TITLE.Application of PTR-MS and 33S Isotope Labelling for Monitoring

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Application of PTR-MS and 33S Isotope Labelling for Monitoring Sulfur Processes in Livestock Waste Frederik Rask Dalby, Michael Jørgen Hansen, and Anders Feilberg Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04570 • Publication Date (Web): 17 Jan 2018 Downloaded from http://pubs.acs.org on January 17, 2018

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Environmental Science & Technology

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TITLE. Application of PTR-MS and 33S Isotope

2

Labelling for Monitoring Sulfur Processes in Livestock

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Waste

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AUTHOR NAMES. Frederik R. Dalby, Michael J. Hansen, Anders Feilberg*

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AUTHOR ADDRESS. Department of Engineering, Aarhus University, Hangøvej 2, 8200 Aarhus N,

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Denmark

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KEYWORDS. Sulfate reduction, Sulfur cycle, Hydrogen sulfide, Manure, Inhibition, Odor

ACS Paragon Plus Environment


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ABSTRACT

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Reduced sulfur compounds emitted from livestock production cause odor nuisance for local residents.

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The microbial processes responsible for this are not well described in swine manure and a method for

11

monitoring the biological processes is necessary to develop strategic abatement technologies. In this

12

study, Proton-Transfer-Reaction Mass Spectrometry and isotope labelled sulfate was combined and

13

applied to elucidate the sulfur processes in swine manure with high time resolution. We successfully

14

monitored reduction of isotope 33S labelled sulfate into corresponding 33S hydrogen sulfide and that

15

some of the 33S hydrogen sulfide was further methylated into 33S methanethiol. The isotope patterns in

16

reduced sulfur compounds together with usage of inhibitors enabled us to calculate a sulfate reduction

17

rate of 1.03 ± 0.18 mM/day equivalent to 76.9 ± 3.0% of total hydrogen sulfide emissions. Cysteine

18

degradation constituted 20.2 ± 2.7% of the total hydrogen sulfide produced and the remaining hydrogen

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sulfide came from demethylation of methanethiol and dimethyl sulfide. Another source to

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methanethiol, besides hydrogen sulfide methylation, was methionine degradation, which contributed

21

with 78.3 ± 2.5% of the methanethiol production, whereas the remaining 21.7 ± 2.5% came from

22

hydrogen sulfide methylation. This study suggests, therefore, that emissions of odorous sulfur

23

compounds from swine manure can be reduced by inhibiting methionine degradation and sulfate

24

reduction.

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INTRODUCTION

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The emission of gases from livestock production in association with handling-and land spreading

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animal manure, contributes to eutrophication (1), greenhouse gas production (2), particle formation (3)

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and cause odor nuisance to the local residents (4, 5). Particularly reduced sulfur compounds have been

30

associated with odor nuisance (6-8) and a better understanding of the factors influencing reduced sulfur

31

compound emissions may lead to strategies and technologies aimed to reduce odor nuisance (7).

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The most significant reduced sulfur compounds in relation to odor from livestock production are

33

hydrogen sulfide, methanethiol and to a lesser extent dimethyl sulfide and dimethyl disulfide.

34

Hydrogen sulfide is most abundant and produced from reduction of sulfate, which comes from urine

35

(9). Small amounts of hydrogen sulfide are methylated by acetogenic bacteria (10) to form

36

methanethiol, which in turn is methylated to dimethyl sulfide (11). Conversely, methanethiol, dimethyl

37

sulfide and dimethyl disulfide can be demethylated by methanogens, to restore hydrogen sulfide. (12,

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13). Cysteine and methionine, which originate from undigested protein in animal faeces, also

39

constribute with reduced sulfur compounds by being degraded directly to hydrogen sulfide and

40

methanethiol respectively (14). The sulfur cycle has been comprehensively researched in marine

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sediments (15-17), but less so in livestock waste. To elucidate the principal mechanisms responsible for

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reduced sulfur compound emissions from livestock waste a high time resolution method is required.

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Proton-Transfer-Reaction Mass Spectrometry (PTR-MS) is a rapid and highly sensitive instrument (18)

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for monitoring air contaminants from e.g. manure over time (6, 7), which has been described in detail

45

elsewhere (19, 20). Consequently, PTR-MS is ideal for estimating the activity of the pathways in the

46

sulfur cycle. In the present study, a new approach by which PTR-MS is combined with isotope

47

labelling is used to distinguish between hydrogen sulfide originating from cysteine degradation,



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methanethiol and dimethyl sulfide demethylation, and sulfate reduction. The method is based on adding

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quantitative amounts of the stable isotope 33S in the form of sulfate to swine manure. Isotope 33S was

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selected over the more abundant isotope 34S, since (H234S)H+ has the same nominal mass to charge

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ratio as that of water clusters (m/z 37, H3O+(H2O)). The atomic ratio of 33S/32S is in this work termed

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R(33/32S) with units of atom percentage (21) and a comparison between R(33/32S) in sulfate, hydrogen

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sulfide, methanethiol and dimethyl sulfide can determine the origin of reduced sulfur compounds. The

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origin and ratio of hydrogen sulfide and methanethiol is particularly important to monitor since

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methanethiol and hydrogen sulfide have high odor activity values and hence a strong impact on odor

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from livestock production (7). Further insight into the sulfur cycle in manure is acquirable by inhibiting

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sulfate-reducing bacteria with molybdate (22) and methanogens with bromoethansulfonate (BES) (23,

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24).

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The method we provide in this study was validated by monitoring H233S production upon 33SO4-2

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addition to swine manure in various concentrations. The impact of cysteine and methionine addition on

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R(33/32S) in hydrogen sulfide and methanethiol are presented to demonstrate the method applicability.

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The method is further used to address how much of methanethiol comes from hydrogen sulfide

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methylation compared to methionine degradation and the relative contribution of sulfate reduction,

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cysteine degradation and methionine to hydrogen sulfide production.

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MATERIALS AND METHODS

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Manure Characterization. Manure from growing-finishing pigs was sampled from a manure storage

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tank at Aarhus University Foulum. The manure was sieved through a metal grid with 6 mm pores to

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separate straw and large particles. The sieved manure was stored in two 10 L aliquots at 5 ºC until

69

processing (up to 6 months after collection). The dry matter content was measured gravimetrically by


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heating 10 g of manure in a B180 (Nabertherm) oven to 105ºC for 24 h (25). The ash content was

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subsequently determined gravimetrically by heating the dried samples at 520ºC for 6 h in a Muffle

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furnace (Nabertherm). Ammonium concentration was measured by spectroscopy with a Spectroquant®

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NOVA 60 (Merck) using an Ammonium Test kit 1.00683 (Merck). Sulfate was measured with ion

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chromatography on a Dionex IonPac AG18 4mm x 50mm and Dionex IonPac AS18 4mm x 250mm

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column and an ED50 Electrochemical detector. Prior to ammonium- and sulfate concentration analysis,

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the manure was diluted 1:50 with milli-Q water (18.2 MΩ, 25 ºC), centrifuged with a Heraeus

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Megafuge 16R (Thermo Scientific®) at 5000 RPM for 30 min, and then filtrated through a 0.20 μm

78

filter. Samples for volatile fatty acids (VFA) were prepared (26) and analysed on a HP 6850 Series GC

79

system (Agilent Technologies, Santa Clara, California, USA) with a flame ionization detector. The GC

80

column was a HP-Innowax with length of 30 m, inner diameter of 0.25mm and stationary phase

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thickness of 0.25μm. The pH-value was measured with a Portamess (Knick) pH sensor and a pH-500

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microsensor (Unisense).

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Experimental setup. A sketch of the experimental setup is presented in Figure 1. Two hundred mL

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sieved swine manure was added to three 850 mL glass bottles. The bottles were closed with GL 45

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screw caps with two Teflon® hose connections. Steady agitation was applied with magnet stirrers to the

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point just before a vortex formed. Gas mass flow controllers (Bronkhorst EL FLOW, Ruurlo,

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Netherlands) were used to continuously apply 200 mL pure nitrogen/min through the Teflon® hose

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connection in the screw caps into the headspace (650 mL) approximately 1 cm from the manure surface

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(43 cm2). The actual flow rate deviated up to 10% from 200 mL/min, which was measured with a TSI

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mass flowmeter series 4100 (TSI) twice a day and accounted for in the calculations. The outlet gas was

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continuously diluted 6-11 times with pure nitrogen before it was directed to a five-way PEEK valve

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(Bio-Chem Valve Inc., Boonton, NJ). The valve switched between the three reactors and a background



93

signal every 4 min resulting in a total cycle time of 16 min. The background signal was charcoal

94

(Supelco, Bellefonte, PA) filtered pure nitrogen. A pressure release hose was connected before the

95

valve, to avoid pressure build up when the valve was closed for a given reactor. The valve outlet led to

96

a High Sensitivity Proton-Transfer-Reaction Mass Spectrometer (HS PTR-MS, Ionicon Analytik,

97

Innsbruck, Austria) for gas composition analysis. All connection tubes were made of Teflon® with

98

3.18- or 6.35 mm inner diameter (Mikrolab A/S, Aarhus Denmark). The experiments lasted 1-4 days

99

and was carried out at room temperature.

100

Chemicals and reagents. During experiments the following chemicals were used in final

101

concentrations of 12 mM sodium 2-bromoethanesulfonate (BES) (Sigma Aldrich, Copenhagen,

102

Denmark), 2.86 mM ammonium molybdate tetrahedrate (Sigma Aldrich), 1 mM L-methionine (Sigma

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Aldrich), 1 mM L-cysteine (Sigma Aldrich) and 0.09-0.374 mM Na233SO4 (Sigma Aldrich). The

104

chemicals were dissolved in 2 mL milli-Q water (18.2 MΩ, 25 ºC) and were injected through the

105

Teflon® hose connections with a syringe.

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Proton-Transfer-Reaction Mass Spectrometry setup. The drift tube settings of the PTR-MS were

107

600 V and 2.1-2.2 mbar yielding an E/N (electric field per gas density (27)) of 138 Townsend. The

108

inlet-and chamber temperature was set to 60 ºC. In Table 1, measured compounds, detection limits and

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dwell times for the multiple ion detection mode are presented. The transmission of the PTR-MS was

110

updated before and after the experimental period (2 months) with a Scott Mini-Mix™ (Restek)

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transmission gas. The hydrogen sulfide concentration was calibrated against the humidity (7) before

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and after the experimental period. Proton transfer rate coefficients were calculated for each individual

113

molecule using capture rate coefficients (28). Peak interference was insignificant, except for dimethyl

114

sulfide, for which this was corrected (see supporting information Figure S1).


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Calculations. The emission rate was described with the ideal gas law in eq 1.

116

𝑟=

117

Where, r is the emission rate (M/day), pi is concentration of compound i (ATM), R is the gas constant

118

(LATM°Kelvin-1mol-1), T is temperature (ºKelvin), Q is the volumetric gas flow rate (L/day) and V is

119

the manure volume (L). The manure volume was considered constant in these calculations, although a

120

maximum volume loss of 10% due to gas and water volatilization was observed over a four day

121

experiment. As such, the calculations are based on the initial manure volume. Models describing

122

R(33/32S) in hydrogen sulfide (the atomic ratio of m/z 36 to m/z 35) were derived from simple

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differential equations assuming complete mixing and equilibrium in eq 2.

124

𝑅(33/32 𝑆)𝑚 = 𝑅(33/32 𝑆)𝑒 − 𝑅(33/32 𝑆)𝑒 ∙ 𝑒 𝑄∙𝑡

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Where t is time (h), R(33/32S)m (atom %) is the modelled R(33/32S) as a function of time and R(33/32S)e

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(atom %) is the expected R(33/32S) when the system is in equilibrium based on 33SO4-2 added and the

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total sulfate present in the manure. The demethylation rate was calculated by comparing the emission

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rates of methanethiol and dimethyl sulfide before and after adding BES in eq 3.

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𝑟𝑑𝑚 = 𝑟𝑀𝑇+𝐵𝐸𝑆 − 𝑟𝑀𝑇

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Where rdm is demethylation rate (M/day), rMT+BES is methanethiol emission rate at its maximum after

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BES addition (M/day) and rMT is methanethiol emission rate just before BES addition (M/day).

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Methanethiol formed by hydrogen sulfide methylation was calculated after injecting BES to secure that

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m/z 50 was sufficiently above the background signal. Equation 4 was used to determine the rate of

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hydrogen sulfide methylation.

𝑝𝑖 𝑅∙𝑇

∙

𝑄

eq 1.

𝑉

eq 2.

eq 3.



𝑅(33/32 𝑆)𝑀𝑇 − 𝑅(33/32 𝑆)𝑏𝑐 𝑀𝑇

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𝑟𝑚 =

136

Where, rm is the methylation rate (M/day), R(33/32S)MT and R(33/32S)H2S is the ratio of m/z 50:49 (atom

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%) and m/z 36:35 (atom %) respectively after adding 33SO4-2. R(33/32S)bcMT and R(33/32S)bcH2S is the ratio

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of m/z 50:49 (atom %) and m/z 36:35 (atom %) respectively without 33SO4-2 addition.

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Hydrogen sulfide emission indirectly caused by methionine degradation was found by eq 5.

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𝑟𝐻2𝑆𝑚 = 𝑟𝑑𝑚 − 𝑟𝑚

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Where rH2Sm was hydrogen sulfide emission indirectly caused by methionine degradation (M/day).

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Cysteine degradation rate was calculated by eq 6.

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𝑖 𝑟𝑐𝑦𝑠 = 𝑟𝐻2𝑆 ∙ [1 −

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Where rcys is the natural degradation rate of cysteine (M/day), riH2S is the hydrogen sulfide emission rate

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when inhibited by molybdate (M/day), cysi was the fractional reduction of maximum cysteine

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degradation rate when inhibited by molybdate and R(33/32S)cH2S was the ratio of m/z 36:35 (atom %) in

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the control replicate.

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RESULTS AND DISCUSSION

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Initially the method was validated by inspecting the uncertainty and repeatability in biological

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triplicates. After method validation, a series of experiments was conducted to assess the rate of various

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sulfur processes in swine manure. The manure characteristics were tested prior to experiments to

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ensure that sulfate and volatile fatty acids were available to the microorganisms. The results and

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conclusions drawn in this study are only valid for the particular swine manure used and may differ if

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the method is applied on other types of livestock waste.

𝑅(33/32 𝑆)𝐻2𝑆 − 𝑅(33/32 𝑆)𝑏𝑐 𝐻2𝑆

∙ 𝑟𝑀𝑇

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eq 4.

eq 5.

𝑅(33/32 𝑆)𝐻2𝑆 − 𝑅(33/32 𝑆)𝑏𝑐 𝐻2𝑆 𝑅(33/32 𝑆)𝑐𝐻2𝑆 − 𝑅(33/32 𝑆)𝑏𝑐 𝐻2𝑆

1

]∙[

1−𝑐𝑦𝑠𝑖

]


eq 6.

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Manure Characterization. The fresh sieved manure had a pH of 6.5 ± 0.1 and a dry matter-and ash

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content of 4.59 ± 0.05% and 1.06 ± 0.01% from triplicates. NH4+ was 3.95 ± 0.17 g/L from triplicates.

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SO4-2 was between 1.1 and 5.5 mM for all experiments. Acetic acid content increased over time from 3-

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to 6 g/L, whereas propionic-, butyric- and hexanoic acid decreased over time confirming acetogenesis

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activity (29, 30).

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Method validation. In order to properly correlate gas emissions to microbial activity in the manure,

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the system was assumed to be in gas-liquid equilibrium and in absence of vertical pH gradients. These

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assumptions were tested and were found acceptable (see supporting information Figure S2 and Figure

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S3). The PTR-MS instrument responded linearly in the concentration range observed in this study (see

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supporting information Figure S4). Table 2 presents an experiment without any chemical

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modifications, which was conducted to assess the experimental setup uncertainty of biological

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triplicates with respect to reduced sulfur compounds emissions.

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In Table 2, the detection limit was well below the working range in this study. The relative standard

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deviation was between 13.3% and 5.4% for all relevant reduced sulfur compounds, which was

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acceptable for biological samples. Based on these findings we carried out the succeeding experiments

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in single replicates and applied standard deviations calculated from Table 2.

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The method relied on isotope-labelled sulfate reduction and hence the ability to trace increased levels

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of H233S upon 33SO4-2 supplementation. Figure 2 presents experiments with three different

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concentrations of 33SO4-2, which was added to separate replicates and the resultant hydrogen sulfide

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(m/z 35) and R(33/32S) in hydrogen sulfide traced in the gas phase. Initial high hydrogen sulfide

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emissions were attributed to stripping of dissolved hydrogen sulfide due to stirring and the low initial

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pH of 6.5, which pushed the acid-base equilibrium of sulfide towards hydrogen sulfide. The manure



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used was 1.3, 1.6 and 1.9 months old, which caused slight differences during the first 5 h. When pH

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stabilized at 7.8 after 19 h, the emission rate of hydrogen sulfide settled at 1.34 ± 0.18 mM/day (eq 1),

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demonstrating repeatability and independence of the manure storage time as long as sulfate was present

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in sufficient concentrations. For the full pH profile, see the supporting information Figure S5. 33SO4-2

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was added after 19 h in final concentrations of 0.38-, 0.19-and 0.09 mM, which increased R(33/32S) in

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hydrogen sulfide. This confirmed isotope labelled sulfate reduction activity in the manure. The

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R(33/32S) increase was slightly above the expected of 11.1-, 5.95 - and 3.37%, respectively. After 45 h,

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R(33/32S) still increased steadily, and this trend was more pronounced when adding more 33SO4-2. Based

185

on the headspace exchange rate and assumption of complete mixing upon 33SO4-2 addition and gas-

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liquid equilibrium, a steady R(33/32S) level would be reached in less than 0.5 h according to the models

187

(eq 2). However, a steady R(33/32S) level was never reached, which was detectable only due to the high

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time resolution of the new method with PTR-MS presented in the present study. Isotope fractionation

189

(31, 32) is one possible explanation, which at high sulfate concentrations will deplete 32SO4-2 slightly

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faster than 33SO4-2 from the manure. However, this effect is expected to be minimal compared to the

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observations in this study (33, 34). Another reason could be high intracellular sulfate concentrations in

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sulfate-reducing bacteria (35, 36), which in some sulfate-reducing bacteria can accumulate up to more

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than 2000 fold, depending on the sulfate concentration (35). Assuming 9.0·107 SRB cells/mL manure

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(37) with a cell size similar to that of the Desulfovibrio species (0.8-1.3 x 0.8-5µm) (38), the SRB

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cellular volume is equivalent to 0.034 % of the manure. Intracellular sulfate accumulation in the range

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of 10 – 2000 fold would thus result in significant time delays before an equilibrium between R(33/32S)

197

in the extracellular sulfate and R(33/32S) in the emitted hydrogen sulfide is reached as seen in Figure 2

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(see supporting information S6 for more details).

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Impact of cysteine and methionine. In Figure 3 and 4, the methodology was further assessed by

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monitoring isotope patterns of hydrogen sulfide and methanethiol upon cysteine and methionine

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addition, respectively. All reactors were supplemented with 33SO4-2 at experiment initiation and

203

cysteine (Figure 3) and methionine (Figure 4) was added after 41 h. Figure 3 presents the immediate

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response in elevated hydrogen sulfide emission upon cysteine addition, which matched perfectly with a

205

parallel drop in R(33/32S) in hydrogen sulfide. This confirmed that the increase in hydrogen sulfide

206

emissions did not originate from sulfate reduction but rather cysteine degradation. Calculations of the

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relative increase of hydrogen sulfide and decrease in R(33/32S) was in agreement when accounting for

208

the increasing R(33/32S) possibly caused by sulfate accumulation. At approximately 60 h, cysteine was

209

completely degraded and both curves returned to their previous course. After 82 h both hydrogen

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sulfide emissions and R(33/32S) dropped. This was a phenomena observed in multiple experiments and

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the time of occurrence was negatively correlated with the storage time of the manure prior to the

212

experiment (results not shown). Sulfate concentrations was measured to be 0.05 ± 0.02 mM when this

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event occurred, which is in approximate accordance with several studies on half saturation constants,

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Ks, for sulfate reduction (39-41). It is plausible that the hydrogen sulfide drop was a consequence of

215

sulfate concentrations decreasing significantly below Ks for a major group of sulfate-reducing bacteria

216

in the swine manure. In Figure 4, methionine addition increased methanethiol and subsequently

217

dimethyl sulfide emissions 150-164 fold and 84-86 fold, respectively. Hydrogen sulfide emissions

218

increased 1.6-1.9 fold, which was clearly observed in the R(33/32S) in hydrogen sulfide. The R(33/32S) in

219

methanethiol increased upon experiment start due to added 33SO4-2 but was problematic to determine

220

already after 15 h due to m/z 50 dropping close to the background signal resulting in large deviation on

221

the R(33/32S) in methanethiol. However, upon methionine addition methanethiol was instantly formed

222

and allowed for accurate R(33/32S) readings, which amounted to 2.00 ± 0.02% equivalent to expected



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background levels (0.87% from 33S + 1.10% from 13C). This confirmed that the produced methanethiol

224

came from methionine degradation rather than hydrogen sulfide methylation. The abrupt decline in

225

methanethiol and dimethyl sulfide emissions after 60 h indicated complete consumption of methionine,

226

which yielded a methionine degradation rate much similar to the rate of cysteine degradation around 1-

227

1.25 mM/day. These results illustrate how both cysteine and methionine addition is observable in the

228

isotope patterns of hydrogen sulfide and methanethiol respectively as long as sulfate reduction is not

229

limited by low sulfate concentrations and m/z 50 is above the detection limit. It was also verified that

230

cysteine and methionine were degraded quickly and that these compounds were naturally present in

231

values much smaller than Ks for cysteine and methionine degradation. This suggest that the bottle neck

232

in methionine and cysteine degradation was protein hydrolysis and stresses the importance of

233

optimizing feeding approaches for livestock animals to prevent surplus of s-amino acids in the animal

234

faeces (42).

235

Methylation rate of hydrogen sulfide and demethylation rate of methanethiol and dimethyl

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sulfide. The methylation of hydrogen sulfide and demethylation rate of methanethiol and dimethyl

237

sulfide was inspected by exploiting the inhibitory effect of BES on methanogens. In Figure 5, BES was

238

added after 18 h triggering a 4.6 fold and 6.8 fold increase in methanethiol and dimethyl sulfide

239

emissions, respectively. Assuming complete demethylation inhibition and comparing before and after

240

emission rates, it was equivalent to demethylation rates of 0.048 ± 0.005 and 0.0049 ± 0.0005 mM/day

241

for methanethiol and dimethyl sulfide demethylation, respectively (eq 3). Hydrogen sulfide emissions

242

were slightly elevated after BES addition. This could imply that sulfate-reducing bacteria to some

243

extent demethylate methanethiol and dimethyl sulfide to produce hydrogen sulfide as earlier described

244

in studies on marine systems (43). In Figure 6, the total methanethiol and methanethiol from hydrogen

245

sulfide methylation is depicted. 33SO4-2 was added at experiment start. Methanethiol from hydrogen


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sulfide methylation rose steadily and amounted to 21.7 ± 2.5% of the total methanethiol emission when

247

peaking. Consequently, 78.3 ± 2.5% of methanethiol came from methionine degradation. Methanethiol

248

from hydrogen sulfide methylation remained constant at 0.0130 ± 0.0015 mM/day (eq 4.) but total

249

methanethiol emissions decreased 10 h after BES addition. This suggested that methionine was slowly

250

consumed and below Ks concentrations. These findings are valuable when attempting various odor

251

abatement strategies, since preventing sulfate reduction is evidently not the major source of

252

methanethiol. Subtracting methanethiol production originating from hydrogen sulfide methylation from

253

the demethylation rates, the methionine contribution to hydrogen sulfide emissions was estimated to

254

0.040 ± 0.005 mM/day or 2.9 ± 0.3% of total hydrogen sulfide emissions (eq 5).

255

Sulfate reduction rate. Hydrogen sulfide originates from sulfate reduction and cysteine degradation.

256

To assess the magnitude of either reaction, molybdate was added as a sulfate reduction inhibitor seen in

257

Figure 7. To test whether molybdate solely inhibited sulfate reduction, cysteine was added prior to -

258

and after molybdate addition in equal amounts. Based on the hydrogen sulfide emissions (eq 1) the

259

maximum cysteine degradation rate was 2.38 ± 0.32-and 0.36 ± 0.05 mM/day before-and after

260

molybdate addition respectively. This was equivalent to an 85% reduction in cysteine degradation rate.

261

This might be explained by the general antimicrobial effect of molybdate in high concentrations (44).

262

In Figure 8, the effect of molybdate on hydrogen sulfide emissions without interference from cysteine

263

addition was inspected while monitoring the R(33/32S) in hydrogen sulfide. A total reduction of 96.5%

264

was observed upon molybdate addition and a coherent drop in R(33/32S) indicated that the inhibition of

265

sulfate reduction was more prominent than that of cysteine degradation. A comparison with the

266

R(33/32S) of a control replicate showed that sulfate reduction was responsible for only 14.8 ± 2.0% of

267

hydrogen sulfide emissions after 56 h (part of eq 6). Combined with the molybdate effect on cysteine

268

degradation, this allowed for estimation of the cysteine degradation rate to be 0.26 ± 0.03 mM/day



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equivalent to 20.2 ± 2.7% of the total hydrogen sulfide emission under non-supplemented conditions

270

(eq 6). Assuming cysteine, methionine and sulfate reduction are the only sources contributing with

271

sulfur, the remaining 76.9 ± 3.0% of the total hydrogen sulfide emissions came from sulfate reduction,

272

which yields a sulfate reduction rate of 1.03 ± 0.04 mM/day. This is six times higher than reported

273

elsewhere (45), which could be associated with the age of the manure used (46). This suggests that

274

odor abatement strategies, in relation to hydrogen sulfide, should focus on minimizing sulfate

275

reduction, which is achievable through acidification technology (47). Methionine degradation indirectly

276

contributed with 2.5 ± 0.3% of the emitted hydrogen sulfide but 78.3 ± 2.5% of the methanethiol

277

production. The importance of dealing with the methionine degradation process is emphasized by the

278

high odor activity value of methanethiol (7). Methionine degradation can partly be controlled by

279

optimizing feeding systems (48), but a focus on inhibiting the biochemical pathway is an alternative

280

option. It remains a challenge to effectively inhibit specific pathways without interfering with general

281

biochemistry in microorganisms or poison the environment. Therefore examining potential inhibitors

282

with the new method presented in our work should be a focus point in future studies.

283 284

ASSOCIATED CONTENT

285

Supporting information. Figure S1, Peak Interference. Figure S2, Assumption of Gas-Liquid

286

equilibrium. Figure S3, Assumption of no pH gradient. Figure S4, Linearity of PTR-MS response.

287

Figure S5, manure pH. Figure S6, sulfate accumulation.

288 289

AUTHOR INFORMATION

290

Corresponding Author* Phone: +45 3089 6099; e-mail: [email protected].

291


Page 15 of 32


292

ACKNOWLEDGEMENT

293

This project was part of the ManUREA project funded by GUDP under the Danish AgriFish Agency,

294

ministry of environment and food Denmark (Grant 34009-15-0934). The authors acknowledge

295

assistance from Heidi Grønbæk Christiansen, Janni Ankerstjerne Sørensen, Jeanette Pedersen, Kai

296

Finster and Hans-Henrik Brinck Friis for helping with laboratory equipment, conducting analysis and

297

sampling manure.

298

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434

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435

10.2134/jeq2009.0400.

436



437

Table 1. Compounds measured by PTR-MS in multiple ion detection mode. Detected mass

Compound

(m/z)

438 439

a

Detection limita

Dwell time

(ppb)

(ms)

18

NH3 (ammonia)

45

200

35

H232S (sulfide)

6.5

500

36

H233S (sulfide)

0.3

2000

32

49

CH3 SH (methanethiol)

0.08

500

50

CH333SH (methanethiol)

0.001

2000

61

CH3COOH (acetic acid)

0.10

500

63

CH332SCH3 (dimethyl sulfide)

0.39

2000

64

CH333SCH3 (dimethyl sulfide)

0.18

2000

32 32

79

CH3 S S (S-fragment).

0.02

500

95

CH332S32SCH3 (dimethyl disulfide)

0.13

500

Calculated as 3 × SD of charcoal filtered background concentration 


Page 22 of 32

Page 23 of 32


440

Table 2. Detection limits, mean emission rates and standard deviations (SD) of biological triplicates on

441

reduced sulfur compounds. m/z

Compound

Emission detection limita

Mean emission rateb

Mean relative SDb

(mM/day)

(mM/day)

(%)

35

Hydrogen sulfide (32S)

3.9 × 10-4

1.4

13.3

36

Hydrogen sulfide (33S)

3.7 × 10-5

5.4 × 10-2

5.4

49

Methanethiol (32S)

4.7 × 10-6

6.7 × 10-3

10.9

50

Methanethiol (33S)

7.6 × 10-7

2.3 × 10-4

11.7

-5

-4

10.5

63

32

Dimethyl sulfide ( S)

2.3 × 10

6.2 × 10

442

a

Calculated as 3 × SD of charcoal filtered background emission rate.

443

b

Based on means of the experimental period between 5-30 h.

444



Page 24 of 32

445 446

Figure 1. The experimental reactor setup. Mass flow controllers (MFC), were used to control the

447

continuous gas flow into the three reactors. A magnet stirrer (M) applied agitation to the manure in the

448

reactors. The reactor gas outlet was diluted with a continuous nitrogen flow controlled by three

449

additional MFCs. The diluted flow was directed to a valve, which was controlled and connected to the

450

PTR-MS instrument.

451


Page 25 of 32


452 453

Figure 2. The hydrogen sulfide emission rates from three replicates with manure age of 1.3 (□) -, 1.6

454

(×) - and 1.9 (○) months. After 19 h, 33SO4-2 was added in concentrations of 11.1% (∆), 5.95% (◊) and

455

3.37% (+), which affected the R(33/32S) in hydrogen sulfide correspondingly. R(33/32S) models (- for ∆, -

456

for ◊ and - for +) stabilized quickly and underestimated R(33/32S).



457 458

Figure 3. The effect of cysteine on the emission rate of hydrogen sulfide (□) and the corresponding

459

R(33/32S) in hydrogen sulfide (×). 33SO4-2 was added at experiment initiation and cysteine was added

460

after 41 h.


Page 26 of 32

Page 27 of 32


461 462

Figure 4. The effect of methionine on the emission rate of hydrogen sulfide (+), methanethiol (∆) and

463

dimethyl sulfide (○) and the corresponding R(33/32S) in hydrogen sulfide (×) and R(33/32S) in

464

methanethiol (-).33SO4-2 was added at experiment initiation and methionine was added after 41 h.



465 466

Figure 5. The effect of BES on emission rates of hydrogen sulfide (◊), methanethiol (∆) and dimethyl

467

sulfide (○). BES was added after 18 h.


Page 28 of 32

Page 29 of 32


468 469

Figure 6. The total methanethiol emission (∆) and the methanethiol emission originating from hydrogen

470

sulfide methylation (□). BES was added after 18 h, and methanethiol originating from hydrogen sulfide

471

methylation is only shown after BES addition.



472 473

Figure 7. The effect of cysteine on hydrogen sulfide emission (□) before and after molybdate addition.

474

Cysteine was added after 19 h and 64 h. Molybdate was supplemented after 41 h.


Page 30 of 32

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475 476

Figure 8. The effect of molybdate on hydrogen sulfide emission (◊) and the corresponding R(33/32S) in

477

hydrogen sulfide (×) was compared with the R(33/32S) in hydrogen sulfide in a control replicate (∆) (no

478

molybdate addition).



479

TOC GRAPHIC

480


Page 32 of 32

TITLE.Application of PTR-MS and 33S Isotope Labelling for Monitoring

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