|
|
|
| List of Illustrations | Part II - Application of Bioremediation
Technology
Appendix A Snug Harbor Test Site
|
Find more information visit these web sites:
|
|
In this paper I will define bioremediation, provide background on how bioremediation works (metabolic pathways) and discuss many factors. I will also discuss the application of this technology in the first year after the Exxon Valdez oil spill.
In the simplest terms, bioremediation is the use of microorganisms (fungi or bacteria) to decompose toxic pollutants into less harmful compounds. In this paper, the term bioremediation is used in the context of promoting the degradation of petroleum hydrocarbons (oil).
To fully understand bioremediation we must discuss the term biodegradation. Biodegradation is a natural process by which microbes alter and break down oil into other substances. The resulting products can be carbon dioxide, water, and simpler compounds that do not affect the environment.
Bioremediation is the optimization of biodegradation. This acceleration can be accomplished by two forms of technology: (1) fetilizing (adding nutients) and/or (2) seeding (adding microbes). These additions are necessary to overcome certain environmental factors that may limit or prevent biodegradation.
Knowlege on bioremediation acquired since 1942 allows us to manipulate environmental factors to enhance natural biodegradation. This knowledge includes:
The Alaskan bioremediation study wanted to prove if bioremediation would work, so a number of experiments were done to accumulate empirical data. The results were very promising. It was estimated that a spill that would normally take five to ten years for natural conditions to return could be returned to natural conditions in as little as two to five years through the use of bioremediation.
Bioremediation is not limited to marine oil spills; it has potential to help clean up land oil spills, pesticides and hazardous waste. There are also benefits to bioremediation such as saving money, being ecologically sound, destroying contaminates (not moving them form one place to another) and treating waste on site.
The application of bioremediation will be an important aspect of waste management now and into the future as more is learned about this technology.
Research on the use of bioremediation to clean up oil spills dates back to 1942 when the American petroleum Institute began subsidizing this research (U.S. Congress 1991a, p.1). A substantial amount of basic knowledge has been acquired since that time. In Part One of my paper I will explain the basic scientific knowledge that has been gained.
Even though we know generally how bioremediation works, little progress has been made in the application of this knowledge to marine oil spills (EPA 1991, p.2). More information about applied bioremediation came from the Alaskan oil spill than from any other incident. I will examine the Alaska oil spill in part two of my paper.
What is bioremediation? In the simplest terms, bioremediation is the use of microorganisms (fungi or bacteria) to decompose toxic pollutants into less harmful compounds. In this paper, the term bioremediation is used in the context of promoting the degradation of petroleum hydrocarbons (oil).
To fully understand bioremediation we must discuss the term biodegradation. Biodegradation is a natural process by which microbes alter and break down petroleum hydrocarbons into other substances. The resulting products can be carbon dioxide, water, and partially oxidized biologically inert by-products (Bragg et al. 1992, p6). Bacteria that consume petroleum are known as "hydrocarbon oxidizers: because they oxidize compounds to bring about degradation (Chianelli et al. 1991).
Bioremediation is the optimization of biodegradation. This acceleration can be accomplished by two forms of technology: (1) fertilizing (adding nutrients) and/or (2) seeding (adding microbes). These additions are necessary to overcome certain environmental factors that may limit or prevent biodegradation.
Certain enzymes produced by microbes attack hydrocarbons molecules, causing degradation. The degradation of oil relies on having sufficient microbes to degrade the oil through the microbes’ metabolic pathways (series of steps by which degradation occurs). Fortunately, nature has evolved many microbes to do this job. Throughout the world there are over 70 genera of microbes that are known to degrade hydrocarbons (See table 1) (U.S. Congress 1991a, p.9). These microbes usually account for less than 1% of natural populations of microbes, but can account for more than 10% of the population in polluted ecosystems (U.S. Congress 1991a, p.9).
If microbes are not present in a system they can be added to help promote bioremediation. The added microbes can be cultures grown from other contaminated areas or they can be microbes genetically engineered to degrade oil. However, even when these microbes are present, degradation of hydrocarbons can take place only if all other basic requirements of the microbes are met.
Microbes are dependent on nutrients for survival. These nutrients are the basic building blocks of life and allow microbes to create the necessary enzymes to break down hydrocarbons. Although nutritional requirements vary among microorganisms (Atlas 1984, p. 333) all of them will need nitrogen, phosphorous and carbon. The survival of a microorganism depends on whether or not it can meet it’s nutritional needs.
Carbon is the most basic structural element of all living forms and is needed in greater quantities than other elements. The nutritional requirements of carbon to nitrogen 10:1 and carbon to phosphorus 30:1 (Atlas and Bartha 1981, p.70). Reduced organic carbon is a source of energy for microbes because it has high energy yeilding bonds in many compounds. In the decomposition of oil, there is plenty of carbon for the microorganism due to the structure of the oil molecule.
Nitrogen is found in the proteins, enzymes, cell wall components, and nucleic acids of microorganisms (Atlas and Bartha 1981, p.70). "Microorganisms must …be supplied nitrogen in some form" (Wistreich and Lechtman 1988, p.90) because without it, the microbial metabolism will be altered (Atlas and Bartha 1981, p.70).
"Because molecular nitrogen can be used by only a few microorganism, most microorganisms require fixed forms of nitrogen, such as organic amino nitrogen, ammonium ions, or nitrate ions" (Atlas 1984, p.333). These other forms of nitrogen can be scarce in certain environments, causing nitrogen to become a limiting factor in the growth of microbial populations.
Phosphorous is needed in the membranes (composed of phospholipids), ATP (energy source of cell) and to link together nucleic acids (Atlas and Bartha 1981, p.70).
Environmental Requirements for Microbial Growth
Along with nutrients, microbes need certain conditions to live. Because microbial growth and enzymatic activity are affected by stress from the following factors, the rate s of biodegradation will also be affected. As the stress increases (less favorable conditions occur) the microbes have a harder time living in their environment. Just as humans need certain conditions to live (like oxygen) so do microbes . There is a certain range of conditions in which microbes can live. As conditions reach the extremes microbial growth slows down, but when conditions are perfect the microbial community can thrive.
Biodegradation is predominantly an oxidation process. "Bacteria enzymes will catalyze the insertion of oxygen into the hydrocarbon so that the molecule can subsequently be consumed by cellular metabolism" "Bragg et al. 1991, p.6). Because of this, oxygen is one of the most important requirements for the biodegradation of oil. There is usually enough oxygen to prevent a lack of it from limiting biodegradation (U.S. Congress 1991a, p.10). The primary source of oxygen for biodegradation is atmospheric oxygen.
When oxygen is limiting, the water can be aerated to allow biodegradation to take place. An example of this is in wastewater treatment plants when oxygen is added in the aeration basin. Oxygen is important in hydrocarbon degradation because the major pathways for both saturates and aromatic hydrocarbons involve molecular oxygen or oxygenases (Atlas 1981, p.195). Theoretical calculations show that 3.5g of oil can be oxidized for every gram of oxygen present (Atlas 1981, p.195).
The current evidence for anaerobic degradation of hydrocarbons is not considered of ecological importance because the rate is so negligible (U.S. Congress 1991a, p.10: Atlas 1981, p.194), even though some microbes have been found to degrade alkanes under anaerobic conditions, when isolated (Atlas 1981, p.194). there have not been many reports of hydrocarbons being anaerobically degraded in natural ecosystems (Atlas 1981, p.194). However, "nitrate of sulfate could serve as an alternative electron acceptor" (Atlas 1981, p.194) instead of oxygen during anaerobic microbial degradation of petroleum hydrocarbons.
Water is needed by microorganisms since it makes up a large proportion of the cell’s cytoplasm. Water is also important because most enzymatic reactions take place in solution. Water is also needed for transport of most materials into and out of the cell (Atlas and Bartha 1981, p.70).
Water is not a limiting factor in marine oil spills, but in bioremediation of oil spilled on land it may be an important factor which needs to be controlled.
"Several variables, including pressure, salinity, and pH, may also have important effects on biodegradation rates" (U.S. Congress 1991a, p.11). In the natural environment, these factors are not a major problem where populations of microorganisms naturally exist, which is good since these variables are not easily controlled in the environment.
Although hydrocarbon degradation has been found to occur at a wide range of temperatures (as low as below 0°C to as high as 70°C) it is an important factor on the rate of biodegradation (Atlas 1981, p.190).
The temperature is so important because "at low temperatures, molecules move relatively slowly, and colliding molecules do not always bring about a reaction" (Atlas 1984, p.339). Raising the temperature will increase the possibility of reactions taking place and increase the rate of diffusion. Without reactions and diffusion life cannot exist. In general the rate of enzymatic reactions can be doubled for every 10°C rise in temperature as long as the enzymes are not denatured (Atlas 1984, p.339). The more enzymatic reactions the faster the biodegradation will occur.
Even though temperature plays an important part in the rate of biodegradation, it does not act alone. The composition of the microbial community and the quality of the oil can affect the rates of biodegradation just as much.
]The concentration of pollutants is an important factor. If the concentration of petroleum hydrocarbons is too high then it will reduce the amount of oxygen, water and nutrients that are available to the microbes. This will create an environment where the microbes are stressed reducing their ability to break down the oil.
Metabolic Pathways for Oil Decomposition
Over the last 20 years complex chemical equations have been derived to describe the metabolic pathways in which oil is broken down. "The general outline bioremediation pathways for aliphatic and aromatic hydrocarbons have been formulated and continues to be developed in greater detail with time" (Glaser, Venosa and Opatken 1991, p.559). All of these pathways will result in the oxidation of at least part of the original hydrocarbon molecule (Bragg et al., p7). The content of a particular petroleum mixture will also influence how each hydrocarbon will degrade (Atlas 1981, p.182) and the type and size of each hydrocarbon molecule will determine the susceptibility to biodegradation (Atlas and Bartha 1993, p.394).
"There are several hundred individual components in every [type of]
crude oil, and the composition of each crude oil varies with its origin"
(Atlas and Bartha 1993, p.394). The difference in composition determines
the quality of any particular oil. Petroleum is a complex mixture of hydrocarbons,
but it can be fractionated into aromatics, aliphatics, asphaltics and a
small portion of non-hydrocarbon compounds (Atlas and Bartha 1993, p.394;
Atlas 1981, p180).
Condensed aromatic rings are attacked one ring at a time if they are
degradable (See Figure 3). The first
ring is opened and reduced to pyruvic acid and CO2, then the next ring
is attacked in the same manner (Atlas and Bartha 1993, p396).
"Some microorganisms attack alkanes subterminaly; that is, oxygen is inserted on a carbon atom within the chain instead of at it’s end" (figure 5) (Atlas and Bartha 1993, p. 394).
Alicyclic hydrocarbons with no terminal methyl groups are biodegraded in a manner similar to the subterminal oxidation. Cyclohexane, an alicycliic hydrocarbon, is degraded as shown in figure 6. (Atlas and Bartha 1991)
once fatty acids (molecules with the general formula CnH2n+1COOH) are formed, the process of beta-oxidation will continue the catabolism. Beta-oxidation will form acetate and a new fatty acid, containing two less carbons then the original (Atlas and Bartha 1993 p.395). This process will repeat itself until the compound is completely broken down. The hydrocarbon will eventually be degraded to CO2 and H2O through the process of hydrocarbon mineralization (Atlas and Bartha 1993 p.395). "The beta-oxidation sequence does not necessarily require the presence of molecular oxygen, fatty acid biodegradation may proceed under anerobic conditions" (Atlas and Bartha 1993 p.395).
By using the science of microbiology and chemistry, scientists have been able to recognize the processes and factors that allow biodegradation to take place. This same knowledge allows us to try to increase these natural processes (bioremediation). By manipulating the environmental factors, we can hope to achieve results faster than occur naturally.
Unfortunately there is no formula that will allow bioremediation to work every single time. Each situation will be unique and will require study to determine its particular needs. In this part of the paper I will examine one such situation and how the knowledge of this technology was used to enhance the the biodegradation of the spilled oil from the Exxon Valdez disaster.
South Central Alaska is beautiful and unique. Prince William Sound, surrounded by land from the Chugach National Forest, has many islands, bays, and fjords, giving it more than 2,000 miles of shoreline and making it one of the nation’s largest relatively undeveloped marine ecosystems (U.S. Congress 1991b, p.75-76). Nearby on the Kenai Peninsula is Keni Fjords National Park and Kachemack Bay State Park. Still further southwest lies the Katmal National Park and Preserve. This entire region also has many national Wildlife Refuges.
Prince William Sound, described as a pristine (unaffected by man) environment, is home to a multitude of organisms. Numerous mammals make their homes in these water, including humpback whales, sea otter and sea lions. Many seabirds also live here including bald eagles, black oyster catchers, common murres and harlequin ducks. Many other organisms live in, on and around the waters of this area.
People also live in this region. Valdez, population about 4,000 (AAA 1993, p.220), is about 110 miles from Anchorage, Alaska’s largest city and home to half of the state’s population. The port of Valdez Alaska, the state’s northernmost ice free port, lies at the southern terminus of the Alyeska pipeline in South Central Alaska.
The state of Alaska is heavily dependent on its natural resources, with 80% of the state treasury connected to oil (Keeble 1991, p.15). Other natural resources are timber, primarily from Chugach and Tongass national forests, and fishing from various small villages. Entire town economies are supported by fishing.
It was here in South Central Alaska on Bligh Reef that the Exxon Valdez oil tanker ran aground on March 24, 1989, a few minutes after midnight. The spill released 20% of the ship’s cargo, amounting to about 11 million gallons (258,000 barrels) of Prudehoe Bay Oil, in about five hours.
A storm on March 26 helped spread the oil to the west, contaminating the western bays and islands of Prince William Sound in the following weeks and eventually spreading to the Gulf of Alaska (Spaulding and Reed 1990, p.426). The spill area eventually covered about 15% of the total shoreline in Prince William Sound and the Gulf of Alaska (Spaulding and Reed 1990, p.426). The spill area eventually covered about 15% of the total shoreline in Prince William Sound and the Gulf of Alaska (Bragg et al. 1992, p.1). The hardest hit areas were those islands of Prince William Sound directly in the path of the oil slick (Bragg et al. 1992), amounting to about 300 miles of rocky coastline (Pritchard 1991). The affected areas where remote and rugged, some having vertical cliffs. The path of the oil traveled to the southwest, as shown in map 1, resulting in the biggest oil spill in U.S. history.
Decisions had to be made. The oil was spreading and contaminating more
and more beaches every day. Clean-up methods had to be decided upon to
prevent further spreading of the oil. Among the clean-up methods available
was the potential use of bioremediation.
 |
Whether to use bioremediation ...
"For several years, the EPA Office of Research and Development (ORD) has been studying the microbial degradation of oil as part of its long-term research program" (EPA 1989, p.7). However, little research had been done on the bioremediation of oil from contaminated beaches. With years of accumulated information, scientists wanted to see if they could apply current knowledge to help them clean up the oil on the beaches from the spill. So a panel of experts in this field was assembled on April 17-18, 1989, to discuss the feasibility of using bioremediation in Alaska, and they 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" (McDonnell 1992, p.102). Their recommendations to the EPA administrator included a recommendation for fertilizer applications (the addition of nutrients) on small scale plots (McDonnell 1992, p.102).
The technology for bioremediation was known. But could the technology be applied to an environment so cold? Were there favorable conditions for degradation of petroleum hydrocarbons? Is Prudehoe Bay Oil of sufficient quality for biodegradation? These issues were important if bioremediation was going to be used, especially since "the complexity of the field applications and the problems associated with demonstrating successful bioremediation on a large scale were substantial" (Pritchard et al. 1992, p.314).
Suitability for Bioremediation
Prudehoe Bay Oil was known to be a high quality crude which would have different rates of decomposition depending on environmental factors (Cook and Westlake 1973, p.89). They knew that this type of oil was degradable from past studies; now scientists needed to know the concentrations of native hydrocarbons degraders.
Hydrocarbons have been naturally added to the environment by pine tree droppings and natural seeps for millions of years. The high energy yield in the carbon-hydrogen bonds of hydrocarbons is an excellent energy source that allowed a complex community to evolve over millions of years to degrade these hydrocarbons (Chianelli et al. 1991).
Numerous oganisms were found in Prince William Sound which worked together
as a complex community to degrade petroleum and it’s products (Chianelli
et al. 1991). For these reasons it was thought that microbes would quickly
colonize the area and begin to degrade the spilled oil (Prichard 1991).
"Research by Atlas and his colleagues supported this assumption" and their
results are shown in Table 2 (Atlas 1991 as referenced in Prichard 1992).
As you can see from table 2, there was a 10,000-fold increase of oil-eating
microbes in contaminated areas over the amount in non-contaminated areas
(Prichard 1992, p.2).
|
|
|
|
|
|
limiting factors were the next concern of the scientist. They knew that biodegradation was occurring, but they needed to find what was limiting the activity of the microbe in order to speed up the process.
"Several investigators have reported that concentrations of available nitrogen and phosphorous in seawater are severely limiting to microbial hydrocarbon degradation", yet others report that nitrogen and phosphorous are not limiting (Tabak et al. 1991, p.581). Tabak explains the difference in opinions on the aims of each researcher’s study (Tabak et al. 1991, p.584). In Prince William Sound fertilizer solution applications had striking results that supported the idea of nitrogen and phosphorous as being limiting nutrients (Prichard 1991, p.115).
Oxygen was not a rate limiting factor in biodegradation in Alaska "because of the generally large size of the sediment, it’s high permeability into sea water and the ample content of dissolved oxygen in seawater flushed through the sediments during each of the two daily tide cycles" (Bragg et al. 1992, p.8).
"Temperature of surface waters are seasonably variable, ranging from 32oF in the winter to as high as 68oF in the summer" (Bragg et al. 1992, p. 13), thus slowing down the degradation in the winter months. With natural oil degrading populations present it was known that the water was not too cold for the organisms to degrade the hydrocarbon throughout the summer.
Alaskan Bioremediation Project
The information on native microbes, limiting factors and Prudehoe Bay Oil was gathered and used to set up the Alaskan Bioremediation Project. This study was designed to determine the feasibility of biodegradation enhancement by adding fertilizers. To accomplish this objective, field studies were necessary. This project is now know as the largest bioremediation study ever done.
To study bioremediation, numerous tests were done in the laboratory "to test the feasibility of applying microbial nutrients to oiled beaches in prince William Sound" (Chianelli et al. 1991, p.550).
EPA and Exxon began field testing of bioremediation in May of 1989 on Knight Island (Chianelli et al. 1991, p.551). Nutrient applications began on June 8, 1989 (EPA 1990, p.6). Snug Harbor, located on the southeastern side of the island (Appendix A), was selected as the first test site (EPA 1990, p.6) because it had a long length of shoreline with several beach materials (Glaser, Venosa and Opatken 1991, p.560). "The Snug Harbor beaches had a moderate degree of oil contamination confined to a broad band within the intertidal zone" (Prichard 1991, p.117). This site was chosen to model conditions after physical washing, since none were available at the time (Prichard 1991, p.117).
Six beach plots with two types of beach materials were chosen for study. On these plots two different fertilizers were tested (See Appendix A): the IBDU briquettes and oleophilic fertilizer (EPA 1990, p.6). To monitor the effects on the environment of the area, an ecological monitoring program was established (U.S. Congress 1989, p.278).
The results of this test were that no detectable nutrients ended up in the waters off the test site and there was no evidence of eutrophication (excess alga growth due to high levels of nutrients)(U.S. Congress 1989, p.280). Approximately 10 to 14 days after the applying oleophilic fertilizers on the cobble beach, visual reductions in the amount of oil covering the rocks were apparent (Pritchard 1992 et al., p.322). Further tests showed that the reduction was due to bioremediation (Pritchard et al. 1992, p.323), although oil remained in the mixed gravel below the rocks (EPA 1990, p.8). There was also a visual reduction in the oil-covered sand and gravel beach treated with oleophilic fertilizers, although it was not as striking as the cobble stone beach (EPA 1990, p.8). All other plots remained oiled; however, "six to eight weeks after fertilizer applications, the contrast between treated and untreated areas on the cobble beaches has lessened (Pritchard et al. 1992, p.323).
Because the oil disappeared from the beaches naturally showing no differences after six to eight weeks, questions have been raised as to whether bioremediation is cost effective (Pritchard et al. 1992, p.323). The major benefit of enhancing biodegradation is the reduced possibility of exposure for wildlife (Pritchard et al. 1992, p.323).
A variety of chemical analyses were done in the laboratory to be sure that the cleaning action was the result of biodegradation and not chemical cleaning (EPA 1990, p.11). "Based on the promising results of the inital field test at Snug Harbor and the absence of any adverse effects on the area’s ecosystem, EPA recommended to Exxon in July that the bioremediation efforts be scaled up during the remainder of the summer" (EPA 1990, p.9).
The next field tests done were at Passage Cove on the northwestern side of Knight Island. Passage Cove on the northwestern side of the Knight Island. Passage Cove had been physically washed by Exxon before bioremediation, but oil was still found as much as two feet below the surface (EPA 1990, p.9). The physical washing was found to facilitate the decomposition by spreading the oil into a thin layer over a large area of gravel and rock (EPA 1990, p.9).
At Passage Cove an aqueous fertilizer solution and a combination of inipol-customblen fertilizer were tested (Bragg et al. 1992, p.21). Within two weeks of application the treated beaches, both the oil beneath the cobblestone, were significantly cleaner than the reference plot which showed no oil loss (EPA 1990, p.9).
By the end of the summer enough data were collected and analyzed to make a decision on what to do. The potential benefits of reducing wildlife exposure to oil allowed the EPA to support a proposal for application of nutrients to oil covered beaches (EPA 1989, p.15). By the end of the summer of 1989, 74 miles of shoreline were treated with nutrient applications (EPA 1990, p.13).
The winter months were used to answer questions that were raised from the summer research in order to improve the effectiveness of bioremediation the following summer. The questions that were examined that winter were:
despite complications, scientists were able to show statistically that bioremediation decomposes oil faster than biodegradation (Pritchard 1991, p.125). The results of all tests showed that biodegradation can be enhanced about two to three fold (Pritchard 1991, p.125). This means that an oil spill that would take five to ten years to degrade can be degraded in as little as two to five years. This acceleration of clean-up time, if proven to be true in other bioremediation efforts, could give bioremediation technology a bright future.
Bioremediation is not only used to clean up after oil spills; the same basic science discussed in part I of this report can be used to remediate many environments. The EPA claims that bioremediation is a technology with enormous promise for the future (EPA 1990, p.20). The process of bioremediation is similar to wastewater treatment where we rely on microbes to clean our water for us. As long as we give them a favorable environment they do a pretty good job.
Some of the areas where bioremediation is still relatively new but it has the potential of saving money, being ecologically sound, destroying contaminates and allowing for the treatment waste on site (EPA 1991, p.7). The application of bioremediation will be an important aspect of waste management now and into the future.
| Top | References |
