cClostridium 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 H 2 , CO, and fructose after 8% O 2 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. Clostridium ljungdahlii is an anaerobic, motile, endosporeforming, Gram-positive, rod-shaped, acetogenic bacterium isolated from chicken yard waste and has application as a biocatalyst to transform syngas components (CO, CO 2 , and H 2 ) into more valuable chemicals (1-4). It was the first bacterium discovered to metabolize these syngas components and to produce ethanol with acetate as its primary fermentation product (1, 4). To reduce the amount of acetate and increase the amount of ethanol produced by C. ljungdahlii for its use in industrial solvent production, different reactor designs and agitation parameters, varied syngas component concentrations, increased gas flow rates and pressures, reduced nitrogen sources, addition of reducing agents to media, adjustment of growth medium pH, and addition of nanoparticles have all been evaluated (1,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Some of these efforts have reportedly improved ethanol/ acetate product ratios from 1:20 to 2:1 in batch cultures and from 1:1 to 21:1 for cultures grown in continuously stirred reactors (8,9,16). More recent sequencing and genetic modification techniques have greatly enhanced our understanding of C. ljungdahlii's metabolism and increased production of ethanol as well as enabled production of other chemicals (e.g., acetone, butyrate, and butanol) (2, 3, 17-21).Despite these advancements, for C. ljungdahlii to be effectively used for industrial syngas transformation, some of its catalytic limitations related to gaseous headspace composition still require evaluation. Although steel mill waste gas was recently used as...

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Syngas fermentation is considered an alternate processing method for biofuel and biochemical production as part of thermochemical biomass conversion. Exposure of syngas fermenting microorganisms to sugars, either in the primary syngas fermentation or through pre-adaptation in the seed culture, has the potential to enhance overall fermentation performance and stress tolerance. In this rapid communication, Clostridium ljungdahlii was grown on different carbon sources including syngas only, syngas-fructose and fructose only to identify ideal pre-adaptation conditions for ethanol and acetate production from subsequent cultures grown in reactors containing syngas only or fructose-syngas substrates. In syngas only reactors, cultures pre-adapted to fructose had faster cell production rates (2X) and at least 83% higher ethanol and 16% higher acetate formation than cells pre-adapted on syngas or syngas-fructose. In syngasfructose reactors, cultures did not show significant growth or acetate production differences under pre-adaptation treatments. Nevertheless, in these syngas-fructose reactors, cultures pre-adapted on syngas and syngas-fructose had nearly 20% higher ethanol production than those pre-adapted on fructose. Among pre-adaptation treatments, fructose had better results in syngas only reactors than syngas-fructose reactors. However, the presence of syngas in preadaptation cultures was better overall for ethanol production.