Changes in structure,activity and metabolism of aerobic granules as a microbial response to high phenol loading

Four column-type sequential aerobic sludge blanket reactors were fed with phenol as the sole carbon and energy source and operated at loading rates of 1.0, 1.5, 2.0 and 2.5 kg phenol m^–3 day^–1. The results indicated that phenol loading exerted a profound influence on the structure, activity and metabolism of the aerobic granules. Compact granules with good settling ability were maintained at loadings up to 2.0 kg phenol m^–3 day^–1, and structurally weakened granules with enhanced production of extracellular polymers and proteins and significantly lower hydrophobicities were observed at the highest loading of 2.5 kg phenol m^–3 day^–1. Specific oxygen uptake rate, catechol 2,3-dioxygenase (C23O) and catechol 1,2-dioxygenase (C12O) activities peaked at a loading of 2.0 kg phenol m^–3 day^–1, and declined thereafter. Granules degraded phenol completely in all four reactors, mainly through the meta cleavage pathway as C23O activities were significantly higher than C12O activities. At the highest loading applied, the anabolism and catabolism of microorganisms were regulated such that phenol degradation proceeded exclusively via the meta pathway, apparently to produce more energy for overstimulation of protein production against phenol toxicity. This work contributes to a better understanding of the ability of aerobic granules to handle high-strength industrial wastewaters containing chemicals that are normally inhibitory to microbial growth.     

The catechol 2,3-dioxygenase gene and toluene monooxygenase genes from Burkholderia sp. AA1, an isolate capable of degrading aliphatic hydrocarbons and toluene

Burkholderia sp. AA1 isolated from a diesel fuel-contaminated site degraded toluene, as well as a wide range of alkanes from decane (C₈) to pentacosane (C₂₅) as sole carbon and energy sources. This strain also utilized m-toluate, p-toluate, o-toluate, and m-cresol as sole carbon and energy sources. Toluene- and toluate-grown cells showed catechol 2,3-dioxygenase activity and indole oxidation activity that is exhibited by some toluene oxygenation enzymes. The catechol 2,3-dioxygenase gene (catB) was cloned and sequenced. Its deduced amino acid sequence is analogous to the extradiol dioxygenases cloned from a variety of microorganisms. A DNA fragment containing the genes for the indole oxidation activity was cloned and sequenced. A seven-gene cluster designated as tbhABCDEFG was identified. Significant similarities were found with multicomponent monooxygenase systems for toluene, benzene and phenol from different bacterial strains. Journal of Industrial Microbiology & Biotechnology (2000) 25, 127–131. Received 28 July 1999/ Accepted in revised form 28 June 2000     

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.     

Biochemical,Transcriptional and Translational Evidences of the Phenol-meta-Degradation Pathway by the Hyperthermophilic Sulfolobus solfataricus 98/2

Phenol is a widespread pollutant and a model molecule to study the biodegradation of monoaromatic compounds. After a first oxidation step leading to catechol in mesophilic and thermophilic microorganisms, two main routes have been identified depending on the cleavage of the aromatic ring: ortho involving a catechol 1,2 dioxygenase (C12D) and meta involving a catechol 2,3 dioxygenase (C23D). Our work aimed at elucidating the phenol-degradation pathway in the hyperthermophilic archaea Sulfolobus solfataricus 98/2. For this purpose, the strain was cultivated in a fermentor under different substrate and oxygenation conditions. Indeed, reducing dissolved-oxygen concentration allowed slowing down phenol catabolism (specific growth and phenol-consumption rates dropped 55% and 39%, respectively) and thus, evidencing intermediate accumulations in the broth. HPLC/Diode Array Detector and LC-MS analyses on culture samples at low dissolved-oxygen concentration (DOC  =  0.06 mg.L⁻¹) suggested, apart for catechol, the presence of 2-hydroxymuconic acid, 4-oxalocrotonate and 4-hydroxy-2-oxovalerate, three intermediates of the meta route. RT-PCR analysis on oxygenase-coding genes of S. solfataricus 98/2 showed that the gene coding for the C23D was expressed only on phenol. In 2D-DIGE/MALDI-TOF analysis, the C23D was found and identified only on phenol. This set of results allowed us concluding that S. solfataricus 98/2 degrade phenol through the meta route.     

Isolation and characterization of a micromycete, a producer of neutral catechol oxidases, from tropical soils with elevated dioxine content

—Samples of South Vietnamese soils intensely treated with Agent Orange defoliant were tested for the presence of fungi and actinomycetes with an elevated phenol oxidase activity. As a result, a fast-growing nonsporulating strain producing neutral phenol oxidases was isolated and identified asMycelia sterilia INBI2-26. The strain formed extracellular phenol oxidases during surface growth on a liquid medium in the presence of guayacol and copper sulfate, as well as during submerged cultivation in liquid medium containing wheat bran and sugar beet pulp. Isoelectric focusing of the culture liquid revealed two major catechol oxidases (PO1 and PO2) with pI 3.5 and 8, respectively. The enzymes were purified by Ultrafiltration, ion exchange chromatography, and exclusion HPLC. Both were stable between pH 3 and 8. At pH 8 and 40°C., they retained at least 50% of activity after incubation for 50 h. At 50°C., PO2 was more stable and retained 40% of activity after 50 h, whereas PO1 was inactivated in 3–6 h. The pH-optimutns for PO1 and PO2 toward catechol were 6 and 6.5; and theK _m values were 1.5±0.35 and 1.25±0.2 mM, respectively. PO1 and PO2 most optimally oxidized 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) at pH 3 withK _m values 1.6±0.18 and 0.045±0.01 mM, respectively, but displayed no activity toward tyrosine. The PO2 absorbance spectrum had a peak at 600 nm, thus indicating the enzyme to be a member of the laccase family.     

Mineralization of phenol and its derivatives by Pseudomonas sp. strain CP4

A Pseudomonas sp. strain, CP4, was isolated that used phenol up to 1.5 g/l as sole source of carbon and energy. Optimal growth on 1.5 g phenol/l was at pH 6.5 to 7.0 and 30°C. Unadapted cells needed 72 h to decrease the chemical oxygen demand (COD) of about 2000 mg/l (from 1 g phenol/l) to about 200 mg/l. Adapted cells, pregrown on phenol, required only 65 h to decrease the COD level to below 100 mg/l. Adaptation of cells to phenol also improved the degradation of cresols. Cell-free extracts of strain CP4 grown on phenol or o-, m- or p-cresol had sp. act. of 0.82, 0.35, 0.54 and 0.32 units of catechol 2,3-dioxygenase and 0.06, 0.05, 0.05 and 0.03 units of catechol 1,2-dioxygenase, respectively. Cells grown on glucose or succinate had neither activity. Benzoate and all isomers of cresol, creosote, hydroxybenzoates, catechol and methyl catechol were utilized by strain CP4. No chloroaromatic was degraded, either as sole substrate or as co-substrate.The authors are with the Department of Microbiology and Bioengineering, Central Food Technological Research Institute, Mysore-570 013, India     

Electron shuttle-stimulated RDX mineralization and biological production of 4-nitro-2,4-diazabutanal (NDAB) in RDX-contaminated aquifer material

The potential for extracellular electron shuttles to stimulate RDX biodegradation was investigated with RDX-contaminated aquifer material. Electron shuttling compounds including anthraquinone-2,6-disulfonate (AQDS) and soluble humic substances stimulated RDX mineralization in aquifer sediment. RDX mass-loss was similar in electron shuttle amended and donor-alone treatments; however, the concentrations of nitroso metabolites, in particular TNX, and ring cleavage products (e.g., HCHO, MEDINA, NDAB, and NH₄ ⁺) were different in shuttle-amended incubations. Nitroso metabolites accumulated in the absence of electron shuttles (i.e., acetate alone). Most notably, 40–50% of [¹⁴C]-RDX was mineralized to ¹⁴CO₂ in shuttle-amended incubations. Mineralization in acetate amended or unamended incubations was less than 12% within the same time frame. The primary differences in the presence of electron shuttles were the increased production of NDAB and formaldehyde. NDAB did not further degrade, but formaldehyde was not present at final time points, suggesting that it was the mineralization precursor for Fe(III)-reducing microorganisms. RDX was reduced concurrently with Fe(III) reduction rather than nitrate or sulfate reduction. Amplified 16S rDNA restriction analysis (ARDRA) indicated that unique Fe(III)-reducing microbial communities (β- and γ-proteobacteria) predominated in shuttle-amended incubations. These results demonstrate that indigenous Fe(III)-reducing microorganisms in RDX-contaminated environments utilize extracellular electron shuttles to enhance RDX mineralization. Electron shuttle-mediated RDX mineralization may become an effective in situ option for contaminated environments.     

beta-Carotene production in sugarcane molasses by a Rhodotorula glutinis mutant

Several wild strains and mutants of Rhodotorula spp. were screened for growth, carotenoid production and the proportion of -carotene produced in sugarcane molasses. A better producer, Rhodotorula glutinis mutant 32, was optimized for carotenoid production with respect to total reducing sugar (TRS) concentration and pH. In shake flasks, when molasses was used as the sole nutrient medium with 40 g l⁻¹ TRS, at pH 6, the carotenoid yield was 14 mg l⁻¹ and -carotene accounted for 70% of the total carotenoids. In a 14-l stirred tank fermenter, a 20% increase in torulene content was observed in plain molasses medium. However, by addition of yeast extract, this effect was reversed and a 31% increase in -carotene content was observed. Dissolved oxygen (DO) stat fed-batch cultivation of mutant 32 in plain molasses medium yielded 71 and 185 mg l⁻¹ total carotenoids in double- and triple-strength medium, respectively. When supplemented with yeast extract, the yields were 97 and 183 mg l⁻¹ total carotenoid with a 30% increase in -carotene and a simultaneous 40% decrease in torulene proportion. Higher cell mass was also achieved by double- and triple-strength fed-batch fermentation. Journal of Industrial Microbiology & Biotechnology (2001) 26, 327–332. Received 18 September 2000/ Accepted in revised form 02 March 2001     

Isolation and characterization of four novel Gram-positive bacteria associated with the rhizosphere of two endemorelict plants capable of degrading a broad range of aromatic substrates

Four new Gram-positive, phenol-degrading strains were isolated from the rhizospheres of endemorelict plants Ramonda serbica and Ramonda nathaliae known to exude high amounts of phenolics in the soil. Isolates were designated Bacillus sp. PS1, Bacillus sp. PS11, Streptomyces sp. PS12, and Streptomyces sp. PN1 based on 16S rDNA sequence and biochemical analysis. In addition to their ability to tolerate and utilize high amounts of phenol of either up to 800 or up to 1,400 mg l⁻¹ without apparent inhibition in growth, all four strains were also able to degrade a broad range of aromatic substrates including benzene, toluene, ethylbenzene, xylenes, styrene, halogenated benzenes, and naphthalene. Isolates were able to grow in pure culture and in defined mixed culture on phenol and on the mixture of BTEX (benzene, toluene, ethylbenzene, and xylenes) compounds as a sole source of carbon and energy. Pure culture of Bacillus sp. PS11 yielded 1.5-fold higher biomass amounts in comparison to mixed culture, under all conditions. Strains successfully degraded phenol in the soil model system (2 g kg⁻¹) within 6 days. Activities of phenol hydroxylase, catechol 1,2-dioxygenase, and catechol 2,3-dioxygenase were detected and analyzed from the crude cell extract of the isolates. While all four strains use ortho degradation pathway, enzyme indicative of meta degradation pathway (catechol 2,3-dioxygenase) was also detected in Bacillus sp. PS11 and Streptomyces sp. PN1. Phenol degradation activities were induced 2 h after supplementation by phenol, but not by catechol. Catechol slightly inhibited activity of catechol 2,3-dioxygenase in strains PS11 and PN1.     

Isolation and Properties of Phenol Utilizing Microorganisms with Special Reference to Catechol 1,2-Oxygenase

Phenol utilizing yeasts were isolated from soil. The relationship were examined between distribution of phenol uptake rate using intact cells and distribution of the activities of catechol 1,2-oxygenase which is one of the key enzymes in phenol metabolism. Two of the isolates showed catechol 1,2-oxygenase activity even when grown in glucose medium, though the enzyme activity was about 1% of the full activity induced by phenol. Partially constitutive mutants for catechol 1,2-oxygenase were obtained by mutagenesis of an inducible strain. The level of mutant enzyme activity was close to that of the isolated constitutive strain. One isolate, Trichosporon cutaneum, preferentially utilized phenol to glucose in medium containing both phenol (200 ppm) and glucose (0.1%), until the concentration of phenol decreased to 10–20 ppm.     

Kinetic study of phenol hydroxylase and catechol 1,2-dioxygenase biosynthesis by <Emphasis Type="Italic">Candida tropicalis</Emphasis> cells grown on different phenolic substrates

When Candida tropicalis was grown on phenol, catechol or resorcinol, the highest levels of specific activity of phenol hydroxylase (EC. 1.14.13.7) and catechol 1,2-dioxygenase (EC. 1.13.11.1) were attained with phenol. With the three aromatic compounds tested, the yeast cells exhibited sharp peaks of specific activity of both enzymes at particular incubation times. Phenol-induced cells containing high levels of both enzymes were capable of degrading rapidly and without delay 4-chlorophenol and 2,6-dichlorophenol, and to a lesser extend pentachlorophenol. However, the yeast could not grow on chlorophenols as major carbon and energy source.     

Phenol degradation performance by isolated Bacillus cereus immobilized in alginate

Phenol degradation by Bacillus cereus AKG1 MTCC9817 and AKG2 MTCC 9818 was investigated and degradation kinetics are reported for the free and Ca-alginate gel-immobilized systems. The optimal pH for maximum phenol degradation by immobilized AKG1 and AKG2 was found to be 6.7 and 6.9, respectively, while 3% alginate was optimum for both the strains. The degradation of phenol by free as well as immobilized cells was comparable at lower concentrations of phenol (100–1000 mg l⁻¹). However, the degradation efficiency of the immobilized strains was higher than that of the free strains at higher phenol concentrations (1500–2000 mg l⁻¹), indicating the improved tolerance of the immobilized cells toward phenol toxicity. More than 50% of 2000 mg l⁻¹ phenol was degraded by immobilized AKG1 and AKG2 within 26 and 36 days, respectively. Degradation kinetics of phenol by free and immobilized cells are well represented by the Haldane and Yano model.     

Phenol degradation by Paenibacillus thiaminolyticus and Bacillus cereus in axenic and mixed conditions

In this study, seven aerobic bacterial strains were screened for phenol tolerance at different concentration of phenol. Bacterial strains were unable to utilize phenol in absence of glucose, indicated the phenomenon of co-metabolism. Among the seven isolated bacterial strains, only ITRC BK-4 and ITRC BK-7 found potential and identified as Paenibacillus thiaminolyticus (DQ435022) and Bacillus cereus (DQ435023), respectively. Phenol degradation was monitored routinely with spectrophotometer and further confirmed by HPLC analysis. ITRC BK-4, ITRC BK-7 and mixed culture degrade 700 ppm phenol up to 51.72, 70.00 and 84.57% respectively in mineral salt medium (MSM) at temperature 37 ± 1°C, pH 7.5 ± 0.2, 120 rpm in presence of 1% glucose (w/v) within 144 h incubation. The mix culture was found more potential for phenol degradation compared to axenic strains. Hence, the axenic and mixed strains of these bacteria would be useful for the removal/mineralization of phenol from industrial waste waters.     

Preferential utilization of phenol rather than glucose by Trichosporon cutaneum possessing a partially constitutive catechol 1,2-oxygenase.

A phenol-utilizing yeast, Trichosporon cutaneum POB 14, which has a partially constitutive activity of catechol 1,2-oxygenase, utilized phenol in preference to glucose in a medium containing both phenol (200 mg/liter) and glucose (0.15%) as carbon sources. The glucose consumption was not observed until the concentration of phenol decreased to around 10 mg/liter. This phenomenon was confirmed by [U-14C]glucose uptake experiments. The intracellular activities of hexokinase (EC 2.7.1.1) and catechol 1,2-oxygenase (EC 1.13.1.1) changed inversely when phenol was added during growth in the glucose medium.     

Degradation of chlorobenzene by strain <Emphasis Type="Italic">Ralstonia pickettii</Emphasis> L2 isolated from a biotrickling filter treating a chlorobenzene-contaminated gas stream

A Ralstonia pickettii species able to degrade chlorobenzene (CB) as the sole source of carbon and energy was isolated from a biotrickling filter used for the removal of CB from waste gases. This organism, strain L2, could degrade CB as high as 220 mg/L completely. Following CB consumption, stoichiometric amounts of chloride were released, and CO₂ production rate up to 80.2% proved that the loss of CB was mainly via mineralization and incorporation into cell material. The Haldane modification of the Monod equation adequately described the relationship between the specific growth rate and substrate concentration. The maximum specific growth rate and yield coefficient were 0.26 h⁻¹ and 0.26 mg of biomass produced/mg of CB consumed, respectively. The pathways for CB degradation were proposed by the identification of metabolites and assay of ring cleavage enzymes in cell extracts. CB was degraded predominantly via 2-chlorophenol to 3-chlorocatechol and also partially via phenol to catechol with subsequent ortho ring cleavage, suggesting partially new pathways for CB-utilizing bacteria.     

Chromium accumulation by two Streptomyces spp. isolated from riverine sediments

Strains designated R22 and R25, isolated from Salí River sediments, Argentina, were highly resistant to chromium. These strains were shown by 16S rRNA sequencing studies to be Streptomyces spp.; this affiliation was consistent with morphological and chemical characteristics. Growth of strains R22 and R25 in medium containing 100 mg l⁻¹ chromate was reduced by only 23% and 34%, respectively, compared with growth in medium without added chromium. Streptomyces sp. strains R22 and R25 both accumulated chromium with yields of 10.0 and 5.6 mg Cr g⁻¹ of dry weight, respectively, and a chromate concentration of 50 mg ml⁻¹. Cell fractionation studies with strain R22 showed that the great majority of the chromium were associated with the cell wall fraction. Streptomyces strains R22 and R25 may have applications in bioremediation of chromium contamination. Journal of Industrial Microbiology & Biotechnology (2001) 26, 210–215. Received 23 June 2000/ Accepted in revised form 24 January 2000     

Isolation and Characterization of Phenol-Degrading Soil Bacterium Xanthobacter flavus

A soil bacterium isolated from a contaminated site degraded phenol when provided as the sole carbon and energy source in the medium. The bacterium was identified as Xanthobacter flavus MTCC 9130. This microbial strain was able to tolerate phenol up to 1000 mg L^?1 concentration. The lag phase increased with the increase in phenol concentration. The optimum growth temperature was 37°C. The organism efficiently utilized phenol and could degrade it completely within 120 h when initial concentration was less than 600 mg L^?1. Degradation of phenol was through ortho pathway, enzyme assay through cell-free extract exhibited the presence of catechol 1,2-dioxygenase. The specific activity was 0.146 μ mol min^?1 mg^?1 protein. However, higher concentrations of phenol in the medium had a negative effect on the growth of the bacterium. Hence this ability of Xanthobacter flavus can be effectively used for bioremediation studies of phenol-contaminated sites.     

Effects of stream order and season on mineralization of [14C]-phenol in streams

Mineralization of trace levels of [¹⁴C]-phenol by heterotrophic microorganisms was quantified at 4 sites along a river continuum in southwestern Virginia. Significant phenol mineralization rates were detected in surface sediment and seston samples at all sites from August 1985 through May 1986. Phenol degradation was strongly affected by season (ANOVA; P < 0.0001). From a baseline rate in August (range: 1.19 × 10^-5 to 897 × 10^-4 mg phenol mineralized mg AFDW^-1 h^-1) phenol mineralization rose to a yearly maximum in October (range: 1.21 × 10^-4 to 1.16 × 10^-3 mg phenol mineralized mg AFDW^-1 h^-1) despite decreasing stream temperatures. This autumnal peak in phenol degradation was attributed to the pulsed input of allochthonous detritus, especially leaf litter, which contains substantial quantities of phenols and related compounds. Although phenol mineralization was significant in these streams, phenols were metabolized at much slower rates than more labile compounds present in the dissolved organic matter (DOM) pool. Estimates of turnover rates for three major components of DOM revealed that glucose and glutamate turnover rates (0.064–0.140 h^-1 mg sediment AFDW^-1 and 0.140–0.610 h^-1 mg sediment AFDW^-1, respectively) were, respectively, 2.2–4.7 × and 9.6–16.9 × greater than phenol turnover rates (0.015–0.064 h^-1 mg sediment AFDW^-1). Although the relatively low rates of utilization of refractory phenolic materials suggest that these compounds may accumulate and become more prevalent components of the DOM pool, phenol concentrations at the 4 study sites remained below detectable levels (i.e., < 1 g 1^-1) throughout the study. Consequently, it seems that although phenolic materials are metabolized more slowly than labile DOM, phenols are degraded at rates which preclude accumulation in the water column.     

Enhancement of phenol biodegradation by Ochrobactrum sp. isolated from industrial wastewaters

Ochrobactrum sp., was tested with regard to its phenol degradation capacity at different pH levels, and with different carbon sources (mineral salt medium with glucose (MSG) and the same medium with 0.5%, 1%, and 2% (v/v) molasses (MSM)) and phenol concentrations. The highest degradation was in mineral salt medium with 1% (v/v) molasses (45.9%), while degradation was 21.1% in mineral salt medium with 5 g l⁻¹ glucose. These data show that the addition of molasses to mineral salt medium enhanced phenol degradation by Ochrobactrum sp. The bacterium can be used effectively to treat wastewaters containing phenol.     

Kinetic studies of phenol degradation by Rhodococcus sp. P1 II. Continuous cultivation

The degradation of phenol by Rhodococcus sp. P1 was studied in continuous culture systems. The organism could be adapted by slowly increasing concentration, step by step, up to 30.0 g · 1^-1 phenol in the influent. The degradation rate reached values of about 0.3 g · g dry mass^-1 ·h^-1. Large step increases in phenol concentration and addition of further substrates (e.g., catechol) were tolerated up to a certain concentration. With increasing dilution rate and increasing inlet phenol concentration the stability of the system decreased.