Direct glucose production from lignocellulose using Clostridium thermocellum cultures supplemented with a thermostable β-glucosidase

Abstract

Background: Cellulases continue to be one of the major costs associated with the lignocellulose hydrolysis process. Clostridium thermocellum is an anaerobic, thermophilic, cellulolytic bacterium that produces cellulosomes capable of efficiently degrading plant cell walls. The end-product cellobiose, however, inhibits degradation. To maximize the cellulolytic ability of C. thermocellum, it is important to eliminate this end-product inhibition.

Results: This work describes a system for biological saccharification that leads to glucose production following hydrolysis of lignocellulosic biomass. C. thermocellum cultures supplemented with thermostable beta-glucosidases make up this system. This approach does not require any supplementation with cellulases and hemicellulases. When C. thermocellum strain S14 was cultured with a Thermoanaerobacter brockii beta-glucosidase (CglT with activity 30 U/g cellulose) in medium containing 100 g/L cellulose (617 mM initial glucose equivalents), we observed not only high degradation of cellulose, but also accumulation of 426 mM glucose in the culture broth. In contrast, cultures without CglT, or with less thermostable beta-glucosidases, did not efficiently hydrolyze cellulose and accumulated high levels of glucose. Glucose production required a cellulose load of over 10 g/L. When alkali-pretreated rice straw containing 100 g/L glucan was used as the lignocellulosic biomass, approximately 72% of the glucan was saccharified, and glucose accumulated to 446 mM in the culture broth. The hydrolysate slurry containing glucose was directly fermented to 694 mM ethanol by addition of Saccharomyces cerevisiae, giving an 85% theoretical yield without any inhibition.

Conclusions: Our process is the first instance of biological saccharification with exclusive production and accumulation of glucose from lignocellulosic biomass. The key to its success was the use of C. thermocellum supplemented with a thermostable beta-glucosidase and cultured under a high cellulose load. We named this approach biological simultaneous enzyme production and saccharification (BSES). BSES may resolve a significant barrier to economical production by providing a platform for production of fermentable sugars with reduced enzyme amounts.

Figure 1

Biological saccharification using a C. thermocellum S14 culture supplemented with CglT. Monitoring of cellulose hydrolysis and free sugar (glucose and cellobiose) accumulation during cultivation of C. thermocellum S14 with or without CglT (+CglT and –CglT, respectively). The units are indicated in mM glucose equivalents. Based on an assumed monomer mass of 162 g/mol of microcrystalline cellulose, a 617 mM glucose equivalent was present initially. Red (+CglT) and blue lines (−CglT) indicate residual cellulose content in culture broth. Error bars represent ± SD (n = 3).

Figure 2

Profiles of C. thermocellum S14 cell growth and released cellulosomal protein in culture broth during biological saccharification. The protein concentration of the pellet was used as an indication of cell mass and cellulosomal protein during cultivation. C. thermocellum was cultured with or without CglT (+CglT and –CglT, respectively) using 100 g/L cellulose (617 mM glucose equivalents). The pH values of each culture are indicated with dotted lines. Red (+CglT) and blue lines (−CglT) indicate residual cellulose content in culture broth. Error bars represent ± SD (n = 3).

Figure 3

Optimization of biological saccharification using C. thermocellum cultures. (A) Effect of CglT activity on biological saccharification. C. thermocellum S14 cultures were supplemented with 10, 30, or 50 U of CglT per g cellulose or left unsupplemented (0 U) at an initial concentration of 100 g/L cellulose. (B) Influence of strain on glucose concentration. C. thermocellum cultures were supplemented with 30 U of CglT per g cellulose at an initial concentration of 100 g/L cellulose. C. thermocellum ATCC 27405 and DSM 1313 were used as type strains for comparison to the originally isolated strain S14. Error bars represent ± SD (n = 3).

Figure 4

Relationship between cellulose saccharification ability and glucose production under various cellulose loads. Biological saccharification was carried out in BM7CO medium containing various concentrations of cellulose: 10 g/L (62 mM glucose equivalent), 30 g/L (185 mM glucose equivalent), 50 g/L (309 mM glucose equivalent), 100 g/L (617 mM glucose equivalent), or 150 g/L (926 mM glucose equivalent). The cellulose saccharification rate was calculated based on the amount of glucose released relative to the dry weight of the input cellulose, respectively. Error bars represent ± SD (n = 3).

Figure 5

Biological saccharification and ethanol fermentation profiles using alkali-pretreated rice straw. (A) Saccharification and (B) fermentation. The units are given in mM glucose and xylose equivalents. Initially, based on an assumed monomer mass of 162 g/mol of glucan and 132 g/mol of xylan, 617 mM glucose and 46 mM xylan equivalents were present. Error bars represent ± SD (n = 3).

Figure 6

Schematic of consecutive biological saccharification method based on recycling the hydrolyzed residue.C. thermocellum supplementation with CBM-CglT was only carried out in the first biological saccharification round, without further culturing or addition of any enzymes. To recover free cellulosomes, CBM-CglT, and C. thermocellum cells, fresh pretreated cellulose substrates were added to the hydrolysis slurry and then reabsorbed from the supernatant. A second round of biological saccharification was performed using enzymes recovered by allowing them to bind to fresh substrate and the hydrolysis residue containing C. thermocellum cells. Consecutive biological saccharification using these recycling procedures may be repeated several times. When recombinant C. thermocellum strains overexpressing CglT or CBM-CglT are used in consecutive biological saccharification, addition of thermostable β-glucosidases may not be required. The left and center images are the relative initial biomass containing 120 g/L alkaline-pretreated rice straw and the culturing slurry. The right image shows the appearance of attached cells and CBM-CglT during recycling. ARS, alkaline-pretreated rice straw.