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Applied and Environmental Microbiology, October 2000, p. 4433-4439, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Possible Interactions within a
Methanotrophic-Heterotrophic Groundwater Community Able To Transform
Linear Alkylbenzenesulfonates
Dubravka
Hr
ak* and
Ana
Begonja
Center for Marine and Environmental Research,
Ru
er Bokovi
Institute, HR-10002 Zagreb, Croatia
Received 10 April 2000/Accepted 27 July 2000
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ABSTRACT |
The relationships and interactions within a
methanotrophic-heterotrophic groundwater community were studied in a
closed system (shake culture) in the presence of methane as the primary
carbon and energy source and with the addition of the pure linear
alkylbenzenesulfonate (LAS) congener 2-[4-(sulfophenyl)]decan as a
cometabolic substrate. When cultured under different conditions, this
community was shown to be a stable association, consisting of one
obligate type II methanotroph and four or five heterotrophs possessing
different nutritional and physiological characteristics. The results of experiments examining growth kinetics and nutritional relationships suggested that a number of complex interactions existed in the community in which the methanotroph was the only member able to grow on
methane and to cometabolically initiate LAS transformation. These
growth and metabolic activities of the methanotroph ensured the supply
of a carbon source and specific nutrients which sustained the growth of
four or five heterotrophs. In addition to the obligatory nutritional
relationships between the methanotroph and heterotrophs, other possible
interactions resulted in the modification of basic growth parameters of
individual populations and a concerted metabolic attack on the complex
LAS molecule. Most of these relationships conferred beneficial effects
on the interacting populations, making the community adaptable to
various environmental conditions and more efficient in LAS
transformation than any of the individual populations alone.
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INTRODUCTION |
A number of microbial communities
have been studied in which coexisted heterotrophs and obligate
methanotrophs, the latter being the unique group of bacteria able to
use methane as the only carbon and energy source (3, 6, 12, 26,
33). Despite the relative ease with which
methanotrophic-heterotrophic communities have been isolated from
different environments (soils, surface layers of sediments, and natural
waters), which suggests their ubiquity in nature, only recently have
more intensive studies been undertaken to establish the possible
significance of these specific associations in the cycling of elements
in the biosphere (16, 20, 26, 34).
In addition to the recognized important role of methane-utilizing
bacteria in global carbon and nitrogen cycling (3, 10, 11, 16, 19,
27), there are an increasing number of reports about their
cometabolic activities in the transformation of multicarbon compounds
(1, 9, 14, 19, 23, 38). These cometabolic activities have
been described as "fortuitous metabolism," suggesting that they are
due to the broad substrate specificity of the methane monooxygenase
enzyme system, whose primary function is to catalyze the oxidation of
methane to carbon dioxide. At present, the high potential of
methanotrophs in the transformation of low-molecular-weight halogenated
hydrocarbons, ubiquitous and toxic pollutants, has been well recognized
(2, 5, 8, 13, 15, 16, 25, 28, 29, 30), while the
significance of these bacteria in the transformation of more complex
compounds, especially aromatic compounds, still awaits recognition.
Although in some cases transformation of complex organic compounds with
methanotrophs involves more extensive or even nonoxidative metabolism,
products of transformations catalyzed by methane monooxygenase are
usually simply hydroxylated derivatives, suggesting that the role of
methanotrophic bacteria is only in the initiation of biodegradative attack (1, 16, 19, 37). On the other hand, oxidative cometabolic transformation has been identified as an important initial
step in the degradation of diverse complex substrates (xenobiotics),
making the substrates more susceptible to further transformation by
heterotrophs (1, 4, 7, 9, 14, 19, 32, 37). It follows that,
besides the generally accepted important role of heterotrophic bacteria
in the mineralization of complex organic compounds, methanotrophs might
also have a significant contribution in at least initiating useful metabolism.
The main objective of this work was to better characterize the
methanotrophic-heterotrophic community originating from an aquifer
material previously characterized to be efficient in cometabolic transformation of trichloroethylene (17, 18), chlorinated biphenyls (1, 37), and linear alkylbenzenesulfonates (LAS) (21, 22, 24). The specific objective was to study possible relationships and interactions between community members during growth
in mineral medium with methane as the only carbon and energy source and
during the transformation of 2-[4-(sulfophenyl)]decan (2C10LAS) added as a cometabolic substrate.
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MATERIALS AND METHODS |
Cultivation of the community and individual populations.
After isolation from aquifer solids of Moffett Field Naval Air Base,
Mountain View, Calif. (17), the methanotrophic-heterotrophic community was stored as a liquid culture at 4°C and subcultured every
6 to 8 weeks in 120-ml sealed serum bottles containing 20 ml of nitrate
mineral salts (NMS) medium (39). The headspace was filled
with a methane-air gas mixture (30:70), and cultures were incubated in
a water bath shaker (180 rpm) at 20°C.
The same shake flask technique and NMS medium without copper were
applied to study the growth characteristics of the community and its
individual populations as well as for 2C10LAS
transformation experiments. To achieve suitable methane and oxygen
concentrations, different amounts of CH4 (5 to 20 ml) were
injected after the withdrawal of an equal volume of air. Methane and
oxygen concentrations were monitored by headspace analyses and the
desired conditions (low oxygen, excess methane, or excess of both
gases) were maintained by the repeated injection of 5 to 20 ml of
CH4 and 5 to 20 ml of O2. Culture growth was
monitored by measuring the optical density at 600 nm
(OD600). The experiments were carried out at either 20 or
30°C.
Specific growth rates (µ) of the community and its methanotrophic
strain for a particular cultivation period (
tn 
1 
tn) were calculated from the
following equation:
where
xn and
xn 1 are the biomass concentration (milligrams [dry weight]
liter
1) at times
tn and
tn 1,
respectively.
For the determination of community structure, separate dilution plating
was carried out on solid NMS medium with 1% (wt/vol)
electrophoresis-grade agarose (GIBCO-BRL, Paisley, Scotland) and
nutrient agar (Difco Laboratories, Detroit, Mich.). The same
NMS-agarose
medium was used for culturing and maintaining the pure
methanotroph
isolated from the community, while for heterotrophic
populations,
oligotrophic medium containing low concentrations of
peptone,
yeast extract, and glucose (PYG) (
35) was applied
instead of
nutrient agar. For the methanotroph, the plates were
incubated
for 10 to 14 days at 30°C, and for heterotrophs, incubation
was
for 2 to 4 days at 20, 25, or 30°C. NMS-agarose plates were kept
in gas-tight jars under a methane and air atmosphere (50:50).
The gas
phase was replaced every 3 to 4 days with a fresh methane-air
mixture.
Both the methanotrophic and heterotrophic isolates were
stored at 4°C
as liquid cultures, and their purity was checked
by several successive
platings on solid
media.
Characterization of individual community populations.
After
isolation and confirmation of purity, individual populations were
further characterized for their nutritional and physiological properties using the methods discussed by Stanier et al.
(36) and by Palleroni and Doudoroff (31). In all
testing, the NMS medium (39), solidified with 1.5% Noble
agar that was essentially free of impurities (Difco Laboratories), was
used as a basal medium, and the selected organic compound as the carbon
and energy source was added at a concentration of 0.1%. For
nutritional screenings of heterotrophic bacteria, oligotrophic media
containing low concentrations of peptone and yeast extract (PY) or PYG
(35) were also used.
For cross-growing experiments in which growth stimulation and
inhibition effects between community populations were studied,
the
inoculum was prepared by using a 1-week-old shake flask culture
of the
methanotrophic strain Met 1 (in early stationary growth
phase) and the
individual heterotrophs grown on solid PYG medium.
Suspensions
(OD
600, approximately 2.5 to 2.8) were prepared either
in
NMS medium (for the experiments with individual populations)
or in
sterile MilliQ water (Millipore, Molsheim, France) (for
the experiments
with individual population lysates). To facilitate
autolysis,
suspensions were incubated for 48 h at 37°C and afterwards
were
filter sterilized (0.22-µm pore diameter; Millipore). Prepared
suspensions or autolysates (1 ml) were added to 20 ml of the
methanotrophic
culture diluted with NMS medium (OD
600,
0.1), giving a proportion
of methanotrophs to individual heterotrophs
of approximately 50:50.
Reagents.
Natural gas containing 98.5% methane was donated
by INA Naftaplin (Zagreb, Croatia), and oxygen (Universal Medical) was
obtained from MG Croatia Plin (Zagreb, Croatia). All chemicals used for the growth media were of analytical grade, and those used for high-performance liquid chromatography (HPLC) analysis were of HPLC
grade (Merck, Darmstadt, Germany). Pure (98%) 2C10LAS was donated by EAWAG (Dübendorf, Switzerland).
Headspace analyses.
Methane and oxygen concentrations during
growth of the community or during cross-feeding experiments with the
methanotrophic and heterotrophic populations were monitored by
headspace analyses using a Fisher-Hamilton gas partitioner equipped
with a thermal conductivity detector as described previously
(22).
2C10LAS analyses.
For quantitative determination
of 2C10LAS during transformation, reversed-phase HPLC was
used by employing an octylsilica column, 250 by 4.4 mm (inner
diameter), 5 µm (Supelcosil LC-18; Supelco Inc., Bellefonte, Pa.),
under isocratic conditions. The eluent used was 38 to 42% (vol/vol)
acetonitrile containing 10 g of sodium perchlorate per liter.
Spectrofluorimetric detection was applied at an excitation wavelength
of 230 nm and an emission wavelength of 295 nm.
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RESULTS |
Growth characteristics of the groundwater community and individual
members.
To better evaluate the metabolic activity and possible
interactions within the methanotrophic-heterotrophic community
originating from an aquifer material, a series of experiments was
performed in which the growth kinetics of the community and its
methanotrophic member (strain Met 1) were compared. The obtained
results (Table 1) illustrated that the
groundwater community was capable of stable growth in shake flasks
under various experimental conditions (different methane and oxygen
concentrations and different temperatures). The general observation was
that although it originated from groundwater where the temperature does
not exceed 15°C, the community grew faster at higher temperatures
(30°C). In accordance with decreased oxygen concentrations relative
to the atmosphere in its natural habitat, this community showed a
preference for low oxygen concentrations (3 to 5%), and the fastest
growth (µ = 0.55 ± 0.03 day1 [mean ± standard deviation]) was achieved when excess methane (10 to 15%) was
maintained in the headspace (Table 1).
Another general observation was that when cultured under various
conditions, the groundwater community was a stable association
consisting of one obligate methanotroph and four heterotrophs.
One
additional heterotrophic population was occasionally observed,
especially when the community was cultured under conditions optimal
for
its growth in shake flasks (CH
4, 10 to 15%;
O
2, 3 to 5%).
Evaluation of the community structure (Table
2) showed that most
of the enriched
communities harvested in the early stationary
phase were dominated by
the methanotroph, which, depending on
methane and oxygen concentrations
(CH
4, 4.5 to 19.4%; O
2, 15.5
to 19%),
represented 50 to 85% of the total population. When cultured
under low
oxygen (3 to 5%) and excess methane (10 to 15%) concentrations,
where
optimal growth of the community was achieved, an even higher
proportion
of methanotrophs (approximately 90% of the total population)
was
obtained. The results presented in Table
2 also illustrate
that among
the heterotrophs, two species dominated and the remaining
three species
formed a small proportion of the total heterotrophic
population. The
community structure was surprisingly stable under
different methane and
oxygen concentrations, and only the relative
proportions of the
dominant heterotrophic populations were changed
significantly by
changing the incubation temperature.
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TABLE 2.
Structure of the methanotrophic-heterotrophic community
enriched under different methane and oxygen concentrations
at 30°Ca
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Comparative growth kinetics experiments performed with the methanotroph
(strain Met 1) as a single culture showed that, similar
to the
community, this strain preferred the conditions of low
oxygen
concentrations (3 to 5%) and higher temperatures (30°C)
over the
conditions prevailing in the natural habitat (Table
1).
However, under
the same experimental conditions, its specific
growth rate (µ = 0.45 ± 0.06 day
1) as a single culture was lower
than when strain Met 1 was grown
as part of the community (µ = 0.55 ± 0.03 day
1). To explore whether an
accumulation of metabolites of methane
oxidation may be one of the
reasons for the impaired growth rate,
further experiments with strain
Met 1 were performed in which
methanol (0.2 or 2.0 mmol
liter
1) was added as a supplementary carbon source. Since
the growth
of strain Met 1 was typically suppressed in the presence of
methanol,
even at a lower concentration (0.2 mmol liter
1)
and especially under conditions where its optimal growth was
achieved
(Fig.
1), the hypothesis of a
self-inhibitory growth
effect due to methanol as a product of methane
oxidation seems
very likely.
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FIG. 1.
Growth of the methanotroph (strain Met 1) in the
presence of methanol as a supplementary carbon source under
microaerobic conditions (O2, 3 to 5%) with excess methane
(CH4, 10 to 15%) at 30°C. Symbols:  , control (without
methanol);  , methanol at 0.2 mM;  , methanol at 2 mM. The data are
means ± standard deviations from triplicate bottles.
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Some general nutritional and physiological properties of the isolated
heterotrophic populations relevant for the evaluation
of possible
relationships and interactions within the methanotrophic-heterotrophic
community are summarized in Table
3. It
is evident that only
one heterotroph (Het 3) exhibited the capability
to grow on NMS
medium, while all other heterotrophs required the
addition of
a carbon source and grew well on oligotrophic media (PY and
PYG)
containing low concentrations (0.025%) of peptone, yeast extract,
and glucose. None of the heterotrophs was able to use methane
as the
only carbon and energy source. These results suggested
that when
cultured in NMS medium with methane as the only carbon
and energy
source, the presence of all heterotrophs except strain
Het 3 was
dependent on the growth and metabolic activity of the
methanotroph.
Furthermore, despite some similarities among the
heterotrophs (all of
them are obligately aerobic and chemo-organotrophic),
individual
heterotrophs differed according to their growth factor
requirements,
utilization of methanol as the only carbon source,
and preferences of
nitrogen sources, which suggested their different
metabolic activities
in the community.
Growth stimulation and inhibition effects between community
populations.
To study possible interactions based on the
modification of growth activities of individual community populations,
a series of experiments was carried out by cross-growing the
methanotrophic strain in the presence of individual heterotrophs and
their lysates. Some of the obtained results are presented in Fig.
2. It is evident that when grown under
microaerobic conditions and with excess methane (O2, 3 to
5%; CH4, 10 to 15%), only one heterotroph (unidentified strain Het 4) stimulated the growth of strain Met 1, while the other
heterotrophs suppressed its growth (Fig. 2A). Similar experiments performed under aerobic conditions (O2, 15 to 18%;
CH4, 15 to 18%) (results not presented) showed that all
heterotrophs stimulated the growth of strain Met 1. In contrast, none
of the filter-sterilized autolysates of the heterotrophic strains
stimulated the growth of strain Met 1 under either microaerobic (Fig.
2B) or aerobic conditions (results not presented). These results
suggested that the growth of the methanotrophic strain may not be
dependent on specific substances (nutrients or growth factors) excreted
by the heterotrophs; however, it was affected by their growth and metabolic activities. Most of these heterotrophic activities stimulated the growth of the methanotrophic strain, while the suppressed growth
under microaerobic conditions suggested that some negative relationships (competition for nutrients and oxygen) may also exist
between the community populations.
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FIG. 2.
Growth of strain Met 1 in the presence of individual
heterotrophic members (A) and their lysates (B). Experimental
conditions are the same as for Fig. 1. Symbols:  , control (strain
Met 1 alone); , Met 1 and Het 4; , Met 1 and Het 5;  , Met 1 and Het 1;  , Met 1 and Het 3;  , Met 1 and Het 2. The data are
means ± standard deviations from triplicate bottles.
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Significantly stimulated growth of four of the five heterotrophs (i.e.,
all except strain Het 3) in the presence of either
strain Met 1 (Fig.
3) or its lysate (results not presented)
suggested
that the most probable role of the methanotrophic strain when
grown in the community may be in providing the heterotrophs with
a
carbon source or specific nutrients necessary for growth in
mineral
medium with addition of methane as the only carbon source.
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FIG. 3.
Growth of individual heterotrophs (Het 1 through 5) in
the presence of the methanotroph (strain Met 1). The initial
concentration of Met 1 was 5 × 108 CFU/ml. For
comparison, growth curves of individual heterotrophic populations in
NMS medium are also shown. The data are means ± standard
deviations from triplicate bottles.
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LAS transformation activity of the groundwater community and its
individual populations.
To explore the hypothesis of concerted
metabolic activity of individual community populations on complex
organic LAS compounds that was suggested in previous studies (21,
22, 24), further comparative transformation experiments with a
pure LAS congener, 2C10LAS, were performed with the
original six-member groundwater community, its methanotrophic strain
(Met 1) as a pure culture, and two-member reconstructed communities
containing strain Met 1 and one of the heterotrophic strains known to
possess the enzyme system for 
-oxidation (Het 1, Het 2, and Het 3).
The results (Fig. 4) confirmed the
previous observation of fast 2C10LAS transformation with
the original community (within 1 day) and very slow 2C10LAS disappearance (within 20 days) with the methanotroph as a pure culture.
Furthermore, although slower than with the original community, LAS
disappearance with all reconstructed communities (Met 1 and Het 1, 5 days; Met 1 and Het 2, 7 days; and Met 1 and Het 3, 4 days) was faster
than LAS disappearance with the methanotroph as a single culture; this
finding supported the hypothesis of combined metabolic attack on the
complex LAS molecule.
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FIG. 4.
Comparison of 2C10LAS transformation
(determined by reversed-phase-HPLC) by the original six-member
groundwater community, two-member reconstructed communities, and the
methanotroph as a single culture. Symbols: , original community;
, Met 1 and Het 3; , Met 1 and Het 1; , Met 1 and Het 2; ,
Met 1 alone. The data are means ± standard deviations from
triplicate bottles; in some cases, the standard deviations were too
small to illustrate.
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DISCUSSION |
The results presented in this work and previous papers (22,
24) showed that the groundwater community was a tight association containing one obligate methanotroph (strain Met 1) and, depending on
experimental conditions, four or five heterotrophic strains (Het 1 through 5), possessing different metabolic activities. Based on cell
morphology, the resting stages formed, the intracytoplasmic membranes,
and some physiological characteristics, strain Met 1 was tentatively
identified as a type II methanotroph. This was confirmed by further
characterization, including the determination of fatty acid methyl
ester profiles and 16S rRNA analyses, which suggested the similarity of
strain Met 1 with the most studied and characterized type II
methanotroph, Methylosinus trichosporium OB3b
(37). On the basis of the nutritional, morphological, and physiological characteristics of the isolated heterotrophic populations presented here and discussed in a previous paper (24), three of the heterotrophs were tentatively identified as members of the
genera Blastobacter (Het 1), Pseudomonas (Het 2),
and Xanthobacter (Het 3), while two still-unidentified
strains (Het 4 and Het 5) showed similarities to prosthecate bacteria.
In addition to the previously reported efficiency in cometabolic
transformation of trichloroethylene (17, 18) and chlorinated biphenyls (1, 37), the methanotrophic-heterotrophic
community also exhibited the capability to transform LAS (21, 22,
23). This metabolic activity was evaluated on the basis of
intensive transformation studies of commercial LAS and pure LAS
congeners in the presence or absence of methane as the primary carbon
and energy source. The methanotrophic-heterotrophic community exhibited transformation activity on different LAS molecules under various environmental conditions (aerobic and microaerobic), with or without methane addition. The main proposed mechanism was the initiation of LAS
transformation on the alkyl side chain by 
-oxidation, followed by
the shortening of the chain by -oxidation. Further experiments, in
which possible LAS transformation activities of individual community
members were determined, suggested that only strain Met 1 had the
capability of LAS transformation (24). The failure of this
strain to transform sulfophenyldecanoic acid as the product of
-oxidation and the observed ability of three heterotrophic strains
(Het 1, Het 2, and Het 3) to transform this first LAS intermediate by
-oxidation suggested that the methanotroph could be involved in the
initiation of LAS transformation, and some of the heterotrophs could be
involved in the continuation of LAS transformation. This hypothesis of
metabolic cooperation between community populations for LAS
transformation was further supported by the results presented in Fig.
4, which illustrated faster LAS disappearance with the original
six-member community and two-member reconstructed communities than with
the methanotroph as a single culture.
Possible interactions within methanotrophic-heterotrophic
community.
Stable growth characteristics of the six-member
groundwater community (Table 1), with almost regular appearance of all
populations under various conditions (Table 2), illustrated the
capacity of the community to self-regulate in response to changing
environmental conditions. This also suggested that the groundwater
community was structured on specific relationships between the obligate methanotroph and the heterotrophs, with their different nutritional requirements and metabolic activities. Furthermore, unstable growth characteristics (occasionally prolonged [1 to 5-days] lag phase, or
oscillations in the growth rate) and impaired specific growth rates of
strain Met 1 when grown as a single culture compared to its growth in
the community (Table 1) suggested interactions which conferred
beneficial effects on the interacting populations. Thus, the most
likely explanation for stimulated growth of strain Met 1 in the
community may be that the heterotrophs removed self-inhibitory organic
compounds excreted by the methanotroph. This is likely, since obligate
methanotrophs are known to be sensitive to amino acids and products of
methane oxidation (16, 33), and growth of strain Met 1 was
found to be inhibited by methanol added as a supplementary carbon
source (Fig. 1). In addition, three of the five isolated heterotrophic
strains (Het 1, Het 4, and Het 5) were able to use methanol and all
heterotrophs used formate, both of which are products of methane
oxidation (Table 3). The accumulation of self-inhibitory products
excreted by the methanotroph may be less expected in an open system,
but according to the available literature (15, 16, 30), much
faster growth of methanotrophs is achieved in an open system than in a
closed system, typically with accumulation of methanol as an
intermediate of methane oxidation.
Another possible explanation for the faster and more stable growth of
the methanotrophic strain in the community rather than
as a single
culture is that specific substances (nutrients or
growth factors)
excreted by the heterotrophs affected the methanotroph's
growth. This
explanation is less probable, since none of the filter-sterilized
autolysates of the heterotrophs showed growth stimulation of this
strain under either microaerobic or aerobic
conditions.
The most probable beneficial relationships between strain Met 1 and the
heterotrophs, when grown in the community in the presence
of methane as
a carbon and energy source, are summarized in Fig.
5. The methanotroph, the only member able
to oxidize methane,
excreted methane oxidation products and metabolites
which may
be used by heterotrophs as a carbon source or as growth
factors.
The heterotrophs in turn removed excreted organic compounds
which
may be inhibitory for the methanotroph. Thus, the removal of
excreted
compounds from culture liquid may be mutually beneficial for
the
methanotroph and heterotrophs. Another probable beneficial effect
of the heterotrophs may be in reducing oxygen tension, thus making
more
favorable conditions for growth of the methanotrophic strain,
which
preferred microaerobic conditions (3 to 5% O
2 in the
headspace).
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FIG. 5.
General scheme of the probable beneficial effects within
the methanotrophic-heterotrophic community when grown in NMS medium
with the addition of methane as the only carbon and energy source.
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The scheme presenting the most likely beneficial interactions within
the methanotrophic-heterotrophic groundwater community
(Fig.
5) seems
rather simple. However, in this community, in which
one obligate
methanotroph and four or five heterotrophs of different
nutritional and
metabolic capabilities coexist, a number of interactions
and
relationships might exist between the populations which enable
their
remaining in the community under conditions favorable only
for the
growth of the methanotroph. The situation was much more
complex when
the groundwater community was cultured in the presence
of both methane
as a primary substrate and LAS as a cometabolic
substrate. In this
case, as schematically presented in Fig.
6,
along with the products of methane
oxidation there were also LAS
intermediates, which may cause additional
nutritional and growth
modification relationships between the
community populations.
Although the relationships and
interactions between individual
populations are not yet fully
understood, it can be proposed that
the methanotroph, as the only
member able to oxidize methane and
cometabolically initiate LAS
transformation, excreted methane
oxidation products and LAS
intermediates which may serve as carbon
sources or growth factors for
the heterotrophs. Based on the nutritional
and physiological
characteristics of individual heterotrophic
populations (Table
3), the
products of methane oxidation might
theoretically sustain the growth of
all five heterotrophs, since
they were able to use formate and at least
three of them (Het
1, Het 4, and Het 5) were able to use methanol as
their carbon
source. On the other hand the growth of the three
heterotrophic
populations (Het 1, Het 2, and Het 3) capable of
transforming
sulfophenyldecanoic acid may be sustained by LAS
intermediates,
suggesting their possible involvement in the
continuation of LAS
transformation.
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FIG. 6.
Scheme of possible interactions within the
methanotrophic-heterotrophic community in the presence of methane as
the carbon source and LAS as a cometabolic substrate.  , substrate
utilization associated with growth; --,
cometabolic transformation not linked to growth.
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A general conclusion which can be drawn from the results of this study
is that in the six-member methanotrophic-heterotrophic
community, a
number of complex interactions may exist between
community populations.
The most probable interactions are those
based on the provision of
specific nutrients, the removal of inhibitory
compounds, the mutual
modification of basic growth parameters,
and the metabolic cooperation
for the combined attack on complex
LAS molecules. Most of these
relationships, some of which are
obligatory and reciprocal for the
interacting populations, confer
beneficial effects, making the
community stable and adaptable
to various environmental conditions and
more efficient in LAS
transformation than any of the individual
populations alone. In
addition to these mutually beneficial effects,
some negative interactions
(competition for oxygen and/or nutrients)
may also exist within
this specific and complex groundwater community
under particular
environmental
conditions.
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ACKNOWLEDGMENTS |
This work was supported by the Croatian Ministry of Science and Technology.
We are grateful to Angela S. Lindner from the Department of
Environmental Engineering Sciences, University of Florida, for her
valuable cooperation in the characterization of the
methanotrophic-heterotrophic community.
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FOOTNOTES |
*
Corresponding author. Mailing address: Center for
Marine and Environmental Research, Ruer Bokovi
Institute, P.O. Box 180, HR-10002 Zagreb, Croatia. Phone: 385-1-46 80 944. Fax: 385-1-46 80 242. E-mail: hrsak{at}rudjer.irb.hr.
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Applied and Environmental Microbiology, October 2000, p. 4433-4439, Vol. 66, No. 10
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Abstract
Introduction
