Protein measurement in solid-state fermentation

Summary Membrane filter culture was used as a model system for the investigation of the growth of Rhizopus oligosporus in solid-state fermentation. The biuret method gave more accurate estimations of protein than the Folin-Lowry method. However, due to the change in the protein content of the biomass during the fermentation, protein cannot be considered to be a reliable index of growth in solid-state fermentation.     

Comparison of solid-state and submerged-state fermentation for the bioprocessing of switchgrass to ethanol and acetate by Clostridium phytofermentans

The conversion of sustainable energy crops using microbiological fermentation to biofuels and bioproducts typically uses submerged-state processes. Alternatively, solid-state fermentation processes have several advantages when compared to the typical submerged-state processes. This study compares the use of solid-state versus submerged-state fermentation using the mesophilic anaerobic bacterium Clostridium phytofermentans in the conversion of switchgrass to the end products of ethanol, acetate, and hydrogen. A shift in the ratio of metabolic products towards more acetate and hydrogen production than ethanol production was observed when C. phytofermentans was grown under solid-state conditions as compared to submerged-state conditions. Results indicated that the end product concentrations (in millimolar) obtained using solid-state fermentation were higher than using submerged-state fermentation. In contrast, the total fermentation products (in weight of product per weight of carbohydrates consumed) and switchgrass conversion were higher for submerged-state fermentation. The conversion of xylan was greater than glucan conversion under both fermentation conditions. An initial pH of 7 and moisture content of 80 % resulted in maximum end products formation. Scanning electron microscopy study showed the presence of biofilm formed by C. phytofermentans growing on switchgrass under submerged-state fermentation whereas bacterial cells attached to surface and no apparent biofilm was observed when grown under solid-state fermentation. To our knowledge, this is the first study reporting consolidated bioprocessing of a lignocellulosic substrate by a mesophilic anaerobic bacterium under solid-state fermentation conditions.     

生物热对传统固态发酵菌群演替及其代谢影响的研究进展

解析传统固态发酵中产生的生物热对微生物菌群代谢的影响,是认识发酵机制、调控发酵过程、保证发酵效率的关键之一。固态发酵过程中,微生物菌群代谢活动所产生的生物热及传热效率低等问题引起微环境温度升高,进而影响微生物的生长与代谢。然而,关于传统固态发酵微生物受生物热的影响及其适应机制仍不明晰。因此,本文以传统固态发酵体系为研究对象,阐述持续生物热介导的高温对固态发酵过程中微生物群落演替和代谢功能的影响,并提出复杂群落中具有多层次调控微生物代谢以适应高温环境的方式,主要从微生物群体与个体层面介绍可能存在的耐热机制。了解生物热对传统固态发酵微生物的影响及潜在的耐热机制,有助于靶向调控发酵过程、强化高温发酵等,以满足未来的工业化需求。     

Solid-state fermentation: a continuous process for fungal tannase production

Truly continuous solid-state fermentations with operating times of 2-3 weeks were conducted in a prototype bioreactor for the production of fungal (Penicillium glabrum) tannase from a tannin-containing model substrate. Substantial quantities of the enzyme were synthesized throughout the operating periods and (imperfect) steady-state conditions seemed to be achieved soon after start-up of the fermentations. This demonstrated for the first time the possibility of conducting solid-state fermentations in the continuous mode and with a constant noninoculated feed. The operating variables and fermentation conditions in the bioreactor were sufficiently well predicted for the basic reinoculation concept to succeed. However, an incomplete understanding of the microbial mechanisms, the experimental system, and their interaction indicated the need for more research in this novel area of solid-state fermentation.     

Development of a model solid-state fermentation system

Summary A model solid substrate was developed, consisting of cassava starch and other nutrients embedded in kappa-carrageenan. A suitable fermentation system and analytical techniques were also developed to cope with the constraints imposed by solid-state fermentation.     

Gibberellic acid production using different solid-state fermentation systems

Gibberellic acid production in liquid fermentation was compared with production of this compound in solid-state fermentation systems using cassava flour, sugar cane bagasse and low density polyurethane. Gibberella fujikuroi produced 23 mg of gibberellin/ml in 120h of liquid fermentation. Solid-state fermentation on bagasse showed excellent growth but presented gibberellin extraction problems. Very low production and growth was observed in solid-state fermentation with low density polyurethane as an inert support. Solid-state fermentation on cassava flour showed high production (250 mg/kg of dry solid medium) in a very short time (36h).     

Biosynthesis of tannin acyl hydrolase from tannin-rich forest residue under different fermentation conditions

Caesalpinia digyna, a tannin-rich forest residue, was used as substrate for production of tannase and gallic acid. Media engineering was carried out under solid-state fermentation, submerged fermentation and modified solid state fermentation conditions for optimum synthesis of tannase and gallic acid (based on 58% tannin content in the raw material). Tannase vis-à-vis gallic acid recovery under modified solid-state fermentation condition was maximum. Conversions of tannin to gallic acid under solid-state fermentation, submerged fermentation and modified solid-state fermentation conditions were 30.5%, 27.5% and 90.9%, respectively. Journal of Industrial Microbiology & Biotechnology (2000) 25, 29–38. Received 02 November 1999/ Accepted in revised form 12 February 2000     

固态发酵技术提高苹果渣附加值的应用研究

能源危机和苹果渣带来的环境问题促进了固态发酵技术的发展.相对于液体深层发酵,固态发酵在生产中具有处理量大,操作简单,成本低等独特的优势.综述了以苹果渣为原料利用固态发酵技术生产酶制剂、柠檬酸、酒精和蛋白饲料等的研究应用进展,以及存在的问题和前景展望.     

A semimechanistic mathematical model for growth of Rhizopus oligosporus in a model solid-state fermentation system

A Semimechanistic mathematical model is developed which describes the growth of Rhizopus oligosporus in a model solid-state fermentation system. Equations are presented for the release of glucoamylase, the diffusion of glucoamylase, the hydrolysis of starch, the generation and diffusion of glucose, and the uptake of glucose and conversion into new biomass. Good agreement of the model with the experimental data was obtained only after the glucoamylase diffusivity and the maximum specific glucose uptake rate were altered from their originally determined values. The model recognizes the distributed nature of the solid-state fermentation and therefore is able to predict the concentration profiles of the system components within the substrate. The model provides an insight into the possible rate-limiting steps in solid-state fermentation-the generation of glucose within the substrate and the resulting availability of glucose at the surface.     

Solid-State Fermentation with Trichoderma reesei for Cellulase Production

Cellulase yields of 250 to 430 IU/g of cellulose were recorded in a new approach to solid-state fermentation of wheat straw with Trichoderma reesei QMY-1. This is an increase of ca. 72% compared with the yields (160 to 250 IU/g of cellulose) in liquid-state fermentation reported in the literature. High cellulase activity (16 to 17 IU/ml) per unit volume of enzyme broth and high yields of cellulases were attributed to the growth of T. reesei on a hemicellulose fraction during its first phase and then on a cellulose fraction of wheat straw during its later phase for cellulase production, as well as to the close contact of hyphae with the substrate in solid-state fermentation. The cellulase system obtained by the solid-state fermentation of wheat straw contained cellulases (17.2 IU/ml), β-glucosidase (21.2 IU/ml), and xylanases (540 IU/ml). This cellulase system was capable of hydrolyzing 78 to 90% of delignified wheat straw (10% concentration) in 96 h, without the addition of complementary enzymes, β-glucosidase, and xylanases.     

Platotex: an innovative and fully automated device for cell growth scale-up of agar-supported solid-state fermentation

Among various factors that influence the production of microbial secondary metabolites (MSM), the method of cultivation is an important one that has not been thoroughly investigated. In order to increase microbial throughput and simplify the extraction and workup steps, we performed a study to compare liquid-state fermentation (LSF) with agar-supported solid-state fermentation (AgSF). We found that AgSF is not only more suitable for our applications but offers, for some microbial strains, a higher yield and broader diversity of secondary metabolites. The main limitation of AgSF is the lack of a system to allow production scale-up. In order to overcome this obstacle we developed Platotex, an original fermentation unit offering 2 m² of cultivation surface that combines automatic sterilization, cultivation, and drying steps. Platotex is also able to support both LSF and solid-state fermentation (SSF). Platotex conforms to international security and quality requirements and benefits from total remote automation through industrial communication and control standards.     

Mass Production of Conidia of Penicillium frequentans,a Biocontrol Agent Against Brown Rot of Stone Fruits

Conidial production of Penicillium frequentans , a biocontrol agent of the fungal pathogen Monilinia laxa , was tested in liquid and solid-state fermentation. Conidial production of P. frequentans in solid-state fermentation was higher than in liquid-state fermentation. Solidstate fermentation was made in specially designed plastic bags (VALMIC ® ) containing peat:vermiculite (1:1 w/w). Addition of nutrients to the peat:vermiculite increased conidial production of P. frequentans , especially when lentil meal was added. The number of conidia obtained in this solid-state fermentation was maintained in the range of 10 8 -10 9 conidia g -1 from 5 to 120 days after inoculation. Germinability of these conidia was > 90% until 90 days of incubation and declined at 120 days. Optimal initial moisture content in the substrate was 30-40% (v/w). At lower moisture contents, significant reductions in conidial production and germinability were observed, particularly at 10% (v/w). Conidial production was similar when the substrate was inoculated with 10 5 , 10 6 or 10 7 conidia g -1 dry substrate. Fresh conidia produced by solid-state fermentation reduced the incidence of brown rot on plums by 75%.     

Modelling of the glucosinolate content in solid-state fermentation of rapeseed meal with fuzzy logic

A fuzzy model was developed to predict the glucosinolate content of rapeseed meal during solid-state fermentation. Process variables such as temperature, relative humidity, fermentation time and the resulting glucosinolate content of the meal were considered. The glucosinolate content calculated by the fuzzy model corresponded with the experimental results obtained by solid-state fermentation.     

固态发酵降低上部低次烟叶中淀粉及蛋白质的含量

利用固态发酵的方法降低上部低次烟叶中淀粉和蛋白质的含量,并对发酵过程中的厌氧细菌和酵母的数量进行检测。采用单因素和正交试验对固态发酵的条件进行优化,结果表明:各因素对发酵上部低次烟叶影响显著性主次次序依次为发酵时间(C)、发酵温度(A)和发酵水分质量分数(B),固态发酵的最佳条件为A2B2C3,即温度45℃、水分质量分数50%、发酵时间9 d。在该发酵条件下,上部低次烟叶的淀粉降解率为20.41%,蛋白质降解率为12.35%。通过固态厌氧发酵的方法可取得较好的、短期内快速降解上部低次烟叶中淀粉和蛋白质含量的效果。     

Production of phytase by Mucor racemosus in solid-state fermentation

Phytase production was studied by three Mucor and eight Rhizopus strains by solid-state fermentation (SSF) on three commonly used natural feed ingredients (canola meal, coconut oil cake, wheat bran). Mucor racemosus NRRL 1994 (ATCC 46129) gave the highest yield (14.5 IU/g dry matter phytase activity) on coconut oil cake. Optimizing the supplementation of coconut oil cake with glucose, casein and (NH(4))(2)SO(4), phytase production in solid-state fermentation was increased to 26 IU/g dry matter (DM). Optimization was carried out by Plackett-Burman and central composite experimental designs. Using the optimized medium phytase, alpha-amylase and lipase production of Mucor racemosus NRRL 1994 was compared in solid-state fermentation and in shake flask (SF) fermentation. SSF yielded higher phytase activity than did SF based on mass of initial substrate. Because this particular isolate is a food-grade fungus that has been used for sufu fermentation in China, the whole SSF material (crude enzyme, in situ enzyme) may be used directly in animal feed rations with enhanced cost efficiency.     

Production and properties of three pectinolytic activities produced byAspergillus niger in submerged and solid-state fermentation

Three extracellular pectinases were produced byAspergillus niger CH4 by submerged and solid-state fermentation, and their physicochemical and kinetic properties were studied. The highest productivities of endo- and exo-pectinase and pectin lyase were obtained with solid-state fermentation. The kinetic and physicochemical properties of these enzymes were influenced by the type of culture method used. All activities were very different in terms of pH and temperature optima, stability at different pH and temperature values and affinity for the substrate (K _m values). In solid-state fermentation, all pectinase activities were more stable at extreme pH and temperature values but theK _m values of endo-pectinase and pectin lyase were higher with respect to those activities obtained by the submerged-culture technique. The pectin lyase activity obtained by the submerged-culture technique showed substrate inhibition but the enzyme obtained by solid-state fermentation did not. Electrophoresis, using sodium dodecyl sulphate/polyacrylamide gel with enzymatic extracts obtained for both culture methods, showed the same number on protein bands but some differences were found in their electrophoretic position. The results obtained in this work suggest that the culture method (submerged or solid-state) may be responsible for inducing changes in some of the pectinolytic enzymes produced byA. niger.     

An enzyme-linked immunosorbent assay for the estimation of fungal biomass during solid-state fermentation

An enzyme-linked immunosorbent assay for sensitive, specific and quantitative estimation of fungal biomass during solid-state fermentation is described. Using this method, differential growth rates and colonization of the substrate can be studied. The assay has potential application for the efficient monitoring of solid-state fermentation involving specific fungus, for which available methods are not adequate. Received: 18 November 1997 / Received last revision: 8 June 1998 / Accepted: 14 June 1998     

Correlation of brightening with cumulative enzyme activity related to lignin biodegradation during biobleaching of kraft pulp by white rot fungi in the solid-state fermentation system.

Biobleaching of hardwood unbleached kraft pulp (UKP) by Phanerochaete chrysosporium and Trametes versicolor was studied in the solid-state fermentation system with different culture media. In this fermentation system with low-nitrogen and high-carbon culture medium, pulp brightness increased by 15 and 30 points after 5 days of treatment with T. versicolor and P. chrysosporium, respectively, and the pulp kappa number decreased with increasing brightness. A comparison of manganese peroxidase (MnP), lignin peroxidase (LiP), and laccase activities assayed by using fungus-treated pulp and the filtrate after homogenizing the fungus-treated pulp in buffer solution indicated that enzymes secreted from fungi were adsorbed onto the UKP and that assays of these enzyme activities should be carried out with the treated pulp. Time course studies of brightness increase and MnP activity during treatment with P. chrysosporium suggested that it was difficult to correlate them on the basis of data obtained on a certain day of incubation, because the MnP activity fluctuated dramatically during the treatment time. When brightness increase and cumulative MnP, LiP, and laccase activities were determined, a linear relationship between brightness increase and cumulative MnP activity was found in the solid-state fermentation system with both P. chrysosporium and T. versicolor. This result suggests that MnP is involved in brightening of UKP by white rot fungi.     

Critical importance of moisture content of the medium in alpha-amylase production by Bacillus licheniformis M27 in a solid-state fermentation system

Summary A large reduction (about 30%–78%) is observed in the production of alpha-amylase by Bacillus licheniformis M27 in standardized wheat bran medium under solid-state fermentation when the moisture content of the medium is higher than the standardized value (65%). However, a surge in enzyme production in the first 24 h of fermentation is observed in media with 75% and 85% moisture. The role of decreased oxygen transfer in reducing enzyme tires by about 78% in the medium containing 95% moisture is evident, since the enzyme tire can be effectively increased by agitating the medium during fermentation. No such limitation in oxygen transfer is evident in medium containing 65% moisture even where incubated under static conditions or when the flask is capped by aluminum foil. The data indicate the critical importance of the moisture content of the medium in -amylase production by B. licheniformis M27 in solid-state fermentation systems. The results also have several implications of scientific and techno-economic importance and are useful in explaining some of the advantages of a solid-state fermentation system over the submerged fermentation process. Offprint requests to: B. K. Lonsane     

Characterization of the Factors that Influence Sinapine Concentration in Rapeseed Meal during Fermentation

We analyzed and compared the difference in sinapine concentration in rapeseed meal between the filamentous fungus, Trametes sp 48424, and the yeast, Saccharomyces cerevisiae, in both liquid and solid-state fermentation. During liquid and solid-state fermentation by Trametes sp 48424, the sinapine concentration decreased significantly. In contrast, the liquid and solid-state fermentation process by Saccharomyces cerevisiae just slightly decreased the sinapine concentration (P ≤ 0.05). After the solid-state fermented samples were dried, the concentration of sinapine in rapeseed meal decreased significantly in Saccharomyces cerevisiae. Based on the measurement of laccase activity, we observed that laccase induced the decrease in the concentration of sinapine during fermentation with Trametes sp 48424. In order to eliminate the influence of microorganisms and the metabolites produced during fermentation, high moisture rapeseed meal and the original rapeseed meal were dried at 90°C and 105°C, respectively. During drying, the concentration of sinapine in high moisture rapeseed meal decreased rapidly and we obtained a high correlation coefficient between the concentration of sinapine and loss of moisture. Our results suggest that drying and enzymes, especially laccase that is produced during the solid-state fermentation process, may be the main factors that affect the concentration of sinapine in rapeseed meal.