Artificial symbiosis for acetone-butanol-ethanol (ABE) fermentation from alkali extracted deshelled corn cobs by co-culture of Clostridium beijerinckii and Clostridium cellulovorans

Abstract

Background: Butanol is an industrial commodity and also considered to be a more promising gasoline substitute compared to ethanol. Renewed attention has been paid to solvents (acetone, butanol and ethanol) production from the renewable and inexpensive substrates, for example, lignocellulose, on account of the depletion of oil resources, increasing gasoline prices and deteriorating environment. Limited to current tools for genetic manipulation, it is difficult to develop a genetically engineered microorganism with combined ability of lignocellulose utilization and solvents production. Mixed culture of cellulolytic microorganisms and solventogenic bacteria provides a more convenient and feasible approach for ABE fermentation due to the potential for synergistic utilization of the metabolic pathways of two organisms. But few bacteria pairs succeeded in producing biobutanol of high titer or high productivity without adding butyrate. The aim of this work was to use Clostridium cellulovorans 743B to saccharify lignocellulose and produce butyric acid, instead of adding cellulase and butyric acid to the medium, so that the soluble sugars and butyric acid generated can be subsequently utilized by Clostridium beijerinckii NCIMB 8052 to produce butanol in one pot reaction.

Results: A stable artificial symbiotic system was constructed by co-culturing a celluloytic, anaerobic, butyrate-producing mesophile (C. cellulovorans 743B) and a non-celluloytic, solventogenic bacterium (C. beijerinckii NCIMB 8052) to produce solvents by consolidated bioprocessing (CBP) with alkali extracted deshelled corn cobs (AECC), a low-cost renewable feedstock, as the sole carbon source. Under optimized conditions, the co-culture degraded 68.6 g/L AECC and produced 11.8 g/L solvents (2.64 g/L acetone, 8.30 g/L butanol and 0.87 g/L ethanol) in less than 80 h. Besides, a real-time PCR assay based on the 16S rRNA gene sequence was performed to study the dynamics of the abundance of each strain during the co-culturing process, which figured out the roles of each strain at different periods in the symbiosis.

Conclusion: Our work illustrated the great potential of artificial symbiosis in biofuel production from lignocellulosic biomass by CBP. The dynamics of the abundance of C. beijerinckii and C. cellulovorans revealed mechanisms of cooperation and competition between the two strains during the co-culture process.

Figure 1

Time courses of single culture ofC. cellulovoranswith AECC as sole carbon source. AECC was decomposed and converted to soluble sugars and organic acids, which could be used to produced solvents in ABE fermentation. (A) Time courses of pH, total cellulase activity, total sugars accumulation and AECC degradation, (B) Time courses of organic acids and solvents production, (C) Time courses of monosaccharides and disaccharides accumulation. ▲AECC, △total sugars, ▼pH, ★ABE, ◁total cellulase activity, ✰accetone, ■butanol, ▽ethanol, ▷acetic acid, □butyric acid, ●cellobiose, ○glucose, ◆xylobiose, ◇xylose, ▶arabinose.

Figure 2

Time courses of co-culture ofC. cellulovoransandC. beijerinckiiwith AECC as sole carbon source. The artificial symbiosis succeeded in ABE production from AECC by CBP. (A) Time courses of pH, total cellulase activity, total sugars accumulation and AECC degradation, (B) Time organic acids and solvents production, (C) Time courses of monosaccharides and disaccharides accumulation. ▲AECC, △total sugars, ▼pH, ★ABE, ◁total cellulase activity, ✰accetone, ■butanol, ▽ethanol, ▷acetic acid, □butyric acid, ●cellobiose, ○glucose, ◆xylobiose, ◇xylose, ▶arabinose.

Figure 3

Effects ofC. beijerinckiiinoculation timing (A), inoculation ratio (B) and pH control strategy on co-culture (C). The co-culture conditions were investigated and optimized as follows: simultaneous inoculation, inoculation ratio of 1:10(v/v) between C. beijerinckii and C. cellulovorans, pH control at 7.0 during the first 24 hours.  butanol,  butyric acid,  AECC degraded,  total sugars,  pH value.

Figure 4

Time courses of co-culture using optimized strategies. The symbiosis degraded 68.6 g/L AECC and produced 11.8 g/L solvents, which was 108% and 46.5% higher than that obtained under the initial co-culture conditions. Besides, a real-time PCR assay based on the 16S rRNA gene sequence revealed the dynamics of the abundance for each strain. (A) Time courses of pH, solvents production total, cellulase activity, total sugars accumulation and utilization, AECC degradation. (B) Time courses of ABE and organic acids production, (C) Time courses of monosaccharides and dissaccharides accumulation and utilization, (D) Dynamics of relative abundace for C. beijerinckii and C. cellulovorans. ▲AECC, △total sugars, ▼pH, ★ABE, ◁total cellulase activity, ✰acetone, ■butanol, ▽ethanol, ▷acetic acid, □butyric acid, ●cellobiose, ○glucose, ◆xylobiose, ◇xylose, ▶arabinose, White, relative abundance of C. beijerinckii, Black, relative abundance of C. cellulovorans.