Wastewater Microbiology

Presentation
WWETT 2015
Presented by: Ryan Hennessy
 Floc Formation
Floc forming bacteria contain an
polysaccharide (slime) layer, known as
a glycocalyx.
The slime is made up of protein and
carbohydrates and helps to cement the
bacteria together
This occurs at low F/M around 2 and
lower
To form irregularly shaped flocs with a
strong backbone, a small abundance of
filamentous bacteria are desirable.
It is possible for both strong and weak
flocs to
exist without filaments
Floc 100x
Role of Higher Life Forms.
These can be an indicator of lack of toxicity and the amount of food
present.
Amoeba and flagellates grow on high prey densities of dispersed bacteria
found when there is soluble cBOD present.
Crawling Ciliates, Stalked Ciliates, and Rotifers are more efficient and grow
on lower dispersed bacteria prey densities associated with good cBOD
removal.
Water Bears indicate good BOD removal and good nitrification (ammonia is
toxic to them).
Bristle worms and nematodes indicate lack of
toxicity and good BOD removal and grow in
longer MCRT systems.
Flagellates
These are small (5-20 um) and more actively due to one or more long whip-like flagellae
Many species of these feed on soluble organic matter (food) and predominance of them can
occur at a high amount of food to bugs (F/M ratio)
Amoeba
These vary in shape and a mobile due to pseudopodia (false feed).
Some species have a hard cell called a test and are known as testate amoeba
They grow well on particulate BOD and are able to grow in low DO
Free Swimming Ciliates
These are round or oval and move due to rows of
short hair-like cilia
Some have cilia fused into spikes that help them
crawl on the surface of flocs (crawling ciliates)
These are found under conditions of good floc formation and
generally good treatment, they are also very sensitive and not seeing
them can be a sign of toxicity
Stalked Ciliates
These occur at conditions similar to free swimming ciliates but are attached
to the flocs by stalks
There are several species of these, some having one organism per stalk and
others which are colonial and have several organisms
Rotifers
These have several shapes and are much larger and complex than protozoa
(rotifers are metazoan)
Most are motile and attach to the flocs with their feet
These occur over a wide range of sludge ages
(4 days or greater)
Higher Intevertebres
These include nematodes, tardigrades, gasteotrichs, bristle worms and
annelids
They have low growth rates and generally only observed in systems with a
longer sludge age
Ammonia is toxic to most of these organisms and they only appear in
systems with good nitrification
Higher Life Forms
Satisfactory performance occurs when theres a balance between free
swimming, attached ciliates and rotifers.
Because there are many other factors for sludge settling besides the
organic loading, process control should not be based on these
Protozoa and Metazoan only make up 5-10% of the MLSS by weight
Higher Life Forms (cont.)
There is no direct link between higher life forms and sludge age
The mix of higher life forms can change rapidly due to conditions in the plant
while identifying the filaments and other bacteria present also provides a look at
conditions over the past several sludge ages
These are still useful to look at from a toxicity standpoint and as part of the big
picture
 Sludge Quality Problems
Cloudiness (turbidity) due to dispersed bacteria
and/or filaments
Cloudiness (turbidity) due to pin flocs that settle
rapidly and leave behind a cloudy supernatant
Increased SVI (slow settling) due to sludge bulking
caused by excess filaments and/ or bacterial slime
 Causes of Dispersed Growth
May occur due to selection of single cells or filaments at high
growth rates (high F/M)
High concentrations of monovalent cations (like potassium and
sodium) relative to the concentrations of divalent cations
(calcium and magnesium)
High temperatures (>40 degrees C) or rapid temperature change
Particularly going through the zone of 35 degrees
C where different thermo-tolerant bacteria are selected
Deflocculation
Poorly biodegradable surfactants
Toxic Materials
 Causes of Pin Floc
Pin floc (<100um) is known to occur at starvation conditions
associated with very low F/M ratio
In these instances small dense flocs settle rapidly
and leave behind a turbid supernatant
Chronic toxicity is also a cause of pin floc
Pin flocs at high MLSS concentration can cause hindered
settling
 Causes of Increased SVI

Filamentous Bulking
Inter-floc bridging, filaments extending from flocs
Filaments within floc causing diffuse structure
Slime Bulking
-Lack of Nutrients
-Excessive zooglea bacteria
 Filamentous Bulking
Filamentous bulking is the number one cause of environmental violations
A bulking sludge settles slowly, and has an SVI >150.
Many sludge thickening and dewatering problems are actually problems
due to a bulking sludge
Filaments can cause bulking due to inter-floc bridging, or open floc
structure.
Bridging and Open Flocs

Bridging Open
Causes of Filament Growth
Low Dissolved oxygen Concentration
Low F/M
Septicity (sulfides and organic acids)
Grease and Oil
Nutrient Deficiency
Low pH
Finding the big picture
Cause Filaments
Low DO Spaeroltius natans
Type 1701
Haliscomenobacter hydrossis
Low F/M ratio Type 0041
Type 0675
Type 1851
Septicity Type 021N
Thiothrix I and II
Nostocoida limicola I, II, III
Type 0411
Type 0961
Type 0581
Type 0092
Oil and Grease Nocardia spp.
Microthrix parvicella
Type 1863
Nutrient Deficiency Nitrogen: Thiothrix I, II, type 021N
Phosphorus: N limicola III, H. hydrossis, S. natans
Low pH Fungi

Source: Dr. Michael G Richard; used with his authorization.

Ranking to determine big picture
Dr. Michael Richards Ranking System

1 (few)- filaments observed in occasional floc

2 (some)- filaments observed in half the flocs
3 (common)- filaments observed in all the flocs, but at low abundance (1-5
filaments per floc)
4 (very common)- filaments observed in all flocs at medium density (5-20
filaments per floc)
5 (abundant)-filaments observed in all flocs at high density (>20 filaments per
floc)
6 (excessive) filaments dominate with little floc
Filament Abundance

Some Common
Zero- Few

Very Common Abundant Excessive

Linking Filaments to Cause
Usually problems occur or begin once filaments reach
the very common level or greater
Floc structure and strength are very important to consider
A common abundance of filaments can confirm a
condition exists, but is not causing a problem
(actually beneficial to floc formation)
Predominant filaments based on the numerical
ranking system can diagnose the cause of a problem.
Dont link filaments to their causes unless common
abundance or greater of that particular filament
Nutrient Deficiency Foaming and Bulking

In general a BOD: Nitrogen: Phosphorus ratio of 100:5:1

is needed for complete BOD removal
Normal sludge contains around 20% polysaccharide on a
dry weight basis, if nutrients are limiting (or sometimes
other stresses such as low DO or high F/M) the
polysaccharide level will increase.
Elevated polysaccharide levels are known to cause
poor settling known as slime bulking as well as
foaming and complications with sludge dewatering
Nutrient Deficient Reverse India Ink Stain
Normal Polysaccharide
Zooglea Foaming and Bulking
Zooglea growth is not related to nutrient deficiency, but zooglea have
a thick slime layer that may cause settling problems, dewatering
problems, and foaming
Zooglea occur due to high F/M conditions and when specific organic
acids and alcohols are elevated due fermentation of the waste-stream
If the reverse India ink stain is elevated the Anthrone test can
separate zooglea growth problems from low nutrient problems
because the amino-sugars in zooglea dont react to polysaccharide
test whereas there are elevated levels (>20%) in nutrient deficient
sludges.
Fingered and Globular Zooglea

Fingered Zooglea 100x Globular Zooglea 1000x

Zooglea India Ink Stain
Diagnosing Foaming Problems
Description Causes
White Surfactants
Start up
High F/M ratio
Dark Nocardioforms
Microthrix Parvicella
Dead bug foam
Denitrification
Slimy- polysaccharide Zooglea bacteria
Nutrient deficiency

Foams should be diagnosed through microscopic evaluation.

Filamentous foams are diagnosed by an increased abundance of the
filament in the foam than the underlying MLSS
Often, in denitrification foams/ scums, the floc size will be 1.5 -2x the size
of the underlying MLSS due to entrapment of Nitrogen gas
*Never assume cause of foam based on appearance
Due to thick concentration of microorganisms in foam, dilution of the sample
may be needed for microscopic evaluation.
Nocardioforms and Microthrix
Microscopic Evaluation
A research grade, phase
contrast microscope with 10x
and 100x (oil immersion)
phase contrast objectives is
necessary.
An ocular scale into one of the
eyepieces to measure filament
diameter, length, and cell size
is needed
References
Jenkins, D., M.G. Richard and G.T. Daigger, Manual on the Causes and
Control of Activated Sludge Bulking, Foaming, and Other Solids
Separation Problems, 3rd Edition., Lewis Publishers (CRC PRESS), Boca
Raton, FL, 2003

Activated Sludge Microbiology Problems and Their Control, 2010

Michael Richard, Ph.D. Michael Richard Wastewater Microbiology LLC
Fort Collins, CO www.mrwwm.com

Water Environment Federation, Operation of Municipal Wastewater

Treatment Plants Volume II: Liquid Processes, Manual of Practice
No.11 McGraw Hill Companies, New York, NY 2008
About the Author
Ryan Hennessy is a consultant and
Technical Service Support Rep for
Microbial Discovery Group.
Expertise in process control, filament
ID, wastewater microscopy and
troubleshooting
Thank you to Dr. Michael Richard for
his guidance and support through the
years in learning how to use the
microscope

Ryan Hennessy
 Thank You

http://www.mdgbio.com/