From BIOL 2P96 Jan 2013 Group 04
 Use of Fungi in Remediation
Hazardous materials produced by human industry are having devastating effects on the environment when these compounds are not disposed of properly. Despite having the knowledge of what these toxic chemicals do to an ecosystem, they are still being introduced into them. The effect on the environment can be devastating.
Bioremediation belongs to the study of biotechnology which uses microorganisms to overcome environmental issues. It involves a wide range of organisms, the most commonly used being fungi. These organisms are known to be tolerant to high concentrations of polluting chemicals, such as chlorophenols, nitrophenols, and polyaromatic hydrocarbons, and are therefore the ideal choice of organism for this study. .
There are a number of alternate methods of dealing with both organic and inorganic materials that have been conventionally used for soil remediation. In some cases, soil is simply removed from its immediate environment and brought to a landfill, but this can be dangerous when working with hazardous wastes. In other cases, the contaminated soil is capped and contained, but not removed from the site. However, this method is only a temporary solution since the contamination is not being dealt with. The cap and contain method is costly, requires maintenance of the soil samples, and could lead to liabilities in any unforeseen circumstances .
Other methods attempt to destroy the various pollutants that are contaminating the soil. These include incineration at high temperatures and treatments with bases or UV radiation. However, these methods also have their disadvantages since they often require complex procedures that can be very costly for the amount of soil being processed .
Over the past twenty years, fungi have been studied for their ability to break down wide varieties of substrates in a safe and simple manner . White rot fungi, which are capable of degrading the plant component lignin, have also been found to utilize many substrates that that are otherwise harmful to the environment, such as wastes from the production of military munitions (such as explosives), TNT, pesticides, DDT, polycyclic aromatic hydrocarbons (PAH), wastes from bleach plants, synthetic dyes, and many more .
Now Fungal bioremediation is often used in combination with phytoremediation as a method to absorb and remove harmful substances from the environment.
Fungi has been used in the past in bioremediation processes for small and large oil spills in masses of water around the world, such as the oil spill in Prince Williams Sound, Alaska, in 1989, as well as in Texas, Rhode Island and Delaware Bay. With fungi being a crucial part of degradation, its importance is increasing with respect to the treatments of freshwater and terrestrial environments. The pollutants that are biodegraded and removed from the environment by fungi include petroleum and hydrocarbons, which when mixed with water, the surface area of the oil increases, allowing for more microbial biodegradation activity to occur.   
 Processes Involved
 In situ
The in situ bioremediation method keeps the contaminated material in its place that it exists and performs the treatment. Some examples of in situ bioremediation include bioventing, bioaugmentation, biosparging, biostimulation, and biodegradation.  
Bioventing is the addition of nutrients and air to contaminated soil through wells in order to trigger the original bacteria within the soil. Bioventing uses as little air flow and oxygen as possible, just enough for biodegradation to take place, at the same time as reducing the conversion of solid or liquid material to gas, and therefore decreasing the amount of contaminants to be spread into the air. This type of ‘’in situ’’ method is not only the most common ‘’in situ’’ bioremediation, but it is also useful for contamination that reaches deeper below the external surface of the soil   
Bioaugmentation involves the insertion of bacterial microorganisms into the contaminated locations. .Bioremediation of petroleum hydrocarbon-contaminated soil by composting in biopiles. ‘’Environmental Pollution’’, 107, 245-254.</ref>  
Biosparging forces pressured air, along with nutrients, into the saturated zone, which can be described as the groundwater, or area below the ground, and is completely filled with water. The method of biosparging increases the concentration of oxygen within the groundwater and improves the process of biodegradation of the contaminants. 
Biostimulation involves the prompting of existing bacteria and fungi in the soil or groundwater that are capable to bioremediation, by adding the essential nutrients for the process to take place. 
Biodegradation is a process in which already existing bacteria and fungi are provoked to destroy contaminated organisms when oxygen and nutrients are provided via the movement of aqueous solutions throughout the contaminated soil. 
In situ bioremediation techniques are low cost options that are not disruptive to the ecosystem as a whole. These methods also provide the added benefit of being able to treat both water and soil .
However, in situ techniques are limited in that the depth of soil that can be affected by the fungi is restricted, with most methods only reaching depths of up to about 30cm. This limitation is due to the diffusion of oxygen through soils, which is required for many in situ techniques .
Other limitations of in situ bioremediation processes include potential difficulties in monitoring the progress of the fungi. Certain environmental constraints also need to be considered since there are certain conditions that cannot be controlled or manipulated .
 Ex situ
The ex situ bioremediation method involves the removal of the contaminated material to perform treatment processes. Some examples of ex situ bioremediation include landfarming, composting, bioreactors, and biopiles  . Ex situ processes are beneficial in that they allow for a greater degree of control over conditions such as pH, aeration, agitation, moistening, and addition of other factors such as electron acceptors, nutrients, solvents or surfactants .
Landfarming involves the excavation of the top contaminated layers of soil, sediment and sludge, approximately 10 to 35cm deep, which is mixed in with the soil and is tilled on an occasional basis. The objective of landfarming is to assist the aerobic damaging of the contaminants. This method is considered to be cost efficient and low maintenance, and is gaining much interest as an alternative for disposal.  
Composting is the combination of contaminated material (soil) with organic matter, such as manure and agricultural wastes, which are not harmful and encourage the growth of large populations of fungi, bacteria, viruses and parasites to degrade the contaminated matter. Aerobic conditions and relatively high temperatures are favoured in the method of composting.  
Bioreactors is a system of containment, in a vessel or container, that is used for the biodegradation of liquids or slurries by processing the contaminated material through the system (machine). Slurries can be defined as a mixture of soil with water, and holds a semi-liquid state.  The biodegradation rate of soil by bioreactor systems is much more rapid than with in situ methods.  
The method of biopiles is a cross between landfarming and composting where ventilated compost piles are made and treated with air and nutrients. This method is generally used for bioremediation of petroleum hydrocarbons via conversion of a solid or liquid into a gas. Biopiles can involve the use of bulking agents, such as woodchips or straw, which increase the bulk of the pile without altering its functions.   .
 Metabolic Processes
Fungi have the capability of transforming organic pollutants through their normal metabolic processes. The pollutants can be fully degraded into carbon dioxide and other simple inorganic components, providing the fungi with a source of energy as the substrate is metabolized. In a process called cometabolism, different organisms act on the pollutant to modify it, and each serves an important role in the breakdown of the pollutant. The same substrate can be acted on by many different organisms to achieve a certain end product. Pollutants can also be used in synthesis reactions by the fungi, where the end product is more complex than the pollutant substrate and can be taken up and used by the cell .
For example, bioremediation can include the degradation of organic substrates, including wood. Plant biomass is a complex network of polysaccharides, proteins, and lignin which can be digested and metabolized by a variety of species of Ascomycota and Basidiomycota fungi . The degradation of lignin by these fungi is considered to be the rate-limiting step in releasing carbon from environments high in lignocellulose compounds. Lignin is a substrate for secondary metabolism, which has to be degraded in order for the fungi to be able to access cellulose as an energy source for primary metabolism. There are three enzymes required to break down lignin: peroxidises, phenoloxidase, and laccase. There are also other enzymes that aid in the digestion of lignin by providing substrates required by the three enzymes named above  Feasibility of bioremediation by white-rot fungi. Applied Microbiology and Biotechnology, 57, 20-33</ref>. These enzymes are involved in demethoxylation (removing a methoxy group), ring hydroxylation and side chain oxidation rather than cleaving the compound as a whole .
Azo is another organic compound, and it is commonly found in dye. Fungal Bioremediation is a process commonly executed on fabric with azo dye for decolourization. Different types of fungi are more effective against certain colours (example:Aspergillus niger and Aspergillus flavus for Remazol Red and Remazol Black respectively.) It is common for fungal bioremediation to be chosen for this process because of it being environmental friendly and low budget. 
Fungal bioremediation can also be used on heavy metals such as chromium. Chromium exists in the natural environment from sewage waste and fertilizers.Chemically treated myceilum can be used to absorb heavy metal and directly remove and protect plants from harmful heavy metal. While heavy metal in water can be absorbaed by certain dead fungal mass.All the processes available from fungal bioremediation are very cost efficient.
 Species Types Involved
It has been observed that a wide variety of fungal species have proven effective in remediation treatment; more notably are those of Basidiomycota and Ascomycota. Although these phyla dominate the majority of the fungi used in remediation, there is evidence that Zygomycota and Glycomycota may also be effective. Within the studies it is reiterated that the reason for the effectiveness of the fungi in remediation lies in the activity of the corresponding enzymes. Many fungi also have adaptive characteristics that allow them to inhabit areas of the world that many would consider inhospitable.
 Glomus mosseae
Ecological problems have given rise to the exploitation of natural resources, causing pollution and degradation of the environment. In turn, this gives rise to unfavourable conditions for plant growth because of the depleted nutrient acquisition. AM, fungi help increase nutrient supply and build resistance to biotic and abiotic stresses. In areas of pollution, plants are extremely dependent on these fungi to enhance their metabolic activity and diminish the effects of environmental stress. 
Belonging to the phylum Glomeromycota, Glomus mosseae is beneficial in encouraging the revegetation of copper (Cu) mine tailings. These tailings are known to damage native vegetation, and contaminate water, land, and air. Metal pollution cause extreme environmental problems for plant growth and development in infected areas. This species is most greatly used as it belongs to arbuscular mycorrhizal fungi, which helps promote plant growth and biomass in metal contaminated soils/substrates. They supply plants with mineral nutrients (especially phosphate and trace elements), improve soil structure (external hyphae and glomalin excreted by external hyphae), and maintain ecosystem stability. Also, they protect host plants against high metal concentrations in soils under metal contamination. Therefore, AM fungi is specifically used to revegetate at mining sites.
The plant species P. vitta and C.Dummundiiinhabit the Cu mine tailings of Tongling southern China, and are extremely dependent on mycorrhizal colonization for growth. These mining sites show a low supply of essential mineral nutrients with excessive metals, and a lower supply of essential plant nutrients. Glomus mussaea help plants obtain more nutrients so resistance to metal contamination can be enhanced. Also, under high metal concentrations in the soil, AM fungi protect host plants against metal toxicity.  Therefore,this species of fungi proves to be extremely useful in the rehabilitation of plant growth in mining areas and are a major contribution to the study of bioremediation.
 Stropharia rugosoannulata
Some species of basidiomycota, such as Stropharia rugosoannulata, is a litter-decaying fungus that has been shown effective in the breakdown of nitroaromatics, including TNT(2,4,6-trinitrotoluene,) by using them as a nitrogen source. Since these compounds are hard to evict from the biosphere where they cause pollution, carcinogenic, and mutanogenic effects, the use of this fungi has been proven useful. S. rugosoannulata cleaves the nitrogen from the aromatic ring under aerobic conditions, using it as a nutrient source. This allows the decomposition of the compound and a decrease of hazardous potential of residues that pollute the environment. 
Although their performance is not as high as white-rot fungi, these basidiomycete fungi also have a heightened ability to decompose PAHs. This is because they are native to the soil environment where these PAHs are found, so this ability is heightened when compared to wood-rot fungi. A study showed that S. rugosoannulata was the most efficient strain in removing PAHs, where 85% of them were successfully decomposed. The results were impoved when managanese (II) was added to the culture, proving that this species of fungi uses manganese peroxidase as an extracellular lignolytic enzyme that is important in degradation. 
This species of fungi has a powerful potential to cleanup contaminated environments to reduce and prevent further pollution.
 Phanerochaete chrysosporium
Species of Basidiomycota, Phanerochaete chrysosporium has proven effective in the decolourization of direct dye wastewater, thus making it a successful remediation tool. The reason for this study resided in the fact that many bodies of water are now affected by the amount of dyes used commercially. The ability of basidiomycota to depolarize and mineralize lignin resulted in the degrading synthetic dyes; treatment demonstrated a 90% decolourization within just 7 days of treatment.
Within P. Chrysosporium are multiple extracellular lignin-modifying enzymes that are responsible for degrading a wide variety of compounds. This is due to their low substance specificity; other fungi lack certain structures and show specification making them unable to decolourize certain dyes. Once again, extracellular lignin-modifying enzymes proved prolific when decolourizing direct dye wastewater. There are multiple enzymes involved in the extracellular lignin-modifying process; the most successful enzyme for decolourizing dyes was manganese peroxidises (MnP).
In addition to the efficient enzymes P. Chrysosporium is also superior at decolourizing dyes due to its high pH value and its complex structures. P. Chrysosporium has a pH value of 9 whereas most other fungi possess a pH value in the acidic range, therefore making it more useful in this process. Also the complex –Trisazo, Polyazo and Stilbene structures further assist in the decolourization for direct dye wastewater.
To summarize Phanerochaete chrysosporium is very effective in remediation and possesses great qualities that allow it to be successful in decolourizing direct dye wastewater. These qualities consist of a high pH value, extracellular lignin-modifying enzymes and complex structures; they work as one to denature and eliminate dyes from water bodies around the world. P. Chrysosporium will continue to be used a remediation tool due to its safe and efficient results. 
 Pleurotus ostreatus
Pleurotus Ostreatus more commonly known as spent white-rot has been used in mycoremediation to eliminate polycyclic aromatic hydrocarbons (PAHs) from oil-based drill cuttings. Sixteen non-substituted PAHs have been identified by the Protection Agency (USPEA) as priority pollutants.
The reason for the high PAH content in drill cuttings is due to the continued use of PAH carrying petroleum fractions which are preferred when working at great depths. Therefore, it is essential that these cuttings are treated before final disposal in order to protect the environment from further damage.
Usually drill cuttings are treated via physico-chemical methods, which are very costly and have environmental implications. Therefore, further research has been conducted to discover a more eco-friendly approach; bioremediation is known to have a relatively low cost and have less impact of the environment. Use of Pleurotus Ostreatus has been discovered to vastly decrease PAH fractions in terms of their properties (molar mass and ring group). For example individual PAH degradation ranged from 97.98% in acenaphthene (3-ring) to 100% in fluorine (3-ring) thus highlighting the extraordinary PAH-removal capacity of spent white-rot fungi.
In conclusion, Pleurotus Ostreatus was discovered to be highly effect in the removal of PAH from oil-based drill cuttings, indicting its effectiveness in remediation. It is also more cost effective than the alternative and has a lower impact on the environment. Spent white-rot fungi utilization in remediation also reduces the amount of the substrate eliminated as waste as well as reducing PAHs in the environment. 
 Pleurotus pulmonarius
The Pleurotus Pulmonarius species are a part of the phylum basidiomycota. Pleurotus Pulmonarius is commonly known as oyster mushrooms which are also classified as white rot fungi and has been proven to be a highly effective and important resource for remediation. The oyster mushroom serves many purposes within the remediation process including processes dealing with wastes waters in crop production and landfill pollutants. 
The use of White rot fungi for bioremediation purposes is of high interest because these species contain a ligninilytic system that allows these species to degrade a wide variety of environmental pollutants.  Pleurotus Pulmonarius is an effective fungus for remediation because it has enzymatic activity that is capable of degrading phenolic related pollutant compounds including chlorinated pesticides like endosulfan and chlorothalonil. Furthermore, it has the capability of degrading cellulose, hemicellulose and ligin. The presence of enzymes including laccase, MnP and phenol oxidase found in Pleurotus Pulmonarius are responsible for the degration of pesticides. The ligninolytic enzymes are one of the most important enzymes present because they are highly efficient in degrading environmental pollutants. The ligninolytic enzymes are the most active enzymes in the remediation process within oyster mushrooms because these enzymes are secreted only in the presence of SMS. Pleurotus Pulmonarius was reported to degrade the pesticides more quickly and were able to completely reduce the original concentration of chlorothalonil. In conclusion, Pleurotus Pulmonarius can be efficiently used in remediation methods to help reduce the effects of the pollutants of organochlorine pesticides.
 Trichoderma harzianum
The Trichoderma Harzianum species are a part of the phylum ascomycota. Trichoderma harzianum has been proven to serve many purposes within the remediation process including processes dealing with seed treatment and soil treatment.  Trichoderma harzianum is a strain of T22 which is rhizosphere that is a fungus that promotes plant growth which is sold commercially.  This species has been show to degrade phosphate, compounds of manganese and metallic zinc.  Trichoderma harzianum has the ability to produce lower pH with seed treatment and soil treatment.  Trichoderma harzianum is the most effective absorbing zinc from charcoal suggesting metabolites responsible for zinc absorption is due to the lactic acid.  Overall Trichoderma harzianum is impotant for removing pollutants with in soil and seed treatment.
 Cunninghamella elegans
The fungi species Cunninghamella elegans is becoming an organism of interest due to its ability to breakdown polycyclic aromatic hydrocarbons (PAHs). These compounds originate from the combustion of fossil fuels and can be toxic to the environment if they are left untreated. More specifically, they will travel to the sediment found in aquatic environments due to the composition of the chemicals.  This makes it challenging to clean up because these toxic compounds are entering into the soil, making them hard to remove. The amount of fossil fuels used around the world signifies the importance of finding ways to breakdown these compounds until less hazardous components.
Cunninghamella elegans is a fungus that inhabits soil, which already makes it a probable organism for coming into contact with PAHs. However, it is important to note that there are many different types of polycyclic aromatic hydrocarbons, all with different structures and components. Cunninghamella elegans has the ability to breakdown 6-nitrochrysene, 6-nitrobenzo[a]pyrene, 3-nitrofluoranthene, 2-nitrofluorene and dibenzofuran to name a few. This species of fungi employs oxidative reactions to break apart these compounds. Metabolites are produced as products that can be used by the fungus. This process occurs relatively quickly in this species. For example, 18.4% of the PAH benzo[a]pyrene was broken down and converted to metabolites over a 96-hour period. Thus, an advantage for using Cunninghamella elegans to detoxify an environment that contains PAHs is the ecosystem can be restored quickly.
 Plectosphaerella cucumerina
Despite the environmental implications, coal mines are still actively being used across the world. One consequence of these sites is related to the drainage system that is normally put in place to carry away waste waters. These waters often leach into the surrounding environment and contain high levels of metals. One specific contaminant in the wastewater of these mines is manganese (II). In high concentrations this element can inhibit growth and possibly kill plants and animals that ingest it.
Several fungi and bacteria species have been noted to have the ability to oxidize manganese compounds. Plectosphaerella cucumerina is a species that has been noted to grow in abandoned mining facilities and inhabit the soil. It has over a 10mM manganese tolerance and can grow at a consistent rate under various concentrations of the metal. Plectosphaerella cucumerina oxidizes manganese (II) into manganese (III) or manganese (IV), which are less toxic than their counterpart. The oxidative mechanisms that are used for manganese are unknown.
It is important to note that the use of fungicides in mining areas will eliminate these fungus species associated with the breakdown of toxic metals.
- ↑ Gadd, G.M. Fungi in Bioremediation. Syndicate of the Univerisity of Cambridge (2001).
- ↑ 2.00 2.01 2.02 2.03 2.04 2.05 2.06 2.07 2.08 2.09 2.10 2.11 2.12 2.13 2.14 2.15 Vidali, M. (2001). Bioremediation. An overview. Pure and Applied Chemistry, 73 (7), 1163-1172
- ↑ 3.0 3.1 3.2 Mougin, C., Boukcim, H., Jolivalt, C. (2009). Soil bioremediation strategies based on the use of fungal enzymes. Soil Biology, 17, 123-149
- ↑ 4.0 4.1 Pointing, S.B. (2001). Feasibility of bioremediation by white-rot fungi. Applied Microbiology and Biotechnology, 57, 20-33
- ↑ 5.0 5.1 Mohanty, M., & Patra, H. (2011). Attenuation of chromium toxicity by bioremediation technology. Reviews Of Environmental Contamination And Toxicology, 2101-34. doi:10.1007/978-1-4419-7615-4_1
- ↑ 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 Boopathy, R. (2000). Factors limiting bioremediation technologies. Bioresource Technology, 74, 63-67
- ↑ Leahy, J.G., Colwell, R.R. (1990). Microbial degradation of hydrocarbons in the environment. Microbiology and Molecular Biology Reviews , 54(3), 305-315
- ↑ Davies, J.S., Westlake, D.W.S. (1979). Crude oil utilization by fungi. Canadian Journal of Microbiology, 25, 146-156
- ↑ 9.0 9.1 9.2 Jørgensen, K.S., Puustinen, J., Suortti, A.-M. (2000). Bioremediation of petroleum hydrocarbon-contaminated soil by composting in biopiles. ‘’Environmental Pollution’’, 107, 245-254.
- ↑ 10.0 10.1 Balba, M.T., Al-Awadhi, N., Al-Daher, R. (1998). Bioremediation of oil-contaminated soil: microbiological methods for feasibility assessment and field evaluation. Journal of Microbiological Methods, 32, 155-164.
- ↑ Filley, T.R., Hatcher, P.G., Shortle, W.C., Praseuth, R.T. (2000). The application of ¹³C-labeled tetramethylammonium hydroxide (¹³C-TMAH) thermochemolysis to the study of fungal degradation of wood. Organic Geochemistry, 31(2-3), 181-198.
- ↑ Moore, D., Robson, G.D., Trinci, A.P.J. (2011). 21st Century Guidebook to Fungi. Cambridge, UK: Cambridge University Press
- ↑ Meharg, A.A. & Cairney, J.W.G. (2000). Ectomycorrhizas - extending the capabilities of rhizosphere remediation? Soil Biology & Biochemistry, 32(11-12), 1475-1484.
- ↑ Baskar, B., & Baskaran, C. C. (2012). Bioremediation of Azo Dyes Using Fungi. International Journal Of Research In Pharmacy & Science, 2(4), 28-37.
- ↑ Azcon, R. Barea, J.M. Biro, B. Ruiz-Lozano, J.M. Vivas, A. Voros, A (2003). Beneficial Effects of Indigenous Cd-tolerant and Cd-sensitive GLomus mosseae Associated with a Cd-adapted Strain of Brevibacillus sp. in Improving Plant Tolerance to Cd Contamination. Applied Soil Mycology 24(3) 177-186.
- ↑ Chen,B.D. Duan, J. Smith S.E. Xiao, X.Y. Zhu, Y.G (2007). Effects of the Arbuscular Mycorrhizal Fungus Glomus mosseae on Growth and Metal Uptake by Four Plant Species in Copper Mine Tailings. Environmental Pollution;(147) 374-380.
- ↑ Geyer, R. Kastner, M. Richnow, H. Russow, R. Weib, M (2004). Fate and Metabolism of [N]2,4,6-Trinitrotoluene in Soil. Environmetal Toxicology and Chemistry 23(8) 1852-1860.
- ↑ Norton, Joanna. Fungi for Bioremediation of Hydrocarbon Pollutants. Biology 499 (10)18-21.
- ↑ 19.0 19.1 19.2 19.3 19.4 19.5 19.6 19.7 19.8 Faraco, V., Giardina, P., Miele, A., Pezzella, C., & Sannia. (2009). Bio-remidiation of coloured inductrial wastewaters by the white-rot fungi Phanerochaete chrysosporium and Pleurotus ostreatus and their enzymes. Bidegradation, 20(2), 209-220.
- ↑ Allagoa, M., Ayotamuno, J., Davis, D., & Okparanma, R. (2011). Mycoremediation of polycyclic aromatic hydrocarbons (PAH) – contaminated oil-based drill-cuttings. African Journal of Biotechnology, 10(26), 5149-5156.
- ↑ 21.0 21.1 21.2 21.3 21.4 Adams, P., Lynch, J., & De Leij, F. (2007). Desorption of zinc by extracellularly produced metabolites of Trichoderma harzianum, Trichoderma reesei and Coriolus versicolor. Journal Of Applied Microbiology, 103(6), 2240-2247.
- ↑ 22.0 22.1 22.2 22.3 Tortella, G.R. & Diez, M.C. (2005). Fungal diversity and use in decomposition of environmental pollutants. Critical Reviews in Microbiology, 31(4), 197-212.
- ↑ 23.0 23.1 Santelli, C.M., Pfister, D.H., Lazarus, D., Sun, L., Burgos, W.D. & Hansel, C.M. (2010). Promotion of Mn(II) oxidation and remediation of coal mine drainage in passive treatment systems by diverse fungal and bacterial communities. Applied and Environmental Microbiology, 76(14), 4871-4875.