Microbial perchlorate reduction with elemental sulfur and other inorganic electron donors

Abstract

${\text{ClO}}_4^{-}$ has recently been recognized as a widespread contaminant of surface and ground water. This research investigated chemolithotrophic perchlorate reduction by bacteria in soils and sludges utilizing inorganic electron-donating substrates such as hydrogen, elemental iron, and elemental sulfur. The bioassays were performed in anaerobic serum bottles with various inocula from anaerobic or aerobic environments. All the tested sludge inocula were capable of reducing perchlorate with H₂ as electron donor. Aerobic activated sludge was evaluated further and it supported perchlorate reduction with Fe⁰ and S⁰ additions under anaerobic conditions. Heat-killed sludge did not convert ${\text{ClO}}_4^{-}$, confirming the reactions were biologically catalyzed. ${\text{ClO}}_4^{-}$ (3 mM) was almost completely removed by the first sampling time on d 8 with H₂ (⩾0.37 mM d⁻¹), after 22 d with S⁰ (0.18 mM d⁻¹) and 84% removed after 37 d with Fe⁰ additions (0.085 mM d⁻¹). Perchlorate-reduction occurred at a much faster rate (1.12 mM d⁻¹), when using an enrichment culture developed from the activated sludge with S⁰ as an electron donor. The enrichment culture also utilized S²⁻ and ${\text{S}}_2{\text{O}}_3^{2-}$ as electron-donating substrates to support ${\text{ClO}}_4^{-}$ reduction. The mixed cultures also catalyzed the disproportionation of S⁰ to S²⁻ and ${\text{SO}}_4^{2-}$. Evidence is presented demonstrating that S⁰ was directly utilized by microorganisms to support perchlorate-reduction. In all the experiments, ${\text{ClO}}_4^{-}$ was stoichiometrically converted to chloride. The study demonstrates that microorganisms present in wastewater sludges can readily use a variety of inorganic compounds to support perchlorate reduction.

Introduction

${\text{ClO}}_4^{-}$ has recently been recognized as a widespread contaminant of surface and ground water in the United States (US). In the Western US, ${\text{ClO}}_4^{-}$ has been found in water supplies of over 15 million people (US-EPA, 1999, US-EPA, 2003). The majority of ${\text{ClO}}_4^{-}$ in the environment comes from the discharge of ammonium ${\text{ClO}}_4^{-}$, an oxidant used as rocket and missile fuel propellant (Motzer, 2001, Urbansky, 2002). ${\text{ClO}}_4^{-}$ has adverse health effect on humans by interfering with the body’s intake of iodine, and thus inhibiting thyroid hormone production (US-EPA, 2002). With its widespread occurrence in water supplies and its potential toxicity, ${\text{ClO}}_4^{-}$ is now on the US EPA drinking water contaminant candidate list with a reference dose of 0.0007 mg kg⁻¹ d⁻¹. Currently, the US EPA Superfund preliminary remediation goal for ${\text{ClO}}_4^{-}$ is 24.5 ppb. In California, the ${\text{ClO}}_4^{-}$ notification level in drinking water is 6 ppb (CDHS, 2005).

${\text{ClO}}_4^{-}$ is chemically inert but it can easily be biotransformed to the environmentally benign end product, Cl⁻. The ${\text{ClO}}_4^{-}/{\text{Cl}}^{-}$ pair has a standard electrode reduction potential (E⁰′) of 1.29 V, which makes ${\text{ClO}}_4^{-}$-reduction thermodynamically feasible with various substrates. Microbial ${\text{ClO}}_4^{-}$-reduction proceeds to O₂ and Cl⁻ via the intermediates ${\text{ClO}}_3^{-}$ and ${\text{ClO}}_2^{-}$, and the reduction of ${\text{ClO}}_4^{-}$ to ${\text{ClO}}_3^{-}$ is the rate-limiting step (Rikken et al., 1996, Logan, 2001). A broad spectrum of facultative microorganisms that can grow utilizing ${\text{ClO}}_4^{-}$ as the sole electron acceptor can be readily isolated from pristine to contaminated environments with simple organic electron-donating substrates such as acetate (Bruce et al., 1999, Coates et al., 1999, Achenbach et al., 2001, Logan et al., 2001). Continuous column bioreactors for ${\text{ClO}}_4^{-}$-reduction utilizing organic substrates have also been evaluated (Herman and Frankenberger, 1999, Giblin et al., 2000a, Kim and Logan, 2001, Min et al., 2004). Although ${\text{ClO}}_4^{-}$ removal was successful in the above-mentioned research, organic residual is a concern because it could stimulate bacterial growth in water distribution systems and interfere with chlorination processes producing disinfection byproducts.

Inorganic electron donors can overcome the disadvantages of organic substrates, and thus are currently the focus of the study for biological reduction of ${\text{ClO}}_4^{-}$. Batch and continuous flow bioreactors have been evaluated utilizing H₂ (Giblin et al., 2000b, Miller and Logan, 2000, Logan et al., 2004, Nerenberg and Rittmann, 2004), and Fe⁰ (Yu et al., 2006). Although H₂ is a good energy substrate, its handling and storage may be a safety issue. Fe⁰ is successfully applied in permeable reactive barriers for the remediation of chlorinated volatile organic compounds (Richardson and Nicklow, 2002, Lai et al., 2006), and could be an alternative for ${\text{ClO}}_4^{-}$-reduction. Fe²⁺ and S²⁻ are examples of other inorganic electron donors used by ${\text{ClO}}_4^{-}$-reducing bacteria (Achenbach et al., 2001, Weber et al., 2006). Granular S⁰ in packed bed bioreactors has been used successfully as electron-donating substrate in autotrophic denitrification processes for drinking water (Flere and Zhang, 1999, Koenig and Liu, 2001, Xu et al., 2003). Granular S⁰ is insoluble and thus provides a slow-release supply of electrons on demand, offering advantages of low maintenance and low cost. Therefore its potential as an electron-substrate for ${\text{ClO}}_4^{-}$-reduction should be explored. The expected stoichiometry of the reaction is as follows:

$$3{\text{ClO}}_4^{-}+4{\text{S}}^0+4{\text{H}}_2\text{O}\to 4{\text{SO}}_4^{2-}+8{\text{H}}^{+}+3{\text{Cl}}^{-}$$

The objective of this research is to evaluate various inoculum sources for their ability to utilize inorganic compounds such as H₂, Fe⁰ and reduced sulfur compounds (i.e., S⁰, S²⁻ and ${\text{S}}_2{\text{O}}_3^{2-}$) as electron-donating substrates for the chemolithotrophic reduction of ${\text{ClO}}_4^{-}$. In this study, we report the first clear evidence of microbial ${\text{ClO}}_4^{-}$-reduction linked to the oxidation of S⁰. The coupling of ${\text{ClO}}_4^{-}$-reduction to chloride with elemental sulfur oxidation to sulfate is an exergonic reaction with standard Gibb’s free energy $(\mathrm{\Delta }{G}^{0\prime })$ of −1146.9 kJ mol⁻¹ ${\text{ClO}}_4^{-}$ (37.6 mol ATP mol⁻¹ ${\text{ClO}}_4^{-}$). This chemolithotrophic reaction is bioenergetically comparable to chemoorganotrophic perchlorate reduction, for example the $\mathrm{\Delta }{G}^{0\prime }$ of perchlorate reduction linked to acetate oxidation is −1210.9 kJ mol⁻¹ ${\text{ClO}}_4^{-}$ (39.7 mol ATP mol⁻¹ ${\text{ClO}}_4^{-}$).