Demonstration of the Crabtree effect in Phaffia rhodozyma during continuous and fed-batch cultivation

By monitoring cell yield and fermentation products during fed-batch and continuous growth, Pfaffia rhodozyma was shown to exhibit the Crabtree effect. In fed-batch culture at feed concentrations of 27 and 55 g glucose/l there was good agreement between the observed biomass formation and that predicted by a mass balance model. At 125 g glucose/l in the feed, biomass formation was less than predicted and fermentation products such as ethanol and acetic acid accumulated in the culture medium. In continuous culture with a feed concentration of 10 g glucose/l, the Crabtree effect became apparent at a dilution rate of 0.1 h -1 . Aerobic fermentation did not occur provided the sugar substrate was maintained at a concentration of less than 0.5 g/l. Although the cell yield coefficient was reduced from 0.5 g/g to 0.16 g/g during aerobic fermentation, the carotenoid content of the cells was unaffected.     

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.     

Production of xylitol by recombinant Saccharomyces cerevisiae containing xylose reductase gene in repeated fed-batch and cell-recycle fermentations

Xylitol is a well-known sugar substitute with low-calorie and anti-cariogenic characteristics. An effort of biological production of xylitol from xylose was made in repeated fed-batch and cell-recycle fermentations of recombinant Saccharomyces cerevisiae BJ3505/δXR harboring the xylose reductase gene from Pichia stipitis. Batch fermentation with 20 g/l xylose and 18 g/l glucose resulted in 9.52 g/l dry cell mass, 20.1 g/l xylitol concentration and approximately 100% conversion yield. Repeated fed-batch operation to remove 10% of culture broth and to supplement an equal volume of 200 g/l xylose was designed to improve xylitol production. In spite of a sudden drop of cell concentration, an increase in dry cell mass led to high accumulation of xylitol at 48.7 g/l. To overcome loss of xylitol-producing biocatalysts in repeated fed-batch fermentation, cell-recycle equipment of hollow fiber membrane was implemented into a xylitol production system. Cell-recycle operation maintained concentration of the recombinant cells high inside a bioreactor. Final dry cell mass of 22.0 g/l, 116 g/l xylitol concentration, 2.34 g/l h overall xylitol productivity were obtained in cell-recycle fermentation supplemented with xylose and yeast extract solution, which were equivalent to 2.3-, 5.8- and 3.8-fold increases compared with the corresponding values of batch-type xylitol production parameters.     

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.     

Production of poly(3-hydroxybutyrate) by high cell density fed-batch culture of Alcaligenes eutrophus with phospate limitation

High cell density fed-batch fermentation of Alcaligenes eutrophus was carried out for the production of poly(3-hydroxybutyrate) (PHB) in a 60-L fermentor. During the fermentation, pH was controlled with NH(4)OH solution and PHB accumulation was induced by phosphate limitation instead of nitrogen limitation. The glucose feeding was controlled by monitoring dissolved oxygen (DO) concentration and glucose concentration in the culture broth. The glucose concentration fluctuated within the range of 0-20 g/L. We have investigated the effect of initial phosphate concentration on the PHB production when the initial volume was fixed. Using an initial phosphate concentration of 5.5 g/L, the fed-batch fermentation resulted in a final cell concentration of 281 g/L, a PHB concentration of 232 g/L, and a PHB productivity of 3.14 g/L . h, which are the highest values ever reported to date. In this case, PHB content, cell yield from glucose, and PHB yield from glucose were 80, 0.46, and 0.38% (w/w), respectively. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 28-32, 1997.     

Intra- and extra-cellular organophosphorus hydrolase production with recombinant <Emphasis Type="Italic">E. coli</Emphasis> using fed-batch fermentation

A fed-batch fermentation process for the production of organophosphorus hydrolase (OPH) (EC 3.1.8.1) by E. coli pET812 was developed in this research. With batch fermentation, the maximum OPH concentrations attained by batch fermentation were as low as 4 × 10⁵ U/l because cell growth and OPH production were inhibited by a high initial concentration of glucose. To develop a fed-batch fermentation process for obtaining higher concentrations of OPH, highly concentrated glucose solution (500 g/l) was added intermittently or continuously to increase the carbon source concentration. Eventually, 3.2 × 10⁶ U/l of OPH was produced with fed-batch fermentation in 24 h. This was eight times higher than the yield with conventional batch fermentation. A total concentration of 399–441 mg of OPH was produced/l, which was four times higher than that reported when using E. coli. Nearly half (44%) of the produced OPH was secreted into the culture solution.     

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 culture of yeast Saccharomyces cerevisiae with a DO-stat method by a fuzzy controller

In this research a fuzzy controller was built to perform fed-batch cultures of Saccharomyces cerevisiae with a DO-stat method. The basic principle of fed-batch culture employing the DO-stat method is that a rapid increase of dissolved oxygen concentration due to a lack of substrate (the DO signal) is used as an indicator for substrate feeding. The proposed fuzzy controller can diagnose the state of fermentation and determine a proper feed rate of substrate for the culture of high density and high yield. The results indicate that cell concentration reached to 110?g/l and residual sugar kept below the level of 0.05?g/l.     

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.     

Influences of yeast extract on specific cellular yield of Ovine growth hormone during fed-batch fermentation of E. coli

In most cases of E. coli high cell density fermentation process, maximizing cell concentration helps in increasing the volumetric productivity of recombinant proteins usually at the cost of lower specific cellular protein yield. In this report, we describe a process for maintaining the specific cellular yield of Ovine growth hormone (oGH) from E. coli by optimal feeding of yeast extract during high cell density fermentation process. Recombinant oGH was produced as inclusion bodies in Escherichia coli. Specific cellular yield of recombinant oGH was maintained by feeding yeast extract along with glucose during fed-batch fermentation. Glucose to yeast extract ratio of 0.75 was found to be optimum for maintaining the specific cellular oGH yield of 66 mg/g of E. coli cells. Continuous feeding of yeast extract along with glucose helped in reducing acetic acid secretion and promoted higher cell growth during fed-batch fermentation. High cell growth of E. coli and high specific yield of recombinant oGH thus helped in achieving high volumetric productivity of the expressed protein. A maximum of 2 g/l of ovine growth hormone was expressed as inclusion bodies in 12 h of fed-batch fermentation.     

D-核糖发酵过程中底物抑制及补料的研究

研究了初始葡萄糖浓度对D -核糖发酵的影响 ,证实了较高的葡萄糖浓度对D -核糖发酵的抑制作用 ,并确定了较为适宜的初始葡萄糖浓度为 1 0 0g·L-1 或 1 5 0g·L-1 。前者条件下D -核糖的转化率和生产强度均达最大 ,分别为 32 8g·kg-1 和0 .6 8g·L-1 ·h-1 ;后者条件下D -核糖的产量达最大值 39.4 8g·L-1 。针对底物抑制现象 ,研究了补料工艺对D -核糖发酵的影响 ,确定发酵 2 4h后补加 5 0g·L-1 的葡萄糖为较优的补料工艺 ,在此工艺条件下最终D -核糖产量相对于对照组提高了 4 8.3%。     

Control of feed rate to a fed-batch culture using a heat-flux sensor

Summary Substrate inhibition in batch fermentations can be avoided by employing the fed-batch technique in which substrate concentration is kept at low levels by a programmed feed rate. This research demonstrates the use of a heat-flux sensor to control substrate addition by continuously monitoring evolving heat which is proportional to fermentation rate. Batch fermentation with 240 g/L glucose in the medium was compared with a fed-batch starting with 20 g/L glucose in the medium and increased, with 500 g/L glucose, to a final equivalent glucose concentration of 240 g/L. The batch fermentation produced 106 g/L ethanol in 39 hr at 2.72 g/L/h, while the best fed-batch produced 114 g/L ethanol in 34 hr at 3.35 g/L/h with the same nutrients.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

营养条件和流加发酵对放射型根瘤菌(Rhizobium radiobacter)产辅酶Q10的影响

利用放射型根瘤菌WSH2 6 0 1(RhizobiumradiobacterWSH2 6 0 1)重点考察了葡萄糖、蔗糖、玉米浆和蛋白胨、添加物以及流加发酵对细胞生长和产辅酶Q1 0 的影响 ,结果表明 ,葡萄糖和蔗糖适合于生产辅酶Q1 0 的最佳浓度分别为 30g L和 40g L ;辅酶Q1 0 发酵时玉米浆和蛋白胨的最适浓度分别为 11g L和 16g L ;添加蕃茄汁、玉米浆能提高发酵液的生物量 ,玉米浆、异戊醇、L 甲硫氨基酸等能促进辅酶Q1 0 的积累 ;与分批发酵相比 ,在 7L罐上流加蔗糖其细胞生物量 (DCW)和辅酶Q1 0 积累量增加 ,若在流加蔗糖的同时流加适当浓度的玉米浆能显著提高辅酶Q1 0 的产量 ,最大产量达到 5 2 .4mg L ;最大生物量 (DCW)和胞内辅酶Q1 0 含量 (C B值 )分别达到 2 6 .4g L和 2 .38mg g DCW ,比不流加的分批发酵分别提高 5 3 %和 33% ,比只流加蔗糖分别提高 2 4%和 2 6 %。     

Application of fuzzy reasoning to control of glucose and ethanol concentrations in baker's yeast culture

Fuzzy reasoning was applied to control both ethanol and glucose concentrations in fed-batch cultures of baker's yeast. This fuzzy controller consisted of three membership functions (concentrations of dissolved oxygen (DO), ethanol and glucose) and 18 production rules. Fuzzy inference was carried out by IF {A is a and B is b,...#x007D;, THEN {C is c} from the on-line measured concentrations of DO, ethanol and glucose. When medium concentrations of ethanol and glucose in fed-batch culture of baker's yeast were set at 2 g/l and 0.2 g/l, both ethanol and glucose concentrations were controlled at 2.67±0.35 g/l and 0.27±0.25 g/l, respectively, ethanol production was reduced from 26 g/l to 34 g/l, cell yield increased from 0.38 to 0.53 g dry cell/g consumed glucose and ethanol yield decreased from 0.50 to 0.14 g ethanol/g consumed glucose, respectively, as compared with those of the glucose only control at 0.2 g/l.     

Production of 2,3-butanediol in a membrane bioreactor with cell recycle

Summary The production of 2,3-butanediol by Enterobacter aerogenes DSM 30053 was studied in a cell recycle system with a microfiltration module. Emphasis was put on the influence of oxygen supply, cell residence time, dilution rate, and pH. Under optimal conditions a productivity as high as 14.6 g butanediol + acetoin/l per hour was achieved with a product concentration of 54 g/l and a product yield of 88%. This productivity is three times higher than that of an ordinary continuous culture. The achievable final product concentration of a cell recycle system was limited by the accumulation of the inhibiting by-product acetic acid, which increased very rapidly at low dilution rate. To maximize product concentration a fed-batch fermentation was carried out with stepwise pH adaption at high cell density. A final product concentration of 110 g/l was obtained with a productivity of 5.4 g/l per hour and a yield of 97%.     

Fed-batch culture ofAeromonas hydrophila for the production of poly(3-hydroxubutyrate-co-3-hydroxyhexanoate) using two carbon sources

To produce polyhydroxyalkanoate (PHA) copolymer which consists of 3-hydroxy-butyrate (3HB) and 3-hydroxyhexanoate (3HHx) by cultivation ofAeromonas hydrophila, fed-batch cultures were done under several nutrient limiting conditions. With the results from flask cultures, fed-batch cultures were carried out to produce large amounts of PHA. In the fed-batch culture, firstly glucose was fed to grow cell, and then, oleic acid fed to stimulate PHA in the cell. The final cell concentration, PHA content, PHA concentration, and 3-hydroxy-hexanoate fraction in 38 hr were 48.9 g/L, 15.05 wt%, 7.36 g/L and 12.2 wt%, respectively, resulting in the productivity of 0.19 g/L-h under phosphate-limiting condition.     

Optimization of fusion proinsulin production by high cell-density fermentation of recombinantE. coli

The optimum conditions for mass production of fusion proinsulin were studied in recombinantEscherichia coli strain BL21 (DE3) [pT7-PI] using fed-batch culture employing pH-stat method. Yeast extract was found to enhance both the growth rate of recombinantE. coli strain BL21 (DE3) [pT7-PI] and its cell mass yield. When the glucose concentration was 10 g/L in the initial medium, 10 g/L concentration of yeast extract was found to be optimal to control the acetate production and to augment both the cell mass yield and the growth rate. Optimum ratio of glucose to yeast extract to minimize the cost of the feeding medium in the fed-batch culture was calculated to be 1.225 and verified by the subsequent experiments. The appropriate inducer concentration and induction time were examined with isopropyl-β-D-thiogalactopyranoside (IPTG). Irrespective of the induction time, IPTG induction resulted in the reduction of growth rate, but the expression level of the fusion protein was maintained at the level of about 20% of the total proteins. Since the volumetric productivity was well maintained in the range between 0.15 and 0.18 g/L.hr at the inducer concentration of above 0.025 mM, the appropriate inducer concentration, in relation to the inducer cost, is considered to be about 0.025 mM.     

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.     

Tower fermentation using Zymomonas mobilis for ethanol production

Summary Flocculation was induced in a pure strain of the bacteria Zymomonas mobilis. When fermenting glucose to ethanol, cell densities of up to 40g/l were achieved and sustained in a 0.92 litre tower fermenter with dilution rates of up to 2.3 hr^-1. A maximum productivity of 100g EtOH/l/hr with 98% conversion of the 105g/l glucose feed was achieved. The limitation to performance with increase in throughput arose from incomplete fermentation of the feed glucose, rather than washout of the flocculated bacteria.     

Rapid ethanol fermentation of cellulose hydrolysate. I. Batch versus continuous systems

Rapid fermentation of bagasse hydrolysate to ethanol under anaerobic conditions by a strain of Saccharomyces cerevisiae has been studied in batch and continuous cultures at pH 4.0 and 30°C temperature with cell recycle. By using a 23.6 g/liter cell concentration, a concentation of 9.7% (w/v)ethanol was developed in a period of 6 hr. The rate of fermentation was found to increase with supplementation of yeast vitamins in the hydrolysate. In continuous culture employing cell recycle and a 0.127 v/v/m air flow rate, a cell mass concentration of 48.5 g/liter has been achieved. The maximum fermentor productivity of ethanol obtained under these conditions was 32.0 g/liter/hr, which is nearly 7.5 times higher than the normal continuous process without cell recycle and air sparging. The ethanol productivity was found to decrease linearly with ethanol concentration. Conversion of glucose in the hydrolysate to ethanol was achieved with a yield of 95 to 97% of theoretical.