Degradation pathways of anaerobic benzene-degrading bacteria

Chiho SUZUKI-MINAKUCHI

Biotechnology Research Center, The University of Tokyo

Research aims
Benzene is a carcinogenic compound, often found in gasoline-contaminatedgroundwater. Under aerobic conditions, benzene can be biodegraded in a short period oftime. However, contamination of subsurface aquifers with benzene often results in thedevelopment of anaerobic zones. Thus, isolation and analysis of anaerobic
benzene-degrading bacteria are necessary for effective bioaugmentation ofbenzene-contaminated groundwater.
Azoarcus sp. strain DN11 is a denitrifying bacterium that is capable of benzene
degradation under aerobic and anaerobic conditions1). Because the use of DN11 has
been approved by both the Ministry of the Environment and the Ministry of Economy,
Trade and Industry, DN11 can be used for environmental bioaugmentation in Japan.
However, DN11 sometimes loses its benzene-degrading ability during cultivation. To
solve this problem, the genes responsible for benzene degradation must be identified.
The objective of this study was to clarify the anaerobic benzene degradation pathway ofAzoarcus sp. strain DN11.
Methods
Three anaerobic benzene degradation pathways have been proposed: (i) an anaerobichydroxylation of benzene, yielding phenol, (ii) a Friedel-Crafts-type methylation ofbenzene, yielding toluene, and (iii) a carboxylation of benzene, yielding benzoate2),although no initial activation enzyme has been identified. Previously, Zhang andcolleagues found that Geobacter metallireducens can degrade toluene, phenol, andbenzoate—and under anaerobic conditions, it can also degrade benzene via phenol3). To

determine the genes (in the draft genome of DN11) responsible for anaerobic benzene
degradation, we used the amino acid sequences of BssA (benzylsuccinate synthase,from the anaerobic toluene degradation pathway), PpsA (phenylphosphate synthase,from the anaerobic phenol degradation pathway), and BamY (benzoate coenzyme Aligase, from the anaerobic benzoate degradation pathway) of G. metallireducens as
query molecules. Primers were constructed for the corresponding homologous genes in
DN11 to check the transcription levels of those genes under anaerobic
benzene-degrading conditions.
The anaerobic benzene degradation assay was performed in an anaerobic chamber
containing 5% hydrogen in nitrogen. Minimal medium containing 15 μM benzene as thesole carbon source was used, and the concentration of benzene in the medium wasmeasured using gas chromatography-mass spectrometry (GC-MS). When benzene
degradation was detected, cells were harvested, RNA was extracted, and cDNA wassynthesized. Reverse transcription (RT)-PCR was performed using the primers
described above.
Results
When DN11 was grown under aerobic conditions, in minimal medium containing 15μM benzene as the sole carbon source, 50–60% of the benzene was degraded, and themedium environment became anaerobic within 24 h. Because small amounts of oxygen
contaminate the medium even under the anaerobic conditions, DN11 initially degrades
benzene using oxygen, following which anaerobic benzene degradation occurs. Under
the anaerobic conditions, the amount of benzene decreased slowly when DN11 wasadded to the medium (Fig. 1). We extracted RNA from the cells incubated in theanaerobic chamber for 16 d.

Fig. 1. Degradation of benzene by DN11 underanaerobic conditions.
DN11 was grown under anaerobic conditions,in minimal medium containing 15 μM benzene as
the sole carbon source. The amount of benzene in
the headspace of each vial was measured usingGC-MS. Each bar shows the average of triplicate
measurements.

From the draft genome of DN11, the proteins ADN11_211, ADN11_33, andADN11_73 were found to be homologous with the proteins encoded by bssA, ppsA, andbamY in G. metallireducens (with 71%, 77%, and 49% identity in the translated aminoacid sequences, respectively). We performed RT-PCR to check the expression levels for
ADN11_211, ADN11_33, and ADN11_73, and found that the expression levels for
ADN11_211 and ADN11_73 were much higher than those for ADN11_33 (Fig. 2).

Fig. 2. Expression levels of ADN11_73, ADN11_33, and ADN11_211 underanaerobic benzene-degrading conditions.
RT-PCR amplification results for (A) ADN11_73, ADN11_33, and ADN11_211
mRNAs, and (B) 16S rRNA. Two independent cultures (indicated as “1” and “2”) wereused for RNA extraction and cDNA synthesis. “3” indicates that genome DNA was used
as a template. “4” indicates that no template was used.

Conclusion
DN11 is known to be capable of degrading toluene and benzoate, but not phenol,
under anaerobic conditions4). Our results suggest that DN11 degrades benzene via
toluene and/or benzoate. Given that G. metallireducens degrades benzene via phenol
under anaerobic conditions, the benzene degradation pathway used by DN11 should bedifferent from that used by G. metallireducens. Further analyses will clarify the detailsof the benzene degradation pathway used by DN11 under anaerobic conditions.
References
1) Kasai, Y., Takahata, Y., Manefield, M., and Watanabe, K. (2006) RNA-based stable
isotope probing and isolation of anaerobic benzene-degrading bacteria fromgasoline-contaminated groundwater. Appl. Environ. Microbiol. 72: 3586–3592.
2) Vogt, C., Kleinsteuber, S., and Richnow, H. H. (2011) Anaerobic benzene degradation
by bacteria. Microb. Biotechnol. 4: 710–724.
3) Zhang, T., Tremblay, P. L., Chaurasia, A. K., Smith, J. A., Bain, T. S., and Lovley, D.
R. (2013) Anaerobic benzene oxidation via phenol in Geobacter metallireducens.
Appl. Environ. Microbiol. 79: 7800–7806.
4) Kasai, Y., Kodama, Y., Takahata, Y., Hoaki, T., and Watanabe, K. (2007) Degradative
capacities and bioaugmentation potential of an anaerobic benzene-degrading
bacterium strain DN11. Environ. Sci. Technol. 41: 6222–6227.