Reaction engineering studies of acetone-butanol-ethanol fermentation with Clostridium acetobutylicum

Acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum has been extensively studied in recent years because the organism is recognized as an excellent butanol producer. A parallel bioreactor system with 48 stirred-tank bioreactors on a 12 mL scale was evaluated for batch cultivations of the strictly anaerobic, butanol-producing C. acetobutylicum ATCC 824. Continuous gassing with nitrogen gas was applied to control anaerobic conditions. Process performances of ABE batch fermentations on a milliliter scale were identical to the liter-scale stirred-tank reactor if reaction conditions were identical on the different scales (e.g., initial medium, pH, temperature, specific evaporation rates, specific power input by the stirrers). The effects of varying initial ammonia concentrations (0.1-4.4 g L(-1) ) were studied in parallel with respect to glucose consumption and butanol production of C. acetobutylicum ATCC 824 as a first application example. The highest butanol yield of 33% (mol mol(-1) ) was observed at initial ammonia concentrations of 0.5 and 1.1 g L(-1) . This is the first report on the successful application of a 48 parallel stirred-tank bioreactor system for reaction engineering studies of strictly anaerobic microorganisms at the milliliter scale.     

Effects of nutritional enrichment on the production of acetone-butanol-ethanol (ABE) by Clostridium acetobutylicum

Clostridium acetobutylicum is an industrially important organism that produces acetone-butanol-ethanol (ABE). The main objective of this study was to characterize the effects of increased cell density on the production of ABE during the phase transition from acidogenesis to solventogenesis in C. acetobutylicum. The increased ABE productivity of C. acetobutylicum was obtained by increasing the cell density using a newly designed medium (designated C. a cetobutylicum medium 1; CAM1). The maximum OD₆₀₀ value of C. acetobutylicum ATCC 824 strain obtained with CAM1 was 19.7, which is 1.8 times higher than that obtained with clostridial growth medium (CGM). The overall ABE productivity obtained in the CAM1-fermetation of the ATCC 824 strain was 0.83 g/L/h, which is 1.5 times higher than that (0.55 g/L/h) obtained with CGM. However, the increased productivity obtained with CAM1 did not result in an increase in the final ABE titer, because phase transition occurred at a high titer of acids.     

Characterization and evaluation of corn steep liquid in acetone-butanol-ethanol production by Clostridium acetobutylicum

Corn steep liquor (CSL) obtained from a commercial starch manufacturing process was analyzed and tested as a complex nutrient source for ABE (acetone, butanol, and ethanol) production by Clostridium acetobutylicum PJC4BK_AdhE1. CSL contained a wealth of trace elements and nitrogenous components, proteins and amino acids. ABE production increased the content of CSL was raised up to 6% (v/v) in medium and then decreased at higher contents. In 6% CSL-containing medium, C. acetobutylicum PJC4BK_AdhE1 produced 21.4 g/L of ABE with a yield of 0.41 g/g in 18 h of fermentation. Although the final titer of ABE was similar in CSL containing media and Clostridial Growth Medium (CGM, containing yeast extract and asparagines as complex nutrients), the yield and productivity of ABE in 6% CSL-containing medium were found to be higher than 32 and 26%, respectively.     

Controlled-ph batch butanol-acetone fermentation by low acid producing Clostridium acetobutylicum B18

Summary C. acetobutylicum B18 produced a large amount of butanol over a wide range of pH (4.5–6.0). At pH 6.0 fermentation and cell growth were most active at pH 6.0, and the highest values of glucose consumption rate (4.37 g/L-h), butanol productivity (1.0 g/L-h), butyric acid recycle rate (0.31 g/L-h), and cell growth rate (0.2 h^-1) were obtained. There existed a critical pH between 6.0 and 6.5 above which cells switched to organic acid producing mode. Clostridial stage appeared essential for solvent production by strain B18 but sporulation was not necessary for solvent formation.     

Pervaporative butanol fermentation by Clostridium acetobutylicum B18

Extractive acetone-butanol-ethanol (ABE) fermentation was carried out successfully using pervaporation and a low-acid-producing Clostridium acetobutylicum B18. A pervaporation module with 0.17 m(2) of surface area was made of silicone membrane of 240 mum thickness. Pervaporation experiments using make-up solutions showed that butanol and acetone fluxes increased linearly with their concentrations in the aqueous phase. Fickian diffusion coefficients were constants for fixed air flow rates, and increased at higher sweep air flow rates. During batch and fed-batch fermentations, pervaporation at an air flow rate of 8 L/min removed butanol and acetone efficiently. Butanol concentration was maintained below 4.5 g/L even though Clostridium acetobutylicum B18 produced butanol steadily. Pervaporation could not remove organic acids efficiently, but organic acids did not accumulate because strain B18 produced little organic acid and recycled added organic acids efficiently. With pervaporation, glucose consumption rate increased compared to without pervaporation, and up to 160 g/L of glucose was consumed during 80 h. Cell growth was not inhibited by possible salt accumulation or oxygen diffusion through the silicone tubing. The culture volume was maintained relatively constant during fed-batch operation because of an offsetting effect of water and product removal by pervaporation and addition of nutrient supplements. (c) 1994 John Wiley & Sons, Inc.     

电子载体对丁醇发酵的影响

研究在培养基中加入不同电子载体对丁醇发酵的影响。结果表明:添加微量的苄基紫精可以促进丁醇的产生,同时可强烈抑制丙酮的合成,丁醇体积分数由66.92%提高到82.35%。苄基紫精可促进菌株快速进入产溶剂期,发酵周期明显缩短,丁醇生产强度显著提高。7%玉米培养基中加入40 mg/L苄基紫精,丁醇产量最高达16.10 g/L,生产强度为0.37 g/(L.h),分别较对照提高10.96%和60.87%。在初始丁醇体积分数较低的条件下,苄基紫精对丁醇合成的促进作用更明显。     

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.     

Investigation of acetone-butanol-ethanol fermentation by fluorescence

Acetone-butanol-ethanol fermentation by Clostridium acetobutylicum was followed by two variations of fluorescence: the intrinsic fluorescence of NADH, related to bacterial metabolism, and the fluorescence polarization of extrinsic 1, 6-diphenyl-1, 3, 5-hexatriene (DPH) related to membrane fluidity. First, NADH fluorescence was correlated to the specific rate production of butyric acid (linear relationship) and to enzymatic activities (acetate kinase, butyrate kinase and aceto-acetate decarboxylase). Second, a simultaneous increase in both DPH anisotropy (order parameter increase) and butanol production was observed. Even though these results seem contradictory, because of the well-known fluidizing effect of butanol on lipids, the apparent changes in fluidity can be the result of adaptive membrane alteration.     

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     

Bacteriocin production by Clostridium acetobutylicum in an industrial fermentation process.

High titers of a noninducible bacteriocin were produced by Clostridium acetobutylicum in a molasses fermentation medium used for the industrial production of solvents. Release of the bacteriocin towards the end of the exponential growth phase was accompanied by lysis of the culture and inhibition of the production of solvents. The producer cells were sensitive to the bacteriocin, which only affected other C. acetobutylicum strains and a Clostridium felsineum strain. The thermolabile bacteriocin was not inactivated by protease enzymes and had no optimum stability between pH 4 and 5. The sedimentation coefficient of the bacteriocin was 6S.     

Bacteriocin production by Clostridium acetobutylicum in an industrial fermentation process.

High titers of a noninducible bacteriocin were produced by Clostridium acetobutylicum in a molasses fermentation medium used for the industrial production of solvents. Release of the bacteriocin towards the end of the exponential growth phase was accompanied by lysis of the culture and inhibition of the production of solvents. The producer cells were sensitive to the bacteriocin, which only affected other C. acetobutylicum strains and a Clostridium felsineum strain. The thermolabile bacteriocin was not inactivated by protease enzymes and had no optimum stability between pH 4 and 5. The sedimentation coefficient of the bacteriocin was 6S.     

Effect of increased hydrogen partial pressure on the acetone-butanol fermentation by Clostridium acetobutylicum

Summary Elevated H₂ partial pressure in the acetone-butanol fermentation increased the butanol and ethanol yields on glucose by an average of 18% and 13%, respectively, while the respective yields of acetone and of the endogenous H₂ decreased by an average of 40% and 30%, and almost no effect was observed on the growth of the culture. The butanol to acetone ratio and the fraction of butanol in the total solvents were also increased with the H₂ pressure. There were no major differences in the observed pattern of change with pressurization at either t=0 or t=18 h. The results demonstrate the importance of H₂ partial pressure in the regulation of the C. acetobutylicum metabolism.     

Metabolic engineering of Clostridium acetobutylicum ATCC 824 for isopropanol-butanol-ethanol fermentation

Clostridium acetobutylicum naturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineered C. acetobutylicum can be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene from Clostridium beijerinckii NRRL B-593 (i.e., adh(B-593)) in C. acetobutylicum ATCC 824. To increase the total alcohol titer, a synthetic acetone operon (act operon; adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in the buk gene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged by in situ removal of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.     

The improvement of glucose/xylose fermentation by Clostridium acetobutylicum using calcium carbonate

Batch fermentation of 60g/l glucose/xylose mixture by Clostridium acetobutylicum ATCC 824 was investigated on complex culture medium. Different proportions of mixtures, ranged between 10 and 50g of each sugar/l, were fermented during pH control at 4.8 (optimum pH for solventogenesis) or during CaCO₃ addition. Using xylose-pregrown cells and pH control, an important amount of xylose was left over at the end of the fermentation when the glucose concentration was higher than that of xylose. The addition of 10g of CaCO₃/l (to prevent the pH dropping below 4.8) increased xylose uptake: a substantial decrease of residual xylose was observed when xylose-pregrown cells as well as glucose-pregrown cells were used as inoculum for all the mixture proportions studied. MgCO₃ (Mg²⁺-containing compound) and CaCl₂ (Ca²⁺-containing compound) reduced residual xylose only during pH control at 4.8 by NaOH addition. As butanol is the major limiting factor of xylose uptake in C. acetobutylicum, fermentations were carried out with or without CaCO₃ in butanol-containing media or in iron deficient media (under iron limitation, butanol synthesis occurred early and could inhibit xylose uptake). Results showed that an excess of CaCOCaCO₃ could increase butanol tolerance which resulted in an increase in xylose utilization. This positive effect seem to be specific to Ca²⁺- or Mg²⁺-containing compounds, going beyond the buffering effect of carbonate.