Microbial decolorization of wastewater

Present invention offers, for the first time, a biological approach through the use of white-rot fungi to the decolorization of dye wastewater. It is also applicable to other colored substances and/or their wastewater such as molasses. Because of its low cost, renewable and regenerative activity, and little or no secondary pollution hazard, biological method is the most widely practiced method in nature and in practice in treating organic refuse and industrial waste. Current invention discloses, specifically, undiscovered activities of the Myrothecium and Ganoderma fungi in removing colored substances from dye solutions and dye wastewater. It is the result of a deliberate process of screening for natural water/soil-born and farm/industrial-waste-derived microorganisms for such specific purpose in our laboratory. The accompanied process invention shows that simple biological treatment could also produce consistently effective results in treating a wide spectrum of dye wastewater under greatly varied conditions. This ability also is not restricted to the dye substances. For instance, it was also effective in decolorizing molasses fermentation wastewater. Since Ganoderma fungi is a well consumed fungi in the Orient particularly for its medicinal value, we have reason to believe that this color transforming and/or removing ability has great potential in facilitating the manufacturing of specialty food and drug products where color may present certain unique consideration.

FIELD OF THE INVENTION 
The present invention relates to microbial decolorization of wastewater, 
and in particular to decolorization of dye molecules or dye-containing 
wastewater by white-rot fungi, or even more specifically, by species 
belonging to the Myrothecium or Ganoderma genus. The above was 
accomplished through a mechanism of dye adsorption, and in certain cases, 
followed by microbial dye degradation. 
BACKGROUND OF THE INVENTION 
In addition to biological treatment, physical and chemical methods are also 
used for removal of colored dye substances in wastewater (Groff and Kim, 
1989). In fact, the latter two have been more widely adopted for their 
effectiveness. Chemical methods often involve coagulation of dye 
substances followed by precipitation of the chemical sludge or oxidation 
process using ozone. Physical methods involve mainly adsorption by 
activated carbon or its like. In hybrid physical/chemical or 
physical/biological processes, ionizing irradiation and ultrafiltration 
are useful methods of pretreatment. Nevertheless, both physical and 
chemical methods have their shortcomings. Coagulation produces excess 
amount of chemical sludge and creates problem of its disposal. Oxidation 
employs costly ozone and is not effective for reductive or sulphur dye 
wastewater. Activated carbon also incurs high operating expenses and 
additional capital on activity regeneration. These are obvious 
opportunities and challenges for biological treatment method to offer 
viable alternatives in treating dye-containing wastewater. 
Bio-decolorization of lignin-containing pulp and paper wastewater using 
white-rot fungi Phanerochaete Chrysosporium and Tinctporia sp. (Eaton, et 
al., 1980; Fukuzumi, et al., 1980) were clear examples of color removal 
thru microbial degradation of the colored substance, i.e., highly 
chlorinated and oxidized polymeric lignin molecules. Similar observation 
was reported later using another white-rot fungus Schizophyllum commune to 
decolorize wastewater from a bagasse pulping plant (Belsare, et al., 
1988). As for dye color removal, a recent review (Groff and kim, 1989) 
described the ability of Rhodococcus, Bacillus cereus and 
Plesiomonas/Achromobacter to degrade soluble dyes, acid red dye and five 
azo-dyes, respectively. The widely practiced biological activated sludge 
method may be useful in removing COD and BOD in dye wastewater. They found 
little information concerning its effectiveness in dye color removal. On 
the other hand, textile dyes were found strongly adsorbed and held by 
wastewater treatment plant sludges that were landfilled. This suggests 
that adsorption may play another key role in biodecolorization. 
Molasses wastewater may present an even tougher color removal task. One 
culture possessing this ability was found belonging to the Basidiomycetes 
family (Hongo, et al., 1973). Further screening utilizing melanoidin, the 
major colored substance in molasses, isolated Coriolus sp. 20 and found 
its color removing activity may be associated with the enzyme sorbose 
oxidase (Watanabe, et al., 1982). Subsequent investigation by Ohmomo, et 
al., at University of Tsukuba, Japan, yielded Coriolus versicolor Ps4a 
(Ohmomo, et al., 1985), Mycelia sterilia D90, Aspergillus fumigatus G-2-6, 
A. oryzae Y-2-32, and Lactobacillus hilgaridii W-NS, all found capable in 
decolorizing molasses. 
Above reviewed bio-decolorization reports limit their studies primarily in 
defined laboratory model systems. Their actual application and 
effectiveness toward colored high strength industrial wastewater were not 
particularly emphasized. Though limited, successful examples of 
bio-decolorization of pulp and paper wastewater (U.S. Pat. No. 4,655,926, 
Chang, et al., 1987) and molasses wastewater (Sirianuntapiboon, et al., 
1988) were reported. The former demonstrated the use of a rotating 
biological contacter and strains of white-rot fungi to remove color in 
waste liquor without giving quantitative results. While the latter claimed 
color removal of 40 percent or higher. 
SUMMARY OF THE INVENTION 
Present invention offers, for the first time, a biological approach thru 
the use of white-rot fungi to the decolorization of dye wastewater. It is 
also applicable to other colored substances and/or their wastewater such 
as molasses. Because of its low cost, renewable and regenerative activity, 
and little or no secondary pollution hazard, biological method is the most 
widely practiced method in nature and in practice in treating organic 
refuse and industrial waste. Current invention discloses, specifically, 
undiscovered activities of the Myrothecium and Ganoderma fungi in removing 
colored substances from dye solutions and dye wastewater. It is the result 
of a deliberate process of screening for natural water/soil-born and 
farm/industrial-waste-derived microorganisms for such specific purpose in 
our laboratory. 
The accompanied process invention shows that simple biological treatment 
could also produce consistently effective results in treating a wide 
spectrum of dye wastewater under greatly varied conditions. This ability 
also is not restricted to the dye substances. For instance, it was also 
effective in decolorizing molasses fermentation wastewater. Since 
Ganoderma fungi is a well consumed fungi in the Orient particularly for 
its medicinal value, we have reason to believe that this color 
transforming and/or removing ability has great potential in facilitating 
the manufacturing of specialty food and drug products where color may 
present certain unique consideration.

DETAILED DESCRIPTION OF THE PRESENT INVENTION 
Process of present invention is as follows: 
I. Source of Wild Type Cultures 
1. Soil from the farms and pastures of National Taiwan University in the 
vicinity of DCB--64 strains 
2. Long chain dicarboxylic-acid-producing culture--74 strains 
3. Pig excrement--168 strains 
4. Wastewater from Taiwan Sugar Corporation--27 strains 
5. Wastwater from China Chemical Corporation--3 strains 
6. Wastwater from the alcohol fermentation factory of Taiwan Sugar 
Corporation--7 strains 
7. Dye wastewater--13 strains 
The total number of strains is 419. 
II. Isolation of Dye Degrading Microorganism 
Nutrient agar (NA) and potato dextrose agar (PDA) petri plates supplemented 
with each of the three dyes molecules listed below were used to screen 
soil and wastewater samples for colonies circled by a clear decolorized 
zone. This procedure followed standard microbiology practices. Antibiotics 
were added to PDA to discourage bacteria growth. 
##STR1## 
Further confirmation used the following dye/media combinations: 
______________________________________ 
Solution Nutrient 
of dyes (100 mg/L) W/2% agar 
______________________________________ 
Orange-II 0.1% glucose 
0.1% peptone 
Orange-II None 
RS 0.1% glucose 
0.1% peptone 
RS None 
10B 0.1% glucose 
0.1% peptone 
10B None 
______________________________________ 
Colonies so isolated were then tested for dye decolorization under 
submerged culture condition at near ambient temperature for up to two 
weeks. One spore-forming filamentous fungus, suspected of the white-rot 
type, showed consistent ability in clearing dye color (10.sup.2 mg/L 
solution) to a degree not detectable by naked eyes. The reaction could 
take place over wide pH range and static incubation condition. In 
addition, the culture propagates rapidly with simple growth requirement. 
For its availability in our lab, Ganoderma species from the Culture 
Collection & Research Center (CCRC) of the Republic of China were also 
included in this dye decolorization screen. Their ability to remove dye 
color was confirmed soon after the identification of the unknown isolate. 
III. Decolorization of Target Dyes with Resting Cells 
The simplest way to carry out the decolorization study was to grow the 
culture first in potato dextrose broth (PDB) to sufficiently high density. 
After centrifugation of the culture broth, packed wet cell cake was then 
removed, weighed and mixed with constant volume of various dye-containing 
sample solutions for the resting cells experiments. 
Degree of decolorization was quantified by measuring reduction of optical 
density (OD) at characteristic wavelength of each dye materials or 
samples. It is the wavelength having maximum light absorbency over a full 
visible spectrum scan. Due to the high OD of untreated samples, standard 
procedure in sample dilution was followed in observing Beer-Lambert law. 
The OD of untreated blank controls were corrected for dilution by adding 
water equal to that trapped in the cell cake. During experiment, they were 
kept in closed containers under refrigerated temperature. 
IV. Culture Identification 
From the observation of the growth of cells and the size, shape, color and 
texture of the spores engendered therefrom, and with reference to 
published reports and papers (Preston, 1943), the isolate is identified as 
Myrothecium verrucaria DCB D-1. M. verrucaria CCRC 31545 (ATCC 9095) 
obtained from CCRC has growth and spore morphology identical to those of 
DCB D-1. Further experiments (the same method as in example 1) porved that 
M. verrucaria 31545 and ATCC 36315 also posses dye color removing 
capability: 
______________________________________ 
Percentage of Color Removal 
Strains RS 10B Orange-II 
______________________________________ 
DCB D-1 98.9 97.6 70.1 
ATCC 9095 96.6 94.5 63.4 
(CCRC 31545) 
ATCC 36315 99.1 98.9 91.2 
______________________________________ 
From these results, our unknown isolate which possesses the novel 
dye-removal capability was identified as M. verrucaria DCB D-1. It is also 
known from published reports and papers that this fungal species produces 
strong cellulase activity (Herr, Luck and Dellweg, 1978; Hulme and 
Stranks, 1971) and synthesizes macrocyclic trichothecene (Jarvis, et al., 
1984; Smitka, et al., 1984; Jarvis and Mazzola, 1980). 
Having identified this filamentous fungus, other fungi belong to the same 
genus and other genuses of the white-rot fungi type deserved further 
examination. Hence, the Ganoderma species were tested and, subsequently, 
confirmed to possess similar color removing ability as M. verrucaria DCB 
D-1 (see example 8). We therefore conclude that this dye adsorption and 
degradation activity is not limited to the Myrothecium and Ganoderma 
genuses. It could be a novel characteristic widely possessed by the 
white-rot fungi. 
V. Examples 
Examples of decolorization of dye solutions and dye wastewater using the 
present invention are described in detail as follows: 
Example 1 
Color removal by M. verrucaria DCB D-1 on the three dye mentioned above 
Dye solutions had the following properties: 
______________________________________ 
Dyes (RS) (10B) (Orange-II) 
______________________________________ 
Color red blue orange 
PH value 7.0 6.7 6.4 
Light ahsorbing 
510 620 485 
wavelength (nm) 
Concentration 
0.25 g/L 0.2 g/L 0.24 g/L 
of solution 
______________________________________ 
Three-day-old shake flask cultured M. verrucaria DCB D-1 cells (in PDB) 
were harvested by centrifugation. Mixed cells (water content: 98.5%) and 
dye solution in a conical flask with a weight ratio of 1:3. Allowed the 
flask to stand for 1 day at a temperature of 28.degree. C. before 
measuring the O.D. value of the supernatants. As for the control group, 
use the same amount of distilled water in place of the cells. The degree 
of decolorization (%)=[(O.D. bdfore treatment - O.D. after treatment)/O.D. 
before treatment].times.100%. Results were recorded as follows: 
______________________________________ 
Dye (RS) (10B) (Orange-II) 
______________________________________ 
O.D. before 5.64 6.02 8.96 
treatment 
O.D. after 0.101 0.451 3.03 
treatment 
percentage of 
98.2 92.5 66.2 
color removal 
______________________________________ 
This decolorization experiment was repeated and results showed good 
reproducibility: 
______________________________________ 
(Percentage of decolorization) 
(Experiments) 
RS 10B Orange-II 
______________________________________ 
1 98.2 92.5 66.2 
2 98.9 97.6 70.1 
______________________________________ 
HPLC method was also developed to quantify the rate of dye removal. Since 
its results showed linear correspondence with the O.D. measurements, we 
adopted the simpler O.D. method for quantification of dye removal. Through 
visual inspection, one saw that the cells adsorbed the dye color first as 
indicated by the quickly colored filamentous mass. The cell bound color 
then gradually degraded, depending on types of the dye molecules, over a 
period of a week or longer. Afterwards, the filamentous mass regained its 
pale white color. 
Higher dye concentration did not hinder color removal and prolonged contact 
between dye and cells produced greater OD reduction. 
Example 2 
Treating dye wastewater samples from a textile dyeing factory 
Samples 
I: basic dye residual solution from yarn dyeing 
II: direct dye residual solution from yarn dyeing 
III: T/R oxidative desizing and disperse dye residual solution from yarn 
dyeing 
IV: reactive dye residual solution 
V: Neolan acid dye residual solution 
VI: sulphide: reactive/vat dye=1:4 
All six samples had wide pH variations and high cholride ion 
concentration-some as high as 15% (W/V) and their general properties were 
as follows: 
______________________________________ 
Wastewater I II III IV V VI 
______________________________________ 
PH value 4.48 9.8 8.2 11 9.3 10.2 
Light absorbing 
567 496 467 439 565 600 
wavelength (nm) 
______________________________________ 
3-5 days old M. verrucaria DCB D-1 in PDB shaker culture were harvested by 
centrifugation. They were mixed with the wastewater samples in different 
weight: volume proportions as follows. Separate sets of the same 
preparation without cells were kept. 
______________________________________ 
Samples I II III IV V VI 
______________________________________ 
Cell/Dye solution 
1:3 1:2.5 1:2.5 1:1.67 
1:2.67 1:2.2 
______________________________________ 
Both sets were incubated at 28.degree. C. for 1 to 2 weeks. Results of O.D. 
removed according to their light absorbing wavelengths were: 
______________________________________ 
Samples I II III IV V VI 
______________________________________ 
Percentage of 
93% 98% 78% 81% 43% 84% 
color removal 
Final pH value 
4.0 4.6 4.8 8.4 3.6 4.7 
______________________________________ 
Visual observation was also recorded: 
Samples 
I: purplish blue.fwdarw.light pink.fwdarw.color is decomposed by cells and 
wastewater became colorless 
II: reddish black.fwdarw.color is adsorbed by cells and wastwater became 
colorless 
III: black.fwdarw.color is adsorbed by cells and wastewater became 
colorless 
IV: reddish black.fwdarw.color is adsorbed by cells and wastewater became 
colorless 
V: crimson red.fwdarw.color is adsorbed by cells and wastewater became pink 
VI: black.fwdarw.color is adsorbed by cells and wastewater became almost 
colorless 
Example 3 
Treating dye wastewater samples from a dye maunfacturing plant 
Sample I: mixed liquor containing reactive, direct, acid and other high 
class dye residues, pH 8.5 
Samples II: reactive red-dye-containing waste liquor, pH 8.3 
Equal weight of the newly isolated bioagent and fresh aeration pond 
activated sludge (from the plant site) were each mixed with same weight of 
raw or 1:1 diluted dye wastewater and incubated for 2 weeks. The activated 
sludge test was under constant aeration, while the new isolate used the 
usual static incubation (same as example 2). 
______________________________________ 
Sample I Sample II 
Method Used (% OD Reduction) 
______________________________________ 
Act. Sludge 3 (pH 8.0) 7 (pH 8.3) 
This Study + 30 37 
1.times. Waste Liquor 
This Study + 94 60 
0.5.times. Waste Liquor 
______________________________________ 
It is evident that the new fungal isolate is far more effective than 
activated sludge in removing dye color from the wastewater. 
Visual inspection yielded the following observation: 
1. Activated sludge process: 
No obvious changes-wastewater was still reddish brown. 
2. Present invention: 
Color is decomposed by mycelia and wastewater showed obvious color 
reduction. 
Example 4 
Molasses fermentation wastewater from monosodium glutamate factory. 
M. verrucaria DCB D-1 cells were added the same way as in previous examples 
to wastewater at a weight:volume ratio of 1:1. They were incubated at 
28.degree. C. Control sample without added cells was stored in the 
refrigerator. After two weeks, O.D. values between sample and control sets 
were compared at 475 nm. 
After treatment, pH changed from the initial 7.3 to 3.7, and the color 
removal percentage was 41%. Similar results were also obtained in treating 
molasses fermentation wastewater from the alcohol distillery. 
Example 5 
Use of heat inactivated cells 
Example 1 was repeated once more using RS dye and 5-day static incubation 
to compare the color removal ability between freshly grown vegetative 
cells and 10 min autoclaved cells. Much to our surprise that autoclaved 
cells were equally effective in decolorizing the dye solution: 
______________________________________ 
(Treatments) (% of color removal) 
______________________________________ 
No heat treatment 96.5 
80.degree. C. for 10 min 
98.4 
100.degree. C. for 20 min 
94.4 
Autoclave at 121.degree. C. for 10 min 
94.9 
______________________________________ 
One can view this as a strong indication of adsorption over degradeation as 
the primary mechanism of color removal. 
Example 6 
Effect of shaking 
Example 1 was repeated to compare results between static and shaker (175 
rpm) incubation. Results after 48-hr were as follows 
______________________________________ 
% O D Reduction 
Dyes Static Shaking 
______________________________________ 
RS 96.5 93.8 
10B 99.5 90.3 
Orange-II 71.5 61.7 
______________________________________ 
The data clearly shows that there is no advantage in shaking and/or 
aeration during the bio-decolorization process. There might even be some 
adverse effect as the data show. 
Example 7 
The color removing ability of various myrothecium species 
To determine the decolorizing effect of different Myrothecium species, 
Example 1 was repeated execpt that the mixtures were allowed to stand for 
2 days, for various Myrothecium species: 
______________________________________ 
Percentage of 
color removal 
Cultures ATCC No. RS 10B Orange-II 
______________________________________ 
M. verrucaria 
(DCB D-1) 98.9 97.6 70.1 
9095(CCRC31545) 
96.6 94.5 63.4 
36315 99.1 98.9 91.2 
M. prestonii 
24427 99.4 97.0 91.6 
M. leucotrichum 
16686 93.7 87.1 58.0 
M. sp 13667 87.9 89.8 -- 
M. pnicilloides 
56896 91.5 -- -- 
M. masonii 24426 89.9 90.8 -- 
M. striatisporum 
18947 97.3 98.9 -- 
M. roridum 16297 81.1 91.5 38.3 
M. cinctum 22270 86.5 58.8 25.5 
______________________________________ 
(- missing figures were due to insufficient cell growth.) 
Above results reveal that, in addition to M. verrucaria strains, other 
Myrothecium cultures also possess the capacity for dye decolorization. 
Moreover, the decolorization effect is wide spread and consistent. 
Example 8 
The color removing ability of various ganoderma species 
Both Ganoderma and Myrothecium are filamentous fungi and possess the 
characteristic ability of decomposing wood-the white-rot type. Therefore, 
in addition to Myrothecium, Ganoderma species were also chosen to 
determine their capacity for dye decolorization. 
Ganoderma cells were grown in PDB shaker culture for 7-14 days. Afterward, 
experiment followed the steps decribed in Example 1, except that the 
mixtures were allowed to stand for 2 days. 
______________________________________ 
Percentage of color removal 
Culture CCRC No. RS 10B Orange-II 
______________________________________ 
G. applanatum 
36066 87.5 68.1 41.4 
36097 90.9 81.8 -- 
36088 93.0 90.4 -- 
36113 83.6 -- -- 
36128 87.6 81.1 -- 
36156 80.0 78.1 -- 
G. lucidum 36143 90.8 90.8 -- 
36123 90.5 85.8 58.2 
36021 95.8 78.8 33.0 
G. subamboinense 
36087 94.0 81.6 40.2 
var. laevisporum 
G. oerstenii 
36293 95.2 94.0 -- 
G. sessile 37028 93.3 -- -- 
G. tropicun 37026 93.5 78.2 45.2 
36041 89.1 77.3 28.4 
37029 91.5 94.4 51.7 
G. resinaceum 
36147 97.5 94.3 -- 
36149 89.6 87.2 -- 
G. weberianum 
36145 95.6 93.1 77.2 
G. colossus 36157 96.5 92.0 -- 
G. sp 36066 91.1 86.8 42.3 
Under 37054 91.3 87.4 -- 
Identification 
37049 93.0 88.2 64.3 
37033 93.0 -- -- 
37053 96.1 94.7 -- 
______________________________________ 
(-- missing figures were due to insufficient cell growth.) 
Example 9 
Kinetics of dye adsorption by M. verrucaria DCB D-1 
The resting cell experiment in Example 1 was repeated using RS dye and 
sampled 5 minutes after addition of cells and every 2 hours thereafter. 
Based on O.D. readings, more than 50% of the dye were removed in the first 
few minutes after contact. Equilibrium for OD reduction was reached in 
about 10 hours 
To probe the limit of dye adsorption/degradation by the mycelial mass, four 
additional shots of dye solution, each contained the same amount of dye as 
the initial shot, were added to the resting cell culture in 24 hours 
intervals. Samples were taken 1 minute and 24 hours after each shot and 
their OD values measured. After adding all five shots together, we found 
that 1 kg of wet mycelial cells could remove close to 4 g of RS dye 
molecules. 
Present invention indicates that microbial decolorization could be a viable 
means in ridding dye wastewater its color related problem. Dye molecule 
adsorption onto cell surface appears to be a quick process and often 
completes in a matter of hours. This does not seem to be a specific 
process--acid, basic, direct, reactive, disperse dyes could all be cleared 
out of solution using the same approach. There is no specific nutrient 
requirement and is insensitivity to high salt concentration or wide 
variation in pH. Once it became clear that even autoclaved cells could 
pick up dye color the same way as vegetative cells, one is less surprised 
at the fact that static incubation of dye and cells alone can produce the 
desired result. Incubation of resting (non-autoclaved) cells beyond the 
dye adsorption stage resulted in visual disappearance of cell bound color 
in about a week time for certain dyes. For others, cell bound dye did not 
disappear even after one month. 
The above described examples have discolsed the proper steps and necessary 
conditions in applying the present invention. Although specific steps and 
conditions are applied in the examples, the purpose is to give a 
comprehensive and descriptive illustration. Therefore, the scope of the 
present invention should not be limited to such illustration.