United States
Environmental Protection
Agency
Municipal Environmental Research ~"^_ 'l^.
Laboratory * &'•*• *• -
Cincinnati OH 45268
'I \
Research and Development
EPA-600/S2-84-017 Mar. 1984
&ERA Project Summary
Biological Countermeasures for
the Control of Hazardous
Material Spills
Neal E. Armstrong, Ernest F. Gloyna, and Orville Wyss
A study was conducted to investigate
the feasibility of using microbiological
processes to mitigate hazardous mate-
rial spills in watercourses. A literature
search, screening tests, laboratory-
scale tests, and small-scale, simulated
spill tests were conducted. Objectives
were to (1) identify microorganisms
capable of degrading selected hazardous
materials, (2) identify their growth
requirements and environmental factors
affecting them, (3) determine the fate
of selected hazardous materials in
water, sediment, and biota, (4) develop
methods for production, storage, recul-
ture, and deployment, (5) investigate
response requirements, and (6) evaluate
the feasibility of biological counter-
measures.
Study results showed that microor-
ganisms can effectively remove certain
hazardous materials since most signifi-
cant ones are biodegradable. Potentially
harmful secondary effects should be
minor since microoganisms are a
natural part of the aquatic environment.
Furthermore, pathogenic bacteria are
not likely to constitute a significant part
(if any) of the countermeasure, and
noxious sludges should not persist
because the microorganisms should
oxidize themselves following consump-
tion of the hazardous material.
This Project Summary was developed
by EPA's Municipal Environmental
Research Laboratory, Cincinnati, OH,
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
The possibility of accidental spills of
hazardous substances poses a constant
threat to the surface waters of the nation.
Effective ways to control such spills and
to mitigate their effects include physical
and chemical techniques; biological
countermeasures have not been thor-
oughly investigated to date. The attrac-
tiveness of a biological countermeasure
for toxic material spills is twofold: (a)
bacteria are natural components of
ecological systems, and their use as a
countermeasure does not constitute the
introduction of a foreign material; and (b)
bacteria can, under optimum conditions,
metabolize organic hazardous materials
to the principal end products, carbon
dioxide and water. But because the use of
a biological countermeasure imposes
special constraints, its use must be
carefully considered. To understand
these special constraints, consideration
must be given to the requirements of a
general countermeasure and to the
information needed to judge the suitability
of a biological countermeausre.
The objective of this study was to
investigate the feasibility of using
microbiological processes to mitigate
hazardous material spills in water-
courses. Toward that objective, a literature
search, screening tests, laboratory-scale
tests, and small-scale, simulated spill
tests were conducted to:
• identify candidate microorganisms
capable of degrading selected haz-
ardous materials;
• determine growth requirements and
environmental factors affecting the
growth of microorganisms that were
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found to break down hazardous
materials successfully;
• determine the fate of selected
hazardous materials, and of their
breakdown products, in water,
sediment, and biota;
• develop production, storage, recul-
ture, and deployment methods;
• investigate response requirements;
and
• evaluate the practical feasibility of
biological countermesures.
Study Description
To begin the biodegradability studies, a
list of 14 hazardous material candidates
was developed. A preliminary review of
the physical and chemical characteristics
of these materials revealed that several
compounds are quite soluble in water and
would be dispersed by natural mixing
processes upon spillage, whereas some,
like xylene and benzene, are relatively
insoluble but rapidly sorb to suspended
particles and thus would remain in
suspension. For experimental purposes,
the list included a variety of hazardous
materials because of their physical or
chemical nature after spillage. Likewise,
the chemical structures of the compounds
were variable enough to provide an
adequate test of biological countermea-
sures on these representative materials.
The final selection of hazardous
materials was made after a literature
review and initial screening tests. The
purpose of the literature review was to
gather information on the candidate
materials regarding (1) physical and
chemical characteristics of the most likely
"spilled" chemicals, (2) previous biode-
gradation and biological treatment inves-
tigations, and (3) toxicity to organisms in
fresh and marine waters. A summary of
the toxicity and biodegradation informa-
tion gathered from the literature review
appears in Table 1.
Screening tests were performed for 11
hazardous materials to examine whether
they were biodegradable and to determine
their suitability for biodegradability
measurement in a standard experimental
test. These tests (Table 2) were conducted
in aerated batch reactors seeded with
municipal sewage sludge. Nitrogen,
phosphorus, an alkalizing agent, and in
some cases glucose and glutamic acid (as
a supplemental food source) were added
to the reactor along with the hazardous
material. (Additions of glucose and
glutamic acid were found to be unneces-
sary when acclimated sludge was used.)
Total and volatile suspended solids (TSS
and VSS), biochemical oxygen demand
Table 1. Biodegradability and Toxicity^ of Hazardous Material Candidates
Hazardous material
Acetone cyanohydrin
Aery lonit rile
Aldrin
Benzene
Cyclic rodenticides
and insecticides
DDT
Isoprene
Methanol
Nitrophenol
Nonyl phenol
Phenol
Styrene
Xylene
Toxaphene
Biodegradation
shown?
Yes2
Yes
Yes
Yes
Yes3
Yes
No4
Yes
Yes
No
Yes
No4
Yes
No
Pathway
known?
Partially
Partially
Partially
Yes
Partially3
Partially
No"
Yes
Partially
No
Yes
No"
Partially
No
Toxicity
defined?
Yes
Yes
Yes
Yes
Yes
Yes
Partially
Yes
Yes
Partially
Yes
Partially
Yes
Yes
' The literature was reviewed to gather information on the physical/chemical characteristics of
these compounds, previous biodegradation and biological treatment investigations, and the
toxicity of the compounds to various organisms in fresh and marine waters.
2Folio wing chemical dissociation.
3For a few compounds.
4No direct evidence, but should be biodegradable; probable pathway known.
Table 2. Screening Tests to Determine Biodegradability
Hazardous
material
Volatilization
demonstrated
Biodegradation
demonstrated
Bacterial
cultures
Aldrin'
Aery lonit rile
Benzene
Isoprene
Methyl alcohol
Nitrophenol
Nonyl phenol
Phenol
Styrene
Toxaphene'
Xylene
X
X
X2
X
X
X
X
X
X
Azotobacter vinelandii
Clostridium pastereonin
Methanomonas methanica
Pseudomonas sp.
Micrococcus sp.
Pseudomonas sp.
Pseudomonas sp.
'Results were inconclusive.
2Although biodegradation was demonstrated, it is not feasible for large-scale field applications.
(BOD), pH, and total organic carbon (TOC)
were monitored throughout the tests. In
addition, microbiological samples were
removed for analysis.
In the screening tests, the behavior of
the hazardous material was sought as a
contaminant in domestic sludge; there-
fore, disappearance of TOC was used to
monitor removal (degradation or volatiliza-
tion). Of the compounds studied, phenol,
methanol, and nitrophenol appeared to
be the most amenable to measurements
related to biodegradation. Although
acrylonitrile and nonyl phenol were also
degraded by microorganisms, the reaction
conditions were viewed as incompatible
for large-scale field use. Because of
volatility, isoprene, benzene, styrene, and
xylene did not appear to be suitable
candidates for conclusive testing under
the experimental conditions used in this
study. Finally, the studies of aldrin and
toxaphene were inconclusive because of
sorption onto the reactor walls and
analytical difficulties.
Following the screening tests, the
three chemicals that appeared to be most
amenable to measurements related to
biological countermeasures (phenol,
methanol, and p-nitrophenol) were
subjected to treatability studies designed
to delineate growth kinetic coefficients,
the effects of environmental variables
(such as pH and temperature), and any
other information necessary to conduct
pilot-scale countermeasure tests. Based
on these treatability tests, the following
kinetic equations satisfactorily describe
the bacterial growth and substrate
removal kinetics:
. = -kXS_
dt
dt
dt Ks +S
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where:
X = biomass concentration, mg/l
S= substrate concentration, mg/l
t=time
a =cell yield coefficient (biological mass
produced/substrate used)
kd = cell decay coefficient, time'1
Ks = Michaelis-Menten constant, mg/l
(substrate concentration at which
the substrate removal rate is one-
half of the maximum rate)
k = substrate removal rate coefficient,
time"1
Table 3 summarizes the values of the
parameters experimentally determined
for phenol and methanol — the two
materials selected for simulated spill
tests.
Additional studies were conducted to
determine the effects of a variety of
experimental parameters, including
temperature, pH, nutrients, and minerals,
on bacterial growth and substrate removal.
Although the cell yield coefficient (a) and
the Michaelis-Menten constant (Ks) did
not change significantly with temper-
ature, the substrate removal rate (k) was
found to be temperature dependent for
both methanol and phenol. But the
modified Arrhenius equation, kj2 = kn
0IT2-TD common|y usec| to define the
effect of temperature on substrate
removal rate, did not accurately describe
the temperature effect on k. The tempera-
ture coefficient, 8, changed with temper-
ature range but fell between 1.0 and 1.4,
depending on substrate, pH, salinity, and
temperature. The substrate removal rate
was also found to be dependent on pH. In
phenol decomposition, which decreases
pH, the buffering capacity of the system
was important, whereas in methanol
decomposition, which does not signifi-
cantly affect pH, the initial pH of the water
was an important factor.
The effects of trace element (Fe++,
Mg++, Mn++, Ca++, and Zn++) and nutrient
(nitrogen and phosphorus) additions
Table 3. Kinetic Parameters
were also investigated. Addition of trace
elements caused a slight increase in the
phenol removal rate. The addition of
nutrients, however, had no effect on
phenol removal, but it did affect bacterial
cell synthesis and decay. Though the cell
yield coefficient (a) increased in the
presence of nutrients, the cell decay
coefficient (kd) decreased. In the methanol
studies, neither nutrients nor trace
elements had any effect on bacterial
growth or substrate removal.
In both methanol and phenol studies,
an initial lag phase was observed in the
substrate removal rate. The average
duration of the lag phase was roughly 4
and 9 hr for phenol and methanol,
respectively. To compensate for the lag
phase, supplemental aeration was re-
quired. Preliminary experiments indi-
cated that the lag phase reflected a lag in
bacterial growth rather than the decom-
position of the substrate into intermediate
compounds. The factors responsible for
the lag phase were not determined, but
the duration of the lag phase generally
appeared to decrease with increasing
temperature.
The oxygen requirements for biode-
gradation were also investigated. One
gram of carbon is assumed to produce
1.88 g of organic cellular material, based
on the approximate formulation of a
bacterial cell (CsHyNOa). Thus, based on
the previously calculated cell yield
coefficients, it was determined that
64.4% of the phenol and 66.5% of the
methanol degraded was used for new cell
synthesis. Given these assumptions, the
oxygen requirements for substrate re-
moval and bacterial growth could be
calculated (Table 4).
Following the kinetic studies, simulated
spill tests were conducted in aquaria and
in ponds to investigate further the
feasibility of using biological counter-
measures. The results of these tests were
used to develop experimental methods
Hazardous material
Parameter
K* mg/l
a, mg/l
k& time'1
Phenol
236 ± 70
1.21 ±0.06
0.066koe7
Methanol
2330 ± 1410
1.25 ± 0.45
0.011 Sk°eM
Table 4. Oxygen Requirements for Substrate Removal and Bacterial Growth
Amount of oxygen
used/g carbon for: Phenol
Methanol
Complete substrate oxidation
Complete oxidation of the cell
Cell synthesis
3.11 g
2.67 g
0.44
(3.11-2.67)
2.67 g
1.33
(4-2.67)
for large-scale biological countermeasure
studies in a 30.5-m diameter, 3.0-m deep
model lake. In addition, flowing-system
tests were carried out in a model river.
Specific objectives of aquaria tests
were to examine the hazardous material
removal rates as a function of bacterial
mass (i.e., the ratio of hazardous material
mass to bacterial mass) and as a function
of the addition of nutrient salts (NaHCO3,
KH2P04, (NH4)2S04). In the phenol tests, a
low phenol/bacteria ratio was desirable,
though precautions had to be taken to
ensure adequate dissolved oxygen levels.
The substrate/bacteria mass ratio studies
with methanol were inconclusive. For
both substrates, the addition of nutrients
(at levels determined from kinetic studies)
caused roughly a two-fold increase in the
biological decay rate. But doubling this
nutrient concentration did not increasethe
decay rate perhaps because the nutrients
reached a saturation level for bacterial
growth.
Pond tests were designed to evaluate
biological countermeasures in larger
systems, and more specifically (1) to
examine the effects of using acclimated
as opposed to unacclimated bacteria, and
(2) to evaluate the use of a portable
treatment unit containing bacteria. These
experiments confirmed the feasibility of
the countermeasure with acclimated
sludge, the possible use of unacclimated
sludge, and the potential utility of a
portable treatment unit.
In a model lake, dye tests were
conducted to determine the dispersion
rates and pattern of the hazardous
material and to develop a method for
calculating mass balance. The use of
Rhodamine B dye was demonstrated to
be a satisfactory method for establishing
a dilution baseline and for tagging the
hazardous material. In addition, the use of
a barrier to contain the hazardous
material and the biological countermeasure
(both acclimated and unacclimated sludge)
was investigated. A semi-rigid but
maneuverable barrier was able to contain
the spilled material successfully. As
expected, the decomposition rate of
phenol within the barrier was greater
with the use of acclimated, rather than
unacclimated, sludge.
A model river was used for material
transport and bulk sludge application
tests, studies of material exchange
through cloth bags, andconfining-barrier
application tests. Based on these investi-
gations, batch treatment systems were
recommended over continuous-stirred-
tank-reactor (CSTR) systems. The former
are simpler to design and construct.
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require less aeration time, and produce
more acclimated sludge than CSTR
systems. For in situ treatment, application
of bulk sludge was not an efficient
method for phenol and methanol because
of sludge settling. Sludge-containing
cloth bags were found to be a useful
means of preventing dispersion of the
spilled material.
Finally, it appears that the practicality
of biological countermeasures using
added organisms (as distinct from using
indigenous organisms) will depend on the
methods available for storage of the
bacterial culture. Preliminary studies
were conducted on freezing and on
lyophilization of phenol-acclimated bac-
terial cultures. Though the feasibility of
these storage techniques was demon-
strated on a small scale, further work is
needed to select appropriate storage and
reconstitution methods, determine the
shelf life of the stored material, evaluate
the need for nutrient and mineral
additions, and identify the amounts of
materials that will be needed for a given
spill (based on the chemical, the volume
of the spill, etc.).
Conclusions
1. Based on criteria for assessing
potential countermeasures for mitigating
hazardous material spills, biological
countermeasures appear useful because:
a. Microorgnisms may be effective
under some conditions for remov-
ing certain hazardous materials.
b. Microorganisms that attack a
variety of hazardous materials
exist (e.g., the Pseudomonads).
c. It should be possible to deploy
microorganisms in situ or in a
portable treatment system in a
fresh liquid state, a powdered
state, or a freshly reconstituted
state.
d. Potentially harmful secondary
effects should be minor because
microorganisms are a natural part
of the aquatic environment. Path-
ogenic bacteria will not likely
constitute a significant part, if
any, of the countermeasure;
noxious sludge should not be
formed; and microorganisms
should not persist, since they
should metabolize their own
protoplasm following consumption
of the hazardous material and
disappearance of the food source.
2. Based on the treatability tests, the
following conclusions are made pertaining
to the use of batch treatment systems as a
countermeasure:
a. Batch treatment systems are
preferred over continuous-stirred
tank-reactor (CSTR) systems for
spills of phenol and methanol,
especially when the spill concen-
trations are high. Batch systems
require much less aeration time to
achieve a certain effluent quality
and produce more acclimated
sludge than CSTR systems.
b. Batch systems can be designed
using numerical methods or using
batch kinetic diagrams.
c. Sludge-containing cloth bags were
found useful for easy containment
of the sludge when consecutive
batch treatments were required.
Floating cloth bags can be used
for in situ treatment methods to
prevent sludge settling.
d. When sludge-containing bags are
used for the removal of spills, the
substrate removal rate by or-
ganisms can be expressed as:
ds =. EkXS
dt Ks+S
e. When sludge-containing cloth
bags are used in a batch treatment
system, the system can be design-
ed in the same manner as a
regular batch system except that
Ek instead of k is used for the
substrate removal rate coefficient.
The aeration time required to
achieve a given removal was
observed to be slightly less than
the theoretically computed time,
probably owing to organisms that
escaped from the cloth bags.
3. Based on spill control tests in a
model river, the following conclusions
can be made:
a. Application of bulk sludge in
streams is not an efficient method
for phenol and methanol removal
because of sludge settling. Floating
cloth bags may be used to prevent
this problem; however, this method
is highly restricted by the reaera-
tion capacity of streams and the
large amount of acclimated sludge
required.
b. Fixed, confining barriers may be
used to prevent the dispersion of
spills. Once pollutants are con-
tained within barriers, they may
be treated in a batch manner.
Cloth bags may be employed
when the mixing intensity is not
sufficient for complete suspension
of sludge. Oxygen need only be
supplied within or near the cloth
bags.
4. Based on model lake tests, the
following conclusions can be made:
a. Phenol spills contained by a
barrier may be removed using
unacclimated sludge from a local
activated sludge domestic waste
treatment plant.
b. Use of biological countermeasures
will result in a significant impact
on the dissolved oxygen resources
in the aquatic system. However,
this impact can be reduced by
mechanical aeration.
Recommendations
The study recommends continuing
development of biological countermea-
sures and a major research emphasis on
the following items:
1. Studies on countermeasure storage
and reconstitution to determine the
shelf-life of the stored material, the
need for additions of mineral salts,
and the amount of material needed
for spills.of a given chemical;
2. Development of techniques for
countermeasure application in quie-
scent and flowing systems;
3. Determination of additional can-
didate chemicals for application of
biological countermeasures; and
4. Further confirmation of methods
for calculating amounts of the
countermeasure needed for a given
spill volume.
The full report was submitted in
fulfillment of Grant No. R802207 by the
University of Texas under the sponsorship
of the U.S. Environmental Protection
Agency.
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Neal E. Armstrong, Ernest F. Gloyna, and Orville Wyss are with the University of
Texas, Austin, TX 78712.
Joseph P. Lafornara was the EPA Project Officer (see below for present contact).
The complete report, entitled "Biological Countermeasures for the Control of
Hazardous Material Spills," (Order No. PB 84-140 276; Cost: $22.00, subject
to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
For further information contact Anthony N. Tafuri at:
Oil and Hazardous Materials Branch
Municipal Environmental Research Laboratory—Cincinnati
U.S. Environmental Protection Agency
Edison, NJ 08837
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Environmental Protection
Agency
for Environmental Research
Information
Cincinnati OH 45268
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