Measurement and regulation of the culture reduction state in Clostridium acetobutylicum

With a constant glucose feed concentration, the change in the continuous culture dillution rate resulted in an altered fermentation profile and the cellular NADH content. The cultures growing at high dillution rates demonstrated an oxidative metabolism low NADH and butanol concentrations. The low specific NADH flourescence (F/X) at high butanol production rates suggested that a rapid regeneration of NADH to NAD is essential for a high solventogenic culture activity. The culture florescence and butanol concentration remained constant in the solventogenic dilution rate range of D = 0.05-0.2 h(-1) with an inverse relationship between the specific flourescence (F/X) and the specific butanol production rate, q(B). Flourometric NADH observations were confirmed by enzymatic NADH determination. The stiochiometric "Fermentation Equation" was used to check the experimental data consistency and to investigate the role of the available biosynthetic and reduction energy on the culture metabolic activities under different growth conditions. The butanol concentration in the broth was stabilized in a fed-batch process when the culture NADH fluorescence was being controlled through the addition of fresh medium.     

Physiological Events in Clostridium acetobutylicum during the Shift from Acidogenesis to Solventogenesis in Continuous Culture and Presentation of a Model for Shift Induction

The pH of continuous cultures of Clostridium acetobutylicum growing at pH 5.6 was allowed to decrease to 4.3 after acid production and thereby to shift the cultures from acetate and butyrate to acetone and butanol formation. Several parameters were determined during the shift. An increase in the intracellular acid concentration to 440 mM was recorded. An excess of undissociated butyric acid but not of acetic acid just before the shift to solventogenesis was followed by a decline in acid production and subsequently by the uptake of acids. The intracellular ATP concentration reached a minimum before the onset of solventogenesis; this presumably reflects the ATP-consuming proton extrusion connected with the increase in the ΔpH from 0.7 to 1.4 units. The pool of NADH plus NADPH exhibited a drastic increase until solventogenesis was induced. The changes in the ATP and ADP and NADH plus NADPH pools during these pH shift experiments were the beginning of a stable metabolic oscillation which could also be recorded as an oscillation of the culture redox potential under steady-state solventogenic conditions. Similar changes were observed when the shift was induced by the addition of butyrate and acetate (50 mM each) to the continuous culture. However, when methyl viologen was added, important differences were found: ATP levels did not reach a minimum, acetoacetate decarboxylase activity could not be measured, and butanol but not acetone was produced. A model for the shift is proposed; it assumes the generation of two signals, one by the changed ATP and ADP levels and the other by the increased NAD(P)H level.     

On-line fluorescence measurements in assessing culture metabolic activities

Summary NADH fluorescence aided by a stoichiometric metabolic pathway model and culture dynamics was used to elucidate the unobservable intracellular physiological state in two metabolically different phases during culture of Clostridium acetobutylicum. The validity of the theoretical model was examined over a range of culture pH regimes and initial sugar concentrations. The H₂/CO₂ gas concentration ratio was found to be an important process parameter. NADH fluorescence detection was compared with simultaneous enzymatic measurements. The specific fluorescence (fluorescence per biomass, F/X) provided a distinction between oxidative and reductive culture metabolism independent of the pH or substrate concentration changes. A good indicator of the type of culture activity proved to be the dF/dt parameter. The net fluorescence measurements correlated with butanol accumulation under all growth conditions suggesting the possible use of the fluorescence probe as a butanol probe in this fermentation.     

Altered Electron Flow in Continuous Cultures of Clostridium acetobutylicum Induced by Viologen Dyes

The physiological response of Clostridium acetobutylicum to methyl and benzyl viologen was investigated. Viologen dyes at low concentrations (at levels of parts per million [micrograms per milliliter]) caused significant metabolic shifts. Altered electron flow appeared to direct carbon flow from acid to alcohol production accompanied by decreased hydrogen evolution. Reducing equivalents normally released as free hydrogen were directed toward formation of NADH which, in turn, resulted in increased alcohol production. In addition, it was shown that solvent production can take place at pH 6.3. Contrary to previous reports, butanol production appears to be independent of high levels of acetate-butyrate and glucose.     

Biobutanol production in a Clostridium acetobutylicum biofilm reactor integrated with simultaneous product recovery by adsorption

Background

Clostridium acetobutylicum can propagate on fibrous matrices and form biofilms that have improved butanol tolerance and a high fermentation rate and can be repeatedly used. Previously, a novel macroporous resin, KA-I, was synthesized in our laboratory and was demonstrated to be a good adsorbent with high selectivity and capacity for butanol recovery from a model solution. Based on these results, we aimed to develop a process integrating a biofilm reactor with simultaneous product recovery using the KA-I resin to maximize the production efficiency of biobutanol.

Results

KA-I showed great affinity for butanol and butyrate and could selectively enhance acetoin production at the expense of acetone during the fermentation. The biofilm reactor exhibited high productivity with considerably low broth turbidity during repeated batch fermentations. By maintaining the butanol level above 6.5 g/L in the biofilm reactor, butyrate adsorption by the KA-I resin was effectively reduced. Co-adsorption of acetone by the resin improved the fermentation performance. By redox modulation with methyl viologen (MV), the butanol-acetone ratio and the total product yield increased. An equivalent solvent titer of 96.5 to 130.7 g/L was achieved with a productivity of 1.0 to 1.5 g?·?L^-1?·?h^-1. The solvent concentration and productivity increased by 4 to 6-fold and 3 to 5-fold, respectively, compared to traditional batch fermentation using planktonic culture.

Conclusions

Compared to the conventional process, the integrated process dramatically improved the productivity and reduced the energy consumption as well as water usage in biobutanol production. While genetic engineering focuses on strain improvement to enhance butanol production, process development can fully exploit the productivity of a strain and maximize the production efficiency.     

Evaluation of high butanol/acetone ratios in ABE fermentations with cassava by graph theory and NADH regeneration analysis

Higher butanol/acetone ratio is always desirable in ABE fermentation, and this ratio is closely associated with the complicated patterns of metabolic reactions and NADH generation rate. The patterns of acetate/butyrate formation and re-assimilation in multiple closed reaction loops, as well as NADH regeneration in ABE fermentation using different substrates varies. In this study, we evaluated butanol/acetone ratio in ABE fermentations utilizing cassava and corn based media by graph theory and NADH regeneration analysis. The theoretical calculations and experimental data revealed that a lower metabolic strength in butyrate loop and enhanced NADH generation rate were responsible for the achievement of higher butanol/acetone ratio when fermenting cassava based substrate. In traditional fermentations and extractive fermentations with oleyl alcohol/bio-diesel as the extractants when using cassava based substrate, butanol/acetone ratios reached 2.24, 2.84, and 2.19 with the increasing increments of 14.9, 61.4, and 6.8% respectively, while butanol productivities stayed at comparably high levels as compared with those of the fermentations when cultivating on corn based substrate.     

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。     

An on-line physiological state recognition system for the lysine fermentation process based on a metabolic reaction model

A metabolic reaction model was developed for the lysine fermentation process by Corynebacterium glutamicum AJ-3462 to estimate the physiological state of the cells-that is, the growth and production activity, and the flux distribution of metabolites-from on-line measurable rates only. First, the extended Kalman filter was applied to eliminate noise in the measured rates. Then, using the metabolic reaction model, the lysine production rate and flux distribution were calculated. The estimation results allowed the physiological state of lysine production to be recognized, and an appropriate measure corresponding to the estimated state, such as intermittent addition of glucose and/or leucine, to be taken to maintain a high level of lysine productivity in batch culture. Finally, application of the recognition system enabled lysine to be produced from glucose at a higher yield than that from glucose- or leucine-limited exponential fed-batch cultures. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 170-181, 1997.     

Redox interactions between catalase and alcohol dehydrogenase pathways of ethanol metabolism in the perfused rat liver

Alcohol metabolism via alcohol dehydrogenase (ADH) and catalase was studied in perfused rat livers by measuring the oxidation of methanol and butanol, selective substrates for catalase and ADH, respectively. In livers from fasted rats, basal rates of methanol uptake of 15 +/- 1 mumol/g/h were decreased significantly to 8 +/- 2 mumol/g/h by addition of butanol. Concomitantly, pyridine nucleotide fluorescence detected from the liver surface was increased by butanol but not methanol. Both effects of butanol were blocked by an inhibitor of ADH, 4-methylpyrazole, consistent with the hypothesis that elevation of the NADH redox state by butanol inhibited H2O2 production via NAD+-requiring peroxisomal beta-oxidation, leading indirectly to diminished rates of catalase-dependent methanol uptake. In support of this idea, both butanol and butyraldehyde inhibited H2O2 generation. The NADH redox state was also elevated by xylitol, causing a 75% decrease in rates of methanol uptake by livers from fasted rats. This effect was not observed in livers from fed rats unless malate-aspartate shuttle activity was reduced by infusion of the transaminase inhibitor aminooxyacetate. Taken together, these data indicate that generation of reducing equivalents from ADH in the cytosol inhibits H2O2 generation leading to significantly diminished rates of peroxidation of alcohols via catalase. This phenomenon may represent an important physiological mechanism of regulation of ethanol oxidation in intact cells.     

Metabolic flexibility of Clostridium acetobutylicum in response to methyl viologen addition

Batch cultures of Clostridium acetobutylicum, were examined with 0, 0.1 and 1 mM methyl viologen addition at four different controlled pH values (between 4.5 and 6.5). Methyl viologen addition diverted the electron flow: reducing equivalents normally released as molecular hydrogen were directed towards NAD(P)H formation. Production of butanol, the most reduced non-gaseous product, was sharply increased (0.65 mol/mol glucose) at the expense of acetone and butyric and acetic acids. In addition to butanol and lactate production, NADH excess induced the formation of glycerol, a product that has never been reported to be formed by C. acetobutylicum. Metabolic perturbation brought about by the electron carrier led to a reduction of the growth rate and an increase of the lag phase. A correlation between the shape of the redox potential curve and the switch from an acidogenic to a solventogenic metabolism is reported.     

Metabolic control of Clostridium thermocellum via inhibition of hydrogenase activity and the glucose transport rate

Clostridium thermocellum has the ability to catabolize cellulosic biomass into ethanol, but acetic acid, lactic acid, carbon dioxide, and hydrogen gas (H₂) are also produced. The effect of hydrogenase inhibitors (H₂, carbon monoxide (CO), and methyl viologen) on product selectivity was investigated. The anticipated effect of these hydrogenase inhibitors was to decrease acetate production. However, shifts to ethanol and lactate production are also observed as a function of cultivation conditions. When the sparge gas of cellobiose-limited chemostat cultures was switched from N₂ to H₂, acetate declined, and ethanol production increased 350%. In resting cell suspensions, lactate increased when H₂ or CO was the inhibitor or when the cells were held at elevated hyperbaric pressure (6.8 atm). In contrast, methyl-viologen-treated resting cells produced twice as much ethanol as the other treatments. The relationship of chemostat physiology to methyl viologen inhibition was revealed by glucose transport experiments, in which methyl viologen decreased the rate of glucose transport by 90%. C. thermocellum produces NAD⁺ from NADH by H₂, lactate, and ethanol production. When the hydrogenases were inhibited, the latter two products increased. However, excess substrate availability causes fructose 1,6-diphosphate, the glycolytic intermediate that triggers lactate production, to increase. Compensatory ethanol production was observed when the chemostat fluid dilution rate or methyl viologen decreased substrate transport. This research highlights the complex effects of high concentrations of dissolved gases in fermentation, which are increasingly envisioned in microbial applications of H₂ production for the conversion of synthetic gases to chemicals.     

Enhanced alcohol yields in batch cultures of Clostridium acetobutylicum using a three-electrode potentiometric system with methyl viologen as electron carrier

Summary Batch cultures of Clostridium acetobutylicum at controlled pH values of 5 and 5.5 were carried out in a three-electrode potentiometric system with methyl viologen (1 mM) as electron carrier. Although an irreversible loss of methyl viologen at the electrode surface was observed, a significant increase in alcohol yield was obtained. In comparison to control fermentation with or without methyl viologen addition, the butanol yield improvements were respectively of 7 or 51% at pH 5, and 56 or 467% at pH 5.5.     

Enhanced butanol production in a microbial electrolysis cell by <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis> IB4

Reducing power such as NADH is an essential factor for acetone/butanol/ethanol (ABE) fermentation using Clostridium spp. The objective of this study was to increase available NADH in Clostridium beijerinckii IB4 by a microbial electrolysis cell (MEC) with an electron carrier to enhance butanol production. First of all, a MEC was performed without electron carrier to study the function of cathodic potential applying. Then, various electron carriers were tested, and neutral red (NR)-amended cultures showed an increase of butanol concentration. Optimal NR concentration (0.1 mM) was used to add in a MEC. Electricity stimulated the cell growth obviously and dramatically diminished the fermentation time from 40 to 28 h. NR and electrically reduced NR improved the final butanol concentration and inhibited the acetone generation. In the MEC with NR, the butanol concentration, yield, proportion and productivity were increased by 12.2, 17.4, 7.2 and 60.3 %, respectively. To further understand the mechanisms of NR, cathodic potential applying and electrically reduced NR, NADH and NAD⁺ levels, ATP levels and hydrogen production were determined. NR and electrically reduced NR also improved ATP levels and the ratio of NADH/NAD⁺, whereas they decreased hydrogen production. Thus, the MEC is an efficient method for enhancing the butanol production.     

Alcohol production by Clostridium acetobutylicum induced by methyl viologen

Summary Controlled batch experiments performed withClostridium acetobutylicum show that methyl viologen induces solvent production at near neutral pH. At a pH of 6.8, significant ethanol production was observed in presence of methyl viologen. At pH 5, production of butanol and ethanol are favored at the expense of acetone.     

Comparative study of ferredoxin-linked and oxygen-metabolizing enzymes of trichomonads

1. The activities of pyruvate:methyl viologen oxidoreductase (EC 1.2.7.1), hydrogenase (EC 1.18.99.1), NADH:methyl viologen oxidoreductase (EC 1.6.99.3), NADPH:methyl viologen oxidoreductase (EC 1.6.99.1), NADH oxidase (EC 1.6.99.3) and NADPH oxidase (EC 1.6.99.1) were determined for Trichomonas vaginalis, Tritrichomonas foetus and Trichomitus batrachorum. 2. The three trichomonad species were found to differ significantly, especially with respect to NADH oxidase and NADH:methyl viologen oxidoreductase activities. 3. The species differences in ferredoxin-linked and oxygen-metabolising enzymes may be related to the ways in which the trichomonads are adapted for growth in their respective hosts.     

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.     

Influence of iron,phosphate and methyl viologen on glycerol fermentation of Clostridium butyricum

 The effect of methyl viologen addition, and iron and phosphate limitation on product distribution during glycerol fermentation of Clostridium butyricum DSM 5431 was investigated in continuous culture. Special attention was paid to the gaseous products H₂ and CO₂, which were measured on-line. In all three cases, an increased yield of 1,3-propanediol linked to a decreased hydrogen release was observed, indicating that a higher proportion of electrons was channelled from reduced ferredoxin towards NADH₂ production. The specific substrate consumption rates and the specific production rates revealed that this increase in propanediol yield was not obtained at the expense of glycolysis products but by an increased substrate conversion (overflow metabolism). The acetate/ butyrate ratio during glycerol fermentation was essentially influenced by the availability of iron. It was substantially increased when the culture turned from iron excess to iron-limited conditions. Therefore iron limitation proved to be a suitable means to achieve high 1,3-propanediol yields and to reduce butyrate formation. Received: 29 August 1995 / Accepted: 20 September 1995     

pH-induced gene regulation of solvent production by Clostridium acetobutylicum in continuous culture: Parameter estimation and sporulation modelling

The acetone–butanol (AB) fermentation process in the anaerobic endospore-forming Gram-positive bacterium Clostridium acetobutylicum is useful as a producer of biofuels, particularly butanol. Recent work has concentrated on trying to improve the efficiency of the fermentation method, either through changes in the environmental conditions or by modifying the genome to selectively favour the production of one particular solvent over others. Fermentation of glucose by C. acetobutylicum occurs in two stages: initially the acids acetate and butyrate are produced and excreted and then, as the external pH falls, acetate and butyrate are ingested and further metabolised into the solvents acetone, butanol and ethanol. In order to optimise butanol production, it is important to understand how pH affects the enzyme-controlled reactions in the metabolism process. We adapt an ordinary differential equation model of the metabolic network with regulation at the genetic level for the required enzymes; parametrising the model using experimental data generated from continuous culture, we improve on previous point predictions (S. Haus, S. Jabbari, T. Millat, H. Janssen, R.-J. Fisher, H. Bahl, J. R. King, O. Wolkenhauer, A systems biology approach to investigate the effect of pH-induced gene regulation on solvent production by Clostridium acetobutylicum in continuous culture, BMC Systems Biology 5 (2011)) [1] both by using a different optimisation approach and by computing confidence intervals and correlation coefficients. We find in particular that the parameters are ill-determined from the data and that two separate clusters of parameters appear correlated, reflecting the importance of two metabolic intermediates. We extend the model further to include another aspect of the clostridial survival mechanism, sporulation, and by computation of the Akaike Information Criterion values find that the there is some evidence for the presence of sporulation during the shift.