Continuous Cultures Of Clostridium Acetobutylicum Research Articles

A shift in the dominant phenotype governs the pH-induced metabolic switch of Clostridium acetobutylicumin phosphate-limited continuous cultures

In response to changing extracellular pH levels, phosphate-limited continuous cultures of Clostridium acetobutylicum reversibly switches its metabolism from the dominant formation of acids to the prevalent production of solvents. Previous experimental and theoretical studies have revealed that this pH-induced metabolic switch involves a rearrangement of the intracellular transcriptomic, proteomic and metabolomic composition of the clostridial cells. However, the influence of the population dynamics on the observations reported has so far been neglected. Here, we present a method for linking the pH shift, clostridial growth and the acetone-butanol-ethanol fermentation metabolic network systematically into a model which combines the dynamics of the external pH and optical density with a metabolic model. Furthermore, the recently found antagonistic expression pattern of the aldehyde/alcohol dehydrogenases AdhE1/2 and pH-dependent enzyme activities have been included into this combined model. Our model predictions reveal that the pH-induced metabolic shift under these experimental conditions is governed by a phenotypic switch of predominantly acidogenic subpopulation towards a predominantly solventogenic subpopulation. This model-driven explanation of the pH-induced shift from acidogenesis to solventogenesis by population dynamics casts an entirely new light on the clostridial response to changing pH levels. Moreover, the results presented here underline that pH-dependent growth and pH-dependent specific enzymatic activity play a crucial role in this adaptation. In particular, the behaviour of AdhE1 and AdhE2 seems to be the key factor for the product formation of the two phenotypes, their pH-dependent growth, and thus, the pH-induced metabolic switch in C. acetobutylicum.

Continuous two stage acetone–butanol–ethanol fermentation with integrated solvent removal using Clostridium acetobutylicum B 5313

The objective of this study was to optimize continuous acetone–butanol–ethanol (ABE) fermentation using a two stage chemostat system integrated with liquid–liquid extraction of solvents produced in the first stage. This minimized end product inhibition by butanol and subsequently enhanced glucose utilization and solvent production in continuous cultures of Clostridium acetobutylicum B 5313. During continuous two-stage ABE fermentation, sugarcane bagasse was used as the cell holding material for the both stages and liquid–liquid extraction was performed using an oleyl alcohol and decanol mixture. An overall solvent production of 25.32 g/L (acetone 5.93 g/L, butanol 16.90 g/L and ethanol 2.48 g/L) was observed as compared to 15.98 g/L in the single stage chemostat with highest solvent productivity and solvent yield of 2.5 g/L h and of 0.35 g/g, respectively. Maximum glucose utilization (83.21%) at a dilution rate of 0.05 1/h was observed as compared to 54.38% in the single stage chemostat.

Genome-Wide Gene Expression Analysis of the Switch between Acidogenesis and Solventogenesis in Continuous Cultures of Clostridium acetobutylicum

Clostridium acetobutylicum is able to switch from acidogenic growth to solventogenic growth. We used phosphate-limited continuous cultures that established acidogenic growth at pH 5.8 and solventogenic growth at pH 4.5. These cultures allowed a detailed transcriptomic study of the switch from acidogenesis to solventogenesis that is not superimposed by sporulation and other growth phase-dependent parameters. These experiments led to new insights into the physiological role of several genes involved in solvent formation. The adc gene for acetone decarboxylase is upregulated well before the rest of the sol locus during the switch, and pyruvate decarboxylase is induced exclusively for the period of this switch. The aldehyde-alcohol dehydrogenase gene adhE1 located in the sol operon is regulated antagonistically to the paralog adhE2 that is expressed during acidogenic conditions. A similar antagonistic pattern can be seen with the two paralogs of thiolase genes, thlA and thlB. Interestingly, the genes coding for the putative cellulosome in C. acetobutylicum are exclusively transcribed throughout solventogenic growth. The genes for stress response are only induced during the shift but not in the course of solventogenesis when butanol is present in the culture. Finally, the data clearly indicate that solventogenesis is independent from sporulation.

Continuous production of butanol by Clostridium acetobutylicum immobilized in a fibrous bed bioreactor.

We explored the influence of dilution rate and pH in continuous cultures of Clostridium acetobutylicum. A 200-mL fibrous bed bioreactor was used to produce high cell density and butyrate concentrations at pH 5.4 and 35degreesC. By feeding glucose and butyrate as a cosubstrate, the fermentation was maintained in the solventogenesis phase, and the optimal butanol productivity of 4.6 g/(L h) and a yield of 0.42 g/g were obtained at a dilution rate of 0.9 h-1 and pH 4.3. Compared to the conventional acetone-butanol-ethanol fermentation, the new fermentation process greatly improved butanol yield, making butanol production from corn an attractive alternative to ethanol fermentation.

Regulation of metabolic shifts inClostridium acetobutylicumATCC 824

Abstract Alcohol formation was initiated in continuous cultures of Clostridium acetobutylicum under distinct steady-state conditions: (i) in glucose-limited cultures established at low operating pH with formation of butanol, ethanol and acetone (induction of the solventogenesis) in which cells contained normal levels of NADH and a high level of ATP and butyric acid; and (ii) by increasing the NADH pressure at neutral pH in glucose-limited cultures after addition of Neutral red, or in glucose-glycerol or glucose-glycerol-pyruvate grown cultures, with a strictly alcohologenic metabolism (no acetone produced) associated with high levels of intracellular NADH and various levels of ATP. These two different metabolic shift systems are correlated with the expression of different genes involved in the solvent-forming pathways and the electron flow distribution. A high NADH level leading to butanol and ethanol formation was accompanied by increased activities of the NADH-dependent alcohol and butyraldehyde dehydrogenases, and ferredoxin:NAD(P) + reductases, and by decreased activities of the NADH:ferredoxin reductase. This last group of enzymes constitutes the key enzymes regulating electron flow, since no change in hydrogenase activity was observed. On the other hand, classical solventogenesis appears to be characterized by high levels of expression of the NADPH-dependent alcohol and butyraldehyde dehydrogenases, and of the two enzymes involved in the acetone-forming pathway, while the ferredoxin:NAD(P) + reductases were not synthesized. A decrease of the in vitro hydrogenase activity explains the lower hydrogen generation. In addition, the regulation of the intracellular pH was different between the alcohologenic culture grown at neutral pH and the solventogenic cultures grown at low pH. An inversion of the transmembrane pH gradient was observed during the production of alcohol at neutral pH and was related to a lower in vivo specific rate of hydrogen production while in the cultures grown at low pH the transmembrane pH generation was not linked to the F 1 F 0 ATPase activity.

Differential induction of genes related to solvent formation during the shift from acidogenesis to solventogenesis in continuous culture of Clostridium acetobutylicum

The expression of all sequenced acetone and butanol formation genes was followed using mRNA analysis during the shift from acidogenesis to solventogenesis in continuous culture of Clostridium acetobutylicum. Differential induction or derepression of the bdhA, bdhB, and adc genes as well as the sol operon was observed during the pH-induced shift. The order of induction of the three different butanol dehydrogenase genes was found to be bdhA-sol operon- bdhB, offering an explanation for the physiological role of the respective enzymes. Peak mRNA synthesis of an autolysin and a heat shock gene at the onset of solventogenesis was detected in addition to the above-mentioned genes. None of the hitherto sequenced genes of butanologenic enzymes was found to be involved in butanol production during the Methyl viologen-induced shift, indicating the presence of yet unknown genes encoding alcohol and aldehyde dehydrogenases.

Carbon dioxide desorption from fermentation broth by use of oxygen vectors

The ability of oxygen vector to extract produced carbon dioxide has been tested in an anaerobic fermentation. During the continuous culture of Clostridium acetobutylicum at pH 4.6 and at a dilution rate of 0.124 h−1, a feed composed of an emulsion of 18.5% by volume of Forane F66E was able to extract about 9% of the total CO2 produced under CO2 partial pressure equal to 0.42 atm. A theoretical evaluation of the extracted amount, based on the hypothesis of total saturation of the vector by carbon dioxide, has lead to very good agreement.

Regulation of metabolic shifts in Clostridium acetobutylicum ATCC 824

Alcohol formation was initiated in continuous cultures of Clostridium acetobutylicum under distinct steady-state conditions: (i) in glucose-limited cultures established at low operating pH with formation of butanol, ethanol and acetone (induction of the solventogenesis) in which cells contained normal levels of NADH and a high level of ATP and butyric acid; and (ii) by increasing the NADH pressure at neutral pH in glucose-limited cultures after addition of Neutral red, or in glucose-glycerol or glucose-glycerol-pyruvate grown cultures, with a strictly alcohologenic metabolism (no acetone produced) associated with high levels of intracellular NADH and various levels of ATP. These two different metabolic shift systems are correlated with the expression of different genes involved in the solvent-forming pathways and the electron flow distribution. A high NADH level leading to butanol and ethanol formation was accompanied by increased activities of the NADH-dependent alcohol and butyraldehyde dehydrogenases, and ferredoxin:NAD(P) + reductases, and by decreased activities of the NADH:ferredoxin reductase. This last group of enzymes constitutes the key enzymes regulating electron flow, since no change in hydrogenase activity was observed. On the other hand, classical solventogenesis appears to be characterized by high levels of expression of the NADPH-dependent alcohol and butyraldehyde dehydrogenases, and of the two enzymes involved in the acetone-forming pathway, while the ferredoxin:NAD(P) + reductases were not synthesized. A decrease of the in vitro hydrogenase activity explains the lower hydrogen generation. In addition, the regulation of the intracellular pH was different between the alcohologenic culture grown at neutral pH and the solventogenic cultures grown at low pH. An inversion of the transmembrane pH gradient was observed during the production of alcohol at neutral pH and was related to a lower in vivo specific rate of hydrogen production while in the cultures grown at low pH the transmembrane pH generation was not linked to the F 1F 0 ATPase activity.

Differential induction of genes related to solvent formation during the shift from acidogenesis to solventogenesis in continuous culture ofClostridium acetobutylicum

The expression of all sequenced acetone and butanol formation genes was followed using mRNA analysis during the shift from acidogenesis to solventogenesis in continuous culture of Clostridium acetobutylicum. Differential induction or derepression of the bdhA, bdhB, and adc genes as well as the sol operon was observed during the pH-induced shift. The order of induction of the three different butanol dehydrogenase genes was found to be bdhA-sol operon-bdhB, offering an explanation for the physiological role of the respective enzymes. Peak mRNA synthesis of an autolysin and a heat shock gene at the onset of solventogenesis was detected in addition to the above-mentioned genes. None of the hitherto sequenced genes of butanologenic enzymes was found to be involved in butanol production during the Methyl viologen-induced shift, indicating the presence of yet unknown genes encoding alcohol and aldehyde dehydrogenases.

Production of acetone and butanol from starch by continuous bioprocess

Conversions of saccharified and non-saccharified starch to acetone, butanol and ethanol (ABE) with a continuous culture of Clostridium acetobutylicum were investigated and compared with regard to products and productivity. It was found, that compared with the microbial process using saccharified starch, the process using non-saccharified starch produced ABE in considerably lower concentrations at dilution rates of >0.15 h −1. Two-stage continuous bioprocess using non-saccharified starch resulted in no appreciable increase in acetone and butanol concentrations. For the reasons given above, preliminary saccharification processing is recommended for the production of ABE from starch using a continuous bioprocess. A two-stage process was developed for continuous production of ABE from saccharified starch. By this process, 15 g l −1 ABE are produced at the rate of 3.3 g l −1 h −1.

Acid in the first stage is a determinant factor for the solvent production in the two-stage continuous fermentation ofClostridium acetobutylicum

To maximise solvent production in a two-stage continuous culture ofClostridium acetobutylicum, a low concentration of solvents had to be maintained in the first vessel. A 19 g/l solvent concentration was achieved, if only 2 g/l acids were present in the first stage. The second stage was dependent on the metabolic state of the cells produced in the first fermentor.

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

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.

Increased levels of ATP and NADH are associated with increased solvent production in continuous cultures of Clostridium acetobutylicum

Levels of intracellular NAD, NADH, ADP, and ATP were measured in batch and continuous cultures of Clostridium acetobutylicum. ATP levels during glucose-sufficient (non-glucose limited), solvent-producing steady states were five to eight times as high as in glucose-limited (acidogenic) steady state cultures (4.8 μmole/g versus 0.9 μmole/g). NADH was also higher in glucose-sufficient cultures, although the increase was not as pronounced as with ATP. When glucose-limited cultures were sparged with CO, the resulting shift to solvent production was accompanied by three- or four-fold increases in NADH and ATP levels. In batch cultures, NADH and ATP levels were relatively high at all times compared to levels in continuous cultures. Although the levels of these nucleotides showed systematic trends during normal batch fermentations, there was no significant change with the onset of solvent production.

Circadian rhythmicity in a fermentation process?

An idea is proposed for the role of the circadian rhythmicity in the control of the oscillatory behavior observed in the growth and product formation during the cell-retention continuous culture of Clostridium acetobutylicum. C. acetobutylicum is highly sensitive to the permeability of the cell membrane. A physical mechanism for the variability of the cytoplasmic membrane has been proposed suggesting that the performance of the cell membrane, due to its liquid crystalline structure, is influenced by the external forces (e.g. earth's magnetic field). A previously developed Physiological State Model was extended by incorporating the effect of external forces on the cell membrane permeability. The new mathematical model could simulate the observed oscillatory behavior of the microbial culture. Some experimental results in support of the theoretical predictions have been presented.

Continuous and biomass recycle fermentations of Clostridium acetobutylicum

The availability and demand of biosynthetic energy (ATP) is an important factor in the regulation of solvent production in steady state continuous cultures of Clostridium acetobutylicum. The effect of biomass recycle at a variety of dilution rates and recycle ratios under both glucose and non-glucose limited conditions on product yields and selectivities has been investigated. Under conditions of non-glucose limitation, when the ATP supply is not growth-limiting, a lower growth rate imposed by biomass recycle leads to a reduced demand for ATP and substantially higher acetone and butanol yields. When the culture is glucose limited, however, biomass recycle results in lower solvent yields and higher acid yields.

Improved stability of the continuous production of acetone-butanol by Clostridium acetobutylicum in a two-stage process

A stable continuous culture has been maintained for 30 days at a high 20 g/l solvent concentration. This substantial increase in the stability of the continuous culture ofClostridium acetobutylicum at the maximal solvent level was achieved by using a two-stage process with a dilution rate of 0.1 h−1 in the first fermentor and 0.04 h−1 in the second fermentor. The two-stage continuous fermentation allows an optimal growth of cells and induction of solvent metabolism in the first stage, and a maximal production yield of solvents in the second stage.

Directed metabolic flow with high butanol yield and selectivity in continuous cultures ofClostridium acetobutylicum

This work addresses the problem of stable butanol formation byClostridium acetobutylicum in continuous culture. Sustained altered electron flow was observed in the presence of benzyl viologen which serves to redirect carbon flow towards primarily butanol formation. A yield of butanol of over 0.28 g.g−1 glucose was obtained and butanol comprised over 90% of the total solvents formed. Additionally, acid formation decreased significantly with butyric acid as the dominant acid end product.

Carbon monoxide gasing leads to alcohol production and butyrate uptake without acetone formation in continuous cultures of Clostridium acetobutylicum

When continuous, steady-state, glucose-limited cultures ofClostridium acetobutylicum were sparged with CO, the completely or almost completely acidogenic fermentations became solventogenic. Alcohol (butanol and ethanol) and lactate production at very high specific production rates were initiated and sustained without acetone, and little or no acetate and butyrate formation. In one fermentation, strong butyrate uptake without acetone formation was observed. Growth could be sustained even with 100% inhibition of H2 formation. Although CO gasing inhibited growth up to 50%, and H2 formation up to 100%, it enhanced the rate of glucose uptake up to 300%. TheYATP was strongly affected and mostly reduced with respect to its steady-state value. The results support the hypothesis that solvent formation is triggered by an altered electron flow.

Carbon monoxide gasing leads to alcohol production and butyrate uptake without acetone formation in continuous cultures ofClostridium acetobutylicum

Carbon monoxide gasing leads to alcohol production and butyrate uptake without acetone formation in continuous cultures ofClostridium acetobutylicum

Carbon monoxide gasing leads to alcohol production and butyrate uptake without acetone formation in continuous cultures ofClostridium acetobutylicum

Carbon monoxide gasing leads to alcohol production and butyrate uptake without acetone formation in continuous cultures ofClostridium acetobutylicum