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Environmental fate & pathways

Biodegradation in soil

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Assimilatory nitrate reductases

Assimilationis the conversion nitrate into ammonium for anabolic reactions. Nitrate is reduced for this purpose by enzymes to nitrite (assimilatory nitrate reductases), and then to ammonia. The assimilatory nitrate reductases are molybdenum-containing enzymes, which are widespread in bacteria, fungi, yeasts, and algae (Cambell, 2001; Inokuch et al, 2002; Joseph-Horne et al, 2001; Siverio, 2001).Nitrate and chlorate are structurally analogous to each other and may potentially be incorporated into the same enzyme active site, as is evidenced by various assimilatory nitrate reductasesof micro-organisms and plants. Chlorate reduction by assimilatory nitrate reductases has been detected in bacteria (Escherichia coli) using a mutant (Motohara et al, 1965). Balch (1987) experimented with36Cl chlorate as a tracer to study nitrate uptake. In Skeletonema costatum and Nitzschia closterium chlorate was transported into the cells. The ability to reduce chlorate in whole cells has been shown in Ankistrodesmus braunii and Chlorella fusca both algae (Rigano, 1970; Tromballa and Broda, 1971). Chlorate is also a substrate for assimilatory nitrate reductase of Chloralla vulgaris (Salmonson and Vennesland, 1972). The assimilatory nitrate reductases convert chlorate to a toxic product chlorite. These results demonstrate that there is a potential for chlorate reduction under aerobic conditions provided that organisms are present capable of utilizing nitrate as nitrogen source.

Dissimilatory nitrate reductases

Denitrification is a process by which bacteria convert nitrate to dinitrogen that is lost to the atmosphere. Denitrifying bacteria use nitrate instead of oxygen in the metabolic processes. Denitrification takes primarily place where oxygen is depleted and where there is ample organic matter to provide energy for bacteria. Two types of dissimilatory nitrate reductases have been found. One of them is coupled to a complete denitrifying pathway (membrane-bound nitrate reductases; nitrate reductase A), and the other is a periplasmic protein whose physiological role seems to be the dissipation of excess reducing power. Periplasmic nitrate reductases, responsible for denitrification under aerobic conditions are specific for nitrate and not capable of reducing chlorate (Berks et al, 1994; McEwan et al, 1987). Chlorate reduction in denitrifying bacteria is primarily due to membrane-bound nitrate reductase (nitrate reductase A) activity (Iobbi et al, 1987; Morpeth and Boxer, 1985). The reduction of nitrate and chlorate in cell-free extracts of nitrate-grown Bacillus cereus was investigated by Hackenthal (1965). Chlorate reduction rates in cell-free extract were approximately twice as high as the nitrate reduction rates. De Groot and Stouthamer (1969) found that Proteus mirabilis formed different reductases including a chlorate reductase (chlorate reductase C). Chlorate reductase purified from Proteus mirabilis could only use chlorate as a substrate (Oltman et al, 1976). It was found that chlorate reductase was produced constitutively while nitrate reductases were produced inductively. However, the chlorate reduction in cell-free extracts of nitrate-grown bacteria is primarily due to membrane-bound nitrate reductases (de Groot and Stouthamer, 1969).

Chlorite is produced from chlorate by denitrifying microorganisms (Quastel et al, 1925; Karki and Kaiser, 1979). It was found that the absorption spectrum of dissimilatory nitrate reductase obtained from Escherichia coli after oxidation by chlorate was different from that of normal oxidized cytochrome. It was assumed that this was due to the oxidative deformation of the haem by the reduction product chlorite (Itagaki et al, 1963). Chlorite formed by denitrifying bacteria is degraded through chemical reactions with reducing agents such as the protein of nitrate reductase. In conclusion, chlorate reduction associated with nitrate-respiring organisms is a cometabolic process. The rate of chlorate reduction by denitrifying bacteria is therefore directly linked to the rate of denitrification.

Growth linked biodegradation (anaerobic)

It is now well-known that bacteria have evolved that can grow by the anaerobic reductive dissimilation of chlorate into innocuous chloride. Bacteria capable of growing with chlorate as electron acceptor are widely spread nature. This has been shown by (per)chlorate reduction with various energy substrates with a number of enrichment cultures (Bryan and Rohlich 1954; van Ginkel et al, 1995; Logan, 1998). The ubiquity of (per)chlorate reducing microorganisms was also shown quantitatively by enumerating the (per)chlorate-reducing bacteria in very diverse environments, including soils, aquatic sediments, sludges, and lagoons. In all of the environments tested, the acetate-oxidizing (per)chlorate reducing bacteria represented a significant population, whose size ranged from 2.3 × 103to 2.4 × 106cells per g of sample (Coates et al, 1999; Wu et al, 2001). Existence of (per)chlorate respiring bacteria have also been demonstrated in marine waters (Logan et al, 2000)

(Per)chlorate reducing microorganisms are easily enriched and isolated from many environments. All of these organisms could grow anaerobically by coupling complete oxidation of reducing agents to reduction of chlorate at high rates (Table). Under fully aerobic conditions, chlorate is not reduced by capable bacteria. Nitrate can also interfere with chlorate reduction (Chaudhuri et al 2002).

 

Table Growth ratesof various cultures capable of reducing chlorate.

Culture

Reducing agent

Rate (h-1)

Reference

Azospira oryzoa GR-1

Acetate

0.1

Rikken et al (1996)

Dechloromonas Agita sp CKB

 

0.28

Bruce et al (1999)

Azospira sp KJ

Acetate

0.26

Logan et al (2001)

Dechlorosomonas sp PDX

Acetate

0.21

Logan et al (2001)

Dechlorosomonas sp PDX

Lactate

0.15

Logan et al (2001)

PDA

Acetate

0.18

Logan et al (2001)

PDB

Actate

0.21

Logan et al (2001)

Mixed culture

Acetate

0.56

Logan et al (1998)

Mixed culture

glucose glutamate

0.12

Logan et al (1998)

Mixed culture

Phenol

0.04

Logan et al (1998)

 

Azospira oryzae strain GR-1 (DSM 11199) isolated from activated sludge was the first bacterium studied in more detail (Rikken et al, 1996; Wolterink et al, 2005). When strain GR-1 was grown on acetate, the release of chloride was proportional to the disappearance of chlorate, showing that this compound was completely reduced. The oxidation of acetate is coupled to the reduction of chlorate, whereas chlorite reduction is not affected by the addition of acetate. Azospira oryzae strain GR-1 disproportionates chlorite into molecular oxygen and chloride. For chlorate reduction by Azospira oryzae strain GR-1 the following biodegradation pathway was formulated: ClO3-  ClO2- Cl-+ O2. The rapid dismutation of chlorite into chloride and molecular oxygen is the key reaction in the reduction of chlorate. All (per)chlorate-reducing bacteria isolated to date have the ability to dismutate chlorite (Coates and Achenbach, 2004). Complete reduction of chlorate into chloride and molecular oxygen is catalysed by two enzymes. Chlorate is reduced to chlorite by (per)chlorate reductase (EC 1.97.1.1) (Kengen et al, 1999). Chlorate respiration at high rates is made possible by the action of the second enzyme which reduces the toxic chlorite to chloride while producing molecular oxygen. This is mediated by chlorite dismutase (EC 1.13.11.49) (van Ginkel et al 1996; Stenklo et al 2001). Chlorite has never been found to accumulate in solution during bacterial respiration of (per)chlorate.

Soils

The high water solubility of chlorate may facilitate its transport in soils from aerobic to anaerobic environments. Microbial chlorate reduction by organisms capabable of growing therefore constitutes a significant sink for chlorate in soils, especially in view of the importance of denitrification and sulphate reduction. Disappearance of chlorate from soils has been known for more than a century (Aslander 1928; Karki and Kaiser, 1979). 

In an attempt to simulate the fate of sodium chlorate used as a herbicide, biodegradation was determined in four soils under aerobic conditions over a period of 120 days using OECD TG 307 (van Ginkel and van der Togt, 2004). Anaerobic degradation was examined in one soil which was kept under aerobic conditions for 28 days and then the soil was water-logged for a period of 120 days. Significant biodegradation of chlorate was noted during the first four weeks under aerobic conditions. This reduction is assumed to take place in anaerobic niches of the soil comparable to nitrate reduction observed in aerobic soils. After four weeks the chlorate reduction leveled off. Nitrate build up detected with a qualitative analysis occurred in the aerobic test systems due nitrification of ammonium in the aerobic niches of the soils. Nitrate is a known inhibitor of chlorate reduction and considered responsible for the leveling off of the chlorate degradation. One soil was water-logged from day 28 to the end of the test. Under these conditions chlorate reduction did not resume immediately but recommenced after the nitrate concentration was significantly reduced. In water-logged loam in the absence of nitrate, remainder of the chlorate was reduced within approximately 6 weeks. The time for 50, 75 and 90% dissipation under aerobic conditions cannot be estimated because after approximately four weeks the reduction of chlorate in all soils decreased significantly due to an artifact of the set-up of the study (increased nitrate concentrations due to nitrification). Nonetheless, kinetic data of chlorate in soils may be determined using the depletion curve from day 0 to 28 of the test period. During this period, the biodegradation of chlorate appears to follow first order kinetics and the half-lives calculated range from 39 to 58 days. These results indicate that chlorate is easily reduced in soils.

Table Half-lives determined in four soils at "environmentally realistic" nitrate concentrations measured in the soils upon arrival.

Soil

Aerobic/anaerobic

DT50 (days)

Loamy sand

Aerobic

55

Loam

Aerobic

39

Sandyloam

Aerobic

58

Clay

Aerobic

47

Loam

Anaerobic

7.5

 

The half-life of chlorate in loam under anaerobic conditions (water-logged soil) was only 7.5 days. This rate of biodegradation is approximately 5 to 8 times higher than rates found in aerobic soils. The rates are probably very conservative because of the high concentrations used are not representative for other uses of sodium chlorate. The establishment of the route of degradation was assessed in loam (van Ginkel and van der Togt, 2004). The stoichiometric release of chloride from chlorate confirms chlorate is entirely reduced without production of other chlorine containing compounds.  Chlorite was not detected in the soil samples during the test period. The stoichiometric release of chloride from chlorate confirms findings with chlorate respiring microorganisms (see above).