Glymph - A Laboratory Program For Wastewater Microbiology

A Laboratory Program for Wastewater Microbiology
Toni Glymph-Martin Senior Environmental Microbiologist
Why Microbiology?
It is important to develop and maintain a process control program that includes wastewater microbiology. Conducting simple analyses, routinely and consistently is key to understanding the microbiological workings of the wastewater treatment plant.
The Problem
Many facilities do not have dedicated laboratory staff The main focus is on permit related compliance monitoring
Typical Process Monitoring

BOD TSS MLSS MLVSS

30-Minute Settling Test

Sludge Volume Index (SVI) Typically used to monitor the settling characteristics The volume (in milliliters) that is occupied by one gram of mixed liquor after 30 minutes of settling.
A Simple Laboratory Program

Floc Color Index Zoogleal Mass Index Total Shelled Protozoa-Metazoa Total Filament Count SVI MLVSS

Wet Mount
Floc Color Index Total Shelled Protozoa Zoogleal Mass Index
Stained Slide
Filament count Zoogleal Mass Index

Always use a well mixed, representative sample of mixed liquor Always use the same volume or number of drops on the slide. Always collect the sample from the same location Use the same sample for all the tests
Wet Mount & Scan
Floc Color Index

Under normal conditions floc should appear brown when observed using phase contrast.
Floc Color Index
-5
+5
Floc Color Index

Low food Low nutrients Under low food/low nutrient conditions bacteria secrete excess lipopolysaccharide The more starved they are, the more they secrete
-5
Floc Color Index

Old Sludge Septic Sludge Older sludge is darker in color. When oxygen is lacking sludge becomes septic and turns dark brown to black in color.
+5
Zoogleal Mass Index

There are basically two types of bacteria in the treatment system.
Those that form floc and those that do not
Floc-formers generally react to negative situations by producing excess amounts of lipopolysaccharide. Non floc-formers generally form zoogleal masses in response to negative conditions.
Zoogleal Mass Index
Zoogleal Mass Index

Total Number of Masses Counted X 5 MLVSS
X 1000
Zoogleal Mass Index/mgVSS
Shelled Protozoa & Metazoa

When conditions are unfavorable protozoa and metazoa that form shells will dominate in the system. Conditions such as low levels of toxicity, changes in pH etc.
Count the protozoa and metazoa (un-shelled) Count the shelled protozoa and metazoa Shelled / Shelled + Un-shelled = % Shelled
<25%
Total shelled protozoa and metazoa
Plant A
Five Batteries 3 Distinct configurations
Battery A and B Battery C Battery E-1 and E-2
SVI
120
100
80
60
SVI
40
20
0 Battery A Battery B Battery C Battery E1 Battery E2
Filament count
8000
7000
6000
5000
4000
Filament count
3000
2000
1000
Floc Color
-1
-1
Floc Color -2
-2
-3
-3
Zoogleal Mass
200
180
160
140
120 Zoogleal Mass
100
80
60
40
20

Total Shelled
300
250
200
150
Total Shelled
100
50
SVI
120 8000 7000 6000 80 60 40 20 0 Battery A Battery B Battery C Battery E1 Battery E2 SVI 5000 4000
Filament count
100
Filament count
3000
2000 1000 0 Battery A Battery B Battery C Battery E1 Battery E2
Total Shelled
300 250 200 150 Total Shelled 0 Battery A -1 -1 -2 -2 -3 Battery A Battery B Battery C Battery E1 Battery E2 -3 Battery B
Floc Color
Battery C Battery E1 Battery E2
Floc Color
100
50 0
Why Microbiology?
Looking at only one parameter does not give you the whole picture.
Why Microbiology?
The SVI or 30-minute settling tests tell you there is a problem but may not necessarily tell you what the problem is.
Filamentous Bacteria Excess Zooglea Slime Bulking
Plant B - SVI
140 120
100
80
Battery B
Battery D 60
40
20
0 Aug Sept Oct Nov Dec Jan Feb Mar
Plant B Filament Count

4000
3500
3000
2500
2000
Battery B
Battery D
1500
1000
500
Plant B Zoogleal Masses

400
350
300
250
200
Battery B
Battery D
150
100
50
Plant B Floc Color

Floc Color
0 Battery A -0.5 Battery B Battery C Battery D
-1 Floc Color -1.5
-2
-2.5
-3
Filamentous Bacteria Identification

For the untrained eye, those that are impatient, or who do not have time, identifying filamentous bacteria can be tedious and time consuming.

Instead of going through the tedious process of determining cell shape, size, length, width, sheaths, branching, attached growth etc. Lump them together based on the conditions best suited for their growth.

Make a smear and Gram stain the slide. Look at the slide using the oil immersion lens and look at the Filament Identification Guide and find a match. If it looks like the picture, then that is probably what it is. If the filament is in the low DO category then that condition most likely contributed to its growth in the system.

Developing the habit of looking at the microorganisms on a regular basis can be hard, particularly given time restraints and dwindling resources. Some observations is better than no observations at all.

If you only have time to look at the floc, then look at the floc; but be consistent. If you choose to do protozoan counts, be consistent.

If you only have time to look at the floc, then look at the floc; but be consistent. If you choose to do protozoan counts, be consistent.

Do it weekly every week, or do it monthly every month. Most of all do it the same way every time. The more you do it the better you get and the easier it will become.

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A Laboratory Program for Wastewater Microbiology

Toni Glymph-Martin Senior Environmental Microbiologist

Typical Process Monitoring

Typical Process Monitoring

Typical Process Monitoring

A Simple Laboratory Program

A Simple Laboratory Program

A Simple Laboratory Program

Wet Mount & Scan

Floc Color Index

Floc Color Index

Floc Color Index

Floc Color Index

Zoogleal Mass Index

Zoogleal Mass Index

Zoogleal Mass Index

Zoogleal Mass Index/mgVSS

Shelled Protozoa & Metazoa

Shelled Protozoa & Metazoa

Shelled Protozoa & Metazoa

Shelled Protozoa & Metazoa

Shelled Protozoa & Metazoa

Shelled Protozoa & Metazoa

Shelled Protozoa & Metazoa

Shelled Protozoa & Metazoa

Shelled Protozoa & Metazoa

Shelled Protozoa & Metazoa

Shelled Protozoa & Metazoa

Shelled Protozoa & Metazoa

Shelled Protozoa & Metazoa

Total shelled protozoa and metazoa

0 Battery A Battery B Battery C Battery E1 Battery E2

0 Battery A Battery B Battery C Battery E1 Battery E2

120 Zoogleal Mass

0 Battery A Battery B Battery C Battery E1 Battery E2

Shelled Protozoa & Metazoa

0 Battery A Battery B Battery C Battery E1 Battery E2

0 Aug Sept Oct Nov Dec Jan Feb Mar

Plant B Filament Count

0 Aug Sept Oct Nov Dec Jan Feb Mar

Plant B Zoogleal Masses

0 Aug Sept Oct Nov Dec Jan Feb Mar

Plant B Floc Color

-1 Floc Color -1.5

Filamentous Bacteria Identification

Filamentous Bacteria Identification

Filamentous Bacteria Identification

A Simple Laboratory Program

A Simple Laboratory Program

A Simple Laboratory Program

A Simple Laboratory Program

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