Isolation Of Extreme Acidithiobacillus Thiooxidans And Bioleaching Of Low-grade Chalcopyrite

With the global decline of rich-ore reserves and increasing metal demand, how toeffectively extract the low-grade tailings has become a worldwide problem. With theadvantages of economical, environmental and efficiently extracting of low-grade ore,bioleaching was a novel and green smelting technique for solving the above problems.However, due to the extremely low-grade and complex composition, the mechanism ofchalcopyrite bioleaching was not perfect and its industrial application was also not satisfied.In this paper, the research was started from the isolation of an extremeacidophilus-Acidithiobacillus thiooxidans (A. thiooxidans) and its extremely acid-resistancemechanism. Combination system and technology were respectively proposed for enhancingthis bioprocess. An identified and quantified method was established with the molecular probe.The community evolutions in different status were further revealed. The bioleachingmechanism model of low-grade chalcopyrite was initially established. The main studycontents were listed as follow:(1) Isolation of the extremely acidophilus A. thiooxidans and its acid-proof mechanism.A double layers culturing method with heterotrophic strain-Rhodotorula sp. as the bottomculture was employed for efficiently isolating the chemoautotroph bioleaching microorganism.The cycle was reduced by1/3when the plating efficiency was improved by3.1times. Anextremely acidophilic sulfur-oxidizer strain was isolated from the leaching solution ofindustrial bioleaching heap in Fujian Zijin Mining. The capacity of inorganic acid-proofreached to pH0.2. The strain was identified as A. thioodidans ZJJN-3with the specialphysiological and double molecular characteristics. The strain ZJJN-3has been preserved inthe China Center for Type Culture Collection-CCTCC M2012104. Then the extremelyacid-proof mechanism was respectively investigated from the intracellular pH, cellmorphology, polysaccharide, membrane fatty acid composition, ATPase activity and thetranscription levels of acid-related proteins. With the increasing acid stress, the cell capsuleexperienced a process of significantly thickening and then gradually shedding off. The cellpolysaccharide content also increased and then dropped along with the evolution process. Theunsaturated fatty acid of cell membrane ratio significantly increased from37.85%to49.06%and cyclopropane fatty acids proportion remained at a high level about12.56%-18.59%. TheATPase activity was greatly strengthened, and the relative transcript levels of acid-proofsystem-GrpE/DnaK/DnaJ also increased to2.2-3.5times of original level. The acid-proofmechanism model in A. thioodidans ZJJN-3was further established basing on the aboveresults.(2) Bioleaching of low-grade chalcopyrite by a synergistic two-strain system. Based onthe repeated fed-batch culture model in the Starkey-sulfide medium, the sulfur conversionpercentage and biomass productivity of A. thioodidans ZJJN-3was improved by31.1%and187.9%. The results of key biochemical parameters, Scanning electron microscope(SEM) andX ray diffraction (XRD) all implied that the biochemical activity was enhanced and thepassiavations were weakened with the synergistic effects of two strains. The bioleachingefficiency was1.81times of the pure Acidithiobacillus ferrooxidans CUMT-1(A. ferrooxidans CUMT-1) system. The pH-stat, Ag+and Cl-were respectively employed forreducing the passivation formation and accelerating the rate of biochemical reactions. Theoptimum combination system was determined as pH1.3-2.0mg·L-1Ag+-2.5g·L-1Cl-. Basedon the higher biomass and chemical reaction rate, the formation of jarosite precipitation andsulfur membrane was effectively weakened in the optimum combination system. The finalCu2+concentration and bioleaching efficiency reached55.5mg·L-1and55.0%, about twice ofthe control system. The exogenous energy substrates, multiple-phases pH-stat control andfed-batch fermentation strategies were respectively determnined to shorten strain-mineraladaptive phase, further weaken the jarosite passivation in the middle-end stage and reduce thesubstrate inhibition in batch bioleaching. The optimum integrated technology was determinedas2g·L-1Fe2++2g·L-1S0, three-stage pH-stat control (1.3-1.0-0.7) and fed-batch strategy(0.5+0.125×4). The verified results in the7L stirred tank fermenter showed that thebiological and chemical effects were better balanced in each bioleaching stage with theassistance of integration technology. The final Cu2+concentration, bioleaching efficiency andaverage production rate of Cu2+were89.1mg·L-1,44.1%and2.23mg·L-1·d-1, which wasimproved by about53%compared to the initial level.(3) Establishment of molecular probe assay for identification and quantification ofbioleaching microorganism. With the assistances of molecular probe, eliminated function ofnuclease S1and fluorescently labeled technique, an assay was established for identifying andquantifying bioleaching microorganism. The specific probes were designed basing on theconserved16S rRNA regions of target species. The blasted result showed that there were atleast two or more bases differences with other closelty related strains. The key operatingconditions of moleacluar probe assay were futher optimized such as the sealing and elutedconditions of low non-specific adsorption (250?L0.1%BSA,30min,0.5%PBST elution),cell disruption (300W,50%duty cycle,4min, lysates secondary crushing), the workingconcentration of probes (primary probe100nM, CP10nM, SP10nM), hybridization time (1h,0.5h,0.5h), hybridization temperature (30?,45?,45?), nuclease S1digestion time (20min) and working concentration of fluorescein antibody (1:5000diluted proportion). Thestandard curve of molecular probe assay was eventually established. The linear coefficientwas98.64%when the detection limitation was low to103cells·mL-1. The verification resultsof pure-strain, mixed-strains and bioleaching systems all showed a good specificity andaccuracy. It could be efficiently applied for analyzing the community structure in thesubsequent bioleaching process.(4) The effects of community evolutions on bioleaching of low-grade chalcopyrite in thetwo-strain system. With the molecular probe technique, the evolution of microbial structure indifferent status and its effects were systematically explored. The effects of attached cell onbiomass of free cell were significant. Although with only3.8%-11.6%of total biomass, highto19.1%-31.2%loss of free cell was caused in the none-attached cell system, especially in thepure strain of A. thiooxidans ZJJN-3system. In the two-strains system, the lack of attachedcells would weaken the community competitiveness of A. thiooxidans ZJJN-3with A.ferrooxidans CUMT-1, which meant more dependence of A. thiooxidans ZJJN-3for attachedcell. The importance of attached cell was also proved by the changes chemical of key parameters. The largest reduction of Fe2+and SO24-concentrations were all high to about40%.The results of XRD and fourier transform infrared spectroscopy (FTIR) analysis of slag allimplied that the sulfur metabolism was closely related with the attached cell.37.5%-50.5%oreven higher efficiency was closely associated with the adsorption process. On the basis of theequilibrium between biological and chemical effect, the techniques and strategies forenhancing chalcopyrite bioleaching was summarized. Futuremore, the bioleaching mechanismmodel of low-grade chalcopyrite was preliminarily established.