United States
Environmental Protection
Agency
Municipal Environmental Research -
Laboratory
Cincinnati OH 45268
Research and Development
EPA-600/S2-83-117 Dec. 1983
Project Summary
Microbial Degradation of
Selected Hazardous Materials:
Pentachlorophenol,
Hexachlorocyclopentadiene, and
Methyl Parathion
Nora K. Thuma, Patricia E. O'Neill, Shirley G. Brownlee, and Ralph S. Valentine
This program was a limited feasibility
study in which a number of pure
microbial cultures were evaluated for
potential in biodegradation of penta-
chlorophenol (PCP), hexachlorocyclo-
pentadiene (HCCP), and methyl para-
thion (M P) in an aqueous medium under
aerobic conditions. Following the initial
screening and selection process, pure
culture organisms identified as having
potential for biodegradation of the
selected chemicals were subjected to
further testing and evaluation. Although
no completely conclusive evidence for
biodegradation of these substances
was obtained, data indicate that a
number of fungi have potential for the
disposal of PCP, HCCP, and MP. One
bacterial culture demonstrated toler-
ance to PCP at 200 ppm in soil and
reduced the PCP concentration in an
aqueous medium when dextrose was
provided. This bacterial isolate, as well
as a fungal isolate, may have potential
for removal of PCP from spill-contami-
nated areas.
In the testing performed, the percent-
disappearance of the challenging haz-
ardous material was corrected for bio-
accumulation and settling out; electron
capture gas chromatographic (GC-EC)
analysis of extracts from several penta-
chlorophenol tests did not indicate the
presence of chlorinated byproducts or
of metabolites. Additional tests are
recommended. Improvements on exist-
ing high performance liquid chromatog-
raphy (HPLC) and GC-EC methods were
achieved.
The limited scope of this project did
not allow for sufficient adaptation of
cultures for complete biological removal
of the selected chemicals. Time con-
straints and budgetary requirements
precluded the use of C-14-labeled
chemicals and the extensive analyses
required for the isolation, identification,
and quantification of potential metabo-
lites or the byproducts of biodegrada-
tion or biotransf ormation of the selected
chemicals.
This Project Summary was developed
by EPA's Municipal Environmental Re-
search 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
Large volumes of hazardous substances
and wastes are transported each year
throughout the United States via high-
ways, railroads, and waterways. In spite
of precautions taken in the transport and
transfer of these substances, accidents
resulting in spills of the materials do
occur. In addition to accidental spills of
hazardous materials, improper or inten-
tional disposal or dumping of chemicals—
both in containers and directly—has con-
taminated much land. Spills pose serious
threats to the environment and, indeed,
to human life. Irv many instances, the
technology for effective cleanup of a
hazardous materials spill or release does
not exist.
Biological degradation is recognized as
an effective process for disposing of or
detoxifying many spilled organic mate-
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rials. Biodegradation may be used either
as a controlled method in biological
reaction systems or as a natural method
acting through indigenous microbial pop-
ulations. Biodegradation of many of the
chemical substances designated as haz-
ardous materials has been demonstrated.
As research further elucidates the meta-
bolic capabilities of microorganisms,
other substances may be found to bio-
degrade under a given set of conditions.
Some chemicals are considered biore-
fractory simply because microorganisms
found in conventional biological treat-
ment systems, or in the soil or water
receiving a spill, are unable to biodegrade
the material. In such cases, the seeding
with microorganisms endogenous to the
site, the use of microbes that have been
adapted in situ or through culturing, or
the alteration of environmental or nutrient
conditions may result in biodegradation
of hazardous materials that resisted bio-
logical destruction or detoxification be-
cause of structure, composition, concen-
tration, or ambient conditions.
Results and Discussion
General
The reported studies were done aero-
bically in a basic salts medium that
commonly had the composition:
KzHPCu 1.0g
KH2PO4 1.0g
NH4N03 1.0g
CaCI2 (0.027 g/ml) 1.0 ml
MgSCu (0.022 g/ml) 5.0 ml
FeCI3 (0.00025 g/ml) 1.0 ml
distilled water (to make). .. 1.0 liter
Depending on the desired test condi-
tions, the pH was adjusted between 6.5
and 8.5 by altering the level of potassium
dihydrogen phosphate or by adding
sodium hydroxide. Most tests included a
carbon food source; dextrose at the 0.1 %
or 0.2% level was most commonly used,
but tests were also run with yeast extract
and with peptones.
A first attempt was always made to
challenge microorganisms with high
levels of hazardous materials (HM) as
aqueous pollutants, namely, 2 g/liter of
PCP, 1 g/liter HCCP, and 1 g/liter of MP
(as Monsanto Methyl Parathion 500* with
47% active ingredient).
'Mention of trade names or commercial products
does not constitute endorsement or recommenda-
tion for use
In practice, the PCP solubility was
strongly dependent on pH, so that most
work was done at pH 8.5 at ca. 200 ppm.
Further, HCCP was very difficult to solu-
bilize. Conventional techniques were
used to achieve dispersion; nonetheless,
settling out remained a major problem so
that probably no more than 500 ppm
actually dissolved or remained suspended
in the medium. Even this concentration
(500 ppm) is suspected of being higher
than the actual concentration; a more
realistic level is less than 50 ppm. Be-
cause of the difficulties encountered in
solubilizing HCCP, the work with that HM
was abandoned; without reasonably high
concentrations of HM in the medium,
analytical data on percent-removal are
too unreliable. In the work with MP, the
initial concentration was 250 ppm. Some
experiments were done on PCP+oil mixed
with soil (this was a simulant for an actual
spill site), where the concentration of
PCP+oil in soil ranged up to 15% (v:w)
with the PCP concentration having a
maximum of 4500 ppm (in sandy soil).
Normally, each HM was challenged by
24 pure culture microorganisms at room
temperature on rotary shaking tables;
there were two controls. The cultures
were obtained in four ways: (1) American
Type Culture Collection (ATCC) (pure,
identified, on slants); (2) Quartermaster
Corps at Natick, MA (same at ATCC); (3,)
contractor cultures and subcultures of
lower purity (not always well-identified
by genus and species and obtained from
soil, sludge, newspapers, bagasse, and
other sources); and (4) subcultures (not
identified by genus and species but
characterized by morphology, color, etc.)
that were obtained by serial subculturing
from screening tests in which microorga-
nisms appeared to have mutated and
were achieving biodegradation.
Not only in the screening tests but in
other work, samples were usually centri-
fuged and the centrifugate and sediment
were analyzed separately. In some cases
(because of time/funding constraints),
only the supernatant was analyzed; in a
few cases during pilot plant runs, the
samples were not centrifuged but the
entire sample was extracted.
PCP was originally analyzed by applying
HPLC to processed hexane extracts. Sub-
sequently, GC-EC procedure was applied
to an acetate derivative of PCP. Similarly,
a hexane extract of HCCP was analyzed
both by HPLC (using acetonitrile) and by
GC-EC. In the case of MP, the commercial
product was first diluted with methanol.
For analysis, a 1-to-1 medium-to-meth-
anol extract was fed to the HPLC. Repro-
ducibility of data was poor. Further, there
was only about 50% recovery (of MP by
HPLC analyses) for known standards. In
screening experiments, for example, 166
ppm of MP was added to the controls but
only 96 ppm was recovered. Additional
work is required to develop an analytical
method that will reliably reflect MP levels
in the medium or in the biomass (bioac-
cumulated or precipitated). Note, how-
ever, that the analytical protocols were
based on published data available in
1977; analytical procedures of improved
reliability are undoubtedly available now.
PCP Screening Experiments
Following incubation at room tempera-
ture on a rotary shaking table, the con-
tents of each flask were centrifuged.
Supernatants and sediments were ex-
tracted and analyzed by HPLC. These
studies indicated that fungal isolates
were the most likely candidate organisms
for removal of PCP from the medium.
Subsequent tests included evaluation of
three bacterial isolates obtained from
PCP-contaminated media and a fungal
contaminant of a HCCP medium. Data
from tests in which the basic salts
medium was maintained at a pH of 8.5
(addition of NaOH) indicated that bacterial
isolate 041 was a candidate organism for
further study. (Organism 041 was a
gram-positive coccus isolated from PCP-
contaminated medium. Colonies of this
microorganism were rough, off-white,
and irregular when subcultured on a
basic salts agar plus yeast extract.) The
final screening studies on biodegradation
of PCP used the basic salts medium
containing 0.1% dextrose for evaluation
of the degradation potential of bacterial
isolate 041 and fungus 044. (The fungus
was isolated from an HCCP-contaminated
medium.) The bacterium (041) reduced
the concentration of PCP in the medium
by 20% to 42%, whereas the fungus
reduced the concentration by 30% to 39%
in the screening experiments. Based on
these results, it appeared that bacterial
isolate 041 and fungus 044 were candi-
date organisms for evaluation of PCP
removal in pilot-scale experiments.
HCCP Screening Experiments
The initial HCCP screening experiments
used methanol as a solvent for addition of
the HCCP to the basic salts medium
containing 0.1% dextrose. Twenty-four
organisms were separately incubated
with HCCP at an original concentration of
1000 ppm. Following incubation at room
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temperature on a rotary shaking table,
the entire contents (medium and biomass
with settled HCCP) of each flask were
extracted separately and analyzed for
HCCP. The HCCP concentration was
reduced in seven of the flasks when
compared to the controls. In the second
screening experiment, the seven most
effective organisms were tested when
HCCP was added to the medium without
the solubilizing agent (methanol). Results
from this comparison indicated that, in
14-day tests, the presence of the solu-
bilizer enhanced the reduction in HCCP
concentration in three of the seven flasks,
but that the other four flasks outper-
formed with no solubilizer. In the third
test, HCCP removal was followed inflasks
containing the seven organisms and 1000
ppm HCCP (in methanol) added to a basic
salts solution with dextrose. The entire
contents of each flask were extracted and
analyzed for HCCP following 6 days of
incubation on a rotary shaking table at
room temperature. The HCCP concentra-
tion was reduced in all test flasks (the
range was 10% to 60%). A bacterial
culture (006), a yeast (369), and two
fungal cultures (022 and 123 (QM-9123))
were identified as the organisms with
greatest potential for HCCP degradation.
Bacterium 006 is a gram-negative rod
isolated from septic tank seed. Organism
369 (ATTC-1369) is a yeast, Candida
tropicalis. The putative fungus (022) was
isolated from soil and produced white,
stringy colonies on Sabouraud's agar.
Gram strains showed huge, oblong, rec-
tangular gram-positive rods with connect-
ing branches. Organism 123 was Tricho-
derma viride.
Maintenance of HCCP in suspension
was recognized as a major problem since
the distribution of HCCP between the
medium and the biomass (including set-
tled HCCP) indicated extensive loss of
HCCPfrom solution. A number of emulsi-
fying agents and techniques were tried
with no marked success. In view of more
promising results in the PCP area and
because of the difficulty of solubilizing
(emulsifying) HCCP, for example, in soil
that had been contaminated by a spill,
work on HCCP was halted. HCCP biode-
gradation can occur; however, the mate-
rial is not irrevocably biorefractory.
MP Screening Experiments
Seven series of experiments were
undertaken to evaluate the potential of
pure cultures for the degradation of MP.
Again, the initial screening test evaluated
24 organisms. Preliminary experiments
indicated that disappearance of MP from
the medium was increased when dextrose
was provided as a nutrient source. Four
microbial cultures were selected in fur-
ther screening studies and monitored for
removal of MP following 6 days of incu-
bation at room temperature. Data from
these studies confirm that the presence
of dextrose in the medium increases the
reduction of MP concentration. A bac-
terium (003) and a fungus (021) were
identified as candidate organisms with
potential for biodegradation of MP. The
003 culture was isolated from septic tank
seed and gram strains showed gram-
positive cocci in clusters. Fungus culture
021 was tentatively identified as Rhizopus
sp.
With dextrose present, the percent-
disappearance of MP with organisms 003
and 021 ranged between 30% and 50%. A
few organisms (010, a fungus isolated
from cotton seed; 016, a soil fungus; 020,
a soil fungus; 123, a fungus, Trichoderma
viride; 645, a fungus, Gliocladium virens)
showed high (40% to 65% removal) over
the first few days but then lost biodegra-
dation capability. Time did not permit
serial subculturing to determine whether
there was: die out, a latency period during
buildup of mutants that could improve
degradation, loss of the organism by
competition (biological selection), or some
other occurrence. As with the HCCP work,
a decision was made to spend the remain-
ing effort on PCP; further work on MP was
halted.
Batch Tests
Only a limited number of batch tests
were conducted. The majority of these
tests was designed to determine the best
level of a bacteriostatic agent to control
the growth of competing bacteria in the
degradation of PCP by a fungus (organism
044). Sodium azide (NaN3) is known to
adversely affect the cell wall of bacteria
(and thus lead to lysis and lack of growth)
but was not known to affect the growth of
fungi. The batch tests were run in aerated
40-liter tanks containing basic salts (pH
8.0), 0.1% dextrose, 100 ppm PCP, and
NaN3 ranging from 0.1% to 0.002%. No
azide was added to the controls. About
0.5 g of PCP was added to each tank daily,
along with dextrose and medium, as
required. The data showed that 0.002%
NaN3 was an appropriate concentration
of bacteriostatic agent for the pilot-scale
PCP studies where the biodegrading
organism was the fungus 044.
PCP Pilot-Scale Tests
The screening studies on PCP, HCCP,
and MP revealed that PCP was the most
acceptable choice for pilot studies. Fur-
ther, there was interest in determining to
what extent a biodegradation process
might be applicable to an actual release/
spill situation involving PCP and fuel oil in
soil.
The pilot scale unit (Figures 1,2, and 3)
was fabricated from methyl methacrylate
sheets and divided into four chambers
that serially overflowed from the first to
the second, etc. The unit was tightly
sealed, as was the feedstock tank. Each
chamber contained 24 liters (6 gal) of
medium and was equipped with wands
for introducing filtered air for aeration
and mixing. Peristaltic pumps were used
to meter the feed and to proportion the
sludge return. Typical pumping rates
were0.25gal/hrof feedandO.1 gal/hrof
sludge return. The chambers were con-
nected by weirs; the overflow (exit) pipe
extended to the top of the fourth chamber.
Depending on the design of each test and
on changes in protocol during the tests
(which were conducted over a period of 2
weeks each), the residence time in the
system ranged from 40 to 100 hours. The
chambers were routinely monitored for
temperature, DO, and pH.
The pilot-scale system was first checked
by filling the storage chamber and tanks
with basic salts and 0.1% dextrose and
then inoculating the chambers with bac-
terium 016 (800, 500, 300, and 300 ml
inoculum in chambers 1 through 4,
respectively). [Organism 016 forms "puff
balls" that are easily detected (Figure 3).]
The air flow and fluid movement patterns
were observed and some modifications in
design were made. In the second run, the
system was cleaned, refilled, and again
inoculated with organism 016. Percent
BODs reduction was measured in each of
the four chambers. Note that this is a
flow-through system and processed about
54 gal/day. The initial DO (feed) was
about 2 ppm, but decreased to about 0.5
ppm in chambers 1 and 2 until rising to 5
to 6 ppm in chambers 3 and 4. BOD of the
feed was about 2000 ppm. Equilibrium
BOD5 reduction in the chambers was
34%, 70%, 92%, and 93%, respectively
(cumulative percents).
Four pilot-scale tests were performed
with PCP. The medium containing dis-
solved and possibly suspended PCP, as
well as PCP in the biomass, was sampled.
Extracts were processed and derivatized
for quantitation by GC-EC. Separate
analyses were performed to correct
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percent-removed for loss by settling or by
incorporation in the biomass.
In pilot test no. 1, the feed contained
100 ppm of PCP at a pH of 8.5. The
inoculum was the fungus 044. An anti-
foaming agent was added. As the degrada-
tion proceeded, the pH dropped and the
rate of degradation decreased. (The solu-
bility of PCP is much greater in basic
solutions.) The maximum percent-
Four Chamber Reactor Unit With Lid
Sampling Port
\
Air Infusion
Control
Effluent
A,r Compressor Overflow
Drain
Peristaltic Pump \ Influent
Cooled
Air
Cooling Water
Sludge
Return Pump
Figure 1. Schematic diagram of continuous pilot-scale reactor.
Figure 2. Continuous pilot plant unit.
removal (corrected for bioaccumulated
PCP) was over 90%. Attempts to regain
the high degradation rate were not suc-
cessful.
In the second test, similar conditions
were used and data obtained. Acid metab-
olites lowered the pH and precipitated
quantities of PCP, such that the total PCP
level in the sludge sometimes approached
500 ppm. Note that the pilot tests were
performed in continuous, flow-through
reactors and that biomass (sludge) had to
be partly voided and partly reintroduced.
In the third test, 0.002% NaN3(bacterio-
stat) was added. The system malfunc-
tioned and, after the problem was cor-
rected, reinoculation (on days 5 and 10 of
the 14-day test) resulted in 70% disap-
pearance.
In test no. 4, organism 041 (bacterium)
was evaluated. Unfortunately, the feed
tank developed overwhelming bacterial
contamination. A new feed system was
developed. Changes were also made in
the nutrient (yeast extract was added to
the dextrose) and in the basic salts
medium (NH4NO3 was eliminated and
replaced by yeast extract). The recycling
of biomass was halted. The analysis
showed unacceptable variability (the bio-
mass, for example, formed an emulsion
with the hexane extractant). The system
may have achieved greater than 70%
disappearance of the PCP. The pilot work
showed definite promise, but had to be
abandoned (limited funding).
Under the best conditions during limited
experiments with PCP in a flow-through,
pilot-scale test chamber, the percent-
disappearance (corrected for assimilation
and precipitation) was about 95%. The
residence time in these experiments
ranged from 40 to 100 hr.
PCP-Spill-Contaminated
Soil Tests
Over a period of years more than a
decade ago, a spent mixture of PCP+fuel
oil, used as a wood preservative, was
routinely pumped into a 25-ft-deep well
at a plant in Haverford, PA. The material
migrated through the soil, entered an
unused, deeply buried drainage pipe, and
polluted a small stream at a housing
development about one-half mile away.
The leachate is being collected and
processed but the question remains as to
whether any pretreatment in the soil
itself might be advantageous.
Microbial test cultures were taken from
the disposal-well bottom liquid, from a
test boring about 300 ft distant, and from
the leachate into the stream. The well
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Figure 3. Continuous pilot plant unit on 8th day of operation with organism 016 (fungus) and
food (no pollutant).
culture showed only 2 colony-forming
units (cf u)/ml, but the other sites showed
levels of 8 x 104 and 3 x 106 cfu/ml,
respectively. The first sample was essen-
tially sterile. The others showed high
levels of bacteria but almost no fungi or
actinomycetes, which commonly account
for 50% of the cfu's in (uncontaminated)
soil.
Lab tests with bacterium 041 and
fungus 044 were conducted in beakers
on separate sterile samples of sand and of
soil that had been contaminated with
levels of the PCP-oil mixture (about 1%
PCP in oil). The sand or soil samples
weighed 200 g and the fraction of PCP+oil
added ranged from 0 (control) to 30 ml
(15% v:w). Basic salts medium with 0.2%
dextrose was added. Both bacterium 041
and fungus 044 grew in the PCP-free
control.
At levels of about 1%PCP+oil in the test
beakers (100-300 ppm PCP in sand),
bacterial growth was strong but fungal
growth (044) was somewhat inhibited.
Tests with sterile soil, repeated up to the
2% level, showed that growth rate de-
creased with increasing PCP+oil level.
Subcultures (agar and also liquid medium)
were taken after 4 days of incubation.
The data showed that the growth of
fungus 044 was significantly inhibited at
PCP+oil levels above 0.5% (v:w) but that
bacterium 041 was more tolerant with
severe inhibition, only occurring beyond
the 2% level.
Determination of the extent of degrada-
tion or of any byproducts or metabolites
formed could not be undertaken (funding
and time constraints).
It is surmised that bacterium 041 and
even fungus 044 could possibly be adap-
ted to consume the PCP+oil contaminant
if aerobic growth were initiated at the
fringe of the contaminated area at Haver-
ford and if nutrients and possibly some
food other than the oil (e.g., dextrose)
were provided.
Conclusions
The following conclusions are drawn
from this study:
1. The limited solubility of PCP, HCCP,
and MP in water makes it difficult to
evaluate microbial assimilation, or
biodegradation, or both in an aqueous
system.
2. Only tentative evidence for biodegra-
dation of PCP, HCCP, or MP by pure
culture organisms was obtained in
these studies from shaker table tests.
3. Data indicate that a fungal isolate
(044) has potential for assimilation of
PCP and may cause degradation. A
bacterial isolate (041) appeared to
reduce the PCP concentration by 20%
to 42% when dextrose was provided
as a carbon source.
4. A flow-through bioreactor (fungal
isolate 044, aerobic, pH 8.5, with
nutrients and dextrose added, and
having a residence time of ca. 50
hours) achieved 95% disappearance
of PCP (100 ppm) corrected for bioac-
cumulation and precipitation and has
putative potential for the degradation
of PCP and other organochlorine
compounds. In severely limited and
incomplete analytical work, no by-
products or partial decomposition
products (metabolites) of PCP were
detected.
5. The bacterial isolate (041) tolerated
higher concentrations of PCP+oil in
sandy soil than did the fungal isolate
(044). The bacterial isolate may have
greater potential than the fungal
isolate for removal of PCP from spill-
contaminated areas.
6. The amount of HCCP that disap-
peared from aqueous suspensions
was increased by the presence of
several of the pure culture organisms
when compared to the controls.
Based on very limited analyses, no
chlorinated byproducts were ob-
served in the aqueous medium as-
sayed for HCCP removal by micro-
organisms.
7. Methyl parathion studies indicated
that two organisms (bacteria 003 and
021) may have potential for removal
of this pesticide when a carbon
source, such as dextrose, is provided.
Recommendations
Based on the results of the study, the
following recommendations are made:
1. Bacterium 041 should be further
evaluated for potential use in the
decontamination of PCP/oil spill
sites.
2. Adaptation of organisms for removal
of PCP, HCCP, and MP should be
evaluated.
3. Future biodegradation studies in this
area should use C-14-labeled com-
pounds or follow other accepted
procedure to facilitate isolation and/
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or identification of metabolites (and
byproducts) and elucidation of mech-
anisms of biotransformation or bio-
degradation.
4. Analytical protocols should be de-
veloped to provide reproducible, if
not 100%, recovery levels of chal-
lenging compounds (such as HCCP
and PCP) from mixtures with fungi
and bacteria. (Such mixtures have a
tendency to promote emulsification
and to yield poor separations with
extractants.)
F Attempts should be made where
practical to identify effective organ-
isms minimally by genus and prefer-
ably by species.
The full report was submitted in fulfill-
ment of Contract No. 68-03-2491 by
Atlantic Research Corporation under the
sponsorship of the U.S. Environmental
Protection Agency.
N. Thuma, P. O'Neill, S. Brownlee, and R. Valentine are with Atlantic Research
Corporation, Alexandria. VA 22314.
John E. Brugger is the EPA Project Officer (see below).
The complete report, entitled "Microbial Degradation of Selected Hazardous
Materials: Pentachlorophenol, Hexachlorocyclopentadiene, and Methyl Para-
thion," (Order No. PB 84-123 934; Cost: $11.50, 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:
Oil and Hazardous Materials Spills Branch
Municipal Environmental Research Laboratory—Cincinnati
U.S. Environmental Protection Agency
Edison, NJ 08837
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United Slates
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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