Sulfur Isotopic Fractionation of Carbonyl Sulfide during Degradation

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Sulfur isotopic fractionation of carbonyl sulfide during degradation by soil bacteria Kazuki Kamezaki, Shohei Hattori, Takahiro Ogawa, Sakae Toyoda, Hiromi Kato, Yoko Katayama, and Naohiro Yoshida Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b05325 • Publication Date (Web): 11 Mar 2016 Downloaded from http://pubs.acs.org on March 21, 2016

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Environmental Science & Technology

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Sulfur isotopic fractionation of carbonyl sulfide during

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degradation by soil bacteria

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Kazuki Kamezakia,*, Shohei Hattoria,*, Takahiro Ogawab, Sakae Toyodac, Hiromi Katod,

5

Yoko Katayamab, and Naohiro Yoshidaa,e

6 7 8

a

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Technology, 4259, Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8502, Japan

Department of Environmental Chemistry and Engineering, Tokyo Institute of

10

b

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Harumicho, Fuchu, Tokyo 183-0057, Japan

12

c

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4259, Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8502, Japan

14

d

15

Sendai 980-8577, Japan

16

e

17

Meguro-ku, Tokyo 152-8550, Japan

Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-8-1

Department of Environmental Science and Technology, Tokyo Institute of Technology, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-Ku,

Earth-Life Science Institute, Tokyo Institute of Technology, 2-12-1-IE-1 Ookayama,

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Corresponding authors:

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Kazuki Kamezaki: [email protected]; Tel: +81-45-924-5506

21

Shohei Hattori: [email protected]; Tel: +81-45-924-5506

22

23

1

ACS Paragon Plus Environment


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ABSTRACT: We performed laboratory incubation experiments on the degradation of

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gaseous phase carbonyl sulfide (OCS) by soil bacteria to determine its sulfur isotopic

26

fractionation constants (34ε). Incubation experiments were conducted using strains

27

belonging to the genera Mycobacterium, Williamsia and Cupriavidus, isolated from

28

natural soil environments. The

29

0.19‰, −3.57 ± 0.22‰ and −3.56 ± 0.23‰ for Mycobacterium spp. strains THI401,

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THI402, THI404 and THI405; −3.74 ± 0.29‰ for Williamsia sp. strain THI410; and

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−2.09 ± 0.07‰ and −2.38 ± 0.35‰ for Cupriavidus spp. strains THI414 and THI415.

32

Although OCS degradation rates divided by cell numbers (cell-specific activity) were

33

different among strains of the same genus, the

34

significant differences. Even though the numbers of bacterial species examined were

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limited, our results suggest that 34ε values for OCS bacterial degradation depend not on

36

cell-specific activities, but on genus-level biological differences, suggesting that

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values are dependent on enzymatic and/or membrane properties. Taking our

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as representative for bacterial OCS degradation, the expected atmospheric changes in

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δ34S values of OCS range from 0.5 to 0.9‰, based on previously reported decreases in

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OCS concentrations at Mt. Fuji, Japan. Consequently, tropospheric observation of δ34S

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values for OCS coupled with 34ε values for OCS bacterial degradation can potentially be

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ε values determined were −3.67 ± 0.33‰, −3.99 ±

34

ε values for same genus showed no

2


34

34

ε

ε values

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used to investigate soil as an OCS sink.

43

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1. Introduction

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Carbonyl sulfide (OCS) is the most abundant gas containing sulfur in the

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ambient atmosphere, with an average mixing ratio of 500 parts per trillion by volume in

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the troposphere.1 OCS average residence time in the troposphere is more than two

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years,2 which enables it to be transported to the stratosphere, where it is converted to

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stratospheric sulfate aerosols (SSA) through atmospheric sink reactions.3 Hence, OCS is

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considered to be an important sulfur source for SSA, playing a significant role in the

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Earth’s radiation budget, and in ozone depletion.4–6 Because of uncertainties in the

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estimates of its various component fluxes, the global budget for OCS is out of

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balance.7,8

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Isotope analysis is useful tool to trace sources and transformations of trace

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gases.9,10 To quantify OCS sources and sinks in natural environments using isotope

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analysis, isotopic fractionation for specific metabolic reactions should be determined in

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laboratory experiments, in addition to ambient measurements. To date, isotopic

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fractionations occurring in the reactions of OCS have been determined only for the OCS

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sink reactions in the stratosphere: OCS photolysis,11–13 as well as its reactions with OH, 3



60

14

and O(3P).15 There are a few reports for tropospheric reactions. Furthermore, these

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determinations are based on absorption cross-section measurements, relative rate

62

methods, and theoretical calculations. There is only one report using isotope ratio mass

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spectrometer (IRMS) measurements, based on an off-line method requiring several

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µmol samples as well as chemical conversion to SO2 or SF6 for measurement. It is

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important to precisely determine the sulfur isotopic fractionation for OCS in the

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troposphere for various processes, including biogeochemical ones. Recently, an online

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method measuring sulfur isotope ratios in OCS on a gas chromatograph (GC)-IRMS

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using S+ fragmentation ions was developed,16 enabling us to easily analyze sulfur

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isotopes in OCS.

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Soil is thought to be important as both a source and a sink of OCS in the

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troposphere.7,8,17–20 In particular, soil has been reported as a large environmental sink

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for atmospheric OCS.7,8,17–20 Bacteria isolated from various soils actively degrade

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OCS,21,22 with various enzymes, such as carbonic anhydrase,23,24 COSase,25 and

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CS2hydrolase26 involved in OCS degradation. In actual soil environments, as well as in

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laboratory experiments, OCS degradation varies with temperature and water content.20

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This degradation contributes to the vertically decreasing gradient of OCS concentration

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from upper atmosphere to ground level.27 OCS degradation by bacteria isolated from 4


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soil is thought to be related to bacterial degradation that occurs at the soil surface.

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However, the magnitude of such contribution in terms of a sink for atmospheric OCS is

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still uncertain. Therefore, it is important to quantitatively evaluate this contribution

81

using OCS sulfur isotope analysis.

82

In this study, we performed incubation experiments using OCS-degrading soil

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bacteria to determine sulfur isotopic fractionation for OCS during such degradation.

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This study provides primary experimental evidence for isotopic fractionation via

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bacterial OCS degradation. The atmospheric implications for the isotopic fractionation

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for OCS measured in this study also are discussed.

87

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2. Materials and methods

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2.1. Strains and medium

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Seven isolates (THI401, THI402, THI404, THI405, THI410, THI414 and

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THI415) known to have OCS degradation activity were used in this study. Strains

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THI401, THI402 and THI404 were isolated from forest soil from Aomori Prefecture,

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Japan. Strains THI405, THI414 and THI415 were isolated from forest soil from

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Yamanashi Prefecture, while strain THI410 was isolated from a cattle-farm soil from

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Tochigi Prefecture.21 Comparative 16S rRNA gene sequence analysis indicated that

5



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strains THI401, THI402, THI404 and THI405 were members of the genus

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Mycobacterium, while strain THI410 belonged to the genus Williamsia, and strains

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THI414 and THI415 were related to the genus Cupriavidus. These isolates were

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streaked on a PYG agar slant with pH 7.2, containing (g L-1): 2.0 polypeptone (Nihon

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Seiyaku, Tokyo, Japan), 1.0 Bacto yeast extract (Difco Laboratories Inc., Detroit, MI,

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USA), 0.5 glucose, and 15.0 Bacto agar (Difco Laboratories Inc.).

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All bacterial strains were grown on a slant at 30 ℃. To reduce the effect of

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hydrolysis of OCS with water,28 all experiments were carried out on the surface of an

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agar-solidified slant medium rather than in a liquid medium, where we confirmed that

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growth of bacterial colonies covered the entire slant. A test tube measuring 20 cm in

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length and 2 cm i.d., containing 10 mL medium to give a headspace volume of 40 mL

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was used to prepare the slant. Within three days of forming colonies, the cap of the test

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tube was changed from a silicone sponge cap to a butyl cap to provide a better seal, and

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the lab air headspace was replaced with N2/O2 (80:20) and 0.03% of CO2.

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Approximately 4000 parts per million by volume (ppmv) OCS gas (104,000 ppmv with

111

N2 as the balance gas; Taiyo Nippon Sanso, Japan) was added to the batch. For THI401,

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we also conducted two batches of experiments using approximately 2000 ppmv. OCS

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gas. In order to test abiotic consumption by slant medium, we prepared the control 6


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batches without bacteria for each day of the preparation of slant medium for incubation

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experiments. Additionally, to observe the biological effects (e.g., cell number) via OCS

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injection to the batches, we also prepared duplicate bacterial batches for each strain with

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no added OCS as control experiments.

118

The time of injection of OCS was defined as t = 0 h. To ensure both thorough

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gas mixing in the headspace and negligible adsorption of gases by the glass, OCS

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concentration measurements and sample collections for OCS sulfur isotopic

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measurements were begun at t = 0.33 h. Concentration measurements and isotope

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measurements of headspace gases were performed on samples collected simultaneously.

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A 20 µL-sample of headspace gas was sampled using a gas-tight syringe and directly

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injected into the GC equipped with a flame photometric detector. An additional 1 mL of

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headspace gas was collected using a gas-tight syringe and transferred into a 5 mL serum

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bottle filled with ultrahigh-purity He (>99.99995% purity; Taiyo Nippon Sanso, Japan)

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for later isotopic analysis. The OCS sampling of headspace gases was conducted at five

128

time points for each batch experiment, until ca. 70% of the initial OCS was degraded.

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On completion of the OCS degradation experiment, the living cell numbers were

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counted as described below. The OCS degradation experiments were carried out over

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several days, with schedules shown in Table 1. 7



132 133

2.2. Carbonyl sulfide mixing ratio measurement

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OCS concentrations were measured using a GC (GC-14B; Shimadzu, Kyoto,

135

Japan) equipped with a flame photometric detector, and a glass column (Sunpak-S,

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Shimadzu; 3 mm i.d., and 2.0 m length). N2 gas (>99.999% purity; Ichimura Sanso,

137

Tokyo, Japan) was used as the carrier gas at a flow rate of 100 mL min-1. The

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temperatures of the injector, the column and the detector were 190 ℃, 60 ℃ and

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190 ℃ , respectively. The coefficient of variation (standard error relative to mean) was a

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maximum of 4%, but averaged 2% (n = 3). Reduction in OCS concentration was

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corrected for by taking into account the amount that was removed by syringe to measure

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OCS concentration and sulfur isotope ratio.

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OCS degradation curves were fitted to the exponential function C(t) = C0e–kt,

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where C(t) is the concentration of OCS at the time t (h), C0 is the initial OCS

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concentration, and k is the rate constant (per hour per test tube). k values were

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determined on the basis of fitting a curve to data on OCS degradation with time.

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8


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2.3. Sulfur isotope analysis of carbonyl sulfide

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Within 14 days of completing the experiment, sulfur isotope compositions of

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OCS were measured using the online GC-IRMS system described in our previous

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study.16 Briefly, the system consists of two parts: a pre-concentration line and a

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GC-IRMS system. The GC was equipped with a capillary column (HP/PLOT Q; Agilent

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Technologies, CA, U.S.A.; with 0.32 mm i.d., 30 m length, and 10 µm thickness) at

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60 ℃. He was used as carrier gas with a flow rate of 1.5 mL min−1, while OCS was

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injected into the IRMS (MAT 253; Thermo Fisher Scientific, Germany) via an open

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split interface (ConFlo IV; Thermo Fisher Scientific). Although the He was

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commercially available ultrahigh-purity He (>99.99995% purity; Taiyo Nippon Sanso,

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Japan), it was further purified using a stainless-steel column having a 7.53 mm i.d., and

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0.7 m length, packed with 5 A Molecular Sieve (Sigma-Aldrich, Japan) and cooled at

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−196 ℃ with liquid N2 to avoid any trace contamination by O2 and N2 gases.16

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OCS was trapped in stainless-steel tubes (10.5 mm i.d., and 150 mm length),

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and cooled with liquid N2 (−196 ℃), while other species were pumped into the

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pre-concentration line. OCS was then trapped in a capillary tube covered by a

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stainless-steel tube containing liquid N2 at −196 ℃ for more than 13 min, before being

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introduced into the GC-IRMS system. Within the IRMS system, the fragment ions 32S+,

9



166

33 +

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used to determine the sulfur isotope ratios in the OCS sample.

S , and

34 +

S produced from electron impact ionization of OCS were measured and

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Once the acquisition process for IRMS measurements was started (t = 0 s),

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liquid N2 for the cryofocusing trap was removed to allow OCS injection to the

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GC-IRMS system. A reference OCS gas was introduced to the system for 20 s intervals

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at three time points: t = 350 s, 585 s, and 1025 s. The reference gas at t = 350 s was used

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as the reference for all calculations of OCS sulfur isotopic compositions. The retention

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time of OCS under these experimental conditions was approximately t = 640 s. From t =

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400 s to 585 s, the effluent from the GC column was kept off the MS line, using a

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back-flushed helium flow. This was because we found an unidentified peak at t = 430 s

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in the samples from the incubation experiments, which is purported to be H2S produced

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by OCS degradation. To avoid the effect of small sample size in our current study, we

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only measured samples larger than 8 nmol, with most closer to 15 nmol in volume. To

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eliminate the influence of the background on each measurement, the column was baked

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at 200 ℃ for 30 min after each run and we did not start the next run until at least 10 min

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after baking of the previous run. Some 11 ppmv of OCS samples (Japan Fine Products,

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Kawasaki, Japan) balanced with He gas were measured between experimental samples

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to confirm the accuracy and precision of measurements. The typical precision (1σ) of 10


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the replicate measurements (n = 3) were 0.42‰, 0.20‰, and 0.32‰ for δ33S, δ34S, and

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∆33S, respectively. In addition, the raw δ value for each sample with respect to the

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reference OCS was calibrated using the 11 ppmv of in-house OCS standard gas,

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previously calibrated against the international sulfur isotopic standard, and measured in

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independent runs on the same day as sample measurements.

189 190

2.4 Cell numbers

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The cell numbers of bacteria were estimated based on their fluorescence under

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microscopic observation, after staining the living cells with Live/Dead reagent

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(LIVE/DEAD BacLight™ Bacterial Viability Kit for microscopy; Thermo Fisher

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Scientific). After completion of our OCS degradation experiment, the whole bacterial

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colony on the slant medium was suspended in 10 mL of 0.85% NaCl. This suspension

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was centrifuged at 10,000 x g for 15 min, and the supernatant was discarded, while the

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pellet was re-suspended thoroughly in 0.85% NaCl, using a vortex. This sequence was

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repeated twice. At this point, 3 µL of Live/Dead reagent was added to 1 mL dilutions of

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the final suspension. After 15 min, the suspended bacterial cells were filtered onto a

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polycarbonate black filter, with 25 mm diameter and 0.2 mm pore size (Plain Black

201

Disks;

Advantech

Corp.,

Taiwan),

and

treated

with

11


0.01%

poly-L-lysine


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(P8920-100ML: Sigma-Aldrich). Cells on the filter disk were transferred to a slide glass,

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and the cell number was counted under an epifluorescence microscope (BZ-8000;

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KEYENCE, Osaka, Japan). This was carried out using green fluorescent protein

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(GFP-B) excitation, in which living cells show green fluorescence derived from the

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SYTO9 stain (Thermo Fisher Scientific), as well as with tetramethylrhodamine (TRITC;

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Thermo Fisher Scientific) excitation in which dead cells show red fluorescence derived

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from propidium iodide. The living cells were counted in ten microscopic view fields per

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sample, while dead cells were counted in three microscopic view fields per sample. At

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the same time, the living cells from batches that were not supplemented with OCS were

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also counted, using the same procedure to evaluate the effects of adding OCS to our

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samples. The cell-specific activity was calculated as the ratio of the rate constant to the

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total number of living cells with a SYTO9 signal. To confirm that the living cell

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numbers were constant during our experiments, we compared living cell numbers in

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bacterial batches just after OCS degradation experiments with those without any OCS

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addition (control experiments).

217 218 219

2.5. Definitions Sulfur isotopic compositions are typically reported using:

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δxS = xRsample / xRreference-1

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(1)

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where xRsample represents the isotope ratios (xS/32S, where x = 33 or 34) of residual OCS

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and Rreference represents the initial OCS composition. The isotope compositions of sulfur

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are often quoted using per mil (‰) notation. In addition to the δ values, capital delta

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notation (∆33S) is used to distinguish mass-independent fractionation (MIF; or

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non-mass-dependent fractionation) of sulfur, which causes deviation from the

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mass-dependent fractionation (MDF) line. The ∆33S value is expressed as: ∆33S = δ33S − [(δ34S + 1)0.515 − 1].

227

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This notation describes the excess or deficiency of 33S relative to a reference MDF line. The magnitude of isotopic fractionation during a single reaction is expressed

229

230

(2)

using the isotopic fractionation factor α, as follows: x

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α = xk / 32k,

(3)

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where xk (x = 33 or 34) and 32k are the reaction rate constants for molecules containing

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the isotopically heavier and lighter isotopes, respectively. Isotopic fractionation constant

234

x

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ε (x = 33 or 34) is also defined as: x

ε = (xα − 1).

(4)

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It is often quoted in ‰ notation. The xε value can be estimated from the relationship

237

between the changes in isotopic ratios and the changes in the substrate concentration 13



238

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(Rayleigh equation),29 as follows: δxS – δxSinitial = xε ln f,

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(5)

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where f =[OCS]/[OCS] initial.

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MIF is often described as the deviation from behavior defined by the

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mass-dependent fractionation law using an equation. We calculated the deviation

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from mass-dependent fractionation in 33S (33E), as follows:

244

33

E = 33ε – 0.515 34ε.

(6)

245

246

3. Results and discussion

247

3.1 Carbonyl sulfide degradation by bacteria

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In batch laboratory experiments, OCS concentrations decreased with time. We

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found that the degradation rates of OCS in bacterial batches were faster than for control

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experiments for all experimental days (Figure 1), indicating that OCS was clearly

251

degraded by the soil bacteria. The living and dead cell numbers of the OCS-degrading

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soil bacteria were not significantly different from numbers of other bacteria

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with/without OCS addition. This shows that the concentration of OCS was not related to

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the growth of OCS-degrading bacteria. Neither does OCS damage the cells of

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OCS-degrading bacteria. The production of H2S during incubation experiments

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corresponds with the decrease in OCS concentrations in any of the bacterial batches (not

257

quantified). H2S was not observed in control batches. This suggests that the decrease in

258

OCS recorded in this study was solely related to bacterial activity. It is worth discussing

259

the observed abiotic consumption of OCS in the control batches in more detail. If the

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OCS degradation in bacterial batches occurred not only because of bacterial OCS

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degradation, but also because of abiotic hydrolysis on slant surfaces and/or a small

262

amount of liquid on the slant, then it is difficult for us to compare bacterial batches

263

performed on different dates (i.e., compare between slants used on different dates).

264

However, the production of H2S during OCS degradation was not observed in all

265

control batches, and this suggests that the decrease in OCS was not related to hydrolysis,

266

because it did not produce H2S. The mechanisms involved in the decrease in OCS are

267

still uncertain, although we surmise it is not related to hydrolysis. It certainly took place

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on the slant surface. In contrast, in our bacterial batches, the surface of the slant was

269

almost covered by bacterial biomass; thus, such abiotic decreases in OCS did not appear

270

to occur. Consequently, it was not necessary to perform additional treatment to consider

271

the contribution of hydrolysis of the OCS for our bacterial batches.

272

To determine k values, OCS degradation curves were fitted to the exponential

273

function C(t) = C0e–kt, according to Kato et al. 2008.21 The fitted plots showed good 15



274

correlation (R2 > 0.87). The average k values obtained (h-1) were 0.31 ± 0.05, 0.13 ±

275

0.00, 0.14 ± 0.00, 0.75 ± 0.05, 0.15 ± 0.01, 0.33 ± 0.07 and 0.11 ± 0.03 for strains

276

THI401, THI402, THI404, THI405, THI410, THI414, and THI415, respectively (Table

277

1). The average cell-specific activities (×1010 h-1 cell-1) were 0.22 ± 0.01, 0.69 ± 0.12,

278

0.18 ± 0.02, 1.12 ± 0.28, 0.27 ± 0.03, 0.83 ± 0.29, and 0.07 ± 0.01 for strains THI401,

279

THI402, THI404, THI405, THI410, THI414, and THI415, respectively (Table 1).

280

We classified cell-specific activity with values over 0.5 h-1 cell-1 as fast, values

281

from 0.1 h-1 cell-1 to 0.5 h-1 cell-1 as moderate, and those under 0.1 h-1 cell-1 as slow. Our

282

results showed that strains THI402, THI405, and THI414 were classified into the fast

283

group, strains THI401, THI404, and THI410 were classified into the moderate group,

284

while strain THI415 was classified as slow. Even though strains THI414 and THI415

285

belong to the same genus, strain THI414 had the fastest cell-specific activity, but strain

286

THI415 had the slowest. Strains THI410 and THI415 showed no significant difference

287

between batches. Consequently, for strains THI410 and THI415, we were able to

288

compare the cell-specific activity among different experiments; experiments conducted

289

on different dates showed no significant differences. Kato et al. 200821 showed that the

290

cell-specific activities (×1010 h-1 c.f.u.-1) were 0.76, 20, 1.7, 2.5, 1.9, 0.77, 0.075 for

291

strains THI401, THI402, THI404, THI405, THI410, THI414, and THI415, respectively. 16


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292

21

293

activity. We found slight differences between our current results and those reported by

294

Kato et al. 2008.21 Although the cell-specific activities of strains THI414 and THI415

295

were consistent with the previous study,21 strains THI401, THI402, THI404, THI405,

296

THI410 had slower activities than reported in Kato et al. 2008.21 The reasons for these

297

differences are not clear; however, the initial concentration of OCS in the previous work

298

was lower (30 ppmv) than in this study, and the method used for cell counts was also

299

different. Such differences in cell-specific activity are not important for comparing 34ε

300

values of OCS, as described below.

They found that strain THI402 had the fastest, and strain THI415 had the slowest

301 302

3.2 Sulfur isotopic fractionation of carbonyl sulfide during bacterial degradation

303

The δ34S values increased as bacterial OCS degradation occurred in all

304

bacterial batches (Figure 2). Correlation between δ34S values and Ln f was over 0.89

305

(Table 1). Consequently, bacterial degradation can be treated as a single step reaction.

306

Thus,

307

correlation curves.

308 309

33

ε and 34ε values for each batch could be determined using the slopes of their

The

34

ε values determined for Mycobacterium spp. and Williamsia sp. were

−3.67 ± 0.33‰, −3.99 ± 0.19‰, −3.57 ± 0.22‰, −3.56 ± 0.23‰, −3.74 ± 0.29‰, for

17



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strains THI401, THI402, THI404, THI405, and THI410, respectively (Table 1). In

311

contrast, the 34ε values for Cupriavidus spp. were smaller than those for the other genera

312

(Figure 2), yielding −2.09 ± 0.07‰ and −2.38 ± 0.35‰ for strains THI414 and THI415,

313

respectively (Table 1). No significant deviations in 34ε values were observed within the

314

same species, even though experiments for strains THI401, THI410, and THI415 were

315

conducted on different dates. Our results suggest that slant surface and period of

316

pre-incubation had no significant effect on fractionation processes. Clearly, distinct

317

isotopic fractionation constants were observed. The

318

Mycobacterium spp. and Williamsia sp. contrast markedly with the

319

−2.2‰ for Cupriavidus spp. Differences in

320

concentrations of OCS, ranging from 2,000 to 4,000 ppmv, based on our comparison of

321

experimental runs THI 401-1 and -2 (4000 ppmv) and THI 401-3 and -4 (2000 ppmv).

322

Furthermore, the

323

even though different cell-specific activities were observed for Mycobacterium spp. and

324

Cupriavidus spp. (Table 1). Thus, although the numbers of bacterial species examined

325

are limited, our results suggest that

326

genus-level biological differences. Possibly, differences in enzyme and/or membrane

327

functions exist between bacterial strains in this study. The isotope fractionation during

34

34

ε values of ca. −3.7‰ for 34

ε value of ca.

34

ε values did not reflect initial

ε values showed no significant differences within the same genus,

34

ε values for OCS bacterial degradation reflect

18


Page 19 of 39


328

OCS degradation by bacteria is described by a pathway, in which fractionation varies as

329

a function of OCS movement into or out of the cell, as well as in the reduction step in

330

Rees’s model.30 We provide a schematic diagram showing bacterial OCS degradation in

331

Figure 3. Thus, the overall net isotopic fractionation (εnet) can be expressed as:

332

εnet = εdif + (εenz − εdif) kout / (kout + kenz)

(7)

333

where εm and εe are the isotopic fractionation constants for the membrane and enzyme

334

processes. Rate constants for diffusion into and out of the cell, as well as for enzyme

335

decomposition are given by kin, kout and kenz. At present, it is difficult to identify which

336

process is dominant, i.e., whether kout and/or kenz mostly affect OCS isotopic

337

fractionation. To understand the metabolic processes involved in OCS degradation by

338

bacteria, further experiments using strains with an isolated enzyme25 are required.

339

The determined sulfur isotopic fractionation for bacterial OCS degradation in

340

this study is relatively small rather than other sulfur isotopic fractionations reported for

341

a variety of other bacterial systems. For example, bacterial sulfate reduction generally

342

produces sulfides depleted in

343

with several factors.31–33 Bacterial disproportionation of sulfur intermediates can also

344

produce large sulfur isotope fractionation with depletion in

345

enrichment in

34

34

S by more than 40‰, although the fractionation vary

34

S by 5–7‰ and

S by 17–21‰ for sulfide and sulfate, respectively.34 Sulfur isotopic 19



Page 20 of 39

346

fractionations during oxidation reactions are limited, because it is difficult to isolate and

347

cultivate the environmentally-relevant sulfur-oxidizing organisms. Previous laboratory

348

experiments with sulfur-oxidizers have shown fractionations in

349

to +5‰, but recent in situ sulfide oxidation showed up to +8‰ fractionation in 34S.35 In

350

contrast to the other bacterial systems, conversion from OCS to H2S does not require the

351

redox change in sulfur. We, therefore, speculate that this might be one of the factors

352

explaining this relatively small fractionation for bacterial OCS degradation.

34

S varying from −6‰

353 354

3.3 The three-isotope plot for carbonyl sulfide during bacterial degradation

355

The results of our isotope measurements are plotted in ln (δ33S + 1) versus ln

356

(δ34S + 1) diagrams (i.e., a three-isotope plot) (Figure 4). The log scale is used here to

357

take into account the power law relationship of MDF. Standard errors are derived from

358

linear regression and depend on residuals of the fits. The slopes ranged from 0.47 to

359

0.61. The ∆33S values estimated from Eq. 2 with their standard error for each bacterial

360

strain were − 0.19 ± 0.24‰, −0.12 ± 0.17‰, −0.10 ± 0.00‰, −0.10 ± 0.03‰, −0.11 ±

361

0.00‰, −0.22 ± 0.06‰, and −0.07 ± 0.06‰ for strains THI401, THI402, THI404,

362

THI405, THI410, THI414, and THI415, respectively (Table 1). These values are

363

equivalent to zero, i.e., they are within the range of measurement error. Our results

20


Page 21 of 39


364

indicate that bacterial OCS degradation processes are a MDF process.

365

MIF signatures in sulfur isotopes found in polar ice core records of sulfate from

366

SSA show initially positive ∆33S values,36,37 although the change in ∆33S values for

367

these background SSA are not known. This suggests that MIF of sulfur occurred in the

368

stratosphere. Positive ∆33S values also have been observed for tropospheric sulfate,38,39

369

suggesting there are contributions from other atmospheric sources (upper troposphere or

370

lower stratosphere).40 It also has been suggested that MIF of sulfur in the atmosphere is

371

derived from CS2 oxidation,41 SO2 photolysis,42,43 photoexcitation,44,45 and intersystem

372

crossing.43,46,47 In contrast, OCS is not expected to produce MIF during its oxidation in

373

atmospheric sink reactions.13-15,45 In this study, experimental bacterial OCS degradation

374

indicated that bacterial processes also are not responsible for the positive ∆33S values

375

found in polar ice/snow and tropospheric sulfate aerosols.

376 377

3.4 Implications

378

The 34ε values for OCS during degradation by soil bacteria in our experiments

379

ranged from −2.2‰ to −3.7‰. If bacteria isolated from soil degrade OC32S faster than

380

OC34S, then residual OCS should be enriched in 34S. Thus, the δ34S values for OCS on

381

the ground surface should increase with respect to atmospheric OCS, as well as sites

21



382

where there is no OCS consumption by soil. OCS concentrations observed on the

383

summit of Mt. Fuji, Japan (ca. 720 ppt; 3776 m a.s.l.; 35°21'N, 138°43'E) are higher

384

than at its foothills (ca. 570 ppt;.1300 m a.s.l.; 35°20'N, 138°48'E).27 Diurnal change in

385

OCS concentration at these sites were not detected.27 This lack of diurnal change

386

suggests that OCS uptake by photosynthesis in plants may be negligible at Mt. Fuji.

387

Therefore, we assumed that the differences in OCS concentrations between summit and

388

foothills were related solely to soil bacterial degradation. In this case, the isotopic

389

fractionation constants determined in this study are representative for such soil bacteria,

390

and can be used to model the fractionation of atmospheric OCS at Mt. Fuji. We

391

estimated an enrichment in

392

atmospheric OCS close to the soil surface using Eq. (5). Based on Eq. (5) using

393

concentration of atmospheric OCS of summit and foothills of Mt. Fuji,27 we expect that

394

the δ34S values for atmospheric OCS will show about 0.5‰ to 0.9‰ difference between

395

the summit and foothills of Mt. Fuji. Given the δ34S value of OCS at Kawasaki of 4.9 ±

396

0.3‰, Japan,16 then we surmise a δ34S value of OCS of 5.4‰ to 5.8‰ for the soil

397

surface at the foothill of Mt. Fuji.

34

S of residual OCS in the δ34S value determined from

398

In summary, we determined the isotopic fractionation constants (33ε and 34ε) for

399

OCS undergoing bacterial OCS degradation for the first time. Although the cell-specific 22


Page 22 of 39

Page 23 of 39


400

activity values were slightly different in strains of the same genus, differences at the

401

generic level were more marked. The

402

(THI401, THI402, THI404 and THI405) and Williamsia sp. (THI410), varied

403

significantly from the ca. −2.2‰ for Cupriavidus spp. (THI414 and THI415). The

404

mechanisms controlling sulfur isotopic fractionation during bacterial OCS degradations

405

are still not known. Thus, the relationship between sulfur isotopic fractionation for both

406

enzymatic and membrane processes should be investigated in future studies to evaluate

407

their

408

chemolithoautotrophic bacterium Thiobacillus thioparus strain THI115 that uses OCS

409

as its sole energy source,48 and in which its OCS-degrading enzyme (COSase) has been

410

purified.25 This would provide a baseline for enzymatic fractionation measurements. In

411

this study, all experiments were conducted with OCS concentrations higher than in the

412

ambient atmosphere, because it is very difficult to conduct low concentration

413

experiments (e.g., they require a high volume). The

414

laboratory study should be further calibrated with field observations. We carried out

415

preliminary estimates for δ34S in soils to evaluate its impact on the atmosphere. We

416

estimated 0.5 to 0.9‰ enrichments occur between summit and foothill sites at Mt. Fuji

417

solely linked to soil bacterial OCS degradation. We conclude that OCS sulfur isotope

respective

contributions.

34

ε values of ca. −3.7‰ for Mycobacterium spp.

This

could

be

better

34

evaluated

using

a

ε values obtained in this

23



418

analysis provides a new tool to investigate soil OCS sinks in the troposphere. In the

419

future, atmospheric observations of δ34S values for OCS will help refine estimates of

420

soil bacterial activity and its contribution to OCS degradation in the troposphere.

421

422

Acknowledgements

423

We would like to thank S. Ishino for her technical assistance with the experiments and

424

fruitful discussion of the data. We are grateful to members of Yoshida’s laboratory and

425

Katayama’s laboratory for their advice and assistance. This work is supported by a

426

Grant-in-Aid for Scientific Research (S) (23224013) from the Ministry of Education,

427

Culture, Sports, Science and Technology (MEXT), Japan.

428

429

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32


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Page 33 of 39


579

TOC art

580 581

582

33



583 584

Figure 1. Time series showing OCS degradation by bacteria grown on a PYG slant (a)

585

Mycobacterium spp., (b) Williamsia sp. and (c) Cupriavidus spp. Control batches are

586

uninoculated sterilized PYG mediums. The concentrations of OCS measured at least

587

five time points per experiment. Experiments were terminated when OCS

588

concentrations were ca. 70% of their initial concentrations.

589 34


Page 34 of 39

Page 35 of 39


590

591 592

593

Figure 2. Changes in δ34S values relative to initial values as a function of the natural

594

logarithm of the fraction of remaining carbonyl sulfide (ln f). Circles (open, closed and

595

colored) represent strains of Mycobacterium spp., triangles (open, closed, colored)

596

represent strains of Williamsia sp., and squares (open, closed, colored) represent strains

597

of Cupriavidus spp.

598 35



599 600

601

Figure 3. Carbonyl sulfide (OCS) uptake and reduction in bacterial cells. Schematic

602

diagram showing OCS uptake and H2S production by bacteria. OCS diffuses in (kin),

603

and out (kout) of the membrane, as well as undergoes decomposition by enzymes (kenz).

604

36


Page 36 of 39

Page 37 of 39


605 606

607

Figure 4. Sulfur isotopic compositions of residual carbonyl sulfide. The dotted line is

608

the mass-dependent fractionation line, with slope (0.515). Circles (open, closed and

609

colored) represent strains of Mycobacterium spp., triangles (open, closed, colored)

610

represent strains of Williamsia sp., and squares (open, closed, colored) represent strains

611

of Cupriavidus spp.

612 37



613 614 615 616 617

Page 38 of 39

Tables

Table 1. Comparison of cell-specific activity and sulfur isotopic fractionations for carbonyl sulfide degradation between various bacterial strains grown on the PYG slants. Note: ID defines the measurement day for given experiments.

33

Bacteria

Mycobacterium sp.

Williamsia sp.

Cupriavidus sp.

ID

a

Strain

A A Bb B

THI401

A A

THI402

C C

c

THI404

C C

THI405

A Dd D

THI410

e

E E

THI414

Batches

Isotope sampling times

1 2 3f 4f average

6 6 6 5

1 2 average 1 2 average 1 2 average 1 2 3 average 1 2

6 6 6 6 6 6 6 6 N.A. 6 6

Rate constant

Cell number#

Cell-specific activity*

(h-1) 0.28 0.27 0.30 0.39 0.31±0.05

(cell) 1.35 1.18 N.D N.D 1.27±0.12

(h-1 cell-1) 0.21 0.23 N.D N.D 0.22±0.01

0.13 0.12 0.13±0.00 0.14 0.14 0.14±0.00 0.70 0.79 0.75±0.05 0.14 0.14 0.16 0.15±0.01 0.40 0.26

0.23 0.15 0.19±0.05 0.86 0.69 0.77±0.12 0.50 0.94 0.72±0.31 0.44 0.58 0.61 0.54±0.09 0.36 0.48

0.56 0.81 0.69±0.12 0.16 0.20 0.18±0.02 1.39 0.84 1.12±0.28 0.31 0.24 0.27 0.27±0.03 1.12 0.54

38


34

ε

‰

−2.01 −2.61 −2.11 −1.60 −2.08±0.36 −1.91 −2.45 −2.18±0.27 −1.83 −2.06 −1.94±0.11 −2.02 −1.85 −1.94±0.09 −2.19 −1.89 N.A.

−2.04±0.15 −1.20 −1.39

33

ε

R2 0.96 0.96 0.85 0.97 0.82 0.97 0.99 0.99 0.93 0.92 0.94 0.94 N.A. 0.97 0.88

‰

−3.74 −4.18 −3.38 −3.39 −3.67±0.33 −3.79 −4.18 −3.99±0.19 −3.35 −3.79 −3.57±0.22 −3.79 −3.33 −3.56±0.23 −4.03 −3.45 N.A.

−3.74±0.29 −2.03 −2.16

Ε

R2 0.98 0.98 0.89 0.97 0.90 0.96 0.98 0.99 0.95 0.98 0.95 0.98 N.A.

‰

−0.08 −0.45 −0.37 0.15 −0.19±0.24 0.04

−0.29 −0.13±0.17 −0.10 −0.10 −0.10±0.00 −0.07 −0.13 −0.10±0.03 −0.11 −0.11 N.A.

−0.11±0.06 0.99 −0.15 0.97 −0.28

Page 39 of 39

618 619 620 621 622 623 624 625


average D THI415 1 6 D 2 6 E 3 6 E 4 5 average # (x10-10); *(x1010); N. A. = not available; N. D. = not detected.

0.33±0.07 0.15 0.13 0.07 0.08 0.11±0.03

0.42±0.09 2.26 1.77 1.04 1.39 1.61±0.52

a

0.83±0.29 0.07 0.07 0.07 0.06 0.07±0.01

4-Mar-15, Pre-incubation period was 7 days. 20-Nov-14, Pre-incubation period was 7 days. c 11-Apr-15, Pre-incubation period was 5 days. d 13-Apr-15, Pre-incubation period was 5 days. e 7-Mar-15, Pre-incubation period was 2 days. f Initial OCS concentration was 2000 ppmv, approximately 4000 ppmv in other bacterial batch. b

39


−1.29±0.09 −1.02 −1.39 −1.42 −1.36 −1.29±0.16

0.93 0.92 1.00 0.95

−2.09±0.07 −1.80 −2.76 −2.51 −2.45 −2.38±0.35

−0.22±0.06 0.96 −0.09 1.00 1.00 0.99

0.03

−0.13 −0.10 −0.07±0.06

Sulfur Isotopic Fractionation of Carbonyl Sulfide during Degradation

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