Verawat Champreda

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.

Synergistic action of recombinant accessory hemicellulolytic and pectinolytic enzymes to Trichoderma reesei cellulase on rice straw degradation

Synergism between core cellulases and accessory hydrolytic/non-hydrolytic enzymes is the basis of efficient hydrolysis of lignocelluloses. In this study, the synergistic action of three recombinant accessory enzymes, namely GH62 α-l-arabinofuranosidase (ARA), CE8 pectin esterase (PET), and GH10 endo-1,4-beta-xylanase (XYL) from Aspergillus aculeatus expressed in Pichia pastoris to a commercial Trichoderma reesei cellulase (Accellerase® 1500; ACR) on hydrolysis of alkaline pretreated rice straw was studied using a mixture design approach. Applying the full cubic model, the optimal ratio of quaternary enzyme mixture was predicted to be ACR:ARA:PET:XYL of 0.171:0.079:0.100:0.150, which showed a glucose releasing efficiency of 0.173 gglc/FPU, higher than the binary ACR:XYL mixture (0.122 gglc/FPU) and ACR alone (0.081 gglc/FPU) leading to a 47.3% increase in glucose yield compared with that from ACR at the same cellulase dosage. The result demonstrates the varying degree of synergism of accessory enzymes to cellulases useful for developing tailor-made enzyme systems for bio-industry.

Isolation and Characterization of a Novel Thermostable Neopullulanase-Like Enzyme from a Hot Spring in Thailand

A gene encoding a thermostable pullulan-hydrolyzing enzyme was isolated from environmental genomic DNA extracted from soil sediments of Bor Khleung hot spring in Thailand. Sequence comparison with related enzymes suggested that the isolated enzyme, designated Env Npu193A, was most likely a neopullulanase-like enzyme. Env Npu193A was expressed in Pichia pastoris as a monomeric recombinant protein. The purified Env Npu193A exhibited pH stability ranging from 3 to 9. More than 60% of enzyme activity was retained after incubation at 60 °C for 1 h. Env Npu193A was found to hydrolyze various substrates, including pullulan, starch, and γ-cyclodextrin. The optimal working condition for Env Npu193A was at pH 7 at 75 °C with K m and V max toward pullulan of 1.22±0.3% and 23.24±1.7 U/mg respectively. Env Npu193A exhibited distinct biochemical characteristics as compared with the previously isolated enzyme from the same source. Thus, a culture-independent approach with sequence-basing was found to be an effective way to discover novel enzymes displaying unique substrate specificity and high thermostability from natural bioresources.

Structure-based protein engineering for thermostable and alkaliphilic enhancement of endo-β-1,4-xylanase for applications in pulp bleaching

In the pulp bleaching industry, enzymes with robust activity at high pH and temperatures are desirable for facilitating the pre-bleaching process with simplified processing and minimal use of chlorinated compounds. To engineer an enzyme for this purpose, we determined the crystal structure of the Xyn12.2 xylanase, a xylan-hydrolyzing enzyme derived from the termite gut symbiont metagenome, as the basis for structure-based protein engineering to improve Xyn12.2 stability in high heat and alkaline conditions. Engineered cysteine pairs that generated exterior disulfide bonds increased the kcat of Xyn12.2 variants and melting temperature at all tested conditions. These improvements led to up to 4.2-fold increases in catalytic efficiency at pH 9.0, 50°C for 1h and up to 3-fold increases at 60°C. The most effective variants, XynTT and XynTTTE, exhibited 2-3-fold increases in bagasse hydrolysis at pH 9.0 and 60°C compared to the wild-type enzyme. Overall, engineering arginines and phenylalanines for increased pKa and hydrogen bonding improved enzyme catalytic efficiency at high stringency conditions. These modifications were the keys to enhancing thermostability and alkaliphilicity in our enzyme variants, with XynTT and XynTTTE being especially promising for their application to the pulp and paper industry.

Optimization of Organosolv Based Fractionation Process for Separation of High Purity Lignin from Bagasse

Lignocellulosic biomass is a renewable source for sustainable production of fuels, chemicals, and other materials with the advantages on its carbon-neutral nature. Fractionation of lignocellulosic materials is a pre-requisite in the biorefinery process in order to convert the cellulose, hemicellulose, and lignin to valuable products with maximized economics prospective. In this work, a modified clean fractionation (CF) process using ternary mixture system of ethyl acetate/methanol/water was studied with the use of acid promoters. H2SO4 was found to be the efficient promoter due to low cost compare to other acid promoters. The optimal fractionation conditions operated in the solvent mixture containing 0.025 M H2SO4 at 160°C for 50 min led to 63.72% recovery of the cellulose in the solid pulp while 90% and 59.94% of hemicellulose-derived products and lignin were recovered in the aqueous-alcohol and organic fractions, respectively. The enzymatic digestibility of the cellulose-enriched pulp was increased, resulting increasing glucose yield from 38.32% of the native biomass to 70.04% using the hydrolysis reaction with Cellic Ctech2® at 15 FPU/g. The work demonstrated the applicability of the modified CF process for fractionation of lignocellulose components for integrated biorefinery process.

Simultaneous saccharification and viscosity reduction of cassava pulp using a multi-component starch- and cell-wall degrading enzyme for bioethanol production

In this study, an efficient ethanol production process using simultaneous saccharification and viscosity reduction of raw cassava pulp with no prior high temperature pre-gelatinization/liquefaction step was developed using a crude starch- and cell wall-degrading enzyme preparation from Aspergillus aculeatus BCC17849. Proteomic analysis revealed that the enzyme comprised a complex mixture of endo- and exo-acting amylases, cellulases, xylanases, and pectina ses belonging to various glycosyl hydrolase families. Enzymatic hydrolysis efficiency was dependent on the initial solid loading in the reaction. Reduction in mixture viscosity was observed with a rapid decrease in complex viscosity from 3785 to 0.45xa0Paxa0s with the enzyme dosage of 2.19xa0mg/g on a dried weight basis within the first 2xa0h, which resulted from partial destruction of the plant cell wall fiber and degradation of the released starch granules by the enzymes as shown by scanning electron microscopy. Saccharification of cassava pulp at an initial solid of 16% (w/v) in a bench-scale bioreactor resulted in 736.4xa0mg glucose/g, which is equivalent to 82.92% glucose yield based on the total starch and glucan in the substrate, after 96xa0h at 40xa0°C. Simultaneous saccharification and fermentation of cassava pulp by Saccharomyces cerevisiae with the uncooked enzymatic process led to a final ethanol concentration of 6.98% w/v, equivalent to 96.7% theoretical yield based on the total starch and cellulose content. The results demonstrated potential of the enzyme for low-energy processing of cassava pulp in biofuel industry.

Effects of acid and alkali promoters on organosolv fractionation of sugarcane bagasse using ternary solvent system

Lignocellulosic plant biomass is a renewable starting material for sustainable production of fuels, chemicals, and materials with advantages on its carbon-neutral nature. Fractionation of lignocellulosic components is a pre-requisite in an integrated biorefinery process in order to convert the cellulose, hemicellulose, and lignin to respective products with maximized economics. In this study, a modified clean fractionation (CF) process using the mixture of ethyl acetate/ethanol/water was studied with the use of acid and alkali promoters at varying temperatures. H2SO4 was found to be the most efficient promoter compared to other acids/alkali the same molarity on yield and homogeneity of the fractionated products. The optimal fractionation conditions operated in the solvent mixture containing 0.025 M H2SO4 at 160°C for 60 min led to 88.79% recovery of the cellulose in the solid pulp while 86.2% and 68.3% of hemicellulose-derived products and lignin were recovered in the aqueous-alcohol and organic fractions, respectively. However, the use of triethylamine (TEA) led to the higher biomass digestibility and glucose recovery of 74.76 and 65.27, respectively at 180°C which higher than those achieved using H2SO4. The work demonstrated the applicability of the modified CF process for fractionation of lignocellulose components for integrated biorefinery process.

Separation of Methanol/Water Mixtures from Dilute Aqueous Solutions Using Pervaporation Technique

The composite polydimethylsiloxane PERVAPTM 4060 was used for separation of methanol/water solutions by using pervaporation technique. The effect of feed concentration, feed temperature, and feed flow rate were investigated for the separation performance. The experimental data showed that increasing of the feed methanol concentration from 0.5 to 10 wt% resulted in an increase in total permeation flux up for to 35 % whilst the separation factor (α) decreased by 85%. The results also showed that increasing operating temperature from 40 to 60 °C caused an increases in methanol permeance up to 130%.

Kinetic study of protein formation and digestion by quartz crystal microbalance

Quartz crystal microbalance technique was used to study kinetic of formation and digestion of bovine serum albumin (BSA) protein on polystyrene-co-maleic acids (PSMA) surface. The kinetic of formation and digestion of BSA was investigated by measuring the frequency shift and resistance shift as a function of time. In order to study pH and concentration effect to layer formation kinetic, the pH of solution was varied from 2.0 to 7.4 while the concentration of BSA was varied in the range of 0.001 to 10 mg/ml. The kinetic of formation appears to be sensitive to the pH of solution and concentration. The formation layer at about pH 3.0 to 3.5 gives the different characteristic from the others. The layer formation increases as the concentration increase then reached the saturate value at the concentration of over 3 mg/ml. The kinetic of digestion was evaluate by applying proteinase enzyme on varies densities of BSA layer. It found that the rate of digestion depends on the density of the molecular BSA packing, modified by varying pH and concentration of BSA.

Effect of pH on the Formation of a Bovine Serum Albumin Layer on a Poly(stryren-co-maleic Acid) Surface

The layer formation of bovine serum albumin (BSA) on a poly(styrene-co-maleic acid) (PSMA) surface was investigated by using quartz crystal microbalance (QCM) technique at various pH values. The formation of a BSA surface was examined by atomic force microscopy (AFM). To study the effect on the layer formation, the pH of solution was varied from 2 to 7.4 while the concentration of BSA was in the range of 0.01 to 5 mg/ml during the layer absorption. It was found that the BSA adsorption strongly depends on the pH of solution, and the concentration of BSA. The absorption layer occurred maximum at the pH value of 3.5 which resulted from the charge of PSMA and BSA molecules. The layer formation reached the saturate value at the concentration higher than 3 mg/ml. The molecular packing of the BSA layer at different pH values was determined by AFM and total mass change of QCM.