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<organicmatter>+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 < organic matter > + 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