Suspended Substances: Microorganisms

OBJECTIVE: When given a practice exam, be able to correctly identify the various types of microbiological species, biofouling, bacterial growth requirements, biocides, and biostats. NOTE: Portions of this material have been previously published in the International Desalination & Water Reuse Quarterly. GLOSSARY: Aerobic Algae Refers to the requirement of atmospheric oxygen for metabolism. A large and diverse group of simple plants that contain chlorophyll (and can therefore carry out photosynthesis) and live in aquatic habitats and in moist environments. Refers to NOT having the requirement of atmospheric oxygen for metabolism. A type of nutrition in which organisms synthesize the organic materials they require from inorganic sources (carbon from CO2, for example). A rod-shaped bacterium. A diverse group of microorganisms all of which consist of only a single cell that lacks a distinct nuclear membrane and has a cell wall of a unique composition. Singular of bacteria. A substance which kills microorganisms. The reduction in RO unit performance due to the presence and activity of microorganisms. A substance which does NOT kill microorganisms but hinders their growth. A sticky coating surrounding certain bacterial cells.

Anaerobic Autotroph

Bacillus Bacteria

Bacterium Biocide Biofouling Biostat Capsule

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Catalase Cellulase Cell Wall Chemoautotroph

An enzyme that catalyzes the breakdown of hydrogen peroxide to water and oxygen. An enzyme which causes the degradation of cellulose. The outer covering of a bacterial cell. An organism which derives its energy from chemical compounds (usually inorganic) and builds its organic compounds using inorganic carbon. An organism which derives its energy from chemical compounds (usually organic) and builds its organic compounds using organic carbon. Organisms which derive their energy requirements from chemical bonds (as opposed to sunlight: See Photosynthesis). A round (spherically) shaped bacterium. A semipermeable membrane beneath the cell wall. Piping containing non-flowing water, which is connected to an actively flowing channel. Bacteria growing in the dead leg send TOC and live organisms into the active stream. The period of exponential decrease in a bacterial growth curve. The resting stage of certain bacteria, formed in response to adverse conditions. The phase of bacteria growth in which the cells are doubling at a genetically predetermined rate. An appendage on a bacterial cell used to attach to a surface (plural = fimbriae)

Chemoheterotroph

Chemosynthetic

Coccus Cytoplasmic Membrane Dead Leg

Death Phase Endospore Exponential Growth Phase Fimbria

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Fungi

A group of single-celled or multicellular organisms which have some similarity with plants but lack chlorophyll and are classified in a separate kingdom. Bacteria which stain red with Grams stain. Bacteria which stain violet with Grams stain. A staining method used to differentiate bacteria. The bacterial sample is smeared on a glass slide, stained with a violet dye, treated with acetone-alcohol (a decolorizer), and finally counter stained with a red dye. An organism which builds its organic compounds from the intake and digestion of organic substances. Bacteria which grow at an optimal temperature range of 30 - 40C. The study of microorganisms. An organism so small it cannot be seen without the aid of a microscope. The tendency of water to pass through a semipermeable membrane into a solution of higher salt concentration. Organisms which derive their energy from sunlight. Bacteria which grow at an optimal temperature range of below 15C. Bacteria which grow at an optimal temperature range of 20 - 30C. A corkscrew (spiral) shaped bacterium. Bacteria, either coccus or bacillus, which form chains of cells.

Gram Negative Gram Positive Grams Stain

Heterotroph Mesophiles Microbiology Microorganism Osmosis Photosynthetic Psycrophiles Psycrotrophs Spiral Strepto-

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StaphyloStationary Phase TDS TOC

Coccus-shaped bacteria which form clusters. The period in a bacterial growth curve when the number of cells dividing equals the number dying. Total Dissolved Solids. A measurement of the weight per unit volume of dissolved materials in a solution. Total Organic Carbon, also Total Oxidizable Carbon. A measurement of the amount of organic material in a sample. An appendage on a bacterial cell used for locomotion. (plural = flagella)

Flagellum

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PURPOSE: The purpose of this lessons material is to understand the types of microbiological species found in water and why they can be a problem in a water treatment system. INTRODUCTION Understanding water microbiology is important to the water treatment professional. Scaling, fouling, chemical attack, and corrosion of water treatment equipment are frequently directly caused by microorganisms or the problems are enhanced by the presence of microorganisms. While much of the detailed structural and physiological information and terminology presented below do not need to be remembered, understanding the structure and physiology allow us to understand how and why microorganisms cause problems. Microbiology is the science of microorganisms. Microorganisms are living organisms which cannot be seen with the unaided eye. A human hair is roughly 100 microns in diameter. The human eye can distinguish particles down to roughly 50 microns. Magnification is required for us to see smaller particles. A microscope with the ability to magnify up to 1000x is commonly used to distinguish aquatic microorganisms which may be as small as 0.2 micron in diameter. All natural water will contain a mixture of microorganisms. The three main categories of microorganisms that we will discuss are bacteria, algae, and fungi. Bacteria will be discussed at length. Algae and fungi will be very briefly described. BACTERIA Bacteria are usually responsible for the majority of problems in reverse osmosis equipment. The thousands of species of bacteria are distinguished by many characteristics including shape, structure, source of energy (sunlight or chemical), and other physiological activities. SHAPE Bacteria come in a lot of sizes. Most individual bacterial bodies are in the size range of 0.2-2.0 microns in diameter and 2.0 to 8.0 microns in length. There are three basic shapes: coccus (round), bacillus (rod), and spiral. Bacterial cells divide by binary fission which is simply a replication of genetic material and a splitting of the cell into two halves, each containing one replica of the genetic material. After splitting, the cells may remain attached or independent of one another. Attachment or independence following cell division is a characteristic of each bacterial species. Coccus-shaped and bacillus-shaped bacteria may remain as individuals, pairs, chains, or clusters after dividing. Individuals are called "single coccus" or "single bacillus". Pairs are called diplococcus or diplobacillus. Chains are called streptococcus or streptobacillus. Clusters are called staphylococcus (bacillus-type bacteria dont form clusters). You may recognize the names of
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human diseases above, such as strept throat and a staph infection being caused, respectively, by streptococcus and staphylococcus types of bacteria. Spiral bacteria do not remain attached after cell division. They are named according to the number of turns and the flexibility of the spiral. If they are shaped like commas (one turn), they are called a vibrio. If they are like a corkscrew with several turns, they are called a spirillum or a spirochete depending upon the flexibility of the spiral. The spirals are not of great importance to us in causing desalination process problems. Figure 13.1 illustrates the three main bacterial shapes and their shapes after cell division.

Shapes
Coccus Bacillus Spiral

Attachments After Cell Division

Single Cocci Single Bacilli Single Spirals

Diplococcus

Diplobacillus

Don't Attach
Streptococcus Streptobacillus

Don't Chain
Staphylococcus

Don't Cluster
Figure 13.1

Don't Cluster

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STRUCTURE Bacteria have the general structure illustrated in Figure 13.2. While a rod shaped bacterium is shown, the main structural components are similar for other shapes. The main structural components are: Cell Wall Cytoplasmic membrane Genetic Material Cytoplasm The outer covering of a bacterial cell A semipermeable membrane beneath the cell wall The chromosomes The jellylike substance contained within the cytoplasmic membrane

Figure 13.2

Cell Wall: The structure of the cell wall distinguishes broad categories of bacteria and plays a role in fouling ability. There are two general types of cell walls in bacteria. These are typically distinguished by the different stains that different cell walls will absorb using a staining procedure called the Gram Stain. Bacteria are designated as gram-positive if they retain a crystal violet stain. Bacteria are designated as gram-negative if the crystal violet stain, is washed out by a decolorizing agent and they accept a counterstain (safranin) which stains them red. The following lists four of the important differences between the cell walls of Gram-positive and Gram-negative bacteria. 1. 2. 3. 4. Thickness of cell wall dense material Resistance to physical disruption Susceptibility to anionic detergents Cell wall lipid content Gram-positive Thick High High Low Gram-negative Thin Low Low High

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ENERGY & CARBON REQUIREMENTS Strains of bacteria, as well as other microorganisms, can grow on just about anything. The carbon needed for growth can be obtained from inorganic or organic sources. If an organism builds its cellular organic compounds from carbon obtained from inorganic carbon dioxide, it is called an autotroph. If it builds its cellular organic compounds from carbon obtained from previously existing organic compounds, its called a heterotroph. The energy needed for growth can be obtained from sunlight or chemicals. If obtained from sunlight, the organism is called photosynthetic. If obtained from chemical bonds, the organism is called chemosynthetic. Humans, as well as all animals, eat plant and/or animal materials to obtain both the carbon and the energy needed for growth, and are classified as chemoheterotrophs. The following lists the nutritional classifications of organisms: NAME PHOTOAUTOTROPHS ENERGY SOURCE Light CARBON SOURCE CO2 Organic Compounds CO2 EXAMPLES Photosynthetic bacteria, algae, and plants Some purple and green bacteria Hydrogen, sulfur, iron, and nitrifying bacteria Most bacteria, all fungi, protozoa, and animals

PHOTOHETEROTROPHS Light CHEMOAUTOTROPHS Usually Inorganic Compounds

CHEMOHETEROTROPHS Usually Organic Compounds TEMPERATURE REQUIREMENTS

Organic Compounds

We can find bacterial species that will grow at almost every temperature. Each strain of bacteria has a minimum, optimum, and maximum temperature requirement for growth. The minimum temperature is the minimum temperature at which the microorganism will grow. The optimum temperature is the temperature at which maximum growth occurs. The maximum temperature is the maximum temperature at which the microorganism will grow. There is generally about a 30 degrees Celsius range between minimum and maximum. Psycrophiles are cold-loving bacteria which can grow at 0C (32F) with an optimum of around 15C (59F) and a maximum of around 20C (68F). These are usually only found at ocean depths or polar regions. 8

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Psycrotrophs are bacteria which can grow at 0C (32F) but have an optimum temperature in the 20 - 30C (68 - 86F) range. These are responsible for a lot of our refrigerated food spoilage. Some of the water bacteria are in this group. Mesophiles are the moderate-temperature-loving bacteria which we will deal with most often. Mesophiles generally have an optimum growth rate in the 30 - 40C (86 - 104F) range. Thermophiles are "hot-loving" bacteria which may have an optimum temperature range of 50 - 60C (122 -140F). Extreme Thermophiles have an optimum temperature ranges 90 - 100C (194 212F) and strains of bacteria have been found thriving near deep ocean volcanic vents where temperatures reach 350C (662F). OXYGEN REQUIREMENT: Some microorganisms use oxygen to produce energy from nutrients; others dont. Those that use oxygen are called aerobic. Those that dont are called anaerobic. Some microorganisms MUST have oxygen in order to survive. These are called obligate aerobes. Those that can use oxygen when it is present but use other substances or other means when oxygen is not present are called facultative anaerobes. Those that cannot use oxygen and are usually harmed by it, are called obligate anaerobes. ALGAE Algae are generally photoautotrophs which use light and CO2 for growth. They may be single celled, for example; ocean plankton. They may be filamentous, for example; green algae mats and fibers attached to surfaces. They may be plant-like, for example; brown ocean algae which can grow to 50 meters (150 feet) in length. Green algae growth may be seen in flowmeters or any other equipment exposed to light. This can cause pluggage. At many water treatment plants, the biggest problem isn't the algae themselves but that the algae die and become food for bacteria. FUNGI Fungi include microscopic yeasts and molds and the macroscopic mushrooms and other fleshy fungi. All fungi are chemoheterotrophs requiring organic compounds for energy and carbon. Fungi can be single celled (example: bread making yeast), filamentous (example: mold and mildew), and fleshy (example: mushrooms). Fungi are responsible for much of the degradation of the cellulosic (hard) parts of plants. Many are able to produce cellulase, an enzyme which decomposes cellulose. This has important consequences for cellulose acetate reverse osmosis membranes.
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BIOFOULING Biofouling is the reduction in RO unit performance due to the presence and activity of microorganisms. Biofouling is usually attributed more to bacteria than to algae and fungi. Algae and/or fungi must be considered, however, because they may contribute their decomposed bodies as food which bacteria feed upon. They may also provide a physical mass which can plug equipment. Regardless of the species, the impacts to the water treatment process are still negative. In most cases, we don't care what the name of the microorganism(s) is (are). We just want to fix the problem and keep it from occurring again. In this discussion, we will not differentiate fouling by algae, fungi, or bacteria. We will assume that biofouling is chiefly caused by bacteria. If there are high levels of bacteria, with or without high levels of fungi or algae, there is a high likelihood of negative impacts to RO unit performance. The negative impacts which biofouling may produce include: 1. 2. 3. 4. Pluggage Scaling Chemical attack Product water quality degradation

Pluggage is caused by the physical mass of microorganisms growing in flow channels. This causes pressure drop problems and water production decline. Scaling is usually an offshoot of pluggage. The lowered flow rates through plugged areas in RO elements can lead to the concentration of feed water above the saturation level of certain compounds. Chemical attack can occur to metals (corrosion) and to membranes. Bacteria attached to a surface produce acidic compounds which can cause the corrosion of the metal or the degradation of cellulose acetate membranes underneath the bacterial layer. The production of organic compounds by bacteria increases the organic compound levels of the product water. This can have an adverse affect on downstream equipment, such as ion exchange units, or on the quality of the final product water. BACTERIAL GROWTH Every problem associated with bacteria is going to be based to some degree on the extraordinary growth and metabolic rates that they can exhibit. If the temperature is optimal, and there is enough

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food present, and if there are no chemicals present that kill them (biocides) or keep them from growing (biostats), they can quickly grow at a exponential rate.

Stationary Phase Exponential Phase Death Phase

Log Number of Bacteria

Lag Phase
Figure 13.3

Time

This extraordinary growth rate usually doesnt begin immediately. There is generally a lag phase in which the microorganisms are metabolically very active but dont reproduce. They are generating enzymes and nuclear material and other cellular products. This is analogous to the production of water from a water treatment plant. There is intense construction and activity in the beginning, with no production of water. Then, at a certain time, production begins and increases rapidly. The lag phase may last for hours or days. Eventually, however, the cells start to divide and enter an exponential growth phase. In this phase, each cell divides into two every so many minutes or hours. The amount of time required to double is genetically determined but is usually between 1 - 3 hours. It may occur as quickly as every 20 minutes, however, or as slowly as every 24 hours. A doubling of every 24 hours would, of course, provide 2 cells for every one in 24 hours. A doubling rate of 20 minutes would provide 4,700,000,000,000,000,000,000 cells, for every one, in 24 hours. At around 1 x 10-14 g/bacteria, this would create a bacterial mass of 5000 tons (4.5 million Kg) per initial bacterium in 24 hours. Fortunately, this doesnt happen. There is a limit to the amount of nutrients available with which to build cell components. Following the exponential growth phase, which usually lasts hours or days, a stationary phase begins. In this phase the death of cells offsets cell division. Cell death is generally caused by the depletion of nutrients and the accumulation of toxic waste products produced by the bacteria.
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The stationary phase may last for relatively long or short periods of time depending on the food supply and waste product removal. Following the stationary phase, comes the death phase in which the death of cells becomes exponential. The death of one species can trigger the exponential phase of another bacterial species which feeds on the organic materials from the first. Thus, we are usually assured of a continuous quantity of living organisms and a continuous supply of dissolved TOC (Total Organic Carbon or Total Oxidizable Carbon) and solid organic fouling bacteria. Water treatment system's filters, valves, pipes, tanks, membranes, etc. can all become habitats for bacteria. If bacteria flow right through the equipment, there will generally be no problem. It is when bacteria colonize (grow in or on) a piece of equipment that problems occur. Bacteria colonization with subsequent exponential growth depend upon the following: 1. Temperature 2. Nutrients 3. pH 4. Osmotic Pressure 5. Oxygen Level 6. Flow Rate 7. Presence of biocides or biostats Temperature: Recall that bacteria have a minimum and maximum temperature range within which growth will occur. Within this range there is an optimum temperature range which promotes the greatest growth. Most biofouling bacteria are mesophiles with an optimum growth rate in the 30 - 40C (86 104F) range. The minimum temperature will be around 10C (50F). The maximum temperature will be around 50C (122F). Biofouling potential, therefore, will be highest at the optimum range, and the lowest at below or above, respectively, the minimum and maximum temperatures. Operating water treatment processes at below the minimum optimum temperature or above the maximum optimum temperature generally limits biofouling significantly. This is generally not feasible except for evaporators which naturally operate at higher temperatures. Most membranes are not operated at temperatures exceeding 50C. Some microchip and pharmaceutical manufacturing plants operate high purity loops at high temperatures in order to minimize bacterial growth. Nutrients: Recall from the previous section that certain bacteria can grow on just about any organic source and some inorganic sources. There will always be enough nutrients available to support some growth. In natural feed waters (lakes, rivers, oceans, groundwaters, etc.), there are usually enough nutrients to support exponential growth.

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pH: Bacteria are as flexible here as with temperature. There are bacteria which can grow at a pH of 1.0. There are bacteria which can grow at a pH of 12. Bacteria, generally, have an optimum growth rate in the pH range of near neutrality, in the 6.5 7.5 range. For most bacterial species, growth is increasingly inhibited with higher or lower pH. Bacterial metabolism produces acidic compounds which diffuse into the surrounding environment, lowering the pH. This lowering of pH is one of the mechanisms that reduces growth rate during the stationary and death phase. Osmotic Pressure: Bacteria are 80% - 90% water. Waterborne bacteria live in an aquatic environment. The cytoplasmic membrane which surrounds the cell contents is semipermeable. Osmosis is the movement of water across a semipermeable membrane, into a solution of higher salt concentration. When most bacteria are placed in a solution of high total dissolved solids content (high TDS), water passes out of the cell and into the solution, due to osmosis. This contracts the cytoplasmic membrane away from the cell wall and greatly inhibits cell growth and replication. This is the mechanism of food preservation by salt or sugar. When most bacteria are placed in high purity water (very low TDS), water passes INTO the cell due to osmosis. This will rupture and kill many types of bacteria. But, as usual, we can find bacteria adapted to all situations. There are bacteria which will grow in the Dead Sea (300,000 mg/L TDS) and in the purest water produced. Oxygen Level: We think of oxygen as a necessity of life causing no problems for living organisms. Oxygen can be considered a poisonous gas in many ways, however. Molecular oxygen did not exist in our atmosphere for most of our planets history. It is speculated that life could not have evolved in the presence of molecular oxygen and that one of the benefits of water being produced from hydrogen and oxygen is that oxygen was removed from the atmosphere. There are several forms of oxygen that are detrimental to bacteria as well as higher life forms. They are: Singlet oxygen: Superoxide free radical: O, which has been boosted to a higher energy state by sunlight and is highly reactive. O2-, formed during normal cell metabolism. Cells must have the enzyme superoxide dismutase in order to destroy this compound.

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Hydrogen Peroxide:

H2O2, which contains the peroxide anion O2-2, which is toxic. Cells must have the enzyme catalase or peroxidase in order to destroy this compound. We have catalase in our cells which is why the solution bubbles when we put hydrogen peroxide on a wound (peroxide anion is converted to molecular oxygen). OH, another intermediate form of oxygen which is highly reactive. Unlike OH- (the ion) OH is formed by ionizing radiation (such as UV lights). O3, another form of oxygen which is a powerful oxidizer (takes electrons from other atoms).

Hydroxyl Free Radical:

Ozone:

Of the above compounds, hydrogen peroxide and ozone are routinely used in many industries to sanitize equipment. Hydrogen peroxide is generally used on a periodic sanitization schedule. Ozone may be added continuously or on a periodic sanitization schedule. As we saw previously, some bacteria grow well in an oxygenated environment and some even require oxygen for growth (aerobes). Some bacteria cannot grow in an oxygen environment (anaerobes). Some bacteria can take it or leave it (facultative anaerobes). Non-polluted, shallow surface waters will generally have at or near saturation levels of oxygen (5 10 mg/L), so the predominate species of bacteria will be aerobic. Polluted waters and deep waters can have much lower oxygen levels, and therefore will have different bacterial species. Groundwaters generally have no dissolved oxygen and favor anaerobic bacteria. Flow Rates: In order to colonize a piece of equipment, the bacteria must attach to a surface either actively or due to settling out in areas of little or no water flow. The higher the velocity of a water stream, the more difficult it is for most bacteria to colonize a piece of equipment. Relatively lowflowing areas, like storage tanks, piping not in use (called dead legs), certain areas in most vessels, etc., have a greater potential for bacterial growth and subsequent biofouling. Wherever there are low flow rates, bacteria will generally flourish, if their other needs are met. Activated carbon beds, with highly porous particles, are ideal for growth. Most vessels, including media filters, ion exchange beds, storage tanks, etc. have low flow areas which promote bacterial growth. Adequate backwashing of media and periodic sanitization of all equipment is generally required to manage bacterial growth. Keeping flow rate as continuous and as high as practical is frequently the most economical way to reduce biofouling. Water treatment equipment in service will grow fewer bacteria than when out of service.

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Biocides & Biostats: Biocides are substances which KILL microorganisms. Biostats are substances which INHIBIT the growth and replication of microorganisms. Chlorine is the most common biocide used in the water treatment industry. There are many forms used, including gaseous chlorine, liquid bleach, and powdered hypochlorites. Chlorine plus ammonia is also commonly used. The resulting chloramines are also biocidal. Chlorine acts by diffusing through the cell walls and cytoplasmic membranes of the bacteria and damaging the genetic material and enzymes. This may cause the immediate death or inactivation of the bacteria, but the effect is time and dosage dependent. Continuous ozonization and/or ultraviolet irradiation are utilized in some industries for bacterial control. Periodic sanitizations are conducted at many facilities using a biocide such as chlorine, ozone, hydrogen peroxide, or a proprietary compound from a vendor or manufacturer. The most common biostats used in industry are sulfur compounds such as sodium bisulfite and sodium metabisulfite. Biostats do not kill the organisms and are only useful for short-term layup of equipment.

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