Microbial degradation and physico-chemical alteration of polyhydroxyalkanoates by a thermophilic <Emphasis Type="Italic">Streptomyces</Emphasis> sp.

A thermophilic Streptomyces sp. capable of degrading various aliphatic polyesters was isolated from a landfill site. The isolate, Streptomyces sp. BCC23167, demonstrated rapid aerobic degradation of several polyesters, including polyhydroxyalkanoate copolymers, poly(ɛ-caprolactone) and polybutylene succinate at 50°C and neutral pH. The degrading activity was repressed by glucose and cellobiose, but tolerant to repression by other carbon substrates. Degradation of a commercial poly[(R)-3-hydroxybutyrate-co-3-hydroxyhexanoate] (PHBHx) by Streptomyces sp. BCC23167 progressed from surface to bulk as suggested by the slight decrease in polymer molecular weight. Differential scanning calorimetry analysis of PHBHx film degradation by Streptomyces sp. BCC23167 showed that relative crystallinity of the film increased slightly in the early stage of degradation, followed by a marked decrease later on. The surface morphology of degraded films was analyzed by scanning electron microscopy, which showed altered surface structure consistent with the changes in crystallinity. The isolate is thus of potential for application in composting technology for bio-plastic degradation.     

Survey of Microbial Diversity in Flood Areas during Thailand 2011 Flood Crisis Using High-Throughput Tagged Amplicon Pyrosequencing

The Thailand flood crisis in 2011 was one of the largest recorded floods in modern history, causing enormous damage to the economy and ecological habitats of the country. In this study, bacterial and fungal diversity in sediments and waters collected from ten flood areas in Bangkok and its suburbs, covering residential and agricultural areas, were analyzed using high-throughput 454 pyrosequencing of 16S rRNA gene and internal transcribed spacer sequences. Analysis of microbial community showed differences in taxa distribution in water and sediment with variations in the diversity of saprophytic microbes and sulfate/nitrate reducers among sampling locations, suggesting differences in microbial activity in the habitats. Overall, Proteobacteria represented a major bacterial group in waters, while this group co-existed with Firmicutes, Bacteroidetes, and Actinobacteria in sediments. Anaeromyxobacter, Steroidobacter, and Geobacter were the dominant bacterial genera in sediments, while Sulfuricurvum, Thiovirga, and Hydrogenophaga predominated in waters. For fungi in sediments, Ascomycota, Glomeromycota, and Basidiomycota, particularly in genera Philipsia, Rozella, and Acaulospora, were most frequently detected. Chytridiomycota and Ascomycota were the major fungal phyla, and Rhizophlyctis and Mortierella were the most frequently detected fungal genera in water. Diversity of sulfate-reducing bacteria, related to odor problems, was further investigated using analysis of the dsrB gene which indicated the presence of sulfate-reducing bacteria of families Desulfobacteraceae, Desulfobulbaceae, Syntrobacteraceae, and Desulfoarculaceae in the flood sediments. The work provides an insight into the diversity and function of microbes related to biological processes in flood areas.     

Optimization of xylanase production from Aspergillus niger for biobleaching of eucalyptus pulp

A crude endo-xylanase produced by Aspergillus niger BCC14405 was investigated for its potential in pre-bleaching of chemical pulp from eucalyptus. The optimal fermentation conditions on the basis of optimization using response surface methodology included cultivation in a complex medium comprising wheat bran, rice bran, and soybean meal supplemented with yeast extract, glucose, peptone, and lactose with a starting pH of 6.0 for 7 d. This resulted in production of 89.5 IU/mL of xylanase with minor cellulase activity. Proteomic analysis using LC/MS/MS revealed that the crude enzyme was a composite of hemicellulolytic enzymes, including endo-β-1,4-xylanase and other hemicellulolytic enzymes attacking arabinoxylan and mannan. Pretreatment of the pulp at a xylanase dosage of 10 IU/g increased the brightness ceiling after the C-Eop-H bleaching step up to 3.0% using a chlorine charge with a C-factor of 0.16-0.20. Xylanase treatment also led to reduction in chlorine charge of at least 20%, with an acceptable brightness level. The enzyme pretreatment resulted in a slight increase in pulp viscosity, suggesting an increase in relative cellulose content. The crude enzyme was potent in the enzyme-aided bleaching of chemical pulp in an environmentally friendly pulping process.     

Production of multi-fiber modifying enzyme from Mamillisphaeria sp. for refining of recycled paper pulp

Enzymatic modification of pulp is receiving increasing interest for energy reduction at the refining step of the paper-making process. In this study, the production of a multi-fiber modifying enzyme from Mamillisphaeria sp. BCC8893 was optimized in submerged fermentation using a response-surface methodology. Maximal production was obtained in a complex medium comprising wheat bran, soybean, and rice bran supplemented with yeast extract at pH 6.0 and a harvest time of 7 d, resulting in 9.2 IU/mL of carboxymethyl cellulase (CMCase), 14.9 IU/mL of filter paper activity (FPase), and 242.7 IU/mL of xylanase. Treatment of old corrugated container pulp at 0.2-0.3 IU of CMCase/g of pulp led to reductions in refining energy of 8.5-14.8%. The major physical properties were retained, including tensile and compression strength. Proteomic analysis showed that the enzyme was a complex composite of endo-glucanases, cellobiohydrolases, beta-1,4-xylanases, and beta-glucanases belonging to various glycosyl hydrolase families, suggestive of cooperative enzyme action in fiber modification, providing the basis for refining efficiency.     

An efficient purification and fractionation of genomic DNA from soil by modified troughing method

AIMS: The aim of this study was to utilize a modified troughing method for purification of large genomic DNA obtained from microbiota in natural environment and for fractionation of genomic DNA into many size ranges that facilitates construction of metagenomic library. METHODS AND RESULTS: Genomic DNA extracted from soil or termite gut was purified by the modified troughing method which utilized gel electrophoresis in the presence of 30% PEG8000. The method performed better than various purification kits and allowed no significant loss in the amount of DNA recovered. In addition, the efficiency of the modified troughing method for DNA size fractionation was investigated. DNA size fractionation was achieved with repetitive rounds of electrophoresis and DNA collection to obtain DNA with many size ranges. CONCLUSIONS: The modified troughing method is a simple and efficient method for purification of genomic DNA and for DNA size fractionation. SIGNIFICANCE AND IMPACT OF THE STUDY: The modified troughing method is a straightforward and inexpensive technique readily available for anyone working with environmental genomic DNA. It facilitates cloning of genomic DNA and enhances rapid discovery of useful bioactive compounds from microbial resources.     

Identification and expression of cellobiohydrolase (CBHI) gene from an endophytic fungus, Fusicoccum sp. (BCC4124) in Pichia pastoris

A gene encoding a cellobiohydrolase (CBHI) was isolated from Fusicoccum sp. (BCC4124), an endophytic fungus belongs in phylum Ascomycota, using 5' and 3' rapid amplification of cDNA end (RACE) technique. This CBHI gene contains 1395 nucleotides and encodes a 465-amino acid protein with a molecular weight of approximately 50 kDa. The deduced amino acid sequence showed significant similarity to those of other fungal CBHI belonging to family 7 of glycosyl hydrolase. Interestingly, the result from the amino acid alignment revealed that this CBHI does not contain the cellulose binding domain nor the linker region. The CBHI gene was successfully expressed in Pichia pastoris KM71. The purified recombinant CBHI has ability to hydrolyze Avicel, filter paper and 4-methylumbelliferyl beta-d-cellobioside (MUC) but not carboxymethylcellulose (CMC). It showed an optimal working condition at 40 degrees C, pH 5 with K(m) and V(max) toward MUC of 0.57 mM and 3.086 nmol/min/mg protein, respectively. The purified enzyme was stable at pH range of 3-11. The enzyme retained approximately 50% of its maximal activity after incubating at 70-90 degrees C for 30 min. Due to its stability through wide range of pH, and moderately stable at high temperature, this enzyme has potential in various biotechnology applications.     

Comparative metagenomic analysis of microcosm structures and lignocellulolytic enzyme systems of symbiotic biomass-degrading consortia

Decomposition of lignocelluloses by cooperative microbial actions is an essential process of carbon cycling in nature and provides a basis for biomass conversion to fuels and chemicals in biorefineries. In this study, structurally stable symbiotic aero-tolerant lignocellulose-degrading microbial consortia were obtained from biodiversified microflora present in industrial sugarcane bagasse pile (BGC-1), cow rumen fluid (CRC-1), and pulp mill activated sludge (ASC-1) by successive subcultivation on rice straw under facultative anoxic conditions. Tagged 16S rRNA gene pyrosequencing revealed that all isolated consortia originated from highly diverse environmental microflora shared similar composite phylum profiles comprising mainly Firmicutes, reflecting convergent adaptation of microcosm structures, however, with substantial differences at refined genus level. BGC-1 comprising cellulolytic Clostridium and Acetanaerobacterium in stable coexistence with ligninolytic Ureibacillus showed the highest capability on degradation of agricultural residues and industrial pulp waste with CMCase, xylanase, and β-glucanase activities in the supernatant. Shotgun pyrosequencing of the BGC-1 metagenome indicated a markedly high relative abundance of genes encoding for glycosyl hydrolases, particularly for lignocellulytic enzymes in 26 families. The enzyme system comprised a unique composition of main-chain degrading and side-chain processing hydrolases, dominated by GH2, 3, 5, 9, 10, and 43, reflecting adaptation of enzyme profiles to the specific substrate. Gene mapping showed metabolic potential of BGC-1 for conversion of biomass sugars to various fermentation products of industrial importance. The symbiotic consortium is a promising simplified model for study of multispecies mechanisms on consolidated bioprocessing and a platform for discovering efficient synergistic enzyme systems for biotechnological application.     

Insights into the Phylogeny and Metabolic Potential of a Primary Tropical Peat Swamp Forest Microbial Community by Metagenomic Analysis

A primary tropical peat swamp forest is a unique ecosystem characterized by long-term accumulation of plant biomass under high humidity and acidic water-logged conditions, and is regarded as an important terrestrial carbon sink in the biosphere. In this study, the microbial community in the surface peat layer in Pru Toh Daeng, a primary tropical peat swamp forest, was studied for its phylogenetic diversity and metabolic potential using direct shotgun pyrosequencing of environmental DNA, together with analysis of 16S rRNA gene library and key metabolic genes. The community was dominated by aerobic microbes together with a significant number of facultative and anaerobic microbial taxa. Acidobacteria and diverse Proteobacteria (mainly Alphaproteobacteria) constituted the major phylogenetic groups, with minor representation of archaea and eukaryotic microbes. Based on comparative pyrosequencing dataset analysis, the microbial community showed high metabolic versatility of plant polysaccharide decomposition. A variety of glycosyl hydrolases targeting lignocellulosic and starch-based polysaccharides from diverse bacterial phyla were annotated, originating mostly from Proteobacteria, and Acidobacteria together with Firmicutes, Bacteroidetes, Chlamydiae/Verrucomicrobia, and Actinobacteria, suggesting the key role of these microbes in plant biomass degradation. Pyrosequencing dataset annotation and direct mcrA gene analysis indicated the presence of methanogenic archaea clustering in the order Methanomicrobiales, suggesting the potential on partial carbon flux from biomass degradation through methanogenesis. The insights on the peat swamp microbial assemblage thus provide a valuable approach for further study on biogeochemical processes in this unique ecosystem.     

Hydrolysis/dehydration/aldol-condensation/hydrogenation of lignocellulosic biomass and biomass-derived carbohydrates in the presence of Pd/WO3-ZrO2 in a single reactor

Hydrolysis/dehydration/aldol-condensation/hydrogenation of lignocellulosic-biomass (corncobs) and biomass-derived carbohydrates (tapioca flour) to produce water-soluble C5-C15 compounds was developed in a single reactor system. WO3-ZrO2 efficiently catalyzed the hydrolysis/dehydration of these feedstocks to 5-hydroxymethylfurfural and furfural, while the impregnation of WO3-ZrO2 with Pd allowed sequential aldolcondensation/hydrogenation of these furans to C5-C15 compounds. The highest C5-C15 yields of 14.8-20.3% were observed at a hydrolysis/dehydration temperature of 573 K for 5 min, an aldol-condensation temperature of 353 K for 30 h, and a hydrogenation temperature of 393 K for 6 h. The C5-C15 yield from tapioca flour was higher than that from corncobs (20.3% compared to 14.8%). Tapioca flour produced more C6/C9/C15, whereas corncobs generated more C5/C8/C13 compounds due to the presence of hemicellulose in the corncobs. These water-soluble organic compounds can be further converted to liquid alkanes with high cetane numbers for replacing diesel fuel in transportation applications.     

Improvement of thermostability of fungal xylanase by using site-directed mutagenesis

Replacing several serine and threonine residues on the Ser/Thr surface of the xylanase from Aspergillus niger BCC14405 with four and five arginines effectively increases the thermostability of the enzyme. The modified enzymes showed 80% of maximal activity after incubating in xylan substrate for 2h at 50 degrees C compared to only 15% activity for wild-type enzyme. The half-life of the mutated enzymes increased to 257+/-16 and 285+/-10 min for the four- and five-arginine mutants, respectively, compared to 14+/-1 min for the wild-type enzyme. Thus, the arginine substitutions effectively increase stability by 18-20-fold. Kinetic parameters of the four-arginine-substitution enzyme were maintained at the level of the wild-type enzyme with the K(m) and V(max) values of 8.3+/-0.1 mgml(-1) and 9556+/-66 (n=3) U mg(-1) protein, respectively. The five-arginine-substitution enzyme showed only slight alteration in K(m) and V(max) with K(m) of 11.7+/-1.7 mgml(-1) and V(max) of 8502+/-65 Umg(-1) protein, indicating lower substrate affinity and catalytic rate. Our study demonstrated that properly introduced arginine residues on the Ser/Thr surface of xylanase family 11 might be very effective in improvement of enzyme thermostability.