Abstract
Clostridium (Ruminiclostridium) thermocellum is a model organism for its ability to deconstruct plant biomass and convert the cellulose into ethanol. The bacterium forms biofilms adherent to lignocellulosic feedstocks in a continuous cell-monolayer in order to efficiently break down and uptake cellulose hydrolysates. We developed a novel bioreactor design to generate separate sessile and planktonic cell populations for omics studies. Sessile cells had significantly greater expression of genes involved in catabolism of carbohydrates by glycolysis and pyruvate fermentation, ATP generation by proton gradient, the anabolism of proteins and lipids and cellular functions critical for cell division consistent with substrate replete conditions. Planktonic cells had notably higher gene expression for flagellar motility and chemotaxis, cellulosomal cellulases and anchoring scaffoldins, and a range of stress induced homeostasis mechanisms such as oxidative stress protection by antioxidants and flavoprotein co-factors, methionine repair, Fe-S cluster assembly and repair in redox proteins, cell growth control through tRNA thiolation, recovery of damaged DNA by nucleotide excision repair and removal of terminal proteins by proteases. This study demonstrates that microbial attachment to cellulose substrate produces widespread gene expression changes for critical functions of this organism and provides physiological insights for two cells populations relevant for engineering of industrially-ready phenotypes.
Highlights
Clostridium (Ruminiclostridium) thermocellum is a fermentative anaerobic thermophile that is being engineered for use in the consolidated bioprocessing of second-generation bio-feedstocks into liquid fuels and other chemicals[1,2]
In C. thermocellum, biofilms specialized for growth on crystalline cellulose and capable of nutrient capture create conditions mimicking continuous carbon limitation for planktonic cells; a phenomenon best exemplified by the low detection of soluble glucans during fermentation[16]
Gene expression in C. thermocellum was described for growth on cellulose[17], switchgrass and poplar[18,19] and after induced chemical stress[20,21] the discrete analysis of the two cell populations has not yet been performed
Summary
Cultures of Clostridium (Ruminiclostridium) thermocellum were grown in batch bioreactors with controlled mixing, pH, temperature and nitrogen gas purge with solid Whatman paper #3 as the cellulose carbon source. Co-existent sessile and planktonic cell fractions were collected (in four independent biological replicate fermentations) for RNA-Seq analysis ~7 hours after exiting the lag phase and shortly before reaching the highest point of culture activity (Fig. 2) at peak alkali titration rate – a common, reliable metric of fermentative product formation[22]. This ensured that biofilms did not reach the growth limitations imposed by maximally colonized cellulose surfaces, as defined in previous studies[8]. Planktonic cells (PL) expressed genes for cell motility, nutrient sensing, the extracellular hydrolysis of polymeric sugars, and for a range of protection mechanisms such as oxidative stress response and repair, cell growth
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