Solid-state Fermentation Cultures Research Articles

Reverse micellar extraction of fungal glucoamylase produced in solid-state fermentation culture.

Partial purification of glucoamylase from solid-state fermentation culture was, firstly, investigated by reverse micellar extraction (RME). To avoid back extraction problems, the glucoamylase was kept in the original aqueous phase, while the other undesired proteins/ enzymes were moved to the reverse micellar organic phase. The individual and interaction effects of main factors (i.e., pH and NaCl concentration in the aqueous phase, and concentration of sodium bis-2-ethyl-hexyl-sulfosuccinate (AOT) in the organic phase) were studied using response surface methodology. The optimum conditions for the maximum recovery of the enzyme were pH 2.75, 100 mM NaCl, and 200 mM AOT. Furthermore, the optimum organic to aqueous volume ratio (Vorg/Vaq) and appropriate number of sequential extraction stages were 2 and 3, respectively. Finally, 60% of the undesired enzymes including proteases and xylanases were removed from the aqueous phase, while 140% of glucoamylase activity was recovered in the aqueous phase and the purification factor of glucoamylase was found to be 3.0-fold.

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.

Use of Agro Waste Biomass for α-Amylase Production by Anoxybacillus amylolyticus: Purification and Properties

Knowledge accumulated from fundamental and applied studies on Anoxybacillus suggests that this genus can serve as a good alternative in many applications related to starch and lignocellulosic biomasses, waste treatment, enzyme technology, and bioenergy production. We investigated the purification, biochemical characterization and immobilization of a thermostable α-amylase from the thermophilic Anoxybacillus amylolyticus, strain MR3CT, isolated in Antarctica and its production on vegetable wastes. In particular, the rhizome from Arundo donax, waste biomass of Cynara cardunculus and potato peels were tested either in Submerged Fermentation (SmF) and Solid State Fermentation (SSF) conditions. The amylase from A. amylolyticus, with a molecular weight of about 60 kDa, displayed an optimum enzyme activity at 60°C and pH 5.6. Moreover, by retaining up to 70% of total activity after 48 h at 60°C, it showed high thermostability in the presence of 2 mM calcium ion. The immobilized enzyme maintained the 48% of its initial activity after the sixth reuse. The optimal conditions for its production in SmF were achieved at 60°C for 24 h with 1% of rhizome from Arundo donax, which was about 2126 U/gds. SSF cultures reached maximum α-amylase yield (102 U/gds) when grown on waste biomass of Cynara cardunculus as substrate, with a substrate-water ratio of 1:1 (w/v), and incubation at 60°C for 4 days. In this study, rhizome of A. donax resulted to be a good substrate for amylase production in SmF thus allowing a cheaper alternative to obtain amylolytic enzymes. Indeed by using rhizomes from A. donax as growth substrate it was possible to recovery an amylase activity level higher than that obtained by synthetic complex medium. Amylase production was also investigated under SSF conditions by using the above listed wastes as sole carbon source for A. amylolyticus growth. Under these conditions, C. cardunculus gave a higher enzyme yield per reactor volume.

Cytochalasins from the endophytic fungus Phomopsis sp. cib-109

A series of cytochalasins (1-6), including a new one (1), were isolated from the ethyl acetate extract of the solid-state fermented rice culture of the fungus Phomopsis sp. cib-109. The structure of compound 1 was elucidated to be 21-acetoxy-6,7,18-trihydroxy-5,6,16,18-tetramethyl[11]cytochalasa-13,19-trans-dien-1-one (cytochalasin R1) on the basis of spectroscopic analysis.

Isochaetomium A2, a new bis(naphthodihydropyran-4-one) with antimicrobial and immunological activities from fungus Chaetomium microcephalum

Isochaetomium A2 (1), a new bis(naphthodihydropyran-4-one), along with chaetochromins A (2) and B (3), was isolated from the solid-state fermented rice culture of Chaetomium microcephalum. The structure of compound 1 was elucidated on the basis of 1D and 2D NMR spectral data, and the relative configuration was confirmed by CD spectrum. Compounds 1-3 possessed significant antimicrobial activity against Escherichia coli 1.044, Staphylococcus aureus 1.252, and Bacillus subtilis 1.079. Moreover, compounds 1-3 showed obvious inhibitory effects on mouse spleen cell proliferation with successive IC50 values of 0.52, 0.19, and 0.24 μM.

Transcriptional and Enzymatic Profiling of Pleurotus ostreatus Laccase Genes in Submerged and Solid-State Fermentation Cultures

The genome of the white rot basidiomycete Pleurotus ostreatus includes 12 phenol oxidase (laccase) genes. In this study, we examined their expression profiles in different fungal strains under different culture conditions (submerged and solid cultures) and in the presence of a wheat straw extract, which was used as an inducer of the laccase gene family. We used a reverse transcription-quantitative PCR (RT-qPCR)-based approach and focused on determining the reaction parameters (in particular, the reference gene set for the normalization and reaction efficiency determinations) used to achieve an accurate estimation of the relative gene expression values. The results suggested that (i) laccase gene transcription is upregulated in the induced submerged fermentation (iSmF) cultures but downregulated in the solid fermentation (SSF) cultures, (ii) the Lacc2 and Lacc10 genes are the main sources of laccase activity in the iSmF cultures upon induction with water-soluble wheat straw extracts, and (iii) an additional, as-yet-uncharacterized activity (Unk1) is specifically induced in SSF cultures that complements the activity of Lacc2 and Lacc10. Moreover, both the enzymatic laccase activities and the Lacc gene family transcription profiles greatly differ between closely related strains. These differences can be targeted for biotechnological breeding programs for enzyme production in submerged fermentation reactors.

Peniciside, a new triterpenoid glycoside, from the fungus Penicillium sp. 169

Peniciside, a new fernene triterpenoid glycoside, was isolated from the EtOAc extract of the solid-state fermented rice culture of the fungus Penicillium sp. 169. Its structure was elucidated on the basis of spectroscopic analysis, and the absolute configuration was determined by X-ray crystallographic analysis and chemical methods. Peniciside is the first example of a fernene triterpenoid glycoside with two hydroxyls at C-19 and C-20.

Co-producing iturin A and poly-γ-glutamic acid from rapeseed meal under solid state fermentation by the newly isolated Bacillus subtilis strain 3-10

The strain 3-10 was isolated from soil and identified as B. subtilis according to morphological and physiological characteristics and nucleotide sequence of 16S rRNA. It co-produced anti-fungal iturin A and fertilizer synergist of poly-γ-glutamic acid (γ-PGA) under solid state fermentation (SSF) with rapeseed meal. The co-production of iturin A and γ-PGA reached 5.3 and 51.3 g/kg-dry weight culture, respectively, and the number of viable cells reached 1.9 × 10(10) CFU/g-dry weight culture. In pot tests, the shoot length and dry weight of watermelon seedlings treated by the SSF culture improved by 48.0 and 30.8%, respectively compared to the control; and its biocontrol effect on watermelon fusarium wilt achieved 89.6%. These results highlight a novel strategy to exploit the low-cost and widely available rapeseed meal as dual-functional bio-organic fertilizer under SSF by B. subtilis.

Environmental, scanning electron and optical microscope image analysis software for determining volume and occupied area of solid‐state fermentation fungal cultures

Here we propose a software for the estimation of the occupied area and volume of fungal cultures. This software was developed using a Matlab platform and allows analysis of high-definition images from optical, electronic or atomic force microscopes. In a first step, a single hypha grown on potato dextrose agar was monitored using optical microscopy to estimate the change in occupied area and volume. Weight measurements were carried out to compare them with the estimated volume, revealing a slight difference of less than 1.5%. Similarly, samples from two different solid-state fermentation cultures were analyzed using images from a scanning electron microscope (SEM) and an environmental SEM (ESEM). Occupied area and volume were calculated for both samples, and the results obtained were correlated with the dry weight of the cultures. The difference between the estimated volume ratio and the dry weight ratio of the two cultures showed a difference of 10%. Therefore, this software is a promising non-invasive technique to determine fungal biomass in solid-state cultures.

Four New Alkaloids, Brevianamides O – R, from the Fungus Aspergillus versicolor

AbstractFour new alkaloids, brevianamides O–R (1–4, resp.), were isolated from the AcOEt extract of the solid‐state fermented rice culture of the fungus Aspergillus versicolor. Their structures were elucidated on the basis of spectroscopic analyses.

Chromones from an ascomycete, Chaetomium aureus

A novel chromone, named chaetoaurin ( 1), along with six known chromone derivatives ( 2– 7), was isolated from the ethyl acetate extract of a solid-state fermented culture of Chaetomium aureus. Their structures were elucidated by extensive spectral analysis. All of these compounds were reported from C. aureus for the first time.

Amylase Production by Saccharomycopsis fibuligera A11 in Solid-State Fermentation for Hydrolysis of Cassava Starch

The optimization of process parameters for high amylase production by Saccharomycopsis fibuligera A11 in solid-state fermentation was carried out using central composite design. Finally, the optimal parameters obtained with the response surface methodology (RSM) were moisture 610.0 ml/kg, inoculum 30.0 ml (OD(600 nm) = 20.0)/kg, the amount ratio of wheat bran to rice husk 0.42, cassava starch concentration 20.0 g/kg, temperature 28 degrees C, and natural pH. Under the optimized conditions, 4,296 U/g of dry substrate of amylase activity was reached in the solid-state fermentation culture of the yeast strain A11 within 160 h, whereas the predicted maximum amylase activity of 4,222 U/g of dry substrate of amylase activity was derived from the RSM regression. It was found that cassava starch can be actively converted into monosaccharides and oligosaccharides by the crude amylase.

Brevianamide J, A New Indole Alkaloid Dimer from FungusAspergillus versicolor

Brevianamide J (1), a new indole alkaloid dimer, was isolated together with four new diketopiperazine alkaloids (brevianamide K-N, 2-5) from the solid-state fermented culture of Aspergillus versicolor. Their structures were elucidated on the basis of spectral data. X-ray crystallographic analysis confirmed the structures of 1 and 4.

Solid state fermentation of lipopeptide antibiotic iturin A by using a novel solid state fermentation reactor system

A new solid state fermentation reactor (SSFR) for solid substrate was used for the production of lipopeptide antibiotic iturin A using Bacillus subtilis RB14-CS. Solid state fermentation (SSF) is the technique of cultivation of microorganisms on solid and moist substrates in the absence of free water. SSF has shown much promise in the development of several bioprocesses and products because of their several advantages like absence of free water that allows simplified downstream processing and low cost. SSFR allows agitation of the SSF culture with improved temperature control and air supply. Interestingly, when okara, the widely available waste product from the tofu industries, was used as the solid substrate for the SSFR, no iturin A production was observed. However, without agitation, production of iturin A was observed in the SSFR but the production level remained low. The low production of iturin A was found to be due to the heat generation and excess temperature rise inside the reactor system during the fermentation process. Maintaining the temperature within a range of 25–30°C, production of iturin A was significantly improved in the SSFR. This was comparable to the laboratory scale production, and signifies the potential application of the SSFR for SSF.

Use of response surface methodology for optimizing process parameters for high inulinase production by the marine yeast Cryptococcus aureus 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 degrees 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.

Effect of hydrolytic and oxidative enzymes produced by solid-state fermentation on greige linen fabric

Solid state fermentation (SSF) was applied for production of fungal enzyme preparations from Phanerochaete chrysosporium, Aspergillus oryzae, Aspergillus giganteus and Trichoderma virens using cotton seed-coat fragment waste as a carbon source and enzyme inducer. Lignin-holocellulose matrix of cotton seed coat fragment proved to be effective in inducing production of ligninolytic, cellulolytic and xylanolytic enzymes in solid-state fermentation. The effect of the enzymes produced by SSF on greige linen fabric is discussed and evaluated. In the first experiment the hydrolytic and accompanying oxidative enzymes in the buffer extract of the whole SSF cultures were used for fabric treatment. In the second trial, the enzymes produced in situ (whole SSF material—mixture of fungal biomass, residual substrate and enzymes) were used for the treatment. Weight loss, reducing sugar liberation and removal of colouring materials were measured. The results showed that at equal enzyme charges the intact SSF materials were more efficient than the enzyme extracts. Of the six strains evaluated, Ph. chrysosporium VKM F-1767 was the most effective in removing colouring matters from greige linen fabric.

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.

Biopreparation of cotton fabric with enzymes produced by solid-state fermentation

Fungal enzyme preparations from Phanerochaete chrysosporium, Aspergillus oryzae, Aspergillus giganteus and Trichoderma virens, produced by solid-state fermentation (SSF) on cotton seed-coat fragment waste as substrate and enzyme inducer were investigated in biopreparation of cotton fabric. Cotton seed-coat fragment is rich in lignin, cellulose and hemicelluloses, therefore enzyme complexes produced by target fungi on such a substrate can be used effectively to degrade impurities in cotton fabrics during biopreparation. Activities of extracellular hydrolytic and ligninolytic enzymes were determined from the SSF extract materials. The potential of the hydrolytic and accompanying oxidative enzymes in the whole SSF cultures was exploited in degradation of seed-coat fragments and other coloring materials of greige cotton fabric. Enzyme assays indicated that many extracellular enzymes have been produced under these conditions including both hydrolytic and oxidative enzymes. A. oryzae NRRL 3485 produced significantly higher amounts of both hydrolytic and oxidative enzymes than other tested fungi. Best results in removal of seed-coat fragments from cotton fabric were obtained by P. chrysosporium NCAIM (=ATCC 34541), P. chrysosporium VKM F-1767 and A. oryzae NRRL 3485 SSF enzyme complexes.

Chaetocochins A−C, Epipolythiodioxopiperazines from Chaetomium cochliodes

Three new epipolythiodioxopiperazines, chaetocochins A (1), B (2), and C (3), along with dethio-tetra (methylthio) chetomin (4) and chetomin (5), were isolated from the ethyl acetate extract of the solid-state fermented rice culture of the fungus Chaetomium cochliodes. Their structures were elucidated on the basis of spectroscopic analysis. Compounds 1, 3, and 4 exhibited significant cytotoxicity in vitro against cancer cell lines Bre-04, Lu-04, and N-04.

Chaetoindicins A−C, Three Isoquinoline Alkaloids from the Fungus Chaetomium Indicum

[structure: see text] Chaetoindicins A-C (1-3), three isoquinolines with novel skeletons, were isolated from the solid-state fermented culture of Chaetomiumindicum. Their structures were elucidated on the basis of spectral data. X-ray crystallographic analysis confirmed the structure of 2.