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