Study On Control Of The Survival And Activity Of Luminescent Bacteria And Toxicity Test

Objective: Aquatic toxicity test is the one of the important methods of the environmental pollution detection and health risk assessment. Fish, daphnia, algae and microorganism are usually used as indicator organism for toxicant, especially luminescent bacteria toxicity test which is representative for microorganism test is simple, rapid and economic. However, stability of luminescent bacteria directly impacts on the accuracy and reliability of the the experimenatal results. For the present, there are freeze drying, continuous culture and immobilization to maintain the stability of activity of biosensor bacteria. The purpose of this study is to find a method that can control the survival and activity of luminescent bacteria, improving the stability and sensitivity of the luminescent bacteria toxicity test, in order to provide technological support to the on-line continuous toxicity test. The toxicity detection and evaluation of source water and landscape water are conducted with luminescent bacteria toxicity test, and the base line values of different water environment are constructed, which can become to be the basic data for water quality early-warning technique.Methods: Photobacterium phosphoreum and Photobacterium leiognathi were chosen as research object, studying the preparation of freez-dried powder by the vacuum freeze-drying technique, and then exploring effect of kinds of protective freeze-drying media on activity of luminescent bacteria in the process of freezing by analyzing the emission intensity of freeze-dried bacteria powder rehydrated. Bioluminescent variation tendency of luminescent bacteria suspensions prepared in different ways and put in 5℃was researched. And the additive recipe of bacteria suspension was optimized. In this paper, The toxicity results of Zn2+, Cr6+, 3,5-dichlorophenol that were determined with luminescent bacteria toxicity test in different osmotic adjustment materials such as NaCl and sucrose were compared. The influence of organic solvents for hydrophobic organic compounds on luminescent bacteria toxicity test was studied. On the conclusion, the toxicities of two kinds of water bodies were determined, and the toxicity base line values of these water bodies were constructed adopting statistical analysis methods.Results: We found that skimmed milk and sugar have a good influence for luminescent bacteria long-term storage, especially with 1%NaCl, 15% skimmed milk, 6%sucrose solution. The RBL of skimmed milk bacteria suspension is about 92% in cold storage with 7 days. Skimmed milk bacteria suspension is superior to fresh bacteria solution and freeze dried bacteria suspension, the RBLs of which are less than 60% in cold storage. In osmotic adjustment liquid test, the results show that sucrose inhances the sensitivity of luminescent bacteria to heavy metal, and decrease the sensitivity to the organic compounds. The EC50 values of chlorobenzene and n-hexane with DMSO or ethanol are respectively 0.047mg.L^-1 and 0.050mg.L^-1, the EC₅0 of that is 0.037mg.L^-1 and 0.048mg.L^-1 without DMSO and ethanol. The base line values of water source areas is 100%-103%, early warning level is 91%. The base line values of landscape water is 97%-101%, early warning level is 84%.Conclusion:The compound protectants made up of NaCl, skimmed milk and sucrose improve the shelf life of freeze-dried luminescect bacteria. And Skimmed milk also improves the stabilization of activity of luminescent bactera in 5℃. The osmotic adjustment liquid including NaCl and sucrose improves the sensitivity of luminescent bacteria to heavy metal. EC50 values of chlorobenzene and n-hexane with DMSO or ethanol are a little lower than that of chlorobenzene and n-hexane without DMSO or ethanol, however, the action is not distinct. The toxicity base line value of water source areas is 100%-103%, and that of landscape water is 97%-101%. The results show luminescent bacteria toxicity test can be applied to contruct the toxicity base line value.