![Neighbor-joining tree of C. phytofermentans and related taxa within the class Clostridia based on 16S rRNA gene sequences.
Taxa with sequenced genomes are marked with an asterisk. Cluster numbers correspond to the cluster system of Collins et al. [68]. Bootstrap values were determined for 1,000 replicates.](https://www.researchgate.net/profile/Andrew-Tolonen/publication/278370229/figure/fig2/AS:340492173758468@1458191068844/Neighbor-joining-tree-of-C-phytofermentans-and-related-taxa-within-the-class-Clostridia_Q320.jpg)
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Genome and Transcriptome of Clostridium phytofermentans, Catalyst for the Direct Conversion of Plant Feedstocks to Fuels
PLOS ONE
Abstract and Figures
Clostridium phytofermentans was isolated from forest soil and is distinguished by its capacity to directly ferment plant cell wall polysaccharides into ethanol as the primary product, suggesting that it possesses unusual catabolic pathways. The objective of the present study was to understand the molecular mechanisms of biomass conversion to ethanol in a single organism, Clostridium phytofermentans, by analyzing its complete genome and transcriptome during growth on plant carbohydrates. The saccharolytic versatility of C. phytofermentans is reflected in a diversity of genes encoding ATP-binding cassette sugar transporters and glycoside hydrolases, many of which may have been acquired through horizontal gene transfer. These genes are frequently organized as operons that may be controlled individually by the many transcriptional regulators identified in the genome. Preferential ethanol production may be due to high levels of expression of multiple ethanol dehydrogenases and additional pathways maximizing ethanol yield. The genome also encodes three different proteinaceous bacterial microcompartments with the capacity to compartmentalize pathways that divert fermentation intermediates to various products. These characteristics make C. phytofermentans an attractive resource for improving the efficiency and speed of biomass conversion to biofuels.
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... The overabundance of Cazymes is not necessarily related to high enzymatic activity, as it depends on the genomic context such as the possible presence of a promoter (Williams-Rhaesa et al. 2018), operon (Chakraborty et al. 2021). Indeed, Clostridia phytofermentas, which is a very good lignocellulolytic degrader (Tolonen et al. 2011) and expresses several high lignocellulolytic enzymatic activities (Petit et al. 2015), secreted approximately 80 Cazymes (among the 161 present in its genome) in silico, which confirms that a very high abundance of Cazymes does not necessarily indicate excellent enzymatic activity. ...
The Acidobacteria phylum is a very abundant group (20–30% of microbial communities in soil ecosystems); however, little is known about these microorganisms and their ability to degrade the biomass and lignocellulose due to the difficulty of culturing them. We, therefore, bioinformatically studied the content of lignocellulolytic enzymes (total and predicted secreted enzymes) and secreted peptidases in an in silico library containing 41 Acidobacteria genomes. The results showed a high abundance and diversity of total and secreted Carbohydrate-Active enzymes (cazyme) families among the Acidobacteria compared to known previous degraders. Indeed, the relative abundance of cazymes in some genomes represented more than 6% of the gene coding proteins with at least 300 cazymes. The same observation was made with the predicted secreted peptidases with several families of secreted peptidases, which represented at least 1.5% of the gene coding proteins in several genomes. These results allowed us to highlight the lignocellulolytic potential of the Acidobacteria phylum in the degradation of lignocellulosic biomass, which could explain its high abundance in the environment.
... 1−4 Clostridium phytofermentans (also called Lachnoclostridium phytofermentans) is a member of the family Lachnospiraceae that is distinguished by its ability to ferment lignocellulosic biomass with ethanol, hydrogen, and acetate as major products. 5,6 Advances in genetic manipulation are needed to exploit the therapeutic and industrial potential of additional Clostridia species. In particular, experimental approaches for in vivo modulation of gene expression have a few prerequisites. ...
Control of gene expression is fundamental to cell engineering. Here we demonstrate a set of approaches to tune gene expression in Clostridia using the model Clostridium phytofermentans. Initially, we develop a simple benchtop electroporation method that we use to identify a set of replicating plasmids and resistance markers that can be cotransformed into C. phytofermentans. We define a series of promoters spanning a >100-fold expression range by testing a promoter library driving the expression of a luminescent reporter. By insertion of tet operator sites upstream of the reporter, its expression can be quantitatively altered using the Tet repressor and anhydrotetracycline (aTc). We integrate these methods into an aTc-regulated dCas12a system with which we show in vivo CRISPRi-mediated repression of reporter and fermentation genes in C. phytofermentans. Together, these approaches advance genetic transformation and experimental control of gene expression in Clostridia.
... In addition to Neurospora crassa, numerous filamentous Ascomycota are known for their prolific cellulase and hemicellulase activity, which make them commonly used in industry (Shah et al., 2017). Such fungal species, including Trichoderma reesei, were detected in our protein datasets, as were a number of bacterial species capable of fermenting complex organic matter, including Clostridium phytofermentans (Petit et al., 2015) and C. cellulolyticum (Desvaux, 2005). Enterobacteriales and Saccharomycetales were also involved in hydrolysis, with Enterobacter previously reported as highly efficient hydrolysers of food waste at low pH (Yan et al., 2014). ...
As the human population grows on the planet so does the generation of waste and particularly that of food waste. In order to tackle the world sustainability crisis, efforts to recover products from waste are critical. Here, we anaerobically recovered volatile fatty acids (VFAs) from food waste and analysed the microbial populations underpinning the process. An increased contribution of fungi relative to bacteria was observed throughout the reactor operation, with both kingdoms implicated into the main three steps of anaerobic digestion occurring within our systems: hydrolysis, acidogenesis and acetogenesis. Overall, Ascomycota, Proteobacteria and Firmicutes were found to drive the anaerobic digestion of food waste, with butyrate as the most abundant VFA likely produced by Clostridium using lactate as a precursor. Taken together we demonstrate that the generation of products of added-value from food waste results from cross-kingdoms microbial activities implicating fungi and bacteria.
... They found that 149.6 mL H 2 /g-VS yield was achieved by the addition of 37.5 mg/L of every nanoparticle. Some researchers used clostridium as a biocatalyst for different applications because clostridium species have a rich tradition in biofuel improvement [207]. Clostridium acetobutylicum has been employed for butanol, acetone and ethanol production from starch and is a profitable established valued bacterium [208]. ...
Global population growth and accelerated urbanisation have resulted in massive amounts of fossil fuel use and waste production. Because of its high energy content, pure nature, and fuel quality, hydrogen fuel is a viable option to fossil fuels. Biohydrogen from agricultural waste, in particular, piques concern because it generates hydrogen while still disposing of waste. This review conducted a bibliometric analysis of biohydrogen production from organic waste to trace the research trends and hotspots based on the literature in the Web of Science (WOS) database from 1970 to 2020. The present review article also focuses on highlighting various processes for converting organic waste into hydrogen, raw materials for biohydrogen production, and catalysts that could distil the latest perceptions that could shed light on a route advancing for successful catalyst design. It also seems that some intentions have been paid on studying waste materials such as pure polysaccharides, disaccharides, and monosaccharides. Among all the catalysts used, non-noble and low-cost active metals over reduced graphene oxide (rGO) support can significantly affect the activity of fermentative hydrogen production from organic waste materials. However, researches focusing on developing anaerobic membrane bioreactors for these technologies are still needed.
... C. phytofermentans is remarkable among the Clostridium genus due to its ability to catabolize a broad range of substrates. Its genome encodes over 169 carbohydrate-active enzymes, the largest number among sequenced clostridia, and its efficient ethanol production makes it a model system for cellulosic biofuel production 21,23,[34][35][36][37] . E. coli is a well studied, facultative anaerobe capable of fermenting a broad range of substrates including glucose and glycerol which is a widely available waste product from biodiesel production 21,23,38 . ...
Planktonic cultures, of a rationally designed consortium, demonstrated emergent properties that exceeded the sums of monoculture properties, including a >200% increase in cellobiose catabolism, a >100% increase in glycerol catabolism, a >800% increase in ethanol production, and a >120% increase in biomass productivity. The consortium was designed to have a primary and secondary-resource specialist that used crossfeeding with a positive feedback mechanism, division of labor, and nutrient and energy transfer via necromass catabolism. The primary resource specialist was Clostridium phytofermentans ( a.k.a. Lachnoclostridium phytofermentans ), a cellulolytic, obligate anaerobe. The secondary-resource specialist was Escherichia coli , a versatile, facultative anaerobe, which can ferment glycerol and byproducts of cellobiose catabolism. The consortium also demonstrated emergent properties of enhanced biomass accumulation when grown as biofilms, which created high cell density communities with gradients of species along the vertical axis. Consortium biofilms were robust to oxic perturbations with E. coli consuming O 2 , creating an anoxic environment for C. phytofermentans . Anoxic/oxic cycling further enhanced biomass productivity of the biofilm consortium, increasing biomass accumulation ~250% over the sum of the monoculture biofilms. Consortium emergent properties were credited to several synergistic mechanisms. E. coli consumed inhibitory byproducts from cellobiose catabolism, driving higher C. phytofermentans growth and higher cellulolytic enzyme production, which in turn provided more substrate for E. coli . E. coli necromass enhanced C. phytofermentans growth while C. phytofermentans necromass aided E. coli growth via the release of peptides and amino acids, respectively. In aggregate, temporal cycling of necromass constituents increased flux of cellulose-derived resources through the consortium. The study establishes a consortia-based, bioprocessing strategy built on naturally occurring interactions for improved conversion of cellulose-derived sugars into bioproducts.
... L. phytofermentans is an obligately anaerobic fibrolytic bacterium that can ferment a wide range of plant polysaccharides [38]. This ability appears to be due to the numerous and diverse range of glycosyl hydrolases encoded within its genome, many of which have been acquired by horizontal gene transfer [40]. Whilst the L. phytofermentans type strain was isolated from forest soil, its optimum temperature for growth was reported to be 37°C with growth observed at pH 6.0-9.0 [38]. ...
Background
Compared to horses and ponies, donkeys have increased degradation of dietary fiber. The longer total mean retention time of feed in the donkey gut has been proposed to be the basis of this, because of the increased time available for feed to be acted upon by enzymes and the gut microbiota. However, differences in terms of microbial concentrations and/or community composition in the hindgut may also underpin the increased degradation of fiber in donkeys. Therefore, a study was conducted to assess if differences existed between the fecal microbiota of pony, donkey and hybrids derived from them (i.e. pony × donkey) when fed the same forage diet.
Results
Fecal community composition of prokaryotes and anaerobic fungi significantly differed between equine types. The relative abundance of two bacterial genera was significantly higher in donkey compared to both pony and pony x donkey: Lachnoclostridium 10 and ‘probable genus 10’ from the Lachnospiraceae family. The relative abundance of Piromyces was significantly lower in donkey compared to pony × donkey, with pony not significantly differing from either of the other equine types. In contrast, the uncultivated genus SK3 was only found in donkey (4 of the 8 animals). The number of anaerobic fungal OTUs was also significantly higher in donkey than in the other two equine types, with no significant differences found between pony and pony × donkey. Equine types did not significantly differ with respect to prokaryotic alpha diversity, fecal dry matter content or fecal concentrations of bacteria, archaea and anaerobic fungi.
Conclusions
Donkey fecal microbiota differed from that of both pony and pony × donkey. These differences related to a higher relative abundance and diversity of taxa with known, or speculated, roles in plant material degradation. These findings are consistent with the previously reported increased fiber degradation in donkeys compared to ponies, and suggest that the hindgut microbiota plays a role. This offers novel opportunities for pony and pony × donkey to extract more energy from dietary fiber via microbial mediated strategies. This could potentially decrease the need for energy dense feeds which are a risk factor for gut-mediated disease.
... T he clostridia are Gram-positive obligately anaerobic bacteria that include human pathogens as well as plant-fermenting species critical for healthy functioning of soil and gut microbiomes. Clostridium (Lachnoclostridium) phytofermentans ISDg (1) is a model plant-fermenting Clostridium that breaks down plant biomass using numerous carbohydrate-active enzymes (CAZymes) and ferments the resulting hexose and pentose sugars into ethanol, hydrogen, and acetate (2)(3)(4). C. phytofermentans is a member of the Lachnospiraceae family that is abundant in soil (5), dominates the rumen (6), and includes human gut commensals that play important roles in nutrition and intestinal health (7). Because of their ability to directly ferment lignocellulose, plant-fermenting clostridia have industrial potential for the transformation of plant biomass into valueadded chemicals. ...
Clostridia are a group of Gram-positive anaerobic bacteria of medical and industrial importance for which limited genetic methods are available. Here, we demonstrate an approach to make large genomic deletions and insertions in the model Clostridium phytofermentans by combining designed group II introns (targetrons) and Cre recombinase. We apply these methods to delete a 50-gene prophage island by programming targetrons to position markerless lox66 and lox71 sites, which mediate deletion of the intervening 39-kb DNA region using Cre recombinase. Gene expression and growth of the deletion strain showed that the prophage genes contribute to fitness on nonpreferred carbon sources. We also inserted an inducible fluorescent reporter gene into a neutral genomic site by recombination-mediated cassette exchange (RMCE) between genomic and plasmid-based tandem lox sites bearing heterospecific spacers to prevent intracassette recombination. These approaches generally enable facile markerless genome engineering in clostridia to study their genome structure and regulation.
IMPORTANCE Clostridia are anaerobic bacteria with important roles in intestinal and soil microbiomes. The inability to experimentally modify the genomes of clostridia has limited their study and application in biotechnology. Here, we developed a targetron-recombinase system to efficiently make large targeted genomic deletions and insertions using the model Clostridium phytofermentans . We applied this approach to reveal the importance of a prophage to host fitness and introduce an inducible reporter by recombination-mediated cassette exchange.
The discovery and characterization of bacterial carbohydrate-active enzymes is a fundamental component of biotechnology innovation, particularly for renewable fuels and chemicals; however, these studies have increasingly transitioned to exploring the complex regulation required for recalcitrant polysaccharide utilization. This pivot is largely due to the current need to engineer and optimize enzymes for maximal degradation in industrial or biomedical applications. Given the structural simplicity of a single cellulose polymer, and the relatively few enzyme classes required for complete bioconversion, the regulation of cellulases in bacteria has been thoroughly discussed in the literature. However, the diversity of hemicelluloses found in plant biomass and the multitude of carbohydrate-active enzymes required for their deconstruction has resulted in a less comprehensive understanding of bacterial hemicellulase-encoding gene regulation. Here we review the mechanisms of this process and common themes found in the transcriptomic response during plant biomass utilization. By comparing regulatory systems from both Gram-negative and Gram-positive bacteria, as well as drawing parallels to cellulase regulation, our goals are to highlight the shared and distinct features of bacterial hemicellulase-encoding gene regulation and provide a set of guiding questions to improve our understanding of bacterial lignocellulose utilization.
Key points
• Canonical regulatory mechanisms for bacterial hemicellulase-encoding gene expression include hybrid two-component systems (HTCS), extracytoplasmic function (ECF)-σ/anti-σ systems, and carbon catabolite repression (CCR).
• Current transcriptomic approaches are increasingly being used to identify hemicellulase-encoding gene regulatory patterns coupled with computational predictions for transcriptional regulators.
• Future work should emphasize genetic approaches to improve systems biology tools available for model bacterial systems and emerging microbes with biotechnology potential. Specifically, optimization of Gram-positive systems will require integration of degradative and fermentative capabilities, while optimization of Gram-negative systems will require bolstering the potency of lignocellulolytic capabilities.
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.
Co-utilization of hexoses and pentoses derived from lignocellulose is an attractive trait in microorganisms considered for consolidated biomass processing to biofuels. This issue was examined for the H2-producing, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus growing on individual monosaccharides (arabinose, fructose, galactose, glucose, mannose and xylose), mixtures of these sugars, as well as on xylan and xyloglucooligosacchrides. C. saccharolyticus grew at approximately the same rate (td approximately 95 min) and to the same final cell density (1-3 x 10(8) cells/ml) on all sugars and sugar mixtures tested. In the monosaccharide mixture, while simultaneous consumption of all monosaccharides was observed, not all were utilized to the same extent (fructose > xylose/arabinose > mannose/glucose/galactose). Transcriptome contrasts for monosaccharide growth revealed minimal changes in some cases (e.g., 31 ORFs changed >/= 2-fold for glucose vs. galactose) while substantial changes occurred for cases involving mannose (e.g., 363 ORFs >/= 2-fold for glucose vs. mannose). Evidence for catabolite repression was noted neither for growth on multi-sugar mixtures nor in the corresponding transcriptomes. Based on the whole-genome transcriptional response analysis and comparative genomics, carbohydrate specificities for transport systems could be proposed for most of the 24 putative carbohydrate ATP-binding cassette (ABC) transporters and single phosphotransferase system (PTS) identified in C. saccharolyticus. While most transporter genes responded to individual monosacchrides and polysaccharides, Csac_0692-0694 was up-regulated only in the monosaccharide mixture. The results here affirm the broad growth substrate preferences of C. saccharolyticus on carbohydrates representative of lignocellulosic biomass and suggest that this bacterium holds promise for biofuels applications
This chapter presents factors that are considered in developing stringently anaerobic techniques and in describing the procedure and rationale of roll-tube method. A roll-tube method was developed in which agar medium was distributed as a thin layer over the internal surface of test tubes charged with an anaerobic atmosphere for the isolation of obligately anaerobic bacteria of the rumen. In the roll-tube method, exposure of bacteria and culture medium to air is avoided by displacing the air in the culture vessel with an oxygen-free gas, such as carbon dioxide, hydrogen, nitrogen, or mixtures of these gases. Carbon dioxide is the gas of choice because it is heavier than air, relatively cheap, and valuable in buffering. Vessels are stoppered under conditions preventing access of air. The cultures require no special incubators and can be removed and examined with no anaerobic precautions if kept stoppered. If opened, anaerobiosis can be continuously maintained during necessary manipulations, and the culture again closed without exposure to oxygen.
Pfam is a large collection of protein multiple sequence alignments and profile hidden Markov models. Pfam is available on
the WWW in the UK at http://www.sanger.ac.uk/Software/Pfam/ , in Sweden at http://www.cgr.ki.se/Pfam/ and in the US at http://pfam.wustl.edu/
. The latest version (4.3) of Pfam contains 1815 families. These Pfam families match 63% of proteins in SWISS-PROT 37 and
TrEMBL 9. For complete genomes Pfam currently matches up to half of the proteins. Genomic DNA can be directly searched against
the Pfam library using the Wise2 package.
Pfam is a large collection of protein multiple sequence alignments and profile hidden Markov models. Pfam is available on
the World Wide Web in the UK at http://www.sanger.ac.uk/Software/Pfam/, in Sweden at http://www.cgb.ki.se/Pfam/, in France at http://pfam.jouy.inra.fr/ and in the US at http://pfam.wustl.edu/. The latest version (6.6) of Pfam contains 3071 families, which match 69% of proteins in SWISS-PROT 39 and TrEMBL 14. Structural
data, where available, have been utilised to ensure that Pfam families correspond with structural domains, and to improve
domain-based annotation. Predictions of non-domain regions are now also included. In addition to secondary structure, Pfam
multiple sequence alignments now contain active site residue mark-up. New search tools, including taxonomy search and domain
query, greatly add to the functionality and usability of the Pfam resource.
The anaerobic acetogenic bacterium Acetobacterium woodii carries out a unique type of Na⁺-motive, anaerobic respiration with caffeate as electron acceptor, termed “caffeate respiration.” Central, and so far the only identified membrane-bound reaction in this respiration pathway, is a ferredoxin:NAD⁺ oxidoreductase (Fno) activity. Here we show that inverted membrane vesicles of A. woodii couple electron transfer from reduced ferredoxin to NAD⁺ with the transport of Na⁺ from the outside into the lumen of the vesicles. Na⁺ transport was electrogenic, and accumulation was inhibited by sodium ionophores but not protonophores, demonstrating a direct coupling of Fno activity to Na⁺ transport. Results from inhibitor studies are consistent with the hypothesis that Fno activity coupled to Na⁺ translocation is catalyzed by the Rnf complex, a membrane-bound, iron–sulfur and flavin-containing electron transport complex encoded by many bacterial and some archaeal genomes. Fno is a unique type of primary Na⁺ pump and represents an early evolutionary mechanism of energy conservation that expands the redox range known to support life. In addition, it explains the lifestyle of many anaerobic bacteria and gives a mechanistic explanation for the enigma of the energetic driving force for the endergonic reduction of ferredoxin with NADH plus H⁺ as reductant in a number of aerobic bacteria.