Bacteria thrive in petroleum jelly

08/28/2010 Degradation of petroleum by bacteria - basic aspects from a microbiological point of view

On the occasion of the oil spill in the Gulf of Mexico on April 20, 2010

An oil disaster poses major technical and organizational challenges for those involved and for environmental protection: leakage and spread must be curbed immediately. The oil that has leaked should be collected as much as possible - a laborious undertaking, especially if the coasts are already affected. But what happens to the oil that has already been distributed, which covers the water and the coastal floor in shimmering dark layers and cannot be collected?

Bacteria break down petroleum
Fundamental aspects from a microbiological point of view

An oil disaster poses major technical and organizational challenges for those responsible, those involved and environmental protection: leakage and spread must be curbed immediately. The oil that has leaked should be collected as much as possible - a laborious undertaking, especially if the coasts are already affected. But what happens to the already widely distributed oil, which covers the water and the coastal floor as a dark, often shimmering layer and can no longer be collected? The fact that oil layers gradually disappear, at least for the most part, that the environment gradually "cleans itself", is thanks to bacteria that break down the oil with oxygen. In the case of sudden, very massive soiling, however, there are problems with degradation. That is why there is a lot of discussion about petroleum-degrading bacteria in an oil disaster: What kind of bacteria are they? Where and how do they live Could it be used specifically to combat oil pollution? Can you breed them? How can oxygen consumption affect the ecosystem? As diverse as the topic may appear, natural oil extraction is based on manageable microbiological and chemical principles.
When we talk about oil fields, oil production and oil spills, it goes without saying that we mean crude oil and not cooking oil. The distinction between edible oil and petroleum is obvious: edible oil serves as food and is accordingly easy to digest. Crude oil is used to produce fuel. In the body it would be indigestible, highly indigestible and harmful to health. The two also differ in color and smell: crude oil is deep brown and smells like petrol, while cooking oils are usually yellowish or slightly greenish and have no or only a slight nutty odor. In view of the origin, the differences are not surprising: While cooking oil is freshly formed by living plants, crude oil was created over millions of years from old biomass through chemical transformations deep underground. Edible oil and crude oil also have something in common: Vegetable oil can in principle also be used to produce fuel. The most noticeable commonality is that both petroleum and cooking oil cannot be mixed with water, swim “on top” and, moreover, do not flow as easily as water, but rather thick, “oily”. All liquids with these properties are colloquially called "oils". The similarities and differences are best explained by the chemical composition.
Illustration 1. Not all oil is created equal. Edible oils have a different chemical composition than petroleum (crude oil) and also behave differently biologically. However, there are also some similarities.
In edible oils, various fatty acid molecules are linked with glycerine as a carrier molecule. If such compounds are solid at room temperature, one does not speak of oils, but of fats. Only when heated do fats become liquid and show their great similarity to oils. Glycerin in free form is a viscous, sweet liquid that is easily miscible with water. In connection with fatty acids, the water solubility of glycerine has disappeared and the compound is extremely water-repellent. The nutritional and health value is determined by the fatty acids. The body uses them primarily as an energy store and source of energy. Fatty acids (Figure 2a) consist to a very high proportion of the elements carbon (symbol: C) and hydrogen (symbol: H). In addition, they contain - and this is crucial for their digestibility - the element oxygen (symbol: O) in bound form. This also enables the chemical connection to the glycerine.
Crude oil and the products obtained from it (diesel oil, heating oil, lubricating oil, paraffin oil) are very complex mixtures of hundreds of substances. The predominant ones in terms of quantity, mostly over 85% by weight, all belong to one chemical class, the hydrocarbons. In other words, petroleum consists largely of hydrocarbons. Chemically, they are made up of the elements carbon and hydrogen, which is why they are so called. Hydrocarbons are indigestible for humans and animals, often incompatible and sometimes also poisonous. Indigestible - albeit non-toxic - are, for example, pure (pharmaceutical) paraffin oil, petroleum jelly and candle paraffin. Benzene and toluene, for example, are indigestible and at the same time toxic.
In hydrocarbons, the atoms of the elements carbon and hydrogen are linked to one another in a variety of ways. Each linkage or combination option represents a very specific hydrocarbon with a chemical name (e.g. propane, hexane, octane, benzene or toluene). A common combination principle is shown in Figure 2b, c. Here the carbon atoms are linked in chains of different lengths. The carbon atoms at the end of the chain are surrounded by three, all others by two hydrogen atoms. The similarity to fatty acids is striking, only the oxygen atoms are missing. These structures are called open-chain saturated hydrocarbons or, in technical terminology, n alkanes for short. n Alkanes with chains of 4 to 17 carbon atoms are liquid at room temperature. With shorter carbon chains, n alkanes are gaseous and with longer carbon chains are solid like wax.
Figure 2:
a. Palmitic acid, a saturated fatty acid that is chemically bound in edible oils and dietary fat.
b. The hydrocarbon hexane evaporates very easily and is accordingly flammable. Light petrol contains a lot of hexane. It smells like petrol from the pharmacy.
c. The hydrocarbon hexadecane evaporates very slowly and is more difficult to ignite. Once ignited, however, it also burns violently. It's almost odorless. Diesel oil and lamp oil contain quite a lot of hexadecane.
If ignited in air, all hydrocarbons burn with intense heat. That is why hydrocarbons are energy carriers. In practical use, hydrocarbons are and remain unsurpassed as energy carriers. With a low specific weight (they are specifically lighter than water) they provide a lot of energy. The combustion is caused by the oxygen (O2) in the air. This combines with the carbon to form carbon dioxide (CO2) and with the hydrogen to form water (H2O). This means that - assuming clean combustion - gaseous carbon dioxide and water vapor are produced, which precipitate as condensation. The high energy content together with oxygen and the combustion to volatile, non-toxic products led to the "triumphant advance" of petroleum hydrocarbons as energy sources in our engineered world. Cars, planes, motor ships as well as gas and oil heating only need hydrocarbons and air to operate.
Petroleum was once formed from small marine organisms, probably mainly algae and cyanobacteria (Figure 3). There must have been epochs in the history of the earth in which marine organisms grew particularly lush in coastal or delimited marine areas. Like their current relatives, they converted CO2 into living biomass and released oxygen, driven by the energy of sunlight. The process is known as photosynthesis. After dying and sinking, some of the dead biomass was broken down again into CO2 by bacteria. However, some of the biomass was buried in the sediment on the sea floor. Part of it, including natural fatty acids, was gradually transformed into petroleum through complex chemical transformations at the elevated temperatures at depth. The oil then slowly migrated from the deep rock layers where it was formed into younger, overlying layers. If it was not let through by a younger layer of rock, it would pool underneath. These are then the typical reservoirs that are opened up by “drilling”. If there was no oil-tight cover, the oil forced its way through channels and crevices to the surface of the sea floor. Sometimes it thickened to an asphalt-like mass, sometimes it floated up in the water. Long before the human era, the environment was constantly confronted with oil, albeit in a very slow process. Even today, such natural oil spills can be found on the sea floor (Figure 4).
The history of its origins makes it clear that crude oil is converted biomass and thus contains the solar energy stored in ancient times. If we use crude oil as an energy source, it may therefore appear "close to nature" and therefore unproblematic, but only at first glance. Because mankind can consume in a period of perhaps not even two centuries what has accumulated in the history of the earth over many millions of years. It's like spending generations' savings in an hour. In addition, the buried biomass was "alienated" during the chemical conversion. Crude oil hardly bears any resemblance to the original biomass and harms most living things, so it is not a purely biological product.
Figure 3. Simplified scheme of petroleum formation.
a. In earlier epochs, photosynthesis, i.e. the use of sunlight, converted carbon dioxide (CO2) and water (H2O) into algae biomass and oxygen (O2).
b. Anything that was not used by other microorganisms after it had died was buried in the sediment.
c. Over the course of millions of years, slow chemical transformations at elevated temperatures in the deep layers resulted in crude oil (shown in red), which either accumulated under dense rock 1 or walked slowly up to the water 2

Where and how do petroleum-degrading bacteria live?

Despite the constant release from natural sources over millions of years, crude oil has not accumulated on the world's oceans. It was and is continuously consumed by bacteria and thus eliminated. Without these, vast coastal landscapes would be covered with crusts of weathered petroleum, and higher life would probably hardly have been able to develop there. Crude oil-degrading bacteria are therefore widespread almost everywhere in the world, in seawater, on beaches, in soils and in inland waters. However, they are usually found in much lower numbers than other bacteria in clean water and soil. They then feed on the small amounts of hydrocarbons that are always present in the environment and on other nutrients. Only when petroleum or petroleum products get into the environment do these special bacteria multiply on a mass scale. They also play a major role at natural escape points such as the “oil wells” and “asphalt volcanoes” in the sea. Here even a very special fauna with special animal forms (Figure 4) is maintained by petroleum-degrading bacteria. Various bacteria feed on petroleum and degradation products, and the animals in turn feed on the bacteria.
Figure 4: Surface of a so-called asphalt volcano, a natural oil source, on the sea floor. Dark asphalt-like lumps can be seen that protrude from the oily sediment and are often colonized by cold-water corals. Other colonists such as the mussel at the bottom left or the crust sponges above can also be seen. The animals feed on bacteria. The bacteria in turn live on petroleum and its breakdown products. Because the crude oil can only be used from the surface by means of oxygen, the lumps and sediment deeper inside are free of oxygen and tarry-oily. Study: Antje Boetius.

From water, sediment and soil samples, especially if these have been contaminated with petroleum or fuel for a long time, petroleum-degrading bacteria can be cultivated using microbiological methods and examined in the laboratory. Petroleum-degrading bacteria were discovered as early as 1900. In the second half of the 20th century, more and more species were found and their growth and metabolism investigated. There are certainly several hundred types of petroleum-degrading bacteria. But even if not all types of petroleum-degrading bacteria have been discovered, it is only a small group of "specialists" compared to the hundreds of thousands of valued bacterial species in nature.
More precisely, one would have to say that the oil-degrading "specialists" mentioned here in our immediate environment are oxygen-breathing bacteria. More recently, petroleum-degrading bacteria that live without oxygen have also been known. However, these grow extremely slowly and hardly play a role in the disposal of oil after accidents. They live on constituents of oil deeper in the sea floor, in coastal sands or in swamps. The remediation of oil pollution in our familiar environment is therefore primarily about oil extraction with oxygen.
Food is basically digested with enzymes. Petroleum-degrading bacteria have special enzymes with which they attack the hydrocarbons and convert them into fatty acids. For organisms that do not have these special enzymes, and this applies to humans, animals and most bacteria, hydrocarbons are indigestible. Figure 2 shows that actually “only” two oxygen atoms have to be built into the hydrocarbon in order to turn it into a completely normal digestible fatty acid. That sounds simple, but it is biochemically difficult and therefore requires special enzymes. The bacteria then breathe in ("burn") the fatty acids formed into carbon dioxide and water and thus receive their energy for life, just as humans and animals breathe in the usual fatty acids from their food. As specialists, petroleum-degrading bacteria can do what otherwise only an internal combustion engine can: use hydrocarbons as energy sources.
When these bacteria are grown in mini-ecosystems in the laboratory, their thriving with hydrocarbons as food is always impressive (Figure 5), provided that the growth conditions such as pH and mineral salt mixture have been set correctly. On the culture medium, a solution of mineral salts including nitrogen, phosphate and iron sources, the petroleum initially forms a cohesive brownish film after the addition. Within a few days, the film (provided it is not too thick) tears into smaller islands and, when shaken, forms small dark droplets that do not reunite to form a film (Figure 6). In the course of further days these droplets become smaller and the water surface becomes almost oil-free. But black small flakes of petroleum components also accumulate, which are probably only broken down very slowly. Under the microscope you can see a lush growth of bacterial cells. Various types of bacteria can be isolated from such cultures (Figure 7).
Figure 5: Degradation attempt in the laboratory aquarium with petroleum with the addition of nitrogen and phosphate fertilizers. A piece of the Wadden Sea surface was covered with a thin (1 millimeter) layer of oil that combined with the mud and flooded. The photos on day 1 (left), after 6 days (middle) and 26 days (right) show the natural "Restoration" of the surface. But underneath there is still a viscous layer of oil that is only very slowly degraded. Photo: Florin Musat.
Figure 6: Scheme of the natural dispersion of petroleum by bacteria. The bacteria live in the water on the surface of the petroleum, but not in the petroleum. The movement of water and its breakdown create smaller and smaller droplets. As a result of the colonization by bacteria, the droplets cannot reunite to form a coherent mass: An emulsion is created.

Figure 7: A strain of bacteria that degrades petroleum under the microscope at 1500x magnification. The rods that appear dark are bacterial cells. The round shape on the right is a droplet of oil - here it appears huge. Photo: Johannes Zedelius.

Why is oil pollution a problem despite biodegradation?

In an oil spill, there are several factors that make the oil a problem, although it can generally be broken down by bacteria.
1) An oil spill in a marine area releases very large quantities into the environment within a short period of time, be it through the leakage of a tanker or through a borehole from the pressurized reservoir.Such a sudden and massive build-up far exceeds natural oil spills, in which the oil gradually forces its way up through channels and cracks in the rock and mud cover. The numbers of bacteria present in the previously clean environment are far too low to break down the oil masses. Measured by the speed at which the oil exits, the reproduction and breakdown of bacteria are too slow, as impressive as they may otherwise be. The oil masses spread out as brown floating carpets, killing animals and polluting the beaches.
2) Crude oil provides plenty of energy for bacterial metabolism, but hardly any other factors that living things also need. The petroleum-degrading bacteria lack vital minerals, especially bound nitrogen (N), phosphorus (P) and iron (Fe). These are in short supply in seawater. In the event of an oil spill, the ratio of energy source to vital minerals is extremely unbalanced. Also, no one could live solely on fats and carbohydrates, that is, "solely on energy".
3) Bacteria need water as a living environment. They cannot grow in petroleum. So they grow where they have both water and oil, and that's on the surface of the oil in the water. However, the greater the amount of oil, the less favorable the ratio of surface area to volume. Therefore, a fine distribution and thus an enlargement of the surface of the petroleum would be very beneficial for bacterial growth and degradation. So decades ago the idea came up to help with chemical dispersants. The operating principle of dispersants is very similar to that of dishwashing detergents and detergents: the water-repellent character of the oil, i.e. its ability to separate itself from water, is reduced.
4) Once the oil on the beaches has thickened through evaporation of the light components and weather conditions, it is no longer broken up by the movement of water, but on the contrary forms tar-like balls and layers connected with sand and other particles. These are far too compact to be broken down by bacteria and will likely remain on and under the surface of the sand for decades or even centuries.
5) The breakdown of petroleum consumes oxygen. In the "open air" on beaches this is the least problem. In water, however, only a small amount of oxygen dissolves from the air, namely about 7 milliliters (ml) in one liter, calculated as a gas. This is quickly consumed if it is not replenished by the impact of waves, currents and mixing. Breaking down just one drop of petroleum (0.2 ml) would use up the oxygen from 80 liters of seawater (Figure 8)! It is true that crude oil is not broken down in an instant, so there is time to supply oxygen. But there is more than one drop per 80 liters of water when there is oil pollution. Aquatic animals can suffocate as a result of a lack of oxygen. Furthermore, completely different bacteria suddenly become active, which live without oxygen and form toxic hydrogen sulphide from sulphate, a seawater mineral. This can be recognized by the black coloration of the marine sediment. The ecosystem is "tipping over".




Figure 8: Little oil - high consumption of oxygen
for dismantling. One drop (0.2 milliliter) is required for the
Bacteria break down oxygen from 80 liters
Sea water.

Can bacteria be grown and used that break down petroleum particularly efficiently?

The wish to make oil layers and oil slicks disappear by spraying them with particularly efficient bacterial breeding strains is understandable. Thoughts of a genetic combination of several useful properties in a "super bacterial strain" may also arise. But it will very likely remain a dream. From an overarching biological point of view, such a concern does not even seem sensible:
1) There is absolutely no shortage of types of bacteria that efficiently break down petroleum. The evolution of life over many hundreds of millions of years has given us a rich range of types of bacteria with the ability to break down various petroleum constituents. Depending on the type of oil and temperature at the affected location, one or the other naturally occurring species multiply. Only the growth conditions have to be favorable, that is, that nutrient salts must be present and that the petroleum must not be present in compact masses.
2) Crude oil-degrading bacteria work in a team. Many species are always active at the same time. Some use, for example, alkanes with short carbon chains, others those with long carbon chains, and in turn other benzene-like hydrocarbons. The interaction of bacteria in a team is also a “tried and tested” principle in the natural recycling of dead biomass. The do-it-all super bacterial species was not naturally created, and there is probably a very fundamental reason for it. In our society, too, we cannot find a person who is equally competent in producing baked goods, cutting hair and processing tax returns.
3) The survivability of special breeding strains outside of the laboratory in nature is very questionable. If, however, one were to "produce" a bacterium through genetic engineering measures that, for example, finely disperses petroleum in the laboratory, which would actually be an advantage, it would most likely not prevail in the harsh reality.
4) Above all, the problem of mineral deficiency would not be solved. Even a laboratory breeding line could not grow without bound nitrogen, phosphate and iron.
Applying petroleum-degrading bacterial mixtures to contaminated areas could, at best, bring about a small time advance in the reproduction at the affected location if the natural numbers of these bacterial cells are initially low. In terms of the duration of the renovation, this lead is very probably insignificant.

Which measures are suitable?

In the case of oil spills, a distinction must be made between two types of measures, acute and long-term.
1) Acute measures are always of a technical (physical) nature. A further leak and the spread of the leaked oil must be prevented. Oil that has reached the coast must be collected as far as possible. Bacteria play no role in these measures. In other words: as little oil as possible into the environment!
However, one tries to prepare the later work of the bacteria here. Dispersants are intended to ensure that the oil is distributed in very fine droplets as a result of the impact of the waves and does not reunite. This way it could be better colonized and broken down by bacteria. The ideal of a dispersant would be that it is non-toxic, inexpensive and also biodegradable itself. However, it should not be broken down too quickly, because it should work as long as the bacteria have not yet taken over the dispersion. There is no such ideal dispersant, which is why the measure is not undisputed. Rather, it is a matter of weighing up between a lesser and a greater evil. Depending on the type and location of the oil accident, it must be clarified whether the oil, which has been finely distributed by means of a chemical, is actually the lesser evil for the living world in the water. There are also biological dispersants that can be obtained from breeding bacteria. However, the available quantities are nowhere near enough and the costs would be immense.
2) Long-term measures are of a biological nature. They follow the technical measures and consist primarily of natural degradation through fertilization with deficient mineral salts (nitrate, ammonium, phosphate and iron salts) and physical processing such as loosening the subsoil support. Treatment methods for optimizing biodegradation can be tried out on small experimental areas and then applied to larger areas. In this way, the oil degradation could sometimes be accelerated up to five times compared to that on untreated surfaces. Still, it's a slow process that can take months or even years. In open water, it is difficult to fertilize oil-polluted areas because the mineral salts are diluted there. There are attempts to bind the mineral salts for bacterial growth to chemical carriers that have an affinity for the oil and thus fertilize the oil slick in a targeted manner; However, there are still no real successes.
Conclusion: There is no magic bullet. Like the technical measures, the biological measures must also be adapted to the respective situations. Finding the appropriate remedy is always experimental.
3) The best measure remains prevention. What may sound trivial at first is urgent. It is also about the protection of remote areas of our planet, about which we only know little, but where oil could still be extracted in the future. The deep sea and polar region are technically and logistically difficult to develop. In the deep sea there is high pressure and low temperature, and in polar regions icy cold and lack of liquid water. How far and how quickly oil can be broken down by microorganisms under these conditions is still largely unknown, but in any case less effective than in our immediate environment. The physical behavior of oil and gas from deep warm reservoirs at depths when mixed with cold water under high pressure is also not well understood. Unknown factors have also created major problems in controlling the oil spill in the Gulf of Mexico. In polar regions such as the Arctic, seasonal darkness and the distance to port logistics would severely limit the ability to react promptly to an oil accident.
Even more security makes further demands on the already technically very complex and impressive oil production. In this way, crude oil is becoming even more of a valuable material, which urges us as daily users to be more conscious about what is currently still the most important of all energy sources.


© Friedrich Widdel, June 25, 2010
Slightly changed on August 1st, 2010

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More information and videos

Oil in the sea - how vulnerable is nature, Deutsche Welle
http://www.dw-world.de/dw/episode/0,,5620167,00.html
Bacteria destroy crude oil, Radio Bremen, buten and inside
http://www.radiobremen.de/mediathek/index.html?id=030779
Oil threatens life in the deep sea, ZDF Mediathek
http://www.zdf.de/ZDFmediathek/beitrag/video/1071056/Oel-bedroht-das-Leben-in-der-Tiefsee
Life in asphalt, DFG Science TV, Marum
http://www.marum.de/DFG_Science_TV_5_Leben_im_Asphalt.html
Expedition to the bottom of the sea, Deutsche Welle TV
http://www.marum.de/Page9105.html
ECHO interview with microbiologist Antje Boetius: Disaster without an emergency plan, echo online
http://www.echo-online.de/wissen/wissenschaftundtechnik/ECHO-Interview-mit-der-Mikrobiologin-Antje-Bo%EBtius-Katastrophe-ohne-Notfallplan;art478,933860
Oil soon in the Gulf Stream ?, IFM-GEOMAR
http://www.ifm-geomar.de/index.php?id=537&tx_ttnews[pointer]=1&tx_ttnews[tt_news]=582&tx_ttnews[backPid Autovermietung=8&cHash=22faf0fe3a
Does the Gulf Stream bring the oil to Europe ?, n-tv.de.
http://www.n-tv.de/panorama/dossier/Bringt-der-Golfstrom-das-Oel-nach-Europa-article933503.html
The deep sea must be protected, sustainability.org
http://www.nachhaltigkeit.org/201005254823/natur-landwirtschaft/interviews/die-tiefsee-muss-geschuetzt- wird
Oil disaster in the Gulf of Mexico, ZMT Bremen
http://www.zmt-bremen.de/Page1424.html
Gulf Oil Blog
http://gulfblog.uga.edu/
Quarks and Co "Oil is your favorite dish" September 14, 2010
http://www.wdr.de/tv/quarks/sendungsbeitraege/2010/0914/004_oel.jsp