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Bioremediation for Marine Oil Spills

May 1991

OTA-BP-O-70

NTIS order #PB91-186197

CONGRESS 01' THE UNITED SH,rwOfFICE OF TECMNOlOOY . l S S E S S ~ E N T

..

•



Recommended Citation:

U.S. Congress, Office of Technology Assessment, Bioremediation for Marine Oil Spills— Background Paper, OTA-BP-O-70 (Washington, DC: U.S. Government Printing Office, May1991).

For sale by the Superintendent of DocumentsU.S. Government Printing Office, Washington, DC 20402-9325

(order form can be found in the back of this report)



Foreword

If anything positive has resulted from the massive Exxon Valdez oil spill, it is that this

environmental calamity increased the Nation’s awareness of the shortcomings of its capabilityto fight oil spills and prompted it to take steps to correct this situation. In the 2 years since thisspill occurred, Congress passed major oil pollution legislation (the Oil Pollution Act of 1990);private industry established and provided significant funding for the Marine Spill ResponseCorp.; and Federal agencies reevaluated their responsibilities, revised contingency plans, andtook steps to improve response technologies.

In addition to concern with refining the efficiency and reliability of the existing responsetechnologies, both the public and private sectors have sought to develop innovative newtechnologies for responding to spills. bioremediation is one such technology. Although the

possibility of using the capabilities of oil-degrading microorganisms to accelerate the naturalbiodegradation of oil has been discussed for years, it is only recently that some of the practicalproblems associated with this idea have begun to be addressed. The Exxon Valdez spill, inparticular, gave researchers a rare opportunity to evaluate the feasibility of using bio-remediation as an oil spill countermeasure.

This OTA background paper evaluates the current state of knowledge and assesses thepotential of bioremediation for responding to marine oil spills. Our basic message is a dualone: we caution that there are still many uncertainties about the use of bioremediation as a

practical oil spill response technology; nevertheless, it could be appropriate in certaincircumstances, and further research and development of bioremediation technologies couldlead to enhancing the Nation’s capability to fight marine oil spills. The request for this studycame from Senator Ted Stevens, a member of OTA’s Technology Assessment Board, and thesenior senator from the State of Alaska, where bioremediation was tested on beaches pollutedby oil from the Exxon Valdez.

w D i r e c t o r

. . .

Ill



OTA Project Staff

bioremediation for Marine Oil Spills

John Andelin, Assistant Director, OTAScience, Information, and Natural Resources Division

Robert W. Niblock, Oceans and Environment Program Manager

William E. Westermeyer, Project Director

ContributorFlorence Poillon, Editor

Administrative Staff

Kathleen Beil

Sally Van Aller



Contents Page

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1SUMMARY AND FIlNDINGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

BACKGROUND . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

The Fate of Oil in the Marine Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Biodegradation and the Chemical Nature of Petroleum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Microbial Processes and the Degradation of Petroleum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Environmental Influences on Biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

General Advantages and Disadvantages of bioremediation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12ALTERNATIVE bioremediation TECHNOLOGIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Nutrient Enrichment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

Seeding With Naturally Occurring Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Seeding With Genetically Engineered Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

ENVIRONMENTAL AND HEALTH ISSUES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18BIOREMEDIATION IN RELATION TO OTHER RESPONSE TECHNOLOGIES . . . . . . . . . 20bioremediation ACTIVITIES IN THE PUBLIC AND PRIVATE SECTORS . . . . . . . . . 23

The Environmental Protection Agency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

NETAC and Commercialization of Innovative Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26The Private Sector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , 27

REGULATORY ISSUES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28APPENDIX A: GLOSSARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29APPENDIX B: ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Boxes Box Page

A. bioremediation v. Biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2B. The Alaska bioremediation Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Figures Figure Page

l. Schematic of Physical, Chemical, and Biological Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62. organization of the bioremediation Action Committee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

TablesTable Page

l. Major Genera of Oil-Degrading Bacteria and Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92. bioremediation: Potential Advantages and Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

3. Principal Features of Alternative bioremediation Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

4. Key Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26



2 q bioremediationfor Marine Oil Spills

Box A—bioremediation v. Biodegradation

Biodegradation refers to the natural processwhereby bacteria or other microorganisms alter andbreak down organic molecules into other sub-stances, such as fatty acids and carbon dioxide.

bioremediation is the act of adding materials tocontaminated environments, such as oil spill sites,to cause an acceleration of the natural biodegrada-tion process.

Fertilization is the bioremediation method of adding nutrients, such as nitrogen and phospho-rus, to a contaminated environment to stimulatethe growth of indigenous microorganisms. Thisapproach is also termed nutrient enrichment.

Seeding refers to the addition of microorgan-isms to a spill site. Such microorganisms mayormay not be accompanied by nutrients. Currentseeding efforts use naturally occurring micro-organisms. Seeding with genetically engineered

microorganisms (GEMs) may also be possible,but this approach is not now being considered forremediating oil spills.

tion of the microorganisms involved in biodegrada-tion had already been developed by the early 1970s.For example, the Office of Naval Research spon-sored about 15 basic and applied research projects in

the late 1960s and early 1970s on oil biodegradationas part of the charge to the Navy at that time to takethe lead in mitigating marine oil pollution. In 1972,a workshop on the microbial degradation of oilpollutants was sponsored by the Office of NavalResearch, the U.S. Coast Guard, and the U.S.Environmental Protection Agency (EPA).

6The 1980s

were a period of rapid advances in knowledge of thegenetics and molecular biology of bacterial degrada-

tion of different hydrocarbons, and of renewedinterest in the microbial ecology of pollution-stressed environments.

Much progress has been made in applying basicknowledge of biodegradation to cleaning up terres-trial and enclosed sites polluted with oil. As long agoas 1967, contractors employed bioremediation toimprove the quality of 800,000 gallons of oilywastewater remaining in the bilge tanks of the

Queen Mary after it was permanently moored inLong Beach Harbor.

7City officials approved the

discharge of this bilge water 6 weeks after treatment.More importantly, a large number of refineries, tank farms, and transfer stations now employ in situbioremediation to restore land contaminated by

accidental spills of fuel oil or other hydrocarbons.8

Much less progress has been made with respect tothe practical problems of applying bioremediationtechnologies to marine oil spills, although advocates

have suggested their use in the wake of several majorspills. The problems associated with using bioreme-diation technologies in marine environments arefundamentally different from those associated withland-based applications. Although bioremediationof oil-contaminated soil is one of the fastest growinguses of this technology, bioremediation applicationson land have all been accomplished in closed orsemi-enclosed environments where microorganisms

have little or no competition and where conditionscan be closely controlled and monitored. The marineenvironment is a dynamic, open system that is muchless susceptible to control, and many additionalvariables exist to compound the difficulties of applying bioremediation techniques. One of themost important series of tests and the first large-scale application of a bioremediation technology ina marine setting was conducted between 1989 and

1991 by the EPA, Exxon, and the State of Alaska onthe Prince William Sound beaches fouled by oilfrom the Exxon Valdez.

SUMMARY AND FINDINGS

Biodegradation is a natural process, and there isno question that, with enough time, microorganismscan eliminate many components of oil from the

environment. The central concern of this study iswhether bioremediation technologies can acceleratethis natural process enough to be considered practi-cal, and, if so, whether they might find a niche asreplacements for, or adjuncts to, other oil spillresponse technologies. The key findings from thisOTA study are summarized below:

. The usefulness of bioremediation for marineoil spills is still being evaluated, and their

6D.G. AIMXUII and S.P. Meyers (eds.), The MicrobialDegradation of Oil Pollutants (proeeedi.ngs of a workshop at Georgia State hiversity, A~Q

December 1972) (Baton Rouge, LA: Center for Wetland Resources, Louisiana State University, 1973).

TApplied BiOtreament Associario@ “Case History Compendiurq” November 1989, p. 34. The compendium also Contains Oher exwples of theuse of bioremediation technologies to address environmental problems.

8T.G. Zitrides, “Bioremediat.ion Comes of Age, ” Pollution Engineering, vol. XXII, No. 5, May 1990, pp. 59-60.



Bioremediation for Marine Oil Spills . 3

q

Photo credit: Environmental Protection Agency

Prince William Sound, Alaska, site of the extensive

bioremediation experiments carried out by theEnvironmental Protection Agency, Exxon, and

the State of Alaska.

ultimate importance relative to other oil spill

response technologies remains uncertain.Recent research and field testing of bioremedi-ation technologies on oiled beaches has pro-duced some encouraging, if not altogetherconclusive, results. Nevertheless, technologiesother than bioremediation (especially mechani-cal ones) are likely to remain the mainstay of the Nation’s response arsenal for now. Incertain non-emergency situations (e.g., forcleaning lightly to moderately oiled beaches),bioremediation could be employed as a primarytechnology. Mechanical methods, dispersants,and possibly in situ burning will most likelyremain more appropriate technologies for theimmediate response to spills at sea.

Potential bioremediation approaches for ma-rine oil spills fall into three major categories:1) stimulation of indigenous microorganismsthrough addition of nutrients (fertilization),2) introduction of special assemblages of natu-rally occurring oil-degrading microorganisms(seeding), and 3) introduction of genetically

engineered microorganisms (GEMs) with spe-cial oil-degrading properties. Stimulation of indigenous organisms by the addition of nutrients is the approach that has been

tested most rigorously. This approach is

viewed by many researchers as the mostpromising one for responding to most types

of marine spills. Recent experiments suggestthat rates of biodegradation in most marineenvironments are constrained by lack of nutri-ents rather than by the absence of oil-degradingmicrobes. The introduction of microbes might be beneficial in areas where native organismsgrow slowly or are unable to degrade a particu-lar hydrocarbon. However, the effectiveness of this approach has not yet been demonstrated.

The wide availability of naturally occurringmicroorganisms capable of degrading compo-nents of petroleum will likely deter considera-tion of GEMs for remediating marine oil spills.Moreover, greater research and developmentneeds, regulatory hurdles, and public percep-tion problems will remain obstacles to thenear-term use of GEMS even if they couldprove useful for degrading some recalcitrant

components of petroleum.

bioremediation technologies for beach cleanuphave thus far received the most attention.Experiments conducted by EPA, Exxon, andthe State of Alaska on cobble beaches fouled byoil from the Exxon Valdez indicated that theaddition of nutrients at least doubled thenatural rate of biodegradation. The efficacy of

commercial microbial products in remediatingbeaches is not yet known. Limited EPA fieldtests using two microbial products on heavilyweathered oil in Alaska were inconclusive.Additional field experiments are required onother types of beaches that involve differentoils and different climatic and marine condi-tions.

bioremediation has not yet been demon-

strated to be an effective response to "at-sea” oil spills. The necessary studies have notbeen done, in part because of the difficulty of conducting controlled experiments and moni-toring in the open ocean. Limited applicationshave been made in the Gulf of Mexico, but theyhave not provided definitive data on effective-ness. The design and validation of open oceanprotocols to test products are necessary before

the efficacy of bioremediation at sea can bedetermined or widely accepted. Even if biore-mediation proves effective in some situations,other, quicker-acting alternatives may be pref-erable as primary response tools.



4 q Bioremediation for Marine Oil Spills

bioremediation may have a role in settings such

as salt marshes and sensitive ecosystems wherethe use of mechanical or other approachesmight do more harm than good. Just as for openwater spills, however, appropriate protocolsneed to be developed for testing and apply-ing bioremediation technologies in thesesituations, and more research is required toprove their effectiveness.

No significant adverse impacts related to the

use of bioremediation technologies for oil spillcleanup have been identified in recent fieldapplications. Effects that have been measuredhave been short-lived and minor. On beaches,in particular, bioremediation may be a lessintrusive approach than other alternatives. How-ever, experience with bioremediation in marinesettings is limited, and it is premature toconclude that the use of bioremediation tech-

nologies will be safe in all circumstances.Regulatory controls to ensure the safe use of bioremediation appear adequate, and thereappear to be no significant Federal regulatoryobstacles to the greater use of bioremediationtechnologies, except those using GEMs. How-ever, more development and testing of bothfertilization and seeding technologies areneeded before on-scene coordinators or oth-

ers responsible for oil spill cleanup would becomfortable advocating their use. Most deci-sionmakers prefer more traditional methods,and usually are not willing to experimentduring a real spill. bioremediation technologiesfor response to marine oil spills, althoughpromising, are still in the experimental phase.One regulatory change that could help stimu-late development of both bioremediation andother oil spill response technologies is for theFederal Government to allow occasional con-trolled oil spills for research and developmentpurposes.

If additional research confirms the effective-ness of bioremediation and leads to the devel-opment of more reliable technologies, oil spilldecisionmakers will have to be educated about

the efficacy of various techniques, the advan-tages and disadvantages of their use, and theavailability of materials and expert assistance.Preliminary efforts to accomplish this haverecently been undertaken by EPA. However,before detailed bioremediation contingency

plans can be developed, uncertainties about the

effectiveness of bioremediation must be ad-dressed. Detailed plans, when and if necessary,would require such information as the oil-degrading capabilities of microorganisms in-digenous to a particular area, the characteristicsof the oil most likely to be spilled in that area,environmental factors constraining oil biodeg-radation, and the circumstances under whichthe use of bioremediation technologies would

be appropriate or allowed.EPA, through its bioremediation Action Com-mittee and research labs, and with the assist-ance of the National Environmental Technol-ogy Applications Corp., is developing proto-cols to determine the efficacy and toxicity of bioremediationproducts in a variety of settings.Testing products against such protocols wouldprovide decisionmakers with the kind of data

needed to determine whether these productscould be used in response to marine oil spills.

A research program to expand the presentknowledge of biodegradation mechanisms, andto improve bioremediation technologies andthe means of applying them to marine oil spills,appears to be justi.tied. Redirecting an apprecia-ble fraction of available marine oil spill re-search funds to bioremediation does not, how-ever, seem warranted. Efforts to improve otheroil spill prevention and response technologiesare also important, and funding is limited.Improving methods for enhancing the growthand activity of petroleum-degrading bacteria isimportant, as is the development of betteranalytical techniques for measuring and moni-toring effectiveness, and field validation of laboratory work. Government and industryshould be encouraged to coordinate their re-search efforts and to share as much informationas possible.

BACKGROUND

Evaluating the effectiveness of bioremediationtechnologies is complicated by several factors. First,biodegradation is only one of the processes at work

removing petroleum from the marine environment;to understand the effect of this process on oilremoval, one must know the effects of otherprocesses. Second, petroleum is not the simplematerial many people presume it to be; rather, itcontains thousands of compounds. Some of these are



Bioremediation for Marine Oil Spills q 5

easily biodegraded; others are relatively resistant to

biodegradation. Third, a large number-not just oneor a few-of species of microorganisms are respon-sible for biodegradation, and these species haveevolved many metabolic pathways to degrade oil.Although the general mechanisms of biodegradationare known, many details remain to be filled in.

Underlying these complications are the basicissues of what constitutes clean and how long one iswilling to wait for results. These are both political

and scientific issues. Can a beach be consideredclean, for example, if its surface appears clean butclose examination reveals oil below the surface, orif unsightly but relatively less harmful constituentsof oil, such as hard-to-degrade asphalt, remain on thebeach after bioremediation is used? How much of animprovement over natural biodegradation rates mustbioremediation technologies offer before their usewould be warranted? A rate increase of a factor of 2

or more would be significant for a spill that mightotherwise persist for 5 or more years, but much lessso for a spill that might be naturally degraded in lessthan a year.

The Fate of Oil in the Marine Environment

The fate of petroleum in marine ecosystems hasbeen intensively studied.

9Crude oil and petroleum

distillate products introduced to the marine environ-ment are immediately subject to a variety of physicaland chemical, as well as biological, changes (figure1).

10Abiological weathering processes include evap-

oration, dissolution, dispersion, photochemical oxi-dation, water-in-oil emulsification, adsorption ontosuspended particulate material, sinking, and sedi-mentation. Biological processes include ingestionby organisms as well as microbial degradation.

ll

These processes occur simultaneously and causeimportant changes in the chemical composition and

physical properties of the original pollutant, whichin turn may affect the rate or effectiveness of biodegradation.

The most important weathering process duringthe first 48 hours of a spill is usually evaporation, theprocess by which low- to medium-weight crude oilcomponents with low boiling points volatilize intothe atmosphere. Evaporation can be responsible forthe loss of one- to two-thirds of an oil spill’s massduring this period,

12with the loss rate decreasing

rapidly over time.13

Roughly one-third of the oilspilled from the Amoco Cadiz, for example, evapo-rated within the frost 3 days. Evaporative loss iscontrolled by the composition of the oil, its surfacearea and physical properties, wind velocity, air andsea temperatures, sea state, and the intensity of solarradiation.

14The material left behind is richer in

metals (mainly nickel and vanadium), waxes, andasphaltenes than the original oil.

15With evapora-

tion, the specific gravity and viscosity of the originaloil also increase. For instance, after several days,spilled crude oil may begin to resemble Bunker C(heavy) oil in composition.

lG

None of the other abiological weathering proc-esses accounts for as significant a proportion of thelosses from a spill. For example, the dissolving, ordissolution, of oil in the water column is a much lessimportant process than evaporation from the per-spective of mass lost from a spill; dissolution of evena few percent of a spill’s mass is unlikely. Dissolu-tion is important, however, because some water-soluble fractions of crude oil (e.g., the light aromaticcompounds) are acutely toxic to various marineorganisms (including microorganisms that may beable to degrade other fractions of oil), and theirimpact on the marine environment is greater thanmass balance considerations might imply.

17

%or example, see references listed in National Research Council, Oil in the Sea: Inputs, Fates, and l?~ecfs (Washington, DC: National AcademyPress, 1985), pp. 335-368; and inG.D. Floodgate, ‘‘The Fate of Petroleum in Marine Environments,’ R.M. Atlas (cd.), Petroleum Microbiology (NewYork, NY: Macmillan Publishing Co., 1984), pp. 355-397.

l~atio~ Rese~ch Council, op. cit., footnote 1, p. 270.

1lJ.R. payne~d G.D. McNabb, Jr., “Weatheringof Petroleum in the Marine Environment,’ Marine Technology Society Journal, vol. 18, No. 3,1984,p. 24.

lzNatio@ Research Council, op. cit., footnote 1, p. 276.

13Flwdgate, op. cit., footnote g, P. 362.

14paPe ~d McNabb, op. cit., footnote 11, p. 26.

ls~oodgate, op. cit., footnote 9, p. 362

115J.E. Mie&e, “oil in the Ocean: The Short- and Imng-T&m Impacts of a Spill,” Congressional Research Service, 90-356 SPR, July 24, 1990, p.11.

ITNatio~ Resemch Council, op. cit., footnote 1, pp. 277-278.



6 q bioremediationfor Marine Oil Spills

Figure l—Schematic of Physical, Chemical, and Biological Processes

Wind

Seasurface

Atmospheric oxidation(photo-oxidation)

Currentm o v e m e n t ~

Water-in-oil

h emulsions + TarSpreading Sea surface oil slick “chocolate mousse” balls

b

~ Dissolution t

I( IGlobular Solubilizing Idispersion

I

chemical Current movement

Oil-in. transformations ~ a n d ~

/water

Chemicaldiffusion

Iemulsion

degradationAttachment

I to DispersedI

particulate+ crude oil

\

matter \

Degradation or \ I

Nonbuoyant assimilation byoil residues fish I

v“ \ !

/Bulksubsurface \ \ Biological /discharge detritus

-- I

/ 1

Sea floor

/ — — ~ wSea f loor sed im ent s

— —assimilation by bottom

— — dwelling organisms —+

SOURCE: National Research Council, Oil in the Sea: Inputs, Fates, and Effects (Washington, DC: National Academy Press, 1985), p. 271. Adapted fromR. Burwood and G.C. Speers, “Photo-oxidation as a Factor in the Environmental Dispersal of Crude Oil,” Estuarine Coastal Marine Science,vol. 2, 1974, pp. 117-135.

Dispersion, the breakup of oil and its transport as have had trouble being accepted in the United States.

small particles from the surface to the water column, The National Research Council has generally ap-is an extremely important process in the disappear- proved their use,19

but effectiveness and, to a lesser

ance of a surface slick.18

Dispersion is controlled degree, toxicity remain concerns. Dispersed oillargely by sea surface turbulence: the more turbu- particles are more susceptible to biological attack lence, the more dispersion. Chemical dispersants than undispersed ones because they have a greaterhave been formulated to enhance this process. Such exposed surface area. Hence, dispersants may en-

dispersants are intended as a first-line defense hance the rate of natural biodegradation.20

against oil spills that threaten beaches and sensitivehabitats such as salt marshes and mangrove swamps. Water-in-oil emulsions, often termed “mousse,”

Although used widely in other countries, dispersants are formed when seawater, through heavy wave

lspawe and McNabb, op. cit., footnote 11, p. 30.

l~atio~ Research COunCil, Marine Board, Using OiZDispersants on the Sea (Washington, DC: National kad~y press, 1989).

%.R. Colwell and J.D. Walker, “Ecological Aspects of Microbial Degradation of Petroleum in the Marine Environment” CRC Critical Reviewsin Microbiology, September 1977, p, 430.




others.29

Branched alkanes are usually more resistant

to biodegradation than normal alkanes but lessresistant than cycloalkanes (naphthenes)-those al-kanes having carbon atoms in ringlike centralstructures.

30Branched alkanes are increasingly re

-

sistant to microbial attack as the number of branchesincreases. At low concentrations, cycloalkanes maybe degraded at moderate rates, but some highlycondensed cycloalkanes can persist for long periodsafter a spill.

31Light oils contain 10 to 40 percent

normal alkanes, but weathered and heavier oils mayhave only a fraction of a percent. Heavier alkanesconstitute 5 to 20 percent of light oils and up to 60percent of heavier oils.

32

Aromatic hydrocarbons are those characterizedby the presence of at least one benzene (or substi-tuted benzene) ring. The low-molecular-weight aro-matic hydrocarbons are subject to evaporation and,although toxic to much marine life, are also rela-

tively easily degraded. Light oils typically containbetween 2 and 20 percent light aromatic compounds,whereas heavy oils contain 2 percent or less.

33As

molecular weight and complexity increase, aromat-ics are less readily degraded. Thus, the degradationrate of polyaromatics is slower than that of monoaro-matics. Aromatics with five or more rings are noteasily attacked and may persist in the environmentfor long periods.

34High-molecular-weight aromatics

comprise 2 to 10 percent of light oils and up to 35percent of heavy oils.

35

The asphaltic fraction contains compounds thateither are not biodegradable or are degraded veryslowly.

36One of the reasons that tar, which is high in

asphaltenes, makes an excellent road paving ma-terial is because it is slow to degrade. Tar balls, likemousse, are difficult to degrade because their low

surface area restricts the availability of oxygen and

other nutrients. Resins include petroleum com-

pounds containing nitrogen, sulfur, and/or oxygen asconstituents. If not highly condensed, they may besubject to limited microbial degradation. Asphal-tenes and resins are difficult to analyze and, to date,little information is available on the biodegradabil-ity of most compounds in these groups.

37Light oils

may contain about 1 to 5 percent of both asphaltenesand resins; heavy or weathered oils may have up to25 percent asphaltenes and 20 percent resins.

38

To summarized biodegradation rates are typicallyhighest for the saturates, followed by the light

aromatics, with high-molecular-weight aromatics,asphaltenes, and resins exhibiting extremely lowrates of degradation.

39As a spill weathers, its

composition changes: the light aromatics and al-kanes dissolve or evaporate rapidly and are metabo-lized by microorganisms. The heavier componentsthat are harder to degrade remain. Weathered

Prudhoe Bay oil contains about 10 percent low-molec-ular-weight alkanes, 45 percent high-molecular-weight alkanes, 5 percent light aromatics, 20 percenthigh-molecular-weight aromatics, 10 percent as-phaltenes, and 10 percent resins.

40

Departures from the typical pattern of biodegrada-tion, however, have been noted by some researchers.For example, extensive losses of asphaltenes and

resins have been observed in some cases. Themicrobial degradation of these relatively recalcitrantfractions has been ascribed to co-oxidation.

41In this

process, a normally refractory hydrocarbon may bepartially degraded in the presence of a second readilydegraded hydrocarbon. Clearly, degradation ratesdepend on many factors, and generalizations aredifficult to make. One conclusion, however, seemsreasonable: no crude oil is subject to complete

biodegradation, and claims that all of a light oil or

zgAtlm, op. cit., footnote 23, p. 212.30Cwney, op. cit., fOOmOte 25.

slA~as, op. cit., footnote 23, p. 212.

32M. HngIs, Environment CanadA personal COIIIIIItiCatiOIL wt. % 1990.

3%id.

34Natio~ Rme~ch Council, op. cit., footnote 1, p. 293.

35Fqy5, 0p. Cit., footnote 32-

36Cooney,

op.cit., fOOtnOte 25, P“ 3“

37Cwney, op. cit., fm~ote 25, p. 3; and National Research Council, op. cit., foo~ote 1, P. 295-

38F~gm, op. cit., fOOtnOte 32.

3WY andColwell, op. cit., footnote 28, p. 305.

@13ngaa, op. cit., footnote 32.

41UY and Colwell, op. cit., footnote 28, p. 306.



Bioremediation for Marine Oil Spills . 9

more than 50 percent of a heavy oil can bebiodegraded in days or weeks are highly suspect.

42

Microbial Processes and the Degradation

of Petroleum

Despite the difficulty of degrading certain frac-tions, some hydrocarbons are among the most easilybiodegradable naturally occurring compounds. Al-together, more than 70 microbial genera are knownto contain organisms that can degrade petroleum

components (table 1). Many more as-yet-uniden-tified strains are likely to occur in nature.43

More-over, these genera are distributed worldwide. Allmarine and freshwater ecosystems contain someoil-degrading bacteria. No one species of micro-organism, however, is capable of degrading all thecomponents of a given oil. Hence, many differentspecies are usually required for significant overalldegradation.

44Both the quantity and the diversity of

microbes are greater in chronically polluted areas. Inwaters that have not been polluted by hydrocarbons,hydrocarbon-degrading bacteria typically make upless than 1 percent of the bacterial population,whereas in most chronically polluted systems (har-bors, for example) they constitute 10 percent or moreof the total population.

45

Microorganisms have evolved their capability todegrade hydrocarbon compounds over millions of

years. These compounds are a rich source of thecarbon and energy that microbes require for growth.Before that carbon is available to microorganisms,however, large hydrocarbon molecules must bemetabolized or broken down into simpler moleculessuitable for use as precursors of cell constituents.The activity of microorganisms at a spill site isgoverned by the organisms’ ability to produceenzymes to catalyze metabolic reactions. This abil-

ity is, in turn, governed by their genetic composition.Enzymes produced by microorganisms in the pres-ence of carbon sources are responsible for attackingthe hydrocarbon molecules. Other enzymes areutilized to break down hydrocarbons further.

%Lack

of an appropriate enzyme either prevents attack or isa barrier to complete hydrocarbon degradation.

Table l—Major Genera of Oil-Degrading Bacteriaand Fungi

BacteriaAchrornobacterAcinetobacterActinomycesAeromonasAlcaligenesArthrobacterBacillusBeneckeaBrevebacteriumCoryneforms

ErwiniaFlavobacteriumKlebsiellaLactobaoillusLeumthrixMoraxellaNocardiaPeptococcusPseudomonasSarcinaSpherotilusSpirillum

StreptomycesVibrioXanthomyces

Fungi AllescheriaAspergillusAureobasidiumBotrytisCandidaCephalosporiumCladosporiumCunninghamellaDebaromycesFusarium

GonytrichumHansenulaHelminthosporiumMucorOidiodendrumPaecylomycesPhialophoraPenicilliumRhodosporidiumRhodotorulaSaccharomycesSaccharomycopisis

ScopulariopsisSporobolomycesTorulopsisTrichodermaTrichosporon

SOURCE: G.D. Floodgate, “The Fate of Petroleum in Marine Ecosystems,”Petroleum Microbiology, R.M. Atlas (ad.) (New York, NY:Macmillan Publishing Co., 1984), p. 373.

The complex series of steps by which biodegrada-tion occurs constitutes a metabolic pathway. Many

different enzymes and metabolic pathways, not all of which can be found in any single species, arerequired to degrade a significant portion of thehydrocarbons contained in petroleum. (Thus, advo-cates of using specially selected mixtures of micro-organisms to bioremediate oil spills or of creating,through recombinant DNA technology, geneticallyengineered organisms are motivated in part by the

desire to combine all the requisite enzymes andpathways.47)

Knowledge of the numerous metabolic pathwaysinvolved in the breakdown of hydrocarbons is farfrom complete. Additional research characterizingthe microbiology and population dynamics of bac-

4~~ney, op. cit., footnote 25, P- 3<

43flmdgate, op. cit., footnote 9, P. 372.~~e and IAWy, op. cit., footnote22, pp. 217-243.45Cmney, op. cit., fOOtnOte 257P. 1.

46Applied Bio@ea@ent A~~~iatioq “me Role of Bio@~~ent of Ofi Spifls,” KW . 2, August 1990, p. 4.

dTAtks, op. cit., footnote 23, P. 213.




terial species capable of degrading oil is critical tounderstanding the biodegradation process.

Environmental Influences on Biodegradation

Environmental variables can also greatly influ-ence the rate and extent of biodegradation. Variablessuch as oxygen and nutrient availability can often bemanipulated at spill sites to enhance natural biodeg-radation (i.e., using bioremediation). Other varia-bles, such as salinity, are not usually controllable.

The great extent to which a given environment caninfluence biodegradation accounts for some of thedifficulty in accurately predicting the success of bioremediation efforts. Lack of sufficient knowl-edge about the effect of various environmentalfactors on the rate and extent of biodegradation isanother source of uncertainty.

Oxygen

Oxygen is one of the most important requirementsfor microbial degradation of hydrocarbons. How-ever, its availability is rarely a rate-limiting factor inthe biodegradation of marine oil spills. Microorgan-isms employ oxygen-incorporating enzymes to initi-ate attack on hydrocarbons. Anaerobic degradationof certain hydrocarbons (i.e., degradation in theabsence of oxygen) also occurs, but usually atnegligible rates. Such degradation follows differentchemical paths, and its ecological significance isgenerally considered minor.

48For example, studies

of sediments impacted by the Amoco Cadiz spillfound that, at best, anaerobic biodegradation isseveral orders of magnitude slower than aerobicbiodegradation.

49Oxygen is generally necessary for

the initial breakdown of hydrocarbons, and subse-quent reactions may also require direct incorporationof oxygen. Requirements can be substantial; 3 to 4parts of dissolved oxygen are necessary to com-pletely oxidize 1 part of hydrocarbon into carbondioxide and water.

50

Oxygen is usually not a factor limiting the rate of biodegradation on or near the surface of the ocean,

where it is plentiful and where oil can spread out toprovide a large, exposed surface area. Oxygen is alsogenerally plentiful on and just below the surface of beaches where wave and tide action constantly assistaeration. When oxygen is less available, however,the rates of biodegradation decrease. Thus, oil thathas sunk to the sea floor and been covered bysediment takes much longer to degrade. Oxygenavailability there is determined by depth in thesediment, height of the water column, and turbu-lence (some oxygen may also become available asthe burrowing of bottom-dwelling organisms helpsaeration) .51 Low-energy beaches and fine-grainedsediments may also be depleted in oxygen; thus, therate of biodegradation may be limited in these areas.Pools of oil are a problem because oxygen is lessavailable below their surfaces. Thus, it may bepreferable to remove large pools of oil on beaches,as was done in Alaska, before attempting bioremedi-

ation.

Nutrients

Nutrients such as nitrogen, phosphorus, and ironplay a much more critical role than oxygen inlimiting the rate of biodegradation in marine waters.Several studies have shown that an inadequate

supply of these nutrients may result in a slow rate of biodegradation.

52Although petroleum is rich in the

carbon required by microorganisms, it is deficient inthe mineral nutrients necessary to support microbialgrowth.

53Marine and other ecosystems are often

deficient in these substances because non-oil-degrading microorganisms (including phytoplank-ton) consume them in competition with the oil-degrading species. Also, phosphorus precipitates as

calcium phosphate at the pH of seawater. Lack of nitrogen and phosphorus is most likely to limitbiodegradation, but lack of iron or other traceminerals may sometimes be important. Iron, forinstance, is more limited in clear offshore watersthan in sediment-rich coastal waters.

54

4S~~y and Colwell, op. cit., footnote 28, P. 307.

%.M. War@ R.M. Atlas, P.D. Boehrn, and J.A. Calder, “Microbial Biodegradation and the Chemical Evolution of Amoco CUdiz Oil Pollu@@”

Ambio, vol. IX, No. 6, 1980, pp. 279.%ee and levy, op. cit., footnote 22, p. 224.

51 CWney, op. cit., fOOmOte 25, P“ 4“

sz~ md UVY, op. cit., footnote 22, p. 223.

5sA&, op. cit., footnote 23, p. 216.

541bid.



Bioremediationfor Marine Oil Spills q 11

Phofo credit: Exxon Corp.

Workman applying fertilizer to the cobble beaches ofPrince William Sound.

Scientists have attempted to adjust nutrient levels(e.g., by adding nitrogen- and phosphorus-richfertilizers) to stimulate biodegradation of petroleumhydrocarbons. This is the experimental bioremediationapproach used recently on about 110 miles of beaches in Prince William Sound, Alaska. Research-ers have also experimented with alternative methodsof applying nutrients. Given the necessity of keepingnutrients in contact with oil, the method of applica-tion is itself likely to be an important factor in thesuccess of bioremediation.

Temperature

The temperature of most seawater is between–2 and 35

oC.

55Biodegradation has been observed in

this entire temperature range, and thus in water

temperatures as different as those of Prince WilliamSound and the Persian Gulf. The rates of biodegrada-tion are fastest at the higher end of this range andusually decrease—sometimes dramatically in verycold climates-with decreasing temperature. One

experiment showed that a temperature drop from 25to 5

oC caused a tenfold decrease in response.

56At

low temperature, the rate of hydrocarbon metabo-lism by microorganisms decreases.57

Also, lighterfractions of petroleum become less volatile, therebyleaving the petroleum constituents that are toxic tomicrobes in the water for a longer time anddepressing microbial activity. Petroleum also be-comes more viscous at low temperature. Hence, lessspreading occurs and less surface area is availablefor colonization by microorganisms. In temperate

regions, seasonal changes in water temperatureaffect the rate of biodegradation, but the processcontinues year-round.

58

Other Factors

Several variables, including pressure, salinity,and pH may also have important effects on biodegra-dation rates. Increasing pressure has been correlated

with decreasing rates of biodegradation; therefore,pressure may be very important in the deep ocean.Oil reaching great ocean depths degrades veryslowly and, although probably of little concern, islikely to persist for a long time.

59

Microorganisms are typically well adapted tocope with the range of salinities common in theworld’s oceans. Estuaries may present a special casebecause salinity values, as well as oxygen and

nutrient levels, are quite different from those incoastal or ocean areas. However, there is littleevidence to suggest that microorganisms are ad-versely affected by other than hypersaline environ-ments.

60

Extremes in pH affect a microbe’s ability todegrade hydrocarbons. However, like salinity, pHdoes not fluctuate much in the oceans-it remains

between 7.6 and 8. l—and does not appear to have animportant effect on biodegradation rates in mostmarine environments. In salt marshes, however, thepH maybe as low as 5.0, and thus may slow the rateof biodegradation in these habitats.

6l

55~oodgate, op. cit., footnote 9, P. 3*1.

‘eIbid.

WU&y and Colwell, op. cit., footnote 28, p. 307.

58moodgate, op. cit., fOO~Ote 9, P. 381-

59J.R. &-hwm, J*D. Wtier, ad R*R4 Colweu, “Deep.sea Bacteria: @owth ad Utibtion of Hy&oc~m at Ambient ~d b Situ PKxXlllV,’

Applied Microbiology, vol. 28, 1974, pp. 982-986.

%e and Levy, op. cit., footnote 22, p. 225.

Glh~y and Colwell, op. cit., footnote 28, p. 308.

292-854 0 - 91 - 2




Table 2—bioremediation: Potential Advantagesand Disadvantages

Advantages: . Usually involves only minimal physical disruption of a site. No significant adverse effects when used correctly. Maybe helpful in removing some of the toxic components of oilqOffers a simpler and more thorough solution than mechanical

technologiesqPossibly less costly than other approaches

Disadvantages:qOf undetermined effectiveness for many types of spillsqMay not be appropriate at seaqTakes time to workqApproach must be specifically tailored for each polluted site. Optimization requires substantial information about spill site

and oil characteristics

SOURCE: Office of Technology Assessment, 1991.

General Advantages and Disadvantages

of bioremediation

bioremediation technologies have several attri-

butes that, depending on the situation and type of sitemay support their use in responding to some oilspills (table 2).

62First, bioremediation usually

involves minimal physical disruption of a site. Thisattribute is especially important on beaches whereother available cleanup technologies (e.g., high- andlow-pressure spraying, steam cleaning, manual scrub-bing, and raking of congealed oil) may causeadditional damage to beach-dwelling biota.

63Appli-

cation of oleophilic (i.e., oil seeking) fertilizersduring the 1989-90 Alaska bioremediation experi-ments was accomplished largely from shallow draftboats located just off the beach. Second, bioremedia-tion technologies appear to have no or only minorand short-lived adverse effects when used correctly.Although research on possible negative impacts iscontinuing, there is so far little evidence to suggestthat potential problems would be significant.

Third, bioremediation may be useful in helpingremove some of the toxic components of petroleum(e.g., low-molecular-weight aromatic hydrocarbons)from a spill site more quickly than they mightotherwise be removed by evaporation alone.

64

Fourth, bioremediation of oil spills is accomplishedon-site, and offers a simpler and more thoroughsolution to polluted areas. In contrast, hot water

Photo credit: Exxon C O r P .

One of the shallow draft boats used by Exxon to applyoleophilic fertilizer to Prince William Sound beaches.

spraying of an oiled beach, for example, flushessome surface oil back into the water, and this oil

must then be recovered by skimmers. The recoveredoil-and-water mixture must be separated, and the oildisposed of or recycled. Also, a significant amountof mechanical equipment and logistical capability isrequired to deal with a large spill.

Because bioremediation equipment and logisticsare usually simpler and less labor intensive, costsmay be lower than for other techniques. At the same

time, the total cost of cleanup is the more importantconcern, and where bioremediation is used as anadjunct or secondary technology, total costs—aswell as total benefits--could be greater. The costs of monitoring bioremediation must also be considered.

bioremediation technologies have several generaldisadvantages. Although bioremediation may work faster-potentially much faster—than natural bio-degradation, it cannot produce significant short-

term results. If beaches are threatened by a largeoffshore spill, for instance, bioremediation is proba-bly not appropriate as an initial defensive measure.In this circumstance, it would usually be moreappropriate to get the oil out of the water as quicklyas possible or, failing this, to disperse or burn itbefore it drifts onto beaches. bioremediation takes

Gz~esea~butw ~e &SCUSSed in the AppliedBiotreatment Association, ‘‘BriefmgPaperonthe Role ofbioremediation of Oil Spills,” rev. 2, August1990.

63M.s. Foster, J*A. Tqley, ~d S.L. Dq “To Cl- or Not lb Clean: The Rationale, Methods, and Consequences of Remov@ Oti From

lkmperate Shores,” The Northwest Environwntal JournuZ, vol. 6, 1990, pp. 105-120.

~J. Gl~r, Envirorlmen~ prot~tion Agency, Risk Reduction Engineering Laboratory, personaJ co~ticatiou Feb. 20, 1991.




off the coast of New Jersey, in Prudhoe Bay, and inseveral ponds near Barrow, Alaska. In each case, the

addition of fertilizer was found to stimulate biodeg-radation by naturally occurring microbial popula-tions.

67

The recent nutrient enrichment experiments inAlaska provided a wealth of experimental data aboutbioremediation in an open environment (box B).Since previous research findings had already dem-onstrated the general value of this approach, the

experiments were intended to determine for one typeof environment how much enhancement of naturalbiodegradation could be expected and to evaluatethe most effective methods of application. Theresults provided additional evidence that applicationof nutrients could significantly enhance the naturalrate of biodegradation on and below the surface of some beaches. As a result, Exxon was authorized bythe Coast Guard on-scene coordinator, in concur-

rence with the Alaska Regional Response Team,68

toapply fertilizers to the oiled beaches in PrinceWilliam Sound. To date, about 110 miles of shoreline have been treated with nutrients, and amonitoring program has been established.

Without additional research, however, it is prema-ture to conclude that nutrient enrichment will beeffective under all conditions or that it will alwaysbe more effective than other bioremediation ap-proaches, other oil spill response technologies, ormerely the operation of natural processes. Theresults of the Alaska experiments were influenced bythe beach characteristics (mostly rocky beaches,well-washed by wave and tide action), the watertemperature (cold), the kind of oil (Prudhoe Baycrude), and the type and quantity of indigenousmicroorganisms in Prince William Sound.

Few detailed analyses or performance data are yetavailable for different sets of circumstances. Onesmaller-scale test using the same fertilizer as inAlaska was recently conducted on beaches inMadeira polluted by the Spanish tanker Aragon.Results in this very different setting and with a

different type of oil were not especially encouraging.Researchers speculated that the unsatisfactory re-

sults could have been due to differences in the typeof oil, the concentration of fertilizer used, the lowerinitial bacterial activity, and/or different climaticconditions.

69At the same time, Exxon recently used

what it learned in Alaska to help degrade subsurfaceno. 2 heating oil spilled in a wildlife refugebordering the Arthur Kill at Prall’s Island, NewJersey. An innovative aspect of this application wasthe use of two trenches parallel to the beach in which

to distribute fertilizer. Nutrients were dissolved withthe incoming tide and pulled down the beach withthe ebb tide, enabling a more even distribution thanpoint sources of fertilizer. Exxon reports that 3months after applying fertilizers, the oil in thetreated zone had been reduced substantially relativeto that in an untreated control zone.

70

Seeding With Naturally Occurring Microorganisms

Seeding (also called inoculation) is the addition of microorganisms to a polluted environment to pro-mote increased rates of biodegradation. The inocu-lum maybe a blend of nonindigenous microbes fromvarious polluted environments, specially selectedand cultivated for their oil-degrading characteristics,or it may be a mix of oil-degrading microbes selectedfrom the site to be remediated and mass-cultured inthe laboratory or in on-site bioreactors. Nutrientswould usually also accompany the seed culture.

The rationale for adding microorganisms to a spillsite is that indigenous microbial populations maynot include the diversity or density of oil-degradersneeded to efficiently degrade the many componentsof a spill. Some companies that advocate seeding

with microorganisms also claim that commercialbacterial blends can be custom-tailored for differenttypes of oil in advance of a spill, that the nutritionalneeds and limitations of seed cultures are wellunderstood, that microbes can easily be produced inlarge quantities for emergency situations, and that

6TR.M. Atlas, “bioremediation of Fossil Fuel Contamma“ ted Sites,” in press, proceedings of Battelle conference on In Situ and On-SiteBiorecZamution, March 1991. Atlas and his colleagues did some of this early work.

~~e most important members of the wka Regional Response lkam are the U.S. Environmental Protection Agency, the A.ktsh Depwtment of Environmental Conservation the U.S. Department of the Interior,he National Oceanic and Atmospheric Administration and the U.S. Forest Service.

@M. Biscoito ~d M. Morei.rii, MUSW Municipal do Funchal, Made@ “Application of In.ipolEAP22 in Porto ?mnto,” repo~ JUIY 1990.

mP.C.Madden, ExxonResearchand Engineering Co., Ietterand KCOmpm@gs~ report onprall’s Island bioremediation to U.S. Coast Guard,Mar. 12, 1991.



Bioremediation for Marine Oil Spills q15

Box B—The Alaska bioremediation Experiments

Following the March 1989 Exxon Valdez oil spill, the U.S. Environmental Protection Agency (EPA), Exxon,and Alaska’s Department of Environmental Conservation (ADEC) undertook what is perhaps the largest and mostcomprehensive series of experiments on oil spill bioremediation to date. The principal objectives of the researchinitiated in May 1989 were to determine if the addition of nutrients to Alaska’s polluted beaches would enhancethe rate and extent of oil biodegradation sufficiently to support widespread use of this technology there and toevaluate which application methods could be most effective.

1Research begun in the summer of 1990 was designed

to evaluate the effectiveness and safety of several microbial products in cleaning Alaska’s beaches.

The Alaska bioremediation work consisted of several discrete elements, including: 1) work begun shortly afterthe spill to determine if nutrient enhancement could be an appropriate technology for mitigating oil pollution of

Prince William Sound’s beaches; 2) application by Exxon of fertilizers to about 110 miles of polluted beaches, afterthe initial studies suggested that fertilizers could be both effective and safe; 3) additional EPA studies to supportExxon’s treatment program and further evaluate application techniques; 4) a long-term program to monitor treatedbeaches, conducted jointly by EPA, Exxon, and ADEC; and 5) evaluation of the potential of adding microbes toAlaska’s beaches to stimulate biodegradation.

Several types of nutrients and application methods were evaluated, including slow-release, water solublefertilizers in both briquette and granular forms; a water soluble fertilizer applied with a sprinkler system; and a liquidoleophilic fertilizer,

2

applied with sprayers, specially formulated to keep nutrients and oil in contact. Visual changesbetween control and experimental plots were observed, and changes in oil chemistry, microbial populations, andoil weight over time were measured. Findings pertain specifically to Prince William Sound, which has a numberof features favorable to nutrient enrichment: a high percentage of naturally occurring hydrocarbon-degradingbacteria, low concentrations of ammonia and phosphate in seawater, highly porous beaches, and large tidal fluxes.Although the work carried out in Alaska is important to bioremediation research and applications in other areas,the same results cannot be expected elsewhere. The major findings follow:

Based on a synthesis of all available evidence, researchers concluded that biodegradation on beach surfaceswas accelerated as much as two- to four-fold by a single application of fertilizer; thus, the addition of nutrients to Alaska’s beaches did significantlystimulate the rate of biodegradation. The watersoluble fertilizer delivered by a sprinkler system

proved the most effective approach, but thismethod was impractical on a large scale. Theoleophilic fertilizer and slow-release granuleswere almost as effective and more practical to use.EPA determined that the most practical approachfor this setting was to apply the oleophilicfertilizer to beaches with surface oiling and to useboth oleophilic fertilizer and fertilizer granuleswhere surface and subsurface oil were found.After several weeks, dramatic visual changes wereobserved in the amount of oil on beaches treatedwith fertilizer. Visual changes do not providequantitative data or prove that biodegradation isoccurring. However, similar changes observed inbeaches treated with the “plain” water solublefertilizer and those treated with oleophilic fertil-izer provided evidence that visually cleaner beaches

Photo credit: Environmental Protection Agency

Visual effect of oleophilic fertilizer on thebiodegradation of surface oil in Snug Harbor, Alaska.

The clear “window” indicates where the fertilizerwas applied.

Iu.s. Environmen~ protection Agency, office of Research and Develo~men~ “InterimRerm_t: Oil SDill bioremediation Project+’ Feb.

28, 1990, 220 pp. See also P.H. Pritch&d ad C.F. Costa, “EPA Alaska Oil Spill Bioremed&tion proj~c~” Environmental S;ience and Technology, March 1991, pp. 372-379. Much of the material in this box was reported in these two citations.

2For more on oleopwc fertilizers see A. Ladousse and B. Tramier, Societe Nationale ELF AQUIT_, “Results of 12 Yem of

Research in Spilled Oil bioremediation: Inipol EAP 22,” 1990.

Continued on next page




Box B—The Alaska bioremediation Experiments-Continued

resulted directly from the addition of nutrients, not from the suggested rock washing effects of oleophilicfertilizers. The oleophilic fertilizers evidently worked as intended, sequestering nutrients at the oil-waterinterface where microorganisms could be effective.

. Changes in oil chemistry provided additional evidence that biodegradation was limited by lack of nutrients.Analysis of fertilizer-treated samples and samples that had been artificially weathered to control forevaporation indicated that many of the easily degradable constituents of petroleum in fertilizer-treatedsamples had decreased substantially over 4 weeks. Some harder-to-degrade fractions of oil also appearedto decrease. No conclusions could be drawn about other difficult-to-degrade fractions because goodmeasurement methods had not been devised for them.

• In early testing, statistically significant increases in the oil degrading microbial population-increases thatwould correlate strongly with the rate of biodegradation-were not observed because of the high variabilityin numbers of bacteria in each sample. However, later results from the joint monitoring program appearedto indicate a sustained three- to four-fold increase in microbial activity.

• No statistically significant conclusions could be made about the rate of biodegradation from “before andafter” measurements of the weight of oil samples. Although a significant decrease in oil residue weight overtime would be evidence of degradation, this might not be a good criterion because bacterial production of high-molecular-weight compounds can occur. Precise measurements were impossible because oil wasdistributed unevenly on the beaches. Although the rate of biodegradation was probably the same in all areas,samples from more heavily oiled areas indicated a slower rate of biodegradation than samples from lightly

oiled areas.. The monitoring program indicated that enhanced microbial activity could be sustained for more than 30 days

from a single fertilizer application. Additional applications were found to increase microbial activity. Inparticular, a second application of fertilizer after 3 to 5 weeks replenished nutrients and stimulated microbialactivity five- to ten-fold.

3

. Fertilizer applications appeared to enhance biodegradation to a depth of at least 50 centimeters on treatedbeaches. Researchers found increased nitrogen nutrients, sufficient dissolved oxygen, and increasedmicrobial numbers and activity at this depth following treatment.

4

. Although evidence was not conclusive, researchers suspected that primary treatment with mechanicalmethods resulted in a more even distribution of oil on the beaches and hence prepared the beaches for

optimum bioremediation.s. Results of the 1990 research on two microbial products were inconclusive, with no statistically significant

enhancement of the rate of biodegradation over natural rates. However, the tests were conducted on oil thathad weathered and degraded naturally during the 18 months since the Exxon Valdez spill. The more easilydegraded components of the oil had already disappeared. The limited testing period-27 days-may alsohave affected the results.

6

SR. prince, J. Clark and J. Lindstrom, ‘‘bioremediation Monitoring Program,’December 1990, pp. 2,85. The authors of this reportrepresent EXXOU EPA, and the Alaska Department of Environmental Conservation respectively.

‘%id.

5wtc~d and COS@ op. cit., footnote 1.

6A. Venos% EPA, Risk Reduction Engineering Laboratov, presentation of research results at EPA-sponsored bioremediation meeting,

Las Vegas, NV, Feb. 20, 1991.

seed cultures can be stored, ready for use, for up to studies have not been conducted in such settings, so3 years.71 no data are available to evaluate the effectiveness of

The value of introducing nonindigenous micro- this approach. Many scientists question the neces-

organisms to marine environments is still being sity of adding microbes to a spill site because most

evaluated. With some exceptions, the scientilc locales have sufficient indigenous oil-degradingcommunity has not been encouraging about the microbes, and in most environments biodegradationpromise of seeding marine oil spills. Controlled is limited more by lack of nutrients than by lack of

7 1A p p f i ~ Biotreatment ~sociatio~ Op . Ci t . , fOOtnOte % Pp. 13-14.




rine oil spills. Experience with bioremediation inmarine settings is still limited, so it is premature to

conclude that its use will always be safe or thatpossible risks will be acceptable in all of thecircumstances in which bioremediation might beemployed. The evidence to date, nevertheless, sug-gests that risks will be unimportant in most situa-tions.

Concerns have been raised about several potential

adverse environmental effects. Among these are the

possibility that the addition of fertilizers could causeeutrophication, leading to algal blooms and oxygendepletion; that components of some fertilizers couldbe toxic to sensitive marine species or harmful tohuman health; that the introduction of nonnativemicroorganisms could be pathogenic to some indig-enous species; that the use of bioremediation tech-nologies could upset ecological balances; and thatsome intermediate products of bioremediation could

be harmful.The possible adverse effects of nutrient enrich-

ment were examined in some detail during the1989-90 Alaska beach bioremediation experiments.

85

To determine the potential for eutrophication inPrince William Sound, researchers measured ammo-nia, phosphate, chlorophyll, bacterial numbers, andprimary productivity in the water column directlyoffshore of fertilizer-treated beaches and in controlareas. They could find no significant differencebetween measurements in control areas and those inexperimental areas.

86There were no indications that

fertilizer application stimulated algal blooms.

The possible toxicity of fertilizer components wasexamined in both laboratory and field tests on anumber of marine species, including sticklebacksfish, Pacific herring, silver salmon, mussels, oysters,

shrimp, and mysids. In the absence of tidal dilution,certain components of the oleophilic fertilizer weremildly toxic to oyster larvae, the most sensitivemarine species.

87However, in the view of research-

ers working in Alaska, such effects were transientand limited to areas immediately adjacent to fertil-ized shorelines.

88The concentration of ammonia,

the only component of fertilizers shown to be

acutely toxic to marine animals, never reached toxiclevels.

The butoxyethanol constituent of the oleophilic

fertilizer is potentially harmful to some mammals.This constituent, however, evaporated from beachsurfaces in less than 24 hours, during which timewildlife deterrent devices were employed. Care hadto be taken, as well, by humans applying theoleophilic fertilizer to avoid inhalation or skincontact. Researchers were also able to show that theoil itself did not wash off the treated beaches and

gssee, for ex~ple, U.S. Environmen~ Protection Agency, OffIce of Research and Development Interim Report:Oil Spill bioremediation Prqkt,Feb. 28, 1990; R.C. Prince, J.R. Clark, and J.E. Lindstrom, bioremediation Monitoring Program, December 1990; and P.H. Pritchard and C.F. COS@

“EPA Alaska Oil Spill bioremediationproject” Environmental Science and Technology, March 1991.

861bid.

8Tu.s. Environmental Protection Agency, “Alaskan Oil bioremediation Project: Update,” EPA/600/13-89/073, July 1990, p. 11.

gg~ce et d., op. cit., footnote 85, p. 72.




accumulate in the tissues of marine test species. Inthis environment, dilution, tidal mixing, and evapo-

ration reduced the potential for significant impacts.In some low-energy environments (e.g., protectedbays), greater impacts might occur. In other environ-ments, the species present, water depth, and watertemperature are all variables to consider in estimat-ing potential impact. The effect of any impact of treatment, however, must be considered in view of the damage already caused by oil.

Evidence is also lacking that introduced orga-nisms might be pathogenic to other life forms. In aseries of experiments with North Slope crude oil, forexample, researchers failed to find any significantlygreater invertebrate mortality with bacterial seeding(or fertilization) than occurred with crude oil alone.

89

However, microorganisms to be considered asseeding candidates must be screened carefully toeliminate potential human or animal pathogens,

including opportunistic pathogens such as Pseudo-monas spp.

The possibility that introduced microbes mightproliferate and upset the ecological balance appearsto be of less concern. If effective at all, suchorganisms should die and be preyed on by protozoaonce they have utilized the oil from a spill.

90Of

greater concern is that microbes introduced fromother environments will not be able to compete aswell as native species and will die before they can dotheir job effectively. EPA’s Office of Pesticides andToxic Substances has been developing proceduresfor evaluating the toxicity of biotechnology prod-ucts. In concert with EPA’s Office of Research andDevelopment, it is establishing tests to evaluate thepotential pathogenicity of nonindigenous microbes.

91

Similar but greater concerns attend the introduc-

tion of genetically engineered organisms. Beforesuch organisms are likely to be introduced to themarine environment (if they have a role to play atall), more basic knowledge of their potential impactson that environment will be required, and regulatoryofficials and the public will have to become morefamiliar with biological mitigation techniques.

An additional concern is that although bacteriamay break down the complex hydrocarbons con-

tained in oil, they could leave behind products of partial biodegradation that are more toxic to marinelife than the original constituents of the oil.

92

However, in time, intermediate products of possibleconcern, such as quinones and naphthalenes, arelikely to be broken down further and thus unlikely toaccumulate in the environment.

93

bioremediation IN RELATION

TO OTHER RESPONSETECHNOLOGIES

Whether bioremediation technologies will beconsidered for use as primary or secondary responsetools, or will be deemed of no use at all, will dependon the circumstances of each oil spill. All responsetechnologies have the common purpose of minimiz-ing the damage caused by a spill. How well atechnology can accomplish this goal indicates itseffectiveness. The perfect response technology hasnot been developed, and numerous uncontrollablevariables may reduce effectiveness far below what itwould be under optimal circumstances. As the sizeof a spill increases, for example, the difficulty of responding to it by any means grows. Adverse seaand weather conditions may greatly reduce theeffectiveness of any open sea response. Even tech-nologies that are adequate in some spill situationswill be much less effective if they cannot bedeployed, operated, and maintained easily.

Before bioremediation is likely to be consideredas a response tool, it must be deemed not onlyeffective for its intended use, but also more effectivethan traditional technologies. The effectiveness andsafety of bioremediation technologies for respond-

ing to different types of spills have not yet beenestablished adequately, and on-scene coordinatorsand other decisionmakers generally are not familiarwith these technologies. Hence, most decision-makers are reluctant to try bioremediation if othertechniques could be effective. More traditionalmethods are preferred, and experimentation during

WR.M. Atlas and M. Busdos@ ‘hlicmbia.l Degra&ionof pdroleum hthi? ArCtiC,” Proceedings of the3rdInternational Biodegr&tion Symposium,

Kingston, RI, August 1975, p. 85. The seeding trials used an organism from the environment being treated.%. Colwell, Maryland Biotechnology Institute, University of Maryland, personalcommunicatio~ February 1991.

glu.s. Environmen~ prot~tion Agency, Office of Res~h and Development, bioremediation Action Committee, ‘ ‘SummarY of June 20, 1990Meeting,” Jtiy 1990.

~M.c. ICenni~~ Geochemical and Environmental Research Group, ‘lkxas A&M University, pfxsOd cOmmurdCatiO% @tok 1990.

93R.M. Aflas, Dep~~t of Biology, University of Louisville, personal Communication February 1991.



22 Bi di ti f M i Oil S ill




Somewhat more is known about the potential forbioremediation of beaches, thanks largely to the

Alaska experiments. The potential use of bioremedi-ation methods on at least some beaches lookspromising, but, as previously noted, results from theAlaska experiments cannot be extrapolated in toto toother types of beaches or spill situations (especiallywithout more precise knowledge of the effect of environmental and microbiological variables on therate and extent of biodegradation).

The greater promise of bioremediation for oiled

beaches is due in part to the fact that bioremediationis more easily controlled and monitored onshorethan it is at sea. Application of nutrients or seedcultures is also easier and less subject to disruptionby adverse conditions. Also, once oil reaches abeach, there is usually more time to consider theapproach to take: certain damage has alreadybeen done, and the emergency response required todeal with oil seeping from a stricken tanker, for

example, is no longer quite so necessary.

Depending on circumstances, bioremediation of beaches may be appropriate sometimes as a primaryresponse tool and sometimes as a secondary tool. Animportant consideration is how heavily oiled a beachis. Heavily oiled beaches may require removal of gross amounts of oil by mechanical means beforebioremediation can be a practical finishing tool.

Some lightly oiled beaches may not require anytreatment. Moderately oiled beaches are likely themain candidates for primary bioremediation treat-ment. After reviewing the Alaska bioremediationexperiments, the Alaska Department of Environ-mental Conservation concluded that bioremediationwas useful as a finishing or polishing tool, but thatpooled oil, tar balls, mousse, asphalt, and otherheavy concentrations of oil should be picked up with

conventional manual and mechanical techniques.

97

The effectiveness of bioremediation on beachesmay also depend on the coarseness of the beach, butthe relationship between the size of oiled sedimentsand the rate of biodegradation has not been evalu-ated thoroughly. The beaches treated in Alaska withsome success were all very coarse, consisting mostlyof cobbles and coarse sand. It is uncertain how

successful bioremediation of freer grained beacheswill be, especially when oil is trapped below the

surface. In very fine-grained sediments, lack of oxygen below the surface may limit the rate of biodegradation.

bioremediation, where effective, may offer apromising option for beach cleanup because theexisting mechanical technologies can cause addi-tional damage to beaches and beach biota.

98This

damage may be unavoidable if the goal is to

“restore” a beach as quickly as possible. Doingnothing (i.e., letting the beach recover naturally at aslower rate) may sometimes be preferable to usingmechanical technologies, but this is seldom politi-cally acceptable. bioremediation offers the possibil-ities of being faster than simply allowing nature totake its course unassisted and of avoiding thenegative impacts of mechanical technologies. More-over, when beaches are inaccessible, the mechanical

equipment that can be brought into use may belimited, but fertilizers or seed cultures can bedispensed without the need for massive machinery.In general, bioremediation is less costly and lessequipment- and labor-intensive than mechanicalcleanup technologies, which suggests a clear advan-tage for bioremediation where it can be usedeffectively instead of other technologies (e.g., mod-erately oiled beaches). The advantage is also evidentwhere it can be used as a secondary technology (i.e.,as a finishing tool), because it offers the possibilityof a more complete solution, more quickly attained(however, the total cost of the cleanup may begreater).

Salt marshes and other sensitive environments,even more than beaches, may be further damaged byintrusive mechanical technologies. For these envi-ronments, bioremediation could be the only feasible

alternative to doing nothing. Little work has beendone in these settings to evaluate the effectiveness orenvironmental impacts of using bioremediation.Biodegradation of oil stranded in salt marshes isgenerally limited by oxygen availability. However,the results of one recent study of waxy crude oils

99

in salt marshes suggest that nutrient enrichment maybe an effective countermeasure, provided that large

%’~id.

98M.S. Foster, J.A. Tarpley, and S.L. D- “To C!leaU or Not lb Clean: The Rationale, Methods, and Consequences of Remov@ Ofi From

‘Rznperate Shores,” The Northwest Environmental Journai, vol. 6, 1990, pp. 105-120.

~axycmde oils tend to spread, evaporate, and naturally disperse very slowly on water, and usually survive in a relatively fresh state considerably

longer than conventional oils.

Bi di ti f M i Oil S ill 23




amounts of oil do not penetrate beneath the aerobicsurface layer. Where oil did penetrate the surface

layer, the researchers observed little degradation.l00

Even bioremediation activities, if they are notcarefully conducted, have the potential for beingintrusive in salt marshes. The scientific supportcoordinator of the National Oceanic and Atmos-pheric Administration who monitored the applica-tion of microorganisms to Marrow Marsh in theHouston Ship Channel noted, for example, thatexcessive foot traffic associated with bioremedia-tion operations caused some unnecessary damage tomarsh grass.

lO1

bioremediation ACTIVITIES IN

THE PUBLIC AND PRIVATE

SECTORS

The Environmental Protection Agency

EPA is the lead Federal agency for oil spillbioremediation research. Both the Department of Energy and the Department of Defense are activelyengaged in research on bioremediation of hazardouswaste, but neither these nor other Federal agenciesare engaged in research on bioremediation of marineoil spills. EPA regards biotechnologies as havingsignificant potential for the prevention, reduction,and treatment of pollution, and the Agency has

placed considerable emphasis on the demonstrationand development of these technologies.

102This

coincides with an important general EPA goal of promoting the development of new and innovativetechnologies to address environmental problems.Agency activities in support of bioremediation formarine oil spills represent a small fraction of itsoverall biotechnology activities. Nevertheless, EPAwould like to establish the technical basis for a

national bioremediation response capability for oilspills.

l03

In February 1990, EPA convened a meeting of interested industry, academic, and government per-

sonnel “to prepare an agenda for action” forincreasing the use of biotechnology. One important

outcome of this meeting was the formation of thebioremediation Action Committee (BAC). The

objective of the Committee is to facilitate the safe

development and use of biotechnology as a solutionto environmental problems. The BAC has now beensubdivided into six subcommittees: Oil Spill Re-

sponse, Treatability Protocol Development, Re-

search, Education, Data Identification and Collec-

tion, and Pollution Prevention (figure 2). Several of these subcommittees have, in turn, been furthersubdivided. All subcommittees report to the assist-

ant administrator of EPA’s Office of Research andDevelopment (ORD), who functions as the chair of the BAC. Many of the same industry, academic, andgovernmental representatives who attended the Feb-ruary meeting are participants on the BAC or itssubcommittees.

The Oil Spill Response Subcommittee is con-

cerned directly with the bioremediation of marine oil

spills. Its major goals are: 1) to evaluate scientificand applied engineering data on the safety andeffectiveness of bioremediation technologies; 2) toassess the information required for bioremediation

decisionmaking by Federal on-scene coordinators

and State oil spill response officials; 3) to prepareinterim guidelines on when and how to use bioreme-diation technologies; and 4) to investigate longer-

term issues for incorporating bioremediation into theNational Spill Response Plan.

l04An eventual result

of deliberations relating to these goals could be thedesign of a national bioremediation oil spill responseplan.

105However, further research and development

of more reliable technologies are required before

EPA is likely to undertake the effort required todevelop such a plan. In the meantime, the Subcom-mittee has prepared interim guidelines to assist

Regional Response Teams in assessing the desirabil-

Im . ~e ~d E.M. tivy, “Bior~e&tion: Waxy Crude Oils Stranded on bw-13nergy Shorelties, ” Proceedings: 1991 Oil Spill Conference, SanDiego, CA, Mar. 4-7, 1991.

101 M-, op. cit., footnote 79.

10ZU.S. EnvironmentalProtection Agency, “SUUMWUY Report on the EPA-Industry Meeting on Environmental Applications of Biotechnology,”

Crystal City, VA, Feb. 22, 1990.103u.s. Enviromn~M Protection Agency, bioremediation #Wion Committee,“S~ of June 20, 1990 Meeting,” p. [email protected]. Env~omenM fiotwtion Agency, officeofReSe=h~ dDevelopment, Bior~@tionAction Committee,“S~ of November7, 1990

Meeting,” Washingto~ DC, Nov. 7, 1990, pp. 4-7.

105F.M. Gregorio, memkr, Oil Spill Response SubCommittee, “Development of a National bioremediation Spill Response Plan,” Aug. 27, 1990.



Figure 2--Organization of the bioremediation Action

~

Fullcommittee

IPollution Oil

prevention spill response I Research

TTreatability

protocol

I I,

E I E I E C I E E E S ISOURCE: U.S. Environmental Protection Agency.

T

I9

User Contributorissues issues

bioremediationfor Marine Oil Spills q 25



bioremediationfor Marine Oil Spills q 25

ity of bioremediation and in planning for its use.l06

Such guidelines will enable decisionmakers to make

quick and defensible decisions about the use of bioremediation technologies.

The Treatability Protocol Development Subcom-mittee has focused its attention on providing techni-cal advice on the development of protocols fortesting the applicability and effectiveness of biore-mediation technologies in different environmentalsettings. Protocols use both chemical analyses and

bioassays to evaluate a bioremediation product’sability to degrade a waste product or pollutant and toensure that the product is safe to introduce into theenvironment.

107Performance criteria included in the

protocols can provide a standard for technologydevelopers against which they can compare theirprocesses.

The development of oil spill protocols is ahigh-priority EPA activity: without them it is not

possible to validate technology or process claimsmade by product vendors. This activity is beingcarried out largely through the National Environ-mental Technology Applications Corp. (NETAC)and EPA’s ORD labs (see below). One laboratory-based protocol has already been developed and usedto evaluate products intended for use on Alaska’sbeaches. Work is in progress on the development of an open water protocol, as well as on protocols forsensitive marine environments (e.g., marshes). TheSubcommittee will also be involved in developingprotocols for bioremediation of hazardous wastes.

The Treatability Protocol Development Subcom-mittee is also addressing several policy issuesrelated to protocols. For example, bioremediationcompanies are concerned about having productsretested that did not do well initially. Another issue

is the recourse available to companies that disagreewith test procedures. A third is the means by whichresults will be reported. (Results will probably bereported as statistically superior to, not statisticallydifferent from, or statistically inferior to a standard;

products are unlikely to be ranked. Also, productswill be judged on different criteria, including

efficacy, shelf life, toxicity, etc. A product that doeswell according to one criterion may not do well onanother.) Finally, there is the question of who paysfor product testing. EPA appears receptive to somecost sharing with product developers, but only forthose products that have met minimum criteria. 108 A

cost sharing program involving EPA, the petroleumindustry, and product vendors might also be ar-ranged so that a broad commercial testing program

could be established.l09

The main objective of the Research Subcommit-tee is to identify high priority needs for generalbioremediation research,

l10not specifically for oil

spill applications, but research advances in priorityareas could directly or indirectly benefit the latter.The Education Subcommittee will evaluate futureneeds for scientists, technicians, and engineers inbioremediation research and applications, as well asways to educate the public about the use of biotechnologies.

Although considerable bioremediation informa-tion has been generated by industry, States, andFederal agencies, this information is not necessarilyeasily accessible, nor has it been certified orstandardized for easy use by others. The DataIdentification and Collection Subcommittee will

focus its efforts on identifying data on field applica-tions and tests of bioremediation technologies (in-cluding marine spill applications such as those inAlaska and Texas), on providing guidance formaking data available without compromising clientor proprietary information, and on establishingroutine procedures for submission of data. TheSubcommittee has recommended that EPA’s Alter-native Treatment Technology Information Center

(ATTIC) database be used as the central database forall biological treatment technologies. Designed pri-marily as a retrieval network for information oninnovative technologies for treating hazardouswastes, ATTIC should have no trouble incorporating

l~u.s. Environmen~ prot~tion Agency, Subcommittee on National bioremediation Spill Response, bioremediation ACtiOn committee, ‘ ‘rnterim

Guidelines for Preparing bioremediation Spill Response Plans” (draft), Feb. 11, 1991.

~07E.B. Bmkey, NatiO@ Environmental lkchnology Applications COW, “Presentation of the Prognxs of the Treatability Protocol Development

Subcommittee,” Washington, DC, Nov. 7, 1990.IOSRe~ks of J. Skinner, Deputy Assistant~“ “strator of the Office of Research and Developmen~ U.S. Environmental Protection Agency, duringNov. 7, 1990 meeting of the bioremediation Action Committee.

109s+ L@ le , D e p u & D & t O r , ~lce of Envfi~enW Engineering and ‘IkchnologyDemonstratio~ U.S. Environmental ProtectionAg~cy, P~on~

communication Feb. 20, 1991.

11 OU.S. Environment Protection Agency, op. cit., footnote 103.



Bioremediation for Marine Oil Spills q 27



f p

Business Daily.113

Thirty-nine proposals were sub-mitted. The NETAC panel evaluated these and

recommended that 11 undergo laboratory testingspecified by the protocol. Effectiveness and toxicitytests were conducted in EPA’s Cincinnati, Ohio Risk Reduction Engineering Laboratory. EPA, again withNETAC’s help, selected two products judged mostappropriate for field testing in Alaska.

The process of identifying promising productsappears to OTA to be both appropriate and fair and,in general, NETAC is performing a valuable service.Nonetheless, a few of the bioremediation firms thathad submitted products contended that the testsspecified in the protocol were not appropriate toassess the true effectiveness of these products. Asmore bioremediation research results become avail-able, it will be possible to refine this frost protocol,if necessary. A NETAC expert panel is currentlyidentifying the kinds of studies and types of testsneeded for an open water bioremediation protocol,and EPA’s ORD labs will again use the resultingframework protocol to develop a complete experi-mental design. Although much skepticism remainsabout the effectiveness of bioremediation at sea, anopen water protocol would be useful whether or notany effective products were identified.

Relative to the new Interim Guidelines publishedby the Oil Spill Response Subcommittee, NETAC is

also preparing to assist Regional Response Teams inplanning for the possible use of bioremediationtechnologies. Specifically, NETAC has begun to:1) systematically compile information on commer-cial bioremediation products, 2) collect samples forpreliminary laboratory evaluation of bioremediationproducts, 3) define a national bioremediation prod-uct evaluation facility to test commercial products,and 4) develop the capability to provide technical

assistance to the States and to regional or nationalresponse teams.

114

The Private Sector

The bioremediation industry is a young industryseeking to develop markets for its products and

expertise. The industry is composed primarily of companies with fewer than 100 employees, and not

many of these companies have been in existence formore than 5 years. Of the companies that havedeveloped bioremediation products, few specializein products for marine oil spills. If a market for suchproducts were to develop, however, many compa-nies would be interested. More than 50 companiesClaiming bioremediation expertise have expressedinterest in supplying products or personnel inresponse to the Persian Gulf oil spill.

115Only a

handful of these products have undergone testing toevaluate their effectiveness on marine spills (seeabove), and none has yet developed a reputationamong experts as an effective response to suchspills.

The Applied Biotreatment Association (ABTA)was established in 1989 to promote the interests of the bioremediation industry. The organization has

55 members and consists of about equal numbers of corporate and adjunct associates. Corporate mem-bers are biotreatrnent companies, and adjunct mem-bers include State biotechnology centers, equipmentcompanies, and university professors. In addition tobioremediation companies, ABTA now includesamong its members two large oil companies. ABTArecently produced a briefing paper on the role of bioremediation in oil spills.

ll6Several of its mem-

bers actively participate in EPA’s bioremediationAction Committee.

For the most part, the oil industry has taken await-and-see attitude toward bioremediation. Fewcompanies are doing research on the bioremediationof marine oil spills, preferring instead to let EPAtake the lead. Recently, the Petroleum Environ-mental Research Forum (PERF), an oil industry

group that sponsors research on environmentalproblems of concern to its members, proposed amass balance study to evaluate the potential of nutrient enrichment and seeding on open water.l

17

This study is expected to begin in mid-1991.Although both the American Petroleum Institute andthe new Marine Spill Response Corp. believe that

1lSU.S. Environmen~ ProtectionAgency, “EPA Seeks Biological Methods for Potential Application to Cleanup of Alaskan Oil Spill,’ Commerce Business Daily, Feb. 12, 1990.

I loNatio~ Environmen~ ‘lkcImology Applications COW., ‘‘NETAC’S Role in Supporting bioremediation Product Selection for Oil Spills, ” Feb.11, 1991,

115K. Devine, Appfi~ Biomatment Association telephone Communication Jan. 28, 1991.

llGApplied BiOtreament &+SociatiO~ “The Role of bioremediation of Oil Spi~S, ” fall 1990.

IITJ. Salanirrq Shell Development Co., personal communication Jan. 29, 1991.




f p

bioremediation warrants further research, neither isplanning a significant research program of its

own.ll8

The Exxon Corp. so far is an importantexception to the general absence of oil industryactivity. Exxon, by virtue of its Alaska and NewJersey experiences, has more familiarity with fertili-zation techniques than other oil companies and, atleast for certain types of environment, has concludedthat their use is merited.

REGULATORY ISSUES

Several provisions of the Federal Water PollutionControl Act (the Clean Water Act (CWA)), asrecently amended by the Oil Pollution Act of 1990(OPA), affect or potentially affect the use of bioremediation products for marine oil spills. TheCWA specified development of a National Contin-gency Plan (NCP) for the removal of oil andhazardous substances. The OPA calls for the revi-sion of parts of this plan. It requires EPA to preparea list of dispersants, other chemicals, and other spill

mitigating devices and substances that may be usedto carry out the plan, and to identify the waters inwhich they may be used and the quantities that canbe used safely.

119Thus, before a bioremediation

product could be considered for use in response to anoil spill, it would, at minimum, have to be on this list.Inclusion on the list implies that certain minimum

safety standards have been met but does not neces-sarily imply that the product is either effective ornontoxic for specific applications. In general, addi-tional testing would be required to evaluate furtherboth efficacy and toxicity.

Subpart J of the National Contingency Plangoverns the use of biological additives (as well asdispersants and other chemical agents) for marine oilspills. It identifies several options that can be used to

obtain authorization for the application of a chemi-cal or biological agent to combat a spill. Section300.910e provides for preauthorization of the use of regulated agents through an advance planning proc-

ess. Thus, on-scene coordinators (OSCs) are author-ized to use biological additives that have been

preapproved by Regional Response Teams. EPAencourages preplanning and believes that the delib-erations of Regional Response Teams provide thebest forum for considering authorizations.

120

If a preauthorized plan has not been established oris not applicable to the specific circumstances of aspill, the OSC must obtain the concurrence of theEPA representative to the Regional Response Team,the affected State(s), and impractical, the Departmentof the Interior and Department of Commerce naturalresource trustees, before using biological or otheragents. An exception occurs when the OSC deter-mines that quick action is necessary to prevent orsubstantially reduce a hazard to human life; in thatcase, the OSC may unilaterally authorize the use of any product, including those not on the NCP product

schedule. Continued use of the product once thethreat has been mitigated is subject to normalconcurrence procedures.

121

Another statute that may have a bearing onbioremediation technologies is the Toxic SubstancesControl Act (TSCA). The TSCA is intended toregulate the manufacture of substances that maypose a risk to human health or the environment. The

Act applies to chemical substances generally, whichEPA has interpreted to include microorganisms.Currently, however, manufacturers of microbialproducts are not required to satisfy Section 5 of TSCA and notify EPA regarding the use of naturallyoccurring microorganisms, and EPA has no plans torequire notification.

122Genetically engineered orga-

nisms, however, would be subject to notification andreview. EPA has not had the opportunity to conduct

product reviews of genetically engineered orga-nisms, but a few companies are considering theiruse. EPA expects to publish a draft biotechnologyrule in the Federal Register by late 1991.

lls~e-e spilI Rmpome Corp. (MSRC), formerly the Petroleum Industry Response Gg anizatioq was established after thel?xxon VaZdez oil spilland is intended to give the private sector an improved capability to respond to major oil spills around the country. MSRC has a sizable research budget.

llgofl pollution Act of 1990, s~tion 4201(b). This schedule maybe obtained from EPA’s EmergencyResponse Division. The list currentlyCOnti30 bioremediation products and includes both chemical fertilizers and microbial products.lmJ. (--gham, U.S. Environmental Protection Agency, presentation to the Treatability Protocol Subcommittee of the bioremediationActiolI

committee, Oct. 15, 1990.

lzl~id.

IZZE. wews~, U.S. EnvkowenM protection Agency, Office for Pesticides and Toxic Substances, pmSOMI communication ~. *6, 1991.

Appendix A



Glossary

Aerobic bacteria: Any bacteria requiring free oxygen forgrowth and cell division.

Aliphatic compound: Any organic compound of hydro-gen and carbon characterized by a linear or branchedchain of carbon atoms. Three subgroups of suchcompounds are alkanes, alkenes, and alkynes.

Anaerobic bacteria: Any bacteria that can grow anddivide in the partial or complete absence of oxygen.

Aromatic: Organic cyclic compounds that contain one or

more benzene rings. These can be monocyclic, bicy-clic, or polycyclic hydrocarbons and their substitutedderivatives. In aromatic ring structures, every ringcarbon atom possesses one double bond.

Assay: Qualitative or (more usually) quantitative deter-mination of the components of a material or system.

Biodegradation: The natural process whereby bacteria orother microorganisms chemically alter and break downorganic molecules.

bioremediation: A treatment technology that uses bio-logical activity to reduce the concentration or toxicityof contaminants: materials are added to contaminatedenvironments to accelerate natural biodegradation.

Catalyst: A substance that alters the rate of a chemicalreaction and may be recovered essentially unaltered inform or amount at the end of the reaction.

Cometabolism: The process by which compounds inpetroleum may be enzymatically attacked by micro-organisms without furnishing carbon for cell growth

and division.Culture: The growth of cells or microorganisms in acontrolled artificial environment.

Dispersant: Solvents and agents for reducing surfacetension used to remove oil slicks from the watersurface.

Emulsification: The process of intimately dispersing oneliquid in a second immiscible liquid (e.g., mayonnaiseis an example of an emulsified product).

Enzyme: Any of a group of catalytic proteins that are

produced by cells and that mediate or promote thechemical processes of life without themselves beingaltered or destroyed.

Fraction: One of the portions of a chemical mixtureseparated by chemical or physical means from theremainder.

Gas chromatography: A separation technique involvingpassage of a gaseous moving phase through a columncontaining a fixed liquid phase; it is used principally asa quantitative analytical technique for compounds that

are volatile or can be converted to volatile forms.

Gravimetric analysis: A technique of quantitative ana-lytical chemistry in which a desired constituent isefficiently recovered and weighed.

Hydrocarbon: One of a very large and diverse group of chemical compounds composed only of carbon andhydrogen; the largest source of hydrocarbons ispetroleum crude oil.

Inoculum: A small amount of material (either liquid or

solid) containing bacteria removed from a culture inorder to start a new culture.

Inorganic: Pertaining to, or composed of, chemicalcompounds that are not organic, that is, contain nocarbon-hydrogen bonds. Examples include chemicalswith no carbon and those with carbon in non-hydrogen-linked forms.

Metabolism: The physical and chemical processes bywhich foodstuffs are synthesized into complex ele-ments, complex substances are transformed into sim-

ple ones, and energy is made available for use by anorganism; thus all biochemical reactions of a cell ortissue, both synthetic and degradative, are included.

Metabolize: A product of metabolism.

Microorganism: Microscopic organisms including bac-teria, yeasts, filamentous fungi, algae, and protozoa.

Mineralization: The biological process of completebreakdown of organic compounds, whereby organicmaterials are converted to inorganic products (e.g., the

conversion of hydrocarbons to carbon dioxide andwater).

Oleophilic: Oil seeking (e.g., nutrients that stick to ordissolve in oil).

Organic: Chemical compounds based on carbon that alsocontain hydrogen, with or without oxygen, nitrogen,and other elements.

Pathogen: An organism that causes disease (e.g., somebacteria or viruses).

Saturated hydrocarbon: A saturated carbon-hydrogencompound with all carbon bonds filled; that is, thereare no double or triple bonds, as in olefins oracetylenes.

Soluble: Capable of being dissolved.

Surface-active agent: A compound that reduces thesurface tension of liquids, or reduces interracialtension between two liquids or a liquid and a solid; alsoknown as surfactant, wetting agent, or detergent.

Volatile: Readily dissipating by evaporation.

–29–

Appendix B



Acknowledgments

We are grateful to the many individuals and organizations in the United States and abroad who shared their special

knowledge, expertise, and information with us in the preparation of this study. We are especially indebted to those whoparticipated in our bioremediation workshop and to those who critiqued our first draft.

Partcipants, OTA Workshop on bioremediation for Marine Oil Spills December 4,1990

James Blackburn

Energy, Environment and Resources CenterUniversity of Tennessee

Al BourquinECOVA Corp.

Peter ChapmanEnvironmental Research LaboratoryU.S. Environmental Protection Agency

Rita ColwellMaryland Biotechnology InstituteUniversity of Maryland

Joseph J. CooneyEnvironmental Sciences programUniversity of Massachusetts

John GlaserRisk Reduction Engineering LaboratoryU.S. Environmental Protection Agency

Jack R. Gould

American Petroleum Institute

Frank M. GregorioEnviroflow

Steven HintonExxon Corporate Research

Stephen LingleOffice of Environmental Engineering and

Technology DemonstrationU.S. Environmental Protection Agency

Margaret MellonNational Wildlife Federation

James MielkeScience Policy Research Division

Congressional Research Service

Clayton PageEnvironmental Remediation, Inc.

Robert G. RokuIBP America

John TealWoods Hole Oceanographic Institution

Edward TennysonMinerals Management Service

B.J. WynneTexas Water Commission

Franz K. HiebertAlpha Environmental, Inc.

Additional Reviewers and Other Contributors

Ronald M. AtlasUniversity of Louisville

Kay AustinU.S. Environmental Protection Agency

Thomas L. BaughOffice of Research and DevelopmentU.S. Environmental Protection Agency

Edgar Berkey

National Environmental Technology Applications Corp.

Rosina BierbaumOffice of Technology Assessment

Neil A. ChallisInternational Tanker Owners Pollution Federation

Jessica M. CogenNational Environmental Technology Applications Corp.

Katherine DevineApplied Biotreatment Association

F. Rainer EngelhardtMarine Spill Response Corp.

Mervin FingasTechnology Development and Tewchnical Services BranchEnvironment Canada

- 3 0 -

Appendix Acknowledgments .31



Mary GadeOffice of Solid Waste and Emergency ResponseU.S. Environmental Protection Agency

Robert HiltabrandResearch and Development CenterU.S. Coast Guard

Rebecca Hoff Hazardous Materials Response BranchNational Oceanic and Atmospheric Administration

Kurt JakobsonU.S. Environmental Protection Agency

Peter JohnsonOffice of Technology Assessment

David KennedyHazardous Materials Response BranchNational Oceanic and Atmospheric Administration

Mahlon C. KennicuttTexas A&M University

Howard Levenson

Office of Technology AssessmentMorris A. LevinMaryland Biotechnology Institute

Alfred W. LindseyOffice of Research and DevelopmentU.S. Environmental Protection Agency

Henry LongestOffice of Solid Waste and Emergency Response

U.S. Environmental Protection AgencyPatrick C. MaddenExxon Research and Engineering

Alan MearnsHazardous Materials Response BranchNational Oceanic and Atmospheric Administration

Tom MerskyNational Environmental Technology Applications Corp.

James G. MuellerSouthern Bio Products, Inc.

Kevin O’Connor

Office of Technology Assessment

Daniel OwenInternational Tanker Owners Pollution Federation

James PayneScience Applications International Corp.

Roger PrinceExxon Research and Engineering

Hap PritchardEnvironmental Research LaboratoryU.S. Environmental Protection Agency

Patrick RocquesTexas Water Commission

Pamela Russell-HarrisOffice of Solid Waste and Emergency ResponseU.S. Environmental Protection Agency

Joseph Salanitro

Shell Development Company

Gary ShigenakaHazardous Materials Response BranchNational Oceanic and Atmospheric Administration

Albert VenosaRisk Reduction Engineering LaboratoryU.S. Environmental Protection Agency

Oskar Zaborsky

National Academy of SciencesRaymond A. ZilinskasMaryland Biotechnology InstituteThe University of Maryland

Bioremediations Oil Spill

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