3.1. Batch trials for methane production form FW in the presence of different concentration of iron
Figure 1 presented biogas production and methane yield with different concentrations of iron. As shown in Fig. 2A, the biogas production was 1725, 1731, 1777, 1821, 1737 and 1543ml at 0.0, 0.5, 1.0, 2.0, 4.0 and 6.0mg /L of iron, respectively. Although the biogas production increased with the increasing of iron concentration between 0.5mg/L to 2.0mg/L, no significant increase was observed. However, when the iron concentration exceeded 2.0mg/L, the biogas production was reduced. Figure 1B illustrated that low concentrations of iron could enhance methane yield, whereas high concentrations of iron had an inhibitory effect on methane yield. And the maximum methane yield achieved at 2.0mg/L of iron was 137.5ml/g·VS, which was approximately 1.17-fold of that in the control. The results are consistent with several previous studies(Choong et al., 2016; Facchin et al., 2013; Meng et al., 2013; Yu et al., 2015). Iron as an indispensable micronutrient could effectively enhance the activity of anaerobes and promote the growth of anaerobes at relatively lower concentration(Choong et al., 2016). However, high concentration of iron is easy to cause a toxicity on anaerobes, inhibit the process of methanogenesis(Choong et al., 2016; Facchin et al., 2013). Therefore, the effect of iron on KW AD exhibited a hormetic trend with 2.0mg /L as the threshold.
3.3. Effect of optimal iron concentration on VFAs in semi-continuous AD experiment
VFAs are the main intermediate products that convert to methane(Tian et al., 2015; Velmurugan et al., 2010). Figure 3 showed the composition and variation of VFAs during AD of KW in semi-continuous AD experiment. As shown in Fig. 3, VFAs were composed of acetic acid, propionic acid and butyric acid. And the acetic acid content is the highest, followed by propionic acid. Besides, the variation of each VFAs was very similar. The concentration of each VFAs in each cycle firstly increased, and then decreased. Notably, as the reaction progresses, the maximum concentration of each VFAs in each cycle decreased. For example, the maximum concentration of acetic acid and propionic acid in last cycle was 0.25g/L and 0.06mg/L, which was about 0.38-fold of that in the first cycle, respectively. After 35 days, there was almost no butyric acid. Hence, the iron supplementation promoted VFAs degradation and prevented acid accumulation.
The bidirectional Wood-Ljungdahl (Reductive Acetyl-Coenzyme A) pathway is main route for many acetoclasts and acetogens to convert acetate to H2 and CO2. Enzymes carbon monoxide dehydrogenaseacetyl-coenzyme A synthase (CODH-ACS) and methyl-transferase-Ac Co-A are two important enzymes for this route, which need sufficient iron(Bender et al., 2011; Dobbek et al., 2001). Besides, it reported that reactions yield included into bidirectional Wood-Ljungdahl pathway was very little energy (e.g. acetate to bicarbonate: +25kJΔG0; propionate to acetate: +18kJ ΔG0)(Thauer et al., 1977), which makes the pathway easy to proceed. Therefore, relatively large amounts of iron are expected to be required for growth in acetoclastic/propionoclastic populations. So iron supplementation was beneficial to these enzymes synthesis.
3.4. Effect of optimal iron concentration on concentrations coenzyme F420 and dehydrogenase concentrations in semi-continuous AD experiment
Dehydrogenase is an essential intracellular microbial enzyme(Salazar et al., 2011) and is widely used as an indicator of overall microbial activity occurred intracellularly in all living microbial cells. Moreover, coenzyme F420 (7, 8-didemethyl-8-hydroxy-5-deazariboflavin derivative), a special coenzyme for electron transport, only presents in methane-producing microorganisms. Therefore, it is feasible to use coenzyme F420 concentration as an indicator of methanogenic activity(Cheng et al., 2007; Whitmore et al., 1986).
Figure 4 shown the time course of coenzyme F420 and dehydrogenase concentration with optimal iron concentration in semi-continuous AD reactors. The variations of coenzyme F420 and dehydrogenase concentration were very similar. As the experiment went on, coenzyme F420 and dehydrogenase concentration both increased, and obtained their maximum at the end 7th cycle. As shown in Fig. 4, the maximum dehydrogenase concentration was 119.71mg/L, which increased by 16.3%. The maximum coenzyme F420 concentration in Fe supplementation groups were 0.24µmol/. And compared to the first cycle, coenzyme F420 concentration in 7th cycle increased by 25.68%. As indispensable micronutrient, addition of iron in semi-continuous AD reactors could enhance the activity of anaerobe and promote the growth of anaerobe, especially methanogens(Choong et al., 2016; Eftaxias et al., 2018b; Molaey et al., 2018). These experimental results were consistent with the previous results of cumulative biogas production and methane yield in iron supplementation semi-continuous AD experiment. Increasing of the coenzyme F420 and dehydrogenase concentration indicated a high activity of anaerobe, especially methanogens. Hence, the methane yield increased.
3.5. Shift of microbial community with optimal iron concentration in semi-continuous AD experiment
The AD operational conditions could change the microbial community structure, which determine methane production(Town et al., 2014). Two biomass samples collected at days 7 and 50 were analyzed via high-throughput sequencing, after which the resulting gene sequences of individual samples were assigned to different taxa levels (from phyla to genus). In this study, only the major groups (genera and phyla) with a relative abundance of no less than 0.1% were assigned to said samples. Changes of anaerobe community structure in semi-continuous reactor with optimal iron concentration shown in Fig. 5. As shown in Fig. 5A, the composition anaerobe community at phyla level was stable. The main anaerobe phyla were Synergistetes, Firmicutes, Euryarchaeota, Bacteroidetes and Chloroflexi, which are known to be involved in different phases of the AD process(Nelson et al., 2011). Other phyla such as Proteobacteria, Actinobacteria and Armatimonadetes were identified in minor proportions, which is consistent with previous studies(Ariesyady et al., 2007; Hernon et al., 2006). In the early stage, Synergistetes (ca. 31.02%) was the most dominant anaerobe at the phylum level, followed by Firmicutes (ca. 23.94%), Euryarchaeota (ca. 16.44%), Bacteroidetes (ca. 16.44%) and Chloroflexi (ca. 4.19%). However, after 50 days, Euryarchaeota grew to be the dominant anaerobe with relative abundances of 32.27%, while the relative abundances of Firmicutes, Synergistetes, Bacteroidetes and Chloroflexi were 22.67%, 16.70%, 16.25% and 6.68%, respectively. The result suggested that iron supplementation stimulated Euryarchaeota growth, which was beneficial to enhance methane yield. Conversely, Synergistetes was inhibited by the iron supplementation.
As shown in Fig. 5B, among the Synergistetes phyla, Synergistaceae_uncultured and Aminobacterium were the major genera during the entire process (42). As anaerobic reaction proceeded, the relative abundance of Synergistaceae_uncultured decreased from 25.49–7.5%, which was the main reason for the decreased of the relative abundance of Synergistetes. However, the relative abundance of Aminobacterium increased from 6.29–9.23%. Meanwhile, the relative abundance of Anaerolineaceae_ (within the phylum of Chloroflexi) and Syntrophomonas (within the phylum of Firmicutes) increased from 3.76% and 1.43–5.19% and 7.05%, respectively. Notably, the enhancement of Syntrophomonas growth was strongest. Aminobacterium as a mesophilic amino-acid-degrading bacteria could grow in syntrophic interactions with the hydrogenotroph Methanobacterium by fermentation of a limited range of amino acids(Baena et al., 1998; Chertkov et al., 2010). Protein is an important component of KW; and it could be hydrolyzed to amino acids by hydrolytic bacteria. Hence, the enhancement of Aminobacterium growth stimulated by iron supplementation would guarantee an effectively syntrophic degradation of protein and improve the methane yield from FW. Syntrophomonas, as a syntrophic metabolizer with hydrogenotrophic methanogens, could convert fatty acids into acetate, H2, formate and CH4(Zhang et al., 2020c). Therefore, the strongest enhancement of Syntrophomonas growth was beneficial to sustain efficient syntrophic communities of bacteria and archaea to avoid over accumulation of intermediate products such as H2 and VFAs(Zhang et al., 2020c), which was in agreement with the change of VFAs shown in Fig. 4. Besides, it was reported that Anaerolineaceae can use H2 as electron carrier to degrade alkane and long-chain fatty acid (LCFA) into methane by cocultivation with hydrogenotrophic methanogens(Liang et al., 2015; Liang et al., 2016). FW contains a certain amount of lipid, which has high methane yield potential. And lipid initially is hydrolyzed to LCFA by extracellular lipases that are excreted by the acidogenic bacteria and then glycerol, then is converted to acetate and hydrogen (H2) through the b-oxidation pathway (syntrophic acetogenesis), and finally to methane (CH4) by methanogenic archaea(Alves et al., 2009; Zhang et al., 2020b). Hence, the enhancement of Anaerolineaceae growth would be conducive to the degradation of lipid into methane.
Figure 5B also shown that no significant change of archaeal community structure with iron supplementation in the semi-continuous AD reactors after 50 days. And the phylum Euryarchaeota was primarily represented by the genus Methanosaeta, Methanosarcina Methanobacterium and Methanospirillum. As anaerobic reaction proceeded, the relative abundance of Methanosaeta, Methanosarcina, Methanobacterium and Methanospirillum increased form 15.08%, 11.46%, 6.34% and 1.43–25.67%, 20.19%, 7.73% and 4.11%, respectively. Obviously, during the AD process, supplementation of iron enhanced these four methanogens growth. And the enhancement of Methanosaeta growth was strongest, followed by Methanosarcina. Notably, Methanosaeta always was the most dominant archaeal genus in the semi-continuous AD reactors. Methanosaeta, an acetic-utilization methanogens, could converted acetate into methane, and its importance for methane production(Conklin et al., 2006; De Vrieze et al., 2012a; De Vrieze et al., 2015). This result is consistent with the observations of Zhang et al. (Zhang et al., 2020c). Methanosarcina as second dominant archaeal genus is a type of both acetoclastic and hydrogenotrophic methanogens. It could produce methane from acetate, methanol, monomethylamine, dimethylamine, trimethylamine, H2/CO2, and CO(Garcia et al., 2000). Thereby, the enhancement of Methanosaeta and Methanosarcina contributed to avoid over accumulation of VFAs. Besides, Methanospirillum and Methanoculleus detected in this process were both hydrogenotrophic methanogens. But the enchantment of them by iron supplementation was relatively low.
According to the results of the shift of microbial community, iron supplementation affect AD performance by forming a high-efficiency microbial community for methane production from KW. As an essential element for anaerobe, iron can synthesize and activate some key coenzymes involved in methanogenesis, such as such as formylmethanofuran dehydrogenase, hydrogenase, acetyl-coenzyme A synthetase, carbon monoxide dehydrogenase, coenzyme M methyltransferase, Ferricytochrome c, F420H2 dehydrogenase and heterodisulfide reductase (Baek et al., 2019; Qi et al., 2021; Schattauer et al., 2011), which are significant form the acid production to methane formation during AD process. For example, carbon monoxide dehydrogenase plays an important role in methane formation form acetate and acetate production from H2, CO2 and methanol(Schattauer et al., 2011). Hydrogenase consumes H2 to offer electrons to CO2 to produce methane by hydrogenotrophic methanogens (Choong et al., 2016). In short, the shift of microbial community suggested a dependence of methanogenic performance on iron.