Microbial growth in water injection systems can lead to many problems, including biofouling, water quality deterioration, injectivity loss, microbial corrosion, and reservoir formation damage. Monitoring of microbial activities is required in any mitigation strategy, enabling operators to apply and adjust countermeasures properly and in due time. In this study, the pre-industrial autonomous microbe sensor (AMS) was constructed with technical improvements from the prototype for increased sensitivity, durability, robustness, and maintainability. The pre-industrial AMS was lab validated, field proven, and deployed at critical locations of seawater injection network for automated detection of microorganisms under the Saudi Arabia’s harsh environment. An excellent correlation between AMS measurement data (fluorescence count) and actual count of microbial cell number under microscope was established (coefficient of determination, R 2 > 0.99) for converting AMS fluorescence count to cell numbers (cell mL -1) in the injection seawater. The pre-industrial AMS only required monthly maintenance with solutions refill, and was able to cope with hot summer months even without protection in an air-conditioned shelter. The study team recommended wider deployment of the online AMS for real-time monitoring of bacteria numbers in the various strategic locations in Saudi Aramco’s complex seawater injection network, as an integral component of pipeline corrosion and leak mitigation program.
Saudi Arabian Oil Company (Saudi Aramco) has the largest seawater injection system in the world. The growth of microorganisms and the formation of biofilm on the pipeline’s inner surfaces and process equipment lead to an array of challenges, including biofouling, water quality deterioration, injectivity loss, reservoir plugging, and microbiologically influenced corrosion (MIC) [
The microbial number and activity are traditionally monitored with conventional growth methods that require manual sampling and handling. Monitoring the microbial activity with conventional methods in this large system, especially at the remote locations, presents a long-standing challenge to Saudi Aramco. In an attempt to ensure the injection water quality and maintain the integrity of injection pipelines network, Saudi Aramco and the Danish Technological Institute (DTI) embarked on a three-phase project with an overall objective of developing and deploying an online sensing technology for automatic and real-time monitoring of microbial numbers in Saudi Aramco’s large injection seawater system.
In the feasibility phase study [
In the subsequent phase, an autonomous microbe sensor (AMS) prototype was constructed, tested, and optimized in the laboratory, followed by a validation in the field for automated detection of microorganisms in the harsh Saudi Arabia desert environment [
The objective of the current phase study was to implement the technical improvements identified in the prototype field validation [
The pre-industrial AMS is an instrument that provides an online and real-time detection of total microbial numbers in seawater pipelines. It is provided with a flow cell that measures the fluorescence intensity of microorganisms in the seawater sample after mixing with DNA staining dye SYBR Green [
The areas for technical improvement and optimization identified in the field testing of the AMS prototype [
1) Optimization of cleaning procedures: The internal AMS cleaning procedures were optimized to minimize the risk of biofouling and iron precipitation. Alkaline (NaOH) and acid (oxalic acid) cleaning procedures were incorporated into the measurement cycle to prevent the biofouling and formation of iron precipitations in liquid handling components.
2) Incorporation of an internal DNA control: The internal DNA control was used to validate the AMS functionality and compensate the temperature effect on fluorescence counts.
3) Optimization of fluorescence detection procedure and software: The sampling procedure and software were optimized in the pre-industrial AMS to minimize the scattering of data points caused by uneven cell distribution.
4) Sensitivity improvement: The detection limit of pre-industrial sensor was improved by optimizing hardware components (spectrophotometer, optical fiber, and flow cell) and microbial staining and measurement procedures.
5) Incorporation of temperature correction factor: The temperature inside the AMS was measured simultaneously with the fluorescence measurements. The inside temperature and the fluorescence count of internal DNA control were used to develop a temperature compensation factor to compensate the temperature effect on the fluorescence dye.
6) Redesigned user interface: The new software interface, layout and data output allowed data export and automatic plotting and display in remote computer.
The design of pre-industrial AMS has incorporated the technical improvement areas identified from AMS prototype. Design changes from the prototype to the pre-industrial AMS are summarized in
The pre-industrial AMS consists of three separate modules: hardware module, consumables module, and power module:
1) Hardware module: includes a custom-made flow cell for achieving flow
Part | Prototype AMS | Pre-industrial AMS |
---|---|---|
Unit layout | Single box | Three modules: hardware, consumables, and battery |
Internal DNA control | N/A | Synthetic DNA in ethyleneiaminetetraacetic acid (EDTA) and ascorbate buffer |
Sample staining | 40 min at ambient temperature | 13 min at 60˚C |
Flow cell | Commercially available flow cell | Custom-built flow cell with minimal volume and large numerical aperture |
Measurement | Sample is static when illuminated | Sample flows slowly when illuminated to avoid bleaching of dye |
Dye dispensing | Fixed volume | Dedicated syringe pump for dye |
Lower limit of field detection | ~105 | Low limit of detection, LLOD |
− AMS 1: 1.9 × 104; AMS 2: 1.2 × 104 | ||
Low limit of quantification, LLOQ | ||
− AMS 1: 5.0 × 104; AMS 2: 3.2 × 104 | ||
Cleaning | NaOH | NaOH and oxalic acid |
Power | AC mains | Battery, charged either by AC mains or solar panels |
Remote communication | Team Viewer through Saudi Aramco intranet when AMS is on | Permanent RS-485 connection. Data retrieval and AMS control through dedicated software on remote computer |
during quantification, a heating chamber for improved staining conditions and two dedicated pumps. Pump A is for dispensing water sample and cleaning solutions; while Pump B is dedicated for SYBR Green dye distribution.
2) Consumables module: includes four containers for water, oxalic acid, sodium hydroxide and waste.
3) Power module: A 24 V DC battery pack was chosen as the power supply. The batteries are connected to three solar panels or 230 V AC mains power.
The modular design of AMS improves the equipment functionality and durability in the challenging Saudi Arabia dessert environment and operation conditions of seawater injection system [
After construction of two pre-industrial AMS units, the AMS was thoroughly tested and validated in the laboratories at DTI premises. The repeatability and functionality of the units were tested with respect to procedures as well as software. The major improvements in lab validation and their effect on AMS performance are summarized in
After implementation of above improvements on AMS units, a correlation of fluorescence signals from AMS and direct counting of microbial cell number
Improvement | Effect |
---|---|
Decrease of Pump A rate to 3 mL・min−1 | Eliminate air from surroundings into syringe A |
New loading procedure for internal DNA control | Improved mixing of internal control (IC) DNA and Milli-Q water |
Increased total volume of diluted internal DNA control to sample volume | Improved loading of IC DNA |
New dye loading procedure. Dead space in Pump B valve used as dye dosing volume | Accurate dye dosing |
Selection of incubation conditions of 13 min at 60˚C | Improved staining of cells with SYBR Green I |
No air is moved through flow cell | Eliminate introduction of air bubbles in flow cell |
Flow direction of flow cell changed | Remove potential air bubbles in flow cell |
New flow rate through flow cell | Optimal flow rate for maximum reading |
Increased rate of dispensing and aspirating with Pump A for all non-critical steps | Minimize total AMS measurement cycle time to 75 min |
Incorporated data export function | For automatic plot of raw and calibrated readings on graph in Excel |
under microscope after DAPI staining was established (
The calibrations show that the AMS units are linear in a wide range of at least two orders of magnitude, with a laboratory detection limit of around 103. From the performed calibrations, the AMS units were accepted for field installation, calibration, and validation.
At the end of lab validation, the optimal AMS measurement settings were determined and implemented in both AMS units (
AMS1 | AMS2 | |||
---|---|---|---|---|
Fluorescence count (AMS) | Cells mL−1 | Fluorescence count (AMS) | Cells mL−1 | |
Sterile filtered system water (blank) | 154 ± 7, n = 3 | 98 ± 3, n = 3 | ||
LLOD | 167 | 6.0 × 103 | 104 | 8.8 × 102 |
LLOQ | 188 | 1.6 × 104 | 114 | 2.3 × 103 |
Regression equation | 473X − 72,788 | 146X − 14,265 | ||
Regression, R2 | 0.9902 | 0.9490 | ||
Linear range | 6.4 × 103 - 5.0 × 106 | 1.1 × 104 - 2.4 × 106 |
LLOD: Lower Limit of Detection; LLOQ: Lower Limit of Quantification.
Improvement | Effect |
---|---|
Volume of sample | 4 ml |
Volume of IC DNA | 4 ml (0.1 ml concentrated IC discarded at first, 0.2 ml IC in 3.8 ml sterile H2O) |
SYBR Green I | Diluted 1:20 in Dimethyl sulfoxide (DMSO). Volume equals to dead space in Pump B (20 μL) |
Incubation of sample | 13 min at 60˚C |
Incubation of IC | Less than one minute at ambient temperature |
Flow rate and time | 3.5 μL・s−1 of 180 sec and 10 sec |
Measurement wavelength interval | 520 - 530 nm |
Fluorescence count | Average of readings every one sec for 180 and 10 sec |
Background reading (auto-fluorescence) | To be subtracted from sample reading. Fluorescence reading of sample without dye, for correction of any auto-fluorescence of the sample water. Background reading is not subtracted from internal control readings. |
Cleaning | Sterile H2O and air between sample and IC measurement. Acid, alkaline, sterile water and air at the end of a cycle. No air through the flow cell. |
Time consumption | 75 min for one full cycle, including washing/cleaning |
After laboratory validation at DTI premises, both AMS units were shipped to Saudi Aramco, and tested for functionality and stability after transportation. After functionality check, AMS1 was connected to the side-stream of a 60" shipping line at an injection facility and AMS2 was installed at an injection plant further downstream of AMS1, connecting to a side-stream of 40" seawater pipeline. The seawater flow continuously passes AMS through a bypass connection to the disposal when AMS is not doing the measurement. During the measurement cycle, a fixed volume of seawater sample is taken into the AMS for staining and measurement of microbial cell number. During the field testing and validation, the sensors were programmed to take two measurements a day, with a monthly maintenance for solutions refill. The data were retrieved by the operators in the Control Room.
The fluorescence intensity from a stained sample depends on the amount of DNA present in the sample, which in turn, depends on the cell size and the metabolic status of the cell, as an actively dividing cell contains more DNA than a dying cell [
The calibration correlates the cell number (cell mL−1) of the system water determined by DAPI staining and counting to AMS fluorescence counts from dilution series of the sample measured in the field. A typical DAPI staining for counting microbial cell number under microscope is shown in
A linear regression between AMS fluorescence measurement and DAPI count for converting AMS fluorescence count to cell numbers in the sample is summarized in
During the field validation of the AMS units, a tendency of higher fluorescence counts with lower AMS inside temperatures has been observed. A full correlation of all internal DNA control readings shows an exponential decrease in fluorescence with increasing temperature, which fits well with an exponential function with both a vertical and horizontal offset [
f ( x ) = A × exp [ B ( x − x 0 ) ] + C (1)
where the fitted parameters are A = 2.4 × 104 s−1, B = −0.10˚C−1, x0 = 4.16˚C, C = 2.49 × 103 s−1, and x = AMS inside temperature (˚C).
The function f(x) between temperature and fluorescence intensity was used to temperature compensate internal control (IC) readings. The compensation is done by multiplying the IC reading with the ratio f(30)/f(T), where 30 is the reference temperature, and T is the AMS inside temperature of the given data point.
AMS1 | AMS2 | |||
---|---|---|---|---|
Fluorescence count (AMS) | Cells mL−1 | Fluorescence count (AMS) | Cells mL−1 | |
Sterile filtered system water (blank) | 114 ± 12, n = 3 | 123 ± 7, n = 3 | ||
LLOD | 136 | 1.9 × 104 | 137 | 1.2 × 104 |
LLOQ | 173 | 5.0 × 104 | 160 | 3.2 × 104 |
Regression equation | 843X − 96,020 | 878X − 107,935 | ||
Regression, R2 | 0.9939 | 0.9940 |
The temperature effect on fluorescence intensity is likely related to the molecular structure of SYBR Green I, as the supplier of SYBR Green (Invitrogen) explained that “the temperature effect is likely caused by less rigid bonds in the fluorescent molecule as the atoms become excited and move around.” Therefore, the observed temperature response is a direct consequence of the reduced SYBR Green fluorescence at elevated temperatures. In this case, the fluorescence intensity of stained IC DNA or stained cells are equally affected by temperature.
An IC measurement is performed for each AMS run with equal DNA content for each run. As the IC and system water samples are affected equally by temperature, the IC can, besides validating the performance of the AMS unit, also be used for an internal temperature compensation.
Other possible causes for the temperature dependency (melting of oligonucleotides DNA and heating of spectrometer) have been considered unlikely. Internal control DNA (a synthetic 55-base pair oligonucleotides with hairpin structure) was tested for the stability at a wide range of temperature. The synthetic DNA in EDTA and ascorbate buffer was found to be stable from 20˚C to 80 ˚C for at least 28 days when stored in a sealed container.
Melting of the double stranded (ds) Oligo DNA (internal control) would result in a lower binding between the SYBR molecule and the DNA, as SYBR Green I only binds to ds DNA [
The spectrometer must be cooled for proper readings, and cooling has been included in the design of the AMS. To test whether the AMS spectrometer is affected by AMS inside temperature, fluorescein was injected manually into the flow cell at high and low temperatures (40˚C and 25˚C, respectively). Fluorescein is a dye that emits fluorescent light with excitation and emission spectra comparable to those of SYBR Green when bound to DNA [
The AMS1 measurement data, from October 2016 to February 2017, on the fluorescence count of the IC DNA and microbial cell number in the seawater are plotted in
Temperature effect on fluorescence intensity is determined to be significant when SYBR Green I dye is used to stain IC DNA and cell DNA in seawater samples. As a result, temperature compensation for all fluorescence counts of both IC DNA and microbial cells is required. The temperature compensation factor was incorporated in the software for converting the raw data to temperature compensated data for data exporting and plotting.
During the field testing and validation, a number of technical challenges have been either encountered or identified, and the solutions have been implemented to address these challenges (
The pre-industrial AMS is equipped with a restrictor to reduce the side-stream pressure from 10 bars to less than 1 bar before water enters the AMS system. The restrictor is equipped with a pressure gauge to monitor inlet pressure. Since the seawater pipeline pressure is much higher, it is important that
another pressure regulator is installed on the side-stream to reduce the pressure to about 10 bars for prevention of damage to AMS hardware. Due to the harsh conditions in the seawater environment, the needle type pressure regulator tends to be easily clogged. To avoid clogging, the ball valve type or spring type pressure regulator is required for both side-stream and AMS. After the right type of pressure regulator is selected and installed, the regulator clogging and potential AMS damage due to high pressure are eliminated.
AMS liquid handling system consists of two syringe pumps, two multi-channel valves, and many tubings to transport various liquids around the AMS system for measurement, cleaning, and disposal. Loose fittings, wear and tear, and corrosion have been noticed during long-term field trials. Replacement of original Delrin fittings with PEEK fittings has solved the corrosion problem caused by oxalic acid and reduced the leaking occurrence. Occasional tightening and close monitoring of the fittings are required when there is large ambient temperature fluctuation. For AMS field deployment, an air conditioned (AC) shelter is recommended to maintain a relatively stable temperature in AMS
Element name | Issue | Lessons Learned | Action Required |
---|---|---|---|
Reagents and solutions | |||
SYBR dye | Experienced inaccurate volume of dye collected by syringe. | Solvent DMSO in the dye will freeze below 12˚C. | Consider insulating SYBR vial or enclosing AMS modules within an air conditioned shelter. |
Reaction and measuring system | |||
Flow cell | Biocide entering flow cell. | Need to coordinate with corrosion engineer for biocide injection time. | Programmed AMS measurement time around the estimated biocide arrival time. |
Liquid handling system | |||
Pressure regulator | Experienced clogging of pressure regulator inside AMS. | Select right type of regulator. | Replaced original needle type pressure regulator with a ball valve to reduce the pressure. Increased sampling tubing size from 1/8" to 6 mm. |
Pressure regulator | Experienced damage of pressure regulator inside AMS. | Avoid overpressure of side-stream. | Installed a pressure regulator on side-stream to reduce the pressure to below 10 bars before connecting side-stream to AMS. Installed a restrictor with a pressure gauge to monitor inlet pressure. |
Corrosion | Experienced corrosion of fittings. | Delrin fittings is prone to oxalic acid corrosion. | Replaced all Delrin fittings with PEEK fittings. |
Leakage | Experienced leakage and precipitates around fittings. | Loosening of fittings due to large temperature fluctuation. | Equipped all fittings with lock nut. Tighten fittings, if necessary. |
Electronics and power system | |||
Solar panel | Experienced lots of dust collection on solar panels. | Required monthly cleaning. | Consider installing solar panels at proper height for easy access during monthly maintenance. |
Power socket | Power socket is not water proof. | Risk of water splash entering power socket. | Replaced power socket with built-in power cable. |
modules, which will have many beneficial effects: eliminate fitting loosening due to large temperature change, prevent SYBR Green dye freezing, prevent temperature-regulated AMS shutdown, and extend the life span of the AMS device.
In general, pre-industrial AMS sampling, conditioning, measurement sequence and method, solution and reagent refilling were found suitable for field application. When in operation, the AMS was subjected to routine maintenance from the operation manual (
Maintenance items | Schedule |
---|---|
Check instrument inlet pressure | Monthly |
Check instrument sample flow rate | Monthly |
Inspect internal sample leak and blockage | Monthly |
Clean solar panels | Monthly |
Refill solutions and reagents | Monthly |
Inspect tubings and fittings, and replace if necessary | Half-year service |
Validate performance and recalibrate if necessary | Half-year service |
The online AMS, based on fluorescent DNA staining technology, is developed in three phases for real-time detection of microbial cell numbers in large seawater injection systems under Saudi Arabian conditions. Numerous technical improvements have been implemented in each developmental phase for increased sensitivity, durability, robustness, and maintainability, while reducing the uncertainties related to AMS operation and function. Two pre-industrial AMS units have been constructed and deployed at critical field locations for automated monitoring of microbial numbers in injection seawater system (salinity ~5.5%). The two units are currently operational, including the hot summer months in Saudi Arabia without AC shelters, requiring only minimal monthly intervention from field operators and engineers. The wider deployment at more strategic locations in large Saudi Aramco sea water injection network is planned. In addition, a renewed market study is on-going to assess the market opportunities, technology landscape, and commercialization strategy.
For long-term reliability and maintainability, it is recommended to house the AMS hardware and solution modules inside an AC shelter. AC shelter is expected to provide two major benefits. One is to prevent solvent DMSO of SYBR Green dye from freezing when temperature in Saudi Arabia winter drops to below 12˚C. DMSO freezing affects the delivery of SYBR Green dye for bacteria staining, and hence the fluorescence signals. For hardware protection, AMS is programmed to shut down automatically when temperature is above 40˚C, resulting in many missing measurements during hot Saudi Arabia summer. An AC shelter increases the flexibility of programming measurement time and frequency throughout the year. It is also recommended to use dual power supply (mains power and solar panel) for battery charge, whenever mains power is available at the installation site. This will ensure the sufficient battery charge to power AMS operation in case of reduced solar panel efficiency caused by adverse weather and environmental conditions (e.g., rain, clouds, dust, and sand storms). The cleaning of solar panels should be maintained even when the mains power is connected.
Calibration is sensitive to the specific water system in question, therefore specific calibration shall be performed during commissioning of each water system, to ensure the accurate conversion of fluorescence count to cell number in the sample. In addition, the performance of the AMS shall be validated after major maintenance and service, and re-calibrated if necessary.
The authors would like to acknowledge the Saudi Arabian Oil Company (Saudi Aramco) and the Danish Technological Institute for granting permission to publish this paper.
Al-Moniee, M.A., Zhu, X.Y., Markfoged, R., Al-Wadei, A.H., Pedersen, P.L., Tuxen, A.K., Al-Nuwaiser, F.I., Tang, L., Roesen, T.S. and Lundgaard, T. (2018) Deployment of Pre-Industrial Autonomous Microbe Sensor in Saudi Arabia’s Injection Seawater System. Journal of Sensor Technology, 8, 1-17. https://doi.org/10.4236/jst.2018.81001