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Final Progress Reports: Michigan State University: Reductive Processes for Bioremediation of Chlorinated Solvent-Metal Mixtures

Superfund Research Program

Reductive Processes for Bioremediation of Chlorinated Solvent-Metal Mixtures

Project Leader: Craig S. Criddle (Stanford University)
Grant Number: P42ES004911
Funding Period: 2000 - 2006

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Final Progress Reports

Year:   2004 

Wild type MR1 is capable of carbon tetrachloride transformation, with concomitant production of chloroform.  Previously Dr. Criddle’s team discovered that this reaction is mediated by supernatant factors.  About 20% of the CT converted by this supernatant is converted to chloroform. Subsequently, the team found that the supernatant factors consist of one or more secreted small biomolecules. Studies with mutants revealed that the active compound is a derivative of DHNA. Based on LC-MS/MS data the scientists tentatively identified one of these compounds as Menaquinone-1.  They have synthesized Menaquinone-1 and confirmed that it does in fact transform CT; they have confirmed that Vitamin K2 (Menaquinone-7,8) also transforms CT.  The project investigators anticipated that Menaquinones are also likely involved in metal and humic acid reduction.  This past year their investigation has focused on the ability of the Menaquione-1 and Vitamin K2’s ability to reduce ferric iron.  The results have shown that both compounds are capable of reducing soluble iron (III).  The team has further investigated the ability to reduce solid form of iron (III) using amorphous iron (III) hydroxide.  Experimental results have clearly demonstrated that Menaquinone-1 reduced solid iron (III) with concurrent formation of ferrous iron (II).  The results also provided strong evidence that the distance electron transfer mechanisms are operative for MR-1 to reduce solid iron (III) oxide for growth.

Pseudomonas stutzeri KC produces and secretes the metal chelating agent pyridine-2,6-bis-thiocarboxylate (PDTC) which degrades and detoxifies carbon tetrachloride (CT).  This transformation is most efficient when PDTC is chelated to copper.   This past year the team has further investigated the CT transformation of the secreted compound PDTC.  The researchers found that a ratio of 1:1 Cu to PDTC resulted in the fastest transformation with only trace amount of chloroform formation and removal of up to 4 mole of carbon tetrachloride.  These results confirm that the PDTC-Cu complex is both a reactant and a catalyst in carbon tetrachloride transformation.  They also indicate that a catalytic mechanism is operative in reducing environments.  A highly sensitive and specific detection method for PDTC was developed using LS/MS/MS to quantitatively measure the real-time production of this compound by Pseudomonas stutzeri KC.  The measurement of PDTC production from an actively growing Pseudomonas stutzeri KC culture indicated also that PDTC production was evidently growth-associated as its concentration peaked at the end of the growth phase and stabilized in the stationary phase.  The result also revealed that the amount of CT reduced greatly exceeds the real-time PDTC concentration by several orders of magnitude indicating that the organism is possibly constantly producing PDTC in the presence of CT.  Currently, the researchers are investigating the PDTC production in the presence and absence of high concentration of CT to confirm whether the PDTC gene is turned on and remained on for the production of PDTC when CT is present. 

An alternative strategy for CT biodegradation is reduction abiotically by reduced iron species.  During the past year Dr. Criddle’s team finished studies on the influence of amine buffers on carbon tetrachloride (CCl4) reductive dechlorination by the iron oxide magnetite (FeIIFeIII2O4).  A baseline was provided by monitoring the reaction in a magnetite suspension containing NaCl as a background electrolyte at pH 8.9.  The baseline reaction rate constant was measured at 7.1x10-5 ±6.3x10-6 Lm-2hr-1.  Carbon monoxide (CO) was the dominant reaction product at 82% followed by chloroform (CHCl3) at 5.2%.  In the presence of 0.01 M Tris(deuteroxymethyl)aminomethane (TRISd), the reaction rate constant nearly tripled to 2.1x10-4 ±6.5x10-6 Lm-2hr-1 but only increased the CHCl3 yield to 11% and did not cause any statistically significant changes to the CO yield.  Reactions in the presence of Triethylammonium (TEAd) (0.01 M) increased the rate constant by 17% to 8.6x10-5 ±8.1x10-6 Lm-2hr-1 but, only increased the CHCl3 yield to 8.8% while leaving the CO yield unchanged.  The same concentration of N,N,N’,N’-tetraethylethylenediamine (TEEN) increased the reaction rate constant by 18% to 8.7x10-5±4.8x10-6 Lm-2hr-1 but enhanced the CHCl3 yield to 34% at the expense of the CO yield that dropped to 35%.  Previous work has shown that CHCl3 can be generated either through hydrogen abstraction by a trichloromethyl radical (.CCl3), or through proton abstraction by the trichlorocarbanion (-:CCl3).  These two possible hydrogenolysis pathways were examined in the presence of deuterated buffers. Deuterium tracking experiments revealed that proton abstraction by the trichlorocarbanion was the dominant hydrogenolysis mechanism in the magnetite buffered, TRISd and TEAd systems.  The only buffer that had minimal influence on both the reaction rate and product distribution was TEAd. Based on these results, the researchers recommend pre-screening any buffers to minimize buffer induced changes in reaction rates and product distributions.  An alternate approach would be to utilize the buffer capacity of the oxide surface and avoid organic buffer interactions entirely.   While this study addresses the impact and practical limitations of using buffers in laboratory studies to control pH it also has implications for translating laboratory data to field studies.  For contaminated systems that contain natural organic material or co-contaminants with functionalities like those in buffers, rates and product distributions may be impacted.

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