Biodegradable azo dyes

A composition comprises an azo dye having a lignin-like substitution pattern and an environmentally common microbe, such as Streptomyces or Phanerochaete chrysosporium. The composition may also comprise an azo dye having a lignin-like substitution pattern, an amount of lignin peroxidase effective to degrade the dye, and an amount of veratryl alcohol effective to recycle lignin peroxidase II to lignin peroxidase. The lignin peroxidase may be provided by an environmentally common microbe. Azo dyes substituted with lignin-like groups are completely mineralized by the environmentally common microbe. The biodegradable azo dye preferably includes a first aromatic ring having a first substituent R1 selected from hydroxy or lower alkoxy, a second substituent R2 selected from lower alkyl or lower alkoxy, and a third substituent R3 selected from lower alkoxy or halogen. In especially preferred embodiments the first substituent R.sub.1 is hydroxy and is para to the azo group, and both R.sub.2 and R.sub.3 are electron-releasing substituents and are ortho to R.sub.1.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention concerns a method of making xenobiotic compounds more 
biodegradable. More specifically, it concerns biodegradable azo dyes. 
2. General Discussion of the Background 
Azo dyes are important synthetic compounds that are widely used in the 
dyestuff and textile industries. Previously, persons skilled in the art 
believed that azo dyes are not biodegradable. Known azo dyes tend to 
persist in the environment unless subjected to costly physical and 
chemical decontamination processes. Compounds such as azo dyes which 
resist biodegradation are known as xenobiotics. The azo linkages or 
aromatic sulfo groups often found in these dyes are generally not 
synthesized by living organisms, which may help explain their 
recalcitrance to degradation. Detailed knowledge about biodegradation of 
these compounds in nature is limited. 
Biologic waste treatment processes are sometimes more efficient and less 
expensive than physical-chemical waste treatment procedures, hence it 
would be desirable to provide a biological process using microorganisms 
that degrade xenobiotic azo dyes. Unfortunately, efforts to isolate such 
microorganisms have been largely unsuccessful in producing a commercially 
suitable process. Azo dye degrading Pseudomonas strains have been isolated 
from chemostat cultures by Kulla, "Aerobic bacterial degradation of azo 
dyes", in Microbial Degradation of Xenobiotic and Recalcitrant Compounds, 
Academic Press, Inc., London, 1981, pages 387-399 (1981). The degradation 
mechanism described for that Pseudomonas involved an oxygen-insensitive 
azoreductase which catalyzed the reductive cleavage of the azo group using 
NAD(P)H as an electron donor. Zimmerman, et al., Eur. J. Biochem., 1982 
129:197-203. Various anaerobic bacteria that degrade azo dyes have also 
been reported by Wuhrman, et al., Eur. J. Appl. Microbiol-Biotechnol., 
1980, 9:325-338 and Meyer, "Biodegradation of synthetic organic 
colorants", in Microbial Degradation of Xenobiotic and Recalcitrant 
Compounds, supra. However, under aerobic conditions these dyes have been 
considered to be essentially non-biodegradable. 
Chang et al.'s U.S. Pat. No. 4,655,926 describes using the white rot fungi 
Phanerochaete chrysosporium to degrade effluent from a pulp or 
paper-making process. The effluent contains lignin or modified lignin. 
Furthermore, Cripps found that Phanerochaete chrysosporium aerobically 
degrades polycyclic hydrocarbons containing azo and sulfo groups. Cripps, 
et al., Appl. Environ. Microbiol., 1990, 56:1114-1118. That paper 
described several unidentified metabolites of microbially degraded 
Tropaeolin O, Congo Red and Orange II after incubation with crude 
ligninase preparations, but the possible mechanism of degradation was not 
explained. Other investigators have shown that P. chrysosporium can 
mineralize chloroaniline/lignin conjugates and xenobiotic molecules bound 
to humic acids. Haider and Martin, Soil Biol. Biochem., 1988, 20:425-249. 
In spite of these advances, the degree of microbial degradation of many azo 
dyes has remained low. Kulla's azo dye degrading Pseudomonas is highly 
substrate specific, and requires extensive screening procedures to isolate 
biodegradative strains. The extreme specificity of Kulla's bacterial 
strains decreases their practical use in industry because industrial 
effluents contain mixtures of dyes. Kulla, et al., "Biodegradation of 
xenobiotics; experimental evolution of azo dye-degrading bacteria", in 
Current Perspectives in Microbial Ecology, (eds. M. J. Klug and C. A. 
Reddy), American Society for Microbiology, Washington, D.C., pages 
663-667. Moreover, the Pseudomonal strains completely and irreversibly 
lose their biodegradative ability when grown with the specific substrate 
for ten generations, as disclosed at page 664 of that publication. 
Finally, sulfonated aromatic groups in the substrate dyes disturbed the 
microbial degradative pathways and limited the usefulness of these 
microorganisms in degrading the vast quantities of industrially produced 
azo dyes. 
Accordingly, it is an object of this invention to provide azo dyes which 
are more completely biodegradable. 
Another object of the invention is to provide such dyes which can be 
degraded more effectively and discarded less expensively than many 
previous azo dyes. 
Yet another object of the invention is to provide such dyes which are less 
harmful to the environment. 
Another object of this invention is to provide azo dyes which can be 
degraded by microorganisms with less substrate specificity. 
Another object is to provide such dyes which are degraded by relatively 
common and genetically stable microorganisms that better retain their 
biodegradative capacity through successive generations. 
Finally it is an object of the invention to provide an improved method of 
treating azo dyes in which sulfonated azo compounds can be degraded. 
These and other objects of the invention will be understood more clearly by 
reference to the following detailed description. 
SUMMARY OF THE INVENTION 
A biodegradable dye compound is disclosed which contains an azo group 
having first and second nitrogen atoms linked to first and second aromatic 
rings. The first aromatic ring has a lignin-like substitution pattern that 
enhances biodegradability of the dye compound. The aromatic ring 
preferably has a substitution pattern that resembles a syringyl or 
guaiacyl moiety. In preferred embodiments, the ring has a first 
substituent R.sub.1 selected from the group consisting of hydroxy, lower 
alkoxy, or amino. In other embodiments, the ring further includes a second 
substituent R.sub.2 selected from the group consisting of hydrogen, alkyl, 
lower alkoxy and halogen. In yet other preferred embodiments, the ring 
includes a third substituent R.sub.3 selected from the group consisting of 
lower alkyl, lower alkoxy and halogen. In especially preferred embodiments 
R.sub.1 is para to the azo linkage. In other preferred embodiments R.sub.2 
is ortho to R.sub.1. In especially preferred embodiments R.sub.1 is para 
to the azo linkage and R.sub.2 is ortho to R.sub.1. 
Especially preferred embodiments of the dye compound have a first 
substituent R.sub.1 on the aromatic ring that is selected from the group 
consisting of hydroxy and lower alkoxy, a second substituent R.sub.2 
selected from the group consisting of lower alkyl and lower alkoxy, and a 
third substituent R.sub.3 selected from the group consisting of lower 
alkoxy and halogen. With three substituents attached to the first aromatic 
ring, especially preferred embodiments have R.sub.1 equal to hydroxy, with 
both R.sub.2 and R.sub.3 ortho to R.sub.1. Especially preferred 
embodiments have R.sub.2 and R.sub.3 as a lower alkoxy, or R.sub.2 lower 
alkyl and R.sub.3 lower alkoxy. The biodegradable dye also may comprise a 
plurality of azo groups linked to first, second and third aromatic rings 
such that the compound is a fully conjugated system, with the first 
aromatic ring having a hydroxy and lower alkoxy group attached thereto. 
These and other compounds can be included in a biodegradable composition in 
which azo dyes according to the present invention are combined with an 
environmentally common microbe capable of degrading the azo dye. A wide 
variety of microorganisms efficiently degrade these dyes, especially 
microorganisms in the soil microflora. Particularly useful are a wide 
variety of Streptomyces species and strains of specific Streptomyces 
species found in soil and elsewhere. Examples of two aerobic 
microorganisms which have been shown to degrade the dyes of the present 
invention are several soil Streptomyces species and the fungus 
Phanerochaete chrysosporium. When used in combination with Streptomyces, 
biodegradation is most enhanced in the disclosed embodiments when R.sub.1 
is a hydroxy group para to the azo linkage, particularly if R.sub.2 is 
ortho to the hydroxy group, and more particularly if R.sub.2 is ortho to 
the hydroxy group and is an electron-releasing group, such as lower alkyl 
or lower alkoxy. 
Biodegradation with Phanerochaete chrysosporium is particularly enhanced in 
some embodiments wherein R.sub.1 is hydroxy para to the azo linkage and 
R.sub.3 is absent, especially if R.sub.2 is a group that does not have a 
high degree of steric hindrance. Methyl, methoxy and halogen are examples 
of small groups with a low steric hindrance. The presence of R.sub.3, 
however, can greatly enhance biodegradation in some embodiments wherein 
R.sub.1 is a hydroxy group para to the azo linkage, and R.sub.2 and 
R.sub.3 are both ortho to R.sub.1. Biodegradation is also enhanced if 
R.sub.2 and/or R.sub.3 are electron-releasing substituents such as lower 
alkyl or lower alkoxy. This enhanced biodegradation is observed with 
R.sub.3 present even in embodiments wherein R.sub.1 is not para to the azo 
linkage. 
With compositions according to the present invention, azo dyes having 
lignin-like substitutions are converted to CO.sub.2. Furthermore, 
commercially available azo dyes can be substituted with lignin-like 
substituents, such as guaiacol-like or syringyl-like functional groups, 
without interfering with the dye characteristics of the compound. 
Compositions according to the present invention may also comprise azo dyes 
having lignin-like substitution patterns, an amount of lignin peroxidase 
effective to degrade the dye, and an amount of veratryl alcohol effective 
to recycle lignin peroxidase II to lignin peroxidase. Especially preferred 
compositions have lignin peroxidase provided by an environmentally common 
microbe, such as Phanerochaete chrysosporium. 
Compositions according to the present invention may also comprise an azo 
dye and an amount of peroxidase effective to degrade the azo dye. The 
peroxidase may be manganese peroxidase and be provided by an 
environmentally common microbe such as Phanerochaete chrysosporium. 
Particularly suitable azo dyes for the composition have a hydroxy group 
para to the nitrogen atoms comprising the azo group. Even more suitable 
dyes are those that have one or two electron releasing substituents, such 
as lower alkyl or lower alkoxy groups, ortho to the hydroxy group. 
In other embodiments of the invention, a preexisting azo dye can be 
modified after use but before disposal to render it more biodegradable by 
these organisms.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
I. OVERVIEW 
Three azo dyes were initially tested as substrates for degradation by 
twelve Streptomyces species and the white rot fungus Phanerochaete 
chrysosporium. The three azo dyes were the commercially available acid 
yellow 9 (4-amino-1,1'-azobenzene-3,4'-disulfonic acid), and two 
synthesized dyes. The two synthesized dyes were azo dye 1 
[4-(3-methoxy-4-hydroxyphenylazo)-azobenzene-3,4'-disulfonic acid] and azo 
dye 2 (3-methoxy-4-hydroxy-azobenzene-4'-sulfonic acid). Sulfanilic acid 
and vanillic acid were also tested as substrates for degradation by the 
twelve Streptomyces species and the white-rot fungus Phanerochaete 
chrysosporium. None of the Streptomyces species degraded acid yellow 9 or 
sulfanilic acid. Linking a guaiacol molecule onto acid yellow 9 or 
sulfanilic acid via azo-linkages produced dyes that were decolorized by 
five of the twelve Streptomyces strains. These Streptomyces were those 
that could also attack vanillic acid, which has the same ring substitution 
pattern (4-hydroxy-3-methoxy) as guaiacol. While P. chrysosporium 
transformed both acid yellow 9 and sulfanilic acid, the two 
guaiacol-substituted azo dyes were decolorized more readily by P. 
chrysosporium than the corresponding unsubstituted molecules. Ligninase 
and manganese peroxidase preparations from the P. chrysosporium culture 
were apparently involved in the degradation. 
Twenty-two azo dyes were then synthesized to further study the influence of 
substituents on azo dye biodegradability, and to explore the possibility 
of enhancing the biodegradability of azo dyes, without affecting their 
properties as dyes, by changing their chemical structures. Decolorization 
of monosulfonated mono azo dye derivatives of azobenzene by the 
Streptomyces spp. was observed with azo dyes 2-5 and 10. These dyes have 
the common structural pattern of a hydroxy group in the para position to 
the azo linkage, and at least one electron-releasing group, such as 
methoxy or one alkyl group, ortho to the hydroxy group. The fungus P. 
chrysosporium attacked Acid Yellow 9 to some extent and extensively 
decolorized azo dyes 1-4 and 9. 
A different pattern was seen for three mono azo dye derivatives of 
naphthol. Streptomyces spp. decolorized 
4-(4-hydroxy-naphthylazo)-benzenesulfonic acid (Orange I) but not 
1-phenylazo-2-hydroxynaphthalinebenzene sulfonic acid (Acid Orange 12) or 
4-(2-hydroxy-1-naphthylazo)benzenesulfonic acid (Orange II). P. 
chrysosporium, though able to transform these three azo dyes, decolorized 
Acid Orange 12 and Orange II more effectively than Orange I. A correlation 
was observed between the rate of decolorization of dyes by Streptomyces 
spp. and the rate of their oxidative cleavage by either a commercial 
preparation of horseradish peroxidase type II, extracellular peroxidase 
preparations of S. chromofuscus A11, or Mn(II)-peroxidase from P. 
chrysosporium. Ligninase of P. chrysosporium showed a dye specificity 
different from that of the other oxidative enzymes. 
P. chrysosporium and Streptomyces spp. also mineralize (convert to 
CO.sub.2) azo dyes having a lignin-like substitution pattern. Five .sup.14 
C radiolabeled azo dyes and sulfanilic acid were synthesized and, along 
with sulfanilic acid, were used to examine the relationship between dye 
substitution patterns and biodegradability (mineralization to CO.sub.2). 
4-Amino-[U-.sup.14 C]benzenesulfonic acid and 
4-(3-sulfo-4-aminophenylazo)-[U-.sup.14 C]benzenesulfonic acid were used 
as representative compounds having sulfo groups or both sulfo and azo 
groups. Such compounds are not known to be present in the biosphere as 
natural products. Lignin-like fragments were introduced into the molecules 
of 4-amino-[U.sup.14 C]benzenesulfonic acid and 
4-(3-sulfo-4-aminophenylazo)-[U-.sup.14 C]benzenesulfonic acid by coupling 
reactions with guaiacol (2-methoxyphenol) to produce azo dye 34, 
4-(3-methoxy-4-hydroxyphenylazo)-[U-.sup.14 C]benzenesulfonic acid and azo 
dye 35, 4-(2-sulfo-3'-methoxy-4'-hydroxyazobenzene-4-azo)[U-.sup.14 
C]benzenesulfonic acid, respectively. Azo dye 36, 
4-(2-hydroxy-1-naphthylazo)-[U-.sup.14 C]benzenesulfonic acid, and azo dye 
37, 4-(4-hydroxy-1-naphthylazo)-[U-.sup.14 C]benzenesulfonic acid, were 
synthesized and used to evaluate the ability of microorganisms to 
mineralize these commercially important compounds. 
Phanerochaete chrysosporium effectively mineralized all of the sulfonated 
azo dyes. In contrast, Streptomyces chromofuscus was unable to mineralize 
aromatics with sulfo groups and both sulfo and azo groups. However, S. 
chromofuscus mediated the mineralization of modified dyes containing 
lignin-like fragments. Lignocellulolytic fungi and bacteria therefore can 
be used for the biodegradation of anionic azo dyes, which thus far have 
been considered to be among the xenobiotic compounds most resistant to 
biodegradation, if the dyes are modified to contain lignin-like fragments. 
Very specific structural changes in the azo dye molecules, i.e. changes 
that resemble lignin-like substitutions, enhanced their biodegradability. 
II. MATERIALS 
The following materials were purchased from Aldrich Chemical Company, Inc., 
1001 West Saint Paul Avenue, Milwaukee, Wis.: sulfanilic acid, guaiacol, 
sodium nitrite, 4-hydroxy-3-methoxybenzoic acid (vanillic acid), phenol, 
substituted phenols, hydrochloric acid (37%, A.C.S. reagent grade), 
aniline, 1-napthol, 2-napthol, p-toluenesulfonyl chloride, 
4-(4-dimethylaminophenylazo)benzenesulfonic acid (Methyl Orange 52, azo 
dye 14), 4-(4-diethylaminophenylazo)benzenesulfonic acid (Ethyl Orange, 
azo dye 15), 4-amino-1,1'-azobenzene-3,4'-disulfonic acid (Acid Yellow 9, 
azo dye 18), 1-phenylazo-2-hydroxynaphthalene-6-sulfonic acid (Acid Orange 
12, azo dye 19), 2-methoxyethanol, Congo Red azo dye 20 (Direct Red No. 
28) [international no. 573-58-01], Acid Red 114, azo dye 21 [6459-94-5], 
Direct blue 51, azo dye 22, Biebrich Scarlet, azo dye 24 [4196-99-0], 
Direct Blue 71, azo dye 25 [4399-55-7], Direct Red 75, azo dye 26 
[2828-43-8], Chrysophenine, azo dye 27 [2870-32-8], Tartrazine, azo dye 28 
[1934-21-0], Direct Yellow 27, azo dye 29, and veratryl alcohol. All 
chemicals were reagent or HPLC grade, unless specified otherwise, and were 
used as purchased. 
Azo dye 4-amino-1,1'-azobenzene-3,4'-disulfonic acid (acid yellow 9), 
4-aminoantipyrine, 2,4-dichlorophenol, [U-.sup.14 C]Aniline-hemisulfate 
and 2,2'-dimethylsuccinic acid were purchased from Sigma Chemical Co. 
III. SYNTHESIS OF AZO DYES 
Azo dyes 1 and 2 were synthesized by attaching guaiacol through an azo 
linkage to acid yellow 9, forming 
4-(3-methoxy-4-hydroxyphenylazo)-azobenzene-3,4'-disulfonic acid (azo dye 
1), or to sulfanilic acid, forming 
3-methoxy-4-hydroxy-azobenzene-4'-sulfonic acid (azo dye 2). 
Vanillylacetone was synthesized as taught by Huynh and Crawford, "Novel 
Extracellular Enzymes (Ligninases) of Phanerochaete chrysosporium," FEMS 
Microbiol. Lett., 28:119-123, 1985. 
A. Synthesis of Azo Dye 1 
Azo dye 1 [4-(3-methoxy-4-hydroxyphenylazo)-azobenzene-3,4'-disulfonic 
acid] was synthesized by dissolving the sodium salt of 
4-Amino-1,1'-azobenzene-3,4'-disulfonic acid (Yellow 9) (0.76 g) in 5% 
sodium hydroxide (8 ml). A solution of sodium nitrite (0.14 g in 0.5 ml of 
water) was added to this mixture. Crushed ice (10 g) and concentrated HCl 
(1.8 ml) was introduced to the solution, which was then vigorously stirred 
for 15 minutes, thereby forming diazotised Yellow 9. A cooled guaiacol 
(2-methoxyphenol) solution was formed (0.25 g dissolved in 3.2 ml 5% 
sodium hydroxide) and the diazotised yellow 9 solution was added to the 
guaiacol solution portionwise over 15 minutes with mechanical stirring. 
Saturated sodium chloride solution was added (15 ml), and the mixture was 
left to crystallize overnight at 5.degree. C. The crystalline product was 
filtered, washed with acetone and ether, and dried in air. Dark brown 
crystals (0.98 g) were collected. (86.6% of theoretical yield). 
B. Synthesis of Azo Dye 2 
Azo dye 2 [3-methoxy-4-hydroxy-azobenzene-4'-sulfonic acid] was synthesized 
by suspending sulfanilic acid [4-amino sulfonic acid] (1.73 g) in 23 ml of 
water. To this suspension was added 8 ml of 5% NaOH. The mixture was 
stirred until the acid dissolved and then sodium nitrite solution (0.7 g 
in 2 ml H.sub.2 O) was added. Diazotised sulfanilic acid was formed by 
pouring the solution with mixing onto a crushed ice (25 g) and 
concentrated HCl (2 ml) mixture until copious precipitation took place (KJ 
starch test was positive). The diazotized sulfanilic acid was added 
portionwise to a cooled guaiacol solution (1.24 g in 20 ml 5% sodium 
hydroxide) with stirring. NaCl (20 g) was added and stirring was continued 
for 30 minutes at room temperature. The crystalline deep-orange 
precipitate was filtered off and washed with ethanol and ether; 2.62 g of 
the product was obtained (64.4% of theoretical yield). 
C. Synthesis of Azo Dyes 3-15, 18-19 
Azo dyes 3-13 were synthesized as follows. Sulfanilic acid (3.8 g, 22 mmol) 
was dissolved in 1N sodium hydroxide solution (22 ml, 22 mmol), and 5N 
sodium nitrite solution (4.4 ml, 22 mmol) was added. Concentrated 
hydrochloric acid (4 ml, 46 mmol) was poured on crushed ice (20 g) and the 
solution of sodium sulfanilate and sodium nitrite was introduced to the 
hydrochloric acid-crushed ice mixture with mechanical stirring over a 
3-min period. The reaction mixture was kept for 2 min in an ice-water 
bath, and the white crystalline precipitate was then filtered off and 
washed with three (5 ml) portions of cold water. To avoid the possibility 
of explosion, the crystalline precipitate, a diazonium betaine, was kept 
wet and no pressure was applied to the filter cake. The wet material was 
transferred to water (20 ml), and the suspension kept at 0.degree. C. for 
the next reaction step. 
Phenol (20 mmol), or a substituted phenol (20 mmol) having the substitution 
pattern of the desired azo dye, was dissolved in 1N sodium hydroxide 
solution (20 ml, 20 mmol), and the solution was stirred vigorously while 
cooled in a salt-ice bath. If sodium phenolate precipitated out, water was 
added to keep the solution homogeneous. The cooled (0.degree. C. to 
5.degree. C.) phenolate solution was stirred mechanically in a salt-ice 
bath, and the suspension of diazonium betaine was added in small portions 
over a 10-min period to couple the phenol and diazonium betaine. The 
coupling product precipitated immediately or after a brief interval. The 
thick crystalline reaction mixture was stirred for 3 h at 0.degree. C. and 
then left overnight at 4.degree. C. 
To dissolve the crystals the reaction mixture was warmed in a water bath at 
60.degree. C. for 3 h. If solubilization was incomplete under these 
conditions, the temperature was increased to 90.degree. C. and, if 
necessary, more water was added. The homogeneous solution was refrigerated 
overnight for slow crystallization. The deposited crystals were filtered 
off and washed three times with 2 ml of cold water. The crystals were 
air-dried in a desiccator over anhydrous calcium chloride, or in a vacuum 
oven. The yield of sodium salt was 14-16 mmol (70-80%) when phenol with 
the free para position was used as a substrate. Additional azo dye could 
be recovered by acidifying the filtrate with concentrated hydrochloric 
acid (5 ml, 60 mmol). The crystals were filtered off, washed with water 
and ethanol, and dried. Usually 2-4 mmol of high-purity azo dye was 
recovered (10-20%) as free acid. When para-substituted phenol was used the 
yield of sodium salt was about 10 mmol (50%) and no further recovery by 
acidification was possible. 
D. Synthesis of Azo Dyes 16 and 17 
Azo dyes 16 and 17 were synthesized by first coupling phenol or guaiacol 
with the diazonium betaine as above. The azo dye precursor (10 mmol) was 
then dissolved in 2N sodium hydroxide solution (15 ml, 30 mmol) and 
treated with dimethyl sulfate (3 ml, 30 ml). This procedure methylated the 
hydroxy group to produce azo dye 16 and azo dye 17. 
E. Synthesis of Azo Dye 20 
Azo dye 20 was prepared as described by Schundehutte's "Methoden Zur 
Herstellung und Umwandlung von Diarylazoverbindungen," p. 236 of Methoden 
der Organischen Chemie, vol. X/3 (1965). Accordingly, diazotised 
sulfanilic acid was coupled with p-toluenesulfonanilide, followed by 
hydrolysis. The crude product was purified by recrystallization of the 
sodium salt from a water-ethanol solution, followed by precipitation by 
acidifying the filtrate with concentrated hydrochloric acid. 
F. Synthesis of Azo Dyes 21 and 22 
Coupling procedures were completed as described for the preparation of 
amino-naphthols in Organic Synthesis, Coll. Vol. 2:33-42. The resulting 
compounds were purified by recrystallization from a water-ethanol acid. 
G. Synthesis of .sup.14 C-Labeled Azo Dyes 33-37 
[U-.sup.14 C]Sulfanilic acid. [U-.sup.14 C]aniline (0.93 g, 10 mmol, 25 
.mu.Ci/mmol) was sulfonated as described elsewhere ("Vogel's Textbook of 
Practical Organic Chemistry," p. 912, 5th Ed., Longman Scientific & 
Technical Pub., John Wiley & Sons, New York(1989)), except that the 
product was not recrystallized. The crude product was dissolved in 1% 
sodium hydroxide solution, treated with decolorizing carbon, filtered, and 
precipitated with dilute hydrochloric acid. The product was colorless 
crystals (1.40 g, 8.1 mmol); specific activity, 3.2.times.10.sup.5 dpm/mg; 
yield, 81%. 
4-Sulfobenzene diazonium betaine. [U-.sup.14 C]Sulfanilic acid (0.17 g, 1 
mmol, 25 .mu.Ci) was diazotized under standard conditions (Vogel, Id., p. 
951) and the crystalline suspension was used immediately in a coupling 
reaction. 
Azo Dye 34 
[4-(3-Methoxy-4-hydroxyphenylazo-[U-.sup.14 C]benzenesulfonic acid] 
4-Sulfobenzenediazonium betaine (prepared from 1 mmol of [U-.sup.14 
C]sulfanilic acid, 3.2.times.10.sup.5 dpm/mg) was coupled with guaiacol 
according to a procedure given elsewhere (Jacobs et al., J. Am. Chem. 
Soc., 41:458-474). The product was metallic green needles (mono- hydrate; 
0.30 g, 0.92 mmol, 23 .mu.Ci); specific activity, 1.7.times.10.sup.5 
dmp/mg; yield, 92%. 
Azo Dye 37 
4-(4-Hydroxynaphthylazo)-[U-.sup.14 C]Benzenesulfonic Acid 
1-naphthol (0.15 g, 1.05 mmol) was coupled with 4-sulfobenzenediazonium 
betaine, prepared from 1 mmol of sulfanilic acid, in an ethanol-water 
solution according to the procedure of Slotta et al., "Zur Konstitution 
der Azo-Indikatoren. I Mitteilung: .alpha.-Napthol-Orange," Chem. Ber. 
68:86-94). The crude product (0.29 g, 88%) was purified by 
recrystallization from water and an ethanol-acetone mixture. The product 
was black-purple needles (0.20 g, 0.61 mmol, 15.2 .mu.Ci); specific 
activity, 1.7.times.10.sup.5 dpm/mg; yield, 61%. 
Azo Dye 36 
4-(2-Hydroxynaphthylazo)-[U-.sup.14 C]Benzenesulfonic Acid Sodium Salt 
2-naphthol (0.15 g, 1.05 mmol) was coupled in an alkaline medium with a 
water suspension of 4-sulfobenzenediazonium betaine prepared from 1.0 mmol 
of [U-.sup.14 C]sulfanilic acid (Vogel, Id., at p. 950). The product was 
orange leaflets (dihydrate; 0.30 g, 0.78 mmol, 19.3 .mu.Ci); specific 
activity, 1.4.times.10.sup.5 dpm/mg; yield, 78%. 
p-Toluenesulfonanilide. Aniline (5 g, 54 mmol) was reacted with 
p-toluenesulfochloride (15 g, 78 mmol) in 10% sodium hydroxide solution 
under standard conditions (Vogel, Id, at p. 1275). The product (8.9 g, 36 
mmol) was twice recrystallized from ethanol; the melting point was 
102.degree.-103.degree. C., yield, 67%. 
4-[4-(p-Tolylsulfamino)phenylazo]-[U-.sup.14 C]benezenesulfonic acid sodium 
salt. p-Toluenesulfonanilide (0.5 g, 2 mmol) was dissolved in 5% sodium 
hydroxide (1.6 ml, 2.1 mmol), ice (3 g) was added, and a suspension of 
p-sulfobenzenediazonium betaine (prepared from 2.0 mmol of [U-.sup.14 
C]sulfanilic acid) was added over a period of 5 min while the reaction 
mixture was mechanically stirred in a salt-ice bath. After 4 hours, the 
yellow crystalline reaction mixture was filtered off, and the product was 
washed on the filter with ice-cold water and air-dried. The crude product 
(0.75 g; yield, 83%) was taken to the next step. 
4-(4-Aminophenylazo)-[U-.sup.14 C]benzenesulfonic acid. 
4-[4-(p-Tolylsulfamino)phenylazo]-[U-.sup.14 C]benzenesulfonic acid (0.75 
g, 1.6 mmol, 40 .mu.Ci) was dissolved in 90% sulfuric acid (2 ml, 3.6 g, 
33 mmol), warmed to 40.degree. C., and kept at this temperature for 4 h. 
The reaction mixture was poured onto ice, and the precipitated product was 
filtered, washed with water, and dried at room temperature. The product 
was cherry-red crystals (0.34 g, 1.2 mmol, 30 .mu.Ci). 
Azo Dye 33 
4-(3-Sulfo-4-Aminophenylazo)-[U-.sup.14 C]Benzenesulfonic Acid 
4-(4-Aminophenylazo)-[U-.sup.14 C]benzenesulfonic acid (0.34 g, 1.2 mmol, 
30 .mu.Ci) was dissolved in fuming (20% SO.sub.3) sulfuric acid (1.0 ml, 
1.93 g, 20.6 mmol), warmed to 60.degree. C., and kept at this temperature 
for 7 hours. After cooling to room temperature, the reaction mixture was 
treated with crushed ice (5 g) and the precipitated product, was filtered 
off. The crude product was dissolved in 5% sodium hydrogen carbonate 
(2-2.5 ml) to reach pH-4, and a small amount of undissolved solid material 
was filtered off and discarded. The filtrate was treated with concentrated 
hydrochloric acid (10 ml) and the purified product, 
4-(3-Sulfo-4-aminophenylazo)-[U-.sup.14 C]benzenesulfonic acid, was 
filtered and washed with ethanol and ether. The product was metallic 
glittering dark-purple needles (0.32 g, 0.90 mmol, 22.5 .mu.Ci); specific 
activity, 1.6.times.10.sup.5 dpm/mg; yield, 75%. 
Azo Dye 35 
4-(2-Sulfo-3'-Methoxy-4'-Hydroxyazobenzene-4-Azo)-[U-.sup.14 
C]Benzenesulfonic Acid Monosodium Salt 
4-(3-Sulfo-4-aminophenylazo)-[U-.sup.14 C]benzenesulfonic acid (0.18 g, 0.5 
mmol, 12.5 .mu.Ci) was dissolved in 2% sodium hydroxide solution (2 ml). 
1N sodium nitrite solution (0.5 ml, 0.5 mmol) was added, and the mixture 
was poured onto ice (3 g) and treated with concentrated HCl (0.2 ml). The 
thick crystalline suspension was kept in an ice-water bath, with 
occasional mixing, for 15 min. Guaiacol (65 mg, 0.52 mmol) was dissolved 
in 2% sodium hydroxide solution (1 ml) and diazonium salt suspension was 
added portion wise over 10 minutes while the reaction mixture was 
mechanically stirred in a salt-ice bath. The thick crystalline paste was 
kept at 0.degree. C. for 3 hours. The reaction product was filtered off, 
washed with acetone, and dried. The product, 
4-(2-Sulfo-3'-methoxy-4'-hydroxyazobenzene-4-azo)-[U-.sup.14 
C]benzenesulfonic acid monosodium salt, was yellow crystals (0.185 g, 0.36 
mmol, 9.0 .mu.Ci); specific activity, 1.1.times.10.sup.5 dpm/mg; yield, 
72%. 
.sup.14 C-labeled dyes were all of 99% or greater radiochemical purity as 
determined by HPLC and/or TLC coupled with the counting of the 
radioactivity associated with dye peaks and/or spots. 
##STR1## 
IV. CHEMICAL CHARACTERIZATION OF THE AZO DYES 
The purity of all synthesized dyes was analyzed by mass spectra, TLC, and 
HPLC. Mass spectra (FAB, glycerol as the sample matrix, negative 
ionization) showed pseudomolecular ions of high intensity, originating 
from proton losses (M-H).sup.-. There were also very characteristic azo 
cleavage ions for all examined compounds at m/z 156 and m/z 171 as 
expected for monosulfonated azo compounds. The mass spectra of azo dye 1 
[4-(3-methoxy-4-hydroxyphenylazo)-azobenzene-3,4'-disulfonic acid] and azo 
dye 2 [3-methoxy-4-hydroxy-azobenzene-4'-sulfonic acid] are shown in FIGS. 
1A and 1B respectively. TLC analysis (silica Gel G, n-butanol-acetic 
acid-water (20:10:50)) confirmed the homogeneity of each of the products. 
The HPLC analysis of the azo dyes was performed by a slightly modified 
version of the method of White and Harbin (White et al. "High Performance 
Liquid Chromatography of Acidic Dyes on a Dynamically Modified 
Polystyrene-divinylbenzene Packing Material with Multi-Wavelength 
Detection and Absorbance Ratio Characterization," Analyst, 114:877-882 
(51)) using as mobile phases (degassed) acetonitrile-water at ratios of 
50:50, 60:40, or 70:30, each containing 0.05M dibasic sodium phosphate and 
0.01M tetrabutylammonium hydrogen sulfate (TBAH). The HPLC analysis showed 
purities of the azo dyes to be from about 97% to about 99%. 
V. MICROORGANISMS AND CULTURE MAINTENANCE 
Twelve wild-type actinomycetes were selected from 20 strains isolated from 
higher termites in Kenya. Pasti and Belli, FEMS Microbiol. Lett., 1985, 
26:107-112. All strains have been identified as Streptomyces, based on the 
key of Williams, et al. J. Gen. Microbiol., 1983, 129:1815-1830. The 
strains listed in Table 3 are those strains identified as Streptomyces by 
Pasti and Belli. Streptomyces viridosporus T7A (ATCC 39115) was isolated 
from soil by D. L. Sinden (M.S. thesis, University of Idaho, Moscow, 
1979). Streptomyces badius 252 (ATCC 39117) was isolated from soil by 
Phelan et al. (Can. J. Microbial, 1979, 27:636-368) and Streptomyces SR-10 
is a protoplast fusion recombinant derived from a cross between 
S.viridosporus T7A and S. setonii 75Vi2. Pette and Crawford, Appl. 
Environ. Microbiol., 1984, 47:439-440. Stock cultures of the Kenyan 
isolates were maintained at 4.degree. C., after growth and sporulation at 
37.degree. C. on the following medium as specified in grams-per-liter of 
deionized water: NH.sub.4 NO.sub.3, 1; KH.sub.2 PO.sub.4, 0.4; yeast 
nitrogen base (Difco), 0.67; yeast extract (Difco), 0.2; lactose, 15; 
bacto-agar (Difco), 18. S. viridosporus T7A, S. badius 252 and S. SR-10 
were maintained at 4.degree. C., after growth and sporulation at 
37.degree. C. on yeast extract-malt extract dextrose agar, as in Pridham 
and Gottlieb, J. Bacteriol., 1948, 56:107-114. Stock cultures were 
subcultered every 2 to 10 weeks, and distilled water suspensions of 
sporulated growth were used as initial inocula in all experiments. S. 
chromofuscus A11 (ATCC 55184) was selected from 20 strains isolated from 
higher termites in Kenya. 
Phanerochaete chrysosporium Burds BKM-C-1767 (ATCC 24725) was used for the 
mineralization of radiolabeled compounds. Phanerochaete chrysosporium 
Burds BKM-1667 (ATCC 24725) was grown in 125-ml flasks. Filter-sterilized 
concentrated stock solutions were used to prepare the final medium. The 
mineral salts stock solution contained 10 g L-asparagine, 5 g NH.sub.4 
NO.sub.3, 20 g KH.sub.2 PO.sub.2, 5 g MgSO.sub.4 .times.7 H.sub.2 O, 1 g 
CaCl.sub.2 .times.2 H.sub.2 O, 0.05 g thiamine, 100 ml trace elements (6), 
and water to 1000 ml. Other stock solutions consisted of 20% glucose; 1M 
sodium 2,2'-dimethylsuccinate (pH 4.5); and 0.75 g phenylalanine and 0.275 
g adenine in 1 liter of distilled water. 
Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) was obtained from The 
Forest Products Laboratory, Madison, Wis. The fungus was maintained and 
spore inocula were prepared as previously described by Huynh and Crawford, 
FFMS Microbiol. Lett., 1985, 28:119-123. 
All these publications describing isolation of the Streptomyces and 
Phanerochaete, and all publications describing culture maintenance, are 
incorporated by reference. 
VI. CULTURE CONDITIONS FOR BIODEGRADATION 
A. Actinomycete Cultures 
Each Streptomyces species was grown in a cotton-plugged 250 ml flask 
containing 25 ml of the following medium: 0.2M Tris buffer (pH 7.6), 100 
ml; vitamin-free Casamino acids (Difco), 1.0 g; thiamine, 100 .mu.g; 
biotin, 100 .mu.g; D-glucose, 2 g; deionized water, 900 ml. Thiamine, 
biotin and D-glucose were filter-sterilized and added to the autoclaved 
medium, as in McCarthy and Broda, J. Gen. Microbiol., 1984, 130:2905-2913. 
The dyes were filter-sterilized and added at 0.005% (w/v) to the autoclaved 
basal medium. Alternatively, azo dye stock solutions were sterilized by 
filtration (pore size 0.2 .mu.m) and added to autoclaved basal medium at 
concentrations of 50 or 100 ppm. Three replicates of every culture were 
incubated, and each strain was grown in media supplemented individually 
with every substrate. Replicate sterile controls also were run in each 
experiment. Cultures were incubated at 37.degree. C. for 14 days with 
shaking (200-250 rpm). Three replicates for each strain growth were 
incubated in only the basal medium as well. 
B. Phanerochaete Chrysosporium 
Phanerochaete chrysosporium was grown in a cotton-plugged 500 ml flask 
containing 250 ml defined medium (Jeffries, et al., Appl. Environ. 
Microbiol., 1981, 42:290-296), with the addition of 75 mg adenine 
(6-aminopurine) and 27 mg L-phenylalanine-per-liter. This addition 
accelerated the growth of the fungus without inhibiting ligninase 
activity. Azo dye stock solutions were sterilized by filtration (pore size 
0.2 .mu.m). Four substrates were initially tested: sulfanilic acid, acid 
yellow 9 and azo dyes 1 and 2. Each substrate was separately added at a 
concentration of about 0.02% (w/v). Cultures were incubated at 37.degree. 
C. for 7 to 15 days with shaking (250 rpm). Solid agar media were also 
employed. The medium was 3.0% (w/v) malt extract (Difco Laboratories) agar 
dispensed in petri plates. Otherwise, azo dye substrates were typically 
added to autoclaved basal medium at concentrations of from about 150 ppm 
to about 300 ppm. 
VII. PROTEIN DETERMINATIONS 
Intracellular protein concentration was used as an index of culture growth. 
Intracellular protein concentration was determined by boiling harvested 
culture pellets for 20 minutes in 1M NaOH. Protein concentration was then 
determined by Sigma calorimetric procedure No. TPRO-562. Extracellular 
protein was determined using culture filtrates and Bio-Rad calorimetric 
procedure No. 500-0006. 
VIII. SPECTROPHOTOMETRIC ASSAY 
A one ml sample of culture medium was centrifuged and then diluted 2.5-5.0 
fold (actinomycete culture) or 5- to 10-fold (fungal culture) with 
distilled water. Alternatively, 1.0 ml of fungal supernatant was 
centrifuged and diluted 5-fold with 10 mM sodium 2,2-dimethylsuccinate 
buffer (DMS) of pH 4.5. Azo dye substrate present was then measured 
spectrophotometrically with a Hewlett-Packard 8452 diode array 
spectrophotometer operated by a PC Vectra computer equipped with HP's 
MS.TM.-DOS/UV-VIS software. To be certain that changes in substrate 
spectra were not due to pH variations, the effects of pH on the visible 
absorption of each compound were also assayed within physiological pH 
range in the culture media. While the spectra of sulfanilic acid (Max abs 
at 250 nm), vanillic acid (Max abs at 252 nm and 286 nm) and acid yellow 9 
(Max abs at 336 nm) were unaffected by pH over the tested pH range, the 
spectra of several novel azo dyes were changed as evidenced by shifts of 
their Abs.sub.max. Thus, the spectrophotometric assays for the dyes were 
typically carried out at their isobestic points. For instance, the 
spectrophotometric assays were carried out at 450 nm for azo dye 1 and 400 
nm for azo dye 2. Spectrophotometric measurements were carried out at the 
absorbance maxima for azo dyes 16, 17, 18, 19, 31, and 32 (See Table 1 
below). Since the spectra of the remaining dyes were affected by pH within 
the physiological pH range, spectrophotometric assays were carried out at 
the isobestic points (See Table 1). The compounds were quantified by first 
developing standard curves of absorbance versus concentrations (0-50 
.mu.g) and then comparing measured absorbance values to the standard 
curves. 
TABLE 1 
______________________________________ 
pH-dependent wavelength shifts of azo dyes tested in 
this biodegradation study. Corresponding e values are included. 
Isobestic 
Azo 0.01M HCl 0.01M Tris 0.01M NaOH 
point 
Dye Max Max Max Max 
No. Wav. Loge Wav. Loge Wav. Loge Wav. Loge 
______________________________________ 
1 394 4.414 400 4.360 
520 4.532 
450 4.263 
2 370 4.304 370 4.290 
466 4.498 
400 4.124 
3 362 4.329 360 4.322 
470 4.493 
394 4.032 
4 380 4.128 380 4.128 
494 4.448 
418 3.967 
5 360 4.310 360 4.296 
458 4.440 
388 4.113 
6 354 4.342 354 4.256 
438 4.209 
384 4.035 
7 338 4.161 338 4.159 
492 3.870 
430 3.669 
8 328 4.296 328 4.290 
492 4.014 
424 3.756 
9 328 4.206 328 4.216 
492 3.931 
420 3.660 
10 348 4.362 348 4.293 
456 4.148 
398 3.944 
11 342 4.216 344 4.231 
354 4.021 
406 3.568 
12 348 4.312 420 4.312 
420 4.327 
376 4.085 
13 352 4.374 436 4.324 
436 4.150 
378 4.150 
14 504 4.569 466 4.339 
466 4.337 
470 4.329 
15 508 4.276 474 4.512 
474 4.512 
514 4.254 
16 352 4.329 352 4.294 
352 4.274 
414 3.563 
17 368 4.052 368 4.245 
368 3.964 
424 3.646 
18 386 4.360 386 4.392 
386 4.395 
446 4.047 
19 484 4.273 484 4.272 
428 3.937 
436 3.929 
30 486 4.075 388 4.324 
388 4.321 
462 3.963 
31 484 4.367 484 4.367 
484 4.146 
525 3.986 
32 475 4.491 475 4.491 
514 4.477 
492 4.415 
______________________________________ 
IX. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ANALYSIS 
Degradation of the dyes and aromatic compounds was confirmed by high 
performance liquid chromatography. A Hewlett-Packard HP 1090 Liquid 
Chromatograph equipped with a HP 40 diode array UV-VIS detector and 
automatic injector was used. The chromatograph was controlled by an HP 
9000 series 300 computer which used HP 7995 A ChemStation software. A 
reverse phase column from Phenomenex (Rancho Palos Verdes Calif., type 
Spherex 5 C 18 size 250.times.2.0 mm, serial number PP/6474A) was used. 
Each 15 minute analysis used a solvent gradient of acetonitrile (solvent 
A) and 10 mM DMS buffer pH 4.5 (solvent B), with the following conditions: 
0 to 5 minutes 100% A; 5 to 12 minutes 25% A 75% B; 12 to 15 minutes 100% 
B; post time 2 minutes injection volume 10 .mu.l. Absorption was measured 
at 250, 325, 350, 400 and 450 nm, and spectra were collected automatically 
by the peak controller. 
X. PREATION OF ENZYMES AND ENZYME ASSAYS 
A. Actinomycetes 
Streptomyces species peroxidases were prepared and assayed using 
2,4-dichlorphenol as a substrate as described in Ramochaondra et al., 
Appl. Environ. Microbiol., 1988, 54:3057-3063. In more detail but without 
limitation, Streptomyces chromofuscus A11, which is generally 
representative of the streptomycetes, was inoculated into 1500 ml of media 
described by Crawford et al, "The Effect of Various Nutrients on 
Extracellular Peroxidases and APPL Production by Streptomyces chromofuscus 
A2 and Streptomyces viridosporus T7A," Appl. Microbiol. Biotechnol., 
34:661-667. Incubation took place at 37.degree. C. with shaking at 250 
rpm. The culture supernatant was collected after 48 h incubation, and 
vacuum-filtered through Whatman no. 1 filter paper. The filtrate was 
concentrated approximately 40- to 50-fold by ultrafiltration through an 
Amicon ultrafiltration stirred cell (series no. 8000, Amicon Corp.) with a 
disc PM-10 filter (diameter, 76 mm). Protein in the concentrated filtrate 
was then precipitated from solution with 80% ammonium sulfate. The 
precipitate was collected by centrifugation (15 min, 10,000 rpm). The 
pellet was resuspended in 10 ml of 20 mM HEPES buffer (pH 8) and dialyzed 
overnight against the same buffer at 4.degree. C. These preparations were 
then stored in 1-ml aliquots at -20.degree. C. until used. Before using, 
the preparation was filtered through a NAP-25 Sephadex G-25 disposable 
column (Pharmacia) in order to separate a yellowish protein fraction from 
a pink low-molecular-weight fraction. The yellowish fraction was used as 
the source of enzyme. 
The peroxidase assay was carried out using a composition having a final 
volume of 1.0 ml. The reaction mixture contained 250 .mu.l of 100 mM HEPES 
buffer (pH 8), 500 .mu.l of 10 mM 2,4-dichlorophenol, 100 .mu.l of 1 mM 
aminoantipyrine, 50 .mu.l enzyme preparation and 100 .mu.l 50 mM H.sub.2 
O.sub.2. The reaction was initiated by the addition of hydrogen peroxide 
at 37.degree. C. One unit of peroxidase activity was expressed as the 
amount of enzyme required for an increase of one 
absorbance-unit-per-minute at 510 nm. Assays of manganese peroxidase and 
of ligninase from P. chrysosporium were carried out as previously 
described by Paszczynski et al in "Manganese peroxidase of Phanerochaete 
chrysosporium: Purification," Methods Enzymol. 161:264-270, incorporated 
herein by reference. One unit of Mn(II)-peroxidase was defined as the 
amount of enzyme required for oxidation of 1 .mu.mol of 
vanillylacetone-per-minute; one unit of ligninase was defined as the 
amount of enzyme required for production of 1 .mu.mol of veratryl 
aldehyde-per-minute. When veratryl alcohol was added, the final 
concentration in the reaction mixture was 1 mM. 
B. Phanerochaete Chrysosporium 
P. chrysosporium BKM-F-1767 was grown in a 20-liter carboy containing one 
liter of nitrogen-limited defined medium (BII-medium), as described by 
Paszczynski, et al., Arch Biochem. Biophys., 1986, 44:750-765. Preparation 
and assay of ligninase and manganese peroxidase from these P. 
chrysosporium cultures were carried out as previously reported by 
Paszczynski, et al., Methods Enzymol., 1988, 161:264-270. 
XI. OXIDATION OF DYES BY ENZYME PREATIONS 
A. Oxidation Conditions 
The oxidation of azo dyes by S. chromofuscus A11 crude enzyme preparation 
was carried in an assay mixture containing 800 .mu.l of 100 mM HEPES (pH 
8), 5 .mu.l of azo dye solution (5 mg/ml), and 100 .mu.l crude enzyme 
preparation (activity: 7.5 absorbance unit/minute/ml; protein content: 
2.88 mg/ml). The reaction was started with the addition of 100 .mu.l of 50 
mM H.sub.2 O.sub.2 and was followed for 1 min at 37.degree. C. Controls 
were run with no addition of H.sub.2 O.sub.2 or with the addition of the 
hemeperoxidase inhibitor KCN (final concentration 25 .mu.M). 
Oxidation of azo dyes by 5 .mu.l of horseradish peroxidase solution 
(activity: 833 IU/minute/ml; protein content; 0.24 mg/ml) were carried out 
similarly. 
The oxidation of azo dyes by 5 .mu.l of P. chrysosporium crude enzyme 
preparation (protein content: 2.3 mg/ml) was carried out for each dye 
under two conditions, one specific for Mn(II)-peroxidase (activity: 735 
IU/minute/ml) and the second specific for ligninase (activity: 694 
IU/minute/ml) Paszczynski et al.'s "Manganese peroxidase of Phanerochaete 
chrysosporium: Purification," Methods Enzymol. 161:264-270. 
B. P. Chrysosporium 
The decolorization of acid yellow 9 azo dye and the oxidation of sulfanilic 
acid by extracellular preparations of Phanerochaete chrysosporium enzymes 
is shown in FIGS. 4A and 4B, respectively. FIG. 4A shows oxidation of acid 
yellow 9 by ligninase. The reaction conditions were 0.2 mM hydrogen 
peroxide, 50 mM sodium tartrate buffer, pH3, 10 .mu.g of dye, and 0.6 
units of enzyme (20 .mu.l) in total volume of 1 ml. Cycle time was 30 
seconds with the last measure after 15 minutes. During a period of 15 
minutes the ligninase exhibited a stable activity which decolorized about 
3 micrograms out of 10 micrograms of acid yellow 9 in the reaction 
mixture. FIG. 4B shows oxidation of sulfanilic acid which was transformed 
slowly by ligninase, with an increase in absorbance at about 480 nm. The 
reaction conditions were the same as for acid yellow 9. No decolorization 
of the synthesized dyes by ligninase was detected. 
FIGS. 4C and 4D show decolorization of azo dyes 1 and 2 by the manganese 
peroxidase of P. chrysosporium. Reaction conditions were 0.2 mM hydrogen 
peroxide, 50 mM sodium tartrate buffer, pH5, 10 .mu.g dye, 1 unit of 
enzyme, 1 mM MnSO.sub.4 in a total volume of 1 ml. Cycle time was 10 
seconds, with the last measure after three minutes of incubation. 
Oxidation of azo dye 1 by manganese peroxidase resulted in new peak 
formation at 355 nm. After one hour incubation of azo dye 1 with manganese 
peroxidase the dye was almost completely degraded comparable to a control 
chromatogram (FIG. 5). No decolorization of acid yellow 9 or oxidation of 
sulfanilic acid by manganese peroxidase was detected. 
C. Streptomyces 
The azo dyes were not decolorized by S. chromofuscus A11 enzyme 
preparations without the addition of H.sub.2 O.sub.2. Furthermore, dye 
decolorization was drastically inhibited by the addition of KCN. Table 2 
shows the result of the spectrophotometric assays using the enzyme 
preparations of S. chromofuscus A11 and P. chrysosporium, and by 
horseradish peroxidase type II. Azo dyes 1-5 and 32 were the only dyes 
decolorized by the streptomycetes and by the commercial preparation of 
horseradish peroxidase. Streptomycetes decolorizes only those dyes that 
were also oxidized mainly by the Mn(II)-peroxidase component of the fungal 
ligninolytic system. FIG. 10 shows the change in spectra of azo dye 3 
during oxidation by horseradish peroxidase (FIG. 10a), by S. chromofuscus 
A11 enzyme preparation (FIG. 10b), and by P. chrysosporium 
Mn(II)-peroxidase (FIG. 10c). Dye 12 was an extraordinarily specific 
substrate for ligninase. When ligninase was involved in oxidizing the azo 
dye, the addition of veratryl alcohol enhanced the oxidation rate of 
virtually all dyes about 100%. See further discussion regarding veratryl 
alcohol below. 
TABLE 2 
______________________________________ 
Oxidation rate of azo dyes (.mu.moles per minute per ml) by 
horseradish peroxidase preparation, by S. chromofuscus A11, 
and by Phanerochaete chrysosporium crude enzyme preparations. 
Enzymes 
Azo Horse- S. 
dye radish chromofuscus 
Mn-perox- 
P. chrysosporium 
No. peroxidase 
peroxidase idase Ligninase 
______________________________________ 
1 8.24 1.78 3.06 0.44 
2 18.19 3.68 8.19 0.72 
3 30.68 17.20 16.37 0.42 
4 27.04 15.19 14.33 0.22 
5 11.94 2.00 3.54 0.00 
6 0.47 0.42 0.02 0.45 
7 0.34 0.19 0.11 1.35 
8 0.07 0.14 0.32 0.49 
9 0.20 0.35 0.07 1.27 
10 0.35 0.78 2.86 0.51 
11 0.15 0.33 1.06 1.28 
12 0.17 0.15 0.00 11.61 
13 0.10 0.13 0.26 0.88 
14 0.07 0.10 0.15 0.32 
15 0.12 0.17 0.23 0.75 
16 0.06 0.06 0.00 0.25 
17 0.32 0.50 0.72 1.03 
18 0.12 0.15 0.09 1.38 
19 0.08 0.35 0.10 2.06 
30 0.40 0.00 0.59 1.58 
31 0.52 0.09 0.47 1.18 
32 3.91 2.62 4.62 0.23 
______________________________________ 
Apparently a hemeperoxidase is responsible for azo dye decolorization 
because S. chromofuscus A11 enzyme preparations failed to decolorize dye 
in the absence of H.sub.2 O.sub.2, and the enzyme preparations were 
drastically inhibited by the addition of KCN, a hemoprotein inhibitor. The 
lignin-degrading system of P. chrysosporium contains a number of 
peroxidases that can catalyze the depolymerization of lignin, as well as 
the initial oxidation of a wide range of other compounds. Streptomycetes 
also oxidatively depolymerize lignin, with extracellular peroxidases 
apparently participating in the lignin solubilization process. 
When an azo dye was a more suitable substrate for fungal ligninase than for 
Mn(II)-peroxidase, the streptomycetes were unable to oxidize the dye 
(Table 2). While Acid Yellow 9 (azo dye 18) remained untouched by the 
streptomycetes cultures and was oxidized by P. chrysosporium lignin 
peroxidase, the synthesized azo dyes 1 and 3 were decolorized by five 
selected streptomycetes cultures and oxidized by P. chrysosporium 
Mn(II)-peroxidase. Apparently, extracellular peroxidases produced by 
lignocellulolytic Streptomyces spp. are similar in substrate specificity 
to horseradish peroxidase type II and to the Mn(II)-peroxidases of P. 
chrysosporium. 
Dye 12 was oxidized only by ligninase. A possible explanation, without 
limitation, is that fluorine atoms withdraw electrons from the aromatic 
ring, making it more difficult for peroxidases with lower oxidation 
potential than that of ligninase [i.e., Mn(II)-peroxidase] to form cation 
radicals. However, azo dye 3, having methyl groups ortho to the 4-hydroxy 
group, which are electron-releasing substituents, was a preferred 
substrate for peroxidases with low oxidation potentials. Hence, azo dyes 3 
and 12 can be used to assay specific bacterial or fungal peroxidases. 
Such an assay would comprise oxidizing each of the azo dyes with peroxidase 
and determining the extent to which each azo dye is oxidized. If azo dye 
12 is extensively oxidized, then a ligninase is involved. If azo dye 3 is 
oxidized, but azo dye 12 is not, then a peroxidase is involved. 
XII. Biotransformation of Microorganisms 
A. Streptomyces 
Table 3 shows the substrate utilization pattern of the Streptomyces species 
after a growth period of 14 days. Only six strains (A4, A10, A11, A12, 
A13, and A14) significantly degraded vanillic acid, while none degraded 
sulfanilic acid or acid yellow 9 to a detectable extent. Significant 
degradation was considered degradation greater than about 10%. This result 
confirms that the compounds characterized by aromatic sulfo group and azo 
linkages are quite recalcitrant. However, 5 strains (A10, A11, A12, A13, 
and A14) significantly degraded both the two new azo dyes. Moreover, azo 
dye 2 was degraded by these strains to a larger extent than azo dye 1. 
TABLE 3 
______________________________________ 
Percent substrate removed by cultures of Streptomyces and 
Phanerochaete during a growth period of 14 
and 7 days respectively 
Acid Azo Axo 
Sulfanilic 
Vanillic Yellow 
dye dye 
Strain acid acid #9 #1 #2 
______________________________________ 
S. chromofuscus A2 
-- -- -- -- -- 
S. diastaticus A3 
-- -- -- -- -- 
S. rochei A4 
-- 100 -- -- -- 
S. chromofuscus A6 
-- -- -- -- -- 
S. cyaneus A7 
-- -- -- -- -- 
S. chromofuscus A8 
-- -- -- -- -- 
S. rochei A10 
-- 91 -- 51 74 
S. chromofuscus A11 
-- 100 -- 56 89 
S. diastaticus A12 
-- 58 -- 27 30 
S. diastaticus A13 
-- 34 -- 15 21 
S. rochei A14 
-- 72 -- 43 72 
S. chromofuscus A20 
-- 5 -- 1 11 
S. viridosporus T7A 
-- 3 -- 1 9 
S. SR-10 -- -- -- -- -- 
S. badius 252 
-- 7 -- 9 18 
P. chrysosporium 
68 n.d. 79 93 94 
______________________________________ 
FIG. 2 shows the pattern of degradation of each compound by strain 
S.chromofuscus A11 versus time, as a typical example. The graph shows the 
degradation of vanillic acid (.quadrature.), sulfanilic acid (.gradient.), 
acid yellow 9 (O), and azo dyes 1 (.circle-solid.) and 2 
(.tangle-soliddn.). The medium contained 0.2M Tris buffer (pH 7.6), 100 
ml; vitamin-free casamino acids, 1.0 g; thiamine, 100 .mu.g; biotin, 100 
.mu.g; D-glucose, 2 g; and deionized water, 900 ml. Starting substrate 
concentrations were 50 ppm. P. chrysosporium degraded sulfanilic acid and 
acid yellow 9, but only to a limited extent. The vanillic acid, in 
contrast, was rapidly and thoroughly degraded, as were azo dyes 1 and 2. 
The ring substitution patterns for vanillic acid, sulfanilic acid, 
guaiacol, and syringyl are shown below: 
##STR2## 
It appears that the linkage of a guaiacol moiety into azo dye yellow 9 
allowed Streptomyces species capable of utilizing vanillic acid to 
decolorize an azo dye that the Streptomyces could not otherwise transform. 
The only vanillic acid degrader that could not attack either azo dyes 1 or 
2 was S. rochei A4, possibly because this strain catabolizes vanillic acid 
by attacking its carboxylic acid group, a substituent absent in the 
guaiacol moiety. Hence, utilization of the two dyes appears to start at 
the guaiacol substituent, but the pathway used by these Streptomyces 
remains to be elucidated. 
S. chromofuscus A11 was chosen as a representative microorganism for 
further study of the degradation pattern. Table 4 below shows the 
degradation of 19 azo dyes and 3 controls (Acid Yellow 9 [dye 17], the 
negative control; and azo dyes 1 and 2, the positive controls) by S. 
chromofuscus A11, as measured by decolorization of the growth medium. Five 
dyes (3, 4, 5, 10 and 32) were significantly decolorized at both 50 and 
100 ppm. Less decolorization of azo dyes 1, 3 and 5 occurred at the higher 
than at the lower concentration (Table 4). This may mean that these dyes 
are more toxic than the remaining azo dyes. For cultures of Streptomyces 
A11, the highest decolorization seemed to occur between day 2 and day 5 
for all dyes at a dye concentration of 50 ppm. The decolorization values 
from spectrophotometric data were checked by HPLC analyses, confirming the 
disappearance of the compounds. 
Significant degradation of the azobenzene derivative dyes (dyes 1-18 and 
30) by actinomycetes occurred when the hydroxy group was in the para 
position to the azo linkage and at least one or two electron-releasing 
substituents were ortho to the hydroxy group (Table 4; azo dyes 1-4, 9). 
Degradation of the naphthol-derivative azo dyes (dyes 19, 31, and 32) 
occurred when the hydroxy group was in the 4-position to the azo linkage. 
The second condensed aromatic ring in naphthalene apparently acts as an 
electron-donating fragment, similar to the electron-releasing substituents 
in the benzene ring of dyes 3-5 and 10. 
TABLE 4 
______________________________________ 
Azo dye decolorization by microorganisms Streptomyces 
chromofuscus A11 and by Phanerochaete chrysosporium over a 
growth period of 14 days. 
% Decolorization 
S. chromofuscus A11 
P. chrysosporium 
Initial concn (ppm) 
Initial concn (ppm) 
Azo dye 50 100 150 300 
______________________________________ 
1.sup.a 82 60 99 62 
2.sup.a 89 83 97 94 
3.sup.a 85 82 96 97 
4.sup.a 20 15 93 92 
5.sup.a -- -- 90 87 
6.sup.b -- -- 72 18 
7.sup.b -- -- 81 55 
8.sup.b -- -- 27 49 
9.sup.a 34 29 89 86 
10.sup.b -- -- 90 92 
11.sup.a -- -- 70 34 
12.sup.a -- -- 86 93 
13.sup.c -- -- 92 88 
14.sup.c -- -- 93 94 
15 -- -- 38 27 
16 -- -- 61 55 
17.sup.c -- -- 79 74 
18.sup.b -- -- 98 15 
19.sup.a 56 44 93 87 
30.sup.c -- -- 85 80 
31.sup.b -- -- 99 53 
32.sup.a 90 88 67 71 
______________________________________ 
.sup.a : auxochrome --OH in para or 1,4 
.sup.b : auxochrome --OH in ortho or 1,2 
.sup.c : auxochrome --NH.sub.2 in para 
B. P. Chrysosporium 
The Phanerochaete fungus almost completely (90%) degraded azo dyes 1 and 2 
after a growth period of seven days as shown in FIG. 3. The graph shows 
decolorization or removal by P. chrysosporium of sulfanilic acid (v), acid 
yellow 9 (O), azo dye 1 (.circle-solid.), and azo dye 2 
(.tangle-soliddn.). Agitated cultures were grown at 37.degree. C. in a 
mineral medium supplemented with phenylalanine, and starting dye 
concentrations were 150-200 ppm. There was a characteristic lag of 80-90 
hours prior to degradation of any of the compounds by the fungus due to 
slower growth compared to the Streptomyces. On the solid medium, P. 
chrysosporium behaved similarly leaving some undegraded color after two 
weeks of growth. P. chrysosporium apparently degrades using its 
ligninolytic enzymes. The ability of this organism to oxidize sulfonated 
azo aromatic compounds was also tested, shown in FIG. 3 and Table 4. The 
maximum rate of decolorization occurred on the fourth day of growth in the 
BII medium for all of the compounds. However, in the cultures with yellow 
9 or sulfanilic acid, as assayed by spectrophotometric and HPLC analysis, 
some undegraded dye remained in the medium after decolorization ended. Yet 
using HPLC, the inventors were not able to detect any residual substrate 
in the culture broth after 1 week of growth, even though color was still 
present in the culture filtrates. One explanation is the finding of Kulla, 
et al., supra, who found that in cultures of Pseudomonas which were 
actively degrading azo dyes, secondary oxidative coupling occurred between 
sulfonated and nonsulfonated phenols, giving dead-end polymers resistant 
to further degradation. 
In testing which, if either, of the ligninolytic peroxidases of P. 
chrysosporium was involved in the degradation of these azo compounds, it 
was found that ligninase oxidized yellow 9 and sulfanilic acid (FIG. 4A 
and B), while manganese peroxidase oxidized azo dyes 1 and 2 (FIG. 4C and 
D). The HPLC analysis of the reaction mixture after incubation of azo dye 
1 with manganese peroxidase revealed polymorphic reaction products (FIG. 
5B). Oxidation of sulfanilic acid by ligninase produced a purple unstable 
product, which upon exposure to air precipitated. During a 15-minute 
incubation period, oxidation of yellow 9 or sulfanilic acid by manganese 
peroxidase was not detected, nor the oxidation of azo dye 1 or 2 by 
ligninase. Thus, it is possible that ligninases may cooperate in the 
degradation of azo dyes 1 and 2. Azo dyes 1 and 2 were decolorized to a 
greater extent by P. chrysosporium than was acid yellow 9 (Table 4). Even 
greater decolorization was noted during the growth of S.chromofuscus A11 
(FIG. 2). 
These results show that linking a guaiacol molecule into the dye structure 
increased its susceptibility to degradation. Azo dye structures are 
typically conjugated multi-unsaturated systems. This makes it possible to 
change only one fragment of the molecule and yet have the entire 
conjugated system become accessible to enzymatic attack, particularly with 
microorganisms like white-rot fungi that use oxidative enzymes that 
generate cation radicals. This finding has general application for 
synthesizing more easily biodegradable azo dyes and other recalcitrant 
compounds in accordance with the present invention. 
To further show the effects of lignin-like substitution P. chrysosporium 
was tested with a number of azo dyes. The fungus was able to decolorize 
all 19 azo dyes tested including the 3 controls. Table 4 shows that the 
extent of decolorization varies for each dye tested. The controls, azo 
dyes 1, 2 and 18, were extensively decolorized at both tested 
concentrations of 50 and 100 ppm. While only 79% of Acid Yellow 9 (dye 17) 
was degraded, most of the newly synthesized dyes were degraded within the 
range of from about 85 to about 99% at a dye concentration of 150 ppm (azo 
dyes 3-6, 8-14, 19, 30 and 31) (Table 4). This enhanced biodegradability 
was also observed at the higher concentration of 300 ppm. At this 
concentration, some dyes (azo dyes 7, 12, 19, and 31) apparently began to 
be toxic for the fungus (Table 4). For cultures of P. chrysosporium, the 
highest decolorization occurs between day 2 and day 5 for all dyes at a 
dye concentration of 50 ppm. Fungal cultures showed a characteristic 
decolorization lag phase of 3-4 days. Decolorization was determined 
spectrophotometrically. HPLC analysis was used to confirm the 
disappearance of the azo dyes. 
XIII. BIOTRANSFORMATION OF AZO DYES 2-19 
Table 5 shows the concentrations of azo dyes 2-19 after cultivation with P. 
chrysosporium for ten days on mineral medium. Table 5 clearly shows that 
the concentration of azo dyes of the present invention are substantially 
reduced after cultivation with P. chrysosporium for a period of ten days. 
Hence, xenobiotic azo dyes can be modified to have lignin-like 
substitution patterns, without affecting the dye characteristics of the 
azo dye, and then degraded by forming a composition comprising P. 
chrysosporium and the azo dye. 
TABLE 5 
______________________________________ 
Concentration of Substrates After Ten Days 
Cultivation with P. chrysosporium 
Azo-comp Beginning Concentrations 
number 100 ppm 150 ppm 200 ppm 
300 ppm 
______________________________________ 
2 2.493 6.350 10.068 7.318 
3 0.358 1.223 1.894 114.772 
4 1.637 3.763 4.218 16.323 
5 6.084 9.870 14.500 22.680 
6 12.909 14.003 33.309 39.326 
7 62.893 41.044 10.200 246.044 
8 16.079 28.401 29.704 135.680 
9 81.911 109.822 32.044 153.733 
10 8.135 15.279 46.588 42.897 
11 6.795 14.416 30.075 21.765 
12 2.368 44.227 6.160 198.240 
13 7.734 20.776 28.216 20.096 
14 4.411 11.843 15.529 36.800 
15 1.901 10.589 17.931 17.791 
16 67.801 92.683 179.300 
219.079 
17 29.803 58.942 93.250 136.057 
18 8.387 20.342 40.875 77.099 
19 2.938 252.592 
______________________________________ 
Table 6 shows the concentration of azo dye substrates after cultivation 
with Streptomyces strains A10, A11, A12, and A13. 
TABLE 6 
______________________________________ 
Percent Degradation of Substrates After Streptomyces 
Cultivation 
Degradation (%) 
Strains 
Substrates 
Wavelengths A10 A11 A12 A13 
______________________________________ 
2 396 77 73 43 18 
3 396 68 73 39 10 
4 416 79 83 39 8 
5 386 9 20 3 8 
6 376 -- -- -- -- 
7 430 -- -- -- -- 
8 422 -- -- -- -- 
9 420 -- -- -- -- 
10 394 11 16 7 6 
11 420 4 9 -- -- 
12 376 -- -- -- -- 
13 376 2 5 1 1 
14 466 -- -- -- -- 
15 474 2 3 1 1 
16 350 -- -- -- -- 
17 408 -- -- -- -- 
18 386 -- -- -- -- 
______________________________________ 
-- = no degradation 
The Streptomyces were grown on the same media and under the same conditions 
as previously described. Spectroscopic analysis of substrates 14, 15, 16, 
18 was unaffected by pH over the tested pH range. However, the Abs.sub.max 
of the substrates 1-12 shifts with pH changes. Thus, the 
spectrophotometric assays for substrates 1-12 were carried out at their 
specific isobastic points. Degradation was calculated as percent of 
substrates removed from culture broth, considering the evaporation factor 
(around 10%). The substrates' concentrations have been calculated versus 
standard curves prepared for each dye (0-50 .mu.g) at the chosen 
wavelength; standard curves of the tested concentrations were linears. 
Table 6 shows that the concentrations of azo dyes having guaicol-like or 
syringyl-like substitutions decrease when cultivated with Streptomyces. 
Hence, xenobiotic azo dyes can be degraded by modifying the dyes to have a 
lignin-like substitution pattern, such as guiacol-like or syringyl-like, 
without changing the dye characteristics, and then forming a composition 
by cultivating the dyes with Streptomyces. 
XIV. EFFECT OF VERATRYL ALCOHOL 
A. Overview 
As shown above, Phanerochaete chrysosporium decolorized polyaromatic azo 
dyes in ligninolytic culture. The oxidation rates of individual dyes 
depended on their structures. Veratryl alcohol (VA) (3,4-dimethoxy benzyl 
alcohol) stimulated azo dye oxidation by pure lignin peroxidase 
(ligninase, LiP) in vitro. Accumulation of compound II of lignin 
peroxidase, an oxidized form of the enzyme, was observed after short 
incubations with azo substrates. When veratryl alcohol was also present, 
only the native form of lignin peroxidase was observed. Azo dyes acted as 
inhibitors of veratryl alcohol oxidation. After an azo dye had been 
degraded, the oxidation rates of veratryl alcohol recovered, confirming 
that these two compounds competed for ligninase during the catalytic 
cycle. Veratryl alcohol acts as a third substrate (with H.sub.2 O.sub.2 
and the azo dye) in the lignin peroxidase cycle during oxidations of azo 
dyes. 
Veratryl alcohol is a secondary metabolite found in ligninolytic cultures 
of Phanerochaete chrysosporium. Veratryl alcohol is synthesized de novo by 
way of phenylalanine, 3,4-dimethoxycinnamyl alcohol, and veratryl 
glycerol. The onset of ligninolytic activity and glucose oxidation leading 
to hydrogen peroxide production by P. chrysosporium appears simultaneously 
with the accumulation of VA in cultures. 
The onset of ligninolytic activity in Phanerochaete chrysosporium requires 
VA, but the relationship between the concentration of VA produced by 
various strains of P. chrysosporium and their mineralization of lignin is 
not clear. Studies with whole cultures of P. chrysosporium indicate that 
the function of VA might be to protect ligninase against inactivation by 
hydrogen peroxide. This has been confirmed by experiments where high 
concentrations of hydrogen peroxide were added to non-protein-synthesizing 
cultures. The concentration of VA necessary for protection of ligninase 
activity was in direct proportion to the rate of hydrogen peroxide 
synthesis by the cultures. 
Using pure ligninase preparations, the degradation of the azo dyes by P. 
chrysosporium was investigated. Oxidations of VA and azo compounds were 
monitored simultaneously since they have considerably different absorption 
maxima. The results suggested that LiPI (lignin peroxidase compound one), 
formed during oxidation of H.sub.2 O.sub.2 by LiP, oxidized polyaromatic 
azo dyes, forming LiPII. LiPII was then reduced back to the native enzyme 
by oxidation of VA. 
Decolorization of some azo dyes by LiP was almost totally dependent on the 
presence of VA. VA significantly increased the oxidation rates of these 
azo dyes. A simultaneous inhibition of LiP-catalyzed VA oxidation by azo 
compounds was observed. When azo dye oxidations were terminated, the rates 
of VA oxidation recovered. These observations suggest that LiPI is able to 
oxidize these dyes, but the LiPII formed requires VA to recycle to the 
native state. Similar enzymatic oxidation interactions could be involved 
during degradation of lignin and other recalcitrant compounds. 
B. Degradation of Azo Dyes 
Decolorization of dyes in cultures were monitored at their absorption 
maxima at pH 4.5, or using HPLC. A Spherisorb ODS2 C18 column was used for 
HPLC analysis with a sequence of DMS buffer at pH 4.5 and acetonitrile as 
solvents (5 min 100% DMS, 15 min 100% acetonitrile, 18 min 100% 
acetonitrile, 19 min 100% DMS, 20 min 100% DMS, 5 min postran 100% DMS). 
Peaks were monitored at 260 and 450 nm. Results from spectrophotometric 
and HPLC methods were compared. 
C. Enzyme Assays 
Lignin peroxidase activity was determined spectrophotometrically at room 
temperature with VA as substrate. One unit (U) of enzyme activity was 
defined as 1 .mu.mole of veratryl aldehyde formed-per-min at pH 3. 
Simultaneous oxidations of azo dyes and veratryl alcohol were measured 
using a Hewlett-Packard 8452 diode array spectrophotometer operated in the 
kinetic mode by a Vectra PC equipped with MS.TM.-DO/UV-VIS software. Azo 
dye concentrations were determined by measuring absorbances at appropriate 
maxima. Dye 24 shows E.sub.(506) =5.07.times.10.sup.4 M.sup.-1 cm.sup.-1, 
and dye 28 shows E.sub.(430) =4.12.times.10.sup.4 M.sup.-1 cm.sup.-1. 
Table 7 shows the decolorization of individual azo dyes by a ligninolytic 
culture of P. chrysosporium after ten days of growth. The initial 
concentration of dye was 200 mg-per-liter. Numbers represent mg-per-liter 
of dye remaining in the cultures. 
TABLE 7 
______________________________________ 
Analytic Methods 
Amount 
Degraded 
Azo Dye HPLC Spectrophotometry 
(av. %) 
______________________________________ 
20 62.20 52.01 71.45 
21 103.62 102.38 48.50 
22 12.28 7.64 95.02 
23 72.43 89.27 59.57 
24 4.43 1.11 98.61 
25 14.86 9.66 93.87 
26 6.98 4.33 97.17 
27 29.42 28.33 85.56 
28 3.00 2.42 98.64 
29 109.39 108.92 45.42 
______________________________________ 
-- Dyes 20 to 26 contain naphthalene. 
-- Cultures with dyes 24 and 28 were completely bleached. 
D. Enzyme Purification 
After six days of growth, when the specific activity of ligninase reached 
about 250 U-per-liter, culture filtrates were separated from mycelial 
debris by filtration. One liter of culture filtrate was concentrated to 
about 10 ml using an Amicon PM-10 membrane and desalted in 10 mM sodium 
acetate buffer (pH 6) using a Sephadex G25 NAP10 column. Ligninase 
isoenzyme was separated from Mn-dependent peroxidases using a Pharmacia 
fast protein liquid chromatography system equipped with a Mono Q HR 5/5 
column. In this study isoforms H2 and H8 were used. The purified proteins 
were electrophoretically homogenous and showed A.sub.408 /A.sub.280 of 
about 4.5. 
E. Oxidation of Azo Dyes 
In vitro studies were limited to dyes 24 and 28. Ligninase without VA 
oxidized azo dyes 24 and 28 to a limited extent. After VA was added to the 
reaction mixtures, oxidation of both substrates began, leading to total 
decolorization of the dyes. FIGS. 6A and 7A show the simultaneous 
oxidation of azo dyes 24 and 28, respectively, and VA by ligninase. 
Without VA, azo dye oxidation was limited and terminated more rapidly than 
in the presence of VA (FIGS. 6B and 7B). When a dye was present in a 
reaction mixture, the rate of VA oxidation decreased due to an apparent 
competition for LiPI between these two compounds. The oxidation rate of VA 
recovered when the azo dye oxidation was completed (FIG. 8). The maximal 
rate of VA oxidation was about 159 AU (absorption change per-min-per-ml of 
ligninase solution). Wavelengths of 310 nm for VA, 506 nm for dye 24, and 
430 nm for dye 28 oxidations were used in monitoring substrate removal. 
The oxidation of azo dyes is stimulated by VA in high (200 .mu.M) and low 
(20 .mu.M) H.sub.2 O.sub.2 concentrations. These results establish that 
ligninase is capable of more effectively oxidizing recalcitrant azo dyes 
when VA is present. 
Ligninases from two parallel reaction mixtures were recovered by Sephadex 
G25 gel filtration, and spectra were recorded (FIG. 9). The first reaction 
mixture contained 50 mM sodium tartrate at pH 3, 18 .mu.M enzyme, 0.2 mM 
H.sub.2 O.sub.2, 15 .mu.g of dye 28, and 1 mM VA. In the second reaction 
mixture, VA was excluded. Spectrum were observed of the native enzyme in 
the reaction mixture that had contained VA, and the spectrum of LiPII in 
the reaction mixture that had not contained VA, in which only the azo dye 
was available as a reductant. Dyes 24 and 28 gave similar results. Without 
limitation it appears that only the highest oxidation stage of ligninase 
(LiPI) is able to attack azo dyes, and that the presence of VA helps to 
complete the catalytic cycle of the enzyme. 
XV. MINERALIZATION OF AZO DYES BY P. CHRYSOSPORIUM AND STREPTOMYCES 
CHROMOFUSCUS 
A. Measurement of the Mineralization of [.sup.14 C]-Labeled Dyes 
Agitated cultures containing 100,000 to 150,000 dpm of azo dye at a 
concentration of 200 mg/liter for P. chrysosporium and 50 mg/liter for S. 
chromofuscus were incubated at 37.degree. C. for 20 days with shaking at 
200 rpm. For each mineralization experiment six replicates-per-culture 
were run for P. chrysosporium and three replicates-per-culture for S. 
chromofuscus. Each culture flask contained a CO.sub.2 trap consisting of a 
small glass cup connected to the bottom of the rubber stopper. The cup 
contained 1 ml of 1N NaOH. Samples were taken every 2 days, and after each 
sampling cultures were flushed with 100% oxygen for 30 s. At the sampling 
time, the stopper was removed and the NaOH containing trapped CO.sub.2 was 
transferred to a 20-ml scintillation vial, and the cup was then rinsed 
twice with 1 ml of water. Two ml of rinse water were also transferred to 
the vial. Finally, 10 ml of Ecolite scintillation cocktail (ICN 
Biochemicals Inc., Irvine, Calif.) were added to each vial. Vials, after 
mixing, were stored in the dark for 24 h. The cpm of trapped radioactivity 
was then determined in a Packard Tri-Carb liquid scintillation analyzer, 
Model 1500, using the .sup.14 C quench standards and SIS numbers. 
B. Measurement of Organic Volatile Compounds 
Either a charcoal-containing polyurethane sponge (Bio-Rad Laboratories, 
Richmond, Calif.) or 2-methoxyethanol was used to trap volatile organic 
compounds released during mineralization experiments. The sponge (1 
cm.sup.3) replaced the 1N NaOH solution in the small glass cap attached to 
the rubber stopper; other conditions were as before for the .sup.14 
CO.sub.2 experiments. For scintillation counting, the charcoal sponges 
were dissolved in 1 ml of hyamine hydroxide contained in scintillation 
vials (1M solution in methanol; NEN Research Products, Boston, Mass.) held 
at 55.degree. C. for 2 h. Ten ml of Ecolite scintillation cocktail were 
then added and the cpm was measured. 
In the second procedure, cultures with CO.sub.2 traps were flushed with 
oxygen for 10 min, and the organic volatiles present were trapped in 10 ml 
of 2-methoxyethanol mixed with 10 ml Ecolite. 
C. Radioactivity in the Medium and Biomass 
The removal of radioactivity from the media was determined by measuring the 
amount of .sup.14 C label in 0.1 ml of the medium at time 0 and 21 days 
for each culture. Uninoculated cultures were used as evaporation controls. 
At the end of each mineralization experiment, culture broths were used as 
evaporation controls. At the end of each mineralization experiment, 
culture broths were acidified with 5 ml of 1M H.sub.3 PO.sub.4, fresh 1N 
NaOH was added to the trap, and the cultures were shaken overnight. No 
additional releases of .sup.14 CO.sub.2 were observed. The radioactivity 
assimilated by the cells was measured by solubilizing biomass harvested 
from each 21-day-old culture in an equal volume of hyamine hydroxide 
solution. Before solubilization, cells were washed three times with 50 ml 
of distilled water to remove adsorbed dyes. Ecolite 10 ml was added to the 
mixture, which was vortexed vigorously. To increase counting efficiency, 
the solution of biomass-hyamine-scintillation cocktail was further diluted 
10 to 20 times with fresh Ecolite before counting. 
P. chrysosporium demonstrated a greater ability than Streptomyces 
chromofuscus to mineralize the azo dyes. S. chromofuscus mineralized 
certain dyes which is significant, since azo dyes are resistant to aerobic 
degradation. About 19% of the radioactivity from guaiacol-substituted azo 
dye 34 was removed from the medium after 21 days growth of S. 
chromofuscus. Of this amount, almost 4% of the .sup.14 C was mineralized 
to CO.sub.2 and 4.6% was assimilated by the cells of the actinomycete. 
Since the dyes were at least 99% radiochemically pure, the recovered 
.sup.14 C in CO.sub.2 and biomass must represent mineralized dye 
molecules. The remaining 9% of the radioactivity was accounted for by dye 
absorbed by the cells. Dyes 35 and 37 showed similar patterns of 
degradation, although mineralization was less efficient. P. chrysosporium 
assimilated similar ranges of degraded dyes and showed smaller amounts of 
absorption to cell mass, with the exception of sulfanilic acid. 
Two methods were used to detect the production of organic volatiles. 
Polyurethane foam containing activated charcoal was a less-sensitive trap 
than the 2-methoxyethanol purging method. After solubilization, the foam 
released activated charcoal, which caused quenching problems during the 
counting of radioactivity. By using 2-methoxyethanol as the trapping 
agent, small but measurable amounts of .sup.14 C released by the cultures 
of P. chrysosporium were detected. 
The benzene ring that is attached directly to the azo linkage and that has 
the sulfonic group in the para position is the most recalcitrant portion 
of the azo dye molecule. This benzene ring was radiolabeled since this 
moiety was common to all the investigated compounds, and therefore the 
results of the degradation and mineralization experiments were comparable. 
In all cases the guaiacol moiety retained its full electronic 
configuration since it is attached to a remote part of the molecule. 
S. Chromofuscus mineralized azo dyes only when the lignin-like guaiacol 
structure was attached to the sulfonated aromatic ring. The results of the 
mineralization experiments with P. chrysosporium (Table 8) showed less of 
a pattern. It appears that the bacterial enzymatic system responsible for 
the degradation are more specific to lignin-like structures than are the 
enzymatic system(s) of the fungus. 
TABLE 8 
______________________________________ 
Percentage of radioactivity recovered in CO.sub.2 and biomass 
and the percentage of radioactivity removed from the medium 
after 21 days' growth of P chrysosporium and Streptomyces A11 
Streptomyces 
P. chrysosporium 
chromofuscus 
Azo dye Medium** CO.sub.2 
cells 
Medium CO.sub.2 
cells 
______________________________________ 
3 31.7 25.7 5.2 19.0 3.6 4.6 
17 33.0 26.9 2.3 0.0 0.0 0.0 
(Acid 
Yellow 9) 
19 41.6 34.8 2.3 0.0 0.0 0.0 
31 (Orange II) 
32 (Orange I) 
29.6 19.7 4.0 22.0 1.1 7.0 
Sulfanilic Acid 
25.0 17.2 1.3 0.0 0.0 0.0 
______________________________________ 
*In addition to CO.sub.2, a small amount (0.1%-0.5%) of organic volatiles 
were detected. 
**C not accounted for as CO.sub.2 or biomass was lost by absorption to 
cell material and could be removed by washing with distilled water. 
Mineralization experiments were conducted with both organisms to determine 
how structural variations in the second ring of the azo dyes affected 
degradation rates over a 21-day incubation period. A progressive 
mineralization of each azo dye was observed (FIGS. 11-13). With P. 
chrysosporium, dye 36 was degraded 2.5 times greater than the dye 37, and 
degradation began 2 days earlier. Dyes 33, 34, and 35 were mineralized to 
a similar extent, although the mineralization rate of dye 34 was twice as 
fast during the first 10 days of incubation. The streptomycetes 
mineralized only the guaiacol derivatives of dyes 34 and 35. A significant 
increase in the mineralization rate of dye 34 could be observed between 
days 6 and 9. The mineralization of sulfanilic acid (FIG. 12) began on the 
tenth day of the fungal culture and reached only 17%. During 
mineralization, a brown oxidation product accumulated at the beginning of 
degradation (14 days). This product was subsequently bleached by the 
culture. This brown material was probably a product of the oxidative 
coupling of benzene-carrying hydroxy and amino groups. 
Both P. chrysosporium and streptomycetes mineralize sulfonated azo dyes, 
including naphthol derivatives, which make up the bulk of commercial dyes. 
Since actinomycetes and fungi are able to decolorize and mineralize azo 
dyes, such compounds can be used as assay compounds to isolate superior 
catabolic microbial strains from natural environments. Peroxidases seem to 
perform an essential role in azo dye transformations. 
XVI. DEGRADATION OF AZO DYES BY SOIL MICROFLORA 
Azo dyes according to the present invention also can be degraded by forming 
a mixture of the azo dye and soil or soil microflora other than 
Phanerochaete chrysosporium and Streptomyces spp. 
The degradation of azo dyes by soil microflora in general was evaluated by 
forming a mixture of azo dyes 33 and 34 with water from a soil sample. The 
soil sample contained soil microflora that had not adapted to using azo 
dyes as substrates. A carbon source was added to the mixture and a 
continuous air flow through the mixture was maintained. The .sup.14 
CO.sub.2 released by the mixture was trapped using a 1N NaOH. 
The results of this study showed that soil microflora other than 
Phanerochaete chrysosporium and Streptomyces spp. are capable of degrading 
azo dyes. Furthermore, attaching a lignin-like substituent to the azo dye 
enhances the degradation of the dye. Specifically, azo dye 33 was 
transformed only 3.4% and mineralized only 1.1% when combined with water 
from a soil sample. Azo dye 34, having a guaiacol substitution pattern, 
was degraded much more effectively, since 46% of the dye was transformed, 
and 14% of the dye was mineralized. 
XVII. Lignin-Like Structures 
As the data in Tables 1-8 demonstrate, the biodegradability of xenobiotics, 
such as azo dyes, can be enhanced by attaching lignin-like structures to 
them. Lignin-like structures are those that are contained in lignin and 
which enhance biodegradability of xenobiotic azo dyes when they are 
attached to them. Lignin-like structures also include analogous chemical 
structures which are not known to be in lignin, yet sufficiently resemble 
lignin structures to provide enhanced biodegradability. 
Chemical and spectrometric studies of softwood lignin indicate that lignin 
is an aromatic polymer in which the monomeric guaiacylpropane units are 
connected by both ether and carbon-carbon linkages. Several substructures 
in lignin macro-molecules include guaiacylglycerol-.beta.-aryl ether 
(.beta.-O-4' substructure 1) which is the most abundant interphenylpropane 
linkage (40-60%) in lignin, followed by phenylcoumaran (.beta.-5' 
substructure 2; 10%), diarylpropane (.beta.-1' substructure 3; 5-10%), 
pinoresinol (.beta.-.beta.' substructure 4; 5%), biphenyl (5-5' 
substructure 5; 10%), diphenyl ether (4-O-5' substructure 6; 5%), and 
others. 
A structure model of softwood lignin is described in Higuchi, Biosynthesis 
and Biodegradation of Wood Components, Wood Research Institute, Kyoto, 
Japan, 1985, page 143, and set forth below to show a variety of ring 
substitutions present in lignin. 
##STR3## 
The composition of lignin varies for different kinds of lignins. The lignin 
of hardwoods such as beech, for example, is composed of approximately 
equal amounts of guaiacyl- and syringylpropane units connected by linkages 
similar to those found in spruce lignin. Grass lignin, such as bamboo 
lignin, is considered to be composed of guaiacyl-, syringyl-, and 
p-hydroxyphenyl. 
The biodegradable azo dyes of the present invention include an azo group 
having a nitrogen atom linked to an aromatic ring, in which the ring has a 
lignin-like substitution pattern. As used herein, the term "lignin-like 
substitution pattern" refers to a ring having substituents which provide a 
lignin-like structure. In its simplest embodiments, the lignin-like 
substitution pattern provides guaiacyl-like or syringyl-like units 
connected by a nitrogen linkage to the remainder of the azo dye. 
Guiacol-like as used herein means, without limitation, that the azo dye 
has a hydroxy group or lower alkoxy group on at least one aromatic ring 
with an electron releasing group, such as a lower alkyl or lower alkoxy 
group, ortho to the hydroxy group. Preferred guiacol substitution patterns 
have the hydroxy group para to the azo group. Especially preferred 
guaicol-like substitution patterns have the hydroxy group para to the azo 
group with the electron-releasing group, comprising methyl or methoxy, 
ortho to the hydroxy group. The aromatic ring can comprise naphthalene, 
wherein the second aromatic ring of the naphthalene system acts as an 
electron-releasing group. Syringyl-like as used herein means, without 
limitation, that the azo dye has a hydroxy group or lower alkoxy group on 
at least one aromatic ring and two electron releasing groups, such as a 
lower alkyl, lower alkoxy, or lower alkyl and lower alkoxy, ortho to the 
hydroxy group. Preferred syringyl-like substitution patterns have the 
hydroxy group para to the azo group. Especially preferred syringyl-like 
substitution patterns have the hydroxy group para to the azo group with 
the electron-releasing groups, comprising methyl, methoxy, or methyl and 
methoxy, ortho to the hydroxy group. The aromatic ring can comprise 
naphthalene wherein the second aromatic ring of the naphthalene system 
acts as an electron-releasing group. 
XVIII. SUMMARY 
The present inventors have found that biodegradability of azo dyes is 
especially enhanced by providing a lignin-like substitution pattern on one 
of the aromatic rings of the azo dye. The lignin-like substitution pattern 
may comprise a first ring substituent R1 selected from the group 
consisting of hydroxy, lower alkoxy or amino, and a second substituent R2 
selected from the group consisting of lower alkyl, lower alkoxy and 
halogen. In especially preferred embodiments, a third ring substituent R3 
is selected from the group consisting of lower alkyl, lower alkoxy and 
halogen. 
It is preferred, although not necessary, that the azo dye be a fully 
conjugated system. In particular embodiments, the dye includes a plurality 
of azo groups having nitrogen atoms linked to aromatic rings such that the 
compound is a fully conjugated system. Diazo or triazo compounds, for 
example, would provide such a fully conjugated system. Such fully 
conjugated systems are both brighter and more susceptible to degradation. 
However, some less than fully conjugated dyes (such as C.I. 25380 direct 
red 75; C.I. 29156 direct orange 102; C.I. 29160 direct red 23; and C.I. 
13950 direct yellow 27) may also be modified by adding lignin-like 
moieties to make them more biodegradable. Modification of a portion of the 
dye molecule will at least make that part of the molecule more degradable, 
and may as a result make the entire molecule more degradable. 
The aromatic ring having the lignin-like substitution pattern can be 
phenyl, naphthyl or other aromatic structures. A naphthyl ring is shown in 
azo dyes 19-26, 31, 32, 36, and 37. The dye may preferably include a 
sulfonic acid group to increase solubility of the dye. The sulfonic acid 
group may be present on either the lignin-like ring or elsewhere in the 
molecule. 
In particular embodiments, R2 is ortho to R1. In other embodiments, R1 is 
hydroxy while R2 is a lower alkoxy, such as methoxy, or a lower alkyl 
group such as methyl or ethyl. In preferred embodiments wherein R1 is 
hydroxy, R2 may preferably be halogen, such as fluorine or chlorine. 
The azo dyes of the present invention preferably include at least one 
sulfonic acid group, on either the lignin-like ring or somewhere else in 
the molecule, to increase the solubility of the azo compound. This 
solubility is important to some dye applications. 
In those embodiments in which R2 is ortho to R1, R2 may preferably be lower 
alkyl, lower alkoxy or halogen. 
The present invention provides a composition that includes Phanerochaete 
chrysosporium and azo dyes according to the present invention. Several 
embodiments of the dyes are degraded by Phanerochaete chrysosporium. These 
embodiments include 4-dimethylamino-azobenzene-4'-sulfonic acid, 
4-diethylamino-azobenzene-4'sulfonic acid, 
3-methyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
3-methoxy-4-hydroxy-azobenzene-4'-sulfonic acid, 
3-chloro-4-hydroxy-azobenzene-4'-sulfonic acid, 
3-sec-butyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
3,5-dimethoxy-4-hydroxy-azobenzene-4'-sulfonic acid, 
3,5-difluoro-4-hydroxy-azobenzene-4-sulfonic acid, 
3,5-dimethyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
2-hydroxy-4,5-dimethyl-azobenzene-4'-sulfonic acid, 
2-hydroxy-3-methoxy-5-methyl-azobenzene-4'-sulfonic acid, and 
4-(3-methoxy-4-hydroxyphenylazo)-azobenzene-3,4'-disulfonic acid. 
Several embodiments of the invention have been found to be particularly 
suitable for significant degradation by Phanerochaete. These embodiments 
include the following azo dyes: 4-dimethylamino-azobenzene-4'-sulfonic 
acid, 4-diethylamino-azobenzene-4'-sulfonic acid, 
3-methyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
3-methoxy-4-hydroxy-azobenzene-4'-sulfonic acid, 
3-chloro-4-hydroxy-azobenzene-4'-sulfonic acid, 
3-sec-butyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
2-hydroxy-5-methyl-azobenzene-4'-sulfonic acid, 
2-hydroxy-5-ethyl-azobenzene-4'-sulfonic acid, 
3,5-dimethyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
3,5-dimethoxy-4-hydroxy-azobenzene-4'-sulfonic acid, 
3,5-difluoro-4-hydroxy-azobenzene-4'-sulfonic acid, 
2-hydroxy-4,5-dimethyl-azobenzene-4'-sulfonic acid, 
2-hydroxy-3-methoxy-5-methyl-azobenzene-4'-sulfonic acid. 
Several embodiments of the invention have been found to be suitable for 
significant degradation by Phanerochaete when the concentration of the azo 
dye is less than about 150 ppm. These embodiments of the azo dyes include 
4-dimethylamino-azobenzene-4'-sulfonic acid, 
4-diethylamino-azobenzene-4'sulfonic acid, 
3-methyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
3-methoxy-4-hydroxy-azobenzene-4'-sulfonic acid, 
3-chloro-4-hydroxy-azobenzene-4'-sulfonic acid, 
3-sec-butyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
3,5-dimethoxy-4-hydroxy-azobenzene-4'-sulfonic acid, 
3,5-difluoro-4-hydroxy-azobenzene-4-sulfonic acid, 
3,5-dimethyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
2-hydroxy-4,5-dimethyl-azobenzene-4'-sulfonic acid, 
2-hydroxy-3-methoxy-5-methyl-azobenzene-4'-sulfonic acid, 
4-(3-methoxy-4-hydroxyphenylazo)-azobenzene-3,4'-disulfonic acid, 
2-hydroxy-5-ethyl-azobenzene-4'-sulfonic acid, 
3,4-dimethoxy-azobenzene-4'-sulfonic acid, 1-(4'-benzenesulfoncic 
acid)-4-hydroxynaphthalene, 4-methoxy-azobenzene-4'-sulfonic acid, 
4-amino-1,1'-azobenzene-3,4'-disulfonic acid, 
1-phenylazo-2-hydroxynaphthalene-6-sulfonic acid, 
4-aminoazobenzene-4'-sulfonic acid and 4-hydroxy-azobenzene-4'-sulfonic 
acid. 
Particularly suitable compositions according to the present invention 
include Phanerochaete chrysosporium and the following embodiments of the 
azo dyes: 3-methyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
3-methoxy-4-hydroxy-azobenzene-4'-sulfonic acid, 
3-sec-butyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
3,5-dimethoxy-4-hydroxy-azobenzene-4'-sulfonic acid, 
3,5-dimethyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
2-hydroxy-4,5-dimethyl-azobenzene-4'-sulfonic acid, 
2-hydroxy-3-methoxy-5-methyl-azobenzene-4'-sulfonic acid, 
4-(3-methoxy-4-hydroxyphenylazo)-azobenzene-3,4'-disulfonic acid, 
2-hydroxy-5-ethyl-azobenzene-4'-sulfonic acid, 
3,4-dimethoxy-azobenzene-4'-sulfonic acid, and 1-(4'-benzenesulfoncic 
acid)-4-hydroxynaphthalene. 
Compositions according to the present invention can include Streptomyces as 
the microbe. Where Streptomyces is the microbe, suitable embodiments of 
the azo dyes include 
4-(3-methoxy-4-hydroxyphenylazo)-azobenzene-3,4'-disulfonic acid, 
3-methoxy-4-hydroxy-azobenzene-4'-sulfonic acid, and 
2-hydroxy-5-ethyl-azobenzene-4'-sulfonic acid. 
Compositions according to the present invention having Streptomyces as the 
microbe may include azo dyes having a third ring substituent R.sub.3 
selected from the group consisting of lower alkyl and lower alkoxy. 
R.sub.2 and R.sub.3 may both be ortho to R.sub.1. Suitable embodiments of 
the azo dyes having both R.sub.2 and R.sub.3 include 
3,5-dimethyl-4-hydroxy-azobenzene-4'-sulfonic acid and 
3,5-dimethoxy-4-hydroxy-azobenzene-4'-sulfonic acid. 
Particularly suitable azo dye compounds comprise an azo group having first 
and second nitrogen atoms linked to first and second aromatic rings, 
wherein the first aromatic ring has a first substituent R.sub.1 selected 
from the group consisting of hydroxy and lower alkoxy, a second 
substituent R.sub.2 selected from the group consisting of lower alkyl, 
lower alkoxy and fluorine, and a lower alkoxy substituent R.sub.3. 
The present invention also provides a biodegradable composition comprising 
(1) an azo dye having first and second nitrogen atoms linked to first and 
second aromatic rings, the first ring having a lignin-like substitution 
pattern, (2) an amount of lignin peroxidase effective to degrade said dye, 
wherein lignin peroxidase is converted to lignin peroxidase II as the dye 
is degraded, and (3) an amount of veratryl alcohol effective to recycle 
lignin peroxidase II to lignin peroxidase. An effective amount of lignin 
peroxidase is defined to mean an amount sufficient to oxidize the azo dye 
to an oxidized state. An amount of veratryl alcohol effective to recycle 
lignin peroxidase to lignin peroxidase II is suitably, for example, at 
least 20 micromoles, and can be 200 micromoles or greater. The lignin 
peroxidase may be provided by a microbe. 
The composition may also comprise an azo dye having first and second 
nitrogen atoms linked to first and second aromatic rings, and an amount of 
peroxidase effective to degrade the dye. The peroxidase may be provided by 
a microbe such as Streptomyces or a fungus such as Phanerochaete 
chrysosporium. Particularly suitable azo dyes have a hydroxy group para to 
the first and second nitrogen atoms comprising the azo group. 
Azo dyes for the composition may have the first ring include a first 
substituent R.sub.1 selected from the group consisting of hydroxy, alkoxy 
and amino, and a second substituent R.sub.2 selected from the group 
consisting of hydrogen, lower alkoxy, lower alkyl and halogen. The first 
aromatic ring may also have a first substituent R.sub.1 selected from the 
group consisting of hydroxy, lower alkoxy, or amino, and a second 
substituent R.sub.2 selected from the group consisting of lower alkyl, 
lower alkoxy and halogen. The first aromatic ring may have a third ring 
substituent R.sub.3 selected from the group consisting of lower alkyl, 
lower alkoxy, and halogen. 
The azo dye may further comprise a plurality of azo groups having nitrogen 
atoms linked to aromatic rings such that the compound is a fully 
conjugated system. Finally, the first aromatic ring may have a first 
substituent R.sub.1 selected from the group consisting of hydroxy and 
lower alkoxy, a second substituent R.sub.2 selected from the group 
consisting of lower alkyl, lower alkoxy and fluorine, and a lower alkoxy 
substituent R.sub.3. 
The present invention also provides a method for degrading xenobiotic azo 
dyes having first and second nitrogen atoms linked to first and second 
aromatic rings, the method comprising the steps of (1) providing a 
lignin-like substitution pattern on the first aromatic ring, and (2) 
exposing the mixture to an amount of lignin peroxidase effective to 
degrade the azo dye, wherein lignin peroxidase is converted to lignin 
peroxidase II as the dye is degraded. The lignin peroxidase may be 
provided by a microbe, such as P. chrysosporium. The method may also 
include the step of combining veratryl alcohol with the azo dye to form a 
mixture and exposing the mixture to lignin peroxidase, wherein the 
veratryl alcohol is added in an amount sufficient to convert lignin 
peroxidase II to lignin peroxidase. 
In a particular embodiment, the biodegradable dye compound comprises an azo 
group having first and second nitrogen atoms linked to first and second 
aromatic rings wherein the first aromatic ring has a first substituent 
R.sub.1 selected from the group consisting of hydroxy and lower alkoxy, a 
second substituent R.sub.2 selected from the group consisting of lower 
alkyl and lower alkoxy, and a third substituent R.sub.3 selected from the 
group consisting of lower alkoxy and halogen. 
In other embodiments, R.sub.3 is halogen or may be selected from the group 
consisting of fluorine and chlorine. 
In other embodiments, the azo dye further includes at least one sulfonic 
acid group. 
In yet other embodiments R.sub.3 is halogen. 
In yet other embodiments, R.sub.1 is hydroxy and R.sub.2 and R.sub.3 are 
both ortho to R.sub.1 ; or R.sub.1 is hydroxy and R.sub.2 and R.sub.3 are 
methoxy; or R.sub.1 is hydroxy and R.sub.2 is methyl and R.sub.3 is 
methoxy. 
In yet other embodiments R3 is lower alkyl or lower alkoxy. 
In still other embodiments, the biodegradable azo dye is selected from the 
group consisting of 3,5-dimethyl-4-hydroxy-azobenzene-4'-sulfonic acid; 
3,5-dimethoxy-4-hydroxy-azobenzene-4'-sulfonic acid; 
3,5-difluoro-4-hydroxy-azobenzene-4'-sulfonic acid; 
2-hydroxy-4,5-dimethyl-azobenzene-4'-sulfonic acid; and 
2-hydroxy-3-methoxy-5-methyl-azobenzene-4'-sulfonic acid. 
In yet other embodiments the biodegradable azo dye compound is selected 
from the group consisting of 3,5-dimethyl-4-hydroxy-azobenzene-4'-sulfonic 
acid, 3,5-dimethoxy-4-hydroxy-azobenzene-4'-sulfonic acid, and 
2-hydroxy-3-methoxy-5-methyl-azobenzene-4'-sulfonic acid. 
In other embodiments, the dye compound comprises an azo group having first 
and second nitrogen atoms linked to first and second aromatic rings, an 
azo group having a nitrogen atom linked to an aromatic ring, the aromatic 
ring having a hydroxy group, and two methoxy groups attached thereto. In 
preferred embodiments, the methoxy groups are both ortho to the hydroxy 
group. 
In other embodiments the compound comprises an azo group having first and 
second nitrogen atoms linked to first and second aromatic rings, an azo 
group having a nitrogen atom linked to an aromatic ring, wherein the 
aromatic ring has a hydroxy group, a methyl group, and a methoxy group 
attached thereto. Preferably the methyl and methoxy groups are both ortho 
to the hydroxy group. 
In some embodiments the biodegradable dye includes an azo group having 
first and second nitrogen atoms linked to first and second aromatic rings, 
an azo group having a nitrogen atom linked to an aromatic ring wherein the 
aromatic ring has a first substituent R1 selected from the group 
consisting of hydroxy and lower alkoxy, a second substituent R.sub.2 
selected from the group consisting of lower alkyl, lower alkoxy and 
fluorine, and a lower alkoxy substituent R.sub.3. In preferred embodiments 
R.sub.1 is hydroxy and R.sub.2 is lower alkyl, with R.sub.2 and R.sub.3 
both ortho to R.sub.1. 
In other embodiments the azo group has first and second nitrogen atoms 
linked to first and second aromatic rings, with the first aromatic ring 
having a hydroxy substituent para to the azo group, and a lower alkyl 
substituent ortho to the hydroxy substituent. In preferred embodiments, 
the lower alkyl substituent is methyl. 
In another embodiment, the azo group has a nitrogen atom linked to an 
aromatic ring, with the ring having a first substituent R.sub.1 and a 
second substituent R.sub.2, wherein both R.sub.1 and R.sub.2 are lower 
alkoxy. Preferably, R.sub.1 and R.sub.2 are both methoxy. In other 
embodiments R.sub.1 and R.sub.2 are ortho to each other, or R.sub.1 is 
para to the azo group. 
In another embodiment, a plurality of azo groups have nitrogen atoms linked 
to first, second and third aromatic rings such that the compound of the 
fully conjugated system, wherein the first aromatic ring has a hydroxy and 
a lower alkoxy group attached thereto. The lower alkoxy group is 
preferably a methoxy. 
In yet another embodiment, the azo group has first and second nitrogen 
atoms linked to first and second aromatic rings, wherein the first 
aromatic ring has a first substituent R.sub.1 para to the nitrogen atom, 
wherein R.sub.1 is selected from the group consisting of hydroxy and lower 
alkoxy, and a second substituent R.sub.2 selected from the group 
consisting of methyl, ethyl and fluorine. In particular embodiments, 
R.sub.2 is ortho to R.sub.1. In yet other embodiments R.sub.2 is methyl. 
In another embodiment the first aromatic ring has a third substituent 
R.sub.3 selected from the group consisting of lower alkyl, lower alkoxy 
and a halogen, particularly wherein R.sub.3 is ortho to R.sub.1. In some 
specific embodiments, R.sub.2 and R.sub.3 are both methyl. 
Several especially preferred embodiments are very completely degraded by 
Phanerochaete, and include 4-dimethylamino-azobenzene-4'-sulfonic acid, 
3-methyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
3-methoxy-4-hydroxy-azobenzene-4'-sulfonic acid, 
3-chloro-4-hydroxy-azobenzene-4'-sulfonic acid, 
3-sec-butyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
3,5-dimethoxy-4-hydroxy-azobenzene-4'-sulfonic acid, 
3,5-difluoro-4-hydroxy-azobenzene-4'-sulfonic acid, 
2-hydroxy-4,5-dimethyl-azobenzene-4'-sulfonic acid, 
2-hydroxy-3-methoxy-5-methyl-azobenzene-4'-sulfonic acid. 
Other compounds show some biodegradability when cultured with the 
Streptomyces strains of the present invention. Examples of such compounds 
having a higher degree of biodegradation with Streptomyces are the 
following: 3-methyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
3-methoxy-4-hydroxy-azobenzene-4'-sulfonic acid, 
3-chloro-4-hydroxy-azobenzene-4'-sulfonic acid, 
3-sec-butyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
3,5-dimethyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
3,5-dimethoxy-4-hydroxy-azobenzene-4'-sulfonic acid, and 
2-hydroxy-3-methoxy-5-methyl-azobenzene-4'-sulfonic acid and 
4-diethylamino-azobenzene-4'-sulfonic acid. 
Especially well degraded dyes with Streptomyces include 
3-methyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
3-methoxy-4-hydroxy-azobenzene-4'-sulfonic acid, 
3-sec-butyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
3,5-dimethyl-4-hydroxy-azobenzene-4'-sulfonic acid, 
3,5-dimethoxy-4-hydroxy-azobenzene-4'-sulfonic acid. These dyes were 
significantly degraded by Streptomyces, that is degraded more than about 
10%. 
Particularly high Streptomyces degradation is observed when R1 is hydroxy, 
and R2 and R3 are methyl, particularly when the methyls are both ortho to 
the hydroxy. Similarly, high Streptomyces degradation is seen when R1 is 
hydroxy and R2 and R3 are both methoxy, particularly if both R2 and R3 are 
ortho to R1. Other Streptomyces degradable compounds include those in 
which R1 is hydroxy, R2 is methyl and R3 is methoxy, especially wherein R2 
and R3 are both ortho to R1. 
The present invention also includes a biodegradable composition containing 
an azo dye having a nitrogen atom linked to an aromatic ring with a 
lignin-like substitution pattern, wherein the composition also includes a 
microbe capable of degrading the dye. The ring has a first substituent R1 
selected from the group consisting of hydroxy, alkoxy and amino, and a 
second substituent R2 selected from the group consisting of hydrogen, 
lower alkoxy, lower alkyl and hydrogen. The amino in preferred embodiments 
is secondary amine. 
In particularly preferred embodiments of the composition, the microbe is 
Phanerochaete chrysosporium. Subspecies of the azo dye that are 
particularly useful in such a composition include those wherein R1 is 
hydroxy, particularly if R2 is a lower alkoxy, lower alkyl or halogen. 
In yet other embodiments of the composition, the microbe is a Streptomyces, 
for example S.rochei, S.chromofuscus, S.diastaticus, S.viridosporus, or 
S.badius. Particularly useful strains of Streptomyces have been found to 
be S.rochei A10, A14, S.chromofuscus A11, A20, S.diastaticus A12, A13, 
S.viridosporus T7A and S.badius 252. Several compounds have been found to 
be particularly biodegradable in combination with Streptomyces. Such 
compounds include those in which R1 is hydroxy, particularly when R2 is 
ortho to the hydroxy. Enhanced biodegradability is also observed when the 
ring includes a third ring substituent R3 selected from the group 
consisting of lower alkyl and lower alkoxy. Biodegradability is 
particularly high when R1 is a hydroxy para to the azo linkage, and R2 is 
ortho to the hydroxy. In such embodiments, R2 is most preferably a lower 
alkoxy or lower alkyl. 
In yet other embodiments of the invention, the biodegradability of 
xenobiotic dyes is increased by introducing a lignin-like aromatic ring 
into a preexisting azo dye. 
Persons skilled in the art will recognize that azo dyes, other than those 
specifically disclosed, are included in the scope of this invention. Other 
microorganisms are also suitable for use in degrading these azo dyes. Soil 
microflora in general are a good source of additional microorganisms, 
which can be tested for biodegradative capacity as described in this 
specification. The dyes can also be degraded in soil itself, which 
contains many species of organisms capable of degrading the lignin-like 
dyes of the present invention. 
The present application describes certain strains of soil Streptomyces 
species which are particularly effective at degrading the disclosed azo 
dyes. Such natural variability is expected, and is not evidence of any 
limitation of the method to use with particular strains of bacteria. Any 
person skilled in the art will be able to select bacteria from soil or 
elsewhere using the biotransformation assays disclosed herein. Actual 
selection of individual biodegradative species and strains is not 
essential because a mixture of soil microflora contains the microorganisms 
sufficient for azo dye biotransformation. 
Table 4 illustrates that higher concentrations of azo dyes are sometimes 
less effectively degraded by P. chrysosporium. Dye 3, for example, becomes 
more toxic to the organism at 300 ppm, in contrast to concentrations below 
300 ppm. Dyes 4 and 5 do not exhibit a similar degree of inhibition. In 
any case, toxic inhibition is not complete even at 300 ppm in sensitive 
organisms. Optimum concentrations of substrate are very specific to the 
substrate and organism of interest, and are subject to the kind of routine 
optimization known to those skilled in the art. 
Having illustrated and described the principles of the invention in several 
preferred embodiments, it should be apparent to those skilled in the art 
that the invention can be modified in arrangement and detail without 
departing from such principles. We claim all modifications coming within 
the spirit and scope of the following claims.