Part 3: Oil Field Microbiology

Question 1

When were microorganisms in oil wells first detected?

Answer 1

Approximately 100 years ago (e.g. H2S identified in the 1920s in wells)

Question 2

Which group of organisms are particularly problematic for oil production and why?

Answer 2

Sulphate reducing bacteria, because:

• oil souring
• production of toxic H2S
• microbially induced corrosion

Question 3

What are the two groups of hydrocarbons?

Answer 3

1) Aliphatic compounds (alkanes, cycloalkanes)

2) Aromatic compounds (6C with alternating single and double bonds)

Question 4

How does boiling point affect the applications of crude oil components?

Answer 4

Boiling point directly correlates to molecule size. This allows oil to be separated by fractional distillation.

e. g. methane (natural gas) =-161 C
e. g. 3 propane (LPG) = -42C

Question 5

What is an isomer?

Answer 5

A molecule with the same molecular formula but a different spatial arrangement of atoms (exluding twisting or rotating around bonds).

Question 6

Give examples of the number of isomers for hydrocarbons.

Answer 6

C3 = 1
C4 = 2
C5 = 3
C6 = 5
C7 = 9
C25 = 36,797,588

Question 7

Why is crude oil so difficult to degrade?

Answer 7

There is a huge variation in the molecules of hydrocarbons. Enzymes are rarely capable of degrading more than one type of compound (including isomers), and so oil is difficult to degrade.

Question 8

What is meant by the “fingerprint” of crude oil?

Answer 8

Each crude oil reservoir has a unique composition of alkanes, cycloalkanes and aromatics (and their isomers).

Question 9

What is gas chromatography and what is it used for?

Answer 9

The identification of the relevant contributions of compounds within an oil sample through volatilising each compound and quantifying.

Used for: 1) identifying the potential uses of crude oil. 2) Identifying source of illegal spillages from tankers.

Question 10

Describe the gas chromatography process,

Answer 10

1) Molecules are volatalised and transported by a carrier gas into a column.
2) Each molecule is retained according to its size and shape to separate them.
3) The samples are passed into a detector
4) The signal produced is dependant on the amount of each compounds present.

Question 11

How much crude oil was spilled during the Amoco Cadiz (Normandy 1978) spill?

Answer 11

223,000 m3 or 233,000,000 litres (Gundlach et al 1983).

Question 12

What proportion of crude oil was degraded by bacteria in the Amoco Cadiz oil spill (Normandy 1978)?

Answer 12

4.5 % - this was a surprise and prompted research into possible applications. (Gundlack et al 1983).

Question 13

Why is microbial degradation of oil ineffective?

Answer 13

It is thought that microbes can only degrade one compound at a time. Therefore a community of specialist bacteria are required.

Question 14

What enzyme is used for aerobic degradation of aliphatic compounds?

Answer 14

Monooxygenase

Question 15

Explain the process of aerobic degradation of aliphatic compounds.

Answer 15

Monooxygenase (enzyme) is used with an oxygen free radical to convert the alkane to an alcohol. The OH bond can then be easily degraded.

Question 16

What enzyme is used for aerobic degradation of aromatic compounds?

Answer 16

Dioxygenase

Question 17

Explain the process of aerobic degradation of aromatic compounds.

Answer 17

Aromatic rings are very stable and hard to degrade. Dioxygenase (enzyme) produces two oxygen radicals which bond to the ring to create an easily degradable di-alcohol.

Question 18

Which degrades faster - aliphatic of aromatic compounds?

Answer 18

Aliphatic (less stable).

Question 19

To what does BTEX refer?

Answer 19

Another name for aromatics - Benzene, Toluene, Ethylbenzene, Xylenes.

Question 20

How do anaerobic microorganisms degrade aromatic rings?

Answer 20

Anaerobes lack oxygen and so do not have a compound reactive enough to break down the aromatic ring. Instead a variety of methods must be used to attack the side chains.

Question 21

Give an example of a method of degrading aromatic rings.

Answer 21

Reduction of the aromatic ring: 2 electrons and 2 ATP are used to remove one of the three double C=C bonds. Therefore the ring is less stable and can be more easily attacked.

Question 22

Has an anaerobic benzene-degrading organism been cultured?

Answer 22

No, although it is known to exist since benzene can be degraded in nature.

Question 23

How does formation of Benzoyl-CoA highlight the need for a number of different organisms to degrade oil?

Answer 23

Benzyl-CoA formation makes degradation easier. However, this can be formed by a number of pathways, meaning that a range of organisms are required. (Fuchs 2008).

Question 24

What are the two methods of increasing the efficiency of microbial degradation of oil?

Answer 24

1) Biosurfectants - emulsifiers which increase the number of oil droplets, thereby increasing surface area and allowing bacteria to degrade more effectively.
2) Biostimulation - adding nutrients to stimulate growth of more bacteria.

Question 25

Which is the smallest biodegradable alkane? (EXAM)

Answer 25

Methane.

Question 26

What did results from the International Ocean Drilling Programme show for the Peru Margin?

Answer 26

The flow of sulphate-rich waters from the ocean crust causes formation of a methane-sulphate transition zone at 90 m depth. This zone has incredibly high cell numbers and must therefore be a zone of synergistic use of sulphate and methane.

Question 27

What is the syntrophic consortium of methane oxidising archaea and sulphate-reducing bacteria?

Answer 27

A methanotrphic archaea and sulphate reducing bacteria work cooperatively to utilise crustal seep at depth. This facilitates anaerobic oxidation of methane (AOM).

Question 28

What is AOM?

Answer 28

Anaerobic oxidation of methane.

Question 29

What sea features does AOM form?

Answer 29

Carbonate sea mounts - examples in the Black Sea have some of the highest biomass concentration in the world (Michaelis et al 2002).

Question 30

What are the findings of Zengler et al (1999)? Hint: hexadecane.

Answer 30

A stable culture was established that could convert hexadecane into methane. Hexadecane was initially converted into acetate and hydrogen and then into methane. Means that 5 or 6 different organisms were working cooperatively to degrade hexadecane without any oxygen or external electron acceptor.

Question 31

Which group of bacteria are especially problematic in oil reservoir contamination?

Answer 31

Sulphate reducing bacteria.

Question 32

What proportion of oil reservoirs experience some level of bacterial contamination?

Answer 32

70 - 80%

Question 33

Which oil reservoirs do not experience bacterial contamination?

Answer 33

Those which are deep enough to effectively be autoclaved due to high pressure and temperature. (Wilhelms et al 2001).

Question 34

What are the ranks of oil bio-degradation?

Answer 34

``` Light - 1 - Short-chain alkanes depleted Moderate - 4 - No n-alkanes remaining Heavy - 6 - Sterones partly degraded Very heavy - 8 - Hopanes partly degraded Severe - 10 - Long-chain aromatics degraded ```

Question 35

How do oil wells become contaminated?

Answer 35

Water injection is necessary to maintain pressure needed for oil recovery. This could be waste water from production or seawater if offshore. Water injection leads to inoculation with microorganisms,

Question 36

Why is seawater injection into oil reservoirs more problematic than freshwater?

Answer 36

Seawater has a higher sulphate concentration (around 30 mM), meaning that sulphur-reducing bacteria are more likely to be stimulated.

Question 37

What type of oil degradation is used in the absence of sulphate? How is this economically useful?

Answer 37

Methanogenic degradation, converting long chain aromatics to methane (and some CO2). Offers a method to economically utilise long-chain hydrocarbons.

Question 38

How does the synthrophic microbial community in oilfields facilitate sulphate-reducing bacteria?

Answer 38

The synthrophic microbial community produces waste hydrogen that can be utilised by SRB.

Question 39

What is "souring" of oil?

Answer 39

The enrichment of oil with H2S.

Question 40

Why is souring of oil a problem?

Answer 40

When burning the oil, any H2S present will become sulphur dioxide which is responsible for acid rain. SO2 may also react further with water to produce sulphurous acid or with oxygen to form SO3 (and then sulphuric acid).

Question 41

Which bacteria are responsible for souring?

Answer 41

Sulphate reducing bacteria.

Question 42

Why is souring particularly problematic in gas reserves?

Answer 42

In summer, excess gas is pumped below ground for storage to be used in winter for heating. H2S forms in these deposits.

Question 43

What chemicals has the gas industry attempted to use to reduce souring?

Answer 43

Methanol Triethylene glycol Formaldehyde

Question 44

Why have methanol, triethylene glycol and formaldehyde been unsuccessful in preventing souring?

Answer 44

Sass and Cypionka (2004) found that SRB could utilise methanol and triethylene glycol as growth substrates e.g. Desulfotomaculem spp. Formaldehyde is very reactive and forms alcohols and other compounds which can then be used by SRB.

Question 45

Why are nitrate reducing bacteria the most effective choice for preventing SRB in oil?

Answer 45

Nitrate has a higher redox potential than sulphate, and so SRB cannot compete against nitrate-reducers. Benefits: 1) Nitrate reducers outcompete SRB for shared electron donors 2) Nitrate reducers oxidise H2S since they are chemolithoautotrophs.

Question 46

What is the name for the commercial product containing nitrate reducing bacteria?

Answer 46

Petrocare

Question 47

What are the problems with nitrate reducing bacteria in oil?

Answer 47

1) Do not exclusively oxidise H2S - also damage oil | - however still better than SRB!

Question 48

Why did microbial communities degrade the Exxon Valdez spill so effectively?

Answer 48

In the Gulf of Mexico, more than 600 natural seeps are known to exist, seeping more than 100 million litres of oil per year. This has meant that bacteria have evolved to effectively degrade oil.

Question 49

What are the two special cases of hydrocarbon seeps?

Answer 49

1) Methane seeps | 2) Asphalt volcanoes.