The unconventional adverse effects of fungal pretreatment on iturin A fermentation by Bacillus amyloliquefaciens CX-20

Fungal pretreatment is the most common strategy for improving the conversion of rapeseed meal (RSM) into value-added microbial products. It was demonstrated that Bacillus amyloliquefaciens CX-20 could directly use RSM as the sole source of all nutrients except the carbon source for iturin A fermentation with high productivity. However, whether fungal pretreatment has an impact on iturin A production is still unknown. In this study, the effects of fungal pretreatment and direct bio-utilization of RSM for iturin A fermentation were comparatively analysed through screening suitable fungal species, and evaluating the relationships between iturin A production and the composition of solid fermented RSM and liquid hydrolysates. Three main unconventional adverse effects were identified. (1) Solid-state fermentation by fungi resulted in a decrease of the total nitrogen for B. amyloliquefaciens CX-20 growth and metabolism, which caused nitrogen waste from RSM. (2) The released free ammonium nitrogen in liquid hydrolysates by fungal pretreatment led to the reduction of iturin A. (3) The insoluble precipitates of hydrolysates, which were mostly ignored and wasted in previous studies, were found to have beneficial effects on producing iturin A. In conclusion, our study verifies the unconventional adverse effects of fungal pretreatment on iturin A production by B. amyloliquefaciens CX-20 compared with direct bio-utilization of RSM.     

Mutagenesis of echinocandin B overproducing Aspergillus nidulans capable of using starch as main carbon source

Abstract

Echinocandin B, a kind of antimycotic with cyclic lipo-hexapeptides, was produced by fermentation with Aspergillus nidulans using fructose as main carbon source. The objective of this study was to screen a high-yield mutant capable of using cheap starch as main carbon source by atmospheric and room temperature plasma (ARTP) treatment in order to decrease the production cost of echinocandin B. A stable mutant A. nidulans ZJB19033, which can use starch as optimal carbon source instead of expensive fructose, was selected from two thousands isolates after several cycles of ARTP mutagenesis. To further increase the production of echinocandin B, the optimization of fermentation medium was performed by response surface methodology (RSM), employing Plackett-Burman design (PBD) followed by Box-Behnken design (BBD). The optimized fermentation medium provided the optimal yield of echinocandin B, 2425.9?±?43.8?mg/L, 1.3-fold compared to unoptimized medium. The results indicated that the mutant could achieve high echinocandin B production using cheap starch as main carbon source, and the cost of carbon sources in fermentation medium reduced dramatically by about 45%.     

Production of the immunosuppressant cyclosporin A by a new soil isolate,Aspergillus fumigatus,in submerged culture

Cyclosporin A (CyA) has received meticulous attention owing to its immunosuppressive and biological activities. In this study, a soil isolate, capable of producing CyA, was named Zag1 strain and identified as Aspergillus fumigatus based on macroscopic and microscopic characteristics, 18S rDNA sequence, and phylogenetic characteristic analysis. To maximize the production of CyA, the fungal culture was grown under various fermentation conditions including selection of the cultivation medium, agitation rate, fermentation time, incubation temperature, pH value, inoculum nature, and medium volume. A simple medium (pH 5.0) containing 5% maltose as a carbon source and 2% potassium nitrate as a nitrogen source favored the highest CyA production when the fermentation process was maintained at 120 rpm for 9 days and at 30 °C using 3% standard inoculum of 5-day-old. The final CyA titer under these conditions was intensified to 2.23–3.31-fold, as compared with the amount obtained with seven types of basal media. A. fumigatus Zag1 appears to possess a good biotechnological potential for CyA production under favorable culture conditions.

     

Construction of a novel recombinant Escherichia coli strain capable of producing 1,3–propanediol and optimization of fermentation parameters by statistical design

Summary The structural gene yqhD from a wild-type Escherichia coli encoding 1,3-propanediol oxidoreductase isoenzyme and the structural gene dhaB from Citrobacter freundii encoding glycerol dehydratase were amplified by using the PCR method. The temperature control expression vector pHsh harboring the yqhD and dhaB genes was transformed into E. coli JM109 to yield the recombinant strain E. coli JM109 (pHsh-dhaB-yqhD). The response surface method (RSM) was then applied to further optimize the fermentation condition of the recombinant strain. A mathematical model was then developed to show the effect of each medium composition and their interactions on the production of 1,3-propanediol by recombinant strain E. coli JM109. The model estimated that a maximal yield of 1,3-propanediol (43.86 g/l) could be obtained when the concentrations of glycerol, yeast extract and vitamin B₁₂ were set at 61.8 g/l, 6.2 g/l and 49 mg/l, respectively; and the fermentation time was 30 h. These predicted values were also verified by validation experiments. Compared with the values obtained by other runs in the experimental design, the optimized medium resulted in a significant increase in the yield of 1,3-propanediol. The yield and productivity under the optimal parameters and process can reach 43.1 g/l and 1.54 g/l/h. Maximum 1,3-propanediol yield of 41.1 g/l was achieved in a 5-l fermenter using the optimized medium. This makes the engineered strain have potential application in the conversion of glycerol to 1,3-propanediol on an industrial scale.     

Production of tyrosinase by Auricularia auricula using low cost fermentation medium

Low cost fermentation media using agricultural by-products (wheat bran extract, rice bran extract and soybean meal extract) as a major nutrient source, were evaluated for the production of tyrosinase from the fungus Auricularia auricula in submerged culture. In single-factor experiments, three components (wheat bran extract, casein and CuSO₄) were chosen to further optimize medium composition using response surface methodology (RSM). The central composite experimental results showed the following optimum medium composition: wheat bran extract 36.0 %, casein 1.1 g/l and CuSO₄ 0.13 g/l. Under these conditions, the highest tyrosinase activity was 17.22 U/ml, which was 2.1 fold higher than that obtained using the non-optimized medium. The present study is the first to report the statistical optimization of medium composition for production of tyrosinase by A. auricula using cheaper wheat bran extract as a major nutrient source. These results might provide a reference for the development of a cost-effective medium for commercial production of tyrosinase.     

Physostigmine production byStreptomyces griseofuscus NRRL 5324: Process development and scale-up studies

A reliable and scalable fermentation process was developed for production of the acetylcholine esterase inhibitor physostigmine employingStreptomyces griseofuscus NRRL 5324. Initial fermentation in small-scale bioreactors reached physostigmine levels of approximately 60 mg L^–1 after 139 h. Optimization of both process operating parameters and production medium composition rapidly yielded a seven-fold increase in physostigmine titer. The scaled up process routinely produced physostigmine titers of approximately 400 mg L^–1 during a fermentation cycle of 180 h, and supported the rapid production of large amounts of physostigmine. A physostigmine production of 500 mg L^–1 representing an eight-fold improvement over the original performance, was achieved using a glucose/ammonium fed-batch process.     

Effect of nanopreparations on development of the populations of <Emphasis Type="Italic">Saccharomyces</Emphasis> brewer’s yeasts

Effect of nanoparticles of different nature on development of the populations of top and bottom fermentation brewers’ yeasts (Saccharomyces cerevisiae) on various media and on ethanol accumulation during wort fermentation was studied. In some cases the presence of nanoparticles in the medium was shown to affect the monitored parameters, primarily the yeast cell titer. Direction and intensity of this effect depend on a number of factors: nanopreparation kind and concentration, medium composition, and cultivation conditions. Inhibition of development of yeast populations was most pronounced in the presence of the silver nanopreparation, while zinc nanoparticles and zinc oxide nanopreparation had the least effect. At the same time, specific concentrations of some nanoparticles stimulated growth of the population and yeast metabolism.     

Effect of culture conditions on growth and sporulation ofBacillus thuringiensis subsp.israelensis and development of media for production of the protein crystal endotoxin

Summary The influence of medium composition on the inoculum and production stages of theBacillus thuringiensis subsp.israelensis bioinsecticide fermentation was investigated. Media which inhibited sporulation were selected for inoculum development stages. Bioinsecticide production media were designed to produce high cell counts and >90% sporulation in a 48h fermentation. Maximum insecticidal activity occurred at the point of maximum bacterial cell lysis/spore release. A process involving two inoculum stages and a 48h production stage in a 40 l fermenter yielded a viable cell count of 6.5 x 10⁹/ml with greater than 95% sporulation. Good correlation existed between spore counts and bioinsecticide activity.     

Generation of high rapamycin producing strain via rational metabolic pathway‐based mutagenesis and further titer improvement with fed‐batch bioprocess optimization

Rapamycin is a triene macrolide antibiotic produced by Streptomyces hygroscopicus. Besides its wide application as an effective immunosuppressive agent, other important bioactivities have made rapamycin a potential drug lead for novel pharmaceutical development. However, the low titer of rapamycin in the original producer strain limits further industrialization efforts and restricts its use for other applications. Predicated on knowledge of the metabolic pathways related to rapamycin biosynthesis in S. hygroscopicus, we have rationally designed approaches to generate a rapamycin high producer strain of S. hygroscopicus HD‐04‐S. These have included alleviation of glucose repression, improved tolerance towards lysine and shikimic acid, and auxotrophy of tryptophan and phenylalanine through the application of stepwise UV mutagenesis. The resultant strain produced rapamycin at 450 mg/L in the shake flask scale. These fermentations were further scaled up in 120 and 20,000 L fermentors, respectively, at the pilot plant. Selected fermentation factors including agitation speed, pH, and on‐line supplementation were systematically evaluated. A fed‐batch strategy was established to maximize rapamycin production. With these efforts, an optimized fermentation process in the larger scale fermentor was developed. The final titer of rapamycin was 812 mg/L in the 120 L fermentor and 783 mg/L in the 20,000 L fermentor. This work highlights a high rapamycin producing strain derived by mutagenesis and subsequent screening, fermentation optimization of which has now made it feasible to produce rapamycin on an industrial scale by fermentation. The strategies developed here should also be applicable to titer improvement of other important microbial natural products on an industrial scale. Biotechnol. Bioeng. 2010;107: 506–515. © 2010 Wiley Periodicals, Inc.     

Deletion of lactate dehydrogenase in Enterobacter aerogenes to enhance 2,3-butanediol production

2,3-Butanediol is an important bio-based chemical product, because it can be converted into several C4 industrial chemicals. In this study, a lactate dehydrogenase-deleted mutant was constructed to improve 2,3-butanediol productivity in Enterobacter aerogenes. To delete the gene encoding lactate dehydrogenase, λ Red recombination method was successfully adapted for E. aerogenes. The resulting strain produced a very small amount of lactate and 16.7% more 2,3-butanediol than that of the wild-type strain in batch fermentation. The mutant and its parental strain were then cultured with six different carbon sources, and the mutant showed higher carbon source consumption and microbial growth rates in all media. The 2,3-butanediol titer reached 69.5 g/l in 54 h during fed-batch fermentation with the mutant,which was 27.4% higher than that with the parental strain.With further optimization of the medium and aeration conditions,118.05 g/l 2,3-butanediol was produced in 54 h during fed-batch fermentation with the mutant. This is by far the highest titer of 2,3-butanediol with E. aerogenes achieved by metabolic pathway engineering.     

Production of schizophyllan using Schizophyllum commune NRCM

In the present work, four strains were screened for schizophyllan production, of which Schizophyllum commune NRCM was selected for further work. The fermentation was carried out for 168 h at 28+/-2 degrees C on an orbital shaker at 180 rpm. In the first step, one factor-at-a-time method was used to investigate the effect of media constituents such as carbon and nitrogen sources on schizophyllan production. Subsequently in the second step, concentration of the medium components was optimized using Response Surface Method (RSM). The yield increased from 3.25+/-0.72 g/l in the unoptimized media to 8.03+/-1.12 g/l in the medium optimized by RSM.     

Enrichment of laccase production by Phoma herbarum isolate KU4 under solid-state fermentation by optimizing RSM coefficients using genetic algorithm

The process parameters were optimized to obtain enhanced enzyme activity from the fungus Phoma herbarum isolate KU4 using rice straw and saw dust as substrate under solid-state fermentation using Response surface methodology (RSM). Genetic algorithm was used to validate the RSM for maximum laccase production. Six variables, viz., pH of the media, initial moisture content, copper sulphate concentration, concentration of tannic acid, inoculum concentration and incubation time were found to be effective and optimized for enhanced production. Maximum laccase production was achieved by RSM at pH 5·0 and 86% of initial moisture content of the culture medium, 150 µmol l⁻¹ of CuSO₄, 1·5% tannic acid and 0·128 g inoculum g⁻¹ dry substrate inoculum size on the fourth day of fermentation. The highest laccase activity was observed as 79 008 U g⁻¹, which is approximately sixfold enhanced production compared to the unoptimized condition (12 085·26 U g⁻¹).     

Medium optimization for pyrroloquinoline quinone (PQQ) production by Methylobacillus sp. zju323 using response surface methodology and artificial neural network–genetic algorithm

Methylobacillus sp. zju323 was adopted to improve the biosynthesis of pyrroloquinoline quinone (PQQ) by systematic optimization of the fermentation medium. The Plackett–Burman design was implemented to screen for the key medium components for the PQQ production. CoCl₂?·?6H₂O, ρ-amino benzoic acid, and MgSO₄?·?7H₂O were found capable of enhancing the PQQ production most significantly. A five-level three-factor central composite design was used to investigate the direct and interactive effects of these variables. Both response surface methodology (RSM) and artificial neural network–genetic algorithm (ANN–GA) were used to predict the PQQ production and to optimize the medium composition. The results showed that the medium optimized by ANN–GA was better than that by RSM in maximizing PQQ production and the experimental PQQ concentration in the ANN–GA-optimized medium was improved by 44.3% compared with that in the unoptimized medium. Further study showed that this ANN–GA-optimized medium was also effective in improving PQQ production by fed-batch mode, reaching the highest PQQ accumulation of 232.0?mg/L, which was about 47.6% increase relative to that in the original medium. The present work provided an optimized medium and developed a fed-batch strategy which might be potentially applicable in industrial PQQ production.     

Optimization of critical medium components using response surface methodology for biomass and extracellular polysaccharide production by <Emphasis Type="Italic">Agaricus blazei</Emphasis>

Response surface methodology (RSM) was applied to optimize the critical medium ingredients of Agaricus blazei. A three-level Box–Behnken factorial design was employed to determine the maximum biomass and extracellular polysaccharide (EPS) yields at optimum levels for glucose, yeast extract (YE), and peptone. A mathematical model was then developed to show the effect of each medium composition and its interactions on the production of mycelial biomass and EPS. The model predicted the maximum biomass yield of 10.86 g/l that appeared at glucose, YE, peptone of 26.3, 6.84, and 6.62 g/l, respectively, while a maximum EPS yield of 348.4 mg/l appeared at glucose, YE, peptone of 28.4, 4.96, 5.60 g/l, respectively. These predicted values were also verified by validation experiments. The excellent correlation between predicted and measured values of each model justifies the validity of both the response models. The results of bioreactor fermentation also show that the optimized culture medium enhanced both biomass (13.91 ± 0.71 g/l) and EPS (363 ± 4.1 mg/l) production by Agaricus blazei in a large-scale fermentation process.     

金霉菌的生理与金霉素的生产 I.接种培养基对于金霉菌的代谢及抗生素产量的影响

将金莓菌的芽孢接种到不同的接种培养基中,24小时后再接种到同一的酦酵培养基上,发现金霉素的产量有显著不同。一个好的接种培养基比一个坏的接种培养基所生的菌体在酦酵过程中产生的金霉素可以大出7至8倍。接种培养基的 pH 值对于以后菌体在酦酵培养基中金霉素的生产也有显著的影响。接种培养基并不显著地影响菌体在酦酵培养基上的生长,但已显然变更了它的代谢作用。由不同接种培养基中萌生的菌体,在同一酦酵培养基中醣的利用率不同,氧化的能力不同,有机酸的形成和堆积也不同,它们对于一些金属元素的反应也不一样。这个研究证明了金霉菌的初期的培养环境,对于以后菌体的发育和金霉素的生产有极大的影响。同时亦指出了金霉菌的发育在不同时期需要不同的环境,有着不同的代谢类型。去掌握这个生物规律——环境条件和发育的过程,是正确的研究方向,是提高抗生素产量的必要途径。     

Development of a defined medium fermentation process for physostigmine production by Streptomyces griseofuscus

Physostigmine is a plant alkaloid of great interest as a therapeutic candidate for the treatment of Alzheimer's disease. Fortunately, this compound is also produced by Streptomyces griseofuscus NRRL 5324 during submerged cultivation. A fermentation process that used chemically defined medium was therefore developed for its production. By means of statistical experimentation, the physostigmine titer was quickly increased from 20 mg/l to 520 mg/l with a culture growth of 19 gl dry cell weight on the shake-flask scale. Further medium optimization resulted in a yield of 790 mg/l in a 23-l bioreactor using a batch process. A titer of 880 mg/l was attained during scale-up in a 800-l fermentor by employing a nutrient-feeding strategy. This production represents a 44-fold increase over the yield from the initial process in shake-flasks. The defined-medium fermentation broth was very amenable to downstream processing.     

Enzyme Production of Pleurotus ostreatus Evaluated by the Three‐Way Principal Component Analysis

The effect of the composition of culture media and fermentation time on the production of β‐glucosidase, xylanase, laccase, manganese‐dependent and independent peroxidases by the edible fungus Pleurotus ostreatus was determined. Culture medium separately containing potato, pepper, and tomato extracts and the enzyme activities were assessed to 31.5 days of fermentation. Three‐dimensional principal component analysis (3D‐PCA) followed by the plots of the first two elements of component matrices was employed for the elucidation of the similarities and dissimilarities between the parameters. It was established that the dimensionality of the original 3D matrix (9, 5, 4) could be reduced to 2, 1, 2 with 4.54 % loss of information. The plots demonstrated that the culture media displayed large differences in their capacity to promote enzyme production and that the presence of pepper and tomato extracts exerted the greatest influence on the enzyme activities. The effect of the fermentation time was manifested only after 14 d of fermentation and the highest differences were observed after 28 and 31.5 d of fermentation. Except laccase and manganese‐dependent peroxidase, the enzyme activities also differed considerably dependent on the composition of the fermentation broth. 3D‐PCA followed by the plots of component matrices is a valuable tool for the simultaneous assessment of three dimensional data matrices in biotechnological processes.     

Enhancement of fructanohydrolase synthesis from <Emphasis Type="Italic">Aspergillus niger</Emphasis> by simultaneous in vitro induction and in vivo acid stress using sucrose ester

This study describes a novel strategy to improve the fructanohydrolase production and growth performance of Aspergillus niger by the addition of sucrose ester to the culture medium in a 3 L fermenter. It was found that in the aerobic and non-pH-controlled condition, the sucrose ester not only acted as a more favorable in vitro inducer than inulin for fructanohydrolase formation, but also significantly forced in vivo carbon and energy flux into enzyme synthesis by acid stress. Response surface methodology (RSM) was used to optimize the composition of the culture medium, hence the fermentation period was shortened (near 50%) and enzyme activity was enhanced (over 4-fold higher), respectively. The results presented here provided a novel way to improve the inducible enzyme production by simultaneous inducement and acid stress.     

The utilization of beet molasses as a novel carbon source for cephalosporin C production by Acremonium chrysogenum: Optimization of process parameters through statistical experimental designs

In this work, cephalosporin C (CPC) production on pilot scale fermenters of 600l capacity with 350l working volume by Acremonium chrysogenum EMCC 904 was performed. The effects of fermentation medium composition, inoculum concentration, initial pH and aeration rate on CPC production by A. chrysogenum strain was investigated by using response surface methodology (RSM). The Plackett-Burman design which involves two concentrations of each nutrient was effective in searching for the major medium components promoting CPC production. Under our experimental conditions; Soya oil, beet molasses and corn steep liquor were found to be the major factors contributing to the antibiotic production. Subsequently, a Box-Behnken design was used for outlining the concentration of the most effective medium constituents. Estimated optimum composition for the production of CPC was as follows: soya oil, 40g/l; beet molasses, 180g/l; and corn steep liquor, 330g/l. The central composite design was used for outlining the optimum values of the fermentation parameters. Estimated optimum values for the production of CPC are as follows: inoculum level, 10(5.5)spores/ml; initial pH, 4.3; and aeration rate, 9364ml/min.