The main bottleneck in the return of industrial butanol production from renewable feedstock through acetone-butanol-ethanol (ABE) fermentation by clostridia, such as Clostridium beijerinckii, is the low final butanol concentration. The problem is caused by the high toxicity of butanol to the production cells, and therefore, understanding the mechanisms by which clostridia react to butanol shock is of key importance. Detailed analyses of transcriptome data that were obtained after butanol shock and their comparison with data from standard ABE fermentation have resulted in new findings, while confirmed expected population responses. Although butanol shock resulted in upregulation of heat shock protein genes, their regulation is different than was assumed based on standard ABE fermentation transcriptome data. While glucose uptake, glycolysis, and acidogenesis genes were downregulated after butanol shock, solventogenesis genes were upregulated. Cyclopropanation of fatty acids and formation of plasmalogens seem to be significant processes involved in cell membrane stabilization in the presence of butanol. Surprisingly, one of the three identified Agr quorum-sensing system genes was upregulated. Upregulation of several putative butanol efflux pumps was described after butanol addition and a large putative polyketide gene cluster was found, the transcription of which seemed to depend on the concentration of butanol.

• Keywords
• Clostridium beijerinckii, ABE fermentation, butanol shock, transcriptome analysis,
• MeSH
• Biological Transport genetics   MeSH
• Bioreactors microbiology   MeSH
• Cell Membrane metabolism   MeSH
• Butanols toxicity   MeSH
• Clostridium beijerinckii drug effects   genetics   metabolism   MeSH
• Stress, Physiological genetics   MeSH
• Glucose metabolism   MeSH
• Glycolysis genetics   physiology   MeSH
• Fatty Acids metabolism   MeSH
• Plasmalogens biosynthesis   MeSH
• Heat-Shock Proteins metabolism   MeSH
• Quorum Sensing genetics   MeSH
• Gene Expression Profiling MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Butanols MeSH
• Glucose MeSH
• Fatty Acids MeSH
• Plasmalogens MeSH
• Heat-Shock Proteins MeSH

N-Butanol, a valuable solvent and potential fuel extender, can be produced via acetone-butanol-ethanol (ABE) fermentation. One of the main drawbacks of ABE fermentation is the high toxicity of butanol to producing cells, leading to cell membrane disruption, low culture viability and, consequently, low produced concentrations of butanol. The goal of this study was to obtain mutant strains of Clostridium beijerinckii NRRL B-598 with improved butanol tolerance using random chemical mutagenesis, describe changes in their phenotypes compared to the wild-type strain and reveal changes in the genome that explain improved tolerance or other phenotypic changes. Nine mutant strains with stable improved features were obtained by three different approaches and, for two of them, ethidium bromide (EB), a known substrate of efflux pumps, was used for either selection or as a mutagenic agent. It is the first utilization of this approach for the development of butanol-tolerant mutants of solventogenic clostridia, for which generally there is a lack of knowledge about butanol efflux or efflux mechanisms and their regulation. Mutant strains exhibited increase in butanol tolerance from 36% up to 127% and the greatest improvement was achieved for the strains for which EB was used as a mutagenic agent. Additionally, increased tolerance to other substrates of efflux pumps, EB and ethanol, was observed in all mutants and higher antibiotic tolerance in some of the strains. The complete genomes of mutant strains were sequenced and revealed that improved butanol tolerance can be attributed to mutations in genes encoding typical stress responses (chemotaxis, autolysis or changes in cell membrane structure), but, also, to mutations in genes X276_07980 and X276_24400, encoding efflux pump regulators. The latter observation confirms the importance of efflux in butanol stress response of the strain and offers new targets for rational strain engineering.

• Keywords
• butanol efflux, butanol tolerance, genome sequence, random chemical mutagenesis, solventogenic Clostridium species,
• Publication type
• Journal Article MeSH

The transient and steady pervaporation of 1-butanol-water mixtures through a poly[1-(trimethylsilyl)-1-propyne] (PTMSP) membrane was studied to observe and elucidate the diffusion phenomena in this high-performing organophilic glassy polymer. Pervaporation was studied in a continuous sequence of experiments under conditions appropriate for the separation of bio-butanol from fermentation broths: feed concentrations of 1.5, 3.0 and 4.5 w/w % of 1-butanol in nutrient-containing (yeast extract) water, temperatures of 37, 50 and 63 °C, and a time period of 80 days. In addition, concentration polarization was assessed. As expected, the total flux and individual component permeabilities declined discernibly over the study period, while the separation factor (average β = 82) and selectivity towards 1-butanol (average α = 2.6) remained practically independent of the process conditions tested. Based on measurements of pervaporation transients, for which a new apparatus and model were developed, we found that the diffusivity of 1-butanol in PTMSP decreased over time due to aging and was comparable to that observed using microgravimetry in pure vapor in 1-butanol. Hence, despite the gradual loss of free volume of the aging polymer, the PTMSP membrane showed high and practically independent selectivity towards 1-butanol. Additionally, a new technique for the measurement and evaluation of pervaporation transients using Fourier transform infrared spectroscopy (FTIR) analysis of permeate was proposed and validated.

• Keywords
• PTMSP, ageing, butanol, diffusivity, pervaporation, water,
• Publication type
• Journal Article MeSH

BACKGROUND: One of the main obstacles preventing solventogenic clostridia from achieving higher yields in biofuel production is the toxicity of produced solvents. Unfortunately, regulatory mechanisms responsible for the shock response are poorly described on the transcriptomic level. Although the strain Clostridium beijerinckii NRRL B-598, a promising butanol producer, has been studied under different conditions in the past, its transcriptional response to a shock caused by butanol in the cultivation medium remains unknown. RESULTS: In this paper, we present a transcriptional response of the strain during a butanol challenge, caused by the addition of butanol to the cultivation medium at the very end of the acidogenic phase, using RNA-Seq. We resequenced and reassembled the genome sequence of the strain and prepared novel genome and gene ontology annotation to provide the most accurate results. When compared to samples under standard cultivation conditions, samples gathered during butanol shock represented a well-distinguished group. Using reference samples gathered directly before the addition of butanol, we identified genes that were differentially expressed in butanol challenge samples. We determined clusters of 293 down-regulated and 301 up-regulated genes whose expression was affected by the cultivation conditions. Enriched term "RNA binding" among down-regulated genes corresponded to the downturn of translation and the cluster contained a group of small acid-soluble spore proteins. This explained phenotype of the culture that had not sporulated. On the other hand, up-regulated genes were characterized by the term "protein binding" which corresponded to activation of heat-shock proteins that were identified within this cluster. CONCLUSIONS: We provided an overall transcriptional response of the strain C. beijerinckii NRRL B-598 to butanol shock, supplemented by auxiliary technologies, including high-pressure liquid chromatography and flow cytometry, to capture the corresponding phenotypic response. We identified genes whose regulation was affected by the addition of butanol to the cultivation medium and inferred related molecular functions that were significantly influenced. Additionally, using high-quality genome assembly and custom-made gene ontology annotation, we demonstrated that this settled terminology, widely used for the analysis of model organisms, could also be applied to non-model organisms and for research in the field of biofuels.

• Keywords
• ABE fermentation, Butanol shock, Clostridium beijerinckii NRRL B-598, RNA-Seq transcriptome,
• Publication type
• Journal Article MeSH

Clostridium beijerinckii NRRL B-598 is a sporulating, butanol and hydrogen producing strain that utilizes carbohydrates by the acetone-butanol-ethanol (ABE) fermentative pathway. The pathway consists of two metabolic phases, acidogenesis and solventogenesis, from which the latter one can be coupled with sporulation. Thorough transcriptomic profiling during a complete life cycle and both metabolic phases completed with flow cytometry, microscopy and a metabolites analysis helped to find out key genes involved in particular cellular events. The description of genes/operons that are closely involved in metabolism or the cell cycle is a necessary condition for metabolic engineering of the strain and will be valuable for all C. beijerinckii strains and other Clostridial species. The study focused on glucose transport and catabolism, hydrogen formation, metabolic stress response, binary fission, motility/chemotaxis and sporulation, which resulted in the composition of the unique image reflecting clostridial population changes. Surprisingly, the main change in expression of individual genes was coupled with the sporulation start and not with the transition from acidogenic to solventogenic metabolism. As expected, solvents formation started at pH decrease and the accumulation of butyric and acetic acids in the cultivation medium.

• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Clostridium beijerinckii cytology   genetics   MeSH
• Fermentation genetics   MeSH
• Stress, Physiological * genetics   MeSH
• Glucose metabolism   MeSH
• Acids metabolism   MeSH
• Fatty Acids metabolism   MeSH
• Heat-Shock Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Bacterial * MeSH
• Solvents metabolism   MeSH
• Spores, Bacterial metabolism   MeSH
• Transcriptome genetics   MeSH
• Hydrogen metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Glucose MeSH
• Acids MeSH
• Fatty Acids MeSH
• Heat-Shock Proteins MeSH
• Solvents MeSH
• Hydrogen MeSH

BACKGROUND: Thinning supplies of natural resources increase attention to sustainable microbial production of bio-based fuels. The strain Clostridium beijerinckii NRRL B-598 is a relatively well-described butanol producer regarding its genotype and phenotype under various conditions. However, a link between these two levels, lying in the description of the gene regulation mechanisms, is missing for this strain, due to the lack of transcriptomic data. RESULTS: In this paper, we present a transcription profile of the strain over the whole fermentation using an RNA-Seq dataset covering six time-points with the current highest dynamic range among solventogenic clostridia. We investigated the accuracy of the genome sequence and particular genome elements, including pseudogenes and prophages. While some pseudogenes were highly expressed, all three identified prophages remained silent. Furthermore, we identified major changes in the transcriptional activity of genes using differential expression analysis between adjacent time-points. We identified functional groups of these significantly regulated genes and together with fermentation and cultivation kinetics captured using liquid chromatography and flow cytometry, we identified basic changes in the metabolism of the strain during fermentation. Interestingly, C. beijerinckii NRRL B-598 demonstrated different behavior in comparison with the closely related strain C. beijerinckii NCIMB 8052 in the latter phases of cultivation. CONCLUSIONS: We provided a complex analysis of the C. beijerinckii NRRL B-598 fermentation profile using several technologies, including RNA-Seq. We described the changes in the global metabolism of the strain and confirmed the uniqueness of its behavior. The whole experiment demonstrated a good reproducibility. Therefore, we will be able to repeat the experiment under selected conditions in order to investigate particular metabolic changes and signaling pathways suitable for following targeted engineering.

• Keywords
• ABE fermentation, Clostridium beijerinckii NRRL B-598, RNA-Seq transcriptome,
• MeSH
• Bacteriophages genetics   MeSH
• Butanols metabolism   MeSH
• Clostridium beijerinckii genetics   metabolism   virology   MeSH
• DNA, Viral genetics   MeSH
• Fermentation MeSH
• Transcription, Genetic MeSH
• Kinetics MeSH
• Pseudogenes genetics   MeSH
• Sequence Analysis, RNA * MeSH
• Gene Expression Profiling * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Butanols MeSH
• DNA, Viral MeSH