Novel Pathway for Alcoholic Fermentation of δ-Gluconolactone in the Yeast Saccharomyces bulderi

Under anaerobic conditions, the yeast Saccharomyces bulderi rapidly ferments delta-gluconolactone to ethanol and carbon dioxide. We propose that a novel pathway for delta-gluconolactone fermentation operates in this yeast. In this pathway, delta-gluconolactone is first reduced to glucose via an NADPH-dependent glucose dehydrogenase (EC 1.1.1.47). After phosphorylation, half of the glucose is metabolized via the pentose phosphate pathway, yielding the NADPH required for the glucose-dehydrogenase reaction. The remaining half of the glucose is dissimilated via glycolysis. Involvement of this novel pathway in delta-gluconolactone fermentation in S. bulderi is supported by several experimental observations. (i) Fermentation of delta-gluconolactone and gluconate occurred only at low pH values, at which a substantial fraction of the substrate is present as delta-gluconolactone. Unlike gluconate, the latter compound is a substrate for glucose dehydrogenase. (ii) High activities of an NADP(+)-dependent glucose dehydrogenase were detected in cell extracts of anaerobic, delta-gluconolactone-grown cultures, but activity of this enzyme was not detected in glucose-grown cells. Gluconate kinase activity in cell extracts was negligible. (iii) During anaerobic growth on delta-gluconolactone, CO(2) production exceeded ethanol production by 35%, indicating that pyruvate decarboxylation was not the sole source of CO(2). (iv) Levels of the pentose phosphate pathway enzymes were 10-fold higher in delta-gluconolactone-grown anaerobic cultures than in glucose-grown cultures, consistent with the proposed involvement of this pathway as a primary dissimilatory route in delta-gluconolactone metabolism.     

Achievements and perspectives to overcome the poor solvent resistance in acetone and butanol-producing microorganisms

Anaerobic bacteria such as the solventogenic clostridia can ferment a wide range of carbon sources (e.g., glucose, galactose, cellobiose, mannose, xylose, and arabinose) to produce carboxylic acids (acetic and butyric) and solvents such as acetone, butanol, and ethanol (ABE). The fermentation process typically proceeds in two phases (acidogenic and solventogenic) in a batch mode. Poor solvent resistance by the solventogenic clostridia and other fermenting microorganisms is a major limiting factor in the profitability of ABE production by fermentation. The toxic effect of solvents, especially butanol, limits the concentration of these solvents in the fermentation broth, limiting solvent yields and adding to the cost of solvent recovery from dilute solutions. The accepted dogma is that toxicity in the ABE fermentation is due to chaotropic effects of butanol on the cell membranes of the fermenting microorganisms, which poses a challenge for the biotechnological whole-cell bio-production of butanol. This mini-review is focused on (1) the effects of solvents on inhibition of cell metabolism (nutrient transport, ion transport, and energy metabolism); (2) cell membrane fluidity, death, and solvent tolerance associated with the ability of cells to tolerate high concentrations of solvents without significant loss of cell function; and (3) strategies for overcoming poor solvent resistance in acetone and butanol-producing microorganisms.     

Determining the inhibition constants in the HCH-1 model of cellulose hydrolysis

The products of cellulose hydrolysis, glucose and cellobiose, caused noncompetitive inhibition patterns to be exhibited when Thermomonospora sp. YX cellulase hydrolyzed dyed cellulose. The glucose binding constant, beta(1), was 0.00683 +/- 0.00031 L/g and the cellobiose binding constant, beta(2), was 0.095 +/- 0.0058 L/g. Thus, cellobiose was about 14 times more inhibitory than glucose.     

Continuous production of acetone and butanol by Clostridium acetobutylicum in a two-stage phosphate limited chemostat

Summary When Clostridium acetobutylicum was grown in continuous culture under phosphate limitation (0.74 mM) at a pH of 4.3, glucose was fermented to butanol, acetone and ethanol as the major products. At a dilution rate of D=0.025 h^–1 and a glucose concentration of 300 mM, the maximal butanol and acetone concentrations were 130 mM and 74 mM, respectively. 20% of the glucose remained in the medium. On the basis of these results a two-stage continuous process was developed in which 87.5% of the glucose was converted into butanol, acetone and ethanol. The cells and minor amounts of acetate and butyrate accounted for the remaining 12.5% of the substrate. The first stage was run at D=0.125 h^–1 and 37° C and the second stage at D=0.04 h^–1 and 33° C. High yields of butanol and acetone were also obtained in batch culture under phosphate limitation.     

Enhanced butanol production in Clostridium acetobutylicum ATCC 824 by double overexpression of 6-phosphofructokinase and pyruvate kinase genes

This study elucidated the importance of two critical enzymes in the regulation of butanol production in Clostridium acetobutylicum ATCC 824. Overexpression of both the 6-phosphofructokinase (pfkA) and pyruvate kinase (pykA) genes increased intracellular concentrations of ATP and NADH and also resistance to butanol toxicity. Marked increases of butanol and ethanol production, but not acetone, were also observed in batch fermentation. The butanol and ethanol concentrations were 29.4 and 85.5 % higher, respectively, in the fermentation by double-overexpressed C. acetobutylicum ATCC 824/pfkA+pykA than the wild-type strain. Furthermore, when fed-batch fermentation using glucose was carried out, the butanol and total solvent (acetone, butanol, and ethanol) concentrations reached as high as 19.12 and 28.02 g/L, respectively. The reason for improved butanol formation was attributed to the enhanced NADH and ATP concentrations and increased tolerance to butanol in the double-overexpressed strain.     

The relationship between hydrogen gas and butanol production by Clostridium saccharoperbutylacetonicum

Two simultaneous fermentations were performed at 26 degrees C with simultaneous inocula using Clostridium saccharoperbutylacetonicum. Fermentation 1 prevented the gas formed by the biomass from escaping the fermentor while 2 allowed the gas formed to escape. Fermentor 1 provided for the production of butanol, acetone, and ethanol, while when the H(2) formed was allowed to escape with fermentor 2, neither butanol nor acetone were produced. Ethanol was also formed in both fermentors and began along with the initial growth of biomass and continued until the fermentations were complete. Butanol and acetone production began after biomass growth had reached a maximum and began to subside. The butanol-acetone-ethanol millimolar yields and ratios were 38:1:14 respectively. The fermentor 2 results show that a yield of 2.1 L H(2), 93 or 370 mmol H(2)/mol glucose, was formed only during the growing stage of growth; neither butanol nor acetone were produced; ethanol was formed throughout the fermentation, reaching a yield of 15.2 mmolar. It appears that hydrogen gas is required for butanol production during the resting stage of growth.     

High-titer n-butanol production by clostridium acetobutylicum JB200 in fed-batch fermentation with intermittent gas stripping

Acetone–butanol–ethanol (ABE) fermentation with a hyper‐butanol producing Clostridium acetobutylicum JB200 was studied for its potential to produce a high titer of butanol that can be readily recovered with gas stripping. In batch fermentation without gas stripping, a final butanol concentration of 19.1 g/L was produced from 86.4 g/L glucose consumed in 78 h, and butanol productivity and yield were 0.24 g/L h and 0.21 g/g, respectively. In contrast, when gas stripping was applied intermittently in fed‐batch fermentation, 172 g/L ABE (113.3 g/L butanol, 49.2 g/L acetone, 9.7 g/L ethanol) were produced from 474.9 g/L glucose in six feeding cycles over 326 h. The overall productivity and yield were 0.53 g/L h and 0.36 g/g for ABE and 0.35 g/L h and 0.24 g/g for butanol, respectively. The higher productivity was attributed to the reduced butanol concentration in the fermentation broth by gas stripping that alleviated butanol inhibition, whereas the increased butanol yield could be attributed to the reduced acids accumulation as most acids produced in acidogenesis were reassimilated by cells for ABE production. The intermittent gas stripping produced a highly concentrated condensate containing 195.9 g/L ABE or 150.5 g/L butanol that far exceeded butanol solubility in water. After liquid–liquid demixing or phase separation, a final product containing ～610 g/L butanol, ～40 g/L acetone, ～10 g/L ethanol, and no acids was obtained. Compared to conventional ABE fermentation, the fed‐batch fermentation with intermittent gas stripping has the potential to reduce at least 90% of energy consumption and water usage in n‐butanol production from glucose. Biotechnol. Bioeng. 2012; 109: 2746–2756. © 2012 Wiley Periodicals, Inc.     

Pilot-scale production of butanol by Clostridium beijerinckii BA101 using a low-cost fermentation medium based on corn steep water

To improve the economic competitiveness of the acetone/butanol/ethanol fermentation process, glucose/corn steep water (CSW) medium was used on a pilot scale for the production of solvents. The production of butanol by the Clostridium beijerinckii NCIMB 8052 parent strain and the solvent-hyperproducing BA101 mutant was compared. In a 20-l fermentation using 5% glucose/CSW medium,  C. beijerinckii 8052 produced 8.5 g butanol/l and 5 g acetone/l, while  C. beijerinckii BA101 produced 16 g butanol/l and 7.5 g acetone/l. Further studies were carried out on a larger scale using an optimized 6% glucose/CSW medium. In a 200-l pilot-scale fermentor,  C. beijerinckii 8052 produced 12.7 g butanol/l and 6 g acetone/l following 96 h of fermentation.  C. beijerinckii BA101 produced 17.8 g/l and 5.5 g/l butanol and acetone respectively, following 130 h of fermentation. These results represent a 40% increase in final butanol concentration by the C. beijerinckii BA101 mutant strain when compared to the 8052 parent strain. The total solvents (acetone, butanol, and ethanol) produced by C. beijerinckii NCIMB 8052 and BA101 in a 200-l fermentation were 19.2 g/l and 23.6 g/l respectively. This is the first report of pilot-scale butanol production by the solvent-hyperproducing C. beijerinckii BA101 mutant employing an inexpensive glucose/CSW medium. Received: 26 May 1998 / Received revision: 21 September 1998 / Accepted: 11 October 1998     

丙酮丁醇梭菌发酵菊芋汁生产丁醇

对丙酮丁醇梭菌Clostridium acetobutylicum L7发酵菊芋汁酸水解液生产丁醇进行了初步研究。实验结果表明,以该水解液为底物生产丁醇,不需要添加氮源和生长因子。当水解液初始糖浓度为48.36 g/L时,其发酵性能与以果糖为碳源的对照组基本相同,发酵终点丁醇浓度为8.67 g/L,丁醇、丙酮和乙醇的比例为0.58∶0.36∶0.06,但与以葡萄糖为碳源的对照组相比,发酵时间明显延长,表明该菌株葡萄糖转运能力强于果糖。当水解液初始糖浓度提高到62.87 g/L时,发酵终点残糖浓度从3.09 g/L增加到3.26 g/L,但丁醇浓度却提高到11.21 g/L,丁醇、丙酮和乙醇的比例相应为0.64∶0.29∶0.05,表明适量糖过剩有助于C.acetobutylicum L7胞内代谢从丙酮合成向丁醇合成途径调节;继续提高水解液初始糖浓度,发酵终点残糖浓度迅速升高,丁醇生产的技术经济指标受到明显影响。     

Effect of cell recycle on continuous butanol-acetone fermentation with Clostridium acetobutylicum under phosphate limitation

Summary An overflow filtration unit for cell recycle with Clostridium acetobutylicum was developed. A cellulose-triacetate ultrafiltration membrane with a cut-off volume of 20 000 MW was found to work best. C. acetobutylicum was grown in continuous culture under phosphate limitation (0.74 mM) at a pH value of 4.4 with cell recycle, the cell dry weight in the culture vessel reached 13.1 g/l at a dilution rate of D=0.10 h^-1 and 37°C. 377 mM of glucose were fermented to 190 mM butanol, 116.2 mM acetone and 25.8 mM ethanol. Total acids were 47.6 mM. The butanol productivity was 1.41 g/l/h. At a dilution rate of 0.40 h^-1 the butanol productivity was increased to 4.1 g/l/h but glucose consumption was decreased to 285 mM and butanol, acetone and ethanol production to 138.2, 97.5, 16.5 mM, respectively.     

Acetone–Butanol–Ethanol Production by Clostridium acetobutylicum ATCC 824 Using Sago Pith Residues Hydrolysate

Sago pith residues (58 % starch, 23 % cellulose, 9.2 % hemicellulose, and 4 % lignin) are one of the abundant lignocellulosic residues generated after starch extraction process in sago mill. In this study, fermentable sugars from enzymatic hydrolysis of sago pith residues were converted to acetone–butanol–ethanol (ABE) by Clostridium acetobutylicum ATCC 824. With an initial concentration of 30 g/L of concentrated sago pith residues hydrolysate containing 23 g/L of glucose and 4.58 g/L of cellobiose, 4.22?±?0.17 g/L of ABE were produced after 72 h of fermentation with yield and productivity of 0.20 g/g glucose and 0.06 g/L/h, respectively. Results are in agreement when synthetic glucose was used as a carbon source. Increasing sago pith residue hydrolysate to 50 g/L (containing 40 g/L glucose) and supplementing with 0.5 g/L yeast extract, approximately 8.84?±?0.20 g/L of ABE (5.41?±?0.10 g/L of butanol) were produced with productivity and yield of 0.12 g/L/h and 0.30 g/g glucose respectively, providing a 52 % improvement.     

Improvements of GC and HPLC analyses in solvent (acetone-butanol-ethanol) fermentation byClostridium saccharobutylicum using a mixture of starch and glycerol as carbon source

A study on the feasibility of using improved computer-controlled HPLC and GC systems was carried out to shorten the time needed for measuring levels of the substrates (glucose, maltose, and glycerol) and products (acetone, butanol ethanol, acetic acid, and butyric acid) produced byClostridium saccharobutylicum DSM 13864 during direct fermentation of sago starch to solvent. The use of HPLC system with a single injection to analyse the composition of culture broth (substrates and products) during solvent fermentation was achieved by raising the column temperature to 80°C. Although good separation of the components in the mixture was achieved, a slight overlap was observed in the peaks for butyric acid and acetone. The shape of the peak obtained and the analysis time of 26.66 min were satisfactory at a fixed flow rate of 0.8 mL/min. An improved GC system was developed, that was able to measure the products of solvent fermentation (acetone, butanol, ethanol, acetic acid, and butyric acid) within 19.28 min. Excellent resolution for each peak was achieved by adjusting the oven temperature to 65°C.     

The effect of fermentable carbohydrate on sporulation and butanol production by Clostridium acetobutylicum P262

Summary This study was conducted to determine whether or not a variation in the type of carbohydrate fermented by Clostridium acetobutylicum could be exploited to inhibit sporulation during the butanol-producing phase of fermentation and thus enhance butanol production. C. acetobutylicum P262 was found to ferment a wide variety of carbohydrates, but butanol production was not necessarily enhanced when percentage sporulation was low. Butanol concentration was more related to the total amount of acidic end-products (acetic and butyric acid) reutilized by the microorganism for solvent production and to the type and amount of carbohydrate utilized. Fermentation of cellobiose led to conditions resulting in complete acid reutilization and the highest butanol concentration (10.4–10.6 g/l). In cultures containing a mixture of glucose and cellobiose, glucose repression of cellobiose utilization resulted in lower butanol concentrations (6.6–7.5 g/l). Sporulation was dependent on the type of carbohydrate utilized by the microorganism. Glucose had a greater enhancing effect on the sporulation process (22–42%) than starch (9–12%) or cellobiose (22–34%). It was concluded that whereas the type of carbohydrate fermented had a specific effect on the extent of sporulation of a culture, conditions of low sporulation did not enhance butanol concentration unless carbohydrate utilization and the reutilization of acidic products were high.Correspondence to: W. M. Ingledew     

Evaluation of recent advances in butanol fermentation, upstream, and downstream processing

Four different processes for butanol production from corn, namely, batch fermentation and distillative recovery (BFDR), batch fermentation and pervaporative recovery (BFPR), fed-batch fermentation and pervaporative recovery (FBFPR), and immobilized cell continuous fermentation and pervaporative recovery (ICCFPR) were evaluated. Pervaporative recovery significantly reduces the cost of butanol production. Depending upon the byproduct credit, which is approximately 3.7 times that of the amount of butanol produced, BFDR, BFPR, FBFPR, and ICCFPR result in a butanol price of 0.55,0.55, 0.14-0.39, 0.12-0.37, and0.12-0.37, and 0.11-0.362kg^-1, respectively. The price of butanol was recently reported at $1.212kg^-1 by Chemical Marketing Reporter. It should be noted that all three components (acetone, butanol, and ethanol: ABE) diffuse through the pervaporation membrane. Further separation and purification of the solvents would require distillation, which has been considered in this exercise. This article also details the impact of byproduct credit, rate of return, and tax on butanol price.     

Production of butanol by Clostridium puniceum in batch and continuous culture

Summary The pink-pigmented, amylolytic and pectinolytic bacterium Clostridium puniceum in anaerobic batch culture at pH 5.5 and 25–30°C produced butan-1-ol as the major product of fermentation of glucose or starch. The alcohol was formed throughout the exponential phase of growth and surprisingly little acetone was simultaneously produced. Furthermore, acetic and butyric acids were only accumulated in low concentrations, and under optimal conditions were completely re-utilised before the fermentation ceased. Thus, in a minimal medium containing 4% w/v glucose as sole source of carbon and energy, after 65 h at 25°C, pH 5.5 all of the glucose had been consumed to yield (g product/100 g glucose utilised) butanol 32, acetone 3 and ethanol 2. Butanol was again the major product of glucose fermentation during phosphate-limited chemostat culture wherein, although the organism eventually lost its capacity to sporulate and to synthesize granulose, production of butanol continued for at least 100 volume changes. Under no growth condition was the organism capable of producing more than 13.3 g l^-1 of butanol. At pH 5.5, growth on pectin was slow and yielded a markedly lesser biomass concentration than when growth was on glucose or starch; acetic acid was the major fermentation product with lower concentrations of methanol, acetone, butanol and butyric acid. At pH 7, growth on all substrates produced virtually no solvents but high concentrations of both acetic and butyric acids.     

Metabolic engineering strategies for butanol production in Escherichia coli

The global market of butanol is increasing due to its growing applications as solvent, flavoring agent, and chemical precursor of several other compounds. Recently, the superior properties of n-butanol as a biofuel over ethanol have stimulated even more interest. (Bio)butanol is natively produced together with ethanol and acetone by Clostridium species through acetone-butanol-ethanol fermentation, at noncompetitive, low titers compared to petrochemical production. Different butanol production pathways have been expressed in Escherichia coli, a more accessible host compared to Clostridium species, to improve butanol titers and rates. The bioproduction of butanol is here reviewed from a historical and theoretical perspective. All tested rational metabolic engineering strategies in E. coli to increase butanol titers are reviewed: manipulation of central carbon metabolism, elimination of competing pathways, cofactor balancing, development of new pathways, expression of homologous enzymes, consumption of different substrates, and molecular biology strategies. The progress in the field of metabolic modeling and pathway generation algorithms and their potential application to butanol production are also summarized here. The main goals are to gather all the strategies, evaluate the respective progress obtained, identify, and exploit the outstanding challenges.     

Bioproduction of butanol in bioreactors: new insights from simultaneous in situ butanol recovery to eliminate product toxicity

Simultaneous acetone butanol ethanol (ABE) fermentation by Clostridium beijerinckii P260 and in situ product recovery was investigated using a vacuum process operated in two modes: continuous and intermittent. Integrated batch fermentations and ABE recovery were conducted at 37 °C using a 14-L bioreactor (7.0 L fermentation volume) containing initial substrate (glucose) concentration of 60 g/L. The bioreactor was connected in series with a condensation system and vacuum pump. Vacuum was applied continuously or intermittently with 1.5 h vacuum sessions separated by 4, 6, and 8 h intervals. A control ABE fermentation experiment was characterized by incomplete glucose utilization due to butanol toxicity to C. beijerinckii P260, while fermentation coupled with in situ recovery by both continuous and intermittent vacuum modes resulted in complete utilization of glucose, greater productivity, improved cell growth, and concentrated recovered ABE stream. These results demonstrate that vacuum technology can be applied to integrated ABE fermentation and recovery even though the boiling point of butanol is greater than that of water.     

Aldehyde-alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations

Metabolic engineering (ME) of Clostridium acetobutylicum has led to increased solvent (butanol, acetone, and ethanol) production and solvent tolerance, thus demonstrating that further efforts have the potential to create strains of industrial importance. With recently developed ME tools, it is now possible to combine genetic modifications and thus implement more advanced ME strategies. We have previously shown that antisense RNA (asRNA)-based downregulation of CoA transferase (CoAT, the first enzyme in the acetone-formation pathway) results in increased butanol to acetone selectivity, but overall reduced butanol yields and titers. In this study the alcohol/aldehyde dehydrogenase (aad) gene (encoding the bifunctional protein AAD responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA, respectively) was expressed from the phosphotransbutyrylase (ptb) promoter to enhance butanol formation and selectivity, while CoAT downregulation was used to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers, overall solvent titers of 30 g/L, and a higher alcohol/acetone ratio. Metabolic flux analysis revealed the likely depletion of butyryl-CoA. In order to increase then the flux towards butyryl-CoA, we examined the impact of thiolase (THL, thl) overexpression. THL converts acetyl-CoA to acetoacetyl-CoA, the first step of the pathway from acetyl-CoA to butyryl-CoA, and thus, combining thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. thl overexpression in strains with asRNA CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to enhance the intracellular butyryl-CoA pool and reduce the acetyl-CoA pool in order to achieve improved butanol titers and selectivity.     

Development and characterization of butanol — Resistant strain ofClostridium acetobutylicum in molasses medium

The production of acetone—butanol—ethanol solvents from cane molasses by locally isolated culture ofClostridium acetobutylicum was limited by butanol toxicity (1.6 mol/L). The butanol tolerance of the isolated culture was increased up to 4.8 mol/L by a serial enrichment method. The butanol-resistant strain had greater efficiency for the conversion of saccharides to mixed solvents and produced 52% more butanol at the expense of acetone and ethanol than the original strain. Moreover, the fermentation profile of parent and butanol-resistant strains in anerobic fermentation of cane molasses demonstrated the superiority of the latter in terms of growth rate, time of onset of butanol production, sugar utilization, final butanol concentration and other parameters.     

限制葡萄糖、葡萄糖/乙酸双底物条件下自由控制丙丁梭菌ABE发酵丙酮浓度和丙酮/丁醇比

提出一种可以提高和自由控制丙丁梭菌ABE发酵丙酮浓度与丙酮/丁醇比的方法。（1）通过控制糖化酶用量、反应时间和温度调节玉米培养基初始葡萄糖浓度,使发酵进入到产溶剂期后,残留葡萄糖浓度降至接近于0 g/L的水平;（2）在葡萄糖受限的条件下,诱导丙丁梭菌合成分泌糖化酶,分解寡糖,将葡萄糖维持于低浓度,进而限制梭菌胞内糖酵解途径的碳代谢和NADH生成速度。与此同时,外添乙酸形成葡萄糖/乙酸双底物环境。在能量代谢基本不受破坏、丁醇未达到抑制浓度的条件下,适度抑制丁醇生产,有效地利用外添乙酸强化丙酮合成;（3）在外添乙酸的基础上,添加适量酿酒酵母,形成丙丁梭菌/酿酒酵母混合发酵体系,提高梭菌对高丁醇浓度的耐受能力。整个发酵体系可以将丙酮浓度和丙酮/丁醇比自由控制在5~12 g/L和0.5~1.0的水平,最大丙酮浓度和丙酮/丁醇比达到11.74 g/L和1.02,并可维持丁醇浓度于10~14 g/L的正常水平,充分满足工业ABE发酵对于丙酮和丁醇产品的不同需求。