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General transcriptomic features of C. beijerinckii DSM 6423, C. beijerinckii NCIMB 8052, and C. acetobutylicum ATCC 824. (a) Number of TSSs found for each strain with a confidence threshold of 25 RPM. (b) Classification of TSSs in 4 categories: intergenic sense (InterS), intragenic sense (IntraS), intergenic antisense (InterA), and intragenic antisense (IntraA). (c) Number of InterS TSSs per gene for each strain. Values correspond to the percentage of genes with detected TSSs. (d) The 235 and 210 motifs found upstream from InterS TSSs of the three strains (e). 59 UTR length distributions, calculated as the distance between an InterS TSS and coding DNA sequence (CDS) starts.
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Innovative processes to transform plant biomass into renewable chemicals are needed to replace fossil fuels and limit climate change. Clostridium acetobutylicum is of industrial interest because it ferments sugars into acetone, butanol and ethanol (ABE). However, this organism is unable to depolymerize cellulose, limiting its use for the direct tra...
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... we compared the data sets for each strain, with (normalized) and without (raw) normalization (Fig. 2, Fig. S2 and 3). Adjusting TSS strength relative to local gene expression significantly changed the data set distribution (Fig. 2a, Fig. S2). When we compared the distribution of the expression of gene subsets with a detected TSS, this further resulted in a significant shift from highly expressed genes (raw data set; .1,000 TPM) to a distribution ...
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... resulted from the higher sensitivity when additional data were considered. We subsequently tested our hypothesis that in the original data set, TSSs tend to accumulate on a few genes (Fig. 2d). A high proportion of reads (.70%) contributed to the detection of TSSs in genes that bore more than 4 TSSs, with a maximum of 244 TSSs for a single gene (Fig. S3). Conversely, expression normalization reduced the proportion of genes with more than 4 TSS to 15%, indicating that (i) that a higher number of genes overall were found with one or several TSSs and (ii) the normalization step improved the data set by removing secondary TSSs which were linked either to pervasive transcription or to ...
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... in 4 categories depending on their orientation and localization relative to the associated genes: InterS (intergenic TSS with downstream gene in same orientation), InterA (intergenic TSS with downstream gene opposite orientation), IntraS (intragenic TSS in gene with same orientation), or IntraA (intragenic TSS in gene with opposite orientation) (Fig. 3b). In the 3 strains, TSS repartition was relatively similar, with most TSSs identified in the sense direction (InterS: 40 to 55%; IntraS: 40 to 55%). Such an abundance of intragenic TSSs has been observed on several occasions using different methodologies (4,12), and this has been hypothesized to mainly be the result of pervasive ...
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... YYYY Volume XX Issue XX 10.1128/spectrum.02288-21depending on the strain; Fig. 3c). As expected, conserved 210 and 235 motifs were found enriched upstream from detected TSSs in all three strains (Fig. 3d), confirming these were bona fide TSSs. Less than 3% of the TSSs were observed in the antisense direction, which supports previous results obtained for C. phytofermentans (3) (Fig. ...
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... YYYY Volume XX Issue XX 10.1128/spectrum.02288-21depending on the strain; Fig. 3c). As expected, conserved 210 and 235 motifs were found enriched upstream from detected TSSs in all three strains (Fig. 3d), confirming these were bona fide TSSs. Less than 3% of the TSSs were observed in the antisense direction, which supports previous results obtained for C. phytofermentans (3) (Fig. ...
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... on the strain; Fig. 3c). As expected, conserved 210 and 235 motifs were found enriched upstream from detected TSSs in all three strains (Fig. 3d), confirming these were bona fide TSSs. Less than 3% of the TSSs were observed in the antisense direction, which supports previous results obtained for C. phytofermentans (3) (Fig. ...
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... in vivo. Confirmation of antisense transcription from some InterA and IntraA TSS was achieved by mapping visualization of forward reads from paired-read RNAseq (Fig. S5). The 59 UTR lengths (measured as distances in bp between InterS TSS positions and corresponding coding DNA sequence [CDS] starts) rarely exceeded 200 bp in the three strains (Fig. 3e), exhibiting no correlation with gene expression (Data Set S8). Most 59 UTRs were between 0 and 100 bp long (with a peak at 232 bp relative to the start codon), with %2 to 3% of transcripts categorized as leaderless (transcripts not bearing an upstream RBS; for this analysis, 59 UTR length of ,6 bp), suggesting that, despite being ...
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... expression data analysis was incorporated into the detection pipeline by performing RNA-seq on the same mRNA samples and using the resulting expression values to normalize Capp-Switch seq data. This additional step limited the gene expression bias and hence enhanced results at the genome scale (expression bias, TSS number/gene) and the gene scale (Fig. S3), underlining the importance of treating Capp-Switch data with a normalization ...
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... genome and gene analyses (Fig. 3, Fig. S3) indicated that, for the three strains, Capp-Switch accurately detects TSSs at the single-nucleotide resolution. Importantly, TSSs have similar features in the three clostridia (total and pro-gene number of TSSs, TSS categories, 59 UTR length, upstream motifs). One interesting feature is the very low number of antisense TSSs, which was ...
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... According to Fig. 4b, to the higher volume of Clac in the inoculum, the higher was the BioH 2 production. Although several anaerobic species of Clostridium can hydrolyze cellulose through the cellulosome secretion, Clac secretes very small quantities of this multi-enzyme complex, therefore, it is only considered a solventogenic strain [57]. Fig. 4c, shows that the lowest pH value (5.5) favored the production of BioH 2 (331.6 mL), thus the increase of pH from 6.5 to 7.5 decreased the gas production. ...
Bovine ruminal fluid (BRF) bioaugmented with Clostridium acetobutylicum (Clac) was assessed for hydrolyzing cellulose and produce biohydrogen (BioH2) simultaneously from pretreated corncob in a single step, without the use of external hydrolytic biocatalysts. The corncob was pretreated using three thermochemical methods: H2SO4 2%, 160 °C; NaOH 2%, 140 °C; NaOCl 2%, 140 °C; autohydrolysis: H2O, 190 °C. Subsequently, BioH2 production was carried out using the pretreated material with the highest digestibility applying a Taguchi experimental array to identify the optimal operating conditions. The results showed a higher glucose released from pretreated corncob with H2SO4 (134.7 g/L) compared to pretreated materials by autohydrolysis, NaOH and NaOCl (123 g/L, 89.8 g/L and 52.9 g/L, respectively). The mixed culture was able to hydrolyze the pretreated corncob and produce 575 mL of H2 (at 35 °C, pH 5.5, 1:2 ratio of BRF:Clac and 5% of solids loading) equivalent to 132 L H2/Kg of biomass.
... Several methods including, the chemical pretreatment, employing commercial enzyme on saccharification, co- culture of two bacterial strain, and simultaneous saccharification by Clostridium are applied to overcome the barrier for releasing fermentable sugar (Table 2). However, previous studies related to saccharification by Clostridium showed the low-solvent yield due to the weak amylase enzyme activity of Clostridia [34,35]. Additionally, in a study by Tran et al., the co-culture of Bacillus subtilis and Clostridium butylicum TISTR 1032 was reported that the highest ABE concentration was measured at 7.40 g/l from 40 g/l starch, which was a 2-fold less than the two-step fermentation [18]. ...
In Asia, uneaten cooked rice is the highest portion amongst many forms of food wastes that are thrown away. In order to make use of the thrown-away rice and potentially use it for liquid fuels, steamed Japanese rice was evaluated on biobutanol production through a two-step fermentation by amylase-producing Aspergillus oryzae, and solvent-producing Clostridium acetobutylicum YM1. The effects of sterilization and providing anaerobic conditions on solvent production in acetone-butanol-ethanol (ABE) fermentation cannot be underestimated. Several conditions, including aerobic, anaerobic, sterile, and non-sterile were investigated concerning the solvent production capability of Clostridium acetobutylicum YM1. The maximum solvent production was 11.02 ± 0.22 g/l butanol and 18.03 ± 0.34 g/l total ABE from 75 g/l dried rice. The results confirmed that the solvent production performance of the YM1 strain was not affected by the sterilization conditions. In particular, 10.91 ± 0.16 g/l butanol and 16.68 ± 0.22 g/l ABE were produced under non-sterile and aerobic conditions, which can reduce industrial-scale production costs.
In the quest for identifying novel renewable energy sources, higher alcohols from fermentative processes have received enormous interest in the last decades. Commercial microbial butanol production through the traditional acetone-butanol-ethanol process was common in the first half of the 20th century, and many attempts are underway to revive this process for butanol production. In addition to butanol, other linear and branched higher alcohols hold great promise as alternative energy sources. Although Clostridium species can naturally produce butanol, most of the other higher alcohols are synthesized in nonnative hosts. This requires the construction of novel pathways, the introduction of heterologous genes, and extensive genetic manipulation of host strains. Therefore, this chapter aims to demonstrate metabolic pathways for the synthesis of various higher alcohols. Moreover, in this chapter, metabolic engineering studies for the production of higher alcohols are reviewed. Recent advances and challenges associated with the microbial synthesis of higher alcohols are discussed.
The second-generation biobutanol production from lignocellulosic material is one of the key interests for research, considering the future of alternative energies based on the circular economy action plans. An important part of the butanol production process depends on the substrate, and therefore on the pretreatment technologies available for decreasing costs and to obtain the highest release of sugars, for a better performance of the microorganism during fermentation. This chapter is a current overview of the diverse pretreatment processes based on their classification, importance, drawbacks, and applications with the objective of facilitating to identify the elements to be considered specifically for butanol production using Clostridium strains.