Engineering Shewanella carassii, a newly isolated exoelectrogen from activated sludge, to enhance methyl orange degradation and bioelectricity harvest

Abstract

Electroactive microorganisms (EAMs) play important roles in biogeochemical redox processes and have been of great interest in the fields of energy recovery, waste treatment, and environmental remediation. However, the currently identified EAMs are difficult to be widely used in complex and diverse environments, due to the existence of poor electron transfer capability, weak environmental adaptability, and difficulty with engineering modifications, etc. Therefore, rapid and efficient screening of high performance EAMs from environments is an effective strategy to facilitate applications of microbial fuel cells (MFCs). In this study, to achieve efficient degradation of methyl orange (MO) by MFC and electricity harvest, a more efficient exoelectrogen Shewanella carassii-D₅ that belongs to Shewanella spp. was first isolated from activated sludge by WO₃ nanocluster probe technique. Physiological properties experiments confirmed that S. carassii-D₅ is a Gram-negative strain with rounded colonies and smooth, slightly reddish surface, which could survive in media containing lactate at 30 °C. Moreover, we found that S. carassii-D₅ exhibited remarkable MO degradation ability, which could degrade 66% of MO within 72 h, 1.7 times higher than that of Shewanella oneidensis MR-1. Electrochemical measurements showed that MFCs inoculated with S. carassii-D₅ could generate a maximum power density of 704.6 mW/m², which was 5.6 times higher than that of S. oneidensis MR-1. Further investigation of the extracellular electron transfer (EET) mechanism found that S. carassii-D₅ strain had high level of c-type cytochromes and strong biofilm formation ability compared with S. oneidensis MR-1, thus facilitating direct EET. Therefore, to enhance indirect electron transfer and MO degradation capacity, a synthetic gene cluster ribADEHC encoding riboflavin synthesis pathway from Bacillus subtilis was heterologously expressed in S. carassii-D₅, increasing riboflavin yield from 1.9 to 9.0 mg/g DCW with 1286.3 mW/m² power density output in lactate fed-MFCs. Furthermore, results showed that the high EET rate endowed a faster degradation efficient of MO from 66% to 86% with a maximum power density of 192.3 mW/m², which was 1.3 and 1.6 times higher than that of S. carassii-D₅, respectively. Our research suggests that screening and engineering high-efficient EAMs from sludge is a feasible strategy in treating organic pollutants.

Keywords: Methyl orange; Microbial fuel cells; Riboflavin; Shewanella carassii; WO3 nanocluster probe.

Graphical abstract

Fig. 1

Phylogenetic tree based on the results of a neighbor-joining analysis of 16S rRNA sequences for the strain Shewanella carassii-D₅ and various members of Shewanella spp. Numbers at nodes indicate bootstrap values > 50% (expressed as percentages of 1000 replications).

Fig. 2

Characterization of biological properties of S. carassii-D₅. (A) Colony morphology photo of the bacteria inoculated on LB agar plate. (B) SEM characterization of the isolated strain. (C) TEM characterization of the bacteria. (D) OD₆₀₀ of inoculated with the bacteria adding acetate, lactate, glycerol, fructose, sucrose, xylose, galactose, and glucose as substrates at concentration ranging from 5 to 20 mM. (E) Optimization of optimum growth temperature of S. carassii-D₅ with 20 mM lactate as the sole carbon source. (F) Comparison of MO degradation ability between S. carassii-D₅ and S. oneidensis MR-1. Three biological replicates were performed. (∗∗: p < 0.01; ∗: p < 0.05).

Fig. 3

Electrochemical characterization of S. carassii-D_5. (A) LSV and the polarization curves and (B) CV curves of S. carassii-D₅ (OD₆₀₀ = 1.0) inoculated in MFCs with lactate as electron donor. (C) Cytochrome level measurement in the LB fermentation broth at OD₆₀₀ = 1.0. (D) The attached biofilm and colony count on anode carbon cloth in MFCs with lactate as electron donor. (E) Water contact angle observation and the affinity of the cells for n-hexadecane. (F) Riboflavin production in the LB fermentation broth at OD₆₀₀ = 1.0. Three biological replicates were performed. (∗∗: p < 0.01; ∗: p < 0.05; ns: no significance; p > 0.05).

Fig. 4

Enhanced production of riboflavin by engineering S. carassii-D₅via synthetic biology approach. (A) Schematic plasmid map of P_tac promoter combined with RibA, RibD, RibE, RibH, and RibC genes expressing vectors. The plasmid was assembled into S. carassii-D₅ to construct recombinant strain S. carassii-D₅-C₅ for the enhanced riboflavin metabolism. (B) The appropriate concentrations of IPTG and kana for growth metabolism of engineered strain. (C) Growth curves of the S. carassii-D₅-C₅ and S. carassii-D₅ in LB fermentation broth with final optimal concentration of 0.2 mM IPTG and 20 mg/L kana at 30 °C for 32 h. (D) Concentration of riboflavin produced by recombinant strain S. carassii-D₅-C₅ and the control strain S. carassii-D₅ in the broth. (E) LSV and the polarization curves and (F) CV curves of recombinant strain S. carassii-D₅-C₅ and the control strain S. carassii-D₅ (OD₆₀₀ = 1.0) inoculated in MFCs with lactate as electron donor. Three biological replicates were performed. (∗∗: p < 0.01; ∗: p < 0.05).

Fig. 5

MO reduction by the recombinant strain S. carassii-D₅-C₅ and S. carassii-D₅. (A) Structural design of MFC for simultaneous MO decolorization and bioelectricity generation. (B) Voltage output obtained from the MFCs operated for one discharge cycle using MO solution as electron acceptor in cathode (pH = 3, initial MO concentration was 50 mg/L). (C) LSV and the polarization curves of the MFCs. (D) The UV–Vis spectra of MO solution in cathode of MFCs inculcated with the recombinant strain S. carassii-D₅-C₅ under illumination at different reaction times. (E) Characterization of MO degradability in cathode. (F) Anaerobic reduction and reduction kinetic curves of MO. Three biological replicates were performed.