RESEARCH LETTER
Isolation and characterization of an obligately chemolithoautotrophic Halothiobacillus strain capable of growth on thiocyanate as an energy source Dimitry Y. Sorokin1,2, Ben Abbas2, Erik van Zessen3 & Gerard Muyzer4 1
Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia; 2Department of Biotechnology, Delft University of Technology, Delft, The Netherlands; 3Paques B.V., Balk, The Netherlands; and 4Department of Aquatic Microbiology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands
Correspondence: Dimitry Y. Sorokin, Winogradsky Institute of Microbiology, Russian Academy of Sciences, Prospect 60-let Octyabrya 7/2, 117312 Moscow, Russia. Tel.: 7 095 1350109; fax: 7 095 1356530; e-mails: [email protected]; [email protected] Received 26 January 2014; revised 12 March 2014; accepted 19 March 2014. Final version published online 11 April 2014. DOI: 10.1111/1574-6968.12432
MICROBIOLOGY LETTERS
Editor: Christiane Dahl Keywords thiocyanate; cyanate; cyanate pathway; halophilic; Halothiobacillus.
Abstract Molecular and microbiological analysis of a laboratory bioreactor biomass oxidizing thiocyanate at autotrophic conditions and at 1 M NaCl showed a domination of a single chemolithoautotrophic sulfur-oxidizing bacterium (SOB) capable of using thiocyanate as an energy source. The bacterium was isolated in pure cultures and identified as a member of the Halothiobacillus halophilus/hydrothermalis clade. This clade includes moderately halophilic chemolithoautotrophic SOB from marine and hypersaline habitats for which the ability to utilize thiocyanate as an electron donor has not been previously demonstrated. Halothiobacillus sp. strain SCN-R1 grew with thiocyanate as the sole energy and nitrogen source oxidizing it to sulfate and ammonium via the cyanate pathway. The pH range for thiocyanate oxidation was within a neutral region between 7 and 8 and the range of salinity was from 0.2 to 1.5 M NaCl, with an optimum at 0.5 M. Despite the close phylogenetic relatedness, none of the tested type strains and other isolates from the H. halophilus/hydrothermalis group exhibited thiocyanate-oxidizing capacity.
Introduction Thiocyanate (NC–S) is a unique inorganic sulfur compound produced naturally in the biological cyanide detoxification processes and as a waste product from cokes and precious metal factories (Wood, 1975; Kelly & Baker, 1990; Hung & Pavlostathis, 1997, 1999; Staib & Lant, 2007). It is a chemically stable compound and only a few bacterial species belonging to chemolithoautotrophic sulfur-oxidizing bacteria (SOB) are capable of utilizing thiocyanate as an energy source. Thiocyanate degradation needs the primary action of specific enzyme(s) to release the reduced sulfane atom for further oxidation. Accordingly, two possible pathways of microbial degradation of thiocyanate are proposed with either carbonyl sulfide (COS) or cyanate (CNO) as intermediate products (Kelly & Baker, 1990): Carbonyl sulfide pathway N≡C-S- + H2O
SCN-hydrolase
O=C=S + NH 3
H2S + CO2
COS-hydrolase
FEMS Microbiol Lett 354 (2014) 69–74
Cyanate pathway N≡C-S- + H2O
?
N≡C-O- + H2 S
NH3 + CO 2
cyanase
In the carbonyl sulfide pathway, the nitrile bond of thiocyanate is hydrolyzed with the formation of COS and ammonia by the enzyme thiocyanate hydrolase found in the chemochemolithoautotrophic betaproteobacterial SOB Thiobacillus thioparus (Katayama et al., 1998), the halophilic chemochemolithoautotrophic gammaproteobacterium Thiohalophilus thiocyanatoxydans (Bezsudnova et al., 2007; Sorokin et al., 2007) and a novel alphaproteobacterium strain THI201 (Hussain et al., 2013). Carbonyl sulfide is further hydrolyzed to hydrogen sulfide, which serves as the actual electron donor. Microbial carbonyl sulfide hydrolysis can be carried out either by a b-carbonic anhydrase homologue of CS2 hydrolase in a thermoacidiphilic archaeon Acidianus sp. (Smeulders et al., 2011) or a ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
D.Y. Sorokin et al.
70
specialized COS hydrolase from the b-carbonic anhydrase family in the betaproteobacterial SOB T. thioparus (Ogawa et al., 2013). Realistic evidences on the existence of an alternative cyanate pathway of primary thiocyanate degradation has, so far, only been obtained during investigation of thiocyanate metabolism in (halo)alkaliphilic SOB, such as Thioalkalivibrio thiocyanoxidans, Thioalkalivibrio paradoxus and Thiohalobacter thiocyanaticus, isolated from soda and salt lakes (Sorokin et al., 2001, 2002, 2010). The identity of the key enzyme in this pathway is still under investigation, but it is already clear that the mechanism is different from anaerobic hydrolysis of the C–S bond as proposed previously (Grant & Sorokin, 2011). The chemochemolithoautotrophic SOB belonging to Halothiobacillus halophilus/hydrothermalis group are dominant culturable forms of aerobic moderately halophilic SOB in hypersaline habitats (Wood & Kelly, 1991; Durand et al., 1993; Kelly & Wood, 2000). Despite this fact, our enrichments from hypersaline lakes with thiocyanate as the sole substrate at moderate salinity never resulted in domination of Halothiobacillus (Sorokin, 2008). In contrast, microbiological analysis of a lab-scale bioreactor community degrading thiocyanate at 1 M NaCl demonstrated that the main organism responsible for thiocyanate oxidation was a representative of the H. halophilus/hydrothermalis cluster. Its isolation and properties are described below.
Materials and methods Isolation source
A 2-L bioreactor working as a chemostat was originally inoculated with a haloalkaliphilic sulfide-oxidizing biomass from the Thiopaq bioreactor in Eerbeek (The Netherlands) with some addition from a salt lake sediment and sludge from an aerobic communal waste water treatment plant (Heerenveen). It was operated at 35 °C, pH 7.9–8.1 and 1 M NaCl with a mixed feed of 20 mM thiosulfate and 5 mM thiocyanate at a dilution rate 0.05 h1. Addition of thiosulfate as a co-substrate increased the thiocyanate removal activity. At the time of biomass sampling the culture supernatant contained a high concentra tion of inorganic N (37 mM NHþ 4 and 7 mM CNO ) which indicated (1) the use of cyanate pathway in thiocyanate degradation and (2) a significant imbalance between the catabolism and biomass growth.
containing 1 M NaCl, 50 mM potassium phosphate buffer (pH 8), 1 mM MgCl2, and 1 mL L1 of trace metals solution (Pfennig & Lippert, 1966) and supplemented with 10–20 mM potassium thiocyanate as a single energy and N-source. The isolation strategy consisted of serial dilution to extinction of the reactor biomass, followed by plating of the maximal positive dilution on solid medium with the same composition made by 1 : 1 mixing of double-strength liquid media and 3% (w/v) washed agar (three successive washes in 50 volumes of distilled water with stirring during 5 h) at 30 °C. It needs to be specifically stressed that the agar-washing step is important in this particular type of isolation because many heterotrophic bacteria can form colonies on mineral agar media due to a presence of small amount of organic contaminants in the agar. Dominating colony types were placed into liquid medium. If the isolates grew in the liquid mineral medium containing thiocyanate as the only energy source, the plating was repeated several times until a single colony type was selected, which would grow with thiocyanate as a the sole energy and nitrogen source. Metabolic profiles of the reactor biomass and pure cultures
The metabolic activity of the reactor biomass and the isolates was investigated by two different ways, either by direct measurements of substrate conversion or by measuring substrate-dependent respiration rates with dO2 electrode, as described previously (Sorokin et al., 2001, 2010). The pH dependence was examined at 0.5–1.0 M total Na+, using the following buffers: for pH 5–8, 0.1 M HEPES/NaCl titrated by 1 M NaHCO3 for pH 8–10, a mixture of sodium bicarbonate/sodium carbonate/NaCl. Influence of NaCl was checked in HEPES buffer at pH 7. All buffers contained 10 mM K+ and 1 mM Mg2+. Analytical procedures
Thiocyanate, cyanate, ammonium and cell protein were analyzed spectrophotometrically as described previously (Sorokin et al., 2001). The cyanase activity was measured in sonacated cell-free extract in 0.1 M NaHCO3 (pH 8) with 5 mM cyanate as substrate. The activity was estimated from the amount of ammonia released in 0.5–2 h incubation time. Molecular and phylogenetic analyses
Enrichment, isolation and cultivation of pure cultures of halophilic SOB
Thiocyanate-oxidizing halophilic SOB were grown from the reactor biomass in a liquid mineral medium ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Genomic DNA was extracted from the cell pellet using the UltraClean Soil DNA Extraction Kit (MoBio Laboratories), following the manufacturer’s instructions. For the pure cultures, the nearly complete 16S rRNA gene was FEMS Microbiol Lett 354 (2014) 69–74
Utilization of thiocyanate as energy source by Halothiobacillus
3.0
Concentrations (mM)
obtained using general bacterial primers GM3f (50 -AGAG TTTGATCCTGGCTCAG-30 ) and GM4r (50 -TACGGTTACCTTGTTACGACTT-30 ). For the DGGE analysis, partial amplification with a primer pair 341F + GC/907R was used (Sch€afer & Muyzer, 2001). DGGE was performed using a denaturing gradient of 20–70% denaturants in 8% polyacrylamide gel. Individual bands were excised, re-amplified, and run again on a denaturing gradient gel to check their purity. PCR products for sequencing were purified using the Qiaquick PCR purification kit (QIAGEN, the Netherlands). The sequences were first compared with sequences stored in GenBank using the BLAST algorithm. Subsequently, the sequences were imported into the ARB software program, automatically aligned, and added to a phylogenetic tree using the Quick-add tool. Sub-trees were then built using the neighbour-joining algorithm with automatic selected correction settings. The 16S rRNA gene sequences of strain SCN-R1 and of the dominant DGGE band were deposited in the GenBank with the accession numbers KC662326 and KC759700, respectively.
71
Degraded SCN–
NH3
CNO–
2.5 2.0 1.5 1.0 0.5 0.0 0
5
10
15
20
Thiosulfate added (mM) Fig. 1. Influence of thiosulfate on thiocyanate degradation by the reactor biomass at pH 8.5 (1 M NaHCO3). Incubation time: 48 h; biomass: OD590 = 0.50; thiocyanate = 5 mM. Of 20 mM thiosulfate was oxidized completely after 13 h of incubation. Mean values from a duplicate experiment with maximum deviation of 15%.
The NaCl range for thiocyanate-oxidizing activity of the reactor biomass was between 0.2 and 1.2 M, while thiosulfate oxidation continued up to 3 M. DGGE analysis of reactor biomass
Potassium thiocyanate, sodium thiosulfate and sodium sulfide (analytical grade) were obtained from SigmaAldrich.
Results and discussion Catalytic properties of the bioreactor biomass
Cells taken directly from the bioreactor degraded thiocyanate only in the presence of thiosulfate. Cyanate was found as a product of thiocyanate degradation (Fig. 1). Concomitant with thiocyanate degradation in the presence of thiosulfate, biomass growth and thiosulfate oxidation was observed. Extensive washing of the cells in a N-free buffer clearly unblocked the thiosulfate-independent thiocyanate oxidation capacity of the cells (Fig. S1). The fact that the bioreactor cells, when placed into an N-free buffer, continued to excrete ammonium during several days, and that external ammonium or cyanate additions did not influence the ability of cells to metabolize thiocyanate, indicated that the thiocyanate biodegradation resulted in unbalanced cell-associated accumulation of inorganic N inhibiting further thiocyanate degradation. Addition of a nitrogen-free energy co-substrate (thiosulfate) seemed to facilitate inorganic N consumption with increasing biomass growth and lifting the inhibitory effect. The same result was achieved by extensive washing of the cells. FEMS Microbiol Lett 354 (2014) 69–74
Molecular analysis of the reactor biomass showed a low genetic diversity with a dominant band affiliated to members of chemolithoautotrophic SOB from the H. halophilus/hydrothermalis clade (Fig. 2). The other detected organisms, such as the two different bacteroidetes and a Nitratireductor, apparently belong to heterotrophic part of the reactor population probably utilizing the organic 40% Uncutured Bacteroidetes AB297398 (96%)
gradient
Chemicals
Halothiobacillus halophilus U58020 (98-99%)
Nitratireductor sp. BJGMM-B15 JQ716222 (99%)
Uncutured Bacteroidetes AB297398 (95%)
70%
Fig. 2. DGGE analysis of the biomass from a thiocyanate-removing lab-scale bioreactor. The gradient was from 30% to 70% and all bands were separate within 40–70% region.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
D.Y. Sorokin et al.
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Serial dilution of washed cells from the reactor in a mineral medium containing 1 M NaCl and 10 mM thiocyanate as a single energy and N source resulted in growth up to a dilution of 108. Plating on the same medium from the maximal positive dilution yielded three different types of colonies. Only one of them designated as strain SCN-R1 (the strain is deposited in the NCCB collection in Utrecht under the number 100459), produced stable growth back on liquid mineral medium with thiocyanate, while the other two only grew when thiocyanate was used as the N-source, in combination with either thiosulfate (identified as a member of the Thiomicrospira pelophila clade) or acetate (identified as a member of the genus Halomonas). Strain SCN-R1, while growing with thiocyanate or thiosulfate as electron donor, had rod-shaped, non-motile cells assembled in chains (Fig. S2). According to the phylogenetic analysis, the isolate belonged to the H. halophilus/hydrothermalis group (Fig. 3) and its 16S rRNA gene sequence was 100% identical to the sequence of the dominant DGGE band from the reactor biomass (see Fig. 2). This indicated that the isolate SCN-R1 was the dominant chemochemolithoautotrophic thiocyanate-oxidizing SOB in the bioreactor. On the other hand, neither the type strains of H. halophilus and H. hydrothermalis nor our halophilic SOB isolates from hypersaline lakes belonging to the same group (Halothiobacillus sp. strains HL1, HL2,
20 18 16 14 12 10 8 6 4 2 0
A-SCN
B-SCN
C-SCN
D-SCN
A-growth
B-growth
C-growth
D-growth
0.3 0.25 0.2 0.15 0.1
Growth, OD590
Isolation and characterization of the dominant halophilic thiocyanate oxidizer
HL6, HL7, HL20 and HL27; Sorokin, 2008) were able to utilize thiocyanate as the enrgy source. Similar to the reactor biomass, growth and concomitant thiocyanate utilization by strain SCN-R1 was not straightforward. After an initial increase of biomass, the growth and thiocyanate consumption stopped and resumed only after the cells were collected and thoroughly washed with an N-free buffer. On the other hand, the addition of thiosulfate only slightly relieved the inhibition in case of the pure culture grown on thiocyanate (Fig. 4). Cyanate was the main nitrogen product of thiocyanate degradation detected in the medium, and the complete oxidation of 20 mM thiocyanate resulted in the release of
SCN–, mM
compounds produced by the dominant chemolithioautotrophic SOB.
0.05 0
5
10
15
20
0 25
Time, h Fig. 4. Growth of strain SCN-R1 with 20 mM thiocyanate at pH 8 and 1 M NaCl. A, Source control culture; B, cells collected after 5 h, washed in 1 M sterile NaCl solution and put back into the same medium; C, cells collected after 5 h, washed twice in 1 M NaCl and put into fresh medium; D, A + 20 mM thiosulfate. Mean values from a duplicate experiment with maximum deviation of 12%.
Fig. 3. Phylogenetic tree based on 16S rRNA gene sequencing, showing the position of the halophilic thiocyanate-oxidizing halophilic SOB isolated from the bioreactor. The tree includes sequences from the gammaproteobacterial (halo)alkaliphilic SOB species from the genus Halothiobacillus and those capable of thiocyanate utilization as the electron donor. Scale bar represent 10% sequence divergence.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Lett 354 (2014) 69–74
Utilization of thiocyanate as energy source by Halothiobacillus
100 (b)
(a)
100 90
90
80
80
Activity, % of maximum
Activity, % of maximum
73
70 60 50 40 30 20
70 60 50 40 30 20 10
10 0 0
0.5
1
1.5
2
2.5
3
3.5
0 6
6.5
7
7.5
NaCl, M
8
8.5
9
9.5
pH
Fig. 5. Influence of pH (at 1 M NaCl) and salt (at pH 8) on the activity of thiocyanate (closed circles) and sulfide (open circles) oxidation by washed cells of strain SCN-R1 pre-grown on thiocyanate. The data were obtained in a single experiment.
2.5 mM NH3 and 9.0 mM cyanate into the culture supernatant. Hence, similar to the source bioreactor biomass, Halothiobacilluis sp. SCN-R1 apparently uses the cyanate pathway for thiocyanate degradation. The results of substrate-dependent respiration tests with cells grown either with thiocyanate or with thiosulfate as an energy source showed that the thiocyanatedegrading system in strain SCN-R1 was inducible [VO2 = 170 nmol (mg protein min)1], and was absent in the cells grown with thiosulfate, similar to other thiocyanate-oxidizing (halo)alkaliphilic SOB described previously (Sorokin et al., 2001, 2007, 2010). However, in contrast to alkaliphilic SOB, cyanase activity, albeit low [35 nmol (mg protein min)1] was present only in thiocyanate-growing cells of strain SCN-R1. A salt response of strain SCN-R1 showed moderate tolerance with an optimum at 0.5 M NaCl and the cells being still active up to 2 M NaCl with thiocyanate and up to 3 M NaCl with sulfide (Fig. 5a). The thiocyanate oxidation had a relatively narrow pH profile in comparison with the sulfide-oxidizing activity, with a sharp decrease below pH 7 and above pH 8 (Fig. 5b). Summarizing the results, we were able to demonstrate that the dominant organism responsible for thiocyanate oxidation in a lab-scale bioreactor operating at 1 M NaCl is a representative of the chemochemolithoautotrophic halophilic SOB of the genus Halothiobacillus. This organism was isolated in pure culture and proven to be capable of chemolithoautotrophic growth with thiocyanate as energy and nitrogen source degrading it via the cyanate pathway. Such potential has never been reported before for members of the genus Halothiobacillus. The microbial oxidation of thiocyanate at high salt concentration might be useful for treatment of saline thiocyanate-containing wastewater generated by, for example, gold cyanidation plants (Stott et al., 2001). However, since the catabolism FEMS Microbiol Lett 354 (2014) 69–74
of thiocyanate by autotrophic SOB generates much more inorganic N than needed for biomass synthesis, a N-free co-substrate or an N-utilizing partner would be necessary for the complete bioremoval of thiocyanate.
Acknowledgements This work was supported by Shell International and Paques B.V. (The Netherlands). Gerard Muyzer was financially supported by the ERC Advanced Grant 322551.
References Bezsudnova EY, Sorokin DY, Tichonova TV & Popov VO (2007) Thiocyanate hydrolase, the primary enzyme initiating thiocyanate degradation in the novel obligately chemolithoautotrophic halophilic sulfur-oxidizing bacterium Thiohalophilus thiocyanatoxidans. Biochim Biophys Acta 1774: 1563–1570. Durand P, Reysenbach A-L, Prieur D & Pace N (1993) Isolation and characterization of Thiobacillus hydrothermalis sp. nov., a mesophilic obligately chemolithotrophic bacterium isolated from a deep-sea hydrothermal vent in Fiji Basin. Arch Microbiol 159: 39–44. Grant WD & Sorokin DY (2011) Distribution and diversity of soda lake alkaliphiles. Extremophiles Handbook, Vol. 1 (Horikoshi K, Antranikian G, Bull AT, Robb FT & Stetter KO, eds), pp. 27–54. Springer, Tokyo. Hung C-H & Pavlostathis S (1997) Aerobic biodegradation of thiocyanate. Water Res 31: 2761–2770. Hung C-H & Pavlostathis S (1999) Kinetics and modelling of autotrophic thiocyanate biodegradation. Biotechnol Bioeng 62: 1–11. Hussain A, Ogawa T, Saito M, Sekine T, Nameki M, Matsushita Y, Hayashi T & Katayama Y (2013) Cloning and expression of a gene encoding a novel thermostable
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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thiocyanate-degrading enzyme from a mesophilic alphaproteobacteria strain THI201. Microbiology 59: 2294– 2302. Katayama Y, Matsushita Y, Kaneko M, Kondo M, Mizuno T & Nyunoya H (1998) Cloning of genes coding for the subunits of thiocyanate hydrolase of Thiobacillus thioparus THI 115 and their evolutionary relationships to nitrile hydratase. J Bacteriol 180: 2583–2589. Kelly DP & Baker SC (1990) The organosulfur cycle: aerobic and anaerobic processes leading to turnover of C1-sulfur compounds. FEMS Microbiol Rev 87: 241–246. Kelly DP & Wood AP (2000) Reclassification of some species of Thiobacillus to the newly designated genera Acidithiobacillus gen. nov., Halothiobacillus gen. nov. and Thermithiobacillus gen. nov. Int J Syst Evol Microbiol 50: 511–516. Ogawa T, Noguchi K, Saito M et al. (2013) Carbonyl sulfide hydrolase from Thiobacillus thioparus strain THI115 is one of the b-carbonic anhydrase family enzymes. J Am Chem Soc 135: 3818–3825. € Pfennig N & Lippert KD (1966) Uber das Vitamin B12-Bed€ urfnis phototropher Schwefelbakterien. Arch Mikrobiol 55: 245–256. Sch€afer H & Muyzer G (2001) Denaturing gradient gel electrophoresis in marine microbial ecology. Methods in Microbiology (Paul JH, ed.), pp. 425–468. Academic, New York, NY. Smeulders MJ, Barends TRM, Pol A et al. (2011) Evolution of a new enzyme for carbon disulphide conversion by an acidothermophilic archaeon. Nature 478: 412–416. Sorokin DY (2008) Diversity of halophilic sulfur-oxidizing bacteria in hypersaline habitats. Microbial Sulfur Metabolism (Dahl C & Friedrich CG, eds), pp. 225–237. Proceedings of the International Symposium on Microbial Sulfur Metabolism 29.06.–02.07.2006. Springer-Berlin, M€ unster, Germany. Sorokin DY, Tourova TP, Lysenko AM & Kuenen JG (2001) Microbial thiocyanate utilization under highly alkaline conditions. Appl Environ Microbiol 67: 528–538. Sorokin DY, Tourova TP, Lysenko AM, Mityushina LL & Kuenen JG (2002) Thioalkalivibrio thiocyanooxidans sp. nov. and Thioalkalivibrio paradoxus sp. nov., novel alkaliphilic, obligately autotrophic, sulfur-oxidizing bacteria from the
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
soda lakes able to grow with thiocyanate. Int J Syst Evol Microbiol 52: 657–664. Sorokin DY, Tourova TP, Bezsoudnova EY, Pol A & Muyzer G (2007) Denitrification in a binary culture and thiocyanate metabolism in Thiohalophilus thiocyanatoxidans gen. nov. sp. nov. – a moderately halophilic chemolithoautotrophic sulfur-oxidizing Gammaproteobacterium from hypersaline lakes. Arch Microbiol 187: 441–450. Sorokin DY, Kovaleva OL, Tourova TP & Muyzer G (2010) Thiohalobacter thiocyanaticus gen. nov., sp. nov., a moderately halophilic, sulfur-oxidizing gammaproteobacterium from hypersaline lakes that utilizes thiocyanate. Int J Syst Evol Microbiol 60: 444–450. Staib C & Lant P (2007) Thiocyanate degradation during activated sludge treatment of coke-ovens wastewater. Biochem Eng J 34: 122–130. Stott MB, Franzmann PD, Zappia LR, Watling HR, Quan LP, Clark BJ, Houchin MR, Miller PC & Williams TL (2001) Thiocyanate removal from saline CIP process water by a rotating biological contactor, with reuse of the water for bioleaching. Hydrometallurgy 62: 93–105. Wood JL (1975) Biochemistry. Chemistry and Biochemistry of Thiocyanic Acid and its Derivatives (Newman AA, ed.), pp. 156–221. Academic Press, New York. Wood AP & Kelly DP (1991) Isolation and characterisation of Thiobacillus halophilus sp. nov., a sulphur-oxidizing autotrophic eubacterium from a Western Australian hypersaline lake. Arch Microbiol 156: 277–280.
Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. Influence of bioreactor cell washing on the activity of SCN-degradation in presence and absence of thiosulfate at pH 8 and 0.6 M NaCl. Fig. S2. Cell morphology (phase-contrast microscopy) of the reactor biomass (a) and of the pure culture of thiocyanate-oxidizing Halothiobacillus strain SCN-R1 growing either on thiocyanate (b) or thiosulfate (c).
FEMS Microbiol Lett 354 (2014) 69–74
Isolation and characterization of an obligately chemolithoautotrophic Halothiobacillus strain capable of growth on thiocyanate as an energy source Dimitry Y. Sorokin1,2, Ben Abbas2, Erik van Zessen3 & Gerard Muyzer4 1
Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia; 2Department of Biotechnology, Delft University of Technology, Delft, The Netherlands; 3Paques B.V., Balk, The Netherlands; and 4Department of Aquatic Microbiology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands
Correspondence: Dimitry Y. Sorokin, Winogradsky Institute of Microbiology, Russian Academy of Sciences, Prospect 60-let Octyabrya 7/2, 117312 Moscow, Russia. Tel.: 7 095 1350109; fax: 7 095 1356530; e-mails: [email protected]; [email protected] Received 26 January 2014; revised 12 March 2014; accepted 19 March 2014. Final version published online 11 April 2014. DOI: 10.1111/1574-6968.12432
MICROBIOLOGY LETTERS
Editor: Christiane Dahl Keywords thiocyanate; cyanate; cyanate pathway; halophilic; Halothiobacillus.
Abstract Molecular and microbiological analysis of a laboratory bioreactor biomass oxidizing thiocyanate at autotrophic conditions and at 1 M NaCl showed a domination of a single chemolithoautotrophic sulfur-oxidizing bacterium (SOB) capable of using thiocyanate as an energy source. The bacterium was isolated in pure cultures and identified as a member of the Halothiobacillus halophilus/hydrothermalis clade. This clade includes moderately halophilic chemolithoautotrophic SOB from marine and hypersaline habitats for which the ability to utilize thiocyanate as an electron donor has not been previously demonstrated. Halothiobacillus sp. strain SCN-R1 grew with thiocyanate as the sole energy and nitrogen source oxidizing it to sulfate and ammonium via the cyanate pathway. The pH range for thiocyanate oxidation was within a neutral region between 7 and 8 and the range of salinity was from 0.2 to 1.5 M NaCl, with an optimum at 0.5 M. Despite the close phylogenetic relatedness, none of the tested type strains and other isolates from the H. halophilus/hydrothermalis group exhibited thiocyanate-oxidizing capacity.
Introduction Thiocyanate (NC–S) is a unique inorganic sulfur compound produced naturally in the biological cyanide detoxification processes and as a waste product from cokes and precious metal factories (Wood, 1975; Kelly & Baker, 1990; Hung & Pavlostathis, 1997, 1999; Staib & Lant, 2007). It is a chemically stable compound and only a few bacterial species belonging to chemolithoautotrophic sulfur-oxidizing bacteria (SOB) are capable of utilizing thiocyanate as an energy source. Thiocyanate degradation needs the primary action of specific enzyme(s) to release the reduced sulfane atom for further oxidation. Accordingly, two possible pathways of microbial degradation of thiocyanate are proposed with either carbonyl sulfide (COS) or cyanate (CNO) as intermediate products (Kelly & Baker, 1990): Carbonyl sulfide pathway N≡C-S- + H2O
SCN-hydrolase
O=C=S + NH 3
H2S + CO2
COS-hydrolase
FEMS Microbiol Lett 354 (2014) 69–74
Cyanate pathway N≡C-S- + H2O
?
N≡C-O- + H2 S
NH3 + CO 2
cyanase
In the carbonyl sulfide pathway, the nitrile bond of thiocyanate is hydrolyzed with the formation of COS and ammonia by the enzyme thiocyanate hydrolase found in the chemochemolithoautotrophic betaproteobacterial SOB Thiobacillus thioparus (Katayama et al., 1998), the halophilic chemochemolithoautotrophic gammaproteobacterium Thiohalophilus thiocyanatoxydans (Bezsudnova et al., 2007; Sorokin et al., 2007) and a novel alphaproteobacterium strain THI201 (Hussain et al., 2013). Carbonyl sulfide is further hydrolyzed to hydrogen sulfide, which serves as the actual electron donor. Microbial carbonyl sulfide hydrolysis can be carried out either by a b-carbonic anhydrase homologue of CS2 hydrolase in a thermoacidiphilic archaeon Acidianus sp. (Smeulders et al., 2011) or a ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
D.Y. Sorokin et al.
70
specialized COS hydrolase from the b-carbonic anhydrase family in the betaproteobacterial SOB T. thioparus (Ogawa et al., 2013). Realistic evidences on the existence of an alternative cyanate pathway of primary thiocyanate degradation has, so far, only been obtained during investigation of thiocyanate metabolism in (halo)alkaliphilic SOB, such as Thioalkalivibrio thiocyanoxidans, Thioalkalivibrio paradoxus and Thiohalobacter thiocyanaticus, isolated from soda and salt lakes (Sorokin et al., 2001, 2002, 2010). The identity of the key enzyme in this pathway is still under investigation, but it is already clear that the mechanism is different from anaerobic hydrolysis of the C–S bond as proposed previously (Grant & Sorokin, 2011). The chemochemolithoautotrophic SOB belonging to Halothiobacillus halophilus/hydrothermalis group are dominant culturable forms of aerobic moderately halophilic SOB in hypersaline habitats (Wood & Kelly, 1991; Durand et al., 1993; Kelly & Wood, 2000). Despite this fact, our enrichments from hypersaline lakes with thiocyanate as the sole substrate at moderate salinity never resulted in domination of Halothiobacillus (Sorokin, 2008). In contrast, microbiological analysis of a lab-scale bioreactor community degrading thiocyanate at 1 M NaCl demonstrated that the main organism responsible for thiocyanate oxidation was a representative of the H. halophilus/hydrothermalis cluster. Its isolation and properties are described below.
Materials and methods Isolation source
A 2-L bioreactor working as a chemostat was originally inoculated with a haloalkaliphilic sulfide-oxidizing biomass from the Thiopaq bioreactor in Eerbeek (The Netherlands) with some addition from a salt lake sediment and sludge from an aerobic communal waste water treatment plant (Heerenveen). It was operated at 35 °C, pH 7.9–8.1 and 1 M NaCl with a mixed feed of 20 mM thiosulfate and 5 mM thiocyanate at a dilution rate 0.05 h1. Addition of thiosulfate as a co-substrate increased the thiocyanate removal activity. At the time of biomass sampling the culture supernatant contained a high concentra tion of inorganic N (37 mM NHþ 4 and 7 mM CNO ) which indicated (1) the use of cyanate pathway in thiocyanate degradation and (2) a significant imbalance between the catabolism and biomass growth.
containing 1 M NaCl, 50 mM potassium phosphate buffer (pH 8), 1 mM MgCl2, and 1 mL L1 of trace metals solution (Pfennig & Lippert, 1966) and supplemented with 10–20 mM potassium thiocyanate as a single energy and N-source. The isolation strategy consisted of serial dilution to extinction of the reactor biomass, followed by plating of the maximal positive dilution on solid medium with the same composition made by 1 : 1 mixing of double-strength liquid media and 3% (w/v) washed agar (three successive washes in 50 volumes of distilled water with stirring during 5 h) at 30 °C. It needs to be specifically stressed that the agar-washing step is important in this particular type of isolation because many heterotrophic bacteria can form colonies on mineral agar media due to a presence of small amount of organic contaminants in the agar. Dominating colony types were placed into liquid medium. If the isolates grew in the liquid mineral medium containing thiocyanate as the only energy source, the plating was repeated several times until a single colony type was selected, which would grow with thiocyanate as a the sole energy and nitrogen source. Metabolic profiles of the reactor biomass and pure cultures
The metabolic activity of the reactor biomass and the isolates was investigated by two different ways, either by direct measurements of substrate conversion or by measuring substrate-dependent respiration rates with dO2 electrode, as described previously (Sorokin et al., 2001, 2010). The pH dependence was examined at 0.5–1.0 M total Na+, using the following buffers: for pH 5–8, 0.1 M HEPES/NaCl titrated by 1 M NaHCO3 for pH 8–10, a mixture of sodium bicarbonate/sodium carbonate/NaCl. Influence of NaCl was checked in HEPES buffer at pH 7. All buffers contained 10 mM K+ and 1 mM Mg2+. Analytical procedures
Thiocyanate, cyanate, ammonium and cell protein were analyzed spectrophotometrically as described previously (Sorokin et al., 2001). The cyanase activity was measured in sonacated cell-free extract in 0.1 M NaHCO3 (pH 8) with 5 mM cyanate as substrate. The activity was estimated from the amount of ammonia released in 0.5–2 h incubation time. Molecular and phylogenetic analyses
Enrichment, isolation and cultivation of pure cultures of halophilic SOB
Thiocyanate-oxidizing halophilic SOB were grown from the reactor biomass in a liquid mineral medium ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Genomic DNA was extracted from the cell pellet using the UltraClean Soil DNA Extraction Kit (MoBio Laboratories), following the manufacturer’s instructions. For the pure cultures, the nearly complete 16S rRNA gene was FEMS Microbiol Lett 354 (2014) 69–74
Utilization of thiocyanate as energy source by Halothiobacillus
3.0
Concentrations (mM)
obtained using general bacterial primers GM3f (50 -AGAG TTTGATCCTGGCTCAG-30 ) and GM4r (50 -TACGGTTACCTTGTTACGACTT-30 ). For the DGGE analysis, partial amplification with a primer pair 341F + GC/907R was used (Sch€afer & Muyzer, 2001). DGGE was performed using a denaturing gradient of 20–70% denaturants in 8% polyacrylamide gel. Individual bands were excised, re-amplified, and run again on a denaturing gradient gel to check their purity. PCR products for sequencing were purified using the Qiaquick PCR purification kit (QIAGEN, the Netherlands). The sequences were first compared with sequences stored in GenBank using the BLAST algorithm. Subsequently, the sequences were imported into the ARB software program, automatically aligned, and added to a phylogenetic tree using the Quick-add tool. Sub-trees were then built using the neighbour-joining algorithm with automatic selected correction settings. The 16S rRNA gene sequences of strain SCN-R1 and of the dominant DGGE band were deposited in the GenBank with the accession numbers KC662326 and KC759700, respectively.
71
Degraded SCN–
NH3
CNO–
2.5 2.0 1.5 1.0 0.5 0.0 0
5
10
15
20
Thiosulfate added (mM) Fig. 1. Influence of thiosulfate on thiocyanate degradation by the reactor biomass at pH 8.5 (1 M NaHCO3). Incubation time: 48 h; biomass: OD590 = 0.50; thiocyanate = 5 mM. Of 20 mM thiosulfate was oxidized completely after 13 h of incubation. Mean values from a duplicate experiment with maximum deviation of 15%.
The NaCl range for thiocyanate-oxidizing activity of the reactor biomass was between 0.2 and 1.2 M, while thiosulfate oxidation continued up to 3 M. DGGE analysis of reactor biomass
Potassium thiocyanate, sodium thiosulfate and sodium sulfide (analytical grade) were obtained from SigmaAldrich.
Results and discussion Catalytic properties of the bioreactor biomass
Cells taken directly from the bioreactor degraded thiocyanate only in the presence of thiosulfate. Cyanate was found as a product of thiocyanate degradation (Fig. 1). Concomitant with thiocyanate degradation in the presence of thiosulfate, biomass growth and thiosulfate oxidation was observed. Extensive washing of the cells in a N-free buffer clearly unblocked the thiosulfate-independent thiocyanate oxidation capacity of the cells (Fig. S1). The fact that the bioreactor cells, when placed into an N-free buffer, continued to excrete ammonium during several days, and that external ammonium or cyanate additions did not influence the ability of cells to metabolize thiocyanate, indicated that the thiocyanate biodegradation resulted in unbalanced cell-associated accumulation of inorganic N inhibiting further thiocyanate degradation. Addition of a nitrogen-free energy co-substrate (thiosulfate) seemed to facilitate inorganic N consumption with increasing biomass growth and lifting the inhibitory effect. The same result was achieved by extensive washing of the cells. FEMS Microbiol Lett 354 (2014) 69–74
Molecular analysis of the reactor biomass showed a low genetic diversity with a dominant band affiliated to members of chemolithoautotrophic SOB from the H. halophilus/hydrothermalis clade (Fig. 2). The other detected organisms, such as the two different bacteroidetes and a Nitratireductor, apparently belong to heterotrophic part of the reactor population probably utilizing the organic 40% Uncutured Bacteroidetes AB297398 (96%)
gradient
Chemicals
Halothiobacillus halophilus U58020 (98-99%)
Nitratireductor sp. BJGMM-B15 JQ716222 (99%)
Uncutured Bacteroidetes AB297398 (95%)
70%
Fig. 2. DGGE analysis of the biomass from a thiocyanate-removing lab-scale bioreactor. The gradient was from 30% to 70% and all bands were separate within 40–70% region.
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Serial dilution of washed cells from the reactor in a mineral medium containing 1 M NaCl and 10 mM thiocyanate as a single energy and N source resulted in growth up to a dilution of 108. Plating on the same medium from the maximal positive dilution yielded three different types of colonies. Only one of them designated as strain SCN-R1 (the strain is deposited in the NCCB collection in Utrecht under the number 100459), produced stable growth back on liquid mineral medium with thiocyanate, while the other two only grew when thiocyanate was used as the N-source, in combination with either thiosulfate (identified as a member of the Thiomicrospira pelophila clade) or acetate (identified as a member of the genus Halomonas). Strain SCN-R1, while growing with thiocyanate or thiosulfate as electron donor, had rod-shaped, non-motile cells assembled in chains (Fig. S2). According to the phylogenetic analysis, the isolate belonged to the H. halophilus/hydrothermalis group (Fig. 3) and its 16S rRNA gene sequence was 100% identical to the sequence of the dominant DGGE band from the reactor biomass (see Fig. 2). This indicated that the isolate SCN-R1 was the dominant chemochemolithoautotrophic thiocyanate-oxidizing SOB in the bioreactor. On the other hand, neither the type strains of H. halophilus and H. hydrothermalis nor our halophilic SOB isolates from hypersaline lakes belonging to the same group (Halothiobacillus sp. strains HL1, HL2,
20 18 16 14 12 10 8 6 4 2 0
A-SCN
B-SCN
C-SCN
D-SCN
A-growth
B-growth
C-growth
D-growth
0.3 0.25 0.2 0.15 0.1
Growth, OD590
Isolation and characterization of the dominant halophilic thiocyanate oxidizer
HL6, HL7, HL20 and HL27; Sorokin, 2008) were able to utilize thiocyanate as the enrgy source. Similar to the reactor biomass, growth and concomitant thiocyanate utilization by strain SCN-R1 was not straightforward. After an initial increase of biomass, the growth and thiocyanate consumption stopped and resumed only after the cells were collected and thoroughly washed with an N-free buffer. On the other hand, the addition of thiosulfate only slightly relieved the inhibition in case of the pure culture grown on thiocyanate (Fig. 4). Cyanate was the main nitrogen product of thiocyanate degradation detected in the medium, and the complete oxidation of 20 mM thiocyanate resulted in the release of
SCN–, mM
compounds produced by the dominant chemolithioautotrophic SOB.
0.05 0
5
10
15
20
0 25
Time, h Fig. 4. Growth of strain SCN-R1 with 20 mM thiocyanate at pH 8 and 1 M NaCl. A, Source control culture; B, cells collected after 5 h, washed in 1 M sterile NaCl solution and put back into the same medium; C, cells collected after 5 h, washed twice in 1 M NaCl and put into fresh medium; D, A + 20 mM thiosulfate. Mean values from a duplicate experiment with maximum deviation of 12%.
Fig. 3. Phylogenetic tree based on 16S rRNA gene sequencing, showing the position of the halophilic thiocyanate-oxidizing halophilic SOB isolated from the bioreactor. The tree includes sequences from the gammaproteobacterial (halo)alkaliphilic SOB species from the genus Halothiobacillus and those capable of thiocyanate utilization as the electron donor. Scale bar represent 10% sequence divergence.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Lett 354 (2014) 69–74
Utilization of thiocyanate as energy source by Halothiobacillus
100 (b)
(a)
100 90
90
80
80
Activity, % of maximum
Activity, % of maximum
73
70 60 50 40 30 20
70 60 50 40 30 20 10
10 0 0
0.5
1
1.5
2
2.5
3
3.5
0 6
6.5
7
7.5
NaCl, M
8
8.5
9
9.5
pH
Fig. 5. Influence of pH (at 1 M NaCl) and salt (at pH 8) on the activity of thiocyanate (closed circles) and sulfide (open circles) oxidation by washed cells of strain SCN-R1 pre-grown on thiocyanate. The data were obtained in a single experiment.
2.5 mM NH3 and 9.0 mM cyanate into the culture supernatant. Hence, similar to the source bioreactor biomass, Halothiobacilluis sp. SCN-R1 apparently uses the cyanate pathway for thiocyanate degradation. The results of substrate-dependent respiration tests with cells grown either with thiocyanate or with thiosulfate as an energy source showed that the thiocyanatedegrading system in strain SCN-R1 was inducible [VO2 = 170 nmol (mg protein min)1], and was absent in the cells grown with thiosulfate, similar to other thiocyanate-oxidizing (halo)alkaliphilic SOB described previously (Sorokin et al., 2001, 2007, 2010). However, in contrast to alkaliphilic SOB, cyanase activity, albeit low [35 nmol (mg protein min)1] was present only in thiocyanate-growing cells of strain SCN-R1. A salt response of strain SCN-R1 showed moderate tolerance with an optimum at 0.5 M NaCl and the cells being still active up to 2 M NaCl with thiocyanate and up to 3 M NaCl with sulfide (Fig. 5a). The thiocyanate oxidation had a relatively narrow pH profile in comparison with the sulfide-oxidizing activity, with a sharp decrease below pH 7 and above pH 8 (Fig. 5b). Summarizing the results, we were able to demonstrate that the dominant organism responsible for thiocyanate oxidation in a lab-scale bioreactor operating at 1 M NaCl is a representative of the chemochemolithoautotrophic halophilic SOB of the genus Halothiobacillus. This organism was isolated in pure culture and proven to be capable of chemolithoautotrophic growth with thiocyanate as energy and nitrogen source degrading it via the cyanate pathway. Such potential has never been reported before for members of the genus Halothiobacillus. The microbial oxidation of thiocyanate at high salt concentration might be useful for treatment of saline thiocyanate-containing wastewater generated by, for example, gold cyanidation plants (Stott et al., 2001). However, since the catabolism FEMS Microbiol Lett 354 (2014) 69–74
of thiocyanate by autotrophic SOB generates much more inorganic N than needed for biomass synthesis, a N-free co-substrate or an N-utilizing partner would be necessary for the complete bioremoval of thiocyanate.
Acknowledgements This work was supported by Shell International and Paques B.V. (The Netherlands). Gerard Muyzer was financially supported by the ERC Advanced Grant 322551.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. Influence of bioreactor cell washing on the activity of SCN-degradation in presence and absence of thiosulfate at pH 8 and 0.6 M NaCl. Fig. S2. Cell morphology (phase-contrast microscopy) of the reactor biomass (a) and of the pure culture of thiocyanate-oxidizing Halothiobacillus strain SCN-R1 growing either on thiocyanate (b) or thiosulfate (c).
FEMS Microbiol Lett 354 (2014) 69–74
