2Microbiology of Environmental Engineering SystemsVolodymyr IvanovCONTENTSMICROBIALGROUPSANDTHEIRQUANTIFICATIONMICROBIALECOSYSTEMSMICROBIALGROWTHANDDEATHDIVERSITYOFMICROORGANISMSFUNCTIONSOFMICROBIALGROUPSINENVIRONMENTALENGINEERING SYSTEMSREFERENCESAbstract Type of energy generation is the major feature in physiological classificationof prokaryotes.Chemotrophs can be separated within four groups bythe type of electronacceptor: (a) anaerobic fermenting prokaryotes, producing biologically available energy byintramolecular oxidation-reduction;(b)anaerobically respiring prokaryotes, using other thanoxygen electron acceptors; (c) microaerophilic bacteria, producing energy by aerobic respi-ration at low concentration of oxygen; (d) obligate aerobes, producing biologically availableenergy with oxygen as electron acceptor.There are also intermediary subgroups, which areusingdifferenttypes of energyproduction,depending on conditions.Phototrophs also canbe classified into related physiological groups by the type of electron donor: (a) electrondonors are products of anaerobic fermentation (organic acids,alcohols, and H2); (b)electrondonors areproducts of anaerobicrespiration (H2S,Fe2+); (c)electron donors areproducts ofmicroaerophilic respiration (S); (d) electron donors are products of aerobic respiration (H2O).To overcome contradiction between the physiological groups and rRNA gene sequencing-based phylogenetic groups,theperiodic table of prokaryotes comprising and explaining theexistenceof allphysiologicalgroupsofprokaryoteswasproposed.Themainfeatureoftheperiodic table of prokaryotes is three parallel phylogenetic lines: (a) prokaryotes with Gram-negative type cell wall, habiting mainly in aquatic systems with stable osmotic pressure;(b)prokaryotes with Gram-positive type cell wall, habiting mainly in terrestrial systems withvaried osmotic pressure; (c) Archaea that lack conventional peptidoglycan and habiting mainlyFromHanbokof EniromentalEngineeringVolme:EmiomentalBiotechnoloyEdited by: L.K. Wang et al, DOI: i0.1007 /978-1-60327-140-0_2 Springer Science+ Business Media, LLC 201019
2 Microbiology of Environmental Engineering Systems Volodymyr Ivanov CONTENTS MICROBIAL GROUPS AND THEIR QUANTIFICATION MICROBIAL ECOSYSTEMS MICROBIAL GROWTH AND DEATH DIVERSITY OF MICROORGANISMS FUNCTIONS OF MICROBIAL GROUPS IN ENVIRONMENTAL ENGINEERING SYSTEMS REFERENCES Abstract Type of energy generation is the major feature in physiological classification of prokaryotes. Chemotrophs can be separated within four groups by the type of electron acceptor: (a) anaerobic fermenting prokaryotes, producing biologically available energy by intramolecular oxidation-reduction; (b) anaerobically respiring prokaryotes, using other than oxygen electron acceptors; (c) microaerophilic bacteria, producing energy by aerobic respiration at low concentration of oxygen; (d) obligate aerobes, producing biologically available energy with oxygen as electron acceptor. There are also intermediary subgroups, which are using different types of energy production, depending on conditions. Phototrophs also can be classified into related physiological groups by the type of electron donor: (a) electron donors are products of anaerobic fermentation (organic acids, alcohols, and H2); (b) electron donors are products of anaerobic respiration (H2S, Fe2+); (c) electron donors are products of microaerophilic respiration (S); (d) electron donors are products of aerobic respiration (H2O). To overcome contradiction between the physiological groups and rRNA gene sequencingbased phylogenetic groups, the periodic table of prokaryotes comprising and explaining the existence of all physiological groups of prokaryotes was proposed. The main feature of the periodic table of prokaryotes is three parallel phylogenetic lines: (a) prokaryotes with Gramnegative type cell wall, habiting mainly in aquatic systems with stable osmotic pressure; (b) prokaryotes with Gram-positive type cell wall, habiting mainly in terrestrial systems with varied osmotic pressure; (c) Archaea that lack conventional peptidoglycan and habiting mainly From: Handbook of Environmental Engineering, Volume 10: Environmental Biotechnology Edited by: L. K. Wang et al., DOI: 10.1007/978-1-60327-140-0_2 c Springer Science + Business Media, LLC 2010 19
20V.Ivanovin extreme environments.There are four periods in the periodic table of prokaryotes: anaer-obicfermentation,anaerobicrespiration,microaerophilicrespiration,andaerobicrespirationThree phylogenetic lines and four periods create 12 groups comprising all chemotrophicand phototrophic prokaryotes.Existence of Gram-positive phototrophic bacteria using prod-ucts of anaerobic,microaerophilic,and aerobic respiration as electron donors was predictedusing this periodic table of prokaryotes.Evolutionary parallelism in phylogenetic lines ofprokaryotes could behypothetically explained by synchronous evolution ofaquatic,terrestrialand extreme ecosystems and horizontal exchange of genes between these ecosystems.Theperiodic table of prokaryotes helps to understand microbial physiological diversity of envi-ronmental engineering systems and can be used in the design of environmental engineeringprocesses.Key Words Prokaryotes·physiological classification·evolutionary parallelism·environ-mental engineering systems.1.MICROBIALGROUPSANDTHEIROUANTIFICATIONMicrobiology is a branch of biology devoted to the study of microorganisms (microbes),which includeboth unicellular and multicellular organisms.Thesemicroorganisms are not vis-ible without the aid of a microscope because they are smaller than 70-100 μm.Microbiologi-cal sciences,such as industrial microbiology,medical microbiology,veterinary microbiologyagricultural microbiology and environmental microbiology,are specified by their objects ofstudy.Environmentalmicrobiology studiesmicrobesinpartsofbiospheresuchaslithospherehydrosphereand atmosphere.Themicrobiology of environmental engineering systems is asubset of environmental microbiology.The objects of this science are the engineering systemsofwater,wastewater, solidwastes,soil andgasbiotreatment.The microbiology of environmental engineering systems pursues practical goals such as:1.Developmentofbiotechnologiesfor themicrobial treatmentofwater,wastewater,solid wastes,soil and gas.2.Development of methods to prevent the outbreaks of water-borne, soil-borne, vector-borne, andairborneinfectiousdiseases.3.Developmentof methods to monitor and control environmental engineering systems.However, achievement of such practical goals is not possible without studying the followinggeneral problems of environmental microbiology:1.Classificationandidentificationofmicroorganisms.2.Physical,chemical and biological interactionsbetween microorganisms and macroorganisms3.Physical and chemical interactionsof microorganisms and environment.4.Biochemical,physiological and cellular adaptations and regulations in microbial systems.This chapter is intended for environmental engineers as well as environmental engineeringstudents who do not possess an in-depth microbiological background. We will address thebasicprinciples ofmicrobiologyofenvironmental engineering systems,with special attentionpaid to the interconnections and diversity of microbial groups as well as their functions in
20 V. Ivanov in extreme environments. There are four periods in the periodic table of prokaryotes: anaerobic fermentation, anaerobic respiration, microaerophilic respiration, and aerobic respiration. Three phylogenetic lines and four periods create 12 groups comprising all chemotrophic and phototrophic prokaryotes. Existence of Gram-positive phototrophic bacteria using products of anaerobic, microaerophilic, and aerobic respiration as electron donors was predicted using this periodic table of prokaryotes. Evolutionary parallelism in phylogenetic lines of prokaryotes could be hypothetically explained by synchronous evolution of aquatic, terrestrial, and extreme ecosystems and horizontal exchange of genes between these ecosystems. The periodic table of prokaryotes helps to understand microbial physiological diversity of environmental engineering systems and can be used in the design of environmental engineering processes. Key Words Prokaryotes physiological classification evolutionary parallelism environmental engineering systems. 1. MICROBIAL GROUPS AND THEIR QUANTIFICATION Microbiology is a branch of biology devoted to the study of microorganisms (microbes), which include both unicellular and multicellular organisms. These microorganisms are not visible without the aid of a microscope because they are smaller than 70–100 μm. Microbiological sciences, such as industrial microbiology, medical microbiology, veterinary microbiology, agricultural microbiology and environmental microbiology, are specified by their objects of study. Environmental microbiology studies microbes in parts of biosphere such as lithosphere, hydrosphere and atmosphere. The microbiology of environmental engineering systems is a subset of environmental microbiology. The objects of this science are the engineering systems of water, wastewater, solid wastes, soil and gas biotreatment. The microbiology of environmental engineering systems pursues practical goals such as: 1. Development of biotechnologies for the microbial treatment of water, wastewater, solid wastes, soil and gas. 2. Development of methods to prevent the outbreaks of water-borne, soil-borne, vector-borne, and airborne infectious diseases. 3. Development of methods to monitor and control environmental engineering systems. However, achievement of such practical goals is not possible without studying the following general problems of environmental microbiology: 1. Classification and identification of microorganisms. 2. Physical, chemical and biological interactions between microorganisms and macroorganisms. 3. Physical and chemical interactions of microorganisms and environment. 4. Biochemical, physiological and cellular adaptations and regulations in microbial systems. This chapter is intended for environmental engineers as well as environmental engineering students who do not possess an in-depth microbiological background. We will address the basic principles of microbiology of environmental engineering systems, with special attention paid to the interconnections and diversity of microbial groups as well as their functions in
21MicrobiologyofEnvironmentalEngineeringSystemsenvironmental engineering systems. A more in-depth description of these topics is given inspecialized chapters of this book.1.1.Groupsof MicroorganismsThe objects of the microbiology of environmental engineering systems include bacteria(prokaryotes), microscopic fungi, microscopic algae, protozoa and other microscopic objectssuch as viruses,metazoa and cysts of thehelminthes.All livingorganisms are composed ofcells. Prokaryotic cells are relatively simple in structure; they lack a true nucleus covered bythe membrane. The most common cell shapes are spherical and rod-shaped. A eukaryoticcell'sstructureismorecomplex because itcontains organellesthatserveas compartmentsforspecialmetabolicfunctions.Viruses are particles assembled from the biopolymers, which are capable of multiplyingand assembling as new virus particles inside living prokaryotic or eukaryotic cells.Viruses arenot traditionally included in biological classifications because they are obligate intracellularparasites of cells,and thus,cannot self-reproduce.Extracellular virus particles are metaboli-cally inert. The typical virus size ranges from 0.02 to 0.2μm. Viruses contain a single typeof nucleic acid, either DNA or RNA.There are known virus-like agents called prions, whichare infectious proteins. Viruses are important for environmental engineering because of thefollowingreasons:1.Pathogenic viruses must be removed, retained or destroyed during water and wastewater treat-ment.2.Viruses of bacteria (bacteriophages)can infect and degrade the bacterial cultures.3.Bacteriophages can beused forthe detection of specific microbial pollution of environment.4.Viruses maybe a vector (carrier)of thegenes in artificial or natural genetic recombinations.Prokaryotes aremicroorganisms with prokaryotic type cells.They consist oftwo phylogeneticgroups: Bacteria and Archaea. The typical size of these cells is between 1 and 2 μm, butthere have been cells known to be smaller or bigger than this range.Prokaryotes are mostactive in the degradation of organic matter and are used in wastewater treatment and soilbioremediation. However, there are many bacteria that are harmful to human, animal andplant health, and the removal or killing of these pathogenic bacteria in water,wastewater orsolidwasteis an importanttask of environmental engineeringEnergy sources for the growth of prokaryotes include:1.Chemical substances (chemotrophy) or light (phototrophy).2.Utilization of organic substances (heterotrophy)or inorganic substances (lithotrophy)Other physiological properties also vary:1.Source ofcarbon may be carbon dioxide (autotrophy)or organic substances (organotrophy).Optimal temperature for growth varies from0°C tohigher than 100°C.2.3.OptimalpHforgrowthvariesfromtwotonine.Relationto oxygen is one of themain features of prokaryotes.Generation of biologically avail-able energy in a conducted cell is due to oxidation-reduction reactions. Oxygen is the mosteffective acceptorof electrons in energygeneration fromoxidation of substances,but not all
Microbiology of Environmental Engineering Systems 21 environmental engineering systems. A more in-depth description of these topics is given in specialized chapters of this book. 1.1. Groups of Microorganisms The objects of the microbiology of environmental engineering systems include bacteria (prokaryotes), microscopic fungi, microscopic algae, protozoa and other microscopic objects such as viruses, metazoa and cysts of the helminthes. All living organisms are composed of cells. Prokaryotic cells are relatively simple in structure; they lack a true nucleus covered by the membrane. The most common cell shapes are spherical and rod-shaped. A eukaryotic cell’s structure is more complex because it contains organelles that serve as compartments for special metabolic functions. Viruses are particles assembled from the biopolymers, which are capable of multiplying and assembling as new virus particles inside living prokaryotic or eukaryotic cells. Viruses are not traditionally included in biological classifications because they are obligate intracellular parasites of cells, and thus, cannot self-reproduce. Extracellular virus particles are metabolically inert. The typical virus size ranges from 0.02 to 0.2 μm. Viruses contain a single type of nucleic acid, either DNA or RNA. There are known virus-like agents called prions, which are infectious proteins. Viruses are important for environmental engineering because of the following reasons: 1. Pathogenic viruses must be removed, retained or destroyed during water and wastewater treatment. 2. Viruses of bacteria (bacteriophages) can infect and degrade the bacterial cultures. 3. Bacteriophages can be used for the detection of specific microbial pollution of environment. 4. Viruses may be a vector (carrier) of the genes in artificial or natural genetic recombinations. Prokaryotes are microorganisms with prokaryotic type cells. They consist of two phylogenetic groups: Bacteria and Archaea. The typical size of these cells is between 1 and 2 μm, but there have been cells known to be smaller or bigger than this range. Prokaryotes are most active in the degradation of organic matter and are used in wastewater treatment and soil bioremediation. However, there are many bacteria that are harmful to human, animal and plant health, and the removal or killing of these pathogenic bacteria in water, wastewater or solid waste is an important task of environmental engineering. Energy sources for the growth of prokaryotes include: 1. Chemical substances (chemotrophy) or light (phototrophy). 2. Utilization of organic substances (heterotrophy) or inorganic substances (lithotrophy). Other physiological properties also vary: 1. Source of carbon may be carbon dioxide (autotrophy) or organic substances (organotrophy). 2. Optimal temperature for growth varies from 0◦C to higher than 100◦C. 3. Optimal pH for growth varies from two to nine. Relation to oxygen is one of the main features of prokaryotes. Generation of biologically available energy in a conducted cell is due to oxidation–reduction reactions. Oxygen is the most effective acceptor of electrons in energy generation from oxidation of substances, but not all
22V.Ivanoumicroorganisms can use it.Thefollowing groups of microorganisms differ in their relation tooxygen:Obligate anaerobic prokaryotes,producing energy by fermentation (it is intramolecular1.oxidation-reduction without an external acceptor ofelectrons);they die after contactwith oxygenbecause they lack protection against oxygen radicals produced during the contact of cells withoxygen.2.Tolerant anaerobes produce energy only by fermentation but survive after contact with oxygendue to protective mechanisms againstoxygen radicals.3.Facultativeanaerobic bacteria,which arecapableto produce energybyfermentation ifoxygen isabsent or by aerobic respiration if oxygen is present.4.Microaerophilic bacteria, which prefer low concentration of dissolved oxygen in a medium.5.Obligate aerobes produceenergy by aerobic respiration only.Anoxic (anaerobic)respiration is typicalfor prokaryotes only and is the oxidation of organicor inorganic substancesbyelectronacceptors otherthanoxygen.Different electron acceptorsare used for energy generation by specific physiological groups of prokaryotes, including:1. Nitrate (NO3-) and nitrite (NO2-) are used by denitrifying bacteria (denitrifiers).2.Sulphate (SO42-) is used by sulphate-reducing bacteria.3.Sulphur (S) is used by sulphur-prokaryotes.4.Ferric ions (Fe3+)is used by iron-reducing bacteria5.Ions of different oxidizedmetals are used as acceptor of electrons.6.Carbondioxide(CO2)isusedbymethanogensFungi are eukaryotic microorganisms,mostly multicellular, which assimilate organic sub-stances and absorb nutrientsthrough the cell surface.Thetypical cell size isbetween 5and20μm.Cells are oftencombined inthebranchedfilaments called hyphaes, which arecombined in a web known as mycelium.Fungi are important degraders of polymers and areused in the composting and biodegradation of toxic organic substances. Fungi are used in envi-ronmental engineeringin composting,soil bioremediation and biodegradation of xenobioticsMycelium effectively penetrates solid wastes and soil. There are five major groups of fungi:1.Oomycetes (watermolds).2.Zygomycetes (molds)3.Ascomycetes (sac fungi and yeasts)reproducedby spores stored in the sac called ascus or sporescalled conidia.4.Basidiomycetes (club fungi and mushrooms).5.Deuteromycetes (or Fungi imperfecti)have no known sexual stage.Molds are filamentous fungi (from Zygomycetes and Ascomycetes) that have widespreadoccurrencein nature.Theyhave a surfacemycelium and aerial hyphae that contain asexualspores (conidia).These spores are airborne allergens in damp or poorly constructed buildingsYeasts (fromAscomycetes)arefungi thatgrow as singlecells,producingdaughter cells eitherby budding (the budding yeasts) or by binary fission (the fission yeasts).Mushrooms arefilamentous fungi that form large above-ground fruiting bodies, although the major portionof the biomass consists of hyphaebelowground
22 V. Ivanov microorganisms can use it. The following groups of microorganisms differ in their relation to oxygen: 1. Obligate anaerobic prokaryotes, producing energy by fermentation (it is intramolecular oxidation–reduction without an external acceptor of electrons); they die after contact with oxygen because they lack protection against oxygen radicals produced during the contact of cells with oxygen. 2. Tolerant anaerobes produce energy only by fermentation but survive after contact with oxygen due to protective mechanisms against oxygen radicals. 3. Facultative anaerobic bacteria, which are capable to produce energy by fermentation if oxygen is absent or by aerobic respiration if oxygen is present. 4. Microaerophilic bacteria, which prefer low concentration of dissolved oxygen in a medium. 5. Obligate aerobes produce energy by aerobic respiration only. Anoxic (anaerobic) respiration is typical for prokaryotes only and is the oxidation of organic or inorganic substances by electron acceptors other than oxygen. Different electron acceptors are used for energy generation by specific physiological groups of prokaryotes, including: 1. Nitrate (NO3 −) and nitrite (NO2 −) are used by denitrifying bacteria (denitrifiers). 2. Sulphate (SO4 2−) is used by sulphate-reducing bacteria. 3. Sulphur (S) is used by sulphur-prokaryotes. 4. Ferric ions (Fe3+) is used by iron-reducing bacteria. 5. Ions of different oxidized metals are used as acceptor of electrons. 6. Carbon dioxide (CO2) is used by methanogens. Fungi are eukaryotic microorganisms, mostly multicellular, which assimilate organic substances and absorb nutrients through the cell surface. The typical cell size is between 5 and 20 μm. Cells are often combined in the branched filaments called hyphaes, which are combined in a web known as mycelium. Fungi are important degraders of polymers and are used in the composting and biodegradation of toxic organic substances. Fungi are used in environmental engineering in composting, soil bioremediation and biodegradation of xenobiotics. Mycelium effectively penetrates solid wastes and soil. There are five major groups of fungi: 1. Oomycetes (water molds). 2. Zygomycetes (molds). 3. Ascomycetes (sac fungi and yeasts) reproduced by spores stored in the sac called ascus or spores called conidia. 4. Basidiomycetes (club fungi and mushrooms). 5. Deuteromycetes (or Fungi imperfecti) have no known sexual stage. Molds are filamentous fungi (from Zygomycetes and Ascomycetes) that have widespread occurrence in nature. They have a surface mycelium and aerial hyphae that contain asexual spores (conidia). These spores are airborne allergens in damp or poorly constructed buildings. Yeasts (from Ascomycetes) are fungi that grow as single cells, producing daughter cells either by budding (the budding yeasts) or by binary fission (the fission yeasts). Mushrooms are filamentous fungi that form large above-ground fruiting bodies, although the major portion of the biomass consists of hyphae below ground
23MicrobiologyofEnvironmentalEngineeringSystemsAlgae arefloating eukaryotic microorganisms that assimilate energyfrom light.Thetypicalsize of a cell is 10-20 μm.Algae carry out oxygenic photosynthesis:(1)CO2+H,O+light→CH,O+OAlgae live primarily in aquatic habitats and on the soil surface.Algae should not be confusedwith cyanobacteria, which are prokaryotes.The classification of algae is based on the type ofchlorophyll and otherpigments,cell wall structure and nature of carbon reserve material:1.Chlorophyta (green algae).2.Chrysophita (golden-brown algae).3.Euglenophyta,haveno cell.4.Pyrrophyta (dinoflagellates).5.Rhodophyta (red algae)6.Phaeophyta (brown algae)Algae are importantfor environmental engineering for the following reasons:1.They remove nutrients from water and are active microorganisms in waste stabilization ponds.2.Some algae arefast-growing in polluted water and produce toxic compounds; these cause the"redtidesinpolluted coastal areas3.Selected species ofmicroscopic algae in natural waters are used for the indication ofwaterquality.4.There may be value-added products, for example, pigments and unsaturated fatty acids from algaegrown in wastewater.Protozoa are unicellular organisms that absorb and digest organic food inside a cell. Thetypical cell sizeisfrom10to50μm.Someprotozoaarepathogenicandmustbe removedfromwater and wastewater.Four major groups of protozoa are distinguished by their mechanism ofmotility: amoebas move by means of false feet; flagellates move by means of flagella; ciliatesuse cilia for locomotion;and some protozoa have no means of locomotion.Examples aregiven in Table 2.1.Protozoa are unicellular organisms that obtain nutrients by ingesting other microbes, orby ingesting macromolecules. The cells form cysts under adverse environmental conditionsTable 2.1Examples of parasitic protozoaGroupExample of parasiticDisease caused by thisspecies from this groupspeciesSarcodina (amoeboids)Entamoeba histolyticaAmebiasisGiardiasisMastigophoraGiardia intestinalis(flagellates)BalantidiasisCiliophora (ciliates)Balantidium coliSporozoa (no means ofPlasmodium vivaxMalaria Cryptosporidiosislocomotion)Cryptosporidium spp.(morethan ten species)
Microbiology of Environmental Engineering Systems 23 Algae are floating eukaryotic microorganisms that assimilate energy from light. The typical size of a cell is 10–20 μm. Algae carry out oxygenic photosynthesis: CO2 + H2O + light → CH2O + O2 (1) Algae live primarily in aquatic habitats and on the soil surface. Algae should not be confused with cyanobacteria, which are prokaryotes. The classification of algae is based on the type of chlorophyll and other pigments, cell wall structure and nature of carbon reserve material: 1. Chlorophyta (green algae). 2. Chrysophita (golden-brown algae). 3. Euglenophyta, have no cell. 4. Pyrrophyta (dinoflagellates). 5. Rhodophyta (red algae). 6. Phaeophyta (brown algae). Algae are important for environmental engineering for the following reasons: 1. They remove nutrients from water and are active microorganisms in waste stabilization ponds. 2. Some algae are fast-growing in polluted water and produce toxic compounds; these cause the “red tides” in polluted coastal areas. 3. Selected species of microscopic algae in natural waters are used for the indication of water quality. 4. There may be value-added products, for example, pigments and unsaturated fatty acids from algae grown in wastewater. Protozoa are unicellular organisms that absorb and digest organic food inside a cell. The typical cell size is from 10 to 50 μm. Some protozoa are pathogenic and must be removed from water and wastewater. Four major groups of protozoa are distinguished by their mechanism of motility: amoebas move by means of false feet; flagellates move by means of flagella; ciliates use cilia for locomotion; and some protozoa have no means of locomotion. Examples are given in Table 2.1. Protozoa are unicellular organisms that obtain nutrients by ingesting other microbes, or by ingesting macromolecules. The cells form cysts under adverse environmental conditions Table 2.1 Examples of parasitic protozoa Group Example of parasitic species from this group Disease caused by this species Sarcodina (amoeboids) Entamoeba histolytica Amebiasis Mastigophora (flagellates) Giardia intestinalis Giardiasis Ciliophora (ciliates) Balantidium coli Balantidiasis Sporozoa (no means of locomotion) Plasmodium vivax Cryptosporidium spp. (more than ten species) Malaria Cryptosporidiosis
24V.Ivanovand are resistant to desiccation, starvation, high temperature and disinfection.Changes in theprotozoan communityreflect the operating conditions of aerobic wastewater treatment:1.Amoebas canbefound in high concentrationsof organic matter (athigh values ofbiochemicaloxygen demandBOD)2.Flagellated protozoa and free-swimming ciliates are associated withhigh bacterial concentrationsinactivated sludgeandmediumconcentrationof BOD values.3.Protozoa contribute significantly to the reduction of bacteria, including pathogens in activatedsludge.4.Stalkedciliatesoccuratlowbacterial andBODconcentrationsinwater.Helminthes areparasitic worms that survive in humans and animals.Many of these parasiticworms have microscopic cysts (seeds). The removal or inactivation of these cysts in water,wastewater and solid wastes is a goal of environmental engineering.Due to the high hydropho-bicity of the cyst surface, cysts can be accumulated in the landfill leachate, foam of aerationtanks, or float up during the storage or primary treatment of sewage.1.2.Microbiological Methods Used inEnvironmental EngineeringSpecific microbiological methods are used to study microorganisms in environmentalengineering systems:Isolation,cultivation,identification and quantification of pure cultures.2.Selection of strainsand constructionofrecombinantmicrobial strains.3.Selectionandquantificationof enrichmentcultures.4.Identification and quantification of microorganisms in environmental sampleswithout cultivation5.Extraction,cloning,enrichmentand identification ofmicrobial genes and theirproducts inenvi-ronmental engineering systems.Isolation of pure culture (microbial strain) is usually performed by spreading a dilutedmicrobial suspension on a Petri dish with a semi-solid medium to produce 10-50 colonieson the dish after several days of cultivation. Cells of one colony are picked up for the nextround of cultivation ona semi-solid orliquid medium.However,thefollowingmethods canalso be effectively used for the isolation of pure microbial culture:1.Mechanical separation of cells bymicromanipulator.2.Sorting of cells or microbeadswith immobilized cell,using flow cytometer.3.Magneticorimmunomagneticseparation.4.Cell chromatography.A microbial population that originates from one colony is called a microbial strain.A micro-bial population that originates from one cell is called a microbial clone.Selection is the screening of microbial variants with specific desirable characteristics withinthe population of one strain. These variants may include:1.Faster ormore efficientgrowth (positive selection)Faster or more efficient biochemical function (positive selection)2.3.Slower or less efficient biochemical function (negative selection).4.Better survival under harmful conditions (positive selection).5.Weaker resistance to some factors of the environment (negative selection)
24 V. Ivanov and are resistant to desiccation, starvation, high temperature and disinfection. Changes in the protozoan community reflect the operating conditions of aerobic wastewater treatment: 1. Amoebas can be found in high concentrations of organic matter (at high values of biochemical oxygen demand – BOD). 2. Flagellated protozoa and free-swimming ciliates are associated with high bacterial concentrations in activated sludge and medium concentration of BOD values. 3. Protozoa contribute significantly to the reduction of bacteria, including pathogens in activated sludge. 4. Stalked ciliates occur at low bacterial and BOD concentrations in water. Helminthes are parasitic worms that survive in humans and animals. Many of these parasitic worms have microscopic cysts (seeds). The removal or inactivation of these cysts in water, wastewater and solid wastes is a goal of environmental engineering. Due to the high hydrophobicity of the cyst surface, cysts can be accumulated in the landfill leachate, foam of aeration tanks, or float up during the storage or primary treatment of sewage. 1.2. Microbiological Methods Used in Environmental Engineering Specific microbiological methods are used to study microorganisms in environmental engineering systems: 1. Isolation, cultivation, identification and quantification of pure cultures. 2. Selection of strains and construction of recombinant microbial strains. 3. Selection and quantification of enrichment cultures. 4. Identification and quantification of microorganisms in environmental samples without cultivation. 5. Extraction, cloning, enrichment and identification of microbial genes and their products in environmental engineering systems. Isolation of pure culture (microbial strain) is usually performed by spreading a diluted microbial suspension on a Petri dish with a semi-solid medium to produce 10–50 colonies on the dish after several days of cultivation. Cells of one colony are picked up for the next round of cultivation on a semi-solid or liquid medium. However, the following methods can also be effectively used for the isolation of pure microbial culture: 1. Mechanical separation of cells by micromanipulator. 2. Sorting of cells or microbeads with immobilized cell, using flow cytometer. 3. Magnetic or immunomagnetic separation. 4. Cell chromatography. A microbial population that originates from one colony is called a microbial strain. A microbial population that originates from one cell is called a microbial clone. Selection is the screening of microbial variants with specific desirable characteristics within the population of one strain. These variants may include: 1. Faster or more efficient growth (positive selection). 2. Faster or more efficient biochemical function (positive selection). 3. Slower or less efficient biochemical function (negative selection). 4. Better survival under harmful conditions (positive selection). 5. Weaker resistance to some factors of the environment (negative selection)
25MicrobiologyofEnvironmentalEngineeringSystemsThe differences between variants are caused by natural spontaneous mutations, i.e.,changesin the DNA sequences of genes.Mutagenic chemicals, ultraviolet rays and ionizing radiationare used to increase the rate of mutagenesis and to increase probability of desirable variantformation.Thescreeningof thedesirablevariantcanbereplaced bythe creationof selectionpressure,i.e., conditions favorablefor growth, survival or development of desirable variant.Therefore, this variant will be accumulated in a microbial population and can be detectedduring the screening.The cultivation of microorganisms is performed under suitable conditions, usually at opti-mal temperature, pH, osmotic pressure and concentration of gases (oxygen, carbon dioxide,hydrogen), on a semi-solid or liquid medium, containing all necessary substances for thegrowth of the strain. The elemental composition of biomass can be shown approximatelybythe formula CHi.gOo.sNo.2,but half of theknown elements are used in the synthesis ofmicrobial biomassand mustbepresent inthemedium.Suitableconditionsand essentialsubstances for the growth of some strains are not known yet and the cultivation of thesemicroorganismshas notbeen successful to date.Additionally,some microorganisms arelivingin strong symbiotic or parasitic relationships with othermicroorganisms or macroorganismsthat cannot be cultivated separately from these organisms. Therefore, not all microorganismscanbeisolatedandcultivatedThe identification of microorganisms involves the determination of relationship of a stud-ied strain (taxon) with some known group, which is then accepted and approved by aninternational scientific committee.Measurement or qualitative evaluation of the relationshipbetween compared microbial groups is performed by the methods of phenotypic classification(conventional taxonomy)and genotypic classification (phylogenetictaxonomy).Phenotypic classification (conventional taxonomy)isbased onthephenotypic characteris-tics, i.e., visible or measured characteristics determined by an organism's interaction with theenvironment:1.Cytological characteristics such as size, shape, cell structure, typical cell aggregates, membranestructures, intracellular structures and cell organelles.One of most important cytological charac-teristics of prokaryotes is the Gram-positive or Gram-negative type of cell wall.A Gram-positivecell well is a thick and rigid 3-D layer of polymer. A Gram-negative cell wall is a thin and moreelastic layerof polymer,whichis covered byan outer membrane and a lipopolysaccharidelayer2.Physiological characteristics such as type ofenergy production,relation to oxygen,pH,tempera-ture,chemicalcontentofcell wall andmembranes,production ofspecificmetabolites andenzymeprofile.Ecological characteristics such as habitats,econiches, colonial structures and interrelationships3.with otherorganisms.Genotypic classification (phylogenetic taxonomy) is based on the analyses of geneticcharacteristics of the organisms, which are stored in the sequences of DNA. Genotypiccharacteristics include:G+CcontentinDNA1.Sequences of genes (the sequences of DNA, which store information on the biopolymers of2.homologous, similar function in different species are compared).3.Sequencesofhomologous (similar)proteins.4.Level ofhybridization between the sequencesof DNA andRNA of compared strains
Microbiology of Environmental Engineering Systems 25 The differences between variants are caused by natural spontaneous mutations, i.e., changes in the DNA sequences of genes. Mutagenic chemicals, ultraviolet rays and ionizing radiation are used to increase the rate of mutagenesis and to increase probability of desirable variant formation. The screening of the desirable variant can be replaced by the creation of selection pressure, i.e., conditions favorable for growth, survival or development of desirable variant. Therefore, this variant will be accumulated in a microbial population and can be detected during the screening. The cultivation of microorganisms is performed under suitable conditions, usually at optimal temperature, pH, osmotic pressure and concentration of gases (oxygen, carbon dioxide, hydrogen), on a semi-solid or liquid medium, containing all necessary substances for the growth of the strain. The elemental composition of biomass can be shown approximately by the formula CH1.8O0.5N0.2, but half of the known elements are used in the synthesis of microbial biomass and must be present in the medium. Suitable conditions and essential substances for the growth of some strains are not known yet and the cultivation of these microorganisms has not been successful to date. Additionally, some microorganisms are living in strong symbiotic or parasitic relationships with other microorganisms or macroorganisms that cannot be cultivated separately from these organisms. Therefore, not all microorganisms can be isolated and cultivated. The identification of microorganisms involves the determination of relationship of a studied strain (taxon) with some known group, which is then accepted and approved by an international scientific committee. Measurement or qualitative evaluation of the relationship between compared microbial groups is performed by the methods of phenotypic classification (conventional taxonomy) and genotypic classification (phylogenetic taxonomy). Phenotypic classification (conventional taxonomy) is based on the phenotypic characteristics, i.e., visible or measured characteristics determined by an organism’s interaction with the environment: 1. Cytological characteristics such as size, shape, cell structure, typical cell aggregates, membrane structures, intracellular structures and cell organelles. One of most important cytological characteristics of prokaryotes is the Gram-positive or Gram-negative type of cell wall. A Gram-positive cell well is a thick and rigid 3-D layer of polymer. A Gram-negative cell wall is a thin and more elastic layer of polymer, which is covered by an outer membrane and a lipopolysaccharide layer. 2. Physiological characteristics such as type of energy production, relation to oxygen, pH, temperature, chemical content of cell wall and membranes, production of specific metabolites and enzyme profile. 3. Ecological characteristics such as habitats, econiches, colonial structures and interrelationships with other organisms. Genotypic classification (phylogenetic taxonomy) is based on the analyses of genetic characteristics of the organisms, which are stored in the sequences of DNA. Genotypic characteristics include: 1. G + C content in DNA. 2. Sequences of genes (the sequences of DNA, which store information on the biopolymers of homologous, similar function in different species are compared). 3. Sequences of homologous (similar) proteins. 4. Level of hybridization between the sequences of DNA and RNA of compared strains
26V.IvanouCollections of strains and clones.The properties of strains and clones are the primary dataused for classification.Strains are stored in microbial collections in the form of suspension,colonies on solid medium, or in dry or frozen state. There are many specialized and nationalcollections of microorganisms.The purpose of such collections is to acquire, authenticate,preserve, develop and distribute biological materials, information, technology, intellectualproperty and standards fortheadvancement,validation and application of scientificknowledgeto private industry, government and academic organizations. For example, large culture collec-tions include the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) andAmerican Type Culture Collection (ATCC).A strain is identified byits assigned number ina microbial collection and the name of the species.For example, Bacillus subtilis ATCC6633refers to a strain of species Bacillus subtilis stored under number 6333 in theATCC.A species is a primary unit of phenotypic classification. It is defined by the phenotypicand genotypic characteristics of a collection of similar strains.The name of any species isgiven and read in Latin and includes the name of the genus (first)and the name of the species(second): Saccharomyces cerevisiae,Bacillus subtilis.Higher levels of phenotypic classification are genus (collection of similar species),family(collection of similar genera),order (collection of similar families)and kingdom (collectionof similar orders).Prokaryotic groups of conventional taxonomy are described in Bergey'sManual of Systematic Bacteriology (1).The manual contains phenotypic characteristicswhich are used to classify prokaryotes by conventional taxonomy. Groups of viruses, fungi,protozoa and algae are described in specific manuals approved by related international scien-tific associations. Prokaryotic groups of phylogenetic taxonomy can be defined through thecomparison of the sequences of ribosomal RNA (rRNA),especially 16S rRNA. It is thoughtthat the number of differences in the sequence reflects the evolutionary distance of the originofcomparedsequencesfromacommonancestorsequenceSelection of enrichment culture refers tothe selection of the population withonedominatedstrainoronedominatedmicrobialcommunity,whichisaccumulatedinthesystemofculti-vation because of the preferred conditions (selection pressure)for this strain or communityEnrichment cultivation is often used in environmental engineeringto select microorganism(s)capable of particular metabolic transformations.Selective conditions (selectionpressure)forthe production of enrichment culture are as follows:Sourceofenergy.1.2.Sourceofcarbon3.Source of nitrogen and phosphorus.4.Temperature.5.pH.6.Concentration of heavymetals.7.Presenceofspecificantibioticinamedium8.Concentration of dissolved oxygen.9.Osmoticpressureofamedium.10.Spectrumandintensityof light,etc.Quantification of microbial biomass,i.e.,determination of cell number(enumeration)orquantity of cell biomass, can beperformed by thefollowing methods:
26 V. Ivanov Collections of strains and clones. The properties of strains and clones are the primary data used for classification. Strains are stored in microbial collections in the form of suspension, colonies on solid medium, or in dry or frozen state. There are many specialized and national collections of microorganisms. The purpose of such collections is to acquire, authenticate, preserve, develop and distribute biological materials, information, technology, intellectual property and standards for the advancement, validation and application of scientific knowledge to private industry, government and academic organizations. For example, large culture collections include the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and American Type Culture Collection (ATCC). A strain is identified by its assigned number in a microbial collection and the name of the species. For example, Bacillus subtilis ATCC6633 refers to a strain of species Bacillus subtilis stored under number 6333 in the ATCC. A species is a primary unit of phenotypic classification. It is defined by the phenotypic and genotypic characteristics of a collection of similar strains. The name of any species is given and read in Latin and includes the name of the genus (first) and the name of the species (second): Saccharomyces cerevisiae, Bacillus subtilis. Higher levels of phenotypic classification are genus (collection of similar species), family (collection of similar genera), order (collection of similar families) and kingdom (collection of similar orders). Prokaryotic groups of conventional taxonomy are described in Bergey’s Manual of Systematic Bacteriology (1). The manual contains phenotypic characteristics, which are used to classify prokaryotes by conventional taxonomy. Groups of viruses, fungi, protozoa and algae are described in specific manuals approved by related international scientific associations. Prokaryotic groups of phylogenetic taxonomy can be defined through the comparison of the sequences of ribosomal RNA (rRNA), especially 16S rRNA. It is thought that the number of differences in the sequence reflects the evolutionary distance of the origin of compared sequences from a common ancestor sequence. Selection of enrichment culture refers to the selection of the population with one dominated strain or one dominated microbial community, which is accumulated in the system of cultivation because of the preferred conditions (selection pressure) for this strain or community. Enrichment cultivation is often used in environmental engineering to select microorganism(s) capable of particular metabolic transformations. Selective conditions (selection pressure) for the production of enrichment culture are as follows: 1. Source of energy. 2. Source of carbon. 3. Source of nitrogen and phosphorus. 4. Temperature. 5. pH. 6. Concentration of heavy metals. 7. Presence of specific antibiotic in a medium. 8. Concentration of dissolved oxygen. 9. Osmotic pressure of a medium. 10. Spectrum and intensity of light, etc. Quantification of microbial biomass, i.e., determination of cell number (enumeration) or quantity of cell biomass, can be performed by the following methods:
27MicrobiologyofEnvironmentalEngineeringSystemsMicroscopic orflowcytometricenumeration ofcells.1.2.Physical measurement of microbial cells and biomass concentration.3.Chemical measurementofmicrobial cellsandbiomassconcentration.4.Biological methods ofcell and virusesenumeration.5.Physiologicalmeasurementofbiomass.6.Molecular-biological methods of cells and viruses enumeration.Factors that affect thechoice of the method include:1.Cost and length of time required for analysis2.Sensitivityand specificityof themethod.3.Availability of the equipment.4.Characteristics of the interest.Microscopicenumeration of cells and virus particles is performed using lightmicroscopes(bright field, phase contrast and fluorescence microscopy), confocal laser scanning micro-scopes(CLSM),transmitted electron microscopes (TEM),scanning electron microscopes(SEM) and otherkinds of microscopes that are able to visualize a number of microbial cellsorvirusparticleson adefined area.Theparticularcell structuremaybe specificallystained.cell or virus surface may be labeled by immunochemical methods and DNA or RNA of cellscan be hybridized with oligonucleotide probes labelled by fluorescence,radioactive or otherlabels.This specific staining can ensure cell enumeration altogether, with cell identificationand measurement of cell physiological state.Flow cytometry enumeration is used to quantify cells by staining specific cells withfluorescent-labelled antibodies,oligonucleotide probes and specific fluorochromes,which areexcited by lasers in the flow of a small diameter (2). The fluorescence of individual cells isthen measured by photomultipliers and the signals are collected and treated by a computer.In addition to cell number, three to six other parameters of thousands of individual microbialcells orvirusparticlescanbeanalyzed inseconds.Physical methods for microbial biomass measurement are based on thedetermination ofweight,optical density,turbidity,fluorescence or radioactivityof microbial suspensions andsolid matter. A convenient method of suspended biomass estimation is turbidity measure-ment.The share of scattered light is proportional to the cell concentration in the sample.Autofluorescenceofmicrobialcellcomponents(chlorophyllofalgae,bacteriochlorophyllandcarotenoidsof cyanobacteria,F42o ofmethanogens)or fluorescenceof stained cellsalso canbe used for the measurement of biomass.Fluorescence spectrometry can be used toquantifymicroorganisms in environmental engineering systems, using determination of the binding ofspecific oligonucleotide probes (3)Chemical methods formicrobial biomass measurement include the analysis of proteinDNA or the components of cell wall,ATP,photopigments,cytochromes, coenzymes NADH2or F420. ATP measurement is a sensitive indicator of a small quantity of viable microorgan-isms.The chemical changes in the mediumcaused bythe microbial growth can be monitoredusing electrochemical sensors and fiber optic sensors
Microbiology of Environmental Engineering Systems 27 1. Microscopic or flow cytometric enumeration of cells. 2. Physical measurement of microbial cells and biomass concentration. 3. Chemical measurement of microbial cells and biomass concentration. 4. Biological methods of cell and viruses enumeration. 5. Physiological measurement of biomass. 6. Molecular-biological methods of cells and viruses enumeration. Factors that affect the choice of the method include: 1. Cost and length of time required for analysis. 2. Sensitivity and specificity of the method. 3. Availability of the equipment. 4. Characteristics of the interest. Microscopic enumeration of cells and virus particles is performed using light microscopes (bright field, phase contrast and fluorescence microscopy), confocal laser scanning microscopes (CLSM), transmitted electron microscopes (TEM), scanning electron microscopes (SEM) and other kinds of microscopes that are able to visualize a number of microbial cells or virus particles on a defined area. The particular cell structure may be specifically stained, cell or virus surface may be labeled by immunochemical methods and DNA or RNA of cells can be hybridized with oligonucleotide probes labelled by fluorescence, radioactive or other labels. This specific staining can ensure cell enumeration altogether, with cell identification and measurement of cell physiological state. Flow cytometry enumeration is used to quantify cells by staining specific cells with fluorescent-labelled antibodies, oligonucleotide probes and specific fluorochromes, which are excited by lasers in the flow of a small diameter (2). The fluorescence of individual cells is then measured by photomultipliers and the signals are collected and treated by a computer. In addition to cell number, three to six other parameters of thousands of individual microbial cells or virus particles can be analyzed in seconds. Physical methods for microbial biomass measurement are based on the determination of weight, optical density, turbidity, fluorescence or radioactivity of microbial suspensions and solid matter. A convenient method of suspended biomass estimation is turbidity measurement. The share of scattered light is proportional to the cell concentration in the sample. Autofluorescence of microbial cell components (chlorophyll of algae, bacteriochlorophyll and carotenoids of cyanobacteria, F420 of methanogens) or fluorescence of stained cells also can be used for the measurement of biomass. Fluorescence spectrometry can be used to quantify microorganisms in environmental engineering systems, using determination of the binding of specific oligonucleotide probes (3). Chemical methods for microbial biomass measurement include the analysis of protein, DNA or the components of cell wall, ATP, photopigments, cytochromes, coenzymes NADH2 or F420. ATP measurement is a sensitive indicator of a small quantity of viable microorganisms. The chemical changes in the medium caused by the microbial growth can be monitored using electrochemical sensors and fiber optic sensors
28V.IvanovPhysiological methods of microbial biomass measurement are based on themeasurementsofphysiological activityofthecells,forexample,respirationrate,biochemical transformationrate and ATP concentration.Biological methods of cell enumeration areas follows:1.Plate count.i.e..cultivation on a semi-solid medium andenumeration of colony-forming units(CFU).Itis assumed thatonecell producesonecolony,butthisassumptionisoften notrightThere may be 103-1012 cells in 1 mL of the sample. Therefore, it should be diluted in a sterilemedium before being spread onto a Petri dish to produce not more than 100-300 colonies perplate.Most probable number count, i.e.identification of the maximum dilution at which thegrowth or2.microbial activity can be easily detected by the colour change,precipitation or formation of gasbubbles.For example, if the maximum dilution to detect microbial activity in 1mL of specificmedium is 5 × 10-4, the most probable number of cells in the sample is 2 × 103 cells/mL. Itis assumed that multiplicationof one cell inthetubewith maximumdilution canproducethedetectableresult (colorchange,gas bubbles),butthis assumptionisoften incorrect.Ifthe studied cells and viral particles havea low cell concentration,theymust beconcentratedusing the following methods:1.Filtration of the sample through a sterile membrane filter having a pore size < 0.45 μm to retainbacterial cellsand<2-5μumtoretaineukaryoticcells.2.Precipitationorcentrifugationofcellsorviralparticlesofthesample.3.Chromatographyofthesample.4.Adsorption of cells and viral particles in the column with a specific adsorbent.Viruses are enumerated biologicallyby spreading a diluted suspension on the surface of a lawnof actively growing cells susceptible to the virus. As a virus particle infects and reproducesthe produced viruses kill surrounding cells, forming a zone of clearing in the cell layer.Molecular-biological methods for microbial biomass quantification are as follows:Immunochemical quantification of microbial biomass due to colour change in the reaction1.betweenspecificantibodie(s)andthecell surface2.Molecular-biological quantification of microbial biomass due to color change in the in situreaction between specific oligonucleotide probe(s)andcell RNAs or DNA (3).3.Quantitative polymerase chain reaction(PCR)called real-time PCR.It involves the extractionof DNA from the sample and amplification of specific genes with its quantification after everycycle of amplification. This method is especially important for bacterial groups that cannot becultivated inthe laboratory becausethe medium orgrowth conditions forthem were notyetdefined,orwhich aresymbiotic orparasitic species.Virusescanbe enumerated by immunochemicalmethods orbyPCRof specificDNA/RNA.1.3.Comparisonof Physical, Chemical,Physico-chemical andMicrobiologicalProcessesEnvironmental engineering problems,i.e.,waste or wastewatertreatment,soil bioremediation and biopurification of exhaust gases, can be usually solved by physical, chemical.physico-chemical and biological/microbiological technologies.An optimal technology can
28 V. Ivanov Physiological methods of microbial biomass measurement are based on the measurements of physiological activity of the cells, for example, respiration rate, biochemical transformation rate and ATP concentration. Biological methods of cell enumeration are as follows: 1. Plate count, i.e., cultivation on a semi-solid medium and enumeration of colony-forming units (CFU). It is assumed that one cell produces one colony, but this assumption is often not right. There may be 103–1012 cells in 1 mL of the sample. Therefore, it should be diluted in a sterile medium before being spread onto a Petri dish to produce not more than 100–300 colonies per plate. 2. Most probable number count, i.e., identification of the maximum dilution at which the growth or microbial activity can be easily detected by the colour change, precipitation or formation of gas bubbles. For example, if the maximum dilution to detect microbial activity in 1 mL of specific medium is 5 × 10−4, the most probable number of cells in the sample is 2 × 103 cells/mL. It is assumed that multiplication of one cell in the tube with maximum dilution can produce the detectable result (color change, gas bubbles), but this assumption is often incorrect. If the studied cells and viral particles have a low cell concentration, they must be concentrated using the following methods: 1. Filtration of the sample through a sterile membrane filter having a pore size < 0.45 μm to retain bacterial cells and < 2–5 μm to retain eukaryotic cells. 2. Precipitation or centrifugation of cells or viral particles of the sample. 3. Chromatography of the sample. 4. Adsorption of cells and viral particles in the column with a specific adsorbent. Viruses are enumerated biologically by spreading a diluted suspension on the surface of a lawn of actively growing cells susceptible to the virus. As a virus particle infects and reproduces, the produced viruses kill surrounding cells, forming a zone of clearing in the cell layer. Molecular-biological methods for microbial biomass quantification are as follows: 1. Immunochemical quantification of microbial biomass due to colour change in the reaction between specific antibodie(s) and the cell surface. 2. Molecular-biological quantification of microbial biomass due to color change in the in situ reaction between specific oligonucleotide probe(s) and cell RNAs or DNA (3). 3. Quantitative polymerase chain reaction (PCR) called real-time PCR. It involves the extraction of DNA from the sample and amplification of specific genes with its quantification after every cycle of amplification. This method is especially important for bacterial groups that cannot be cultivated in the laboratory because the medium or growth conditions for them were not yet defined, or which are symbiotic or parasitic species. Viruses can be enumerated by immunochemical methods or by PCR of specific DNA/RNA. 1.3. Comparison of Physical, Chemical, Physico-chemical and Microbiological Processes Environmental engineering problems, i.e., waste or wastewater treatment, soil bioremediation and biopurification of exhaust gases, can be usually solved by physical, chemical, physico-chemical and biological/microbiological technologies. An optimal technology can