Allopurinol-mediated lignocellulose-derived microbial inhibitor tolerance by Clostridium beijerinckii during acetone–butanol–ethanol (ABE) fermentation

Abstract

In addition to glucans, xylans, and arabinans, lignocellulosic biomass hydrolysates contain significant levels of nonsugar components that are toxic to the microbes that are typically used to convert biomass to biofuels and chemicals. To enhance the tolerance of acetone-butanol-ethanol (ABE)-generating Clostridium beijerinckii NCIMB 8052 to these lignocellulose-derived microbial inhibitory compounds (LDMICs; e.g., furfural), we have been examining different metabolic perturbation strategies to increase the cellular reductant pools and thereby facilitate detoxification of LDMICs. As part of these efforts, we evaluated the effect of allopurinol, an inhibitor of NAD(P)H-generating xanthine dehydrogenase (XDH), on C. beijerinckii grown in furfural-supplemented medium and found that it unexpectedly increased the rate of detoxification of furfural by 1.4-fold and promoted growth, butanol, and ABE production by 1.2-, 2.5-, and 2-fold, respectively. Since NAD(P)H/NAD(P)(+) levels in C. beijerinckii were largely unchanged upon allopurinol treatment, we postulated and validated a possible basis in DNA repair to account for the solventogenic gains with allopurinol. Following the observation that supplementation of allopurinol in the C. beijerinckii growth media mitigates the toxic effects of nalidixic acid, a DNA-damaging antibiotic, we found that allopurinol elicited 2.4- and 6.7-fold increase in the messenger RNA (mRNA) levels of xanthine and hypoxanthine phosphoribosyltransferases, key purine-salvage enzymes. Consistent with this finding, addition of inosine (a precursor of hypoxanthine) and xanthine led to 1.4- and 1.7-fold increase in butanol production in furfural-challenged cultures of C. beijerinckii. Taken together, our results provide a purine salvage-based rationale for the unanticipated effect of allopurinol in improving furfural tolerance of the ABE-fermenting C. beijerinckii.

The pangenome of the genus Clostridium

“…Some species of the genus have the ability to produce acetone, butanol and ethanol simultaneously through the ABE fermentation process. Clostridium can use a wide range of substrates via ABE fermentation, including simple carbohydrates or glycerol (Gao and Rehmann, 2014;Gu et al, 2014;Ujor et al, 2014;Aristilde, 2017;Herman et al, 2016), and complex polysaccharides such as lignocellulosic materials (Wen et al, 2014;Ramos et al, 2015;Ujor et al, 2015;Aristilde, 2017). Other valuable fermentable chemicals produced by Clostridium are diols (i.e., butanediol, propanediol), gases (i.e., carbon dioxide, hydrogen, methane) and organic acids (i.e., lactic, acetic, butyric and fumaric) (Kim et al, 2005).…”

Development of a high-throughput assay for rapid screening of butanologenic strains

“…As proof of principle, three random ABE fermentation supernatants generated from Cb fermentation of glucose media 11 were screened for butanol using our Th ADH_ae-based assay. Butanol concentrations were determined from a calibration curve of known butanol concentrations versus absorbance, and compared with data obtained from gas chromatography analysis that is a proven benchmark.…”

“…Electrocompetent Cb cells were transformed to generate two recombinant strains: Cb -pWUR460_ dhaD1 + gldA1 and Cb -pWUR460_ gldA1 + dhaK . To prepare electro-competent Cb cells, 200 µL of Cb spores were heat-shocked at 75 °C for 10 min, cooled on ice for 2 min, and then inoculated into 10 mL anoxic tryptone–glucose–yeast extract (TGY) medium 11 . The culture was grown at 35 °C in an anaerobic chamber with a modified atmosphere of 82% N 2 , 15% CO 2 , and 3% H 2 as previously described 11 until it reached an OD 600 ~ 0.9–1.1 (usually ~12 h from time of inoculation).…”

“…Gas chromatography analysis of n-butanol in Cb culture supernatants was performed using an Agilent 7890 A gas chromatograph (Agilent Technologies, Inc., Wilmington, DE, USA) equipped with a flame ionization detector (FID) and a J&W capillary column [19091 N-213; 30 m (length) × 320 μm (internal diameter) × 0.50 μm (HP-INNOWax film)] 11 .…”

Efficient Catalytic Dehydration of High-Concentration 1-Butanol with Zn-Mn-Co Modified γ-Al2O3 in Jet Fuel Production

“…Considerable researches have focused on the conversion of sugars obtained from biomass to 1-butanol, where sugars can be fermented to 'acetone-butanol-ethanol' (ABE) using Clostridium acetobutylicum or Clostridium beijerinckii [3,4]. Fermented 1-butanol can be separated from the fermentation broth by techniques such as gas-stripping, distillation, membrane separation, and extraction.…”