Degradation of phenanthrene on plant leaves by phyllosphere bacteria

The activity of phyllosphere bacteria in the degradation of phenanthrene was investigated as a mechanism for the removal of atmospheric phenanthrene after its deposition on plant leaves. Initially, leaf samples of six plant species were collected from two roadsides in Bangkok to determine the presence of phenanthrene-degrading bacteria. The numbers of phenanthrene-degrading phyllosphere bacteria were varied and ranged from 3.5 x 10(4) to 1.95 x 10(7) CFU/g, in which the highest number was found from Ixora sp. Further studies were carried out in the laboratory by spraying phenanthrene on Ixora sp. leaves and then monitoring the amount of deposited phenanthrene and number of phenanthrene-degrading bacteria after incubation. The results showed that the amount of phenanthrene was significantly reduced on leaves containing phenanthrene-degrading bacteria. These were detected along with a rapid increase in the number of bacteria on leaves. The results indicated that many phyllosphere bacteria could utilize phenanthrene to support their growth and thereby reduce the amount of deposited phenanthrene on leaf surfaces. Several phenanthrene-degrading bacteria were later isolated from the leaves and identified with a high 16S rDNA sequence similarity to the genera Pseudomonas, Microbacterium, Rhizobium, and Deinococcus.     

Isolation and Description of a Stable Carbazole-Degrading Microbial Consortium Consisting of <Emphasis Type="Italic">Chryseobacterium</Emphasis> sp. NCY and <Emphasis Type="Italic">Achromobacter</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> NCW

A stable microbial consortium, separated from a refinery wastewater sample, was able to utilize carbazole as the sole source of carbon, nitrogen, and energy, and liberated ammonia from excess nitrogen. Two bacterial strains (NCY and NCW) were isolated from the microbial consortium using a nutrient agar plate. Based on the 16S rDNA sequence analysis, the two bacteria were identified as Chryseobacterium sp. NCY and Achromobacter sp. NCW, respectively. No intermediates of carbazole degradation were detected by high-performance liquid chromatography. The substrate specificity assay showed that the consortium could utilize compounds similar to carbazole, such as phenanthrene, naphthalene, and imidazole. Neither the pure strain NCY nor NCW could degrade carbazole after domestication for several times. It was suggested that the two bacteria formed a microbial consortium capable of metabolizing carbazole.     

Phenanthrene degradation by estuarine surface microlayer and bulk water microbial populations

Paired surface microlayer and bulk water samples from five sites in the Great Bay Estuary, New Hampshire, were examined with regard to numbers of bacteria,¹⁴C-phenanthrene biodegradation potentials, and organic and inorganic chemical characteristics. Microlayer samples were generally enriched in nutrients (N and P), dissolved organic matter, and culturable heterotrophic bacteria compared with their corresponding bulk waters. Microlayer samples from marina environments were also enriched in aromatic hydrocarbons, as determined by UV spectrophotometric and fluorometric analyses, and demonstrated substantial phenanthrene biodegradation activity in the assay employed. Biodegradation activity of marina bulk water samples ranged from nil to levels exceeding those exhibited by microlayer samples. No diminution of biodegradation activity was observed after filtration (1.2 m effective retention) of microlayer water, indicating that the responsible organisms were not particle-associated. Phenanthrene-degrading bacteria, enumerated by counting clearing zones in a crystalline phenanthrene overlay after colony development on a phenanthrene/toluene agar (PTA) medium, were superior to epifluorescence direct counts or standard plate counts on PTA or estuarine nutrient agar in predicting¹⁴C-phenanthrene biodegradative activity.     

Bioavailability of solid and non-aqueous phase liquid (NAPL)-dissolved phenanthrene to the biosurfactant-producing bacterium Pseudomonas aeruginosa 19SJ

The biodegradation of phenanthrene by the biosurfactant-producing strain Pseudomonas aeruginosa 19SJ was investigated in experiments with the compound present either as crystals or dissolved in non-aqueous phase liquids (NAPLs). Growth on solid phenanthrene exhibited an initial phase not limited by dissolution rate and a subsequent, carbon-limited phase caused by exhaustion of the carbon source. Rhamnolipid biosurfactants were produced from solid phenanthrene and appeared in solution and particulate material (cells and phenanthrene crystals). During the carbon-limited phase, the concentration of rhamnolipids detected in culture exceeded the critical micelle concentration (CMC) determined with purified rhamnolipids. The biosurfactants caused a significant increase in dissolution rate and pseudosolubility of phenanthrene, but only at concentrations above the CMC. Externally added rhamnolipids at a concentration higher than the CMC increased the biodegradation rate of solid phenanthrene. Mineralization curves of low concentrations of phenanthrene initially dissolved in two NAPLs [2,2,4,4,6,8,8-heptamethylnonane and di(2-ethylhexyl)phthalate] were S-shaped, although no growth was observed in the population of suspended bacteria. Biosurfactants were not detected in solution under these conditions. The observed mineralization was attributed not only to suspended bacteria, but also to bacterial populations growing at the NAPL–water interface, mineralizing the compound at higher rates than predicted by abiotic partitioning. We suggest that rhamnolipid production and attachment increased the bioavailability of phenanthrene, so promoting biodegradation activity.     

Yields of Bacterial Cells from Hydrocarbons

A strain of Nocardia and one of Pseudomonas, both isolated on pristane (2,6,10,14-tetramethylpentadecane), gave cell yields of approximately 100% on n-octadecane and pristane. Both organisms grew more rapidly on the n-octadecane than on the pristane. A mixed culture, isolated on 3-methylheptane, whose two components were identified as species of Pseudomonas and of Nocardia, gave approximately 100% cell yields and grew with generation times of about 5 hr on n-heptane, n-octane, and 2-methylheptane. The generation time on 3-methylheptane was 8.6 hr and the cell yield was only 79%. A strain of Pseudomonas isolated from naphthalene enrichments and one from phenanthrene enrichments both gave a cell yield of 50% on naphthalene. The phenanthrene isolate gave a cell yield of 40% on phenanthrene. A Nocardia species isolated on benzene gave a 79% cell yield on benzene. The generation times of the bacteria isolated on aromatic hydrocarbons were related to the solubility of the aromatic hydrocarbons on which they were grown; the more insoluble hydrocarbons gave slower growth.     

Physical State in Which Naphthalene and Bibenzyl are Utilized by Bacteria

The generation times of a strain of Pseudomonas grown on a mineral salts medium in the presence of various amounts of naphthalene did not vary with the amount of solid present, and these generation times were the same as the generation time on mineral salts medium containing only dissolved naphthalene. The generation time of a soil isolate grown on mineral salts medium in the presence of 0.5 g of solid bibenzyl per liter was the same as the generation time on a mineral salts medium saturated with bibenzyl. The evidence indicates that naphthalene and bibenzyl are utilized in the dissolved state.     

Physiologo-biochemical properties of a strain of Beijerinckia mobilis 1phi Phn+--a degrader of polycyclic aromatic hydrocarbons

Beijerinckia mobilis 1f capable of degrading polycyclic aromatic hydrocarbons (PAHs) was isolated from a soil contaminated with creosote. Strain 1f could utilize phenanthrene and naphthalene as the sole sources of carbon. The mean rate of phenanthrene degradation during culture growth was 7-8 micrograms/(ml h). After cultivation under nonselective conditions, strain 1f retained its ability to degrade phenanthrene. Cometabolism considerably widened the range of PAHs that could be transformed by strain 1f. The strain was able to grow in a mineral medium with creosote as the sole source of carbon. After 30 days of cultivation in this medium, the total concentration of PAHs decreased from 665.5 mg/l to 170 mg/l.     

Numerical taxonomy of phenanthrene-degrading bacteria isolated from the Chesapeake Bay

Phenanthrene-degrading bacteria were isolated from Chesapeake Bay samples by the use of a solid medium which had been overlaid with an ethanol solution of phenanthrene before inoculation. Eighteen representative strains of phenanthrene-degrading bacteria with 21 type and reference bacteria were examined for 123 characteristics representing physiological, biochemical, and nutritional properties. Relationships between strains were computed with several similarity coefficients. The phenogram constructed by unweighted-pair-group arithmetic average linkage and use of the simple Jaccard (SJ) coefficient was used to identify seven phena. Phenanthrene-degrading bacteria were identified as Vibrio parahaemolyticus and Vibrio fluvialis by their clustering with type and reference strains. Several phenanthrene-degrading bacteria resembled Enterobacteriaceae family members, although some Vibrio-like phenanthrene degraders could not be identified.     

Numerical taxonomy of phenanthrene-degrading bacteria isolated from the Chesapeake Bay.

Phenanthrene-degrading bacteria were isolated from Chesapeake Bay samples by the use of a solid medium which had been overlaid with an ethanol solution of phenanthrene before inoculation. Eighteen representative strains of phenanthrene-degrading bacteria with 21 type and reference bacteria were examined for 123 characteristics representing physiological, biochemical, and nutritional properties. Relationships between strains were computed with several similarity coefficients. The phenogram constructed by unweighted-pair-group arithmetic average linkage and use of the simple Jaccard (SJ) coefficient was used to identify seven phena. Phenanthrene-degrading bacteria were identified as Vibrio parahaemolyticus and Vibrio fluvialis by their clustering with type and reference strains. Several phenanthrene-degrading bacteria resembled Enterobacteriaceae family members, although some Vibrio-like phenanthrene degraders could not be identified.     

Effect of a commercial alcohol ethoxylate surfactant (C<Subscript>11-15</Subscript>E<Subscript>7</Subscript>) on

Biodegradation of poorly soluble polycyclic aromatic hydrocarbons (PAHs) has been a challenge in bioremediation. In recent years, surfactant-enhanced bioremediation of PAH contaminants has attracted great attention in research. In this study, biodegradation of phenanthrene as a model PAHs solubilized in saline micellar solutions of a biodegradable commercial alcohol ethoxylate nonionic surfactant was investigated. The critical micelle concentration (CMC) of the surfactant and its solubilization capacity for phenanthrene were examined in an artificial saline water medium, and a type of marine bacteria, Neptunomonas naphthovorans, was studied for the biodegradation of phenanthrene solubilized in the surfactant micellar solutions of the saline medium. It is found that the solubility of phenanthrene in the surfactant micellar solutions increased linearly with the surfactant concentrations, but, at a fixed phenanthrene concentration, the biodegradability of phenanthrene in the micellar solutions decreased with the increase of the surfactant concentrations. This was attributed to the reduced bioavailability of phenanthrene, due to its increased solubilization extent in the micellar phase and possibly lowered mass transfer rate from the micellar phase into the aqueous phase or into the bacterial cells. In addition, an inhibitory effect of the surfactant on the bacterial growth at high surfactant concentrations may also play a role. It is concluded that the surfactant largely enhanced the solubilization of phenanthrene in the saline water medium, but excess existence of the surfactant in the medium should be minimized or avoided for the biodegradation of phenanthrene by Neptunomonas naphthovorans.     

Enhanced biodegradation of phenanthrene in a biphasic culture system

Degradation of phenanthrene byPseudomonas aeruginosa AK1 was examined in (i) an aqueous mineral salts medium to which phenanthrene particles of varying size (i.e. diameter) were added, and (ii) an aqueous/organic biphasic culture system consisting of mineral salts medium supplemented with 2,2,4,4,6,8,8-heptamethylnonane (HMN) as the phenanthrene-carrying organic phase. In both systems, the rate of phenanthrene biodegradation could be significantly enhanced by manipulations leading to improved phenanthrene mass transfer into the aqueous phase. With crystalline phenanthrene, the rate of biodegradation was found to be directly correlated to the particle surface area, whereas in the biphasic system the rate of biodegradation of the dissolved phenanthrene was mainly governed by the HMN/water interface area. In the latter system, exponential growth with a doubling time t _d of 6–8 hours has been achieved under conditions of intensive agitation of the medium indicating that phenanthrene degradation by strain AK1 is limited mainly by physicochemical parameters. Addition of selected surfactants to the culture medium was found to accelerate phenanthrene degradation by strain AK1 only under conditions of low agitation (in the presence of HMN) and after pretreatment of phenanthrene crystals by ultrasonication (in the absence of HMN). Evidence is presented that the stimulating effect of the surfactants was primarily due to improved dispersion of phenanthrene particle agglomerates (in the aqueous mineral salts medium supplemented with phenanthrene crystals) or of the phenanthrene-carrying lipophilic solvent drops (in the aqueous/organic biphasic culture system) whereas the solubilizing activity towards phenanthrene was neglectible. Under conditions of intensive mixing of the culture medium (i.e. if a high particle surface area or HMN/water interface area, respectively, is provided), the addition of surfactants did not enhance phenanthrene biodegradation.     

Hydrocarbon utilization by nodule bacteria and plant growth-promoting rhizobacteria

Standard and locally isolated nodule bacteria and plant growth-promoting rhizobacteria (PGPR) were grown on crude oil and individual pure hydrocarbons as sole sources of carbon and energy. The nodule bacteria included two standard Rhizobium leguminosarum strains, two standard Bradyrhizobium japonicum strains, and one unknown nodule bacterial strain that was locally isolated from Vicia faba nodules. The PGPR included one standard Serratia liquefaciens strain and two locally isolated strains of Pseudomonas aeruginosa and Flavobacterium sp. The pure hydrocarbons tested included n-alkanes with chain lengths from C9 to C40 and the aromatic hydrocarbons benzene, biphenyle, naphthalene, phenanthrene, and toluene. Quantitative gas liquid chromatographic analyses confirmed that pure cultures of representative nodule bacteria and PGPR could attenuate n-octadecane and phenanthrene in the surrounding nutrient medium. Further, intact nodules of V. faba containing bacteria immobilized on and within those nodules reduced hydrocarbon levels in a medium in which those nodules were shaken. It was concluded that legume crops are suitable phytoremediation tools for oily soil, since they enrich such soils not only with fixed nitrogen, but also with hydrocarbon-utilizing microorganisms. Further, legume nodules may have biotechnological value as materials for cleaning oily liquid wastes.     

Utilization of gas oil by a yeast culture

A mixed yeast culture (Culture 4) was grown on representative gas oil samples as well as paraffin wax. Culture 4 was found to utilize n-paraffinic hydrocarbons almost quantitatively from most gas oil fractions; significant alteration of other hydrocarbon components was not detected. Generation times of 4.0–9.0hr. were typical during the exponential growth phase in fermentations with various gas oil fractions. Cell yields were 70–90% based on n-paraffin utilization. The culture appeared to exhibit maximum efficiency of n-alkane removal in the C₁₉ to C₂₄ range. The cells recovered from the fermentations contained 8.8–9.3% nitrogen. Paraffin wax also served as a suitable carbon source when dissolved in 2,6,10,14-tertramethylpentadecane (pristane). However, substrate utilization appeared to be incomplete.     

Biosurfactant Production by a Soil Pseudomonas Strain Growing on Polycyclic Aromatic Hydrocarbons

The capacity of polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria to produce biosurfactants was investigated. Twenty-three bacteria isolated from a soil contaminated with petroleum wastes were able to form clearing zones on mineral salt agar plates sprayed with solutions of PAHs. Naphthalene and phenanthrene were utilized as sole substrates. Biosurfactant production was detected by surface tension lowering and emulsifying activities from 10 of these strains grown in an iron-limited salt medium supplemented with high concentrations of dextrose or mannitol, as well as with naphthalene or phenanthrene. Glycolipid determinations showed that in cultures of Pseudomonas aeruginosa 19SJ on naphthalene, the maximal productivity of biosurfactants was delayed compared with that in cultures grown on mannitol. However, when small amounts of biosurfactants and naphthalene degradation intermediates were present at the onset of the cultivation, the delay was markedly shortened. Production of biosurfactants was accompanied by an increase in the aqueous concentration of naphthalene, indicating that the microorganism was promoting the solubility of its substrate. Detectable amounts of glycolipids were also produced on phenanthrene. This is the first report of biosurfactant production resulting from PAH metabolism.     

Biodegradation of Phenanthrene by the Green Alga Scenedesmus obliquus ES‐55

While the degradation of polycyclic aromatic hydrocarbons by bacteria and fungi has been broadly investigated, less is known about the metabolism of these compounds by algae. The goal of the experiments was to test the degradability of phenanthrene by the green alga Scenedesmus obliquus ES‐55 (Chlorophyceae) and to identify the metabolites. It was shown that S. obliquus ES‐55 metabolized phenanthrene. Under light conditions, phenanthrene (14 mg/L) inhibits cell division by more than twice. However, the metabolic processes in the cells affected by phenanthrene continued because the content of chlorophyll increased. In the exponential phase under phototrophic conditions the alga degraded phenanthrene. Phenanthrene was removed by algae up to 42 % in BBM medium and up to 24 % in Kuhl medium. Dihydroxy‐dihydro‐phenanthrene, a degradation metabolite in fungi, bacteria and cyanobacteria, could also be detected as a transformation product of S. obliquus ES‐55. Further detected common metabolites foster the assumption that both phototrophic and non‐photothrophic organisms metabolize phenanthrene via a similar pathway. The present study is the first evidence of the ability of an axenic culture of the green alga S. obliquus to biotransform phenanthrene into other metabolites.     

Role of dissolution rate and solubility in biodegradation of aromatic compounds

Strains of Moraxella sp., Pseudomonas sp., and Flavobacterium sp. able to grow on biphenyl were isolated from sewage. The bacteria produced 2.3 to 4.5 g of protein per mol of biphenyl carbon, and similar protein yields were obtained when the isolates were grown on succinate. Mineralization of biphenyl was exponential during the phase of exponential growth of Moraxella sp. and Pseudomonas sp. In biphenyl-supplemented media, Flavobacterium sp. had one exponential phase of growth apparently at the expense of contaminating dissolved carbon in the solution and a second exponential phase during which it mineralized the hydrocarbon. Phase-contrast microscopy did not show significant numbers of cells of these three species on the surface of the solid substrate as it underwent decomposition. Pseudomonas sp. did not form products that affected the solubility of biphenyl, although its excretions did increase the dissolution rate. It was calculated that Pseudomonas sp. consumed 29 nmol of biphenyl per ml in the 1 h after the end of the exponential phase of growth, but 32 nmol of substrate per ml went into solution in that period when the growth rate had declined. In a medium with anthracene as the sole added carbon source, Flavobacterium sp. converted 90% of the substrate to water-soluble products, and a slow mineralization was detected when the cell numbers were not increasing. Flavobacterium sp. and Beijerinckia sp. initially grew exponentially and then arithmetically in media with phenanthrene as the sole carbon source. Calculations based on the growth rates of these bacteria and the rates of dissolution of phenanthrene suggest that the dissolution rate of the hydrocarbon may limit the rate of its biodegradation.     

Role of dissolution rate and solubility in biodegradation of aromatic compounds.

Strains of Moraxella sp., Pseudomonas sp., and Flavobacterium sp. able to grow on biphenyl were isolated from sewage. The bacteria produced 2.3 to 4.5 g of protein per mol of biphenyl carbon, and similar protein yields were obtained when the isolates were grown on succinate. Mineralization of biphenyl was exponential during the phase of exponential growth of Moraxella sp. and Pseudomonas sp. In biphenyl-supplemented media, Flavobacterium sp. had one exponential phase of growth apparently at the expense of contaminating dissolved carbon in the solution and a second exponential phase during which it mineralized the hydrocarbon. Phase-contrast microscopy did not show significant numbers of cells of these three species on the surface of the solid substrate as it underwent decomposition. Pseudomonas sp. did not form products that affected the solubility of biphenyl, although its excretions did increase the dissolution rate. It was calculated that Pseudomonas sp. consumed 29 nmol of biphenyl per ml in the 1 h after the end of the exponential phase of growth, but 32 nmol of substrate per ml went into solution in that period when the growth rate had declined. In a medium with anthracene as the sole added carbon source, Flavobacterium sp. converted 90% of the substrate to water-soluble products, and a slow mineralization was detected when the cell numbers were not increasing. Flavobacterium sp. and Beijerinckia sp. initially grew exponentially and then arithmetically in media with phenanthrene as the sole carbon source. Calculations based on the growth rates of these bacteria and the rates of dissolution of phenanthrene suggest that the dissolution rate of the hydrocarbon may limit the rate of its biodegradation.     

Methanotrophic Bacteria and Facilitated Transport of Pollutants in Aquifer Material

In situ stimulation of methanotrophic bacteria has been considered as a methodology for aquifer remediation. Chlorinated aliphatic hydrocarbons such as trichloroethylene are fortuitously oxidized by the methane monooxygenase produced by methanotrophic bacteria. Experimental results are presented that indicate that both colloidal suspensions containing methanotrophic cells and the soluble extracellular polymers produced by methanotrophic cells have the potential to enhance the transport and removal of other environmental contaminants such as polynuclear aromatic hydrocarbons and transition metals in aquifer material. Three well-characterized methanotrophic bacteria were used in the experiments: Methylomonas albus BG8 (a type I methanotroph), Methylosinus trichosporium OB3b (a type II methanotroph), and Methylocystis parvus OBBP (a type II methanotroph). Isotherms were obtained for sorption of two radiolabeled pollutants, [¹⁴C] phenanthrene and ¹⁰⁹Cd, onto an aquifer sand in the presence and absence of washed cells and their extracellular polymer. Column transport experiments were performed with the washed methanotrophic cells and phenanthrene. The distribution coefficients for Cd with extracellular polymers were of the same order as that obtained with the aquifer sand, indicating that polymers from the methanotrophic bacteria could act to increase the transport of Cd in a porous medium. Polymer from BG8 significantly reduced the apparent distribution coefficient for Cd with an aquifer sand. [¹⁴C] phenanthrene also sorbed to extracellular polymer and to washed, suspended methanotrophic cells. The exopolymer of BG8 and OBBP significantly reduced the apparent distribution coefficient (K_d) for phenanthrene with aquifer sand. The distribution coefficients for phenanthrene with the methanotrophic cells were an order of magnitude greater than those previously reported for other heterotrophic bacteria. Cells of the methanotrophs also significantly reduced the apparent K_d for phenanthrene with an aquifer sand. The three strains of methanotrophs tested displayed mobility in a column of packed sand, and strain OBBP reduced the retardation coefficient of phenanthrene with an aquifer sand by 27%. These data indicate that both extracellular polymer and mobile cells of methanotrophic bacteria display a capacity to facilitate the mobility of pollutant metals and polynuclear aromatic hydrocarbons in aquifer material.     

Degradation of phenanthrene, fluorene, fluoranthene, and pyrene by a Mycobacterium sp.

Mycobacterium sp. strain BB1 was isolated from a former coal gasification site. It was able to utilize phenanthrene, pyrene, and fluoranthene as sole sources of carbon and energy and to degrade fluorene cometabolically. Exponential growth with solid phenanthrene, pyrene, and fluoranthene was obtained in fermentor cultures. The growth rates were 0.069, 0.056, and 0.040 h-1, respectively. Several metabolites of phenanthrene and fluorene metabolism were identified.     

Some Growth Characteristics of a Hyphomicrobium sp. in Batch Culture

S ummary : A Hyphomicrobium sp. isolated from a mixed culture of methane utilizing micro-organisms grew rapidly with generation times as short as 4·7 h in pure culture with methanol as the carbon substrate. The stoichiometry of oxygen utilization by this micro-organism, as indicated by the cell yield with respect to oxygen varied at low levels of dissolved oxygen in the culture medium. In addition, the initial methanol concentration in the medium affected the maximum specific growth rate of the Hyphomicrobium sp.