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)