MARINEBIOTECH.ORG | Environmental Research

Introduction

It has been long-known that ecological systems possess a degree of innate capacity for breaking down pollutants or contaminants that enter them. The biological agents responsible for much of this self-cleaning are most often microbial in nature. The breakdown and elimination of environmental contaminants by living organisms is termed bioremediation.

Microbially mediated removal of contaminants from natural systems over time can occur entirely in the absence of human intervention. The process can also be given a 'jump-start,' through human management or resource input. Both of these bioremediation scenarios will be considered here.

Oil spills or other petroleum product contamination is most commonly reported as the target of bioremediation efforts in the marine environment. But, the strategy is also currently used or applicable in cleaning up contaminated groundwater, polluted industrial sites, pesticide-loaded estuaries, and other similar scenarios. The following material presents general marine biotech-based bioremediation strategies primarily as they are applied to the cleanup of oil spills in marine systems.

Historical Perspective

As far back as the early 1940s, the American Petroleum Institute was funding research into the use of microbial systems to clean up oil spills. More recently, the EPA Office of Research and Development (ORD), the US Geological Survey and others have focused considerable attention on the science of bioremediation. Much of this work was undertaken in earnest by USGS starting in the 1980s when Congress directed the agency to investigate the dynamics of toxic wastes within contaminated aquatic systems. A key finding of this research was that naturally occurring microorganisms play a central role in the transport and degradation of not only petroleum products, but also of nearly every toxic compound that enters the environment. Moreover, the research indicated that microbial breakdown of toxins could be greatly accelerated through the addition of nutrients.

Although significant insight into the bioremediation process had been gained over the years, bioremediation was not attempted on a large scale until the 1989 Exxon Valdez Alaskan oil spill (see below).

Select from the five broad bioremediation topics presented below to explore the role marine biotechnology is playing in advancing this critically important field.

Bioremediation Basics

While it is apparent from the above that bioremediation is not the only tool, it is increasingly utilized as part of an integrated cleanup response. A key appeal of biotech-based bioremediation in coastal systems is that it can be incredibly cost effective compared to alternative cleanup strategies.

One major positive aspect is that contamination is treated on site. Traditional cleanup of contaminated shoreline systems like mudflats and marshes usually entails the physical removal and safe disposal of large volumes of contaminated sediment. This accounts for most of the costs associated with traditional cleanup efforts in these habitats. Moreover, bioremediation actually breaks down the target contaminants, versus traditional strategies that simply remove them to another location. An additional benefit is that site disturbance is minimized and therefore so is the amount of disturbance-related environmental stress placed on adjacent habitats. For example, accidental smothering of large numbers of benthic organisms by sediments kicked up during dredging will not occur if polluted sediments no longer need to be removed for disposal.

The speed and efficiency with which bioremediated contaminants are broken down depends primarily on three variables: temperature, the concentration of potentially limiting nutrients, and the amount of oxygen available in the environment. Each of these is briefly described here.

Temperature: As with nearly all metabolic processes, contaminant biodegradation occurs more rapidly at warmer temperatures. Biologically mediated breakdown of pollutant spills is therefore expected to be more efficient in warmer environments.
Dissolved Oxygen: Except in the case of very large plankton blooms, the water column environment of the open ocean tends not to be oxygen limited. The same cannot be said for many marine sedimentary (soft bottom) communities that are often deficient in dissolved oxygen. As a consequence, aerobic (oxygen-requiring) metabolic processes responsible for much of the breakdown of oil and other contaminants are greatly curtailed in these systems. Material must instead be broken down through slower anaerobic (non-oxygen-requiring) microbial metabolic pathways. Intertidal mudflats, vegetated marshes and other coastal wetlands are marine environments where sediment oxygen concentrations can be limiting to microbial breakdown processes. These systems are examined in more detail in the following subsections.
Nutrients: Just as the metabolic functions of plants and animals can be negatively impacted by nutrient deficiencies, so too can the ability of natural microbial communities to biodegrade contaminants be compromised if essential nutrients are in short supply. The macronutrient nitrogen is usually the limiting nutrient for marine microbes, followed by phosphorus. If both of these nutrients are present in excess, it is possible that micronutrients like copper or iron then become limiting.

Micronutrients rarely become limiting in natural microbial systems, however, as only minute amounts are needed to sustain proper functions. One notable exception is found in the phytoplankton community in the Southern Ocean circling Antarctica. The Southern Ocean is an iron-deficient system, and the scarcity of naturally occurring iron is known to limit water column primary production and uptake of atmospheric CO₂ to supply photosynthesis. Some researchers have suggested that fertilizing the Southern Ocean through the addition of massive amounts of iron may be a viable strategy for removing excess atmospheric CO₂, the principal 'greenhouse gas' implicated in global warming. As yet, this controversial proposition remains the largest bioremediation project never attempted

Giving Nature a Hand

As previously noted, some degree of intrinsic bioremediation occurs in contaminated marine systems with no direct management or input from humans. This passive (other than perhaps an environmental monitoring component) bioremediation option is appealing from a practical and economic standpoint, and might even be the preferred approach if contamination is expected to remain localized and if it does not threaten human or environmental health beyond the immediate impact zone.

Of significance, however, is the fact that human intervention can enhance and accelerate the bioremediation process. If intrinsic bioremediation rates are too slow, for example, it may be possible to manipulate environmental conditions to stimulate contaminant biodegradation by the natural microbial community. Understanding that the natural system may need just a little nudge—in the form of added nutrients or oxygen, for example—makes the prospect of cleaning up large, long-impacted areas perhaps less intimidating.

The possible bioremediation strategies used in response to something like a marine oil spill can be divided into three approaches:

Intrinsic bioremediation is the removal through biotic means of contaminants from natural systems over time in the absence of human intervention.
Biostimulation entails letting the native microbial populations biodegrade the contamination, but 'jump-starting' these populations in some manner. This is most often through the addition of nutrient fertilizer. Providing supplemental oxygen or adding chemical electron donors or acceptors (e.g., nitrate or sulfate as electron acceptor alternatives to oxygen in anaerobic systems) are other possible avenues for enhancing natural biodegradation rates.
Bioaugmentation is the least common approach. This is essentially the 'superbug' strategy of introducing novel oil-degrading microbiota to supplement the breakdown capacity of the native populations. The added microbiota would be strains rare or absent from the natural microbial community at the remediation site, and may possibly be genetically engineered for their role as oil eaters as well. Not widely practiced, there are reasonable concerns surrounding this remediation strategy.

While decidedly less high-tech than many other applications of marine biotechnology, environmental remediation, particularly through biostimulation, is promising nevertheless. In truth, rarely do specialized microorganisms of any sort need to be introduced to oil-contaminated sites, as most natural systems are already home to microbial species known capable of breaking down spill components. For this reason, the biostimulation strategy is very often the one that makes sense.

Although naturally occurring oil-degrading communities rarely require augmentation (adding more or different biota), they apparently are population-limited under natural low-oil conditions. This contention is supported by research findings demonstrating that population sizes of such microbes exhibit a natural increase in the presence of oil contamination. Oil-degrading microbes usually account for less than 1% of the natural marine microbial community. In contrast, they can multiply to represent more than 10% of the population in contaminated locations.

Proof of Concept: Bioremediation of the Exxon Valdez Spill

Bioremediation protocols employed during cleanup of the infamous 1989 Exxon Valdez oil spill in Prince William Sound, Alaska, effectively demonstrated that application of fertilizer (nitrogen and phosphorus) led to a significant increase in the rate of biodegradation. Oil-contaminated shorelines treated with fertilizer appeared significantly cleaner within just a few weeks of the application.

Low level inputs of hydrocarbons to the oceans, in the form of geological seeps, pine droppings, etc., exist as natural marine ecosystem components. It is not surprising, therefore, that some lineages within the marine microbial community have evolved the ability to break down hydrocarbons and exploit the considerable energy stored in the chemical bonds of these compounds.

In the spring following the Valdez spill, a review panel concluded, "that the Alaska oil spill situation should be treated as a laboratory to increase the nation's knowledge and readiness for action in future oil spills." Recommendations were given to the EPA that called for experimental fertilizer application on small-scale test sites on oil-impacted shorelines to attempt to biostimulate the natural breakdown capacity of the system. Preliminary studies carried out in Prince William Sound revealed a diverse community of microbiota capable of degrading petrochemicals. This finding suggested that there was an ample natural population from which contaminated shorelines could be colonized. Surveys also indicated that populations of oil-eating microbes were elevated by several orders of magnitude on contaminated versus unimpacted shoreline sites.

With the determination made that natural microbial populations were not likely to need augmentation (addition of organisms), attention turned to the role of the environmental variables of dissolved oxygen, temperature, and nutrient availability. Oxygen was found not to be a limiting factor on contaminated shorelines, owing to the high permeability of pore spaces between the large (cobble and pebble) sediment size classes and the location of the shoreline in the well-mixed intertidal zone. The seasonal variability of sea surface temperatures suggested that biodegradation would proceed very slowly, if at all, during the winter months when surface temperatures reached 0°C (32°F), In the summer, however, surface temperatures climb as high as 20°C (68°F), allowing hydrocarbon breakdown to proceed apace during the warmest months of the year. Finally, significantly positive results from pilot studies that applied fertilizer to selected contaminated Prince William Sound shorelines implicated nitrogen and phosphorous as being limiting nutrients in the natural environment.

The results from these first summer field trials were very encouraging. Within two weeks of fertilizer application, cobble beaches on contaminated shorelines had visibly less oil coating them than untreated sites. Equally encouraging, water samples collected off of treatment sites showed no trace of nutrient elevation or eutrophication (algal growth due to nutrient enrichment), suggesting that fertilizer application was not impacting adjacent habitats. Follow-up tests confirmed that biodegradation (and not a physical or chemical weathering process) was the cause of the reduction in oil present on treated beaches. Based on positive early findings, bioremediation efforts were scaled up so that by the end of the summer of 1989, 74 miles of oil-contaminated shoreline had been treated with biostimulating nutrient fertilizer, with considerably more shoreline treated the following year. Since this time, biostimulation has been used with success in other oil-impacted sites as well.

Biostimulated remediation after Valdez, not surprisingly, did not completely remove oil contamination. The mixed gravel layers below the larger cobble rocks were still coated, and sites with smaller (sand and gravel) particle sizes exhibited less complete oil degradation than cobble beaches. Additionally, within approximately two months the visible contrast between sites that had been fertilized and the untreated control sites had diminished.

The apparent key benefit of biostimulation in the case of Valdez, then, was that it sped up the natural recovery process and reduced the possibility of exposure of wildlife to dangerous oil contamination. The current view is that biostimulation can shorten the recovery time for severely oil-impacted shorelines to as little as 2-5 years, compared with 5-10 years if sites are left untreated. With the ability to cut the time to site recovery in half, a strong argument exists in favor of biostimulated remediation as a cost effective component of a marine spill response strategy.

Bioremediation Strategies in Marshes

The abundance of indigenous microbial marsh life adapted to break down and consume organic hydrocarbon products in carbon-rich, vegetated systems makes biostimulation a cleanup option worth pursuing. Often, however, addition of nutrients to oil-impacted coastal wetlands site does not substantially enhance the rate of biodegradation. In such cases, soil anoxia (lack of oxygen), not nutrients, is the likely limiting factor.

Breakdown of oil contamination in marine sediments is often confounded by a lack of sufficient dissolved oxygen. This may be particularly true where there is not a large, healthy marsh plant community that can aerate the soil through its root/rhizome systems. Marsh remediation sites with an established plant community, by contrast, benefit from the oxygenation of sediments through permeable root and rhizome systems. This may permit aerobic degradation of contaminants to proceed at a reasonable pace.

In unvegetated sediments, or in impact sites where proper functioning of the vegetation has been compromised, the addition of soil oxidants like calcium peroxide, manganese oxide, and nitrate might be considered.

Ralph Portier of the LSU Department of Environmental Studies notes that there also has been success using surfactant shoreline cleaners that lift oil out of the oxygen-deficient sediments without actively dispersing it. Such managed use of surfactants (and possibly dispersants) to maximize oil biodegradation in shoreline habitats merits continued investigation.

In impacted marsh sites where oxygen is limited and biostimulation is not attempted, some natural microbial degradation of petrochemical contaminants still occurs, albeit slowly. In the absence of oxygen, anaerobic microbes use other inorganic electron acceptors and metabolic pathways to break down pollutants over time. Though the rate of natural breakdown is slow (months to years), the cumulative beneficial impact on the habitat over time is likely important. It may be possible, however, to accelerate the process in anaerobic soil conditions through the addition of alternative electron acceptors. For example, sulphate is an important electron acceptor in the chemical ecology of anoxic marine sediments. As long as sulphate remains abundant, sulphate-reducing bacteria like Desulfobacula and Desulfobacterium can make slow but steady progress in the oxidation of sediment hydrocarbons.

An added benefit to employing the biostimulation strategy in wetlands is that fertilization stimulates growth of the marsh vegetation along with the microbiota. Healthy wetland vegetation removes some of the soil contamination through direct uptake, and it also enhances the environment for the soil microbes by leaking oxygen from root/rhizome systems and increasing the capacity for aerobic biodegradation as previously noted.

Phytoremediation

Effective as a healthy marsh plant community can be in promoting self-cleansing of the habitat, phytoremediation is another marine biotechnology-based shoreline contamination remediation strategy that warrants consideration. Phytoremediation is the planting of suitable marsh vegetation to facilitate in situ bioremediation and also to aid in recovery and restoration of the impacted habitat. When practiced, phytoremediation is most often carried out in combination with fertilizer addition (biostimulation), which benefits both the natural and planted vegetation as well as the oil-degrading soil microbiota.

The Future of Bioremediation

There is still a lot of work to be done so that the bioremediation methods can be perfected. When and how to apply fertilizer, how much to apply, whether to add micronutrients, whether to use slow-release or highly soluble forms, etc., are all questions that bioremediation experts are currently examining.

The biggest problem with the current state-of-the-art of bioremediation is that it is still very much an empirical, trial-and-error endeavor. Protocols developed for one scenario in a particular locale may not prove effective in other locations or under different conditions. The reasons underlying the variable success rates are usually not apparent. But, they point out the problem that microbial metabolism and growth in contaminated environments are still poorly understood. Marine biotechnology can assist in improving this situation.

As the specific genes involved in bioremediation are identified there emerges the possibility of using specific mRNA probes to determine the degree to which a microbial community is actively transcribing the enzymes involved in bioremediation. Combining such probes with techniques for sequencing highly conserved microbial genes (eg, the 16S ribosomal RNA gene, variants of which are present in all microbes.) should allow the eventual generation of data sets relating which microbial lines are most involved in bioremediation processes.

Detailed knowledge about the physiological aspects and bioremediation capacity of specific microbial strains may also begin to emerge as whole-genome sequencing studies are initiated for key biodegrading taxa. Once entire genomes are elucidated, whole-genome DNA microarray analyses would allow detailed examination of the expression of all the genes in the genome under a variety of environmental conditions.

Filling in the knowledge gaps in this manner should allow the development of multivariate mathmatical models that will aid in predicting how diverse assemblages of biodegrading microorganisms will perform under defined sets of conditions, and how they will respond to changes in those parameters.

REFERENCES

Atlas, RM 1981. Microbial Degradation of Petroleum Hydrocarbons: an Environmental Perspective. Microbiological Reviews 45:180-209.

Chanelli, RR. 1991. Bioremediation technology development and application to the Alaskan spill. Proceedings, International Oil Spill Conference, March 4-7 1991, San Diego CA. U.S. Coast Guard, American Petroleum Institute, and U.S. Environmental Protection Agency 1991:549-558.

EPA, Office of Research and Development. 1990. Alaskan Oil Spill Bioremediation Project (EPA/600/8-89/073).

Lovley, DR. 2003. C leaning up with genomics: applying molecular biology to bioremediation. Nature Reviews Microbiology 2003:35-44.

Portier, RJ. 2000. Contributions of marine biotechnology to marsh oil spill restoration. Pp 61-67 In: Opportunities for Environmental Applications of Marine Biotechnology: Proceedings of the October 5-6 1999 Workshop. National Academy Press, Washington, DC.

Pritchard, PH. 1991. Bioremediation as a technology: experiences with the Exxon Valdez oil spill. Journal of Hazardous Materials 28:115-130.

Pritchard, PH, Mueller JG, Rogers JC, Kremer FV, and Glaser JA. 1992 Oil spill bioremediation: experiences, lessons and results from the Exxon Valdez Oil Spill in Alaska. Biodegradation 3:315-335.

U.S. Congress, House of Representatives. 1989. Investigation of the Exxon Valdez oil spill, Prince William Sound, Alaska. Oversight hearing before the Subcommittee on Water, Power and Offshore Energy Resources of the Committee on the Insular Affairs. One Hundred First Congress, First Session on Oil Spill Cleanup Technology (Serial No, 101-5 Part II).