Cyclohexane is a highly volatile and colorless cyclic alkane mainly produced by hydrogenation of benzene. Cyclohexane can be widespread in the environment through petroleum and fuel spills. The United States Environmental Protection Agency (US EPA, 1994) reported several environmental issues related to gas emissions containing cyclohexane. Moreover, the toxicity of cyclohexane is related to its octanol-water partition coefficient (log KOW) with a value of 3.44 causing high cell damage. The saturated cyclic structure of cyclohexane makes it more recalcitrant compared to alkanes or monoaromatic compounds.
In this study, two strains capable of degrading cyclohexane were isolated from the soil and sludge of the wastewater treatment plant of University of Stuttgart and from a biotrickling filter system. The strains were classified as gram-negative and identified as Acidovorax sp. CHX100 and Chelatococcus sp. CHX1100. Both strains demonstrated the capability to degrade cycloalkanes (C5 - C8), while only the strain CHX1100 was able to use short n-alkanes (C5 - C8) as a sole source of carbon and energy. Growth of Acidovorax sp. CHX100 using cyclohexane was much faster compared to Chelatococcus sp. CHX1100. The strains were able to degrade 99.5 % of 7.5 mM cyclohexane in minimal medium. The results demonstrated the potential applicability of Acidovorax sp. CHX100 and Chelatococcus sp. CHX1100 for the treatment of waste gas containing cycloalkanes (C5 - C8) and other recalcitrant compounds.
The research determined the complete pathway for cyclohexane degradation through mutagenesis, gene expression, induction studies, genome analysis, and analysis of metabolites. Transposon mutagenesis of Acidovorax sp. CHX100 revealed a novel cytochrome P450chx monooxygenase which catalyzes the transformation of cyclohexane to cyclohexanol. Primer walking method categorized CYP450chx as cytochrome P450 class I taking into account its operon structure: monooxygenase, FAD oxidoreductase, and ferredoxin. CYP450chx was successfully cloned and expressed in E. coli JM109. The indole co-oxidation and CO-difference spectrum provided evidence regarding the induction and activity of CYP450chx using heterogeneous hosts. Biotransformation properties of CYP450chx were confirmed through the catalysis of cycloalkanes (C5 - C8) to their respective cycloalkanols. Moreover, degenerated primers were designed and optimized from a set of known sequences of cyclohexanol dehydrogenase genes (chnA) as well as cyclohexanone monooxygenases (chnB) and used to amplify and identify the gene cluster, which encoded the conversion of cyclohexanol to caprolactone in the strain Acidovorax sp. CHX100. Finally, the genome sequencing analysis verified that the strain Acidovorax sp. CHX100 has the genes encoding enzymes for the degradation of cyclohexanol to adipic acid. The gene cluster was composed by the following genes: a 1-oxa-2-oxocycloheptane lactonase (chnC), a cyclohexanone monooxygenase (chnB), a cyclohexanol dehydrogenase (chnA), a putative signal peptide, a 6-hydroxyhexanoate dehydrogenase (chnD), and a 6-oxohexanoate dehydrogenase (chnE).
A Biotrickling filter was operated using cyclohexane as waste air contaminant. Acivodorax sp. CHX100 was chosen as inoculum due to its ability to degrade cyclohexane. Performance was evaluated by means of different resident times from 37 s to 18 s. Removal efficiency laid in the range of 80 % to 99 %, the concentrations varied from 60 mg·C·m-3 to 480 mg·C·m-3 respectively. Elimination capacities laid between 5.4 g C·m-3·h-1 and 38 g C·m-3·h-1. Removal efficiency was decreased to 40 % by concentrations higher than 720 mg·C·m-3. The results of this research propose a novel approach for cleaning waste air containing cyclohexane by means of a biotrickling filter.