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Department of Environmental and Natural Resource Science, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Correspondence
Yoko Katayama
katayama{at}cc.tuat.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Tochigi Prefectural Agricultural Experiment Station, Japan.
Present address: Department of Biochemistry & Integrative Medical Biology, School of Medicine, Keio University, Japan.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Bandy et al., 1992; Chin & Davis, 1995). COS is regarded as a dominant source of stratospheric sulfate aerosol, which influences the earth's radiation budget, and therefore the climate (Crutzen, 1976). Therefore, attention is being focused on both sources and sinks of COS (Andreae & Crutzen, 1997; Watts, 2000).
Soil is a major sink for atmospheric COS (Castro & Galloway, 1991; Andreae & Crutzen, 1997; Kesselmeier et al., 1999; Kuhn et al., 1999; Simmons et al., 1999; Watts, 2000). Changes in temperature and water content, as well as repeated exposure of soil to COS, affect the rate of COS consumption by soil (Bremner & Banwart, 1976; Lehmann & Conrad 1996; Kesselmeier et al., 1999; Saito et al., 2002). Therefore, soil micro-organisms carrying COS-degrading activity have been regarded as having a dominant role as a sink for atmospheric COS.
Biological degradation of COS has been examined on an enzyme level in several organisms that consume CO2. Carbonic anhydrase (CA), a ubiquitous enzyme that catalyses reversible hydration between CO2 and 
, reacts with COS (COS+H2O
CO2+H2S) because of the structural analogy of CO2 and COS. This process has been confirmed in rat hepatocytes and erythrocytes (Chengelis & Neal, 1979), higher plants (Protoschill-Krebs & Kesselmeier, 1992), algae (Protoschill-Krebs et al., 1995), lichens (Gries et al., 1994), and cyanobacteria (Miller et al., 1989; Badger & Price, 1990). Micro-organisms possessing the ability to metabolize CO2 are considered to be involved in COS consumption (Conrad, 1996). Nevertheless, it is unclear which pathways soil micro-organisms employ to degrade atmospheric COS because there have been few reports on isolation of COS-degrading microbes from soil environments. Furthermore, most studies on COS degradation by micro-organisms have been conducted under favourable growth conditions for the micro-organism tested. However, soil environments differ from such favourable conditions in both nutrient and physico-chemical aspects. Most of the COS mixing ratios used for experiments of microbial degradation have been higher than the ambient levels of COS. Conrad & Meuser (2000) showed that COS is consumed by a different biological activity at an ambient mixing ratio than that used at higher mixing ratios, indicating that bacteria utilizing COS at higher mixing ratios are not always able to use the gas at ambient levels.
In this study, we report COS-degrading activity in heterotrophic bacteria that were isolated from soil environments. The degradation of COS was confirmed at an ambient mixing ratio, using sterilized soil as the incubation medium for the isolates. The results provide experimental evidence for the role of soil bacteria as a sink for atmospheric COS.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Isolated bacteria were maintained on a PYG slant, pH 7.2, containing (g l–1): 2.0 polypeptone (Nihon Seiyaku), 1.0 Bacto yeast extract (Difco), 0.5 glucose, and 15.0 Bacto agar.
Degradation of 30 p.p.m.v. COS on a PYG slant.
The isolated bacterium was streaked on a PYG slant. The PYG slant was prepared in a glass tube of 20 cm length and 2 cm i.d., and it contained 10 ml PYG medium to make a headspace volume of 40 ml. When colonies were formed, the cap of the test tube was changed from a silicone sponge cap to a butyl cap, and 11.5 µl COS gas [105 000 parts per million by volume (p.p.m.v.), with N2 as the balance gas; Nissan Tanaka] was added to the headspace to make a final COS mixing ratio of 30 p.p.m.v. At the times indicated (Figs 1 and 2), 50 µl headspace gas was obtained using a gas-tight microsyringe, and then injected into a gas chromatograph to measure the mixing ratio of COS, as described below. The moist condition of a sealed test tube containing agar medium gradually decreases the COS mixing ratio due to hydrolysis of COS with water (Ferm, 1957); therefore, bacterial COS degradation was evaluated by comparing the chemical degradation of COS based on a control slant that was not inoculated. Experiments were carried out in duplicate, under aerobic conditions, because anaerobic conditions have been known to inhibit COS consumption by soil (Lehmann & Conrad, 1996).
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Soil (Andosol) used for the incubation of cells was obtained from the surface layer of soil (0–5 cm) at the university farm, air-dried for 17 days at room temperature, sieved with a 2 mm mesh, and then stored at 4 °C. Organic carbon content, pH and maximum water-holding capacity of this soil were 8.2 % (w/w, dry soil), 6.2 and 90 % (water weight/dry soil weight), respectively. The stored soil was sterilized by autoclaving at 121 °C for 30 min before use. The sterility of the soil was confirmed by streaking the soil suspension onto PYG agar medium.
Comparison of bacterial COS degradation between the slant and the soil.
COS degradation curves of the isolates at 25 °C were fitted to the exponential function C(t)=C0e–kt (R2 >0.8), where C(t) is the mixing ratio of COS given at the time t (h), C0 is the initial COS mixing ratio, and k is the rate constant (per hour per test tube). The rate constants of COS degradation obtained by using the cells grown on the slant and in the soil were compared to determine the effect of incubation in soil on the COS-degrading activity of the bacteria.
The number of bacterial cells was estimated based on the c.f.u. method. The cells on the slant medium were suspended in 10 ml sterilized distilled water, and serial dilutions were plated onto PYG agar medium. The cells inoculated in the soil (4 g soil plus 1.5 ml cell suspension) were suspended in 14.5 ml sterilized distilled water, and mixed thoroughly by vortexing for 2 min. Then, the dilutions of the resultant suspension were plated onto PYG agar medium. The number of cells in the soil was counted at the time of inoculation, and then again after a 10 day incubation.
Degradation of ambient levels of COS in the soil.
For the experiment carried out at the ambient mixing ratio of COS, a 5 l gas-sampling bag (aluminized polyethylene bag; GL Science) was used to prepare a sufficient volume of headspace for air sampling. The bacterium was pre-incubated in PYG-Low medium, diluted with sterilized water, and dropped onto the sterilized soil (5.3 ml suspension per 14 g soil) in a glass Petri dish (8.8 cm i.d.). The initial cell density and the water content were 106 c.f.u. (g wet soil)–1 and 58 % (water weight/dry soil weight), respectively. After an incubation of 10 days, the slightly hardened soil was tilled with a sterilized spatula, and then the Petri dish with the soil, but without the lid, was placed in the gas-sampling bag. One corner of the bag had been cut off, and the inside was sterilized with 70 % ethanol. At times 0, 10, 20 and 30 h, a 300 ml sample of air was taken from the bag with a syringe, and loaded onto a pre-cryo column under liquid oxygen. After the cryogenic concentration of COS, the column was heated to 100 °C in 2 min, using a Flash Sampler (FLS-1; Shimadzu) to introduce the sample into the gas chromatograph. Measurement was performed twice to ensure accurate determination of the low level of COS. We obtained the reproducibility of the bacterial degradation of the ambient level of COS by performing repeated experiments.
COS analysis.
COS was measured by a gas chromatograph (GC-14B; Shimadzu) equipped with a flame photometric detector, and a glass column packed with Porapak QS (50–80 mesh; Waters Associates), as described previously (Katayama et al., 1992). Nitrogen was used as the carrier gas, and the flow rate was 43 ml min–1. The temperatures of the injector, the column and the detector were 150, 110 and 150 °C, respectively. The pre-cryo column for the analysis of ambient COS was packed with 1,2,3-Tris(2-cyanoethoxy)propane (GL Science). Deviations from the mean of duplicate samples were found to deviate from the mean by less than 10 and 20 % for the analyses of p.p.m.v. and p.p.t.v. levels, respectively. The detection limits for COS with direct injection to the gas chromatograph, and for cryogenic concentration, were 3.9 p.p.m.v. and 260 p.p.t.v., respectively.
Phylogenetic analysis.
The isolate was grown in PYG liquid medium, and harvested by centrifugation. DNA was extracted from the cell pellet by using an Isoplant DNA extraction kit (Nippon Gene). Partial 16S rRNA between the 357–907 nt region (Escherichia coli numbering) (Muyzer et al., 1995) was amplified by PCR. The primers used here were 16S357F (5'-CCTACGGGAGGCAGGCAG-3') and 16S907R (5'-CCCCGTCAATTCCTTTGAGTTT-3'). PCR products were purified using an UltraClean 15 DNA purification kit (Mo Bio Laboratory). Sequencing was performed with a BigDye Terminator Cycle Sequencing Ready Reaction DNA sequencing kit (Applied Biosystems) and an ABI Prism 377 DNA sequencer (Applied Biosystems). The 16S rRNA gene sequences were compared with sequences in the GenBank database using BLAST (Altschul et al., 1990). The nucleotide sequences of 16S rRNA gene of COS-degrading bacteria isolated in this study have been deposited in the DNA Data Bank of Japan under the following accession numbers: THI401, AB206556; THI402, AB206557; THI404, AB206559; THI405, AB206560; THI410, AB206564; THI414, AB206569; THI415 and AB206568.
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RESULTS |
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Seven isolates (THI401, THI402, THI404, THI405, THI410, THI414 and THI415) were used for the further experiments because they showed both stable COS degradation activity and growth. THI401, THI402 and THI404 were isolated from forest soil in Aomori Prefecture, THI405, THI414 and THI415 were isolated from forest soil in Yamanashi Prefecture, and THI410 was isolated from cattle-farm soil in Tochigi Prefecture. Comparative 16S rRNA gene sequence analysis indicated that THI401, THI402, THI404 and THI405 were rapid-growing strains of Mycobacterium spp., and that THI410 belonged to the genus Williamsia. THI414 and THI415 were related to the genus Cupriavidus.
COS degradation in the PYG slant and the soil
We compared COS degradation by the bacterial isolates grown on the slant and in the soil to determine whether the bacteria were able to exhibit COS degradation in a soil environment. Figs 1
and 2 show COS degradation by bacteria grown on the PYG slant and in the soil. Initially, 30 p.p.m.v. COS in the test tube containing an uninoculated slant was reduced slowly to about 60 % of the original level in 30 h (Fig. 2a); this was probably caused by abiotic and irreversible hydrolysis of COS (Ferm, 1957; Elliott et al., 1989) on the surface of the agar medium. Mycobacterium spp. strains THI401, THI402, THI404 and THI405 degraded 30 p.p.m.v. COS to the detection limit within 2 h (Fig. 1a). These four strains exhibited a level of COS degradation activity in soil that was similar to that on the PYG slant; they degraded COS to the detection limit within 3 h (Fig. 1b). The rate constants of COS degradation observed in the PYG slant tube and the soil are summarized in Table 1. The mycobacterial strains showed lower rate constants in the soil than in the PYG tube. Growth of these isolates was estimated based on cell number, and the results indicated that THI402, THI404 and THI405 exhibited a level of growth in soil that was similar to that in the slant culture.
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, Fig. 2a). However, in soil, these three strains were unable to degrade COS (Fig. 2b). THI410 and THI414 showed large reductions in the rate constants for COS degradation in soil, to 1.3 % and 3.0 % of the original, respectively. Based on specific activity of COS degradation (Table 1), culturing the bacteria in soil appeared to have a negative effect on the growth of THI410, and on the COS-degradation activity of THI414 and THI415, while it did not have a negative effect on COS degradation by the mycobacterial isolates.
Production of H2S during COS degradation was observed for several isolates, while it was not observed in the uninoculated control. Fig. 3 shows H2S production by Mycobacterium sp. THI401, and Williamsia sp. THI410, both of which were grown on PYG agar slants. According to the decrease in the level of COS, a volume of H2S that was equivalent to about 8–10 % of the initially added COS was detected in the headspace, and this then decreased upon further incubation. Similar levels of H2S production were observed when Mycobacterium spp. THI402, THI404 and THI405 were grown on the PYG slant. Although Cupriavidus spp. THI414 and THI415 did not produce a detectable level (>0.39 p.p.m.v.) of H2S during degradation of 30 p.p.m.v COS, around 2 p.p.m.v. H2S was detected when 300 p.p.m.v. COS was added. These results indicate that H2S is an intermediate of COS degradation in the strains tested. When the experiment was carried out in soil, H2S was not detected in the headspace gas.
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shows the time courses of the COS mixing ratios in the bag. The initial COS mixing ratio (560 p.p.t.v.) was maintained throughout the experiment when a Petri dish, without soil, was placed in the bag. Thus, absorption of COS to the inner surface of the bag, and to the glass, was negligible. The COS mixing ratio in the bag that contained the sterilized soil gradually increased from 540 p.p.t.v. to a level of 1910 p.p.t.v. at 30 h, indicating that sterilization of the soil by autoclaving resulted in abiotic emission of COS. Therefore, the degradation of ambient levels of COS was evaluated based on the overall balance between emission from the sterilized soil and degradation by the bacterium.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), indicating that soil microbes are ready to degrade COS, despite no acclimation period to an elevated level of COS.
The ability to degrade an ambient mixing ratio of COS was examined when the isolate was incubated in soil. However, autoclave sterilization of soil resulted in COS emission from the soil. Nguyen et al. (1995) reported COS emission from biomass burning in field experiments, and it is likely that heat treatment by autoclaving brought about COS emission from the soil. On the other hand, Mycobacterium spp. showed a significant decrease in the ambient level of COS. To compare these COS-uptake rates obtained in soil with previously reported data, the COS degradation rates of the bacteria were calculated on the basis of the difference in the COS mixing ratio at times 0 and 10 h (Fig. 4). The net rates of COS degradation by THI401 and THI405, for example, were 0.14 and 0.24 pmol m–2 s–1, respectively. Because the rate of COS emission from the soil was 0.38 pmol m–2 s–1, the gross levels of reduction in COS were 0.52 and 0.62 pmol m–2 s–1 for THI401 and THI405, respectively. These values are of a similar order of magnitude to the COS uptake rate of 0.81 pmol m–2 s–1 that was obtained from a soil experiment (Steinbacher et al., 2004). These results indicate that when there are ambient levels of COS, soil bacteria seem to have the potential to contribute to COS consumption in a soil environment.
Of the isolates examined so far, those that exhibited the ability to degrade ambient levels of COS in soil were found to belong to the genus Mycobacterium. Considering the abundance of mycobacteria in the soil environment (Iivanainen et al., 1997), it is important to understand the mechanism and the prevalence of COS-degradation activity by Mycobacterium spp. CA has been purified from Mycobacterium tuberculosis H37Rv (Suarez Covarrubias et al., 2005), which is a slow-growing strain; however, conversion of an ambient mixing ratio of COS by bacteria carrying CA has not yet been examined. A chemolithoautotrophic bacterium, Thiobacillus thioparus, can utilize COS as a sole energy source (Smith & Kelly, 1988), and a relevant COS-degrading enzyme has been purified from T. thioparus (unpublished results). The role of COS metabolism in the microbial growth and/or energetics of the chemo-organotrophic bacteria isolated here remains to be examined.
In this study, we isolated COS-degrading bacteria, and compared their COS-degradation activities in different cultural conditions of artificial medium and soil, under different mixing ratios of COS at 30 p.p.m.v. and ambient levels. Further studies are in progress to elucidate the enzymic characteristics of these COS-degrading bacteria.
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ACKNOWLEDGEMENTS |
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Edited by: H. L. Drake
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REFERENCES |
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Andreae, M. O. & Crutzen, P. J. (1997). Atmospheric aerosols: biogeochemical sources and role in atmospheric chemistry. Science 276, 1052–1058.
Badger, M. R. & Price, G. D. (1990). Carbon oxysulfide is an inhibitor of both CO2 and 
uptake in the cyanobacterium Synechococcus PCC7942. Plant Physiol 94, 35–39.
Bandy, A. R., Thornton, D. C., Scott, D. L., Lalevic, M., Lewin, E. E. & Driedger, A. R., III (1992). A time series for carbonyl sulfide in the northern hemisphere. J Atmos Chem 14, 527–534.[CrossRef]
Bremner, J. M. & Banwart, W. L. (1976). Sorption of sulfur gases by soils. Soil Biol Biochem 8, 79–83.[CrossRef]
Castro, M. S. & Galloway, J. N. (1991). A comparison of sulfur-free and ambient air enclosure techniques for measuring the exchange of reduced sulfur gases between soils and the atmosphere. J Geophys Res 96, 15427–15437.
Chengelis, C. P. & Neal, R. A. (1979). Hepatic carbonyl sulfide metabolism. Biochem Biophys Res Commun 90, 993–999.[CrossRef][Medline]
Chin, M. & Davis, D. D. (1995). A reanalysis of carbonyl sulfide as a source of stratospheric background sulfur aerosol. J Geophys Res 100, 8993–9006.[CrossRef]
Conrad, R. (1996). Soil microorganisms as controllers of atmospheric trace gases (H2, CO, CH4, OCS, N2O and NO). Microbiol Rev 60, 609–640.
Conrad, R. & Meuser, K. (2000). Soils contain more than one activity consuming carbonyl sulfide. Atmos Environ 34, 3635–3639.
Crutzen, P. J. (1976). The possible importance of CSO for the sulfate layer of the stratosphere. Geophys Res Lett 3, 73–76.
Elliott, S., Lu, E. & Rowland, F. S. (1989). Rates and mechanisms for the hydrolysis of carbonyl sulfide in natural waters. Environ Sci Technol 23, 458–461.
Ferm, R. J. (1957). The chemistry of carbonyl sulfide. Chem Rev 57, 621–640.[CrossRef]
Gries, C., Nash, T. H., III & Kesselmeier, J. (1994). Exchange of reduced sulfur gases between lichens and the atmosphere. Biogeochemistry 26, 25–39.
Iivanainen, E. K., Martikainen, P. J., Räisänen, M. L. & Katila, M. L. (1997). Mycobacteria in boreal coniferous forest soils. FEMS Microbiol Ecol 23, 325–332.[CrossRef]
Katayama, Y., Narahara, Y., Inoue, Y., Amano, F., Kanagawa, T. & Kuraishi, H. (1992). A thiocyanate hydrolase of Thiobacillus thioparus. A novel enzyme catalyzing the formation of carbonyl sulfide from thiocyanate. J Biol Chem 267, 9170–9175.
Kesselmeier, J., Teusch, N. & Kuhn, U. (1999). Controlling variables for the uptake of atmospheric carbonyl sulfide by soil. J Geophys Res 104, 11577–11584.[CrossRef]
Kuhn, U., Ammann, C., Wolf, A., Meixner, F. X., Andreae, M. O. & Kesselmeier, J. (1999). Carbonyl sulfide exchange on an ecosystem scale: soil represents a dominant sink for atmospheric COS. Atmos Environ 33, 995–1008.
Lehmann, S. & Conrad, R. (1996). Characteristics of turnover of carbonyl sulfide in four different soils. J Atmos Chem 23, 193–207.[CrossRef]
Miller, A. G., Espie, G. S. & Canvin, D. T. (1989). Use of carbon oxysulfide, a structural analog of CO2, to study active CO2 transport in the cyanobacterium Synechococcus UTEX 625. Plant Physiol 90, 1221–1231.
Muyzer, G., Teske, A., Wirsen, C. O. & Jannasch, H. W. (1995). Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments. Arch Microbiol 164, 165–172.[CrossRef][Medline]
Nguyen, B. C., Mihalopoulos, N., Putaud, J. P. & Bonsang, B. (1995). Carbonyl sulfide emissions from biomass burning in the tropics. J Atmos Chem 22, 55–65.[CrossRef]
Protoschill-Krebs, G. & Kesselmeier, J. (1992). Enzymatic pathways for the consumption of carbonyl sulphide (COS) by higher plants. Bot Acta 105, 206–212.
Protoschill-Krebs, G., Wilhelm, C. & Kesselmeier, J. (1995). Consumption of carbonyl sulphide by Chlamydomonas reinhardtii with different activities of carbonic anhydrase (CA) induced by different CO2 growing regimes. Bot Acta 108, 445–448.
Saito, M., Honna, T., Kanagawa, T. & Katayama, Y. (2002). Microbial degradation of carbonyl sulfide in soils. Microbes Environ 17, 32–38.[CrossRef]
Simmons, J. S., Klemedtsson, L., Hultberg, H. & Hines, M. E. (1999). Consumption of atmospheric carbonyl sulfide by coniferous boreal forest soils. J Geophys Res 104, 11569–11576.[CrossRef]
Smith, N. A. & Kelly, D. P. (1988). Oxidation of carbon disulphide as the sole source of energy for the autotrophic growth of Thiobacillus thioparus strain TK-m. J Gen Microbiol 134, 3041–3048.
Steinbacher, M., Bingemer, H. G. & Schmidt, U. (2004). Measurements of the exchange of carbonyl sulfide (OCS) and carbon disulfide (CS2) between soil and atmosphere in a spruce forest in central Germany. Atmos Environ 38, 6043–6052.
Suarez Covarrubias, A., Larsson, A. M., Högbom, M., Lindberg, J., Bergfors, T., Björkelid, C., Mowbray, S. L., Unge, T. & Jones, T. A. (2005). Structure and function of carbonic anhydrases from Mycobacterium tuberculosis. J Biol Chem 280, 18782–18789.
Torres, A. L., Maroulis, P. J., Goldberg, A. B. & Bandy, A. R. (1980). Atmospheric OCS measurements on project Gametag. J Geophys Res 85, 7357–7360.
Watts, S. F. (2000). The mass budgets of carbonyl sulfide, dimethyl sulfide, carbon disulfide and hydrogen sulfide. Atmos Environ 34, 761–779.
Received 3 July 2007;
revised 26 September 2007;
accepted 5 October 2007.
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, THI401; 
, THI402; 
, THI404; 
, THI405. 
, Negative control [uninoculated sterilized PYG medium (a), or soil (b)]. The points on the curves represent mean values of duplicates – deviation from the mean was less than 10 %.
) and THI415 (
, Williamsia sp. THI410; 
, Cupriavidus sp. THI414; X, Cupriavidus sp. THI415;