Universidad del Turabo

Efficiency of White-Rot Fungi on the Biodegradation of Hydrocarbon

Contaminated Soil

By

Rita Ellen Koett Rosas,

B.S., Chemistry, University of Puerto Rico

M.M.C., Quality Management, Polytechnic University of PR

Dissertation

Submitted to the School of Science and Technology

in partial fulfillment of the requirements for

the degree of Doctor of Philosophy

in Environmental Science

(Management)

Gurabo, Puerto Rico

May, 2014

ii

Universidad del Turabo

A dissertation submitted in partial fulfillment of

the requirements for the degree of

Doctor of Philosophy

___April 03, 2014___

date of defense

Efficiency of White-Rot Fungi on the Biodegradation of Hydrocarbon

Contaminated Soil

Rita Ellen Koett Rosas

Approved

Betsaida Torres García, PhD Carlos J. Olivo Delgado, EdD

Research Advisor Supervisor/Advisor

Angel L. Rivera, PhD Ramón Martínez Pacheco, PhD

Member Member

Teresa Lipsett Ruiz, PhD

Dean

© Copyright 2014

Rita Ellen Koett Rosas. All Rights Reserved

iii

Acknowledgements

The journey may become easier when you are with people who are supportive. I have

experienced the same kind of atmosphere during the completion of my dissertation work. It is a

pleasant aspect to have the privilege to evince a word of thanks to everyone who made this

journey possible in a smooth manner.

I would like to express my deep, sincere and loving thanks to my advisor Dr.

Betsaida Torres. Her valuable advice and guidance towards the writing skills and overall

completion of this investigation have been very helpful for this study. Her understanding and

personal guidance have provided a good basis for the present investigation. Especially the strict

and extensive comments and many discussions and the interactions with Dr. Torres had a direct

impact on the final form and quality of this dissertation.

I owe my sincere thanks to Dr. Carlos Olivo as the supervisor advisor he has provided

valuable advice and guidance towards the writing skills and overall completion of this

investigation. His extensive discussions around my work have been very helpful for this study.

I owe my deepest thanks to Dr. Paul Bayman, Head of Department of Biology from the

University of Puerto Rico, for his support during my investigation while storing the fungus at his

laboratory and preparing the subcultures to maintain the fungus during my investigation.

I warmly thank Maritza Vázquez and Altol Labs for her kind support and guidance and

allowing to use the lab to perform the analytical testing section. I wish to thank all the research

scholars and lab assistants for their help.

I have regard and wish to extend my warmest thanks to my colleagues Jorge Hernandez,

Iris Cosme, Ivette Rivera, Adalberto Bosque for their encouragement and understanding.

I feel a deep sense of gratitude for my family who formed part of my vision and gave me

support along this path providing a sense of pride for the good things that really matter in life.

iv

Above all, I’m grateful to almighty God for blessing me to complete this work

successfully.

Rita Ellen Koett Rosas

v

Vitae

Rita Ellen Koett Rosas, born in New York, USA, came to Puerto Rico at an early age and

established on the east coast, Fajardo, with her parents. She completed university studies at the

University of Puerto Rico, Río Piedras campus obtaining a bachelor degree in Science in 1980

with major in chemistry. After almost twenty years working for different pharmaceuticals and

medical devices industries she decided to pursue a master degree in Manufacturing

Competitiveness with a major in quality management from Polytechnic University of PR in 2005.

Actually in pursue of a doctoral degree in Environmental Science from the Universidad del

Turabo. Since 1978 she has been working for different pharmaceutical industries, medical

devices and consumer products companies thus occupying managerial positions within in the

quality department of each company. As of August 2012, and during the past four semesters,

have been offering courses in the areas of Good Manufacturing Practices (GMPs), Validations

and Team Work to students registered to complete an Associate Degree in Biotechnology at the

Universidad del Turabo, Gurabo campus.

vi

Table of Contents

Page

List of Tables ........................................................................................................................... viii

List of Figures .......................................................................................................................... ix

List of Appendix ...................................................................................................................... x

Abstract .................................................................................................................................... xi

Resumen en español ................................................................................................................. xii

Chapter One. Introduction ...................................................................................................... 1

Section I. Bioremediation Executed ........................................................................... 1

Section II. Waste Disposal .......................................................................................... 3

Section III. Contaminants ........................................................................................... 4

Section IV. Soil Microorganisms ................................................................................ 10

Section V. Lignin ........................................................................................................ 13

Section VI. Fungi Bioremediation Techniques ........................................................... 17

Section VII. Intellectual Merit .................................................................................... 24

Chapter Two. Literature Review ............................................................................................. 27

Section I. Waste Production ........................................................................................ 27

Section II. Bioremediation .......................................................................................... 29

Section III. Microorganisms in Bioremediation ......................................................... 32

Section IV. Soil in Bioremediation ............................................................................. 33

Section V. Fungi in Bioremediation ........................................................................... 37

Chapter Three. Methods .......................................................................................................... 44

Section I. Introduction ................................................................................................ 44

Section II. Soil Profile ................................................................................................ 45

vii

Section III. Fungi ........................................................................................................ 60

Section IV. Stock TPH Standard ................................................................................ 62

Section V. TPH working standard solution ............................................................... 62

Section VI. Preparation of soil/water/TPH working standard solution with five different

fungi ............................................................................................................................ 62

Section VII. Soil/water/TPH working standard solution with most effective degrading

fungi ............................................................................................................................ 65

Section VIII. Selection of optimum conditions .......................................................... 66

Section IX. Statistical analysis .................................................................................... 66

Chapter Four. Discussion ........................................................................................................ 68

Section I. Introduction ................................................................................................ 68

Section II. Results ....................................................................................................... 69

Chapter Five. Conclusion and Recommendations .................................................................. 93

Section I. Introduction ................................................................................................ 93

Section II. Conclusion ................................................................................................. 93

Section III. Recommendations .................................................................................... 95

Literature Cited… .................................................................................................................... 97

Appendix One. Abbreviations ................................................................................................ 118

viii

List of Tables

Page

Table 1. LD50 values of some representative PAHs ............................................................ 8

Table 2. Environmental factors and optimum condition for microbial activity

for soil bioremediation ......................................................................................... 35

Table 3. Sample size for TKN determination ..................................................................... 48

Table 4. Atomic absorption concentration ranges with direct aspiration atomic absorption.

.............................................................................................................................. 56

Table 5. Soil Profile, Coto clay, non-sterile ....................................................................... 70

Table 6. Soil Profile, Coto clay, sterile .............................................................................. 71

Table 7. TPH degradation rate for WRF Phanerochaete chrysosporium

BKM-1767 isolate ................................................................................................ 72

Table 8. TPH degradation rate for WRF Pleurotus djamor TU-7659 isolate .................... 74

Table 9. TPH degradation rate for WRF Pleurotus djamor native strain

PR-1562 isolate .................................................................................................... 74

Table 10. TPH degradation rate for WRF Trametes elegans FP-105038 isolate ................. 75

Table 11. TPH degradation rate for WRF Trametes elegans native strain

PR-1151 isolate .................................................................................................... 76

Table 12. TPH degradation rate for most effective selected WRF Trametes

elegans native strain PR-1151 isolate .................................................................. 77

Table 13. Factor and Model Summary for the five fungi tested ........................................... 81

Table 14. Analysis of Variance for the five fungi tested ...................................................... 81

Table 15. Factor and Model Summary for the three fungi tested ......................................... 86

Table 16. Analysis of Variance for the three fungi tested .................................................... 86

ix

List of Figures

page

Figure 1. Pleurotus djamor ............................................................................................... 21

Figure 2. Phanerochaete chrysosporium .......................................................................... 23

Figure 3. Trametes elegans ............................................................................................... 24

Figure 4. Summary of different bioremediation strategies for typical hazardous wastes . 31

Figure 5. Timeframe of Trametes elegans native strain PR-1151 .................................... 78

Figure 6. Fungi relationship ............................................................................................. 80

Figure 7. Timeframe relationship ..................................................................................... 81

Figure 8. Fungi interaction of the same species ............................................................... 83

Figure 9. Time interaction of native isolates .................................................................... 83

Figure 10. Normal probability Plot for five fungi Two-Way ANOVA test ....................... 84

Figure 11. Timeframe relationship ..................................................................................... 85

Figure 12. Fungi relationship ............................................................................................. 86

Figure 13. Time interaction of native isolate ...................................................................... 88

Figure 14. Fungi interaction of native isolate ..................................................................... 88

Figure 15. Normal probability Plot for three fungi Two-Way ANOVA test ..................... 89

x

List of Appendix

Page

Appendix One. Abbreviations .......................................................................................... 118

xi

Abstract

Rita Ellen Koett Rosas (PhD, Environmental Science)

Efficiency of White-Rot Fungi on the biodegradation of hydrocarbon contaminated soil

(May/2014)

Abstract of a doctoral dissertation at Universidad del Turabo.

Dissertation mentor: Betsaida Torres, PhD.

No. of pages in text 119

The pollution of soil and water by industrial effluents that contain oil-waste is a problem

in our Island. This problem requires our full attention since the waste treatment has a negative

impact on health, natural terrestrial ecosystem and natural aquatic environment. Solutions that

might be used to solve this problem include, not limited to, chemical treatments, degradation of

wastes, physico-chemical treatment to wastewater, and, in some cases, even bioremediation.

The white-rot fungi (WRF), among which are Phanerochaete chrysosporium, Pleurotus

djamor, and Trametes elegans, have been investigated by several scientists due to their capability

for the degradation of hazardous wastes. They are able to mineralize a wide variety of

chlorinated organics, polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons

(PAHs), total petroleum hydrocarbons (TPHs), pesticides and others.

In this investigation we will expose these WRF Phanerochaete chrysosporium, Pleurotus

djamor, and Trametes elegans and their native species to a contaminated soil and determine the

most effective WRF in soil remediation. Effects of different inoculation times of the most

effective WRF found will be measured. Results will further provide insights into the

development of contaminated soil biodegradation processes for food safety and agriculture

sustainability.

xii

Resumen

Rita Ellen Koett Rosas (PhD, Environmental Science)

Efficiency of White-Rot Fungi on the biodegradation of hydrocarbon contaminated soil

(May/2014)

Resumen de disertación doctoral en Universidad del Turabo

Disertación fue supervisada por la Dra. Betsaida Torres

Total de paginas 119

La contaminación de suelos y aguas por efluentes industriales que contienen aceite de los

residuos es un problema en nuestra isla. Este problema requiere de toda nuestra atención ya que

el tratamiento de los residuos tiene un impacto negativo en la salud, los ecosistemas terrestres

naturales y el medio ambiente acuático natural. Soluciones que podrían ser utilizados para

resolver este problema incluyen, sin limitarse a, tratamientos químicos, la degradación de los

desechos, tratamiento físico-químico de aguas residuales, y, en algunos casos, incluso

biorremediación.

Los hongos de podredumbre blanca (WRF), entre los que se Phanerochaete

chrysosporium, Pleurotus djamor y Trametes elegans, han sido investigados por varios científicos

debido a su capacidad para la degradación de los desechos peligrosos. Son capaces de

mineralizar una amplia variedad de compuestos orgánicos clorados, bifenilos policlorados (BPC),

hidrocarburos aromáticos policíclicos (HAP), hidrocarburos totales de petróleo (HTP), pesticidas

y otros.

En esta investigación vamos a exponer estos chrysosporium WRF Phanerochaete,

Pleurotus djamor y Trametes elegans y sus especies nativas de un suelo contaminado y

determinar el WRF más eficaz en la recuperación de suelos. Efectos de diferentes tiempos de

inoculación del WRF más efectivo encontrado se medirán. Los resultados serán proporcionar

xiii

más conocimientos sobre el desarrollo de los procesos de biodegradación de suelos contaminados

para la seguridad alimentaria y la agricultura sostenibilidad.

1

Chapter One

Introduction

Section I. Bioremediation Executed

Bioremediation is a technique used to remediate a contaminated area, either soil or water

by means of use of biological processes. It can be used where hazardous waste products are

degraded or detoxified by microorganisms or microbial processes (Soetan 2011); it is very

popular and publicly accepted to remediate contaminated soil due to its cost-effectiveness (Davis

et al. 1993) as it brings along environmental safety aspects. This technique requires the presence

of a contaminant, an electron acceptor and the presence of indigenous and/or augmented

microorganisms capable of degrading a specific contaminant. Treating xenobiotic- contaminated

soils, for instance, requires the identification and implementation of physico-chemical and/or

nutritional conditions to favor optimum growth, and xenobiotic-degrading activities of indigenous

(biostimulation) or inoculated (bioaugmentation) xenobiotic-degrading microbes (Lamar et al.

1999). Augmenting the contaminated site by using an appropriate microorganism inoculum is a

technique that can enhance the biodegradation process of hydrocarbons (Kristanti et al. 2011).

This approach allows for the selection of microbes based on additional physiological,

biochemical, or ecological characteristics that may confer bioremediation performance (Lamar et

al. 1999). Whereas, using indigenous microorganism consortium ensures organisms with higher

tolerance to hydrocarbon toxicity to withstand variations on environmental conditions (Kristanti

et al. 2011). It involves the selection of microbes based primarily on their xenobiotic-degrading

activities (Lamar et al. 1999). There are many natural populations (consortia) and

microorganisms that can perform different biodegradation activities known for several decades

(Soetan 2011) including bacteria, yeast, fungi or algae.

2

Whole microorganisms, either naturally occurring or introduced, or isolated enzymes

which degrade persistent contaminants into none or less toxic compounds in combination with

complementary physical, chemical or mechanical processes, can improve the reliability and

effectiveness of detoxification (Alcalde et al. 2006). These organisms can detoxify the

environment, consuming and breaking down pollutants until their mineralization, a complete

transformation into CO2 and H2O (Jurado et al. 2011). Using their own enzymatic pathways

(Ulfig et al. 2001), conversion into harmless byproducts such as simpler organic compounds, or

immobilizing them into soil humic substances (Jurado et al. 2011) can help avoid problems of

contaminants in the soil.

Microbial biodegradation factors affecting success of the process and enzymatic reaction

rate are nutrient availability, moisture content, pH, and temperature of the soil matrix.

Bioremediation can use any kind of microorganism to clean-up hazardous waste sites as it is the

safest, rapid, least disruptive, ecologically responsible method, and the most cost-effective

treatment (Solomon et al. 1996; and Alcalde et al. 2006). In the bioremediation process, a

contaminated site is exposed to microorganisms which produce enzymes that can break down the

toxins, leaving behind harmless metabolic byproducts such as CO2 and chlorides (Solomon et al.

1996).

More than 1,000 different species of bacteria and fungi can be used to clean-up various

forms of pollution. These microorganisms contain enzymes involved in lignin-metabolism.

Laccase (Lac), lignin peroxidase (LiP) and manganese peroxidase (MnP) from white-rot fungi

(WRF) are among the most studied enzymes in bioremediation, where Lac is involved in

detoxification of phenols, triclorophenols, and PAHs (polycyclic aromatic hydrocarbons)

distributed in terrestrial and aquatic environments (Alcalde et al. 2006). These enzymes,

peroxidases and laccase, are the key lignin degrading enzymes with great potential in industrial

applications (Sumit and Vimala 2012). Upreti and Srivastava (2003) propose two possible

3

approaches to reduce the “vices” of such polymeric materials: develop biodegradable commodity

plastic or identify potential microorganisms and a develop protocol to effectively biodegrade the

polymeric materials. This investigation pursues the latter approach.

Section II. Waste Disposal

Presently, there is increasing knowledge and awareness of the health risks and

environmental impact associated with the waste handling process. It is our responsibility to take

action in order to reduce this impact for the benefit of our health and well being of future

generations.

Waste is classified in three basic groups: medical, infectious and domestic waste.

Medical waste is accumulated by the result of patient’s diagnosis, treatment or immunization of

human beings as stated by Altin et al. (2003); whereas the infectious waste is waste that has been

in contact with a patient diagnosed with an infectious disease and could eventually produce an

infectious disease as well (Altin et al. 2003). The United States Environmental Protection

Agency (USEPA 1989B) defines domestic waste, as garbage or trash consisting of everyday

items as product packaging, bottles, food scraps, and newspaper, among others.

The USEPA (1989B) states “hospital waste”, also known as solid waste, as any waste,

either biological or non-biological, that has been discarded and not considered for further use. It

also includes, and not limited to, hazardous chemicals, infectious, pathological, radioactive

wastes, stock cultures, blood and blood products, pharmaceutical wastes, batteries, plastics,

disposable needles, syringes, scalpels, and sharp items (Altin et al. 2003; and Oyeleke et al.

2008). The waste of interest in this investigation is the one caused by petroleum spillage and/or

run-off oil caused during rainfall.

It was until the public awareness of blood borne diseases raised in the early 1980s, when

the HIV (Human Immunodeficiency virus) and HBV (Hepatitis B virus) caught our attention, that

serious concern arised on proper waste disposal directly in landfills. For example, the amount of

4

waste generated in US (United States) hospitals is approximately 6,670 tons per day, or about 1%

of the 158 million tons of MSW (Municipal Solid Waste) produced annually (Rutala and Mayhall

1992). Today, it is required to pre-treat hospital waste avoiding direct contact with any MSW in

the landfill. Treatments for these types of wastes include autoclaves, retorts, microwave/chemical

disinfection systems, burial in covered pits, and encapsulation by immobilizing compounds (Díaz

et al. 2005). Other treatments considered are point-of-use needle destruction technology and

mechanical compaction/reduction system. However, all of this “pre-treated” waste will still end

in the landfill, mixed with MSW, and producing an inadequate final disposal of waste resulting in

negative impact on public health, the environment and the ecosystems (Díaz et al. 2005).

Final waste disposal sites, known as landfills, are dumps where the MSW is deposited

until it reaches a height that for esthetics or technical reasons is considered to be the desirable

maximum and, upon closure, some soil is deposited on top (Themelis and Ulloa 2007). This

accumulation of waste is a fundamental step in any current waste management strategy causing

water and soil pollution, generating a large amount of leachates into our ecosystems (Ellouze et

al. 2007) as does occur with run-off of fuel during rainfall weather conditions.

Section III. Contaminants

Soil contamination is defined as the presence on or over any land of any liquid or solid

waste of such quantity, nature or duration or under such conditions that can affect or could affect

the human health or well being, animal or vegetation, or even may interfere with the freedom of

life or the property (Oyeleke et al. 2008). Improper handling of non-hazardous solid waste can

contaminate the soil with liquid leachate depending on the type of waste, soil conditions and

water content; as a hazardous solid waste like fuel spillage due to run-off during rainfall, if

improperly handled, can represent damage irreversible to the soil for future agricultural use

(Oyeleke et al. 2008).

5

Examples of main soil contaminants are insecticides, paints, battery waste, hydraulic oil,

rupture of underground storage tanks, oil and fuel dumping or spillage, and even pathogenic

waste, as part of the leachate. These pollutants can contaminate the soil and water systems with

heavy metals that in turn produce ecological impacts on water bodies and soil sites which lead to

an increased nutrient load, especially if they are essential metals (Oyeleke et al. 2008). Pollutants

of these categories can broadly be classified as metal, nonmetal, metalloid, inorganic, and organic

compounds (Brar et al. 2006). The organic contaminants comprise aliphatic, alicyclic, aromatic,

and PAHs, halogenated and non-halogenated compounds, pesticides, and explosives; while

inorganic pollutants can include metals such as Ag, Al, As, Be, Cd, Cr, Cu, Hg, Fe, Ni, Pb, Sb,

Se, Zn, and radioactive elements and their derivatives (Brar et al. 2006). Population increase and

industrial growth negatively impact on the production of domestic, municipal and industrial

wastes, affecting its disposal to the landfill and water bodies.

Leachates may contain heavy loads of organic chemicals and ammonia nitrogen causing a

major source of contamination to surface water, fauna and flora (Ellouze et al. 2007), overall

surrounding soil and ground water (Guo et al. 2009). The leachate can produce excess of nutrients

and phosphates, consequently causing eutrophication, where a body of water can develop a dense

growth of algae (Asamundo et al. 2005). As the dense growth of algae and other organisms are

formed, their eventual decay depletes the shallow waters of oxygen causing the death of aquatic

life and affecting the physico-chemical properties of the soil and water systems such as pH, BOD

(Biochemical Oxygen Demand) and COD (Chemical Oxygen Demand).

Overall, the principal concern associated with hydrocarbon waste contaminants in soil

and water systems is the toxicity leading to health risk to humans (Brar et al. 2006). For example,

vegetable consumption grown in soil with trace heavy metals will be hazardous in the human

food chain producing an adverse effect on human and animal health, causing defects or even

death (Soen 2011). As this pollution affects the microorganisms living in these ecosystems, it

http://dictionary.reference.com/browse/algae

6

creates an overall disparity in the Earth. Endocrine disruptors, for instance, PAHs, PAEs

(phthalates), and polychlorinated biphenyls (PCBs), are detected in landfill leachates (Lü et al.

2008). The degraded PCBs congeners with ortho, meta, and/or para substitutions may contain up

to 209 possible combinations with a number of chlorine substitutions in the biphenyl nucleus

varying from 1 to 10 (Federici et al. 2012), chemically and physically stable, and resistant to

degradation in the natural environment (Borazjani et al. 2005). The PCBs lipophilic properties

make them slightly soluble in water, readily soluble in oils, causing bioaccumulation in the fatty

tissues of fish, birds, animals, and humans (Borazjani et al. 2005). Therefore, the exposure to

PCBs can be through ingestion of food or drinking water known to be contaminated with this

product. Meanwhile, pollution as transportation, industrial activities, incineration of refuse and

waste burning (Cerniglia 1997), gasification, and even, plastic waste incineration, are some of the

sources of PAHs.

These chemicals and their derivatives are pollutants produced via natural and

anthropogenic sources (Bishnoi et al. 2008), found in most terrestrial ecosystems that arise from

industrial operations (Eibes et al. 2005), product of organic materials occurring from natural

combustion as forest fires (Levin et al. 2003), and volcanic eruptions (Romero et al. 2010). This

is a reason to confirm why USEPA has classified PAHs among priority pollutants (Bishnoi et al.

2008; and Pozdnyakova 2012). The resultant products contain hydrogen and carbon, well known

as hydrocarbons, which are a health concern for their toxic, mutagenic, and carcinogenic

properties (Martens and Frankenberger, Jr. 1995; Bezalel et al. 1997; Daane et al. 2001; and

Levin et al. 2003).

Petroleum hydrocarbons (PHCs) are common site contaminants, not generally regulated

as hazardous wastes. During environmental studies the family of PHCs is known as TPHs. One

of the most common contaminants found in soil, known as xenobiotic chemical constituents, are

categorized into four fractions of complex mixtures as saturates (aliphatics), aromatics, resins and

7

asphaltenes (Ogbo and Okhuoya 2008) plus constituents that may contain N, S or O in addition to

H and C (Robertson et al. 2007). These chemicals are purposely released into the environment

via the dumping of petroleum waste or wastewater or as a result of accidents during the

production, transport and use of chemicals (Jayasinghe et al. 2008). TPHs are complex mixtures

of hundreds of hydrocarbon compounds, ranging from light, volatile, short-branched organic

compounds (aliphatic), which comprise the light fraction of refined oil, to heavy, long-chained,

branched compounds. The aliphatic compounds primary source of entry into the environment is

via emissions from combustion processes, spillage of petroleum products (Rockne and Reddy

2003) by surface spills or leaks from pipelines, flow lines, delivery line failures (Olusola and

Ejiro 2011), storage tanks (Robertson et al. 2007) and run-off waters. The chemical composition

of the product can be affected by weathering and/or biological modification upon release to the

environment. Therefore, the contamination changes the physico-chemical and biological

properties of the soil being toxic to some soil microorganisms, plants (Ogbo and Okhuoya 2008),

animals and, even, people (Mohsenzadeh et al. 2012). Hydrocarbons have been found to bio-

accumulate in aquatic organisms constituting a significant health risk for people living in

industrialized areas of the world (Cerniglia 1992). To obtain an effective TPHs aerobic

biodegradation the soil needs to have sufficient oxygen and enough soil water to provide a habitat

for adequate populations of active microorganisms. If oxygen is present, these organisms will

generally consume available TPHs. Furthermore, aerobic biodegradation of petroleum

compounds can occur relatively quickly, with degradation half-lives as short as hours or days

under some conditions (DeVaull, 2007).

Table 1. LD50 values of some representative PAHs (adapted from Bamforth and Singleton 2005).

Material Number of

carbon rings

LD50 value

(mgkg-1

)

Test subject Exposure route

8

Naphthalene 2 533-710 Male/female

mice respectively

Oral

Phenanthrene 3 750 Mice Oral

Anthracene 3 >430 Mice Intraperitoneal

Fluoranthene 4 100 Mice Intravenous

Pyrene 4 514 Mice Intraperitoneal

Benzo[a]pyrene 5 232 Mice Intraperitoneal

Generally, toxicity increases with an increase in number of benzene rings, but data should be examined using careful

consideration of the exposure route, etc (data taken from the Risk Assessment Information System (RAIS)

http://risk.lsd.oml.gov).

Table 1 depicts chemical compounds that are composed of fused, aromatic rings whose

biochemical persistence arises from dense clouds of π-electrons on both sides of the ring

structures, making them resistant to nucleophilic attack (Johnsen et al. 2005). Materials with a 5-

ring hydrocarbon structure known as BaP (Benzo[a]pyrene), a petroleum, coal tar and fuel-oil

component, are very recalcitrant and resistant to microbial degradation formed upon the pyrolisis

of organics (Romero et al. 2010). Even though PAHs are persistent, due to their high

hydrophobicity, low water solubility (Bishnoi et al. 2008; and Okparanma et al. 2011), and high

solid-water distribution ratios, they are easily adsorbed onto organic matter as soils and sediments

(Eibes et al. 2005), standing against their ready microbial utilization, and promotion of their

accumulation in the solid phases of the terrestrial environment (Johnsen et al. 2005), a great

variety of microorganisms (bacteria and fungi) can degrade them.

There also exist xenobiotics compounds in waste that are man-made synthetic organic

chemicals very difficult to degrade, foreign to the biosphere, as they accumulate in the

environment and cause harmful, deleterious effects on the living system (Sharma 2010).

Xenobiotic compounds are recalcitrant toxic pesticides, hydrocarbon pollutants of the soil; the

main sources of contamination are the spills and leaks of petroleum products (George-Okafor et

http://risk.lsd.oml.gov/

9

al. 2009). These halogenated aliphatic and aromatic compounds like DDT (1,1,1-trichloro-2,2-

bis(4-chlorophenyl) ethane), BHC (benzene hexachloride), PCBs, polyethylene, polystyrene and

PVC (polyvinyl chloride) are not easily biodegradable due to the extensive branching of the

molecule or introduction of halogen, nitro or sulphonyl group (Sharma 2010). The same occurs

with the gamma isomer of hexachlorocyclohexane (γ-HCH), commonly known as lindane, an

organochlorine compound widely used in the past in the agricultural sector for its insecticidal

properties; however, its stability and hydrophobicity make them persistent pollutants and their

toxicity also creates environmental and human exposure concerns (Rigas et al. 2008). Another

xenobiotic compound is known as atrazine, 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-

triazine (ATZ), a non-selective herbicide for vegetation control in non-crop land, and one of the

most widely used herbicides in the world for the control of annual grasses. It has led to the

contamination of terrestrial ecosystems, and can be measured in ground and surface water in

many countries, constituting a public health and environmental concern of pesticide

contamination (Mougin et al. 1994). Finally, fenthion’s degradation products, particularly the

sulphone and sulphoxide derivatives, can be persistent depending on environmental conditions

when applied to an affected area. This organophosphorous pesticide was used to control bird

pests in agricultural systems, (Zacchi et al. 2000).

Waste leachate may also contain BTEX compounds (benzene, toluene, ethylbenzene, and

o-, m- and p-xylenes), an important family of organic pollutants, components of gasoline and

aviation fuels. They and their nitro derivatives are widely used in industrial syntheses, considered

carcinogenic and neurotoxic, and classified as priority pollutants (Levin et al. 2003). Another

contaminant released into the environment, 2,4,6-Trinitritoluene (TNT), predominates in soil and

groundwater at many sites of TNT production. It is a recalcitrant, mutagenic compound shown to

be toxic to gram-positive bacteria, yeasts, and fungi as well as green algae (Michels and

Gottschalk 1994).

10

It is essential to contain or mitigate these organic and inorganic contaminants so as to

prevent them from contaminating surface and groundwater by dissolution or dispersion (Brar et

al. 2006). Control methods which are less damaging to the environment are urgently sought, with

remediation of contaminated sites being a priority (Zacchi et al. 2000).

Section IV. Soil Microorganisms

USEPA has indicated that soil pollution is a threat to the environment, to food safety and

to a sustainable agriculture as there is a direct link between the health of the ecological

environment and the health of humanity (Bonaventura and Johnson 1997). Nevertheless,

pollution prevention and environmental remediation are interwoven into strategies proposed for

sustaining human and environmental health; it involves enhanced degradation of toxic

compounds by transforming them into innocuous substances (Brar et al. 2006), the primary

stimulant in bioremediation of contaminated environments (Bonaventura and Johnson 1997).

The variety of contaminants, their synergistic and antagonistic effects, and their physico-

chemical properties make them serious toxicants, which may survive different treatments. Soils

possess great diversity of microorganisms in terms of their inherent physical, chemical and

biological characteristics (Jones et al. 2009). These ecosystems retain a set of intrinsic biological

functions independent from location as the major input of organic matter is plant residues,

composed mainly of the same primary building blocks (cellulose, hemicellulose, protein, lignin

and lipids), leading to the formation of a soil organic matter of similar chemical structure (Jones

et al. 2009).

In the majority of the cases, soils are classified according to the primary factors that

regulate their formation (time, climate, parent material, topography and vegetation), thus

implying they behave differently from one another (Jones et al. 2009). Even though they possess

different sets of organisms, all soil microbial communities might be expected to retain a core set

of functions such as protein and cellulose degradation. The microorganisms require carbon, an

11

energy source, to sustain the metabolic functions, growth and reproduction (Sen and Chakrabarti

2009), and to digest the contaminated material as nutrient, hazard to humans, converting them

into non-hazardous products as CO2 and water. Biological decomposition techniques include the

use of natural microorganisms such as bacteria, yeast, fungi and algae to degrade the toxic

material into a less toxic one. The successful use of microbial inocula in soils requires the

microorganism to have contact with the contaminant as physical adsorption to soil particles or

filtration through the soil small pores limits the transport of organisms (Márquez-Rocha et al.

2001).

For instance, both aerobic and anaerobic bacteria have been shown to degrade BTEX

compounds, but most of these studies on bacterial degradation of BTEX have used microbial

consortia and no native strain of bacterium is known to degrade all the components efficiently as

o-xylene, particularly, has been shown to be recalcitrant to bacterial degradation (Levin et al.

2003). Therefore, the biological treatment using WRF and their enzymes, prove to be a success

in the elimination of non-stabilized organic matter and toxic compounds in the range of

xenobiotic environmental pollutants (Ellouze et al. 2007). Many xenobiotics chemical

constituents (TPHs) are structurally analogous to compounds naturally found in the soil

environment (plant material, fungal and root exudates and allelophatic chemicals) and appear to

be biodegraded through the same biochemical pathways (Robertson et al. 2007). There is

evidence that bioremediation of hydrocarbon contaminated soil by the use of microorganisms is

an efficient, inexpensive, adaptable, and an environmentally sound treatment method

(Adenipekun et al. 2011). These treatments are a major source of renewable organic matter such

as lignocelluloses since its chemical properties allows it to be a substrate of great

biotechnological value, consisting of lignin, hemicellulose and cellulose, found in woody plants

(Jafari et al. 2007), dead plant materials, and is the most abundant biomass on Earth (Kalmiş et al.

2008).

12

Indigenous microorganisms are used as a bioremediation strategy at polluted sites to

degrade PAHs by improving their in-situ degradation capability through the optimization of

temperature, pH, water-content, and oxygen concentration (Bishnoi et al. 2008). Whereas

introduced species, as the ones used for this investigation, may be presumed to be involved in

PAHs degradation techniques as possible bioremediation processes (Bishnoi et al. 2008). The

degradation of PAHs is favored at nitrogen-limiting conditions and low pH (about 4.5) making

bioremediation technique an efficient, economic, versatile, and environmentally sound treatment

(Bishnoi et al. 2008). Biodegradation of PAHs with WRF occurs similar to the lignin polymer

degradation process of the carbon cycle of the biosphere.

Section V. Lignin

Lignin has its origin in the Latin word “lignum” which means wood (Wong 2009). This

compound is a three-dimensional biopolymer with a high molecular weight considered the most

abundant renewable source of aromatic polymer on the earth, important constituent of the living

terrestrial biomass and, therefore, a resistant material to microbial degradation (Magan et al.

2010). It can be synthesized in plants by linking hydroxycinnamyl, coniferyl and sinapyl alcohols

to obtain p-hydroxyphenol (Tišma et al. 2010). Lignin (the main component of wood) cellulose

and hemicellulose are the main constituents of the chemical substance called lignocellulose

(Tišma et al. 2010). Bioconversion of lignin is one of the most important processes occurring in

the carbon cycle of the biosphere (Rogalski et al. 1991). This polymer is highly oxidized,

therefore, difficult to oxidize further as a complex hetero-polymer with no stereo-chemical

regularity due to its free radical mechanism of synthesis. WRF are the most efficient lignin

degraders in nature, due to their ability to degrade complex and recalcitrant organic molecules.

This property makes them attractive microorganisms for bioremediation of soil contaminated by

organic pollutants. Xenobiotics chemicals, for instance, can only be degraded by the unspecific

extracellular ligninolytic enzymes WRF can produce.

13

These microorganisms can eventually degrade lignin when the mycelia of the organisms

penetrate the cell cavity and release ligninolytic enzymes to be able to decompose xylon, a small

fungi grown on trees, to a white sponge-like mass (Gao et al. 2010). They are the only ones

known to be able to degrade the highly recalcitrant natural polymer lignin or lignin model

compounds, by producing three types of extracellular enzymes known as lignin-modifying

enzymes (LMEs); LiP, MnP and Lac (Gao et al. 2010). Subsequently, the lignin biodegradation

must involve a non-specific and non-stereo-selective mechanism in soil like the biodegradative

mechanisms found in these WRF (Aust 1995).

The mechanism of the lignin depolymerization applicable to peroxidases is one in which

numbers of cationic radicals are generated during oxidation of the lignin complex functioning as

lignin-degrading peroxidases (LDPs) substrates (Singh and Chen 2008). The ferric form of the

enzyme, referred as resting enzyme, is oxidized by two-electrons using hydrogen peroxide (H2O2)

to a form of peroxidases known as compound I (Aust 1995). This compound can be reduced by

one-electron by means of chemicals having a suitable reduction potential (Aust 1995). Then, the

enzyme is reduced to a form called compound II whereas the chemical is oxidized by one-

electron (Aust 1995). This one-electron oxidized chemical, or the original chemical, can

eventually reduce compound II back to resting enzyme (Aust 1995). In order to be oxidized by

two-electrons (compound I), one-electron is removed from ferric iron to form ferryl while the

second electron is withdrawn from the porphyrin ring. The latter is reduced first to form

compound II so the reduction potential for compound I is slightly higher than for compound II

(Aust 1995).

The WRF Trametes (Coriolus, Polyporus) versicolor, Phanerochaete chrysosporium and

Phlebia radiata are among the most efficient lignin-degrading microorganisms (Rogalski et al.

1991). T. versicolor produces all the enzymes and the same refer to P. radiata, while P.

chrysosporium has been by far the most popular fungus in lignin biodegradation research, it

14

produces only LiP and MnP, not Lac. Typical for the occurrence of lignin-modifying enzymes in

T. versicolor seems to be the large variability between strains which may reflect inherent

heterogeneity of this species. The production of Lac of T. versicolor is stimulated by starvation

or by some phenolic compounds added to the growth medium. Culture conditions are also

important for the production of lignin-modifying enzymes by other fungi. The ligninolytic

activity of P. chrysosporium can only be seen after the primary growth phase when carbon,

nitrogen or sulphur limitation occurs. Aromatic substances such as veratryl alcohol are added to

the medium as stimulators, as the secondary metabolite, involved in lignin degrading systems,

which increases the production of LiP in P. chrysosporium and in P. radiata.

LiP acts in the oxidation of non-phenolic phenylpropanoid units which lead to the

deconstruction of lignin polymer oxidizing aromatic nuclei to aryl cation radicals which

eventually undergo several non-enzymatic reactions, including cleavage of C-C and C-O linkages

(Singh and Chen 2008). This enzyme oxidizes non-phenolic lignin substructure of aromatic

nuclei attracting one-electron in the presence of H2O2 generating cation radicals whose

spontaneous reactions lead to extensive degradation of lignin (Periasamy and Natarajan 2004)

while in the oxidation of non-phenolic phenylpropanoid units it can lead to polymer

fragmentation (Kalmiş et al. 2008).

Whereas, MnP, one of the important enzymes of fungi, oxidizes Mn2+

to Mn3+

in the

presence of H2O2, allowing the attack of phenolic structures in lignin with low redox potential

stabilized by suitable metal chelators secreted by the fungi (Kalmiş et al. 2008). Initially, Mn2+

is

oxidized by both MnP I and MnP II compounds, generating Mn3+

. Then, Mn3+

is stabilized by

chelation with organic acids such as oxalate and malonate produced by the fungal cells, and

diffuses from the surface of the enzyme as Mn3+

complex which in the end oxidizes the organic

substrates (Singh and Chen 2008).

15

Finally, Lac enzymes have the capacity to oxidize phenolic and non-phenolic compounds

and the ability to decolorize dyes in wastewaters (Ellouze et al. 2007). These are the most widely

present enzymes in the nature, the oldest and most studied enzymatic systems, as it contains 15-

30% carbohydrate and have a molecule mass of 60-90 kDa (Shraddha et al. 2011). Glycosylated

polyphenol oxidases contain 4 copper ions per molecule, carry out 1 electron oxidation of

phenolic and its related compound, and reduce oxygen to water, so the copper containing, 1,4-

benzenediol: oxygen oxidoreductases (EC 1.10.3.2) can be found in higher plants and

microorganisms.

When a substrate is oxidized by a laccase, it loses a single electron and forms a free

radical that may undergo oxidation or non-enzymatic reactions including hydration,

disproportionation, and polymerization. This enzyme plays an important role in soil

bioremediation, biodegradation of environmental phenolic pollutant, and removal of endocrine

disruptors. It is also used in nanotechnology, due to its ability to catalyze electron transfer

reactions without additional co-factor (Shraddha et al. 2011). Catalysis can occur when reduction

of one oxygen molecule to water is accompanied with the oxidation of one-electron in a wide

range of aromatic compounds including polyphenols, and aromatic amines. Lac enzymes are

secreted out in the medium extracellularly, and temperature and pH are limiting factors for these

enzymes, as they differ from one strain to another. The optimal temperature of Lac enzymatic

production is found to be 25ºC in presence of light, while, in the dark the optimal temperature is

30ºC, and the pH will occur within 4.5 and 6.0 (Shraddha et al. 2011). Lac is widely distributed

in higher plants, bacteria, fungi, and insects and has been isolated from Ascomyceteous,

Deuteromycteous and Basidiomycetous fungi. The white-rot Basidiomycetes fungi efficiently

degrade the lignin which oxidizes phenolic compounds to give phenoxy radicals and quinines.

Microorganisms inoculated into the solid-phase remediation of soil (Davis et al. 1993)

can release enzymes, which play an active key role in the biological, and biochemical

16

transformation of the matrix. The enzymes are responsible for the breakdown of several organic

compounds characterized by a complex structure finally producing simple water-soluble

compounds (Zeng et al. 2010). There may be pollutants bound to soil particles less available to

enzymatic processes being much more resistant to degradation (Davis et al. 1993).

Conclusively, there can be a biotechnology application to improve the contaminated soil

by means of whole microorganisms or their enzymes to synthesize or bioconvert the

contaminated material into new product (Jafari et al. 2007). The use of WRF for soil

bioremediation can potentially contribute to a safer environment by decreasing or completely

eliminating harmful contaminants, while improving soil conditions for cultivation purposes.

This investigation aims to provide evidence of degradation effectiveness for selected

WRF; Phanerochaete chrysosporium BKM-1767, Pleurotus djamor TU-7659, Trametes elegans

FP-105038, Pleurotus djamor native strain PR-1562 and Trametes elegans native strain PR-1151

and will not investigate enzymatic actions.

Section VI. Fungi Bioremediation Techniques

Fungi are an important contributor to the ecological balance of the world (Solomon et al.

1996). Like bacteria, most fungi are saprotrophs (also called saprobes), decomposers that absorb

nutrients from organic waste and dead organisms. Once fungi degrade wastes and dead

organisms, water, carbon (as CO2) and mineral components of organic compounds are released,

and these elements are recycled (Solomon et al. 1996). If this continuous decomposition does not

occur, essential nutrients can be locked-up in huge mounds of dead animals, feces, branches, logs,

and even leaves. Then, the nutrients are not available for use by new generations or organisms;

therefore, life can cease (Solomon et al. 1996). In Puerto Rico there are forests which contain

fungi playing an important role in ecosystems to help decompose organic matter by releasing

nutrients that may become available to plants (Lodge et al. 2007). For this reason WRF native

strains will be used for biodegradation purposes of a hydrocarbon contaminated soil.

17

Fungi contain genetic information that can make them very efficient in the adaptability to

extreme environmental conditions of water, temperature, pH and salinity as indicated by Lacina

et al. (2003) and Cantrell et al. (2006). The enzymes released by the fungi will help in the

remediation process of the soil portion affected by the polluted-waste leachate compounds as

mentioned in studies by Davis et al. (1993) when using lignin-degrading fungi to degrade

chemicals as creosote and/or PCP (pentachlorophenol). Other compounds that can be oxidized by

white rot fungi are chlorinated phenols, PCBs, dioxins, pesticides, explosives, TPHs and, even,

dyes (Levin et al. 2003). These fungi cause the “white-rot” of wood, as well as litter

decomposition, by breaking down lignin which require extracellular enzymes and are better

candidates for the bioremediation of highly apolar pollutants than non-ligninolytic

microorganisms (Levin et al. 2003).

The WRF are unable to supply all their carbon and energy requirements from lignin.

Therefore there is a need of substrates as cellulose or other carbon sources for their growth and

delignification to make them a highly potential material for degradation of various xenobiotic

compounds and dyes (Kalmiş et al. 2008), as to ease degradation of high molecular-mass organic

pollutants (extracellular fungal enzymes) (Ellouze et al. 2007). In this investigation we

incorporated hay into the soil mixture as a source of carbon content to support the growth, and to

facilitate the degradation process by WRF. If we were to compare the degradation ratio of WRF

versus the bacteria, there are not all microbial bacteria known that can degrade all components of

BTEX compounds (Levin et al. 2003) while Phanerochaete chrysosporium simultaneously has

degraded all the BTEX components (Levin et al. 2003). This WRF was the focus of our

investigation as there are studies indicating the degrading effectiveness on contaminated sites.

Other WRF as Pleurotus tuber-regium (Ejoh et al. 2012), Polyporus sp. S133 (Hadibarata and

Tachibana 2009), and Lentinus subnudus (Adenipekun and Fasidi 2005) have been evaluated with

notable differences regarding to the extent of their pollutant transformation ability. The

18

intracellular process used by bacteria does not have access to degrade pollutants as does the

extracellular enzymes produced by WRF.

The observation that white-rot fungi can oxidize PAHs rapidly with their extracellular

ligninolytic enzyme systems has therefore raised interest in the use of these organisms for

bioremediation of PAH-polluted soils as mentioned by Levin et al (2003). The non-specific

oxidases involved in lignin degradation (mainly laccases, lignin- and manganese-peroxidases) are

able to transform by oxidation the PAHs to a more water-soluble product with much greater

bioavailability (Levin et al. 2003). The use of white-rot fungi for bioremediation requires an

understanding of the factors that enhance their ability to detoxify, and clarification of the enzyme

mechanisms used (Levin et al. 2003).

During a bioremediation process the enzymatic degradation is selected as an alternative

to bacterial processes as the biological degradation requires long periods of treatment (from 2 to 4

weeks) and presents lag phases (2 days) until degradation can be observed (Eibes et al. 2005).

The optimal growth of WRF occurs at 102ºF, pH range of 4.0 to 4.5, and high oxygen content.

Lignin degradation using pure oxygen is 2 to 3 times greater than with air. The growth rate of

WRF increases significantly when the water potential increases from 1.5 to 0.03 MPa

(MegaPascal=106 Pa), and growth in soil increases directly with nitrogen content and optimal

moisture content of 40-45% (Eibes et al. 2005).

The first steps in the biotransformation of PAHs were investigated by Bezalel et al (1997)

using two phase I enzymes, cytochrome P-450 monooxygenase and epoxide hydrolase.

Intracellular systems generally present in most fungi, such as cytochrome P-450 monooxygenase,

can also be involved in organopollutant degradation (Levin et al. 2003). There is a potential for

phase II biotransformations (formation of sulfates, glucuronides, glucosides, or glutathione

conjugates from phenolic derivatives of the PAHs), since there is water-soluble metabolite, which

could be conjugation products (Bezalel et al. 1997). While for xenobiotics compounds such as

19

2,4-dinitrotoluene and 2,4,5-trichlorophenol, the multi-step catabolic pathways were shown to

involve cycles of intracellular reductions and extracellular oxidations, terminating in intracellular

oxidative ring cleavage prior to complete mineralization (Zacchi et al. 2000).

The WRF belonging to the basidomycetes as Pleurotus species (Periasamy and Natarajan

2004) (Figure 1) produce various extracellular LMEs with very low substrate specificity as LiP,

MnP, H2O2 producer enzymes, aryl alcohol oxidase and Lac. These species produce protein rich

biomass of fruiting bodies, enabling to mineralize a wide range of highly recalcitrant organo-

pollutants structurally similar to lignin (Kalmiş et al. 2008) and a wide range of xenobiotic

compounds (Faraco et al. 2009). Pleurotus species possess the capacity to accelerate the

biodegradation of organic pollutants (Ellouze et al. 2007) and reduce more than 95% of PAHs

inoculated into test soil to non-toxic components (Adenipekun and Isikhuemhen 2008).

Figure 1. Pleurotus djamor (The Hidden Forest, 2005).

The biosynthetic potentialities of these species are the production and secretion of a wide

spectrum of enzymes enabling to thrive over a wide range of plant wastes, since changes of

extracellular enzyme activities of cellulases and laccases are directly correlated with growth and

fruit body formation (Periasamy and Natarajan 2004). To establish a different relationship

20

between LiP and MnP, the first is characterized by oxidation of high redox-potential aromatic

compounds as veratryl alcohol while MnP requires Mn2+

to complete the catalytic cycle forming

Mn3+

chelates which act as diffusing oxidasers (Periasamy and Natarajan 2004), thereby

generating aromatic radicals (Sánchez 2010). The LiP activity may be defined as the enzyme

amount leading to produce 1 μmol veratryl aldehyde by the oxidation of veratryl alcohol per

minute. Whereas MnP activity is expressed as the enzyme amount to produce 1 μmol of Mn3+

by

the oxidation of Mn

2+ per minute.

For Lac the activity is expressed as the enzyme amount which leads to the oxidation of 1

μmol of 2,2-azino-bis-[3[ethyltiazoline-6-sulfonate] (ABTS) per minute (Zeng et al. 2010). The

specific activities of the enzymes may be expressed as units per gram of dry medium. These

WRF species also are able to breakdown all components of lignocellulose, fraction of the total

organic matter, from composted agricultural waste, including lignin, being more refractory to

microbial attack, once lignocellulolytic microorganisms are inoculated in the soil as a strategy to

enhance lignocellulose degradation (Zeng et al. 2010). As the fungi have the ability to degrade

several lignocellulose substrates it can be produced on natural materials from agriculture,

woodland, animal husbandry, and manufacturing industries (Sánchez 2010).

Another well-known WRF is Phanerochaete chrysosporium (Figure 2), the most active

ligninolytic organism for its ability to degrade/mineralize lignin including soil humic substances

(Winquist et al. 2009). Additionally, it degrades a wide variety of aromatic compounds as

chlorinated organics, PCBs, PAHs, pesticides and others (Aust et al. 1994), by its non-specific

oxidative and extracellular multi-enzyme system (Ballaminut and Matheus 2007), involving MnP,

LiP and Lac (Zeng et al. 2010) and other enzymes. This fungi is capable of degrading starch,

cellulose, pectin, lignin, lignocelluloses, and can decolorize azo-triphenyl methane dyes

(Asamundo et al. 2005) and complex organic substances as halogenated aromatics (dioxin), and

many others (Singh and Chen 2008). This WRF also degrades a wide range of PAHs including

21

acenaphthene, acenaphthylene, anthracene, benzo(a)anthracene,

enzo(a)pyrene,benzo(b)fluoranthene, benzo(g,h,i)perylene, benzo(k)fluoranthene, chrysene,

fluorene, indeno(1,2,3-c,d)pyrene, naphthalene, phenanthrene, and pyrene (Kristanti et al. 2011).

Figure 2. Phanerochaete chrysosporium (microbewiki.kenyon.edu227)

P. chrysosporium is the model of WRF for the production of peroxidases attracting a

growing interest for the bio-treatment (removal or destruction) of wastewater ingredients such as

metals, inorganic nutrients and organic compounds (Lacina et al. 2003). Physiological level of

oxalate in Phanerochaete chrysosporium cultures stimulates the MnP activity (Singh and Chen

2008). This species is capable of degrading PCBs by transforming a variety of cogeners with

varying degrees and position of chlorine substitutions (Borazjani et al. 2005).

Finally, the genus Trametes (Figure 3) is probably the most actively investigated in the

phylum of Basidiomycota for lignolytic enzyme formation and application (Nyanhongo et al.

2007). The use of this WRF for remediation purposes is the constitutive, extracellular secretion

and the non-specific nature of the lignolytic enzymes, which obviates the need for adaptation to

the target molecule (Nyanhongo et al. 2007).

https://www.google.com.pr/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&uact=8&docid=bYwpo1q8nvKBYM&tbnid=38oyQ1EMtswLSM:&ved=0CAQQjB0&url=https%3A%2F%2Fmicrobewiki.kenyon.edu%2Findex.php%2FPhanerochaete_chrysosporium&ei=ZW9NU5rDFqehsQTiloHwCg&bvm=bv.64764171,d.dmQ&psig=AFQjCNEg5ZrICRR8khn1O5AZMxV12CjeAA&ust=1397669974976657https://www.google.com.pr/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&uact=8&docid=bYwpo1q8nvKBYM&tbnid=38oyQ1EMtswLSM:&ved=0CAQQjB0&url=https%3A%2F%2Fmicrobewiki.kenyon.edu%2Findex.php%2FPhanerochaete_chrysosporium&ei=ZW9NU5rDFqehsQTiloHwCg&bvm=bv.64764171,d.dmQ&psig=AFQjCNEg5ZrICRR8khn1O5AZMxV12CjeAA&ust=1397669974976657

22

These species grow in inexpensive media such as straw or sawdust and their hyphal

growth form allows them to extend far from their original starting point, very attractive for soil

bioremediation purposes. The lignolytic enzymes available in this species are Lac and MnP as

LiP is rarely reported and additional oxidoreductases as cellobiose dehydrogenase (CDH) and

pyranose 2-oxidase (P2O) are also found in this species.

Figure 3. Trametes elegans (www.wisconsinmushrooms.com)

Section VII. Intellectual Merit

Soil contamination is a threat to the health of ecological systems (alter plant metabolism

by reducing crop yields), the environment and human population. The main objective of this

research was to determine the efficiency of a WRF from Phanerochaete chrysosporium,

Pleurotus djamor, and Trametes elegans spp. as a viable strategy for bioremediation of

contaminated soil and as a means to restore it to a healthful state conducive to its use for safe

agricultural purposes. This research pursued the use of WRF’s natural biological ability to

http://www.wisconsinmushrooms.com/

23

destroy contaminant pollutants. The ultimate goal was to investigate a remediation process that

could contribute both to food safety and sustainable agriculture.

As the WRF can be used for soil bioremediation, the hypothesis of this study was to

demonstrate the degrading effectiveness on the hydrocarbon waste pollutant in a soil.

a) Null hypothesis (Ho): μ1- μ2- μ3- μ4 = 0; WRF- Phanerochaete chrysosporium,

Pleurotus djamor, and Trametes elegans or native fungi have the same degrading

effectiveness on hydrocarbon waste pollutant in soil.

b) Alternative hypothesis (Ha): μ1- μ2- μ3- μ4 ≠ 0; WRF- Phanerochaete

chrysosporium, Pleurotus djamor, and Trametes elegans or native fungi do not

have the same degrading effectiveness on hydrocarbon waste pollutant in soil.

As part of the investigation, we would like to provide significant evidence for a

bioremediation technique that can potentially enable a contaminated site to be re-conditioned as a

contaminant free-soil for use in agriculture and farming. Soil pollution is a threat to the

environment, to food safety and to a sustainable agriculture. WRF, Phanerochaete

chrysosporium, Pleurotus djamor, and Trametes elegans, were introduced to a contaminated soil

affected by hydrocarbon waste pollutant. These microorganisms are capable to degrade the

contaminant measuring the effects at different inoculation intervals.

This investigation is a ground breaking study using native WRF from Puerto Rico as a

soil restoration technology, in order to develop innovative, biologically safe and cost-effective

treatments for removing hydrocarbon waste pollutants from soil that cannot be performed by

existing chemical processes; accomplished by using rainfall and adding TPH contaminant to a

known soil.

Topics to be explored during the completion of this investigation are:

1) What are the ideal conditions for effective degradation process of WRF-

Phanerochaete chrysosporium, Pleurotus djamor, and Trametes elegans or their

24

native fungi to reduce or eliminate the hydrocarbon pollutant contamination in

soil produced by uncontrolled waste management?

2) Which WRF species is the best candidate for a scale-up bioremediation study

based on the degrading effectiveness in a soil used for agricultural purposes?

3) What is the degradation rate at which the most effective WRF degrades a

petroleum related contaminant in soil?

4) Can a bioremediation strategy to mitigate soil hydrocarbon contamination caused

by petroleum related waste leachate be implemented?

25

Chapter Two

Literature Review

Section I. Waste Production

Bio-medical waste is any solid and fluid including its container and any intermediate

product, generated during the diagnosis, treatment or immunization of human beings or animals,

in research activity (Sarojini et al. 2007), biological production or testing. There are two

categories for bio-waste known as non-hazardous (85%), where you can find plastic, cardboard

(USEPA 2005), packaging material, and paper, and bio-hazardous, divided in two different types;

infectious or regulated waste (10%) as sharps, plastic disposables, liquid waste, blood and blood

products, pathological waste (USEPA 2005) and non-infectious waste (5%) as discarded glass,

chemical waste, incinerated waste, and radioactive waste (Razdan and Cheema 2009; and Radha

et al. 2009).

The waste produced in the course of health-care activities carries a higher potential for

infection and injury than any other type of waste by its inadequate and inappropriate handling

causing serious health consequences and a significant impact on the environment (Katoch 2007).

This waste should be classified according to source, typology and risk factor associated with

handling, storage and ultimate disposal. While some hospitals or pathological laboratory waste

may contain toxic chemicals, like mercury, xylene and formalin (Dwivedi et al. 2009), requiring

special treatment as incineration or even hazardous waste landfill facilities, the same may occur

to the waste produced by environmental pollution caused by: spills during the industrial

production process, disposal of toxic compounds or excessive treatment of agricultural surfaces

also disposed in landfill.

The industrial wastes can be comprised of organic compounds such as aliphatic and

aromatic hydrocarbons, TPHs, derived from petroleum, charcoal and wood, as well as natural

26

products, halogenated solvents, pesticides, herbicides and explosives (Csutak et al. 2010).

Intensification of agriculture and manufacturing industries has resulted in increased release of a

wide range of xenobiotic compounds to the environment (Kumar et al. 2011). For instance,

PAHs are a class of organic compounds that consist of two or more fused benzene rings and/or

pentacyclic molecules arranged in various structural configurations, highly recalcitrant due to

their hydrophobicity and low water solubility, ubiquitous in the natural environment, which

originate from two main sources: natural (biogenic and geochemical) and anthropogenic, major

source of environmental pollution (Bamforth and Singleton 2005). These compounds naturally

occur in fossil fuels such as coal and petroleum, but are also formed during the incomplete

combustion of organic materials such as diesel, wood and vegetation, widely distributed in soils

and sediments, groundwater and the atmosphere (Bamforth and Singleton 2005). Persistence of

PAHs in the environment depends on a variety of factors, as its chemical structure, concentration

and dispersion, and bioavailability of the contaminant (Bamforth and Singleton 2005). PAHs

have five fates in the environment; volatilization, leaching, degradation, bioaccumulation and

sequestration (Maiti et al. 2012). The pollution caused by TPHs compounds and their derivatives

is considered the most prevalent and worldwide environmental contamination adversely affecting

human health along with a threat to environment (Kristanti et al. 2011). Reason to say the excess

loading of hazardous waste has led to scarcity of clean water and disturbances of soil thus

limiting crop production (Kumar et al. 2011).

TPHs are released into the environment through accidents, as managed releases, or

unintended by-products of industrial, commercial or private actions. Consequently, there is a need

to understand the changes that may occur over time in the composition of hydrocarbons found in

soil, water, or air in order to assess public health issues for TPH. Continuous spills or prolong

history of disposal at specific sites can lead to concerns; oil dumped onto soils can saturate the

soil matrix, affecting its use for agricultural purposes. The goals of the ecosystems’

27

decontamination are recovery of soil health and fertility, detoxification of ground water, re-

utilization of wastewater, removal of negative effects on human and animal health, and

production of healthy air by means of bioremediation techniques.

Section II. Bioremediation

Bioremediation is the elimination, attenuation or transformation of polluting or

contaminating substances by use of biological processes (Shukla et al. 2010). This process is an

interesting alternative for restoring the ecological equilibrium in polluted environments through

immobilization, chemical transformation and mineralization (Robertson et al. 2007). It involves

mainly non-invasive technologies with rather low costs and less disruption of the contaminated

environment, with a high public acceptance, carried out on site (Shukla et al. 2010) if compared

to other remediation technologies (Rigas et al. 2008) with only rarely the addition of some

degradation enhancers (Csutak et al. 2010).

It is based on biodegradation processes related to microbial population dynamics in soil

or water and its ability to consume xenobiotics as carbon source (Csutak et al. 2010). The clean-

up of contaminated environment by means of exploiting the diverse metabolic activities of

microorganisms is achieved by converting the contaminants to harmless products as carbon IV

oxide and water, or by conversion into microbial biomass (Rigas et al. 2008: and Ejoh et al.

2012).

There exist many techniques of dispersal, collection, removal, landfill disposal and

incineration that simply dilute or sequester the contaminants or transfer them to another

environmental medium (Rockne and Reddy 2003). In contrast, bioremediation can be regarded

as a more effective and environmentally friendly strategy since it results in the partial or complete

biotransformation of organo-xenobiotics to microbial biomass and stable, innocuous end

products. It is a technique where biological treatments are used, for the clean-up of hazardous

chemicals in the environment, to transform and/or degrade toxic compounds or otherwise render

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them harmless (Vidali 2001; and Bennet et al. 2002), to disarm noxious chemicals without

forming new toxins, treat contaminants on site with relatively little disturbance to the

contaminated matrix (Head 1998).

The acceptance of bioremediation as a viable clean-up strategy, however, in many cases

also depends on cost (cannot be more expensive than existing chemical and physical treatments).

As a biological remediation alone or in combination with other methods, it has gained an

established place as a soil remediation technology (Rockne and Reddy 2003). This technique can

be applied for the complete destruction of a wide variety of contaminants (Rigas et al. 2008) such

as industrial wastes (solid and liquid), municipal/urban wastes (sewage), mining wastes

(including effluents containing heavy metals, etc), chemical spills and hazardous wastes (Sen and

Chakrabarti 2009).

Bioremediation processes use biological agents, mainly microorganisms (yeast, fungi or

bacteria) to clean-up contaminated soils and water. These microorganisms are most important for

reclamation, immobilization or detoxification of metallic pollutants (Gadd 2010), by using the

contaminants as nutrient or energy sources (Shukla et al. 2010; and Kumar et al. 2011). The use

of living organism, microorganisms, and their enzymatic set is considered an effective

remediation technology for the partial or total recovery of polluted sites (Rao et al. 2010). Usually

bioremediation systems run under aerobic conditions, if the process runs under anaerobic

conditions it may permit microbial organisms to degrade otherwise recalcitrant molecules (Shukla

et al. 2010).

For instance, the bioremediation process to degrade PCBs (Figure 4) needs two steps;

dechlorination, mediated by anaerobic microorganisms (Borazjani et al. 2005), and oxidative ring

cleavage, using aerobic organisms contained within the Earth’s crust (De et al. 2004). Attributes

that can affect bioremediation process are superior growth rates, competitive ecological strategies

and tolerances to high contaminant concentrations (Lamar et al. 1999). The types of

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microorganisms present stimulated by supplementing nutrients (nitrogen and phosphorous),

electron acceptors (oxygen), and substrates (methane, phenol, and toluene), soil structure, pH,

moisture, temperature, and introducing microorganisms with desired catalytic capabilities (Shukla

et al. 2010) are means that can enhance microbial degradation (Sen and Chakrabarti 2009) and,

eventually, control the time other contaminants as PAHs persist in the environment (Bamforth

and Singleton 2005).

Figure 4. Summary of different bioremediation strategies for typical hazardous wastes (adapted

from Brar et al. 2006).

Section III. Microorganisms in Bioremediation

The origins and sources of pollution are different: industrial activities as mining and

metal processing, petrochemical and industrial complexes, industry effluents, chemical weapons

production, pulp and paper industries, dye industries and industrial manufacturing; and

anthropogenic activities as traffic, agricultural activities, and others (Rao et al. 2010). Population

increase also impacts on the environment when using natural resources, producing wastes,

causing environmental stress as loss of biodiversity, and negative effects on air, soil and water

30

pollution. These pollutants can affect the health of humans, animals and the environment as

inhibit respiration, provoke reduced reproduction of fish-eating birds, and even, contribute to the

birth of premature babies or children with genetic defects as downs syndrome, anencephaly, and

spina bifida (Rao et al. 2010). Increase in contaminated sites poses major environmental and

human health problems so there exists a need to implement decontamination strategies mandatory

in order to properly decontaminate the environment (Rao et al. 2010).

Substances with high polluting potential are present in the environment and affect soil,

sediments, water, air, microbial organisms, plants, animals, and humans (Rao et al. 2010). These

substances may be distributed in one or all environmental compartments not only as mixtures of

different organic compounds but also of organic and inorganic independent chemicals (Rao et al.

2010). The use of microorganisms, during the remediation process, is the most effective method

for dealing with widespread pollution (Tachibana et al. 2005; Miyoshi et al. 2005; Tachibana et

al. 2006; and Tachibana et al. 2007). The effectiveness of bioremediation process is often a

function of the microbial population or consortium and how it can be enriched and maintained in

an environment (Márquez-Rocha et al. 2001). In most cases, the treatment of all contaminated

environment has involved bio-stimulation, addition of nutrients to stimulate the spontaneous

enrichment of the indigenous hydrocarbon oxidizing microbial population based primarily on

their xenobiotic-degrading abilities, or bio-augmentation containing a consortium of

microorganisms and inorganic nutrients, nitrogen, phosphate and potassium (NPK) (Sharma and

Rehman 2009), with superior pollutant-degrading abilities that may confer with exceptional

bioremediation performance (Lamar et al. 1999).

These transformations by microorganisms involve a complete destruction or

immobilization of contaminants instead of transfer from one environment to another as does

occur by extraction or incineration, which entails the transfer of pollutant from soil to atmosphere

(Sharma and Rehman 2009). The ex-situ treatments involve the physical removal of the

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contaminated matrix in the terrestrial ecosystem while the in-situ treatments propose a relatively

low cost and ecologically friendly procedure on site for the ability to re-use the land, providing

for a less toxic soil contamination using either the bio-stimulation or the bio-augmentation

microorganism process.

Section IV. Soil in Bioremediation

In terrestrial ecosystems, soil is the major sink for metal contaminants, while sediments

are the major sink for metals in aquatic systems affected through run-off, leaching and transport

via mobile colloids (Gadd 2010). Metals are significant natural components of all soils, present in

organic fractions, frequently in bound forms, with some metal recycling occurring as a result of

organic matter degradation. Their presence in the mineral fraction constitutes a pool of

potentially mobile metal species, many essential nutrients for plants and microbes, and important

solid components, with a fundamental effect on soil geo-chemical processes (Table 2), as occur

with clays, minerals, iron and manganese oxides (Gadd 2010). Soil is “the biogeochemical

engine of Earth’s life support system” (Valentín et al. 2013). It provides us with food, fodder,

fibre and fuel, readily rateable agriculture and forestry goods, delivers ecosystem services not

easily traded in markets (Valentín et al. 2013). Life-supporting functions include, recycling of

carbon and essential nutrients of all living materials, filtering and storage of water, regulation of

the atmosphere and biological control of pests among others. Continuous urbanization,

desertification intensified by the global change and short-sighted agricultural practices threaten

the natural soil capital overall affecting the food safety and agriculture sustainability for our

future generations.

The soil possesses properties as color, structure, texture and pH with positive influence in

the bioremediation techniques. For instance, the color in soil provides important clues about

organic matter, drainage, and other soil conditions. A darker color can indicate more organic

matter is present compared to soil of a lighter color. Often the surface horizon, called the

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epipedon, has a darker color than the subsoil due to higher organic matter content. There are six

basic types of soil structure: platy, prismatic, blocky, granular, loose structure, and compacted

structure but not necessarily one will observe these exact forms in the field. For the composition

of the soil we consider the texture as the relative proportion of sand, silt and clay particles where

the ones with moderate amount of each of the above are called loams. There are 12 soil texture

classes: sand (S), loamy sand (LS), sandy loam (SL), loam (L), silt loam (SiL), silt (Si), sandy

clay loam (SCL), clay loam (CL), silty clay loam (SiCL), sandy clay (SC), silty clay (SiC), and

clay (C) (Staff 1993). Finally, the pH is determined to measure how acidic (pH7) the soil is.

Table 2. Environmental factors and optimum condition for microbial activity for soil

bioremediation (adapted from Shukla et al. 2010).

Environmental Factor Optimum conditions Condition required

for microbial

activity

Available soil moisture 25-85% water holding

capacity

25-28% of water

holding capacity

Oxygen >0.2 mg/L DO, >10% air-

filled pore space for

aerobic degradation

Aerobic, minimum

air-filled pore space

of 10%

Redox potential Eh > 50 mill volts -

Nutrients C:N:P= 120:10:1 molar N and P for microbial

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ratio growth

pH 6.5-8.0 5.5 to 8.5

Temperature 20-30ºC 15-45ºC

Contaminants Hydrocarbon 5-10% of

dry weight of soil

Not too toxic

Heavy metals 700 ppm Total content

2000ppm

Type of soil Coto-clay- Sand/Silt/Clay:

35:19:46

Low clay or silt

content

As soil ages biological processes increasing the nitrogen contents, as leaching will reduce

phosphorous and become more acidic, will be distinctive in the layers (Plaster 2003) determined

by the soil properties. There are five layers known as master horizons, O, A, E, B, and C.

The O-horizon is an organic layer made of wholly or partially decayed plant and animal

debris (undisturbed soil). This is the uppermost layer and is usually found only in areas that have

some sort of permanent vegetation. It is a layer of plant litter lying on the surface of the mineral

soil where considerable decomposition or organic matter takes place, giving its characteristic dark

color.

The A-horizon, known as topsoil, is the surface mineral layer where organic matter

accumulates, and leaches clay and iron material (eluviation) down to other layer while sand

remains. When present, it lies beneath the O-horizon and is darker than the underling horizons

(although not as dark as the O) due to accumulation of organic matter in the soil surface.

Leaching is most intense in the soil surface and causes solutes to move down the profile to the

underlying horizons.

The E-horizon, is the greatest eluviation zone, it possesses leached clay, chemicals an