Efecto de Algas en Determinacion de Dbo - Fitzgerald

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
Stable URL: http://www.jstor.org/stable/25035197
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THE EFFECTOF ALGAEON
BOD MEASUREMENTS
George P. Fitzgerald
A study has been made to measure As soon as some of the preliminary

the BOD of various species of algae studies on the effect of algae on the
alone and in association with bacteria various measurements involved and the
under incubation conditions of 5-days' effects of mixing algae and bacteria
time in the dark and at a temperature were known, studies were made of the
of 20 ?C to determine if algae have an factors affecting the activity of the
effect on BOD measurements under the algae during the BOD tests. The vari
standard conditions. ous factors studied have been the
Initial studies have been carried out amount of algae, the source of algae
using the green alga, Chlorella pyre (i.e., the medium in which the algae
noidosa (Wis. 2005). This alga is es were cultured), and the age of the
pecially adaptable to various culture algal cultures at the time of use in
conditions and can be used as a basis the BOD test (i.e., the factors causing
for comparison with other algae having senility in algal cultures using two
more restricted culture conditions. media, Gorham's and secondary sewage
The culture procedures used to sup effluent).
ply Chlorella for the BOD tests are As an extension of the data collected
described and the growth rates of this using Chlorella as a test organism, fur
alga in four different media were meas ther studies were carried out compar
ured. Because there is little informa ing the activity of the blue-green,
tion in the literature on the measure bloom-forming alga, Microcystis aeru
ment of dissolved oxygen (DO) in the ginosa (Wis. 1036) to that of Chlo
presence of algae, tests were carried rella. Also, the activity of other green
out to observe if algae affected this and blue-green was measured:
algae
measurement. Another series of tests Scenedesmus Nostoc mus
dimorpha,
was made to determine if the algae
corum, and Gloeotrichia echinulata.
used in BOD tests under the standard
Studies on the influence of the dilu
conditions would survive the 5 days
tion medium in the BOD test have
of darkness in the presence of bacteria.
been made. Comparison of the BOD
The effect of adding sewage bacteria to
cultures of algae was also determined, of Chlorella in dilution water, algal
both on the activity of the algae and culture media, and in lake waters, as
the effect of the culture supernatant in well as seasonal changes in the influ
the BOD test. ence of lake waters are reported.
Materials and Methods

The Chlorella pyrenoidosa (Wis. culture and a volume of about 15 ml
2005) used was maintained as a pure was used to inoculate each experi
mental culture. A 2-1 Erlenmeyer
George P. Fitzgerald is associated with the
flask with 1,500 ml of medium was used
Hydraulic and Sanitary Lahoratones, College
as a culture vessel. A cotton
of Engineering, University of Wisconsin, plug sup
Madison, Wis. ported a 6-mm diam glass tube with a
1524
Vol. 36, No. 12 BOD MEASUREMENTS 1525
constricted end that was used for bub The composition of the four media
bling a 1.25-percent C02-air mixture used for culturing Chlorella are listed
through the culture. Each bubbler in Table I. The dilution water me
was attached by surgical rubber tub dium is BOD dilution water (1) plus
ing to a 25- X 200-mm glass tube con 30 mg/1 of nitrogen from NaN03. The
taining cotton as a filter. A 50-mm pH of this medium is near 7.4 at har
teflon-covered magnet was placed in vest time. Allen's medium (2) is also
each flask. The culture vessel, media, a neutral medium, the final pH being
magnet, and filter were sterilized by near 7.0. Myers' medium (3) is more
autoclaving. Inoculations were made acidic, the pH being near 6.5. Gor
with sterile syringes. Motors (1/100 ham's medium (4) is alkaline, with a
hp shaded pole motor, Dayton Elec. final pH of near 9.0.
Mfg. Co., Chicago, 111.) with magnets The algae to be used in an experi
attached to their shafts were mounted ment were centrifuged from their cul
vertically on wooden frames below the ture solution after their concentration
culture vessels and the speed of 15 wTas
measured, the supernatant was
magnetically stirred cultures was con discarded, the cells were washed with
trolled with one variable transformer. pH 7 P04-buffer, and recentrifuged.
The algae were cultured under continu The cells then were suspended in a
ous light [180 ft-c (1,940 lumen/sq convenient volume of PO^buffer and
m)] in a constant temperature (23 ? measured samples added to seeded di
1?C) culture room. lution water for mixing and filling of
TABLE I.?Media Used for Culture of Chlorella for BOD Experiments

A. Concentration of salts used in media (mg/1)
Salt Dilution Water Gorham's Allen's Myers'
NH4CI 1.7 50
NaN03 182.0 496 1,000
KN03 1,210
KH2PO4 8.5 1,230
K2HP04 21.75 39 250
Na2HP04-7H20 33.4
Fe Citrate
FeCls 0.15
Fe2(S04)3 52
MgS04-7H20 22.5 75 513 2,460
CaCl2 27.5 27 50
Na2Si03-9H20 58
Citric Acid 6
EDTA 1
Na Citrate 195
Na2COs 20
Total Salts 297.5 728 1,866 5,147
B. Concentration of essential ions in media (mg/1)
Ion Dilution Water Gorham's Allen's Myers'
N 30 82 178 168
P 10 7 45 295
Fe 0.05 1 1 15
Mg 2 7 50 243
Ca 10 10 18
S 3 10 67 323
K 12 17 112 353
1526
JOURNAL WPCF December 1964
BOD bottles. Duplicate bottles were The procedure used to measure the
used to determine the initial DO con amount of oxygen absorbed by algae
centrations and triplicate bottles were the 5-day BOD determinations
during
used for after incuba
analysis 5-days' was to measure the oxygen used by
tion at 20 ?C in a dark room. Bottles
seeded dilution water (24-hr settled
containing algae wrere covered with
sewage seed) and seeded dilution wa
black hoods as a further precaution to
ter plus algae. The difference in the
prevent light from entering during the
incubation Measurements were oxygen depletion was referred to as
period.
made as suggested in Standard Meth being caused by the algae and reported
ods (1). as A DO per mg algae.
Results
Growth of Chlorella in Different added, it was found that up to two
Media hour intervals between the addition of
the sulfuric acid or titration did not
Because there is no one growth meas
common to all algae labora affect the DO measured in the pres
urement
ence of Chlorella.
tories, growth measurements were
made on cultures as suspended solids, Effect of Seed Volume on
volatile solids, COD [dichromate
Activity of Algae
method with Ag2S04 catalyst (1)],
cell counts, and packed volume. The Experiments were carried out in
between these which the oxygen absorbed by different
general relationships
measurements of Chlorella cultures of volumes (3.0 to 30 ml/1) of 20- to 24-hr
10 of are settled sewage seed in dilution water
approximately days age
in Table II. was measured as well as the total
presented
amount of oxygen absorbed when a
Effect of Algae on Dissolved definite amount of Chlorella from a
Oxygen Measurement dilution water culture was added to
The effect of the presence of algae each seed volume in dilution water.
in measuring dissolved The results of these tests indicated
oxygen (DO)
that a variation of 10 fold in seed vol
by the Alsterberg (azide) modification
of the Winkler ume or 7 fold in oxygen absorption due
method (1) was de
termined.
to the seed had little or no effect on
It was found that concentrations of the amount of oxygen absorbed by the
Chlorella as high as 5 X 106 cells/ml added amount of algae.
did not appreciably affect the meas
urement was Effect of Contaminating Chlorella
of DO. The end-point
Cultures with Sewage on Subse
slightly obscured, but experience with
the measurement overcame this dis quent BOD Measurements
advantage. Once the manganous sul Settled sewage was added to cultures
fate and alkaline iodide reagents were of Chlorella (one to five ml sewage
TABLE IL?Relative Growth of Chlorella after 10 Days in Different Media
Culture ss vs COD Cell Count

Medium (mg/1) (mg/l) (mg/1) (cell/ml XI06)
Dilution water 125 100 175 12.5

Gorham's 175 200 250 5.0
Allen's 375 350 550 50.0
Myers' 375 350 550 50.0
per liter of culture) that previously had been autoclaved, and cells which
had been sampled for BOD measure had been freeze-dried.
ments. After about five additional Experiments were first carried out
days of incubation in the culture room to test the viability of Chlorella cells
under normal growth conditions the which had (a) no treatment, (b) had
cultures were again sampled for BOD been frozen only, and (c) which had
measurements. The effects of both been freeze-dried (3.6-percent final
washed algal cells and of the culture moisture content as compared to 83
supernatants were measured in seeded percent moisture in fresh cells). Tests
dilution water tests. indicated that cells suspended in either
A total of 45 tests was carried out distilled water or autoclaved 8-wk old
on algae from dilution water, Allen's culture supernatant, and then frozen
and Myers' media, that had been in for the same period of time required
cubated with sewage. It was found in the freeze-drying process (1 to 3
that the average activity of the algae days), had a viability of about 20 per
from contaminated cultures was within cent as compared to unfrozen controls.
the experimental limits for that me No cells were
viable in freeze-dried
dium as compared with algae from samples of up
to 10,000 cells but, in
pure cultures. From these data it was one experiment, 4 algae cells out of
concluded that algae from either pure 500,000 cells survived the freeze-drying
or contaminated cultures would have treatment. Therefore, it may be con
essentially the same effect on the BOD cluded that freeze-dried Chlorella cells
measurement. for these tests are, by and large, non
There a very different
was effect of viable, but may still be considered to
contaminating Chlorella cultures with be chemically intact much the same
sewage on the results in BOD measure as live cells, as compared to cells which
ments produced by the supernatants have been killed by autoclaving.
from the cultures. It was found in The results of 8 BOD tests each with
12 tests with cultures of Chlorella from fresh, freeze-dried, and autoclaved
'
dilution water, Allen's and Myers me Chlorella cells indicated that the aver
dia, that the effect of contamination age activity factors (A DO/mg SS)
with the sewage caused a decrease in were 0.13, 0.44, and 0.48, respectively.
the BOD of the supernatant to the Thus, it has been confirmed that dead
extent that the BOD was 1/5 or less algal cells exert higher BOD's than
after incubation with sewage for 5 live cells, and the BOD of cells killed
days as compared with the supernatant by freeze-drying are nearly as great
of the cultures prior to contamination. as the BOD of cells killed by auto
This is to be expected, of course, be claving.
cause the organic matter accumulated
in algal culture supernatants had been Comparison of Viability and
found to increase the activity of sew Photosynthetic Capacity
age seed and, thus, was further proof tests of the mixture of al
Viability
that some of this material could be dilution water
gae and seeded BOD
broken down by sewage organisms. before and after incubation were made
by diluting samples of the mixture
Comparison of Live and Dead Algae buffer
using sterile, pH 7.2 phosphate
The literature states (5) (6) (7) (8) (1) and plating in petri dishes with
that deadalgae had considerably tryptone glucose extract agar. The
higher oxygen demands than live algae. viable algal cells forming colonies un
In order to check this a series of BOD der these conditions were counted after
tests was made with live algae from incubation in the constant-light culture
Allen's medium, similar cells which room for at least 10 days. The algal
1528
cell counts in some plates were less the two-hr period. The average rate
than in duplicate plates or algal colo of 35 tests of algae from Allen's me
nies were smaller in some duplicate dium measured at the start of the
plates, inhibition resulting because of BOD tests was 0.093A DO/mg SS,
the presence of certain types of bac while it was 0.083A DO/mg SS when
teria. tested after 5-days' incubation, a de
The results of viability tests com crease of 11 percent. The average of
paring the number of cells viable at these two rates of oxygen production,
the time BOD tests were started (zero 0.088A DO/mg SS, for a 2-hr period
time) with the number of viable cells of light exposure is equivalent to 5.3
found in BOD bottles after 5-days' mg of oxygen production/1 for a 5-day
incubation in the dark indicated that period, or a considerably higher rate
88 percent of the cells from Allen's than the average respiratory rate for
medium (17 tests) and 76 percent of algae from this source (0.10A DO/mg
cells from dilution water medium (34 SS). (See also reference 6.)
tests) were alive after the incubation The photosynthetic capacity of algae
period. However, in comparing data from dilution water medium (32 tests)
of individual experiments it was found had an average rate of 0.056A DO/mg
that the amount of oxygen used per mg SS at zero time and 0.024A DO/mg SS
of suspended solids (A DO/mg SS) after 5-days' incubation, a decrease, in
was not necessarily related to the re 5-days' time, in photosynthetic ca
sults of viability tests; essentially the pacity of 57 percent. Therefore, it
same A DO/mg SS was obtained from would seem that the higher activity
experiments which had 100-percent re in the BOD test of algae from dilution
covery of cells as from experiments in water medium may be due to the
which there was only 52-percent re death of some cells from dilution water
covery (dilution water algae). medium and the resulting stimulation
The photosynthetic capacity of the of bacterial respiration.
algae-seed mixtures was measured at
zero time and after 5 days' incubation Comparison of Algal Activity Rates
DO analyses Under Bacterial-Free Conditions
by comparing duplicate
of samples held in the dark with The procedure used to measure the
samples exposed to 350 ft-c (3,770 lu amount of oxygen that algae would use
men/sq m) of light for 2 hr just before in five days, in the absence of bacteria,
analysis. The amount of oxygen that was to add algae from pure cultures to
the algae produced in the light in ex sterile BOD dilution water and siphon
cess of that used in the dark was con the mixture to sterile BOD bottles.
trolled by the physical conditions of At the same time as these tests were
the medium at the time of analysis, tests were also run in
run, duplicate
such as C02 content of the water in seeded dilution water using identical
the BOD bottles, as well as the physio
samples of algae.
logical state of the algae. However, The results of the bacterial-free and
since this test was used to compare seeded BOD tests indicated that 12
algae from different sources but in more was
percent oxygen consumed
the same medium after the same in
per mg suspended solids in seeded
cubation times, it was considered to BOD tests than in bacterial-free BOD
be a reliable comparative test for the tests with algae from Allen's medium
activity of the different algae at that and 34 percent more with
(8 tests),
particular time.
algae from the dilution water medium
The amount or rate of oxygen pro (12 tests). The average BOD of algae
duced was expressed as the change in from Allen's medium (bacterial-free)
oxygen per mg suspended solids for was 0.12 as compared to 0.10A
DO/mg
SS for the average for all seeded BOD bacterial-free conditions indicated that
tests with algae from Allen's medium. there was little difference in the photo
However, the average BOD of dilution synthetic activity of algae from Allen 's
water algae in the bacterial-free tests medium at zero time (0.093 seeded vs.
was 0.13 as compared to 0.19A DO/mg 0.095 sterile A DO/mg SS). However,
SS for all seeded tests. The similarity in the case of algae from the dilution
of the activity of these algae under water medium, the average zero time
bacterial-free conditions (Allen's, 0.12; photosynthetic capacity increased from
and dilution water, 0.13 A DO/mg SS) 0.056A DO/mg SS for the seeded tests
should be noted. to 0.071A DO/mg SS for bacterial-free
Comparisons of measurements of the tests. The average photosynthetic ca
photosynthetic capacity of the algae pacity of all seeded tests after 5-days?
from the two media under seeded and incubation for dilution water algae
Seed only
Seed and Algae (13 mg/1)

_ 6h
Seed and Algae (27 mg/1)
Time (Days)
FIGURE 1.?Oxygen utilization by seed and 13 mg/1 Chlorella and

27 mg/1 Chlorella in seeded dilution water.
1530 JOURNAL WPCF December 1964
Algae (Dark plus 2-hr

light before analysis)
Algae (Initial 2-hr light)
Algae( Dark Only)
Time (Days)
FIGURE 2.?Oxygen utilization by Chlorella (13 mg/1) from Allen's

medium in seeded dilution water [Light: 2 hr, 350 ft-c (3,770 lumen/
sqm)].
(32 tests) was 0.024A DO/mg SS. In measurements except that duplicate
contrast, the photosynthetic capacity samples were analyzed at the times re
of algae from this medium under con ported.
ditions of sterile incubation for 5 days Figure 1 presents data from an ex
was 0.054A DO/mg SS, or twice the periment showing the daily change in
rate of tests run in seeded BOD bottles. dissolved oxygen in BOD bottles of
seeded dilution water, seed plus 13
Daily and Long-Term BOD Studies and seed plus 27 mg/1
mg/1 algae,
A series of BOD tests has been run algae from Allen's medium. These
to establish the rate of oxygen used data indicate that oxygen utilization
by algae on a daily basis and for long by the algae is fairly uniform over the
periods of time, up to 16 days. The 5-day period in the dark.
tests were set up as for normal BOD Figure 2 presents data obtained
when algae from Allen's medium was the rate of oxygen utilization from
added to seeded dilution water (13 mg samples not exposed to an initial light
algae/1) and some bottles were either treatment (there was an average of
increase in of six
kept in the dark until analyzed (in five-percent activity
exposed to 2 hr of 350 ft-c samples exposed to light as compared
duplicate),
(3,770 lumens/sq m) of light and then to samples without light treatment).
kept in the dark, or exposed to 2 hr The data showing the level to which
of 350 ft-c m) of the DO concentrations are raised in
(3,770 lumens/sq
light just before analysis after incuba samples exposed to light just before
tion in the dark. The initial DO con analysis on a daily basis (Figure 2)
centrations of samples can be increased indicate that for the 5-day period with
by exposure of the samples with algae algae from Allen's medium there is
to light with little significant change in little change in the photosynthetic ca
6X Seed Only
Algae (Allen's Medium)
Algae (Dilution Water Medium)
Time (Days)
FIGURE 3.?Oxygen utilization by 6X seed, Chlorella (14 mg/1) from

Allen's medium, and Chlorella (16 mg/1) from dilution water medium in
seeded dilution water.
Algae (Dark plus 2hr

light before analysis )
Algae (Dark Only)
Time (Days)
FIGURE 4.?Oxygen utilization by Chlorella (28 mg/1) from Allen's

medium in seeded dilution water [Light: 2 hr, 350 ft-c (3,770 lumen/
sqm)].
pacity of these algae (average oxygen tests) and the rate of oxygen utiliza
production of 35 samples at zero time tion by algae from Allen's medium (14
was 0.093A DO/mg SS and 0.083A mg/1) as compared with that of algae
DO/mg SS for 5-day samples). from dilution water medium (16
BOD tests were also run for as long mg/1).
as 16 days to determine the rate at Figure 4 presents the rate at which
which oxygen was consumed by algae oxygen was used by 28 mg/1 of algae
over periods of time in excess of the from Allen's medium over a 16-day
usual 5-days' time used in BOD tests. period, as well as the amount of oxy
Figure 3 shows the rate at which oxy gen produced by exposure of samples
gen was consumed by sewage seed (at to 350 ft-c (3,770 lumen/sq m) of light
6 times the amount used to seed the for 2 hr prior to analysis. It can be
dilution water used with the algae seen that the BOD bottles became
anaerobic (DO exhausted) in about 9 from the dilution water medium over
'
days' time. However, the algae pres a period of 5-days time.
ent continued to produce oxygen when A re-evaluation of the effect of the
exposed to light, but at a decreasing different culture media on the activity
rate. Viability tests of samples from of Chlorella in the BOD test was car
these bottles at zero, 5, 12, 14, and 16 ried out by comparing the apparent
days indicated no loss in viability respiration of the algae over the 5-day
(average percent viable cells were 100, period with the photosynthetic capac
94, 120, 123, and 115, respectively). ity of the algae as measured after 5
Thus, these data confirm the results of days' incubation in the dark. The
Golueke et al. (9) who found Chlorella results are presented in Table III.
and Scenedesmus to be relatively re These data indicate that algae grown
sistant to anaerobic digestion and of in the three media, Myers ',Allen's, and
Finkle et al. (10) who found Chlorella Gorham's, have about the same photo
to be viable after 70 days in the dark. synthetic capacity after 5-days' incu
bation in the dark, but there is about
The Effect of the Culture Medium a 50-percent increase in the activity of
the algae in the BOD test. The algae
Preliminary experiments indicated
from the dilution water medium have
that the activity of Chlorella in the
the highest activity in the BOD test,
BOD test could range from 0.06 to
but demonstrate the lowest activity in
0.31 mg 02 used per mg suspended the photosynthetic capacity test.
solids per 1 (A DO/mg SS), depend
ing on the medium in which the The Effect of the Age of the
algae had been cultured. The distress Culture on Algal Activity
ing observation from these data was
that the apparent of the algae In order to measure the effect the
activity
from the different media to age of a culture had on the activity of
appeared
be inversely correlated to the potential algae in the BOD test, algae were used
from cultures considered young or old,
growth rate of Chlorella in the media;
that is, algae from the two more con depending on the growth rate of
centrated media, Myers' and Allen's, the algae in the particular medium. A
showed less activity in the BOD test comparison was made of these two
than algae from the very dilute dilu "ages" of cultures for dilution water,
tion water medium. Other data, such Allen's, Myers', and Gorham's (air for
as the results of bacterial-free BOD aeration) media. The results of these
indicated that perhaps tests are summarized in Figure 5 as
tests, however,
the measured BOD of algae from the the amount of oxygen used by the
dilution water medium was really a algae per mg of suspended solids (A
combination of algal respiration and DO/mg SS) for the different ages of
stimulated bacterial the cultures. The circles indicate the
respiration.
Because cell counts and viability average activity factor for each age
tests by the dilution of the culture media tested and the
technique gave
little correlation to other data, meas heavy lines running through the cir
cles indicate the range of ages used.
urements of the photosynthetic capac
Light lines are used to make the lines
ity of algae from Allen's and dilution
for each medium continuous. The
water media at the start and end of
number of samples used to determine
BOD tests were made. These data
the average value is indicated by the
indicated that the photosynthetic ca
figure in parentheses at each dot. The
pacity of algae from Allen's medium ranges of pH of the media are also
decreased 11 percent whereas there The pH values presented
presented.
was a decrease of 57 percent with algae are for the cultures at the time of har
TABLE III.?The Effect of the Culture Medium on the Activity of Chlorella

in the BOD Test
BOD* Photosynthetic Capacity!

Culture No. of
Medium pH Tests
Range Avg. Range Avg.
Myers' 6.5 23 0.069-0.12 0.090 0.087-0.18 0.14

Allen's 32 0.065-0.22 0.11 0.030-0.26 0.13
Gorham's 34 0.044-0.23 0.14 0.031-0.22 0.13
Dil. water 14 0.12 -0.31 0.20 0.014-0.039 0.027
*A
DO/mg SS/5 days.
f A DO/mg SS/2 hr, 350 ft-c (3,770 lumens/sq m) light,
vesting. There was little change in mg of cell material as the age of the
the pH of the cultures throughout the culture increases within the limits
growth period. tested. However, in the case of algae
It is evident that the average algal cultured in the other two media there
activity factor for young and old cul was a definite loss in activity in older
tures of dilution water (0.18 and 0.18A cultures as compared with young cul
DO/mg SS) and Allen's medium (0.11 tures. The average value for young
and 0.12A DO/mg SS) indicate com and old cultures was 0.094 and 0.063A
paratively little change in activity per DO/mg SS for algae from Myers' me
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).
dium and 0.16 and 0.12A DO/mg SS cultures (0.15 vs. 0.17AV DO/mg SS).
for algae from Gorham's medium. When expressed as a comparison of
Inasmuch as the two media (dilu cultures of similar ages, at different
tion water and Allen's) that had the pH values, there was only a 4-percent
least decrease in activity with age of difference between young cultures at
the culture were approximately neu pH 7.0 to 7.8 and pH 8.7 to 9.1;
tral in pH value, it might be possible whereas, algae from old cultures of
that pH values further removed from pH 7.0 to 7.8 were 18 percent more
the neutral value may be the principal active than algae from similar cultures
cause of the senility of the cultures. of pH 8.7 to 9.1.
Thus, it is indicated that the pH of
Effect of the pH of Gorham's the culture medium will affect the ac
Medium on Algal Activity tivity of the algae, the effect being
more pronounced as the age of the
Because the data of the previous sec
culture increases. However, this effect
tion had pointed out that there might
may not be the result of the difference
be a relation between the pH of the
in pH but may have been caused by
culture medium and the activity of the
other factors accompanying these pH
algae, further studies were carried out
changes, such as a lack of dissolved
using Gorham's medium.
CO2 at pH 9, or a change in the
It had been found that the range in
pH values for cultures in Gorham 's
nitrogen utilization at the different pH
medium was 8.7 to 9.1 when these cul
values.
tures were aerated with air, and pH
7.0 to 7.8 when aerated with the 1.25 Studies with Chlorella Grown in
percent C02-air mixture. Therefore, Secondary Sewage Effluent
aside from changes in the soluble min Because the data obtained from stud
eral composition because of changes in ies with the previously discussed syn
pH, this medium could be used to thetic media indicated that differences
study the effect of near neutral and in the activity of the algae could be
alkaline pH values on the activity of caused by variations in the medium,
Chlorella. studies were made on Chlorella grown
A series of 34 tests was made with in different variations of autoclaved
algae from the alkaline Gorham's me secondary sewage effluent. The gen
dium (aerated with air), 15 tests using eral composition of this effluent was
cultures 3 to 8 days old and 19 tests approximately 20 mg NH3-N/l, 7 mg
with cultures 19 to 23 days old. A P04-P/l, 950 mg total solids/l, 380 mg
second series of 34 tests was made methyl orange alkalinity (as CaCO3)
with the neutral Gorham 's medium /1, 10 to 30 mg BOD/l, and a pH of
(aerated with a 1.25-pereent C02-air 7.8.
mixture), 18 tests using cultures 3 to Autoclaved Madison, Wisconsin, sec
7 days old and 16 tests using cultures ondary sewage effluent, referred to as
16 to 23 days old. The average values sewage effluent or S.E., was used as
obtained for each medium and range in the culture medium for Chlorella in
ages are summarized in Figure 6 as the same manner as the synthetic me
the amount of oxygen used per mg of dia. The cultures were aerated with
suspended solids (A DO/mg SS). a 1.25-pereent C02-air mixture at least
There was a loss of 24 percent in until growth was well established, usu
the activity of algae from older cul ally 2 days. The cultures continuously
tures at pH 8.7 to 9.1 as compared to aerated with the 1.25-pereent CO2-air
younger cultures (0.12 vs. 0.16A DO/ mixture maintained a pH of 7.3 to 7.8
mg SS), whereas algae from cultures (original pH of sewage effluent was
at pH 7.0 to 7.8 had a difference of 7.8, while the pH of autoclaved effluent
only 11 percent between old and young was about 9). Cultures which were
153(; JOURNAL WPCF December 1964
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 12 16 20 24
Age (Days)
FIGURE 6.-Effect of pH of Gorham's medium on activity of Chorella.
aerated with only air after the initial cultured under conditions of C02-lim
period of growth had a range of pH ited growth (pH 9) as compared to
from 8.8 to 9.4. At these high pH sufficient CO2 (pH 7.5). The average
values considerable salts could be pre activity factor, A DO/mg SS, for all
cipitated (40 to 90 mg/l of suspended sewage effluent cultures with added
solids and about 75 percent of soluble CO2 was 0.29 (41 samples), whereas
P04-P) (11) (12) ; also, there was it was 0.27 (41 samples) for algae
little or no dissolved CO2 present. The grown under C 02 deficiency and a
amount of suspended solids formed higher pH (ratio of 1.1). The aver
when sewage effluent was autoclaved age factor A DO/109 cells, was 1.5 for
and then aerated for six days with pH 7.5 cultures and 1.6 for pH 9 cul
the 1.25-percent C02-air mixture was tures (ratio of 0.93).
about 40 mg/l, whereas about 65 mg/l These results with algae cultured in
were formed when only air was used. modifications of sewage effluent have
Corrections for these amounts of in indicated, therefore, that, as in the
organic suspended solids were made case of the synthetic media tested, al
in calculating the supended solids of gae grown under conditions of suffi
the algal cultures. cient CO2 (pH 7 to 7.5) were more
The tests run with the algae grown active per mg of suspended solids in
in sewage effluent were designed to in the BOD test than algae from C02
dicate whether the activity of algae deficient cultures (pH 9) . Other tests
in the BOD test was different when indicated that this difference is ap
parently enhanced by increased age five tests with fructose as an added

of the cultures but does not appear substrate have indicated that the pres
to be related to the supply of nitrogen ence of Chlorella had no effect on the
as NH3-N, or to the presence or ab bacterial BOD of fructose (less than
sence of salts which might be removed 10-percent variation with controls).
from solution at the higher pH value. Also, stimulation of bacterial respira
tion by the presence of fructose had
Effect of Glucose and Fructose on no effect on the BOD of Chlorella (less
BOD of Chlorella than 5-percent variation). In effect,
the BOD of Chlorella in the presence
The data reported thus far have
of fructose was merely additive to the
dealt with the BOD of Chlorella when
BOD of fructose. In contrast, in the
measured in an inorganic medium with
studies in which glucose was used as an
the addition of minor amounts of dis
added substrate, the algae had a de
solved or suspended organic matter in
cided effect on the bacterial BOD.
the form of settled sewage (the BOD of
The presence of Chlorella decreased the
seeded dilution water being about 0.5
bacterial BOD of glucose by about
mg/1). In order to demonstrate the
50 percent. This effect could be in
complexity of the relationship between
terpreted to indicate that the algae
the BOD of algae in such an environ
absorbed some of the glucose from solu
ment to the BOD of algae in an en
vironment mate tion, but the effect of this absorbed
containing organic
glucose on the apparent BOD of the
rials in appreciable quantities, a series
algae was not as great as an
of five tests was carried out using glu equivalent
cose and fructose. amount of glucose absorbed by sewage

These two organic
bacteria.
compounds represent readily available
compounds to bacteria while being se
Comparative Studies with Chlorella
lectively available to Chlorella. Glu
and Microcystis
cose is known to be an available source
of energy Growth Rate Studies
(13) (14), whereas fructose
has been shown to be relatively un Studies have been carried out com
available to certain species of algae, paring the rate of growth of Chlorella
including those used in these tests pyrenoidosa (Wis. 2005) and the blue
(14). green bloom producing alga, Micro
These tests were carried out in the cystis aeruginosa (Wis. 1036) when
normal manner of testing seeded dilu these two algae are cultured in Gor
tion water with or without algae and ham's medium.
seeded dilution water containing 4.3 The rate of growth of the algae was
mg of glucose or fructose per liter with followed by counts of the number of
or without algae. The results of the cells/ml and calculated as follows:
__ / Log cell number at ?_2\ . w. i

K = / ~
(U ^ (m days)"
{ Log cell number at <_J/
Two different periods of time were ent rates of growth and could be used
used to compare the rate of growth: to compare the algae under different
3 to 5 days after inoculation (increase physiological conditions.
in cell counts approximately 3 mil In two experiments with aerated
cells/ml to 10 mil cells/ml) and 5 Gorham's medium (pH 9) the growth
to 12 days after inoculation (increase rates for Chlorella were 0.22 and 0.25
in cell counts approximately 10 mil for the younger cultures and 0.10 and
cells/ml to 28 mil cells/ml). It was 0.09 for the older cultures. In the
found in preliminary experiments that same experiments, the growth rates for
these two periods generally had differ Microcystis were 0.22 and 0.19 for the
TABLE IV.-Summary of the Effect of Age and pH on the BOD and Photosynthetic
Capacity of Chlorella and Microcystis Grown in Gorham's Medium
(Averages of at Least 16 Tests per Condition)
Chlorella Microcystis
Medium Ages
(days)
BOD* Photosynthetic BOD* Photosynthetic
Capacityf Capacity!
Gorham's pH 7 3-8 0.17 0.15 0.16 0.085

Gorham's pH 7 19-24 0.15 0.11 0.075 0.073
Gorham's pH 9 3-8 0.16 0.093 0.13 0.16
Gorham's pH 9 19-24 0.12 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.
younger cultures and 0.09 for the older decrease in activity with age is more
cultures. When Gorham's medium pronounced with algae from the pH
was gassed with 1.25-percent C02 in 9 medium as compared to algae from
air (pH 7), the rates of growth of the pH 7 medium. In contrast, it was
Chlorella and Microcystis in young found with Microcystis that, whereas
cultures were 0.33 and 0.24, respec the activity of the algae in the BOD
tively, while in cultures 5- to 7-days test and the photosynthetic capacity
old the rates of growth were 0.15, and of the algae decreased with the age
0.20, respectively. of the cultures, the photosynthetic ca
Measurements of the suspended pacity of Microcystis from the more
solids in the cultures during the period alkaline, pH 9, medium was much
of 3- to 5-days' growth from the time greater than that from the neutral
of inoculation showed that the sus medium.
pended solids increased from approxi

40 to 110 mg/1. the Effect of Mixing Chlorella and Micro
mately During
of 5 to 7 days in the pH 7 cystis
period
medium the suspended solids increased Since some workers have been able
from 110 to 240 mg/1, whereas in to show antagonism between various
the pH 9 medium the suspended solids species of algae (15) (16), a series of
increase for the 5- to 12-days' period 16 tests was carried out in which dif
was only 110 to 300 mg/1. ferent relative concentrations of Chlo
rella from Allen's medium and Micro
Effects of pH and Age cystis from Gorham's (pH 9) medium
A series of tests was carried out in were used separately and in combina
which cultures of Chlorella and Micro tions in the BOD and photosynthetic
cystis of different ages from Gorham's capacity tests.
medium were used in the BOD test (at From the activity of the algae when
least 16 samples per condition). The measured separately in these tests, cal
cultures were either aerated and were culations were made of expected ac
alkaline (pH range 8.9 to 10.6) or tivity rates of the various mixtures,
were gassed with 1.25-percent C02 in depending on the relative concen
air and were neutral (pH range 6.5 tration of each alga. The results of
to 7.8). 16 BOD tests of mixtures of these
A summary of the data from BOD algae indicated an average A DO/mg
tests and tests of the photosynthetic SS of 0.10, the range being 0.069 to
capacity after five-days' incubation in 0.13, or well within the range of ac
the dark is presented in Table IV. tivity measured for the algae sepa
The activity of Chlorella decreases rately. The results of the photosyn
with the age of the cultures and this thetic capacity tests, after 5-days' in
cubation in the dark, separately and chosen for study as a representative

in mixtures indicated that the meas species of the types of green algae
ured rates for the mixtures were 99 found in such installations.
to 108 percent of theoretical rates for It was found that Scenedesmus could
such mixtures, calculated from rates be cultured readily in Allen's medium
measured when algae wrere tested sepa for the BOD tests. The average effect
rately. Thus, one can say that the on the BOD test in seeded dilution wa
activities of Chorella and Microcystis, ter of 37 tests indicated it had an ac
when mixed in these tests, were addi tivity factor of 0.13A DO/mg SS and
tive and not antagonistic. an average photosynthetic capacity
after 5 days in the dark of 0.13A
Long-Term BOD Studies SS.
DO/mg
The amount of oxygen used (BOD) A few tests (a total of eight) were
and photosynthetic capacity of either run with the blue-green alga, Nostoc
Chlorella or Microcystis in seeded BOD muscorum (Wis. 1013). This alga is
dilution water have been followed at well known for the many studies of
1- to 3-day intervals for as long as its ability to fix atmospheric nitrogen
28 days. Four tests were run with (17) (18). The average activity factor
Chlorella from 3 different media and in the BOD test of this alga was 0.093A
3 tests with Microcystis from Gorham's DO/mg SS and the average photosyn
(pH 9) medium. In all tests with thetic capacity after 5 days in the
either alga, there was a gradual de dark was 0.081A DO/mg SS.
crease in DO in the BOD bottles until The alga, Gloeotrichia echinulata
zero DO was reached (7- to 12-days' (Wis. 1052), was chosen as a repre
incubation). The photosynthetic ca sentative of the blue-green algae that
pacity of the algae was measured up cause obnoxious blooms in lakes and
to and after the time the bottles be are also nitrogen fixers. This alga was
came anaerobic. cultured in three different media, Gor
It was found that, in the samples ham's, Gorham's minus the nitrogen
wTith Chlorella, the photosynthetic ca source, and Allen's. The average ac
pacity of the algae gradually decreased tivity factors in the BOD test were:
after the bottles became anaerobic, but from Gorham's medium (16 tests),
wTas still measureable for periods as 0.091A DO/mg SS; from Gorham's
long as eight days under anaerobic minus nitrogen (6 tests), 0.13A DO/mg
conditions. Photosynthetic capacity of SS; and from Allen's (6 tests), 0.048A
Chlorella decreased from 0.18 to 0.078A DO/mg SS. The photosynthetic ca
DO/mg SS during 8 days under anae pacity of these algae after 5 days in
robic conditions in a typical experi the dark was generally found to be
ment. In the tests with Microcystis, small for the majority of the samples
the photosynthetic capacity of the alga tested.
was maintained at a higher rate for a As a result of this observation of
longer period of time. Photosynthetic the low photosynthetic capacity of
capacity of Microcystis decreased from a series of BOD
Gloeotrichia, daily
0.12 to 0.079A DO/mg SS during 19 and photosynthetic tests was
capacity
days under anaerobic conditions in one carried out. In the first two tests ?.n
experiment. seeded dilution water of Gloeotrichia
cultured in Gorham's medium, it was
BOD of Other Algae noted that the algae ceased to evolve
The effects of three other algal spe oxygen on the second or third day
cies on the BOD test have been studied. when exposed to light. Within 48 hr
The first, Scenedesmus dimorpha, was of the time when the algae ceased to
isolated from a sewage oxidation pond photosynthesize, there was a sudden
at Lancaster, California, and was increase in the rate at which oxygen
1540
was consumed as compared to the lake water were 0.14, 0.15, and 0.59A
rate of the first 3 to 4 days and to the DO/mg SS/5 days, respectively.
rate of oxygen consumed in Chlorella From the data of Figure 7, Gloeo
tests run at the same time. trichia seems to die after three to four
A third series of tests with Gloeo days under the conditions of the BOD
trichia from Gorham's (pH 9) medium test (the point at which the algae cease
was carried out with seeded dilution to photosynthesize), a situation that
water, seeded Gorham's medium, and previously had been found to be the
raw lake water as the BOD test me case in studies of marine diatoms (8).
dium. The results of the tests in Subsequently, the organic matter of
seeded dilution water and seeded Gor the algae apparently becomes avail
ham's medium are plotted in Figure able to the bacteria present and an
7. The results of the tests in raw lake increased BOD activity results (a cal
water were similar to these results culation of the projected BOD of the
except the rate of oxygen used was algae in dilution water during the
greater; BOD activity factors for di period, 5 to 10 days, would give a BOD
lution water, Gorham's medium, and rate of about 0.38A DO/mg SS).
o 6
3 4
Time (Days)
FIGURE 7.?Oxygen utilization by Gloeotrichia (10 mg/1) in seeded

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.
Summary and Conclusions

1. The rate of growth of Chlorella 9. Daily and long-term studies of
in different media has been followed Chlorella in the BOD test showed that
by measurements in the culture of the rate of oxygen utilization was quite
suspended solids, volatile solids, COD, uniform until the BOD bottles became
cell counts, and packed volume. anaerobic. It was found that Chlorella
2. It was found that DO could be could continue to produce oxygen by
determined readily using the Winkler photosynthesis for at least seven days
method in solutions containing Chlo after the BOD bottles became anae
rella in concentrations at least as high robic.
as 5 X 106 cells per ml. 10. The activity of Chlorella in the
3. The activity of Chorella in seeded BOD test was found to vary consider
dilution water in the BOD test appears ably, averages of activity ranging from
to be independent of the volume of 0.09- to 0.19-mg 02 used per mg sus
seed added (in the range of 3 to 30 pended solids per liter, depending on
ml seed/1). the medium in which the algae had
4. There appeared to be no differ been cultured.
ence in the activity of Chlorella in the 11. The average and range of values
BOD test whether the original culture obtained in the BOD test with Chlo
was bacterial-free or contaminated rella from different culture media are
with sewage bacteria (one to five ml presented in tabular form.
sewage/1). However, the addition of 12. It was found that the activity of
sewage to cultures caused a five-fold algae from Myers' and Gorham's me
loss in the activity due to the super dia decreased with increasing age of
natant of the algal cultures when the cultures.
tested in BOD tests. 13. Studies of the effect of pH of
5. The BOD of autoclaved or freeze Gorham's medium on the activity
dried Chlorella cells were approxi of Chlorella in the BOD test indicated
mately four fold that of living cells. that algae from the more alkaline (pH
6. No consistent relationship could 9) medium exhibited a greater de
be obtained between the amount of crease in activity with increasing age
oxygen used by Chlorella in the BOD of the culture than algae from neutral
test and the number of viable cells (pH 7.0 to 7.8) medium.
measured by dilution and plating of 14. Chlorella cultured in autoclaved
samples after five-days' incubation. secondary sewage effluent exhibited a
7. The photosynthetic capacity of higher activity in the BOD test (an
the algae before and after incubation average of 0.27-mg O2 used per mg
in the BOD test indicated the physio suspended solids) than algae from the
logical state of the algae. Tests showed synthetic culture media. Also, algae
that this measurement could be used from neutral pH cultures were more
to indicate if algae were still active active than algae from C02-deficient
after different periods of dark incuba cultures (pH 9).
tion and the relative activity of algae 15. The rate of oxygen utilization by
from different cultures. Chlorella in the BOD test was found
8. Tests of the BOD of Chlorella to be independent of any increase in
under seeded and bacterial-free condi bacterial respiration due to the pres
tions indicated the effect of algae from ence of fructose. However, in the
Allen's medium were merely additive presence of glucose in the BOD test,
to the respiration of bacteria, whereas the algae had a decided effect on the
algae from the more dilute dilution bacterial BOD of this organic com
water medium might have caused a pound.
slight stimulation to the respiration of 16. When comparative tests with
bacteria present. Chlorella and Microcystis were made
1542
it was found that the rate of growth were merely additive and not antago
of these algae in the same medium nistic to each other.
(Gorham's pH 9) was remarkably 19. Microcystis was found to be
similar. able to photosynthesize after long pe
17. Both Chlorella and Microcystis riods (up to 19 days) of anaerobic
exhibited a decrease in activity with conditions as well or better than
Chlorella.
increasing age of cultures. However,
20. The effects of Scenedesmus di
Microcystis from Gorham's pH 9 me
morpha and Nostoc muscorum on the
dium had a higher photosynthetic ca
BOD test were similar to those of
pacity after 5-days' incubation than
Chlorella and Microcystis.
the same alga from Gorham's pH 7 21. Tests with Gloeotrichia echinu
medium.
lata indicated that this alga appears
18. When Chlorella and Microcystis to die under the conditions of the
were mixed for the BOD test it was BOD test, thereby increased
causing
found that their separate activities bacterial respiration.
Acknowledgment
This research has been supported by tional Institutes of Health, U. S. Pub
a research grant (K-15) from the Na lic Health Service.
References
" "
1. Standard Methods for the Examination Algae. Applied Microoiol., 5, 47
''
of Water and Wastewater. 11th Ed., (1957).
Amer. Pub. Health Assn., New York 10. Finkle, B. J., Appleman, D., and Flei
"
(1960). scher, E. K., Growth of Chlorella vuh
2. Allen, M. B., "The Cultivation of Myxo garis in the Dark." Science, 111, 309
phyceae." Arch. Mikrooiol. (Ger (1950).
34 11. Kaneshige, H. M., Fitzgerald, G. P.,
many), 17,
(1952).
3. Burlew, J. Culture: From and Rohlich, G. A., "Stabilization
S.,
"Algal
to Pilot Plant." Pond Studies.'' Univ. of Wisconsin
Laboratory Carnegie
No. 600 Engineering Experiment Station, 1
Inst., Washington, (1953).
4. Hughes, E. O., Gorham, P. R., and Zehn (1957).
12. Fitzgerald, G. P., and Rohlich, G. A.,
der, A., "Toxicity of a Unialgal Cul
"An Evaluation of Stabilization Pond
ture of Microcystis aeruginosa." Can. ''
Literature. Sewage and Industrial
Jour. Microoiol., 4, 225 (1958).
5. Oswald, W. Wastes, 30, 10, 1213 (Oct. 1958).
J., and Gotaas, H. B., "Pho
13. Cramer, M., and Myers, J., "Nitrate
tosynthesis in Sewage Treatment."
Reduction and Assimilation in Chlo
Proc. San. Eng. Div., Amer. Soc. Civil
rella.f' Jour. Gen. Physiol., 32, 93
Engr., 81, Sep. No. 686 (1955).
6. Wisniewski, T. F., and Their
(1948).
"Algae 14. Griffiths, D. C. L., and
Effects on Dissolved J., Thresher,
Oxygen and Bio <'
'' H. The Heterotrophic Nu
chemical Oxygen Demand. In " Oxy Street, E.,
trition of Chlorella vulgaris." Ann.
gen Relationships in Streams." U. S.
Bot. (Brit.), 24, 1 (1960).
Pub. Health Serv., Seminar, R. A.
15. Swanson, C. A., "The Effect of Culture
Taft San. Eng. Center, Cincinnati, Filtrates on Respiration in Chlorella
Ohio (Mar. 1958).
vulgaris." Amer. Jour. Bot., 30, 8
7. Bartsch, A. F., "Algae and Their Effects
on DO and BOD."
(1943).
Discussion in 16. Jorgensen, E. G., "Growth Inhibiting
"Oxygen Relationships in Streams," "
Substances Formed by Algae. Phys
IT. S. Pub. Health Serv., Seminar, R. iol. Plantarum (Denmark), 9, 712
A. Taft San. Eng. Center, Cincinnati,
Ohio
(1956).
(Mar. 1958). 17. Allison, F. S. R., and Morris,
E., Hoover,
8. Waksman, S. A., Stokes, L. J., and But H. with
J., "Physiological Studies
ler, M. R., "Relation of Bacteria to the Nitrogen-Fixing Alga, Nostoc mus
Diatoms in Sea Water." Jour. Ma corum." Bot. 433
Gazette, 98, (1937).
rine Biol. Assn. United Kingdom 18. De, P. "The Role of Blue-Green Al
K.,
(Brit.), 22, 359 (1937). gae in Nitrogen Fixation in Rice
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Efecto de Algas en Determinacion de Dbo - Fitzgerald

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Efecto de Algas en Determinacion de Dbo - Fitzgerald

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The Effect of Algae on BOD Measurements

Author(s): George P. Fitzgerald

A study has been made to measure As soon as some of the preliminary

Materials and Methods

TABLE I.?Media Used for Culture of Chlorella for BOD Experiments

Salt Dilution Water Gorham's Allen's Myers'

Total Salts 297.5 728 1,866 5,147

B. Concentration of essential ions in media (mg/1)

Ion Dilution Water Gorham's Allen's Myers'

TABLE IL?Relative Growth of Chlorella after 10 Days in Different Media

Culture ss vs COD Cell Count

Dilution water 125 100 175 12.5

Seed and Algae (13 mg/1)

Seed and Algae (27 mg/1)

FIGURE 1.?Oxygen utilization by seed and 13 mg/1 Chlorella and

Algae (Dark plus 2-hr

Algae (Initial 2-hr light)

Algae( Dark Only)

FIGURE 2.?Oxygen utilization by Chlorella (13 mg/1) from Allen's

Algae (Allen's Medium)

Algae (Dilution Water Medium)

FIGURE 3.?Oxygen utilization by 6X seed, Chlorella (14 mg/1) from

Algae (Dark plus 2hr

Algae (Dark Only)

FIGURE 4.?Oxygen utilization by Chlorella (28 mg/1) from Allen's

TABLE III.?The Effect of the Culture Medium on the Activity of Chlorella

BOD* Photosynthetic Capacity!

Myers' 6.5 23 0.069-0.12 0.090 0.087-0.18 0.14

FIGURE 5.?Effect of age of culture on activity of Chlorella (four media).

FIGURE 6.-Effect of pH of Gorham's medium on activity of Chorella.

parently enhanced by increased age five tests with fructose as an added

cose and fructose. amount of glucose absorbed by sewage

__ / Log cell number at ?_2\ . w. i

(Averages of at Least 16 Tests per Condition)

Gorham's pH 7 3-8 0.17 0.15 0.16 0.085

pended solids increased from approxi

cubation in the dark, separately and chosen for study as a representative

FIGURE 7.?Oxygen utilization by Gloeotrichia (10 mg/1) in seeded

Summary and Conclusions

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