Minnan Long

Purification and Properties of Endoglucanase from a Sugar Cane Bagasse Hydrolyzing Strain, Aspergillus glaucus XC9

An endoglucanase (EG) from Aspergillus glaucus XC9 grown on 0.3% sugar cane bagasse as a carbon source was purified from the culture filtrate using ammonium sulfate, an anion exchange DEAE Sepharose fast flow column, and a Sephadex G-100 column, with a purification fold of 21.5 and a recovery of 22.3%. The ideal time for EG production is on the fourth day at 30 degrees C using bagasse as a substrate. Results obtained indicate that the enzyme was a monomer protein, and the molecular weight was determined to be 31 kDa. The optimum pH and temperature of EG for the hydrolysis of carboxymethylcellulose sodium (CMC-Na) were pH 4.0 and 50 degrees C, respectively. EG was stable over the pH range from 3.5 to 7.5 and at temperatures below 55 degrees C. Kinetic behavior of EG in the hydrolysis of CMC-Na followed Michaelis-Menten kinetics with constant K(m) of 5.0 mg/mL at pH 4.0 and 50 degrees C. The enzyme activity was stimulated by Fe(2+) and Mn(2+) but inhibited by Cd(2+), Pb(2+), and Cu(2+). The EDC chemical modification suggested that at least one carboxyl group probably acted as a proton donor in the enzyme active site.

Enhanced H2 gas production from bagasse using adhE inactivated Klebsiella oxytoca HP1 by sequential dark-photo fermentations.

Sequential dark-photo fermentations (SDPF) was used for hydrogen production from bagasse, an acetaldehyde dehydrogenase (adhE) gene inactivated Klebsiella oxytoca HP1 (DeltaadhE HP1) mutant was used to reduce the alcohol content in dark fermentation (DF) broths and to further enhance the hydrogen yield during the photo fermentation (PF) stage. Compared with that of the wild strain, the ethanol concentration in DF broths of DeltaadhE HP1 decreased 69.4%, which resulted in a hydrogen yield in the PF stage and the total hydrogen yield over the two steps increased by 54.7% and 23.5%, respectively. The culture conditions for hydrogen production from acid pretreated bagasse by SDPF were optimized as culture temperature 37.5 degrees C, initial pH 7.0, and cellulase loading 20 FPA/g in the DF stage, with initial pH 6.5, temperature 30 degrees C and photo intensity 5,000 lux in the PF stage. Under optimum conditions, by using DeltaadhE HP1 and wild type strain, the H(2) yields were 107.8+/-5.3 mL H(2)/g-bagasse, 96.2+/-4.4 mL H(2)/g-bagasse in DF and 54.3+/-2.2 mL H(2)/g-bagasse, 35.1+/-2.0 mL H(2)/g-bagasse in PF, respectively. The special hydrogen production rate (SHPR) were 5.51+/-0.34 mL H(2)/g-bagasseh, 4.95+/-0.22 mL H(2)/g-bagasseh in DF and 0.93+/-0.12 mL H(2)/g-bagasseh, 0.59+/-0.07 mL H(2)/g-bagasseh in PF, respectively. The total hydrogen yield from bagasse over two steps was 162.1+/-7.5 mL H(2)/g-bagasse by using DeltaadhE HP1, which was 50.4% higher than that from dark fermentation only. These results indicate that reducing ethanol content during dark fermentation by using an adhE inactivated strain can significantly enhance hydrogen production from bagasse in the SDPF system. This work also proved that SDPF was an effective way to improve hydrogen production from bagasse.

Cellulase production in a new mutant strain of Penicillium decumbens ML-017 by solid state fermentation with rice bran

To produce cellulolytic enzyme efficiently, Penicillium decumbens strain L-06 was used to prepare mutants with ethyl methane sulfonate (EMS) and UV-irradiation. A mutant strain ML-017 is shown to have a higher cellulase activity than others. Box-Behnkens design (BBD) and response surface methodology (RSM) were adopted to optimize the conditions of cellulase (filter paper activity, FPA) production in strain ML-017 by solid-state fermentation (SSF) with rice bran as the substrate. And the result shows that the initial pH, moisture content and culture temperature all have significant effect on the production of cellulase. The optimized condition shall be initial pH 5.7, moisture content 72% and culture temperature 30°C. The maximum cellulase (FPA) production was obtained under the optimized condition, which is 5.76 IU g(-1), increased by 44.12% to its original strain. It corresponded well with the calculated results (5.15 IU g(-1)) by model prediction. The result shows that both BBD and RSM are the cellulase optimization methods with good prospects.

Cellulase production by solid state fermentation using bagasse with Penicillium decumbens L-06

The cellulase production byPenicillium decumbens L-06 in solid state fermentation (SSF) was investigated using bagasse as the substrate in this paper. The optimum conditions for cellulase production achieved by single factor testing were: the ratio of bagasse to wheat bran 1∶1 (w/w), the ratio of water to material 3∶1 (v/w), culture temperature 30 °C, initial pH 5.0, ammonium sulphate as nitrogen source with the concentration of 1%, 6 day’s fermentation period. BoxuBehnken factorial design (BBD) and response surface methodology (RSM) were further used to optimize conditions for cellulase (Filter paper activity) production. The maximal cellulase (Filter paper activity) production (3.89 FPu g−1) was obtained under the optimized conditions (ratio of water to material 2.38∶1, initial pH 5.28, cultivation time 150.5 h). It was well corresponded to the calculated results (3.97 FPu g−1) by model prediction.

Purification and characterization of beta-1,4-glucosidase from Aspergillus glaucus

National Key Basic Research Program (NKBRP) [2010CB732201]; Natural Science Foundation of Fujian Province [2009J01196]; National Foundation for fostering talents of basic science [J1030626]

Increased biological hydrogen production by deletion of hydrogen-uptake system in photosynthetic bacteria.

Hydrogenases are the key enzymes for the biological hydrogen production, which can be classified as H(2)-uptake hydrogenase and H(2)-production hydrogenase. The genes encoding a membrane-bound [NiFe]-hydrogenase (MBH), which is mainly responsible for hydrogen uptake, from the photosynthetic bacterium Allochromatium vinosum was cloned and sequenced. It consist of two structural genes (hydS, hydL) and two intergenic genes (isp1, isp2), which are therefore organized as hydS-isp1-isp2-hydL. This is different from the arrangement of other typical hydrogenase gene clusters. A deletion mutant-strain PhihydSL, lacking isp1, isp2, partial hydS and hydL genes, was constructed by marker-exchange mutagenesis. Under dark fermentative conditions, the hydrogen production yield by this mutant increased by 62%. The result suggests that the disruption of MBH could greatly improve the hydrogen production in the cells by decreasing the hydrogen uptake.

Enzymatic saccharification of cassava residues and glucose inhibitory kinetics on β-glucosidase from Hypocrea orientalis

Cassava residues are byproducts of the starch industry containing abundant cellulose for bioproduction of green fuel. To obtain maximum sugar yields from cassava residues, the optimal conditions for hydrolyzing the residues were determined using cellulase prepared from a novel Hypocrea orientalis strain. The optimal pH value and optimal temperature for the cellulase hydrolysis were 5.0 and 50 °C, respectively. The concentration of NaOH was determined to be 1% for pretreatment of cassava residues to gain enough soluble sugars suitably. The yield of released sugars was 10 mg/mL in the optimal conditions after 24 h of reaction, which was similar to that of bagasse and wheat grass. Inhibition kinetics of H. orientalis β-glucosidase (BG) by glucose was first studied using the progress-of-substrate-reaction method as described by Tsou (Tsou, C. L. Adv. Enzymol. Related Areas Mol. Biol. 1988, 61, 381-436), and the microscopic inhibition rate constants of glucose were determined. The results showed that glucose could inhibit BG reversibly and competitively. The rate constants of forward (k(+0)) and reverse (k(-0)) reaction were measured to be 4.88 × 10(-4) (mM·s)(-1) and 2.7 × 10(-4) s(-1), respectively. Meanwhile, the inhibition was more significant than that of L-glucose, D-mannose, D-galactose, D-aminoglucose, acetyl-D-glucose, and D-fructose. This work reveals how to increase sugar yields and reduce product inhibition during enzymatic saccharification of cellulose.

Defluviimonas indica sp. nov., a marine bacterium isolated from a deep-sea hydrothermal vent environment.

A Gram-stain-negative, strictly aerobic, chemoheterotrophic marine bacterium, designated 20V17(T), was isolated from a deep-sea hydrothermal vent chimney collected from the South-west Indian Ridge. Cells of strain 20V17(T) were motile, short rods, 1.2-1.8 µm in length and 0.5-0.7 µm in width. Growth was observed at between 20 and 37 °C (optimum 25 °C-28 °C), pH 5.0 and 8.0 (optimum pH 7.0) and 0.5 and 8% (w/v) NaCl (optimum 1.5-2.0% NaCl). The major fatty acids were C(18 : 1)ω7c (74.4%), C(19 : 0) cyclo ω8c (11%), C(18 : 0) (5.1%) and C(18 : 0) 3-OH (2.8%), and the polar lipid profile comprised diphosphatidylglycerol, phosphatidylethanolamine, an unidentified glycolipid and four unidentified phospholipids. Ubiquinone 10 was the major quinone. The G+C content of genomic DNA was 66.3 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain 20V17(T) belonged to the genus Defluviimonas and shared 96.5 and 96.1% sequence similarity with Defluviimonas denitrificans D9-3(T) and Defluviimonas aestuarii BS14(T), respectively. On the basis of the taxonomic data obtained in this study, strain 20V17(T) represents a novel species of the genus Defluviimonas, for which the name Defluviimonas indica sp. nov. is proposed. The type strain is 20V17(T) (CGMCC 1.10859(T) = JCM 17871(T) = MCCC 1A01802(T)).

Enhanced photo-fermentative hydrogen production from different organic substrate using hupL inactivated Rhodopseudomonas palustris.

National Nature Science Foundation of China [30870051, 31170760]; Key Project of International Collaboration of Science and Technology [2009DFA60930]; Natural Science Foundation of Fujian Province, China [2011J01219]; Inventions and Innovation of Fujian, China [(2009)111]

Thermophilic hydrogen-producing bacteria inhabiting deep-sea hydrothermal environments represented by Caloranaerobacter.

Hydrogen is an important energy source for deep-sea hydrothermal vent ecosystems. However, little is known about microbes and their role in hydrogen turnover in the environment. In this study, the diversity and physiological characteristics of fermentative hydrogen-producing microbes from deep-sea hydrothermal vent fields were described for the first time. Seven enrichments were obtained from hydrothermal vent sulfides collected from the Southwest Indian Ocean, East Pacific and South Atlantic. 16S rRNA gene analysis revealed that members of the Caloranaerobacter genus were the dominant component in these enrichments. Subsequently, three thermophilic hydrogen producers, strains H363, H53214 and DY22619, were isolated. They were phylogenetically related to species of the genus Caloranaerobacter. The H2 yields of strains H363, H53214, DY22619 and MV107, which was the type species of genus Caloranaerobacter, were 0.11, 1.21, 3.13 and 2.85 mol H2/mol glucose, respectively. Determination of the main soluble metabolites revealed that strains H363, H53214 and MV107 performed heterolactic fermentations, while strain DY22619 performed butyric acid fermentation, indicating distinct fermentation patterns among members of the genus. Finally, a diversity of forms of [FeFe]-hydrogenase with different modular structures was revealed based on draft genomic data of Caloranaerobacter strains. This highlights the complexity of hydrogen metabolism in Caloranaerobacter, reflecting adaptations to environmental conditions in hydrothermal vent systems. Collectively, results suggested that Caloranaerobacter species might be ubiquitous and play a role in biological hydrogen generation in deep-sea hydrothermal vent fields.