Complete Genome Sequence of Pseudomonas Stutzeri S116 Provides Insights into the Mechanism of Microbial Fuel Cells


 To identify suitable biocatalysts applied in microbial fuel cells (MFCs), Pseudomonas stutzeri S116 isolated from marine sludge was investigated, which possessed excellcent bioelectricity generation ability (BGA). Herein, P. stutzeri as a bioanode and biocathode achieved maximum output voltage (254.2 mV and 226.0 mV), and power density of (765 mW/m2 and 656.6 mW/m2). Complete genome sequencing of P. stutzeri was performed to reveal its potential microbial functions. The results exhibited that the strain was the ecologically dominant Pseudomonas, and its primary annotations were associated with energy production and conversion (6.84%), amino acid transport and metabolism (6.82%) and inorganic ion transport and metabolism (6.77%). The thirty-six genes involved in oxidative phosphorylation indicate that strain possesses an integrated electron transport chain. Moreover, many genes encoding redox mediators (mainly riboflavin and phenazine) were detected in the databases. Simultaneously, thiosulfate oxidization and dissimilatory nitrate reduction were annotated in the sulfur metabolism and nitrogen metabolism pathway. Gene function and cyclic voltammetry (CV) analysis indicated BGA of P. stutzeri probably was attributed to its cytochrome c and redox mediators, which enhance extracellular electron transfer (EET) rate.


Introduction
Sulfur-oxidizing bacteria (SOB) can oxidize sulfur compounds as energy sources and utilize inorganic carbon (CO 2 ) for their growth (Kelly et al. 1997). Therefore, they play an important role in environmental remediation, which can remove pollutants containing reduced sul de and x CO 2 (Pokorna et al. 2015). Generally, the mechanism of electricity generation in MFCs is as follows: Electroactive microorganisms in the anodic chamber oxidize substrates (pollutants or organic substances) to generate electrons, subsequently, the electrons are carried by an external electric circuit to a cathode, nally, current ows and electrical energy generates (Chadwick et (Park et al. 1999).
Based on the striking bifunctional biocatalysis of P. stutzeri S116, CV and electrochemical impedance spectroscopy (EIS) method were performed to investigate its electrochemical activity, complete genome sequencing was used to determine its genetic functions, which partly interpret the mechanism of bioanodic and biocathodic catalysis in MFCs.

MFC performance analysis
The output voltage of the MFC was recorded by a data acquisition system. Polarization curves and power density curves were calculated by Ohm's law, which was obtained by changing external resistors. Ohm's law was described as follows: I (A/m 2 ) =U/(RA) and P (W/m 2 ) =U 2 /(RA), where I is the current density, R is the resistance, P is the power density, U is the voltage, and A is the area of the cathode (Wu et al. 2016).
Cyclic voltammetry (CV) measurement of electrodes were operated by the three-electrode system using an electrochemical workstation (Bio-Logic, SP-300, France). The carbon cloth, platinum electrode and saturated calomel electrode were used as the working, reference and counter electrodes, respectively. CV was performed at a scanning speed of 50 mV/s from -1 to 1.0 V in Reactor 1 (-1 to 0.2 V in Reactor 2). EIS was carried out at a sinusoidal perturbation amplitude of 5 mV in a frequency range from 100 kHz to 5 mHz.
Identi cation of bacterial species The puri ed strain was identi ed using 16S rRNA gene sequencing. The DNA was extracted by a bacterial genome DNA extraction kit (Ezup, Sangon Biotech, Shanghai), and the 16S rRNA gene was ampli ed by PCR (2720 thermal cycler, Applied Biosystems) with universal primers (7F: 5'-CAGAGTTTGATCCTGGCT-3', 1540R: 5'-AGGAGGTGATCCAGCCGCA -3'). The loop condition of PCR was as follows: predenaturation for 4 min at 94 °C, 30 cycles of denaturation at 94 °C for 45 s, annealing at 55 °C for 45 s, elongation at 72 °C for 60 s, repair extension at 72 °C for 8 min, and termination reaction at 4 °C. PCR products were puri ed using 1% agarose gel electrophoresis and subjected to Sanger sequencing (Sangon Biotech (Shanghai) Co., Ltd.). The sequencing results were aligned using BLAST, and phylogenetic trees were constructed by MEGA (MEGA version 7.0) to analyze the obtained gene sequences (Herbold et al. 2014).
Complete genome sequence and functional annotation of P. stutzeri High-quality genomic DNA of P. stutzeri was extracted using a QIAGEN Genomic tip (Biomarker Technologies Co., Ltd.). The concentration and purity of DNA were detected using a NanoDrop and Qubit (Thermo Scienti c, USA), and large segments were ltered using the BluePippin system (Sage Science, USA). A library was prepared using the large segments DNA, Oxford Nanopore Technologies (ONT) Template prep kit (SQK-LSK109) and NEB Next FFPE DNA Repair Mix kit. The high-quality library was sequenced on the ONT PromethION platform, and the raw sequencing data were obtained.
For genome assembly, quality control of the sequencing data was performed by Guppy3.2.6 software to lter low-quality fragments of the reads. The obtained subreads were assembled using Canu v1.5/ wtdbg v2.

Results
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 bio lm was surveyed among carbon bers (Fig. 1d).
16S rRNA molecular identi cation and general features description The isolated strain was identi ed and con rmed 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.  (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 re ects 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/m 2 (Reactor 1) and 656.6 mW/m 2 (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.

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%).
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 ve most abundant genes were annotated in the VFDB (Figure S4), including type IV pili (61 genes), capsule (49 genes), agella (44 genes), pyoverdine (38 genes) and polar agella (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-de cient 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).
Generally, there are two oxidation pathways from thiosulfate to SO 4 2- Ribo avin can freely shuttle cell membranes and capture electrons from the respiratory chain, which plays an important role in EET. Ribo avin metabolism (ko00740 8 genes) for P. stutzeri indicates that ribulose 5-phosphate is metabolized into ribo avin. In addtion, ribo avin, as a redox active compound, is secreted by many bacteria (Abbas et al. 2011). COG0307 and COG0196 encoding ribo avin synthase and ribo avin kinase are annotated in COG, which are essential enzymes related to the biosynthesis of ribo avin.
Pilus are generally detected in gram-negative bacteria and closely related to bacterial activity, bio lm 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:2.7.13.3, 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 nally entered the nitrogen metabolism pathway (ko00910, 36 genes). The predicted metabolic pathways in P. stutzeri were shown in Fig. 6.

Discussion
Pseudomonas is a typical electrogenic microorganism applied in bioanode MFCs (Arkatkar et al. 2021). However, for biocathode MFCs, few studies have described it comprehensively and completely. Here, the strain S116 exhibited excellent performance as a biocathode catalyst. The low Rct (11.8 Ω) values of biocathode indicates that S116 is a highly e cient catalyst between the bio lm and the cathode electrode. Moreover, the cost of biocathode MFCs are distinctly lower than abiotic MFCs (such as transition metal elements, Pt-coated metals, and ferricyanide). Simultaneously, biocathodes can improve MFCs sustainability due to consumption of electron mediator is solved (Bergel et al. 2005). In a word, P. stutzeri S116 is a promising electrogenic microorganism possessing bifunctional catalysis applied in MFCs.
Many genes encoding cytochrome c annotated for COG function analysis, which can form a complex extracellular electron transport network and realize the transmembrane transport of electrons (Li et al. 2018;Shelobolina et al. 2007;Orellana et al. 2013). Simultaneously, type IV pilus as "Nanowires" were detected. However, the truncated pilus protein (pilA encoding) was not founded in databases, which is closely related to the pilus with highly electrical conductivity (Campos et al. 2013). Redox mediators such as ribo avin can intercept electrons from the respiratory chain, and transfer them outside the cell membrane (Lovely 2012). Ribo avin metabolism pathway indicated 8 critical genes involved in ribo avin synthesis. The critical ribo avin synthase (EC:2.5.1.9) in the reaction process is detected, which catalyzes the last step of ribo avin biosynthesis in microorganisms. Subsequently, ribo avin is synthesized into dimethyl-benzimidazole (entering porphyrin and chlorophyll metabolism) or FAD. In the VFDB, six genes (phzF1, phzC1, phzG1, phzH, phzE1 and phzD1) are responsible for phenazine biosynthesis. Phenazine secreted by P. aeruginosa is a heterocyclic compound containing nitrogen, which plays an important role in EET as a physiological electron transfer mediator of electricigens (Zee et al. 2009). Herein, the CV curve indicated that the de nite reduction and oxidation peaks were detected in the range of -0.7 V~ 0 V (vs. Ag/AgCl electrode), which approaches the redox potential of phenazine and ribo avin (Zhang et al. 2011 The study provided a promising bifunctional biocatalyst appiled in MFCs. Complete genome sequence of Pseudomonas stutzeri S116 and CV data represent the redox mediators secreted by P. stutzeri S116 were probably responsible for performance of MFCs. The critical genes and metabolic pathways involved in thiosulfate oxide and nitrate reduction were detected, which indicated that the strain can e ciently treat wastewater containing sul de and nitrite.   Schematic of the complete genome of P. stutzeri S116 isolated from marine activated sludge samples. The rst circle (outermost) indicates genomic numbers, with each tick representing 5 kb; genes on forward and reverse chains with different colors based on COG categories are represented at the second and third circles; repetitive sequences (fourth circle); tRNA with bule and rRNA with purple ( fth circle); GC skew (sixth circle). The light yellow region indicates that the GC content is higher than the average in the genome; nevertheless, the bule region represents the opposite. The dark gray region represents G content greater than C, and the red region represents C content greater than G.

Figure 4
Functional categories of P. stutzeri S116 annotated by clusters of orthologous groups of proteins (COGs). GO Classi cation for P. stutzeri S116 isolated from marine activated sludge. The chart shows the enriched genes with secondary-level functions in all genes against GO.

Figure 6
Metabolic pathways for P. stutzeri S116 involved the electron respiratory chain, nitrate reduction pathway, thiosulfate oxidation pathway, ribo avin metabolism and the predicted EET pathway between the electronic mediators and the electrodes.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Supplementarymaterials.pdf