Dialysis Continuous Process for Ammonium-Lactate Fermentation of Whey: Experimental Tests

Laboratory experiments were conducted to validate theoretical predictions describing a dialysis continuous process for the fermentation of whey lactose to ammonium lactate, in which the fermentor contents are poised at a constant pH by adding ammonia solution and dialyzed through a membrane against water. Dried sweet-cheese whey was rehydrated to contain 230 mg of lactose per ml, supplemented with 8 mg of yeast extract per ml, charged into a 5-liter fermentor without sterilization, adjusted in pH (5.3) and temperature (44°C), and inoculated with Lactobacillus bulgaricus. The fermentor and dialysate circuits were connected, and steady-state conditions were established. A series of such conditions was managed nonaseptically for 94 days to study the process and to demonstrate efficiency and productivity. As time progressed, the fermentation remained homofermentative and increased in conversion efficiency, although membrane fouling necessitated dialyzer cleaning about every 4 weeks. With a retention time of 19 h, 97% of the substrate was converted into products. Relative to nondialysis continuous or batch processes for the fermentation, the dialysis continuous process enabled the use of more concentrated substrate, was more efficient in the rate of substrate conversion, and additionally produced a second effluent of less concentrated but purer ammonium lactate.     

Dialysis continuous process for ammonium lactate fermentation: Simulated and experimental dialysate-feed,immobilized-cell systems

A generalized mathematical model, previously developed and experimentally validated, was modified and used to computer-simulate two dialysate-feed systems for operating a dialysis continuous process for the ammonium lactate fermentation. The simulations predicted that the feeding of substrate into the dialysate circuit and thence into the fermentor circuit via dialysis should greatly improve the production of cell mass and metabolite product. Experiments were conducted to test the system in which the fermentor is operated without an effluent, thus immobilizing the cells. Dried cheese whey ultrafiltrate was rehydrated to contain a normal concentration of lactose (62 mg/ml), supplemented with yeast with an adapted culture of Lactobacillus bulgaricus. The system was operated without interruption for 26 days. Results during steady-state conditions showed that the system is a new and useful way to immobilize living cells for the purpose of producing a metabolite at a high rate for a prolonged time. The substrate consumed by the cells is converted to product via maintenance metabolism only and is sterilized by dialysis.     

Dialysis Continuous Process for Ammonium-Lactate Fermentation of Whey: Mathematical Model and Computer Simulation

A mathematical model was developed to describe a dialysis process for the continuous fermentation of whey lactose to lactic acid, with neutralization to a constant pH by ammonia. In the process, whey of a relatively high concentration is fed into the fermentor circuit at a relatively low rate so that the residual concentration of lactose is low. The fermentor effluent contains ammonium lactate, bacterial cells, and residual whey solids and could be used as a nitrogen-enriched feedstuff for ruminant animals. Only water is fed into the dialysate circuit at a relatively high rate. The dialysate effluent contains purified ammonium lactate and could be converted to lactic acid and ammonium sulfate for industry. The fermentation was specifically modeled as a set of equations representing material balances and rate relationships in the two circuits. Dialysis continuous fermentations, in general, were modeled by combining these equations and by using dimensionless parameters. The generalized model was then solved for the steady state and used to simulate the specific fermentation on a digital computer. The results showed the effects of various material and operational and kinetic parameters on the process and predicted that it could be operated efficiently.     

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.     

Dialysis Continuous Process for Ammonium-Lactate Fermentation: Improved Mathematical Model and Use of Deproteinized Whey

Separate terms for substrate limitation and product inhibition were incorporated into an equation describing the rate of cell growth for the steady-state fermentation of lactose to lactic acid with neutralization to a constant pH by ammonia. The equation was incorporated into a generalized mathematical model of a dialysis continuous process for the fermentation, developed previously, in which the substrate is fed into the fermentor and the fermentor contents are dialyzed through a membrane against water. The improved model was used to simulate the fermentation on a digital computer, and the results agreed with previous experimental tests using whole whey as the substrate. Further simulations were then made to guide experimental tests using deproteinized whey as the substrate. Dried cheese-whey ultrafiltrate was rehydrated with tap water to contain 242 mg of lactose per ml, supplemented with 8 mg of yeast extract per ml, charged into a 5-liter fermentor without sterilization, adjusted in pH (5.5) and temperature (44°C), and inoculated with an adapted culture of Lactobacillus bulgaricus. The fermentor and dialysate circuits were connected, and a series of steady-state conditions was managed nonaseptically for 71 days. The fermentation of deproteinized whey relative to whole whey, with both highly concentrated, resulted in similar extents of product accumulation but at a lesser rate.     

自絮凝颗粒酵母乙醇连续发酵耦合酵母回用工艺的研究

模拟现有酒精发酵行业普遍采用的多级发酵罐串联系统,建立了一套由三级串联操作的搅拌式发酵罐和两个沉降罐组成的反应器系统,以脱胚脱皮玉米粉双酶法制备的糖化液为发酵底物,培养基初始还原糖浓度为220g/L,添加(NH4)2HPO41.5g/L和KH2PO42.5g/L,以0.057h-1的恒定稀释速率流加,将自沉降浓缩后的酵母乳先后经活化和不活化两种方式处理并循环至第一级发酵罐,系统在两种操作条件下分别达到了拟稳态。实验结果表明活化处理对改善发酵工艺技术指标方面发挥了显著的作用,发酵终点乙醇浓度达到101g/L,还原糖和残总糖分别在3.2和7.7g/L左右,发酵系统的设备生产强度指标为5.77g/(L.h),与无酵母回用的搅拌式反应器系统中自絮凝颗粒酵母乙醇发酵工艺相比,提高了70%。     

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.     

Studies on continuous ethanol fermentation of sugar cane molasses

Summary A new laboratory system for continuous fermentation is described. It is well suited for fermenting concentrated substrates such as moderately dilute molasses. A rotating microporous filter, which is annexed to the fermentor vessel, allows the free escape of metabolic products while retaining yeast in the fermentor.The slop is recirculated after removal of ethanol by distillation leading to a build-up of non-fermentables. The concentration of these and of yeast cells is checked by a controlled bleed. The described system is a useful tool for small-scale experiments on continuous ethanol fermentation.     

Performance evaluation of ethanol fermentor systems using a vector-valued objective function

It is shown that the performance evaluation using a vector-valued objection function whose components are the product productivity, the product concentration, and the substrate conversion is quite useful in getting deeper insight into the development of new processes and in determining the operating point. Particular attention is focused on the ethanol fermentation using variety of systems such as the conventional chemostat system, multiple fermentor system, cell recycle system, extractive fermentor system, cell recycle system, extractive fermentor system, and immobilized cell system. The contour map and the projection of the noninferior set are used in investigating the performance improvement and the trade-offs among performance indexes.     

自絮凝酵母SPSC01在组合反应器系统中酒精连续发酵的研究

建立了一套由四级磁力搅拌发酵罐串联组成、总有效容积4000mL的小型组合生物反应器系统 ,其中一级罐作为种子培养罐。以脱胚脱皮玉米粉双酶法制备的糖化液为种子培养基和发酵底物 ,进行了自絮凝颗粒酵母酒精连续发酵的研究。种子罐培养基还原糖浓度为100g L ,添加 (NH₄)₂HPO₄ 和KH₂PO₄ 各 20g L ,以0.017h^-1 的恒定稀释速率流加 ,并溢流至后续酒精发酵系统。发酵底物初始还原糖浓度 220g/L ,添加 (NH₄)₂HPO₄ 15g/L和KH₂PO₄2 5g/L ,流加至第一级发酵罐 ,稀释速率分别为 0.017、0.025、0.033、0.040和0.05 0h^-1。实验数据表明 ,自絮凝颗粒酵母在各发酵罐中呈部分固定化状态 ,在稀释速率0.040h^-1 条件下 ,发酵系统呈一定的振荡行为 ,其他四个稀释速率实验组均能够达拟稳态。当稀释速率不超过 0 0 33h^-1 ,流出末级发酵罐的发酵液中酒精浓度可以达到 12 % (V/V)以上 ,残还原糖和残总糖分别在 0 11%和 0 35 % h^-1,流出末级发酵罐的发酵液中酒精浓度可以达到12%（V/V）以上,残还原糖和残总糖分别在0.11%和0.35%（W/V）以下。在稀释速率为0.033h^-1时,计算发酵系统酒精的设备生产强度指标为3.32（g·L^-1·h^-1）,与游离酵母细胞传统酒精发酵工艺相比,增加约1倍。     

Effect of nutrient limitation and two-stage continuous fermentor design on productivities during "Clostridium ragsdalei" syngas fermentation

The effect of three limiting nutrients, calcium pantothenate, vitamin B₁₂ and cobalt chloride (CoCl₂), on syngas fermentation using “Clostridium ragsdalei” was determined using serum bottle fermentation studies. Significant results from the bottle studies were translated into single- and two-stage continuous fermentor designs. Studies indicated that three-way interactions between the three limiting nutrients, and two-way interactions between vitamin B₁₂ and CoCl₂ had a significant positive effect on ethanol and acetic acid formation. In general, ethanol and acetic acid production ceased at the end of 9 days corresponding to the production of 2.01 and 1.95 g L⁻¹ for the above interactions. Reactor studies indicated the three-way nutrient limitation in two-stage fermentor showed improved acetic acid (17.51 g g⁻¹ cells) and ethanol (14.74 g g⁻¹ cells) yield compared to treatments in single-stage fermentors. These results further support the hypothesis that it is possible to modulate the product formation by limiting key nutrients during C. ragsdalei syngas fermentation.     

Continuous acetone-butanol-ethanol (ABE) fermentation using immobilized cells of Clostridium acetobutylicum in a packed bed reactor and integration with product removal by pervaporation

An integrated solvent (ABE) fermentation and product removal process was investigated. A stable solvent productivity of 3.5 g/L h was achieved by using cells of Clostridium acetobutylicum immobilized onto a packed bed of bonechar, coupled with continuous product removal by pervaporation. Using a concentrated feed solution containing lactose at 130g/L, a lactose value of 97.9% was observed. The integrated fermentation and product removal system, with recycling of the treated fermentor effluent containing only low amount of solvents (/but lactose and acids), leads to only low acid losses. Therefore, most of the acids are converted to solvents, and this results in a high solvent yield of 0.39 g solvents/g lactose utilized. The pervaporation system provided a high product removal rate even at low solvent concentrations. A solvent membrane flux of 7.1 g/m(2) h with a selectivity of 5 was achieved during these investigations. The system proved to be very reliable.     

一步法发酵菊芋生产乙醇

利用马克斯克鲁维酵母(Kluyveromyces marxianus)YX01具有菊粉酶生产能力且乙醇发酵性能良好的特点,直接发酵菊粉生成乙醇.在摇瓶中考察了该菌株最适发酵温度,进而在2.5L发酵罐中考察了通气量和底物浓度的影响.实验结果表明:该菌株最适发酵温度为35℃;在通气量为50 mL/min和100 mL/min时菌体生长加快,发酵时间缩短,但在不通气条件下糖醇转化率明显提高;在菊粉浓度235 g/L时,发酵终点乙醇浓度达到92.2 g/L,乙醇对糖的得率为0.436,为理论值的85.5%.在此基础上,使用近海滩涂种植海水灌溉收获的菊芋为底物,以批式补料方式直接发酵菊芋干粉浓度为280 g/L的底物,发酵终点乙醇浓度为84.0 g/L,乙醇对糖的得率为0.405,为理论值的80.0%.这些研究工作,为以菊芋为原料的燃料乙醇技术开发奠定了基础.     

Application of multistage continuous fermentation for production of fuel alcohol by very-high-gravity fermentation technology

A fermentation system to test the merging of very-high-gravity (VHG) and multistage continuous culture fermentation (MCCF) technologies was constructed and evaluated for fuel ethanol production. Simulated mashes ranging from 15% to 32% w/v glucose were fermented by Saccharomyces cerevisiae and the dilution rates were adjusted for each glucose concentration to provide an effluent containing less than 0.3% w/v glucose (greater than 99% consumption of glucose). The MCCF can be operated with glucose concentrations up to 32% w/v, which indicates that the system can successfully operate under VHG conditions. With 32% w/v glucose in the medium reservoir, a maximum of 16.73% v/v ethanol was produced in the MCCF. The introduction of VHG fermentation into continuous culture technology allows an improvement in ethanol productivity while producing ethanol continuously. In comparing the viability of yeast by methylene blue and plate count procedures, the results in this work indicate that the methylene blue procedure may overestimate the proportion of dead cells in the population. Ethanol productivity (Yps) increased from the first to the last fermentor in the sequence at all glucose concentrations used. This indicated that ethanol is more effectively produced in later fermentors in the MCCF, and that the notion of a constant Yps is not a valid assumption for use in mathematical modeling of MCCFs. Journal of Industrial Microbiology & Biotechnology (2001) 27, 87–93. Received 20 January 2001/ Accepted in revised form 28 April 2001     

Rapid ethanol fermentations using vacuum and cell recycle

Cell recycle and vacuum fermentation systems were developed for continuous ethanol production. Cell recycle was employed in both atmospheric pressure and vacuum fermentations to achieve high cell densities and rapid ethanol fermentation rates. Studies were conducted with Saccharomyces cerevisiae (ATCC No. 4126) at a fermentation temperature of 35°C. Employing a 10% glucose feed, a cell density of 50 g dry wt/liter was obtained in atmospheric-cell recycle fermentations which produced a fermentor ethanol productivity of 29.0 g/liter-hr. The vacuum fermentor eliminated ethanol inhibition by boiling away ethanol from the fermenting beer as it was formed. This permitted the rapid and complete fermentation of concentrated sugar solutions. At a total pressure of 50 mmHg and using a 33.4% glucose feed, ethanol productivities of 82 and 40 g/liter-hr were achieved with the vacuum system with and without cell recycle, respectively. Fermentor ethanol productivities were thus increased as much as twelvefold over conventional continuous fermentations. In order to maintain a viable yeast culture in the vacuum fermentor, a bleed of fermented broth had to be continuously withdrawn to remove nonvolatile compounds. It was also necessary to sparge the vacuum fermentor with pure oxygen to satisfy the trace oxygen requirement of the fermenting yeast.     

Ethanol fermentation of raw cassava starch with Rhizopus koji in a gas circulation type fermentor

Studies have been conducted in a gas circulation type fermentor in order to characterize the ethanol fermentation of uncooked cassava starch with Rhizopus koji. Results showed that ethanol concentration reached 13-14% (v/v) in 4-day broth, and the maximum productivity of ethanol was 2.3 g ethanol/L broth h. This productivity was about 50% compared to the productivity of a glucose-yeast system. Ethanol yield reached 83.5-72.3% of the theoretical yield for the cassava starch used. The fermentor used in the present work has been proven by experiment to be suitable for ethanol fermentation of the broth with solid substrate.     

Ethanol from Whey: Continuous Fermentation with a Catabolite Repression-Resistant Saccharomyces cerevisiae Mutant

An alternative method for the conversion of cheese whey lactose into ethanol has been demonstrated. With the help of continuous-culture technology, a catabolite repression-resistant mutant of Saccharomyces cerevisiae completely fermented equimolar mixtures of glucose and galactose into ethanol. The first step in this process was a computer-controlled fed-batch operation based on the carbon dioxide evolution rate of the culture. In the absence of inhibitory ethanol concentrations, this step allowed us to obtain high biomass concentrations before continuous fermentation. The continuous anaerobic process successfully incorporated a cell-recycle system to optimize the fermentor productivity. Under conditions permitting a low residual sugar concentration (≤1%), maximum productivity (13.6 g liter⁻¹ h⁻¹) was gained from 15% substrate in the continuous feed at a dilution rate of 0.2 h⁻¹. Complete fermentation of highly concentrated feed solutions (20%) was also demonstrated, but only with greatly diminished fermentor productivity (5.5 g liter⁻¹ h⁻¹).     

Ethanol production from xylose with the yeast Pichia stipitis and simultaneous product recovery by gas stripping using a gas-lift loop fermentor with attached side-arm (GLSA)

The bioconversion of xylose into ethanol with the yeast Pichia stipitis CBS 5773 is inhibited when 20 g/L of ethanol are present in the fermentation broth. In order to avoid this limitation, the fermentation was carried out with simultaneous recovery of product by CO(2) stripping. The fermentation was also improved by attaching a side-arm to the main body of a classical gas-lift loop fermentor. This side-arm increases the liquid circulation, mass transfer, and gas distribution, reducing the amount of oxygen in the inlet gas necessary to perform the fermentation of xylose under microaerobic conditions (K(L)a approximately 16 h(-1)). The continuous stripping of ethanol from the fermentation broth in this new bioreactor system allowed the consumption of higher xylose concentrations than using Erlenmeyer shaker flasks, improved significantly the process productivity and provided a clean ethanol solution by using an ice-cooled condenser system. Finally, a fed-batch fermentation was carried out with a K(L)a = 15.8 h(-1). Starting with 248.2 g of xylose, 237.6 g of xylose was consumed to produce 88.1 g of ethanol which represents 72.6% of the theoretical yield (47.2 g/L of ethanol was recovered in the condenser, while 9.6 g/L remained in the fermentation broth).     

Continuous conversion of D-xylose to ethanol by immobilized pachysolen tannophilus

The yeast Pachysolen tannophilus was entrapped in calcium alginate beads to ferment D-xylose on a continous basis in the presence of high cell densities. Experimental operating variables included the feed D-xylose concentration, the dilution rate, and the fermentor biomass concentration. Under favorable operating conditions, cultures retained at least 50% of their initial productivity after 26 days of operation. The specific ehanol production rate was dependent on the substrate level in the fermentor, passing through an optimum when the D-xylose concentration was between 28 and 35 g/L. Consequently, reactor productivity increased with dilution rate and feed D-xylose concentration until a maximum was reached. The ethanol content of the effluent always decreased with increasing dilution rate, but excessive dilution rates diminished the ethanol content without increasing productivity. Unlike production rate, ethanol yield declined monotonically from 0.35 g/g as the fermentor substrate concentration increased. The yield was 69% of that theoretically possible when the D-xylose concentration was near zero, as opposed to 42% when it was in the range supporting the optimum specific rate of ethanol production. As long as D-xylose was supplied to cells faster than they could consume it, productivity increased with the mass of cells immobilized. The effectiveness factor associated with the calcium alginte beads used in this system was 0.4, indicating that only 40% of the entrapped biomass was effective in converting D-xylose to ethanol because of diffusion limitations.     

Continuous ethanol production from sugar beet molasses using an osmotolerant mutant strain of Zymomonas mobilis

A laboratory process was established for ethanol production by fermentation of sugar beet molasses with the bacterium Zymomonas mobilis. Sucrose in the molasses was hydrolyzed enzymatically to prevent levan formation. A continuous system was adopted to reduce sorbitol formation and a two-stage fermentor was used to enhance sugar conversion and the final ethanol concentration. This two-stage fermentor operated stably for as long as 18 d. An ethanol concentration of 59.9 g/l was obtained at 97% sugar conversion and at high ethanol yield (0.48 g/g, 94% of theoretical). The volumetric ethanol productivity (3.0 g/l·h) was superior to that of batch fermentation but inferior to that of a single-stage continuous system with the same medium. However, the thanol concentration was increased to a level acceptable for economical recovery. The process proposed in this paper is the first report of successful fermentation of sugar beet molasses in the continuous mode using the bacterium Z. mobilis.