K. Chandraraj

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.

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.

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.

Xylanase and xylooligosaccharides (XOS) are employed in food and feed industries. Though xylanase production from lignocellulosic materials (LCMs) by solid-state fermentation (SSF) is well known, the XOS formed during growth is not recovered due to its conversion to xylose by β-xylosidase and subsequent bacterial metabolism. A new strain, Bacillus subtilis KCX006, was exceptionally found to synthesize β-xylosidase-free endo-xylanase and multiple xylan debranching enzymes constitutively in the presence of LCMs. Absence of β-xylosidase resulted in accumulation of XOS during growth of KCX006 on LCMs. Therefore, this strain was used for simultaneous production of xylanase and XOS from agro-residues in solid-state fermentation (SSF). Partial purification of XOS from culture supernatant using activated charcoal followed by high-performance liquid chromatography (HPLC) analysis showed xylobiose to xylotetraose formed as the major products. Among various LCM substrates, wheat bran and groundnut oil-cake supported highest xylanase and XOS production at 2158 IU/gdw and 24.92 mg/gdw, respectively. The levels of xylanase and XOS were improved by 1.5-fold (3102 IU/gdw) and 1.9-fold (48 mg/gdw), respectively, by optimization of culture conditions.

In Bacillus subtilis KCC103, α-amylase is hyper-produced and α-amylase synthesis is not subject to catabolite repression. The α-amylase was produced from KCC103 by solid-state fermentation (SSF) using agro-residues and oil cakes as growth substrates. The KCC103 was also tested for its resistance to repression by hyper level (>10% w/w) of glucose and xylose on α-amylase production in SSF. Among growth media containing various combinations of agro-residues, the medium with wheat bran and sunflower oil cake supported highest enzyme production (20700 IU (g dry wt)-1). The α-amylase production was enhanced (4.2 folds) by optimizing the growth substrate and the process parameters: the optimal conditions were wheat bran:sun flower oil cake ratio-1:1 (w/w), substrate particle size-500 μm, substrate to flask volume-1:100 (w/v), initial substrate moisture content-90% (v/w), inoculum size-35%, initial medium pH-7.0, growth temperature-37 °C and cultivation time-48 h. α-Amylase production was further enhanced up to 1.7 folds when SSF was carried out using optimized medium supplemented with sugars or yeast extract (1% w/v) under optimized conditions. Supplementation of biomass sugars, glucose or xylose at 20% (w/w), did not repress the synthesis of α-amylase showing the hyper-tolerance of KCC103 to repression by simple sugars on α-amylase production in SSF. © 2010 WILEY-VCH Verlag GmbH &Co. KGaA, Weinheim.

High solids loading hydrolysis (HSLEH) of sugarcane bagasse (SCB) pretreated by low temperature aqueous ammonia soaking (AAS) was performed to obtain high concentrations of glucose and further fermentation to high concentration of ethanol. HSLEH was performed by fed batch addition of substrate and one-time enzyme feed at starting point. When the solids loading at 40% was tried in fed-batch hydrolysis, the observed concentrations of glucose and ethanol were 119 g/l and 58.4 g/l respectively. When laccase (200 U/g solids) with 1-HBT (25 mg/g solids) as a mediator was added to cellulase in HSLEH, the glucose and ethanol concentrations were further enhanced to 157 g/l and 72.4 g/l. The present glucan conversion of 74% in HSLEH was higher when compared with the reported values for biomass pretreated by high temperature steam explosion, acid and alkali pretreatments. The present ethanol concentration (72.4 g/l) was also higher than the reported levels of ethanol in the second-generation bioethanol production. As HSLEH of biomass by low temperature AAS has not been reported so far, the present results clearly demonstrates that LMS assisted HSLEH of biomass pretreated by low temperature AAS is promising for cellulosic ethanol production.

Sugarcane is one of the major agricultural crops cultivated in tropical climate regions of the world. Each tonne of raw cane production is associated with the generation of 130 kg dry weight of bagasse after juice extraction and 250 kg dry weight of cane leaf residue postharvest. The annual world production of sugarcane is ~1.6 billion tones, generating 279 MMT tones of biomass residues (bagasse and cane leaf matter) that would be available for cellulosic ethanol production. Here, we investigated the production of cellulosic ethanol from sugar cane bagasse and sugar cane leaf residue using an alkaline pretreatment: ammonia fiber expansion (AFEX). The AFEX pretreatment improved the accessibility of cellulose and hemicelluloses to enzymes during hydrolysis by breaking down the ester linkages and other lignin carbohydrate complex (LCC) bonds and the sugar produced by this process is found to be highly fermentable. The maximum glucan conversion of AFEX pretreated bagasse and cane leaf residue by cellulases was ~85%. Supplementation with hemicellulases during enzymatic hydrolysis improved the xylan conversion up to 95-98%. Xylanase supplementation also contributed to a marginal improvement in the glucan conversion. AFEX-treated cane leaf residue was found to have a greater enzymatic digestibility compared to AFEX-treated bagasse. Co-fermentation of glucose and xylose, produced from high solid loading (6% glucan) hydrolysis of AFEX-treated bagasse and cane leaf residue, using the recombinant Saccharomyces cerevisiae (424A LNH-ST) produced 34-36 g/L of ethanol with 92% theoretical yield. These results demonstrate that AFEX pretreatment is a viable process for conversion of bagasse and cane leaf residue into cellulosic ethanol. © 2010 Wiley Periodicals, Inc.