Isolation and characterisation of chitosanase from Bacillus sp. 739 strain

The specific nature of the chitosanase activity of the strain Bacillus sp. 739 has been determined. Maximum enzyme activity was observed in a medium containing the biomass of the fruiting bodies of the fungus Macrolepiota procera. The chitosanase was purified to homogeneity using chromatography on DEAE-Sephadex A-50 and Toyopearl HW-50. The molecular weight of the enzyme, assessed by electrophoresis (the Laemmli procedure) approximated 46 kDa. Temperature and pH optima of the purified chitosanase were in the ranges 45-55 degrees C and 6.0-6.5, respectively. Time to half-maximum inactivation of the enzyme at 50 degrees C was equal to 1 h. With colloidal chitosan as the substrate, the value of K(M) of the purified chitosanase was equal to 25 mg/ml. The enzyme also exhibited a weak ability to hydrolyze colloidal chitin.     

Physiological characterization of Mycobacterium sp. strain 1B isolated from a bacterial culture able to degrade high-molecular-weight polycyclic aromatic hydrocarbons

AIM: The aim of this study was to further characterize a bacterial culture (VUN 10,010) capable of benzo[a]pyrene cometabolism. METHODS AND RESULTS: The bacterial culture, previously characterized as a pure culture of Stenotrophomonas maltophilia (VUN 10,010), was found to also contain another bacterial species (Mycobacterium sp. strain 1B), capable of degrading a similar range of PAH substrates. Analysis of its 16S rRNA gene sequence and growth characteristics revealed the strain to be a fast-growing Mycobacterium sp., closely related to other previously isolated PAH and xenobiotic-degrading mycobacterial strains. Comparison of the PAH-degrading characteristics of Mycobacterium sp. strain 1B with those of S. maltophilia indicated some similarities (ability to degrade phenanthrene and pyrene), but some differences were also noted (S. maltophilia able to degrade fluorene, but not fluoranthene, whereas Mycobacterium sp. strain 1B can degrade fluoranthene, but not fluorene). Unlike the S. maltophilia culture, there was no evidence of benzo[a]pyrene degradation by Mycobacterium sp. strain 1B, even in the presence of other PAHs (ie pyrene) as co-metabolic substrates. Growth of Mycobacterium sp. strain 1B on other organic carbon sources was also limited compared with the S. maltophilia culture. CONCLUSIONS: This study isolated a Mycobacterium strain from a bacterial culture capable of benzo[a]pyrene cometabolism. The Mycobacterium strain displays different PAH-degrading characteristics to those described previously for the PAH-degrading bacterial culture. It is unclear what role the two bacterial strains play in benzo[a]pyrene cometabolism, as the Mycobacterium strain does not appear to have endogenous benzo[a]pyrene degrading ability. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the isolation and characterization of a novel PAH-degrading Mycobacterium strain from a PAH-degrading culture. Further studies utilizing this strain alone, and in combination with other members of the consortium, will provide insight into the diverse roles different bacteria may play in PAH degradation in mixed cultures and in the environment.     

Monocyclic aromatic hydrocarbon degradation by Rhodococcus sp. strain DK17

Rhodococcus sp. strain DK17 was isolated from soil and analyzed for the ability to grow on o-xylene as the sole carbon and energy source. Although DK17 cannot grow on m- and p-xylene, it is capable of growth on benzene, phenol, toluene, ethylbenzene, isopropylbenzene, and other alkylbenzene isomers. One UV-generated mutant strain, DK176, simultaneously lost the ability to grow on o-xylene, ethylbenzene, isopropylbenzene, toluene, and benzene, although it could still grow on phenol. The mutant strain was also unable to oxidize indole to indigo following growth in the presence of o-xylene. This observation suggests the loss of an oxygenase that is involved in the initial oxidation of the (alkyl)benzenes tested. Another mutant strain, DK180, isolated for the inability to grow on o-xylene, retained the ability to grow on benzene but was unable to grow on alkylbenzenes due to loss of a meta-cleavage dioxygenase needed for metabolism of methyl-substituted catechols. Further experiments showed that DK180 as well as the wild-type strain DK17 have an ortho-cleavage pathway which is specifically induced by benzene but not by o-xylene. These results indicate that DK17 possesses two different ring-cleavage pathways for the degradation of aromatic compounds, although the initial oxidation reactions may be catalyzed by a common oxygenase. Gas chromatography-mass spectrometry and 300-MHz proton nuclear magnetic resonance spectrometry clearly show that DK180 accumulates 3,4-dimethylcatechol from o-xylene and both 3- and 4-methylcatechol from toluene. This means that there are two initial routes of oxidation of toluene by the strain. Pulsed-field gel electrophoresis analysis demonstrated the presence of two large megaplasmids in the wild-type strain DK17, one of which (pDK2) was lost in the mutant strain DK176. Since several other independently derived mutant strains unable to grow on alkylbenzenes are also missing pDK2, the genes encoding the initial steps in alkylbenzene metabolism (but not phenol metabolism) appear to be present on this approximately 330-kb plasmid.     

Co-metabolic degradation of chlorinated hydrocarbons by Pseudomonas sp. strain DCA1

Pseudomonas sp. strain DCA1, which is capable of utilizing 1,2-dichloroethane (DCA) as sole carbon and energy source, was used to oxidize chlorinated methanes, ethanes, propanes, and ethenes. Chloroacetic acid, an intermediate in the DCA degradation pathway of strain DCA1, was used as a co-substrate since it was readily oxidized by DCA-grown cells of strain DCAI and did not compete for the monooxygenase. All of the tested compounds except tetrachloroethylene (PER) were oxidized by cells expressing DCA monooxygenase. Strain DCAI could not utilize any of these compounds as a growth substrate. Co-metabolic oxidation during growth on DCA was studied with 1,2-dichloropropane. Although growth on this mixture occurred, 1,2-dichloropropane strongly inhibited growth of strain DCAI. This inhibition was not caused by competition for the monooxygenase. It was shown that the oxidation of 1,2dichloropropane resulted in the accumulation of 2,3-dichloro-1-propanol and 2-chloroethanol.     

Polycyclic aromatic hydrocarbons degradation by Agrocybe sp. CU-43 and its fluorene transformation

Agrocybe sp. CU-43, a white-rot fungus isolated from Thailand, showed a high potential for degrading both low- and high-molecular weight polycyclic aromatic hydrocarbons. At 100 ppm fluorene was degraded by 99% within six days while at the same concentration 99 and 92% degradation of phenanthrene and anthracene, respectively, occurred in 21 days, and fluoranthene and pyrene were reduced by 80 and 75%, respectively, in 30 days. In a soil model, Agrocybe sp. CU-43 completely degraded 250 ppm fluorene at room temperature within four weeks. Laccase and manganese peroxidase activities, but not lignin peroxidase activity, were detected during the biodegradation of fluorene. Two of the metabolites from fluorene degradation by the fungus were identified via reversed-phase HPLC as 9-fluorenol and 9-fluorenone, the less toxic intermediates of fluorene. However, 9-fluorenol is not an end product for the degradation. These results suggest that fluorene degradation by Agrocybe sp. CU-43 may take place via the same pathway(s) employed by other ligninolytic and non-ligninolytic fungi. This is the first report of fluorene biodegradation by a fungus belonging to the genus Agrocybe.     

Isolation and characterization of polycyclic aromatic hydrocarbons-degrading Sphingomonas sp. strain ZL5

A bacterial strain ZL5, capable of growing on phenanthrene as a sole carbon and energy source but not naphthalene, was isolated by selective enrichment from crude-oil-contaminated soil of Liaohe Oil Field in China. The isolate was identified as a Sphingomonas sp. strain on the basis of 16S ribosomal DNA analysis. Strain ZL5 grown on phenanthrene exhibited catechol 2,3-dioxygenase (C23O) activity but no catechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, protocatechuate 3,4-dioxygenase and protocatechuate 4,5-dioxygenase activities. This suggests that the mode of cleavage of phenanthrene by strain ZL5 could be meta via the intermediate catechol, which is different from the protocatechuate way of other two bacteria, Alcaligenes faecelis AFK2 and Nocardioides sp. strain KP7, also capable of growing on phenanthrene but not naphthalene. A resident plasmid (approximately 60 kb in size), designated as pZL, was detected from strain ZL5. Curing the plasmid with mitomycin C and transferring the plasmid to E. coli revealed that pZL was responsible for polycyclic aromatic hydrocarbons degradation. The C23O gene located on plasmid pZL was cloned and overexpressed in E. coli JM109(DE3). The ring-fission activity of the purified C23O from the recombinant E. coli on dihydroxylated aromatics was in order of catechol > 4-methylcatechol > 3-methylcatechol > 4-chlorocatechol > 3,4-dihydroxyphenanthrene > 3-chlorocatechol.     

Isolation and expansion of the catabolic potential of a Pseudomonas putida strain able to grow in the presence of high concentrations of aromatic hydrocarbons.

Pseudomonas putida DOT-T1 was isolated after enrichment on minimal medium with 1% (vol/vol) toluene as the sole C source. The strain was able to grow in the presence of 90% (vol/vol) toluene and was tolerant to organic solvents whose log P(ow) (octanol/water partition coefficient) was higher than 2.3. Solvent tolerance was inducible, as bacteria grown in the absence of toluene required an adaptation period before growth restarted. Mg2+ ions in the culture medium improved solvent tolerance. Electron micrographs showed that cells growing on high concentrations of toluene exhibited a wider periplasmic space than cells growing in the absence of toluene and preserved the outer membrane integrity. Polarographic studies and the accumulation of pathway intermediates showed that the strain used the toluene-4-monooxygenase pathway to catabolyze toluene. Although the strain also thrived in high concentrations of m- and p-xylene, these hydrocarbons could not be used as the sole C source for growth. The catabolic potential of the isolate was expanded to include m- and p-xylene and related hydrocarbons by transfer of the TOL plasmid pWW0-Km.     

Anaerobic degradation of non-substituted aromatic hydrocarbons

Aromatic hydrocarbons are among the most prevalent organic pollutants in the environment. Their removal from contaminated systems is of great concern because of the high toxicity effect on living organisms including humans. Aerobic degradation of aromatic hydrocarbons has been intensively studied and is well understood. However, many aromatics end up in habitats devoid of molecular oxygen. Nevertheless, anaerobic degradation using alternative electron acceptors is much less investigated. Here, we review the recent literature and very early progress in the elucidation of anaerobic degradation of non-substituted monocyclic (i.e. benzene) and polycyclic aromatic hydrocarbons (PAH such as naphthalene and phenanthrene). A focus will be on benzene and naphthalene as model compounds. This review concerns the microbes involved, the biochemistry of the initial activation and subsequent enzyme reactions involved in the pathway.     

Anaerobic degradation of ethylbenzene and other aromatic hydrocarbons by new denitrifying bacteria

Anaerobic degradation of alkylbenzenes with side chains longer than that of toluene was studied in freshwater mud samples in the presence of nitrate. Two new denitrifying strains, EbN1 and PbN1, were isolated on ethylbenzene and n-propylbenzene, respectively. For comparison, two further denitrifying strains, ToN1 and mXyN1, were isolated from the same mud with toluene and m-xylene, respectively. Sequencing of 16SrDNA revealed a close relationship of the new isolates to Thauera selenatis. The strains exhibited different specific capacities for degradation of alkylbenzenes. In addition to ethylbenzene, strain EbN1 utilized toluence, but not propylbenzene. In contrast, propylbenzene-degrading strain PbN1 did not grow on toluene, but was able to utilize ethylbenzene. Strain ToN1 used toluene as the only hydrocarbon substrate, whereas strain mXyN1 utilized both toluene and m-xylene. Measurement of the degradation balance demonstrated complete oxidation of ethylbenzene to CO₂ by strain EbN1. Further characteristic substrates of strains EbN1 and PbN1 were 1-phenylethanol and acetophenone. In contrast to the other isolates, strain mXyN1 did not grow on benzyl alcohol. Benzyl alcohol (also m-methylbenzyl alcohol) was even a specific inhibitor of toluene and m-xylene utilization by strain mXyN1. None of the strains was able to grow on any of the alkylbenzenes with oxygen as electron acceptor. However, polar aromatic compounds such as benzoate were utilized under both oxic and anoxic conditions. All four isolates grew anaerobically on crude oil. Gas chromatographic analysis of crude oil after growth of strain ToN1 revealed specific depletion of toluene.     

Differential degradation of bicyclics with aromatic and alicyclic rings by Rhodococcus sp. strain DK17

The metabolically versatile Rhodococcus sp. strain DK17 is able to grow on tetralin and indan but cannot use their respective desaturated counterparts, 1,2-dihydronaphthalene and indene, as sole carbon and energy sources. Metabolite analyses by gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry clearly show that (i) the meta-cleavage dioxygenase mutant strain DK180 accumulates 5,6,7,8-tetrahydro-1,2-naphthalene diol, 1,2-indene diol, and 3,4-dihydro-naphthalene-1,2-diol from tetralin, indene, and 1,2-dihydronaphthalene, respectively, and (ii) when expressed in Escherichia coli, the DK17 o-xylene dioxygenase transforms tetralin, indene, and 1,2-dihydronaphthalene into tetralin cis-dihydrodiol, indan-1,2-diol, and cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, respectively. Tetralin, which is activated by aromatic hydroxylation, is degraded successfully via the ring cleavage pathway to support growth of DK17. Indene and 1,2-dihydronaphthalene do not serve as growth substrates because DK17 hydroxylates them on the alicyclic ring and further metabolism results in a dead-end metabolite. This study reveals that aromatic hydroxylation is a prerequisite for proper degradation of bicyclics with aromatic and alicyclic rings by DK17 and confirms the unique ability of the DK17 o-xylene dioxygenase to perform distinct regioselective hydroxylations.     

Isolation and characterization of a gene cluster for dibenzofuran degradation in a new dibenzofuran-utilizing bacterium, Paenibacillus sp. strain YK5

Spore-forming bacterial strains capable of utilizing dibenzofuran (DF) as a sole source of carbon and energy were isolated. Characteristics of the isolates justified their classification into the genus Paenibacillus, and their closest relative was P. naphthalenovorans. Degenerate primers for aromatic hydrocarbon dioxygenase alpha subunit (AhDOa) genes and genomic DNA of the strain YK5 were used for gene isolation. The nucleotide sequences of clones of the PCR products revealed that the strain YK5 carries at least five different AhDOa genes. Northern hybridization analysis showed that one of the AhDOa genes was transcribed under DF-containing culture conditions. A gene cluster encoding the AhDOa was isolated. The genes predicted to encode extradiol dioxygenase (dbfB) and hydrolase (dbfC) were found to be an upstream of genes encoding the alpha and beta subunit of the AhDO (dbfA1 and dbfA2, respectively); the latter two gene products showed 60 and 53% identity to the amino acid sequences of DbfA1 and DbfA2 of Terrabacter sp. DBF63, respectively. Two Paenibacillus validus JCM 9077 strains transformed with the dbf gene clusters acquired the ability to convert DF to 2,2′,3-trihydroxybiphenyl (THBP) and salicylic acid (SAL). These results suggest that the enzymes encoded by the gene cluster isolated in this study are involved in DF metabolism in YK5.     

一株高浓度多环芳烃降解菌的鉴定和降解特性

采用选择性富集培养方法,从沈抚灌区土壤中分离得到多环芳烃(PAHs)高效降解菌NI2,应用此降解菌制备固定化菌剂,修复焦化厂内高浓度PAHs污染土壤,并通过生理生化和16S rDNA测序进行微生物鉴定.经过30 d的降解实验,菌N12对污染土壤中各PAH的去除率＞66％,总去除率为80％.生理生化和16S rDNA测序分析表明,分离得到的菌株N12为分支杆菌属(Mycobacterium sp.),该菌具有与其他分枝杆菌同源的双加氧酶基因nidA和pdoA2.结果表明,从土壤中筛选获得的分枝杆菌可以修复高浓度PAHs污染工业土壤.     

Effects of co-occurring aromatic hydrocarbons on degradation of individual polycyclic aromatic hydrocarbons in marine sediment slurries.

Rates of polycyclic aromatic hydrocarbon (PAH) degradation and mineralization were influenced by preexposure to alternate PAHs and a monoaromatic hydrocarbon at relatively high (100 ppm) concentrations in organic-rich aerobic marine sediments. Prior exposure to three PAHs and benzene resulted in enhanced [14C]naphthalene mineralization, while [14C]anthracene mineralization was stimulated only by benzene and anthracene preexposure. Preexposure of sediment slurries to phenanthrene stimulated the initial degradation of anthracene. Prior exposure to naphthalene stimulated the initial degradation of phenanthrene but had no effect on either the initial degradation or mineralization of anthracene. For those compounds which stimulated [14C]anthracene or [14C]naphthalene mineralization, longer preexposures (2 weeks) to alternative aromatic hydrocarbons resulted in an even greater stimulation response. Enrichment with individual PAHs followed by subsequent incubation with one or two PAHs showed no alteration in degradation patterns due to the simultaneous presence of PAHs. The evidence suggests that exposure of marine sediments to a particular PAH or benzene results in the enhanced ability of these sediments to subsequently degrade that PAH as well as certain other PAHs. The enhanced degradation of a particular PAH after sediments have been exposed to it may result from the selection and proliferation of specific microbial populations capable of degrading it. The enhanced degradation of other PAHs after exposure to a single PAH suggests that the populations selected have either broad specificity for PAHs, common pathways of PAH degradation, or both.     

Draft genome sequence of Rhodococcus sp. strain P14, a biodegrader of high-molecular-weight polycyclic aromatic hydrocarbons

The genus Rhodococcus is known for its ability to degrade various xenobiotic compounds. Rhodococcus sp. strain P14 isolated from crude oil-contaminated sediments can degrade mineral oil and polycyclic aromatic hydrocarbons (PAHs). The draft genome sequence of Rhodococcus sp. P14 was obtained using Solexa technology, which provided an invaluable genetic background for further investigation of the ability of P14 to degrade xenobiotic compounds.