Effect of soil/contaminant interactions on the biodegradation of naphthalene in flooded soil under denitrifying conditions

Summary The mineralization of ¹⁴C-labelled naphthalene was studied in pristine and oil-contaminated soil slurry (30% solids) under denitrifying conditions using a range of concentrations from below to above the aqueous phase saturation level. Results from sorption-desorption experiments indicated that naphthalene desorption was highly irreversible and decreased with an increase in the soil organic content, thus influencing the availability for microbial consumption. Under denitrifying conditions, the mineralization of naphthalene to CO₂ occurred in parallel with the consumption of nitrate and an increase in pH from 7.0 to 8.6. When the initial substrate concentration was 50 ppm (i.e. close to the aqueous phase saturation level), about 90% of the total naphthalene was mineralized within 50 days, and a maximum mineralization rate of 1.3 ppm day^–1 was achieved after a lag period of approx. 18 days. When added at concentrations higher than the aqueous phase saturation level (200 and 500 ppm), similar mineralization rates (1.8 ppm day^–1) occurred until about 50 ppm of the naphthalene was mineralized. After that the mineralization rates decreased logarithmically to a minimum of 0.24 ppm day^–1 for the rest of the 160 days of the experiments. For both of these higher concentrations, the reaction kinetics were independent of the concentration, indicating that desorption of the substrate governs the mineralization rate. Other results indicated that pre-exposure of soil to oil contamination did not improve the degradation rates nor reduce the lag periods. This study clearly shows the potential of denitrifying conditions for the biodegradation of low molecular weight PAHs. Offprint requests to: R. Samson     

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.     

The abundance of nahAc genes correlates with the 14C-naphthalene mineralization potential in petroleum hydrocarbon-contaminated oxic soil layers

In this study, we evaluated whether the abundance of the functional gene nahAc reflects aerobic naphthalene degradation potential in subsurface and surface samples taken from three petroleum hydrocarbon contaminated sites in southern Finland. The type of the contamination at the sites varied from lightweight diesel oil to high molecular weight residuals of crude oil. Samples were collected from both oxic and anoxic soil layers. The naphthalene dioxygenase gene nahAc was quantified using a replicate limiting dilution-polymerase chain reaction (RLD-PCR) method with a degenerate primer pair. In the non-contaminated samples nahAc genes were not detected. In the petroleum hydrocarbon-contaminated oxic soil samples nahAc gene abundance [range 3 x 10(1)-9 x 10(4) copies (g dry wt soil)(-1)] was correlated (Kendall non-parametric correlation r2=0.459, p<0.01) with the aerobic 14C-naphthalene mineralization potential (range 1 x 10(-5)-0.1 d(-1)) measured in microcosms at in situ temperatures (8 degrees C for subsurface and 20 degrees C for surface soil samples). In these samples nahAc gene abundance was also correlated with total microbial cell counts (r2=0.471, p<0.01), respiration rate (r2=0.401, p<0.01) and organic matter content (r2=0.341, p<0.05). NahAc genes were amplified from anoxic soil layers indicating that, although involved in aerobic biodegradation of naphthalene, these genes or related sequences were also present in the anoxic subsurface. In the samples taken from the anoxic layers, the aerobic 14C-naphthalene mineralization rates were not correlated with nahAc gene abundance. In conclusion, current sequence information provides the basis for a robust tool to estimate the naphthalene degradation potential at oxic zones of different petroleum hydrocarbon-contaminated sites undergoing in situ bioremediation.     

Biodegradation of phenanthrene and pyrene by Ganoderma lucidum

Biodegradation of two polycyclic aromatic hydrocarbons (PAHs), phenanthrene and pyrene, by a white rot fungus, Ganoderma lucidum, in broth cultures was investigated. It was found that the biomass of the organism decreased with the increase of PAH concentration in the cultures. In the cultures with 2 to 50 mg l⁻¹ PAHs, the degradation rate constants (k₁) increased with the PAH concentration, whereas, at the level of 100 mg l⁻¹, the degradation rate constants decreased. In the presence of 20 mg l⁻¹ PAHs, the highest degradation rates of both PAHs occurred in cultures with an initial pH of 4.0 at 30 °C. The addition of CuSO₄, citric acid, gallic acid, tartaric acid, veratryl alcohol, guaiacol, 2,2′-azino-bis-(3- ethylbenzothazoline-6-sulfonate) (ABTS) enhanced the degradation of both PAHs and laccase activities; whereas the supplement of oxalate, di-n-butyl phthalate (DBP), and nonylphenol (NP) decreased the degradation of both PAHs and inhibited laccase production. In conclusion, G. lucidum is a promising white rot fungus to degrade PAHs such as phenanthrene and pyrene in the environment.     

One-step process for debromination and aerobic mineralization of tetrabromobisphenol-A by a novel Ochrobactrum sp. T isolated from an e-waste recycling site

A novel bacterium, Ochrobactrum sp. T, capable of simultaneous debromination and aerobic mineralization of tetrabromobisphenol-A (TBBPA), was isolated from a sludge sample collected from an electronic-waste recycling site. The bacterium exhibited maximal debrominase activity at pH 6.5, 35 °C, and 200 rpm in Luria-Bertani culture medium. Initial TBBPA concentration and pH had more significant effects on degradation efficiency than those of temperature and inoculum size. Degradation and debromination efficiencies of 91.8% and 86.7%, respectively, were achieved within 72 h under optimized conditions of 35 °C, pH 7.0, inoculum volume of 25 mL, and TBBPA concentration of 3 mg L⁻¹. In addition, a 35.6% decrease in total organic carbon was observed after the degradation of 5 mg L⁻¹ TBBPA for 120 h. Eight metabolic intermediates were identified during the biodegradation of TBBPA. This study is the first report to propose a one-step process for TBBPA debromination and mineralization by a single bacterial strain.     

Aerobic biodegradation characteristics and metabolic products of quinoline by a Pseudomonas strain

A bacterial strain, BW003, which utilized quinoline as its sole C, N and energy source, was isolated and identified as Pseudomonas sp. BW003 degraded 192–911 mg/l quinoline within 3–8 h with removal rates ranging from 96% to 98%. The optimum conditions for the degradation were 30 °C and pH 8. In the process of biodegradation, at least 43% of quinoline was transformed into 2-hydroxyquinoline, then 0.69% of 2-hydroxyquinoline was transformed into 2,8-dihydroxyquinoline, and then, presumably, into 8-hydroxycoumarin. Meanwhile, at least 48% of the nitrogen in quinoline was directly transformed into ammonia-N. An extra carbon source enhanced the nitrogen transformation from ammonia-N. Further experiments showed that, besides cell synthesis, BW003 transformed less than 6% of ammonia-N into nitrate through heterotrophic nitrification. In addition, BW003 contained a large plasmid, which may be involved in quinoline metabolism. The study indicates that quinoline and its metabolic products can be eliminated from wastewater by controlling the C/N ratio using BW003 as the bioaugmentation inoculum.     

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.     

Effect of biosurfactant and fertilizer on biodegradation of crude oil by marine isolates of Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa

This study was conducted to investigate the effects of fertilizers and biosurfactants on biodegradation of crude oil by three marine bacterial isolates; Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa. Five sets of experiments were carried out in shake flask and microcosm conditions with crude oil as follows: Set 1-only bacterial cells added (no fertilizer and biosurfactant), Set 2-with additional fertilizer only, Set 3-with additional biosurfactant only, Set 4-with added biosurfactant + fertilizer, Set 5-with no bacterial cells added (control), all the above experimental sets were incubated for 168 h. The biosurfactant + fertilizer added Set 4, resulted in maximum crude oil degradation within shake flask and microcosm conditions. Among the three bacterial isolates, P. aeruginosa and biosurfactant produced by this strain resulted in maximum crude oil degradation compared to the other two bacterial strains investigated. Interestingly, when biosurfactant and bacterial cells were used (Set 3), significant oil biodegradation activity occurred and the difference between this treatment and that in Set 4 with added fertilizer + biosurfactant were only 4-5% higher degradation level in shake flask and 3.2-7% in microcosm experiments for all three bacterial strains used. It is concluded that, biosurfactants alone capable of promoting biodegradation to a large extent without added fertilizers, which will reduce the cost of bioremediation process and minimizes the dilution or wash away problems encountered when water soluble fertilizers used during bioremediation of aquatic environments.     

Versatility of fluorene metabolite (phenol) in fluorene biodegradation by a sulfate reducing culture

Biodegradability of fluorene and the versatility of fluorene metabolite (i.e. phenol) in fluorene biodegradation by a sulfate-reducing enrichment culture were investigated. Batch experiments (with 5 mg l⁻¹ fluorene) were designed via the central composite design to examine the effects of sulfate (5-35 mM) and biomass (5-50 mg l⁻¹) concentrations (variables) on fluorene degradation (response). The experimental results revealed that fluorene removal was more influenced by the biomass concentration than the sulfate concentration. The optimal sulfate and biomass concentrations for fluorene biodegradation (90% removal) were found to be 14.4 mM and 37.8 mg l⁻¹, respectively. Under the optimal conditions, a set of biodegradation experiments were repeated to evaluate both the biodegradability of fluorene metabolite and the potential effect of phenol accumulation on fluorene degradation. The outcomes indicated a slow phenol degradation rate, i.e. 0.02 mg l⁻¹ d⁻¹. Moreover, a small reduction in the fluorene biodegradation efficiency was observed in the presence and accumulation of phenol. However, this sulfate reducing culture is a valuable resource for the simultaneous degradation of fluorene and phenol.