Biogas Upgrading via Hydrogenotrophic Methanogenesis in Two

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Biogas upgrading via hydrogenotrophic methanogenesis in two-stage Continuous Stirred Tank Reactors at mesophilic and thermophilic conditions Ilaria Bassani, Panagiotis G Kougias, Laura Treu, and Irini Angelidaki Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03451 • Publication Date (Web): 21 Sep 2015 Downloaded from http://pubs.acs.org on September 24, 2015

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Environmental Science & Technology

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Biogas upgrading via hydrogenotrophic

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methanogenesis in two-stage Continuous Stirred

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Tank Reactors at mesophilic and thermophilic

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conditions

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Ilaria Bassani, Panagiotis G. Kougias, Laura Treu, Irini Angelidaki*

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Department of Environmental Engineering, Technical University of Denmark, Kgs. Lyngby,

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Denmark

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KEYWORDS biogas upgrading, methane, anaerobic digestion, methanogens, metagenomics,

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random DNA sequencing, full-length 16S rRNA, hydrogenotrophic archaea

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ACS Paragon Plus Environment

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ABSTRACT This study proposes an innovative setup composed by two stage reactors to

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achieve biogas upgrading coupling the CO2 in the biogas with external H2 and subsequent

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conversion into CH4 by hydrogenotrophic methanogenesis. In this configuration, the biogas

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produced in the first reactor was transferred to the second one, where H2 was injected. This

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configuration was tested at both mesophilic and thermophilic conditions. After H2 addition, the

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produced biogas was upgraded to average CH4 content of 89% in the mesophilic reactor and

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85% in the thermophilic. At thermophilic conditions, a higher efficiency of CH4 production and

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CO2 conversion was recorded. The consequent increase of pH did not inhibit the process

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indicating adaptation of microorganisms to higher pH levels. The effects of H2 on the microbial

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community were studied using high-throughput Illumina random sequences and full-length 16S

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rRNA genes extracted from the total sequences. The relative abundance of archaeal community

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markedly increased upon H2 addition with Methanoculleus as dominant genus. The increase of

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hydrogenotrophic methanogens and syntrophic Desulfovibrio and the decrease of aceticlastic

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methanogens indicate a H2-mediated shift towards the hydrogenotrophic pathway enhancing

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biogas upgrading. Moreover, Thermoanaerobacteraceae were likely involved in syntrophic

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acetate oxidation with hydrogenotrophic methanogen in absence of aceticlastic methanogenesis.

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INTRODUCTION

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Wind and biomass are promoted worldwide as sustainable forms of energy. The Danish energy

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policy has set a goal to cover 50% of electricity demand in 2020 by exploitation of wind energy

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(1). Moreover, it is a goal to use up to 50% of the manure for bioenergy production. With the

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expansion of the wind mill sector, the necessity for electricity storage has arisen. This study

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proposes an interesting solution to store the electricity as CH4 and, together, upgrade biogas to

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higher CH4 content. Anaerobic digestion (AD) of biomass produces biogas with ~50-70% CH4


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and 30-50% CO2. Biogas containing higher concentrations of CH4 (>90%) has higher heating

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value and can be used as fuel for cars or transported through the national gas grid (2).

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Nevertheless, although several biogas upgrading methods exist, their cost is commonly high (3).

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Renewable electricity utilization is expanding worldwide and uneven production of wind and

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solar energy can result in excess of resources. This surplus can be used to electrolyze water to

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produce H2. Nevertheless, H2 as fuel presents some drawbacks related to its low volumetric

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energy content and difficulty in storage and transport (4).

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Biological biogas upgrading, coupling the H2, produced by water electrolysis, with the CO2 in

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biogas and converting it to CH4, has been recently reported (5). Further studies showed that the

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H2 injection into the reactor can convert >40% of the CO2 present in biogas (6). Although

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biological biogas upgrading has several advantages, the direct H2 injection in the reactor (in-situ

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upgrading) can cause technical challenges. CO2 removal could lead to a substantial increase of

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pH negatively affecting the process. In addition, H2 low gas-liquid mass transfer rate is a

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process’ limiting factor (7), because it must be dissolved in reactor’s liquid phase to be utilized

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by microorganisms.

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AD process occurs by a combination of pathways assigned to an extremely complex and

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specialized microbial consortium determined by bacteria-archaea interactions, resilient to process

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variations (8). Specifically, bacteria hydrolyze polymers into monomers, and then ferment them

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to lactate, volatile fatty acids (VFA) and alcohols. These products are further fermented by

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syntrophic bacteria to acetate, formate, H2, and CO2 that are used as substrates by methanogens.

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Syntrophic interactions are necessary because these reactions are thermodynamically

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unfavorable unless their products are kept at low concentrations by a second microorganism,

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such as methanogens, that utilizes them (9). For example, dissolved H2 concentration has a role


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in products’ levels regulation. High H2 partial pressure leads to propionate and butyrate

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accumulation, while low H2 partial pressure enhances CO2 and CH4 production (9). Normally,

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most of the CH4 is formed from acetate (70%), mainly by aceticlastic methanogenesis, e.g.

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Methanosarcinales, and bacterial syntrophic acetate oxidation (SAO, i.e. oxidation of acetate to

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CO2 and H2) and only the remaining 30% is produced directly from H2/CO2 (8, 10).

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Methanogenesis from H2/CO2 is mainly carried out by hydrogenotrophic methanogens that

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reduce CO2 with H2 to produce CH4. Therefore, we hypothesized that the H2 addition changes the

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microbial community composition promoting the hydrogenotrophic methanogenic pathway and

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the CO2 consumption. Previous studies reported that, in H2-mediated upgraded reactors,

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dominant archaeal species belonged to Methanobacteriales, i.e. Methanothermobacter

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thermautotrophicus (11). However, because of the complexity of the microbial community, most

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of the biogas reactor population is still unidentified and poorly characterized.

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In this experiment a novel reactor configuration was designed to investigate the effect of the

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H2 addition on AD process performance. Moreover, different bioinformatics approaches were

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combined to achieve a more complete insight into the microbial community composition. The

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setup was composed by two serial-connected continuous stirred-tank reactors (CSTRs), treating

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cattle manure. The first reactor was the main biogas producer, while the second one, where H2

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was injected, was treating the effluent from the first reactor and serving as upgrading chamber,

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responsible for the conversion of CO2 to CH4. Mesophilic and thermophilic conditions were

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applied to investigate the effect of the temperature on the process. The microbial community was

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analyzed with three innovative metagenomic approaches: (I) taxonomy assignment performed

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directly on Total Random Sequences (TRS), (II) 16S rRNA Shotgun Reads (16S SR) extracted

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from TRS, (III) Assembled Full-Length 16S rRNA gene sequences (16S AFL) from 16S SR.


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TRS were mapped against unique clade-specific marker genes to achieve an accurate

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classification level and relative abundance estimation. Moreover, the recently developed method

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(12) that aligns short-read DNA sequences to a large 16S rRNA database, to reconstruct the

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complete 16S rRNA gene, was, for the first time, applied to the study of the biogas reactor

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community.

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MATERIALS AND METHODS

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Substrate characteristics and feedstock preparation

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The cattle manure substrate was obtained from the Hashøj biogas plant, Denmark, sieved

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through a 2 mm net to remove large particles, such as barley residues, and stored at -20°C, in 5 L

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bottles. Before usage, the substrate was thawed at 4°C for 1-2 days. The manure had a pH of

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7.44, total solids (TS) and volatile solids (VS) content of 47.40±1.86 and 34.56±1.40 g/L,

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respectively. The total Kjeldahl Nitrogen (TKN) and ammonium nitrogen NH4+ (NH4–N) were

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3.03 ± 0.10 and 2.07 ± 0.01 g-N/L, respectively. The concentration of total VFA was 6.83 ± 0.48

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g/L.

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Reactor’s setup and operation

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The setup consisted of two analogous two-stage CSTR, each with a total working volume of

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3.5 L. The working volume of primary (R1 and R2) and secondary (SR1 and SR2) reactors were

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1.5 L and 2 L, respectively. Both primary and secondary reactors were filled with inoculum,

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obtained from Hashøj (mesophilic) and Snertinge (thermophilic) biogas plants, Denmark. Main

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substrates treated by these biogas plants are animal slurry (pig and cattle) and wastes from food

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industries. Each reactor was mixed by magnetic stirrers. The temperature of R1 and SR1 was


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maintained at mesophilic conditions (35 ± 1 °C), while R2 and SR2 were kept at thermophilic

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conditions (55 ± 1 °C) using thermal jackets. Primary reactors were fed twice per day with cattle

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manure, while the secondary were fed with the effluent from the primary. Moreover, the biogas

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produced in the primary reactors was transferred to the secondary ones. The hydraulic retention

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times (HRTs) were selected to simulate full-scale applications conditions and set to 25 days for

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R1, 33 days for SR1, 15 days for R2 and 20 days for SR2. The total organic loading rate (OLR)

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was 0.6 and 1 gVS/Lday for mesophilic and the thermophilic reactor system, respectively.

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Although the inocula were originating from reactors operated at same temperature, the initial

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HRT was dedicated to allow adaptation of the inocula to the operational conditions. The

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reactors’ working conditions were ensured during the whole experiment, avoiding leakages and

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filtering substrate and inoculum, to minimize fibers accumulation of consequent working volume

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reduction. Once steady state conditions were achieved, H2 was continuously injected to the

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secondary reactors through a diffuser placed at the bottom of the reactor. The H2 flow rate was

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established according to the stoichiometry of hydrogenotrophic methanogenesis reaction: per

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each mole of CO2 contained in the biogas before the H2 addition, 4 moles of H2 were added, i.e.

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~192 and 510 mL/Lday for mesophilic and thermophilic reactor, respectively.

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Analytical methods

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The biogas production of both reactors was measured daily by an automated gas meter with a

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100 mL reversible cycle and registration (13). TS, VS, NH4–N and TKN were measured

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according to the Standard Methods for Examination of Water and Wastewater (14). Samples

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from primary and secondary reactors were collected for pH and VFA analysis twice per week.

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The pH was measured immediately after the collection to avoid the CO2 removal from the liquid


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phase, by a digital PHM210 pH meter connected to the Gel pH electrode (pHC3105–8;

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Radiometer analytical). VFA samples were prepared according to Kougias and coworkers (15).

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VFA concentration was determined using a gas chromatograph (GC-2010; Shimadzu) with a

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flame ionization detector and FFAP column as described previously (15) The biogas composition

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was measured twice per week by a Gas Chromatograph equipped with a Thermal Conductivity

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Detector (GC-TCD) as described previously (16). For batches assays, the CH4 production was

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measured regularly by a gas Chromatograph with a Flame Ionization Detector (FID), as

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described previously (17). Detention limits for the measurement of CH4, CO2 and H2 by GC

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were defined by the calibration curve (5-100%), while the detection limits for VFA were 5-1500

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mg/L.

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Effect of pH on specific hydrogenotrophic methanogenic activity

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A batch assay was performed to determine the hydrogenotrophic methanogenic activity of the

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microbial community of SR1 and SR2 at different pH values, at steady state after the H2

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addition. Inoculum from the secondary reactors was obtained and immediately transferred to 118

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mL serum bottles (20 mL of inoculum each batch), flushing with N2. The pH was adjusted to 6.0,

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7.0, 8.0, 8.5 and 10.0 using N2:CO2 (80:20) or HCl or NaOH 2 M. All the batches were prepared

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in triplicates, sealed with rubber stoppers (Rubber B.V., Hilversum, NL) and aluminum caps

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(Wheaton, Millville, NJ). 1 atm of H2 and CO2 (80:20) were injected in the batches and they

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were incubated at 35°C and 55°C and 200 rpm. The results were expressed as CH4 yield

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(mL/gVS).

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Microbial Community Composition


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Samples were obtained during reactors’ steady state operation to ensure representative process

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conditions and microbial community stability. Genomic DNA was extracted from the secondary

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reactors with RNA PowerSoil® DNA Elution Accessory Kit (MO BIO Laboratories, Carlsbad,

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CA). An initial filtration step, through a 100 µm nylon cell strainer filter, was introduced to

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remove the plant residues originating from animal feed (i.e. barley plant). The sample was

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centrifuged at 2500 g for 10 min and the supernatant was discarded recovering ~2 g of pellet.

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Quality and quantity of the DNA extracted were determined using NanoDrop (ThermoFisher

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Scientific, Waltham, MA) and Qbit fluorimeter (Life Technologies, Carlsbad, CA). Samples

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were sequenced by the Ramaciotti Centre for Gene Function Analysis (UNSW, Sydney), using

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NextSeq 500 sequencing technology and Nextera XT kit (Illumina, San Diego, CA) for library

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preparation (150+150 bp).

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Rarefaction curves and alpha diversity indexes were calculated with MG-RAST toolkit (18) on

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16S SR as described by Kougias and co-workers (19), except for the minimum alignment length

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that was 150 bp.

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Total raw reads were filtered to remove the low quality sequences using Trimmomatic (20).

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The rRNA-like sequences were extracted using riboPicker (21), by aligning the TRS with

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SILVA and Ribosomal Database Project (RDP) 16S rRNA gene sequences databases. Extracted

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sequences were assembled into the complete 16S rRNA using EMIRGE (12). 16S AFL were

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taxonomically assigned by RDP classifier (cutoff 0.8; 22). A further alignment was performed

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using BLASTN against NCBI 16S rRNA database (23). The taxonomic level was assigned

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according to the identity thresholds reported by Yarza and co-workers (24). Phylogenetic trees

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representing complete BLAST results were drawn using MEGAN software (25).


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The composition of the microbial community was further determined from TRS using

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Metagenomic Phylogenetic Analysis (MetaPhlAn) tool (distance function "braycurtis",

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"correlation" method; 26). The minimum value of relative abundance considered was 0.01%. The

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microbial relative abundance is indicated in the results as percentage of the total community. The

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phylogenetic tree representing the microbial community was drawn using GraPhlAn

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(https://bitbucket.org/nsegata/graphlan). Finally, heat maps representing the relative abundance

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and the folds change of microorganisms were drawn using Multiexperiment viewer (MeV; 27).

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Raw Illumina sequences were submitted to the NCBI sequence read archive database (SRA)

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with accession number SRP058235. 16S AFL data were submitted to MG-RAST with accession

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numbers

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(thermophilic preH2) and 4623917.3 (thermophilic postH2).

4623916.3

(mesophilic

preH2),

4623915.3

(mesophilic

postH2),

4624043.3

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RESULTS AND DISCUSSION

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Process monitoring and biogas upgrade

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Operational data from the reactors under steady state conditions before and after the H2

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addition are summarized in Table 1. The biogas produced by the primary reactor alone

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constituted 97% and 74% of the total biogas, in mesophilic and thermophilic reactor,

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respectively. Before the H2 addition, the CH4 content of the mesophilic reactor was ~70%. After

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the H2 addition, the CH4 production rate increased by 53% resulting in an average CH4 content of

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~89% (with a maximum of 92%). Correspondingly, the CO2 production rate decreased by 65%,

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due to the conversion of CO2 to CH4 resulting in an average CO2 content of 9% (with a minimum

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of 5%) (Figure S1a). Similarly, in the thermophilic reactor before the H2 addition, the CH4

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content was ~67%. After the H2 addition, the CH4 production rate increased by 45% leading to an


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average CH4 content of ~85% (with a maximum of 91%). Correspondingly, the CO2 production

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rate decreased by 77% leading to an average CO2 content of 7% (with a minimum of 6%) (Figure

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S1b). In both reactors, the decrease of CO2 production rate in the biogas was higher than the

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increase of CH4 production rate. This was likely due to the concomitant pH increase, resulting in

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a larger portion of CO2 dissolved in the reactor liquid phase, as bicarbonate.

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Notably, after the H2 addition, the CH4 yield derived from the H2-mediated CO2 conversion to

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CH4 represented ~25% of the total CH4 yield, in both reactors. Moreover, the CH4 yield resulting

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from the degradation of manure increased by 14% and by 7% in mesophilic and thermophilic

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reactor, respectively (Figure S2a and b).

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The biogas quality achieved fulfills the objective of >90% CH4 content, at both temperature

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conditions, increasing biogas heating value and extending its possible usage as energy carrier (2).

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In the mesophilic reactor, 99% of the injected H2 (with a maximum of 100%) was utilized and

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54% of the CO2 (with a maximum of 77%) was converted to CH4 (Figure S3a). Similarly, in the

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thermophilic, 92% of the H2 injected (with a maximum of 100%) was utilized and the 62% of the

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CO2 (with a maximum of 73%) was converted to CH4 (Figure S3b). The incomplete conversion

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of CO2 could be due to inadequate amount of injected H2. As previously described the H2 flow

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rate was set according to the stoichiometry of hydrogenotrophic methanogenesis reaction.

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However, an excess of biogas, and thus CO2, was produced after the H2 addition, compared to

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the pre H2 phase, resulting in a surplus of unconverted CO2 (Table 1).

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Besides the incomplete removal of CO2, the unutilized H2 found in the gas phase was likely

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due to the low hydrogen gas-liquid mass transfer rate (7). As previously observed the gas transfer

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to the liquid phase plays an important role for H2 microbial uptake and is also influenced by the


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stirring speed (28). In this experiment, the H2 gas transfer was not fast enough leading to the

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accumulation of not dissolved H2 to the reactor’s head space.

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In both primary reactors pH and VFA levels remained stable for the whole experiment (Figure

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S4a and c). In the secondary, VFA levels remained stable, while the pH, upon the H2 addition,

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increased to approximately 8.2 in SR1 and more markedly to 8.5 in SR2 (Figure S4b and d). The

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lower increase found in SR1 is likely due to the lower conversion efficiency observed in the

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mesophilic reactor. Although methanogenesis normally occurs in a pH range between 6.5 and 8.5

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and the process can be severely affected if the pH is below 6 or above 8.5 (29), in this

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experiment, no process inhibition or reduction in the conversion of CO2 to CH4 were observed.

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To verify this finding a batch assay was performed, in which the hydrogenotrophic

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methanogenic activity of inocula from SR1 and SR2 at steady state after the H2 addition was

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tested at different pH values. The results from this test confirmed the feasibility of

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biomethanation process at a maximum pH of 8.5 for both temperature conditions (although with

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significantly decreased CH4 yields) stating the adaptation of microorganisms to the increased pH

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values. Conversely, pH levels above 8.5 resulted in fatal deterioration of the process (Figure 1).

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Comparison of mesophilic versus thermophilic reactor performances

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Upon the H2 addition, a significant increase in CH4 content was achieved at both temperature

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conditions. Nevertheless, although in the mesophilic reactor a higher percentage of H2 was

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utilized, the CO2 converted was lower (Table 1 and Figure S3a and b).

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Thermophilic conditions are commonly associated with higher CH4 yields and production rates

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(30). By comparing VS of the primary reactors, 20% less organic material was detected at

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thermophilic conditions, meaning that at higher temperature 20% more biomass was degraded


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(data not shown). This is in accordance with previous report where degradation of organic matter

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at thermophilic conditions was more efficient than at mesophilic (30). This result partially

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validates the different performances of continuous reactors. However, because the OLR was

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different, a direct comparison of the performances was not feasible. The methanogenic activity

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of mesophilic and thermophilic inocula used in the continuous reactors was tested in a batch

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assay with glucose as substrate (Supporting Information). By comparing the resulting CH4

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production rates, the CH4 potential of the glucose, at standard temperature and pressure

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conditions, resulted in 54% higher CH4 productivity at 55°C than 37°C (data not shown).

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Therefore, these results attribute to the inoculum an important role in the discrepancies observed

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in reactors’ process stating its importance in process performances.

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Microbial Community Composition

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Rarefaction curves and alpha diversity indexes showed high community dynamicity, with

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higher diversity in mesophilic reactor (Table 2 and Figure S5). After the H2 addition, the

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diversity decreased at both temperature conditions resulting into a more specialized community.

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Although the taxonomic assignment based on a single gene has various disadvantages (PCR

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biases, ambiguous assignments), the 16S rRNA gene is currently the only extensively used and

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sufficiently informative marker available in high-quality databases (24). In this study, the full-

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length 16S rRNA gene was reconstructed providing a perspective of the taxonomic diversity,

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including undiscovered taxa, together with microorganisms’ richness and relative abundances

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(12).

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Interestingly, from the present application of the 16S rRNA gene assembly on average 70

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sequences per sample were obtained (Table 2). Because only the 16S rRNA sequences of the


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most abundant microorganisms are expected to be assembled, they can be considered as the most

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relevant community members. Complete results of microbial community taxonomy according to

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16S AFL are reported in Figure S6 and Datasets S1-S6. Because most of biogas reactor’s

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community is still uncharacterized, the taxonomic assignment provided by the used algorithms

266

was, for some microorganisms involved in biogas upgrading, uncertain, asserting the necessity

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for further investigation. In particular, in the thermophilic reactor, the classification of the most

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abundant microorganism, accounting for the half of the community, was unclear. While RDP

269

classifier pointed out a similarity to Acetomicrobium (phylum Bacteroidetes) with a low

270

threshold (0.37), BLAST assigned it to order Clostridiales (86% similarity).

271

The low phylogenetic resolution observed underlines the limits of single gene sequencing

272

analysis and the necessity of TRS information. From the application of the TRS strategy, higher

273

microbial community richness was detected at every classification level, displaying, for instance,

274

numerous phyla not detected by 16S AFL analysis (Figure S7). In accordance with previous

275

studies, Bacteroidetes, Proteobacteria, Firmicutes and Actinobacteria were dominant in the AD

276

process, likely involved in polysaccharides and proteins hydrolysis (31-34). Additionally,

277

Firmicutes and Proteobacteria include acetogenic and syntrophic bacteria that can degrade VFA

278

(32). 16S AFL analysis indicated only Firmicutes and Bacteroidetes as dominant phyla

279

presenting more than 40% relative abundance (Figure S8). According to TRS analysis,

280

Proteobacteria represented the most abundant phylum in both reactors (~21 and 36%,

281

respectively), while Firmicutes did not account for more than 11% of the total community

282

(Figure S7).

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In particular, an unclassified genus of Desulfobulbaceae was found to be very abundant and

284

further increasing after the H2 addition to 28% (2-folds) in the thermophilic reactor (Figures 2c


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and S9). Relevant species such as Desulfobulbus propionicus and Desulfurivibrio alkaliphilus

286

were also detected, though in low abundance. The former decreased ~3-folds at both temperature

287

conditions, the latter, coherently with the higher pH observed, increased at thermophilic

288

conditions. The decrease of D. propionicus can be explained with its ability to use H2 in absence

289

of sulfate to convert acetate and CO2 to propionate (35). In fact, the selective stimulation of

290

hydrogenotrophic methanogens could have caused a competition for the H2.

291

Several species of Desulfovibrio were identified, with D. desulfuricans as the most abundant

292

(1.5-2%). Under sulfate limited conditions, these species can produce acetate, H2 and CO2 in co-

293

occurrence with a hydrogenotrophic methanogens (36, 37). The concomitant increase in

294

abundance of Desulfovibrio spp. and of methanogens (>1-fold) observed in the thermophilic

295

reactor (Figures 2c and S9) highlights a possible syntrophic association of great importance for

296

biogas production and upgrading.

297

Moreover, microorganisms similar to Pseudomonas and Acinetobacter were identified by both

298

16S AFL and TRS analysis (0.5-3%; Figures 2c, 3, S6 and S9). These genera, together with

299

phylum Actinobacteria and genus Advenella, were previously detected in anaerobic reactors (19,

300

38, 39) and are involved in recalcitrant compounds decomposition producing enzymes for

301

lignocellulose degradation (40).

302

Among Bacteroidetes, the most representative genus was an unclassified member of

303

Sphingobacteriaceae accounting for 15% and 5% of the total community, in mesophilic and

304

thermophilic reactor respectively, prior the H2 addition. This genus was found to decrease after

305

the addition of H2. Unclassified species of Bacteroides, Cellulophaga and Flavobacterium were

306

found with >3% relative abundance in the mesophilic reactor and 18% relative abundance, is also involved in synergistic

311

cellulose degradation (43).

312

Firmicutes were mainly represented by Clostridiales belonging to genus Clostridium and to an

313

unclassified species of Alkaliphilus. While in the thermophilic reactor their relative abundance

314

remained quite stable (~7%) they halved in the mesophilic (from 8 to 4%). Because of the

315

difficulty in taxonomic classification of Clostridia (21), TRS results are in disagreement with

316

16S AFL analysis, where Clostridiales were found to double in the mesophilic reactor and

317

halved in the thermophilic (Figure 2). These microorganisms are known to play a fundamental

318

role in cellulosome-mediated cellulose hydrolysis, but they do not present the β-sugar

319

consumption pathway.

320

Sphingobacteriales acting synergistically with Clostridiales and whose function is crucial for

321

cellulose hydrolysis (44, 45). This hypothesis can reasonably explain their concomitant change in

322

abundance observed in this experiment.

323 324

This reaction is, thus, carried out by Thermotogales and

Further interesting microorganisms, found by TRS, were Aminobacterium colombiense and Mycoplasma, which role still remains unclear.

325

Families Porphyromonadaceae, Rikenellaceae and order Cytophagales, abundant in 16S AFL

326

results, were underrepresented in TRS, maybe due to their low occurrence within the database

327

used or to misassignments. These microorganisms, together with Erysipelotrichacales, are

328

involved in carbohydrates and proteins degradation and VFA production (41, 46, 47). Their

329

relative abundance changed maintening levels of VFA degraders and producers constant.


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Moreover, 16S AFL analysis revealed in the mesophilic reactor, after the H2 addition, a species

331

similar to Candidatus Cloacamonas acidaminovorans (92% identity) accounting for 1.7% of the

332

community. This recently characterized microorganism, might oxidize propionate and ferment

333

sugars and aminoacids to produce H2 and CO2 indicating this species as a possible syntrophic

334

bacterium (46, 48).

335

Other abundant microorganisms found were identified by 16S AFL analysis as similar to

336

genera Halocella and Sedimentibacter and species Advenella faeciporci (99% identity) and

337

Tissierella creatinini (95% identity).

338

Generally, bacteria involved in the first steps of the AD decreased, while hydrogenotrophic

339

methanogens and syntrophic bacteria increased. These findings, together with the decreased

340

microbial diversity, state the role of the H2 driving the AD towards the final steps enhancing the

341

methanogenic process.

342

Concerning the archaeal community, 16S AFL analysis attributed to Euryarchaeota a relative

343

abundance of 1-2%. Moreover, TRS clearly ascertained their predominant position as their

344

relative abundance increased from 17 to 45% (~3-folds) and from 27 to 36%, in mesophilic and

345

thermophilic reactor, respectively (Figure S7). In accordance with a previous study,

346

Methanoculleus was found as predominant genus (46). In particular, according to TRS it

347

increased from 8 to 36% (4.5-folds) in the mesophilic reactor and from 17 to 24% (1.5-folds) in

348

the thermophilic (Figures 2, 3 and S9). Interestingly, in both TRS and 16S AFL analysis it was

349

identified at species level as M. marisnigri (>97% similarity).

350

The known hydrogenotrophic methanogens (49, 50), Methanocorpusculum labreanum,

351

Methanogenium sp., (1-3%) and an unknown genus of Methanoregulaceae (6-7%), were also

352

found (Figures 2, 3 and S9).


16

Page 17 of 30


353

Genus Methanosarcina was detected only by TRS (1% of the total community). M. barkeri and M. acetivorans were also found, but less

355

represented (Figures 2c and 3). The low abundance of genus Methanosarcina can be explained

356

by the low VFA concentration and particularly acetate in the reactors. High acetate levels are, in

357

fact, known to favor the selective proliferation of aceticlastic methanogens (34). Moreover,

358

aceticlastic methanogens are more sensitive to pH and ammonia levels than hydrogenotrophic

359

(50). In their absence, acetate consumption and CH4 formation are mainly carried out through

360

SAO and reduction of CO2/H2 to CH4 by a hydrogenotrophic methanogen (10). A possible

361

candidate for SAO could belong to Thermoanaerobacteraceae (51), which, according to 16S

362

AFL, was very abundant in both reactors (>20% and >7%, respectively) after the H2 addition

363

(Figure 2a and b). At both temperature conditions aceticlastic methanogens decreased, although

364

more markedly at mesophilic conditions.

365

The increase of Methanoculleus and Mehanoregulaceae confirms our hypothesis delineating a

366

selective action of the H2 on hydrogenotrophic methanogens stimulating CO2 consumption and

367

biogas upgrading.

368

In conclusion, the results clearly state the feasibility of H2-mediated biogas upgrading, at both

369

mesophilic and thermophilic conditions with higher biomethanation and CO2 conversion

370

efficiency at thermophilic. Moreover, H2 transfer to the liquid phase was an important factor

371

limiting the H2 availability for microorganisms. Concerning the effect of H2 on microbiome, the

372

innovative metagenomic methods complemented each other providing a better characterization

373

of the microbial community and an understanding of its complexity. Specifically, the decrease of

374

hydrolytic and fermentative bacteria and aceticlastic methanogens and the increase of

375

hydrogenotrophic methanogens and syntrophic bacteria assert the selective pressure of the H2


17


Page 18 of 30

376

towards the hydrogenotrophic pathway enhancing the CO2 consumption and consequently the

377

biogas upgrading.

378


18

Page 19 of 30


379

Table 1: Mesophilic and thermophilic reactor performances at steady state conditions before and

380

after the H2 addition Mesophilic

Thermophilic

Pre H2

Post H2

Pre H2

Post H2

94±20

110±13

368±42

385±24

n.a.

91

n.a.

274

CH4

69.7±0.3

88.9±2.4

67.1±0.8

85.1±3.7

CO2

30.3±0.3

8.8±3.2

32.9±0.9

6.6±0.9

0

2.3±1.8

0

8.3±3.6

CH4 yield manure (mL/gVS)

111±24

130±23

249±27

267±24

Total CH4 yield (mL/gVS)

111±24

168±21

249±27

359±22

CH4 rate (mL/Lday)

66±14

100±12

247±27

359±20

CO2 rate (mL/Lday)

29±6

10±3

121±15

28±5

CO2 conversion (mL/Lday)

0

23±3

0

93±5

H2 flow rate (mL/Lday)

0

192±28

0

510±32

H2 consumption (mL/Lday)

0

178±26

0

470±35

Biogas rate (mL/Lday) Biogas rate R1 (mL/Lday) Biogas composition (%)

H2

pH of the R1 and R2

7.74±0.16

7.78±0.04 7.82±0.16 7.95±0.03

pH of the SR1 and SR2

7.73±0.15

8.17±0.13 7.89±0.17 8.49±0.04

total VFA R1 and R2 (g/L)

0.16± 0.04

0.17±0.04 1.18±0.84 0.19±0.07

total VFA SR1 and SR2 (g/L)

0.09±0.05

0.16±0.03 0.28±0.17 0.38±0.07


19


Page 20 of 30

381 382

Table 2: Summary of sequencing results and alpha diversity indexes. Mesophilic

Thermophilic

Pre H2

Post H2

Pre H2

Post H2

87,819,274

65,063,014

69,103,700

73,977,902

High quality sequences

80%

80%

81%

81%

Extracted rRNA-like sequences

171,275

143,980

178,823

207,687

Full-length 16S rRNA

79

70

70

62

70.45

86.09

58.95

49.60

Illumina total random sequences

assembled sequences Alpha diversity 383 384

ASSOCIATED CONTENT

385

Supporting Information. Reactor’s performances (Figures S1-S4), microbial community

386

composition (Figures S5-S9 and Datasets S1-S6), and additional material and methods are

387

available free of charge via the Internet at http://pubs.acs.org.

388

AUTHOR INFORMATION

389

Corresponding Author

390

*e-mail: [email protected] ; phone: +45 45 25 14 29; fax: +45 45 93 28 50

391

Author Contributions


20

Page 21 of 30


392

The manuscript was written through contributions of all authors. All authors have given approval

393

to the final version of the manuscript.

394

ACKNOWLEDGMENT

395

We thank Hector Garcia and Hector Diaz for technical assistance and Hugo Maxwell Connery

396

for the IT support. This work was supported by the Danish Council for Strategic Research under

397

the project “SYMBIO – Integration of biomass and wind power for biogas enhancement and

398

upgrading via hydrogen assisted anaerobic digestion”, contract 12-132654.

399 400

ABBREVIATIONS

401

AD, anaerobic digestion; VFA, volatile fatty acids; CSTR, continuous stirred-tank reactors; TRS,

402

Random Sequences; 16S SR, 16S rRNA Shotgun Reads; 16S AFL, Assembled Full-Length 16S

403

rRNA gene; R1, primary mesophilic reactor; R2, primary thermophilic reactor; SR1, secondary

404

mesophilic reactor; SR2, secondary thermophilic reactor.

405

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406

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Page 29 of 30


Figure 1: CH4 yield of the secondary mesophilic (grey column) and thermophilic reactor (black column) at steady state after the H2 addition, measured in batch experiment at different initial pH values.

Figure 2: Heat maps of relative abundance (>1%; left part of each panel) and folds change (log2>1; right part of each panel) of the most interesting microorganisms populating mesophilic and



Page 30 of 30

thermophilic reactors at steady state before and after the H2 addition. Correspondence between colors and relative abundance or fold change is reported in the scale at the top of each panel. Folds change is represented in red and green for increased and decreased microorganisms, respectively. The results are obtained according to 16S AFL (a and b) and TRS analysis (c).

Figure 3: Phylogenetic tree of the whole AD microbial community both of mesophilic and thermophilic reactors at steady states, according to TRS analysis. In color are reported microorganism names identified at family and genus level, while, in black, those identified at inferior classification levels. The size of the bullets indicates the abundance of each taxon considering together all samples. Black branches represent microorganisms with an occurrence lower than the threshold imposed (max_annot_clades 50).


Biogas Upgrading via Hydrogenotrophic Methanogenesis in Two

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