| Metal nanoparticles have been widely used in the fields of ceramics, chemical industry, communication and biomedicine for their huge specific surface area, special small size effect, good photoelectric performance and other excellent physical and chemical properties. With the wide use of metal nanoparticles in various fields, their wastes are inevitably produced and enter into the nature. At the same time, organic colloids from natural sources, dust aerosols from volcanic eruptions and other metal nanoparticles also widely exist in nature. Natural and artificial metal nanoparticles in these environments can be transferred directly or indirectly into the ocean through sewage dumping, air subsidence, surface runoff, etc., and threaten the marine environment. Marine bacteria are the most abundant microbial group in the marine ecosystem, and they play an important role in the matter circulation, energy flow and maintenance of the diversity of the marine ecosystem. With the increase of the concentration of metal nanoparticles in the marine environment, whose impaction on the physiological ecology of marine bacteria needs to be furtherly studied. In recent years, a new type of automated phenotype method, the non-contact conductivity sensor (CCS) method, has been created and applied to obtain the data of the toxic effect of nanomaterials on bacteria. The improved capacitance-coupled non-contact conductivity detector is mainly used for online and real-time monitoring of the conductivity value of microbial culture fluid. The response values obtained are proportional to the concentration and mobility of ionic current in the culture medium. Since the uncharged or weakly charged substrate will be converted into highly charged small molecule substances during the growth and proliferation of bacteria, thus increasing the conductivity of the culture medium, the growth process of bacteria can be recorded quickly and accurately by detecting the change of the conductivity of the culture medium.Because bacteria in the environment are divided into Gram-positive and Gram-negative bacteria according to their different cell structures, the cell wall of Gram-negative bacteria has more outer membrane composed of tightly packed lipopolysaccharide (LPS) molecules than that of Gram-positive bacteria, which leads to different resistance effects of Gram-positive and Gram-negative bacteria to external stress. At the same time, Bacillus subtilis and Vibrio parahaemolyticus, as Gram-positive and Gram-negative bacteria widely existing in Marine environment, represent two important microbial categories respectively. Among them, Bacillus subtilis is a typical probiotic in Marine environment, which plays a key role in promoting host health and environmental restoration. Vibrio parahaemolyticus is a representative of pathogenic bacteria in the Marine environment, which has a significant impact on causing foodborne diseases. Based on the ecological roles and functions of these two bacteria in Marine microbial communities, this study took Bacillus subtilis (gram-positive bacteria) and Vibrio parahaemolyticus (Gram-negative bacteria) isolated from the environment of Bohai Bay as test organisms, and took common metal nanoparticles as research objects, and used non-contact conductivity sensor (CCS) method to study their growth inhibition effects on Bacillus subtilis and Vibrio parahaemolyticus. The research process includes:(1) Preparation of bacterial solution: Vibrio parahaemolyticus was inoculated in TCBS liquid medium at 28℃ for 12 h; The bacteria solution was dipped and streaked on the TCBS plate and cultured overnight. The single colonies on the plate were selected and inoculated into the new TCBS liquid medium at 28℃ for 12 h. The cultured bacterial solution was put into a centrifuge for centrifugation, the supernatant was poured out, washed and centrifuged twice with normal saline (0.85% NaCl), and the bacterial precipitation was re-suspended in normal saline for subsequent study. The preparation method of Bacillus subtilis is the same as above, and the medium used is LB medium.(2) Preparation of metal nanoparticle suspension. (3) Growth toxicity test: Taking Bacillus subtilis and AgNPs as an example, 10 mL of the prepared Ag NPs suspension was measured in sterilized glass bottles. The prepared 100 μL Bacillus subtilis solution was inoculated into it and mixed evenly. The 3 mL mixed system was absorbed with a sterile syringe and added into the NMR tube, with 3 tubes for each concentration, and 3 tubes for each positive and negative control (positive control: 10 mL medium and 100 μL bacterial solution were added into the NMR tube; Negative control: Added the same amount of medium into NMR tube); The NMR tube was put into the instrument for measurement. The voltage at the excitation electrode of the instrument was 16 V and the frequency was 2 MHz. The instrument was set to collect data every 1 minute, and the experiment lasted for 12 h.The results showed that:(1) Nano-gold (Au NPs), nano-silver (Ag NPs), nano-silver oxide (Ag2O NPs) and nano-titanium dioxide (TiO2 NPs) could inhibit the growth of Bacillus subtilis and Vibrio parahaemolyticus, and the 12 h-EC20 values of the nano-gold (Au NPS), nano-silver (Ag NPs), nano-silver oxide (Ag2O NPS) and nano-titanium dioxide (TiO2 NPS) against Bacillus subtilis were 1.81, 0.03, 1.71 and 54.43 mg/L, respectively. The 12 h-EC20 values of Vibrio parahaemolyticus were 8.11, 0.16, 2.97 and 81.55 mg/L, respectively.(2) In the concentration range set in this study, nano-zinc oxide (ZnO NPs) and nano-iron oxide (Fe2O3 NPs) showed a promotion effect on the growth of Vibrio parahemolyticus, but showed an inhibitory effect on Bacillus subtilis. In this paper, the toxic effects of six common metal nanoparticles on Bacillus subtilis and Vibrio parahemolyticus were studied and analyzed by CCS method, and the EC20 values of these six metal nanoparticles on the two bacteria were obtained. The EC20 values can provide a theoretical basis for the environmental risk assessment of the construction of metal nanomaterials in the Marine ecosystem in China. |
