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1:": EJ'LTT‘FEI’TTETFI. STUDY OF THE: EE‘F'TECTS OF" STEAGE SJEDING

0’! T' ’3 WIiV°¥Iﬂ5I"“-.L 0(Ym7‘5 3:77;??? 07“ .s" S'T’ERILE IT-IDUSTRIAL

117M GMT?
"1 iAJA. 1.21.

A THESIS S'E3 TafII‘T 7.3 TO

THE FA SULTY 0}“

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of
AGE-lICULTUfiTS MD $~-.}"‘I..I‘T.3 ""133."
by

Kenneth W. Cos 13

CAZ'IDI ELITE FUR T1" DYE-Gt E OF

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L“‘1,5LJWJ'\)WO any L5 Akin.

JU‘IET‘Z, 1938

THESIS

mrnrv'ﬂ. *‘* “WW; "1"
pO‘J...‘ \-"'~a.~.|-v:)v;:,5 I‘TL

The writer wished to express his appreciation to

Mr. E. F. Eldridge and Dr. W. L. Valimsnn whose thoughtful

assistance and helﬁful guidance made this thesis possible.

1168-15

INTRO DUCT ION

The importance of the biochemical oxygen demand
(B. O. D.) test cannot be questioned in its application to
sewaze treatment plant Operation, water treatment plant
operation, stream surveys and the treatment of industrial
wastes. The test is used to determine the efficiency of a
waste treatment plant or of a unit of such a plant, to
determine the extent of pollution of a stream and to compare
the strengths of different domestic and industrial wastes.

This thesis is chiefly concerned with the application
of the test to industrial waste analyses. In the design
of sewage treatment plants the engineers are often
confronted with the problem of determining the population
equivalent of some contributing industrial waste. This is
usually done by determining the H. O. D. of the waste and
calculating the population equivalent in terms of domestic
sewage having the same B. O. D. requirements. Large
contributions from industries have an important bearing on
the capacities of sewage treatment plants and it is
important that the 3. O. D. determination of any industrial
waste be accurate.

The biochemical oxygen demand determination is a measure
of the oxygen required to oxidize the ornanic matter of
a certain sample (1). The oxidation is brought about by
bacteria in a samnle which is held at some predetermined
temperature for the required incubation period; frequently

for five days. Eizht ounce glass stoppered bottles are

-2-

used for incubating the sample which is prepared by
making a proper dilution of the waste being used. Several
dilutions are usually required so that after the incubation
period a few parts per million of oxygen will be left in
the bottles. Sterile industrial wastes must be inoculated
with the proper organisms. To do this, various amounts of
sewage are added as seeding to the bottles and then incubated
for the desired period at some constant temperature.
Bottles containing the various amounts of seeding and
diluting water with no waste are also incubated to
eliminate the 3. O. D. of the seeding material that has
been added. Similar sets of incubation bottles are set
up for each day that the B. O. D. is to he determined.
After the incubation has proceeded the proper number of days,
one set of bottles is removed from the incubator and the
dissolved oxygen contents of the ineuhations are determined
by chemical methods. The difference between the oxygen
content of the diluting water and the dilution is a
measure of the B. O. D. of the waste.

however, the test as it is now made, has not proven
entirely satisfactory for the determination of the 8. O. D.
of industrial wastes. vany discrepencies are apparent due
to several factors among which the most important is the

seeding given to the dilution prior to incubation.

HICTCRICiL

The biochemical oxygen demand determination is by no
means a new test for it was used as early as 1370 by a
British Rivers Pollution Commission. In Germany it was
used as early as 1900 and in France as early as 1335. The
test in its present modified form has been used in the
United States since about 1715.(2)

From experiments conducted by Theriault and confirmed
by others, it has been found that the deoxygenation of an
organic waste proceeds in two distinct stages-~first the
carbonaceous staze where the carbonaceous matter is
oxidized and then the second or nitrification stage where
the nitrOgenous matter is oxidized. The general course of
the deoxygenation curve is shown in the accomnanying graph
which is taken from Theriault. The curves are plotted from
data of the same sample incubated at different temperatures
for varying lengths of time.

Phelps proposed a formula many years ago which fits
with reasonable accuracy the eXperinental results of
deoxygenation. The formula is based on the assumption
that the rate of deoxygenation at any instant is directly
proportional to the amount of organic matter present in the
sample and is applicable only to the first stage of
deoxygenation.

Without going into the derivation of Phelps formula

which can be found elsewhere(3) it may be stated as follows:

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Xt = L — Lt a L(l - lo-Klt)

Where Lt equals the B. O. D. in p.p.m. remaining after
time t. Where L equals the B. O. D. in p.p.m. exerted by
the organic matter during first stage.

L - Lt 3 Xt oxygen in p.p.m. used up in t days, as
determined by the B. O. D. test.

K1 8 deoxygenation constant.

The deoxygenation constant for any temoerature has
been found to vary with the 20° temperature constant as
the following formula;

Klan) ‘ Kuzmll-W‘T " 20’]

Where K1(20) . 0.100

The temperature also affects the magnitude of the
first stage B. O. D., and the first stage demand at any
temperature T bears the following relation to the 20°
demand: LI. :- L 20 [1+ 0.02m - 20)]

These formulas can be used to change any oxygen value
for one temperature and a given period of incubation into
terms of the oxygen value under an entirely different set of
conditions. They are applicable to recently polluted samples.

There are a number of factors which affect the 3. O. D.
determination: the pH of the medium, the amount and kind
of bacteria present and the mineral content of the sample
.and/or diluting water.

In general the advantage of the test over a strictly
chemical test like the potassium permanganate oxygen consumed

test is that it is very similar to the natural stream

-6-

conditions or conditions in the diluting body of water.
The disadvantages of the test are that there are often toxic
substances which inhibit bacterial growth especially in
some industrial wastes, long periods of incubations at
constant temperatures which require a high degree of mechu
anical control and rather close attentionl5) and also
there may be, particularly in industrial wastes, certain
chemical constituents which will interfere with the
dissolved oxygen determination hy the Winkler method.
Theriault(6)(7) and Theriault and Kchaneelal have success-
fully modified the Winkler method to give correct values
for dissolved oxygen when interference is encountered
due to iron salts, nitrites, organic matter, hypochlorites
and sulphite wastes.‘ Eldridge and hallmannlhl have found
that a synthetic diluting water with a mineral content very
similar to the mineral content of river waters will give
higher 3. O. D. values than distilled, bicarbonate, carbon.
ate or phosphate waters. They also found that the pH of
their dilutions dropped slightly during the oxidation period
in dilutions made with tap water and distilled water, but
in dilutions with other waters there was practically no
change. Their results show that the initial pH has some
effect on the B. O. D. results, but that it is not necessar-
ily a limiting factor.

Eldridge(9) has found that in making dilutions of

sterile material and seeding with sewage that the B. O. D.

varies almost directly with the amount of seeding up to
about seven days incubation.

Holderby and Lea(10) have found that the B. 0. D. is
substantially affected by the ratio of organic car on to
total nitrogen. With ratios of cnr\on to nitrogen as large
as 120 to l the rate of oxidation is ver slow, but if the
ratio be reduced to 5.7 to l the five day oxygen demand
values would he at a maximum.

Tuch uncertainity surrounds the test esvecially when
seeding material must be added as pointed out by ﬁldridgelll)
who shows that the five day 3. O. D. of a paper mill white
water increases in hither dilutions and increases with an
increasing amount of seeding. These same results have been
found to apnly to a sterile milk waste as is shown in the

accompanying eXperinental and data portions of this report.

This study was made to determine, if possible, the
correct amount of sewage seeding to be added to a sterile
industrial waste sample to get the proper B. 0. D. of the
waste. The experiment was divided into three trials, each
of which extended over a twenty-day incubation period using
an incubation temperature of 20°1=1° Centigrade.

milk waste was selected because it eliminated many
factors which affect the B. O. D. determination. This waste
is homogeneous, has an optimum ph value, is easily

obtainable and easily made sterily. A factory waste of milk

-8-

diluted with distilled water and containing 0.5 per cent
whole milk was made up and sutoclsved for'sach trial. In
trial No. l, 1.0 per cent and 0.5 per cent dilutions were
made of this factory waste and in trials No. 2 and No. 3
0.75 per cent and 0.5 per cent dilutions were used.

Eight ounce glass stoppered‘bottlss were calibrated
and used for the incubation of the samples. Bottles were
selected which did not vary more than five cubic centimeters
from 250 cubic centimeters capacity. The samples were
incubated in a water bath at 20°C. for periods of 2, 5,
10, 15 and 20 days. At the and of these periods the N
dissolved oxygen content was determined by the Winklcr method.

The sodium bicarbonate diluting wateril) used was
prepared by adding six grams of sodium‘bicarbonsts to five
gallons of distilled water and bubbling air through the
contents until the dissolved oxygen content is above 7.0
p.p.m. It has been pointed out‘by Eldridge and Hallmsnnc‘)
that this diluting water does not give as high 8. 0. D.
values as other waters.‘but it has been temporarily
accepted as a standard in sewage treatment plants and
gives results comparable to practice in this part of the
country.

The effluent of the East Lansing Imhoff tanks was
filtered and used as s seeding material.

The following dilutions and seedings were made for the
2, 5, 10, 15 and 20 day B. O. D. determinations.

TRIIK I“? o 1
1"Diluting - 1.0 per cent _ 0.5 per cent - each with no need.
Water waste waste
2- n a» u o l «- it a 1000 “
3- a - n - n - n n. 360. n
4- ﬂ ‘ ﬂ - ﬂ .. N N 530. It

TRIALS ﬁO. 2 AN? N0. 3

I‘Diluting-O.75 per cent -005 per cent - each with no seed.

Water waste waste
2- n - n d. n - a N 100. n
3- n ‘. ﬂ - ﬂ - n " 300. a
4‘ n . it - n - u N 500. I

Bacterial counts were made of the sewage seeding
material and of the dilutions Just before the chemicals were
added to make the dissolved oxygen test. These give the
number of organisms added to each B. O. D. bottle and the
bacterial population of the samples after the incubation
periods of 2, 5, 10, 15 and 20 days. These bacterial counts
are plotted against the days incubation for trial No. 2 on
graphs no. 9 and to. 10. Graph No. 11 compares the
bacterial count and the B. O. D. of the same sample. Graph
No. 12 gives the daily B. O. D. values plotted against the
days incubation.

Graph 30. 5 shows the average effects of sewage seeding
on the B. O. D. of a 0.5 per cent milk waste using various
periods of incubation and is a composite of graphs No. 6,
no. 7 and we. 8 which in turn are the reswective graphs of

trials ﬁo,~1, no. 2 and No. 3.

 

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- -10..

DISCUSEIOE

It is clearly shown by graph 30. l, which is a composite
of graphs So. 2, K0. 3 and he. 4, that for the 2, 5 and
10 day incubation ncriods there is a marked increase in the
B. O. D. of the waste with an increase in the amount of seed-
ing materials. The curves for the 15 and 20 day incdbationl
have shown a tendency to flatten out and even to decline
with the amount of seeding. Such decline cannot be explained
by the writer.

Graph no. 5. being a composite of graphs No. 6, No. 7
and he. 8, shows again that the B. O. D. of a 0.5 per cent
milk waste varies directly as the amount of needing nearly up
to the tenth day of incubation. From this point on, the
curves tend to come closer together and some even cross each
other. The fact that the curve for the five cc. of seeding
declines between the tenth and fifteenth days of incubation
cannot be eXplained. It is apparent that the five day
B. O. D. results are not dependable because of the wide
variations in values from 345 to 635 P.P.m. for the can.
waste with amounts of sewage seeding varying from nothing to
5 cc.

Graph no. 9 shows the number of bacteria per cc. present
in the B. 0. D.'bott1es after the incubation period for the
'bottles which contained only diluting water and seeding
material. The curves representing the growth in the bottles
with.no seed and with 1 cc. of seed show the typical lag

in.the growth to maximum number of organisms, while the other

curves show a rapid increase in the first two days and a
gradual decline over the remainder of the period. The lag
in the development of growth in the samples with no seed
and a small amount of seed is present but not so pronounced
in the dilutions of the 0.5 per cent milk waste as shown
by graph ho. lO.

Graph 30. ll compares the number of bacteria with the
B. O. D. of a sample containing 1 cc. of sewage seeding, over
the twenty-day incubation period. It shows how the number of
organisms present in a B. O. D.‘bottle increases rapidly to
a maximum in the first one or two days, then as the relative
amount of food per organism decreases the bacterial pepula-
tion must and does decrease steadily over he remainder of
the incubation period. As the num er of organisms decrease
because of a decrease of food or oxidizable organic matter
the daily B. O. D. values decrease. In other works the
increment of increase in B. O. D. values decreases during
the incubation period. This fact is clearly shown by graph
Ho. 12 which gives the increment of increase of B. O. D.
values for each day, over the value for the preceeding

period of incubation.

CIT? CLUSIOTIS
1. With this waste selected it was found that the
B. O. D. increased in direct proportion to the aspunt of
sewage seeding.

2. The bacterial population of the B. 0. D. samples

was found to increase rapidly in the first one or two days
to a maximum then gradually drop as the incubation period
proceeded.

3. From the results obtained no definite amount of
seeﬂing can be said to be the Draper amount to give the
exact value of the B. O. D. of this waste.

4. It is quite certain that by using the 5 day B. O. D.
values we are liable to a wide variation in results as clearly
shown by granh 30. 5.

5. It was fauna that the daily B. O. 9. curves follow
the same general trend as do the curves fer the number of
organisms uresent in the B. 0. D. bottles as Shawn‘by

centering greens ﬁe. 10 anl Ho 12.

1

TARULATED DATA
Trial Ho. 1.
Seeding materiel count - 33g000 bacteria per ce-

O.5 per cent milk waste

Days B. 0.. De *' 13.13.12).
ineu- no lee. See. See.
bation eeed seed seed seed
2 44 323 230 368
5 420 552 533 520
10 722 608 636 704
15 723 812 556 412

20 734 604 732 560

Trial U0. 2
Seeding material count - 110,000 bacteria per cc.

0.5 per cent milk west-

Days B. 0'. Do'popomo
incuc ﬁe 100. 300. Sec.
betion seed seed seed seed
2 36 253 272 300
5 132 055 540 752
10 416 516 392 356
15 696 716 726 603
20 6&3 732 734 33
Days Ho. of Bacteria per 00.
incu- E0 100. 300. 500.
bation seed seed seed seed
2 84,000 550,000 900,000 590, 00
5 30,000 40,000 53,000 45,000

10 150,000 0:,000 51,300 40,000
0

15 6,900 ,600 1,600 3, 00
23 1,500 2,000 1,000 3,000

Trial ”0 o 3

Seeding Fatericl Count - 93,000 bacteria per cc.

0.5 per cent milk waste

3378 B. O. D. "" IND.“-
incu- No 103. 300. Sec.
bstion seed seed seed seed
2 200 312 296 33b
5 433 512 533 604
10 534 615 623 720
15 743 0&0 564 543

20 724 576 734 560

-13-

33 I LIGC-TL' THY

1. Theroux, Eldridge and fellnnnw. nnelysis of
Water an; Sewage".

2. Theriault, E. J. "The Rate of Deoxyﬁenation of
Iolluted Yeters" from Tublic Keelth Report, Vol. 41, No.6
Feb. 5, 1226.

3. Tetcnlf and Eddy. "Sewn e and Sewage Disposal"
7?. 375 - 373.

A. Eldridge, E. F. and “allmann, W. L. "Influence
of Dilutiog Vater on the Biochemical ﬁxygen :enend".

5. Ruchhoft, C. C. "The B. 0. D. test and Oxidation
in Seraﬁe Treatnent". U. 0. Public Henlth Service.

6. Theriault, :. J. "Detailei Instructions for the
ferfornnnce of the Discolvcl Oxygen end Biochemical Oxygen
Delana Torts" 3upolemant Ho. 90 to 2ublic ﬁenlth Reports
of 1731.

7. Theriault, E. J. "The Determination of Dissolved
Oxygen by the Winkler ﬁethod. Fublic eolth Bull. ﬁe. 151.

3. Theriault, E. J. and icVemee, P. D. "Dissolved
Oxygen in Freoence of Qrganic 7etter, Hypochlorites and
Sulphitee." Industrial and Eng. Chem. Anal. Ed. 4, 57-64
(June 15, 133?) also Puolic Health Reports, V01. AB, No.

A5 (Tovenber 10, 1133) and Reprint to. 1501.
9. Eldridge, E. 2. "Here About the B. 0. D. Determin-

—~

ation" Sewerage Works Journal V31. 5, ?o. 5 (Sent. 1733).

10.

Uitrogen“
11.

tastes."

.

Holderby, J. H. and Lea, W. L. "The B. O. D. test

as Infliencod by the Ratio of 0 ganio Carbon to Total

Sewerage Works Jour. V01. 7, No. 1. (Jan. 1935).
Eldridge. E. F. "The B. U. D. of Industrial

Kichigan Engineering Exp. Sta. Bull. No. 73

(Jan. 1933}.

 

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