DNA Analysis of Microbial Contamination in Water



Microbial Contamination of Water

Traditionally, indicator micro-organisms have been used to suggest the presence of pathogens (Berg 1978). Today, however, we understand a myriad of possible reasons for indicator presence and pathogen absence, or vice versa. http://www.who.int/water_sanitation_health/dwq/iwachap13.pdf

Microbial contamination of drinking water

Ground-water microbiology is a relatively new field of study.


Until the 1970’s, scientific concepts and methods limited our knowledge of groundwater microbiology. First, it was common to assume that the ground- water environment was devoid of life. Second, methods for sampling ground- water environments for microbes were very limited. Third, it was generally assumed that water passing through the soil was purified by active microbial processes and by filtration; therefore, there was little concern with ground- water contamination. As ground-water contamination became more and more evident during the 1980’s, the motivation for understanding ground-water environments increased. In addition, new methods in microbiology, based on advances in molecular biology, provided microbiologists with new tools to explore this difficult-to-sample microbial habitat.

Some general concepts in ground-water microbiology.

Since 1990, several reviews of ground-water microbiology have been published. Madsen and Ghiorse (1993) explored the suitability of ground-water habitats for microbial growth, and compared ground-water environments to other aquatic habitats (lakes, rivers, streams, wetalnds) where microbes are abundant. Chapelle (1993) related microbial activities in ground water to subsurface geochemistry. And Fyfe (1996) has recently proposed that the term “biosphere” be extended to include deep subterranean habitats, based on recent research demonstrating the presence of bacteria in deep subsurface oil and gas deposits, and their role in mineral formation. Recent research, summarized in these reviews, leads to several general statements that can be made about ground-water microbiology:

  1. Most subsurface materials contain bacteria which can be cultured.
  2. Most of the bacterial types found in soils and surface waters have also been found in shallow unconfined and confined aquifers.
  3. The ground-water environment is different from other aquatic environments in that organic carbon is not replenished by photosynthetic organisms, but must be supplied from the surface or from the aquifer materials themselves.
  4. The ground-water environment is also different from other aquatic environments in that bacteria are the dominant inhabitants, although protozoa may also be common, and subterranean caves may harbor unique invertebrate faunas.
  5. The majority of the numbers and types of microbes in ground-water environments are found attached to the aquifer solids, and not free in the ground water itself.
  6. Many ground-water quality parameters, such as pH, oxidation/reduction (redox) status, dissolved oxygen, or the presence of specific mineral constituents, may be influenced by microbial activity in the aquifer. This is especially true when the aquifer is contaminated with substances that bacteria can use for growth.

Natural activities of bacteria in ground water.

Over a century of research on naturally-occurring bacteria and their activities allows us to interpret some of the roles of bacteria in ground- water environments. We know that bacteria are found everywhere in our environment. They are common in air, soil, water and in the habitats of our daily lives. Bacteria are commonly present in soil at numbers of about 108-109 cells per gram. Bacterial slime (biofilms) on rocks in streams and rivers may contain 109 bacteria per square centimeter. Pristine lake waters contain many thousands of naturally- occurring bacteria per liter. These naturally-occurring bacteria maintain the fertility of soil, they transform minerals and nutrients in water and sediments, and degrade leaf litter and other plant materials producing materials useful to other organisms. In addition, naturally-occurring bacteria carry out activities useful to humans by degrading wastes in our landfills and compost piles, and cleansing water of the pollutants we add. We purposefully use some bacteria to make food (cheese, beer, sauerkraut), we put bacteria to work in sewage treatment plants, and we use them in biotechnology to produce chemicals. Therefore, microbiologists have learned a great deal about the types and activities of naturally-occurring bacteria. Based on principles learned from other environments, we would expect bacteria in ground water to be able to:

  • transform organic carbon to carbon dioxide (CO2)
  • use up oxygen when sufficient carbon is available for growth
  • transform nitrogen between oxidized (e.g., nitrate – NO3) and reduced (e.g., ammonium – NH4 or nitrogen gas – N2) forms
  • transform iron between oxidized [Fe(III)] and reduced [Fe(II)] forms
  • transform sulfur between oxidized (e.g., sulfate – SO4) and reduced (e.g., sulfide – H2S) forms
  • produce methane
  • degrade pesticides, fuels and other organic contaminants
  • affect the distribution and solubility of some metals (e.g., arsenic, uranium, etc.)

Indeed, there is evidence for each of these activities in ground water. The review articles cited above are good resources for more information and case examples. In addition, Norris et al. (1994) provides a general review of the role of bacteria in natural and augmented bioremediation of fuels and solvents in ground water.

Most of the activities of bacteria in ground water are the direct result of the astounding metabolic versatility of bacteria. Although humans and other vertebrate and invertebrate animals are primarily dependent on respiration using oxygen, some bacteria may respire using NO3, SO4, oxidized (ferric) iron [Fe(III)] or a variety of metals (such as arsenic or uranium) as the oxidant. In addition, in the absence of oxygen, bacteria may carry out processes such as methane production or fermentation. Finally, bacteria may be capable of growth on some organic compounds which are toxic to other organisms. The combination of these unique metabolic capabilities suggests that bacteria play important roles in pristine and contaminated ground water environments. Nevertheless, bacteria are limited, as are all living things, by extremes of pH and temperature, by lack of nutrients to support growth, and by toxicity of some compounds. In addition, bacteria are subject to predation by larger microorganisms, such as protozoa. Each of these environmental features must be assessed when interpreting the role of bacteria in a particular ground water process.

Health effects of microbes in ground water

Although there are some bacteria in all ground waters, and in general they carry out beneficial processes, some bacteria or other microorganisms (e.g., protozoa, viruses) may cause disease in humans. Naturally some microorganisms have learned to live on or in the human body. Many of these microorganisms do no harm, and are even beneficial because they compete with other microorganisms that might cause disease if they could become established in or on our bodies. A few microorganisms (called pathogens) can cause disease in humans. Some of these disease-causing microorganism are closely associated with humans and other warm-blooded animals. These pathogens are transmitted from one organism to another by direct contact, or by contamination of food or water. Many of the pathogens which cause gastrointestinal disease are in this category. Several human gastrointestinal pathogens produce toxins which act on the small intestine, causing secretion of fluid which results in diarrhea. Cells of the pathogen are shed in the feces, and if these cells contaminate food or water which is then consumed by another person, the disease spreads. Other pathogens are “opportunists” : they may not be closely associated with humans or other mammals and they rarely cause disease in healthy adults. Instead, these may be common bacteria or fungi which exist in soil or water, but may cause disease in persons already weakened by a pre-exisiting disease.

The fecal indicator bacteria (Escherichia coli, fecal coliforms, fecal streptococci) are typically used to measure the sanitary quality of water for recreational, industrial, agricultural and water supply purposes. The fecal indicator bacteria are natural inhabitants of the gastrointestinal tracts of humans and other warm-blooded animals. These bacteria in general cause no harm. They are released into the environment with feces, and are then exposed to a variety of environmental conditions that eventually cause their death. In general, it is believed that the fecal indicator cannot grow in natural environments, since they are adapted to live in the gastrointestinal tract. Studies have shown that fecal indicator bacteria survive from a few hours up to several days in surface water, but may survive for days or months in lake sediments, where they may be protected from sunlight and predators. In ground water, temperature, competition with bacteria found naturally in the water, predation by protozoa and other small organisms, and entrapment in pore spaces may all contribute to their demise. We assume that pathogens similar to the fecal indicator bacteria die at the same rate as fecal indicator bacteria. Therefore, if we find relatively high numbers of fecal indicator bacteria in an environment, we assume that there is an increased likelihood of pathogens being present as well. Unfortunately, some pathogenic bacteria, viruses and protozoans may have special survival mechanisms, such as cyst formation in Cryptosporidium, or attachment of viruses to particles, so that waters free of fecal indicator bacteria may still harbor these microorganisms. This is even true of water which has undergone treatment for drinking water purposes.

There is no clear way to associate risk of disease with the bacteriological quality of ground water and measured by the presence of fecal indicator bacteria. First, there is no direct association between the presence of fecal indicator bacteria and the presence of specific pathogens. Second, individuals are not equally susceptible to pathogens. Whether or not a pathogen is successful in causing disease is related to the health of the exposed individual and the state of his or her immune system, as well as to the number of pathogen cells required to make the person ill. Some pathogens can cause disease when only a few cells are present. In other cases, many cells are required to make a person ill. Children, elderly persons and persons with pre-existing illnesses are more susceptible to many pathogens than are healthy young or middle-aged adults. Third, it would be difficult to monitor for every possible pathogen. Each type of pathogen requires a specific test and many of these tests are time-consuming or expensive. Monitoring for each type of known pathogen would be prohibitively expensive. Finally, new pathogens are still being discovered. It was only about 5 years ago that a specific bacterium was identified as a cause of stomach ulcers in humans. In addition, “old” bacteria are acquiring new “tricks” in that they are becoming resistant to antibiotics and are re-emerging as serious pathogens. The issue of emerging infectious disease, and a call for the strengthening of our public health knowledge base and infrastructure was made by the Centers for Disease Control (CDC) in 1994.

What factors may lead to microbial contamination of ground water?

Ground water has traditionally been considered to be the water source least susceptible to contamination by indicator bacteria or human pathogens. This is certainly true of ground water from deep, confined aquifers. Geldreich (1990) reviewed the microbiological quality of source waters for drinking water supply, the sources of contamination to ground water environments, and the instances of waterborne disease outbreaks attributed to untreated or poorly-treated ground water which contained pathogens. If fecal indicator bacteria or pathogens commonly associated with humans are present in ground water in measureable numbers, there is most likely a nearby connection with a contaminated surface environment, such as a seepage from a waste lagoon or a contaminated surface water, or a subsurface source of contamination such as a septic tank, a broken or leaking sewer line, or an old or improperly designed landfilll.

It is important to recognize that in spite of what we do know about bacteria and other microorganisms, we still know relatively little about their types, activities and habitats. For example, the ability of certain bacteria to grow by carrying out the reduction of Fe(III), arsenic or uranium was first demonstrated conclusively in the early 1990’s. Likewise, the discovery of new pathogens, the association of common bacteria or protozoa with specific diseases, occurs on a relatively frequent basis. Bacteria, viruses and protozoa are difficult to study, and most microbiologists believe that we have identified fewer than 10% of the types of bacteria actually present in nature. We also have only a very rudimentary understanding of what types of activities bacteria carry out in nature, and the environmental factors which influence their activities and survival. We have even less information on bacteria in ground water, since this field of study is so recent. It is likely that we will learn much more about the prevalence, activities, and significance of microorganisms in ground water in the coming decade.

Selected References:

CDC (Centers for Disease Control and Prevention). 1994. Addressing emerging infectious disease threats: a strategy for the United States. Atlanta, GA: U.S. Department of Health and Human Services. This report can be obtained via the World Wide Web at http://www.cdc.gov.

Chapelle, F.H. 1993. Ground-water microbiology and geochemistry. John Wiley and Sons, New York.

Fyfe, W.S. 1996. The biosphere is going deep. Science 273:448.

Geldreich, E.E. 1990. Microbiological quality of source waters for water supply. pp. 3-31 in G. A. McFeters (ed.) Drinking Water Microbiology: Progress and Recent Developments. Springer-Verlag, New York.

Madsen, E. L. and W. C. Ghiorse. 1993. Groundwater microbiology: subsurface ecosystem processes. pp. 167-214 in T.E. Ford (ed.) Aquatic Microbiology. Blackwell Scientific Publications, Boston.

Norris, R.D. et al. 1994. Handbook of Bioremediation. U.S. EPA Robert S. Kerr Environmental Research Laboratory. Lewis Publishers, Ann Arbor.


The contamination of drinking water by pathogens causing diarrhoeal disease is the most important aspect of drinking water quality. The problem arises as a consequence of contamination of water by faecal matter, particularly human faecal matter, containing pathogenic organisms. One of the great scourges of cities in Europe and North America in the 19th century was outbreaks of waterborne diseases such as cholera and typhoid. In many parts of the developing world it remains a major cause of disease. It is therefore essential to break the faecal–oral cycle by preventing faecal matter from entering water sources and/or by treating drinking water to kill the pathogens. However, these approaches need to operate alongside hygiene practices such as hand washing, which reduce the level of person-to-person infection.

Detection and enumeration of pathogens in water are not appropriate under most circumstances in view of the difficulties and resources required so Escherichia coli and faecal streptococci are used as indicators of faecal contamination. The assumption is that if the indicators are detected, pathogens, including viruses, could also be present and therefore appropriate action is required. However, the time taken to carry out the analysis means that if contamination is detected, the contaminated water will be well on the way to the consumer and probably drunk by the time the result has been obtained. In addition the small volume of water sampled (typically 100 ml) means that such check monitoring on its own is not an adequate means of assuring drinking water safety. It is also essential to ensure that the multiple barriers are not only in place but working efficiently at all times, whatever the size of the supply. Drinking water is not, however, sterile and bacteria can be found in the distribution system and at the tap. Most of these organisms are harmless, but some opportunist pathogens such as Pseudomonas aeruginosa and Aeromonas spp. may multiply during distribution given suitable conditions3. Currently there is some debate as to whether these organisms are responsible for any waterborne, gastrointestinal disease in the community but P. aeruginosa is known to cause infections in immunocompromised patients and weakened patients in hospitals.

A number of organisms are emerging as potential waterborne pathogens and some are recognized as significant pathogens that do give rise to detectable waterborne outbreaks of infection. The most important of these is Cryptosporidium parvum, a protozoan, gastrointestinal parasite which gives rise to severe, self limiting diarrhoea and for which there is, currently, no specific treatment. Cryptosporidium is excreted as oocysts from infected animals, including humans, which enables the organism to survive in the environment until ingested by a new host3. This organism has given rise to a number of waterborne or water associated outbreaks in the UK, and an outbreak of cryptosporidiosis in Milwaukee in the USA resulted in many thousands of cases4, and probably a number of deaths among the portion of the population which were immunocompromised5. The most important barriers to infection are those that remove particles, including coagulation, sedimentation and filtration. However, water is not the only source of infection. It is probable that person-to-person spread following contact with faecal matter from infected animals is more important and there have been outbreaks involving milk and swimming pools6. Currently, there is no scientifically based standard for Cryptosporidium in drinking water. A similar parasite, Giardi, has been responsible for a number of cases of gastrointestinal illness and in the USA, illness was referred to as beaver fever because beavers were shown to be a source in some areas. As with Cryptosporidium, water is not the only source but, unlike Cryptosporidium, it is reasonably susceptible to chlorine and because of its larger size can be more easily removed by particle removal processes3.

Although the common waterborne diseases of the 19th century are now almost unknown in developed countries, it is vital that vigilance is maintained at a high level because these diseases are still common in many parts of the world. The seventh cholera pandemic, which started in 1961, arrived in South America in 1991 and caused 4700 deaths in 1 year7. According to the WHO World Health Report 1998, over 1 billion people do not have an adequate and safe water supply of which 800 million are in rural areas. WHO also estimate that there are 2.5 million deaths and 4 billion cases due to diarrhoeal disease, including dysentery, to which waterborne pathogens are a major contributor. There are still an estimated 12.5 million cases of Salmonella typhi per year and waterborne disease is endemic in many developing countries. In this age of rapid global travel, the potential for the reintroduction of waterborne pathogens in developed countries still remains. In addition, as our knowledge of microbial pathogens improves, we are able to identify other organisms that cause waterborne disease. The Norwalk-like viruses are named after a major waterborne outbreak in North America, and there is a range of emerging pathogens including Campylobacter, a major cause of food poisoning, and E. coli O157, which has caused deaths in North America where chlorination was not present, or failed, and other barriers were inadequate3.

Microbial contamination of drinking water thus remains a significant threat and constant vigilance is essential, even in the most developed countries.

Chorus I, Bartram J. Toxic cyanobacteria in water. A Guide to their Public Health Consequences, Monitoring and Management. Published on behalf of WHO by E & FN Spon, London and New York, 1999
↵ World Health Organization. Guidelines for Drinking-Water Quality, 3rd edn. www.who.int/water_sanitation_health/GDWQ/draftchemicals/list.htm. Last accessed June 2003. Geneva: WHO, 2003
↵ Hunter P. Waterborne Disease. Epidemiology and Ecology. Chichester: Wiley, 1997


Wastewater Microbiology


Biology is the heart of wastewater treatment technology, and the microbial community is the heart of the wastewater treatment facility. In order to solve wastewater treatment problems, it is critical to fix the root cause of the problem (a microbial community that is not optimized) rather than just symptoms.

Of course, the first step to optimizing a microbial community is identifying and understanding the microorganisms that make up the biomass. That’s why Novozymes’ Wastewater Team created a Guide to Wastewater Microbiology, a 15-page free download that provides an introduction to microorganisms, growth requirements, and microbial processes related to wastewater treatment.

Surface Water Microbial Contamination


EPA develops criteria to protect people from microbial organisms (sometimes referred to as pathogens) such as bacteria and viruses in water bodies (e.g., lakes, rivers, beaches).  Pathogens can make our waters unsafe for humans. Swimming and other recreational activities in water contaminated with pathogens can make people ill. People can also become exposed by drinking untreated water from contaminated water bodies.

EPA recommends criteria for limiting microbial organisms in water bodies to protect human health. State and tribal governments can use the criteria as guidance when setting their own water quality standards to protect human health.

Microbiologically influenced corrosion (MIC)


Microbiologically influenced corrosion (MIC) is a serious problem that impacts nearly all industries and exacts a severe toll in terms of operating costs, loss of production, deterioration of capital equipment and the consequences of corrosion related failures. While a proactive step, monitoring for MIC associated microorganisms is often hindered by the fact that sampling methods may not capture key members of the microbial community involved in MIC (sampling biases) and current reliance on conventional culture-dependent methods which may underestimate and oversimplify the problem (cultivation bias). Molecular biological tools (MBTs) based on analysis of DNA extracted directly from field samples circumvent the limitations of culture-depend methods. However, sampling biases must also be addressed in order to ensure that molecular analyses truly provide more accurate and comprehensive MIC evaluation. Eckert et al (2003) have developed a method for embedding corrosion products and biofilms formed on corrosion coupons in a cured resin conserving their spatial orientation for a variety of analytical techniques. In the current study, a procedure was developed for DNA extraction from discrete sections of the resin embedded biofilm. Quantitative polymerase chain reaction (qPCR) was then used to quantify total bacteria and specific microorganisms commonly implicated in MIC. The development of the extraction procedure along with improvements in sectioning will permit the DNA based analyses (qPCR, microarrays, and even high-throughput sequencing) for the most direct characterization of microbial communities of corrosive biofilms.