Microbial degradation of n-alkyl tetrahydrothiophenes found in petroleum

Although n-alkyl-substituted tetrahydrothiophenes are found in nonbiodegraded petroleums, they are not found in petroleums which have undergone biodegradation in their reservoirs. These observations suggested that this group of compounds with alkyl chain lengths from approximately C10 to at least C30 is biodegradable. Two of these sulfides, 2-n-dodecyltetrahydrothiophene (DTHT) and 2-n-undecyltetrahydrothiophene, were synthesized, and their biodegradabilities were tested by using five gram-positive, n-alkane-degrading bacterial isolates. The alkyl side chains of these compounds were oxidized, and the major intermediates found in 2-n-undecyltetrahydrothiophene- and DTHT-metabolizing cultures were 2-tetrahydrothiophenecarboxylic acid (THTC) and 2-tetrahydrothiopheneacetic acid (THTA), respectively. Four n-alkane-degrading fungi were also shown to degrade DTHT, yielding both THTA and THTC. Quantitation of tetrahydrothiophene ring-containing products in 28-day-old bacterial and fungal cultures suggested that THTC and THTA were metabolized further to unidentified products. In addition, two of the bacterial isolates were shown to degrade a mixture of n-alkyl tetrahydrothiophenes isolated from Bellshill Lake crude oil.     

Biodegradation of alkylpyridines by bacteria isolated from a polluted subsurface

Ten bacterial strains were isolated from alkylpyridine polluted sediments 7.6 m below the surface. These strains were able to degrade 11 different alkylpyridine isomers. Degradation rates depended on number and position of the alkyl group. Isomers with an alkyl group at position 3 were more resistant to microbial attack. Of the 10 strains, 6 isolates were selected for detailed study. These isolates mineralized the isomers to CO2, NH4+, and biomass. All strains were gram-negative rods with a strict aerobic metabolism. Characterization of physiological and biochemical properties revealed similarity between strains. Eeach strain however, had a limited substrate range which enabled it to degrade no more than 2 to 3 compounds of the 14 alkylpyridine isomers tested. Examination of the genetic variability among cultures with the randomly amplified polymorphic DNA technique revealed high levels of genomic DNA polymorphism. The highest similarity between 2 strains (0.653) was observed between 2-picoline and 3-picoline degrading cultures. The molecular basis of the differences in substrate specificity is under investigation.     

Biodegradation of alkylpyridines by bacteria isolated from a polluted subsurface

Ten bacterial strains were isolated fromalkylpyridine polluted sediments 7.6 m below thesurface. These strains were able to degrade 11different alkylpyridine isomers. Degradation ratesdepended on number and position of the alkyl group. Isomers with an alkyl group at position 3 were moreresistant to microbial attack. Of the 10 strains, 6isolates were selected for detailed study. Theseisolates mineralized the isomers to CO₂,NH₄ ⁺, and biomass. All strains weregram-negative rods with a strict aerobic metabolism. Characterization of physiological and biochemicalproperties revealed similarity between strains. Eeachstrain however, had a limited substrate range whichenabled it to degrade no more than 2 to 3 compounds ofthe 14 alkylpyridine isomers tested. Examination ofthe genetic variability among cultures with therandomly amplified polymorphic DNA technique revealedhigh levels of genomic DNA polymorphism. The highestsimilarity between 2 strains (0.653) was observedbetween 2-picoline and 3-picoline degrading cultures. The molecular basis of the differences in substratespecificity is under investigation.     

The aerobic dechlorination activities of two bacterial species isolated from a refuse dumpsite in Nigeria

Two bacterial species isolated using enrichment culture techniques from the topsoil of a main refuse dumpsite in Nigeria were assessed for their dehalogenation potentials. The bacterial isolates were identified as belonging to the Bacillus and Pseudomonas genera. Axenic cultures of the isolates utilized monochloroacetic acid (MCA), trichloroacetic acid (TCA), trichloromethane (CHCl₃) and tetrachloromethane (CCl₄) as the sole source of carbon for growth up to a final substrate concentration of 0.1% (w/v). The mean generation times of the isolates in all the growth media ranged significantly (P<0.05) from 2.41 to 10.04 h and were generally higher than that observed in glucose medium (1.46–1.51 h). The numbers of the chloride atoms in the different organochlorides were negatively correlated with the ability of the organisms to degrade the compounds. Dehalogenase specific activities of the cell-mediated cultures ranged from 0.1 to 0.96 μg ml^–1 chloride release (mg protein)^–1 h^–1 and were significantly (P <0.05) higher than that of the cell-free extract [0.09–0.8 μg ml^–1 chloride release (mg protein)^–1 h^–1]. The optimal pH of the dehalogenase activity was found to be 8.0, and the optimal temperature was between 30 and 35 °C. Electronic Publication     

Isolation and characterization of anaerobic indole- and skatole-degrading bacteria from composting animal wastes

Four species of indole-degrading Clostridium and 3 species of skatole-degrading Clostridium were isolated from piggery or chicken manure composting processes. Since type strains of respective isolates did not degrade these compounds, the degradability of the compounds was a novel characteristic. All isolates were mesophilic. The maximum growth allowance concentrations of these isolates were 300 to 800 mg/l in indole and 100 to 300 mg/l in skatole. All isolates showed better growth and utilization of indolic compounds in nutrient-rich medium than in minimal medium. Skatole-degrading isolates degraded some substituted indoles tested, 3-indoleacetic acid, indole and oxindole, but did not degrade 1-methylindole, 2-methylindole, isatin or anthranilic acid. On the other hand, indole-degrading isolates degraded only oxindole. The growth of Clostridium malenominatum A-3 was inhibited by a low concentration (0.005%) of indole or skatole, even when 200-fold excess glucose was present in the medium. When 0.03% indole or skatole was added to the medium, C. malenominatum A-3 showed a lag phase for about 10 and 70 h, respectively. When 0.01% of these compounds was added to the medium, the uptake of glucose was inhibited. C. malenominatum A-3 degraded these compounds under nutrient-rich and minimal conditions.     

Modification of Water-Soluble Coal-Derived Products by Dibenzothiophene-Degrading Microorganisms

To study mechanisms by which microorganisms oxidize thiophenic sulfur in coal, we tested bacterial cultures for the ability to degrade dibenzothiophene (DBT), DBT-5-oxide, and DBT-sulfone and to modify water-soluble coal products derived from Illinois no. 6 and Ugljevik coals. In yeast extract medium, the majority of selected isolates degraded DBT and accumulated DBT-5-oxide in culture fluids; all but one of the cultures degraded DBT-5-oxide, and none of them degraded DBT-sulfone. Elemental analysis data indicated that the microbial cultures were able to decrease the amount of sulfur in soluble coal products derived from Illinois no. 6 and Ugljevik coals. However, these data suggested that microbially mediated sulfur removal from soluble Ugljevik coal occurred by nonspecific mechanisms. That is, extensive degradation of the carbon structure was concurrent with the loss of sulfur. This conclusion was supported by X-ray photoelectron spectroscopic data which indicated that the reduced sulfur forms in the soluble Ugljevik coal product was not oxidized by microbial treatment.     

THTA, a thermotolerance gene of Aspergillus fumigatus

Aspergillus fumigatus grows optimally from 37 to 42 degrees C but can grow at temperatures up to 55 degrees C. To study the genetic basis of thermotolerance and its role in virulence of A. fumigatus, temperature sensitive mutants were isolated. One of the mutants that grew at 42 degrees C but not at 48 degrees C was complemented and the gene, THTA, was identified. Deletion of THTA showed the same temperature sensitivity as the original mutant. THTA encodes a putative protein of 141 kDa with unknown function and the HA-tagged ThtAp accumulated to similar levels in cultures grown at either 37 or 48 degrees C. Southern blot analysis and database searches revealed the presence of THTA-related sequences in several other ascomycetous fungi. No difference in virulence was observed between the deltathtA and wild-type strains. Thus, THTA is essential for growth of A. fumigatus at high temperatures but does not contribute to the pathogenicity of the species.     

Utilization of Iron Gallate and Other Organic Iron Complexes by Bacteria from Water Supplies

The degradation of four soluble organic iron compounds by bacteria isolated from surface waters and the precipitation of iron from these complexes by the isolates was studied. All eight isolates brought about the precipitation of iron when grown on ferric ammonium citrate agar. Three isolates were able to degrade ferric malonate, and three others degraded ferric malate with iron precipitation. Only three isolates, two strains of Pseudomonas and one of Moraxella, were able to degrade gallic acid when this was supplied as the sole carbon source. One strain of Pseudomonas was found to be active in degrading ferric gallate. Electron microscopy of cells of this bacterium after growth in ferric gallate as the sole carbon source yielded results indicating uniform deposition of the iron on or in the bacterial cells. Seven of the isolates could degrade the iron gallate complex if supplied with additional carbon in the form of yeast extract.     

A comparative study of the biodegradation of the surfactant sodium dodecyltriethoxy sulphate by four detergent-degrading bacteria

The 35S-labelled metabolites produced during biodegradation of sodium dodecyltriethoxy [35S]sulphate (SDTES) by four bacterial isolates were identified and quantified. All four isolates used ether-cleavage as the predominant primary degradation pathway. In two of the organisms, the etherase system (responsible for approx. 60-70% of primary biodegradation) liberated mono-, di- and triethylene glycol monosulphates in substantial proportions, the last two esters undergoing some further oxidation to acetic acid 2-(ethoxy sulphate) and acetic acid 2-(diethoxy sulphate), respectively. For these isolates, liberation of SO4(2-) directly from SDTES was also significant (30-40%) and the organisms were shown to contain alkyl sulphatases active towards SDTES. For the remaining two isolates, etherase action was even more important (responsible for greater than 80% of primary biodegradation) and was restricted almost totally to the alkyl-ether bond to generate mainly triethylene glycol sulphate, some of which was further oxidized. Very small amounts of diethylene glycol monosulphate were also produced, but its mono-homologue, and the oxidation products of both these esters, were absent. Small amounts of inorganic sulphate (approx. 10%) were liberated by these isolates and one of them also produced compounds tentatively identified as intermediates of omega-/beta-oxidation.     

Identification and characterization of humic substances-degrading bacterial isolates from an estuarine environment

Bacterial isolates were obtained from enrichment cultures containing humic substances extracted from estuarine water using an XAD-8 resin. Eighteen isolates were chosen for phylogenetic and physiological characterization based on numerical importance in serial dilutions of the enrichment culture and unique colony morphology. Partial sequences of the 16S rRNA genes indicated that six of the isolates were associated with the alpha subclass of Proteobacteria, three with the gamma-Proteobacteria, and nine with the Gram-positive bacteria. Ten isolates degraded at least one (and up to six) selected aromatic single-ring compounds. Six isolates showed ability to degrade [(14)C]humic substances derived from the dominant salt marsh grass in the estuary from which they were isolated (Spartina alterniflora), mineralizing 0.4-1.1% of the humic substances over 4 weeks. A mixture of all 18 isolates did not degrade humic substances significantly faster than any of the individual strains, however, and no isolate degraded humic substances to the same extent as the natural marine bacterial community (3.0%). Similar studies with a radiolabeled synthetic lignin ([beta-(14)C]dehydropolymerisate) showed measurable levels of degradation by all 18 bacteria (3.0-8.8% in 4 weeks), but mineralization levels were again lower than that observed for the natural marine bacterial community (28.2%). Metabolic capabilities of the 18 isolates were highly variable and generally did not map to phylogenetic affiliation.     

Selection and isolation of bacteria capable of degrading dinoseb (2-sec-buty-4,6-dinitrophenol)

Dinoseb (2-sec-butyl-4,6-dinitrophenol) has been a widely used herbicide that persists in some contaminated soils, and has been found in groundwaters, causing health and environmental hazards. Persistence in some soils may stem from a lack of dinoseb-degrading organisms. We established a chemostat environment that was strongly selective for aerobic (liquid phase) and anaerobic (sediment phase) bacteria able to degrade dinoseb. The chemostat yielded five taxonomically diverse aerobic isolates that could transform dinoseb to reduced products under microaerophilic or denitrifying conditions, but these organisms were unable to degrade the entire dinoseb molecule, and the transformed products formed multimeric material. The chemostat also yielded an anaerobic consortium of bacteria that could completely degrade dinoseb to acetate and CO₂ when the Eh of the medium was less than-200 mV. The consortium contained at least three morphologically different bacterial species. HPLC analysis indicated that dinoseb was degraded sequentially via several as yet unidentified products. Degradation of these intermediates was inhibited by addition of bromoethane sulfonic acid. GC-MS analysis of metabolites in culture medium suggested that regiospecific attacks occurred non-sequentially on both the nitro groups and the side-chain of dinoseb. The consortium was also able to degrade 4,6-dinitro-o-cresol, 3,5-dinitrobenzoic acid, 2,4-dinitrotoluene, and 2,6-dinitrotoluene via a similar series of intermediate products. The consortium was not able to degrade 2,4-dinitrophenol. To our knowledge, this is the first report of strictly anaerobic biodegradation of an aromatic compound containing a multicarbon, saturated hydrocarbon side chain.Abbreviations BESA bromoethane sulfonic acid - RAMM reduced anaerobic mineral medium     

Phytol Degradation by Marine Bacteria

Microbial degradation of phytol is often proposed to be the primary source of the acyclic isoprenoid acids observed in sediments, yet only a limited number of these acids have been found in bacterial cultures grown on phytol. This study reports detailed capillary gas chromatography and gas chromatography-mass spectrometry analyses of the products resulting from growth of marine bacteria on phytol as the sole carbon source. We examined two strains of bacteria which were able to oxidize phytol to phytenic acid but were unable to further degrade phytol. The third isolate studied converted phytol to a mixture of five saturated isoprenoid acids. The C₁₇ isoprenoid acid produced was of particular interest, since its genesis from phytol would have involved several unusual intermediates. It is suggested that this acid is produced by bacterial metabolism of the C₁₈ isoprenoid ketone (produced from phytol abiologically under oxic conditions) and that its abundance is thus a sensitive indicator of sedimentary depositional conditions.     

Bacterial biodegradation of aliphatic sulfides under aerobic carbon- or sulfur-limited growth conditions

AIMS: To isolate bacteria capable of cleaving aliphatic carbon-sulfur bonds as potential biological upgrading catalysts for the reduction of molecular weight and viscosity in heavy crude oil. METHODS AND RESULTS: Thirty-one bacterial strains isolated from enrichment cultures were able to biotransform model compounds representing the aliphatic sulfide bridges found in asphaltenes. Using gas chromatography and mass spectrometry, three types of attack were identified: alkyl chain degradation, allowing use as a carbon source; nonspecific sulfur oxidation; and sulfur-specific oxidation and carbon-sulfur bond cleavage, allowing use as a sulfur source. Di-n-octyl sulfide degradation produced octylthio- and octylsulfonyl-alkanoic acids, consistent with terminal oxidation followed by beta-oxidation reactions. Utilization of dibenzyl sulfide or 1,4-dithiane as a sulfur source was regulated by sulfate, indicating a sulfur-specific activity rather than nonspecific oxidation. Finally, several isolates were also able to use dibenzothiophene as a sulfur source, and this was the preferred organic sulfur substrate for one isolate. CONCLUSIONS: The use of commercially available alkyl sulfides in enrichment cultures gave isolates that followed a range of metabolic pathways, not just sulfur-specific attack. SIGNIFICANCE AND IMPACT OF THE STUDY: These results give new insight into biodegradation of organosulfur compounds from petroleum and for biotreatment of such compounds in chemical munitions.     

A Structure-Activity Study with Aryl Acylamidases

We examined the relationship between chemical structure and biodegradability of acylanilide herbicides by using a set of model compounds. Four bacterial isolates (one gram-negative and three gram-positive) that grew on acetanilide were used. These soil isolates cleaved the amide bond of acetanilide via an aryl acylamidase reaction, producing aniline and the organic acid acetate. A series of acetanilide analogs with alkyl substitutions on the nitrogen atom or the aromatic ring were tested for their ability to induce aryl acylamidase activity and act as substrates for the enzyme. The substrate range, in general, was limited to those analogs not disubstituted in the ortho position of the benzene ring or which did not contain an alkyl group on the nitrogen atom. These same N-substituted compounds did not induce enzyme activity either, whereas the ortho-substituted compounds could in some cases.     

Degradation of Endrin, Aldrin, and DDT by Soil Microorganisms

Twenty microbial cultures which had been shown to degrade dieldrin were tested to determine their ability to degrade endrin, aldrin, DDT, gamma isomers of benzenehexachloride (gamma-BHC), and Baygon. All isolates were able to degrade DDT and endrin, whereas 13 degraded aldrin. However, none of them was able to degrade Baygon or gamma-BHC.     

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.     

Degradation of some phenoxy acid herbicides by mixed cultures of bacteria isolated from soil treated with 2-(2-methyl-4-chloro)phenoxypropionic acid

Mixed bacterial cultures capable of using 2-methyl-4-chIorophenoxyacetic acid (MCPA) and 2, 4-dichlorophenoxyacetic acid (2, 4-D) as the sole source of carbon and energy were isolated from field soil treated with the herbicide (±)2-(2-methyl-4-chloro)phenoxypropionic acid (mecoprop). An enrichment technique with two aromatic compounds as sources of carbon was used. Effects of temperature and substrate concentration were studied. The mixed cultures retained their ability to degrade MCPA although the bacteria were grown for 3 months (32 successive passages) with glucose as the sole source of carbon and energy. With benzoic acid as co-substrate, one of the cultures was also able to degrade mecoprop and (±)2-(2, 4-dichloro)phenoxypropionic acid (dichlorprop). This ability was not maintained, however, over more than 10 passages.     

Involvement of fungi and bacteria in enhanced and nonenhanced biodegradation of carbendazim and other benzimidazole compounds in soil

The relationship between chemical structure and the enhancement of microbial degradation of three benzimidazole compounds in soil was determined. Preapplication of methyl benzimidazole-2-ylcarbamate (carbendazim or MBC), 2-aminobenzimidazole (2AB), and benzimidazole enhanced their degradation upon repeated application (self-enhanced degradation). MBC and 2AB cross-enhanced the degradation of each of these two compounds, whereas benzimidazole did not enhance the degradation of MBC. Thiabendazole (TBZ) did not enhance its own degradation or cross-enhance the degradation of MBC. No increase in the number of MBC-degrading fungi or in the capacity of soilborne fungi to degrade MBC was detected in soil exhibiting enhanced MBC degradation (MBC-history). A sharp increase in esterolytic activity in the microsomal fraction of Alternaria alternata capable of degrading MBC in culture was induced by the presence of MBC in the growth medium. 2AB was the main metabolite of MBC that accumulated in A. alternata cultures and in cell-free preparations. MBC was degraded much faster by mixed bacterial cultures that originated from MBC-history soil than in cultures from MBC-nonhistory soil. Fluctuations in the MBC degrading capacity of mixed bacterial cultures occurred during repeated subculturing of the mixed culture. Inoculation of nonhistory soil with mixed bacterial cultures resulted in enhanced MBC degradation, whereas inoculation with A. alternata did not enhance MBC degradation. It is suggested that while fungi contribute to MBC dissipation in soil, bacteria have a greater role in enhanced biodegradation of MBC in soil.     

Aerobic microbial metabolism of some alkylthiophenes found in petroleum

Six alkylthiophenes, 2-hexadecyl-5-methylthiophene (I), 2-methyl-5-tridecylthiophene (II) and 2-butyl-5-tridecylthiophene (III), 2-(3,7-dimethyloctyl)-5-methylthiophene (IV), 2-methyl-5-(3,7,11,15-tetramethyl-hexadecyl)thiophene (V) and 2-ethyl-5-(3,7,11,15-tetramethylhexadecyl)thiophene (VI) were synthesized and used as substrates in biodegradation studies. The products of their aerobic metabolism by pure bacterial cultures were identified. In most cases, the long alkyl chains of these thiophenes were preferentially attacked and in pure cultures of alkane-degrading bacteria, the major metabolites that accumulated in the medium were 5-methyl-2-thiopheneacetic acid from (I), 5-methyl-2-thiophenecarboxylic acid from (II) and occasionally from (V), 5-butyl-2-thiophenecarboxylic acid from (III) and 5-ethyl-2-thiopheneacetic acid from (VI). These transformations are consistent with the metabolism of the alkyl side chains via the beta-oxidation pathway. In contrast, 5-(3,7-dimethyloctyl)-2-thiophenecarboxylic acid was produced from (IV). Because it was available in greatest supply, (I) was studied most thoroughly. It supported growth of the six n-alkanedegrading bacteria tested and (I) was degraded more quickly than pristance but not as quickly as n-hexadecance in mixtures of these three compounds. In the presence of Prudhoe Bay crude oil and a mixed culture of petroleum-degrading bacteria, the acid metabolites from (I), (II) and (III) underwent further biotransformations to products that were not detected by the analytical methods used. The addition of n-hexadecane to the mixed culture of petroleum-degrading bacteria also enhanced the further biotransformations of the metabolites from (I).