Mechanism Of Heavy Metal Biosorption By The Recombinant Photosynthetic Bacteria And Their Reuse Of Cadmium

To develop low-cost,high-efficiency,and recyclable methods for metal remediation is important and necessary for the prevalence of its contamination.Compared with traditional physical/chemical methods,biosorption has its advantages in controlling and treating metal pollution because of its economic and energy-saving features,high treatment efficiency,and easy separation and recovery of the adsorbates.At present,although there are some studies on the genetic modification of autotrophic microorganisms（photosynthetic bacteria）to improve their ability to adsorb and reclaim metals,the biological mechanisms associated are not fully understood.In this thesis research,we constructed a recombinant Rhodopseudomonas palustris expressing with the metallothionein（MT）from the freshwater crab Sinopotamon henanense.The recombinant R.palustris/pSCMT was tested for its tolerance to Cd²⁺,Zn²⁺and Cu²⁺,and its corresponding biosorption performance patterns and influencing factors was explored.The characteristics and application potential of the biosynthetic CdSe-QDs by the bacteria were also assessed and analyzed.The main findings are as follows:1.The recombinant R.palustris/pSCMT was successfully constructed with stable expression of ShMT.The optimal electroshock transformation conditions such as resistance,electric field strength,capacitance,electroshock time and amount of plasmid DNA were 100Ω,15-20 k V/cm,25μF,5 ms and 0.1μg,respectively.And the ideal induction conditions for ShMT expression were 30℃,0.5 m M IPTG and4-fold dilution.2.Characterization of heavy metals biosorption by the recombinant R.palustris/pSCMT.The sensitivity order of the recombinant to the metal ions tested was Cd²⁺>Cu²⁺>Zn²⁺;the maximum tolerance concentrations were 21.98,81.03 and97.54 mg/L,respectively,with its sensitivity toward Cd²⁺and Cu²⁺being higher than the wild-type.The maximum biosorption of Cd²⁺and Cu²⁺by the recombinant was224.22 and 233.64 mg/g,respectively,which was about twice higher than the wild-type;while the maximum biosorption of Zn²⁺was 99.80 mg/g.The pseudo-second order and Langmuir models were more suitable for the recombinant to describe the metals biosorption processes.Unlike the wild-type,the biosorption process of Cu²⁺and Zn²⁺by the recombinant were also characterized by particle diffusion,in which Cd²⁺was also controlled by temperature and non-homogeneous sorption.Moreover,the recombinant was more adaptable to the changes of environmental factors,and its biosorption capacity and removal rate of metals were relatively stable.3.Mechanism of heavy metals biosorption by the recombinant R.palustris/pSCMT.The proportion of intracellular biosorption of Cd²⁺and Cu²⁺by the recombinant was significantly increased about 2 times higher than those by the wild-type,while no significant change was observed for Zn²⁺.Compared to the wild-type,the functional groups involved in the biosorption of Zn²⁺and Cu²⁺were increased in the recombinant,but for Cd²⁺it was decreased.The participating functional groups in the recombinant cells were more specific,where the-SO₃ group of proteins,the PO₂^-group on phospholipids or the C=O and C-N chemical bonds in amide I were associated with Cd²⁺and Cu²⁺adsorption;the-OH or-NH bonds on carbohydrates were associated with Zn²⁺biosorption.After biosorption of metals,the surface structure of the recombinant became rough and wrinkled with nanoparticles.Different from the wild-type,the recombinant SOD activity decreased and GSH content increased after biosorption of Cd²⁺,Zn²⁺,but it was opposite for Cu²⁺.Unlike the wild-type,the recombinant sodC expression was up-regulated 2.3～4.3-fold but its ftsH expression was down-regulated 44.3～54.9%after biosorption of Cd²⁺and Cu²⁺,which were opposite for Zn²⁺.The relative expression changes of above genes were opposite to that of the wild-type.After biosorption of heavy metals,Shmt and suf S expression were up-regulated 2～5.3-fold,and cadA and copC expression were down-regulated 22.1～74.5%,while those in the wild-type was up-regulated1.3～6.5-fold.This indicated that the recombinant have elevated antioxidant and biosynthetic capacity with decreased cellular exocytosis.4.Reuse of heavy metal cadmium by the recombinant R.palustris/pSCMT.The biosynthesis of CdSe-QDs by the recombinant was successful with the synthetic yields and quantum yields of the products being 15.32±1.25 mg/g and 3.85±0.32%,respectively,which were about 3～4 times higher than those of the wild-type.The fluorescence characteristic peaks of the biogenic CdSe-QDs appeared at 460 and 530nm,and their average particle size was 5.37±0.06 nm.They were cubic crystal structures with lattice spacing d of 3.6 and 2.1（?）.The mechanism of its biosynthesis may be related to ion exchange,protein binding and bacterial cell division,where the-SH and-OH groups may be crucial to maintain the binding stability of the bacteria.During the synthesis process,their SOD activity was decreased,but GSH and total protein contents were increased.Their Shmt and suf S expression were up-regulated2.82～3.58-fold,which was 6-fold higher than that of the wild-type,while the ftsH,sodC,cadA and copC expression were down-regulated by about 20～45%,which was the opposite of those found in the wild-type.Therefore,the biogenic CdSe-QDs were less toxic to HCT116 cells with an IC₅₀ of 389.01μM,which could be used for cell imaging assays.In conclusion,this study constructed the recombinant R.palustris/pSCMT and used it to synthesize biogenic CdSe-QDs with success.It was also confirmed that the recombinant has better biosorption and reclamation capacities of the metals studied.The expression of ShMT in the recombinant could have influences on the biosorption pattern,functional groups involved,biochemical indicators and gene responses which also regulated the processes of cellular exocytosis,oxidative stress and reduction reactions.Therefore,the results of this study could provide the scientific basis and information regarding future application of the recombinant in the treatment and reuse of metal.