Development of Biosensors for Phenol Determination from Bacteria Found in Petroleum Fields of West Siberia

Nine gram-negative bacterial strains, selected from 300 strains isolated from soils of the West Siberian petroliferous basin and growing on oil and oil products, consume phenol as a single carbon and energy source. The strains were used for the development of a sensor bioreceptor. The most active 32-I strain was shown to bear a plasmid responsible for phenol degradation. The plasmid-free derivative of this strain, 32-I-1, did not grow on phenol. The possibility of creating a model biosensor for phenol based on the plasmid-containing 32-I strain is considered. The detection limit for phenol was 5 M. The optimum conditions for the sensor operation are: pH 7.4, 35°C, and operation time 30 h.     

Amperometric biosensors for detection of phenol using chemically modified electrodes containing immobilized bacteria

Eight strains of Pseudomonas were studied for development of phenol sensor. The immobilization of cells was performed by absorbing them on the working part of mediator-modified screen-printed electrodes (SPEs). Only three Pseudomonas strains were able to transfer electrons resulting from specific oxidation of phenol to the electrode by means of mediators; ferrocene, duroquinone and dimethyferrocene were successfully used with the strains 394 (p20), 74-III and 83-IV (working names), respectively. The lower limits for detection of phenol were 1 micro M for the strain 74-III and 10 micro M for the strain 83-IV and 394 (p20). Calibrations were obtained as the dependencies of logarithm of current changes (log deltaI) on logarithm of concentration (logC), log delta I vs. logC. Among all substrates tested (phenol, catechol, hydroquinone, ethanol, methanol, propanol, isopropanol, isobutanol, isoamylalcohol, acetate, glucose, xylose, vanillin, 2,4,6-trichlorphenol, 2,3,6-trichlorphenol, 4-hydroxy-3-methoxybenzoic acid, coumarin, pentafluorophenol), bacterial sensor demonstrated a good selectivity with respect to phenol and lower responses to catechol and hydroquinone (10-times lower). The dependence of signals on operating conditions was studied. The biosensor should be used during the day of preparation. The operational stability was satisfactory to perform up to 10 consecutive measurements. Low cost and very simple manufacturing procedure allow for bacterial sensor to be applied as disposable devices.     

Utilization of phenol by hydrocarbon assimilating yeasts

77 Ascomycetous, basidiomycetous as well as imperfect yeast strains of 46 different species and 20 genera were tested for growth with the substrates n-octane, n-hexadecane, and phenol. Of 59 yeast strains with ascomycetous cell wall structure 33 grew on hydrocarbons and 32 on phenol. No yeast strain out of 26 which are unable to use n-alkanes as a source of carbon and energy grew on phenol. In comparison with the latter 32 out of 33 n-hexadecane assimilating yeasts were also capable of using phenol. All n-octane utilizing yeasts of this group also assimilate phenol as a carbon source for growth.The correlation of the hydrocarbon assimilation with the phenol assimilation seems to be not so strong in the basidiomycetous yeasts. 7 out of 18 strains from this group grew on n-hexadecane and 13 on phenol.Furthermore, it could be shown that the use of hydrocarbons and phenol (as well as methanol) is strongly correlated with the coenzyme Q structure of the respective yeast strain.The results are discussed with respect to the particular chemical properties of the substrates used and the fact that coenzyme Q structure is considered to be an important marker of evolutionary relationships among yeasts.     

Discovery of rare and highly toxic microcystins from lichen-associated cyanobacterium Nostoc sp. strain IO-102-I

The production of hepatotoxic cyclic heptapeptides, microcystins, is almost exclusively reported from planktonic cyanobacteria. Here we show that a terrestrial cyanobacterium Nostoc sp. strain IO-102-I isolated from a lichen association produces six different microcystins. Microcystins were identified with liquid chromatography-UV mass spectrometry by their retention times, UV spectra, mass fragmentation, and comparison to microcystins from the aquatic Nostoc sp. strain 152. The dominant microcystin produced by Nostoc sp. strain IO-102-I was the highly toxic [ADMAdda(5)]microcystin-LR, which accounted for ca. 80% of the total microcystins. We assigned a structure of [DMAdda(5)]microcystin-LR and [d-Asp(3),ADMAdda(5)]microcystin-LR and a partial structure of three new [ADMAdda(5)]-XR type of microcystin variants. Interestingly, Nostoc spp. strains IO-102-I and 152 synthesized only the rare ADMAdda and DMAdda subfamilies of microcystin variants. Phylogenetic analyses demonstrated congruence between genes involved directly in microcystin biosynthesis and the 16S rRNA and rpoC1 genes of Nostoc sp. strain IO-102-I. Nostoc sp. strain 152 and the Nostoc sp. strain IO-102-I are distantly related, revealing a sporadic distribution of toxin production in the genus Nostoc. Nostoc sp. strain IO-102-I is closely related to Nostoc punctiforme PCC 73102 and other symbiotic Nostoc strains and most likely belongs to this species. Together, this suggests that other terrestrial and aquatic strains of the genus Nostoc may have retained the genes necessary for microcystin biosynthesis.     

Phylogenetic analysis of proteolytic Acinetobacter strains based on the sequence of genes encoding aminoglycoside 6'-N-acetyltransferases

The sequence of seven aac(6')-I genes encoding aminoglycoside 6'-N-acetyltransferases from proteolytic Acinetobacter strains including genomic species 14, 15, 16, and 17 and from ungrouped proteolytic strains 631, 640, and BM2722 was determined. Pulsed-field gel electrophoresis of genomic DNA of these strains and of Acinetobacter sp. 6 CIP A165 digested with SfiI followed by hybridization with rRNA and aac(6')-I specific probes indicated that these genes were located in the chromosome. Phylogenetic analysis of the genes indicated that aac(6')-I of A. baumannii, Acinetobacter ungrouped strain 631, and Acinetobacter sp. 16 formed a cluster (91.5 to 92.3% identity) whereas aac(6')-I of Acinetobacter sp. 15, sp. 17, and Acinetobacter ungrouped strain BM2722 formed another cluster (90.7 to 94.6% identity). A third cluster was constituted by A. haemolyticus and Acinetobacter sp. 6 (83.6% identity). The phylogeny drawn from aac(6')-I sequences was consistent with that based on DNA-DNA hybridization and phenotype comparison. The aac(6')-I genes were all species specific except for aac(6')-Ih located in a 13.7-kb non conjugative plasmid from A. baumannii BM2686. We conclude that aac(6')-I genes may be suitable for identification at the species level and for analysis of the phylogenetic relationships of Acinetobacter.     

Mutation of Candida tropicalis by irradiation with a He-Ne laser to increase its ability to degrade phenol

Candida tropicalis isolated from acclimated activated sludge was used in this study. Cell suspensions with 5 x 10(7) cells ml(-1) were irradiated by using a He-Ne laser. After mutagenesis, the irradiated cell suspension was diluted and plated on yeast extract-peptone-dextrose (YEPD) medium. Plates with approximately 20 individual colonies were selected, and all individual colonies were harvested for phenol biodegradation. The phenol biodegradation stabilities for 70 phenol biodegradation-positive mutants, mutant strains CTM 1 to 70, ranked according to their original phenol biodegradation potentials, were tested continuously during transfers. Finally, mutant strain CTM 2, which degraded 2,600 mg liter(-1) phenol within 70.5 h, was obtained on the basis of its capacity and hereditary stability for phenol biodegradation. The phenol hydroxylase gene sequences were cloned in wild and mutant strains. The results showed that four amino acids were mutated by irradiation with a laser. In order to compare the activity of phenol hydroxylase in wild and mutant strains, their genes were expressed in Escherichia coli BL21(DE3) and enzyme activities were spectrophotometrically determined. It was clear that the activity of phenol hydroxylase was promoted after irradiation with a He-Ne laser. In addition, the cell growth and intrinsic phenol biodegradation kinetics of mutant strain CTM 2 in batch cultures were also described by Haldane's kinetic equation with a wide range of initial phenol concentrations from 0 to 2,600 mg liter(-1). The specific growth and degradation rates further demonstrated that the CTM 2 mutant strain possessed a higher capacity to resist phenol toxicity than wild C. tropicalis did.     

Phenol degradation by yeasts isolated from industrial effluents

Yeast strains of the genera Aureobasidium, Rhodotorula and Trichosporon were isolated from stainless steel effluents and tested for their ability to utilize phenol as the sole carbon source. Fourteen strains grew in the presence of up to 10 mm phenol. Only the strain Trichosporon sp. LE3 was able to grow in the presence of up to 20 mm phenol. An inhibitory effect was observed at concentrations higher than 11 mm, resulting in reduction of specific growth rates. Phenol degradation was a function of strain, time of incubation and initial phenol concentration. All strains exhibited activity of catechol 1,2-dioxygenase and phenol hydroxylase in free cell extracts from cells grown on phenol, suggesting that catechol was oxidized by the ortho type of ring fission. Addition of glucose and benzoate reduced the phenol consumption rate, and both substrates were used simultaneously. Glucose concentrations higher than 0.25% inhibited the induction of phenol oxidation by non-proliferating cells and inhibited phenol oxidation by pre-induced cells.     

Treatment of paper factory effluent using a phenol degrading Alcaligenes sp. under free and immobilized conditions

Treatment of the paper factory effluent was done with free and immobilized cells of a phenol degrading Alcaligenes sp. d(2). The free cells could bring a maximum of 99% reduction in phenol and 40% reduction in chemical oxygen demand (COD) after 32 and 20 h of treatment, respectively. In the case of immobilized cells, a maximum of 99% phenol reduction and 70% COD reduction was attained after 20 h of treatment under batch process. In the continuous mode of operation using packed bed reactor, the strain was able to give 99% phenol removal and 92% COD reduction in 8h of residence time The optimum flow rate was 2.5 ml/h and the half life period was 76 h. Even after the complete removal of phenol, the strain could further enhance reduction in chemical oxygen demand, which clearly indicated that in the paper factory effluent, this strain could also oxidize organic matter other than phenol.     

Biodegradation of phenol by bacterial strains from petroleum-refining wastewater purification plant

The ability of four strains of bacteria derived from a biological petroleum-refining wastewater purification plant to carry out the biodegradation of phenol was studied. Two of the strains belonging to the genus Pseudomonas were found to be characterised by high effectiveness of the removal of phenol which was used as sole carbon and energy source (the strains were designated P1 and P2). In turn the effect of inoculum size, initial concentration of substrate (500 and 1,000 mg phenol/L) and temperature (10, 20 and 30 degrees C) on the rate of phenol degradation by strains P1, P2 and mixture of both was investigated. It was found that strain P1 which was identified as Pseudomonas fluorescens degraded phenol better than strain P2--Pseudomonas cepacia. The rate of phenol biodegradation was significantly affected by size of inoculum and temperature of incubation. Phenol was removed the fastest with the highest inoculum used. The optimal temperature was about 20 degrees C. At 10 and 30 degrees C the process of biodegradation was visibly inhibited. The rate of phenol utilisation was also found to decrease with increased concentration of substrate.     

Phenol utilisation by fungi isolated from activated sludge

The paper presents the efficiency of phenol removal (concentrations from 500 to 2000 mg/l) by fungi isolated from activated sludge purifying wastewater with high phenol concentration. Five fungal strains were isolated and identified. All isolated strains appeared to be Moniliales from the class of Fungi Imperfecti (Candida sp., Monosporium sp., Trichosporon sp.) Stationary cultures of the individual strains and their mixtures were maintained in Czapek medium containing phenol in concentration from 500 to 2000 mg/l. All isolated strains (except one) were capable of utilising phenol up to a concentration of 1500 mg/l. Depending on investigated strain, phenol in concentration of 500 mg/l was decomposed during 4-25 days, 750 mg/l during 4-14 days. After 20 days, a phenol decline of 1000 mg/l was observed. After 16 days, the phenol decline was 1500 mg/l. Higher phenol concentrations (1500 mg/l) were utilised only by a mixture of two strains. The investigated fungal strains showed good efficiency of phenol removal from high phenol concentration in wastewater and they may be proposed for use in the process of purifying wastewater of this type.     

Batch phenol degradation by candida tropicalis and its fusant

Phenol degradation by Candida tropicalis and its fusant, which is produced using protoplast fusion as a selective technique, is evaluated under batch and high concentration conditions. The respirometric data show that oxygen uptake activities of both yeast strains peak at pH 7.0 and 32 degrees C, but the fusant is more active than the control strain. Although the data show that both yeast strains are capable of sustaining discernible degradation in the presence of phenol inhibition, however, the C. tropicalis fusant is capable of attaining better phenol degradation than the control strain and it is less susceptible to phenol inhibition. Under the conditions tested, C. tropicalis is completely inhibited at phenol concentrations >/=3,300 mg/L, whereas for the C. tropicalis fusant complete inhibition is absent until phenol concentrations are >/=4, 000 mg/L. The observed cell yields of both yeast strains are virtually identical and remain fairly constant at approximately 0.5 mg MLVSS/mg C6H5OH (MLVSS: mixed liquor volatile suspended solids). Copyright 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 391-395, 1998.     

Ability of intestinal lactic bacteria to bind or/and metabolise phenol and p-cresol

Intestinal microflora can contribute to colon cancer by the production of substances playing a role in carcinogenesis. Metabolites of protein fermentation in the colon, such as ammonia, H₂S, indole, phenol, skatole are toxic. Lactic bacteria existing in the colon may exert an anti-carcinogenic action, but the mechanism is poorly understood. In the present study the ability of intestin|al lactobacilli to bind or metabolise phenol and p-cresolin vitro was determined.Lactobacillus strains were cultivated in MRS and in a modified MRS broth with reduced concentrations of carbon source. Phenol and p-cresol content in the media were from 2 to 10 μg/ml. In MRS medium lactobacilli could decrease the concentration of phenol and p-cresol and it was 0.2-5.8 μg/ml for phenol and 0.2-1.4 μg/ml for p-cresol. After cultivation in a modified MRS broth, the decrease was 0.5-2.0 μg/ml for phenol and 0.5-2.4 μg/ml for p-cresol. The binding capacity of bacterial cells was rather low. After incubation of non-growing bacteria the decrease of phenol concentration was 0.1-0.5 μg/ml and p-cresol 0.1-2.8 μg/ml. But the ability of growing lactobacilli to metabolise the compounds cannot be excluded. After interaction of lactobacilli with 10 μg/ml of phenol they displayed a lower genotoxicity, as evaluated by the alkaline comet assay. The phenomenon not always depended on the decrease of phenol concentration, but on the medium, the strain of bacteria and for phenol it ranged from 32 to 48%.Lactobacillus strains tested did not lower the genotoxicity of p-cresol.     

Metabolism of Phenol and Cresols by Mutants of Pseudomonas putida

Mutant strains of Pseudomonas putida strain U have been obtained which are deficient in enzymes of the degradative pathways of phenol and cresols. Mutant strains deficient in catechol 2, 3-oxygenase accumulated the appropriate catechol derivative from cresols. A mutant strain which would not grow on either phenol or a cresol was shown to be deficient in both 2-hydroxymuconic semialdehyde hydrolase and a nicotinamide adenine dinucleotide, oxidized form, (NAD(+))-dependent aldehyde dehydrogenase. When this strain was grown in the presence of phenol or a cresol, the appropriate product of meta fission of these compounds accumulated in the growth medium. A partial revertant of this mutant strain, which was able to grow on ortho- and meta-cresol but not para-cresol, was shown to have regained only the hydrolase activity. This strain was used to show that the products of meta ring fission of the cresols and phenol are metabolized as follows: (i) ortho- and meta-cresol exclusively by a hydrolase; (ii) para-cresol exclusively by a NAD(+)-dependent aldehyde dehydrogenase; (iii) phenol by both a NAD(+)-dependent dehydrogenase and a hydrolase in the approximate ratio of 5 to 1. This conclusion is supported by the substrate specificity and enzymatic activity of the hydrolase and NAD(+)-dependent aldehyde dehydrogenase enzymes of the wild-type strain. The results are discussed in terms of the physiological significance of the pathway. Properties of some of the mutant strains isolated are discussed.     

两株高效苯酚降解菌的筛选、鉴定及生物强化-DTRO组合工艺初步验证

【目的】从煤化工废水中分离、筛选苯酚高效降解微生物,初步考察微生物与DTRO技术联用,构建含酚废水生物强化处理工艺的可行性。【方法】采用苯酚浓度梯度培养基对苯酚降解微生物进行分离和筛选;根据菌体形态电子显微镜观察、菌株生理生化特性考察和16S r RNA基因系统发育树构建,对菌株进行初步生物学鉴定;将筛选出的高效苯酚降解菌制备成相应的菌剂与碟管式反渗透(DTRO)技术组合形成"生物强化-DTRO"工艺,并试用于含酚废水的处理。【结果】共获得7株纯化细菌,其中Phe-03和Phe-05为高效苯酚降解菌;该2株菌均可以苯酚为唯一碳源生长。经鉴定Phe-03为壤霉菌属(Agromyces)菌株;Phe-05为棒杆菌属(Corynebacterium)菌株。到目前为止,壤霉菌属(Agromyces)菌株降解苯酚尚未见报道。在初始苯酚浓度达到1 300 mg/L条件下,Phe-03和Phe-05菌株44 h内对苯酚降解率均达到70%以上;76 h后苯酚降解率均超过90%。组合形成的"生物强化-DTRO"工艺不仅可以有效去除废水中的酚类化合物,而且还能减少反渗透膜污染,以及增加膜的通透性。【结论】研究表明微生物技术可与DTRO技术联用,构建含酚废水生物强化处理工艺,可为含酚废水处理技术研究提供一种选择思路。     

Mutation of Candida tropicalis by Irradiation with a He-Ne Laser To Increase Its Ability To Degrade Phenol

Candida tropicalis isolated from acclimated activated sludge was used in this study. Cell suspensions with 5 × 10⁷ cells ml⁻¹ were irradiated by using a He-Ne laser. After mutagenesis, the irradiated cell suspension was diluted and plated on yeast extract-peptone-dextrose (YEPD) medium. Plates with approximately 20 individual colonies were selected, and all individual colonies were harvested for phenol biodegradation. The phenol biodegradation stabilities for 70 phenol biodegradation-positive mutants, mutant strains CTM 1 to 70, ranked according to their original phenol biodegradation potentials, were tested continuously during transfers. Finally, mutant strain CTM 2, which degraded 2,600 mg liter⁻¹ phenol within 70.5 h, was obtained on the basis of its capacity and hereditary stability for phenol biodegradation. The phenol hydroxylase gene sequences were cloned in wild and mutant strains. The results showed that four amino acids were mutated by irradiation with a laser. In order to compare the activity of phenol hydroxylase in wild and mutant strains, their genes were expressed in Escherichia coli BL21(DE3) and enzyme activities were spectrophotometrically determined. It was clear that the activity of phenol hydroxylase was promoted after irradiation with a He-Ne laser. In addition, the cell growth and intrinsic phenol biodegradation kinetics of mutant strain CTM 2 in batch cultures were also described by Haldane's kinetic equation with a wide range of initial phenol concentrations from 0 to 2,600 mg liter⁻¹. The specific growth and degradation rates further demonstrated that the CTM 2 mutant strain possessed a higher capacity to resist phenol toxicity than wild C. tropicalis did.     

Phenol degradation by immobilized cold-adapted yeast strains of Cryptococcus terreus and Rhodotorula creatinivora

Three strains were isolated from hydrocarbon-polluted alpine habitats and were representatives of Cryptococcus terreus (strain PB4) and Rhodotorula creatinivora (strains PB7, PB12). All three strains synthesized and accumulated glycogen (both acid- and alkali-soluble) and trehalose during growth in complex medium containing glucose as carbon source and in minimal salt medium (MSM) with phenol as sole carbon and energy source. C. terreus strain PB4 showed a lower total accumulation level of storage compounds and a lower extracellular polysaccharides (EPS) production than the two R. creatinivora strains, PB7 and PB12. Biofilm formation and phenol degradation by yeast strains attached to solid carriers of zeolite or filter sand were studied at 10°C. Phenol degradation by immobilized yeast strains was always higher on zeolite compared with filter sand under normal osmotic growth conditions. The transfer of cells immobilized on both solid supports to a high osmotic environment decreased phenol degradation activity by all strains. However, both R. creatinivora PB7 and PB12 strains maintained higher ability to degrade phenol compared with C. terreus strain PB4, which almost completely lost its phenol degradation activity. Moreover, R. creatinivora strain PB7 showed the highest ability to form biofilm on both carriers under high osmotic conditions of cultivation.     

Isolation and characterization of phenol-degrading yeasts from an oil refinery wastewater in Brazil

This study investigated the aerobic degradation of phenol by yeast strains isolated from an oil refinery wastewater from the Northeast of Brazil. The samples displayed low fungal diversity, as only yeast colonies were detected on Sabouraud dextrose agar containing chloramphenicol 0.05% (w/v). Among the isolates, three yeast strains were selected to be evaluated for their potential for degrading high phenol concentrations. These species were identified through morphological and biochemical characteristics as Candida tropicalis, C. rugosa, and Pichia membranaefaciens. Although the strains were able to degrade the phenol concentration present in the wastewater, which was 7 mg l⁻¹, only C. tropicalis was capable of growing at high concentrations of phenol such as 500 mg l⁻¹ and 1,000 mg l⁻¹ in a mineral medium containing this pollutant as the only carbon source. C. rugosa and P. membranaefaciens were inhibited in the presence of 500 mg l⁻¹ of phenol. However, a longer incubation time was needed for C. tropicalis strain to degrade 1,000 mg l⁻¹ of phenol compared to the time required to degrade 500 mg l⁻¹. Moreover, the strain released a significant amount of polysaccharide biosurfactant in the medium probably to minimize the toxic effect of the high phenol concentration. When challenged with 1,500 and 2,000 mg l⁻¹ of phenol, C. tropicalis was unable to grow at the tested conditions. The results indicate that this strain of C. tropicalis can be considered both a good phenol-degrader and biosurfactant-producer. Application of this strain might be useful in bioremediation activities or treatment of phenol-polluted wastewater.     

The Effects of Cyanobacterial Exudates on Bacterial Growth and Biodegradation of Organic Contaminants

The pulp and paper industry largely depends on the biodegradation activities of heterotrophic bacteria to remove organic contaminants in wastewater prior to discharge. Our recent discovery of extensive cyanobacterial communities in pulp and paper waste treatment systems led us to investigate the potential impacts of cyanobacterial exudates on growth and biodegradation efficiency of three bacterial heterotrophs. Each of the three assessed bacteria represented different taxa commonly found in pulp and paper waste treatment systems: a fluorescent Pseudomonad, an Ancylobacter aquaticus strain, and a Ralstonia eutropha strain. They were capable of utilizing phenol, dichloroacetate (DCA), or 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. Exudates from all 12 cyanobacterial strains studied supported the growth of each bacterial strain to varying degrees. Maximum biomass of two bacterial strains positively correlated with the total organic carbon content of exudate treatments. The combined availability of exudate and a known growth substrate (i.e., phenol, DCA, or 2,4-D) generally had a synergistic affect on the growth of the Ancylobacter strain, whereas mixed effects were seen on the other two strains. Exudates from four representative cyanobacterial strains were assessed for their impacts on phenol and DCA biodegradation by the Pseudomonas and Ancylobacter strains, respectively. Exudates from three of the four cyanobacterial taxa repressed phenol biodegradation, but enhanced DCA biodegradation. These dissimilar impacts of cyanobacterial exudates on bacterial degradation of contaminants suggest a species-specific association, as well as a significant role for cyanobacteria during the biological treatment of wastewaters.     

Growth of Pure and Mixed Cultures of Micro-organisms Concerned in the Treatment of Carbonization Waste Liquors

S ummary : Three strains of bacteria responsible for the destruction of the major constituents of carbonization waste liquor were isolated from a laboratory scale, activated sludge plant successfully treating such a liquor. Of the 3 strains one was able to grow on thiocyanate; the other 2 strains grew well on phenol. Behaviour of these organisms in pure and mixed culture showed marked differences: in pure culture, growth of the thiocyanate-degrading strain was unaffected by the presence of 100 mg of phenol/l, but in mixed culture, active growth of another organism on the phenol completely inhibited growth on the thiocyanate. Batch and continuous culture experiments were made with 2 organisms competing for phenol. Both stimulation and inhibition of growth were found, dependent on the ratio between the concentrations of organisms present.     

Specific ratio and survival of Pseudomonas CF600 as co-culture for phenol degradation in continuous cultivation

Studies were carried out to understand parallel survival of two strains when cultivated as co-culture on a single carbon source in continuous cultivation. Strains used were Pseudomonas sp. strain CF600 that is reported for degradation of phenol; and HKR1 a lab strain, which was isolated from a site contaminated with phenol. In continuous cultivation Pseudomonas sp. CF600 showed an accumulation of colored intermediate, 2-hydroxy muconic semialdehyde (HMS), when fed with phenol as a sole source of carbon under dissolved oxygen limiting condition (40% saturation level). Under the same cultivation condition when it was co-cultured with strain HKR1, complete degradation of phenol was observed with no accumulation of intermediate. Different dilution rates (0.03, 0.15, and 0.30) were set in the bioreactor during cultivation. It was also observed that both the strains follow a typical cell density ratio of 1:18 as strain HKR1: Pseudomonas sp. CF600 irrespective of the dilution rates used in the study to favor degradation of phenol. Pseudomonas sp. CF600 is reported to degrade phenol via a plasmid-encoded pathway (pVI150). The enzymes for this meta-cleavage pathway are clustered on 15 genes encoded by a single operon, the dmp operon. PCR using primers from the different catabolic loci of dmp operon, demonstrated that the strain HKR1 follows a different metabolic pathway for intermediate utilization.