Cells Of Clostridium Acetobutylicum Research Articles

A novel integrated fermentation/recovery system for butanol production by Clostridium acetobutylicum

Intensive butanol production was carried out by immobilized cells of Clostridium acetobutylicum in a novel continuous fermentation system. The fermentation system consisted of four packed bed biofilm reactors (PBBR) connected in series. The novelty of the proposed system is: i) the use of a novel stagewise simulated moving bed (SMB) design/operational mode; ii) the integration of the reaction and the product recovery sections. The SMB operational mode was accomplished by equipping the PBBR with manifolds and time-actuated switching valves. PBBR integration with the product recovery section was also tested. An adsorption column was operated between the 3rd and 4th reactor of the series.The tuning of the PBBR system overall dilution rate (DOV) allowed to maximize: i) sugar conversion (DOV <0.150 h−1); ii) butanol concentration (0.650 h−1<DOV< 0.900 h−1); iii) butanol productivity (13.0 g/Lh at DOV = 0.900 h−1).The PBBR system was upgraded by integrating it with an in-line adsorption column and it guaranteed a twofold increase in butanol productivity (up to 22 g/Lh) and an average butanol concentration close to 24.0 g/L.

Fed-batch acetone-butanol-ethanol fermentation using immobilized Clostridium acetobutylicum in calcium alginate beads

Butanol fermentation has garnered renewed interest due to the search for renewable sources of energy. Although acetone-butanol-ethanol (ABE) fermentation has been studied for a long time, the methods utilized yield either a high butanol/ABE productivity and low titer or high butanol/ABE titer at a low productivity. In this work, we report the utilization of a highly dense calcium alginate immobilized Clostridium acetobutylicum cells in combination with fed-batch mode of fermentation to attain a high butanol/ABE productivity and concentration. A butanol concentration of up to 21.6 g/L was attained with a productivity of 0.40 g/L·hr, which is a 65% and 192% improvement to the conventional batch fermentation.

Intracellular metabolic changes of Clostridium acetobutylicum and promotion to butanol tolerance during biobutanol fermentation

During the fermentation process, Clostridium acetobutylicum cells are often inhibited by the accumulated butanol. However, the mechanism underlying response of C. acetobutylicum to butanol stress remains poorly understood. This study was performed to clarify such mechanism through investigating the butanol stress-associated intracellular biochemical changes at acidogenesis phase (i.e., middle exponential phase) and solventogenesis phase (i.e., early stationary phase) by a gas chromatography-mass spectrometry-based metabolomics strategy. With the aid of partial least-squares-discriminant analysis, a pairwise discrimination between control group and butanol-treated groups was revealed, and 27 metabolites with variable importance in the projection value greater than 1 were identified. Under butanol stress, the glycolysis might be inhibited while TCA cycle might be promoted. Moreover, changes of lipids and fatty acids compositions, amino acid metabolism and osmoregulator concentrations might be the key factors involved in C. acetobutylicum metabolic response to butanol stress. It was suggested that C. acetobutylicum cells might change the levels of long acyl chain saturated fatty acids and branched-chain amino acids to maintain the integrity of cell membrane through adjusting membrane fluidity under butanol stress. The increased level of glycerol was considered to be correlated with osmoregulation and regulating redox balance. In addition, increased levels of some amino acids (i.e., threonine, glycine, alanine, phenylalanine, tyrosine, tryptophan, aspartate and glutamate) might also confer butanol tolerance to C. acetobutylicum. These results highlighted our knowledge about the response or adaptation of C. acetobutylicum to butanol stress, and would contribute to the construction of feasible butanologenic strains with higher butanol tolerance.

Biobutanol production using unhydrolyzed waste acorn as a novel substrate

Clostridium acetobutylicumcells did not grow on untreated acorn powder but they grew and produced acetone, butanol, and ethanol on tannin-free acorn powder.

Direct production of acetone–butanol–ethanol from waste starch by free and immobilized Clostridium acetobutylicum

Production of acetone–butanol–ethanol (ABE) from potato waste starch using free and immobilized cells of Clostridium acetobutylicum was investigated. The starch was directly fermented to ABE by C. acetobutylicum without hydrolysis, and the results were compared with those obtained from glucose as a reference. Maximum butanol yields from waste starch and glucose were 0.21 and 0.26g/g of initial carbon source (20g/L), respectively. Batch fermentation of 60g/L of waste starch in a 5L bioreactor with free cells resulted in production of 9.9g/L butanol. In a repeated batch fermentation, using immobilized cells of the bacterium in calcium alginate–polyvinyl alcohol (PVA)–boric acid beads and 60g/L of the waste starch, a final butanol concentration of 15.3g/L was achieved.

Developing controllable hypermutable Clostridium cells through manipulating its methyl-directed mismatch repair system.

Development of controllable hypermutable cells can greatly benefit understanding and harnessing microbial evolution. However, there have not been any similar systems developed for Clostridium, an important bacterial genus. Here we report a novel two-step strategy for developing controllable hypermutable cells of Clostridium acetobutylicum, an important and representative industrial strain. Firstly, the mutS/L operon essential for methyldirected mismatch repair (MMR) activity was inactivated from the genome of C. acetobutylicum to generate hypermutable cells with over 250-fold increased mutation rates. Secondly, a proofreading control system carrying an inducibly expressed mutS/L operon was constructed. The hypermutable cells and the proofreading control system were integrated to form a controllable hypermutable system SMBMutC, of which the mutation rates can be regulated by the concentration of anhydrotetracycline (aTc). Duplication of the miniPthl-tetR module of the proofreading control system further significantly expanded the regulatory space of the mutation rates, demonstrating hypermutable Clostridium cells with controllable mutation rates are generated. The developed C. acetobutylicum strain SMBMutC2 showed higher survival capacities than the control strain facing butanol-stress, indicating greatly increased evolvability and adaptability of the controllable hypermutable cells under environmental challenges.

Production of butanol from glucose and xylose with immobilized cells of Clostridium acetobutylicum

Pretreated cotton towels were used as carriers to immobilize Clostridium acetobutylicum CGMCC 5234 cells for butanol or ABE production from glucose and xylose. Results showed that cell immobilization was a promising method to increase butanol concentration, yield and productivity regardless of the sugar sources compared with cell suspension. In this study, a high butanol concentration of 10.02 g/L with a yield of 0.20 g/g was obtained from 60 g/L xylose with 9.9 g/L residual xylose using immobilized cells compared with 8.48 g/L butanol and a yield of 0.141 g/g with 20.2 g/L residual xylose from 60 g/L xylose using suspended cells. In mixed-sugar fermentation (30 g/L glucose plus 30 g/L xylose), the immobilized cultures produced 11.1 g/L butanol with a yield of 0.190 g/g, which were 28.3% higher than with suspended cells (8.65 g/L) during which 30 g/L glucose was utilized completely using both immobilized and suspended cells while 3.46 and 13.1 g/L xylose maintained untilized for immobilized and suspended cells, respectively. Based on the results, we speculated that immobilized cells showed enhanced tolerance to butanol toxicity and the cultures preferred glucose to xylose during ABE fermentation. Moreover, the cultures showed obvious difference when grown between high initial concentrations of glucose and those of xylose. Repeated-batch fermentations from glucose with immobilized cells showed better long-term stability than from xylose. At last, the morphologies of free and immobilized cells adsorbed on pretreated cotton towels during the growth cycle were examined by SEM.

A transcriptional study of acidogenic chemostat cells of Clostridium acetobutylicum – Cellular behavior in adaptation to n-butanol

To gain more insight into the butanol stress response of Clostridium acetobutylicum the transcriptional response of a steady state acidogenic culture to different levels of n-butanol (0.25–1%) was investigated. No effect was observed on the fermentation pattern and expression of typical solvent genes (aad, ctfA/B, adc, bdhA/B, ptb, buk). Elevated levels of butanol mainly affected class I heat-shock genes (hrcA, grpE, dnaK, dnaJ, groES, groEL, hsp90), which were upregulated in a dose- and time-dependent manner, and genes encoding proteins involved in the membrane composition (fab and fad or glycerophospholipid related genes) and various ABC-transporters of unknown specificity. Interestingly, fab and fad genes were embedded in a large, entirely repressed cluster (CAC1988–CAC2019), which inter alia encoded an iron-specific ABC-transporter and molybdenum-cofactor synthesis proteins. Of the glycerophospholipid metabolism, the glycerol-3-phosphate dehydrogenase (glpA) gene was highly upregulated, whereas a glycerophosphodiester ABC-transporter (ugpAEBC) and a phosphodiesterase (ugpC) were repressed. On the megaplasmid, only a few genes showed differential expression, e.g. a rare lipoprotein (CAP0058, repressed) and a membrane protein (CAP0102, upregulated) gene. Observed transcriptional responses suggest that C. acetobutylicum reacts to butanol stress by induction of the general stress response and changing its cell envelope and transporter composition, but leaving the central catabolism unaffected.

Production of biofuels from pretreated microalgae biomass by anaerobic fermentation with immobilized Clostridium acetobutylicum cells

The purpose of this work was to study the possible use of pretreated biomass of various microalgae and cyanobacteria as substrates for acetone–butanol–ethanol (ABE) fermentation by Clostridium acetobutylicum cells immobilized into poly(vinyl alcohol) cryogel. To this end, the biochemical composition of photosynthetic microorganisms cultivated under various conditions was studied. The most efficient technique for pretreating microalgal biomass for its subsequent conversion into biofuels appeared to be thermal decomposition at 108°C. For the first time the maximum productivity of the ABE fermentation in terms of hydrogen (8.5mmol/L medium/day) was obtained using pretreated biomass of Nannochloropsis sp. Maximum yields of butanol and ethanol were observed with Arthrospira platensis biomass used as the substrate. Immobilized Clostridium cells were demonstrated to be suitable for multiple reuses (for a minimum of five cycles) in ABE fermentation for producing biofuels from pretreated microalgal biomass.

U(VI) reduction by spores of Clostridium acetobutylicum

Vegetative cells of Clostridium acetobutylicum are known to reduce hexavalent uranium (U(VI)). We investigated the ability of spores of this organism to drive the same reaction. We found that spores were able to remove U(VI) from solution when H2 was provided as an electron donor and to form a U(IV) precipitate. We tested several environmental conditions and found that spent vegetative cell growth medium was required for the process. Electron microscopy showed the product of reduction to accumulate outside the exosporium. Our results point towards a novel U(VI) reduction mechanism, driven by spores, that is distinct from the thoroughly studied reactions in metal-reducing Proteobacteria.

A proteomic and transcriptional view of acidogenic and solventogenic steady-state cells of Clostridium acetobutylicum in a chemostat culture

The complex changes in the life cycle of Clostridium acetobutylicum, a promising biofuel producer, are not well understood. During exponential growth, sugars are fermented to acetate and butyrate, and in the transition phase, the metabolism switches to the production of the solvents acetone and butanol accompanied by the initiation of endospore formation. Using phosphate-limited chemostat cultures at pH 5.7, C. acetobutylicum was kept at a steady state of acidogenic metabolism, whereas at pH 4.5, the cells showed stable solvent production without sporulation. Novel proteome reference maps of cytosolic proteins from both acidogenesis and solventogenesis with a high degree of reproducibility were generated. Yielding a 21% coverage, 15 protein spots were specifically assigned to the acidogenic phase, and 29 protein spots exhibited a significantly higher abundance in the solventogenic phase. Besides well-known metabolic proteins, unexpected proteins were also identified. Among these, the two proteins CAP0036 and CAP0037 of unknown function were found as major striking indicator proteins in acidogenic cells. Proteome data were confirmed by genome-wide DNA microarray analyses of the identical cultures. Thus, a first systematic study of acidogenic and solventogenic chemostat cultures is presented, and similarities as well as differences to previous studies of batch cultures are discussed.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-010-2741-x) contains supplementary material, which is available to authorized users.

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.

Continuous acetone-ethanol—butanol fermentation by immobilized cells of Clostridium acetobutylicum

Continuous acetone-ethanol—butanol fermentation by immobilized cells of Clostridium acetobutylicum

Continuous acetone–ethanol–butanol fermentation by immobilized cells of Clostridium acetobutylicum

Eight Clostridium acetobutylicum strains were examined for α-amylase and strains B-591, B-594 and P-262 had the highest activities. Defibered-sweet-potato-slurry (DSPS), containing 39.7 g starch l −1, supplemented with potassium phosphate (1.0 g l −1) , cysteine-HCl (5.0 g l −1 ), the antifoam (polypropylene glycol, 0.1 mg ml −1 ), was used as continuous feedstock (FS) to a multistage bioreactor system for acetone–ethanol–butanol (AEB) fermentation. The system consisted of four columns (three vertical and one near horizontal) packed with beads containing immobilized cells of C. acetobutylicum P-262. When DSPS was pumped into the bioreactor system, at a flow rate of 2.36 ml min −1 , the effluent had 7.73 g solvents l −1 (1.56, acetone; 0.65, ethanol; 5.52 g, butanol) and no starch. Productivity of total solvents synthesized during continuous operation were 1.0 g l −1 h −1 and 19.5% yield compared to 0.12 g l −1 h −1 with 29% yield using the batch system. We propose using DSPS for AEB fermentation in a continuous mode with immobilized P-262 cells that are active amylase producers which will lead to cost reduction compared to the batch system.

Continuous production of acetone-butanol-ethanol using immobilized cells of Clostridium acetobutylicum and integration with product removal by liquid-liquid extraction

Cells of Clostridium acetobutylicum were immobilized by adsorption onto bonechar, and used in a packed bed or fluidized bed reactor for continuous production of ABE from whey permeate. At dilution rates in the range 0.35–1.10 h −1, ABE productivity values of 3.0 to 4.0 g/ l·h were observed, but lactose utilization values were poor. When operated in an integrated system with product removal by liquid-liquid extraction, there was a decrease in productivity but lactose utilization was increased markedly. Of the three extractants tested, oleyl alcohol was superior to both benzyl benzoate and dibutyl phthalate.

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.

Protein phosphorylation in response to stress in Clostridium acetobutylicum

The possible involvement of protein phosphorylation in the clostridial stress response was investigated by radioactively labeling growing cells of Clostridium acetobutylicum with 32Pi or cell extracts with [gamma-32P]ATP. Several phosphoproteins were identified; these were not affected by the growth stage of the culture. Although the extent of protein phosphorylation was increased by heat stress, the phosphoproteins did not correspond to known stress proteins seen in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified clostridial DnaK, a stress protein, acted as a kinase catalyzing the phosphorylation of a 50-kilodalton protein. The phosphorylation of this protein was enhanced in extracts prepared from heat-stressed cells. Diadenosine-5',5"'-P1,P4-tetraphosphate had no influence on protein phosphorylation.

Integration of continuous production and recovery of solvents from whey permeate: use of immobilized cells of Clostridium acetobutylicum in a flutilized bed reactor coupled with gas stripping

An investigation was performed into the operation of an integrated system for continuous production and product recovery of solvents (acetone-butanol-ethanol) from the ABE fermentation process. Cells of Clostridium acetobutylicum were immobilized by adsorption onto bonechar, and used in a fluidized bed reactor for continuous solvent production from whey permeate. The reactor effluent was stripped of the solvents using nitrogen gas, and was recycled to the reactor. This relieved product inhibition and allowed further sugar utilization. At a dilution rate of 1.37 h−1 a reactor productivity of 5.1 kg/(m3 · h) was achieved. The solvents in the stripping gas were condensed to give a solution of 53.7 kg/m3. This system has the advantages of relieving product inhibition, and providing a more concentrated solution for recovery by distillation. Residual sugar and non-volatile reaction intermediates are not removed by gas stripping and this contributes to high solvent yields.

A mathematical model of a fluidized bed reactor for the continuous production of solvents by immobilized Clostridium acetobutylicum

AbstractA fluidized bed reactor has been employed for the continuous production of solvents from whey permeate using cells of Clostridium acetobutylicum immobilized by adsorption onto bonechar. Substrate diffusion equations have been developed for the bioparticles, and a mathematical model has been advanced to describe the operation of the reactor. The model was fitted to the experimental data using the concept that not all of the biomass within the reactor was active in solvent production. On this basis, less than 5% was ‘active’ biomass.

Introduction of plasmids into whole cells of Clostridium acetobutylicum by electroporation

Introduction of plasmids into whole cells of Clostridium acetobutylicum by electroporation