Facultative Anaerobe Caldibacillus debilis GB1: Characterization and Use in a Designed Aerotolerant, Cellulose-Degrading Coculture with Clostridium thermocellum

Abstract

bDevelopment of a designed coculture that can achieve aerotolerant ethanogenic biofuel production from cellulose can reduce the costs of maintaining anaerobic conditions during industrial consolidated bioprocessing (CBP). To this end, a strain of Caldibacillus debilis isolated from an air-tolerant cellulolytic consortium which included a Clostridium thermocellum strain was characterized and compared with the C. debilis type strain. Characterization of isolate C. debilis GB1 and comparisons with the type strain of C. debilis revealed significant physiological differences, including (i) the absence of anaerobic metabolism in the type strain and (ii) different end product synthesis profiles under the experimental conditions used. The designed cocultures displayed unique responses to oxidative conditions, including an increase in lactate production. We show here that when the two species were cultured together, the noncellulolytic facultative anaerobe C. debilis GB1 provided respiratory protection for C. thermocellum, allowing the synergistic utilization of cellulose even under an aerobic atmosphere. Isolation and characterization of organisms with the ability to degrade and ferment cellulose are essential steps toward enhancing biofuel production via consolidated bioprocessing (CBP) (1). Designing a culture that could achieve both cellulose degradation and ethanol production under nonreduced conditions would reduce the costs and complexity of culturing strictly anaerobic bacteria, as this would eliminate the need for maintaining a reduced environment. Clostridium thermocellum is perhaps the best-studied model organism capable of CBP under anaerobic conditions (2, 3). Coculturing a cellulolytic organism with a noncellulolytic partner capable of producing the desired end products at high rates and yields has been shown to improve the overall biofuel production capability via CBP (4) and increased cellulose hydrolysis rates (4, 5). Many biofuel-producing cellulolytic bacteria, including C. thermocellum, lack aerotolerance and have not been shown to be able to tolerate greater than 2% O 2 in the atmosphere (6, 7). While members of the genus Clostridium are typically described to be strict anaerobes, the genomes of some members, including Clostridium straminisolvens, Clostridium acetobutylicum, and Clostridium intestinalis, encode genes required for aerotolerance, and these organisms have been shown to grow in microaerobic environments (6,8,9). Moreover, genetic modification of C. acetobutylicum resulted in a significant increase in aerotolerance, such that the recombinant strain was able to grow in the presence of atmospheric concentrations of oxygen (10).Previously, it was reported that cocultures containing a Clostridium species paired with facultative aerobes can lead to oxygen removal in a sealed vessel followed by fermentation (11-13) or achieve aerotolerance via respiratory protection under microaerobic conditions (14). The importance of biofilms and the use of mixed cultures to help lignocellulosic degradation h...

Genome-Centric Analysis of a Thermophilic and Cellulolytic Bacterial Consortium Derived from Composting

“…G. thermoglucosidasius C56-YS93 is known as a biomass degrader and was isolated from the Obsidian hot spring in Yellowstone National Park (Brumm et al, 2015 ). A noncellulolytic facultative anaerobe C. debilis strain (GB1) isolated from an air-tolerant cellulolytic consortium has been studied for its ability to supply respiratory protection for cellulolytic bacteria, such as Clostridium thermocellum , when co-cultured (Wushke et al, 2015 ).…”

Advances and Applications of Clostridium Co-culture Systems in Biotechnology

“…The fourth advantage is the improvement of system robustness. On the one hand, co-cultures with facultative anaerobic or aerobic bacteria will consume oxygen during the co-culture and provide anaerobic conditions for Clostridium ( Zuroff et al, 2013 ; Wushke et al, 2015 ; Mai et al, 2016 ; Ebrahimi et al, 2019 ; Oliva-Rodríguez et al, 2019 ). On the other hand, metabolites that inhibit Clostridium growth may be removed by co-culture partners.…”

“…Among the co-cultures, Thermoanaerobacterium saccharolyticum DSM571 could utilize pentose and hexose, which were degraded and not used by C. thermocellum ATCC27405 to produce ethanol, but which could increase substrate utilization and product yield. In addition, the co-culture of C. thermocellum DSM1237 and Caldibacillus debilis GB1 achieved aerotolerant ethanogenic bio-fuel production of 5.5 mmol/L cell culture from cellulose ( Wushke et al, 2015 ). At the same time, co-culturing the cellulolytic mesophile C. phytofermentans and Candida molischiana or S. cerevisiae cdt-1 achieved a more stable obligate mutualism for consortia-mediated lignocellulosic ethanol production by controlling the volumetric transport rate of oxygen.…”

“…This type of microbial co-culture system provides protection from environmental influences ( Bader et al, 2010 ). It has been reported that many types of associated bacteria such as Bacillus , Enterobacter , Saccharomyces cerevisiae ( Figure 1B ) ( Zuroff et al, 2013 ), Candida molischiana ( Zuroff et al, 2013 ), Caldibacillus debilis ( Wushke et al, 2015 ), and Nesterenkonia sp. F ( Ebrahimi et al, 2019 ) have the ability to remove oxygen, creating anaerobic conditions for Clostridium .…”

Digestibility of Wheat and Cattail Biomass Using a Co-culture of Thermophilic Anaerobes for Consolidated Bioprocessing

“…The lower hydrogen concentrations, due to continuous uptake, then causes a thermodynamic shift, making hydrogen production more thermodynamically feasible, resulting in an increase in acetate production by C. thermocellum [263,264]. The use of facultatively aerobic organisms in co-cultures can even allow the strictly anaerobic C. thermocellum to degrade lignocellulose under aerobic conditions, presumably due to the aerobes using up the oxygen to respire the sugars released from lignocellulose hydrolysis [265][266][267].…”