Wastewater Microbiology

1. Wastewater Microbiology Bob Rawson

2. Recommended Text Books • Manual of instruction for Wastewater Treatment Plant Operators two Volume New York State Department of Environmental Conservation LC control OL220555M. • Operation of Wastewater Treatment Plants Ken Kerri. • Wastewater Engineering Treatment Disposal /Reuse. Metcalf & Eddy Dr. George Tchobanoglous, U.C. Davis. • Sewerage Organisms a Color Atlas, J Cox, Chicago.

3. Recommended References continued • Standard Methods for the Examination of Water and Wastewater, 21st Edition or most recent available. • MOP 11. WEF. • EPA Technology Transfer see internet web pages. • White’s Manual of Chlorination. • Any good recent Microbiology text book

4. Topics we will discuss • Structure of the Cell. Plant Animal Bacterial. • Fixed Film and Suspended Growth. (Examples) • Biological Mechanisms that affect water Quality and accomplish WW Treatment like: • Photosynthesis • Respiration Aerobic, Anaerobic and Facultative. • Nutrient Assimilation, importance & Cycles of • Carbon, Nitrogen, [phosphorous, sulfur.

5. Discussion Topics continued • Description of Wastewater Microbes. Structures Pathogens: Disease causing organism Microorganisms used in Wastewater Stabalization Obnoxious organisms causeing Odor, Corrosion toxicity. • Use of the Microscope. • Common Laboratory test methods. • TSS • Coliform Tests • BOD • Bioasays

6. Three Domains Six Kingdoms • Bacteria (Monera) • Archea • Eucaryota • Plants • Animals • Fungi • Protista (The Protozoans) • Protophyta • Protozoa • Protomycota

7. Phylogenetic Tree of Life

8. Taxonomy • Domain • Kingdom • Phylum (Division) • Subphylum • Class • Order • Family Binomial Genus Species

9. Example of Taxonomy • Eucaryota • Kingdom Animalia • Phylum Chordata • Subphylum Terrapoda • Class Mammalia • Order Carnivora • Family Canidae • Genus Canis • Species Familiarias (The Familiar Dog)

10. Microbial TaxonomyNaming, classifying microorganisms • 1. Naming-Linnaeus introduced binomial nomenclature • -Every organism is given a unique two-part name • Genus + specific epithet = species name • Ex. Escherichia coli, Yersinia pestis, Bacillus anthracis • 2. Classification-Organisms with similar characteristics are grouped together • Ex. Genus Bacillus • Bacillus anthracis, Bacillus subtilus, Bacillus cerus

11. Microbial TaxonomyNaming, classifying microorganisms • -Higher levels of organization are used to group species having similar characteristics • Ex. Bacillaceae is the name of a Family that contains several different species of rod-shaped, spore-forming, Gram positive, aerobic bacteria such as Bacillus anthracis,Bacillus subtilus and Bacillus cereus • 3. Identification • -Determination of the name of an organism that has been isolated by observing its characteristics

14. Differences Between Plant and Animal Cells. • Plant Cell • Rigid support and Protection • Cellulose a fiberous complex polysaccharide allows H2o and air to pass through. • Lignin a hard phenolic compound. Will react with chlorine or fire to form dioxin. • Plastids: • leucoplasts Starch

15. Plant Cell continued • Chromoplasts like Chloroplasts • Chloroplasts contain Chlorophyll • Responsible for photosynthesis by acting as a catalyst to split H2O in the presence of light. • We will discuss Photosynthesis further.

16. Plant Cell

17. Animal Cell

18. Typical Animal cell • Cell Wall not absence of cell wall. • Cytoplasm. The intercellular contents • Organelles including: • Plastids . • Mitochondria. The power house ATP transactions. • Golgi Complex. Stores enzymesd • Cilia or flagella. Movement • Food Vacuole

19. Microorganisms and Their Impacts • Microorganisms were the first living organisms on the planet • Prokaryotes (bacteria) 1st appeared ~3.5 billion years ago • Can be found nearly everywhere on earth in large numbers • -Soil, oceans, deep underground, extreme environments, on and in higher organisms

20. ~100 trillion (1014) microorganisms per person • 10 X number of cells of the human body ! • Most are in the intestinal tract • ~1 trillion bacteria on the skin • Normal flora aids food digestion and synthesizes vitamins in intestinal tract, inhibit growth of pathogenic microorgansisms by competiting for space and nutrients

21. Importance of Microbes • Major affect on the composition of gases in earth's atmosphere • -Formation of oxygen by photosynthesis (required for survival by many organisms) First source of Oxygen was Cyanobacteria. • -Formation of carbon dioxide and methane during decomposition (Greenhouse gasses) • Transformations of nitrogen (major plant nutrient) • Fix Carbon and Decompose organic matter. • Agriculture, Wastewater Treatment, Bioremediation Phytoremediation and Mycoremediation. • Recycle nutrients • -Carbon, nitrogen, phosphorus, sulfur • Microorganisms also cause diseases. Pathogenic organisms

22. -There are two main types of microbial cells • 1. Prokaryotic • 2. Eukaryotic • 1. Prokaryotic • Small • Lack a nucleus or organelles • Often exist as single cells (unicellular) • All bacteria are prokaryotes • 2. Eukaryotic • Larger than prokaryotes • Have a nucleus and organelles • May be unicellular or multicellular • Yeast, fungi, protozoa, algae, helminths

23. Main types of microbial cells Cont. • 2. Eukaryotic • Larger than prokaryotes • Have a nucleus and organelles • May be unicellular or multicellular • Yeast, fungi, protozoa, algae, helminths

24. Typical Animal Cell Continued • Endoplasmic Reticulum with nodular lysosomes. • Lysosomes containing digestive enzymes

25. Virus • -Viruses are small particles composed of protein and nucleic acid (DNA or RNA) • Do not have the characteristics that define cellular organisms (see below) • Only able to reproduce when inside a cell • Obligate parasites that may have arisen as a degenerate form of early bacteria.

26. Bacteria Cell

27. Bacteria Cell • Capsule or Slime layer • Slimes are Sticky Gelatinous Material secreted by bacteria Cell • Functions of slime layer. • Allow bacteria to stick together onto substrate or flocculate • Protection from toxic environment like chlorine UV

28. Further functions of Slime Layer • Bacteria Stick together to a substrate or floculate. • Protection from hostile environment • Trap Food which is adsorbed to the cell surface by: • Adsorb =Adhere to surface, in preparation for absorption. • Slimes contain enzymes which Digest food. • Slimes contain endotoxins and exotoxins that cause disease reactions.

29. Sizes of Microorganisms • The micrometer, mm, is the unit of measure often used to describe the size of microorganisms • 1 mm = 1/1,000,000 meter = 0.000001 m = 1 x 10-6 m • Typical sizes • Viruses 0.01 to 0.2 mm • Bacteria 0.2 to 5 mm • Yeast 5 to 10 mm • Algae 10 to 100 mm • Protozoa 50 to 1000 mm

33. Activated Sludge • A suspended Growth treatment Process using microorganisms including; bacteria; fungi; and protozoans. • Three steps: • Transfer of food • Conversion of food to Cell Mass • Flocculation or settling of organisms that are stuck together.

34. Activate Sludge Transfer Continued • Adsorption: Particles and colloids stick to the slime layer • Absorption: Nutrients passing through the cell wall. • Phagocytosis/ Pinocytosis • Digestion: • Osmosis solute moves from more concentrated location to less concentrated location down gradient.

35. Transfer continued • Dialysis. Opposite of osmosis requires energy • Active Transport. Water and oil don’t mix until we add soap, or an emulsifier that acts as a carrier. • Cell membranes contain phospholipid • Large molecules that are on the outside like glucose cannot get in like small uncharged particles. To get in a carrier molecule attaches to the particle making it soluble in the lipid layer just like soap makes oil soluble in water. This requires energy provided by ATP.

36. Further steps in Activated Sludge Process • Second Step is the Conversion processes: • Respiration. • Aerobic • Anaerobic • Facultative • Flocculation: • Happy well fed microbes stick together and settle out taking nutrients colloids etc with them. They become sludge (Bio Solids).

37. ATP • ATP is Adenosine Triphosphate • High energy phosphate bonded to a five carbon sugar. • Source of all energy for cell activity • Primary reason for the importance of phosphorous as a growth limiting factor in wastewater and environmental systems. (Algae blooms)

38. Photosynthesis • Light Phase • Dark Phase • Light Phase: • Light strikes water (H2O • Water splits into H2 and O2 and an electron. • The O2 is given off and Hydrogen is transferred to a hydrogen acceptor TNP or Tri-phosphopyridine nucleotide.

39. Light Phase of Photosynthesis continued • The electron is used by the mitochondria in the cell to convert ADP into ATPand energy is stored in the high energy phosphate chemical bond. • This ATP is used in the Dark Phase f Photosynthesis.

40. Photosynthesis Dark Phase • ATP + CO2 +H20 = PGAL • PGAL is Phosphoglyceraldehyde a three carbon sugar. • 2 PGAL combine to form one six carbon sugar called Glucose. • Glucose is stored in the cell leucoplast for future respiration. • Water is produced as the final Hydrogen acceptor.

41. Respiration, The flip side of Photosynthesis • Respiration is the reverse of photosynthesis • Two types of Respiration: • Aerobic Respiration. Free atmospheric O2 • Anaerobic Respiration. Chemically bound O2 • In aerobic Respiration Glucose is chemically burned with oxygen in the cell to produce water+ carbon dioxide + Energy.

42. Photosynthesis vs Respiration

43. Anaerobic Respiration • When Free oxygen is not available other hydrogen acceptors are used. CO2, SO4,NO2,NO3 • In anaerobic respiration the carbon substrate is incompletely oxidized producing unstable metabolites. • In Plants and Yeasts : Alcohol and CO2 produced when glucose is metabolized. (Beer and Bread). • In Animals Glucose is converted to lactic acid in muscles as a temporary hydrogen acceptor.

44. Digesters are Anaerobic processes • Two Stages of Digesters: • Acid Stage: Fast. • Gas Stage: Slow and sensitive to temperature. • Acid Stage: Acid forming bacteria convert food substrate into odorous volatile acids including: • Acetic • Caproic • Valeric • Butyric • Formic

45. Gas Stage of anaerobic Digestion. • Gas forming bacteria convert volatile acids into water and gases. • The gases include: • Carbon Dioxide CO2 35% Plus or Minus • Ch4, Methane: 70 Percent + or - • Hydrogen Sulfide: • Argon:

46. Bacteria Vital Statistics • Rigid unicellular microbes lacking chlorophyll • Size 2 X 0.7 Micron.; • Present everywhere. • Mostly Non-Pathogenic. • Necessary for higher forms of life to exist because they recycle nutrients.

48. Methods of identifying Bacteria • Multiple Fermentation Tube. • Membrane Filter • Enzyme Substrate. • Gram Staining • Specific media.

49. Bacteria Characterized • Gross Appearance Growth and Morphology including spore forming non-spore forming. • Morphology is shape: • Rod Shaped is called Bacillus : Coli, Typhoid. • Sphere Shaped is called Coccus: Streptococci. • Spirelus is corkscrew shaped: Cholera

50. Bacteria Characterized • Gross Appearance: Spore former, Non-spore Former Spore a walled resting stage harder to kill. • Morphology- Form: Rod, Sphere, Spiral. • Temperature Range: Cold, Medium or hot loving. • Oxygen Requirement: Aerobic, anaerobic, Facultative.