The sulfur metabolism during Acetogenesis and Solventogenesis of Clostridium acetobutylicum
ABSTRACT: The batch fermentation of Clostridium acetobutylicum is characterized by an acetogenic growth phase during exponential growth when mainly acetate and butyrate are fermentation products. Then, at the end of exponential growth and during stationary phase, the organism switches to solventogenic growth and large amounts of acetone, ethanol and butanol are produced. These growth phases can be studied independent from each other in a phosphate-limited continuous culture. In transcription analysis of continuous cultures using DNA microarrays it became evident that, among others, operons involved in sulfur assimilation are strongly up-regulated during solventogenesis. Using the ClosTron technique we constructed two knock-out mutants in the genes CAC0105 and CAC0930 annotated as involved in sulfate reduction and cysteine biosynthesis. Complementation experiments were carried out with sulfite and cysteine to prove the predicted function. The fermentation experiments of wild type and mutants using phosphate-limited and sulfur-limited continuous culture demonstrated that less sulfur source was consumed in solventogenic phase and the efficiency of cysteine uptake became lower. DNA microarrays were performed to study the difference of transcriptional expression when the wild type was challenged with insufficient sulfur source and the mccB (CAC0930) mutant was inactivated in the continuous culture. The result provided insights into understanding the sulfur metabolism regulatory.
Project description:Oxidative stress is harmful for organism and occurs when the cells exposed to superoxid, hydrogen peroxide and alkylhydroperoxides. In microorganism, the glutathione- and thioredoxin-dependent reduction systems are universal and play an important role in response to defending oxidative stress. The _-glutamylcysteine synthetase (_-GCS) is an essential enzyme to biosynthesize the tripeptide glutathione (GSH) in organism. Similarly, thioredoxin reductase is an important enzyme in thioredoxin-dependent reduction system. In Clostridium acetobutylicum, the _-glutamylcysteine synthetase (encoded by CAC1539, gcs) and thioredoxin reductase (encoded by CAC1548, trxB) were inactivated using ClosTron technology. The gcs mutant grew insufficiently and consumed less glucose in the phosphate-limited continuous culture and exhibited more sensitive to oxidative stress. The trxB mutant just exhibited lower growth rate and less glucose uptake in the solventogenic phase, compared to wild type. The DNA microarrays were performed to investigate the transcripome difference between wild type and the mutants. In gcs mutant, the genes related to chemotaxis and flagella biosynthesis proteins were induced significantly and in the trxB mutant, the sporulation genes were induced largely. Based on the phenotypes and transcriptome comparison results, the relationship between GSH- and Trx-dependent induction systems was discussed in Clostridium acetobutylicum.
Project description:Artificial electron carriers have been widely used to shift the solvent ratio towards butanol in acetone-butanol-ethanol (ABE) fermentation of solventogenic clostridia according to decreased hydrogen production. In this study, first insights on the molecular level were gained to explore the effect of methyl viologen addition to cultures of Clostridium acetobutylicum. Employing batch fermentation in mineral salts medium, the butanol:acetone ratio was successively increased from 2.3 to 12.4 on a 100 ml scale in serum bottles and from 1.4 to 16.5 on a 1,300 ml scale in bioreactors, respectively. The latter cultures were used for DNA microarray analyses to provide new information on the transcriptional changes referring to methyl viologen exposure and thus, exhibing gene expression patterns according to the manipulation of the cellular redox balance.
Project description:The fermentation culture of Clostridium beijerinckii mutant BA101 was monitored from exponential growth to stationary phase. During this period the culture underwent a shift from acidogenesis to solventogenesis. Acetone and butanol production was initiated with the onset of the solventogenic phase. Using DNA microarray changes in gene expression were examined during the transitional period. RNA samples were taken from Clostridium beijerinckii mutant BA101 fermentation culture at individual time points during the acidogenic phase and the solventogenic phase. The samples were used for microarray hybridization.
Project description:Solventogenic Clostridium species ferment carbohydrates to acetone, butanol and ethanol which are well-known next-generation biofuels. However, repeated subculture of or continuous fermentation by Clostridium often decreases and eventually terminates the solvent production and spore formation, which is a process called strain degeneration. Supplementation of CaCO3 to fermentation medium could partially recover metabolism of degenerated strain by more than 50% increase of cell growth and solvent production. The transcriptome profile of Clostridium beijerinckii NCIMB 8052 (DG-8052) and its response to CaCO3 treatment were analysed by microarray. Since fermentation by C. beijerinckii NCIMB 8052 is a biphasic process, gene expressions of two fermentations were compared at each stage, i.e. 12h and 24h fermentation time representing acidogenic phase and solventogenic phase, respectively. This study examined expression of 5168 genes capturing 98.6% of the C. beijerinckii NCIMB 8052 genome. With the addition of CaCO3, DG-8052 had 565 and 916 genes significantly up-regulated at acidogenic phase and solventogenic phase, respectively. According to the enrichment analysis of pathway and Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, these genes were significantly overrepresented in cellular functions such as Amino acid transport and metabolism, organic acid biosynthetic process, bacteria chemotaxis and defense mechanisms. On the other hand, there were 704 and 1044 genes significantly down-regulated at acidogenic phase and solventogenic phase, respectively. These repressed genes were mainly enriched in functions such as ion transmembrane transport, ATP synthesis, oxidative phosphorylation. Overall design: Clostridium beijerinckii NCIMB8052 degenerated strain cells in P2 medium vs. Clostridium beijerinckii NCIMB8052 degenerated strain cells in P2 medium with 4g/L CaCO3 Two-fermentation time points (12h and 24h) experiments (degenerated strain cells in P2 vs.degenerated strain cells in P2 with CaCO3);Biological replicates: 3 replicates of degenerated strain cells in P2 at 12h; 3 replicates of degenerated strain cells in P2 at 24h;3 replicates of degenerated cells in P2 with CaCO3 at 12h;3 replicates of degenerated cells with CaCO3 at 24h.
Project description:The fermentation culture of Clostridium beijerinckii mutant BA101 was monitored from exponential growth to stationary phase. During this period the culture underwent a shift from acidogenesis to solventogenesis. Acetone and butanol production was initiated with the onset of the solventogenic phase. Using DNA microarray changes in gene expression were examined during the transitional period. Overall design: RNA samples were taken from Clostridium beijerinckii mutant BA101 fermentation culture at individual time points during the acidogenic phase and the solventogenic phase. The samples were used for microarray hybridization.
Project description:Clostridium acetobutylicum is characterized by its acetone-butanol (AB) fermentation which <br>can be reproducibly established under continuous grow conditions in a chemostat. <br>At pH 5.7 cells show typical acidogenic metabolism and mainly produce the acids <br>acetate and butyrate. After lowering and further control the external pH at 4.5 <br>the exponentially growing cells switch towards stable solvent production with the <br>dominating fermentation products acetone and butanol. <br>Here we present a comprehensive comparison of proteome and transcriptome <br>data of continuously growing cells of C. acetobutylicum in a chemostat culture <br>under phosphate limitation at pH 5.7 (acidogenesis) and pH 4.5 (solventogenesis).
Project description:The Clostridium beijerinckii NCIMB 8052 wild-type culture was monitored from exponential growth to stationary phase. During this period the culture underwent a shift from acidogenesis to solventogenesis. Acetone and butanol production was initiated with the onset of the solventogenic phase. Using DNA microarray changes in gene expression were examined during the transitional period. RNA samples were taken from Clostridium beijerinckii NCIMB 8052 wild-type fermentation culture at individual time points during the acidogenic phase and the solventogenic phase. The samples were used for microarray hybridization.
Project description:Furfural is the prevalent microbial inhibitor generated during pretreatment and hydrolysis of lignocellulosic biomass to monomeric sugars, but the molecular response of Clostridium beijerinckii NCIMB 8052 to this compound is unknown. To discern the effect of furfural on C. beijerinckii and to gain insights into the molecular mechanisms of action and detoxification, we studied the physiological changes of furfural-stressed cultures during acetone-butanol-ethanol (ABE) fermentation, and profiled differentially expressed genes by genome-wide transcriptional analysis. C. beijerinckii exposed to furfural stress during the acidogenic growth phase produced 13% more ABE than the unstressed control. The growth and ABE by C. beijerinckii ceased following exposure to furfural stress during the solventogenic growth phase. By comparing gene expression of furfural-stressed cultures to that of the unstressed control, at both the acidogenic and solventogenic phases, we ascertained that furfural induces expression of several genes including those that code for heat shock proteins, redox enzymes and cofactor associated proteins, and ATP-binding cassette transporters, and represses genes belonging to the phosphotransferase system, two-component system, chemotaxis and cell motility. Based on these results, we discuss the underpinning for furfural-mediated change in ABE fermentation by the solventogenic Clostridium species. C. beijerinckii 8052 pre-culture was incubated anaerobically to attain acidogenic or solventogenic growth phase. The culture was then subdivided into two bottles. One bottle was challenged with furfural and the other bottle was left unchallenged. After 3 h growth, C. beijerinckii 8052 samples were collected, during which the original concentration of furfural in the growth medium was reduced to more than half. Total RNA was isolated, purified, converted to enriched mRNA, and dye-coupled (Alexa Fluor 555) complementary cRNA. This was followed by hybridization and microarray data analysis.
Project description:This study describes a transcriptome-phenotype matching approach in which the starter L. lactis MG1363 was fermented under a variety of conditions that differed in the levels of oxygen and/or salt, as well as the fermentation pH and temperature. Samples derived from these fermentations in the exponential phase of bacterial growth were analyzed by full-genome transcriptomics and the assessment of heat and oxidative stress phenotypes. Variations in the fermentation conditions resulted in up to 1000-fold differences in survival during heat and oxidative stress. More specifically, aeration during fermentation induced protection against heat stress, whereas a relatively high fermentation temperature resulted in enhanced robustness towards oxidative stress. Concomitantly, oxygen levels and fermentation temperature induced differential expression of markedly more genes when compared with the other fermentation parameters. Correlation analysis of robustness phenotypes and gene expression levels revealed transcriptome signatures for oxidative and/or heat stress survival, including the metC-cysK operon involved in methionine and cysteine metabolism. To validate this transcriptome-phenotype association we grew L. lactis MG1363 in the absence of cysteine which led to enhanced robustness towards oxidative stress. Conclusions Overall, we demonstrated the importance of careful selection of fermentation parameters prior to industrial processing of starter cultures. Furthermore, established stress genes as well as novel genes were associated with robustness towards heat and/or oxidative stress. Assessment of the expression levels of this group of genes could function as an indicator for enhanced selection of fermentation parameters resulting in improved robustness during spray drying. The increased robustness after growth without cysteine appeared to confirm the role of expression of the metC-cysK operon as an indicator of robustness and suggests that sulfur amino acid metabolism plays a pivotal role in oxidative stress survival. two connected loops, both containing samples derived on a single day (sample 1-6, sample 7-13)
Project description:Clostridium acetobutylicum was grown in a batch-culture with minimal medium containing glucose and xylose as substrate. Diauxie growth was observed after glucose was consumed. Following the organism grows on xylose. Transcriptional analysis was done to pursue the cellular processes during the switch from growth on glucose to growth on xylose. We compared DNA-Microarray data from cells grown during the exponential phase on glucose (A), with cells growing during the start of diauxie growth lag (B), during the end of diauxie growth lag (C) and during exponential growth on xylose (D). We used cells grown in a continuous culture with glucose as substrate as common reference for the samples A-D.