The purpose of the present study was to purify and characterize the catechol 1,2-dioxygenase (EC 1.13.11.1; catechol-oxygen 1,2-oxidoreductase; C12O) enzyme from the local isolate of Pseudomonas putida. This enzyme catalyzes the initial reaction in the ortho-pathway for phenol degradation in various gram-negative bacteria, including the genus of Pseudomonas. Pseudomonads are commonly used in the biodegradation of xenobiotics due to their versatility in degrading a wide range of chemical compounds. Eighty-nine soil samples were taken from the contaminated soil of the Midland Refineries Company (MRC) of Al-Daura refinery area at Baghdad from April to August 2021. The samples were grown in a mineral salt medium containing 250 mg per L of phenol to test their ability to biodegrade phenol. The pH was adjusted to 8.0 at 30 °C using a shaking incubator for 24-48 h. A number of 62 (69.6%) isolates of the total number were able to degrade phenol efficiently. The findings of the VITEK system and the housekeeping gene 16S rDNA confirmed that out of the positive isolates for phenol degradation, 36 from 62 (58.06%) were identified as Pseudomonas spp. isolates. Those isolates were distributed as P. aeruginosa 30 (83.3%) and P. putida 6 (16.6%). The enzyme production capabilities of the isolates were evaluated, and the highest activity was 2.39 U per mg for the isolate No. 15 which it was identified as P. putida. The previous isolate was selected for enzyme production, purification, and characterization. The enzyme was purified using ion exchange and gel filtration chromatography, with a combined yield of 36.12% and purification fold of 15.42 folds. Using a gel filtration column, the enzyme's molar mass was calculated to be 69 kDa after purification. The purified enzyme was stable at 35 °C and a pH of 6.0.
- MeSH
- bakteriální proteiny metabolismus genetika chemie izolace a purifikace MeSH
- biodegradace * MeSH
- fenol * metabolismus MeSH
- fylogeneze MeSH
- katechol-1,2-dioxygenasa * metabolismus genetika MeSH
- koncentrace vodíkových iontů MeSH
- Pseudomonas putida * enzymologie genetika metabolismus MeSH
- půdní mikrobiologie * MeSH
- RNA ribozomální 16S genetika MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
The potential of the culturable bacterial community from an Alpine coniferous forest site for the degradation of organic polymers and pollutants at low (5 °C) and moderate (20 °C) temperatures was evaluated. The majority of the 68 strains belonged to the phylum Proteobacteria (77%). Other strains were related to Bacteroidetes (12%), Alphaproteobacteria (4%), Actinobacteria (3%), and Firmicutes (3%). The strains were grouped into 42 different OTUs. The highest bacterial diversity was found within the phylum Bacteroidetes. All strains, except one, could grow at temperatures from 5 to 25 °C. The production of enzyme activities involved in the degradation of organic polymers present in plant litter (carboxymethyl cellulose, microgranular cellulose, xylan, polygalacturonic acid) was almost comparable at 5 °C (68%) and 20 °C (63%). Utilizers of lignin compounds (lignosulfonic acid, lignin alkali) as sole carbon source were found to a higher extent at 20 °C (57%) than at 5 °C (24%), but the relative fractions among positively tested strains utilizing these compounds were almost identical at the two temperatures. Similar results were noted for utilizers of organic pollutants (n-hexadecane, diesel oil, phenol, glyphosate) as sole carbon source. More than two-thirds showed constitutively expressed catechol-1,2-dioxygenase activity both at 5 °C (74%) and 20 °C (66%). Complete phenol (2.5 mmol/L) degradation by strain Paraburkholderia aromaticivorans AR20-38 was demonstrated at 0-30 °C, amounts up to 7.5 mmol/L phenol were fully degraded at 10-30 °C. These results are useful to better understand the effect of changing temperatures on microorganisms involved in litter degradation and nutrient turnover in Alpine forest soils.
- MeSH
- Bacteria klasifikace genetika izolace a purifikace metabolismus MeSH
- bakteriální proteiny metabolismus MeSH
- biodegradace MeSH
- biodiverzita MeSH
- biopolymery metabolismus MeSH
- cévnaté rostliny mikrobiologie MeSH
- fenol metabolismus MeSH
- fylogeneze MeSH
- látky znečišťující životní prostředí metabolismus MeSH
- lesy * MeSH
- lignin metabolismus MeSH
- půdní mikrobiologie MeSH
- RNA ribozomální 16S genetika MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
The microbial fuel cells (MFCs) are recognized to be highly effective for the biodegradation of phenol. For isolating the phenol-degrading bacteria, the sample containing 500 mg/L phenol was collected from the MFCs. The strain (WL027) was identified basing on the 16S rRNA gene analysis and phylogenetic analysis as Bacillus cereus. The effects of pH, temperature, concentrations of phenol, heavy metal ions, and salt on the growth of strain as well as the degradation of phenol have been carefully studied. The WL027-strain exhibited favorable tolerance for the metal cations including Cr2+, Co2+, Pb2+, and Cu2+ with the concentration of 0.2 mg/L and NaCl solution with a high concentration of 30 g/L. In 41 h, 86.44% of 500 mg/L phenol has been degraded at the initial pH at 6 and the temperature of 30 °C. The strain was highly active electrogenesis bacteria and the coulombic efficiency reached 64.25%, which showed significant advantage on the efficient energy conversion. Therefore, due to the highly efficient degradation of phenol, WL027-strain could be used in the treatment of phenol-containing wastewater.
- MeSH
- Bacteria klasifikace genetika izolace a purifikace metabolismus MeSH
- biodegradace MeSH
- fenol metabolismus MeSH
- fylogeneze MeSH
- koncentrace vodíkových iontů MeSH
- teplota MeSH
- těžké kovy analýza metabolismus MeSH
- zdroje bioelektrické energie mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
The aim of this study was to design an effective method for the bioremediation of coking wastewaters, specifically for the concurrent elimination of their highly toxic components - cyanide and phenols. Almost full degradation of free cyanide (0.32-20 mM; 8.3-520 mg L(-1)) in the model and the real coking wastewaters was achieved by using a recombinant cyanide hydratase in the first step. The removal of cyanide, a strong inhibitor of tyrosinase, enabled an effective degradation of phenols by this enzyme in the second step. Phenol (16.5 mM, 1,552 mg L(-1)) was completely removed from a real coking wastewater within 20 h and cresols (5.0 mM, 540 mg L(-1)) were removed by 66% under the same conditions. The integration of cyanide hydratase and tyrosinase open up new possibilities for the bioremediation of wastewaters with complex pollution.
- MeSH
- fenol metabolismus MeSH
- fenoly metabolismus MeSH
- koks MeSH
- kyanidy metabolismus MeSH
- odpadní voda * MeSH
- tyrosinasa MeSH
- Publikační typ
- časopisecké články MeSH
Phenol and its derivatives (alkylphenols, halogenated phenols, nitrophenols) are natural or man-made aromatic compounds that are ubiquitous in nature and in human-polluted environments. Many of these substances are toxic and/or suspected of mutagenic, carcinogenic, and teratogenic effects. Bioremediation of the polluted soil and water using various bacteria has proved to be a promising option for the removal of these compounds. In this review, we describe a number of peripheral pathways of aerobic and anaerobic catabolism of various natural and xenobiotic phenolic compounds, which funnel these substances into a smaller number of central catabolic pathways. Finally, the metabolites are used as carbon and energy sources in the citric acid cycle. We provide here the characteristics of the enzymes that convert the phenolic compounds and their catabolites, show their genes, and describe regulatory features. The genes, which encode these enzymes, are organized on chromosomes and plasmids of the natural bacterial degraders in various patterns. The accumulated data on similarities and the differences of the genes, their varied organization, and particularly, an astonishingly broad range of intricate regulatory mechanism may be read as an exciting adventurous book on divergent evolutionary processes and horizontal gene transfer events inscribed in the bacterial genomes. In the end, the use of this wealth of bacterial biodegradation potential and the manipulation of its genetic basis for purposes of bioremediation is exemplified. It is envisioned that the integrated high-throughput techniques and genome-level approaches will enable us to manipulate systems rather than separated genes, which will give birth to systems biotechnology.
Rhodococcus erythropolis CCM2595 is able to efficiently utilize phenol and other aromatic compounds. We cloned and sequenced its complete gene cluster - catA, catB, catC, catR, pheR, pheA2, pheA1 - involved in the ortho-cleavage pathway of phenol. The activity of the key enzyme of the phenol degradation pathway, two-component phenol hydroxylase, was found to be induced by phenol. When both phenol and succinate were present in the medium, phenol hydroxylase activity decreased substantially. To analyze the regulation of phenol degradation at the transcriptional level, the transcriptional fusions of the divergently oriented promoters PpheA2 and PpheR with the gfpuv reporter gene were constructed. The promoters driving expression of the genes of the pheR-pheA2pheA1 cluster were localized by determining the respective transcriptional start points. Measurements of GFP fluorescence as well as quantitative RT-PCR revealed that expression of the phe genes is induced by phenol at the transcriptional level. The transcription of pheA2A1 and pheR was repressed by succinate, whereas no repression by glucose or glycerol was observed. Activation of the R. erythropolis CCM2595 pheA2 promoter by PheR, an AraC-type transcriptional regulator, was demonstrated by overexpression of the pheR gene. Analysis of the transcriptional regulation of two similar phe clusters from R. jostii RHA1 by various substrates showed that the type of carbon catabolite repression and the temporal transcriptional pattern during cultivation are different in each of the three phe clusters analyzed.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- biodegradace MeSH
- fenol metabolismus MeSH
- katabolická represe * MeSH
- multigenová rodina MeSH
- oxygenasy se smíšenou funkcí genetika metabolismus MeSH
- promotorové oblasti (genetika) MeSH
- regulace genové exprese u bakterií MeSH
- Rhodococcus enzymologie genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The aim of this study was to evaluate the impact of short-term repeated exposure to a static magnetic field (induction 370 mT) on the Rhodococcus erythropolis cells. Specifically, it was ascertained the magnetic field's potential to influence degradation of a phenol substrate, cell growth and respiration activity (oxygen consumption) during substrate biodegradation. The experiment took place over 3 days, with R. erythropolis exposed to the magnetic field for the first day. During the experiment, different recirculation rates between the reactor and the magnetic contactor has been tested. Use of the magnetic field at higher recirculation rates (residence time in contactor was less than 7 min) stimulated substrate (phenol) oxidation by around 34%; which, in turn, promoted R. erythropolis growth by around 28% by shortening the lag- and exponential-phases and increasing bacterial respiration activity by around 10%.
- MeSH
- aerobióza MeSH
- biodegradace MeSH
- bioreaktory mikrobiologie MeSH
- fenol metabolismus MeSH
- magnetické pole * MeSH
- počítačová simulace MeSH
- Rhodococcus růst a vývoj metabolismus MeSH
- techniky vsádkové kultivace přístrojové vybavení MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Pollutant interactions during the aerobic biodegradation of phenolic mixtures with either 2-nitrophenol (2-NP) or 3-nitrophenol (3-NP) by a NP-adapted microbial consortium in simulated wastewater were studied in a packed-bed bench scale bioreactor continuously operated in a flow mode. Phenol/2-NP and phenol/3-NP mixtures with varied phenol/nitrophenol ratios were shown to exhibit different biodegradability patterns. The presence of 2-NP led to a much lower overall elimination capacity and lower process stability in comparison to mixtures with 3-NP. In contrast to the expected greater degradation of a more biodegradable substrate in mixtures, phenol was degraded with a lower efficiency at higher phenol concentrations than NPs, although this difference became less pronounced with the gradual biofilm adaptation to phenol. This unusual substrate interaction, which appears to be common in the biotreatment of substituted phenol mixtures, was explained by prior biofilm adaptation to less degradable substrates, NPs. The biofilm composition was significantly altered during the long-term reactor operation. Although eukaryotes were not present in the inoculum, four fungal species were isolated from the biofilm after 1.5 years of operation. Of the initially present strains, only Chryseobacterium sp. and several Pseudomonas species persisted till the end of operation.
- MeSH
- biodegradace MeSH
- biofilmy * MeSH
- bioreaktory mikrobiologie MeSH
- chemické látky znečišťující vodu metabolismus MeSH
- fenol metabolismus MeSH
- fyziologie bakterií * MeSH
- nitrofenoly metabolismus MeSH
- odpad tekutý - odstraňování metody MeSH
- odpadní voda analýza MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
We used reversed phase liquid chromatography-electrospray ionization tandem mass spectrometry for direct analysis of mycolic acids (MAs) from four different cultivations of Rhodococcus erythropolis. This technique enabled us to identify and quantify the specific molecular species of MAs directly from lipid extracts of the bacterium, including the determination of their basic characteristics such as retention time and mass spectra. We identified a total of 60 molecular species of MAs by means of LC/MS. In collision-induced dissociation tandem mass spectrometry, the [M-H](-) ions eliminated two residues, i.e., meroaldehyde and carboxylate anions containing α-alkyl chains. The structural information from these fragment ions affords structural assignment of the mycolic acids, including the lengths and number of double bond(s). Two strains, i.e., R. erythropolis CCM 2595 and genetically modified strain CCM 2595 pSRK 21 phe were cultivated on two different substrates (phenol and phenol with addition of humic acids as a sole carbon source). The addition of humic acids showed that there is a marked increase of unsaturated mycolic acids, mostly in the range of 20-100 %. This effect is more pronounced in the R. erythropolis CCM 2595 strain.
- MeSH
- biotransformace MeSH
- chromatografie kapalinová MeSH
- fenol metabolismus MeSH
- huminové látky MeSH
- kultivační média chemie MeSH
- kyseliny mykolové chemie metabolismus MeSH
- Rhodococcus chemie metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- uhlík metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVE: Comamonas testosteroni Pb50 is a microorganism that possesses high tolerance for phenol and shows strong phenol degrading activity. This bacterial strain is capable of utilizing phenol as the sole carbon and energy source. Although examples are known in which the C. testosteroni utilizes phenol for growth or metabolism, much less information are known on the nature of the phenol-oxidizing enzymes in this microorganism. Therefore, the occurrence and cellular location of phenol hydroxylase (EC 1.14.13.7), the enzyme participating in the first step of phenol degradation, catalyzing its hydroxylation to catechol in a bacterial Comamonas testosteroni Pb50 strain grown in the presence of phenol as a sole carbon and energy source are the aims of this study. METHODS: Combination of fractionation with polyethylene glycol 6000 and gel permeation chromatography on columns of Sepharose 4B and Sephacryl S-300 was used for isolation of phenol hydroxylase detectable in the medium in which C. testosteroni was cultivated. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel chromatography on Sephacryl S-300 were used to evaluate the molecular mass of the enzyme. The enzyme activity was followed by HPLC (phenol consumption and/or catechol formation). RESULTS: Whereas low activity of phenol hydroxylase was detected in cytosol isolated from C. testosteroni, more than 16-fold higher activity of this enzyme was found in the medium in which C. testosteroni was cultivated. The presence of phenol hydroxylase extracellular activity suggests that this microorganism may secrete the enzyme into the extracellular medium. Using the procedure consisting of fractionation with polyethylene glycol 6000 and gel permeation chromatography on columns of Sepharose 4B and Sephacryl S-300, the enzyme was isolated from the medium to homogeneity. The formation of catechol mediated by purified phenol hydroxylase is strictly dependent on the presence of NADPH, which indicates that this enzyme is the NADPH-dependent phenol hydroxylase. The enzyme is a homotetramer having a molecular mass of 240 000, consisting of four subunits having a molecular mass of 60 000. The optimum pH of the enzyme for the phenol oxidation is pH 7.6. CONCLUSION: The results are the first report showing isolation and partial characterization of extracellular NADPH-dependent phenol hydroxylase of a bacterial C. testosteroni Pb50 strain capable of oxidizing phenol to catechol. The data demonstrate the progress in resolving the enzymes responsible for the first step of phenol degradation by bacteria.
- MeSH
- časové faktory MeSH
- Comamonas testosteroni enzymologie genetika MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- extracelulární prostor enzymologie genetika MeSH
- fenol metabolismus MeSH
- katalýza MeSH
- katecholy metabolismus MeSH
- klonování DNA MeSH
- koncentrace vodíkových iontů MeSH
- NADP metabolismus MeSH
- oxidace-redukce MeSH
- oxygenasy se smíšenou funkcí genetika izolace a purifikace metabolismus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
