The Potential of the Fungi Trichoderma Harzianum as a Biodegradable Solution in Decomposing Waste Plastics

The Potential of Trichoderma harzianum as a Biodegradable Solution in Decomposing Low Density and High Density Polyethylene Waste Plastics

A research proposal presented to the Faculty of College of Arts and Sciences Cagayan State University Carig Campus, Tuguegarao City

In partial fulfillment of the requirements of the Degree of Bachelor of Science in Biology

Lina Riza N. Montero

Prof. Osmond Narag Research Instructor

Chapter I Introduction Plastics are ubiquitous in the modern world. Polyethylene is the world's most common plastic. It finds innumerable applications in everything from bottles and jugs to shopping bags and children's toys. It owes its surprising versatility to its properties and molecular structure. There are different kinds of polyethylene available; two of the most common are highdensity polyethylene or HDPE and low-density polyethylene or LDPE. LDPE was the first PE to be developed. It has low-density levels and only a small amount of branching. It is very flexible and easy to clean. It is often used to make plastic film wrap and plastic bags. Additionally, it is used to make plastic items that need to be molded, such as plastic bottles used in labs. While HDPE has higher density levels; it is characterized by a linear structure consisting of no branching. That makes HDPE stronger and more resistant to chemicals. It is most commonly used for items requiring blow molding techniques, such as toys, automobile parts and bottles. It is also used to create cutting boards since it meets FDA food service standards. Although most of these plastics can be recycled, much of it ends up in our trash cans. There are concerns in the environmental industry that, since these plastics do not break down easily in a landfill, there will be many future negative effects due to our overuse of these products. According to the American Chemistry Council's Resin Production and Sales Stats report, released in November 2009, HDPE is the most common plastic and LDPE is the fifth most common plastic produced in the U.S. The slow rate at which plastic degrades vs. the

amount produced makes the disposal of it an environmental issue. To overcome this everincreasing serious problem, decomposition of waste plastics seems to be a fruitful solution. Certain bacterial strains had been discovered to degrade these plastics, however, seeing that there are various fungal strains that have the potential to be involved in the process of decomposition that can contribute to the adamant pollution and equilibrium in nature. This study will provide information regarding the potency of Trichoderma particularly the T. harzianum as a natural and valuable decomposing agent in non- biodegradable matter in order to reduce the utilization of chemicals capable of decomposition. Trichoderma is a genus of fungi that is present in all soils, decaying wood and vegetable matter. Their dominance in soil may be attributed to their diverse metabolic capability and aggressive competitive nature (Lewis and Papavizas, 1991). These characteristics make them significant decomposers of woody and herbaceous material and are also necrotrophic against other decomposers. Many species in this genus can be characterized as opportunistic a virulent plant symbionts, the ability of several Trichoderma species to form mutualistic endophytic relationships with several plant species. One of which species is the Trichoderma harzianum, the most frequent Trichoderma species cultivated from soil worldwide. That displays a remarkable diversity of lifestyles ranging from saprotrophy in free soil and dead wood, in rhizosphere and on dead fungal biomass to biotrophy in necrotrophic mycoparasisitic attacks of other fungi and endophytic associations with plants (http://genome.jgi.doe.gov). In addition to, it is reported that T. harzianum may have the capability of degrading organochlorine pesticides such as DDT, dieldrin, endosulfan, pentachloronitrobenzene, pentachlorophenol and hence has potential applications for bioremediation (Kelley, 1976). These pesticides contain petroleum oils that are refined from crude oil which is the main ingredient of plastic.

Objectives of the Study Generally, this study aims to understand the microphysical potency of T. harzianum as an effective biodegradable solution in decomposing waste plastics. It specifically aims to: 

To describe the potential of T. harzianum as potential decomposer,



To observe the physical manifestations of exposing LPDE and HPDE plastics to T. harzianum under experimental condition,



To determine the efficiency of T. harzianum as a decomposer,



To determine the rate of decomposition made by T. harzianum to LPDE and HPDE plastics.

Scope and Delimitation of the Study This study is intended only for the determination and provision of Trichoderma specifically the Trichoderma harzianum as potential and effective decomposer of nonbiodegradable plastics (HDPE and LDPE).

Time and Place of the Study Cagayan State University, Carig Campus, Tuguegarao City, DOST Lab from September to November 2014.

Definition of terms

Composting is the decomposition of plant remains and other materials to reduce the volume of garbage needlessly sent to landfills for disposal. Decomposer organisms that break down unused dead material that carry out the natural process of decomposition. Fungi any member of a large group of eukaryotic organisms that includes microorganisms such as yeasts and molds, as well as the more familiar mushrooms. High-density polyethylene (HDPE) or polyethylene high-density (PEHD) is a polyethylene thermoplastic made from petroleum. Low-density polyethylene (LDPE) is a thermoplastic made from the monomer ethylene Mycoparasitic is a parasitic fungus whose host is another fungus. Necrotrophic a parasite that kills its host, then feeds on the dead matter. Polyethylene is the most common plastic the polymer that makes grocery bags Potato Dextrose Agar is a nonselective medium for the cultivation of yeasts and molds. Trichoderma a genus of fungi that is present in all soils, where they are the most prevalent culturable fungi. Many species in this genus can be characterized as opportunistic avirulent plant symbionts. T. harzianum are the most frequent Trichoderma species cultivated from soil worldwide that may have the potential to degrade plastics.

Chapter II

Review of Related Literature The Nature and Effect of Plastics Plastics have become a necessary commodity in today’s world. Everyone knowingly or unknowingly uses plastic substances. Karki (2008) discusses that plastic is used not only for making plastic bags but also for producing products that cover parts of vehicles that need to be protected. The word plastic comes from the Greek word plastikos, which means, ‘able to be molded into different shapes (Joel FR., 1995). The plastics we use are made from, inorganic and organic raw material such as carbon, silicon, nitrogen, oxygen, chloride and hydrogen. Basic material used for making plastic are extracted from coal, oil and natural gas. Plastics are defined as the polymers which become mobile on heating and thus can be cast into moulds. Plastics are nonmetallic mouldable compounds and the materials made from them, can be pushed into almost any desirable shape and then retain that shape (Seymour RB., 1989) Commodity plastics are used in packaging, disposable diaper backing, fishing nets and agricultural film. They include polymers such as polyethylene, polypropylene, polystyrene, polyvinylchloride, polyurethane, polyethyleneterepthalate, nylon (Shah, 2007). Improperly disposed plastic materials are a significant source of environmental pollution, potentially harming life. The plastic sheets or bags do not allow water and air to go into earth which causes infertility of soil, preventing degradation of other normal substances, depletion of underground water source and danger to animal life. In seas also plastic rubbish from ropes and nets to the plastic bands from beer packs chokes and entangles marine mammals (Cooper and Vaughan, 1967). According to the municipal administrators carry bags are the main cause of blocked drains and thus municipal wastes cannot be incinerated leading to accumulated garage, sludge, junk (Roff and Scott, 1971). On this living

planet, a biosphere, plastic is a raging parasite that devours and pollutes everything (http://www.mnn.com/greentech/research-innovations/blogs/boydiscovers-microbe-that-eatsplastic). Fungi as a Decomposer Fungi are any member of a large group of eukaryotic organisms that includes microorganisms such as yeasts and molds, as well as the more familiar mushrooms. Abundant worldwide, most fungi are inconspicuous because of the small size of their structures, and their cryptic lifestyles in soil, on dead matter, and as symbionts of plants, animals, or other fungi. Fungi perform an essential role in the decomposition of organic matter and have fundamental roles in nutrient cycling and exchange (http://en.wikipedia.org). Most fungi are decomposers. Fungi break down, or decompose, the complex carbon compounds that are part of living matter. They absorb nutrients and leave behind simpler compounds. Fungi are heterotrophs. They get their energy from living or once living matter. They, along with bacteria, decompose the bodies of dead plants and animals. They also decompose materials left behind by organisms, such as fallen leaves, shed skin, and animal droppings. One interesting application of a mold is the use of the fungus Trichoderma. This mold grows in soil. The digestive chemicals it produces are used to

give

blue

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look

(www.d123.org/olhms/ebarlos/documents/Fungi2.4C3.pdf). In addition to, several reports appeared that fungi don’t just decompose organic matter but also degrade inorganic ones like Polyurethanes (PU) that are present in many aspects of modern life. They represent a class of polymers that have found a widespread use in the medical, automotive and industrial fields. Studies revealed that polyester-type PUs is more susceptible to fungal attack than other forms (Kaplan et al., 1968).

Studies Related to the Decomposition of Waste plastics using Fungi When UV rays strike plastic, they break the bonds holding the long molecular chain together. Over time, this can turn a big piece of plastic into lots of little pieces. However, plastic buried in a landfill rarely sees the light of the day, but in the ocean which is where a lot of plastic wastes end up. Plastics are bathed in as much light as water. In 2009, researchers from Nihon University in Chiba Japan, found that in warm ocean water can degrade in as little as a year. This doesn’t sound so bad until you realize those small bits of plastic are toxic chemicals such as bisphenol A (BPA) and PS oligomer (Harris and William, 2009). In the early 1980s the research on degradability of plastics began. Past research has isolated and identified the use of Trichoderma spp. for bioconversion of solid wastes (kitchen waste, humus, compost and soil). In the study, a total of 135 isolates of Trichoderma were isolated. These 135 isolates were divided into 5 aggregate groups. Representative isolates from each group were sent for identification. It is then concluded that the most frequently isolated species was T. harzianum and was identified as an effective agent for solid waste conversion using spore suspension. A same study targeted the potential of fungal isolates as a potential bioconversion agent of municipal solid waste. Samples of fungal strains were collected from different waste disposal site. Overall among the 5 fungal strains used in the study Tricoderma spp. were the most effective strain for the solid waste decomposition. Bari (2007), reported that T. harzianum was the most effective strain for solid waste decomposition and showed the highest weight loss (31.8%) when the culture disc approach was used but present results are partially in accordance with findings of Zheng and Shetty and Martin and Dale. These plastics differ in degradation rate, application, and price. In one development, plastics' inertness and resistance to microbial attack was reduced by incorporating starch and later prooxidants (transition metals and oil) (Griffin, 1973). Three types of

degradation of polyethylene in these degradable starch-polyethylene polymers can occur by different molecular mechanisms: chemical degradation, photodegradation and biological degradation. Chemical degradation occurs when the prooxidants catalyze the formation of free radicals in polyethylene, which react with molecular oxygen to attack the polyethylene matrix (Johnson et al.). Heat and oxygen accelerate this chain scission of the polyethylene. Photodegradation also occurs within the polyethylene matrix whereby UV light catalyzes the autoxidation and generation of free radicals (David et al., 1992). Biological degradation of these polyethylene films has been reported in pureculture studies with various microorganisms such as Streptomyces sp. (Lee et al., 1991), Phanerochaete sp.(Ali et al., 2009), Penicillium, Fusarium,, Alternaria, Spicaria spp., Aspergillus (Ibrahim et al., 2011), Aureobasidium, Poecilomyces (Mehdi et al., 2010) after chemical degradation was initiated and with their corresponding extracellular enzymes. The Mechanism of Trichoderma spp. Trichoderma spp. are free- living that are highly interactive in root, soil and foliar environments. It has been known for many years that they produce a wide range of antibiotic substances and that they parasitize other fungi. The first description of a fungus named Trichoderma dates back to 17941 (Persoon, 1794), and in 1865 a link to the state of a Hypocrea species was suggested (Tulasne, 1865). But the different species assigned to the genus Trichoderma hypocrea were difficult to distinguish morphologically. It was even proposed to reduce taxonomy to only a single species, Trichoderma viride. Hence, it took until 1969 that development of a concept for identification was initiated (Rifai, 1969; Samuels, 2006). Thereafter, numerous new species of Trichodermal hypocrea were discovered, and by 2006 the genus already comprised more than 100 phylogenetically defined species (Druzhininae et al.,

2006). Trichoderma spp. are ubiquitous colonizers of cellulosic materials and are often found wherever decaying plant material is available (Kubicek et al. 2008), as well as in the rhizosphere of plants where they can induce systemic resistance against pathogens (Harman, 2000). The search for potent biomass regarding enzymes and organisms also led to isolation of these fungi from unexpected sources such as cockroaches (Yoder et al. 2008), marine mussels and shellfish (Sallenave et al. 1999). Trichoderma spp. are characterized by rapid growth, mostly bright green conidia and a repetitively branched condiophore structure (Gams and Bissett, 1998). The sexual stage when found is within the Ascomycetes in the genus Hypocrea. Trichoderma spp. possesses innate resistance to most agricultural chemicals, including fungicides, although individual strains differ in their resistance. Some lines have been selected or modified to be resistant to specific chemicals. Composting is the most suitable option among the wastes management strategies with economic and environmental profits since this process reduces the bulk volume of organic materials, eliminates the risk of spreading of pathogens, weed seeds or parasites associated with direct land application of manure and leads to final stabilized products which can improve and sustain soil fertility. However, composting of lignocellulosic EFB takes a longer period of time which is considered as the most blocking stump of this eco-friendly disposal technique (Chen et al., 1992). Trichoderma spp. are widely known as a lignocellulose decomposer because they are filamentous and have the ability to produce profilic spores which can invade substrates quickly (Tengerdy and Szakacs, 2003). Various studies have shown that composting of lignocellulosic materials preinoculated with potential Trichoderma spp. can reduce the time of biodegradation (Mohammed et al., 2012). However, for an economically competitive process an increase in efficiency of more than 40 fold would be necessary, which is a formidable challenge for research with Trichoderma. Besides these major applications of Trichoderma spp. the fields of green and

white biotechnology become increasingly important for environmentally safe production of enzymes and antibiotics. The extensive studies on diverse physiological traits available and still progressing for Trichoderma make these fungi versatile model organisms for research on both industrial fermentations as well as natural phenomena. Trichoderma harzainum The filamentous fungus Trichoderma harzianum is the genetically distinct temperate agamospecies belonging to the group of closely related (cryptic), albeit diverse, species of the Harzianum clade of Trichoderma (teleomorph Hypocrea, Ascomycota, Dikarya). In the broad taxonomic sense these fungi (T. harzianum) are the most frequent Trichoderma species cultivated from soil worldwide. They display a remarkable diversity of lifestyles ranging from saprotrophy in free soil and dead wood, in rhizosphere and on dead fungal biomass to biotrophy in necrotrophic mycoparasisitic attacks of other fungi and endophytic associations with plants. Because of its mycotrophyc ability T. harzianum has often been set equal to Trichoderma-based biocontrol agents in general, as it is the principal component in several commercial biofungicide formulations. It is used for foliar application, seed and soil treatments for suppression of various diseases causing by such pathogens as Botrytis, Fusarium and Penicillium sp. Although T. harzianum is not a causative agent of the green mold disease on mushroom farms it is frequently isolated from infected cultures of Agaricus and Pleurotus and respective substrata. Interestingly, the causative agents of the mushroom green mold diseases (T. aggressivum, T. pleurotum and T. pleuroticola, respectively) also belong to the Harzianum clade, i.e. are closely related to T. harzianum. Similar to T. virens (teleomorph Hypocrea virens), a rhizosphere-competent T. harzianum may not only grow on plant roots, but its hyphae penetrate root epidermis (endophytism), which enhances plant growth and immune system. Some molecular mechanisms

of Trichoderma mycotrophy and interactions with plants - such as the role and regulation of formation of cell wall hydrolytic enzymes and antagonistic secondary metabolites - have been intensively investigated in T. harzianum. It is reported that T. harzianum is capable of degrading organochlorine pesticides such as DDT, dieldrin, endosulfan, pentachloronitrobenzene, pentachlorophenol and hence has potential applications for bioremediation (Kelley, 1976). As a mycoparasitic and antagonistic fungus T. harzianum is suggested to be a powerful environmental opportunist, which is able to interplay in communities of invasive Trichoderma spp. in various disturbed ecosystems and thus replace or suppress the local mycofauna. Hence the genome sequence of such an outstanding opportunistic fungus as T. harzianum is expected to provide a platform to identify genetic resource to be used in pest control, development of biofungicides, improvement of plant health, decomposition of plastics and environmental monitoring (http://genome.jgi.doe.gov/).

Chapter III Materials and Methods

Materials The equipment, supplies and materials that will be used in this study are the following: cultured stock of T. harzianum (culture pellets & spore suspension), petri dish, LDPE and HDPE plastics, Erlenmayer flask (150 mL), 2% Tween 20, centrifuge, test tubes, scissors, incubator, autoclave, PDA (potato dextrose agar), Potato Dextrose Broth, distilled water and record notebook.

Methods Treatments The following treatments that will be used to conduct the study are: T1 – Control1 LDPE (no inoculation) T2 – Control 2 HDPE (no inoculation) T3 – 50g of Low Density Polyethylene (LDPE) cut into 2-3mm with spore suspension of T. harzianum T4 – 50g of Low Density Polyethylene (LDPE) cut into 2-3mm with culture pellet of T. harzianum T5 – 50g of High Density Polyethylene (HDPE) cut into 2-3mm with spore suspension of T. harzianum T6 – 50g of High Density Polyethylene (HDPE) cut into 2-3mm with culture pellet of T. harzianum

Procedures A. Obtaining of Subculture and Treatments

T. harzianum, will be obtained from the culture stock of the University of the Philippines –Los Banos, Campus Laboratory. The fungus will be maintained in Petri dishes of PDA (potato dextrose agar). After inoculating from the original slant culture stock, the petri dishes will be incubated at 280C for 4-7 days and subsequently stored at 50C. Spore suspension will be obtained by washing the Petri dish cultures with a sterile aqueous solution of 2% Tween 20. The resulting suspension will be centrifuged (∼2800×g, 5 min). Fungal pellets will be obtained from the germination of spores that are suspended in shake flasks in the preliminary cultivation stage. B. Preparation of waste plastics for decomposition LDPE and HDPE plastics will be collected from the garbage center of the CSU, Carig Campus. Aseptic condition will be maintained as far as possible during the collection. The samples will be cut into 2-3 mm pieces and 50 g of each will be aliquotted into a 150 mL Erlenmayer flask, which will then be sealed with a cotton plug. The Erlenmayer flask containing waste plastic will then be autoclaved at 121 °C for 15 min. C. Liquid shaking culture test method A 50g plastic bag cut into 2-3mm will be added to Erlenmeyer flask (150 mL)containing 10 mL Potato Dextrose Broth, sterilized by autoclave at 121 °C for 20 min. The test isolate (3 culture pellets/50g) will be added to the test medium, properly stoppered, and incubated at 30 °C with reciprocal shaking (120 oscillations/min). The degradation of the LDPE and HDPE plastics will be monitored by measuring the weight of the plastics before and after incubation. At the end of the incubation period, the pieces of plastics will be taken out and washed several times with distilled water. Next, they will be dried overnight at 80°C and the changes will be observed.

D. Petri dish test method Potato Dextrose Agar will be prepared by adding 15 g of agar to 1 L of basal medium, followed by autoclaving for 20 min and pouring it into petri dishes. Sterilized pieces of plastic bag will be overlaid on the medium surface, and the test isolate (1.0 mL of spore suspension) will be added over the plastic bag pieces. At the end of incubation period, the pieces of plastic bag will be taken out and will be washed several times with distilled water. They will be next dried over night at 80 °C and the changes will be observed. E. Data Collection Procedures The data will be collected based on the observations that will be made during the experiment like changes in color, odor, volume loss and weight loss of waste plastic bag. It will be observed at 10-day intervals up to 60 days. 

For measurement of volume loss (%), the following formula will be used:



Volume loss (%) = V-V1/V × 100 where V is initial volume, V1 is final volume.



Weight loss (%) of plastic bag will be calculated employing the formula:



Weight loss (%) = W-W1/W × 100 where W is initial weight, W1 is final weight.

F. Statistical Tools and Analysis The experiment will be conducted using a complete randomized design (CRD) with 3 treatments replicated 3 times. Correlations will be calculated between the sample’s physiochemical characteristics and cfus. Results of all analyses will be judged for significance at the 5% level. The data will be statistically analyzed with the help of the computer package MSTATC.

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