Effect of ammonia on methane production pathways and reaction rates in acetate-fed biogas processes

Anaerobic digestion (AD) process is an important technology for the production of renewable energy in the form of CH₄ from organic waste streams (De Vrieze et al. 2012; Narihiro et al. 2015). In this process, energy-rich methane is mainly generated from acetate and H₂/CO₂ through Acetoclastic (AM) and Hydrogenotrophic Methanogenesis (HM) as catalyzed respectively by acetotrophic and hydrogenotrophic methanogens. Acetoclastic activity has always been generally considered to be the dominant pathway (Zinder & Koch 1984). In the past decade, a second metabolism was reported for the formation of CH₄ from acetate precursor, that occurs via syntrophic acetate oxidation (SAO) in which acetate is oxidized to H₂ and CO₂ by acetate oxidizing bacteria, followed with consumption of H₂/CO₂ by the hydrogenotrophs (SAO-HM). SAO has been found in anaerobic digesters with diverse environments, especially under high ammonia concentrations (Karakashev et al. 2006; Westerholm et al. 2011; Sun et al. 2014; Werner et al. 2014; Hao et al. 2016), indicating the importance of this pathway and relevant functional microorganisms for commercial biogas production when encountering ammonia inhibition problems.

Total ammonium (in the form of free molecule NH₃ and ion NH₄⁺) can be a methanogenesis inhibitor when accumulated to high concentrations (up to tens of g/L) in AD of nitrogen-rich wastes (Westerholm et al. 2011; Westerholm 2012; Fotidis et al. 2013a, 2013b; Lv et al. 2014). The toxicity has been ascribed to proton imbalance and/or potassium deficiency induced by diffusion of NH₃ into cells (Westerholm 2012). The bioprocess can be sometimes deteriorated due to the interaction between ammonium, acids and pH, or in some cases an ‘inhibited pseudo-steady state’ may appear, a condition where the process is running stably but with a lower methane yield (Chen et al. 2008; Lü et al. 2013).

In the biogas processes, various functional microbes and pathways demonstrated different responses to ammonia stress. Generally, acetotrophic methanogens are considered to be less tolerant than the hydrogenotrophic ones (Chen et al. 2008; Demirel & Scherer 2008). The opposite was, however, also reported (Wiegant & Zeeman 1986; Fujishima et al. 2000). Considering the SAO metabolism, NH₄⁺-N ≥ 3 g/L was suggested to initiate the activity of acetate oxidizing syntrophs (Schnürer & Nordberg 2008), as a shift of predominant pathway from AM to HM occurred with elevated NH₄⁺-N concentration. Behavior of the versatile Methanosarcinaceae under ammonia stress is still a ‘mystery’. AM activity performed by its members was observed at 5–7 g/L of NH₄⁺-N (Lü et al. 2013; Fotidis et al. 2013a; Hao et al. 2015). However, H₂/CO₂ utilization or even acetate oxidation by this methanogen family cannot be excluded (De Vrieze et al. 2012; Lü et al. 2013). The threshold of NH₄⁺ and NH₃ concentration as a warning of methanogenesis inhibition or pathway shift is therefore important for digester operators to take efficient measure. A clear relationship between NH₃ concentration and methanogenic pathways/reaction rates is, however, still lacking in previous studies.

Temperature is another factor that can significantly influence the microbial community structure and methanogenic pathways (De Vrieze et al. 2015). Thermophilic conditions can favor hydrogenotrophic methanogens, but not Methanosaetaceae (Demirel & Scherer 2008). Although acetate-oxidizing bacteria were found under both mesophilic and thermophilic conditions (Hattori 2008; Westerholm 2012), higher temperature is energetically favorable to the SAO reaction (Hattori 2008), which has been established as a predominant pathway in several thermophilic digesters (Zinder & Koch 1984; Karakashev et al. 2006; Sun et al. 2014). Furthermore, a fraction of NH₃ can increase with elevated temperature (Chen et al. 2008), that may also influence the inhibitory effect of ammonia on methanogenesis. Thus, a detailed link between NH₃, the contribution of various pathways and functional microorganisms under both temperature regimes should be studied in order to relieve ammonia stress and optimize biogas production when AD process encounters ammonia inhibition problems.

In this study, the methane production process was compared in a wide range of total ammonium nitrogen (TAN) concentrations from 0.14 to 9 g/L at thermophilic and mesophilic conditions. Three methanogenic sludges that originated from different anaerobic reactors in China and France were used. The aim was to make proposals for operation of biogas reactors under high ammonia levels by revealing the relationship between methane production pathway, reaction rates, free ammonia concentration and functional microorganisms.

Three types of inocula were used for a comparison between thermophilic and mesophilic methanogenic microbiome of different origins: ‘TCS’ was taken from a thermophilic anaerobic reactor in China treating synthetic ‘wastewater’ made from glucose and acetate; ‘MCS’ and ‘MFS’ originated from two mesophilic anaerobic digesters in China and France, treating industrial wastewater (Table 1; Supplemental Methods, Section 1, available with the online version of this paper). Freshly collected methanogenic sludges were added to 100 mL BMP medium in serum bottles to reach a volatile solids concentration of 4 g/L. TAN concentration was regulated to 0.14, 3.0, 5.0, 7.0 and 9.0 g/L (calculated as NH₄⁺-N) in different reactors by adding NH₄Cl, to induce ‘inhibitory effects’ of different degrees. Most experiments were conducted in duplicate, while only one reactor was set up for 3.0 g/L of TAN. 100 mmol/L sodium acetate was used as the substrate. The initial pH was adjusted to 7.0 (±0.1) and controlled below 8.0 during the process by adding 1 mol/L HCl solution. Inocula of MCS and MFS were incubated statically at 35 °C and inoculum TCS was incubated at 55 °C (refer to Section 1 in Supplemental Methods for more details).

Methane yield was calculated by using the gas pressure and gas composition values, which was assimilated to an ideal gas (Hao et al. 2015). Gas pressure in the headspace was periodically measured using a differential manometer (Digitron 2082P). Biogas composition was analyzed using a micro GC (CP4900, Varian) equipped with four parallel chromatographic lines (two molecular sieve 5A, one poraplot Q and one volamine columns), coupled with a thermoconductivity detector, as described by Chapleur et al. (2014).

Liquid samples were taken using a sterile syringe and centrifuged at 16,000 g for 5 min (at 4 °C). Supernatant was separated and stored at −20 °C. Acetate concentration in the supernatant was measured by conductimetric detection, using a Dionex 120 ion chromatography system equipped with an IonPac ICE-AS1 analytical column. Dissolved organic carbon was detected by using a BIORITECH 700 analyzer (Bioritech, France). pH was measured immediately after sampling using a Mettler Inlab 427 probe (Mettler Toledo Ltd).

The instant CH₄ production rate (R_CH4) was calculated from the measurements of CH₄ accumulation, using Origin Pro 10.1 (Origin Lab Corp, USA). The specific methane production rate (μ_CH4) was calculated as mol-CH₄ produced from per mol acetate added per day [mol/(mol-acetate·d) or d⁻¹], obtained from dividing the R_CH4 by the total amount of acetate initially added. The ‘instant’ lag phase was defined as the time to the first observation of methane production exceeding 1% in the headspace of the bottles.

Fluorescence in situ hybridization (FISH) was performed as described by Qu et al. (2009), using probes EUB338, ARC915, MX825, MS1414 and MG1200 to respectively target bacteria, archaea, Methanosaetaceae, Methanosarcinaceae and Methanomicrobiales. Detailed probes information, sample fixation and hybridization procedure are described in Section 2 of Supplemental Methods (available with the online version of this paper).

To better characterize CH₄ production activities at various ammonia concentrations, the whole process can be divided into three phases according to the instant methane production rate (μ_CH4), which were: lag phase (0 ≤ μ_CH4 < 0.002 d⁻¹), slow CH₄ production phase (0.002 d⁻¹ ≤ μ_CH4 ≤ 0.010 d⁻¹) and fast CH₄ production phase (μ_CH4 > 0.010 d⁻¹). Three CH₄ production patterns can be then described: (1) at relatively low TAN levels (0.14–3 g/L for MCS and MFS, 0.14–5 g/L for TCS), CH₄ was quickly produced after a short initial lag phase (<10 d); (2) at higher TAN levels (5 g/L for MCS and MFS, 7 g/L for TCS), a second lag or slow CH₄ production phase appeared between the two fast CH₄ production phases, indicating unstable and inhibited methanogenesis at these ‘breakthrough’ TAN concentrations; (3) at quite high TAN levels (7 g/L of TAN for MCS and MFS, 9 g/L of TAN for TCS), CH₄ was actively produced after a long initial lag phase (>50 d). At 9 g/L of TAN for MCS and MFS, active methanogenesis only started after a lag phase >80 d, which is much longer than that for TCS. It suggested slower activity recovery or growth of ammonia-tolerant microbes under mesophilic conditions. This regular pattern, as demonstrated by CH₄ curves, was also observed for the change of acetate and inorganic-carbon dissolved in the liquid phase (Figure S1, available with the online version of this paper).

The dynamics of methane production pathways were evaluated by stable carbon isotopic signature of biogas (Figure 1(d)–1(l)). αc and δ¹³CH₄ act as pathway indicators, with αc <1.055, δ¹³CH₄ > –60‰ suggesting predominance of AM pathway; and αc > 1.065, δ¹³CH₄ < –60‰ representing predominance of HM pathway (Whiticar et al. 1986), which has been applied in previous studies (Qu et al. 2009; Lü et al. 2013; Leite et al. 2016; Vaughn et al. 2016). As acetate was used as the only organic substrate, a predominance of HM pathway also indicates active SAO reaction for decomposition of acetate to CO₂ and H₂.

Surprisingly, AM pathway could still recover at quite high NH₃ levels up to 1 g/L in the MFS-inoculated system, as indicated by several α_c values lower than 1.030 under these conditions. It updated the record on the tolerance of acetotrophic Methanosarcinaceae to NH₃ (De Vrieze et al. 2012).

Reaction rates of the two methanogenic pathways were compared. SAO-HM demonstrated lower reaction rates than AM pathway at high TAN levels, which was also lower than that of the recovered AM pathway under similar conditions. Thermophilic condition favored SAO-HM compared to mesophilic condition.

Generally, methane production rate quickly decreased with increasing TAN concentration. At breakthrough, TAN levels, the maximum instant rate μ_CH4-max (appeared in the first fast methane production phase) decreased 6–9-fold (0.012–0.031 d⁻¹) compared with that at TAN of 0.14 g/L (Table 2), which expressed the reaction rate for AM before the pathway shift. At maximum TAN levels, μ_CH4-max for TCS (0.023–0.024 d⁻¹) was about one-sixth of that at TAN of 0.14 g/L (0.141–0.149 d⁻¹), and for MCS it was one-tenth (0.019–0.020 d⁻¹). These values demonstrated the reaction rate of HM which followed the SAO after the pathway shift. At TAN of 7 g/L, μ_CH4-max for MFS recovered to 0.022–0.031 d⁻¹, about one-fourth to one-third of that at TAN of 0.14 g/L (0.091–0.096 d⁻¹), displaying AM reaction rates performed by Methanosarcinaceae under different TAN levels.

Methane production curves were fitted to the modified Gompertz model (Table 2), generally, the simulated maximum methane production rate R_max demonstrated similar tendency with μ_CH4-max. The lower methane production rates via SAO agree with previous reports (Karakashev et al. 2006; Schnürer & Nordberg 2008), and might explain low biogas production rates in digesters at ‘inhibited pseudo-steady state’ induced by accumulation of ammonia (Westerholm 2012; Sun et al. 2014).

Methane production rates via SAO-HM under thermophilic conditions [R_max 0.027–0.031 mol/(mol-acetate·d) for TCS at TAN of 9 g/L] were higher than that under mesophilic conditions [R_max 0.011–0.020 mol/(mol-acetate·d) for MCS at TAN of 7 g/L]. This should be explained as higher temperature favors SAO reaction energetically (Hattori 2008). Regarding the duration of lag phase, initiation of SAO-HM in TCS and MCS took 49–54 d and 62–70 d, respectively; the recovery of AM activity in MFS took 64–77 d. This long lag phase should result from the slow growth of the ammonia-tolerant microorganisms performing SAO, HM or AM. As reported previously, the co-culture of syntrophic acetate oxidizing bacteria (SAOB) and hydrogenotrophic methanogens usually had a long doubling time of 9–78 d (Westerholm 2012). Growth of SAOB under thermophilic conditions was faster than that under mesophilic conditions; predominance of ammonia-tolerant Methanosarcinaceae, however, took a longer period. The even longer lag phase and lower reaction rate for MCS and MFS at TAN of 9 g/L indicated that quite high ammonia concentration can also inhibit SAOB and ammonia-tolerant Methanosarcinaceae, which was reported by Lü et al. (2013).

This study suggested that stable performance can be obtained via SAO-HM pathway in biogas reactors operated at high ammonia concentrations (up to 1.2 g/L of NH₃ and 9 g/L of TAN). Since the growth and reaction rate of the functional microorganisms under high-ammonia conditions are much slower than the acetotrophic methanogens growing at lower-ammonia levels, a long hydraulic retention time (HRT) (>50 d) is required to maintain the dominance of these microorganisms, and the biogas production rate is lower compared with that of the AM-dominated reactors. Besides the SAOB, some members of Methanosarcinaceae can also prevail under such conditions as observed in the mesophilic MFS and previous studies (Fotidis et al. 2013a; Lü et al. 2013; Hao et al. 2015). The AM activity of these microbes was even higher than that of acetate-oxidizing syntrophs at similar conditions. Factors influencing the competition between SAOB and Methanosarcinaceae under high ammonia concentrations are still not clear. Temperature may play some role, since thermophilic operation seems to favor the growth of SAOB, as indicated by the higher methane production rate and shorter lag period under this condition. It is also demonstrated that, when NH₃ concentration reached 200 mg/L, the operation of the anaerobic digesters should be re-examined and adjusted to improve the establishment of ammonia-tolerant microbial communities. HRT and temperature should be particularly considered, as well as the mixing conditions which may influence the contact between SAOB and the hydrogenotrophs.

Methane production pathways and their specific rates were studied at TAN concentrations of 0.14–9 g/L in acetate-fed biogas reactors inoculated with three methanogenic sludges. High levels of TAN showed significant inhibition of methanogenesis. This could, however, be recovered via SAO coupled with HM performed by acetate oxidizing syntrophs or through Acetoclastic Methanogenesis catalyzed by Methanosarcinaceae, after a long lag phase >50 d. NH₃ was the active component for this inhibition, of which 200 mg/L is suggested as a threshold for the shift of predominant pathway to SAO-HM. Methane production rate via SAO-HM at TAN of 7–9 g/L was about 5–9-fold lower than that of AM performed at TAN of 0.14 g/L, which was also lower than that of AM recovered at TAN of 7 g/L. Thermophilic condition favored the establishment of SAO-catalyzing microbial community, as indicated by the higher reaction rate and shorter lag phase. The operational strategy is thus suggested to be adjusted when NH₃ exceeds 200 mg/L, regarding HRT, temperature and mixing conditions.

The experimental work was performed using scientific equipment acquired with the support DRRT Ile de France in the framework of the CPER-LABE (2007–2013) contract. The research is partially supported by the China National Science Foundation (Nos 51622809, 51378375) and China 111 Project. We also thank Chrystelle Bureau, Angéline Guenne, Lénaïck Rouillac, Nadine Derlet and Céline Madigou for their assistance during the analytical work.