Solventogenic Phase Research Articles

Bioenergy II: An Assessment of the Kinetics of Butanol Production by Clostridium acetobutylicum

Results of a research activity aiming at investigating the feasibility of the acetone-butanol-ethanol (ABE) production by Clostridium acetobutylicum ATCC824 are reported. The kinetics of the ABE production process by free C. acetobutylicum in batch reactors has been assessed. A CSTR equipped with a microfiltration unit has been set-up and operated to assess the kinetics in the solventogenic phase. Solutions of lactose—concentration ranging between 10 and 100 g/L—were adopted as medium with the aim of emulating cheese whey. The conversion process was characterized in terms of cells, acids, solvents, pH and total organic compounds as a function of time. Tests carried out under batch conditions show that: i) cells growth is constant for lactose concentration (CL) smaller than 100 g/L; ii) the butanol specific production rate - estimated at the onset of the solventogenic phase - as a function of CL may be represented by a Monod-like kinetics; iii) the lactose conversion—measured at the end of the solventogenesis phase—decreases with CL; iv) the selectivity of butanol with respect to total solvents increases with CL and stabilizes at about 72%W for CL larger than 30 g/L. Preliminary tests carried out under continuous conditions show that the process takes advantage from the steady state operation under solventogenic conditions. In particular, the butanol conversion rate is larger in the CSTR than in the STR, despite the lower final lactose concentration experienced in the CSTR.

Semi-continuous propagation and synchronous differentiation of hyper-solventogenic cells of Clostridium thermosaccharolyticum in a xylan medium at 56 °C

Synchronized sporangia of Clostridium thermosaccharolyticum were prepared by diluting a Basal Xylan (BX) culture 10 times at each generation for two generations followed by 100 times dilution for unrestricted growth in a BX medium enriched with Ca-gluconate or CaCO 3. This was a technique that enhanced the starter in which division synchrony and mass synchrony once established for synchrony in differentiation continued for almost 10 cycles, as long as the O 2 purging was provided. Following the multi-step growth that correlated with the repetitive spikes of calcium incorporation up to 3.8 × 10 −4 nmol/cell, cell density approached the stationary phase. These synchronized sporangia were used as a starter in the semi-continuous operation by repeating 50% draw-offs at each generation in the presence of 50 ppm O 2 for nine generations, during which the percentage of sporangia increased from 60% to 88%. Traditional binary fission (TBF) of elongated sporangia (longer than 4×) must then be stopped by the addition of chemical growth inhibitors or temperature shift-down to 35 °C for steep solventogenesis that also promoted an increase of sporangia to 99%. It was evident that the stressful conditions were essential in the propagation process, and for retaining the highest percentage of sporangia, which had to be arrested at the solventogenic phase (without acidogenesis) to reach 7.05% final concentration in 27 h at 56 °C, or 10.57% in 105 h at 35 °C, giving a recovery yield of 97.9%. Differentiation and modified division without TBF required O 2 purging to continue.

Genome-scale reconstruction and in silico analysis of the Clostridium acetobutylicum ATCC 824 metabolic network

To understand the metabolic characteristics of Clostridium acetobutylicum and to examine the potential for enhanced butanol production, we reconstructed the genome-scale metabolic network from its annotated genomic sequence and analyzed strategies to improve its butanol production. The generated reconstructed network consists of 502 reactions and 479 metabolites and was used as the basis for an in silico model that could compute metabolic and growth performance for comparison with fermentation data. The in silico model successfully predicted metabolic fluxes during the acidogenic phase using classical flux balance analysis. Nonlinear programming was used to predict metabolic fluxes during the solventogenic phase. In addition, essential genes were predicted via single gene deletion studies. This genome-scale in silico metabolic model of C. acetobutylicum should be useful for genome-wide metabolic analysis as well as strain development for improving production of biochemicals, including butanol.

Metabolic engineering for solvent productivity by downregulation of the hydrogenase gene cluster hupCBA in Clostridium saccharoperbutylacetonicum strain N1-4

The selective production of acetone and butanol is highly desirable from the viewpoint of biofuel production. We have manipulated the activity level of a hydrogenase for this purpose because hydrogen and solvent production are closely correlated with each other. First, we cloned the hydrogenase gene cluster from Clostridium saccharoperbutylacetonicum strain N1-4 and downregulated its expression using an antisense RNA strategy. The cloned hydrogenase gene cluster contained three adjacent open reading frames, designated hupC, hupB, and hupA. Sequence analysis revealed that HupA could accommodate an H-cluster, which is the catalytic domain of the Fe-hydrogenase. HupB and HupC contained no H-cluster but could accommodate several Fe-S clusters. The hupCBA genes were co-transcribed, and the level of the transcript was maximized in the solventogenic phase. When the antisense RNA of the hupC upstream region (180 bp) was expressed under the bdh (encoding butanol dehydrogenase) promoter, significant reduction of hupC translation was observed, indicating that this antisense RNA is effective in strain N1-4. Production of hydrogen in the antisense transformant increased 3.1-fold. Hydrogen-evolving activity was comparable in both the control and antisense strains, but hydrogen uptake activity significantly decreased in the antisense strain (13% remaining). These results indicate that the HupCBA proteins are involved in hydrogen uptake. Importantly, the level of acetone in the antisense transformant increased 1.6-fold, and butanol production decreased to 75.6% compared to the control strain. Thus, we successfully altered solvent productivity by controlling electron flow in an acetone/butanol-producing Clostridium species.

The profile of enzymes relevant to solvent production during direct fermentation of sago starch by Clostridium saccharobutylicum P262 utilizing different pH control strategies

The profile of enzymes relevant to solvent production during direct fermentation of sago starch by Clostridium saccharobutylicum P262 in a 2 L stirred tank fermenter was determined utilizing different pH control strategies. During fermentation without pH control (initial pH of 6), the specific activity of crotonase, thiolase, and β-hydroxybutyryl-CoA dehydrogenase increased proportionally with solvent production. The highest crotonase (3,450.7 kat) and phosphotransbutyrylase activity (1,475.6 kat) was observed in fermentation where pH was maintained at 5 during the acidogenic phase and corresponded to a fairly high acid accumulation but low solvent production. During fermentation with a controlled pH of 5.25 during the sol-ventogenic phase, the highest thiolase specific activity (255.7 kat) was obtained and corresponded to the highest production of acetone. On the other hand, the highest specific activities of crotonase, β-hydroxybutyryl-CoA dehydrogenase, and phosphotransbutyrylase were observed at pH 5.5 and corresponded to the highest production of ethanol and butanol. Butyryl-CoA dehydrogenase had no significance role in solvent fermentation. These results suggested that pH control strategies were important for improvement of solvent production during direct fermentation of sago starch by C. saccharobutylicum.

Electrooptical measurements for monitoring metabolite fluxes in acetone–butanol–ethanol fermentations

Anisotropy of electrical polarizability in Clostridium acetobutylicum cells during pH 5 controlled acetone butanol ethanol fermentations was observed. Cell length was determined from the electrooptical data. Mean length was determined as being 2.5 microm in the growth phase and 3.5 microm in the early stationary phase. Based on the obtained frequency dispersion of polarizability anisotropy (FDPA) in the range of 190 to 2,100 kHz, the switch from the acidogenic to the solventogenic phase could be monitored. The slope of polarizability versus the frequency made it possible to differentiate between phases of dominating acid and solvent production. Metabolite fluxes determined from concentration measurements correlated well to the polarizability. A partial least-squares (PLS) model was established and validated by applying data from several fermentations. The root mean square error of calibration (RMSEC) was 0.09 for the acid fluxes and 0.11 for the solvent fluxes. The root mean square error of prediction (RMSEP) was 0.20 for acid fluxes and 0.24 for solvent fluxes. The ratio of polarizability at high and low frequencies correlated to the ongoing sporulation process. At ratios below 0.25, spore formation in the cells became visible under the microscope. The advantage of using electrooptical measurements is the ability to observe metabolite fluxes rather than concentrations, which provides useful information on productivity during a bioprocess.

Nitrogen-fixation genes and nitrogenase activity in Clostridium acetobutylicum and Clostridium beijerinckii

Several solvent-producing clostridia, including Clostridium acetobutylicum and C. beijerinckii, were previously shown to be nitrogen-fixing organisms based on the incorporation of 15N2 into cellular material. The key nitrogen-fixation (nif) genes, including nifH, nifD, and nifK for nitrogenase component proteins as well as nifE, nifN, nifB and nifV for synthesis of the iron-molybdenum cofactor (FeMoco) of nitrogenase, have now been identified in C. acetobutylicum or C. beijerinckii or both. The organization of these genes is similar to the distinctive pattern that was first observed in Clostridium pasteurianum, with the nifN and nifB genes fused into the nifN-B gene and with the nifV gene split into the nifVomega and nifValpha genes. The corresponding nif genes of these three clostridial species are highly related to each other. However, in the two solvent-producing clostridia, the nifH and nifD genes are interspersed by two glnB-like genes, which are absent in the corresponding region in C. pasteurianum. However, the nifN-B and nifVomega genes of C. pasteurianum are interspersed by the putative modA and modB genes (for molybdate transport), which are absent in the corresponding region in C. acetobutylicum. C. acetobutylicum and C. beijerinckii grew well under nitrogen-fixing conditions, and the acetylene-reducing activity of nitrogenase was measured in the two species. Acetone, butanol, and isopropanol production occurred in nitrogen-fixing cultures, but the peak of nitrogen-fixing activity preceded the active solventogenic phase.

Development of markers for product formation and cell cycle in batch cultivation of Clostridium acetobutylicum ATCC 824

Experiments were performed to identify relationships between morphological and physiological events during batch fermentation of Clostridium acetobutylicum ATCC 824. Differing fermentation conditions were obtained by manipulation of the culture pH value during the process. The bacterium showed marked changes in morphology during its cultivation, similar to those previously observed for other strains. However, although the acidogenic phase was characterized by the presence of rod-shaped cells, and the solventogenic phase by clostridial forms, there was no simple relationship between the proportion of clostridial forms present and the ratio of solvents to acids. Nevertheless, the shift from acidogenesis to solventogenesis was always coincident with the presence of some clostridial forms and with the accumulation of granulose within the cells. In addition, the “solvent shift” was associated with major changes in the cellular protein pattern, as analysed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Hence, the potential solventogenic ability of any particular culture may be recognised by its morphological appearance and/or its cellular protein pattern.

Regulation of nitrogen metabolism, starch utilisation and the β-hbd—adh1gene cluster inClostridium acetobutylicum

The successful genetic manipulation of Clostridium acetobutylicum for the increased production of solvents will depend on an understanding of gene structure and regulation in the bacterium. The glutamine synthetase (glnA) gene is regulated by antisense RNA, transcribed from a downstream promoter, in the opposite direction to the glnA gene. An open reading frame (ORF) was detected downstream of the glnA gene, which has sequence homology to response regulators with anti-termination activity and may be involved in sensing nitrogen conditions. The expression of the linked beta-hbd, adh1 and fixB genes was investigated throughout the bacterial growth cycle by RNA hybridisation techniques. The adh1 gene was independently expressed as a 2.4-kb transcript which peaked at 12 h, immediately prior to the solventogenic phase. The beta-hbd and fixB genes were transcribed throughout the acidogenic and solventogenic phases. A regulator gene, regA, which complements a Bacillus subtilis ccpA mutant, has been identified and sequenced from C. acetobutylicum P262. The regA gene repressed the degradation of starch by an uncharacterised C. acetobutylicum gene, and may therefore play a role in the utilisation of carbohydrate substrates in this organism.

Structure and transcription of genes within the β- hbd-adh1 region of Clostridium acetobutylicum P262

The 1.2-kb DNA fragment upstream of the linked β- hbd (3-hydroxybutyryl-CoA dehydrogenase) and adh1 (NADPH-dependent alcohol dehydrogenase) genes from Clostridium acetobutylicum P262 was sequenced. The upstream region contained an open reading frame (ORFB) which was found to have 44% amino acid identity to the fixB gene products of yRhizobium and Azorhizobium. The β- hbd and ORFB genes were expressed during the acidogenic and solventogenic phases. The β- hbd gene was transcribed on a single mRNA species of 2.0 kb, whereas the ORFB gene was transcribed on two species of mRNA of 2.0 and 3.5 kb, respectively. The adh1 gene was induced or derepressed at the pH breakpoint before the onset of solventogenesis and was transcribed on a single species of mRNA of 2.4 kb.

Regulation of nitrogen metabolism, starch utilisation and the β- hbd— adh1 gene cluster in Clostridium acetobutylicum

The successful genetic manipulation of Clostridium acetobutylicum for the increased production of solvents will depend on an understanding of gene structure and regulation in the bacterium. The glutamine synthetase ( glnA) gene is regulated by antisense RNA, transcribed from a downstream promoter, in the opposite direction to the glnA gene. An open reading frame (ORF) was detected downstream of the glnA gene, which has sequence homology to response regulators with anti-termination activity and may be involved in sensing nitrogen conditions. The expression of the linked β- hbd, adh1 and fixB genes was investigated throughout the bacterial growth cycle by RNA hybridisation techniques. The adh1 gene was independently expressed as a 2.4-kb transcript which peaked at 12 h, immediately prior to the solventogenic phase. The β- hbd and fixB genes were transcribed throughout the acidogenic and solventogenic phases. A regulator gene, regA, which complements a Bacillus subtilis ccpA mutant, has been identified and sequenced from C. acetobutylicum P262. The regA gene repressed the degradation of starch by an uncharacterised C. acetobutylicum gene, and may therefore play a role in the utilisation of carbohydrate substrates in this organism.

NADH fluorescence in a fed batch fermentation by C.acetobutylicum

The existing process kinetics model of the acetone-butanol fermentation was adjusted and adapted for the fed-batch mode of operation. The optimal sugar feeding strategy for maximum solvent production was determined from the model and implemented in an experiment where the fluorescent features of the culture were also determined. While the model predictions agreed with experimentally observed trends, the actual data did not compare well. The characterisation of the culture physiological state under dynamic growth conditions was attempted by utilizing on-line fluorescence measurements and the concept of stoichiometric metabolic pathway rates. This methodology served well in assessing the NADH related culture metabolic activity and the substrate consumption rate in the solventogenic phase. Le modèle de cinétique des procédés existant pour la fermentation acétone-butanol a été adapté pour une alimentation discontinue. On a déterminé la stratégie d'alimentation en sucre optimale pour une production de solvant maximum à partir du modèle et on l'a appliquée dans une expérience, pour laquelle les caractéristiques de fluorescence de la culture ont également été déterminées. Tandis que les prédictions du modèle concordent bien avec les tendances observées expérimentalement, les données réelles se comparent mal. On a tenté de caractériser l'état physiologique de la culture dans des conditions de croissance dynamiques en utilisant des mesures de fluorescence directes et le concept des vitesses des trajectoires métaboliques stoechiométriques. Cette méthodologie est utile pour l'évaluation de l'activité métabolique des cultures associées au NADH et de la vitesse de consommation du substrat dans la phase solvantogène.

Effects of extractive fermentation on butyric acid production byClostridium acetobutylicum

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 ΔpH ofC. acetobutylicum and its importance with regard to the production of butyric acid.

Comparison of Nucleoside Triphosphate Levels in the Wild-type Strain with Those in Sporulation-deficient and Solvent-deficient Mutants of Clostridium acetobutylicum P262

The role of GTP and other ribonucleoside phosphates in the differentiation of Clostridium acetobutylicum and two sporulation-deficient mutants was investigated. The clostridial stage (cls-1) mutant was unable to produce the clostridial stage, solvents, granulose, capsules or endospores, whereas the sporulation-deficient (spo-1) mutant produced the clostridial stage, solvents, granulose and capsules, but did not form a forespore septum. The nucleoside triphosphate profiles of the wild-type and spo-1 mutant were similar and were characterized by a trough in nucleotide levels which occurred just prior to the pH break point, the onset of the stationary growth phase, clostridial stage formation and the transition from the acidogenic to the solventogenic phase. The nucleoside triphosphate concentrations during the exponential growth phase were much lower than those found during the stationary phase. The nucleotide levels in the cls-1 mutant during the exponential phase were comparable to those observed in the wild-type and spo-1 mutant. However, the nucleotide levels in the cls-1 mutant did not increase during the stationary growth phase. The involvement of nucleotide levels, particularly that of GTP, in the differentiation of C. acetobutylicum was indicated by the effect of inhibitors, which have been shown to decrease ribonucleotide levels in other organisms and cause an increase in sporulation.

Metabolism of adenylylated nucleotides in Clostridium acetobutylicum.

In response to the stresses imposed by temperature upshift or addition of butanol, Clostridium acetobutylicum cultures accumulated diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) and adenosine 5'-P1,P4-tetraphospho-5'-guanosine (Ap4G) to high levels. The two adenylylated nucleotides were also accumulated in batch culture in the absence of imposed stresses when the clostridia switched from the acidogenic phase of growth to the solventogenic phase. Most of the adenylylated nucleotides were extracellular. The intracellular concentrations of these compounds were low throughout batch growth and in cells stressed by added butanol. In contrast to other procaryotes, these clostridia did not possess enzymes to degrade the dinucleotides, as shown with both intact cells and cell-free preparations. Our findings are consistent with the hypothesis that endogenously produced solvents are stressful to the cells, stimulating the synthesis of adenylylated nucleotides. The nucleotides accumulate extracellularly because they cannot be degraded and because the cell membranes are permeabilized by the solvents produced.

Effect of pyruvate on glucose metabolism in Clostridium acetobutylicum

Pyruvate effects on the metabolism of Clostridium acetobutylicum during glucose fermentation were studied. After addition to the culture medium, the pyruvate was rapidly used, provoking several changes in the metabolic pattern of the bacteria. When pyruvate addition occurred early in the fermentation, the glucose utilization decreased and the solventogenic phase was not induced. When pyruvate was added during solventogenesis, glucose consumption was slightly affected and the cells fermented both substrates simultaneously: however, the acidogenic phase started again to the detriment of solvent formation. Usually, during the solvent phase, the cells remetabolized acetic and butyric acids into solvents, but when pyruvate was added, the utilization of acids was stopped and the specific rates of acetate and butyrate formation increased immediately. The acidogenic growth phase was characterized by high levels of acetate and butyrate kinase which dropped during the solvent phase. Addition of pyruvate limited the down shift of these two enzymes and the levels of the activities remained constant during the course of the fermentation. Conversely, the acetoacetate decarboxylase, which is characteristic of the solvent phase, decreased sharply in the presence of pyruvate. The fact that the specific rate of glucose consumption was not decreased by the pyruvate metabolism, a cosubstrate, proves that the phosphoroclastic reaction is not a limiting step. Furthermore, the pyruvate utilization represented a promising approach to obtain useful data on the intracellular compounds implicated in the mechanism for switching from the acidogenic to the solventogenic phase.

Culture fluorescence characteristics and its metabolic significance in batch cultures of Clostridium acetobutylicum

Qualitative characteristics of changes in culture fluorescence during batch growth ofClostridium acetobutylicum [ATCC 824] have been determined. A solventogenic phase culture shows higher changes in normalized fluorescence levels per unit change in cell concentration compared with an acidogenic phase culture. This suggests that during solvent formation intracellular NADH levels are higher than in the acidogenic phase and that NADH availability may influence the onset of solventogenesis.

Phosphotransferase Activity in Clostridium acetobutylicum from Acidogenic and Solventogenic Phases of Growth

Clostridium acetobutylicum cells, when energized with fructose, transported and phosphorylated the glucose analog 2-deoxyglucose by a phosphoenolpyruvate-dependent phosphotransferase (PT) system. Butanol up to 2% did not inhibit PT activity, although its chaotropic effect on the cell membrane caused cellular phosphoenolpyruvate and the 2-deoxyglucose-6-phosphate to leak out. Cells harvested from the solventogenic phase of batch growth had a significantly lower PT activity than did cells from the acidogenic phase.

Immobilized Clostridium acetobutylicum P262 Mutants for Solvent Production

The production of acetone, butanol, and ethanol by two immobilized, sporulation-deficient (spo) Clostridium acetobutylicum P262 mutants which were held in the solventogenic phase was investigated. The spoA2 mutant, which was an early-sporulation mutant and did not form a forespore septum, produced higher solvent yields than did the spoB mutant which was a late-sporulation mutant and was blocked at a stage after forespore septum formation. The spoA2 mutant was also granulose and capsule negative. In a conventional batch fermentation, the wild-type strain produced 15.44 g of solvents per liter after 50 h at a productivity of 7.41 g of solvents per liter per day. The spoA2 mutant produced 15.42 g of solvents per liter at a productivity of 72.4 g of solvents per liter per day, with a retention time of 2.4 h in a continuous immobilized cell system employing a fluidized bed reactor. This represents a major advance, since the immobilization of wild-type cells showed similar increases in productivity but a ca. fivefold reduction in final product concentrations.