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Analysis of redox responses during TNT transformation by Clostridium acetobutylicum ATCC 824 and mutants exhibiting altered metabolism

  • Environmental Biotechnology
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Abstract

The transformation of trinitrotoluene (TNT) by several mutant strains of Clostridium acetobutylicum has been examined to analyze the maximal rate of initial transformation, determine the effects of metabolic mutations of the host on transformation rate, and to assess the cell metabolic changes brought about during TNT transformation. Little difference in the maximal rate of TNT degradation in early acid phase cultures was found between the parental ATCC 824 strain and strains altered in the acid forming pathways (phosphotransacetylase, or butyrate kinase) or in a high-solvent-producing strain (mutant B). This result is in agreement with the previous findings of a similar degradation rate in a degenerate strain (M5) that had lost the ability to produce solvent. A series of antisense constructs were made that reduced the expression of hydA, encoding the Fe-hydrogenase, or hydE and hydF, genes encoding hydrogenase maturating proteins. While the antisense hydA strain had only ∼30 % of the activity of wild type, the antisense hydE strain exhibited a TNT degradation rate around 70 % that of the parent. Overexpression of hydA modestly increased the TNT degradation rate in acid phase cells, suggesting the amount of reductant flowing into hydrogenase rather than the hydrogenase level itself was a limiting factor in many situations. The redox potential, hydrogen evolution, and organic acid metabolites produced during rapid TNT transformation in early log phase cultures were measured. The redox potential of the acid-producing culture decreased from −370 to −200 mV immediately after addition of TNT and the hydrogen evolution rate decreased, lowering the hydrogen to carbon dioxide ratio from 1.4 to around 1.1 for 15 min. During the time of TNT transformation, the treated acidogenic cells produced less acetate and more butyrate. The results show that during TNT transformation, the cells shift metabolism away from hydrogen formation to reduction of TNT and the resulting effects on cell redox cofactors generate a higher proportion of butyrate.

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Acknowledgments

This project was supported by the Army Research Office, grant number W911NF0910119. Also, we would like to thank Mary Harrison and David French for assistance with this research and data management.

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  1. Xianpeng Cai

    Present address: Conagen Inc, Suite 238, 1005 North Warson Road, St. Louis, MO, 63132, USA

Authors and Affiliations

  1. Department of Biochemistry & Cell Biology, Rice University, MS 140, 6100 Main Street, Houston, TX, 77005-1892, USA

    George N. Bennett

  2. US Army Research Laboratory, 2800 Powder Mill Road, Adelphi, MD, 20783, USA

    Matthew Servinsky, James Kiel & Christian Sund

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  1. Xianpeng Cai
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  2. Matthew Servinsky
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  3. James Kiel
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  5. George N. Bennett
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Correspondence to George N. Bennett.

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Cai, X., Servinsky, M., Kiel, J. et al. Analysis of redox responses during TNT transformation by Clostridium acetobutylicum ATCC 824 and mutants exhibiting altered metabolism. Appl Microbiol Biotechnol 97, 4651–4663 (2013). https://doi.org/10.1007/s00253-012-4253-3

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  • DOI: https://doi.org/10.1007/s00253-012-4253-3

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