Combined ozonation and biodegradation for remediationof mixtures of polycyclic aromatic hydrocarbons in soil

A study was conducted to investigate the feasibilityof a combined treatment (i.e., ozonation andbiodegradation) to overcome the inherent bacterialbioavailability limitation, and hence bioremediationlimitation, of polycyclic aromatic hydrocarbons insoil. Ozonation was very efficient in the removal ofnaphthalene, fluorene, phenanthrene, and anthracene,but not for pyrene, chrysene, and benzo(a)pyrene fromsoil freshly spiked with the hydrocarbons. A similarresult was obtained from coal tar-contaminated soil.Elimination of polycyclic aromatic hydrocarbonsincreased appreciably in sand containing 0.03%organic carbon, indicating the adverse effect oforganic carbon on the efficiency of ozone treatment.In spiked and coal tar-contaminated soils, ozonationfollowed by biodegradation significantly increased thedegradation of various polycyclic aromatichydrocarbons including chrysene and benzo(a)pyrenewhich were not degraded by the test bacterialconsortium alone. In particular, the effect of thecombined treatment was more pronounced in coaltar-contaminated soil than in sterile soil spiked withhydrocarbons, probably due to the augmented biologicalactivity of the introduced consortium. The resultssuggest that a combined treatment including ozonationand biodegradation may be a promising bioremediationtechnology in soil contaminated with mixtures ofpolycyclic aromatic hydrocarbons such as formermanufactured gas plant sites.     

Kinetic studies of biodegradation of insoluble compounds by continuous determination of oxygen consumption

Continuous determination of oxygen consumption by electrolytic respirometry has been experimented as a means to study the biodegradation kinetics of scarcely soluble environmental pollutants. The substrates used were the polycyclic aromatic hydrocarbons (PAH), naphthalene, phenanthrene, anthracene, fluoranthene and pyrene. The definition of an appropriate mode of PAH supply, either as crystals or more generally as a solution in a water non-miscible solvent, was found essential for yielding reproducible biodegradation kinetics. In these conditions, for all compounds tested, oxygen determination was found suitable for quantitative evaluation of PAH biodegradation and formation of biomass and soluble metabolites. The study of biodegradation kinetics with this methodology showed that a first phase of exponential growth could be characterized in most cases, followed by a phase of limited growth. Possible mechanisms involved in insoluble substrate uptake are discussed. During exponential growth, the bacteria utilized (although not necessarily exclusively) the PAH solubilized in the aqueous medium.     

海洋石油降解微生物及其降解机理

微生物修复作为一种新型环保的生物修复技术,已成为海洋石油污染生物修复的核心技术。对海洋石油降解微生物的种类即细菌、蓝藻、真菌以及藻类进行了总结,对微生物对石油烃的降解途径与降解机理进行了综述。微生物降解烷烃的过程包括末端氧化、烷基氢过氧化物以及环己烷降解3种形式。微生物对芳香烃的降解是通过芳香烃被氧化酶氧化导致苯环开环来实现的。微生物对多环芳烃的降解是在单加氧酶或双加氧酶的催化作用下被最终降解为二氧化碳和水而被分解。并对影响石油烃降解微生物的因素包括温度、营养物质等因素进行了分析。     

The microbiological fate of polycyclic aromatic hydrocarbons: carbon and oxygen balances for bacterial degradation of model compounds

A series of pure bacterial strains belonging mainly to theRhodococcus andPseudomonas genera were grown on one of the following polycyclic aromatic hydrocarbons (PAH) supplied as sole carbon and energy source: naphthalene, fluorene, phenanthrene, anthracene, fluoranthene and pyrene. In each case, a quantitative evaluation of the carbon repartition of the PAH degraded into CO₂, biomass and water-soluble metabolites was carried out. In addition, the kinetics of oxygen consumption and of water-soluble metabolite accumulation during PAH biodegradation was followed with respirometric equipment. Satisfactory carbon balances were obtained and the data correlated well with oxygen consumption values. The results show that growth on PAH presents high mineralization yields (from 56% to 77% of carbon) and sizeable production of biomass (from 16% to 35% of carbon) and limited but significant accumulation of metabolites (from 5% to 23% of carbon). The mineralization yields were higher and biomass yields lower in the case of higher PAH. Some differences between strains were also observed.     

Degradation of polycyclic aromatic hydrocarbons by pure strains and by defined strain associations: inhibition phenomena and cometabolism

Six bacterial strains capable of using, as sole carbon and energy source, at least one of the following polycyclic aromatic hydrocarbons (PAH), naphthalene, fluorene, phenanthrene, anthracene, fluoranthene and pyrene, were isolated. The interactions between these PAH during their biodegradation were studied in experiments involving PAH pairs, one PAH at least being used as a carbon source. All individual strains were found capable of cometabolic degradation of PAH in a range varying among strains. Inhibition phenomena, sometimes drastic, were often observed but synergistic interactions were also detected. Naphthalene was toxic to all strains not isolated on this compound. Strain associations were found efficient in relieving inhibition phenomena, including the toxic effect of naphthalene. Accumulation of water-soluble metabolites was consistently observed during PAH degradation.     

Degradation of polycyclic aromatic hydrocarbons in the presence of synthetic surfactants.

The biodegradation of polycyclic aromatic hydrocarbons (PAH) often is limited by low water solubility and dissolution rate. Nonionic surfactants and sodium dodecyl sulfate increased the concentration of PAH in the water phase because of solubilization. The degradation of PAH was inhibited by sodium dodecyl sulfate because this surfactant was preferred as a growth substrate. Growth of mixed cultures with phenanthrene and fluoranthene solubilized by a nonionic surfactant prior to inoculation was exponential, indicating a high bioavailability of the solubilized hydrocarbons. Nonionic surfactants of the alkylethoxylate type and the alkylphenolethoxylate type with an average ethoxylate chain length of 9 to 12 monomers were toxic to a PAH-degrading Mycobacterium sp. and to several PAH-degrading mixed cultures. Toxicity of the surfactants decreased with increasing hydrophilicity, i.e., with increasing ethoxylate chain length. Nontoxic surfactants enhanced the degradation of fluorene, phenanthrene, anthracene, fluoranthene, and pyrene.     

Utilization of solubilized and crystalline mixtures of polycyclic aromatic hydrocarbons by a Mycobacterium sp.

A strain of Mycobacterium, that is able to degrade fluorene, phenanthrene, fluoranthene and pyrene was grown on various mixtures of these substrates. The polycyclic aromatic hydrocarbons (PAH) were provided either as crystals or solubilized by a surfactant. Mixed PAH were degraded simultaneously, but not in parallel, indicating that the degradation pathways were not incompatible. Certain interactions of the substrates were observed. For example, the degradation of solubilized pyrene was delayed in the presence of fluorene and enhanced in the presence of phenanthrene. Fluorene was degraded cometabolically with the other PAH serving as growth substrates, but not as the only source of carbon. The utilization of phenanthrene occurred at the fastest rate and was not affected by the presence of fluorene, pyrene or fluoranthene.     

Biodegradation of polycyclic aromatic hydrocarbons

The intent of this review is to provide an outline of the microbial degradation of polycyclic aromatic hydrocarbons. A catabolically diverse microbial community, consisting of bacteria, fungi and algae, metabolizes aromatic compounds. Molecular oxygen is essential for the initial hydroxylation of polycyclic aromatic hydrocarbons by microorganisms. In contrast to bacteria, filamentous fungi use hydroxylation as a prelude to detoxification rather than to catabolism and assimilation. The biochemical principles underlying the degradation of polycyclic aromatic hydrocarbons are examined in some detail. The pathways of polycyclic aromatic hydrocarbon catabolism are discussed. Studies are presented on the relationship between the chemical structure of the polycyclic aromatic hydrocarbon and the rate of polycyclic aromatic hydrocarbon biodegradation in aquatic and terrestrial ecosystems.     

Degradation of polycyclic aromatic hydrocarbons at low temperature under aerobic and nitrate-reducing conditions in enrichment cultures from northern soils

The potential for biodegradation of polycyclic aromatic hydrocarbons (PAHs)at low temperature and under anaerobic conditions is not well understood, but such biodegradation would be very useful for remediation of polluted sites. Biodegradation of a mixture of 11 different PAHs with two to five aromatic rings, each at a concentration of 10 micro g/ml, was studied in enrichment cultures inoculated with samples of four northern soils. Under aerobic conditions, low temperature severely limited PAH biodegradation. After 90 days, aerobic cultures at 20 degrees C removed 52 to 88% of the PAHs. The most extensive PAH degradation under aerobic conditions at 7 degrees C,53% removal, occurred in a culture from creosote-contaminated soil. Low temperature did not substantially limit PAH biodegradation under nitrate-reducing conditions. Under nitrate-reducing conditions,naphthalene, 2-methylnaphthalene, fluorene, and phenanthrene were degraded. The most extensive PAH degradation under nitrate-reducing conditions at 7 degrees C, 39% removal, occurred in a culture from fuel-contaminated Arctic soil. In separate transfer cultures from the above Arctic soil, incubated anaerobically at 7 degrees C, removal of 2-methylnaphthalene and fluorene was stoichiometrically coupled to nitrate removal. Ribosomal intergenic spacer analysis suggested that enrichment resulted in a few predominant bacterial populations,including members of the genera Acidovorax,Bordetella, Pseudomonas, Sphingomonas, and Variovorax. Predominant populations from different soils often included phylotypes with nearly identical partial 16S rRNA gene sequences (i.e., same genus) but never included phylotypes with identical ribosomal intergenic spacers (i.e., different species or subspecies). The composition of the enriched communities appeared to be more affected by presence of oxygen, than by temperature or source of the inoculum.     

Polyphasic approach for assessing changes in an autochthonous marine bacterial community in the presence of Prestige fuel oil and its biodegradation potential

A laboratory experiment was conducted to identify key hydrocarbon degraders from a marine oil spill sample (Prestige fuel oil), to ascertain their role in the degradation of different hydrocarbons, and to assess their biodegradation potential for this complex heavy oil. After a 17-month enrichment in weathered fuel, the bacterial community, initially consisting mainly of Methylophaga species, underwent a major selective pressure in favor of obligate hydrocarbonoclastic microorganisms, such as Alcanivorax and Marinobacter spp. and other hydrocarbon-degrading taxa (Thalassospira and Alcaligenes), and showed strong biodegradation potential. This ranged from >99% for all low- and medium-molecular-weight alkanes (C₁₅–C₂₇) and polycyclic aromatic hydrocarbons (C₀- to C₂- naphthalene, anthracene, phenanthrene, dibenzothiophene, and carbazole), to 75–98% for higher molecular-weight alkanes (C₂₈–C₄₀) and to 55–80% for the C₃ derivatives of tricyclic and tetracyclic polycyclic aromatic hydrocarbons (PAHs) (e.g., C₃-chrysenes), in 60 days. The numbers of total heterotrophs and of n-alkane-, aliphatic-, and PAH degraders, as well as the structures of these populations, were monitored throughout the biodegradation process. The salinity of the counting medium affects the counts of PAH degraders, while the carbon source (n-hexadecane vs. a mixture of aliphatic hydrocarbons) is a key factor when counting aliphatic degraders. These limitations notwithstanding, some bacterial genera associated with hydrocarbon degradation (mainly belonging to α- and γ-Proteobacteria, including the hydrocarbonoclastic Alcanivorax and Marinobacter) were identified. We conclude that Thalassospira and Roseobacter contribute to the degradation of aliphatic hydrocarbons, whereas Mesorhizobium and Muricauda participate in the degradation of PAHs.     

Advances in the field of high‐molecular‐weight polycyclic aromatic hydrocarbon biodegradation by bacteria

Interest in understanding prokaryotic biotransformation of high-molecular-weight polycyclic aromatic hydrocarbons (HMW PAHs) has continued to grow and the scientific literature shows that studies in this field are originating from research groups from many different locations throughout the world. In the last 10 years, research in regard to HMW PAH biodegradation by bacteria has been further advanced through the documentation of new isolates that represent diverse bacterial types that have been isolated from different environments and that possess different metabolic capabilities. This has occurred in addition to the continuation of in-depth comprehensive characterizations of previously isolated organisms, such as Mycobacterium vanbaalenii PYR-1. New metabolites derived from prokaryotic biodegradation of four- and five-ring PAHs have been characterized, our knowledge of the enzymes involved in these transformations has been advanced and HMW PAH biodegradation pathways have been further developed, expanded upon and refined. At the same time, investigation of prokaryotic consortia has furthered our understanding of the capabilities of microorganisms functioning as communities during HMW PAH biodegradation.     

Degradation of Polycyclic Aromatic Hydrocarbons at Low Temperature under Aerobic and Nitrate-Reducing Conditions in Enrichment Cultures from Northern Soils

The potential for biodegradation of polycyclic aromatic hydrocarbons (PAHs) at low temperature and under anaerobic conditions is not well understood, but such biodegradation would be very useful for remediation of polluted sites. Biodegradation of a mixture of 11 different PAHs with two to five aromatic rings, each at a concentration of 10 μg/ml, was studied in enrichment cultures inoculated with samples of four northern soils. Under aerobic conditions, low temperature severely limited PAH biodegradation. After 90 days, aerobic cultures at 20°C removed 52 to 88% of the PAHs. The most extensive PAH degradation under aerobic conditions at 7°C, 53% removal, occurred in a culture from creosote-contaminated soil. Low temperature did not substantially limit PAH biodegradation under nitrate-reducing conditions. Under nitrate-reducing conditions, naphthalene, 2-methylnaphthalene, fluorene, and phenanthrene were degraded. The most extensive PAH degradation under nitrate-reducing conditions at 7°C, 39% removal, occurred in a culture from fuel-contaminated Arctic soil. In separate transfer cultures from the above Arctic soil, incubated anaerobically at 7°C, removal of 2-methylnaphthalene and fluorene was stoichiometrically coupled to nitrate removal. Ribosomal intergenic spacer analysis suggested that enrichment resulted in a few predominant bacterial populations, including members of the genera Acidovorax, Bordetella, Pseudomonas, Sphingomonas, and Variovorax. Predominant populations from different soils often included phylotypes with nearly identical partial 16S rRNA gene sequences (i.e., same genus) but never included phylotypes with identical ribosomal intergenic spacers (i.e., different species or subspecies). The composition of the enriched communities appeared to be more affected by presence of oxygen, than by temperature or source of the inoculum.     

Rhizospheric degradation of phenanthrene is a function of proximity to roots

Rhizodegradation of recalcitrant organic pollutants, such as polycyclic aromatic hydrocarbons (PAH), may benefit from the major role that root exudates have on rhizospheric microbial processes. We investigated the influence of the proximity to ryegrass (Lolium perenneL.) roots on microbial populations and their biodegradation of phenanthrene (PHE) using compartmented pots. PAH degrading bacteria, total heterotrophic bacteria and PHE biodegradation were quantified in three consecutive sections at different distance (0–3, 3–6, 6–9 mm) from a mat of actively exuding roots. A bacterial gradient was observed with higher numbers of heterotrophs and PAH degrading bacteria closest to the roots. In parallel, a PHE biodegradation gradient was evident in the presence of roots. The biodegradation reached 86%, 48% and 36% of initially added PHE, respectively, in the layers 0–3 mm, 3–6 and 6–9 mm from the roots. The biodegradation rate was similar throughout the three layers of the non planted control. The present experimental system seems well suited for spatial and dynamic studies of PAH rhizoremediation.     

Degradation of polycyclic aromatic hydrocarbons and long chain alkanes at 6070 °C by Thermus and Bacillus spp

Although polycyclic aromatic hydrocarbons (PAH) and alkanesare biodegradable at ambient temperature, in some cases low bioavailabilities are thereason for slow biodegradation. Considerably higher mass transfer rates and PAH solubilities and hence bioavailabilities can be obtained at higher temperatures. Mixed and pure cultures of aerobic, extreme thermophilic microorganisms (Bacillus spp., Thermus sp.) were used to degrade PAH compounds and PAH/alkane mixtures at 65 °C. The microorganismsused grew on hydrocarbons as sole carbon and energy source. Optimal growthtemperatures were in the range of 60–70 °C at pH values of 6–7. The conversion of PAH with 3–5 rings (acenaphthene, fluoranthene, pyrene, benzo[e]pyrene) was demonstrated. Efficient PAH biodegradation required a second, degradable liquid phase. Thermus brockii Hamburg metabolized up to 40 mg (l h)^-1 pyrene and 1000 mg(1 h)^-1 hexadecane at 70 °C. Specific growth rates of 0.43 h^-1 were measured for this strain with hexadecane/pyrene mixtures as the sole carbon and energy source in a 2-liter stirred bioreactor. About 0.7 g cell dry weight were formed from 1 g hydrocarbon. The experiments demonstrate the feasibility and efficiency of extreme thermophilic PAH and alkane biodegradation.     

Biodegradation of soluble aromatic compounds of jet fuel under anaerobic conditions: laboratory batch experiments

Laboratory batch experiments were performed with contaminated aquifer sediments and four soluble aromatic components of jet fuel to assess their biodegradation under anaerobic conditions. The biodegradation of four aromatic compounds, toluene, o-xylene, 1,2,4-trimethylbenzene (TMB), and naphthalene, separately or together, was investigated under strictly anaerobic conditions in the dark for a period of 160 days. Of the aromatic compounds, toluene and o-xylene were degraded both as a single substrate and in a mixture with the other aromatic compounds, while TMB was not biodegraded as a single substrate, but was biodegraded in the presence of the other aromatic hydrocarbons. Substrate interaction is thus significant in the biodegradation of TMB. Biodegradation of naphthalene was not observed, either as a single substrate or in a mixture of other aromatic hydrocarbons. Although redox conditions were dominated by iron reduction, a clear relation between degradation and sulfate reduction was observed. Methanogenesis took place during the later stages of incubation. However, the large background of Fe(II) masked the increase of Fe(II) concentration due to iron reduction. Thus, although microbial reduction of Fe(III) is an important process, the evidence is not conclusive. Our results have shown that a better understanding of the degradation of complex mixtures of hydrocarbons under anaerobic conditions is important in the application of natural attenuation as a remedial method for soil and groundwater contamination.     

Measurement of biodegradability parameters for single unsubstituted and methylated polycyclic aromatic hydrocarbons in liquid bacterial suspensions

Substrate depletion experiments were conducted to characterize aerobic biodegradation of 20 single polycyclic aromatic hydrocarbons (PAHs) by induced Sphingomonas paucimobilis strain EPA505 in liquid suspensions. PAHs consisted of low molecular weight, unsubstituted, and methyl-substituted homologs. A material balance equation containing the Andrews kinetic model, an extension of the Monod model accounting for substrate inhibition, was numerically fitted to batch depletion data to estimate extant kinetic parameters including the maximal specific uptake rates, q(max), the affinity coefficients, K(S), and the substrate inhibition coefficients, K(I). Strain EPA505 degraded all PAHs tested. Applied kinetic models adequately simulated experimental data. A cell proliferation assay involving reduction of the tetrazolium dye WST-1 was used to evaluate the ability of strain EPA505 to utilize individual PAHs as sole energy and carbon sources. Of the 22 PAHs tested, 9 supported bacterial growth. Evaluation of the biokinetic data showed that q(max) correlated highly with transmembrane flux as theoretically estimated by a diffusion model, pointing to transmembrane transport as a potential rate-determining process. The biodegradability data generated in this study is essential for the development of quantitative structure-activity relationships (QSARs) for biodegradability and for modeling biodegradation of simple PAH mixtures.     

Nutrient-limited biodegradation of PAH in various soil strata at a creosote contaminated site

The effects of nutrient addition on the in situ biodegradation of polycyclic aromatic hydrocarbons in creosote contaminated soil were studied in soil columns taken from various soil strata at a wood preserving plant in Norway. Three samples were used: one from the topsoil (0–0.5 m), one from an organic rich layer (2–2.5 m) and one from the sandy aquifer (4.5–5 m). The addition of inorganic nitrogen and phosphorous stimulated the degradation of polycyclic aromatic hydrocarbons (PAHs) in the top soil and the aquifer sand. These two soils, which differed strongly in contamination levels, responded similarly to nutrient addition with the corresponding degradation of 4-ring PAHs. The ratio between available nitrogen (N) and phosphorous (P) might explain the degree of degradation observed for the 4-ring PAHs. However, the degree of degradation of 3-ring PAHs did not significantly increase after nutrient addition. An increase in the respiration rate, after nutrient addition, could only be observed in the topsoil. In the aquifer sand, 4-ring PAH degradation was not accompanied by an increase in the respiration rate or the number of heterotrophic micro-organisms. PAH degradation in the organic layer did not respond to nutrient addition. This was probably due to the low availability of the contaminants for micro-organisms, as a result of sorption to the soil organic matter. Our data illustrate the need for a better understanding of the role of nutrients in the degradation of high molecular weight hydrocarbons for the successful application of bioremediation at PAH contaminated sites.     

Revealing potential functions of VBNC bacteria in polycyclic aromatic hydrocarbons biodegradation

The bioremediation of polycyclic aromatic hydrocarbon (PAH)‐contaminated sites is not running smoothly, because of the lower activity of PAH‐degrading bacteria in actual bioremediation applications. The phenomenon of “viable but nonculturable” (VBNC) state may be a main limiting factor for their poor biodegradation capabilities of PAHs. Due to their abilities of entering into the VBNC state, most of bacterial populations with PAH‐degradation potential remain unculturable. Resuscitation of VBNC bacteria will enhance the degradation capability of indigenous bacteria which will eventually obtain their better capabilities in environmental bioremediation. Although evidences have been presented indicating that resuscitation of VBNC bacteria in polychlorinated biphenyl (PCB)‐contaminated environments not only significantly enhanced PCB degradation, but also obtained novel highly efficient PCB‐degrading bacteria, scanty information is available on the VBNC bacteria in PAH‐contaminated sites. VBNC bacteria, as a vast majority of potential microbial resource could be the repository of novel highly efficient PAH‐biodegraders. Therefore, studies need to be done on resuscitation of VBNC bacteria to overcome key bottlenecks in bioremediation of PAH‐contaminated sites. This mini‐review provides a new insight into the potential functions of VBNC bacteria in PAHs biodegradation.

Significance and Impact of the Study

As the vast majority microbial resource, viable but nonculturable (VBNC) bacteria, which showed their potential functions in polycyclic aromatic hydrocarbons (PAHs) biodegradation, can be of great significance in environmental bioremediation. It is therefore important to resuscitate VBNC bacteria for their better capabilities. Meanwhile, preventing the indigenous functional community from entering into the VBNC state will also maintain the high activity of PAH‐degrading bacteria in actual bioremediation applications. Undoubtedly, much more work needs to be done to reveal indigenous micro‐organisms in the VBNC state from the perspective of environmental functions.     

Anthracene biodegradation and surface activity by an iron-stimulated Pseudomonas sp

Iron may enhance polycyclic aromatic hydrocarbons (PAHs) degradation directly by increasing the activity of the enzymes involved in the aerobic biodegradation pathways for hydrocarbons, and indirectly by increasing the PAHs bioavailability due to the stimulation of biosurfactant production. In the present work, the PAH anthracene was used in order to study the effect of different forms and concentrations of iron on its biodegradation and surfactant production by Pseudomonas spp. isolates from a 14-years old petrochemical sludge landfarm site. Among the iron forms, iron nitrate was chosen based on its high solubility and effect on the increase in the growth of the isolate. Iron concentration of 0.1mM was selected as the limit between deficiency and toxicity for isolates growth and anthracene degradation. After 48 days Pseudomonas citronellolis isolate 222A degraded 72% of anthracene related to iron stimulation and surface tension decrease, indicating surfactant production. Pseudomonas aeruginosa isolate 332C was iron-stimulated but did not reduce surface tension while P. aeruginosa isolate 312A exhibited a noniron and surfactant dependence to degrade 72% of anthracene. Isolate 222A showed a direct dependence on iron to stimulate surfactant activity, which probably increased anthracene bioavailability. To our knowledge, this is the first report about the iron effect on anthracene degradation and surfactant production by a Pseudomonas sp. Based on the iron requirement and surfactant activity, the Pseudomonas isolates may be useful for bioremediation of PAHs.