GB2373512A

GB2373512A - A method of killing bacteria

Info

Publication number: GB2373512A
Application number: GB0127146A
Authority: GB
Inventors: Timothy Frank Illson
Original assignee: Lattice Intellectual Property Ltd
Current assignee: Lattice Intellectual Property Ltd
Priority date: 2000-11-15
Filing date: 2001-11-13
Publication date: 2002-09-25
Also published as: GB0127146D0; WO2002040642A1; AU2002214155A1; GB0027824D0

Abstract

A method for controlling bacteria comprising stressing a bacterium so that it lyses and produces bacteriophages which then kill other bacteria. The method may be applied to kill sulphate reducing bacteria in oil and gas processing and production facilities. The phages may be naturally present in the bacteria or they may be added to and infect the bacteria, producing lysogens.. The bacteria <I>e.g Desulfovibrio</I> or <I>Desulfotomaculum</I> species, may be stressed by applying ultra violet, (u.v), light, heat, stress inducing toxic chemicals or antibiotics to them. The prophages are induced to enter the lytic life cycle and the cell is ruptured releasing new virus particles which infect other bacteria. Under harsh environmental conditions the bacteriophage continue to follow the lytic life cycle so lysing more microbes. The method is intended to kill corrosion causing microorganisms in petroleum production plants.

Description

23735 1 2

BACTERIAL CONTROL

The present invention relates to the control of bacteria, particularly bacteria which can cause corrosion or other undesired effects.

Bacteria present in process plant and gas production facilities such as oil and gas wells, pipelines and sewage plants can cause microbiologically influenced corrosion which can lead to severe problems. The bacteria can convert sulphate generally found in such facilities into hydrogen sulphide (HIS) which causes localised acidification and thus localized corrosion of carbon and stainless steels when in the presence of water. Furthermore, hydrogen sulphide is toxic and when generated in oil and gas wells can poison operators at the top of the well.

In order to avoid rnicrobiologically influenced corrosion, water can be treated with biocidal chemicals such as aldehydes or quaternary amines. These chemicals can be effective against free floating bacteria but are considerably less effective against sessile bacteria covered by a biofilm. Furthermore, the chemicals are toxic to both humans and marine life and regulatory authorities are reluctant to allow their use.

An object of the present invention is to control the spread of undesired bacteria whilst reducing one or more of the undesired effects described above.

According to the present invention there is provided a method for controlling bacteria, the method comprising stressing a bacterium so that it lyses and produces bacteriophages that are then used to kill other bacteria.

Stressing bacteria so that they Iyse or rupture and die producing viruses called bacteriophages which then kill other bacteria is a very efficient way of killing bacteria without the large scale use of biocidal chemicals which can be damaging to the environment. The bacteriophages produced by the lysing bacteria are specific to other bacteria of the same type.

The bacteria can be stressed by any suitable method such as the application of an appropriate amount of ultra-violet light, heat, antibiotics or chemicals that are toxic to the bacteria. The amount of ultra-violet light, heat, toxic or stress inducing chemicals or antibiotics to which the bacteria are exposed is predetermined in experiments to ensure that it stresses a bacterium but does not irrunediately kill it before it produces bacteriophages which can then be used to kill other bacteria.

The invention is particularly suitable for use with low density concentrations of bacteria which are particularly susceptible to being stressed. Such relatively low density concentrations of bacteria generally exist in process plant and gas wells due to the relatively harsh conditions.

The bacteriophages may already exist latent within the host bacterial cell and Iysing of the bacteria is induced by stressing the bacteria. Alternatively or additionally bacteriophages may be introduced into a bacterium before it is stressed.

An example of the present invention will now be described with reference to the accompanying drawings in which: Figure 1 shows one form of bacteriophage multiplication; Figure 2 shows two forms of bacteriophage multiplication; Figure 3 schematically shows steps performed in the present invention; and Figure 4 shows the results of an experiment performed to illustrate the present invention. Bacteriophage multiplication takes place by either of two mechanisms respectively called the lytic cycle in which the host bacterium lyses and is killed and the Iysogenic cycle in which the host bacterial cell may remain alive following invasion of the bacteriophage. Each of these mechanisms will be described below: 1. In the lytic cycle a bacteriophage injects its nucleic acid into the host cell and immediately directs synthesis of its own viral components using host cell resources.

At maturation of the components are assembled into new viral particles and the host cell lyses (ruptures) and dies.

The consequence of the lytic cycle is an immediate switch of the bacterial metabolism to meet the needs of the infective bacteriophage with cell rupture and bacteriophage dispersion following sometimes in a matter of minutes.

Figure 1 illustrates such a lytic cycle in which at stage A a bacteriophage 10 attaches itself to a host bacterium 20 having a cell wall 21 and a chromosome 22. The bacteriophage 10 has a capsid or head 11 containing DNA 12 and a tail 13 comprising a sheath 14, several tail fibres IS, and a base plate 16. At stage B part of the tail of the bacteriophage 10 penetrates the cell wall 21 of the bacterium 20, contracts its sheath 14 and injects its DNA 12. At stage C the bacteriophage DNA 12 directs synthesis of bacteriophage components by the host cell. At stage D the bacteriophage components are assembled into new bacteriophages and at stage E the host cell lyses and releases the new bacteriophages. 2. In the lysogenic cycle the host bacterium may remain alive following invasion by the bacteriophage. The bacteriophage injects its DNA into the host cell and the bacteriophage DNA is able to insert itself into the DNA of the host cell (the inserted bacteriophage DNA is referred to as a prophage). Every time the

host bacterial cell machinery replicates the bacterial chromosome it also replicates the prophage DNA. The prophage remains latent in the host cell DNA. Figure 2 illustrates how a host bacterial cell injected with bacteriophage DNA may follow either of the lytic or Iysogenic cycles. At stage (i) a bacteriophage 10 attaches to a host cell 20 and injects its DNA 12. The host cell 20 already contains its own chromosome 22. At stage (ii) the bacteriophage DNA circularises and may enter either the lyric cycle illustrated on the left hand side of figure 2 or the lysogenic cycle illustrated on the right hand side of figure 2. If the cell follows the lytic cycle, at stage (iii) new bacteriophage DNA and proteins are synthesized and assembled into new bacteriophages and at stage (iv) the host cell 20 lyses releasing the new bacteriophages at least some of which may attach to new host cells.

If at stage (ii) instead of following the lytic cycle the host cell 20 follows the lysogenic cycle it will proceed from stage (ii) to stage (v) in which the bacteriophage DNA integrates within the bacterial chromosome by recombination to form a prophage 23. At stage (vi) when the lysogenic bacterium reproduces normally it will also reproduce its chromosome 22 with the prophage 23 so that the bacteriophage DNA is also reproduced. The host cell 20 and reproduced cells with the prophage 23 may continue reproducing indefinitely. However, occasionally the prophage may excise itself from the bacterial chromosome by another recombination event initiating a Iytic cycle as shown in stage (vii).

As shown in Figure 3, it has been found that by subjecting a bacteria cell 20 containing bacteriophage DNA to stress (step S) that the cell 20 then follows the lytic cycle, Iysing the cell and releasing numerous bacteriophages 10 to attach to other bacteria. When bacteriophages attach to these other bacteria they will also follow the lytic cycle and lyse, killing the bacteria and releasing more bacteriophages (step K) especially if the bacteria are not thriving which is generally the case in the unfavourable environment down oil and gas production facilities and in process plant.

The bacteria may be stressed by any of a plurality of influences such as an appropriate preferably predetermined dose of ultra-violet light, heat, antibiotics or chemicals. The dose must be large enough to stress the bacteria but not too large to kill all the bacteria without at least some of the bacteria being able to lyse and release further bacteriophages. The bacteria 20 to be killed will generally already contain latent bacteriophages but if necessary or desired bacteriophages can be added to the bacteria cells 20 before they are stressed by any well known method (step B).

Experiments were performed with a well known sulphate reducing bacterium called Desulfovibrio desulfuricans which is readily available from production wells and many culture collections well known to those skilled in the art including strains such as ATCC 13541 and ATCC 14563 (ATCCAmerican Type Culture Collection).

Samples of the bacteria were grown in suitable nutrients well known to those skilled in the art in an incubator for 20 hours at 30 C. 10 ml aliquots of growing culture were withdrawn and placed in Petri dishes in an anaerobic cabinet. Some Petri dishes

containing samples were exposed to ultra-violet light for between 0 to 5 minutes by a 300gWcm 2 W light source spaced 15.2 cm from the sample. Irradiated samples were put into anaerobic, empty, sterile vials and incubated at 30 C. 1 ml samples were removed after 0, 24 and 48 hours and the optical density of the samples was measured. Since bacteria block the passage of light, a sample with more bacteria will have a greater optical density (OD). As bacteria multiply the OD will continuously increase. Figure 4 shows the marked reduction in post treatment growth rate of W treated samples (i.e. reduction in increase of optical density 1D).

Stressing the sulphate reducing bacteria with other influences such as heat, chemicals or antibiotics produces similar effects. Other sulphate reducing bacteria are also affected in a similar way.

Bacteria which may usefully be controlled by the method of the invention include those generally found in process plant and oil and gas production facilities.

Frequently these are sulphate reducing bacteria such as those belonging to the genera Desulfovibrio and Desulfotomaculum. In particular the method may be used to control growth of bacteria such as Desulfovibrio salexigens, Desulfovibrio gigas and Desulfotomaculum nigrificans. Examples of particular strains include ATCC 13541, NCIMB 8303, ATCC 14944, ATCC 19364 and ATCC 49858. (NCIMB = National Collections of Industrial and Marine Bacteria).

Very usefully the method of the invention can be applied to the control of sulphate reducing bacteria by stressing these bacteria and isolating bacteriophages produced.

The produced bacteriophages are then added to water contained within oil and gas production facilities, process plant or hydrocarbon storage or to water intended for use in hydrotesting pipelines and other pressurised equipment to control sulphate reducing bacteria infestation.

Claims

1. A method for controlling bacteria, the method comprising stressing a bacterium so that it lyses and produces bacteriophages which then kill other bacteria.

2. A method according to claim 1, wherein the bacterium is subjected to a predetermined level of stress.

3. A method according to claim 2, wherein the predetermined level of stress is sufficient to stress the bacterium but not so great that it kills the bacterium before it produces bacteriophages to kill other bacteria.

4. A method according to claim 2 or claim 3, wherein the bacterium is stressed by the application of ultra-violet light.

5. A method according to claim 2 or claim 3, wherein the bacterium is stressed by the application of heat.

6. A method according to claim 2 or claim 3, wherein the bacterium is stressed by the application of a chemical.

7. A method according to claim 2 or 3, wherein the bacterium is stressed by the application of an antibiotic.

8. A method according to any of the preceding claims, wherein bacteriophages are added to the bacterium before it is stressed.

9. A method according to any of the preceding claims, wherein the bacteria to be stressed are in process plant or oil and gas production facilities.

10. A method according to any of the preceding claims, wherein the bacterium to be stressed is a sulphate reducing bacterium.

11. A method according to any of the preceding claims, wherein the produced bacteriphages are isolated and the isolated bacteriophages are added to a medium containing further bacteria.

12. A method according to claim 11, wherein the medium containing further bacteria is water contained within oil and gas production facilities, process plant or hydrocarbon storage or water intended for use in hydrotesting pipelines and other pressurised equipment.

13. A method substantially as hereinbefore described with particular reference to the accompanying drawings.

14. A method for controlling sulphate reducing bacteria, the method comprising stressing a sulphate reducing bacterium so that it lyses and produces bacteriophages that kill other sulphate reducing bacteria and the method including the following steps: (i) Stressing sulphate reducing bacteria and isolation of bacteriophage; (ii) Adding bacteriophage to water contained within oil and gas production facilities, process plant or hydrocarbon storage to control sulphate reducing bacteria infestation.

15. A method for controlling sulphate reducing bacteria, the method comprising stressing a sulphate reducing bacterium so that it lyses and produces bacteriophages that kill other sulphate reducing bacteria and the method including the following steps: (i) Stressing sulphate reducing bacteria and isolation of bacteriophage; (ii) Adding bacteriophage to water intended for use in hydrotesting pipelines and other pressurised equipment.

16. A method for controlling sulphate reducing bacteria, the method comprising stressing a sulphate reducing bacterium so that it lyses and produces bacteriophages that kill other sulphate reducing bacteria and the method including the following steps: (i) Stressing sulphate reducing bacteria within oil and gas production facilities or process plant to produce bacteriophage, said bacteriophages then control sulphate reducing bacteria infestation.