The Effect of Algae on BOD Measurements
Author(s): George P. Fitzgerald
Source: Journal (Water Pollution Control Federation), Vol. 36, No. 12 (Dec., 1964), pp. 1524-1542
Published by: Water Environment Federation
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THE EFFECTOF ALGAEON
BOD MEASUREMENTS
George
P. Fitzgerald
to measure
A study has been made
of various
the BOD
species of algae
with bacteria
alone and in association
of 5-days'
conditions
under incubation
time in the dark and at a temperature
if algae have an
of 20 ?C to determine
under the
effect on BOD measurements
conditions.
standard
Initial studies have been carried out
the green alga, Chlorella
pyre
using
This alga is es
noidosa
(Wis. 2005).
to various
culture
pecially
adaptable
and can be used as a basis
conditions
for comparison with other algae having
more restricted
conditions.
culture
used to sup
The culture procedures
tests are
the
BOD
for
Chlorella
ply
and the growth rates of this
described
alga in four different media were meas
there is little informa
Because
ured.
on the measure
tion in the literature
ment of dissolved
oxygen
(DO) in the
presence
of
algae,
tests
were
carried
this
out to observe
if algae affected
measurement.
Another
series of tests
was made
if the algae
to determine
used in BOD tests under the standard
the 5 days
conditions
would
survive
in the presence of bacteria.
of darkness
The effect of adding sewage bacteria to
cultures of algae was also determined,
of the algae and
both on the activity
in
the effect of the culture supernatant
the BOD test.
Materials
The
2005)
As soon as some of the preliminary
studies on the effect of algae on the
various measurements
involved and the
effects of mixing
algae and bacteria
were known, studies were made of the
factors
the activity
of the
affecting
algae during the BOD tests. The vari
ous factors
have
studied
been
the
amount
of algae, the source of algae
in which
the algae
(i.e., the medium
were
and
the
age of the
cultured),
at the time of use in
algal cultures
the BOD test (i.e., the factors causing
in algal
cultures
two
senility
using
and secondary sewage
media, Gorham's
effluent).
As an extension of the data collected
as a test organism, fur
using Chlorella
ther studies were carried out compar
of the blue-green,
ing the activity
bloom-forming
alga,
that
to
ginosa
(Wis. 1036)
rella. Also, the activity
and
blue-green
aeru
Microcystis
of other green
was
algae
of Chlo
measured:
mus
Nostoc
dimorpha,
echinulata.
corum, and Gloeotrichia
Studies on the influence of the dilu
Scenedesmus
tion medium
in
the BOD
test
have
been made.
of the BOD
Comparison
in dilution
of Chlorella
water,
algal
culture media,
and in lake waters,
as
well as seasonal
in
the
influ
changes
ence
of
lake
are
waters
reported.
and Methods
Chlorella
(Wis.
pyrenoidosa
as a pure
used was maintained
the
with
is associated
P. Fitzgerald
George
and Sanitary
College
Lahoratones,
Hydraulic
of
University
of Wisconsin,
Engineering,
Wis.
Madison,
culture and a volume of about 15 ml
was
to inoculate
used
each experi
mental
A
culture.
2-1 Erlenmeyer
flask with 1,500 ml of medium was used
as
a culture
ported
vessel.
a 6-mm diam
A
cotton
glass
plug
sup
tube with
1524
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a
12
Vol. 36, No.
BOD MEASUREMENTS 1525
end that was used for bub
constricted
a
C02-air mixture
bling
1.25-percent
the
Each
bubbler
culture.
through
was attached
rubber tub
by surgical
ing to a 25- X 200-mm glass tube con
A 50-mm
cotton as a filter.
taining
was
in
teflon-covered
magnet
placed
each flask. The culture vessel, media,
and filter were
sterilized
magnet,
by
were
Inoculations
autoclaving.
The composition
of the four media
are listed
used for culturing
Chlorella
in Table
water me
I. The dilution
dium is BOD dilution water
(1) plus
30 mg/1 of nitrogen
from NaN03.
The
is near 7.4 at har
pH of this medium
vest time. Allen's medium
(2) is also
a neutral medium,
the final pH being
near 7.0. Myers' medium
(3) is more
the pH being near 6.5. Gor
acidic,
ham's medium
(4) is alkaline, with a
final pH of near 9.0.
The algae to be used in an experi
ment were centrifuged
from their cul
ture solution after their concentration
made
with
sterile syringes.
Motors
(1/100
Elec.
hp shaded pole motor, Dayton
with
magnets
Mfg. Co., Chicago,
111.)
attached
to their shafts were mounted
on wooden
frames below the
vertically
and the speed of 15
culture
vessels
stirred cultures was con
magnetically
trolled with one variable
transformer.
The algae were cultured under continu
ous light
[180 ft-c
(1,940
lumen/sq
in a constant
m)]
(23 ?
temperature
culture
1?C)
wTas
room.
TABLE
I.?Media
A.
Salt
Used
for Culture
Concentration
Dilution Water
NH4CI
of Chlorella
of salts
used
for BOD
in media
Gorham's
Experiments
Myers'
50
496
1,000
KN03
1,210
8.5
KH2PO4
K2HP04
1,230
21.75
39
250
75
27
58
6
1
513
50
33.4
Na2HP04-7H20
Fe Citrate
0.15
FeCls
Fe2(S04)3
52
22.5
MgS04-7H20
CaCl2
27.5
Na2Si03-9H20
Citric Acid
EDTA
Na
2,460
195
Citrate
20
Na2COs
Total
was
supernatant
(mg/1)
Allen's
1.7
182.0
NaN03
the
measured,
the cells were washed with
discarded,
and recentrifuged.
pH 7 P04-buffer,
in a
The cells then were
suspended
volume
of PO^buffer
and
convenient
measured
added
to
di
seeded
samples
lution water for mixing
and filling of
297.5
Salts
B.
Ion
N
P
Fe
Mg
Ca
S
K
Concentration
Dilution Water
30
10
0.05
2
10
728
of essential
1,866
ions
Gorham's
82
7
1
7
10
3
10
12
17
in media
5,147
(mg/1)
Allen's
Myers'
178
45
1
50
18
67
112
168
295
15
243
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323
353
1526
JOURNAL WPCF
BOD
bottles.
bottles were
Duplicate
the initial DO con
used to determine
and triplicate
centrations
bottles were
used
for
after
analysis
containing
black
room.
wrere
algae
incuba
5-days'
tion at 20 ?C in a dark
Bottles
covered
as a further precaution
light from entering during
incubation
to
the
were
Measurements
period.
made as suggested
ods (1).
in Standard
used to measure
the
procedure
absorbed
of oxygen
by algae
the 5-day BOD determinations
during
was to measure
used by
the oxygen
The
amount
seeded
Meth
water
dilution
with
hoods
prevent
December 1964
and
seeded
(24-hr settled
dilution wa
sewage seed)
ter plus algae.
in the
The difference
was
referred
to as
oxygen
depletion
being caused by the algae and reported
as A DO per mg algae.
Results
Growth
of Chlorella
Media
in Different
Because
there is no one growth meas
common to all algae labora
urement
growth
tories,
were
measurements
made on cultures as suspended
solids,
volatile
COD
[dichromate
solids,
method
with Ag2S04
catalyst
(1)],
cell counts, and packed volume.
The
between
these
general
relationships
measurements
of Chlorella
cultures
of
are
10
of
age
approximately
days
in Table II.
presented
on Dissolved
of Algae
Measurement
Oxygen
Effect
The effect of the presence
of algae
in measuring
dissolved
oxygen
(DO)
by the Alsterberg
(azide) modification
of the Winkler
method
(1) was de
termined.
It was found that concentrations
of
as high as 5 X 106 cells/ml
Chlorella
did not appreciably
affect the meas
was
urement
of DO.
The end-point
with
but
slightly
obscured,
experience
the
overcame
measurement
Once
advantage.
fate
and alkaline
TABLE
Culture
Medium
Dilution
Gorham's
Allen's
Myers'
water
the
this
manganous
iodide
reagents
IL?Relative
Growth
dis
sul
were
of Chlorella
vs
it was found that up to two
added,
hour intervals between
the addition
of
acid or titration
the sulfuric
did not
in the pres
affect the DO measured
ence of Chlorella.
Effect
of Seed
Activity
Volume
of Algae
on
were
carried
out in
Experiments
which the oxygen absorbed by different
volumes
of 20- to 24-hr
(3.0 to 30 ml/1)
settled sewage seed in dilution water
was measured
as well
as the total
a
amount
of oxygen
absorbed when
amount
definite
of Chlorella
from a
dilution
water
culture was added
to
each seed volume
in dilution water.
The results of these tests indicated
that a variation
of 10 fold in seed vol
ume or 7 fold in oxygen absorption
due
to the seed had little or no effect on
the amount of oxygen absorbed by the
added amount of algae.
of Contaminating
Effect
Chlorella
on
Cultures with
Subse
Sewage
quent BOD Measurements
Settled sewage was added to cultures
of Chlorella
(one to five ml sewage
after
10 Days
in Different
Media
ss
(mg/1)
(mg/l)
COD
(mg/1)
Cell Count
(cell/ml XI06)
125
175
375
375
100
200
350
350
175
250
550
550
12.5
5.0
50.0
50.0
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Vol. 36, No.
BOD MEASUREMENTS 1527
12
that previously
per liter of culture)
for BOD measure
had been sampled
about five additional
ments.
After
in the culture room
days of incubation
the
normal
conditions
under
growth
cultures were again sampled for BOD
The
effects
of both
measurements.
washed
algal cells and of the culture
were measured
in seeded
supernatants
dilution water tests.
A total of 45 tests was carried out
on algae from dilution water, Allen's
that had been in
and Myers'
media,
cubated with
It was found
sewage.
that the average activity
of the algae
cultures was within
from contaminated
the experimental
limits for that me
as compared
from
dium
with
algae
was
data
it
these
cultures.
From
pure
that algae from either pure
concluded
have
or contaminated
cultures would
the same effect on the BOD
essentially
measurement.
a very different
effect of
cultures
with
Chlorella
contaminating
sewage on the results in BOD measure
There
was
ments
by the supernatants
produced
in
It was found
from the cultures.
from
12 tests with cultures of Chlorella
'
me
and Myers
dilution water, Allen's
of
contamination
effect
that
the
dia,
in
the sewage caused a decrease
with
to the
of the supernatant
the BOD
extent that the BOD was 1/5 or less
for 5
with
after
incubation
sewage
as
the
with
supernatant
days
compared
of the cultures prior to contamination.
of course, be
This is to be expected,
cause the organic matter
accumulated
had been
in algal culture supernatants
of sew
found to increase the activity
was
further
seed
age
proof
and, thus,
could be
that some of this material
broken down by sewage organisms.
Comparison
of Live
and Dead
Algae
The literature states (5) (6) (7) (8)
had
considerably
algae
than live algae.
demands
higher oxygen
In order to check this a series of BOD
live algae from
tests was made with
cells which
similar
Allen's
medium,
that
dead
and cells which
autoclaved,
freeze-dried.
were first carried out
Experiments
of Chlorella
cells
to test the viability
which had (a) no treatment,
(b) had
been frozen only, and (c) which had
final
freeze-dried
been
(3.6-percent
as compared
content
to 83
moisture
in fresh cells).
Tests
percent moisture
in either
indicated that cells suspended
8-wk old
distilled water or autoclaved
culture
and
then
frozen
supernatant,
of time required
for the same period
in the freeze-drying
process
(1 to 3
of about 20 per
days), had a viability
had
had
been
been
as
cent
compared
to
unfrozen
controls.
were
of up
in freeze-dried
viable
to
10,000 cells but, in
samples
one experiment,
4 algae cells out of
500,000 cells survived the freeze-drying
treatment.
it may be con
Therefore,
cluded that freeze-dried
Chlorella
cells
for these tests are, by and large, non
to
viable, but may still be considered
be chemically
intact much
the same
as live cells, as compared to cells which
have been killed by autoclaving.
The results of 8 BOD tests each with
and
autoclaved
fresh,
freeze-dried,
Chlorella
cells indicated
that the aver
factors
age activity
(A DO/mg
SS)
were 0.13, 0.44, and 0.48, respectively.
Thus, it has been confirmed that dead
than
algal cells exert higher BOD's
live cells, and the BOD of cells killed
are nearly
as great
by freeze-drying
as the BOD
of cells killed
by auto
No
cells
claving.
of Viability
and
Comparison
Capacity
Photosynthetic
of al
tests of the mixture
Viability
dilution
water
gae and seeded BOD
before and after incubation were made
of the mixture
samples
by diluting
buffer
using sterile, pH 7.2 phosphate
in
dishes
and
with
petri
plating
(1)
extract
The
agar.
glucose
tryptone
colonies un
viable algal cells forming
der these conditions were counted after
in the constant-light
culture
incubation
room for at least 10 days.
The algal
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1528
JOURNAL WPCF
cell counts
in some plates were
less
than in duplicate
plates or algal colo
nies were
in some duplicate
smaller
inhibition
because
of
plates,
resulting
the presence
of certain
bac
of
types
teria.
The results
of viability
tests com
the number
of cells viable at
paring
the time BOD tests were started
(zero
the number
of viable cells
time) with
found
in BOD
after
bottles
5-days'
incubation
in the dark indicated
that
88 percent
of the cells from Allen's
medium
of
(17 tests) and 76 percent
cells from dilution water medium
(34
tests) were alive after the incubation
in comparing
data
period.
However,
of individual
was
it
found
experiments
that the amount of oxygen used per mg
of suspended
solids
(A DO/mg
SS)
was
not
to
related
necessarily
the
re
sults of viability
the
tests; essentially
same A DO/mg
SS was obtained from
re
which had 100-percent
experiments
in
covery of cells as from experiments
re
which
there was only 52-percent
covery
(dilution water
algae).
The photosynthetic
of the
capacity
algae-seed
was
mixtures
measured
5 days' incubation
DO analyses
by comparing
duplicate
of samples
in the dark with
held
samples exposed to 350 ft-c (3,770 lu
men/sq m) of light for 2 hr just before
The amount of oxygen
that
analysis.
in the light in ex
the algae produced
cess of that used in the dark was con
trolled by the physical
of
conditions
the medium
at the time of analysis,
such as C02 content of the water
in
the BOD bottles, as well as the physio
logical state of the algae.
However,
since this test was used
to compare
sources but
from different
in
algae
same
medium
after
the
same
in
cubation
it was
to
considered
times,
be a reliable
test for the
comparative
of the different
activity
algae at that
time.
particular
The amount or rate of oxygen pro
as the change in
duced was expressed
oxygen
per mg
suspended
solids
the two-hr period.
The average
rate
of 35 tests of algae from Allen's
me
at the start of the
dium measured
BOD
tests was
0.093A DO/mg
SS,
while
it was 0.083A DO/mg
SS when
tested after 5-days'
a de
incubation,
crease of 11 percent.
The average of
these two rates of oxygen production,
0.088A DO/mg
SS, for a 2-hr period
of light exposure
is equivalent
to 5.3
for a 5-day
mg of oxygen production/1
or a considerably
rate
period,
higher
than the average
rate for
respiratory
algae from this source (0.10A DO/mg
SS).
(See also reference
6.)
The photosynthetic
capacity of algae
from dilution water medium
(32 tests)
had an average rate of 0.056A DO/mg
SS at zero time and 0.024A DO/mg
SS
a decrease,
after 5-days'
in
incubation,
ca
in photosynthetic
5-days'
time,
of 57 percent.
it
pacity
Therefore,
seem that the higher
would
activity
in the BOD test of algae from dilution
water
medium
be due
to the
may
death of some cells from dilution water
medium
and the resulting
stimulation
of bacterial
respiration.
at
zero time and after
the
1964
December
for
of Algal
Comparison
Under
Bacterial-Free
Rates
Activity
Conditions
The procedure
used to measure
the
amount of oxygen that algae would use
in five days, in the absence of bacteria,
was to add algae from pure cultures to
sterile BOD dilution water and siphon
the mixture
to sterile BOD
bottles.
At the same time as these tests were
tests were also run in
run, duplicate
seeded dilution
water
identical
using
samples of algae.
The results of the bacterial-free
and
seeded BOD
tests indicated
that 12
percent
more
oxygen
was
consumed
in seeded
solids
per mg
suspended
BOD tests than in bacterial-free
BOD
tests with algae from Allen's
medium
and 34 percent more with
(8 tests),
algae from the dilution water medium
The average BOD of algae
(12 tests).
from Allen's
medium
(bacterial-free)
was 0.12 as compared to 0.10A
DO/mg
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Vol. 36, No.
12
BOD MEASUREMENTS 1529
SS for the average for all seeded BOD
tests with algae from Allen's medium.
the average BOD of dilution
However,
water algae in the bacterial-free
tests
was 0.13 as compared to 0.19A DO/mg
SS for all seeded tests.
The similarity
of the activity
of these algae under
bacterial-free
conditions
(Allen's, 0.12;
and dilution water, 0.13 A DO/mg
SS)
should be noted.
of measurements
of the
Comparisons
of the algae
photosynthetic
capacity
from the two media under seeded and
bacterial-free
conditions
that
indicated
there was little difference
in the photo
synthetic activity of algae from Allen 's
medium
at zero time (0.093 seeded vs.
0.095 sterile A DO/mg
SS).
However,
in the case of algae from the dilution
water
the
medium,
average
zero
Seed only
_
Seed and Algae (13 mg/1)
6h
Seed and Algae (27 mg/1)
Time (Days)
FIGURE
1.?Oxygen
27 mg/1
time
increased from
photosynthetic
capacity
0.056A DO/mg
SS for the seeded tests
to 0.071A DO/mg
SS for bacterial-free
tests.
ca
The average photosynthetic
pacity of all seeded tests after 5-days?
water
incubation
for dilution
algae
utilization
by seed and
Chlorella
in seeded
dilution
13 mg/1
Chlorella
and
water.
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1530
JOURNAL WPCF
December
1964
Algae (Dark plus 2-hr
light before analysis)
Algae (Initial 2-hr light)
Algae( Dark Only)
Time (Days)
utilization by Chlorella (13 mg/1) from Allen's
FIGURE
2.?Oxygen
medium in seeded dilution water [Light: 2 hr, 350 ft-c (3,770 lumen/
sqm)].
In
SS.
(32 tests) was 0.024A DO/mg
the
contrast,
capacity
photosynthetic
under con
of algae from this medium
for 5 days
ditions of sterile incubation
was 0.054A DO/mg
SS, or twice the
rate of tests run in seeded BOD bottles.
Daily
and Long-Term
BOD
Studies
A series of BOD
tests has been run
to establish
used
the rate of oxygen
by algae on a daily basis and for long
The
of time, up to 16 days.
periods
tests were set up as for normal BOD
measurements
samples were
except
analyzed
that duplicate
at the times re
ported.
1 presents
data from an ex
Figure
the daily change in
periment
showing
dissolved
in BOD
bottles
of
oxygen
seeded dilution
seed
13
water,
plus
and seed plus 27 mg/1
algae,
mg/1
from Allen's
medium.
These
algae
data
indicate
that oxygen
utilization
over the
by the algae is fairly uniform
in the dark.
5-day period
2 presents
data
obtained
Figure
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BOD MEASUREMENTS 1531
12
Vol. 36, No.
was
medium
when algae from Allen's
added to seeded dilution water
(13 mg
and some bottles were either
algae/1)
(in
analyzed
kept in the dark until
exposed to 2 hr of 350 ft-c
duplicate),
(3,770 lumens/sq m) of light and then
to 2 hr
kept in the dark, or exposed
of
of 350 ft-c
m)
(3,770
lumens/sq
light just before analysis after incuba
The initial DO con
tion in the dark.
of
centrations
samples can be increased
of
the samples with algae
by exposure
to light with little significant
change in
from
utilization
rate of oxygen
samples not exposed to an initial light
of
treatment
(there was an average
six
of
in
increase
activity
five-percent
to light as compared
samples exposed
to samples without
light treatment).
The data showing the level to which
are raised
in
the DO
concentrations
to
light just before
samples
exposed
on a daily basis
2)
(Figure
analysis
indicate that for the 5-day period with
there is
medium
algae from Allen's
ca
little change in the photosynthetic
the
6X Seed Only
Algae (Allen's Medium)
Algae (Dilution Water Medium)
Time
FIGURE
Allen's
seeded
medium,
dilution
3.?Oxygen
and
(Days)
utilization by 6X seed, Chlorella (14 mg/1)
Chlorella
(16 mg/1)
from
dilution
water
from
medium
water.
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in
1532
JOURNAL WPCF
December
1964
Algae (Dark plus 2hr
light before analysis )
Algae (Dark Only)
Time (Days)
utilization by Chlorella (28 mg/1) from Allen's
FIGURE
4.?Oxygen
medium in seeded dilution water [Light: 2 hr, 350 ft-c (3,770 lumen/
sqm)].
(average oxygen
pacity of these algae
of 35 samples at zero time
production
was
SS and 0.083A
0.093A DO/mg
SS for 5-day samples).
DO/mg
BOD tests were also run for as long
as 16 days to determine
the rate at
which
oxygen was consumed by algae
over periods
of time in excess of the
usual 5-days'
time used in BOD tests.
3 shows the rate at which oxy
Figure
gen was consumed by sewage seed (at
6 times the amount used to seed the
water
the algae
dilution
used with
tests) and the rate of oxygen utiliza
tion by algae from Allen's medium
(14
as compared with that of algae
mg/1)
water
medium
from
dilution
(16
mg/1).
4 presents
the rate at which
Figure
of algae
oxygen was used by 28 mg/1
over a 16-day
medium
from Allen's
period, as well as the amount of oxy
of samples
gen produced
by exposure
to 350 ft-c (3,770 lumen/sq m) of light
It can be
for 2 hr prior to analysis.
seen that
bottles
became
the BOD
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Vol. 36, No.
12
BOD MEASUREMENTS 1533
in about 9
anaerobic
(DO exhausted)
the algae pres
days' time. However,
ent continued
to produce oxygen when
to
exposed
light, but at a decreasing
rate. Viability
tests of samples from
these bottles at zero, 5, 12, 14, and 16
no
loss in viability
indicated
days
(average percent viable cells were 100,
94, 120, 123, and 115, respectively).
Thus, these data confirm the results of
et al. (9) who found Chlorella
Golueke
re
and Scenedesmus
to be relatively
to anaerobic
sistant
and of
digestion
et al. (10) who found Chlorella
Finkle
to be viable after 70 days in the dark.
The
Effect
of
the Culture
Medium
indicated
Preliminary
experiments
that the activity
in the
of Chlorella
test could range
BOD
from 0.06 to
0.31 mg 02 used per mg
suspended
solids per 1 (A DO/mg
SS), depend
in which
the
ing on the medium
The distress
algae had been cultured.
from these data was
ing observation
that the apparent
of the algae
activity
from the different media
to
appeared
be inversely
to the potential
correlated
in the media;
growth rate of Chlorella
that is, algae from the two more con
centrated media, Myers'
and Allen's,
showed less activity
in the BOD
test
than algae from the very dilute dilu
tion water medium.
Other data, such
as the results
of bacterial-free
BOD
indicated
that perhaps
tests, however,
the measured
BOD of algae from the
water medium
a
dilution
was really
combination
of algal respiration
and
stimulated
bacterial
respiration.
Because
cell counts
and viability
tests by the dilution
gave
technique
little correlation
to other data, meas
urements
of the photosynthetic
capac
and dilution
ity of algae from Allen's
water media
at the start and end of
tests were made.
BOD
These
data
ca
indicated
that the photosynthetic
of algae from Allen's
medium
pacity
11 percent
decreased
whereas
there
was a decrease of 57 percent with algae
over
from the dilution water medium
'
a period of 5-days
time.
A re-evaluation
of the effect of the
different
culture media on the activity
in the BOD
of Chlorella
test was car
ried out by comparing
the apparent
of the algae over the 5-day
respiration
the photosynthetic
capac
period with
after 5
ity of the algae as measured
in the dark.
incubation
The
days'
results are presented
in Table III.
These data indicate that algae grown
in the three media, Myers ',Allen's,
and
have about the same photo
Gorham's,
after 5-days'
incu
synthetic
capacity
bation in the dark, but there is about
a 50-percent
increase in the activity of
the algae in the BOD test. The algae
from the dilution water medium
have
the highest
in the BOD
test,
activity
but demonstrate
in
the lowest activity
the photosynthetic
test.
capacity
The Effect
of the Age
of
on Algal Activity
Culture
the
In order to measure
the effect the
of
age of a culture had on the activity
algae in the BOD test, algae were used
from cultures considered young or old,
on the growth
rate
of
depending
the algae in the particular medium.
A
was made
of these two
comparison
of cultures for dilution water,
"ages"
and Gorham's
Allen's, Myers',
(air for
The results of these
aeration) media.
tests are summarized
in Figure
5 as
the amount
of oxygen
used by the
solids
algae per mg of suspended
(A
ages of
SS) for the different
DO/mg
the cultures.
The circles indicate the
factor
for each age
average
activity
of the culture media
tested and the
lines running
the cir
heavy
through
cles indicate
the range of ages used.
lines are used to make the lines
Light
for each medium
continuous.
The
number
of samples used to determine
the average value
is indicated
by the
at each dot. The
figure in parentheses
are also
ranges of pH of the media
The pH values presented
presented.
are for the cultures at the time of har
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1534
JOURNAL WPCF
TABLE
III.?The
Effect
of the Culture
1964
December
on
Medium
the Activity
of Chlorella
in the BOD Test
BOD*
Culture
Medium
pH
6.5
Myers'
Allen's
Capacity!
Range
Avg.
Range
23
0.069-0.12
0.090
0.065-0.22
0.044-0.23
0.12 -0.31
0.11
0.14
0.087-0.18
0.030-0.26
0.031-0.22
0.14
32
0.20
0.014-0.039
0.027
34
14
Gorham's
Dil. water
Photosynthetic
No. of
Tests
Avg.
0.13
0.13
*A
DO/mg SS/5 days.
f A DO/mg SS/2 hr, 350 ft-c (3,770 lumens/sq m) light,
in
There was
little change
vesting.
the pH of the cultures
the
throughout
growth period.
It is evident
that the average algal
factor for young and old cul
activity
tures of dilution water
(0.18 and 0.18A
and
Allen's
medium
(0.11
SS)
DO/mg
com
and 0.12A DO/mg
indicate
SS)
little change in activity per
paratively
as the age of the
mg of cell material
culture
increases
within
the
limits
in the case of algae
tested.
However,
in the other two media
cultured
there
was a definite
in older
loss in activity
cultures as compared with young
cul
tures.
The average value for young
and old cultures was 0.094 and 0.063A
SS for algae from Myers' me
DO/mg
0.20
Dilution Water
(pH 7.4)
0.15 h
Allen's
(pH 7.2-7A)
Gorham's
0.10
(pH 8.7-9.1)
0.05 h"
Myer's
(pH 6.0-6.7)
12
16
20
24
Age (Days)
FIGURE
5.?Effect
of age
of
culture
on
activity
of Chlorella
(four
media).
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SS
dium and 0.16 and 0.12A DO/mg
medium.
for algae from Gorham's
(dilu
as the two media
Inasmuch
that had the
and Allen's)
tion water
age of
in activity with
least decrease
neu
approximately
the culture were
be possible
tral in pH value, it might
removed from
that pH values further
the neutral value may be the principal
cause of the senility of the cultures.
Effect
of
Medium
1535
BOD MEASUREMENTS
Vol. 36, No. 12
the pH of Gorham's
on Algal Activity
sec
Because
the data of the previous
tion had pointed out that there might
the pH of the
be a relation between
and the activity of the
culture medium
algae, further studies were carried out
using Gorham's medium.
It had been found that the range in
in Gorham 's
for cultures
pH values
these cul
medium was 8.7 to 9.1 when
tures were aerated with
air, and pH
the 1.25
7.0 to 7.8 when aerated with
percent C02-air mixture.
Therefore,
aside from changes in the soluble min
because of changes in
eral composition
to
could be used
pH,
this medium
and
the effect of near neutral
study
of
alkaline pH values on the activity
Chlorella.
A series of 34 tests was made with
me
algae from the alkaline Gorham's
dium (aerated with air), 15 tests using
3 to 8 days old and 19 tests
cultures
A
19 to 23 days old.
cultures
with
second
series of 34 tests was made
the neutral
Gorham 's medium
with
a 1.25-pereent
C02-air
(aerated with
3 to
mixture),
18 tests using cultures
7 days old and 16 tests using cultures
16 to 23 days old. The average values
and range in
obtained for each medium
6 as
in Figure
ages are summarized
the amount of oxygen used per mg of
suspended solids (A DO/mg SS).
in
There was a loss of 24 percent
the activity
of algae from older cul
to
tures at pH 8.7 to 9.1 as compared
cultures
(0.12 vs. 0.16A DO/
younger
mg SS), whereas
algae from cultures
of
at pH 7.0 to 7.8 had a difference
only 11 percent between old and young
cultures
(0.15 vs. 0.17AV DO/mg
SS).
as a comparison
of
When
expressed
cultures
of similar
ages, at different
pH values,
there was only a 4-percent
cultures
at
difference
between
young
pH 7.0 to 7.8 and pH 8.7 to 9.1;
of
from old cultures
whereas,
algae
pH 7.0 to 7.8 were
18 percent more
active than algae from similar cultures
of pH 8.7 to 9.1.
Thus, it is indicated
that the pH of
the culture medium will affect the ac
the effect being
tivity of the algae,
more
pronounced
as the age of the
culture increases. However, this effect
may not be the result of the difference
in pH but may have been caused by
these pH
other factors accompanying
such as a lack of dissolved
changes,
in the
CO2 at pH 9, or a change
nitrogen utilization at the different pH
values.
Studies with Chlorella Grown in
Secondary Sewage Effluent
Because
the data obtained
from stud
ies with the previously discussed syn
indicated
thetic media
that differences
in the activity
of the algae could be
in the medium,
caused by variations
studies were made on Chlorella
grown
in different variations of autoclaved
secondary
sewage effluent.
The gen
eral composition
of this effluent was
approximately
20 mg NH3-N/l,
7 mg
950 mg total solids/l,
P04-P/l,
380 mg
methyl orange alkalinity (as CaCO3)
/1, 10 to 30 mg BOD/l,
and
a pH
of
7.8.
Autoclaved Madison, Wisconsin, sec
ondary sewage effluent, referred to as
sewage
effluent
or S.E.,
was
used
as
the culture medium for Chlorella in
the same manner
as the synthetic
dia.
The cultures were aerated
a 1.25-pereent
C02-air mixture
at
until growth was well established,
me
with
least
usu
ally 2 days. The cultures continuously
aerated with
the 1.25-pereent
CO2-air
mixture maintained
a pH of 7.3 to 7.8
pH of sewage
effluent was
(original
7.8, while the pH of autoclaved
effluent
was about 9).
Cultures
which were
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December 1964
JOURNAL WPCF
153(;
0.20
0.15 -
Gorham's
7.0-7.8)
~~~~~~~(pH
\
Gorham's
(pH 8.7 - 9,1)
0.10 _
CL)
cn
E
0.05
0
4
8
24
20
16
12
Age (Days)
FIGURE
6.-Effect
of pH of Gorham's medium
aerated with only air after the initial
period of growth had a range of pH
these high pH
from 8.8 to 9.4. At
values considerable salts could be pre
of suspended
cipitated
(40 to 90 mg/l
solids and about 75 percent of soluble
(12) ; also, there was
P04-P)
(11)
little or no dissolved CO2 present. The
amount of suspended solids formed
when
and
sewage
effluent was
then aerated
for six
autoclaved
days with
the 1.25-percent C02-air mixture was
about 40 mg/l, whereas
about 65 mg/l
were formed when only air was used.
of in
for these amounts
Corrections
organic suspended solids were made
in calculating the supended solids of
the algal cultures.
the algae grown
The tests run with
to in
in sewage effluent were designed
the activity
of algae
dicate whether
in the BOD
test was different
when
on activity of Chorella.
cultured under conditions of C02-lim
ited growth
(pH 9) as compared
to
The average
sufficient CO2 (pH 7.5).
activity
factor, A DO/mg
SS, for all
added
with
sewage
effluent
cultures
whereas
CO2 was 0.29
(41 samples),
it was
0.27
(41 samples)
for algae
grown
under
C 02 deficiency
and a
higher pH (ratio of 1.1).
The aver
age factor A DO/109
cells, was 1.5 for
pH 7.5 cultures and 1.6 for pH 9 cul
tures (ratio of 0.93).
These results with algae cultured in
modifications of sewage effluent have
indicated,
therefore,
that, as in the
case of the synthetic media
tested, al
conditions
gae grown under
of suffi
cient CO2 (pH 7 to 7.5) were more
active per mg of suspended
solids in
the BOD
test than algae from C02
deficient cultures (pH 9) . Other tests
indicated
that
this
difference
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is ap
Vol.
36, No.
BOD MEASUREMENTS 1537
12
enhanced
age
by increased
parently
but does not appear
of the cultures
to be related to the supply of nitrogen
as
NH3-N,
or
to
the
presence
or
ab
sence of salts which might
be removed
from solution at the higher pH value.
Effect
on
of Glucose
and Fructose
of Chlorella
BOD
thus far have
The data
reported
dealt with the BOD of Chlorella when
in an inorganic medium with
measured
amounts of dis
the addition
of minor
in
solved or suspended
organic matter
the form of settled sewage (the BOD of
seeded dilution water being about 0.5
In order to demonstrate
the
mg/1).
between
of the relationship
complexity
the BOD of algae in such an environ
ment
to the BOD
of algae in an en
vironment
containing
organic
mate
a series
rials in appreciable
quantities,
of five tests was carried out using glu
cose and fructose.
These two organic
represent
compounds
readily available
to
bacteria
while being se
compounds
to Chlorella.
available
Glu
lectively
cose is known to be an available
source
of energy
fructose
(13) (14), whereas
un
has been shown to be relatively
available
to certain
of
species
algae,
in these
those used
tests
including
(14).
These tests were carried out in the
normal manner
of testing seeded dilu
tion water with or without
algae and
seeded dilution
water
4.3
containing
mg of glucose or fructose per liter with
or without
The results of the
algae.
__
K =
/ Log cell number
as an added
five tests with
fructose
that the pres
substrate have indicated
ence of Chlorella
had no effect on the
bacterial BOD
of fructose
(less than
variation
with
10-percent
controls).
of bacterial
stimulation
respira
Also,
tion by the presence
of fructose
had
no effect on the BOD of Chlorella
(less
In effect,
than 5-percent
variation).
in the presence
the BOD of Chlorella
to the
of fructose was merely
additive
In contrast,
in the
BOD of fructose.
studies in which glucose was used as an
the algae had a de
added substrate,
cided
effect on the bacterial
BOD.
The presence of Chlorella decreased
the
bacterial
BOD
of glucose
by about
50 percent.
This effect could be in
to
indicate
that the algae
terpreted
absorbed some of the glucose from solu
tion, but the effect of this absorbed
on the apparent
BOD
of the
glucose
amount
not
as
as
great
of glucose
an
absorbed
equivalent
by sewage
bacteria.
Comparative
Growth
Studies
with
and Microcystis
Rate
Chlorella
Studies
Studies
have been carried out com
the rate of growth of Chlorella
paring
pyrenoidosa
(Wis. 2005) and the blue
bloom
green
producing
alga, Micro
cystis aeruginosa
(Wis. 1036) when
these two algae are cultured
in Gor
ham's
medium.
The rate of growth of the algae was
followed
of
by counts of the number
as follows:
calculated
and
cells/ml
at ?_2\
{ Log cell number at <_J/
of time were
Two different
periods
used to compare
the rate of growth:
3 to 5 days after inoculation
(increase
in cell counts
3 mil
approximately
to 10 mil
and 5
cells/ml
cells/ml)
to 12 days after inoculation
(increase
in cell counts
10 mil
approximately
to
28
mil
It was
cells/ml
cells/ml).
found in preliminary
that
experiments
these two periods generally
had differ
was
algae
/
.
~
(U ^
w.
i
(m days)"
ent rates of growth and could be used
to compare
the algae under different
conditions.
physiological
In two experiments
with
aerated
Gorham's medium
(pH 9) the growth
rates for Chlorella were 0.22 and 0.25
for the younger
cultures and 0.10 and
0.09 for the older cultures.
In the
same experiments,
the growth rates for
were 0.22 and 0.19 for the
Microcystis
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1538
JOURNAL WPCF
of Age
of the Effect
IV.-Summary
and Microcystis
of Chlorella
Capacity
TABLE
(Averages
of at Least
December
pH on the BOD
in Gorham's
Grown
and
16 Tests
and Photosynthetic
Medium
per Condition)
Chlorella
Ages
(days)
Medium
Gorham's
pH
7
Gorham's
pH
pH
7
9
pH
9
Gorham's
Gorham's
3-8
19-24
0.17
0.15
3-8
19-24
0.16
0.12
Microcystis
Photosynthetic
Capacityf
BOD*
1964
Photosynthetic
Capacity!
BOD*
0.15
0.16
0.085
0.11
0.073
0.093
0.075
0.13
0.16
0.042
0.083
0.11
*A
DO/mg SS/5 days.
f A DO/mg SS/2 hr, 350 ft-c (3,770 lumens/sq m) light.
cultures and 0.09 for the older
younger
cultures.
When
medium
Gorham's
was gassed with
C02 in
1.25-percent
of
air
(pH 7), the rates of growth
in young
and Microcystis
Chlorella
cultures
were
tively, while
old the rates
0.20,
0.33
and
0.24,
respec
5- to 7-days
in cultures
of growth were 0.15, and
respectively.
the
of
Measurements
suspended
solids in the cultures during the period
of 3- to 5-days' growth from the time
of inoculation
that the sus
showed
solids increased
from approxi
pended
40 to 110 mg/1.
the
mately
During
in the pH 7
of 5 to 7 days
period
medium
the suspended
solids increased
in
110 to 240 mg/1,
from
whereas
the pH 9 medium
the suspended
solids
increase for the 5- to 12-days' period
was only 110 to 300 mg/1.
Effects of pH and Age
A series of tests was carried out in
and Micro
which cultures of Chlorella
ages from Gorham's
cystis of different
medium were used in the BOD test (at
least 16 samples per condition).
The
cultures were either aerated and were
or
alkaline
8.9 to 10.6)
(pH range
were gassed with
in
C02
1.25-percent
air and were neutral
(pH range 6.5
to 7.8).
A summary
of the data from BOD
tests and tests of the photosynthetic
in
incubation
after five-days'
capacity
in Table IV.
the dark is presented
The activity
of Chlorella
decreases
the age of the cultures and this
with
in activity with age is more
decrease
with
pronounced
algae from the pH
as compared
9 medium
to algae from
the pH 7 medium.
In contrast,
it was
found with Microcystis
whereas
that,
the activity
of the algae in the BOD
test and the photosynthetic
capacity
of the algae decreased
with
the age
ca
of the cultures,
the photosynthetic
more
from
of
the
pacity
Microcystis
was much
9, medium
alkaline,
pH
than
that
from
the
neutral
greater
medium.
Effect
of Mixing
cystis
Since
to
show
Chlorella
some workers
and Micro
have
between
antagonism
been
able
various
species of algae
(15) (16), a series of
16 tests was carried out in which dif
ferent relative
concentrations
of Chlo
rella from Allen's medium
and Micro
cystis from Gorham's
(pH 9) medium
were used separately
and in combina
tions in the BOD and photosynthetic
tests.
capacity
From the activity
of the algae when
in these tests, cal
measured
separately
were made
ac
culations
of expected
rates
of
the
various
tivity
mixtures,
depending
on
the
relative
concen
tration
of each alga.
The results
of
16 BOD
tests of mixtures
of these
an average A DO/mg
algae indicated
SS of 0.10, the range being 0.069 to
the range of ac
0.13, or well within
measured
for
the algae
sepa
tivity
The results
of the photosyn
rately.
thetic capacity
in
tests, after 5-days'
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BOD MEASUREMENTS 1539
12
Vol. 36, No.
in the dark, separately
cubation
and
that the meas
indicated
in mixtures
were
99
ured rates for the mixtures
rates
for
of
theoretical
to 108 percent
such
calculated
mixtures,
when
measured
algae
wrere
from
rates
tested
sepa
one can say that the
Thus,
rately.
and Microcystis,
of Chorella
activities
in these tests, were addi
when mixed
tive and not antagonistic.
BOD
Long-Term
Studies
of oxygen used
The
(BOD)
of either
and photosynthetic
capacity
or Microcystis
in seeded BOD
Chlorella
at
have been followed
water
dilution
for as long as
1- to 3-day
intervals
run with
tests were
28 days.
Four
and
media
different
from
3
Chlorella
from Gorham's
3 tests with Microcystis
In all tests with
(pH 9) medium.
de
either alga, there was a gradual
crease in DO in the BOD bottles until
zero DO was reached
(7- to 12-days'
ca
The photosynthetic
incubation).
was
measured
of
the
up
algae
pacity
to and after
the time the bottles be
amount
came
anaerobic.
It was found
that, in the samples
ca
the photosynthetic
wTith Chlorella,
decreased
pacity of the algae gradually
after the bottles became anaerobic, but
as
wTas still measureable
for periods
as
anaerobic
under
long
eight days
conditions.
capacity of
Photosynthetic
from 0.18 to 0.078A
Chlorella decreased
SS during 8 days under anae
DO/mg
in a typical
robic conditions
experi
ment.
In the tests with Microcystis,
the photosynthetic
capacity of the alga
was maintained
at a higher rate for a
longer period of time. Photosynthetic
from
decreased
capacity of Microcystis
19
0.12 to 0.079A DO/mg
SS during
in one
days under anaerobic conditions
experiment.
BOD
of Other
Algae
The effects of three other algal spe
cies on the BOD test have been studied.
was
The first, Scenedesmus
dimorpha,
isolated from a sewage oxidation
pond
at Lancaster,
and was
California,
for study as a representative
of the types of green
algae
species
found in such installations.
It was found that Scenedesmus
could
in Allen's medium
be cultured
readily
The average effect
for the BOD tests.
on the BOD test in seeded dilution wa
ter of 37 tests indicated
it had an ac
SS and
tivity factor of 0.13A DO/mg
an average
capacity
photosynthetic
in the dark of 0.13A
after
5 days
SS.
DO/mg
A few tests (a total of eight) were
run with
the blue-green
alga, Nostoc
muscorum
This
alga is
(Wis. 1013).
well known
studies
of
for the many
to fix atmospheric
its ability
nitrogen
factor
(17) (18). The average activity
in the BOD test of this alga was 0.093A
SS and the average photosyn
DO/mg
in the
after
5 days
thetic
capacity
dark was 0.081A DO/mg
SS.
chosen
The
echinulata
Gloeotrichia
alga,
(Wis. 1052), was chosen as a repre
sentative
of the blue-green
algae that
cause obnoxious
in lakes and
blooms
are also nitrogen fixers.
This alga was
in three different media, Gor
cultured
minus
the nitrogen
ham's, Gorham's
source,
and
Allen's.
The
average
ac
in the BOD
test were:
tivity factors
from Gorham's
medium
(16 tests),
0.091A DO/mg
from Gorham's
SS;
minus nitrogen
(6 tests), 0.13A DO/mg
(6 tests), 0.048A
SS; and from Allen's
ca
SS.
The
photosynthetic
DO/mg
of these algae after 5 days in
pacity
to be
the dark was generally
found
small for the majority
of the samples
tested.
As a result of this observation
of
the low photosynthetic
of
capacity
a
series
of
BOD
Gloeotrichia,
daily
and photosynthetic
tests was
capacity
In the first two tests ?.n
carried out.
water
seeded dilution
of Gloeotrichia
in Gorham's
cultured
it was
medium,
noted that the algae ceased to evolve
on the second or third day
oxygen
when exposed to light. Within
48 hr
of the time when
the algae ceased to
a sudden
there was
photosynthesize,
in the rate at which
increase
oxygen
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1540
JOURNAL WPCF
was
as compared
to the
consumed
rate of the first 3 to 4 days and to the
rate of oxygen
in Chlorella
consumed
tests run at the same time.
A third series of tests with Gloeo
trichia from Gorham's
(pH 9) medium
was carried out with
seeded dilution
seeded Gorham's
and
water,
medium,
raw lake water
as the BOD
test me
dium.
The
in
results
of the tests
seeded dilution water and seeded Gor
are plotted
ham's medium
in Figure
7. The results of the tests in raw lake
were
water
similar
to these results
the rate of oxygen
used was
except
factors for di
greater; BOD
activity
lution water, Gorham's
and
medium,
o
December 1964
lake water were 0.14, 0.15, and 0.59A
days, respectively.
DO/mg
SS/5
From
the data of Figure
7, Gloeo
trichia seems to die after three to four
of the BOD
days under the conditions
test (the point at which the algae cease
a situation
to photosynthesize),
that
had been found to be the
previously
case in studies of marine
diatoms
(8).
the organic matter
of
Subsequently,
the algae
becomes
avail
apparently
able to the bacteria
and an
present
increased BOD activity
results
(a cal
culation
of the projected
BOD of the
water
in dilution
the
algae
during
period, 5 to 10 days, would give a BOD
rate of about 0.38A DO/mg
SS).
6
3
Time
4
(Days)
FIGURE
utilization by Gloeotrichia (10 mg/1)
in seeded
7.?Oxygen
dilution water or seeded Gorham's medium [Light: 2 hr, 350 ft-c (3,770
lumen/sq
m)].
Solid
line,
dark
only;
dotted
line,
dark
plus
2-hr
light.
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Vol. 36, No.
12
BOD MEASUREMENTS 1541
Summary
and Conclusions
1. The rate of growth
of Chlorella
in different
media
has been followed
in the culture
of
by measurements
volatile
solids,
suspended
solids, COD,
cell counts, and packed volume.
2. It was found
that DO could be
determined
readily using the Winkler
in solutions
method
Chlo
containing
at least as high
rella in concentrations
as 5 X 106 cells per ml.
in seeded
3. The activity
of Chorella
dilution water in the BOD test appears
to be independent
of the volume
of
seed added
(in the range of 3 to 30
ml seed/1).
4. There appeared
to be no differ
ence in the activity
in the
of Chlorella
the original
culture
BOD test whether
or
was
bacterial-free
contaminated
with
sewage bacteria
(one to five ml
the addition
of
However,
sewage/1).
to cultures
caused a five-fold
sewage
loss in the activity
due to the super
natant
cultures
when
of the algal
tested in BOD
tests.
or freeze
5. The BOD of autoclaved
dried
cells were
Chlorella
approxi
four fold that of living cells.
mately
6. No
consistent
could
relationship
be obtained
between
the amount
of
in the BOD
oxygen used by Chlorella
test and the number
of viable
cells
measured
and plating
of
by dilution
incubation.
samples after five-days'
7. The photosynthetic
of
capacity
the algae before and after incubation
in the BOD
test indicated
the physio
logical state of the algae. Tests showed
that this measurement
could be used
to indicate
if algae were
still active
after different
periods of dark incuba
tion and the relative activity
of algae
from different
cultures.
8. Tests
of the BOD
of Chlorella
under seeded and bacterial-free
condi
the effect of algae from
tions indicated
were merely
medium
Allen's
additive
to the respiration
of bacteria, whereas
the more
dilute
dilution
algae from
water medium
have
caused a
might
to the respiration
of
slight stimulation
bacteria
present.
9. Daily
and long-term
of
studies
Chlorella
in the BOD test showed that
was quite
the rate of oxygen utilization
uniform until the BOD bottles became
anaerobic.
It was found that Chlorella
to produce
could continue
oxygen
by
for at least seven days
photosynthesis
anae
the BOD
became
bottles
after
robic.
in the
10. The activity
of Chlorella
BOD test was found to vary consider
ably, averages of activity ranging from
0.09- to 0.19-mg 02 used per mg sus
on
solids per liter, depending
pended
in which
the algae had
the medium
been
cultured.
11. The average and range of values
in the BOD
obtained
test with Chlo
rella from different
culture media are
in tabular form.
presented
12. It was found that the activity of
and Gorham's me
algae from Myers'
dia decreased
with
age of
increasing
the cultures.
13. Studies
of the effect of pH of
on
Gorham's
medium
the activity
in the BOD test indicated
of Chlorella
that algae from the more alkaline
(pH
a greater
exhibited
de
9) medium
crease in activity with
age
increasing
of the culture than algae from neutral
(pH 7.0 to 7.8) medium.
14. Chlorella
in autoclaved
cultured
a
sewage effluent exhibited
secondary
in
the
BOD
test
higher
activity
(an
of 0.27-mg O2 used per mg
average
than algae from the
suspended
solids)
culture media.
synthetic
algae
Also,
from neutral
pH cultures were more
than algae from C02-deficient
active
cultures
(pH 9).
15. The rate of oxygen utilization
by
in the BOD
Chlorella
test was found
to be independent
of any increase
in
bacterial
due to the pres
respiration
ence of fructose.
in the
However,
of glucose
in the BOD
presence
test,
the algae had a decided
effect on the
com
bacterial
BOD
of this organic
pound.
16. When
comparative
Chlorella
and Microcystis
tests with
were made
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1542
JOURNAL WPCF
were
it was found that the rate of growth
in the same medium
of these algae
(Gorham's
pH
9) was
remarkably
similar.
17. Both
exhibited
Chlorella
a decrease
and Microcystis
in activity
with
age of cultures.
However,
from Gorham's
pH 9 me
a higher photosynthetic
ca
increasing
Microcystis
dium had
after
pacity
5-days'
the same alga from
incubation
than
Gorham's
7
pH
medium.
18. When
were mixed
found
that
Chlorella
for
and Microcystis
test it was
the BOD
their
activities
separate
1964
December
additive
and not antago
merely
to each other.
nistic
was
19. Microcystis
to be
found
able to photosynthesize
after long pe
riods
of anaerobic
(up to 19 days)
or better
as well
conditions
than
Chlorella.
20. The effects of Scenedesmus
di
on the
and Nostoc muscorum
morpha
to those of
BOD
test were
similar
and Microcystis.
Chlorella
21. Tests with Gloeotrichia
echinu
lata indicated
that this alga appears
to die under
the conditions
of the
BOD
increased
test, thereby
causing
bacterial
respiration.
Acknowledgment
This research has been supported
by
a research grant
(K-15) from the Na
tional Institutes
of Health,
lic Health
Service.
U.
S. Pub
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