Morphological characteristics of P. stutzeri
The morphology of the isolated strain was characterized by scanning electron microscopy (SEM). SEM (Fig. 1a) showed that the strain adhered to the surface of the electrode. The strain was a short rod without spores, its surface was wrinkled, and the pili was observed (Fig. 1b and 1c). The biofilm was surveyed among carbon fibers (Fig. 1d).
16S rRNA molecular identification and general features description
The isolated strain was identified and confirmed by 16S rRNA gene sequencing. Sequence alignment were performed using BLAST. Sequences with similarities greater than 99% were selected to construct phylogenetic trees using MEGA 7.0. The sequence data of P. stutzeri S116 are publicly available in the NCBI database (GenBank accession number MZ220459). The phylogenetic tree showed that the 16S rRNA gene sequence of S116 has 99% homology with Pseudomonas stutzeri IHBB 9574 and Pseudomonas stutzeri (shown in Figure S1). The phylogeny data (including alignments) are available in the Treebase repository (http://purl.org/phylo/treebase/phylows/study/TB2:S28911?x-access-code=52f486836b34e93c0c34658911e7e960&format=html). The general features of S116 was described in Table S1. The strain was deposited in China General Microbiological Culture Collection Center (CGMCC) with the deposit number CGMCC 1.19374.
Electrochemical property of the bioanode and biocathode
Thirty milliliters of the medium was replaced by fresh solution when the output voltage of the MFC decreased to approximately 50 mV. After the MFC was operated for 30 h, the Reactor 2 reached the stable generation voltage in the first cycle with peak voltage at 170.5 mV, and for 80 h the Reactor 1 reached the highest peak voltage at 254.2 mV. During the second and third cycles, the Reactor 1 and 2 reached the highest output voltages of 228.3 mV and 225.5 mV, respectively. It took less than 10 hours for the Reactor 1 to produce an output voltage from the lowest voltage to the highest voltage (Fig. 2a).
To investigate the electrochemical activity of electrogenic microorganism in MFCs, CV analysis of bioanode and biocathode were performed (shown in Fig. 2b and c). The bioelectrode compared with the bare carbon cloth electrode possessed distinct redox peaks in the CV spectra, which indicated that the electrocatalytic activity of P. stutzeri S116 was associated with the electrode. The bioanode exhibited two distinct reduction peaks (-0.92 mA at -0.504 V, -0.845 mA at -0.665 V) and the highest oxidative peak current of 0.595 mA at 0.308 V. The biocathode exhibited three distinct oxidation peaks (-0.13 mA at -0.488 V, 0.246 mA at -0.354 V, 0.105 mA at -0.24 V) and reduction peaks (-1.1 mA at -0.795 V, -0.743 mA at -0.62 V, -0.584 mA at -0.52 V). Simultaneously, for bare bioanode, no distinct redox reaction was measured. The position of the redox peak reflects the redox potential of components involved in ETT (Feng et al. 2010). In addition, the size of the redox peak represents the electrochemical activity of P. stutzeri.
Polarization and power density curves of the MFCs were tested during the third cycle when the Reactors generated voltage at the highest point (shown in Fig. 3d). The obtained maximum power was 765 mW/m2 (Reactor 1) and 656.6 mW/m2 (Reactor 2), respectively.
The interaction between the electrogenic microbe and the electrodes in MFCs was analyzed by EIS. The Nyquist plot (Figure S2) indicated that the bioelectrodes had a similar semicircle diameter, the Rct values in the MFCs were approximately 11.8 Ω (cathode) and 17.0 Ω (anode), respectively, which represented a low charge-transfer resistance (Rct) and rapid electron transfer.
Genomic features of P. stutzeri S116
The filtered subreads of the P. stutzeri S116 genome were assembled and rectified into a scaffold length of 4,756,665 bp with a GC content of 63.47%. Gene prediction indicated a total gene length of 4,224,096 bp with 4402 CDSs. Noncoding RNA prediction showed that the numbers of rRNAs, tRNAs and ncRNAs were 15, 63, and 76, respectively. Gene annotation in general databases is described as follows: eggNOG (COG) 3842, GO 3371, KEGG 2493, NR 4385, SwissProt 2805. Moreover, CAZy (121), TCDB (1343), and VFDB (887) were annotated in special databases (Fig. 3). Transmembrane protein and secreted protein prediction indicated that the genes were 1111 and 470, respectively. The genome sequence are publicly available in the NCBI database (BioProject accession PRJNA743140, https://www.ncbi.nlm.nih.gov/bioproject/743140/).
Gene function analysis
The protein sequences of genes were aligned against Nr database by BLAST, species distribution was exhibited in Figure S3. 3744 genes are responsible for Pseudomonas stutzeri withthe highest proportion (85.38%).
In the COG categories, energy production and conversion (268 genes), amino acid transport and metabolism (267 genes), and inorganic ion transport and metabolism (265 genes) had higher abundances, with proportions of 6.84%, 6.82%, and 6.77%, respectively (shown in Fig. 4). To detect the potential roles of P. stutzeri, specific COGs involved in bioelectricity generation were analyzed. Forenergy production and conversion, dehydrogenase (COG0508, COG1012, COG1052, COG1063, COG1071, COG1319, NOG00108, NOG02207), cytochrome c (COG3258, COG2010, COG3909, NOG62129, NOG18013) and electron transport complex (COG2878, COG4657, COG4658, COG4659, COG4660) were the three most abundant gene function class, which are all involved in electron transport (Logan et al., 2010). Simultaneously, the important components of the respiratory chain, such as complex I (NOG31185, NOG34255), Fe-S protein (COG2975, COG3313), NADH dehydrogenase (COG1252), succinate dehydrogenase (COG0479, COG1053), cytochrome b561 (COG3038), and complex III (COG0723, COG1290), were annotated in COGs. Moreover, cytochrome c oxidase (COG2993, COG4736), playing an important component of complex IV, had been annotated, which reduces oxygen to water as the terminal electron acceptor in the respiratory chain (Cai et al., 2020). For amino acid transport and metabolism function, ABC transporter and aminotransferase were relatively higher abundant. With respect to inorganic ion transport and metabolism, ABC transporter, binding-protein-dependent transport systems inner membrane component were two most abundant function.
Genes of P. stutzeri were categorized by GO into three functional nodes to determine the biological relevance of the strain: (1) cellular component, which is used to describe subcellular structure, location, and macromolecular complexes; (2) molecular function describes the function of a gene or gene product; (3) biological process describes biological processes of the encoded gene products. Among the three GO categories, biological process was the most abundant (Fig. 5).
In the biological process category, genes involved in metabolic processes (1737 genes) made up the highest proportion (51.5%) of the total genes (3372 genes), cellular process (1497 genes) was 44.4%, single-organism process possessed 38.9% proportion with1312 genes, and localization (536 genes; 15.9%). In the molecular function category, most genes of 1918 were involved in catalytic activity, with a proportion of 56.9%, and in binding, with a proportion of 43.5% (1467 genes). In the cellular component category, 1140 genes was involved in membrane with the highest proportion of 33.8%, membrane part (1040) 30.8%, cell (1030) 30.5%, and cell part (1007) 29.9%.
For P. stutzeri S116, the five most abundant genes were annotated in the VFDB (Figure S4), including type IV pili (61 genes), capsule (49 genes), flagella (44 genes), pyoverdine (38 genes) and polar flagella (37 genes). Pili, as the conductive appendages distributed on the surface of bacteria, can transfer electrons directly to the anode, the conductive pilus of electrogenic microorganisms is one of the important mechanisms of EET (Reguera et al. 2005; Lovley et al. 2006). In addition, pyoverdine contributes to the survival of microbes in nutrient-deficient soil (Ignacio et al. 2018).
Critical metabolic pathways
Genes were annotated against the KEGG databases to investigate the critical metabolic pathways involved in bioelectricity generation and bioelectrode catalysis in MFCs. For P. stutzeri S116, energy metabolism and a two-component system are the two essential functions in KEGG annotations (shown in Figure S5).
The respiratory chain on the membrane of P. stutzeri S116 is an important pathway for electron transport and energy production. Oxidative phosphorylation (ko00190, 36 genes) indicated that the strain S116 possessed an integrated electron transport chain. The critical enzymes including succinate dehydrogenase (EC:126.96.36.199), ubiquinol-cytochrome c reductase (EC:188.8.131.52) and cbb3-type cytochrome c oxidase (EC:184.108.40.206, cytochrome aa3) were detected, which are all annotated in COGs. In complex II, sdhC (K00241), sdhD (K00242), sdhA (K00239) and sdhB (K00240) encode cytochrome b, membrane anchor subunit, iron-sulfur subunit and flavoprotein subunit, respectively, where succinate is dehydrogenized into fumarate. Complex III primarily contains ubiquinol-cytochrome c reductase iron-sulfur subunit (EC:220.127.116.11), ubiquinol-cytochrome c reductase cytochrome b subunit (K00412) and ubiquinol-cytochrome c reductase cytochrome c1 subunit (K00413). The electrons are transported from complex III to cytochrome c oxidase (complex IV) via cytochrome c, where oxygen is reduced into H2O and energy is generated. Nevertheless, annotated type 2 NADH dehydrogenase (K03885, EC:18.104.22.168) is involved in regulation rather than respiration (Howitt et al. 1999). Therefore, electron transport in P. stutzeri S116 forms a succinate pathway with high probability (Figure S6).
Generally, there are two oxidation pathways from thiosulfate to SO42- or S4O62- for SOB: (1) S2O32- is oxidized to SO42- by the Sox multienzyme complex (Fiedrich et al. 2005). (2) Thiosulfate dehydrogenase (EC:22.214.171.124, tsdA) catalyzes S2O32- to S4O62- (Brito et al. 2015). Simultaneously, the pathway of sulfur metabolism (ko00920, 34 genes) indicates that thiosulfate is catalyzed by thiosulfate sulfurtransferase (EC:126.96.36.199) into sulfite. Moreover, the sqr gene encoding sulfide:quinone oxidoreductase (EC:188.8.131.52) was detected, which can oxidate H2S into S0.
Riboflavin can freely shuttle cell membranes and capture electrons from the respiratory chain, which plays an important role in EET. Riboflavin metabolism (ko00740 8 genes) for P. stutzeri indicates that ribulose 5-phosphate is metabolized into riboflavin. In addtion, riboflavin, as a redox active compound, is secreted by many bacteria (Abbas et al. 2011). COG0307 and COG0196 encoding riboflavin synthase and riboflavin kinase are annotated in COG, which are essential enzymes related to the biosynthesis of riboflavin.
Pilus are generally detected in gram-negative bacteria and closely related to bacterial activity, biofilm formation, surface adhesion, DNA acquisition and signal transduction (Reardon et al. 2013). Genes encoding type IV pilus-assembly proteins, such as pilB, pilC, pilE, pilW, pilZ, pilV, pilO, pilM, pilN, pilQ, pilY, pilV and pilP, were detected in the COG and KEGG databases. Two-component system (ko02020, 153 genes) for P. stutzeri involved in chemotaxis primarily includes twitching motility proteins encoded by genes such as pilG, pilH, pilI, pilJ, and pilK. Moreover, the redox signal is transmitted by the annotated critical sensor histidine kinase (EC:184.108.40.206, K15011) into an electron transfer system and aerobic respiration. Simultaneously, the system indicated that nitrate and nitrite were phosphorylated and transported to nitrate reductase, and finally entered the nitrogen metabolism pathway (ko00910, 36 genes). The predicted metabolic pathways in P. stutzeri were shown in Fig. 6.