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Applied and Environmental Microbiology, December 2001, p. 5740-5749, Vol. 67, No. 12
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.12.5740-5749.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
In Situ Detection, Isolation, and Physiological
Properties of a Thin Filamentous Microorganism Abundant in Methanogenic
Granular Sludges: a Novel Isolate Affiliated with a Clone Cluster, the
Green Non-Sulfur Bacteria, Subdivision I
Yuji
Sekiguchi,1,2,*
Hiroki
Takahashi,1
Yoichi
Kamagata,2
Akiyoshi
Ohashi,1 and
Hideki
Harada1
Department of Environmental Systems
Engineering, Nagaoka University of Technology, Nagaoka, Niigata
940-2188,1 and Research Institute of
Biological Resources, National Institute of Advanced Industrial
Science and Technology (AIST), Tsukuba, Ibaraki
305-8566,2 Japan
Received 13 July 2001/Accepted 26 September 2001
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ABSTRACT |
We previously showed that very thin filamentous bacteria affiliated
with the division green non-sulfur bacteria were abundant in the
outermost layer of thermophilic methanogenic sludge granules fed with
sucrose and several low-molecular-weight fatty acids (Y. Sekiguchi, Y. Kamagata, K. Nakamura, A. Ohashi, H. Harada, Appl. Environ. Microbiol.
65:1280-1288, 1999). Further 16S ribosomal DNA (rDNA)
cloning-based analysis revealed that the microbes were classified
within a unique clade, green non-sulfur bacteria (GNSB) subdivision I,
which contains a number of 16S rDNA clone sequences from various
environmental samples but no cultured representatives. To investigate
their function in the community and physiological traits, we attempted
to isolate the yet-to-be-cultured microbes from the original granular
sludge. The first attempt at isolation from the granules was, however,
not successful. In the other thermophilic reactor that had been
treating fried soybean curd-manufacturing wastewater, we found
filamentous microorganisms to outgrow, resulting in the formation of
projection-like structures on the surface of granules, making the
granules look like sea urchins. 16S rDNA-cloning analysis combined with
fluorescent in situ hybridization revealed that the projections were
comprised of the uncultured filamentous cells affiliated with the GNSB
subdivision I and Methanothermobacter-like cells and the
very ends of the projections were comprised solely of the filamentous
cells. By using the tip of the projection as the inoculum for primary
enrichment, a thermophilic, strictly anaerobic, filamentous bacterium,
designated strain UNI-1, was successfully isolated with a medium
supplemented with sucrose and yeast extract. The strain was a very slow
growing bacterium which is capable of utilizing only a limited range of
carbohydrates in the presence of yeast extract and produced hydrogen
from these substrates. The growth was found to be significantly
stimulated when the strain was cocultured with a hydrogen-utilizing
methanogen, Methanothermobacter thermautotrophicus,
suggesting that the strain is a sugar-fermenting bacterium, the growth
of which is dependent on hydrogen consumers in the granules.
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INTRODUCTION |
Upflow anaerobic sludge blanket
(UASB) processes have been used worldwide over the past decades mainly
for the treatment of medium- and high-strength organic wastewaters
(22, 31). Although thermophilic (50 to 60°C) UASB
processes could be applied to high-temperature wastewaters discharged
from certain manufacturing processes in some industries (22, 42,
51), almost all practical UASB processes constructed so far have
been operated at mesophilic (30 to 40°C) or ambient temperatures. At
present, there are only a few practical UASB processes running at
thermophilic temperatures. One of the primary reasons for the lack of
popularity of thermophilic UASB processes is the difficulty or lower
reliability in accomplishing granulation of sludge under thermophilic
conditions (40, 42).
For start-up of UASB processes, successful formation of stable granules
from seed sludge is undoubtedly the major premise. Several factors are
known to affect granulation, i.e., physicochemical properties of
constituents in sludge such as surface hydrophobicity of cells
(4, 11, 30), the presence of extracellular polymers (30, 46), and the composition of microorganisms (8,
16, 30, 52, 53). Among these, the microbial constituents are thought to be the decisive factor in many cases.
In general, filamentous and aggregating types of
Methanosaeta cells are commonly observed other than
Methanosarcina cells in mesophilic UASB granules (8,
12, 20, 23, 24, 32, 53). They are considered important for
making cores of sludge granules by constructing web-like structures
(53). In contrast, the long-filament type of
Methanosaeta cells is seldom observed in thermophilic UASB
granules, whereas the short-filament type (dispersing type) of
Methanosaeta cells can frequently be found (18, 19,
23, 37, 39, 40, 45). Instead of Methanosaeta, very
thin filamentous microorganisms, which are morphologically different
from Methanosaeta, have been considered essential for the
formation of well-settleable thermophilic granules. This type of
microbe was frequently (or almost always) observed and found to
entirely cover the thermophilic granules, forming a web-like coating on
granules (32, 37, 39, 40). However, no detailed information on the microbes is available.
We previously analyzed the community structure of the thermophilic
(55°C) methanogenic granular sludge in a UASB reactor treating an
artificial wastewater (34) and determined the phylogenetic position and spatial distribution of the thin-filamentous organisms through the full-cycle rRNA approach (32). One of the
interesting findings in the community structure analysis was that
unidentifiable clones within the division green non-sulfur bacteria
(GNSB) were frequently obtained from the sludge. Subsequent
fluorescence in situ hybridization using an oligonucleotide probe
targeting 16S rRNAs of the uncultured GNSB revealed that the GNSB in
the reactors were thin-filamentous cells and predominated only in the
outermost layers of thermophilic sludge granules as one of the
significant populations (32).
To elucidate their physiology and metabolic function, we have attempted
to cultivate the thin-filament microorganisms belonging to the GNSB
group from several anaerobic wastewater sludges. In this paper, we
report the isolation and partial characterization of a microorganism
that is affiliated with the GNSB group and suggest its ecological
importance in anaerobic wastewater treatment processes.
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MATERIALS AND METHODS |
Operation of UASB reactors.
Granules were collected from two
lab-scale UASB reactors (reactor I and reactor II, 13-liter capacity),
both of which had been operated at a thermophilic (55°C) temperature.
Reactor I was fed with a synthetic substrate containing sucrose,
acetate, propionate, and yeast extract (4.5:2.25:2.25:1, chemical
oxygen demand [COD] ratio) over 3 years of operation
(34). Reactor II had received the actual organic
wastewater which had been discharged from a fried soy bean
curd-manufacturing factory. Both reactors exhibited good performance
for COD removal, with 90 to 95% removal efficiencies, and good methane
formation throughout the experimental period.
Microorganisms, media, and cultivation.
The following
organisms were used in this study. Thin-filamentous bacterium strain
UNI-1 was enriched and isolated in this study.
Methanothermobacter thermautotrophicus (formerly
Methanobacterium thermoautotrophicum
[49]) strain 
H (DSM 1053) was obtained from the
Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ,
Braunschweig, Germany). The culture medium used for enrichment and
isolation of strain UNI-1 was prepared as described previously
(17, 33). All cultivations were carried out at 55°C in
50-ml serum vials containing 20 ml of medium (pH25°C, 7.2) under an atmosphere of 80% N2-20% CO2
(vol/vol) without shaking. Neutralized substrates were added to the
vials from stock solutions prior to inoculation. M. thermautotrophicus H was cultivated at 55°C using the medium
mentioned above except that hydrogen was added to the gas phase (80%
N2-20% CO2, vol/vol) in the vials as the
energy source. For cocultivation, M. thermautotrophicus H
and strain UNI-1 were inoculated into the medium supplemented with
sucrose (20 mM) and yeast extract (0.1%) (5% inoculum of each).
The purity of the strain isolated in this study was routinely examined
by microscopy and incubation of the cultures with the medium containing
0.1% yeast extract and a mixture of carbohydrates (sucrose, glucose,
arabinose, and fructose, each at 2 mM) at 35 or 55°C.
In situ hybridization.
Fixation of granules was done as
described previously (32). Whole-cell in situ
hybridization was performed by the method described elsewhere
(32, 34). For in situ hybridization with spine-like
projections on sea urchin-like granules, we carefully cut the fixed
projections with a surgical knife under a binocular microscope and
immobilized the projections on glass slides coated with Vectabond
(Vector Laboratories).
The following 16S rRNA-targeted oligonucleotide probes were used in
this study: GNSB633, specific for clones MUG9 and TUG8,
-9, and -10, which were classified in the GNSB group as described
previously
(
32); EUB338 for the domain
Bacteria
(
2); ARC915
for the domain
Archaea
(
36); MX825 for the genus
Methanosaeta (
27); and MB1174 for the family
Methanobacteriaceae (
27).
For in situ
hybridization, we adjusted the stringency of hybridization
by adding
formamide to the hybridization buffer (20% [vol/vol]
for EUB338,
MX825, and GNSB633, 35% for MB1174 and ARC915). For
double staining of
the granule sections, indodicarbocyanine (Cy5)-
and rhodamine-labeled
probes were used
simultaneously.
Construction of 16S rDNA clone library from spine-like structures
formed on granules.
DNA extraction, PCR amplification, cloning,
and sequencing procedures for constructing a 16S ribosomal DNA (rDNA)
clone library were performed as previously reported (34)
with slight modifications. For DNA extraction from spine-like
structures on granules, we washed the granules with phosphate-buffered
saline (PBS; 0.13 M NaCl, 10 mM sodium phosphate buffer, pH 7.2)
several times and carefully cut and collected the projections with a
surgical knife under a binocular microscope, dispersed the projections
by weak sonication (50 W, 5 to 10 s), and subjected them to DNA extraction.
For construction of the 16S rDNA clone library from the projections, we
used the following primer set for the PCR amplification
of bacterial
16S rRNA genes:
Bacteria-specific primer EUB341F
(5'-GGTTACCTTGTTACGACTT-3', positions 341 to 357 in
Escherichia coli) (
25) and prokaryote-specific
primer 1490R (5'-GGTTACCTTGTTACGACTT-3',
1491 to 1509 in
E. coli) (
50). The PCR products were purified
with a MicroSpin column (Amersham Pharmacia Biotech), followed
by
cloning into plasmids using the TA cloning kit (Novagen). Twenty
clonal
rDNAs were randomly picked and subjected to
sequencing.
DNA extraction and amplification of 16S rDNA from a pure
culture.
DNA extraction from pure culture of several isolates was
performed by the method of Hiraishi (13), and 16S rDNA was
amplified by PCR as described above. In this PCR, the following primers were used: the Bacteria-specific primer 8F
(5'-AGAGTTTGATCCTGGCTCAG-3', 8 to 27 in E. coli)
and universal primer 1490R (50). The PCR products were
purified with a MicroSpin column (Amersham Pharmacia Biotech) and
subjected to further analysis.
Sequencing and phylogenetic analysis.
Sequences of
representative rDNA clones as well as the 16S rDNA of pure cultures
were determined by Thermo Sequenase fluorescent-labeled primer cycle
sequencing kit (Amersham Pharmacia Biotech) and an automated sequence
analyzer (DSQ-1000L; Shimadzu) as described previously
(17). Sequence data were aligned with the Clustal X
package (38) and corrected by manual inspection.
Phylogenetic trees were constructed by the neighbor-joining method
(28) with the MEGA version 2 package (Kumar et al.,
submitted for publication). Bootstrap resampling analysis
(10) for 100 replicates was performed for the estimation
of confidence of tree topologies.
Microscopy and analytical methods.
Cells immobilized and
hybridized on glass slides were viewed with a fluorescent microscope
(Olympus BX50F), and the sections and spine-like projections hybridized
with the probes were examined under a confocal laser scanning
microscope (Olympus Fluoview BX50). Scanning electron microscopy (SEM)
was performed as described previously (40). Short-chain
fatty acids were determined with a gas chromatograph (Shimadzu GC-14A;
flame ionization detector; packing material, FAL-M (G. L. Science); column temperature, 125°C). Alcohols and other
compounds were determined by high-pressure liquid chromatography (HPLC)
using an RSpak KC-811 column (Shodex; eluent, 3 mM HClO4;
column temperature, 50°C) and a UV detector (210 nm; Shimadzu
SPD-10A). Carbohydrates such as sucrose and glucose were determined by
HPLC using an SCR101-H column (Shimadzu; eluent, H2O;
column temperature, 60°C) and a refractive index detector
(Shimadzu RID-10A). Methane, hydrogen, and carbon dioxide were
determined by gas chromatography (GL Science model 370; detector type,
thermal conductivity detection; packing material, Unibeads C;
column temperature, 145°C).
Nucleotide sequence accession number.
The 16S rDNA sequence
of strain UNI-1 was deposited in the EMBL/GenBank/DDBJ databases under
accession no. AB046413.
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RESULTS |
Detailed analysis of phylogenetic positions of uncultured clones
recovered from UASB processes.
In our previous study, we recovered
several types of uncultured clones affiliated with GNSB from mesophilic
(35°C) and thermophilic (55°C) UASB granular sludges
(34). The rDNA cloning-based analysis indicated that these
clones accounted for approximately 20% of the total clones in
libraries of both mesophilic (110 clones) and thermophilic (115 clones)
sludges (34). In addition, we had designed a fluorescently
labeled oligonucleotide probe (GNSB633) specific to those clones (MUG9,
TUG8, TUG9, and TUG10 clones, referred to as thermophilic UASB cluster
in Fig. 1) to elucidate their in situ
morphology and spatial distribution within granular sludges, resulting
in the detection of only filamentous cells in the outermost layer of
thermophilic sludge granules (32).
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FIG. 1.
Phylogenetic tree of GNSB, including environmental
clones. The 16S rDNA sequences obtained in the previous study (MUG and
TUG clones) and reference sequences were aligned, and phylogenetic
trees were constructed by the neighbor-joining method (pairwise
analysis). Bar represents 5 nucleotide substitutions per 100 nucleotides in 16S rDNA sequences. The sequence of Aquifex
pyrophilus was used to root the tree. Bootstrap values above 50%
are indicated at the branch points. The accession number of each
reference sequence and the origin of environmental clones are also
shown in parentheses. Names of cultivated organisms are shown in bold
and italic. Our UASB clones, referred to as thermophilic UASB cluster
and strain UNI-1, are shown in a bracket.
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To precisely determine the phylogenetic positions of those GNSB clones,
we performed phylogenetic analysis of the clonal sequences
with all
known 16S rDNA/RNAs of cultured and uncultured organisms
belonging to
GNSB (Fig.
1). Through this analysis, we found our
GNSB clones to be
classified within GNSB subdivision I (
14),
which comprises
a number of environmental 16S rDNA sequences but
contains no cultured
microorganisms. Since the specific DNA probe
(GNSB633) reacted with the
filamentous microorganisms at the outermost
layer of thermophilic
granules, we focused on these organisms
for further
studies.
Attempts to cultivate GNSB filaments from UASB granules in reactor
I.
To elucidate the physiology and metabolic function of the probe
GNSB633-positive uncultured filaments, we attempted to isolate these
microorganisms from granules in reactor I, which was the same
thermophilic reactor used in the previous studies (17, 32,
34), and had been fed with sucrose, acetate, and propionate. Since the filamentous organisms were observed only in the outermost layer of the thermophilic methanogenic granules (32), it
was very likely that these cells could utilize primary substrates such
as carbohydrates. We therefore used sucrose, glucose, and several other
carbohydrates as carbon and energy sources for primary enrichment. The
surface layer of the granules was carefully scraped using a
micromanipulator under binocular microscopy, sonicated briefly, and
then inoculated into several media containing carbohydrate and yeast extract.
To judge whether targeted cells were grown in the media, fluorescence
in situ hybridization with GNSB633 probe was applied
to the cells in
the cultures. We also tried to enrich the filaments
in both liquid and
solid media in which serially diluted samples
were inoculated. However,
no cultures contained the targeted cells.
In many cases, irrelevant
fast-growing microbes such as
Thermoanaerobacterium and
Thermoanaerobacter species (data not shown) outcompeted the
target
organisms.
Morphological changes in sludge granules in a thermophilic UASB
reactor (reactor II).
During the attempts, we unexpectedly found a
unique phenomenon in the other thermophilic UASB reactor (reactor II).
Reactor II had been treating actual organic wastewater from a fried
soybean curd-manufacturing factory under thermophilic (55°C)
conditions, exhibiting sufficient COD removal throughout the operation
of the reactor. During the first operational period (days 0 to 100), the reactor contained normal granular sludge which had a good settleability (Fig. 2A). However, after
100 days of operation, we found changes in the configuration of sludge
granules, i.e., the sludge granules became fluffy (Fig. 2B). The sludge
still retained the granular structure, but a number of filaments were found to be sticking out of the surface of the granules, just like sea
urchins (Fig. 2C). Over time, the number of sea urchin-like granules
gradually increased, and finally they became predominant in the
reactor. The formation of the fluffy granules did not significantly affect COD removal and methane production in the reactor, but the
fluffy granules had far less settling ability than the normal granules,
indicating anaerobic bulking of granular sludge. SEM observations of
the spine-like projections on the granules showed that the projections
comprised mainly filamentous organisms (Fig. 3).
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FIG. 2.
Photographs of granules formed in reactor II. (A) Sludge
granules at day 91 of operation. (B) Fluffy granules formed at day 133. (C) Magnified view of a fluffy granule.
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FIG. 3.
Scanning electron micrographs of spine-like projections
sticking out of the fluffy granules (sea urchin granules) in reactor
II. (A) Whole view of a projection (bar, 150 µm). (B) Higher
magnification of the bottom part of the projection (bar, 20 µm). (C)
Higher magnification of the tip of the projection (bar, 30 µm)
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To identify the populations within the projections, we performed 16S
rRNA-based analysis. Whole cell in situ hybridization
analysis with a
universal bacterial probe (EUB338) (
2) showed
that only a
few cells within the projections reacted with the
probe; instead,
almost all filamentous populations hybridized
with the GNSB633 probe
(Fig.
4). Inside the projections, some
archaeal cells could be detected using a universal archaeal probe
(ARC915) (
36) (Fig.
4) and were stained with the
Methanosaeta-specific
probe MX825 or the
Methanobacteriaceae-specific probe MB1174 (
27)
(data not shown).
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FIG. 4.
In situ hybridization of a projection from the fluffy
thermophilic granules viewed by confocal laser scanning microscopy
(digitally produced images with pseudocolors). The projection was
simultaneously hybridized with Cy5-labeled archaeal probe ARC915
(indicated as red) and rhodamine-labeled probe GNSB633 for thermophilic
UASB cluster of GNSB subdivision I (indicated as green). (A) Magnified
view of the bottom part of the projection (bar, 50 µm). (B) Magnified
view of the tip portion of the projection (bar, 50 µm).
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To determine the phylogenetic position of the GNSB-like organisms
present in the projections, an rDNA clone library was constructed
from
DNAs extracted from the projection part using a bacterial
universal
primer set. Through this analysis, we found that half
of the total
bacterial clones were identical, and the identical
sequence was
assigned to the thermophilic UASB cluster of GNSB
subdivision I. Moreover, the clonal sequence was identical to
that of clone TUG8,
which was obtained in our previous cloning
analysis (Fig.
1). Remaining
clones were clustered within other
clades, such as the gram-positive
bacteria
Clostridium/Bacillus group and the OP9 division
(
15) (data not
shown).
Cultivation of GNSB-like filaments from spine-like projections of
sea urchin granules in reactor II.
Since the probe
GNSB633-positive filamentous cells were found to be highly abundant in
the spine-like projections, particularly at the tips of the projections
(Fig. 4B), we again attempted to cultivate the GNSB subdivision I
filaments using the tips as the inoculum. We washed the fluffy granules
and carefully cut the tip of the projections with a surgical knife
under a binocular microscope, dispersed the cells by sonication,
serially diluted them, and placed them into a liquid medium
supplemented with several substrates. Of a number of substrates tested,
we found that medium containing sucrose (20 mM) plus yeast extract
(0.1%) or glucose (20 mM) plus yeast extract (0.1%) supported the
growth of the filamentous cells after 2 weeks of incubation at 55°C.
Although the fast-growing anaerobes outgrew the filamentous cells at
lower dilutions, the filamentous cells grew slowly at
higher dilutions.
Cotton-like dense flocs appeared in the liquid
cultures receiving the
highest dilution, and the floc-like aggregates
contained a number of
thin-filamentous cells and F
420-autofluorescent
rods
resembling
Methanothermobacter, associating together (data
not shown). The thin-filamentous microbes were reactive with the
GNSB633
probe.
By using the highest dilutions, we attempted to isolate the filamentous
cells by the roll-tube method with the addition of
bromoethane
sulfonate (final concentration, 1 mM in culture) to
inhibit the growth
of the concomitant methanogens. Colonies that
were only 0.1 to 0.2 mm
in diameter appeared in solid medium after
1 month of incubation, and
the same isolation procedure was repeated
several times. The
filamentous strain designated UNI-1 was eventually
obtained in pure
culture.
Physiological and genetic properties of strain UNI-1.
The
strain was a strictly anaerobic, thin (0.2 to 0.3 mm)-filamentous
bacterium, as shown in Fig. 5. The strain
was moderately thermophilic (optimum temperature, 55°C). It required
yeast extract for growth, and only a limited range of substrates, such
as sucrose, glucose and arabinose, could be utilized in the presence of
yeast extract. The strain was a slow-growing, fermentative bacterium which produced hydrogen, acetate, and carbon dioxide from these substrates (the approximate doubling time estimated from the production of hydrogen in the medium with glucose and yeast extract was 3 days).
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FIG. 5.
In situ hybridization of strain UNI-1 cells isolated in
this study. The cells were hybridized with the rhodamine-labeled
GNSB633 probe. (A) Phase-contrast micrograph of strain UNI-1 cells
(bar, 10 µm). (B) Fluorescent micrograph of the same field as panel
A, showing that all cells were stained with the thermophilic UASB
cluster-specific probe GNSB633.
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Growth was found to stagnate after a certain amount of hydrogen had
accumulated. However, significant improvement of growth
was observed
when the strain was cocultivated with a hydrogen-utilizing
methanogen,
Methanothermobacter thermautotrophicus (Fig.
6). This
coculture converted 223 µmol
of glucose to 150 µmol of methane,
408 µmol of carbon dioxide, and
355 µmol of acetate in the presence
of 0.1% yeast extract (94%
carbon recovery and 75% electron recovery
on the basis of glucose
degraded).
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FIG. 6.
Fermentation of glucose by strain UNI-1 in the presence
(A) and the absence (B) of the hydrogenotrophic methanogen
Methanothermobacter thermautotrophicus strain H. The
amounts of gases are shown as millimoles of gas produced per liter of
culture.
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Phylogenetic analysis of strain UNI-1 based on 16S rDNA sequences
revealed that the strain was clustered with subdivision
I of GNSB,
which had the same sequence as the clone TUG8 (see
Fig.
1). Through
detailed analysis, the 16S rDNA sequence of strain
UNI-1 was found to
have two mismatches with the EUB338 probe sequence
(Fig.
7). In fact, this strain could not be
stained by in situ
hybridization with the EUB338 probe at the standard
hybridization
stringency (data not shown). Moreover, the bacterial 16S
rDNA-specific
primer EUB341F (
25), which was used in the
construction of the
16S rDNA clone library for the projections in this
study, also
contained two mismatches with the 16S rDNA sequence of
strain
UNI-1. This was probably why only half of the total clones
analyzed
from the projections of the granules were recovered as GNSB
clones,
although the projections comprised primarily this type of
organism,
as shown in in situ hybridization (see above).
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FIG. 7.
Alignment of target sequences of the EUB338 probe suite
with the corresponding regions of 16S rRNA of members of the GNSB
group, showing mismatches with the Bacteria universal probe
EUB338 and its sequence variations (EUB338-II and EUB338-III).
Mismatches with the EUB338 probe sequence are shaded.
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DISCUSSION |
Contribution of UNI-1-type filamentous cells to granulation of
anaerobic sludge.
Our data presented in this study show that at
least one of the filamentous populations on the surface of the
thermophilic granules was a slow-growing bacterium affiliated with GNSB
subdivision I, which is able to utilize only limited types of
carbohydrates in the presence of yeast extract. It has been reported
that the granulation of thermophilic sludges was difficult to achieve
when only volatile fatty-acid mixtures were used as the sole substrate, while the addition of sucrose or glucose to the influent wastewater resulted in the formation of granular sludge with good settleability (40, 42, 43, 45, 54). In thermophilic UASB processes having well-settleable sludge granules, it was always observed that
UNI-1 type of thin-filamentous microbes predominated on the surface of
the sludge granules (32, 37, 39, 40). Considering these
findings together with the physiological properties of strain UNI-1,
the strain UNI-1 type of filamentous organism is one of the
indispensable organisms for the granulation of thermophilic UASB sludges.
In liquid culture, strain UNI-1 cells grew and formed well-settleable
dense, cotton-like flocs. This trait seems to contribute
to granule
formation and preservation of the structure, as mesophilic
Methanosaeta cells do in mesophilic UASB granules. In the
mesophilic
UASB processes, UNI-1-like thin-filamentous cells were
occasionally
observed in addition to filamentous
Methanosaeta cells (
32,
57). The detailed
phylogenetic positions and physiological properties
of the mesophilic,
thin-filamentous populations are not known.
However, considering the
facts that GNSB subdivision I-type clones
were frequently recovered
from several mesophilic UASB granules
(
34,
56) and that
mesophilic filamentous cells probably require
carbohydrates, it is
strongly suggested that such microorganisms
are also affiliated with
the GNSB subdivision I group. In terms
of sludge granule formation
under mesophilic conditions, the thin-filamentous
microorganisms seem
to be less significant than the filamentous
bacteria in thermophilic
processes, since well-settleable granules
could be made in the absence
of the thinner filamentous cells
(
57).
Besides the importance of the UNI-1-type organisms in thermophilic UASB
reactors, our study also shows the other aspect of
this type of
microorganism as a potential causative agent of the
bulking of granular
sludges. Endo and Tohya previously found anaerobic
bulking in an
anaerobic mesophilic (30°C) contact process, which
was also caused by
the outbreak of thin filamentous cells (
9).
These
filamentous microbes were thought to be heterotrophic bacteria,
because
they could be eliminated by introducing acidified wastewater,
similar
to the case of Wu et al. (
57). In addition, Alphenaar
observed similar fluffy anaerobic granules with hairy surface
structures as the result of the outgrowth of filamentous cells
in
mesophilic UASB reactors (
1). A similar observation was
also reported in a high-rate thermophilic UASB reactor
(
44).
These observations, together with our data, suggest
that it is
important to control the growth of these filamentous cells
not
only to enhance granule formation but also to prevent the bulking
of sludge
granules.
In our experiment, from day 100 of the operation of the soybean
wastewater-treating UASB process, we used diluted wastewater
to reduce
the COD loading for the reactor from 70 kg of COD/m
3/day to
30 kg of COD/m
3/day. In addition, the influent wastewater
has been changed from
day 100; in the earlier stage of operation, we
used wastewater
directly discharged from the manufacturer, which
contained large
amounts of suspended organic solids, but we used the
supernatant
of wastewater as the feed to eliminate the suspended solids
from
day 100 of the operation, since the severe formation of scum
inside
the reactor was found to occur due to the suspended solids in
the wastewater. The major difference between the two wastewaters
was
that the latter wastewater contained relatively high concentrations
of
carbohydrates compared with the previous one (data not
shown).
It is not clear which factors caused the outbreak of UNI-1-like
filaments during the process. However, considering the physiological
properties of strain UNI-1, the decrease in COD loading together
with
the increase in the carbohydrate content in the wastewater
might have
triggered the outbreak, since (i) the low COD loading
might have
enhanced the autolysis of microbial cells in the sludge,
resulting in
providing a yeast extract-like nutrient that is essential
for the
growth of strain UNI-1, and (ii) relatively high concentrations
of
carbohydrates, which can be substrates for UNI-1 cells, might
have
enhanced the growth of the strain. As suggested by Alphenaar
(
1), two-phase separation, in which a controlled
acidifying
phase was installed prior to the methanogenic UASB process,
will
be helpful to maintain an appropriate concentration of
carbohydrates
in the influent wastewater to prevent the anaerobic
bulking of
granular sludge as well as to enhance
granulation.
Partial characterization of strain UNI-1.
Based on 16S
rRNA/DNA sequences, the GNSB group has been divided into four
subdivisions, I, II, III, and IV (Fig. 1) (14). Subdivision I contains the most diverse environmental clones among the
four subdivisions; those clones were derived from sediments (55), subsurface (3), hot spring
(15), aquifer samples (7), anaerobic
dechlorinating consortia (29, 47, 48), a sulfate-reducing
consortium (26), and anaerobic sludges (6, 34,
56). Some of these clones came from virtually aerobic environments like fresh water (ultraoligotrophic lake)
(41), activated sludge (35), and an aerated
lagoon (58). Apparently, strain UNI-1 belongs to
subdivision I, but much more information on the tangible microbes
within this subdivision must be accumulated to address the question of
why these microorganisms can be so elusive, refusing isolation in the laboratory.
Interestingly, the strain was found to be sensitive to some extent to
hydrogen, and growth could be greatly improved in coculture
with a
hydrogenotrophic methanogen similar to "
Syntrophococcus sucromutans," which is known as a syntroph which utilizes
various
carbohydrates in the presence of hydrogenotrophic methanogens
(
21). As a matter of fact, we found that
Methanothermobacter-like
hydrogen-consuming methanogens were
associating with this microorganism
in the projections of the granules.
In addition to this characteristic
feature, the strain is a very
slow-growing organism and hence
is easily outcompeted by fast-growing
heterotrophs. This was probably
the primary reason why our first
attempts to isolate the organism
from the surface layer of healthy
granules in reactor I
failed.
The other interesting aspect of strain UNI-1 is that its 16S rRNA
sequence had two mismatches with the EUB338 probe, which
is a 16S
rRNA-targeted oligonucleotide probe and has been used
widely to detect
many members of the domain
Bacteria by in situ
hybridization
(
2). Since several other known microbes and environmental
clones were found to have those mismatches, two supplementary
versions
of the EUB338 probe (EUB338-II and EUB338-III probes)
have recently
been developed by Daims et al. (
5). One of the
modified
probes (EUB338-III) was fully complementary to the 16S
rRNA of strain
UNI-1 (see Fig.
7), and hence it was possible to
detect the strain with
the modified universal probe suite (data
not shown). In addition,
bacterial 16S rDNA-specific primer EUB341F,
which is a widely used
forward primer for 16S rDNA-based microbial
community analysis,
particularly for denaturing gradient gel electrophoresis
(
25), also contained two mismatches with the corresponding
sequence
of strain UNI-1. These findings indicate that those microbes
have
not been recognizable by conventional universal probes or primers
commonly used in hybridization and PCR
detection.
In summary, it is not appropriate to hasten the conclusion that
subdivision I of GNSB comprises solely heterotrophic, anaerobic
microbes, but our results suggest that unseen microbes in the
GNSB
subdivision I group may be relatively slower growers than
commonly
cultivatable microbes and/or need to be associated with
other microbes
(syntrophy) for growth. Our results also indicate
that it would be
still feasible to apply traditional techniques
to the isolation of
unseen microbes if the investigators use them
thoughtfully in
combination with molecular tools and carefully
select good inocula
which contain sufficient amounts of targeted
cells.
| |
ACKNOWLEDGMENTS |
We thank Takeshi Yamada and Hiroyuki Imachi at Nagaoka University
of Technology for help with cultivation of strain UNI-1.
This study was financially supported by research grant 11794002 of the
Grant-in-aid for University and Society Collaboration subsidized by the
Ministry of Education, Culture, Sports, Science and Technology, Japan.
| |
FOOTNOTES |
*
Corresponding author. Present address: Microbial and
Genetic Resources Research Group, Research Institute of Biological
Resources, National Institute of Advanced Industrial Science and
Technology, Central 6, Tsukuba, Ibaraki 305-8566, Japan. Phone:
81-298-61-6590. Fax: 81-298-61-6587. E-mail:
y.sekiguchi{at}aist.go.jp.
| |
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Applied and Environmental Microbiology, December 2001, p. 5740-5749, Vol. 67, No. 12
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.12.5740-5749.2001
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Abstract
Introduction
