|Other Abstract||Electrochemically active bacteria (EAB) are capable of transferring electrons generated by intracellular organic matter oxidation, to extracellular electron acceptors by conductive pills (e-pili) and the cytochrome c on the outmost membranes, this process is defined as microbial extracellular electron transport (EET). Microbial EET is a new manner of anaerobic energy metabolism, which has broad applied prospects in environmental restorations and clean energy generation. In recent years, the EAB presenting extracellular electron transfer capability have been widely reported in the intestines of human beings and other mammals. EAB play an important role in maintaining the gut microbiota homeostasis and health of hosts. Urechis unicinctus is a marine organism of national geographical possessing dozens of nutrients and high research values. However, the existence and applied potentials of EAB in the gut of Urechis unicinctus has not been reported. Here, Urechis unicinctus was selected as experimental samples. Subsequently, the EAB in the intestine was screened and isolated. In addition, the basic physiological and electrochemical activities of the EAB was depicted. Furthermore, the degradation capacity of chloramphenicol by the intestine EAB was investigated. This study underlights the essential functions of EAB in the intestine gut of Urechis unicinctus and provides an insight into the interactions between EAB and hosts.
(1) Isolation and identification of gut microbes from Urechis unicinctus. The intestinal enrichments were obtained by dissecting fresh Urechis unicinctus under anaerobic conditions. Five single colonies with distinct differences in morphology, size, color and transparency were then successfully isolated from the intestinal enrichment by dilution coating and plate streaking. 16S rRNA sequence alignment and phylogenetic tree analysis show that the 5 isolates were from 4 different genera, including Shewanella, Staphylococcus (2 strains), Lysinibacillus, and Bacillus. Moreover, the environmental distribution, electrochemical activity, and applied potential of the 5 species were analyzed. Shewanella marisflavi and Lysinibacillus macrolides showing the closest relationship with the 5 isolated intestinal bacteria, had been proved to possess EET ability. Shewanella is used as a model genus to study EAB, therefore, in this study Shewanella marisflavi UU-3-2 was chosen as a key species in the following experimental processes.
(2) Electrochemical and physiological characters of Shewanella marisflavi UU-3-2 isolated from Urechis unicinctus. The basic physiological and electrochemical activity of UU-3-2 were tested. Firstly, the isolate is gram-negative identified by gram staining, and then the morphology was depicted by scanning electron microscope (SEM), The species is a short rod, about 2 and 0.5μm in length and width, respectively accompanied by flagella on one of its ends. Secondly, high performance liquid chromatography (HPLC) was used to analyze the metabolites, the results show that, sodium acetate and succinic acid were the terminal metabolic products as sodium lactate and fumaric acid acting as the electron donor and acceptor, respectively. Subsequent ferrozine assays demonstrate that the strain is capable of reducing the Fe(III) in soluble ferric citrate(FeC6H5O7) and insoluble Fe2O3 under anaerobic conditions. Meanwhile, the formaldoxime assays show that Mn(IV) in insoluble MnO2 could be reduced by this species. These results indicate that UU-3-2 has the capacity in dissimilatory metal reduction. Finally, a maximum current density (146 mA/m2) and secreted redox substances were detected using single-chamber microbial fuel cells (SCMFCs) and cyclic voltammetry (CV) detection, respectively. Therefore, a line of the above evidence implies that UU-3-2 is an intestinal EAB presenting electricity production and dissimilatory metal reduction.
(3) Study on chloramphenicol (CAP) degradation potential by Shewanella marisflavi UU-3-2. The variations of CAP concentrations were monitored by HPLC. The results manifest that chloramphenicol was degraded by the EAB S. marisflavi UU-3-2, and the removal rate of chloramphenicol (10 mg/L) peaked at 78.7% within 12 h. In addition, the effects of pH, the concentrations of salt, electron donor and electron acceptor on the chloramphenicol degradation and bacterial growth were exploited. The results show that, (1) The optimum pH value for bacterial growth and CAP degradation was at pH 7.2 while the species still could degrade chloramphenicol under acidic or alkaline conditions; (2) the salt concentration assay imply that the strain presented the best CAP degradation efficiency (77.4%) at salinity of 3%. Though CAP degradation was inhibited at low (1%) or high (8%) salinity, the strain still kept reduced CAP degradation and physiological activity, indicating that the strain can tolerate to some extent severe environmental conditions; (3) the CAP degradation curve show that the degradation rate was proportional to the increased concentrations of electron donors and acceptors; 4) the antibiotic sensitivity and biosafety were analyzed by the drug sensitivity test, the results show that UU-3-2 was sensitive to scores of G- bacteria antibiotics, testifying to that although this isolate had the ability to degrade CAP it may had a high biological safety. In conclusion, UU-3-2 is an EAB with the capacity of CAP degradation, indicating its applied potential in antibiotic pollution remediation.
In this study, five intestinal microbes from Urechis unicinctus were successfully isolated and identified. Among them, UU-3-2 was confirmed to be an EAB with a maximum current density about 146 mA/m2. Furthermore, UU-3-2 show to some extent applied potential in antibiotics pollution remediation as the capability in CAP degradation. This work extends EAB from terrestrial niches to marine ecosystems, providing basic physiology and pathology data on the studying of EAB in the intestinal gut of marine organisms.|