Isolation And Identification Of An Acid-tolerant Microalgae And Its Physiological Response To Metal Ion Stress

Microalgae is an important primary producer in Aacid mine drainage（AMD）,which can adapt to the stress of environmental conditions such as low pH,high sulfate and high concentration of dissolved toxic metals,and has the ability to remove heavy metals,improve environmental pH and increase the concentration of dissolved oxygen,so it is a potential resource for AMD in situ remediation.In this study,an acid-tolerant microalgae was isolated from the waste reservoir of an acid mine in a pyrite mine in Anhui Province.The effects of pH and concentration of typical metal ions Mn²⁺and Cu²⁺on its growth characteristics were investigated.The changes of biomass,photosynthetic pigment,malondialdehyde（MDA）,sulfhydryl group and reduced glutathione（GSH）contents,and the specific activities of superoxide dismutase（SOD）and ascorbate peroxidase（APX）of the microalgae were determined to analyze the physiological response mechanism of the microalgae to Mn²⁺and Cu²⁺stress.（1）A purified algal strain was isolated and identified as Graesiella sp.MA1 by morphological observation and 18S rDNA sequencing.It was found that the alga grew well under the condition of pH 7.0 to 5.0,and the lowest pH it can tolerate was 3.5.The changes of growth process,alkali-producing capacity and oxygen-increasing capacity under different initial inoculation concentrations were studied at the initial pH of 3.5.It was found that the specific growth rate(0.3d^-1)was the highest in each group when the initial inoculation OD value was 0.3.The final dissolved oxygen and pH values of all experimental groups were stable around 21 mg/L and 10.0,which indicated that the alga had significant capacity of increasing oxygen and producing alkali.（2）The maximum tolerance concentrations of Graesiella sp.MA1 to Mn²⁺and Cu²⁺under acidic conditions were 55 mg/L and 6 mg/L,respectively.The removal rates of 55mg/L Mn²⁺and 6 mg/L Cu²⁺by Graesiella sp.MA1 at pH 3.5 were 18.55%and 18.23%,respectively,and 44.89%and 87.98%at pH 7.0,respectively.（3）Under the condition of pH 3.5,the results of Mn²⁺and Cu²⁺concentration stress tests showed that the biomass and alkali production capacity of Graesiella sp.MA1 at24 d were inhibited with the increase of concentration of heavy metals,and the chlorophyll a/b value was significantly decreased,indicating that the photosynthetic process was affected.The increase of MDA content indicated that the alga was under the oxidative stress of Mn²⁺and Cu²⁺,and the increase of soluble protein,soluble polysaccharide,carotenoid,SOD and APX activities indicated that the alga could reduce the toxic effect of heavy metals by regulating cell osmotic capacity and antioxidant capacity.Mn²⁺concentration significantly upregulated the contents of sulfhydryl and GSH,while Cu²⁺showed opposite effects on sulfhydryl and GSH.（4）On the 6th day,compared with the control group at pH 7.0,the biomass, chlorophyll a/b,carotenoid,and sulfhydryl group content,SOD and APX activities of the alga in the control group at pH 3.5 were significantly inhibited,while the contents of soluble protein,polysaccharide and GSH were not significantly changed.In conclusion,Graesiella sp.MA1 isolated and purified from acidic mine waste reservoir can tolerate low pH,Mn²⁺and Cu²⁺within a certain concentration range.Under such extreme environment,its intracellular antioxidant system can reduce membrane lipid peroxidation,thus playing an important role in tolerance to heavy metal ions and detoxification.Since Graesiella sp.MA1 can tolerate low pH,heavy metal ions and increase oxygen and alkali production,this study provides a theoretical basis for in situ bioremediation of acidic mine wastewater using microalgae.