Primary Researches On Environmental Microbiological Communities Based On Sequences Analysis Of 16S RRNA/rDNA Fragments

Microorganisms are key organisms in the biological treatment systems,such as wastewater biochemical treatment systems, solid waste landfilling orcomposting systems. So, studying the microbial communities in these systemsis of great importance for technique improvement and efficiency enhancement.And more, knowledgements of microbes in polutted environments can providesome useful information for pollution recovery for that native microbes areimportant actors for the self-purification of pollution. However, technologiesfor environmental microbiology researches mainly are traditional techniquesbased on culture and separation, and it could not satisfy nowadayenvironmental microbiology researches which require analysis andidentification more directly and more exactly. Using molecular thechnologiesbased on 16S rRNA or rDNA sequence comparison, researchers can studymicrobes without separation and culture. And more, researchers even canexpect direct and exact results of conformation or function of microbialcommunities 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 orimproved for DNA extract from environmental samples, and it wascomplicated compost samples in this study. Using cell direct count, it wasshowed that cell lysis efficiencies of the three methods were higher than 94 %.Concentrations of humic acids in purified DNA were lower than 20 ng/μldetermined with spectrophotometer, and the valuss of A260/280 were between1.7 and 1.8. Diffirential PCR amplified products were produced by using 16SrDNA targeted primer pairs of 27F/1495R and GC341F/907R. Results ofrestriction fragment length polymorphism analysis with PCR products usingprimer pair of 27F/1495R and denaturing gradient gel electrophoresis withPCR products using primer pair of GC341F/907R showed that identicalgenetic diversity was detected from DNA extracted by the three differentmethods. So, all the results indicated that these three different methods could extract DNA of high quality from compost. Since that compost is morecomplicated than other environmental samples, these DNA extraction methodscould be adapted to satisfy DNA extraction from most of environmentalsamples.For getting a primary kownledgement of bacterial community and itsdynamic in compost, a lab scal compost lasting 18 days was performed withcunalinary waste. There were 7 days during which temperature was higherthan 50℃, and the highest temperature during the composing was as high as65℃. DNA was extracted from even days' and thermaophilic period samplesby protease K-CTAB protocol. PCR products, amplified 16S rDNA usingprimer pair GC341F/907R, were used for DGGE analysis. Bacteriacommunities were then studied by statistical analysis combined withmolecular techniques of sequencing and phylogengtic analysis. The resultsshowed that the bacterial diversity in compost declined along with the fise oftemperature during composting, and it was distinguished by the differences ofdominant bacteria in different days. And more, it was thermophilic bacteria ofBacillus dominating the compost during thermophilic period.For studying community comformation and species dynamic of bacteriaworked for nitrogen removal from landfill leachate in sequesing biofilm batchreactor, a 3-L reactor was operated normally for 1 month after a 3-monthacclimation. Total DNA was extracted with lysozyme protocol from 8 samples,including that one was from landfill, one was from acclimated biofilm and sicwere from a single running cycle. PCR products, amplified 16S rDNA usingprimer pair GC341F/907R, were used for DGGE analysis. Bacteriacommunities were then studied by statistical analysis combined withmolecular techniques of sequencing and phylogengtic analysis. Resultsshowed that a mature biofilm, a combination of aerobic denitrifying bacteria,anaerobic ammonia-oxidizing bacteria, a mass of aerobic nitrifyingbacteriaand anaerobic denitrifying bacteria, was acclimated in the reactor and threedifferent nitrogen removal modes, conventional denitrification, simultaneousnitrification and denitrification and anaerobic ammonia oxidation, may coexistin the reactor. Members in the biofilm changed little during nomal run, and bacteria in mature biofilm may mainly come from landfill leachate not frominoculation since that most of bands found in biofilm could also be found inleachate.