Direct conversion of cellulose to L-lactic acid by a novel thermophilic Caldicellulosiruptor strain

Abstract

Background: Consolidated bioprocessing (CBP) of lignocellulosic biomass to L-lactic acid using thermophilic cellulolytic/hemicellulolytic bacteria provides a promising solution for efficient lignocellulose conversion without the need for additional cellulolytic/hemicellulolytic enzymes. Most studies on the mesophilic and thermophilic CBP of lignocellulose to lactic acid concentrate on cultivation of non-cellulolytic mesophilic and thermophilic bacteria at temperatures of 30-55 °C with external addition of cellulases/hemicellulases for saccharification of substrates.

Results: L-Lactic acid was generated by fermenting microcrystalline cellulose or lignocellulosic substrates with a novel thermophilic anaerobic bacterium Caldicellulosiruptor sp. DIB 104C without adding externally produced cellulolytic/hemicellulolytic enzymes. Selection of this novel bacterium strain for lactic acid production is described as well as the adaptive evolution towards increasing the L-lactic acid concentration from 6 to 70 g/l on microcrystalline cellulose. The evolved strains grown on microcrystalline cellulose show a maximum lactic acid production rate of 1.0 g/l*h and a lactic acid ratio in the total organic fermentation products of 96 wt%. The enantiomeric purity of the L-lactic acid generated is 99.4%. In addition, the lactic acid production by these strains on several other types of cellulose and lignocellulosic feedstocks is also reported.

Conclusions: The evolved strains originating from Caldicellulosiruptor sp. DIB 104C were capable of producing unexpectedly large amounts of L-lactic acid from microcrystalline cellulose in fermenters. These strains produce L-lactic acid also from lignocellulosic feedstocks and thus represent an ideal starting point for development of a highly integrated commercial L-lactic acid production process from such feedstocks.

Keywords: Anaerobic; Caldicellulosiruptor; Consolidated bioprocessing; High temperature; L-Lactic acid; Lignocellulose; Thermophilic bacteria.

Fig. 1

Lactic acid production by Caldicellulosiruptor strains DIB 004C, DIB 041C, DIB 087C, DIB 101C, DIB 103C, DIB 104C and DIB 107C. Bacteria were grown in flasks at 72 °C and 100 rpm on 10 g/l microcrystalline cellulose (A, B) or 5,9 g/l pretreated miscanthus (C, D). The media contained 10 g/l MOPS as buffer. Lactic acid concentration (g/l) (A, C) and lactic acid ratio (mol%) (B, D) in formed fermentation products (lactate + acetate + ethanol) are presented

Fig. 2

Lactic acid production by Caldicellulosiruptor DIB 104C cultures generated during adaptive evolution. A Strain DIB 104C was applied in sequential repeated batch transfers using media containing microcrystalline cellulose as substrate and with or without externally added lactic acid as stress factor. The transfers in flasks without externally added lactic acid (referred to as “w/o LA”) and in fermenters with externally added lactic acid (referred to as “with LA”) were not continued (indicated by the symbol “x”) because the improvement in lactic acid production was lower as compared to the transfers in fermenters without externally added lactic acid. The numbers of sequential transfers in fermenters are shown in dotted line boxes. Occasionally during the repeated batch transfers, a significant increase in lactic acid production was observed (referred to as “steps”) and an aliquot of the respective cultures was stored for preservation and isolation of single-cell colonies (referred to as “SCC”). Culture names with an asterisk represent pure, isogenic strains; culture names without an asterisk represent mixed populations. B The mixed populations and isogenic strains with significantly improved lactic acid production (referred to as steps 1 to 7) were grown in fermenters on up to 200 g/l microcrystalline cellulose at 70 °C with pH stabilized at 6.4. Lactic acid production with cultures from evolution steps 1 to 7 are presented

Fig. 3

Effect of externally added lactic acid on growth of Caldicellulosiruptor DIB 104C wild-type strain and evolved strain clone 45. Bacteria were grown at 70 °C in 16 ml Hungate tubes with 9 ml medium containing 5 g/l glucose and 10 g/l MOPS. Lactic acid was added as sodium lactate to initial concentrations of 4.0 g/l (A), 15.5 g/l (B), 19.2 g/l (C) or 22.8 g/l (D). Growth was monitored by measurement of the optical density (McFarland units) of cultures directly in tubes using a densitometer DEN-1. One McFarland unit corresponds to an optical density OD₅₅₀ of 1 when measured in 1-cm cuvettes

Fig. 4

Lactic acid, acetic acid and ethanol production by Caldicellulosiruptor DIB 104C wild-type strain and evolved strain clone 45. Wild-type strain (A, B) and evolved clone 45 (C, D) were grown in fermenters on microcrystalline cellulose at 70 °C with pH stabilized at 6.4. Lactic acid, acetic acid and ethanol concentrations (g/l) (A, C) and products ratio (wt%) (B, D) in formed fermentation products (lactate + acetate + ethanol) are presented