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.     

一株法夫酵母虾青素高产菌株的生产性能

为了评价虾青素高产菌株-法夫酵母JMU-MVP14的生产性能及建立虾青素高产发酵技术,通过测定糖、生物量、虾青素产量、总类胡萝卜素产量等发酵参数,用摇瓶试验对比了法夫酵母JMU-MVP14和出发菌株的差异,用7 L罐试验对比了pH值调控方式及补料培养基成分对发酵的影响,用1 m3罐试验评估了法夫酵母JMU-MVP14高密度发酵虾青素的产量水平。摇瓶发酵结果表明,法夫酵母JMU-MVP14虾青素及总类胡萝卜素的细胞产率分别达到6.01 mg/g及10.38 mg/g;7 L罐分批发酵试验结果表明,自动流加调     

Butyric acid production from brown algae using <Emphasis Type="Italic">Clostridium tyrobutyricum</Emphasis> ATCC 25755

Butyric acid fermentation by Clostridium tyrobutyricum ATCC 25755 using glucose or brown algae as a carbon source was carried out. Initially, different fermentation modes (batch, fed-batch, and semi-continuous) at pH 6 and 37°C were compared using a model medium containing glucose as a carbon source. By feeding the whole medium containing 40 ∼ 50 and 30 g/L of glucose into the fed-batch and semi-continuous fermentations, very similar butyrate yields (0.274 and 0.252 g butyrate/g glucose, respectively) and productivities (0.362 and 0.355 g/L/h, respectively) were achieved. The highest butyrate concentration was about 50 g/L, which was observed in the fed-batch fermentation with whole medium feeding. However, semi-continuous fermentation sustained a longer fermentation cycle than the fed-batch fermentation due to end-product and metabolic waste inhibition. The established conditions were then applied to the fermentation using brown algae, Laminaria japonica and Undaria pinnatifida, as substrates for butyric acid fermentation. To hydrolyze brown algae, 7.5 ∼ 10% (w/v) dried brown algae powder was suspended in 1% (w/v) NaOH or 0.5 ∼ 2.5% (w/v) H₂SO₄ and then autoclaved at 121°C for 30 ∼ 90 min. The resulting butyrate concentration was about 11 g/L, which was produced from 100 g/L of L. japonica autoclaved for 60 min in 1.5% H₂SO₄ acid solution.     

甘蔗渣和糖蜜混合发酵制备燃料丁醇

以抗逆突变株Clostridium beijerinckii IB4为研究对象,葡萄糖为C源,对其进行补料分批发酵过程的优化,同时将该优化工艺应用于甘蔗渣和糖蜜混合发酵制备燃料丁醇。结果表明:在5 L发酵罐中,先加入作为还原糖的甘蔗渣酸解糖液10 g/L,16 h后补加甘蔗糖蜜30 g/L,于35℃、100 r/min发酵50 h,丁醇和总溶剂产量分别达到11.1和15.3 g/L,丁醇比例高达72.5%。     

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.     

可溶性TRAIL蛋白的高密度培养及补料策略研究

采用分批补料的方法高密度培养重组大肠杆菌C600/PbvTRAIL制备人可溶性TRAIL蛋白,优化发酵工艺,探索简单高效的分离纯化方法并测定蛋白生物活性。通过比较几种不同的补料策略：间歇流加、Dostat、pHstat,摸索了一种流加策略,即DOstatpHstat组合流加,有效的避免了发酵过程中,尤其是诱导表达阶段乙酸积累的增加,使TRAIL蛋白在高密度培养条件下,得到高效表达。菌体密度最终达到300g/L（WCW）以上,可溶性TRAIL蛋白占菌体总蛋白的4.2%,含量为1.1g/L。在整个发酵过程中,乙酸浓度接近于0,且未使用任何特殊手段,如纯氧、加压等,简化了发酵工艺,降低了发酵成本,为TRAIL的工业化生产创造了条件。     

Effect of amino acid supplement on cell yield and gene product in Escherichia coli harboring plasmid

Escherichia coli harboring a recombinant plasmid was cultivated in fed-batch culture to enhance production of a gene product. Expression of the leucine gene from Thermus thermophilus in the recombinant plasmid was examined by the assay of beta-isopropylmalate dehydrogenase activity at 75 degrees C. When E. coli was cultivated in medium without leucine, biomass concentration reached 15 g/L and the specific activity became 0.082 U/mg protein. When leucine was fed in the medium throughout cultivation, although biomass concentration reached 63 g/L, the specific activity decreased to 0.016 U/mg protein. When E. coli was cultivated in medium containing 1 g leucine/L, the specific activity remained virtually constant (about 0.13 U/mg protein) and biomass concentration reached 32 g dry cells/L. In these cultivations, growth yields of several amino acids and glucose were examined. When leucine was not added to the medium, growth yields except for histidine were lowest. When leucine was fed throughout the cultivation, growth yields of glucose and tryptophan were highest. The pH-stat was useful for feeding amino acids.     

Fed-batch mode in shake flasks by slow-release technique

Most industrial production processes are performed in fed-batch operational mode. In contrast, the screenings for microbial production strains are run in batch mode which results in completely different physiological conditions than relevant for production conditions. This may lead to wrong selections of strains. Silicone elastomer discs containing glucose crystals were developed to realize fed-batch fermentation in shake flasks. No other device for feeding was required. Glucose was fed in this way to Hansenula polymorpha cultures controlled by diffusion. Two strains of H. polymorpha were investigated in shake flasks: the wild-type strain (DSM 70277) and a recombinant strain pC10-FMD (P(FMD)-GFP). The oxygen transfer rate (OTR) and respiratory quotient (RQ) of the cultures were monitored online in shake flasks with a Respiration Activity Monitoring System (RAMOS). Formation of biomass and green fluorescent protein (GFP), pH-drift and the metabolite dynamics of glucose, ethanol and acetic acid were measured offline. With the slow-release technique overflow metabolism could be reduced leading to an increase of 85% in biomass yield. To date, 23.4 g/L cell dry weight of H. polymorpha could be achieved in shake flask. Biomass yields of 0.38-0.47 were obtained which are in the same magnitude of laboratory scale fermentors equipped with a substrate feed pump. GFP yield could be increased by a factor of 35 in Syn6-MES mineral medium. In fed-batch mode 88 mg/L GFP was synthesized with 35.9 g/L fed glucose. In contrast, only 2.5 mg/L with 40 g/L metabolized glucose was revealed in batch mode. In YNB mineral medium over 420-fold improvement in fed-batch mode was achieved with 421 mg/L GFP at 41.3 g/L fed glucose in comparison to less than 1 mg/L in batch mode with 40 g/L glucose.     

High-cell-density fed-batch cultivation of the docosahexaenoic acid producing marine alga Crypthecodinium cohnii

The heterotrophic marine alga Crypthecodinium cohnii is known to produce docosahexaenoic acid (DHA), a polyunsaturated fatty acid with food and pharmaceutical applications, during batch cultivation on complex media containing sea salt, yeast extract, and glucose. In the present study, fed-batch cultivation was studied as an alternative fermentation strategy for DHA production. Glucose and acetic acid were compared as carbon sources. For both substrates, the feed rate was adapted to the maximum specific consumption rate of C. cohnii. In glucose-grown cultures, this was done by maintaining a significant glucose concentration (between 5 and 20 g/L) throughout fermentation. In acetic acid-grown cultures, the medium feed was automatically controlled via the culture pH. A feed consisting of acetic acid (50% w/w) resulted in a higher overall volumetric productivity of DHA (r(DHA)) than a feed consisting of 50% (w/v) glucose (38 and 14 mg/L/h, respectively). The r(DHA) was further increased to 48 mg/L/h using a feed consisting of pure acetic acid. The latter fermentation strategy resulted in final concentrations of 109 g/L dry biomass, 61 g/L lipid, and 19 g/L DHA. These are the highest biomass, lipid, and DHA concentrations reported to date for a heterotrophic alga. Vigorous mixing was required to sustain aerobic conditions during high-cell-density cultivation. This was complicated by culture viscosity, which resulted from the production of viscous extracellular polysaccharides. These may present a problem for large-scale industrial production of DHA. Addition of a commercial polysaccharide-hydrolase preparation could decrease the viscosity of the culture and the required stirring.     

Production of the macrolide antibiotic tylosin in cyclic fed-batch culture

Tylosin-producing Streptomyces fradiae was cultured on a synthetic medium with a high glutamate-glucose ratio. Tylosin batch fermentations with this medium were characterized by a high initial specific production rate of tylosin (q(tylosin), mg/g h) that decreased as the fermentation progressed. Continuous feeding of glutamate, glucose, and methyloleate at a constant feed rate initiated during the period of high q(tylosin) had been shown to produce some increase in tylosin productivity. By using a cyclic feeding strategy, it was possible to increase tylosin productivity further. Tylosin fed-batch fermentations with glutamate and glucose being fed to the culture in cyclic square-wave profiles with methyloleate in excess showed several-fold increase in final q(tylosin) and tylosin titers. By varying cycle amplitudes and period of the substrates, it was found that maximum tylosin productivity occurred when the glutamate cycle amplitude was 600 mg/L and that of glucose was 42.5 mg/L per cycle period of 24 h. With these cycle amplitudes of glutamate and glucose, the tylosin cyclic fed-batch culture also showed high cellular uptake of methyloleate. Decreasing or increasing glucose cycle amplitude at fixed glutamate amplitude lowered tylosin production, and no further stimulation of tylosin synthesis was observed when alpha-ketoglutarate was supplemented to the cyclic substrate feeds. Under optimum cyclic conditions it was possible to maintain linear tylosin accretion and a constant value of q(tylosin) up to 240 h.     

Enhanced 2,3-butanediol production in fed-batch cultures of free and immobilized Bacillus licheniformis DSM 8785

2,3-Butanediol (2,3-BD) is a valuable bulk chemical with particular use in industry. 2,3-BD has a potential as solvent and fuel additive, as carrier for pharmaceuticals, or as feedstock for the production of synthetic rubber. Until now, the highest 2,3-BD concentrations were obtained with risk group 2 microorganisms (e.g., Klebsiella oxytoca). In this study, the nonpathogenic bacterium Bacillus licheniformis DSM 8785 was used for 2,3-BD production from glucose. In batch experiments, a maximum 2,3-BD concentration of 72.6 g/L was reached from 180 g/L glucose after 86 h. The yield was 0.42 g/g glucose and the productivity was 0.86 g/(L h). During fed-batch cultivation, 2,3-BD production could be increased up to 144.7 g/L, with a productivity of 1.14 g/(L h). Additionally, repeated batch/fed-batch experiments were conducted using immobilized B. licheniformis in the form of LentiKats®. Results showed a high activity and stability of the immobilizates even after multiple medium replacements, as well as 2,3-BD concentrations, yields, and productivities similar to those obtained with free cells. To our knowledge, these results show the highest 2,3-BD concentration reported so far using a risk group 1 microorganism in general and B. licheniformis in particular. Furthermore, productivity lies in the same range with data reported from risk group 2 strains, which makes B. licheniformis DSM 8785 a suitable candidate for large-scale fermentation processes.     

2,3-Butanediol production using Klebsiella oxytoca ATCC 8724: Evaluation of biomass derived sugars and fed-batch fermentation process

For this study, 2,3-butanediol (BD) fermentation from pure and biomass-derived sugar were optimized in shake-flask and 5-L bioreactor levels using Klebsiella oxytoca ATCC 8724. The results showed that 70 g/L of single sugar (glucose or xylose) and 90 g/L of mixed-sugar (glucose:xylose = 2:1) were optimum concentrations for efficient 2,3-BD fermentation. At optimum sugar concentrations, 2,3-BD productivities were 1.03, 0.64 and 0.50 gL⁻¹ h⁻¹, and yields were 0.43, 0.36 and 0.35 g/g in glucose, xylose and mixed-sugar medium, respectively. The lack of simultaneous utilization of glucose and xylose led to the lowest productivity in the mixed-sugar medium. Detoxification of biomass hydrolyzates was necessary for efficient 2,3-BD fermentation when sugar concentrations in the medium was 90 g/L or higher, but not with sugar concentrations of 30 g/L or less. A fed-batch fermentation using glucose medium led to an increase 2,3-BD titer to 79.4 g/L and yields 0.47 g/g, while productivity decreased to 0.79 gL⁻¹ h⁻¹. However, the fed-batch process was inefficient using mixed-sugar and biomass hydrolyzates because of poor xylose utilization. These results indicated that appropriate biomass processing technologies must be developed to generate separate glucose and xylose streams to produce high 2,3-BD titer from biomass-derived sugar using a fed-batch process.     

Enhanced phenylpyruvic acid production with Proteus vulgaris in fed-batch and continuous fermentation

Phenylpyruvic acid is a deaminated form of phenylalanine and is used in various areas such as development of cheese and wine flavors, diagnosis of phenylketonuria, and to decrease excessive nitrogen accumulation in the manure of farm animals. However, reported phenylpyruvic acid fermentation studies in the literature have been usually performed at shake-flask scale with low production. In this study, phenylpyruvic acid production was evaluated in bench-top bioreactors by conducting fed-batch and continuous fermentation for the first time. As a result, maximum phenylpyruvic acid concentrations increased from 1350 mg/L (batch fermentation) to 2958 mg/L utilizing fed-batch fermentation. Furthermore, phenylpyruvic acid productivity was increased from 48 mg/L/hr (batch fermentation) to 104 and 259 mg/L/hr by conducting fed-batch and continuous fermentation, respectively. Overall, this study demonstrated that fed-batch and continuous fermentation significantly improved phenylpyruvic acid production in bench-scale bioreactor production.     

过量表达自身hemA基因的重组大肠杆菌发酵生产5-氨基乙酰丙酸的研究

研究了优化重组大肠杆菌产5-氨基乙酰丙酸（ALA）的条件,提高大肠杆菌发酵生产AL气的产量。在测定重组大肠杆菌GT48的生长曲线的基础上,确定诱导时间,优化摇瓶发酵条件。然后,进一步在5L发酵罐上进行间歇和流加发酵研究。摇瓶实验表明,细胞培养最佳初始pH为6．5,最佳诱导时间为稳定期前期,最佳接种量为2％,过高的葡萄糖浓度对细胞生长和产物合成均有一定的抑制作用。在5L发酵罐间歇发酵中,重组菌产ALA能力达到47．8mg／L。采用流加发酵可以进一步将产物产量提高到63．8mg／L。构建的过量表达自身的hemA基因的大肠杆菌具有较高的产ALA能力,通过发酵条件优化和采用流加发酵可以提高AL气产量。     

Acetate production from whey lactose using co-immobilized cells of homolactic and homoacetic bacteria in a fibrous-bed bioreactor

Acetate was produced from whey lactose in batch and fed-batch fermentations using co-immobilized cells of Clostridium formicoaceticum and Lactococcus lactis. The cells were immobilized in a spirally wound fibrous sheet packed in a 0.45-L column reactor, with liquid circulated through a 5-L stirred-tank fermentor. Industrial-grade nitrogen sources, including corn steep liquor, casein hydrolysate, and yeast hydrolysate, were studied as inexpensive nutrient supplements to whey permeate and acid whey. Supplementation with either 2.5% (v/v) corn steep liquor or 1.5 g/L casein hydrolysate was adequate for the cocultured fermentation. The overall acetic acid yield from lactose was 0.9 g/g, and the productivity was 0.25 g/(L h). Both lactate and acetate at high concentrations inhibited the homoacetic fermentation. To overcome these inhibitions, fed-batch fermentations were used to keep lactate concentration low and to adapt cells to high-concentration acetate. The final acetate concentration obtained in the fed-batch fermentation was 75 g/L, which was the highest acetate concentration ever produced by C. formicoaceticum. Even at this high acetate concentration, the overall productivity was 0.18 g/(L h) based on the total medium volume and 1.23 g/(L h) based on the fibrous-bed reactor volume. The cells isolated from the fibrous-bed bioreactor at the end of this study were more tolerant to acetic acid than the original culture used to seed the bioreactor, indicating that adaptation and natural selection of acetate-tolerant strains occurred. This cocultured fermentation process could be used to produce a low-cost acetate deicer from whey permeate and acid whey.     

分批补料对木糖发酵生产乙醇的影响

以树干毕赤酵母为发酵菌种,纯木糖为发酵底物,通过分批补料来提高糖利用率以及乙醇得率。结果表明,在24h内,最佳初始木糖浓度为80g/L,在28h的发酵周期中,可以将木糖浓度提高至90g/L,在32h发酵周期内可以将木糖浓度提高至100g/L。通过分批补料,乙醇浓度得到明显提高。当总糖浓度分别为80g/L、90g/L时,24h发酵周期内,分批补料次数以1次为宜,乙醇浓度分别达30.95g/L、32.60g/L,相比于不补料即一次性投料,乙醇浓度分别提高了9.36%、9.18%。总糖浓度100g/L,28h发酵周期内,补料2次效果最佳,乙醇浓度达37.49g/L,比一次性投料下提高了10.36%,较一次性投料达到相同发酵效果缩短了4h。     

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.     

Fed-batch sorbose fermentation using pulse and multiple feeding strategies for productivity improvement

Microbial oxidation of D-sorbitol tol-sorbose byAcetobacter suboxydans is of commercial importance since it is the only biochemical process in vitamin C synthesis. The main bottleneck in the batch oxidation of sorbitol to sorbose is that the process is severely inhibited by sorbitol. Suitable fed-batch fermentation designs can eliminate the inherent substrate inhibition and improve sorbose productivity. Fed-batch sorbose fermentations were conducted by using two nutrient feeding strategies. For fed-batch fermentation with pulse feeding highly concentrated sorbitol (600 g/L) along with other nutrients were fed intermittently in four pulses of 0.5 liter in response to the increased DO signal. The fed-batch fermentation was over in 24 h with a sorbose productivity of 13.40 g/L/h and a final sorbose concentration of 320.48 g/L. On the other hand, in fed-batch fermentation with multiple feeds, two pulse feeds of 0.5 liter nutrient medium containing 600 g/L sorbitol was followed by the addition of 1.5 liter nutrient medium containing 600 g/L sorbitol at a constant feed rate of 0.36 L/h till the full working capacity of the reactor. The fermentation was completed in 24 h with an enhanced sorbose productivity of 15.09 g/L/h and a sorbose concentration of 332.60 g/L. The sorbose concentration and productivity obtained by multiple feeding of nutrients was found to be higher than that obtained by pulse feeding and was therefore a better strategy for fed-batch sorbose fermentation.     

High-level production of ethanol during fed-batch ethanol fermentation with a controlled aeration rate and non-sterile glucose powder feeding of Saccharomyces cerevisiae

In this study, we utilized a unique strategy for fed-batch fermentation using ethanol-tolerant Saccharomyces cerevisiae to achieve a high-level of ethanol production that could be practically applied on an industrial scale. During this study, the aeration rate was controlled at 0.0, 0.13, 0.33, and 0.8 vvm to determine the optimal aeration conditions for the production of ethanol. Additionally, non-sterile glucose powder was fed during fed-batch ethanol fermentation and corn-steep liquor (CSL) in the medium was used as an organic N-source. When aeration was conducted, the ethanol production and productivity were superior to that when aeration was not conducted. Specifically, the maximum ethanol production reached approximately 160 g/L, when the fermentor was aerated at 0.13 vvm. These findings indicate that the use of a much less expensive C-source may enable the fermentation process to be directed towards the improvement of overall ethanol production and productivity in fermentors that are aerated at 0.13 vvm. Furthermore, if a repeated fed-batch process in which the withdrawal and fill is conducted prior to 36 h can be employed, aeration at a rate of 0.33 and/or 0.8 vvm may improve the overall ethanol productivity     

Optimization of nutritional requirements and feeding strategies for clavulanic acid production by Streptomyces clavuligerus

The present work reports the nutritional requirements and environmental conditions for submerged culture of Streptomyces clavuligerus for clavulanic acid production using orthogonal matrix method (Taguchi L(16) design) and also fed-batch fermentation for clavulanic acid production by feeding glycerol, arginine and threonoine to the fermentation medium intermittently. Clavulanic acid production was increased by 18% with the span of feeding glycerol and reached a maximum at 1.30mg/ml with 120h glycerol feeding as compared to 1.10mg/ml in the control. The production also increased with the span of feeding amino acids and reached a maximum of 1.31 and 1.86mg/ml with feeding arginine and threonine, respectively in 120h. There was an overall increase of 18% and 9% in clavulanic acid production with arginine and threonine feeding as compared to the respective controls (1.10 and 1.70mg/ml, respectively).