The document discusses the potential for white rot fungi, such as Phanerochaete sordida YK-624 and P. chrysosporium IFO31249, to bioremediate polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzo-furans (PCDFs). Experiments showed that both fungi were capable of degrading between 70-75% of various PCDDs and PCDFs within 14 days when supplemented with glucose and oxygen. The degradation occurred through non-specific extracellular enzymes and showed no clear dependence on substrate structure. Care was taken to prevent evaporation and exposure during the experiments.

1. MYCOREMIDIATION
POTENTIAL OF
WHITE ROT FUNGI
By:
Mugdha Nigam
ME Biotech

2.  Bioremediation is the use of micro-organisms- naturally present
or genetically engineered to remove pollutants.
 They can be applied on site (in situ) or off site ( ex situ) mediated by
mixed microbial consortia or pure microbial strains and plants.
 They include several processes – bioventing, biostimulation,
biosparging, bioaugmentation, bioleaching, phytoremediation,
fungal bioremediation and biosorption.

4.  Mycoremediation, a phrase coined by Paul Stamets, is a form of bioremediation, using fungi to
degrade or sequester contaminants in the environment.

5.  They are capable of mineralizing a wide variety of toxic xenobiotics.
 They occur ubiquitously in the natural environment.
 They have the potential to oxidize substrates that have low solubility because the
key enzymes involved in the oxidation of several pollutants are extracellular.
 The constitutive nature of the key enzymes involved in lignin degradation obviates
the need for these organisms to be adapted to the chemical being degraded.
 The preferred substrates for the growth of white-rot fungi, such as corn cobs, straw,
peanut shells, and sawdust, are inexpensive and easily added as nutrients to the
contaminated site.
 The key LDEs are expressed under nutrient-deficient conditions, which are
prevalent in many soils. Nitrogen serves as the main limiting factor.
 Four main genera of white rot fungi have shown potential for bioremediation:
Phanerochaete, Trametes, Bjerkandera, and Pleurotus.

6.  Lignin peroxidase is a glycosylated heme protein that catalyzes
hydrogen peroxide-dependent oxidation of lignin-related aromatic
compounds. They have a higher redox potential than most peroxidases
and so are able to oxidize a wide range of chemicals, including some
non-phenolic aromatic compounds.
 Mn-dependent peroxidase also requires hydrogen peroxide to oxidize
Mn2+ to Mn3+. The Mn3+ state of the enzyme then mediates the
oxidation of phenolic substrates.
 Laccase, a multicopper oxidase enzyme, is the primary enzyme
involved in the degradation process. It uses dioxygen as an oxidant,
reducing it to water and it has the ability to catalyze the oxidation of a
widerange of dihydroxy and diamino aromatic compounds. It is most
stable at a pH of 5-6 and temperature of 45°C. However, this enzyme is
still active at pH levels as low as 4 and as high as 7. This is beneficial
in contaminated field sites with very low pH levels.

9.  SOURCES - Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated
dibenzo-furans (PCDFs) are unintentionally formed in the process of:
- producing chlorine-containing herbicides,
- in the bleaching of paper pulp by using chlorine compounds
- during combustion of domestic and industrial waste
- during burning of petrochemicals and PAHs.
 EFFECTS –
- PCDDs and PCDFs have been a public concern for several decades because of
their strong toxicity in animal tests. These hazardous compounds tend to
accumulate in the body fat of animals since they are relatively lipophilic and
chemically stable.
- They have been released into the environment as recalcitrant contaminants and
have been found in many environmental matrices such as air, soil, and plants.
- Studies of the degradation of PCDDs and PCDFs in the environment have shown
these rates to be extremely low, the half-life of 2,3,7,8-tetrachlorodibenzo-p-dioxin
(2,3,7,8-tetraCDD) in an outdoor pond and soil being in the order of 1 year.

10. BIODEGRADATION METHODS –
 P. sordida YK-624 and P. chrysosporium IFO31249 culture were prepared on
Low-nitrogen basal III medium containing 1% glucose, 1.2 mM ammonium
tartrate, and 20 mM dimethylsuccinate (pH 4.5). After incubation for 7 days, 10%
glucose was added to each inoculated flask and the headspaces were flushed with
oxygen.
 Ethyl acetate solution of PCDDs-PCDFs (500 pg each) was added, and each flask
was sealed with a glass stopper and sealing tape. The cultures were incubated for 3,
7, 10, and 14 days (each in triplicate). For 10- and 14-day incubation samples, 10%
glucose was added to the cultures, and the flasks were oxygenated on day 7.
 At the end of the incubation, hexane and 13C-labeled PCDDs-PCDFs (500 pg each)
was added to the cultures. To recover the PCDDs and PCDFs adsorbed to the
mycelia and to dissolve the mycelia thoroughly, concentrated sulphuric acid was
added.

11.  The cultures were extracted twice with hexane. All of the hexane extracts
were washed with water.
 The hexane layer was evaporated, and polar compounds were removed
with silica gel chromatography.
 Uninoculated medium controls were treated in the way.
 Concentrations of PCDDs-PCDFs were determined by high-resolution gas
chromatography and high-resolution mass spectrometry (HRGC-HRMS)
(selected ion-monitoring mode [SIM]).

12. RESULTS –
 For all compounds, the glucose-supplemented culture led to a higher
percent degradation.

13.  In both PCDDs and PCDFs, hexa-CDD and -CDF showed the highest
degradation values, i.e., ;75% and ;70%, respectively.
 The degradation of PCDDs-PCDFs by P. Chrysosporium IFO31249 was
also carried out for 14 days under conditions similar to those for YK-624
(Table 1). The results show almost the same degradation rate as that for
YK-624.
 Time courses for the degradation of PCDDs and PCDFs by the fungus
YK-624 were plotted. All of the PCDDs and PCDFs were partially
degraded. The percent degradation values were promoted by the addition
of glucose and oxygenation on days 0 and 7; however, the effect
continued only for 3 days, as indicated by the fact that the slopes of
percent degradation of days 0 to 3 and 7 to 10 were steeper than those of
days 3 to 7 and 10 to 14 – (figure 4 and 5).

14. Degradation of PCDDs by P. sordida YK-624 under low-nitrogen
medium. Glucose was added to each culture, and headspaces were purged with
oxygen.

15. Degradation of PCDFs by P. sordida YK-624 under low-nitrogen
Medium. Glucose was added to each culture, and headspaces were purged with
oxygen.

16. CONCLUSION –
 The degradation method was developed carefully to avoid the evaporation
of dioxins and consequent human exposure. The solubilities of PCDDs are
extremely low. The 500 pg of these compounds used in this experiment did
not dissolve in 10 ml of the aqueous culture and remained largely in solid
or vapour states. Consequently, biodegradation was carried out in flasks by
sealing with glass stoppers and, in addition, sealing with sealing tape in
order to protect against the loss of these compounds.
 P. sordida YK-624 and P. chrysosporium IFO31249 were capable of
substantial degradation of the mixtures of the 2,3,7,8-substituted tetra- to
octa-CDDs and CDFs tested, as determined by substrate disappearances.
 These fungi showed no clear structural dependence for degradation of
PCDDs and PCDFs, verifying that the degradation of these substrates is
indeed, a free-radical process showing little specificity.