Heavy Metal Bioaccumulation Characteristics And Mechanisms Of The Improved Cadmium Tolerance At High NaCl Concentrations Or Low PH In Pichia Kudriavzevii

Heavy metal pollution in aquatic environment leads to an increasing risk of heavy metal contamination in aquatic product, causing significant threats to human health. In recent years, a great deal of attention has been paid to the bioaccumulation of heavy metals by microorganisms, which is considered as a potential technique for heavy metal removal from complex food material environment including liquid aquatic products. Good heavy metal resistance and bioaccumulation characteristics are the necessary premise of the method and also the important assurance for the heavy metal removal ability of microorganism. This study investigated the heavy metal (cadmium, copper and zinc) tolerance, bioaccumulation characteristics and removal ability of growing multi-stress-tolerant Pichia kudriavzevii A16. The effects of high NaCl concentrations or low pH on the heavy metal tolerance, bioaccumulation characteristics and removal ability of growing P. kudriavzevii A16 were also investigated. The mechanisms of the improved cadmium tolerance at high NaCl concentrations or low pH were mainly revealed.1. The heavy metal (cadmium, copper and zinc) tolerance, bioaccumulation characteristics and removal ability of growing P. kudriavzevii A16 were studied. In all study, Saccharomyces cerevisiae CICC1211 was used as a reference for comparison. P. kudriavzevii A16 showed high tolerance and bioaccumulation of cadmium, copper and zinc. P. kudriavzevii A16 exhibited high removal abilities of cadmium and zinc: the removal rate of above 95% was achieved for cadmium and zinc in P. kudriavzevii A16 when initial cadmium and zinc concentrations were respectively less than 0.04 and 0.1 mmol/L. The heavy metal bioaccumulation of both yeasts was significantly affected by other heavy metals:the cadmium bioaccumulation per unit weight of biomass was obviously inhibited by copper and zinc; the copper bioaccumulation per unit weight of biomass was obviously enhanced by cadmium and zinc; the zinc bioaccumulation per unit weight of biomass was inhibited by cadmium and copper for P. kudriavzevii A16 but improved for S. cerevisiae CICC1211. P. kudriavzevii A16 possessed more powerful heavy metal removal ability compared with S. cerevisiae CICC1211 at the same bioaccumulation time, initial heavy metal concentaions and initial biomass concentrations or in the presence of other heavy metal ions.2. In order to elucidate the possibility of employing P. kudriavzevii A16 for heavy metal (cadmium, copper and zinc) removal at high NaCl concentrations or low pH, the effects of high NaCl concentrations or low pH on the heavy metal tolerance, bioaccumulation characteristics and removal ability of growing P. kudriavzevii A16 and S. cerevisiae CICC1211 (as a reference for comparison) were systematicly investigated. The results showed that the cadmium tolerance of both yeasts was significantly improved at high NaCl concentrations and low pH probably due to the decrease of cadmium bioaccumulation per unit weight of biomass, especially the intracellular cadmium bioaccumulation. Differently from the effect on the cadmium tolerance, the copper tolerance was obviously inhibited at high NaCl concentrations and low pH. The enhanced copper bioaccumulation per unit weight of biomass, especially the intracellular copper bioaccumulation might be an important reason for the decrease of copper tolerance. Although no obvious effect on the zinc tolerance of both yeasts was observed, the zinc bioaccumulation increased at high NaCl concentrations and low pH. Differently from S. cerevisiae CICC1211, high NaCl concentrations and low pH did not overwhelmingly inhibit the heavy metal removal rate of P. kudriavzevii A16 but improved its removal ability of copper and zinc. The heavy metal removal ability of P. kudriavzevii A16 was much higher than S. cerevisiae CICC1211 at high NaCl concentrations and low pH.3. To reveal the mechnisam of the improved cadmium tolerance at high NaCl concentrations or low pH, the RNA-Seq with de novo assembling was used to study the differential gene expression of P. kudriavzevii A16 between CdNaCl group and Cd group, and between CdHCl group and Cd group on the transcriptome level. The transcripts with FDR<0.001 and|log2(FPKM foldchange)|>2 were assigned as differentially expressed and used for functional annotation and analysis. There were 62 differentially expressed transcripts between CdNaCl group and Cd group with 23 up-regulated transcripts in CdNaCl group mainly for the function of antioxidant activity, response to stress, glycerol transport and electron carrier activity, and 39 up-regulated transcripts in Cd group mainly for the function of transmembrane transporter, ribosome biogenesis, transcription and cell wall biogenesis. For CdHCl group and Cd group, there were 61 differentially expressed transcripts with 32 up-regulated transcripts in CdHCl group with main function including response to stress, glycerol transport, electron carrier activity and antioxidant activity, and 39 up-regulated transcripts in Cd group with main function including transmembrane transporter, ribosome biogenesis, transcription and cell wall biogenesis.4. The effects of high NaCl concentrations or low pH on the proteome of P. kudriavzevii A16 under cadmium stress were studied by using two-dimensional electrophoresis. The proteins|log2(foldchange)|>1 were assigned as differentially expressed and then identified by mass spectrometrymass. There were 39 differentially expressed proteins between CdNaCl group and Cd group with 28 up-regulated proteins in CdNaCl group mainly for the function of material metabolism, energy generation, ribosome biogenesis, transcription and antioxidant activity, and 11 up-regulated proteins in Cd group mainly for the function of material metabolism and transmembrane transporter. There were 21 differentially expressed proteins between CdHCl group and Cd group with 11 up-regulated proteins in CdHCl group with main function including material metabolism and antioxidant activity, and 10 up-regulated proteins in Cd group with main function including material metabolism and transmembrane transporter.5. The key genes related to the function for the improvement of cadmium tolerance of P. kudriavzevii A16 were quantitatively measured by using SYBR Green qRT-PCR. It was shown that high NaCl concentrations or low pH significantly improved the expression of antioxidant enzyme genes including SODC, SODM, PRX1, PRX2D and CATA in P. kudriavzevii A16 under cadmium stress. The changes of antioxidant enzyme activities were consistent with that of the expression of antioxidant enzyme genes that the activities of T-SOD, POD and CAT in P. kudriavzevii A16 under cadmium stress were all improved at high NaCl concentrations or low pH. The enhanced expression of antioxidant enzyme genes might be an important reason for the decrease of the reactive oxygen species (ROS) accumulation, cell death and oxidative damage of proteins and lipids induced by cadmium, contributing to the improvement of the yeast growth. Similarly to the expression of antioxidant enzyme genes, the expression of YCF1, GST, HSP12 and RAD57 in P. kudriavzevii A16 under cadmium stress was also significantly enhanced at high NaCl concentrations or low pH, which might play an important role in the decrease of cadmium toxity and oxidative stress and maintenance of cell stability.Heavy metal pollution is one of the most serious safety problems in aquatic products. Recently, there is no feasible way for solving the problem of heavy metal polution in aquatic products. This study is important to provide significant clues for appling heavy metal bioaccumulation of microorganisms in liquid aquatic products, new ideas for improving the heavy metal tolerance and bioaccumulation of microorganisms, and important biological information for fundamental biological research and application of P. kudriavzevii.