Lily Eurwilaichitr

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.

Identification and Characterization of a Cellulase-Encoding Gene from the Buffalo Rumen Metagenomic Library

Microorganisms residing in the rumens of cattle represent a rich source of lignocellulose-degrading enzymes, since their diet consists of plant-based materials that are high in cellulose and hemicellulose. In this study, a metagenomic library was constructed from buffalo rumen contents using pCC1FOS fosmid vector. Ninety-three clones from the pooled library of approximately 10,000 clones showed degrading activity against AZCL-HE-Cellulose, whereas four other clones showed activity against AZCL-Xylan. Contig analysis of pyrosequencing data derived from the selected strongly positive clones revealed 15 ORFs that were closely related to lignocellulose–degrading enzymes belonging to several glycosyl hydrolase families. Glycosyl hydrolase family 5 (GHF5) was the most abundant glycosyl hydrolase found, and a majority of the GHF5s in our metagenomes were closely related to several ruminal bacteria, especially ones from other buffalo rumen metagenomes. Characterization of BT-01, a selected clone with highest cellulase activity from the primary plate screening assay, revealed a cellulase encoding gene with optimal working conditions at pH 5.5 at 50 °C. Along with its stability over acidic pH, the capability efficiently to hydrolyze cellulose in feed for broiler chickens, as exhibited in an in vitro digestibility test, suggests that BT-01 has potential application as a feed supplement.

Identification and Characterization of Lipolytic Enzymes from a Peat-Swamp Forest Soil Metagenome

In this work, a metagenomic library was generated from peat-swamp forest soil obtained from Narathiwat Province, Thailand. From a fosmid library of approximately 15,000 clones, six independent clones were found to possess lipolytic activity at acidic pH. Analysis of pyrosequencing data revealed six ORFs, which exhibited 34–71% protein similarity to known lipases/esterases. A fosmid clone, designated LP8, which demonstrated the highest level of lipolytic activity under acidic conditions and demonstrated extracellular activity, was subsequently subcloned and sequenced. The full-length lipase/esterase gene, estPS2, was identified. Its deduced amino acid was closely related to a lipolytic enzyme of an uncultured bacterium, and contained the highly conserved motif of a hormone-sensitive family IV lipase. The EstPS2 enzyme exhibited highest activity toward p-nitrophenyl butyrate (C4) at 37 °C at pH 5, indicating that it was an esterase with activity and secretion characteristics suitable for commercial development.

Biochemical characterization and in vitro digestibility assay of Eupenicillium parvum (BCC17694) phytase expressed in Pichia pastoris

A mature phytase cDNA, encoding 441 amino acids, from Eupenicillium parvum (BCC17694) was cloned into a Pichia pastoris expression vector, pPICZ alpha A, and was successfully expressed as active extracellular glycosylated protein. The recombinant phytase contained the active site RHGXRXP and HD sequence motifs, a large alpha/beta domain and a small alpha-domain that are typical of histidine acid phosphatase. Glycosylation was found to be important for enzyme activity which is most active at 50 degrees C and pH 5.5. The recombinant phytase displayed broad substrate specificity toward p-nitrophenyl phosphate, sodium-, calcium-, and potassium-phytate. The enzyme lost its activity after incubating at 50 degrees C for 5 min and is 50% inhibited by 5mM Cu(2+). However, the enzyme exhibits broad pH stability from 2.5 to 8.0 and is resistant to pepsin. In vitro digestibility test suggested that BCC17694 phytase is at least as effective as another recombinant phytase (r-A170) which is comparable to Natuphos, a commercial phytase, in releasing phosphate from corn-based animal feed, suggesting that BCC17694 phytase is suitable for use as phytase supplement in the animal diet.

Culture-independent phylogenetic analysis of the microbial community in industrial sugarcane bagasse feedstock piles.

Sugarcane bagasse is an important lignocellulosic by-product with potential for conversion to biofuels and chemicals in biorefinery. As a step towards an understanding of microbial diversity and the processes existing in bagasse collection sites, the microbial community in industrial bagasse feedstock piles was investigated. Molecular biodiversity analysis of 16S rDNA sequences revealed the presence of a complex bacterial community. A diverse group of mainly aerobic and facultative anaerobic bacteria was identified reflecting the aerobic and high temperature microenvironmental conditions under the pile surface. The major bacterial taxa present were identified as Firmicutes, Alpha- and Gammaproteobacteria, Acidobacteria, Bacteroidetes, and Actinobacteria. Analysis of the eukaryotic microbial assemblage based on an internal transcribed spacer revealed the predominance of diverse cellulolytic and hemicellulolytic ascomycota. A microbial interaction model is proposed, focusing on lignocellulose degradation and methane metabolism. The insights into the microbial community in this study provide a basis for efficient utilization of bagasse in lignocellulosic biomass-based industries.

Expression of Biologically Active Crustacean Hyperglycemic Hormone (CHH) of Penaeus monodon in Pichia pastoris

Crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) are members of a major peptide family produced from the X-organ sinus gland complex in the eyestalk of crustaceans. This peptide family plays important roles in controlling several physiologic processes such as regulation of growth and reproduction. In this study the complementary DNA encoding a peptide related to the CHH/MIH/GIH family (so-called Pem-CMG) of the black tiger prawn Penaeus monodon was successfully expressed in the yeast Pichia pastoris under the control of the AOX1 promoter. The recombinant Pem-CMG was secreted into the culture medium using the α-factor signal sequence; of Saccharomyces cerevisiae without the Glu-Ala-Glu-Ala spacer peptide. The amino terminus of the recombinant Pem-CMG was correctly processed as evidenced by amino-terminal peptide sequencing. The recombinant Pem-CMG was purified by reverse-phase high-performance liquid chromotography and used in a biological assay for CHH activity. The final yield of the recombinant Pem-CMG after purification was 260 µg/L of the culture medium. Both crude and purified recombinant Pem-CMG produced from P. pastoris showed the ability to elevate the glucose level in the hemolymph of eyestalk-ablated P. monodon, which demonstrates that Pem-CMG peptide functions as hyperglycemic hormone in P. monodon.

Binding characteristics and synergistic effects of bacterial expansins on cellulosic and hemicellulosic substrates

Expansins are non-catalytic proteins which loosen plant cell wall structure. In this study, binding kinetics and synergistic action of five bacterial expansins on cellulosic and hemicellulosic polysaccharides were studied. The expansins differed in binding capacity (Bmax) and affinity (Kd) for different substrates. A common pattern of binding efficiency (Bmax/Kd) was found among the expansins tested, in which efficiency was greatest for the phosphoric acid-swollen cellulose (PASC), then the hemicellulose arabinoxylan followed by the microcrystalline cellulose (Avicel PH101). The expansins enhanced the action of Trichoderma reesei cellulase/hemicellulase mixture for degrading all three substrates to varying degrees. Among the substrates and expansins tested, BpEX from Bacillus pumilus and CmEX from Clavibacter michiganensis showed the greatest enhancement effect on arabinoxylan with 11.4 and 12.2-fold greater reducing sugar yield than the reaction with enzyme alone. The work gives insights into the wider application of expansins on enhancing polysaccharide hydrolysis, particularly on hemicellulosic substrates.

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 beaching of chemical pulp in an environmentally friendly pulping process.

Identification of novel bacterial expansins and their synergistic actions on cellulose degradation.

Novel expansins, non-catalytic proteins which induce weakening of the rigid cellulose structure, have been identified in this study. A pipeline of bioinformatics was implemented for sequence and structure-based prediction of putative bacterial expansin-like group × family from NR databases. All putative expansins had no detectable activity against cellulosic and hemicellulosic substrates but showed varying degrees of synergy (2.0-7.6 folds) with the commercial Trichoderma reesei cellulase (Celluclast™ 1.5L) on degradation of filter paper in order of BpEX ≈ CmEX > MaEX > PcEX > SaEX. A mixture design with full cubic model predicted optimal formulation comprising Celluclast™: CmEX from Clavibacter michiganensis = 72.4%: 27.6%, with no synergy of β-glucosidase on degradation of alkaline pretreated rice straw. Under these conditions, the reducing sugar yield was 163.6% compared with the reaction containing cellulase alone. This work demonstrated the potential benefit of novel bacterial expansins on enhancing cellulose degradation efficiency in lignocellulosic biomass 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.