| Although, the synergetic interactions between chemolithoautotroph At. ferrooxidans and heterotroph Aph. acidophilum in bioleaching and acid mine drainage (AMD) environment have drawn a share of attention, in-depth research regarding synergetic interactions are still unknown on physiological and transcriptional level. To gain a better understanding of the synergic interactions and ecological functions between these two species that commonly occurred in bioleaching system and AMD environment, a series of research regarding the co-culture of these two species have been conducted.The content of researches included:(1) evaluation of the accuracy of RT-qPCR quantified growth dynamics of At. ferrooxidans and Aph. acidophilum;(2) a co-culture composed of At. ferrooxidans and Aph. acidophilum were successfully acclimated in this study, the growth dynamics and physiological activity were monitored;(3) the expression difference of carbon and iron metabolism related genes between At. ferrooxidans pure culture and its co-culture with Aph. Acidophilum was studied;(4) the stability of co-culture which consists of Aph. acidophilum and At. ferrooxidans separately exposed to four metal ions (Cd2+, Cu2-, Ni2+and Mg2+) was tested;(5) the growth dynamics and physiological activity of At. ferrooxidans and its natural co-culture with Aph. acidophilum in media with or without glucose were measured respectively;(6) this co-culture was also applied to bioleaching of pyrite and low grade chalcopyrite.The results of these researches are listed as follow:(1) for accurate relative cell number quantification of the At. ferrooxidans and Aph. acidophilum, the total cell number in culture sample should be between6.52×109cells and2.61×1010cells; and the specific primers of these two species ensured the specificity of the results respectively;(2) A co-culture composed of At. ferrooxidans and Aph. acidophilum has been successfully acclimated in this study, and depending on the RT-qPCR quantified growth dynamics, the At. ferrooxidans in co-culture entered earlier and had2days longer exponential phase, obtained5times more cell number than that in pure culture.(3) the ferrous iron concentration in culture medium and the expression of iron oxidation related genes revealed that the energy acquisition of At. ferrooxidans in co-culture was more efficient than that in pure culture. Furthermore, the analysis of CO2fixation related genes in At. ferrooxidans indicated that the second copy of RuBisCO encoding genes cbbLS-2and the positive regulator encoding gene cbbR were up-regulated in co-culture system;(4) In the Cd2+, Cu2+, Ni2+and Mg2+metal resistance experiment, heterotrophic bacteria Aph. acidophilum facilitated the ferrous iron oxidation by At. ferrooxidans and improved its efficiency of energy utilization. The maximum tolerant concentration (MTC) of At. ferrooxidans to Cu2+was improved from2.0g/L to5.0g/L by Aph. acidophilum, and the cell density of co-culture in5.0g/L Cu2+was almost the same as purely cultured At. ferrooxidans in2.0g/L Cu2+. In addition, the MTC of co-cultured At. ferrooxidans to Mg2+was also improved from12.0g/L to17.0g/L by Aph. acidophilum.(5) whether glucose was added in culture media or not, the Fe2+oxidation efficiency of At. ferrooxidans is higher in co-culture than that in pure culture. When the concentration of glucose is5g/L, pure culture of At. ferrooxidans couldn’t oxidize Fe2+while the co-culture could finish the Fe2+oxidation in100h, and the pH is higher when more glucose was added in both cultures. Without glucose, the cell number ratio of At. ferrooxidans to Aph. acidophilum in co-culture was about100:1, which suggested the usual cell number ratio between autotrophic bacteria and heterotrophic bacteria in AMD environment. In both pure and co-culture condition, the cell number of At. ferrooxidans decreased and the lag phase prolonged with the increase of glucose concentration; while in the case of Aph. acidophilum in co-culture, the cell number and lag phase showed a reverse trend;(6) In bioleaching experiment, the pyrite bioleaching efficiency of co-culture increased by22.70%as compared with that of purely cultured At. ferrooxidans. While in the low grade chalcopyrite bioleaching system with few iron, the bioleaching efficiency of both At. ferrooxidans and its co-culture with Aph. acidophilum were lower than33%. In the low grade chalcopyrite bioleaching system with pre-added2g/L Fe2+, the bioleaching efficiency of At. ferrooxidans and its co-culture with Aph. acidophilum were raised to41.27%and52.22%, respectively.In conclusion, a co-culture composed of At. ferrooxidans and Aph. acidophilum were successfully acclimated in this study and the relative cell number can be quantified accurately. Aph. acidophilum could heterotrophically grow with At. ferrooxidans and promote the growth of it. By means of activating iron oxidation related genes and the2nd set of cbbLS genes in At. ferrooxidans, the Aph. acidophilum facilitated the iron oxidation and CO2fixation by At. ferrooxidans. Since Aph. acidophilum facilitated the Fe2+oxidation by At. ferrooxidans and reduced the inhibition by glucose, the addition of organic compounds such as glucose may not be a good AMD bio-remediation strategy. For efficient Fe2+oxidiation, the proper cell number of At. ferrooxidans should be100times higher than that of Aph. acidophilum. At. ferrooxidans and Aph. acidophilum in co-culture could maintain their physiological stability and sustain their ecological function under environmental stress. The bioleaching results suggested that acidophilic heterotrophic bacteria Aph. acidophilum should be applied to the bioleaching system with high iron concentration, in which it could collaborate with iron oxidation bacteria to improve the bioleaching efficiency. |
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