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C. phytofermentans growth in GS2 glucose medium containing different concentrations of acetate (A), formate (B), 5-hydroxymethylfurfural (C), furfural (D), coumarate (E), ferulate (F), vanillate (G), 4-hydroxybenzoic acid (H), vanillin (I), benzaldehyde (J), syringaldehyde (K), and catechol (L). Colors show inhibitor concentrations (g liter⁻¹): gray (0), purple (1), blue (2), dark green (3), light green (5), yellow (10), orange (20), and red (30). Data show mean cell density (OD600) of 4 cultures ± standard deviation (SD).
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Increasing the resistance of plant-fermenting bacteria to lignocellulosic inhibitors is useful to understand microbial adaptation and to develop candidate strains for consolidated bioprocessing. Here, we study and improve inhibitor resistance in Clostridium phytofermentans (also called Lachnoclostridium phytofermentans), a model anaerobe that ferme...
Citations
... Existing molecular tools derived from Lachnospiraceae are promising candidates to advance genome engineering in these bacteria, including Cas proteins with broadened PAM recognition (Tran et al., 2021), anti-CRISPR proteins (Forsberg et al., 2019), and cytosine deaminases (Guo et al., 2023). Moreover, adaptive laboratory evolution can be used to generate Lachnospiraceae strains with complex, multigenic traits such as inhibitor tolerance that are intractable by rational genome engineering (Cerisy et al., 2017). ...
The Lachnospiraceae is a family of anaerobic bacteria in the class Clostridia with potential to advance the bio-economy and intestinal therapeutics. Some species of Lachnospiraceae metabolize abundant, low-cost feedstocks such as lignocellulose and carbon dioxide into value-added chemicals. Others are among the dominant species of the human colon and animal rumen, where they ferment dietary fiber to promote healthy gut and immune function. Here, we summarize recent studies of the physiology, cultivation, and genetics of Lachnospiraceae, highlighting their wide substrate utilization and metabolic products with industrial applications. We examine studies of these bacteria as Live Biotherapeutic Products (LBPs), focusing on in vivo disease models and clinical studies using them to treat infection, inflammation, metabolic syndrome, and cancer. We discuss key research areas including elucidation of intra-specific diversity and genetic modification of candidate strains that will facilitate the exploitation of Lachnospiraceae in industry and medicine.
... That the hydrolysate system exhibited a low butanol concentration could have been caused by some inhibitors (e.g. phenolic compounds and organic acids) in the hydrolysate that can decrease the microorganism's activity [53], whereas the whole slurry system showed the highest concentration of butanol due to the advantage of cell immobilization [54]. ...
Water hyacinth (WH; Eichhornia crassipes) is an invasive and free-floating aquatic plant that is rapidly spreading
over the world causing various environmental problems. The utilization of WH as a feedstock for biofuel production
is a challenging solution to overcome the environmental problem as well as address the energy shortage.
Due to the complex structure of lignocellulosic materials, pretreatment is an inevitable step to maximize the
sugar yield before being used as a substrate in fermentation. The aim of this study was to investigate the utilization
of WH for biobutanol production via acetone-butanol-ethanol fermentation by Clostridium beijerinckii JCM
8026. The WH was pretreated with sodium hydroxide (NaOH) at various concentrations [0, 2, and 4% (w/v)],
reaction times (30, 60, and 90 min), and temperatures (80, 100, and 120 ◦C). The pretreatment results were
optimized using response surface methodology with a Box-Behnken design and Minitab software analysis. The
maximum total sugar concentration (21.05 g/L) following cellulose digestion was obtained after a 2% (w/v)
NaOH for 90 min at 100 ◦C. This hydrolysate slurry gave a subsequent butanol concentration, yield, and productivity
of 2.09 g/L, 0.15 g/g, and 0.012 g/L/h, respectively, following C. beijerinckii JCM 8026 fermentation.
To help understand the effect of the pretreatments, changes in the pretreated samples were observed by surface
area analyses, scanning electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, and
thermogravimetric analysis.
... In addition, it is unexpected that so many low-abundance OTUs positively correlated with degradation activities. For examples, the low-abundance bacteria belonging to family Veillonellaceae (OTU33 and OTU34), family Lachnospiraceae (OTU14) and genus Lachnoclostridium (OTU16) were reported to contribute to the plant fiber degradation (Cerisy et al. 2017;Vahidi et al. 2021); OTU41 (genus Faecalicatena) and OTU42 (genus Clostridium) showed positive correlation with xylan degradation rate but not cellulose degradation rate, suggesting their preference of decomposing hemicellulose; OTU44 belonging to genus Hydrogenoanaerobacterium was also highly positively correlated with the overall degradation rate, suggesting its beneficial role in lignocellulose decomposition which is previously unknown. These findings suggested that many non-dominant bacteria that were enriched during the revival conduced a lot to the lignocellulose degradation besides certain dominant ones. ...
Bacterial consortium is an important source of lignocellulolytic strains, but it is still a challenge to distinguish the direct decomposers of lignocellulose from other bacteria in such a complex community. This study aims at addressing this issue by focusing on the dynamic changes in community structure and degradation activity of MMBC-1, an established and stable lignocellulolytic bacterial consortium, during its subculturing revival. MMBC-1 was cryopreserved with glycerol as a protective agent and then inoculated for revival. Its enzyme activities for degradation recovered to the maximum level after two rounds of subculturing. Correspondingly, the cellulose and hemicellulose in lignocellulosic carbon source were gradually decomposed during the revival. Meanwhile, the initial dominant bacteria represented by genus Clostridium were replaced by the bacteria belonging to Lachnospira , Enterococcus , Bacillus , Haloimpatiens genera and family Lachnospiraceae. However, only three high-abundance (> 1%) operational taxonomic units (OTUs) ( Lachnospira , Enterococcus and Haloimpatiens genera) were suggested to directly engage in lignocellulose degradation according to correlation analysis. By comparison, many low-abundance OTUs, such as the ones belonging to Flavonifractor and Anaerotruncus genera, may play an important role in degradation. These findings showed the dramatic changes in community structure that occurred during the subculturing revival, and paved the way for the discovery of direct decomposers in a stable consortium.
Graphical Abstract
... Due to the presence of lignin, the phenolic compounds from lignin (e.g. aliphatic acids, furfural, 5-hydroxymethylfurfural, etc.) can impede the microorganism's activity (Cerisy et al. 2017;Shi et al. 2020). Therefore, the pretreatment of the VG biomass enhanced its potential as a carrier in the ABE fermentation. ...
Immobilization of microbe cells has been considered as a potential alternative for enhancing biobutanol productivity through acetone-butanol-ethanol (ABE) fermentation. Vetiver grass (VG; Vetiveria zizanoides) pretreated by sodium hydroxide (NaOH) or sulphuric acid was used as carrier for immobilized Clostridium beijerinckii TISTR 1461 in ABE fermentation. The removal of the lignin and hemicellulose fractions of VG during the pretreatment resulted in structural changes, especially an increased crystallinity and surface area. The VG pretreated with 6% (w/v) NaOH at 120 °C for 6 h had the best potential as a carrier in ABE fermentation, giving a butanol and total ABE concentration of 11.6 g/L and 19.8 g/L, respectively, approximately 1.3-fold greater than that of the free-cell system. The VG samples were characterized by X-ray diffraction, Fourier-transform infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy, and surface area analyses.
... However, the abundance of Acetobacter increases. Clostridium_sensu_stricto_12, among the dominant strains of S3, was a typical fermenting microorganism, which can use sugars as a substrate for electricity generation in MFC (Guo et al., 2016;Cerisy et al., 2017;Vanitha et al., 2017). In addition, the abundance of Burkholderia-Caballeronia-Paraburkholderia, which was known for rapid electronic collection and biodegradability, increased to 3.33%. ...
A vertically configured photocatalytic-microbial fuel cell (photo-MFC) is developed by combining a nanodiamond-decorated ZnO (ZnO/ND) photocathode with a bioanode. The system can effectively couple the light energy with bioenergy to enhance the degradation of volatile organic compounds (VOCs) and boost electricity output. Results show that the composite system exhibits increased performance for toluene removal (60.65%), higher than those of individual parts (ZnO/ND-photocatalysis: 37.16%, MFC: 17.81%). Furthermore, its electrochemical performance is dramatically increased. The peak power density of 120 mW/m² and the current density of 1.07 A/m² are generated under light illumination, which are about 1.57-fold and 1.37-fold higher than that under dark (76 mW/m², 0.78 A/m²), respectively. Microbial community analysis demonstrates Proteobacteria and Firmicute are dominant phyla, implying they play important roles on accelerating the extracellular-electron transfer and toluene degradation. In addition, the underlying mechanism for toluene degradation in the photo-MFC system is preliminary explored. Our results suggest that the photo-MFC has great potential for simultaneous treatment of VOCs with energy recovery.
... Characterization of evolved strains increases our understanding of how to achieve desired phenotypes (Portnoy et al., 2011;Zhang et al., 2009). Such characterization has identified changes to the membrane as contributing to the evolved phenotype (Cerisy et al., 2017;Chen et al., 2020;Royce et al., 2013), leading to the proposition of the cell membrane as a target for rational strain engineering Jezierska and Van Bogaert, 2017;Liu et al., 2013;Qi et al., 2019;Sandoval and Papoutsakis, 2016). Such engineering strategies could be proposed from previous data or modeling results (Monje-Galvan et al., 2019). ...
The cell membrane plays a central role in the fitness and performance of microbial cell factories and therefore it is an attractive engineering target. The goal of this work is to develop a systematic framework for identifying membrane features for use as engineering targets. The metrics that describe the composition of the membrane can be visualized as “knobs” that modulate various “outcomes”, such as physical properties of the membrane and metabolic activity in the form of growth and productivity, with these relationships varying depending on the condition. We generated a set of strains with altered membrane lipid composition via expression of des, fabA and fabB and performed a rigorous characterization of these knobs and outcomes across several individual inhibitory conditions. Here, the knobs are the relative abundance of unsaturated lipids and lipids containing cyclic rings; the average lipid length, and the ratio of linear and non-linear lipids (L/nL ratio). The outcomes are membrane permeability, hydrophobicity, fluidity, and specific growth rate. This characterization identified significant correlations between knobs and outcomes that were specific to individual inhibitors, but also were significant across all tested conditions. For example, across all conditions, the L/nL ratio is positively correlated with the cell surface hydrophobicity, and the average lipid length is positively correlated with specific growth rate. A subsequent analysis of the data with the individual inhibitors identified pairs of lipid metrics and membrane properties that were predicted to impact cell growth in seven modeled scenarios with two or more inhibitors. The L/nL ratio and the membrane hydrophobicity were predicted to impact cell growth with the highest frequency. We experimentally validated this prediction in the combined condition of 42 °C, 2.5 mM furfural and 2% v/v ethanol in minimal media. Membrane hydrophobicity was confirmed to be a significant predictor of ethanol production. This work demonstrates that membrane physical properties can be used to predict the performance of biocatalysts in single and multiple inhibitory conditions, and possibly as an engineering target. In this manner, membrane properties can possibly be used as screening or selection metrics for library- or evolution-based strain engineering.
... Second, metabolic engineered strains of C. acetobutylicum are able to produce a wide variety of interesting platform molecules [44][45][46]. Third, Clostridium bacteria present an important microbial adaptation and a good resistance to various inhibitors [47,48]. ...
The coupling of thermochemical and biological conversion of biomass is a promising strategy to produce chemicals in future integrated biorefineries. Indeed, thermochemical conversion such as pyrolysis is a fast process without any solvent or enzyme for the depolymerisation of biomass. In this work, cellulose was pyrolyzed to produce sugars which have been then fermented by bacteria (Clostridium acetobutylicum) to produce acetone and butanol. This type of bacteria presents an interesting biological platform: it is resilient, easily up-scalable and Clostridium can be genetically engineered to target various other chemicals. Pyrolysis of cellulose was performed in a continuous fluidized bed reactor equipped with a staged condensation system, including a warm electrostatic precipitator. Different bio-oil fractions rich in levoglucosan (LVG) and with different concentrations in inhibitors for the fermentation stage were produced. LVG was found to be non-fermentable by C. acetobutylicum. Therefore, the bio-oil fractions were hydrolysed to obtain fermentable glucose. The mechanisms of acid hydrolysis (with diluted H2SO4) of LVG and cellobiosan have been revealed by high resolution mass spectrometry. The microorganisms were not able to grow with all hydrolysed bio-oil fractions depending on the concentration in inhibitors (aldehydes and organic acids). The fractions rich in LVG (and then glucose) lead to normal bacterial growth and normal fermentation products pattern without the need of detoxification. These results show the importance of a pyrolysis process with a staged condensation as a preliminary step for fermentation. It opens the road to production of various cellulose-derived chemicals by bacteria.
... Remarkably, examining the heatmap of environment factors, Lachnoclostridium, a representative biomarker of harmful bacteria, was positively correlated with both the expression of inflammatory cytokines and infection rates, while it was negatively correlated with SCFAs and mucin expression in our experiments. Few studies have addressed species of this genus, especially its function in gut health, and have only concerned the decomposition of lignin and metabolism of tryptophan [41,42]. Alistipes, Ruminiclostridium, Bilophila, Mucispirillum, and Eubacterium xylanophilum were similarly closely associated with environmental factors, so concerns need to be raised regarding them. ...
As the richest component in human milk oligosaccharides (HMOs), 2’-fucosyllactose (2’-FL) can reduce the colonization of harmful microbiota in vivo, thus lowering the risk of infection; however, the mechanism for this is still unclear. In this study, a model of Escherichia coli O157 infection in healthy adult mice was established to explore the effect of 2’-FL intervention on E. coli O157 colonization and its protective effects on mice. The results showed that 2’-FL intake reduced E. coli O157 colonization in mice intestine by more than 90% (p < 0.001), and it also reduced intestinal inflammation, increased the content of fecal short-chain fatty acids, and enhanced intestinal barrier function. These beneficial effects were attributed to the increased expression of mucins such as MUC2 (increased by more than 20%, p < 0.001), and inhibition of E. coli O157 cell adhesion (about 30% reduction, p < 0.001), and were associated with the modulation of gut microbiota composition. 2’-FL significantly increased the abundance of Akkermansia, a potential probiotic, which may represent the fundamental means by which 2’-FL enhances the expression of mucin and reduces the colonization of harmful bacteria. The current study may support the use of 2’-FL in the prevention of foodborne pathogen infections in human.
... Recently, Lin et al. showed that low-molecular weight phenolic compounds are able to diffuse into the cell more rapidly, causing more pronounced inhibition on glucose assimilation and thus more toxicity than high-molecular weight phenolic compounds (Lin et al., 2015). Exposure of Clostridium phytofermentans to ferulic acid was found to induce expression of cphy1055-cphy1056 (Cerisy et al., 2017), an efflux pump gene similar to mdlAB found in E. coli, which is known to confer resistance to organic solvents (Foo et al., 2014). Pattrick et al. identified 147 proteins that displayed a significant change in abundance in E. coli cells exposed to vanillin. ...
The rapid development in the field of metabolic engineering has enabled complex modifications of metabolic pathways to generate a diverse product portfolio. Manipulating substrate uptake and product export is an important research area in metabolic engineering. Optimization of transport systems has the potential to enhance microbial production of renewable fuels and chemicals. This chapter comprehensively reviews the transport systems critical for microbial production as well as current genetic engineering strategies to improve transport functions and thus production metrics. In addition, this chapter highlights recent advancements in engineering microbial efflux systems to enhance cellular tolerance to industrially relevant chemical stress. Lastly, future directions to address current technological gaps are discussed.
... C. phytofermentans ISDg (ATCC 700394) was cultured anaerobically at 30°C in GS2 medium (44) containing 3 g liter Ϫ1 of either glucose (G5767; Sigma), xylose (X3877; Sigma), cellobiose (C7252; Sigma), or galactan (P-GALLU; Megazyme). Growth on different carbon sources was measured in 400-l cultures in 100-well microtiter plates (9502550; Bioscreen) that were sealed in the anaerobic chamber (2% H 2 , 98% N 2 ) as previously described (45). Cell densities were measured using a Thermo Scientific Bioscreen C as the optical density at 600 nm (OD 600 ) with 30 s of shaking before each reading. ...
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.
