A Laboratory Program for Wastewater Microbiology

TONI GLYMPH-MARTIN

SENIOR ENVIRONMENTAL

MICROBIOLOGIST

Wastewater

Microbiology &

Process Control

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.

WHY MICROBIOLOGY?

• THE WASTEWATER TREATMENT PROCESS IS A

BIOLOGICAL ZOO HOUSING BACTERIA,

PROTOZOA, METAZOA AND OTHER

MICROORGANISMS.

• MICROORGANISMS USE THE ORGANIC MATERIAL

AS FOOD SOURCES FOR ENERGY, TO CREATE NEW

CELL COMPONENTS, AND TO MAINTAIN LIFE

PROCESSES.

WHY MICROBIOLOGY?

• THE MICROORGANISMS ARE THE VIPS OF THE

TREATMENT PROCESS

• THE HEALTH AND WELL-BRING OF THESE

MICROORGANISMS ARE CRITICAL TO THE ADEQUATE

TREATMENT OF SEWAGE.

• THERE ARE DIFFERENT TYPES OF TREATMENT

PROCESSES, BUT THEY ALL RELY ON

MICROORGANISMS TO BREAK DOWN AND REMOVE

ORGANIC WASTES.

Bar screens

trap large

debris

Water is slowed down to

allow grit to settle out

Primary tank floats oil &

grease and removes

settleable solids

Solids

Handling

Microorganisms mixed with wastewater

(mixed liquor) in the presence of oxygen,

consume biodegradable materials

Wastewater from homes, industry, and sometimes

storm water enter the WRP via interceptors

Primary solids are

pumped to solids

handling/processing.

Mixed liquor is

pumped to

secondary clarifiers

where biological

solids, containing

microorganisms is

separated from the

liquid.

Most of the

microorganisms

are returned to

aeration to feed

on the

continuous

inflow of

wastewater.

Some secondary solids are wasted to

solids handling/processing.

Primary

Tank

Grit

Removal

Aeration Basin

Secondary Clarifier

Final treated

water is

discharged

to the

waterbody.

Wastewater

Aeration Basin

Microbes

WASTEWATER MICROBIOLOGY

• THE WASTEWATER TREATMENT

PROCESS IS A BIOLOGICAL

PROCESS

• IN ORDER TO PROPERLY

EVALUATE THIS PROCESS WE

SHOULD INCORPORATE

BIOLOGICAL TOOLS.

• ONE OF THOSE BIOLOGICAL

TOOLS IS THE MICROSCOPE.

THE PROBLEM

• MANY FACILITIES DO NOT

HAVE DEDICATED STAFF.

• THE MAIN FOCUS IS ON

NPDES PERMIT RELATED

COMPLIANCE

MONITORING.

TYPICAL PROCESS MONITORING

• BOD

• TSS

• MLSS

• MLVSS

TYPICAL PROCESS MONITORING

• 30-MINUTE SETTLING

TEST

• SLUDGE VOLUME INDEX

(SVI)

• TYPICALLY USED TO

MONITOR THE SETTLING

CHARACTERISTICS

Wastewater

Aeration Basin Sample

Microbes

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


• FLOC COLOR INDEX

• ZOOGLEAL MASS INDEX

• TOTAL SHELLED PROTOZOA-

METAZOA

• TOTAL FILAMENT COUNT


• WET MOUNT

• FLOC COLOR INDEX

• TOTAL SHELLED PROTOZOA

• ZOOGLEAL MASS INDEX

• STAINED SLIDE

• FILAMENT COUNT

WET MOUNT & SCAN

2 cm

Wax marks

WET MOUNT

PUKE

FLOC COLOR INDEX

• UNDER NORMAL

CONDITIONS FLOC

SHOULD APPEAR BROWN

WHEN OBSERVED USING

PHASE CONTRAST.

FLOC COLOR INDEX

-5 0 +5

FLOC COLOR INDEX

-5

• Low food/nutrients

• Under low food/low

nutrient conditions

bacteria secrete excess

amounts of exocellular

lipopolysaccharide

• The more starved they

are, the more they

secrete

FLOC COLOR INDEX

+5

• Old Floc (sludge)

• Septic

• Older floc is darker in

color.

• When oxygen is lacking

the floc becomes septic

and turns dark brown to

black in color.

FOOD : MICROORGANISM RATIO (F:M)

M (Determined by the MLVSS or MLSS)

F (Determined by the BOD or COD test)

• The color of the floc is directly related to

the Food:Microorganism ratio.

FLOC COLOR INDEX

• FLOC COLOR CAN ALSO BE AN INDICATOR OF

STRESS TO THE FLOC-FORMING

MICROORGANISMS.

• STRESS CAN BE CAUSED BY LOW LEVELS OF

TOXICITY, CHANGES IN PH ETC.

JOHN E. EGAN WRP (50 MGD)

• BEGINNING AUGUST 1, 2011 THROUGH DECEMBER 31, 2012

• 137 SAMPLES COLLECTED

• SOUTH BATTERY: 55 SAMPLES

0

0.05

0.1

0.15

0.2

0.25

0.3

-4.5

-4

-3.5

-3

-2.5

-2

-1.5

-1

-0.5

0

Comparison of Floc Color with F:M Ratio - Egan South Battery August 2011-September 2012

Floc color

F/M Ratio

2 per. Mov. Avg. (Floc color)

2 per. Mov. Avg. (F/M Ratio)

O’BRIEN WATER RECLAMATION PLANT


0.00

0.05

0.10

0.15

0.20

0.25

0.30

Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

F/M

Ratio

Monthly Average F/M RatiosAugust 2011 through December 2012

Battery A Battery B Battery C Battery D

0.00

0.05

0.10

0.15

0.20

0.25

-3.00

-2.50

-2.00

-1.50

-1.00

-0.50

0.00


Slu

dge C

olo

r In

dex

Comparison of Sludge Color Index and Average F/M Ratios

SCI F/M Ratio

CALUMET WATER RECLAMATION PLANT

• FIVE BATTERIES

• 3 DISTINCT

CONFIGURATIONS

• BATTERY A AND B

• BATTERY C

• BATTERY E-1 AND

E-2

0

20

40

60

80

100

120

Battery A Battery B Battery C Battery E1 Battery E2

Sludge Volume Index

-3

-3

-2

-2

-1

-1

0


Floc Color

MICROBIOLOGY OF ACTIVATED SLUDGE

• BACTERIA (95%)

• PROTOZOA (4%)

• METAZOA (1%)

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.

NAKED AMOEBA

SHELLED AMOEBAE

SHELLED AMOEBA

TUBE-DWELLERS

SHELLED ROTIFER


• COUNT ALL THE SHELLED PROTOZOA AND

METAZOA


• THE GROWTH OR DOMINANCE OF SHELLED

SPECIES IS USUALLY THE RESULT OF CHANGES TO

THE AERATION BASIN INFLUENT CHARACTERISTICS

(I.E. CHANGES IN PH, LOW LEVELS OF TOXICITY)

THAT CREATES AN UNFAVORABLE ENVIRONMENT

FOR THE MICROORGANISMS.


• THIS PARAMETER IS BEST USED AS AN INDICATOR

OF LOW LEVELS OF TOXICITY.

• COMMON IN LARGE, URBAN CITIES

• COUNTS TRENDING WELL ABOVE AVERAGE CAN

INDICATE OF AN INCREASE IN TOXIC

SUBSTANCES OR A MORE HARSH

ENVIRONMENT.

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

ZOOGLEA

• SIMILAR TO INCREASES IN THE SHELLED SPECIES POPULATION,

INCREASES IN THE NUMBER OF ZOOGLEA MASSES IS

USUALLY THE RESULT OF CHANGES TO THE INFLUENT

CHARACTERISTICS THAT CREATES AN UNFAVORABLE

ENVIRONMENT FOR THE BACTERIA. THIS TOO IS USUALLY

BEYOND THE OPERATOR’S CONTROL.

• AN INCREASE IN THE NUMBER OF ZOOGLEA MASSES

COUPLED WITH AN INCREASE IN SHELLED SPECIES IS A

STRONG INDICATION OF TOXIC OR HARSH CONDITIONS.

O’BRIEN WATER RECLAMATION PLANT


O’BRIEN WRP – SHELLED SPECIES

Average Shelled

Protozoa/Metazoa

Count (mgVSS)

Percent Shelled

Species (%)

Battery A 439 48

Battery B 434 50

Battery C 441 50

Battery D 295 37

O’BRIEN WRP - ZOOGLEA

Average ZMI

(#/mgVSS)

Range

(#/mgVSS)

Battery A 162 20-609

Battery B 256 13-857

Battery C 211 30-817

Battery D 82 9-332

-10

10

30

50

70

90

110

130

0

50

100

150

200

250

300

350

400

450

500


Zoogle

a M

ass

Index

Comparison of Average Zooglea Mass Index (ZMI) with Sludge Volume Index (SVI)

0

20

40

60

80

100

120

140

160

180

200


Calumet - Zoogleal Mass

0

50

100

150

200

250

300


Total Shelled Species

0

1000

2000

3000

4000

5000

6000

7000

8000


Calumet - Filament Count

WHY MICROBIOLOGY?

• LOOKING AT ONLY

ONE PARAMETER DOES

NOT GIVE YOU THE

WHOLE PICTURE.


• 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.


• 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.

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 COUNT

• COLLECT A REPRESENTATIVE SAMPLE FROM THE DISCHARGE END

OF THE AERATION BASIN

• SPLIT A PORTION OF EACH SAMPLE FOR MLVSS ANALYSIS.

• EACH SLIDE USED FOR STAINING IS MARKED WITH 2 LINES

SPACED 2 CM APART USING A WAX PENCIL. THE WAX

MARKINGS SERVED AS A BORDER TO CONTAIN AND

CONCENTRATE THE SAMPLE WITHIN A FIXED AREA.

2 cm

Wax marks


• USING A PIPETTE, PLACE 200ΜL (4 DROPS)OF WELL

MIXED SAMPLE ONTO EACH MARKED SLIDE AND

SPREAD EVENLY WITHIN THE MARKED AREA.

• SET ASIDE TO DRY FOR 2 HOURS.

2 cm

Wax marks


• GRAM STAIN:

• CRYSTAL VIOLET – 1 MINUTE

• GRAM’S IODINE – 1 MINUTE

• DECOLORIZER – 25 SEC (OR UNTIL YOU NO

LONGER SEE STAIN RUNNING OFF)

• SAFRANIN – 1 MINUTE


• MAKE DUPLICATE SLIDES FOR EACH SAMPLE OF MIXED

LIQUOR AND SCAN AT 100X MAGNIFICATION USING OIL

IMMERSION SCANNING 3-5 PASSES PER SLIDE.


Count the number of intersects =8


NUMBER OF INTERSECTS X 5 = FILAMENTS/ML

FILAMENTS/ML X 1000 = FILAMENTS/MGVSS

MLVSS

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 Laboratory Program for Wastewater Microbiology

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