Iron oxide alleviates acids stress by facilitating syntrophic metabolism between Syntrophomonas and methanogens

Chemosphere 247 (2020) 125866

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Iron oxide alleviates acids stress by facilitating syntrophic metabolism between Syntrophomonas and methanogens Tugui Yuan a, Jae Hac Ko b, Lili Zhou a, Xuemeng Gao a, Ying Liu c, Xiaoyu Shi a, Qiyong Xu a, * a Shenzhen Engineering Laboratory for Eco-efficient Recycled Materials, School of Environment and Energy, Peking University Shenzhen Graduate School, University Town, Xili, Nanshan District, Shenzhen, 518055, PR China b Department of Environmental Engineering, College of Ocean Sciences, Jeju National University, Jeju Special Self-Governing Province, 63243, Republic of Korea c College of Life Sciences and Oceanography, Shenzhen University, Guangdong, 518055, PR China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


ZVI and IO could shorten the lag phase of food waste anaerobic digestion.
IO showed higher methane production rate and faster VFAs reduction.
IO could ensure successful methanogenesis under high substrate to inoculum ratio.
Abundance of Syntrophomonas and methanogens were greatly enriched by IO.
IO might promote electron transfer between Syntrophomonas and methanogens.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 October 2019 Received in revised form 6 January 2020 Accepted 6 January 2020 Available online 8 January 2020

Anaerobic digestion (AD) is a promising technology for food waste management, but frequently restricted with long lag phase as a consequent of acidification. Two laboratory experiments were conducted to investigate the effects of iron materials on food waste AD. Experiment 1 compared the effects of iron oxide (IO) and zero valent iron (ZVI) on AD performance. The results showed that both IO and ZVI could enhance methane (CH4) generation, but IO showed better performance regarding the reduction of lag phase. The lag phase of the reactor supplemented with IO was 17.4% and 42.7% shorter than that of the reactor supplemented with ZVI and the control, respectively. Based on these results, experiment 2 was designed to examine the role of IO in alleviation of acid stress at high substrate to inoculum (SI) ratio. The results showed that supplemented IO into reactor could ensure a successful methanogenesis when operating at high SI ratio, while IO-free reactor was failed to generate CH4 although operating for 77 days. Supplementing IO into the reactor after 48 h of digestion could restore the CH4 generation, though its lag phase was 2.6 times of the reactor supplemented with IO at the beginning of the digestion. Microbial community structure analysis revealed that IO could simultaneously enrich Syntrophomonas and methanogens (i.e. Methanobacterium, Methanofollis and Methanosarcina), and might promote electron

Handling Editor: Yongmei Li Keywords: Food waste Anaerobic digestion Zero valent iron Iron oxide Methane production Microbial community structure

* Corresponding author. E-mail address: [email protected] (Q. Xu). https://doi.org/10.1016/j.chemosphere.2020.125866 0045-6535/© 2020 Elsevier Ltd. All rights reserved.

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transfer between those two types of microbes, which were critical for achieving an effective methanogenesis. © 2020 Elsevier Ltd. All rights reserved.

1. Introduction Proper management of food waste (FW) is of a great concern in many municipalities, especially in developing countries, because of their potential environmental pollution and food safety issues. As the largest fraction of municipal solid waste (MSW) components, FW accounts for 50%e70% of the total MSW collected by weight in China (Mo et al., 2018; Tai et al., 2011). Compared with landfilling and incineration, anaerobic digestion (AD) is a promising technology because it can simultaneously treat FW and generate methane (CH4) as an energy source (Chowdhury et al., 2019; Gaur and Suthar, 2017; Yuan et al., 2019). In 2018, there were over 150 food waste treatment facilities constructed in China, with a total treatment capacity of 28,000 tons per day. Among these, AD was the most commonly used approach, accounting for over 80% of all treatment capacities. However, there are many challenges associated with food waste AD, such as long lag phase and low CH4 generation rate, etc.(Braguglia et al., 2018; Ko et al., 2018). It has been reported that supplementation of iron materials could promote AD performance in terms of lag phase reduction and CH4 generation rate improvement (Baniamerian et al., 2019; Feng et al., 2014; Hu et al., 2015; Zhen et al., 2015). Zero valent iron (ZVI), a strong reducing agent, has been widely used as a promoter for AD process. For example, Meng et al., (2013) observed that supplementing ZVI powder into an acidogenic reactor increased the propionate conversion rate, as compared with the reactor without ZVI addition. They ascribed the enhancement effect to the improved enzymatic activities of the acetogenesis by the addition of ZVI. While, Zhen et al. (2015) concluded that ZVI could stimulate CH4 generation from waste activated sludge by acting as a source of electron donor. In addition, multiple lines of evidence suggested that conductive iron oxide (IO) minerals could facilitate syntrophic metabolism of the organic matter during digestion (Dang et al., 2016; Jiang et al., 2018; Kouzuma et al., 2015; Stams and Plugge, 2009). Kato et al., (2012) reported that the supplementation of IO minerals (i.e. hematite or magnetite) could shorten the lag time and increase the CH4 production rate by creating unique interspecies interactions and facilitating methanogenesis. In addition, Yamada et al., (2015) proved that the supplementation of IO (magnetite) promoted thermophilic methanogenesis of acetate and propionate by inducing electric syntrophy between organic acid-oxidizing bacteria and methanogenic archaea. The above studies suggested that the supplementation of iron materials could be a potential alternative to improve the efficiency of FW anaerobic digestion. However, Yang et al., (2013) reported that nano ZVI (average size ¼ 55 ± 11 nm) had suppressive effect on methanogenesis by disrupting cell integrity and increasing H2 production. The contradictory result indicated that further investigations are still needed to evaluate the effects of iron materials on AD. Moreover, most studies have been done with pure culture and simple substrates in the presence/absence of IO or ZVI (Kato et al., 2012; Yamada et al., 2015; Zhou et al., 2014; Zhuang et al., 2015). And only limited research has been conducted in the complicated anaerobic systems containing multiple microbial communities and/or mixed organic waste. Based on the above discussion, laboratory batch experiments were conducted to investigate the effects of the supplementation of iron materials on food waste AD system. In one experiment, the

impacts of ZVI and IO on the performance of the AD reactor were compared. In a second experiment, the roles of IO in alleviation of acids stress under high substrate to inoculum ratio condition were further investigated, by comparing the performances of the reactors supplemented with IO at the beginning of the digestion and after 48 h acidification. For both two experiments, the reactors without iron materials supplementation were used as the control. Temporal evolution of microbial community structures was examined to illuminate the enhancement effects of the iron materials on AD performance. The results may provide insight into food waste anaerobic digestion in the presence of iron materials. 2. Materials and methods 2.1. Preparation of food waste, seed sludge and iron materials Food waste was collected from a canteen at the campus of Shenzhen University Town, China. The oil slick was first washed out and then the FW was grounded to reduce the particle size with a household disposer (with a size less than 5 mm). The processed FW was stored in a refrigerator at 4  C. Mesophilic anaerobic sewage sludge collected from a wastewater treatment plant (Shenzhen, Guangdong province, China) was used as seed sludge. The chemical characteristics of the collected FW and seed sludge are shown in Table 1. Iron powder (99% metal basis, 20 mesh) purchased from Alfa Aesar was used as ZVI in this study. While IO was prepared according to the following procedures: deionized water was firstly mixed with the iron powder and then heated in an oven containing air at 50  C for 12 h. And the process was repeated for 5 times until the surface of the iron powder was covered by iron rust. Finally, the rusty iron powder (i.e. IO) was dried in an oven at 100  C for 5 h, and then stored in a desiccator. The chemical states of the rusty iron powder were determined through the XPS test by using ESCALAB 250Xi instrument (Thermo Fisher, UK). The XPS spectra of Fe 2p region for the rusty iron powder showed a typical Fe2O3 XPS spectrum (Fig. S1). 2.2. Reactor setup and operation 2.2.1. Experiment 1 Experiment 1 was designed to compare the effects of ZVI and IO on the performance of FW anaerobic digestion. Batch experiments were carried out in three parallel 2.5 L glass reactors with a working volume of 1.8 L (Fig. S 2). Every reactor contained 230 g FW and 1415 g sewage sludge, with a substrate to inoculum (SI) ratio of 3:1 (g-VS FW/g-VS sludge), as presented in Table 2. Based on our preliminary experiments, a total amount of 54 g ZVI or IO was added into the glass reactors, hereinafter referred as ZVI group and IO

Table 1 Characteristics of food waste and seed sludge. Parameter Total solids (% ww) Total volatile solids (% ww) pH

Food waste E1 20.86 ± 0.05 19.02 ± 0.18 e

E2 17.40 ± 0.30 14.40 ± 0.20 e

Seed sludge E1 2.51 ± 0.01 1.03 ± 0.006 7.06 ± 0.07

Note: ww: wet weight; E 1: Experiment 1; E 2: Experiment 2.

E2 1.90 1.10 6.70

T. Yuan et al. / Chemosphere 247 (2020) 125866 Table 2 Experimental design and set-up. Groups

Food waste (g VS) Seed sludge (g VS) SI ratio (g-VS/g-VS) ZVI addition (g) IO addition (g)

Experiment 1

Experiment 2

Control

ZVI

IO

Control

IO

230 1415 3:1 e e

230 1415 3:1 54 e

230 1415 3:1 e 54

804 1500 7:1 e e

804 1500 7:1 e 54

(0 h)

IO

(after 48 h)

804 1500 7:1 e 54

Note: SI ratio: substrate to inoculum ratio; ZVI: zero valent iron; IO: iron oxide; IO group: adding IO at the beginning of digestion; IO (after 48 h) group: adding IO after 48 h of digestion. －: no addition. (0 h)

group, respectively (Table 2). The reactor without supplementation of iron materials was used as the control (hereinafter referred as control group). Each reactor was purged with nitrogen gas to remove oxygen and then tightly sealed with a rubber stopper connected to a 3-L aluminum gas pack (Dalian Delin gas packing co., Ltd, China). The reactors were operated in a mesophilic temperature range (35 ± 0.2  C). 2.2.2. Experiment 2 Based on the results of the Experiment 1, the roles of IO in alleviation of acidification stress under high SI ratio were further investigated in Experiment 2. The reactor configuration and working volume were as same as the Experiment 1 (Fig. S 2). The performances of the reactors supplemented with IO at the beginning of the digestion (hereinafter referred as IO (0 h) group) and after 48 h of the digestion (hereinafter referred as IO (after 48 h) group) were compared. The reactor without IO supplementation was used as the control. Based on the results of preliminary experiments (Table S1 and Fig. S 3), each reactor was added with 804 g FW and 1500 g sewage sludge to obtain a SI ratio of 7:1 (g-VS FW/g-VS sludge), as presented in Table 2. The amount of IO supplemented was 54 g, and the reactor operation conditions were same as the Experiment 1. 2.3. Sample collection and analysis Leachate samples were collected 2 to 4 times per week from the leachate sampling port for the analyses of pH value and volatile fatty acids (VFAs) during the experiments. The pH value was measured using a pH meter (Sartorius, PB-10, German). While VFAs (acetic acid, propionic acid, butyric acid, and valeric acid) were measured on a gas chromatograph (Agilent, 7890A, USA) equipped with a capillary column (HP INNOWAX 15 m  0.530 mm 

3

1.00 mm) and a flame ionization detector (FID). The detector temperature was 300  C and the flow rate of carrier gas (H2) was 3.2 mL min1. Biogas volume was measured by a 100-mL glass syringe and its composition was analyzed by a gas chromatograph (7890B, Agilent, USA) with a molecular sieve column (80/100 Molecular Sieve 5A, G3591-80020) equipped with a thermal conductivity detector (TCD). The GC was operated at the oven temperature of 80  C and detector temperature of 250  C. Helium was used as the carrier gas and the reference flow and make up flow was 45 mL min1 and 5 mL min1, respectively. Solid samples taken at days 10, 18, 20 and 33 of the Experiment 1 and days10, 18, 31, 35, 40 and 50 of the Experiment 2 were used for microbial analysis. Details of the analysis including DNA extraction, PCR amplification, Illumina MiSeq sequencing, and the processing of sequencing data are described in the Supporting Information. To distinguish the significance of a difference between two treatments, the Analysis of Variance (ANOVA) was applied to the cumulative CH4 production curves using SPSS Statistics v20.0 with a confidence level of 95%. The value of p < 0.05 was considered to be statistically significant. The statistical analysis results are presented in the Supporting Information. 3. Results and discussion 3.1. Experiment 1 3.1.1. Methane generation The CH4 production of the reactors supplemented with and without the iron materials were different (Fig. 1), as evidenced by the results of the ANOVA analysis (p < 0.05). It was noticed that a large amount of CH4 was generated from all the reactors within the first 3 days, indicating that some readily useable substrates (such as acetic acid, see Section 3.1.2) existed in the FW were directly used by methanogens at the beginning of the operation. Since then, the daily CH4 production rate decreased gradually and the lag phase of methanogenesis was observed due to the drop of pH (Fig. S4) as a consequence of the hydrolysis-acidification of FW. The shortest lag phase was observed in the IO group (13.3 days), followed by the ZVI group (16.1 days) and the control group (23.2 days). This indicated that both IO and ZVI could shorten the lag phase by 42.7% and 30.6%, respectively. In terms of the maximum CH4 production rate, the IO group showed the largest value of 28.2 mL g1 VS day1, followed by the ZVI group (22.0 mL g1 VS day1) and the control group (19.1 mL g1 VS day1). However, ZVI group showed slightly higher cumulative CH4 yield (431.6 mL g1 VS) than that of the IO group (416.4 mL g1 VS) and the control group (410.5 mL g1 VS).

Fig. 1. Daily methane yield (a) and cumulative methane yield (b) (Experiment 1).

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This was due to the dissolution of ZVI under acid condition during the digestion, resulting in hydrogen generation (Eq. (1)). The hydrogen generated was then further converted to CH4 by hydrogenotrophic methanogens (Eq. (2)), resulting in more CH4 generation in the ZVI group. Similar observations were also reported in previous studies (Liu et al., 2012; Zhen et al., 2015), where CH4 production increased with the addition of ZVI in anerobic reactor. Nevertheless, IO had shorter lag phase and faster maximum methane production rate than those of ZVI, as discussed above. Therefore, from the perspective of practical demand in engineering, this study suggested to adopt IO as an additive rather than ZVI to achieve effective AD performance.

Fe0 þ 2Hþ /Fe2þ þ H2

(1)

4H2 þ CO2 /CH4 þ 2H2 O

(2)

“IO”the reactor added with iron oxide; “ZVI” the reactor added with zero valent iron; “Control” the reactor without addition of iron materials.

3.1.2. Variation of volatile fatty acids (VFAs) Fig. 2 presents the variation of VFAs components during the digestion. Acetic and propionic acids were the two dominating types of VFAs, accounting for about 80% of the total VFAs concentration. A large amount of acetic acid (around 1500 mg L1) was detected at the beginning of the experiment (Fig. 2 a). Then the acetic acid was quickly consumed up after 3 days, corresponding to a shark increase of CH4 production (Fig. 1). While the concentrations of propionic acid (Fig. 2 b), butyric acid (Fig. 2 c) and valeric acid (Fig. 2 d) in all the groups were firstly increased up to peak

values and then decreased gradually below the detection limit. Basically, the IO group showed the fastest reduction rates for all VFAs components, followed by the ZVI group, which was in line with the results of CH4 generation. For example, it took around 16, 21 and 26 days for the IO group, ZVI group and Control group, respectively, to consume the propionic acid from their corresponding peak values. These results suggested that adding iron materials accelerated VFAs consumption, where IO showed better performance.

3.1.3. Effects of IO and ZVI on microbial community distribution The microbial community structures of all the groups were analyzed and compared. As illustrated in Fig. 3a, the supplementation of IO or ZVI could significantly enrich the abundance of methanogens during the digestion. At day 33, the relative abundance of methanogens found in the IO group and the ZVI group was 3.7 and 1.5 times of the Control group, respectively. It was noted that the relative abundance of methanogens of the IO group was 101%e147% higher than that of the ZVI group during days 18e33, which was in line with the results of CH4 generation (Fig. 1). The dominant genus of methanogen was Methanosarcina and Methanobacterium, which are two common methanogen genera in anaerobic digester (Luo et al., 2015). The genus Methanosarcina has been reported as a crucial contributor to CH4 production, which could grow on both H2/CO2 and acetic acid (Kurade et al., 2019). As showed in Fig. 3a, the relative abundance of Methanosarcina observed in the IO group increased from 1.5% at day 18 to 7.3% at day 33, which was 2.6e2.9 and 4.8e6.3 times of the ZVI group and the Control group, respectively. This result indicated that IO showed better performance in terms of enriching the genus Methanosarcina, as compared with ZVI. Similarly, higher relative

Fig. 2. Variation of VFAs component concentrations: (a) acetic acid, (b) propionic acid, (c) butyric acid, and (d) valeric acid (Experiment 1).

T. Yuan et al. / Chemosphere 247 (2020) 125866

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Fig. 3. Effect of IO and ZVI on microbial community distribution (Experiment 1).

abundance of Methanobacterium was also found in the IO group during days 18e33 (Fig. 3c). The genus syntrophomonas is known as a kind of syntrophic bacteria, which could degrade non-acetic VFAs such as propionic acids and butyric acids (Zhao et al., 2018). As illustrated in Fig. 3d, the proportion of syntrophomonas in all the groups were increased during digestion as the consequent of VFAs degradation (Fig. 2). The relative abundance of syntrophomonas of the IO group at day 24 was 7.7%, which was 81% and 85% higher than those of the ZVI group and the Control group, respectively. However, its relative abundance decreased to 4.4% at day 33 due to the consumption of nonacetic VFAs, and thus less available food to support their growth. While, the relative abundance of Syntrophomonas of the ZVI group (6.9%) and the Control group (7.7%) at day 33 was similar to that of the IO group at day 24. This result indicated that IO could greatly facilitate the temporal evolution of syntrophomonas, which was beneficial for the conversion of non-acetic acids. In addition, it was reported that syntrophomonas can grow syntrophically with hydrogenotrophic methanogens via interspecies H2 transfer (IHT) (Lei et al., 2016). In the present study, the addition of IO might promote the IHT between methanogens and syntrophomonas, and consequently stimulated the methanogenesis. 3.2. Experiment 2 3.2.1. Methane generation and variation of pH values As presented in Fig. 4 a, the CH4 production was distinctly different among all the reactors tested (p < 0.05). As expected, the

CH4 generation of the Control group was negligible although after 77 days operation, indicating that the activity of methanogens was severely inhibited during the whole experiment. It has been reported that the SI ratio was a critical parameter for achieving a successful methanogenesis, and for alleviating the acid stress under high organic shock (Braz et al., 2019; He et al., 2017; Yuan et al., 2019). In the present study, the SI ratio used was 7:1, which was not enough to alleviate the acids stress. As a consequent, the pH values of the control group were commonly found around 4.0e5.0 during the whole digestion (Fig. 4 b), and thus resulted in methanogenesis failure. “IO(0 h)”the reactor added iron oxide at the begging of digestion; IO (after 48 h)the reactor added iron oxide after 48 h of digestion; “Control”  no addition. However, successful methanogenesis was found in the reactors supplemented with IO (Fig. 4 a), and its supplementation strategies greatly affected the CH4 production (p < 0.05). The lag phase of reactor with IO supplemented at the beginning of the digestion (i.e. IO (0 h)) was 61.5% shorter than that of the reactor added with IO after 48 h of the digestion (i.e. IO (after 48 h)). Furthermore, IO (0 h) group had higher maximum CH4 production rate and potential than those of the IO (after 48 h) group. The pH values in both IO added reactors were increased gradually from around 4.2e4.7 at the beginning to around 8.6e8.7 at the end of digestion (Fig. 4 b). Basically, the IO (0 h) group had higher pH values than those of the IO (48 h) group, which was in line with the results of CH4 production (Fig. 4 a) and the change of VFAs concentration (Fig. S 5). As expected, the VFAs concentrations in both IO added reactors were

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Fig. 4. Cumulative methane yield (a) and variation of pH values (b) at different IO supplementation strategies (Experiment 2).

reduced as the result of methane generation. However, the VFAs of the Control group were kept in a high level during the whole digestion due to the failure of methanogenesis. These results indicated that IO could alleviate the acid stress and ensure successful methanogenesis, even supplementing it after 48 h of acidification. 3.2.2. Temporal evolution of microbial communities at different IO supplementary strategies The microbial communities were analyzed on days 10, 18, 31, 35,

40 and 50 to investigate the effects of IO on the microbial community structure. As illustrated in Fig. 5 a, different IO addition strategies resulted in diverse dynamic patterns of methanogens abundance. Basically, the IO (0 h) group showed highest abundance of methanogens followed by the IO (after 48 h) group and the control group. This result indicated that IO could promote the growth of methanogens, and supplementing IO at the beginning of the digestion showed better performance. Furthermore, greater proportions of syntrophomonas were observed in the reactors added with IO, as compared with the control (Fig. 5 a), which was in line

Fig. 5. Temporal evolution of relative abundance of total methanogens and Syntrophomonas(a), Methanobacterium (b), Methanofollis (c) and Methanosarcina (d) (Experiment 2).

T. Yuan et al. / Chemosphere 247 (2020) 125866

with the observation of Experiment 1 (Section 3.1.3). To understand the effects of IO on the specific pathway of methanogenesis, methanogens at genus level were further analyzed. Three genera, i.e. Methanobacterium (Fig. 5 b), Methanofollis (Fig. 5 c) and Methanosarcina (Fig. 5 d) were detected in all the samples, and their relative abundance in each reactor showed notable different. The proportion of Methanobacterium, whose energy metabolism relies exclusively on H2/CO2 (Thauer et al., 2008), was 2.9e5.6 and 2.8e4.5 times of the IO(0 h) group at days 10, as compared with the IO(after 48 h) group and the control group, respectively. It has been reported that the scavenging of H2 was critical for metabolizing organic acids to acetate, thus avoided the cumulation of acids, leading to successful methanogenesis (Park et al., 2018). In this study, the enhancement of Methanobacterium with IO addition was beneficial for the removal of H2 at the beginning of the digestion, and thus expedite CH4 production. While the proportion of Methanofollis and Methanosarcina were found higher in the reactors supplemented with IO during the whole digestion, as compared with the control. Especially, the proportion of Methanofollis remarkably increased from 0.3% to 0.4% at days 10 and 18 to 6.5% at day 31, and kept at 5.7%e6.8% until day 40, which was in accordance with the results of CH4 generation (Fig. 4 a). The genus Methanofollis, another type of hydrogenotrophic methanogens, was normally detected in anaerobic digestor treating high loading wastewater (Kong et al., 2018). However, noticed that this genus was found much lower at the beginning of digestion, as compared with Methanobacterium. This result indicated that IO enriched firstly genus Methanobacterium and then genus Methanofollis, those of which played important role in maintaining low H2 concentration during the digestion. In addition, compared to the IO (after 48 h) group, the proportion of these two genera was found commonly higher in the IO (0 h) group, which was in line with results of CH4. This indicated that the addition of IO at the beginning of the digestion was vital to timely scavenge the H2 by hydrogenotrophic methanogens, which enable a shorter lag phase of methanogenesis. Over all, high abundance of hydrogenotrophic methanogen (including genera Methanobacterium and Methanofollis) proved that IO could enhance the removal of H2, which was favorable for conversion of VFAs and methane production. As regard to genus Methanosarcina, its relative abundance was also distinctly higher in the reactors added with IO than that of the control. In summary, the supplementation of IO enriched greatly the abundance of methanogens and genus Syntrophomonas, which was critical for achieving an effective methanogenesis.

7

though higher pH value and higher methane production were observed in the ZVI group. In addition, the IO had higher relative abundance of methanogens at the same pH condition as compared with the control and the ZVI group. For example, the pH values of the IO (0 h) group at day 24 (pH ¼ 7.87) and the Control group at day 33 (pH ¼ 7.85) were comparable. However, the corresponding relative abundance of methanogens in the IO (0 h) group was 34.1% higher than that of the Control group. These results suggested that the pH buffer was not considered as the main promotion mechanism for maintaining the activity of methanogens by iron materials. Does IHT and/or direct interspecies electron transfer (DIET) contribute to iron’s alleviating effect on acids stress inhibition? It has been reported that Ferroferric oxide could promote DIET between Syntrophomonas and Methanosaeta (Li et al., 2019; Zhao et al., 2018), or between Geobacter spp. and methanogens (Kato et al., 2012), and thus promoted methanogenesis. However, in the present study, DIET improvement was not considered as the main reason for the enhanced VFAs conversion to methane, as neither Methanosaeta nor Geobacter spp was not found during the digestion. It has been suggested that rusty iron scrap could enrich iron reducing bacteria such as Pseudomonas, which could accelerate electron transfer during methanogenesis and thus beneficial for VFAs degradation (Baniamerian et al., 2019; Ding et al., 2017; Zhang et al., 2014). However, the only iron reducing bacteria found in the present study was Pseudomonas, and its proportions were extremely low and comparable between the reactors with or without iron materials with a range of 0.01%e0.89%. Thus, enriched iron reducing bacteria was also not considered as the main promotion mechanism of iron materials in the present study. It’s well known that methanogens could not directly utilize the non-acetic acids (e.g. propionic and butyric acids), and those of which must be firstly decomposed to acetic acid (so called acetogenesis) by syntrophic bacteria (Arif et al., 2018). However, those acetogenesis reactions could not occur under standard conditions as the DG ’ is positive (e.g. Eqs. (3) and (4)), and were only thermodynamically feasible when H2 partial pressure was low enough (Wang et al., 2018). Since H2 served as an electron carrier for interspecies electron transfer, the improved IHT could be considered as the first reason to counteract the acid stress inhibition during digestion. In the present study, the enrichment of syntrophic acetogens Syntrophomonas and dominant hydrogenotrophic methanogens (including Methanobacterium and Methanofollis), implying that IHT was improved by IO, as illustrated also in the Graphical Abstract.

CH3 CH2 COO þ 2H2 O / CH3 COO þ CO2 þ 3H2 ðDG ’ ¼ þ 76:1 kJ = molÞ

(3)

3.3. Possible promotion mechanisms of iron materials The present study examined the protective effect of iron materials (including IO and ZVI) for functional microbes under inhibition stress of acids. As discussed above, IO was demonstrated to be able to alleviate the acid stress and enhance methane production under varied organic loading conditions. It then raised the question that what was the main possible promotion mechanism of iron materials on mitigating the acids stress for methanogenesis？ Does IO/ZVI act through being as pH buffer? Since the iron materials could react chemically with protons (Hwang et al., 2019; Yang et al., 2013; Zhen et al., 2015), the first impression on iron’s function in an anaerobic process might be that iron increased the pH for methanogens. It was noticed in Experiment 1 that the pH value of the reactors added with iron materials were slightly higher than that of the control during the lag phase (Fig. S4), which suggested that the iron materials could contribute to pH buffer during digestion. However, the relative abundance of methanogens at day 18 in the ZVI group and the Control group was comparable (Fig. 3a),

CH3 CH2 CH2 COO þ 2H2 O / 2CH3 COO þ Hþ þ 2H2 ðDG ’ ¼ þ 48:1 kJ = molÞ

(4)

4. Conclusions Two laboratory experiments were conducted to investigate the effects of iron materials on food waste anaerobic digestion. The results of Experiment 1 showed that iron oxide (IO) had faster methane production rate than that of zero valent iron (ZVI). Further results achieved in Experiment 2 demonstrated that IO could ensure successful methanogenesis when operating at high substrate to inoculum ratio condition, by raising the abundance of methanogens and Syntrophomonas and promoting electron transfer between those two types of microbes. This study suggested that IO can be used as an effective way to promote CH4 generation from

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food waste anaerobic digestion. CRediT authorship contribution statement Tugui Yuan: Writing - original draft. Jae Hac Ko: Conceptualization, Methodology. Lili Zhou: Formal analysis, Methodology. Xuemeng Gao: Formal analysis, Data curation. Ying Liu: Formal analysis. Xiaoyu Shi: Visualization. Qiyong Xu: Writing - review & editing, Supervision. Acknowledgements authors would like to thank the National Key Research and Development Program of China (2018YFC1902903) and Shenzhen Science and Technology Innovation Committee (KJYY20171012103638606 and JSGG20170822164024506) for their funding support for this study. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2020.125866. References Arif, S., Liaquat, R., Adil, M., 2018. Applications of materials as additives in anaerobic digestion technology. Renew. Sustain. Energy Rev. 97, 354e366. https://doi.org/ 10.1016/J.RSER.2018.08.039. Baniamerian, H., Isfahani, P.G., Tsapekos, P., Alvarado-Morales, M., Shahrokhi, M., Vossoughi, M., Angelidaki, I., 2019. Application of nano-structured materials in anaerobic digestion: current status and perspectives. Chemosphere 229, 188e199. https://doi.org/10.1016/j.chemosphere.2019.04.193. Braguglia, C.M., Gallipoli, A., Gianico, A., Pagliaccia, P., 2018. Anaerobic bioconversion of food waste into energy: a critical review. Bioresour. Technol. 248, 37e56. https://doi.org/10.1016/J.BIORTECH.2017.06.145. Braz, G.H.R., Fernandez-Gonzalez, N., Lema, J.M., Carballa, M., 2019. Organic overloading affects the microbial interactions during anaerobic digestion in sewage sludge reactors. Chemosphere 222, 323e332. https://doi.org/10.1016/ j.chemosphere.2019.01.124. Chowdhury, B., Lin, L., Dhar, B.R., Islam, M.N., McCartney, D., Kumar, A., 2019. Enhanced biomethane recovery from fat, oil, and grease through co-digestion with food waste and addition of conductive materials. Chemosphere 236, 124362. https://doi.org/10.1016/j.chemosphere.2019.124362. Dang, Y., Holmes, D.E., Zhao, Z., Woodard, T.L., Zhang, Y., Sun, D., Wang, L.Y., Nevin, K.P., Lovley, D.R., 2016. Enhancing anaerobic digestion of complex organic waste with carbon-based conductive materials. Bioresour. Technol. 220, 516e522. https://doi.org/10.1016/j.biortech.2016.08.114. Ding, B., Li, Z., Qin, Y., 2017. Nitrogen loss from anaerobic ammonium oxidation coupled to Iron(III) reduction in a riparian zone. Environ. Pollut. 231, 379e386. https://doi.org/10.1016/j.envpol.2017.08.027. Feng, Y., Zhang, Y., Quan, X., Chen, S., 2014. Enhanced anaerobic digestion of waste activated sludge digestion by the addition of zero valent iron. Water Res. 52, 242e250. https://doi.org/10.1016/j.watres.2013.10.072. Gaur, R.Z., Suthar, S., 2017. Anaerobic digestion of activated sludge, anaerobic granular sludge and cow dung with food waste for enhanced methane production. J. Clean. Prod. 164, 557e566. https://doi.org/10.1016/ j.jclepro.2017.06.201. He, Q., Li, L., Peng, X., 2017. Early warning indicators and microbial mechanisms for process failure due to organic overloading in food waste digesters. J. Environ. Eng. 143, 04017077 https://doi.org/10.1061/(ASCE)EE.1943-7870.0001280. Hu, Y., Hao, X., Zhao, D., Fu, K., 2015. https://doi.org/10.1016/j.chemosphere.2014.10.022. Chemosphere 140, 34-39. https://doi.org/10.1016/ j.chemosphere.2014.10.022 Hwang, Y., Sivagurunathan, P., Lee, M.K., Yun, Y.M., Song, Y.C., Kim, D.H., 2019. Enhanced hydrogen fermentation by zero valent iron addition. Int. J. Hydrogen Energy 44, 3387e3394. https://doi.org/10.1016/j.ijhydene.2018.06.015. Jiang, J., Li, L., Cui, M., Zhang, F., Liu, Yuxian, Liu, Yonghui, Long, J., Guo, Y., 2018. Anaerobic digestion of kitchen waste: the effects of source, concentration, and temperature. Biochem. Eng. J. 135, 91e97. https://doi.org/10.1016/ j.bej.2018.04.004. Kato, S., Hashimoto, K., Watanabe, K., 2012. Methanogenesis facilitated by electric

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