| Special acidophilic species are usually introduced into the bioleaching systems for constructing and optimizing microbial community during bioleaching process. However, little is known about the mechanism of species introduction into original bioleaching consortia. The built co-culture system or nature bioleaching microbial community, after adaptation, functioned as the original bacterial consortium. In this study, exogenous bacteria, belonging to different metabolic type, were introduced into original bioleaching systems at different growth periods. The introduction mechanism was studied in order to determine the most appropriate introduction type and time for the original bioleaching microbial community, as well as provide some theoretical basis.Four vibrio or spiral-shaped bacteria strains were isolated from acid mineral drainage (AMD) using liquor gradient dilution method. Based on the analysis of purity, physiological property,16S rDNA and gyrB gene sequence, morphology, and bioleaching capacity towards pyrite, the four strains were identified as L. ferriphilum.A co-culture system consisted of At. caldus,L. ferriphilum, Ferroplasma,Acidiphillum spp.,S. thermosulfidooxidans and At. ferrooxidans was built. It was found that the co-culture system demonstrated superior pyrite bioleaching capacity under the condition (40℃, pH1.5and0.5%(g/100ml) pulp density). Besides, At. caldus,L. ferriphilum and Ferroplasma were the dominant groups compared with At. ferrooxidans,Acidiphillum spp. and S. thermosulfidooxidans. The growth period can be divided into four phases:adaptation phase from0to48hours, rapid-growth phase from48to96hours, stable growth phase from96to192hours, and decline phase from192to240hours.The above co-culture system served as the indigenous community. At. thiooxidans A01, the exogenous species, was introduced into the original consortium at66and138hours, and the pyrite bioleaching rate increased by10%and13%, respectively. Based on the real-time PCR analysis, the introduction of At. thiooxidans A01promoted the growth of L. ferriphilum, Ferroplasma and Acidiphillum spp., inhibited the growth of At. Caldus, and demonstrated inconspicuous effect on the growth of At. ferrooxidans and S. thermosulfidooxidans. Based on the functional gene arrays data, protoheme ferrolyase, formate hydrogenlyase,ADP heptose, phosphoheptose isomerase and glycosyltransferase genes of L. ferriphilum, ferredoxin oxidoreductase gene of Ferroplasma sp. and acetyl-CoA carboxylase gene of Acidiphillum spp. were up-regulated from0to20hours after the introduction. In contrast, the doxD gene of At. caldus and ribulose-1,5-bisphosphate carboxylase/oxygenase gene of S. thermosulfidooxidans were down-regulated from0to20hours after the introduction. The ADP-heptose Synthase gene of At. ferrooxidans was up-regulated from0to12hours after the introduction, and then down-regulated from12to20hours.The composition of the above co-culture was slightly adjusted in the following experiment. To be specific, along with the removal of L. ferriphilum, At. thiooxidans was added in the system. Under the same experimental condition, L. ferriphilum YSK serving as the exogenous species, was introduced into the indigenous consortium at0,66and138hours, and the pyrite bioleaching rate increased by9%,14%and19%, respectively. Based on the real-time PCR analysis, the introduction of L. ferriphilum YSK promoted the growth of At. caldus, At. thiooxidans and Acidiphillum spp., inhibited the growth of Ferroplasma and S. thermosulfidooxidans, and demonstrated inconspicuous effect on the growth of At. ferrooxidans. The shift of functional gene expression level from "138hours introduction" was analyzed using the functional gene arrays. It was found that ADP heptose,phosphoheptose isomerase, glycosyltransferase,Biotin carboxylase and protoheme ferrolyase genes of L. ferriphilum, acetyl-CoA carboxylase gene of Acidiphillum spp. and doxD gene of At. caldus were up-regulated from0to20hours after the introduction. In addition, lipid A disaccharide synthase LpxB,glycosyl transferase and ADP-heptose Synthase genes of At. ferrooxidans showed up-regulated expression from0to8hours after the introduction, and then down-regulated expression from8to20hours. The situation was more complex with ferredoxin oxidoreductase gene of Ferroplasma sp. In detail, this gene was up-regulated from0to4hours, down-regulated from16to20hours, and then up-regulated again from16to20hours after the introduction. CbbS gene of At. ferrooxidans showed down-regulated expression from0to20hours after the introduction.The acidophilic microorganism mixture from different AMD environment was adapted for five generations in chalcopyrite bioleaching system, and then was used in chalcopyrite bioleaching. An increase by45%of the bioleaching rate was observed. According to the RFLP analysis, the microbial community consisted of eight common bioleaching microorganisms, including At. ferrooxidans, At. caldus, Sulfobacillus sp., L. ferriphilum, S. thermosulfidooxidans, Acidiphilium sp., Alicyclobacillus sp. and Pseudomonas sp., however, only the existence of At. ferrooxidans, At. caldus, Sulfobacillus sp., L ferriphilum and Acidiphilium was detected after adaptation. Based on the analysis of functional gene arrays, functional gene abundance of At. Caldus, L.ferriphilum, Sulfobacillus sp., Sulfolobus sp. and Acidiphilium sp. was increased along with the adaptation, however, the abundance of S. thermosulfidooxidans,Alicyclobacillus sp. and At. ferrooxidans was through a decline. The functional genes of At. caldus and Sulfolobus sp. involved in sulfur metabolism, demonstrated increasing gene abundance, while the functional genes of At. ferrooxidans involved in sulfur metabolism showed decreasing abundance. In addition, protoheme ferrolyase gene, functional genes involved in iron metabolism of L. ferriphilum showed increasing abundance. The functional genes involved in metal resistance of At. ferrooxidans, At. caldus, L. ferriphilum, Sulfolobus spp. and Acidiphilium sp. also demonstrated increasing abundance.At. thiooxidans A01, the exogenous species, was introduced into the indigenous consortium, the adapted microbial community from AMD environment, respectively at the0th,24th and36th day (bioleaching cycle spanned48days). The results showed that the copper ion concentration was respectively204.3,215.1,230.8and251.5mg/L in the bioleaching system without and with introduction of At. thiooxidans A01at0th,24th and36th day. Microbial community structure was analyzed using RFLP method. After the inroduciton of At. thiooxidans A01, it suggested that L. ferriphilum, At. ferrooxidans, At. caldus, Sulfobacillus sp., At. albertensis and Acidiphilium were together detected in bioleaching all the way. Moreover, the proportion of dominant groups, namely, At. caldus,L. ferriphilum and At. ferrooxidans, in these two systems, changed from38.4%,30.4%and17.6%at36th day to29.6%,40%and12%at48th day, respectively. However, only five microbial species except for At. albertensis were detected in the control group without introduction of At. thiooxidans A01. The real-time PCR and functional gene arrays data suggested that At. thiooxidans A01, introduced at the the36th day, primarily attached on the mineral surface, and the adsorbance reached the peak at the42th day, followed by the decreasing amount. For the supernatant At. thiooxidans A01, the cell number was initially low, reached the peak at the45th day, and then turned into a decreasing tendency. The abundance of both attached and free At. caldus and Sulfobacillus sp. was decreased after the introduction, while, that of L. ferriphilum, At. ferrooxidans and Acidiphilium sp. were all increased. The abundance of protoheme ferrolyase gene of L. ferriphilum and sulfide-quinone reductase gene of At. ferrooxidans were increased after introduction in both attached and free bacteria. However, the genes involved in sulfur metabolism, such as doxD gene, showed decreasing abundance. Moreover, the metal resistance genes of At. ferrooxidans, L. ferriphilum and Acidiphilium sp. showed an increasing abundance.
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