Enzymatic production of N-acetyl-D-glucosamine by solid state fermentation of chitinase by Penicillium ochrochloron MTCC 517 using agricultural residues

The optimization studies for production of chitinase were carried out by response surface methodology (RSM) based on statistics experimental design using three substrates, which were wheat, rice and red gram bran. 2⁴ full factorial central composite design was applied to evaluate optimal combinations of variables. These variables were chitin concentration, initial moisture content, inoculum level, and incubation time. The results of second order polynomial showed that all four variables had significant effect on chitinase production. Maximum chitinase activity was recorded for wheat bran (2443.23 U g⁻¹) than rice (1216.65 U g⁻¹) and red gram bran (961.32 U g⁻¹). An overall 3-fold increase in chitinase activity was achieved using optimized strategies of RSM. Growth of the fungus on all bran particles have been visualized by scanning electron microscopy. These results indicated the potential of Penicillium ochrochloron for economical production of chitinase using agricultural residues. TLC and HPLC analysis of colloidal chitin hydrolysate with partially purified chitinases revealed that the major reaction product was monomeric GlcNAc indicating the potential of these enzymes for efficient production of GlcNAc.     

Production and optimization of thermophilic alkaline protease in solid-state fermentation by <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CN902

The purpose of the present research is to study the production of thermophilic alkaline protease by a local isolate, Streptomyces sp. CN902, under solid state fermentation (SSF). Optimum SSF parameters for enzyme production have been determined. Various locally available agro-industrial residues have been screened individually or as mixtures for alkaline protease production in SSF. The combination of wheat bran (WB) with chopped date stones (CDS) (5:5) proved to be an efficient mixture for protease production as it gave the highest enzyme activity (90.50 U g⁻¹) when compared to individual WB (74.50 U g⁻¹) or CDS (69.50 U g⁻¹) substrates. This mixed solid substrate was used for the production of protease from Streptomyces sp. CN902 under SSF. Maximal protease production (220.50 U g⁻¹) was obtained with an initial moisture content of 60%, an inoculum level of 1 × 10⁸ (spore g⁻¹ substrate) when incubated at 45°C for 5 days. Supplementation of WB and CDS mixtures with yeast extract as a nitrogen source further increased protease production to 245.50 U g⁻¹ under SSF. Our data demonstrated the usefulness of solid-state fermentation in the production of alkaline protease using WB and CDS mixtures as substrate. Moreover, this approach offered significant benefits due to abundant agro-industrial substrate availability and cheaper cost.     

Chitinase Production in Solid-State Fermentation by Enterobacter sp. NRG4 Using Statistical Experimental Design

The optimization of nutrient levels for chitinase production by Enterobacter sp. NRG4 in solid-state fermentation conditions (SSF) was carried out using response surface methodology (RSM) based on central composite design (CCD). The design was employed by selecting wheat bran-to-flake chitin ratio, moisture level, inoculum size, and incubation time as model factors. The results of first-order factorial design experiments showed that all four independent variables have significant effects on chitinase production. The optimum concentrations for chitinase production were wheat bran-to-flake chitin ratio, 1; moisture level, 80%; inoculum size, 2.6 mL; and incubation time, 168 h. Using this statistical optimization method, chitinase production was found to increase from 616 U · g⁻¹ dry weight of solid substrate to 1475 U · g⁻¹ dry weight of solid substrate.     

Production of cephamycin C by <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis> NT4 using solid-state fermentation

Cephamycin C is an extracellular broad spectrum β-lactam antibiotic produced by Streptomyces clavuligerus, S. cattleya and Nocardia lactamdurans. In the present study, different substrates for solid-state fermentation were screened for maximum cephamycin C production by S. clavuligerus NT4. The fermentation parameters such as substrate concentration, moisture content, potassium dihydrogen phosphate, inoculum size and ammonium oxalate were optimized by response surface methodology (RSM). The optimized conditions yielded 21.68 ± 0.76 mg gds⁻¹ of cephamycin C as compared to 10.50 ± 1.04 mg gds⁻¹ before optimization. Effect of various amino acids on cephamycin C production was further studied by using RSM, which resulted in increased yield of 27.41 ± 0.65 mg gds⁻¹.     

Concentration of fungal ligninolytic enzymes by ultrafiltration and their use in distillery effluent decolorization

Extracellular ligninolytic enzymes secreted by seven different fungi grown under solid-state fermentation using agro-residue as substrate were extracted through successive extractions. In general, most of the enzymes were recovered during first and second extractions. These extractants were then subjected to ultrafiltration using a 10 kDa membrane for further concentration. The permeates collected after every filtration and the final retentate were analyzed for protein and enzymes. In all the isolates, enzyme concentration was maximum in first retentate, which reduced significantly in second, third, and fourth retentate. Maximum per unit laccase (14.44 U g⁻¹) and MnP production (142.2 U g⁻¹) was observed in Fusarium verticillioides TERIDB16 while maximum LiP production (137.42 U g⁻¹) was in Alternaria gaisen TERIDB6. The retentate was further used for checking its decolorization efficiency of undiluted distillery effluent. The maximum decolorization (37%) was obtained using the enzyme extract of Pleurotus florida EM1303.     

Use of response surface methodology for optimizing process parameters for high inulinase production by the marine yeast <Emphasis Type="Italic">Cryptococcus aureus</Emphasis> G7a in solid-state fermentation and hydrolysis of inulin

The optimization of process parameters for high inulinase production by the marine yeast strain Cryptococcus aureus G7a in solid-state fermentation (SSF) was carried out using central composite design (CCD), one of the response surface methodologies (RSMs). We found that moisture, inoculation size, the amount ratio of wheat bran to rice husk, temperature and pH had great influence on inulinase production by strain G7a. Therefore, the CCD was used to evaluate the influence of the five factors on the inulinase production by strain G7a. Then, five levels of the five factors above were further optimized using the CCD. Finally, the optimal parameters obtained with the RSM were the initial moisture 61.5%, inoculum 2.75%, the amount ratio of wheat bran to rice husk 0.42, temperature 29 °C, pH 5.5. Under the optimized conditions, 420.9 U g⁻¹ of dry substrate of inulinase activity was reached in the solid-state fermentation culture of strain G7a within 120 h whereas the predicted maximum inulinase activity of 436.2 U g⁻¹ of inulinase activity of 436.2 U g⁻¹ of dry weight was derived from the RSM regression. This is the highest inulinase activity produced by the yeast strain reported so far. A large amount of monosaccharides and oligosaccharides were detected after inulin hydrolysis by the crude inulinase.     

De-colourization of textile effluent using immobilized Geotrichum candidum: an insight into mycoremediation

Textile effluent is generally complicated to manage because of its extremely noxious and recalcitrant coloured compositions. Mycoremediation is an extensively used strategy for the competent degradation of hazardous pollutants present in textile effluent. Fungus could be immobilized in synthetic or natural matrices. The current study shows the decolourization of the textile effluent by 85·5 and 98·5% within 6 h using suspended and immobilized fungus, Geotrichum candidum with optimized parameters like inoculum size (5%), pH (4·5), and temperature (30°C). To maintain a high biomass of fungal population and enhance the retention of fungal strain in the contaminated sites, the fungi need to be immobilized. Hence, the fungus was immobilized naturally onto the selected inert support that is, coconut fibres by the means of adsorption, where they grew as active films on the fibres after being grown in the culture broth. The optimized process parameters of inoculum size, fibre quantity and agitation speed for immobilized G. candidum were 5%, 2·2 g l⁻¹ of effluent and 100 rev min⁻¹ respectively. High level of laccase (22 and 25 U l⁻¹ in suspended and immobilized fungal cells treatment respectively) was observed during the process of decolourization and it was found that decolourization was directly proportional to the laccase activity. The UV–vis, FTIR, ¹H NMR and GC-MS analyses of treated textile industrial wastewater revealed the degradation of toxic pollutants in the textile effluent and formation of lower molecular weight intermediates. The study revealed a higher efficacy of immobilized G. candidum in comparison to suspended fungal culture, employing ligninolytic enzyme laccase, which catalyzes the degradation/transformation of aromatic dyes in the textile effluent thus decolourizing it.     

Solid state bioreactor production of transglutaminase by Amazonian Bacillus circulans BL32 strain

In this work, we investigated the production of transglutaminase (TGase) by an Amazonian isolated strain of Bacillus circulans by solid-state cultivation (SSC). Several agro-industrial residues, such as untreated corn grits, milled brewers rice, industrial fibrous soy residue, soy hull, and malt bagasse, were used as substrates for microbial growth and enzyme production. Growth on industrial fibrous soy residue, which is rich in protein and hemicellulose, produced the highest TGase activity (0.74 U g⁻¹ of dried substrate after 48 h of incubation). A 2³ central composite design was applied to determine the optimal conditions of aeration, cultivation temperature and inoculum cell concentration to TGase production. The best culture conditions were determined as being 0.6 L air min⁻¹, 33 °C and 10 log ₁₀ CFU g⁻¹ of dried substrate, respectively. Under the proposed optimized conditions, the model predicted an enzyme production of 1.16 U g⁻¹ of dried substrate, closely matching the experimental activity of 1.25 U g⁻¹. Results presented in this work point to the use of this newly isolated B. circulans strain as a potential alternative of microbial source for TGase production by SSC, using inexpensive culture media.     

Evaluation of alpha-galactosidase biosynthesis by Streptomyces griseoloalbus in solid-state fermentation using response surface methodology

Aim: The present study was aimed at evaluating the effects of the three crucial factors, galactose concentration, inoculum size and moisture content, on α‐galactosidase production by the filamentous actinobacterium Streptomyces griseoloalbus in solid‐state fermentation. Methods and Results: Central Composite design was adopted to derive a statistical model for the optimization of fermentation conditions. Maximum α‐galactosidase yield (117 U g^–1 of dry fermented substrate) was obtained when soya bean flour supplemented with 1·5% galactose and with initial moisture content of 40% was inoculated with 1·9 × 10⁶ CFU g^?1 initial dry substrate. Conclusions: The model was valid and could result in considerably enhanced enzyme yield. Significance and Impact of the Study: The results indicated a cost effective method for the production of α‐galactosidase using soya bean flour. This is the first report on exploitation of the potential of filamentous bacterium for the production of α‐galactosidase, an enzyme having versatile applications.     

Enhanced inulinase production in solid state fermentation by a mutant of the marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> using surface response methodology and inulin hydrolysis

In order to isolate inulinase overproducers of the marine yeast Pichia guilliermondii, strain 1, cells were mutated by using UV light and LiCl₂. One mutant (M-30) with enhanced inulinase production was obtained. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant in solid-state fermentation. The initial moisture, inoculum, the amount ratio of wheat bran to rice bran, temperature, pH for the maximum inulinase production by the mutant M-30 were found to be 60.5%, 2.5%, 0.42, 30°C and 6.50, respectively. Under the optimized conditions, 455.9 U/grams of dry substrate (gds) of inulinase activity was reached in the solid state fermentation culture of the mutant M-30 whereas the predicted maximum inulinase activity of 459.2 U/gds was derived from RSM regression. Under the same conditions, its parent strain only produced 291.0 U/gds of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far.     

Effect of growth substrate,method of fermentation,and nitrogen source on lignocellulose-degrading enzymes production by white-rot basidiomycetes

The exploration of seven physiologically different white rot fungi potential to produce cellulase, xylanase, laccase, and manganese peroxidase (MnP) showed that the enzyme yield and their ratio in enzyme preparations significantly depends on the fungus species, lignocellulosic growth substrate, and cultivation method. The fruit residues were appropriate growth substrates for the production of hydrolytic enzymes and laccase. The highest endoglucanase (111 U ml⁻¹) and xylanase (135 U ml⁻¹) activities were revealed in submerged fermentation (SF) of banana peels by Pycnoporus coccineus. In the same cultivation conditions Cerrena maxima accumulated the highest level of laccase activity (7,620 U l⁻¹). The lignified materials (wheat straw and tree leaves) appeared to be appropriate for the MnP secretion by majority basidiomycetes. With few exceptions, SF favored to hydrolases and laccase production by fungi tested whereas SSF was appropriate for the MnP accumulation. Thus, the Coriolopsis polyzona hydrolases activity increased more than threefold, while laccase yield increased 15-fold when tree leaves were undergone to SF instead SSF. The supplementation of nitrogen to the control medium seemed to have a negative effect on all enzyme production in SSF of wheat straw and tree leaves by Pleurotus ostreatus. In SF peptone and ammonium containing salts significantly increased C. polyzona and Trametes versicolor hydrolases and laccase yields. However, in most cases the supplementation of media with additional nitrogen lowered the fungi specific enzyme activities. Especially strong repression of T. versicolor MnP production was revealed.     

Cellulase production from agricultural residues by recombinant fusant strain of a fungal endophyte of the marine sponge Latrunculia corticata for production of ethanol

Several fungal endophytes of the Egyptian marine sponge Latrunculia corticata were isolated, including strains Trichoderma sp. Merv6, Penicillium sp. Merv2 and Aspergillus sp. Merv70. These fungi exhibited high cellulase activity using different lignocellulosic substrates in solid state fermentations (SSF). By applying mutagenesis and intergeneric protoplast fusion, we have obtained a recombinant strain (Tahrir-25) that overproduced cellulases (exo-β-1,4-glucanase, endo-β-1,4-glucanase and β-1,4-glucosidase) that facilitated complete cellulolysis of agricultural residues. The process parameters for cellulase production by strain Tahrir-25 were optimized in SSF. The highest cellulase recovery from fermentation slurries was achieved with 0.2% Tween 80 as leaching agent. Enzyme production was optimized under the following conditions: initial moisture content of 60% (v/w), inoculum size of 10⁶ spores ml⁻¹, average substrate particle size of 1.0 mm, mixture of sugarcane bagasse and corncob (2:1) as the carbon source supplemented with carboxymethyl cellulose (CMC) and corn steep solids, fermentation time of 7 days, medium pH of 5.5 at 30°C. These optimized conditions yielded 450, 191, and 225 units/gram dry substrate (U gds⁻¹) of carboxylmethyl cellulase, filter-paperase (FPase), and β-glucosidase, respectively. Subsequent fermentation by the yeast, Saccharomyces cerevisiae NRC2, using lignocellulose hydrolysates obtained from the optimized cellulase process produced the highest amount of ethanol (58 g l⁻¹). This study has revealed the potential of exploiting marine fungi for cost-effective production of cellulases for second generation bioethanol processes.     

Indigestible dextrin is an excellent inducer for α-amylase, α-glucosidase and glucoamylase production in a submerged culture of Aspergillus oryzae

α-Amylase activities of Aspergillus oryzae grown on dextrin or indigestible dextrin were 7·8 and 27·7 U ml⁻¹, respectively. Glucoamylase activities of the cultures grown on dextrin or indigestible dextrin were 5·4 and 301 mU ml⁻¹, respectively. The specific glucoamylase production rate in indigestible dextrin batch culture reached 1·35 U g DW⁻¹ h⁻¹. In contrast, biomass concentration of A. oryzae in indigestible dextrin culture was 35% of that in dextrin culture. Thus, the culture method using indigestible dextrin has the potential to improve amylolytic enzyme production and fungal fermentation broth rheology.     

Enhanced solid‐state citric acid bio‐production using apple pomace waste through surface response methodology

Aims: To evaluate the potential of apple pomace (AP) supplemented with rice husk for hyper citric acid production through solid‐state fermentation by Aspergillus niger NRRL‐567. Optimization of two key parameters, such as moisture content and inducer (ethanol and methanol) concentration was carried out by response surface methodology. Methods and Results: In this study, the effect of two crucial process parameters for solid‐state citric acid fermentation by A. niger using AP waste supplemented with rice husk were thoroughly investigated in Erlenmeyer flasks through response surface methodology. Moisture and methanol had significant positive effect on citric acid production by A. niger grown on AP (P < 0·05). Higher values of citric acid on AP by A. niger (342·41 g kg^?1 and 248·42 g kg^?1 dry substrate) were obtained with 75% (v/w) moisture along with two inducers [3% (v/w) methanol and 3% (v/w) ethanol] with fermentation efficiency of 93·90% and 66·42%, respectively depending upon the total carbon utilized after 144 h of incubation period. With the same optimized parameters, conventional tray fermentation was conducted. The citric acid concentration of 187·96 g kg^?1 dry substrate with 3% (v/w) ethanol and 303·34 g kg^?1 dry substrate with 3% (v/w) methanol were achieved representing fermentation efficiency of 50·80% and 82·89% in tray fermentation depending upon carbon utilization after 120 h of incubation period. Conclusions: Apple pomace proved to be the promising substrate for the hyper production of citric acid through solid‐state tray fermentation, which is an economical technique and does not require any sophisticated instrumentation. Significance and Impact of the Study: The study established that the utilization of agro‐industrial wastes have positive repercussions on the economy and will help to meet the increasing demands of citric acid and moreover will help to alleviate the environmental problems resulting from the disposal of agro‐industrial wastes.     

Optimization of fermentation conditions for production of xylanase by a newly isolated strain,Penicillium thiersii ZH-19

The objective of this study was to maximize production of xylanase by a newly isolated strain Penicillium thiersii ZH-19. Response surface methodology was employed to study the effects of significant factors such as pH, temperature, xylan concentration, and cultivation time, on the production of xylanase by Penicillium thiersii ZH-19. The optimal fermentation parameters for enhanced xylanase production were found to be pH 7.72, temperature 24.8°C, xylan 13.2 g l⁻¹ and the fermentation time 125.8 h. The model predicted a xylanase activity of 75.24 U ml⁻¹. Verification of the optimization showed that the maximum xylanase production reached 73.50 U mL⁻¹ in the flask experiments and 80.23 U mL⁻¹ in the scale of 15-L fermenter under the optimal condition.     

Studies on the production of nigerloxin using agro-industrial residues by solid-state fermentation

Nigerloxin, a new and potent lipoxygenase inhibitor, was discovered in our laboratory through solid-state fermentation of wheat bran by Aspergillus niger V. Teigh (MTCC-5166). The aim of this study is to investigate the possibility of using different agro-industrial residues as nutritional supplements along with wheat bran to enhance the production of nigerloxin. Nigerloxin produced by SSF was quantified spectrophotometrically at 292 nm. The results indicate that the inhibitor production was influenced by the type of solid substrate supplemented, moisture content, pH and size of the inoculum. Individually optimized supplements were tested in different combinations to determine their effects on nigerloxin production. A twofold increase in the production of nigerloxin (4.9 ± 0.3 mg gds⁻¹) was achieved by supplementing wheat bran with 10% w/w sweet lemon peel and 5% v/w methanol at optimized process parameters, that is, an initial moisture content of 65% v/w and incubation period of 6 days with an initial inoculum size of 2 ml (8 × 10⁵ spores gds⁻¹). Nigerloxin production was stable between pH of 4 and 5.     

Impact of process parameters on chitinase production by an alkalophilic marine Beauveria bassiana in solid state fermentation

A chitinolytic fungus, Beauveria bassiana was isolated from marine sediment and significant process parameters influencing chitinase production in solid state fermentation using wheat bran were optimised. The organism was strongly alkalophilic and produced maximum chitinase at pH 9·20. The NaCl and colloidal chitin requirements varied with the type of moistening medium used. Vegetative (mycelial) inoculum was more suitable than conidial inoculum for obtaining maximal enzyme yield. The addition of phosphate and yeast extract resulted in enhancement of chitinase yield. After optimisation, the maximum enzyme yield was 246·6 units g⁻¹ initial dry substrate (U gIDS⁻¹). This is the first report of the production of chitinase from a marine fungus.     

Thermostable cellulase production of Aspergillus fumigatus Z5 under solid-state fermentation and its application in degradation of agricultural wastes

A lignocellulosic decomposing fungus Z5 was isolated and identified as Aspergillus fumigatus, its capacity to produce cellulase was assessed under solid-state fermentation (SSF) using lignocellulosic materials as substrates. Cultivation conditions of A. fumigatus Z5 for cellulase production were optimized, results showed that for carboxymethyl cellulase (CMCase) and filter paper enzyme (FPase), the best condition was 50 °C, 80% initial moisture, initial pH 4.0 and 7% initial inoculum, the average activity of CMCase activity, FPase activity reached 526.3 and 144.6 U g⁻¹ dry weight (dw) respectively, much higher than most of previous reports of this genus. Optimal temperature and pH for the CMCase activity of the crude enzyme were found to be 50 °C and 5.0, respectively. Zymogram analysis showed that eight kinds of CMCase were secreted by A. fumigatus Z5 when cellulose-containing materials were supplied in the culture. The crude enzyme secreted by the strain was further applied to hydrolyze pretreated corn stover and the enzymatic hydrolysate was used as substrate for ethanol production by Saccharomyces cerevisiae. The yield of bio-ethanol was 0.112 g g⁻¹ dry substrate (gDS), suggesting that it is a promising fungus in the bio-ethanol production process.     

Lignocellulose-degrading enzyme production by white-rot <Emphasis Type="Italic">Basidiomycetes</Emphasis> isolated from the forests of Georgia

The production of lignocellulolytic enzymes by eleven basidiomycetes species isolated from two ecosystems of Georgia was investigated for the first time under submerged (SF) and solid-state fermentation (SSF) of lignocellulosic by-products. Notable intergeneric and intrageneric differences were revealed with regard to the extent of hydrolase and oxidase activity. Several fungi produced laccase along with hydrolases in parallel with growth during the trophophase, showing that the synthesis of this enzyme is not connected with secondary metabolism. The lignocellulosic substrate type had the greatest impact on enzyme secretion. Some of the substrates significantly stimulated lignocellulolytic enzyme synthesis without supplementation of the culture medium with specific inducers. Exceptionally high carboxymethyl cellulase (CMCase, 122 U ml⁻¹) and xylanase (195 U ml⁻¹) activities were revealed in SF of mandarin peelings by Pseudotremella gibbosa IBB 22 and of residue after ethanol production (REP) by Fomes fomentarius IBB 38, respectively. The SSF of REP by T. pubescens IBB 11 ensured the highest level of laccase activity (24,690 U l⁻¹), whereas the SSF of wheat bran and SF of mandarin peels provided the highest manganese peroxidase activity (570–620 U l⁻¹) of Trichaptum biforme IBB 117. Moreover, the variation of lignocellulosic growth substrate provides an opportunity to obtain enzyme preparations containing different ratios of individual enzymes.     

Production of an anti-MUC1 C595 dbFv antibody fragment in recombinant Escherichia coli

The production of C595 diabody fragment (dbFv) in Escherichia coli (E. coli) HB2151 clone has been explored. The comparison of fermentation processes mode demonstrated that a higher biomass inoculum operation enhanced C595 dbFv production. It was demonstrated that a concentration of 12.1 mg l⁻¹ broth of dbFv and a cell concentration of 23.6 g l⁻¹ broth were achieved at the end of 75 l fermentation.