Microbial Communities In Acid Mine Waters And Their Adsorption On Pyrite

Microorganisms such as Acidithiobacillus ferrooxidans and Leptospirillumferrooxidans are the most studied acid mine drainage (AMD) organisms, they areiron-oxidizing species, catalyze ferrous iron oxidization, thus determine the rate ofsulfides dissolution, and play very important roles in AMD generation. To present, amore complete picture of microbially mediated AMD production and the mechanismof interfacial interaction between microbes and minerals, the composition ofmicrobial communities in five acid mine water samples were studied, usingculture-independent 16S rDNA based cloning approach- restriction fragment lengthpolymorphism (RFLP) analysis method, isolation, scanning electronic microscopy(SEM) and fluorescent in situ hybridization (FISH) methods.Three samples, named SX1, SX2 and SX3, were obtained from three separateAMD sites of Zhongtiaoshan copper mine in Shanxi Province, China. The rest twosamples, named K1 and K2, were gained from two separate bioleaching systems ofTongshankou copper mine, Hubei Province, China. Phylogenetic analysis revealedthat the bacteria in these five samples fell into 4 major groups: Proteobacteria(γ-proteobacteria,β-proteobacteria,α-proteobacteria andδ-proteobacteria),Nitrospira (Leptospirillum), Firmicutes and Bacteroidetes. Proteobacteria organismssuch as A. ferrooxidans appeared in samples SX3, K1 and K2, but was scarce insamples SX1 and SX2; Nitrospira organisms such as Leptospirillum ferrooxidans,Leptospirillum ferriphilum and Leptospirillum groupⅢ, were prevalent in samplesSX1, K1 and K2, but a few in samples SX2 and SX3; Firmicutes organisms were onlyfound in samples SX3 and K1; Bacteroidetes organisms were only found in sampleSX3. Archaea were only detected in two samples K1 and K2 from Tongshankoucopper mine. Thermoplasma and Ferroplasma lineages were detected abundantly inthese two samples. Unexpectedly, sequences affiliated with Sulfolobales andMethanothermus genus were also detected in small amount. Additionally, highconcentrations of some chemical elements such as S, Fe, Cu, Zn and Ag seemed to bekey factors resulting in the diverse distribution of typical iron-oxidizers (such asAcidithiobacillus ferrooxidans and Leptospirillum species) among water samples.A new simple method for isolation of L. ferriphilum has been created. One strain ofL. ferriphilum and three strains of Acidiphilium sp. have been isolated from the acid mine waters. These results further proved the truth previously revealed by RFLP, andalso supplied some new ways for isolation of L. ferriphilum and Acidiphiliurn sp..The adsorption of microbes on pyrite surface has been studied mainly by SEM.After adsorption for a long time (42d), A. fwould set themselves as nucleus adsorbingferroalumen precipitation, and Leptospirillum species adsorbed on the pyrite surfacewere likely to accumulate together and form biofilm. The ferroalumen precipitationlayer covering the A. fsurface may decrease the oxidation rate of pyrite by A. fin thelater leaching period. While the Leptospirillum species' surfaces always keep smoothwithout any precipitation adsorbed. They may always have the ability of oxidizing thepyrite from the beginning to the end, especially, in the later leaching period, they maybe the main oxidizer.A. f bacteria had been detected relatively abundant adsorbed on the pyrite surfaceby FISH, this may imply they were likely to adsorb on pyrite and also the adsorptionwas a strong adsorption. A small amount of Archaea, Ferroplasma and Acidiphiliumspecies also have been detected adsorbing on pyrite surface. The thick biofilm mainlyformed by Leptospirillum species may make the cells' permeability become poor, sodecrease the hybridization efficiency of probe LF655. These results may prove someresults previously revealed by RFLP and also may imply that not only A. for L. finteract with the pyrite singly, but some other microbes such as Archaea, Ferroplasmaand Acidiphilium species may also play important roles in the processing of oxidationof pyrite. All of them may be synergistic interaction with the pyrite.