AEM Accepted Manuscript Posted Online 26 February 2016 Appl. Environ. Microbiol. doi:10.1128/AEM.03502-15 Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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Carbon isotope fractionation during catabolism and anabolism in

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acetogenic bacteria growing on different substrates

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Christoph Freude, Martin Blaser

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Department of Biogeochemistry, Max-Planck-Institute for Terrestrial Microbiology, Karl-

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von-Frisch-Str. 10, 35043 Marburg, Germany

8 9 10

Keywords:

acetogen,

homoacetogen,

chemolithotroph,

fractionation into biomass.

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Corresponding author:

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Martin B. Blaser

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Max Planck Institute for Terrestrial Microbiology

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Karl-von-Frisch-Str. 10,

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35043 Marburg, Germany

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Tel:

+49-6421-178 870

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Fax:

+49-6421-178 999

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Email: [email protected]

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acetate,

formate,

methanol,

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Abstract

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Homoacetogenic bacteria are versatile microbes, which use the acetyl-CoA pathway to

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synthesize acetate from CO2 and hydrogen. Likewise the acetyl-CoA pathway may be used to

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incorporate other one carbon substrates (e.g. methanol or formate) into acetate or to

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homoferment monosaccharides completely to acetate. In this study we analyzed the

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fractionation of acetogenic pure cultures grown on different carbon substrates: While the

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fractionation of Sporomusa sphaeroides grown on C1 compounds was strong εC1 = -49 to -64

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‰, the fractionation of Moorella thermoacetica and Thermoanaerobacter kivui using glucose

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(εGlu = -14.1 ‰) was roughly 1/3 of these values suggesting a contribution of less depleted

30

acetate from fermentative processes. For M. thermoacetica this could indeed be validated by

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the addition of nitrate, which inhibited the acetyl-CoA pathway resulting in a fractionation

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during fermentation of εferm = -0.4‰. In addition we determined the fractionation into the

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microbial biomass of T. kivui grown on H2/CO2 (εanabol. = -28.6 ‰) as well as glucose (εanabol.

34

= +2.9 ‰).

35

36

1. Introduction

37

Acetogenic bacteria are a widespread microbial group unified by their ability to use the

38

reductive acetyl-CoA pathway which allows chemolithoautotrophic growth on hydrogen and

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carbon dioxide and is the only known pathway that combines carbon dioxide fixation with

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energy conservation (1-6). Most acetogens can use the reductive acetyl-CoA pathway for

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autotrophic growth reducing two carbon dioxide molecules with four hydrogen to liberate one

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acetate. Under these conditions CO2 reduction with H2 yields a ΔG0’ of – 95 kJ/mol. This is

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barely enough to generate one ATP using either a sodium or proton-dependent electron

44

motive force (7). On the other hand, the acetyl-CoA pathway can be accompanied by

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fermentation of carbon substrates. Under these autotrophic conditions the energy yield for e.g.

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glucose fermentation to three acetate is ΔG0’ = - 310.9 kJ/mol. In contrast to most other

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anaerobe fermentations of glucose which yield only one to three ATP via substrate level

48

phosphorylation during glycolysis, acetogens can redirect the reducing equivalents to the

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reductive acectyl-CoA pathway, which allows the generation of an additional ATP as

50

described above (3, 8).

51

One approach to evaluate the contribution of different bacterial pathway to certain

52

compound pools in environmental systems is to use the natural abundance of stable carbon

53

isotopes to track their metabolic activity in the environment(9, 10): In principle each

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metabolic pathway is characterized by a specific preference for a certain carbon isotope

55

(usually the lighter 12C) during catalysis, resulting in a characteristic depletion of 13C between

56

substrate and end product. Mathematically this depletion is characterized by the so called

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apparent fractionation factor (α) or enrichment factor (ε = 10³[1-α]). For a reaction A → B,

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the fractionation factor is defined as αA,B = (δ13CA + 103)/(δ13CB +103) (11).

59

There are two types of isotope effects: the equilibrium isotope effect and the kinetic

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isotope effect. In the first case equilibrium is attained in a closed, mixed system in which a

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bidirectional reaction occurs. The associated isotope effects are typically small e.g. the

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dissolution of inorganic carbon (DIC) in seawater results in unequal isotope distribution

63

between the atmospheric CO2 being -7.9 ‰ depleted in 13C compared to the dissolved

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bicarbonate (12, 13). In contrast biological reactions usually display a kinetic isotope effect,

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which generally discriminates against the heavy isotope, so that the isotopic value of the

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product is lower than that of the substrate. Biochemical pathways usually consist of a

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sequence of irreversible or unidirectional reactions which influence the overall fractionation

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factor to various extents (14-16). Most biochemical reactions that interconvert C1 compounds

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have large fractionation effects; in contrast, most heterotrophic reactions involving complex

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organic substrates have small fractionations (see below). Usually the fractionation factor are

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determined in microbial pure cultures and can be derived from either substrate depletion or

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product accumulation (17). Well documented example for carbon isotope fractionations

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comprise e.g. methanogenesis, showing fractionation factors between -25 to -69‰ for

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methanogenesis from CO2 (18-21), -73 to -83‰ from methanol (22, 23), and -7 to -35‰ for

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methanogenesis from acetate (21, 24-27), respectively. In environmental samples

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fractionation factors can be used to discriminate different pathways, if the difference in

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fractionation is strong enough e.g. E.g., the fractionation factor for hydrogenotrophic

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methanogenesis can be determined by incubation of soil samples in the presence of CH3F (28-

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31), which specifically inhibits aceticlastic methanogenesis (32, 33).

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The acetyl-CoA pathway imprints a strong isotopic enrichment of -55 to -60 ‰ during

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acetate formation using H2/CO2 as substrate (34-36). In contrast the fractionation associated

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with acetate fermentation by microbial glycolytic fermentation is small ( 1 CH3COOH + 2 CO2 + 2 H2O

(2)

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1 HCOOH + 3 H2 + CO2=> 1 CH3COOH + 2 H2O

(3)

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4 CH3OH + 2 CO2 => 3 CH3COOH + 2 H2O

(4)

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1 C6H12O6 => 3 CH3COOH

(5)

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Cultures were grown in 120 mL serum bottles, which contained 50 mL medium. At the

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times indicated 100 µL gas samples were taken with a gas tight syringe (VICI, Baton Rouge,

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LA USA) for analysis of concentration and δ13C of both CO2 and CH4; 2 mL of liquid

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samples were taken for analysis of concentration and δ13C of glucose and short chained fatty

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acids (SCFA).

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2.2. Chemical and isotopic analyses: CH4 was analyzed by gas chromatography (GC)

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using a flame ionization detector (Shimadzu, Kyoto, Japan). CO2 was analyzed after

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conversion to CH4 with a methanizer (Ni-catalyst at 350 °C, Chrompack, Middelburg,

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Netherlands). Isotope measurements of 13C/12C in gas samples were performed on a gas

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chromatograph combustion isotope ratio mass spectrometer (GC-C–IRMS) system (Thermo

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Fisher Scientific, Bremen, Germany). The principle operation was described by Brand (51).

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The gaseous compounds were first separated in a Hewlett Packard 6890 GC using a Pora Plot

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Q column (27.5 m length, 0.32 mm internal diameter, and 10 µm film thickness; Chromopack

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Frankfurt, Germany) at 30 °C and He (99.996 % purity; 2.6 ml/min) as carrier gas. The

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sample was run through the Finnigan Standard GC Combustion Interface III and the isotope

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ratio of 13C/12C was analyzed in the IRMS (Finnigan MAT Deltaplus). The reference gas was

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CO2 (99.998 % purity) (Air Liquide, Düsseldorf, Germany), calibrated with the working

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standard methylstearate (Merck). The latter was intercalibrated at the Max Planck Institute for

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Biogeochemistry, Jena, Germany (courtesy of W. A. Brand) against the NBS-22 and USGS-

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24 standards and reported in the delta notation versus Vienna Pee Dee Belemnite.

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δ 13C = 103(Rsample/Rstandard-1)

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with R = 13C/12C of sample and standard, respectively.

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Isotopic analysis and quantification of glucose and SCFA were performed on a high

(6)

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pressure liquid chromatography (HPLC) system (Spectra System P1000, Thermo Fisher

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Scientific, San Jose, CA, USA; Mistral, Spark, Emmen, the Netherlands) equipped with an

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ion-exclusion column (Aminex HPX-87-H, BioRad, München, Germany) and coupled to

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Finnigan LC IsoLink (Thermo Fisher Scientific, Bremen, Germany) as described (52). Isotope

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ratios were detected on an Isotope Ratio Mass Spectrometer (IRMS) (Finnigan MAT

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Deltaplus Advantage). The precision of the GC-IRMS was ±0.2 ‰, and that of the HPLC-

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IRMS was ±0.3 ‰. For further details on determination of acetate concentration via HPLC-

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IRMS compare the supplementary material (Fig. S9, Tab. S2).

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The isotopic fractionation into the biomass was evaluated by harvesting three replicate

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samples (120 mL serum bottles) each containing 50 mL culture for each time point.The cells

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were harvested by centrifugation (10 minutes at 6.000 rpm (9.700g)). The resulting pellet was

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dried in tin capsules and sent to the isotope centre in Göttingen. There the isotopic values

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were determined using an elemental analyser coupled to an IRMS (Kompetenzzentrum

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Stabile Isotope at the University of Göttingen, Germany). Pure acetate was used as a reference

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to compare the results with our HPLC-IRMS measurements.

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2.3. Calculations:

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The apparent fractionation factor α for a reaction A → B is defined after (11) as:

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αA/B =(δA + 1000)/(δB + 1000)

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also expressed as ε ≡ 103 (1 – α). The isotope enrichment factor ε associated with

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(7)

homoacetogenesis was determined as described by Mariotti (17) from the residual reactant

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δr = δri + ε[ln(1− f)]

(8)

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and from the product formed

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δp = δri − ε(1− f)[ln(1− f)]/ f

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where δri is the isotope composition of the reactant (CO2) at the beginning, δr and δp are the

(9)

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isotope compositions of the residual CO2 and the pooled acetate, respectively, at the instant

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when f was determined. Linear regression of δr against ln(1 – f) and of δp against (1 – f)[ln(1 –

201

f )]/f gives ε as the slope of best-fit lines. The fractional yield f of a reaction is usually based

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on the consumption of the substrate CO2 (0 < f < 1); in a closed system the amount of used

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substrate (f) can be derived from the measured isotopic composition (35).

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fdelta= (δri − δr)/( δp – δr)

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In contrast to the isotope enrichment factor ε derived from microbial pure cultures as

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(10)

described above, many environmental studies express the apparent fractionation Δ as:

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Δ = δprod – δsubst

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Where δsubst is the δ13C value of the substrate and the δprod is the δ13C value of the product.

209

While this estimated fractionation is valid for open systems like environmental samples, it

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is only valid at the very beginning of a reaction in a closed system with limited substrate

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concentrations (17, 53, 54). Nevertheless we used this estimation to calculate the apparent

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fractionation for the mixotroph experiment to estimate the fractionation into acetate vs.

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biomass for cells grown on formate or H2/CO2.

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(11)

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Results

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Growth on C1 compounds

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In order to evaluate the fractionation factor in the acetyl-CoA pathway, we measured the

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production of acetate during the consumption of different C1 compounds which are either

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direct intermediates (CO2, formate) or can be easily incorporated into the acetyl-CoA pathway

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(methanol) (compare Fig. 1). These experiments were conducted using Sporomusa

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sphaeroides. The substrate depletion and product formation was analyzed over time and the

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delta 13C of all carbon containing compounds was measured during the reaction (Fig. 2).

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Despite different 13C values of the initial substrates methanol δmethanol = -38 ‰, formate δformate

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= -31 ‰ or H2/CO2 δCO2 = -17 ‰ the released acetate eventually reached the same value

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δacetate = -67 ‰. The delta 13C of all three substrates became enriched in heavy carbon with

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roughly the same rate. When the apparent fractionation factors were calculated based on the

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substrate data the following values were obtained: methanol εmethanol = -56.9 ‰, formate

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εformate = -56.5 ‰ and H2/CO2 εCO2 = -63.8 ‰. The delta 13C of CO2 was not changing much

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when methanol or formate served as substrates due to the large background of bicarbonate

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(4.5 mmol/bottle NaHCO3) in the culture medium. Details on the acetate formation for the

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individual substrates are given in Fig. S2. Details and further discussion on the isotope values

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of acetate are given in Fig. S3 and supplementary discussion.

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In a different approach the influence of different C1 compounds was tested using the

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thermophilic acetogen Thermoanaerobacter kivui grown under a headspace of N2/CO2 on

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formate (1 mmol per bottle). In parallel T. kivui was inoculated under a headspace of H2/CO2

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(2.5 mmol and 0.6 mmol per bottle (70 mL headspace in 120 mL)) either alone or

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mixotrophic in the presence of low (0.25 mmol per bottle (50 ml medium per 120 mL bottle))

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or high (1 mmol per bottle (50 ml medium per 120 mL)) formate concentrations (Fig. 3, Tab.

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1). The bicarbonate concentration was 4.5 mmol per bottle in all incubations. Details on the

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carbon turnover are given in Fig. S4. Using hydrogen (equ. 1) and formate (equ. 3)

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consumption as proxies for the substrate turnover into acetate an almost complete turnover

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could be documented for all experiments (Tab. 1). This was used to calculate the relative

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contribution of both substrates under mixotrophic conditions and to estimate the apparent

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fractionation Δ between substrate (t0) and product (acetate, tend) or substrate (t0) and biomass

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(tend). A certain statistically not significant trend towards 13C depleted acetate as well as

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biomass could be observed for increasing contributions of formate vs. CO2 (Tab. 1). The

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calculated enrichment factor using the linear regression given in equ. 13 resulted in εCO2 = -

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48.7 ‰ for the incubations under H2/CO2 and εform = -51.9 ‰ for the formate incubations

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under N2/CO2.

250 251

Growth on glucose and nitrate inhibition

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In a further set of experiments the fractionation was determined when acetogenic pure

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cultures were grown on complex carbon substrates. As an example Moorella thermoacetica

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grown on glucose (200 µmol), glucose (200 µmol) plus nitrate (300 µmol), or H2/CO2 (2.5

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mmol / 625 µmol) was used. Indeed our isotopic measurements revealed almost no

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fractionation with glucose and nitrate εGlu+NO3 = -0.4 ‰, strong fractionation with H2/CO2

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εH2/CO2 = -55.8 ‰ and an intermediate fractionation with glucose εGlu = -18.5 ‰ reflecting the

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mixing of both acetate generating pathways (Fig. 4). Additional data on the nitrate depletion

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are given in Fig. S5. Similar results could be obtained for T. kivui where the fractionation

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under glucose (εGlu = -14.1 ‰) was roughly 1/3 of the fractionation under H2/CO2 (εH2/CO2 = -

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53.0 ‰) (compare Fig. S6) and also for Acetobacterium carbinolium (εGlu = -18.8 ‰ vs.

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εH2/CO2 = -54.0 ‰) (data not shown).

263 264

Fractionation into biomass

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In contrast to aerobe bacteria anaerobic bacteria can only use a small amount of the

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available energy for anabolism (usually around 10 %) and hence only a small portion of the

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available carbon is fixed into biomass. However it was observed that T. kivui grew to dense

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cultures when grown on glucose but only faint turbidity was observed for cells grown on

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H2/CO2. Therefore it was questioned how substrate usage affects the delta 13C values of the

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microbial biomass. Therefore, Thermoanaerobacter kivui was grown on glucose (200 µmol

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under N2/CO2) or on H2/CO2 (2.5 mmol / 625 µmol) in the described bicarbonate buffered

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minimal medium. Hence we expected that under both conditions roughly the same amount of

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acetate would be produced. The delta 13C of substrate and product as well as biomass was

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followed over time (Fig. 5). The fractionation during catabolism was calculated using the 13C

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values of the substrate together with the 13C values of the acetate using eq. 8; the fractionation

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during anabolism was calculated respectively using the 13C values of the substrate together

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with the 13C values of the biomass. While growth on glucose resulted in a small positive

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fractionation during catabolism εcatabol. = + 4.2 ‰ as well as anabolism εanabol. = + 2.9 ‰,

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growth on H2/CO2 was accompanied by a stronger fractionation caused by catabolism εcatabol.

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= -48.6 ‰ than anabolism εanabol. = -28.6 ‰.

281

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Discussion

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Growth on C1 compounds

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The data presented in this manuscript confirmed the overall strong fractionation of

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acetogens using the acetyl-CoA pathway when H2/CO2 was the substrate (34, 35); in addition

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it could be shown, that the fractionation of cells grown on different C1-compounds likewise

287

resulted in a very strong fractionation and was largely independent from substrate usage

288

(different C1 compounds). This is in contrast to several published examples where the rate

289

limiting step (usually the initial step of a reaction cascade) is primarily determining the

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overall fractionation.

291

One prominent example that the initial step of a reaction cascade is determining the overall

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fractionation can be found in the plant system, where the initial CO2 fixing reaction of C3 and

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C4 plants (RubisCO vs. phosphoenolpyruvate carboxylase) dictates strong differences in the

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resulting plant material (55) which is largely unchanged throughout the food chain (56, 57). In

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the microbial world a similar behavior is well documented for methanogenic pure cultures on

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different substrates, where each substrate is accompanied by a unique fractionation factor: e.g.

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methanogenic pure cultures of M. barkeri grown on different carbon substrates show highly

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distinct carbon isotope fractionations from substrate to product when grown on H2/CO2 (-45.4

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to -46.3 ‰), methanol (-72.5 to -83.4 ‰) or acetate (-21.2 to -34.8 ‰) (22, 58). These results

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have been independently validated for other methanogenic pure cultures on H2/CO2 (20, 21),

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methanol (23) and acetate (25, 27). Further on, it was shown for hydrogenotrophic

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methanogens (using H2/CO2) that the fractionation is highly affected by the hydrogen

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(energy) level of the incubations resulting in stronger fractionation under H2 limiting

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conditions (20-22). For a sulfate reducing Desulfovibrio the fractionation of sulfur isotopes

305

was also affected by the electron donors and negatively correlated to the sell-specific sulfate

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reduction rate (59). Recent evidence suggest that also for sulfate reducing bacteria the overall

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isotope fractionation during sulfate reduction is influenced by all steps in the dissimilatory

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pathway (16, 60).

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By contrast, differences in the substrate ratios of H2 and CO2 using acetogenic cultures did

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not affect the fractionation (61). Furthermore, it has been shown that there is no

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intramolecular fractionation during acetate formation: isotope values of the methyl-group

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were similar to the delta 13C values of the overall acetate signal (35). The range of

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fractionation for different C1 compounds reported in this study is well within the previous

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reported range (-38 to -68 ‰) for acetogenic cultures grown on H2/CO2 (34, 35). And now

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our data on the conversion of different C1 compound to acetate suggest a similar

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discrimination against 13C irrespective of the individual C1 compound.

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Our results of the S. sphaeroides incubations can be interpreted in two ways: 1.) One

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explanation is that the initial fractionation of all possible initial enzymes is highly similar. 2.)

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Alternatively our results can be explained if we assume that the rate limiting step determining

320

the overall fractionation is the C-C bond formation by the CODH/ACS complex. We will

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discuss both possibilities:

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1.) The individual entry points for the different C1 compounds are schematically given in

323

Fig. 1. In detail they involve the following four (a-d) enzymes: methanol is incorporated using

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a methanol cobalamin methyltransferase (a) (62). While one molecule of CO2 is reduced via

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CO to the carboxyl-group of acetate by the enzyme-complex CO dehydrogenase / acetyl-CoA

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synthase (b) a second molecule of CO2 is reduced to formate by the formate dehydrogenase

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(c). Hence formate is a direct intermediate and can either be oxidized to CO2 or further

328

reduced in an ATP-dependent reaction catalyzed by the formyl-tetrahydrofolate synthetase (d)

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(3). In principle it is possible that all these four entry points exhibit the same fractionation.

330

However, fractionation of methanol and CO2 in methanogenic pure cultures renders

331

completely different fractionation factors using homologous enzymes for the initial

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incorporation. Therefore we think that in acetogenic bacteria the fractionation is mainly

333

controlled by a later step during the reductive acetyl-CoA pathway; presumably the

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condensation of the two carbons in the CODH / ACS system:

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2.) All the substrates we used were incorporated into the methyl branch of the acetyl-CoA

336

pathway (Fig. 1) yielding the methyl-group of the released acetate. However, the reduction of

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formate is accompanied by the release of CO2 (eq. 2); while one part of the methanol is

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oxidized to CO2 to provide reducing equivalents three molecules of methanol and CO2 are

339

reduced to acetate resulting in a net consumption of CO2 (eq. 4). In any case our experimental

340

set up does not allow the discrimination whether the carboxyl-group is directly formed from

341

external CO2 reduction (e.g. carbonate buffer) or originates from an intracellular CO2 pool

342

generated through oxidation of methanol or formate.

343

The acetyl-CoA pathway can operate in both directions; while most acetogens use it for

344

carbon fixation from CO2 and energy conservation via an Ech of Rnf system (7) others can

345

operate in the reverse direction together with an syntrophic partner to perform syntrophic

346

acetate oxidation (63, 64). As a result most reactions of the acetyl-CoA pathway can be seen

347

as highly reversible and hence can easily equilibrate different isotopic δ13C values. Therefore

348

it is plausible that not the initial step but the first condensing step is the critical point for the

349

fractionation. Indeed this explanation would be in agreement with the results obtained for the

350

C3 and C4 plants where the initial step is a C-C forming condensation reaction.

351

When T. kivui is grown on the expense of a methylated aromatic compound (vanillic acid)

352

the initial step (O-demethylation of vanillic acid) may be less reversible. Indeed initial trials

353

resulted in less depleted acetate for T. kivui grown on the expense of vanillic acid (δac =-50

354

‰; compare Fig. S7) compared to δac =-67 ‰ derived from cultures grown on C1 compounds

355

(compare Fig. 1). This data could however not be further evaluated since the 13C value of the

356

substrate vanillic acid could not be measured using our HPLC-IRMS approach.

357 358

Growth on glucose and nitrate inhibition

359

Both investigated cultures (M. thermoacetica and T. kivui) showed a strong fractionation

360

(ca. -50 to -55 ‰) during acetate formation when the acetyl-CoA pathway was the only

361

operative pathway (34). As soon as glucose was used as a substrate the apparent fractionation

362

was a mixture of fermentation and acetyl-Co pathway resulting in a fractionation of less than

363

< -20 ‰.

364

These findings have environmental implications: The strong fractionation during acetate

365

production via the acetyl-CoA pathway can easily be tracked in natural environments. Ideally

366

it can be used to estimate the contribution of acetogens to the acetate pool, when the

367

fractionation of all other acetate generating and depleting reactions is known (9). The mixed

368

fractionation of acetogens grown on larger molecules like glucose (less than < -20 ‰) may be

369

difficult to differentiate in environmental systems where the acetate signal deviates by ± 10 ‰

370

from the delta13C value of the total organic carbon (65). Part of this variation may originate

371

from the contribution of the acetogenically formed 13C depleted acetate. Indeed incubations of

372

lake sediments only resulted in 13C depleted acetate under H2/CO2 addition and not in control

373

experiments under N2 (66), suggesting that the acetogens in the later samples are either less

374

active or grow on complex substrates.

375 376

Fractionation into biomass

377

Many studies use published pure culture fractionation factors to deduce the importance of a

378

certain reaction in environmental settings. Most of these fractionation factors have been

379

calculated using a mass balance equation based on the substrate to product conversion but

380

neglecting the buildup of biomass. Hence we aimed to validate if this approach is reasonable.

381

Comparing the fractionation during catabolism and anabolism shows that fractionation into

382

the biomass is usually lower than the discrimination during catabolism. E.g. the fractionation

383

into the biomass is roughly 1/3 of the fractionation into methane for M. barkeri grown on

384

H2/CO2 (-13.9 vs. -45.3 ‰), acetate (-7.3 vs. -34.4 ‰), methanol (-31.1 vs. -83.4 ‰) and

385

trimethylamine (-24.8 vs. -66.5 ‰) (22).

386

So far the fractionation into biomass of bacterial pure cultures has mainly been documented

387

using endpoint measurements of the microbial biomass (compare Table S1). In contrast we

388

wanted to calculate the apparent fractionation into the biomass following a time series

389

experiment. Indeed we found that the fractionation during anabolism was roughly 2/3 of the

390

fractionation during catabolism. We discuss some possible reasons for this ration in the

391

supplementary discussion together with Fig. S8.

392

In general we would conclude that the fractionation into biomass is usually smaller than

393

the fractionation during catabolism. While oxidation of glucose yields ΔG0’ = -2.870 kJ mol-1

394

while anaerobic fermentation to CO2 and CH4 liberates only ΔG0’ = -390 kJ mol-1 (67). Hence

395

it can be assumed that under anaerobe conditions most of the available carbon is used for

396

catabolism and only a small portion can be used for anabolism. Therefore it is unlikely that

397

the bias caused by the fractionation into the biomass will impact on the apparent fractionation

398

of the catabolic reactions.

399

Another way to rationalize the fractionation into the biomass becomes apparent when we

400

compare different substrates (Table 2). The strongest fractionation can be found for C1

401

compounds (CO2, methanol) (-12.9 to -23 ‰). In comparison the biomass of organisms using

402

acetate ranges from 1.4 to -6.5 ‰; while the organisms utilizing larger molecules like glucose

403

have almost no discriminatory power regarding the different carbon isotopes (-0.4 to -3-3 ‰).

404

The isotope signal of organic carbon in environmental samples is rather constant; as is the

405

signal in important carbon pools (65). Usually the small differences observed can be

406

explained the small fractionation factors reported for fermentative processes (catabolic as well

407

as anabolic). On the other hand a strong fractionation into biomass has been reported for

408

methanogens using end point measurements (22), and was observed for acetogens grown on

409

H2/CO2 (this study). Indeed our results suggest that fractionation of autotrophic organisms

410

into their respective biomass is relatively strong and may in principle affect the organic

411

carbon pool in environmental samples. However the microbial communities responsible for

412

e.g. methanogenesis usually comprise only a few percent of the total microbial community

413

and hence it may be difficult to follow the isotopic differences imprinted by C1 metabolisms

414

in environmental settings.

415 416

Acknowledgements

417

The authors thank Ralf Conrad (MPI Marburg) for critically reading the manuscript. We

418

would like to thank three anonymous referees for valuable comments. We thank Rolf Thauer

419

(MPI Marburg) and Wolfgang Buckel (MPI Marburg) for helpful discussions on the isotopic

420

fractionation into biomass.

421

References

422

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Drake HL, Gößner AS, Daniel SL. 2008. Old acetogens, new light. Annals of the New York Academy of Sciences 1125:100-128.

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615 616

617 618

Table 2 average fractionation into biomass for different organismic groups and substrates. Given are average values ± DS. Details can be found in Table S1. organismic group Acetogen Methanogen Sulfate reducer Phototroph Methanogen Methanogen Sulfate reducer Fermentating bacterium Acetogen

619

Substrate CO2 CO2 CO2 CO2 methanol acetate acetate glucose glucose

Δsubstrate-biomass -12.9 ± 5.0 -18.5 ± 6.3 -20.4 ± 13.9 -23.0 ± 8.4 -20.5 ± 11.9 1.4 ± 6.7 -6.5 ± 1.3 -0.4 ± 1.9 -3.3 ± 3.0

n = 18 n = 23 n=5 n=8 n=5 n=4 n=2 n=6 n=6

620 621 622

Figure 1. Schematic representation of the reductive acetyl CoA pathway and entering points

623

of possible substrates. In black the reduction of CO2 to either the methyl or the carboxyl-

624

group is highlighted; the reverse reaction (oxidation) is given in grey arrows. For simplicity,

625

reducing-equivalents are given as H2. The entrance points of different C1 substrates is given

626

in dark grey. Note that the carboxyl-group can be formed by reduction of CO2 or indirectly by

627

oxidation of the respective C1-compound. The enzymes acting on the different C1 compounds

628

are listed: Methanol-cobalamin methyltransferase (1); CO dehydrogenase / acetyl-CoA

629

synthase (2); formate dehydrogenase (3); formyl-tetrahydrofolate synthetase (4). Details on

630

the reactions are given in equations 1 – 6 and in the text.

631 632

10 0 -10

delta 13C

-20 -30 -40 -50 -60 -70 -80 0

633

5

10

15

20 25 time (days)

30

35

40

CO2 (H2/CO2)

acetate (H2/CO2)

CO2 (methanol)

acetate (methanol)

methanol (methanol)

CO2 (formate)

acetate (formate)

formate (formate)

45

634

Figure 2. Sporomusa sphaeroides DSM 2875 grown on different C1 compounds (substrates

635

are given in brackets behind the measured analyte as: H2/CO2, formate or methanol). Depicted

636

is the 13C value of substrate (black filled symbols) and product (acetate; open symbols); CO2

637

for incubations with methanol or formate are given as open grey symbols. Values are given as

638

average of three independent replicates ± standard deviation. Individual plots for the different

639

substrates are given as Fig. S.1

640

20 10 0

delta 13C [‰]

-10 -20 -30 -40 -50 -60 -70 0

5

10

15

20

25

30

time (days) H₂/CO₂ (CO₂)

641

H₂/CO₂ (Ac)

low mix (CO₂)

high mix (CO₂)

formate (CO₂)

low mix (Form)

high mix (Form)

formate (Form)

low mix (Ac)

high mix (Ac)

formate (Ac)

642

Figure 3. Mixotrophic growth of Thermoanaerobacter kivui DSM 2030 on formate and

643

H2/CO2 leads to incorporation of both substrates. The measured analytes CO2, formate (Form)

644

or acetate (Ac) are given in brackets. H2/CO2 concentrations were 2.5 mmol/bottle (H2) and

645

0.6 mmol/bottle (CO2) in the H2/CO2 treatment as well as in the low and high mix treatment.

646

Formate concentrations were 0.25 mmol/bottle in the low mix and 1 mmol/bottle in the high

647

mix and formate treatment respectively. The δ13C value of the released acetate is a direct

648

result from the mixing of the δ13C values of the substrates while the fractionation of the

649

individual substrates is quite constant. Values are given as average of three independent

650

replicates ± SD.

651

10

A

0

delta 13C (‰)

-10 -20 -30 -40 -50 -60

CO₂ (H₂/CO₂)

Acetate (H₂/CO₂)

Glucose (Glu)

Acetate (Glu)

Glucose (Glu + NO₃)

Acetate (Glu+NO₃)

-70 0

5

10

15

20

25

time (days)

652 20

B

delta 13C of Substrate (‰)

15 10

H2/CO2

ɛ=-55.8‰

Glu

ɛ=-18.5‰

Glu + NO3 ɛ=-0.4‰

5 0 -5 -10 -15 -0.6

653

-0.5

-0.4

-0.3

-0.2

-0.1

0.0

ln(1-f)

654

Figure 4. Moorella thermoacetica DSM 2955 grown on H2/CO2 () or glucose () or

655

glucose with nitrate (). A) δ13C values of substrate (CO2 and glucose) and product (acetate)

656

data. The different treatments H2/CO2, glucose (Glu) and glucose plus nitrate (Glu + NO3) are

657

given in brackets. B) regression analysis of the respective incubations. The δ13C value of the

658

acetate reflects the stoichiometry of the producing pathway: e.g. on glucose 2/3 of the

659

released acetate originate from glycolysis and decarboxylation (glucose + nitrate ɛ = -0.4 ‰)

660

and 1/3 from the acetyl-CoA pathway (H2/CO2 ɛ = -55.8 ‰); the coordinated action of both

661

pathways results in a mixed fractionation reflecting the contribution of both pathways: ɛ = -

662

18.5 ‰.

20

A

10

delta 13C (‰)

0 -10 -20 -30 -40 -50 -60 0

5

10

15

20

25

30

35

time (days)

663 10

glucose (Glu)

acetate (Glu)

cells (Glu)

CO₂ (H₂/CO₂)

acetate (H₂/CO₂)

cells (H₂/CO₂)

B

y = -48.569x - 14.03 R² = 0.9959

5 0 delta 13C (‰)

-5

y = -28.559x - 14.459 R² = 0.9599

-10 -15 -20 y = 4.2214x - 26.207 R² = 0.9755

-25 -30

y = 2.8678x - 26.153 R² = 0.9732

-35 -1.2

-1

-0.8

-0.6

-0.4

-0.2

0

ln(1-f)

664 665

Figure 5. Fractionation into biomass: A) Delta 13C of Thermoanaerobacter kivui grown on

666

either H2/CO2 or glucose n = 3. Substrate is given in brackets. B) Regression analysis of CO2

667

conversion into acetate  or biomass  for cells grown on H2/CO2 and regression analysis of

668

glucose into acetate  or biomass for cells grown on glucose.