Study On Resistance Characteristics And Bioaccumulation Mechanisms Of Rhodotorula Mucilaginosa J5 To Antimony

Microbial adsorption is one of the potential technologies for the pollution remediation due to its safety,economy and environmental protection.It has also attracted widespread attention at home and abroad.With this research background,the selection,breeding,and application of heavy metal antimony adsorptive microorganisms were investigated in this thesis.The resistance characteristics and adsorptive mechanisms were discussed.The results of this study are beneficial for providing excellent microbial resources and powerful technical support for the industrial application of antimoy-containing waste water treatment by biosorption method.The main researches and the results are summarized as follows.1.The selection and resistance level testing of antimony-resistant strains was carried out.Polluted soils of different land properties among smelting area,road nearby ore,mining area and ore tailing area of antimony mine area in Xikuangshan,Hunan were collected as the source of strains.A Gram-positive,aerobic,budding reproduction fungus was isolated from an agricultural soil samples near the smelter of northern mine,named as J5.The 18S rRNA sequence homology analysis and NCBI database comparison showed that the strain belongs to the Genus Rhodotorula,and its gene bank registration number is MH611511.The growth rate of J5 in liquid medium containing 50mg/L Sb（Ⅲ）was better than that of the control group,and the minimum inhibitory concentration was 1200mg/L,denoting the Rhodotorula mucilaginosa J5 has a good application in the antimony pollution repair.2.The mechanism of resistance of R.mucilaginosa J5 to Sb（Ⅲ）was studied.The results showed that the anti-Sb（Ⅲ）ability of the strain was not strong and unstable,and the plasmid extraction test did not find any plasmid bands.Therefore,we initially proposed the idea that the resistance gene may be located on the chromosome.After that,the cDNA library of the liquid sample was constructed using two yeast liquid samples containing Sb（Ⅲ）0 and 300mg/L,and the transcriptome sequencing analysis was performed using the Illumina-Hiseq2500 sequencing platform.The results are as follows:A total of 1311 significantly differently expressed genes（DEGs）before and after Sb（Ⅲ）stress.Among them,Sb（Ⅲ）stress promotes the synthesis of glutathione（GSH）and Fe-S clusters by stimulating the up-regulated expression of cysteine（Cys）and iron-sulfur cluster assembly protein,and Fe-S cluster oxidizes a part of Sb（Ⅲ）to a less cytotoxic Sb（V）,GSH binds to antimony and excretes it out of the cell,which may be an important role in inducing the enhancement of antimonial resistance.Under the stress of Sb（Ⅲ）,the expression of cationic antiporter（CAXs）was up-regulated,which promoted the transport of antimony to vacuole,thus the detoxification ability was improved In addition,heavy metal causes oxidative stress,resulting in lipid peroxidation.The peroxidase（POD）gene inhibits it by down-regulation and protects cells from damage.Heat shock proteins（HSPs）play an important role in protecting organisms from heavy metal toxicity by up-regulating expression.3.Optimization for the adsorption of Sb（Ⅲ）from aqueous solutions by Rhodotorula mucilaginosa Strain J5.The single factor experiment,Plackett–Burman experiment,steepest ascent design and Central Composite Design（CCD）methodology were employed to study the adsorption rate of Sb（Ⅲ）from aqueous solution by Rhodotorula mucilaginosa J5.The 13influencing factors such as the carbon source were set up,and a quadratic model for predicting the optimum adsorption conditions was derived which determined the best conditions of adsorption.The adsorption mechanism was also studied.The study showed that pH,Fe²⁺concentration,and temperature of Sb（Ⅲ）as significant factors.The adsorption rate reached 56.83%at the optimized conditions of glucose 2%（m/V）,CaCl₂ 0.5%（m/V）,beef extract 0.75%（m/V）,peptone 0.75%（m/V）,Fe²⁺concentration 1.51g/L,inoculation dose4%（V/V）,pH=3.50,temperature 29℃,agitation speed 150r/min and Sb（Ⅲ）initial concentration 10¹2mg/L.4.The adsorption mechanism of growth strain R.mucilaginosa J5 on Sb（Ⅲ）was discussed.Firstly,SEM-EDS showed that physical adsorption occurred readily on the loose and porous surface of Rhodotorula mucilaginosa J5,accompanied by ion exchange of Na⁺,Ca²⁺ions with Sb（Ⅲ）simultaneously.Secondly,FTIR spectrum showed that active groups such as carboxyl,amidogen,phosphate group,and polysaccharide C-O participated absorption by complexation with Sb（Ⅲ）.XRD analysis indicated that the growth strain R.mucilaginosa J5 adsorbed Sb（Ⅲ）and formed new compounds:Ca₂Fe（SbO₆）,Ca₁₁Sb₁₀,FeSb₂O₆ and FeSbO₄,suggesting that the addition of Ca²⁺is beneficial to enhance the adsorption capacity.Lastly,when the liquid medium contained DCC inhibiting ATP activities,the type of the growing cell not only improved the removal efficiency,but also prevented intracellular Sb（Ⅲ）re-released into the environment,to avoid the secondary pollution.It is revealed that Rhodotorula mucilaginosa is a potentially green and low-cost absorbent for the treatment of Sb（Ⅲ）contaminated water.