Growth of Pseudomonas chloritidismutans AW-1T on n-alkanes with chlorate as electron acceptor

Applied Microbiology and Biotechnology, Jun 2009

Microbial (per)chlorate reduction is a unique process in which molecular oxygen is formed during the dismutation of chlorite. The oxygen thus formed may be used to degrade hydrocarbons by means of oxygenases under seemingly anoxic conditions. Up to now, no bacterium has been described that grows on aliphatic hydrocarbons with chlorate. Here, we report that Pseudomonas chloritidismutans AW-1T grows on n-alkanes (ranging from C7 until C12) with chlorate as electron acceptor. Strain AW-1T also grows on the intermediates of the presumed n-alkane degradation pathway. The specific growth rates on n-decane and chlorate and n-decane and oxygen were 0.5 ± 0.1 and 0.4 ± 0.02 day−1, respectively. The key enzymes chlorate reductase and chlorite dismutase were assayed and found to be present. The oxygen-dependent alkane oxidation was demonstrated in whole-cell suspensions. The strain degrades n-alkanes with oxygen and chlorate but not with nitrate, thus suggesting that the strain employs oxygenase-dependent pathways for the breakdown of n-alkanes.

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Growth of Pseudomonas chloritidismutans AW-1T on n-alkanes with chlorate as electron acceptor

Farrakh Mehboob 0 Howard Junca 0 Gosse Schraa 0 Alfons J. M. Stams 0 0 H. Junca AG Biodegradation, Helmholtz-Zentrum fr Infektionsforschung, Inhoffenstrae 7, 38124 Braunschweig, Germany 1 ) Laboratory of Microbiology, Wageningen University , Dreijenplein 10, 6703 HB Wageningen, The Netherlands Microbial (per)chlorate reduction is a unique process in which molecular oxygen is formed during the dismutation of chlorite. The oxygen thus formed may be used to degrade hydrocarbons by means of oxygenases under seemingly anoxic conditions. Up to now, no bacterium has been described that grows on aliphatic hydrocarbons with chlorate. Here, we report that Pseudomonas chloritidismutans AW-1T grows on n-alkanes (ranging from C7 until C12) with chlorate as electron acceptor. Strain AW-1T also grows on the intermediates of the presumed n-alkane degradation pathway. The specific growth rates on n-decane and chlorate and n-decane and oxygen were 0.5 0.1 and 0.4 0.02 day1, respectively. The key enzymes chlorate reductase and chlorite dismutase were assayed and found to be present. The oxygendependent alkane oxidation was demonstrated in wholecell suspensions. The strain degrades n-alkanes with oxygen and chlorate but not with nitrate, thus suggesting that the strain employs oxygenase-dependent pathways for the breakdown of n-alkanes. - Petroleum, a complex mixture of aromatic and aliphatic hydrocarbons, is one of the most common environmental contaminants. On average, saturated and aromatic hydrocarbons together make 80% of the oil constituents (Widdel and Rabus 2001). Since the saturated hydrocarbon fraction is the most abundant in crude oil, its biodegradation is quantitatively most important in oil bioremediation (Head et al. 2006). n-Alkanes are relatively stable due to lack of functional groups, presence of only sigma bonds, nonpolar nature, and low solubility in water. Aerobic microbial degradation of n-alkanes is known since almost a century (Shngen 1913), and the mechanisms of degradation, with the enzymes and genes involved, are rather well understood (Berthe-Corti and Fetzner 2002; Head et al. 2006; van Beilen and Funhoff 2007; Wentzel et al. 2007). During aerobic degradation, molecular oxygen acts as a cosubstrate and as a terminal electron acceptor (Berthe-Corti and Fetzner 2002; Chayabutra and Ju 2000). Oxygenases incorporate molecular oxygen into the n-alkanes to form the corresponding alcohols, which are further degraded by beta-oxidation (Wentzel et al. 2007). Since intermediates do not accumulate, the initial step of oxygen incorporation seems to be the rate-limiting step (Chayabutra and Ju 2000). Insight into anaerobic degradation of n-alkanes is limited. The first step of anaerobic degradation of n-alkanes is thermodynamically difficult and has been proposed to occur in the sulfate-reducing bacterium strain Hxd3 via carboxylation (So et al. 2003). Molecular evidence for a mechanism of n-alkane activation through fumarate addition was obtained recently (Callaghan et al. 2008; Grundmann et al. 2008). Anaerobic degradation of n-alkanes is slow compared to aerobic degradation (Wentzel et al. 2007), and only a few denitrifying and sulfate-reducing bacteria have been isolated (Ehrenreich et al. 2000; So and Young 1999). Microbial (per)chlorate reduction is a process that yields molecular oxygen, a property that has application possibilities in the bioremediation of polluted anoxic soils (Coates et al. 1998; Tan et al. 2006; Weelink et al. 2008). During chlorate reduction, chlorate (ClO3) is reduced to chlorite (ClO2) by the enzyme chlorate reductase. Chlorite is then split into Cl and O2 by chlorite dismutase (Rikken et al. 1996; Wolterink et al. 2002). The oxygen released during chlorate reduction might be used to degrade n-alkanes by oxygenases. Here, we report the finding that Pseudomonas chloritidismutans AW-1T, a chlorate-reducing bacterium, that was previously isolated in our laboratory with acetate as carbon and energy source is able to grow on n-decane with oxygen or chlorate as electron acceptor. This finding suggests that an additional function of chlorite dismutation is to generate molecular oxygen to perform oxygenasedependent reactions to support growth on n-alkanes. Materials and methods Inoculum, media, cultivation, and counting P. chloritidismutans strain AW-1T (DSM 13592T) was isolated in our laboratory (Wolterink et al. 2002) and was kindly provided by Serv Kengen. For experiments with nitrate, it was adapted to nitrate by repeated subculturing on acetate and nitrate, while gradually decreasing the oxygen concentration according to Cladera et al. (2006). The medium for P. chloritidismutans strain AW-1T was based on the medium described by Dorn et al. (1974). The composition of the medium (in grams per liter of anaerobic demineralized water) was as follows: Na2HPO42H2O, 3.48; KH2PO4 1; resazurin, 0.005; CaCl2, 0.009; ammonium iron (III) citrate, 0.01; NH4SO4, 1; MgSO47H2O, 0.04. Vit (...truncated)


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Farrakh Mehboob, Howard Junca, Gosse Schraa, Alfons J. M. Stams. Growth of Pseudomonas chloritidismutans AW-1T on n-alkanes with chlorate as electron acceptor, Applied Microbiology and Biotechnology, 2009, pp. 739-747, Volume 83, Issue 4, DOI: 10.1007/s00253-009-1985-9