Development And Application Of The Microarray Technology During The Research On The Structure And Function Of Microbial Communities For Acid Mine Drainages From The Copper Mines

The bio-oxidation of metal sulfide minerals has produced huge amount of acidicmineral drainage (AMD), causing severe environmental problems. The existence ofvarious acidophilic microorganisms was observed in AMD systems. Not only do thesemicroorganisms contribute to the formation of AMD, but also they have significantimportance in researches concerning the bioleaching of various metals and thebioremediation of environmental problems. In this paper, the diversity and thefunction of microbial community of AMD system in Dexing copper mine werestudied.In the first place, 16SrRNA genes and gyrB genes were used as molecularmarker to evaluate the diversity of microbial communities from six AMD in Dexingcopper mine. The result showed that the predominant microorganism in Dexingcopper mine were Proteobacteria, Acidobacteria, Actinobacteria, Nitospira,Firmicute and Chlorella. PCA studies revealed that the geochemistry parameter of thesample may greatly influence the microbial community structure. The composition ofmicrobial community tends to be closer for samples that have similar geochemicalproperty. CCA studies showed that the concentration of Fe and S were the majorfactor that lead to diversification of microbial community structure. But thepredominant factor that determines microbial community structure may change underdifferent environmental condition.The comparison of results by 16SrRNA gene and gyrB gene analysis showsthat gyrB gene analysis may provide more accurate information about the microbialcommunity structure than 16S rRNA gene analysis and thus more likely to revealedthe true nature of a microbial community. In this paper, we evaluated the applicationof 16S rRNA gene and gyrB gene analysis on AMD sample from two different miningareas. The result produced by the two molecular markers showed that the microbialcommunity composition was similar to each other, but the microbial communityabundance acquired from gyrB analysis was extremely higher than obtained through16S rRNA analysis in terms of Shannon diversity index. Through gyrB gene, we willbe able to differentiate closely related microorganisms in phylogenetic analysis, andthis was further demonstrated through analysis on pure culture A.ferrooxidans. Basedon results above, we studied four AMD samples from Dexing copper mine through16SrRNA gene analysis and gyrB gene analysis; the results also supported our conjecture. Besides, we also found that gyrB shows better performance in researchconcerning the correlation between community structure and geographic chemicalparameters.Microarray technology has been widely used in researches concerning thefunction and structure of microbial community due to its unique advantages. But sofar there have been rare report about application of microarray technology in AMDsystem. In order to effectively monitor the change in acidic system and the microbialcommunity activity, we had developed a 50mer oligonucleiotide microarray based onthe gene sequence obtained beforehand. The microarray contains 1072 unique probes,including 571 16S rRNA gene probes and 501 functional gene probes. Among thefunctional genes contained in the microarray there are genes related to the metabolismof carbon (158), nitrogen (72), sulfide (39), iro (68) as well as genes related togenetics (97), metal ion resistance (27), membrane function (16), transposon (13)and IST sequence (11). The specificity, sensitivity and quantitative potential of themicroarray we fabricated were evaluated. Hybridization results showed that theprobes were highly specific to the PCR products of the corresponding gene. Themicroarray was capable of detecting genome DNA as little as 5ng. Strong linearrelationship between the signal intensity and target DNA concentration(r2=0.98) wasobserved. Besides, hybridization of the microarray fabricated with natural acidicsample revealed that the microarray we fabricated is capable of effectivelydifferentiating the function and structure of microbial communities in AMD systems.Using the microarray we fabricated, studies have been done on AMD systems inDexing copper mine. The results of 16SrRNA were in accord with that obtained fromtraditional analysis. For instance, the dominate species in sample SLS and YTWbelongs to A.ferrooxidans, and we had obtained very strong hybridization signal fromA.ferrooxidans strains. Cluster analysis of 16SrRNA showed that sample SLS andYTW, which possess similar community structure, gathered together. This result wasalso coincides with results of traditional molecular analysis. Cluster analysis offunctional gene revealed that some genes related to energy metabolism were gatheredtogether and showed strong hybridization signal. This phenomenon indicated that theenergy metabolism of microorganism was very active in AMD systems. This mayresulted from the fact that microorganisms in AMD systems typically utilize Fe2+ andS as their main energy source.