Mycoremediation: A Biological Approach to Remediating Contaminated and Polluted Soils
By: Tess Burzynski
ABSTRACT
The increasing rates of organic and inorganic pollutants arising in the environment have posed great threat to the ecological system. These pollutants interact with soil habitats creating a toxic environment for biological activity. Due to the bioavailability of these pollutants, the agricultural field is also of major concern. Because the high contamination posing threat to biological health, remediation techniques of soil are of elevating interest. In this work, mycoremediation, is analyzed. This biological approach to remediating the soil is an economic and environmental efficient strategy. The Saprophytic fungi, known as white-rot fungi, have been the backbone of this bioremediation study. Specific strains of fungal species are known to degrade pollutants such as polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), synthetic dyes and polymers, and heavy metals. Their complex extracellular enzyme system is reason for this superb biodegradation of contaminants. Several experimental studies have been executed to explain the degradation mechanisms of these organisms and how further studies can improve soil rehabilitation and health overall.
INTRODUCTION
Environmental pollutants are abundant in today’s modern society, threatening wildlife and human health, becoming an ecological hazard. These toxins are derived from naturally occurring and anthropogenic sources. Natural occurring sources of environmental pollutants include forest fires, volcanic activity, dust storms, etc. Anthropogenic sources are mainly derived from the burning of fossil fuels and wood, construction, vehicle exhaust, excess fertilization, pesticides and more. These toxins, once leached into the environment, can greatly contaminate the soil, in turn threatening food supplies and water quality, causing life threatening illnesses. Modern pesticides are known to cause reproductive issues and even death. Mutagenicity, reprotoxicity and endocrine disruption have been reported in both humans and wildlife (Plant et al., 2011). Due to the harmful effects environmental pollutants have on the planet and its inhabitants, it is vital to develop and execute remediation techniques.
Soil plays a dynamic role in the environment because it acts as a sink for pollutants. The fate of organic and inorganic chemicals in soil organic matter must be understood and considered for successful bioremediation. Soil is a heterogeneous mixture of organic and mineral matter, air and water (Pendias 2010). Soil organic matter manages much of a soils functionality including its interaction with the pollutants within the soil. A soil is not considered polluted until a present substance is greater than the normal concentrations in result of human activity and has detrimental environmental effects. Soil contamination is where a soil state veers from the normal composition but does not have detrimental effects on organisms (Pendias 2010). Soil contamination and pollution are increasing due to anthropogenic activity. The movement of pollutants through the soil and the bioavailability of certain substances are reasons for remediation concern.
Mycology is the study of Fungi which are ubiquitous in the environment and thrive in various soil habitats. Fungi are vital for a healthy soil system due to their underground fungal network consisting of complex branched hyphae known as mycelium. This fungal network acts as a transporter of nutrients through the soil. Some fungi are symbiotic in which they form in symbiosis with 70% of plants (Stamets 2005). These are known as mycorrhizal fungi. The mycelium attaches to the plant roots, increasing their surface area and uptake of nutrients. Because mycelium is very tenacious, holding the forest floor together, it is able to reach great depths. By doing so, nutrients that would otherwise be out of reach to most plant roots are made available. In return, plants provide the fungi with the carbohydrates needed for survival. Fungi are main decomposers in the environment on an annual basis, restoring the soil longevity. Saprophytic fungi can be placed into three different decomposing groups; primary, secondary and tertiary decomposers (Stamets 2005). Primary decomposers are fast growing, sending out vast protruding strands of mycelium. Pleurotus species, Lentinula edodes and Grifola frondosa are just a few of the primary decomposing fungal species. They each use different enzymes to readily decompose plant tissue. Secondary decomposers operate in sync with other microorganisms in the soil. The composting process of primary decomposers gives rise to these secondary decomposers as they grow from composting piles. Tertiary decomposers develop at the end of the decomposition process. Species include Conocybe, Agrocybe, Mycena, Armillaria Pluteus, and Agaricus (Stamets 2005). Some of these species can grow both parasitically and saprophytically.
DISCUSSION: Mycoremediation
Mycoremediation is a biological approach which utilizes Saprophytic fungi to degrade toxins in the environment (Stamets 2012). Fungi are superb molecular disassemblers; they break down recalcitrant long chained toxins into simpler, less toxic forms (Stamets 2005). Saprophytic fungi digest dead organic matter. Mycelium assists in this cost efficient biological solution to remediating soil. Because fungi are superb adapters they are ubiquitous in the environment. Bacterial growth is limited in certain environmental conditions while fungi are able to grow in various soil conditions some of which include low pH and nutrient availability, varying temperature and moisture situations and even contaminated sites (Stamets 2005). Fungi, unlike plants, are unable to obtain nutrients via photosynthesis. Instead, they excrete extracellular enzymes to digest macromolecules in the environment and harness them as sustenance. Some of these extracellular enzymes aid in the mycoremediation process by metabolizing xenobiotic compounds including polychlorinated biphenyls (PCBs), persistent organophosphates (POPs), polycyclic aromatic hydrocarbons (PAHs), trinitrotoluene (TNT), and synthetic dyes and polymers (Cenek et al., 2004). Mycoremediation can also immobilize heavy metals such as lead, uranium, and mercury in situ (Stamets 2005).
Mycoremediation concentrates on white rot-fungi, which are vigorous degraders of lignin to CO2. Lignin is a complex aromatic plant polymer found in all vascular plants providing them with a fibrous and rigid texture (Reddy 1995). White-rot fungi are the only decomposers of lignin thus playing a vital role in life’s natural recycling process. These fungi employ their lignin-degrading enzyme system (LDE) to mineralize a variety of recalcitrant compounds in the environment. The LDE system is comprised of extracellular peroxidases; lignin peroxidase (LIP), manganese-dependent peroxidase (MnP) and laccases (Reddy 1995). These enzymes are crucial to the mycoremedation process. Phanerochaete chrysosporium, Trametes versicolor, and Pleurotus ostreatus are but of a few white-rot fungi that have been analyzed for remediation.
Economic Sufficiency
The cultivation of white-rot fungi is a cost effective technique. Mycelium is able to cultivate and thrive on several types of agricultural wastes like corn husks, coffee grounds, wheat straw, cottonseed hulls and hardwood chips. Obtaining these agricultural wastes from local farmers is inexpensive and environmentally beneficial by putting wastes to constructive use. The technique for applying mycoremediation to contaminated soils is carried out by inoculating the choice of agricultural waste with a specified saprophytic fungi strain. Once the mycelium has fully cultivated the agricultural wastes, about one week depending on conditions, it will then be tilled into the site of contamination. Large scale applications have yet to be carried out; however, in vitro and small scale applications have been successful.
Polycyclic aromatic hydrocarbons (PAHs)
Hydrocarbons, like petroleum products, are similar to lignin in that they share an analogous type of bond. The manganese-dependent peroxidase enzyme that takes part in breaking down lignin can also break down bonds of hydrocarbons. Once the mycelial enzymes break the hydrocarbon bonds there are non-solid byproducts of 50 % gaseous carbon dioxide and 10-20% water (Stamets 2005). Polycyclic aromatic hydrocarbons (PAHs) are a class of hydrocarbons that are widespread in the environment. This group of pollutants is derived from fossil fuel burning, oil drilling and coal mining (Reddy 1995). PAHs are known to be potential carcinogens and have shown to be resistant to biodegradation in the past, making them a considerable concern for mycoremediation. A study was done involving the white-rot fungi, Pleurotus ostreatus and an abandoned wood preservation site (Stamets 2005). Using plastic containers, drill-cutting samples were collected from the site. Four samples were obtained with one acting as a control. The 2000 gram samples were combined with 500 grams of top soil and varying masses of P. ostreatus fungal substrate. The first sample consisted of 500g of substrate, 1000g for the second and 2000g of fungal substrate for sample 3. Each sample was replicated 3 times for viable results. The bioremediation took place for duration of 56 days at 30 degrees Celsius. After this period, levels of the PAHs were analyzed using the USEPA (1996) method 8270B (Okparanma et al., 2011). Results varied depending on the amount of fungal substrate added to the samples and the differing properties of the PAHs including ring structure and molar mass. Findings resulted in a decrease of the total amount of PAHs between 19.75 and 7.62% (Okparanma et al., 2011). Degradation of the individual PAHs was 97.98% for the acenapthene, 100% in fluorene, phenanthrene and anthracene which are all a 3-ring structure. These results suggest potential for mycoremediation in removal of oil-based hydrocarbons from contaminated soil. This could be a constructive technique in bringing down PAHs levels to an environmentally safe level.
Figure 1. Degradation rates of PAHs in drill cuttings. (Okparanma et. al., 2014)
Nitroaromatics
Nitroaromatics are widely used in the industrial production of pesticides, pharmaceutical products, explosives and artificial dyes (Khadar et al., 1991). Production sites of these nitroaromatic based products are highly contaminated in both groundwater and soils. 2,4 and 2,6- dinitrotoluene are the major residue from industrial production of these products and are known to be mutagenic in bacteria and mammals (Khadar et al., 1991). According to the Environmental Protection Agency, dinitrotoluene, when chronically exposed to humans, can cause damage to the central nervous system and blood. Because of the harmful effects of dinitrotoluene and its extensive use, the importance of remediating this pollutant is high. A study was done by examining the biodegradation of 2, 4 and 2, 6-dinitrotoulenes by the white-rot fungus, P. chrysosporium. After a 24 day incubation period results were detected. Extracellular enzymes of P. chrysosporium successfully mineralized the 2, 4 dinitrotoluene. In nitrogen-limited cultures, 34% of the original dinitrotoluene was mineralized to CO2 while only 7% of the dinitrotoluene was mineralized in nitrogen-sufficient cultures (Valli 1991). The lignin-degrading enzyme system of P. chrysosporium is responsible for the mineralization of this pollutant. This suggests positive results in using the fungi strain of P. chrysosporium as a bioremediation technique to rid of nitroaromatic pollutants in the environment.
Polychlorinated biphenyls (PCBs)
Polychlorinated biphenyls (PCBs) are man-made organic chemical compounds with a wide range of industrial and commercial uses. These pollutants are highly toxic and have been produced in the United States since 1930 (Lucile et al., 2014). A series of health effects can be caused from PCBs as they affect ones immune system, reproductive system, nervous and endocrine systems (Lucile et al., 2014). Once released into the environment due to leaks, illegal dumping, incineration and poorly controlled hazardous waste strategies, they do not readily break down and cycle between air, water and soil. PCB degradation rates were analyzed in an experiment using Trametes versicolor, Ph. chrysosporium and Pleurotus ostreatus fungal strains. A commercial PCB mixture Delor 103 was combined with a brown soil which contained 0.8% organic carbon and nitrogen with a pH of 5.3 (Lucile et al., 2014). The experiment took place in special tube reactors where the mycelium grew over a two month period. Following this duration the PCBs were analyzed using a high-resolution gas chromatograph. Results detected there was little to no degradation of PCBs by P. chrysosporium and T. versicolor leading them to be non-significant. Quite the reverse, P. ostreatus was able to degrade 40% of the Delor 103 PCB mixture. This percentage of degradation was also true for the aerated control where other microorganisms played part in degradation. Although results detected degradation of PCBs, the mechanism is yet to be known. This is different for polyaromatic hydrocarbons and chlorophenols where in these cases, the degradation process is due to the ligninolytic enzymes lignin-peroxidase and manganese-dependent peroxidase. Though the mechanism is still unclear, there are positive degradation results which offer reason for further studies regarding the degradation of polychlorinated biphenyls by the fungal strand Pleurotus ostreatus.
Heavy Metals
Heavy metals such as uranium, lead, mercury, cadmium, and arsenic are ample pollutants in our environment. These metals often occur together in soil sediments and pose threat to biological systems where they magnify in the food chain (Pendias 2010). Oxidation stress is a side effect from exposure to these heavy metals, leading to DNA damage. Plants, animals and humans undergo bioaccumulation of heavy metals via the food chain which leads to the importance of ridding these toxins from the source. Uptake of heavy metals by fungal species has been studied and metal transformation by these species has been detected (Stamets 2005). Studies have revealed the high oxide tolerance displayed by fungi giving them the ability to solubilize uranium trioxide and triuranium octaoxide, where the mycelium accumulates the uranium. Fungi can also solubilize cadmium, copper, lead and zinc. Considerations such as pH and growth tolerance were observed, linking these traits with the fungal ability to solubilize these metals. A relatively recent study utilized P. chrysogenum on rice straw for remediation of copper and cadmium contaminated agricultural soil. Surface soil was collected from an agricultural contaminated loamy soil (38.7% sand, 39.2% silt, 22.1% clay) (Ming et. al., 2014). The levels of contamination of the Cu and Cd respectively read 5.0 and 440.43 mg kg -1. The experiment conducted 24 pots of 2.0 kg of contaminated air-dried soil combined with P. chrysogenum inoculated rice straw. The control pot consisted of only contaminated soil and non-inoculated rice straw. In each pot, 15 lettuce seeds were sown. After 45 days, the lettuce was harvest and divided into shoots and roots which were detected for Cd and Cu by graphite-furnace atomic absorption spectrometry (Ming et al.,2014). Soil samples were taken from each pot for chemical analysis. Results showed that the P. chrysogenum rice straw decreased the acid-extractable and reducible Cu and Cd and increased the oxidizable Cu and Cd fractions (Ming et al., 2014). The Cu and Cd contents in the lettuce were also reduced compared to the control. This suggests restrained uptake of the Cu and Cd by the lettuce due to the introduction of the fungal species. Due to these results, certain fungal species can be considered for stabilizing heavy metals in contaminated soils.
CONCLUSION
The concern for remediation of polluted soils is on the rise. Due to the negative impacts on biological systems and the ecological world as a whole, owing to these increasingly toxic environments, has caused worry for the future. The potential of ubiquitous microorganisms like fungal mycelium to transform these toxic wastelands is a promising solution. Although there are gaps in previous studies, preventing mycoremediation to be utilized fully, there are ongoing studies in expectation to fill these holes and discover more about this environmentally sufficient bioremediation technique. The primary goal of this system is to apply the mycorestoration idea on a small grass-roots level. With the ability of these microorganisms to work with the natural environment, it can also be used as a preventative technique. Perhaps it would be beneficial for landowners to install a mycorestoration project to their site of concern. By doing so, pollution is rehabilitated one area at a time, eventually cumulating in a positive way to the entire system. Further studies on large scale mycorestoration techniques would be beneficial as well as the interactions of fungal mycelium in competition with other microorganisms such as bacteria. By better understanding the mutual relationships fungi and its complex lignin-degrading enzymatic system have within the soil system, the closer mycoremediation will come to rehabilitating contaminated and polluted soil.
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