Microbially mediated degradation of nitrogen-containing phenol compounds

Nitrogen-containing phenol compounds are biodegradable by a consortium of microorganisms. The consortium was isolated from waste sludge by successive subculturing into medium containing picric acid as the only carbon source. During the period of degradation, picric acid was seen to degrade to a colored intermediate which later disappeared. UV-Vis spectrometry, HPLC and GC-mass spectrophotometry showed that the entire ring structure was eventually destroyed. The consortium contains microorganisms from the genera Arthrobacter, Avrobacterium and Pseudomonas.

FIELD OF INVENTION 
The present invention relates to the microbial-mediated degradation of 
picric acid and other nitrogen substituted phenols by a consortium of 
microbes. More specifically, a defined mixture of bacteria have been 
isolated that have the ability to use picric acid as a sole carbon source 
and completely degrade picric acid to the level where no aromatic 
degradation products can be detected. 
BACKGROUND 
Picric acid (2,4,6-trinitrophenol) is a compound used in a variety of 
industrial applications including the manufacture of explosives, aniline, 
color fast dyes, pharmaceuticals and in steel etching. Picric acid and 
ammonium picrate were first obtained as fast dyes for silk and wool. 
However, the unstable nature of picric acid was soon exploited for use as 
an explosive and explosive boosters where it is the primary component of 
blasting caps which are used for the detonation of 2,4,6-Trinitrotoluene 
(TNT). Because of its explosive nature, disposal of waste picric acid 
poses unique hazards not generally associated with other environmental 
toxicants. 
Mounting public concern and increasing government regulations have provided 
the impetus for a safe, effective means to remediate picric acid 
contaminated environments. Past methods of disposing of munitions and 
other wastes containing picric acid have included dumping at specified 
land-fill areas, isolation in suitable, reinforced containers, land based 
deep-welling, dumping in deep water at sea and incineration. All of these 
methods carry some potential for harm to the environment. For example, 
incineration creates a problem of air pollution and disposal on land risks 
the possibility that toxic substances will elute or leach into locations 
where they may threaten aquatic life forms, animals or humans. A more 
desirable disposal method might incorporate a chemical or enzymatic 
degradative process. 
The metabolic reduction of organic nitrogen groups has been known for some 
time. Westfall, J. Pharmacol. Exp. Therap., 78:386 (1943) reported that 
liver, kidney and heart tissue are active in the reduction of 
trinitrotoluene, however, was not able to identify the specific enzyme 
system responsible. Westerfield et al., J. Biol. Chem., 227:379 (1957) 
further disclosed that purified xanthine oxidase is capable of reducing 
organic nitrogen groups and demonstrated that the molybdenum (Mo) 
co-factor was essential in the degradative process. 
Microbial degradation of organic nitrogen compounds has been limited to a 
handful of organisms. Erickson, J. Bact., 41:277 (1941) reported that 
certain strains of Micromonospora were able to utilize picric acid and 
trinitro-resorcinol as a carbon source and Moore. J. Gen. Microbiol., 
3:143 (1949) described two unspecified proactinomnycetes as being capable 
of using nitrobenzene as a simultaneous source of carbon and nitrogen. 
Gundersden et al., Acta. Agric. Scand., 6:100 (1956) described the 
metabolism of picric acid by Corynebacterium simplex which was isolated 
from soil as a 4,6-dinitro-2-methylphenol-degrading organism. Degradation 
was determined by measuring the amount of nitrate produced when the 
organism was contacted with an organic nitrogen compound. The extent of 
degradation and the identification of specific degradation products were 
not reported. Later, Wyman et al., Appl. Environ. Microbiol., 37(2):222 
(1979) found that a strain of pseudomonas aeruginosa reduced picric acid 
to 2-amino-4,6-dinitrophenol (picramic acid) under anaerobic conditions. 
Wyman further determined that degradation products from both picric and 
picramic acid produced by this strain demonstrated mutagenicity as assayed 
by the standard AMES test. Another pseudomonas sp., P. putida, has been 
shown to be able to use picric acid as a carbon source and achieve some 
bio-conversion of the compound to 1,3,5-trinitro benzene, 
2,4,6-trinitroaldehyde, and 3,5-dinitrophenol. Kearney et al., 
Chemosphere, 12 (11-12):1583 (1983). 
Most recently, Rhodococcus erythropolis has been identified as a picric 
acid degrading bacteria. Lenke et al., Appl. Environ. Microbiol., 
58(9):2933 (1992) teach that R. erythropolis, under aerobic conditions, 
can incompletely utilize picric acid as a nitrogen source producing 
nitrite and 2,4,6-trinitro-cyclohexanone, which cannot be degraded 
further. 
In spite of the investigative activity in the area of microbial degradation 
of picric acid and other organic nitrogen compounds, there remain several 
difficulties to overcome before any of the above mentioned microbial 
systems can be used for the effective remediation of contaminated 
environments. All of the microbes investigated are isolated organisms and, 
although they show picric acid degrading activity in vitro, there is 
little evidence that these organisms will function under in situ 
conditions. Additionally, no organism or group of organisms has been 
isolated that demonstrate complete degradation of picric acid. At present 
the art teaches that only partial degradation is possible and that some of 
the degradation products may also be harmful to the environment as 
mutagens. There remains a need, therefore, for an effective degradative 
process for picric acid and related compounds that will degrade those 
compounds completely and be effective in both the in vitro and in situ 
remediation of contaminated environments. 
SUMMARY OF THE INVENTION 
The present invention provides a consortium of microorganisms able to use 
nitrogen-containing phenol compounds as a sole carbon source and of 
completely degrading them so that no aromatic degradation products are 
detectable. Nitrogen-containing phenol compounds may include, but are not 
limited to ammonium picrate, picramic acid, 2,4-dinitrophenol (2,4-DNP), 
2,5-dinitrophenol (2,5-DNP), 2,6-dinitrophenol (2,6-DNP), 3-aminophenol, 
2-aminophenol, 4-aminophenol, 2,4,6-trinitrotoluene, mononitrophenols, and 
nitroaromatics. 
The present invention further provides a method for degrading picric acid 
and related compounds by growing a consortium of microorganisms capable of 
the complete degradation of nitrogen-containing phenol compounds under 
suitable conditions.

As used herein, "ATCC" refers to the American Type Culture Collection 
international depository located at 12301 Parklawn Drive, Rockville Md. 
20852 U.S.A. 
DETAILED DESCRIPTION OF THE INVENTION 
As used herein the following terms may be used for interpretation of the 
claims and specification. 
The term "contaminated environments" will refer to any environment 
contaminated with picric acid or related compounds. Typical contaminated 
environments may include, but are not limited to, soil, ground water, air, 
waste disposal sites, and waste streams. 
The term "picric acid" will refer to the compound 2,4,6-trinitrophenol. 
The term "nitrogen-containing phenol compounds" will refer to any phenol 
ring compound substituted with at least one NO.sub.2 or NH.sub.2 group or 
salts of the substituted compound. The phenol ring may also contain any 
other non-nitrogen chemical substitutions. Typical related compounds may 
include but are not limited to, ammonium picrate, picramic acid, 
2,4-dinitrophenol (2,4-DNP), 2,5-dinitrophenol (2,5-DNP), 
2,6-dinitrophenol (2,6-DNP), 3-aminophenol, 2-aminophenol, 4-aminophenol, 
2,4,6-trinitrotoluene, mononitrophenols, and nitroaromatics. 
The term "complete degradation" refers to the degradation of picric acid 
and related compounds to a point where no aromatic degradation products 
may be detectable. 
The terms "microbial consortium" or "consortium" refers to any collection 
of microorganisms having at least two different species, capable of 
completely degrading picric acid and related compounds only when they 
occur together. The consortium may consist of many species of the same 
genera, many different genera of the same family or even members of 
different families of microorganisms. 
The present invention relates to a novel consortium of microorganisms 
capable of the complete degradation of picric acid and related compounds 
and the use of said consortium for the remediation of picric acid from 
contaminated environments. 
The microbial consortium of the present invention may consist of any 
combination of microorganisms where at least two different species are 
present. Most preferred is a consortium that includes, but is not limited 
to the bacterial species Arthrobacter uratoxydans, Aurobacterium saperdae, 
Bacilllus cereus, Flavobacterium esteroaromaticum, Micrococcus luteus, 
Microccus varians, Methylobacterium mesophilicum, Pseudomonas putida, and 
Ochrobacterium anthropi. 
The consortium of the present invention was isolated from a waste treatment 
facility of an industrial site and selected by two methods. In the first 
method, a sample of waste material was inoculated into minimal medium 
supplemented with picric acid. The culture was subcultured into fresh 
medium three times at 100 h of culture. Each subculture was tested for its 
ability to degrade picric acid and related compounds. 
In the second method, a sample of waste stream material was inoculated into 
minimal medium containing aniline waste stream effluent which contained 
about 2500 ppm picric acid. The final concentration of picric acid in the 
culture was 125 ppm. The culture was subcultured three times at 100 h of 
culture. Each subculture was tested for its ability to degrade picric acid 
and related compounds. 
The concentration of picric acid was monitored during culture over a 66 h 
period by HPLC using a diode-array detector. Scanning of picric acid over 
a wavelength range of 200-600 nm (FIG. 2) indicated a strong absorbance at 
354.6 nm. Changes in absorbance at this wavelength were used to determine 
changes in concentration of picric acid. As can be seen in FIG. 3a, 
cultures containing inoculum from cells selected for growth in the 
presence of picric acid were able to completely degrade picric acid over a 
66 h period. In similar fashion, data, illustrated in FIG. 3b, indicate 
that cultures containing cells grown in the presence of aniline waste 
stream effluent containing 2500 ppm picric acid (final concentration in 
culture, 125 ppm) also demonstrated the ability to completely degrade 
picric acid over a 66 h period. 
Analysis of the isolates indicated that there were at least 9 different 
bacterial species present in the consortium including the species, 
Arthrobacter uratoxydans, Aurobacterium saperdae, Bacilllus cereus, 
Flavobacterium esteroaromaticum, Micrococcus luteus, Microccus varians, 
Methylobacterium mesophilicum, Pseudomonas putida, and Ochrobacterium 
anthropi. 
No single member of the consortium demonstrated the ability to degrade 
picric acid when inoculated into medium containing either picric acid or 
aniline waste stream effluent as sole carbon source. It appears that it is 
necessary for a consortium of two or more of these microorganisms to 
remain together for complete picric acid degradation to occur. 
The following examples are meant to illustrate the invention but should not 
be construed as limiting. 
EXAMPLES 
MATERIALS AND METHODS 
Picric acid was obtained from Sigma as a 1% solution in water. HPLC 
separations were performed using an Hewlett Packard high performance 
liquid chromatograph (HPLC) model 1090 (Hewlett Packard, Valley Forge, 
Pa.). HPLC mobile phases were obtained from the Millipore Corporation 
(Bedford, Mass.) and were used as recommended by the manufacturer for the 
separation of organic nitrogen compounds. Spectrophotometric 
determinations were performed using a Perkin Elmer lambda 5 
spectrophotometer, (Perkin-Elmer Corp., Greenwich, Conn.). 
Example 1 
ISOLATION OF BACTERIAL CONSORTIUM AND SELECTION FOR GROWTH ON PICRIC ACID 
Samples of sludge which contained microorganisms were recovered from 
aerators at an industrial waste treatment facility. A first culture was 
constructed by inoculating a 5 ml sample into 25 ml of minimal medium 
(Table I) and adding 1.25 ml picric acid (1% solution in water). The 
culture was maintained at 30.degree. C. with shaking for 100 h. The 
culture, 5 ml, was withdrawn and subcultured in 25 ml minimal medium 
supplemented with 1.25 ml picric acid as described above. Three successive 
subcultures were grown at 30.degree. C. with shaking for 100 h. Cells 
collected from the third subculture were designated PIC002. 
A second culture was constructed by inoculated a 5 ml sample into 25 ml of 
minimal medium and adding 1.5 ml of aniline waste stream effluent. The 
aniline waste stream effluent contained 2500 parts per million (ppm) of 
picric acid. The addition of aniline waste stream effluent to the culture 
medium resulted in a picric acid concentration of 125 ppm. The culture was 
maintained at 30.degree. C. with shaking for 100 h. Five ml of culture 
were withdrawn and subcultured in 25 ml of minimal medium supplemented 
with 1.5 ml of aniline waste stream effluent. Three successive subcultures 
were grown at 30.degree. C. with shaking for 100 h. Cells collected from 
the third subculture were designated Bm002. Pic002 and Bm002 were both 
found to contain a variety of different microorganisms. 
Consortium designations Pic002 and Bm002 have been deposited with the 
American Type Culture Collection under the terms of the Budapest Treaty. 
Bacterial consortium Pic002 is assigned ATCC 55381. Bacterial consortium 
Bm002 is assigned ATCC 55382. 
TABLE I 
______________________________________ 
Minimal medium (1) Trace Elements (3) 
Wt in Yeast extract (2) Wt in 
Compound 
grams (10% Soln.) Compound grams 
______________________________________ 
K.sub.2 HPO.sub.4 
6 MgSO.sub.4.7H.sub.2 O 
2 
KH.sub.2 PO.sub.4 
4 CaCl.sub.2 
0.4 
NH.sub.4 Cl 
3.2 MnSO.sub.4 
0.08 
(Make to 1 liter FeSO.sub.4.7H.sub.2 O 
0.05 
with double Na.sub.2 MoO.sub.4.3H.sub.2 O 
0.15 
distilled water, (Make to 100 ml 
pH 7.2-7.3) with double 
distilled water and 
add 1 ml conc. HCl) 
______________________________________ 
To 1 L of (1) add 5 ml of (2) and 10 ml of (3). Add appropriate amounts o 
carbon source. 
TABLE II 
______________________________________ 
Approximate Aniline Waste Stream Effluent Composition 
Compound Concentration 
______________________________________ 
Ammonium picrate 2500 ppm 
Ammonium dinitrophenol 
300 ppm 
Nitrobenzene &lt;50 ppm 
Benzene &lt;10 ppm 
Aniline &lt;50 ppm 
______________________________________ 
Example 2 
DEGRADATION OF PICRIC ACID BY CONSORTIUM 
HPLC analysis and detection: Identification of picric acid and related 
compounds was accomplished using Hewlett Packard HPLC, model 1090 (Hewlett 
Packard, Valley Forge, Pa.) with an attached diode-array detector. 
Separations of picric acid and break-down products were carried out on a 
ZORBAX SB-C8 column (E. I. du Pont de Nemours and Company, Wilmington, 
Del.) with column guard employing a gradient with two mobile phases. 
Mobile phase A consisted of PIC A low UV reagent 0.005M solution in double 
distilled water (Tetrabutyl Ammonium Phosphate) (Millipore Corp., Bedford, 
Mass.). The second mobile phase, B, was HPLC grade methanol. Mobile phases 
were combined for a standard separation run according the following 
protocol: 
______________________________________ 
Minutes % A % B 
______________________________________ 
1 55 45 
7 55 45 
20 20 80 
10 55 45 
______________________________________ 
Samples of cell free medium were analyzed by a diode-array detector using a 
sample wavelength of 254 nm and a reference wavelength of 450 nm. Using 
this protocol it was possible to separate and quantify picric acid, 
2,4-dinitrophenol and nitrobenzene. A typical HPLC elution is shown in 
FIG. 1. As can be seen by the data in FIG. 1, 2,4-dinitrophenol elutes at 
a retention time of about 9.4 min., nitrobenzene elutes at a retention 
time of about 12.5 min. and picric acid elutes at a retention time of 
about 14 min. 
Spectral analysis on cell free medium samples was done to confirm the 
change in picric acid concentration. It was determined that picric acid 
absorbed maximally at a wavelength of 354.6 nm. A typical spectral scan of 
picric acid over a wavelength range of 200-600 nm is shown in FIG. 2. 
Picric acid degradation: 
Bacterial culture Pic002 was grown up in medium containing 125 ppm of 
picric acid. Bacterial culture Bm002 was grown in medium containing a 20 
fold dilution of aniline waste stream effluent (final concentration of 
picric acid, 125 ppm) as a carbon source. Both sets of cultures were grown 
at 30.degree. C. with shaking for 100 h. Five ml of each culture was 
inoculated into 25 ml of fresh medium and allowed to grow over a 66 h 
period at 30.degree. C. Control media containing no inoculum of cell were 
incubated under identical conditions. Samples were removed at 0, 18, 42, 
and 66 h and analyzed by HPLC and by spectrophotometry over a range of 
ultraviolet and visual wavelengths for the presence of picric acid. 
Results are shown in FIGS. 3a and 3b. FIG. 3a shows a scan over 200-600 nm 
of samples taken from Pic002 cultures grown in the presence of picric acid 
and demonstrates a decline in the absorbance spectrum at 354.6 nm from 1.8 
at 0 h to baseline at 66 h indicating complete degradation of picric acid 
in the cultures. FIG. 3b shows a similar scan of samples taken from Bm002 
cultures grown in the presence of aniline waste stream effluent containing 
a final concentration of 125 ppm picric acid and also demonstrates 
complete degradation of picric acid in the sample by the inoculum. 
To determine if degradation of picric acid was complete, samples of the 66 
h cultures from both Pic002 and Bm002 cultures were analyzed on a 
Micromass mass spectrophotometer coupled to a Varian Vista 600 gas 
chromatograph equipped with 30 meter DB-1 (methylsilicon Megabore, 0.53 mm 
i.d.) column, programmed at a temperature gradient of 
100.degree.-275.degree. C. (10.degree. C./min.) for the following 
potential picric acid degradation products: nitrocatechol, picramic acid, 
2,4-DNP, 2,5-DNP, 2,6-DNP, nitrobenzene, and aminophenol. 
The analysis indicated that none of the above picric acid degradation 
products could be detected in the 66 h culture of either Pic002 or Bm002. 
This indicated the surprising result that the consortium of microorganisms 
is able not only to denitrify picric acid but also to perform ring opening 
reactions and degrade the compound completely using the breakdown products 
as alternate carbon sources. 
Example 3 
ANALYSIS OF BIOCHEMICAL ABILITY OF ISOLATES WITH RESPECT TO PICRIC ACID 
Cultures of the bacterial consortia, Pic002 and Bm002 were streaked on R2A 
(Difco) medium and individual colonies were picked and re-plated on the 
same medium to confirm the individuality of the colonies. Each colony was 
subjected to analysis of fatty acid composition by gas chromatography for 
identification of individual species. Cultures were grown on a standard 
trypticase soy broth base in the presence of brain-heart infusions with 
supplements. Following culture in broth, the microorganisms were subjected 
to saponification in sodium hydroxide, followed by methylation in HCl and 
methanol, and finally fatty acid extraction into hexane and methyl 
tert-butyl ether. Gas chromatography of the extracted fatty acids reveals 
profiles of 9-20 carbon fatty acids in patterns typical of various genera 
and species of bacteria. Nine different bacterial species were identified 
in the consortium corresponding to individual colonies and were identified 
as, Arthrobacter uratoxydans, Aurobacterium saperdae, Bacilllus cereus, 
Flavobacterium esteroaromaticum, Micrococcus luteus, Microccus varians, 
Methylobacterium mesophilicum, Pseudomonas putida, and Ochrobacterium 
anthropi. Each species was tested for its ability to degrade picric acid 
in isolated form, away from the other members of the consortium. 
One colony from each species, isolated as above, was inoculated into 10 ml 
of the medium described in Table I, supplemented with picric acid to a 
final concentration of 125 ppm. Cultures were incubated for 72 h at 
30.degree. C. and inspected visually for metabolism of picric acid. At a 
final concentration of 125 ppm in medium, picric acid gives a strong 
yellow color. The first indication of the metabolism of picric acid by 
bacterial cultures is a decrease in the intensity and quality of the 
yellow color. After a 72 h incubation none of the cultures of isolates 
demonstrated any color variation, indicating that picric acid was not 
being metabolized. Samples of the medium were subjected to HPLC and UV 
photospectrometric analysis as described in Example 2 and it was 
determined that no degradation of picric acid had taken place. These data 
suggest that at least some of the members of the consortia need to be 
cultured together for the degradation of picric acid to occur and that 
none of the individual members of the consortium are able to degrade 
picric acid, in isolation from the other members of the consortium.