Project description:Clostridium acetobutylicum is a Gram-positive, endospore-forming bacterium that is considered as a strict anaerobe. It ferments sugars to the organic acids acetate and butyrate or shifts to formation of the solvents - ethanol, butanol and acetone. In most bacteria the major regulator of iron homeostasis is Fur (ferric uptake regulator). Analysis of the genome of Clostridium acetobutylicum has revealed three genes encoding Fur-like proteins. The amino acid sequece of one of them showed 70% similarity to the Fur protein of the closely related Bacillus subtilis.<br>Thus, to gain insight into the role of Fur and the mechanisms for maintenance of iron homeostasis in this strict anaerobic organism, we determined its transcriptional profile in response to iron limitation and inactivation of fur.
Project description:Clostridium carboxidivorans P7 (DSM 15243) is a bacterium that converts syngas (a mixture of CO, H2, and CO2) into hexanol. An optimized and scaled-up industrial process could therefore provide a renewable source of fuels and chemicals while consuming industry waste gases. However, the genetic engineering of this bacterium is hindered by its multiple restriction-modification (RM) systems: the genome of C. carboxidivorans encodes at least ten restriction enzymes and eight methyltransferases (MTases). To gain insight into the complex RM systems of C. carboxidivorans, we analyzed genomic methylation patterns using single-molecule real-time (SMRT) sequencing and bisulfite sequencing. We identified six methylated sequence motifs. To match the methylation sites to the predicted MTases of C. carboxidivorans, we expressed them individually in Escherichia coli for functional characterization. Recognition motifs were identified for all three Type I MTases (CAYNNNNNCTGC/GCAGNNNNNRTG, CCANNNNNNNNTCG/CGANNNNNNNNTGG and GCANNNNNNNTNNCG/CGNNANNNNNNNTGC), two Type II MTases (GATAAT and CRAAAAR), and a single Type III MTase (GAAAT). However, no methylated recognition motif was found for one of the three Type II enzymes. One recognition motif that was methylated in C. carboxidivorans but not in E. coli (AGAAGC) was matched to the remaining Type III MTase through a process of elimination. Understanding these enzymes and the corresponding recognition sites will facilitate the development of genetic tools for C. carboxidivorans that can accelerate the industrial exploitation of this strain.
Project description:Transcriptomic analysis by AmpliSeq Ion Torrent to explore the effects of the long non-coding RNA TGFB2-AS1 on gene expression in human keratinocytes
Project description:Syngas fermentation with acetogens is known to produce mainly acetate and ethanol efficiently. Co-cultures with chain elongating bacteria making use of these products are a promising approach to produce longer-chain alcohols. Synthetic co-cultures with identical initial cell concentrations of Clostridium carboxidivorans and Clostridium kluyveri were studied in batch-operated stirred-tank bioreactors with continuous CO/CO2 -gassing and monitoring of the cell counts of both clostridia by flow cytometry after fluorescence in situ hybridization (FISH-FC). At 800 mbar CO, chain elongation activity was observed at pH 6.0, although growth of C. kluyveri was restricted. Organic acids produced by C. kluyveri were reduced by C. carboxidivorans to the corresponding alcohols butanol and hexanol. This resulted in a threefold increase in final butanol concentration and enabled hexanol production compared with a mono-culture of C. carboxidivorans. At 100 mbar CO, growth of C. kluyveri was improved; however, the capacity of C. carboxidivorans to form alcohols was reduced. Because of the accumulation of organic acids, a constant decay of C. carboxidivorans was observed. The measurement of individual cell concentrations in co-culture established in this study may serve as an effective tool for knowledge-based identification of optimum process conditions for enhanced formation of longer-chain alcohols by clostridial co-cultures.
Project description:The growing need for sustainable biotechnological solutions to address environmental challenges, such as climate change and resource depletion, has intensified interest in microbial-based production systems. Synthetic biofilms, which mimic natural microbial consortia, offer a promising platform for optimizing complex metabolic processes that can convert renewable feedstocks into valuable chemicals. In this context, understanding and harnessing the interactions between co-immobilized microorganisms are critical for advancing bioprocesses that contribute to circular bioeconomy goals. In this study, we investigated the viability and metabolic activity of Clostridium carboxidivorans and Clostridium kluyveri within a synthetic, dual-layered biofilm composed of agar hydrogel. This setup compartmentalized each bacterial species. Embedding the bacteria in a structured biofilm offers numerous opportunities for bioproduction, but the inability to monitor cell growth or movement within the immobilization matrix limits process insights. To address this, we adapted a fluorescence in situ hybridization (FISH) protocol, enabling precise, species-specific visualization of bacterial distribution and growth within the gel matrix. Batch processes with the dual-layered biofilm in anaerobic flasks, designed with a metabolic advantage for C. kluyveri, revealed distinct growth dynamics. C. kluyveri exhibited significant metabolic activity, forming clusters at low initial cell concentrations and converting ethanol and acetate into 1-butyrate and 1-hexanoate, indicating viability and cell growth. C. carboxidivorans remained evenly distributed without significant growth or product formation, suggesting that while the cells were viable, they were not metabolically active under the experimental conditions. Both bacterial species were confined to their respective compartments throughout the process, with C. kluyveri showing enhanced substrate conversion at higher initial cell densities in the hydrogel. The pH drop throughout the batch experiment likely contributed to incomplete substrate consumption, particularly for C. kluyveri, which thrives within a narrow pH range. These findings highlight synthetic biofilms as a promising platform for optimizing microbial interactions and improving bioprocess efficiency, especially in applications involving complex metabolic exchanges between co-immobilized microorganisms. Further research will focus on applying conditions to support the growth and metabolic activity of C. carboxidivorans to explore spatial dynamics of bacterial migration and cooperative relationships in the synthetic biofilm.
Project description:Co-cultures of clostridia with distinct physiological properties have emerged as an alternative to increase the production of butanol and other added-value compounds from biomass. The optimal performance of mixed tandem cultures may depend on the stability and fitness of each species in the consortium, making the development of specific quantification methods to separate their members crucial. In this study, we developed and tested a multiplex qPCR method targeting the 16S rRNA gene for the simultaneous quantification of Clostridium acetobutylicum, Clostridium carboxidivorans and Clostridium cellulovorans in co-cultures. Designed primer pairs and probes could specifically quantify the three Clostridium species with no cross-reactions thus allowing significant changes in their growth kinetics in the consortia to be detected and correlated with productivity. The method was used to test a suitable medium composition for simultaneous growth of the three species. We show that higher alcohol productions were obtained when combining C. carboxidivorans and C. acetobutylicum compared to individual cultures, and further improved (> 90%) in the triplet consortium. Altogether, the methodology could be applied to fermentation processes targeting butanol productions from lignocellulosic feedstocks with a higher substrate conversion efficiency.
Project description:BackgroundClostridium carboxidivorans P7 is capable of producing ethanol and butanol from inexpensive and non-food feedstock, such as syngas. Achieving improved ethanol and butanol production in the strain for industrial application depends on the energetics and biomass, especially ATP availability.ResultsThis study found that exogenous addition of citrulline promoted accumulation of ATP, increased specific growth rate, and reduced the doubling time of C. carboxidivorans P7. In heterotrophic fermentation experiments, the addition of citrulline increased intracellular ATP by 3.39-fold, significantly enhancing the production of total alcohol (ethanol + butanol) by 20%. Moreover, in the syngas fermentation experiments, the addition of citrulline improved the level of intracellular ATP and the biomass by 80.5% and 31.6%, respectively, resulting in an 18.6% and 60.3% increase in ethanol and the alcohol/acid production ratio, respectively.ConclusionsThis is the first report that citrulline could promote the growth of C. carboxidivorans P7 and increase the level of intracellular ATP, which is of great significance for the use of C. carboxidivorans P7 to synthesize biofuels.
Project description:Purpose: Analyze gene expression during C. perfringens colonization in the chicken Transcriptomic profile of mRNA from C. perfrinegns from in vivo and in vitro conditions were determined in biological duplicates by RNA-Seq using Illumina HiSeq 2500