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.     

Bioconversion of Butyric Acid to Butanol by Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) in a Limited Nutrient Medium

This study was designed to investigate the ability of Clostridium saccharoperbutylacetonicum N1-4 to produce butanol in a limited nutrient medium using mixtures of glucose and butyric acid as substrates. Specific combinations of glucose and butyric acid were found to influence the enhancement and retardation of butanol production as well as the reduction and modulation of the number of bacterial cells. Increasing the butyric acid concentration leads to the inhibition of bacterial growth, whereas the presence of (0?C5?g/L) butyric acid and (0?C10?g/L) glucose enhances the butanol production. The combination of 5?g/L butyric acid with 5 and 10?g/L of glucose was found to be the most suitable, but the use of glucose at concentrations greater than 10?g/L shifted the optimal butyric acid concentrations to 10 and 15?g/L for maximum butanol production signifying the requirement of a specific combination of glucose and butyric acid for enhanced butanol production in the fermentation process. C. saccharoperbutylacetonicum N1-4 demonstrated the ability to produce butanol in the absence of glucose, but no acetone or ethanol was produced under these conditions, reflecting the nature of the pathways involved in the production of butanol using only butyric acid. Ten grams per litre of butyric acid was found able to produce 13?g/L of butanol in the presence of 20?g/L of glucose, and 0.7?g/L butanol was produced in the absence of glucose. This study indicates the importance of the glucose to butyric acid ratio to the enhancement of butanol production.     

Intracellular Conditions Required for Initiation of Solvent Production by Clostridium acetobutylicum

We investigated the intracellular physiological conditions associated with the induction of butanol-producing enzymes in Clostridium acetobutylicum. During the acidogenic phase of growth, the internal pH decreased in parallel with the decrease in the external pH, but the internal pH did not go below 5.5 throughout batch growth. Butanol was found to dissipate the proton motive force of fermenting C. acetobutylicum cells by decreasing the transmembrane pH gradient, whereas the membrane potential was affected only slightly. In growing cells, the switch from acid to solvent production occurred when the internal undissociated butyric acid concentration reached 13 mM and the total intracellular undissociated acid concentration (acetic plus butyric acids) was at least 40 to 45 mM. Similar values were obtained when cultures were supplemented with 50 mM butyric acid initially or when a phosphate-buffered medium was used instead of an acetate-buffered medium. To measure the induction of the enzymes involved in solvent synthesis, we determined the rates of conversion of butyrate to butanol in growing cells. The rate of butanol formation reached a maximum in the mid-solvent phase, when the butanol concentration was 50 mM. Although more solvent accumulated later, de novo enzyme synthesis decreased and then ceased.     

Characteristics of butanol fermentation by a low-acid-producing Clostridium acetobutylicum B18

Fermentation characteristics of Clostridium acetobutylicum B18 were studied in batch experiments with and without pH control. This strain is shown to be potentially useful in simultaneous acetone-butanol-ethanol fermentation-separation systems because of its low acid production. In a pH-uncontrolled batch culture this strain produced mostly solvents, including 15 g/l of butanol. Ethanol production was low. Strain B18 recycled organic acids more efficiently than other strains. In particular, butyric acid was completely recycled when glucose was not limiting. Yield of liquid products (solvents plus organic acids) and carbon recovery in total products (gas plus liquid) were 33.1–36.4 wt% and 90–91 mol%, respectively, for 20–80 g/l of initial glucose. Glucose consumption and the percentage of butanol among solvents were higher at 32°C than at 37°C. Strain B18 required approximately 0.4 g/l of undissociated butyric acid at the onset of solvent production in pH-uncontrolled batch culture. The low undissociated butyric acid requirement enabled this strain to produce 13.8 g/l of butanol at a controlled pH of 6.0.Contribution no. 19998 of the Minnesota Agricultural Experiment Station Correspondence to: C.-H. Park     

The acetone butanol fermentation on glucose and xylose. II. Regulation and kinetics in fed-batch cultures

The kinetics in fed-batch cultures of acetone butanol fermentation by Clostridium acetobutylicum is compared on glucose, xylose, and mixtures of both sugars. The final conversion yield of sugars into solvents always increases with the sugar feeding rate. At low feeding rates, the sugar concentration in the medium becomes limiting, which results in a slower cellular growth, a slower metabolic transition from an acid to a solvent fermentation and, thus, a higher accumulation of acids. It is only at sufficiently high feeding rates that fed-batch fermentations yield kinetic results comparable to those of batch fermentations. With mixtures of glucose and xylose, because of a maintained low glucose level, both sugars are taken up at the same rate during a first fermentation period. An earlier accumulation of xylose when the fermentation becomes inhibited suggest that xylose utilization is inhibited when the catabolic flux of glucose alone can satisfy the metabolic activity of the cell. Kinetic results with batch and fed-batch fermentations indicate several important features of the regulation of C. acetobutylicum metabolism: an early inhibition by the produced acids; an initiation of solvent formation between 4 and 6 g/L acetic and butyric acid depending on the metabolic activity of the cell; a metabolic transition from acids to solvents production at a rate closely related to the rate of sugar uptake; during solvent production, a reassimilation of acids above a minimal rate of sugar consumption of 0.2 h(-1); a final inhibition of the fermentation at a total butanol and acids concentration of ca. 20 g/L.     

Efficient conversion of lactic acid to butanol with pH-stat continuous lactic acid and glucose feeding method by Clostridium saccharoperbutylacetonicum

In order to achieve high butanol production by Clostridium saccharoperbutylacetonicum N1-4, the effect of lactic acid on acetone–butanol–ethanol fermentation and several fed-batch cultures in which lactic acid is fed have been investigated. When a medium containing 20 g/l glucose was supplemented with 5 g/l of closely racemic lactic acid, both the concentration and yield of butanol increased; however, supplementation with more than 10 g/l lactic acid did not increase the butanol concentration. It was found that when fed a mixture of lactic acid and glucose, the final concentration of butanol produced by a fed-batch culture was greater than that produced by a batch culture. In addition, a pH-controlled fed-batch culture resulted in not only acceleration of lactic acid consumption but also a further increase in butanol production. Finally, we obtained 15.5 g/l butanol at a production rate of 1.76 g/l/h using a fed-batch culture with a pH-stat continuous lactic acid and glucose feeding method. To confirm whether lactic acid was converted to butanol by the N1-4 strain, we performed gas chromatography–mass spectroscopy (GC-MS) analysis of butanol produced by a batch culture during fermentation in a medium containing [1,2,3-¹³C₃] lactic acid as the initial substrate. The results of the GC-MS analysis confirmed the bioconversion of lactic acid to butanol.     

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.     

The role of acids on the production of acetone and butanol by Clostridium acetobutylicum

Summary The production of solvent by Clostridium acetobutylicum was studied, using fed-batch fermentations. Different specific rates of carbohydrate utilisation were obtained by variations in feeding rates of sugar. At slow catabolic rates of sugar, addition of acetic acid or butyric acid, alone or together, increased the rate of the metabolic transition by a factor 10 to 20, the amount of solvents by a factor 6 and the percentage of fermented glucose to solvents by a factor 3. The same results were obtained with both glucose and xylose fermentations. Depending on the rates of growth, butanol production began at acid levels of 3–4 g·l^-1 for fast metabolism and at acid levels of 8–10 g·l^-1 for slow metabolism. Associated with slow metabolism, reassimilation of acids required values as high as 6.5 g·l^-1 of acetic acid and 7.5 g·l^-1 of butyric acid. At a high rate of metabolism, acetic and butyric acids were reassimilated at concentrations of 4.5 g·l^-1.     

Enzymes limiting butanol and acetone formation in continuous and batch cultures of Clostridium acetobutylicum

Summary The formation of butanol in continuous cultures of Clostridium acetobutylicum is regulated at the genetic level via expression of butyraldehyde dehydrogenase since increased in vitro activities of this key enzyme are associated with increased in vivo butanol formation rates in both acidogenic and solventogenic fermentations. Addition of glucose, butyric acid and carbon monoxide results in induction of butyraldehyde dehydrogenase. The production of acetone in continuous fermentation is also controlled at the genetic level through expression of coenzyme A (CoA)-transferase; this enzyme is induced by glucose. Carbon monoxide inactivates acetoacetate decarboxylase. In controlled-pH batch fermentation solventogenesis does not correlate with in vitro activities of butyraldehyde dehydrogenase. Instead, initiation of alcohol formation is accompanied by increased activities of both reduced nicotine adenine dinucleotide (NADH)- and reduced nicotine adenine dinucleotide phosphate (NADPH)-specific alcohol dehydrogenases. The production of acetone in batch fermentation is regulated at the genetic level through combined induction of both CoA-transferase and acetoacetate decarboxylase. These two enzymes are not detected in either batch or continuous culture at or above pH 6.0. This finding explains the inability of the cells to produce acetone at elevated culture pH.     

Enhanced Butanol Production by Clostridium beijerinckii BA101 Grown in Semidefined P2 Medium Containing 6 Percent Maltodextrin or Glucose

Dramatically elevated levels of butanol and acetone resulted in higher butanol and total solvent yields for hyperamylolytic Clostridium beijerinckii BA101 relative to the NCIMB 8052 parent strain grown in semidefined P2 medium containing either 6% glucose or STAR-DRI 5 maltodextrin. C. beijerinckii BA101 consistently produced on the order of 19 g of butanol per liter in 20-liter batch fermentations. This represents a greater than 100% increase in butanol concentration by the BA101 strain compared to the parent NCIMB 8052 strain. The kinetics of butanol production over time also indicate a more rapid rate of butanol production by BA101 in semidefined P2 medium containing glucose or maltodextrin. The lower levels of butyric and acetic acids produced over the course of the fermentation carried out by BA101 are consistent with an enhanced capacity for uptake and recycling of these acids. C. beijerinckii BA101 appears to more completely utilize carbohydrate compared to the 8052 strain. Carbon balance following fermentation by C. beijerinckii 8052 and BA101 indicates that sufficient carbon is available for the twofold increase in butanol concentration observed during BA101 fermentations. C. beijerinckii BA101 also has superior solvent production capacity during continuous culture fermentation in P2 medium containing 6% glucose. Volumetric solvent yields of 0.78 and 1.74 g/liter/h for BA101 and 0.34 and 1.17 g/liter/h for NCIMB 8052 were obtained at dilution rates of 0.05 and 0.20 h(sup-1), respectively. No drift towards acid synthesis (strain degeneration) was observed for up to 200 h (d = 0.05 h(sup-1)) and 100 h (d = 0.20 h(sup-1)).     

Influence of pH and undissociated butyric acid on the production of acetone and butanol in batch cultures of Clostridium acetobutylicum

Summary The kinetics of growth and acid and solvent production are examined in batch fermentation of Clostridium acetobutylicum at pH between 4.5 and 6.0. At the lower pH, growth occurs in two consecutive phases and solvents are the main excreted metabolites. At the higher pH, there is a single growth phase with only acid formation. The influence of the pH can be correlated with a critical role of the concentration of undissociated butyric acid in the medium: cellular growth is inhibited above 0.5 g/l and solvent production starts at an undissociated acid level of 1.5 g/l. Reducing the intracellular acid dissociation by lowering the intracellular pH also favours the production of acetone and butanol.     

Improved n-butanol production by a non-acetone producing Clostridium pasteurianum DSMZ 525 in mixed substrate fermentation

The kinetics of growth, acid and solvent production in batch culture of Clostridium pasteurianum DSMZ 525 were examined in mixed or mono-substrate fermentations. In pH-uncontrolled batch cultures, the addition of butyric acid or glucose significantly enhanced n-butanol production and the ratio of butanol/1,3-propanediol. In pH-controlled batch culture at pH?=?6, butyric acid addition had a negative effect on growth and did not lead to a higher n-butanol productivity. On the other hand, mixed substrate fermentation using glucose and glycerol enhanced the growth and acid production significantly. Glucose limitation in the mixed substrate fermentation led to the reduction or inhibition of the glycerol consumption by the growing bacteria. Therefore, for the optimal growth and n-butanol production by C. pasteurianum, a limitation of either substrate should be avoided. Under optimized batch conditions, n-butanol concentration and maximum productivity achieved were 21 g/L, and 0.96 g/L?×?h, respectively. In comparison, mixed substrate fermentation using biomass hydrolysate and glycerol gave a n-butanol concentration of 17 g/L with a maximum productivity of 1.1 g/L?×?h. In terms of productivity and final n-butanol concentration, the results demonstrated that C. pasteurianum DSMZ 525 is well suitable for n-butanol production from mixed substrates of biomass hydrolysate and glycerol and represents an alternative promising production strain.     

Acetone-butanol fermentation and its variants

Recent intensive research on the acetone-butanol-ethanol and the isopropanol-butanol-ethanol fermentation has increased the basic understanding of these processes substantially. Metabolic investigations on Clostridium acetobutylicum, and Clostridium beijerinkii show that enzyme activities necessary for solvent production are induced only in solvent-producing cells. Although produced, or added, acetic and butyric acid have significant effects on the metabolic activities, the transition from acid to solvent production cannot as yet be fully explained. Based on studies in continuous cultures, the kinetics of product formation can be described. Knowledge of the mechanism of butanol toxicity is accumulating but no dramatic increase in butanol tolerance has so far been obtained. Successful results, approaching the limitations determined by biological and technological possibilities, have been obtained in batch and continuous cultures, and in continuous processes based on immobilized cells. Continuous processes are superior to batch cultures in respect of their productivity.     

Enhanced butanol production by optimization of medium parameters using Clostridium acetobutylicum YM1

A new isolate of the solvent-producing Clostridium acetobutylicum YM1 was used to produce butanol in batch culture fermentation. The effects of glucose concentration, butyric acid addition and C/N ratio were studied conventionally (one-factor-at-a-time). Moreover, the interactions between glucose concentration, butyric acid addition and C/N ratio were further investigated to optimize butanol production using response surface methodology (RSM). A central composite design was applied, and a polynomial regression model with a quadratic term was used to analyze the experimental data using analysis of variance (ANOVA). ANOVA revealed that the model was highly significant (p < 0.0001) and the effects of the glucose and butyric acid concentrations on butanol production were significant. The model validation experiment showed 13.82 g/L butanol was produced under optimum conditions. Scale up fermentation in optimized medium resulted in 17 g/L of butanol and 21.71 g/L of ABE. The experimental data of scale up in 5 L bioreactor and flask scale were fitted to kinetic mathematical models published in the literature to estimate the kinetic parameters of the fermentation. The models used gave the best fit for butanol production, biomass and glucose consumption for both flask scale and bioreactor scale up.     

Improvements of metabolites tolerance in <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> by micronutrient zinc supplementation

Micronutrient zinc is of great importance for acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum. The effect of zinc supplementation on toxic metabolites (formic, acetic, butyric acid and butanol) tolerance during ABE fermentation was investigated under various stress-shock conditions without pH control. Great improvements on cell growth, glucose utilization and butanol production were achieved. In the presence of 0.45 g/L formic acid, zinc contributed to 11.28 g/L butanol produced from 55.24 g/L glucose compared to only 5.27 g/L butanol from 29.49 g/L glucose in the control without zinc supplementation. More importantly, relatively higher levels of 7.5 g/L acetic acid, 5.5 g/L butyric acid and 18 g/L butanol could be tolerated by C. acetobutylicum with zinc supplementation while no fermentation was observed under the same stress-shock condition respectively, suggesting that the acids and butanol tolerance in C. acetobutylicum could be significantly facilitated by pleiotropic regulation of micronutrient zinc. Thus, this paper provides an efficient bioprocess engineering strategy for improving stress tolerance in Clostridium species.     

Effects of extractive fermentation on butyric acid production byClostridium acetobutylicum

Summary The addition of an oleyl alcohol extractant to a batch fermentation of glucose byClostridium acetobutylicum resulted in a concentration profile that was distinctly different from the non-extractive control fermentation. The concentration of butyric acid increased and subsequently decreased in the control fermentation. The concentration of butyric acid increased but did not subsequently decrease in the oleyl alcohol extractive fermentation. The production of butyric acid was found to have been prolonged into the solventogenic phase in the oleyl alcohol extractive fermentation. Butyric acid was continually replenished from glucose while it was being converted to butanol. Supplementation of exogenous acetic and butyric acids, the metabolic uncoupler carbonyl cyanide 3-chlorophenylhydrazone, or decanol to the oleyl alcohol extractive fermentation helped to reinstate the normal butyric acid concentration profile. These findings are discussed with respect to the effects of these additives on the pH ofC. acetobutylicum and its importance with regard to the production of butyric acid.     

Growth rate dependence of solventogenesis and solvents produced byClostridium beijerinckii

Summary Acidogenesis and solventogenesis byClostridium beijerinckii NRRL B-593 has been studied in batch growth, and in sucrose-limited chemostat and recycling fermentor growth. Cells grown in batch culture without pH control primarily produced either butyric and acetic acids, or these acids plus butanol, ethanol and isopropanol in ratios depending on the medium's content of reducing agent, calcium and iron. Cells in chemostat-culture at a mass doubling time (t_d) of 5.8 h produced primarily butyric and acetic acids at pH 6.8 and these acids plus butanol, ethanol and isopropanol at pH 4.8. Cells grown in a recycling fermentor (in which the t_d continuously increases) at pH 6.8 entered solventogenesis at a t_d of 43 h, producing primarily propanol, ethanol and butanol, along with butyric acid, but with greatly decreased production of acetic acid. Although clostridial form morphology, succeeded by sporulation, usually accompanied solventogenesis, the association was not invariant so that solventogenesis and sporogenesis can occur separately in this species.     

The effect of pH on nitrogen supply, cell lysis, and solvent production in fermentations of Clostridium acetobutylicum

In batch fermentations of C. acetobutylicum, with 5 g/L yeast extract and 50mM glucose, the ratio of ammonium to glucose affected solvent production when the pH was left to vary uncontrolled from 4.5 to 3.65. High solvent production was observed for a low ratio. When the pH was controlled at 4.5, only acids were produced for all ratio values. At a low ammonium-to-glucose ratio, solvents were produced when the pH was controlled at 3.7. Acids only were produced for a low ratio value at pH 4.0 or for a high ratio value at pH 3.7. In continuous cultures, mostly acids were produced under glucose limitation, but solvents were produced under nitrogen limitation. It was concluded that the nitrogen availability controls solvent production and that the pH affects the availability of organic nitrogen. Biomass autolysis at the stationary phase of batch cultures was reversibly inhibited at pH values less than 3.8. In batch fermentations, the overall molar growth yields on ATP (Y(ATP)) varied from 5.5 to 9.0 and the transient yields from 5.5 to 15.5. In continuous cultures, the Y(ATP) values varied from 5.5 to 14.7 under glucose limitation, and from 6.1 to 9.3 under nitrogen limitation. The Y(ATP) depended on the ammonium to glucose ratio and the culture pH, but did not show the usual dependence on the specific growth rate in batch cultures. The experiments seem to confirm the hypothesis that solvent production is controlled by the demand and availability of ATP.     

Role of Chemotaxis in Solvent Production by Clostridium acetobutylicum

The motility of Clostridium acetobutylicum has been investigated during a typical batch fermentation process for solvent production. The motility is characterized by “runs” during the early phase of sugar utilization and acid production, but this changes to “tumbles” during the onset of solventogenesis. Sugars and undissociated acetic and butyric acids have been shown to be attractants for the bacterium, while acetone, butanol, ethanol, and dissociated acetate and butyrate are repellents. It is suggested that chemotactic responses explain why highly motile cells are strongly solventogenic.     

Adsorbents for extractive bioconversion applied to the acetone-butanol fermentation

Summary Four different polymeric resins were tested as adsorbents in extractive bioconversion applied to the fermentative production of acetone and butanol by Clostridium acetobutylicum. The polymers were tested for their ability to adsorb butanol from pure solutions, and fermentation broths. Furthermore, the effect on the fermentability of the media was tested. The pH was increased to prevent adsorption of intermediates such as acetic and butyric acids. Bonopore, the polymer giving the best adsorption pattern with no undesirable effects, was tested in repeated batch cultures with C. acetobutylicum.