Biodegradation of phenol and chlorophenols with defined mixed culture in shake-flasks and a packed bed reactor

Pseudomonas testosteroni CPW301 degraded phenol and 4-chlorophenol simultaneously, but degradation rates of these compounds were affected by 4-chlorophenol. Phenol increased the cell concentration and therefore the degradation efficiency of 4-chlorophenol was improved. Pseudomonas solanacearum TCP114 could degrade only 2,4,6-trichlorophenol. A defined mixed culture of P. testosteroni CPW301 and P. solanacearum TCP114 could treat phenol, 4-chlorophenol, and 2,4,6-trichlorophenol completely and overcome the inhibition of substrates to other microorganisms. The degradation capacity of the packed bed reactor (PBR) was higher than that of the continuous stirred tank reactor, but the PBR was unsuitable for oxygen-sensitive microorganisms.     

Degradation of phenol by a coimmobilized entrapped mixed culture

Summary The degradation of phenol by a defined mixed culture, consisting of Pseudomonas putida P8 and Cryptococcus elinovii H1, was studied. The microorganisms were entrapped either in 30 g·l^-1 calcium-alginate or in chitosan-alginate. Chitosan-alginate entrapment was suitable for a continuous culture. The coimmobilized mixed culture of Cryptococcus elinovii H1 which degrades phenol via an ortho pathway and of Pseudomonas putida P8 which uses the meta cleavage pathway was able to degrade high phenol concentrations up to 3.2 g·l^-1 in semicontinuous cultures. The degradation performance in continuous cultures could reach a maximum of 0.41 g·l^-1·h^-1 phenol. The mixed culture could be stored for up to six months without loss of phenol degradation capacity.Dedicated to Professor Dr. Dr. h. c. K. Esser on the occasion of his 65th birthday     

Metabolism of phenol and cresols by Bacillus stearothermophilus.

An obligate thermophilic strain of Bacillus stearothermophilus, strain PH24, isolated from industrial sediment by elective culture, grew readily at 55 C on phenol or on one of the isomers of cresol as the major carbon source. Intact cells grown in the presence of phenol, o-cresol, m-cresol, or p-cresol were induced to oxidize, without lag, these substrates together with catechol, 3-methylcatechol, and 4-methylcatechol. Cell extracts prepared from B. stearothermophilus PH24 after growth in the presence of phenol converted phenol to catechol with a concomitant uptake of 1 mol of oxygen per mol of substrate in reaction mixtures supplemented with reduced nicotinamide adenine dinucleotide. These preparations also catalyzed the oxidation of o-cresol to 3-methylcatechol and of m-cresol and p-cresol to 4-methylcatechol. Enzyme activity was inhibited by 1 mM p-chloromercuribenzoate and by 0.1 mM 0-phenanthroline. Catechol and the corresponding methylcatechol intermediates were further dissimilated by cell extracts of phenol-grown cells via the meta-cleavage route to yield 2-hydroxymuconic semialdehyde and the respective methylated derivatives.     

Anaerobic degradation of phenol using an acclimated mixed culture

Summary Anaerobic methanogenesis of phenol using mixed cultures derived from cow dung and municipal sewage sludge and adapted to phenol was done in batch reactors. The phenol degradation rate depended on the period in which the culture was acclimated to phenol. Interference in phenol uptake by glucose was observed. Consumption of both phenol and acetic acid was observed when an acetate-adapted culure was used. A phenol-acclimated culture was able to degrade dihydroxy phenols thus indicating the feasibility of cross-acclimation. Offprint requests to: P. Ghosh     

Biodegradation of the mixture of 2,4,6-trichlorophenol, 4-chlorophenol,and phenol by a defined mixed culture

Two new strains, Pseudomonas sp. TCP114 degrading 2,4,6-trichlorophenol (TCP) and Arthrobacter sp. CPR706 degrading 4-chlorophenol (4-CP), were isolated through a selective enrichment procedure. Both strains could also degrade phenol. The degradability of one component by a pure culture was strongly affected by the presence of other compounds in the medium. For example, when all three components (TCP, 4-CP, and phenol) were present in the medium, a pure culture of CPR706 could not degrade any of the components present. This restriction on degradability could be overcome by employing a defined mixed culture of the two strains. The mixed culture could degrade all three components in the mixture through cooperative activity. It was also demonstrated that the mixed culture could be immobilized by using calcium alginate for the semi-continuous degradation of the three-component mixture. Immobilization not only accelerates the degradation rate, but also enables reuse of the cell mass several times without losing the cells' degrading capabilities.     

Degradation of chlorophenols by a defined mixed microbial community

Synthetic sewage containing phenol, acetone, and alkanols plus 4-chlorophenol or a mixture of isomeric chlorophenols is completely degraded by a defined mixed culture with Pseudomonas sp. strain B13 as a chlorocatechol-dissimilating member of the community. Total degradation of the organic carbon was indicated by release of stoichiometric amounts of chloride and low content of dissolved organic carbon in the cell-free effluents. During adaptation to high loads of chlorophenols the initial meta-cleavage activity was completely replaced by ortho-cleavage activity of type I and II. In the fully acclimated culture, hybrid strains such as Alcaligenes sp. strain A7-2 were detected, which are more competitive than Pseudomonas sp. strain B13 with respect to chlorophenol degradation.     

Changes in phenol oxidation rate of a mixed microbial culture caused by sol-gel immobilization

A mixed microbial culture was entrapped into porous silica gel prepared by two different sol-gel methods. The immobilization of cells into prepolymerized tetraethoxysilane was more stressful to living microbial cells than the entrapment into colloidal SiO₂. Our experimental equipment operating in a sensor mode was able to detect 0.5 mg phenol l^–1 and had a linear response in the range from 2 to 10 mg phenol l^–1.     

Toxicity and kinetic parameters of the aerobic biodegradation of the phenol and alkylphenols by a mixed culture

A mixed culture aerobically metabolized phenol, cresol isomers (o-,m-,p-), 2-ethylphenol and xylenol isomers (2,5-DMP and 3,4-DMP) as the sole carbon and energy source. This culture had a high tolerance towards phenol with values of maximum degradation rate (V_\max) of 47 M phenol mg^–1 protein h^–1 and inhibition substrate constant (Ki) of 10 mM. These kinetic parameters were considerably diminished and the toxicity increased with the alkylphenols. For example with 2,5-xylenol, V_\max and Ki values of 0.8 M 2,5-xylenol mg^–1 protein h^–1 and 1.3 mM, respectively, were obtained. The cresols were 5-fold more toxic than phenol, whereas 2-ethylphenol and 3,4-xylenol were 11-fold more toxic, and 2,5-xylenol was 34-fold more toxic than phenol.     

Degradation of chlorophenols by a defined mixed microbial community.

Synthetic sewage containing phenol, acetone, and alkanols plus 4-chlorophenol or a mixture of isomeric chlorophenols is completely degraded by a defined mixed culture with Pseudomonas sp. strain B13 as a chlorocatechol-dissimilating member of the community. Total degradation of the organic carbon was indicated by release of stoichiometric amounts of chloride and low content of dissolved organic carbon in the cell-free effluents. During adaptation to high loads of chlorophenols the initial meta-cleavage activity was completely replaced by ortho-cleavage activity of type I and II. In the fully acclimated culture, hybrid strains such as Alcaligenes sp. strain A7-2 were detected, which are more competitive than Pseudomonas sp. strain B13 with respect to chlorophenol degradation.     

Degradation of phenol by a mixed culture of Pseudomonas putida and Cryptococcus elinovii adsorbed on activated carbon

Summary Phenol degradation by a defined mixed culture of Pseudomonas putida P8 and Cryptococcus elinovii H 1, which were immobilized by adsorption on activated carbon, was studied.The immobilized mixed culture was able to degrade phenol up to 17 g/l and degraded it faster than the pure cultures, depending on a complementary metabolism of the two microorganisms.Storage experiments revealed an excellent longterm storage capability of the biocatalyst: activated carbon with adsorbed cells of Pseudomonas putida P8 and Cryptococcus elinovii H1 could be stored up to 12 months without decrease on degradation capacity.Scanning electron micrographs showed that Pseudomonas putida P8 had grown through the pore system of the activated carbon into the inside of the carbon particles.     

Theoretical evaluation of isotopic fractionation factors in oxidation reactions of benzene,phenol and chlorophenols

We have studied theoretically the rate determining steps of reactions of benzene with permanganate, perchlorate, ozone and dioxygen in the gas phase and aqueous solution as well as phenol and dichlorophenol in protonated and unprotonated forms in aqueous solution. Kinetic isotope effects were then calculated for all carbon atoms and based on their values isotopic fractionation factors corresponding to compound specific isotopic analysis have been evaluated. The influence of the oxidant, substituents, environment and protonation on the isotopic fractionation factors has been analyzed.     

Biodegradation of chlorophenols by mixed and pure cultures from a fluidized-bed reactor

An aerobic, continuous-flow fluidized-bed reactor was established with inoculum from activated sludge, and fed a mixture of 2,4,6-trichlorophenol (TCP), 2,3,4,6-tetrachlorophenol (TeCP) and pentachlorophenol (PCP) as the sole sources of carbon and energy for 2 years. Experiments with the enrichment were performed with material from the reactor. Later, degradation experiments were completed using pure cultures of bacteria that were isolated from suspended samples of the carrier biofilm. In batch-bottle bioassays, the reactor enrichment degraded PCP, TeCP and TCP both in mineral salts (MS) and tryptone-yeast extract-glucose (TGY) media. ortho-Methoxylated chlorophenols including 4,5-dichloroguaiacol (4,5-DCG), tetrachloroguaiacol (TeCG) and trichlorosyringol (TCS) resisted biodegradation by the enrichment both in MS and TGY media, whereas 5,6-dichlorovanillin (5,6-DCV) was readily transformed to an unidentified metabolite. Experiments with ¹⁴C labeled chlorophenols showed mineralization of 2,4-dichlorophenol (DCP) and 2,3,5-TCP to ¹⁴CO₂ by the enrichment. Material from the suspended biofilm after continuous chlorophenol feeding for 2 years was inoculated onto TGY-agar plates, and showed predominantly two colony, types accounting for over 99% of the total colony counts. The two colony types, were equal in abundance. Six Gram-negative, oxidase- and catalase-positive, non-fermentative small rods were isolated in TGY agar media supplemented with 10 mg/l of TeCP or PCP. All isolates formed colonies in TGY plus 150 mg/l of PCP. The isolates degraded TCP and TeCP but not PCP. In mixtures of isolated bacteria the rates of chlorophenol degradation were similar to those observed with individual isolates. Three isolates were identified as Pseudomonas saccharophila and three were an unidentified species of Pseudomonas.     

Degradation of phenol by a defined mixed culture immobilized by adsorption on activated carbon and sintered glass

Summary A defined mixed culture of the yeast Cryptococcus elinovii H1 and the bacterium Pseudomonas putida P8 was immobilized by adsorption on activated carbon and sintered glass, respectively. Depending on its adsorption capacity for phenol the activated carbon system could completely degrade 17 g/l in batch culture, whereas the sintered glass system was able to degrade phenol up to 4 g/l. During semicontinuous degradation of phenol (1 g/l) both systems reached constant degradation times with the fourth batch that lasted 8 h when using the activated carbon system and 10 h in the sintered glass system. In the course of continuous degradation of phenol the activated carbon system reached a maximum degradation rate of 9.2 g l^–1 day^–1 compared to 6.4 g l^–1 day^–1degraded by the sintered glass system. 2-Hydroxymuconic acid semialdehyde could be identified and quantitatively determined as a metabolite of phenol degradation by P. putida P8. Increased membrane permeability under the influence of phenol was demonstrated by the examination of K⁺ efflux from P. putida P8. Offprint requests to: H.-J. Rehm     

Influence of position of substituent groups on removal of chlorophenols and cresols by horseradish peroxidase and determination of optimum conditions

Enzymatic treatment of o-, m-, and p-chlorophenols and o-, m-, and p-cresols from artificial wastewater was undertaken through the enzymatic conversion into the corresponding phenoxy radicals with horseradish peroxidase (HRP) and nonenzymatic radical coupling reaction. The concentration of chlorophenols and cresols decreased sharply over the reaction time and water-insoluble oligomer precipitates were generated. The optimum conditions were determined to be the H2O2 concentration of 2.5 mM and poly(ethylene glycol) with molecular mass of 1.0 x 10(4) (10K-PEG) of 0.10 mg/cm3 at 30 degrees C for treatment of p-chlorophenol at 2.5 mM. The optimum pH values depended on the relative position of a chlorine atom for chlorophenols and on a methyl group for cresols. Concentrations of HRP and 10K-PEG were increased to 1.0 U/cm3 and 1.0 mg/cm3 respectively to treat m-chlorophenol highly. For o-chlorophenol, a decrease in the pH value to 3.0 after the enzymatic treatment led to the enhancement of the aggregation of oligomer precipitates. The % residual value for o-cresol effectively decreased by absorbing water-soluble intermediates on the chitosan films. These results indicate that chlorophenols and cresols were removed to a great degree by this technique, although the detailed procedure depended on the position of substituent groups of chlorophenols and cresols.     

Studies on composition and stability of a large membered bacterial consortium degrading phenol

A ten member microbial consortium (AS) consisting of eight phenol-degrading and two non-phenol-degrading strains of bacteria was developed and maintained in a fed-batch reactor by feeding 500 mg l⁻¹ phenol for four years at 28 ± 3 °C. The consortium could degrade 99% of 500 mg l⁻¹ phenol after 24 hours incubation with a biomass increase of 2.6 × 10⁷ to 4 × 10¹² CFU ml⁻¹. Characterization of the members revealed that it consisted of 4 principal genera, Bacillus, Pseudomonas, Rhodococcus, Streptomyces and an unidentified bacterium. Phenol degradation by the mixed culture and Bacillus subtilis, an isolate from the consortium was compared using a range of phenol concentrations (400 to 700 mg l⁻¹) and by mixing with either 160 mg l⁻¹ glucose or 50 mg l⁻¹ of 2,4-dichlorophenol in the medium. Simultaneous utilization of unrelated mixed substrates (glucose/2,4-dichlorophenol) by the consortium and Bacillus subtilis, indicated the diauxic growth pattern of the organisms. A unique characteristic of the members of the consortia was their ability to oxidize chloro aromatic compounds via meta pathway and methyl aromatic compounds via ortho cleavage pathway. The ability of a large membered microbial consortia to maintain its stability with respect to its composition and effectiveness in phenol degradation indicated its suitability for bioremediation applications.     

苯酚降解菌的分离及培养特性研究

对南充市郊炼油厂活性污泥进行富集,驯化筛选得到2株能以苯酚作为唯一碳源和能源生长的菌株,编号为S1,S2,两菌株可耐10,000mg/L左右的苯酚浓度,实验得出其最佳生长条件为pH7-8,温度25℃-30℃,在适宜条件下,对苯酚有较好的降解能力,而且苯对两菌株的生长表现为抑制作用。     

Response to oxygen of diazotrophic Azospirillum brasilense — Arthrobacter giacomelloi mixed batch culture

Azospirillum brasilense and Arthrobacter giacomelloi were grown together in batch culture under different oxygen pressures. The response to oxygen of growth, nitrogenase activity and respiration rate was determined. The two microorganisms were found to be able to coexist all over the range of partial oxygen pressures examined, that is from 0.004–0.20 bar. Nitrogenase activity by mixed culture of A. brasilense and A. giacomelloi always appeared higher than that of A. brasilense pure culture. Low respiratory activity at partial oxygen pressures higher than 0.02 bar by both pure and mixed cultures seemed not to account for the high nitrogenase activity and improved oxygen tolerance of the mixed culture.Abbreviations pO₂ partial oxygen pressure     

Mutualistic interaction between dichloromethane‐ and chloromethane‐degrading bacteria in an anaerobic mixed culture

The microbial mixed culture RM grows with dichloromethane (DCM) as the sole energy source generating acetate, methane, chloride and biomass as products. Chloromethane (CM) was not an intermediate during DCM utilization consistent with the observation that CM could not replace DCM as a growth substrate. Interestingly, cultures that received DCM and CM together degraded both compounds concomitantly. Transient hydrogen (H₂) formation reaching a maximum concentration of 205 ± 13 ppmv was observed in cultures growing with DCM, and the addition of exogenous H₂ at concentrations exceeding 3000 ppmv impeded DCM degradation. In contrast, CM degradation in culture RM had a strict requirement for H₂. Following five consecutive transfers on CM and H₂, Acetobacterium 16S rRNA gene sequences dominated the culture and the DCM‐degrader Candidatus Dichloromethanomonas elyunquensis was eliminated, consistent with the observation that the culture lost the ability to degrade DCM. These findings demonstrate that culture RM harbours different populations responsible for anaerobic DCM and CM metabolism, and further imply that the DCM and CM degradation pathways are mechanistically distinct. H₂ generated during DCM degradation is consumed by the hydrogenotrophic CM degrader, or may fuel other hydrogenotrophic processes, including organohalide respiration, methanogenesis and H₂/CO₂ reductive acetogenesis.