(Per)chlorate reduction by an acetogenic bacterium, Sporomusa sp., isolated from an underground gas storage

A mesophilic bacterium, strain An4, was isolated from an underground gas storage reservoir with methanol as substrate and perchlorate as electron acceptor. Cells were Gram-negative, spore-forming, straight to curved rods, 0.5–0.8 μm in diameter, and 2–8 μm in length, growing as single cells or in pairs. The cells grew optimally at 37°C, and the pH optimum was around 7. Strain An4 converted various alcohols, organic acids, fructose, acetoin, and H₂/CO₂ to acetate, usually as the only product. Succinate was decarboxylated to propionate. The isolate was able to respire with (per)chlorate, nitrate, and CO₂. The G+C content of the DNA was 42.6 mol%. Based on the 16S rRNA gene sequence analysis, strain An4 was most closely related to Sporomusa ovata (98% similarity). The bacterium reduced perchlorate and chlorate completely to chloride. Key enzymes, perchlorate reductase and chlorite dismutase, were detected in cell-free extracts.     

Purification and Characterization of (Per)Chlorate Reductase from the Chlorate-Respiring Strain GR-1

Strain GR-1 is one of several recently isolated bacterial species that are able to respire by using chlorate or perchlorate as the terminal electron acceptor. The organism performs a complete reduction of chlorate or perchlorate to chloride and oxygen, with the intermediate formation of chlorite. This study describes the purification and characterization of the key enzyme of the reductive pathway, the chlorate and perchlorate reductase. A single enzyme was found to catalyze both the chlorate- and perchlorate-reducing activity. The oxygen-sensitive enzyme was located in the periplasm and had an apparent molecular mass of 420 kDa, with subunits of 95 and 40 kDa in an alpha(3)beta(3) composition. Metal analysis showed the presence of 11 mol of iron, 1 mol of molybdenum, and 1 mol of selenium per mol of heterodimer. In accordance, quantitative electron paramagnetic resonance spectroscopy showed the presence of one [3Fe-4S] cluster and two [4Fe-4S] clusters. Furthermore, two different signals were ascribed to Mo(V). The K(m) values for perchlorate and chlorate were 27 and <5 microM, respectively. Besides perchlorate and chlorate, nitrate, iodate, and bromate were also reduced at considerable rates. The resemblance of the enzyme to nitrate reductases, formate dehydrogenases, and selenate reductase is discussed.     

Growth of <Emphasis Type="Italic">Pseudomonas chloritidismutans</Emphasis> AW-1<Superscript>T</Superscript> on <Emphasis Type="Italic">n</Emphasis>-alkanes with chlorate as electron acceptor

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-1^T grows on n-alkanes (ranging from C7 until C12) with chlorate as electron acceptor. Strain AW-1^T 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.     

Isolation,Characterization and Kinetics of Perchlorate Reducing Magnetospirillum Species

Perchlorate reducing bacteria reduce perchlorate to chlorate (ClO₃?), which, in turn, is reduced to chlorite (ClO₂?) and ultimately to chloride (Cl^?). Magnetospirillum strains are reported to use chlorate/perchlorate as electron acceptors. This study describes the perchlorate reducing property of strain VITRJS5, a Magnetopsirillum isolated from freshwater sediment collected from Chelur freshwater lake, Kerala, India. The strain was microaerophile and was phylogenetically related to a Magnetospirillum sp., a member of the α-subclass of the class Proteobacteria. The placement of the isolate in the genus Magnetospirillum has further confirmed the presence of four key magnetosome membrane genes. PCR amplification and phylogenetic analysis of central metabolic genes such as nifH (nitrogenase) and cbbM (type II RubisCo) displayed the highest similarity (97% and 81%, respectively) with Magnetospirillum sp. BB-1 The growth kinetic parameters of the isolate were studied with acetate as the electron donor in batch experiments. Monod's substrate utilization model has been established with oxygen, nitrate and perchlorate as electron acceptors separately. The maximum specific growth rate (µ_max) and half-saturation constant (k_s^conc) for the bacterium varied while utilizing different electron acceptors. The maximum specific growth rate was 0.226, 0.190 and 0.096 per hour and half-velocity constant K_s was 25.09, 33.36 and 65.37 mg acetate/l for oxygen, nitrate and perchlorate, respectively. The reduction of perchlorate has been analyzed using kinetic studies of the substrate uptake by the bacteria and the half-velocity constant K_s was found to be 52.8 mg/l. The results indicate that the strain VITRJS5 effectively reduces perchlorate by using it as an electron acceptor.     

Reduction of (per)chlorate by a novel organism isolated from paper mill waste

As part of a study on the microbiology of chlorate reduction, several new dissimilatory chlorate-reducing bacteria were isolated from a broad diversity of environments. One of these, strain CKB, was selected for a more complete characterization. Strain CKB was enriched and isolated from paper mill waste with acetate as the sole electron donor and chlorate as the sole electron acceptor. Strain CKB is a completely oxidizing, non-fermentative, Gram-negative, facultative anaerobe. Cells of strain CKB are 0.5 x 2 microm and are highly motile, with a single polar flagellum. In addition to acetate, strain CKB can use propionate, butyrate, lactate, succinate, fumarate, malate or yeast extract as electron donors, with chlorate as the sole electron acceptor. Strain CKB can also couple chlorate reduction to the oxidation of ferrous iron, sulphide, or the reduced form of the humic substances analogue 2,6-anthrahydroquinone disulphonate. Fe(II) is oxidized to insoluble amorphous Fe(II) oxide, whereas sulphide is oxidized to elemental sulphur. Growth is not associated with this metabolism, even when small quantities of acetate are added as a potential carbon source. In addition to chlorate, strain CKB can also couple acetate oxidation to the reduction of oxygen or perchlorate. Chlorate is completely reduced to chloride. Strain CKB has an optimum temperature of 35 degrees C, a pH optimum of 7.5 and a salinity optimum of 1% NaCl. Strain CKB can grow in chlorate and perchlorate concentrations of 80 or 20 mM respectively. Under anaerobic conditions, strain CKB can dismutate chlorite into chloride and O2, and is only the second organism shown to be capable of this metabolism. Oxidized minus reduced spectra of whole-cell suspensions of strain CKB showed absorbance maxima at 423, 523 and 552nm, which are indicative of the presence of c-type cytochrome(s). Analysis of the complete sequence of the 16S rDNA indicates that strain CKB is a member of the beta subclass of the Proteobacteria. The phototroph Rhodocyclus tenuis is the closest known relative. When tested, strain CKB could not grow by phototrophy and did not contain bacteriochlorophyll. Phenotypically and phylogenetically, strain CKB differs from all other described bacteria and represents the type strain of a new genus and species.     

(Per)chlorate Reduction by the Thermophilic Bacterium Moorella perchloratireducens sp. nov., Isolated from Underground Gas Storage

A thermophilic bacterium, strain An10, was isolated from underground gas storage with methanol as a substrate and perchlorate as an electron acceptor. Cells were gram-positive straight rods, 0.4 to 0.6 μm in diameter and 2 to 8 μm in length, growing as single cells or in pairs. Spores were terminal with a bulged sporangium. The temperature range for growth was 40 to 70°C, with an optimum at 55 to 60°C. The pH optimum was around 7. The salinity range for growth was between 0 and 40 g NaCl liter⁻¹ with an optimum at 10 g liter⁻¹. Strain An10 was able to grow on CO, methanol, pyruvate, glucose, fructose, cellobiose, mannose, xylose, and pectin. The isolate was able to respire with (per)chlorate, nitrate, thiosulfate, neutralized Fe(III) complexes, and anthraquinone-2,6-disulfonate. The G+C content of the DNA was 57.6 mol%. On the basis of 16S rRNA analysis, strain An10 was most closely related to Moorella thermoacetica and Moorella thermoautotrophica. The bacterium reduced perchlorate and chlorate completely to chloride. Key enzymes, perchlorate reductase and chlorite dismutase, were detected in cell extracts. Strain An10 is the first thermophilic and gram-positive bacterium with the ability to use (per)chlorate as a terminal electron acceptor.     

Identification of an anaerobic bacterium which reduces perchlorate and chlorate asWolinella succinogenes

A bacterium coded as strain HAP-1 was isolated from a municipal anaerobic digestor for its ability to reduce >7000 ppm perchlorate in wastewaters. The organism is capable of the dissimilatory reduction of perchlorate on chlorate to chloride for energy and growth. It is a Gram-negative, non-sporeforming, obligately anaerobic, motile thin rod. Antibiotic resistance, utilization of carbon substrates and utilization of electron acceptors by bacterium HAP-1 were similar toWolinella succinogenes. The organism's 16S rRNA sequence was 0.75% different from that of the type strain ofW. succinogenes. The fatty acid compositions of the two organisms are very similar. The morphological, physiological and 16S rRNA sequence data indicated that bacterium HAP-1 is a strain ofW. succinogenes that can utilize perchlorate or chlorate as a terminal electron acceptor.     

Purification and characterization of chlorite dismutase: a novel oxygen-generating enzyme

A novel enzyme that catalyzes the disproportionation of chlorite into chloride and oxygen was purified from a gram-negative bacterium, strain GR-1 to homogeneity. A four-step purification procedure comprising Q-Sepharose, hydroxyapatite, and phenyl-Superose chromatography and ultrafiltration resulted in a 13.7-fold purified enzyme with a final specific activity of 2.0 mmol min^–1 (mg protein)^–1. The dismutase obeyed Michaelis-Menten kinetics. The V _max and K _m calculated for chlorite were 2,200 U (mg protein)^–1 and 170 μM, respectively. Dismutase activity was inhibited by hydroxylamine, cyanide, and azide, but not by 3-amino-1,2,4-triazole. Chlorite dismutase had a molecular mass of 140 kDa and consisted of four 32-kDa subunits. The enzyme was red-colored and had a Soret peak at 392 nm. Per subunit, it contained 0.9 molecule of protoheme IX and 0.7 molecule of iron. Chlorite dismutase displayed maxima for activity at pH 6.0 and 30° C. Received: 9 April 1996 / Accepted: 12 August 1996     

Kinetics of Perchlorate- and Chlorate-Respiring Bacteria

Ten chlorate-respiring bacteria were isolated from wastewater and a perchlorate-degrading bioreactor. Eight of the isolates were able to degrade perchlorate, and all isolates used oxygen and chlorate as terminal electron acceptors. The growth kinetics of two perchlorate-degrading isolates, designated “Dechlorosoma” sp. strains KJ and PDX, were examined with acetate as the electron donor in batch tests. The maximum observed aerobic growth rates of KJ and PDX (0.27 and 0.28 h⁻¹, respectively) were only slightly higher than the anoxic growth rates obtained by these isolates during growth with chlorate (0.26 and 0.21 h⁻¹, respectively). The maximum observed growth rates of the two non-perchlorate-utilizing isolates (PDA and PDB) were much higher under aerobic conditions (0.64 and 0.41 h⁻¹, respectively) than under anoxic (chlorate-reducing) conditions (0.18 and 0.21 h⁻¹, respectively). The maximum growth rates of PDX on perchlorate and chlorate were identical (0.21 h⁻¹) and exceeded that of strain KJ on perchlorate (0.14 h⁻¹). Growth of one isolate (PDX) was more rapid on acetate than on lactate. There were substantial differences in the half-saturation constants measured for anoxic growth of isolates on acetate with excess perchlorate (470 mg/liter for KJ and 45 mg/liter for PDX). Biomass yields (grams of cells per gram of acetate) for strain KJ were not statistically different in the presence of the electron acceptors oxygen (0.46 ± 0.07 [n = 7]), chlorate (0.44 ± 0.05 [n = 7]), and perchlorate (0.50 ± 0.08 [n = 7]). These studies provide evidence that facultative microorganisms with the capability for perchlorate and chlorate respiration exist, that not all chlorate-respiring microorganisms are capable of anoxic growth on perchlorate, and that isolates have dissimilar growth kinetics using different electron donors and acceptors.     

Crystal Structure of Chlorite Dismutase, a Detoxifying Enzyme Producing Molecular Oxygen

Chlorite dismutase (Cld) is a key enzyme of perchlorate and chlorate respiration. This heme-based protein reduces the toxic compound chlorite into the innocuous chloride anion in a very efficient way while producing molecular oxygen. A sequence comparison between Cld homologues shows a highly conserved family. The crystal structure of Azospira oryzae strain GR-1 Cld is reported to 2.1 Å resolution. The structure reveals a hexameric organization of the Cld, while each monomer exhibits a ferredoxin-like fold. The six subunits are organized in a ring structure with a maximal diameter of 9 nm and an inner diameter of 2 nm. The heme active-site pocket is solvent accessible both from the inside and the outside of the ring. Moreover, a second anion binding site that could accommodate the assumed reaction intermediate ClO‾ for further transformation has been identified near the active site.The environment of the heme cofactor was investigated with electron paramagnetic resonance spectroscopy. Apart from the high-spin ferric signal of the five-coordinate resting-state enzyme, two low-spin signals were found corresponding to six-coordinate species. The current crystal structure confirms and complements a recently proposed catalytic mechanism that proceeds via a ferryl species and a ClO‾ anion. Our structural data exclude cooperativity between the iron centers.     

Kinetics of perchlorate- and chlorate-respiring bacteria

Ten chlorate-respiring bacteria were isolated from wastewater and a perchlorate-degrading bioreactor. Eight of the isolates were able to degrade perchlorate, and all isolates used oxygen and chlorate as terminal electron acceptors. The growth kinetics of two perchlorate-degrading isolates, designated "Dechlorosoma" sp. strains KJ and PDX, were examined with acetate as the electron donor in batch tests. The maximum observed aerobic growth rates of KJ and PDX (0.27 and 0.28 h(-1), respectively) were only slightly higher than the anoxic growth rates obtained by these isolates during growth with chlorate (0.26 and 0.21 h(-1), respectively). The maximum observed growth rates of the two non-perchlorate-utilizing isolates (PDA and PDB) were much higher under aerobic conditions (0.64 and 0.41 h(-1), respectively) than under anoxic (chlorate-reducing) conditions (0.18 and 0.21 h(-1), respectively). The maximum growth rates of PDX on perchlorate and chlorate were identical (0.21 h(-1)) and exceeded that of strain KJ on perchlorate (0.14 h(-1)). Growth of one isolate (PDX) was more rapid on acetate than on lactate. There were substantial differences in the half-saturation constants measured for anoxic growth of isolates on acetate with excess perchlorate (470 mg/liter for KJ and 45 mg/liter for PDX). Biomass yields (grams of cells per gram of acetate) for strain KJ were not statistically different in the presence of the electron acceptors oxygen (0.46 +/- 0.07 [n = 7]), chlorate (0.44 +/- 0.05 [n = 7]), and perchlorate (0.50 +/- 0.08 [n = 7]). These studies provide evidence that facultative microorganisms with the capability for perchlorate and chlorate respiration exist, that not all chlorate-respiring microorganisms are capable of anoxic growth on perchlorate, and that isolates have dissimilar growth kinetics using different electron donors and acceptors.     

Isolation and Characterization of Alicycliphilus denitrificans Strain BC, Which Grows on Benzene with Chlorate as the Electron Acceptor

A bacterium, strain BC, was isolated from a benzene-degrading chlorate-reducing enrichment culture. Strain BC degrades benzene in conjunction with chlorate reduction. Cells of strain BC are short rods that are 0.6 μm wide and 1 to 2 μm long, are motile, and stain gram negative. Strain BC grows on benzene and some other aromatic compounds with oxygen or in the absence of oxygen with chlorate as the electron acceptor. Strain BC is a denitrifying bacterium, but it is not able to grow on benzene with nitrate. The closest cultured relative is Alicycliphilus denitrificans type strain K601, a cyclohexanol-degrading nitrate-reducing betaproteobacterium. Chlorate reductase (0.4 U/mg protein) and chlorite dismutase (5.7 U/mg protein) activities in cell extracts of strain BC were determined. Gene sequences encoding a known chlorite dismutase (cld) were not detected in strain BC by using the PCR primers described in previous studies. As physiological and biochemical data indicated that there was oxygenation of benzene during growth with chlorate, a strategy was developed to detect genes encoding monooxygenase and dioxygenase enzymes potentially involved in benzene degradation in strain BC. Using primer sets designed to amplify members of distinct evolutionary branches in the catabolic families involved in benzene biodegradation, two oxygenase genes putatively encoding the enzymes performing the initial successive monooxygenations (BC-BMOa) and the cleavage of catechol (BC-C23O) were detected. Our findings suggest that oxygen formed by dismutation of chlorite can be used to attack organic molecules by means of oxygenases, as exemplified with benzene. Thus, aerobic pathways can be employed under conditions in which no external oxygen is supplied.     

The role of an NAD-independent lactate dehydrogenase and acetate in the utilization of lactate byClostridium acetobutylicum strain P262

Clostridium acetobutylicum strain P262 utilized lactate at a rapid rate [600 nmol min^–1 (mg protein)^–1], but lactate could not serve as the sole energy source. When acetate was provided as a co-substrate, the growth rate was 0.05 h^–1. Butyrate, carbon dioxide and hydrogen were the end products of lactate and acetate utilization, and the stoichiometry was 1 lactate + 0.4 acetate → 0.7 butyrate + 0.6 H₂ + 1 CO₂. Lactate-grown cells had twofold lower hydrogenase than glucose-grown cells, and the lactate-grown cells used acetate as an alternative electron acceptor. The cells had a poor affinity for lactate (K_s = 1.1 mM), and there was no evidence for active transport. Lactate utilization was catabolyzed by an inducible NAD-independent lactate dehydrogenase (iLDH) that had a pH optimum of 7.5. The iLDH was fivefold more active with d-lactate than l-lactate, and the K _m for d-lactate was 3.2 mM. Lactate-grown cells had little butyraldehyde dehydrogenase activity, and this defect did not allow the conversion of lactate to butanol. Received: 17 October 1994 / Accepted: 30 January 1995     

Behavioral response of dissimilatory perchlorate-reducing bacteria to different electron acceptors

The response behavior of three dissimilatory perchlorate-reducing bacteria to different electron acceptors (nitrate, chlorate, and perchlorate) was investigated with two different assays. The observed response was species-specific, dependent on the prior growth conditions, and was inhibited by oxygen. We observed attraction toward nitrate when Dechloromonas aromatica strain RCB and Azospira suillum strain PS were grown with nitrate. When D. aromatica and Dechloromonas agitata strain CKB were grown with perchlorate, both responded to nitrate, chlorate, and perchlorate. When A. suillum was grown with perchlorate, the organism responded to chlorate and perchlorate but not nitrate. A gene replacement mutant in the perchlorate reductase subunit (pcrA) of D. aromatica resulted in a loss of the attraction response toward perchlorate but had no impact on the nitrate response. Washed-cell suspension studies revealed that the perchlorate grown cells of D. aromatica reduced both perchlorate and nitrate, while A. suillum cells reduced perchlorate only. Based on these observations, energy taxis was proposed as the underlying mechanism for the responses to (per)chlorate by D. aromatica. To the best of our knowledge, this study represents the first investigation of the response behavior of perchlorate-reducing bacteria to environmental stimuli. It clearly demonstrates attraction toward chlorine oxyanions and the unique ability of these organisms to distinguish structurally analogous compounds, nitrate, chlorate, and perchlorate and respond accordingly.     

Perchlorate reduction by a novel chemolithoautotrophic,hydrogen-oxidizing bacterium

Water treatment technologies are needed that can remove perchlorate from drinking water without introducing organic chemicals that stimulate bacterial growth in water distribution systems. Hydrogen is an ideal energy source for bacterial degradation of perchlorate as it leaves no organic residue and is sparingly soluble. We describe here the isolation of a perchlorate-respiring, hydrogen-oxidizing bacterium (Dechloromonas sp. strain HZ) that grows with carbon dioxide as sole carbon source. Strain HZ is a Gram-negative, rod-shaped facultative anaerobe that was isolated from a gas-phase anaerobic packed-bed biofilm reactor treating perchlorate-contaminated groundwater. The ability of strain HZ to grow autotrophically with carbon dioxide as the sole carbon source was confirmed by demonstrating that biomass carbon (100.9%) was derived from CO2. Chemolithotrophic growth with hydrogen was coupled with complete reduction of perchlorate (10 mM) to chloride with a maximum doubling time of 8.9 h. Strain HZ also grew using acetate as the electron donor and chlorate, nitrate, or oxygen (but not sulphate) as an electron acceptor. Phylogenetic analysis of the 16S rRNA sequence placed strain HZ in the genus Dechloromonas within the beta subgroup of the Proteobacteria. The study of this and other novel perchlorate-reducing bacteria may lead to new, safe technologies for removing perchlorate and other chemical pollutants from drinking water.     

Bioremediation of Chlorate or Perchlorate Contaminated Water Using Permeable Barriers Containing Vegetable Oil

A scale model of an in situ permeable barrier, formed by injecting vegetable oil onto laboratory soil columns, was used to remove chlorate and perchlorate from flowing groundwater. The hypothesis that trapped oil would serve as a substrate enabling native microorganisms to reduce chlorate or perchlorate to chloride as water flowed through the oil-rich zone had merit. Approximately 96% of the 0.2 mM chlorate and 99% of the 0.2 mM perchlorate present in the water was removed as water was pumped through columns containing vegetable oil barriers. The product formed was chloride. When nitrate at 1.4 mM was added to the water, both nitrate and chlorate were removed. High concentrations of chlorate or perchlorate can be treated; 24 mM chlorate and 6 mM perchlorate were completely reduced to chloride during microcosm incubations. Microorganisms capable of reducing perchlorate are plentiful in the environment. Received: 19 December 2001 / Accepted: 25 January 2002     

Anaerobic Growth of Microorganisms with Chlorate as an Electron Acceptor

The ability of microorganisms to use chlorate (ClO₃^-) as an electron acceptor for respiration under anaerobic conditions was studied in batch and continuous tests. Complex microbial communities were cultivated anaerobically in defined media containing chlorate, all essential minerals, and acetate as the sole energy and carbon source. It was shown that chlorate was reduced to chloride, while acetate was oxidized to carbon dioxide and water and used as the carbon source for synthesis of new biomass. A biomass yield of 1.9 to 3.8 g of volatile suspended solids per equivalent of available electrons was obtained, showing that anaerobic growth with chlorate as an electron acceptor gives a high energy yield. This indicates that microbial reduction of chlorate to chloride in anaerobic systems is coupled with electron transport phosphorylation.     

Role of Thiobacillus ferrooxidans and sulphur (sulphide)-dependent ferric-ion-reducing activity in the oxidation of sulphide minerals

 Thiobacillus ferrooxidans oxidized the sulphide minerals e.g., pyrite, pyrrhotite and copper concentrate under anaerobic conditions in the presence of ferric ion as sole electron acceptor. Copper and iron were solubilized from sulphide ores by the sulphur (sulphide)-dependent ferric-ion oxidoreductase activity. Treatment of resting cells of T. ferrooxidans with 0.5% phenol for 30 min completely destroyed the iron- and copper-solubilizing activity. The above treatment destroyed the sulphur(sulphide)-dependent ferric-ion-reducing activity completely but did not affect the iron-oxidizing activity. The results suggest that sulphur(sulphide)-dependent ferric-ion-reducing activity actively participates in the oxidation of sulphide minerals under anaerobic conditions. The activity of sulphur(sulphide)-dependent ferric ion reduction in the solubilization of iron and copper from the sulphide ores were also observed under aerobic conditions in presence of sodium azide (0.1 μmol), which completely inhibits the iron-oxidizing activity. Received: 23 May 1995/Received revision: 10 October 1995/Accepted: 16 October 1995     

Studies on methyl chloride dehalogenase and O-demethylase in cell extracts of the homoacetogen strain MC based on a newly developed coupled enzyme assay

An enzyme assay was developed to determine the activities of methyl chloride dehalogenase and O-demethylase of the homoacetogen strain MC. The formation of methyl tetrahydrofolate from tetrahydrofolate and methyl chloride or from tetrahydrofolate and vanillate was coupled to the oxidation of methyl tetrahydrofolate to methylene tetrahydrofolate mediated by methylene tetrahydrofolate reductase purified from Peptostreptococcus productus (strain Marburg) and to the subsequent oxidation of methylene tetrahydrofolate to methenyl tetrahydrofolate catalyzed by methylene tetrahydrofolate dehydrogenase purified from the same organism. To drive the endergonic methyl tetrahydrofolate oxidation with NAD⁺ as an electron acceptor, the NADH formed in this reaction was reoxidized in the exergonic lactate dehydrogenase reaction. The formation of NADPH and methenyl tetrahydrofolate in the methylene tetrahydrofolate dehydrogenase reaction was followed photometrically at 350 nm; ε₃₅₀ was about 29.5 mM^–1cm^–1 (pH 6.5). Using the coupled enzyme assay, the cofactor requirements, the apparent kinetic parameters, the pH and temperature optima of both enzymes, and the effect of inhibitors were determined. The activity of methyl chloride dehalogenase and of O-demethylase was dependent on the presence of ATP; arsenate severely inhibited both enzyme activities in the absence of ATP. The coupled enzyme assay described allows purification and characterization of methyl chloride dehalogenase and O-demethylase and is also appropriate for the enzymatic determination of methyl tetrahydrofolate. Received: 2 August 1995 / Accepted: 28 September 1995     

Benzene Degradation Coupled with Chlorate Reduction in a Soil Column Study

Perchlorate and chlorate are electron acceptors that during reduction result in the formation of molecular oxygen. The produced oxygen can be used for activation of anaerobic persistent pollutants, like benzene. In this study chlorate was tested as potential electron acceptor to stimulate benzene degradation in anoxic polluted soil column. A chlorate amended benzene polluted soil column was operated over a period of 500 days. Benzene was immediately degraded in the column after start up, and benzene removal recovered completely after omission of chlorate or a too high influent chlorate concentration (22 mM). Mass balance calculations showed that per mole of benzene five mole of chlorate were reduced. At the end of the experiment higher loading rates were applied to measure the maximal benzene degradation rate in this system; a breakthrough of benzene was not observed. The average benzene degradation rate over this period was 31 μmol l⁻¹ h⁻¹ with a maximal of 78 μmol l⁻¹ h⁻¹. The high degradation rate and the necessity of chlorate indicate that oxygen produced during chlorate reduction indeed is used for the activation of benzene. This is the first column study where benzene biodegradation at a high rate coupled with anaerobic chlorate reduction is observed.