Research - Jan6.docx

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Chapter IINTRODUCTIONBackground of the Study Due to increase in knowledge regarding new methods of protecting plants from pests and enhancement of plant nutrition, people have overlooked the possible results that may lead to even more disastrous outcomes such as infertility of soil.Soil contaminated with pesticide decreases the soils fertility. It also decreases the biodiversity in the soil for the pesticide not only kills the intended pests, but also the other organisms living there. Bodies of water near agricultural fields are contaminated because of runoffs which causes widespread pollution in the area. Pesticide disrupts the plants cycle, making it become more dependent on the chemicals provided by the pesticide. Crops absorb the pesticide and the toxins are being distributed to the plant cells therefore making it toxic and unsafe to eat. People, especially children, who consume these infected plants, are put at a vulnerable state for acquiring diseases. Thus, humans required nourishment and soil fertility are compromised (Dudeja and Singh, 2008).Mycoremediation is a form of bioremediation that enlists the biological activity of fungi to restore polluted environments to a less polluted state (Singh, 2006). The term 'mycoremediation' was coined by mycologist Paul Stamets and relates specifically to the ecological functionality of fungal mycelium rather than 'mushrooms' which are the fruiting bodies of the mycelium. Bioremediation has been common practice for a very long time, and can be traced back as far as 600bc when the Romans used bacteria to treat their waste water (Singh, 2006). Mycoremediation is however a younger branch of the science of bioremediation and has only been practiced since the beginning of the 20th century. There is a lack of fundamental knowledge within the field which has translated to slow advancement of the science (Sasek, 2003).Working with fungi can lead to hastened process of soil recovery. The natural behavior of some fungi to work within and support a larger community of organisms seems to be the key part of their role. Fungi with known abilities in breaking down complex plant cell structures, the lignin decomposing (white-rot) Basidiomycotina, have been the primary case studies thus far in mycoremediation, but a much wider array of soil borne fungi and endophytes with lesser understood natural roles are becoming more important subjects of inquiry. (Potera, Carol)Since the role of fungi in the environment is widely recognized, the researchers have inferred that fungi species found in mangrove areas are possible species that can be used in remediating soil contaminated with pesticide because there are already several fungi species that were identified to have successfully remediated pollutants. The present study was therefore, designed to evaluate the mycoremediation potential of endophytic fungi on Chlorpyrifos.Statement of the Problems This study aimed to assess the mycoremediation potential on the pesticide chlorpyrifos of fungal isolates from mangrove areas in Sagay, Camarines Sur. It also aims to answer the following questions:1. Was there any growth of fungi observed upon applying them with the pesticide (5 ml, 10 ml, and 15 ml)?2. Did the three endophytic fungi show a change in its color and in its medias color?Statement of the HypothesesFor the main problem:H:Fungal isolatestaken from mangrove areas cannot be used on the mycoremediation of pesticide.H:Fungal isolates taken from mangrove areas can be used on the mycoremediation of pesticide.Based on the two specific problems, four hypotheses were stated.H1: There was no growth of fungi observed after the pesticide of 15 ml was applied with fungi.Ha1: There was fungi growth observed after the pesticide of 5 ml was applied with fungi.H2: The three endophytic fungi did not show a change in its color and in its medias.H2: The three endophytic fungi showed a change in its color and in its medias color.

Significance of the StudyAgricultural FieldsMaintaining soil fertility is an important factor in agricultural fields for soil plays a vital role in promoting plant growth. Since most fields are being contaminated with pesticide, the researchers have sought of a method to lessen the toxicity of the soil by degrading the pesticide. Since Brodan, which contains chlorpyrifos is one of the most common pesticides used; the researchers may help farmers in lessening the pesticide in their soil.Departments and Agencies in the Philippines which focus on AgricultureDifferent agricultural agencies such as the Department of Environmental and Natural Resources (DENR), Department of Agriculture (DA), and Bureau of Agricultural Research (BAR) may be helped by this study for it will give them further knowledge on the importance of biotechnology in the country which they may use to spread to the Filipino citizens.

Field of BioremediationThis study also adds further knowledge in the field of bioremediation for fungal species that is isolated in the mangrove tree and it is put into consideration in remediating soil by lessening pesticide. Scope and DelimitationThe study will utilize three different endophytic fungi species obtained from the roots of mangrove trees in Sagay, Camarines Sur and will be isolated in three separate petri dishes and will then be cultured using Potato Dextrose Agar (PDA) because it is a nutrient rich media used for culturing a wide variety of microorganisms. The culturing of the isolates will be done in the Biology Laboratory of Philippine Science High School Bicol Region Campus.This study focuses if the fungi isolates have a mycoremediation potential on the pesticide chlorpyrifos. The pesticide with the commercial name, Brodan together with Potato Dextrose Broth will be used as treatment. The pesticide will have three different volumes, 5 ml, 10 ml and 15 ml. Potato Dextrose Broth will have the same volume in each flask which is 55 ml. The nine treatments will be analyzed by qualitative evaluation after the experiment to observe the changes made. Mycelial growth, change in the fungis color, and change in the medias color will be observed during the qualitative evaluation. The extraneous factors in the set-up are the rate of proliferation and fungal growth.

Chapter IIREVIEW OF RELATED LITERATURE AND STUDIESRelated LiteratureMycoremediationMycoremediation is an innovative biotechnology that uses conditioned fungi and fungal mycelium applied to surface soils to remove and degrade contaminants (Aston, L.M., Cullinan, V.I., Thomas, S.A., Woodruff, D.L., 2009).According to Stamets, P. (2005), also known as the renaissance mycologist, mushroom suffered from biological racism. Due to this observation, he propelled the higher fungi into the 21st century. In alliance with several public and private organizations, he pioneered the use of mycoremediation and mycofiltration technologies. Baker, L. (2002) expounded that these comprised the cultivation of mushrooms to clean up contaminated waste sites, and upgrade ecological and human health.Stamets, P. (2005) explained that mycoremediation is the use of various species of fungus as a technique to clean toxins and excess nutrients out of an environment. The word mycoremediation came from the prefix myco-, fungus and remediation, the action of remedying. This method works because of the mycelium. The mycelium is a loose network of the delicate filaments hyphae that form the body of a fungus. Mycelium is the one responsible for the production of extacellular enzymes and acids that can dismantle long chains of hydrogen and carbon, the base structure common to pesticides.FungiFungi are adept molecules that are able to break down long chained toxins into simpler less toxic chemicals. They essentially use and assimilate these toxins as nutrients. Mycelial enzymes can decompose some of the most impervious and resistant materials made by humans or nature. This is due to the fact that many of the bonds that hold the plant material together are similar to the bonds found in petroleum products. These products include diesel, oil, herbicides and pesticides. Toxins such as textile dyes and estrogen-based pharmaceuticals are susceptible to the enzymes secreted by the mycelia. Some mushroom species can reduce the toxicity of several of these, while others are more selective (Stamets, 2005).In 2002, Caryn Sykes tackled about the effectiveness of fungi in the bioremediation field. According to her, this remediation process is inexpensive, easy to use, requires no liability for secondary waste, and even allows the treated material to be reused afterwards. Since it is a natural system, and does not introduce any corrosive or other chemicals for cleanup, there would be a higher probability that the public would accept this method. Also, the use of fungi only requires minimal handling and low maintenance of sites. In terms of safety, it is expected to be safer than most other alternatives for it does not require digging up contaminated products, and later disposing it off at waste sites. It restores value to depleted land. Most of all, its upshot is nontoxic. As stated by Sykes, C. (2002), although no matter how beneficial it is, it also has a few drawbacks. There are certain approaches to this remediation wherein they are only suitable for particular situations. Some organizations are finding it hard to convince others of this method for often times, people prefer to rely on proven and existing technologies.Mangrove FungiIn the study of Chandralata in 2005, he stated that mangrove fungi usually possess several metabolites of industrial and environmental importance. It has a potential on the bioremediation field and can possibly be used against harmful substances such as pesticides which is described by the National Institute of Environmental Health Sciences as any substance used to kill, repel, or control certain forms of plant or animal life that are considered to be pests.ChlorpyrifosThe wide application of organophosphorous (OP) insecticides such as Chlorpyrifos are employed for plant protection against insect pests. This organophosphorous pesticide is one of the major chemicals responsible for the contamination and deterioration of soil and groundwater, particularly in the close vicinities of agricultural fields (Jayashree and Vasudevan, 2006).A decline in the population of beneficial soil microorganisms can be instigated due to heavy treatment of soil with pesticides. According to soil scientist Dr. Elaine Ingham, If we lose both bacteria and fungi, then the soil degrades. Overuse of chemical fertilizers and pesticides have effects on the soil organisms that are similar to human overuse of antibiotics (Savonen, 1997).SoilSoil is a natural body of mineral and organic constituents differentiated into horizons usually unconsolidated of variable depth which differs among themselves as well as from the underlying parent material in morphology, physical makeup, chemical properties and composition and biological characteristics (Dokuchaiev, 1900; Hilgard, 1892; Joffe, 1936; Whitney, 1982).

Related Studies In the study of Kumari et al (2011) entitled Fungal degradation of chlorpyrifos by Acremoniumsp. strain (GFRC-1) isolated from a laboratory-enriched red agricultural soil, an enrichment technique was used to isolate the mixed and the pure fungi from three soils. The enriched mixed fungal culture was capable of biodegrading chlorpyrifos (300 mg L -1) when cultivated in Czapek Dox medium. The identified pure fungal strain,Acremoniumsp., utilized chlorpyrifos as a source of carbon and nitrogen. The highest chlorpyrifos degradation (83.9%) byAcremoniumsp. strain GFRC-1 was found when cultivated in the nutrient medium with full nutrients. Desdiethyl chlorpyrifos was detected as a major biodegradation product of chlorpyrifos. The isolated fungal strain will be used for developing bioremediation strategy for chlorpyrifos-polluted soils. The study Enzymatic degradation of organophosphorus insecticidechlorpyrifosby fungus WZ-I by Xie et al (2005) stated that the degradation characteristics ofchlorpyrifosinsecticides was determined by the crude enzyme extracted from the isolated strain WZ-I ( Fusarium LK. ex Fx). The best separating condition and the degrading characteristic ofchlorpyrifoswere studied. Rate of degradation forchlorpyrifosby its intracellular enzyme, extracellular enzyme and cell fragment was 60.8%, 11.3% and 48%, respectively. The degrading enzyme was extracted after this fungus was incubated for 8 generations in the condition of noninducement, and its enzymic activity lost less, the results show that this enzyme is an intracellular and connatural enzyme.According to Fang et al with their study entitled Fungal degradation of chlorpyrifos byVerticilliumsp. DSP in pure cultures and its use in bioremediation of contaminated soil and pakchoi, the degradation characteristics of chlorpyrifos by an isolated fungal strainVerticilliumsp. DSP in pure cultures, soil, and on pakchoi (Brassica chinensisL.) were investigated. Degradation rate of chlorpyrifos in the mineral salts medium was proportional to the concentrations of chlorpyrifos ranging from 1 to 100mgl1. The rate of degradation for chlorpyrifos (1mgl1) in the mineral salts medium was 1.12 and 1.04 times faster at pH 7.0 than those at pHs 5.0 and 9.0, and the degradation at 35C was 1.15 and 1.12 times faster, respectively, than those at 15 and 20C. The addition of the fungal strain DSP into the contaminated soils was found to significantly increase the degradation of chlorpyrifos.Most of the study used the same method in knowing the degradation of chlorpyrifos. In the study of Harish et al (2013) Biodegradation of Organophosphate Pesticide by Soil Fungi, two fungal isolates isolated by enrichment technique were studied in Burkes liquid mineral medium supplemented with ethion andchloropyrifios at concentration of 100 PPM respectively, to quantify cellgrowth. The growth of Rhizopus nodosus and Trichoderma harznaium was monitored by observing the growth and turbidity of the media. The growth was maximum after 48 h. which was visualized with increase in their cell size and mass. The absence of turbidity was an indication of their growth utilizing pesticide as sole carbon source. It was observed that Chlorpyrifos and Ethion gradually declined over period of 21 days. After gathering relevant information regarding the currently being asserted topic, the researchers believe that the relevant information collected strongly support the feasibility of the proposed topic.

Chapter IIIMETHODOLOGYPresented in this chapter were the methods conducted in the aforementioned study which aimed to assess the mycoremediation potential on the pesticide chlorpyrifos of fungal isolates from mangrove areas in Sagay, Camarines Sur.Causal Model of the Study Degradation by fungi

Mangrove endophytes utilized in the degradation; amount of pesticide

Change in volume and color of pesticide

Rate of proliferation; fungal growth

Figure 1. Interactions among the variables in the experiment

In Figure 1, the type of fungi used in the degradation of the contaminant served as the independent variables. The fungal species were utilized in the degradation of chlorpyrifos and were isolated by the researchers as part of their methodology. Nine Erlenmeyer flasks that contained Lorsban, which was the brand name of the pesticide used, were used as treatment. The effects of these factors in the degradation of contaminants were measured by comparing if there was a significant change in the chemical contents of the pesticide. The rate of degradation of the fungi was the linking variable. The rate of proliferation of the fungal species was the extraneous variable because this could not be manipulated by the researchers. The natural life cycle of the fungi which followed the exponential growth curve could not be held back by the researchers but it could have affected the rate of degradation.

Materials and EquipmentThe materials and equipment used included the following: incubator, analytical balance, autoclave, graduated cylinder, beaker, cotton, aluminum foil, nine Erlenmeyer flasks, distilled water, thermometer, pH meter, shaking incubator, Lorsban, Potato Dextrose Agar. Potato Dextrose Agar was the medium used in the culturing of fungi and as substrate in the degradation process.

Procedure A. Isolation of MicrobesFungal strains were isolated from mangrove areas in Sagay, Camarines Sur. Repeated sub culturing was done to get pure cultures. Strains were maintained in the Potato Dextrose Agar.B. Sterilization of MaterialsNine Erlenmeyer flasks, two 100 ml beakers, stirring rod, and graduated cylinder were autoclaved before use.C. Preparation of MediaA mixture of 13.5 g of Potato Dextrose Agar and 500 ml distilled water were divided and put into nine Erlenmeyer flasks each containing 55 ml of the broth. D. Inoculation of Pure Cultures in the MediaThe pure cultures were mixed with the pesticide in nine Erlenmeyer flasks. The diluted pesticide was divided into nine treatments. Each treatment contained 5 ml, 10 ml, and 15 ml of the 10% active ingredient Chlorpyrifos. This part of the methodology was based mainly on the study of Harish published on March 2013.E. Qualitative AnalysisThe nine treatments were analyzed by qualitative evaluation after the experiment to observe the changes made. Mycelial growth, change in the fungis color, and change in the medias color were observed during the qualitative evaluation. The data that were gathered from the observation were recorded.F. Disinfection of MaterialsThe materials were all cleaned before and after use. The media were properly disposed along with the fungi and petri dishes. The laboratory and the equipment were also cleaned before and after the experimentation.

Schematic Representation of the Methodology Isolation of mangrove fungal endophytes

Identification of Microbes

Extraction of Chlorpyrifos

Inoculation of Microbes on the Pesticide

Qualitative analysis

Chapter IVRESULTS AND DISCUSSIONThree different fungal endophytes obtained from mangrove areas in Sagay, Camarines Sur were each subjected to three different volumes of pesticide: 5 ml, 10 ml, and 15 ml to assess their mycoremediation potential on the pesticide chlorpyrifos. Mycoremediation is an innovative biotechnology that uses conditioned fungi and fungal mycelium applied to surface soils to remove and degrade contaminants (Aston, L.M., Cullinan, V.I., Thomas, S.A., Woodruff, D.L., 2009). Qualitative analysis, which is comprised of observing the mycelial growth, change in the fungis color, and change in the medias color, was done to assess the experiment.The tables below show the data gathered through the experiment conducted.Table 1. Corresponding volume of diluted pesticide (ml) in each fungusVolume of diluted pesticide (ml)

Fungus 151015

Fungus 251015

Fungus 351015

Table 2. Observations in the three fungi using qualitative analysisMycelial GrowthChange in Fungis ColorChange in Medias Color

Fungus 1NoneNoneNoneNoneNoneNoneNoneNoneNone

Fungus 2NoneNoneNoneNoneNoneNoneNoneNoneNone

Fungus 3NoneNoneNoneNoneNoneNoneNoneNoneNone

There are no changes in the color of the fungi and media nor is there any presence of mycelial growth. This shows that none of the three fungi obtained from Sagay, Camarines Sur has a mycoremediation potential.

Lorsban: Pesticide used which contains chlorpyrifos as the main active ingredient

Obtaining of the pure cultures

Fungi in different volumes

Chapter VCONCLUSIONSoil contaminated with pesticide decreases the soils fertility and also affects the organisms living there. This study then aimed to assess the mycoremediation potential on the pesticide chlorpyrifos of fungal isolates from mangrove areas in Sagay, Camarines Sur.Fungal strains were first isolated from mangrove areas in Sagay, Camarines Sur and repeated sub culturing was done to get pure cultures. Strains were maintained in Petri dishes containing Potato Dextrose Agar. A mixture of 13.5 g of Potato Dextrose Agar and 500 ml distilled water were divided and put into nine Erlenmeyer flasks each containing 55 ml of the broth. The pure cultures were mixed with the pesticide in nine Erlenmeyer flasks. The diluted pesticide was divided into nine treatments. Each treatment contained 5 ml, 10 ml, and 15 ml of the 10% active ingredient Chlorpyrifos. Using qualitative analysis, which is comprised of observing for changes in mycelial growth, fungis color, and medias color, data were gathered and recorded.From the qualitative evaluation conducted, it was observed that there was neither any change in the color of the fungi and media, nor in the mycelial growth. This then resulted to an acceptance of the null hypotheses. Thus, it is concluded that the three fungal isolates obtained from Sagay, Camarines Sur have no mycoremediation potential on the pesticide chlorpyrifos.BIBLIOGRAPHYBaker, L. (2002, November 25). How mushrooms will save the world.Salon.com. RetrievedFebruary23, 2013, from http://wordspy.com/words/mycoremediation.aspDefinition of Soil, Approaches of Soil Study. (n.d.).My Agriculture information bank. Retrieved from http://www.agriinfo.in/default.aspx?page=topic&superid=4&topicid=246Enhanced solubilization of rock phosphate by Penicillium bilaiae in pH-buffered solution culture. (2006, November 1). RetrievedFebruary28, 2013, from http://www.highbeam.com/doc/1G1-164595964.htmlfacts about Penicillium - Evi. (n.d.).Evi | Ask me anything. Retrieved from http://www.evi.com/q/facts_about__penicilliumFriesen, T. J., Holloway, G., Hill, G. A., & Pugsley, T. S. (2007, January 18). Biocontrol Science and Technology. RetrievedFebruary28, 2013, from http://www.tandfonline.com/doi/abs/10.1080/09583150500258263?journalCode=cbst20Manimegalai, Selvaraj, & Ambikapathy (2011). Studies on Isolation and Identification of VAM Fungi in Solanum viarum Dunal ofMedicinal Plants.Archives homepage. Retrieved fromhttp://archives.evergreen.edu/masterstheses/Accession86-10MES/Rogers_TMES_2012.pdfMycoremediation : The sustainability wiki. (n.d.).Appropedia. Retrieved from http://www.appropedia.org/MycoremediationObire, & Putheti (1996). FUNGI IN BIOREMEDIATION OF OIL POLLUTED ENVIRONMENTS.Year of Energy 2009. Retrieved fromhttp://energy.sigmaxi.org/wp-content/uploads/2009/09/obire_putheti_bioremediation.pdfParkinsons (n.d.). A method for isolating fungi from soil microhabitats - Springer.Home - Springer. Retrieved fromhttp://link.springer.com/article/10.1007%2FBF01394646Rogers, T. (2012).Archives homepage. Retrieved from http://archives.evergreen.edu/masterstheses/Accession86-10MES/Rogers_TMES_2012.pdfSoil Management. (n.d.).Welcome to College of Tropical Agriculture and Human Resources, University of Hawaii. Retrieved from http://www.ctahr.hawaii.edu/mauisoil/a_comp.aspxSridhar, K. R., ed.Frontiers in Fungal Ecology, Diversity and Metabolites. I. K. International Publishing House Pvt. Ltd., n.d. Web. 28June2013. Sykes, C. (2002, February 26). Magical Mushrooms: Mycoremediation. RetrievedFebruary23, 2013, from http://www.frost.com/sublib/display-market-insight-top.do?id=SSAI-57QV3KThomas (n.d.).People ForPuget Sound: Our vision is a clean and healthy Puget Sound. People For Puget Sound. Retrieved1999, fromhttp://pugetsound.org/science/puget-sound-science/reports/mycoremediation-pilot-project-near-the-dungeness-riverWord Spy - mycoremediation. (n.d.).Word Spy. Retrieved from http://wordspy.com/words/mycoremediation.aspPotera,C. (2007). Pesticides Disrupt Nitrogen Fixation.Environmental Health Perspectives,115(12). Retrieved from http://www.questia.com/library/journal/1G1-173712890/pesticides-disrupt-nitrogen-fixation#articleDetails

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