Molecular Mechanism Of Bioremediation Of Triphenyl Phosphate And Copper Polluted Water By Brevibacillus Brevis

Organophosphate flame retardants（OPFRs）and heavy metals are both common environmental pollutants.Their accumulation and dispersion in the environment can severely affect the stability and function of ecosystems.Heavy metals can enter humans and other organisms through air,water,and soil,causing negative effects.Microbial remediation technology can use microbial metabolic activity to remove organic pollutants and heavy metal ions,effectively reducing the economic costs and social impacts of environmental pollution and ecological destruction.A large amount of research has shown that microorganisms have strong degradation capabilities for OPFRs,but there are few reports on the removal performance mechanism of strains for the compound pollution of OPFRs and heavy metals and the strain stress response.Therefore,this study used triphenyl phosphate（TPHP）,a common OPFR in contaminated water,and copper（Cu）as the research objects,selecting Brevibacillus brevis as the experimental strain.The removal effect,cell physiological response mechanism,and protein expression differences of the strain for TPHP and Cu²⁺compound pollution were studied.The main research results are as follows:（1）The removal efficiency of the degradation system is influenced by factors such as p H,temperature,and initial concentrations of the two pollutants.At p H 7 and 30℃,the microorganisms’TPHP removal rate in the compound pollution system can reach up to67.3%.Low p H and low temperature have a greater inhibitory effect on the removal of pollutants by the bacteria than high p H and high temperature.Under the same conditions,when the p H is 4,the removal rate of TPHP by B.brevis is as low as 38.9%,and when the temperature is 20℃,the removal rate of TPHP by B.brevis is as low as 30.1%.When the TPHP concentration increases to 20 mg/L,its degradation rate drops to 20.0%,and when the Cu²⁺concentration increases to 100 mg/L,the TPHP degradation rate drops to 19.0%.It is speculated that the degradation and transformation pathways include hydroxylation and other three pathways.When the Cu²⁺concentration exceeds 10 mg/L,the removal efficiency of the bacteria for TPHP and Cu²⁺both significantly decrease.（2）Through observations using transmission electron microscopy（TEM）,scanning electron microscopy（SEM）,and laser confocal microscopy,it was found that under single TPHP stress,the surface of B.brevis appeared slightly wrinkled.The single Cu²⁺and TPHP-Cu²⁺complex systems caused more obvious deformation and even rupture of the B.brevis cells,leading to the outflow of cell contents.The degree of damage to the bacterial cytoplasmic membrane and the induction of cell apoptosis caused by the TPHP-Cu²⁺complex system was greater than that of the single TPHP and single Cu²⁺systems.Fourier transform infrared（FTIR）spectroscopy detected stronger responses of carboxyl,amide groups,glycoproteins,fatty acids,and other components on the cell surface,and the higher the Cu²⁺concentration in the system,the greater the cell damage and the higher the cell apoptosis rate.When the Cu²⁺concentration increased to 20 mg/L,the proportion of cell apoptosis exceeded 50%.（3）After protein component analysis,compared to the control group,46,315,and 202differentially expressed upregulated proteins were detected in B.brevis cells exposed to TPHP,Cu²⁺,and TPHP-Cu²⁺systems,respectively,along with 90,69,and 127 differentially expressed downregulated proteins.COG annotation analysis showed that the amino acid transport metabolism function proteins appeared significantly different in the three systems.The functional proteins of the differentially expressed proteins in the TPHP system were related to inorganic ion transport metabolism,while the Cu²⁺system’s functional proteins were related to nuclear substance structure and dynamic indication.The differentially expressed proteins in the TPHP-Cu²⁺system were related to signal transduction.GO analysis showed that the biological processes involved in the differentially expressed proteins in the TPHP system were mainly related to ribosome synthesis,while the TPHP-Cu²⁺and Cu²⁺system’s differentially expressed proteins were mainly related to cell amino acid metabolism and five other processes.Under TPHP stress,the upregulation of D50S ribosomal protein stimulated cell translation processes,while under Cu²⁺stress,the action of glutamate dehydrogenase promoted the production of extracellular polysaccharides and surface membrane proteins in bacteria,which had a positive effect on pollutant removal.During TPHP-Cu²⁺stress,proteins such as isoleucine t RNA ligase were key enzymes involved in pollutant metabolism and transport,and were all significantly upregulated.