This paper will appear in the next issue of the journal "Chemical Speciation & Bioavailability" http://www.sciencereviews2000.co.uk/view/journal/chemical-speciation-and-bioavailability
Some of the prospects of using fungi, principally white-rot fungi, for cleaning contaminated land are surveyed. That white-rot fungi are so effective in degrading a wide range of organic molecules is due to their release of extra-cellular lignin-modifying enzymes, with a low substrate-specificity, so they can act upon various molecules that are broadly similar to lignin. The enzymes present in the system employed for degrading lignin include lignin-peroxidase (LiP), manganese peroxidase (MnP), various H2O2 producing enzymes and laccase. The degradation processes can be augmented by adding carbon sources such as sawdust, straw and corn cob at polluted sites.
1. Introduction to fungi.
Fungi feature among Nature’s most vigorous agents for the decomposition of waste matter, and are an essential component of the soil food web (Rhodes, 2012), providing nourishment for the other biota that live in the soil. The forest floor is covered with leaf litter from the previous season, which plants cannot use directly to grow on, because the fallen leaves are too tough to be broken down and digested; thus, any nutrients they contain are locked within them. The key organism for breaking down the leaf litter is fungus: strictly, mycelium – the vegetative part of the fungus – which we often observe as fine, white threads that grow out from dead wood, and leaves etc. Indeed, fungi are the only organisms on Earth that can decompose wood. The mycelium exudes powerful extracellular enzymes and acids that are able to decompose lignin and cellulose, the two essential components of plant fibre. As the fungus breaks down wood and leaves, a rich material called humus is formed. In the natural ecosystem, a realm of organisms from different kingdoms make their assault on those different substrates that are present, and the rate of degradation becomes maximal when there is a good supply of nutrients in the soil, e.g. N, P, K and other essential inorganic elements (Rhodes, 2013). Aspergullus and other moulds are highly efficient in decomposing starches, hemicelluloses, celluloses, pectins and other sugar polymers, and some aspergilli can degrade such intractable substrates as fats, oils, chitin, and keratin. Substrates of human origin, such as paper and textiles (cotton, jute and linen) are readily degraded by these moulds, when the process is often referred to as biodeterioration. In 1969, when the Italian city of Florence (Firenza) flooded, it was found that 74% of the isolates from a damaged Ghirlandaio fresco in the Ognissanti church were Aspergillus versicolor (Rhodes, 2013). To achieve a successful mycoremediation, the correct fungal species must be selected to target a particular pollutant, for which a simple screening procedure has been described (Matsubara et al., 2006). An encyclopedic overview of the research literature on the action of fungi on organic pollutants, up to 2006 is available (Singh, 2006), along with the beautifully illustrated “Mycelium Running: How Mushrooms Can Help Save the World” (Stamets, 2005), which serves to provide a hands-on guide to growing fungi and applying them to remediating contamination, e.g. from oil spills and chemical toxins, on the practical scale.
2. The issue of contaminated land.
The decontamination of soil and water from pollutants using microorganisms (bioremediators) is known as bioremediation (Rhodes, 2013). There are essentially two approaches, described as in situ and ex situ. In situ methods are those in which the contaminated material is treated on-site, whereas when the material is physically removed to be treated elsewhere it is referred to as ex situ. To excavate and remove contaminated soil is a relatively costly procedure, as is compounded when this is cleaned using chemical methods, or by incineration. In contrast, if the soil can be left where it is and decontaminated there, the overall expense is far less. Moreover, in washing or extracting toxic materials from soil, the contamination is simply moved from one place to another, and is not eradicated per se, while incineration may cause problems in its own right, e.g. dioxin formation, as well as being energy intensive. Methods of bioremediation offer means to degrade toxic organic materials, e.g. from oil spills, pesticides, and industrial waste, at the molecular level, converting them to more innocuous compounds. The ultimate goal of bioremediation is the full mineralization of contaminants, i.e. their transformation to CO2, H2O, N2, HCl, etc. Heavy metal and radioactive cations, of course, cannot be decomposed but can be rendered into forms of low solubility, e.g. by a change in oxidation state, such as U (IV) (in UO2) (Singh et al., 2014), so that they remain less harmfully in the ground, or might be physically removed by phytoremediation or mycoremediation, which involves harvesting the plant or fungus.
3.Bioremediation using fungi.
White-rot fungi digest lignin by the secretion of enzymes and give a bleached appearance to wood, from undissolved cellulose, hence their name. In contrast, brown-rot fungi degrade cellulose, leaving lignin as a typically brownish deposit. These fungi also cause chequered, cubical cracking and shrinking in wood, which is frequently apparent on felled confer trees (Stamets, 2005). It has been estimated that some 30% of the literature on fungal bioremediation is concerned with white-rot fungi (Singh, 2006). As we shall see, there are particular mechanisms implicit to white-rot over other kinds of fungi, which offer advantages, e.g. over the use of bacteria, as a means for bioremediation. In particular, bacteria need to be pre-exposed to the particular pollutant they are intended to degrade, in order to induce those enzymes that are required to accomplish the task. There is, furthermore, a pollutant concentration level below which the enzymes are not expressed in bacteria, thus limiting the technology (Adenipekun and Lawal, 2012). A very large range of organic molecules are susceptible to the actions of various strains of white-rot fungi, to varying degrees, and even normally highly intractable and persistent substances, including polyaromatic hydrocarbons (PAH), may be degraded by them (Singh, 2006). The white-rot fungus Phanerochaete chrysosporium is an ideal model for bioremediation by fungi, since it is more efficient than other fungi or microorganisms in degrading toxic or insoluble materials. It presents simultaneous oxidative and reductive mechanisms which permit its use in many different situations, regarding the type of contamination, its degree, and the nature of the site itself. A number of other white-rot fungi also can degrade persistent xenobiotic compounds, e.g. Pleurotus ostreatus, Trametes versicolor, Bjerkandera adusta, Lentinula edodes, Irpex lacteus, Agaricus bisporus, Pleurotus tuber-regium, Pleurotus pulmonarius. (Singh, 2006; Adenipekun and Lawal, 2012). Soils may also be decontaminated from crude oil, with the requirement that lignocellulosic substrates (e.g. sawdust straw and corn cob) are also provided, to support the growth of fungal species in the soil (Lang et al., 1995). Other toxic materials that have been successfully degraded using white-rot fungi are: polychlorinated biphenyls and dioxins, pesticides, phenols and chlorophenols, effluents from pulp and paper mills, dyestuffs and heavy metals (Singh, 2006). It has been proposed that fungi might be deployed in the biodegradation of sites that are polluted by complex mixtures of PAH, for example from creosote, coal tar and crude oil (Loske et al., 1990). However, it has been shown that the degradation of Benzo[a]pyrene by Pleurotus ostreatus is strongly influenced by the presence of heavy metal cations and mediators such as vanillin and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate). A 15 mM concentration of copper was found to best enhance the degradation (74.2%), which was progressively worsened as the Cu concentration increased. The extent of degradation was increased to 83.6 % when 5 mM of vanillin was included in the medium (Bhattacharya et al., 2014). The possibility is offered, therefore, that the presence of vanillin (a breakdown product of lignin) might augment the process of mycoremediation using white-rot fungi in actual field-applications. It has been demonstrated (Isikhuemhen et al., 2011) that L. Squarrosulus can degrade cornstalks significantly after 30 days, with a maximum lignocellulolytic enzyme activity being achieved on day 6 of cultivation, to generate exopolysaccharides. Thus, L. squarrosulus might prove very effective in the industrial pretreatment and biodelignification of lignocellulosic biomass. The main reason that white-rot fungi are active to such a wide range of compounds is their release of extra-cellular lignin-modifying enzymes, with a low substrate-specificity, so they can act upon various molecules that are broadly similar to lignin (Adenipekun and Lawal, 2012). The enzymes present in the system employed for degrading lignin include lignin-peroxidase (LiP), manganese peroxidase (MnP), various H2O2 producing enzymes (Kirk and Farrell, 1987) and laccase, although the three types of enzymatic activity are not present in all lignolytic fungi.
4. Practical implementation of mycoremediation using white-rot fungi.
In order to use white-rot fungi successfully for bioremediation, knowledge must be taken from fungal physiology, biochemistry, enzymology, ecology, genetics, molecular biology, and engineering, among other cognate subjects. A four-phase strategy has been advocated (Lamar and White, 2001): bench-scale treatability, on-site pilot testing, production of inoculum, and finally full-scale application. Substrates such as wood chips, wheat straw, peat, corn cobs, sawdust, a nutrient-fortified mixture of grain and sawdust, bark, rice, annual plant stems and wood, fish oil, alfalfa, spent mushroom compost, sugarcane bagasse, coffee pulp, sugar beet pulp, okra, canola meal, cyclodextrins, and surfactants can be used in inoculum production both off-site or on-site, or as mixed with contaminated soils to improve the processes of degradation (Singh, 2006). It is critical to attain the correct nitrogen/carbon ratio in the substrates used, so to avoid any impeding effect on the efficiency of the fungi in the bioremediation process. Fungal inocula coated with alginate, gelatin, agarose, carrageenan, chitosan, etc., in the form of pellets, may offer a better outcome than with inocula produced using bulk substrates.
The latter approach, termed encapsulation, is derived from the mushroom spawn industry, and both preserves the viability of the inoculum and contributes nutrients to maximally support the degradation of pollutants. This, furthermore, increases the survival and effectiveness of the introduced species. Fungal inoculum may also be obtained by solid state fermentation. Such inoculum preparation methods improve the likelihood of success in the first phase (above), while good technical and engineering vitalise the second phase. Success in stages three and four depends on the exact remediation practices employed for the monitoring, optimisation, continuity and maintenance of the process overall. Native microbial populations will also provide a potential competition to mycoremediation process, but there is, as yet, a lack of defined protocols to eliminate such influences. There are some patents available which refer to the subject of remediation using white-rot fungi (Singh, 2006).
Clearly, there is scope for the use of fungi in decomposing in situ intractable, persistent, and highly toxic pollutants, including TNT (2,4,6-trinitrotoluene) http://www.defmin.fi/files/2461/Steffen_Kari.pdf. and the nerve gases VX and sarin (Stamets, 2005). By inocculating a plot of soil contaminated by diesel oil, with mycelia from oyster mushrooms (Pleurotus ostreatus), it was found that after 4 weeks, 95% of many of the PAHs had been converted to non-toxic compounds. It seems that the naturally present community of microbes acts in concert with the fungi to decompose the contaminants, finally to CO2 plus H2O (full mineralisation). In 2007, a cargo ship spilled 58,000 gallons of fuel along the San Francisco shoreline. Mats woven from human hair (resembling doormats) were used as sponges to soak up the spilled oil https://www.youtube.com/watch?v=WscZJ2Dh0R. These were then collected and layered with oyster mushrooms and straw: the mushrooms broke down the oil and after several weeks the resulting soil was clean enough to be used for roadside landscaping. Wood-degrading fungi are extremely effective in decomposing toxic aromatic pollutants from petroleum and also chlorinated persistent pesticides (Rhodes, 2013). Mycofiltration is a similar procedure, in which mycelia are used as a filter to remove toxic materials and microorganisms from water in the soil. A major protagonist of mycoremediation is Paul Stamets, who has proposed (Stamets, 2005) that there should be “Mycological Response Teams”, who would employ fungi to recycle and rebuild healthy soil in the area following any contamination incident (oil spill, chemical leak, radiation egress, e.g. at Fukushima). It has been suggested that edible mushrooms might be grown for the purposes of mycoremediation, and the prospects of whether they would be safe to eat afterwards are considered (Kulshreshtha et al., 2014). Naturally this depends on the exact nature of the pollutants, so that heavy metals are likely be a problem (if they are absorbed and concentrated into the mushroom), while some organic soil contaminants might be decomposed without so imparting toxicity. In the latter case, the benefit is offered that land that is contaminated and unfit for agriculture could be both cleaned and made to yield a nutritious food crop.
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Photo credit: Oyster mushroom image via flickr/kqedquest. Creative Commons 2.0 license.