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Applied and Environmental Microbiology, May 1999, p. 2209-2216, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Molecular Phylogenetic and Biogeochemical Studies of
Sulfate-Reducing Bacteria in the Rhizosphere of
Spartina alterniflora
Mark E.
Hines,1,*
Robert S.
Evans,2
Barbara R.
Sharak
Genthner,3
Stephanie G.
Willis,2
Stephanie
Friedman,4
Juliette N.
Rooney-Varga,2,
and
Richard
Devereux4
Department of Biological Sciences, University
of Alaska Anchorage, Anchorage, Alaska 995081;
Institute for the Study of Earth, Oceans and Space, University
of New Hampshire, Durham, New Hampshire
038242; Center for Environmental
Diagnostics and Bioremediation, University of West Florida,
Pensacola, Florida 325143; and
National Health and Environmental Effects Research
Laboratory, U.S. Environmental Protection Agency, Gulf Breeze,
Florida 325614
Received 22 October 1998/Accepted 1 March 1999
 |
ABSTRACT |
The population composition and biogeochemistry of sulfate-reducing
bacteria (SRB) in the rhizosphere of the marsh grass Spartina alterniflora was investigated over two growing seasons by
molecular probing, enumerations of culturable SRB, and measurements of
SO42
reduction rates and geochemical
parameters. SO42 reduction was rapid in marsh
sediments with rates up to 3.5 µmol ml1
day1. Rates increased greatly when plant growth began in
April and decreased again when plants flowered in late July. Results
with nucleic acid probes revealed that SRB rRNA accounted for up to 43% of the rRNA from members of the domain Bacteria in
marsh sediments, with the highest percentages occurring in bacteria
physically associated with root surfaces. The relative abundance (RA)
of SRB rRNA in whole-sediment samples compared to that of
Bacteria rRNA did not vary greatly throughout the year,
despite large temporal changes in SO42
reduction activity. However, the RA of root-associated SRB did increase
from <10 to >30% when plants were actively growing. rRNA from
members of the family Desulfobacteriaceae comprised the
majority of the SRB rRNA at 3 to 34% of Bacteria rRNA,
with Desulfobulbus spp. accounting for 1 to 16%. The RA of
Desulfovibrio rRNA generally comprised from <1 to 3% of
the Bacteria rRNA. The highest
Desulfobacteriaceae RA in whole sediments was 26% and was
found in the deepest sediment samples (6 to 8 cm). Culturable SRB
abundance, determined by most-probable-number analyses, was high at
>107 ml1. Ethanol utilizers were most
abundant, followed by acetate utilizers. The high numbers of culturable
SRB and the high RA of SRB rRNA compared to that of
Bacteria rRNA may be due to the release of SRB substrates
in plant root exudates, creating a microbial food web that circumvents fermentation.
| |
INTRODUCTION |
Temperate salt marshes are among the
most productive ecosystems on Earth with carbon fixation rates
exceeding 1,000 g of C m2 year1
(50). A large portion of this carbon is decomposed within
marsh sediments, and sulfate (SO42) reduction
accounts for more than half of this decomposition (24). The
presence of a dense root or rhizome system in salt marsh sediments and
the ability of this root system to deliver organic materials and
oxidants below ground produce a dynamic subsurface redox
biogeochemistry capable of supporting steep chemical gradients and a
diverse microflora (18). Hence, the rhizosphere is an ideal
microhabitat for bacterial proliferation and is important for plant health.
The dynamics of interactions between the salt marsh rhizosphere and
bacteria can be regulated strongly by plant growth stage and the
release of materials from roots. Organic matter supplied as root
exudates may account for the majority of growing season SO42 reduction in salt marsh sediments
(22). Changes in plant growth, i.e., initiation of active
elongation (vegetative growth) and commencement of reproduction, affect
SO42 reduction in sediments inhabited by the
common cordgrass Spartina alterniflora (18, 22).
Therefore, the marsh is a habitat with high rates of microbial activity
that are strongly affected by plant activities.
Considerable effort has been devoted to studies of
SO42 reduction in salt marsh sediments
(15, 22, 25, 26). However, populations of sulfate-reducing
bacteria (SRB) and how they might change in response to plant activity
are not well known. Molecular approaches, especially those using
phylogeny-based methods, offer the ability to investigate bacterial
population dynamics of specific phylogenetic groups which often share
physiological traits. The 16S rRNA phylogeny of the SRB is well
described, and hybridization probes which target each of the known
major SRB groups and several individual species have been developed
(10, 11). These phylogenetic groups correspond with distinct
physiological assemblages (11), so rRNA-based methods can
provide information on the types of electron donors that might be used
by SRB in the S. alterniflora rhizosphere. The present study
was conducted to characterize SO42 reduction
and SRB populations in the rhizosphere of S. alterniflora by
utilizing hybridization probing techniques together with activity and
enumeration approaches. The site chosen for the work was a marsh that
we had studied in detail previously and which provided a fundamental
baseline of information on the distribution of bacterial activity rates
(19, 22), geochemistry (18, 22, 44, 61, 62), and
diversity of SRB (47, 48).
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MATERIALS AND METHODS |
Study site.
Samples were collected for 18 months (two
growing seasons) from 1993 to 1994 from a tall-form (2.0-m), creekside
stand of S. alterniflora in Chapman's Marsh in southeastern
New Hampshire (22). The tall form was studied because this
actively growing form is capable of releasing larger quantities of
O2 and dissolved organic carbon from roots than the short
form (22). In addition, during reproductive periods, all of
the tall-form culms simultaneously produce reproductive organs, while
only about 30% of the culms in the short-form stands do so
(29). Hence, the tall form was best suited for an
investigation of the effects of changes in plant growth stage on
below-ground microbial activity. The sediments were organically rich,
i.e., composed primarily of living and dead plant roots and rhizomes,
but they also contained detrital clay and silt-sized particles
delivered from upstream terrestrial sources. To avoid disturbing the
vegetation and sediment, boardwalks were used to access sampling sites.
Sample handling.
Sediment cores (5-cm diameter) were
collected by using a handheld corer with a polycarbonate liner
(47). Cores were flushed with N2 immediately
after collection, capped, and held anoxically on ice for transport to
the laboratory. Cores were either processed within 1 to 2 h of
sample collection (for rate and most chemical analyses) or stored at
80°C until used for further manipulations (for RNA extractions).
Samples for enumeration of SRB by culturing techniques were shipped
cold overnight to the U.S. Environmental Protection Agency laboratory
in Gulf Breeze, Fla. All handling in the laboratory was conducted anoxically.
Pore water samples for sulfate analyses were collected with in situ
sippers (22), which were deployed during the spring each
year and removed in the fall prior to ice formation. These devices did
not cause any unusual sediment erosion. Pore water samples were
collected, filtered, and dispensed anoxically within 1 or 2 min in the
field. Because the sippers were left in place for several months at a
time, we were able to study temporal changes at several exact
locations. In addition, the placement of the sippers prior to plant
growth in the spring allowed for nondestructive sampling that prevented
artifacts due to root damage (22, 27).
Sulfate reduction.
Rates of SO42
reduction (SRR) were determined by a 35S reduction assay
using chromium (21). Briefly, duplicate sediment cores were
sliced into sections in a N2-filled glove bag and portions were placed into 5-ml plastic syringes which were sealed with serum
stoppers. Syringes and stoppers were preincubated for 2 weeks under
N2 to prevent diffusion of O2 into samples
(6). Subsamples were not homogenized prior to use. One µCi
of 35SO42 was injected into each
syringe, and samples were incubated in a dark N2-filled jar
overnight at ambient temperature. Activity was stopped by freezing to
80°C. The concentration of 35S present in dissolved
sulfide, acid-volatile sulfides, pyrite, and elemental sulfur was
determined by reducing these chemical species to sulfide with reduced
acidic chromium (21).
rRNA extraction and hybridization.
Nucleic acids were
extracted from bulk sediments collected over 18 months from three
depths: 0 to 2, 2 to 4, and 6 to 8 cm. During the second year (1994),
cores were also sliced in half vertically, the upper two depth sections
in one half of the core were combined, and the sediment was removed by
gentle rinses with salinity-adjusted buffer (47). The rinsed
roots were considered rhizosphere samples that contained bacteria
closely associated with or attached to root material. RNA was extracted
from all sediments and rhizosphere samples by a bead-beating technique (13, 47), and nucleic acids were further purified with
Sephadex G25 spin columns (43).
RNA was denatured by adding 3 volumes of 2% (vol/vol) glutaraldehyde
in 50 mM sodium phosphate (pH 7.0) to 1 volume of RNA
extract and
incubating at room temperature for 10 min (
55).
Denatured
RNA was then diluted with sterile distilled H
2O containing
0.0002% (wt/vol) bromophenol blue and 1 µg of poly(A)
ml
1. Using a slot blot device (Minifold II; Schleicher
and Schuell,
Inc., Keene, N.H.) under slight vacuum, the various
dilutions
of sample and standard RNAs (in a volume of 100 µl) were
applied
to Immobilon-N membranes (Millipore Corp., Bedford, Mass.) that
had been prewetted in 95% (vol/vol) ethanol and rinsed in distilled
H
2O. Membranes were then dried at room temperature and
baked at
80°C for 1 h prior to prehybridization and subsequent
hybridization
(
56).
Oligonucleotide probes were end labeled with
32P
(
13), purified with Nensorb 20 cartridges (Dupont Corp.,
Wilmington, Del.)
(
55), and hybridized at 40°C overnight.
After the membranes
were washed (
56) (washing temperatures
given in Table
1), they
were air dried
briefly and the amount of probe was quantified
with a gas proportional
radioisotope detection system (Ambis,
Inc., San Diego, Calif.).
Two sets of hybridization membranes were used for slot blot analysis
(
55). One membrane was hybridized with a probe specific
for
a particular bacterial group or genus, while the other utilized
a
general probe designed to hybridize with 16S rRNA of almost
all species
in the domain
Bacteria (EUB338) (
56) (Table
1).
The specific probes utilized were SRB probes 687 (primarily the
family
Desulfovibrionaceae), 660 (
Desulfobulbus
spp.), and 804
(most members of the family
Desulfobacteriaceae). In addition,
several samples collected
during the first few months of the 1993
season were analyzed with
probes that target phylogenetic groups
within the
Desulfobacteriaceae, including probes 129 (
Desulfobacter spp.), 221 (
Desulfobacterium
spp.), and 814 (
Desulfosarcina,
Desulfococcus,
and
Desulfobotulus spp.) (
13). Samples were added
to membranes
at three concentrations, with 50 to 200 ng per slot used
for membranes
assayed with the
Bacteria probe and 600 to
1,800 ng per slot used
on membranes for specific probes. Membranes
assayed with the specific
probes received reference rRNA extracted from
pure cultures (Table
1) at a range of ~0.78 to 25 ng per slot to
generate a standard
curve. Membranes hybridized with the general
Bacteria probe (EUB338)
received ~1.56 to 200 ng per blot
of reference rRNA that was the
same reference material used for the
specific
probes.
The relative abundances (RA) of the specific probe targets as a
function of total
Bacteria rRNA were determined by first
quantifying
radioactive signal per slot and correcting for background.
Next,
the following equation was used to calculate RA: RA (percent)
= [(
mss ×
m
sr)/(
mes ×
mer)] × 100, where
mss
is the slope of
specific probe signal per unit of sample rRNA,
msr is the slope
of the specific probe signal
per unit of reference rRNA,
mes is
the slope of
the
Bacteria probe signal per unit of sample rRNA,
and
mer is the slope of the
Bacteria
probe signal per unit of
reference rRNA (
16). Samples for
which the slope of probe signal
per unit of rRNA was not linear (i.e.,
r2 < 0.90) were omitted from
analyses.
MPN analyses.
SRB in sediment cores were enumerated by the
most-probable-number (MPN) technique. Three types of samples were
analyzed: bulk sediments from the top 3 cm and from a depth of 12 to 15 cm and the rhizosphere of the top 3 cm. Duplicate cores were
aseptically extruded and cut horizontally, and sections were separated
with a sterile razor in an anaerobic glove box. The section from the upper 3 cm was also cut vertically to provide subsamples for
rhizosphere analysis. Sections from duplicate cores were combined,
weighed, and transferred to a sterile Waring blender inside an
anaerobic chamber. The medium of Widdel and Pfennig (64),
without Na2SO4 or an electron donor, was used
to rinse residual sample into the blender and to prepare a
101 dilution (wt/vol) of the sediments. This dilution was
homogenized by blending for 5 min, and the homogenate was aseptically
transferred to a sterile serum bottle, sealed, and reduced. This
dilution was the inoculum for a triplicate MPN dilution series
(102 to 109) in tubes sealed with serum
stoppers. To prepare rhizosphere samples, the half-core duplicate
sections from the upper 3 cm were combined and aseptically rinsed by
gently immersing in a series of beakers containing the sterile rinse
medium (100 ml) until all visible sediment was removed. The rinsed
roots were weighed, and a 101 dilution was prepared as
described above.
The medium of Widdel and Pfennig (
64) prepared as described
previously (
52) was used for MPN determination. The salinity
of the medium was adjusted to that of the overlying marsh water
at the
time of sampling with the addition of NaCl and MgCl · 6H
2O
in appropriate ratios (
64). The completed
medium contained one
of the following electron donors: acetate, 20 mM;
ethanol, 20
mM; benzoate, 5 mM; butyrate, 10 mM; malate, 10 mM; or
propionate,
10 mM. All incubations were at 20°C. Growth was
determined as
the increase in optical density at a wavelength of 600 nm. Sulfate
removal was confirmed by ion chromatography
(
51). Tubes showing
both growth and
SO
42 consumption were considered positive and
used to calculate the
abundance of SRB per gram of
sample.
| |
RESULTS |
Sulfate reduction.
Temperatures in surficial marsh sediments
displayed a typical seasonal cycle with highs near 25°C in summer and
below 0°C in winter (Fig. 1a). In
general, SRR followed the seasonal temperature, with low rates in
winter and very high rates at over 2,000 nmol ml1
day1 in summer (Fig. 1b). However, temporal changes in
SRR corresponded better with changes in plant growth than with
temperature, i.e., SRR increased rapidly at the commencement of
vegetative growth (aerial elongation) and decreased upon the initiation
of reproductive growth (flowering) in late July. This phenomenon was
noted previously in this marsh (22). In 1993, plant growth
displayed a bimodal pattern in which aerial growth slowed in the middle
of the season (late June to early July) and then increased again. The
SRR displayed a similar bimodal pattern that year (Fig. 1b). In 1994 when plant growth was continuous, SRR increased rapidly upon the
initiation of plant growth and remained high until mid-August. SRR were
most rapid near the sediment surface where activities reached ~3,500 nmol ml1 day1 in July (Fig.
2). Rates below 5 cm were generally <500
nmol ml1 day1.
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FIG. 1.
(a) Sedimentary temperature and (b) plant height ( )
and SRR ( ) in marsh sediments over time. Rates are averages of data
from the upper 0- to 2-cm and 2- to 4-cm depths. The data are plotted
over time for two growing seasons (in 1993, April [A], June [J],
and October [O] are shown; in 1994, January [J], April [A], June
[J], and October [O] are shown in the x axis).
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Application of probes to environmental rRNA.
Selected samples
were analyzed for variability in the probe assay by comparing (i)
triplicate cores, (ii) cores that were divided into two sections
vertically, and (iii) individual rRNA samples analyzed two or three
times. These comparisons showed that variability was usually less than
10% and often less than 5% of the mean (data not shown). This
variability increased to as much as 21% in a few cases where the
signal was weak and the RA was low.
The sum of the probe data for rRNA from
Desulfovibrio (probe
687),
Desulfobacteriaceae (probe 804), and
Desulfobulbus (probe
660) accounted for up to ~30% of the
total
Bacteria rRNA in the
marsh sediments (Fig.
3). Some of the highest RAs were noted in
winter. The highest RAs occurred in the deepest samples (6 to
8 cm),
which in 1994 accounted for 30% of the
Bacteria rRNA. This
depth yielded much higher values in 1994 than during the previous
year.
However, samples from 6 to 8 cm were not analyzed during
each sampling
period. In general, the lowest RAs were noted in
surficial samples (0 to 2 cm), yet these still accounted for ~5
to 12% of the total
Bacteria rRNA throughout the study period.
The data for 0 to
2 cm were the most variable and displayed two
large maxima during the
summer of 1993. RA at the 2- to 4-cm depth
interval displayed smoother
trends that included a decrease during
the middle of the growing season
in both years. Other than this
two- to fourfold decrease in RA at 2 to
4 cm, dramatic changes
in RA corresponding to plant growth variations
were not observed
in bulk sediment samples despite the fact that SRR
did vary greatly
(Fig.
1).
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FIG. 3.
Temporal changes at three depths in the RA of species of
Desulfobacteriaceae (probe 804) (),
Desulfobulbus (probe 660) (), and
Desulfovibrio (probe 687) ( ) in marsh sediments. Stippled
area indicates period of plant vegetative growth. The data are plotted
over time for two growing seasons (in 1993, April [A], June [J],
August [A], October [O], and December [D] are shown; in 1994, February [F], April [A], June [J], August [A], and October
[O] are shown on the x axis).
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|
The
Desulfobacteriaceae accounted for up to 25% of the
Bacteria rRNA and exhibited a much higher RA than the
Desulfovibrio and
Desulfobulbus spp. (Fig.
3).
The abundance of the
Desulfobacteriaceae rRNA was most
pronounced below the upper 2 cm where it was usually
10- to 20-fold
higher than the other groups. The
Desulfovibrio rRNA was
poorly represented in all samples and accounted for less
than 1 to 2%
of the
Bacteria rRNA, while the
Desulfobulbus
rRNA
generally represented from 1 to 5%. In the middle of both growing
seasons, the RA of the
Desulfobacteriaceae rRNA from
sediments
2 to 4 cm down decreased significantly (Fig.
3). The
Desulfobulbus rRNA increased during this same period in 1993 from sediments
0 to 2 and 2 to 4 cm down and in sediments 0 to 2 cm
down in 1994.
The signals from probes 129, 221, and 814 were too low to
accurately
quantify in many instances and, when summed, represented a
small
portion of the RA of the
Desulfobacteriaceae.
Root-associated SRB exhibited higher RAs than did samples of bulk
sediments, with the former accounting for up to 40% of the
Bacteria rRNA (Fig.
4). The
data in Fig.
4 are compared to the
average for the data from the upper
0 to 2 and 2 to 4 cm combined,
since subsamples for root analyses
consisted of the 0- to 4-cm
depth interval. The RA of root-associated
Desulfobacteriaceae dominated the SRB, as was noted for bulk
sediment samples. However,
in contrast to bulk sediment, the RA of the
root-associated SRB
increased during vegetative growth. This was seen
most clearly
for the
Desulfobacteriaceae (probe 804), which
increased nearly
fourfold to over 30%.
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FIG. 4.
Temporal changes in the RA of species of
Desulfobacteriaceae (probe 804) (),
Desulfobulbus (probe 600) (), and
Desulfovibrio (probe 687) () both in bulk sediments and
on roots. Stippled area indicates period of plant vegatative growth.
The data are plotted over time for most of 1994 as follows: January
(J), February (F), March (M), April (A), May (M), June (J), July (J),
August (A), September (S), and October (O).
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|
Enumeration of SRB by MPN.
SRB physiological types were
enumerated in sediment and isolated rhizosphere samples on three
occasions during 1993. In addition, the upper 2 cm of sediment was
assayed similarly in April 1994 (Table
2). Electron donors were chosen as those
that might be released by roots (e.g., ethanol and malate
[42]) or produced by anaerobic cellulolytic
microorganisms (7). In almost all cases, the highest numbers
of SRB were recovered with ethanol-containing medium with abundances
reaching nearly 5 × 107 g1 (wet
weight). In fact, ethanol-utilizing SRB outnumbered the other types by
nearly an order of magnitude in most instances. The second most
abundant group was the acetate-utilizing species which were usually
greater than 106 g1, followed by butyrate
utilizers, which reached 2.5 × 106 g1.
In June 1993, surficial sediments (0 to 3 cm), deeper sediments (12 to
15 cm), and roots rinsed free of sediment contained
similar numbers of
SRB on a weight basis (Table
2). Acetate-utilizing
SRB were slightly
higher in root samples at 2.4 × 10
6 g
1
compared to 1 × 10
6 g
1 in bulk
sediments. In August, acetate-utilizing SRB were threefold
more
abundant in surficial sediments and on roots than in the
deeper
sediment samples. Butyrate-utilizing SRB were also more
abundant in
surficial sediments in August and displayed a sharp
decrease in deeper
sediments. Propionate-utilizing SRB were relatively
low in all samples
at 10
5 to 10
6 g
1 but did not show
a significant decrease in abundance with depth,
as was seen for many of
the other SRB groups. In October, ethanol-,
propionate-, and
butyrate-utilizing species decreased with depth
by about a factor of
10, while acetate and benzoate utilizers
did not
decrease.
| |
DISCUSSION |
Sulfate reduction.
The seasonal changes in the biogeochemical
conditions in marsh sediments followed changes in plant growth activity
and physiology as inferred previously by other studies in this marsh
(18, 19, 22). SO42 reduction
activity responded rapidly to changes in plant physiology. During
active vegetative growth, SO42 reduction was
rapid, whereas commencement of plant flowering near the end of July
each year resulted in sharp decreases in rate. A likely explanation for
this was that during vegetative growth, plants leaked dissolved organic
compounds into the sediment, fueling anaerobic bacterial metabolism
(22). This process ceases upon flowering as plants allocate
carbon to reproductive organs (39). It is also possible that
bacterial activity was inhibited by release of organic compounds
(phenolic compounds) with bacteriostatic properties (3, 9).
Blaabjerg et al. (1) noted a similar finding that a
significant portion of the SO42 reduction
activity in the rhizosphere of the sea grass Zostera marina
was fueled by organic exudates rather than by sedimentary organic
matter. SRR in the marsh were extremely high and similar in magnitude
to those reported for microbial mats (57, 60).
SRB RA.
SRB rRNA comprised a very large percentage of
Bacteria rRNA in marsh sediments throughout both 1993 and
1994. One would not expect SRB to dominate Bacteria, since
SRB are situated at the terminal end of the decomposition hierarchy
where they use end products of other bacteria for energy and growth
(45, 54, 63). Devereux et al. (12), using
methodology identical to that used here, reported that SRB accounted
for <3% of the Bacteria in an unvegetated marine sediment.
Trimmer et al. (58) found that <3.5% of the
Bacteria rRNA in an estuarine sediment was attributable to
SRB, but they did note SRB RA as high as 12% in brackish sediment (salinity, <1.3%). Purdy et al. (46) utilized probe
techniques and reported that SRB comprised a relatively minor portion
of the bacteria in estuarine sediment slurries. Because the methods used here measured rRNA RA and not cell numbers, the high SRB RA in
marsh sediments indicated that SRB were either unusually abundant
compared to other bacteria or were more active on a per cell basis and
contained more rRNA. The marsh sediments exhibited SRR that were
approximately an order of magnitude higher than those determined by
Devereux et al. (12) and Trimmer et al. (58).
However, high rates alone do not necessarily explain high SRB RA, since
the latter represents the proportion of Bacteria rRNA that
is from SRB which is a function of the microbial food web configuration.
An explanation for the high relative proportion of SRB rRNA in the
marsh sediments is that a portion of the substrates utilized
directly
by SRB, i.e., fermentation products, were provided directly
by roots
during root fermentation activity. We did note active
alcohol
dehydrogenase activity by root tissues (unpublished data)
which would
provide ethanol for use by SRB (
41,
42).
S. alterniflora roots also produce malate (
41) and perhaps
acetate (
19). Hence,
SRB using these root exudates would not
require a synergistic
relationship with fermentative bacteria. In
addition, other types
of substrates released from roots, such as
carbohydrate monomers
(
38), would require a less diverse
bacterial consortium to degrade
them compared to that needed to degrade
solid-phase and polymeric
compounds. Such a simplified food web would
permit SRB to be a
larger proportion of the bacterial community. This
premise is
supported by the finding that root-associated SRB displayed
a
higher RA than SRB in bulk sediments (Fig.
4). In addition, Purdy
et
al. (
46) noted increases over time in the RA of SRB in
sediment
slurries amended with SRB electron donors, which underscores
the
above
premise.
The RA and composition of SRB in bulk sediment samples did not vary
greatly in response to plant growth stage despite the
changes in SRR.
One would assume that the rapid increases and
decreases in SRR, which
were noted during the commencement of
plant growth and immediately
after flowering, would result from
a change in the quantity and
composition of substrates used by
SRB. It has been noted previously in
this marsh that the redox
status of the sediment changes dramatically
during the growing
season due to rapid changes in the delivery of
oxidants by roots
(
22). These changes could also cause a
shift in the relative
importance of particular physiological groups of
SRB. However,
probe data for bulk sediments displayed little variation
throughout
the year compared to SRR. Since the SRB RAs in the marsh
were
generally high, it appeared that the marsh harbored a large and
active population of SRB that continued to dominate sediment bacterial
biomass throughout the year, regardless of
SO
42 reduction and how rapidly it changed
over time. In fact, the
general increase in the RA of the SRB during
winter suggested
that the SRB were able to survive long periods of cold
better
than other bacteria in the
marsh.
Probe data for bulk sediment samples did correspond with changes in
plant growth on some occasions. In particular, the
Desulfobacteriaceae at the 2- to 4-cm depth during both
years decreased by as much
as 75% in the middle of the vegetative
growth period (Fig.
4).
One explanation for this decrease is that the
delivery of organic
material and oxidants to sediments by plants
enhanced the growth
of other types of bacteria capable of taking
advantage of the
changing habitat. Although anaerobic bacterial
activity increased
greatly during vegetative growth due to dissolved
organic carbon
input, it has been shown in this marsh that
S. alterniflora also
supplies oxygen to the sediments simultaneously
(
22). O
2 exudation
(in conjunction with organic
matter release) would favor growth
of aerobic bacteria and other
bacteria capable of using alternate
electron acceptors such as metals
and nitrate regenerated from
subsurface redox cycling. Reduced S (solid
and dissolved) in these
sediments exhibited a sharp decrease in
concentration in June
1993, indicating a significant increase in
oxidant input (
23a).
That decrease corresponded exactly with
the decrease in RA of
the
Desulfobacteriaceae. Since SRR
increased greatly during summer,
it is likely that total SRB rRNA also
increased but that non-SRB
rRNA increased even more, resulting in a
decrease in SRB
RA.
The RA of the
Desulfobulbus spp. increased in June 1993 as
the
Desulfobacteriaceae decreased (Fig.
3). In fact, the
highest
RA noted for the
Desulfobulbus spp. occurred in the
0- to 2- and
2- to 4-cm-deep sections in mid June. Apparently, members
of this
genus were stimulated by the increased introduction of
O
2, perhaps
due in part to their ability to
disproportionate S
0 generated from the oxidation of reduced
S in the subsurface (
36).
In contrast to bulk sediments, the RA of all the root-associated SRB
groups, especially the
Desulfobacteriaceae group, increased
greatly during active plant growth. We propose that exudation
of
organic matter from roots stimulated these SRB relative to
other
bacterial groups even though the opposite occurred in bulk
sediments.
Despite the summer increase in the RA of SRB on roots,
the bulk of the
total marsh SRB rRNA decreased during summer relative
to total rRNA.
However, root bacterial populations were unique
in that a large portion
of the total
Bacteria rRNA was SRB rRNA
and that this rRNA
increased further when plants were growing
above ground. This result
was intriguing, since one would envisage
that the root surface would be
subjected to higher levels of O
2 than the remaining
sediment if O
2 were released from roots. Our
previous work
in this marsh demonstrated that large quantities
of O
2 are
released by roots during vegetative growth (
22). One
would
anticipate that bacteria other than SRB would flourish on
the root,
causing the SRB to make up a smaller portion of the
total
Bacteria. The opposite result may be due to microcolonies
of
SRB on roots, which respond to organic exudates, and the fact
that
organic release is separated spatially from O
2 release. If
O
2 exudation is due primarily to movement of air through
plant
tissue when pore water is being removed by transpiration at low
tide (
8), then it is conceivable that organic exudation
would
not necessarily occur at the same sites as passive O
2
exudation.
In addition, the metabolic activity of root-associated
bacteria
on a cell basis may be extremely high compared to bulk
sedimentary
bacteria, since the former are juxtaposed at the source of
dissolved
organic material. Hence, the root SRB may be capable of
creating
a microenvironment conducive to growth through rapid
production
of sulfide-causing abiotic removal of O
2.
Furthermore, root-associated
bacteria may be better adapted to the
strong biogeochemical gradients
on roots by possessing the ability to
metabolize O
2 and other
electron acceptors and the ability
to use a larger array of substrates
than their bulk sediment
counterparts. High SRR have been reported
in the oxic layers of
microbial mats (
5,
14).
The marsh SRB population was dominated by members of the
Desulfobacteriaceae and to a lesser extent by
Desulfobulbus spp.
These results suggest that the
Desulfobacteriaceae and
Desulfobulbus spp. may be
well adapted to the marsh because they are better
suited for a habitat
that tends to rapidly change temporally and
spatially. For example,
Desulfobulbus propionicus is capable of
conserving energy
for growth from the disproportionation of elemental
sulfur
(
36) which is abundant in marsh sediments (
37).
Members
of the
Desulfobacteriaceae are capable of utilizing
a diverse
array of electron donors including formate, lactate, ethanol,
acetate, C
3 to C
16 fatty acids (
63),
secondary alcohols such
as 2-propanol and 2-butanol, isobutyrate
(
17), H
2, fumarate,
malate, and benzoate
(
63). Members of this group are capable
of complete
oxidation of organic carbon to CO
2, and aerobic respiration
has been reported (
14). Such nutritional versatility could
be
advantageous in a complex environment such as the salt marsh
sediment
and
rhizosphere.
The RA of
Desulfovibrio spp. in comparison was low and
accounted for less than 11% on average of the total SRB rRNA detected,
with a maximum of 27%. In the brackish sediments studied by Trimmer
et
al. (
58),
Desulfovibrio spp. accounted for up to
90% of the
detected SRB, while estuarine sediments contained ~35 to
65%.
Devereux et al. (
12) noted that
Desulfovibrio spp. were 49 to
68% of the SRB rRNA in
shallow marine sediments. Perhaps
Desulfovibrio spp. are
better adapted to habitats that tend to be more
reduced.
The increase with depth in the RA of members of the
Desulfobacteriaceae contrasts with observations in
subtidal unvegetated
sediments where
Desulfovibrio
spp. exhibited an increase with
depth (
12). In fact,
the highest RA of
Desulfobacteriaceae in
the marsh was found
in the deepest sediments sampled (Fig.
4).
Living
S. alterniflora roots occur at the deepest depths sampled
(
2,
28,
30), so the roots may have been influential at
those depths.
However, even though SRB consisted of a large portion
of the total
Bacteria rRNA at these depths, the actual amounts
of rRNA
extracted from sediments below the surface were quite
small. Hence, the
actual SRB biomass at depth was at least 10-fold
lower than at the
surface (data not
shown).
The hypothesis that dynamic biogeochemical cycling selects for the
enrichment of the
Desulfobacteriaceae is challenged by
the
fact that this group remains dominant throughout the year
even when
plants are inactive. Apparently, the rapid activity
of specific SRB in
the summer is sufficient to maintain the population
even in winter when
rates are orders of magnitude lower. We conducted
a preliminary study
in a plot where plants had died from deposition
of wrack (dead plant
material) and in which we had severed plant
roots along the periphery
down to >40 cm to prevent lateral root
and rhizome growth. However,
even these sediments harbored a SRB
population that 2 years later had a
composition similar to sediments
inhabited by living plants (data not
shown). The decrease in the
RA of
Desulfobacteriaceae during
the growing season also indicated
that the populations of the non-SRB
that diluted the
Desulfobacteriaceae did respond temporally
to plant growth while the SRB apparently
remained. Therefore, the
finding that the
Desulfobacteriaceae persisted at these
depths and in winter may simply indicate their
ability to survive
longer than other
bacteria.
Data from probes which target subgroups within the
Desulfobacteriaceae (probes 129, 814, and 221) were barely
detectable,
indicating that unknown members of this family not targeted
by
these probes are important in marsh sediments. In fact, Rooney-Varga
et al. (
47) found an uncultivated organism closely related
to
Desulfococcus multivorans that accounted for 24 to 40%
of the
Desulfobacteriaceae rRNA in the same extracts
described here.
This percentage was higher than the sum of the results
using probes
129, 814, and 221 indicating that unknown SRB species are
important
in marsh
sediments.
Abundance of culturable SRB.
High numbers of culturable SRB
were measured by the MPN technique, which is not surprising,
considering the high SRR. SRB viable counts from other studies were
much lower than those reported here, often by several orders of
magnitude (53). Viable counting methods are considered to
underestimate bacterial densities due to the inability of the growth
medium to allow for growth of all members of the community. Although
SRB counts by MPN methods often do not correlate well with SRR
(53), there are studies that do show good correlation
(23, 31, 33, 40, 49). In subtidal estuarine, marine, and
lake sediments, SRB counts vary from 103 to 106
per ml of whole sediment (4, 20, 33, 35). The marsh sediments studied here consistently contained over 107 SRB
per ml. These abundances are slightly greater than those reported for
very active cyanobacterial mats (57). This result provides
further evidence for the predominance of SRB in salt marsh sediments
and supports the premise that these SRB use substrates provided by
plants as root exudates.
Vester and Ingvorsen (
59) reported much higher SRB MPNs when
using media made from natural sediment or sludge compared to
those
using synthetic media. However, our measurements using synthetic
media
yielded similar or higher SRB numbers. When our data were
compared to
SRR in the marsh, we calculated specific SRR
(qSO
42) of ~10
14 mol of
SO
42 cell
1 day
1,
which are similar to those reported for pure cultures (
32),
cyanobacterial mats (
57), and for enrichment cultures using
natural media (
59). Therefore, our MPN estimations may be
approaching
the natural SRB densities but are likely still somewhat
low.
SRB viable counts did not vary temporally with SRR. However, MPN data
did agree with the RA of SRB determined with probes.
Both methods
revealed that SRB were abundant during periods when
activity rates were
low. These results underscore the premise
that SRR may change
dramatically in response to seasonal temperature
and plant growth
changes, yet the population composition and perhaps
even the actual
density of SRB may not change significantly throughout
the
year.
Densities of SRB on roots were similar in magnitude to those found in
bulk sediments on a weight basis. However, root mass
was minor compared
to total sediment (<20%). Therefore, although
the association of SRB
with
S. alterniflora roots is significant,
the
root-associated SRB probably accounted for less than 20% of
the total
sedimentary
bacteria.
The ethanol-utilizing SRB outnumbered the other types on most
occasions. Ethanol utilization is a trait found in many SRB
groups
including virtually all of the
Desulfovibrio spp.,
Desulfobulbus spp., and many of the species of other groups
(
63). Members
of the
Desulfomicrobium and
Desulfonema genera do not have this
ability (
10).
S. alterniflora releases ethanol from roots when
metabolizing anaerobically (
42), and it is tempting to
speculate
that the high abundance of ethanol-utilizing SRB in the marsh
sediments is a result of the preferential use of these exudates.
However, MPN techniques may support growth of certain physiological
types of SRB better than others (
34). During the present
study,
pure cultures isolated from MPN tubes containing ethanol were
always
Desulfovibrio sp. even though two of the isolates
were
unique (
48). Probe results demonstrated that
Desulfobulbus spp.
were generally more abundant than
Desulfovibrio spp. (Fig.
4),
yet MPN values for
propionate-utilizing
Desulfobulbus spp. were
lower than
those for corresponding desulfovibrios from ethanol-containing
media
(Table
2). One new
Desulfobulbus species was isolated from
a
butyrate-containing MPN tube (
48). Hence, it is likely that
the high estimates of ethanol-utilizing desulfovibrios were simply
due
to the fact that ethanol-utilizing SRB (probably
Desulfovibrio spp.) grew well in the MPN medium used, while
the medium is less
than optimal for
Desulfobulbus spp.
Acetate-utilizing enrichments
and MPNs tend to grow slowly, so the fact
that the acetate-utilizing
SRB MPNs were relatively high
(>10
6 ml
1) suggests that acetate-utilizing
SRB are also a significant component
of the marsh sediment and
rhizosphere.
In conclusion, probe results demonstrated that SRB were abundant in
marsh sediments and represented a high percentage of the
bacteria
present. Population abundance and composition did not
vary temporally
nearly as much as rates of activity did in response
to changes in plant
growth. However, SRB on root surfaces increased
during plant vegetative
growth while those in bulk sediments decreased.
This variation
suggested that release of dissolved organic material
from roots is
capable of stimulating a variety of bacterial groups
throughout the
sediment but especially rhizoplane SRB. The rapidly
changing habitat in
marsh sediments selects for members of the
Desulfobacteriaceae, a metabolically versatile
group.
| |
ACKNOWLEDGMENTS |
This work was supported in part by the U.S. Environmental
Protection Agency cooperative agreement CR-820062 and the National Science Foundation Ecology Program grant DEB-9520272.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, University of Alaska at Anchorage, 3211 Providence Dr., Anchorage, AK 99577. Phone: (907) 786-7762. Fax: (907) 786-4607. E-mail: afmeh{at}uaa.alaska.edu.
Present address: Biological Sciences Department, University of
Massachusetts Lowell, Lowell, MA 01854.
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Applied and Environmental Microbiology, May 1999, p. 2209-2216, Vol. 65, No. 5
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
