Sorbitol as a non-repressing carbon source for fed-batch fermentation of recombinant Pichia pastoris

Glycerol/methanol and sorbitol/methanol mixed-feed fermentation strategies for the production of recombinant proteins by Pichia pastoris were compared in order to examine sorbitol's potential as a carbon source. Although P. pastoris does have a lower cell yield on sorbitol than on glycerol, the specific rate of product formation is higher (60 g protein g^–1 dry wth for sorbitol/methanol, vs 45 g protein g^–1 dry wth for glycerol/methanol), resulting in comparable final recombinant expression levels. Importantly, the presence of residual sorbitol in the growth medium appears to be less repressive to the alcohol oxidase promoter in this organism, providing a more forgiving means of operating mixed-feed fed-batch recombinant P. pastoris fermentations.     

Production of rhamnolipid biosurfactant by fed-batch culture of Pseudomonas aeruginosa using glucose as a sole carbon source.

The pH-stat fed-batch culture of Pseudomonas aeruginosa YPJ-80 was done to produce a rhamnolipid biosurfactant. With glucose as the sole carbon source, the final concentrations of cells and rhamnolipid biosurfactant obtained in 25 h were 25 g cell dry weight/l and 4.4 g/l, respectively.     

Production of pullulan by a fed-batch fermentation

Summary In pullulan production from sucrose byAureobasidium pullulans, a sugar concentration higher than 5% (w/v) inhibited cell growth and the production of exopolysaccharide. By a fed-batch fermentation, the inhibitory effects of the high sugar concentration were overcome and 58.0 g/1 of exopolysaccharide were obtained from 10% sucrose.Abbreviations m, n relationship parameters for the growth and non-growth associated product formation - X, Xmax biomass and maximum biomass concentration (g cell/1) - P product concentration (g exopolysaccharide/1) - specific growth rate of cell (hr^–1)     

Production of polyhydroxyalkanoates (PHAs) with canola oil as carbon source

Wautersia eutropha was able to synthesize medium chain length polyhydroxyalkanoates (PHAs) when canola oil was used as carbon source. W. eutropha was cultivated using fructose and ammonium sulphate as carbon and nitrogen sources, respectively, for growth and inoculum development. The experiments were done in a laboratory scale bioreactor in three stages. Initially, the biomass was adapted in a batch culture. Secondly, a fed-batch was used to increase the cell dry weight and PHA concentration to 4.36 g L(-1) and 0.36 g L(-1), respectively. Finally, after the addition of canola oil as carbon source a final concentration of 18.27 g L(-1) PHA was obtained after 40 h of fermentation. With canola oil as carbon source, the polymer content of the cell dry matter was 90%. The polymer was purified from dried cells and analyzed by FTIR, NMR and DSC using PHB as reference. The polymer produced by W. eutropha from canola oil had four carbon monomers in the structure of the PHA and identified by 1H and 13C NMR analysis as 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxyoctanoate (3HO), and 3-hydroxydodecanoate (3HDD).     

Alcohol fermentation using lignin-related compounds as a carbon source

Clostridium indolis JCM 1380 produced 0.24 g/l of ethanol from protocatechuic acid with consumption of about 40% of the substrate (1.2 g/l) at 96 h of cultivation, while Clostridium sphenoides JCM 1415 produced 0.37 g/l of ethanol from p-hydroxybenzoic acid with consumption of about 30% of the substrate (1.5 g/l) at 60 h of cultivation. However, a long lag time was necessary to produce ehtanol from protocatechuic acid by C. indolis, whereas ethanol from p-hydroxybenzoic acid by C. sphenoides was produced without a lag time.     

Production of emulsan in a fermentation process using soybean oil (SBO) in a carbon-nitrogen coordinated feed

A fermentation process for the microbial production of an amphipatic lipopolysaccharide, emulsan, has been established using a triglyceride carbon source in a coordinated carbon-nitrogen feed strategy. The polysaccharide was produced by the Acinetobacter strain at a productivity of about 0.5 g emulsan/L h while utilizing only the fatty acids (FA) portion of the triglycerides or free fatty acids that were fed into the medium.A useful correlation between the utilization of the fatty acids and the consumption of the nitrogen was found and employed for the practical feed strategy using an appropriate C--N ratio (7.7 g C/g N) of the soybeanoil (SBO) (carbon source) to the ammonium hydroxide base (nitrogen source). Either the pH control or the supervising computer system could accomplish the adequate balanced feed in to the fermentor. Lipolysis slowdown was overcome by switching from a triglyceride carbon source to a free fatty acids one. A yield of about 0.2 g emulsan/g fatty acids was obtained and a final concentration of over 20 g/L was reached. The polymeric product was found to be partially associated with the cell-oil complexes in the fermentation broth unless the oily carbon source was efficiently exhausted. A fedbatch fermentation that employed a shift of the carbon source feed from triglycerides to free fatty acids appeared to be an appropriate and feasible way of producing the polymer.     

Enhanced production of exopolysaccharides using industrial grade starch as sole carbon source

Bioprocess and Biosystems Engineering - Industrial grade soluble corn starch was used directly and effectively as the fermentation substrate for microbial exopolysaccharides production. Bacillus...     

Production of alkaline protease with Bacillus licheniformis in a controlled fed-batch process

Summary A production method for alkaline serine protease with Bacillus licheniformis in a synthetic medium was developed. Employing closed-loop control of oxygen, nitrogen and carbon source the pO₂ was held at 5%, the ammonium concentration kept below 1 mM and the glycerol concentration was maintained between 20 and 100 mM. Protease production was monitored by flow injection analysis. Thus, in a fed-batch procedure production could be increased 4.6-fold in comparison to an uncontrolled batch process. Offprint requests to: G. Bierbaum     

Experimental optimization of a real time fed-batch fermentation process using Markov decision process

This article describes a methodology that implements a Markov decision process (MDP) optimization technique in a real time fed-batch experiment. Biological systems can be better modeled under the stochastic framework and MDP is shown to be a suitable technique for their optimization. A nonlinear input/output model is used to calculate the probability transitions. All elements of the MDP are identified according to physical parameters. Finally, this study compares the results obtained when optimizing ethanol production using the infinite horizon problem, with total expected discount policy, to previous experimental results aimed at optimizing ethanol production using a recombinant Escherichia coli fed-batch cultivation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 317-327, 1997.     

补料分批发酵过程中两阶段发酵生产1,3-丙二醇

在补料分批发酵过程中提高比生长速率不仅减少乙醇、甲酸的生成,而且提高1,3-丙二醇的得率和比生产速率.发酵后期甘油的浓度在15～26 g/L时有利于提高1,3-丙二醇的生产.采取在发酵前期控制菌体较高比生长速率和发酵后期控制适宜甘油浓度相结合的策略,有效地提高了1,3-丙二醇的生产,降低副产物乳酸和乙醇的生成.     

以甘蔗糖蜜为碳源发酵生产海藻糖的研究

对以甘蔗糖蜜为碳源生产海藻糖的工艺条件进行了研究,结果表明,甘蔗糖蜜完全可以提供酵母菌体生长和海藻糖合成所需的碳源,以28g鲜酵母/L的接种量,流加培养14d(其中培养11h后调pH至5.0,加入1.5%NaCl,升温至39℃继续培养3h),可使海藻糖的产量达9.4g/L。     

Upstream process optimization of polyhydroxybutyrate (PHB) by Alcaligenes latus using two-stage batch and fed-batch fermentation strategies

This research focused on optimizing the upstream process time for production of polyhydroxybutyrate (PHB) from sucrose by two-stage batch and fed-batch fermentation with Alcaligenes latus ATCC 29714. The study included selection of strain, two-stage batch fermentations with different time points for switching to nitrogen limited media (14, 16 or 18?h) and fed-batch fermentations with varied time points (similar to two stage) for introducing nitrogen limited media. The optimal strain to produce PHB using sucrose as carbon source was A. latus ATCC 29714 with maximum-specific growth rate of 0.38?±?0.01?h^?1 and doubling time of 1.80?±?0.05?h. Inducing nitrogen limitation at 16?h and ending second stage at 26?h gave optimal performance for PHB production, resulting in a PHB content of 46.7?±?12.2?% (g PHB per g dry cell weight) at the end of fermentation. This was significantly higher (P?≤?0.05) (approximately 7?%) than the corresponding fed batch run in which nitrogen limitation was initiated at 16?h.     

Lipase production by Aspergillus terreus using mustard seed oil cake as a carbon source

An extracellular lipase-producing fungus was isolated from the garden soil of the Post Graduate Department of Botany, Utkal University, Bhubaneswar, Odisha, India and identified as Aspergillus terreus. The A. terreus strain isolated was found to be capable of producing lipase in both solid state culture and liquid static surface culture. Experiments aimed at evaluating and improving the production of lipase and at studying the culture conditions revealed that of the many different materials tested as substrates, mustard oil cake (MoC) was the best substrate for extracellular lipase production. A correlation was found between the lipase production profile and biomass development. In a study aimed at continuing this line of research, we have investigated the influence of various culture conditions, such as environmental (i.e. temperature and pH), nutritional (i.e. carbon, nitrogen, metal ions, vitamins, combined agro-wastes and growth regulators) and other factors (inoculum size and initial moisture content) on the production of lipase by A. terreus in solid state and liquid static surface cultures. We observed that optimum lipase biosynthesis occurred under the following conditions: initial pH of 6.0, 30 °C, a 96-h incubation, lactose and ammonium persulphate as the carbon and nitrogen source respectively and 80 % moisture content. Changes in the vitamins (vitamin C, riboflavin, folic acid and vitamin E) and growth regulators (gibberellic acid, kinetin, 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid) did not support enhanced lipase production. MoC and neem oil cake (NoC) added to the media at a ratio of 9:1 respectively, supported maximum lipase production. Based on these results, we concluded that controlling the various culture conditions, supplementing MoC as a substrate and nutrient source modification of the medium can spectacularly enhance lipase biosynthesis by A. terreus.     

Repeated fed-batch cultivation of Arthrospira (Spirulina) platensis using urea as nitrogen source

Arthrospira (Spirulina) platensis (Nordstedt) Gomont was autotrophically cultivated for biomass production in repeated fed-batch process using urea as nitrogen source, with the aim of making large-scale production easier, increasing cell productivity and then reducing the production costs. It was investigated the influence of the ratio of renewed volume to total volume (R), the urea feeding time (t_f) and the number of successive repeated fed-batch cycles on the maximum cell concentration (X_m), cell productivity (P_x), nitrogen-to-cell conversion yield (Y_x/n), maximum specific growth rate (μ_m) and protein content of dry biomass. The experimental results demonstrated that R = 0.80 and t_f = 6 d were the best cultivation conditions, being able to simultaneously ensure, throughout the three fed-batch cycles, the highest average values of three of the five responses (X_m = 2101 ± 113 mg L^?1, P_x = 219 ± 13 mg L^?1 d^?1 and Y_x/n = 10.3 ± 0.8 g g^?1).     

Utilization of a wood-based syrup as a fermentation carbon source

Summary An industrial by-product stream consisting primarily of - and -methyl xylosides supported microbial growth provided the treated syrup was diluted such that the osmotic pressure was within the range tolerated by the particular microorganism used. A culture screening study identified two organisms that efficiently utilized both - and -methyl xyloside. Xylose, -, and -methyl xylosides were all utilized simultaneously during batch growth ofArthrobacter sp. in a controlled-pH fermentor, although xylose was consumed faster than either xyloside. In addition,Arthrobacter sp. produced glucose isomerase when grown on the syrup.     

Evaluation of bacterial contamination in a fed-batch alcoholic fermentation process

Alcoholic fermentation by a commercial baker's yeast in a fed-batch process with cell recycling and high-test molasses as substrate was strongly inhibited by Lactobacillus fermentum CCT 1407 after a few recycles. When total acidity (mainly lactic acid) exceeded 4.8 g/l broth it seriously interfered with yeast bud formation and viability and above 6.0 g/l it decreased alcoholic efficiency.     

Production of targeted poly(3-hydroxyalkanoates) copolymers by glycogen accumulating organisms using acetate as sole carbon source

One of the main limitations in bacterial polyhydroxyalkanoate (PHA) production with mixed cultures is the fact that primarily polyhydroxybutyrate (PHB) homopolymers are generated from acetate as the main carbon source, which is brittle and quite fragile. The incorporation of different 3-hydroxyalkanoate (HA) components into the polymers requires the addition of additional carbon sources, leading to extra costs and complexity. In this study, the production of poly(3-hydroxybutyrate (3HB)-co-3-hydroxyvalerate (3HV)-co-3-hydroxy-2-methylvalerate (3HMV)), with 7-35C-mol% of 3HV fractions from acetate as the only carbon source was achieved with the use of glycogen accumulating organisms (GAOs). An enriched GAO culture was obtained in a lab-scale reactor operated under alternating anaerobic and aerobic conditions with acetate fed at the beginning of the anaerobic period. The production of PHAs utilizing the enriched GAO culture was investigated under both aerobic and anaerobic conditions. A polymer content of 14-41% of dry cell weight was obtained. The PHA product accumulated by GAOs under anaerobic conditions contained a relatively constant proportion of non-3HB monomers (30+/-5C-mol%), irrespective of the amount of acetate assimilated. In contrast, under aerobic conditions, GAOs only produced 3HB monomers from acetate causing a gradually decreasing 3HV fraction during this aerobic feeding period. The PHAs were characterized by gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). The data demonstrated that the copolymers possessed similar characteristics to those of commercially available poly(3HB-co-3HV) (PHBV) products. The PHAs produced under solely anaerobic conditions possessed lower melting points and crystallinity, higher molecular weights, and narrower molecular-weight distributions, compared to the aerobically produced polymers. This paper hence demonstrates the significant potential of GAOs to produce high quality polymers from a simple and cheap carbon source, contributing considerably to the growing research body on bacterial PHA production by mixed cultures.