A Mathematical model for ethanol production by extractive fermentation in a continuous stirred tank fermentor

Extractive fermentation is a technique that can be used to reduce the effect of end product inhibition through the use of a water-immiscible phase that removes fermentation products in situ. This has the beneficial effect of not only removing inhibitory products as they are formed (thus keeping reaction rates high) but also has the potential for reducing product recovery costs. We have chosen to examine the ethanol fermentation as a model system for end product inhibition and extractive fermentation and have developed a computer model predicting the productivity enhancement possible with this technique together with other key parameters such as extraction efficiency and residual glucose concentration. The model accommodates variable liquid flowrates entering and leaving the system, since it was found that the aqueous outlet flowrate could be up to 35% lower than the inlet flowrate during extractive fermentation of concentrated glucose feeds due to the continuous removal of ethanol from the fermentation broth by solvent extraction. The model predicts a total ethanol productivity of 82.6 g/L h if a glucose feed of 750 g/L is fermented with a solvent having a distribution coefficient of 0.5 at a solvent dilution rate of 5.0 h(-1). This is more than 10 times higher than for a conventional chemostat fermentation of a 250 g/L glucose feed. The model has furthermore illustrated the possible trade-offs that exist between obtaining a high extraction efficiency and a low residual glucose concentration.     

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.     

Mathematical model of microporous hollow-fiber membrane extractive fermentor

A mathematical model is presented for a microporous hollow-fiber membrane extractive fermentor (HFEF). The model is based on the continuous flow of the aqueous nutrient phase and cells through the shell space of the fermentor where the fermentation reaction occurs. The product diffuses from the shell space through the hollow-fiber membrane where it is continuously removed by solvent flowing concurrently through the fiber lumen. Results for ethanol production show that the HFEF has a volumetric productivity significantly higher than that possible using conventional methods. The model predicts the existence of an optimum volume fraction of hollow fibers in the fermentor that maximizes the total volumetric productivity. This optimum is the result of a classic trade-off between the volume fraction of the fermentor required for fermentation and that required for efficient removal of the ethanol product to minimize product inhibition.     

Continuous fermentation of high-strength glucose feeds to ethanol

Summary Aqueous feeds of 413 and 495 g/L glucose were fermented to ethanol at 90–95% conversion in a continuous flow extractive fermentation system with cell recycle. Compared to the continuous conventional fermentation of a 195 g/L glucose medium, the volumetric productivity was more than doubled in extractive mode, with no deleterious effects on cell viability, specific glucose consumption rate or ethanol yield. The use of an effective, biocompatible and stable in situ extractant with flash vaporization can also produce a concentrated ethanol vapour stream, reducing distillation costs of the product.     

Continuous SSCF of AFEX™ pretreated corn stover for enhanced ethanol productivity using commercial enzymes and Saccharomyces cerevisiae 424A (LNH‐ST)

High productivity processes are critical for commercial production of cellulosic ethanol. One high productivity process—continuous hydrolysis and fermentation—has been applied in corn ethanol industry. However, little research related to this process has been conducted on cellulosic ethanol production. Here, we report and compare the kinetics of both batch SHF (separate hydrolysis and co‐fermentation) and SSCF (simultaneous saccharification and co‐fermentation) of AFEX? (Ammonia Fiber Expansion) pretreated corn stover (AFEX?‐CS). Subsequently, we designed a SSCF process to evaluate continuous hydrolysis and fermentation performance on AFEX?‐CS in a series of continuous stirred tank reactors (CSTRs). Based on similar sugar to ethanol conversions (around 80% glucose‐to‐ethanol conversion and 47% xylose‐to‐ethanol conversion), the overall process ethanol productivity for continuous SSCF was 2.3‐ and 1.8‐fold higher than batch SHF and SSCF, respectively. Slow xylose fermentation and high concentrations of xylose oligomers were the major factors limiting further enhancement of productivity. Biotechnol. Bioeng. 2013; 110: 1302–1311. © 2012 Wiley Periodicals, Inc.     

Mathematical model of extractive fermentation: application to the production of ethanol

A mathematical model has been developed for the process of extractive fermentation. The model rigorously treats the material balance, reaction kinetics, and liquid-liquid equilibrium relationships. Convergence is promoted through use of the Quasi-Newton Method. Extractive fermentation is particularly attractive for those bioreactions where the cell growth and product formation is inhibited by the product or other secondary cellular products. The model is illustrated for the production of ethanol. The results show an increase in specific productivity and the ability to process a more concentrated feed. However, volumetric productivity is reduced in the presence of a low capacity solvent.     

Ethanol fermentation of crude acid hydrolyzate of cellulose using high-level yeast inocula

High-level yeast inocula was investigated as a means of overcoming the toxicity problem in ethanol fermentation of acid hydrolyzate of wood cellulose. When the inoculum level exceeded 10(8) initial cells/mL, 50% of the yeast cells survived the initial cell death period during which furfural and HMF were depleted. The fermentation thus proceeded to completion by virtue of cell regrowth. The specific ethanol productivity in batch fermentation on the basis of viable cells was comparable to that of pure glucose fermentation. Continuous fermentation with cell recycle was superior to batch fermentation in that there was no overall cell decline and the ethanol yield was substantially higher. The maximum ethanol productivity in continuous fermentation was 4.9 g/L h and it occurred at a dilution rate of 0.24 hr(-1).     

Extractive fermentation-integrated reaction and product recovery

Summary Refinements have been made to a prototype process for the production of ethanol by extractive fermentation. The process is characterized by thein situ extraction of ethanol from a 7 L continuous stirred tank fermenter and the thermal recovery of ethanol from the extracting solvent, which is circulated in a closed loop through the process. Data are provided to show the efficacy and stability of the process under various operating conditions, and the near complete (>96%) continuous conversion of a 300 g/L glucose feed.     

Evaluation of cell recycling in continuous fermentation of enzymatic hydrolysates of spruce with Saccharomyces cerevisiae and on-line monitoring of glucose and ethanol

The maximum growth rate of Saccharomyces cerevisiae ATCC 96581, adapted to fermentation of spent sulphite liquor (SSL), was 7 times higher in SSL of hardwood than the maximum growth rate of bakers' yeast. ATCC 96581 was studied in the continuous fermentation of spruce hydrolysate without and with cell recycling. Ethanol productivity by ATCC 96581 in continuous fermentation of an enzymatic hydrolysate of spruce was increased 4.6 times by employing cell recycling. On-line analysis of CO₂, glucose and ethanol (using a microdialysis probe) was used to investigate the effect of fermentation pH on cell growth and ethanol production, and to set the dilution rate. Cell growth in the spruce hydrolysates was strongly influenced by fermentation pH. The fermentation was operated in continuous mode for 210 h and a theoretical ethanol yield on fermentable sugars was obtained. Received: 25 May 1998 / Received revision: 11 August 1998 / Accepted: 12 August 1998     

Enhanced acetone-butanol fermentation using repeated fed-batch operation coupled with cell recycle by membrane and simultaneous removal of inhibitory products by adsorption

A novel acetone-butanol production process was developed which integrates a repeated fed-batch fermentation with continuous product removal and cell recycle. The inhibitory product concentrations of the fermentation by Clostridium acetobutylicum were reduced by the simultaneous extraction process using polyvinylpyridine (PVP) as an adsorbent. Because of the reduced inhibition effect, a higher specific cell growth rate and thus a higher product formation rate was achieved. The cell recycle using membrane separation increased the total cell mass density and, therefore, enhanced the reactor productivity. The repeated fed-batchoperation overcame the drawbacks typically associated with a batch operation such as down times, long lag period, and the limitation on the maximum initial substrate concentration allowed due to the substrate inhibition. Unlike a continuous operation, the repeated fed-batch operation could beoperated for a long time at a relatively higher substrate concentration without sacrificing the substrate loss in the effluent. As a result, the integrated process reached 47.2 g/L in the equivalent solvent concentration (including acetone, butanol, and ethanol) and 1.69 g/L . h in the fermentor productivity, on average, over a 239.5-h period. Compared with a controlled traditional batch acetone-butanol fermentation, the equivalent solvent concentration and the tormentor productivity were increased by 140% and 320%, respectively. (c) 1995 John Wiley & Sons Inc.     

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.     

A numerical evaluation of a hollow fiber extractive fermentor process for the production of ethanol

The economics of a process for the production of ethanol employing a hollow fiber extractive fermentor have been investigated. A computer simulation of the process incorporating a mathematical model of the fermentor was used to calculate the mass and energy balances. The results of the process simulation were read into a computer spreadsheet programmed with the economic calculations from which a final ethanol product cost was obtained. The process was found to be as competitive as conventional fermentation processes even at the currently high cost--$4/sq ft--of hollow fibers. It was determined that the 1986 price of 46.2 cents/L of ethanol produced by the process would be reduced by 1.8 cents/L for every $1/sq foot drop in the price of hollow fibers. A comparison of this process with conventional fermentation processes indicates that its potential savings lie in its ability to use a concentrated sugar feed, and the fermentor's increased productivity and ability to produce a concentrated ethanol stream which is removed by the extracting solvent.     

Long-Term Continuous Cultivation of Clostridium beijerinckii in a Two-Stage Chemostat with On-Line Solvent Removal

A two-stage continuous cultivation experiment with Clostridium beijerinckii NRRL B592 is described. This strain maintained its ability to produce neutral solvents (acetone, n-butanol, and ethanol) at an overall dilution rate of 0.13 h(sup-1) and achieved an average overall solvent concentration of 9.27 g/liter and an overall solvent productivity of 1.24 g/liter/h for more than 100 overall retention times. The experiment was performed without pH control on a semisynthetic medium containing yeast extract, and product inhibition was the limiting factor. Solid carrier material was present in both stages, and the solvent productivity in both stages was similar. A membrane evaporation module integrated into the recirculation loop of a second-stage bioreactor after 2,166 h increased solvent productivity and improved the yield of solvents by about 40%. The membrane reduced the concentration of solvents, which would otherwise inhibit the fermentation. Additionally, the integrated membrane evaporation dampened metabolic oscillations, which are characteristic of continuous cultivation of clostridia. It was also demonstrated that a moderate concentration buildup (approximately 30% of bioreactor inflow) caused by water flux through the membrane caused no detrimental effects to the bacterial cells. However, much higher water fluxes through the membrane, associated with a much more dramatic increase in the concentration of salts in the medium, did appear to favor cell degeneration.     

Production of ethanol by coupling fermentation and solvent extraction

Summary A new technology of fermentation is proposed. The inhibitor product is removed continuously by coupling fermentation and solvent extraction. Applied to ethanol fermentation this technology is suitable to any case where the terminal product is inhibitory.The proposed technology uses both plug flow reactor and liquid-liquid extraction to achieve continuously the extractive fermentation of ethanol. The solvent used for liquid-liquid extraction is dodecanol. A new reactor was used. It is a column packed with a porous material . The fermentation broth is pulsed (a) to increase the interfacial area between the liquid medium and the dodecanol, and (b) to: decrease the gas hold up.Alcoholic fermentations were performed on glucose syrup at 35°C using Saccharomyces cerevisiae, with adsorbed cells as reference, with adsorbed cells and extractive fermentation. The results show that the fermentation is substantially improved. By this new method the ethanol productivity was multiplied by 5 and a solution of 407 g/l of glucose was totally fermented with a yeast which cannot normally transform more than 200 g/l glucose.     

Ethanol fermentation from molasses at high temperature by thermotolerant yeast Kluyveromyces sp. IIPE453 and energy assessment for recovery

High temperature ethanol fermentation from sugarcane molasses B using thermophilic Crabtree-positive yeast Kluyveromyces sp. IIPE453 was carried out in batch bioreactor system. Strain was found to have a maximum specific ethanol productivity of 0.688 g/g/h with 92 % theoretical ethanol yield. Aeration and initial sugar concentration were tuning parameters to regulate metabolic pathways of the strain for either cell mass or higher ethanol production during growth with an optimum sugar to cell ratio 33:1 requisite for fermentation. An assessment of ethanol recovery from fermentation broth via simulation study illustrated that distillation-based conventional recovery was significantly better in terms of energy efficiency and overall mass recovery in comparison to coupled solvent extraction–azeotropic distillation technique for the same.     

Performance of batch, fed-batch, and continuous A-B-E fermentation with pH-control

Batch, fed-batch, and continuous A-B-E fermentations were conducted and compared with pH controlled at 4.5, the optimal range for solvent production. While the batch mode provides the highest solvent yield, the continuous mode was preferred in terms of butanol yield and productivity. The highest butanol yield and productivity found in the continuous fermentation at dilution rate of 0.1 h⁻¹ were 0.21 g-butanol/g-glucose and 0.81 g/L/h, respectively. In the continuous and fed-batch fermentation, the time needed for passing acidogenesis to solventogenesis was an intrinsic hindrance to higher butanol productivity. Therefore, a low dilution rate is suggested for the continuous A-B-E fermentation, while the fed-batch mode is not suggested for solvent production. While 3:6:1 ratio of acetone, butanol, and ethanol is commonly observed from A-B-E batch fermentation by Clostridium acetobutylicum when the pH is uncontrolled, up to 94% of the produced solvent was butanol in the chemostat with pH controlled at 4.5.     

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 ethanol by immobilized Saccharomyces bayanus in an extractive fermentation system

An extractive fermentation system using immobilized yeast cells was developed to study the ethanol production at high sugar concentrations. Organic acids were used as extracting solvents of ethanol and their toxicity was tested in free and k-carrageenan entrapped cell preparations. Immobilization seems to protect cells against solvent toxicity, when long-chain organic acids, e.g., oleic acid, were used, probably due to steric and diffusional limitations, the free cells not being viable at high oleic acid concentrations. The entrapped cells also present a higher metabolic activity than their free counterparts at high glucose concentrations. A solution of 300 g/L of glucose was totally fermented by the immobilized yeast cells, which when free cannot normally convert more than 200 g/L. In situ recovery of ethanol by oleic acid in a batch immobilized cell system led to higher ethanol productivities and to the fermentation of 400 g/L, when an oleic acid/medium ratio of 5 was used.     

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.     

Optimization of batch alcoholic fermentation of glucose syrup substrate

The quantitative effects of substrate concentration, yeast concentration, and nutrient supplementation on ethanol content, fermentation time, and ethanol productivity were investigated in a Box–Wilson central composite design experiment, consisting of five levels of each variable, High substrate concentration, up to 30° Brix, resulted in higher ethanol content (i.e., up to 15.7% w/v or 19.6% v/v) but longer fermentation time and hence lower ethanol productivity. Increasing yeast concentration, on the other hand, resulted in shorter fermentation time and higher productivity. The highest ethanol productivity of about 21 g EiOH/L h was obtained at low substrate concentration (i.e., 12° Brix), low alcohol content (i.e., 6% by weight), high yeast concentration (i.e., 4.4%), and high supplementation of yeast extract (i.e., 2.8). Productivity of this magnitude is substantially higher that that of the traditional batch fermentation of fed-batch fermentation. It is comparable to the results of continuous fermentation but lower than those of vacuum fermentation but lower than those of vacuum fermentation. Optimal conditions for maximal ethanol productivity can be established by a multiple regression analysis technique and by plotting the contours of constant response to conform to the constraints of individual operations.