APPuED MICROBIOLOGY, Sept. 1967, p. 1211-1215 Vol. 15, No.

5
Copyright @ 1967 American Society for Microbiology Printed in U.S.A.

Oxygen-Nitrogen Relationships in Autotrophic

Nitrification
C. T. WEZERNAK AND J. J. GANNON
Department ofEnvironmental Health, School ofPublic Health, University of Michigan, Ann Arbor, Michigan 48104
Received for publication 10 May 1967

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Oxygen utilization by the autotrophic nitrifiers Nitrosomonas and Nitrobacter was
studied. Experimental evidence is presented which reflects the effect of carbon diox-
ide fixation on overall oxygen utilization in autotrophic nitrification. Measurement
of dissolved oxygen and inorganic nitrogen changes indicates that oxygen-nitrogen
ratios in inorganic nitrogen oxidation are equal to 3.22 parts (expressed in milligrams
per liter) of oxygen per part of ammonia nitrogen oxidized to nitrite nitrogen and
1.11 parts of oxygen per part of nitrite nitrogen oxidized to nitrate nitrogen. These
values rather than the stoichiometric ratios should be used in nitrogenous oxygen
demand calculations.

A number of reports have appeared recently
dealing with the subject of nitrification and its
implications and position in the total BOD
(biochemical oxygen demand) of waste water and
the effect of nitrogenous compounds on the
oxygen balance of streams. A study of the Grand
River by the Michigan Water Resources Com-
mission (12) demonstrated the necessity of in-
cluding inorganic nitrogen oxidation in oxygen
balance calculations.
It is generally accepted that the biological
oxidation of the ammonium ion to nitrite and
nitrate is largely carried out in an aquatic en-
vironment by certain specific autotrophic bacteria
belonging to the family Nitrobacteraceae, and
that these organisms obtain their energy for
growth and cell synthesis from the oxidation of
ammonia and nitrite nitrogen. Bergey's Manual
of Determinative Bacteriology (7th ed.) lists seven
genera in this family, which are divided into
nitrite-formers and nitrate-formers. The principal
genera in each group are Nitrosomonas and Nitro-
bacter, respectively. All members of the family
are considered aerobic autotrophs, and are
found widely distributed in nature.
Several recent studies (4, 5, 9, 14) seem to
indicate that the strict autotrophic nature of these
organisms is somewhat overemphasized, and that
certain organic compounds have an influence on
growth. However, Delwiche and Finstein (5)
reported that none of the organic compounds
studied could substitute for carbon dioxide as a
sole carbon source.
The oxidation reactions as carried out by the
Nitrobacteraceae are summarized as follows: for
Nitrosomonas sp. and related bacteria,
1211
NH4+ + 1I .O2 -* 2H+ + NO- + H20 (1)
for Nitrobacter sp. and related bacteria,
NO- + iO2 -- NO (2)
Based on the above equations, it is generally
assumed that 3.43 mg of oxygen is required to
oxidize 1 mg of ammonia nitrogen to nitrite
nitrogen, and that 1.14 mg of oxygen is utilized
for the oxidation of 1 mg of nitrite nitrogen to
nitrate nitrogen. However, since this is a biolog-
ical oxidation in which carbon dioxide is fixed, it
follows that the nitrogenous oxygen demand
(NOD) of inorganic nitrogen would vary some-
what from the above stoichiometric relationships.
This fact was pointed out by Buswell and Pagano
(3) and by Montgomery and Borne (13). Never-
theless, the above stoichiometric relationships are
currently used in the literature, largely because of
the fact that there is a paucity of direct evidence
which demonstrates that the amount of oxygen
utilized in autotrophic nitrification is different
from the above.
The present report presents oxygen utilization
data together with cell yield information, which
demonstrate the overall oxygen utilization ratios
as they occur in autotrophic nitrification.
MATERIALS AND METHODS
Organisms. The following organisms were used:
Nitrobacter agilis, from the American Type Culture
Collection, and Nitrosomonas sp., isolated from acti-
vated sludge by use of the technique described by
Lewis and Pramer (11).
Medium. An inorganic medium containing the fol-
lowing (in milligrams per liter) was used for N. agilis
studies: KNO2 (as N), 8; KH2PO4, 70; MgSO4, 25;
1212 WEZERNAK AND GANNON
CaCl2, 19; FeSO4 (as Fe), 0.1; Na2MoO4 , 0.025; and
distilled water to bring the volume to 1 liter; 5%
Na2CO, solution was used to adjust to pH 7.9, and
phosphate buffers were used. For Nitrosomonas sp.
studies, (NH4)2S04 (as N) at a concentration of 5 mg
per liter was substituted for the KNO2 in the above
formulation. All other constituents were the same.
Counting technique. Direct counts of organisms were
made under the microscope by use of a Petroff-Hausser
counting chamber.
Equipment and testing techniques. Standard BOD
APPL. MICROBIOL.
four final determinations. The nitrite-nitrogen
values for each experiment were obtained by
subtracting the average of two initial determina-
tions from the average of two final determinations.
As indicated earlier, the overall energy-yielding
reaction is summarized as follows:
NH4+ + 1½g 02
2H+ + NO2- + H20 + energy (3)

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bottles were used in the bottle studies. A 2-liter Erlen-
meyer flask fitted with a Gelman galvanic cell oxygen
probe (7), together with a strip chart recorder, was
used in the oxygen probe studies. All glassware was
rinsed with acid and distilled water and was sterilized.
Oxygen determinations were made by use of the
azide modification of the iodometric method (2) with
additional sodium azide. Ammonia-nitrogen (direct
nesslerization) and nitrite-nitrogen determinations
were made by use of standard procedures (2). The
ultraviolet absorption technique described by Gold-
man and Jacobs (8) was used for nitrate-nitrogen
determination.
All test samples were incubated at 30 C for the
period of study. For each test, 12 BOD bottles were
used, 6 for initial determinations and 6 for the final
analysis, and the results were averaged.
RESULTS AND DIscussioN
BOD bottle studies. The aim of these experi-
ments was to measure oxygen utilization directly
and to relate these results to the amounts of
ammonia nitrogen oxidized. Ten experiments
with Nitrosomonas sp. as the inoculum were
carried out in standard BOD bottles. The results
are summarized in Table 1 and represent analysis
of 120 bottles. The dissolved oxygen (DO)
depletion values shown for each experiment
represent the difference between the average of
four initial dissolved oxygen determinations and
TABLE 1. Oxygen utilization by Nitrosomonas sp. in
standard BOD bottles
DOa depeitn
(mg/lPer)
Nitrite N
O/N
Expt (mg/liter) fornation
(mg/liter)
In the interest of completeness, it should be
recognized that the above oxidation of the am-
monium ion to nitrite involves a valence change
from -3 to +3. There is evidence in the literature
(1) which shows that the oxidation proceeds in
two electron steps involving two intermediates,
one of which is NH20H. The conversion of these
intermediates appears to be fairly rapid with
little, if any, accumulation. The energy thus
derived in the above oxidation is used for carbon
dioxide fixation. The result is that in the process
of protoplasm synthesis a small amount of oxygen
is produced, as follows:
5CO2 + NH3 + 2H20 -+ C5H702N + 502 (4)
The oxygen thus produced would reduce the
overall oxygen utilization ratio to something less
than the ratio indicated by equation 3. The
average of the results shown in Table 1 indicates
that 3.22 mg of oxygen is required per mg of
ammonia nitrogen oxidized to nitrite nitrogen,
rather than the theoretical value of 3.43 mg.
The results of five experiments (60 bottles)
with N. agilis as the innoculum are summarized
in Table 2. The values shown for each experiment
were obtained from the averages of four initial
and four final dissolved-oxygen determinations,
and from the averages of two initial and two final
nitrate-nitrogen determinations. Here again,
because of CO2 fixation, the oxygen to nitrogen
ratio in the oxidation of nitrite nitrogen to nitrate
nitrogen is less than the theoretical. The average
of the results (Table 2) indicates that 1.11 mg of

1 3.37 1.06 3.18 TABLE 2. Oxygen utilization by Nitrobacter agilis in

2 3.65 1.16 3.15 standard BOD bottles
3 4.64 1.41 3.30
4 3.65 1.12 3.25 DO de letion Nitrate
fornationN 0/N
5 4.40 1.34 3.28
Expt '/ltr)
1j(Mgwilte (mg/liter)
6 5.27 1.67 3.15
7 4.85 1.47 3.30 1 4.00 3.55 1.12
8 4.32 1.35 3.20 2 3.90 3.50 1.11
9 4.45 1.37 3.25 3 3.60 3.30 1.09
10 4.90 1.54 3.18 4 6.42 5.80 1.11
5 6.68 6.05 1.10
= 43.51 2 = 13.49 Avg = 3.22
= 24.60 2 = 22.20 Avg = 1.11
a DO = dissolved oxygen.
VOL. 15, 1967 OXYGEN UTILIZATION IN AUTOTROPHIC NITRIFICATION 1213

GALVANIC CELL
,OXYGEN PROBEEI

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RECORDER MAG-MIXER
FIG. 1. Experimental apparatus.
oxygen is utilized per mg of nitrite nitrogen In all of the above bottle experiments, the fina
oxidized to nitrate nitrogen, which is slightly less dissolved oxygen concentration was 1.5 mg per
than the theoretical value of 1.14 based on equa- liter or greater.
tion 2. Oxygen probe studies. To confirm the ratios

7 7
6.2 MG./L. NO; - N FORMED
2.0 MG./L. NO, -N FORMED

6-

5-

J
v
4 .4

0
ci 3 3
ci
6i
0~

0 10 20 30 40 50
HOURS HOURS
FIG. 2. NOD due to nitrite-nitrogen oxidation by FIG. 3. NOD due to ammonia-nitrogen oxidation
Nitrobacter agilis. by Nitrosomonas sp.
1214 WEZERNAK AND GANNON APPL. MIcRoBIoL.

given above and to observe nitrification progres-
sion directly, oxygen probe experiments were
carried out. The equipment used is illustrated in
Fig. 1. Experiments were conducted at a tempera-
ture of 30 C and a pH of 8.0. Initial concentrations
of organisms were 500 X 103/ml.
The results of these experiments fell within the
ranges given in Tables 1 and 2 and served to con-
firm the values observed in the BOD bottle
studies. Typical curves are shown in Fig. 2 and 3.
Cell yield data. Concurrent with the above
studies, flask experiments were carried out in
which changes in population numbers and nitro-
gen component changes were observed (Fig. 4 and
5). Figure 4 shows that, at the end of the log
phase of growth, 245 mg of ammonia nitrogen
per liter was oxidized, resulting in a population

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density increase of 100 x 106/ml. Assuming that
the average cell weight for Nitrosomonas is
2.4 X 10-13 g per cell and that the cells contain
50.3% carbon, as reported by Engel (6), an
estimate of the amount of oxygen produced as a
result of CO2 fixation can be made by use of
z equation 4, in which the cell formula is taken to
be C5H702N. The calculations for Nitrosomonas
sp. based on the above assumptions are as follows:
z

I0
x
2 5CO2 + NH3 + 2H20 -) C5H702N + 502
In
ITa
NH4+N oxidized = 245 mg/liter
2
N
Cell yield = 100 x 106/ml = 100 x 109/
liter
Ld
-i
Cell weight = 2.4 X 10-13 g/cell = 2.4 x

10-10 mg/cell
mg cells/liter = 2.4 X 10-10 x 10 x 1010 =
24 mg cells
mg cells/mg NH4+ N oxidized = 24/245 =
0.098
mg carbon/mg NH4+ N oxidized = 0.098 X
HOU RS 0.503 = 0.0493
FIG. 4. Ammonia nitrogen oxidized during growth mg oxygen produced/mg NH4+ N oxidized =
of Nitrosomonas sp. 0.0493 x 160/60 0.13 =

Net oxygen utilized oxygen utilized for

=

inorganic nitrogen oxidization minus

oxygen produced in CO2 fixation.
Net oxygen utilized = 3.43 0.13 = 3.30
-

mg/liter
This gives an overall oxygen to nitrogen ratio of
3.30, which is consistent with the values obtained
380 MG./L 775 MG./L. in the BOD bottle studies.
I0
N02 - N N02 -N In a similar manner, an estimate of the oxygen
I
'0

produced in CO2 fixation by N. agilis can be

2
-i
made. At the end of the log phase of growth,
N

I
380 mg of nitrite nitrogen was oxidized, yielding a
cell increase of 64 X 106/ml. In this calculation,
4-i the assumption is that carbon forms 50% of the
cell and that the average cell weight is 10-1' g,
as reported by Lees and Simpson (10). Thus,
6.4/380 x 0.50 x 160/60 0.023 mg of oxygen
=

produced per mg of nitrite nitrogen oxidized.

Based on the above calculation, the overall
20 40 60 8o100 120 oxygen to nitrogen ratio is 1.117, which agrees
HOURS
very well with the value of 1.11 obtained in the
FIG. 5. Nitrite nitrogen oxidiz ed during growth of BOD bottle studies.
Nitrobacter agilis. Based on the results of direct-measurement
VOL. 15, 1967 OXYGEN UTILIZATION IN AUTOTROPHIC NITRIFICATION
experiments of oxygen utilization and supported
by calculations derived from cell yield experi-
ments, the results of this investigation can be
summarized as follows:
(i) The amount of oxygen produced as the
result of CO2 fixation in the process of autotrophic
nitrification is measurable and must be considered
in calculating NOD.
(ii) The oxygen-nitrogen ratios in autotrophic
nitrification are equal to the amount of oxygen
utilized for inorganic nitrogen oxidation minus the
amount of oxygen produced in CO2 fixation.
Experimental evidence indicates these ratios are
as follows: 3.22 mg of oxygen per liter per 1 mg of
ammonia nitrogen per liter oxidized to nitrite
nitrogen, and 1.11 mg of oxygen per liter per 1
mg of nitrite nitrogen per liter oxidized to nitrate
nitrogen.
The evidence and results presented herein con-
firm the estimates made by Montgomery and
Borne (13).
ACKNOWLEDGMENTS
This investigation was supported by Public Health
Service general research support grant NIH 1 SOL
FR5447-05 GRS, project 70, and by training grant
WP-15 from the Federal Water Pollution Control
Administration.
LITERATURE CITED
1. ALEEM, M. I. H., AND A. NASON. 1963. Metabolic
pathways of bacterial nitrification, p. 392-409.
In C. H. Oppenheimer [ed ], Symposium on
marine microbiology. Charles C Thomas, Pub-
lisher, Springfield, Ill.
2. AMERICAN PUBLIC HEALTH ASSOCIATION. 1965.
Standard methods for the examination of water
and wastewater, 12th ed. American Public
Health Association, Inc., New York.
1215
3. BUSWELL, A. M., AND J. F. PAGANO. 1952. Reduc-
tion and oxidation of nitrogen compounds in
polluted streams. Sewage Ind. Wastes 24:897-
903.
4. CLARK, C., AND E. L. SCHMIDT. 1966. Effect of
mixed culture on Nitrosomonas europaea simu-
lated by uptake and utilization of pyruvate. J.
Bacteriol. 91:367-373.
5. DELWICHE, C. C., AND M. S. FINSTEIN. 1965.
Carbon and energy sources for the nitrifying
autotroph Nitrobacter. J. Bacteriol. 90:102-107.
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6. ENGEL, M. S. 1959. The growth and autotrophic
metabolism of Nitrosomonas europaea. Uni-
versity Microfilms, Inc., Ann Arbor, Mich.
7. GANNON, J. J., J. R. PELTON, AND J. WESTFIELD.
1965. Respirometer assembly for BOD meas-
urement of river waters and biologically treated
effluents. Intern. J. Air Water Pollution 9:
27-40.
8. GOLDMAN, E., AND R. JACOBs. 1961. Determina-
tion of nitrates by ultraviolet absorption. J.
Am. Water Works Assoc. 53:187-191.
9. IDA, S., AND M. ALEXANDER. 1965. Permeability
of Nitrobacter agilis to organic compounds. J.
Bacteriol. 90:151-156.
10. LEES, H., AND J. R. SIMPSON. 1957. The biochemis-
try of the nitrifying organisms. 5. Nitrite oxida-
tion by Nitrobacter. Biochem. J. 65:297-305.
11. LEWIS, R. F., AND D. PRAMER. 1958. Isolation of
Nitrosomonas in pure culture. J. Bacteriol.
76:524-528.
12. MICHIGAN WATER RESOURCES COMMISSION. 1962.
Oxygen relationships of Grand River, Lansing
to Grand Ledge, 1960 Survey. Lansing, Michi-
gan.
13. MONTGOMERY, H. A. C., AND B. J. BoRNE. 1966.
The inhibition of nitrification in the BOD test.
J. Proc. Inst. Sewage Purif. 4:357-368.
14. VAN GOOL, A., AND H. LAUDELOUT. 1966. For-
mate utilization by Nitrobacter winogradskyi.
Biochim. Biophys. Acta 127:295-301.