Abstract
BackgroundClostridium (Ruminiclostridium) thermocellum is a model fermentative anaerobic thermophile being studied and engineered for consolidated bioprocessing of lignocellulosic feedstocks into fuels and chemicals. Engineering efforts have resulted in significant improvements in ethanol yields and titers although further advances are required to make the bacterium industry-ready. For instance, fermentations at lower pH could enable co-culturing with microbes that have lower pH optima, augment productivity, and reduce buffering cost. C. thermocellum is typically grown at neutral pH, and little is known about its pH limits or pH homeostasis mechanisms. To better understand C. thermocellum pH homeostasis we grew strain LL1210 (C. thermocellum DSM1313 Δhpt ΔhydG Δldh Δpfl Δpta-ack), currently the highest ethanol producing strain of C. thermocellum, at different pH values in chemostat culture and applied systems biology tools.ResultsClostridium thermocellum LL1210 was found to be growth-limited below pH 6.24 at a dilution rate of 0.1 h−1. F1F0-ATPase gene expression was upregulated while many ATP-utilizing enzymes and pathways were downregulated at pH 6.24. These included most flagella biosynthesis genes, genes for chemotaxis, and other motility-related genes (> 50) as well as sulfate transport and reduction, nitrate transport and nitrogen fixation, and fatty acid biosynthesis genes. Clustering and enrichment of differentially expressed genes at pH values 6.48, pH 6.24 and pH 6.12 (washout conditions) compared to pH 6.98 showed inverse differential expression patterns between the F1F0-ATPase and genes for other ATP-utilizing enzymes. At and below pH 6.24, amino acids including glutamate and valine; long-chain fatty acids, their iso-counterparts and glycerol conjugates; glycolysis intermediates 3-phosphoglycerate, glucose 6-phosphate, and glucose accumulated intracellularly. Glutamate was 267 times more abundant in cells at pH 6.24 compared to pH 6.98, and intercellular concentration reached 1.8 μmol/g pellet at pH 5.80 (stopped flow).ConclusionsClostridium thermocellum LL1210 can grow under slightly acidic conditions, similar to limits reported for other strains. This foundational study provides a detailed characterization of a relatively acid-intolerant bacterium and provides genetic targets for strain improvement. Future studies should examine adding gene functions used by more acid-tolerant bacteria for improved pH homeostasis at acidic pH values.
Highlights
Clostridium (Ruminiclostridium) thermocellum is a model fermentative anaerobic thermophile being studied and engineered for consolidated bioprocessing of lignocellulosic feedstocks into fuels and chemicals
C. thermocellum LL1210 chemostat growth and lower pH limits in MTC medium Clostridium thermocellum LL1210 was cultured in duplicate chemostats fed with defined medium (MTC) containing 3 g/L cellobiose for 1058 h (Fig. 1, Additional file 1)
At pH values at or below 6.24, we observed gene expression patterns and metabolite levels consistent with five pH homeostasis mechanisms used by C. thermocellum LL1210, namely (i) proton pumping by F1F0-ATPase, (ii) macromolecule protection by chaperones, and (iii) prevention of proton influx through MotAB were mechanisms supported by changes in gene expression (Fig. 2, Additional file 2); (iv) proton pumping by PPi-ases was supported by metabolomics (Table 1); and (v) changes in membrane fatty acid composition was supported by both techniques (Fig. 2, Additional file 2, Table 1)
Summary
Clostridium (Ruminiclostridium) thermocellum is a model fermentative anaerobic thermophile being studied and engineered for consolidated bioprocessing of lignocellulosic feedstocks into fuels and chemicals. In the highest yielding C. thermocellum strain published to date, strain LL1210, pathways for the synthesis of lactic acid, acetic acid, formic acid, and most H 2 production were eliminated [12], followed by adaptive laboratory evolution to improve growth rate and ethanol titer [18]. This allowed the strain to produce 22 g/L ethanol from 60 g/L cellulose, with a maximum theoretical yield for ethanol of 75% further advances are required for the bacterium to be industry-ready. While these strains synthesize essentially no organic acids as end products, fermentation of sugars to reduced compounds such as ethanol or butanol results in the production of a more oxidized compound such as bicarbonate, which will result in acidification of the medium
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