Fuel oil spills resulting from storage tank leaks, overfills or catastrophic floods pose a sizable risk to human health. Hydrocarbons get entrapped along with water inside the pore spaces of solids thus forming so called “ganglia.” This problem emerged after a catastrophic flood that occurred in Grand Forks, N. Dak. in April 1997. During the flood, a number of fuel oil tanks in residential basements were ruptured, and the spilled hydrocarbons mixed with water and absorbed in concrete walls. Afterward, slow evaporation exposed residents to hydrocarbon vapors for years. Unfortunately, common remediation techniques, such as heating and pump-and-treat technologies, prove to be inefficient. For instance, heating caused pollutants to penetrate deeper within concrete blocks, which merely effected a delay in the release of hydrocarbon vapors into the ambient air. Treating surfaces with soap did not work, because surfactants could not reach the oil trapped in the ganglia.
Most of the research on bioremediation of solids addresses the biodegradation of hydrocarbons in soils. Some research describes the removal of hydrocarbons from other low-porosity solid media, such as sand or metal filings. The feasibility of biotreatment has been postulated for construction debris. Concrete bioremediation has been thoroughly documented only for organochlorine herbicides in stirring reactors suitable only for application on concrete debris. Therefore, there is a need for an efficient method of removing hydrocarbons from the pore spaces of solids.
The present invention is a method of removing pollutants from porous, solid materials. A biomass, which is able to degrade at least one pollutant, is applied on to the porous, solid material. Environmental conditions are sustained until a desired amount of pollutant removal is achieved.
New evidence suggests that an overlay bioremediation method efficiently removes biodegradable compounds from the pores of solid surfaces. The present invention is designed to take advantage of this finding. Naphthalene removal from concrete and n-hexadecane removal from concrete and wood serve as model systems for studying the findings that are the basis for this invention.
Concrete samples were chipped from a single standard 3000 psi concrete tile manufactured at Concrete, Inc., wood samples were commercial-grade Southern Yellow Pine purchased from Menards (both of Grand Forks, N. Dak., U.S.A.), and reagent grade chemicals were used. 14C-labeled n-hexadecane and naphthalene were purchased from Sigma and American Radiolabeled Chemicals (St. Louis, Mo.), respectively. Unless stated otherwise, radiolabeled n-hexadecane and naphthalene were used throughout the experiments. All chemicals, solutions, and tools were steam-sterilized by autoclaving for one hour at 2.5 atm.
Scintillation counting was performed on a Beckman 6800 counter in plastic vials using 5 ml of Econo-safe scintillation cocktail (Research Products International, Mount Prospect, Ill., U.S.A.). Biomass concentration was monitored by either optical density or protein assay. Biomass disruption by sodium dodecyl sulfate (SDS) was followed by protein assay using bicinchoninic acid. Cell counts were obtained upon calibration.
The experiments were conducted using Pseudomonas aeruginosa PG 201 and two other unidentified strains isolated from oil-contaminated soil that consume naphthalene and n-hexadecane. Expression of hydrocarbon-degrading enzymes by the bacteria may be constitutive or induceable. Biomass was grown in an aqueous mineral medium containing 3.4 g/L KH2PO4, 4.3 g/L K2HPO4, 2.0 g/L (NH4)2SO4, 0.8 g/L MgSO4, 0.04 g/L CaCl2, 0.03 g/L FeSO4 and 25 ml/L of a trace mineral solution containing 40 mg/L MnCl2, 80 mg/L Na2MoO4, 6 mg/L CuSO4, 13 mg/L H3BO3 and 60 mg/L ZnSO4. When growing bacteria for bioremediation experiments, 50–70 ml of mineral medium were inoculated with the desired strain, 0.3 g of a hydrocarbon was added, and the flask was incubated at 30° C. Once growth slowed, the suspension having a cell concentration adjusted to (4±2)×1010/ml (the bacterial suspension) was used either in shaking flasks or overlay procedures.
Hydrocarbons present in the liquid phase or adsorbed on flask surfaces were extracted with 1.0 ml of n-decane for 1 min. 10 ml of 2-propanol per a 1.5 g piece of concrete extracted for over 80 hours was used to extract hydrocarbons from concrete. Complete extraction was verified by scintillation counting.
To analyze the mass balance of samples, the biomass was separated by centrifugation at 2,000 rpm for 15 min. 100 μl of 1.0 M ethanolic KOH and 0.9 ml of 2% aqueous SDS solution were added to the pellet and boiled in a water bath for 7 min. to lyse the bacterial cells. The alkali was neutralized with 20 μl of 6.0 M acetic acid, and an aliquot was taken for scintillation counting. The radioactivity of the supernatant was also measured to account for the conversion of the hydrocarbons into water-soluble metabolites.
5, 25, 50 and 100 μl aliquots of neat-form n-hexadecane were applied to 1.2±0.3 g concrete samples. After a 5 minute incubation at room temperature to allow the hydrocarbon to be absorbed into the concrete, the samples were placed in 100 ml flasks containing 10 ml of the sterile mineral medium and shaken on an orbital rotator at 100 rpm at room temperature. After 120 hours, the samples were withdrawn from the mineral medium and extracted with 2-propanol to recover the hydrocarbon retained in the concrete. The contents of the flasks were extracted with 1.0 ml of n-decane to recover the leached, non-retained n-hexadecane. Radioactivity of the extracts was measured to yield the percentage of hydrocarbon retention and mass balance.
As seen in the table of
Subsequent bioremediation experiments use 5 μl aliquots of hydrocarbons since nearly quantitative pollutant absorption is observed even for five times larger amounts. 5 μl aliquots guarantee that all n-hexadecane is retained in the concrete.
Only moderate removal of concrete-absorbed n-hexadecane was observed after a 7 day incubation. Depending on the strain used, 8%–19% or 10%–17% of n-hexadecane was removed. The addition of surfactants did not increase the efficiency of hydrocarbon degradation. This observation may serve as evidence that substrates must diffuse from the depth of concrete pores. If the hydrocarbons were released from near-surface sites, hydrocarbon diffusion would be facilitated by surfactants resulting in greater removal efficiency. Significant surfactant-induced enhancement of the biodegradation of hydrocarbons absorbed in sand particles was previously demonstrated, and the discrepancy in results should be ascribed to the significant difference in pore volumes and/or structures of the two materials.
Longer incubation times, up to 30 days, were also carried out (data not shown). The values shown in
Biodegradation kinetics were also different for neat-form and concrete-absorbed n-hexadecane. Statistically significant removal of concrete-absorbed n-hexadecane was observed only after 100–120 hours of incubation, whereas the consumption of neat-form n-hexadecane was detected in 48–55 hours. These differences in both the final degradation efficiency and kinetics for neat-form and concrete-absorbed n-hexadecane may be explained by slow substrate diffusion in the pores toward the surface. Therefore, the rate-limiting step appears to switch from biochemical factors in the neat-form n-hexadecane to mass transfer/diffusion factors in the concrete-absorbed samples.
The results show that most of the labeled carbon was converted to CO2 rather than accumulating in the biomass, thus indicating that the bacteria removing concrete-absorbed n-hexadecane did not exhibit any significant growth. Since hydrocarbon diffusion in concrete appears to be rate-limiting, this observation may be explained by a slow n-hexadecane release rate from concrete samples. Therefore, bacterial growth is severely limited by the carbon/energy source, such that the bacteria maintain themselves but do not reproduce.
Conversely, for neat-form n-hexadecane, which is more readily accessible to bacteria, substantial biomass growth was observed. These results also show that bacteria remove concrete-absorbed n-hexadecane via biodegradation rather than facilitating desorption—both mechanisms having been observed for bioremediation of solids.
Accumulation of radioactivity in the aqueous phase, and its disappearance from the n-decane extract indicated the biotransformation of naphthalene into more polar chemicals. As seen in the table of
The dynamics for concrete-absorbed naphthalene differed in that removal of the bulk of the concrete-absorbed naphthalene took days instead of hours. The removal efficiency was also lower compared to the neat-form substrate with 15%–20% of initial naphthalene remaining absorbed in the concrete. This is consistent with a diffusion-controlled process, which was observed for n-hexadecane.
The graph of
Contrary to n-hexadecane, naphthalene removal was similar either with or without biomass. This suggests that either there was no naphthalene biodegradation, or naphthalene was simply leached from the concrete where the bacteria then degraded some of the naphthalene.
To clarify the issue, 14C partitioning and mass balance was carried out for removal of concrete-absorbed naphthalene in shaking flasks. The results both with (Runs with biomass) and without (Blanks) soil bacteria are summarized in
During the first six hours of incubation with biomass, the mass balance was as high as 96% indicating that very little radioactivity was lost due to evaporation. A significant fraction of radioactivity simultaneously accumulated in the aqueous phase implying biotransformation of naphthalene. Nearly one-third (28%–35%) of the initial naphthalene was biotransformed to some water-soluble products in six hours of incubation. This value is similar to the observed naphthalene removal efficiency from concrete shown in
By contrast, the distribution of labeled carbon in blanks was different. Most of the naphthalene evaporated from the system with little 14C detected in the liquid. For incubations longer than six hours, mass balance converged poorly, presumably due to a partial 14C conversion to CO2.
Since removal of concrete-absorbed hydrocarbons in shaking flasks is impractical for real world applications, biodegradation of hydrocarbons using overlay techniques was evaluated. The graph of
To inoculate the agar plates for overlay experiments, 5 μl of the bacterial suspension described previously was spread on the surface of a mineral medium agar plate with an inoculation loop such that two-thirds of the plate surface was covered. The bacteria were then grown at 30° C. for 4 days resulting in cell counts on the plates of about 1010 cells/cm2.
1.5±0.1 g concrete samples having at least one flat surface were contaminated with a radiolabeled hydrocarbon. The samples were incubated for 5 min. to allow the hydrocarbon to be imbibed by the concrete, and the samples were submerged in 20 ml of sterile mineral medium to create ganglia. The samples were then removed from the liquid and placed flat side down (hydrocarbon having been applied to flat side) on the bacterial biomass adhered to the agar plate. The plates were incubated at room temperature in plastic bags to minimize dessication. Sterile plates with no bacteria were used as blanks. For analysis, the hydrocarbon absorbed in concrete was extracted with 2-propanol and quantified by scintillation counting.
As shown in
The results show that n-hexadecane is efficiently removed from wood using the agar overlay procedure. Notably, as the percentage of agar increased in the overlay, the degradation efficiency also increased.
Even at the suboptimal growth conditions of 23° C., the removal of more than 80% of n-hexadecane is achieved in 15–22 days. This is comparable to results at 37° C., which is the optimal growth temperature. These results confirm that the process rate is controlled by diffusion.
As with shaking flasks, experiments with concrete-absorbed naphthalene were complicated by its evaporation. Comparison of the experiments with and without biomass adhered to agar plates showed that, by contrast with shaking flasks, bacterial biomass speeded naphthalene removal from concrete. 16%–32% of naphthalene removal was due to the addition of biomass. It is noteworthy that, as shown in
To gain insight into using this technology for practical applications, filter paper was used as a support for bacteria. Filter paper overlays were prepared by adding 6 ml of the bacterial suspension to a Petri dish containing four layers of filter paper. The filter paper was kept moist throughout the experiment with periodic additions of mineral medium applied with a pipette.
As shown in
Degradation of n-hexadecane was also tested using filter paper overlays.
The results of two series of experiments show that n-hexadecane degradation with filter paper overlays was less efficient than for naphthalene. This difference may be explained by a difference in moisture content of the concrete and the relative solubilities of the hydrocarbons.
Water is believed to hinder diffusion of chemicals within porous materials. This trend has been observed for noble gases and volatile organic compounds in concrete and soil and for NaCl in meat. As to volatile chemicals, it is thought that the liquid phase that fills the pores affects the gas phase diffusional resistance. To verify this effect for the present systems, the water content of concrete pieces was measured under conditions characteristic for all three bioremediation protocols.
The water content of concrete samples in shaking flasks after a 24 hr. incubation was 0.104±0.015 g/l g of concrete. For overlays incubated for 7–15 days, the water content was 0.06±0.02 and 0.103±0.012 g/l g concrete for agar plate overlays and filter paper overlays, respectively. The values obtained for both shaking flasks and filter paper overlays were roughly twice that of agar plate overlays. The poor reproducibility of filter paper overlays is likely due to the periodic application of mineral medium making the water content vary between each sample.
The difference in water content of concrete pores may explain the greater removal efficiency of n-hexadecane with agar plate overlays than with filter paper overlays and the greater removal efficiency of naphthalene in shaking flasks. This is consistent with the results of
The difference in the final biodegradation efficiency in overlay experiments for n-hexadecane and naphthalene may be explained by fast naphthalene evaporation due to its relatively high volatility. Had the 40%–50% of initial naphthalene which had evaporated stayed in the system, it would have been metabolized by bacteria resulting in a removal efficiency similar to n-hexadecane.
The data obtained on naphthalene removal may also be compared with bioremediation of concrete contaminated with herbicides. Removal of herbicides was previously conducted in batch reactors filled with aqueous phase, which in terms of contact and transfer, is comparable to shaking flasks. It was found that herbicides were nearly quantitatively removed from concrete in four weeks. The dynamics of naphthalene removal was similar. This makes sense, because the water solubility of polar herbicides (chlorinated phenols and carboxylic acids) is at least as high as that of naphthalene. However, in contrast to the herbicides, quantitative removal of either naphthalene or n-hexadecane was not observed in our study. At least 15%–20% of either hydrocarbon remained absorbed in concrete, even in long-term experiments, both in shaking flasks and overlays. Perhaps those 15%–20% of hydrocarbons exhibiting very strong absorption in concrete are adsorbed on very hydrophobic surface sites within the pores. A similar observation was made for hydrocarbon absorption in soil.
Preliminary data also indicate that, under conditions similar to removal of hydrocarbons described above, 90% of dinitrotoluene (DNT) is removed by DNT-degrading microorganisms, for example bacteria and fungi, in about 20–40 days from both wood and concrete. Removal efficiencies are similar to those of naphthalene as DNT is even more water-soluble than naphthalene by an order of magnitude.
Hydrocarbons having a molecular formula as high as C20HX, where X varies depending on the level of saturation, have been successfully removed using this technique. Thus, the method of the present invention may be used to remove a variety of pollutants from any porous material—wood and concrete being only two examples.
In practice, there are a number of embodiments by which this method may be performed. The biomass may be sprayed onto the contaminated structure with the support subsequently being applied. The contaminated structure itself may also act as the support for the bacterial biomass. Alternatively, the biomass may be loaded to the support, which is then applied to the contaminated structure. The support may be in the form of a gelatinous material, such as agar; an absorbant paper, such as filter paper; or a liquid having enough viscosity to adhere to the contaminated support.
A gelatinous material is applied by heating the material to a point where it is liquified but will not kill the biomass, pouring the material over the support structure and allowing the material to solidify as it spreads over the structure. Alternatively, a liquid may be used that polymerizes to a gelatinous material.
Wet absorbant paper is hung similar to wall paper. The absorbant paper has an adhesive quality, so that it may stick to the contaminated structure without any other form of adhesive. To insure that the paper remains adhered to the contaminated structure, however, the paper may have areas of adhesive applied so that once wet it loosely sticks to the contaminated structure. Other viscous liquid materials may be sprayed or brushed onto the contaminated structure, similar to applying paint.
The moisture level of the various supports may be maintained by periodic spraying with water or an aqueous mineral medium. Alternatively, a humidifier-type apparatus could be operated that produces a mist. For optimal removal efficiency, the moisture level may be monitored and adjusted to achieve peak pollutant removal consistent with the findings described above.
The nutrients and minerals required for maintenance and/or growth of the biomass will vary depending on the particular biomass used. The biomass may be bacterial or any other type of microorganism. The nutrients and minerals may be added directly to the biomass before its application to the contaminated structure or support. It may also be impregnated within the various supports so that plain water is all that is needed to maintain moisture levels. Alternatively, nutrients and minerals may be added to the water used to maintain moisture levels.
Additionally, ambient temperatures must be within a range that maintains viability of the microorganisms. Generally, the temperature range should be kept between 5° C. and 40° C.
The length of time needed for pollutant removal varies depending on the characteristics of the pollutant, the composition of the contaminated structure, and which embodiment of the present invention is used. Generally, the process will require about one to two months.
Once the desired amount of pollutant is removed from the contaminated structure, the support, if applicable, is removed, and the contaminated structure is cleaned with detergent and water and then with bleach. Most of the microorganisms used for this type of bioremediation are harmless, so a simple clean-up is all that is required.
Although the present invention has been described with reference to preferred embodiments, workers skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope of the invention.
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Number | Date | Country | |
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20040175818 A1 | Sep 2004 | US |