Process of degrading chloro-organics by white-rot fungi

A process of degrading chloro-organics contained in liquid waste or effluent utilizes a white-rot fungus as the active ingredient in the chloro-organic degradation. The white-rot fungus is grown in the presence of certain nutrients including nutrient nitrogen and is then caused to enter a secondary metabolic state through nitrogen starvation. The fungus is then immersed in the liquid containing chloro-organics for a time period sufficient for the fungus to degrade the chloro-organics. At least periodically during the degradation period, the fungus is exposed to an oxygen enriched atmosphere. The efficacy and active lifetime of the fungus may be increased by the addition to the liquid of at least one member of the class consisting of nitrogen, a mixture of nutrient minerals, and a biological detergent.

This invention relates to biological treatment of waste to improve the 
environmental character thereof, and particularly to the degradation of 
chloro-organics contained in certain liquid wastes, by-products or 
effluents by using a white-rot fungus to convert the chloro-organics from 
aromatics to aliphatics. 
BACKGROUND OF THE INVENTION 
The discharge of chlorinated organics, particularly chlorophenols, to the 
environment is a major public health concern. In addition to causing taste 
and odor problems in potable water supplies, some chloro-organics are 
believed to be carcinogenic or mutagenic. Because chlorinated organics are 
inherently recalcitrant to biological degradation, these compounds pass 
through conventional wastewater treatment plants largely uneffected. To 
our knowledge, no effective method of biologically degrading 
chloro-organics has heretofore been recognized or proposed. 
There have been recent attempts to use biological processes for treating 
effluents from papermaking operations to decolorize such effluents. Nova 
Scotia Research Foundation, Environment Canada, Cooperative Pollution 
Abatement Research Project Report No. 410-1 (1976). One of these attempts 
proposes the use of white-rot fungi to decolorize such effluents. 
Fukuzumi, Nishida, Aoshima, and Minami, Decolorization of Kraft Waste 
Liquor with White Rot Fungi (pt. 1) 23 (6) Journal of the Japan Wood 
Research Society 290 (1977). However, none of these attempts propose, or 
to our knowledge even recognize, that white rot fungi will degrade 
chloro-organics. Since chlorinated organics are used as fungicides, the 
degradation of chlorinated organics by the fungus was unexpected and 
surprising. 
With the foregoing in mind, it is an object of the present invention to 
provide an effective process of biologically degrading chloro-organics 
contained in certain liquids to improve the environmental character 
thereof. 
A more specific object of the present invention is to provide a process of 
degrading chloro-organics and particularly chlorophenols by using white 
rot fungi to convert the aromatics to aliphatics. 
SUMMARY OF THE INVENTION 
The present invention accomplishes these objects by growing a white-rot 
fungus on the surface of a carrier in the presence of certain nutrients 
including nitrogen to form a mycelial film on the carrier surface, 
inducing the fungus into a secondary metabolic state by nitrogen 
starvation, degrading the chloro-organics contained in a liquid by 
immersing the fungus while in its secondary metabolic state in the liquid 
for a sufficient time period for the fungus to convert the aromatics into 
aliphatics, while at least periodically exposing the fungus to an oxygen 
enriched atmosphere. It has been determined that the efficacy of the 
fungus in degrading the chloro-organics and the active lifetime of the 
fungus may be increased by adding to the liquid containing the 
chloro-organics a small amount of at least one of a class consisting of 
nitrogen, a mixture of nutrient minerals, or a biological detergent.

DESCRIPTION OF THE PREFERRED EMBODIMENT 
Most microorganisms, including those used in conventional wastewater 
treatment plants or systems, cannot degrade chlorinated organics and 
particularly chlorophenols. White-rot fungi, on the other hand, have the 
capability of degrading such chloro-organics by converting the aromatics 
to aliphatics. Though the process of this invention can use any of many 
white-rot fungi, Phanerochaete chrysosporium strain BKM F-1767, isolated 
in east central Russia in 1968, is preferred because of its vigorous 
growth and rapid degradation of the chloro-organics at the relatively high 
temperature of 39.degree.-40.degree. C. and because of its formation of 
abundant conida (asexual spores) which facilitates inoculation and 
handling. 
The first step in the present process is the growing of the white-rot 
fungus to provide a readily available source of the active ingredient of 
the chloro-organic degradation process. Such growth should take place 
under sterile or semi-sterile conditions in a stationary liquid medium 
containing nitrogen and sufficient basal nutrients for rapid germination 
and growth. 
The following nutrients were used in our experiments in the amounts stated 
in grams per liter of growth medium: 
______________________________________ 
Nutrient Amount 
______________________________________ 
KH.sub.2 PO.sub.4 2 g/l 
MgSO.sub.4.H.sub.2 O 0.5 g/l 
CaCl.sub.2 0.1 g/l 
NH.sub.4 Cl 0.12 g/l 
Thiamine 0.001 g/l 
Glucose 10 g/l 
______________________________________ 
The temperature of the growth medium should be 30.degree.-40.degree. C., 
the pH should be 4.0-5.0 and the surrounding atmosphere should have an 
oxygen content of 20%-100%. 
While any suitable apparatus or equipment for growing the fungus and 
degrading the chloro-organics may be used, a rotating biological contact 
reactor has proven to be effective in bench scale experiments and some 
similar production scale equipment is anticipated to be equally effective 
when the present process is scaled up to production capacity. Such a 
rotating biological contact reactor is shown in FIG. 1 and is generally 
referred to as 10. Reactor 10 offers unique operating features including 
high surface area per unit volume, low maintenance costs, low energy 
requirements, simple construction and operation, and commercial 
availability. 
The reactor 10 comprises a tank 11 defined by four walls 12, 13, 14 and 15 
and a bottom 16, and three partitions 17, 18 and 19 extend between walls 
14 and 15 to divide the tank 11 into four compartments. A cover 20 is 
provided so that the atmosphere within reactor 10 may be controlled. A 
shaft 21 is journal for rotation in suitable bearings 22 (only one of 
which is shown) mounted in walls 12 and 13. Shaft 21 drivingly mounts 
eight plastic discs 24, 25, 26, 27, 28, 29, 30, and 31 with two discs 
being located in each of the four compartments of tank 11. A suitable 
motor 32 is connected to shaft 21 for driving shaft 21 and disc 24-31 in 
rotation for reasons to be explained. 
The reactor 10 may be operated as a batch reactor or as a continuous, 
plug-flow reactor. The reactor 10 used in our experiments had a capacity 
of 2.5 liters of liquid being treated. The tank 11 and discs 24-31 were 
constructed of such size that the discs were 40% submerged in the liquid 
when tank 11 had 2.5 liters of liquid therein. 
An oxygen source 33 is connected to tank 11 above the level of liquid 
therein by tubing 34 to maintain an oxygen enriched atmosphere, i.e. with 
an oxygen content higher than air, above the liquid and in contact with 
60% of discs 24-31. Preferably, the oxygen content in the atmosphere above 
the liquid is between about 20% and 100%. The desired temperature of 
approximately 40.degree. C. is maintained by circulating heated water 
through a jacket around tank 11 from a source of hot water (not shown) 
connected to tank 11 by tubing 35 and 36. Suitable drains 37 for removal 
of the effluent once the chloro-organics have been degraded are connected 
to each compartment of tank 11. 
A carbon/energy source is a nutritional requirement for both fungal growth 
and degradation of the chloro-organics, and the liquid to be treated does 
not normally provide such nutritional requirements. Glucose, cellulose, 
and inexpensive, commercially available, corn syrup work equally well. In 
fact, our study has demonstrated that primary sludge from a paper mill may 
be used to provide such nutritional requirements. 
To grow the fungus, a spore suspension is inoculated into a growth medium 
in tank 11 containing growth nutrients at pH 4.5. The spores readily 
germinate and grow rapidly to form mycelial mats or films on the roughened 
discs 24-31. During the first two days, the growing mycelium consumes 
nutrients and attaches to the roughened disc as a thin film. After these 
first two days, the growth medium is drained from tank 11 and tank 11 is 
refilled with liquid containing additional growth medium. After two more 
days of growth, the mycelial mats on discs 24-31 have consumed the 
available nitrogen and the fungus enters a secondary metabolic state 
capable of degrading chloro-organics. Throughout this growth period, an 
oxygen enriched atmosphere has been maintained above the effluent 
containing growth medium, and the discs have been slowly rotated at a 
speed at the periphery of preferably 2 feet per minute. 
With the fungus in a secondary metabolic state, degradation of 
chloro-organics contained in a liquid such as effluent or other wastewater 
can now begin. The rate of degradation is higher at a peripheral speed 2 
feet per minute than at 30 feet per minute and consequently a low 
peripheral speed is preferred during degradation of the chloro-organics. 
Experiments were conducted with a peripheral speed of 2 feet per minute 
with excellent results. 
The concentration of oxygen in the enclosed space above the liquid in tank 
11 is an important factor affecting the rate of degradation of the 
chloro-organics and the active lifetime of the fungus. Preferably, the 
oxygen concentration in this atmosphere should be between about 20% and 
100% and more preferably between about 50% and 100%. 
The degradation of chloro-organics by the white rot fungus was discovered 
by us during experiments in treating effluents from the first alkaline 
extraction stage of a chlorine bleach plant from a pulp and papermaking 
operation. This discovery was made in our efforts to assess the effect of 
low molecular weight compounds in such effluents on the white rot fungus 
being used to treat these effluents. 
In making this assessment, the effluents before and after exposure to the 
white rot fungus were extracted with chloroform after adjusting the pH to 
2.5. These extracts were subsequently examined by gas chromatography/mass 
spectrometry. The gas chromatagrams of the chloroform extracts before and 
after exposure of the effluents to the white rot fungus and the major 
components that have been identified by the GC/MS analysis are shown in 
FIG. 2. Chlorinated phenols were dominant low molecular weight components 
in the effluents before exposure to the white rot fungus. Surprisingly, 
these chlorinated phenols were substantially reduced completely 
disappeared after exposure to the white-rot fungus as is shown in Table 1 
below. This result is considered to be highly significant because many of 
the identified chlorinated phenols are known to be toxic to aquatic life. 
In the effluent after exposure to the white rot fungus, veratryl alcohol, a 
well-known metabolite of the white rot fungus used in these experiments, 
was the principal product. Other components were veratraldehyde, 
6-chloroveratryl alcohol, and vanillyl alcohol. Acetoveratrone and 
4,5-dichloroveratrole were also detected. Some of these, like veratryl 
alcohol, may also be metabolites of the fungus. It is believed that the 
aromatic compounds found in the effluent before exposure to the white-rot 
fungus were converted to aliphatic compounds since some aliphatic 
chlorinated compounds were found to be present in the effluent after 
exposure to the white-rot fungus. 
TABLE 1 
______________________________________ 
Quantitation of chloroform-extractable aromatics 
obtained from bleach plant effluent (E-1) 
before and after exposure to the white-rot fungus 
Before After 
Exposure Exposure 
Compound RTa ppm 
______________________________________ 
Benzoic acid 5.40 .sup. 0.12-0.45.sup.b 
none 
Dichlorobenzoic acid.sup.c 
5.48 none 
2,4,6-Triclorophenol 
7.47 0.15-0.33 trace-0.01 
Vanillin 8.05 0.01-0.02 0.05-0.10 
4,5-Dichloroguaiacol 
9.18 0.08-0.25 trace 
Acetoguaiacone 9.22 trace none 
6-Chlorovanillin 
10.22 0.05-0.10 trace 
Trichloroguaiacol.sup.c,d 
10.67 0.15-0.34 trace 
5-Chlorovanillin 
11.05 trace none 
4,5,6-Trichloroguaiacol 
12.74 0.04-0.10 trace 
Tetrachloroguaiacol 
14.06 0.08-0.15 trace 
Vanillyl alcohol 
8.67 none 0.05-1.20 
Veratraldehyde 9.11 none 0.08-1.30 
Veratryl alcohol 
9.37 none 10-20 
4,5-Dichloroveratrole 
9.59 none 0.10-0.30 
4,5,6-Trichloroveratrole 
11.66 none trace-0.02 
3,4-Dimethoxyaetophenone 
10.39 none trace-0.02 
5-Chloroveratryl alcohol 
11.84 none trace 
6-Chloroveratryl alcohol 
12.20 none 0.50-0.80 
______________________________________ 
.sup.a Retention time on gas chromatogram/capillary column30M with DB5 as 
the liquid phase; oven temperature was held at 60.degree. C. for 1 minute 
then programmed at a rate of 15.degree. C./min to 140.degree. C., and 
5.degree. C./min. to 255.degree. C.: FI D: 
.sup.b Includes dichlorobenzoic acid. 
.sup.c Isomer unknown. 
.sup.d Quantitation of this compound was based on the GC response factor 
of 4,5,6trichloroguaiacol. 
In our experiments, we discovered that the efficacy and active lifespan of 
the fungus is markedly increased by the addition of a small amount of 
nitrogen to the effluent or liquid being treated by the present process. 
Care must be taken, however, because the addition of too much nitrogen 
decreases the efficacy thereof and may even cause the fungus to leave its 
secondary metabolic state and therefore terminate all degradation of the 
chloro-organics. It has been determined that the addition to the effluent 
of an amount of nutrient nitrogen (NH.sub.4 Cl) equal to 20-30% of the 
original growth nitrogen or about 0.024 to 0.036 grams per liter resulted 
in a substantive increase in the rate of fungal activity and in the 
duration of the active lifetime of the fungus. 
We have also discovered that the addition to the effluent or liquid of a 
small amount of nutrient minerals (micro-nutrients or micro-minerals) has 
a very similar effect to that produced by the addition of nitrogen. It is 
not known whether all of these minerals are needed or whether some lesser 
number may be used or whether lower amounts will suffice, but the present 
invention contemplates the use of only those nutrient minerals which are 
necessary to produce the desired effect. In our experiments, we used a 
mixture of nutrient minerals including: 
______________________________________ 
Nitrilotriacetic acid 0.135 g/l 
MgSO.sub.4.H.sub.2 O 0.27 g/l 
NaCl 0.09 g/l 
MnSO.sub.4.H.sub.2 O 0.045 g/l 
CaCl.sub.2 0.007 g/l 
FeSO.sub.4.7H.sub.2 O 0.009 g/l 
ZnSO.sub.4.H.sub.2 O 0.009 g/l 
CoCl.sub.2.6H.sub.2 O 0.009 g/l 
CaSO.sub.4.5H.sub.2 O 0.0009 g/l 
Na.sub.2 MO.sub.4..sub.2 H.sub.2 O 
0.0009 g/l 
AlK(SO.sub.4).sub.2.12H.sub.2 O 
0.0009 g/l 
H.sub.3 BO.sub.3 0.0009 g/l 
Total 0.58 g/l 
______________________________________ 
Also, the addition to the effluent or liquid of a small amount of 
detergent, i.e., 0.3% by volume, produces a marked increase in the 
efficacy and active lifetime of the fungus. An illustrative example of 
such a biological detergent is a complex of polyoxyethylene ethers of 
mixed partial oleic esters of sorbital anhydrides sold under the brand 
name "Tween-80" by Fisher Scientific Company. 
The greatest improvement in efficacy and active lifetime of the fungus were 
achieved when both a mixture of nutrient minerals and a detergent were 
added to the effluent or liquid. Surprisingly, the effect of the minerals 
and of the detergent were additive, producing up to a fourfold increase. 
This additive effect was not realized when nitrogen and minerals were 
added to the effluent nor when nitrogen and detergent were added. 
In the drawings and specifications, there have been set forth preferred 
embodiments of the invention, and although specific terms are employed, 
they are used in a generic and descriptive sense only and not for purposes 
of limitation.