Researches On Environmental Microbiological Communities And Their Function Based On Molecular Ecology

Microorganisms are key organisms in the biological treatment systems,such as wastewater biochemical treatment systems,solid waste landfilling or composting systems.So,studying the microbial communities in these systems is of great importance for technique improvement and efficiency enhancement.And more, knowledgements of microbes in polutted environments can provide some useful information for pollution recovery for that native microbes are important actors for the self-purification of pollution.However,technologies for environmental microbiology researches mainly are traditional techniques based on culture and separation,and it could not satisfy nowaday environmental microbiology researches which require analysis and identification more directly and more exactly.Using molecular thechnologies based on 16S rRNA or rDNA sequence comparison,researchers can study microbes without separation and culture.And more,researchers even can expect direct and exact results of conformation or function of microbial communities with in situ hybridization and in situ PCR.Three different methods,which were resignated as lysozyme protocol,ultrasonic lysis protocol and protease K-CTAB protocol,were designed or improved for DNA extract from environmental samples,and it was complicated compost samples in this study.Using cell direct count,it was showed that cell lysis efficiencies of the three methods were higher than 94%.Concentrations of humic acids in purified DNA were lower than 20 ng/μl determined with spectrophotometer,and the valuss of A_260/280were between 1.7 and 1.8.Diffirential PCR amplified products were produced by using 16S rDNA targeted primer pairs of 27F/1495R and GC341F/907R.Results of restriction fragment length polymorphism analysis with PCR products using primer pair of 27F/1495R and denaturing gradient gel electrophoresis with PCR products using primer pair of GC341F/907R showed that identical genetic diversity was detected from DNA extracted by the three different methods.So,all the results indicated that these three different methods could extract DNA of high quality from compost.Since that compost is more complicated than other environmental samples,these DNA extraction methods could be adapted to satisfy DNA extraction from most of environmental samples.For getting a primary kownledgement of bacterial community and its dynamic in compost,a lab scal compost lasting 18 days was performed with cunalinary waste. There were 7 days during which temperature was higher than 50℃,and the highest temperature during the composing was as high as 65℃.DNA was extracted from even days' and thermaophilic period samples by protease K-CTAB protocol.PCR products,amplified 16S rDNA using primer pair GC341F/907R,were used for DGGE analysis.Bacteria communities were then studied by statistical analysis combined with molecular techniques of sequencing and phylogengtic analysis.The results showed that the bacterial diversity in compost declined along with the fise of temperature during composting,and it was distinguished by the differences of dominant bacteria in different days.And more,it was thermophilic bacteria of Bacillus dominating the compost during thermophilic period.At the same time,it was studied and analysed on the mechanism on the nitrogen removal when treating the leachate with highly concentrated ammonia and nitrogen by sequencing batch biofilm reactor(SBBR)designed by ourselves.Under the ambient temperature of(32±0.4)℃,and after a 58-day-long period of domestication and a 33-day-long period of stability,the ammonia nitrogen removal efficiency reached 95% in the SBBR reactor.The way of high frequency micro aeration suppresses the activity of nitrobacteria,and also eliminates the influence of nitrous acid and pH undulation to the activity of anaerobic ammonia oxidation bacteria and nitrous acid bacteria.In the period of aeration,the dissolve oxygen concentration was controlled at 1.2-1.4 mg.L^-1; the nitrous acid bacteria became the majority,and nitrite accumulated.In the period of hypoxia,anaerobic ammonia oxidation bacteria became the majority,nitrite and ammonia nitrogen accumulated in the period of aeration was removed at the same time.For studying community comformation and species dynamic of bacteria worked for nitrogen removal from landfill leachate and the mechanism on the nitrogen removal in sequesing biofilm batch reactor,Bacteria communities were then studied by statistical analysis combined with molecular techniques of sequencing and phylogengtic analysis.Results showed that a mature biofilm,a combination of aerobic denitrifying bacteria,anaerobic ammonia-oxidizing bacteria,a mass of aerobic nitrifyingbacteria and anaerobic denitrifying bacteria,was acclimated in the reactor and denitrification and anaerobic ammonia oxidation,may coexist in the reactor. Members in the biofilm changed little during nomal run,and bacteria in mature biofilm may mainly come from landfill leachate not from inoculation since that most of bands found in biofilm could also be found in leachate.In this study,the main approach of biological nitrogen removal,no less than 65% of the total NH₄⁺-N was removed in this approach,was composed of partial nitrification,anaerobic ammonium oxidation and denitrification,the second approach included twain processes such as partial nitrification and denitrification,and the third one was conventional nitrogen removal process(nitrification and denitrification).All approaches accomplished by the way which the processes carried on simultaneous or asynchronous.When the approach occurred in the simultaneous way,it performed as nitrogen-loss.The accomplisher of asynchronous way depended on the microbe's different activity between aerated phase and anaerobic phase.In addition,in view of economization of the running cost,under three different kinds of low temperature domestication strategy,three sets of the anaerobic sequence biofilm reactors(ASBBR)with the same specification were applied.The traditional analytical method was used to analyse the nitrogen change in the process of operation, simultaneously techniques of denaturing gradient gel electrophoresis(DGGE)was also used to analyse the species group structure on the biofilm,and the activity of anaerobic ammonia oxidation under different low temperature domestication strategy was preliminary investigated.The result showed that corresponding to the three different kinds of low temperature domestication strategy;a1,a2 and a3,A1,A2 and A3 showed different anaerobic ammonia oxidation activity after domestication in 62d,56b,and 70d respectively.Taking the nitrogen transformation efficiency as a measurement criteria,its activities from high to low in turn are;A3＞A1＞A2.The DGGE analysis further explained that under different domestication strategies the reactors would display different specie-diversities,but the population structure maintained almost the same.And in the strategy with the same inoculums,it had the uniformly influence to the population diversity and the anaerobic ammonia oxidation activity produced by the temperature decreasing range.