Green gram husk--an inexpensive substrate for alkaline protease production by Bacillus sp. in solid-state fermentation

Alkaline protease production under solid-state fermentation was investigated using isolated alkalophilic Bacillus sp. Among all agro-industrial waste material evaluated, green gram husk supported maximum protease production. Solid material particle size regulated the enzyme production and yield was improved with the supplementation of carbon and nitrogen sources to the solid medium. Optimum enzyme production was achieved with 1.5% maltose and 2.0% yeast extract with 371% increase than control. Glucose did not repressed enzyme production but inorganic nitrogen sources showed little negative impact. The physiological fermentation factors such as pH of the medium (pH 9.0), moisture content (140%), incubation time (60 h) and inoculum level played a vital role in alkaline protease production. The enzyme production was found to be associated with the growth of the bacterial culture.     

Production of protease and lipase by solvent tolerant Pseudomonas aeruginosa PseA in solid-state fermentation using Jatropha curcas seed cake as substrate

Deoiled Jatropha seed cake was assessed for its suitability as substrate for enzyme production by solid-state fermentation (SSF). Solvent tolerant Pseudomonas aeruginosa PseA strain previously reported by us was used for fermentation. The seed cake supported good bacterial growth and enzyme production (protease, 1818 U/g of substrate and lipase, 625 U/g of substrate) as evident by its chemical composition. Maximum protease and lipase production was observed at 50% substrate moisture, a growth period of 72 and 120 h, and a substrate pH of 6.0 and 7.0, respectively. Enrichment with maltose as carbon source increased protease and lipase production by 6.3- and 1.6-fold, respectively. Nitrogen supplementation with peptone for protease and NaNO(3) for lipase production also enhanced the enzyme yield reaching 11,376 U protease activity and 1084 U lipase activity per gram of Jatropha seed cake. These results demonstrated viable approach for utilization of this huge biomass by solid-state fermentation for the production of industrial enzymes. This offers significant benefit due to low cost and abundant availability of cake during biodiesel production.     

Coconut cake – a potent substrate for the production of lipase by Candida rugosa in solid-state fermentation

Investigations were conducted with the aim of producing extracellular lipase from Candida rugosa by solid-state fermentation (SSF), using coconut oil cake (COC) as a solid substrate. To optimize production, various modifications were made to enrich the substrate by supplementing it with mineral solution, different carbon sources and several inorganic as well as organic nitrogen sources. Among them, urea (1%), peptone (3%) and maltose (5%) were found to be most suitable. Addition of olive oil (10%) encouraged lipase synthesis. The maximum lipase activity in the enriched substrate was 87.76 units per gram of dry fermented substrate [U/gds] compared to 25.81 U/gds in the raw cake at 96 h of fermentation, and growth was as high as 14.44 mg/gds of glucosamine. This was reached at 72 h in the enriched substrate. C. rugosa growth was calculated indirectly by estimating the glucosamine content in the cell wall after its hydrolysis. The enzyme yield was far better than any values reported as yet.     

Utilization of Jatropha deoiled seed cake for production of cellulases under solid-state fermentation

Toxic waste generated by Jatropha seed cake after utilization of biodiesel on one hand has stimulated the need to develop new technologies to treat the waste and on the other, forced us to reevaluate the efficient utilization of its nutritive potential for production of various high-value compounds and its conversion to non-toxic forms which could be used as animal feed stock. In this study, Jatropha seed cake was used for production of cellulases by new isolate of Thermoascus aurantiacus under solid-state fermentation. The interaction of nitrogen source concentration, moisture ratio, initial pH of the medium and inoculum size was investigated and modelled using response surface methodology (RSM) using Box-Behnken Design (BBD). Under optimized conditions endo-β-1,4-glucanase, β-glucosidase and filter paper activities were found to be 124.44, 28.86, 4.87?U/g of substrate, respectively. Characterization of endo-β-1,4-glucanase, β-glucosidase was done after partial purification by ammonium sulfate fractionation followed by desalting. The endo-β-1,4-glucanase and β-glucosidase showed maximum activity at 70?°C and pH 4. Saccharification studies performed with different lignocellulosic substrates showed that sugar cane bagasse was most susceptible to enzymatic hydrolysis. The study suggests that Jatropha seed cake can be used as a viable nutrient source for cellulase production without any pretreatment under solid-state fermentation by T. aurantiacus.     

Olive-mill wastewaters: a promising substrate for microbial lipase production

The present study investigated the valorization of olive-mill wastewater (OMW) by its use as a possible growth medium for the microbial production of extra-cellular lipase. To this end, strains of Geotrichum candidum (NRRL Y-552 and Y-553), Rhizopus arrhizus (NRRL 2286 and ISRIM 383), Rhizopus oryzae (NRRL 6431), Aspergillus oryzae (NRRL 1988 and 495), Aspergillus niger (NRRL 334), Candida cylindracea (NRRL Y-17506) and Penicillium citrinum (NRRL 1841 and 3754, ISRIM 118) were screened. All strains were able to grow on the undiluted OMW, producing extra-cellular lipase activity. C. cylindracea NRRL Y-17506 showed the highest lipase activity on all the typologies of OMW used. Its lipase production on OMW was markedly affected by the type of nitrogen source and was induced by the addition of olive oil. The highest activity (9.23 IU ml(-1)) of the yeast was obtained on OMW supplemented with NH(4)Cl (2.4 g l(-1)) and olive oil (3.0 g l(-1)).     

Xylanase production using agro-residue in solid-state fermentation from Bacillus pumilus ASH for biodelignification of wheat straw pulp

Two stage statistical design was used to optimize xylanase production from Bacillus pumilus ASH under solid-state fermentation. Initially, Plackett-Burman designing (PB) was used for the selection of crucial production parameters. Peptone, yeast extract, incubation time, moisture level and pH were found to be the crucial factors for the xylanase production. Crucial variables were further processed through central composite designing (CCD) of response surface methodology (RSM) to maximize the xylanase yield. Each significant factor was investigated at five different levels to study their influence on enzyme production. Statistical approach resulted in 2.19-fold increase in xylanase yield over conventional strategy. The determination coefficient (R (2)) as shown by analysis of variance (ANOVA) was 0.9992, which shows the adequate credibility of the model. Potential of cellulase-free xylanase was further investigated for biobleaching of wheat straw pulp. Xylanase aided bleaching through XCDED(1)D(2) sequence resulted in 20 and 17% reduction in chlorine and chlorine dioxide consumption as compared to control. Significant increase in pulp brightness (%ISO), whiteness and improvement in various pulp properties was also observed.     

Microwave assisted alkali-catalyzed transesterification of Pongamia pinnata seed oil for biodiesel production

In this study, microwave assisted transesterification of Pongamia pinnata seed oil was carried out for the production of biodiesel. The experiments were carried out using methanol and two alkali catalysts i.e., sodium hydroxide (NaOH) and potassium hydroxide (KOH). The experiments were carried out at 6:1 alcohol/oil molar ratio and 60 °C reaction temperature. The effect of catalyst concentration and reaction time on the yield and quality of biodiesel was studied. The result of the study suggested that 0.5% sodium hydroxide and 1.0% potassium hydroxide catalyst concentration were optimum for biodiesel production from P. pinnata oil under microwave heating. There was a significant reduction in reaction time for microwave induced transesterification as compared to conventional heating.     

Bioprocess optimization for production of thermoalkali-stable protease from Bacillus subtilis K-1 under solid-state fermentation

Cost-effective production of proteases, which are robust enough to function under harsh process conditions, is always sought after due to their wide industrial application spectra. Solid-state production of enzymes using agro-industrial wastes as substrates is an environment-friendly approach, and it has several advantages such as high productivity, cost-effectiveness, being less labor-intensive, and less effluent production, among others. In the current study, different agro-wastes were employed for thermoalkali-stable protease production from Bacillus subtilis K-1 under solid-state fermentation. Agricultural residues such as cotton seed cake supported maximum protease production (728?U?ml^?1), which was followed by gram husk (714?U?ml^?1), mustard cake (680?U?ml^?1), and soybean meal (653?U?ml^?1). Plackett–Burman design of experiment showed that peptone, moisture content, temperature, phosphates, and inoculum size were the significant variables that influenced the protease production. Furthermore, statistical optimization of three variables, namely peptone, moisture content, and incubation temperature, by response surface methodology resulted in 40% enhanced protease production as compared to that under unoptimized conditions (from initial 728 to 1020?U?ml^?1). Thus, solid-state fermentation coupled with design of experiment tools represents a cost-effective strategy for production of industrial enzymes.     

Sugar-cane pressmud as a novel and inexpensive substrate for production of lactic acid in a solid-state fermentation system

Lactobacillus casei subsp. casei CFTRI 2022 produced a higher concentration of lactic acid (5.27 g/100 g dry sugar-cane pressmud) in a solid-state fermentation (SSF) system as compared to L. helveticus CFTRI 2026 and Streptococcus thermophilus CFTRI 2034. The lactic acid production by L. casei subsp. casei CFTRI 2022 was found to be significantly influenced by the initial moisture content, initial pH and initial sugar concentration of the medium. Studies on four inert materials to reduce the initial sugar concentration in the medium showed the high potential of microcrystalline cellulose whereas the use of diatomaceous earth, acid-washed river sand and washed pith bagasse posed problems. The data indicate the potential of lactic acid production from sugar-cane pressmud in an SSF system.     

Coffee husk: an inexpensive substrate for production of citric acid by Aspergillus niger in a solid-state fermentation system

Aspergillus niger CFTRI 30 produced 1.3 g citric acid/10 g dry coffee husk in 72 h solid-state fermentation when the substrate was moistened with 0.075 M NaOH solution. Production was increased by 17% by adding a mixture of iron, copper and zinc to the medium but enrichment of the moist solid medium with (NH₄)₂SO₄, sucrose or any of four enzymes did not improve production. The production of about 1.5 g citric acid/10 g dry coffee husk at a conversion of 82% (based on sugar consumed) under standardized conditions demonstrates the commercial potential of using the husk in this way.The authors are with the Department of Microbiology and Bioengineering, Central Food Technological Research Institute, Mysore-570 013, India;     

Bacillus pumilus SG2 isolated from saline conditions produces and secretes two chitinases

AIMS: Isolation and characterization of chitinases from a halotolerant Bacillus pumilus. METHODS AND RESULTS: Bacillus pumilus strain SG2 was isolated from saline conditions. It is able to produce chitinase activity at high salt concentration. SDS-PAGE analysis of the B. pumilus SG2 culture supernatant showed two major bands that were induced by chitin. The amino acid sequence of the two proteins, designated ChiS and ChiL, showed a high homology with the chitinase of B. subtilis CHU26, and chitinase A of B. licheniformis, respectively. N-terminal signal peptide of both proteins was also determined. The molecular weight and isoelectric point of the chitinases were determined to be 63 and 74 kDa, and 4.5 and 5.1, for ChiS and ChiL respectively. The genes encoding for both chitinases were isolated and their sequence determined. The regulation of the chitinase genes is under the control of the catabolite repression system. CONCLUSIONS: Secreted chitinase genes and their flanking region on the genome of B. pumilus SG2 have been identified and sequenced. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a multiple chitinases-producing B. pumilus halotolerant strain. We have identified two chitinases by using a reverse genetics approach. The chitinases show resistance to salt.     

Optimization of xylanase production using inexpensive agro-residues by alkalophilic Bacillus subtilis ASH in solid-state fermentation

Alkalophilic Bacillus subtilis ASH produced high levels of xylanase using easily available inexpensive agricultural waste residues such as wheat bran, wheat straw, rice husk, sawdust, gram bran, groundnut and maize bran in solid-state fermentation (SSF). Among these, wheat bran was found to be best substrate. Xylanase production was highest after 72 h of incubation at 37 °C and at a substrate to moisture ratio of 1:2 (w/v). The inoculum level of 15% resulted in maximum production of xylanase. The enzyme production was stimulated by the addition of nutrients such as yeast extract, peptone and beef extract. In contrast, addition of glucose and xylose repressed the production of xylanase. The extent of repression by glucose (10%, w/v) was 81% and it was concentration-dependent. Supplementation of the medium with 4% xylose caused 59% repression. Under optimized conditions, xylanase production in SSF (8,964 U of xylanase/g dry wheat bran) was about twofold greater than in submerged fermentation. Thus, B. subtilis produced a very high level of xylanase in SSF using inexpensive agro-residues, a level which is much higher than that reported by any other bacterial isolate. Furthermore, the enzyme was produced at room temperature and with tap water without the addition of any mineral salt in SSF, leading to a marked decrease in the cost of xylanase production, which enhances its industrial potential.     

Pigeon pea waste as a novel, inexpensive, substrate for production of a thermostable alkaline protease from thermoalkalophilic Bacillus sp. JB-99

Thermoalkaliphilic Bacillus sp. JB-99 was grown in a 250 ml Erlenmeyer flask containing 50 ml medium containing (g/l) Pigeon pea waste 10; NaNO3, 5.0; K2HPO4, 5.0; MgSO4 x 2H2O, 0.2 and Na2CO3, 10.0. Incubations were carried out at 50 degrees C on a rotary incubator shaker for 15 h. A high level of extra cellular thermostable protease activity was observed after 24 h incubation. The optimum temperature and pH for activity were 70 degrees C and 11, respectively, so this enzyme showed stable activity at high temperature and under alkaline conditions.     

First report of a bifunctional chitinase/lysozyme produced by Bacillus pumilus SG2

Bacillus pumilus SG2 isolated from high salinity ecosystem in Iran produces two chitinases (ChiS and ChiL) and secretes them into the medium. In this study, chiS and chiL genes were cloned in pQE-30 expression vector and were expressed in the cytoplasm of Escherichia coli strain M15. The recombinant proteins were purified using Ni-NTA column. The optimum pH and optimum temperature for enzyme activity of ChiS were pH 6, 50°C; those of ChiL were pH 6.5, 40°C. The purified chitinases showed antifungal activity against Fusarium graminearum, Rhizoctonia solani, Magnaporthe grisea, Sclerotinia sclerotiorum, Trichoderma reesei, Botrytis cinerea and Bipolaris sp. Moreover, purified ChiS was identified as chitinase/lysozyme, which are capable of degrading the chitin component of fungal cell walls and the peptidoglycan component of cell walls with many kinds of bacteria (Xanthomonas translucens pv. hordei, Xanthomonas axonopodis pv. citri, Bacillus licheniformis, E. coli C600, E. coli TOP10, Pseudomonas aeruginosa and Pseudomonas putida). Strong homology was found between the three-dimensional structures of ChiS and a chitinase/lysozyme from Bacillus circulans WL-12. This is the first report of a bifunctional chitinase/lysozyme from B. pumilus.     

Purification and characterization of a thermostable alkaline protease from alkalophilic Bacillus pumilus

AIMS: An investigation was carried out on the purification and characterization of an alkaline protease from Bacillus pumilus MK6-5. METHODS AND RESULTS: An alkalophilic Bacillus pumilus MK6-5 was grown in a laboratory fermenter containing 1% reverse osmosis concentrated cheese whey powder, 0.25% corn steep liquor, 1% glucose, 0.5% tryptone, 1% sodium citrate, 0.02% MgSO4.7H2O and 0.65% Na2CO3 at 35 degrees C and pH 9.6, agitation at 250 rev min(-1) and aeration of 1 vvm for 60 h. When the enzyme was purified using ammonium sulphate precipitation, ion exchange and gel filtration chromatographies, a 26.2% recovery of enzyme with 36.6-fold purification was recorded. The purified protease was found to be homogenous by SDS-PAGE with molecular mass estimate of 28 kDa. The enzyme was optimally active at pH 11.5 and temperature of 55-60 degrees C. The Km and kcat values observed with synthetic substrates at 37 degrees C and pH 8.0 were 1.1 mmol l(-1) and 624 s(-1) for Glu-Gly-Ala-Phe-pNA and 3.7 mmol l(-1) and 826 s(-1) for Glu-Ala-Ala-Ala-pNA, respectively. The kinetic data revealed that small aliphatic and aromatic residues were the preferred residues at the P1 position. Inhibition profile exhibited by PMSF suggested the B. pumilus protease to be an alkaline serine protease. CONCLUSIONS: Bacillus pumilus MK6-5 produced a calcium-dependent, thermostable alkaline serine protease. SIGNIFICANCE AND IMPACT OF THE STUDY: The thermostable alkaline protease from Bacillus pumilus MK6-5 will be extremely useful in ultrafiltration membrane cleaning due to its ability to work in broad pH and temperature ranges, and tolerance to detergents, unlike the mesophilic proteases which face these limitations.     

短小芽孢杆菌2080碱性蛋白酶的纯化与性质

短小芽孢杆菌（Bacillus pumilus）2080碱性蛋白酶的发酵液经超滤、硫酸铵沉淀、CM Sepharose Fast Flow和DEAE Sepharose Fast Flow离子交换层析得到了纯化的组分。SDS-PAGE电泳分析显示其分子量约为61kDa。酶学性质研究表明,该纯化酶的最适pH为10．5,最适温度为50℃。     

Sugarcane-pressmud as a novel substrate for production of citric acid by solid-state fermentation

Sugarcane-pressmud, a by-product of cane-sugar manufacture, was used as a substrate for production of citric acid by Aspergillus niger CFTRI 30, in a solid-state fermentation system. Of the 170 g of sugar supplied, 131 g were consumed, with a 79% yield of citric acid over 120 h. Potassium ferrocyanide improved the conversion to about 88% and lowered the fermentation time by 24 h. Enrichment with sugar and NH₄NO₃ was essential to improve productivity. About 174 g citric acid/kg dry sugarcane-pressmud were produced after 120 h in ferrocyanid-treated medium which initially contained 12.5% (w/w) effective sugar and 0.1% (w/w) NH₄NO₃. About 3% (w/w) of the original sugar present in the sugarcane-pressmud was non-utilizable. This is the first report on the potential of sugarcane-pressmud for citric acid production.V.S. Shankaranand and B.K. Lonsane are with the Fermentation Technology and Bioengineering Discipline, Central Food Technological Research Institute, Mysore-570 013, India     

Pineapple waste - a novel substrate for citric acid production by solid-state fermentation

Summary Citric acid production in solid-state fermentation by Aspergillus foetidus ACM3996 was better on pineapple waste than on apple pomace, wheat bran or rice bran. The highest citric acid content achieved on pineapple waste was 16.1 g per 100 g dried pineapple waste, with a moisture content of 70% and in the presence of 3% methanol. This represents a yield of 62.4% based on the sugar consumed.     

Alkaline protease production by an isolated Bacillus circulans under solid-state fermentation using agroindustrial waste: process parameters optimization

Alkaline protease production using isolated Bacillus circulans under solid-state fermentation environment was optimized by using Taguchi orthogonal array (OA) experimental design (DOE) methodology to understand the interaction of a large number of variables spanned by factors and their settings with a small number of experiments in order to economize the process optimization. The software-designed experiments with an OA worksheet of L-27 was selected to optimize fermentation (temperature, particle size, moisture content and pH), nutrition (yeast extract and maltose), and biomaterial-related (inoculum size and incubation time) factors for the best production yields. Analysis of experimental data using Qualitek-4 methodology showed significant variation in enzyme production levels (32,000-73,000 units per gram material) and dependence on the selected factors and their assigned levels. Validation of experimental results on alkaline protease production by this bacterial strain based on DOE methodology revealed 51% enhanced protease production compared to average performance of the fermentation, indicating the importance of this methodology in the evaluation of main and interaction effects of the selected factors individually and in combination for bioprocess optimization.