Earth Planet. Sci. Lett. 624 (2023) 118446

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Large sulfur isotope fractionation during abiotic consumption of hydrogensulfide after cessation of bacterial growth in batch culture experiments ofsulfate reducing bacteria
Fumihiro Matsu’ura a, b, *, Hiroko Makita c, d, Ken Takai d, Mayuko Nakagawa b, e,
Yuichiro Ueno b, d, eaInternational Center for Isotope Effects Research, Nanjing University, No.163 Xianlin Avenue, Qixia District, Nanjing, Jiangsu Province 210023, China
Earth-Life Science Institute (ELSI), Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro-ku, Tokyo 152-8550, Japanc
Department of Ocean Sciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japand
Institute for Extra-Cutting-Edge Science and Technology Avant-Garde Research, (X-star), Super-cutting-edge Grand and Advanced Research (SUGAR) Program, Japan
Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, 237-0061, Japane
Department of Earth and Planetary Sciences, Tokyo Institute of Technology, Meguro, Tokyo 152-8551, Japanb

A R T I C L E I N F O

A B S T R A C T

Keywords:
Large sulfur isotope fractionation
Sulfate reducing bacteria
Abiotic sulfide consumption
Organic sulfur
Sulfide mineral precipitation

Microbial sulfate reduction is a key process in the oceanic sulfur cycle and imparts a large sulfur isotope frac­tionation. The mechanism of sulfur isotope fractionation during microbial sulfate reduction has been studied
from the geochemical and biochemical aspects since the 1950s. Recently, however, the large sulfur isotope
fractionation, exceeding 47‰, has been observed in some pure culture experiments of sulfate reducing bacteria
and the tentative understanding of microbial sulfur isotope fractionation has not well explained the mechanism.
Here we quantified growth phase dependent sulfur isotope fractionation of a type sulfate reducer Desulfovibrio
desulfuricans (DSM642) and revealed that the magnitude of isotope fractionation increased from -13.4 ± 3.6‰ inearly exponential phase to -65.9 ± 21.0‰ in later exponential phase having clear negative correlation with cell
specific sulfate reduction rate. Our results show the importance of cells growth phase and states that control cell
specific sulfate reduction rate and sulfur isotope fractionation. Although microbial sulfate reduction likely
continued to stationary phase of cells, microbial sulfur isotope fractionation could not be quantified because of
the decreasing total dissolved sulfide (ΣH2S: H2S, HS− , and S2− ) concentration after stationary phase. A nonmicrobial ΣH2S-consuming reaction is interpreted to have occurred during the stationary phase and this hadlarge sulfur isotope fractionations, -10.5 ± 1.1‰ and -45.6 ± 12.4‰. Besides, the non-microbial ΣH2Sconsuming reaction decreased Δ33S’ value of the ΣH2S. While the end-product of the non-microbial ΣH2Sconsuming reaction in the stationary phase remains unidentified, we observed precipitates of some sulfide
minerals and organic sulfur in the stationary phase media. Regardless of the end-product, the reaction increases
the sulfur isotopic composition of dissolved ΣH2S, which can account for the high sulfur isotopic compositions of
ΣH2S compared to pyrite and organic sulfur observed in some modern marine sediments.

1. Introduction
Sulfur isotope fractionation by sulfate-reducing microbes is a key tounderstand biogeochemical sulfur cycle in the past and present Earth(Johnston, 2011). The sulfur isotope fractionation has been well known
to change with sulfate concentration, species and concentration ofelectron donor, temperature, and so on (Sim et al., 2011a, 2011b;

Leavitt et al., 2013; Bradley et al., 2016; Pellerin et al., 2020). However,
the mechanism of the sulfur isotope fractionation has not been fully
understood despite of many studies conducted (Sim et al. 2017; Bertranet al., 2018).
The mechanism has been discussed based on the steady state model
developed by Rees (1973). The steady state model provides an insightthat the net sulfur isotope fractionation during microbial sulfate* Corresponding author at: International Center for Isotope Effects Research, Nanjing University, No.163 Xianlin Avenue, Qixia District, Nanjing, Jiangsu Province
210023, China.
E-mail address: matsuura.f@nju.edu.cn (F. Matsu’ura).
https://doi.org/10.1016/j.epsl.2023.118446
Received 8 June 2022; Received in revised form 13 October 2023; Accepted 15 October 2023
Available online 31 October 2023
0012-821X/© 2023 Elsevier B.V. All rights reserved.

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Earth and Planetary Science Letters 624 (2023) 118446

reduction approaches the equilibrium isotope fractionation of sulfate
and total dissolved sulfide (ΣH2S: H2S, HS− , and S2− ) at ambient tem­
perature when the rates of all intracellular enzymatic reactions become
slow enough to be approximated as reaching equilibrium. The results ofincubation experiments by Sim et al. (2011a) provided a good fit to thesteady state model that the fractionations obtained from their experi­ments approached the equilibrium value at room temperature. Howev­
er, the sulfur isotope fractionation and reaction at each enzymatic step
have been just indicated and the whole processes and interactions ofeach enzymatic step have not been completely understood (Santos et al.,2015; Leavitt et al., 2019).
Recently Sim et al. (2017) for the first time indicatedgrowth-phase-dependent concentration and isotopic composition of
intracellular sulfur species, and suggested that a closed system effect on
the intracellular sulfate would be important to account for the sulfur
isotope fractionation of microbial sulfate reduction. Although growthphases and states of microbes alter the chemical composition of intra­
cellular sulfur species, only a few studies documented the influence ofthe growth phases and states on sulfur isotope fractionation during
microbial sulfate reduction (Matsu’ura et al., 2016a; Pellerin et al.,2018). The small amount of ΣH2S produced during stationary phase (thestationary phase is defined as a growth phase where cell growth rate isnull) of microbial growth may have imposed a limitation on quantifying
the isotope fractionation during maintenance metabolism especially inprevious batch culture experiments. Here, we carried out batch typeglucose culture experiment of Desulfovibrio desulfuricans (DSM642) toquantify sulfur isotope fractionation during stationary phase. We founda drastic change of the sulfur isotope fractionation from early to late
exponential cells (the exponential phase is defined as a growth phase
where cell numbers increase exponentially with constant growth rate),
although the much lower ΣH2S concentration during the stationary
phase prevented us from quantifying the sulfur isotope fractionation
during stationary phase. Instead, we found that non-microbial
ΣH2S-consuming reactions became dominant during the stationary
phase, which imparts large sulfur isotope fractionations and increases
the sulfur isotopic composition of ΣH2S. The ΣH2S-consuming reaction
can account for high δ34S values of ΣH2S compared to pyrite or organic
sulfur observed in some modern marine sediments.

start experiment. The inoculum of sulfate-reducing bacterium waspre-cultivated in the same medium at 30 ◦ C. Series-1 experiment was
conducted without shaking and series-2 experiment was conducted withshaking at 180 rpm. The shaking experiment was conducted to confirm
the results of Matsu’ura (2016b).
2.2. Cell growth and chemical analyses
Growth of D. desulfuricans (DSM642) was measured by direct cellcounting after staining with 4,6-diamidino-2-phenylindole (DAPI) usinga Leica AF6000 advanced fluorescence imaging system with Leica
DM5500B microscope. In series-1 experiment, cell counting was con­ducted using 100 μL of medium of harvesting 2 vials (Table S1). In
series-2 experiment, cell counting was conducted using 100 μL of me­
dium of vials prepared for cell counting, H’-1 and H’-2 (Table S1).
Cultures were collected alphabetically at 21, 43, 70, 140, 360, 645,
and 1801 h after inoculation of series-1 experiment and at 20, 44, 70,
116, 210, 402, and 760 h after inoculation of series-2 experiment(Table S2). ΣH2S concentration was analyzed for all three vials at eachcollection time, and sulfate concentration, isotopic composition of sul­fate and ΣH2S were analyzed for 2 of 3 vials at each collection time.
ΣH2S concentration was determined by the colorimetric methyleneblue method (Cline, 1969) with 400 μL of each collected medium filteredthrough 0.22 μm syringe filter, and the uncertainties were based on themethod of linear regression. ΣH2S in each vial was collected as silver
sulfide by following procedure. At first 1 mL of 45.6 mM of alkaline zincacetate solution was added to each vial to precipitate ΣH2S as ZnS andterminate cell activity, followed by injection of 1 mL of 6N HCl to thevial to acidify the medium and convert ZnS to H2S. The H2S gas was
scrubbed with distilled water by purging with N2 gas and finally trapped
as Ag2S by reaction with silver nitrate solution. After completion ofpurging H2S, 1 mL of 1 M BaCl2 solution was added to the vial to collectsulfate as BaSO4. The collected Ag2S and BaSO4 were then washed byrepeated centrifugation using distilled water. The collected BaSO4 wasweighed and further reduced to H2S by the reaction with tin (II)-Strong
phosphoric acid solution (Kiba et al., 1955). The evolved H2S wascollected as Ag2S in the same way as described above. Sulfate concen­
tration was determined from the weight of collected BaSO4 with theuncertainty of 3 % for series-1and 1.5 % for series-2 experiments (1σ).
Cell specific sulfate reduction rates (fmol/cell/day) are described as
Eq. (1) (Detmers et al., 2001))/ ) ())/( ( (csSRR = (MH2S(i+1) − − MH2S(i) )C(i+1) + C(i) 2 × T(i+1) − T(i) .

2. Method
2.1. Batch culture experiments(1)

We used a sulfate-reducing bacterium, Desulfovibrio desulfuricans(DSM 642) for the batch culture experiments in the same as Matsu’ura
et al., (2016a). We conducted 2 series of experiments (series-1 andseries-2) for isotope analysis and 1 series of supplemental experiments(series-3: see supplemental information) for detailed chemical analysis
and microscopic observation of precipitates in incubation media. We
prepared 3 L of medium and about 30 bottles of 100 ml glass vials foreach series of experiment. We named each vial as A-1 to J-3 for theseries-1 experiment and A’-1 to H’-3 for the series-2 experiment(Tables S1 and S2). Three vials were harvested at each collection time
for both series of experiments. In each glass vial, 40 mL of culture me­dium for the sulfate reducing bacteria (SRB) [modified DSMZ 63] wasinjected, and purged with N2 gas. The vial was plugged with a butyl
rubber stopper. Modified DSMZ 63 contains K2HPO4, 0.5 g; NH4Cl, 1.0 g;
Na2SO4, 1.0 g; CaCl2 × 2H2O, 0.1 g; MgSO4 × 7H2O, 2.0 g; D(+) glucose,
2.7 g; yeast extract, 1.0 g; resazurin, 1.0 mg; ascorbic acid, 0.1 g;
Na-thioglycolate, 0.1 g; distilled water, 1 L. The pH value was adjusted
at 7.0. Before adding ascorbic acid and Na-thioglycolate, the 3L medium
was autoclaved for degassing, which may have resulted in some water
loss due to evaporation. Glucose was used both as an electron donor andas a carbon source. After autoclaving and storing the glass vials at
experimental temperature, 30 ◦ C, 100 μL of bacterial culture was inoc­
ulated into fresh medium of all 30 vials except for 3 control samples tohere C and MH2S refer to cell density and concentration of ΣH2S at ith
and (i+1)th of time of harvesting vials. The uncertainties on all the
calculated values in this study are denoted as σ and calculated byanalytical values, analytical uncertainties, and equation of uncertainty
propagation (eqs. S1, S2, S3, S5, S7, S9, and S10), respectively.
2.3. Cell free experiments
A cell free experiment was carried out to confirm whether thedecreasing ΣH2S in the stationary phase was related to microbial activity
or not. In addition, a supplemental cell free experiment was conductedto confirm whether glucose was involved in the reaction (see supple­
mental information). To prepare the ΣH2S solution, 0.312 g of
Na2S•9H2O and resazurin was dissolved in 300 mL of degassed distilledwater while continuing N2 purging of the water. The pH was adjusted to
7.0. The N2 purge of the solution was continued for 40 min after pH
adjustment to deoxidize the solution, although at the same time H2S gas
leaked from the solution. After the N2 purge, 5 mL of the ΣH2S solution
was added to each of 6 uninoculated medium, which are same as used inseries-2 experiment. The 6 vials were immediately set to incubator at
30 ◦ C without shaking. Two vials were collected at each collection time2

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Earth and Planetary Science Letters 624 (2023) 118446

Fig. 1. Cell densities, sulfate concentrations, and ΣH2S concentrations of series-1 and series-2 experiments. (A) Cell growth of entire series of experiments. (B) Cell
growth of early stage of incubation. (C) Sulfate concentration throughout the experiments. (D) Sulfate concentration of early stage of the incubation. (E) ΣH2S
concentration throughout the experiments. (F) ΣH2S concentration of early stage of the incubation. Blue and green plots are series-1 and series-2 experiments,
respectively.

of 22 h, 335 h, and 743 h after setting the vials in the incubator. ΣH2S
concentration of each collected medium was determined same as
described above. The pH of all collected medium was checked andranged from 6.62 to 6.80.

removed by a cold trap maintained at -90 ◦ C. The pre-treated SF6 wasfurther purified by gas chromatography (GC8A, Shimadzu), equippedwith the first 4 m column packed with Porapack-Q (1/8″ OD, 80-100mesh) connected with the second 3 m Molecular Sieves 5A column(1/8″ OD, 60-80 mesh) at 50 ◦ C oven temperature and a 25 mL/min of
He flow rate. Purified SF6 was introduced into a mass spectrometer
33 +
SF5 ,
(Thermo Fisher Scientific, MAT253) and abundances of 32SF+
5,
34 +
36 +
SF5 , and SF5 were measured in a dual inlet mode.
Sulfur isotopic compositions are reported as below.
()(2)
δ3x S = 3x Rsample /3x Rstandard − 1 × 1000

2.4. Sulfur isotope analyses
Multiple sulfur isotope analysis was carried out via a flash heating
method with CoF3 (Ueno et al., 2015). The Ag2S samples from ΣH2S andsulfate were wrapped with CoF3 in a thin foil that consists ofiron-nickel-cobalt alloy (pyrofoil) and heated to 590 ◦ C for 3 seconds by
Curie-point pyrolyzer (JHP-22, Japan Analytical Industry Co., Ltd.). The
Ag2S was converted to SF6, followed by collection in a cold trap at-196 ◦ C (Trap-1) by using liquid nitrogen. Subsequently, HF was)(δ3x S′ = ln 3x Rsample /3x Rstandard × 1000

3(3)

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Earth and Planetary Science Letters 624 (2023) 118446

Fig. 2. (A) ΣH2S concentration in the stationary phase cells and the cell free experiment. The time of 360 h in series-1 experiment and 210 h in series-2 experiments
are set to 0h for the two series of experiments. The ΣH2S decreasing rate of the cell free experiment was obtained from the slope of the linear regression line as -0.081
μM/h. (B) Evolution of δ34S’ and Δ33S’ values in the stationary phase in series-1 experiment with calculated Rayleigh distillation equation model. The data align wellwith model calculation. (C) Evolution of δ34S’ and Δ33S’ values in the stationary phase in series-2 experiment with calculated Rayleigh distillation equation model.
The data deviate largely from the model calculation with original 34εst and 33λst values, -45.6‰ and 0.5079, respectively (gray lines). (D) Cross plots of ΣH2S
concentration and δ34SSO4 value in the stationary phase of the two series of experiments. The calculated slope of series-2 experiment (-2.99 μM/‰), was used for the
calculation of the impact of the ΣH2S-consuming reaction on the 34εSR values (see supplement).

Δ33 S′ = δ33 S′ − 0.515 × δ34 S′

where 3xαSR is sulfur isotope fractionation factor of microbial sulfate
reduction. The sulfur isotope fractionation factor is described as()/( 3x)3xαSR =3x RH2S Inst /3x RSO4 = δ3x SH2S Inst + 1000
δ SSO4 + 1000 .
(6)

(4)

where 3x denotes 33 or 34, 3xRsample denotes isotopic ratio of samples(3xS/32S). All values are reported relative to V-CDT assuming that IAEAS1 has a composition on the V-CDT scale of δ34S = -0.3‰ and Δ33S’ =+0.100‰ (Ding et al., 2001; Ono et al., 2006). The analytical uncer­
tainty (1σ) on δ34S and Δ33S’ for series-1 experiment are ±0.78‰ and ±0.005‰ and are ±0.26‰ and ± 0.009‰ for series-2 experiment. The
uncertainties are based on replicate analyses of IAEA-S1.

where xRH2S_Inst and δxSH2S_Inst denote isotopic ratio and isotopic
composition of ΣH2S produced between ith and (i+1)th of time of har­vesting vials, respectively. The 3xRSO4 and δ3xSSO4 denote the average ofisotopic ratio and isotopic composition of sulfate at the (i+1)th har­vesting vials. Equations for calculating the values and uncertainties on
δ3xSH2S_Inst, δ3xS SO4, and δ3xSTS (isotopic composition of total sulfur ineach vial) are described in supplemental information.
Sulfur isotope fractionation of the ΣH2S-consuming reaction in sta­tionary phase is described as()
3xεSt = 1000 × 3x αSt − 1(7)2.5. Isotope fractionation calculations
In this study, sulfur isotope fractionation of microbial sulfate
reduction in exponential phase is described as()
3xεSR = 1000 × 3x αSR − 1(5)4

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Earth and Planetary Science Letters 624 (2023) 118446(caption on next page)5

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Earth and Planetary Science Letters 624 (2023) 118446

Fig. 3. Temporal variations of the isotopic compositions of sulfate and ΣH2S, sulfur isotope fractionation of microbial sulfate reduction in exponential
phase—34εSR—, and isotopic composition of total sulfur—δ34STS—. (A) Temporal variation of the δ34SSO4 values throughout the experiments. (B) Enlarged figure of(A) in early stage of the experiments. (C) Temporal variation of the δ34SH2S values throughout the experiments. (D) Enlarged figure of (C) in early stage of theexperiments. (E) Temporal variation of the Δ33S’SO4 values throughout the experiments. (F) Enlarged figure of (E) in early stage of the experiments. (G) Temporalvariation of the Δ33S’H2S values throughout the experiments. (H) Enlarged figure of (G) in early stage of the experiments. (I) Temporal variations of δ34SSO4 andδ34SH2S_Inst in the exponential phase of the two series experiments. Circles and squares denote δ34SSO4 and δ34SH2S_Inst, respectively. Blue and green colors denoteseries-1 and series-2 experiments, respectively. The 34εSR value was obtained from these values. (J) Temporal variation of 34εSR value in the exponential phase of thetwo series of experiments. (K) Temporal variation of the δ34STS values throughout the experiments. Blue and green plots are series-1 and series-2 experiments,
respectively.

deceasing after 140 h to 69.9 ± 3.7 μM at 1801 h during the stationary
phase in series-1 experiment (Fig. 1, Table S2). The average ΣH2S con­centration increased from 64.3 ± 6.3 μM at 44 h to 139.0 ± 2.9 μM at
116 h during the exponential phase and started decreasing after 210 h to
95.7 ± 5.1 μM at 762 h during the stationary phase in series-2 experi­
ment (Fig. 1, Table S2).
The average ΣH2S concentration decreased from 120.5 ± 3.7 μM at
22 h to 60.3 ± 3.9 μM at 743 h in the cell free experiment (Fig. 2A). The
decreasing ΣH2S concentration in the cell free experiment is consistent
with those of the stationary phase of the series-1 and -2 experiments(Fig. 2A).

where 3xαSt is sulfur isotope fractionation factor of the ΣH2S-consuming
reaction and described as
3xαSt =3x Rproduct /3x RH2S .

(8)

where 3xRproduct and 3xRH2S denote isotopic ratio of the product of thestationary phase reaction and of ΣH2S, respectively. The isotope frac­tionation factor and the uncertainty of the reaction of the stationary
phase is calculated from Rayleigh distillation equation described as()
δ3x S′H2S(j) = δ3x S′H2S(j− 0) + 1000 × 3x αSt − 1 × lnf(9)where δ3xS’H2S(j) denotes the isotopic composition of ΣH2S in eachcollected sample during the stationary phase. The f value denotes frac­tion of ΣH2S concentration relative to the initial concentrations of ΣH2S
in stationary phase of each series of experiment; the initial ΣH2S con­centrations are fixed as the concentration of E-2 (173.1 μM) in series-1
experiment and E’-2 (148.9 μM) in series-2 experiment. The linear
regression of the plots of δ3xS’H2S(j) and lnf provide the isotope frac­tionation factor, the isotopic composition of initial ΣH2S (δ3xS’H2S(j-0)),
and uncertainties of them (Eq. 9, Fig. S1). Because of the large uncer­
tainty on the lnf value, we employed straight line fitting with un­
certainties present in both the x and y variables (Fig. S1, see Appendix A
in Matsu’ura et al., 2016a).
The mass dependence of the isotope fractionation during the reactionof the stationary phase is described as
)/ (
)
(
33
(10)
λSt = ln 33 αSt ln 34 αSt .

3.3. δ34S values of sulfate and ΣH2S
The average δ34S value of sulfate in the series-1 experiment increased
from -2.5 ± 0.2‰ at 21 h to -1.7 ± 0.2‰ at 140 h during the exponential
phase and continued increasing to -1.0 ± 0.2‰ at 1801 h during thestationary phase (Fig. 3A, Table S2). The average δ34S value of sulfate in
the series-2 experiment increased from -1.7 ± 0.2‰ at 20 h to -1.2 ±0.15‰ at 116 h during the exponential phase and became almost con­stant after 116 h (Fig. 3A, Table S2). The δ34S value of C’-1 was - 3.7‰,
which is low compared to other samples (Fig. 3B, Table S2).
The average δ34S value of ΣH2S in the series-1 experiment decreased
from -0.3 ± 0.8‰ at 21 h to -29.4 ± 0.5‰ at 140 h during the expo­
nential phase and was increasing after 140 h to -15.5 ± 0.1‰ at 1801 hduring the stationary phase (Figs. 3C and 3D, Table S2). The average
δ34S value of ΣH2S in the series-2 experiment decreased from -6.9 ±0.3‰ at 44 h to -27.2 ± 0.3‰ at 116 h during the exponential phase andwas increasing after 210 h to -18.7 ± 0.2‰ at 762 h during the sta­tionary phase (Figs. 3C and 3D, Table S2).
The δ34STS value continuously increased from -2.5 ± 0.2‰ at 21 h to
-1.1 ± 0.2‰ at 1801 h concomitant with the increase of δ34S value of
sulfate in series-1 experiment (Fig. 3K). The δ34STS value in series-2
experiment also continuously increased from -1.8 ± 0.2‰ at 44 h to
-1.2 ± 0.2‰ at 762 h (Fig. 3K).

The equation of calculation on the uncertainty of 33λSt value(including the difference of the calculation method from previous
studies) is described in supplemental information.
3. Results
3.1. Cell growth
In the series-1 experiment, cell density increased from 2.0 × 105
cells/mL at inoculation to 2.2 × 107 cells/mL by 211 h and becamealmost constant after 211 h (Fig. 1; Table S1). In the series-2 experiment,
cell density increased from 2.0 × 105 cells/mL at 17 h to 2.9 × 107 cells/
mL at 164 h, fluctuated around 3.0 × 107 cells/mL until 401 h, anddecreased to 2.1 × 107 cells/mL at 760 h (Fig. 1; Table S1). Cell growthrate during the exponential phase in series-2 experiment is higher thanthat of the series-1 experiment (Fig. 1; Table S1).

3.4. Δ33S’ values of sulfate and ΣH2S
The average Δ33S’ value of sulfate in the series-1 experiment wasalmost constant at about +0.035‰ until 140 h and then decreased to+0.020‰ at 360 h followed by an increase to +0.041‰ at 1801 h(Figs. 3E and 3F, Table S2). The average Δ33S’ value of sulfate in theseries-2 experiment was almost constant at about +0.020‰ until 116 h,which is lower than that of the series-1 experiment in the exponential
phase (Figs. 3E and 3F, Table S2).
The average Δ33S’ value of ΣH2S in the series-1 experiment increased
from +0.046 ± 0.005‰ at 21 h to +0.077 ± 0.004‰ at 140 h during the
exponential phase and was decreasing after 140 h to -0.002 ± 0.007‰ at
1801 h during the stationary phase (Figs. 3G and 3H, Table S2). The
average Δ33S’ value of ΣH2S in the series-2 experiment increased from+0.079‰ at 70 h to +0.132 ± 0.009‰ at 116 h during the exponential
phase and was decreasing after 210 h to +0.024 ± 0.009‰ at 762 hduring the stationary phase (Figs. 3G and 3H, Table S2).

3.2. Concentration of sulfate and ΣH2S
The sulfate concentration was between 15 mM and 16 mM
throughout in the series-1 experiment (Fig. 1, Table S2). In the series-2
experiment, the sulfate concentration decreased from 16.9 mM at 20 h to
16.0 mM at 116 h and became almost constant after 116 h. The sulfate
concentration of C’-1 (a vial collected at 70 h of series-2 experiment)
was 14.7 mM, which is clearly low compared to other samples (Fig. 1,
Table S2).
The average ΣH2S concentration increased from 19.3 ± 9.1 μM at 21
h to 174.2 ± 9.3 μM at 140 h during the exponential phase and was
6

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Earth and Planetary Science Letters 624 (2023) 118446

concentration analyzed by ion-chromatography in the series-3 experi­
ment decreased from 15.6 mM to 15.2 mM during the exponential phase(Fig. S2C and S2D). Although the decreased sulfate concentration was
μM level in all series experiments, the decreased sulfate concentration
was larger than the analyzed ΣH2S concentration (Figs. 1C and 1E;
Figs. S2C and S2E). The discrepancy indicates sulfur reservoirs otherthan extracellular sulfate and ΣH2S exist.
Some previous studies advocated the inconsistency of δ34STS is owing
to cell’s internal sulfur reservoir (Rees et al., 1973; Stögbauer et al.,2004). We calculated the mass of the internal sulfur at 70 h of theseries-2 experiment where cell density was 1.4 × 107 cells/mL assuming
mass of internal sulfur reservoir is 10− 12 mgS/cell (Rees, 1973). The
calculated mass of the internal sulfur reservoir was 0.6 μg, which cor­
responds to only 0.4 μM of sulfur in the medium. Thus, another sulfur
reservoir is required to account for the discrepancy. Previous studies
showed accumulation of extracellular intermediate sulfur species suchas sulfite or thiosulfate (Chambers and Trudinger, 1979; Davidson et al.,2009). The thiosulfate concentration analyzed in the series-3 experi­
ment using ion-chromatography shows thiosulfate did not increase andwas under the detection level in almost all samples (Fig. S3A), whichshows thiosulfate cannot account for the mass-imbalance. Finally, we
carried out SEM-EDS observation of precipitates in the media of theseries-3 experiment. We observed 4 visible grains (>10μm) of zinc sul­
fide even in an uninoculated medium (Fig. S4A), which of the sulfur
source is likely derived from yeast extract and sulfur-containing
reducing agent. Although we did not conduct SEM observation for
exponential phase samples in the series-1 and -2 experiments, most of
the sulfur-containing mineral was considered to be zinc sulfide(Fig. S4B-D). The size and number of the zinc sulfide crystals seems tohave increased throughout the experiment (7 visible grains at 476 h and6 visible grains at 593 h; Fig. S4B-D). However, the calculated amount of
one visible ZnS grain assuming the volume of 253μm3 was only 6.4 ×
10− 10 mol, which corresponds to 16 nM of sulfur in the medium. Evenconsidering all visible grain in the medium at 476 h (7 × 40 mL/10 mL= 28), the sulfur in visible ZnS is equivalent to 0.45 μM, which also
cannot explain the discrepancy of mass balance. Thus, the mass andisotope imbalances in exponential phase are still elusive. Notably, theΣH2S concentration carried over by the 100 μL injection medium wastrivial and corresponds to <0.5 μM.
The δ34STS value in the stationary phase increased from -1.96 ± 0.2‰
at 140 h to -1.11 ± 0.2‰ at 1801 h in series-1 experiment associatedwith the increase of δ34SSO4 value (Figs. 3A and 3K). Contrary to theseries-1 experiment, the δ34STS value in the stationary phase is almost
within the error range in series-2 experiment (Fig. 3K). The factor which
increases the δ34SSO4 is likely the microbial sulfate reduction in the caseof series-1 experiment and the microbial sulfate reduction likely
occurred even during the stationary phase cells in the series-1 experi­ment. However, the occurrence of microbial sulfate reduction is notevident during the stationary phase of the series-2 experiment.

Table 1
Calculated sulfur isotope fractionations and csSRRs of both series of experi­ments. The time of (i+1)th of harvesting vials is used to express the time interval
between ith and (i+1)th collection time.

Series-1 43 h
Series-1 70 h
Series-1 140 h
Series-2 70 h
Series-2 116 h34

σ34εSR

csSRR(fmol/cell/day)(fmol/cell/day)

(‰)

(‰)

28.1
4.1
2.1
4.0
1.0

7.2
2.3
0.5
1.1
0.3

-13.4
-39.3
-44.3
-25.1
-65.9

3.6
25.6
10.5
7.4
21.0

σcsSRR

εSR

3.5. Sulfur isotope fractionation
3.5.1. Sulfur isotope fractionation during microbial sulfate reduction: 34εSRThe δ34SH2S_Inst decreased from -15.7 ± 3.6‰ at 43 h to -45.9 ± 9.6‰
at 140 h in the series-1 experiment (Fig. 3I). The magnitude of 34εSRvalue calculated from the δ34SH2S_Inst of the series-1 experiment
increased from -13.4 ± 3.7‰ at 43 h to -44.3 ± 10.5‰ at 140 h (Fig. 3J;
Table 1). The δ34SH2S_Inst decreased from -26.6 ± 7.4‰ at 70 h to -67.0 ±18.3‰ at 116 h in the series-2 experiment (Fig. 3I). The magnitude of34
εSR value calculated from the δ34SH2S_Inst of the series-2 experiment
increased from -25.1 ± 7.8‰ at 70 h to -65.9 ± 21.0‰ at 116 h (Fig. 3J;
Table 1).
3.5.2. Sulfur isotope fractionation of the ΣH2S-consuming reaction in
stationary phase: 34εSt
The calculated 34εSt and δ34S’H2S(j-0) values in the series-1 experimentare -10.5 ± 1.1‰ and -24.8 ± 0.7‰, respectively (Fig. S1); the calcu­
lated 34εSt and δ34S’H2S(j-0) values in the series-2 experiment are -45.6 ±12.4‰ and -34.2 ± 2.7‰, respectively (Fig. S1). The mass dependenceof the reaction (33λSt) in the series-1 and -2 experiments are 0.507986 ±
0.000004 and 0.5079 ± 0.0175, respectively (Fig. 2). In the series-1
experiment, the δ34S’H2S and Δ33S’H2S values of samples fit well withthe model of Rayleigh distillation equation with the obtained values of34
εSt = -10.5‰, 33λSt = 0.507986, δ34S’H2S(j-0) = -24.8‰, and Δ33SH2S(j-0)’= +0.051‰ (Fig. 2B). In the series-2 experiment, the δ34S’H2S and
Δ33S’H2S values of samples deviate from the model of Rayleigh distilla­tion equation with the obtained values of 34εSt =-45.6‰, 33λSt = 0.5079,
δ34S’H2S(j-0) = -34.2‰, and Δ33SH2S(j-0)’ = +0.105‰ (gray lines inFig. 2C). The black lines in Fig. 2C were annotated with the model
calculation with modified 33λSt value as 0.5035 to adjust the data. The
difference of slope of the linear regression line of ΣH2S concentrationand δ34SH2S value in the stationary phase shows the isotope fractionation
factors of the two series experiments are different (Fig. 2D).
4. Discussion
4.1. Inconsistent δ34STS values in exponential and stationary phase
The δ34STS values in the exponential phase increased c.a. 0.5‰ inboth series of experiments (Fig. 3K). If we could quantify concentrationand isotopic composition of all sulfur species in the vials correctly, δ34STS
must have been constant. In the series-1 experiment, ΣH2S concentra­tion and δ34SSO4 increased c.a. 170 μM and 1‰, respectively (Figs. 1E
and 3A), which shows clear evidence of microbial sulfate reduction. On
the other hand, sulfate concentration was almost constant throughout
the experiment, which indicates the analytic uncertainty on the sulfate
concentration is too large to quantify the decreased sulfate concentra­tion for series-1 experiment (Fig. 1C). The uncertainty on the sulfate
concentration of the series-1 experiment is likely attributed to extractionof aliquot of medium for cell counting before chemical analysis(Table S1). The extraction of medium is unlikely to affect the ΣH2S
concentration because we employed colorimetric method. In the series-2
experiment, the sulfate concentration decreased from 16.9 mM to 16.0
mM during the exponential phase (Fig. 1C). Besides, the sulfate

4.2. Sulfur isotope fractionation by microbial sulfate reduction (34εSR)and cell growth phase and states
The magnitude of microbial sulfur isotope fractionation, 34εSR, andcsSRR have a clear negative correlation (Fig. 4; Table 1). The largest34
εSR value of -65.9 ± 21.0‰ at 116 h of series-2 experiment is achieved
when the csSRR was the lowest, 1.0 ± 0.3 fmol/cell/day (Table 1).
These were the values that obtained from the latest phase of growth inboth the series of experiments. On the other hand, the smallest 34εSRvalue, -13.4 ± 3.6‰, was achieved at 43 h of series-1 experiment when
the csSRR was the highest, 28.1 ± 7.2 fmol/cell/day (Table 1). Our re­sults show the 34εSR value varies over 50‰ by changing growth phaseand states of cells. The difference of the largest 34εSR value between theseries-1 and -2 experiments was also controlled by csSRR (Fig. 4;
Table 1). Since the largest 34εSR value was different in the two series ofexperiment, any of the conditions is involved in the difference between
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Earth and Planetary Science Letters 624 (2023) 118446

ΣH2S-consuming reaction in the stationary phase on the 34εSR values are
trivial and discussed in supplemental information.
Although there is no report that D. desulfuricans utilizes glucose as anelectron donor and our HPLC-based glucose analysis failed to detectdecreasing glucose in the series-3 experiment (Fig. S3B), we confirmedthat D. desulfuricans oxidized glucose instead of yeast extract as theelectron donor in the present study. When D. desulfuricans was cultivatedin the medium with or without glucose in series-3 experiment, thespecific growth rate was greater in the medium with glucose than in the
glucose-free medium in series-3 experiment (Figs. S2A and S2B). In
addition, we observed all sulfate and glucose were consumed and mM
level of ΣH2S was produced when we modified the inoculum volume andthe reducing agents of bacterial culture (See supplemental information).
The influence of ΣH2S released from the decomposition of cysteine inyeast extract and Na-thioglycolate on the 34εSR values is minimal and isdiscussed in the supplemental information.
As for the decrease of csSRR according to the shift of growth phaseand cell states, there are two possible cases. One is the case that almost
all the cells in the cultures decreased their SRR in a similar manner likely
due to the depletion of electron donor. The other is the case that most of
the cells ceased the sulfate reduction activity but only a limited numberof acclimated cells under the varying conditions kept the high sulfate
reduction. The microscopic observation of cell morphology indicatedthat the cell size in our experiments gradually decreased in a similarmanner from the exponential and the stationary phases of growth(Fig. 5). This implies that the decrease of total sulfate reduction rateaccording to the varying growth phases results from the decrease of
sulfate reduction rate in each cell.

Fig. 4. Sulfur isotope fractionation of microbial sulfate reduction in the
exponential phase (34εSR) and cell specific sulfate reduction rate. The magnitudeof 34εSR value and csSRR have clear negative correlation consistent with pre­vious studies. Blue and green squares denote series-1 and series-2 experimentsof this study, respectively. Black and gray small squares denote the data fromSim et al. (2011a, 2011b) and Leavitt et al. (2013), respectively.

the two series of experiments (Fig. 4). The much lower csSRR was
observed at 116 h of series-2 (with shaking) experiments than those of
the series-1 (without shaking) experiment, which resulted from muchhigher cell density at that time of series-2 experiments than those of theseries-1 experiment (Fig. 1B). Further, total sulfate reduction rate waslower at 116 h of the series-2 experiments than those of the series-1
experiment (Fig. 1F). The shaking condition may have provided afaster cell specific growth rate (Figs. 1A and 1B) and subsequently
introduced the faster entry to the stationary phase of growth and sulfate
reduction than the static condition, which may have led to the smallest
csSRR value at 116 h of the series-2 experiments. The influence of the

4.3. Intracellular sulfur isotope fractionation mechanism of sulfate
reducing bacteria
We carried out a model calculation according to Wing and Halevy(2014) to compare the results with other studies (Fig. 6). The relatively
good fit of our results with the model indicates intracellular metabolite
concentration shape the variation of the 34εSR value (Wing and Halevy,2014). However, the 34εSR values of the experimental results of thisFig. 5. Fluorescence photomicrograph of D. desulfuricans in various growth phase and states. (a) Cells at 43.2 h of series-1 experiment. (b) Cells at 91.2 h of series-1
experiment. (c) Cells at 163.8 h of series-2 experiment. (d) Cells at 359.8 h of series-2 experiment. The sizes of all pictures are 40 × 40 μm. The cell size gradually
decreased from the early exponential phase to the stationary phase.
8F. Matsu’ura et al.

Earth and Planetary Science Letters 624 (2023) 118446

4.4. The ΣH2S-consuming reaction in stationary phase
We observed the decreasing ΣH2S concentration during the station­
ary phase, which involves large sulfur isotope fractionations, 34εSt =-10.5 ± 1.1‰ and -45.6 ± 12.4‰, and decrease Δ33S’H2S values (Figs. 2Band 2C). Despite the large uncertainty, the 34εSt value in series-2
experiment must be larger than that of the series-1 experimentbecause of the steep increase in the slope of the linear regression in
Fig. 2D, which may be owing to the shaking experimental condition. The
lack of decreasing ΣH2S concentration in the stationary phase of same
experiments in Matsu’ura (2016b) is likely due to undetailed collection
time and inaccurate quantification of ΣH2S concentration. Further, weobserved decreasing ΣH2S in the cell-free experiment, which suggests
the decrease in ΣH2S concentration in the series-1 and series-2 experi­ments was not mediated by bacterial cells but mediated bynon-microbiological reactions (Fig. 2A). Although the detail mechanism
is still unidentified owing to the lack of identification of end-products,
there are several possible explanations of non-microbiological re­actions as follow.
One model is ΣH2S oxidation with inorganic oxidants such as oxygen,iron, or manganese. However, this seems unlikely because the medium
contains reducing agents such as ascorbic acid and Na-thioglycolate. In
addition, the medium color was transparent throughout the experimentwith resazurin indicator, which indicates contamination of oxygen into
the medium was negligible.
The second model is precipitation as sulfide minerals. As mentioned
in section 4.1, zinc sulfide was observed as the major sulfur-containing
precipitates even in uninoculated samples and increased in number andsize throughout the incubation experiment (Figs. S4A-D). Further, anickel sulfide was observed in the medium of the supplemental cell-freeexperiment after 504 h of H2S injection (Fig. S4E). However, as dis­cussed about δ34STS in the exponential phase, the calculated amount ofsulfur in these visible precipitates is trivial as compared to the decreased
amount of ΣH2S. In addition, for measurement of ΣH2S isotope
composition, we collected zinc sulfide precipitates from media including
ΣH2S (section 2.2). Thus, sulfide precipitates, especially zinc sulfide,
during the incubation experiments were included in the measured
values of ΣH2S isotope composition.
Finally, we inspected the possibility of sulfurization of organic
matter with ΣH2S. It is well known that glucose as well as other organic
matter is sulfurized with ΣH2S in aquatic solution in ambient tempera­ture and relatively short time scale (Amrani, 2014; Pohlabeln et al.,2017). We detected one visible (>10μm) sulfurized organic matter grainin the medium of series-3 experiment at 593 h using SEM (Fig. S4F),
showing that organic matter sulfurization occurred in the incubation
medium. The amount of sulfur in the organic sulfur grain was calculated
as 9.0 × 10− 11 mol assuming that the organic sulfur grain possesses a
volume of 203μm3 and is primarily composed of thiolane (CH2)4S. The
calculated amount of the sulfur corresponds to 2.3 × 10− 3 μM in our
growth medium, which is 2 orders of magnitude lower than that of thezinc sulfide (0.45 μM). In addition, the decreasing rate of the ΣH2S
concentration was similar between the media with and without glucose(Fig. S3C). However, our incubation medium contains organic com­pounds other than glucose such as yeast extract and Na-thioglycolate.
Besides, the majority of organic sulfur compounds exist in liquid form
at our experimental condition. Thus, organic matter sulfurization may
explain non-microbiological reaction of decreasing ΣH2S concentration
during the stationary phase but would be promoted by organic com­pounds other than glucose such as yeast extract and Na-thioglycolate in
the medium.
In summary, we observed precipitates of sulfide minerals andorganic sulfur as the possible sinks of the decreasing ΣH2S concentration
during stationary phase. However, the calculated sulfur amount in theobserved grains of sulfide minerals and organic sulfur seemed to be notenough to account for the decreased ΣH2S amount during the stationary
phase. Nevertheless, there may be abundant invisible small grains andFig. 6. Steady state model calculation according to Wing and Halevy, (2014).
We applied scaling factor of 2.1 × csSRR + 70.0 for the calculation of the modelof this study because the model with the value best fitted our results. The outersulfate concentration, ΣH2S concentration, and temperature were fixed to 16.0
mM, actual experimental values, and 30 ◦ C, respectively for the calculation ofthis study. The model calculation for the data of Leavitt et al. (2013) and Sim
et al. (2011a, 2011b) are from Wing and Halevy, (2014).

study at low csSRR area are high compared to those of the model (Fig. 6).
Two possibilities can be considered to account for the discrepancy.
The first possibility is inconsistency of calculated intracellular
metabolite concentrations with those of actual cells. The deviation of thedata in this study from the model at low csSRR area may be attributed to
low intracellular ΣH2S concentration assumed for the model calculation.
Wing and Halevy (2014) assumed the intracellular and extracellularΣH2S concentrations are equal and showed that model results are sen­sitive to the intracellular ΣH2S concentration. The extracellular ΣH2S
concentration was set to 1 mM and c.a. 0.1 mM for the models of Leavitt
et al. (2013) and this study, respectively (Fig. 6). Thus, if low csSRRs
resulted in the buildup of intracellular ΣH2S in the experiment of thisstudy, the calculated 34εSR value by the model of this study become
higher and may align well with the data at low csSRR area. Besides, Sim
et al. (2017) showed from the experiments of extracting intracellular
metabolites of sulfate reducing bacteria that ratio of intracellular sulfite
to APS is low compared to the model. Sim et al. (2017) suggested thediscrepancy is likely owing to the employment of high redox potential
electron carrier—menaquinone—in the model calculation and employ­
ing a low redox potential electron carrier may cancel the discrepancy
between the experimental results and the model.
The second possibility is that the 34εSR value is determined only fromthe isotopic composition of intracellular sulfate (Sim et al., 2017). Sim
et al. (2017) showed intracellular sulfate was not accumulated and hadhigh δ34S value (+48.7‰) during the middle exponential phase of cells
owing to high sulfate reduction rate. On the other hand, intracellularsulfate was accumulated and had low δ34S value of +5.3‰ in the later
exponential to stationary phases cells. They interpreted the change ofthe δ34S value of intracellular sulfate resulted from a closed system effectowing to the change of sulfate reduction rate, and sulfur isotope frac­tionation during intracellular sulfate reduction to ΣH2S always accom­
panies large isotope fractionation over 50‰. If this is the case, the 34εSRvalue should not follow the calculation of steady state models including
Wing and Halevy (2014), and our experimental results should not fit
with the model (Fig. 6). Further experimental quantification of, andisotope analyses of intracellular metabolites seems to be a powerful way
to elucidate the mechanism of intracellular sulfur isotope fractionationof microbial sulfate reduction.

9F. Matsu’ura et al.

Earth and Planetary Science Letters 624 (2023) 118446

Fig. 7. Schematics of plausible sulfur cycling of some modern marine sediments in where ΣH2S is accumulated. Blue line denotes δ34S value of seawater andporewater sulfate. Red dotted and solid lines represent the δ34S values of ΣH2S generated through microbial sulfate reduction and modified by the ΣH2S-consuming
reaction, respectively. 34εSR denotes sulfur isotope fractionation of microbial sulfate reduction same as defined in this study. Green line denotes δ34S values of the
products (metal sulfide or organic sulfur) of the ΣH2S-consuming reaction. The 34εproduct denotes sulfur isotope fractionation of the ΣH2S-consuming reaction, which isequal to 34εst in this study. The pictures of nickel sulfide and organic sulfur are from Fig. S4. SWI denotes sediment water interface.

pyrite with low δ34S value (-30 to -40‰) was formed in microenviron­ments such as microbial aggregates or biofilms. This interpretation isbased on the observation that equilibrium isotope fractionation between
most metal sulfides and ΣH2S is negligible and occurs in the opposite
direction (δ34Sproduct > δ34SH2S) (Seal, 2006). A large kinetic isotope
fractionation is necessary to produce metal sulfide with a lower δ34S
value compared to that of ΣH2S. Although Böttcher et al. (1998)
demonstrated the occurrence of kinetic isotope fractionations in thenormal direction during iron-sulfide precipitation from ΣH2S, the sulfur
isotope fractionations are minimal, approximately -3‰. In addition to
the metal sulfide, Raven et al. (2015) showed that the δ34S values ofsome organic sulfur species in the sediments of Cariaco Basin are lower
than those of the coexisting ΣH2S nadir to -43.6‰, which is ca 14‰
lighter than the coexisting ΣH2S. Besides, the δ34S values of the organic
sulfur is lower than the coexisting pyrite (chromium reducible sulfur)
whose δ34S value is about -33‰. These low δ34S value of organic sulfur
was enigmatic because no large kinetic isotope effect has been reported
for organic matter sulfurization (Raven et al. 2015). Furthermore, when
considering the pyrite and organic sulfur were formed from same ΣH2S
pool, organic matter sulfurization is expected to involve a larger kinetic
isotope effect than pyrite formation in the sediments of Cariaco Basin.
Raven et al. (2015) proposed the kinetic isotope effect during organic
matter sulfurization is a consequence of the irreversible reaction be­
tween HS− (bisulfide) and organic matter, leading to the formation of
thiol, instead of the reversible reaction between S2−
x (polysulfide) andorganic matter, leading to the formation of sulfur catenation. Assuming
the reaction occurring in the stationary phase of this study involves
organic matter sulfurization with a kinetic isotope effect, this could
account for the observed low δ34S values in organic sulfur within Car­
iaco Basin sediments (Fig. 7). At the same time, the formation of organic
sulfur species with low δ34S value increases the δ34S value of ΣH2S,
potentially explaining the higher δ34SH2S values observed compared tothose of coexisting pyrite in some modern marine sediments. Large ki­
netic isotope fractionation observed in the stationary phase cells of ourstudy is necessary to account for the elevated δ34SH2S values observed insome modern marine sediments including Cariaco Basin. Identificationof the end-product of the reaction of the stationary phase in this study isnecessary for further discussion.

dissolved forms of both inorganic and organic sulfur materials in themedium during the stationary phase. Although the detail mechanism isstill uncertain, it seems likely that non-microbiological consumptions of
ΣH2S during the stationary phase by minerals and organic matterdecreased the amount of accumulated ΣH2S produced by sulfate
reducing bacteria.
4.5. Implication for natural environments
The results of this study reinforce the importance of cell growthphase and states on 34εSR value in natural environments. The csSRR in
natural oceanic sediments is controlled by the organic matter flux to thesediments (D’Hondt et al., 2002); however, the csSRRs observed in thisstudy and previous incubation experiments (10− 1 to 102 fmol/cell/day)are logarithmically higher than those observed in the natural environ­ments (10− 7 to 10◦ fmol/cell/day), which hesitate us to apply results ofincubation experiments to elucidate the sulfur cycling of natural envi­
ronments (Jørgensen 2021; Turchyn and Druhan, 2022). Indeed, Mas­
terson et al. (2022) indicated the 34εSR value in marine sediments isalways ca. -70‰ regardless of the flux of organic matter as well as
methane, and csSRR in marine sediments is always lower than that
achieved in incubation experiments. One of the exceptional sites for theconsistent 34εSR value in natural environments are microbial mats andmethane seep sediments where copious amount of organic matter ormethane load increase csSRR and decrease the extent of 34εSR value(Habicht and Canfield, 1997; Gong et al., 2018).
In addition to the microbial sulfur isotope fractionation, we showed
non-microbiological isotope fractionations by ΣH2S-consuming reactionin the stationary phase, 34εSt = -10.5 ± 1.1‰ and -45.6 ± 12.4‰(Fig. 2), which are likely owing to the precipitation of acid insoluble
sulfide minerals or sulfurization of organic matter. In both cases, theproduct has lower δ34S value compared to ΣH2S and increase δ34SH2S
value in where hydrogen sulfide is accumulated in water column orsediments. In particular, high δ34SH2S value compared to that of coex­
isting pyrite or organic sulfur was reported in several modern marine
sediments (Werne et al., 2003; Raven et al., 2016). Raven et al. (2016)
reported the δ34SH2S value in Santa Barbara Basin porewater is uniformly
5–30‰ higher than that of coexisting pyrite and interpreted that the10

F. Matsu’ura et al.

Earth and Planetary Science Letters 624 (2023) 118446

5. Conclusion

References

Two series of pure culture incubation experiments of a type sulfate
reducing bacterium, D. desulfuricans, were carried out from the early
exponential phase to the stationary phase. The magnitude of the 34εSRvalues increased from -13.4 ± 3.6‰ in the early exponential phase to-65.9 ± 21.0‰ in the later exponential phase concomitant with thedecrease of csSRR from 28.1 ± 7.2 to 1.0 ± 0.3 fmol/cell/day. The
decrease of csSRR is likely owing to the increase in the small cells withlow sulfate reduction rate. The large variation of 34εSR value shows theimportance of cell’s growth phase and states. The shift of cell growthphase likely changed the concentration and isotopic composition of
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large negative sulfur isotope fractionation and increase δ34S value ofΣH2S, our finding can account for high δ34SH2S value compared to that ofcoexisting pyrite or organic sulfur reported in several modern marine
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CRediT authorship contribution statement
Fumihiro Matsu’ura: Writing – original draft, Methodology,
Investigation, Conceptualization. Hiroko Makita: Supervision, Meth­odology, Investigation. Ken Takai: Writing – review & editing, Super­
vision. Mayuko Nakagawa: Investigation. Yuichiro Ueno: Writing –review & editing, Investigation, Conceptualization.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
All experimental data in this study are attached in supplemental
tables.
Acknowledgment
The authors are grateful to the associate editors, Prof. Boswell Wing
and Prof. Laurence Coogan for handling this paper. Constructive com­ments by two anonymous reviewers helped improving the manuscript.
We are grateful to Prof. Shawn McGlynn with the help of cell counting at
Tokyo Tech. We are grateful to Nanjing University for their support ofthe corresponding author’s travel for the experiments in revision stage
conducted in Japan.
Supplementary materials
Supplementary material associated with this article can be found, inthe online version, at doi:10.1016/j.epsl.2023.118446.
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Earth and Planetary Science Letters 624 (2023) 118446

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