Continuous 2-keto-gluconic acid (2KGA) production from corn starch hydrolysate by Pseudomonas fluorescens AR4

A continuous fermentation process for 2-keto-gluconic acid (2KGA) production from cheap raw material corn starch hydrolysate was developed using the strain Pseudomonas fluorescens AR4. The dilution rate and feeding glucose concentration had a significant effect on the cell concentrations, glucose utilization and 2KGA production performance. The optimal operating factors were obtained as: 0.065 h⁻¹ of dilution rate, 180 g/L of feeding glucose concentration, and 16 h of batch fermentation time as the starting point. Under these conditions, the steady state had the 135.92 g/L of produced 2KGA concentration, 8.83 g/L.h of average volumetric productivity, and 0.9510 g/g of yield. In conclusion, the proposed efficient and stable continuous fermentation process for 2KGA production by the strain P. fluorescens AR4 is potentially competitive for industrial production from corn starch hydrolysate in terms of 2KGA productivity and yield.     

Continuous 2-keto-<Emphasis Type="SmallCaps">l</Emphasis>-gulonic acid fermentation from <Emphasis Type="SmallCaps">l</Emphasis>-sorbose by <Emphasis Type="Italic">Ketogulonigenium vulgare</Emphasis> DSM 4025

A single-stage continuous fermentation process for the production of 2-keto-l-gulonic acid (2KGA) from l-sorbose using Ketogulonigenium vulgare DSM 4025 was developed. The chemostat culture with the dilution rate that was calculated based on the relationship between the 2KGA production rate and the 2KGA concentration was feasible for production with high concentration of 2KGA. In this system, 112.2 g/L of 2KGA on the average was continuously produced from 114 g/L of l-sorbose. A steady state of the fermentation was maintained for the duration of more than 110 h. The dilution rate was kept in the range of 0.035 and 0.043 h⁻¹, and the 2KGA productivity was 3.90 to 4.80 g/L/h. The average molar conversion yield of 2KGA from l-sorbose was 91.3%. Under the optimal conditions, l-sorbose concentration was kept at 0 g/L. Meanwhile, the dissolved oxygen level was changing in response to the dilution rate and 2KGA concentration. In the dissolved oxygen (DO) range of 16% to 58%, it was revealed that the relationship between DO and D possessed high degree of positive correlation under the l-sorbose limiting condition (complete consumption of l-sorbose). Increasing D closer to the critical value for washing out point of the continuous fermentation, DO value tended to be gradually increased up to 58%. In conclusion, an efficient and reproducible continuous fermentation process for 2KGA production by K. vulgare DSM 4025 could be developed using a medium containing baker’s yeast without using a second helper microorganism.     

Continuous 2-Keto-l-gulonic acid fermentation by mixed culture of Ketogulonicigenium vulgare DSM 4025 and Bacillus megaterium or Xanthomonas maltophilia

The fermentation process of 2-keto-L-gulonic acid (2KGA) from L-sorbose was developed using a two-stage continuous fermentation system. The mixed culture of Ketogulonicigenium vulgare DSM 4025 and Bacillus megaterium DSM 4026 produced 90 g/L of 2KGA from 120 g/L of L-sorbose at the dilution rate of 0.01 h⁻¹ in a single-stage continuous fermentation process. But after the production period was beyond 150 h, the significant decrease of 2KGA productivity was observed. When the non-spore forming bacteria Xanthomonas maltophilia IFO 12692 was used instead of B. megaterium DSM 4026 as a partner strain for K. vulgare DSM 4025, the 2KGA productivity was significantly improved in a two-stage continuous culture mode, in which two fermentors of the same size and volume were connected in series. In this mode, with two sets of 3-L jar fermentors, the steady state could be continued to over 1,331.5 h at least, when the dilution rates were 0.0382 h⁻¹ and 0.0380 hour⁻¹, respectively, for the first and second fermentors. The overall productivity was calculated to be 2.15 g/L/h at 113.1 g/L and a molar conversion yield of 90.1%. In the up-scaling fermentation to 30-L jar fermentors, 118.5 g/L of 2KGA was produced when dilution rates in both stages were 0.0430 hour⁻¹, and the overall productivity was calculated to be 2.55 g/L/h.     

碳源和氮源对5-酮基-葡萄糖酸生成的影响

氧化葡萄糖杆菌Gluconobacter oxydans可以将葡萄糖氧化成葡萄糖酸,并进一步氧化成2-酮基-葡萄糖酸(2KGA)和5-酮基-葡萄糖酸(5KGA),其中5KGA在催化剂的作用下能够转化为L(+)-酒石酸。为了提高5-酮基-葡萄糖酸产量,以仅生成5KGA的氧化葡萄糖杆菌Gluconobacter oxydans HGI-1为出发菌株,研究不同碳源(蔗糖、乳糖、麦芽糖、淀粉、葡萄糖)和有机氮源(酵母浸粉、鱼粉、玉米浆、黄豆饼粉、棉籽饼粉)对5KGA产量的影响。500 mL摇瓶试验结果表明,当葡萄糖浓度为100 g/L时,5KGA产量最高为98.20 g/L;当有机氮源为酵母浸粉、鱼粉和玉米浆,其添加量的蛋白含量为1.60%时,5KGA产量分别为100.20 g/L、109.10 g/L和99.83 g/L,其中,使用鱼粉的5KGA产量最高,使用玉米浆的5KGA产量比酵母浸粉略低。出于经济考虑,文中选择玉米浆作有机氮源,并在5 L发酵罐中进行分批发酵放大试验,5KGA的产量为93.80 g/L,最大生成速率为3.48 g/(L·h),平均生成速率为1.56 g/(L·h)。结果表明,葡萄糖和玉米浆分别为Gluconobacter oxydans HGI-1规模化生产5KGA的最适碳源和氮源,可利用葡萄糖几乎全部(85.93%)转化为5KGA。     

Controlling substrate concentration in fed-batch candida magnoliae culture increases mannitol production

Candida magnoliae HH-01, a yeast strain that is currently used for the industrial production of mannitol, has the highest mannitol production ever reported for a mannitol-producing microorganism. However, when the fructose concentration exceeds 150 g/L, the volumetric mannitol production rate decreases because of a lag in mannitol production, and the yield decreases as a result of the formation of side products. In fed-batch culture, the volumetric production rate and mannitol yield from fructose vary substantially with the fructose concentration and are maximal at a controlled fructose concentration of 50 g/L. In continuous feeding experiments, the maximum mannitol yield was 85% (g/g) at a glucose/fructose feeding ratio of 1/20. A high glucose concentration in the production phase resulted in the formation of ethanol followed by a decrease in yield and productivity. NAD(P)H-dependent mannitol dehydrogenase was purified to homogeneity from C. magnoliae. In vitro, mannitol dehydrogenase was inhibited by increasing ethanol concentration. Mannitol product was also found to be inhibitory with a K(i) of 183 mM. Under optimum conditions, a final mannitol production of 213 g/L was obtained from 250 g fructose/L after 110 h.     

Fermentation process for continuous production of succinic acid in a fibrous bed bioreactor

Succinic acid (SA) was produced from Actinobacillus succinogenes with high cell density by continuous fermentation using fibrous bed bioreactor (FBB). The effects of feeding glucose concentration, dilution rate, and pH on continuous production of SA were examined to achieve an efficient and economical bioprocess. The optimum feeding glucose concentration, dilution rate, and pH were 80 g/L, 0.05 1/h, and 6.0–6.5, respectively. A SA concentration of 55.3 ± 0.8 g/L, productivity of 2.77 ± 0.04 g/L/h, and yield of 0.8 ± 0.02 g/g were obtained, and the continuous fermentation exhibited long-term stability for as long as 18 days (440 h) with no obvious fluctuations in both SA and biomass levels. The Jerusalimsky equation for the specific rate of SA production presented the inhibition phenomenon of the product, demonstrating that 60 g/L SA might be a critical concentration in this continuous FBB system. The results obtained could be beneficial for future fermentor designs and improvements in SA production.     

l-Lactic acid production by Bacillus subtilis MUR1 in continuous culture

A novel UV-induced mutant strain of recombinant Bacillus subtilis MUR1 was used for the production of l-LA in continuous cultures with a variety of culture conditions. The maximal productivity of 17.6 g/L/h was obtained with a l-LA concentration of 44.1 g/L at the dilution rate of 0.4 h⁻¹. The highest concentration of l-LA (77.1 g/L) was produced at the dilution rate of 0.05 h⁻¹. This study showed that the maximum l-LA productivity of B. subtilis MUR1 which can only last for a very short period of time during the exponential phase in fed-batch cultures, can be extended indefinitely at steady state in continuous cultures. l-LA production increased with the increase of yeast extract concentrations in the medium. Moreover, temperature, agitation rate and various glucose concentrations in the feed were compared in continuous cultures. Different nitrogen sources (lysine, glutamine, ammonium sulphate and corn steep liquor) were studied to partly or completely replace yeast extract in the medium, most of them showed positive effects on l-LA production and cell growth. The l-LA productivities from continuous cultures in this study are higher than the productivity of current microbial industrial processes which use Lactobacillus to produce l-LA.     

Ethanol fermentation in a magnetically fluidized bed reactor with immobilized Saccharomyces cerevisiae in magnetic particles

Ethanol fermentation by immobilized Saccharomyces cerevisiae cells in magnetic particles was successfully carried out in a magnetically stabilized fluidized bed reactor (MSFBR). These immobilized magnetic particles solidified in a 2 % CaCl(2) solution were stable and had high ethanol fermentation activity. The performance of ethanol fermentation of glucose in the MSFBR was affected by initial particle loading rate, feed sugar concentration and dilution rate. The ethanol theoretical yield, productivity and concentration reached 95.3%, 26.7 g/L h and 66 g/L, respectively, at a particle loading rate of 41% and a feed dilution rate of 0.4 h(-1) with a glucose concentration of 150 g/L when the magnetic field intensity was kept in the range of 85-120 Oe. In order to use this developed MSFBR system for ethanol production from cheap raw materials, cane molasses was used as the main fermentation substrate for continuous ethanol fermentation with the immobilized S. cerevisiae cells in the reactor system. Molasses gave comparative ethanol productivity in comparison with glucose in the MSFBR, and the higher ethanol production was observed in the MSFBR than in a fluidized bed reactor (FBR) without a magnetic field.     

Succinic acid production from continuous fermentation process using Mannheimia succiniciproducens LPK7

To achieve a higher succinic acid productivity and evaluate the industrial applicability, this study used Mannheimia succiniciproducens LPK7 (knock-out: lahA, pflB, pta-ackA), which was recently designed to enhance the productivity of succinic acid and reduce by-product secretion. Anaerobic continuous fermentation of Mannheimia succiniciproducens LPK7 was carried out at different glucose feed concentrations and dilution rates. After extensive fermentation experiments, a succinic acid yield and productivity of 0.38 mol/mol and 1.77 g/l/h, respectively, were achieved with a glucose feed concentration of 18.0 g/l and 0.2 h-1 dilution rate. A similar amount of succinic acid production was also produced in batch culture experiments. Therefore, these optimal conditions can be industrially applied for the continuous production of succinic acid. To examine the quantitative balance of the metabolism, a flux distribution analysis was also performed using the metabolic network model of glycolysis and the pentose phosphate pathway.     

A rational approach to improving productivity in recombinant Pichia pastoris fermentation

A Mut(S) Pichia pastoris strain that had been genetically modified to produce and secrete sea raven antifreeze protein was used as a model system to demonstrate the implementation of a rational, model-based approach to improve process productivity. A set of glycerol/methanol mixed-feed continuous stirred-tank reactor (CSTR) experiments was performed at the 5-L scale to characterize the relationship between the specific growth rate and the cell yield on methanol, the specific methanol consumption rate, the specific recombinant protein formation rate, and the productivity based on secreted protein levels. The range of dilution rates studied was 0. 01 to 0.10 h(-1), and the residual methanol concentration was kept constant at approximately 2 g/L (below the inhibitory level). With the assumption that the cell yield on glycerol was constant, the cell yield on methanol increased from approximately 0.5 to 1.5 over the range studied. A maximum specific methanol consumption rate of 20 mg/g. h was achieved at a dilution rate of 0.06 h(-1). The specific product formation rate and the volumetric productivity based on product continued to increase over the range of dilution rates studied, and the maximum values were 0.06 mg/g. h and 1.7 mg/L. h, respectively. Therefore, no evidence of repression by glycerol was observed over this range, and operating at the highest dilution rate studied maximized productivity. Fed-batch mass balance equations, based on Monod-type kinetics and parameters derived from data collected during the CSTR work, were then used to predict cell growth and recombinant protein production and to develop an exponential feeding strategy using two carbon sources. Two exponential fed-batch fermentations were conducted according to the predicted feeding strategy at specific growth rates of 0.03 h(-1) and 0.07 h(-1) to verify the accuracy of the model. Cell growth was accurately predicted in both fed-batch runs; however, the model underestimated recombinant product concentration. The overall volumetric productivity of both runs was approximately 2.2 mg/L. h, representing a tenfold increase in the productivity compared with a heuristic feeding strategy.     

Multi-stage high cell continuous fermentation for high productivity and titer

We carried out the first simulation on multi-stage continuous high cell density culture (MSC-HCDC) to show that the MSC-HCDC can achieve batch/fed-batch product titer with much higher productivity to the fed-batch productivity using published fermentation kinetics of lactic acid, penicillin and ethanol. The system under consideration consists of n-serially connected continuous stirred-tank reactors (CSTRs) with either hollow fiber cell recycling or cell immobilization for high cell-density culture. In each CSTR substrate supply and product removal are possible. Penicillin production is severely limited by glucose metabolite repression that requires multi-CSTR glucose feeding. An 8-stage C-HCDC lactic acid fermentation resulted in 212.9 g/L of titer and 10.6 g/L/h of productivity, corresponding to 101 and 429% of the comparable lactic acid fed-batch, respectively. The penicillin production model predicted 149% (0.085 g/L/h) of productivity in 8-stage C-HCDC with 40 g/L of cell density and 289% of productivity (0.165 g/L/h) in 7-stage C-HCDC with 60 g/L of cell density compared with referring batch cultivations. A 2-stage C-HCDC ethanol experimental run showed 107% titer and 257% productivity of the batch system having 88.8 g/L of titer and 3.7 g/L/h of productivity. MSC-HCDC can give much higher productivity than batch/fed-batch system, and yield a several percentage higher titer as well. The productivity ratio of MSC-HCDC over batch/fed-batch system is given as a multiplication of system dilution rate of MSC-HCDC and cycle time of batch/fed-batch system. We suggest MSC-HCDC as a new production platform for various fermentation products including monoclonal antibody.     

Production of galacto-oligosaccharides by immobilized recombinant beta-galactosidase from Aspergillus candidus

The preparation of galacto-oligosaccharides (GOSs) was studied using the immobilized recombinant beta-galactosidase from Aspergillus candidus CGMCC3.2919. The optimal pH and temperature for the immobilized enzyme were observed at pH 6.5 and 40 degrees C, respectively. Increasing the initial lactose concentration increased the yield of GOSs. The dilution rate was found to be a key factor during the continuous production of GOSs. The maximum productivity, 87 g/L.h was reached when 400 g/L lactose was fed at dilution rate of 0.8/h. The maximum GOS yield reached 37% at dilution rate of 0.5/h. Continuous operation was maintained for 20 days in a packed-bed reactor without apparent decrease in GOS production. The average yield of GOSs was 32%, corresponding to the average productivity of 64 g/L.h, which implied that the immobilized recombinant beta-galactosidase has potential application for GOS production.     

Continuous acetone-butanol-ethanol fermentation using SO2-ethanol-water spent liquor from spruce

SO₂–ethanol–water (SEW) spent liquor from spruce chips was successfully used for batch and continuous production of acetone, butanol and ethanol (ABE). Initially, batch experiments were performed using spent liquor to check the suitability for production of ABE. Maximum concentration of total ABE was found to be 8.79 g/l using 4-fold diluted SEW liquor supplemented with 35 g/l of glucose. The effect of dilution rate on solvent production, productivity and yield was studied in column reactor consisting of immobilized Clostridium acetobutylicum DSM 792 on wood pulp. Total solvent concentration of 12 g/l was obtained at a dilution rate of 0.21 h⁻¹. The maximum solvent productivity (4.86 g/l h) with yield of 0.27 g/g was obtained at dilution rate of 0.64 h⁻¹. Further, to increase the solvent yield, the unutilized sugars were subjected to batch fermentation.     

Effect of air supplement on the performance of continuous ethanol fermentation system

For the purpose of improving ethanol productivity, the effect of air supplement on the performance of continuous ethanol fermentation system was studied. The effect of oxygen supplement on yeast concentration, cell yield, cell viability, extracellular ethanol concentration, ethanol yield, maintenance coefficient, specific rates of glucose assimilation, ethanol production, and ethanol productivity have been evaluated, using a high alcohol tolerant Saccharomyces cerevisiae STV89 strain and employing a continuous fermentor equipped with an accurate air metering system in the flow rate range 0-11 mL air/L/h. It was found that, when a small amount of oxygen up to about 80mu mol oxygen/L/h was supplied, the ethanol productivity was significantly enhanced as compared to the productivity of the culture without any air supplement. It was also found that the oxygen supplement improved cell viability considerably as well as the ethanol tolerance level of yeast. As the air supply rate was increased, from 0 to 11 mL air/L/h while maintaining a constant dilution rate at about 0.06 h(-1), the cell concentration increased from 2.3 to 8.2 g/L and the ethanol productivity increased from 1.7 to 4.1 g ethanol/L/h, although the specific ethanol production rate decreased slightly from 0.75 to 0.5 g ethanol/g cell/h. The ethanol yield was slightly improved also with an increase in air supply rate, from about 0.37 to 0.45 ethanol/g glucose. The maintenance coefficient increased by only a small amount with the air supplement. This kind of air supplement technique may very well prove to be of practical importance to a development of a highly productive ethanol fermentation process system especially as a combined system with a high density cell culture technique.     

Production of ethanol by Clostridium thermosaccharolyticum: I. Effect of cell recycle and environmental parameters

The direct microbial conversion (DMC) process for the production of ethanol from lignocellulosic biomass is limited by low volumetric ethanol production rates due to the low cell densities of Clostridium thermosaccharolyticum which is a key organism for ethanol production in this process. Hence, this study focuses on the use of a continuous- culture cell recycle system to improve the volumetric ethanol productivity and yield of the fermentation of xylose by C. thermosaccharolyticum. Early experiments with the continuous-culture cell recycle system showed a two-fold improvement in volumetric ethanol productivity. However, the ethanol yield at the higher dilution rates suffered because of the large amount of lactate produced. The manipulation of two environmental parameters-iron concentration in the nutrient medium and the N(2) purge rate of the fermentor headspace-allowed a dramatic reduction in the lactate production and a simultaneous improvement in the ethanol titer and yield. Under the improved conditions of increased iron concentration (12.5 mg/L FeSO(4) . 7H(2)O) and decreased N(2) purge rate (0.1 L/min), a continuous culture of C. thermosaccharolyticum operating at a dilution rate of 0.24 h(-1) and 50% cell recycle produced 8.6 g/L ethanol and less than 1 g/L each of acetate and lactate. The volumetric ethanol productivity was 2.2 g/L/h, which is 8 times larger than obtained for a continuous culture operated with no cell recycle and the same specific growth rate.     

Production of Streptokinase in Continuous Culture

A method for continuous cultivation of a β-hemolytic streptococcus, strain H 64, is described. The production of cells and streptokinase at various dilution rates, pH, and temperature were studied in a complex medium supplied with excess glucose. At pH 7.0, productivity of cells and streptokinase, as well as the yield constant with respect to glucose, all increased with increasing dilution rate in the range of 0.1 to 0.5 hr^-1. The production of streptokinase was found to be a function of both growth rate and cell concentration. Although higher concentrations of streptokinase were obtained in experiments with batch cultures, the production of streptokinase in continuous cultures was found to be 2.3 times higher. The possible industrial application of a continuous production method is considered.     

Evaluation ofl-lactic acid production in batch, fed-batch, and continuous cultures ofRhizopus sp. MK-96-1196 using an airlift bioreactor

Various processes which producel-lactic acid using ammonia-tolerant mutant strain,Rhizopus sp. MK-96-1196, in a 3 L airlift bioreactor were evaluated. When the fed-batch culture was carried out by keeping the glucose concentration at 30 g/l, more than 140 g/l ofl-lactic acid was produced with a product yield of 83%. In the case of the batch culture with 200 g/l of initial glucose concentration, 121 g/L ofl-lactic acid was obtained but the low product yield based on the amount of glucose consumed. In the case of a continuous culture, 1.5 g/l/h of the volumetric productivity with a product yield of 71% was achieved at dilution rate of 0.024 h⁻¹. Basis on these results three processes were evaluated by simple variable cost estimation including carbon source, steam, and waste treatment costs. The total variable costs of the fed-batch and continuous cultures were 88% and 140%, respectively, compared to that of batch culture. The fed-batch culture with highl-lactic acid concentration and high product yield decreased variable costs, and was the best-suited for the industrial production ofl-lactic acid.     

Wood pulp as an immobilization matrix for the continuous production of isopropanol and butanol

The study was focused on developing a continuous method to produce an alcohol mixture suitable to be used as a gasoline supplement. The immobilized column reactor with wood pulp fibers was successfully used for the continuous production of butanol and isopropanol using Clostridium beijerinckii DSM 6423. A sugar mixture (glucose, mannose, galactose, arabinose and xylose) representing lignocellulose hydrolysate was used as a substrate for the production of solvents. The effect of dilution rate on solvent production was studied during continuous operation. The maximum total solvent concentration of 11.99 g/l was obtained at a dilution rate of 0.16 h^?1. The maximum solvent productivity (5.58 g/l h) was obtained at a dilution rate of 1.5 h^?1. The maximum solvent yield of 0.45 g/g from sugar mixture was observed at 0.25 h^?1. The system will be further used for the solvent production using wood hydrolysate as a substrate.     

Improved production of live cells of Lactobacillus rhamnosus by continuous cultivation using glucose-yeast extract medium

In this study, the growth kinetics of Lactobacillus rhamnosus and lactic acid production in continuous culture were assessed at a range of dilution rates (0.05 h(-1) to 0.40 h(-1)) using a 2 L stirred tank fermenter with a working volume of 600 ml. Unstructured models, predicated on the Monod and Luedeking-Piret equations, were employed to simulate the growth of the bacterium, glucose consumption, and lactic acid production at different dilution rates in continuous cultures. The maximum specific growth rate of L. rhamnosus, mu-max, was estimated at 0.40 h(-1), and the Monod cell growth saturation constant, Ks, at approximately 0.25 g/L. Maximum cell viability (1.3 x 10(10) CFU/ml) was achieved in the dilution rate range of D = 0.28 h(-1) to 0.35 h(-1). Both maximum viable cell yield and productivity were achieved at D = 0.35 h(-1). The continuous cultivation of L. rhamnosus at D = 0.35 h(-1) resulted in substantial improvements in cell productivity, of 267% (viable cell count) that achieved via batch cultivation.     

Simultaneous saccharification and continuous fermentation of sludge-containing mash for bioethanol production by Saccharomyces cerevisiae CHFY0321

A continuous process was employed to improve the volumetric productivity of bioethanol production from cassava mash containing sludge and to simplify the process of ethanol production from cassava. After raw cassava powder was liquefied, it was used directly in a continuous process without sludge filtration or saccharification. A fermentor consisting of four linked stirrer tanks was used for simultaneous saccharification and continuous fermentation (SSCF). Although the mash contained sludge, continuous fermentation was successfully achieved. We chose the dilution rate on the basis of the maximum saccharification time; the highest volumetric productivity and ethanol yield were observed at a dilution rate of 0.028 h?1. The volumetric productivity, final ethanol concentration, and % of theoretical ethanol yield were 2.41 g/Lh, 86.1g/L, and 91%, respectively. This SSCF process using the self-flocculating yeast Saccharomyces cerevisiae CHFY0321 illustrates the possibility of realizing cost-effective bioethanol production by eliminating additional saccharification and filtration processes. In addition, flocculent CHFY0321, which our group developed, showed excellent fermentation results under continuous ethanol production.