Oxidation of methyl halides by the facultative methylotroph strain IMB-1.

Washed cell suspensions of the facultative methylotroph strain IMB-1 grown on methyl bromide (MeBr) were able to consume methyl chloride (MeCl) and methyl iodide (MeI) as well as MeBr. Consumption of >100 microM MeBr by cells grown on glucose, acetate, or monomethylamine required induction. Induction was inhibited by chloramphenicol. However, cells had a constitutive ability to consume low concentrations (<20 nM) of MeBr. Glucose-grown cells were able to readily oxidize [(14)C]formaldehyde to (14)CO(2) but had only a small capacity for oxidation of [(14)C]methanol. Preincubation of cells with MeBr did not affect either activity, but MeBr-induced cells had a greater capacity for [(14)C]MeBr oxidation than did cells without preincubation. Consumption of MeBr was inhibited by MeI, and MeCl consumption was inhibited by MeBr. No inhibition of MeBr consumption occurred with methyl fluoride, propyl iodide, dibromomethane, dichloromethane, or difluoromethane, and in addition cells did not oxidize any of these compounds. Cells displayed Michaelis-Menten kinetics for the various methyl halides, with apparent K(s) values of 190, 280, and 6,100 nM for MeBr, MeI, and MeCl, respectively. These results suggest the presence of a single oxidation enzyme system specific for methyl halides (other than methyl fluoride) which runs through formaldehyde to CO(2). The ease of induction of methyl halide oxidation in strain IMB-1 should facilitate its mass culture for the purpose of reducing MeBr emissions to the atmosphere from fumigated soils.     

Consumption of Tropospheric Levels of Methyl Bromide by C1 Compound-Utilizing Bacteria and Comparison to Saturation Kinetics

Pure cultures of methylotrophs and methanotrophs are known to oxidize methyl bromide (MeBr); however, their ability to oxidize tropospheric concentrations (parts per trillion by volume [pptv]) has not been tested. Methylotrophs and methanotrophs were able to consume MeBr provided at levels that mimicked the tropospheric mixing ratio of MeBr (12 pptv) at equilibrium with surface waters (≈2 pM). Kinetic investigations using picomolar concentrations of MeBr in a continuously stirred tank reactor (CSTR) were performed using strain IMB-1 and Leisingeria methylohalidivorans strain MB2^T — terrestrial and marine methylotrophs capable of halorespiration. First-order uptake of MeBr with no indication of threshold was observed for both strains. Strain MB2^T displayed saturation kinetics in batch experiments using micromolar MeBr concentrations, with an apparent K_s of 2.4 μM MeBr and a V_max of 1.6 nmol h⁻¹ (10⁶ cells)⁻¹. Apparent first-order degradation rate constants measured with the CSTR were consistent with kinetic parameters determined in batch experiments, which used 35- to 1 × 10⁷-fold-higher MeBr concentrations. Ruegeria algicola (a phylogenetic relative of strain MB2^T), the common heterotrophs Escherichia coli and Bacillus pumilus, and a toluene oxidizer, Pseudomonas mendocina KR1, were also tested. These bacteria showed no significant consumption of 12 pptv MeBr; thus, the ability to consume ambient mixing ratios of MeBr was limited to C₁ compound-oxidizing bacteria in this study. Aerobic C₁ bacteria may provide model organisms for the biological oxidation of tropospheric MeBr in soils and waters.     

Rapid Consumption of Low Concentrations of Methyl Bromide by Soil Bacteria

A dynamic dilution system for producing low mixing ratios of methyl bromide (MeBr) and a sensitive analytical technique were used to study the uptake of MeBr by various soils. MeBr was removed within minutes from vials incubated with soils and ~10 parts per billion by volume of MeBr. Killed controls did not consume MeBr, and a mixture of the broad-spectrum antibiotics chloramphenicol and tetracycline inhibited MeBr uptake by 98%, indicating that all of the uptake of MeBr was biological and by bacteria. Temperature optima for MeBr uptake suggested a biological sink, yet soil moisture and temperature optima varied for different soils, implying that MeBr consumption activity by soil bacteria is diverse. The eucaryotic antibiotic cycloheximide had no effect on MeBr uptake, indicating that soil fungi were not involved in MeBr removal. MeBr consumption did not occur anaerobically. A dynamic flowthrough vial system was used to incubate soils at MeBr mixing ratios as low as those found in the remote atmosphere (5 to 15 parts per trillion by volume [pptv]). Soils consumed MeBr at all mixing ratios tested. Temperate forest and grassy lawn soils consumed MeBr most rapidly (rate constant [k] = 0.5 min⁻¹), yet sandy temperate, boreal, and tropical forest soils also readily consumed MeBr. Amendments of CH₄ up to 5% had no effect on MeBr uptake even at CH₄:MeBr ratios of 10⁷, and depth profiles of MeBr and CH₄ consumption exhibited very different vertical rate optima, suggesting that methanotrophic bacteria, like those presently in culture, do not utilize MeBr when it is at atmospheric mixing ratios. Data acquired with gas flux chambers in the field demonstrated the very rapid in situ consumption of MeBr by soils. Uptake of MeBr at mixing ratios found in the remote atmosphere occurs via aerobic bacterial activity, displays first-order kinetics at mixing ratios from 5 pptv to ~1 part per million per volume, and is rapid enough to account for 25% of the global annual loss of atmospheric MeBr.     

Anaerobic Mineralization of Stable-Isotope-Labeled 2-Methylnaphthalene

An active sulfate-reducing consortium that degrades 2-methylnaphthalene (2-MNAP) at rates of up to 25 μM day⁻¹ was established. Degradation was inhibited in the presence of molybdate and ceased in the absence of sulfate. As much as 87% of 2-[¹⁴C]MNAP was mineralized to ¹⁴CO₂. 2-Naphthoic acid (2-NA) was detected as a metabolite, and incubation with either deuterated 2-MNAP or [¹³C]bicarbonate indicates that 2-NA is the result of oxidation of the methyl group. Also detected were carboxylated 2-MNAPs, suggesting the presence of an alternative pathway for 2-MNAP degradation.     

Strain IMB-1, a Novel Bacterium for the Removal of Methyl Bromide in Fumigated Agricultural Soils

A facultatively methylotrophic bacterium, strain IMB-1, that has been isolated from agricultural soil grows on methyl bromide (MeBr), methyl iodide, methyl chloride, and methylated amines, as well as on glucose, pyruvate, or acetate. Phylogenetic analysis of its 16S rRNA gene sequence indicates that strain IMB-1 classes in the alpha subgroup of the class Proteobacteria and is closely related to members of the genus Rhizobium. The ability of strain IMB-1 to oxidize MeBr to CO₂ is constitutive in cells regardless of the growth substrate. Addition of cell suspensions of strain IMB-1 to soils greatly accelerates the oxidation of MeBr, as does pretreatment of soils with low concentrations of methyl iodide. These results suggest that soil treatment strategies can be devised whereby bacteria can effectively consume MeBr during field fumigations, which would diminish or eliminate the outward flux of MeBr to the atmosphere.Methyl bromide (MeBr) is a fumigant used in the cultivation of selected fruits, vegetables, and flowers and in the preservation of stored grains and structures. Use of MeBr as a pesticide increases the yield and quality of crops without leaving behind toxic residues characteristic of more complex organopesticides. However, because bromine released from MeBr destroys stratospheric ozone (18, 22, 29, 33), its use will be eliminated in the United States and elsewhere under the auspices of the Clean Air Act and the Montreal Protocol unless effective mechanisms which prevent its escape to the atmosphere can be found (36). Currently, much uncertainty exists with regard to the tropospheric residence time (τ) of MeBr, a factor which is used to calculate its ozone degradation potential (2). Estimates of τ range from ∼1.7 years when only oxidation by tropospheric OH radicals is considered (22) to less than 1.2 years when oceanic sinks are factored in (20). The discovery that soil bacteria oxidize MeBr from the atmosphere, when quantified and combined with the two preceding sinks, lowers τ to ∼0.8 years (32). Chemical destruction of MeBr occurs by hydrolysis, exchange with other halides, and reaction with organic matter (8, 9, 12), but its destruction by microorganisms has been noted in soils and aquatic environments (3, 16, 17a, 19, 23, 27, 28, 32). In aerobic environments, MeBr is oxidized to CO₂ and Br⁻ (3, 16, 23, 27).Bacterial oxidation of MeBr in soils has been reported both at very low (∼5 to 15 parts per trillion) ambient atmospheric mixing ratios (17a) and at the very high concentrations employed for field fumigation (23). The relative contributions that chemical reactions and bacterial oxidation make to the destruction of MeBr during agricultural fumigation are not yet known, but their combined effect will constrain the emissions of MeBr from soils. Reported destruction of MeBr within the soil matrix, as evidenced by the accumulation of Br⁻, can be substantial and account for as much as 39 to 70% of the applied MeBr in some cases (39, 40). Physical manipulations (e.g., soil compaction and deeper injection of MeBr) have been proposed to increase the retention time of MeBr within the soil matrix, thereby allowing for its more extensive degradation and subsequent decrease in its outward flux to the atmosphere (13). In addition, use of thicker, impermeable covering tarps has been proposed to reduce losses (14, 37), as has the substitution of methyl iodide for MeBr (11, 25). However, enhancement of microbial degradation of MeBr while it is present in the soil matrix may also be a means to eliminate emissions. This could be achieved by exploiting the ability of certain soil bacteria that use MeBr as a carbon and energy source (23). Here, we report further details on the characteristics of such an isolate (23), which we designate strain IMB-1. We demonstrate how the properties of IMB-1 can be used to greatly accelerate the oxidation of MeBr in fumigated soils. Because agricultural field fumigation represents the largest source of anthropogenic emissions of MeBr to the atmosphere, it is at least possible in theory that the overall goal of eliminating most human-derived emission of MeBr could be achieved by in situ biodegradation of this substance.     

Uptake of Choline and Its Conversion to Glycine Betaine by Bacteria in Estuarine Waters

The uptake and degradation of nanomolar levels of [methyl-¹⁴C]choline in estuarine water samples and in seawater filtrate cultures composed mainly of natural free-living bacteria was studied. Uptake of [¹⁴C]choline exhibited Michaelis-Menten kinetics, with K_t + S_n values of 1.7 to 2.9 nM in filtrate cultures and 1.7 to 4.1 nM in estuarine-water samples. V_max values ranged from 0.5 to 3.3 nM · h⁻¹. The uptake system for choline in natural microbial assemblages therefore displays very high affinity and appears able to scavenge this compound at the concentrations expected in seawater. Uptake of choline was inhibited by some natural structural analogs and p-chloromercuribenzoate, indicating that the transporter may be multifunctional and may involve a thiol binding site. When 11 nM [¹⁴C]choline was added to water samples, a significant fraction (>50%) of the methyl carbon was respired to CO₂ in incubations lasting 10 to 53 h. Cells taking up [¹⁴C]choline produced [¹⁴C]glycine betaine ([¹⁴C]GBT), and up to 80% of the radioactivity retained by cells was in the form of GBT, a well-known osmolyte. Alteration of the salinity in filtrate cultures affected the relative proportion of [¹⁴C]choline degraded or converted to [¹⁴C]GBT, without substantially affecting the total metabolism of choline. Increasing the salinity from 14 to 25 or 35 ppt caused more [¹⁴C]GBT to be produced from choline but less ¹⁴CO₂ to be produced than in the controls. Lowering the salinity to 7 ppt decreased [¹⁴C]GBT production and increased ¹⁴CO₂ production slightly. Intracellular accumulations of [¹⁴C]GBT in the salt-stressed cultures were osmotically significant (34 mM). Choline may be used as an energy substrate by estuarine bacteria and may also serve as a precursor of the osmoprotectant GBT, particularly as bacteria are mixed into higher-salinity waters.     

New Insights into Methyl Bromide Cooxidation by Nitrosomonas europaea Obtained by Experimenting with Moderately Low Density Cell Suspensions

We examined the rates and sustainability of methyl bromide (MeBr) oxidation in moderately low density cell suspensions (~6 × 10⁷ cells ml⁻¹) of the NH₃-oxidizing bacterium Nitrosomonas europaea. In the presence of 10 mM NH₄⁺ and 0.44, 0.22, and 0.11 mM MeBr, the initial rates of MeBr oxidation were sustained for 12, 12, and 24 h, respectively, despite the fact that only 10% of the NH₄⁺, 18% of the NH₄⁺, and 35% of the NH₄⁺, respectively, were consumed. Although the duration of active MeBr oxidation generally decreased as the MeBr concentration increased, similar amounts of MeBr were oxidized with a large number of the NH₄⁺-MeBr combinations examined (10 to 20 μmol mg [dry weight] of cells⁻¹). Approximately 90% of the NH₃-dependent O₂ uptake activity and the NO₂⁻-producing activity were lost after N. europaea was exposed to 0.44 mM MeBr for 24 h. After MeBr was removed and the cells were resuspended in fresh growth medium, NO₂⁻ production increased exponentially, and 48 to 60 h was required to reach the level of activity observed initially in control cells that were not exposed to MeBr. It is not clear what percentage of the cells were capable of cell division after MeBr oxidation because NO₂⁻ accumulated more slowly in the exposed cells than in the unexposed cells despite the fact that the latter were diluted 10-fold to create inocula which exhibited equal initial activities. The decreases in NO₂⁻-producing and MeBr-oxidizing activities could not be attributed directly to NH₄⁺ or NH₃ limitation, to a decrease in the pH, to the composition of the incubation medium, or to toxic effects caused by accumulation of the end products of oxidation (NO₂⁻ and formaldehyde) in the medium. Additional cooxidation-related studies of N. europaea are needed to identify the mechanism(s) responsible for the MeBr-induced loss of cell activity and/or viability, to determine what percentages of cells damaged by cooxidative activities are culturable, and to determine if cooxidative activity interferes with the regulation of NH₃-oxidizing activity.     

Humic Acids as Electron Acceptors for Anaerobic Microbial Oxidation of Vinyl Chloride and Dichloroethene

Anaerobic oxidation of [1,2-¹⁴C]vinyl chloride and [1,2-¹⁴C]dichloroethene to ¹⁴CO₂ under humic acid-reducing conditions was demonstrated. The results indicate that waterborne contaminants can be oxidized by using humic acid compounds as electron acceptors and suggest that natural aquatic systems have a much larger capacity for contaminant oxidation than previously thought.     

Enzymatic Combustion of Aromatic and Aliphatic Compounds by Manganese Peroxidase from Nematoloma frowardii

The direct involvement of manganese peroxidase (MnP) in the mineralization of natural and xenobiotic compounds was evaluated. A broad spectrum of aromatic substances were partially mineralized by the MnP system of the white rot fungus Nematoloma frowardii. The cell-free MnP system partially converted several aromatic compounds, including [U-¹⁴C]pentachlorophenol ([U-¹⁴C]PCP), [U-¹⁴C]catechol, [U-¹⁴C]tyrosine, [U-¹⁴C]tryptophan, [4,5,9,10-¹⁴C]pyrene, and [ring U-¹⁴C]2-amino-4,6-dinitrotoluene ([¹⁴C]2-AmDNT), to ¹⁴CO₂. Mineralization was dependent on the ratio of MnP activity to concentration of reduced glutathione (thiol-mediated oxidation), a finding which was demonstrated by using [¹⁴C]2-AmDNT as an example. At [¹⁴C]2-AmDNT concentrations ranging from 2 to 120 μM, the amount of released ¹⁴CO₂ was directly proportional to the concentration of [¹⁴C]2-AmDNT. The formation of highly polar products was also observed with [¹⁴C]2-AmDNT and [U-¹⁴C]PCP; these products were probably low-molecular-weight carboxylic acids. Among the aliphatic compounds tested, glyoxalate was mineralized to the greatest extent. Eighty-six percent of the ¹⁴COOH-glyoxalate and 9% of the ¹⁴CHO-glyoxalate were converted to ¹⁴CO₂, indicating that decarboxylation reactions may be the final step in MnP-catalyzed mineralization. The extracellular enzymatic combustion catalyzed by MnP could represent an important pathway for the formation of carbon dioxide from recalcitrant xenobiotic compounds and may also have general significance in the overall biodegradation of resistant natural macromolecules, such as lignins and humic substances.     

Consumption of tropospheric levels of methyl bromide by C(1) compound-utilizing bacteria and comparison to saturation kinetics.

Pure cultures of methylotrophs and methanotrophs are known to oxidize methyl bromide (MeBr); however, their ability to oxidize tropospheric concentrations (parts per trillion by volume [pptv]) has not been tested. Methylotrophs and methanotrophs were able to consume MeBr provided at levels that mimicked the tropospheric mixing ratio of MeBr (12 pptv) at equilibrium with surface waters ( approximately 2 pM). Kinetic investigations using picomolar concentrations of MeBr in a continuously stirred tank reactor (CSTR) were performed using strain IMB-1 and Leisingeria methylohalidivorans strain MB2(T) - terrestrial and marine methylotrophs capable of halorespiration. First-order uptake of MeBr with no indication of threshold was observed for both strains. Strain MB2(T) displayed saturation kinetics in batch experiments using micromolar MeBr concentrations, with an apparent K(s) of 2.4 microM MeBr and a V(max) of 1.6 nmol h(-1) (10(6) cells)(-1). Apparent first-order degradation rate constants measured with the CSTR were consistent with kinetic parameters determined in batch experiments, which used 35- to 1 x 10(7)-fold-higher MeBr concentrations. Ruegeria algicola (a phylogenetic relative of strain MB2(T)), the common heterotrophs Escherichia coli and Bacillus pumilus, and a toluene oxidizer, Pseudomonas mendocina KR1, were also tested. These bacteria showed no significant consumption of 12 pptv MeBr; thus, the ability to consume ambient mixing ratios of MeBr was limited to C(1) compound-oxidizing bacteria in this study. Aerobic C(1) bacteria may provide model organisms for the biological oxidation of tropospheric MeBr in soils and waters.     

An in Vitro System from Maize Seedlings for Tryptophan-Independent Indole-3-Acetic Acid Biosynthesis

The enzymatic synthesis of indole-3-acetic acid (IAA) from indole by an in vitro preparation from maize (Zea mays L.) that does not use tryptophan (Trp) as an intermediate is described. Light-grown seedlings of normal maize and the maize mutant orange pericarp were shown to contain the necessary enzymes to convert [¹⁴C]indole to IAA. The reaction was not inhibited by unlabeled Trp and neither [¹⁴C]Trp nor [¹⁴C]serine substituted for [¹⁴C]indole in this in vitro system. The reaction had a pH optimum greater than 8.0, required a reducing environment, and had an oxidation potential near that of ascorbate. The results obtained with this in vitro enzyme preparation provide strong, additional evidence for the presence of a Trp-independent IAA biosynthesis pathway in plants.     

Co-Permeability of 3H-Labeled Water and 14C-Labeled Organic Acids across Isolated Plant Cuticles : Investigating Cuticular Paths of Diffusion and Predicting Cuticular Transpiration

Penetration of ³H-labeled water (³H₂O) and the ¹⁴C-labeled organic acids benzoic acid ([¹⁴C]BA), salicylic acid ([¹⁴C]SA), and 2,4-dichlorophenoxyacetic acid ([¹⁴C]2,4-D) were measured simultaneously in isolated cuticular membranes of Prunus laurocerasus L., Ginkgo biloba L., and Juglans regia L. For each of the three pairs of compounds (³H₂O/[¹⁴C]BA, ³H₂O/[¹⁴C]SA, and ³H₂O/[¹⁴C]2,4-D) rates of cuticular water penetration were highly correlated with the rates of penetration of the organic acids. Therefore, water and organic acids penetrated the cuticles by the same routes. With the combination ³H₂O/[¹⁴C]BA, co-permeability was measured with isolated cuticles of nine other plant species. Permeances of ³H₂O of all 12 investigated species were highly correlated with the permeances of [¹⁴C]BA (r² = 0.95). Thus, cuticular transpiration can be predicted from BA permeance. The application of this experimental method, together with the established prediction equation, offers the opportunity to answer several important questions about cuticular transport physiology in future investigations.     

Carbon Monoxide Oxidation by Clostridium thermoaceticum and Clostridium formicoaceticum

Cultures of Clostridium formicoaceticum and C. thermoaceticum growing on fructose and glucose, respectively, were shown to rapidly oxidize CO to CO₂. Rates up to 0.4 μmol min⁻¹ mg of wet cells⁻¹ were observed. Carbon monoxide oxidation by cell suspensions was found (i) to be dependent on pyruvate, (ii) to be inhibited by alkyl halides and arsenate, and (iii) to stimulate CO₂ reduction to acetate. Cell extracts catalyzed the oxidation of carbon monoxide with methyl viologen at specific rates up to 10 μmol min⁻¹ mg of protein⁻¹ (35°C, pH 7.2). Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and ferredoxin from C. pasteurianum were ineffective as electron acceptors. The catalytic mechanism of carbon monoxide oxidation was “ping-pong,” indicating that the enzyme catalyzing carbon monoxide oxidation can be present in an oxidized and a reduced form. The oxidized form was shown to react reversibly with cyanide, and the reduced form was shown to react reversibly with alkyl halides: cyanide inactivated the enzyme only in the absence of carbon monoxide, and alkyl halides inactivated it only in the presence of carbon monoxide. Extracts inactivated by alkyl halides were reactivated by photolysis. The findings are interpreted to indicate that carbon monoxide oxidation in the two bacteria is catalyzed by a corrinoid enzyme and that in vivo the reaction is coupled with the reduction of CO₂ to acetate. Cultures of C. acidi-urici and C. cylindrosporum growing on hypoxanthine were found not to oxidize CO, indicating that clostridia mediating a corrinoid-independent total synthesis of acetate from CO₂ do not possess a CO-oxidizing system.     

Successive Mineralization and Detoxification of Benzo[a]pyrene by the White Rot Fungus Bjerkandera sp. Strain BOS55 and Indigenous Microflora

White rot fungi can oxidize high-molecular-weight polycyclic aromatic hydrocarbons (PAH) rapidly to polar metabolites, but only limited mineralization takes place. The objectives of this study were to determine if the polar metabolites can be readily mineralized by indigenous microflora from several inoculum sources, such as activated sludge, forest soils, and PAH-adapted sediment sludge, and to determine if such metabolites have decreased mutagenicity compared to the mutagenicity of the parent PAH. ¹⁴C-radiolabeled benzo[a]pyrene was subjected to oxidation by the white rot fungus Bjerkandera sp. strain BOS55. After 15 days, up to 8.5% of the [¹⁴C]benzo[a]pyrene was recovered as ¹⁴CO₂ in fungal cultures, up to 73% was recovered as water-soluble metabolites, and only 4% remained soluble in dibutyl ether. Thin-layer chromatography analysis revealed that many polar fluorescent metabolites accumulated. Addition of indigenous microflora to fungal cultures with oxidized benzo[a]pyrene on day 15 resulted in an initially rapid increase in the level of ¹⁴CO₂ recovery to a maximal value of 34% by the end of the experiments (>150 days), and the level of water-soluble label decreased to 16% of the initial level. In fungal cultures not inoculated with microflora, the level of ¹⁴CO₂ recovery increased to 13.5%, while the level of recovery of water-soluble metabolites remained as high as 61%. No large differences in ¹⁴CO₂ production were observed with several inocula, showing that some polar metabolites of fungal benzo[a]pyrene oxidation were readily degraded by indigenous microorganisms, while other metabolites were not. Of the inocula tested, only PAH-adapted sediment sludge was capable of directly mineralizing intact benzo[a]pyrene, albeit at a lower rate and to a lesser extent than the mineralization observed after combined treatment with white rot fungi and indigenous microflora. Fungal oxidation of benzo[a]pyrene resulted in rapid and almost complete elimination of its high mutagenic potential, as observed in the Salmonella typhimurium revertant test performed with strains TA100 and TA98. Moreover, no direct mutagenic metabolite could be detected during fungal oxidation. The remaining weak mutagenic activity of fungal cultures containing benzo[a]pyrene metabolites towards strain TA98 was further decreased by subsequent incubations with indigenous microflora.     

Measurement of Monosaccharides and Conversion of Glucose to Acetate in Anoxic Rice Field Soil

Degradation of glucose has been implicated in acetate production in rice field soil, but the abundance of glucose, the temporal change of glucose turnover, and the relationship between glucose and acetate catabolism are not well understood. We therefore measured the pool sizes of glucose and acetate in rice field soil and investigated the turnover of [U-¹⁴C]glucose and [2-¹⁴C]acetate. Acetate accumulated up to about 2 mM during days 5 to 10 after flooding of the soil. Subsequently, methanogenesis started and the acetate concentration decreased to about 100 to 200 μM. Glucose always made up >50% of the total monosaccharides detected. Glucose concentrations decreased during the first 10 days from 90 μM initially to about 3 μM after 40 days of incubation. With the exception at day 0 when glucose consumption was slow, the glucose turnover time was in the range of minutes, while the acetate turnover time was in the range of hours. Anaerobic degradation of [U-¹⁴C]glucose released [¹⁴C]acetate and ¹⁴CO₂ as the main products, with [¹⁴C]acetate being released faster than ¹⁴CO₂. The products of [2-¹⁴C]acetate metabolism, on the other hand, were ¹⁴CO₂ during the reduction phase of soil incubation (days 0 to 15) and ¹⁴CH₄ during the methanogenic phase (after day 15). Except during the accumulation period of acetate (days 5 to 10), approximately 50 to 80% of the acetate consumed was produced from glucose catabolism. However, during the accumulation period of acetate, the rate of acetate production from glucose greatly exceeded that of acetate consumption. Under steady-state conditions, up to 67% of the CH₄ was produced from acetate, of which up to 56% was produced from glucose degradation.     

Manganese Peroxidase-Dependent Oxidation of Glyoxylic and Oxalic Acids Synthesized by Ceriporiopsis subvermispora Produces Extracellular Hydrogen Peroxide

The ligninolytic system of the basidiomycete Ceriporiopsis subvermispora is composed of manganese peroxidase (MnP) and laccase. In this work, the source of extracellular hydrogen peroxide required for MnP activity was investigated. Our attention was focused on the possibility that hydrogen peroxide might be generated by MnP itself through the oxidation of organic acids secreted by the fungus. Both oxalate and glyoxylate were found in the extracellular fluid of C. subvermispora cultures grown in chemically defined media, where MnP is also secreted. The in vivo oxidation of oxalate was measured; ¹⁴CO₂ evolution was monitored after addition of exogenous [¹⁴C]oxalate to cultures at constant specific activity. In standard cultures, evolution of CO₂ from oxalate was maximal at day 6, although the MnP titers were highest at day 12, the oxalate concentration was maximal (2.5 mM) at day 10, and the glyoxylate concentration was maximal (0.24 mM) at day 5. However, in cultures containing low nitrogen levels, in which the pH is more stable, a better correlation between MnP titers and mineralization of oxalate was observed. Both MnP activity and oxidation of [¹⁴C]oxalate were negligible in cultures lacking Mn(II). In vitro assays confirmed that Mn(II)-dependent oxidation of [¹⁴C]oxalate by MnP occurs and that this reaction is stimulated by glyoxylate at the concentrations found in cultures. In addition, both organic acids supported phenol red oxidation by MnP without added hydrogen peroxide, and glyoxylate was more reactive than oxalate in this reaction. Based on these results, a model is proposed for the extracellular production of hydrogen peroxide by C. subvermispora.     

Nitrogen, Carbon, and Sulfur Metabolism in Natural Thioploca Samples

Filamentous sulfur bacteria of the genus Thioploca occur as dense mats on the continental shelf off the coast of Chile and Peru. Since little is known about their nitrogen, sulfur, and carbon metabolism, this study was undertaken to investigate their (eco)physiology. Thioploca is able to store internally high concentrations of sulfur globules and nitrate. It has been previously hypothesized that these large vacuolated bacteria can oxidize sulfide by reducing their internally stored nitrate. We examined this nitrate reduction by incubation experiments of washed Thioploca sheaths with trichomes in combination with ¹⁵N compounds and mass spectrometry and found that these Thioploca samples produce ammonium at a rate of 1 nmol min⁻¹ mg of protein⁻¹. Controls showed no significant activity. Sulfate was shown to be the end product of sulfide oxidation and was observed at a rate of 2 to 3 nmol min⁻¹ mg of protein⁻¹. The ammonium and sulfate production rates were not influenced by the addition of sulfide, suggesting that sulfide is first oxidized to elemental sulfur, and in a second independent step elemental sulfur is oxidized to sulfate. The average sulfide oxidation rate measured was 5 nmol min⁻¹ mg of protein⁻¹ and could be increased to 10.7 nmol min⁻¹ mg of protein⁻¹ after the trichomes were starved for 45 h. Incorporation of ¹⁴CO₂ was at a rate of 0.4 to 0.8 nmol min⁻¹ mg of protein⁻¹, which is half the rate calculated from sulfide oxidation. [2-¹⁴C]acetate incorporation was 0.4 nmol min⁻¹ mg of protein⁻¹, which is equal to the CO₂ fixation rate, and no ¹⁴CO₂ production was detected. These results suggest that Thioploca species are facultative chemolithoautotrophs capable of mixotrophic growth. Microautoradiography confirmed that Thioploca cells assimilated the majority of the radiocarbon from [2-¹⁴C]acetate, with only a minor contribution by epibiontic bacteria present in the samples.     

Anaerobic Oxidation of [1,2-14C]Dichloroethene under Mn(IV)-Reducing Conditions

Anaerobic oxidation of [1,2-¹⁴C]dichloroethene to ¹⁴CO₂ under Mn(IV)-reducing conditions was demonstrated. The results indicate that oxidative degradation of partially chlorinated solvents like dichloroethene can be significant even under anoxic conditions and demonstrate the potential importance of Mn(IV) reduction for remediation of chlorinated groundwater contaminants.     

Metabolism of Polyamines in Transgenic Cells of Carrot Expressing a Mouse Ornithine Decarboxylase cDNA

The metabolisms of arginine (Arg), ornithine (Orn), and putrescine were compared in a nontransgenic and a transgenic cell line of carrot (Daucus carota L.) expressing a mouse Orn decarboxylase cDNA. [¹⁴C]Arg, [¹⁴C]Orn, and [¹⁴C]putrescine were fed to cells and their rates of decarboxylation, uptake, metabolism into polyamines, and incorporation into acid-insoluble material were determined. Transgenic cells showed higher decarboxylation rates for labeled Orn than the nontransgenic cells. This was correlated positively with higher amounts of labeled putrescine production from labeled Orn. With labeled Arg, both the transgenic and the nontransgenic cells exhibited similar rates of decarboxylation and conversion into labeled putrescine. When [¹⁴C]putrescine was fed, higher rates of degradation were observed in transgenic cells as compared with the nontransgenic cells. It is concluded that (a) increased production of putrescine via the Orn decarboxylase pathway has no compensatory effects on the Arg decarboxylase pathway, and (b) higher rates of putrescine production in the transgenic cells are accompanied by higher rates of putrescine conversion into spermidine and spermine as well as the catabolism of putrescine.     

Methyl t-Butyl Ether Mineralization in Surface-Water Sediment Microcosms under Denitrifying Conditions

Mineralization of [U-¹⁴C]methyl t-butyl ether (MTBE) to ¹⁴CO₂ without accumulation of t-butyl alcohol (TBA) was observed in surface-water sediment microcosms under denitrifying conditions. Methanogenic activity and limited transformation of MTBE to TBA were observed in the absence of denitrification. Results indicate that bed sediment microorganisms can effectively degrade MTBE to nontoxic products under denitrifying conditions.