Subculturing of a polychlorinated biphenyl-dechlorinating anaerobic enrichment on solid media.

An anaerobic culture capable of dechlorinating polychlorinated biphenyls was subcultured under strict anaerobic conditions on solid media containing sterilized river sediment. The dechlorination activity was transferred as a bacterial colony on a solid medium three times. After two transfers on solid medium, the culture was no longer methanogenic but still dechlorinated a mixture of tri- and tetrachlorobiphenyls. This demonstrates that anaerobic bacteria are responsible for the polychlorinated biphenyl dechlorination and can be grown without polychlorinated biphenyl on solid media.     

Tenax-GC Extraction Technique for Residual Polychlorinated Biphenyl and Polyaromatic Hydrocarbon Analysis in Biodegradation Assays

A rapid Tenax-GC extraction technique has been evaluated for use in conjunction with aqueous biodegradation assays for polyaromatic hydrocarbons and polychlorinated biphenyls. The method was quantitatively efficient and reproducible for phenanthrene, but variable and not quantitative for Aroclor 1254 (polychlorinated biphenyls). Aqueous sample volumes and varying concentrations of organic matter influenced polychlorinated biphenyl and polyaromatic hydrocarbon extraction efficiency. Phenanthrene recovery was decreased by soil extract but unaffected by spent bacteriological culture medium. Both types of organic matter caused significant reduction of Aroclor 1254 recovery. Polyaromatic hydrocarbon and polychlorinated biphenyl biodegradation assays, performed with reservoir samples, supported the laboratory evaluation. The study demonstrated the utility of the Tenax-GC extraction technique for phenanthrene analysis in biodegradation assessment; however, Tenax-GC extraction was not appropriate for Aroclor 1254 biodegradation studies.     

Anaerobic degradation of biphenyl by the facultative anaerobic strain Citrobacter freundi BS2211

Using a synthetic medium supplemented with biphenyl (a polycyclic aromatic hydrocarbon), a new bacterial strain of Citrobacter freundii was isolated from enrichment cultures containing soil and industrial wastewater samples of the Serpukhov Condenser Factory. This strain was found to be capable of degrading biphenyl under anaerobic conditions in the course of nitrate reduction. When the initial concentration of biphenyl in culture medium equaled 150 mg/l, the culture with a titer of 10(9) cells/ml degraded up to 26-28% of biphenyl in 3 days (28 degrees C). At 250 mg/l, the culture with a titer of 10(7) cells/ml degraded 15% of biphenyl in 21 days. Approximately 10% of the substrate consumed was utilized completely, whereas the remainder underwent transformation.     

Anaerobic Degradation of Biphenyl by the Facultative Anaerobic Strain Citrobacter freundii BS2211

Using a synthetic medium supplemented with biphenyl (a polycyclic aromatic hydrocarbon), a new bacterial strain of Citrobacter freundiiwas isolated from enrichment cultures containing soil and industrial wastewater samples of the Serpukhov Condenser Factory. This strain was found to be capable of degrading biphenyl under anaerobic conditions in the course of nitrate reduction. When the initial concentration of biphenyl in the culture medium equaled 150 mg/ml, the culture with a titer of 10⁹ cells/ml degraded up to 26–28% of biphenyl in 3 days (28°C). At 250 mg/ml, the culture with a titer of 10⁷ cells/ml degraded 15% of biphenyl in 21 days. Approximately 10% of the substrate consumed was utilized completely, whereas the remainder underwent transformation.     

Anaerobic ortho Dechlorination of Polychlorinated Biphenyls by Estuarine Sediments from Baltimore Harbor

Reductive dechlorination of the ortho moiety of polychlorinated biphenyls (PCBs) as well as of meta and para moieties is shown to occur in anaerobic enrichments of Baltimore Harbor sediments. These estuarine sediments ortho dechlorinated 2,3,5,6-chlorinated biphenyl (CB), 2,3,5-CB, and 2,3,6-CB in freshwater or estuarine media within a relatively short period of 25 to 44 days. ortho dechlorination developed within 77 days in marine medium. High levels of ortho dechlorination (>90%) occurred when harbor sediments were supplied with only 2,3,5-CB. Incubation with 2,3,4,5,6-CB or 2,3,4,5-CB resulted in the formation of the ortho dechlorination product 3,5-CB; however, para dechlorination of these congeners always preceded ortho chlorine removal. ortho dechlorination of PCBs is an exceedingly rare event that has not been reported previously for marine or estuarine conditions. The activity was reproducible and could be sustained through sequential transfers. In contrast, freshwater sediments incubated under the same conditions exhibited only meta and para dechlorinations. The results indicate that unique anaerobic dechlorinating activity is catalyzed by microorganisms in the estuarine sediments from Baltimore Harbor.     

Heterotrophic bacterial guild structure: Relationship to biodegradative populations

Numerical taxonomic analysis of a freshwater bacterial guild demonstrated that the bacteria capable of growth on phenanthrene and polychlorinated biphenyl media were representative of the taxa obtained from low nutrient oligotrophic media. The diversity of heterotrophic bacteria and members of new taxa recovered from the guild followed a poisson distribution relative to the number of isolation media used. Moderately high nutrient, yeast extract peptone and glucose agar was found to be the most selective isolation medium relative to the total number of taxa recovered whereas low nutrient, lake water agar was the least selective medium used. Carbon source utilization patterns of the isolated taxa indicated that taxa within the guild had broad niche ranges and could potentially occupy many niches within a dynamic environment. The structure of the bacterial guild was dominated by mesophilic oligotrophs. The results of this investigation demonstrate that potential biodegradative populations are representative of the diverse taxa found in uncontaminated freshwater environments.     

Isolation and Characterization of a Mixed Culture That Degrades Polychlorinated Biphenyls

A mixed culture composed of two Pseudomonas strains, designated as KKL101 and KKS102, was isolated from soil. This mixed culture had an enhanced ability to degrade various polychlorinated biphenyls (PCBs) which include highly chlorinated components. They did not grow individually on the mineral salts medium supplemented with a highly chlorinated PCB (PCB48, a mixture of mainly tetrachlorobiphenyl) and biphenyl. When the spent medium of KKL101 was added to the washed cell preparation of KKS102, however, the latter grew on these carbon sources, producing yellow compounds which were identified as metabolic intermediates of the carbon sources, biphenyl and PCBs. These results suggest that KKL101 produces a growth factor(s) essential for KKS102 to grow on PCBs and that the growth of KKL101 is supported by the metabolic intermediates produced by KKS102. It appears that these two bacterial strains have a symbiotic relationship. From the analysis of the degradation products of various PCB congeners, it was found that strain KKS102 degrades a wide range of PCBs which have been considered to be refractory to biological degradation.     

Anaerobic degradation of the aromatic hydrocarbon biphenyl by a sulfate-reducing enrichment culture

The aromatic hydrocarbon biphenyl is a widely distributed environmental pollutant. Whereas the aerobic degradation of biphenyl has been extensively studied, knowledge of the anaerobic biphenyl-oxidizing bacteria and their biochemical degradation pathway is scarce. Here, we report on an enrichment culture that oxidized biphenyl completely to carbon dioxide under sulfate-reducing conditions. The biphenyl-degrading culture was dominated by two distinct bacterial species distantly affiliated with the Gram-positive genus Desulfotomaculum . Moreover, the enrichment culture has the ability to grow with benzene and a mixture of anthracene and phenanthrene as the sole source of carbon, but here the microbial community composition differed substantially from the biphenyl-grown culture. Biphenyl-4-carboxylic acid was identified as an intermediate in the biphenyl-degrading culture. Moreover, 4-fluorobiphenyl was converted cometabolically with biphenyl because in addition to the biphenyl-4-carboxylic acid, a compound identified as its fluorinated analog was observed. These findings are consistent with the general pattern in the anaerobic catabolism of many aromatic hydrocarbons where carboxylic acids are found to be central metabolites.     

Degradation of polychlorinated biphenyl mixtures (Aroclors 1242, 1254, and 1260) by the white rot fungus Phanerochaete chrysosporium as evidenced by congener-specific analysis.

Evidence for substantial degradation of polychlorinated biphenyl mixtures Aroclor 1242, 1254, and 1260 by the white rot fungus Phanerochaete chrysosporium, based on congener-specific gas chromatographic analysis, is presented. Maximal degradation (percent by weight) of Aroclors 1242, 1254, and 1260 was 60.9, 30.5, and 17.6%, respectively. Most of the congeners in Aroclors 1242 and 1254 were degraded extensively both in low-N (ligninolytic) as well as high-N (nonligninolytic) defined media. Even more extensive degradation of the congeners was observed in malt extract medium. Congeners with varying numbers of ortho, meta, and para chlorines were extensively degraded, indicating relative nonspecificity for the position of chlorine substitutions on the biphenyl ring. Aroclor 1260, which has not been conclusively shown to undergo aerobic microbial degradation, was shown to undergo substantial net degradation by P. chrysosporium. Maximal degradation of Aroclor 1260 was observed in malt extract medium (18.4% on a molar basis), in which most of the individual congeners were degraded.     

Effect of hydrogen peroxide on the biodegradation of PCBs in anaerobically dechlorinated river sediments

The ability to initiate aerobic conditions in dechlorinated anaerobic sediments was tested using hydrogen peroxide as an oxygenation agent. Hydrogen peroxide additions to the sediment induced aerobic polychlorinated biphenyl (PCB) degraders as indicated first, by an increase in bacterial count and second by a decline in PCB concentration from 135 µg/g to 20 µg/g over a 96-day period. Dechlorinated anaerobic sediment seems also to harbor indigenous anaerobic and aerobic microorganisms with high PCB degradation abilities. Those results support the potential ofin situ degradation of PCBs using a sequential anaerobic-aerobic technique.     

'Super bugs' for bioremediation

Chlorinated organic compounds are among the most significant pollutants in the world. Sequential use of anaerobic halorespiring bacteria, which are the key players in biological dehalogenation processes, and aerobic bacteria whose oxygenases are modified by directed evolution could lead to efficient and total degradation of highly chlorinated organic pollutants. Recently three interesting papers on halorespiration and polychlorinated biphenyl biodegradation were published.     

The degradation of biphenyl and chlorobiphenyls by mixed bacterial cultures

Abstract Pseudomonas sp. HV3 grows on naphthalene but not on biphenyl, as the sole source of carbon. When the cells of Pseudomonas sp. HV3 grown on naphthalene were shaken with biphenyl as the carbon source in a mineral salt solution, a yellow metabolite identified as the meta -cleavage product of biphenyl was excreted. The degradation of biphenyl stopped here, but was completed if either 2-methyl-4-chlorophenoxy acetic acid (MCPA)-degrading mixed culture or a Nocardia strain was added to the growth solution. Neither of these uses naphthalene or biphenyl as growth substrate. The mixed culture of Pseudomonas sp. HV3 and Nocardia sp. also degrades the commercial polychlorinated biphenyl (PCB) mixture Aroclor 1221. A yellow metabolite was likewise produced in the degradation, and sometimes two different peaks of the yellow metabolite were observed. The gas chromatography-mass spectrometry (GC-MS) analyses showed that 40–87% of Aroclor 1221 was degraded during an incubation time of 6–21 days. Chlorobenzoic acids were found as metabolites.     

High-performance liquid chromatographic assay of biphenyl metabolism by hepatocytes cultured in an embryo/hepatocyte co-culture medium

A high-performance liquid chromatographic method has been modified for the evaluation of both Phase I and II metabolism of biphenyl by hepatocytes maintained in an embryo/hepatocyte co-culture medium. Extracts of the media, before and after hydrolysis of conjugates, are directly injected onto the HPLC and the major hydroxylated metabolites plus unmetabolized biphenyl are detected by fluorescence after separation under gradient or isocratic conditions. The method is almost free of interferences and is relatively simple and rapid. In the case of the monohydroxylated derivatives, the minimum media concentrations which can be measured are 7 to 20 nM (0.07 to 0.2 pmol on-column). Recoveries from culture medium to which known amounts of biphenyl and metabolites had been added were quantitative (90-103%) and the reproducibility good (interassay CV less than 5%). The assay was applied to cultures of hepatocytes derived from rabbit and from phenobarbital induced and noninduced rat.     

Reductive dechlorination of weathered Aroclor 1260 during anaerobic biotreatment of Arctic soils

We investigated the microbial reductive dechlorination of both weathered (aged) and nonweathered (freshly added) Aroclor 1260 in aerobic soil from Resolution Island, Nunavut, Canada. Initial polychlorinated biphenyl (PCB) concentrations were 106 and 100 ppm, respectively. The aerobic soil samples were inoculated with anaerobic sediment, incubated at 30 degrees C until methanogenic, inoculated with a dechlorinating enrichment culture, and incubated a further 8 weeks. The average number of chlorine substituents per biphenyl molecule was biologically reduced from 6.6 to 5.1 and from 6.2 to 4.5 for weathered and nonweathered Aroclor 1260, respectively. Removal of hexa- and heptachlorobiphenyls (CBs), the major homolog groups present, was significantly greater for nonweathered than for weathered Aroclor 1260. Formation of dechlorination products, primarily 2,2',4,4'- and 2,2',4,6'-tetraCBs, was also significantly greater for nonweathered than for weathered Aroclor 1260. We additionally compared the dechlorination at 21 degrees C of weathered Aroclor 1260 in soils from Resolution Island and Saglek, Labrador, Canada. The average number of chlorine substituents per biphenyl molecule was biologically reduced from 6.7 to 5.1 and from 6.5 to 4.6, respectively. This study demonstrated the potential for bioremediation of aerobic soil contaminated with Aroclor 1260 and showed that weathering may limit such treatment to an extent variable among different soils.     

Reductive debromination of the commercial polybrominated biphenyl mixture firemaster BP6 by anaerobic microorganisms from sediments.

Anaerobic microorganisms eluted from three sediments, one contaminated with polybrominated biphenyls (PBBs) and two contaminated with polychlorinated biphenyls, were compared for their ability to debrominate the commercial PBB mixture Firemaster. These microorganisms were incubated with reduced anaerobic mineral medium and noncontaminated sediment amended with Firemaster. Firemaster averages six bromines per biphenyl molecule; four of the bromines are substituted in the meta or para position. The inocula from all three sources were able to debrominate the meta and para positions. Microorganisms from the Pine River (St. Louis, Mich.) contaminated with Firemaster, the Hudson River (Hudson Falls, N.Y.) contaminated with Aroclor 1242, and Silver Lake (Pittsfield, Mass.) contaminated with Aroclor 1260 removed 32, 12, and 3% of the meta plus para bromines, respectively, after 32 weeks of incubation. This suggests that previous environmental exposure to PBBs enhances the debromination capability of the sediment microbial community through selection for different strains of microorganisms. The Pine River inoculum removed an average of 1.25 bromines per biphenyl molecule during a 32-week incubation period, resulting in a mixture potentially more accessible to aerobic degradation processes. No ortho bromine removal was observed. However, when Firemaster was incubated with Hudson River microorganisms that had been repeatedly transferred on a pyruvate medium amended with Aroclor 1242, 17% of the meta and para bromines were removed after 16 weeks of incubation and additional debromination products, including 2-bromobiphenyl and biphenyl, were detected. This suggests the possibility for ortho debromination, since all components of the Firemaster mixture have at least one ortho-substituted bromine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reductive debromination of the commercial polybrominated biphenyl mixture firemaster BP6 by anaerobic microorganisms from sediments.

Anaerobic microorganisms eluted from three sediments, one contaminated with polybrominated biphenyls (PBBs) and two contaminated with polychlorinated biphenyls, were compared for their ability to debrominate the commercial PBB mixture Firemaster. These microorganisms were incubated with reduced anaerobic mineral medium and noncontaminated sediment amended with Firemaster. Firemaster averages six bromines per biphenyl molecule; four of the bromines are substituted in the meta or para position. The inocula from all three sources were able to debrominate the meta and para positions. Microorganisms from the Pine River (St. Louis, Mich.) contaminated with Firemaster, the Hudson River (Hudson Falls, N.Y.) contaminated with Aroclor 1242, and Silver Lake (Pittsfield, Mass.) contaminated with Aroclor 1260 removed 32, 12, and 3% of the meta plus para bromines, respectively, after 32 weeks of incubation. This suggests that previous environmental exposure to PBBs enhances the debromination capability of the sediment microbial community through selection for different strains of microorganisms. The Pine River inoculum removed an average of 1.25 bromines per biphenyl molecule during a 32-week incubation period, resulting in a mixture potentially more accessible to aerobic degradation processes. No ortho bromine removal was observed. However, when Firemaster was incubated with Hudson River microorganisms that had been repeatedly transferred on a pyruvate medium amended with Aroclor 1242, 17% of the meta and para bromines were removed after 16 weeks of incubation and additional debromination products, including 2-bromobiphenyl and biphenyl, were detected. This suggests the possibility for ortho debromination, since all components of the Firemaster mixture have at least one ortho-substituted bromine.(ABSTRACT TRUNCATED AT 250 WORDS)     

利用基本培养基初步筛选牛瘤胃中纤维素降解菌

目的初步筛选牛瘤胃中纤维素降解菌。方法分别采用基本培养基（牛肉膏蛋白胨培养基、马丁培养基）,利用好氧、兼性和厌氧3种不同的培养方法进行初选,初步分离牛瘤胃中的细菌与真菌,再通过复选培养基（加入微晶纤维素）,筛选降解纤维素的菌种。结果筛选分离出降解纤维素的1株厌氧细菌和1株厌氧真菌。结论此实验方法简单易行,能够有效地从牛瘤胃中筛选出生长良好的纤维素降解菌。     

Reductive ortho and meta Dechlorination of a Polychlorinated Biphenyl Congener by Anaerobic Microorganisms

We used gas chromatography-mass spectrometry to study the metabolic fate of 2,3,5,6-tetrachlorobiphenyl (2356-CB) (350 μM) incubated with unacclimated methanogenic pond sediment. The 2356-CB was dechlorinated to 25-CB (21%), 26-CB (63%), and 236-CB (16%) in 37 weeks. This is the first experimental demonstration of ortho dechlorination of a polychlorinated biphenyl by anaerobic microorganisms.     

Lippia dulcis shoot cultures as a source of the sweet sesquiterpene hernandulcin

The axenic shoot culture of Lippia dulcis Trev., Verbenaceae, was established on hormone-free Murashige-Skoog solid medium containing 3% sucrose. Shoots were cultured in various liquid or solid media. Woody Plant liquid medium was best for shoot multiplication, but the production of hernandulcin was relatively low. The highest hernandulcin content (2.9% dry wt) was obtained after 28 days of culture on Murashige-Skoog solid medium containing 2% sucrose. The addition of chitosan to the culture media enhanced the growth of shoots as well as the production of hernandulcin, especially with the liquid medium.Abbreviations MS(2%) Murashige-Skoog medium containing 2 % sucrose - MS(3%) Murashige-Skoog medium containing 3 % sucrose - 1/2MS half strength Murashige-Skoog medium containing 2% sucrose - B5 Gamborg B5 medium containing 2% sucrose - WP Woody Plant medium containing 2% sucrose