Ethanol production from lactose by extractive fermentation

Summary The suitability of extractive fermentation as a technique for the production of ethanol from lactose by Candida pseudotropicalis was examined as a potential improvement over conventional methods. A biocompatible solvent was selected through determination of the critical log P (octanol-water distribution coefficient) of the fermentation organism. Using Adol 85 NF, the selected solvent, extractive fed-batch and conventional fed-batch systems were operated for 160 h. The extractive system showed a 60% improvement in lactose consumption and ethanol production, as well as a 75% higher volumetric productivity.     

Closed-loop control of fed-batch cultures of recombinant Escherichia coli using on-line HPLC

This article describes a fully automated system for the on-line monitoring and closed-loop control of a fed-batch fermentation of recombinant Escherichia coli, and presents two case studies of its used in limiting production of unwanted byproducts such as acetic in fed-batch fermentations. The system had two components. The first components, on-line monitoring, comprised an aseptic sampling device, a microcentrifuge, and HPLC System. These instruments removed a Sample from a fermentor, spun it at high speed to separate solid and liquid components, and then automatically injected the supernatant onto an HPLC column for analysis. The second component consisted of control algorithms programmed using the LabView visual programming environment in a control computer that was linked via a remote components were linked so that results from the on-line HPLC were captured and used by the control algorithm was designed to demonstrate coarse feedback control to confirm the operability of the controller. The second case study showed how the system could be used in a more sophisticated feedings strategy providing fine control and limiting acetate concentration to a low level throughout the fermentation. (c) 1994 John Wiley & Sons, Inc.     

Monitoring cell concentration and activity by multiple excitation fluorometry.

Four key cellular metabolic fluorophores--tryptophan, pyridoxine, NAD(P)H, and riboflavin--were monitored on-line by a multiple excitation fluorometric system (MEFS) and a modified SLM 8000C scanning spectrofluorometer in three model yeast fermentation systems--bakers' yeast growing on glucose, Candida utilis growing on ethanol, and Saccharomyces cerevisiae RTY110/pRB58 growing on glucose. The measured fluorescence signals were compared with cell concentration, protein concentration, and cellular activity. The results indicate that the behavior and fluorescence intensity of various fluorophores differ in the various fermentation systems. Tryptophan fluorescence is the best signal for the monitoring of cell concentration in bakers' yeast and C. utilis fermentations. Pyridoxine fluoresce is the best signal for the monitoring of cell concentration in the S. cerevisiae RTY110/pRB58 fermentation. In bakers' yeast fermentations the pyridoxine fluorescence signal can be used to monitor cellular activity. The NAD(P)H fluorescence signal is a good indicator of cellular activity in the C. utilis fermentation. For this fermentation NAD(P)H fluorescence can be used to control ethanol feeding in a fed-batch process.     

An Algorithm for operating a fed-batch fermentation using quasi-steady state

The determination of an optimum feeding profile of a fed-batch fermentation requires the solution of a singular optimum control problem, which is often complicated by changes in the process kinetics during the fermentation. The procedure of optimization may be sufficiently simple, if the feeding part of fermentation is carried out in the quasi-steady state. In this work an algorithm for operating a fed-batch fermentation using mentioned regime is offered. The algorithm supposes a periodical correction of the feeding strategy. Applying to fed-batch lysine fermentation demonstrate efficacy of this algorithm over frequently used strategies.     

Acetate yield increased by gas circulation and fed-batch fermentation in a novel syntrophic acetogenesis and homoacetogenesis coupling system

Gas circulation and fed-batch fermentation were applied for enhancing acetate production by mixed culture in a novel syntrophic acetogenesis and homoacetogenesis coupling system. The results show that the acetate yield in the fed-batch test with gas circulation is about 47% higher than that in the batch test without gas circulation. The fed-batch method helps to increase acetate yield by balancing hydrogen production in the acetogenesis phase (the 1st phase) and hydrogen consumption in the homoacetogenesis phase (the 2nd phase) of the coupling system. Gas circulation enhances mass transfer between different phases of the coupling system, hence resulting in increased homoacetogenesis in the 2nd phase and relief of the products (H2) inhibition to syntrophic acetogenesis in the 1st phase. The effects of gas circulation and fed-batch fermentation on direct glucose conversion to acetate were also investigated.     

Optimization of human serum albumin production in methylotrophic yeast Pichia pastoris by repeated fed-batch fermentation

An optimization method for repeated fed-batch fermentation was established with the aim of improving the recombinant human serum albumin (rHSA) production in Pichia pastoris. A simulation model for fed-batch fermentation was formulated and the optimal methanol-feeding policy calculated by dynamic programming method using five different methanol-feeding periods. The necessary state variables were collected from the calculated results and used for further optimization of repeated fed-batch fermentation. The optimal operation policy was investigated using the pre-collected state variables by estimating the overall profit per total methanol-feeding time. The calculated results indicated that the initial cell mass from the 2nd fed-batch fermentation on should be set at 35 or 40 g and methanol-feeding time at 264 h. In repeated fed-batch fermentation using the optimal operation policy, actual culture volume was in good agreement with the values simulated by model equations, but some discrepancy was observed in rHSA production. Minimum experiments were therefore carried out to re-evaluate rHSA production levels, which were then applied in re-calculations to determine the optimal operation policy. The optimal policy for repeated fed-batch fermentation established in the present study (i.e., 4-times-repeated fed-batch fermentation) achieved a 47% increase in annual rHSA production. Optimization of the culture period also brought about a 28% increase in annual rHSA production even in simple (not repeated) fed-batch fermentation.     

Principal component analysis for minimal model identification of a noise-affected fermentation: application to streptokinase

Principal component analysis (PCA) has been applied to a fed-batch fermentation for the production of streptokinase to identify the variables which are essential to formulate an adequate model. To mimic an industrial situation, Gaussian noise was introduced in the feed rate of the substrate. Both in the presence and in the absence of noise, the same five variables out of seven were selected by PCA. The minimal model trained separately without and with noise was able to predict satisfactorily the course of the fermentation for a condition not employed in training. These observations attest the suitability of PCA to formulate minimal models for industrial scale fermentations.     

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.     

Effect of setpoint on L-lysine fermentation

In control of a bioprocess, setpoint of fed-batch fermentation processes has a great influence on both the cost and the operating efficiency. Determination of a setpoint depends on the system and objective function. This work investigates two operating conditions for a fed-batch culture of L-lysine production. One is to maintain the reducing sugar concentration (RSC) on a constant setpoint, whereas the other has a piecewise variation of the RSC setpoint. Productivity, yield, and a cost function are employed to evaluate the performance of different setpoints on the fermentation process. Constant setpoint which is commonly used in fed-batch culture is not the best approach in L-lysine fermentation. Piecewise variation of setpoint shows that the better policy is to set the RSC at a higher concentration in the early cell growth stage then to decrease the RSC to a lower level.     

Growth monitoring and control through computer-aided on-line mass balancing in a fed-batch penicillin fermentation

A computer-aided methodology is developed for on-line monitoring and control of cell growth in fed-batch penicillin fermentation using a semidefined medium containing low corn steep liquor concentration (5.7 g/L). Cell growth is monitored and controlled with the use of experimental correlation and carbon-balancing equatiions on a real-time basis throughout the fermentation. Through a combination of feed-forward and feedback control of sugar addition, residual glucose concentration in the broth was maintained below 1 g/L and cell-growth rate was kept at constant at preset vaiues. The accuracy and reproducibility of this technique are demonstrated. The use of real-time control of cell growth is expected to aid future investigations of this antibiotic fermentation.     

Efficient production of l-lactic acid from hydrolysate of Jerusalem artichoke with immobilized cells of Lactococcus lactis in fibrous bed bioreactors

Hydrolysate of Jerusalem artichoke was applied for the production of l-lactic acid by immobilized Lactococcus lactis cells in a fibrous bed bioreactor system. Preliminary experiments had indicated that the high quality hydrolysate, which was derived from the 40min acid treatment at 95°C and pH 1.8, was sufficient to support the cell growth and synthesis of l-lactic acid. With the addition of 5g/l yeast extract, the fermentative performance of free cell system was evidently improved. After the basal settlement of hydrolysate based fermentation, the batch mode and the fed-batch mode fermentation were carried out in the free cell system and the fibrous bed bioreactor system, respectively. In all cases the immobilized cells presented the superior ability to produce l-lactic acid. The comparison of batch mode and fed-batch mode also indicated that the growth-limiting feeding strategy could reduce the lag phase of fermentation process and enhance the production of l-lactic acid. The achieved maximum concentration of l-lactic acid was 142g/l in the fed-batch mode. Subsequent repeated-batch fermentation of the fibrous bed bioreactor system had further exhibited the persistence and stability of this system for the high production of l-lactic acid in a long term. Our work suggested the great potential of the fibrous bed bioreactor system and hydrolysate of J. artichoke in the economical production of l-lactic acid at industrial scale.     

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.     

An algorithm for operating a fed-batch fermentor at optimum specific-growth rate

An algorithm for operating a fed-batch fermentor at an optimum specific fermentation rate is proposed. It does not require on-line measurement of nutrient concentration in the culture medium. An on-line estimate of the specific fermentation rate is sufficient for implementation of this scheme. The algorithm is model independent and works well even with poor estimates of the product yields and the specific fermentation rate. Results of a detailed simulation study are presented for a simple case of optimization of cell-mass production in a fed-batch fermentor. The results clearly demonstrate the efficacy of this algorithm under a wide range of fermentation situations.     

Dissolved-oxygen-stat fed-batch fermentation of 1,3-dihydroxyacetone from glycerol by Gluconobacter oxydans ZJB09112

This study investigated the effects of DO concentration on DHA fermentation and of DO-stat fed-batch fermentation using a pH control strategy, on 1,3-dihydroxyacetone (DHA) production. The results showed that DO-stat fed-batch fermentation with pH-shift control was the optimal bioprocess for DHA production. DO-stat fed-batch fermentation was carried out at 30% air saturation, and the culture pH was automatically maintained at pH 6.0 during the first 20 h and then shifted to pH 5.0 until the end of the fermentation. An optimal DHA concentration of 175.9 ± 6.7 g/L, with a production yield to glycerol of 0.87 ± 0.04 g/g, was obtained at 72 h of DO-stat fed-batch fermentation at 30°C in a 15 L fermenter.     

鸟苷补料分批发酵的研究

目的:以枯草芽孢杆菌TA208为出发菌株,研究了补料分批发酵方式下各种参数对鸟苷产量的影响。方法:采用补料分批发酵工艺,利用纸层析法测定发酵液中鸟苷的产量。结果:确定了葡萄糖、酵母粉和次黄嘌呤的最优补料方式,使鸟苷产量达到32.05g/L,较分批发酵方式提高了36.3%。结论:发酵工艺过程控制对发酵生产鸟苷具有重大影响。     

Effects of fed-batch fermentation and pH profiles on nisin production in suspended-cell and biofilm reactors

A biofilm reactor not only shortens the lag phase of nisin production, but also enhances nisin production when combined with an appropriate pH profile. Due to the substrate inhibition that takes place at high levels of carbon source, fed-batch fermentation was proposed as a better alternative for nisin production. In this study, the combined effects of fed-batch fermentation and various pH profiles on nisin production in a biofilm reactor were evaluated. The tested pH profiles include 1) a constant pH profile at 6.8 (profile 1), 2) a constant pH profile with an autoacidification after 4 h (profile 2), and 3) a step-wise pH profile with pH adjustment every 2 h (profile 3). When profile 1 was applied, fed-batch fermentation enhanced nisin production for both suspended-cell (4,188 IU ml⁻¹) and biofilm (4,314 IU ml⁻¹) reactors, yielded 1.8- and 2.3-fold higher nisin titer than their respective batch fermentation. On the other hand, pH profiles that include periods of autoacidification (profiles 2 and 3) resulted in a significantly lower nisin production in fed-batch fermentation (2,494 and 1,861 IU ml⁻¹ for biofilm reactor using profile 2 and 3, respectively) due to toxicity of excess lactic acid produced during the fermentation. Overall, this study suggested that fed-batch fermentation can be successfully used to enhance nisin production for both suspended-cell and biofilm reactors.     

Inline noninvasive Raman monitoring and feedback control of glucose concentration during ethanol fermentation

Raman spectroscopy as a process analytical technology tool was implemented for the monitoring and control of ethanol fermentation carried out with Saccharomyces cerevisiae. The need for the optimization of bioprocesses such as ethanol production, to increase product yield, enhanced the development of control strategies. The control system developed by the authors utilized noninvasive Raman measurements to avoid possible sterilization problems. Real-time data analysis was applied using partial least squares regression (PLS) method. With the aid of spectral pretreatment and multivariate data analysis, the monitoring of glucose and ethanol concentration was successful during yeast fermentation with the prediction error of 4.42 g/L for glucose and 2.40 g/L for ethanol. By Raman spectroscopy-based feedback control, the glucose concentration was maintained at 100 g/L by the automatic feeding of concentrated glucose solution. The control of glucose concentration during fed-batch fermentation resulted in increased ethanol production. Ethanol yield of 86% was achieved compared to the batch fermentation when 75 % yield was obtained. The results show that the use of Raman spectroscopy for the monitoring and control of yeast fermentation is a promising way to enhance process understanding and achieve consistently high production yield.     

On-line monitoring and controlling system for fermentation processes

A personal computer-based on-line monitoring and controlling system was developed for the fermentation of microorganism. The on-line HPLC system for the analysis of glucose and ethanol in the fermentation broth was connected to the fermenter via an auto-sampling equipment, which could perform the pipetting, filtration and dilution of the sample and final injection onto the HPLC through automation based on a programmed procedure. The A/D and D/A interfaces were equipped in order to process the signals from electrodes and from the detector of HPLC, and to direct the feed pumps, the motor of stirrer and gas flow-rate controller. The software that supervised the control of the stirring speed, gas flow-rate, pH value, feed flow-rate of medium, and the on-line measurement of glucose and ethanol concentration was programmed by using Microsoft Visual Basic under Microsoft Windows. The signal for chromatographic peaks from on-line HPLC was well captured and processed using an RC filter and a smoothing algorithm. This monitoring and control system was demonstrated to be effective in the ethanol fermentation of Zymomonas mobilis operated in both batch and fed-batch modes. In addition to substrate and product concentrations determined by on-line HPLC, the biomass concentration in Z. mobilis fermentation could also be on-line estimated by using the pH control and an implemented software sensor. The substrate concentration profile in the fed-back fermentation followed well the set point profile due to the fed-back action of feed flow-rate control.     

Batch, fed-batch and repeated fed-batch fermentation processes of the marine thraustochytrid Schizochytrium sp. for producing docosahexaenoic acid

Different fermentation processes, including batch, fed-batch and repeated fed-batch processes by Schizochytrium sp., were studied and compared for the effective DHA-rich microbial lipids production. The comparison between different fermentation processes showed that fed-batch process was a more efficient cultivation strategy than the batch process. Among the four different feeding strategies, the glucose concentration feed-back feeding strategy had achieved the highest fermentation results of final cell dry weight, total lipids content, DHA content and DHA productivity of 72.37, 48.86, 18.38 g l^?1 and 138.8 mg l^?1 h^?1, respectively. The repeated fed-batch process had the advantages of reducing the time and cost for seed culture and inoculation between each fermentation cycles. The results of fermentation characteristics and lipid characterization of the repeated fed-batch process indicated that this repeated fed-batch process had promising industrialization prospect for the production of DHA-rich microbial lipids.     

Rapid quantitation and monitoring of plasmid DNA using an ultrasensitive DNA-binding dye

A sensitive fluorescence-based method for monitoring plasmid DNA during production was investigated. This simple method of assaying for plasmid DNA allows rapid monitoring of plasmid yields from a recombinant Escherichia coli fed-batch fermentation. The assay has several advantages over traditional methods of plasmid DNA measurement. The fluorescent dye is highly specific and can measure total plasmid DNA concentration in about 5 min. The assay is sensitive over a wide range of plasmid concentrations of between 15 and 280 ng/mL, even in the presence of impurities that occur within alkaline lysate preparations. The technique can also be applied to monitoring fermentation and downstream purification steps.