FUNDAMENTALS OF MICROBIOLOGY

Lesson 31. MICROBIOLOGY OF AQUATIC WATER

Module 7. Environmental microbiology

Lesson 31

MICROBIOLOGY OF AQUATIC WATER

31.1 Bacteria

Bacteria that occur in water habitats may be divided into the following:

• Autochthonous (native) constantly occupying water habitats
• Allochthonous (foreign) finding their way from the soil or the air as well as microorganisms that get into the water basins along with municipal and industrial sewage.

31.1.1 Autochthonous bacteria

We can distinguish photoautotrophs, chemoautotrophs and chemoorganoautotrophs as follows:

a) photosynthesizing bacteria (photoautotrophs)

Purple and green bacteria are among the photosynthesizing autotrophs. Due to their metabolism these bacteria can be divided into the following groups:

• Filiform green bacteria (Chloroflexaceae),
• Sulfuric green bacteria (Chlorobiaceae),
• Sulfuric purple bacteria (Chromatiaceae and Ectothiorhodaceae),
• Non-sulfuric purple bacteria (Rhodospirillaceae),
• Heliobacteria (Heliobacteriaceae).

The photosynthesis of bacteria is carried out slightly differently to that of plants. Most importantly, it is an oxygen-free process which requires the presence of reduced mineral compounds and it is not accompanied by a release of oxygen but by a production of oxidized non-organic or organic compounds. The assimilating pigments of bacteria are categorized by the ability to absorb infrared light that is not absorbed by green plants. The photosynthesis in surface waters is conducted mainly by algae and plants and the role of the bacterial photosynthesis is less important.

b) Chemosynthesizing bacteria (chemoautotrophs)

Chemoautotrophs get energy from the oxidation processes of non-organic compounds.

Depending on the nature of the oxidized substrate the following can be distinguished: nitrifying, ferruginous, sulfuric and hydrogen bacteria.

• The role of the nitrifying bacteria in surface waters is the oxidation of ammonia and nitrite to nitrate. In greater concentrations the above compounds may be harmful to water organisms as well as to humans (in cases when such water is utilized for water supply systems). Moreover, the production of nitrate is a fundamental process that supplies water plants with a source of nitrogen.
• Ferruginous bacteria grow in waters when the content of bivalent iron ranges between 0.15-8.5 mg/dm³. Their negative influence includes corrosion and fouling of plumbing, sewage systems and different metal constructions. The most common ferruginous bacteria are the Leptothrix ochracea and Crenothrix polyspora and they belong to the filamentous bacteria which are categorized by the fact that the single cells form thread-like forms surrounded by a gelatinous sheath of various thickness. Stored ferruginous substances in cells change the coloration of cells threads into a yellow or dark-brown shade. The ferruginous bacteria are very common in fresh bodies of water. Especially in waters from wells and springs where it is possible to observe their clusters with the naked eye. Moreover, they occur abundantly in muddy streams, marshes and ponds.

• Sulfuric bacteria occur mainly in waters containing hydrogen sulfide which is toxic for most microorganisms, whereas for this group it is one of the crucial compounds for survival. These bacteria can be found in mineral springs that contain hydrogen sulfide of geological origin as well as in highly polluted waters where it is produced as a result of oxygen-free protein decomposition or desulfurication processes. The typical representatives of the sulfuric bacteria are: bacteria that move in sliding motions Beggiatoa alba and fixed to the bottom Thiothrix nivea. The forms of individual sulfuric bacteria are:

-Thiobacillus thioparus - stores sulfur derived from oxidation of thiosulfate

-Thiobacillus thiooxidans - grows in acidic habitats of pH 1.0 - 4.0

-Thiobacillus ferroxidans - besides thiosulfates and tetrationans, it possesses the ability to decompose ferruginous salts.

-Thiobacillus denitrificans - is a relative anaerobe and it has ability to utilizenitrates as the electron acceptor during the oxidation of hydrogen sulfide. In aerobic conditions the above function is performed by the oxygen.

• Hydrogen bacteria have ability to oxidize hydrogen using oxygen as a final acceptor of electrons. Most often they feed heterotrophically and switch to autotrophic feeding when hydrogen is present in the habitat. The most widespread species belong to the genus Hydrogenomonas. Micrococcus denitrificans belongs to a group of the hydrogen bacteria and they conduct the oxidation of hydrogen while simultaneously reducing nitrate down to molecular nitrogen. Desulfovibrio desulfuricans also oxidizes hydrogen while reducing sulphate down to hydrogen sulphide.

• Heterotrophic bacteria (chemoorganotrophs) A predominant part of autochthonous bacteria which occur in water basins are the chemoorganotrophic bacteria which belong to a group of saprophytes that feed upon dead plant and animal organic matter. Typical bacteria plankton that occupy an entire water mass are the cilliated Gramnegative rods and they represent the following genera: Pseudomonas, Achromobacter Alcaligenes, Vibrio and Aeromonas, as well as the Gram-positive cocci that belong to the Micrococcus genus, Treponema and spiral bacteria of the Spirillum genus. The underwater parts of higher plants and the underwater fixed particles are colonized by numerous stem-like bacteria (e.g. Caulobacter), sheathed, filiform, and gemmating bacteria (e.g. Hyphomicrobium), which are one of the microorganisms forming the periphyton. Organisms which usually grow in bottom sediments are oxygen-free putrefactive bacteria, then oxygen-free cellulolytic bacteria and finally oxygen-free chemoorganotrophs such as Desulfovibrio genus that reduce sulfate down to the hydrogen sulphide. In addition there are some less numerous oxygen-free methane generating bacteria which reduce organic compounds down to methane

31.1.2 Allochthonous bacteria

Waters of high fertility and also highly polluted surface waters are abundant in saprophytes and parasitic bacteria from among which, the following are predominant: Gram negative intestinal rods of Escherichia coli as well as the Proteus genus, Klebsiella and Enterobacter, and also rods of Pseudomonas aeruginosa and of the Arthrobacter genus. Moreover, Gram-positive rods (bacilli) of the Bacillus, Corynebacterium and Clostridium genera, which are washed out from the soil and get into the bodies of water during heavy rainfalls, also belong to the allochthonous bacteria. Municipal wastes are the main source of pathogenic bacteria. Moreover, during the infiltration processes and surface run-offs, soil bacteria find their way into the waters as well. The role of air in water contamination is significant in densely populated areas of cities and industrial regions.

31.2 Water Fungi

In contrast to bacteria which grow best in waters of pH between 6 and 8, fungi occur only in waters below pH 6.0. Usually fungi occur in shallow waters, right on or just below the surface, which is closely connected to the fact that the organisms require significant amounts of oxygen). The predominant fungi in water environments are represented by mold fungi which belong to the Oomycota class (Leptomitus, Phytophthora) and to the class of Zygomycota (Mucor and Rhizopus). Relatively frequently fungi belonging to Ascomycota as well as the Deuteromycota are found in surface waters. Almost all fungi are heterotrophs that decompose organic matter; waters are occupied by both saprophytes and parasites which colonize water plants and animals. They have more diverse shapes than bacteria and they differentiate into larger cells and more complicated structures. In addition to unicellular ones there are also multi-cellular fungi with large mycelium.

Fungi usually do not occur in clean waters. They grow in abundance on the bottom of waters polluted by sewage (e.g. Leptomitus lacteus).

31.3 Blue-Green Algae

Blue-green algae are a group of organisms previously considered to be algae. Currently they are classified to the Procaryota kingdom and the sub-kingdom of Eubacteria. There are unicellular, colonial (loose cells connected with a single mucus envelope) and filamentous forms. The prokaryotic organisms contain a nucleoid instead of an isolated nucleus. In contrast to other bacteria they are capable of conducting oxygen photosynthesis. They contain chlorophyll and sometimes disguise it in other photosynthesizing pigments: ficocyanine and alloficocyanine. Characteristically the blue-green coloring of blue-green algae comes from the combination of chlorophyll and ficocyanine. Blue-green algae reproduce mainly through proliferation by cell fission. Their characteristic trait is that they possess gaseous vacuoles which allow movement in water to places of better illumination. Some (Anabaena) are capable of binding atmospheric nitrogen in structures called heterocysts. Due to their resistance to extreme environmental conditions they are ubiquitous. They can be found in deserts and in hot springs. Blue-green algae can cause blooming in lakes and other water reservoirs. Some of them produce toxic metabolites.

31.4 Algae

Algae are the simplest autotrophic eukaryotes that incorporate over 20 thousand species. Algae occur in fresh and sea waters. They are important producers of organic matter and oxygen. Algae live in the form of single cells or they create multicellular body of various shapes called thallus (threads, spheres, multilayer clusters). The composition of algae community changes significantly with respect to quality and quantity, depending on the content of the mineral salts in any given reservoir as well as on the characteristics of the substances that make up the main pollutant.

The following are the characteristic algae that occur in oligotrophic waters: diatoms of the following genera: Asterionella, Tabellaria, Melosira and some other algae (Dinobrion). In eutrophic waters the content of algae is completely different. Most of all, such waters contain only a vestigial number of diatoms, and instead of them the algae from the Dinophyta class as well as the Spirogyra genus appear.

31.5 Water Protozoa

Protozoa live in all types of waters, from small puddles, to inland waters, to the seas. They feed heterotrophically absorbing the dissolved organic compounds or feeding upon bacteria. They are most numerous in highly polluted waters and are the element of activated sludge. When the pollution level is not too high ciliates become predominant, and that concerns both the free-swimming ones (e.g. Paramecium) and the settled ones (e.g. Vorticella). Protozoa can be subdivided into four classes:

31.5.1 Flagellata – flagellates

These move utilizing long flagella. They feed heterotrophically and occur in polluted waters or in inefficiently functioning activated sludge. Besides dissolved substances, they may also absorb bacteria or unicellular algae. Flagellates live individually or in colonies. There are parasitic forms among them too. This is exemplified by a human parasite Giardia lamblia and the Trypanosoma gambiense which is transferred on to humans by the Tsetse fly causing African sleeping-sickness and neurological disturbances.

31.5.2 Ciliata–ciliates

Most of the representatives lead a free-swimming life style (Paramaecium, Euplotes), others crawl or are attached to the bottom. They feed upon bacteria, algae and organic substances. Ciliates occur in large numbers in polluted waters and in activated sludge. Some, such as Balantidum coli which causes dysentery, are parasites of animals and humans.

31.5.3 Rhizopoda-amoebae

The cells move around utilizing the plasmatic pseudopodia which are used for locomotion and for capture of food. Some amoebae have a changeable shape, others however, have a constant shape as they are equipped with a mini-skeleton or an outer shell. Some amoebae lead a parasitic life (Entamoeba histolytica).

31.5.4 Sporozoa

Only parasites belong to this class and representatives are Cryptosporidium parvum causing intestinal diseases and Plasmodium malariae causing the malaria. The second parasite attacks the red blood cells. This pathogen is carried by the Anopheles mosquito.

31.6 Water Transmitted Pathogenic Microorganisms

31.6.1 Bacteria

The groups of obligate pathogenic bacteria, which occur in polluted surface waters, contain rods causing typhoid fever (Salmonella typhi), as well as other Gram negative bacteria of the Salmonella genus (the cause of various infections of the digestive tract). Bacterial dysentery caused by Gram negative rods of the Shigella genus are not as common as the above. In surface waters of tropical countries, bacteria of the Vibrio genus (cholera) frequently occur. Moreover, Mycobacterium tuberculosis causing tuberculosis and treponema of the Leptospira can be also found in polluted waters. The latter bacteria cause bacterial jaundice. In surface waters which are described as opportunistic microorganisms (facultatively, pathogenic). These belong to the Pseudomonas, Aeromonas, Klebsiella, Flavobacterium, Enterobacter, Citrobacter, Serratia, Acinetobacter, Proteus and Providencia genera. All of the rods are part of the usual flora of the intestine and are not typically pathogenic for as long as they occur in human or animal digestive tracts. In some cases though, these bacteria find their way into other organs becoming a potential cause of different illnesses such as inflammation of urinary and respiratory systems and also sepsis which is a general infection of all internal organs.

31.6.2 Viruses

Besides pathogenic bacteria of surface waters, into which municipal and industrial sewage is disposed, the waters also contain significant amounts of other pathogenic microorganisms such as the polio virus. They are responsible for causing the Heine-Medina disease (polio). Enteroviruses, which cause intestinal infections, occur even in slightly polluted rivers.

31.6.3 Protozoa

Infections of the digestive tract caused by protozoa may come from contaminated water. Most parasitic protozoa produce cysts which are able to survive inside their host in unfavorable conditions. When the conditions improve, cysts transform into so called trophozoits, the vegetative form occurring in humans.

31.6.4 Parasitic fungi

In polluted surface waters parasitic fungi can also occur, for example Microsporum sp., Trichophyton sp. and Epidermophyton sp. They are dermatophytes causing ringworm and other cutaneous infections.

A list of water borne bacteria and viruses along with associated diseases is provided in (Table 31.1 – 31.2) respectively.

31.7 Indicator Organisms

In routine laboratory work, which conducts sanitary-epidemiological supervision, it is impossible to constantly monitor water for all pathogenic and potentially pathogenic microorganisms, which may be found in water. Therefore, routine monitoring concentrates mainly on detecting bacteria that indicate faecal contamination of water. The sanitary quality of water may be checked by utilizing the saprophytic microflora that occupy the human large intestine. The following indicators of water contamination have been adopted:

• Coliforms
• Faecal coliforms
• Faecal streptococci
• Bacilli of Clostridium genus, sulphite-reducing bacteria
• Staphylococci – coagulase positive
• Pseudomonas aeruginosa

31.7.1 Coliforms

Bacteria of the coli group are mainly made up of strains of Escherichia coli as well as the genera: Enterobacter, Citrobacter and Klebsiella. They are detected on media containing lactose at 37°C.

Faecal coliforms (thermotolerant) are mainly strains of Escherichia coli and only some of the strains of Enterobacter, Citrobacter and Klebsiella, which have an ability to ferment lactose at 44°C.

The presence of coliforms or faecal coliforms in a water sample indicates relatively recent contamination of water with faecal matter, sewage, soil or with decaying plants. For most types of waters a quantitative determination of both groups of coliforms is recommended.

31.7.2 Faecal streptococci

While in a water environment, faecal streptococci are characterized by a slightly longer period of survival and resistance to most disinfecting products than the coliforms. Faecal streptococci include microorganisms of Enterococcus and Streptococcus genera, which belong to the serological group of Lancefield D. Detection of faecal streptococci in a test sample, significantly exceeding the coli group bacteria, may suggest water contamination with animal faecal matter or sewage from animal farms.

31.7.3 Bacilli of clostridium genus

The detection of sulphite reducing bacteria (mainly strains of Clostridium perfringens) may suggest less recent contamination with faecal matter; their endospores are able to survive for many years in unfavourable conditions. Sulphite reducing clostridia are a good indicator of properly conducted water treatment processes - coagulation, sedimentation, and filtration. Endospores of these bacteria as well as the cysts of parasitic protozoa (Cryptosporidium parvum, Giardia lamblia) ought to be eliminated in those stages of water treatment, because they are especially resistant to the disinfecting agents. Conducting analysis of a water sample, in order to detect bacteria of the Clostridium genus, is technically less complicated than searching for parasitic protozoa and it ensures that the treated water is free from protozoa and from the eggs of pathogenic worms (Helminthes).

31.7.4 Pseudomonas aeruginosa

Currently, detection of Pseudomonas aeruginosa bacteria in drinking water, running water, swimming pools and surface waters is recommended in addition to the above elements of sanitary analysis. They are Gram-negative rods that do not produce spores. Their characteristic trait is the ability to produce a blue-green pigment - pyocyanin as well as a fluorescent pigment – fluorescein. Representatives of this species were isolated from human faeces and in cases of infection from urinary tracts, inner ear, suppurating wounds etc. These bacteria pose a potential pathogenic danger for both humans and animals. In addition, they are widely distributed in surface waters and soil. It is also important that the species may live in chlorinated water because it is, to some extent, resistant to disinfection.

31.7.5 Staphylococci

The Staphylococcus genus is mainly used to assess sanitary quality of swimming pools. Recreation waters are the cause of infections of respiratory tracts, skin and eyes. For this reason microbiological analysis based on standard indicators (coliforms) is insufficient. Some researchers have recommended Staphylococcus aureus to be used as an additional indicator of sanitary quality of recreational waters, because its presence is associated with human activity in these waters.

31.8 Total Number of Bacteria

In routine analysis the total number of bacteria present in 1 ml of water is also determined by an agar plate method. One set of plates is incubated at 37°C for 48 h (mesophilic bacteria). Another set of plates is incubated at 22°C for 72 h (psychrophilic bacteria). After incubation the colonies are counted and the amount of cfu/ml (colony forming units) can be calculated.

31.8.1 Total number of psychrophilic bacteria

Non pathogenic water bacteria grow mainly at lower temperatures. It is important that Gram-negative bacteria in water produce lipopolysaccharides in their cell wall which can be toxic – like endotoxins of pathogenic bacteria. Because of this, their numbers in water should be constantly monitored. A large increase in their numbers is evidence of the presence of easily available organic compounds in the water. Theoretically, the presence of 0.1 mg organic carbon in water can result in an increase of bacteria up to 108 cfu in 1 ml. Phosphorus is also a factor which stimulates the growth of microorganisms. Adding even small amounts of this element (<50 mg/l) causes 10 times the acceleration of bacterial growth in a water treatment plant.

31.8.2 Total number of mesophilic bacteria

More dangerous are high numbers of bacteria growing at 37°C, because among this high population, pathogenic forms may be found which are dangerous for human health.

High number of bacteria in samples of water can prove that water treatment processes proceed badly or that polluted water is siphoned.

Table 31.1 Bacterial infections

Table 31.2 Intestinal viruses which may be transmitted by water and diseases caused by them