Accelerated CO2 reduction to methane for energy by zero valent iron in oil reservoir production waters

Accepted Manuscript Accelerated CO2 reduction to methane for energy by zero valent iron in oil reservoir production waters Lei Ma, Lei Zhou, Serge Maurice Mbadinga, Ji-Dong Gu, Bo-Zhong Mu PII:

S0360-5442(18)30105-1

DOI:

10.1016/j.energy.2018.01.087

Reference:

EGY 12199

To appear in:

Energy

Received Date: 13 September 2017 Revised Date:

22 November 2017

Accepted Date: 18 January 2018

Please cite this article as: Ma L, Zhou L, Mbadinga SM, Gu J-D, Mu B-Z, Accelerated CO2 reduction to methane for energy by zero valent iron in oil reservoir production waters, Energy (2018), doi: 10.1016/ j.energy.2018.01.087. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Accelerated CO2 Reduction to Methane for Energy by Zero Valent Iron in

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Oil Reservoir Production Waters Lei Ma1, Lei Zhou1,3, Serge Maurice Mbadinga1,3,Ji-Dong Gu2, Bo-Zhong Mu1,3*

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China University of Science and Technology, 130 Meilong Road, Shanghai 200237, P.R.

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China

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SAR, P.R. China

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School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong

Engineering Research Center for Microbial Enhanced Energy Recovery, Shanghai 200237,

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State Key Laboratory of Bioreactor Engineering and Institute of Applied Chemistry, East

P.R. China

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*

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E-mail: [email protected]

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Phone: +86 21 64252063; Fax: +86 21 64252485

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Corresponding Author: Bo-Zhong Mu

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Abstract We assessed the microbiological reduction of carbon dioxide into methane bioenergy

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through biochemical reaction by addition of zero valent iron (ZVI) as an alternative electron

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donor in oil reservoir production waters under strictly anaerobic conditions. Enhanced

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methane production was observed in all the treatments amended with ZVI compared with the

20

controls. The outcome of the microbial community (Illumina Next Generation Sequencing)

21

analysis

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Methanothermobacter spp. responsible for the production of methane. Moreover, the

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detection of FeCO3 in the culture medium amended with ZVI at the end of the experiment,

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characterized by X-ray Photoelectron Spectroscopy (XPS), indicated a potential CO2

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transformation via mineralization under the investigation conditions with simultaneous

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methane production. This study offers an alternative strategy for carbon dioxide reduction

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into methane for energy and also a potential possibility for carbon dioxide reducing and

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subsequently clean bioenergy recovery in oil reservoirs.

that

CO2-reducing

methanogens

were

closely

related

to

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Keywords: CO2 biotransformation; Methanogenesis; ZVI; Oil reservoir; Greenhouse gas;

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Energy recovery

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Graphic abstract

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1 Introduction Petroleum reservoirs are special deep subsurface environment with variety of specialized

37

microorganisms coexisting and interacting with multiphase fluids of crude oil, water and gas,

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and can also be considered as natural geobioreactors for industrial operation [1]. Crude-oil

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hydrocarbon biodegradation into methane occurs widely in petroleum reservoirs [2]. CO2 is

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an important microbial metabolic product of crude-oil from anaerobic degradation and also a

41

primary substrate for methanogenesis [2, 3]. In addition, the amount of CO2 required to

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recover a barrel of oil ranges from 3000 to more than 20000 cubic feet at standard conditions

43

in the enhanced oil recovery project (EOR) [4, 5], and most CO2 from EOR or carbon capture

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and storage (CCS) appears to be dissolved in formation fluid and gas-phase trapping [6, 7]

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and is a potential carbon feedstock for fuels and energy regeneration.

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Biological transformation of CO2 into value-added products such as methane as energy

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and other organic compounds that can be used as sustainable and clean energy sources in

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petroleum reservoirs, as one of value-added carbon management technologies without

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secondary pollution, attracts considerable attention in recent years and plays a significant role

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in the field of biogeochemistry, energy recovery and offsetting economic costs associated

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with EOR or CCS [8-12]. Diversity microorganisms related to bioconversion of CO2 such as

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acetogenic microorganisms and methanogens have been reported from various oil reservoirs

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[13, 14], indicating the potential for biotransformation of CO2 in subsurface petroleum

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reservoirs. The functional gene-based analysis of microbial communities in different oil

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reservoirs further confirms the above possibility for CO2 conversion [15, 16]. In particular,

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variety of methanogens have been detected and partly isolated from both high- and

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low-temperature

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approaches [17-20]. Microbial methanogenesis has also been confirmed to be widely

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distributed in petroleum reservoirs through the combination of molecular approaches and

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radioisotopic tracers [21, 22]. In particular, the employment of indigenous microorganisms as

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biocatalysts for methane production from CO2 effectively avoids requirements of high

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temperature and pressures by conventional gas-phase catalytic conversion methods [10].

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reservoirs

based

upon

culture-dependent

and

culture-independent

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In the simulated oil reservoir bioreactors through incubations of the production water

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and crude oil in the laboratory, CO2 can be transformed into methane by hydrogenotrophic 3

ACCEPTED MANUSCRIPT methanogens as the dominant methanogenic biochemical pathway in high-temperature oil

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reservoirs [21], whereas high pressure of CO2 invokes acetotrophic methanogenesis in place

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of syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis [23].

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Methanogenic archaea are also expected to fix CO2 and produce small organic molecules

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(formate) in addition to methane [24]. Homoacetogenesis with the substrate of CO2 and H2

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rather than methanogenesis is preferentially stimulated under simulating the in situ pressure

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and temperature in the imitated CO2-injection systems [25]. Carbonate and bicarbonate ions

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generated from dissolved CO2 can also react to dissolved calcium carbonate, silicate minerals

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or other trace metals and immobilized the CO2 in the form of minerals [26].

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Among different strategies, bioconversion of CO2 into methane for energy in petroleum

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reservoirs is regarded as a value-added option for regulating global carbon cycle and

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sustainable energy recovery with near-zero net CO2 emission [8, 27]. Available electron

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donors are essential and necessary for the occurrence of this biochemical pathway. Several

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elemental metals (particularly for ZVI) have been examined as potential electron donors for

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microbial methanogenesis from CO2 [28]. In fact, elemental iron could be served as the sole

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source of electrons for methanogenesis and growth from CO2 as early as 1987 [29]. In recent

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years, ZVI as well as various forms of iron and waste iron scrap has been widely studied in

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the process of anaerobic digestion and environmental contaminant remediation commonly

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along with effectively enhanced methane production due to high efficiency, improved

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conditions for anaerobic degradation and relatively cost-effective [30-36], which focus on the

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transformation and degradation of organic matters. Methane generation with iron by a

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Methanobacterium-like isolate is faster than known hydrogen-using methanogens [37].

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However, whether biotransformation of CO2 into methane is stimulated by ZVI as an

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alternative electron donor in oil reservoir production waters has never been assessed.

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Based upon the background information we proposed that ZVI can be potentially

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alternative electron donors for transformation of CO2, and promotes and facilitates the

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microbial methane production as bioenergy in petroleum reservoir production waters.

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Therefore, three different high-temperature petroleum reservoir production waters with

93

favorable condition for methanogenesis were selected as source of microbes and substrates

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amended with ZVI as an alternative electron donor to access the bioconversion of CO2 into 4

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methane for energy and the feasibility of microbial ecological transformation process.

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2 Materials and Methods

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2.1 Enrichment Cultures The inoculum for enrichment cultures was collected

from three different

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high-temperature petroleum reservoir production waters of Shengli Oilfield in China as the

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source of microbes and substrates, namely Z3-13, Z3-26 and Z3-X251. About 50 ml (100%)

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of inoculum were transferred into an autoclaved serum bottle (internal volume 120 ml) with

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amendment of resazurin as an indicator of oxygen under a stream of N2 gas and then sealed

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with a butyl rubber stopper (Bellco Glass, Inc., Vineland, NJ) and aluminum crimp seal.

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Active enrichment cultures were amended with ZVI (Sigma-Aldrich, Milwaukee, WI).

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Background controls were prepared without addition of ZVI in parallel. The specific surface

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area of the ZVI powder was measured by using Brunauer-Emmett-Teller (BET). All

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treatments of experiments were conducted in triplicate. About 2.0 g of ZVI powder were

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added to each empty serum bottle of active enrichment cultures under a stream of N2 gas after

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sterilization. All of the cultures were incubated under anaerobic conditions at 55 oC in the

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dark for 16 days.

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2.2 Chemical analysis

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Gas chromatography (GC) was used to measure the production of methane, hydrogen

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and carbon dioxide in the headspace of serum bottles during the incubation. 200 µl headspace

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gas taken by gastight syringe were injected onto GC by a micro-syringe for analysis. Program

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setting of the GC analysis was as follows: the initial column temperature at 50 oC for 2 min,

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the increased to 130 at a rate of 15 oC/min, the temperature at 130 oC sustained for 1 min. The

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second increase was conducted at a rate of 30 oC/min to 180 oC, the final temperature at 180

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o

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conductivity detector (TCD) was maintained at 200 oC and the temperature of reburner used

121

to detect the concentration of carbon dioxide was maintained at 350 oC. Three external

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standard curves of the different gases (CH4, H2 and CO2) were used for converting peak areas

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of samples into their respective concentrations (r2, 0.9974, 0.9980, 0.9958, respectively. n=5).

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Gas chromatography-mass spectrometer (GC-MS) (Agilent Technologies, Inc.) was used

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C for 30 min. Temperature of injector, flame ionization detector (FID) and thermal

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ACCEPTED MANUSCRIPT for the detection of intermediate metabolites (volatile fatty acids (VFAs)) in the liquid phase

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through the anaerobic incubation. For the analysis of VFAs, 10 ml of culture liquid were

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taken from the bottles and the pH was adjusted with ammonia solution to > 12 and then dried

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in an oven at 110 oC. Esterification was carried out by adding 0.5 ml of 10% butanol/sulphate

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solution at 90 oC for 60 min. Extraction of VFAs was conducted with 0.5 ml of n-dodecane

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for three times and the extracts were injected onto the GC-MS. The program setting was as

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follows: oven temperature was maintained at 60 oC for 1 min and then increased at the rate of

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15 oC/min to 130 oC.

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The ferrozine-spectrophotometric method was used for detecting the concentration of

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Fe(II) and total Fe (i.e. Fe(II) + Fe(III) species) after reduction of Fe(III) to Fe(II) by using

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hydroxylamine hydrochloride and C-18 column as the reductant and filter respectively in the

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liquid phase through the anaerobic incubation. The measurement was carried out on the

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wavelength of 562 nm by ultraviolet and visible spectrophotometer.

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2.3 SEM-EDS and XPS analysis

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Scanning electron microscopy (SEM, FEIQ45) coupled with energy dispersive X-ray

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spectroscopy (EDS, BRUKER X-Flash-30) was applied to determine the chemical

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composition and superficial morphology of the ZVI powder after incubation and the original

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sample. Each sample was freeze-dried and then directly put onto a specimen stub for

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SEM-EDS analysis.

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The ZVI solids from culture medium were collected, freeze dried and washed with

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ultrapure water and ethanol for three times, then further dried at 60 oC under vacuum

146

overnight. X-ray photoelectron spectroscopy (XPS) was carried out on the prepared samples

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using Thermo Scientific Escalab 250Xi with monochromatic Al Kalpha X-ray source. The

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pass energy was set to 20 eV for scanning the Fe 2p, O 1s and C 1s regions. All spectra were

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calibrated to the main C 1s peak at 284.8 eV.

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2.4 DNA extraction and Illumina MiSeq sequencing

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At the end of incubation period, 10 ml of enrichment liquid were taken from both

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ZVI-amended cultures (three replicates) and the background controls (three replicates), then

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centrifuged at 12000× g for 20 min. The biomass pellet after centrifugation was used for

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DNA extraction by using AxyPrepTM Bacterial Genomic DNA Maxiprep Kit (Axygen 6

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Biosciences, USA) followed by the manufacturer’ instructions.

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Archaeal and bacterial 16S rRNA was sequenced using identified DNA by 1% agarose

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gel by primer sets 344F/915R [38] and 515F/907R [39], including archaeal primer sets 344F

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(5’-ACGGGGYGCAGCAGGCGCGA-3’)/915R

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and

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(5’-CCGTCAATTCMTTTRAGTTT-3’). Polymerase chain reaction (PCR) amplification was

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performed in a 20 µl reaction volume containing 5× FastPfu Buffer (4 µl), 2.5 mM of dNTPs

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(2 µl), 5 µM of each primer (0.8 µl), FastPfu Polymerase (0.4 µl), BSA (0.2 µl), ~10 ng of

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template DNA in an ABI GeneAmp®9700. For archaeal 16S rRNA gene, PCR amplification

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reaction was performed according to the followings: 3 min for initial denaturation at 95 oC,

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followed by 38 cycles of 95 oC for 30 s, 51 oC for 30 s, 72 oC for 45 s, and final elongation

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step 72 oC for 10 min. For bacterial 16S rRNA gene, PCR amplification conditions were as

167

follows: 3 min for initial denaturation at 95 oC, followed by 29 cycles of 95 oC for 30 s, 55 oC

168

for 30 s, 72 oC for 45 s, and final elongation step 72 oC for 10 min. The obtained PCR

169

products were sequenced by using Illumina Next Generation Sequencing. The valid archaeal

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and

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(http://rdp.cme.msu.edu/classifier/classifier.jsp) on the level of genus and phylum,

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respectively. The data of archaea and bacteria generated from Illumina MiSeq were

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respectively deposited into NCBI SRA database under the accession number SRP116087 and

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SRP116068.

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2.5 Carbon balance and thermodynamics calculations

primer

sets

515F

(5’-

GTGCCAGCMGCCGCGG-3’)/907R

16S

rRNA sequences

were

classified

using

RDP CLASSIFIED

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bacterial

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bacterial

(5’-GTGCTCCCCCGCCAATTCCT-3’)

The total amount of carbon dioxide ((ƩCO2) = liquid CO2 + gas CO2 in ZVI-amended

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cultures) was obtained by summation of CO32-, HCO3- and H2CO3 in the liquid phase and

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CO2 in the gas phase. ∆CO2 was used to express the amount of transformed CO2 driven by

179

addition of ZVI, which was obtained by subtracting ƩCO2 in ZVI-amended cultures from

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corresponding background controls, and the conversion rate of CO2 in ZVI-amended cultures

181

was obtained by ∆CO2 / ƩCO2 in background controls.

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Gibbs free energy data for all compounds and Gibbs free energy calculations with

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different pH value at 55 oC were obtained by methods described elsewhere [40] and the

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changes of Gibbs free energy value related to anaerobic bioconversion of CO2 and methane 7

ACCEPTED MANUSCRIPT production in different cultures were calculated. ∆G°′T is calculated and modified with the

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protons and pH values from ∆G°T, which is the standard Gibbs free energy at temperature T

187

(∆G°′T =∆G°T +m2.303RTlog10-pH, m is the net number of protons formed in the equation

188

and pH is the pH value in the anaerobic enrichment cultures). Thermodynamics analysis for

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possible pathways of CO2/H2 utilization was conducted with the concentration of acetate and

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hydrogen as thermodynamic constrains. All the [H2] was in atmospheric pressure and

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[CH3COO-] was in M.

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3 Results and Discussion

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3.1 Enhanced methanogenesis by addition of ZVI

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The fundamental physicochemical parameters of three selected production waters from

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Shengli oil reservoirs with a depth of 1192.6 m below ground and in situ temperature

197

estimated at 63 oC collected for anaerobic methanogenic incubation amended with ZVI were

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summarized in Table 1. The geochemical conditions of all three production waters are

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favorable for microbial methanogenesis, with <2 mM of SO42-, <3 M of Cl- and neutral pH

200

(7.0) [41]. The amount of VFAs as the important intermediates of anaerobic crude-oil

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degradation as well as the substrates for microbial metabolism in the production waters was

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low.

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Table 1 Physicochemical characteristics of the production waters used in this study

Parameter Temperature (oC) pH Depth (m) CO32- (mM) SO42- (mM) Cl- (mM) Formate (mM) Acetate (mM) Propionate (mM) Butyrate (mM) Water cut (%) Oil viscosity (mPa·s) Mineralization (mg/l)

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Z3-13 63 7.0 1192.6 17.96 0.03 106.90 0.54 ND 0.05 0.17 97.2 1885.0 7370.0 8

Z3-26 63 6.4 1192.6 8.05 0.03 129.56 ND ND ND 0.16 88.3 1885.0 8616.0

Z3-X251 63 6.7 1192.6 14.83 0.03 109.84 0.052 0.04 ND 0.22 93.8 1885.0 8400.0

ACCEPTED MANUSCRIPT 166.48 1.56 1.80 2.90 0.33 ND ND ND ND

65.14 1.50 2.11 11.26 0.53 ND ND ND 0.14

170.20 1.98 2.25 1.61 0.53 ND ND ND ND

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Na+ (mM) Mg2+ (mM) Ca2+ (mM) NH4+ (mM) K+ (mM) Mn2+ (mM) PO43- (mM) S2- (mM) NO3- (mM)

Anions and cations were analyzed by ion chromatography and ICP-AES (Inductively Coupled Plasma-Atomic Emission

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Spectrometry), respectively; Volatile fatty acids were determined by GC-MS after butanol esterification; ND, not detected.

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Methane production rates of the three sets of enrichment cultures including

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ZVI-amended active cultures and background controls for 16 days of anaerobic incubation

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are shown in Fig. 1. In the microcosms amended with ZVI solely, methane production rate

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reached to 61.67, 64.73 and 78.49 µmol/(l·d) in Z3-13+ZVI, Z3-26+ZVI and Z3-X251+ZVI,

210

respectively, while in the corresponding background controls without addition of ZVI only

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approximate 0.60, 0.71 and 0.12 µmol/(l·d) was detected correspondingly. The rate of

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methane generation in ZVI-amended cultures was about 91-654 times higher than ZVI

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non-amended controls. Enhanced methane production was observed in all the cultures

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amended with ZVI (specific surface area of 0.0741 m2/g at micrometer level) as an available

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alternative electron donor. At the same time, only low level of methane was detected in the

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controls corresponding to no ZVI amended background, indicating the methanogenic activity

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was stimulated by the addition of ZVI in production waters from petroleum reservoirs.

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Fig. 1. Methane production rates of different enrichment cultures incubated at 55 oC for 16 days of anaerobic

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incubation. (■) incubated with 2.0 g ZVI powder as the electron donor (3 replicates), and (■) without any electron

221

donors as the background controls (3 replicates).

Carbon dioxide consumption and hydrogen generation in ZVI-amended enrichment

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cultures and background controls after anaerobic incubation were calculated and are shown in

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Table 2. Similarly, an obvious hydrogen production was detected in all ZVI-amended cultures,

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and very little hydrogen was found in background controls without addition of ZVI indicating

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hydrogen could be generated effectively by addition of ZVI. The concentration of hydrogen

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in Z3-13+ZVI, Z3-26+ZVI and Z3-X251+ZVI was approximately 4356, 2256 and 1296

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µmol/l, while only about 1.0, 0.9 and 0.6 µmol/l was detected in the corresponding

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background controls without addition of ZVI, respectively. The theoretical amount hydrogen

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generated from 2.0 g of ZVI in active cultures was about 0.51 mol/l, which was enough for

231

microbial methanogenesis with existing CO2 and VFAs as the substrate detected in all initial

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inoculum (Table 1).

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Table 2 Carbon dioxide consumption and hydrogen generation in ZVI-amended enrichment cultures and background

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controls after anaerobic incubation.

(µmol)

Sample

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Liquid CO2

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Gas CO2 (µmol)

ƩCO2

(µmol)

Gas H2

by ZVI

(µmol/l)

(µmol) (%)

+ ZVI

CK

332.8

49.2

4355.6

1.0

635.4

266.0

41.9

2255.6

0.9

729.6

423.2

58.0

1295.8

0.6

+ ZVI

CK

+ ZVI

CK

+ ZVI

CK

Z3-13

310.3

373.4

33.6

303.3

343.9

676.7

Z3-26

352.4

252.5

17.0

382.9

369.4

Z3-X251

284.1

399.7

22.3

329.9

306.4

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Conversion rate ∆CO2

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Liquid CO2: the sum amount of CO2, CO32- and HCO3- in the liquid phase of the incubation systems;

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Gas CO2: the amount of CO2 in the gas phase of the incubation systems;

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ƩCO2: the sum amount of CO2, CO32- and HCO3- in both liquid phase and gas phase;

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∆CO2: ƩCO2 in the background control without ZVI - ƩCO2 in ZVI-amended enrichment culture;

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Conversion rate %: ∆CO2 / ƩCO2 in the background control;

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Gas H2: the concentration of H2 in the gas phase of incubation systems;

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+ ZVI: active enrichment systems incubated with 2.0 g ZVI powder as the electron donor in 50 ml petroleum production

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water (3 replications);

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CK: background controls incubated without any electron donors in 50 ml petroleum production water (3 replications).

Petroleum reservoir can be regarded as a “geobioreactor” [1], in which the

245

bioconversion of CO2 into value-added compounds by various CO2-utilizing microorganisms

246

(especially methanogens) inhabiting these environments was feasible [15]. In particular,

247

available electron donors were essential and necessary for biotransformation of CO2 into

248

methane according the reaction: 4H2 + CO2 → CH4 + 2H2O. In the present study, ZVI was

249

selected as an alternative electron donor for the above-mentioned process owing to variety of

250

advantages such as its low cost, high efficiency and favorable condition for methanogens [42],

251

and enhanced methane production were detected in all ZVI-amended cultures. The

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ZVI-driven methanogenesis was consistent with H2/[H] generation from dissolution of ZVI

253

with water (Fe0 + 2H2O = Fe2+ + H2 + 2OH-) in the ZVI-amended cultures [29]. Generated

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available H2 could be subsequently used by hydrogenotrophic methanogens [43], and it is

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therefore not surprising to detect the decrease of CO2 in ZVI-amended cultures in comparison

256

with background controls (Table 2). Based upon the above biochemical pathway, H2

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production could also be served as another indicator for the reactivity and corrosion of ZVI

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[33], and ZVI oxidation would not occur without hydrogen consumption by the methanogens

259

from thermodynamics calculation perspective [29]. In addition to be considered as the

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electron donor, the capability of ZVI to serve as an important trace metal to stimulate

261

methanogens has also been confirmed in the previous research [36]. In fact, ZVI as well as its

262

analogue has been widely observed in the process of anaerobic digestion and environmental

263

remediation [30, 31, 33, 36, 42, 44], and stimulation of methane production was found in

264

almost all the existing reports. It is also generally accepted that the favorable microbial

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methanogenic condition for better growth of organisms associated with addition of ZVI might

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be another essential reason for enhanced methanogenesis, in which the reaction of ZVI with

267

available protons from water dissociation accompanied by rising pH could balance the

268

decrease in pH in the anaerobic cultures that resulted naturally from the acetogenesis by

269

alkane degradation and homoacetogenesis. At the same time, a larger variation in pH

270

observed in ZVI-amended cultures suggested that ZVI had much higher reactivity for

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hydrogen generation under strictly anaerobic conditions (Table S1). Further enhancement of

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methane production as energy might be focused on reducing particle size and effectiveness of

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ZVI to obtain large specific surface areas for reactivity [45]. The results obtained in this study clearly demonstrated that microbial transformation of

275

CO2 into methane for energy could be stimulated and enhanced by the addition of ZVI as an

276

alternative electron donor in oil reservoir production waters and also present a sustainable

277

strategy for the value-added carbon dioxide management along with fuel methane generation,

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which could also be regarded as an environmental friendly and energy recovery strategy for

279

remediation of waste water.

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3.2 ZVI-induced shift of microbial community

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Microbial community of archaea (genus) and bacteria (phylum) in ZVI-amended

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enrichment cultures and background controls after anaerobic incubation are shown in Figs. 2

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and S1. 10 ml enrichment cultures taken out from both ZVI-amended enrichment cultures and

284

background controls after anaerobic incubation were subjected for archaeal and bacterial

285

community analysis influenced by ZVI through 16S rRNA high-throughput sequencing,

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showing that the archaeal and bacterial community composition on the level of genus and

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phylum have been changed by addition of ZVI in ZVI-amended cultures. The dominant

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archaeal members (>99% occupation of detected sequences) in ZVI-amended enrichment

289

cultures were all affiliated with Methanothermobacter belong to the order of

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Methanobacteriales, which was prevalent thermophilic hydrogenotrophic methanogens found

291

in

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Methanothermobacter could produce methane from CO2/H2 and formate and some species

293

also need acetate as required growth factor in basal medium. The decrease of Methanosarcina,

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Methanothrix, Methanofollis and Methanocella was also observed in ZVI-amended cultures

295

compared with background controls, in which those affiliating with Methanofollis and

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Methanocella are CO2-reducing methanogens while Methanothrix could produce methane

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with acetate as the substrate and Methanosarcina have a variety of methanogenic biochemical

298

pathways. In contrast, archaeal community showed more diversity in background controls

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and was partly occupied by the genus of Methanosarcina, Methanothrix and

300

Methanobacterium. For bacterial community composition, the phylum of Firmicutes and

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α-Proteobacteria possibly related to homoacetogenesis decreased in all ZVI-amended cultures

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(except Firmicutes in Z3-X251+ZVI), indicating acetogenic metabolic activity was inactive

petroleum

reservoirs

[20,

21].

The

members

belong

to

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ACCEPTED MANUSCRIPT in the presence of ZVI in petroleum reservoir production water (Fig. S1).

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Fig. 2. Microbial community of archaea on genus level in ZVI-amended enrichment cultures and background

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controls after anaerobic incubation. The archaeal community in ZVI-amended cultures and blank controls were

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represented by the left and right column in each sample respectively.

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It is generally accepted that syntrophic acetate oxidation coupled with hydrogenotrophic

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methanogenesis is probably the predominant pathway for methane production in

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high-temperature oil reservoirs [21]. In the present study, Methanothermobacter spp. as a

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typical hydrogenotrophic methanogen was the most encounters and possibly responsible for

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methane production with energetically favorable condition in ZVI-amended cultures (Fig. 2).

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Meanwhile, accumulation of formate as an important intermediate in syntrophic butyrate or

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propionate oxidation or generated by some methanogens in ZVI-amended cultures [24, 46]

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implying that formate methanogenesis with thermodynamics feasibility would turn to be

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another essential methanogenic pathway possibly carried out by Methanothermobacter,

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Methanosarcina and Methanothrix [47, 48] in comparison with background controls without

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detection of formate. The decrease property of the typical acetoclastic methanogens

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(Methanothrix) compared with background controls might be related to limited substrate and

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inhibition from CO production associated with high partial pressure of H2 induced by ZVI

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[49]. In fact, enrichment of hydrogenotrophic methanogens in incubation cultures has been

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proven as an attractive option for up-grading oil reservoirs for methane production as energy

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[50].

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3.3 A transformation potential from ZVI and CO2 in ZVI-amended cultures

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cultures and ZVI non-amended controls measured via ferrozine-spectrophotometric after

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anaerobic incubation, and confirmed significant accumulation in active treatments. The

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amount of Fe3+ were obtained by subtracting Fe2+ detected in the bottles and ΣFe2+ after the

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reduction of Fe3+ to Fe2+. The higher amount of Fe2+ and Fe3+ in Z3-13+ZVI, Z3-26+ZVI and

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Z3-X251+ZVI were observed in contrast with the corresponding controls respectively. In the

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presence of ferric iron, the competition for available electrons between methane production

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and Fe(III) reduction might also restrict methanogenesis from CO2 by specific methanogens

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[51, 52]. By the time Fe(III) bioreduction and methanogenesis reached stability, these two

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pathways were mutually beneficial possibly because bioreduction of Fe(III) could reduce the

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reduction potential of the incubation culture so that methanogenesis become favorable, and in

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turn methanogenesis stimulated the growth and metabolic of methanogens, which further

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improved Fe(III) bioreduction [53]. It was therefore consistent with phenomenon of low

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concentration of Fe3+. In fact, the theoretical H2 production from Fe2+ and Fe3+ existing in the

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liquid phase based on charge conservation was much lower than detected H2 in the headspace

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of the ZVI-amended cultures, indicating the dissolved ZVI was not the only form in this

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environment.

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XPS investigations containing XPS scan spectrums of Fe 2p, O 1s and C 1s of ZVI solid

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powder in production water amended with ZVI after anaerobic incubation and original ZVI

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without incubation as the background control are shown in Figs. 3, S2 and Table S2. ZVI

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solids from three ZVI-amended treatments at the end of anaerobic incubation and original

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sample were collected, dried and analyzed by XPS. XPS scan spectrums of Fe 2p, O 1s and C

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1s showed CO2 can also be removed via mineralization with ZVI as the reducing agent that

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could conduct an oxidation/reduction reaction with water and CO2 to generate iron carbonate

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and hydrogen according the reaction: Fe0 + CO2 +H2O = FeCO3 + H2 as indicated by C 1s

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peak at 289.1 eV, Fe 2p3/2 peak at 710.4 eV, Fe 2p3/2 satellite at 714.5 eV and O 1s peak at

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531.3 eV [42, 54], which present another alternative way for conversion of CO2 under strictly

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anaerobic conditions. Apart from FeCO3, FeOOH was also detected in active enrichment

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cultures.

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Fig. 3. XPS investigation (XPS scan spectrum of Fe 2p, O 1s and C 1s) of ZVI solid powder in Z3-X251 amended with

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ZVI after anaerobic incubation and original ZVI without incubation as the background control. Other two samples

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(Z3-13 and Z3-26) showed similar results with Z3-X251 and were given in Fig. S2. Detailed peak positions and information

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of generated peaks by XPS-peak-differentiation-imitating analysis were listed in Supplementary Table S2 and black dotted

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lines were used to characterize peak positions of Fe 2p3/2 satellite, Fe 2p3/2, O 1s and C 1s of FeCO3 from left to right.

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SEM images with different magnifications as well as EDS analysis of ZVI solid powder

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in production water amended with ZVI after anaerobic incubation and original ZVI powder

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without anaerobic incubation were shown in Figs. 4 and S3. SEM images showed that many

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scraggy and rough hollows on the anaerobic corrosive ZVI surface and smooth texture on the

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original ZVI powder, which confirmed the slow dissolution of ZVI through the whole

365

anaerobic incubation [44] resulting in the relative more iron ion and higher pH in

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ZVI-amended cultures in contrast with background controls (Table S1). Hydrogen-utilizing

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organisms were provided more tendency for substrate uptake owing to increased surface area

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of corrosive ZVI driven by microbial dissolution, which might involve methanogenesis

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promotion factors by ZVI. Meanwhile, the prismatic and needle-like crystals observed in the

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SEM images with higher magnification might comprised of iron (hydr)oxides clusters (e.g.,

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FeCO3, Fe3O4, Fe2O3, Fe(OH)3 and FeOOH) [54, 55], which suggested the microbe-driven

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dissolution of ZVI during the anaerobic incubation. Charge and intermediate form H2/[H]

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transfer through generated conductive iron compounds would occur more easily and directly

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[42], and further enhanced hydrogenotrophic methanogenesis such as the predominant

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members affiliated with the genus of Methanothermobacter in ZVI-amended cultures [37, 43]

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and original background control. The silicate peak with relatively large area in element

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composition profiles from ZVI-amended enrichment cultures compared with the background

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control suggested that silicate existed in oil reservoir production water might be adsorbed to

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ZVI or complexed with iron at high pH value at the end of incubation [56], and subsequently

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might be served as a possible sink for capture of CO2 in the form of magnesium, iron or

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calcium carbonates. The data obtained in this study demonstrated that ZVI might be

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considered as CO2 sink under the strictly anaerobic conditions in petroleum reservoir

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production waters.

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Fig. 4. Scanning electron microscopic micrographs with different magnifications and EDS analysis (red arrows) of

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ZVI solid powder in Z3-X251 amended with ZVI after anaerobic incubation (a-c) and original ZVI powder without

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anaerobic incubation (d-f). SEM-EDS analysis for other two samples (Z3-13 and Z3-26) were shown in Fig. S3.

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3.4 Transformation pathways of CO2 in the anaerobic enrichment cultures

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The total amount of CO2 (ƩCO2) in Z3-13+ZVI was 343.9 µmol, while in the

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corresponding background control without any electron donors about 676.7 µmol CO2 (ƩCO2)

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was detected, which meant almost 50% of CO2 (∆CO2) was (bio)transformed driven by ZVI.

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The same trend with the treatment of Z3-13 was shown in other two oil reservoir production

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waters (Table 2). Accumulation of VFAs especially formate were detected in the

395

ZVI-amended cultures apart from methane production (Fig. 5). Thermodynamics analysis for

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possible pathways of CO2/H2 utilization in anaerobic enrichment cultures with the

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concentration of acetate and hydrogen as thermodynamic constrains are shown in Figs. 6 and 16

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Fig. 5. Composition of VFAs in both ZVI-amended enrichment cultures and background controls after anaerobic

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incubation at 55 oC. (■) incubated with 2.0 g ZVI powder as the electron donor, and (■) without any electron donors

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as the background controls.

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Fig. 6. Thermodynamics analysis for possible pathways of CO2/H2 utilization in Z3-X251 amended with ZVI

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enrichment culture with the concentration of acetate and hydrogen as thermodynamic constrains. (

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Z3-X251 amended with ZVI enrichment culture and (

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incubation. Other two samples (Z3-13 and Z3-26) showed the similar results with Z3-X251 and were presented in Fig.

408

S4.

) represented

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) was showed state of background control after anaerobic

The transformation of CO2 into methane in ZVI-amended enrichment cultures was

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efficiently stimulated by addition of ZVI in petroleum reservoir production waters by contrast

411

with background controls even at a much higher ZVI concentration of 46.6 g/l [45] (Table 2).

412

In EOR project, most residual CO2 after EOR was dissolved in the formation fluids and

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maintained gas phase after more than 30 years in petroleum reservoirs [6, 7], and thus

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available electron donors are essential to stimulate the conversion of CO2 for carbon reducing

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as well as energy recovery in the petroleum reservoir production water.

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Based upon the analysis of Gibbs free energy, hydrogenotrophic methanogenesis and

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homoacetogenesis were presumably the dominant pathways for the consumption of available 18

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methanogenesis played an essential role in variety of biochemical pathways of CO2

420

conversion on account of the predominant members affiliated with Methanothermobacter in

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ZVI-amended enrichment cultures, and the rate of methane production detected in this study

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reached at 61.67, 64.73 and 78.49 µmol/(l·d). Methanogenic archaea are also expected to fix

423

CO2 and produce small organic molecules (formate) [24], which might be related to formate

424

accumulation in ZVI-amended enrichment cultures.

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Although homoacetogenesis (H2 threshold < 200 Pa) was more competitive than

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methanogenesis (H2 threshold < 2 Pa) at high partial pressure of hydrogen [57] and the

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generation of OH- from dissolution of ZVI could facilitate acetate formation with H2 and CO2

428

as the substrate, the results showed that acetate was not visibly accumulated and the

429

occupation of Methanothrix and Methanosarcina with acetate as the substrate for

430

methanogenesis in archaeal community decreased in ZVI-amended cultures compared with

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background controls (Fig. 2), possibly due to the utilization of acetate for the growth of

432

microorganisms such as the members belong to Methanothermobacter and Methanoculleus

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[58] and the decreased property of Firmicutes related to acetogenesis from CO2 and H2 in

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petroleum reservoir production waters [15] (Fig. S1). In addition, generated acetate could

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also be utilized for methanogenesis through acetate oxidation coupled with hydrogenotrophic

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methanogenesis [21].

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4 Conclusion

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A promising strategy to stimulate and accelerate biological transformation of CO2 into

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methane as energy with ZVI as the alternative electron donor in oil reservoir production

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waters was achieved in this study. Enrichment of Methanothermobacter spp. supported its

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competitive role in biomethane production via CO2-reducing methanogenesis and formate

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methanogenesis in ZVI-amended cultures. The detection of FeCO3 mineral also presents an

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alternative potential for immobilization of CO2 in the presence of ZVI under the anaerobic

445

conditions. Methane production at high rates (> 61.67 µmol/(l·d)) with amendment of ZVI in

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this study provided an opportunity for value-added CO2 management technologies and

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further bioenergy regeneration from CO2 in projects of EOR and CCS in oil reservoirs.

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Acknowledgements The authors are grateful to the management of Shengli Oilfield for sampling support.

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This work was supported by the National Science Foundation of China (No. 41530318,

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41403066), the Research Foundation of Shanghai (No. 15JC1401400), and the Fundamental

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Research Funds for the Central Universities of China (No. 222201717017, 222201414029).

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Highlights Accelerated biotransformation of CO2 into methane as energy was achieved by addition of zero valent iron (ZVI).

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Methane production rate reached as high as 60 µmol/(l·d).

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Methanothermobacter spp. was responsible for the methanogenesis.

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This strategy suggested a great potential on energy recovery from mitigation of CO2.

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