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Applied and Environmental Microbiology, August 1999, p. 3526-3533, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Culturable Populations of Sporomusa spp.
and Desulfovibrio spp. in the Anoxic Bulk Soil of Flooded
Rice Microcosms
Dirk
Rosencrantz,1
Frederick A.
Rainey,2,
and
Peter H.
Janssen1,*
Max-Planck-Institut für terrestrische
Mikrobiologie, D-35043 Marburg,1 and
Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,
D-38124 Braunschweig,2 Germany
Received 2 February 1999/Accepted 18 May 1999
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ABSTRACT |
Most-probable-number (MPN) counts were made of homoacetogenic and
other bacteria present in the anoxic flooded bulk soil of laboratory
microcosms containing 90- to 95-day-old rice plants. MPN counts with
substrates known to be useful for the selective enrichment or the
cultivation of homoacetogenic bacteria (betaine, ethylene glycol,
2,3-butanediol, and 3,4,5-trimethoxybenzoate) gave counts of 2.3 × 103 to 2.8 × 105 cells per g of dry
soil. Homoacetogens isolated from the terminal positive steps of these
dilution cultures belonged to the genus Sporomusa. Counts
with succinate, ethanol, and lactate gave much higher MPNs of 5.9 × 105 to 3.4 × 107 cells per g of dry
soil and led to the isolation of Desulfovibrio spp.
Counting experiments on lactate and ethanol which included Methanospirillum hungatei in the medium gave MPNs of
2.3 × 106 to 7.5 × 108 cells per g
of dry soil and led to the isolation of Sporomusa spp. The
latter strains could grow with betaine, ethylene glycol, 2,3-butanediol, and/or 3,4,5-trimethoxybenzoate, but apparently most
cells of Sporomusa spp. did not initiate growth in counting experiments with those substrates. Spores apparently accounted for
2.2% or less of the culturable bacteria. It appears that culturable Desulfovibrio spp. and Sporomusa spp. were
present in approximately equal numbers in the bulk soil. Multiple,
phylogenetically-distinct, phenotypically-different, strains of each
genus were found in the same soil system.
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INTRODUCTION |
Processes leading to methane
emission from rice paddy soils have been quantified (9, 37)
and their importance to world climate recognized (14). Since
these soils are flooded, they are largely anoxic and typically (but not
exclusively) methanogenic. In the oxic zones associated with the plant
root system, the chemical reoxidation of reduced inorganic sulfur and
iron compounds leads to a significant flow of electrons to sulfate and
ferric iron as electron acceptors (19, 42, 51). In the bulk
soil, the flow of carbon and electrons is mainly fermentative, with
methane being the major reduced end product of organic matter breakdown (4, 42). Plant polymers and root exudates are important
sources of carbon and energy for microbial activity, leading to methane formation through a trophic web of largely uncharacterized microbial populations.
Based on 14C-glucose (30) and
14CO2 and 14C-acetate
(42) turnover studies and mass balance calculations on
slurries of rice paddy soil (4), it is known that some 80%
of the methane formed is derived from aceticlastic methanogenesis. This
is higher than the 67% expected from a normal methanogenic trophic
web. To explain this, it has been suggested that homoacetogenic
bacteria may play a significant role in acetate production in anoxic
rice paddy soil (4, 10, 30, 45). The utilization by
homoacetogenic bacteria of intermediates generated by the initial
fermentative degradation of organic matter may result in a higher flow
of carbon and electrons through acetate to methane. Homoacetogenic
bacteria have also been shown to be active in other soils when they
become anoxic (32, 47).
Conrad et al. (10) enriched Acetobacterium spp.
from Italian rice paddy soil using hydrogen as the growth substrate by
incubating enrichment cultures at low temperatures to select against
hydrogenotrophic methanogens. Enrichment cultures show the presence of
microorganisms in the sample material but do not tell us about the
sizes of the populations. Molecular techniques have been used to detect
homoacetogenic bacteria based on DNA probes targeting the gene for
formyltetrahydrofolate synthetase (34), a key enzyme of
their central metabolic pathway, but this has not been applied to rice
paddy soils. The homoacetogenic bacteria do not represent a
phylogenetically coherent group (49), and so 16S rRNA-based
approaches are not applicable. We have attempted to enumerate the
homoacetogenic bacteria by utilizing substrates favoring their
selective enrichment and have developed new approaches which were
useful in obtaining high viable counts of homoacetogens. To obtain a
better understanding of the microbial community structure of the anoxic
bulk soil of the rice paddy system, we have identified and partially
characterized numerically significant representative strains isolated
by the extinction dilution method.
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MATERIALS AND METHODS |
Medium preparation.
Two sulfide-reduced,
bicarbonate-buffered mineral media, supplemented with vitamins, were
used in this study, DM and FM (26). Medium DM was used for
the enumeration experiments, while medium FM was used for experiments
with the pure cultures. Screw-cap bottles were filled, leaving a small
gas bubble, or tubes or serum bottles were partially filled (with the
headspace gas composed of N2 plus CO2 [80:20
{vol/vol}]), and closed with butyl rubber stoppers.
L isomers of organic acids and D isomers of
sugars were used. 2,3-Butanediol (Fluka, Buchs, Switzerland) was a mix
of racemic and meso forms. Substrates and other supplements were
prepared as neutralized (with NaOH or HCl, as required) 200-mM to 2-M
stock solutions and sterilized by autoclaving or, in the case of
heat-labile compounds and sugars, by sterile filtration (0.2-µm pore
size). The substrates were added to sterile media just before
inoculation to a final concentration of 10 mM, except for
monosaccharides (4 mM), 3,4,5-trimethoxybenzoate (5 mM), formate (20 mM), and succinate (20 mM). Nitrate and sulfate were added as sodium
salts to a 20-mM final concentration. Sulfur was prepared as an aqueous slurry (26) and added at approximately 100 mmol/liter.
Hydrogen was added to the N2-CO2 headspace of
partially filled serum bottles or tubes to an overpressure of 0.6 bar,
with the addition of 1 mM acetate as a supplementary carbon source
unless otherwise noted.
Microbial strains.
Desulfovibrio sulfodismutans ThAc01
(DSM 3696T), Acetobacterium woodii WB1 (DSM
1030T), Pelobacter acetylenicus WoAcy1 (DSM
3246T), and Methanospirillum hungatei JF1 (DSM
864T) were from the Deutsche Sammlung von Mikroorganismen
und Zellkulturen GmbH (Braunschweig, Germany). A. woodii and
P. acetylenicus were cultivated in screw-cap bottles in FM
medium with 10 mM ethylene glycol and 10 mM acetoin, respectively.
M. hungatei was cultivated in FM medium in partially filled
serum bottles, closed with butyl rubber stoppers, under a headspace gas
composed of N2 plus CO2 (80:20 [vol/vol]) and
supplemented with 0.6 bar of H2 and 5 mM acetate.
Enumeration and isolation of bacteria.
Rice (Oryza
sativa var. Roma type japonica) was grown in plastic containers in
the laboratory, as described by Frenzel et al. (20), in
flooded soil obtained from wetland rice fields of the Italian Rice
Research Institute in Vercelli, Italy. Soil cores were taken from
laboratory rice cultures in which the plants were 90 to 95 days old by
pressing a plastic tube into the soil to a depth of about 15 cm. Only
the lower 10 cm of the cores was used. Three-tube most-probable-number
(MPN) counts were made as described previously with DM as the growth
medium (26). The MPN tubes were incubated in the dark at
25°C (unless otherwise noted) for 13 weeks or at 15°C for 33 weeks.
Growth was considered positive if the substrate had been consumed (this
could not be assessed in the case of betaine) and products had been
formed (organic acids, gallate, CH4, or sulfide, as
appropriate). The MPN was calculated from the dry weight of the soil
(by drying it to constant weight at 105°C), the dilution factor, and
tables for three parallel dilution series based on a statistical
treatment of such counting methods (1). The significance of
differences between MPN values was tested as described by Cochran
(8).
Some soil samples were pasteurized by heating them in a water bath at
80°C for 15 min prior to making the dilution series. M. hungatei was added to some MPN series at 1 ml of a well-grown culture per 9 ml of inoculated medium immediately after the dilution series had been prepared.
Isolation and cultivation of pure cultures.
The agar deep
method for isolating pure cultures was described by Pfennig
(38). The tubes were incubated at 25°C. Culture purity was
checked microscopically by growth tests with various growth substrates;
by growth in a complex medium consisting of DM or FM medium with (per
liter) 0.5 g of yeast extract, 2 mmol of glucose, 5 mmol of
fumarate, 5 mmol of pyruvate, and 2 mmol of acetate; and by testing
growth on nutrient agar (Difco Laboratories, Detroit, Mich.) plates
supplemented with 4 mM glucose.
Pure cultures were normally grown at 25°C in completely filled
screw-cap bottles, with the addition of 100 µM (final concentration)
sodium dithionite from sterile stocks (
48). Defined
cocultures
with
M. hungatei and cultures in which gases were
to be measured
were grown in aliquots of 50 ml of medium in 120-ml
serum bottles
with N
2 plus CO
2 (80:20
[vol/vol]) gas and closed with butyl rubber
stoppers.
Characterization and analytical methods.
Genomic DNA was
extracted, and the mol% G+C ratio was determined by reversed-phase
high-performance liquid chromatography (HPLC) as described by Janssen
et al. (25). Phase-contrast photomicrographs were made after
immobilizing the cells on an agar-coated microscope slide
(39). Gram staining was carried out as described by
Süßmuth et al. (44), employing 95% isopropanol as
the decolorizing agent and with A. woodii and P. acetylenicus as controls. A fluorescence test (40) was
used to detect the presence of desulfoviridin.
Sporulation was tested by adding 1 ml of sterile soil extract to 50 ml
of medium supplemented with 25 mg of thiamine, 25 mg
of
CaCl
2 · 2H
2O, and 15 mg of
MnCl
2 · 4H
2O. The medium was then
inoculated, and spores were looked for in the stationary phase
by
phase-contrast microscopy. Soil extract was prepared from dried
rice
paddy soil as described by Cote and Gherna (
13).
The concentrations of organic growth substrates and organic end
products of metabolism were measured by ion exclusion HPLC
(
30), except for betaine, which was not measured, and
3,4,5-trimethoxybenzoate
and gallate, which were measured by
reversed-phase HPLC (
28).
Hydrogen production was determined
by gas chromatography (
26).
Sulfide production was
qualitatively assessed by a copper precipitation
test (
11)
and quantitatively assessed by a colorimetric assay
(
7).
Methane was measured by gas chromatography (
18). The
increase of ammonium due to nitrate reduction was tested with
Merckoquant ammonium test strips (Merck, Darmstadt,
Germany).
Enzyme assays.
Cultures (50 ml) were grown in serum bottles
and harvested by centrifugation at 3,000 × g for 30 min at 4°C in the bottles. The supernatants were expelled by
inverting the bottles and introducing a stream of N2 via a
hypodermic needle through the stopper while simultaneously allowing the
supernatant to be expelled via a second hypodermic needle. The cells
were resuspended in 1 ml of anoxic 50 mM potassium phosphate buffer (pH
7.2) and used directly in the enzyme assays. The presence of carbon
monoxide dehydrogenase and formate dehydrogenase was assayed
spectrophotometrically (24) on whole cells permeabilized in
the cuvette by adding 10 µl of a toluene-ethanol mixture (1:9
[vol/vol]) to the 1-ml assay. A. woodii and P. acetylenicus were used as positive and negative controls, respectively.
The protein content of the cell concentrates was estimated by boiling
200 µl of diluted (in potassium phosphate buffer) cell
suspension
with 50 µl of 10 M NaOH. Standards of bovine serum
albumin were
treated the same way. The samples and standards were
then centrifuged
at 13,000 ×
g for 5 min, and 200 µl of the
supernatant
was added to 600 µl of phosphate buffer. The protein
concentrations
in these samples and standards were determined by the
method of
Bradford (
2).
16S rDNA sequence determination and analyses.
Genomic DNA
extraction, PCR-mediated amplification of the 16S ribosomal DNA (rDNA),
and purification of PCR products were carried out by procedures
described previously (41). Purified PCR products were
sequenced with Taq DyeDeoxy terminator cycle sequencing kits
(Applied Biosystems, Weiterstadt, Germany) as directed in the
manufacturer's protocol. The Applied Biosystems 373A DNA sequencer was
used for the electrophoresis of the sequence reaction products. The ae2
editor (36) was used to align the 16S rDNA sequences
determined in this study against the 16S rDNA sequences of
representatives of the main bacterial lineages available from public
databases. Pairwise evolutionary distances were computed by using the
correction of Jukes and Cantor (27). Phylogenetic dendrograms were reconstructed from distance matrices by the least squares distance method of De Soete (16).
Nucleotide sequence accession numbers.
The accession numbers
of the reference strains used in the phylogenetic analyses are as
follows: Acetonema longum, M61919; Acidaminococcus
fermentans, X78017; Clostridium quercicolum, M59110;
Desulfomicrobium baculatus, M37311; Desulfomonas pigra, M34404; Desulfovibrio acrylicus, U32578;
Desulfovibrio africanus, X99236; Desulfovibrio
desulfuricans subsp. desulfuricans, M34113;
Desulfovibrio gabonensis, U31080; Desulfovibrio gigas, M34400; Desulfovibrio halophilus, U48243;
Desulfovibrio longus, X63623; Desulfovibrio
longreachensis, Z24450; Desulfovibrio profundis,
U90726; Desulfovibrio salexigens, M34401;
Desulfovibrio termitidis, X87409; Desulfovibrio
"vietnamensis," X93994; Desulfovibrio
vulgaris subsp. vulgaris, M34399; Pectinatus cerevisiiphilus, ARB_2608; Phascolarctobacterium
faecium, X72865; Quinella ovalis, M62701;
Selenomonas lacticifex, ARB_533F; Selenomonas
ruminantium subsp. ruminantium, ARB_A646;
Sporomusa paucivorans, M59117; Sporomusa
silvacetica, Y09976; Sporomusa termitida, M61920;
Zymophilus paucivorans, ARB_B5A2.
The 16S rDNA sequences determined in this study have been deposited in
the EMBL database under the following accession numbers:
strain DR1,
Y17758; strain DR5,
Y17761; strain DR6,
Y17760;
strain DR10,
Y17757;
strain DR14,
Y17756; strain DR15,
Y17762;
strain DR1/8,
Y17763; strain
VeLac3,
Y17755; and
D. sulfodismutans,
Y17764.
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RESULTS |
Counts by the MPN method.
Serial liquid dilutions were made of
anoxic soil from various flooded microcosms in which rice had been
grown for 90 to 95 days. The culturable-cell numbers varied, depending
on the growth substrate used (Table 1),
with counts of between about 104 and 107 cells
per g of dry soil. The cell counts obtained with lactate, ethanol, and
succinate were not significantly different from each other
(P < 0.05), nor were the counts obtained with betaine,
ethylene glycol, 2,3-butanediol, and 3,4,5-trimethoxybenzoate
significantly different from each other. There were, however,
significant differences (P < 0.05) between the cell
counts obtained with lactate, ethanol, and succinate and those obtained
with the other substrates used (with the exception that there was no
significant difference between the counts on succinate and betaine). To
test the variability of the counts with soil from different microcosms,
the experiments were repeated with selected substrates (noted in Table
1), but the numbers of culturable bacteria recovered with any one
growth substrate did not vary significantly (P < 0.05). MPN counts incubated at 15°C (noted in Table 1) were not
significantly different (P < 0.05) from those obtained
at 25°C.
To simulate the low-hydrogen partial pressures occurring in the flooded
rice paddy soil (
29), counts were made in which
all tubes
were additionally inoculated with a well-grown culture
of
M. hungatei. This had no significant effect on the cell numbers
able
to be recovered (
P < 0.05) except in one experiment
where
the number of lactate-utilizing bacteria able to be cultivated
was 100 times higher than when the methanogen was not added (Table
2). In an independent replicate
experiment with soil from a different
microcosm, the addition of
M. hungatei had little effect on the
recovery of
lactate-utilizing bacteria (Table
2).
Counts of hydrogenotrophic bacteria.
The MPN of culturable
hydrogenotrophic microorganisms (in the absence of sulfate) was
2.7 × 107 per g of dry soil (95% confidence
interval, 5.3 × 106 to 1.0 × 108)
when the incubations were carried out at 25°C. The terminal positive
tubes of these dilution series contained autofluorescing rod-shaped
bacteria, and the culture headspaces contained large amounts (>10%
[vol/vol]) of methane. Control tubes containing no added hydrogen
produced no methane. Incubation of the counting experiments at 15°C
resulted in much lower counts (P < 0.05) of methanogenic organisms: 1.3 × 103 cells per g of dry
soil (95% confidence interval, 2.5 × 102 to 5.3 × 103), based on the production of methane in the tubes.
Based on the appearance of acetate in the tubes incubated at 15°C, a
culturable population of 7.1 × 103 hydrogenotrophic
homoacetogens per g of dry soil was estimated (95% confidence
interval, 1.2 × 103 to 3.4 × 104).
MPN counting of hydrogenotrophic sulfate-reducing bacteria revealed a
culturable population size of 3.3 × 104 cells per g
of dry soil (95% confidence interval, 5.5 × 103 to
1.6 × 105).
Determination of spore numbers.
Soil slurries used to
inoculate the MPN dilution series were pasteurized to kill cells not
present as heat-resistant spores. Dilutions were first made from the
soil slurry to determine the total population size, and then the same
slurries were incubated at 80°C for 15 min. After this step, the
slurries were used to inoculate another series of MPN dilutions. On all
four substrates used, the cell counts with the pasteurized soil
slurries were significantly lower (P < 0.05) than with
the untreated slurries. We define the MPN after pasteurization as the
number of spores able to initiate growth in our media, and thus
estimate the proportion of spores of those culturable bacteria
enumerated by comparison to counting experiments without
pasteurization. Dormant heat-resistant spores were estimated to
represent 2.2% or less of the populations of organisms able to be
cultivated on these substrates (Table 3).
Isolation of representative strains.
We isolated pure cultures
from the terminal positive tubes of the liquid dilution series. Based
on the theory of liquid dilution series, the organisms growing in the
tubes receiving the most-diluted inoculum should represent numerically
significant members of the original community able to grow with that
substrate under the conditions used. Ten strains were isolated in pure
culture, each from a different MPN series.
Comparative 16S rRNA gene sequence analysis showed that strain DR7 was
closely related to members of the genus
Desulfobacterium (data not shown). Growth was always slow and difficult to reproduce,
and the strain was not characterized any
further.
The remaining nine strains were characterized phenotypically and
phylogenetically.
Characterization of strains of the genus Desulfovibrio.
Strains DR1, DR10, and DR14 were isolated from MPN dilution series on
succinate, lactate, and ethanol, respectively, without the addition of
M. hungatei (Table 1). These strains were isolated on their
respective growth substrates in agar deep cultures. Strain VeLac3 was
isolated from the MPN dilution series on hydrogen plus sulfate, using
10 mM lactate plus 20 mM sulfate in agar deep cultures. All four
strains showed the same morphology as the dominant cell type in the
terminal positive tubes of the MPN series from which they were isolated.
Strains DR1, DR10, DR14, and VeLac3 were "vibrio"-shaped,
desulfoviridin-containing, non-spore-forming, gram-negative bacteria,
with genomic DNA with mol% G+C contents of between 58 and 66.
All were
motile. The cells were 2 to 8 µm long, depending on the
strain, and
about 1 µm in diameter. All four strains were able
to grow with
sulfate as a terminal electron acceptor, with hydrogen,
formate,
lactate, and ethanol as electron donors but not with
acetate or
propionate. Acetate was produced from lactate, and
sulfate was reduced
to sulfide. Strains DR1, DR10, and DR14 were
able to ferment, albeit
weakly, ethanol and lactate, while strain
VeLac3 grew well with lactate
without an added electron acceptor
but not with ethanol. Strains DR1
and DR14 grew slowly but reproducibly
on succinate, producing acetate.
We did not investigate this any
further. All four strains were unable
to grow with hydrogen plus
sulfate in the absence of added acetate,
although a number of
passages in acetate-free medium were required to
dilute out acetate
introduced with the inoculum. In addition to
sulfate, nitrate
and elemental sulfur could be used as terminal
electron
acceptors.
All four strains grew rapidly with lactate or ethanol in coculture with
M. hungatei in the absence of sulfate. This was investigated
in more detail with strain DR1. The balances of substrate and
product
show that the electron equivalents from the fermentation
of lactate and
ethanol were recovered as methane. In the absence
of both sulfate and
M. hungatei, strain DR1 fermented lactate
and ethanol only
poorly, and hydrogen was produced (Table
4).
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TABLE 4.
End products of lactate or ethanol metabolism by
Desulfovibrio sp. strain DR1 and Sporomusa sp.
strain DR1/8 with the addition of sulfate, co-inoculation of M. hungatei JF1, or without additiona
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16S rDNA sequences comprising 1,477 nucleotides were determined for
strains DR1, DR10, DR14, and VeLac3, and a 1,479-nucleotide
sequence
was determined for
D. sulfodismutans ThAc01. The
phylogenetic
positions of these strains, based on 16S rDNA sequence
comparisons,
are shown in Fig.
1. The new
strains clearly fall within the radiation
of the genus
Desulfovibrio and cluster with the species
D. sulfodismutans,
to which they have 16S rDNA sequence similarities
in the range
of 92.9 to 93.3%. The four isolates DR1, DR10, DR14, and
VeLac3
have 16S rDNA sequence similarities to each other in the range
98.5 to 99.7%, indicating that they are closely related strains
that
may represent one or more new species of the genus
Desulfovibrio.
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FIG. 1.
16S rDNA-based phylogenetic dendrogram showing the
positions of strains DR1, DR10, DR14, and VeLac3 within the radiation
of the genus Desulfovibrio and related taxa. The scale bar
represents 5 inferred nucleotide changes per 100 nucleotides
analyzed.
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Characterization of strains of the genus Sporomusa.
Strains DR1/8 and DR15 were isolated from MPN dilution series on
lactate and ethanol, respectively, to which M. hungatei had been added (Table 2). Strains DR5, DR6, and DR16 were isolated from MPN
dilution series on 3,4,5-trimethoxybenzoate, ethylene glycol, and
2,3-butanediol, respectively (Table 1). All five strains were isolated
by using their respective growth substrates in agar deep cultures,
without the addition of M. hungatei.
Strains DR1/8, DR5, DR6, DR15, and DR16 were all motile curved rods
which formed spores. Gram staining was difficult to control,
but
usually a gram-positive reaction was obtained. The mol% G+C
ratios of
the genomic DNA were 40.8 to 42.2. The strains varied
in cell shape and
size, the proportion of spore-containing cells
in cultures grown under
the same conditions, and the shapes and
positions of the spores (Fig.
2). All five strains
could grow
with hydrogen plus carbon dioxide, ethanol, 2,3-butanediol,
betaine,
and glycerol, and they varied in their abilities to grow with
formate, lactate, methanol, ethylene glycol, 3,4,5-trimethoxybenzoate,
succinate, glucose, and fructose (Table
5). Gallate and acetate
were formed from
3,4,5-trimethoxybenzoate. Products other than
acetate were not
determined for betaine. With all other growth
substrates, acetate was
the sole organic end product. High activities
of carbon monoxide
dehydrogenase and formate dehydrogenase, key
enzymes of the
Wood-Ljungdahl acetyl-coenzyme A pathway (
52),
were detected
in pyruvate-grown cells of strains DR1/8 (16.2 and
28.5 µmol · min
1 · mg of protein
1, respectively)
and DR5 (39.1 and 17.6 µmol · min
1 · mg
of protein
1, respectively). The balance of substrates and
products for strain
DR1/8 growing with ethanol and with lactate shows
that about 1.5
mol of acetate were formed per mol of substrate (Table
4).
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FIG. 2.
Phase-contrast photomicrographs of cells of
strains of Sporomusa spp. Vegetative cells (A) and a
sporulating cell (B) of strain DR1/8, vegetative cells (C) and a
sporulating cell (D) of strain DR5, vegetative cells (E) and
sporulating cells (F) of strain DR6, vegetative cells (G) and
sporulating cells (H) of strain DR15, and vegetative cells (I) and a
sporulating cell (J) of strain DR16 are shown. Bar = 10 µm (all
panels).
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All five strains could grow syntrophically with
M. hungatei,
in which case less acetate was formed and methane was produced.
This
was investigated in more detail with strain DR1/8. The balance
of
substrate turnover and product (Table
4) shows that the syntrophic
coupling was incomplete and suggests that hydrogen evolution (recovered
as methane) and acetate formation by the Wood-Ljungdahl acetyl-coenzyme
A pathway occurred
simultaneously.
16S rDNA sequences ranging in length from 1,429 to 1,504 nucleotides
were determined for strains DR5, DR6, DR15, DR16, and
DR1/8.
Comparative analyses of these sequences with those available
from the
public databases showed these strains to be most closely
related to
members of the genus
Sporomusa within the low-G+C lineage
of
the gram-positive bacteria (Fig.
3). The
16S rDNA sequence
similarity values between the sequences of the
strains DR5, DR6,
DR15, and DR1/8 are in the range of 97.2 to 99.8%.
Strains DR15
and DR16 have 16S rDNA sequences which are identical over
the
1,450 nucleotides determined. Strains DR6 and DR15 are very closely
related, showing 99.8% 16S rDNA sequence similarity and between
94.4 and 97.7% similarity to the other
Sporomusa species,
indicating
that, together with strain DR16, they could represent a
novel
species. Strain DR5 has 99.1% 16S rDNA sequence similarity with
S. silvacetica, a homoacetogen isolated from a forest soil
(
31).
Strain DR1/8 represents a distinct lineage within the
Sporomusa group, with 95.7 to 97.6% 16S rDNA sequence
similarity to the
described species and 97.2 to 97.6% similarity to
the other
Sporomusa strains isolated in this study.
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FIG. 3.
16S rDNA-based phylogenetic dendrogram showing the
positions of strains DR1/8, DR5, DR6, and DR15 within the radiation of
the genus Sporomusa and related taxa. The scale bar
represents 5 inferred nucleotide changes per 100 nucleotides
analyzed.
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DISCUSSION |
Viable cell counts.
We used the MPN viable count technique to
estimate the population sizes of homoacetogenic bacteria in the anoxic
bulk soil of microcosms containing 90- to 95-day-old rice plants. This
technique is associated with a number of difficulties (33),
including the ability to detect only those organisms able to grow on
the growth media used. The theory of dilution culture predicts that the
organisms growing in the terminal positive tubes of such extinction dilution series were present in higher numbers in the original sample
than might be the case for organisms recovered from lower dilutions.
We used a number of approaches to try to maximize the viable counts of
homoacetogenic bacteria. We carried out MPN counts
with organic growth
substrates which support the growth of or
favor the enrichment of
homoacetogenic bacteria. We attempted
to modify the hydrogen partial
pressure in the culture medium
so that it remained at levels similar to
those found in rice paddy
soil throughout the incubation
period.
Betaine, ethylene glycol, 2,3-butanediol, and 3,4,5-trimethoxybenzoate
all gave counts of
2.8 × 10
5 cells per g of dry
soil. Except with betaine, the use of these
substrates led us to
isolate
Sporomusa spp. (strains DR5, DR6,
and DR16) from the
terminal positive tubes of these dilution series.
These substrates,
which are reportedly useful for cultivating
and enriching homoacetogens
(
17), thus favored the growth of
homoacetogens, as expected.
It appears that homoacetogenic bacteria
play a significant role in the
carbon and electron flow in rice
paddy soil (
4,
10,
30,
45).
The counts obtained with
2,3-butanediol, ethylene glycol, and
3,4,5-trimethoxybenzoate
appear to be much lower than we expected,
being maximally some
0.06% of the total microbial cell count
(
6) in this paddy soil
system. These substrates, therefore,
did not appear to be useful
for assessing the population size of
homoacetogens when compared
with other results (see
below).
Counts with succinate, ethanol, and lactate gave much higher MPNs of
5.9 × 10
5 to 3.4 × 10
7 cells per g
of dry soil. Counts in soils from different microcosms
were not
significantly different, suggesting that there was little
variation
between microcosms. From the terminal positive tubes
of these dilution
series, we isolated
Desulfovibrio spp. (strains
DR1, DR10,
and DR14) rather than homoacetogens. Based on their
phenotypic
characteristics and the results of comparative 16S
rDNA sequence
comparisons, strains DR1, DR10, DR14, and VeLac3
are typical members of
the genus
Desulfovibrio. Sulfate-reducing
bacteria belonging
to the genus
Desulfovibrio have previously
been isolated
from the same rice paddy soil from Italy (
15,
50).
Counting experiments on lactate and ethanol which included
M. hungatei in the medium gave counts of 2.3 × 10
6
to 7.5 × 10
8 cells per g of dry soil. From the
terminal positive tubes of
these dilution series, we isolated
Sporomusa spp. (strains DR1/8
and DR15). The one very high
count on lactate in the presence
of
M. hungatei may be the
result of the heterogeneity in the soil,
although we attempted to
produce homogeneous suspensions to inoculate
our counting experiments.
The general reproducibility of our MPN
estimates shows we were mostly
successful. Why the addition of
M. hungatei to counting
experiments resulted in the isolation
of
Sporomusa spp. when
the same substrates without the addition
of the methanogen led to the
isolation of
Desulfovibrio spp. is
not known. The addition
of large inocula of
M. hungatei to cultures
of
hydrogen-producing bacteria successfully maintains the hydrogen
partial
pressure at levels similar to those found in rice paddy
soil
(
5). Of course, cultures which were not coinoculated with
M. hungatei also received methanogens via the soil. The same
rice
paddy soil contained up to 2.7 × 10
7
hydrogenotrophic methanogens per g of dry soil, in agreement
with
earlier findings (
21). However, the numbers added to each
tube of the MPN dilution series, relative to the number of cells
of
Sporomusa spp., was apparently not high enough to modify the
growth conditions to allow the homoacetogens to dominate the cultures
on lactate and ethanol in the absence of added
M. hungatei.
Interestingly,
the strains of
Sporomusa spp. isolated from
the experiments containing
M. hungatei could grow with
betaine, ethylene glycol, 2,3-butanediol,
and/or
3,4,5-trimethoxybenzoate (Table
5), but apparently most
cells of
Sporomusa spp. did not initiate growth in counting
experiments
with these
substrates.
The use of hydrogen with sulfate gave counts of only 3.3 × 10
4 hydrogenotrophic sulfate-reducing bacteria per g of dry
soil,
even though the estimate of
Desulfovibrio spp. counted
with other
growth substrates, and subsequently shown to be able to grow
with
hydrogen, was significantly (
P < 0.05) greater.
Similarly, incubation
of MPN counting experiments at 15°C with
hydrogen in the absence
of sulfate did not allow the cultivation of
high numbers of hydrogenotrophic
homoacetogens, although under these
conditions homoacetogens have
been shown to outcompete methanogens
(
10). As with our other
attempts to count homoacetogens with
organic growth substrates,
our present knowledge of the metabolic
capabilities of these organisms
did not permit us to predict which
conditions are best for initiation
of growth in microbiological
media.
We conclude that
Desulfovibrio spp. and
Sporomusa
spp. were present in culturable population sizes, each of about 3 × 10
6 to 3 × 10
7 cells per g of dry
soil. The total microbial community size of
this system was estimated
at about 5 × 10
8
4',6-diamidino-2-phenylindole-stainable cells per g of dry soil
(
6).
Spore numbers.
Homoacetogenic bacteria belonging to the genera
Sporomusa, Clostridium, and Acetonema
are able to form heat-resistant spores (43). Spores will be
counted as active cells, after they germinate, in the viable cell
counts. We carried out cell counts on soil samples before and after
pasteurization. On four different substrates, the active proportion of
the total population was estimated to be 97.8% or greater. This meant
that either spores did not germinate under the cultivation conditions
used, and therefore were not counted, or only very low numbers of
spores were present in the soil. In either case, the population sizes
counted in our experiments represent mainly active cells rather than a
significant number of spores.
Competition.
The nine strains isolated and characterized in
more detail belong to two genera able to grow on a range of common
substrates. Those substrates which may be significant in the anoxic
soil habitat include hydrogen, formate, ethanol, and lactate. This
suggests that there may be competition for growth substrates within and between members of these genera.
At the typical hydrogen concentrations in anoxic rice paddy soil, about
7 to 12 Pa (
29), only sulfate-reducing bacteria
or
methanogenic archaea can be expected to be able to utilize
hydrogen,
since they have threshold concentrations for hydrogen
utilization
(
12) that are lower than this.
Sporomusa spp.,
with
threshold concentrations for hydrogen utilization of about 40
to
80 Pa (
12), would not be expected to utilize hydrogen at
these concentrations. At low sulfate concentrations (<30 µM),
sulfate-reducing bacteria cannot compete with methanogens for
hydrogen
(
35). Sulfate is present at concentrations of about
10 µM
in the anoxic bulk soil of rice microcosms (
51). Thus,
neither
Desulfovibrio spp. nor
Sporomusa spp.
could be expected
to utilize
hydrogen.
Both
Desulfovibrio spp. and
Sporomusa spp. can
grow as syntrophs on lactate, ethanol, and other substrates in
conjunction
with hydrogenotrophic methanogens (
3,
22,
23).
From thermodynamic
considerations, a methanogenic coculture growing on
lactate or
ethanol should have lower threshold concentrations for
organic
substrates than a fermenting bacterium alone. This may explain
how
Desulfovibrio spp. and
Sporomusa spp. can
exist in the anoxic
bulk soil. Both groups also have alternative
strategies for growth,
depending on local conditions: either sulfate
reduction or
homoacetogenesis.
Comparative analysis of 16S rDNA and morphological characteristics
suggested that the five strains of
Sporomusa spp. probably
represent three different species. The five strains varied in
cell size
and shape, shape and position of the spores, and the
range of
substrates used. The phenotypic differences suggest slight
differences
which may allow them to occupy different niches, although
the
differences found in this study may not be those that are
significant
in nature. Similarly, the four
Desulfovibrio spp.
isolated
(three originated from one sample) differ slightly from
one another and
may occupy slightly different niches within the
soil habitat. In a
homogeneous system with a single limiting factor,
one strain would be
expected to outcompete the others. Soil is,
however, highly
heterogeneous. This high heterogeneity possibly
allows a wider range of
apparently similar organisms to coexist
within one ecosystem
(
46).
| |
ACKNOWLEDGMENT |
We thank Oliver Kappler for carrying out the analyses of gallate
and 3,4,5-trimethoxybenzoate.
| |
FOOTNOTES |
*
Corresponding author. Present address: Department of
Microbiology and Immunology, University of Melbourne, Parkville,
Victoria 3052, Australia. Phone: 61(3) 9344 5706. Fax: 61 (3) 9347 1540. E-mail:
p.janssen{at}microbiology.unimelb.edu.au.
Present address: Department of Biological Sciences, Louisiana State
University, Baton Rouge, LA 70803.
| |
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Abstract
Introduction
