United States
Environmental Protection
Agency
Industrial Environmental Research
Laboratory
Research Triangle Park NC 27711 *x/ STT
Research and Development
EPA-600/S2-83-111 Jan. 1984
Project Summary
Nitrification Inhibition Biokinetics
R. D. Neufeld, J. H. Greenfield, A. J. Hill, C. B. Rieder, and D. O. Adekoya
The report gives results of basic
studies (supported by both the American
Iron and Steel Institute and the U.S.
Environmental Protection Agency) into
causes of nitrification process instability
as often observed in steel industry
wastewaters. The experimental ap-
proach taken was to evaluate the
influence of elevated free ammonia
levels, pH, elevated temperatures,
cyanides, and certain trace organics
that tend to pass through the carbona-
ceous reactor zone on the kinetic
parameters that quantify biological
nitrification. Theoretical calculations
based on laboratory defined parameters
suggest allowable concentrations of
trace inhibitors and operational strat-
egies for stable nitrification of industrial
wastewaters.
Based on data obtained, the toxic
inhibition to biological nitrification
decreased in the order of free cyanide,
coal tar acid phenolics, phenol, 2,3,6-
trimethylphenol, 2-ethylpyridine, 2,4,6-
trimethylphenol, complexed cyanides,
and thiocyanate. All except free cyanide
appeared to follow a "shoulder effect:"
low levels of inhibitor had no influence
on rates of biological nitrification, but
higher levels had profound effects. The
upper temperature limit for stable
optimum nitrification is about 30°C,
with decreasing rates of nitrification on
either side of this optimum. Nitrification
rates approach zero as wastewater
temperatures approach 45°C. Free or
unionized ammonia is inhibitory at levels
of 10 mg/L or more. Free ammonia is a
function of watewater total ammonia,
pH, and temperature; thus, pH and
temperature may be varied indepen-
dently to ensure stable nitrification in
wastewaters containing significant
total ammonia levels.
This Project Summary was developed
by EPA's Industrial Environmental
Research Laboratory, Research Triangle
Park. NC. to announce key findings of
the research project that is fully
documented in a separate report of the
same title (see Project Report ordering
information at back).
Introduction
Many "advanced wastewater" treat-
ment processes are based on physical-
chemical and biological techniques for
the removal of wastewater ammonia;
however, these processes are not usually
employed in industry due to economics,
marginal overall removal efficiencies,
and questionable technological application
to specific industrial operations. Of the
processes available for the removal of
ammonia from steel industry wastewaters,
engineered biological systems are the
most pragmatic. Data from steel industry
and other industrial operations indicate
that a properly designed and operated
biological nitrification facility can reduce
ammonia levels to less than 10 mg/L
over extended periods. Biological nitrifi-
cation processes, however, are known to
exhibit unaccounted for upsets, and thus
are considered unreliable in some
industrial sectors.
This research investigated possible
causes of biological nitrification process
instabilities as currently observed in coke
plant and other industrial plant waste-
water treatment facilities. Nitrification
process instability, as studied in this
research, occurs as a result of toxic
inhibition as caused by an interaction of
organics, free ammonia, elevated tem-
peratures, pH, complexed and free
cyanides, phenolics, and non-biodegrad-
able organics that pass through carbona-
ceous removal stages.
From laboratory data obtained on
studies of nitrification process instability,
conclusions with engineering calculations
are presented to illustrate design and
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operational considerations to assist
industry in meeting EPA effluent restric-
tions for ammonia-nitrogen and phenolic
compounds. The conditions utilized in
this study were aimed at assisting the
coke plant segment of the iron and steel
industry.
Table 1 summarizes the Best Available
Technology (BAT) limitations, a technology
level to be implemented by July 1, 1984,
and the prior Best Practicable Technology
(BPT) effluent limitations for the by-
product coke-making segment of the iron
and steel industry.
Overview of Results
Experimental results to date show that
unionized ammonia acts as a toxic
inhibitor at levels in excess of 10 mg/L.
Quantification of the influence of union-
ized ammonia on biokinetics yields an
expression similar to classical substrate
inhibition models. Lower pH values
(around 7.0) are superior systems treating
high levels (greater than 250 mg/L) of
total ammonia or systems experiencing
large variations of total ammonia. Al-
though biokinetics are slower at this pH
value, enhanced stability due to ammonia/
ammonium ion equilibria favor this
operating decision.
Nitrification biokinetics are highly
sensitive to elevated temperatures: rates
of nitrification increase as temperature
Table 1. Effluent L imitation Guidelines for the By-Product Cokemaking Subcategory of the Iron
and Steel Industry*
BA T Effluent Limitations
increases to a maximum at about 30°C,
beyond which the overall rate of nitrifica-
tion decreases.
Free cyanide (CNa) was the most toxic
inhibitor investigated. Concentrations of
free cyanide greater than 0.11 mg/L
must be avoided for stable operation of
biological nitrification processes.
A comparison of the toxic effects of
coke plant tar acid phenolics showed that
this substance was of greater toxicity to
nitrifiers than reagent grade phenol on an
equal phenol concentration basis. The
apparently more severe toxicity of the
coal tar acids may be due to a few
substances which may be in the liquid
matrix, or a synergistic approach repre-
senting combinations of large numbers of
substances as being toxic to nitrification.
Experimental Approach
Nitrification biokinetics has been
shown to follow classical Monod kinetics
of the form:
v = VmaxS/ (Km+S) (1)
where, v = rate of nitrification, t 1
S= ammonia level in solution,
mg/L
Vmax, Km = system constants.
In addition, for biological systems, the
following system equation may be derived:
= av-b (2)
Pollutant
Parameter
Ammonia -N
Cyanide
Phenols (4 AA-P)
Benzene
Naphthalene
Benzo(a)pyrene
Max/mum for Any 1 Day
Discharge Load
(kg/Mg)
(lb/10OO Ib)
O.0543
0.00638
O.OOO0638
0.0000319
0.0000319
O.OOO0319
Discharge
Cone.
mg/Lh
65.1
7.6
0.08
0.04
0.04
0.04
Average of Daily Values
for 30 Consecutive Days
Discharge Load
(kg/Mg)
(lb/1000 Ib)
0.0160
0.00351
0.0000319
Discharge
Cone.
mg/L
19.2
4.2
0.04
BPT Effluent Limitations
Discharge Load Discharge
(kg/Mg) Cone.
(Ib/IOOO Ib) mg/L"
TSS°
Oil and Grease
Ammonia-N
Cyanide
Phenols (4 AA-P)
pH within the range
of 6.0-9.0
0.253
0.0327
0.274
0.0657
0.00451
303.4
39.2
328.5
78.8
5.4
Discharge Load
(kg/Mg)
(lb/1000 Ib)
0.131
0.0109
0.0912
0.0219
0.00150
Discharge
Cone.
mg/L
157.1
13.1
109.4
26.3
1.8
"Federal Register, 1982.
^Discharge concentration (for comparative purposes) is based on effluent flows of 200 gal. /ton
(835 L/Mgl of coke for BA T and BPT.
"Total suspended solids.
where, 0 = sludge age, days
a = observed yield coefficient, g/g
b = endogenous decay coefficient,
day"1
The approach taken in this research
was to quantify the influence of inhibitors
typical of coke plant wastewaters on the
biokinetic parameters for nitrification.
Toxic or biokinetic inhibition tends to alter
the shape of the effluent ammonia vs.
sludge age design curve which results
from the combination of Equations (1) and
(2).
The data from this research are in the
final form of summary plots of the in-
fluence of parameters typical of coke
plant waste waters on the biokinetic
constants of nitrification, and on the
system design curve for process nitrifica-
tion. The conditions considered are free
(or unionized) ammonia which acts as a
"substrate inhibitor," free and complexed
cyanides and thiocyanate, phenol and tri-
methylated phenolics, pyridine, and ele-
vated temperatures. For comparison
purposes, a sample of coal tar acid was
obtained from a local coke plant which, as
indicated above, was found to be more
toxic to nitrifiers when compared on an
equal phenol (measured by 4-AA) basis.
Results of free ammonia inhibition are
given in detail in the project report.
Details of trace substance interaction
and temperature interactions are included
in prior reports to the American Iron and
Steel Institute. These results are sum-
marized below.
Experimental Results of
Elevated Temperatures on
Nitrification Biokinetics
The experiments conducted were
developed to quantify the influence of
elevated temperatures on the defining
parameters at a pH of 8.0. (Note that the
data developed were for nitrifiers that
were acclimated to the elevated tempera-
ture in continuous cultures; the data
reflects, not the shock effects of tempera-
ture, but steady-state biokinetics for
essentially pure nitrifiers.)
Figure 1 is a summary plot of maximum
nitrification rate (Vmax) as a function of
temperature. It shows that, at pH = 8.0,
Vmax is constant at 1.256 g NH3/g
volatile suspended solids (VSS)-day in the
range of 22-30°C and then decreases to
about zero at 45°C. This decrease in Vmax
was found to be described by:
Vmax - 3.78 - 0.084 (T) (3)
for 30°C < T°C < 45°C.
The numerical value for the Michaelis-
Menten parameter Km was also evaluated
with temperature as shown in Figure 2. In
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1.4
1.2
•§ 1.0
to
}"
$ 0.6
j 0.4
0.2
0
rVm« =1.256. S* = 0.072
" /or22° 30°C
20 24 28 32 36 40 44
Temperature, °C
Figure 1. Vmat versus temperature.
100
80
60
40
30,
. 20
10
8
6
4
3
log Km = -1.8829 + 0.08228(T)i
forT>30°C r
= 1.53-0.0315
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10.0,
I i.o
0.1
0.01
I'ma, = 1-2
log Vm.» = 0.853 - 1.117 log /TMP)
200
0 1.0 10.0 100.0
2,3.6-Trimethylphenol, mg/L
Figure 5. l/max versus concentration of 2,3,6-trimethy/phenol.
1000.0
10.0
I
0.1
Km = 3.3
log Km = 7.839-7.069 log (TMP)
1.0 10.0
2,3.6-Trimethylphenol, mg/L
100.0
1000.0
Figure 6. Km versus concentration of 2.3,6-trimethylphenol.
thiocyanate should be multiplied by 10.)
Figure 8 shows that coal tar acids are more
inhibitory than reagent phenol, low levels
of 2,3,6-trimethylphenol are far more
inhibitory to nitrification than levels of
2,4,6-tnmethylphenol, and free cyanide
is the most toxic to nitrifiers of all the
chemical species studied. Complexed
cyanides and thiocyanates exhibited
toxicities to nitrification only at levels far
greater than those for the other species
studied and may be considered "non toxic"
to nitrifiers at usual levels found in coke
plant effluents.
For practical purposes, by drawing
a horizontal line on Figure 8 at a sludge age
of 10 days, the intersections of this line
with the curves for individual compounds
provide insight into allowable levels of
these substances (assuming no syner-
gism) for nitrification. Table 3 lists these
levels. They should be considered as
20 40 60 80 100 120
Sludge Age, Days
Figure 7. Effect of 2.3,6-trimethylphenol
level on sludge age as a design
and operational parameter for
nitrification.
allowable levels of such trace substances
in effluents so that biological nitrification
may take place in a stable manner.
Summary of Findings
Acquired laboratory data lead to the
following conclusions concerning the
effect of inhibition on biological nitrifica-
tion.
(1) Toxic inhibition of biological nitrifi-
cation decreased in the order of free
cyanide, tar acid phenolics, phenol, 2,3,6-
trimethylphenol, 2-ethylpyridine, 2,4,6-
trimethylphenol, complexed cyanide, and
thiocyanate.
(2) All substances, except free cyanide
and phenol, appeared to follow a "shoul-
der effect:" low levels of inhibitor had no
influence on the rates of biological
nitrification, but higher levels had
profound effects. The absence of the
shoulder effect indicates that low levels
of free cyanide should be avoided for
stable operation of biological nitrification
processes.
(3) Calculations based on laboratory
data show maximum levels of contami-
nants that will still permit stable nitrifica-
tion at a 10 day sludge age at pH = 8 and
effluent ammonia levels of 10 mg/L.
(4) The upper temperature limit for
stable optimum nitrification is 30°C.
(5) Free (or unionized) ammonia is
inhibitory at levels of 10 mg/L or more.
Free ammonia is mainly a function of
wastewater total ammonia, pH, and
temperature.
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Table 2. Values Below Which Toxicant Has No Effect on Nitrification Kinetic Parameter
Compound
"Free Cyanide
"Phenol
Coal tar acids
(as 4-AA phenol)
2,3.6-TMP
2-Ethylpyridine
2.4.6-TMP
Fe(CN)i3
SCN-
(Shoulder values at pH = 8.0}
V^gNHs/gVSS-day
Toxic at all values
Toxic at all values
1.2 mg/L
4.9 mg/L
10 mg/L
30 mg/L
80 mg/L
236 mg/L
Km mg NHs/L
No effect on Km
No effect on Km
5 mg/L
17. 2 mg/L
42 mg/L
50 mg/L
No effect on Km
No effect on Km
"Nitrification inhibition is proportional to (CN) 3 and (phenolf*5; free cyanide is far more toxic than
phenol.
10O
2,3,6-Trimethylphenol
O)
I
10
Thiocyanate
(concentrations x 10)
C/VC
40
60
80 100 120
Concentration, mg/L
Figure 8. Required sludge age to meet 10 mg/L NH3effluent versus concentration of inhibitor.
Table 3.
Maximum Levels of Contaminants to Permit Nitrification at a 10-Day Sludge Age
(effluent NH3 = 10 mg/L at pH = 8.O)
Compound
Concentration, mg/L
Free cyanide
Coal tar acids (as 4-AA phenol}
Phenol
2.3.6 TMP
2-Ethylpyridine
2.4.6 TMP
SCAT
0.11
1.7
5.5
7.8
22
39
190
660
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R. D. Neufeld, J. H. Greenfield, A. J. Hill. C. B. Rieder, andD. O. Adekoya are with
the University of Pittsburgh, Pittsburgh, PA 15261.
J. S. Ruppersberger is the EPA Project Officer (see below).
The complete report, entitled "Nitrification Inhibition Biokinetics," (Order No. PB
84-110 162; Cost: $17.50, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Industrial Environmental Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC27711
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United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
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I'll
US,OFT:<.'..V-M
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U.S. GOVERNMENT PRINTING OFFICE: 1984-759-102/834
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