A bio-augmented system with Methylosarcina sp. LC-4 immobilized on bio-carriers: Towards an integrated approach to mitigate and valorize methane emissions from landfills to biodiesel.

Bio-augmented systems based on methanotrophs are indispensable in curbing anthropogenic methane emissions from engineered landfills or dumpsites to curtail rising levels of greenhouse gases. Using a defined methanotroph culture immobilized on an inert material-based bio-carrier makes it possible to harness these methane emissions for creating value-added products, thus contributing to the circular bio-economy. The methane oxidation capacity of the model methanotroph Methylosarcina sp. LC-4, a prospective organism for biodiesel production using methane present in landfill gas, immobilized on several inert bio-carriers, was evaluated to identify a bio-carrier that provided optimum conditions for the process. Among the several bio-carriers evaluated, perlite and vermiculite were selected due to their high specific surface area and superior water-holding capacity, which result in the retention of nutrients and biomass and higher methane elimination capacity. While perlite showed high biomass holding capacity and methane transport, vermiculite supported a high growth of methanotrophs. LC-4 immobilized on perlite and vermiculite as the bio-carrier showed maximum methane elimination capacity (MEC) of 291.3 g m-2 day-1 and 155.5 g m-2 day-1, respectively. The low bed height of only 0.13 m and a short start-up period of 2-4 days are promising for use as alternate daily cover in a landfill. The recovered biomass had 12% (w/w) fatty acid methyl ester (FAME), with a high fraction of (∼85%) of C14-C18 saturated and monounsaturated fatty acids, suitable for biodiesel production. The combination of perlite and vermiculite increased MEC and FAME content levels. The current study demonstrated a new bio-augmented system designed with a pure methanotroph for methane elimination with a short start-up time and the valorization of the assimilated methane.

Organosolv Pretreatment of Sorghum Stalks Using Glycerol.

Glycerol is a promising low-cost solvent for biomass pretreatment since a large amount of glycerol is generated as a by-product in the biodiesel industry. Pretreatment is a method of disintegration of the recalcitrant structure of biomass to enhance the accessibility of cellulose and hemicelluloses to enzymes for complete saccharification. During pretreatment, glycerol breaks the lignin carbohydrate complex and selectively solubilizes lignin. Thus, the glycerol pretreatment improves the accessibility of cellulose to cellulases leading to higher sugar yields. The glycerol pretreatment is carried out at high temperature (>190 °C) to disintegrate the structure of biomass. The glycerol pretreatment in the presence of acid or base catalyst such as H2SO4 or NaOH results in lower pretreatment temperature and higher glucan hydrolysis. This chapter describes the methodology to carry out glycerol pretreatment of sorghum biomass with or without acid/alkali as catalyst and the basic calculations to determine the efficiency of the pretreatment.

Production of xylooligosaccharides in SSF by Bacillus subtilis KCX006 producing ß-xylosidase-free endo-xylanase and multiple xylan debranching enzymes.

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 ß-xylosidase and subsequent bacterial metabolism. A new strain, Bacillus subtilis KCX006, was exceptionally found to synthesize ß-xylosidase-free endo-xylanase and multiple xylan debranching enzymes constitutively in the presence of LCMs. Absence of ß-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.

Detoxification of hexavalent chromium by Leucobacter sp. uses a reductase with specificity for dihydrolipoamide.

Leucobacter sp. belongs to the metal stressed community and possesses higher tolerance to metals including chromium and can detoxify toxic hexavalent chromium by reduction to less toxic trivalent chromium. But, the mechanism of reduction of hexavalent chromium by Leucobacter sp. has not been studied. Understanding the enzyme catalyzing reduction of chromium is important to improve the species for application in bioremediation. Hence, a soluble reductase catalyzing the reduction of hexavalent chromium was purified from a Leucobacter sp. and characterized. The pure chromate reductase was obtained from the cell-free extract through hydrophobic interaction and gel filtration column chromatographic methods. It was a monomeric enzyme and showed similar molecular weights in both gel filtration (∼68 KDa) and SDS-PAGE (64 KDa). It reduced Cr(VI) using both NADH and NADPH as the electron donor, but exhibited higher activity with NADH. The optimal activity was found at pH 5.5 and 30 °C. The K(m) and V(max) for Cr(VI) reduction with NADH were 46.57 µM and 0.37 µmol min(-1) (mg protein) (-1), respectively. The activity was inhibited by p-hydroxy mercury benzoate, Ag(2+) and Hg(2+) indicating the role of thiol groups in the catalysis. The spectrophotometric analysis of the purified enzyme showed the absence of bound flavin in the enzyme. The N-terminal amino acid sequence and LC/MS analysis of trypsin digested purified enzyme showed similarity to dihydrolipoyl dehydrogenase. The purified enzyme had dihydrolipoyl dehydrogenase activity with dihydrolipoamide as the substrate, which suggested that Leucobacter sp. uses reductase with multiple substrate specificity for reduction of Cr(VI) detoxification.

Hyper-production of alpha-amylase from agro-residual medium with high-glucose in SSF using catabolite derepressed Bacillus subtilis KCC103.

In Bacillus subtilis KCC103, alpha-amylase is hyper-produced and alpha-amylase synthesis is not subject to catabolite repression. The alpha-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 alpha-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 alpha-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 mum, 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 degrees C and cultivation time-48 h. alpha-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 alpha-amylase showing the hyper-tolerance of KCC103 to repression by simple sugars on alpha-amylase production in SSF.

Enzymatic digestibility and ethanol fermentability of AFEX-treated starch-rich lignocellulosics such as corn silage and whole corn plant.

BACKGROUND: Corn grain is an important renewable source for bioethanol production in the USA. Corn ethanol is currently produced by steam liquefaction of starch-rich grains followed by enzymatic saccharification and fermentation. Corn stover (the non-grain parts of the plant) is a potential feedstock to produce cellulosic ethanol in second-generation biorefineries. At present, corn grain is harvested by removing the grain from the living plant while leaving the stover behind on the field. Alternatively, whole corn plants can be harvested to cohydrolyze both starch and cellulose after a suitable thermochemical pretreatment to produce fermentable monomeric sugars. In this study, we used physiologically immature corn silage (CS) and matured whole corn plants (WCP) as feedstocks to produce ethanol using ammonia fiber expansion (AFEX) pretreatment followed by enzymatic hydrolysis (at low enzyme loadings) and cofermentation (for both glucose and xylose) using a cellulase-amylase-based cocktail and a recombinant Saccharomyces cerevisiae 424A (LNH-ST) strain, respectively. The effect on hydrolysis yields of AFEX pretreatment conditions and a starch/cellulose-degrading enzyme addition sequence for both substrates was also studied. RESULTS: AFEX-pretreated starch-rich substrates (for example, corn grain, soluble starch) had a 1.5-3-fold higher enzymatic hydrolysis yield compared with the untreated substrates. Sequential addition of cellulases after hydrolysis of starch within WCP resulted in 15-20% higher hydrolysis yield compared with simultaneous addition of hydrolytic enzymes. AFEX-pretreated CS gave 70% glucan conversion after 72 h of hydrolysis for 6% glucan loading (at 8 mg total enzyme loading per gram glucan). Microbial inoculation of CS before ensilation yielded a 10-15% lower glucose hydrolysis yield for the pretreated substrate, due to loss in starch content. Ethanol fermentation of AFEX-treated (at 6% w/w glucan loading) CS hydrolyzate (resulting in 28 g/L ethanol at 93% metabolic yield) and WCP (resulting in 30 g/L ethanol at 89% metabolic yield) is reported in this work. CONCLUSIONS: The current results indicate the feasibility of co-utilization of whole plants (that is, starchy grains plus cellulosic residues) using an ammonia-based (AFEX) pretreatment to increase bioethanol yield and reduce overall production cost.

Alkali-based AFEX pretreatment for the conversion of sugarcane bagasse and cane leaf residues to ethanol.

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 approximately 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 approximately 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.

Mixture optimization of six core glycosyl hydrolases for maximizing saccharification of ammonia fiber expansion (AFEX) pretreated corn stover.

In this work, six core glycosyl hydrolases (GH) were isolated and purified from various sources to help rationally optimize an enzyme cocktail to digest ammonia fiber expansion (AFEX) treated corn stover. The four core cellulases were endoglucanase I (EG I, GH family 7B), cellobiohydrolase I (CBH I, GH family 7A), cellobiohydrolase II (CBH II, GH family 6A) and beta-glucosidase (betaG, GH family 3). The two core hemicellulases were an endo-xylanase (EX, GH family 11) and a beta-xylosidase (betaX, GH family 3). Enzyme family and purity were confirmed by proteomics. Synergistic interactions among the six core enzymes for varying relative and total protein loading (8.25, 16.5 and 33 mg/g glucan) during hydrolysis of AFEX-treated corn stover was studied using a high-throughput microplate based protocol. The optimal composition (based on% protein mass loading) of the cocktail mixture was CBH I (28.4%): CBH II (18.0%): EG I (31.0%): EX (14.1%): betaG (4.7%): betaX (3.8%). These results demonstrate a rational strategy for the development of a minimal, synergistic enzymes cocktail that could reduce enzyme usage and maximize the fermentable sugar yields from pretreated lignocellulosics.

Use of a new catabolite repression resistant promoter isolated from Bacillus subtilis KCC103 for hyper-production of recombinant enzymes.

Bacillus subtilis KCC103 hyper-produces alpha-amylase and the synthesis is resistant to carbon catabolite repression. The strain efficiently produced alpha-amylase in low cost agro-biomass based medium rich in simple sugars without catabolite repression. Here, the catabolite repression resistant promoter (amyR4) of alpha-amylase was isolated from KCC103 and used to synthesize recombinant enzymes in B. subtilis. When the bgaB gene encoding beta-galactosidase of Bacillus stearothermophilus was cloned and expressed under the amyR4 promoter, high level of beta-galactosidase activity was found in Escherichia coli (28 U/ml)) and B. subtilis (19 U/ml). Further, the genes encoding endoxylanase (xynA) and carboxymethyl cellulase (bglC) from B. subtilis were cloned with signal peptides and expressed with CCR-resistant amyR4 promoter. In E. coli, the expression was intracellular with activities of cellulase and xylanase at 76 and 105IU/ml respectively. The expression was extracellular in B. subtilis with activities at 17 and 17 IU/ml of cellulase and xylanase respectively in LB medium. When recombinant B. subtilis was cultured in LB-glucose medium, the synthesis of recombinant enzymes was not subject to catabolite repression and the expression was observed throughout the growth. This is important as glucose in the medium can prevent sporulation of the Bacillus and prevent activation of the other scavenger pathways that leads to degradation of recombinant proteins. The catabolite derepressed promoter of alpha-amylase from B. subtilis KCC103 can be efficiently used for overexpression of various industrial enzymes.

Optimization of biobleaching of paper pulp in an expanded bed bioreactor with immobilized alkali stable xylanase by using response surface methodology.

Purified alkali stable xylanase from Aspergillus fischeri was immobilized on polystyrene beads using diazotization method. An expanded bed bioreactor was developed with these immobilized beads to biobleach the paper pulp in continuous mode. Response surface methodology was applied to optimize the biobleaching conditions. Temperature (degrees C), flow rate of pulp (ml/min) and concentration of the pulp (%) were selected as variables in this study. Optimal conditions for biobleaching process were reaction temperature 60 degrees C, flow rate of 2 ml/min and 5% (w/v) of pulp. The kappa number reduced from 66 in the unbleached pulp to 20 (reduction of 87%). This system proves to be a better option for the conventional chlorine based pulp bleaching.

Comparison of in vitro Cr(VI) reduction by CFEs of chromate resistant bacteria isolated from chromate contaminated soil.

Chromate resistant and reducing strains were isolated from chromium contaminated soil and identified as Bacillus sp. (KCH2 and KCH3), Leucobacter sp. (KCH4) and Exiguobacterium sp. (KCH5). KCH3 and KCH4 showed higher Cr(VI) tolerance (2 mM) and Cr(VI) reduction (1.5 mM) than KCH5 (1.5 mM and 0.75 mM, respectively). Cr(VI) reduction by CFEs of KCH3 and KCH4 showed NAD(P)H dependence, optimum activity at pH 5.5, low K(m) (45-55 microM) and substrate inhibition by Cr(VI) (>75 microM), whereas that of KCH5 showed NADH dependence, pH optimum at 6.0, high K(m) (200 microM) and no inhibition by Cr(VI). Cr(VI) reduction was optimum at 35 degrees C for CFEs of KCH3 and KCH5 and 30 degrees C for that of KCH3. Cr(VI) reduction by CFEs of all the strains were inhibited by Hg(2+) and enhanced by Cu(2+). Activity enhancement by Cu(2+) was more predominant (290%) for KCH4. The characterization of Cr(VI) reduction by CFEs of chromate resistant isolates of different genera is useful for development of Cr(VI) bioremediation.

Alpha-amylase production from catabolite derepressed Bacillus subtilis KCC103 utilizing sugarcane bagasse hydrolysate.

A catabolite derepressed Bacillus subtilis strain KCC103 was used to produce alpha-amylase in medium containing sugarcane bagasse hydrolysate (SBH). Addition of SBH (1% reducing sugar (w/v)) to the nutrient medium supported maximum alpha-amylase production of 67.4 Um l(-1). HPLC analysis of SBH showed the presence of glucose, xylose and arabinose in the ratio of 0.9:1.0:0.16 (w/w/w). In SBH-medium glucose and xylose were consumed completely while arabinose remained unutilized. Uptake rate of glucose was 2-folds higher than xylose but rate of alpha-amylase production with xylose was 1.5-folds higher than glucose. Arabinose had no effect on growth and alpha-amylase synthesis. Further, alpha-amylase production in SBH-medium was enhanced to 144.5 Um l(-1) (2.2-fold) by response surface methodology where the levels of SBH, and other media components were varied. The modified medium consisted of (in gl(-1)) SBH: 24; peptone: 17.43; yeast extract: 1.32 and beef extract: 1.82. High level of SBH showed no significant inhibition of alpha-amylase synthesis. The derepressed strain KCC103 is useful to produce alpha-amylase economically in short time (30-36 h).

Purification and characterization of a maltooligosaccharide-forming alpha-amylase from a new Bacillus subtilis KCC103.

A maltooligosaccharide-forming alpha-amylase was produced by a new soil isolate Bacillus subtilis KCC103. In contrast to other Bacillus species, the synthesis of alpha-amylase in KCC103 was not catabolite-repressed. The alpha-amylase was purified in one step using anion exchange chromatography after concentration of crude enzyme by acetone precipitation. The purified alpha-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 degrees C, it was rapidly deactivated at 70 degrees C with a half-life of 7 min and at 50 degrees C, the half-life was 94 min. The K (m) and V (max) for starch hydrolysis were 2.6 mg ml(-1) and 909 U mg(-1), respectively. Ca(2+) did not enhance the activity and stability of the enzyme; however, EDTA (50 mM) abolished 50% of the activity. Hg(2+), Ag(2+), and p-hydroxymercurybenzoate severely inhibited the activity indicating the role of sulfydryl group in catalysis. The alpha-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-alpha-amylase is useful in bread making as an antistaling agent and it can be produced economically using low-cost sugarcane bagasse.