Journal of Bioscience and Bioengineering VOL. 127 No. 3, 345e352, 2019 www.elsevier.com/locate/jbiosc Effects of ammonium and/or sulfide on methane production from acetate or propionate using biochemical methane potential tests Li Tan,1,2,3 Qiu-Shi Cheng,3 Zhao-Yong Sun,3,* Yue-Qin Tang,3 and Kenji Kida3 Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China,1 Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu 610041, China,2 and College of Architecture and Environment, Sichuan University, Chengdu 610065, China3 Received 22 April 2018; accepted 25 August 2018 Available online 19 September 2018 The inhibitory effects of ammonium and sulfide on the methane production using acetate or propionate as a carbon source were investigated under different pH and temperature conditions. The methane production rate, duration of the lag phase, and inhibition threshold limit during methane production were estimated using the Gompertz equation and inhibitor mathematical model. The methane production rates at 53C were 2.3e2.7 times higher than those at 35Cin D 2L the case of non-inhibitors. Increasing the NH4 and/or S concentration decreased the methane production rate and D increased the duration of the lag phase. For methane fermentation that was not acclimated to high NH4 concentration, D the critical NH4 concentration beyond which methane fermentation would stop was 4000e5650 mg/L, depending on the D 2L D pH, temperature, and carbon source. When NH4 and S were coexistent, the critical NH4 concentration decreased to approximately 3800 mg/L when propionate was used and to approximately 4450 mg/L when acetate was used. However, D 2L D no synergistic effect of NH4 and S on the methane production rate was found at an NH4 concentration of < 5000 mg/L L and S2 concentration of 50 mg/L. Ó 2018, The Society for Biotechnology, Japan. All rights reserved. [Key words: Methane fermentation; Biogas; Ammonia; Sulfide; Inhibition] À Conversion of organic waste to biogas via methane fermentation inhibition of methanogens in the presence of S2 inhibition alone þ 2À is an effective approach for minimizing environmental pollution (2). These results indicate that NH4 and S have a potential from the untreated waste and conserving fossil fuel. From the synergistic inhibitory effect on anaerobic digestion. À perspective of environmental sustainability, methane fermentation To alleviate inhibition of methane fermentation by S2 and in- is regarded as a promising process for renewable energy generation crease the biogas quality, a methane fermentation process with and waste stabilization (1). micro-aeration was developed in our previous study (9), and the When treating organic waste with a low carbon to nitrogen (C/ phylogenetic diversity of microorganisms in the fermenter was N) ratio and/or low carbon to sulfur (C/S) ratio, methane analyzed (10). Furthermore, a methane fermentation process with þ fermentation is easily inhibited by ammonium (NH4 )and/or micro-aeration and biogas recirculation was developed to simul- 2À þ 2À sulfide (S ) ions because free ammonia (NH3)andhydrogen taneously alleviate the inhibitory effects of NH4 and S (11,12). sulfide (H2S) can diffuse into the cell membrane (2e5). Moreover, During the methane fermentation process with micro-aeration and the quality of biogas is reduced by the presence of NH3 and H2S biogas recirculation, H2S in biogas was primarily oxidized to sulfur (6). The anaerobic digestion process will be more difficult when and/or sulfate with micro-aeration. The biogas exhausted was þ 2À NH4 and S are coexistent (7,8).Hansenetal.(7) studied the washed with water before recirculation, and NH3 was fixed in the anaerobic digestion of swine manure, and found that when the water tank mainly as NH4HCO3 by reaction of NH3 and CO2. Recir- ammonia concentration was 4.6 g-N/L, even a very low concen- culation of the water-washed biogas into the fermenter enhanced 2À þ tration of sulfide (23 mg S /L) has an inhibitory effect on the stripping of NH3 and H2S. Therefore, the inhibitory effects of NH4 2À anaerobic digestion process. Lauterbock et al. (8) found that when and/or S were alleviated (11). The state of NH3 and H2S in the the ammonia concentration in the reactor was 5.7 g-N/L, only liquid phase is pH- and temperature-dependent. Generally, NH3 2À 28e37 mg/L S resulted in a 50% reduction (IC50) in the methane preferentially exists at high pH, while H2S preferentially exists at 2À þ 2À yield. However, the S concentration 50 mg/L may cause IC50 low pH. Therefore, the inhibitory effects of NH4 and/or S could potentially be alleviated by controlling the pH and temperature during the methane fermentation process (13). In our previous study, the efficiency of NH3 removal increased 1.23-fold with an * Corresponding author at: College of Architecture and Environment, Sichuan increase of the temperature of the methane fermentation process University, No. 24 South Section 1, First Ring Road, Chengdu 610065, China. Tel.: from 53Cto60C (12). However, methane fermentation is easily þ86 28 85990936; fax: þ86 28 85990936. E-mail addresses: [email protected] (L. Tan), [email protected] affected by pH and/or temperature changes. Moreover, volatile fatty (Q.-S. Cheng), [email protected] (Z.-Y. Sun), [email protected] (Y.-Q. Tang), kida@ acids (VFAs), especially acetic acid and propionic acid, were accu- þ 2À gpo.kumamoto-u.ac.jp (K. Kida). mulated when the inhibitory effects of NH4 and/or S were 1389-1723/$ e see front matter Ó 2018, The Society for Biotechnology, Japan. All rights reserved. https://doi.org/10.1016/j.jbiosc.2018.08.011 346 TAN ET AL. J. BIOSCI.BIOENG., expressed (11,14,15), causing failure of methane fermentation. To BMP test under each condition was independently carried out twice, and the data þ 2À presented are the average values. directly alleviate the inhibitory effects of NH4 and/or S ,itis þ 2À Data analysis The modified Gompertz equation (Eq. 1) was used to fit the important to investigate the effects of NH4 and/or S on methane experimental methane production curve to determine the methane production production under various pH and temperature conditions. performance (17). In this study, biochemical methane potential (BMP) tests are V$e carried out to investigate the effects of the individual species and PðtÞ¼Pm$exp À exp ðl À tÞþ1 (1) þ 2À P synergistic inhibition by NH4 and S on methane production un- der different pH and temperature conditions using acetate or pro- where P(t) (mL/L) is the accumulated methane production at time t (h); Pm (mL/L) is pionate as a carbon source. The Gompertz equation and inhibitor the maximum accumulated methane production during the test period, which was fi mathematical model are used to estimate the methane production xed at P (185) at 35 C and P (50) at 53 C for model prediction; V (mL/L/h) is the methane production rate, which was defined as the volume of methane produced rate, duration of the lag phase, and inhibition threshold limit for per liter sludge in 1 h; and l (h) is the duration of the lag phase. þ 2À methane fermentation. The effect of the NH4 or S concentration on the methane production rate under specific pH and temperature conditions was determined by using the inhib- itor mathematical model (Eq. 2) (17): MATERIALS AND METHODS C n V ¼ V 1 À (2) C 0 C* Acclimation of inoculum sludge Sludge from a thermophilic anaerobic þ À where C (mg/L) is the inhibitor (NH or S2 ) concentration; V (mL/L/h) is the digester for treating the stillage of ethanol fermentation was acclimated in an 4 C methane production rate at an inhibitor concentration C; V (mL/L/h) is the methane anaerobic digester with a working volume of 8 L (MDL-10L; B.E. Marubishi, Chiba, 0 production rate in the absence of an added inhibitor; C* (mg/L) is the critical in- Japan) at 53C and 85 rpm by using canteen garbage (volatile total solid (VTS), þ 2À hibitor concentration, beyond which methane fermentation would stop. Origin Pro 201 g/kg, wet base) as a feed. The VFAs, gas evolution, NH4 ,SO4 ,andpHwere 8.0 was used for curve-fitting with a minimum residual sum of squared errors be- monitored during acclimation. Sludge at steady-state was used for the BMP tests. tween the experimental data and model curve. Experimental set-up The BMP tests used an Automatic Methane Potential The inhibition ratio (R)isdefined as follows (Eq. 3): Analysis System II (AMPTS II; Bioprocess Control Sweden AB, Sweden) and were carried out in a 600 mL vial with a working volume of 400 mL. The conditions used for ðV À V Þ R ¼ Con E Â 100% (3) each experimental vial are summarized in Table 1. A 230 mL aliquot of nutrient VCon solution, 0.1 mol/L phosphate buffer (60 mL; pH 7.0 or 8.0), and inoculum sludge þ where V (mL/L/h) is the methane production rate of the control vial (in the (20 mL) were mixed. NH4Cl solution (0e50 mL; [NH4 ] ¼ 40 g/L) and/or Na2S Con À solution (0e60 mL; [S2 ] ¼ 2 g/L) were added according to the designated inhibitor absence of an added inhibitor at pH 7.0 and 53 C); VE (mL/L/h) is the methane concentrations. Sodium acetate or sodium propionate solution (10 mL), with a total production rate for the experimental vials with different inhibitor concentrations. þ 2À organic carbon (TOC) concentration of 40 g/L, was added as a carbon source.