Solid substrate fermentation of wheat straw to fungal protein

Steam-treated wheat straw at a 70% (w/w) moisture level was subjected to solid substrate fermentation (SSF) with Trichoderma reesei (Riga, USSR) or a mixed culture of T. reesei and Endomycopsis fibuliger (R-574) in fermentation equipment of various design: some with mixing, some with stationary layers, including a mixedlayer 1.5-m(3) pilot plant scale fermenter. The best protein productivity was obtained in stationary layer fermenters with a product containing 13% protein. The main limitations of lignocellulose SSF, such as hindrance of fungal growth, limiting accessibility and availability of substrate, and difficulty in moisture and heat control, were analyzed. The technological parameters of SSF, submerged fermentation, and alternate lignocellulose conversion processes were compared. The SSF had lower overall efficiency but higher product concentration per reaction volume than other conversion schemes.     

The enzymatic hydrolysis and fermentation of pretreated wheat straw to ethanol

Autohydrolysis and ethanol-alkali pulping were used as pretreatment methods of wheat straw for its subsequent saccharification by Trichoderma reesei cellulase. The basic hydrolysis parameters, i.e., reaction time, pH, temperature, and enzyme and substrate concentration, were optimized to maximize sugar yields from ethanol-alkali modified straw. Thus, a 93% conversion of 2.5% straw material to sugar syrup containing 73% glucose was reached in 48 h using 40 filter paper units/g hydrolyzed substrate. The pretreated wheat straw was then fermented to ethanol at 43 degrees C in the simultaneous saccharification and fermentation (SSF) process using T. reesei cellulase and Kluyveromyces fragilis cells. From 10% (w/v) of chemically treated straw (dry matter), 2.4% (w/v) ethanol was obtained after 48 h. When the T. reesei cellulase system was supplemented with beta-glucosidase from Aspergillus niger, the ethanol yield in the SSF process increased to 3% (w/v) and the reaction time was shortened to 24 h.     

The use of cellulases from a beta-glucosidase-hyperproducing mutant of Trichoderma reesei in simultaneous saccharification and fermentation of wheat straw

Conidia of the cellulolytic strain Trichoderma reesei F522 were mutagenized with UV irradiation and N-methyl|-N'-nitro-N-nitrosoguanidine (NTG). A visual agar plate detection system was developed, using esculin and ferric ions, to identify mutants of T. reesei with increased beta-glucosidase activity. Selected mutants were tested for production of extracellular cellulases in shake flasks on autohydrolyzed wheat straw as carbon source. The most active mutant V-7 showed about 6-times higher activity of beta-glucosidase than the parent strain F-522, whereas the filter paper degrading and endo-1,4-beta-D-glucanase activities increased by 45% and by almost 31%, respectively. Cellulase preparations obtained from the parent and mutant strains were then used along with Kluyveromyces fragilis cells for ethanol production from ethanol-alkali pulped straw in the simultaneous saccharification and fermentation (SSF) process. From 10% (w/v) of straw pulp (dry matter), 2.5% (w/v) ethanol was obtained at 43 degrees C after 48 h using cellulase derived from the parent strain of T. reesei. When the beta-glucosidase-hyperproducing mutant V-7 was employed, the ethanol yield in the SSF process increased to 3.4% (w/v), the reaction time was shortened to 24 h and no cellobiose was detected in straw hydrolyzates.     

Dynamic modeling and analyses of simultaneous saccharification and fermentation process to produce bio-ethanol from rice straw

The rice straw, an agricultural waste from Asians’ main provision, was collected as feedstock to convert cellulose into ethanol through the enzymatic hydrolysis and followed by the fermentation process. When the two process steps are performed sequentially, it is referred to as separate hydrolysis and fermentation (SHF). The steps can also be performed simultaneously, i.e., simultaneous saccharification and fermentation (SSF). In this research, the kinetic model parameters of the cellulose saccharification process step using the rice straw as feedstock is obtained from real experimental data of cellulase hydrolysis. Furthermore, this model can be combined with a fermentation model at high glucose and ethanol concentrations to form a SSF model. The fermentation model is based on cybernetic approach from a paper in the literature with an extension of including both the glucose and ethanol inhibition terms to approach more to the actual plants. Dynamic effects of the operating variables in the enzymatic hydrolysis and the fermentation models will be analyzed. The operation of the SSF process will be compared to the SHF process. It is shown that the SSF process is better in reducing the processing time when the product (ethanol) concentration is high. The means to improve the productivity of the overall SSF process, by properly using aeration during the batch operation will also be discussed.     

Bioconversion of corn straw by coupling ensiling and solid-state fermentation

A two-stage process that combined solid-state fermentation (SSF) and ensiling was used for bioconversion of corn straw, in order to increase nutritional value and palatability for animal feed. SSF of corn straw increased the level of protein from 6.7% to 14.7% and decreased the cellulose by 38.0% and hemicellulose by 21.2%. Cellulase and xylanase were produced during SSF. After SSF, the fermented substrate was directly ensiled by inoculating with lactic acid bacteria (LAB). In situ produced enzymes and bacterial inoculation resulted in a rapid drop in pH, a high level of lactic acid production, partial degradation of cell wall components and generation of reducing sugars (RSs). Efficiency of ensiling at 25 degrees C, 30 degrees C, 35 degrees C, 40 degrees C was evaluated. Temperature influenced the effect of ensiling; the higher the temperature, the shorter the ensiling period. The combined fermentation upgraded the nutritional value, enhanced the efficiency of ensiling and reduced bioprocessing costs.     

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.     

Standardization of the filter paper activity assay for solid substrate fermentation

The conditions of the filter paper activity (FPA) assay were standardized for solid substrate fermentation (SSF). The FPA is a relative measure of the overall cellulose hydrolysing capacity of microbial cellulase preparations, thus reliable and comparable data may be obtained only under standardized conditions. The standardization developed for submerged fermentation (SF) cannot be translated directly to SSF. In SSF, the FPA is strongly dependent on the extraction volume and on the dilution of the enzyme in the assay. The optimal extraction volume was substrate dependent in SSF of corn fiber, spent brewing grains and wheat straw for cellulase production by Trichoderma reesei Rut C30. Other cellulolytic enzyme assays (endoglucanase, beta-glucosidase and xylanase) were much less sensitive to the extraction volume.     

Steric hindrance of growth of filamentous fungi in solid substrate fermentation of wheat straw

Cellulolytic fungi, such as Trichoderma reesei, T. lignorum, and Chaetomium cellulolyticum reach a low packing density of mycelia when grown on straw under conditions of solid substrate fermentation. The low packing density is shown to be caused partially by the geometric limitation of the growth of mycelia in the substrate and particularly in its pores, but the exact contribution of this limitation and other limitations such as mass transfer and substrate availability cannot be easily distinguished. The combined effect of such limitations is called steric hindrance. This steric hindrance is associated with and may be the principal cause of low biomass concentration in SSF.     

Effect of culture phasing and a polysaccharide on production of xylanase by mixed culture of Trichoderma reesei D1-6 and Aspergillus wentii Pt 2804

A significant increase in extracellular xylanase activity was observed in the mixed culture fermentation of Trichoderma reesei D1-6 and Aspergillus wentii Pt 2804 when A. wentii inoculation was phased by 15 h. A. wentii produced a polysaccharide, chiefly consisting of glucose monomeric units, which was required for expression of maximum xylanase activity. Expression of high activity of xylanase in the A. wentii phased mixed culture compared to that in either T. reesei or A. wentii single cultures appeared to be controlled by the combined action of a polysaccharide produced by A. wentii and the relative growth of the two fungi in the mixed culture.     

Extractive fermentation for improved production of endoglucanase by an intergeneric fusant of Trichoderma reesei/Saccharomyces cerevisiae using aqueous two-phase system

Extractive aqueous two-phase fermentation of endoglucanase, a key enzyme for the conversion of cellulosic substances to fermentable sugars, from an intergeneric fusant of Trichoderma reesei/Saccharomyces cerevisiae is a meaningful approach for better production and simple recovery of this enzyme. A phase composition of 6.5% (w/w) dextran and 7.5% (w/w) polyethylene glycol 6000, having a partition coefficient of 2.89 and 1.31 for endoglucanase from an intergeneric fusant of T. reesei/S. cerevisiae and T. reesei (WT) (being a control in this study), respectively, was chosen for extractive fermentation of the enzyme. Endoglucanase production is higher in medium containing polyethylene glycol (PEG) 6000 than in medium without PEG 6000. Comparative analysis of endoglucanase fermentation by fusant and T. reesei was carried out in shake culture and environment-controlled bioreactor conditions. The fusant produced 0.43U of endoglucanase (overall production: 0.34U) in the top phase of an aqueous two-phase system (ATPS), compared to 0.3U in medium without the phase system in shake culture. In a batch reactor, the endoglucanase level for the fusant in the top phase of ATPS was 0.49U (overall production: 0.40U), compared to 0.38U produced in medium without aqueous two-phase components. To corroborate this study, T. reesei produced 8.41U of endoglucanase (overall production: 5.96U) in the top phase of ATPS, compared to 7.18U in the medium without the phase system in shake culture. On the other hand, in a batch bioreactor, T. reesei produced 10.13U of endoglucanase (overall production: 6.90U) in the top phase of ATPS, compared to 8.56U of the enzyme in medium without aqueous two-phase components. The lower overall enzyme production by T. reesei in the two-phase system might be due to limitation in oxygen transfer to the dispersed phase where the enzyme is produced. A higher cell concentration and a reduced lag phase was obtained in ATPS, compared to a similar medium without phase forming polymers for both the intergeneric fusant of T. reesei/S. cerevisiae and T. reesei.     

Designing simultaneous saccharification and fermentation for improved xylose conversion by a recombinant strain of Saccharomyces cerevisiae

Wheat straw is an abundant agricultural residue which can be used as a raw material for bioethanol production. Due to the high xylan content in wheat straw, fermentation of both xylose and glucose is crucial to meet desired overall yields of ethanol. In the present work a recombinant xylose fermenting strain of Saccharomyces cerevisiae, TMB3400, cultivated aerobically on wheat straw hydrolysate, was used in simultaneous saccharification and fermentation (SSF) of steam pretreated wheat straw. The influence of fermentation strategy and temperature was studied in relation to xylose consumption, ethanol formation and by-product formation. In addition, model SSF experiments were made to further investigate the influence of temperature on xylose fermentation and by-product formation. In particular for SSF at the highest value of fibre content tested (9% water insoluble substance, WIS), it was found that a fed-batch strategy was clearly superior to the batch process in terms of ethanol yield, where the fed-batch gave 71% of the theoretical yield (based on all available sugars) in comparison to merely 59% for the batch. Higher ethanol yields, close to 80%, were obtained at a WIS-content of 7%. Xylose fermentation significantly contributed to the overall ethanol yields. The choice of temperature in the range 30-37 degrees C was found to be important, especially at higher contents of water insoluble solids (WIS). The optimum temperature was found to be 34 degrees C for the raw material and yeast strain studied. Model SSF experiments with defined medium showed strong temperature effects on the xylose uptake rate and xylitol yield.     

Oxygen uptake kinetics during solid state fermentation with Rhizopus oligosporus

Membrane filter culture was used to relate O2 uptake with direct biomass measurement of Rhizopus oligosporus in solid-state fermentation (SSF). Overall values of YX/O and mO were 0.782 mg biomass mg O2–1 and 0.0413 mg O2 mg biomass–1 h–1, respectively. However, these values were not constant during the fermentation, which makes the use of O2 uptake to estimate biomass during SSF problematic. Despite these problems, measuring bioreactor off-gases is the only practical method which allows on-line monitoring of bioreactor performance.     

Production of Xylanase by Thermophilic Melanocarpus albomyces IIS-68

Melanocarpus albomyces IIS-68, a thermophilic fungus was used for the production of extracellular xylanase on various agroresidues in solid-state fermentation (SSF). Growth on untreated wheat straw and sugar cane bagasse supported xylanase production, while rice straw and rice husk did not. Alkali treatment and acid chlorite treatment of these latter substrates, which lead to extensive delignification, enhanced xylanase production. In contrast, these treatments caused a decline in xylanase activity on wheat straw and bagasse. Acetyl esterase was produced concurrently with xylanase, maximal activity being produced on bagasse. Enzyme production was higher in SSF than in submerged fermentation (SmF). Studies with electron micrographs indicated that culture filtrate proteins were able to degrade wall polymers.     

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

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

Heat transfer simulation in solid substrate fermentation

A mathematical model was developed and tested to simulate the generation and transfer of heat in solid substrate fermentation (SSF). The experimental studies were realized in a 1-L static bioreactor packed with cassava wet meal and inoculated with Aspergillus niger. A simplified pseudohomogeneous monodimensional dynamic model was used for the energy balance. Kinetic equations taking into account biomass formation (logistic), sugar consumption (with maintenance), and carbon dioxide formation were used. Model verification was achieved by comparison of calculated and experimental temperatures. Heat transfer was evaluated by the estimation of Biot and Peclet heat dimensionless numbers 5-10 and 2550-2750, respectively. It was shown that conduction through the fermentation fixed bed was the main heat transfer resistance. This model intends to reach a better understanding of transport phenomena in SSF, a fact which could be used to evaluate various alternatives for temperature control of SSF, i.e., changing air flow rates and increasing water content. Dimensionless numbers could be used as scale-up criteria of large fermentors, since in those ratios are described the operating conditions, geometry, and size of the bioreactor. It could lead to improved solid reactor systems. The model can be used as a basis for automatic control of SSF for the production of valuable metabolites in static fermentors.     

Caffeic acid production by simultaneous saccharification and fermentation of kraft pulp using recombinant Escherichia coli

Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and a precursor for biologically active compounds and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by introducing the genes encoding a 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa and tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233 mg/L) far exceeded that by SHF (37.9 mg/L). In the SSF with low cellulase loads (≤2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. In SHF, the formation of by-product acetate and the accumulation of potential fermentation inhibitors increased significantly with kraft pulp hydrolysate than filter paper hydrolysate. The combination of these inhibitors had synergistic effects on caffeic acid fermentation at low concentrations. With lower loads of cellulase in SSF, less potential fermentation inhibitors (furfural, 5-hydroxymethyfurfural, and 4-hydroxylbenzoic acid) accumulated in the medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.

     

螺旋纤维床固定化反应器两步法生产壳聚糖酶

采用了菌体生长与产酶分步的新工艺利用里氏木霉（Trichoderma reesei） ATCC56764生产壳聚糖酶,酶活力比在相同条件下进行的一步法产酶提高了1.7倍。采用此工艺在螺旋纤维床生物反应器中进行产酶试验,酶活比采用游离细胞培养又提高了39%,达到0.246U/mL。固定化菌丝还能够长期保持活性,在重复分批操作中,经过10批共15天的产酶实验,平均每批的酶活保持在0.235U/mL左右。     

Microbial production of a biofuel (acetone–butanol–ethanol) in a continuous bioreactor: impact of bleed and simultaneous product removal

Acetone butanol ethanol (ABE) was produced in an integrated continuous one-stage fermentation and gas stripping product recovery system using Clostridium beijerinckii BA101 and fermentation gases (CO₂ and H₂). In this system, the bioreactor was fed with a concentrated sugar solution (250–500 g L^?1 glucose). The bioreactor was bled semi-continuously to avoid accumulation of inhibitory chemicals and products. The continuous system was operated for 504 h (21 days) after which the fermentation was intentionally terminated. The bioreactor produced 461.3 g ABE from 1,125.0 g total sugar in 1 L culture volume as compared to a control batch process in which 18.4 g ABE was produced from 47.3 g sugar. These results demonstrate that ABE fermentation can be operated in an integrated continuous one-stage fermentation and product recovery system for a long period of time, if butanol and other microbial metabolites in the bioreactor are kept below threshold of toxicity.     

Morphological characterization and viability assessment of Trichoderma reesei by image analysis

The production of cellulase from the filamentous fungus Trichoderma reesei is a critical step in the industrial process leading to cellulose ethanol. As a result of the lack of quantitative analysis tools, the intimate relationship that exists between the morphological and physiological states of the microorganism, the shear field in the bioreactor, and the process performance is not yet fully understood. A semiautomatic image analysis protocol was developed to characterize the mycelium morphology and to estimate its percentage viability during the fermentation process based on four morphological types (unbranched, branched, entangled, and clumped microorganisms). Pictures taken under bright field microscopy combined with images of fluorescein diacetate stained fungi were used to assess the morphological parameters and the percentage viability of microorganisms simultaneously. The method was tested during the course of fed-batch fermentation in a reciprocating plate bioreactor. The use of the image analysis protocol was found to be successful in quantifying the variations in the morphology and the viability of T. reesei throughout the fermentation.