Sutipa Tanapongpipat

Simultaneous non-thermal saccharification of cassava pulp by multi-enzyme activity and ethanol fermentation by Candida tropicalis

Cassava pulp, a solid by-product from starch processing, is a promising and underused biomass that can be converted to biofuels and other value-added bio-products. In this study, an alternative cassava pulp saccharification process, which utilizes the multi-activity enzyme from Aspergillus niger BCC17849 and obviates the need for a pre-gelatinization step, was developed. The crude multi-enzyme composed of non-starch polysaccharide hydrolyzing enzyme activities, including cellulase, pectinase and hemicellulase act cooperatively to release the trapped starch granules from the fibrous cell wall structure for subsequent saccharification by raw starch degrading activity. A high yield of fermentable sugars, equivalent to 716 mg glucose and 67 mg xylose/g of cassava pulp, was obtained after 48 h incubation at 40 degrees C and pH 5 using the multi-enzyme, which was greater than the yield obtained from the optimized combinations of the corresponding commercial enzymes. The multi-enzyme saccharification reaction can be performed simultaneously with the ethanol fermentation process using a thermotolerant yeast Candida tropicalis BCC7755. The combined process produced 14.3 g/l ethanol from 4% (w/v) cassava pulp after 30 h of fermentation. The productivity rate of 0.48 g/l/h is equivalent to 93.7% of the theoretical yield based on total starch and cellulose, or 85.4% based on total fermentable sugars. The non-thermal enzymatic saccharification process described is more energy efficient and yields more fermentable sugar than the conventional enzymatic process. Furthermore, the process is applicable for production of various bio-products of economic importance.

Bioethanol production from ball milled bagasse using an on-site produced fungal enzyme cocktail and xylose-fermenting Pichia stipitis.

Sugarcane bagasse is one of the most promising agricultural by-products for conversion to biofuels. Here, ethanol fermentation from bagasse has been achieved using an integrated process combining mechanical pretreatment by ball milling, with enzymatic hydrolysis and fermentation. Ball milling for 2 h was sufficient for nearly complete cellulose structural transformation to an accessible amorphous form. The pretreated cellulosic residues were hydrolyzed by a crude enzyme preparation from Penicillium chrysogenum BCC4504 containing cellulase activity combined with Aspergillus flavus BCC7179 preparation containing complementary beta-glucosidase activity. Saccharification yields of 84.0% and 70.4% for glucose and xylose, respectively, were obtained after hydrolysis at 45 degrees C, pH 5 for 72 h, which were slightly higher than those obtained with a commercial enzyme mixture containing Acremonium cellulase and Optimash BG. A high conversion yield of undetoxified pretreated bagasse (5%, w/v) hydrolysate to ethanol was attained by separate hydrolysis and fermentation processes using Pichia stipitis BCC15191, at pH 5.5, 30 degrees C for 24 h resulting in an ethanol concentration of 8.4 g/l, corresponding to a conversion yield of 0.29 g ethanol/g available fermentable sugars. Comparable ethanol conversion efficiency was obtained by a simultaneous saccharification and fermentation process which led to production of 8.0 g/l ethanol after 72 h fermentation under the same conditions. This study thus demonstrated the potential use of a simple integrated process with minimal environmental impact with the use of promising alternative on-site enzymes and yeast for the production of ethanol from this potent lignocellulosic biomass.

Microbial degradation and physico-chemical alteration of polyhydroxyalkanoates by a thermophilic Streptomyces 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.

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

Aims:u2002 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.

Purification, Biochemical Characterization, and Gene Cloning of a New Extracellular Thermotolerant and Glucose Tolerant Maltooligosaccharide-Forming α-Amylase from an Endophytic Ascomycete Fusicoccum sp. BCC4124

An endophytic fungus, Fusicoccum sp. BCC4124, showed strong amylolytic activity when cultivated on multi-enzyme induction enriched medium and agro-industry substrates. α-Amylase and α-glucosidase activities were highly induced in the presence of maltose and starch. The purified target α-amylase, Amy-FC1, showed strong hydrolytic activity on soluble starch (kcat/Km=6.47×103 min−1(ml/mg)) and selective activity on γ- and β-cyclodextrins, but not on α-cyclodextrin. The enzyme worked optimally at 70 °C in a neutral pH range with t1/2 of 240 min in the presence of Ca2+ and starch. Maltose, matotriose, and maltotetraose were the major products from starch hydrolysis but prolonged reaction led to the production of glucose, maltose, and maltotriose from starch, cyclodextrins, and maltooligosaccharides (G3–G7). The amylase showed remarkable glucose tolerance up to 1 M, but was more sensitive to inhibition by maltose. The deduced protein primary structure from the putative gene revealed that the enzyme shared moderate homology between α-amylases from Aspergilli and Lipomyces sp. This thermotolerant, glucose tolerant maltooligosaccharide-forming α-amylase is potent for biotechnological application.

Cell‐surface phytase on Pichia pastoris cell wall offers great potential as a feed supplement

Cell-surface expression of phytase allows the enzyme to be expressed and anchored on the cell surface of Pichia pastoris. This avoids tedious downstream processes such as purification and separation involved with extracellular expression. In addition, yeast cells with anchored proteins can be used as a whole-cell biocatalyst with high value added. In this work, the phytase was expressed on the cell surface of P. pastoris with a glycosylphosphatidylinositol anchoring system. The recombinant phytase was shown to be located at the cell surface. The cell-surface phytase exhibited high activity with an optimal temperature at 50-55 degrees C and two optimal pH peaks of 3 and 5.5. The surface-displayed phytase also exhibited similar pH stability and pepsin resistance to the native and secreted phytase. In vitro digestibility test showed that P. pastoris containing cell-surface phytase released phosphorus from feedstuff at a level similar to secreted phytase. Yeast cells expressing phytase also provide additional nutrients, especially biotin and niacin. Thus, P. pastoris with phytase displayed on its surface has a great potential as a whole-cell supplement to animal feed.

Selective Fluorescent Detection of Aspartic Acid and Glutamic Acid Employing Dansyl Hydrazine Dextran Conjugate

AbstractHighly water soluble polymer (DD) was prepared and evaluated for its fluorescence response towards various amino acids. The polymer consists of dansyl hydrazine unit conjugated into dextran template. The conjugation enhances higher water solubility of dansyl hydrazine moiety. Of screened amino acids, DD exhibited selective fluorescence quenching in the presence of aspartic acid (Asp) and glutamic acid (Glu). A plot of fluorescence intensity change of DD against the concentration of corresponding amino acids gave a good linear relationship in the range of 1u2009×u200910−4xa0M to 25u2009×u200910−3xa0M. This establishes DD as a potential polymeric sensor for selective sensing of Asp and Glu.n Figure 1Fluorescence quenching of dansyl hydrazine dextran conjugate (DD) in the presence of L-aspartic or L-glutamic acids