29Microbiologyof EnvironmentalEngineering SystemsTable 2.2Advantages and disadvantages ofdifferent environmental engineeringtechnologiesType of technologyAdvantagesDisadvantagesPhysical technologiesRequired time is from someLow specificity and highenergy demand(sedimentation,filtrationseconds to some minutes;volatilization,fixationhigh predictability of theevaporation, heat treatment,systemradiation, etc.)Chemical technologiesRequired time is from someHigh expenses for reagents,(oxidation, incineration,seconds to some minutes;energy,and equipment; airreduction, chemicalhigh predictability of thepollution due toincineration,formation ofimmobilization,chelatingsystemchemicaltransformation)secondary wastesPhysico-chemical treatmentRequired time is from someHigh expenses forminutes to some hoursadsorbents; formation of(adsorption,absorptionchromatography)secondary wasteLow volume or absence ofMicrobiological technologiesHigh expenses for aeration,(biooxidationsecondary hazardousnutrients, and maintenancebiotransformation.wastes;process can beofoptimal conditions;biodegradation)initiated by naturalrequired time is from somemicroorganisms or smallhours to days; unexpectedor negativeeffects ofquantity of added microbialmicroorganisms-biomass;highprocessdestructors;lowspecificity; wide spectrumof degradable substancespredictability of the systemand diverse methods ofbecause ofcomplexity andbiodegradationhigh sensitivity ofbiological systemsbe selected, based on economical or environmental criteria.Some general advantages anddisadvantagesofdifferentenvironmental engineeringtechnologiesareshown inTable2.22.MICROBIALECOSYSTEMS2.1. Structure of EcosystemsAn ecosystem comprisesbiotic(biological)and abiotic(physical, chemical)components,interacting with each other and isolated from the environment by a boundary.The hierarchy of life units in microbial ecosystems can be represented in order of increasingspatial andbiological complexity of ecosystems and the sequenceof their combination:Suspended cells (unicellular organisms) of one species.1.2.Suspended cells (unicellular organisms) of microbial community3.Aggregatedcells and multicellularmicroorganisms.4.Ecosystems of located biotop.5.Ecosystemsofwholebiosphere
Microbiology of Environmental Engineering Systems 29 Table 2.2 Advantages and disadvantages of different environmental engineering technologies Type of technology Advantages Disadvantages Physical technologies (sedimentation, filtration, volatilization, fixation, evaporation, heat treatment, radiation, etc.) Required time is from some seconds to some minutes; high predictability of the system Low specificity and high energy demand Chemical technologies (oxidation, incineration, reduction, chemical immobilization, chelating, chemical transformation) Required time is from some seconds to some minutes; high predictability of the system High expenses for reagents, energy, and equipment; air pollution due to incineration, formation of secondary wastes Physico-chemical treatment (adsorption, absorption, chromatography) Required time is from some minutes to some hours High expenses for adsorbents; formation of secondary waste Microbiological technologies (biooxidation, biotransformation, biodegradation) Low volume or absence of secondary hazardous wastes; process can be initiated by natural microorganisms or small quantity of added microbial biomass; high process specificity; wide spectrum of degradable substances and diverse methods of biodegradation High expenses for aeration, nutrients, and maintenance of optimal conditions; required time is from some hours to days; unexpected or negative effects of microorganismsdestructors; low predictability of the system because of complexity and high sensitivity of biological systems be selected, based on economical or environmental criteria. Some general advantages and disadvantages of different environmental engineering technologies are shown in Table 2.2. 2. MICROBIAL ECOSYSTEMS 2.1. Structure of Ecosystems An ecosystem comprises biotic (biological) and abiotic (physical, chemical) components, interacting with each other and isolated from the environment by a boundary. The hierarchy of life units in microbial ecosystems can be represented in order of increasing spatial and biological complexity of ecosystems and the sequence of their combination: 1. Suspended cells (unicellular organisms) of one species. 2. Suspended cells (unicellular organisms) of microbial community. 3. Aggregated cells and multicellular microorganisms. 4. Ecosystems of located biotop. 5. Ecosystems of whole biosphere
30V.IvanouThe boundary between an ecosystem and its surrounding environment is a steep gradientofphysical and/or chemical properties.Thephysical boundaryisformedby aninterphasebetween solid and liquid phases, solid and gas phases, liquid and gas phases.For example.the microbial ecosystem of an aerobictank for wastewatertreatment is separated from theenvironment by the reactor walls and air-water interphase.The steep gradient of chemicalsubstances, for example, oxygen, ferrous, hydrogen sulphide, etc., forms a chemical barrier.Such barriers separate, for example,aerobic and anaerobic ecosystems in a lake.The steepgradient of conditions can also be created by cell aggregation in flocs, granules or biofilms.The main function of the boundaryis tomaintain integrity of an ecosystembycontrolledisolation from the environment, and to protect an ecosystem from the destructive effects ofthe environment.Theboundariesofunicellularorganismareasfollows:1.Thecell membrane(cytoplasmicmembrane)performs selective andcontrolledexchangeofmolecules between cell and environment.It isthemostsensitiveboundary becauseeven a smallbreak in the cell membrane will destroy the isolating and energy-generating properties of a cellmembrane. Surface-active substances, organic solvents, oxidants and high temperature destroytheintegrityofacell membrane.2.The cell wall protects a cell from changes in osmotic pressure and mechanical impulses. Bacteriawith a thick cell wall are stained as Gram-positive cells.Bacteria that are stained as Gram-negative cells have a thin cell wall covered by an outer3.membrane. Lipopolysaccharides of outer membrane of Gram-negative bacteria are very specific.These molecules interact with the human body's immune system and are often toxic or allergenicfor humans.Gram staining is just one,and not always reliable method to differentiate bacteriawith Gram-positive and Gram-negativetypes of cell walls.Some prokaryotes,for example,mycoplasmas,have no cell wall.4.5.Fungi and algae often have cell walls containing polysaccharides such as cellulose or chitin. Somealgae have inorganic compounds such as calcium carbonate or silica in their rigid walls. Animalcells often have no cell walls.The glycocalyx (capsule) is an extracellular polysaccharide, covering microbial cells of some6.species.Itsfunctions includeattachmentofthecells tothe surface;aggregation ofcells;protectionof cells against drying, oxidants, heavy metals and antibiotics.A multicellular aggregate is formed and separated from its surrounding environment due to:1.Aggregation byhydrophobicforce,electrostatic interactions or salt bridges.Loose polysaccharide or inorganic matrix (iron hydroxide, for example), combining the cells2.altogether bymechanical embedding,chemical bonds,hydrogen bonds,electrostatic forces orhydrophobic interactions.3.Formation of mycelia, which is a net of branched cell filaments.4.Polysaccharide matrix with a filamentous frame.5.Structured matrix with layers parallel to the boundary or subaggregates, which are perpendicularto the boundary (4)6.Coverage by a common sheath of organic (polysaccharides,proteins) or inorganic origin (ironhydroxide, silica, calcium carbonate).7.Coverage by a common sheath ("skin"of microbial aggregate)consisting of dead cells
30 V. Ivanov The boundary between an ecosystem and its surrounding environment is a steep gradient of physical and/or chemical properties. The physical boundary is formed by an interphase between solid and liquid phases, solid and gas phases, liquid and gas phases. For example, the microbial ecosystem of an aerobic tank for wastewater treatment is separated from the environment by the reactor walls and air–water interphase. The steep gradient of chemical substances, for example, oxygen, ferrous, hydrogen sulphide, etc., forms a chemical barrier. Such barriers separate, for example, aerobic and anaerobic ecosystems in a lake. The steep gradient of conditions can also be created by cell aggregation in flocs, granules or biofilms. The main function of the boundary is to maintain integrity of an ecosystem by controlled isolation from the environment, and to protect an ecosystem from the destructive effects of the environment. The boundaries of unicellular organism are as follows: 1. The cell membrane (cytoplasmic membrane) performs selective and controlled exchange of molecules between cell and environment. It is the most sensitive boundary because even a small break in the cell membrane will destroy the isolating and energy-generating properties of a cell membrane. Surface-active substances, organic solvents, oxidants and high temperature destroy the integrity of a cell membrane. 2. The cell wall protects a cell from changes in osmotic pressure and mechanical impulses. Bacteria with a thick cell wall are stained as Gram-positive cells. 3. Bacteria that are stained as Gram-negative cells have a thin cell wall covered by an outer membrane. Lipopolysaccharides of outer membrane of Gram-negative bacteria are very specific. These molecules interact with the human body’s immune system and are often toxic or allergenic for humans. Gram staining is just one, and not always reliable method to differentiate bacteria with Gram-positive and Gram-negative types of cell walls. 4. Some prokaryotes, for example, mycoplasmas, have no cell wall. 5. Fungi and algae often have cell walls containing polysaccharidessuch as cellulose or chitin. Some algae have inorganic compounds such as calcium carbonate or silica in their rigid walls. Animal cells often have no cell walls. 6. The glycocalyx (capsule) is an extracellular polysaccharide, covering microbial cells of some species. Its functions include attachment of the cellsto the surface; aggregation of cells; protection of cells against drying, oxidants, heavy metals and antibiotics. A multicellular aggregate is formed and separated from its surrounding environment due to: 1. Aggregation by hydrophobic force, electrostatic interactions or salt bridges. 2. Loose polysaccharide or inorganic matrix (iron hydroxide, for example), combining the cells altogether by mechanical embedding, chemical bonds, hydrogen bonds, electrostatic forces or hydrophobic interactions. 3. Formation of mycelia, which is a net of branched cell filaments. 4. Polysaccharide matrix with a filamentous frame. 5. Structured matrix with layers parallel to the boundary or subaggregates, which are perpendicular to the boundary (4). 6. Coverage by a common sheath of organic (polysaccharides, proteins) or inorganic origin (iron hydroxide, silica, calcium carbonate). 7. Coverage by a common sheath (“skin” of microbial aggregate) consisting of dead cells
31MicrobiologyofEnvironmental EngineeringSystemsAmicrobial aggregate can beconsidered as a multicellular organism if its parts have differentcoordinated or synchronized physiological functions, i.e., growth, motility, sexual interac-tions,assimilation of atmospheric nitrogen,production ofextracellular polysaccharides, trans-port and distribution of nutrients and reduction of oxygen.Microbial communities of environmental engineering systems are usually suspended oradhered to surfacecells and microbial aggregates such as fixed biofilms and suspended flocsorgranules.Theboundariesof theseecosystemsareasfollows:1.Side walls of theequipment with a fixed microbial biofilm.2.Bottomof the equipment with the sediment of microbial aggregates.Gas-liquid interphasewith accumulated hydrophobic substances (lipids,hydrocarbons,aromatic3.aminoacids) and cells or aggregates with high hydrophobicity of their surface or cells andaggregates containing gas vesicles (5).Diversity of a microbial ecosystem refers to the heterogeneity of genotypes (diversity ofstrains, species, physiological groups), in space (different zones, layers, aggregates and chem-ical or physical gradients), and in time (temporal changes in diversity of genotypes and spatialstructure of ecosystem).Successionrefers to the typical sequence of temporal changes in anecosystem.Stagnationorclimaxis a stateof ecosystemcharacterizedbyweakchangescausedbypoorenvironment,degenerationorageingofthesystemThere areknown numerous mathematical expressions toquantify diversity.For example,Shannon-Weaver index (H) is:(2)H = [pi - In(p:)]-0where p; is the proportion of the i-th group in the community, and S is a number of the groupsinthecommunity.Evennessindex(E)isameasureofhow similartheabundancesof differentgroups are:E=H/InS(3)When there are similar proportions of all groups, then the evenness index is one. The evennessindex is larger than onewhen the abundances are very dissimilar.An example of quantitativecharacterization of microbial diversity in an anaerobic digester of activated sludge is givenbelow.Example:diversity in an anaerobic digester.There areat least fivemicrobial groupsinvolved in anaerobic digestion:Hydrolytic bacteria degrading polymers (polysaccharides, proteins, nucleic acids) to monomers1.(glucose,aminoacids,nucleosides).Acidogenic bacteria fermenting monomers to organic acids and alcohols.2.3.Acetogenic bacteria producing acetate from other organic acids and alcohols.4.Acetotrophicmethanogens,producing methanefrom acetate.5.Hydrogenotrophic methanogens, producing methane from hydrogen and carbon dioxide
Microbiology of Environmental Engineering Systems 31 A microbial aggregate can be considered as a multicellular organism if its parts have different coordinated or synchronized physiological functions, i.e., growth, motility, sexual interactions, assimilation of atmospheric nitrogen, production of extracellular polysaccharides, transport and distribution of nutrients and reduction of oxygen. Microbial communities of environmental engineering systems are usually suspended or adhered to surface cells and microbial aggregates such as fixed biofilms and suspended flocs or granules. The boundaries of these ecosystems are as follows: 1. Side walls of the equipment with a fixed microbial biofilm. 2. Bottom of the equipment with the sediment of microbial aggregates. 3. Gas–liquid interphase with accumulated hydrophobic substances (lipids, hydrocarbons, aromatic aminoacids) and cells or aggregates with high hydrophobicity of their surface or cells and aggregates containing gas vesicles (5). Diversity of a microbial ecosystem refers to the heterogeneity of genotypes (diversity of strains, species, physiological groups), in space (different zones, layers, aggregates and chemical or physical gradients), and in time (temporal changes in diversity of genotypes and spatial structure of ecosystem). Succession refers to the typical sequence of temporal changes in an ecosystem. Stagnation or climax is a state of ecosystem characterized by weak changes caused by poor environment, degeneration or ageing of the system. There are known numerous mathematical expressions to quantify diversity. For example, Shannon-Weaver index (H) is: H = i=s i=0 pi − ln(pi) (2) where pi is the proportion of the i-th group in the community, and S is a number of the groups in the community. Evenness index (E) is a measure of how similar the abundances of different groups are: E = H/ ln S (3) When there are similar proportions of all groups, then the evenness index is one. The evenness index is larger than one when the abundances are very dissimilar. An example of quantitative characterization of microbial diversity in an anaerobic digester of activated sludge is given below. Example: diversity in an anaerobic digester. There are at least five microbial groups involved in anaerobic digestion: 1. Hydrolytic bacteria degrading polymers (polysaccharides, proteins, nucleic acids) to monomers (glucose, aminoacids, nucleosides). 2. Acidogenic bacteria fermenting monomers to organic acids and alcohols. 3. Acetogenic bacteria producing acetate from other organic acids and alcohols. 4. Acetotrophic methanogens, producing methane from acetate. 5. Hydrogenotrophic methanogens, producing methane from hydrogen and carbon dioxide.
32V.IvanouIfthecellconcentrationoftheseorganismsper1mLis4×107,7×108,2×107,5×10°and1 × 18, respectively, the Shannon-Weaver index (H) of microbial diversity by physiologicalfunctions will be 13, and the evenness index (E) will be 8.1. The diversity indices are related tothe process efficiency and stability and can be used in environmental engineering to comparetheprocesseswithdifferentoperationalparameters.2.2.InteractionsinMicrobial EcosystemsThe types of interactions between the biotic elements of a microbial ecosystem (cellsof microbialpopulation,microbialpopulations,microorganisms andmacroorganisms)arepositive and negative.Positive interactions are as follows:Commensalism (onlyonebioticelementhasbenefits)1.2.Cooperation,mutualism(bothelementshavebenefits)3.Essential mutualism, symbiosis (both elements cannot live separately)Negative interactions are as follows:1.Neutral competition (organisms compete in the rateand efficiency of nutrients consumption,growth rateor in theresistanceto unfavourableforgrowth environmental factors).2.Antagonism(bothabioticelements sufferfrominteractionbecausetheyproducespecificfactorsthat negatively affect growth rate or other physiological or biochemical properties of competitors)3.Amensalism(onlyoneelementsuffersfromtheinteraction).4.Predation and parasitism; it is interaction when one element (prey) suffers and the other element(predator) benefits.There may be neutralism, i.e., absence of positive or negative interactions between bioticelements.Thepopulationdensity or average distancebetween biotic elements determines thetypeofinteraction (Fig.2.1),Positive interactionsNegative interactionsAveragedistancebetweencellsor(cellconcentration)-1Fig.2.1.Microbial interactions depending on cell concentration in ecosystem or the distance betweencellsincommunity
32 V. Ivanov If the cell concentration of these organisms per 1 mL is 4 × 107, 7 × 108, 2 × 107, 5 × 108 and 1 × 108, respectively, the Shannon-Weaver index (H) of microbial diversity by physiological functions will be 13, and the evenness index (E) will be 8.1. The diversity indices are related to the process efficiency and stability and can be used in environmental engineering to compare the processes with different operational parameters. 2.2. Interactions in Microbial Ecosystems The types of interactions between the biotic elements of a microbial ecosystem (cells of microbial population, microbial populations, microorganisms and macroorganisms) are positive and negative. Positive interactions are as follows: 1. Commensalism (only one biotic element has benefits). 2. Cooperation, mutualism (both elements have benefits). 3. Essential mutualism, symbiosis (both elements cannot live separately). Negative interactions are as follows: 1. Neutral competition (organisms compete in the rate and efficiency of nutrients consumption, growth rate or in the resistance to unfavourable for growth environmental factors). 2. Antagonism (both abiotic elements suffer from interaction because they produce specific factors that negatively affect growth rate or other physiological or biochemical properties of competitors). 3. Amensalism (only one element suffers from the interaction). 4. Predation and parasitism; it is interaction when one element (prey) suffers and the other element (predator) benefits. There may be neutralism, i.e., absence of positive or negative interactions between biotic elements. The population density or average distance between biotic elements determines the type of interaction (Fig. 2.1). Positive interactions Average distance between cells or (cell concentration)–1 Negative interactions Fig. 2.1. Microbial interactions depending on cell concentration in ecosystem or the distance between cells in community
33Microbiologyof EnvironmentalEngineeringSystemsaBioticelement2Bioticelement1Factor(s) of mediumbBiotic element 2Biotic element1Factor(s) of mediumFig.2.2.Examples of microbial commensalism: (a), one group of microorganisms produces growthfactor(s)essentialforanothergroup:(b),facultativeanaerobes useoxygen and create anaerobichabitatsuitableforthegrowthofobligateanaerobesWhen the population density is low, organisms have neither positive nor negative interac-tions.When the population density is medium, organisms competeamong themselves for theavailability ofresources,byrate or efficiencyof growth,andbyproduction of metabolites,which negatively affect the growth of competitors.When the population density is highcells usually aggregate and cooperate between themselves.Both competition and cooperationare carried outmainly because of thechanges of chemical factors of environment such asconcentration of nutrients,pH and redox potential of the medium, excretion of antibiotics.extracellular digestive enzymes, or heavy metals binding exopolysaccharides and simultane-ousbiodegradationof substances.Commensalism,a microbial system relationshipinwhich onlyonebioticelementbenefitsis realized by different ways (Fig.2.2).There are thousands of examples of this interaction inenvironmental biotechnological systems.Some of them are as follows:1.Facultative anaerobes use oxygen and create the conditions for the growth of obligate anaerobes:this interaction is important in the formation of anaerobic layer in microbial aggregates existingunder aerobic conditions (6).2Onegroup ofmicroorganisms produces a growthfactor essential for another group;this interac-tion is an obvious condition for the outbreak of pathogenic Legionella pneumophila, originatedfrom such engineering systems as airconditioners,cooling towers and fountains.Sequential biodegradation of xenobiotics by different groups of microorganisms; the microbial3.group performing biodegradation does not depend on the activity of the groups degrading itsproductofmetabolism.Mutualism is a type of interaction in which both biotic elements (microbial groups) haveadvantagesfromtheirinteraction(Fig.2.3)
Microbiology of Environmental Engineering Systems 33 Factor(s) of medium Biotic element 1 Biotic element 2 a b Factor(s) of medium Biotic element 1 Biotic element 2 Fig. 2.2. Examples of microbial commensalism: (a), one group of microorganisms produces growth factor(s) essential for another group; (b), facultative anaerobes use oxygen and create anaerobic habitat suitable for the growth of obligate anaerobes. When the population density is low, organisms have neither positive nor negative interactions. When the population density is medium, organisms compete among themselves for the availability of resources, by rate or efficiency of growth, and by production of metabolites, which negatively affect the growth of competitors. When the population density is high, cells usually aggregate and cooperate between themselves. Both competition and cooperation are carried out mainly because of the changes of chemical factors of environment such as concentration of nutrients, pH and redox potential of the medium, excretion of antibiotics, extracellular digestive enzymes, or heavy metals binding exopolysaccharides and simultaneous biodegradation of substances. Commensalism, a microbial system relationship in which only one biotic element benefits, is realized by different ways (Fig. 2.2). There are thousands of examples of this interaction in environmental biotechnological systems. Some of them are as follows: 1. Facultative anaerobes use oxygen and create the conditions for the growth of obligate anaerobes; this interaction is important in the formation of anaerobic layer in microbial aggregates existing under aerobic conditions (6). 2. One group of microorganisms produces a growth factor essential for another group; this interaction is an obvious condition for the outbreak of pathogenic Legionella pneumophila, originated from such engineering systems as air conditioners, cooling towers and fountains. 3. Sequential biodegradation of xenobiotics by different groups of microorganisms; the microbial group performing biodegradation does not depend on the activity of the groups degrading its product of metabolism. Mutualism is a type of interaction in which both biotic elements (microbial groups) have advantages from their interaction (Fig. 2.3)