Production and optimization of process parameters for alkaline protease production by a newly isolated Bacillus sp. under solid state fermentation

Production of alkaline protease employing the laboratory isolate, Bacillus sp. under solid state fermentation (SSF) was optimized. The effect of wheat bran and lentil husk was examined. Wheat bran showed highest enzyme production. The appropriate incubation time, inoculum size, moisture level and buffer solution level were determined. Maximum yields of 429.041 and 168.640 U g⁻¹ were achieved by employing wheat bran and lentil husk as substrates in 0.1 M carbonate/bicarbonate buffer at pH 10 with 30 and 40% initial moisture level at 24 h. Inoculum size and buffer solution level were found to be 20 and 25% and 0.5:1 for wheat bran and lentil husk, respectively.     

Production of Extracellular Alkaline α‐Amylase by Solid State Fermentation with a Newly Isolated Bacillus sp.

Abstract

Production of alkaline α‐amylase employing our laboratory isolate, Bacillus sp., under solid state fermentation, was optimized. The effect of wheat bran and lentil husk was examined. Lentil husk exhibited the highest enzyme production. The appropriate incubation time, inoculum size, moisture level, and buffer solution level were determined. Maximum yields of 216,000 and 172,800 U/g were achieved by employing lentil husk and wheat bran as substrates in 0.1 M carbonate/bicarbonate buffer at pH 10.0 with 30% initial moisture level at 24 h. Inoculum size and buffer solution level were found to be 20% and 1:0.5 for two solid substrates.     

Solid state fermentation for production of α-amylase by a thermotolerant Bacillus subtilis from hot-spring water

The production of extracellular α-amylase by thermotolerant Bacillus subtilis was studied in solid state fermentation (SSF). The effect of wheat bran (WB) and rice husk (RH) was examined. The appropriate incubation period, moisture level, particle size and inoculum concentration was determined. Maximum yields of 159,520 and 21,760 U g⁻¹ were achieved by employing WB and RH as substrates in 0.1 M phosphate buffer at pH 7 with 30% initial moisture content at 24 and 48 h. Particle size and inoculum concentration were found to be 1000 μm, 20% and 500 μm, 15% for WB and RH, respectively. Enzyme yield was 7.3-fold higher with WB medium compared with RH.     

Parametric optimization of feruloyl esterase production from Aspergillus terreus strain GA2 isolated from tropical agro-ecosystems cultivating sweet sorghum

A fungal strain, Aspergillus terreus strain GA2, isolated from an agricultural field cultivating sweet sorghum, produced feruloyl esterase using maize bran. In order to obtain maximum yields of feruloyl esterase, the solid state fermentation (SSF) conditions for enzyme production were standardized. Effective feruloyl esterase production was observed with maize bran as substrate followed by wheat bran, coconut husk, and rice husk among the tested agro-waste crop residues. Optimum particle size of 0.71- 0.3 mm and moisture content of 80% favored enzyme production. Moreover, optimum feruloyl esterase production was observed at pH 6.0 and a temperature of 30 degrees C. Supplementation of potato starch (0.6%) as the carbon source and casein (1%) as the nitrogen source favored enzyme production. Furthermore, the culture produced the enzyme after 7 days of incubation when the C:N ratio was 5. Optimization of the SSF conditions revealed that maximum enzyme activity (1,162 U/gds) was observed after 7 days in a production medium of 80% moisture content and pH 6.0 containing 16 g maize bran [25% (w/v)] of particle size of 0.71-0.3 mm, 0.6% potato starch, 3.0% casein, and 64 ml of formulated basal salt solution. Overall, the enzyme production was enhanced by 3.2-fold as compared with un-optimized conditions.     

L-glutaminase production by <Emphasis Type="Italic">Trichoderma koningii</Emphasis> under solid-state fermentation

Solid state fermentation was conducted for the production of L-glutaminase by Trichoderma koningii Oud.aggr. using different agro-industrial byproducts inlcuding wheat bran, groundnut residues, rice hulls, soya bean meal, corn steep, sesamum oil cake, cotton seed residues and lentil industrial residues as solid substrates. Wheat bran was the best substrate for induction of L-glutaminase (12.1 U/mg protein) by T. koningii. The maximum productivity (23.2 U/mg protein) and yield (45.0 U/gds) of L-glutaminase by T. koningii occurred using wheat bran of 70% initial moisture content, initial pH 7.0, supplemented with D-glucose (1.0%) and L-glutamine (2.0% w/v), inoculated with 3 ml of 6 day old fungal culture and incubated at 30°C for 7 days. After optimization, the productivity of L-glutaminase by the solid cultures of T. koningii was increased by 2.2 fold regarding to the submerged culture.     

Acid Protease Production by Fungi Used in Soybean Food Fermentation

Growth conditions for maximum protease production by Rhizopus oligosporus, Mucor dispersus, and Actinomucor elegans, used in Oriental food fermentations, were investigated. Enzyme yields by all three fungi were higher in solid substrate fermentations than in submerged culture. The level of moisture in solid substrate must be at about 50 to 60%. Very little growth of these fungi was noted when the moisture of substrate was below 35%, whereas many fungi including most storage fungi generally grow well on solid substrate with that level of moisture. Among the three substrates tested—wheat bran, wheat, and soybeans—wheat bran was the most satisfactory one for enzyme production. The optimal conditions for maximum enzyme production of the three fungi grown on wheat bran were: R. oligosporus, 50% moisture at 25 C for 3 to 4 days; M. dispersus, 50 to 63% moisture at 25 C for 3 to 4 days; A. elegans, 50 to 63% moisture at 20 C for 3 days. Because these fungi are fast growing and require high moisture for growth and for enzyme synthesis, the danger of contamination by toxin-producing fungi would be minimal.     

Production of Aspergillus xylanase by lignocellulosic waste fermentation and its application

Strains of Aspergillus terreus and A. niger, known to produce xylanase with undetectable amounts of cellulase, were studied for xylanase (EC 3.2.1.8) production on various lignocellulosic substrates using solid state fermentation. Of the lignocellulosic substrates used, wheat bran was the best for xylanase production. The effects of various parameters, such as moistening agent, level of initial moisture content, temperature of incubation, inoculum size and incubation time, on xylanase production were studied. The best medium for A. terreus was wheat bran moistened with 1:5 Mandels and Strenberg mineral solution containing 0.1% tryptone, at 35 degrees C, and at inoculum concentration 2x107-2x108 spores 5 g-1 substrate; for A. niger, the best medium was wheat bran moistened with 1:5 Mandels and Strenberg mineral solution containing 0.1% yeast extract, at 35 degrees C, and at an inoculum concentration of 2x107-2x108 spores 5 g-1 substrate. Under these conditions, A. terreus produced 68.9 IU ml-1 of xylanase, and A. niger, 74.5 IU ml-1, after 4 d of incubation. A crude culture filtrate of the two Aspergillus strains was used for the hydrolysis of various lignocellulosic materials. Xylanase preparations from the two strains selectively removed the hemicellulose fraction from all lignocellulosic materials tested.     

Evaluation of solid substrates for enzyme production by Coriolus versicolor, for use in bioremediation of chlorophenols in aqueous effluents

In the development of a system for the removal of chlorophenols from aqueous effluents, a range of solid substrates for the growth of Coriolus versicolor were investigated. Substrates included wood chips, cereal grain, wheat husk and wheat bran. Suitability for transformation of chlorophenols depended on laccase production by the fungus. The greatest amount of laccase (<25 Units g⁻¹ substrate) was produced on wheat husk and wheat bran over 30 days colonisation. Aqueous extracts of laccase from wheat husk and wheat bran cultures removed 100% of 2,4-dichlorophenol (50 ppm) from solution within 5 h and 75–80% of pentachlorophenol (50 ppm) within 24 h. Wheat bran was formulated into pellets with biscuit flour to provide a compact substrate for fungal immobilisation. Addition of 8–12% yeast extract to the pellets increased laccase production five-fold. Colonised pellets were added to chlorophenol solutions in 200–4000-ml bioreactors, resulting in >90% removal of chlorophenols within 100 min. Received: 10 April 2000 / Received revision: 4 July 2000 / Accepted: 10 July 2000     

Production of alpha amylase by Bacillus licheniformis using an economical medium

The present study is concerned with the selection of new medium for the production of alpha amylase by Bacillus licheniformis. Different agricultural by-products such as wheat bran, sunflower meal, cotton seed meal, soybean meal, rice husk or rice bran were tested for the production of alpha amylase. Among different agricultural by-products evaluated, wheat bran was found to be the best basal and standardized medium for optimal production of alpha amylase. The production was increased 2-folds when soluble starch was replaced with pearl millet starch at 1% level and nutrient broth concentrations was reduced from 1% level to 0.5%. The newly selected fermentation medium containing (% w/v) wheat bran 1.25, nutrient broth 0.5, pearl millet starch 1.0, lactose 0.5, NaCl 0.5, CaCl2 0.2 in 100 ml of phosphate buffer. The kinetic values of Y(p/x), Y(p/s), and Q(p) indicated that the production of enzyme was greater in newly selected medium than the conventional more expensive medium.     

Mixture design as first step for improved glutaminase production in solid-state fermentation by isolated Bacillus

Aim: Investigation of mixture‐design impact on glutaminase production by isolated Bacillus sp. Methods and Results: An augmented simplex centroid design was used to optimize a three (wheat bran, Bengal gram husk and palm seed fibre) component mixture for glutaminase production. Selected substrate materials showed impact on glutaminase production values at individual level by Bengal gram husk [2789 U gds^?1 (gram dry substrate] and in two‐level combination with wheat bran and Bengal gram husk (maximum of 3300 U gds^?1). Conclusion: Bengal gram husk is the most suitable substrate medium for glutaminase production by Bacillus sp. Maximum glutaminase production is achieved using solid‐substrate mixture at two‐level combinations in the ratio of 66 : 34 for Bengal gram husk and wheat bran, respectively. Significance and Impact of the Study: The present study has significance in large‐scale production of glutaminase at commercial level with the use of multi‐substrate rather than single‐substrate/support material.     

Production of raw-starch digesting amyloglucosidase by Aspergillussp GP-21 in solid state fermentation

Aspergillus sp GP-21 produced a raw-starch digesting amyloglucosidase which showed optimum activity at 65°C and pH 5.0–5.5. At 50°C the enzyme converted about 40% of raw corn starch to glucose within 48 h. Enzyme production was studied in solid state fermentation using wheat bran. Productivity was affected by the level of moisture, incubation temperature and the presence or absence of supplements. Maximum enzyme production was observed at a moisture level of 75% and at 30°C. Enzyme production was stimulated by supplementing wheat bran with 0.25% proteose peptone, 1% trace mineral solution, 0.01% CaCl₂ and 0.01% MgSO₄. Received 27 October 1998/ Accepted in revised form 15 March 1999     

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.     

Production of xylanase by Emericella nidulans NK-62 on low-value lignocellulosic substrates

Thermotolerant Emericella nidulans NK-62 was isolated from bird nesting material and was tested for its ability to produce xylanase. The fungus when grown on a medium containing wheat bran (2% w/v) supplemented with Czapek's mineral salt solution at 45 °C for 7 days produced 362 IU/ml of xylanase (EC 3.2.1.8). The specific activity of E. nidulans NK-62 xylanase was found to be 275 IU/mg of total protein. The enzyme was found to be active over a broad temperature and pH range with 60 °C as optimum temperature for enzyme activity. The enzyme was stable at 50 °C and its half-life at 55 °C was 45 min. -xylosidase (EC 3.2.1.37) and carboxymethylcellulase (EC 3.2.1.4) activities, 0.018 and 0.21 IU/ml respectively, were also noticed. The fungus was screened for its ability to produce xylanase on four different lignocellulosic substrates. It produced 318.9 IU/ml of cellulase-free xylanase on corn cobs. The fungus could also utilize lentil bran (seed husk of Lens esculentus) and meal of groundnut shells to produce 84.8 and 17.3 IU/ml xylanase respectively.     

Optimizing some factors affecting protease production under solid state fermentation

Production of extracellular alkaline protease by a locally isolated fungal species, Rhizopus oryzae, under solid state fermentation was optimized. The maximum enzyme activity under the optimum conditions of temperature (32?°C), relative humidity (90%–95%), spore count (～2?×?10⁵/g wheat bran), moisture content of solid substrate (140%) adjusted suitably with salt solution (M-9) of pH?5.5 was 341 unit/g wheat bran.     

嗜热真菌DSM10635生产耐热木聚糖酶的小试研究

应用嗜热真菌Thermomyces lanuginosus DSM10635,采用固体发酵的方法探索耐热木聚糖酶的优化生产条件。在研究玉米芯,玉米皮,玉米秆,麸皮,松树屑,桦树屑等不同底物,在不同温度、玉米芯颗粒大小以及料水比条件下培养比较酶产量后,发现该嗜热真菌产耐热木聚糖酶的最佳底物为玉米芯或玉米皮,最佳培养温度为50℃--55℃,在加水量为1份玉米芯：2．8份水,玉米芯的颗粒直径大约为1mm时产酶量最高。实验结果显示,嗜热真菌DSM10635在优化后的培养条件下木聚糖酶产量可达到12525．80IU／g玉米芯。     

Exoglucanase production by Aspergillus niger grown on wheat bran

β-Exoglucanase production on the lignocellulosic material, wheat bran, by Aspergillus niger under solid state fermentation (SSF) on a laboratory scale was investigated. Different fermentation parameters, such as moisture content, initial pH, temperature, depth of the substrate, and inoculum size on exoglucanase production were optimized. Moisture content of 40 %, pH of 7.0, substrate depth of 1.0 cm, inoculum size of 2?×?10⁶ spores/g of wheat bran, and temperature at 30 °C were optimal for maximum production of exoglucanase. Maximum yields of exoglucanase with 28.60 FPU/g of wheat bran were obtained within 3 days of incubation under optimal conditions.     

Enhanced production of pectinase by Bacillus sp. DT7 using solid state fermentation

Bacillus sp. DT7 produced very high levels of alkaline and thermotolerant pectinase by solid state fermentation. Production of this enzyme was affected by nature of solid substrate, level of moisture content, presence or absence of carbon, nitrogen, mineral and vitamin supplements. Maximum enzyme production of 8050 U/g dry substrate was obtained in wheat bran supplemented with polygalacturonic acid (PGA; 1%, w/v) and neurobion (a multivitamin additive; 27 micro l/g dry substrate) with distilled water at 75% moisture level, after 36 h of incubation at 37 degrees C.     

黑曲霉固态发酵及酶解玉米皮

以玉米提取淀粉后的玉米皮渣为主要原料,采用黑曲霉固态发酵法产酶再酶解的二步法降解玉米皮中纤维素类物质。经Plackett-Burman法及响应面设计优化发酵条件得:温度30℃,接种量10%,初始水分体积分数60%,物料厚度2.47 cm,初始pH 5.79,发酵时间6 d;滤纸比酶活可达11.01 U/g,较原始酶活提高了40.61%;产酶结束后加入pH 4.8醋酸-醋酸钠缓冲液,置于50℃下酶解144 h,中性洗涤纤维与酸性洗涤纤维降解率分别为46.09%、48.82%,还原糖质量分数达到9.02%。     

Production of mosquitocidal <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> by solid state fermentation using agricultural wastes

In this study, Bacillus sphaericus NRC 69 was grown in culture media, in which 12 agricultural wastes were tested as the main carbon, nitrogen and energy sources under solid state fermentation. Of the 12 tested agricultural by-products, wheat bran was the most efficient substrate for the production of B. sphaericus mosquitocidal toxins against larvae of Culex pipiens (LC₅₀ 1.2 ppm). Mixtures of tested agricultural wastes separately with wheat bran enhanced the produced toxicity several folds and decreased LC₅₀ between 3.7- and 50-fold in comparison with that of agricultural wastes without mixing. The toxicity of B. sphaericus grown in wheat bran/rice hull at 8/2 (g/g) and wheat bran/barley straw at 1/4 (g/g) showed the same toxicity as that in wheat bran medium (LC₅₀ decreased 17- and 16-fold, in comparison with that in rice hull or barely straw media, respectively). In wheat bran medium, the maximum toxicity of the tested organism obtained at 50% moisture content, inoculum size 84 × 10⁶ CFU/g wheat bran and incubation for 6 days at 30°C. Addition of cheese whey permeate at 10% to wheat bran medium enhanced the toxicity of B. sphaericus NRC 69 about 46%.     

Citric acid production by solid state fermentation using sugarcane bagasse

A solid state fermentation (SSF) method was used to produce citric acid by Aspergillus niger DS 1 using sugarcane bagasse as a carrier and sucrose or molasses based medium as a moistening agent. Initially bagasse and wheat bran were compared as carrier. Bagasse was the most suitable carrier, as it did not show agglomeration after moistening with medium, resulting in better heat and mass transfer during fermentation and higher product yield. Different parameters such as moisture content, particle size, sugar level and methanol concentration of the medium were optimised and 75% moisture level, 31.8 g sugar/100 g dry solid, 4% (v/w) methanol and particles of the size between 1.2 and 1.6 mm were found to be optimal. Sucrose and clarified and non-clarified molasses medium were also tested as moistening agents for SSF and under optimised conditions, 20.2, 19.8 and 17.9 g citric acid /100 g of dry solid with yield of 69.6, 64.5 and 62.4% (based on sugar consumed) was obtained in sucrose, clarified and non-clarified molasses medium respectively, after 9 days of fermentation.