Mechanism Of Chromium Isotope Fractionation During Microbial Cr(?) Reduction

Hexavalent chromium[Cr(VI)]is a major contaminant found at many industrial sites,such as metal plating,paints and pigments,leather and wood preservation.Cr(VI)is soluble and exhibit toxic and mutagenic effects,while Cr(?)is relatively insoluble and considerably less toxic.Microbial activities can effectively and cost-effectively reduce Cr(?)into Cr(?)in groundwater.Cr isotope analysis is a promising approach for monitoring and quantifying microbial reduction of Cr(VI)at polluted sites.Most physical processes,such as sorption,dilution,and volatilization do not exhibit significant Cr isotope fractionation.Meanwhile,Cr isotope data is indicative of Cr(VI)reduction on a relatively large scale in groundwater.The measurements of Cr isotope fractionation factors and the understanding of mechanism of fractionation are key steps to use the Cr isotopes to assess the extent of microbial Cr(VI)reduction.Therefore,this dissertation systematically investigated mechanism of microbially induced Cr isotope fractionation.The results of this dissertation were summarized as follows:1.The influence of critical factors on observed Cr isotope fractionation during Cr(VI)reduction by Shewanella oneidensis MR-1In temperature-control experiments,Cr isotope fractionation showed two-stage behavior:during Stage I,values of the enrichment factors(?)were-2.81 ± 0.19‰ and-2.60 ± 0.14‰ at 180C and 34?,respectively;during Stage ?,as Cr(VI)reduction progressed,Cr isotope fractionation was significantly masked,and the ? values decreased to-0.98 ± 0.49‰ and-1.01 ± 0.11‰ at 18? and 34?,respectively.Similar two-stage isotope fractionation behaviors were observed in pH-control experiments(pH=6.0 and 7.2)and in experiments with or without the addition of a competing electron acceptor(nitrate).These results indicated that the extent of Cr isotope fractionation was not affected by changes in ambient environmental conditions.The masking of isotope fractionation in Stage ? indicated restrictions on the bioavailability of Cr(?)and mass-transfer limitations.The observable Cr isotope fractionation can be used as a promising diagnostic tool to detect the bioavailability indices.2.Cr isotope fractionation during Cr(VI)uptake process by S.oneidensis MR-1 and the impact of bioavailability on Cr isotope fractionationDuring Cr(VI)reduction by S.oneidensis MR-1,Cr(VI)reduction mainly occurs on the cell surface by C-type cytochrome(OmcA and MtrC)or Cr(VI)is transported into cells by energy-dependent sulfate uptake pathway and reduces inside the cytoplasm.The mutants deficient in MtrC and OmcA(?mtrC-?omcA)are not capable of extracellular reduction and reduce Cr(?)inside the cytoplasm.Therefore,?mtrC-?omcA is a good candidate to investigate Cr isotope fractionation during Cr(?)uptake process.We investigated the relationship between isotope fractionation,bioavailability,and initial Cr(?)concentration.We attempted to observe Cr isotope fractionation during Cr(?)uptake process on the condition of extremely constrained bioavailability by changing the electron donor concentration and initial Cr(?)concentration.When isotope fractionation of bond reconfiguration was completely masked by bioavailability restriction,the observed fractionation represent the fractionation during microbial Cr(?)uptake.When the initial Cr(?)concentrations were 100 ?M,50 ?M and 7 ?M,the values of ? were-1.87 ± 0.06‰,-1.27 ± 0.76‰ and-0.22 ± 0.13‰,respectively.The ? value of-0.22 ± 0.13‰ means that the step of Cr(?)uptake became rate-limiting and the isotope fractionation for bond reconfiguration was almost completely masked.We applied the Kampare model to illustrated the relationship between isotope fractionation and bioavailability and further our understanding of Cr isotope fractionation during the Cr(?)uptake through the cell membrane.3.Mechanism of Chromium isotope fractionation during Cr(?)reduction by the extreme thermophile,Caldicellulosiruptor saccharolyticusWe investigated the effects of different metabolic pathways and various environmental parameters on Cr isotope fractionation during Cr(?)reduction via the extremely thermophilic bacterium C.saccharolyticus.Cr(?)reduction mainly occurred on the cell surface by Ni-Fe hydrogenase during Cr(?)reduction by C.saccharolyticus.The activity of Ni-Fe hydrogenase was inhibited in the presence of CuCl2 and Cr(?)was transported into cells.Despite different Cr(?)reduction locations,Cr isotope enrichment factors(?)were identical within uncertainty for experiments either with or without CuCl2.The addition of neutral red(NR)functioned as electron carrier that enabled bacteria to drive the electron transfer processes.The ?values were also similar for experiments with or without NR,respectively.By comparing these ? values,the effects of different electron transport pathways and Cr(?)transfer processes,including Cr(?)transport across cell envelope and binding to reacting enzymes on Cr isotope fractionation were discussed.The step of Cr(?)uptake across the cell envelope was probably not an additional rate-limiting mass transfer barrier.Furthermore,the effects of environmental parameters on Cr isotope fractionation were tested,including high temperatures(50 and 70?),different hydrogen concentrations,and variable electron donors(glucose and hydrogen).Changes in the tested temperature and hydrogen concentration did not affect the e value.However,the e value was slightly higher with hydrogen as electron donors compared to the value observed with glucose as the electron donor.