ISSN 0026-2617, Microbiology, 2018, Vol. 87, No. 1, pp. 66–78. © Pleiades Publishing, Ltd., 2018. Original Russian Text © T.A. Kanapatskiy, O.S. Samylina, A.O. Plotnikov, E.A. Selivanova, Yu.A. Khlopko, A.I. Kuznetsova, I.I. Rusanov, E.E. Zakharova, N.V. Pimenov, 2018, published in Mikrobiologiya, 2018, Vol. 87, No. 1, pp. 56–69.
EXPERIMENTAL ARTICLES
Microbial Processes of Organic Matter Production and Decomposition in Saline Rivers of the Lake Elton Area (Volgograd Oblast, Russia) T. A. Kanapatskiya, *, O. S. Samylinaa, A. O. Plotnikovb, c, E. A. Selivanovab, Yu. A. Khlopkob, A. I. Kuznetsovaa, I. I. Rusanova, E. E. Zakharovaa, and N. V. Pimenova aWinogradsky
Institute of Microbiology, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, Russia Institute for Cellular and Intracellular Symbiosis, Ural Branch, Russian Academy of Sciences, Orenburg, Russia c Orenburg State Medical University, Orenburg, Russia *e-mail:
[email protected]
b
Received June 26, 2017
Abstract—The rates of microbial processes and phylogenetic diversity of the microorganisms responsible for organic matter production and decomposition in the benthic communities and bottom sediments of the rivers Solyanka, Lantsug, Khara, Chernavka, and Bol’shaya Smorogda (Lake Elton area, Volgograd oblast, Russia) were studied. The biomass and primary production of cyano–bacterial communities varied significantly within the ranges of 20–903 mg Chl a/m2 and 0.2–21 mg C/(m2 h), respectively. Depending on the season, the share of anoxygenic CO2 fixation varied from 20% to the values comparable to the rate of oxygenic photosynthesis. The total heterotrophic activity of microbial communities determined as the rate of dark CO2 assimilation varied from 31 to 750 μmol/(dm3 day) in the mats and from 3 to 137 μmol/(dm3 day) in the sediments. The rates of sulfate reduction and hydrogenotrophic methanogenesis varied from 10 to 2621 μmol S/dm3 day) and from 1.5 to 323 nmol CH4/(dm3 day), respectively. High-throughput sequencing of the 16S rRNA genes in cyano–bacterial mats revealed microorganisms belonging to 20 phyla, with the sequences of Cyanobacteria, Proteobacteria, and Bacteroidetes being the most numerous. Keywords: primary production, sulfate reduction, cyano–bacterial mats, saline rivers, high-throughput sequencing, 16S rRNA gene DOI: 10.1134/S0026261718010095
Lake Elton is fed by seven rivers (Solyanka, Lantsug, Khara, Chernavka, Bol’shaya Smorogda, Malaya Smorogda, and Karantinka), which are typical water courses of the plain with asymmetrical valleys, meandering beds, and low flow rates (in summer, flow rates in the mouth reach do not exceed 0.2–0.4 m/s). These rivers are mainly fed by ground waters and atmospheric precipitation. The peak discharge occurs during the spring snow melting; in winter, the discharge is very low, and in the summer–autumn period it depends on the intensity of rains. The domination of saliferous and carbonate sedimentary rocks, solonetz and solonchak soils in the drainage area determines the level of water salinity in the rivers that inflow Elton. The salinities increase from the rivers’ headstream to the mouth. The total content of phosphorus and inorganic nitrogen (0.03–2.5 and 7.3–53.1 mg/L, respectively) is similar to that of hypertrophic-type waters (Nekrutkina, 2006). River mouth areas on Lake Elton form vast shallow basins with low water flow rates and elevated salinity due to admixture of the lake’s brine. These conditions
Lake Elton, the largest saline lake in Europe, is located in Volgograd oblast (Russia) close to its western border with Kazakhstan. These territories are characterized by saline dome tectonics generating saline domes and compensatory basins, one of which is occupied by Lake Elton. The Lake Elton area lies in the zone of extreme continental climate with a long summer period. Typical regional landscapes surrounding the lake are desert steppes. Under local climatic conditions, annual evaporation from the lake surface is 2–3 times higher than annual precipitation. The temperatures in the Lake Elton area exhibit a dramatic range of variation: from –36.1°C in January to 45°C in August. During the dry period of the year (August–September), it is only in the deeper northern part of the lake that any water remains, and salt precipitation occurs on the territories that dry out. The water of the lake is a brine, a saturated sodium/magnesium chloride solution, oily to touch, with salinities ranging from 200 to 863 g/L (Gusakov and Gagarin, 2012). 66
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(a)
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(b) L1 L2
Ch1 Ch2 Kh2
S1 S2
BS2
Fig. 1. Lake Elton and inflowing saline rivers: (a) map of the sample collection sites (S, Solyanka; L, Lantsug; Kh, Khara; Ch, Chernavka; BS, Bol’shaya Smorogda; 1, middle reach; 2, mouth reach); (b) growth of cyanobacterial mats in the Lantsug mouth reach in 2014.
are favorable for extensive development of benthic cyanobacterial communities (mats), which occurs during spring and autumn periods. Several previous studies have addressed ecological and hydrobiological issues concerning planktonic and benthic communities of the Lake Elton area (Zinchenko and Golovatyuk, 2010; Gusakov and Gagarin, 2012; Nomokonova et al., 2013). However, phylogenetic diversity of microorganisms that mediate production and degradation in mats and sediments, as well as the rates of microbial processes in these rivers have not been assessed so far. The goal of the present work was to investigate the geochemical activity and the phylogenetic diversity of prokaryotes of the benthic microbial communities of the rivers feeding Lake Elton. MATERIALS AND METHODS Biogeochemical and microbiological studies were performed at several stations located in the middle and mouth reach of the rivers Solyanka, Lantsug, Khara, Chernavka, and Bol’shaya Smorogda. The location of sampling sites is shown on Fig. 1a. Water and sediment samples were collected in August 2013, August 2014, and May 2015. Water salinity was measured with an ATAGO ATCS/Mill-E portable refractometer (Japan). Water alkalinity (Alk, mg-eq/L) was determined by titrimetry using a standard reagent set (Merck, Germany). Pore waters were collected by sediment centrifugation. Sulfate content was determined on a Stayer ion chromatograph (Russia); methane content was assessed using partition equilibrium analysis on a Kristall 2000 gas chromatograph with a flame ionization detector (Russia). The redox potential (Eh, mV) was measured using MICROBIOLOGY
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a pH320/Set-1 field potentiometer (WTW, Germany). Corg content was determined using a TOC Vcph automated analyzer with an SSM 5000A unit (Shimadzu, Japan). The carbon isotope composition in methane (δ13C) was determined using a Trace GC Ultra gas chromatograph (Тhermo Electron Corporation, Germany) coupled to a Delta plus mass spectrometer (ThermoFinnigan, Germany). The biomass of phototrophic communities was assessed by chlorophyll a content. For this purpose, three replicates of 4-cm2-large mat samples were collected. Pigments were extracted from the biomass with 80% acetone, and their total absorption spectra were determined using a Carry 100Bio spectrophotometer (Varian, United States). Calculations were performed using the standard formulas (Namsaraev, 2009). Primary production levels of phototrophic communities were determined directly on site by measuring NaH14CO3 fixation rates. For this purpose, biomass samples were collected and gently mixed to homogeneity with an automated pipette. In a penicillin vial, a 1-mL aliquot of the biomass homogenate was mixed with 3 mL water from the sample collection site, and supplemented with 150 μL NaH14CO3 solution (5 μCi per vial). The contribution of anoxygenic photosynthesis was determined in the vials supplemented with 50 μL of 3-(3',4'-dichlorophenyl)-1,1dimethylurea (diuron, an inhibitor of oxygenic photosynthesis) to the final concentration of 10–7 mM. The experiment included the following variants of incubation: on light, on light with diuron, and in the dark, two replicates for each variant. The vials were incubated in water under ambient conditions for 1 h, and then the samples were fixed with 0.5 mL formalin
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solution or with 0.5 mL of 2 M KOH. To determine the level of H14CO3– incorporation into the biomass, the samples were transferred onto 0.45-μm Millipore filters (United States) and washed with 10% NaCl (pH 2, adjusted with HCl). The filters with biomass samples were air-dried, immersed into 5 mL of Ultima Gold LLT scintillation liquid (Perkin Elmer, United States), and examined using a Packard TRI-Carb TR counter (United States). The productivity of communities was calculated in mg C per m2 per hour based on the area and thickness of the phototrophic part of the mat. The rates of dark carbon dioxide assimilation (DCA), methane oxidation, methanogenesis, and sulfate reduction in bottom sediments were determined using radiotracer analysis. A 3-mL sediment sample was placed into a cut-off 5-mL plastic syringe and sealed with a gas-tight butyl rubber plug. Following addition of 0.2 mL of the relevant labeled substrate through the stopper, the samples were incubated for 1–2 days and then fixed with 0.5 mL of 2 M KOH. Methane oxidation rates were determined using 14CH4 dissolved in gas-free distilled water (1 μCi per sample); sulfate reduction rates were measured using Na235SO4 (10 μCi per sample), and the rates of methanogenesis and DCA were measured using NaH14CO3 (10 μCi per sample). Alkali-fixed samples that stored at 4°C for 2 h before addition of the labeled substrate served as the controls. Further treatment of the samples was performed as described previously (Pimenov and Bonch-Osmolovskaya, 2006). The composition of prokaryotic communities was studied by high-throughput sequencing of the 16S rRNA gene fragments. For this purpose, 1-mL mat samples containing water and sediments were collected in sterile Eppendorf tubes in the mouth reach of the Chernavka and Bol’shaya Smorogda rivers. The cells were collected by centrifugation, and total DNA was isolated using a modified enzymatic lysis technique (Bel’kova, 2009), which included an additional step of incubation with lysozyme for efficient destruction of gram-positive bacterial cells. A negative control specimen of 100 μL deionized sterile water was treated along with other samples to rule out potential contamination. DNA purity was verified by photometry on a NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific, United States) and by electrophoresis in 1.5% agarose gels. DNA concentration was determined on a Quantus fluorimeter (Promega, United States) using the Quanti Fluor dsDNA kit (Promega). The variable V3–V4 fragment of the 16S rRNA gene was sequenced with the primers SD-Bact-0341-b-S-17 and S-D-Bact-0785-a-A-21 (Klindworth et al., 2013) using DNA material isolated from cyanobacterial mat samples from the rivers Chernavka and Bol’shaya Smorogda, which gave rise to the libraries nos. 36 and 38, respectively. Sequencing was performed using the MiSeq Reagent Kit V3 (Illumina, United States) on a MiSeq analyzer according to the pro-
tocol proposed by Illumina (http://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide15044223-b.pdf) in the Microbial Persistence Collective Use Center of the Institute of Cellular and Intracellular Symbiosis, Ural Branch, Russian Academy of Sciences. Bioinformatic analysis of the obtained 16S rRNA gene sequence fragments (reads) was performed with USEARCH v8.0.1623_win32 software (Edgar, 2010). The analysis involved fusion of paired reads, read selection by quality and length (minimal size, 300 bp), elimination of chimerical reads, doubletons and singletons, and read clustering into operational taxonomic units (OTUs) at the similarity level of 97%. Taxonomic classification of the OTUs was performed using the VAMPS interactive platform (Huse, 2014). Some OTUs were aligned against the nr database (GenBank) using the BLAST algorithm (http:// blast.ncbi.nlm.nih.gov/ Blast.cgi). Altogether, cyanobacterial mat samples from the rivers Chernavka and Bol’shaya Smorogda produced 156236 and 248043 prokaryotic 16S rRNA gene fragments clustered into 1176 and 816 OTUs, respectively. After removal of singletons and doubletons, the number of OTUs decreased to 915 and 604, respectively. The OTUs corresponding to chloroplast DNA were also removed from the libraries prior to analysis. To evaluate specific representation of higher taxa (classes of protobacteria and phyla of other microbial groups), representative sequences of dominating OTUs (amounting to at least 0.1% of the total read number) were selected from each library. The same sequences were used to construct a phylogenetic tree, which also included the sequences from other minor higher-rank taxa (phyla and classes of proteobacteria). If a representative sequence had at least 98% homology to a 16S rRNA gene sequence from a known cultivable microorganism, the cluster was classified as the relevant species; otherwise, an array of the 16S rRNA gene sequences from the most closely related microorganisms, both cultured and uncultured, was generated. Next, phylogenetic trees were constructed based on these samples using the maximum likelihood approach with Unipro UGENE software (Okonechnikov et al., 2012). The sequencing data obtained for DNA libraries of cyanobacterial mats from the rivers Chernavka (no. 36) and Bol’shaya Smorogda (no. 38) were deposited in the NCBI SRA database under accession numbers SRR5856409 and SRR5856408, respectively. RESULTS Physicochemical characteristics of the water samples collected at different sites are presented in Table 1. The water salinity in the rivers ranged from 5–16‰ (Lantsug and Bol’shaya Smorogda) to 29–33‰ (Solyanka and Chernavka) and increased from the river head to the mouth reach. Water samples from the MICROBIOLOGY
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Table 1. Physicochemical characteristics of the water collected at the sampling sites on the rivers of the Lake Elton area Total salinity, ‰ River
Alk, mg-eq/L
Eh, mV
Station August 2013
Solyanka
S1 S2 Lantsug L1 L2 Chernavka Ch1 Ch2 Bol’shaya Smorogda BS2 Khara Kh2
25 25 5 13.5 27 29.5 11 12.5
August 2014 27 29 6 16.5 29.4 31 16 20
May 2015 25 26 7 27 29 33 15 13
August 2014 4.6 4.5 5.6 3.4 8 5.6 4.7 2.1
May 2015 7.5 5.6 4.7 3.2 4.6 4 13 6
August 2014 80 9 ND 92 86 ND ND ND
ND stands for no data.
rivers Solyanka, Lantsug, and Chernavka had pH values close to neutral (7.3–7.9); water samples from the Bol’shaya Smorogda and Khara rivers, as well from the Lantsug mouth reach, were weakly alkaline (8– 8.5). Alkalinity of the water samples tested ranged from 2 to 13 mg-eq/L, and water temperature ranged from 24 to 32.5°C. Physicochemical characteristics of the bottom sediments are presented in Table 2. The topmost horizon of sediment samples was composed of oxidized yellowish gray warp sheet up to 0.8 cm thick. It was lying over a layer of strongly reduced sediments with Eh from –210 to –450 mV composed of pelitic or aleuropelitic silts with an admixture of fine sand. This horizon was 4 to 15 cm thick, depending on its location (flow rate, course width, etc.) and on the sample time (in May, its thickness was lower than in August). It was underlain by silted fine gray sand merging into clay. At stations S1, S2, and Kh2, the topmost 10-cm layer contained coarse organic debris. Frequently, the sediments had an odor of oil products and hydrogen sulfide. Organic carbon content in the sediments (Corg) exhibited a broad range of variation, from 0.2 to 5.9%, with a mean value of 0.82%. The alkalinity of pore waters of the bottom sediments ranged from 2.5 to 23 mg-eq/L and decreased with depth. The salinities were within the range of 7– 114‰. Pore waters were found to contain high sulfate concentrations, reaching 656 and 257 mmol/dm3 in the mouths of Lantsug and Khara, respectively. In most cases, high sulfate levels were observed in the 5– 10-cm horizon (on the average, approximately 20 mmol/dm3). Thus, sediments in the mouth reach of the rivers of the Lake Elton area had sulfate content levels comparable to those found in the upper sediments of highly productive marine coastal zones (7– 19 mmol/dm3) and in the sediments of hypersaline MICROBIOLOGY
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lakes with salinity 125–330‰ (118–1468 mmol/dm3) (Lein et al., 2002; Sorokin et al., 2012). Methane content in bottom sediments was heterogeneous and ranged from 1 to 1286 μmol/dm3. Low methane content (up to 60 μmol/dm3) was observed in the sediment samples from the mouth reach of the rivers Chernavka, Lantsug, Khara, and Bol’shaya Smorogda. These habitats were characterized with river bed broadening, washout of sand, and elevated salinity of pore waters (27–114‰). Aleuropelitic sediments found at the stations Ch1, S1, S2, and L1 (Table 2) exhibited methane content levels of over 100 μmol/dm3. The methane present in mouth reach sediments of the rivers Solyanka and Bol’shaya Smorogda had a carbon isotope composition of –61.2 and –88.2‰, respectively, which indicated its microbial origin. Biomass and production processes in benthic phototrophic communities. Benthic phototrophic communities of saline rivers of the Lake Elton area are cyanobacterial films and annual mats, which develop extensively at mouth reach sites characterized by small depths (1–5 cm), slow water flow rates, and large areas. Under these conditions, mats may cover up to 100% of sediment surfaces (Fig. 1b). The biomass and primary production (PP) of these communities varied strongly: 20–903 mg chlorophyll a per m2 and 0.2–21 mg C/(m2 h), respectively (Table 3). In spring (May), mat productivity was higher than in the end of summer (August). In August, the share of anoxygenic H14CO3− fixation, as measured with diuron addition, reached the levels comparable with the rate of oxygenic photosynthesis, while in May it was lower and did not exceed 20% of the oxygenic photosynthesis level. Dark CO2 assimilation. To assess the total heterotrophic activity of microbial communities, the rates of dark CO2 assimilation (DCA) were measured in mats and benthic sediments. In August 2013 and August 2014, DCA rates in the mats were 456–750 and 133–
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Table 2. Physicochemical characteristics of the sediments collected at the sampling sites on the rivers of the Lake Elton area
SO 24 − , mmol/dm3
CH4, Station
S1 S2
L1
L2
Kh2 Ch1
Ch2 BS2
Horizon, cm
Eh, mV
Alk, mg-eq/L
S, ‰
0–5 5–10 0–5 5–12 11–20 0–5 5–10 12–20 0–5 5–10 10–15 0–5 5–10 0–5 5–10 10–20 0–5 5–10 0–5 5–10 10–15
–210 –240 –350 –350 ND –250 –430 –350 –450 –420 ND –400 ND –270 –460 ND –280 –390 ND ND ND
8.5 4.5 11.5 5.5 6 8 6 8.5 14 11.5 9 23 10.5 22 3.5 7.5 11.5 6.5 12 10 2.5
26 25 27 27 25 7 7 6 30 40 114 26 57 26 26 15 27 20 27 53 60
μmol/dm3
Corg, %
August 2014
May 2015
August 2014
May 2015
August 2014
May 2015
592 246 108 200 ND 390 650 ND 24 11 10 3.5 3 310 1286 300 32 50 5.5 10 3
694 550 530.5 610 565 163 620 756 3 6 13 60 10 529 176 ND 6 24 1 1.8 3
1.4 61.3 0.2 ND ND ND ND ND 3.6 160 ND 152.5 257.1 0.3 3.4 ND 2.9 12.1 28.4 41.8 ND
0.5 12.9 4.5 6.6 1.1 28.6 34.6 24.3 34.7 39.4 656 68 107.8 0.3 19.3 ND 6 10.1 25 30.6 50
1.4 0.67 0.8 0.2 ND ND ND ND ND ND ND 0.76 0.37 5.88 1.9 ND 0.4 0.3 0.3 1.2 0.3
0.74 0.67 1.1 1.8 0.4 1.2 0.8 1.2 1.1 1 0.7 2.4 1.3 3.6 0.32 0.08 1 0.47 0.36 0.15 0.8
Designations: S, total salinity; Eh, redox potential; Alk, water alkalinity; ND, no data.
Table 3. Biomass and primary production of cyanobacterial communities in the rivers of the Lake Elton area
Station Kh2 L2
Ch2
BS2 S1 S2
Season August 2013 August 2013 August 2014 May 2015 August 2013 August 2014 May 2015 August 2014 May 2015 August 2014 May 2015 August 2014 May 2015
Biomass, mg Chl a per m2 697–829 23–37 60–194 743.5–903 20.4–24.8 101–138 138–205 78.7–89 146–220.5 252–348.5 53–246 296–595
Photosynthetic production, mg C/(m2 h) oxygenic
anoxygenic
12 0.7 1.1 9.5 0.2 3.5 Site dried out 14.6 21 11.1 17.6 0.7 5.2
7.3 0.5 1.5 0.65 0.3 2.9
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Table 4. Levels of dark CO2 assimilation (DCA) in mats and sediments of the rivers of the Lake Elton area DCA in the sediments, μmol/(dm3 day) (2 cm–7 cm–15 cm)
3
DCA in the mats, μmol/(dm day)
Station
August 2013
August 2014
ND ND 456 613 750
628 133 430 665 ND
S1 S2 L2 Ch2 Kh2
May 2015 39 31 32 ND 63
16–5–3.6 137–10–ND 32–9–3 133.5–27–18 64–13.5–ND
ND stands for no data.
665 μmol/(dm3 day), respectively, while in May 2015 they were the lowest and ranged from 31 to 63 μmol/(dm3 day). In underlying silts, DCA values were low and decreased with sediment depth (Table 4). The observed DCA values were similar to those in the silts of the Baltic Sea (2–47 μmol/(dm3 day) at the salinity 11‰), shallow bays (10–464 μmol/(dm3 day) at the salinity 2–5‰), and soda lakes of Mongolia (8–300 μmol/(dm3 day) at the salinity 3–390‰) (Sorokin et al., 2004; Pimenov et al., 2008, 2013). It was found that DCA levels were lower in spring than in August, which may be explained by the early stage of mat development and extensive growth of cyanobacteria acting as phototrophic edificators. The high DCA levels observed in August were due to extensive degradation of the accumulated biomass mediated by heterotrophic microorganisms. Table 5. Rates of sulfate reduction in the surface sediments of the rivers of the Lake Elton area, mmol S/dm3 day Station S1 S2 L2 Kh2 Ch1 Ch2 BS2
Horizon, cm August 2014 0–5 5–10 0–5 5–10 0–5 5–10 0–5 5–10 0–5 5–10 0–5 5–10 0–5 5–10
ND ND 0.08 0.6 0.039 1.25 ND ND 0.26 2.09 0.42 0.14 1.076 0.7
0.05 0.53 0.2 0.83 0.55 1.75 0.1 0.01 0.05 2.6 0.3 0.03 0.48 0.02
ND stands for no data. MICROBIOLOGY
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Terminal degradation processes. In sulfate-containing habitats, the principal process of organic matter degradation is sulfate reduction (SR), which dominates over methanogenesis. The rates of these processes were measured in the upper horizons (0–5 and 5–10 cm) of river sediments in the Lake Elton area. SR rates ranged from 39 to 2090 μmol S/(dm3 day) in August 2014 and from 10 to 2621 μmol S/(dm3 day) in May 2015 (Table 5). Because of the limited number of samples in which this parameter was studied, it was impossible to determine whether SR rates depended on the season, salinity, sediment depth, sulfate content, or organic matter concentration. In May 2015, the rates of hydrogenotrophic methanogenesis were expectedly lower than SR rates and ranged from 1.5 to 323 nmol CH4/(dm3 day) with a mean of 45 nmol CH4/(dm3 day). The lowest rates of methanogenesis were observed in sediments samples from the rivers Solyanka, Lantsug, and Chernavka: 1.5–22 nmol CH4/(dm3 day), while the highest were found in the upper 10-cm sediment layer in the Khara and the Bol’shaya Smorogda rivers: 40– 323 nmol/(dm3 day). These results agree with the previously published values of 20–570 nmol/(dm3 day) for habitats with the salinity 11–35‰ (Ivanov et al., 2001; Pimenov et al., 2003; Sorokin et al., 2004; Savvichev et al., 2005). Taxonomic composition of benthic prokaryotic communities was analyzed using high-throughput sequencing of the 16S rRNA gene fragments in mat samples collected in the mouth reach of the rivers Chernavka (Ch2) and Bol’shaya Smorogda (BS2). On the higher-taxon levels, these communities were similar in composition. In particular, sequences representing the domain Bacteria were absolutely predominating over those of Archaea (over 99.7% of the total read number). Archaeal sequences belonged to unidentified members of the phylum Euryarchaeota. The sample from Chernavka also contained methanogenic archaea of the class Methanomicrobia (Methanolobus sp.). In the libraries from the rivers Chernavka
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18(0.2) 17(0.2) 16(0.1) 15(0.6) 14(0.3) 13(0.3) 12(0.4) 11(0.4) 10(8.7)
20(1.8) 21(14.3)
1(6.7) 2(26.0)
20(4.8) 19(0.1) 11(0.2)
21(6.8) 1(26.5)
10(5.9) 9(1.8) 8(2.6)
9(3.5)
7(12.8)
2(8.9)
8(2.6) 6(2.5) 3(14.7)
7(15.8) 4(0.1)
6(1.0) (a)
5(0.5)
5(2.2)
3(26.5) (b)
Fig. 2. Specific abundance of predominant OTUs combined to phyla (for proteobacteria, to classes) in the DNA libraries from mat samples from the rivers Chernavka (a) and Bol’shaya Smorogda (b). Notation: 1, Cyanobacteria; 2, Alphaproteobacteria; 3, Gammaproteobacteria; 4, Betaproteobacteria; 5, Deltaproteobacteria; 6, other classes of Proteobacteria; 7, Bacteroidetes + Balneolaeota; 8, Verrucomicrobia; 9, Chloroflexi; 10, Planctomycetes; 11, Spirochaetes; 12, Candidatus Saccharibacteria; 13, Chlamydiae; 14, Deinococcus-Thermus; 15, Hydrogenedentes; 16, Parcubacteria; 17, Acidobacteria; 18, Actinobacteria; 19, Lentisphaerae; 20, OTUs that do not correspond to any known phylum; 21, OTUs whose share is less than 0.1%. The share of all reads is given in parentheses.
and Bol’shaya Smorogda, identified sequences belonging to the domain Bacteria represented 20 and 18 different phyla, respectively (Figs. 2, 3). The highest taxonomic diversity was observed for the bacterial phyla Proteobacteria, Planctomycetes, Bacteroidetes, Chloroflexi, Verrucomicrobia, Actinobacteria, and Cyanobacteria. Among proteobacteria, there were members of the classes Alpha-, Beta-, Gamma-, and Deltaproteobacteria. Bacterial sequences of the classes Alphaproteobacteria and Gammaproteobacteria and of the phyla Bacteroidetes, Balneolaeota, and Cyanobacteria were the most numerous in both libraries studied (Fig. 2, Table 6). Both communities also included members of the phyla Chlamydiae, Lentisphaerae, Spirochaetae, Ignavibacteria, Gemmatimonadetes, Firmicutes, Deinococcus-Thermus, Deferribacteres, Acidobacteria, Chlorobi, and Armatimonadetes (Fig. 2).
Filamentous cyanobacteria, which are the principal components forming the physical structure of the mat, were numerous and diverse (Fig. 3, Table 6). Cyanobacteria present in the mat sample from Chernavka (9 OTUs) included members of the genera Arthrospira and Phormidium (order Oscillatoriales), as well as Jaaginema sp. (order Synechococcales). In the library derived from mat sample from Bol’shaya Smorogda (16 OTUs), the most abundant sequences belonged to the genera Lyngbya and Phormidium (order Oscillatoriales), as well as Nodosilinea and Leptolyngbya (order Synechococcales). In the library from the Chernavka mat, a significant portion of the sequences of Alphaproteobacteria (over 30–50%) belonged to the recently described genus Aestuariispira of the order Rhodospirillales (Park, 2014) (over 10% of all reads), while in the
Fig. 3. Combined phylogenetic similarity cladogram of the 16S rRNA gene fragment sequences of predominant OTUs from the libraries of cyanobacterial mats of the rivers Chernavka and Bol’shaya Smorogda. The clusters corresponding to phyla (for proteobacteria, to classes) are separated by a vertical line, with the phylum name given beside. Notation: s, branches with bootstrap support of ≥90% (1000 replicates); d, branches with bootstrap support of
