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Applied and Environmental Microbiology, February 1999, p. 381-388, Vol. 65, No. 2
0099-2240/99/$00.00+0
Monitoring Methanotrophic Bacteria in Hybrid Anaerobic-Aerobic
Reactors with PCR and a Catabolic Gene Probe
Carlos B.
Miguez,1
Chun F.
Shen,2
Denis
Bourque,1
Serge R.
Guiot,2 and
Denis
Groleau1,*
Microbial and Enzymatic Technology
Group1 and
Environmental Bioengineering
Group,2 Biotechnology Research Institute,
National Research Council of Canada, Montreal, Quebec, Canada H4P
2R2
Received 26 August 1998/Accepted 4 November 1998
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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.
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INTRODUCTION |
In nature, methane-oxidizing
bacteria (methanotrophs), as expected, live in close association with
methane-producing microorganisms (methanogens); as reported by Buchholz
et al. (5), methanotrophs are strongly associated with the
oxic-anoxic interface in water columns or sediments. In eutrophic
lakes, maximal rates of methane oxidation have been shown to occur in
the oxic-anoxic interfaces of the water column, where the oxygen
concentrations may not exceed 1 ppm (14, 15).
In a few studies workers have attempted to mimic in bioreactors the
close spatial association of the two trophic microbial groups by using
sequential anaerobic-aerobic processes, and in some cases, there has
been an attempt to apply this association to the biodegradation of some
pollutants (11, 20, 30). This concept could be applied to
the biodegradation of tetrachloroethylene (PCE) and trichloroethylene
(TCE). Biodegradation of PCE to TCE can occur in anaerobic environments
(10, 29), whereas TCE derived from PCE can be further
degraded aerobically by methanotrophic bacteria that contain the
soluble methane monooxygenase (sMMO) (4, 6, 7, 9, 16, 22,
27). In simple bioprocessing terms, the procedure involves
transferring the effluent of an anaerobic reactor into an aerobic
reactor enriched with a methanotrophic consortium and supplying
oxygen and methane to the latter reactor to sustain and maximize
the methanotrophic metabolic activity.
Upflow anaerobic sludge blanket (UASB) reactors have been
characterized well, and the usefulness of these reactors for treatment of municipal and industrial wastes has been well-documented
(28). UASB reactors can accommodate low concentrations of
oxygen without deleterious effects on the integrity or metabolic
activity of the granular biomass (13, 23). Thus, a partially
aerated UASB reactor contains the substrates required by methanotrophic
bacteria (i.e., indigenously produced methane and exogenously added
oxygen) and may, therefore, be an ideal system for maintaining
consortia composed of methanogens and methanotrophs. A bioprocess in
which a hybrid aerobic-anaerobic sludge reactor is used has been
designed recently in our laboratory (23).
In this study, as a further step in the development of a
generic bioprocess, we evaluated the ability of methanotrophic bacteria to grow and/or survive in UASB reactors. To do this,
coaggregation of methanotrophs with anaerobic granular sludge
dominated by methanogens was studied in laboratory-scale UASB
reactors under different oxygenation conditions and with
different hydraulic retention times (HRTs). Granular sludge
was inoculated with methanotrophic cultures known to contain the sMMO.
In this work, special emphasis was put on monitoring various
methanotrophic populations, particularly those associated with the
granular sludge. The density of the methanotrophic population that
contained the sMMO gene cluster was monitored by using molecular biology tools developed in a previous study (21) and other
more traditional methods. The results obtained in these investigations are described below.
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MATERIALS AND METHODS |
Bacterial strains and media.
Methylosinus
trichosporium OB3b (= ATCC 35070) and Methylosinus
sporium (= ATCC 35069) were obtained from the American Type Culture Collection, Rockville, Md.). These strains were grown on
copper-free low-nitrate mineral (LN-NMS) medium (3) at
30°C in an atmosphere containing 50% methane and 50% air.
UASB reactors.
The design of the UASB reactors used in this
study has been described by Shen and Guiot (23). Recycled
effluent was oxygenated by placing an oxygen-bearing diffuser on top of
the granular bed or by incorporating an aeration column coupled to the
UASB reactor (Fig. 1). Reactors of
different sizes (1 and 5 liters) were used in this study. The 1-liter
reactors were inoculated with 200 ml of granular sludge (about
13.4 g of volatile suspended solids [VSS]) originating from a
full-scale UASB reactor treating wastewater from a baby food plant
(Champlain Industry, Cornwall, Ontario, Canada), whereas the 5-liter
reactors were inoculated with 1,000 ml (about 50 g of VSS) of the
same sludge. After the granular sludge was added, the reactors were
acclimatized to predetermined oxygenation levels for 5 days at an HRT
of 72 h. The operation of the reactor, the chemical composition of
the feed, and rate of addition of the feed were as described by Shen
and Guiot (23).
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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.
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Inoculation of reactors with M. trichosporium and
M. sporium.
The cultures used to inoculate reactors were
grown in 6-liter Erlenmeyer flasks containing 3 liters of LN-NMS
medium. Each inoculum consisted of cells obtained after growth on
LN-NMS medium plates (one plate per liter of medium) in modified
"anaerobic jars" flushed with a 50% methane-50% air mixture and
incubated at 30°C. The inocula used for the flasks were obtained by
recovering the biomass on LN-NMS medium plates with about 5 ml of
LN-NMS medium. During growth, the headspaces of the cultures were
flushed twice daily with a 50% methane-50% air mixture for 3 days;
the cultures were agitated at 250 rpm and incubated at 30°C, and then they were harvested and concentrated 100-fold by using a
0.1-µm-pore-size polysulfone hollow fiber membrane filter (Amicon).
Viable bacterial populations in granular sludge.
Granular
sludge was added to a preweighed sterile tube containing 3.5 g of
3-mm-diameter glass beads. The weight of each sample was determined,
and a volume of a sterile saline solution (0.85% [wt/vol] NaCl)
equivalent to three times the weight of the granular sample was added.
The samples were kept on ice, vortexed vigorously for 2 min, and then
serially diluted (10-fold dilutions) in the sterile saline solution.
Aliquots (0.1 ml) of each dilution were plated onto solid LN-NMS agar
plates, and the plates were incubated for 2 to 3 weeks at 30°C either
under a 50% methane-50% air atmosphere or under a 100% air
atmosphere. All determinations were made in triplicate. Colonies
obtained from the serial dilutions were tested for the ability to
express sMMO activity and the presence of the sMMO gene. Colonies that
exhibited sMMO activity were identified by using the naphthalene
oxidation assay (4). Colonies that expressed the sMMO
genotype were identified by a colony hybridization technique
(12). The colonies were transferred onto nylon membrane filters and were examined by performing a Southern analysis with gene
probes specific for the mmoX gene of the sMMO gene cluster, essentially as described by Sotsky et al. (25). The sMMO
gene probe was prepared as described by Miguez et al. (21).
Determination of the numbers of strictly aerobic and
facultatively aerobic microorganisms.
The numbers of cells were
determined by using the Standard Methods for the Examination of
Water and Wastewater (1). Sludge (granular material)
samples obtained from predetermined ports in the reactor were removed,
mixed, and blended in a sterile Kinematica blender. Aliquots of the
disrupted sludge were then serially diluted, plated onto plate count
agar (Difco), and incubated at 30°C for 24 to 48 h. The colonies
were counted, and the number of colonies was considered to represent
the total number of facultatively aerobic microorganisms. Colonies
obtained from plates that were incubated aerobically and contained 50 to 100 CFU were transferred onto sterile plate count agar and incubated
anaerobically in an anaerobic jar (GasPack system) at 30°C for 1 month. Growth under these conditions revealed the presence of
facultatively aerobic microorganisms. The colonies that were not able
to grow represented the strictly aerobic microorganisms.
DNA extraction from granular sludge for PCR.
DNA was
extracted from 1-g (wet weight) granule samples by using the
freeze-thaw method of Tsai and Olson (26). DNA was further
purified by using Sephadex G-200 saturated with TE buffer (21). All PCR procedures were performed as previously
described (21).
Sensitivity of detection of the sMMO gene in granular sludge by
PCR.
The sMMO gene in granular sludge was detected by PCR
amplification of the mmoX gene fragment (21). The
sensitivity of the method was estimated as follows: (i) serial 10-fold
dilutions of a 48-h culture of M. sporium ATCC 35069 were
prepared by using 120 mM sodium phosphate buffer; (ii) prior to DNA
extraction (see above), a 1-ml aliquot of each serial dilution was
added to 1 g of a granular sludge sample; and (iii) PCR
amplification was performed as described previously (21).
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RESULTS |
Detection of sMMO activity and of the sMMO gene in granular
sludge.
Before inoculation of the reactors, efforts were made to
evaluate the concentration of sMMO-containing methanotrophic bacteria in the granular sludge originating from a full-scale UASB reactor treating wastewater from the baby food plant. The results indicated that the granular sludge contained at least 2,000 indigenous
methanotrophic cells having sMMO activity per g (wet weight) of
granular sludge, as determined by the naphthalene oxidation assay
(4).
As a second step, we attempted to detect the sMMO gene in the granular
sludge by the PCR method. Early investigations involving
inoculation of
granular sludge with a serially diluted culture
of
M. sporium (1.4 × 10
8 viable cells/ml) showed that
the PCR method could detect as few
as about 2,000 sMMO gene-containing
methanotrophic cells per g
(wet weight) of granular sludge (Fig.
2, lanes 6 and 7). Considering
that
1 g (wet weight) of granular sludge may contain between
10
11 and 10
12 viable cells, this detection
limit represented a detection capability
of approximately 2 sMMO
gene-containing cells per 10
8 to 10
9 viable
microbial cells. Interestingly, autoclaving granular sludge
for 40 min
did not prevent amplification of the extracted DNA
by the
mmoX primer set (results not shown).
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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.
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Isolation of indigenous sMMO-containing methanotrophs.
In
order to obtain initial information on the natural population of
sMMO-containing methanotrophs in the granular sludge, the
methanotrophic colonies obtained from the initial granular sludge
samples were divided into subgroups on the basis of differences in
colony appearance and morphology, and representative cultures were
further purified to homogeneity and maintained at 4°C. The fresh
isolates obtained were then fingerprinted by using a modified randomly
amplified polymorphic DNA (RAPD)-PCR method to verify their genetic
similarity to some well-characterized methanotrophic cultures. The
results (Fig. 3) revealed strong
similarities among the fresh isolates and M. sporium
ATCC 35069.
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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.
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Consequently,
M. sporium ATCC 35069 and another
closely related sMMO-containing methanotroph,
M. trichosporium OB3b (= ATCC
35070), were used as test organisms in
studies aimed at determining
the feasibility of coupling
sMMO-containing methanotrophs with
methanogens in UASB reactors under
O
2-limiting
conditions.
Survival of M. trichosporium and M. sporium in UASB reactors operated with various levels of
oxygenation.
This study was done in two phases, phase I and phase
II (Table 1). In phase I we used 1-liter
reactors, and the reactors were inoculated with either M. trichosporium OB3b (= ATCC 35070) or M. sporium
ATCC 35069. In phase II we used 5-liter reactors, and the reactors were
inoculated with only M. sporium ATCC 35069. As
indicated in Table 1, growth and survival of methanotrophs was
evaluated under various oxygen feeding and hydraulic regimes.
Phase I.
The objective of phase I was to determine which of
the two well-characterized sMMO-containing methanotrophs used exhibited greater growth and/or survival potential during cocultivation with
anaerobic consortia (granular sludge) in the UASB reactors. M. trichosporium (6.9% inoculum based on the total
sludge VSS) was inoculated into two 1-liter UASB reactors that were
operated under two different oxygenation regimes, 1.5 and 3 liters of
O2/day. M. sporium (6.5% inoculum based on
the total sludge VSS), on the other hand, was inoculated into only one
1-liter UASB reactor that was oxygenated at a rate of 1.5 liters/day;
therefore, M. sporium was not tested at the
3.0-liter/day oxygenation rate as M. trichosporium was.
Equal numbers of M. trichosporium viable cells were
inoculated into the two reactors to a final concentration of 2 × 107 cells/g of VSS. M. sporium was
inoculated to a concentration of 109 cells/g of VSS (Fig.
4). The reactors were operated for 72 days (Table 1) at three different HRTs. The results obtained with M. trichosporium OB3b are shown in Fig. 4A. With the
two reactors, as expected, an immediate 5-log increase in the total
number of methanotrophs was observed. The size of the methanotrophic
population gradually decreased during reactor operation and with
changes in the HRT until it was around 107 cells/g of VSS,
which was about 2 logs less than the size of the methanotrophic
population right after inoculation. In spite of this significant
decrease, the size of the total methanotrophic population in the
granular sludge after 72 days of reactor operation was at least 2 logs
greater than the size of the population prior to inoculation,
suggesting that inoculation with M. trichosporium contributed to establishing a significant methanotrophic population. The level of oxygenation (either 1.5 or 3.0 liters/day) did not appear
to influence this finding. The size of the population of methanotrophs
that exhibited sMMO activity (i.e., had the ability to oxidize
naphthalene) followed a pattern very similar to that of the total
methanotrophic population for the first 40 days of operation (Fig. 4A).
However, the size of this population decreased rapidly after 40 days to
almost undetectable levels, probably in part as a result of the change
in the HRT from 1 to 0.5 day. The decrease in the number of
methanotrophs that exhibited sMMO activity appeared to be more rapid in
the reactor aerated at a rate of 3.0 liters/day than in the other
reactor. Not surprisingly, with the two reactors, the profile obtained
for sMMO probe-positive methanotrophs generally resembled the profile
obtained for methanotrophs that exhibited sMMO activity.
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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.
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The results obtained for
M. sporium are presented in
Fig.
4B. As expected, the size of the total methanotrophic population
increased by at least 5 logs immediately after inoculation with
M. sporium and, as described above for
M. trichosporium, decreased
gradually during reactor operation until
it was about 10
7 cells/g of VSS after 72 days, a value that
still was at least
2 logs greater than the value prior to inoculation.
The numbers
of methanotrophs that exhibited sMMO activity and the
numbers
of methanotrophs that contained the sMMO gene were quite
similar;
the profile for each of these two populations resembled the
profile
for the total methanotrophic population. In contrast to the
results
obtained with the reactors inoculated with
M. trichosporium (Fig.
4A), the number of methanotrophs that
exhibited sMMO activity
or contained the sMMO gene did not decreased
significantly after
the change in the HRT from 1 to 0.5 day. The sizes
of these populations
were approximately 10
6 cells/g of VSS
by day
72.
Interestingly, for several granular sludge samples, the number of
methanotrophs that exhibited sMMO activity was greater than
the number of methanotrophs that contained the sMMO gene. This
might indicate that some of the colonies that exhibited sMMO activity
did not hybridize with our
mmoX probe.
Finally, with all three reactors, visual observation indicated that the
physical characteristics of the granular sludge changed
during reactor
operation; the granular sludge became more fluffy
upon exposure to
oxygen, suggesting that colonization by a different
microbial
population occurred. In spite of these changes in physical
characteristics, the overall performance of all of the bioreactors,
as
measured by the volumetric production of methane, was identical
to that
of control bioreactors not fed any oxygen (results not
shown).
During phase I, efforts were made to estimate the sizes of the
populations of
mmoX probe-positive methanotrophic bacteria
present in the aqueous phases of the reactors (Table
2). A general
pattern was observed;
following inoculation of the three reactors,
a significant increase in
the size of the extragranular methanotrophic
population was always
observed, although the significance varied
greatly. With all three
reactors, the size of the extragranular
methanotrophic population
decreased very significantly with time
but generally remained greater
than 10
5 cells/g of VSS for the first 40 days of operation
in spite of
the changes in reactor operation. The effluent from all
UASB type
reactors, including ours, is rarely a clear homogeneous
aqueous
solution. It is usually murky and full of particulate suspended
matter which adheres to glass walls and tubing. Periodically and
unpredictably, the suspended matter is released and is discharged
with
the rest of the effluent. For these reasons, the numbers
of
mmoX probe-positive methanotrophs in the effluent varied.
Our
results show that during operation of the reactors
mmoX
probe-positive
methanotrophs were indeed lost in the effluent.
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TABLE 2.
Concentration of mmoX probe-positive
methanotrophic bacteria not associated with granular sludge in Phase I
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Phase II.
The main objective of phase II was to improve the
survival and retention of sMMO-containing methanotrophs in the granular biomass. To do this, certain modifications to the design and operation of the reactors were introduced, and 5-liter reactors were used instead
of 1-liter reactors. To ensure that the dissolved oxygen concentration
in the influent reaching the granular sludge was proper and constant,
the 5-liter reactors were connected to aeration columns as described by
Shen and Guiot (23). Three dissolved oxygen concentrations
were tested, 2, 5, and 8.5 ppm. In addition, the nitrogen components of
the feed, NH4HCO3 and
(NH4)2SO4, were replaced with
urea (109 mg/liter) and NaNO3 (425 mg/liter) in order
to provide sufficient nitrogen for methanotrophic growth (2) while avoiding the potential inhibition of methane
oxidation by ammonium ions (18). Finally, since
M. sporium ATCC 35069 appeared to exhibit better
survival characteristics during phase I (Fig. 4), this organism was
used as the inoculum in the phase II investigations.
As shown in Table
1 and discussed above, six 5-liter reactors that
contained 1,000 ml of granular sludge (50 g of VSS) each
were first
acclimatized to predetermined levels of dissolved oxygen
(i.e., 2, 5, and 8.5 ppm) for 5 days prior to inoculation with
M. sporium. Three of the six reactors were each inoculated with
a
culture of
M. sporium (10
9 cells/g of VSS)
at a level equivalent to a 2.4% inoculum based
on the total sludge
VSS. The three control reactors (which were
not inoculated with
methanotrophs) were operated as follows: one
reactor was maintained
under strictly anaerobic conditions, and
the remaining two reactors
were maintained at influent dissolved
oxygen concentrations of 5 and
8.5 ppm. The following results
were obtained (Fig.
5).
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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.
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(i) Just prior to inoculation with
M. sporium, the
granular sludge contained 2.3 × 10
6 methanotrophs per
g of VSS; only 2.6% of these methanotrophs
(0.6 × 10
5 cells/g of VSS) exhibited sMMO activity, and 6.5%
(1.5 × 10
5 cells/g of VSS) proved to be probe
positive (Fig.
5A).
(ii) Right after inoculation, the granular sludge contained
approximately 10
9 methanotrophs per g of VSS; all of these
methanotrophs exhibited
sMMO activity and responded positively to the
mmoX probe (Fig.
5A). Inoculation, therefore, increased
the size of the total methanotrophic
population about 500-fold.
(iii) After 35 days of operation (Fig.
5B), the total number of viable
methanotrophs in the inoculated reactors had decreased
by at least 2 logs and varied between 2.1 × 10
6 and 7 × 10
6 cells per g of granular sludge. A similar pattern was
observed
for the number of bacteria that exhibited sMMO activity or
were
probe positive (Fig.
5B).
(iv) After 35 days of operation of all three inoculated reactors, the
numbers of probe-positive bacteria and bacteria with
sMMO activity were
very similar to the total methanotroph numbers,
unlike the situation
observed in the reactors before inoculation.
It was obvious that
inoculation was instrumental in helping establish
a relatively stable
population of methanotrophic bacteria that
exhibited sMMO
activity.
(v) Maintaining the dissolved oxygen concentration at either 5 or 8.5 ppm increased the size of the methanotrophic population
by a factor of
about three compared with the population incubated
in the presence of 2 ppm of dissolved oxygen (Fig.
5B).
(vi) Figure
5C shows that the number of strictly aerobic
microorganisms per gram of granular sludge was relatively
constant
at each level of dissolved oxygen but that the size of the
population
of facultative microorganisms varied more with the
dissolved oxygen
concentration and, in particular, increased at
the lowest oxygen
level used (2
ppm).
(vii) In all cases, the size of the total population of methanotrophs
measured on day 35 never exceeded 0.7 × 10
7 cells per
g of granular sludge, while the size of the total strictly
aerobic
microbial population ranged from 10
9 to 10
10
cells per g. Such population sizes for methanotrophs may be considered
significant, although they are relatively
small.
The results obtained with the control (uninoculated) reactors are shown
in Fig.
5D and E. These results may be summarized
as
follows.
(i) After 35 days of operation of the control reactors, the numbers of
methanotrophs were highly variable. The numbers of
viable methanotrophs
were approximately 10
4 cells per g of granular sludge in
reactors maintained at either
0 or 5 ppm of dissolved oxygen but
reached 10
7 cells per g in the reactor maintained at 8.5 ppm of dissolved
oxygen (Fig.
5D).
(ii) Although measurable populations of methanotrophs with sMMO
activity were detected in the reactors maintained at 0 or
5 ppm of
dissolved oxygen, interestingly, no methanotrophs that
responded to
our probe were detected (Fig.
5D). However, there
was a very
significant population of probe-positive methanotrophs
in the reactor
maintained at 8.5 ppm of dissolved
oxygen.
(iii) In the control (uninoculated) reactors, the size of the
population of strictly aerobic microorganisms, as expected,
increased
greatly with the level of dissolved oxygen (Fig.
5E).
Similar, although
less pronounced, results were obtained for the
population of
facultative
microorganisms.
As was the case during phase I, the granular sludge present in the
aerated (oxygenated) reactors acquired floccular characteristics
during
operation of the reactors. This was probably attributable
to
significant changes in the microbial composition of the populations
in
the oxygenated reactors, such as a significant increase in
the size of
the population of strictly aerobic and facultative
microorganisms (Fig.
5E) and the establishment of a relatively
small but significant
methanotrophic
population.
Aeration of the anaerobic reactors used during the phase II study did
not seriously affect methanogenesis, as shown previously
(
24). Whether the bioreactors were inoculated with
methanotrophs
or not and whether the dissolved oxygen concentration
maintained
in the bioreactors was 2 or 8.5 mg/liter (Table
1), the
methane
production rate was 61 to 65% of the rate measured in the
bioreactor
that was operated completely
anaerobically.
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DISCUSSION |
This study had the following two main goals: (i) to evaluate the
ability of well-known methanotrophic cultures to grow and/or survive in
UASB reactors in the form of coaggregates containing methanogens and
methanotrophs as part of the granular sludge; and (ii) to validate the
use of PCR and catabolic gene probes for monitoring the populations of
sMMO-related methanotrophs associated with the granular sludge in
hybrid anaerobic-aerobic bioreactors operated under various
regimes. In the PCR method used in this study we used a
primer set designated mmoX1-mmoX2 which has been described recently (21) and was derived from the
mmoX gene encoding the alpha subunit of the sMMO.
sMMO-containing methanotrophs associated with granular sludge.
As indicated above, the source of the granular sludge inoculum
used for bioreactor inoculation was a full-scale UASB
reactor treating wastewater from a baby food plant. Our results
indicated that before inoculation this anaerobic granular
sludge contained at least 2,000 indigenous methanotrophic bacteria
exhibiting sMMO activity per g (wet weight) of sludge; however,
the number of bacteria was generally 104 to 105
cells per g. Given the industrial nature of the granular
sludge (i.e., anaerobic granular sludge that was present in an open
system with methane and was exposed to wastewater containing some
dissolved oxygen), it was not surprising to find such levels of
sMMO-containing methanotrophs. Consistent with this finding, Kato et
al. (17) obtained strong evidence that methane-oxidizing
microorganisms were present in granular sludge intentionally exposed to
oxygen in laboratory experiments.
The PCR method used in this study could detect as few as about
2,000 sMMO gene-containing methanotrophs per g (wet weight)
of granular
sludge (Fig.
2). This detection limit was, therefore,
equivalent to the
lowest population levels of sMMO-related methanotrophs
measured in
our granular sludge (see above) by the traditional
plate count
method. According to our calculations, our PCR method
was able to
detect approximately 2 sMMO gene-containing cells
per 10
8
to 10
9 viable microbial cells. This detection ability is
very interesting;
it is equivalent to detecting one "contaminating"
bacterial cell
in a 1-ml aliquot of a low-density bacterial
culture.
The cultivable sMMO-containing methanotrophs obtained from the granular
sludge exhibited significant genetic similarities
to
M. sporium ATCC 35069 after RAPD-PCR analysis. Based on the
fact that
methanotrophs are known to be difficult to isolate from
environmental
samples, we concluded that a variable and sometimes
significant
population of
M. sporium-like bacteria was
present
in the granular sludge without necessarily assuming that this
population was the dominant sMMO-containing population.
Nevertheless,
the results justified our decision to inoculate anaerobic
reactors
with
M. sporium ATCC 35069 and with another
closely related methanotroph,
M. trichosporium OB3b
(= ATCC 35070), since similar organisms
were naturally present in the
granular
sludge.
Inoculation of reactors with known methanotrophic cultures.
Our results show conclusively that anaerobic reactors operated at
various levels of aeration can maintain for up to 72 days a significant
sMMO-containing microbial population following inoculation of the
reactors with known methanotrophic cultures (Fig. 4 and 5). These
initial results are promising. In all of the cases studied, the size of
the total methanotrophic population at the end of the reactor
experiment was always at least 2 logs greater than the size of the
initial population. In this regard, the sMMO-containing populations
resulting from inoculation with M. sporium appeared to
be more stable and more resistant to changes in the HRT than the
equivalent populations associated with reactors inoculated with
M. trichosporium (Fig. 4).
Correlation between the various population levels.
In general,
there was a good correlation between the sMMO-containing population
levels measured with molecular tools (colony hybridization to an sMMO
gene-specific probe) and the population levels measured with the
naphthalene oxidation assay. This indicated that most of the sMMO
hybridizing colonies also produced an active sMMO. At times, the number
of methanotrophs that exhibited sMMO activity was greater than the
number of methanotrophs that contained the sMMO gene. This could
indicate that some methanotrophs did not respond to our mmoX
gene probe, a possibility that should not be forgotten.
Methanotrophs not associated with the granular sludge.
As
indicated in Table 2, significant populations of sMMO probe-positive
methanotrophs that were not associated with the granular sludge were
present following inoculation of the reactors during phase I. The size
of the methanotrophic population decreased significantly with time and
with changes in the HRT but nevertheless always remained greater than
105 cells per g for the first 40 days of reactor operation.
Since the reactors were operated in a continuous mode, this suggested either that the extragranular methanotrophic fraction was able to
survive and grow up to a certain point and/or that there was continuous
release of sMMO probe-positive bacteria from the granules. Note that
for two of the three reactors inoculated (Table 2) no sMMO
probe-positive methanotrophic population was detected in the
extragranular fraction before inoculation. This suggests that
inoculation of the reactors may also have been beneficial to the
extragranular methanotrophic fraction, a phenomenon that could also
help increase reactor performance in subsequent biotreatment studies.
Methanogenic-methanotrophic hybrid reactors.
In recent years,
the concept of coupling methanogens and methanotrophs, either
sequentially or synchronously, in order to enhance the biodegradation
of pollutants has attracted the attention of a few groups of scientists
(8, 11, 19, 24). This concept is relatively new, and process
development based on this concept is still in its infancy. In this
study, we show that significant methanotrophic populations can be
maintained in aerobic-anaerobic bioreactors that are initially
inoculated with a pure culture of a methanotrophic bacterium and are
operated under oxygen-limited conditions. Most of the cultivable
methanotrophic bacteria obtained from our reactors not only were sMMO
probe positive but also, and more importantly, exhibited sMMO
activity. Our initial results appear quite promising. In
this study, the bioreactors were inoculated with a methanotrophic
culture only once. It is likely that larger populations of
methanotrophic bacteria could be maintained in hybrid bioreactors if
repeated pulse inoculation was used, a hypothesis that should be tested
in the near future.
| |
ACKNOWLEDGMENTS |
We thank the following former or present Biotechnology Research
Institute colleagues for their collaboration: J. Al-Hawari for
scientific support in analytical chemistry, Alain Corriveau and Louise
Paquet for analytical technical support, and Jennifer Sealy, a former
co-op student, for technical and scientific assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Microbial and
Enzymatic Technology Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec,
Canada H4P 2R2. Phone: (514) 496-6186. Fax: (514) 496-5485. E-mail:
Denis.Groleau{at}nrc.ca.
| |
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Abstract
Introduction
