Monitoring methanotrophic bacteria in hybrid anaerobic-aerobic reactors with PCR and a catabolic gene probe

We attempted to mimic in small upflow anaerobic sludge bed (UASB) bioreacto rs the metabolic association found in nature between methanogens and methan otrophs. UASB bioreactors were inoculated with pure cultures of methanotrop hs, and the bioreactors were operated by using continuous low-level oxygena tion in order to favor growth and/or survival of methanotrophs. Unlike the reactors in other similar studies, the hybrid anaerobic-aerobic bioreactors which we used were operated synchronously, not sequentially. Here, emphasi s was placed on monitoring various methanotrophic populations by using clas sical methods and also a PCR amplification assay based on the mmoX gene fra gment of the soluble methane monooxygenase (sMMO). The following results we re obtained: (i) under the conditions used, Methylosinus sporium appeared t o survive better than Methylosinus trichosporium; (ii) the PCR method which we used could detect as few as about 2,000 sMMO gene-containing methanotro phs per g (wet weight) of granular sludge; (iii) inoculation of the bioreac tors with pure cultures of methanotrophs contributed greatly to increases i n the sMMO-containing population (although the sMMO-containing population d ecreased gradually with time, at the end of an experiment it was always at least 2 logs larger than the initial population before inoculation); (it) i n general, there was a good correlation between populations with the sMMO g ene and populations that exhibited sMMO activity; and (v) inoculation with sMMO-positive cultures helped increase significantly the proportion of sMMO -positive methanotrophs in reactors, even after several weeks of operation under various regimes. At some point, anaerobic-aerobic bioreactors like th ose described here might be used for biodegradation of various chlorinated pollutants.