LECTURE PRESENTATIONS For BROCK BIOLOGY OF MICROORGANISMS, THIRTEENTH EDITION Michael T. Madigan, John M. Martinko, David A. Stahl, David P. Clark Lectures by John Zamora Middle Tennessee State University Chapter 35 Wastewater Treatment, Water Purification, and Waterborne Microbial Diseases 2012 Pearson Education, Inc. I. Wastewater Microbiology and Water Purification 35.1 Public Health and Water Quality 35.2 Wastewater and Sewage Treatment 35.3 Drinking Water Purification 1
I. Wastewater Microbiology and Water Purification Water Potential common source of infectious diseases Can also be a source for chemically induced intoxications Ensuring water purity is essential for public health Treatment and purification schemes use microorganisms to identify, remove, and degrade pollutants 35.1 Public Health and Water Quality Coliforms and Water Quality Water can be sampled for the presence of specific indicator microorganisms The coliform group of microorganisms Includes Enterobacter, Escherichia coli, and Klebsiella pneumoniae They are a widely used indicator for fecal contamination in water 2
35.1 Public Health and Water Quality Testing for Coliforms and E. coli All tests assay the growth of organisms recovered from the water samples Common methods for enumerating the samples: Most-probable-number (MPN) procedure Membrane filter (MF) procedures (Figure 35.1) E. coli can be distinguished from total coliforms with defined substrates (e.g., MUG, IBDG; Figure 35.2) Figure 35.1 Coliform colonies growing on a membrane filter A drinking water sample was passed through the filter. The filter was then placed on eosin methylene blue (EMB) medium that is both selective and differential for lactosefermenting bacteria (coliforms). The dark, shiny appearance of the colonies is characteristic of coliforms. Each colony developed from one viable coliform cell present in the original sample. 3
Figure 35.2 Total coliforms and Escherichia coli. A filter exposed to a drinking water sample was incubated at 35 o C for 24 hours on MI media and examined under UV light. The single E. coli colony appears dark blue (arrow). The other colonies are coliforms that fluoresce and appear white to light blue. 35.1 Public Health and Water Quality A commonly used method for performing coliform counts is the IDEXX Colilert test system (Figure 35.3) Utilizes proprietary substrates Sample with no coliforms remains clear Coliforms turn media yellow E. coli turns media fluorescent blue 4
Figure 35.3 The IDEXX Colilert water quality test system Colilert reagents are added to 100-ml water samples. After incubation for 24 h at 35-37 o C, the samples develop yellow color if they contain coliform bacteria (right). Samples containing Escherichia coli develop yellow color and also develop blue fluorescence (left). Samples negative for coliform bacteria remain clear (center). 35.1 Public Health and Water Quality Public Health and Drinking Water Purification Effective water treatment practices that reduced the incidence of waterborne disease were not in place until the twentieth century (Figure 35.4) Coliform-counting methods were developed and adapted around 1906 Water purification started with filtration About 1913, chlorine came into use as a disinfectant for large water supplies 5
Number of typhoid cases 30.12.2012 Figure 35.4 The effect of water purification on the incidence of waterborne disease 10,000 Filtration, 1906 1000 Chlorination, 1913 100 1885 1895 1905 1915 1925 1935 1945 Year The graph shows the incidence of typhoid fever in Philadelphia, Pennsylvania. Note the dramatic reduction in the incidence of typhoid fever after the introduction of both filtration and chlorination. 35.2 Wastewater and Sewage Treatment Wastewater Domestic sewage or liquid industrial waste Wastewater treatment Relies on industrial-scale use of microbes for bioconversion Following treatment, the discharged treated wastewater (effluent water) is suitable for Release into surface waters Release to drinking water purification facilities 6
35.2 Wastewater and Sewage Treatment Wastewater treatment facility Its goal is to reduce organic and inorganic materials in wastewater to a level that no longer supports microbial growth and to eliminate other potentially toxic materials The efficiency of treatment is expressed in terms of a reduction in the biochemical oxygen demand (BOD) Amount of dissolved oxygen consumed by microbes to completely oxidize all organic and inorganic matter in a water sample 35.2 Wastewater and Sewage Treatment Wastewater treatment is a multistep operation employing both physical and biological processes (Figure 35.5) Primary, secondary and sometimes tertiary treatments are used Primary treatment Uses physical separation methods to separate solid and particulate organic and inorganic materials from wastewater 7
Figure 35.5 Wastewater treatment processes WASTEWATER Screening Sedimentation PRIMARY treatment Anaerobic digestion Aerobic oxidation Activated sludge/aeration Trickling filter SECONDARY treatment Digested sludge: drying; incineration; use as fertilizer, or burial Disinfection Treated effluent to discharge Effective water treatment plants use the primary and secondary treatment methods shown here. Tertiary treatment may also be used to reduce BOD levels in effluent water to undetectable levels. 35.2 Wastewater and Sewage Treatment Secondary treatment Anaerobic secondary treatment involves a series of digestive and fermentative reactions carried out by various microbes under anoxic conditions (Figure 35.7) The process is carried out in large enclosed tanks (sludge digesters or bioreactors) 8
Figure 35.7 Anaerobic treatment Sludge inlet Anaerobic sludge digester. Only the top of the tank is shown; the remainder is underground. Gas outlet CH 4 /CO 2 Scum layer Supernatant Actively digesting sludge Stabilized sludge Sludge outlet Scum removal Supernatant removal Inner workings of a sludge digester Complex polymers (polysaccharides, lipids, proteins) Monomers (sugars, fatty acids, amino acids) Fermentation Acetate Methanogenesis Hydrolysis by microbial enzymes Fermentation H 2 CO 2 CH 4 CO 2 CH 4 H 2 O Major microbial processes in anaerobic sludge digestion. Methane (CH 4 ) and carbon dioxide (CO 2 ) are the major products of anaerobic biodegradation. 35.2 Wastewater and Sewage Treatment Secondary Treatment (cont d) Aerobic secondary treatment uses digestive reactions carried out by microbes under aerobic conditions to treat wastewater with low levels of organic materials Activated sludge (Figure 35.8a & b) and the trickling filter (Figure 35.8c) are the most common decomposition processes 9
Figure 35.8a Aerobic wastewater treatment processes (aeration tank) Aeration tank of an activated sludge system in a metropolitan wastewater treatment plant. The tank is 30 m long, 10 m wide and 5 m deep. Figure 35.8b Aerobic wastewater treatment processes (activated sludge process) Wastewater from primary treatment Aeration tank Settling tank Clear effluent Air Activated sludge return Activated sludge Excess sludge Anaerobic sludge digester Wastewater flow through an activated sludge system. Recirculation of activated sludge to the aeration tank introduces microorganisms responsible for oxidative degradation of the organic components of the wastewater 10
Figure 35.8c Aerobic wastewater treatment processes (trickling filter) Trickling filter process. The booms rotate, distributing wastewater slowly and evenly on the rock bed. The rocks are 10-15 cm in diameter and the bed is 2 m deep. 35.2 Wastewater and Sewage Treatment In the activated sludge process, wastewater is mixed and aerated in large tanks and slimeforming bacteria (e.g., Zoogloea ramigera; Figure 35.9) grow and form flocs Most treatment plants chlorinate the effluent after secondary treatment to further reduce the possibility of biological contamination 11
Figure 35.9 A wastewater floc formed by the bacterium Zoogloea ramigera Floc formed in the activated sludge process consists of a large number of small, rodshaped cells of Z. ramigera surrounded by a polysaccharide slime layer, arranged in characteristic fingerlike projections in this negative stain with India ink. 35.2 Wastewater and Sewage Treatment Tertiary treatment Any physiochemical or biological process employing bioreactors, precipitation, filtration, or chlorination procedures similar to those used for purification of drinking water Reduces the levels of inorganic nutrients (e.g., phosphate, nitrate, nitrite) Most complete method of treating sewage but not widely adopted due to costs 12
35.3 Drinking Water Purification Wastewater treated by secondary methods is not yet potable, or safe for human consumption It requires further treatment to remove pathogens, eliminate taste and odor, reduce chemicals (e.g., iron, manganese), and decrease turbidity A typical drinking water treatment installation purifies raw (untreated) water (Figure 35.10a) Figure 35.10a Water treatment plant Ohio River River pumping station Coagulation basin Sedimentation basins Underground clear-water reservoir Chlorination Filter buildings Aerial view of a water treatment plant. The arrows indicate direction of flow of water through the plant. 13
35.3 Drinking Water Purification Purification involves many steps (Figure 35.10b) Sedimentation to remove particles Coagulation and flocculation form additional aggregates, which settle out Filtration Disinfection (typically with chlorine gas or UV radiation) Figure 35.10b Water treatment plant Remove sand, gravel, large particulates Coagulation Filtration Raw water Sedimentation Form and remove floc, containing insoluble material and microorganisms Remove remaining particulates and most organic and inorganic compounds Chlorination Storage Kill remaining microorganisms and prevent growth of new inocula Finished water Distribution Schematic overview of a typical community water purification system 14
II. Waterborne Microbial Diseases 35.4 Sources of Waterborne Infection 35.5 Cholera 35.6 Giardiasis and Cryptosporidiosis 35.7 Legionellosis (Legionnaires Disease) 35.8 Typhoid Fever and Other Waterborne Diseases 35.4 Sources of Waterborne Infection Common sources of waterborne disease transmission include Potable water used for drinking and cooking (Figure 35.11a) May be improperly treated or of low quality May be from nonregulated sources (e.g., private wells) Recreational water from public ponds, lakes, swimming pools, etc. (Figure 35.11b) 15
Number of outbreaks Number of outbreaks 30.12.2012 Figure 35.11a Waterborne disease outbreaks 60 50 40 30 20 10 Drinking water outbreaks 0 1974 1977 1980 1983 1986 1989 1992 1995 1998 2001 2004 Year Data were provided by the Centers for Disease Control and Prevention, Atlanta, Georgia, USA. Reported drinking water disease outbreaks from 1974 to 2006. Of 729 outbreaks, about 90% were due to biological agents (bacteria, viruses and protists) Figure 35.11b Waterborne disease outbreaks 45 Recreational water outbreaks 30 15 0 1980 1984 1988 1992 1996 2000 2004 Year Reported recreational water outbreaks from 1980 to 2006. Of 544 total outbreaks, almost all were due to biological agents. 16
35.4 Sources of Waterborne Infection Microorganisms transmitted in water generally grow in the intestines and leave the body in feces Feces then pollute water Numerous bacterial and protozoan pathogens can be transmitted in drinking water In the U.S. the number of disease outbreaks due to drinking or recreational water contamination is low Lack of adequate water treatment facilities and access to clean water contribute to the spread of infectious disease 35.5 Cholera Cholera Severe diarrhea Caused by Vibrio cholerae (Figure 35.12) Typically transmitted through ingestion of contaminated water Largely restricted to developing countries Endemic in Africa, Southeast Asia, the Indian subcontinent, and Central and South America Can be controlled by application of water treatment 17
Figure 35.12 Cells of Vibrio cholerae This colorized scanning electron micrograph shows a rod to curved rod morphology. The organism is about 0.3 µm in diameter and up to 2 µm in length. 35.5 Cholera Pathogenesis V. cholerae attaches to epithelial cells in the small intestine, where it grows and releases cholera toxin This enterotoxin causes severe diarrhea Can result in dehydration and death unless treated 18
35.5 Cholera Diagnosis Presence of V. cholerae bacilli in the feces of patients Prevention Immunization not recommended Public health measures are very important Treatment Oral rehydration and electrolyte replacement therapy 35.6 Giardiasis and Cryptosporidiosis Giardia intestinalis causes giardiasis, an acute gastroenteritis Cryptosporidium parvum causes cryptosporidiosis These organisms are found in nearly all surface waters and are highly resistant to chlorine due to cyst formation 19
35.6 Giardiasis and Cryptosporidiosis Giardia intestinalis (Figure 35.16) A flagellated protist that is usually transmitted to humans in fecally contaminated water The protist cells produce a resting stage (cyst) that is resistant to desiccation and chemical disinfection Following ingestion, cysts germinate and attach to the intestinal wall, causing symptoms of infection Many individuals exhibit no symptoms and can act as carriers Figure 35.16 The parasite Giardia Scanning electron micrographs. A motile trophozoite*. The trophozoite is about 15 µm in length. A giardial cyst. The cyst is about 11 µm in length. 20
35.6 Giardiasis and Cryptosporidiosis Cryptosporidium parvum (Figure 35.17) Protist that lives as a parasite in warm-blooded animals Produces thick-walled cells (oocysts) that are shed in the feces of infected animals The oocysts are transmitted in fecally contaminated water Oocysts are highly resistant to chlorine and UV radiation; thus sedimentation and filtration methods are most effective at removal Figure 35.17 Cryptosporidium The arrows point to two of the many intracellular trophozoites embedded in human gastrointestinal epithelium. The trophozoites are 2-5 µm. The thick-walled oocysts are 3-5 µm in diameter in this fecal sample. 21
35.6 Giardiasis and Cryptosporidiosis Cryptosporidiosis is usually a self-limiting mild diarrhea But can lead to more serious disease in compromised individuals 35.7 Legionellosis (Legionnaires Disease) Legionella pneumophila Causes legionellosis Normally transmitted in aerosols Gram-negative bacterium with complex nutritional requirements (Figure 35.18) Common in terrestrial and aquatic habitats Relatively resistant to heating and chlorination 22
Figure 35.18 Legionella pneumophila Colorized scanning electron micrograph of L. pneumophila cells. Cells are 0.3-0.6 µm in diameter and up to 2 m in length. 35.7 Legionellosis (Legionnaires Disease) Legionella pneumophila (cont d) Common in cooling towers and evaporative condensers of large air conditioning systems The pathogen grows in the water and is disseminated in humidified aerosols Infection is not spread from person to person 23
Cases per one million people 30.12.2012 35.7 Legionellosis (Legionnaires Disease) The prevalence of legionellosis is increasing and infections are often underreported (Figure 35.19) Treatment with antibiotics is effective Prevention can be accomplished by improving maintenance and design of water-dependent cooling and heating systems and water delivery systems Figure 35.19 Incidence of Legionnaire s disease in the United States 12 10 8 6 4 2 0 1992 1997 2002 2007 Year In 2007, there were 2716 reported cases. Data are from the Centers for Disease Control and Prevention, Atlanta, Georgia 24
35.8 Typhoid Fever and Other Waterborne Diseases Worldwide, the most important pathogenic bacteria transmitted by water are Salmonella enterica serovar Typhi and Vibrio cholerae S. enterica ser. Typhi causes typhoid fever and has been virtually eliminated in developed countries due to water treatment 35.8 Typhoid Fever and Other Waterborne Diseases Viruses can also be transmitted in water and cause human disease For example, enteroviruses such as poliovirus, norovirus, and hepatitis A are shed into water from feces Viruses can survive in water for relatively long periods but are inactivated by disinfection with chlorine 25
35.8 Typhoid Fever and Other Waterborne Diseases Entamoeba histolytica (Figure 35.20) A pathogenic protist transmitted to humans primarily through contaminated water and sometimes food Anaerobic and produces resistant cysts Infection can be asymptomatic or lead to diarrhea and/or dysentery If untreated, invasive cells can invade the liver and occasionally the lungs and brain Amoebicidal drugs are not universally effective Figure 35.20 The trophozoite of Entamoeba histolytica, the amoeba that causes amebiasis The small red structures are red blood cells. The trophozoites range from 12 to 60 µm in length. 26
35.8 Typhoid Fever and Other Waterborne Diseases Naegleria fowleri (Figure 35.21) A free-living amoeba found in soil and water runoff that can also cause amebiasis Infections usually result from swimming in warm, soil-contaminated water sources (e.g., hot springs or lakes) Enters the human body through the nose and burrows directly into the brain, causing extensive hemorrhage and brain damage (meningoencephalitis) Drug treatment is effective if infections are identified early Figure 35.21 Trophozoites of Naegleria fowleri in brain tissue This amoeba causes meningoencephalitis. Oval to round and amoeboid (irregularly shaped) trophozoites (arrows) are present as dark-stained structures with densely stained nuclei. There is extensive destruction of the surrounding brain tissue. Individual trophozoites are 10 35 µm long. 27