylitol productivity using low cost medium formulation. This approach may allow the future development of xylitol production from xylose present in lignocellulosic biomass, with additional potential for implementation in biorefinery strategies.
Keywords: Candida guilliermondii ; Permeabilization; Triton X-100; Biotransformation; d-xylose; Xylitol

Mesophilic Acidogenesis of Food Waste-Recycling Wastewater: Effects of Hydraulic Retention Time, pH, and Temperature by Gyuseong Han; Seung Gu Shin; Joonyeob Lee; Changsoo Lee; Minho Jo; Seokhwan Hwang (980-999).
The effects of hydraulic retention time (HRT), pH, and operating temperature (T OP) on the degradation of food waste-recycling wastewater (FRW) were investigated in laboratory-scale hydrolysis/acidogenesis reactors. Response surface analysis was used to approximate the production of volatile organic acids and degradation of volatile suspended solids (VSS), carbohydrate, protein, and lipid with regard to the independent variables (1 ≤ HRT ≤ 3 days, 4 ≤ pH ≤ 6, 25 ≤ T OP ≤ 45 °C). Partial cubic models adequately approximated the corresponding response surfaces at α < 5 %. The physiological conditions for maximum acidification (0.4 g TVFA + EtOH/g VSadded) and the maximal degradation of VSS (47.5 %), carbohydrate (92.0 %), protein (17.7 %), and lipid (73.7 %) were different. Analysis of variance suggested that pH had a great effect on the responses in most cases, while T OP and HRT, and their interaction, were significant in some cases. Denaturing gradient gel electrophoresis analysis revealed that Sporanaerobacter acetigenes, Lactobacillus sp., and Eubacterium pyruvivorans-like microorganisms might be main contributors to the hydrolysis and acidogenesis of FRW. Biochemical methane potential test confirmed higher methane yield (538.2 mL CH4/g VSadded) from an acidogenic effluent than from raw FRW.
Keywords: Hydrolysis; Acidogenesis; Particulate organic matters; Response surface analysis; Volatile fatty acid

Single-Step Partial Purification of Intracellular β-Galactosidase from Kluyveromyces lactis Using Microemulsion Droplets by Bekir G. Mazı; Haluk Hamamcı; David M. Ogrydziak; Stephanie R. Dungan (1000-1015).
Partial purification of β-galactosidase from the crude extract of Kluyveromyces lactis was carried out using water-in-isooctane microemulsions formed by the anionic surfactant, sodium di-ethylhexyl sulfosuccinate (Aerosol OT). In order to obtain the crude extract, yeast cells of K. lactis were disrupted by a cell disrupter and separated. The purification of β-galactosidase from the extract by a recently developed one-step reversed micellar (i.e., microemulsion-based) extraction method was then tested, by measuring total protein mass and enzyme activity in the product stream and by analyzing its composition using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. Effects of salt concentration, protein concentration, and pH on the extraction were investigated. Using this approach, a 5.4-fold purification of β-galactosidase was achieved with 96 % total activity recovery, using a feed containing crude extract and 50 mM K-phosphate buffer (pH 7.5) and 50 mM KCl. Gel filtration chromatography showed that the single extraction was successful at removing low molecular weight impurity proteins (molecular weight (MW) < 42 kDa) from the crude extract.
Keywords: Microemulsion; Enzymes; Purification; β-Galactosidase; Extraction; Reversed micelles

Sugar Beet Pulp as Leuconostoc mesenteroides T3 Support for Enhanced Dextransucrase Production on Molasses by Miona G. Miljković; Slađana Z. Davidović; Milica B. Carević; Đorđe N. Veljović; Dragana D. Mladenović; Mirjana D. Rajilić-Stojanović; Suzana I. Dimitrijević-Branković (1016-1027).
Sugar beet pulp (SBP) and molasses, as an agro industrial waste material, are produced in large amounts annually. Thus, a major challenge nowadays is to develop procedures that could increase the value of the generated waste. In this study, SBP as a support for cell immobilization and molasses as a source of nutrients were used for a dextransucrase (DS) production by Leuconostoc mesenteroides T3. The influence of SBP in native form (SBP-N) and after treatment with NaOH (SBP-NaOH) on DS production was investigated. The optimal medium composition for the maximum DS production was determined by varying the concentration of molasses, SBP, and sucrose. The maximum DS yield of 2.02 U/ml was obtained in the medium with 2.5 % of molasses, 2.5 % SBP-NaOH, and 4 % of sucrose concentration. Scanning electron microscopy (SEM) showed immobilization of Lc. mesenteroides T3 cells onto SBP-NaOH. According to the obtained results, the production of DS on molasses could be improved by using NaOH-treated SBP as a carrier for whole-cell immobilization. Our study reveals the basis for the development of process for DS production with additional reduction of expenses by using waste materials for obtaining the valuable biotechnological product.
Keywords: Molasses; Sugar beep pulp; Leuconostoc mesenteroides T3; Immobilization; Dextransucrase