Aquatic Bacteria & Fungi

1. Aquatic Bacteria & Fungi • Objective • To know the main cellular features, physiology and function of bacteria & fungi in water and wastewater environments • To know the species interactions in anaerobic digestion • to understand how substrate conditions and nutritional requirements determine the competitive success of these microbes in pollutant degradation processes. • References • Gray N.F. Biology of Wastewater Treatment • Lester J.N. & Birkett J.W. Microbiology and Chemistry for Environmental Scientists & Engineers • Madigan M.T., Martinko J.M., Parker J. Brock - Biology of Microorganisms • Stanier R.Y. General Microbiology • Kiely G Environmental Engineering • Lecture Outline • Bacteria - Cell Structure Physiology & Function • Fungi- Cell Structure Physiology & Function

2. Bacteria • What are they? • Prokaryotic organisms • Bacteria (eubacteria), Archaea (archaebacteria) • Importance in Environmental Engineering • Biodegradation • Nutrient Cycling • Pathogens in Contaminated Waters

3. Bacterial Cell Structure • Size • smallest living organisms, 1m. • Shape • typically cocci or rods (bacilli), spiral, stalked, filamentous. • multicellular swarms (gliding myxobacteria, myxococcus) • DNA • circular, supercoiled, no nuclear membrane. • Extranuclear DNA or Plasmids. • Reproduction • Asexual = Binary fission, Conjugation via Pili.

4. Cell Structure • Cell Wall • Two types, Gram Positive, Gram Negative • Both have Peptidoglycan • Gram Negatives also have Lipopolysaccharide (LPS) • Archaea • similar to G+ve, have pseudopeptidoglycan

5. Cell Structure • Flagellum • May be present - Motile • Polar or peritricious • Driven by Proton motive Force (PMF) • Chemotaxis - tumble frequency increases. • Cytoplasm • complex subcellular organelles usually absent. • vesicular and lamellar structures (mesosomes) form by invagination of cytoplasmic membrane (e.g. N-fixing, Nitrifying, and Phototrophic bacteria). • cytoplasmic membrane essential (maintains PMF). • Ribosomes - Protein synthesis • Enzymes - metabolism • Granules (Inclusions) • Gas Vesicles (buoyancy, e.g. cyanobacteria)

6. Characteristics • Oxygen Requirements • Aerobic • Microaerophilic • Facultative (aerobe) • Anaerobic (strict) • Growth Requirements - Organic substrates • Heterotrophic (Chemoorganotrophs) • Pseudomonas, Bacillus, Zoogloea, etc. • Key role in Nutrient Cycling • Biodegradation of Organic Detritus • Soluble low molecular weight substrates e.g. acetate, methanol, sugars. • Polymers degraded by extracellular hydrolytic Enzymes.

7. Metabolism • Growth Requirements - Inorganic substrates • Autotrophic (Chemolithotrophic, Phototrophic) • Nitrosomonas, Nitrobacter, Methanococcus, Chlorobium, etc. • Reduced forms of sulphur H2S, S0, S2O32-, SO3- • Reduced forms of nitrogen NH3 • Hydrogen H2 • Iron Fe2+ • Growth Requirements - Light • Photosynthetic (phototrophic) • light and CO2 • oxygenic blue-green (cyanobacteria) • anoxygenic green-sulphur (Chlorobium sp.)

8. Bacteria in Aquatic Environments • Natural Waters • Energy source (depends on metabolism and dissolved species) • Cellular Nutrient Requirements • C, H, O, N, P, S. • vitamins, growth factors, trace elements. • Dissolved Gases (O2, CO2, H2S) • Nitrogen is usually limiting in oligotrophic waters. • Origin of Nutrients • Algal secretions, death. • Zooplankton feeding, death. • Soil run-off • discharge of treated (& untreated) effluents.

9. Bacteria in Aquatic Environments • Planktonic • suspended free cells • vertical movement • O2 • stratified nutrients (in anoxic zone) • Particulate • associated with POM • Biofilms • surfaces of stones and plants (epiphytic) • can be slow growing, psychrophilic environments.

10. Methanogenesis • Methanogenic Bacteria (Archaea) • Chemolithotrophic (autotrophs) • H2 and CO2 • e.g. Methanobacterium, Methanococcus, Methanospirillum • 4H2 + H+ + HCO3- CH4 + 3H2O • Energy -136kJ (but as low concentrations = -30kJ) • Low pE (anaerobic) environments • Inhibited in Marine sediments • Other substrates include Acetate, Methanol, Formate etc.

11. METHANOGENESIS Complex polymers Protein, Cellulose Hydrolysis Sugars, amino acids Monomers Fermentation Propionate Butyrate Alcohols H2 + CO2 Acetate Acetogenesis Syntrophs Fermentation Acetate H2 + CO2 Acetate METHANE Methanogens Acetoclastic Methanogens H2-Utilising, Acetoclastic

12. Methanogenesis • Methanogenesis involves Co-operation • Inter-species Hydrogen transfer • Several Steps from a complex substrate (Cellulose) 1. Hydrolysis (depolymerisation) to cellobiose (G-G) 2. Fermentation of Glucose to Fatty acids, H2 and CO2 3. Fatty acids oxidised to H2 and CO2 (SYNTROPHS) 4. Methanogens produce CH4 • Syntrophs require H2 to be consumed • Typically H2 < 10-4 M

13. Fungal Cells • Size • Typically 5m diameter filament, variable length • Structure • Filamentous – hyphae bundled as Mycelia (moulds) Usually branched • Rods (Yeasts ) • Chitin and cellulose cell walls • DNA • chromosomes, nuclear membrane. • Reproduction • Asexual = tip cell, sexual = spores called conidia.

14. Physiology of Fungi • No chlorophyll, produce extra-cellular enzymes. • Heterotrophic nutrition. Parasitic or Saprophytic • Very slow rate of growth cf. bacteria. Tolerate low DO, low pH, High C:Nratios. Dairy & Trade wastes Environmental Requirements 1. Nutrients - Only organic C C10H17O6N - or Organic C + N } i.e. low ratio N:C and some need therefore tolerate vitamins N deficiency. 2. Moisture relatively low concentration H2O (75-80%) (Usually 95-98% in bacteria etc.) Therefore can grow on moist and aquatic environments.

15. Physiology of Fungi 3. pH Normally prefer low pH (produce acid themselves) 4. Oxygen Normally prefer O2 (i.e. aerobic) although some species can tolerate anaerobic conditions temporarily. Aerobic respiration: C6H12O6 6CO2 + 6H2O Anaerobic respiration fermentation: C6H12O6 2C2H5OH + 2 CO2 (Yeasts) 5. Temperature Grow in range 2 - 25oC, optimum = 15oC i.e. psychrophilic - cold-loving

16. Importance of Fungi in Freshwater 1. Fungi play similar role to bacteria. Very important in breakdown of complex organics to simpler substances for algae (i.e. NH3 mineralization) White rot fungi (Phenaerochete) degrade lignin and produce enzymes that degrade complex pollutant molecules Associated with polluted waters because of high nutrient requirements. 2. Indicators of pollution Fusarium, Leptomitisand Geotrichum associated with a mesosaprobic zone in Saprobian system.

17. Importance of Fungi in Freshwater 3. Actinomycetes and Fungi Give Taste and Odour problems in treated water. a) Grow on reservoir walls, and release complex organic compounds when dead. (TASTE AND ODOUR). Also grow on dead algae. Very common after algal blooms. Saprophytic b) Grow in cold water systems in buildings, especially where cold and hot water pipes are adjacent. 4. Sewage Fungus growths in rivers receiving certain industrial wastes (e.g. wood pulping and dairy wastes). 5. Marine Waters

18. Sewage Fungus WRC Survey of 90 Sewage Fungus Associations thick, slimy growths on river bed pulp mill, dairy or strong sewage Leptomitis lacteus 4% Geotrichum 7% FUNGI Fusarium aqueductum 3% Other fungi 10% Sphaerotilus natans 89% BACTERIA* Zoogloea 94% Stigeoclonium 10% Diatoms 18% ALGAE Ulothrix 4% Sewage Fungus - therefore a misnomer Mainly bacteria

19. Fungi in Activated Sludge Rare, unless high proportion of trade wastes (e.g. Canneries, Dairies, Distilleries) High C:N ratio Low pH Low DO Overloading Under aeration Give rise to BULKING SLUDGE. *Geotrichum Pullularia pullulans Sporotrichum Also filamentous bacteria give rise to same problem e.g. Nocardia, Sphaerotilus natans, Thiothrix, Microthrix+ many others

20. Fungi in Trickling Filters Leptomitis lacteus often present in feed channels. Fusarium aqueductum Colonise surface of filter Geotrichum candidum Able to withstand impact of sewage. Sepedonium spp. Subbaromyces speldens Common in sub-surface Ascoidea rubescens zone. Phoma, Saprolegnia, Leptomitis lacteus occasionally present. In winter, species with low optimum temperature e.g. Sepedonium dominate.

21. Fungi in Trickling Filters Industrial Wastes e.g. from Canneries, Dairies, Distilleries etc. encourage growth of fungi (High C:N ratio) Problems caused by Fungi: Heavy growth causes PONDING, especially in winter. Operational procedures: Film accumulation controlled by • Recirculation, • Alternating double filtration • low frequency dosing.

22. Colonisation of Trickling Filters Fungi, high energy of maintenance (40-50 mg/l BOD) Bacteria have much lower saturation constants than fungi (Ks = 0-20 mg/l BOD for sewage bacteria) Therefore bacteria continue to grow at low substrate concentrations i.e. Bacteria have a competitive advantage over fungi at low substrate concentrations. Vice versa at high substrate concs.