SRB enumeration by serial dilution Code: PRB-PRN-PR45

REVISION

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01 06/04/2016 All section Insert picture and detail on work step

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SRB enumeration by serial dilution Code: PRB-PRN-PR45

TABLE OF CONTENTS

1 PURPOSE ..................................................................................................................................4
2 SCOPE .......................................................................................................................................4
3 REFERRENCES ........................................................................................................................4
4 DEFINITION AND ABBREVIATIONS ..................................................................................4
5 DESCRIPTION OF THE METHOD API RP38 .......................................................................4
6 SAFETY AND ENVIRONMENT ............................................................................................5
7 PROCEDURES..........................................................................................................................5
7.1 Equipment required for the test...................................................................................................5
7.2 Taking water samples .................................................................................................................5
7.2.1 Taking water sample from process system ................................................................................5
7.2.2 Taking produced water sample from Slop tanks........................................................................6
7.3 Testing ........................................................................................................................................6
7.4 Results of the test........................................................................................................................7
7.5 Report of the results form (see attached report form) .................................................................8
7.6 SRB treatment ............................................................................................................................8
7.7 Housekeeping after test completes ..............................................................................................8
8 APPENDIX ................................................................................................................................8

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SRB enumeration by serial dilution Code: PRB-PRN-PR45

1 PURPOSE
This operating procedure is to describe the steps of enumerating SRB by the serial dilution
method. The presence of bacteria will indicate that biocide dosing will be required upstream
of the location where the bacteria was found.
2 SCOPE
This method will apply to the produced water on the process system and slop tanks of FPSO
Ruby II
Sampling Point Location:
- Downstream of Produced Water Degasser V-2003
- Produced water from slop tanks
Test Frequency:
- The test for water sample from slop tanks will be every three months
- The test for water sample from Produced Water Degasser V-2003 will be every six
months
3 REFERRENCES
- API RP38 Biological Analysis of Subsurface Injection Waters (Recommends that the
cultures be incubated at a temperature within 5°C of the recorded temperature of the water at
the time of sampling).
- PRB-MDK-PR28 Biocide batch treatment to lower H2S ppm content in slop tanks
- 103-MP-DC-047 Vessel Produced water Pumping system P&ID
- P1-RFT-M1-0203 Produced water degasser vessel V-2003 P&ID
4 DEFINITION AND ABBREVIATIONS
FPSO Floating Production, Storage and Offloading
SRB Sulphate Reducing Bacteria
MSDS Material Safety Data Sheet
PPE Personal Protective Equipment
API American Petroleum Institute
5 DESCRIPTION OF THE METHOD API RP38
Anaerobic sulphate reducing bacteria (SRB) are everywhere in the environment. They are
found in secondary oil recovery effluents, estuarine waters, deep wells, cooling waters, metal
working fluids, and water based hydraulic fluids, and petroleum based products that in some
way become contaminated with water. During their growth they are responsible for fouling,
objectionable hydrogen sulphide odours, corrosion of metals, and pitting of concrete. It is
essential that these organisms be monitored and controlled to prevent damage cause by their
metabolic activities. This is the usual procedure for detection of sulphate reducing bacteria
and estimation of their population has been the serial dilution method described in API RP38,
“biological analysis of subsurface injection waters”.
The process of enumeration is based on the ability of sulphate reducing bacteria to obtain
oxygen from sulphate ions. They release sulphide ions which combine with ferrous ions
to form a black precipitate of ferrous sulphide. The sample bottle used for SRB enumeration
will turn black if there is a single SRB introduced but the process occurs faster if there are
more SRB’s to digest the sulphate ions.
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SRB enumeration by serial dilution Code: PRB-PRN-PR45

6 SAFETY AND ENVIRONMENT

6.1 MSDS of all chemical are available on board FPSO; Personnel working with any chemical
should read and understand the MSDS to ensure that he knows the hazards and control
measures applied in case of accident with the chemical spill, gas release or personal affected
by the chemical
6.2 Full PPE must be worn when carry out SRB sample analysis including gas mask and
mandatory PPE’s.
6.3 Use fume hood for the chemicals that is volatile, flammable, hazardous to health, etc. in order
to avoid affecting the entire Lab Room.
6.4 Lab Room area must always be clean from any chemical spillage and the utensils/equipment
used must be well organized so as not to contaminate with each other.
6.5 No waste or chemical is allowed to discard overboard during and after sampling completes.
Strictly follow company waste management procedures.
6.6 If any unsafe condition/unsafe act taking places during these sampling, personnel are allowed
to stop the work, report to Production supervisor for prompt actions and control measures.
7 PROCEDURES
7.1 Equipment required for the test
+ SRB media kits according to API RP38
+ 250ml glass sample bottle.
+ 1 ml disposable sterile syringe with needle
+ Incubator

7.2 Taking water samples

7.2.1 Taking water sample from process system
- Inform to CCR whenever taking the Produced water sample.
- Location of sampling point is at downstream of produced water degasser V-2003 as marked
up in attached P&ID
- Flush the sampling point into the open drain trap before taking sample to ensure the line is
displaced and fresh liquid is obtained until sample line is warm.
- Slowly open isolation valves on sample line to collect the liquid sample by clean glass bottle
(250 ml).
- Close isolation valves fully and immediately cap the sample bottle until obtained 80%
volume and put the plug on the end of sampling line.
- Bring the sample to laboratory for testing.
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SRB enumeration by serial dilution Code: PRB-PRN-PR45

7.2.2 Taking produced water sample from Slop tanks

- Cargo Operator will inform to OS whenever taking the Produced water sample from Slop
tanks at pump room.
- Location of sampling point is at produced water from Slop tanks as marked up in attached
P&ID
- Flush the sampling point into the bucket before taking sample to ensure that the line is
displaced and fresh liquid is obtained until sample line is warm.
- Slowly open isolation valve on sample line to collect the liquid sample by clean glass bottle
(250 ml).
- Close isolation valve fully and immediately cap the sample bottle until obtained 80% volume
and put the plug on the end of sampling line.
- Bring the sample to laboratory for testing.
7.3 Testing

Step Description Pictures

 Put 06 SRB growth media bottles together

in a row with transparent tape.
 Remove a sterile syringe from its plastic
1
container.
 Immerse the tip in the water sample and fill
the syringe to exactly 1 ml.

 Point the tip upward and force out exactly 1

ml of water, make sure that no air is trapped
in the syringe.
2
 Invert the taped bottles, insert the needle
through the rubber stopper in the first bottle,
and squirt into SRB growth media bottles.

 Without pulling out the syringe, shake the

bottles to mix the broth with the injection
water
3
Note: This avoid air ingress the SBR growth
media bottles

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SRB enumeration by serial dilution Code: PRB-PRN-PR45

 With draw exactly 1 ml of the mixture in the

4 first bottle into syringe and inject it into the
second bottle.

 Shake and draw exactly 1 ml of mixture in

5 the second bottle into syringe and inject it
into the third bottle.

 Continue the same steps until the last bottle has received 1 ml from the previous bottle.
6 If the duplicate dilution series is required, an identical amount of the original sample
should be taken and processed in a similar manner.

 Keep the bottles in the Incubator at a

7 temperature within 5oC of water at the time
of sampling.

 Daily observer up to 28 days. Positive results

8 (bottles will turn from colorless to black) are
indicated as described in the table of the
interpretation of results.

7.4 Results of the test

Table of interpretation results
Number of positive Bacteria/cc of
Rating of Results
bottles Original Sample
1 1-10 Satisfactory
2 10-100 Satisfactory
3 100-1.000 Low
4 1.000-10.000 Moderate- system needs watching
5 10.000-100.000 High – may cause formation plugging
6 100.000-1.000.000 Very high- system need immediate attention
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SRB enumeration by serial dilution Code: PRB-PRN-PR45

7.5 Report of the results form (see attached report form)

Reading Sample No of Bac. /CC Bac. /cm2

Sample Rating of
result taken Time Method positive of original of original
point results
date date bottles sample sample

API

API

API

API

API

API

API

API

API

API

API

API

7.6 SRB treatment

When test result of SRB above 1.000 Bacteria/cc of original sample, then Biocide will be
considered to be injected in to the system. Injecting location are inlet of HP and Ruby Pre-
heater for topside system and dump valves for slop tanks.
Biocide dosing rate will be advised by chemist based on test results. Normally, dosing rate
should be 200-500 ppm if biocide treatment is required.
Refer to procedure PRB-MDK-PR28 Biocide batch treatment to lower H2S ppm content in
slop tanks for more information.
7.7 Housekeeping after test completes
- Dispose of the syringe and needles in the sharps container in the laboratory. When the
container is ready for disposal it shall be taken to hazardous waste bin.
- Wash the sample bottles with water and lab detergent
- Rinse equipment with distilled water and sterile
8 APPENDIX

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