ISSN 0026-2617, Microbiology, 2016, Vol. 85, No. 4, pp. 471–480. © Pleiades Publishing, Ltd., 2016. Original Russian Text © E.N. Frolov, A.Y. Merkel, N.V. Pimenov, A.A. Khvashchevskaya, E.A. Bonch-Osmolovskaya, N.A. Chernykh, 2016, published in Mikrobiologiya, 2016, Vol. 85, No. 4, pp. 446–457.
EXPERIMENTAL ARTICLES
Sulfate Reduction and Inorganic Carbon Assimilation in Acidic Thermal Springs of the Kamchatka Peninsula E. N. Frolova, 1, A. Y. Merkela, N. V. Pimenova, A. A. Khvashchevskayab, E. A. Bonch-Osmolovskayaa, and N. A. Chernykha aWinogradsky
Institute of Microbiology, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, Russia “Water” Production Centre, Institute of Natural Resources, Tomsk Polytechnic University, Tomsk, Russia 1e-mail:
[email protected]
b
Received December 21, 2015
Abstract—Thermoacidophilic sulfate reduction, which remains a poorly studied process, was investigated in the present work. Radioisotope analysis with 35S-labeled sulfate was used to determine the rates of dissimilatory sulfate reduction in acidic thermal springs of Kamchatka, Russia. Sulfate reduction rates were found to vary from 0.054 to 12.9 nmol SO4/(cm3 day). The Oil Site spring (Uzon caldera, 60°C, pH 4.2) and Oreshek spring (Mutnovskii volcano, 91°C, pH 3.5) exhibited the highest activity of sulfate-reducing prokaryotes. Stable enrichment cultures reducing sulfate at pH and temperature values close to the environmental ones were obtained from these springs. Analysis of the 16S rRNA gene sequences revealed that a chemolithoautotrophic bacterium Thermodesulfobium sp. 3127-1 was responsible for sulfate reduction in the enrichment from the Oil Site spring. A chemoorganoheterotrophic archaeon Vulcanisaeta sp. 3102-1 (phylum Crenarchaeota) was identified in the enrichment from Oreshek spring. Thus, dissimilatory sulfate reduction under thermoacidophilic conditions was demonstrated and the agents responsible for this process were revealed. Keywords: dissimilatory sulfate reduction, thermoacidophilic environments, sulfate-reducing prokaryotes, radioisotope techniques DOI: 10.1134/S0026261716040068
In the course of dissimilatory sulfate reduction, sulfate, which acts as the terminal electron acceptor, is reduced to sulfide. This process involves various organic compounds, usually of low molecular mass, as well as hydrogen, as electron donors; all of them are produced at initial stages of organic matter (OM) degradation by anaerobic microbial communities (Pimenov et al., 2014). For this reason, sulfate-reducing prokaryotes (SRP) not only play a key role in the sulfur cycle, but are also an indispensable component of the global carbon cycle. The vast variety of currently known SRP includes species belonging to five phylogenetic lineages of bacteria (Deltaproteobacteria, Clostridia, Thermodesulfobiaceae, Nitrospiriae, and Thermodesulfobacteria), and two phyla of archaea: Euryarchaeota and Crenarchaeota (Muyzer and Stams, 2008). SRP occur in diverse anaerobic environments that vary strongly in their physicochemical characteristics (Muyzer and Stams, 2008). Although the physiology and ecology of SRP attract considerable attention, there is currently hardly any information concerning dissimilatory sulfate reduction under thermoacidophilic conditions, which, however, could be very interesting, since such microorganisms are exposed to two
extreme environmental factors simultaneously: high temperatures and low pH values. While the existence of thermophilic sulfate reduction is beyond doubt, the possibility of acidophilic sulfate reduction has long been considered critically, based on the assumption that sulfate reduction is possible only at near-neutral pH values (Widdel, 1988; Hao et al., 1996; Koschorreck, 2008). Nevertheless, during the past decade, a substantial body of ecological data on sulfate reduction under acidic conditions has been accumulated; moreover, a number of mesophilic acidophilic sulfatereducing microorganisms have been isolated as pure cultures (Alazard et al., 2010; Sánchez-Andrea et al., 2014). There are few known thermoacidophilic SRP. According to available publications, among the presently known thermoacidophilic SRP are only three members of the phylum Crenarchaeota and one bacterial species of the phylum Firmicutes. The first species that was described as capable of sulfate reduction under thermoacidophilic conditions was Caldivirga maquilingensis, a hyperthermophilic archaeon growing at the temperatures of 60–92°C and in the pH range of 2.3–6.4, with an optimum at 85°C and pH of 3.7–4.2 (Itoh et al., 1999). Later, based on genomic
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Table 1. Sulfate reduction rates in acidic thermal springs studied
Spring name
Coordinates
Oreshek (no. 3102) 52°31.818′ N 158°11.499′ E 823 m no. 3105M 52°32.150′ N 158°11.653′ E 794 m no. 3106M 52°32.454′ N 158°11.083′ E 796 m no. 3105U 54°30.384′ N 160°00.087′ E 661 m no. 3112U 54°30.413′ N 160°00.043′ E 663 m Oil Site (no. 3127) 54°30.023′ N 160°00.088′ E 654 m
Temperature, °C
pH
Sulfate concentration, mg/L
Sulfate reduction rate, nmol SO4/ (cm3 day)
91
3.5
378
1.094
84
4.1
165
0.083
91
3.2
15
0.054
65
4.0
433
0.147
83
3.0
635
0.110
60
4.2
220
12.900
data, it was suggested that another hyperthermophilic archaeon, Thermoproteus tenax, may also be capable of growth by means of sulfate respiration at the temperatures of 55–96°C with an optimum at 86°C and in the pH range of 1.7–6.7 with an optimum at pH 5.6 (Zillig et al., 1981; Fischer et al., 1983; Siebers et al., 2011). Based on genomic analysis, it was also speculated that “Vulcanisaeta moutnovskia” 768-28 (Gumerov et al., 2011), which exhibits optimal growth at 85°C and pH 5‒5.5 (Prokofeva et al., 2005), might also be capable of sulfate reduction. However, there has been no experimental evidence of dissimilatory sulfate reduction activity in these microorganisms. Another thermoacidophilic species capable of sulfate reduction is Thermodesulfobium narugense of the phylum Firmicutes, which grows in the temperature range of 37– 65°C and the pH range of 4.0–6.5, with the optimum at 55°C and pH 5.5–6.0. This organism is a chemolithoautotroph oxidizing hydrogen and formate and reducing sulfate or nitrate (Mori et al., 2003). The goals of the present study were as follows: (1) to detect and investigate dissimilatory sulfate reduction and inorganic carbon assimilation in acidic thermal springs of Mutnovskii volcano and Uzon caldera; (2) to isolate the microorganisms responsible for this process; and (3) to characterize them. MATERIALS AND METHODS Sampling and measurements of the rates of sulfate reductions and inorganic carbon assimilation. The samples were collected in 2014‒2015 at the foot of
Mutnovskii volcano and in the Uzon caldera. Specimens from thermal springs were collected into 50-mL glass vials with gas-tight butyl rubber stoppers and aluminum screw caps, which were filled to capacity, hermetically sealed, and transported to the laboratory at ambient temperature. Sample characteristics are provided in Table 1. Sulfate reduction rates were measured by means of the radioisotope technique using 35S-sulfate. The rates of autotrophic CO2 fixation were determined using 14C-bicarbonate. Radioisotope experiments were performed in the laboratory 10‒14 days after sampling using 15-mL Hungate tubes with screw caps. Each tube contained a 6-mL specimen aliquot with 1 : 1 sediment to liquid phase ratio. The headspace was filled with 100% CO2, and 0.2 mL 35SO 24 − (37 × 10 4 Bq) or 14C-bicarbonate (37 × 10 4 Bq) were injected into each tube with a syringe through the stopper. The tubes were incubated in thermostats at temperatures corresponding to those of the springs for 3 days; after incubation, the samples were fixed 1 mL of 2 M NaOH. The subsequent treatment was performed as described previously (Pimenov and BonchOsmolovskaya, 2006). Analytical techniques. Sulfide formation was determined using the colorimetric method with N,Ndimethyl-para-phenylenediamine as proposed by Trüper and Schlegel (1964); the developing blue coloration was subsequently quantified by spectrophotometry at λ = 670 nm. Sulfate ion concentrations MICROBIOLOGY
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were determined on a Stayer ion chromatographer (Russia). Hydrogen concentrations were determined on a Chromatech Crystal 5000.1 chromatograph using a Hayesep N 80/100 column at the temperature of 40°C; argon was used as the carrier gas. Elements of the carbonate system (hydrocarbonate ion and free carbon dioxide) were determined by titrimetry. Hydrocarbonate ion was determined by titration with 0.01 N HCl solution in the presence of methyl orange, and free carbon dioxide was determined by titration with 0.1 N NaOH solution in the presence of phenolphthalein. Cations and anions (NH 4+, NO 2−, NO 3−, SO 24 −, Cl–, PO34−, Br–, Ca2+, Mg2+, Na+, K+, and Li+) were determined by ion chromatography using a two-channel reagent-free Тhermo Scientific Dionex ICS-5000 complex (United States) with a conductivity detector and a common autosampler. Anions were determined on an IonPac AS19 analytic column (2 × 250 mm) and an IonPac AG19 guard column (2 × 50 mm). Measurements were performed under the following operation conditions: eluent feed rate, 0.36 cm3/min; working pressure in the pump, no more than 2000 psi; cell temperature, 35°C; column temperature, 30°C. Potassium hydroxide solution produced with a concentration gradient was used as the eluent with complete peak separation within 45 min. Cations were determined in the isocratic elution mode on an IonPac CS 16 analytical column with complete peak separation within 30 min. The loading volume was 25 mL. The chromatograph was calibrated using the basic combined calibration solutions (Dionex, United States). Total organic carbon and soluble nitrogen were determined by means of high-temperature catalytic oxidation of carbon and nitrogen compounds to CO2 and NO, respectively, using a Liquid TOC analyzer (Elementar, Germany) with an infrared radiation detector. The analysis is based on two-step sample evaporation and degradation, with subsequent vapor oxidation on the catalytic layer. Oxidation was performed in the presence of oxygen at 850°C. Concentrations of chemical elements in water—Li, Be, B, Na, Mg, Al, Si, P, K, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, As, Se, Rb, Sr, Y, Zr, Nb, Mo, Ru, Rh, Pd, Ag, Cd, In, Sn, Sb, Te, Cs, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hf, Ta, W, Re, Os, Ir, Pt, Au, Hg, Tl, Pb, Bi, Th, and U—were determined using inductively coupled plasma mass spectrometry (ICP–MS). The ICP–MS technique involves the use of inductively coupled argon plasma as a source of ions and their separation and subsequent detection by mass spectrometry. Analysis was performed on a NexION 300D mass spectrometer (Perkin Elmer, United States) with the following operation parameters: plasma power, 1600 W; spraying argon flow, 0.86–0.9 L/min; orifice gas flow, MICROBIOLOGY
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16 L/min; auxiliary gas flow, 1.2 L/min. Mass spectrometry was performed in the standard and collision modes; in the latter case, helium flow was 2.8 mL/min. Polyatomic interference was suppressed by decreasing the kinetic energy of ions using the kinetic energy discrimination mode in the collision cell in the presence of an inert gas (helium). Element concentrations in the samples were calculated taking into account the detection limit characterizing the system in question and the calibration parameters of the mass spectrometer. All laboratory measurements were performed at 20 ± 5°C. Cultures of thermoacidophilic sulfate reducers. SRP enrichment cultures were obtained in liquid Pfennig medium (Pfennig, 1965) supplemented with vitamins (Kevbrin and Zavarzin, 1992), micronutrients (Wolin et al., 1963), and sodium sulfate (10 mM). Yeast extract (1 g/L), acetate (10 mM), lactate (10 mM), pyruvate (10 mM), ethanol (20 mM), methanol (20 mM), or H2 as a mixture with CO2 (1 : 1) were used as growth substrates; pH and the temperature of incubation corresponded to those of the sampling sites. The media were prepared using the Hungate anaerobic technique (Hungate, 1969). SRP growth was assessed by increasing hydrogen sulfide concentrations in comparison to control, as well as by light microscopy. Pure culture of strain 3127-1 was obtained on a solid medium with 1.5% agar in a CO2 : H2 (1 : 1) atmosphere. Colonies were transferred into liquid growth medium with a glass capillary tube in a CO2 atmosphere, and subsequent purification was carried out by tenfold dilutions. DNA isolation. Specimen aliquots of 2–4 mL were centrifuged at 14100 g 10 min at 4°C. The resulting pellet was resuspended in TNE buffer (20 mM Tris, 15 mM NaCl, 20 mM EDTA; pH 7.4) containing lysozyme (5 μg/mL) and RNase (200 μg/mL) and incubated at 37°C for 30 min. Next, proteinase K (5– 10 μg/mL) and SDS (0.5%) were added; the mixture was incubated at 54°C for 30 min, and cooled. After adding an equal volume of cold phenol–chloroform– isoamyl alcohol (50 : 50 : 1), the mixture was agitated for 10 min and then centrifuged for 10 min at 14100 g. The aqueous phase was transferred into a fresh tube, mixed with an equal volume of chloroform, and centrifuged for 5 min at 14100 g. This step was repeated twice. Finally, after addition of 0.1 volume of 3 M sodium citrate (pH 5.2) and two volumes of cold 96% ethanol, the aqueous phase was incubated at 20°C for 60 min; DNA was collected by centrifugation for 5 min at 14100 g, washed subsequently with 70 and 96% ethanol, air-dried, and dissolved in TE buffer (10 mM Tris, 1 mM EDTA; pH 7.4). DNA was visualized by electrophoresis in a 1% agarose gel. Polymerase chain reaction. Bacterial and archaean 16S rRNA genes were amplified using the following
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primers: U515F (5'-GTGBCAGCMGCCGCGGTAA-3', this forward primer was used for both bacteria and archaea), Bac907R (5'-CCGTCAATTCMTTTGAGTTT-3'; Muyzer et al., 1993), and Arch915R (5'GTGCTCCCCCGCCAATTCCT-3'; Casamayor et al., 2000). Thermoproteus uzoniensis was identified with the primers TuzF1068F (5'-GACCCCCACCCCTAGTTGCTTCCCCGCT-3') and Tuz1446R (5'-GAGTTCTCTGCTCGTCCCCCCACCCG-3'). For the purposes of DGGE, the U515F primer was modified with a 5'-GC-clamp (5′-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCG-3′). Amplification was performed in two steps, using normal primers at the first step, and primers with the GC-clamp at the second step. The PCR mixture (20 μL) contained the following components: 2 μL 10× buffer with 1.5 mM MgCl2 (Eurogen, Russia), 12.5 mM each dNTP (Eurogen), 20 pM each primer, 1.2 Е Taq DNA polymerase (Eurogen), and approximately 10 ng DNA. Amplification was performed in a single-channel thermal cycler (Perkin Elmer Cetus, United States). The PCR protocol was as follows: 5 min of initial denaturation for at 94°C; 33 cycles of 30 s denaturation at 94°C, 30 s primer annealing at 52°C (for bacterial primers), 62°C (for archaean primers), or 75°C (for DGGE-adapted primers), and 1 min chain elongation; 10 min of final elongation at 72°C. For PCR with TuzF1068F and Tuz1446R, the protocol was as follows: 3 min of initial denaturation, 95°C; 33 cycles of 20 s denaturation at 93°C, 10 s annealing at 68°C, and 30 s elongation at 72°C; 15 min final elongation at 72°C. Amplification of the reaction mixture containing no DNA served as a negative control. PCR products were visualized in a 1% agarose gel. DGGE analysis. PCR products were separated in an 8% acrylamide gel (vol/vol) with an acrylamide gradient from 35 to 70% in 0.5× TAE buffer, using 7 M urea (BioRad) and 40% formamide (Fluca) as denaturing agents. DGGE was performed in a SCIE-PLAS chamber (Great Britain) for 17 h at 70 V and 60°C. After electrophoresis, the gels were washed with distilled water and stained with SYBR® Gold (Molecular probes, Netherlands) for 40 min in the dark. Bands containing amplification products were visualized on a transilluminator, excised, and incubated in tubes containing 20 μL distilled water to elute the DNA. The products were then reamplified with the relevant primers, visualized in 1.5% agarose gel, and purified using a Wizard® SV Gel and PCR Clean-Up System (Promega, United States). Nucleotide sequences of the obtained PCR products were determined using a Big Dye Terminator kit v. 3.1 on an ABI 3730 automated DNA sequencer (Applied Biosystems Inc., United States) as proposed by the manufacturer. Phylogenetic analysis. Phylogenetic trees based on comparison of the 16S rRNA gene sequences were constructed using MEGA 6 software (Tamura et al.,
2013). The analysis was performed using the maximum likelihood method based on the Tamura–Nei model (Tamura and Nei, 1993). RESULTS Sampling sites. The numerous thermal springs of the Uzon caldera and the foot of Mutnovskii volcano vary significantly in size and physicochemical characteristics. Some of the springs function constantly for several years and even decades, while some others are transient. In this work, processes of thermoacidophilic sulfate reduction were investigated in three springs located at the foot of Mutnovskii volcano: 3102M (Oreshek), 3105M, and 3106M, and in three springs of the Uzon caldera: 3105U, 3112U, and 3127U (Oil Site). The lowest water temperature was 60°C, and the highest, 91°C; the minimal and the maximal pH values were 3.0 and 4.2, respectively. Sulfate content ranged from 15 to 635 mg/L (Table 1). The results of hydrochemical analysis of water from springs 3102M and 3127U are shown in Table 2. Rates of sulfate reduction and inorganic carbon assimilation. Using radioisotope analysis, we determined sulfate reduction rates in the springs, which varied from 0.054 to 12.9 nmol SO4/(cm3 day) (Table 1). Only in two springs the rates definitely indicated occurrence of sulfate reduction: Oreshek and Oil Site with 1.094 nmol SO4/(cm3 day) and 12.9 nmol SO4/(cm3 day), respectively. The rates of CO2 assimilation determined for the same springs were 7.56 nmol CO2/(mL day) for Oreshek and 1.44 nmol CO2/(mL day) for Oil Site. Cultivation of thermoacidophilic sulfate reducers. To identify the agents of dissimilatory sulfate reduction in Oreshek and Oil Site springs, we obtained and analyzed enrichment cultures of SRP. The stable microbial association isolated from Oil Site was capable of reducing sulfate under chemolithoautotrophic conditions in the presence of hydrogen as an electron donor, while cultivation with yeast extract, acetate, lactate, pyruvate, methanol, or ethanol was unsuccessful. DGGE analysis of the 16S rRNA genes retrieved from the enrichment culture showed that it comprised two bacterial components: a strain related to Thermodesulfobium narugense (Fig. 1) and another one related to Desulfurella acetivorans. Growth of the binary culture required strictly anoxic conditions and occurred only in the presence of sulfide as a reducing agent. A pure culture of Thermodesulfobium sp. 3127-1 was obtained by cultivation on a solid medium in the presence of hydrogen. After 2-week-long incubation at 60°C and pH 4.8, well-isolated red-brown colonies 2– 3 mm in diameter were formed. The colonies were transferred into liquid medium and further purified by tenfold dilutions. As a result of several sequential dilution passages, a pure culture was obtained. The isoMICROBIOLOGY
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Table 2. Data of a hydrochemical water analysis for the springs Oreshek (no. 3202M) and Oil Site (no. 3127U) Oreshek
CO2, mg/L
Oil Site
229
53
CO3, mg/L
Not found
Not found
HCO3−, mg/L
Not found
3
2−
378
220
Cl–, mg/L
1
188
Total hardness, mg eq./L
4.45
SO 4 , mg/L
Ca2+, mg/L 2+,
Mg Na
+,
1.98
53
25
22
mg/L
8.9
15
mg/L
K+, mg/L
147
5.2
Fetotal, mg/L
9.3 Not found
24.7
Mineralization, mg/L
506
633
Conductivity, μSm/cm
1340
1270
NH 4+ , mg/L
32
31.6
NO32 −, mg/L
