A Novel Parasitism Between Cellulolytic Clostridium Thermocellum And Non-cellulolytic Thermoanaerobacterium Thermosaccharolyticum

Gram-positive thermophilic Clostridium thermocellum is an anaerobic bacterium with a highly efficient cellulytic system. The hallmark of the system is an extracellular multienzyme complex, termed the cellusome. The cellusome can degrade microcrystalline cellulose to produce glucose and cellobiose, which are two inhibitors for cellulase synthesis and cannot be utilize completely by Clostridium thermocellum.In an early report, Clostridium thermocellum has always been constantly contaminated by non-cellulolytic bacteria that remove glucose and cellobiose producing by cellulose, which enhanced end-product formation and cellulose utilization. So the author reasoned a mutualism relationship between the two species. In this work, we reconstructed C. thermocellum-T. thermosaccharolyticum coculture, and analyze the levels of cellulase inhibitors and transcription of lignocellulose degradation related genes in mono-and co-cultures, cellulose utilization, end-product formation and so on. We further identified the relationship between C. thermocellum and T. thermosaccharolyticum as a parasitic relationship, showing T. thermosaccharolyticum removes the inhibition of transcription of lignocellulose degradation related genes by cellobiose and a novel repressor glucose.We first analyze levels of cellulase inhibitors and transcription of lignocellulose degradation related genes and end-product formation in mono-and co-cultures, which showing both the rate of formation and final concentration of these end-products of Avicel fermentation was clearly enhanced in the coculture. Consequently, the transcriptional levels of lignocellulose degradation related genes in C. thermocellum were significantly higher in the coculture than the monoculture.The analysis of residual cellulose in Avicel-grown C. thermocellum monoculture and the coculture of C. thermocellum/T. thermosaccharolyticum was carried out by either degrading residual cellulose in cultures with acid and analyzing evolved glucose, or degrading residual cellulose in cultures with Penicillium cellulases and analyzing evolved glucose. Surprisingly, from both methodology, the coculture degrades cellulose at the same rate with the coculture, in contrary to our original expectation and the mutualism model. To further identify the interactions between C. thermocellum and T. thermosaccharolyticum in the coculture, we performed a biomass analysis of these microbes. Using total protein content to resemble total biomass an improvement of total biomass could be observed in the coculture than the monoculture during growth on Avicel. We further identified the ratio of C. thermocellum to T. thermosaccharolyticum cells by quantifying 16S rDNA for each microbe in extracted genomic DNA in the coculture with high throughput sequencing, which showing that C. thermocellum growth is damaged by coculturing with T. thermosaccharolylicum during growth on both Avicel and corncob while T. thermosaccharolyticum cannot survive independently.The capability of C. thermocellum and T. thermosaccharolyticum to degrade glucose and cellobiose, as well as to produce end products lactate, acetate and ethanol was compared. T. thermosaccharolylicum clearly has a competitive advantage over C. thermocellum on glucose and cellobiose metabolism.In conclusion, the interaction between symbiotic C. thermocellum JN4 and T. thermosaccharolyticum GD17 was investigated, showing T. thermosaccharolyticum removes the inhibition of transcription of lignocellulose degradation related genes by cellobiose and a novel repressor glucose. The inability of T. thermosaccharolyticum to survive on both cellulose and natural substrate corncob, and the damaged growth of C. thermocellum during interaction with T. thermosaccharolyticum suggest a novel microbe-microbe parasitism between cellulolytic and non-cellulolytic bacteria. Discussion on this novel parasitism suggest this is a more stable relationship and interaction between cellulolytic and non-cellulolytic bacteria, backed up by our results showing a significant competitive advantage of T. thermosaccharolyticum over C. thermocellum on consumption of glucose and cellobiose, the primary energy and nutrient sources. This competitive advantage (and also the advantage in forming end-products) of non-cellulolytic bacteria also explains the improved end-products formation during coculturing with cellulolytic bacteria examplied by the coculture of C. thermocellum and T. thermosaccharolyticum, although the cellulolytic bacteria were damaged during the interactions.Last, we also researched the molocular mechanism of hydrogen production in C. thermocellum, and successfully expressed the three key subunits of Ech hydrogenase in Escherichia coli. Because of the time constraint, we not yet investigate the biochemical properties and physiological function of the membrane-bound Ech hydrogenase in C. thermocellum.