AS Microbiology – Good Actors and Bad Seth Terry, Ph.D.

2 – AS Microbiology, Vail PWO Conference 2006 Good

3 – AS Microbiology, Vail PWO Conference 2006 Bad

4 – AS Microbiology, Vail PWO Conference 2006 UGLY!

5 – AS Microbiology, Vail PWO Conference 2006 A key question for operators Of course, settleability represents only one aspect of ‘goodness’ So, as an operator, you have to ask yourself the question: “Do you stand for ‘goodness’, or - for ‘badness’?” Judge Smails, Caddyshack (1980)

6 – AS Microbiology, Vail PWO Conference 2006 What makes a sludge “good” Controllable settling Appropriate microbiology

7 – AS Microbiology, Vail PWO Conference 2006 Controllability Indicated by an ABSENCE of filaments ?????? NO! A widespread misconception Sludges without filaments generally settle too quickly, leaving behind turbid effluent Sludges with too many filaments generally produce crystal clear supernatants, but settle at rates incompatible with secondary clarifiers The ‘sweet spot’ lies somewhere in between

8 – AS Microbiology, Vail PWO Conference 2006 Appropriate Microbiology Growing the right bugs To control settleability Relative proliferation of filaments To achieve permit compliance BOD oxidizers Nitrifiers Denitrifiers Polyphosphate-accumulating organisms (PAOs) Fermenting facultative organisms To avoid odor Minimizing sulfate reducing bacteria (SRBs)

9 – AS Microbiology, Vail PWO Conference 2006 Population Control Clearly, we would like to control the bacterial population in our aeration tanks We do so indirectly Bacteria are very effective at exploiting environmental niches Food source, DO level, temperature, water chemistry, metabolites from other bacteria Environment controls population

Microbiology Basics

11 – AS Microbiology, Vail PWO Conference 2006 ‘Bugs’ Are Not ‘True Bugs’ Two pairs of wings Partially hardened first pair Jointed piercing and sucking mouthparts Series of nymphal stages in development Phylum Arthropoda, class Insecta, order Hemiptera

12 – AS Microbiology, Vail PWO Conference 2006 Bugs in Wastewater Treatment Bacteria – Single-celled organisms Prokaryotes Lack discrete nuclear membrane Protozoa – Single-celled animals Eukaryotes DNA housed in nuclear membrane Metazoa – Multi-celled animals Specialization of cellular functions

13 – AS Microbiology, Vail PWO Conference 2006 Bacterial Types Origin Enteric Soil Feed Heterotrophic (organic) Autotrophic (inorganic) Growth Pattern Floc former Filament Final Electron Acceptor Aerobic respiration O 2 Anaerobic respiration NO 3 - SO 4 2- CO 3 2- Anaerobic fermentation Organic acids & alcohols Methane

14 – AS Microbiology, Vail PWO Conference 2006 Phylogenetic Classification Tending to replace out-dated system based on phenotypic relationships, particularly in microbiology Latest system uses genetic differences detected by molecular techniques (molecular chronometer) Root = primeval organism, the ‘Universal Ancestor’ Domains: Bacteria, Archaea, Eukarya, (Korarchaeota) Kingdoms: at least 14, but probably >50! (Class) (Order) (Family) Genus: usually 93% to 95% molecular similarity Species: isolated monoculture (single bacteria culture) } Academic work!

15 – AS Microbiology, Vail PWO Conference 2006 Metabolic Classification All Organisms Chemotrophs: Light not required Chemolithotrophs: Electrons from inorganic chemicals Chemolithoautotrophs: C from CO2 (‘AUTOTROPHS’) Mixotrophs: C from organic source (‘HETEROTROPHS’) Chemoorganotrophs: Electrons AND C from organic chemicals (‘HETEROTROPHS’) Phototrophs: Light required Photoautotrophs: C from CO2 (ALGEA) Photoheterotrophs: C from organic source (purple non-sulfur bacteria) First distinction is made on relation to light Second distinction may be made on electron source Third distinction is made on carbon source for cell growth Adapted from Brock, 9 th Ed.

16 – AS Microbiology, Vail PWO Conference 2006 Classification Based on e - -acceptor Distinction based upon relationship to oxygen Adapted from Brock, 10 th Ed.

17 – AS Microbiology, Vail PWO Conference 2006 Floc-Forming Bacterial Species Pseudomonas Flavobacterium Achromobacter Bacillus Alcaligenes Micrococcus

18 – AS Microbiology, Vail PWO Conference 2006 Electron Photo of a “Floccy” Floc

19 – AS Microbiology, Vail PWO Conference 2006 Electron Photo of a Filamentous Floc a.k.a “The Sponge”

20 – AS Microbiology, Vail PWO Conference 2006 Filamentous Bacterial Species Sphaerotilus natans Beggiatoa Haliscomenobacter hydrossis Microthrix parvicella Nocardia-forms Nostocoida limicola Thiothrix

21 – AS Microbiology, Vail PWO Conference 2006 Common Eikelboom “Type” Filaments N

22 – AS Microbiology, Vail PWO Conference 2006 Filament Growth Environments Low DO Septic/Sulfide Organic loading rate and biodegradability pH Nutrient deficiency Completely mixed, continuously fed

23 – AS Microbiology, Vail PWO Conference 2006 F/M vs. Protozoa/Metazoa Populations F/M lb BOD/lb MLSS (Eikelboom, 2000) Metazoa Shelled Am. Carnivores Stalks Crawlers Free-Swim. Flagellates Amoeba Range Most Common

Elements of Control

25 – AS Microbiology, Vail PWO Conference 2006 Environmental Factors Physical Chemical Biological Controlled Uncontrolled

26 – AS Microbiology, Vail PWO Conference 2006 Physical Factors Temperature Mixing DO Hydraulics Detention time Mixing

27 – AS Microbiology, Vail PWO Conference 2006 Chemical Factors BOD Form of food Availability of food Basic water chemistry pH Generally between 7.0 and 7.5 Alkalinity Nitrification requirement Nutrients Toxins

28 – AS Microbiology, Vail PWO Conference 2006 Biological Factors Terminal electron acceptor Aerobic Anoxic Anaerobic Sludge age Mean cell residence time (MCRT)

29 – AS Microbiology, Vail PWO Conference 2006 Energy Production/Utilization Organic Matter Electrons Bacteria

30 – AS Microbiology, Vail PWO Conference 2006 Final Electron Acceptors O 2 – aerobic NO 3 - – anoxic SO 4 2- – anaerobic CO 3 2- – anaerobic Organic compounds Fermentation Alcohols Acids

31 – AS Microbiology, Vail PWO Conference 2006 Energy Distribution Aerobic 33%  Heat 67% maintenance & growth Maintenance 50% high load 70% low load Growth 50% high load 30% low load Anaerobic 4.6% heat 95.4% maintenance & growth Maintenance 88.5% Growth 6.9%

32 – AS Microbiology, Vail PWO Conference 2006 Food to Microorganism Ratio F/M or F:M lb BOD removed per lb MLVSS in system Basis System F/M – 24 hour period Instantaneous F/M – Right now Highest at head of tank, gets smaller down the tank Related to MCRT

33 – AS Microbiology, Vail PWO Conference 2006 Log # Viable Organisms Time Heterotrophic Growth Curve Autotrophic Growth Curve Log Stationary Death Lag Bacterial Growth Curve Declining Growth Accelerated Growth

34 – AS Microbiology, Vail PWO Conference 2006 Active Microorganism Numbers Time or Distance Down the Aeration Tank # Viable Microorganisms Log growth phase Declining growth phase Endogenous phase

35 – AS Microbiology, Vail PWO Conference 2006 Changing F/M Q RAS F/M M F

36 – AS Microbiology, Vail PWO Conference 2006 Growth Curve with Feed Aeration period # Active Microorganisms Raw waste Length of Aeration Tank Return to Aeration Tank

37 – AS Microbiology, Vail PWO Conference 2006 Primary and Secondary Growth Time Numbers of microorganisms Secondary predominancePrimary predominance Secondary organisms feed upon cell-lysis products, primarily protein

38 – AS Microbiology, Vail PWO Conference 2006 Soluble BOD vs. Time Aeration period 5-day B.O.D. B.O.D. determined on settled supernatant Biosorption zone

39 – AS Microbiology, Vail PWO Conference 2006 Primary and Secondary Organisms Primary Carbohydrates Metabolized by many different genera Organic acids, aldehydes, ketones, alcohols Pseudomonas Micrococcus Bacillus Achromobacter

40 – AS Microbiology, Vail PWO Conference 2006 Primary and Secondary Organisms Secondary Proteins Lysis of bacterial cells Cell contents primarily protein Alcaligenes Flavobacterium

41 – AS Microbiology, Vail PWO Conference 2006 Measures of Metabolism Direct growth methods Micro-counting Particle counting Indirect methods SOUR/Respiration Rate ORP Direct biochemical methods NADH

42 – AS Microbiology, Vail PWO Conference 2006 SOUR vs. A-Tank Length Distance along aeration tank Oxygen utilization

Questions