RESEARCH ARTICLE
Enrichment and cultivation of pelagic bacteria from a humic lake
using phenol and humic matter additions
Kristine Michelle L. Hutalle-Schmelzer1,2, Elke Zwirnmann3, Angela Krüger3 & Hans-Peter Grossart1
1
Department of Limnology of Stratified Lakes, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Stechlin, Germany; 2Department of
Biological Sciences, College of Science, University of Santo Tomas, Manila, Philippines; and 3Leibniz-Institute of Freshwater Ecology and Inland Fisheries,
Berlin, Germany
Correspondence: Hans-Peter Grossart,
Department of Limnology of Stratified Lakes,
Leibniz-Institute of Freshwater Ecology and
Inland Fisheries, Alte Fischerhuette 2,
D-16775 Stechlin, Germany. Tel.: 149 33082
699 91; fax: 149 33082 699 17; e-mail:
[email protected]
Received 25 July 2009; revised 2 December
2009; accepted 11 December 2009.
Final version published online 20 January 2010.
DOI:10.1111/j.1574-6941.2009.00831.x
MICROBIOLOGY ECOLOGY
Editor: Riks Laanbroek
Keywords
bacterial community; Lake Grosse Fuchskuhle;
humic matter; phenol; denaturing gradient gel
electrophoresis.
Abstract
Individual bacterial populations are known to respond differently toward substrate
availability. To test how the availability of either pure phenol or natural humic
matter (HM) selects for specific pelagic bacteria phylotypes from a humic lake
(Lake Grosse Fuchskuhle, northeastern Germany), we used culture-dependent and
-independent approaches. Using a batch approach, the bacterial community
composition (BCC) differed depending on both the quantity and the quality of
added substrates. Using a dilution-to-extinction approach, distinct BCC were
detected by eliminating less abundant species. Most bacteria that were common in
the lake were favored by phenol, and yet different subsets of the native BCC were
enriched by HM. Specific bacterial groups with different growth requirements
were consistently present, negatively influenced, or positively enriched following
substrate additions. This study comprises the first explicit demonstration that
bacteria such as Methylobacterium, Methylophilus, and Methylosinus spp. can be
enriched on phenol or HM. Our isolation approaches led to the successful
cultivation of a variety of native bacteria from the lake, such as Novosphingobium
(Alphaproteobacteria) and Flexibacter (Bacteroidetes), or phenol-utilizing bacteria
such as members of Actinobacteria or Burkholderia (Betaproteobacteria). Enrichment and cultivation on phenol and HM as substrates revealed highly specialized
bacterial communities that resemble those found in many HM-rich lakes.
Introduction
Individual bacterial populations are known to differ in their
response to shifts in dissolved organic matter (DOM)
availability, for example humic matter (HM; Burkert et al.,
2003). In a study on the structure and function of aquatic
bacteria, Langenheder et al. (2005) have shown that DOM
availability selects for ‘generalists’ growing within a wide
range of substrates, as well as ‘specialists’ growing on specific
substrates only. In a later study, the same authors (Langenheder et al., 2006) suggest that changes in bacterial community composition (BCC) reflect differences in specific
function, rather than aggregated microbial functions such
as community biomass and respiration.
It has been shown that bacterial communities in HM-rich
lakes are unable to adapt rapidly to changes in environmental conditions such as pH and substrate quality, suggest2010 Federation of European Microbiological Societies
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c
ing a distinctly different ‘humic (bacteria) cluster’ in terms
of community function and composition (Langenheder
et al., 2005). Until now, however, it remains unclear whether
adaptation of distinct bacterial communities, for example of
humic lakes, to specific environmental parameters is important for lake-specific differences in bacterial functions. In
humic lakes, specific bacterial functions in aquatic systems
include the utilization of aromatic DOM (such as phenol
and dissolved HM; Szabó et al., 2007). Therefore, it is of
great interest to evaluate the effects of increased input of
phenol and HM on organic matter turnover and BCC in
these lakes. Phenolic compounds are common byproducts
of HM degradation in lakes or are introduced into lakes by
drainage of humified terrestrial detritus from cultivated
land, forests, and humic lakes (Thurman, 1985). Various
dissolved organic carbon (DOC) fractions such as lowmolecular-weight (LMW) organic acids are an important
FEMS Microbiol Ecol 72 (2010) 58–73
59
Phenol and humic matter additions
substrate for heterotrophic bacteria (Szabó et al., 2007).
Whereas humic substances (HS) account for 50–90% of the
total DOC in HM-rich lakes (Thurman, 1985; Amador et al.,
1990), LMW compounds (including dissolved amino acids,
carbohydrates and fatty acids, vitamins, nucleotides, pigments, and steroids) comprise only a small fraction of DOC
in these lakes (Münster et al., 1999). In this study, changes in
the quantity and quality of the DOC pool following microbial degradation were examined. We monitored quantitative
changes of important components of the chromatogramable
DOC (cDOC) portion pool including HS, polysaccharides
(PS), LMW organic acids, and other fractions (e.g. proteins
and amino acids). This led to detailed insights into substrate
utilization by specific bacterial communities.
Batch and dilution-to-extinction approaches have often
been used in microbial ecology to manipulate microbial
communities. By growing bacteria under controlled conditions or by eliminating rare organisms from mixed microbial assemblages, different insights can be gained into
bacterial dynamics or substrate utilization (Franklin et al.,
2001). Our aim was to determine the relationship between
DOC availability and BCC. We hypothesize that common
pelagic bacteria from Lake Grosse Fuchskuhle can grow
when exposed to phenol and HM, even at high concentrations. Therefore, we used additions of phenol and HM in
combination with the dilution-to-extinction approach to
enrich common bacteria, for example Actinobacteria and
Betaproteobacteria (Burkert et al., 2003; Grossart et al.,
2008), from humic and acidic Lake Grosse Fuchskuhle. In
addition, we applied molecular methods for the phylogenetic characterization of the BCC and isolates obtained.
Materials and methods
Overview of the experimental protocol
This study is based on three experimental approaches: batch
treatment, dilution-to-extinction treatment, and bacterial
cultivation using agar media. In both batch and dilution-toextinction treatments, PCR-denaturing gradient gel electrophoresis (DGGE) analyses were performed before and 21
days after addition of phenol or HM to water samples taken
in December 2004 from the oxic zone (0–2 m, pooled
sample) of the humic-rich SW basin of Lake Grosse Fuchskuhle. Bacterial cultivation on various agar media followed
all incubations. Phenol concentrations as well as the quality
and quantity of HM were measured, and DGGE bands or
isolates were phylogenetically characterized using 16S rRNA
gene fragments as a marker.
Study site
Lake Grosse Fuchskuhle is a dystrophic and eutrophic lake
in the Brandenburg-Mecklenburg Lake District in northFEMS Microbiol Ecol 72 (2010) 58–73
eastern Germany and has an area of 0.02 km2 and a
maximum depth of 5.6 m. It is connected to a fen of LedoPinetum vegetation, and Myrtillo-Pinetum vegetation surrounds the lake and fen. The lake has no inlet or outlet, but
is fed by rain and groundwater (Sachse et al., 2001). It is
artificially divided into four basins, which have different
catchment areas, physical and chemical parameters, microbial activities, and microbial food web structures (Kasprzak,
1993; Koschel, 1995; Grossart et al., 2008). In December
2004, the temperature was 4.2 1C in both basins. However,
DOC concentrations ranged from 10.3 mg L1 in the NE
basin to 28.3 mg L1 in the SW basin, and pH values from
6.1 in the NE basin to 4.9 in the SW basin. The largest
fraction of the cDOC pool comprised of HS (Z66 2% in
the SW basin; Burkert et al., 2003). Chlorophyll a concentrations varied between 32 mg L1 in the NE basin and
12 mg L1 in the SW basin.
Phenol and HM treatments
In both phenol and HM treatments, 100 mL of lake surface
water with a bacterial abundance of 1.79 0.14 106 cells mL1 was used as an inoculum and diluted to
obtain an initial bacterial density of c. 1 106 cells mL1. As
a medium for each treatment, surface lake water was
collected in December 2004 from 0–2 m depth from the
SW basin, prefiltered through 0.2 mm Nuclepore filters
(Sartorius AG, Göttingen, Germany), and sterilized by
autoclaving. Phenol as well as HM were added to a small
volume of sterile lake water and prefiltered through 0.22 mm
filters (Carl Roth GmbH, Karlsruhe, Germany) before
adding them to the medium in both batch and dilution
treatments. The final substrate concentrations were measured to verify that all added substrates remained in the
dissolved state. All incubations were performed in triplicate
and lasted for 21 days.
(1) For batch treatments in 500-mL flasks, 5 mL of inoculum was added to 250 mL of sterile lake water. Phenol
(carbolic acid, Merck, final concentration of 0, 10, 20, and
50 mg L1) or HM (final concentration of 0, 100, 200, and
400 mg L1) was supplemented into sterile lake water in
triplicate. For HM, we used a lyophilized HM extract from
the lake prepared in late November 2003 by reverse osmosis
(Sachse et al., 2001).
(2) For dilution-to-extinction treatments in microtiter
plates, a 100-mL inoculum was serially diluted (101–1011)
to 900 mL of sterile lake water in triplicate, supplemented
with phenol and HM (final concentration as in batch
treatment). One hundred microliters were discarded from
the last wells for phylogenetic analyses.
(3) To acquire bacterial isolates at the end of the incubation,
100-mL subsamples from all treatments were incubated on
agar plates (1.5% w/v) prepared from sterile lake water
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60
supplemented with phenol or HM using the ‘spread-plate’
technique. In addition, five other media were used to recover
isolates: NSY medium (Hahn et al., 2003), half strength
nutrient agar (Cleseri et al., 1998), half strength Saboraud
agar (Rice et al., 2000), agar with distilled water, and HM
(6% w/v) agar. All isolates were screened by PCR using
primers specific for Bacteria (primer pair 341f and 907r;
Muyzer et al., 1993). All plates were kept in complete
darkness at 15 1C for 4 weeks to prevent autotrophy and to
prevent photolysis of HM (Anesio et al., 2005). In all
treatments, pH varied between 4.7 and 4.8 during the entire
incubation. Nutrients were in the same range as has been
measured previously in Lake Grosse Fuchkuhle (Allgaier &
Grossart, 2006b).
Analysis of phenol and HM
Each week, a 5-mL subsample was taken from each flask in
batch treatments and filtered through a 0.22-mm syringe
filter (Carl Roth GmbH). From these subsamples, the
concentrations of phenol were directly determined in duplicate by HPLC analysis and UV detection (DIONEX) at
272 nm. Chromatographic separation was carried out on an
UltraSep ES Phen 1 column (250 3 mm inner diameter,
SEPSERV, Germany) at 35 1C, and phenol was monitored at
272 nm. The eluent consisted of 40% acetonitrile and 60%
H3PO4 (pH 4), and the flow rate was set at 0.6 mL min1.
This method had a lower detection limit of 50 mg L1 and a
precision of 5–10%.
From another set of subsamples, the quantity of
DOC was directly measured in duplicate using
liquid chromatography-organic carbon detection, in combination with UV detection (254 nm; Huber & Frimmel,
1996). Three different cDOC fractions were quantified: HS,
PS, and LMW compounds, which included small proteins
and amino acids.
To check for abiotic processes such as adsorption and
complexation of phenol and HM, we have run a control
each in which microbial activity was inhibited by addition of
HgCl2. There was no noteworthy removal of phenol or HM
by abiotic processes.
Bacterial abundance
After 21 days of incubation, a 1-mL sample was taken from
each flask in batch treatments. Total bacterial abundance
and specific abundances of Bacteria, Betaproteobacteria, and
Actinobacteria were determined using an improved catabolized reporter deposition FISH protocol (CARD-FISH;
Sekar et al., 2003) using probe mix EUB I–III (Daims et al.,
1999), probe BET42a (Manz et al., 1992), and probe
HGC69a (Roller et al., 1994). We specifically focused on
Betaproteobacteria and Actinobacteria as both bacterial
groups are prominent members of the natural bacterial
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K.M.L. Hutalle-Schmelzer et al.
community in Lake Grosse Fuchskuhle (Burkert et al.,
2003; Allgaier & Grossart, 2006a, b; Buck et al., 2009).
Bacterial counting was performed as described previously
(Hutalle-Schmelzer & Grossart, 2009).
Analysis of BCC before and after phenol or HM
additions
At the beginning of the experiment (T0), 100 mL of lake
water were filtered through 5.0 mm and then through 0.2 mm
Nuclepore membranes (Sartorius AG). This allowed for
separation of particle-associated (PA) from free-living (FL)
bacteria before DNA extraction. We separated the initial
sample into both bacteria fractions to yield better comparability with our previous results from the lake (e.g. Allgaier
& Grossart, 2006a, b). At the end of the experiment, 100-mL
aliquots in batch treatments and 100-mL aliquots in dilution-to-extinction treatments were used for DNA extraction. Extraction, amplification, and purification of genomic
DNA were performed according to Hutalle-Schmelzer &
Grossart (2009).
DGGE analyses
For DGGE analyses, primer pairs specific for Bacteria
(primer pair 341f and 907r, Muyzer et al., 1993) and
Actinobacteria (primer pairs HGC236f and HGC664r,
Glöckner et al., 2000) were used. At the 5 0 -end of each
forward primer, a 40 bp GC-rich nucleotide sequence (GCclamp) was added to stabilize migration of the DNA
fragments in the DGGE gel (Muyzer et al., 1993). Two
replicates and negative controls with no substrate additions
were analyzed. For DGGE, we used a 7% v/v polyacrylamide
gel with a denaturing gradient of urea and formamide
ranging from 40% to 70% (Bacteria) and from 55% to 70%
(Actinobacteria). The amplified DNA was quantified on
agarose gels using the Low DNA mass ladder (Invitrogen).
Approximately 20 ng of amplified DNA 50 mL1 were used
for DGGE analyses. After electrophoresis at 100 V for 18 h,
DGGE bands were stained, documented, and DNA of
excised bands was eluted and purified (Hutalle-Schmelzer
& Grossart, 2009). The eluted DNA was reamplified with
primers 341f (Bacteria) or HGC236f (Actinobacteria) without a GC-clamp.
DGGE banding patterns were analyzed by nonmetric
multidimensioning scaling (NMS) ordinations using GELCOMPAR II, version 3.5 (Applied Maths). Within GELCOMPAR II,
a band-based binary presence/absence table was calculated
by applying the Dice correlation coefficient.
Phylogenetic analysis
DNA sequences obtained with an ABI Prism 3100-Avant
Genetic Analyzer (Applied Biosystems) were checked
FEMS Microbiol Ecol 72 (2010) 58–73
61
Phenol and humic matter additions
manually using the CHROMAS program (version 1.45; Technelysium) and compared with sequences in the NCBI BLAST
database (http://www.ncbi.nlm.nih.gov/blast). Phylogenetic
analysis of the retrieved sequences was performed as described previously (Hutalle-Schmelzer & Grossart, 2009).
Phylogenetic inferences were based on groupings carried out
by Glöckner et al. (2000), Urbach et al. (2001), Zwart et al.
(2002), Gich et al. (2005), and Newton et al. (2006).
Nucleotide sequence accession numbers
All sequences of 16S rRNA gene fragments that we obtained
were deposited in GenBank with the following accession
numbers: EU391179–EU391264, EU409443–EU409523, and
EU414842–EU414908.
Statistical analysis
All statistical analyses were performed using the SPSS software (version 4.0.1; SPSS Inc., Chicago, IL). Temporal
changes of phenol or DOC and its fractions were tested by
regression analysis, followed by ANOVA to test for the
significance of the slope (b). To compare the fractions of
Betaproteobacteria and Actinobacteria of total Bacteria
in all treatments, the Welsh test was used because variances
were nonhomogenous. To compare different concentrations
of phenol or DOC and its fractions among treatments,
ANCOVA was used with time as a covariate. Concentrations
were standardized as the percent of the initial values of
each treatment. For phenol treatments, the percent of the
initial value was square root transformed to comply with
homoskedasticity. To test for significance of differences
between DGGE banding patterns of the treatments, ANOSIM
(Clarke & Green, 1988) was applied using the software
PRIMER 6, version 6.1.9 (PRIMER-E). ANOSIM generates a
test statistic (R), which is an indication of the degree of
separation between groups: a score of 1 indicates complete
separation, whereas a score of 0 indicates no separation.
Furthermore, a significance level is calculated based on
a maximum of 999 (or all possible) permutations of the
data set.
Results
Temporal changes of phenol and HM in batch
treatments
In natural lake water, the concentration of phenol was below
the detection limit (50 mg L1). In nonpoisoned batch treatments with added phenol, phenol significantly decreased
over time in all treatments, indicating rapid bacterial
degradation (P o 0.025, data not shown). During incubation, the decrease in phenol was significantly higher in the
10 mg L1 treatment than in the 20 and 50 mg L1 treatments
(P = 0.010, data not shown). In the natural lake water, the
initial concentrations of total DOC and HM were 28.3 and
19.1 mg L1, respectively. After addition of HM, total DOC
increased, but the concentrations of DOC and its fractions
did not change significantly during incubation, except for
the cDOC fraction of the 400 mg L1 HM treatment, which
decreased significantly with time (P = 0.043, Supporting
Information, Table S1).
Bacterial abundance
At 21 days of incubation, bacteria in the control (without any
substrate addition) had increased from their initial concentration (1 106 cells mL1) to 2.203 0.26 106 cells mL1.
Bacterial abundance in all phenol and the 100 mg L1 HM
treatment also increased over time, but much less than that in
the control (Table 1). In contrast, bacterial numbers in the 200
and 400 mg L1 HM treatments slightly decreased over time.
After 21 days of incubation, Actinobacteria accounted for
o 1.2% of the total bacteria (0.009 106 mL1) in both
phenol and HM treatments, and Betaproteobacteria for
17.3–29.8% (0.129–0.209 106 mL1) in the phenol and
1.2–37.7% (0.010–0.187 106 mL1) in the HM treatments
(Table 1, Fig. 1). Betaproteobacteria were significantly more
abundant than Actinobacteria in all treatments after 21 days of
incubation (Welsh test, t = 2.983, d.f. = 6.010, P = 0.024).
BCC in batch cultures
As indicated in Figs 2 and 3, our incubation conditions led
to changes in BCC over time. However, all control samples
Table 1. Bacterial cell counts (106 cells mL1 SD) of Bacteria, Betaproteobacteria, and Actinobacteria in control cultures (without any addition) and in
cultures with addition of phenol and HM after 21 days of incubation
Control
Total cells
Bacteria
Betaproteobacteria
Actinobacteria
Phenol (mg L1)
Humic matter (mg L1)
0
10
20
50
100
200
400
2.023 0.257
1.823 0.177
0.159 0.019
0.021 0.015
1.080 0.159
0.701 0.083
0.209 0.022
0.007 0.009
1.132 0.107
0.776 0.082
0.198 0.032
0.007 0.010
1.137 0.148
0.747 0.091
0.129 0.019
0.009 0.011
1.233 0.124
0.828 0.099
0.010 0.003
0.005 0.006
0.976 0.046
0.457 0.056
0.038 0.005
0.002 0.004
0.848 0.114
0.496 0.077
0.187 0.024
0.002 0.003
FEMS Microbiol Ecol 72 (2010) 58–73
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62
K.M.L. Hutalle-Schmelzer et al.
50
Betaproteobacteria
Actinobacteria
% Bacteria
40
30
20
10
0
0
Control
10
20
50
100
mg L–1 Phenol
200
400
mgL–1 HM
Fig. 1. CARD-FISH counts of Betaproteobacteria and Actinobacteria 21
days after addition of phenol and HM. Counts are given as percent of
total Bacteria. Error bars show SDs.
(without phenol and HM addition) were different from
those in the respective treatments (phenol and HM additions) (Figs S1 and S2). This indicates that in all treatments,
addition of phenol or HM lead to shifts in BCC that are
different from those caused solely by ‘containment’ in our
culture vessels. Furthermore, BCC of lake water changed in
batch cultures with additions of phenol or HM in a
concentration-dependent manner as indicated by NMS
ordinations (Fig. S1) and cluster analyses of DGGE banding
patterns (Fig. S2). ANOSIM revealed highly significant differences between the DGGE banding patterns in both phenol
and HM treatments (R = 0.981, P = 0.01), as well as between
attached and FL bacteria communities (R = 1, P = 0.01).
Dominant DGGE bands in the initial sample (T0) represented typical freshwater phylotypes (Zwart et al., 2002) (Fig.
2a and b, Table S2), for example members of Novosphingobium
(Alphaproteobacteria), Acidovorax, Comamonas, Methylophilus, and Polynucleobacter (Betaproteobacteria), uncultured
Actinobacteria, and Flavobacterium (Bacteroidetes).
BCC in dilution-to-extinction cultures
With increasing dilution, a gradual decrease in the number of
bands was observed in all treatments. An abrupt decrease in
band number was observed in dilutions from 103 to 105 (Figs
2a, b and 3a, b). In addition, different subsets of the original
BCC were enriched after phenol and HM additions as shown
by DGGE analyses and subsequent sequencing of excised
DGGE bands (Tables S2 and S3 for Figs 2a, b and 3a, b,
respectively). After addition of phenol (Fig. 2a and b), persistent natural lake water phylotypes included Alphaproteobacteria
(Novosphingobium) and Betaproteobacteria (Acidovorax, Methylophilus, and Polynucleobacter). Uncultured Actinobacteria
(FSW11-4, LSF_001), Betaproteobacteria (Comamonas, FNE1110), and Bacteroidetes (MA_43_2003DFb_F02) disappeared
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after phenol addition. In contrast, Betaproteobacteria
(Herbaspirillum, clone NE45 and Pigmentiphaga, QLWP2DMWB-4) appeared only after phenol addition, whereas
other Betaproteobacteria (Duganella), Gammaproteobacteria
(Alkalindiges), and Bacteroidetes (Flavobacterium, Flexibacter, Chitinophaga, and Pedobacter) appeared only sporadically during incubation. Dominant bands frequently
detected by DGGE in both treatments are shown in Table 2.
In the HM treatments, relatively few dominant bands
belonged to native lake bacteria (Fig. 3a and b, Table 2). The
frequently detected Alphaproteobacteria (Novosphingobium)
and Betaproteobacteria (Polynucleobacter) did not form distinct and persistent bands after addition of HM. Betaproteobacteria (Methylophilus) were present only with weak bands
at higher concentrations of HM. The bacterial phylotypes
that produced major and persistent bands after HM addition belonged to Betaproteobacteria related to Oxalobacter
sp. (Duganella) and Bacteroidetes related to Flexibacter CF1
(Chitinophaga), with the latter forming the brightest DGGE
bands at the highest dilutions in the 200 mg HM L1 treatment. Other bacteria detected only in HM additions were
Alphaproteobacteria related to Methylobacterium and to
clones LrhB44 (Methylosinus), KTS111 (Rhodopila), and
WD236 (Roseomonas). Three bacteria that appeared sporadically after HM addition were Actinobacteria almost
identical to clone FSW11-4, gammaproteobacterium related
to Legionella, and Bacteroidetes related to clone PLF_064
(Flexibacter).
Actinobacteria -specific DGGE banding patterns
The Actinobacteria-specific DGGE revealed a much higher
diversity of Actinobacteria than the Bacteria-specific DGGE
(Fig. S3). It also revealed that the Actinobacterium related to
clone FSW11-4 was present in almost all treatments. Cluster
analysis showed that FL Actinobacteria of the untreated
initial lake water clustered closely to FL Actinobacteria of all
HM treatments, suggesting that HM is an important actinobacterial substrate in the lake. However, DGGE banding
patterns of PA Actinobacteria after HM additions, and FL
and PA Actinobacteria after phenol additions, were intermixed and did not form separate clusters (data not shown).
Cultivation of bacterial isolates from
the treatments
A total number of 168 isolates were obtained, and of these,
76 were successfully analyzed (Table S4). Isolates that were
detected as dominant bands in DGGE analyses of BCC in
natural lake water and in phenol and HM additions include
Alphaproteobacteria (Novosphingobium; clones 61-0300c543 and FSW11-5) and Bacteroidetes (Flexibacter
sp. CF1). Among the isolates that were not detected as
dominant bands in the treatments are bacteria belonging to
FEMS Microbiol Ecol 72 (2010) 58–73
63
Phenol and humic matter additions
(a)
15
9
10
11
12
1
2
13
16
19
24
14
3
4
5
6
7
8
20
21
22
23
18
FL
31
26
27
28
29
PA 100 10–1 10–2 10–3 10–4 10–5 10–6 100 10–1 10–2 10–3 10–4 10–5 10–6
10 mg L–1
0 mg L–1
T0
(b)
41
37
40
51
46
50
45
58
39
34
33
38
64
44
57
48
42
63
49
67
53
59
Fig. 2. DGGE gels (16S rRNA gene fingerprint) of
Bacteria 21 days after phenol addition: (a) 0 and
10 mg phenol L1, (b) 20 and 50 mg phenol L1.
The phylogenetic affiliation of the sequenced
bands is given in Fig. 4a–e and Tables S2 and S3.
FL and PA = free-living and particle-associated
bacteria of the lake sample (December 2004).
32
47
FL
PA
T0
Phylogeny of sequenced DGGE bands
and isolates
Bacterial and actinobacterial 16S rRNA gene sequences
obtained by DGGE or from isolates were mostly affiliated
to the following phylogenetic groups: Alpha-, Beta-, and
Gammaproteobacteria, Actinobacteria, and Bacteroidetes.
100 10–1 10–2 10–3 10–4 10–5
20 mg
70
60
52
Alphaproteobacteria (Azospirillum, Bradyrhizobium, Rhodopseudomonas, and Sphingomonas), Betaproteobacteria
(Burkholderia, Collimonas, Mitsuaria, and Ralstonia), Gammaproteobacteria (Nevskia and Pseudomonas), Actinobacteria
(Mycobacterium and Frankiaceae), and Bacteroidetes (Cytophagales). The phylogenetic characterization of all isolates is
given in Table S4.
FEMS Microbiol Ecol 72 (2010) 58–73
55
L–1
62
100 10–1
66
10–2 10–3 10–4
50 mg L–1
The phylogenetic relationship of all sequenced DGGE bands
and isolates from this study is given in Fig. 4a–e.
Discussion
Bacterial degradation of phenol and HM
Although we did not detect any free phenol (4 50 mg L1) in
Lake Grosse Fuchskuhle during winter, phenolic compounds can represent 15–25% of leachate DOC in forest
lakes such as Lake Grosse Fuchskuhle (Ossipova et al., 2001),
and contribute up to 37–42% of the total DOC inputs
during peak litter input in autumn (Meyer et al., 1998;
Wiegner et al., 2005). Our phenol enrichment experiment
demonstrates that even at high concentrations (50 mg L1),
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64
K.M.L. Hutalle-Schmelzer et al.
(a)
71
77
82
72
79
80
84
83
86
78
FL PA 10 10
87
10
10
T0
10
10
10
10
10 10
0 mg L–1
10
10
10
10
10
100 mg L–1
FL PA
T0
(b)
105
106
107
100
110
111
103
92
112
113
115
109
104
FL PA 10 10
T0
10
10 10
10
10 10
200 mg L–1
10 10
10 10
116
10 10
400 mg L–1
pure phenol is degraded by a number of bacteria in the lake
(data not shown). Thus, consistent microbial degradation of
the phenolic constituents of leaf leachates and HM in Lake
Grosse Fuchskuhle is very likely and may explain its low in
situ concentrations (Clair et al., 1989). DOC in the SW basin
of Lake Grosse Fuchskuhle reaches up to 50 mg L1 and
mainly consists of HM from an adjacent fen (Sachse et al.,
2001). In none of the HM addition experiments was a
significant decrease in the concentrations of cDOC or HS
observed over time. However, the chemical composition of
the added HM can be modified by microbial activity, as
shown by fluctuations in the concentrations of the three
measured cDOC fractions (data not shown). Because all
treatments were performed in complete darkness, photoautotrophic growth and generation of bacterial substrates
such as LMW compounds by photodecomposition (e.g.
Goldstone et al., 2002) can be ruled out.
Enrichment of specific bacteria in dilution-toextinction treatments
Alphaproteobacteria are an important bacterial component
in nearly all environments (Nold & Zwart, 1998). Their
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117
118
10 10
FL PA
T0
Fig. 3. DGGE gels (16S rRNA gene fingerprint) of
Bacteria 21 days after HM addition of: (a) 0 and
100 mg HM L1, (b) 200 and 400 mg HM L1. The
phylogenetic affiliation of the sequenced bands is
given in Fig. 4a–e and Tables S2 and S3. FL and
PA = free-living and particle-associated bacteria of
the natural sample (December 2004).
detection in the initial water sample from Lake Grosse
Fuchskuhle as well as in the phenol treatments indicates
that these bacteria are well adapted to the environmental
conditions of the acidic SW basin and presumably degrade
phenol. In particular, members of Sphingomonadaceae have
been frequently found in our study and are known for their
important role in the breakdown of refractory DOC and
aromatic compounds (Basta et al., 2005). The genus Novosphingobium – common in the SW basin of Lake Grosse
Fuchskuhle – includes bacteria that degrade phenolic contaminants of groundwater (Tiirola et al., 2002), and are
often found in HM-rich lakes (Kent et al., 2006). In this
study, bacteria belonging to Novosphingobium were favored
by the addition of phenol. Betaproteobacteria are a major
fraction of limnetic bacterioplankton (Zwart et al., 2002),
including that of Lake Grosse Fuchskuhle (see our CARDFISH results of sample T0, Burkert et al., 2003; Allgaier &
Grossart, 2006a, b; Buck et al., 2009). Acidovorax, Methylophilus, and Polynucleobacter formed frequent DGGE bands
of both initial untreated lake water and all phenol treatments. Acidovorax species are associated with the degradation of aromatic substrates (Eriksson et al., 2002), and have
been detected in an acid-impacted lake (Percent et al., 2008).
FEMS Microbiol Ecol 72 (2010) 58–73
65
Phenol and humic matter additions
Table 2. Frequent DGGE band sequences (the GenBank accession no. of the nearest relative is given)
III
Phylogenetic group
Acidovorax
Clone Kas105B
Clone Sta1-17
Aphanizomenon; clone PRD18H03
Comamonas; clone FNE11-10
Croceibacter; clone A43_2003DFb_F02
Herbaspirillum
Clone NE45
H12
Intrasporangium
Clone FSW11-2
Clone FSW11-4
Methylophilus
Clone PRD18A09
Clone PRD18C03
Nostoc; clone PRD18E08
Novosphingobium
N. resinovorum; NCIMB
Pigmentiphaga; QLW-P2DMWB-4
Polynucleobacter
KF023/clone MoIso1
Sphingopyxis
Clone 61-03-00c543
Clone FSW11-5
a
b
g
A
B
C
Accession #
1
EF203185
AJ416187
AY948067
DQ501302
EF378368
1
1
1
1
FL (T0)
1
1
1
1
PA (T0)
HM
1a,b
1b,d
1e,f
1g
1
1
1a,c,d
1a,c,d
AJ575695
AY345556
1
1
Phenol
DQ316351
DQ316353
1
1
AY947994
AY948011
AY948039
1
1
EF029110
AJ938031
1
1
1
AB269809
1
1b,d
DQ316835
DQ501324
1
1
1b,c,d
1b,c,d
1
1
1
1
1
1
1
1
1c,d
1a
1e,f
1
1b,c,d
a, Alphaproteobacteria; b, Betaproteobacteria; g, Gammaproteobacteria; A, Actinobacteria; B, Bacteroidetes; C, Cyanobacteria; a, 0 mg L1 phenol/
HM; b, 10 mg L1 phenol; c, 20 mg L1 phenol; d, 50 mg L1 phenol; e, 100 mg L1 HM; f, 200 mg L1 HM; g, 400 mg L1 HM.
Methylophilus is a methylotrophic bacterium that is found in
various freshwater habitats, sediments, and soil (De Marco
et al., 2004). To our knowledge, the present study is the first
to demonstrate that members of the Methylophilus group
isolated from a lake can be enriched on phenol and may thus
be useful for bioremediation. Betaproteobacteria of the
beta-II (Polynucleobacter) cluster occur in several types of
freshwater habitats (Hahn, 2003). They form the dominant
bacterial group in the SW basin of Lake Grosse Fuchskuhle
in summer not only in terms of abundance but also
metabolic activity (Grossart et al., 2008). In this previous
study, we suggested that this bacterium may be involved in
phenol degradation. However, it has recently been shown
that these bacteria can utilize acetate (Buck et al., 2009;
Hahn et al., 2009a, b), a common byproduct of organic
matter degradation (including HM, see below). Bacteroidetes are usually found in sediments and are known for
their ability to degrade lignocellulosic plant materials
(Das et al., 2007). Several Bacteroidetes were isolated from
acidic Sphagnum-dominated wetlands (Pankratov et al.,
2006) and an acid-impacted lake (Percent et al., 2008).
Detection of members of Bacteroidetes such as Cytophagales
or Flavobacterium after phenol addition suggests that they
might be able to utilize phenol as a substrate. The observed
FEMS Microbiol Ecol 72 (2010) 58–73
shifts in BCC after phenol additions might be due to phenol
toxicity (Dean-Ross & Rahimi, 1995) or phenol resistance
(Szabó et al., 2007). However, persistent and dominant
existence of native bacterial phylotypes in phenol additions
indicates that phenol is an important bacterial substrate,
in particular in humic and acidic lakes (Yannarell et al.,
2003).
In contrast to the phenol treatments, addition of HM
resulted in a markedly different BCC. Based on our results
from DGGE fingerprinting analyses, it is likely that dominant bacteria in the phenol treatments, for example Novosphingobium spp. and members of the Polynucleobacter
necessarius cluster, cannot degrade HM themselves, but
might be favored by various HM degradation products, for
example acetate and phenol, which are also common constituents of leaf litter leachates (Peña-Méndez et al., 2005).
To our knowledge, Alphaproteobacteria such as Methylobacterium and Methylosinus spp. have not previously been
related to the presence of HM; however, in our study both
genera occurred at 400 mg L1 HM in both 105 and 107
dilutions. Members of Methylobacterium are facultative
methylotrophs that are commonly found in association with
plants, and have been isolated from soil, dust, and lake
sediments (Sy et al., 2005). Methylosinus are obligate
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66
K.M.L. Hutalle-Schmelzer et al.
(a)
Fig. 4. Phylogenetic trees of 16S rRNA gene sequences of all DGGE bands and isolates from the natural sample at time T0, untreated surface water
samples (control), and from those enriched with phenol and HM. Sequences obtained in the present study are given in bold letters, with their GenBank
accession numbers in parentheses. Bootstrap values at the main branching points are given. (a) Alphaproteobacteria, (b) Betaproteobacteria, (c)
Gammaproteobacteria, (d) Actinobacteria, and (e) Bacteroidetes. Fuku2 identifies all sequences from this study, whereas ‘ISO’ denotes all isolates and
‘SW’ indicates DGGE bands. For each DGGE band, PH and HM stand for phenol and HM additions, respectively. The following numbers denote specific
DGGE bands and isolates given in Tables S2 and S3, respectively. After each accession number, the sample from which DGGE bands or isolates have
been retrieved are given (to 0 initial lake sample, Phe = phenol addition (0, 10, 20, and 50 mg L1, HM = humic matter addition (100, 200, and
400 mg L1).
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c
FEMS Microbiol Ecol 72 (2010) 58–73
67
Phenol and humic matter additions
(b)
Fig. 4b. Continued.
FEMS Microbiol Ecol 72 (2010) 58–73
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68
K.M.L. Hutalle-Schmelzer et al.
(c)
Fig. 4c. Continued.
methanotrophs that are widespread in nature including lake
sediments (Costello & Lidstrom, 1999). Furthermore, they
are known to cometabolize a number of environmental
aromatic contaminants (Hanson & Hanson, 1996). Therefore, it is very likely that they degrade HM also in lakes. A
betaproteobacterium that was present in all HM treatments
is related to Oxalobacter. Its cultured representatives degrade
oxalate, but obtain some of their carbon from acetate
(Cornick & Allison, 1996). Oxalate and acetate are degradation products of complex organic material, such as HM (e.g.
Goldstone et al., 2002); thus growth of Oxalobacter species is
potentially favored by HM or its byproducts. Actinobacteria
are an autochthonous component of many lakes (Warnecke
et al., 2005), including Lake Grosse Fuchskuhle (Burkert
et al., 2003; Allgaier & Grossart, 2006a, b; Buck et al., 2009).
Our results, based on Bacteria-specific primers and DGGE,
suggest that Actinobacteria have a limited capability of
growing in the presence of phenol or HM. However, based
on Actinobacteria-specific primers and DGGE, they are
present and relatively diverse in all HM treatments (Fig.
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S5). A single DGGE band identical to clone FSW11-4
(Allgaier & Grossart, 2006a) was detected in all phenol and
HM batch treatments. Thus, specific Actinobacteria of the Ac
clusters presumably grow on HM, but are likely outcompeted in number by other bacterial phylotypes. Because we
do not have any of these Actinobacteria in pure culture, their
role in HM degradation remains speculative. However,
Actinobacteria are potential producers of extracellular peroxidases in lakes similar to their relatives in soil (Buck et al.,
2008), and they are involved in riverine leaf litter decomposition (Das et al., 2007). Bacteroidetes are supposed to take
up and degrade high-molecular-weight DOC fractions
(Kirchman, 2002). In this study, Bacteroidetes related to
Flexibacter sp. (CF1) are also favored by HM. Flexibacter
have been detected from algal mucilage (Fischer et al., 1998)
and are associated with leaf litter processing in streams (Das
et al., 2007). Their frequent detection after HM addition
indicates the utilization of HM and/or its constituents.
Therefore, it is not surprising that most of the bacteria
detected after addition of HM belong to bacterial groups
FEMS Microbiol Ecol 72 (2010) 58–73
69
Phenol and humic matter additions
(d)
Fig. 4d. Continued.
affiliated with leaf litter degradation or aromatic contaminant bioremediation. In humic lakes, extracellular enzyme
activities are highly correlated with aromatic carbon uptake
(Münster et al., 1999). This notion suggests that extracelluFEMS Microbiol Ecol 72 (2010) 58–73
lar enzyme activities (Buck et al., 2008) and other bacterial
processes such as aerobic mineralization and methane
oxidation may be important features of the BCC in Lake
Grosse Fuchskuhle.
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70
K.M.L. Hutalle-Schmelzer et al.
(e)
Fig. 4e. Continued.
Cultivation of specific bacteria
Several potentially important phenol-degrading bacteria
could be isolated, for example members of Actinobacteria
and Betaproteobacteria (Burkholderia). Frequently detected
isolates on HM-rich media include Sphingomonas (Alphaproteobacteria), which are known for degrading polyaromatic
compounds (Basta et al., 2005). Another isolate (Flexibacter
CF1) is known to degrade a variety of polymeric substances
(see above). Other isolates such as Alphaproteobacteria
related to bacteria BAC47 (Devosia), LS-1 (Magnetospirillum), and S07 (Azospirillum), and gammaproteobacterium
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related to Soe1 (Nevskia) have only 94–97% sequence
similarity to the next-described isolate (Table S4). Thus, a
combination of dilution-to-extinction treatments and our
cultivation approaches is suitable to capture new and ecologically important bacteria of humic lakes. The in-depth
physiological characterization of these isolates, however,
remains an important future task.
Summary
This study shows that addition of phenol and HM at
increasing concentrations led to enrichment of specific
pelagic bacteria from an HM-rich lake. Our phylogenetic
FEMS Microbiol Ecol 72 (2010) 58–73
71
Phenol and humic matter additions
analyses show a clear replacement of bacteria detected at
different concentrations along the dilution gradient, which
suggests a selective response of bacteria toward addition of
phenol or HM. The observed changes in BCC following
phenol and HM additions indicate physiological differences
among specific bacteria, for example their tolerance to
phenol toxicity vs. their ability to degrade phenolic compounds. Our phylogenetic analysis of bacteria enriched and
cultivated on phenol and HM as substrates points to highly
specialized bacterial communities that resemble those found
in many HM-rich lakes. Hence, these bacterial communities
seem to account for a major part of DOM degradation in
such lakes including Lake Grosse Fuchskuhle. The isolates
obtained are an important basis for further physiological
characterization of a number of bacterial phylotypes commonly found in HM-rich lakes.
Acknowledgements
We thank E. Mach for technical assistance during sampling
and for measurement of DOC, K. Pohlmann for helping
with the statistical analyses, the Deutscher Akademischer
Austauschdienst (DAAD) for the PhD scholarship given to
K.M.L.H.-S., as well as the Boehringer Ingelheim Fonds for a
course travel grant awarded to K.M.L.H.-S. This study was
also supported by a DFG grant (PA 1655/1-1) given to A.P.
and H.-P.G.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Fig. S1. NMS ordination plots of DGGE banding patterns of
FL (open symbols) and PA (filled symbols) bacteria from
batch treatments 21 days after addition of phenol or HM.
Fig. S2. Dendrogram of cluster analyses from DGGE gels
(16S rRNA gene fingerprint) of Bacteria 21 days after phenol
and HM additions in batch treatments.
Fig. S3. DGGE banding pattern of Actinobacteria in incubations with phenol and HM.
Table S1. Linear regression analysis of the temporal changes
in concentration of (a) phenol in the phenol treatment and
(b) DOC and its fractions in the HM treatment.
Table S2. Phylogenetic affiliation of sequenced DGGE bands
(Bacteria) in dilution-to-extinction incubations with
phenol.
Table S3. Phylogenetic affiliation of sequenced DGGE bands
(Bacteria) in dilution-to-extinction incubations with HM.
Table S4. Phylogenetic affiliation of sequenced isolates from
treatments with phenol (0, 10, 20, and 50 mg L1) and HM
(0, 100, 200, and 400 mg L1).
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