Metabolic response of Clostridium ljungdahlii to oxygen exposure

Abstract

Clostridium ljungdahlii is an important synthesis gas-fermenting bacterium used in the biofuels industry, and a preliminary investigation showed that it has some tolerance to oxygen when cultured in rich mixotrophic medium. Batch cultures not only continue to grow and consume H2, CO, and fructose after 8% O2 exposure, but fermentation product analysis revealed an increase in ethanol concentration and decreased acetate concentration compared to non-oxygen-exposed cultures. In this study, the mechanisms for higher ethanol production and oxygen/reactive oxygen species (ROS) detoxification were identified using a combination of fermentation, transcriptome sequencing (RNA-seq) differential expression, and enzyme activity analyses. The results indicate that the higher ethanol and lower acetate concentrations were due to the carboxylic acid reductase activity of a more highly expressed predicted aldehyde oxidoreductase (CLJU_c24130) and that C. ljungdahlii's primary defense upon oxygen exposure is a predicted rubrerythrin (CLJU_c39340). The metabolic responses of higher ethanol production and oxygen/ROS detoxification were found to be linked by cofactor management and substrate and energy metabolism. This study contributes new insights into the physiology and metabolism of C. ljungdahlii and provides new genetic targets to generate C. ljungdahlii strains that produce more ethanol and are more tolerant to syngas contaminants.

FIG 1

C. ljungdahlii cell growth (A) and ethanol (B) and acetate (C) production when cultures are exposed to 0 (circles) and 8% (squares) O₂. One-hundred-sixty-milliliter batch reactors have a 110-ml headspace volume. Eight percent O₂ was added to the headspace at 12 h (arrows). The data represent an average from three biological replicates per condition. Error bars indicate standard deviations.

FIG 2

C. ljungdahlii CO (squares), H₂ (circles), and fructose (triangles) utilization in response to 0% O₂ (anaerobically grown [solid symbols]) and 8% O₂ (open symbols) supplementation of the headspace gas. The data represent an average from six biological replicates per condition (A). Total CO₂ concentration (grams per liter) in 14-h 0%-O₂-exposed, 14-h 8%-O₂-exposed, 36-h 0%-O₂-exposed, and 36-h 8%-O₂-exposed C. ljungdahlii batch cultures. tCO₂ = pCO₂ headspace + [HCO₃⁻] + α × pCO₂ medium, where α (estimated solubility coefficient) = 1.3511 mg/liter/mm Hg. Uninoculated bottles were used as controls. The data represent an average from three biological replicates per condition (B). Concentration of dissolved oxygen (parts per million) at the bottom of 8%-O₂-exposed C. ljungdahlii batch cultures. Zero-percent-O₂-exposed cultures were used as blanks, and uninoculated bottles were used as controls. Reducing agents (Cys-HCl and Na₂S) were omitted in unreduced medium. The data represent an average of three biological replicates per condition (C). Eight percent O₂ was added to the headspace at 12 h (arrow). One-hundred-sixty-milliliter batch reactors have a 110-ml headspace volume. Error bars indicate standard deviations.

FIG 3

(A) KEGG image of the C. ljungdahlii cofactor repair and management (crm) gene cluster. (B) Visual comparison of read alignment to a locus on the C. ljungdahlii genome containing the crm gene cluster of FastQ reads derived from RNA sequencing of 0%-O₂-exposed (anaerobically grown) and 8%-O₂-exposed cultures. Images were generated using the sequence assembly visualization software Tablet. Sample 1 is one of three 14-h-time point anaerobic cell transcriptomes, and sample 7 is one of three 14-h-time point 8%-O₂-exposed cell transcriptomes. Arrows 1, 2, 3, 4, and 5 point to positions 2107698 (beginning of the CLJU_c19400 ORF), 2111421 (end of the CLJU_c19440 ORF), 2111708 (beginning of the CLJU_c19450 ORF), 2115295 (end of the CLJU_c19470 ORF), and 2116834 on the genome (beginning of the CLJU_c19490 ORF).

FIG 4

(A) Predicted C. ljungdahlii anabolic pathways leading to ethanol. Aldehyde oxidoreductase (AOR), acetate, acetaldehyde, oxidized and reduced ferredoxin (Fd_ox/FD_red), anaerobic-type CODH complex, CO, CO₂, and ethanol are in boldface to indicate metabolic flux proposed to account for higher ethanol and lower acetate production by 8%-O₂-exposed C. ljungdahlii batch cultures. Pta, phosphotransacetylase; Ack, acetate kinase; AdhE, bifunctional aldehyde/alcohol dehydrogenase; 2 [H], reduced form of NAD [phosphate]; PFOR, pyruvate:ferredoxin oxidoreductase; CODH, carbon monoxide dehydrogenase. (B) AOR (methyl viologen acetate oxidoreductase activity) enzyme activity assay results for cell extract from 14-h 0%-O₂-exposed (anaerobically grown), 14-h 8%-O₂-exposed, 36-h 0%-O₂-exposed, and 36-h 8%-O₂-exposed C. ljungdahlii batch cultures. Activity (units per milligram) is defined as micromoles of methyl viologen oxidized per minute. The data represent an average from six biological replicates per condition. Error bars indicate standard deviations.

FIG 5

(A) NAD(P)H hydrogen peroxidase enzyme activity assay results for 14-h 0%-O₂-exposed (anaerobically grown), 14-h 8%-O₂-exposed, 36-h 0%-O₂-exposed, and 36-h 8%-O₂-exposed samples. Activity (units per milligram) is defined as micromoles of NAD(P)H oxidized per minute. The data represent an average of six biological replicates per condition. Error bars indicate standard deviations. (B) SDS-PAGE analysis of C. ljungdahlii cell-free cell extract (5 μg) from 14-h 0%-O₂-exposed (lane 2), 14-h 8%-O₂-exposed (lane 3), 36-h 0%-O₂-exposed (lane 4), and 36-h 8%-O₂-exposed (lane 5) cultures. Five microliters of PageRuler prestained protein ladder was loaded into lanes 1 and 6. The image was modified to remove irrelevant lanes. (C) Protein identification by MALDI-TOF/TOF and Mascot analysis of O₂-induced band (denoted by an arrow in panel B). The significant score identification threshold (P < 0.05) for the C. ljungdahlii protein database is 72. (MS/MS and peptide sequenced ion scores higher than 72 are considered a significant identification.) The MS/MS score and peptide sequenced ion score for CLJU_c39340 (the locus tag for a predicted rubrerythrin) are 726 and 697, respectively.