K. Chandraraj

A maltooligosaccharide-forming α-amylase was produced by a new soil isolate Bacillus subtilis KCC103. In contrast to other Bacillus species, the synthesis of α-amylase in KCC103 was not catabolite-repressed. The α-amylase was purified in one step using anion exchange chromatography after concentration of crude enzyme by acetone precipitation. The purified α-amylase had a molecular mass of 53 kDa. It was highly active over a broad pH range from 5 to 7 and stable in a wide pH range between 4 and 9. Though optimum temperature was 65-70°C, it was rapidly deactivated at 70°C with a half-life of 7 min and at 50°C, the half-life was 94 min. The K m and Vmax for starch hydrolysis were 2.6 mg ml -1 and 909 U mg-1, respectively. Ca2+ did not enhance the activity and stability of the enzyme; however, EDTA (50 mM) abolished 50% of the activity. Hg2+, Ag2+, and p-hydroxymercurybenzoate severely inhibited the activity indicating the role of sulfydryl group in catalysis. The α-amylase displayed endolytic activity and formed maltooligosaccharides on hydrolysis of soluble starch at pH 4 and 7. Small maltooligosaccharides (D2-D4) were formed more predominantly than larger maltooligosaccharides (D5-D7). This maltooligosaccharide forming endo-α-amylase is useful in bread making as an antistaling agent and it can be produced economically using low-cost sugarcane bagasse. © Springer-Verlag 2006.

Rice straw is a promising feedstock for sustained production of ethanol and value-added products. In this study, production of ethanol with xylitol from rice straw was improved by employing moderate aqueous ammonia pretreatment and sequential fermentation. The pretreatment removed significant amount of lignin and enhanced the enzymatic digestibility of rice straw by four fold. The analysis of the pretreated rice straw by SEM, XRD and FTIR showed significant changes in physicochemical structure, favoring enzymatic hydrolysis. The fermentability of the sugar hydrolyzate to ethanol was evaluated in repeated batch fermentation with cell recycling. Ethanol yield of 98% was achieved up to three batches of recycling of cells of Candida tropicalis. Fermentation of glucose and xylose in the hydrolyzate to ethanol and xylitol was studied using C. tropicalis in single and two-stage sequential fermentations. While ethanol yield was same, xylitol yield was higher in the two-stage fermentation than single batch fermentation. The results suggest that the sequential fermentation of sugar hydrolyzate of ammonia pretreated rice straw would be a promising process for production of ethanol and xylitol.

A sequential electrochemical oxidation (EO) and algal bubble column photobioreactor (BPBR) system was proposed to treat distillery wastewater (DWW). EO was carried out in a 2 L reactor with Ti-RuO2 anodes. Electrochemically oxidised DWW (EO-DWW) was then supplied to the microalgae Asterarcys quadricellulare. The EO operating current, treatment time and post-treatment dilution were optimised with a central composite design (CCD) with algal specific growth rate, lipid accumulation and photosynthetic quantum yield (Fv/Fm) as dependent variables. The optimal treatment conditions for the growth of A. quadricellulare were 27 A for 26 h and a post-treatment dilution of 8. Under optimal conditions, A. quadricellulare grew at a specific growth rate of 1.06 d−1 with a lipid accumulation of 12.7% and an Fv/Fm of 0.7. The optimal conditions were validated, and a 1.6 L bubble column photobioreactor was designed to treat the EO-DWW sequentially. The sequential EO-BPBR system removed 92% COD, 76% TOC and 82% TN from DWW. The algal biomass productivity was 0.96 g/L/d with a carbon sequestration of 550–700 mg/L/d and an aqueous carbon capture of 240–280 mg C/L/d. Additionally, the flue gas evolved from the EO reactor was analysed and contained 68% H2, 18% O2 and 12.5% CO2. The H2 in the flue gas can compensate for 26.5% of the energy spent for the EO process. The algal biomass produced in the sequential process can compensate for 6% of the total energy consumed for EO. Therefore, 32% of the energy spent on EO can be reclaimed by sequential EO-BPBR treatment.

Functional oligosaccharides (FOs) are indispensable components in food, feed, pharmaceuticals, cosmetics, and nutraceuticals preparations. They are polysaccharides containing the degree of polymerization of 2-10, not digested (or only partially digested) by gut enzymes, having no or less caloric values, stable at acidic pH, thermostable (>50 °C), and intact in bile salts. Consequently, FOs are neither digested nor degraded in the upper alimentary canal and reach the large intestine of the host. FOs confer several health benefits, such as proliferation of probiotic microbes, elimination of pathogen colonization, enhancement of mineral absorption, evacuation of heavy metal ions by increasing bowel movements, and selective proliferation of specific probiotic microbes in the gut. FOs can be categorized as sucrose based (fructo-oligosaccharides), lactose based (galacto-oligosaccharides and lacto-sucrose), starch related (isomalto/malto-oligosaccharides, trehalose, and cyclodextrins), soy-based oligosaccharides, and non-starch based (xylo/arabino-oligosaccharides, and pectin- and chitosan-based oligosaccharides). FOs are produced by chemical, enzymatic, and chemoenzymatic techniques, but the enzymatic method is preferred because it produces specific FOs under mild, environmentally friendly conditions. FOs are purified and characterized using membrane filtration, chromatography, and spectroscopy. The prebiotic function of FOs in the host's health has been studied using animal models and a simulated human intestinal microbial ecosystem.

Sorghum biomass is a potential feedstock for lignocellulosic bioethanol production. The selection of suitable sorghum variety is essential to obtain high ethanol yield. In this paper we screened sorghum varieties belonging to sweet sorghum, post rainy sorghum, and hybrid sorghum. These varieties were screened based on their agronomic traits, amenability to pretreatment methods, and enzymatic digestibility. The sorghum biomass was pretreated using glycerol (60 %) at 190 ̊C for 60 min and aqueous ammonia (15 %) at 120 ̊C for 60 min. The digestibility of the pretreated biomass was determined using commercial cellulase (Cellic CTec2) at 10U/g loading, and the structural changes in the pretreated biomass were analyzed by spectroscopy and scanning electron microscopy. Sweet sorghum varieties showed significant variations in phenotypic traits such as fresh stalk yield, dry fodder yield, and juice yield. The cellulose digestibility among the sorghum varieties after the pretreatment also differed significantly. The cellulose digestibility levels of glycerol range from 64 % to 89 % and ammonia pretreated sorghum from 63 % to 81 %. The total sugar yields varied from 227 mg/g to 356 mg/g and 209 mg/g to 313 mg/g for sorghum pretreated with ammonia and glycerol, respectively. Although the delignification of sorghum varieties was higher (31%–65%) after ammonia pretreatment than glycerol pretreatment, the cellulose digestibility was higher for the glycerol pretreated biomass. These results indicated that effect of delignification on cellulose digestibility is trivial. This study explores factors affecting pretreatment and cellulose digestibility of sorghum varieties for maximum sugar yield in the cellulosic ethanol process.

Keratinase, a protease was produced from keratinolytic bacterium, Bacillus paralicheniformis MKU3, using poultry feather as substrate. Statistical analyses were carried out to enhance the activity of keratinase produced as a result of which enzyme activity was improved 6-fold yielding a maximum of 1872.5 U/ml and a specific activity of 1143.19 U/mg in 72 h. The keratinase produced was used to hydrolyze poultry feather waste which resulted in an almost 90% degradation by measurement of dry weight. Large scale production of keratinase was performed from 5-L to 500-L and it has been observed that an increase in the volume of bioreactor greatly improved the rate of production of keratinase along with a sharp decrease in production time. The keratinase produced was used to study dehairing on sheepskin and the efficiency was observed to be 100%. SEM and histochemical analysis on the skin confirmed efficient dehairing. The processed leather crust was further tested for physical strength and histochemistry wherein it has been proved that the quality of the leather crust produced is good. The environmental impact of the effluent produced as a result of dehairing was also assessed.

Xylooligosaccharides (XOS) are used as prebiotics in food industries. XOS with high purity is required for effective prebiotic function. All crude xylanases exhibit β-xylosidase, which forms significant amount of xylose and reduces the yield and purity of XOS. Here, we describe the use of crude xylanase of Bacillus subtilis devoid of β-xylosidase to produce high-pure XOS from ammonia pretreated sugarcane bagasse. MALDI-TOF-MS and HPLC analysis of XOS showed the formation of xylobiose, xylotriose and xylotetraose with xylobiose as the major product. The conversion of bagasse to XOS was >99% with negligible amount of xylose (0.4%). NMR analysis of XOS mixture indicated the presence of arabinosyl and glucuronyl substituted XOS. The relative content of substituted XOS in the mixture was 32%. The XOS mixture supported growth of probiotic bifidobacterial strains under anaerobic conditions. The bifidobacterial strains completely utilized XOS with production of short chain fatty acids indicating their prebiotic function. The concentration of fatty acids formed was in the order of acetate, formate and lactate. Hence, the present method would be useful for further development of low-cost process for production of high-pure prebiotic XOS from lignocellulosic materials.

Sugarcane (Saccharum officinarum) bagasse (SCB) is a promising feedstock for cellulosic ethanol production. Combination of low energy pretreatments with efficient enzymatic saccharification is desirable to reduce process cost. SCB was converted into fermentable sugars with high yield by aqueous ammonia soaking (AAS) pretreatment and laccase-mediator assisted enzymatic hydrolysis. The optimal process conditions for AAS were determined as 20% aqueous ammonia, 50 °C, 48 h residence time and 1.2 mm particle size by employing response surface method. Hydrolysis of pretreated SCB by cellulases resulted in release of 57.30% of total sugars in the raw SCB. Addition of laccase to cellulase enzyme mixture enhanced the total sugar yield to 68.2%. When the mediator 1-hydroxybenzotriazole was added to the cellulase-laccase cocktail, the total sugar yield was improved to 78.4%. The mediator also reduced the amount of laccase required for maximum sugar yield. The glucan conversion of pretreated SCB with cellulase-laccase-mediator cocktail was 86%, which is higher than the reported levels of glucan conversion for SCB pretreated by AAS at high temperature. Therefore, the combination of low-temperature AAS and laccase-mediator assisted enzymatic hydrolysis would be useful to develop low-energy biomass conversion process.

Bioethanol is a potential alternative fuel to fossil fuels. Bioethanol as a fuel has several economic and environmental benefits. Though bioethanol is produced using starch and sugarcane juice, these materials are in conflict with food availability. To avoid food–fuel conflict, the second-generation bioethanol production by utilizing nonfood lignocellulosic materials has been extensively investigated. However, due to the complexity of lignocellulose architecture, the process is complicated and not economically competitive. The cultivation of lignocellulosic energy crops indirectly affects the food supplies by extensive land use. Marine algae have attracted attention to replace the lignocellulosic feedstock for bioethanol production, since the algae grow fast, do not use land, avoid food–fuel conflict and have several varieties to suit the cultivation environment. The composition of algae is not as complex as lignocellulose due to the absence of lignin, which renders easy hydrolysis of polysaccharides to fermentable sugars. Marine organisms also produce cold-active enzymes for hydrolysis of starch, cellulose, and algal polysaccharides, which can be employed in bioethanol process. Marine microoorganisms are also capable of fermenting sugars under high salt environment. Therefore, marine biocatalysts are promising for development of efficient processes for bioethanol production.