Ability of a solid state fermentation technique to significantly minimize catabolic repression of α-amylase production by Bacillus licheniformis M27

Summary The production of -amylase by Bacillus licheniformis M27 in submerged fermentation was completely inhibited due to catabolic repression in medium containing 1% glucose. In contrast, the enzyme production in a solid state fermentation system was 19,550 units/ml extract even when the medium contained 15% glucose. The peak in enzyme titre was, however, shifted from 48 to 72 h. The ability of the solid state fermentation system to significantly overcome catabolic repression was not known earlier and is probably conferred by various physico-chemical factors and culture conditions specific to the system. Offprint requests to: B. K. Lonsane     

Enhanced production of raw starch degrading enzyme using agro-industrial waste mixtures by thermotolerant Rhizopus microsporus for raw cassava chip saccharification in ethanol production

In the present study, solid-state fermentation for the production of raw starch degrading enzyme was investigated by thermotolerant Rhizopus microsporus TISTR 3531 using a combination of agro-industrial wastes as substrates. The obtained crude enzyme was applied for hydrolysis of raw cassava starch and chips at low temperature and subjected to nonsterile ethanol production using raw cassava chips. The agro-industrial waste ratio was optimized using a simplex axial mixture design. The results showed that the substrate mixture consisting of rice bran:corncob:cassava bagasse at 8?g:10?g:2?g yielded the highest enzyme production of 201.6?U/g dry solid. The optimized condition for solid-state fermentation was found as 65% initial moisture content, 35°C, initial pH of 6.0, and 5?×?10⁶ spores/mL inoculum, which gave the highest enzyme activity of 389.5?U/g dry solid. The enzyme showed high efficiency on saccharification of raw cassava starch and chips with synergistic activities of commercial α-amylase at 50°C, which promotes low-temperature bioethanol production. A high ethanol concentration of 102.2?g/L with 78% fermentation efficiency was achieved from modified simultaneous saccharification and fermentation using cofermentation of the enzymatic hydrolysate of 300?g raw cassava chips/L with cane molasses.     

Kinetics of beta-mannanase fermentation by Bacillus licheniformis

Bacterial growth, konjac powder utilization and -mannanase production by Bacillus licheniformis NK-27 in batch fermentation were used to develop a model of the process. The optimal set of parameters was estimated by fitting the model to experimental data. The results predicted by the model were in good agreement with the experimental data.     

Regulation of alpha-amylase production inBacillus licheniformis M27 by enzyme end-products in submerged fermentation and its overcoming in solid state fermentation system

Summary Alpha-amylase production byBacillus licheniformis M27 in submerged fermentation was reduced from 480 to 30 units/ml when soluble starch concentration in medium was increased from 0.2 to 1.0%. In contrast, the enzyme production increased by 29 times even with 42 fold increase in the concentration of soluble starch and other starchy substrates in solid state fermentation system. The data establish regulation of the enzyme formation by enzyme end-product in submerged fermentation and ability of solid state fermentation to minimize it significantly. These features were not known earlier.     

β-Glucosidase production by Aspergillus phoenicis in solid state fermentation

Summary -glucosidase production was studied in solid-state cultivation by Aspergillus phoenicis on sugar beet pulps. The experiments were carried out in column incubators aerated with humidified air, at 30 °C. Results of physiological studies showed that the production of -glucosidase from beet pulps in the solid state fermentation required : 70% moisture content, initial pH of about 4, and 10⁷ spores/g substrate. -glucosidase produced under these conditions could be removed from the solid medium with water, or stored in the form of a dried fermented product, for a direct use in the saccharification reactions.     

Streptomyces sp. TEM 33 possesses high lipolytic activity in solid-state fermentation in comparison with submerged fermentation

Solid-state fermentation (SSF) is a bioprocess that doesn’t need an excess of free water, and it offers potential benefits for microbial cultivation for bioprocesses and product development. In comparing the antibiotic production, few detailed reports could be found with lipolytic enzyme production by Streptomycetes in SSF. Taking this knowledge into consideration, we prefer to purify Actinomycetes species as a new source for lipase production. The lipase-producing strain Streptomyces sp. TEM 33 was isolated from soil and lipase production was managed by solid-state fermentation (SSF) in comparison with submerged fermentation (SmF). Bioprocess-affecting factors like initial moisture content, incubation time, and various carbon and nitrogen additives and the other enzymes secreted into the media were optimized. Lipase activity was measured as 1.74 ± 0.0005 U/g dry substrate (gds) by the p-nitrophenylpalmitate (pNPP) method on day 6 of fermentation with 71.43% final substrate moisture content. In order to understand the metabolic priority in SSF, cellulase and xylanase activity of Streptomyces sp. TEM33 was also measured. The microorganism degrades the wheat bran to its usable form by excreting cellulases and xylanases; then it secretes the lipase that is necessary for degrading the oil in the medium.     

Phytase production by the thermophilic fungus Rhizomucor pusillus

A thermophilic fungus, Rhizomucor pusillus, isolated from composting soil, was studied for phytase production using solid-state fermentation. The optimization of phytase production was carried out by Box–Behnken design of experiments, using three independent variables (pH of medium, culture age and incubation period), resulting in a maximal level of phytase (9.18 units/g substrate). The partially purified phytase was optimally active at 70 °C and pH 5.4, though the enzyme showed 80% activity over a wide pH range, 3.0–8.0. The phytase was found to have broad substrate specificity.     

Comparative studies on the production of cellulases by thermophilic fungi in submerged and solid-state fermentation

Summary Six thermophilic fungi were examined for their ability to produce cellulolytic enzymes in liquid (LF) and solid-state fermentation (SSF). The best cellulase activities were achieved by Thermoascus aurantiacus and Sporotrichum thermophile. Taking into consideration that solid-state medium obtained from 100 g of dry sugar-beet pulp occupies about 11 of fermentor volume equivalent to 11 of LF, it was confirmed that enzyme productivity per unit volume from both fungi was greater in SSF than in LF. The cellulase system obtained by SSF with T. aurantiacus contained 1.322 IU/1 of exo--d-glucanase, 53.269 IU/1 of endo--d-glucanase and 8.974 IU/1 of -d-glucosidase. The thermal and pH characteristics of cellulases from solid-state fermentation of T. aurantiacus and S. thermophile are described.     

Fermentation production of keratinase from Bacillus licheniformisPWD-1 and a recombinant B. subtilis FDB-29

Bacillus licheniformis PWD-1, the parent strain, and B. subtilis FDB-29, a recombinant strain. In both strains, keratinase was induced by proteinaceous media, and repressed by carbohydrates. A seed culture of B. licheniformis PWD-1 at early age, 6–10 h, is crucial to keratinase production during fermentation, but B. subtilis FDB-29 is insensitive to the seed culture age. During the batch fermentation by both strains, the pH changed from 7.0 to 8.5 while the keratinase activity and productivity stayed at high levels. Control of pH, therefore, is not necessary. The temperature for maximum keratinase production is 37°C for both strains, though B. licheniformis is thermophilic and grows best at 50°C. Optimal levels of dissolved oxygen are 10% and 20% for B. licheniformis and B. subtilis respectively. A scale-up procedure using constant temperature at 37°C was adopted for B. subtilis. On the other hand, a temperature-shift procedure by which an 8-h fermentation at 50°C for growth followed by a shift to 37°C for enzyme production was used for B. licheniformis to shorten the fermentation time and increase enzyme productivity. Production of keratinase by B. licheniformis increased by ten-fold following this new procedure. After respective optimization of fermentation conditions, keratinase production by B. licheniformis PWD-1 is approximately 40% higher than that by B. subtilis FDB-29. Received 16 July 1998/ Accepted in revised form 07 March 1999     

Statistical optimization of a thermostable and neutral glucoamylase production by a thermophilic mold <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis> in solid-state fermentation

Glucoamylase production by a thermophilic mold Thermomucor indicae-seudaticae was optimized in solid-state fermentation (SSF) by conventional one variable at a time approach and further statistically using response surface methodology (RSM). Glucoamylase secretion was strongly affected by three variables (moisture ratio, inoculum level and incubation time), and therefore, these three factors were further optimized using response surface methodology. The glucoamylase production in flasks containing wheat bran, under the conditions optimized by RSM, was 455 ± 23 U/g of dry moldy bran (DMB), while the predicted value by a polynomial model was 433.30 U/g DMB. The enzyme titre (455 ± 23 U/g DMB) attained in the validation experiment of this investigation is higher than those reported in the literature. When the large-scale production was attempted in enamel trays, a marginally lower enzyme titres were attained. An overall 1.8-fold increase in glucoamylase production was achieved in SSF due to statistical optimization in comparison with that of one variable at a time approach (250 ± 13 U/g DMB). A 10-fold enhancement in glucoamylase production was recorded in SSF as compared to that in submerged fermentation.     

Effect of additional carbon source and moisture level on xylanase production by <Emphasis Type="Italic">Cochliobolus sativus</Emphasis> in solid fermentation

The fungus Cochliobolus sativus has been shown to be an efficient producer of xylanase from an industrial point of view. The addition of extra carbon sources and the initial moisture content of the solid-state fermentation were found to have a marked influence on the xylanase production by C. sativus Cs6 strain. Xylan and starch resulted in an increased xylanase production (1469.4 and 1396.56 U/g, respectively) after 8 days of incubation. Optimal initial moisture content for xylanase production was 80%. The cultivation systems can easily be modified to enhance the productivity of the enzyme formation by C. sativus Cs6, which will facilitate the scale up processes for mass production.     

Solid-state cultivation ofPenicillium capsulatum on beet pulp

Summary Extracellular enzyme production byP. capsulatum during solid-state fermentation on beet pulp is maximal at 30°C with initial moisture contents of 60–75% and when growth medium is supplemented with corn steep liquor, ammonium sulphate and yeast extract. On balance, solid-state co-cultures ofP. capsulatum withT. reesei orT. emersonii do not yield greater overall enzyme activity thanP. capsulatum alone.     

Potential of Aspergillus oryzae CFTRI 1480 for producing proteinase in high titres by solid state fermentation

Aspergillus oryzae CFTRI 1480, an isolate from a spoiled moist sample of casein, produced 59,105 units of an extracellular proteinase/g dry mouldy bran (DMB) at 72 h in an arbitrarily formulated wheat bran medium in a solid state fermentation system. The enzyme production was significantly affected by mineral salt content and pH of the liquid used for moistening the wheat bran. Enzyme titres were enhanced 1.34-fold with the addition of 0.4% corn starch. Optimization of key parameters, i.e., initial moisture content, age and size of inoculum, increased the enzyme production to 191,869 units/g DMB and reduced the fermentation time to 48 h. Such high titres in a simple medium, surpassing most of the literature reports, indicate the industrial importance of the culture. The properties of acetone-precipitated enzyme, viz, the optimum pH of 10.0, more than 95% activity between pH 7.0 and 10.0, temperature optimum at 55° C and more than 90% activity between 10 and 27°C, are similar to those of commercially available fungal proteinases employed in animal feed, leather and other industries. Correspondence to: B. K. Lonsane     

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.     

Bacterial diversity in thermophilic aerobic sewage sludge

Summary Using -amylase as an example, extremely thermophilic Bacilli isolated from heat-treated sewage sludge are shown to be a source for enzymes stable and active at high temperatures. The isolates which are classified as subspecies of Bacillus stearothermophilus differ from each other in protein composition indicating the heterogeneiety of that subspecies. Media are evaluated for good growth and high enzyme productivity. Best media are those composed of three or four different complex components like combinations of peptone, soy grist, and malt extract, -amylase production on simple carbon sources is negligible. From the cultivation supernatants crude -amylase extracts are prepared and their behaviour at high temperatures is described. The optimal temperature of all tested enzymes is 80°C. They are stable at suboptimal temperatures for over 20 h and at 95° C 50% of their activity is lost within 2 h. The activity at 95° C is however preserved for over 3 h in presence of starch. The products of the starch digestion are maltotriose, maltose, and some glucose. The amylases can therefore compete in activity and stability with commercially available -amylases from Bacillus licheniformis.     

Enrichment of laccase production by Phoma herbarum isolate KU4 under solid-state fermentation by optimizing RSM coefficients using genetic algorithm

The process parameters were optimized to obtain enhanced enzyme activity from the fungus Phoma herbarum isolate KU4 using rice straw and saw dust as substrate under solid-state fermentation using Response surface methodology (RSM). Genetic algorithm was used to validate the RSM for maximum laccase production. Six variables, viz., pH of the media, initial moisture content, copper sulphate concentration, concentration of tannic acid, inoculum concentration and incubation time were found to be effective and optimized for enhanced production. Maximum laccase production was achieved by RSM at pH 5·0 and 86% of initial moisture content of the culture medium, 150 µmol l⁻¹ of CuSO₄, 1·5% tannic acid and 0·128 g inoculum g⁻¹ dry substrate inoculum size on the fourth day of fermentation. The highest laccase activity was observed as 79 008 U g⁻¹, which is approximately sixfold enhanced production compared to the unoptimized condition (12 085·26 U g⁻¹).     

Production of deuterated β-carotene by metabolic labelling of Spirulina platensis

Method for production of deuterated -carotene for the bioavailability studies of vitamin A has been developed using Spirulina platensis in culture. Suspension cultures of Spirulina in heavy water (99.4% D₂O) medium produced maximum biomass and -carotene in 28 to 30 days. Of the total carotenoids, lutein constituted 30 to 35% while -carotene was about 24%. MS showed that 60 to 65% H atoms in -carotene were deuterated. 100% replacement of H atom with deuterium was achieved by preventing exchange with atmospheric moisture. The medium could be used in several cycles for metabolic labelling of carotenoids whereby the cost of production is reduced.     

Production of protease with Bacillus licheniformis mutants insensitive to repression of exoenzyme biosynthesis

Two mutant strains of Bacillus licheniformis insensitive to catabolite repression were selected by classical mutagenesis in connection with the development of a fed-batch procedure for protease production. B. licheniformis 4a produced up to 20 U (Anson-Units) subtilisin Carlsberg/ml in fed-batch experiments in the presence of up to 1.5 m glycerol, but was inhibited by excess ammonium. Formation of spores, excretion of -amylase and the biosynthesis of citrate synthase and isocitrate dehydrogenase were likewise not repressed by glycerol. The strain was characterized by unusually low activity of the -oxoglutarate dehydrogenase complex and increased biosynthesis of polyglutamic acid in the presence and excretion of -oxoglutarate in the absence of ammonium, respectively. The results are discussed in view of a possible connection between the defect in the -oxoglutarate dehydrogenase complex and insensitivity to catabolite repression. The second strain B. licheniformis 114 was able to synthesize 11.5 U protease/ml independently of the glycerol and ammonium concentration in the medium. Correspondence to: G. Bierbaum     

Phage lambda pL promoter controlled α-amylase expression inEscherichia coli during fermentation

Summary Phage lambda p_L promoter controlled expression of theBacillus stearothermophilus gene coding for a thermostable -amylase inE. coli was studied in shake flask cultures and in a laboratory fermenter. At an inducible temperature (40 °C) the final cell density was lower, but the total enzyme activity produced ca. 80% higher than at a non-inducible temperature (30 °C). Moreover, 17% of the total enzyme activity was found in the culture medium. The -amylase yield, production rate and proportion secreted were further increased by shifting the fermentation temperature after certain period of bacterial growth rather than at the beginning of fermentation.     

Utilization of rice straw for laccase production by <Emphasis Type="Italic">Streptomyces psammoticus</Emphasis> in solid-state fermentation

Laccase production from a novel actinobacterial strain, Streptomyces psammoticus, MTCC 7334 was optimized in solid-state fermentation. The process parameters were initially optimized by the conventional “one factor at a time” approach, and the optimal levels of the factors that had considerable influence on enzyme production were identified by response surface methodology. Rice straw was identified as a suitable substrate for laccase production (17.3 U/g), followed by coffee pulp (15.8 U/g). Other optimized conditions were particle size, 500–1,000 μm (21.2 U/g); initial moisture content, 65% (26.8 U/g); pH of moistening solution, 8.0 (26.9 U/g); incubation temperature, 32°C (27.6 U/g) and inoculum size, 1.5 × 10⁷ CFU (33.8 U/g). Yeast extract served as the best nitrogen source (34.8 U/g). No enhancement in enzyme yield was observed with carbon supplementation. The level of yeast extract, inoculum size and copper sulphate were optimized statistically. Statistical optimization performed using a central composite design resulted in threefold increase in laccase activity (55.4 U/g) as compared to the unoptimized medium (17.3 U/g). The upgrading of fermented rice straw for fodder enhancement is also discussed briefly.