Degradation and mineralization of high-molecular-weight polycyclic aromatic hydrocarbons by defined fungal-bacterial cocultures

This study investigated the biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs) in liquid media and soil by bacteria (Stenotrophomonas maltophilia VUN 10,010 and bacterial consortium VUN 10,009) and a fungus (Penicillium janthinellum VUO 10, 201) that were isolated from separate creosote- and manufactured-gas plant-contaminated soils. The bacteria could use pyrene as their sole carbon and energy source in a basal salts medium (BSM) and mineralized significant amounts of benzo[a]pyrene cometabolically when pyrene was also present in BSM. P. janthinellum VUO 10,201 could not utilize any high-molecular-weight PAH as sole carbon and energy source but could partially degrade these if cultured in a nutrient broth. Although small amounts of chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene were degraded by axenic cultures of these isolates in BSM containing a single PAH, such conditions did not support significant microbial growth or PAH mineralization. However, significant degradation of, and microbial growth on, pyrene, chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene, each as a single PAH in BSM, occurred when P. janthinellum VUO 10,201 and either bacterial consortium VUN 10,009 or S. maltophilia VUN 10,010 were combined in the one culture, i.e., fungal-bacterial cocultures: 25% of the benzo[a]pyrene was mineralized to CO(2) by these cocultures over 49 days, accompanied by transient accumulation and disappearance of intermediates detected by high-pressure liquid chromatography. Inoculation of fungal-bacterial cocultures into PAH-contaminated soil resulted in significantly improved degradation of high-molecular-weight PAHs, benzo[a]pyrene mineralization (53% of added [(14)C]benzo[a]pyrene was recovered as (14)CO(2) in 100 days), and reduction in the mutagenicity of organic soil extracts, compared with the indigenous microbes and soil amended with only axenic inocula.     

Isolation of poly(3-hydroxybutyrate) (PHB)-degrading microorganisms and characterization of PHB-depolymerase from Arthrobacter sp. strain W6.

Microbial degraders of poly(3-hydroxybutyrate) (PHB) were isolated from soil. Arthrobacter sp. strain W6 used not only PHB as a carbon source, but also PHAs such as poly(3-hydroxybutyrate-co-[5%]3-hydroxyvalerate), poly(3-hydroxybutyrate-co-[14%]3-hydroxyvalerate), and poly(3-hydroxybutyrate-co-[22%]3-hydroxyvalerate). PHB-depolymerase was purified to homogeneity from the culture broth of Arthrobacter sp. strain W6 by a procedure involving DEAE- and butyl-Toyopearl column chromatographies. The Mr of the enzyme was estimated to be about 47,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 8.5 and 50 degrees C, and was inhibited by phenylmethylsulfonyl fluoride, Hg2+, Ag+, and Pb2+.     

Biodegradation of TNT (2,4,6-trinitrotoluene) by Phanerochaete chrysosporium.

Extensive biodegradation of TNT (2,4,6-trinitrotoluene) by the white rot fungus Phanerochaete chrysosporium was observed. At an initial concentration of 1.3 mg/liter, 35.4 +/- 3.6% of the [14C]TNT was degraded to 14CO2 in 18 days. The addition of glucose 12 days after the addition of TNT did not stimulate mineralization, and, after 18 days of incubation with TNT only, about 3.3% of the initial TNT could be recovered. Mineralization of [14C]TNT adsorbed on soil was also examined. Ground corncobs served as the nutrient for slow but sustained degradation of [14C]TNT to 14CO2 such that 6.3 +/- 0.6% of the [14C]TNT initially present was converted to 14CO2 during the 30-day incubation period. Mass balance analysis of liquid cultures and of soil-corncob cultures revealed that polar [14C]TNT metabolites are formed in both systems, and high-performance liquid chromatography analyses revealed that less than 5% of the radioactivity remained as undegraded [14C]TNT following incubation with the fungus in soil or liquid cultures. When the concentration of TNT in cultures (both liquid and soil) was adjusted to contamination levels that might be found in the environment, i.e., 10,000 mg/kg in soil and 100 mg/liter in water, mineralization studies showed that 18.4 +/- 2.9% and 19.6 +/- 3.5% of the initial TNT was converted to 14CO2 in 90 days in soil and liquid cultures, respectively. In both cases (90 days in water at 100 mg/liter and in soil at 10,000 mg/kg) approximately 85% of the TNT was degraded. These results suggest that this fungus may be useful for the decontamination of sites in the environment contaminated with TNT.     

Biodegradation of TNT (2,4,6-trinitrotoluene) by Phanerochaete chrysosporium

Extensive biodegradation of TNT (2,4,6-trinitrotoluene) by the white rot fungus Phanerochaete chrysosporium was observed. At an initial concentration of 1.3 mg/liter, 35.4 +/- 3.6% of the [14C]TNT was degraded to 14CO2 in 18 days. The addition of glucose 12 days after the addition of TNT did not stimulate mineralization, and, after 18 days of incubation with TNT only, about 3.3% of the initial TNT could be recovered. Mineralization of [14C]TNT adsorbed on soil was also examined. Ground corncobs served as the nutrient for slow but sustained degradation of [14C]TNT to 14CO2 such that 6.3 +/- 0.6% of the [14C]TNT initially present was converted to 14CO2 during the 30-day incubation period. Mass balance analysis of liquid cultures and of soil-corncob cultures revealed that polar [14C]TNT metabolites are formed in both systems, and high-performance liquid chromatography analyses revealed that less than 5% of the radioactivity remained as undegraded [14C]TNT following incubation with the fungus in soil or liquid cultures. When the concentration of TNT in cultures (both liquid and soil) was adjusted to contamination levels that might be found in the environment, i.e., 10,000 mg/kg in soil and 100 mg/liter in water, mineralization studies showed that 18.4 +/- 2.9% and 19.6 +/- 3.5% of the initial TNT was converted to 14CO2 in 90 days in soil and liquid cultures, respectively. In both cases (90 days in water at 100 mg/liter and in soil at 10,000 mg/kg) approximately 85% of the TNT was degraded. These results suggest that this fungus may be useful for the decontamination of sites in the environment contaminated with TNT.     

Utilization of l-threonine by a species of Arthrobacter. A novel catabolic role for `aminoacetone synthase''

1. A species of Arthrobacter (designated Arthrobacter 9759) was isolated from soil by its ability to grow aerobically on l-threonine as sole source of carbon atoms, nitrogen atoms and energy; the organism also grew well on other sources of carbon atoms including glycine, but no growth was obtainable on aminoacetone or dl-1-aminopropan-2-ol. 2. During growth on threonine, (14)C from l-[U-(14)C]threonine was rapidly incorporated into glycine and citrate, and thereafter into serine, alanine, aspartate and glutamate. 3. With extracts of threonine-grown cells supplied with l-[U-(14)C]threonine, evidence was obtained of the NAD and CoA-dependent catabolism of l-threonine to produce acetyl-CoA plus glycine. Short-term incorporation studies in which [2-(14)C]acetate and [2-(14)C]glycine were supplied (a) to cultures growing on threonine, and (b) to extracts of threonine-grown cells, showed that the acetyl-CoA was metabolized via the tricarboxylic acid cycle and glyoxylate cycle whereas the glycine was converted into pyruvate via the folate-dependent ;serine pathway'. 4. The threonine-grown organism contained ;biosynthetic' threonine dehydratase and a potent NAD-linked l-threonine dehydrogenase but possessed no l-threonine aldolase activity. 5. Evidence was obtained that the acetyl-CoA and glycine produced from l-threonine had their immediate origin in the alpha-amino-beta-oxobutyrate formed by the threonine dehydrogenase; the CoA-dependent cleavage of this compound was catalysed by an alpha-amino-beta-oxobutyrate CoA-ligase, which was identified with ;aminoacetone synthase'. A continuous spectrophotometric assay of this enzyme was developed, and it was found to be inducibly synthesized only during growth on threonine and not during growth on acetate plus glycine. 6. By using a reconstituted mixture of separately purified l-threonine dehydrogenase and alpha-amino-beta-oxobutyrate CoA-ligase (i.e. ;aminoacetone synthase'), l-[U-(14)C]threonine was broken down to [(14)C]glycine plus [(14)C]acetyl-CoA (trapped as [(14)C]citrate). 7. There was no evidence of aminoacetone metabolism by Arthrobacter 9759 even though a small amount of this amino ketone appeared in the culture medium during growth on threonine.     

Biodegradation of pentachlorophenol in soil: the response to physical, chemical, and biological treatments

The effects of physical, chemical, and biological treatments on biodegradation of pentachlorophenol (PCP) were studied in a silt-loam soil contaminated with 175 mg PCP/kg and uniformly 14C-labelled PCP. Biodegradation of 14C-labelled PCP and technical-grade PCP were monitored over 210 days incubation. Mineralization of labelled PCP was significantly (p=0.05) influenced by soil treatments. Negligible biodegradation occurred in either the sterile control soil or the uninoculated control soil, with less than 1% of added 14C recovered as 14 CO2. Inoculation of unamended soil with a strain of Flavobacterium (ATCC 39723) known to degrade PCP increased biodegradation of PCP; approximately 60% of the [14C]PCP was recovered as 14CO2. Increased soil water content (60% versus 30% w/w) enhanced biodegradation (67% recovery of 14C as CO2), while increased chloride ion concentration and anoxic conditions were inhibitory (20 and 1% recoveries, respectively). Residual soil PCP concentrations were also influenced by various treatments. In the sterile control soil and noninoculated control, after 210 days incubation, concentrations of PCP were 143 and 1223 mg/kg, respectively, while the PCP concentration in the inoculated soil was 21 mg/kg. When soil organic matter was increased by adding finely ground red clover leaf and stem material, the residual PCP concentration was reduced to 6 mg/kg after 210 days. Increased soil water content resulted in a residual PCP concentration of 5 mg/kg. High-pressure liquid chromatography of soil extracts revealed no accumulation of partial PCP degradation products. The results indicated that biodegradation of PCP in soil was significantly influenced by various soil amendments.     

Biodegradation of 1,2,3- and 1,2,4-trichlorobenzene in soil and in liquid enrichment culture.

The biodegradation of radiochemically pure (99%) 1,2,3- and 1,2,4-trichlorobenzene (TCB) in soil was investigated. Experimental difficulties posed by the high volatility and slow biodegradation rate of the TCBs were partially overcome by using a specially designed incubation and trapping apparatus. Evolution of (14)CO(2) from active versus poisoned soil dosed with 50 mug of the individual TCBs per g gave conclusive proof that both isomers are biodegradable. At 20 degrees C, 1,2,4-TCB was mineralized at an approximate rate of 1 nmol/day per 20 g of soil sample, and 1,2,3-TCB was mineralized at one-half to one-third that rate. Mineral fertilizers or cosubstrates failed to increase TCB mineralization rates in soil. Anaerobic conditions had a negative effect on mineralization, and increased temperatures had a positive effect. With increasing 1,2,4-TCB concentrations, (14)CO(2) evolution exhibited saturation kinetics with an apparent K(m) of 55.5 nmol per g of soil. Recovery of total radioactivity was good from soil containing high organic matter concentrations. From low-organic-matter soil, some of the radioactivity was recovered only on combustion, and overall recovery was lower. In soil-inoculated liquid culture, the cosubstrates glucose and benzene caused a slight stimulation of 1,2,4-TCB mineralization. Cochromatography of known standards with the extracts of soil pretreated with [(14)C]TCBs indicated that 3,4,5-trichlorophenol, 2,6-dichlorophenol and, to a lesser degree, 2,3-dichlorophenol were present in soils incubated with 1,2,3-TCB. 2,4-, 2,5-, and 3,4-dichlorophenol were present in soils incubated with 1,2,4-TCB.     

Pristine soils mineralize 3-chlorobenzoate and 2,4-dichlorophenoxyacetate via different microbial populations.

Biodegradation of two chlorinated aromatic compounds was found to be a common capability of the microorganisms found in the soils of undisturbed, pristine ecosystems. We used 2,4-dichlorophenoxyacetate (2,4-D) and 3-chlorobenzoate (3CBA) as enrichment substrates to compare populations of degrading bacteria from six different regions making up two ecosystems. We collected soil samples from four Mediterranean (California, central Chile, the Cape region of South Africa, and southwestern Australia) and two boreal (northern Saskatchewan and northwestern Russia) ecosystems that had no direct exposure to pesticides or to human disturbance. Between 96 and 120 samples from each of the six regions were incubated with 50 ppm of [U-14C]2,4-D or [U-14C]3CBA. Soils from all regions samples mineralized both 2,4-D and 3CBA, but 3CBA was mineralized without a lag period, while 2,4-D was generally not mineralized until the second week. 3CBA degradative capabilities were more evenly distributed spatially than those for 2,4-D. The degradative capabilities of the soils were readily transferred to fresh liquid medium. 3CBA degraders were easily isolated from most soils. We recovered 610 strains that could release carbon dioxide from ring-labeled 3CBA. Of these, 144 strains released chloride and degraded over 80% of 1 mM 3CBA in 3 weeks or less. In contrast, only five 2,4-D degraders could be isolated, although a variety of methods were used in an attempt to culture the degraders. The differences in the distribution and culturability of the bacteria responsible for 3CBA and 2,4-D degradation in these ecosystems suggest that the two substrates are degraded by different populations. We also describe a 14C-based microtiter plate method that allows efficient screening of a large number of samples for biodegradation activity.     

Comparative biodegradation of alkyl halide insecticides by the white rot fungus, Phanerochaete chrysosporium (BKM-F-1767).

The ability of Phanerochaete chrysosporium to degrade six alkyl halide insecticides (aldrin, dieldrin, heptachlor, chlordane, lindane, and mirex) in liquid and soil-corncob matrices was compared by using 14C-labeled compounds. Of these, only [14C]lindane and [14C]chlordane underwent extensive biodegradation, as evidenced by the fact that 9.4 to 23.4% of these compounds were degraded to 14CO2 in 30 days in liquid cultures and 60 days in soil-corncob cultures inoculated with P. chrysosporium. Although [14C]aldrin, [14C]dieldrin, [14C]heptachlor, and [14D]mirex were poorly mineralized, substantial bioconversion occurred, as determined by substrate disappearance and metabolite formation. Nonbiological disappearance was observed only with chlordane and heptachlor.     

Comparative biodegradation of alkyl halide insecticides by the white rot fungus, Phanerochaete chrysosporium (BKM-F-1767).

The ability of Phanerochaete chrysosporium to degrade six alkyl halide insecticides (aldrin, dieldrin, heptachlor, chlordane, lindane, and mirex) in liquid and soil-corncob matrices was compared by using 14C-labeled compounds. Of these, only [14C]lindane and [14C]chlordane underwent extensive biodegradation, as evidenced by the fact that 9.4 to 23.4% of these compounds were degraded to 14CO2 in 30 days in liquid cultures and 60 days in soil-corncob cultures inoculated with P. chrysosporium. Although [14C]aldrin, [14C]dieldrin, [14C]heptachlor, and [14D]mirex were poorly mineralized, substantial bioconversion occurred, as determined by substrate disappearance and metabolite formation. Nonbiological disappearance was observed only with chlordane and heptachlor.     

Dimethylsulfone as a growth substrate for novel methylotrophic species of Hyphomicrobium and Arthrobacter

Dimethylsulfone is a major product of the chemical oxidation in the atmosphere of the principal biogenic sulfur gas, dimethylsulfide, but no studies have been reported on the mechanisms for its microbiological degradation. Three novel strains of bacteria have been isolated from enrichment cultures provided with dimethylsulfone as the only carbon and energy substrate. These are novel facultatively methylotrophic species of Hyphonmicrobium and Arthobacter, capable of growth on a range of one-carbon substrates. Cell-free extracts contained activities of enzymes necessary for a reductive/oxidative pathway for dimethylsulfone degradation: membrane-bound-dimethylsulfone and dimethylsulfoxide reductases, dimethylsulfide monooxygenase, and methanethiol oxidase. Enzymatic evidence is also presented for the subsequent oxidation of formaldehyde by formaldehyde and formate dehydrogenases in the Hyphomicrobium strain and by a dissimilatory ribulose monophosphate cycle in the Arthrobacter strains. The strains also grew on dimethylsulfoxide and dimethylsulfide, and dimethylsulfide-grown bacteria oxidized dimethylsulfide and dimethylsulfoxide but not dimethylsulfone. Formaldehyde assimilation was effected in the Hyphomicrobium strain by the serine pathway, but enzymes of the ribulose monophosphate cycle for formaldehyde assimilation were present in the Arthrobacter strains grown on dimethylsulfone. In contrast, one of the Arthrobacter strains was shown to switch to the serine pathway during growth on methanol. Growth yields on dimethylsulfone and formaldehyde were consistent with the occurrence of the serine pathway in Hyphomicrobium strain S1 and the ribulose monophosphate cycle in Arthrobacter strain TGA, and with the proposed reductive pathway for dimethylsulfone degradation in both.     

Biodegradation of nitro-substituted explosives 2,4,6-trinitrotoluene, hexahydro-1,3,5-trinitro-1,3,5-triazine, and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine by a phytosymbiotic Methylobacterium sp. associated with poplar tissues (Populus deltoides x nigra DN34)

A pink-pigmented symbiotic bacterium was isolated from hybrid poplar tissues (Populus deltoides x nigra DN34). The bacterium was identified by 16S and 16S-23S intergenic spacer ribosomal DNA analysis as a Methylobacterium sp. (strain BJ001). The isolated bacterium was able to use methanol as the sole source of carbon and energy, which is a specific attribute of the genus Methylobacterium. The bacterium in pure culture was shown to degrade the toxic explosives 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine (HMX). [U-ring-(14)C]TNT (25 mg liter(-1)) was fully transformed in less than 10 days. Metabolites included the reduction derivatives amino-dinitrotoluenes and diamino-nitrotoluenes. No significant release of (14)CO(2) was recorded from [(14)C]TNT. In addition, the isolated methylotroph was shown to transform [U-(14)C]RDX (20 mg liter(-1)) and [U-(14)C]HMX (2.5 mg liter(-1)) in less than 40 days. After 55 days of incubation, 58.0% of initial [(14)C]RDX and 61.4% of initial [(14)C]HMX were mineralized into (14)CO(2). The radioactivity remaining in solution accounted for 12.8 and 12.7% of initial [(14)C]RDX and [(14)C]HMX, respectively. Metabolites detected from RDX transformation included a mononitroso RDX derivative and a polar compound tentatively identified as methylenedinitramine. Since members of the genus Methylobacterium are distributed in a wide diversity of natural environments and are very often associated with plants, Methylobacterium sp. strain BJ001 may be involved in natural attenuation or in situ biodegradation (including phytoremediation) of explosive-contaminated sites.     

Biomineralization of an organophosphorus pesticide,Monocrotophos, by soil bacteria

AIMS: To study biomineralization of Monocrotophos (MCP) and identify the metabolites formed during biodegradation. METHODS AND RESULTS: Two cultures, namely Arthrobacter atrocyaneus MCM B-425 and Bacillus megaterium MCM B-423, were isolated by enrichment and adaptation culture technique from soil exposed to MCP. The isolates were able to degrade MCP to the extent of 93% and 83%, respectively, from synthetic medium containing MCP at the concentration of 1000 mg x l(-1), within 8 d, under shake culture condition at 30 degrees C. The cultures degraded MCP to carbon dioxide, ammonia and phosphates through formation of one unknown compound--Metabolite I, valeric or acetic acid and methylamine, as intermediate metabolites. The enzymes phosphatase and esterase, reported to be involved in biodegradation of organophosphorus compounds, were detected in both the organisms. CONCLUSIONS:Arthrobacter atrocyaneus MCM B-425 and B. megaterium MCM B-423 isolated from soil exposed to MCP were able to mineralize MCP to carbon dioxide, ammonia and phosphates. SIGNIFICANCE AND IMPACT OF THE STUDY: Pathway for biodegradation of MCP in plants and animals has been reported. A microbial metabolic pathway of degradation involving phosphatase and esterase enzymes has been proposed. The microbial cultures could be used for bioremediation of wastewater or soil contaminated with Monocrotophos.     

Degradation and mineralization of atrazine by a soil bacterial isolate.

An atrazine-degrading bacterial culture was isolated from an agricultural soil previously impacted by herbicide spills. The organism was capable of using atrazine under aerobic conditions as the sole source of C and N. Cyanuric acid could replace atrazine as the sole source of N, indicating that the organism was capable of ring cleavage. Ring cleavage was confirmed in 14CO2 evolution experiments with [U-14C-ring]atrazine. Between 40 and 50% of ring-14C was mineralized to 14CO2. [14C]biuret and [14C]urea were detected in spent culture media. Cellular assimilation of 14C was negligible, in keeping with the fully oxidized valence of the ring carbon. Chloride release was stoichiometric. The formation of ammonium during atrazine degradation was below the stoichiometric amount, suggesting a deficit due to cellular assimilation and metabolite-N accumulation. With excess glucose and with atrazine as the sole N source, free ammonium was not detected, suggesting assimilation into biomass. The organism degraded atrazine anaerobically in media which contained (i) atrazine only, (ii) atrazine and glucose, and (iii) atrazine, glucose, and nitrate. To date, this is the first report of a pure bacterial isolate with the ability to cleave the s-triazine ring structure of atrazine. It was also concluded that this bacterium was capable of dealkylation, dechlorination, and deamination in addition to ring cleavage.     

Microbial transformations of styrene and [14C] styrene in soil and enrichment cultures.

Two different mechanisms were responsible for the disappearance of styrene in enrichment cultures: (i) a mixed population of microorganisms, capable of utilizing styrene as a sole carbon source, oxidized this substrate to phenylethanol and phenylacetic acid; (ii) the culture also mediated polymerization of the monomer to low-molecular-weight styrene oligomers. This chemical reaction probably occurred as the result of microbial degradation of butylcatechol, an antioxidant polymerization inhibitor present in commercial styrene. The resultant polymer material was subsequently metabolized. In soil incubation studies, 14CO2 evolution from applied [8-14C] styrene was used to estimate microbial degradation. Approximately 90 percent of the labeled carbon was evolved from a 0.2 percent addition, and about 75 percent was lost from the 0.5 percent application over a 16-week period.     

Microbial transformations of styrene and [14C] styrene in soil and enrichment cultures.

Two different mechanisms were responsible for the disappearance of styrene in enrichment cultures: (i) a mixed population of microorganisms, capable of utilizing styrene as a sole carbon source, oxidized this substrate to phenylethanol and phenylacetic acid; (ii) the culture also mediated polymerization of the monomer to low-molecular-weight styrene oligomers. This chemical reaction probably occurred as the result of microbial degradation of butylcatechol, an antioxidant polymerization inhibitor present in commercial styrene. The resultant polymer material was subsequently metabolized. In soil incubation studies, 14CO2 evolution from applied [8-14C] styrene was used to estimate microbial degradation. Approximately 90 percent of the labeled carbon was evolved from a 0.2 percent addition, and about 75 percent was lost from the 0.5 percent application over a 16-week period.     

Thiocyanate utilization by an Arthrobacter.

An Arthrobacter species, which utilized thiocyanate (SCN-) as a nitrogen source, was isolated from soil by the enrichment culture method. The organism tolerated SCN- concentrations up to 0.1 M. On addition of nitrate or ammonium ion to cultures of the isolate growing in the presence of SCN-, the organism continued to degrade SCN-. Degradation could be followed by release of 14CO2 from SCN-(14C). The SCN- -degrading activity diminished to low levels as the stationary phase of growth was appraoched.     

Formation of bound residues during microbial degradation of [14C]anthracene in soil

Carbon partitioning and residue formation during microbial degradation of polycyclic aromatic hydrocarbons (PAH) in soil and soil-compost mixtures were examined by using [14C]anthracenes labeled at different positions. In native soil 43.8% of [9-14C]anthracene was mineralized by the autochthonous microflora and 45.4% was transformed into bound residues within 176 days. Addition of compost increased the metabolism (67.2% of the anthracene was mineralized) and decreased the residue formation (20. 7% of the anthracene was transformed). Thus, the higher organic carbon content after compost was added did not increase the level of residue formation. [14C]anthracene labeled at position 1,2,3,4,4a,5a was metabolized more rapidly and resulted in formation of higher levels of residues (28.5%) by the soil-compost mixture than [14C]anthracene radiolabeled at position C-9 (20.7%). Two phases of residue formation were observed in the experiments. In the first phase the original compound was sequestered in the soil, as indicated by its limited extractability. In the second phase metabolites were incorporated into humic substances after microbial degradation of the PAH (biogenic residue formation). PAH metabolites undergo oxidative coupling to phenolic compounds to form nonhydrolyzable humic substance-like macromolecules. We found indications that monomeric educts are coupled by C-C- or either bonds. Hydrolyzable ester bonds or sorption of the parent compounds plays a minor role in residue formation. Moreover, experiments performed with 14CO2 revealed that residues may arise from CO2 in the soil in amounts typical for anthracene biodegradation. The extent of residue formation depends on the metabolic capacity of the soil microflora and the characteristics of the soil. The position of the 14C label is another important factor which controls mineralization and residue formation from metabolized compounds.     

Mineralization of the cyclic nitramine explosive hexahydro-1,3,5-trinitro-1,3,5-triazine by Gordonia and Williamsia spp

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a cyclic nitroamine explosive that is a major component in many military high-explosive formulations. In this study, two aerobic bacteria that are capable of using RDX as the sole source of carbon and nitrogen to support their growth were isolated from surface soil. These bacterial strains were identified by their fatty acid profiles and 16S ribosomal gene sequences as Williamsia sp. KTR4 and Gordonia sp. KTR9. The physiology of each strain was characterized with respect to the rates of RDX degradation and [U-14C]RDX mineralization when RDX was supplied as a sole carbon and nitrogen source in the presence and absence of competing carbon and nitrogen sources. Strains KTR4 and KTR9 degraded 180 microM RDX within 72 h when RDX served as the only added carbon and nitrogen source while growing to total protein concentrations of 18.6 and 16.5 microg/ml, respectively. Mineralization of [U-14C]RDX to 14CO2 was 30% by strain KTR4 and 27% by KTR9 when RDX was the only added source of carbon and nitrogen. The addition of (NH4)2SO4- greatly inhibited KTR9's degradation of RDX but had little effect on that of KTR4. These are the first two pure bacterial cultures isolated that are able to use RDX as a sole carbon and nitrogen source. These two genera possess different physiologies with respect to RDX mineralization, and each can serve as a useful microbiological model for the study of RDX biodegradation with regard to physiology, biochemistry, and genetics.     

Simultaneous degradation of acetonitrile and biphenyl by Pseudomonas aeruginosa

A bacterium capable of utilizing either acetonitrile as the sole source of carbon and nitrogen or biphenyl as the sole source of carbon was isolated from soil and identified as Pseudomonas aeruginosa. The bacterium also utilized other nitriles, amides, and polychlorinated biphenyls (PCBs) as growth substrates. Acetonitrile- or biphenyl-grown cells oxidized these substrates without a lag. In studies with [14C]acetonitrile, nearly 74% of the carbon was recovered as 14CO2 and 8% was associated with the biomass. In studies with [14C]biphenyl, nearly 68% of the carbon was recovered as 14CO2 and nearly 6% was associated with the biomass. Although higher concentrations of acetonitrile as the sole sources of nitrogen inhibited the rates of [14C]biphenyl mineralization, lower concentrations (0.05%, w/v) gave a 77% stimulation in 14CO2 recovery. Pseudomonas aeruginosa metabolized acetonitrile to ammonia and acetic acid and biphenyl to benzoic acid. The bacterium also simultaneously utilized biphenyl as the sole carbon source and acetonitrile as the sole nitrogen source. However, biphenyl utilization increased only after the depletion of acetonitrile. Metabolites of the mixed substrate were ammonia and benzoic acid, which completely disappeared in the later stages of incubation. Nitrile hydratase and amidase were responsible for the transformation of acetonitrile to acetic acid and ammonia.