Effects Of Disinfectant On Bacterial Community Structure Of Microcosm: A Temperature Gradient Gel Electrophoresis (TGGE) Analysis

Temperature Gradient Gel Electrophoresis (TGGE) is a derivative technique of Denatured Gradient Gel Electrophoresis (DGGE). Due to effective separation of DNA fragments with identical length and different base sequences, this technique was initially used to study point mutation of gene. It was first used for the study on microbial ecology in 1993, analyzing the 16S rDNA gene fragments with identical molecular size and different base sequences. According to the differences in base sequences of variable region of small subunit gene of ribosome in bacteria, we may give a more detailed description of population diversity, quantitative proportion and phyletic evolutional information of bacteria. TGGE technique is used not only for analyzing community structure of microorganisms, but also for rapid and simultaneous analysis of the differences in microbial communities, and for researches on variations of microbial community with the change of time and pressure of external environment. Another important advantage of this technique is its high resolution, controllable electrophoretic conditions, good reproducibility, and convenient and rapid operation. Thus, TGGE technique has been quickly and extensively applied to all kinds of studies on microbial ecology since it was invented. Now it has been developed as one of major analysis methods of environmental microbial community structure.Because Severe Acute Respiratory Syndrome (SARS) and highly pathogenic avian influenza have broken out in some cities of our country in recent years, massive disinfectants were used to prevent and control epidemic situation. Though persistent use of disinfectant in a large scale can enhance disinfection effect, it also increased environmental burden, caused different degrees of environmental and ecological pollution and even destroyed ecological balance. Destruction of ecological balance often begins with an irreversible change of microbial ecology. Now we rarely know about the effects of disinfectant on microbial ecology in environment. So it is very essential to study the microbial ecological effects caused by disinfectant after its release into environment. This research was supported by SARS projects of Ministry of Science and Technology of PRC (No. 2003A07).Objectives: TGGE fingerprinting was used to reveal the diversity of bacterial population of microcosm and to gain a basic understanding of effects of disinfectant on bacterial community structure in microcosm. This research will provide scientific evidences for reasonable use of disinfectant and minimize the damage to microbial ecology in environment caused by disinfectant.Methods: Six pond microcosms were constructed to simulate aquatic ecosystems, including one for control. Sodium hypochlorite was added to five experimental microcosms every 24 hours for 13 consecutive days and followed by 10 days'observation. The doses of sodium hypochlorite added were 1.0 mg/l, 2.5 mg/l, 5.0 mg/l, 10.0 mg/l and 20.0 mg/l, respectively. Water sample was collected from every microcosm over 8 time points at 0, 1st, 5th, 9th, 13th, 15th, 19th, 23rd day during the experiment. Meanwhile, free chlorine residual and total bacterial counts were determined. Spread plate method was processed with incubation at 37℃for 48 hours and the total genomic DNA of bacteria was extracted. The 16S rDNA V3 region fragments were amplified from the total DNA using general primers. TGGE analysis of 16S rDNA V3 region was conducted to reveal the effects of disinfectant on bacterial community in microcosm by comparative analysis of TGGE fingerprinting in 8 time points from different treated microcosms. Several major bands from TGGE profiles were recovered from gel, recovery DNA was reamplified, cloned and libraries were constructed. Four clones were picked from each library and sent to invitrogen (Shanghai) for sequencing. Sequences analysis was performed to study the polymorphism of TGGE bands and diversity of bacterial community in microcosm.Results: Concentration of free chlorine was in direct proportion to doses and time from addition of disinfectant and descended markedly after addition stopped. Total bacterial counts showed an obvious increase instead of decrease under low doses (1.0, 2.5 mg/l) of disinfectant. While the doses of disinfectant were higher (10.0, 20.0 mg/l), total bacterial counts were obviously decreased or even down to zero during disinfection treatment but restored after stopping addition of disinfectant. TGGE fingerprinting in 8 time points from five experimental microcosms showed different patterns. One strong and one weak band in the TGGE profiles of low dose treatments were inverted at the beginning of addition, followed by recovery of the profiles. The TGGE profiles of high dose treatments changed indistinctively in the beginning, but the strong and weak band were inverted and one band disappeared after stopping addition of disinfectant. Sequences analysis of TGGE bands demonstrated each band was composed of more than one sequence type. The 21 sequences were aligned and divided into 10 OTUs (operational taxonomic unit) at 97% homology cutoff using the DNAMAN program. All the OTUs shared 97%～100% homology with known sequences of closest relatives in the GenBank database. A phylogenetic analysis classified 10 OTUs into three major groups: Bacillus, Pseudomonas and Drinking water bacterium MB16, and the two formers were dominant microbial population.Conclusions: This research indicated disinfectant would affect bacterial community structure in environment, but the effects caused by disinfectant in different doses varied markedly. There was a clear dose-effect relationship between doses of disinfectant and bacterial community change in microcosms. The effects of disinfectant in low doses on bacterial community structure were limited and reversible. After microcosm system gradually adapted to disinfectant, the community structure would be recovered. However, the destructions of disinfectant in high doses on bacterial community structure were severe and irreversible. The community structure would be impossible to recover even if addition of disinfectant was stopped. The result of research suggested that blind and excessive application of disinfectant would severely destroy microbial ecology in environment.