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

Abstract

We attempted to mimic in small upflow anaerobic sludge bed (UASB) bioreactors the metabolic association found in nature between methanogens and methanotrophs. UASB bioreactors were inoculated with pure cultures of methanotrophs, and the bioreactors were operated by using continuous low-level oxygenation 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, emphasis was placed on monitoring various methanotrophic populations by using classical methods and also a PCR amplification assay based on the mmoX gene fragment of the soluble methane monooxygenase (sMMO). The following results were obtained: (i) under the conditions used, Methylosinus sporium appeared to survive better than Methylosinus trichosporium; (ii) the PCR method which we used could detect as few as about 2,000 sMMO gene-containing methanotrophs per g (wet weight) of granular sludge; (iii) inoculation of the bioreactors with pure cultures of methanotrophs contributed greatly to increases in the sMMO-containing population (although the sMMO-containing population decreased gradually with time, at the end of an experiment it was always at least 2 logs larger than the initial population before inoculation); (iv) in general, there was a good correlation between populations with the sMMO gene 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 those described here might be used for biodegradation of various chlorinated pollutants.

FIG. 1

Schematic diagram of the bioreactor setup used. The bioreactor volume was 1 liter during phase I and 5 liters during phase II. The culture was aerated with an immersed sparger during phase I, whereas the culture was aerated by using a separate aeration column (with a sparger) during phase II.

FIG. 2

Sensitivity of detection of the sMMO gene in enriched granular sludge by PCR when the mmoX1-mmoX2 primer set was used. For details see Materials and Methods. Identical granular sludge samples were spiked with different numbers of viable M. sporium ATCC 35069 cells, and the samples were analyzed by PCR. Lanes 1 and 9, 100-bp size ladder marker; lane 2, granular sludge sample containing 1.4 × 10⁸ cells of M. sporium; lane 3, granular sludge sample containing 1.4 × 10⁷ cells; lane 4, granular sludge sample containing 1,400 cells; lane 5, granular sludge sample containing 14 cells; lane 6, granular sludge sample containing 1.4 M. sporium cells; lane 7, granular sludge sample containing no M. sporium cells (there is a faint band); lane 8, water. Since the granular sludge sample used was found to contain about 2,000 sMMO-positive cells per g (wet weight), the sensitivity of our PCR method was calculated to be about 2,000 mmoX probe-positive cells per g (wet weight) of sludge.

FIG. 3

RAPD-PCR analysis of representative fresh methanotrophic isolates obtained early in this study. Lane 1, 100-bp size ladder marker; lane 2, M. sporium ATCC 35069; lane 3, UASB isolate M5; lane 4, Ville Mercier isolate M1; lane 5, UASB isolate M6; lane 6, Ville Mercier isolate M10; lane 7, UASB isolate M3.

FIG. 4

Phase I study. (A) Survival of various methanotrophic populations in granular sludge in 1-liter reactors aerated at two different oxygenation levels and inoculated with M. trichosporium OB3b (= ATCC 35070). Symbols: •, total methanotrophic population; □, methanotrophs exhibiting sMMO activity; ◊, methanotrophs containing the sMMO gene. For details see Materials and Methods. The graph at the bottom shows the HRT profile used in these experiments. (B) Survival of various methanotrophic populations (see above) in granular sludge in a 1-liter reactor aerated at a rate of 1.5 liters of oxygen per day and inoculated with M. sporium ATCC 35069. l, liters; d, days.

FIG. 5

Phase II study: survival of various microbial populations in granular sludge in 5-liter reactors aerated at various oxygenation levels and inoculated or not inoculated with M. sporium ATCC 35069. (A) Various methanotrophic populations right before and after inoculation. (B through E) Results obtained after 35 days of reactor operation. (B) Various methanotrophic populations in the granular sludge of the inoculated reactors. (C) strictly aerobic and facultatively aerobic microbial populations associated with the granular sludge in the inoculated reactors. (D) Various methanotrophic populations in the noninoculated reactors. (E) Strictly aerobic and facultatively aerobic microbial populations in the granular sludge of the noninoculated reactors. N.D., not detected.