9 Anaerobic Bio Tests

Anaerobic Wastewater Treatment
9-Anaerobic laboratory tests: SMA, BMP,

Prof.Dr.ir. Jules B. van Lier
Toxicity
11-15 July 2011
Content of this presentation
• Sludge or substrate characteristic?
• Quick overview of some characteristics
• Biogas measurements (quantity and quality)
• Activity test
• Degradability test
• Toxicity test
Anaerobic lab tests 2

Substrate or sludge characteristic?
• For anaerobic treatment one has to know the characteristics of the

substrate(s) to be treated and of the sludge (similar to BOD..)
• Different tests exist for both types of characteristics
• During a test wastewater and sludge interact
• Keep this in mind when setting up tests
Wastewater or sludge characteristic?

Wastewater Sludge
• Composition • Specific Methanogenic Activity

• Degradability • Composition
• Toxicity • Settleability
• Appearance

Substrate characteristics
(focus on wastewater)
Chemical characterisation
• The more is known about wastewater composition, the better insight
you get in the possibilities for treatment
• Commonly measured parameters for anaerobic treatment

• pH
• Chemical Oxygen Demand (COD)
• Volatile Fatty Acids (VFA)
• Total Suspended Solids (TSS)
• Volatile Suspended Solids (VSS)
• Nitrogen compounds (Total N, NH4, Organic N, N-kjeldahl,…)
• Phosphorous (Total P, PO4, Organic P,…)
• Oil and Grease (O&G)
• Conductivity (E)

Chemical characterisation
What do they tell you??
• pH
• Alkalinity → Buffer capacity
• COD → Organic “strength”

• VFA → Degree of acidification
• TSS → Solids (excluding salts)
• VSS → Organic solids (=part of TSS)
• N, P → Nutrients
• O&G → Usually adverse effects on treatment
•E → Salt content
Anaerobic degradability
• The maximum anaerobic degradability of a substrate depends on

its composition
• But: the degradation rate depends on both the substrate

composition and on the properties of the used sludge
• Anaerobic degradability is most often expressed as:

• Maximum obtainable methane production
(e.g. in litres CH4 / m3 of wastewater)
• Maximum obtainable COD reduction
(e.g. in %COD removed)

Toxicity
• Household sewage is usually not toxic
• Some industrial wastewaters are toxic
• Toxicity is usually assessed by doing

• Degradability tests at different wastewater dilutions
• Sludge activity tests at different wastewater dilutions
• Solutions for treatment of toxic wastewaters (e.g.)

• Make changes in factory to diminish toxicity / isolate toxic streams
• “Dilute” wastewater to a non-toxic level (internal / external dilution)
• Sludge can sometimes adapt to toxic compounds
Sludge characteristics

Specific Methanogenic Activity (SMA)
• Measure for the activity of methanogenic sludge under ideal

conditions
• Maximum methane production rate that can be obtained with a

particular sludge sample:
• Measured with acetate, not with wastewater
• The SMA can vary for sludge from the same source taken at different
times (or of a different storage time)
• When designing anaerobic tests, preferably the SMA of the sludge

is measured first
• If SMA can not be measured a value has to be assumed
Composition
• Basic composition
• Dry matter content (=TSS)
• Organic matter content (=VSS, ≈bacteria content)
• Microbial techniques make it possible to analyse for

specific microorganisms
• In most cases that information is not needed
• Chemical analysis can be done for content of e.g.

calcium, heavy metals, etc.
• In most cases that information is not needed

Settleability
• In order to maintain sludge in a reactor it has to have good

settleability
• Can vary in time, because of (e.g.)

• entrapment of gas bubbles
• changes in sludge composition
• Easily measured
Appearance
• Appearance of sludge gives valuable information
• Sludge types: granular, flocculent or dispersed
Different appearances
within one type…
(just human beings…)

Appearance – Changing
Original sludge …after some time in lab-scale

reactor with high-fat wastewater
Laboratory tests
- Activity -

Anaerobic tests for wastewater need:
• Gas tight, pressure resistant bottles
• Gas tight caps with good septa

• They will be pierced multiple times and should not start to leak
• Sludge
• Biogas volume measurement
• Biogas quality measurement
Outcome of tests is influenced by:
• Anaerobic conditions in bottles

• Flush with N2 or add a little sulphide to capture O2
• pH
• Temperature
• Should be kept constant
• Usually incubators or climate rooms are used
• Nutrients
• Shaking

The maximum amount of methane gas produced per day,

calculated per gram of bacteria present
produced under optimum conditions.
Unit: gCH4-COD/gVSS.d
Transform ml or
pressure into Sludge VSS!
CH4-COD! (~bacteria content)
Basics of the test:
• Performed in duplicate or triplicate

• pH buffer and nutrients added to each bottle
• Acetate used a substrate
• Blanks without substrate are used to correct
for endogenous biogas production
• Tests should take 2 weeks max.
• Sometimes 1 day is enough
• Growth is neglected
• Tests are normally performed at 30°C

Calculation:
G  CF
SMA 
VSS  W  T
SMA = specific methanogenic activity (gCOD-CH4/gVSS.d);

G = methane produced (mL)
CF = conversion factor (gCOD-CH4 STP/mL-CH4 local conditions)
VSS = volatile suspended solids in the sludge (gVSS/g sludge)
W = sludge added to the bottle (g)
T = time interval (d)
STP = Standard Temperature (273K) and Pressure (1 atm)

Data collection example:
Date and time Cum time (d) CH4 production (mL)
Duplicate a Duplicate b
Daily Cummulative Daily Cummulative
24-Feb-02
26-Feb-02 2.19 55.0 55.00 74.5 74.50
27-Feb-02 3.10 26.0 81.00 24.0 98.50
1-Mar-02 4.81 68.5 149.50 74.0 172.50
3-Mar-02 6.98 77.0 226.50 93.5 266.00
4-Mar-02 8.25 76.0 302.50 92.0 358.00
5-Mar-02 9.23 51.0 353.50 61.0 419.00
6-Mar-02 10.04 50.0 403.50 59.0 478.00
8-Mar-02 12.32 105.0 508.50 16.5 494.50
10-Mar-02 13.95 44.0 552.50 123.0 617.50
12-Mar-02 16.31 98.0 650.50 97.0 714.50
14-Mar-02 18.35 72.5 723.00 84.5 799.00

G  CF
SMA 
(gCH4-COD/gVSS)
VSS  W  T
600
production
500
y = 49.3142x - 100.2904
de metano (mL)
2
R = 0.9931
400
ProducciónMethane
300 Environment:
temperature
200
& pressure
100
0
0 2 4 6 8 10 12 14
Tiempo (d) Time (d)
Laboratory tests
- Degradability -

Biochemical Methane Potential
(BMP)
The amount of methane gas produced from a substrate,
calculated per unit of substrate present,
under optimum conditions.
Unit: ml CH4 / ml substrate
Or other unit for Or other unit for

gas production substrate quantity
%COD transformed into CH4

Alternatives: g CH4-COD / litre wastewater
ml CH4 / litre wastewater
ml CH4 / g OM
BMP test
Basics of the test:

• Performed in duplicate or triplicate
• Biogas quantity and quality measured like in SMA test
• Blanks without substrate are used to correct for endogenous biogas
production from the sludge
• Usually takes several weeks (sometimes months), if test will run until
gas production ceases. Or shorter if a fixed time is set.
• Distinguish between:
• Maximum theoretical degradability (ideal conditions, diluted, etc.)
• “Real” degradability

BMP – Anaerobic biodegradability
• BMP result indicates the potential biodegradability of a substrate
under anaerobic conditions
450
• Important processes:
ml CH4 / g substrate-COD
400
• Hydrolysis of particles (for wastewaters with high SS)
350
• Acidogenesis (acidification? check pH/VFA regularly)
300
• Methanogenesis
250
200
150
100
50
0
0 5 10 15 20 25 30
time (d)
Homogeneous substrates
• Most substrates can be tested in bottles

Larger reactors can be considered
• Larger reactors can be used instead of bottles
• inhomogeneous substrates, up-scaling, …
“Real” biodegradability
• In practice conditions are different from BMP test
• For example:
• Substrate concentration
• Temperature
• Absence of certain nutrients (or different concentrations)
• pH
• Tests can done at “real” conditions give good indication

• Do not expect to see the exact laboratory results in the full scale
installation
• Conditions will always be different from laboratory

Toxicity
Toxicity
• Inhibition of the biological processes

(Biodegradation does not take place, or is slower)
• Different degrees of toxicity

• Concentration dependent
• Lethal
• Sometimes adaptation possible

Two groups of toxic compounds
• Compounds normally found in biological systems

• substrates, (intermediate) products, nutrients, trace elements, etc.
 Concentration is an important factor in this case
• Compounds not normally found in biological systems

• chlorophorm, cyanide, etc.
 Mostly related to industrial wastewaters
Toxicity test
• Toxicity can be measured using

• SMA test
• Degradability test
• Wastewater is tested in different concentrations for effect on:

• SMA of sludge
• Obtainable degradability
• Similarly, the effect of other influencing factors can be tested by

performing activity tests and degradability tests under different
conditions
• Think of: temperature, nutrients, pH, etc.

Example of toxicity test
• Granular sludge was exposed to different wastewaters from a

coffee processing plant for a period of 35 days
(Incubated in duplicate, at 30°C, under continuous mixing)
• Before the test the sludge was activated using a VFA mixture
(acetate, propionate and butyrate)
• Every week the wastewater was renewed
• Activity tests were done with the sludge before and after
exposure
(Incubated in duplicate, at 30°C, under continuous mixing)
Toxicity test example – Test set up

• VFA-COD was similar in all 4 wastewaters (ca. 1100 mg/l)
• Control: standard medium with 1100 mg acetate-COD/l
• Buffer and nutrients were added to all bottles

Toxicity test example – Test set up
• Activity tests were done with the wastewaters

• SMA test is usually performed in standard medium
• Presence of wastewater can make interpretation more difficult
• VFA concentration in wastewaters and control was adjusted to be

around 1500 mg/l in all cases
• Sludge concentration 2 g VSS/l
• Activity was measured in gCH4-COD/gVSS.d
Toxicity test example - Results

0.5 0.5
g CH4-COD / g VSS
g CH4-COD / g VSS
Wastewater 1 - Before Wastewater 1 - After

0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0.0 0.0
0.0 0.5 t(d) 1.0 0.0 0.5 t(d) 1.0
0.5 0.5
g CH4-COD / g VSS
g CH4-COD / g VSS
Wastewater 3 - Before Wastewater 3 - After

0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0.0 0.0
0.0 0.5 t (d) 1.0 0.0 0.5 t(d) 1.0

0.5 0.5
g CH4-COD / g VSS
g CH4-COD / g VSS Wastewater 4 - Before Wastewater 4 - After
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0.0 0.0
0.0 0.5 t (d) 1.0 0.0 0.5 t(d) 1.0
0.5 0.5
g CH4-COD / g VSS
g CH4-COD / g VSS
Control - Before Control - After
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0.0 0.0
0.0 0.5 t (d) 1.0 0.0 0.5 t(d) 1.0
Activity
(g CH4-COD/gVSS.d) (% of activity in control)
Before After Before After
Wastewater 1 0.27 0.09 64 16
Wastewater 2 0.36 0.70 86 129
Wastewater 3 0.47 0.24 112 44
Wastewater 4 0.34 0.74 81 137
Control 0.42 0.54 - -

Biogas measurement techniques
Key measurement – Biogas

production
• Biogas is most important measurement in anaerobic testing

• Quantity: litres of methane per g of sludge organic matter
• Quality: methane content of biogas
• Different methods exist
• All biogas quantity measuring methods depend on gas-tight

containers!

Biogas – Quantity measurements
Liquid displacement: Pressure measurement:
Produced gas is led from test bottle Produced gas is maintained in the
into other bottle with liquid test bottle
This liquid is displaced by the gas Pressure in test bottle is measured
The displaced liquid volume is equal Increase in pressure is recalculated

to the volume of produced gas. to volume of produced gas.
(and there are some other methods)
Biogas quantity – Liquid

displacement method
5% NaOH solution
for displacement and CO2 capture
Biogas produced
Screw cap with rubber septum
Nutrients, substrate, buffer, water
Displaced NaOH solution

Anaerobic sludge
displacement method

displacement method
• Leakage possible Points of thought…

• Test bottle septum and cap
• Liquid bottle septum and cap
• Tubes
• Connection of needles and tubes
• Connection of needle and septum
• NaOH should be prevented from entering the test bottle
• It is best to connect and disconnect the system for each measurement

• Always overpressure so no backflow of NaOH
• No evaporation of collected NaOH

Biogas quantity – Pressure method
Handheld meter
Biogas quantity - Pressure method
Pressure sensors directly connected to computer

Oxitop® System

Points of thought…
• Leakage easier to prevent
• Calculations needed to transform pressure in volume
• Separate methane measurement needed
• Take care with increasing pressure:

Bottles might start to leak,
or worse: they might break!
• Equipment more expensive than for liquid displacement

Biogas quantity - Other methods
Gas bags
Gas meter (only for larger volumes)
Biogas quality
• Produced biogas mainly consists of methane and carbon dioxide:
60-80% CH4 and 20-40% CO2
• Usually also present: H2S, H2 (maybe others as well)

• Low concentrations, unless special wastewater
• For all anaerobic tests: what matters most is the methane!
• CH4-content can be measured in different ways

Biogas quality
• Liquid displacement test with NaOH solution: as CO2 is captured

in the solution, no CH4-measurement is needed.
• If the biogas volume is monitored (pressure method or other

methods), then the CH4 content of the gas should be measured
• Usually no other gas measurements are needed

• With specific wastewaters other gases might be monitored, such as
H2S
Biogas quality – Liquid displacement
• CO2 can be removed from the gas using the liquid displacement
method:
• A large biogas sample (e.g. in a 100 ml syringe) is led slowly through

a liquid displacement system
• The measured volume of CH4 is then divided by the volume of
injected biogas
• This gives the fraction of CH4 in the biogas
• Other gases are not taken into account

Biogas quality – Gas
chromatography
• Biogas composition can be measured on a GC

• In some cases this can be used for quantification as well. Bottles
should be pressure resistant, %CH4 will increase in time
• Depending on exact method used it is possible to measure

different (combinations of) gases:
• % of methane in biogas
• % of methane and % of carbon dioxide in biogas
• % CH4, CO2, O2 and N2 in biogas
• % H2S in biogas
• etc.
Take home message

Summary
• Analysis of sludge and wastewater
Tests:
• Sludge activity Tools for toxicity test
• Wastewater degradability
• Different ways of measuring biogas quantity and quality
Before setting up any tests:
what is it you want to know?

9 Anaerobic Bio Tests

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Anaerobic Wastewater Treatment

9-Anaerobic laboratory tests: SMA, BMP,

Content of this presentation

• Sludge or substrate characteristic?

• Quick overview of some characteristics

• Biogas measurements (quantity and quality)

Anaerobic lab tests 2

• For anaerobic treatment one has to know the characteristics of the

• Different tests exist for both types of characteristics

• During a test wastewater and sludge interact

• Keep this in mind when setting up tests

Anaerobic lab tests 3

Wastewater or sludge characteristic?

• Composition • Specific Methanogenic Activity

Anaerobic lab tests 4

Anaerobic lab tests 5

• Commonly measured parameters for anaerobic treatment

Anaerobic lab tests 6

• Alkalinity → Buffer capacity

• COD → Organic “strength”

Anaerobic lab tests 7

• The maximum anaerobic degradability of a substrate depends on

• But: the degradation rate depends on both the substrate

• Anaerobic degradability is most often expressed as:

Anaerobic lab tests 8

• Household sewage is usually not toxic

• Some industrial wastewaters are toxic

• Toxicity is usually assessed by doing

• Solutions for treatment of toxic wastewaters (e.g.)

Anaerobic lab tests 9

Anaerobic lab tests 10

• Measure for the activity of methanogenic sludge under ideal

• Maximum methane production rate that can be obtained with a

• When designing anaerobic tests, preferably the SMA of the sludge

Anaerobic lab tests 11

• Microbial techniques make it possible to analyse for

• Chemical analysis can be done for content of e.g.

Anaerobic lab tests 12

• In order to maintain sludge in a reactor it has to have good

• Can vary in time, because of (e.g.)

Anaerobic lab tests 13

• Appearance of sludge gives valuable information

• Sludge types: granular, flocculent or dispersed

Anaerobic lab tests 14

Original sludge …after some time in lab-scale

Anaerobic lab tests 15

Anaerobic lab tests 16

• Gas tight, pressure resistant bottles

• Gas tight caps with good septa

• Biogas volume measurement

• Biogas quality measurement

Anaerobic lab tests 17

Outcome of tests is influenced by:

• Anaerobic conditions in bottles

Anaerobic lab tests 18

The maximum amount of methane gas produced per day,

Anaerobic lab tests 19

Specific Methanogenic Activity (SMA)

Basics of the test:

• Performed in duplicate or triplicate

Anaerobic lab tests 20

SMA = specific methanogenic activity (gCOD-CH4/gVSS.d);

Anaerobic lab tests 21

Specific Methanogenic Activity (SMA)