A genetic engineering strategy to enhance outer membrane vesicle-mediated extracellular electron transfer of Geobacter sulfurreducens.

Bioelectrochemical systems (BESs) are unique devices that harness the metabolic activity of electroactive microorganisms (EAMs) to convert chemical energy stored in organic substrates into electrical energy. Enhancing electron transfer efficiency between EAMs and electrodes is the key to practical implementation of BESs. Considering the role of outer membrane vesicles (OMVs) in mediating electron transfer of EAMs, a genetic engineering strategy to achieve OMVs overproduction was explored to enhance electron transfer efficiency and the underlying mechanisms were investigated. This study constructed a mutant strain of Geobacter sulfurreducens that lacked the ompA gene encoding an outer membrane protein. Experimental results showed that the mutant strain produced more OMVs and possessed higher electron transfer efficiency in Fe(III) reduction, dye degradation and current generation in BESs than the wild-type strain. More cargoes such as c-type cytochromes, functional proteins, eDNA, polysaccharides and signaling molecules that might be favorable for electron transfer and biofilm formation were found in OMVs produced by ompA-deficient anodic biofilm, which possibly contributed to the improved electron transfer efficiency of ompA-deficient biofilm. The results indicate that overproduction of OMVs in EAMs might be a potential strategy to enhance BESs performance.

Sub-MIC antibiotics affect microbial ferrihydrite reduction by extracellular membrane vesicles.

Environmental concentrations of antibiotics, usually below MIC, have significant biological effects on bacterial cells. Sub-MIC antibiotics exposure induces bacteria to produce outer membrane vesicles (OMVs). Recently, OMVs is discovered as a novel pathway for dissimilatory iron reducing bacteria (DIRB) to mediate extracellular electron transfer (EET). Whether and how the antibiotic-induced OMVs modulate iron oxides reduction by DIRB have not been studied. This study showed the sub-MIC antibiotics (ampicillin or ciprofloxacin) increased OMVs secretion in Geobacter sulfurreducens, and the antibiotic-induced OMVs contained more redox active cytochromes facilitating iron oxides reduction, especially for the ciprofloxacin-induced OMVs. Deduced from a combination of electron microscopy and proteomic analysis, the influence of ciprofloxacin on SOS response triggered prophage induction and led to the formation of outer-inner membrane vesicles (OIMVs) in, which was a first report in Geobacter species. While ampicillin disrupting cell membrane integrity resulted in more formation of classic OMVs from outer membrane blebbing. The results indicated that the different structure and composition of vesicles were responsible for the antibiotic-dependent regulation on iron oxides reduction. This newly identified regulation on EET-mediated redox reactions by sub-MIC antibiotics expands our knowledge about the impact of antibiotics on microbial processes or "non-target" organisms.

Accelerating the start-up of the cathodic biofilm by adding acyl-homoserine lactone signaling molecules.

Electroactive biofilms (EABs) are essential for bioelectrochemical systems, however, the formation of cathodic EABs is more time-consuming than anodic EABs. This study aims to evaluate whether acyl-homoserine lactones (AHLs) could facilitate the start-up of cathodic Geobacter soli EABs. With AHL addition, the biomass, cell viability, and extracellular polymeric substance (EPS) abundance of cathode-associated G. soli EABs were increased. Likewise, redox activities of EPS and outermost proteins in the cathodic EABs were enhanced in the presence of AHLs, which consequently led to better start-up performance of biofilms. Compared to the control without AHLs, start-up lag periods were reduced by approximately 50%, electron uptakes were enhanced by 1.3-2.0 times, and denitrification rates were more than doubled with AHL addition in the start-up cycle, which were comparable to those of mature G. soli cathodic EABs. These findings open up an opportunity for accelerating the start-up of cathodic biofilms via AHLs.

Enhanced electrosynthesis performance of Moorella thermoautotrophica by improving cell permeability.

Microbial electrosynthesis systems (MES) are promising devices in which microbes obtain electrons from electrodes to produce extracellular multicarbon compounds. This study investigates whether improvement in cell permeability can enhance electrosynthesis performance of Gram-positive Moorella thermoautotrophica in MES. Results showed that when ≤30mg/L penicillin was added, the cell permeability was doubled, and the electron uptake per biomass (including both cathode-associated biomass and suspended biomass) was 1.84 times that of the control, while formate and acetate production rates per biomass were 1.96 and 2.23 times those of the control, respectively. Enhanced cell permeability caused higher redox activities of outmost cytochrome C and increased release of redox electron shuttles, both of which were beneficial to extracellular electron uptake. Coulombic efficiencies increased from 73%±3% to 88%±3% with better cell permeability, demonstrating that higher proportion of electrical energy recovered in the chemical-production reaction. This research demonstrates that making a moderate decrease in peptidoglycan of cell walls to improve cell permeability can enhance electron uptakes and chemical production rates of Gram-positive microbes in MES, which would serve as a base for the future genetic modification study of superior electrosynthesis strains.

Quorum sensing signals enhance the electrochemical activity and energy recovery of mixed-culture electroactive biofilms.

The impacts of exogenous or endogenous quorum sensing (QS) signaling molecules on mixed-culture electroactive biofilms (EABs), especially extracellular polymeric substances (EPS) and exoelectrogens using direct electron transfer mechanism inside EABs are poorly understood. This research focuses on the influence of acylhomoserine lactones (AHLs), the most common QS signaling molecules for gram negative bacteria, on mixed-culture EABs. Results indicated that both exogenous and endogenous AHLs played the role as regulators to improve the electrochemical activities of EABs. The energy recovery of MFCs increased from 20.5% ± 3.9% to 28.3% ± 4.1% with endogenous AHLs and further rose to 36.2% ± 5.1% with exogenous AHLs, and the start-up period of MFCs shortened from 13 days to 10 days with endogenous AHLs and further reduced to 4 days in the presence of exogenous AHLs. The influences of exogenous and endogenous AHLs were non-instantaneous. They improved some intrinsic properties, i.e. the electrode-associated biomass, the biofilm compactness and the ratio of live/dead cells to obtain superior EABs. Meanwhile, both endogenous and exogenous AHLs increased the concentration and redox activities of EPS. Besides, endogenous AHLs enhanced the diversity of EPS components. Noteworthily, the relative abundance of Geoboacter sp. which is the typical microbe using direct electron transfer mechanism is raised by exogenous AHLs, though so far neither known chemical QS-related gene nor protein has been reported in this genus. These findings will increase the current understanding of QS in EABs and open up an opportunity for regulating mixed-culture MFCs via QS.