Metatranscriptomic insights into the microbial electrosynthesis of acetate by Fe 2+ /Ni 2+ addition

As important components of enzymes and coenzymes involved in energy transfer and Wood-Ljungdahl (WL) pathways, Fe 2 + and Ni 2 + supplementation may promote the acetate synthesis through CO 2 reduction by the microbial electrosynthesis (MES). However, the effect of Fe 2 + and Ni 2 + addition on acetate production in MES and corresponding microbial mechanisms have not been fully studied. Therefore, this study investigated the effect of Fe 2 + and Ni 2 + addition on acetate production in MES, and explored the underlying microbial mechanism from the metatranscriptomic perspective. Both Fe 2 + and Ni 2 + addition enhanced acetate production of the MES, which was 76.9% and 110.9% higher than that of control, respectively. Little effect on phylum level and small changes in genus-level micro - bial composition was caused by Fe 2 + and Ni 2 + addition. Gene expression of ‘Energy metabolism’, especially in ‘Carbon fixation pathways in prokaryotes’ was up-regulated by Fe 2 + and Ni 2 + addition. Hydrogenase was found as an important energy transfer mediator for CO 2 reduction and acetate synthesis. Fe 2 + addition and Ni 2 + addition respectively enhanced the expression of methyl branch and carboxyl branch of the WL pathway, and thus promoted acetate production. The study provided a metatranscriptomic insight into the effect of Fe 2 + and Ni 2 + on acetate production by CO 2 reduction in MES. Graphical Abstract Highlights


Introduction
CO 2 is the primary greenhouse gases and the large emission of CO 2 is the main cause of global warming. Although CO 2 emission reduction measures are continuously strengthened, global CO 2 emission still keeps increasing and reached 36.3 gigatonnes (Gt) in 2021 (Agency 2021). Carbon capture, utilization and storage (CCUS) are proposed as important and indispensable strategy for slowing the increase of atmospheric CO 2 concentration and alleviating global warming. Microbial electrosynthesis (MES) is a novel technology of carbon utilization, which applied electroactive microorganisms to reduce CO 2 and synthesize organic chemicals, such as acetate, ethanol, and butyrate, by taking electrons from cathodes (Ammam et al. 2016;Mohanakrishna et al. 2018;Tahir et al. 2021;Fontmorin et al. 2021;Martens et al. 2017;Batlle-Vilanova et al. 2017). Reduction of CO 2 to acetate by MES through Wood-Ljungdahl (WL) pathway is the most energy efficient carbon fixation pathway (Bar-Even et al. 2012), and the production of acetate is the foundation for the bioelectrosynthesis of other chemical products, such as medium chain fatty acids (Vassilev et al. 2018). Therefore, acetate production is a preferred option of MES application in carbon capture. However, previous studies have shown the low efficiency of reducing CO 2 to acetate in MES (Prevoteau et al. 2020), owing to the difficulties in establishing high-performance acetogenic microbial community on biocathodes and the low efficiency of electron transfer from cathodes to intracellular circumstances (Prevoteau et al. 2020;Chiranjeevi and Patil 2020). Different strategies, such as polarity reversal and heterotrophic pre-cultivation, have been proposed to accelerate the establishment and enhance the performance of biocathodes (Bajracharya et al. 2017;Zhang et al. 2022). Parameters influencing the performance of microbial electrosynthesis were also investigated, which revealed the importance of applied potential and inorganic carbon source (Izadi et al. 2020).
Modification of electrodes have been studied to improve the electron transfer capacity of the cathodes (Aryal et al. 2017a;Wu et al. 2020;Zhang et al. 2013;Wang et al. 2020;Cui et al. 2017). Moreover, addition of electron shuttle substances has also been applied for enhancing electron transfer in MES (Choi et al. 2012;Liang et al. 2020). After electrons reach the surface of acetogens, electron transfer by intracellular electron transport chains is required to achieve the utilization of electrons by WL pathway for CO 2 reduction and acetate production. The metabolic pathway, coupled with microbial electron transport and energy conservation, are the key foundation for optimizing microbial electrosynthesis, which have been received lots of research attention (Marshall et al. 2016;Kracke et al. 2015;Wenzel et al. 2018). It involves many enzymes and coenzymes for intracellular electron transfer and combined energy transfer, such as hydrogenases (H 2 ases), ATP synthase (ATPase), Rhodobacter nitrogen fixation (Rnf), Nicotinamide adenine dinucleotide (NADH) and so on (Hirose et al. 2018;Buckel and Thauer 2018;Tremblay et al. 2013;Schuchmann and Muller 2014). Therefore, enhancing the synthesis of corresponding enzymes and coenzymes, and improving the efficiency of intracellular electron and energy transfer are also essential to improve the efficiency of CO 2 reduction and acetate production in MES. In previous studies (Marshall et al. 2016;Kracke et al. 2015;Wenzel et al. 2018), the involvement of H 2 ases, Rnf and NADH in intracellular electron and energy transfer has been proposed, which provided the theoretical foundation for promoting intracellular electron and energy transfer. However, specific measures for improving H 2 ases, Rnf, and NADH involved intracellular electron and energy transfer have not been established. It is worth noting that a large part of the related enzymes and coenzymes, such as H 2 ases, Rnf and NADH, require metal elements as components, especially Fe and Ni (Schuchmann and Muller 2012;Muller et al. 2008;Velazquez et al. 2005). The Fe and Ni are important components for the active site of H 2 ases, which are divided into iron-iron type and iron-nickel type according to the presence of iron-iron and iron-nickel in their active sites. Moreover, the subunits of NADH and Rnf also contain Fe-S clusters (Velazquez et al. 2005;Muller et al. 2008;Hreha et al. 2015). In addition, some enzymes in the WL pathway also recruited Fe and Ni as their components, such as CO dehydrogenase (CODH), formate dehydrogenase (FDH) and acetyl-CoA synthase (ACS) (Ragsdale 2009;Boyington et al. 1997;Doukov et al. 2008).
Therefore, abundant exogenous Fe and Ni might be an important requirement for the high efficiency of acetate production. A previous study has found the increased enzyme activities of FDH, CODH, hydrogenase, and NADH in acetogen Clostridium ragsdalei with Fe or Ni addition (Saxena and Tanner 2011). Although the role of Fe and Ni addition in MES for acetate synthesis has not been reported, a reasonable hypothesis is that it is a potential way to enhance MES for acetate production by Fe and Ni addition, probably via improving intracellular electron and energy transfer with enhanced synthesis of related enzymes.
Based on this hypothesis, the aim of this study is to explore (i) the effect of Fe 2+ and Ni 2+ addition on acetate production in MES, (ii) the underlying mechanism of the effect of Fe 2+ and Ni 2+ addition on acetate production in MES from the metatranscriptomic perspective. For this purpose, the performance of MES for acetate synthesis was evaluated under control, Fe 2+ added, and Ni 2+ added conditions. The variation of metatranscriptome under the three conditions were comprehensively analyzed along with the structure of microbial communities. Through this study, a metatranscriptomic insight into the effect of Fe 2+ and Ni 2+ on acetate production in MES could be provided.

MES setup and operation
Six H-type MES reactors were constructed as described previously (Zhang et al. 2022). Each H-type MESs was constructed with two media bottles as the two chambers. The proton exchange membrane (PEM) of Nafion 117 (Dupont, USA) was used to spatially separate the two chambers, and ensure the proton transfer between the two chambers. The operating volume of the chambers was 300 mL and the headspace volume in each chamber was 10 mL. Carbon fiber brushes with a diameter of 5.5 cm and a length of 7.5 cm, which were fabricated by Xinlong Brand brush making factory (Harbin, China) with carbon fibers from TOHO TENAX, Co., Ltd., Japan. The components of the medium for acetate production in the cathode chambers was NaHCO 3 2.5 g/L, KH 2 PO 4 0.3 g/L, NH 4 Cl 0.3 g/L, CaCl 2 ·2H 2 O 0.02 g/L, MgSO 4 ·7H 2 O 0.2 g/L, which was supplemented with vitamin solution (DSMZ-Medium 141) of 10 mL/L, trace element solution (DSMZ-Medium 141) of 10 mL/L and yeast powder of 0.1 g/L. The composition of element solution and vitamin solution are shown in Text S1. NaHCO 3 was served as the inorganic carbon source for acetate synthesis. To inhibit methane production, 10 mM Sodium 2-bromoethanesulfonate (NaBES) was added into the medium. The anode operated with 300 mL 25 mM PBS and 20 g/L K 4 Fe(CN) 6 ·3 H 2 O. A standard calomel electrode (SCE) (Rex 217, Shanghai INESA Scientific Instrument Co., Ltd), which is 0.241 V vs. standard hydrogen electrode (SHE), was installed in the cathode chamber of each MES reactor as the reference electrode. The potential of the cathode was controlled at -1.0 V vs. SCE with a potentiostat (CHI1030C, CH Instruments Ins, China).
The inoculum for cathode chambers was collected from the effluent of an acetate producing MES reactor (Zhang et al. 2022). The OD600 was controlled at approximate 0.5 after inoculation. The reactors were operated in batch mode under 37 °C, with each batch operated for exactly 10 days. Inoculation was repeated at the beginning of the first 3 cycles. Subsequently, the reactors were operated parallelly for acetate production. After three cycles operation, the reactors were divided into three groups, i.e. Fe addition group, Ni addition group, and control group, with two reactors each. The Fe addition group and Ni addition group was supplemented with 50 mg/L Fe 2+ (FeCl 2 ·4H 2 O) and 50 mg/L Ni 2+ (NiCl 2 ·6H 2 O), respectively, which is a concentration that showed no inhibition on anaerobic microbial community (Guo et al. 2019). The pH in cathode chamber was controlled around 7.0 by adjustment with 2 M HCl every 12 h. The concentration of produced acetate was measured every two days. Microbial community and metatranscriptomic analysis were conducted after three cycles operation.

Analyses methods
Acetate was determined by a gas chromatography (GC-2010, Shimadzu, Japan) as described previously (Zhang et al. 2022). Scanning electron microscopy (SEM) analysis were conducted by microscope of Gemini 300a (ZEISS, Germany). The concentration of total ferrous and nickel ions was quantified using an atomic absorption spectrophotometer AAF-7000 F (Shimadzu, Japan). X-ray diffraction (XRD) analysis was conducted on a D2 Phaser (Bruker AXS GMBH, Germany). The electron recovery efficiency (ERE) of the produced acetate was calculated based on EREin% = n t ×fe×F t 0 Idt . Where n t is the number of moles of acetate analyzed at time t, f e represent 8 electron equivalents per mol for acetate, F is Faraday constant (96,485 C mol − 1 electron) and I is current (A).

DNA extraction and 16 S rRNA gene high throughput sequencing
Microbial community on the biocathodes of the MES reactors were analyzed with 16S rRNA gene high throughput sequencing. After operation, the samples of microorganisms were collected from the entire cathodes of each reactor by ultrasonic treatment filtration with a 0.22 µm membrane. The microbial samples collected from the replicate reactors were mixed prior to further analysis. DNA extraction was conducted with a DNeasy Power Soil Kit (Qiagen, Germany). DNA concentration was measured with a NanoDrop 2000 (Thermo Scientific, Wilmington, USA). The primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') were adopted to amplify the V3-V4 regions of bacterial 16 S rRNA gene on a thermocycler PCR system (GeneAmp 9700, ABI, USA). The amplicons were gel purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, expressed genes (DEG) analysis, the no expression of genes in samples were removed via the edge R package. Total 103,872 residual transcripts were used to analyze the DEGs with falsediscoveryrate (FDR) < 0.05 and |log2 (FC)| ≥ 1, where FC means the fold change of genes. GO enrichment annotation of the up-regulated and down-regulated genes was carried out by WEGO, and the top 25 enrichment of GO categories were displayed. Raw metatranscriptomic data have been deposited into the NCBI Sequence Read Achieve database (Accession Number: PRJNA833493).

Acetate production
Acetate production performance of the MES reactors are shown in Fig. 1a. The cumulative acetate concentrations of the control in three cycles were 88.2 mg/L, 92.4 mg/L, and 119.3 mg/L, respectively. The gradually increasing acetate production indicated the developing performance of the reactors. By supplement of ferrous and nickel ions, acetate production was significantly improved. The cumulative acetate concentrations of Fe group were 121.11 mg/L, 145.02 mg/L, and 186.51 mg/L, respectively in the three cycles, which were 37%, 57%, and 56%, higher than that in the control. The accumulated acetate concentration in cycle 3 of the Fe group was 79% higher than that before Fe 2+ addition ( Figure S1). The promotion of nickel ion addition was even better than ferrous ion addition. The cumulative acetate concentration of Ni group were 187.69 mg/L, 222.28 mg/L, and 264.13 mg/L, respectively, in the three cycles, and 113%, 140%, and 121% higher than that of control. The accumulated acetate concentration in cycle 3 of the Ni group was 118% higher than before Ni 2+ addition, much higher than that in the control (121%). The significant improvement of acetate synthesis of the MES reactors by Fe 2+ and Ni 2+ addition was observed.
With the improved acetate synthesis, the electron recovery efficiency was also significantly increased with Fe 2+ and Ni 2+ addition (Fig. 1b). The electron recovery efficiency for the acetate production of the Fe group were 47.47%, 47.72% and 68.3%, respectively, in the three cycles, substantially higher than that of control (28.9%, 32.6% and 45.9%). With nickel ions addition, the electron recovery efficiency for the acetate production was enhanced to 63.44%, 75.40%, and 86.49%, respectively. Acetate synthesis was aided by the addition of Fe 2+ and Ni 2+ , which also resulted in an increase in electron recovery efficiency, showing that the addition of Fe 2+ and Ni 2+ were favorable to the flow of electrons to WL pathway and acetate synthesis. USA), quantified using a QuantiFluor ™ -ST (Promega, USA), followed by pooled in equimolar, and paired-end sequenced on an Illumina MiSeq platform (Illumina, San Diego, USA). Microbial composition analysis and functional prediction by Tax4Fun were conducted based on I-Sanger platform (Majorbio, Shanghai, China). Raw 16 S rRNA gene sequencing data have been deposited into the NCBI Sequence Read Achieve database (Accession Number: PRJNA833556).

RNA extraction and metatranscriptomic analysis
Total RNA was extracted from the biocathode microorganisms using a E.Z.N.A.® Soil RNA Midi Kit (Omega Biotek, Norcross, GA, U.S.) according to the manufacturer's protocols. RNA concentration was quantified with a Nano-Drop2000 (Thermo Fisher Scientific, U.S.), and RNA quality was evaluated with 1% agarose gels electrophoresis.
Ribosomal RNA was removed from the extracted total RNA using a Ribo-zero Magnetic kit (Epicentre, San Diego, USA). cDNA libraries were constructed using TruSeq™ RNA sample prep kit (Illumina, San Diego, USA). The barcoded libraries were paired end sequenced on the Illumina Hiseq 2500 platform at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China), using a HiSeq 4000 PE Cluster Kit and a HiSeq 4000 SBS Kits.
After sequencing, the overlapping of paired end Illumina reads were merged into a single longer read using the SeqPrep (https://github.com/jstjohn/SeqPrep). Low-quality reads (length < 50 bp or with a quality value < 20 or having N bases) were removed by the Sickle (https://github.com/ najoshi/sickle). Residual rRNA reads were removed using the SortMeRNA (Kopylova et al. 2012) by aligning to the SILVA 128 version database. Open reading frames (ORFs) from each sample were predicted using the TransGeneScan (Ismail et al. 2014). All sequences with a 95% sequence identity (90% coverage) were clustered as the non-redundant gene catalog by the CD-HIT (Li and Godzik 2006). Reads after quality control were mapped to the representative genes with a 95% identity. Fragments per kilobase of exon model per million mapped fragments (FPKM) were evaluated using the RSEM (Li and Dewey 2011).
BLASTP (Version 2.2.28 + ) was employed for taxonomic annotations by aligning non-redundant gene catalogs against NCBI NR database with e-value cutoff of 1-e − 5 . Gene Ontology (GO) annotation was performed using the Blast2go, with aligning sequences to the GO database. Clusters of Orthologous Groups (COG) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation was performed using the BLASTP against eggNOG database (V4.5) and KEGG database, respectively. The e-value cutoff of all the blasting was set at 1-e − 5 . For differentially addition of Fe 2+ and Ni 2+ . However, the Shannon index and the Simpson index showed that the microbial diversity of Ni group was the highest and the Fe group was the lowest, a relatively higher and lower microbial diversity in Ni group and Fe group than that in the control group were revealed.
Venn map (Fig. 2a) showed that the three groups had 53 shared OTUs, which accounted 67.1 − 70.7% of the total OTUs in each library. The high proportion of shared OTUs indicated the similar microbial communities in the three group reactors. The main phyla in the three group reactors were Synergistetes, Proteobacteria, Firmicutes, Desulfobacterota, Bacteroidetes and Thermotogae, among which the total proportion of Synergistetes, Proteobacteria, Firmicutes and Desulfobacterota accounted for more than 80% (Fig. 2b). Fe group showed the increased relative abundance of Synergistetes, Proteobacteria and Firmicutes, and decreased the proportion of Desulfobacterota. Meanwhile, Synergistetes was increased by Ni 2+ addition.
The main genera in the three group reactors were Desulfovibrio, Rhodocyclaceae, Aminobacterium, Blvii28_wastewater-sludge_group, and Clostridium_sensu_stricto_1, with total proportion of more than 60% (Fig. 2c). Relative abundance of Desulfovibrio and Rhodocyclaceae in control were 27.8% and 18.6%, respectively. In the Fe group, the relative abundance of Desulfovibrio decreased to 7.4%, while the relative abundance of Rhodocyclaceae increased to 34.7%. A large part of Desulfovibrio and Rhodocyclaceae are H 2 producers (Perona-Vico et al. 2020;Xiang et

Microbial community of the biocathode
The concentration of extracted total DNA and the microbial sequencing diversity of biofilms of the biocathodes in different groups are shown in Table 1. The concentration of total DNA extracted from the biocathodes of control, Fe and Ni groups were 531.3 ng/µL, 587.5 ng/µL, and 397.7 ng/ µL, respectively, indicating the biomass on the cathodes of the Fe group was the highest, while that in the Ni group was relatively lower than the control. SEM also showed the relatively abundance biomass on the cathode of Fe group, along with many precipitates on the cathode surface ( Figure  S2). On the surface of the cathode from Ni group, biomass along with some precipitates were also observed. However, no precipitate was observed on the cathode of the control group. The abundant precipitates on the biocathode of Fe and Ni group were probably caused by deposition of added ferrous and nickel ions during operation. The ferrous ion and nickel ions concentration did drop gradually during the batch operation, and the precipitates were identified as the crystals of iron and nickel contained compounds ( Figure  S2).
The sequencing coverage of each sample reached 99.9%, indicated that the sequencing results were representative ( Table 1). The Ace index and Chao 1 index were the highest in Ni group, and the lowest in the Control, which indicated that the richness of microbial community increased with the addition was higher than that by Ni 2+ addition. Fe 2+ addition and Ni 2+ addition showed different effect on the metatranscriptomics of the microbial community in the MES. The generation of methane was inhibited by the BES, however, the large amounts of the methanobacteria were found in the cultures. The BES is a structural analog of coenzyme M that involves in the final step of methane biosynthesis. The transcriptional expression of upstream genes may still occur, and the growth of methanobacteria may not inhibited by BES. Therefore, the methanogens still existed in the reactors with methanogensis inhibited by BES.

Gene annotation
The distribution of transcripts to general functional categories was assessed based on the BLAST against KEGG database (Fig. 4). KEGG classifies different metabolic pathways from three levels of level 1, level 2, and level 3. Figure 4 is showing the differences of metabolic pathways of different reactors at level 2 and level 3. The ordinates are different metabolic pathways, and the abscissa is the proportion of al. 2017). The low potential of the cathode is beneficial to the survival of H 2 producers, and resulted in the relatively high abundance of Desulfovibrio and Rhodocyclaceae. The relatively high abundance of Desulfovibrio and Rhodocyclaceae might affect the H 2 production capacity of biochatode, and therefore promote the acetate synthesis with H 2 mediated (indirect electron transfer). In the Ni group, the relative abundance of Desulfovibrio did not change significantly, while the relative abundance of Rhodocyclaceae decreased to 6.7%. Meanwhile, the relative abundance of Clostridium_sensu_stricto_1 decreased to 3.9%. Overall, Fe 2+ and Ni 2+ addition had little effect on phylum level, and caused small changes in genus-level composition.

Basic information of metatranscriptomic libraries
Total 152 million clean reads (approximate 20 Gb) were produced from the three group MES reactors based on metatranscriptomic analysis (  Fig. 3. Azonexus, Methanobacterium, and Desulfovibrio were the three genera with the highest transcriptomic expression. Acetogen genera, such as Clostridium and Acetoanaerobium, located in the top 20 compared to that of Control (2.2% ). The high expression of "methane metabolism" was consistent with the predicted high expression genus of "Methanobacterium" (Fig. 3). The expression abundance of "Methane metabolism" in the control group was much higher than that of Fe group and Ni group.
The distribution of transcripts to general functional categories was assessed based on the BLAST against the Egg-NOG database. 'Energy production and conversion' was also one of the top abundant expressed COG functional annotation ( Figure S3). The order of expression abundance of 'Energy production and conversion' in the three groups based on COG annotation was consistent with the order of 'Energy metabolism' based on KEGG annotation, which mutual verified the reliability of the annotations.

GO enrichment analysis of DEGs
Total of 49,212 DEGs between the Fe or Ni group and the control were identified. The volcano maps of the identified DEGs are shown in Figure S4. Compared to the control, 6633 genes up-regulated and 4496 genes down-regulated with ferrous ion addition, and the proportion of significantly various metabolic pathways in the metatranscriptome. The top three transcriptional at level 2 pathways were 'Energy metabolism', 'Global and overview maps', and 'Carbohydrate metabolism'. The relative abundance of 'Energy metabolism' in Control, Fe group, and Ni group were 15.9%, 10.2% and 14.9%, respectively. The results indicated the importance of 'Energy metabolism' function for acetate production of MES, as inferred from the essential intracellular electron transfer that accompanied by energy metabolism. The top 5 expression categories at level 3 pathways in 'Global and overview maps', 'Energy metabolism' and 'Carbohydrate metabolism' were shown in Fig. 4b. The dominant expressed categories at level 3 pathways in 'Global and overview maps' were 'Carbon metabolism', and 'Biosynthesis of amino acids', which are the basic metabolism for the survival and growth of microorganism. In 'Energy metabolism', 'Carbon fixation pathways in prokaryotes' was one of the dominant at level 3 pathways, which was consistent with the performance of the MES reactors for acetate production. 'Carbon fixation pathways in prokaryotes' includes the WL pathway, which is the primary pathway for CO 2 fixation in MES, and the proportion of 'Carbon fixation pathways in prokaryotes' transcriptional expression was altered by the addition of Fe 2+ (1.9%) and Ni 2+ (2.3%), Fig. 3 The top 20 expressed genera based on transcript abundance from the biocathodes group. The common enriched gene categories in Fe and Ni group were peptidase activity, acting on L-amino acid peptides (GO:0070011), peptidase activity (GO:0008233), oxidoreductase activity (GO:0016491), hydrolase activity (GO:0016787), catalytic activity (GO:0003824), anion binding (GO:0043168), ion binding (GO:0043167), molecular function (GO:0003674), purine nucleotide binding (GO:0017076), small molecule binding (GO:0036094), and nucleotide binding (GO:0001882). Thereinto, the oxidoreductase activity and catalytic activity were the catalysis function of an oxidation-reduction in biochemical reactions, which were related to electron transfer. In the Ni group, the enrichment ratio of oxidoreductase activity and catalytic activity were 19.5% and 15.8%, respectively, higher than that of 15.9% and 14.2% in the Fe group. The addition of up-regulated and down regulated genes were 13.5% and 9.1% of the total genes. Meanwhile, 6365 genes up-regulated and 4879 genes down-regulated with nickel ions addition, and the proportion of significantly up-regulated and down regulated genes were 12.9% and 9.9% of the total genes.
The top 25 enriched GO categories with Fe 2+ and Ni 2+ addition were shown in Fig. 5. Go functional classification is mainly divided into three parts, Molecular Function (MF), Biological Process (BP) and Cellular Component (CC). The addition of Fe 2+ and Ni 2+ were a certain enrichment effect on GO function, and Fig. 5 is showing the enrichment of GO categories. The genes were mainly classified in 'molecular function' in both Fe group and Ni group, and the enrichment ratio of Ni group was slightly higher than that of Fe  Figure 6 is showing the impact of Fe 2+ or Ni 2+ addition on the expression of typical energy conversion complex KOs. H 2 ase is an important energy metabolism enzyme, which use electron bifurcation to drive the endergonic ferredoxin reduction by coupling it to the exergonic NAD + reduction (Lohner et al. 2014;Feng et al. 2022;Tai et al. 2021;Schuchmann and Muller 2012). In the Fe group, a large part of H 2 ase KEGG Orthogroups (KOs) met the standard (F DR < 0.05 and |log2FC| ≥ 1), such as K03618 (hydrogenase-1 operon protein HyaF), K03620 (Ni/Fe-hydrogenase 1 B-type cytochrome subunit), K04651 (hydrogenase nickel incorporation protein HypA/HybF), K04654 (hydrogenase expression/formation protein HypD), and K05796 (electron Ni 2+ promoted the enrichment of oxidoreductase activity and catalytic activity in MES, which was conducive to electron transfer and energy conversion.

Expression differences of genes in energy conservation system and Wood-Ljungdahl (WL) pathway
Expression differences of main genes for energy conversion are shown in Fig. 6, with the definition of analyzed KOs shown in Table S2. In the KEGG database, energy conversion-related KOs were discovered and examined. K03612 (H + /Na + -translocating ferredoxin: NAD + oxidoreductase subunit G) in the Ni group.
The KOs of theWL pathway was found in the metatranscriptomic libraries in all samples. The differentially expressed KOs with F DR < 0.05 and |log2FC| ≥ 1 are shown in Fig. 7, with the definition of analyzed KOs shown in Table S3. Figure 7 is showing the impact of Fe 2+ or Ni 2+ addition on the expression of WL pathway KOs. In Fe group, the log2 FC of K05299 (formate dehydrogenase (NADP + ) alpha subunit) and K15023 (5-methyltetrahydrofolate corrinoid/iron sulfur protein methyltransferase) was 2.6 and 3.1. While, in Ni group, the log2 FC of K00198 (anaerobic carbon-monoxide dehydrogenase catalytic subunit), K05299, K15022 (formate dehydrogenase (NADP + ) beta subunit), and K15023 was 1.5, -2.0, 2.1 and 1.4, respectively.

Discussion
In the current study, the effects of Fe 2+ and Ni 2+ ions on the acetate synthesis in MES, and explored the microbial mechanism through metatranscriptomic perspective. In previous studies, acetate production by microbial electrosysthesis with pure cultures have been revealed (Nevin et al. 2010;Bajracharya et al. 2015). However, it is technical difficulty and economically expensive to construct and operate full scale microbial electrosynthesis reactors with pure culture. Open culture is a reasonable and feasible alternative for microbial electrosynthesis reactor operation, which were adopted by most reported studies (Arends et al. 2017;Bajracharya et al. 2017). Therefore, this study investigated the effect of Fe 2+ and Ni 2+ addition on acetate production in MES with open culture. The same with most of previous studies (Ammam et al. 2016;Nimbalkar et al. 2018;Liu et al. 2019), trace element solution (DSMZ-Medium 141) was added in the medium in this study, with a final concentration of 0.2 mg/L and 0.07 mg/L for Fe 2+ and Ni 2+ , respectively. DSMZ-Medium 141 was designed for conventional microbial culture and was not specifically designed for microbial electrosynthesis. The final concentration of Fe 2+ and Ni 2+ at 0.2 mg/L and 0.07 mg/L respectively are supposed to be relatively sufficient for the requirement of conventional microbial culture. However, the reduction potential of the cathode in MES is much lower than that in conventional microbial culture, the sulfate in the medium is easily reduced to sulfide, and thus causing the formation of Fe-S and Ni-S precipitates and the sharp decrease of Fe 2+ and Ni 2+ concentrations. It might result in the insufficiency of Fe 2+ and Ni 2+ in the MES. Therefore, 50 mg/L Fe 2+ and 50 mg/L Ni 2+ was respectively supplemented into the Fe group and Ni group, to investigate the effect of Fe 2+ and Ni 2+ addition on acetate production in MES, and explore transport protein HydN) (Fig. 6a). The log2 FC of K03618, K03620, K04651, K04654, and K05796 in Fe group were 4.6, 2.8, 5.5, 1.6, 1.1, and 2.3, respectively. Moreover, in the Ni group, more H 2 ase KOs met the standard compare with Fe group, such as K00437 (Ni-Fe hydrogenase large subunit), K03618, K04651, K04653 (hydrogenase expression/formation protein HypC), K05586 (bidirectional Ni-Fe hydrogenase diaphorase subunit, hoxE), K05587 (bidirectional Ni-Fe hydrogenase diaphorase subunit, hoxF), K05588 (bidirectional Ni-Fe hydrogenase diaphorase subunit, hoxU), K05922 (quinone-reactive Ni/Fe-hydrogenase large subunit), and K05927 (quinone-reactive Ni/ Fe-hydrogenase small subunit), with log2 FC of 2.8, 2.2, 4.1, 1.1, 3.6, 1.4, 3.0, 5.1, and 4.9, respectively. ATPase is a membrane-bound electron transport system, with sodium pump as the motive force (Mayer and Muller 2014). The expression of K02110 (F-type H + -transporting ATPase subunit c) and K02114 (F-type H + -transporting ATPase subunit epsilon) in ATPase were promoted, and the expression of K01535 (H + -transporting ATPase), K02107 (V/A-type H + /Na + -transporting ATPase subunit G/H), K02108 (F-type H + -transporting ATPase subunit a), K02119 (V/A-type H + /Na + -transporting ATPase subunit C), K02120 (V/A-type H + /Na + -transporting ATPase subunit D), K02121 (V/A-type H + /Na + -transporting ATPase subunit E), K02123 (V/Atype H + /Na + -transporting ATPase subunit I), and K06865 (ATPase) were decreased with ferrous ion addition (Fig. 6b). While in Ni group, the expression of K02108, K02110, K02111 (F-type H + /Na + -transporting ATPase subunit alpha), K02112 (F-type H + /Na + -transporting ATPase subunit beta), K02113 (F-type H + -transporting ATPase subunit delta), K02114, and K02115 (F-type H + -transporting ATPase subunit gamma) were promoted, only K01535 and K02116 (ATP synthase protein I) expression were depressed. NADH oxidoreductase complexes are essential cofactors for metabolism and intercellular electrons transfer in microorganisms. The expression of K00340 (NADH-quinone oxidoreductase subunit K), K00356 (NADH dehydrogenase) and K13378 (NADH-quinone oxidoreductase subunit C/D) in NADH oxidoreductase complex were promoted with ferrous ion addition, while the expression of K00330 (NADH-quinone oxidoreductase subunit A), K00333 (NADH-quinone oxidoreductase subunit D) and K00343 (NADH-quinone oxidoreductase subunit N) were depressed. While, the nickel ions addition promoted the expression of K00338 (NADHquinone oxidoreductase subunit I) and K13378. Rnf complex is important in energy conservation in acetogens Tremblay et al. 2013). The expression of K03613 (H + /Na + -translocating ferredoxin: NAD + oxidoreductase subunit E) in Rnf complex was promoted in the Fe group. While, the nickel ions addition depressed the expression of abundant electroactive microorganisms and acetogens were reported in Proteobacteria and Firmicutes (Mateos et al. 2018;Zhang et al. 2022;Drake et al. 2008), the increased proportion of Proteobacteria and Firmicutes indicated the potential of acetogenesis enhancement in the MES by Fe 2+ addition. In addition, Azonexus, Thauera and Dechloromonas are typical hydrogen producing bacteria (Jourdin et al. 2015), as well as Desulfovibrio and Rhodocyclaceae, which are important for acetate synthesis in the reactors via H 2 mediated indirect electron transfer (Perona-Vico et al. 2020;Xiang et al. 2017). Their relatively high proportion in the microbial community probably indicated the participation of H 2 mediated electron transfer in acetate synthesis in the MES of this study. In addition, though relative abundance was relatively low in the reactors (3.9-9.0%), Clostridium_sensu_stricto_1 might be essential microorganisms for acetate production in the reactors, as they feature WL pathway in a high possibility (Aryal et al. 2017b;Aklujkar et al. 2017). The high expressed microorganisms, such as acetogens and hydrogen-producing microorganisms, indicated their key roles in acetate production in the MES. Though methanogenesis was inhibited in the MES reactors by BES addition, high transcriptional expression of Methanobacterium and "methane metabolism" were found. It was owing to that the BES is a structural analog of coenzyme M, which is a cofactor involving in the terminal step of methane biosynthesis (Liu et al. 2011). Therefore, the generation of methane can be inhibited by the BES, however, the transcriptional expression of upstream genes may still occur, owing to the suitable environmental conditions the underlying mechanism of the effect of Fe 2+ and Ni 2+ addition on acetate production in the MES from the metatranscriptomic perspective. In the Fe and Ni group, the concentration of Fe 2+ and Ni 2+ decreased rapidly from more than 50 mg/L to 2.3 mg/L and 1.1 mg/L respectively, in the first 2 days operation (Fig. S2d), which was owing to the precipitation under the low potential at the cathodes. After 4 days operation, the concentration of Fe 2+ and Ni 2+ was relatively stable at around 1.5 mg/L and 0.2 mg/L, respectively. Though the Fe 2+ and Ni 2+ concentrations have decreased dramatically during operation, the final concentrations of Fe 2+ and Ni 2+ were still much higher than that in the basic medium (Fe 2+ and Ni 2+ concentrations). The improved acetate production and electron recovery efficiency in Fe and Ni group, indicating the significant promotion of electron utilization for acetate synthesis of Fe 2+ and Ni 2+ addition in MES. The acetogenic electron recovery efficiency was the ratio of the electrons required for acetate production by microorganisms to the total electrons, which mean the electron conversion efficiency. The results showed that both Fe 2+ and Ni 2+ addition could promote the flow of electrons to WL pathway and acetate synthesis.
The fewer observed microorganisms on the cathode of the control by SEM might be owing to the more detached biomass during sample preparation for SEM observation, as the precipitates layer on the cathodes in Fe and Ni group might protect the biomass from detach.
Fe group showed the increased relative abundance of Synergistetes (26.5%), Proteobacteria (35.4%) and Firmicutes (16.2%), based on 16 S rRNA sequencing. As of K03618, K04651, and K05588 were promoted with ferrous or nickle ion addition, while the expression of K03620, K04654, and K05796 were promoted only with ferrous ion addition. K03620 was a subunit of periplasmic Ni-hydrogenases, which usually connects to the haem B (Dross et al. 1992). Haem B componens of cytochrome b contain the Rieske FeS protein (Sena et al. 2009), which require ferrous ion for synthesis and activity. K04654 was a subunit of hydrogenases, which combines with K04651 and K04653 to form an iron-nickel hydrogenases. The activity of hydrogenase complex mainly depends on K04651, and the addition of ferrous ion may increase the expression of K04654 . K05796 was also a subunit of hydrogenase and coupled with formate dehydrogenase, which showed similaritie to subunits of formate dehydrogenase . While, the expression of K00437, K04653, K05586, K05587, K05922, K05927, K14086, and K14088 were only promoted in Ni group, which were ironnickel type subunits of hydrogenase. With relatively higher concentration of Fe 2+ (0.2 mg/L) than Ni 2+ (0.07 mg/L) concentration in the basic medium, Ni 2+ addition affected more on the expression of these hydrogenase subunites than Fe 2+ addition. NADH complex could interconvert redox energy and electrochemical energy, and serves to prevent the overreduction of the quinone pool and to provide cellular reducing equivalents (Spero et al. 2015). The WL pathway connected energy currencies by using protons to generate a proton motive force (PMF), which is subsequently used for ATP synthesis. The expression of ATP synthase was largely influenced by proton gradient. The growth and metabolism of microorganisms were actived and consumed a large amount of protons in the Ni group, forming a large proton gradient.
Formate dehydrogenase, Carbon-monoxide dehydrogenase and methyltransferase were the key to energy transfer coupling with WL and typical iron or iron-nickel proteins in the WL pathway (Lemaire and Wagner 2021;Seravalli et al. 1999;Niks and Hille 2019). The coupling of energy conversion and the WL pathway could be enhanced by the addition of ferrous and nickel ions, resulting in a better conversion of energy to acetate, and the electron recovery efficiency of acetate in Fe and Ni group were higher than that in control. However, ferrous ions only have significant effects on K05299 and K15023, which were less than of in Ni group, indicating that the energy conversion and WL pathway coupling were weaker than those in Ni group. Formate dehydrogenase contains tungsten, selenium, iron, and inorganic sulfur, and the expression of formate dehydrogenase was promoted in Fe group (Yamamoto et al. 1983). The formate dehydrogenase is coupled with hydrogenase, and its expression is also affected by hydrogenase (Schuchmann and Müller 2013). Meanwhile, carbon-monoxide dehydrogenase is in the reactors for methane production. As the expression abundance of "Carbon fixation pathways in prokaryotes" in Ni group was slightly higher, and that in Fe group was even slightly lower, than that in the control group, the high expression of "Methane metabolism" in the control group might also be one of the reasons for its low acetate production and low electron recovery.
The GO enrichment analysis of DEGs up-regulated by the addition of Fe 2+ and Ni 2+ was higher than the number of genes down-regulated, though the proportions of upregulated and down-regulated genes were relatively low. A possible explanation for this might be that the metabolic pathway was basically the same in the three group reactors, however different metabolism expression was caused by Fe 2+ and Ni 2+ addition. The top 25 enriched GO categories with Fe 2+ and Ni 2+ addition were indicated that both Fe 2+ and Ni 2+ addition stimulated the gene expression of the Molecular Function (MF), i.e. biochemical activity, in the acetate production MES reactors. The higher enrichment ratio in Ni group was consistent with the higher acetate production in Ni group, which combined indicating the better promotion of acetate synthesis in MES by Ni 2+ addition. In addition, a part of enrichment genes in the Fe group were biological process (BP), indicating that some more electron might be consumed by other metabolic processes in the Fe group.
Nickle ions addition enriched more on the expression of H 2 ase than ferrous ion addition. This discrepancy could be attributed to these H 2 ase were all nickel-iron proteins, and nickel ions may be the main factor affecting the synthesis and activity of these proteins, especially the Fe concentration in the basic medium was slighter higher than Ni. However, the most of ATPase subunits were decreased with ferrous ion addition and the most of ATPase subunits were promoted in the Ni group, indicating that nickel ions addition promoted the expression of F-type H + -transporting ATPase and beneficial to the ATP synthesis. In NADH and RNF complexes, the addition of ferrous and nickel ions had different expression of their subunits. Generally, the expression abundance of above four energy conversion complex in the Ni group were higher than that of the Fe group. The nickel ion addition promoted the expression of H 2 ase, ATPase, and NADH oxidoreductase complex, which may increase the intercellular electron transfer efficiency in microorganisms. However, the improvement of KOs expression of energy conversion in Fe group was relatively low, which was consistent with the lower electron recovery efficiency in Fe group than that in Ni group. The promotion of ferrous ion addition on the expression of H 2 ase, ATPase and NADH oxidoreductase complex were lower than that of nickel ion addition.
The KOs of H 2 ase changed significantly in the process of energy conservation in Fe and Ni group. The expression a typical iron-nickel protein, which contained 10 irons and 1 nickel per monomer, and its expression was up-regulated in Ni group (Ragsdale 2009). Interestingly, the ferrous ions mainly promoted the methyl branch to increase the production of acetate, while the nickel ions promoted the carbonyl branch to increase the production of acetate. The results indicated that hydrogenase is the main mediator of energy transfer for reducing CO 2 to acetate in the MES, and the expression of different hydrogenase subunits were promoted with Fe 2+ or Ni 2+ addition. It achieved the increase of microbial acceptance of electrons and acetate synthesis in both Fe and Ni groups, however, via different pathways, which was methyl branch in Fe group, and carboxyl branch in Ni group.

Conclusion
This study investigated the effects of Fe 2+ and Ni 2+ ions on the acetate synthesis in MES, and explored the microbial mechanism through metatranscriptomic perspective. Both Fe 2+ and Ni 2+ promoted acetate production in MES, while Ni 2+ addition achieved better promotion than Fe 2+ addition. Gene expression of 'Energy metabolism', especially in 'Carbon fixation pathways in prokaryotes' was up-regulated by Fe 2+ and Ni 2+ addition. Both Fe 2+ and Ni 2+ addition up-regulated the expression of hydrogenase, and Ni 2+ addition up-regulated the expression of ATP synthase, in energy-conserving systems. Fe 2+ and Ni 2+ addition respectively enhanced the expression of methyl branch and carboxyl branch of the WL pathway, and thus promoted acetate production. By increasing the expression of genes related to the electron transfer and WL pathway, the addition of Fe 2+ and Ni 2+ could efficiently boost the performance of acetate production. Higher acetate production acieved with Ni 2+ addition, sugessting that Ni 2+ addition is a more efficient measure for the improvement of MES than Fe 2+ addition.