Bioremediation of marine environment

contaminated with petroleum

hydrocarbon

Richard Mannion

Martin Casserly

Martin Gallagher

Table of Contents

• Introduction

• Environmental factors

• Fate of contaminant

• Chemical composition of petroleum hydrocarbons

• Hydrocarbon degrading microbes

• Biodegradation of hydrocarbon components

• Factors that determine cost of oil spill

• Two approaches to oil spill bioremediation

Bioremediation is defined as the act of adding or improving the availability of

materials (e.g., nutrients, micro organisms, or oxygen) to contaminated environments

to cause an acceleration of natural biodegradative processes. The results of field

experiments and trials following actual spill incidents have been reviewed to evaluate

the feasibility of this approach as a treatment for oil contamination in the marine

environment. The ubiquity of oil-degrading micro organisms in the marine

environment is well established, and research has demonstrated the capability of the

indigenous micro flora to degrade many components of petroleum shortly after

exposure. Studies have identified numerous factors which affect the natural

biodegradation rates of oil, such as the origin and concentration of oil, the availability

of oil-degrading micro organisms, nutrient concentrations, oxygen, climatic

conditions, and sediment characteristics.

Total petroleum hydrocarbons (TPH) are a term used to describe a large family of

several hundred chemical compounds that originally come from crude oil. Crude oil is

used to make petroleum products, which can contaminate the environment. TPH is a

mixture of chemicals, but they are all made mainly from hydrogen and carbon, called

hydrocarbons. TPH may enter the environment through accidents, from industrial

releases, or as by-products from commercial or private uses. TPH may be released

directly into water through spills or leaks.

Petroleum means "rock oil". The term petroleum is nowadays used as a common

denotation for crude oil and natural gas. Petroleum, then, is a collective term for

hydrocarbons, whether solid, liquid or gaseous. Reserves of natural gas and crude oil

have formed over millions of years as plants and animals have been broken down and

undergone chemical change at high temperature and pressure. That is why oil and

natural gas (and coal) are referred to as "fossil fuels". Petroleum is found in porous

rock-formed in large sedimentary basins, where the oil and gas has been trapped by

some kind of barrier thereby forming a reservoir. Some TPH fractions will float on

the water and form surface films. Other TPH fractions will sink to the bottom

sediments. Some TPH fractions will move into the soil where they may stay for a long

time. It is therefore the work of these microbes to degrade the hydrocarbons.

Altogether, more than 70 microbial genera are known to contain organisms that can

degrade petroleum components. The micro organisms transform contaminants to less

harmful compounds through aerobic and anaerobic respiration, fermentation, co

metabolism and reductive dehalogenation. There are certain environmental factors

that affect the bioremediation processes such as temperature, oxygen, pH, turbulence,

background concentration of inorganic nutrients and the type of contaminants. The

resulting products can be carbon dioxide, water, and partially oxidized biologically

inert by-products. Certain enzymes produced by microbes attack hydrocarbons

molecules, causing degradation. The degradation of oil relies on having sufficient

microbes to degrade the oil through the microbe’s metabolic pathways. If microbes

are not present in a system they can be added to help promote bioremediation. The

added microbes can be cultures grown from other contaminated areas or they can be

microbes genetically engineered to degrade oil. However, even when these microbes

are present, degradation of hydrocarbons can take place only if all other basic

requirements of the microbes are met.

Environmental factors

Carbon

Carbon is the most basic structural element of all living forms and is needed in greater

quantities than other elements. The nutritional requirements of carbon to nitrogen are

10:1 and carbon to phosphorus 30:1 (Atlas and Bartha 1981, p.70). Reduced organic

carbon is a source of energy for microbes because it has high energy yielding bonds in

many compounds. In the decomposition of oil, there is plenty of carbon for the micro

organism due to the structure of the oil molecule.

Nitrogen

Nitrogen is found in the proteins, enzymes, cell wall components. (Atlas and Bartha

1981, p.70).The microbes must be supplied with nitrogen in some form. Without

sufficient nitrogen the metabolism of the microbes will be altered. The main source of

nitrogen is from the atmosphere. Only a few microbes can use this molecular nitrogen

so most micro organisms require fixed nitrogen such as organic nitrogen, ammonium

ions or nitrate ions. These other forms of nitrogen can be scarce in certain

environments, causing nitrogen to become a limiting factor in the growth of microbial

populations.

Oxygen

Biodegradation is predominantly an oxidation process. Not only do the aerobic

microbes need the oxygen to survive, bacteria enzymes catalyze the insertion of

oxygen into the hydrocarbon so that the molecule can subsequently be consumed by

cellular metabolism. Because of this, oxygen is one of the most important

requirements for the biodegradation of oil. There is usually enough oxygen to prevent

a lack of it from limiting biodegradation. The primary source of oxygen for

biodegradation is atmospheric oxygen. When oxygen is limiting the water can be

aerated.

Water

Water is an important factor as it is needed by the microbes. It makes up a large

portion of the cells cytoplasm and because most enzymatic reactions take place in

solution. Water is also needed for transport of most materials into and out of the cell.

Water is not a limiting factor in marine oil spills, but in bioremediation of oil spilled

on land it may be an important factor which needs to be controlled.

Fate of contaminant

When petrol contaminants enter an aquatic system they are subject to some physical,

chemical and biological changes. These changes contribute to the loss or alteration of

some of the components.

Toxicity

Some oils have toxic hydrocarbons. These may prevent or delay microbial attack and

can slow down the process of degradation. Under favourable conditions microbes will

degrade 30-50% of a crude oil residue. However with favourable conditions and the

proper microbes all type of crude oils can be broken down, straight chained, branched

chained, crylic, aromatic, polynuclear aromatic. In most natural ecosystems the

numbers of hydrocarbon utilising microbes will initially limit the rate of hydrocarbon

degradation. After a short period of exposure to the petroleum pollutant the number of

utilising microbes increases and is no longer a problem.

Sorption

Solid surfaces can act by adsorption which is the retention of solutes in solution by

the surface of the solid material, or by absorption which is the retention of the solute

within the mass of the solid rather then on its surface. Sorption reflects the rate of

volatilisation. Adsorption may involve van der walls forces or hydrogen bonding.

Cationic molecules may be absorbed to the ion exchange sites of clay minerals or

organic matter present.

Volatilisation

Some hydrocarbons are very volatile and are lost to the atmosphere by evaporation.

Evaporation is the most important natural cleansing process during the early stages of

an oil spill, and it results in the removal of lighter-weight components in oil.

Depending on the composition of the oil spilled, up to 50 percent of the more toxic,

lighter weight components of oil may evaporate within the first 12 hours following a

spill (U.S. EPA, 1999). In terms of environmental impacts, evaporation is the most

important weathering process during the early stages of an oil spill in that it can be

responsible for the removal of a large fraction of the oil including the more toxic,

lower molecular weight components.

For oil on water, evaporation removes virtually all the normal alkanes smaller than

C15 within 1 to 10 days. Volatile aromatic compounds, such as benzene and toluene,

can also be rapidly removed from an oil slick through evaporation. However, these

volatile oil components may be more persistent when oil is stranded in sediments. The

volatile components make up 20-50% of most crude oils, about 75% of No. 2 fuel oil,

and about 100% of gasoline and kerosene. As a result, the physical properties of the

remaining slick change significantly (e.g., increased density and viscosity). Major

factors influencing the rate of evaporation include composition and physical

properties of the oil, wave action, wind velocity, and water temperature.

Tempeture

Hydrocarbon degradation has been found to occur at a wide range of temperatures.

Degradation has occurred at tempetures as low as 0oc and as high as 700c.It is an

important factor on the rate of biodegradation (Atlas 1981, p.190).It is so important

because at low temperatures, molecules move relatively slowly and colliding

molecules do not always bring about a reaction" (Atlas 1984, p.339). Raising the

temperature will increase the possibility of reactions taking place. In general the rate

of enzymatic reactions can be doubled for every 10°C. The more enzymatic reactions

the faster the biodegradation will occur. Even though temperature plays an important

part in the rate of biodegradation, it does not act alone. Concentration of the

hydrocarbon is also an important factor.

Spreading

The spreading of oil on water is one of the most important processes during the first

hours of a spill, provided that the oil pour point is lower than the ambient temperature.

The principal forces influencing the spreading of oil include gravity, inertia, friction,

viscosity and surface tension. This process increases the overall surface area of the

spill, thus enhancing mass transfer via evaporation, dissolution, and later

biodegradation.

Dispersion

Dispersion, or formation of oil-in-water emulsions, involves incorporating small

droplets of oil into the water column, resulting in an increase in surface area of the oil.

In general, oil-in-water emulsions are not stable. However, they can be maintained by

continuous agitation, interaction with suspended particulates, and the addition of

chemical dispersants. Dispersion may influence oil biodegradation rates by increasing

the contact between oil and the microbes.

Emulsification

The process of emulsification of oils involves a change of state from an oil-on-water

slick or an oil-in-water dispersion to a water-in-oil emulsion, with the eventual

possible formation of a thick, sticky mixture that may contain up to 80% water,

commonly called “chocolate mousse”. The formation and stability of emulsions are

primarily related to the chemical composition of the oils and are enhanced by wax and

asphaltic materials. Surface-active materials generated through photochemical and

biological processes are also involved in formation of the emulsions. The formation of

emulsions makes oil clean-up operations more difficult by decreasing the

effectiveness of physical oil spill recovery procedures and suppressing the natural

rates of oil biodegradation.

Photo oxidation

Photo oxidation occurs when oxygen under sunlight reacts with oil components.

Photo oxidation leads to the breakdown of more complex compounds into simpler

compounds, which tend to be lighter in weight and more soluble in water, allowing

them to be removed further through other processes.

Concentration

The concentration of pollutants is an important factor. If the concentration of

petroleum hydrocarbons is too high then it will reduce the amount of oxygen, water

and nutrients that are available to the microbes. This will create an environment where

the microbes are stressed reducing their ability to break down the oil. The

concentration of the hydrocarbon will affect the biodegradability and toxicity to the

degrading organisms. The concentration at which inhibition occurs depends on the

compound. Concentrations in the range of 1 to 100µg/ml of water are not said to be

very toxic. The concentration of the hydrocarbons will have two effects. At low

concentrations all fractions of the hydrocarbon will be attacked. At high

concentrations only fractions susceptible to degradation will be attacked. The

concentration of the contaminant will affect the number of microbes present. It has

been found that high concentrations of gasoline in contaminated water were related to

higher counts of microbes.

Viscosity

Viscosity is the property of the fluid that describes how it resists a change in shape or

movement. The lower the viscosity a fluid has, the more easily it flows. The viscosity

of petroleum is related to oil compositions and the ambient temperature. It is an

important index of the spreading rate of spilled oil.

Solubility

The solubility of oil in water is extremely low and depends on the chemical

composition of the petroleum hydrocarbon in question and temperature. For a typical

crude oil, solubility is around 30 mg/L (NAS, 1985). The most soluble oil components

are the low molecular weight aromatics such as benzene, toluene and xylene. This

property is important with respect to oil fate, oil toxicity and bioremediation processes

Soil sediment

Depending on where the contamination has occurred some of the hydrocarbon may

get washed up along the shore line and become trapped in the sand sediments and in

and between rocks. The type of soil present will determine whether not insitu

treatment is possible.  Effective treatment of sub soils requires continual access to

nutrients by microbes to promote growth.  Tight soils, those with high clay content,

are more likely to plug up and restrict the free flow of nutrients to the microbes.

However soils with high sand content which are common in a marine environment

allow nutrients and oxygen to flow and are therefore more tractable to bioremediation.

The Chemical composition of petroleum hydrocarbons

Petroleum Hydrocarbon based products are quite common today with many uses in

transport, heating and machinery function. There are three different types of

petroleum hydrocarbons products, they are; Crude oil, petroleum components and

refined oil products.

Crude oil

Crude oil is the most well known for marine destruction due to spills, it is so

destructive as it is extremely thick and heavy and just suffocates the environment

whether it be on the seashore or out to sea.

Crude oil is made up of both hydrocarbon compounds (accounting for 50-98% of its

total formation) and non hydrocarbon compounds (which may contain sulphur

nitrogen oxygen and some trace metals) in a wide range of combinations

Petroleum components

Petroleum components may be broken up into four major groups due to their

differential solubility in organic solvents. They are Saturated Hydrocarbons, Aromatic

Hydrocarbons, Resins and Asphaltines.

Saturated Hydrocarbons

Saturated Hydrocarbons include normal and branched alkanes with aliphatic

structures and cyclic alkanes with alicyclic structures.

Saturated Hydrocarbons can range in chain length from one carbon to over 40

Carbons. Saturates usually are the most abundant material in crude oil.

Aromatic Hydrocarbons

Aromatic Hydrocarbons include monocyclic aromatics examples of such aromatics

include Benzine, Toluene and Xylene and polycyclic aromatic Hydrocarbons (PAH’s)

examples of such PAH’s include Napthalene, Anthracene and Phenanthrene and

which have two or more fused aromatic rings.

PAH’s are of particular environmental concern because of their potential

carcinogenicity or which maybe converted into carcinogens through microbial

metabolic reactions

Resins

Resins include polar compounds containing nitrogen, sulphur and oxygen examples of

these include pyridines and thiophenes and these are often referred to as NSO

compounds

Asphaltines

Asphaltines consist of poorly characterised hydrocarbons with high molecular weight

compounds and possible NSO group connections also metals such as nickel,

vanadium and iron are also associated with asphaltines.

Refined oil products

Refined oil products such as petrol, kerosene, fuel oils, lubricating oils are all derived

from crude oil through certain processes such as catalytic cracking and fractional

distillation. These products have physical and chemical characteristics according to

the type of crude oil used and the process of which the product was formed. In the

catalytic cracking process unsaturated compounds or olefins (Alkenes and Cyclo-

Alkenes) which are not generally present in crude oil that can easily are formed. For

example the concentrations of Olefins are as high as 30% in petrol while about only

1% in jet fuel.

Table 1 Chemical compositions of refined petroleum products (adapted from Clark and Brown, 1977)       Distillation Fraction Hydrocarbon types

Range of carbon atoms

Typical Refined products

Gasoline & naphtha Saturates 04--12 Gasoline  olefins  

  Aromatics    Middle Distillate Saturates 10--20 Kerosene  olefins Jet Fuel  Aromatics Heating oil      Diesel oilsWide cut gas oil Saturates 18--45 Wax   Aromatics   Lubricating oilResidium Resins >40 Residual oils  Asphaltines   Asphalt

Hydrocarbon Degrading Microbes

These Micro organisms which are capable of degrading petroleum hydrocarbons and

related compounds are ubiquitous in marine habitats. There are up to 200 species of

bacteria, yeasts and fungi that have been shown to degrade hydrocarbons ranging

from methane to compounds of over a 40 carbon chain length. In the marine

environment bacterial are considered to be the most dominant of the hydrocarbon

degraders with a range of distribution that even covers extreme cold environments

such as the Antarctic and arctic regions.

The Distribution of hydrocarbon degraders or utilizing micro organisms is

predominantly related to historical exposure of the environment to petroleum

hydrocarbons. These environments with a recent or continuous oil contamination will

have a higher concentration of hydrocarbon degraders compared to a low percentage

of hydrocarbon degraders in unpolluted areas. In clean or free from the presence of

petroleum hydrocarbons the percentage of hydrocarbon degraders will make up less

than 0.1% of the total microbial community, while as in areas of oil pollution they can

constitute up to 100% of the viable micro organisms present.

It should be known that there is no single strain of bacteria capable or that has the

metabolic capacity to degrade all the hydrocarbon components. In nature

biodegradation of crude oil or its by-products typically involve a succession of species

within the microbes present.

Micro organisms classifies as they utilise non hydrocarbon that may also play a very

important role in the full removal of petroleum eventually from the environment.

Degradation of petroleum involves a progressive of sequential (in sequence)

reactions, in which certain organisms may carry out an initial attack on the petroleum

compound and turn this produces an intermediate compounds that are subsequently

utilized by a different group of organisms this leads to further degradation and

possible removal after a number of these processes have occurred.

Micro organisms capable of degrading petroleum hydrocarbons

Bacteria Yeast and Fungi

Achromobacter Aspergillus Acinetobacter CandidaAlcaligenes CladosporiumAnthrobacter PenicilliumBacillus RhodotorulaBrevibacterium SporobolomycesCornybacterium CandidaFlavobacterium TrichodermaNocardiaPseudimonasVibro

Biodegradation of Hydrocarbon components

Petroleum components have been classified into four major groups as mentioned

earlier they are Saturates, Aromatics, Resins and Asphaltines.

Saturates

In general the n group alkanes are the easiest degradable component of saturated

hydrocarbons.

Biodegradation of the n group alkanes with molecular weights up to C44 has been

demonstrated while as Alkanes in the C10 to C 26 are considered the most frequently

and readily utilized hydrocarbons.

The predominant mechanism of the n group Alkanes degradation involves the

terminal oxidation to the corresponding alcohol, aldehydes or fatty acid functional

group.

Highly branched iso-prenoid alkanes such as pristine and phytane which were earlier

thought to be resistant to degradation through study have been found to be readily

degradable.

Complex alicyclic compounds such as hopanes and steranes are among the most

persistant petroleum compounds in the environment.

Aromatics

Aromatics are generally more resistant to biodegradation although but some low

molecular weight aromatics such as naphthalene may actually be oxidised before

many saturates.

Mono aromatic hydrocarbons are toxic to some micro organisms due to their solvent

action on the cell membranes, but mono aromatic hydrocarbons in low concentrations

are extremely easily biodegraded through aerobic respiration.

Polycyclic aromatic hydrocarbons with between 2-4 rings are less toxic and

biodegradable with the level of formation complexity.

Polycyclic aromatic hydrocarbons with five or more rings can be degraded only by

co-metabolism, which the microbes fortuitously (accidentally) transform non growth

substrates while metabolising hydrocarbons of a simpler combination or other primary

substrates found within the oil.

The bacterial degradation of aromatics normally involves the formation of a diol

followed by a ringed cleavage and finally the formation of a di-carboxylic acid.

Fungi and other types of eukaryotes normally oxidise aromatics by mono-oxygenases

an enzyme produced that acts somewhat similar to the bacterial metabolism although

a trans-diol is formed instead.

Asphaltines and Resins

Up until very recently very little was known about the biodegradation of both Resins

and Asphaltines, as this was due to their complex structures which were extremely

difficult to analyse although with new technology ever improving this has made it

easier to analyse such complex compounds to gain a better understanding into the

structure and biodegradation of these compounds.

They have been considered to be refractory (stubborn) to biodegradation, however

recently evidence has shown that Asphaltine components can be biodegraded through

co-metabolism.

Some Resins particularly with a low molecular weight Resin fraction can also be

biodegraded at low concentrations.

Summary

To summarise the susceptibility of petroleum hydrocarbons to microbial degradation

generally occurs in the following order:

n-alkanes<branched alkanes<low molecular weight aromatics<cyclic alkanes

although this pattern is not universal. The composition of oils may greatly differ in

heterogeneity (combinations) which in turn will affect the rate of biodegradation by

the microbial population present.

The degradation rate for the constituents may vary significantly for different oils.

Many of the complex branching, cyclic, and aromatic hydrocarbons, which otherwise

would not be biodegraded individually, although can be oxidised through co-

metabolism in an oil mixture due to the abundance of other substrates which can be

easily metabolised within the mixture of components present within the oil.

The biological fate of the oil components in an oil mixture still requires further

research. Particularly efforts should be made to establish a data base regarding the

biodegradability of different types of oils and their components such as petroleum

products.

Factors That Determine The Cost Of Oil Spills

There is a general agreement that the main factors influencing the cost of spills are :

Type of oil

Amount spilled

Pattern of spillage

Location

Clean-up response

Termination of Clean-up

The interactions between these factors are complex.

Type of Oil Spilled

One of the most important factors that determine the seriousness and therefore the

ultimate cost of an oil spill, one of the most important is the type of oil.

Generally light refined products (e.g. petrol, diesel) and light crude oils do not persist

on the surface of the sea for a long time due to rapid evaporation of the volatile

components and because they disperse and dissipate naturally, especially in rough

seas. This occurred during the Braer incident in the Shetland Isles, UK in January

1993. A combination of light crude oil and severe weather conditions resulted in the

entire cargo of 85,000 tonnes was lost. The spill had little contact with the shoreline,

even though the tanker was stranded on the coast. Clean-up costs in this case were

extremely low (about US $ 0.5m), especially due to the large quantity of oil involved.

At the other end of the spectrum of oil types are heavy crudes and heavy fuel oils.

These oils are highly persistent when spilled due to the high proportion of non-

volatile

components and high visocity. These oils have the potential, to travel great distances

from the original spill location. The downside to this, is that the clean-up of heavy oil

spills can be very difficult, and be very costly. This is illustrated by two of the most

expensive tanker spills of all time, the Erika off France and the Nakhodka off Japan.

Both involved relatively small amounts of oil (17,500 tonnes in the case of the

Nakhodka and about 20,000 tonnes in the Erika) spilled a distance from the coast.

Severe weather affected offshore recovery operations, allowing the highly persistent

oil to spread over a large area of sea, which lead to extensive coastal contamination.

The cost of cleaning up of heavy fuel oil relative to the quantity spilled is also

demonstrated by the Tanio, which crashed off the north coast of Brittany, France in

1980. In this case the clean-up of the 14,500 tonnes of heavy fuel oil cargo

contaminated over 200 km of the Brittany coastline was just as difficult and as costly

to clean up as the 223,000 tonnes of crude oil from the Amoco Cadiz which had

contaminated the same area almost exactly two years earlier.

The nature of the damage caused by a spill will also vary due to the type of oil. Light

refined products may constitute a fire and explosive hazard if spilled in confined

situations, which may lead to a wide variety of third party claims due, for example, to

temporary closure of port areas or nearby industry. These oils also tend to be more

toxic than heavier oils. This can lead to mortalities of marine plants and animals if

high

concentrations of light oil enter the water through wave action and then are rapidly

diluted by natural sea movements. Also these oils may cause the tainting of edible

fish, shellfish and other marine products, as occurred in the Braer where the main

affected product was of high value farmed fish. This was the major part of the US $

50m compensation claims. All these effects will usually be localized and in the case

of light oils since the toxic components are also the ones that evaporate most rapidly.

Fish and shellfish also lose the oil components that cause taint once clean water

conditions return.

Heavy crude, emulsified crude and heavy fuel oils, have generally lower toxicity, and

can become a threat to seabirds and other wildlife that become physically coated or

smothered. More problems can arise if the overall density of the oil increases further

(e.g. due to mixing with sediment in coastal waters) it gets to the stage that residues

sink. This can lead to a the prolonged contamination of the sea beds. All these

problems can result in extended clean-up costs and large third party damage claims

for economic loss, as illustrated by the spills of heavy fuel oil cargo from the

Nakhodka and Erika.

Amount Spilled

The amount of oil spilled is a very important factor in determining costs. If there is no

variation in other factors, a 100,000 tonne spill will result in far wider contamination,

and will require a far more extensive clean-up response, because greater damage will

result in much higher costs than, say, a 10,000 tonne spill. However, the relationship

is not linear. Etkin (1999), believes that the clean-up costs on a per a tonne basis

decreased significantly with increasing amounts of oil spilled. Which means the cost

of cleaning up small spills is much grater than for large spills.

This trend makes it tempting to conclude that it is alright to calculate average costs of

spills of different sizes. This is a simplistic approach ignores the complexity and inter-

relations between the factors that cause the considerable variation in the cost of

similar sized incidents. This show that simple comparisons between the costs of

individual spills based on the single parameter of the cost per unit of spill can be

highly misleading.

Pattern of Spillage

As well as total spill volume, the pattern of oil loss can be important as well. For

example, the clean-up operation for a single large release of oil may be high but may

be completed in a matter of weeks. The resulting damage to marine resources and

amenities may also be short-term. But the same quantity of oil lost over several

months from a damaged tanker close to the coast may require a major clean-up effort,

resulting in repeated cleaning of amenity areas and long-term effects on fishery

resources and tourism. The best example of this is the Betelgeuse, a tanker that

exploded and sank at a terminal at Whiddy Island in Bantry, County Cork on the 8th

January 1979 with a 42 crew members killed along with 7 oil terminal workers also

killed. Because of the on going release from the various parts of the wreck it was

necessary to maintain the clean-up response consisting of oil collection and chemical

dispersal at sea, defensive booming of sensitive shorelines and regular beach clean-up

for some 21 months. It must be remembered that the total amount of oil spilled during

this period probably amounted to no more than 1,500tonnes, it is clear that the cost

(US $120m) of the response was far high than it would have been had the same

quantity of oil been spilled in a single release.

Location

The location of a spill can have a huge bearing on the costs of an incident as it will

determine the requirements for the clean-up response, as well as the degree of damage

to the environment and economic resources. All oils, if they remain at sea long

enough, will dissipate through natural processes. When a tanker spills oil far from the

coast the response will more often than not often be confined to aerial surveillance of

the slick to monitor its movement. The cost of responding to oil spills can be low.

This is illustrated by the fact that the three largest tanker spills of all time – Atlantic

Empress off Tobago, West Indies in 1979 (287,000 tonnes), Castillo De Bellver off

South Africa in 1983 (252,000 tonnes) and Abt Summer off Angola in 1991 (260,000

tonnes) – these resulted in very low clean-up and damage costs because no major

quantities of oil reached coastlines. Had a similar volume and type of oil been spilled

near a sensitive coastlines (as, for example, occurred in the Amoco Cadiz in France in

1978), the clean-up requirements would have been totally different, as would have

been the impact on fisheries, tourism and other sensitive economic and environment

resources. The costs would have alsoe been much greater.

The physical characteristics of the spill site (e.g. prevailing winds, tidal range,

currents, water depth) as well as its distance from the coast are important as they have

a major bearing on the clean-up response at sea and a successful salvage operation.

Shoreline contamination will also be determined. The high cost of the shoreline clean-

up in both the Erika and Nakhodka incidents was due to the extensive coastal

contamination (some 400 km in the Erika and over 1,000 km in the Nakhodka), which

was a result of the highly persistent nature of the oil and its spread from the incident

location that was some distance offshore.

Other site-specific factors influence the cost of oil spill clean-up

1. the vulnerability of different shoreline types,

2. the extent to which they are self-cleansing,

3. the feasibility of undertaking manual clean-up (e.g. accessibility, likelihood of

clean-up causing more damage than the oil itself),

4. the availability and cost of local labour and many.

Clean-up Response

As a general rule, considerable effort and money is devoted to trying to deal with oil

spills at sea, in an attempt to prevent the damage and public outcry often associated

with extensive pollution of inshore waters and shorelines.

As already discussed, oil spills will on occasions dissipate naturally and not pose a

threat to sensitive coastal resources. On other occasions there may be little that can be

done due to bad weather or other particular circumstances. The decision not to

respond, is a difficult one, because it will be viewed by politicians, public and the

media as unacceptable. An active response is sometimes used even when technical

opinion is agreed that it is unlikely to have a major benefit. This is usually due to the

fact that oil spilled on the surface of the sea spreads, rapidly, and will extend over an

area that is too great to be cleaned-up. Also there could be holding and collection

systems imposed by winds and waves, and there could be reduced effectiveness of

chemical dispersants on high viscosity oils. Response in such circumstances can lead

to high clean-up costs for little or no benefit in stopping the oil’s impact on coastlines.

However, there are exceptions: the spill of 2,450 tonnes of heavy fuel oil cargo from

the Baltic Carrier off Denmark showed that considerable success can be achieved

offshore when conditions are favourable and the recovery operation is well co-

ordinated. In this case, approximately 900 tonnes, around one third of it’s volume,

was collected by a fleet of twelve recovery vessels from three countries. This greatly

reduced the extent of shoreline contamination. Also, in the Sea Empress incident in

Wales, UK in 1996, a combination of natural and chemical dispersion, 450 tonnes in

total of chemical dispersant was applied from a plane, this was claimed to have a

major impact in removing at least 18,000 tonnes of crude oil from the sea surface.

It is often stated that shore clean-up is much more costly than offshore clean-up. But

offshore clean-up is almost always incomplete leaving the bulk of the oil to be dealt

with on the shore. One reason why shore clean-up is often mostly cheaper than an

offshore clean-up is that it usually relies on manual recovery methods and locally-

available equipment. In contrast, offshore clean-up requires considerable amounts of

expensive equipment, vessels, aircraft and trained operators, which might have to

begotten from a distant location. However, a more important factor determining the

cost of shoreline clean-up is the amount of cleaning that is required before the

contaminated area will be considered acceptable. The removal of bulk oil from a

heavily contaminated shoreline is relatively straight forward and can often be done

quickly, depending on the type of shoreline (e.g. rock, sand, mud), ease of access and

other incident and site-specific factors.

As the amount of shoreline contamination is gradually reduces more and more effort

is required to effect an improvement. The operation has little returns with rapidly

escalating costs as the operation moves into the secondary and final clean-up stages

phases.

Termination of Clean-up

All shore clean-up activities should be constantly evaluated to ensure that they remain

appropriate as circumstances change. Any operation should be stopped once if it has

been shown to be ineffective, or likely to cause unacceptable damage to

environmental or economic resources.

The standards set for clean-up vary from country to country and depend on national

attitudes. For example, amenity beaches oiled just before or during the holiday season

will usually need to be cleaned rapidly to a high level to permit their use in order to

minimise lost income by hoteliers and others involved in the tourism industry. This

may require the use of ‘aggressive’ clean-up techniques such as bulldozers on sandy

beaches and high pressure washing of nearby rocks, even at the risk of causing

additional environmental damage. On the other hand, areas like salt marshes and

mangrove swamps that are of great ecological importance may be better left to clean

themselves naturally in view of their sensitivity to physical disturbance. Similarly, it

will usually be appropriate and least damaging to the flora and fauna to leave natural

processes such as wave action and scouring to deal with any residual oil on rocky

shores in remote areas.

Bioremediation Technologies

There are two main approaches to oil spill bioremediation. 1) bioaugmentation

(seeding), in which oil-degrading microbes are added to supplement the existing

microbial population, and 2) biostimulation, in which the growth of indigenous oil

degraders is stimulated by the addition of nutrients or other growth-limiting co-

substrates and/or habitat alteration.

Bioaugmentation

What is a microbe ? A microbe is a microscopic organism or bacteria. The microbes

used in bioaugmentation are found naturally throughout the world within the

environment. The microbes are chosen and custom blended based on their ability to

degrade and remediate various hydrocarbons This option for oil-bioremediation has

been used since the 1970s. The principal for adding oil-degrading microbes is that

indigenous microbial populations may not be able to degrading the wide range of

potential substrates which are present in the complex mixtures such as petroleum

(Leahy and Colwell, 1990). Bioaugmentation may also be used when the indigenous

hydrocarbon-degrading population is low, when speed of decontamination is the

primary factor, and when seeding may reduce the lag period to start the

bioremediation process (Forsyth et al., 1995).

Laboratory studies on bioaugmentation have had mixed results. Aldrett et al. (1997)

tested 12 commercial microbial cultures for bioremediation of Alaska North Slope

crude oil in the lab. After 28 days, four products showed an enhancement of oil

biodegradation with significantly higher degradation rates of alkanes and aromatics

when compared to a nutrient control. In another shaker-flask experiment, Hozumi et

al. (2000) investigated the effectiveness of a microbial product in treating a heavy oil

spilled from Nakhodka using the thin layer chromatography-flame ionization

detection (TLC-FID) analysis. They found that around 35% of the oil was degraded

with addition of the microbial product. A major surprise,was the asphaltene fraction

had the highest loss among the four major oil components, this may show that oil loss

was actually due to biodegradation rather than some quality control problem with the

chemical analysis. Some laboratory studies found that microbial seeding may enhance

oil degradation in seawater but not in freshwater environments (Leahy and Colwell,

1990). Bioaugmentation may be effective in laboratory studies where environmental

conditions are well controlled, but its effectiveness is not guaranteed in the field.

The creation of a “superbug” that combines the genetic information from many

organisms and that has the ability to degrade a variety of different types of

hydrocarbons has also been considered. Friello et al. (1976) successfully produced a

multiplasmid-containing Pseudomonas strain capable of oxidizing aliphatic, aromatic,

terpenic, and polyaromatic hydrocarbons. Thibault and Elliot (1980) also developed a

multiplasmid P. putida strain that can simultaneously degrade some lighter alkanes

and aromatics. However, the survival of this strain in the environment has a question

mark over it. The issues of safety, containment, and the potential for ecological

damage must be fully thought out before field testing of these organisms can be

conducted (Leahy and Colwell, 1990). There is also the problem of public perception

over the release of “foreign” and especially “genetically engineered” microorganisms

into the environment.

Biostimulation

Biostimulation involves the addition of rate-limiting nutrients to accelerate the

biodegradation process. In most shoreline ecosystems that have been heavily

contaminated with hydrocarbons, nutrients are likely the limiting factors in oil

biodegradation.

What are the nutrients? In the bioremediation area nutrients are sometimes called

biocatalysts or biostimulants. These include products or ingredients which support the

stimulation and growth or reproduction of microbes, namely nitrogen and phosphorus.

The nutrient types and concentrations change depending on the oil properties and the

environmental conditions. Wrenn et al. (1994) studied the effects of different forms of

nitrogen on biodegradation of light Arabian crude oil in respirometers. They found

that in poorly buffered seawater, nitrate is a better nitrogen source than ammonia

because acid production associated with ammonia metabolism may inhibit oil

biodegradation. It should also be remembered that ammonia is less likely to be lost

from the system by washout due to its higher adsorptive capacity to organic matter.

But the disadvantage of ammonia is that it is toxic to many marine species. Using

nitrate as a biostimulation agent, Venosa et al. (1994) determined that approximately

1.5 to 2.0 mg N/L supported near maximal biodegradation of heptadecane

immobilized onto sand particles in a microcosm study. Du et al. (1999) investigated

the optimal nitrogen concentration for the biodegradation of Alaska North Slope

crude oil in continuous flow beach microcosms at a loading of 5g-oil/kg sand. The

results showed that nitrate concentrations below approximately 10 mgN/L limited the

rate of oil biodegradation. The higher nutrient requirement was attributed to the more

complex substrate (crude oil).

(Rosenberg and Ron, 1996). Stated that approximately 150 mg of nitrogen and 30 mg

phosphorus are consumed in the conversion of 1 g of hydrocarbon to cell material.

Therefore, a commonly used strategy has been to add nutrients at concentrations that

approaches a stoichiometric ratio of C:N:P of 100:5:1. This theory suggests that

manipulating the N:P ratio may result in the enrichment of different microbial

populations, and the optimal N:P ratio can be different for degradation of different

compounds (such as hydrocarbons mixed in with other biogenic compounds in soil).

However, the practical use of these ratio-based theories remains a challenge.

Particularly, in marine shorelines, maintaining a certain nutrient ratio is impossible

because of the dynamic washout of nutrients resulting from the action of tides and

waves. Oil biodegradation largely takes place at the interface between oil and water.

Therefore, the effectiveness of biostimulation depends on the nutrient concentration in

the interstitial pore water of oily sediments (Atlas and Bartha, 1992; Bragg et al.,

1994). The nutrient concentration should be maintained at a level high enough to

facilitate bacterial growth.

Commonly used nutrients include water-soluble nutrients, solid slow-release

nutrients, and oleophilic fertilizers. Each type of nutrient has its advantages and

disadvantages. General characteristics of these nutrients and important factors

affecting their persistence in the field, such as waves and tides, and physical intrusion

effects, can be seen in table below.

Water-soluble nutrients

Commonly used water-soluble nutrient products include mineral nutrient salts (e.g.

KNO3, NaNO3, NH3NO3, K2HPO4, MgNH4PO4), and many commercial inorganic

fertilizers (e.g. the 23:2 N:P garden fertilizer used in Exxon Valdez case). They are

usually applied in the field through the spraying of nutrient solutions or spreading of

dry granules. This approach has been effective in enhancing oil biodegradation in

many field trials (Swannell et al., 1996; Venosa et al., 1996). Compared to other types

of nutrients, water-soluble nutrients are more readily available and easier to

manipulate to maintain target nutrient concentrations in interstitial pore water.

Another advantage of this type of nutrient over organic fertilizers is that the use of

inorganic nutrients eliminates the possible competition of carbon sources. The field

study by Lee et al. (1995a) indicated that although organic fertilizers had a greater

effect on total heterotrophic microbial growth and activity, the inorganic nutrients

were much more effective in stimulating crude oil degradation. However, water-

soluble nutrients also have several potential disadvantages. First, they are more likely

to be washed away by the actions of tides and waves because of their water-solubility.

The field study in Maine demonstrated that water-soluble nutrients can be washed out

within a single tidal cycle in high-energy beaches (Wrenn, 2000, see section 2.6.2).

Second, inorganic nutrients, ammonia in particular, should be added carefully to avoid

reaching toxic levels. Existing field trials, however, have not observed acute toxicity

to sensitive species resulting from the addition of excess water-soluble nutrients

(Mearns et al., 1997; Prince et al., 1994). Third, water-soluble nutrients may have to

be added more frequently than slow release nutrients or organic nutrients, resulting in

more labor-intensive, costly, and physical intrusive applications.

Granular nutrients (slow-release)

Many attempts have been made to design nutrient delivery systems that overcome the

washout problems characteristic of intertidal environments (Prince, 1993). Use of

slow release fertilizers is one of the approaches used to provide continuous sources of

nutrients to oil contaminated areas. Slow release fertilizers are normally in solid forms

that consist of inorganic nutrients coated with hydrophobic materials like paraffin or

vegetable oils. This approach may also cost less than adding water-soluble nutrients

due to less frequent applications. Slow release fertilizers have shown some promises

from oil bioremediation studies and applications. For example, Olivieri et al.(1976)

found that the biodegradation of a crude oil was considerably enhanced by addition of

a paraffin coated MgNH4PO4. Another slow-release fertilizer, Customblen (vegetable

oil coated calcium phosphate, ammonium phosphate, and ammonium nitrate),

performed well on some of the shorelines of Prince William Sound, particularly in

combination with an oleophilic fertilizer (Atlas, 1995a; Pritchard et al., 1992;

Swannell et al., 1996). Lee et al. (1993) also showed that oil biodegradation rates

increased with the use of a slow release fertilizer (sulfur-coated urea) compared to

water-soluble fertilizers.

However, the major challenge for this technology is control of the release rates so that

optimal nutrient concentrations can be maintained in the pore water over long time

periods. For example, if the nutrients are released too quickly, they will be subject to

rapid washout and will not act as a long-term source. On the other hand, if they are

released too slowly, the concentration will never build up to a level that is sufficient to

support rapid biodegradation rates, and the resulting stimulation will be less effective

than it could be. The field trials on of the shorelines of Prince William Sound showed

that on certain beaches, Customblen granules were apparently washed away before

any significant enhancement of bioremediation was recorded (Swannell et al., 1996).

Several recent studies have shown that a slow release nutrient (Max Bac, a product

similar to Customblen) failed to demonstrate enhancement of oil degradation because

the nutrient release rate was too slow to affect oil biodegradation (Croft et al., 1995;

Sveum and Ramstad, 1995).

Oleophilic nutrients

Another approach to overcome the problem of water-soluble nutrients being rapidly

washed out was to utilize oleophilic organic nutrients (Atlas and Bartha, 1973;

Ladousse and Tramier, 1991). The rationale for this strategy is that oil biodegradation

mainly occurred at the oil-water interface; since oleophilic fertilizers are able to

adhere to oil and provide nutrients at the oil-water interface, enhanced biodegradation

should result without the need to increase nutrient concentrations in the bulk pore

water. A well-known oleophilic fertilizer is Inipol EAP 22, a microemulsion

containing urea as a nitrogen source, lauryl phosphate (the phosphorus source), 2-

butoxy-1-ethanol as a surfactant, and oleic acid to give the material its

hydrophobicity. This fertilizer has been subjected to extensive studies under various

shoreline conditions and was successfully used in oil bioremediation on of the

shorelines of Prince William Sound. Other oleophilic organic fertilizers include

polymerized urea and formaldehyde, and some organic fertilizers derived from natural

products such as fishmeal (Lee et al., 1995a; Rosenberg et al., 1992; Sveum and

Ramstad, 1995). The effectiveness of oleophilic fertilizers also depends on the

characteristics of the contaminated environment such as action of wave and tide, and

sediment types. Based on several earlier studies, Sveum et al. (1994) indicated that

oleophilic fertilizers proved to be more effective than water-soluble fertilizers when

the spilled oil resided in the intertidal zone. But they have no advantages in enhancing

oil biodegradation in the supralittoral zone where water transport is limited. Inipol

EAP 22 was found to be more effective in coarse sediments than in fine sediments

due to the difficulty in penetration for the oleophilic fertilizer in fine sediments

(Sveum and Ladousse,1989). Variable results have also been produced regarding the

persistence of oleophilic fertilizers. Some studies showed that Inipol EAP 22 can

persist in a sandy beach for a long time under simulated tide and wave actions (Santas

and Santas, 2000; Swannell et al. 1995). Others found that Inipol EAP22 was rapidly

washed out before becoming available to hydrocarbon-degrading bacteria (Lee and

Levy, 1987; Safferman, 1991). Another disadvantage with oleophilic fertilizers is that

they contain organic carbon which may be biodegraded by microorganisms in

preference to petroleum hydrocarbons (Lee et al., 1995a; Swannell et al., 1996), and

may also result in undesirable anoxic conditions (Lee et al., 1995b; Sveum and

Ramstad, 1995). In summary, the effectiveness of these various types of nutrients will

depend on the characteristics of the contaminated environment. Slow-release

fertilizers may be ideal nutrient sources if the nutrient release rates can be well

controlled. Water-soluble fertilizers are likely more cost-effective in low-energy and

fine-grained shorelines where water transport is limited. And oleophilic fertilizers

may be more suitable for use in high-energy and coarse-grained beaches. However,

successful application of bioremediation products will always require appropriate

testing and evaluation based on the specific conditions of each contaminated site.

Type of nutrients Advantages Disadvantages

Water soluble Readily available. Easy to manipulate for target nutrient concentrations. No complicated effect of organic matter

Rapidly washed out by wave and tide. Labor-intensive, and physical intrusive applications. Potential toxic effect

Slow release Provide continuous sources of nutrients and may be more cost effective than other types of nutrients

Maintaining optimal nutrient release rates could be a challenge

Oleophilic Able to adhere to oil and provide nutrients at the oil-water interface

Expensive. Effectiveness isVariable.Containing organiccarbon, which maycompete with oildegradation and resultin undesirable anoxicconditions

Bioaugmentation appears less effective than biostimulation because:

1) hydrocarbon degraders are ubiquitous in nature and, when an oil spill occurs, the

influx of oil will cause an immediate increased response in the hydrocarbon-

degrading populations; but,

2) if nutrients are in limited supply, the rate of oil biodegradation will be less than

optimal; thus,

3) supplying nutrients will enhance the process initiated by the

spill, but adding microorganisms will not, because they still lack the

necessary nitrogen and phosphorus to support growth.