Solid-state Fermentation Medium Research Articles

Kinetic Study of a Glucose Tolerant β-Glucosidase from Aspergillus fumigatus ABK9 Entrapped into Alginate Beads

A β-glucosidase from Aspergillus fumigatus ABK9 was purified from a pre-optimized solid state fermentation medium. The molecular weight of the purified enzyme (62.78 kDa) was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, zymogram analysis and confirmed by MALDI-TOF mass spectrometry. The purified enzyme was entrapped in 4 % alginate beads and some physico-chemical properties of free and immobilized β-glucosidase were analyzed. The immobilized enzyme displayed higher Km and Vmax values but lower Kcat/Km value in comparison to its free counterpart. The pH and temperature stability of the enzyme were enhanced after immobilization. Increased thermostability of the immobilized enzyme was evidenced by the high activation energy (48.80 kJ mol−1) for thermal denaturation, longer half-life (T1/2) (1,037 min at 50 °C), higher melting temperature (Tm) (85 °C), and temperature coefficient (Q10) values (1.0). Besides superior thermodynamic properties, increased storage stability (80 % after 30 days), glucose tolerance (Ki = 430 mM) and reusability of the immobilized enzyme (nine cycles until E1/2) confirmed its promising industrial applicability.

Enzymatic Hydrolysis of Sugarcane Bagasse Using Enzyme Extract and Whole Solid-state Fermentation Medium of Two Newly Isolated Strains of Aspergillus Oryzae

Enzymatic conversion of lignocellulosic biomass into fuels and chemicals will be a key technology in the future. However, in order to make this process economically feasible, it is necessary to improve the efficiency of enzyme production, since the cost of the enzymatic cocktails significantly influences the viability of the overall process. In this sense, the use of solid-state fermentation (SSF) is particularly advantageous for enzyme production. Here, a comparative study on the enzymatic hydrolysis of steam- exploded sugarcane bagasse (SESB) using enzymatic extract (EE) and whole solid-state fermentation medium (WM) of two newly isolated strains of Aspergillus oryzae (P6B2 and P27C3A) from the Amazon Rainforest was carried out. The biomass conversion using WM from A. oryzae P6B2 was more efficient when compared with the EE, while for A. oryzae P27C3A the conversion yields were similar. The WM from P27C3A supplemented with a low dosage of commercial enzyme resulted in a final conversion of 45% of the theoretical value. Furthermore, the combination of the enzymes from both strains, allowed up to 5 folds improvement in SESB conversion. These results showed that the in-house enzyme production by wild-typeoryzae strains cultivated under SSF can be very advantageous for the enzymatic hydrolysis of biomass and, thus, it can be considered as a potential biotechnological configuration for the production of biofuels.

Ultrasound-assisted extraction and characterization of hydrolytic and oxidative enzymes produced by solid state fermentation

Ligninolytic and hydrolytic enzymes were produced with six selected fungi on flax substrate by solid state fermentation (SSF). The extracellular enzyme production of the organisms in two SSF media was evaluated by measuring the soluble protein concentration and the filter paper, endoxylanase, 1,4-β-d-glucosidase, 1,4-β-d-endoglucanase, polygalacturonase, lignin peroxidase, manganese peroxidase and laccase activities of the clear culture solutions produced by conventional extraction from the SSF materials. The SSF material of the best enzyme producer (Trichoderma virens TUB F-498) was further investigated to enhance the enzyme recovery by low frequency ultrasound treatment. Performance of both the original and ultrasound macerated crude enzyme mixtures was evaluated in degradation of the colored lignin-containing and waxy materials of raw linen fabric. Results proved that sonication (at 40%, 60% and 80% amplitudes, for 60min) did not result in reduction in the filter paper, lignin peroxidase and laccase activities of the crude enzyme solution, but has a significant positive effect on the efficiency of enzyme extraction from the SSF material. Depending on the parameters of sonication, the enzyme activities in the extracts obtained can be increased up to 129–413% of the original activities measured in the control extracts recovered by a common magnetic stirrer. Sonication also has an effect on both the enzymatic removal of the lignin-containing color materials and hydrophobic surface layer from the raw linen.

Use of Spent Mushroom Substrate for Production of <I>Bacillus thuringiensis</I> by Solid-State Fermentation

The aim of this study was to explore a cost-effective method for the mass production of Bacillus thuringiensis (Bt) by solid-state fermentation. As a locally available agroindustrial byproduct, spent mushroom substrate (SMS) was used as raw material for Bt cultivation, and four combinations of SMS-based media were designed. Fermentation conditions were optimized on the best medium and the optimal conditions were determined as follows: temperature 32 degrees C, initial pH value 6, moisture content 50%, the ratio of sieved material to initial material 1:3, and inoculum volume 0.5 ml. Large scale production of B. thuringiensis subsp. israelensis (Bti) LLP29 was conducted on the optimal medium at optimal conditions. High toxicity (1,487 international toxic units/milligram) and long larvicidal persistence of the product were observed in the study, which illustrated that SMS-based solid-state fermentation medium was efficient and economical for large scale industrial production of Bt-based biopesticides. The cost of production of 1 kg of Bt was approximately US$0.075.

Optimization of Culture Conditions for the Production of Extracellular Cellulases via Solid State Fermentation

Aim: The aims of the present study were to screen different filamentous fungi for extracellular cellulases production and to optimize solid-state fermentation medium and culture conditions to enhance cellulases production. Study Design: Using agro-industrial waste as raw material for the production of cellulases by a hyper cellulase producing fungus and evaluating the influence of various parameters to design a suitable SSF process for cellulase production. Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and Biotechnology Division, National Research Centre (NRC), Cairo, Egypt, between January 2013 and October 2013. Methodology: Different filamentous fungi were grown and maintained on potato dextrose agar slants at 28oC for 7 days. The spores were washed down by distilled water. Then, 2.0 ml aliquots were used to inoculate 250 ml Erlenmeyer flasks, containing rice straw as the only carbon source. The inoculated flasks were incubated for 5 days at 28oC. The enzymes were extracted by mixing homogenously the fermented substrate with 50 ml citrate phosphate buffer (0.1 M, pH 5.0) and agitated (150 rpm) for 1 hr. Pooled extracts were centrifuged at 5000 rpm for 15min and the clear supernatant was used as a source of extracellular enzyme. Results: Aspergillus oryzae NRRL 3484 exhibited relatively higher cellulases production. Original Research Article British Microbiology Research Journal, 4(6): 698-714, 2014 699 The optimum incubation period, temperature, and initial moisture level were reported on the 7 day, at 28°C, and 70%, respectively. Peptone proved to be the suitable nitrogen source followed by yeast extract, while pH 5.0 was ideal for cellulases production. Conclusion: Using ligninolytic fungi, including their enzymes, may be one potential alternative to provide a more practical and environmental-friendly approach for enhancing the nutritive value of rice straw. Moreover, the application of ligninolytic fungi or their enzymes combined with chemical pre-treatments to rice straw may be an alternative way to shorten the period of the incubation times and (or) decrease the amount of chemicals, effecting some synergy.

Induced Production of Exoglucanase, and <i>β</i>-Glucosidase from Fungal Co-Culture of T. <i>viride</i> and G. <i>lucidum</i>

In the present study, a co-culture technique was adopted with an aim to investigate a hyper production of exoglucanase, and β-glucosidase using cheap and easily available agro-industrial residue corn stover as growth supporting substrate. Various physio-chemical and nutritional variables were optimized using classical and completely randomized designs for induced production of exoglucanase, and β-glucosidase from the co-culture of Trichoderma viride and Ganoderma lucidum in solid state fermentation (SSF). Analysis profile showed that when the conditions of the SSF medium containing 15 g corn stover substrate (50% w/w moisture) inoculated with 6 mL of inoculum were optimal, the maximum productions of exoglucanase (485 ± 6.5 U/mL) and β-glucosidase (255 ± 3.3 U/mL) were recorded after 5 days of incubation at pH 6 and 35°C.

Optimization of Cultural Conditions for Solid State Fermentation of Amylase Production by Aspergillus species

Amylase is an amylolytic enzyme used in food industry which is generally produced by Aspergillus spp. under solid state fermentation. The present study is concerned with the isolation, screening and selection of suitable strains of Aspergillus spp. and optimization of cultural conditions for the biosynthesis of amylase. Rice and wheat brans were used as substrates which are readily available inexpensive raw materials for amylase production. From 85 samples of rice and wheat grains, 55 colonies obtained on potato dextrose agar (PDA) were suspected to be Aspergillus oryzae and only 35 colonies possessed the morphological characteristics similar to that of A. oryzae indicating the isolates were most likely the strains of A. oryzae. Of all the fungal isolates of Aspergillus spps., Asp.31 gave maximum production of amylase (720.782 IUgds-1) in solid state fermentation media. This strain was selected as a parental strain for optimization for cultural conditions. The obtained data were analyzed using SPSS- 11.5 program. Of all the substrates (rice bran, wheat bran and their mixture), rice bran was the best for producing amylase of highest activity 611.614 IUgds-1.The highest enzyme activity of 698.749 IUgds-1 was observed at 50% initial moisture level of the substrate. The optimum temperature was 25°C for producing the crude amylase enzyme with amylase activity of 577.757 IUgds-1. Nepal Journal of Science and Technology Vol. 14, No. 1 (2013) 67-74 DOI: http://dx.doi.org/10.3126/njst.v14i1.8924

Extracellular endo-mannanase from Bacillus sp. CFR1601: Economical production using response surface methodology and downstream processing using aqueous two phase system

Bacillus sp. CFR1601, isolated from decaying plant litter, produced an extra-cellular endo-mannanase (198.0IU/g) under solid state fermentation (SSF) using defatted coconut residue as the prime solid substrate. In order to enhance endo-mannanase production, three component, five level central composite design (CCD) of response surface methodology (RSM) was used. Based on contour plots and variance analysis, optimum conditions for endo-mannanase production from Bacillus sp. CFR1601 were attained when defatted coconut residue was supplemented with sesame oil meal (10.0, w/w), Tween-80 (0.2%, v/v) and inoculated with bacterial cells from log phase (12h old; OD600nm≈3.6). The empirical model developed through RSM brought about 4.04–4.39-fold (800.0–870.0IU/g) improvement in endo-mannanase yield as compared to un-optimized growth conditions. Downstream processing of endo-mannanase from SSF media was carried out for the first time using polyethylene glycol (PEG)/salt aqueous two phase system (ATPS). ATPS system consisting of a combination of PEG 3350 12.0% (w/w), Na2SO4 12.0% (w/w), protein load 10.0% (w/w) and pH 5.0 resulted in one-sided partitioning of endo-mannanase towards bottom phase with 3.8-fold purification and 95.4% recovery. Second stage ATPS with fresh top phase further improved purification of endo-mannanase to 12.32-fold. Our overall results suggest a cost-effective and integrated process for production and downstream processing of endo-mannanase.

Medium optimization for keratinase production by a local Streptomyces sp. NRC 13S under solid state fermentation

Thirteen different Streptomyces isolates were evaluated for their ability to produce keratinase using chicken feather as a sole carbon and nitrogen sources under solid state fermentation (SSF). Streptomyces sp. NRC 13S produced the highest keratinase activity (1,792 U/g fermented substrate (fs)). The phenotypic characterization and analysis of 16S rDNA sequencing of the isolate were studied. Optimization of SSF medium for keratinase production by the local isolate, Streptomyces sp. NRC13S, was carried out using the one-variable-at-a-time and the statistical approaches. In the first optimization step, the effect of incubation period, initial moisture content, initial pH value of the fermentation medium, and supplementation of some agro-industrial by-products on keratinase production were evaluated. The strain produced about 2,310 U/gfs when it grew on chicken feather with moisture content of 75% (w/w), feather: fodder yeast ratio of 70:30 (w/w), and initial pH 7 using phosphate buffer after 8 days. Based on these results, the Box-Behnken design and response surface methodology were applied to find out the optimal conditions for the enzyme production. The corresponding maximal production of keratinase was about 2,569.38 U/gfs.

Purification and characterization of an extracellular laccase from solid-state culture of Pleurotus ostreatus HP-1.

A native isolate of Pleurotus ostreatus HP-1 (Genbank Accession No. EU420068) was found to have an excellent laccase producing ability. The extracellular laccase was purified to electrophoretic homogeneity from copper sulphate induced solid-state fermentation medium by ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was determined to be monomeric protein with an apparent molecular mass of 68,420 kDa, and an isoelectric point (pI) of 3.5. The inductively coupled plasma spectroscopy showed a presence of iron, zinc and copper in the purified enzyme. The absorption spectrum in the range of 200–700 nm showed the maximum absorption at 610 nm characteristic of fungal laccase and corresponding to the presence of type I copper atom. The laccase was stable at different temperatures up to 70 °C and retained 61 % activity at 50 °C. The enzyme reaction was inhibited by cysteine; sodium azide and EDTA. The enzyme oxidized various known laccase substrates, its lowest Km value being for ortho-dianisidine and highest Kcat and Kcat/Km for ABTS. The purified laccase exhibited different pH optima for different substrates. The N-terminal sequence did not show any similarity with N-terminal sequence of other species of genera Pleurotus.

Multiresponse Optimization of Inoculum Conditions for the Production of Amylases and Proteases by Aspergillus awamori in Solid-State Fermentation of Babassu Cake

This work aimed at investigating the simultaneous production of amylases and proteases by solid-state fermentation (SSF) of babassu cake using Aspergillus awamori IOC-3914. By means of experimental design techniques and the desirability function, optimum inoculum conditions (C/N ratio of propagation medium, inoculum age, and concentration of inoculum added to SSF medium) for the production of both groups of enzymes were found to be 25.8, 28.4 h, and 9.1 mg g−1, respectively. Significant influence of both initial C/N ratio and inoculum concentration was observed. Optimum amylolytic activities predicted by this multiresponse analysis were validated by independent experiments, thus indicating the efficacy of this approach.

Optimization of the production of poly-γ-glutamic acid by Bacillus amyloliquefaciens C1 in solid-state fermentation using dairy manure compost and monosodium glutamate production residues as basic substrates

Poly-γ-glutamic acid (γ-PGA) is a polymer with uses in foods, cosmetics, medicine and agriculture. The medium for the production of γ-PGA by Bacillus amyloliquefaciens C1 was optimized by response surface methodology using agro-industrial wastes in solid-state fermentation (SSF). The optimal SSF medium (20 g substrates with 50% initial moisture) for producing γ-PGA was determined to contain 5.51 g dairy manure compost, 1.91 g soybean cake, 0.57 g corn flour, 2.15 g monosodium glutamate production residues, 1.5 g wheat bran, 0.5 g rapeseed cake, 0.1 g citric acid, 0.05 g MgSO 4·7H 2O and 0.03 g MnSO 4·H 2O. In this medium the strain produced up to 0.0437 g γ-PGA per gram of substrates when cultured for 48 h at 37 °C. SDS–PAGE showed that the molecular weight of the γ-PGA was more than 130 kDa. Due to the high-yields observed and the low-cost nature of the optimal medium, this study indicates a possibility to establish economical large-scale production of γ-PGA.

Optimization of physical and nutritional factors for synthesis of lignin degrading enzymes by a novel strain of Trametes vericolor

This paper reports the production of ligninase enzymes by a new strain of Trametes versicolor IBL-04 producing a novel pattern of ligninolytic enzymes with highest MnP activities followed by LiP and laccase. In previous studies Trametes versicolor has been reported to produce higher activities of MnP, followed by laccase and LiP. Lignocellulosic substrates including wheat straw, rice straw, banana stalks, corncobs, corn stover, and sugarcane bagasse were used in solid state fermentation (SSF) for the production of ligninases including peroxidase (LiP), manganese peroxidase (MnP), and laccase by Trametes versicolor IBL-04. Maximum production of MnP (998 U/mL), LiP (620 U/mL), and Laccase (49.7 U/mL) was observed after 5 days in the SSF medium containing 5g rice straw (60% w/w moisture) in still culture SSF. Moisture, pH, temperature, inoculums size, additional carbon and nitrogen sources, and surfactants had a significant influence on ligninase synthesis by the fungus. Production of ligninases was substantially enhanced by optimizing SSF production process. Maximum MnP (1775 U/mL), LiP (1663 U/mL), and laccase (99 U/mL) were produced when rice straw (5g) at 66.6 % moisture (w/w) receiving 5ml inoculum was incubated at pH 4.0 and 30oC in the presence of maltose (1% w/w) as carbon source, urea (0.2% w/w) as nitrogen source and 1mM Tween-80 (0.3 ml) as surfactant.

Detection of the Typical Condition of chitinase Production from the Yeast Saccharomyces cerevisiae S4

Five Saccharomyces cerevisiae isolated from the ability of chitinase production from the isolates were studied. Quantitative screening appeared that Saccharomyces cerevisiae S4 was the highest chitinase producer specific activity 1.9 unit/mg protein. The yeast was culture in liquid and solid state fermentation media (SSF). Different plant obstanases were used for (SSF) with the chitine, while liquid media contained chitine with the diffrented nitrogen source. The favorable condition for chitinase producers were incubated at 30 ºC at pH 6 and 1% colloidal chitine.

Detection of the Typical Condition of chitinase Production from the Yeast Saccharomyces cerevisiae S4

Five Saccharomyces cerevisiae isolated from the ability of chitinase production from the isolates were studied. Quantitative screening appeared that Saccharomyces cerevisiae S4 was the highest chitinase producer specific activity 1.9 unit/mg protein. The yeast was culture in liquid and solid state fermentation media (SSF). Different plant obstanases were used for (SSF) with the chitine, while liquid media contained chitine with the diffrented nitrogen source. The favorable condition for chitinase producers were incubated at 30 ºC at pH 6 and 1% colloidal chitine.

Enhanced phytase production through interspecific protoplast fusion of Aspergillus niger CFR 335 and Aspergillus ficuum SGA 01 auxotrophic mutants

Interspecific protoplast fusion between Aspergillus niger CFR 335 and Aspergillus ficuum SGA 01 auxotrophic mutant strains was carried out in the presence of PEG-3500 in order to enhance the phytase production. The protoplasting frequency found was 2.64 × 10 −3 and 3.72 × 10 −3 while the regeneration frequency was 32 × 10 −2 and 47 × 10 −2 in A. niger CFR 335 and A. ficuum SGA 01 respectively. Fusion frequency of 90 × 10 −2 was obtained when equal numbers of two protoplasts were induced to fuse. Sixteen stable hybrids were characterized for phytase production, their biomass and DNA contents. The hybrid biomasses were subjected to proximate analysis such as estimation of total sugars, proteins, total lipids and ash. H1 was found promising with high stability and delayed sporulation with the onset after 10 days of incubation while the maximum enzyme production of 126.2 U/gds was attained by 6–7 days, in solid-state fermentation medium. As there are no such studies reported till date, the present work opens a new area in phytase research in developing stable hybrids with desirable characteristics to meet present market demand for the enzyme.

A new green technology for direct production of low molecular weight chitosan

Low molecular weight chitosan (LMW-chitosan) in the molecular range 5–10 kDa was firstly prepared directly from the Absidia coerulea mycelia. To improve the LMW-chitosan production, the solid-state fermentation media were optimized to investigate the influence of substrate and supplemental medium components on LMW-chitosan production. The LMW-chitosan was obtained after treatments with 2% NaOH and 10% acetic acid. Maximal LMW-chitosan production was 6.12 g/kg substrate. Gel permeation chromatography combined with laser light scattering gave a M w of 6.4 kDa with M w/ M n of 1.09. FT-IR, X-ray diffraction and 13 C NMR spectra of the product showed typical peak distributions the same as those of standard chitosan which confirmed the extracted product to be chitosan-like. The method provided a new, simple and green technology to produce LMW-chitosan directly.

Production and Application of Novel Sterol Esterases from Aspergillus Strains by Solid State Fermentation

AbstractAmong numerous mesophilic fungi screened for sterol esterase activity followed by the esterification reaction between plant β‐sitosterol and lauric acid in organic solvent, six Aspergillus strains were selected as the most active producers. These fungi had not been studied previously for sterol esterase production. The fungi were cultivated under solid state fermentation (SSF) conditions. The gently dried SSF cultures as such were tested in the esterification reactions, without any special enzyme isolation and purification (downstream) processes. All the six Aspergillus SSF preparations were able to synthesize sterol esters. Sterol esterase activity of these GRAS cleared Aspergillus strains was inducible by sterol ester supplementation to the SSF medium and showed remarkably different moisture optimum during growth as compared to the production of lipase (determined by pNP‐palmitate). Genome analysis revealed that sterol esterase production might be a common feature of many Aspergillus species. The synthetic usefulness of the best SSF preparations of A. oryzae NRRL 6270 and A. sojae NRRL 6271 was demonstrated by synthesis of esters of plant sterols with lauric acid resulting in 45–63% conversions (GC) and 27–38% isolated yields of steryl laurates. The isomer preference of A. oryzae NRRL 6270 towards the 10E,12Z isomer of conjugated linoleic acid (CLA) in the esterification reaction with plant sterols was also determined.

Enhanced lignin peroxidase synthesis by Phanerochaete Chrysosporium in solid state bioprocessing of a lignocellulosic substrate

A solid state fermentation (SSF) process for the production of lignin peroxidase was optimized to enhance enzyme production by Phanerochaete chrysosporium. Optimization of the corncob SSF medium caused a significant reduction in fermentation time to give maximum lignin peroxidase yield. Supplementation of the SSF medium by low concentrations of peptone, yeast extract and Tween-80 enhanced lignin peroxidase production. Maximum yield of lignin peroxidase was 13.7 U/gds (units per gram dry substrate) noted after 5 days of SSF with 70% moisture and 20% (v/w) inoculum.

Medium optimization by response surface methodology for poly-γ-glutamic acid production using dairy manure as the basis of a solid substrate

Dairy manure, supplemented with agro-industrial materials, was used as the solid substrate for high yield of poly-gamma-glutamic acid (gamma-PGA) by Bacillus subtilis CCTCC202048. The solid-state fermentation medium was optimized by response surface methodology. In the first optimization step, a Plackett-Burman design was used to evaluate the influence of related factors. Wheat bran, soybean cake and glutamic acid were found to be more compatible supplement with dairy manure and positively influenced on gamma-PGA production. In the second step, the concentrations of the three supplemental nutrients above were further optimized using a Box-Behnken design. The average gamma-PGA yield (4.70%) in triplicate under optimal conditions was obtained on the laboratory scale, whereas it was 3.58% at compost experiment. These would lay a foundation for lessening the pollution of dairy manure, increasing fertilizer efficiency and exploring a late-model organic fertilizer that retains water and nutrients.