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
Typical Process Monitoring
• 30-Minute
Settling Test
Typical Process Monitoring
• 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
A Simple Laboratory Program
• Wet Mount
– Floc Color Index
– Total Shelled Protozoa
– Zoogleal Mass Index
• Stained Slide
– Filament count
– Zoogleal Mass Index
A Simple Laboratory Program
• 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
0
+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
Zoogleal Mass Index/mgVSS
X 1000
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.
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
• 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
SVI
60
40
20
0
Battery A
Battery B
Battery C
Battery E1
Battery E2
Filament count
8000
7000
6000
5000
Filament count
4000
3000
2000
1000
0
Battery A
Battery B
Battery C
Battery E1
Battery E2
Floc Color
0
Battery A
Battery B
Battery C
Battery E1
Battery E2
-1
-1
Floc Color
-2
-2
-3
-3
Zoogleal Mass
200
180
160
140
120
Zoogleal Mass
100
80
60
40
20
0
Battery A
Battery B
Battery C
Battery E1
Battery E2
Shelled Protozoa & Metazoa
Total Shelled
300
250
200
Total Shelled
150
100
50
0
Battery A
Battery B
Battery C
Battery E1
Battery E2
SVI
Filament count
8000
120
7000
100
6000
80
5000
SVI
60
4000
Filament count
3000
40
2000
20
1000
0
0
Battery A
Battery B
Battery C
Battery A Battery B Battery C Battery
E1
Battery E1 Battery E2
Total Shelled
Battery
E2
Floc Color
300
0
Battery A
250
Battery B
Battery C Battery E1 Battery E2
-1
200
-1
150
Total Shelled
-2
100
-2
50
-3
0
Battery
A
Battery
B
Battery
C
Battery
E1
Battery
E2
-3
Floc Color
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
Battery B
2000
Battery D
1500
1000
500
0
Aug
Sept
Oct
Nov
Dec
Jan
Feb
Mar
Plant B – Zoogleal Masses
400
350
300
250
Battery B
200
Battery D
150
100
50
0
Aug
Sept
Oct
Nov
Dec
Jan
Feb
Mar
Plant B – Floc Color
Floc Color
0
Battery A
Battery B
Battery C
Battery D
-0.5
-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.
Filamentous Bacteria Identification
• 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.
Filamentous Bacteria Identification
• 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.
A Simple Laboratory Program
• 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.
A Simple Laboratory Program
• 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.
A Simple Laboratory Program
• 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.
A Simple Laboratory Program
• 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.