White rot fungi are basidiomycetes that are capable of degrading a lignocellulose substrate.  There are other fungi capable of doing this, such as brown rot fungi, but they do not produce the same enzymes that are portentous for the research into pollution removal.  They are called white rot because the degradation process results in a bleaching of the wood substrate.  Fungi are robust organisms that have a high tolerance to toxic environments, making them ideal to use for bioremedial purposes.  They can also withstand high temperatures and a wide range of pH, further enhancing their hardy capabilities. 

The picture shows Phanerochaete chrysosporium, the principal fungus for bioremediation, from Tom Volk's fungi pages

White Rot Fungi Degradation System:

Three types of extracellular enzymes are produced by white rot fungi that are non-selective yet effective in attacking lignin.  These are often referred to as Lignin Modifying Enzymes (LMEs), and they are Lignin Peroxidase (LiP), Manganese-Dependent Peroxidase (MnP) and Laccase (Lac).  Whilst they effectively break down lignin, these fungi cannot utilise it as an energy source and it is assumed that they degrade it for access to the cellulose in the cell wall.

LiP:  Not all white rot fungi produce LiP, but it is a key component for the fungi that are being investigated for use.  It is a haem-containing enzyme with a high redox potential that needs two further metabolites to function.   These are produced by the fungus itself; the first is Hydrogen Peroxide (H₂O₂) which is also needed by MnP, and the second is Veratryl Alcohol (VA) which is used as a mediator for the redox reactions.  LiP oxidises methoxyl groups on aromatic rings, and can work on substrates with quite high redox potentials.

MnP:  MnP is another haem containing peroxidase, and uses H₂O₂ to catalyse oxidation of Mn²⁺ to Mn³⁺, this in turn oxidises phenolic substrates.  Although similar in action to LiP, it does not have the same ability to oxidize substances with higher redox potentials.

Lac: Laccase is a multi copper oxidase which has the ability to oxidise phenolic compounds.  In the presence of oxygen, it converts phenolic compounds into quinine radicals and then further converts them to quinones.  It also produces some co-substrates which can be useful for degradation.

What is lignin ? A short explanation

LME Mode of Action

A series of redox reactions are initiated by the LMEs which degrade the lignin.  Redox reactions are either reductions or oxidations, which essentially is the adding or removing of electrons from the molecule.  As electrons are negative, an oxidation reaction is the removal of electrons and a reduction (whilst sounding like it is decreasing) reaction is the addition of electrons.  These often occur together hence redox reactions.  A redox potential, which is important in influencing which substrates the enzymes work on, is the ability of a substance to be oxidised, therefore the higher the potential the more oxidising the substance is.  The LMEs work by oxidising the aromatic compounds until they cleave the aromatic ring structure, which then allows further degradation.

LiP as shown in the diagram, has Veratryl alcohol as a mediator and catalyses the oxidation of H₂O₂ which in turn generates VA⁺, a diffusible radical which in turn can oxidise lignin aromatic compounds to radical cations.  VA⁺ is then reduced back to VA and can be used again.  The oxidised aromatic radical is then susceptible to further oxidation in the presence of O₂ which is basically an electron cascade. The diagram below shows how the electron cascade works.

 MnP works in much the same way, but instead of using VA as a mediator, it uses the Mn³⁺ chelate created when H₂O₂ oxidised Mn²⁺, which works in much the same way to diffuse into complex compounds.

Laccase follows the path shown in the figure below.  It catalyses a single-electron oxidation of phenolic compounds by using a mediator, but this has not yet been elucidated.  The radicals created by this then react further with other compounds depending on what is present in the substrate.  The general accepted degradation pathway is as follows:

In the presence of oxygen:

Phenolic groupà quinone radicalà quinonesà further degradation by other enzymes.

So how does this affect organopollutants? 

It is the ability of the fungi to degrade aromatic ring containing compounds such as lignin which make their enzymes applicable to organopollutants.  As the enzymes are non-specific they are capable of degrading most aromatic complexes.

 This diagram shows a simplified degradation path for polycyclic aromatic hydrocarbons by fungi, and this is the accepted method today.  Each LME affects the organopollutants differently, and together they act on different parts to break down the entire substrate into carbon dioxide. 

Laccase can catalyse both depolymerising reactions and also polymerising reactions.  Whilst the depolymerising is obviously useful for the breakdown of the pollutants, polymerisation can also be useful, even though larger compounds are created.  This is because sequestration is acceptable as a bioremedial method.  Whilst forming a larger compound does not remove it from the environment, it can make the substance non-toxic, and thus negates the need for its removal.  The depolymerisation occurs by the oxidation of phenolic compounds in the pollutant which can result in an alkyl-phenyl cleavage, and consequently the aromatic ring is broken.  The co-substrates produced assist due to their small size.  This gives them the ability to access areas deep inside organopollutant matrices which are otherwise non-accessible to the enzymes themselves, thus improving the area available for degradation.

 MnP produces a Mn³⁺ chelate oxalate which is small enough to diffuse into areas inaccessible to enzymes, which helps with organopollutants that are buried deep in soil which is not necessarily available to the fungal enzymes , even with the hyphae that are produced by the mushrooms.

LiP appears to be the crucial enzyme for degradation of organopollutants, as some of the most successful experiments have used white rot fungi which produce LiP.  This is likely to be due to its ability to degrade substances with higher redox potentials.

What fungi are used?

The main fungus studied is Phanerochaete chrysosporium,  as it was during an experiment using it that ligninases were first discovered, and henceforth has become the focus of research.

Also studied quite extensively are Trametes versicolor, Pleurotus ostreatus, Phanerochaete sordida, Trametes hirsutus, and Fusarium culmorum. There are many other white rot fungi studied, and also some non-ligninolytic fungi.   Whilst they all have similar methods of degradation, it is possible that they differ slightly in the enzymes and cofactors involved.

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