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Pathogenic Wastewater Microbiology
Biological Wastewater Treatment
Fundamentals of Biological Treatment
Microbiology of Onsite Systems
The purpose of this module is to provide a fundamental background on the relationship between microbes, wastewater, and wastewater treatment, i.e., why are we concerned about microbes in wastewater, what role do they play in wastewater treatment, and what happens when "clean" water is released into the environment.
Wastewater, by its nature, is teaming with microbes. Many of these microbes are necessary for the degradation and stabilization of organic matter and thus are beneficial. On the other hand, wastewater may also contain pathogenic or potentially pathogenic microorganisms, which pose a threat to public health. By definition, a pathogen is an organism capable of inflicting damage on its host. Waterborne and water-related diseases caused by pathogenic microbes are among the most serious threats to public health today. Up to 35% of the potential productivity of developing nations is lost because of waterborne disease. Waterborne diseases whose pathogens are spread by the fecal-oral route (with water as the intermediate medium) can be caused by bacteria, viruses, and parasites (including protozoa, worms, and rotifers). Many of the most common pathogenic microorganisms are described in the Bad Bug Book website maintained by the Food and Drug Administration. Dr. Charles Hagedorn at Virginia Polytechnic Institute has several informative websites including Wastewater Testing and Wastewater Microbes where many of the pathogenic microorganisms discussed are more fully described.
Bacteria are defined as any of the one-celled prokaryotic organisms, which vary in morphology and nutritional requirements and may be free-living, saprophytic, or pathogenic. The major bacterial pathogens and their associated diseases are:
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Bacterial Pathogens |
Related Disease |
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Salmonella |
Salmonellosis |
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S. typhimurium |
Typhoid fever |
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Shigella |
Shigellosis |
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Enterococcus (Fecal Streptococci) |
Diarrhea |
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E. coli ( Fecal Coliform) |
Diarrhea |
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Vibro cholerae |
Cholera |
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Camplyobacter jejuni |
Gastroenteritis |
Viruses are defined as genetic elements, containing either DNA or RNA and a protein capsid membrane, which are able to alternate between intracellular and extracellular states, the latter being the infectious state. Over 100,000 different viral types have been identified in human feces, and therefore, there is a direct correlation between contact with improperly disposed of treated waste and diarrheal disease. Among the viral pathogens listed below are over 120 enteric viruses, all pathogenic to humans requiring low infectious doses. The major viral pathogens include:
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Viral Pathogens |
Related Disease |
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Hepatitis A |
Hepatitis |
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Norwalk-like agents |
Gastroenteritis |
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Virus-like 27 nanometer particles |
Gastroenteritis |
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Rotavirus |
Gastroenteritis and polio |
Diarrhea is one of the most common features of waterborne disease. Fecal pollution is one of the primary contributors to diarrhea. In this country, we tend to think of diarrhea as primarily a nuisance. Diarrhea, however, causes dehydration that if not properly treated, can ultimately lead to death. In fact globally, 4,200,000 deaths per year are attributed to diarrhea caused mainly by bacteria and viruses. A person infected with a disease-causing virus may excrete up to 106 (1,000,000) infectious particles per gram of feces. When you stop to consider that potentially, it only takes 1 virus particle to cause disease, the capacity for disaster with untreated or improperly treated waste is enormous. Examples of bacteria commonly associated with diarrheal disease are Shigella dysenteriae and Salmonella typhi. Two protozoans commonly associated with diarrheal disease are Giardia lamblia (responsible for the most widespread protozoan caused disease in the world) and members of the genus Cryptosporidium.
Parasites are defined as organisms that grow, feed, and live on or in another organism to whose survival it contributes nothing. The three most important protozoal pathogens in temperate zone countries are:
Water quality, and its threat to public health, has inspired development of tests designed to measure its suitability for drinking, bathing, and release back to the environment. Water that looks clear and pure may be contaminated with pathogenic microorganisms. For example, 105 (100,000) bacteria per milliliter of water is invisible to the naked eye. Therefore, even water that appears “pure” must be tested to ensure that it contains no microorganisms that might cause disease. On the other hand there are so many potential pathogens that it is impractical to test for them all. Because of this, tests have been developed for indicator organisms. These are organisms that are present in feces (or sewage), survive as long as pathogenic organisms, and are easy to test for at relatively low cost.
Indicator organisms indicate that fecal pollution has occurred and microbial pathogens might be present. Total and fecal coliforms, and the enterocci - fecal streptocci are the indicator organisms currently used in the public health arena. Coliform bacteria include all aerobic and facultative anaerobic, gram-negative, nonspore-forming, rod-shaped bacteria that ferment lactose with gas formation. There are three groupings of coliform bacteria used as standards: total coliforms (TC), fecal coliforms (FC) and Escherichia coli. Total coliforms are the broadest grouping including Escherichia, Enterobacter, Klebsiella, and Citrobacter. These are found naturally in the soil, as well as in feces. Fecal coliforms are the next widest grouping, which includes many species of bacteria commonly found in the human intestinal tract. Usually between 60% and 90% of total coliforms are fecal coliforms. E. coli are a particular species of bacteria that may or may not be pathogenic but are ubiquitous in the human intestinal tract. Generally more than 90% of the fecal coliform are Escherichia (usually written as E. coli)
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Protozoal Pathogens |
Related Disease |
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cryptosporidiosis |
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giardiasis |
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amoebic dysentery |
It was mentioned earlier that many of the microbes present in wastewater are beneficial. In fact, many wastewater treatment technologies are dependent on these beneficial microorganisms for remediation of wastewater so that it won’t detrimentally impact the environment. One of the primary goals of biological treatment is the removal of organic material from wastewater so that excessive oxygen consumption won’t become a problem when it is released to the environment.
Another goal of biological treatment is nitrification/denitrification. Nitrification is an aerobic process in which bacteria oxidize reduced forms of nitrogen (NH4+®NO2-®NO3-). Denitrification is an anaerobic process by which oxidized forms of nitrogen are reduced to gaseous forms (NO3-®NO2-® N2O or N2), which can then escape into the atmosphere. This is important because the release of nitrogen to the aquatic environment can also cause eutrophication.
Immobilization of phosphate (PO43-) through bacterial assimilation or precipitation is important for the same reason. Another goal of biological treatment is elimination of pathogenic microorganisms either through predation or out-competition. The oxidation/stabilization of organic sludge is also of importance in biological treatment of wastewater.
Organic material in wastewater originates from microorganisms, plants, animals, and synthetic organic compounds. Organic materials enter wastewater in human wastes, paper products, detergents, cosmetics, and foods. They are typically a combination of carbon, hydrogen, oxygen, and nitrogen and may contain other elements. Typical wastewater contains organic matter in the forms of proteins (40 to 60 percent), carbohydrates (25 to 50 percent), and oils and fats (8 to 12 percent) (Crites and Tchobanoglous, 1998). Wastewater may also contain small amounts of synthetic organic molecules (i.e., pesticides and solvents) which may range from simple to complex in structure.
The oxidation of organic materials in the environment can have profound effects on the maintenance of aquatic life and the aesthetic quality of waters. Biochemical oxidation reactions involve the conversion of organic material using oxygen and nutrients into carbon dioxide, water, and new cells. The equation that expresses this is:
Organic material + O2 + nutrients ® CO2 + H2O + new cells + nutrients + energy
It can be seen from this equation that organisms use oxygen to breakdown carbon-based materials for assimilation into new cell mass and energy. A common measure of this oxygen use is biochemical oxygen demand (BOD). BOD is the amount of oxygen used in the metabolism of biodegradable organics. If water with a large amount of BOD is discharged into the environment, it can deplete the natural oxygen resources. Heterotrophic bacteria utilize deposited organics and O2 at rates that exceed the oxygen-transfer rates across the water surface. This can cause anaerobic conditions, which leads to noxious odors. It can also be detrimental to aquatic life by reducing dissolved oxygen concentrations to levels that cause fish to suffocate. The end result is an overall degradation of water quality. Typical wastewater contains 110-400 mg/L BOD (Crites and Tchobanoglous, 1998).
Wastewater often contains large amounts of the nutrients, particularly nitrogen and phosphorous (as phosphate- PO43-). Typical wastewater values range from 20-85 mg/L total nitrogen and 4-15 mg/L total phosphorous (Crites and Tchobanoglous, 1998). Nitrogen and phosphorous are essential for growth of all organisms and are typically limiting in the environment. Nitrogen is a complex element existing both in organic and inorganic forms. The forms of most interest from a water quality perspective are organic nitrogen (often as proteins or urea), ammonia, nitrite, nitrate, and di-nitrogen. Phosphorous is found in synthetic detergents and is used for corrosion control in water supplies. Because of its increased usage, its concentration has risen from 3-4 mg/L to 10-20 mg/L since the introduction of synthetic detergents.
The introduction of large concentrations of these nutrients from untreated or improperly treated wastewater can lead to eutrophication. Eutrophication is the process by which bodies of water become rich in mineral and organic nutrients causing plant life, especially algae, to proliferate, then die and decompose thereby reducing the dissolved oxygen content and often killing off other organisms. A specific health problem associated with increased levels of nitrogen is methemoglobinemia or blue-baby syndrome. This disease is a direct result of elevated concentrations of nitrite.
The basic mechanisms of biological treatment are the same for all treatment processes. Microorganisms, principally bacteria, metabolize organic material and inorganic ions present in wastewater during growth. Which brings us to the fundamental differences between catabolic and anabolic processes. Catabolic processes are those biochemical processes involved in the breakdown of organic products for the production of energy or for use in anabolism. Catabolic processes are dissimilar because the reactants and products in the reaction are not incorporated into new cell mass. These reactions can be thought of as redox reactions because they involve the transfer of electrons resulting in the generation of energy to be used in cell metabolism. In contrast, anabolic processes are the biochemical processes involved in the synthesis of cell constituents from simpler molecules. These processes usually require energy and are assimilatory. That is the processes result in the incorporation of the reacting molecules or compounds into new cell mass.
A bacterial perspective of contaminant removal involves several steps. First, there must be a liquid-phase transport of the contaminant to the cell surface. Next, there must be a sorption of the contaminant to the cell surface. Third, if necessary, extracellular enzymes will hydrolyze the contaminant into subunits, which can then be transported by diffusion to and through the cytoplasmic membrane. This is the point at which dissimilatory or assimilatory processes take place resulting in a gain in energy and/or an increase in biomass. It is important to realize that biological wastewater treatment alone does not result in the regeneration of potable water.
The growth of bacteria in pure culture has been the mainstay of microbiology, specifically the mainstay of microbiological technique. Solid media techniques (and selective/differential media) have allowed the isolation of individual species from complex natural populations. The study of individual strains of bacteria in nutrient rich batch cultures is still the basis of microbiology today. In natural environments and in pathogenic relationships, bacteria are different than the same organisms grown in vitro. In natural systems, bacterial consortia (mixed populations) grow as biofilms.
Bacteria in the environment are described as planktonic or sessile. Planktonic bacteria are free in the environment and are committed to motility and colonization of new surfaces. Sessile bacteria are bound within or to a surface structure. These bacteria have more active reproduction and general metabolism. Because of this, they have an increased heterotrophic potential. In other words, sessile bacteria have greater activity than the same microorganisms dispersed in the biofilm (planktonic). This has been proven by studies describing the release of increased concentrations of 14CO2 from a radio-labeled organic substrate by sessile microorganisms. Another important concept relative to microorganisms and biofilm formation is phenotypic plasticity. This concept describes the ability of bacterial species to change their morphologies in response to the situation. To illustrate this concept, we will describe the genus Caulobacter, which is commonly found in oligotrophic aquatic environments. Caulobacter binds to surfaces via a stalk possessing a holdfast. Spread of the biofilm occurs via motile swarmer cells which bind to surfaces, lose their flagella, and form a stalk to complete the cell cycle.
The surfaces that bacteria bind to are not homogenous. Even on a single particle of clay, on a micrometer scale, there will be differences in charge density. In fact, the range of ionic interactions and hydrophobicity seems to be important in terms of both the bacteria forming the biofilm and for the surface upon which the biofilm is formed.
There are three steps necessary for the formation of biofilms. First, there must be a macromolecular conditioning of the surface to be colonized. This is a purely chemical process that occurs on the order of microseconds. If you put any clean surface into the environment, low molecular weight compounds possessing their own unique hydrophilic and hydrophobic character will bind to that surface. Step two, microbial binding, is a two-step process. First, there is reversible binding (colonization) by bacteria. Next, if the cell senses the proper conditions, irreversible binding takes place, often triggering capsule formation. Finally, there is further permanent attachment of cells and cell division leading to microcolony formation and biofilm generation.
A biofilm consists of cells immobilized at a substratum and frequently embedded in an organic polymer matrix of microbial origin (capsule). The coverage is either uniform or non-uniform (because of microcolony formation). In the natural environment (e.g., pristine alpine streams), sessile bacterial populations are 103 to 104 times higher than planktonic bacteria. The biofilm may consist of a single monolayer up to multiple layers of bacterial cells. An example of this is algal mats which can be 300 to 400 millimeters thick. Because bacteria grow as microcolonies in biofilms (i.e., as clumps of cells) they are protected from bacteriophage, phagocytic eukaryotic microorganisms, antibiotics, toxic heavy metals, and xenobiotics. The diffusion gradients through these microcolonies protect the inner cells from pollutants in the water, and the outer cells of the microcolony are sacrificed for the good of the population.
One important distinction of biofilms is that they can provide a variety of microenvironments i.e., they are chemically heterogeneous throughout. They establish their own gradients of nutrients, oxygen saturation, and pH relative to the bulk environment. Because the capsule is hydrated, biofilms are greater than 95% water and thus they will trap inorganic and organic material that is soluble or particulate in nature. The solid/liquid interface between the biofilm and the environment is important as well to current/flow rates. There is a critical role of transport and transfer processes which are generally rate controlling in biofilm systems. For example, high flow rates in oligotrophic environments will be well nourished due to high transfer rates across the interface.
In natural systems, biofilms are responsible for the removal of dissolved and particulate contaminants and are important in the cycling of chemical elements. These concepts are equally important in wastewater treatment systems. Also, in the natural environment, enhanced growth may result from nutrient trapping. Another important property of biofilms is that the capsule, the bulk matrix of biofilms, acts as an ion exchange resin because it consists of anionic polymers which will bind Mg2+, Ca2+, and Fe2+/3+cations. Examples of naturally occurring biofilms include mineral, metal, and wood degrading consortia of bacteria.
We have discussed the presence of beneficial and disease-causing microorganisms associated with wastewater. Another topic of importance when discussing the fundamental microbiology of wastewater is the ability (or lack thereof) of pathogens to move through and survive in the subsurface. There are many reasons that this is important. In the United States, approximately 25% of all water used is groundwater and approximately 50% of the population relies on groundwater for drinking. The good news is that bacteria don’t usually move large distances in fine textured soils (generally less than a few meters). The bad news is that they can move larger distances in coarse-textured or fractured materials. Fortunately, pathogenic microorganisms not native to the subsurface generally don’t multiply underground and will eventually die. Despite these facts, they can move far enough distances and live long enough to be of concern around wastewater disposal areas. Of special concern, saturated flow conditions lead to horizontal movement of microbes. Unsaturated conditions are optimal and lead to greater attenuation.
There are two factors that significantly affect mobility of bacteria and viruses through the subsurface. First, the size of existing water filled pores (including cracks, fissures, and solution channels) will affect mobility. Second, the velocity of water through these pores plays an important role in microorganism mobility. There are also two mechanisms of retention of bacteria in the subsurface.
The first mechanism is filtration. This is the trapping of particles and bacteria in pore spaces. Larger suspended particles are trapped first. These then act as a filter for progressively smaller particles and bacteria. Eventually this system will become clogged and block further transport.
The second mechanism of retention of bacteria is adsorption. This is the adhesion of bacteria (or viruses), in an extremely thin layer, to the surfaces of solid bodies. Clays are ideal for this type of bacterial retention because of their small size, layered structure, and large surface-to-volume ratio. Thus, adsorption plays a more important role in soils that contain clays.
Bacterial size also plays a role in determining whether they are more likely to be retained by adsorption or filtration. Larger bacteria are more likely to be removed by filtration, whereas smaller bacteria are more likely to be removed by adsorption.
Because of the smaller size of viruses (they are approximately 100 nanometers as opposed to the approximate 1 micrometer length of the “typical” bacterial cell), their retention is mainly by adsorption. The isoelectric point of viruses usually ranges from pH 3 to pH 7. Below pH 3, they are generally positively charged and will be immobilized by negatively charged surfaces, and above pH 7 they are generally negatively charged and will be immobilized by positively charged surfaces. Increased salt concentrations and the presence of divalent and trivalent cations will also increase adsorption. Having said all this, rainfall can mobilize previously retained bacteria and viruses.
The septic tank works by a combination of sedimentation and anaerobic (without molecular oxygen) digestion. Anaerobic bacteria are responsible for the digestion. Anaerobic bacteria are non-pathogenic and are present in large numbers in the human intestine. A new supply of these bacteria are regularly added to the septic tank with each flush of human fecal material. Anaerobic digestion represents an incomplete digestion. Methane, hydrogen sulfide, and sulfur dioxide gases are produced, as well as a sludge of high molecular weight hydrocarbons. This sludge will readily decompose further when exposed to oxygen and aerobic bacteria. This further decomposition will take place in the municipal sewage treatment plant or landfill if either of these places is used to dispose of sludge pumped periodically from septic tanks. Anaerobic digestion achieves the following reduction of the contaminant load:
| Water Quality Parameter | % Removal In A Septic Tank |
| BOD (Biochemical Oxygen Demand) | 15% to 50% |
| TSS (Total Suspended Solids) | 25% to 45% |
| Settleable Solids | > 90% |
| Enteric Bacteria | 10% to 40% |
| Enteroviruses | No Significant Reductions |
| Protozoa | No Significant Reductions |
Aerobic digestion (with molecular oxygen) is far more complete. Aerobic digestion takes place in a properly constructed and maintained drainfield, as well as in an aerobic treatment device (ATU). In an ATU, the aerobic bacteria are selected out from any remaining aerobic bacteria which survive the trip through the septic tank, or are facultative bacteria which can exist both with and without molecular oxygen, or are random seed bacteria which are everywhere in our environment. In the soil, there are hundreds of different types of organisms that proliferate in the trenches where there is a regular supply of nutrients (septic tank effluent). Biological mats develop on the sides and bottoms of the trenches and add to a biological filtration of the effluent passing through it into the soil environment. The structure of these mats are due in part to the long filaments often growing out of several common strains of soil bacteria. If biomats are improperly managed, the growth can become so thick that the pores in the soil structure surrounding the disposal trench can become clogged. With the right balance of molecular oxygen to influent, the biological mat can be maintained as a benefit to the water treatment, and the wastes can be degraded completely to carbon dioxide and water allowing the aerobic treatment to go to completion.
Aerobic treatment in a trench or in an ATU is complete digestion and can achieve the following reductions of influent contaminant levels:
| Water Quality Parameter | % Removal In A Septic Tank |
| BOD (Biochemical Oxygen Demand) | 75% to 90% |
| COD (Chemical Oxygen Demand) | 75% to 90% |
| TSS (Total Suspended Solids) | 75% to 90% |
| Ammonia (Changed to Nitrate - N) | 80% to 90% |
| Enteric Bacteria | generally high but variable |
| Enteroviruses | generally high but variable |
| Protozoa | generally high but variable |