5 WATER TREATMENT MICROBIOLOGY INTRODUCTION Some knowledge of the behavior of microorganisms in water is extremely important since their presence can cause corrosion or plugging of equipment or the injection wellbore. They are simply another source of plugging solids or conditions which result in corrosion. Biology can be defined as that branch of knowledge that deals with living organisms and their vital processes. Thus, in its broadest definition, it is that part of science which is concerned with the life processes of plants and animals. Microbiology is one branch of biology which concentrates on microscopic forms of life known as microorganisms. Of primary concern in oilfield operations is the behavior of microscopic, single- celled organisms which are capable of living under all sorts of conditions and multiply with incredible speed. Microorganisms Found in Oilfield Water Systems Bacteria Bacteria comprise the broad class of microorganisms of greatest interest to us in water handling. ‘The bacterial cell has little visible structure, even when examined with an electron microscope. Around the outside there is a cell wall which gives the cell its shape. Inside the cell wall is a thin semipermeable membrane which surrounds the contents of the bacterial cell and selectively controls the passage of substances between the cell and its external environment. The cell is filled with water containing different minerals and chemicals. Most species are motile, meaning that the bacteria propel themselves through water by means of one or more whip-like flagella (singular: flagellum) which rotate and function like little outboard mo- tors. A generalized diagram of a bacterial cell is shown in Figure 5.1. CHAPTER 5 181WATER TREATMENT MICROBIOLOGY Semipermeable Membrane Celi Wall DNA Figure 5.1 Generalized Diagram of Typical Bacterial Cell Eucarya This group of micro-organisms includes algae, fungi and protozoa. Algae Algae are single-cell plants which contain chlorophyll. Algae manufacture their food by photo- synthesis using light as a source of energy and CO> as a source of carbon for all growth. They need sunlight for normal growth, although they can grow (slowly) in the dark. 52) Algae can form slime on the water surface which is easily identified by its green or blue-green color. It is often observed in stagnant water and in cooling towers. Algae are present in seawater, although usually not as slimes. There are significant numbers in ‘most oceans and they can contribute substantially to the plugging solids in the water, especially during algae “blooms” which occur at certain times of the year. Fungi Fungi generally grow better in aerobic systems and are thought to cause few problems in most ‘water injection systems. Protozoa Protozoa are the simplest form of animal life. They are found in both fresh and salt water and require oxygen to live. In water injection systems they are found in open tanks or pits. They are also found in filters when the water is aerated. Generally, control of the other microbial population suffices to control the protozoan population. Macro-Organisms ‘The oceans of the world contain creatures which are referred to as plankton. The term plankton is applied to all those animals and plants which live freely in the water and which, because of their limited powers of movement, just drift along with the water currents. This is not to say that they cannot swim. Some, such as crustacea and fish larvae, can swim very well, but they may not be able to swim faster than the water moves. Plankton may be grouped by sizes. Very large animals such as jellyfish are called megaloplank- ton. Those organisms which are readily visible with the naked eye and down to a size which is re- tained by a net with I mm holes are referred to as “coarse net” or macroplankton. Organisms smaller 182 APPLIED WATER TECHNOLOGYBACTERIA CLASSIFICATION than 1 mm in size, but larger than 75 Hm are microplankion. The smallest size is called nanoplank- ton. It includes tiny plants, bacteria, and creatures called flagellates, ‘The smallest flagellates, that is those below 5 jum, are sometimes called ultraplankton. Plankton are important to us because they make up a significant portion of the plugging solids in most seawaters. Also, they serve as food for larger creatures which may in turn contribute to plugging. BACTERIA CLASSIFICATION Bacteria may be classified in a number of ways. A description of some of the most common classification criteria follow. Size And Shape Bacteria are extremely small (about 0.5 um in diameter) and exist in literally thousands of spe- cies. True bacteria are shaped like spheres, straight rods, or curved rods. The shapes are named as follows: 1, Assingle spherical bacterium: coccus ‘occ Several spherical bacteria: As a matter of interest, a string or chain of cocci is called a streptococcus, while a sheet or plane of cocci is called a staphylococcus. 2. Straight rod: bacillus 3. Curved rod: Vibrio — single curve in the form of a “C” Sigmoid — shaped like an “S” Spirillum — two or more curves in the shape of a screw or spiral Growth The reason that bacteria can create so much trouble is that they can multiply with incredible speed. Some can double their population in 20 minutes under ideal conditions, which means that a single bacterium can become a thriving colony of millions of bacteria in a very few hours. A handful of slime from a water may contain as many bacteria as there are people in the world. Bacteria can withstand an extremely wide range of temperatures (at least 14-210°F[-10 to 99°C), pH values (about 0 to 10.5), and oxygen concentrations (0 to almost 100% oxygen).52) However, in water systems, they grow best in the pH range of 5-9 and at temperatures less than 180°F [82°C].°4 They also prefer fresh water, but can do nicely in brines. They are extremely adaptable and hardy. Bacteria can live either in groups or “colonies” attached to solid surfaces or suspended in water. Bacteria attached to a surface are called “sessile” bacteria. When they are suspended in water, they are termed “planktonic” bacteria, or sometimes simply “swimmers” or “floaters.” The majority of bacteria are sessile. It has been reported that in a typical system, there are 1000 to 10.000 times as many bacteria attached to a surface as there are floating in the water.°) It has also been shown that as sessile bacteria grow they produce a sticky substance called a polysaccharide, which the bacteria utilize to cement themselves to a solid surface. Continued CHAPTER 5 183WATER TREATMENT MICROBIOLOGY production of the polysaccharide results in the formation of a “biofilm” which surrounds and covers the bacteria. The biofilm can become quite thick (200-250 cells, and 1 mm in thickness).°® Within the layers of polysaccharide, there can be a whole community of bacteria. Cells of one species often exist in their own protected state beside cells of another, creating a mixed adherent population. Oxygen Requirements ‘One method of classification of bacteria which is of interest in oilfield systems is whether or not specific bacteria require oxygen to live. They fall into three categories: 1 2. 3. Aerobic bacteria — require oxygen to grow. Anaerobic bacteria — grow best in the absence of oxygen. Facultative bacteria — grow in either the presence or absence of oxygen. BACTERIA WHICH CAUSE PROBLEMS As previously stated, bacteria can contribute to both corrosion and plugging. Bacteria can affect the corrosion process in oilfield systems in several ways. They can: 1 2. 4 o Plugging can result from bacterial act tion of corrosion product (such as iron sulfide) or the precipi Generate hydrogen sulfide, thereby increasing the corrosivity of the water. Produce organic acids that initiate or accelerate corrosion on the metal surface beneath the colonies. Produce enzymes that can increase the corrosion rate by direct participation in the electro- chemical corrosion process. Oxidize soluble iron in the water, causing it to precipitate and form deposits (called “tuber- cles” ) that accelerate corrosion through the formation of concentration cells. A combination of the preceding. ivity due to the formation of bacterial biomass, the genera- tion of soluble iron from the water. Sulfate Reducing Bacteria (SRB) Sulfate reducers probably cause more serious problems in oilfield injection systems than any other bacteria. They reduce sulfate or sulfite ions in the water to sulfide ions, resulting in HS as a by-product. Four types of problems can result from sulfate reducer activity in an injection system: 1 They can participate directly in the corrosion reaction and cause pitting directly beneath the bacterial colony, as shown in Figure 5.2. ‘The generation of HzS by bacteria can increase the corrosivity of the water. If the system is already sour, the additional HzS generated by the bacteria may have little or no effect. However, if the system was originally sweet, the addition of H2S to the system by bacterial activity can substantially increase corrosion rates and result in a pitting attack throughout the system. The presence of sulfate reducing bacteria in a system which was originally free of HS creates the possibility of sulfide cracking and blistering of carbon steels. 184 ‘APPLIED WATER TECHNOLOGYBACTERIA WHICH CAUSE PROBLEMS Figure 5.2 Corrosion Pit Beneath Sulfate Reducing Bacteria Colony 4. Sour corrosion results in the formation of insoluble iron sulfide which is an excellent plug- ging material. Sulfate reducing bacteria are most likely to be found in stagnant or low velocity areas, and be- neath scales or sludges. Common places for bacterial activity in injection systems are tanks, filters and the rat hole in injection and water source wells. Types of Sulfate Reducing Bacteria ‘About nine families, or genera (plural of genus), of sulfate reducing bacteria are known. How- ever, most SRB corrosion problems are attributed to members of two families: Desulfovibrio and Desulfotomaculum. Some of the species of each which are known to contribute to corrosion are listed in Table 5.1.67 TABLE 5.1 SRB Families (Genus Species ‘Shape Desulfovibrio | africanus | Sigmoid rod desuifuricans | Vibrio salexigens | Vibrio vulgaris Vibrio Desulfotomaculum | nigrificans* | Rod orientis Curved rod “ormerly Clostridium nigrificans ig organisms most commonly detected in the oilfield belong to the genus De- The suifate-reduc sulfovibrio!9) Desulfotomaculum can form spores. A bacterial spore is a structure formed within the body of a bacterium. Spores are resistant to temperature, acids, alcohols, disinfectants, drying, freezing, and many other adverse conditions. Spores may last for hundreds of years and then germinate in favorable conditions.’ A spore has many of the characteristics of a seed but is not a reproductive structure. 54) CHAPTER 5 185WATER TREATMENT MICROBIOLOGY Effect of Salinity Sulfate reducing bacteria are found in natural waters of all salinities from near zero to satura- tion! Many Desulfovibria are salt tolerant and can grow in NaCl concentrations as high as 100 000 ppm. Higher concentrations tend to limit growth. Among Desulfovibria, two species have specific salt requirements. Desulfovibrio salexigens has an absolute minimum requirement of 25 000 ppm NaCl, while Desulfovibrio vulgaris is a fresh water strain requiring NaCl concentrations of less than 10 000 ppm.*7) Desulfotomacula are seldom found in waters containing more than 20 000 ppm NaCl. Above 20 000 ppm, the population is almost always Desulfovibria.(5! Temperature, Pressure and pH Sulfate reducing bacteria as _a group are reported to tolerate temperatures from 40-170°F [4-77°C], a pH range of about 5 to 9, and pressures of at least 14 500 psi [100 000 kPa]:5) However, absolute values of temperature, pressure and pH required for the growth of sulfate reducing bacteria in natural environments are impossible to state with any degree of certainty. For example, sulfate re- ducers isolated from wells with bottom hole temperatures in excess of 250°F [121°C] have been cul- tured in the laboratory at lower temperatures, but would not grow at temperatures greater than 190°F [88°C] at atmospheric pressure.) Furthermore, the maximum temperature at which sulfate reducing bacteria grow apparently increases with pressure. The following statements apply to growth in the laboratory in artificial media: + Desulfovibrio: The optimum temperature range for growth is approximately 77-110°F [25-43°C}, with an upper temperature limit of 120°F [49°C].°7) + Desulfotomaculum nigrificans: The optimum temperature for growth is 130°F [54°C]. They exhibit slow growth at 150-160°F [66-71°C], and can survive at 170°F (77°C].°7) * Desulforomaculum orientis: Exhibits optimum growth at temperatures of 85-100°F [30-38°C]. They are killed when the temperature exceeds 108°F [42°C],57) Nutrition Bacteria absorb their nutrients directly from the environment around them. Each cell contains proteins called enzymes that help break down nutrient molecules and enable the bacteria to extract energy from them. ©!) A single living cell contains hundreds of different enzymes, each of which is an effective catalyst for a specific chemical reaction. However, the enzymes work together in a coordinated manner to produce the materials required for normal cell growth and metabolism. Although all enzymes are initially produced in the cell, some are secreted through the cell wall and function in the cell's environment. This type of enzyme enables the cell to assimilate large mole- cules by breaking them down outside the cell into smaller molecules which can be absorbed through the cell wall. 52) Sulfate reducing bacteria require a number of nutrients in order to sustain growth. Some of the primary ones are: * Carbon. Sulfate reducing bacteria are heterotrophic, meaning that all or most of their cell carbon is derived from organic substances and that they generate carbon dioxide when they 186 APPLIED WATER TECHNOLOGYBACTERIA WHICH CAUSE PROBLEMS grow. They utilize organic materials dissolved in the water such as organic acids ot alco- hols as a source of carbon. Apparently they cannot utilize petroleum hydrocarbons'® 252) + Nitrogen and Phosphorus. + Dissolved Iron. Sulfate reducing bacteria have an absolute requirement for relatively high concentrations of dissolved iron.5-!” + Sulfate, Suifite, Bisulfite or Thiosulfate Ions. Although the primary diagnostic character of sulfate reducing bacteria is that they grow with sulfate, reducing it to sulfide, they can also grow with sulfite and other reduced sulfur compounds. *-! A shortage of any of these materials can limit SRB growth. ‘The addition of oxygen scavengers or phosphorus containing compounds such as scale inhibitors could enhance growth if the concentrations of phosphorus or sulfate in the system are so low they are limiting growth. However, this is not likely, as most injection waters contain sufficient nutrients for an abundant growth of bacteria.°4) A source of nitrogen is rarely if ever a limiting factor on the growth of sulfate-reducing bacteria in waterflooding activities!°" Therefore, it is unlikely that nitrogen containing compounds such as corrosion inhibitors would stimulate bacterial growth, Effect of Dissolved Oxygen and Hydrogen Sulfide Sulfate reducing bacteria are found in oxygenated surface waters such as oceans. They are usu- ally present in very small numbers and do not multiply at an appreciable rate. However, if the water is deaerated prior to injection, they then have an oxygen-free environment which is ideal for growth. Sulfate reducing bacterial activity is a common occurrence in deaerated seawater injection systems. It is also possible to have growth in an aerated system with the assistance of aerobic bacteria. ‘The aerobes establish themselves on the walls of the system and consume oxygen as they grow. An anaerobic environment is created beneath the aerobic bacteria, thereby providing an ideal breeding ground for sulfate reducers. Hydrogen sulfide decreases the growth rate and can, at high concentration, slow the growth rate to zero. It is believed that the decreased growth rate is caused by a reduction of the dissolved iron ‘concentration due to reaction with hydrogen sulfide and subsequent precipitation as iron sulfide.*/ Reservoir Souring There have been a significant number of cases of “reservoir souring” due to sulfate reducing bacterial activity. 95) In each case, the reservoir fluids are initially sweet. Injection of a sulfate- bearing water is initiated. Sometime after breakthrough of injection water in the producing wells, HS appears. The concentration gradually rises, and has been reported as high as 3100 mg/L in the gas phase.(53) In most cases, this phenomenon is associated with the injection of seawater. However, it has not ‘occurred in every field where seawater has been injected. It has been reported in seawater floods in California, Alaska, and the North Sea. ‘The mechanism(s) by which reservoir souring occurs is not completely understood. However, reservoir contamination by thermophilic sulfate reducing bacteria in the injection water is considered to be the most probable explanation. CHAPTER 5 187WATER TREATMENT MICROBIOLOGY In shallow reservoirs the temperature is favorable for bacterial growth. However, even in deeper, hotter zones, the reservoir rock in the vicinity of the wellbore is cooled by the injection of large volumes of water, creating a favorable temperature regime for their growth. Once established in the reservoir, sulfate reducing bacteria produce hydrogen sulfide which then travels with the injection water to the producing wells. Even if an effective biocide is being used, a 100% kill is unlikely, and once bacterial growth is established in a rock matrix, it is almost impossible to control with chemicals. This is due to the difficulty of contacting every square inch of surface arca within the rock with adequate quantities of the chemical. Zobell calculated that sulfate reducing bacteria could travel at between 2 and 40 feet per year through oil bearing sands.°/5) Therefore, although reservoir souring could occur in this way, one ‘would not expect bacterial transport from the injection wells to the producing wells to be the primary cause of reservoir souring. Too many reservoirs have soured before bacteria traveling at these speeds could have reached the producing wells. Bacterial contamination of the producing wells is also a possibility. However, it would be antici- pated that this type of problem would be a more or less random event, and would occur in a few wells rather than throughout the entire field. The net result of reservoir souring is increased corrosion, and more important, the possibility of sulfide stress cracking and/or hydrogen blistering in the production facilities. This makes it necessary to evaluate the probability of souring, and to select downhole tubulars for the producing wells and surface production equipment accordingly. lron-Oxidizing Bacteria Iron-oxidizing bacteria are able to oxidize soluble ferrous ions in the water to form a sheath of ferric hydroxide around them as they grow. Examples of iron bacteria are Siderocapsa, Gallionella and Sphaerotilus. They are classified as aerobic bacteria, although they can apparently grow well with only trace amounts of oxygen (< 0.5 ppm).5%! Iron bacteria can cause both corrosion and plugging. Although they do not directly participate in the corrosion reaction, corrosion can result either from the activity of sulfate reducers under the hy- Groxide sheath or by the creation of an oxygen concentration cell. Large numbers of iron bacteria can precipitate a sufficient quantity of ferric hydroxide to cause severe plugging problems. Iron bacteria are usually found in fresh waters but they may occur in brine. Slime-Forming Bacteria Slime-forming bacteria are a general class of bacteria capable of producing dense masses of slime on solid surfaces. Examples are Pseudomonas, Flavobacterium, Enterobacter, Escherichia, and Bacil- Jus. They cause plugging and contribute to corrosion in the same ways as iron bacteria by shielding part of the surface. Slime can be expected in either brine or fresh water. or anaerobic systems. However, they are more common in low salinity aerobic systems. Other Types of Bacteria There are many other types of bacteria present in water injection systems which are not discussed here. The sulfate reducers, iron bacteria, and slime formers constitute the major sources of microbio- 188 APPLIED WATER TECHNOLOGYCULTURING, IDENTIFYING AND COUNTING BACTERIA logical problems, and some knowledge of their behavior will generally be sufficient to avoid serious microbiological problems. CULTURING, IDENTIFYING AND COUNTING BACTERIA Monitoring a system for bacterial activity entails sampling (which is covered in the following section), identification and counting of the bacteria. Culturing Bacteria Culturing bacteria is analogous to culturing flowers, potatoes, or green beans. The object is to make them grow. Bacterial culturing in artificial growth media is the standard technique for the esti- mation of bacterial populations. A water sample thought to contain bacteria is placed in a liquid known as a “culture medium” which is a solution consisting of water and food that will make the bacteria of interest grow and multiply. In addition, many media contain a growth indicator. For example, culture media for sulfate reducing bacteria contain iron. When SRB’s grow, they produce HS, which reacts with the iron to create an insoluble black precipitate, iron sulfide. Different types of bacteria require different culture media, and some bacteria refuse to grow at all in artificial media. However, most bacteria of interest can be cultivated in a particular medium. ‘The fact that media can be formulated in which only specific types of bacteria will grow makes it possible to identify the bacteria simply by noting the media in which growth occurred. Furthermore, by running the sample at several different dilutions, the number of each type of bacteria can be estimated. The composition of the water used in the formulation of culture media may be arbitrary (e.g. 10 000 ppm NaCl) or it may be tailored to more closely match a specific water. There are several possibilities for the water used to make up the media: * Use a sodium chloride solution which matches the chloride ion concentration of the water of interest. ‘+ Make up a synthetic brine approximating the composition of the water. + Use sterilized field water. This option is recommended when feasible. The water composi- tion, including micronutrients, is as close to that in the actual system as possible. There are many different versions of laboratory identification and counting techniques, but they will not be covered here.“ The general principles already outlined are followed with varying de- grees of sophistication. API RP 38 describes a recommended method for examination of waterflood injection waters which is used by many laboratories, Extinction Dilution Technique /6517) ‘This is a field technique, sometimes called serial dilution, which can be used to detect different kinds of bacteria. Detection of each class or type of bacteria requires a specific culture medium. Sampling ‘Sampling procedures are covered later in this chapter. CHAPTER 5 199WATER TREATMENT MICROBIOLOGY Procedure ‘The extinction dilution procedure is illustrated in Figure 5.3 and is performed as follows: 1, Line up a series of serum bottles, each containing 9 mL of sterile growth medium. A 4 to 6 bottle series is common. 2. Inject 1 mL of the water sample into the first bottle and shake well. ‘Throw the syringe away. 3. Withdraw 1 mL of solution from the first bottle with a new disposable sterile syringe and inject it into the second bottle. Shake the bottle. Throw the syringe away. 4. Withdraw 1 mL of solution from the second bottle with a new sterile syringe and inject it into the third bottle. Shake the bottle well and throw the syringe away. 5. Repeat this procedure until all bottles have been inoculated. 1 ml Water ‘Sample Bottle oe 2 3 4 5 6 Dilution man 4:10 4900 1:1000-—1:10,000-—+1:100,000 11,000,000 Figure 5.3 Extinction Dilution Technique ‘The point of this procedure is to dilute the sample to the point that the final 1 mL of solution that ‘you inject into the last bottle has no bacteria in it; hence, the name extinction dilution technique. You have diluted the sample to the point of extinction of any bacteria present. ‘The fact that the dilution is performed in a series of fixed dilution ratios allows you to estimate the bacterial population of the original 1 mL water sample. ‘The rules of the game state that you cannot transfer part of a bacterium from one serum bottle 10 another. This means that when you withdraw 1 mL from a serum bottle containing 10 mL of liquid, there must be at least 10 bacteria in a bottle, or an average of at least one bacteria per mL, before a transfer is allowed. For example, if there were only 8 bacteria in the bottle, the average population would be 0.8 bacterium per mL, and transfer cannot occur according to the rules. Similarly if there are 15 bacteria in a bottle, the average population is 1.5 bacteria per mL. Since only whole-number transfers are allowed, a one mL withdrawal of liquid will net you 1 bacterium for transfer to the next bottle, Additional examples are given in Table 5.2. 190 ‘APPLIED WATER TECHNOLOGYCULTURING, IDENTIFYING AND COUNTING BACTERIA TABLE 5.2 Serial Dilution Examples Series No. => ‘Original 1 mL Sample First Bottle Second Bottle ‘Third Bottle Fourth Bottle Types of Cultures In practice, two types of culture media are normally utilized and, therefore, two separate series of dilutions are run: one for sulfate reducing bacteria (SRB) and the other for general bacteria. Sulfate Reducing Bacteria ‘The SRB series utilizes a growth medium which is specific to sulfate reducing bacteria. ‘The bacteria counts obtained using this medium include Desulfovibrio. Desulfotomaculum may also be detected. However, if system conditions appear to be favorable to the growth of Desulfotomaculum, it may be desirable to inoculate an additional media designed especially for their detection as an addi- tional precaution. Commonly used media include those described in API RP 38, as well as a number of other media proposed by Postgate and others.°9510.5.16) Once the bottles have been inoculated, they are set aside and allowed to “incubate” for a fixed time period. An incubation period of 28 days is recommended. However, shorter incubation periods may be used when it can be demonstrated that all growth occurs in less than 28 days. ‘A constant temperature is desirable during the incubation period and growth rates are temperature sensitive. The cultures should be incubated at a temperature within 9°F (5°C] of the recorded tempera- ture of the water at the time of sampling if possible. If not, keep the temperature between 77°F and 100°F [25-38°C} Growth is indicated when the bottle turns black. The SRB media contain soluble ferrous iron or a sterile iron nail. When SRB's grow, they produce H2S which reacts with the iron to form insoluble, black iron sulfide. General Bacteria Count The “general” series employs a different growth medium which promotes the growth of “general” heterotrophic bacteria as well as facultative bacteria. An incubation period of 5 days is common, The “general” count includes the general aerobic bacteria, primarily the slime formers, and can also include anaerobic bacteria as well as facultative bacteria. It does not include iron bacteria, which are difficult to culture in an artificial medium, They are usually detected by microscopic means. Three types of media are in common use for the detection of general heterotrophic bacteria: 1, Standard Bacteriological Nutrient Broth Growth is indicated by the development of turbidity. ‘The turbidity is caused by the bacte- rial cells themselves, and is usually evident when the cell count exceeds 1 000 000 per mL. CHAPTER 5 191WATER TREATMENT MICROBIOLOGY 2. Phenol Red Dextrose Broth ‘This medium contains sugar and phenol red, which is and acid-base indicator which turns from red to yellow when the pH of the culture medium drops below 6.6. When the sugar is fermented or oxidized by bacteria, various organic acids are produced. The resulting acid- ity causes the pH to drop, which causes the color of the medium to change from red to yellow59) Growth is indicated by a color change from red to yellow, the development of turbidity, or both. Turbidity indicates general bacterial growth, and a color change indicates the pres- ence of acid-producing bacteri 3. Thioglycolate Medium. This medium is used to detect anaerobic heterotrophic bacteria. Growth is indicated by the development of turbidity. Once the bottles have been inoculated, they are set aside and allowed to “incubate” for 5 days at the same temperature as the SRB bottles. Final Count As a rule of thumb, growth will usually occur within 3 days for general bacteria, but two weeks to a month may be required for sulfate reducer growth. The bottles should be examined daily for signs of growth and the results recorded. ‘The final reading for general bacteria should be taken after 5 days, and the final count on the SRB’s should be taken after 28 days unless experience indicates that shorter periods are acceptable. Interpretation of Results ‘The number of bacteria present in the original 1 mL. of water injected into the first bottle can be estimated using Table 5. TABLE 5.3 Bacterial Growth Interpretation No. of Bacteria Number of Bacteria Bottle Showing | Dilution Indicated Reported Growth Factor (Bacteria/mL) (Bacteria/mL) 10099 100 to 999 1000 to 9999 10,000 to 99,999 100,000 to 999,999 For example, if bottles 1, 2, and 3 show growth, but bottles 4 through 6 remain clear, then the water contains 100-999 bacteria per milliliter, and a count of 1000 per mL is reported. If only one bottle shows growth, and the rest remain clear, then the water contains 1-9 bacteria per milliliter, and a count of 10 bacteria per mL is reported. 192 APPLIED WATER TECHNOLOGYCULTURING, IDENTIFYING AND COUNTING BACTERIA Common Growth Interpretation Problems ‘There are a number of common occurrences which can interfere with interpretation of the results obtained using the serial dilution technique. Some of the more common ones are 1. All Bottles Show Growth If all bottles show growth, then it is not possible to estimate the population. For example, if all 6 bottes in a series are positive, you would have to report the population as “equal to or greater than 1 000 000 per mL.” 2. Immediate Positives A point of caution: If the first bottle of SRB media turns black within 2 hours after the one mL water sample has been injected into the bottle, itis not the result of bacterial growth in the bottle. They grow pretty fast, but not that fast. The black color is iron sulfide resulting from high levels of HS in the water sample reacting with dissolved iron in the bacterial culture medium. If blackening of the first bottle occurs within 2 hours, one of the following courses of action is common: a. Proceed with the serial dilution. Bottles 2 through 6 will usually remain clear and can be used to detect growth. Set them aside for observation as usual. b. Collect at least 50 mL of water in a sterile bottle and add an Alkaseltzer tablet. ‘The Alkaseltzer will strip most of the H,S from the sample. When the tablet has stopped fizzing, remove one mL of water with a sterile syringe and perform the serial dilution as usual. Similarly, if the first bottle of general media turns turbid (or yellow in the case of the Phenol red indicator) within one hour of inoculation, this is not due to bacterial growth, but is the result of natural turbidity in the water or an excessively low pH which causes the color change. 3. Bottle Skipping ‘Sometimes one of the bottles in the middle of a series remains clear while bottles on either side of it show growth. If this occurs, the skip should be noted, and the number of bacteria Corresponding to the highest numbered bottle in the series which showed growth should be reported. For example, assume that growth is indicated in bottles 1, 2, and 4, while bottles 3, 5 and 6 remain clear after being incubated for the required time period. There are a number of Possible explanations for this event, including accidental contamination of bottle 4. How- ever, you seldom know why it occurred, and have to simply make the best of a question- able situation. In this example, if you are reasonably confident that the bottles are num. bered properly, you should report 10 000 per mL., and note which bottle was skipped. Significance of Results This method permits the estimation of the number of planktonic bacteria floating in the water, Since most of the bacteria in a system will be sessile bacteria attached to solid surfaces, it is a very oor way fo assess the number of bacteria actualy living in the system. Nevertheless, itis a simple ‘est to perform, and has proven to be a very useful indicator of the level of bacterial activity. Often the absolute number of bacteria in a given water sample is less important than changes in Population. If the number of bacteria increases as you move across the system from the water source CHAPTER 5 193WATER TREATMENT MICROBIOLOGY to the injection wells, active bacterial growth within the system is almost certain. Similarly, increased bacterial counts with time at any given point in the system is a good indicator of active growth. 1. General Count Bacterial counts of less than 10 000 organisms per mL are not considered significant in untreated waters. As a rule of thumb, a count of 100 000 per mL indicates a strong possi- bility of plugging and a need for biocide treatment. Start examining injectivity for any decrease and look for any evidence of increased injection pressures or filter plugging. Some slime forming bacteria will only grow on a solid surface and will not be detected by this technique. 2. Sulfate Reducing Bacteria The presence of a single sulfate reducing bacteria is considered to represent a potential Problem.*/° Remember that SRB are sessile bacteria and that this method only detects planktonic bacteria. Hence, the odds are very high that there are many bacteria clinging to the wall of the system for every one which is floating in the water. Conversely, low bacterial counts do not necessarily mean that there is no bacterial problem. ‘They may be so securely established under deposits that very few bacteria are dislodged from the sessile community, resulting in low counts in the water. Other evidence of the severity of the problem are H2S levels and “black water” due to iron sulfide in the water. Once sulfate reducing bacteria are detected, they should be monitored very closely, and treatment should be instituted if bacterial counts and or H2S levels increase. Rapid Bacterial Detection and Counting Methods Culturing techniques rely upon growth of bacteria for their detection. Bacteria may be present in 4 given sample, but it they do not grow in the selected artificial media, they go undetected. In addition, growth takes time, and the use of culturing as a bacterial detection and counting technique is not quick. ‘There are a number of methods which allow one to rapidly gather information about microbial Populations in oilfield systems. They directly detect bacteria or enzymes which are associated with Particular types of bacteria, Some of the more common rapid assessment techniques are summarized in the following para- graphs. For a more complete summary, see NACE TM0194, Field Monitoring of Bacterial Growth in Oilfield Systems, 19) Microscopic Examination Microscopic techniques are extremely useful to trained microbiologists. For example, iron and slime bacteria can be identified quickly with a phase-contrast microscope. However, it is often difficult to distinguish bacteria from suspended solids. Fluorescence Microscopy? ‘One way to make bacteria stand out from their surroundings is to treat them with a fluorescent dye, or “stain.” which will make them fluoresce when exposed to UV light of the proper wavelength, ‘They can be easily distinguished from non-living particles of similar size and shape by looking at them 194 ‘APPLIED WATER TECHNOLOGYCULTURING, IDENTIFYING AND COUNTING BACTERIA through a microscope under UV light. This technique is called fluorescence microscopy allows direct counting of bacteria within a very short time. Celis are usually concentrated by filtration through a polycarbonate membrane filter, which will retain the cells on a flat surface rather than in the mesh surface of cellulose acetate membranes (the ‘ones usually used to collect suspended solids for TSS determinations). Cell Stains Total cell counts can be obtained using stains which combine with cell material present in both living and dead bacteria. Common stains used for this purpose include acridine orange, and DAPI (4, 6 diamidio-2-phenylindole). The total number of living cells can be determined using FDA (fluorescein diacetate) stain. Antibody Stain The fluorescent dye is bound to anitbodies which will seek out sulfate-reducing bacteria cells. Consequently, only those bacteria recognized by the antibodies will fluoresce under the microscope. The major advantage of the method is speed, as results are obtained within two hours. ‘The major limitation of the method is that the antibodies are specific to the type of SRB used in their manufacture. However, a large number of SRB antibodies can be combined to make the test fairly general. The method detects both living and dead cells. Unfortunately, in addition to requiring trained personnel, microscopic techniques are not practical for routine use in the field. ATP Photometry‘5205.21) ATP analysis provides a means for rapidly determining the quantity of living organisms present in an injection water. It is not specific to bacteria, but since the majority of the organisms present in many oilfield waters are bacteria, it can be a very useful technique. It is particularly helpful in the evaluation of biocide treatment programs. The basis for the method is as follows: 1, ATP (adenosine triphosphate) is present in the cells of all living organisms. 2. ATP is destroyed within 20 seconds of cell death through release of ATP-destroying en- zymes, 3. The amount of ATP per cell is a linear function of cell volume, e.g., twice the cell volume results in twice as much ATP. ATP analysis is a method of measuring the amount of ATP present in a given sample. This enables a quantitative measurement of the amount of living organisms in the sample. The method is based on the fact that ATP will react with a mixture of luciferin and luciferase in the presence of ‘oxygen to produce light (bioluminescence). The amount of light emitted from the reaction is measured with a photometer and the number of organisms determined from calibration data. ‘A summary of the procedure is as follows: 1. The water sample is filtered through a 0.45 um pore size membrane filter. Bacteria and other organisms are thus removed from the water and retained on the filter surface. This concentrates the organisms, thus increasing measurement sensitivity. Also, it is required when the water contains NaCl (as in the case of most oilfield waters) because NaCI inter- CHAPTER 5 195 Ieee NeWATER TREATMENT MICROBIOLOGY Jeres with the ATP-luciferin/luciferase reaction. It should be noted that sulfide also inter- feres with the reaction. 2. The ATP must then be quickly extracted from the cells prior to its destruction by enzymes soon after cell death. Several methods are available, but a common method is to drop the membrane filter into a boiling solution of 0.05 molar tris hydroxy nitromethane buffer solu- tion. The heat destroys the ATP-destroying enzymes, preventing their interference. It also kills the bacteria and causes them to burst and release their cellular contents, including ATP, into the buffer solution. 3. Luciferin/luciferase is then added to the buffer solution and the emitted light measured with photometer. The number of bacteria present is then obtained from calibration data. ATP analysis has one overwhelmingly positive feature: it provides a very quick indication of the total planktonic bacterial activity in a system. You don't have to wait through long incubation periods to get results as in the case of conventional culturing techniques. However, it does not identify the type of bacteria present. As previously stated, the presence of a few sulfate reducing bacteria can cause trouble, whereas a general count of 10 000 bacteria per mL can usually be tolerated. Therefore, if ATP analysis were to indicate a total count of 5000 bacteria per mL in a particular sample, this would be acceptable provid- ing none were sulfate reducers. However, the situation would be quite different if 2000 of those bacte- ria were sulfate reducing bacteria! Because of the need to specifically identify sulfate reducers, it is ‘common to use conventional culturing techniques to determine their population in conjunction with ATP analysis as a rapid means of measuring total bacterial activity. Enzyme Determinations®2 Hydrogenase Measurement The test is used to detect the presence of the hydrogenase enzyme. This enzyme is produced by bacteria which are able to use hydrogen as an energy source. Hydrogen is generated at the cathode in many corrosion reactions. The utilization of this hydro- gen by bacteria such as SRB depolarizes the cathode and accelerates the corrosion reaction. Therefore, the presence of hydrogenase indicates the presence of bacteria which can accelerate the corrosion reac- tion by cathodic depolarization. Hydrogenase testing is usually performed using sessile samples. The sample is exposed to an enzyme-extracting solution, and the degree of hydrogen oxidation in an oxygen-free atmosphere is indicated by a color reaction with a dye. A response can be expected in 30 minutes to 4 hours. A 12-hour exposure is generally used for comparison purposes. The test can be carried out in the field or laboratory. APS-Reductase Measurement Co Aviy Crvlece ) This method is based on the functional definition of sulfate-reducing bacteria, which includes any bacteria capable of anaerobically reducing sulfate to sulfide. A unique requirement for this process is the presence of an enzyme, APS-reductase. Since this internal enzyme is common to all SRB’s, meas- urement of its concentration in a sample permits the estimation of the total number of SRB present. The test requires about 20 minutes and can be carried out in the field or laboratory using commer- cially available test kits. The lower limit of detection is typically about 1000 cells/mL, although sensi- tivity can be increased by concentrating the sample. 196 ‘APPLIED WATER TECHNOLOGYCULTURING, IDENTIFYING AND COUNTING BACTERIA Water Sampling for Bacterial Analysis ‘Samples are taken for the determination of both the planktonic and sessile populations. Sampling the water to determine the number of planktonic bacteria present is by far the most common practice. Sampling Points ‘Sample the following points in an injection system: 1. Water Source The wellhead, pond, stream, lake, ocean, or produced water. 2. Vessels, Tanks and Filters Sample both the inlet and outlet, as well as the backwash discharge on filters. 3. Injection Wells ‘Sample at several injection wellheads located at various distances from the injection plant. Sampling for Planktonic Bacterial Analysis ‘The sampling technique is basically the same as described in an earlier chapter. However, there are some additional requirements: 1, If a sample container is used, it should be initially sterile, or free of bacteria, 2. The use of a plastic or rubber sampling hose which has been previously used is discour- aged, as it is a possible source of contamination. You don’t want to contaminate the sam- ple and find a bacterial problem which doesn’t really exist in the system. 3. Sample in such a way that oxygen is excluded when culturing sulfate reducing bacteria, 4, An alternate procedure for collecting a water sample is to insert the tip of the syringe directly into the stream of water flowing from the sample valve, then immediately inocu- late the first bottle of media. This method is ideal for on-site analysis. Timing The water sample should be injected into the growth media or subjected to other analysis imme- diately after sampling. Changes in the bacterial population occur rapidly and old samples can give a false picture of the population in the water system. Sulfide Concentration Sniff the sample for HzS, then measure the total sulfide concentration. If the water entering the system is sweet, but becomes sour somewhere in the system, this is a very good sign that sulfate reducers are the source of the HS. Also note the color, clarity and amount of suspended solids. Severe SRB problems often cause the water to turn black due to the formation of iron sulfide. Sampling Sessile Bacteria Robbins Device The Robbins Device was developed at the University of Calgary by Robbins and Costerton as a means of estimating the population of sessile bacteria in a water system.*7) The basic concept of the CHAPTER 5 197WATER TREATMENT MICROBIOLOGY device is to insert small, sterile, mild steel coupons or “studs” through the wall of a piece of 1/2-inch diameter pipe such that the face of the studs are flush with the internal pipe wall. In effect, they become a part of the pipe wall and furnish a site for the growth of sessile bacteria. A sidestream of water from the system is flowed through the device, and after a given exposure period, the studs are removed, and the number of bacteria on each stud is evaluated by one of several methods. Commercial biological probes based on the principles of the Robbins Device are marketed by several vendors. These probes consist of a holder containing five to eight, small, mild steel studs or coupons. The probe is available in two versions: + High Pressure Access Systems This type of probe contains either five or six studs and is designed for connection to a high Pressure access fitting.(Figure 5.4) This enables the probe to be installed and retrieved without depressurizing the system by using one of the high pressure access systems dis- cussed in Chapter 4. Left Hand ‘Screw Thread Bacteria 6 Each oo 6320 (Petri) Setscrews Bio Bullets (Mild Stoo! ‘or Brass) Figure 5.4 Cortest Biological Sample Probe Assembly 198 APPLIED WATER TECHNOLOGYCHEMICAL CONTROL OF MICROORGANISMS + Low Pressure Systems ‘The probe contains eight studs and is attached to a two-inch threaded plug. The portion of the system where the probe is located must be isolated by valves and depressured for probe insertion and removal. The general procedure for using a biological probe is as follows: 1, Sterilize the studs by soaking in ethyl alcohol, and insert into the plastic holder with sterile tweezers. (Don’t touch the studs!) 2. Install the probe in the system, preferably on the bottom of a line at the 5 or 7 o'clock position. 3. After the desired exposure period (typically 2-6 weeks) remove the probe from the system and quickly extract the desired number of studs. Be careful not to contaminate the studs. Don’t touch them! 4. Determine the number of bacteria attached to the surface of each stud. Three methods are in common use: a. Each stud is placed in a plastic test tube containing a special solution and glass beads. The bacteria are then dislodged from the stud by placing the test tube on a vortex mixer or by vigorous hand shaking for two minutes. A portion of the solu- tion is then removed and the bacterial content evaluated by the ATP or serial dilu- tion methods. b. The second technique involves scraping the biofilm from the stud surface with a sterile scalpel blade. The scrapings, the stud, and the blade are placed in a special solution, which is then put in an ultrasonic cleaning device. A sample of the solu- tion is then evaluated by serial dilution or ATP methods.2?) ¢. The bacterial population on a stud surface can also be determined by direct exami- nation using fluorescence microscopy. Use of Solids Scrapings It is often desirable to scrape scale or solids from the pipe wall or from a pit and examine it for the presence of bacteria. Culturing can be carried out using media in bottles equipped with screw-on caps. A consulting laboratory or chemical company should be contacted for help in the event you decide to culture scale samples, Direct examination of solids can be carried out using some of the rapid bacterial and detection methods previously discussed. CHEMICAL CONTROL OF MICROORGANISMS Types of Chemicals ‘Chemicals used for bacterial control can be broadly classed in several ways. Function 1. Bactericide: A chemical which kills bacteria, 2. Bacteriostat: A chemical which inhibits or retards growth of bacteria, CHAPTER 5 199WATER TREATMENT MICROBIOLOGY 3. Biocide: A chemical which kills other forms of life in addition to bacteria, A more univer- sal killer. 4. Biostat: A chemical which retards or inhibits the growth of other life forms in addition to bacteria. Chemical Composition Inorganic Chemicals Chlorine Chlorine is the most widely used inorganic biocide for water injection systems. Its use is confined to surface waters such as seawater and fresh water from supply wells. Its use is covered in a sub- sequent section of this chapter. Chlorine Dioxide ‘Chlorine dioxide (ClO2) is used as a bactericide in industrial waters.) It is typically generated on-site by mixing either sodium chlorate (NaC1O;) or sodium chlorite (NaCIOz) with hydrochloric acid to produce ClO and has successfully been used in oilfield systems. It can also be created by reacting sodium chlorite with chlorine. Itis a powerful oxidizer and reacts with sulfides in water to form sulfates. ‘The primary concems with the use of chlorine dioxide are its corrosivity to steel and the possibil- ity of creating a combustible mixture when it comes in contact with crude oil. 28) These concerns have limited its use as a biocide in production operations. Organic Chemicals Aldehydes, quaternary ammonium compounds and amines are common examples of organic bac- tericides. Most bactericides sold by chemical companies, other than chlorine, are organic. Aldehydes Glutaraldehydes are very widely used for bacterial control in the oilfield, Acrolein is also used, but much more rarely. (See Chapter 6 for more information on Acrolein) Formaldehyde is rarely used because of concerns about its role as a carcinogen, Aldehydes are not very good at penetrating biofilms and are often blended with other chemicals, such as quaternary ammonium compounds to increase their penetration efficiency. “Quats” and Amines Quaternary ammonium compounds and quaternary phosphonium compounds are commonly re- ferred to as “Quats.” They are very surface active and effectively act as detergents. Their use is limited to waters with fairly low salinity such as seawater and fresher. Amines are quite similar to quats. They are very surface active. When applied continuously they behave as corrosion inhibitors. 200 ‘APPLIED WATER TECHNOLOGYCHEMICAL CONTROL OF MICROORGANISMS Chlorination Chlorination is very widely used because it is one of the least expensive and most effective bactericides. When added to water, chlorine hydrolyzes to form hypochlorous and hydrochloric acids:/515) Ch + H,0—> H* + Cl” + HOI ‘The hypochlorous acid then ionizes to form hydrogen ions and hypochlorite ions: HOCI— H* +01- Effect of pH The degree of ionization is dependent on pH. Below a pH of 5, molecular chlorine is present. Above this pH, HOCI and OCI” are the species present. The relative percentages are shown as a func- tion of pH in Figure 5.5. = ° *” i— cl ® % = » » © « ge Th »§ 100 56 7 8 6 0 W A Figure 5.5 Effect of pH on the Concentration of Hypochlorous Acid‘-24) The effectiveness of chlorine depends on which of these species are present and is therefore a function of pH. For instance, the killing power of HOCI is much greater than that of OCI’. Hence, the higher the pH, the less effective a given quantity of chlorine. It is only 1 to 10% active at a pH of 8-9. However, in waters containing bromide ions (such as seawater), chlorine oxidizes the bromide ions to form HOBr, which is still an effective biocide at pH 8-9. The amount of chlorine required to effect a kill is a function of time and temperature as well as PH. The rate of kill increases with temperature and decreases with pH. CHAPTER 5 201WATER TREATMENT MICROBIOLOGY Some typical data showing the relationship between pH and the chlorine residual required for a 10-minute kill is shown in Table 5.4, TABLE 5.4 Required Chlorine Residual ‘The chlorine residual or “free” chlorine is the total amount of chlorine present as Cl2, HOCI, and ocr. Continuous injection of chlorine should be sufficient to maintain a chlorine residual of 0.2-0.5 ppm at the injection wellhead. Chlorine Demand Since chlorine is a very strong oxidizing agent, it reacts with many materials. Once it reacts it is no longer available to kill bacteria. Hence, it is necessary to establish how much chlorine will be used up by reaction with other materials in order to establish the total amount of chlorine needed. The amount used up by the system is called the chlorine demand. The amount of chlorine which must be injected to kill bacteria is: Chlorine Required for Bacterial Control = Total Chlorine Injected ~ Chlorine Demand Some of the things which react with chlorine are: 1. Ferrous Iron One ppm chlorine will oxidize 1.6 ppm ferrous iron (Fe**) to ferric iron (Fe***), 2. Hydrogen Sulfide Chlorine will react with HS as previously mentioned in Chapter 4, 3. Organic Compounds Chlorine will react with some corrosion inhibitors and scale control chemicals. 4, Sulfite fon Chlorine will react with the sulfite oxygen scavengers. This is covered in Chapter 6. This means that the use of chlorine is limited if there is a lot of HjS or ferrous iron to cope with in the system. Chlorine is not normally used in systems which have long, bare steel injection lines. If the line is several miles long, it will be necessary to inject large quantities of chlorine in order to obtain a residual of 0.2-0.5 ppm at the end of the line due to the loss of chlorine by reaction with the steel. This can result in increased corrosion rates as well as increased treatment costs. Compatibility The compatibility with any other chemicals to be used in the system must also be checked. In the case of oxygen scavengers, either of two approaches may be followed: 202 ‘APPLIED WATER TECHNOLOGYCHEMICAL CONTROL OF MICROORGANISMS 1. The chlorine may be added a sufficient distance downstream to permit the oxygen scaveng- ing reaction to go to completion prior to reaching the chlorine injection point. 2. If chlorine is to be injected upstream of oxygen scavenger injection, additional scavenger ‘must be injected so that there will be enough present to react with both the chlorine and the oxygen. Since this results in removal of the chlorine, it is necessary to inject chlorine or some other biocide downstream of oxygen scavenging if a biocide is required in the down- stream portion of the system. Forms of Chlorine “Chlorine” is available in several forms. However, from the standpoint of bacterial control, it really doesn’t matter which one you pick, since all will form the same relative amounts of Cls, HOCI and OCI" once added to the water, depending on the pH. Liquid Chlorine Liquid chlorine is supplied in steel cylinders ranging in size from 105 Ibs to 1 ton. The chlorine vaporizes as it leaves the cylinder and is added to the water as a gas with a chlorinator. A typical vacuum operated, solution feed chlorinator is shown in Figure 5.6. ‘Chlorine Trap Figure 5.6 Flow Diagram of a Gas Chlorinator (Courtesy Capital Controls) Water flowing through the ejector at high velocity creates a vacuum which opens a spring-op- posed diaphragm check valve in the ejector body. When the check valve opens, a vacuum is transmit- ted to the vacuum regulator, causing the diaphragm to open the chlorine inlet valve. Chlorine gas passes through the flow meter and rate control valve to the ejector, where it mixes and dissolves in the water. Caution: Chlorine is very poisonous and must be handled with care. Hypochlorite Generators Hypochlorite (OCI) can be produced in chloride-containing waters by electrolysis. ‘The brine is passed through a special electrolytic cell called a hypochlorite generator, which contains two electrodes, an anode and a cathode. An impressed DC electrical current flows from the anode to the cathode, CHAPTER 5 beWATER TREATMENT MICROBIOLOGY Figure 5.7 Chemical Reactions in a Hypochlorite Generating Cell through the brine, Hydrogen ions are converted to hydrogen gas at the cathode and chloride ions are converted to OCI” ions at the anode as shown in Figure 5.7. The OCI solution/H gas mixture leaving the generator is usually passed through a gas-water separator to separate the hydrogen gas from the hypochlorite solution prior to its addition to the water system, The hydrogen is separated and vented for safety reasons, since the lower limit of flammability for hydrogen in air is 4.1%.°25) Hypochlorite generators are commonly used in seawater systems to eliminate the need for trans- porting and storing liquid chlorine. Hypochlorite Solutions Liquid hypochlorite solutions such as sodium hypochlorite or calcium hypochlorite are available and can be injected with a conventional chemical pump. They are seldom used because of their high cost relative to the other sources of chlorine. 204 APPLIED WATER TECHNOLOGYCHEMICAL CONTROL OF MICROORGANISMS Chemical Selection and Evaluation Chemical selection for bacterial control is analogous to the selection of a chemical for the control of corrosion or scale. First the problem (the type of bacterial problem) must be identified, then chemicals chosen which will deal with it effectively. On-site time-kill testing of different bactericides is recommended as an initial step in chemical selection. ‘Several items must be considered in selection: Kill or Control? The decision whether to use a bactericide or a bacteriostat is dictated by the type of bacteria involved. Sulfate reducers require a bactericide, since a total kill is desired. Since a reasonable number of slime formers can be tolerated without serious problems, a bacteriostat is often used for their control, “Resistant Strains” We often hear about the development of “resistant strains,” which become resistant to a particular biocide. This seems to infer genetic mutation which results in the evolution of a bacteria which is extremely difficult to kill. In practice, this is not thought to occur. ‘The apparent creation of a “resistant strain” is evidenced by the fact that a given biocide gradu- ally ceases to be effective. The most common explanation is often referred to as “selective adaptation.” This simply means that certain members of the bacterial population are easily killed by a given biocide, while other types of bacteria are not. Over a period of time the bacteria which are resistant to the biocide gradually become dominant in the bacterial community, and the effectiveness of the chemical ‘treatment declines. This phenomena does not always occur. However, because it is possible, it is usually a good idea to select a minimum of two biocides. Then, if a given chemical starts to show decreased effectiveness, alternating treatment with a second, generically-different chemical will usually solve the problem. Bacteria do not develop an immunity to chlorine.*7) However, at normal treating concentrations, chlorine has limited power to penetrate deposits and organic masses, and may not be effective in dirty systems. Effectiveness can often be improved by the injection of an appropriate surfactant along with the chlorine. Compatibility with Water and Other Chemicals Make sure that the chemical is compatible with your water. Some will precipitate or “salt out” in higher brines. Also, compatibility with oxygen scavengers and corrosion or scale inhibitors must be established. Time-Kill Tests It takes time for any bactericide to kill the bacteria present. Therefore, it is wise to determine what length of time is necessary for a particular chemical to do its job at several chemical concentra- tions. A specific method for sulfate reducers is spelled out in NACE Standard TM0194./9) Ics important to know the time-kill relationship, especially where batch treatment is utilized. CHAPTER 5. 205WATER TREATMENT MICROBIOLOGY Treatment Method The treatment method must also be considered. If the bactericide is to be continuously injected an initially high concentration is usually required to bring the bacterial population under control; then lower chemical concentrations can usually be effective once the bacterial population has been brought under control. If batch treatment is to be used, a high concentration slug is periodically pumped through the system, and the object is a total kill. Here the concentration, slug size and contact time can be initially sized by time-Kill tests, then adjusted by actual experience. In most cases, organic biocides are injected on a batch basis because of improved cost-effective- ness over continuous injection. Also, selective adaptation is more likely when bacteria are exposed to continuous, low-level concentrations than when subjected to periodic high concentration shocks. Since immunity is not a problem with chlorine, it is usually injected on a continuous basis. Con- centrations are kept as low as possible in order to minimize any increased corrosivity, as well as for economic reasons. Chemical Application Clean the System The first rule of successful use of bactericides is to clean the system. A thorough cleaning job is necessary and has only one purpose: to remove all obstacles between the bactericide and the bacteria. ‘No chemical can kill bacteria if it cannot contact them! (4 1, Clean out the injection lines and tubing as outlined in Chapter 3, using solvents, acid and line scrapers if necessary. 2. Open all tanks, vessels and filters, and manually clean out all accumulated sludge and scale. 3. Backflow all injection wells. If the wellbores are plugged with bacterial deposits or slimes, a strong oxidizing agent such as sodium hypochlorite is normally used to attack them. In order to achieve maximum effectiveness, the sodium hypochlorite is usually followed by hydrochloric acid. The sodium hypochlorite solution kills bacteria and completely dissolves the polysaccharide biofilm.° The acid dissolves any acid-soluble inorganic material and neutralizes the basic sodium hypochlorite solution. This prevents the precipitation of compounds from the formation water which are less soluble at basic pH values, such as calcium carbonate. Sodium hypochlorite solutions are quite corrosive and must be inhibited prior to use. In tests conducted by Dowell, exposure of N-80 tubing to an uninhibited 5% sodium hypochlorite solution at 175°F [80°C]and atmospheric pressure resulted in a corrosion rate of 664 mpy.°26) However, inhibi- tors are available which will reduce the corrosion rate to acceptable levels. In systems where large diameter lines run for several miles it is highly advisable to install perma- ent pigging facilities and pig the lines on a regular basis. This act alone will help to decrease bacterial problems by removing deposits and at least part of the biofilm. If the line scraper is followed by a batch of biocide, the effectiveness of the biocide is greatly increased due to the fact that the chemical can readily contact the remaining biofilm. This normally results in a substantial reduction in chemical costs. 206 APPLIED WATER TECHNOLOGYCHEMICAL CONTROL OF MICROORGANISMS Of course, bactericides contain a certain amount of detergency. If the system is not too dirty, there may be enough detergency to clean up the system. However, this is seldom true and a systematic cleaning operation prior to initiation of treatment is strongly recommended. Also, remember that if you rely strictly on the detergency of the chemical, try to prevent the solids which may be washed loose from being injected into the formation. This can be accomplished by installation of wellhead cartridge filters. Eliminate Stagnant or Low Velocity Areas Examine the possibility of eliminating stagnant or low velocity areas by changes in system de- sign. Since bacteria grow more rapidly in slowly moving or stagnant waters, system modification to minimize the number of low velocity areas will make bacterial control an easier task. Sterilize the System Once the system has been cleaned, it should be “sterilized.” This means that the bacteria must now be killed. “A high concentration slug of bactericide, with sufficient residence time to kill the bacteria, is used. Don’t forget that this means the lines, tanks, filters and injection wells, However, it also means the water source. This frequently involves producing wells if produced water is used. Institute Treatment At this point “normal” treatment, either continuous or batch, can be instituted. ‘The chemical should be added as near to the water source as possible. It should be added in an area where good mixing is possible. Injection into the water intake line (surface waters), down the annulus of the supply well, or just downstream of the supply pump are common locations. Evaluation of Chemical Treatment Programs As in any other situation, the ultimate question is whether or not the chemical(s) selected actually works in your system. This can only be determined by closely monitoring the system. A good monitoring program will include some combination of the following procedures, carried ut across the system on a regular schedule: 1. Inspection of the chemical injection points to ensure that the chemical is the one you se- lected, and that it is being injected into the system at the proper concentration. 2. Estimation of specific families of planktonic bacterial population in water samples using serial dilution or other culturing methods. 3. Estimation of total planktonic population in the water using ATP or Fluorescence Micros- copy. 4. Estimation of the sessile bacterial population using a Robbins Device for collection, ATP or Fluorescence Microscopy for estimation of the total population, and culturing to identify the types of bacteria. 5. Measurement of HyS concentrations. 6. Visual examination of the water and suspended solids measurements, 7. Internal inspection of the system for deposits and corrosion. CHAPTER 5 207WATER TREATMENT MICROBIOLOGY It should be emphasized that you may be able to kill the planktonic bacteria, yet fail to kill the sessile bacteria which are actually doing the damage to the system. When this happens, it is typically due to the presence of scale, corrosion products, or deposits shielding the bacteria from the chemical, and/or the inability of the chemical to penetrate the biofilm. The success of a treating program should be judged not only by a decrease in the bacterial popu- lation, but also by a decrease in leak frequency, plugging, and suspended solids, coupled with increased water clarity. ULTRAVIOLET RADIATION It is well known that ultraviolet light will kill bacteria. It halts growth by producing chemical alterations in the cell which prevents it from dividing. However, commercial ultraviolet units capable of treating large volumes of water were not available until the mid 1980's. ‘When water containing bacteria flows through an ultraviolet light unit, the probability of killing a given bacterium is dependent on the length of time the cell is exposed to the ultraviolet light and the intensity of the light reaching the cell. The intensity reaching the cell will depend upon: 1. The output of the lamp(s), which is usually measured in milliwatts/em?, 2. The distance from the UV light source to the bacterium, 3. The nature and amount of suspended matter in the water. Undissolved particles will scatter some of the UV light, thereby reducing the amount penetrating the water. 4. Any material dissolved in the water which can absorb ultraviolet energy will reduce the intensity. Films can build up on the quartz windows between the water and the UV lamp and reduce light intensity, Quartz windows are used because quartz is transparent to the ultraviolet wave lengths. A typical dose-response curve is given in Figure 5.8. The dosage is expressed as milliwatt-sec- ‘onds/cm®, which is the product of the output of the lamp (milliwatts/ cm?) multiplied by the exposure time in seconds. ‘The relative Kill is expressed both as the surviving fraction, which is the number of surviving cells divided by the original number of cells, and as the percent survivors. Results given for one field test demonstrated 99.94% kill of sulfate reducing bacteria. Further- more, it was stated that the cost of using ultraviolet light was significantly less expensive than using a biocide treatment in the case reported.(57”) Ultraviolet light units are intended to prevent entry of planktonic bacteria into a particular portion of a system by killing them as they are carried through the unit in the water. Any bacteria that escape death in the UV unit will be free to multiply in the system, since there is no residual effect from the radiation. Therefore, it is likely that some form of chemical treatment will still be required in most systems where UV radiation is utilized. It should be noted that the use of ultraviolet light is limited to clean waters containing very small amounts of suspended solids and little if any dissolved or suspended oil, due to the ability of these materials to reduce the ultraviolet light intensity transmitted through water. 208 APPLIED WATER TECHNOLOGYULTRAVIOLET RADIATION LUV Dosage (miliwatt-sec Jom") Figure 5.8 Typical Ultraviolet Light Dose-Response Curve for a Bacterium 27) CHAPTER 5Sa 52 53 34 55 56 57 58 59 5.10 sal 52 5.13 sd sas 5.16 5.17 518. 5.19 5.20 52k 5.22 523 5.24 $25 5.26 $27 WATER TREATMENT MICROBIOLOGY REFERENCES Patent, DLH.: Bacteria, How they Affect Other Living Things, Holiday House, New York (1980). Pelezar, M. J., Jt Chan, E. C. S.,& Krieg, N.R.: Microbiology, McGraw-Hill, New York (1986) Pope, D.H., Duquette, D.J.. Johannes, A.H. and Wayner, P.C.: “Microbiologically Influenced Corrosion of Industrial Alloys,” Materials Performance (April 1984) 14-18. Ostroff, A. G.: Introduction to Oilfield Water Technology, National Association of Corrosion Engineers, Houston, TX (1979). Watkins, L. and Costerton, J. W.: “Growth and Biocide Resistance of Bacterial Biofilms in Industrial Systems,” Chemical Times & Trends (Oct. 1984) 35-40. Costerton, J. W. and Lashen, E. S.: “Influence of Biofilm on Efficacy of Biocides on Corrosion-Causing Bacteria,” Materials Performance (Feb.1984) 13-17. Stoecket, J. G.i “Guide for the Investigation of Microbiologically Induced Corrosion,” Materials Perform- ‘ance (August 1984) 48-55, Sharpley, JM.: Applied Petroleum Microbiology, Buckman Laboratories, Inc.. Memphis, TN (1961) NACE: TPC Publication 3. Microbiologically Influenced Corrosion and Biofouling in Oilfield Equipment, NACE Intemational, Houston, TX (1990), Posigate, J. R.: The Sulphate-Reducing Bacteria, Cambridge University Press, Cambridge, England (1979). Private communication. Cragnolino, G.: “The Role of Sulfate-Reducing and Sulfur-Oxidizing Bacteria on Localized Corrs Paper No. 244, presented at NACE Corrosion/83, Anaheim, California (April 18-22, 1983). Smith, R.S., Landes, S.H., and Thurlow, M.T.: “Guidelines Help Counter SRB Activity in Injection Water,” Oil & Gas J. (Dec. 4, 1978) 87-91. NACE: The Role of Bacteria in the Corrosion of Oil Field Equipment, National Association of Corrosion Engineers, Houston, TX (1976). Zobell, C. E: “Ecology of Sulfate-Reducing Bacteria,” Producers Monthly (May 1958) 12-29. NACE: Review of Current Practices for Monitoring Bacterial Growth in Oilfield Systems, NACE Interna- tional, Houston, TX (1976) APL: Recommended Practice for Biological Analysis of Water Flood Injection Waters, API RP 38, Ameri- ‘can Petroleum Institute (May 1959). Whitesell, L. B.: “Field Evaluation of Microbial Problems and Their Control,” Paper No. SPE-64, SPE. Rocky Mountain Section Meeting (1961). NACE: TMO194-Field Monitoring of Bacterial Growth in Oilfield Systems, NACE Intemational, Houston (1994). NACE: Review of Non-Conventional and Supplemental Methods for the Detection of Sulphate-Reducing Bacteria in Oilfield Waters, NACE International, Houston, TX (1989). Litman, E.C.: “Oilfield Bactericide Parameters as Measured by ATP Analysis,” SPE Paper 5312 (Jan. 1975). Ruseska, I, Robbins, J., Costerton, J.W. and Lashen, E.S.: “Biocide Testing Against Corrosion-Causing, Oil-Field Bacteria Helps Control Plugging,” Oil & Gas J. (March 8, 1982) 253-64. Pues, W., Lee, E. S., and Kissell, C. L: “Chemical Mitigation of Corrosion by Chlorine Dioxide in Oilfield Waterfloods,” Materials Performance (May 1985) 45-50. Grier, J. C. and Christensen, R. J. “Biocides Give Flexibility in Water Treatment,” Hydrocarbon Process- ing (Nov. 1975) 283. Coward, H. F. and Jones, G. W.: Limits of Flammability of Gases and Vapors, Bureau of Mines, Bulletin $03. Crowe, C. W.: “New Treating Technique to Remove Bacterial Residues from Water- Injection Wells,” J Patr. Tech. (May 1968) 475, Clark, 1.B., Luppens, JC, and Tucker, P-T.: “Using Ultraviolet Radiation for Controlling Sulfate-Redue- ing Bacteria in Injection Water,” SPE Paper 13245 presented at the 59th Annual Technical Conference and Exhibition of the Soc. of Pett. Engrs., Houston, TX (Sept. 16-19, 1984). 210 APPLIED WATER TECHNOLOGY