RESEARCH ARTICLE
E¡ective isolation of bacterioplankton genus Polynucleobacter
from freshwater environments grown on photochemically
degraded dissolved organic matter
Keiji Watanabe, Nobuyuki Komatsu, Yuichi Ishii & Masami Negishi
Ibaraki Kasumigaura Environmental Science Center, Tsuchiura, Ibaraki, Japan
Correspondence: Keiji Watanabe, Ibaraki
Kasumigaura Environmental Science Center,
Tsuchiura, Ibaraki 300-0023, Japan. Tel.: 181
29 828 0963; fax: 181 29 828 0968; e-mail:
[email protected]
Received 30 April 2008; revised 28 August
2008; accepted 2 September 2008.
First published online 27 November 2008.
DOI:10.1111/j.1574-6941.2008.00606.x
Editor: Gary King
Keywords
freshwater bacterioplankton; effective
isolation; Polynucleobacter ; dissolved organic
matter; photochemical products; microbial
loop.
Abstract
Effective isolation of freshwater bacterioplankton belonging to genus Polynucleobacter from a shallow eutrophic lake and its tributary was achieved by size-selective
filtration with a 0.7-mm pore filter and cultivation on R2A agar medium. Partial
16S rRNA gene analysis showed that over 80% of all the strains were highly similar
to the Polynucleobacter cluster. Essential medium components for effective
cultivation are pyruvate, yeast extract and peptone, whereas soluble starch and
glucose are not necessary. Isolate KF001 (affiliated with Polynucleobacter subcluster
D) has a strict requirement for organic acids as carbon sources, and we hypothesize
that the Polynucleobacter cluster of bacteria could utilize compounds formed via
photochemically dissolved organic matter (DOM) degradation for growth.
Because organic acids form from solar radiation of DOM in aquatic environments,
carbon sources that are typical products of DOM photochemical degradation were
added to the medium. These compounds were readily utilized by KF001 in this
study. Finally, we observed the stimulation of strain KF001 activity by photochemical degradation of natural lake water. Our findings suggest a carbon flow of
DOM photoproducts to Polynucleobacter in the freshwater microbial loop.
Introduction
Freshwater bacterioplankton belonging to the genus Polynucleobacter (Betaproteobacteria) are widely detected in
freshwater environments of various climatic regions around
the world using culture-independent methods (Glöckner
et al., 2000; Šimek et al., 2001; Wu & Hahn, 2006a).
Polynucleobacter [Polynucleobacter necessarius (AM397067)]
was discovered as an endosymbiont of the benthic ciliate
Euplotes aediculatus without pure culture isolation (Heckmann & Schmidt, 1987). Recent work on the lifestyle of
Polynucleobacter, subcluster C (PnecC), has shown that the
bacteria are either obligately free-living or obligately endosymbiotic (Vannini et al., 2007). Since their discovery, freeliving bacteria of the Polynucleobacter cluster have been
widely detected in freshwater environments, and the Polynucleobacter cluster of bacteria sometimes numerically dominates bacterioplankton groups found in humic freshwater
environments. Based on assessments using Betaproteobacteria (b II)-targeted FISH probes or probes exclusive to
FEMS Microbiol Ecol 67 (2009) 57–68
Polynucleobacter subcluster C (PnecC), these bacteria have
been shown to account for 6–13% (b II) or 4 60% (PnecC)
of the total number of bacterioplankton cells (Burkert et al.,
2003; Hahn et al., 2005). However, these bacteria could not
be isolated and maintained in pure culture until recently.
One principal method has been used in previous studies
to isolate members of the Polynucleobacter genus. Hahn
(2003) succeeded in isolating free-living Polynucleobactercluster bacteria by the filtration–acclimatization method
(FAM) and the novel dilution–acclimatization method
(DAM) (Hahn et al., 2004, 2005). In FAM, very small
volume (o 0.1 mm3) samples are first passed through
0.2-mm pore filters for isolation of freshwater bacterioplankton cells, followed by an acclimatization medium to provide
a slow transition from low environmental substrate concentrations to high concentrations of standard microbial media.
This approach prevents overgrowth of certain bacterial
strains. On the other hand, DAM uses dilution steps for the
separation of the most abundant bacteria from the less
abundant ones using three kinds of additional media instead
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58
of the filtration step of FAM. Using the FAM method, many
previously uncultured bacterial strains, including free-living
Polynucleobacter-cluster bacteria, have been isolated from
several climatic zones worldwide and maintained in pure
culture. However, FAM requires many steps to achieve
acclimatization.
Previous studies have phylogenetically subdivided the
Polynucleobacter cluster into four subclusters: PnecA, PnecB,
PnecC and PnecD. FAM has also been used successfully to
isolate free-living PnecB, PnecC and PnecD bacteria (Hahn,
2003; Wu & Hahn, 2006a). Wu & Hahn (2006a) have
investigated the ecological aspects of Polynucleobacter-cluster bacteria, and they have described that pH and water
temperature have a strong effect on the dynamics of PnecB
and PnecC populations, as well as on the entire Polynucleobacter community. Further, the PnecB population dynamics
were found to be influenced by water temperature, as well as
phytoplankton and metazooplankton successions (Wu &
Hahn, 2006b). However, intimate knowledge of the bacteria’s role in biogeochemical cycles or food web dynamics in
freshwater environments is still lacking. Only one report
of its ecology, concerning predator (nanoflagellate)–prey
(Polynucleobacter) interactions, has been published (Boenigk
et al., 2004).
Here, we report studies of a newly designed, one-step
method for effective isolation and cultivation of members
of the genera Polynucleobacter from freshwater environments. We also discuss the ecology of the Polynucleobactercluster bacteria with respect to utilization of dissolved
organic matter (DOM) photoproducts generated by solar
radiation.
Materials and methods
Sampling and environmental characteristics
Lake Kasumigaura, composed of three smaller lakes, is the
second largest lake in Japan; it is predominantly shallow and
eutrophic (area, 219.9 km2; mean depth, 4 m; maximum
depth, 10 m; and retention time, 200 days). Surface water
was sampled on June 30, 2006 from the lake and its
main tributary (the Sakura River) (Fig. 1): L1 (36102 0 N,
140124 0 E), L2 (36100 0 N, 140134 0 E), L3 (35154 0 N, 140134 0 E),
R1 (36119 0 N, 140111 0 E) and R2 (36106 0 N, 140108 0 E).
Depth, temperature, pH, electric conductivity (EC), dissolved oxygen (DO) and Eh (oxidation–reduction potential)
were measured simultaneously at the time of sampling.
Subsurface water samples (depth, 0–0.5 m) were collected
in contamination-free, 1-L polypropylene bottles and preserved in cool boxes until the end of the sampling, when
they were stored at 4 1C for up to 2 days until analysis of
environmental characteristics. Finally, the samples were
stored frozen at 20 1C.
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K. Watanabe et al.
R1
140°30′E
N
R2
Pacific Ocean
L1
L2
36°00′N
L3
Lake Kasumigaura
10 km
Fig. 1. Location of sampling sites in Lake Kasumigaura and its main
tributary.
Chlorophyll a concentrations, chemical oxygen demand
(CODMn), dissolved organic carbon (DOC), dissolved total
nitrogen (DTN), dissolved total phosphorus (DTP), NO
31
3
2
were measured
N, NO
2 -N, NH4 -N, PO4 -P and SO4
within 2 days of sampling. Chlorophyll a concentrations
were measured using the modified standard method that
uses methanol as the extraction solvent (Kerr & Subba Rao,
1966). The CODMn analysis method was performed according to Japanese standard methods (Japanese Industrial
Standards Committee, 1998). Before chemical analysis of
the dissolved fraction, water samples were passed through
precombusted (450 1C for 4 h) GF/F glass-fiber filters (0.7mm pore size, Whatman, Middlesex, UK) and secondarily
filtered through 0.1-mm pore size, hydrophilic Durapores
membrane filters (Millipore, Billerica, MA) under slightly
reduced pressure (o 200 mm Hg). DOC was measured by a
TOC-V/CSN (Shimadzu, Shiga, Japan), total nitrogen (TN)
and total phosphorus (TP) by an Auto Analyzer 3 (Bran1
1
Luebbe, Tokyo, Japan), NO
3 -N, NO2 -N, NH4 -N and
3
2
PO4 -P by an AACS-II (Bran1Luebbe), and SO4 using an
ion chromatograph ICS-2000 (Dionex, Tokyo, Japan)
equipped with an IonPac AS19 column and an IonPac
AG19 guard column (Dionex).
Isolation and cultivation
Water samples from each sampling site were filtered axenically through disposable syringes equipped with 0.7-mm
glass-fiber filters (PradiscTM 25 GF/F disposable filter device;
Whatman), and 100-mL aliquots of filtrate were spread onto
R2A agar plates (Becton Dickinson, Franklin Lakes, NJ)
(Reasoner & Geldreich, 1985) and incubated at 25 1C for 1
FEMS Microbiol Ecol 67 (2009) 57–68
59
Isolation and ecological aspects of Polynucleobacter
week. After cultivation, a single bacterial colony randomly
picked from each agar plate was inoculated into 10 mL of
sterilized R2A liquid medium in a silicone-stoppered test
tube, and the sample was incubated at 120 s.p.m. (strokes
per minute) and at 25 1C for 1 week. The R2A liquid
medium was prepared in our laboratory with the following
concentrations t (in g L1): yeast extract (BD), 0.5; proteose
peptone no. 3 (BD), 0.5; casamino acids (BD), 0.5; dextrose
(glucose) (BD), 0.5; soluble starch (BD), 0.5; sodium
pyruvate (MP Biomedicals, Solon, OH), 0.3; K2HPO4
(Wako Pure Chemical Industries, Osaka, Japan), 0.3; and
MgSO4 7H2O (Wako), 0.05. The pH was adjusted to 7.2.
For analysis, the cultivation broth was centrifuged (10 000 g
for 15 min at 4 1C) and the supernatant was discarded.
Several bacterial pellets were used for the following DNA
extraction and PCR amplification steps.
16S rRNA gene analysis
Bacterial DNA was extracted from the pellet using the
DNeasy kit (Qiagen, Hilden, Germany). Bacterial 16S rRNA
genes were amplified from DNA extracts using the 27f (5 0 AGAGTTTGATCMTGGCTCAG-3 0 ) (Lane, 1991) and 1492r
(5 0 -ACGGYTACCTTGTTACG-3 0 ) primers (Liu et al.,
2001). For the amplification reaction, TaKaRa Ex Taq
polymerase (Takara Bio, Shiga, Japan) was used. Reactions
were carried out using a GeneAmps PCR system 9700
(Applied Biosystems, Tokyo, Japan) as follows: 10 min at
95 1C, 30 cycles of 30 s at 95 1C, 3 min at 60 1C, and 1 min at
72 1C, with a final 7 min at 72 1C and 4 1C after cycling was
completed. PCR products were purified using a QIAquic
PCR product purification kit (Qiagen).
The PCR products were sequenced directly on a CEQ2000
sequencer using a Dye Terminator Cycle Sequencing kit
(Beckman Coulter, Fullerton, CA). The 16S rRNA gene sequence primers 27f, 341f (50 -CCTACGGGAGGCAGCAG-3 0 )
(Muyzer et al., 1993), 518r (50 -ATTACCGCGGCTGCTGG-3 0 )
(Muyzer et al., 1993), 911r (5 0 -CCGTCAATTCATTT
GAGTTT-3 0 ), 1100r (5 0 -GGGTTGCGCTCGTTG-3 0 ) (Lane,
1991), 1390r (5 0 -ACGGGCGGTGTGTRCAA-3 0 ) (Liu et al.,
2001) and 1492r were used.
The sequences obtained were compared with known
sequences using the BLAST service.
The nucleotide sequences of partial 16S rRNA genes have
been deposited in the DNA Data Bank of Japan (DDBJ)
(http://www.ddbj.nig.ac.jp) under the following accession
numbers: AB269792–AB269814 and AB278120–AB278122.
described above; then it was cultivated in 10 mL of R2A
liquid medium in a silicone-stoppered test tube at 25 1C for
1 week. One milliliter of culture broth (2.2 109 cells mL1)
was centrifuged (10 000 g for 15 min at 4 1C), washed with
phosphate buffer (pH 7.4) three times, and resuspended
in 500 mL of phosphate buffer. The cell count was performed with the 4 0 ,6 0 -diamidino-2-phenylindole (DAPI)
stain (Porter & Feig, 1980; Turley & Hughes, 1992). The
sample was fixed as described previously (Imase et al., 2008).
This fixed sample was dried in a freeze-drier (ES-2030;
Hitachi, Tokyo, Japan), and coated with Pt–Pd using an ion
sputterer (E-1030; Hitachi). The sample was examined with
a field emission SEM (JSM-6330F; JEOL, Tokyo, Japan) at
magnification 23 000 and acceleration voltage 5 kV.
Measurement of isolate KF001’s cell diameter
using laser diffractometry
Measurement of cell diameter as the equivalent volume
diameter of isolate KF001 was performed with a laser
diffraction particle size analyzer SALD-2200 (Shimadzu,
Shiga, Japan). Strain KF001 was cultivated in a 500-mL
Erlenmeyer flask containing 100 mL of R2A liquid medium,
sealed with a silicone stopper, at 25 1C for 2 weeks in a rotary
shaker (120 r.p.m.). After cultivation, the culture broth
(5.7 109 cells mL1) was centrifuged (10 000 g for 15 min
at 4 1C) and the precipitate was resuspended in 20 mL of
newly autoclaved R2A liquid medium. The index of refraction was set at 1.60–0.10i, and this experiment was performed in triplicate.
Analysis of essential medium components
Isolate KF001 was inoculated in 10 mL of sterilized R2A
liquid medium and precultured at 25 1C for 1 week. After
preculture, 100 mL of the cultivation broth (2.2 109 cells mL1) was inoculated in 10-mL aliquots of R2A
liquid media, each of which lacked one component of the
medium. These samples were cultivated on a shaker at
120 s.p.m. at 25 1C for 500 h. Control samples contained
complete R2A liquid medium. The growth on each liquid
medium was monitored at A600 nm using a UV/VIS Spectrophotometer V-560 equipped with an EMC-418 cell holder
(Jasco International Co. Ltd, Tokyo, Japan). This system
allows measurements using at least 50 mL of sample volume.
Five replicates of this experiment were performed.
Biolog assay for substrate utilization screening
Observation of isolate KF001 using a scanning
electron microscope (SEM)
Isolate KF001 [which exhibited high similarity to Polynucleobacter subcluster D (PnecD)] was isolated and purecultured from the freshwater environment using the method
FEMS Microbiol Ecol 67 (2009) 57–68
The substrate utilization patterns of isolated strains (KF001,
KF003, KF016, KF022, KF023, KF029 and KF032) were
determined using Biolog GN2 and AN Microplates (Biolog
Inc., Hayward, CA). The GN2 and AN Microplate test
panels contain 95 wells, each with a different carbon2008 Federation of European Microbiological Societies
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60
containing compound, and one well with water. Cell suspensions in GN/GP and AN Inoculating Fluid (Biolog) had
A600 nm = 0.6, measured on a UV/VIS spectrophotometer
V-560 (Jasco). Microplates were incubated at 27 1C for
72 h, after which the carbon source utilization within each
well was quantified by measuring A595 nm on a microplate
reader, model 680XR (Bio-Rad, Hercules, CA). The analysis
of each well was carried out as follows: raw A595 nm values of
each plate at 0 and 72 h were first normalized by comparison
with the appropriate control well containing water and no
carbon source, and then the difference between A595 nm data
at 72 h and the A595 nm data at 0 h was calculated. The cutoff
point between negative results and positive results occurred
at an A595 nm value of 0.15. Each strain was analyzed in at
least four independent experiments using separately prepared inocula.
Effect of filtration on microbial culturability and
abundance
A water sample from the L1 site was immediately and
separately filtered through a 5.0-mm Millex-SV filter unit
(Millipore), a 2.7-mm 13-mm GF/D syringe filter (Whatman), a 0.7-mm Puradisc 25 (Whatman), a 0.22-mm MillexGV filter unit (Millipore) and a 0.1-mm pore Millex-W filter
unit (Millipore). The control sample was not filtered. The
control and 5.0-mm pore filtrate samples were diluted
axenically 10-fold using sterile phosphate buffer (pH 7.0),
whereas the other filtrated samples (2.7-, 0.7-, 0.22- and
0.1-mm filtrate) were not diluted. Each 100-mL sample was
spread with bent glass rods onto R2A agar plates. Five
replicates of each sample were created. After inoculation,
plates were incubated at 25 1C for 1 week, and bacterial
number was quantified as CFU mL1.
ATP concentrations in the filtered water samples generated using several filters as described above were quantified
by a bioluminescence assay for evaluation of the total
microbial content. Five 2-mL replicate samples from each
fraction were centrifuged (10 000 g for 15 min at 4 1C) and
the supernatant was discarded. Precipitates were suspended
in 100 mL of 0.1 mol L1 Tris-acetate buffer (pH 7.7) containing 0.5% (v/v) trichloroacetic acid (TCA) and incubated
at room temperature for 15 min to extract ATP from the
microbial cells. The Tris-acetate buffer containing 0.5% TCA
was used as the blank value in the ATP standard curve. ATPbioluminescence assays were carried out on these extracts
using an ENLITENs ATP assay system bioluminescence
detection kit (Promega, Madison, WI) by a GLOMAX 20/20
Luminometer (Promega). rL/L Reagent (150-mL aliquot,
included in the kit) was injected into each extracted sample,
which was then gently mixed for 2 s with a vortex mixer. A
10-s relative light unit (RLU) signal integration time was
used for each sample.
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K. Watanabe et al.
Growth potential of isolate KF001 using model
photoproducts from DOM
This experiment performed on isolate KF001 was fundamentally similar to the method described above (see
Analysis of essential medium components). Thirteen reagents, including sodium pyruvate, acetaldehyde, trisodium
citrate, sodium formate, formaldehyde solution, propionaldehyde, glyoxylic acid monohydrate, sodium oxalate, disodium malonate, glyoxal solution, sodium acetate, levulinic
acid and acetone, were purchased from Wako Pure Chemical
Industries in Japan for use as model compounds of products
formed via photochemical DOM degradation (Kieber et al.,
1989, 1990; Mopper et al., 1991; Moran & Zepp, 1997).
These reagents were exchanged for sodium pyruvate
in modified R2A (MR2A: no glucose and starch) liquid
medium as the carbon source, which had a final concentration of 2.7 mmol L1. The control sample did not include
these model compounds.
Utilization of actual DOM photoproducts
Water samples were collected from the surface (0–0.5 m)
and at 6-m depth at sampling site L1 on August 28, 2007 in
order to study the effect of photochemically degraded DOM
on Polynucleobacter-cluster bacterial activity. The samples
were filtered using precombusted GF/F filters and secondarily filtered with 0.1-mm pore size filters (hydrophilic
Durapores membrane filter; Millipore). Finally, the samples were filtered axenically using sterilized NALGENEs
filterware equipped with 0.2-mm pore polyethersulfone
membranes (Thermo Fisher Scientific, Waltham, MA) and
the filtrates were added to autoclaved quart bottles (100 mL)
on a clean bench. Samples in the dark treatment were
covered with an aluminum foil. All bottles (light treatment
and dark treatment) were exposed to sunlight under clear
weather conditions for 9 h in a water-circulated bath
equipped with a cooler (LX-110GX; Reisea, Tokyo, Japan)
at 27 1C. Then 10-mL aliquots of each bottle were axenically
added to autoclaved, silicone-stoppered test tubes, which
had been precultured for 5 days before adding KF001 isolate
solution at a final concentration of 2.5 105 cells mL1.
Blank samples were made by adding an equal volume of
sterilized distilled water. The test tubes were incubated on a
120 s.p.m. shaker at 25 1C for 64 h in the dark. ATP
concentrations of isolate KF001 using actual DOM photoproducts were monitored using a BacTiter-GloTM microbial
cell viability assay kit (Promega) and a GLOMAX 20/20
Luminometer (Promega). After cultivation, the total bacterial numbers in each culture broth were determined using
DAPI stain. Statistical analysis was performed using SPSS 13.0
for Windows. Five replicates of this experiment were performed.
FEMS Microbiol Ecol 67 (2009) 57–68
61
Isolation and ecological aspects of Polynucleobacter
Results
Chemical characteristics of each sampling site
The chemical characteristics of our sampling sites are shown
in Table 1. Sampling sites L1, L2 and L3 exhibited almost the
same chemical characteristics. However, sampling site R1
(the headwater) had remarkably low DOC, EC and SO2
4
3
concentrations and high NO
3 -N and PO4 -P concentrations
compared with the lake sites (L1, L2 and L3).
Isolation and cultivation of bacterioplankton
passed through a 0.7-lm filter and partial 16S
rRNA gene analysis
The number of bacterial colonies that were passed through
0.7-mm glass-fiber filters and cultivated on R2A agar plates
from each sampling site were assessed by CFU 100 mL1 as
follows: L1, 127; L2, 3; L3, 13; R1, 3; and R2, 61. A large
number of the colonies were very small in size and had no
color (clear).
A total of 26 colonies (L1, 11 colonies; L2, three colonies;
L3, six colonies; R1, one colony; and R2, five colonies) were
randomly picked from agar plates, and partial 16S rRNA
gene analysis was carried out (Table 2). Isolates KF001 and
KF003, KF004, KF005, KF006, KF009, KF010, KF012,
KF013, KF014, KF015, KF017, KF018, KF019, KF021,
KF024, KF025, KF026 and KF027 exhibited high similarity
to betaproteobacterium MWH-MoNR2 (AJ550650) (100%
identity) and betaproteobacterium MWH-MoIso2 (AJ550672)
(99–100% identity). These strains belong to the genus Poly-
nucleobacter, subcluster D (PnecD) (Hahn, 2003). Isolates
KF022, KF023 and KF032 were similar to betaproteobacterium
MWH-LF2-54B (AJ964893) (99% and 100% identities) and
Polynucleobacter sp. MWH-Teich-2B6 (AM110109) (99%
identity), and these strains belonged to genus Polynucleobacter, subcluster C (PnecC). Isolates KF016, KF020, KF029
and KF030 were close to Sphingomonas wittichii RW1
(CP000699) (97% identity), Actinobacterium MWHHuqW11 (AJ630368) (99% identity), Microbacteriaceae
bacterium MWH-Vic1 (AJ565413) (99% identity), and
symbiont c.f. Flavobacterium of Tetraponera binghami
(AF459795) (93% identity), respectively. Sequences obtained from 84.6% (22/26) of the total isolated strains (not
representative of the percent composition of the bacterial
community) were highly similar to those of Polynucleobacter-cluster bacteria (affiliated with subclusters PnecC and
PnecD), and these were widely distributed around Lake
Kasumigaura (L1, L2 and L3) and its tributary (R2).
However, only sampling site R1 (headwater) did not have
detectable Polynucleobacter-cluster bacteria.
Morphological and physiological observation
After cultivation, the morphological characteristics of isolate
KF001 (exhibiting high similarity with PnecD) were observed using a SEM. Isolate KF001 had a small cell size and
C-shaped cells (Fig. 2). All cells observable by SEM were
o 1 mm in size.
Cell diameter analysis using laser diffractometry showed
via the equivalent volume diameter that the average cell
diameter of strain KF001 was 0.59 0.10 mm.
Table 1. Chemical characteristics of Lake Kasumigaura and its tributaries at each sampling site
Sampling site
Parameters
L1 (June 30)
L2 (June 30)
L3 (June 30)
R1 (June 30)
R2 (June 30)
L1 (August 28)
Depth (m)
Water temperature ( 1C)
pH
EC (mS cm1)
DO (mg O2 L1)
Eh (mV)
Chlorophyll a (mg L1)
CODMn (mg O L1)
DON (mg N L1)
DOC (mg C L1)
TN (mg N L1)
TP (mg N L1)
1
NO
3 -N (mg N L )
NO2-N (mg N L1)
1
NH1
4 -N (mg N L )
1
3
PO4 -P (mg P L )
1
SO2
4 (mg L )
5.8
27.2
8.9
304
9.8
371
25
7.8
243
3.24
510
67
ND
ND
9
2
27.3
6.7
26.0
8.9
307
9.7
333
23
8.1
278
3.24
560
75.4
ND
ND
9
ND
16.2
3.4
26.4
8.2
392
7.7
331
34
8.8
274
3.54
599
98.7
ND
ND
16
6
25.5
0.1
17.5
7.6
91
9.2
381
1
6.0
576
0.86
2645
61.6
2131
ND
4
28
2.7
0.5
27.8
8.4
347
11.1
382
39
6.9
142
2.42
1240
83.9
822
11
50
ND
30.9
5.8
29.8
9.0
298
10.1
302
85
10.8
432
3.62
892
90.1
ND
ND
33
4
21.0
Samples were taken from surface water of each site on June 30, 2006 and August 28, 2007.
ND, not detected; COD, chemical oxygen demand; DON, dissolved organic nitrogen.
FEMS Microbiol Ecol 67 (2009) 57–68
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62
K. Watanabe et al.
Table 2. Phylogenetic affiliations of isolated and pure-cultured microbes passed through a 0.7 mm-pore filter and cultivated on R2A agar plate, based
on partial 16S rRNA gene analysis
Sampling
sites
Isolate name
(Accession no.)
Length
(bp)
Top match (Accession no.)
Identity
(%)
Taxonomic affiliation
L1
L1
L1
L1
L1
L1
L1
L1
L1
L1
L1
L2
L2
L2
R2
R2
R2
L3
L3
L3
L3
L3
R1
R2
R2
L3
KF001 (AB278120)
KF003 (AB269792)
KF004 (AB269793)
KF005 (AB269794)
KF006 (AB269795)
KF009 (AB269796)
KF010 (AB269797)
KF012 (AB269798)
KF013 (AB269799)
KF014 (AB269800)
KF015 (AB269801)
KF017 (AB269803)
KF018 (AB269804)
KF019 (AB269805)
KF021 (AB269807)
KF022 (AB269808)
KF023 (AB269809)
KF024 (AB269810)
KF025 (AB269811)
KF026 (AB269812)
KF027 (AB269813)
KF032 (AB278122)
KF016 (AB269802)
KF020 (AB269806)
KF029 (AB278121)
KF030 (AB269814)
1438
1430
1428
1430
1431
1434
1438
1431
1434
1426
1426
1435
1428
1433
1435
1435
1435
1435
1432
1434
1434
1459
1395
1434
1433
1421
Betaproteobacterium MWH-MoNR2 (AJ550650)
Betaproteobacterium MWH-MoIso2 (AJ550672)
Betaproteobacterium MWH-MoIso2 (AJ550672)
Betaproteobacterium MWH-MoIso2 (AJ550672)
Betaproteobacterium MWH-MoIso2 (AJ550672)
Betaproteobacterium MWH-MoIso2 (AJ550672)
Betaproteobacterium MWH-MoIso2 (AJ550672)
Betaproteobacterium MWH-MoIso2 (AJ550672)
Betaproteobacterium MWH-MoIso2 (AJ550672)
Betaproteobacterium MWH-MoIso2 (AJ550672)
Betaproteobacterium MWH-MoIso2 (AJ550672)
Betaproteobacterium MWH-MoIso2 (AJ550672)
Betaproteobacterium MWH-MoIso2 (AJ550672)
Betaproteobacterium MWH-MoIso2 (AJ550672)
Betaproteobacterium MWH-MoIso2 (AJ550672)
Betaproteobacterium MWH-LF2-54B (AJ964893)
Betaproteobacterium MWH-LF2-54B (AJ964893)
Betaproteobacterium MWH-MoIso2 (AJ550672)
Betaproteobacterium MWH-MoIso2 (AJ550672)
Betaproteobacterium MWH-MoIso2 (AJ550672)
Betaproteobacterium MWH-MoIso2 (AJ550672)
Polynucleobacter sp. MWH-Teich-2B6 (AM110109)
Sphingomonas wittichii RW1 (CP000699)
Actinobacterium MWH-HuqW11 (AJ630368)
Microbacteriaceae bacterium MWH-VicE1 (AJ565413)
Symbiont cf. Flavobacterium (AF459795)
100
100
100
100
100
100
100
100
100
100
100
100
100
100
99
99
100
100
100
100
100
99
97
99
99
93
Polynucleobacter (PnecD)
Polynucleobacter (PnecD)
Polynucleobacter (PnecD)
Polynucleobacter (PnecD)
Polynucleobacter (PnecD)
Polynucleobacter (PnecD)
Polynucleobacter (PnecD)
Polynucleobacter (PnecD)
Polynucleobacter (PnecD)
Polynucleobacter (PnecD)
Polynucleobacter (PnecD)
Polynucleobacter (PnecD)
Polynucleobacter (PnecD)
Polynucleobacter (PnecD)
Polynucleobacter (PnecD)
Polynucleobacter (PnecC)
Polynucleobacter (PnecC)
Polynucleobacter (PnecD)
Polynucleobacter (PnecD)
Polynucleobacter (PnecD)
Polynucleobacter (PnecD)
Polynucleobacter (PnecC)
Sphingomonas
Actinobacteria
Actinobacteria
Flavobacteriaceae
0.20
A600 nm
0.16
Control
Casamino acid (–)
Pyruvate (–)
Yeast (–)
Glucose (–)
Potassium (–)
Peptone (–)
Soluble starch (–)
Magnesium (–)
200
400
0.12
0.08
0.04
0
0
Fig. 2. SEM image of isolated KF001 strain after 1 week of cultivation.
The length of the white bar is 1 mm.
Investigation of the essential components in R2A medium for isolate KF001 growth (Fig. 3) showed that glucose,
soluble starch and potassium were not utilized for growth,
while yeast extract, peptone and pyruvate were essential.
Lack of casamino acid or magnesium caused a time lag in
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c
100
300
500
Time (h)
Fig. 3. Growth of isolate KF001 in R2A liquid medium with one component
missing from each trial. The medium in the control contains all components.
Means 1 SD shown (n = 5 replicates of each of nine samples).
reaching the maximum growth point (the highest value of
A600 nm).
Carbon source utilization tests of isolated strains KF001,
KF003, KF016, KF022, KF023, KF029 and KF030 were
FEMS Microbiol Ecol 67 (2009) 57–68
63
Isolation and ecological aspects of Polynucleobacter
Table 3. Substrate metabolism profiles of isolated strains using Biolog analysis
Substrate
KF001
KF003
KF022
KF023
KF032
KF016
KF029
D-Fructose
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
a-D-Glucose
D-Mannose
a-Methyl-D-glucoside
Sucrose
Turanose
D-Psicose
Acetic acid
Formic acid
Glyoxylic acid
a-Hydroxybutyric acid
b-Hydroxybutyric acid
D,L-Lactic acid
L-Lactic acid
D-Lactic acid methyl ester
Propionic acid
Pyruvic acid
Pyruvic acid methyl ester
Succinamic acid
Succinic acid
Succinamic acid mono-methyl ester
a-Ketovaleric acid
L-Aspartic acid
L-Glutamic acid
Alaninamide
L-Alanyl-glycine
L-Proline
Glycerol
D,L-a-Glycerol phosphate
The cutoff point between negative and positive responses in the Biolog assay (using GN2 and AN microplates) was 0.15.
These analyses were done in at least four independent experiments using separately prepared inocula.
Isolates KF001 and KF003, KF022, KF023 and KF032, KF016, and KF029, were affiliated with Polynucleobacter subcluster D, subcluster C,
Sphingomonas and Actinobacteria, respectively.
carried out using the Biolog GN2 and AN Microplates
(Table 3). Isolates KF001, KF003, KF022, KF023 and KF032
(which were highly similar to PnecC and PnecD) exhibited a
remarkable specificity towards organic acids, whereas KF016
and KF029 (similar to Sphingomonas and Actinobacteria)
were broadly specific to carbohydrates, organic acids, and
amino acids.
Effect of filtration
The effect of the filtration treatment on microbial culturability in water samples was estimated using several different
pore-size filters and is shown in Fig. 4a. The unfiltered
control sample reached 25.3 103 9.9 103 CFU mL1
after 1 week of cultivation on an R2A agar plate. Filtrates
using 5-, 2.7- and 0.7-mm pore filter units reached
2.1 103 1.3 103, 2.9 103 2.5 103 and 1.0 103
0.9 103 CFU mL1, respectively. When filtrates were created using 0.22- or 0.1-mm pore filter units, bacterial
FEMS Microbiol Ecol 67 (2009) 57–68
colonies could not be observed on R2A agar plates. CFU
counts for filtered samples were remarkably reduced compared with unfiltered samples.
Bioluminescence-based ATP assays of each filtrate (Fig.
4b) showed that ATP concentrations were 119.0 16.6
(control), 3.23 1.16 (5 mm), 0.88 0.09 (2.7 mm),
0.08 0.01 (0.7 mm), 0.04 0.02 (0.22 mm) and 0.03 0.01 pmol L1 (0.1 mm). Similar to the aforementioned culturability test, the filtrate treatments markedly decreased the
ATP concentrations.
Model compounds formed via photochemical
degradation from DOM as carbon sources for
bacterial growth
The isolated KF001 strain exhibited growth potential on
pyruvate, acetate, formate, acetone, malonate and oxalate
(Fig. 5). The maximum growth values (highest values of
A600 nm) of strain KF001 on these photochemical breakdown
2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
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64
K. Watanabe et al.
0.16
40
35
25
20
Glyoxylate
Propanal
Acetaldehyde
Glyoxal
Levulinate
Citrate
Formaldehyde
Control
0.08
15
0.04
10
5
0
0
(b)
100
200
300
400
500
Time (h)
140
ATP concentration (×10 pmol L–1)
Oxalate
Acetate
Malonate
0
Fig. 5. Growth of isolate KF001 using model compounds formed from
photochemical DOM degradation as carbon sources in MR2A medium.
Means 1 SD shown (n = 5 replicates of each of 13 samples).
120
100
8
6
4
2
fil
tra
0.
te
1µm
fil
tra
te
te
tra
-µ
m
fil
0.
22
m
0.
7µ
m
2.
7µ
m
fil
fil
tra
tra
te
l
tro
on
5µ
C
te
0
Fig. 4. Filtration effects on microbial activity using different pore size
filters. (a) Microbial culturability is represented by CFU. (b) Microbial
activity is represented by ATP concentration. Means 1 SD shown (n = 5
replicates of each of 12 samples).
products were: pyruvate, 0.102 0.003; acetate, 0.077 0.003;
formate, 0.042 0.01; acetone, 0.045 0.001; malonate,
0.042 0.017; and oxalate, 0.031 0.003. However, isolate
KF001 could not be grown on propanal, acetoaldehyde,
citrate, formaldehyde, glyoxylate, glyoxsal or levulinate.
Utilization of actual DOM photoproducts
The effects of photochemical degradation products from
DOM in natural lake water using surface (0–0.5 m) or deep
(6 m) water collected from sampling site L1 on strain KF001
activity (ATP concentration) were investigated using a
bioluminescence assay, and the results are shown in Fig. 6a
and b. Light treatment samples showed a remarkable
increase in ATP concentrations compared with dark samples
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Published by Blackwell Publishing Ltd. All rights reserved
c
Pyruvate
Formate
Acetone
0.12
30
A600 nm
Culturability (×103 CFU mL–1)
(a)
after a 10-h incubation in both surface and deepwater
samples, and quickly decreased after 14 h (surface) or 18 h
(deep). In contrast, the ATP concentration in the dark
treatment increased slowly and reached a peak at 32 h in
samples from both depths. After 24 h, the ATP concentration was constant and the dark treatment showed higher
ATP than in the light treatment. Curves (light and dark
treatment) showed the same trends between surface and
deepwater samples. However, deepwater samples maintained slightly higher ATP concentrations (about
47 pmol L1) for 8 h compared with surface water under the
light treatments. The total cell number in light treatment
samples of strain KF001 after 64 h of incubation was
significantly higher (both surface and deepwater samples:
P = 0.008) than in the dark treatment (Fig. 7).
Discussion
Although the Polynucleobacter-cluster bacteria have been
observed frequently in freshwater environments using culture-independent methods (Crump et al., 2003; Cottrell
et al., 2005; Boucher et al., 2006), their isolation from several
climatic zones worldwide has been achieved only recently,
mainly via the multistep FAM and partially via the novel
DAM, which includes a dilution step instead of a filtration
step (Hahn, 2003; Hahn et al., 2004, 2005; Wu & Hahn,
2006a, b). Our study was successful in isolating and pureculturing the bacterioplankton using a simpler method
having a filtration and a cultivation step. A random screening from a shallow eutrophic lake and its tributary using our
method and partial 16S rRNA gene analysis (Table 2) shows
that 4 80% of the total isolated strains belonged to the
FEMS Microbiol Ecol 67 (2009) 57–68
65
Isolation and ecological aspects of Polynucleobacter
30
50
Cell number (× 105 cells mL–1)
Light
Dark
45
40
35
30
25
20
*
25
20
15
10
5
(b)
(L
ig
ht
)
(D
ar
k)
D
W
Light
Dark
45
SW
(L
50
D
W
ig
h
t)
(D
ar
k)
0
SW
ATP concentration (pmol L–1)
*
(a)
Fig. 7. Total cell numbers of KF001 isolate in natural lake water after a
64-h incubation. Water samples used in the light treatment (white bars)
were pre-exposed to natural sunlight and those in the dark treatment
(gray bars) were covered with an aluminum foil. (SW) Surface water
samples were collected from 0 to 50 cm depth; (DW) deepwater samples
were collected from a 6-m depth at site L1. Means 1 SD shown (n = 5
replicates of each of four samples). Indicates a significant change
(P o 0.05; two-sample t-test).
40
35
30
25
20
0
16
32
48
64
Time (h)
Fig. 6. ATP concentrations determined using a bioluminescence assay
of KF001 isolate incubated with photoproducts from natural lake water.
Samples in the light condition were exposed to natural sunlight and
those in the dark condition were covered with an aluminum foil. (a)
Surface water sample collected from 0 to 50 cm depth; (b) deepwater
sample collected from a 6-m depth at site L1. Means 1 SD shown (n = 5
replicates of each of four samples).
genus Polynucleobacter (affiliated with subclusters PnecC
and PnecD).
Our method was effective in isolating Polynucleobactercluster bacteria partly because we used size-selective filtration by a 0.7-mm pore filter. Because the average cell
diameter of the KF001 monoculture strain (which is highly
similar to PnecD) was 0.59 0.10 mm, determined as an
equivalent volume diameter using laser diffractometry, it
passed through a 0.7-mm pore filter. On the other hand,
large bacteria (4 0.7-mm cell diameter) were excluded by
this filtration step, and resulted in a few aggressively growing
bacteria (o 0.7-mm cell diameter) reaching the agar plate,
where they would be cultivated. In the analysis of the
filtration effects of filters of different pore sizes, subsequent
assays showed that bacterial colonies could be cultured from
FEMS Microbiol Ecol 67 (2009) 57–68
unfiltered samples to 0.7-mm pore filtrate samples (Fig. 4a),
and ATP concentrations were remarkably reduced in filtrates
passing through pore sizes o 0.7 mm (Fig. 4b). Thus, in our
study, we found two aspects of the 0.7-mm pore cutoff: the
detection limit of CFU by direct plating of filtrate and the
point where bacterial cells are remarkably excluded. On the
other hand, Porter et al. (2004) showed that in the case of
size fractionation through filters of different pore sizes (80,
5.0, 3.0, 1.0, 0.65 and 0.45 mm) in samples from lakes of
different trophic statuses (oligotrophic, mesotrophic, eutrophic and hypereutrophic), a significant decrease in the
total cell numbers was observed in 0.65- and 0.45-mm
filtrates and, apart from the oligotrophic lake sample, cells
from each lake exhibited significantly less culturability
below the 5.0-mm size fraction. These trends are similar to
our results, but the fraction size at which the cells exhibited
significantly less culturability and the point of a remarkable
loss in the total cell number were different. Moreover, the
previous study could not reveal a detection limit of the
culturability for a 100-fold concentration of the samples
using a tangential flow filtration. Our results raise the
possibility that the effective isolation of Polynucleobactercluster bacteria is required to reduce other bacterial numbers by threshold size-exclusion filtration (e.g. 0.7-mm pore
cutoff), and this implies that these bacterioplankton compete weakly with other environmental microorganisms
when forming colonies on agar plates. In fact, for no
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66
filtration, we could not isolate Polynucleobacter-cluster bacteria from surface water collected from sampling site L1
(data not shown).
The other reason for the success of our method was that
R2A medium is suitable for the growth of Polynucleobactercluster bacteria (PnecC and PnecD), as reported previously
(Hahn, 2003). The essential-component assay in R2A medium showed that pyruvate, yeast extract and peptone were
strictly necessary, whereas glucose and soluble starch were
not utilized as carbon sources for growth (Fig. 3). Generally,
the free dissolved carbohydrate (monomeric saccharide)
level is low or undetectable in aquatic environments because
the turnover of monomers is very fast (Kaplan & Newbold,
2003). Jørgensen et al. (1998) reported that measurable
quantities of monosaccharides were included in DOM
photoproducts. However, we could not detect monomeric
carbohydrates in our samples (both DOM photoproducts
and whole DOM) using high-performance anion-exchange
chromatography with pulsed amperometric detection
(HPAEC-PAD) (data not shown). These contrasting results
are likely due to the different types of DOM in different
samples. On the other hand, in carbon source utilization
tests using the Biolog microplates, isolates KF001, KF003,
KF022, KF023 and KF032 (affiliated with PnecC and PnecD)
could not utilize all the tested carbohydrates (Table 3).
Therefore, it has been assumed that carbohydrates that are
derived from photosynthesis in algal or aquatic vegetation
and immediately incorporated into several bacteria in the
freshwater environment will not be essential carbon sources
for the growth of PnecC and PnecD bacteria. Thus, these
bacterioplankton and other carbohydrate-utilizing bacteria
might have different ecological niches. These findings suggest that R2A medium without glucose and soluble starch
allows more selective isolation of Polynucleobacter-cluster
bacteria by excluding utilizable carbohydrates that support
the growth of carbohydrate-utilizing bacteria. Therefore, we
designed the MR2A medium.
Photochemical DOM degradation in aquatic environments generates carbonyl compounds, primarily fatty acids
and keto acids, which play an important role in the bacterial
food web of the aquatic carbon cycle (Kieber & Mopper,
1987; Kieber et al., 1989, 1990; Mopper et al., 1991; Wetzel
et al., 1995). In Biolog tests, isolates KF001, KF003, KF022,
KF023 and KF032 were strictly dependent on organic acids
(Table 3). Because of these findings and our own study
results, we assume that photoproducts of DOM (e.g. organic
acids) will be good carbon sources for free-living Polynucleobacter bacterioplankton in freshwater environments.
Based on this hypothesis, we added model compounds that
can be formed via photochemical DOM breakdown, and
isolate KF001 utilized these compounds as growth substrates
(Fig. 5). On the other hand, Hahn (2003) succeeded in
isolating and culturing pure strains of the bacteria using
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K. Watanabe et al.
NSY (nutrient broth, soytone, yeast extract) medium, which
does not include organic acids as carbon sources. We
confirmed that isolate KF001 utilized soytone or beef extract
(included in the NSY medium) as a carbon source instead of
pyruvate in the MR2A medium (data not shown). Soytone
or beef extract includes essential carbon sources (e.g.
organic acids) for the bacteria. However, this study did not
clarify the essential nitrogen sources; both peptone and yeast
extract were obligately required to isolate KF001. The
bacteria could not utilize inorganic nitrogen as their sole
nitrogen source (data not shown; KNO3, NH4Cl,
[NH4]2SO4 and NaNO3 of 1.0 g L1 were individually added
to MR2A liquid medium instead of yeast extract, proteose
peptone no. 3, and casamino acids, and the growth of isolate
KF001 were monitored by A600 nm for 500 h cultivation. In
all cases, an increase in the A600 nm value was not observed,
compared with the control sample for which the nitrogen
source was excluded. Five replicates of this experiment were
performed.), and inorganic nitrogen concentrations were
low in lake samples (Table 1), suggesting that organic
nitrogen (whose specifics are beyond the scope of this study)
is likely important for the bacterial growth. A number of
studies have detected amino acids in DOM photoproducts
(Jørgensen et al., 1998; Tarr et al., 2001).
In humic ponds, Polynucleobcter (PnecC) comprise
4 60% of the total bacterial community (Hahn et al.,
2005). In Lake Mondsee (a deep oligo-mesotrophic lake),
PnecB bacteria were detected at the highest numbers in the
epilimnion (with maxima at 2- and 4-m depths), and their
numbers decreased with depth to values close to the detection limit (Wu & Hahn, 2006b). We could not isolate the
bacteria from sampling site R1 (headwater), which is
encircled by many needle leaf trees and is not directly
exposed to sunlight, and this site showed a remarkably low
DOC concentration in all samples (Table 1). Additionally,
we observed that isolate KF001’s bacterial activity (ATP
concentration) was stimulated by photochemical DOM
degradation (when lake water samples were exposed to
natural sunlight) using the bioluminescence assay. It has
been observed that photochemical degradation of humic
substances in freshwater samples enhances bacterial growth
(Lindell et al., 1995; Bano et al., 1998; De Lange et al., 2003;
Anesio et al., 2005). Although these reports and our results
are complex phenomena of PnecB, PnecC and PnecD, it may
suggest that the Polynucleobacter-cluster bacteria show a
preference for high concentrations of DOM (e.g. humic
substances) and photic zone (i.e. epilimnion).
Under the conditions of our experiment, the ATP concentration increased to its peak value within 14 h of solar
radiation (Fig. 6a and b), and a similar pattern (short-time
response) was observed in the case of cell-specific protein
synthesis ([3H]leucine incorporation) of freshwater bacteria, which was inoculated into filtrated and solar-radiated
FEMS Microbiol Ecol 67 (2009) 57–68
67
Isolation and ecological aspects of Polynucleobacter
lake water (Jørgensen et al., 1998). In our case, the ATP
concentrations slowly reached a plateau (about 75% of the
peak value of solar-radiated samples) in nonexposed samples. These results may suggest that our isolate, KF001
(PnecD), utilizes whole DOM as well as DOM photoproducts.
In natural environments, organic acids are supplied by
processes including DOM photolysis (Moran & Zepp,
1997), precipitation (Kawamura et al., 1996; Fornaro &
Gutz, 2003), bacterial fermentation, and algal or aquatic
vegetational photosynthesis (Hellebust, 1974; Kamilova
et al., 2006; Chen et al., 2007). However, precipitation is
not a constant event and almost all strains of Polynucleobacter-cluster bacteria, which were pure cultured in previous
studies and in this study, had been isolated from aerobic
zones (i.e. surface water). Thus, these pathways (rainfall or
bacterial fermentation) would not be the main source. On
the other hand, Wu & Hahn (2006b) reported that PnecB
bacterial abundance was negatively correlated with the
chlorophyll a concentration caused by Planktonthrix spp. (a
weak contributor to primary production), whereas statistical analysis indicated a correlation with phytoplankton
biomass in Lake Mondsee. Nonetheless, photosynthetic
excretion of organic acids by phytoplankton and its utilization by bacterioplankton remain poorly understood. Consequently, we suspect that organic acids supplied by DOM
photolysis might be one of the important pathways for
Polynucleobacter-cluster bacteria (e.g. isolate KF001). However, more detailed confirmation about direct observation of
DOM photoproducts’ (e.g. organic acids) utilization by
Plynucleobacter-cluster bacteria at the subcluster level is
required to clarify the ecological aspects of the cluster
because Wu & Hahn (2006a) showed that the contribution
of the subcluster populations to Polynucleobacter communities varied in the trophic status of the lake.
In past studies, the bioavailability of DOM photoproducts generated by solar radiation was usually measured
using a natural bacterial community (not a monocultural
strain), which was collected from water samples as a
mixture. In this study, we have attempted to clarify part of
the freshwater environmental microbial loop by examining
carbon flow from photochemical DOM degradation to
bacterioplankton isolated in monoculture.
Acknowledgements
We are grateful to Dr Masato Imase for SEM observation.
This work was supported by the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) of Japan.
FEMS Microbiol Ecol 67 (2009) 57–68
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