Abstract
BackgroundConsolidated bioprocessing (CBP) of lignocellulosic biomass to ethanol using thermophilic bacteria provides a promising solution for efficient lignocellulose conversion without the need for additional cellulolytic enzymes. Most studies on the thermophilic CBP concentrate on co-cultivation of the thermophilic cellulolytic bacterium Clostridium thermocellum with non-cellulolytic thermophilic anaerobes at temperatures of 55°C-60°C.ResultsWe have specifically screened for cellulolytic bacteria growing at temperatures >70°C to enable direct conversion of lignocellulosic materials into ethanol. Seven new strains of extremely thermophilic anaerobic cellulolytic bacteria of the genus Caldicellulosiruptor and eight new strains of extremely thermophilic xylanolytic/saccharolytic bacteria of the genus Thermoanaerobacter isolated from environmental samples exhibited fast growth at 72°C, extensive lignocellulose degradation and high yield ethanol production on cellulose and pretreated lignocellulosic biomass. Monocultures of Caldicellulosiruptor strains degraded up to 89-97% of the cellulose and hemicellulose polymers in pretreated biomass and produced up to 72 mM ethanol on cellulose without addition of exogenous enzymes. In dual co-cultures of Caldicellulosiruptor strains with Thermoanaerobacter strains the ethanol concentrations rose 2- to 8.2-fold compared to cellulolytic monocultures. A co-culture of Caldicellulosiruptor DIB 087C and Thermoanaerobacter DIB 097X was particularly effective in the conversion of cellulose to ethanol, ethanol comprising 34.8 mol% of the total organic products. In contrast, a co-culture of Caldicellulosiruptor saccharolyticus DSM 8903 and Thermoanaerobacter mathranii subsp. mathranii DSM 11426 produced only low amounts of ethanol.ConclusionsThe newly discovered Caldicellulosiruptor sp. strain DIB 004C was capable of producing unexpectedly large amounts of ethanol from lignocellulose in fermentors. The established co-cultures of new Caldicellulosiruptor strains with new Thermoanaerobacter strains underline the importance of using specific strain combinations for high ethanol yields. These co-cultures provide an efficient CBP pathway for ethanol production and represent an ideal starting point for development of a highly integrated commercial ethanol production process.
Highlights
Consolidated bioprocessing (CBP) of lignocellulosic biomass to ethanol using thermophilic bacteria provides a promising solution for efficient lignocellulose conversion without the need for additional cellulolytic enzymes
Co-cultures of engineered C. thermocellum and Thermoanaerobacterium saccharolyticum produced 38.1 g/l ethanol from Crystalline cellulose (Avicel), which is the highest ethanol concentration reported for a thermophilic, cellulolytic co-culture to date [8]
For the enrichment of cellulolytic bacteria resistant against inhibitors present in pretreated lignocellulosic biomass [22], the cultures were grown on media containing unwashed dilute acid steam-explosion -pretreated poplar wood followed by serial dilutions
Summary
Consolidated bioprocessing (CBP) of lignocellulosic biomass to ethanol using thermophilic bacteria provides a promising solution for efficient lignocellulose conversion without the need for additional cellulolytic enzymes. Ethanol is an established alternative fuel from renewable resources [1] Today it is mainly produced from sugar or starchy biomass, limiting the environmental benefit [2] and posing a competition for the raw materials with food industry. In CBP an organism or a mixed culture of organisms produces enzymes for hydrolysis of cellulose and hemicellulose in lignocellulosic biomass and ferments the C5 and C6 sugars into ethanol or other valuable products without addition of cellulolytic or hemicellulolytic enzymes. Co-cultures of engineered C. thermocellum and Thermoanaerobacterium saccharolyticum produced 38.1 g/l ethanol from Avicel (crystalline cellulose), which is the highest ethanol concentration reported for a thermophilic, cellulolytic co-culture to date [8]. The performance of these monocultures and co-cultures was not evaluated with real lignocellulosic substrates under industrial conditions and few data have been reported yet for C. thermocellum co-cultures that would support the process viability using pretreated and untreated lignocellulosic substrates under laboratory conditions [11,13]
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