Effects of mixing energy on the sedimentation of vegetable oil spills by clay.

The effects of clay dose and mixing energy on the efficiency of vegetable oil sedimentation by clay are investigated. The sedimentation efficiency increased with increasing clay dose to a maximum of about 80% of added oil. The maximum sedimentation efficiency was achieved at a lower clay dose, and the sedimentation efficiency was greater for a given clay dose when the oil was present as a thick oil film rather than as a thinner film. Sedimentation efficiency was relatively constant for mixing energies less than about 0.01 m2 s(-3) (0.01 W kg(-1)) but decreased dramatically at higher energy dissipation rates. Mixing energy may not be an important factor in determining the effectiveness of this response alternative because energy dissipation rates in natural surface water bodies under most typical conditions are less than 0.01 m2 s(-3). The effects of oil film thickness and mixing energy on the efficiency of vegetable oil sedimentation suggests that vegetable oil-mineral aggregates (VOMA) form through a different mechanism to that of petroleum oil-mineral aggregates (OMA). One consequence of the different formation mechanisms is that VOMA are much larger than petroleum OMA.

Monitoring oil spill bioremediation using marsh foraminifera as indicators.

A controlled experiment was conducted in June 2000 to identify the environmental impacts of weathered crude oil on an Atlantic coastal salt marsh to help evaluate in situ biological remediation techniques for restoring the environment. Foraminifera, marsh microfossils known to be sensitive to a range of environmental stress factors, were used to monitor the effects of the residual oil and the experimental treatments. Results show that the foraminifera responded quickly to the oil and that the oil had a statistically significant, negative impact, as demonstrated by a dramatic increase in deformities in the tests of Miliammina fusca, compared to specimens from the non-oiled control plots. The results clearly show that foraminifera can be excellent indicators of oil pollution using only the percent of deformed tests. The advantages that foraminifera provide are the ease of sampling, processing and examination, with the added benefit that these organisms leave a fossil record.

Dispersibility of crude oil in fresh water.

The effects of surfactant composition on the ability of chemical dispersants to disperse crude oil in fresh water were investigated. The objective of this research was to determine whether effective fresh water dispersants can be designed in case this technology is ever considered for use in fresh water environments. Previous studies on the chemical dispersion of crude oil in fresh water neither identified the dispersants that were investigated nor described the chemistry of the surfactants used. This information is necessary for developing a more fundamental understanding of chemical dispersion of crude oil at low salinity. Therefore, we evaluated the relationship between surfactant chemistry and dispersion effectiveness. We found that dispersants can be designed to drive an oil slick into the freshwater column with the same efficiency as in salt water as long as the hydrophilic-lipophilic balance is optimum.

Biodegradability of dispersed crude oil at two different temperatures.

Laboratory experiments were initiated to study the biodegradability of oil after dispersants were applied. Two experiments were conducted, one at 20 degrees C and the other at 5 degrees C. In both experiments, only the dispersed oil fraction was investigated. Each experiment required treatment flasks containing 3.5% artificial seawater and crude oil previously dispersed by either Corexit 9500 or JD2000 at a dispersant-to-oil ratio of 1:25. Two different concentrations of dispersed oil were prepared, the dispersed oil then transferred to shake flasks, which were inoculated with a bacterial culture and shaken on a rotary shaker at 200 rpm for several weeks. Periodically, triplicate flasks were removed and sacrificed to determine the residual oil concentration remaining at that time. Oil compositional analysis was performed by gas chromatography/mass spectrometry (GC/MS) to quantify the biodegradability. Dispersed oil biodegraded rapidly at 20 degrees C and less rapidly at 5 degrees C, in line with the hypothesis that the ultimate fate of dispersed oil in the sea is rapid loss by biodegradation.

Challenges in biodegradation of trace organic contaminants--gasoline oxygenates and sex hormones.

Advances in analytical methods have led to the identification of several classes of organic chemicals that are associated with adverse environmental effects. Two such classes of organic chemicals, gasoline oxygenates and sex hormones, are used to illustrate challenges associated with the biodegradation of trace organic contaminants. Gasoline oxygenates can be present in groundwater, alone, or commingled with xylene, at appreciable concentrations. However, target-treated water standards dictate that gasoline oxygenates be reduced to the microgram-per-liter concentration range before consumption. Sex hormones, on the other hand, are present in wastewater matrixes in the part-per-trillion concentration range, and the biggest challenge that must be met, before optimizing their removal, is facilitating their detection.

Protocol for laboratory testing of crude-oil bioremediation products in freshwater conditions.

In 1993, the Environmental Protection Agency, National Risk Management Research Laboratory (EPA, NRMRL), with the National Environmental Technology Application Center (NETAC), developed a protocol for evaluation of bioremediation products in marine environments [18]. The marine protocol was adapted for application in freshwater environments by using a chemically defined medium and an oil-degrading consortium as a positive control. Four products were tested using the modified protocol: two with nutrients and an oleophilic component; one with nutrients, sorbent, and organisms; and one microbial stimulant. A separate experiment evaluated the use of HEPES and MOPSO buffers as replacements for phosphate buffer. The oleophilic nutrient products yielded oil degradation similar to the positive control, with an average alkane removal of 97.1+/-2.3% and an aromatic hydrocarbon removal of 64.8+/-1.2%. The positive control, which received inoculum plus nutrients, demonstrated alkane degradation of 98.9+/-0.1% and aromatic degradation of 52.9+/-0.1%. The sorbent-based product with inoculum failed to demonstrate oil degradation, while the microbial stimulant showed less oil degradation than the positive control. Replacement of phosphate buffer with other buffers had no significant effect on one product's performance. Differences in product performance were easily distinguishable using the protocol, and performance targets for alkane and aromatic hydrocarbon degradation are suggested.

Biodegradation of methyl tert-butyl ether under various substrate conditions.

Five aerobic enrichments efficient at degrading methyl tert-butyl ether (MTBE) under different substrate conditions were developed in well-mixed reactors containing a polyethlene porous pot for biomass retention. The five substrate conditions were as follows: MTBE alone; MTBE and diethyl ether (DEE); MTBE and diisopropyl ether (DIPE); MTBE and ethanol (EtOH); and MTBE with benzene, toluene, ethylbenzene, and xylene (BTEX). All five cultures demonstrated greater than 99.9% removal of MTBE. Addition of alternative substrate was found to have no effect on the performance of the reactors. The bacterial communities of the reactors were monitored periodically by denaturing gradient gel electrophoresis (DGGE) to determine when homeostasis was achieved. Phylogenetic analysis of the excised DGGE bands was done in order to compare the bacterial community compositions of the reactors. All cultures were found to be mixed cultures, and each enrichment was shown to have a unique composition. A majority of the bands in all reactors represented a group of organisms belonging to the Cytophaga-Flexibacter-Bacterioides (C-F-B) Phylum of bacteria. This was also the only group found in all of the reactors. This study demonstrates that MTBE can be degraded effectively in bioreactors under several substrate conditions and gives insight into the microorganisms potentially involved in the process.

Aerobic biodegradation of gasoline oxygenates MTBE and TBA.

MTBE degradation was investigated using a continuously stirred tank reactor (CSTR) with biomass retention (porous pot reactor) operated under aerobic conditions. MTBE was fed to the reactor at an influent concentration of 150 mg/l (1.70 mmol/l). A second identical reactor was operated as a control under the same conditions with the addition of 2.66 g/l of sodium azide, to kill any biological activity. Results from these experiments suggest that biomass retention is critical to the degradation of MTBE. The rate of MTBE removal was shown to be related to the VSS concentration. MTBE removal exceeded 99.99% when the VSS concentration in the reactor was over 600 mg/l. Results obtained from batch experiments conducted on mixed liquor samples from the porous pot reactor indicate that the individual rates of biodegradation of MTBE and TBA were higher for initial concentrations of 15 mg/l than for concentrations of 5 mg/l. The presence of TBA at lower concentrations did not effect the rate of MTBE degradation, however higher concentrations of TBA did reduce the rate of biodegradation of MTBE. Denaturing Gradient Gel Electrophoresis (DGGE) analysis reveals that the culture consisted of a community of bacterial organisms of about 6 species.

Phylogenetic analysis of aerobic freshwater and marine enrichment cultures efficient in hydrocarbon degradation: effect of profiling method.

Aerobically grown enrichment cultures derived from hydrocarbon-contaminated seawater and freshwater sediments were generated by growth on crude oil as sole carbon source. Both cultures displayed a high rate of degradation for a wide range of hydrocarbon compounds. The bacterial species composition of these cultures was investigated by PCR of the 16S rDNA gene using multiple primer combinations. Near full-length 16S rDNA clone libraries were generated and screened by restriction analysis prior to sequence analysis. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was carried out using two other PCR primer sets targeting either the V3 or V6-V8 regions, and sequences derived from prominent DGGE bands were compared to sequences obtained via cloning. All data sets suggested that the seawater culture was dominated by alpha-subgroup proteobacteria, whereas the freshwater culture was dominated by members of the beta- and gamma-proteobacteria. However, the V6-V8 primer pair was deficient in the recovery of Sphingomonas-like 16S rDNA due to a 3' terminal mismatch with the reverse primer. Most 16S rDNA sequences recovered from the marine enrichment were not closely related to genera containing known oil-degrading organisms, although some were detected. All methods suggested that the freshwater enrichment was dominated by genera containing known hydrocarbon-degrading species.

Microbial population changes during bioremediation of an experimental oil spill.

Three crude oil bioremediation techniques were applied in a randomized block field experiment simulating a coastal oil spill. Four treatments (no oil control, oil alone, oil plus nutrients, and oil plus nutrients plus an indigenous inoculum) were applied. In situ microbial community structures were monitored by phospholipid fatty acid (PLFA) analysis and 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) to (i) identify the bacterial community members responsible for the decontamination of the site and (ii) define an end point for the removal of the hydrocarbon substrate. The results of PLFA analysis demonstrated a community shift in all plots from primarily eukaryotic biomass to gram-negative bacterial biomass with time. PLFA profiles from the oiled plots suggested increased gram-negative biomass and adaptation to metabolic stress compared to unoiled controls. DGGE analysis of untreated control plots revealed a simple, dynamic dominant population structure throughout the experiment. This banding pattern disappeared in all oiled plots, indicating that the structure and diversity of the dominant bacterial community changed substantially. No consistent differences were detected between nutrient-amended and indigenous inoculum-treated plots, but both differed from the oil-only plots. Prominent bands were excised for sequence analysis and indicated that oil treatment encouraged the growth of gram-negative microorganisms within the alpha-proteobacteria and Flexibacter-Cytophaga-Bacteroides phylum. alpha-Proteobacteria were never detected in unoiled controls. PLFA analysis indicated that by week 14 the microbial community structures of the oiled plots were becoming similar to those of the unoiled controls from the same time point, but DGGE analysis suggested that major differences in the bacterial communities remained.

A model for diffusion controlled bioavailability of crude oil components.

Crude oil is a complex mixture of several different structural classes of compounds including alkanes, aromatics, heterocyclic polar compounds, and asphaltenes. The rate and extent of microbial degradation of crude oil depends on the interaction between the physical and biochemical properties of the biodegradable compounds and their interactions with the non-biodegradable fraction. In this study we have systematically altered the concentration of non-biodegradable material in the crude oil and analyzed its impact on transport of the biodegradable components of crude oil to the microorganisms. We have also developed a mathematical model that explains and accounts for the dependence of biodegradation of crude oil through a putative bioavailability parameter. Experimental results indicate that as the asphaltene concentration in oil increases, the maximum oxygen uptake in respirometers decreases. The mathematically fitted bioavailability parameter of degradable components of oil also decreases as the asphaltene concentration increases.

Selective enumeration of aromatic and aliphatic hydrocarbon degrading bacteria by a most-probable-number procedure.

A most-probable-number (MPN) procedure was developed to separately enumerate aliphatic and aromatic hydrocarbon degrading bacteria, because most of the currently available methods are unable to distinguish between these two groups. Separate 96-well microtiter plates are used to estimate the sizes of these two populations. The alkane-degrader MPN method uses hexadecane as the selective growth substrate and positive wells are detected by reduction of iodonitrotetrazolium violet, which is added after incubation for 2 weeks at 20 degrees C. Polycyclic aromatic hydrocarbon degraders are grown on a mixture of phenanthrene, anthracene, fluorene, and dibenzothiophene in a second plate. Positive wells turn yellow to greenish-brown from accumulation of the partial oxidation products of the aromatic substrates and they can be scored after a 3-week incubation period. These MPN procedures are accurate and selective. For pure cultures, heterotrophic plate counts on a nonselective medium and the appropriate MPN procedure provide similar estimates of the population density. Bacteria that cannot grow on the selective substrates do not produce false positive responses even when the inoculum density is very high. Thus, this method, which is simple enough for use in the field, provides reliable estimates for the density and composition of hydrocarbon-degrading microbial populations.

Measurement of hydrocarbon-degrading microbial populations by a 96-well plate most-probable-number procedure.

A 96-well microtiter plate most-probable-number (MPN) procedure was developed to enumerate hydrocarbon-degrading microorganisms. The performance of this method, which uses number 2 fuel oil (F2) as the selective growth substrate and reduction of iodonitrotetrazolium violet (INT) to detect positive wells, was evaluated by comparison with an established 24-well microtiter plate MPN procedure (the Sheen Screen), which uses weathered North Slope crude oil as the selective substrate and detects positive wells by emulsification or dispersion of the oil. Both procedures gave similar estimates of the hydrocarbon-degrader population densities in several oil-degrading enrichment cultures and sand samples from a variety of coastal sites. Although several oils were effective substrates for the 96-well procedure, the combination of F2 with INT was best, because the color change associated with INT reduction was more easily detected in the small wells than was disruption of the crude oil slick. The method's accuracy was evaluated by comparing hydrocarbon-degrader MPNs with heterotrophic plate counts for several pure and mixed cultures. For some organisms, it seems likely that a single cell cannot initiate sufficient growth to produce a positive result. Thus, this and other hydrocarbon-degrader MPN procedures might underestimate the hydrocarbon-degrading population, even for culturable organisms.

Low-temperature stability of viruses in sludges.

Enteroviruses survived for up to 38 days without diminishing in numbers in extended-aeration sludges maintained at 5 degrees C. In oxidation ditch sludges similarly maintained, enteroviruses survived for up to 17 days without diminishing in numbers. The pHs of the sludges in this study were well inside the pH 6 to 8 corridor in which destruction of enteroviruses by the detergents and ammonia present in sludges reportedly does not occur. Unexplained, however, was the survival of large numbers of enteroviruses in sludges at pH 3.5, a pH at which some anionic detergents commonly present in sewage are rapidly virucidal. The long survival of enteroviruses in these sludges at 5 degrees C indicates that such sludges can probably be stored under refrigeration in the laboratory for extended periods while awaiting processing without suffering significant losses in enterovirus numbers.

Lysis of Sphaerotilus natans swarm cells by Bdellovibrio bacteriovorus.

Six strains of Sphaerotilus natans (smooth form) were lysed by five parasitic strains of Bdellovibrio bacteriovorus. The possible use of Bdellovibrio to control the proliferation of S. natans in the environment was hypothesized.

Bactericidal effect of various combinations of gamma radiation and chloramine on aqueous suspensions of Escherichia coli.

Methods of combining gamma radiation with chloramine to disinfect aqueous suspensions of Escherichia coli were investigated. Logarithmically grown cells were exposed to the bactericidal agents sequentially (i.e., radiation followed by chloramine, and chloramine followed by radiation) and simultaneously. Regardless of which combination was used, the bactericidal effect was always less than additive. During the phase of work involving the simultaneous addition of both agents, it was observed that chloramine was destroyed more rapidly by radiation than were the organisms. Since an increase in the bactericidal effectiveness of either disinfectant by prior or simultaneous treatment of the cells with the other disinfectant was not achieved in buffered distilled water, it was concluded that disinfection of wastewater effluents by combining ionizing radiation with chloramine would not be economically feasible.