Potential of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> as a bioremediation organism

Summary The capability of Rhodococcus erythropolis CCM 2595(ATCC 11048) to utilize phenol, pyrocatechol, resorcinol, p-nitrophenol, p-chlorophenol, hydroquinone and hydroxybenzoate, respectively, or as respective binary mixtures with phenol, was described. This capability was found to depend on the substrate and its initial concentration. Some monoaromatic compounds had a suppressive effect on the strain’s ability to utilize phenol in a binary mixture and easily utilizable monoaromatics were strong inducers of the phenol 2-monooxygenase (EC 1.14.13.7). The capacity of R. erythropolis to colonize a synthetic zeolite was demonstrated and the enhancement of phenol tolerance of biofilms utilizing phenol was observed. The effect of humic acids on phenol killing was described and discussed as well. To allow use of recombinant DNA technology for strain improvement, methods of genetic transfer (transformation and conjugation) in R. erythropolis were established.     

Phenol Degradation by Immobilized Cells of Arthrobacter citreus

An aerobic microorganism with an ability to utilize phenol as carbon and energy source was isolated from a hydrocarbon contamination site by employing selective enrichment culture technique. The isolate was identified as Arthrobacter citreus based on morphological, physiological and biochemical tests. This mesophilic organism showed optimal growth at 25°C and at pH of 7.0. The phenol utilization studies with Arthrobacter citreus showed that the complete assimilation occurred in 24 hours. The organism metabolized phenol up to 22 mM concentrations whereas higher levels were inhibitory. Thin layer chromatography, UV spectral and enzyme analysis were suggestive of catechol, as a key intermediate of phenol metabolism. The enzyme activities of phenol hydroxylase and catechol 2,3-dioxygenase in cell free extracts of Arthrobacter citreus were indicative of operation of a meta-cleavage pathway for phenol degradation. The organism had additional ability to degrade catechol, cresols and naphthol. The degradation rates of phenol by alginate and agar immobilized cells in batch fermentations showed continuous phenol metabolism for a period of eight days.     

Effects and fate of phenol in simulated landfill sites

Phenol was administered to landfill waste in concentrations from 150 to 1,000 ppm via the feed-liquor of lysimeter systems over an 18-week incubation period. Biotic contributions to phenol removal in the landfill waste were of greater significance than abiotic removal. The addition of phenol did not cause the isolation of thermophilic phenol degraders. Plates inoculated from the test lysimeter receiving phenol were eventually predominated by mesophilic phenol-degradingMicrococcus, Nocardia, andArthrobacter spp.; plates inoculated from the control lysimeter, receiving water, were predominated by species incapable of utilizing phenol.     

Silica-immobilized Methylobacterium sp. NP3 and Acinetobacter sp. PK1 degrade high concentrations of phenol

Aims: To immobilize Methylobacterium sp. NP3 and Acinetobacter sp. PK1 to silica and determine the ability of the immobilized bacteria to degrade high concentrations of phenol. Methods and Results: The phenol degradation activity of suspended and immobilized Methylobacterium sp. NP3 and Acinetobacter sp. PK1 bacteria was investigated in batch experiments with various concentrations of phenol. The bacterial cells were immobilized by attachment to or encapsulation in silica. The encapsulated bacteria had the highest phenol degradation rate, especially at initial phenol concentrations between 7500 and 10 000 mg l^?1. Additionally, the immobilized cells could continuously degrade phenol for up to 55 days. Conclusions: The encapsulation of a mixed culture of Methylobacterium sp. NP3 and Acinetobacter sp. PK1 is an effective and easy technique that can be used to improve bacterial stability and phenol degradation. Significance and Impact of the Study: Wastewater from various industries contains high concentrations of phenol, which can cause wastewater treatment failure. Silica‐immobilized bacteria could be applied in bioreactors to initially remove the phenol, thereby preventing phenol shock loads to the wastewater treatment system.     

Characterization of the phenol monooxygenase gene from Chromobacterium violaceum: Potential use for phenol biodegradation

In this work, the biodegradation mechanism of phenol and sub products (such as catechol and hydroquinone) in Chromobacterium violaceum was investigated by cloning and molecular characterization of a phenol monooxygenase gene in Escherichia coli. This gene (Cvmp) is very similar (74 and 59% of similarity and identity, respectively) to the ortholog from Ralstonia eutropha bacteria capable of utilizing phenol as the sole carbon source. The phenol biodegradation ability of E. coli recombinant strains was tested by cell-growth in a minimal medium containing phenol as the sole source of carbon and release of intermediary metabolites (catechol and hydroquinone). Interestingly, during the growth of these strains on phenol, catechol, and hydroquinone accumulated transiently in the medium. These metabolites were further analyzed by HPLC. These results indicated that phenol can be initially orto or para hydroxylated to produce cathecol or hydroquinone, respectively, followed by meta-cleavage of aromatic rings. To verify this information, the metabolites obtained from HPLC were submitted to LC/MS to confirm their chemical structure, thereby indicating that the recombinant strains utilize two different routes simultaneously, leading to different ring-fission substrates for the metabolism of phenol.     

Identification and Genetic Characterization of Phenol-Degrading Bacteria from Leaf Microbial Communities

Microbial communities on aerial plant leaves may contribute to the degradation of organic air pollutants such as phenol. Epiphytic bacteria capable of phenol degradation were isolated from the leaves of green ash trees grown at a site rich in airborne pollutants. Bacteria from these communities were subjected, in parallel, to serial enrichments with increasing concentrations of phenol and to direct plating followed by a colony autoradiography screen in the presence of radiolabeled phenol. Ten isolates capable of phenol mineralization were identified. Based on 16S rDNA sequence analysis, these isolates included members of the genera Acinetobacter, Alcaligenes, and Rhodococcus. The sequences of the genes encoding the large subunit of a multicomponent phenol hydroxylase (mPH) in these isolates indicated that the mPHs of the gram-negative isolates belonged to a single kinetic class, and that is one with a moderate affinity for phenol; this affinity was consistent with the predicted phenol levels in the phyllosphere. PCR amplification of genes for catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) in combination with a functional assay for C23O activity provided evidence that the gram-negative strains had the C12O−, but not the C23O−, phenol catabolic pathway. Similarly, the Rhodococcus isolates lacked C23O activity, although consensus primers to the C12O and C23O genes of Rhodococcus could not be identified. Collectively, these results demonstrate that these leaf surface communities contained several taxonomically distinct phenol-degrading bacteria that exhibited diversity in their mPH genes but little diversity in the catabolic pathways they employ for phenol degradation.     

Biodegradation of phenol and <Emphasis Type="Italic">m</Emphasis>-cresol by <Emphasis Type="Italic">Candida albicans</Emphasis> PDY-07 under anaerobic condition

Strain Candida albicans PDY-07 was used to study the anaerobic biodegradation of phenol and m-cresol as single and dual substrates in batch cultures. The strain had a higher potential to degrade phenol than m-cresol. The cell growth kinetics of batch cultures with various initial m-cresol concentrations was investigated, and the Haldane kinetic model adequately described the dynamic behavior of cell growth on m-cresol. When cells grew on the mixture of phenol and m-cresol, substrate interactions were observed. Phenol inhibited the utilization of m-cresol; on the other hand, m-cresol also inhibited the degradation of phenol. However, the presence of low-concentration phenol enhanced m-cresol biodegradation; 100 mg/l m-cresol could be completely degraded within a shorter period of time than m-cresol alone in the presence of 150–300 mg/l phenol. The maximum m-cresol biodegradation rate was obtained at the existence of 200 mg/l phenol. Phenol was preferably utilized by the strain as a carbon and energy source. In addition, a sum kinetics model was used to describe the cell growth behavior in binary mixture of phenol and m-cresol, and the interaction parameters were determined. The model adequately predicted the growth kinetics and the interaction between the substrates.     

Phenol-induced membrane changes in free and immobilized Escherichia coli

Summary Membranes of Escherichia coli cells grown in the presence of phenol were examined after isolation of the cytoplasmic and outer membrane fractions. Both membrane types showed reduced lipid-to-protein ratios compared to cells grown without phenol. Phenol-induced differences in the expression of individual proteins of the outer membrane, probably involved in the uptake of iron, were expressed in smaller quantities after phenol addition. Growth in the presence of phenol increased the respiratory activity of the cytoplasmic membrane, whereas the direct inhibition of O₂ consumption by phenol was not affected by the presence of this compound in the growth medium. E. coli cells grown entrapped in calcium alginate showed low lipid-to-protein ratios even without phenol in the growth medium. Immobilization of cells also markedly changed the protein pattern of the outer membrane. Offprint requests to: H.-J. Rehm     

Kinetics of phenol oxidation by washed cells

The uptake of phenol by pure cultures of Pseudomonas putida growing on phenol in continuous culture has been studied. The purpose of the experiments was to determine the kinetic parameters governing uptake of phenol by organisms growing on phenol in the high-conversion range by measuring uptake rates per unit biomass per unit time at various phenol concentrations. The microorganisms used were taken from a chemostat at residence times of 8, 5.25, 3.85, 3.2, 3, and 2.7h. The Monod–Haldane model and modifications of it were applied to the data and the best kinetic parameters were determined by nonlinear least-squares techniques. The best model was a two-parameters simplification of Monod–Haldane in which μ = K₁S/(K₂ + S²). The value of K₁ was found to increase monotonically with the value of phenol concentration in the original chemostat with an apparent induction “threshold” of 0.1 mg/L.     

Protective Effect of Immobilized Ammonia Oxidizers and Phenol‐degrading Bacteria on Nitrification in Ammonia– and Phenol‐containing Wastewater

Phenol present in wastewaters from various industries has an inhibitory effect on nitrification even at low concentrations. Hence, the biological treatment of wastewater containing both phenol and ammonia involves a series of treatment steps. It is difficult to achieve nitrification capability in an activated sludge system that contains phenol at concentrations above the inhibitory level. Batch treatment of wastewater containing various concentrations of phenol showed that the ammonia oxidation capability of suspended Nitrosomonas europaea cells, an ammonia oxidizer, was completely inhibited in the presence of more than 5.0 mg/L phenol. To protect the ammonia oxidizer from the inhibitory effect of phenol and to achieve ammonia oxidation capability in the wastewater containing phenol at concentrations above the inhibitory level, a simple bacterial consortium composed of an ammonia oxidizer (N. europaea) and a phenol‐degrading bacterial strain (Acinetobacter sp.) was used. Ammonia oxidation did not occur in the presence of phenol at concentrations above the inhibitory level when suspended or immobilized N. europaea and Acinetobacter sp. cells were used in batch treatment. Following the acclimatization of the immobilized cells, accumulation of nitrite was observed, even when the wastewater contained phenol at concentrations above the inhibitory level. These results showed that immobilization was effective in protecting N. europaea cells from the inhibitory effect of phenol present in the wastewater.     

Evidence of two pathways for the metabolism of phenol by Aspergillus fumigatus

Aspergillus fumigatus (ATCC 28282), a thermotolerant fungus, has been shown to be capable of growth on phenol as the sole carbon and energy source. During growth of the organism on phenol, catechol and hydroquinone accumulated transiently in the medium; cells grown on phenol oxidised these compounds without a lag period. Two different routes operating simultaneously, leading to different ring-fission substrates, are proposed for the metabolism of phenol. In one route, phenol undergoes ortho-hydroxylation to give catechol, which is then cleaved by an intradiol mechanism leading to 3-oxoadipate. In the other route, phenol is hydroxylated in the para-position to produce hydroquinone, which is then converted into 1,2,4-trihydroxybenzene for ring fission by ortho-cleavage to give maleylacetate. Cell-free extracts of phenol-grown mycelia were found to contain enzymic activities for the proposed steps. Two ring-fission dioxygenases, one active towards 1,2,4-trihydroxybenzene, but not catechol, and one active towards both ring-fission substrates, were separated by FPLC. Succinate-grown mycelia did not oxidise any of the intermediates until a clear lag period had elapsed and did not contain any of the enzymic activities for phenol metabolism.     

Phosphonium ionic liquids for degradation of phenol in a two-phase partitioning bioreactor

Six ionic liquids (ILs), which are organic salts that are liquid at room temperature, were tested for their biocompatibility with three xenobiotic-degrading bacteria, Pseudomonas putida, Achromobacter xylosoxidans, and Sphingomonas aromaticivorans. Of the 18 pairings, seven were found to demonstrate biocompatibility, with one IL (trihexyl(tetradecyl)phosphonium bis(trifluoromethylsulfonyl) amide) being biocompatible with all three organisms. This IL was then used in a two-phase partitioning bioreactor (TPPB), consisting of 1 l aqueous phase loaded with 1,580 mg phenol and 0.25 l IL, inoculated with the phenol degrader P. putida. This initially toxic aqueous level of phenol was substantially reduced by phenol partitioning into the IL phase, allowing the cells to utilize the reduced phenol concentration. The partitioning of phenol from the IL to the aqueous phase was driven by cellular demand and thermodynamic equilibrium. All of the phenol was consumed at a rate comparable to that of previously used organic-aqueous TPPB systems, demonstrating the first successful use of an IL with a cell-based system. A quantitative ³¹P NMR spectroscopic assay for estimating the log P values of ILs is under development.     

Development of a microtitre plate method for determination of phenol utilization, biofilm formation and respiratory activity by environmental bacterial isolates

Twenty-five aerobic phenol-degrading bacteria, isolated from different environmental samples on phenol agar after several subcultures in phenol broth, utilized phenol (0.2 g l⁻¹) within 24 h, but removal of phenol was more rapid when other carbon sources were also present. A microtitre plate method was developed to determine growth rate, biofilm formation and respiratory activity of the strains isolated. Pseudomonas putida strains C5 and D6 showed maximum growth (as O.D. at 600 nm), P. putida D6 and unidentified bacterial strain M1 were more stable at high concentrations of phenol (0.8 g l⁻¹), and P. putida C5 formed the greatest amount of biofilm in 0.5 g phenol l⁻¹ medium. Measurement of dehydrogenase activity as reduction of triphenyl tetrazolium chloride supported data on growth rate and biofilm formation. The microtitre plate method provided a selective method for detection of the best phenol degrading and biofilm-forming microorganisms, and was also a rapid, convenient means of studying the effect of phenol concentration on growth rate and biofilm formation.     

Isolation and Characterization of Phenol-catabolizing Bacteria from a Coking Plant

New phenol degrading bacteria with high biodegradation activity and high tolerance were isolated as Burkholderia cepacia PW3 and Pseudomonas aeruginosa AT2. Both isolates could grow aerobically on phenol as a sole carbon source even at 3 g/l. The whole-cell kinetic properties for phenol degradation by strains PW3 and AT2 showed a V_max of 0.321 and 0.253 mg/l/min/(mg protein), respectively. The metabolic pathways for phenol biodegradation in both strains were assigned to the meta-cleavage activity of catechol 2,3-dioxygenase.     

Single-culture aerobic granules with <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis>

Aerobic granules are cultivated by a single bacterial strain, Acinetobacter calcoaceticus, in a sequencing batch reactor (SBR). This strain presents as a good phenol reducer and an efficient auto coagulator in the presence of phenol, mediated by heat-sensitive adhesins proteins. Stable 2.3-mm granules were formed in the SBR following a 7-week cultivation. These granules exhibit excellent settling attributes and degrade phenol efficiently at concentrations of 250–2,000 mg l⁻¹. The corresponding phenol degradation rate reached 993.6 mg phenol g⁻¹ volatile suspended solids (VSS) day⁻¹ at 250 mg l⁻¹ phenol and 519.3 mg phenol g⁻¹ VSS day⁻¹ at 2,000 mg l⁻¹ phenol concentration. Meanwhile, free A. calcoaceticus cells were fully inhibited at phenol >1,500 mg l⁻¹. Denaturing gradient gel electrophoresis fingerprint profile demonstrated no genetic modification in the strain during aerobic granulation. The present single-strain granules showed long-term structural stability and performed high phenol degrading capacity and high phenol tolerance. The confocal laser scanning microscopic test revealed that live A. calcoaceticus cells principally distributed at 200–250 μm beneath the outer surface, with an extracellular polymeric substance layer covering them to defend phenol toxicity. Autoaggregation assay tests demonstrated the possibly significant role of secreted proteins on the formation of single-culture A. calcoaceticus granules.     

Growth on Phenol at Chemostress Levels Amplifies the Expression of the Phenol Degradation Pathway in Acinetobacter calcoaceticus

Bacteria often use pollutants as sole carbon and energy sources. However, if they are toxic and the concentrations are high these compounds inhibit growth, and eventually poison the biocatalysts. In order to identify mechanisms contributing to stability, the protein patterns of Acinetobacter calcoaceticus during growth on potentially toxic phenol were analysed using two‐dimensional gel electrophoresis. Enzymes involved in the catabolism or assimilation of phenol, such as phenol monooxygenase and catechol 1,2‐dioxygenase, were induced at a more than twofold level in response to long‐term exposure to high concentrations of the compound serving as the sole carbon and energy source. This would have clear adaptive benefits, since increased rates of consumption of phenol would reduce the susceptibility of Acinetobacter calcoaceticus to phenol poisoning. In contrast, transient induction of only one heat shock protein and one oxidative stress protein was detected during long‐term exposure to high concentrations of phenol.     

Anaerobic carboxylation of phenol to benzoate: use of deuterated phenols revealed carboxylation exclusively in the C4-position

Summary [U-D]Phenol and [4-D]phenol were used to rule out carboxylation of phenol in the C1-position by a strictly anaerobic, defined mixed culture. By mass spectrometric analysis of deuterated phenol species and of benzoate, which were formed from [U-D]phenol by D/H-exchange or by carboxylation from cell suspensions, it was shown that only one deuterium (D) from the aromatic nucleus was replaced with a least 97% efficiency. This excluded benzoate synthesis by carboxylation in the C1-position of phenol. Finally, carboxylation in the para-position of phenol was demonstrated with [4-D]phenol by gas chromatography/mass spectroscopy of the products. Since direct measurement of phenol carboxylase activity was impossible due to a very active interfering decarboxylase activity, the optimal pH range and ion strength, as well as the requirement of cations in crude cell-free extracts was characterized by means of D/H-exchange from deuterated phenol.Dedicated to Prof. R. S. Wolfe on the occasion of his 70th birthday Offsprint requests to: J. Winter     

Phenol and <Emphasis Type="Italic">n</Emphasis>-alkanes (C<Subscript>12</Subscript> and C<Subscript>16</Subscript>) utilization: influence on yeast cell surface hydrophobicity

This study was focused on the role of two types of diametrically different carbon sources, n-alkanes represented by a mixture of dodecane–hexadecane, and phenol on modification of the cell surface hydrophobicity. Capabilities of using either solely hydrocarbons or hydrocarbons in the mixture with phenol as well as phenol itself by yeast species Candida maltosa, Yarrowia lipolytica and Pichia guilliermondii were investigated. Studies were complemented by cell biomass formation measurements. The corresponding cell surface hydrophobicity was assessed by microbial adhesion to the hydrocarbon test (MATH). Degradation of phenol was examined using GC-SPE technique, whereas hydrocarbons were extracted prior to gravimetric determination. Results obtained indicated that the hydrophobic or hydrophilic nature of the carbon source had significant influence on the cell surface hydrophobicity. Although the results differed for some individual yeast strains, the generalization can be made that there is the correlation between the best hydrocarbon and phenol degradation and corresponding cell wall properties of the yeast examined.     

The importance of phenol oxidase activity in lignin degradation by the white-rot fungus Sporotrichum pulverulentum

The lignin degradation abilities of wildtype, a phenol oxidase-less mutant and a phenol oxidase-positive revertant of Sporotrichum pulverulentum were compared to determine if phenol oxidase activity is necessary for lignin degradation by white-rot fungi. The phenol oxidase-less mutant was unable to degrade kraft lignin or wood. The phenol oxidase-positive revertant, however, regained the ability of the wildtype to degrade kraft lignin and all of the major components of wood. It was found that kraft lignin and lignin-related phenols decreased cellulase and xylanase production by the phenol oxidase-less mutant. Addition of highly purified laccase increased the production of endo-1,4--glucanase in the phenol oxidase-less mutant in the presence of vanillic acid and kraft lignin. After addition of laccase to kraft lignin agar plates, the phenol oxidase-less mutant could degrade kraft lignin.It is proposed that phenol oxidase function in regulating the production of both lignin-and polysaccharide-degrading enzymes by oxidation of lignin and lignin-related phenols when S. pulverulentum is growing on wood.Abbreviation WT wildtype Sporotrichum pulverulentum Research supported by a grant from Stiftelsen Nils and Dorthi Troëdssons forskningsfond