25 Lecture Wastewater

7/27/2019 25 Lecture Wastewater

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Microbiology of WastewaterTreatment

Lecture 25

Other Sources:

http://www.splammo.net/JLbactsite.html

Black, Microbiology Principles and Exploration 7th Ed

Madigan, Brock Biology of Microorganisms, 11th Ed.

Lecture Topics

Why treat wastewater

The wastewatertreatment plant

Important microbialprocesses

Microbial monitoring

What is wastewater? Industrial sources:

Petrochemical, dairy, food, pharmaceutical,metallurgical, etc.

Domestic sources:

Form households and non-industrial businesses

Domestic sewage:

Sinks, toilets, and showers

US domestic sewage varies little from community tocommunity across the country

Our waste is not unique. We even flush our toilets atthe same time.

Whats in domesticwastewater?

Also called sewage: it looks likespent dishwater

The chemists only care aboutthe organic content,specifically carbohydrates,fats, and proteins.

Sewage is 99.9% water and0.02-0.04% solids

Example: Washington DC, 200tons of solids per day isproduced

40-50% is proteins, 40-50%

carbohydrates, 5-10% fats

What would happen if this wasreleased into the surroundingenvironment?

http://nsm1.nsm.iup.edu/tsimmons


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Environmentalconsequences of not

treating sewage:

People can get sick frompathogen contaminatedwater. Big problem indeveloping nations.

Releasing wastewaterdirectly into a streamleads to oxygen depletion. This is caused by aerobic

respiration linked to highorganic carbon loads inthe waste.

Low oxygen in water cancause fish kills.

The degree of oxygenconsumption in wastewatercan be quantified. This is called the

Biological Oxygen Demand(BOD) http://ian.umces.edu

How tomeasure BOD

Five day bioassay for oxygenconsumption, BOD5

300 ml bioassay in special bottles.

Uses an oxygen meter to measuredissolved oxygen (DO)consumption in a five day period

The BOD5 is calculated by:

BOD5 (mg/L) = D1-D2/P D1= initial DO (mg/L) of the

sample

D2= sample DO (mg/L) after 5days

P= decimal volumetric fraction ofsample used.

Domestic sewage: 150-200 mg/L

Milk processing/cannery waste: 5000-6000

Pulping operations: 10,000-15,000 mg/L

Why the different numbers?

The need for wastewater treatment plants

Goal of wastewater treatment: Protect health

Preserve natural resources Prevent ecological damage

How to accomplishes thesegoals: Use wastewater treatment plants

(WWTP)

The WWTP removes energy-richorganic matter before dischargeinto the environment.

And uses technology toprevent/lower the occurrence ofwater borne diseases.


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Aerobic secondarywaste treatment:

trickle filter

Trickling filter is a bed ofcrushed rocksthe 1-treated sewage is trickledover it. Lots of surfaces formicroorganisms to attach to.

Complete mineralization ofwaste to CO2, ammonia,nitrate, sulfate, and phosphate

Same process occurring in afish aquarium.

www.unclestu.com

Aerobic secondary wastetreatments: activated sludge

Air bubbled through waste

water. Bacteria form large flocs.

Zoogloea ramigerais oneof the key species thatforms a slime and is thebase of the floc.

After the flocs form theyare allowed to settle out

Filamentous bacteria cancause sludge bulkingproblems--sludge thickens

Aerobic Activated sludge

Much of the organic material binds to the floc and is

eventually taken up by the microbial biofilm.

95% of the BOD is reduced during this stage.

Important microbes in thesewage treatment plant

Nitrifying bacteria

Aerobes Convert nitrogenous

waste into nitrate

Denitrifying bacteria Anaerobes

Convert nitrate to N2 Methanogens

Generate methane fromacetate

Or use H2 and CO2 tomake methane

Mostly archaea


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Nitrifying bacteria Ammonia is converted into nitrate

Ammonia has a high BOD because NH3 oxidationrequires oxygen.

Two groups of microbes are involved: Ammonia oxidizing bacteria (AOB)

Nitrite oxidizing bacteria (NOB)

AOB oxidize NH3 to NO2- in two steps: Ammonia monooxygenase (AMO)

Hydroxylamine oxidoreductase (HAO)

NOB oxidize NO2- to NO3-

Uses the Nor enzyme complex

Both AOB and NOB respire oxygen

Nitrification Enzymes

AMO= converts ammonia to hydroxylamine (toxic)HOA = converts toxic hydroxylamine to nitrite

NOR = nitrite oxidoreductase

NO3-

NH3 NH2OH NO2-

NO2-

1/2O2 H2O H2O

1/2O2 H2O

PMF

AMO HOA NOR

G = -275 kJ/mol G = -76 kJ/mol

e- e-

H+ H+

Ammonia Oxidizing Bacteria (AOB):(A,B) Nitrsomonas; (C,D) Nitrosolobus

Nitrite Oxidizing Bacteria (NOB):(E, F) Nitrospira; (G, H) Ntirococcus


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Sewage treatment andenvironmental monitoring

Monitoring effluents and the surrounding

environment is important (Why?): Asses the efficacy of the treatment process

C, N, P, metals, microbes, effluent toxicity

It is often too difficult to directlymonitor a specific pathogen or virus/phage(Why?).

Instead, monitoring is usually done forindicator organisms.

What is an indicator organism? An organism that can be readily cultured that

indicates the presence of a pathogenic

microorganism or correlates to a health problem. Five criteria for an indicator organism:

Consistently present in feces and at higher concentrations thanpathogens.

Should not multiply outside the human intestinal tract.

Should be as resistant or more resistant than the pathogen toenvironmental conditions and to disinfection.

Easy to assay (culture and quantify) and differentiate from otherorganisms.

Environmental concentrations should correlate with pathogens ormeasurable health hazards.

Common indicator bacteria Coliforms:

Facultatively aerobic, gram-negative, nonspore-

forming, rod-shaped bacteria; ferment lactosewith gas formation at 35C within 48 hrs.

Usually enteric bacterial group (E. coli,Klebsiella, Citrobacter, Enterobacter,Serratia, Yersinia)

Poor indicator: often found outside of theintestinal tract

Fecal coliforms

Thermotolerant coliforms (44.5C), 20% oftotal coliforms.

Monitoring ofindicators isdone usingculturing

techniques andselectivemedia.

Gram positives


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Most probable number (MPN) analysisfor quantifying coliforms

Quantifies total coliform bacteria in watersamples by three sub-tests

Presumptive, Confirmed, and Completed The MPN is used to monitor waste effluents,

drinking water systems, and recreational waters.

High MPN results can lead to beach closures.

In a drinking water system, a positive forcoliform is a HUGE deal. This would set off aseries of events to find the source of coliformcontamination. Would you want to be drinking water with coliforms in it?

Growth:Yes No

Fermentation:Yes Yes NoGas

MPN: Presumptive Test Determines presence of coliforms (gas producers).

Serial dilution of replicate lactose broth tubes

MPN index reportsdata as per 100 ml


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MPN: Confirmed Test Part two of the MPN test

Positive tubes in the presumptive test areinoculated into brilliant green lactose bile broth(BGLB) These tubes will confirm acid and gas production

(fermentation end products).

Bile will inhibit gram positive organisms

MPN: Completed Test

Positive tubes from theconfirmed test are analyzedby streak plating on eosin

methylene blue (EMB) plates. Incubated at 35C for 24-

48 hr

Only coliforms will turn darkwith a metallic green sheen

Gram + inhibited by eosin/MB

Coliform colonies are gramstained and to verify thatthey are gram negative andnon-spore forming (seedefinition of coliforms).

http://www.microbelibrary.org/

Low pH: dye

turns metallic

green

Example MPN problem:Glucose fermenters in lake water:

Dilution of lake water: 1st dilution(101)

2nd dilution(103)

3rd dilution(105)

Amount inoculated into each of three

tubes of Glucose Fermentation Broth1 ml 0.1 ml 1 ml 0.1 ml 1 ml 0.1 ml

Set of tubes (designations used

below)

A B C D E F

# of tubes showing growth 3 3 3 3 2 0

# of tubes showing acid production 3 3 3 1 0 0

Choose numbers with three consecutive sets of dilution to

extinction for sugar fermenting organisms (tubes: C, D, E)

The MPN table for 3-1-0 is 0.43 organisms (next slide).

0.43 organisms were inoculated into the middle tube (D).

Use the dilution to calculate the MPN per ml

Tube D had 0.1 ml of a 10-3 dilution of lake water.

Now calculate the MPN in the original sample:

0.43 organisms X 103 0.1 ml = 4.3 x 103 glucose fermenters per ml

The MPN index would be 4.3 x 103 per ml 100 = 43 organisms per 100 ml

First set

of tubes

Middle set

of tubes

Last set of

tubes

MPN in

inoculum of the

middle set

0 0 0 0.03

0 0 1 0.03

0 0 2 0.06

0 0 3 0.09

0 1 0 0.03

0 1 1 0.061

0 1 2 0.092

0 1 3 0.12

0 2 0 0.062

0 2 1 0.093

0 2 2 0.12

0 2 3 0.16

0 3 0 0.094

0 3 1 0.13

0 3 2 0.16

0 3 3 0.19

1 0 0 0.036

1 0 1 0.072

1 0 2 0.11

1 0 3 0.15

1 1 0 0.073

1 1 1 0.11

1 1 2 0.15

1 1 3 0.19

1 2 0 0.11

1 2 1 0.15

1 2 2 0.20

1 2 3 0.24

1 3 0 0.16

1 3 1 0.20

1 3 2 0.24

1 3 3 0.29

2 0 0 0.091

2 0 1 0.14

2 0 2 0.20

2 0 3 0.26

2 1 0 0.15

2 1 1 0.20

2 1 2 0.27

2 1 3 0.34

2 2 0 0.21

2 2 1 0.28

2 2 2 0.35

2 2 3 0.42

2 3 0 0.29

2 3 1 0.36

2 3 2 0.44

2 3 3 0.53

3 0 0 0.23

3 0 1 0.39

3 0 2 0.64

3 0 3 0.95

3 1 0 0.43

3 1 1 0.75

3 1 2 1.2

3 1 3 1.6

3 2 0 0.93

3 2 1 1.5

3 2 2 2.1

3 2 3 2.9

3 3 0 2.4

3 3 1 4.6

3 3 2 11

3 3 3 24

Three tube MPN table


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Molecular techniques formonitoring indicators

Molecular methods can be very sensitive.

In theory, one gene copy can be detected via PCR. Molecular methods can be rapid:

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