Effect of ammonia concentration on hythane (H2 and CH4) production in two-phase anaerobic digestion

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Effect of ammonia concentration on hythane (H2 and CH4) production in two-phase anaerobic digestion Ahmed M. Mustafa a,c, Xiang Chen b, Hongjian Lin a, Kuichuan Sheng a,* a

College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China Zhejiang WangNeng Ecological Science and Technology Co., Ltd, Hangzhou, 310000, China c Department of Agricultural Engineering, Faculty of Agriculture, Suez Canal University, Ismailia, 41522, Egypt b

highlights
Effect of ammonia on two-phase anaerobic digestion of food waste was investigated.
A solution was explored to mitigate the total ammonia nitrogen (TAN) toxicity.
TAN concentration of 4044 mg/L corresponded to a threshold in hydrogen reactor.
Recovery achieved in methane reactor after acute inhibition of TAN below 5800 mg/L.
Methane reactor long subjected to TAN above 6200 mg/L revealed chronic inhibition.

article info

abstract

Article history:

Co-production of hydrogen and methane by two-phase anaerobic digestion (AD) may offer

Received 10 January 2019

a sustainable solution for the centralized treatment of food waste (FW), while ammonia

Received in revised form

accumulation is potentially encountered. A mesophilic two-phase AD was investigated for

20 August 2019

hydrogen and methane production from FW at varying ammonia concentrations. The

Accepted 28 August 2019

process achieved a hydrogen yield of 47.7 mL/g VS and a methane yield of 335 mL/g VS by

Available online 26 September 2019

optimizing the organic loading rate (OLR) and recirculation ratio. Total ammonia nitrogen (TAN) concentration of 4044 mg/L corresponded to a threshold in the hydrogen reactor,

Keywords:

above which ammonia would initiate inhibition of hydrogenogenesis and acidogenesis.

Anaerobic digestion

Methane yield was recovered in the methane reactor after acute inhibiting effects of TAN

Ammonia inhibition

below 5800 mg/L, while TAN above 6200 mg/L caused chronic inhibition of methanogens.

Hydrogenogenesis

Adjusting hydraulic retention time (HRT) and recirculation ratio in hydrogen and methane

Methanogenesis

reactors reduced TAN to 960 and 2105 mg/L respectively, resulting in successful recovery

Hythane

was achieved in the hydrogen reactor but not in the methane reactor. The two-phase AD

Food waste

for methane and hydrogen production can be a promising solution for ammonia accumulation in AD from FW. © 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

Abbreviations: Anaerobic digestion, AD; Hydraulic retention time, HRT; Organic loading rate, OLR; Specific hydrogen production, SHP; Specific methane production, SMP; Total ammonia nitrogen, TAN; Volumetric biogas production, VBP; Volatile fatty acids, VFA; Volumetric hydrogen production, VHP; Volumetric methane production, VMP. * Corresponding author. E-mail address: [email protected] (K. Sheng). https://doi.org/10.1016/j.ijhydene.2019.08.229 0360-3199/© 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

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Introduction FW is the most considerable fraction of municipal solid wastes; it typically represents 50% or more of municipal solid waste, especially in developing countries [1]. FW is a biodegradable material which has attracted attention due to its higher energy potential and inexhaustibility [2,3]. AD of FW to recover bioenergy is promising due to its sustainable nature [4,5]. Previous studies have focused on using a single-phase AD. Recently, the two-phase AD process, that produces hydrogen gas in the first phase, followed by the methane production in the second phase, is drawing more attention due to many of its advantages [6e9]. The mixture of the two gaseous products in the two-phase process is termed hythane that a promising vehicle fuel because of its superior combustion efficiency [10,11]. Biohythane can be described as a mixture of several gases of 50e55% CH4, 10e15% H2, and 30e40% CO2 and traces of H2S, ammonia (NH3), etc. yielded through two-stage AD of organic wastes [10]. In the two-phase process, one mol of glucose could theoretically be converted to two mol of methane gas and four mol of hydrogen gas, achieving energy efficiency of 89.0% [12]. This energy efficiency is higher than either theoretical hydrogen production efficiency of 33.5% via single-phase hydrogen AD, or theoretical methane production efficiency of 83.2% via single-phase methane AD [12]. Therefore, the co-production of hydrogen and methane in the form of hythane by two-phase AD may offer a sustainable solution for the centralized treatment of FW. Ammonia plays a vital role in the performance and stability of AD of N-rich feedstock [13,14]. Optimal ammonia concentration ensures sufficient buffering capacity in the AD reactor, thus enhancing the stability of the AD process, while high ammonia concentration is reported as the primary cause of process failure [15,16]. Ammonia would accumulate in anaerobic digesters due to the catabolism of protein-rich FW and recirculation of the digestate [17]. AD of FW is prone to ammonia accumulation that poses a risk of inhibition to the biological process due to its direction inhibition of microbial activity and restricting the use of FW as a substrate for industrial biogas production [17,18]. Sheng et al. [19] evaluated the effect of ammonia on the AD of FW over an added TAN concentration up to 4500 mg/L. The results showed that a threshold TAN concentration for ammonia inhibition to methanogenesis was found to be 1540 mg/L. The risk of ammonia accumulation during AD of FW makes the gas production process potentially unstable. However, numerous research works on inhibition of the AD process by ammonia were mainly focused on single-phase methane AD [20]. Limited studies were carried out to evaluate the effect of ammonia on single-phase hydrogen AD [21,22], let alone twophase AD. Moreover, the two-phase AD system was found to be more sensitive to elevated concentrations of ammonia than the single-phase system [23]. Therefore, further research efforts are required to investigate the stability and inhibition of two-phase AD on FW with a wide variation of ammonia. Several mechanisms of ammonia inhibition have been proposed, such as the direct inhibition of methane-producing enzymes, a change in intracellular pH of methanogens, and

proton imbalance or potassium deficiency [24,25]. There is still no simple strategy available to counteract ammonia inhibition when it exceeds the threshold inhibition concentration. Over the past few decades, recovery strategies have been proposed to prevail over the ammonia inhibition, including the acclimation of microflora, the control of pH and temperature, the dilution of reactor contents, the adjustment of carbon to nitrogen (C/N) ratio of feedstock, and the immobilizing the microorganisms [13]. Nevertheless, some of these control techniques either had negative effects on methane production or economically not feasible. Despite considerable research works on the subject, ammonia inhibition is still a common threat in the AD process, and operational instability caused by ammonia is frequently reported [26,27]. Research on the effect of a wide range of ammonia concentration on hydrogen and methane co-production from FW via two-phase AD, however, is very limited. The objectives of this study were, therefore, to (1) evaluate the effect of a wide range of TAN concentration on process stability and inhibition occurrence in both hydrogen- and methane-producing reactors (2) investigate the effect of adjusting HRT and recirculation ratio of the digestate on TAN concentration, pH, hydrogen and methane production during two-phase AD to mitigate the ammonia toxicity, (3) simulate and predict the effect of TAN concentration on hydrogen and methane production through the two-phase AD.

Materials and methods Feedstock and inoculum The FW of 50 kg was obtained from a student cafeteria at Zhejiang University in Hangzhou, China. Impurities contained in the FW sample, such as animal bones, eggshell, wastepaper, and plastics, were removed manually. The sample (one batch) was then crushed into small particles (<5 mm) via a blender. The ground FW sample as one batch was sealed in plastic bags and stored at 20  C. The substrates were thawed overnight under ambient conditions before usage. Effluent from a mesophilic anaerobic digester fed with livestock manure was used as inoculum. The effluent sludge was kept in air-tight buckets under ambient conditions (about 25  C). The characteristics of the FW and inoculum sludge are shown in Table 1.

Table 1 e Characteristics of FW and inoculum sludge. Parameters TS (%, w.b.) VS (%, w.b.) VS/TS (%) C (%, d.b.) N (%, d.b.) C/N pH TAN (mg/L, w.b.)

FW

Inoculum sludge

26.2 ± 0.4 24.6 ± 0.3 93.8 ± 0.1 47.6 ± 0.5 2.4 ± 0.6 19.8 ± 0.9 4.7 ± 0.01 ND

7.0 ± 0.1 3.7 ± 0.1 53.1 ± 0.3 25.4 ± 0.3 3.5 ± 0.2 7.3 ± 0.5 7.6 ± 0.01 1960 ± 15

Note: w.b., wet base; d.b., dry base; ND, not determined.

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Reactor set-up and operation The two-phase AD system designed in this study is shown in Fig. 1. The hydrogen reactor was a 10-L reactor with a working volume of 6 L, and the methane reactor was a 30-L reactor with a working volume of 20 L. The hydrogen reactor was placed in an incubator in which the temperature was kept at 35 ± 1  C. The methane reactor was incubated at 35 ± 1  C through warm water recycling. The hydrogen reactor was shaken up manually 3 to 4 times per day. The methane reactor was stirred automatically with a motor for 20 min per 2 h (35 rpm). The hydrogen reactor was fed with the FW slurry, and the digestate from the hydrogen reactor was fed directly to the methane reactor. Part of the digestate from the methane reactor was recycled to the hydrogen reactor manually once per day according to the pre-determined recirculation ratio (shown in Table 2), making up a recirculated two-phase AD system. For both of the hydrogen and methane reactors, digestate was discharged from the bottom of the reactors, and fresh feed was added via ports on the top once per day. The reactors continued to run for 126 days, and the whole experiment was divided into four stages. In stage I (days 1e28), 6 L and 20 L of inoculum sludge were loaded into the hydrogen and methane reactors, respectively, before feeding substrates. The daily feeding was

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started after three days of acclimation, and day 0 was defined as the day that the reactors were fed for the first time. The initial OLR for the hydrogen reactor was 18.8 g VS/(L$d), and the HRT in the hydrogen reactor was set at four days, and the HRT in the methane reactor was set at 20 days. In practice, 1.5 L of digestate was drained from the hydrogen reactor, and a calculated amount of FW was fed in according to the prescribed OLR. The reactor was then made up to working volume using tap water. In stage II (days 29e60), the OLR for the hydrogen reactor was adjusted from 18.8 g VS/(L$d) to 9.4 gVS/ (L$d), and the digestate from the methane reactor was recycled to the hydrogen reactor at a recirculation ratio of 1.0 (Qr/ Qi). The HRT in the hydrogen reactor was kept at four days, and the HRT in the methane reactor was kept at 20 days. In stage III (days 61e100), NH4Cl was added gradually to the methane reactor for increasing TAN concentrations. In stage IV (days 101e126), the addition of NH4Cl to the methane reactor was stopped since day 101, and OLR and HRT in the hydrogen reactor were still kept at 9.4 g VS/(L$d) and 4 d, respectively. The recirculation ratio of digestate from the methane reactor was adjusted to 0.5, while HRT in the methane reactor was adjusted to 16 d, to accelerate the transfer and outflow of TAN in the reactors. The operational conditions of four stages (stages I, II, III, and IV) of the reactors are shown in Table 2. The monitoring profiles of volumetric

Fig. 1 e Two-phase AD system with digestate recirculation for hydrogen and methane production from biomass wastes.

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Table 2 e Operating conditions applied during the two-phase experimental test. Operating time (d)

Stage Stage Stage Stage

I II III IV

1e28 29e60 61e100 101e126

Hydrogen reactor

Methane reactor

OLR gVS/(L$d)

HRT (d)

Feed rate (L/d)

Recirculation ratio ()

HRT (d)

Feed rate (L/d)

18.8 9.4 9.4 9.4

4 4 4 4

1.5 1.5 1.5 1.5

0 1.0 1.0 0.5

20 20 20 16

1.0 1.0 1.0 1.25

Note: OLR, organic loading rate; HRT, hydraulic retention time.

biogas production (VBP), volumetric hydrogen production (VHP), specific hydrogen production (SHP), hydrogen content, TAN, pH, and VFA are shown in Fig. 2.

has been applied to describe the reaction kinetics of methane production by batch or continuous anaerobic fermentation [28,33], but this model has not been used to fit two phases for hydrogen and methane production in the process.

Analytical methods

 n I R ¼ Rm 1  * I

The total solids (TS) and volatile solids (VS) were determined according to Standard Methods [28]. Total carbon and nitrogen contents were measured by an elemental analyzer (EA 1112, CarloErba, Italy). The samples were centrifuged at 10,000 rpm for 15 min and then filtered through qualitative filter paper. The filtrate was used for NHþ 4 -N, VFA, and alkalinity determination. The pH was determined by a pH meter (PHS-3D, Shanghai Jinghong, China). The NHþ 4-N was analyzed by spectrophotometry according to the Standard Methods [28]. NH3-N concentration was calculated according to Anthonisen et al. [29] equation. Total VFAs and alkalinity were determined following a two-step titration procedure [30]. For the analysis of VFA, the filtrate was acidified using 25% metaphosphoric acid with an acid-to-filtrate ratio of 1:10, and subsequently the mixture was placed at 4  C for 30 min’ standing. The mixing was further filtered through membrane filters (0.45 mm), and the filtrate was measured for VFA using a gas chromatograph (GC 2014, Shimadzu, Japan). The GC was equipped with a 30 m  0.25 mm  0.25 mm DB-FFAP capillary column and a flame ionization detector. The temperatures of the injector port and detector were 250 and 280  C, respectively. Argon was used as carrier gas at a total flow rate of 78 mL/min and a column flow rate of 1.47 mL/min. The temperature of the column oven was set at 100  C initially and kept for 2 min, then was raised to 220  C at a heating rate of 20  C/min. The temperature was then maintained at 220  C for 2 min. During the analysis, a sample volume of 0.4 mL was injected, and the split ratio was 1:50 [31]. The daily biogas production was measured by wet gas meters (LML-2, Changchun Automotive Filter, China). The recorded biogas volume was converted to its volume at standard conditions of 0  C and 0.1 MPa. The biogas composition (CH4, CO2, H2, and N2) was analyzed using a gas chromatograph (GC 2014, Shimadzu, Japan) equipped with a Carbosphere column and a thermal conductivity detector. The temperatures of the column oven, injector port, and detector were 100, 120, and 120  C, respectively. Argon at a flow rate of 30 mL/min was used as carrier gas [31].

Extended Monod equation The extended Monod equation (1) can be used to simulate and predict the effect of TAN on methane production. This model

(1)

where I is the TAN concentration, in mg/L; R is the hydrogen or methane yield at the TAN concentration of I, in mL/g VS; Rm is the maximum hydrogen or methane production (no inhibition), mL/g VS; I * is the fatal TAN concentration, beyond which the reaction will not continue, mg/L; and n is a constant to fit. In this model, the inhibitory effect of TAN is described by Rm, n and I*, where the constant n determines the shape of the fitted curve, thereby reflecting the acute or chronic inhibition pattern. In this paper, the Matlab R2010b software is used to estimate the model parameters (Rm, n and I*) by nonlinear least-squares fitting by the Gauss-Newton method.

Results and discussion Hydrogen reactor performances Biogas production started immediately from day one, as shown in Fig. 2, accompanied by rapid increases of the VBP and VHP. Then a peak VBP of 1.47 L/(L$d) and a peak VHP of 0.40 L/(L$d) were observed after two days of digestion. However, biogas production dropped immediately after the peak. Both of VBP and VHP dropped to below 0.1 L/(L$d) at day 10, and the hydrogen production process ceased. Meanwhile, pH in the hydrogen reactor fell to 3.62, indicating severe acidification. The optimum pH interval of 4.5e6.0 was recommended for the microbial consortium during hydrogenogenesis [33], while in this study the pH continued dropping at start-up of stage I. Alkalinity was insufficient to keep the pH at an appropriate pH interval, resulting in the falloff of hydrogen production. From day 11 to the end of stage I (days 11e28), VBP fluctuated at low levels of 0.01e0.14 L/(L$d). Meanwhile, VHP value remained below 0.02 L/(L$d), and the SHP was less than 1.0 mL/ g VS since hydrogen content was relatively low (<20%). In this case, however, there was an excess accumulation of VFAs within the hydrogen reactor, where concentrations of propionate and acetate were as high as 4660 mg/L and 3090 mg/L, respectively (Fig. 3). In the acidogenic fermentation, acetate or butyrate and ATP can be produced by phosphorylation of the acetyl-CoA. In addition to acetate and butyrate pathways,

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Fig. 2 e Profiles of VBP, TAN concentration, VHP, hydrogen content, SHP, and pH in hydrogen reactor of a two-phase process.

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Fig. 3 e Profiles of VFA concentration in the hydrogen reactor of the two-phase process. propionate could be generated to maintain microbial growth but resulting in reduced hydrogen production, the generation of propionate indicates the consumption of hydrogen [34]. High concentrations of propionate also caused inhibition of H2-producing acetogenic bacteria, resulting in low degradation rates of propionate and other acids [35,36]. Propionate is formed at high H2 partial pressure when methanogens are not able to consume hydrogen from acidogenesis and acetogenesis fast enough under inhibiting conditions. The inhibition effect would be significantly enhanced when the propionate concentration reached higher than 3000 mg/L. The accumulation of propionate could be attributed to high organic loading, VFAs inhibition, and the lack of some macroand micro-nutrients in the feedstock [37]. In stage II (days 29e60), considering the observed serious inhibition of hydrogen production in stage I, the OLR for the hydrogen reactor was adjusted from 18.8 g VS/(L$d) to 9.4 g VS/ (L$d). Moreover, the digestate from the methane reactor (Qr) was recycled to the hydrogen reactor at a recirculation ratio of 1.0 (Qr/Qi), where Qi is the feed rate of the raw material (FW þ tap water). As a result of recirculation of the digestate from the methane reactor to the hydrogen reactor, the pH in the hydrogen reactor quickly increased from 3.62 to 5.08 at day 30, accompanied with rapid increases of VBP and VHP. The recycled digestate introduced alkalinity, which can adjust or maintain the pH in the H2 reactor [38]. Peak gas production was observed at day 38, with peak VBP of 1.29 L/(L$d), peak VHP of 0.50 L/(L$d), and peak SHP of 53.1 mL/g VS. Meanwhile, the hydrogen content increased from 3.7 to 38.7%. From day 39 to day 48, the gas production fluctuated. During this period, concentrations of propionate and acetate decreased rapidly. At day 42, propionate concentration decreased to 391 mg/L and stabilized at approximately 500 mg/L in the remaining period of stage II. Acetate concentration decreased to 1084 mg/ L at day 48. Results indicated that low VFAs degradation rates and the inhibitory effect on hydrogen production caused by propionate accumulation were disinhibited. From day 49, VBP and VHP stabilized with the variation of daily biogas production less than 10%, indicating that a

steady-state was achieved in the hydrogen reactor. The stable values of VBP, VHP, SHP, and hydrogen content corresponded to 0.90e0.96 L/(L$d), 0.43e0.46 L/(L$d), 46.2e48.7 mL/g VS, and 47.3e48.3%, respectively. The pH also ranged between 5.22 and 5.30, well within the optimum interval of 4.5e6.0. The methanogens (H2-consuming archaea) introduced by the recirculation of digestate from the methane reactor did not disturb the hydrogen production process. The methane content in the hydrogen reactor kept below 0.5% during the steady-state. A study by Lee et al. [39] also found that independent hydrogen production was achieved by HRT control and digestate recirculation without membrane filtration or heat treatment of H2-consuming bacteria present in the returned digestate. During the steady-state of stage II, the main liquid metabolite within the hydrogen reactor was butyrate. The theoretical production of H2 is two mol per mole of glucose consumed when butyric acid is the highest output of fermentation [34]. Luo et al. [37] investigated a two-stage anaerobic hydrogen and methane production from organic wastes. The results also showed that the dominant metabolite for hydrogen production was butyrate. In stage III (days 61e100), The TAN concentration was increased by adding of NH4Cl in the methane reactor from day 61. Since part of the digestate from the methane reactor was recycled to the hydrogen reactor, TAN in the hydrogen reactor gradually climbed to 4971 mg/L. From day 61 to day 68, VBP was the same as that in the steady-state of stage II. The average VHP and SHP were both increased by 5.5% compared to those in the steady-state of stage II, due to the elevated hydrogen content. Subsequently, there was a fluctuation of gas production from day 69 to day 87, while in general the VHP and SHP were still higher than those in the steady-state of stage II. From day 88, with the continuous rise of TAN concentration in the hydrogen reactor (>4044 mg/L), the VBP and VHP started to decrease rapidly, and both dropped to below 0.05 L/(L$d) at day 94. Meanwhile, the SHP decreased to 1.8 mL/g VS on day 94. Since then, the hydrogen production process almost ceased. Besides the inhibiting effect of TAN, the sudden decline of

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hydrogen production may occur as a result of the pH drop and alkalinity in the recycled digestate from the methane reactor. On day 88, pH in the methane reactor fell to 6.0 while VFA/ alkalinity was as high as 3.1, indicating that digestate from the methane reactor was already lack of enough alkalinity to buffer pH. It was clear that hydrogen production was highly associated with the butyrate concentration (Figs. 2 and 3). During the steady-state of stage II, the concentration of butyrate was stabilized at 2300 mg/L; in the early phase of stage III (days 61e87), TAN in the reactor gradually increased and hydrogen production was increased by a certain extent compared with that during the steady-state of stage II. The concentration of butyrate gradually increased in this period correspondingly. Hydrogen production quickly decreased since day 88. Correspondingly, a dramatic decrease in butyrate concentration was simultaneously observed. Monitoring of the butyrate concentration may be used as a guiding parameter for assessing process imbalance because of the high positive correlation between hydrogen production and butyrate concentration. In the final phase of stage III, acids in the hydrogen reactor all kept at low levels, and hydrogen production was moreover basically stopped. These indicated that H2-producing and acetogenic bacterial communities were both severely inhibited.

Methane reactor performances The monitoring profiles of VBP, VMP, SMP, methane content, TAN, pH, and VFA in the methane reactor are shown in Figs. 4 and 5. In stage I (days 1e28), gas production increased initially slowly in the first two days, while the rapid increase was found after day 3. Methane production became stable from day 15, and the reactor achieved average VBP of 1.47 L/(L$d), VMP of 0.86 L/(L$d) and SMP of 228.6 mL/g VS with an average methane content of 58.5%, indicated that a successful startup and good methane production performance were achieved in this stage. In stage II (days 29e60), VMP and SMP dropped immediately from day 29 resulting from OLR lowering. Then there was a 14-days process of gas production fluctuations from day 31 to day 44. After day 45, the gas production was relatively stable with VMP and SMP around 0.89e0.97 L/(L$d) and 0.60e0.65 L/ (L$d), respectively. However, the average VMP and SMP were 36.2% and 26.7% lower than those in the steady-state of stage I, respectively. The methane content in the steady-state of stage II remained relatively constant at about 67.3%, which was 14.9% higher than stage I. The SMP also increased from 228.6 mL/g VS in stage I to 335.0 mL/g VS, indicating that higher substrate degradation and higher conversion rates were achieved in stage II due to the adjustment of OLR and proper recirculation ratio. In a study conducted by Wang et al. [40], effects of end-products during hydrogenogenesis (i.e., ethanol, acetate, propionate, and butyrate) on methane production performances during methanogenesis were evaluated. The results showed that butyrate had the highest conversion rate of fatty acid to methane, and butyrate fermentation pathway was the favorable fermentation pathway of hydrogenogenesis. In our study, the dominant metabolite of hydrogen reactor in stage I was propionate,

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while butyrate in stage II (shown in Fig. 5). Higher methane content and methane production were achieved in stage II compared to stage I, confirming that butyrate was the more suitable intermediate for the methane reactor. During the steady-state of stage II, a stable pH was maintained in the range of 7.03e7.10 in the methane reactor, while the steady-state was also achieved in the hydrogen reactor from day 49 with a stable pH range of 5.22e5.30. This result suggests that pH control and continuous hydrogen and methane co-production via two-phase AD process can be successfully achieved by setting suitable OLR and recirculation ratio, without any added external alkalinity. Furthermore, the main liquid metabolite within the methane reactor was propionate in the steady-state of stage II, while all the residual acids remained at low concentration levels. In a comparison of single-phase and two-phase digesters treating swine manure [36], it was found that propionate fermentation pathway was dominant both in the single-phase digester and in the methane reactor of the two-phase system. High propionate was reported as an inhibitor of methane production [36]. Furthermore, it may cause instability in the anaerobic digesters. The inhibitory propionate concentrations were reported to vary from 500 to 2000 mg/L, which was attributed to the differences in environmental conditions. Though the propionate concentration in this study was higher (ca. 2600 mg/L) in the methane reactor in stage II, it did not affect the system stability, and the SMP was still high (>330 mL/g VS). This result was consistent with a previous study by Luo et al. [37]. They also found that propionate was the predominant metabolite in the methane reactor of the two-phase system, and the anaerobic process was still stable under high propionate concentration (ca. 30 mM). In stage III (days 61e100), NH4Cl was added to the methane reactor in order to increase the TAN to high concentrations. The addition of NH4Cl was divided into 4 phases. For the first 3 phases, different amounts of NH4Cl were added with the substrate at day 61, 68 and 74, which adjusted the TAN concentrations to approximately 4000, 5500 and 6000 mg/L, respectively to study the acute inhibitory or to promote a characteristic of TAN against two-phase AD system. NH4Cl was continuously added to the methane reactor once per day from day 78 to the end of stage III (day 100), to regulate the continuous rise of TAN concentration to study the chronic inhibitory or to promote a characteristic of TAN. On the first day after the addition of TAN for the first time (i.e., day 61), VBP began to decline. It declined to 0.76 L/(L$d) on day 62, which was reduced by 19.1% compared with that in the steady-state of stage II. Meanwhile, VMP and SMP also declined to 0.51 L/(L$d) and 269.5 mL/g VS respectively, indicating a specific inhibiting effect of TAN on the methane production. However, VBP and VMP experienced a gradually recovering process from day 63. Both VBP and VMP recovered to levels equivalent to those during the steady-state of stage II on day 67. Subsequently, during instantaneous increases of TAN concentration on day 68 and day 74, similar gas production conditions were observed, i.e., a shift from gas production declining phase to gas production recovery phase. Results indicated that the sudden rise of TAN concentration in the methane reactor had an immediate inhibiting effect on methanogens. However,

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Fig. 4 e Profiles of VBP, TAN concentration, VMP, methane content, SMP, and pH in the methane reactor of a two-phase process.

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Fig. 5 e Profiles of VFA concentration in the methane reactor of the two-phase process.

since methanogens could quickly adapt to the relatively high TAN concentration, normal gas production performance could be recovered. The resistance of the methane reactor against the relatively high TAN concentration might result from the followings: The TAN concentration in the reactor was stabilized at approximately 1900 mg/L during the steadystate of stage II, and methanogens had already experienced a certain degree of acclimation towards TAN, thus strengthening the resistance capacity of methanogens against TAN impact. However, different from the full recovery of VBP and VMP after addition of NH4Cl for the first two times, VBP and VMP recovered to approximately 0.86 and 0.56 L/(L$d) respectively after addition of NH4Cl for the third time (TAN > 6200 mg/L). They declined by 8.5% and 11.1% respectively compared with those during the steady-state of stage II. NH4Cl was continuously added to the methane reactor from day 78. With the gradual rise of TAN concentration, VBP and VMP began to decline continuously since day 81. The TAN climbed up to 9836 mg/L on day 100, while SMP decreased to 156.7 mL/g VS, 53.2% lower than that in the steady-state of stage II. From day 61 to day 94, the methane content remained relatively stable and varied within a range of 66.3%e70.5%, which was consistent with that during the steady-state of stage II. However, the methane content declined sharply from day 95. The lowest methane content of 45.5% was observed on day 97. Table 3 shows a summary of different studies on the role of TAN in the AD of organic feedstock's. From the data in Table 3, it can be concluded that the inhibiting range for AD of Organic Fraction of Municipal Solid Waste (OFMSW) is 3000e3700 mg/L as TAN, and CH4 decreased by 50% [24]. Whilst for swine manure, cattle manure, soluble non-fat dry milk, and food waste it is from 4000 to 7000 mg/L of TAN, and the reduction in CH4 yield ranged between 25 and 96% [32,41e45], while for pig manure is 11,000 mg/L of TAN, and CH4 decreased by 50% [46]. Meanwhile, pH in the methane reactor declined to 5.5, which was much lower than the acceptable pH interval of 6.5e8.5 suitable for methane production [47]. The current pH already fell into the optimum pH interval of 4.5e6.0 for the H2-

producing bacteria. Hydrogen component was initially detected in the methane reactor from day 95. The hydrogen content on day 95 was determined to be 0.9%, and it even reached 4.9% on day 97. The methane content on day 97 was 45.5%, indicating that the activity of methanogens was inhibited and it failed to timely convert hydrogen to methane. However, since the current pH condition in the methane reactor was usually suitable for H2-producing acetogenic bacteria while the gaseous metabolite was still mainly methane, it thus indicated that the methanogens showed relatively strong resistance against the acidic environment. In stage III, with the rise of TAN concentration, the concentration of propionate gradually decreased, while the concentration of acetate experienced a fluctuating growing process. Besides, a sharp accumulation of butyrate occurred from day 74; the principal liquid metabolite was found to be butyrate from day 92, indicating that the fermentation pathway was already shifted to butyrate pathway. There are several studies related to the metabolism rate of methanogens in the methane reactor over the main end products (i.e., VFAs) from the hydrogen reactor. Wang et al. [48] found out that the degradation rates for acids in the methane reactor were sorted as follows: butyrate > acetate > propionate, while a study by Ren et al. [49] reported that the degradation rates for organic acids were as follows: acetate > ethanol > butyrate > propionate. Although the results drawn by different studies slightly differed, butyrate has favorable degradation performance in a usually running methane AD system. Besides, as an intermediate metabolite, butyrate was mainly converted to acetate and hydrogen, which would be further converted to methane by methanogens. During the steady-state of stage II, the concentration of butyrate in the methane reactor stabilized at approximately 300 mg/L, as shown in Fig. 5. In stage III, the concentration of butyrate sharply increased, and it reached as high as 2432 mg/ L on day 96. Since butyrate could be a suitable substrate for methanogens while the methane production gradually decreased, it thus indicated that high TAN concentration might cause a severe inhibition of butyrate degradation to impose a negative influence on methane production.

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53 64 25 72 e 70 70 96 50 50 50 >6200 5770 >4000 7000 5700 4400 e e 3000e3700 3400e3500 11,000

Simulation the effect of TAN on gas production

5.5e6.9 6.4 7.9 7.9 >7.5 7.9 8.0 8.2 7.6 7.6 7.6e8.1

1 L/d from H2 reactor e 6.5 (4.5)% 7.0 (5.0)% e e (4.5)% (4.5)% e e 15.6 (12.6)%

9.4 g VS/L d 4 g COD/L d e e 1.5 g VS/L d 1.1 g COD/L d e e 1e9.7 g VS/L d 1e9.7 g VS/L d 9.4 g VS/L d

e 53 900 995 >1000 62 1600 2600 220e280 680e690 1450

The effect of TAN concentration on hydrogen and methane production are shown in Fig. 6. The parameters of the hydrogen-producing phase were obtained by fitting the extended Monod equation to be Rm ¼ 60.0 ± 9.6, I* ¼ 4792 ± 17, n ¼ 0.38 ± 0.18; the parameters of the methane-producing phase were, Rm ¼ 350.2 ± 11.9, I * ¼ 10,350 ± 588, n ¼ 0.24 ± 0.07. Additionally, the model predicted the IC10 and IC50 of TAN concentrations (at which the hydrogen production was reduced by 10% or 50%, respectively) to be 1204 mg/L and 4170 mg/L, respectively. For methane production, the IC10 and IC50 TAN concentrations were 3725 mg/L and 9800 mg/L,

f

e

d

c

SNFDM: Soluble non-fat dry milk. CSTR, continuously stirrer tank reactor. CFR, continuously fed reactor. UASB, up-flow anaerobic sludge blanket reactor. ASBR, anaerobic sequencing batch reactor. FAN, free ammonia nitrogen. b

a

20 7 15 15 180 30 15 15 93e19 93e19 13.3 Food waste þ NH4Cl SNFDMa þ NH4Cl Cattle Manure Cattle Manure Food waste Swine manure Swine manure Swine manure OFMSW OFMSW Pig manure

CSTRb CSTRb CFRc UASBd CSTRb ASBRe CSTRb CSTRb CSTRb CSTRb CSTRb

35 55 55 55 e 10 55 60 37 55 55

Reduction in CH4 yield% TAN mg/L FANf mg/L

Inhibition limit Loading rate Feed rate TS (VS) pH Temp.  C HRT d Reactor type Feedstock

Table 3 e Comparison of ammonia inhibition and the reduction in methane yield during AD of various feedstock’s.

Nakakubo et al. [50] analyzed the effect of ammonia on methane production from pig manure via thermophilic singlephase AD. Results showed that iso-butyrate, butyrate, and isovalerate were significantly accumulated in the reactor when TAN concentration exceeded 7700 mg/L. Moreover, these three acids could be used individually as indicators of ammonia inhibition. Butyrate, in particular, was severely accumulated and its concentration was over five times higher than a control reactor fed only raw material without NH4Cl pulsing. Although the accumulation of iso-butyrate and isovalerate was not observed in our study, the severe accumulation of butyrate indicated that the methane reactor was severely inhibited by TAN.

This study [32] [41] [42] [43] [44] [45] [45] [24] [24] [46]

Reference

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 2 7 2 9 7 e2 7 3 1 0

Fig. 6 e Simulation of TAN effects on the (a) hydrogen and (b) methane production.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 2 7 2 9 7 e2 7 3 1 0

respectively. It can be seen from Fig. 6 that the extended Monod equation well fit the effect of TAN concentration on methane production in the two-phase process (R2 ¼ 0.959), but the fitting of the hydrogen production is not sufficient (R2 ¼ 0.709). It can be seen from Fig. 6a that according to the curve obtained by the extended Monod equation, the hydrogen production in the hydrogen-producing phase is inhibited after the TAN concentration >1100 mg/L and the hydrogen production continues to decline with the increase of the TAN concentration. However, the actual experimental data showed that the hydrogen production fluctuated significantly when the TAN concentration was <4044 mg/L, but the hydrogen production was not observed to be significantly inhibited as a whole, and the gas production was stabilized compared with the phase II. The hydrogen production was increased by 57.3% when the added TAN concentration was 3.5 g/L, but the high concentration of ammonia nitrogen (>6.0 g/L) has a significant inhibitory effect.

Recovery performance of the two-phase AD system The addition of NH4Cl to the methane reactor was stopped from day 101, and possible control strategies for recovery from ammonia inhibition were started to be investigated. OLR and HRT in the hydrogen reactor were still kept at 9.4 g VS/(L$d) and 4 d, respectively. The recirculation ratio of digestate from the methane reactor was adjusted to 0.5, and HRT in the methane reactor was adjusted to 16 d, to accelerate the transfer and outflow of TAN in the reactors. After the adjustment of OLR, HRT and recirculation ratio, TAN concentrations in both hydrogen and methane reactors immediately dropped. On day 106, TAN concentrations in the hydrogen and methane reactors dropped to 3148 mg/L and 6321 mg/L, respectively. VBP in the methane reactor increased significantly from day 101 to day 107, possibly resulting from the quick decline of TAN concentration. It recovered to 0.86 L/(L$d) on day 107. However, the methane content in the methane reactor presented a gradually decreasing process during this period. Methane contents on day 106 and day 107 were only 43.4% and 42.5%, respectively. Consequently, VMP and SMP only presented slight rises. Meanwhile, hydrogen production in the hydrogen reactor still ceased, and no trace of recovery was observed. Besides, VFAs were also maintained at low concentrations, indicating that H2-producing and acetogenic bacteria were still inhibited and failed to recover their activities although TAN concentration already declined promptly. Since both the hydrogen and methane reactors failed to recover to their normal gas production performances quickly, digested from the methane reactor preserved during the steady-state of stage II was used to displace reactor contents in the hydrogen and methane reactors on day 106, 110 and 114, respectively. The objectives of this approach were in an attempt to enhance microbial biomass and activities in the reactors and also enhance buffering capacity through the supplement of alkalinity. For each displacement, 1.5 L of reactor content in the hydrogen reactor was displaced, while 3.0 L of reactor content was displaced in the methane reactor. Besides, discharging and feeding were conducted as usual on

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the day of displacement. The preserved methane phase digestate was stored in air-tight buckets under ambient conditions before re-use. The characteristics of the preserved methane phase digestate were determined as follows: TS, 1.5 ± 0.1% (w.b.); VS, 0.7 ± 0.02% (w.b.); pH, 7.6 ± 0.04; TAN, 2348 ± 36 mg/L; VFA/alkalinity, 0.68 ± 0.07; acetate, 342 mg/L; propionate, 2931 mg/L; butyrate, not determined; iso-butyrate, 50 mg/L; valerate, 12 mg/L; iso-valerate, 51 mg/L. Upon the exact day of initial displacement (day 106), the concentration of propionate in the hydrogen reactor immediately increased to 1027 mg/L, and it kept a rising trend during the subsequent days 107e110. However, the recovery of hydrogen production was still not observed, indicating that acetogenic bacteria initially recovered activities compared with H2-producing bacteria. With the ongoing of the experiment, TAN concentration in the hydrogen reactor continuously decreased. After the displacement of reactor content for the third time (day 114), TAN concentration in the hydrogen reactor dropped to 1593 mg/L. On day 115, pH in the reactor increased to 5.3, and correspondingly, quick rises of VBP, VHP, and SHP were observed. Besides, on day 116, VBP, VHP, and SHP reached their peak values of 1.64 L/(L$d), 0.83 L/(L$d) and 88.6 mL/g VS, respectively. The H2-producing bacteria in the hydrogen reactor also recovered activities. From day 119, gas production stabilized, indicating that steady-state was achieved in the hydrogen reactor. During the period from day 119 to the end of stage IV, average VBP, VHP, SHP, and hydrogen content was 1.15 L/(L$d), 0.49 L/(L$d), 51.8 mL/g VS and 42.5%, respectively (Table 4). Besides, the pH in the reactor ranged from 5.18 to 5.27. On day 116, the concentration of propionate decreased to 124 mg/L, while concentrations of acetate and butyrate began to increase rapidly. On day 122, the concentration of butyrate increased to 2320 mg/L, which was equivalent to the average concentration (2300 mg/L) of butyrate during the steady-state of stage II. Besides, the liquid metabolites in the hydrogen reactor were mainly butyrate and acetate since day 122, indicating butyrate fermentation pathway. As previously mentioned, the butyrate fermentation pathway was the favorable pathway during the hydrogenogenesis and acidogenesis phases. Also, SHP after recovery was even improved compared with that before ammonia inhibition (Table 4). Results indicated that successful recovery from ammonia inhibition could be achieved in the hydrogen reactor by adjusting HRT and recirculation ratio and displacing reactor contents with the preserved digeatate. Upon the end of stage IV (day 126), TAN concentration in the hydrogen reactor decreased to 960 mg/L, which was already lower than the average TAN concentration (1122 mg/L) during the steady-state before inhibition. For the methane reactor, pH began to gradually increase since the day of initial displacement (day 106). After the displacement of reactor content for the third time (day 114), pH in the reactor increased to 6.4, and TAN concentration dropped to 3351 mg/L. However, gas production performance failed to recover gradually. Volumetric gas production and SMP presented a gradually declining process instead since day 107. Although the methane content gradually increased since day 108 and it reached 64.9% on day 126, the gas production was maintained at low levels. When stage IV ended,

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i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 2 7 2 9 7 e2 7 3 1 0

Table 4 e Average performances of the hydrogen and methane reactors at the steady stage and recovery stage. Parameters

Gas yield (mL/g VS) Gas content (%) pH Acetate (mg/L) Propionate (mg/L) Butyrate (mg/L) Iso-butyrate (mg/L) Valerate (mg/L) Iso-valerate (mg/L) TAN (mg/L) Energy (kJ/g VS) Total energy (kJ/g VS) H2/(H2 þ CH4) (v/v, %)

Steady-state of stage II

Recovery stage

H2 reactor (H2)

CH4 reactor (CH4)

H2 reactor (H2)

CH4 reactor (CH4)

47.7 ± 0.8 47.8 ± 0.3 5.3 ± 0.1 1140 ± 27 521 ± 13 2503 ± 90 162 ± 10 842 ± 10 253 ± 11 1122 ± 19 0.52 ± 0.01 12.50 ± 0.28 17.6 ± 0.4

335.0 ± 7.6 67.3 ± 0.5 7.1 ± 0.1 1246 ± 70 2627 ± 28 292 ± 10 327 ± 7 834 ± 15 503 ± 11 1894 ± 22 11.98 ± 0.27

51.8 ± 1.8 42.5 ± 0.7 5.2 ± 0.04 1180 ± 268 82 ± 15 2041 ± 209 50 ± 5 340 ± 24 87 ± 3 981 ± 46 0.56 ± 0.02 5.31 ± 0.28 32.6 ± 0.8

132.8 ± 7.5 57.6 ± 5.7 6.2 ± 0.1 2229 ± 61 621 ± 28 1300 ± 226 119 ± 4 593 ± 67 215 ± 3 2144 ± 37 4.75 ± 0.27

volumetric gas production was still under level equivalent to that when the inhibition phase of stage III ended. Moreover, no trace of recovery was observed. The SMP even decreased by 16.6% compared with that on day 100. On day 126, TAN concentration decreased to 2105 mg/L, which was close to the average TAN concentration (1894 mg/L) during the steady-state of stage II. Results indicated that activities of methanogens were still under low levels, although TAN concentration in the methane reactor was adequately controlled. Different from that the methane reactor could still recover to normal gas production levels after suffering from the acute inhibiting effect of TAN, the reactor failed to recover with the reduction of TAN concentration after chronic inhibition of TAN. Several studies revealed that exposing methanogens to a step-wise increase of TAN concentration could gradually enhance the adaptation of anaerobes against TAN, and the methanogens could retain viability at concentrations far exceeding the initial inhibiting threshold once adapted [15,32]. However, this adaptation has not always been followed by full recovery of the methane production before inhibition [13]. In this study, the acute TAN inhibition state was shifted to chronic inhibition state. TAN concentration in the methane reactor also experienced a step-wise increasing process. However, by the end of the experiment, recovery of gas production performance still failed to be observed, which may be due to that an appropriate time was required for the adaptation [13]. Besides, with the increase of TAN levels, longer acclimation time may be needed. Adequate acclimation of bacterial communities may take two months or even longer. The short or inadequate acclimation time in this study may fail successful adaptation in the methane reactor.

Conclusions The TAN concentration is lower than 4044 mg/L improved hydrogen production in the hydrogen reactor of the twophase digestion process, while TAN above 4044 mg/L caused obvious inhibition. The hydrogen production was increased by 57.3% when the added TAN concentration was 3.5 g/L. Complete recovery was achieved in the methane reactor after acute inhibitory effects of TAN below 5800 mg/

L. Nevertheless, incomplete recovery to a level lower than normal methane yield was followed after being subjected to TAN above 6200 mg/L. The methane reactor long subjected to TAN concentration over 6200 mg/L was chronically inhibited.

Acknowledgments This work was financially supported by the National Key Research and Development Program of China, China (No. 2016YFD0800804) and the National Science and Technology Pillar Program of China, China (No. 2012BAC17B02) and, a special fund for the agro-technique extension of the New Countryside Development Institute at Zhejiang University, China (No. 2017ZDNT015).

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