Effects of CO on hydrogenotrophic methanogenesis under thermophilic and extreme-thermophilic conditions: Microbial community and biomethanation pathways

Highlights

• 5% (v/v) CO supplementation enhanced CH₄ production at both 55 °C and 70 °C.

• Methanothermobacter thermoautotrophicus was the dominant methanogen.

• Lag phase significantly shortened after acclimation with 5% (v/v) CO.

• Biomethanation processes followed the order: H₂/CO₂, H₂/CO and CO/H₂O.

Abstract

Coke oven gas is considered as a potential hydrogen source for biogas bio-upgrading. In this study, the effects of CO on biomethanation performance and microbial community structure of hydrogenotrophic mixed cultures were investigated under thermophilic (55 °C) and extreme-thermophilic (70 °C) conditions. 5% (v/v) CO did not inhibit hydrogenotrophic methanogenesis during semi-continuous operation, and 83–97% CO conversion to CH₄ was achieved. Methanothermobacter thermoautotrophicus was the dominant methanogen at both temperatures and was the main functional archaea associated with CO biomethanation. Specific methanogenic activity test results showed that long-term 5% CO acclimation shortened the lag phase from 5 h to 1 h at 55 °C and 15 h to 3 h at 70 °C. CO₂ was the preferred carbon source over CO for hydrogenotrophic methanogens and CO consumption only started when CO₂ was completely depleted. M. thermoautotrophicus dominated mixed cultures showed a great potential in simultaneous hydrogenotrophic methanogenesis and CO biomethanation.

Introduction

Biogas produced via anaerobic digestion (AD) consists of (% by volume) 50–75% CH₄ and 25–40% CO₂, and can be used mainly for on-site heat and power generation (Li and Khanal, 2016). The low CH₄ content in biogas, however, limits its potential applications (Angenent et al., 2018). One strategy to improve the methane content in biogas is via upgrading (Bassani et al., 2017, Kougias et al., 2017), which has several inherent merits, such as use as a natural gas for direct injection into gas grid, use as a transportation fuel (compressed natural gas) or in the synthesis of methanol (Duan et al., 2011, Deng and Hagg, 2010). In recent years, biological upgrading via hydrogenotrophic methanogenesis, involving the bioconversion of H₂/CO₂ to CH₄, has attracted significant research attention (Ryckebosch et al., 2011, Sun et al., 2015). The external H₂ needed in biological upgrading process could be generated through water electrolysis (Gong et al., 2014) and biomass-derived ethanol electrolysis (Chen et al., 2014). However, ex-situ hydrogen production via these electrolysis processes is expensive. Moreover, methane is a low-value energy carrier compared to pure hydrogen (Midilli et al., 2005). Thus, there is a need to find a cheaper H₂ source for cost effective biogas upgrading.

There are several low-cost sources of H₂ gas, such as coke oven gas (54–59% H₂, 24–31% CH₄ and 5.5–7% CO) produced as a by-product in the coke making process (Wang et al., 2013), and synthesis gas, also known as syngas (consists of primarily CO, H₂ and CO₂) produced via gasification of agri-residues and waste biomass (Wainaina et al., 2018). As the world’s largest coke producer, China annually produces 70 billion Nm³ coke oven gas; however, only 20% of the gas produced is utilized as fuel (Razzaq et al., 2013). These H₂-rich industrial gases could serve as a potential low-cost H₂ source for biological biogas upgrading. Wang et al. (2013) tested the simulated coke oven gas (H₂:CO (v/v) ratio of 92:8) for biogas upgrading in an anaerobic reactor that combined sludge digestion and in-situ biogas upgrading. The authors reported CH₄ content as high as 99% in the gas phase without inhibition of anaerobic digestion process due to CO under mesophilic condition. Several methanogenic species have been found to utilize CO for their growth, namely Methanothermobacter thermoautotrophicus (Daniels et al., 1977), Methanosarcina barkeri (Bott et al., 1986), and Methanosarcina acetivorans (Rother and Metcalf, 2004). The CO consuming potential of methanogens in the anaerobic granules was also demonstrated by Guiot et al. (2011) in which the authors reported that methanogens were able to grow on CO alone. In the subsequent study by the research group, evolution of the archaeal population in the anaerobic sludge during adaptation to 100% CO atmosphere at mesophilic condition (35 °C) showed the presence of microorganisms belonging to the orders Methanomicrobiales and Methanobacteriales in the consortium, suggesting a shift toward dominance of hydrogenotrophic methanogens (Navarro et al., 2016). Moreover, Luo et al. (2013) also investigated simultaneous CO biomethanation and sewage sludge co-digestion in a single-stage reactor under thermophilic condition and found that the introduction of CO enhanced the hydrogenotrophic methanogenic activity.

However, most studies applied CO as feed gas and mainly focused on the biomethanation of CO. When syngas or coke oven gas is used as hydrogen source for biogas upgrading, the presence of CO could contribute to additional methane production. There is also a need to examine the competition between CO₂ and CO for H₂, bioconversion pathways of CO under H₂-rich/-poor conditions, and the specific methanogenic activity (SMA).

Hydrogenotrophs were reported to function well at a wide temperature ranges (15–98 °C) (Liu and Whitman, 2008). Study has shown that thermophilic condition results in significantly higher CO biomethanation potential than mesophilic condition in spite of alleviated gas-liquid mass transfer limitation (Guiot et al., 2011). Moreover, our previous study showed that hydrogenotrophic methanogenesis was more favourable under thermophilic (55 °C) and extreme-thermophilic (65 and 70 °C) conditions than mesophilic conditions (Xie et al., 2017). To the best of our knowledge, there is lack of study that examined the effects of CO on hydrogenotrophic methanogenesis under extreme-thermophilic conditions (70 °C) and the associated microbial communities.

Based on the above rationales, the objectives of this study were to evaluate the CO and CO₂ biomethanation process of enriched hydrogenotrophs under thermophilic and extreme-thermophilic conditions. The competition between CO₂ and CO for H₂, biomethanation pathways of CO, and effects of CO on the SMA were also examined. To further understand the effect of CO on microbial communities, the changes in archaea and bacterial community diversities and structures were also examined using 16S rRNA high throughput sequencing of the V3-V4 region.