Biochemical Oxygen Demand

Term Project for Biochemical Engineering Completed by Linda S. Magenis

Elementary Background Material

Biochemical Oxygen Demand is a common, environmental procedure for

determining the extent to which oxygen within a sample can support
microbial life. The following tutorial explores the theory and basics of
performing this test when one has little or no prior experience. This
method is popular in many environmental laboratories analyzing
waste water, compost, sludge, and soil samples. Although methods
for each matrix are similar, this tutorial focuses on the method
associated with only waste water effluents.

The main details of this method are taken specifically from Standard
Methods for the Examination of Water and Wastewater (Method 507:
1985, and Method 218B: 1971) and the United States Environmental
Protection Agency of 1979 (Method 405.1). Slight variations and
additional insight are added from my experience as an analyst, and
modifications will be noted. Other methods may exist amongst
laboratories performing this test, so it must be stressed that this
method, although approved, is not all inclusive. In addition, this
procedure is only suitable for samples void of serious matrix
interferences. To gain a broader appreciation of oxygen demand,
additional avenues of interest may be explored including CBOD
(carbonaceous oxygen demand), COD (chemical oxygen demand),
and TOC (total organic carbon).

The test for Biochemical Oxygen Demand is especially important in waste

water treatment, food manufacturing, and filtration facilities where
the concentration of oxygen is crucial to the overall process and end
products. High concentrations of dissolved oxygen (DO) predict that
oxygen uptake by microorganisms is low along with the required
break down of nutrient sources in the medium (sample). On the
other hand, low DO readings signify high oxygen demand from
microorganisms, and can lead to possible sources of contamination
depending on the process.
Performing the test for Biochemical Oxygen Demand requires a significant
time commitment for preparation and analysis. The entire process
requires five days, and it is not until the last day where data is
collected and evaluated. During this time, samples are initially
seeded with microorganisms and supplied with a carbon nutrient
source of glucose-glutamic acid. The sample is then introduced to
an environment suitable for bacterial growth at reproducible
temperatures, nutrient sources, and light within a 20 degree Celsius
incubator such that oxygen will be consumed. Quality controls,
standards and dilutions are also run to test for accuracy and
precision. Determination of the dissolved oxygen within the sample
can be determined through Winkler titration methods. The difference
in initial DO readings (prior to incubation) and final DO readings
(after 5 days of incubation) predicts the BOD of the sample. A
suitable detection limit as per environmental QC is 1 mg/L.

Click on to the following links for this procedure:

 Equipment

 Reagents

 Sample Preparation

 Procedure

 Analysis

 Calculations

Equipment

The following is a list of necessary materials and equipment for starting the
procedure:

Series of 250-300 ml BOD bottles with ground glass stoppers, and caps

Incubator set at 20 degrees Celsius

Two large carboys (20 liter capacity depending on sample amount)

Series of class A pipets (0.2 ml-10 ml)

Aeration device

pH meter

Six 500 ml ehrlenmeyer flasks

Millipore water

Phosphate buffer pillows for every 6 liter carboy volume

DO meter with membrane electrode

Seed

50 ml class A buret

Stir plates and stir bars

Series of volumetric flasks

Series of beakers

Deionized water

Reagents

The following is a list of reagents used in this method that are

commercially prepared:

Concentrated Sulfuric Acid

0.0375 N Sodium Thiosulfate

Starch indicator

Crystalline D-glucose and glutamic acid

Bleach (5.25%)

Acetic Acid

Potassium Iodide

Sodium hydroxide pellets

Manganese sulfate
Sodium sulfite

The remaining reagents are prepared within the laboratory. Caution must be
taken since the shelf life of these reagents should not exceed 6 months
unless otherwise noted.

1. 0.1 N Sulfuric Acid: Add 2.8 mls concentrated acid to 1 liter distilled
water.

2. 0.1 N Sodium Hydroxide: Add 4 grams sodium hydroxide pellets to 1

liter distilled water.

3. Sodium Sulfite solution: Prepare daily. Dissolve 1.575 g sodium sulfite

up to one liter deionized water.

4. Manganese Sulfate solution: Dissolve 364 grams manganese sulfate up

to one liter deionized water. Slight heating and filtration may be
necessary.

5. Glucose-Glutamic solution: Dissolve 75 mg glucose and 75 mg glutamic

acid up to 500 ml deionized water. Sufficient stirring is required.

6. Alkali Azide solution: In a 1 liter volumetric, dissolve 500 gram sodium

hrdroxide pellets with 150 grams potassium iodide. When
dissoliution is complete, add an additional 40 mls distilled water with
10 grams sodium azide. Caution must be taken when handling this
solution.

7. 1+1 Acetic Acid: 500 ml pure grade acetic acid is added to 500 ml
deionized water with stirring. Caution must be taken since the
temperature of the solution will increase rapidly.

8. Potassium Iodide solution: Dissolve 10 grams potassium iodide in 100

ml volumetric flask with distilled water.

Sample Preparation

Handling of the sample is critical to this procedure. The sample must be

incubated within 48 hours of its original sampling time. Analysis after
this point will have significant effects on the oxygen concentration
within the sample and may often lead to less than accurate results.
Usually, the DO of the sample will tend to decrease.
When the sample is first brought in for analysis, it must be maintained at a
temperature of approximatley 4 degrees Celsius. This is to ensure
the fact that the oxygen concentration will remain constant and will
also inhibit the further growth of organisms. Once in the lab, the pH
of the sample must be adjusted for analysis. The desired pH for this
procedure is between 6.5 and 7.5 where bacterial growth is possible.
A 100 ml sample is usually adjusted with 0.1 N sulfuric acid or 0.1 N
sodium hydroxide depending on the original pH of the sample. Once
obtained, the sample content is checked to deterimine chlorine
content. Chlorine must be removed from the sample because it
introduces an interference with the dissolved oxygen.

Chlorine Content

The chlorine content on a 100 ml neutralized sample can be determined by

adding 10 ml potassium iodide solution and 10 ml 1+1 acetic acid as
mentioned in the reagents section. When fully mixed, the addition of
a starch indicator will denote the presence of chlorine if the sample
turns a greyish-black. At this point, dropwise addition of freshly
prepared sodium sulfite will diminsh the chlorine within the 100 ml
sample. Once the chlorine has been dissipated, a new 100 ml
sample must be neutralized and the same number of sodium sulfite
drops must be added. At this point, the sample is ready for analysis.
If no chlorine is detected in the sample after the 20 ml reagent
addition, the addition of sodium sulfite is not necessary, and the
sample must be neutralized again from a fresh sample.

Procedure

Once reagents have been properly prepared, the sample is ready for
analysis. The BOD procedure including the DO analysis is actually
quite lengthy, so time maintenance is important. This portion can be
carried out in four separate steps including carboy setup, adjustment
of DO, preparation of seed inoculum, and BOD sample preparation.
It is best to set up the carboy as soon as possible to ensure proper
results. In addition, the BOD sample preparation is the last step of
the procedure.

Carboy Set Up
Seed Preparation

DO Preparation

BOD Sample Preparation

Carboy Set Up

The BOD procedure calls for two carboys to be used each time the
procedure is carried out. The carboys will contain the water
necessary for the procedure, and two are used instead of one to
serve as a source of comparison amongst carboys. Also, it is a good
idea to have two sources of water in case something goes terribly
wrong with one of the carboys, there will be some sort of back up.

To ensure that the two carboys involved in the procedure are free of
contaminants, the carboys must first be rinsed with acid and water.
Sufficient water should be rinsed to eliminate all traces of acid. Once
the acid is flushed from the carboys, a small amount of bleach is
added to eliminate excess organisms that may serve as sources of
interference. The carboys are again flushed with adequate deionized
water until the presence of bleach is eliminated.

The carboys are then filled with pure water that has been finely filtered.
Millipore water seems to work best in this procedure. The carboys
should be filled up to supply atleast six liters for the first sample, and
three liters for each successive sample. The six liters for the first
sample are necessary because quality controls will also be included
as part of the run. Once filled, one phosphate buffer pillow is added
to the carboy per six liters water. These pillows are commercially
available, and save the analyst time from preparing the reagents.
After addition, the water solutions must be aerated until the point of
saturation. Commonly, many laboratories will prepare this water the
day before the procedure, but five hours prior to analysis appears to
be sufficient. After aeration, the water is ready to be used to fill the
BOD bottles, and must be capped until then.

Seed Preparation
The seed solution must be set up some time before the BOD bottle are
filled with water. This is done by adding 0.045 grams of a polyseed
inoculum (or sometimes a BOD sample can be used as the
inoculum) to 250 mls distilled water. The seed will not dissolve but it
is important that the seed is stirred continously at moderate speed
for about two hours. At approxiamtley 30 minutes prior to use, the
seed solution is allowed to settle undisturbed. Caution must be taken
not to disturb the seed particles because the liquid portion of the
solution is used in the procedure. Remnants of actual seed within
the prepared BOD bottles could greatly effect the results.

DO Preparation

The DO (dissolved oxygen) of the prepared carboy water must be

determined to serve as a reference to all other sample and standard
readings. This is most often accomplished by performing a Winkler
titration on the carboy water, and adjusting the DO meter to this
reading. This method is not contained in the BOD procedure, but
rather comes from Standard Methods for the Examination of Water
and Wastewater (Method 218B: Azide Modification: 1971) In my
experience, we often used a pH meter with an electrode that could
also determine the DO of the solution as our DO meter.

The Winkler titration is carried out by first withdrawing three samples of

water from each carboy. Caution must be taken that no air bubbles
become trapped in the bottle. As a result, it is best to withdraw the
water slowly while the bottles are tilted slightly. Once filled, one
bottle from each carboy is set aside until later. The remaining four
bottles are used in the actual titration. 2 mls manganese sulfate is
added into each bottle under the surface of the water, followed by 2
mls alkali azide solution. The bottles are stoppered and shaken until
a brown floc appears. The bottles are allowed to settle until the floc
is halfway settled. The bottles are then shaken again and allowed to
settle. Once settled, 2 mls concentrated sulfuric acid is added down
the neck of each bottle and the bottles are shaken again. At this
point, the floc will disappear and the solutions should be amber in
color. Now, the solutions are ready for titration.
The solutions are first transfered to 500 ml ehrlenmeyer flasks. Each
solution is titrated with 0.0375 N sodium thiosulfate until the solution
turns a pale yellow. (Standard Methods suggests using 0.025 N
sodium thiosulfate for a different volume of sample.) At this point, a
few drops of starch indicator are added to turn the solution a dark
blue. Titration continues until the solution turns clear. The volume of
titrant used in mls directly corresponds to the DO of the sample. The
average DO reading from each carboy is calculated and recorded. If
the DO readings from each carboy set are not relatively close to
each other (within 0.1 mls) the process must be repeated until
consistent DO readings are obtained. The DO readings should
normally fall between 6.0 and 9.0 mg/L. If values are greater than
9.0 mg/L, the carboy water must be aerated again to reduce the DO.

Once the DO readings are calculated, the two bottles that were withdrawn
at the beginning are now used. The carboy bottle from which the
samples will be made up from is used to adjust the DO meter (in our
case, the pH meter). Once adjusted, the DO of the other bottle is
measured to determine whether the meter is reading correctly. If the
meter reading comes within 0.1 of the Winkler reading, the
calibration is complete. If not, the entire Winkler procedure must be
completed for both carboys.

BOD Sample Preparation

Finally, the last step in the BOD procedure involves inoculating the sample
with various dilutions along with standards and blanks for quality
control. The following quality control must always accompany each
BOD run:

- 2 carboy water blanks

- 4 standards

- 2 seeded blanks

- optional control sample

Usually, many laboratories will include one set of QC for every ten
samples. Additional QC is necessary for more than 10 samples.

Preparation:

1. Water blanks - carboy water is withdrawn to the rim of the bottles.

2. Standards - 4, 6, 6, and 8 mls of standard solution are added to
separate bottles. An additional 2 mls of seed solution is added to
each bottle and the bottles are then filled to the rim with carboy
water.

3. Seeded blanks - 2mls of seed solution are added to each of 2 BOD

bottles. The bottles are filled to the rim with carboy water.

4. Samples- 4-5 bottles are usually necessary for each sample. Some
samples may have to be diluted in order for the DO range to be
detected by the meter. Observation of the sample will usually give
an indication to its dilution. Clean samples usually require small
dilutions whereas wastewater samples will need high dilutions due to
their high BOD values. Once reasonable dilutions have been
determined, the specific volume of sample is added to each bottle
along with 2 mls polyseed. The bottles are filled to the rim with
carboy water.

Once all the bottles have been filled, the initial DO's of each solution is
determined on the meter and recorded. Once recorded, the bottles
are capped with ground glass stoppers to avoid excess bubbles and
capped. The bottles are placed in an incubator at approxiamtely 20
degrees celsius where they will remian for five days.

Analysis

After five days of incubation, the samples are ready to be analysed. The
samples are removed from the incubator and allowed to equilibrate
to room temperature. In the meantime, the analyst should calibrate
the meter again with the carboy water as discussed in the DO
Sample Preparation section. It may be desired to use fresh carboy
water for the calibration as discussed in the Carboy Set Up page.

Once the meter is calibrated, the samples are read starting with the blanks
and ending with the actual samples. The final DO of each solution is
recorded and the initial and final readings will be used to calculate
the BOD. The best results come about when the initial and final DO
values for the blanks are similar indicating the absence of organisms
and reliable equipment. The blank DO should normally be less than
0.2 mg/L

BOD Calculations
The calculations for BOD take into account the unseeded blanks and the
seeded solutions. These values must be subtracted out in order to
obtain reasonable BOD results. The following calculations are taken
from Standard Methods (Method 507: 1985, p. 531).

A. The BOD of the blanks are calculated by subtracting the final DO from
the initial DO:

BOD blank=DO1-DO2;

B. The BOD of the seeds are calculated by subtracting the final DO from
the initial DO and multiplying this number by the dilution factor:

BOD seed=(DO1-DO2) * Dilution factor per 300 ml

C. The BOD of sample and standards are calculated by subtracting the

final DO from the initial DO and multiplying this number by the
dilution factor. The final value is determined by substracting out the
BOD blank and the seed blank for each delta DO.

If the testing procedure was carried out correctly and the dilutions of the
sample were made appropriately, the analyst should have obtained
BOD values that are within a reasonable percent error and relative
percent difference. Generally, values are discarded for a specific
sample dilution if the final DO of the sample is < 1.0 mg/L of if delta
DO is < 1.0 mg/L. This also stresses the importance of using
different dilutions for each sample to key in on the appropraite BOD
when little is known about how the sample will react and how high its
BOD levels are.

Why 5 days ?

It can take as long as 25 days before no further changes can be detected in a

bottle in which a BOD test is being conducted. Depending on the
nature of the sample, the test may be near completion in a few days. A
reasonable compromise between waiting too long to get results and
getting unreliable answers is 5 days. As long as the samples are pretty
much the same from one sampling period to another, the 5-day test
works fairly well. For example, samples from a process in a waste
treatment plant will have essentially the same nature over long
periods. The 5-day BOD will be highly meaningful in showing
variations in plant performance.
The following figure shows simulation of the BOD test with different rate
coefficients:

Note the vertical line for 5 days. If the samples are quite different in their
composition, the error in comparing them at 5 days will be great, and
a longer time for the test would be better. This must be balanced by
a long wait before having results, and delay in making adjustments
based on these results may be costly.

DO Preparation

The DO (dissolved oxygen) of the prepared carboy water must be

determined to serve as a reference to all other sample and standard
readings. This is most often accomplished by performing a Winkler
titration on the carboy water, and adjusting the DO meter to this
reading. This method is not contained in the BOD procedure, but
rather comes from Standard Methods for the Examination of Water
and Wastewater (Method 218B: Azide Modification: 1971) In my
experience, we often used a pH meter with an electrode that could
also determine the DO of the solution as our DO meter.
The Winkler titration is carried out by first withdrawing three samples of
water from each carboy. Caution must be taken that no air bubbles
become trapped in the bottle. As a result, it is best to withdraw the
water slowly while the bottles are tilted slightly. Once filled, one
bottle from each carboy is set aside until later. The remaining four
bottles are used in the actual titration. 2 mls manganese sulfate is
added into each bottle under the surface of the water, followed by 2
mls alkali azide solution. The bottles are stoppered and shaken until
a brown floc appears. The bottles are allowed to settle until the floc
is halfway settled. The bottles are then shaken again and allowed to
settle. Once settled, 2 mls concentrated sulfuric acid is added down
the neck of each bottle and the bottles are shaken again. At this
point, the floc will disappear and the solutions should be amber in
color. Now, the solutions are ready for titration.

The solutions are first transfered to 500 ml ehrlenmeyer flasks. Each

solution is titrated with 0.0375 N sodium thiosulfate until the solution
turns a pale yellow. (Standard Methods suggests using 0.025 N
sodium thiosulfate for a different volume of sample.) At this point, a
few drops of starch indicator are added to turn the solution a dark
blue. Titration continues until the solution turns clear. The volume of
titrant used in mls directly corresponds to the DO of the sample. The
average DO reading from each carboy is calculated and recorded. If
the DO readings from each carboy set are not relatively close to
each other (within 0.1 mls) the process must be repeated until
consistent DO readings are obtained. The DO readings should
normally fall between 6.0 and 9.0 mg/L. If values are greater than
9.0 mg/L, the carboy water must be aerated again to reduce the DO.

Once the DO readings are calculated, the two bottles that were withdrawn
at the beginning are now used. The carboy bottle from which the
samples will be made up from is used to adjust the DO meter (in our
case, the pH meter). Once adjusted, the DO of the other bottle is
measured to determine whether the meter is reading correctly. If the
meter reading comes within 0.1 of the Winkler reading, the
calibration is complete. If not, the entire Winkler procedure must be
completed for both carboys.