Enhancement of 5-aminolevulinate production with recombinant Escherichia coli using batch and fed-batch culture system

5-Aminolevulinate (ALA) production with recombinant Escherichia coli Rosetta (DE3)/pET28a(+)-hemA was studied. In batch fermentation, the addition of glucose and glycine was effective to improve ALA production. Then the fed-batch fermentation was conducted with continuous feeding of precursors. When the concentrations of succinic acid and glycine were 7.0 g/l and 4.0 g/l, respectively, in the feeding, the ALA yield reached 4.1g/l. But the molar yield (ALA/glycine) was decreased in the fed-batch fermentation compared to batch fermentation. And it was found that the pH control during fed-batch cultivation was very important for the cell growth and ALA production. A two-stage pH value controlling strategy was suggested, in which, the pH value in the first 6h was regulated at pH 5.9, after then at pH 6.2, and the ALA yield was as high as 6.6g/l via fed-batch fermentation.     

Fed-batch fermentation studies with Bacillus thuringiensis H-14 synthesising endotoxin.

Cell yield and toxicity of B. thuringiensis H-14 was improved markedly by adopting a simple fed-batch fermentation technique based on controlling glucose concentration. Maintenance of steady glucose concentration (0.3-0.5%) in the culture medium was achieved by the continuous addition of concentrated glucose solution. Addition of glucose at 3 g/hr/l of culture starting from 3rd hr till 16th hr of fermentation was found to yield cell densities of 80 g/l (wet weight) which represented a nearly 3-fold increase over the batch mode. A fivefold increase in toxicity was obtained by fed-batch fermentation. Cultivation of B. thuringiensis H-14 to high cell densities had no negative effect on sporulation and toxin synthesis. The rate of pH drop and dissolved oxygen level were within manageable limits.     

Mass production of thiostrepton by fed-batch culture of Streptomyces laurentii with pH-stat modal feeding of multi-substrate

Summary High concentration production of an antibiotic, thiostrepton, was achieved by the fed-batch culture of Streptomyces laurentii. To produce thiostrepton efficiently, the pH of the medium had to be maintained in a very narrow range between 6.0 and 6.2. As the substrates, not only glucose but also natural nutrients such as defatted soy bean meal and corn steep liquor were demanded. All of these substrates (multi-substrate) had to be supplied during the cultivation. pH was used as the indicator to detect the deficiency of substrates. When the glucose in the medium had been exhaustively consumed, the pH increased immediately; the multi-substrate solution (MS-solution) was then supplied. The composition of the feeding solution was determined from data obtained in batch cultures. During the fed-batch culture, glucose concentration was kept lower than 5 g/l by regulating the feed amount of MS-solution with this pH-stat modal control. By this control strategy a high concentration of thiostrepton, 10.5 g/l, was obtained, while total cell mass concentration reached 157 g/l. The productivity of thiostrepton was greatly increased compared with the conventional batch culture.     

Feed-control development for succinic acid production with Anaerobiospirillum succiniciproducens

The aim of this work was the development of a feed-control for a succinic acid production fed-batch process. The performed batch trials indicated a correlation between succinic acid production and base consumption pH control. Based on the metabolism of Anaerobiospirillum succiniciproducens, a theoretical correlation between base consumption and glucose feed was established and proved in cultivation trials. With the established fed-batch process, the succinic acid yield could be increased to 0.875 (g/g glucose) in comparison to batch processes (0.60) with similar glucose concentrations. Additionally, the results indicate that the osmolarity of the medium has a significant influence on succinic acid production.     

Enhanced production of exopolysaccharides by fed-batch culture of Ganoderma resinaceum DG-6556

The objectives of this study were to optimize submerged culture conditions of a new fungal isolate, Ganorderma resinaceum, and to enhance the production of bioactive mycelial biomass and exopolysaccharides (EPS) by fed-batch culture. The maximum mycelial growth and EPS production in batch culture were achieved in a medium containing 10 g/l glucose, 8 g/l soy peptone, and 5 mM MnCl(2) at an initial pH 6.0 and temperature 31 degrees C. After optimization of culture medium and environmental conditions in batch cultures, a fed-batch culture strategy was employed to enhance production of mycelial biomass and EPS. Five different EPS with molecular weights ranging from 53,000 to 5,257,000 g/mole were obtained from either top or bottom fractions of ethanol precipitate of culture filtrate. A fed-batch culture of G. resinaceum led to enhanced production of both mycelial biomass and EPS. The maximum concentrations of mycelial biomass (42.2 g/l) and EPS (4.6 g/l) were obtained when 50 g/l of glucose was fed at day 6 into an initial 10 g/l of glucose medium. It may be worth attempting with other mushroom fermentation processes for enhanced production of mushroom polysaccharides, particularly those with industrial potential.     

Fed-batch fermentation of glucose to acetate by an improved strain ofClostridium thermoaceticum

The fed-batch approach to the production of acetate from glucose by an improved strain ofClostridium thermoaceticum resulted in better performance than the batch fermentation, especially in media containing an excess (3X) of nutrients and trace salts. At pH 6.6, 46 g/l acetic acid was produced in 192 hours with 93% substrate utilization. In contrast, batch fermentation under similar conditions resulted in a maximum of 35 g/l acetic acid with less than 82% substrate utilization.     

Optimisation of L-lysine production byCorynebacterium sp in fed-batch cultures

Summary The influence of initial concentration of glucose from 60 to 233 g/l on the production of L-lysine byCorynebacterium sp was studied first in batch culture. The maximum conversion rate into L-lysine was obtained at 165 g/l and the best specific production rate for L-lysine was observed at 65 g/l of glucose. In fed-batch fermentations, better conversion and the specific production rates were obtained. Maintaining of a high glucose concentration in the fed-batch technique allowed a 54% increase of the L-lysine production compared to the batch culture.     

Lipid production in batch and fed-batch cultures of Rhodosporidium toruloides from 5 and 6 carbon carbohydrates

ABSTRACT: BACKGROUND: Microbial lipids are a potential source of bio- or renewable diesel and the red yeast Rhodosporidium toruloides is interesting not only because it can accumulate over 50% of its dry biomass as lipid, but also because it utilises both five and six carbon carbohydrates, which are present in plant biomass hydrolysates. METHODS: R. toruloides was grown in batch and fed-batch cultures in 0.5 l bioreactors at pH 4 in chemically defined, nitrogen restricted (C/N 40 to 100) media containing glucose, xylose, arabinose, or all three carbohydrates as carbon source. Lipid was extracted from the biomass using chloroform-methanol, measured gravimetrically and analysed by GC. RESULTS: R. toruloides was grown on glucose, xylose, arabinose or mixtures of these carbohydrates in batch and fed-batch, nitrogen restricted conditions. Lipid production was most efficient with glucose (up to 25 g lipid L1, 48 to 75% lipid in the biomass, at up to 0.21 g lipid L1h1) as the sole carbon source, but high lipid concentrations were also produced from xylose (36 to 45% lipid in biomass). Lipid production was low (15-19% lipid in biomass) with arabinose as sole carbon source and was lower than expected (30% lipid in biomass) when glucose, xylose and arabinose were provided simultaneously. The presence of arabinose and/or xylose in the medium increased the proportion of palmitic and linoleic acid and reduced the proportion of oleic acid in the fatty acids, compared to glucose-grown cells. High cell densities were obtained in both batch (37 g L1, with 49% lipid in the biomass) and fed-batch (35 to 47 g L1, with 50 to 75% lipid in the biomass) cultures. The highest proportion of lipid in the biomass was observed in cultures given nitrogen during the batch phase but none with the feed. However, carbohydrate consumption was incomplete when the feed did not contain nitrogen and the highest total lipid and best substrate consumption were observed in cultures which received a constant low nitrogen supply. CONCLUSIONS: Lipid production in R. toruloides was lower from arabinose and mixed carbohydrates than from glucose or xylose. Although high biomass and lipid production were achieved in both batch and fed-batch cultures with glucose as carbon source, for lipid production from mixtures of carbohydrates fed-batch cultivation was preferable. Constant feeding was better than intermittent feeding. The feeding strategy did not affect the relative proportion of different fatty acids in the lipid, but the presence of C5 sugars did.     

Growth and fatty acid composition of Nannochloropsis sp. grown mixotrophically in fed-batch culture

The cell growth and eicosapentaenoic acid (EPA) yields of Nannochloropsis sp. were enhanced in the fed-batch cultures. With feeding glucose solution, the biomass reached 1.1 g dry wt l(-1) after 10 days' culture, which was 40% higher than that obtained in the batch culture (0.8 g dry wt l(-1)). With supplement of nitrate solution, the biomass reached 1 g dry wt l(-1), and reached the stationary phase 2 days earlier than the others. The maximum of biomass (1.2 g dry wt l(-1)) was obtained with the supplement of the mixture of glucose and nitrate solution. The EPA yields of Nannochloropsis sp. after 10 days' growth in the fed-batch cultures were 52 mg l(-1), 43 mg l(-1) and 56 mg l(-1) with, respectively, addition of nitrate, glucose and both together. In batch culture only 35 mg EPA l(-1) was obtained.     

Repeated pH-stat fed-batch fermentation for rhamnolipid production with indigenous Pseudomonas aeruginosa S2

Rhamnolipid is one of the most commonly used biosurfactants with the ability to reduce the surface tension of water from 72 to 30 mN/m. An indigenous isolate Pseudomonas aeruginosa S2 possessing excellent ability to produce rhamnolipid was used as a model strain to explore fermentation technology for rhamnolipid production. Using optimal medium and operating conditions (37°C, pH 6.8, and 250 rpm agitation) obtained from batch fermentation, P. aeruginosa S2 was able to produce up to 5.31 g/l of rhamnolipid from glucose-based medium. To further improve the rhamnolipid yield, a pH-stat fed-batch culture was performed by maintaining a constant pH of 6.8 through manipulating glucose feeding. The effect of influent glucose concentration on rhamnolipid yield and productivity was investigated. Using the pH-stat culture, a maximum rhamnolipid concentration (6.06 g/l) and production rate (172.5 ml/h/l) was obtained with 6% glucose in the feed. Moreover, combining pH-stat culture with fill-and-draw operation allowed a stable repeated fed-batch operation for approximately 500 h. A marked increase in rhamnolipid production was achieved, leading to the best rhamnolipid yield of approximately 9.4 g/l during the second repeated run.     

Intracellular pH-based controlled cultivation of yeast cells: II. cultivation methodology

Intracellular pH (pH(i)) was measured on-line in a bioreactor using a fluorescent pH(i) indicator, 9-aminoacridine, and controlled fed-batch cultivations of yeast cells based on pH(i) (FB-pH(i)) were performed. In FB-pH(i) cultivations, automated glucose additions were made to the culture in response to culture pH(i). The average ethanol (an-aerobic product) yield was significantly lower [0.12 g g(-1) glucose in fed-batch pH(i) cultivations with 100 ppm glucose additions (FB-pH(i)-100 cultivation) vs. 0.48 g g(-1) glucose in batch] and cell yield was higher (0.54 g g(-1) glucose in FB-pH(i)-100 cultivation vs. 0.3 g g(-1) glucose in batch) compared to batch cultivation. An expression has been derived to calculate changes in pH(i) from measured fluorescence values when the cell concentration increases during growth. Cultivations based on pH(i), performed with different magnitudes of glucose addition (100, 50, and 10 ppm additions), showed that lower magnitudes of glucose addition resulted in lower ethanol yields while cell yield remained unaffected. The ratio of specific oxygen uptake rate to specific glucose uptake rate (OUR/GUR) increased with decreased in magnitude of glucose additions in FB-pH(i) cultivations, suggesting that the culture aerobic state was higher when the magnitude of glucose addition was lower. The average cell productivity in FB-pH(i) cultivations was 29% higher than in batch cultivation. Cells were also cultivated at high OUR conditions, and the results are compared with other cultivations. (c) 1993 John Wiley & Sons, Inc.     

一株高产脯氨酸的嗜醋酸棒杆菌的选育及发酵条件优化

以嗜醋酸棒杆菌为出发菌株,经过片段化全基因组体外诱变、重组和连续的磺胺胍抗性筛选,获得一株L-脯氨酸的高产菌株。摇瓶发酵优化结果表明,葡萄糖、生物素和硫胺素的最适用量分别为16%、300μg/L、400μg/L,最适pH为6.8~7.0,装液量为25ml/500ml摇瓶,发酵培养72h后L-脯氨酸产率高达到75.6g/L,与对照相比提高了5%。考察了50L发酵罐中细胞生长对L-脯氨酸产量的影响,补料分批发酵结果表明(比生长速率分别为0.06/h、0.08/h和0.1/h),比生长速率在0.08/h左右时L-脯氨酸的产率最高,L-脯氨酸的比生产速率Q_P达到0.091 g/(g.h),产率高达82.1 g/L,比优化前提高了14%。     

Production of poly(4-hydroxybutyric acid) by fed-batch cultures of recombinant strains of Escherichia coli

A recombinant strain of Escherichia coli was used to produce poly(4-hydroxybutyric acid), P(4HB), homopolyester by fed-batch culture in M9 mineral salts medium containing glucose and 4-hydroxybutyric acid as carbon sources. The final cell dry weight, P(4HB) concentration and P(4HB) content were 12.6 g/l, 4.4 g/l, and 36% of cell dry weight, respectively, in a 27-l stirred and aerated fermenter after 60 h of fed-batch fermentation at constant pH.     

Study of mycelial growth and bioactive polysaccharide production in batch and fed-batch culture of Grifola frondosa

The fermentation of Grifola frondosa was investigated in the shake flasks and a 5-L jar fermenter in batch and fed-batch modes. In the shake-flask experiments, the preferable mycelial growth and exopolysaccharide (EPS) production was observed at relatively low pH; maltose and glucose were preferred carbon sources for high mycelial production. The EPS was doubled after 13 d of cultivation when glucose was increased from 2% to 4%. Yeast extract (YE) (0.4%) in combination with corn steep powder (CSP) (0.6%) and YE (0.8%) in combination with CSP (1.2%) were preferred nitrogen sources for high mycelial production and EPS production, respectively. All plant oils tested significantly stimulate cell growth of G. frondosa but they failed to enhance EPS production. The EPS products usually consisted of two fractions of different molecular sizes varied by the plant oils used. The fed-batch fermentation by glucose feeding was performed when the glucose concentration in the medium was lower than 0.5% (5g/L), which greatly enhanced the accumulation of mycelial biomass and EPS; the mycelial biomass and EPS were 3.97g/L and 1.04g/L before glucose feeding, which reached 8.23g/L and 3.88g/L at 13 d of cultivation. In contrast, the mycelial biomass and EPS in the batch fermentation were 6.7g/L and 3.3g/L at 13 d of cultivation.     

Fed-batch two-phase production of alanine by a metabolically engineered Escherichia coli

dl-Alanine was produced from glucose in an Escherichia coli pfl pps poxB ldhA aceEF pTrc99A-alaD strain which lacked pyruvate-formate lyase, phosphoenolpyruvate (PEP) synthase, pyruvate oxidase, lactate dehydogenase, components of the pyruvate dehydogenase complex and over-produced alanine dehydrogenase (ALD). A two-phase process was developed with cell growth under aerobic conditions followed by alanine production under anaerobic conditions. Using the batch mode, cells grew to 5.3 g/l in 9 h with the accumulation of 6–10 g acetate/l, and under subsequent anaerobic conditions achieved 34 g alanine/l in 13 h with a yield of 0.86 g/g glucose. Using the fed-batch mode at μ = 0.15 h⁻¹, only about 1 g acetate/l formed in the 25 h required for the cells to reach 5.6 g/l, and 88 g alanine/l accumulated during the subsequent 23 h. This fed-batch process attained an alanine volumetric productivity of 4 g/lh during the production phase, and a yield that was essentially 1 g/g.     

High production of alkaline protease byBacillus licheniformis in a fed-batch fermentation using a synthetic medium

Summary High production (9016 U/ml) of alkaline protease byBacillus licheniformis has been achieved. A 49% increase in production was achieved by the method used as compared with a batch process. By using a synthetic medium and a fed-batch operation controlled by the Advanced Fermentation Software (AFS) package, it was found that the keys to high production of protease are: (i) to maintain a low concentration of glucose (<0.43 g/l) in the medium; (ii) to control pH at a certain level (pH 6.50) in the culture; and (iii) to use rough type colonies as the starting culture. Our fed-batch fermentation process successfully simulates and surpasses ordinary batch fermentation processes. By using ammonium sulfate instead of soy bean flour as the only nitrogen source, an expected benefit was the elimination of unpleasant odors caused by natural organic nitrogenous components in the media. This would improve the industrial production environment.     

Carotenoid production from hydrolyzed molasses by Dietzia natronolimnaea HS-1 using batch,fed-batch and continuous culture

The different cultivation strategies of batch, fed-batch and continuous culture for the synthesis of biomass and carotenoids by Dietzia natronolimnaea HS-1 from waste molasses and its hydrolysate were compared. The efficiency of three various pretreatments (enzymatic, acidic and acidic at high temperature) for the determination of the best hydrolysate was also studied by evaluating the conversion rate of sucrose. The analytical procedures initially showed that canthaxanthin (CTX) and enzymatic hydrolysis were the most abundant pigment biosynthesized and the most suitable process for the substrate production, respectively. An increase in reducing sugar concentration of the enzymatic hydrolysate molasses (EHM) from 25 to 50 g/l led to a drastic decrease in biomass formation and substrate utilization. EHM (25 g/l) was a better substrate for the cell growth and product formation than the waste molasses (25 g/l). The application of EHM instead of molasses enhanced the biomass production in fed-batch culture more than batch and continuous cultures. However, the continuous cultivation had the highest biomass (12.98 g/l), carotenoid (27.33 mg/l) and CTX (25.04 mg/l) yields with 25 g/l of EHM. The CTX isolated from D. natronolimnaea HS-1 may be used as a natural antioxidant for possible production of healthy-functional foods in the future.     

Efficient conversion of lactic acid to butanol with pH-stat continuous lactic acid and glucose feeding method by Clostridium saccharoperbutylacetonicum

In order to achieve high butanol production by Clostridium saccharoperbutylacetonicum N1-4, the effect of lactic acid on acetone–butanol–ethanol fermentation and several fed-batch cultures in which lactic acid is fed have been investigated. When a medium containing 20 g/l glucose was supplemented with 5 g/l of closely racemic lactic acid, both the concentration and yield of butanol increased; however, supplementation with more than 10 g/l lactic acid did not increase the butanol concentration. It was found that when fed a mixture of lactic acid and glucose, the final concentration of butanol produced by a fed-batch culture was greater than that produced by a batch culture. In addition, a pH-controlled fed-batch culture resulted in not only acceleration of lactic acid consumption but also a further increase in butanol production. Finally, we obtained 15.5 g/l butanol at a production rate of 1.76 g/l/h using a fed-batch culture with a pH-stat continuous lactic acid and glucose feeding method. To confirm whether lactic acid was converted to butanol by the N1-4 strain, we performed gas chromatography–mass spectroscopy (GC-MS) analysis of butanol produced by a batch culture during fermentation in a medium containing [1,2,3-¹³C₃] lactic acid as the initial substrate. The results of the GC-MS analysis confirmed the bioconversion of lactic acid to butanol.     

Distribution of substrates carbon in sophorose lipid production by Torulopsis bombicola

Summary Torulopsis bombicola (ATCC 22214) produced sophorose lipid to 80 g/l in batch culture containing 11% glucose and 10% soybean oil as carbon and energy sources. According to the carbon mass balance analysis, 13% and 37% of input carbon were channeled to cells and to products, respectively, and 50% of the total input carbon was channeled to CO₂ gas in batch culture. In fed-batch culture with intermittent oil feeding, however, the carbon fractions incorporated into sophorose lipid and cells were 60% and 12%, respectively, and the carbon fraction evolved as CO₂ gas was 30%. In conclusion, yield of sophorose lipid based on total input carbon substrates was increased from 0.37 g/g-substrate in batch culture to 0.6 g/ g-substrate by employing a fed-batch culture.     

Kinetics of sugars consumption and ethanol inhibition in carob pulp fermentation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in batch and fed-batch cultures

The waste materials from the carob processing industry are a potential resource for second-generation bioethanol production. These by-products are small carob kibbles with a high content of soluble sugars (45–50%). Batch and fed-batch Saccharomyces cerevisiae fermentations of high density sugar from carob pods were analyzed in terms of the kinetics of sugars consumption and ethanol inhibition. In all the batch runs, 90–95% of the total sugar was consumed and transformed into ethanol with a yield close to the theoretical maximum (0.47–0.50 g/g), and a final ethanol concentration of 100–110 g/l. In fed-batch runs, fresh carob extract was added when glucose had been consumed. This addition and the subsequent decrease of ethanol concentrations by dilution increased the final ethanol production up to 130 g/l. It seems that invertase activity and yeast tolerance to ethanol are the main factors to be controlled in carob fermentations. The efficiency of highly concentrated carob fermentation makes it a very promising process for use in a second-generation ethanol biorefinery.