World J Microbiol Biotechnol DOI 10.1007/s11274-013-1530-y
ORIGINAL PAPER
Herbicides induce change in metabolic and genetic diversity of bacterial community from a cold oligotrophic lake P. Aguayo • C. Gonza´lez • R. Barra J. Becerra • M. Martı´nez
•
Received: 12 July 2013 / Accepted: 16 October 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Pristine cold oligotrophic lakes show unique physical and chemical characteristics with permanent fluctuation in temperature and carbon source availability. Incorporation of organic toxic matters to these ecosystems could alter the bacterial community composition. Our goal was to assess the effects of simazine (Sz) and 2,4 dichlorophenoxyacetic acid (2,4-D) upon the metabolic and genetic diversity of the bacterial community in sediment samples from a pristine cold oligotrophic lake. Sediment samples were collected in winter and summer season, and microcosms were prepared using a ration 1:10 (sediments:water). The microcosms were supplemented with 0.1 mM 2,4-D or 0.5 mM Sz and incubated for 20 days at 10 °C. Metabolic diversity was evaluated by using the Biolog EcoplateTM system and genetic diversity by 16S rDNA amplification followed by denaturing gradient gel electrophoresis analysis. Total bacterial counts and live/ dead ratio were determined by epifluorescence microscopy. The control microcosms showed no significant differences (P [ 0.05) in both metabolic and genetic diversity between summer and winter samples. On the other hand, the addition
P. Aguayo C. Gonza´lez M. Martı´nez (&) Laboratorio de Microbiologı´a Ba´sica y Bioremediacio´n, Departamento de Microbiologı´a, Facultad de Ciencias Biolo´gicas, Universidad de Concepcio´n, Casilla 160-C, Concepcio´n, Chile e-mail: [email protected] R. Barra Centro EULA-Chile, Unidad de Sistemas de Acua´ticos, Casilla 160-C, Concepcio´n, Chile J. Becerra Laboratorio de Quı´mica de Productos Naturales, Facultad de Ciencias Naturales y Oceanogra´ficas, Universidad de Concepcio´n, Casilla 160-C, Concepcio´n, Chile
of 2,4-D or Sz to microcosms induces statistical significant differences (P \ 0.05) in metabolic and genetic diversity showing the prevalence of Actinobacteria group which are usually not detected in the sediments of these non-contaminated lacustrine systems. The obtained results suggest that contaminations of cold pristine lakes with organic toxic compounds of anthropic origin alter their homeostasis by inhibiting specific susceptible bacterial groups. The concomitant increase of usually low representative bacterial groups modifies the bacterial composition commonly found in this pristine lake. Keywords Bacterial diversity Cold oligotrophic pristine lake Herbicides
Introduction In aquatic environments physical and chemical fluctuations modulate bacterial diversity and metabolic activity of resident microorganisms living in these ecosystems (Cavicchioli et al. 2003; Venkatesharaju et al. 2010). In addition, human activity like agriculture or forestry close to aquatic environment may also impact the homeostasis of these water systems (Gunderson 2000; Johnsen et al. 2001; Kritzberg et al. 2005; Venkatesharaju et al. 2010). Simazine (Sz) and 2,4-dichlorophenoxyacetic acid herbicides (2,4-D) are examples of toxic organic compounds of anthropic origin that are incorporated as contaminant to pristine lakes, which modify bacterial diversity by inhibiting some resident bacterial groups allowing the proliferations of others (Johnsen et al. 2001). Sz may alter the nitrogen cycle in lake systems (McCarty 1999) while 2,4-D may interfere the turnover of organic matters (Prado and Airoldi 2000). The impact of herbicides upon bacterial
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diversity of soil is variable as stated by Johnsen et al. (2001). The authors also suggested the relevance of molecular methods like PCR-denaturing gradient gel electrophoresis (DGGE) for establishing the genetic structure of the bacterial community. Because only a minor part of aquatic microbial communities are cultivable, quantification of changes induced by pesticides upon bacterial communities present in natural environments are complex and difficult to achieve by conventional microbiological methods (Nakatsu et al. 2000). Thus, to establish the structure and composition of microbial communities in these ecosystems conventional analysis must be complemented with DGGE and 16S rRNA gene sequence analysis (Barra-Caracciolo et al. 2005; Morgante et al. 2010). Molecular analysis and metabolic approaches have been particularly useful for determining the effect of pollutants on microbial communities of various environments (Alexander 1994; Kozdro´j and van-Elsas 2001). Using these approaches our research group has previously reported that although some Patagonian lakes had a unique bacterial community structure, the resident bacterial groups may be able to perform similar metabolic functions (Mackenzie et al. 2011). These lakes have been described as unpolluted environments since pesticides contamination has not been detected. However, in the last decade an increased antropic activity close to these no-contaminated water bodies increased the risk of pesticide contamination of these lakes. The aim of this work was to evaluate if Sz and 2,4-D can induce changes in the metabolic and genetic diversity of the bacterial community present in sediments samples from a pristine cold oligotrophic lake.
Materials and methods Study sites and sampling Surface sediment samples were collected during winter (August 2010) and summer (January 2011) season from Lake Te´mpanos located in Queulat National Park at the Chilean Patagonia (44°–46°S, 72°–73°W). Superficial sediments (500 g) were collected by a with Van Veen drag, placed in sterile polyethylene bags, refrigerated at 4 °C, and transported to laboratory for further processing. Physico-chemical analyses In order to perform the grain size analysis, sediment samples were sieved at 1.0 and 4.0 A units (A = 2log2 particle diameter in mm) and separated into fine (mud) and coarse (sand) fractions. Grain size was analyzed using an Elzone 282 PC coulter counter particle analyzer. Total organic matter (% TOC) in each sample was estimated by
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the loss on ignition (LOI) technique following the method described by Boyle (2002). Total nitrogen, nitrates, total phosphorous, ortho-phosphate, polychlorinated biphenyls and organochloride pesticides, were determined according to standard methods (APHA-AWWA-WPCF 1985). Microcosms preparation Each sediment sample was diluted 1:10 in filtered lake water and sonicated with ultrasound at nonlethal frequency 40 kHz for 5 min and let settled for 10 min. Aliquots of 100 ml were distributed into 500 ml Erlenmeyer flasks to prepare microcosms as described by Aguayo et al. (2009). Microcosms were supplemented with 0.1 mM 2,4-D or 0.5 mM Sz and incubated at 10 °C for 20 days. A non supplemented microcosm was used as a control. Five milliliters per sample were obtained at the beginning of the experiment and after 20 days of incubation to determinate the total bacterial counts by using the BacLight Viability Kit (Molecular Probes, Eugene, USA). In addition, cultivable heterotrophic bacterial populations were estimated by the microdrops method using R2A agar (Herbert 1990). Metabolic profile analysis Metabolic profile of bacterial communities from each sediment or microcosm sample was determined by using the Biolog EcoplateTM system (Biolog Inc., CA, USA). 100 ll bacterial suspension [1 9 105 cells ml-1] was inoculated in each well, and microplates were incubated at 10 °C for 15 days (Garland 1996). The optical density (590 nm) was recorded with a microplate reader (EL800 Biotek). Richness index (R) was estimated as the number of oxidized carbon substrates, while the relative functional diversity (RFD) was determined by the Shannon–Weaver coefficient (H), as follow: [(Hmax) = ln (S)], where S is the number of evaluated substrates in Biolog EcoplateTM system (Gomez et al. 2006). Microbial activity (MA) was estimated using the average well color development (AWCD) index, according to Gomez et al. (2006). All the assays were performed in triplicate, using sterile distilled water as a control. DNA extraction, PCR and DGGE The DNA was purified using the Power Soil DNA Isolation Kit (Mo Bio Laboratories) following the protocol provided by the manufacturer. Total DNA was amplified with 16S rRNA universal primers EUB 9–27 (50 -GAGTTTGATCC TGGCTCAG-30 ) and EUB 1542 (50 -AGAAAGGAGGTG ATCCAGCC-30 ) (Brosius et al. 1978). Nested PCR was performed using the primer pair 341f-GC clamp (5-0 CCTA CGGGAGGCAGCAG-30 ) and 534r (5-0 ATTACCGCGGC TGCTGG-30 ) (Muyzer et al. 1993). The genetic profile was
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determined by DGGE (Ferris et al. 1996) performed on DGGE-2001 system (CBS Scientific Company, CA, USA), for 16 h at 70 V using a 40–80 % urea-formamide gradient and 7.5 % acrylamide/bis-acrylamide (Boon et al. 2002). After electrophoresis, sediment DGGE gel was stained for 40 min with SYBR Gold nucleic acid gel stain (Molecular Probes) and visualized under UV-light. Microcosms DGGE gels were stained with silver nitrate. Each band from the DGGE gels was excised and placed in 50 ll of HypureTM molecular biology grade water (nuclease-free) (Thermo scientific), and 1 ll of which was amplified again by PCR using the primers 341f and 534r (Zwisler et al. 2003). Nucleotide sequencing was performed at Macrogen (Macrogen Inc., Seoul, Korea). Taxonomic Units (OTUs) were defined as described by Bent and Forney (2008) and the number of DGGE bands present in the gel were considered as the number of phylotypes in each sample (Dı´ez et al. 2007). Nucleotide sequences were analyzed by using the available programs at the NCBI web page (http://www.ncbi.nlm.nih.gov).
analysis of the differences among microcosms was also performed by ANOSIM test.
Results Physico-chemical characterization of lake Te´mpanos In water samples phosphorous and orthophosphate concentration detected in lake Te´mpanos was \0. 12 mg/L in summer and\0.14 mg/L in winter, while TOC was 0.54 mg/ L in summer and 3.37 mg/L in winter. Close to the coast this lake becomes frozen on its surface during winter and spring for short periods, and has an average temperature of c.a. 4° C in winter and c.a. 8 °C in summer (Table 1). Pesticides analysis indicated that these compounds were not present in water samples. The grain size analysis of sediment showed a muddy texture and fine sediment material (5.51 A) allowing its classification as medium mud sediment. Total bacterial and viable cell counts in sediments and microcosms
Gel and sequence analysis Gel images were analyzed using a Gel-Pro Analyzer 4.0 software and a binary matrices were built (presence = 1, absence = 0). A Pairwise calculation was made using Bray–Curtis’s similarity coefficient. Similarity matrix was generated and used for constructing a multidimensional scaling diagram (MDS) for further analysis with PRIMER V.6 software package (Clarke and Gorley 2001) Statistical
The total bacterial counts in sediment samples were 4.4 9 106 cells g-1 in winter and 3.3 9 107 cells g-1 in summer. The viable bacterial counts were 4.2 9 105 CFUg-1 in winter and 1.7 9 106 CFUg-1 in summer, with approximately 9.5 and 5.3 % of cultivable bacteria in R2A agar, respectively (Table 2).
Table 1 Physico-chemical characteristic of lake Te´mpanos Season
Physicochemical parameters Temp. (°C)
pH
Total phosphorus (mg/L)
Ortho phosphate (mg/L)
Total Nitrogen (mg/L)
Nitrate (mg/L)
TOC (mg/L)
Winter
4.0
4.0
\0.14
\0.14
0.08
0.080
3.37
Summer
8.3
4.0
\0.12
\0.12
0.10
0.008
0.54
TOC total organic carbon
Table 2 Total (living and dead cells) and culturable cell counts in sediment and microcosm samples Total (living and dead cells) and culturable cells count
Te´mpanos lake Microcosm control c
Microcosm Sz
Microcosm 2,4-D a
c
c
Total count (cells/g)a
Living cells (cells/g)a
Culturable (CFU/g)
Winter
Winter
Winter
Summer
Summer
4.7 9 106
3.3 9 107
4.4 9 106
3.2 9 107
7.5 9 10
6
1.4 9 10
7
3.3 9 10
7
9.2 9 10
6
5.5 9 10
6
9.1 9 10
6
5.0 9 10
6
6.5 9 10
6
7.4 9 10
6
1.0 9 10
7
6.3 9 10
6
7.8 9 10
6
Summer
4.2 9 105 (9.5)b
1.7 9 106 (5.3)b
6
1.4 9 106 (1.5)
5
1.4 9 106 (21.5)
6
2.7 9 106 (34.5)
1.5 9 10 (4.5) 3.2 9 10 (6.4) 1.3 9 10 (20.6)
Criterion of membrane integrity
b
Values in parenthesis correspond to percentage of culturable bacteria related to a live cells
c
Values obtained after 20 days of incubation
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After 20 days of incubation, microcosms showed no significant differences in their total bacterial counts (P [ 0.05) as compared with those observed in sediment samples. However, cultivable bacterial counts showed differences between winter and summer samples in microcosms control and microcosms supplemented with 2,4-D showing an increase of one logarithm. Microcosms supplemented with Sz showed similar cultivable bacterial count as compared to the sediment sample. Bacterial metabolic profile from sediments and microcosms Bacterial community in sediment samples obtained in winter and summer showed similar substrate use richness (R) as detected by the Biolog EcoplateTM system (28 carbon sources). Microcosms assay showed variable substrate use regarding the season of the sampling (data not shown). In The control microcosms 22 different carbon sources in winter were used and 24 in summer. The addition of Sz to microcosms increased number of the carbon sources used as substrate to 24 in winter and decreased it to 16 in summer. The supplementation with 2,4-D was associated with an increase of substrate use richness, because 26 carbon sources were used in winter samples and 24, in summer samples. On the other hand, no significant difference in the intensity of use of the substrates was observed in microcosms (Fig. 1). The AWCD index showed lower values in summer microcosms supplemented with 2,4-D and Sz than those obtained for sediments and the control microcosm (Table 3), but in winter microcosms both herbicides increased the AWCD values. Bacterial genetic profile from sediments and microcosms The number of bands (OTUs) observed in the native sediments was 15 in winter and 19 in summer. In microcosms the number of bands detected was 14 in winter and 18 in summer. In contrast, in microcosm supplemented with 2,4-D the number of bands was 18 in winter and 23 in summer and in microcosms supplemented with Sz was 24 and 22 in winter and summer respectively. The genetic structure of the bacterial community detected in summer and winter shows 80 % similarity (Figs. 2, 3). However, the supplementation in microcosms with 2,4-D decreased the similarity value to 40 % (Fig. 4). Phylogenetic analysis of the excised bands showed that all bacterial groups detected in sediment samples were present in microcosms. The main groups detected in sediment samples included bacteria belonging to the Beta, Gamma and Delta Proteobacteria (Table 4). Microcosms also showed the presence of Alphaproteobacteria that were undetected in sediment samples (Table 5). On the other
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Fig. 1 Relative AWCD calculated from absorbance values of Biolog Ecoplate for the lake and microcosms in study. a Substrates utilization by sediment communities in lakes, b Substrates utilization in microcosm (Winter), c Substrates utilization in microcosm (Summer). The absorbance was measure after 96 h incubation. The substrate were divided in six categories; Carbohydrates (n = 10), Amines (n = 2), amino acids (n = 6), Carboxylic acids (n = 7), Polymers (n = 4), Phenolic compounds (n = 2)
hand, microcosms supplemented with 2,4-D showed an increase in Actinobacteria, although this genus was of low representation in the control microcosm and non detected in the sediment samples.
Discussion Cold oligothrophic lakes have been analyzed mainly from an ecological point of view (Mackenzie et al. 2011) but few reports describe the impact of anthropic contaminant upon their stability (Williamson et al. 1999). In this study, the sediment from the Patagonian cold lake Te´mpanos was evaluated in microcosm assays after been challenged with
World J Microbiol Biotechnol Table 3 Relative functional diversity and metabolic activity of bacterial communities in Patagonian lakes Functional diversity (H’)
Metabolic activity (AWCD)
Winter
Summer
Winter
Summer 1.22 ± 0.11
Te´mpanos lake
3.24 ± 0.06
3.32 ± 0.11
0.87 ± 0.06
Microcosm control
3.18 ± 0.08
2.99 ± 0.07
0.88 ± 0.08
0.95 ± 0.07
Microcosm 2,4-D
3.25 ± 0.09
3.27 ± 0.13
1.10 ± 0.09
0.75 ± 0.13
Microcosm simazine
3.18 ± 0.10
3.26 ± 0.12
1.02 ± 0.10
0.69 ± 0.12
The metabolic activity in the microcosm was determined after 20 days of incubation at 10 °C Mean ± SD H’ Shannon–Weaver diversity index, AWCD average wells color development
Denaturing Gradient (40-80%)
Fig. 2 Denaturing gradient gel electrophoresis of native bacterial communities in Sediment lake. Similarity between native bacterial communities was calculated, using Jaccard’s index. Lw = Te´mpanos lake Winter, Ls = Te´mpanos lake Summer. Acrilamide gel stain with SYBR Gold
two herbicides—Sz and 2,4-D—as a model. Our purpose was to evaluate how the bacterial community from a pristine aquatic environment responds to chemical perturbation. lake Te´mpanos exhibits short periods of surface freezing close to
Denaturing Gradient (40-80%)
Fig. 3 Denaturing gradient gel electrophoresis of bacterial communities in microcosm with toxic compounds. Similarity between bacterial communities in microcosm was calculated, using Jaccard’s index. Cw = Control microcosm (Winter), Cs = Control microcosm (Summer), SzW = Simazine microcosm (Winter), Simazine microcosm (Summer), 2,4-Dw = 2,4-D microcosm (Winter), 2,4Ds = 2,4-D microcosm (Summer). Acrilamide gel staining with silver nitrate
the coast. Physicochemical analysis showed pristine water and a mud texture with fine sediment material classified as medium mud sediment according the Wentworth (1922). Dissolved nitrogen and total phosphorus levels were \1.7 mgL-1, values typically found in oligothrophic lake systems (Woelfl et al. 2003; McGee et al. 2010; Rojas and Le Roux 2005). The increment of dissolved carbon in lake Te´mpanos during winter could be the consequence of allochthonous input because the region is characterized by strong raining periods (1,205.9 mm of rainfall per year) concentrated mainly during winter and spring. The low nitrogen, phosphorous and dissolved carbon level detected suggests that this lake should be considered as an ultraoligothrophic (Soto 2002; Balseiro et al. 2007). Organic-chloride pesticides were not detected in the samples, confirming the pristine quality of this water body. The low pH of water (average 4.0) could be explained by the volcanic activity of the Andes (Ribeiro Guevara et al. 2010; Romero 2012). Thus, the physico-chemical properties detected and the observed oligotrophic conditions make the lake Te´mpanos an adequate model for evaluating the stability of the bacterial communities against contamination with organic toxic compounds.
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World J Microbiol Biotechnol Fig. 4 Multi-dimensional scaling (MDS) plot of the assemblages bands from Te´mpanos lake (a) and microcosms (b). MDS analysis conducted on a Bray–Curtis analysis of similarity for bands. Contours represent the degree of similarity (%) between samples in each cluster. Cw = Control microcosm (Winter), Cs = Control microcosm (Summer), SzW = Simazine microcosm (Winter), Simazine microcosm (Summer), 2,4Dw = 2,4-D microcosm (Winter), 2,4-Ds = 2,4-D microcosm (Summer). Similarity index was evaluated for percentage
Sediment samples did not show significant differences in total bacterial counts between seasons, suggesting that this kind of lacustrine system does not modify the resident bacterial community along the year. Similar results have been reported by Jiang et al. (2006) for sediments of other oligotrophic lakes, suggesting that resident bacterial populations from this type of environments are stable. As estimated by epifluorescence microscopy 90 % of bacterial cells present in the sediment should be considered as dormant or dead bacteria (Buerger et al. 2012). On the other hand, the percentages of cultivable bacterial cells with respect to live cells in summer and winter samples oscillated 5.3–9.5 %, and these values are similar to those reported for English lakes by Porter et al. (2004). The small fraction of bacteria recovered in cultures should belong to facultative oligotrophic groups which are able to grow under a wide range of organic matter, as compared to strict oligotrophic groups (Mackenzie et al. 2011; Vasileska 2002). It is known that complex rich media inhibit the growth of oligotrophic bacteria (Giovanonni and Stingl
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2007; Hoppe et al. 1988), hence, the recovering of bacteria in culture from oligotrophic lakes depends on the selective media used (Porter et al. 2004). Metabolic analysis showed that the bacterial community of Te´mpanos lake sediment in summer and winter reaches the highest score value calculated by Biolog EcoplateTM system. This score was consistent with those observed in undisturbed environment by Gomez et al. (2006). Denaturing gradient gel electrophoresis analysis of the amplified V3 region from rDNA16S showed 80 % of similarity between samples obtained in winter and summer suggesting that bacterial structure and diversity did not change with seasons in this lacustrine system. The groups detected included cultivable and non cultivable bacteria belonging to the Beta, Gamma and Delta Proteobacteria. Similar results were also reported in pristine lacustrine systems (Imhoff 2006; Mackenzie et al. 2011). On the other hand, OTUs referred to from Alphaproteobacteria were detected in all microcosms while less number of OTUs of this group was present in sediment samples
World J Microbiol Biotechnol Table 4 Assignment of taxonomic groups to band sequences extracted from DGGE gel based on 180 bp and the closest sequence match of know phylogenetic affiliation in sediment samples Band
Closest sequences related
Taxonomic group
% Similarity
Samples
1
Uncultured bacterium clone ESBAC05B09
ND
100
Lw–Ls
2
Uncultured bacterium isolate DGGE band 5S2
ND
79
Lw–Ls
3
Uncultured bacterium clone MN82
ND
89
Lw–Ls
4
Uncultured bacterium clone MEB2_685
ND
78
Lw
5
Uncultured Klebsiella sp. clone S8
Gammaproteobacteria
97
Lw–Ls
6
Uncultured soil bacterium clone CRS5570T-1
ND
81
Lw–Ls
7 8
Uncultured bacterium clone 13C-HEW-092 Uncultured Herbaspirillum sp. clone AS84P1 16S
ND Betaproteobacteria
92 93
Lw–Ls Lw–Ls
9
Janthinobacterium sp.
Betaproteobacteria
89
Lw–Ls
10
Herminiimonas glaciei strain UMB49
Betaproteobacteria
89
Lw
11
Uncultured Acidobacteria bacterium clone KBS_T8
Acidobacteria
87
Lw–Ls
12
Silvimonas terrae strain KM-45 16S
Betaproteobacteria
91
Lw
13
Thiomonas sp. enrichment culture clone ‘DGGE gel band B2’
Betaproteobacteria
91
Ls
14
Methylibium fulvum strain Gsoil 322
Gammaproteobacteria
97
Lw–Ls
15
Pelomonas aquatica strain : CCUG 52575
Betaproteobacteria
93
Lw–Ls
16
Methylibium petroleiphilum PM1 strain PM1
Betaproteobacteria
87
Lw–Ls
17
Pelomonas puraquae strain : CCUG 52769
Betaproteobacteria
89
Lw–Ls
18
Uncultured bacterium clone MFBC4H11
ND
82
Lw–Ls
19
Pelobacter propionicus DSM 2379 strain
Deltaproteobacteria
93
Lw–Ls
20
Zymophilus raffinosivorans strain VTT E-90406
Firmicutes
91
Ls
21
Brachymonas denitrificans strain AS-P1
Betaproteobacteria
90
Ls
Samples obtained for sequencing analysis
suggesting that Alphaproteobacteria have low representation among the bacterial community in lake Te´mpanos. Not significant differences (P [ 0.05) in total and cultivable bacterial counts of microcosms were observed independently of the presence of 2,4-D or simazine. On the other hand, microcosms supplemented with Sz used less substrates offered by Biolog EcoplateTM system than those supplemented with 2,4-D, suggesting that Sz is more effective than 2,4-D as metabolic interfering compound of oligotrophic bacterial communities. Similar results have been observed by Ros et al. (2006) using molecules analogous to Sz. The addition of 2,4-D or Sz modifies the richness of substrates used, the relative functional diversity (H’) and the microbial activity (AWCD) of the microbial community but different responses were observed in microbial activity during summer and winter. MDS analysis of OTUs obtained in DGGE also suggests that genetic bacterial diversity did not change in summer and winter (80 % similarity), but microcosms supplemented with 2,4-D or Sz show differences with the control (40 % similarity). The ANOSIM analysis (P \ 0.05) confirmed this finding. The results support the idea that organic toxic compounds could modify the dynamic of biogeochemical process by decreasing the ability of the bacterial communities to use a particular carbon substrate as has also been suggested by Lesan and Bhandai (2003).
No degradation of Sz or 2,4-D was detected by spectrometric UV-analysis (data not shown) although an increase in the cultivable bacterial fraction was observed suggesting that the increment of recoverable bacterial population could be due to the presence of an intrinsically tolerant subpopulations among the sediment bacterial community of lake Te´mpanos. We speculate that these subpopulations should be able to grow in the presence of Sz or 2,4-D by using both lysed dead bacteria as carbon source (Pinchuk et al. 2008) or cellular endogenous substrates like poly-hydroxy-alkanoates (Godoy et al. 2003; Pavez et al. 2009). Genetic analysis of microcosms showed predominance of bacteria belonging to phyla Alpha, Beta and Gamma Proteobacteria, but in microcosms supplemented with 2,4D predominated the phylum Actinobacteria which was not detected in the sediments of lake Tempanos and was of low representation in the control microcosm. The change in the genetic diversity observed with the addition of pesticides to microcosms could be caused by some specific bacterial group—usually none detected in DGGE—that increase their proportion in the presence of the allocthonous organic compounds as described by Cottrell and Kirchman (2000). Interestingly, previous results at our laboratory (Mackenzie et al. 2011) also showed that Actinobacteria group was
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World J Microbiol Biotechnol Table 5 Assignment of taxonomic groups to band sequences extracted from DGGE gel based on 180 bp and the closest sequence match of know phylogenetic affiliation in Microcosms lake Band
Closest sequences related
Taxonomic group
% Similarity
Samples
1
Methylobacterium populi
Alphaproteobacteria
82
Cw
2
Duganella zoogloeoides
Betaproteobacteria
92
Cw
3
Uncultured alpha proteobacterium clone
Alphaproteobacteria
96
Cw
4
Methylobacterium populi
Alphaproteobacteria
96
Cw
5
Uncultured Curvibacter sp.
Betaproteobacteria
92
Cw
6
Uncultured bacterium clone
ND
95
Cw–Cs
7 8
Sphingomonas japonica Uncultured Lysobacter sp.
Alphaproteobacteria Gammaproteobacteria
97 75
Cw–Cs–2,4s, 2,4w Cs
9
Sphingomonas sp.
Alphaproteobacteria
97
Cs–Cw
10
Sphingomonadaceae bacterium
Alphaproteobacteria
97
Cs–Cw
11
Novosphingobium sp.
Alphaproteobacteria
91
Cs–Cw–Szs
12
Duganella sp.
Betaproteobacteria
98
Cs–Cw–Szs–2,4s
13
Uncultured beta proteobacterium clone A3-67
Betaproteobacteria
87
Cs–Szs–2,4w
14
Massilia timonae
Betaproteobacteria
92
Szw
15
Uncultured alpha proteobacterium clone B3-79
Alphaproteobacteria
89
Szw–Szs
16
Uncultured beta proteobacterium clone A3-67
Betaproteobacteria
95
Szw
17
Arthrobacter tecti
Actinobacteria
91
Szw–Szs
18
Uncultured bacterium clone 0300_02
ND
90
Szw
19
Uncultured bacterium clone Transfer_3C08
ND
93
Szw Szs
20
Magnetospirillum sp.
Alphaproteobacteria
95
21
Uncultured bacterium clone Bbsn10-02E6
ND
89
Szs–Szw–Cw–Cs–2,4s
22 23
Uncultured Gemmatimonadales bacterium clone Uncultured bacterium isolate DGGE gel band
Gemmatimonadetes ND
89 90
Szs–Cs–2,4s Szs–Szw–Cw–Cs–2,4s
24
Uncultured Alicycliphilus sp.
Betaproteobacteria
99
2,4s–Szs–Cw–Cs
25
Delftia acidovorans
Betaproteobacteria
94
2,4s–Cw–Cs
26
Xylophilus ampelinus
Gammaproteobacteria
89
2,4s–Szs–Cs
27
Bosea minatitlanensis
Alphaproteobacteria
89
2,4s–2.4w–Szs–Cs–Cw
28
Streptomyces sp.
Actinobacteria
92
2,4w–2,4s–Szs–Cs
29
Mycobacterium sp.
Actinobacteria
72
2,4w–2,4s–Szs–Szw–Cw–Cs
30
Uncultured actinobacterium clone
Actinobacteria
93
2,4w
31
Cellulosimicrobium cellulans
Actinobacteria
95
2,4w
32
Uncultured soil bacterium clone B18-3
ND
68
2,4w
33
Aeromicrobium fastidiosum
Actinobacteria
61
2,4w
34
Bosea minatitlanensis strain AMX51
Alphaproteobacteria
90
2,4s
Samples obtained for sequencing analysis
unusually detected in water samples from various other Patagonian lakes, confirming that this bacterial group is of low representation in pristine cold oligotrophic environments. Therefore, the changes in metabolic and genetic diversity observed in the presence of 2,4-D or Sz could be the consequence of a negative impact of this kind of compounds upon specific susceptible bacterial groups and, thus, they modify the bacterial diversity commonly found in this pristine lake. The results allow us to propose that the detection of an increased proportion of Actinobacteria group in a particular sample obtained from pristine lakes
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could be indicative of homeostasis alteration as consequence of toxic organic matter contamination. Acknowledgments This work was supported by FONDECYT, Grant No. 1100462. Paulina Aguayo V. is a CONICYT Fellowship. The Authors thank to Miss Ruth Contreras for her technical support.
References Aguayo J, Barra R, Becerra J, Martı´nez M (2009) Degradation of 2, 4, 6 tribromophenol and 2, 4, 6 trichlorophenol by aerobic
World J Microbiol Biotechnol heterotrophic bacteria present in psychrophilic lakes. World J Microbiol Biotechnol 25:553–560 Alexander M (1994) Biodegradation and biorremediation, 2nd edn. Academic Press, New York APHA-AWWA-WPCF (1985) Standard methods for the examination of water and wastewater, 16th edn. EUA, Washington Balseiro G, Modenutti B, Queimalin˜os C, Reissig M (2007) Daphnia distribution in Andean Patagonian lakes: effect of low food quality and fish predation. Aquat Ecol 41:599–609 Barra-Caracciolo A, Grenni P, Ciccoli R, Di Landa G, Cremisini C (2005) Simazine biodegradation in soil: analysis of bacterial community structure by in situ hybridization. Pest Manag Sci 61:863–869 Bent J, Forney L (2008) The tragedy of the uncommon: understanding limitations in the analysis of microbial diversity. ISME J 2:689–695 Boon N, De Windt W, Verstraete W, Top M (2002) Evaluation of nested PCR-DGGE (Denaturing gradient gel electrophoresis) with group-specific 16s rRNA primers for the analysis of bacterial communities from different wastewater treatment plants. FEMS Microbiol Ecol 39:101–112 Boyle J (2002) Mineralogical and geochemical indicator techniques. In: Last W, Smol JP (eds) Tracking environmental change using lake sediments. Springer, New York, pp 83–141 Brosius J, Palmer M, Kennedy P, Nollerm H (1978) Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci USA 75:4801–4805 Buerger S, Spoering A, Gavrish E, Leslin C, Ling L, Epstein S (2012) Microbial scout hypothesis, stochastic exit from dormancy, and the nature of slow growers. Appl Environ Microbiol 78: 3221–3228 Cavicchioli R, Ostrowski M, Fegatela F, Goodchild A, Boixereus G (2003) Life under nutrient limitation in oligotrophic marine environments: an eco/physiological perspective of Sphingopyxis alaskiensis (Formerly Sphingomonas alaskiensis). Microbial Ecol 45:203–217 Clarke K, Gorley R (2001) PRIMER v5: User manual/tutorial PRIMER-E. Plymouth, UK Cottrell M, Kirchman D (2000) Natural assemblages of marine proteobacteria and members of the cytophaga-flavobacter cluster consuming low- and high-molecular-weight dissolved organic matter. Appl Environ Microbiol 66:1692–1697 Dı´ez B, Bauer K, Bergman B (2007) Epilithic cyanobacterial communities of a marine tropical beach rock (Heron Island, Great Barrier Reef): diversity and diazotrophy. Appl Environ Microbiol 73:3656–3668 Ferris M, Muyzer G, Ward D (1996) Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl Environ Microbiol 62:340–346 Garland J (1996) Analytical approaches to the characterization of samples of microbial communities using patterns of potential source utilization. Soil Biol Biochem 28:213–221 Giovanonni S, Stingl U (2007) The importance of culturing bacterioplankton in the omics age. Nature Rev Microbiol 5:820–826 Godoy F, Bunster M, Matus V, Aranda C, Gonzalez B, Martı´nez M (2003) Poly-b-hydroxyalkanoates consumption during degradation of 2,4,6-trichlorophenol by Sphingopyxis chilensis S37. Lett Appl Microbiol 36:315–320 Gomez E, Ferreras L, Toresani S (2006) Soil bacteria functional diversity as influenced by organic amendment application. Bioresource Technol 97:1484–1489 Gunderson L (2000) Ecological resilience in theory and application. Annu Rev Ecol Syst 31:425–439 Herbert RA (1990) Methods for enumerating microorganisms and determining biomass in natural environments. In: Grigovora R,
Norris JR (eds) Methods in microbiology: techniques in microbial ecology, vol 22. Academic Press, London p 619 Hoppe H, Kim S, Gocke K (1988) Microbial decomposition in aquatic environments: combined process of extracellular enzyme activity and substrate uptake. Appl Environ Microbiol 54:784–790 Imhoff J (2006) The phototrophic alpha-proteobacteria. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K, Stackebrandt E (eds) The prokaryotes, 3rd edn. Springer, Minneapolis, pp 41–64 Jiang H, Dong H, Zhang G, Yu B, Chapman L, Matthew F (2006) Microbial diversity in water and sediment of lake Chaka, an Athalassohaline lake in Northwestern China. Appl Environ Microbiol 72:3832–3845 Johnsen K, Jacobsen S, Torsvik V, Sørensen J (2001) Pesticide effects on bacterial diversity in agricultural soils: a review. Biol Fert Soils 33:443–453 Kozdro´j J, van-Elsas J (2001) Structural diversity of microorganisms in chemically perturbed soil assessed by molecular and cytochemical approaches. J Microbiol Methods 43:197–212 Kritzberg E, Cole J, Pace M, Graneli W (2005) Does autochthonous primary production drive variability in bacterial metabolism and growth efficiency in lakes dominated by terrestrial C inputs. Aquat Microb Ecol 38:103–111 Lesan H, Bhandai A (2003) Isotherms for Atrazine desorption from two surface soils. Environ Eng Sci 3:257–263 Mackenzie R, Barros J, Martı´nez M (2011) Metabolic and molecular analysis of aerobic heterotrophic bacteria in cold-oligotrophic freshwater ecosystems. World J Microbiol Biotechnol 27: 2499–2504 McCarty G (1999) Modes of action of nitrification inhibitors. Biol Fertil Soils 29:1–9 McGee D, Broecker W, Winckler G (2010) Gustiness: the driver of glacial dustiness? Quaternary Sci Rev 29:2340–2350 Morgante V, Lo´pez-Lo´pez A, Flores C, Gonza´lez M, Gonza´lez B, Va´squez M, Rossello´-Mora R, Seeger M (2010) Bioaugmentation with Pseudomonas sp. strain MHP41 promotes simazine attenuation and bacterial community changes in agricultural soils. FEMS Microbiol Ecol 71:114–126 Muyzer G, Dewaal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for rRNA. Appl Environ Microbiol 59:695–700 Nakatsu C, Torsvik V, Øvrea˚s L (2000) Soil community analysis using DGGE of 16S rDNA polymerase chain reaction products. Soil Sci Soc Am J 64:1382–1388 Pavez P, Castillo J, Gonza´lez C, Martı´nez M (2009) Poly-bHydroxyalkanoate exert a protective effect against carbon starvation and frozen conditions in Sphingopyxis chilensis. Curr Microbiol 59:636–640 Pinchuk G, Ammons C, Culley D, Li S, McLean J, Romine M, Nealson K, Fredrickson J, Beliaev A (2008) Utilization of DNA as a sole source of phosphorus, carbon, and energy by Shewanella spp.: ecological and physiological implications for dissimilatory metal reduction. Appl Environ Microbiol 74:1198–1208 Porter J, Morris S, Pickup R (2004) Effect to trophic status on the culturability and activity of bacteria from a range of lakes in the English lake district. Appl Environ Microbiol 70:2072–2078 Prado A, Airoldi C (2000) Effect of the pesticide 2,4-D on microbial activity of the soil monitored by microcalorimetry. Thermochim Acta 349:17–22 Ribeiro Guevara S, Meili M, Rizzo A, Daga R, Arribere M (2010) Sediment records of highly variable mercury inputs to mountain lakes in Patagonia during the past millennium. Atmos Chem Phys 10:3443–3453 Rojas E, Le Roux J (2005) Sedimentary processes on a Gilbert-type delta in lake Llanquihue southern Chile. Rev Geol Chile 32:19–31
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World J Microbiol Biotechnol Romero J (2012) Some backgrounds on the short October 2011 eruption at Mt. Hudson, 45°300 S, Southern Andes, Pyroclastic flow. J Geol 1:1–8 Ros M, Goberna M, Moreno J, Herna´ndez T, Garcı´a C, Insam H, Pascual J (2006) Molecular and physiological bacterial diversity of a semi-arid soil contaminated with different levels of formulated atrazine. Appl Soil Ecol 34:93–102 Soto D (2002) Oligotrophic patterns in southern Chile lakes: the relevance of nutrients and mixing depth. Rev Chil Hist Nat 75:377–393 Vasileska A (2002) Fosfatase activity of lake Ohrid waters as an indicator of it’s ecophysiological condition. Master Thesis, University ‘‘Sts Kiril and Metodij’’, Institute of biology, Skopje (In Macedonian Eng. Summary) Venkatesharaju K, Ravikumar P, Somashekar R, Prakash K (2010) Physico-chemical and bacteriological investigation on the river
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Cauvery of kollegal stretch in Karnataka. J Sci Eng Technol 6:50–59 Wentworth C (1922) A scale of grade and class terms for clastic sediments. J Geol 30:377–392 Williamson C, Donald P, Morris D, Pace M, Olson O (1999) Dissolved organic carbon and nutrients as regulators of lake ecosystems: resurrection of a more integrated paradigm Littitiol. J Ocean 44:795–803 Woelfl S, Villalobos L, Parra O (2003) Trophic parameters and method validation in lake Rin˜ihue (North Patagonia: Chile). Rev Chil Hist Nat 76:459–474 Zwisler W, Selje N, Simon M (2003) Seasonal patterns of the bacterioplankton community composition in a large mesotrophic lake. Aquat Microb Ecol 31:211–225
ORIGINAL PAPER
Herbicides induce change in metabolic and genetic diversity of bacterial community from a cold oligotrophic lake P. Aguayo • C. Gonza´lez • R. Barra J. Becerra • M. Martı´nez
•
Received: 12 July 2013 / Accepted: 16 October 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Pristine cold oligotrophic lakes show unique physical and chemical characteristics with permanent fluctuation in temperature and carbon source availability. Incorporation of organic toxic matters to these ecosystems could alter the bacterial community composition. Our goal was to assess the effects of simazine (Sz) and 2,4 dichlorophenoxyacetic acid (2,4-D) upon the metabolic and genetic diversity of the bacterial community in sediment samples from a pristine cold oligotrophic lake. Sediment samples were collected in winter and summer season, and microcosms were prepared using a ration 1:10 (sediments:water). The microcosms were supplemented with 0.1 mM 2,4-D or 0.5 mM Sz and incubated for 20 days at 10 °C. Metabolic diversity was evaluated by using the Biolog EcoplateTM system and genetic diversity by 16S rDNA amplification followed by denaturing gradient gel electrophoresis analysis. Total bacterial counts and live/ dead ratio were determined by epifluorescence microscopy. The control microcosms showed no significant differences (P [ 0.05) in both metabolic and genetic diversity between summer and winter samples. On the other hand, the addition
P. Aguayo C. Gonza´lez M. Martı´nez (&) Laboratorio de Microbiologı´a Ba´sica y Bioremediacio´n, Departamento de Microbiologı´a, Facultad de Ciencias Biolo´gicas, Universidad de Concepcio´n, Casilla 160-C, Concepcio´n, Chile e-mail: [email protected] R. Barra Centro EULA-Chile, Unidad de Sistemas de Acua´ticos, Casilla 160-C, Concepcio´n, Chile J. Becerra Laboratorio de Quı´mica de Productos Naturales, Facultad de Ciencias Naturales y Oceanogra´ficas, Universidad de Concepcio´n, Casilla 160-C, Concepcio´n, Chile
of 2,4-D or Sz to microcosms induces statistical significant differences (P \ 0.05) in metabolic and genetic diversity showing the prevalence of Actinobacteria group which are usually not detected in the sediments of these non-contaminated lacustrine systems. The obtained results suggest that contaminations of cold pristine lakes with organic toxic compounds of anthropic origin alter their homeostasis by inhibiting specific susceptible bacterial groups. The concomitant increase of usually low representative bacterial groups modifies the bacterial composition commonly found in this pristine lake. Keywords Bacterial diversity Cold oligotrophic pristine lake Herbicides
Introduction In aquatic environments physical and chemical fluctuations modulate bacterial diversity and metabolic activity of resident microorganisms living in these ecosystems (Cavicchioli et al. 2003; Venkatesharaju et al. 2010). In addition, human activity like agriculture or forestry close to aquatic environment may also impact the homeostasis of these water systems (Gunderson 2000; Johnsen et al. 2001; Kritzberg et al. 2005; Venkatesharaju et al. 2010). Simazine (Sz) and 2,4-dichlorophenoxyacetic acid herbicides (2,4-D) are examples of toxic organic compounds of anthropic origin that are incorporated as contaminant to pristine lakes, which modify bacterial diversity by inhibiting some resident bacterial groups allowing the proliferations of others (Johnsen et al. 2001). Sz may alter the nitrogen cycle in lake systems (McCarty 1999) while 2,4-D may interfere the turnover of organic matters (Prado and Airoldi 2000). The impact of herbicides upon bacterial
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diversity of soil is variable as stated by Johnsen et al. (2001). The authors also suggested the relevance of molecular methods like PCR-denaturing gradient gel electrophoresis (DGGE) for establishing the genetic structure of the bacterial community. Because only a minor part of aquatic microbial communities are cultivable, quantification of changes induced by pesticides upon bacterial communities present in natural environments are complex and difficult to achieve by conventional microbiological methods (Nakatsu et al. 2000). Thus, to establish the structure and composition of microbial communities in these ecosystems conventional analysis must be complemented with DGGE and 16S rRNA gene sequence analysis (Barra-Caracciolo et al. 2005; Morgante et al. 2010). Molecular analysis and metabolic approaches have been particularly useful for determining the effect of pollutants on microbial communities of various environments (Alexander 1994; Kozdro´j and van-Elsas 2001). Using these approaches our research group has previously reported that although some Patagonian lakes had a unique bacterial community structure, the resident bacterial groups may be able to perform similar metabolic functions (Mackenzie et al. 2011). These lakes have been described as unpolluted environments since pesticides contamination has not been detected. However, in the last decade an increased antropic activity close to these no-contaminated water bodies increased the risk of pesticide contamination of these lakes. The aim of this work was to evaluate if Sz and 2,4-D can induce changes in the metabolic and genetic diversity of the bacterial community present in sediments samples from a pristine cold oligotrophic lake.
Materials and methods Study sites and sampling Surface sediment samples were collected during winter (August 2010) and summer (January 2011) season from Lake Te´mpanos located in Queulat National Park at the Chilean Patagonia (44°–46°S, 72°–73°W). Superficial sediments (500 g) were collected by a with Van Veen drag, placed in sterile polyethylene bags, refrigerated at 4 °C, and transported to laboratory for further processing. Physico-chemical analyses In order to perform the grain size analysis, sediment samples were sieved at 1.0 and 4.0 A units (A = 2log2 particle diameter in mm) and separated into fine (mud) and coarse (sand) fractions. Grain size was analyzed using an Elzone 282 PC coulter counter particle analyzer. Total organic matter (% TOC) in each sample was estimated by
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the loss on ignition (LOI) technique following the method described by Boyle (2002). Total nitrogen, nitrates, total phosphorous, ortho-phosphate, polychlorinated biphenyls and organochloride pesticides, were determined according to standard methods (APHA-AWWA-WPCF 1985). Microcosms preparation Each sediment sample was diluted 1:10 in filtered lake water and sonicated with ultrasound at nonlethal frequency 40 kHz for 5 min and let settled for 10 min. Aliquots of 100 ml were distributed into 500 ml Erlenmeyer flasks to prepare microcosms as described by Aguayo et al. (2009). Microcosms were supplemented with 0.1 mM 2,4-D or 0.5 mM Sz and incubated at 10 °C for 20 days. A non supplemented microcosm was used as a control. Five milliliters per sample were obtained at the beginning of the experiment and after 20 days of incubation to determinate the total bacterial counts by using the BacLight Viability Kit (Molecular Probes, Eugene, USA). In addition, cultivable heterotrophic bacterial populations were estimated by the microdrops method using R2A agar (Herbert 1990). Metabolic profile analysis Metabolic profile of bacterial communities from each sediment or microcosm sample was determined by using the Biolog EcoplateTM system (Biolog Inc., CA, USA). 100 ll bacterial suspension [1 9 105 cells ml-1] was inoculated in each well, and microplates were incubated at 10 °C for 15 days (Garland 1996). The optical density (590 nm) was recorded with a microplate reader (EL800 Biotek). Richness index (R) was estimated as the number of oxidized carbon substrates, while the relative functional diversity (RFD) was determined by the Shannon–Weaver coefficient (H), as follow: [(Hmax) = ln (S)], where S is the number of evaluated substrates in Biolog EcoplateTM system (Gomez et al. 2006). Microbial activity (MA) was estimated using the average well color development (AWCD) index, according to Gomez et al. (2006). All the assays were performed in triplicate, using sterile distilled water as a control. DNA extraction, PCR and DGGE The DNA was purified using the Power Soil DNA Isolation Kit (Mo Bio Laboratories) following the protocol provided by the manufacturer. Total DNA was amplified with 16S rRNA universal primers EUB 9–27 (50 -GAGTTTGATCC TGGCTCAG-30 ) and EUB 1542 (50 -AGAAAGGAGGTG ATCCAGCC-30 ) (Brosius et al. 1978). Nested PCR was performed using the primer pair 341f-GC clamp (5-0 CCTA CGGGAGGCAGCAG-30 ) and 534r (5-0 ATTACCGCGGC TGCTGG-30 ) (Muyzer et al. 1993). The genetic profile was
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determined by DGGE (Ferris et al. 1996) performed on DGGE-2001 system (CBS Scientific Company, CA, USA), for 16 h at 70 V using a 40–80 % urea-formamide gradient and 7.5 % acrylamide/bis-acrylamide (Boon et al. 2002). After electrophoresis, sediment DGGE gel was stained for 40 min with SYBR Gold nucleic acid gel stain (Molecular Probes) and visualized under UV-light. Microcosms DGGE gels were stained with silver nitrate. Each band from the DGGE gels was excised and placed in 50 ll of HypureTM molecular biology grade water (nuclease-free) (Thermo scientific), and 1 ll of which was amplified again by PCR using the primers 341f and 534r (Zwisler et al. 2003). Nucleotide sequencing was performed at Macrogen (Macrogen Inc., Seoul, Korea). Taxonomic Units (OTUs) were defined as described by Bent and Forney (2008) and the number of DGGE bands present in the gel were considered as the number of phylotypes in each sample (Dı´ez et al. 2007). Nucleotide sequences were analyzed by using the available programs at the NCBI web page (http://www.ncbi.nlm.nih.gov).
analysis of the differences among microcosms was also performed by ANOSIM test.
Results Physico-chemical characterization of lake Te´mpanos In water samples phosphorous and orthophosphate concentration detected in lake Te´mpanos was \0. 12 mg/L in summer and\0.14 mg/L in winter, while TOC was 0.54 mg/ L in summer and 3.37 mg/L in winter. Close to the coast this lake becomes frozen on its surface during winter and spring for short periods, and has an average temperature of c.a. 4° C in winter and c.a. 8 °C in summer (Table 1). Pesticides analysis indicated that these compounds were not present in water samples. The grain size analysis of sediment showed a muddy texture and fine sediment material (5.51 A) allowing its classification as medium mud sediment. Total bacterial and viable cell counts in sediments and microcosms
Gel and sequence analysis Gel images were analyzed using a Gel-Pro Analyzer 4.0 software and a binary matrices were built (presence = 1, absence = 0). A Pairwise calculation was made using Bray–Curtis’s similarity coefficient. Similarity matrix was generated and used for constructing a multidimensional scaling diagram (MDS) for further analysis with PRIMER V.6 software package (Clarke and Gorley 2001) Statistical
The total bacterial counts in sediment samples were 4.4 9 106 cells g-1 in winter and 3.3 9 107 cells g-1 in summer. The viable bacterial counts were 4.2 9 105 CFUg-1 in winter and 1.7 9 106 CFUg-1 in summer, with approximately 9.5 and 5.3 % of cultivable bacteria in R2A agar, respectively (Table 2).
Table 1 Physico-chemical characteristic of lake Te´mpanos Season
Physicochemical parameters Temp. (°C)
pH
Total phosphorus (mg/L)
Ortho phosphate (mg/L)
Total Nitrogen (mg/L)
Nitrate (mg/L)
TOC (mg/L)
Winter
4.0
4.0
\0.14
\0.14
0.08
0.080
3.37
Summer
8.3
4.0
\0.12
\0.12
0.10
0.008
0.54
TOC total organic carbon
Table 2 Total (living and dead cells) and culturable cell counts in sediment and microcosm samples Total (living and dead cells) and culturable cells count
Te´mpanos lake Microcosm control c
Microcosm Sz
Microcosm 2,4-D a
c
c
Total count (cells/g)a
Living cells (cells/g)a
Culturable (CFU/g)
Winter
Winter
Winter
Summer
Summer
4.7 9 106
3.3 9 107
4.4 9 106
3.2 9 107
7.5 9 10
6
1.4 9 10
7
3.3 9 10
7
9.2 9 10
6
5.5 9 10
6
9.1 9 10
6
5.0 9 10
6
6.5 9 10
6
7.4 9 10
6
1.0 9 10
7
6.3 9 10
6
7.8 9 10
6
Summer
4.2 9 105 (9.5)b
1.7 9 106 (5.3)b
6
1.4 9 106 (1.5)
5
1.4 9 106 (21.5)
6
2.7 9 106 (34.5)
1.5 9 10 (4.5) 3.2 9 10 (6.4) 1.3 9 10 (20.6)
Criterion of membrane integrity
b
Values in parenthesis correspond to percentage of culturable bacteria related to a live cells
c
Values obtained after 20 days of incubation
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After 20 days of incubation, microcosms showed no significant differences in their total bacterial counts (P [ 0.05) as compared with those observed in sediment samples. However, cultivable bacterial counts showed differences between winter and summer samples in microcosms control and microcosms supplemented with 2,4-D showing an increase of one logarithm. Microcosms supplemented with Sz showed similar cultivable bacterial count as compared to the sediment sample. Bacterial metabolic profile from sediments and microcosms Bacterial community in sediment samples obtained in winter and summer showed similar substrate use richness (R) as detected by the Biolog EcoplateTM system (28 carbon sources). Microcosms assay showed variable substrate use regarding the season of the sampling (data not shown). In The control microcosms 22 different carbon sources in winter were used and 24 in summer. The addition of Sz to microcosms increased number of the carbon sources used as substrate to 24 in winter and decreased it to 16 in summer. The supplementation with 2,4-D was associated with an increase of substrate use richness, because 26 carbon sources were used in winter samples and 24, in summer samples. On the other hand, no significant difference in the intensity of use of the substrates was observed in microcosms (Fig. 1). The AWCD index showed lower values in summer microcosms supplemented with 2,4-D and Sz than those obtained for sediments and the control microcosm (Table 3), but in winter microcosms both herbicides increased the AWCD values. Bacterial genetic profile from sediments and microcosms The number of bands (OTUs) observed in the native sediments was 15 in winter and 19 in summer. In microcosms the number of bands detected was 14 in winter and 18 in summer. In contrast, in microcosm supplemented with 2,4-D the number of bands was 18 in winter and 23 in summer and in microcosms supplemented with Sz was 24 and 22 in winter and summer respectively. The genetic structure of the bacterial community detected in summer and winter shows 80 % similarity (Figs. 2, 3). However, the supplementation in microcosms with 2,4-D decreased the similarity value to 40 % (Fig. 4). Phylogenetic analysis of the excised bands showed that all bacterial groups detected in sediment samples were present in microcosms. The main groups detected in sediment samples included bacteria belonging to the Beta, Gamma and Delta Proteobacteria (Table 4). Microcosms also showed the presence of Alphaproteobacteria that were undetected in sediment samples (Table 5). On the other
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Fig. 1 Relative AWCD calculated from absorbance values of Biolog Ecoplate for the lake and microcosms in study. a Substrates utilization by sediment communities in lakes, b Substrates utilization in microcosm (Winter), c Substrates utilization in microcosm (Summer). The absorbance was measure after 96 h incubation. The substrate were divided in six categories; Carbohydrates (n = 10), Amines (n = 2), amino acids (n = 6), Carboxylic acids (n = 7), Polymers (n = 4), Phenolic compounds (n = 2)
hand, microcosms supplemented with 2,4-D showed an increase in Actinobacteria, although this genus was of low representation in the control microcosm and non detected in the sediment samples.
Discussion Cold oligothrophic lakes have been analyzed mainly from an ecological point of view (Mackenzie et al. 2011) but few reports describe the impact of anthropic contaminant upon their stability (Williamson et al. 1999). In this study, the sediment from the Patagonian cold lake Te´mpanos was evaluated in microcosm assays after been challenged with
World J Microbiol Biotechnol Table 3 Relative functional diversity and metabolic activity of bacterial communities in Patagonian lakes Functional diversity (H’)
Metabolic activity (AWCD)
Winter
Summer
Winter
Summer 1.22 ± 0.11
Te´mpanos lake
3.24 ± 0.06
3.32 ± 0.11
0.87 ± 0.06
Microcosm control
3.18 ± 0.08
2.99 ± 0.07
0.88 ± 0.08
0.95 ± 0.07
Microcosm 2,4-D
3.25 ± 0.09
3.27 ± 0.13
1.10 ± 0.09
0.75 ± 0.13
Microcosm simazine
3.18 ± 0.10
3.26 ± 0.12
1.02 ± 0.10
0.69 ± 0.12
The metabolic activity in the microcosm was determined after 20 days of incubation at 10 °C Mean ± SD H’ Shannon–Weaver diversity index, AWCD average wells color development
Denaturing Gradient (40-80%)
Fig. 2 Denaturing gradient gel electrophoresis of native bacterial communities in Sediment lake. Similarity between native bacterial communities was calculated, using Jaccard’s index. Lw = Te´mpanos lake Winter, Ls = Te´mpanos lake Summer. Acrilamide gel stain with SYBR Gold
two herbicides—Sz and 2,4-D—as a model. Our purpose was to evaluate how the bacterial community from a pristine aquatic environment responds to chemical perturbation. lake Te´mpanos exhibits short periods of surface freezing close to
Denaturing Gradient (40-80%)
Fig. 3 Denaturing gradient gel electrophoresis of bacterial communities in microcosm with toxic compounds. Similarity between bacterial communities in microcosm was calculated, using Jaccard’s index. Cw = Control microcosm (Winter), Cs = Control microcosm (Summer), SzW = Simazine microcosm (Winter), Simazine microcosm (Summer), 2,4-Dw = 2,4-D microcosm (Winter), 2,4Ds = 2,4-D microcosm (Summer). Acrilamide gel staining with silver nitrate
the coast. Physicochemical analysis showed pristine water and a mud texture with fine sediment material classified as medium mud sediment according the Wentworth (1922). Dissolved nitrogen and total phosphorus levels were \1.7 mgL-1, values typically found in oligothrophic lake systems (Woelfl et al. 2003; McGee et al. 2010; Rojas and Le Roux 2005). The increment of dissolved carbon in lake Te´mpanos during winter could be the consequence of allochthonous input because the region is characterized by strong raining periods (1,205.9 mm of rainfall per year) concentrated mainly during winter and spring. The low nitrogen, phosphorous and dissolved carbon level detected suggests that this lake should be considered as an ultraoligothrophic (Soto 2002; Balseiro et al. 2007). Organic-chloride pesticides were not detected in the samples, confirming the pristine quality of this water body. The low pH of water (average 4.0) could be explained by the volcanic activity of the Andes (Ribeiro Guevara et al. 2010; Romero 2012). Thus, the physico-chemical properties detected and the observed oligotrophic conditions make the lake Te´mpanos an adequate model for evaluating the stability of the bacterial communities against contamination with organic toxic compounds.
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World J Microbiol Biotechnol Fig. 4 Multi-dimensional scaling (MDS) plot of the assemblages bands from Te´mpanos lake (a) and microcosms (b). MDS analysis conducted on a Bray–Curtis analysis of similarity for bands. Contours represent the degree of similarity (%) between samples in each cluster. Cw = Control microcosm (Winter), Cs = Control microcosm (Summer), SzW = Simazine microcosm (Winter), Simazine microcosm (Summer), 2,4Dw = 2,4-D microcosm (Winter), 2,4-Ds = 2,4-D microcosm (Summer). Similarity index was evaluated for percentage
Sediment samples did not show significant differences in total bacterial counts between seasons, suggesting that this kind of lacustrine system does not modify the resident bacterial community along the year. Similar results have been reported by Jiang et al. (2006) for sediments of other oligotrophic lakes, suggesting that resident bacterial populations from this type of environments are stable. As estimated by epifluorescence microscopy 90 % of bacterial cells present in the sediment should be considered as dormant or dead bacteria (Buerger et al. 2012). On the other hand, the percentages of cultivable bacterial cells with respect to live cells in summer and winter samples oscillated 5.3–9.5 %, and these values are similar to those reported for English lakes by Porter et al. (2004). The small fraction of bacteria recovered in cultures should belong to facultative oligotrophic groups which are able to grow under a wide range of organic matter, as compared to strict oligotrophic groups (Mackenzie et al. 2011; Vasileska 2002). It is known that complex rich media inhibit the growth of oligotrophic bacteria (Giovanonni and Stingl
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2007; Hoppe et al. 1988), hence, the recovering of bacteria in culture from oligotrophic lakes depends on the selective media used (Porter et al. 2004). Metabolic analysis showed that the bacterial community of Te´mpanos lake sediment in summer and winter reaches the highest score value calculated by Biolog EcoplateTM system. This score was consistent with those observed in undisturbed environment by Gomez et al. (2006). Denaturing gradient gel electrophoresis analysis of the amplified V3 region from rDNA16S showed 80 % of similarity between samples obtained in winter and summer suggesting that bacterial structure and diversity did not change with seasons in this lacustrine system. The groups detected included cultivable and non cultivable bacteria belonging to the Beta, Gamma and Delta Proteobacteria. Similar results were also reported in pristine lacustrine systems (Imhoff 2006; Mackenzie et al. 2011). On the other hand, OTUs referred to from Alphaproteobacteria were detected in all microcosms while less number of OTUs of this group was present in sediment samples
World J Microbiol Biotechnol Table 4 Assignment of taxonomic groups to band sequences extracted from DGGE gel based on 180 bp and the closest sequence match of know phylogenetic affiliation in sediment samples Band
Closest sequences related
Taxonomic group
% Similarity
Samples
1
Uncultured bacterium clone ESBAC05B09
ND
100
Lw–Ls
2
Uncultured bacterium isolate DGGE band 5S2
ND
79
Lw–Ls
3
Uncultured bacterium clone MN82
ND
89
Lw–Ls
4
Uncultured bacterium clone MEB2_685
ND
78
Lw
5
Uncultured Klebsiella sp. clone S8
Gammaproteobacteria
97
Lw–Ls
6
Uncultured soil bacterium clone CRS5570T-1
ND
81
Lw–Ls
7 8
Uncultured bacterium clone 13C-HEW-092 Uncultured Herbaspirillum sp. clone AS84P1 16S
ND Betaproteobacteria
92 93
Lw–Ls Lw–Ls
9
Janthinobacterium sp.
Betaproteobacteria
89
Lw–Ls
10
Herminiimonas glaciei strain UMB49
Betaproteobacteria
89
Lw
11
Uncultured Acidobacteria bacterium clone KBS_T8
Acidobacteria
87
Lw–Ls
12
Silvimonas terrae strain KM-45 16S
Betaproteobacteria
91
Lw
13
Thiomonas sp. enrichment culture clone ‘DGGE gel band B2’
Betaproteobacteria
91
Ls
14
Methylibium fulvum strain Gsoil 322
Gammaproteobacteria
97
Lw–Ls
15
Pelomonas aquatica strain : CCUG 52575
Betaproteobacteria
93
Lw–Ls
16
Methylibium petroleiphilum PM1 strain PM1
Betaproteobacteria
87
Lw–Ls
17
Pelomonas puraquae strain : CCUG 52769
Betaproteobacteria
89
Lw–Ls
18
Uncultured bacterium clone MFBC4H11
ND
82
Lw–Ls
19
Pelobacter propionicus DSM 2379 strain
Deltaproteobacteria
93
Lw–Ls
20
Zymophilus raffinosivorans strain VTT E-90406
Firmicutes
91
Ls
21
Brachymonas denitrificans strain AS-P1
Betaproteobacteria
90
Ls
Samples obtained for sequencing analysis
suggesting that Alphaproteobacteria have low representation among the bacterial community in lake Te´mpanos. Not significant differences (P [ 0.05) in total and cultivable bacterial counts of microcosms were observed independently of the presence of 2,4-D or simazine. On the other hand, microcosms supplemented with Sz used less substrates offered by Biolog EcoplateTM system than those supplemented with 2,4-D, suggesting that Sz is more effective than 2,4-D as metabolic interfering compound of oligotrophic bacterial communities. Similar results have been observed by Ros et al. (2006) using molecules analogous to Sz. The addition of 2,4-D or Sz modifies the richness of substrates used, the relative functional diversity (H’) and the microbial activity (AWCD) of the microbial community but different responses were observed in microbial activity during summer and winter. MDS analysis of OTUs obtained in DGGE also suggests that genetic bacterial diversity did not change in summer and winter (80 % similarity), but microcosms supplemented with 2,4-D or Sz show differences with the control (40 % similarity). The ANOSIM analysis (P \ 0.05) confirmed this finding. The results support the idea that organic toxic compounds could modify the dynamic of biogeochemical process by decreasing the ability of the bacterial communities to use a particular carbon substrate as has also been suggested by Lesan and Bhandai (2003).
No degradation of Sz or 2,4-D was detected by spectrometric UV-analysis (data not shown) although an increase in the cultivable bacterial fraction was observed suggesting that the increment of recoverable bacterial population could be due to the presence of an intrinsically tolerant subpopulations among the sediment bacterial community of lake Te´mpanos. We speculate that these subpopulations should be able to grow in the presence of Sz or 2,4-D by using both lysed dead bacteria as carbon source (Pinchuk et al. 2008) or cellular endogenous substrates like poly-hydroxy-alkanoates (Godoy et al. 2003; Pavez et al. 2009). Genetic analysis of microcosms showed predominance of bacteria belonging to phyla Alpha, Beta and Gamma Proteobacteria, but in microcosms supplemented with 2,4D predominated the phylum Actinobacteria which was not detected in the sediments of lake Tempanos and was of low representation in the control microcosm. The change in the genetic diversity observed with the addition of pesticides to microcosms could be caused by some specific bacterial group—usually none detected in DGGE—that increase their proportion in the presence of the allocthonous organic compounds as described by Cottrell and Kirchman (2000). Interestingly, previous results at our laboratory (Mackenzie et al. 2011) also showed that Actinobacteria group was
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World J Microbiol Biotechnol Table 5 Assignment of taxonomic groups to band sequences extracted from DGGE gel based on 180 bp and the closest sequence match of know phylogenetic affiliation in Microcosms lake Band
Closest sequences related
Taxonomic group
% Similarity
Samples
1
Methylobacterium populi
Alphaproteobacteria
82
Cw
2
Duganella zoogloeoides
Betaproteobacteria
92
Cw
3
Uncultured alpha proteobacterium clone
Alphaproteobacteria
96
Cw
4
Methylobacterium populi
Alphaproteobacteria
96
Cw
5
Uncultured Curvibacter sp.
Betaproteobacteria
92
Cw
6
Uncultured bacterium clone
ND
95
Cw–Cs
7 8
Sphingomonas japonica Uncultured Lysobacter sp.
Alphaproteobacteria Gammaproteobacteria
97 75
Cw–Cs–2,4s, 2,4w Cs
9
Sphingomonas sp.
Alphaproteobacteria
97
Cs–Cw
10
Sphingomonadaceae bacterium
Alphaproteobacteria
97
Cs–Cw
11
Novosphingobium sp.
Alphaproteobacteria
91
Cs–Cw–Szs
12
Duganella sp.
Betaproteobacteria
98
Cs–Cw–Szs–2,4s
13
Uncultured beta proteobacterium clone A3-67
Betaproteobacteria
87
Cs–Szs–2,4w
14
Massilia timonae
Betaproteobacteria
92
Szw
15
Uncultured alpha proteobacterium clone B3-79
Alphaproteobacteria
89
Szw–Szs
16
Uncultured beta proteobacterium clone A3-67
Betaproteobacteria
95
Szw
17
Arthrobacter tecti
Actinobacteria
91
Szw–Szs
18
Uncultured bacterium clone 0300_02
ND
90
Szw
19
Uncultured bacterium clone Transfer_3C08
ND
93
Szw Szs
20
Magnetospirillum sp.
Alphaproteobacteria
95
21
Uncultured bacterium clone Bbsn10-02E6
ND
89
Szs–Szw–Cw–Cs–2,4s
22 23
Uncultured Gemmatimonadales bacterium clone Uncultured bacterium isolate DGGE gel band
Gemmatimonadetes ND
89 90
Szs–Cs–2,4s Szs–Szw–Cw–Cs–2,4s
24
Uncultured Alicycliphilus sp.
Betaproteobacteria
99
2,4s–Szs–Cw–Cs
25
Delftia acidovorans
Betaproteobacteria
94
2,4s–Cw–Cs
26
Xylophilus ampelinus
Gammaproteobacteria
89
2,4s–Szs–Cs
27
Bosea minatitlanensis
Alphaproteobacteria
89
2,4s–2.4w–Szs–Cs–Cw
28
Streptomyces sp.
Actinobacteria
92
2,4w–2,4s–Szs–Cs
29
Mycobacterium sp.
Actinobacteria
72
2,4w–2,4s–Szs–Szw–Cw–Cs
30
Uncultured actinobacterium clone
Actinobacteria
93
2,4w
31
Cellulosimicrobium cellulans
Actinobacteria
95
2,4w
32
Uncultured soil bacterium clone B18-3
ND
68
2,4w
33
Aeromicrobium fastidiosum
Actinobacteria
61
2,4w
34
Bosea minatitlanensis strain AMX51
Alphaproteobacteria
90
2,4s
Samples obtained for sequencing analysis
unusually detected in water samples from various other Patagonian lakes, confirming that this bacterial group is of low representation in pristine cold oligotrophic environments. Therefore, the changes in metabolic and genetic diversity observed in the presence of 2,4-D or Sz could be the consequence of a negative impact of this kind of compounds upon specific susceptible bacterial groups and, thus, they modify the bacterial diversity commonly found in this pristine lake. The results allow us to propose that the detection of an increased proportion of Actinobacteria group in a particular sample obtained from pristine lakes
123
could be indicative of homeostasis alteration as consequence of toxic organic matter contamination. Acknowledgments This work was supported by FONDECYT, Grant No. 1100462. Paulina Aguayo V. is a CONICYT Fellowship. The Authors thank to Miss Ruth Contreras for her technical support.
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