Abstract
Clostridium thermocellum is a major candidate for bioethanol production via consolidated bioprocessing. However, the low ethanol tolerance of the organism dramatically impedes its usage in industry. To explore the mechanism of ethanol tolerance in this microorganism, systematic metabolomics was adopted to analyse the metabolic phenotypes of a C. thermocellum wild-type (WT) strain and an ethanol-tolerant strain cultivated without (ET0) or with (ET3) 3% (v/v) exogenous ethanol. Metabolomics analysis elucidated that the levels of numerous metabolites in different pathways were changed for the metabolic adaption of ethanol-tolerant C. thermocellum. The most interesting phenomenon was that cellodextrin was significantly more accumulated in the ethanol-tolerant strain compared with the WT strain, although cellobiose was completely consumed in both the ethanol-tolerant and wild-type strains. These results suggest that the cellodextrin synthesis was active, which might be a potential mechanism for stress resistance. Moreover, the overflow of many intermediate metabolites, which indicates the metabolic imbalance, in the ET0 cultivation was more significant than in the WT and ET3 cultivations. This indicates that the metabolic balance of the ethanol-tolerant strain was adapted better to the condition of ethanol stress. This study provides additional insight into the mechanism of ethanol tolerance and is valuable for further metabolic engineering aimed at higher bioethanol production.
Highlights
Bioethanol produced from lignocellulosic biomass is a prospective and attractive substitute for petroleum-based liquid fuels as a sustainable and renewable source of energy [1]
Both the ET0 and ET3 cultivations displayed longer lag phases and lower maximal biomass yields than that of WT, whereas the maximal biomass of ET3 was only around half of that of ET0. These results indicate that cell growth was inhibited by ethanol, which was proven in a previous report [10]
The level of NAD in the ET3 cultivation decreased to approximately one-ninth of that in the ET0 cultivation, whereas the level of nicotinamide adenine dinucleotide phosphate (NADP) in the ET3 cultivation was approximately one-third of that in ET0 cultivation. These results indicate that the biosynthesis of NAD, compared to the biosynthesis of NADP, was obviously more affected by ethanol
Summary
Bioethanol produced from lignocellulosic biomass is a prospective and attractive substitute for petroleum-based liquid fuels as a sustainable and renewable source of energy [1]. Clostridium thermocellum, a gram-positive thermophilic anaerobic bacterium, has been proposed as a potential candidate microorganism for bioethanol CBP production due to its high efficiency of cellulose degradation and direct production of ethanol [2,3]. The industrial application of C. thermocellum has been hampered because of its low hemicellulose utilisation, low ethanol productivity and titre, and low ethanol tolerance [4,5,6]. Various strategies, including co-cultivation with other bacteria and metabolic engineering, have been developed to improve the ethanol production and hemicellulose utilization of C. thermocellum [7,8,9]. The low ethanol tolerance of C. thermocellum is still one of the major bottlenecks of its bioethanol industrialisation
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