Solid-state Culture Research Articles

The ascomycete Xylaria polymorpha produces an acetyl esterase that solubilises beech wood material to release water-soluble lignin fragments

Three ascomycetous soft-rot fungi belonging to the family Xylariaceae were found to produce high levels of a p-nitrophenyl acetate-hydrolyzing activity during growth on lignocellulosic materials, i.e., wheat straw and beech wood chips. Accordingly, Kretzschmaria deusta, Morchella elata, Xylaria polymorpha were seemingly most noteworthy acetyl esterase producers and, of which, X. polymorpha (strain A35) was chosen for further studies. Induction study indicated that raw carbohydrate sources, such as beech wood, rape straw, birch wood, and wheat straw, were extremely important for acetyl esterase production. Acetyl esterase of X. polymorpha was produced in solid-state culture on wheat straw and purified by different steps of anion-exchange and size-exclusion chromatography. This purified enzyme (M W = 44 kDa and pI values of 3.5–3.6) exhibited the capability to solubilise in vitro beech wood to release water-soluble lignin fragments with molecular masses of 1–3 kDa as analyzed by high performance size exclusion chromatography.

Cellulose-degrading enzymes from Aspergillus terreus D34 and enzymatic saccharification of mild-alkali and dilute-acid pretreated lignocellulosic biomass residues

Production of cellulose-degrading enzymes from Aspergillus terreus D34 using different growth substrates was studied under solid-state cultivation. We have tested two lignocellulosic biomass residues viz., rice straw (RS) and sugarcane bagasse (BG), both separately and in combinations, and crystalline cellulose as a sole source of carbon for cellulase production. We also demonstrated different cellulase cocktail formulations and enzymatic saccharification studies on mild-alkali and dilute-acid pretreated RS- and BG-biomass residues. Substrate-specific non-denaturing native gels showed two exoglucanases, four endoglucanases, three β-glucosidases, and four xylanases in the microbial culture extract of RS-grown cells. While in the BG-culture extract, two exoglucanases, five endoglucanases, three β-glucosidases, and four xylanases were detected. Similarly, in crystalline cellulose-grown culture extract, three exoglucanases, four endoglucanases, one β-glucosidase, and two xylanases were detected. However, the cellulase compositions were highly varied with the culture extracts obtained from the mixed biomass (RSBG) growth substrate. We found that few enzymes were specifically induced while others were repressed in RSBG-grown cultures. Enzymatic saccharification resulted in the production of maximum reducing sugars of 0.733 and 0.498 g g−1 with mild-alkali treated RS- and BG-biomass residues with saccharification yields reaching up to 82.8% ± 1.0% and 59.3% ± 1.7%, respectively. The cellulase activities, namely FPase, CMCase, avicelase, β-glucosidase, and endoxylanase, were significantly higher in the BG-grown culture extract. Optimization of microbial growth carbon sources produced an efficient cellulase enzyme cocktail mixture with an approximately twofold higher total cellulase (FPase) activity that drastically reduced the required amount of enzyme (in terms of unit volumes) for enzymatic hydrolysis studies.

English

The present study was carried out to investigate the optimal culture conditions for β-mannanase production by Trichosporonoides oedocephalis using cheap and easily available agro-wastes as the carbon source in solid state cultivation. The effect of process parameters like incubation time, carbon source, incubation temperature, pH, percentage moisture content and inoculum concentration were optimized for enhanced mannanase production. Maximum β-mannanase activity was observed after 96 h of incubation. Different agricultural wastes were screened as substrates for β-mannanase production in comparison with locust bean gum (LBG). Among tested carbon sources, orange peels proved to the best for β-mannanase production. Initial pH of the culture medium was also optimized and a pH of 8.0 was found to be the optimum pH value for β-mannanase production. The optimum temperature, percentage moisture content and inoculum concentration were 30°C, 60% and 1×107 (spores/ml), respectively. Key words: Beta-mannanase, process parameters, solid state fermentation, agricultural wastes, Trichosporonoides oedocephalis.

Variations in oxygen concentration cause differential antioxidant response and expression of related genes in Beauveria bassiana

The entomopathogenic fungus Beauveria bassiana is widely used in pest biocontrol strategies. We evaluated both the antioxidant response mediated by compatible solutes, trehalose or mannitol, and the expression of related genes using oxygen pulses at three oxygen concentrations in solid state culture (SSC): normal atmosphere (21 % O2), low oxygen (16 % O2) and enriched oxygen (26 % O2). Trehalose concentration decreased 75 % after atmospheric modifications in the cultures, whereas mannitol synthesis was three-fold higher under the 16 % O2 pulses relative to normal atmosphere (100 and 30 μg mannitol mg−1 biomass, respectively). Confirming this result, expression of the mpd gene, coding for mannitol-1-P dehydrogenase (MPD), increased up to 1.4 times after O2 pulses. The expression of the bbrgs1 gene, encoding a regulatory G protein related to conidiation, was analysed to explain previously reported differences in conidial production. Surprisingly, expression of bbrgs1 decreased after atmospheric modification. Finally, principal component analysis (PCA) indicated that 83.39 % of the variability in the data could be explained by two components. This analysis corroborated the positive correlation between mannitol concentration and mpd gene expression, as well as the negative correlation between conidial production and bbrgs1 gene expression. This study contributes to understanding of antioxidant and molecular response of B. bassiana induced under oxidant conditions.

PRODUKSI BIOINSEKTISIDA oleh Bacillus thuringiensis MENGGUNAKAN KULTIVASI MEDIA PADAT

*Penulis untuk korespondensi In bio insecticide production, the use of agricultural by products as a substrate to produce the starter has the role important on the efficient production processes, as well as the use of single isolate and production media. The bio insecticide production was carried out by the solid state cultivation, by examining the effect of substrate thick nessoncell growth and bio insecticide toxicity of bio insecticide produced. The bacterial of localisolates used were obtained from carrion worm of Attacus atlas, and identified as Bacillus thuringiens is and had important potential as bio insecticide. Tofu whey was used for starter media, while mixture of dregs sago and iles-iles as its production media. During the cultivation the media thickness affected on pH, cell growth, spores and substrate consumption. The increased of substrate thickness caused the decrease of pH, and simultaneously the decrease of cell growth, spores and substrate consumption. The product with highes toxicity capability against dipteralarvae (LC 50 of 0,24 mg/mL) and lepidoptera larvae (LC 50 of 3,3 mg/mL) was obtained under cultivation process with substrate thicknes of 1 cm. Keywords : Bacillus thuringiensis, bioinsecticide, solid-state cultivation, solid waste sago, solid waste iles-iles

Characterization of a novel Aspergillus niger beta-glucosidase tolerant to saccharification of lignocellulosic biomass products and fermentation inhibitors

AbstractProperties of beta-glucosidase produced by Aspergillus niger URM 6642 recently isolated from the Atlantic rainforest biome and its potential tolerance to saccharification of lignocellulosic biomass products and fermentation inhibitors was evaluated. The fungus was cultivated under solid state culture conditions at 37°C with different agro-industrial wastes. High levels of beta-glucosidase (3778.9 U g

Protein production by Arthrospira (Spirulina) platensis in solid state cultivation using sugarcane bagasse as support

The genus Arthrospira comprises a group of filamentous multicellular cyanobacteria and can be used for animal feed and human food. Solid state fermentation or cultivation (SSF) involves the use of a culture medium composed of solid material with given moisture content. No studies have been published about the cultivation of microalgae or cyanobacteria on solid medium. Furthermore, although sugar-cane bagasse is used as source of energy in alcohol distilleries in Brazil, the excess could be a support to photosynthetic microorganism growth. The experimental design methodology was used to evaluate the protein production by Arthrospira platensis under SSF using sugarcane bagasse as support, taking into account the moisture content of the medium, light intensity and inoculum concentration. Moisture was found to have a strong influence on the performance of the process. The best conditions were: moisture of 98.8%; inoculum concentration of 0.15g biomass·kg wet culture medium−1 and light intensity of 6.0klx.

Transcriptomic analysis of temperature responses of Aspergillus kawachii during barley koji production.

The koji mold Aspergillus kawachii is used for making the Japanese distilled spirit shochu. During shochu production, A. kawachii is grown in solid-state culture (koji) on steamed grains, such as rice or barley, to convert the grain starch to glucose and produce citric acid. During this process, the cultivation temperature of A. kawachii is gradually increased to 40 °C and is then lowered to 30 °C. This temperature modulation is important for stimulating amylase activity and the accumulation of citric acid. However, the effects of temperature on A. kawachii at the gene expression level have not been elucidated. In this study, we investigated the effect of solid-state cultivation temperature on gene expression for A. kawachii grown on barley. The results of DNA microarray and gene ontology analyses showed that the expression of genes involved in the glycerol, trehalose, and pentose phosphate metabolic pathways, which function downstream of glycolysis, was downregulated by shifting the cultivation temperature from 40 to 30 °C. In addition, significantly reduced expression of genes related to heat shock responses and increased expression of genes related with amino acid transport were also observed. These results suggest that solid-state cultivation at 40 °C is stressful for A. kawachii and that heat adaptation leads to reduced citric acid accumulation through activation of pathways branching from glycolysis. The gene expression profile of A. kawachii elucidated in this study is expected to contribute to the understanding of gene regulation during koji production and optimization of the industrially desirable characteristics of A. kawachii.

Open and continuous fermentation: products, conditions and bioprocess economy.

Microbial fermentation is the key to industrial biotechnology. Most fermentation processes are sensitive to microbial contamination and require an energy intensive sterilization process. The majority of microbial fermentations can only be conducted over a short period of time in a batch or fed-batch culture, further increasing energy consumption and process complexity, and these factors contribute to the high costs of bio-products. In an effort to make bio-products more economically competitive, increased attention has been paid to developing open (unsterile) and continuous processes. If well conducted, continuous fermentation processes will lead to the reduced cost of industrial bio-products. To achieve cost-efficient open and continuous fermentations, the feeding of raw materials and the removal of products must be conducted in a continuous manner without the risk of contamination, even under 'open' conditions. Factors such as the stability of the biological system as a whole during long cultivations, as well as the yield and productivity of the process, are also important. Microorganisms that grow under extreme conditions such as high or low pH, high osmotic pressure, and high or low temperature, as well as under conditions of mixed culturing, cell immobilization, and solid state cultivation, are of interest for developing open and continuous fermentation processes.

Influence of Additives on the Yield and Pathogenicity of Conidia Produced by Solid State Cultivation of an Isaria javanica Isolate

Recently, the Q biotype of tobacco whitefly has been recognized as the most hazardous strain of Bemisia tabaci worldwide, because of its increased resistance to some insecticide groups. As an alternative control agent, we selected an Isaria javanica isolate as a candidate for the development of a mycopesticide against the Q biotype of sweet potato whitefly. To select optimal mass production media for solid-state fermentation, we compared the production yield and virulence of conidia between 2 substrates (barley and brown rice), and we also compared the effects of various additives on conidia production and virulence. Barley was a better substrate for conidia production, producing 3.43 × 1010 conidia/g, compared with 3.05 × 1010 conidia/g for brown rice. The addition of 2% CaCO3 + 2% CaSO4 to barley significantly increased conidia production. Addition of yeast extract, casein, or gluten also improved conidia production on barley. Gluten addition (3% and 1.32%) to brown rice improved conidia production by 14 and 6 times, respectively, relative to brown rice without additives. Conidia cultivated on barley produced a mortality rate of 62% in the sweet potato whitefly after 4-day treatment, compared with 53% for conidia cultivated on brown rice. The amendment of solid substrate cultivation with additives changed the virulence of the conidia produced; the median lethal time (LT50) was shorter for conidia produced on barley and brown rice with added yeast extract (1.32% and 3%, respectively), KNO3 (0.6% and 1%), or gluten (1.32% and 3%) compared with conidia produced on substrates without additives.

Efficient polygalacturonase production from agricultural and agro-industrial residues by solid-state culture of Aspergillus sojae under optimized conditions.

Previously identified fungal pectinase producers of the species Aspergillus sojae were used for optimization of polygalacturonase production in solid-state fermentation applying Design of Experiment. The effects of media composition and several process parameters, like inoculum size, moisture level, incubation time and temperature on polygalacturonase activity were studied in screening and optimization investigations. Utilization of agricultural and agro-industrial by-products provided the establishment of a cost-efficient and sustainable process for enzyme production. Comparison of pectinase production by A. sojae ATCC 20235 and A. sojae CBS 100928 under optimized conditions yielded 6.9 times higher polygalacturonase activity by A. sojae ATCC 20235. Highest enzyme yield (909.5 ± 2.7 U/g) was obtained by A. sojae ATCC 20235 after 8 days at 30°C applying 30% sugar beet pulp as inducer substrate in combination with wheat bran as medium wetted at 160% with 0.2 M HCl. Furthermore, an overview of pectinolytic enzyme activities present in the extracts of both strains is provided. Protein profiles of both strains are given by SDS-PAGE electrophoresis, as well as zymograms for pectinolytic enzymes in comparison to commercial pectinase preparations.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-742) contains supplementary material, which is available to authorized users.

The hydrophobicity of the support in solid state culture affected the production of hydrophobins from Lecanicillium lecanii

Lecanicillium lecanii has been successfully employed to produce hydrophobin-like proteins (HfbL) in solid state and submerged cultures varying the type of solid inert support. This study shows the results on the effect of hydrophilic Perlite and hydrophobic Polyurethane inert supports in solid state cultures for production of HfbLs by L. lecanii. The hydrophobicity of the support employed in solid state cultures influenced the yields and surface activities of the class I and class II HfbLs produced by L. lecanii. Class I HfbL was only produced using the hydrophobic polyurethane foam support, showing high surface activity that reduced ca. 50% hydrophobicity of Teflon, whereas class II HfbLs were produced on both polyurethane foam and perlite supports, and they reduced ca. 50% the surface tension of water and ca. 25% reduction of the hydrophobicity of Teflon.

Production and characterization of β-glucosidase obtained by the solid-state cultivation of the thermophilic fungus Thermomucor indicae-seudaticae N31.

In this paper, several agro-industrial wastes (soybean meal and wheat straw, rice and peanut husks, corn cob and corn stover, and sugarcane bagasse) were tested for the production of β-glucosidase by the cultivation of thermophilic fungus Thermomucor indicae-seudaticae N31 in solid-state fermentation (SSF). Among the tested substrates, the highest yields were obtained in soybean meal. Other fermentation parameters were also evaluated, such as initial pH, merge substrates, and fermentation time, as well as the physicochemical characterization of the enzyme. The best results were obtained after 192 h of fermentation with the initial pH adjusted to 6.0. The substrate mixture did not improve the enzyme production by microorganism. The β-glucosidase showed best catalytic activity at pH 4.5 and at 75 °C and remained stable in the pH range from 4.5 to 9.5 and the temperature range 40-75 °C. The enzyme showed 80 % of its activity at a concentration of 15 mM glucose and remained stable up to 20 % ethanol.

Electric field as pretreatment to enhance the activity of a whole-cell biocatalyst for hydrocarbon degradation in contaminated water

Abstract In this paper, a method using two successive cultures, solid then liquid, for the production and use of a hydrocarbon degrading biocatalyst (BC) is proposed. Hexadecane (HXD) was used as a model hydrocarbon. An electric field was applied during the solid state culture (SSC) as a novel approach, denoted the electric field pretreatment, to enhance the HXD degrading activity. Afterward, the catalytic activity (CA) was determined in liquid culture. The CA of the biocatalyst pretreated and untreated was evaluated, discriminating between sorption capacity and HXD degradation rate; biomass production on the electric field pretreated biocatalyst was only 20% of that on the untreated biocatalyst, but the maximum biocatalyst sorption capacity was enhanced from 119 ± 41 mg (g BC)−1 to 207 ± 23 mg (g BC)−1 by the effect of the electric field pretreatment. The activity of the biocatalyst was mainly associated with the pretreated fungal biomass; its activity was 9-fold higher than that of the untreated biomass. Linear model was used to obtain the affinity constant and Langmuir to adjust maximum sorption capacity. This enhancement in sorption capacity was associated with the high HXD degradation rate observed here; 86% of the initial HXD was eliminated in 42 h by the pretreated biocatalyst, whilst 53% was eliminated in 48 h by the untreated biocatalyst.

Agar-supported cultivation of Halorubrum sp. SSR, and production of halocin C8 on the scale-up prototype Platotex

Halorubrum sp. SSR was isolated from a solar saltern in Algeria. The strain exhibited a high antibiotic activity against the indicator strain Natronorubrum aibiense G23, and the bioactive compound showed thermal, acid and alkali stability. SSR was grown on agar-supported cultivation (AgSF) to compare yields and applicability with traditional submerged cultivation. AgSF scale-up was implemented taking benefit from the solid-state cultivation prototype Platotex. This technology leads to high amounts of the target Halocin and facilitate the downstream steps. The antibiotic compound was purified according to a fast efficient procedure including ion exchange chromatography followed by a fractionation on C18 Sep-Pack cartridge. The compound was identified as Halocin C8 according to N-terminal amino acid sequencing and high-resolution mass spectrometry.

Isolation, characterization, and antitumor activity of a novel heteroglycan from cultured mycelia of Cordyceps sinensis.

A novel heteroglycan, Cordyceps sinensis polysaccharide 1 (molecular weight 1 17 × 10(5) Da), was isolated and purified from mycelia of the fungus C. sinensis obtained by solid-state culture. Structural characterization by chemical analysis, GC-MS, FTIR, and NMR spectroscopy showed that C. sinensis polysaccharide 1 was mainly composed of (1 → 6)-linked α-D-Glc and α-D-Gal, with minor β-(1 → 4)-D-Xyl and β-(1 → 4)-D-Man residues probably located in the side chains with a trace amount of α-(1 → 3)-L-Rha residue. In biological assays, C. sinensis polysaccharide 1 significantly inhibited proliferation of sarcoma 180 cells and induced apoptosis in a dose-dependent manner. Further studies will elucidate the antitumor mechanism of C. sinensis polysaccharide 1 and promote its utilization for the development of novel, effective anticancer drugs.

Screening and optimization of parameters affecting fungal pretreatment of oil palm empty fruit bunch (EFB) by experimental design

In the present study, various white-rot fungi were used for the pretreatment of oil palm empty fruit bunch (EFB) using solid-state cultivation. The results showed that Trametes versicolor TISTR 3224 gave the highest selectivity value (the ratio of lignin degradation to cellulose degradation) of 1.57. In comparison, Trametes sp. BCC 8729, Phanerochaete chrysosporium ATCC 24725, Marasmius sp. BCC 9542 and Xylaria sp. BCC 7749 gave selectivity of 0.60, 0.59, 0.30 and 0.06, respectively. Screening parameters for the fungal pretreatment of EFB using T. versicolor TISTR 3224 was studied by Plackett–Burman design (PBD). It indicated that the moisture content and co-substrate gave a positive effect on the lignin degradation, while EFB concentration had a negative effect on cellulose degradation. The optimum conditions for lignin degradation obtained from Box–Behnken statistical experimental design (BBD) were 80 % moisture content, 2.29 % wheat flour and 23.3 % EFB. Under this condition, 15.6 % of delignification was obtained. After an enzymatic hydrolysis, the digestibility of fungal treated EFB under the optimum condition achieved 1.34-fold compared with untreated EFB.

Oxygen transfer in solid-state cultivation under controlled moisture conditions.

The aim of this work was to study oxygen transfer as a function of the initial moisture content in solid-state cultivation under controlled moisture conditions. The use of controlled moisture conditions prevents drastic changes in the medium during cultivation, allowing the use of a pseudo-steady-state model to estimate the overall oxygen mass transfer coefficient (K L a) in the biofilm around the solid particles. Drechslera (Helminthosporium) monoceras, an aerobic mold that produces allergenic proteins, was cultured on wheat bran in a packed bed column bioreactor. The bed height (30 mm) and air flow rate (0.4 L/min) were selected to implement moisture control. The results show that there is an optimal moisture content (35 %) at which a lower biofilm thickness and packing of the bed improves K L a. However, a higher biomass growth was obtained at 45 % moisture. The different patterns of biomass growth demonstrate the importance of the balance between aerial and film growth in solid-state cultivation. These results contribute to the understanding of oxygen transfer in solid fermentation, optimization of processes, and production of allergen extracts from D. (Helminthosporium) monoceras biomass.

Intraspecific Diversity in the Production and Characterization of Laccase within Ganoderma lucidum

Ganoderma lucidum has a well-developed ligninolytic enzyme system, where laccase is the dominant and sometimes only synthesizing enzyme, and therefore could find an application in the delignification of abundant plant raw materials and in food, feed, paper, and biofuel production. The questions that provided the goals for the present study were whether the profile of G. lucidum laccase depends on cultivation type and carbon source, as well as whether intraspecific diversity exists. Conditions of submerged cultivation proved more preferable for laccase activity compared with solid-state cultivations in all studied strains, while oak sawdust provided a better carbon source than wheat straw. Maximum laccase activity (7241.0 U/L) was measured on day 14 of oak sawdust submerged fermentation by strain BEOFB 431. Intraspecific diversity in synthesized proteins was more significant in wheat straw than in oak sawdust submerged fermentation. The profile of laccase isoforms was dependent on strain, plant residue, type, and period of cultivation. Four acidic laccase isoforms (pI 3.6) were detected in G. lucidum BEOFB 431 at the same cultivation point where maximal enzyme activity was measured.

Upgrading the antioxidant potential of cereals by their fungal fermentation under solid-state cultivation conditions.

Solid-state fermentation (SSF) at 30°C for 72h with four generally recognized as safe (GRAS) filamentous fungi (Aspergillus oryzae NCIM 1212, Aspergillus awamori MTCC No. 548, Rhizopus oligosporus NCIM 1215 and Rhizopus oryzae RCK2012) showed high efficiency for the improvement of water-soluble total phenolic content (TPC) and antioxidant properties including ABTS(●+) [2,2'-azinobis (3-ethylbenzothiazoline-6-sulphonic acid)] and DPPH(●) (2,2'-diphenyl-1-picrylhydrazyl) scavenging capacities of four whole grain cereals, namely wheat, brown rice, maize and oat. A maximum 14-fold improvement in TPC (11·61mg gallic acid equivalent g(-1) grain) was observed in A.oryzae fermented wheat, while extract of R.oryzae fermented wheat (ROFW) showed maximum of 6·6-fold and fivefold enhancement of DPPH(●) scavenging property (8·54μmol Trolox equivalent g(-1) grain) and ABTS(●+) scavenging activity (19·5μmol Trolox equivalent g(-1) grain), respectively. The study demonstrates that SSF is an efficient method for the improvement of antioxidant potentials of cereals and R.oryzae RCK2012 fermented wheat can be a powerful source of natural antioxidants. Antioxidant-rich food products are getting popularity day by day. In this study, potential of solid-state fermentation (SSF) has been studied for the improvement of antioxidant potential of different cereals by GRAS micro-organisms. The comparative evaluation of the antioxidant potential of various fungal fermented products derived from whole grain cereals, such as wheat, brown rice, oat and maize, has been carried out. Among these, Rhizopus oryzae RCK2012-fermented wheat was observed as a potent source of natural antioxidants. A diet containing fermented cereals would be useful for the prevention of free radical-mediated diseases.