MWEA Process Seminar
2013
Happy Microbiology = Happy Operators
Brian J. Ross, Genesee County Water & Waste Services
November 6, 2013
The Activated Sludge Process
Microorganisms
– How does an operator see them?
How do you use the Microscope?
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Ocular Lens = 10x
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Objective Lens = 10x, 40x, 100x
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Light Source
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Condenser
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Adjustment Knob
How do you use the Microscope?
1) Prepare the Slide
2) Smear Preparation & Staining
Simple Staining vs Differential Staining
1) Gram Positive
2) Gram Negative
3) Neisser Stain
Activated Sludge Microorganisms
5 Major Groups
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Bacteria
Protozoa
Metazoa
Filamentous bacteria
Algae and fungi
Bacteria are complex, even though each has only a single cell.
Bacteria are among the most abundant organisms on Earth.
Bacteria make up approximately 95% of all organisms in the Activated Sludge Process.
Bacteria – 3 Classifications
• Aerobic
• Anaerobic
• Facultative
The most important microorganisms in Activated Sludge are aerobic.
Bacteria are frequently classified based on how they respond to oxygen.
Structure of Bacteria – 3 Shapes
• Coccus (spherical or oval)
• Bacillus (rectangular or rod-like)
• Spirillum (spiral or corkscrew)
Structure of Bacteria
• 1 ) Coccus (spherical or oval)
occurring singly or in pairs – called diplococci
attached in short chains – called streptococci
in groups of four (4) – called tetrads
in three dimensional cubes of eight – called sarcinae
in grape-like clusters – called staphlyococci
Structure of Bacteria
• 2) Bacillus (rectangular or rod-like)
occurring in pairs – called diplobacilli
in short chains – called streptobacilli
occurring in short oval rods – called coccobacilli
Structure of Bacteria
• 3) Spirillum (spiral or corkscrew)
look like commas – called vibrios
rigid body shaped like a corkscrew – called spirilla
flexible body shaped like a corkscrew – called spirochetes
How is a Bacterium Built?
• Slime layer
excreted from the inside to the outside
used for adsorption of larger particles
ultimately used to form “floc”
• Cell wall
semi-rigid
used to give it shape
designed to protect internal parts
• Cell membrane
functions as an entrance and an exit to the cell
secretes enzymes
• Enzymes
proteins that cause biochemical reactions
they breakdown larger particles into smaller ones
smaller units can transport across membrane
process call absorption
• Method of locomotion
wiggle or squirm
glide through medium
propel with flagella
Single cell
How do Bacteria Eat?
What do Bacteria need?
• Organic compounds
human wastes
• Inorganic substances
iron, sulfur, nitrogen
• Essential elements
carbon, nitrogen, phosphorous, sulfur, oxygen
• Other nutrients
carbohydrates, sugars, fats (used for energy)
• Miscellaneous
substances
Ions: magnesium, calcium, potassium, iron, and
chloride
How do Bacteria Multiply?
How do Bacteria Multiply?
Growth in Activated Sludge
Bacteria - The Growth Curve
Lag phase
Accelerated growth phase
Declining growth phase
Stationary phase
Death phase
How do Bacteria Multiply?
Process Control- What can we control?
• It’s all about the operator maintaining
favorable conditions for bacteria
• Can we control? ….
How?.....
pH
dissolved oxygen levels
detention times
sludge age (MCRT)
food to microorganism (F/M) ratio
temperature
Industrial discharges
Protozoa – 3 Classifications
• Amoebae
• Flagellates
• Ciliates
Free-swimming ciliates
Crawling (grazing) ciliates
Stalked ciliates
Protozoa play a critical role in the treatment process, digesting dispersed bacteria
Protozoa make up approximately 3% of all organisms in Activated Sludge
Protozoa are also single-celled microorganisms
Protozoa
1. Amoebae (“naked”, called Proteus)
Amoebae are the most primitive Protozoa.
Amoebae feed on small organic particles, algae, bacteria, and other small protozoa.
Amoebae contribute very little to the overall treatment of wastewater.
Protozoa
1. Amoebae (how it feeds on small organic particles - pseudopods)
Protozoa
1. Amoebae (“testate”, called Arcella)
Protozoa
2) Flagellates (called Flagellate Bodo) – most common
Flagellates are the most widespread of the Protozoa.
Flagellates compete with bacteria for soluble nutrients.
Flagellates also feed on bacteria and small algae.
Protozoa
• Ciliates
Free-swimming ciliates
Crawling (grazing) ciliates
Stalked ciliates
Comes from the Latin word “eyelash”
Ciliates feed on mostly bacteria, yeast, and algae
They do not contribute to the overall treatment by actively removing organic matter
They do contribute to the overall clarity of the wastewater by removing suspended bacteria
Abundance of ciliates indicates that most organic material has been removed from the wastewater
Protozoa
• Ciliates
Free-swimming ciliates
Protozoa
• Ciliates
Free-swimming ciliates
Protozoa
• Ciliates
Free-swimming ciliates
Protozoa
• Ciliates
Crawling (grazing) ciliates
Crawlers are not good swimmers.
Common in Activated Sludge Process. Their dominance indicates good treatment
Crawling ciliates gain their dominance after most soluble nutrients are removed
At this stage, most of the dispersed bacteria have begun to clump together
Protozoa
• Ciliates Stalked ciliates
They have no cilia on their bodies
The cilia around their mouth is used to gather food
They rarely swim freely
Found attached to almost anything
Carchesium colony can contain hundreds of heads
Factors that affect Protozoa
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Level of Organic Nutrients
Dissolved Oxygen
Competition influence
Temperature
These factors influence the abundance and types of Protozoa present in the wastewater
A lack of DO will severely limit the number and type of Protozoa in the system
If there is a high level of organic nutrients the bacteria will out compete the Protozoa
Protozoa grow best in ambient temperatures around 15-25 degrees C (+10C =2x)
Relative Numbers of Microorganisms vs Time
in Activated Sludge
Metazoa – 3 Classifications
• Rotifers
• Nematodes (also bristle worms)
• Tartigrades (water bear)
Their presence indicates treatment system conditions
Metazoa are multi-cellular organisms and include all animals except Protozoa.
They are larger than Protozoa.
Very little to do with removal of organic matter in the Activated Sludge process.
Metazoa
• Rotifers
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The word rotifer comes from the Greek word meaning “wheel bearing animal”
The “wheels” of the rotifer are called corona, which is covered in cilia
The corona provides locomotion and also brings food to the rotifer
The female is larger than the male. Lifespan of male is short. His purpose is to fertilize
Metazoa
• Rotifers
Metazoa
• Nematodes
Nematode is simple structure, even though it is multi-cellular.
Nematodes possess a digestive, reproductive, and nervous system.
They feed on bacteria, fungi, small protozoa, and sometimes other nematodes
Some have teeth like a spear they use to stick prey and then use it like a straw to suck out contents
Metazoa
• Nematodes
Metazoa
• Tartigrades (water bear)
The word Taritigrade means “slow walker”. There movement is clumsy and slow.
Water Bears have developed ways to survive extreme environmental swings.
If they lack DO, they will swell up and float for days. If they lack water, they will shrivel like a raisin.
Tartigrades are very sensitive to “toxic” conditions.
They feed on algae and small protozoa. They feed like nematodes (pierce food – suck out parts)
Metazoa
• Tartigrades (pregnant water bear)
The Food Web
Filamentous – 2 Classifications
• Foaming filaments
• Bulking filaments
Filamentous
• Foaming filaments
Foaming filaments – caused by:
• Excess greases, oils, & fats
• Low F/M ratio
• Temperature changes
Filamentous
• Bulking filaments
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The presence of some filamentous bacteria is helpful. They provide support structure for floc
When they gain dominance they may cause settling problems
Associated with changes in: temperature, DO, sludge age, nutrients, and grease.
Bulking filaments - caused by:
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Low DO
Low F/M Ratios
Longer Sludge ages
Septic wastewater
Nutrient deficient incoming wastewater
Changes in pH
Excess greases, oils, and fats
Identifying types of filamentous
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Filament shape and length
Cell shape and length
Cell septa
Sheath
Attached growth
Branching
Sulfur granules
Motility
Identifying types of filamentous
• Filament shape and length
Identifying types of filamentous
• Cell shape and length
Identifying types of filamentous
• Cell septa
Identifying types of filamentous
• Sheath
Identifying types of filamentous
• Attached growth
Identifying types of filamentous
• Branching
Identifying types of filamentous
• Sulfur granules
Identifying types of filamentous
• Motility
Algae and Fungi
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Algae contains chlorophyll
photosynthesis occurs
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Fungi do not contain chlorophyll
Nitrification
• Nitrosomonas
Converts Ammonia-Nitrogen (NH4+) to
Nitrite Nitrogen (NO2-)
• Nitrobacter
Converts Nitrite Nitrogen (NO2-) to Nitrate
Nitrogen (NO3-)
Once cBOD is used up, Nitrification can begin
Very sensitive organisms
Who can Identify these bugs?
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Who can Identify these bugs?
Who can Identify these bugs?
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Who can Identify these bugs?
Who can Identify these bugs?
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Who can Identify these bugs?
MWEA Process Seminar
2013
Conclusion
MWEA Process Seminar
2013
Question
&
Answer