USE OF BIOCHAR TO ENHANCE BIOREMEDIATION OF AN OXISOL CONTAMINATED WITH DIESEL OIL BY ABEKA HAMMOND (10230116) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF M.PHIL DEGREE IN SOIL SCIENCE JULY, 2014 University of Ghana http://ugspace.ug.edu.gh i DECLARATION I do hereby declare that this thesis has been written by me and that it is the record of my own research work. It has not been presented for another degree elsewhere. Works of other researchers have been duly cited by references to the authors. All assistance received has also been acknowledged. Sign: ……………………. Abeka Hammond (Student) Sign: ……………………. Dr. Innocent Y.D. Lawson (Principal Supervisor) Sign: ……………………. Prof. S.K.A Danso (Co-Supervisor) University of Ghana http://ugspace.ug.edu.gh ii DEDICATION This work is affectionately and humbly dedicated to my caring Mother and her sisters, my cousin Thomas Oyom and to all those who took an interest and encouraged me in my academic pursuit. University of Ghana http://ugspace.ug.edu.gh iii ACKNOWLEDGEMENT Glory be to God for bringing me this far in my academic pursuit. I wish to express my sincere gratitude first and foremost to my family, for every form of support and sacrifice throughout all these years of my education. I wish to also express my sincere gratitude to my principal supervisor Dr. Innocent Y. D. Lawson who never gave up on me during this challenging period but kept guiding and encouraging me. I also acknowledge the tireless efforts of Prof. S. K. A Danso, my co-supervisor, for all the pieces of advice and contributions towards the shaping of this thesis. Not forgetting all the other lecturers in the Soil Science Department who in one way or the other contributed to the outcome of this thesis. I thank Mr. Julius Addo, Mr Edmund Anum, Mr Martin Aggrey, and most especially Mr Victor Adusei and all the other laboratory technicians of the department for their support and assistance during the laboratory phase of the work. I am most grateful for the enormous support, encouragement and motivation I got from Dr. Edward Yeboah (SRI-Kumasi) during the biochar preparation period. My appreciation also goes to the Leventis Foundation for the scholarship they gave me for my research work. My heartfelt thanks also go to my colleague students in the Soil Science Department, for their friendship and as well as every ounce of support and contributions in making this thesis a reality. I cannot forget the support of my pastors and the entire congregation of I.C.C-Narrow Gate Cathedral, who helped me both spiritually and physically. University of Ghana http://ugspace.ug.edu.gh iv ABSTRACT Oil pollution is a worldwide threat to the environment, especially in oil producing countries, and the remediation of oil-contaminated soils is a major challenge for environmental research. Bioremediation is a useful method for restoring oil contaminated soils because of its cost effectiveness and environmental friendliness. However, the process is very slow in soils with low pH. Soils of the Western Region of Ghana where most of the country‘s oil activities take place are classified as Oxisols. These soils are acidic in nature and have soil conditions unfavourable for effective biodegradation of petroleum and its products. Application of biochar to soils is currently gaining considerable interest globally due to its potential to serve as a liming agent and in raising soil pH in different soil types. It is against this background that the present study was carried out to investigate the effectiveness of biochar as a soil conditioner in enhancing microbial degradation of diesel oil in theAnkasa series (Plinthic acrudox) sampled from the Western Region of Ghana and the subsequent growth of cowpea in the bioremediated soils. The acidic soil was contaminated with diesel oil at 100 mL/kg soil. Two biochar types, from rice straw (RB) and from saw dust (SB), were applied to the contaminated soils at 0, 65, 130, 195 and 260 Mg/ha. The treated soils were incubated and sampled for determination of the hydrocarbon utilizing bacteria (HUB) population, total aerobic heterotrophic bacteria (HET) population, change in soil pH and the amount of oil degraded at 10 days interval for 40 days. In another experiment the soil was contaminated with diesel oil at 100 mL/kg soil, amended with RB at 195 Mg/ha and fertilized with N and/or P in the form of ammonium nitrate and single super phosphate, respectively at 60 kg/ha. These treated soils were also incubated and sampled for analysis of hydrocarbon utilizing bacteria (HUB) population and the amount of oil degraded at 10 days interval. Cowpea was sown into the residual soils and harvested 6 weeks after planting for the determination of the number of nodule formed, shoot and root dry weights. In the first University of Ghana http://ugspace.ug.edu.gh v experiment, results showed that all the biochar treatments significantly (p < 0.05) increased the amount of diesel oil degraded, HUB and HET populations when compared to the control.The RB treatments significantly (p < 0.05) enhanced diesel oil degradation more than the SB treatments. Results also showed that RB at 260 Mg/ha resulted in the highest amount of diesel oil degraded but was not significantly (p > 0.05) different from RB at 195 Mg/ha. Soil pH, soil organic carbon, total exchangeable bases, effective cation exchange capacity, and base saturation were also significantly (p < 0.05) increased by the biochar treatments. Soil available P increased significantly (p < 0.05) for the RB treatments. However, total N and exchangeable acidity significantly (p < 0.05) decreased when amended with biochar. X-ray diffraction analysis showed that these biochars contained large quantities of carbonates and oxides.Fertilizing RB (195 Mg/ha) with N and/or P significantly (p < 0.05) increased amount of diesel oil degraded and HUB population 40 days after incubation when compared to RB only treatment. Fertilization with N and/or P enhanced shoots and roots dry weights of cowpea. Addition of N was inhibitory to nodulation, however, P fertilization enhanced nodulation. In conclusion, the enhanced degradation was attributed to the presence of large quantities of carbonates and oxides in the biochars which might have served as liming agents, improved the soilmicrobiology and other chemical properties. Further studies should be conducted on the application of combination of rice straw biochar, nitrogen and phosphorus, in the bioremediation of oil under acidic condition should be conducted in the field to confirm its effectiveness for future recommendation. University of Ghana http://ugspace.ug.edu.gh vi TABLE OF CONTENTS DECLARATION…………………………………………………………………………………..i DEDICATION…………………………………………………………………………………….ii ACKNOWLEDGEMENT………………………………………………………………………..iii ABSTRACT……………………………………………………………………………………...iv TABLE OF CONTENTS…………………………………………………………………………vi LIST OF TABLE……………………………………………………………………………….xiv LIST OF FIGURES……………………………………………………………………………...xv CHAPTER ONE…………………………………………………………………………………..1 INTRODUCTION ...................................................................................................................... 1 CHAPTER TWO………………………………………………………………………………….6 LITERATURE REVIEW ........................................................................................................... 6 2.1 What is biochar? ............................................................................................................... 6 2.1.1 Physical properties of biochar .................................................................................... 7 2.1.2 Chemical properties of biochar .................................................................................. 7 2.1.3 Structural composition of biochar .............................................................................. 8 2.1.4 Chemical composition and surface chemistry of biochar ........................................ 10 2.1.5 Pore size distribution and connectivity of biochar ................................................... 13 2.1.6 Feedstock and its influence on biochar characteristics ............................................ 14 2.1.7 Temperature and its influence on biochar characteristics ........................................ 16 University of Ghana http://ugspace.ug.edu.gh vii 2.2 Biochar‘s Impact on Soil Performance ........................................................................... 17 2.2.1 Improvement of Nutrient Availability, Storage and CEC ....................................... 18 2.2.1.1 Biochar effect on soil N .................................................................................... 18 2.2.1.2 Biochar effect on P availability......................................................................... 19 2.2.1.3 Biochar effect on other nutrients ....................................................................... 21 2.2.1.4 Biochar effect on CEC ...................................................................................... 21 2.2.2 Biochar effect on soil organic carbon ...................................................................... 22 2.2.3 Biochars‘ effect on water holding capacity of the soil ............................................ 23 2.2.4 Biochar effect on pH and aluminium toxicity .......................................................... 24 2.2.5 Biochar effect on soil biology .................................................................................. 26 2.2.6 Biochar effect on climate change ............................................................................. 28 2.2.7 Biochar effect on sorption of hydrophobic organic compounds (HOCS) and others ........................................................................................................................................... 30 2.2.8 Impact of biochar on crop productivity ................................................................... 33 2.3 Petroleum and its‘ products ............................................................................................ 34 2.3.1 Origin and formation of petroleum oil. .................................................................... 34 2.3.2 Composition and classification of petroleum hydrocarbons .................................... 37 2.3.3 Sources of petroleum in the environment ................................................................ 39 2.3.4 Distribution and methods of enumerating petroleum degrading microorganisms... 40 2.3.5 Reasons for persistence of hydrocarbons in the environment ................................. 43 University of Ghana http://ugspace.ug.edu.gh viii 2.3.6 Effect of petroleum on the soil................................................................................. 44 2.3.6.1 Physical effects ................................................................................................. 44 2.3.6.2 Chemical effects................................................................................................ 45 2.3.6.3 Biological effects .............................................................................................. 46 2.4 Remediation of contaminated soils ......................................................................... 46 2.4.1 In-situ methods for soil remediation .................................................................... 46 2.4.1.1 Volatilization..................................................................................................... 47 2.4.1.2 Phytoremediation .............................................................................................. 47 2.4.1.3 Leaching ............................................................................................................ 48 2.4.1.4 Vitrification ....................................................................................................... 49 2.4.1.5 Isolation or containment ................................................................................... 49 2.4.1.6 Passive remediation .......................................................................................... 49 2.4.2 Non in-situ methods of soil remediation .................................................................. 50 2.4.2.1 Land treatment .................................................................................................. 50 2.4.2.2 Thermal treatment ............................................................................................. 51 2.4.2.3 Asphalt incorporation........................................................................................ 51 2.4.2.4 Solidification or stabilization ............................................................................ 51 2.4.2.5 Chemical extraction .......................................................................................... 51 2.4.2.6 Excavation......................................................................................................... 52 2.4.3 In-situ microbial bioremediation.............................................................................. 52 University of Ghana http://ugspace.ug.edu.gh ix 2.4.3.1 Basic understanding of bioremediation principles ............................................ 53 2.4.3.2 Bioremediation technologies for crude oil contaminated sites ......................... 55 2.4.3.3 Principle of aerobic degradation of hydrocarbons ............................................ 61 2.4.3.4 Mixed versus pure cultures in microbial degradation of crude oil ................... 63 2.4.3.5 Bioaugmentation in microbial degradation of crude oil ................................... 64 2.4.3.6 Effect of additives on biodegradation (biostimulation) .................................... 66 2.4.3.7 Factors affecting biodegradation of oil in soil .................................................. 68 2.4.3.8 How fast is the oil being consumed? ................................................................ 72 2.4.3.9 Fate of microbes after degradation ................................................................... 73 2.5 Oxisols and low pH (soil acidity) ................................................................................... 74 2.5.1 Major constraints of soil acidification ..................................................................... 75 2.5.1.1 Aluminum and hydrogen toxicity ..................................................................... 75 2.5.1.2 Manganese toxicity ........................................................................................... 76 2.5.1.3 Calcium deficiency ........................................................................................... 77 2.5.1.4 Magnesium deficiency ...................................................................................... 77 2.5.1.5 Phosphorus deficiency ...................................................................................... 78 2.5.2 Types (pools) of soil acidity .................................................................................... 79 2.5.2.2 Exchangeable or salt replaceable acidity .......................................................... 81 2.5.2.3 Residual acidity ................................................................................................. 81 2.5.3 Causes of soil acidity ............................................................................................... 81 University of Ghana http://ugspace.ug.edu.gh x 2.5.3.1 Weathering and leaching................................................................................... 81 2.5.3.2 Organic matter decomposition .......................................................................... 82 2.5.3.3 Acid rain............................................................................................................ 83 2.5.3.4 Crop production and crop removal ................................................................... 83 2.5.3.5 Application of acid forming fertilizers ............................................................. 84 2.5.3.6 Oxidation of elemental sulphur ......................................................................... 85 2.5.3.7 Hydrolysis of Al 3+ ............................................................................................. 85 2.5.4 Effects of soil acidity on crop production ................................................................ 85 2.5.5 Management of soil acidity ...................................................................................... 86 2.5.5.1 Liming ............................................................................................................... 87 2.5.5.2 Application of organic materials ....................................................................... 87 CHAPTER THREE……………………………………………………………………………...89 MATERIALS AND METHODS .............................................................................................. 89 3.1 Soil sample collection ..................................................................................................... 89 3.2 Laboratory Analyses ....................................................................................................... 90 3.2.1 Particle size distribution ........................................................................................... 90 3.2.2 Bulk density ............................................................................................................. 91 3.2.3 Soil pH ..................................................................................................................... 92 3.2.4 Organic carbon ......................................................................................................... 92 3.2.5 Total nitrogen ........................................................................................................... 93 University of Ghana http://ugspace.ug.edu.gh xi 3.2.6 Available phosphorus............................................................................................... 94 3.2.7 Exchangeable cations ............................................................................................... 95 3.2.7.1 Potassium (K) determination ............................................................................ 95 3.2.7.2 Sodium (Na) determination............................................................................... 96 3.2.7.3 Calcium (Ca) determination .............................................................................. 96 3.2.7.4 Magnesium (Mg) determination ....................................................................... 97 3.2.8 Exchangeable acidity (H + and Al 3+ )......................................................................... 97 3.2.9 Effective Cation Exchange Capacity (ECEC) ......................................................... 98 3.3 Biochar production.......................................................................................................... 98 3.3.1 Biochar pH ............................................................................................................... 98 3.3.2 Ca, Mg, P, Na and K content of biochar .................................................................. 99 3.3.3 Mineral composition of biochar ............................................................................. 100 3.4 Experiment 1 ................................................................................................................. 100 3.4.1 Amount of diesel oil degraded ............................................................................... 100 3.4.2 Hydrocarbon-utilizing bacterial count ................................................................... 101 3.4.3 Heterotrophic bacterial count ................................................................................. 101 3.5 Experiment 2 ................................................................................................................. 102 3.6 Experiment 3 ................................................................................................................. 102 3.7 Statistical analysis ......................................................................................................... 103 University of Ghana http://ugspace.ug.edu.gh xii RESULTS ............................................................................................................................... 104 4.1 Soil characteristics ........................................................................................................ 104 4.2 Some soil properties after biochar amendment ............................................................. 104 4.2.1 Soil pH ................................................................................................................... 104 4.2.2 Organic Carbon (OC), Total Exchangeable Bases (TEB), Base Saturation (BS), Effective Cation Exchange Capacity (ECEC), Total Nitrogen (TN), Available P and Exchangeable acidity ...................................................................................................... 106 4.3 Amount of oil degraded ................................................................................................ 108 4.4 Hydrocarbon utilizing bacterial population .................................................................. 111 4.5 Heterotrophic bacterial count ........................................................................................ 114 4.6 Percentage of HUB present in total heterotrophic bacterial population (HET) ............ 115 4.7 Amount of oil remaining (%) in the soil after the treatment period (Exp. 2) ............... 117 4.8 Percent germination of cowpea ..................................................................................... 118 4.9.1 Nodule count .............................................................................................................. 119 4.9.2 Shoot dry weight and root dry weight per plant…………………………………..120 CHAPTER FIVE……………………………………………………………………………….122 DISCUSSION ......................................................................................................................... 122 5.1 Changes in Some Soil Properties after Biochar Amendment ....................................... 122 University of Ghana http://ugspace.ug.edu.gh xiii 5.1.1 Changes in soil pH after biochar amendment ........................................................ 122 5.1.2 Changes in OC, TEB, BS and ECEC after biochar amendment ............................ 123 5.1.3 Changes in total N after biochar amendment ......................................................... 124 5.1.4 Changes in soil available P after biochar amendment ........................................... 124 5.1.5 Changes in soil exchangeable acidity after biochar amendment ........................... 125 5.2 Hydrocarbon utilizing bacteria population ................................................................... 125 5.3 Amount of oil degraded ................................................................................................ 126 5.4 Growth and nodulation of cowpea ................................................................................ 127 CHAPTER SIX…………………………………………………………………………………131 CONCLUSION AND RECOMMENDATION .......................................................................... 131 REFERENCE…………………………………………………………………………………...132 APPENDIX……………………………………………………………………………………..165 University of Ghana http://ugspace.ug.edu.gh xiv LIST OF TABLES Table Page Table 2.1 Relative proportion range of the four main components of biochar (weight percentage) as commonly found for a variety of source materials and pyrolysis conditions ........................... 11 Table 2.2 Sources and estimates of crude oil or its products released into the environment ....... 40 Table 2.3 Successfully used microbial system or strains in bioremediation of oil polluted environment .................................................................................................................................. 60 Table 3.1 Some chemical properties of the biochar used………………………………………99 Table 4.1 Some physio-chemical properties of the soil used …………………………………..105 Table 4.2 Changes in soil pH after biochar amendment ............................................................. 106 Table 4.3 Some chemical properties of the treated soils at the end of experiment ..................... 107 University of Ghana http://ugspace.ug.edu.gh xv LIST OF FIGURES Figures Page Fig. 2.1 Putative structure of charcoal . .......................................................................................... 9 Fig. 2.2 Effect of biochar on soil ................................................................................................. 19 Fig. 2.3 Principle of aerobic degradation of hydrocarbon ............................................................ 62 Fig. 2.4 Major Constraints of Soil Acidification ......................................................................... 75 Fig. 2.5 Most Nutrients are Highest and Most Toxins are Lower at pH 5.5-7.0 .......................... 79 Fig. 2.6 Pools of soil acidity . ...................................................................................................... 79 Fig. 2.7 Types of soil acidity . ..................................................................................................... 80 Fig.4.1 A graph of cumulative amount of diesel oil degraded in experiment 1………………..109 Fig.4.2 A graph of cumulative amount of diesel oil degraded in experiment 2………………..111 Fig.4.3 A graph of HUB populations of the contaminated soils amended with biochar ............ 112 Fig.4.4 A graph of HUB populations of contaminated soils treated with biochar, N and P ....... 113 Fig.4.5 A graph of the total heterotrophic bacterial population in the contaminated soil amended with biochar. ............................................................................................................................... 115 Fig.4.6 A graph of % HUB in HET population in experiment 1 ................................................ 116 Fig.4.7 A graph of the amount of oil remaining in the treated soils at the end of Expt. 2 .......... 117 Fig.4.8 A graph of percent germination of cowpea per pot ........................................................ 118 University of Ghana http://ugspace.ug.edu.gh file:///L:/MPhil%20Thesis%20Abeka%20Hammond(Final.docx%23_Toc394337924 file:///L:/MPhil%20Thesis%20Abeka%20Hammond(Final.docx%23_Toc394337925 xvi Fig.4.9 A graph of nodule count per plant .................................................................................. 119 Fig.4.9.1 A graph of shoot dry weight per plant (g)……………………………………………120 Fig.4.9. 2 A graph of root dry weight per plant (g) .................................................................... 121 University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE INTRODUCTION The increase in urbanisation and mechanised agriculture has resulted in an increase in use of petroleum and its products (Ekpo and Nya, 2012).Accidental and deliberate crude oil spills have been and still continue to be a significant source of environmental pollution, and pose a serious environmental problem, due to the possibility of air, water and soil contamination (Trindade et al., 2005). For example, approximately 6 ×10 7 barrels of oil was spread over 2 ×10 7 m 3 soil and 320 oil lakes were created across the desert during the first Gulf War in Kuwait (Al Saleh and Obuekwe, 2005).Other sources of oil contamination include leakage from storage containers, refuelling of vehicles, wrecks of oil tankers carrying oil and improper disposal by mechanics when cleaning tankers (Hill and Moxey, 1980). Crude oil contamination of land negatively affects certain soil parameters such as the mineral and organic matter content, the cation exchange capacity, redox properties, available basic cations, available N and P and pH value (Wyszkowski and Ziolkowska, 2008). As crude oil creates anaerobic condition in the soil, coupled with water logging and acidic metabolites, the result is high accumulation of aluminum and manganese ions, which are toxic to plant and microbial growth (Odu, 1981).Pollution of the soil environment by crude oil can limit its protective function, upset metabolic activity, unfavourably affect soil chemical characteristics, reduce fertility and negatively influence plant production (Gong et al., 1996). Crude oil can bioaccumulate in food chains where they disrupt biochemical or physiological activities of many organisms thus, causing carcinogenesis of some organs, mutagenesis in the genetic material, and impairment in reproductive capacity and/or causing hemorrhage in the exposed population (Onwurah et al., 2007). Crude oil was found to reduce growth, photosynthetic rate, stem height, University of Ghana http://ugspace.ug.edu.gh 2 density, and above ground biomass of Spartina alterniflora and S. patens and may cause their death (Krebs and Tamer, 1981). Severe crude oil spill in Cross-River state, Nigeria, has forced some farmers to migrate out of their traditional home, especially those that depend solely on agriculture (Onwurah et al., 2007). This is because petroleum hydrocarbons ‗sterilize‘the soil and prevent crop and microbial growth and yield for a long period of time (Onwurah 1999a). The negative impact of oil spillages remains the major cause of depletion of the Niger Delta of Nigeria‘s vegetative cover and the mangrove ecosystem (Odu, 1981). Many published articles have documented the potentials of native microorganisms to degrade oil both in the laboratory (eg. Lawson et al., 2012) and in field trials (eg. Bragg et al., 1994).However, these potentials are to a large extent curtailed in unfavourable soil conditions.There are 30 or more different genera of bacteria and fungi known to degrade hydrocarbons intrinsically and are found in almost any soil or aquatic environment, but they need help to degrade oil effectively (Bragg et al., 1994). Help is needed more especially in Oxisols, where biodegradation is usually slower.Oxisolsare acidic in nature and are characterized by low nitrogen, phosphorus, organic carbon, basic cations (Ca, Mg, K, Na), and microbial activities(Beinroth et al., 1990). According to Walworth et al. (2005),Oxisols create such unfavourable conditions for microbial degradation that, there is about near zero degradation in the soil. Ghana discovered crude oil in 2007 at Cape Three Points in the Western Region and commercial production started in late 2010. However, on December 26, 2009, the country experienced its first spillage of about 584 barrels of low-based mud drilling fluid into the sea and the second mud spill of seven barrels occurred on March 23, 2010 (Daily Graphic, 2010). Even though these spillages were not into soils, many people have concerns about future soil University of Ghana http://ugspace.ug.edu.gh 3 contamination.Besides, soils of the Western Region of Ghana, where most of the oil activities take place are classified as Oxisols with low pH, because of the leaching of basic cations as a result of high rainfall. These acidic soilshave high levels of Fe and other heavy metals, and are dominated by acidic cations like Al 3+ and H + . These acidic soils also do not create favourable conditions for the cultivation of legumes because nodule initiation and formation, nitrogen fixation and growth are adversely affected due to low pH and unavailable phosphorus.It is well established that nitrogen fixing plants, either legumes or actinorhizal plants, require phosphate for adequate growth and root nodulation (Huss-Danell, 1997 and Marschner, 1995). The processes leading to the eventual removal of hydrocarbon pollutants from the environment have been extensively documented and involve the trio of physical, chemical and biological alternatives. However, bioremediation which is defined as any process that uses microorganisms or their enzymes to return the environment altered by contaminants to its original condition, is an attractive process due to its cost effectiveness and the benefit of pollutant mineralization to CO2 and H2O (da Cunha,1996). It also provides highly efficient and environmentally safe clean-up tools (Margesin, 2000). This technology accelerates the naturally occurring biodegradation under optimized conditions such as oxygen supply, temperature, pH, the presence or addition of suitable microbial population (bioaugmentation), nutrients (biostimulation) and water content (Trindade et al., 2005). However, there is the need for further studies towards optimizing the process conditions for the application of bioremediation strategies in diverse climatic zones especially in extreme environments (such as acidic soils).This is so because, the effectiveness of bioremediation is often a function of the microbial population and how it can be enriched and maintained in an environment such as acidic oil polluted soil. University of Ghana http://ugspace.ug.edu.gh 4 The need to modify unfavourable soil conditions to expedite microbial degradation has therefore become necessary. Biochar applications have been shown toincrease soil pH, improve nutrient storage, ECEC, increase soil carbon content, increase water holding capacity, decrease aluminum toxicity, decrease tensile strength, change microbiology of the soil, decrease greenhouse gases (N2O and CH4)emissions from the soil, improve soil conditions for earthworm populations and improve fertilizer use efficiency (Downie et al., 2009; Sohi et al. 2009; Mbagwu and Piccolo 1997; Piccolo et al., 1996; Piccolo and Mbagwu, 1990).Most literature have concluded that, the greatest positive effects of biochar were seen on acidic, free-draining soils, with other soil types, specifically calcarosols showing no significant effect (either positive or negative). Literature have also shown that biochar can stimulate soil microbial activities (Jones et al., 2011a, Jones et al., 2011b and Lehmann et al., 2011). It has a higher sorption affinity for a range of organic and inorganic compounds, and higher nutrient retention ability compared to other forms of soil organic matter (Bucheli and Gustafsson 2000, 2003; Allen-King et al. 2002). These multiple potential benefits of biochar, combined with the fact that it can potentially be a relatively low-cost and environmentally friendly tool for soil reclamation, provide incentive for more research. Hence, the present study seeks to rely on these numerous benefits of biochar in acidic soils, to create a favourable condition for the microorganisms involved in crude oil degradation to thrive and work effectively at a faster rate as well as provide favourable soil condition for crops like grain legumes that are sensitive to low pH.Although the use of biochar to enhance crude oil degradation is not a new development in Ghana especially, specific attention has not been focused on its use in acidic soils in the Western Region which has most of its soils dominated by Oxisols. The use of biochar has become necessary because, experts argue that the other physiochemical cleaning methods such as burying, evaporation, dispersion and washing of University of Ghana http://ugspace.ug.edu.gh http://www.sciencedirect.com/science/article/pii/S0038071711003865#bib29 http://www.sciencedirect.com/science/article/pii/S0038071711003865#bib30 http://www.sciencedirect.com/science/article/pii/S0038071711003865#bib30 http://www.sciencedirect.com/science/article/pii/S0038071711003865#bib38 5 contaminated soils cause geological damage which might even exceed the damage caused by the polluting oil (Bartha,1986), and they are also very expensive and not environmentally friendly. It would therefore be appropriate to research into the use of biochar in oil contaminated soils in Ghana that has just discovered oil in commercial quantities. The objectives of the present study therefore are to investigate 1. the effects of biochar on microbial degradation of diesel oil in acidic soils. 2. the ffects of supplementing biochar with nutrients on microbial degradation of diesel oil. 3. the growth and nodulation response of cowpea in oil-remediated soil. Hypothesis HO: Amendment of acidicsoil with biochar does not enhance microbial diesel oil degradation and subsequent crop growth. HA: Amendment of acidic soils with biochar enhances microbial diesel oil degradation and subsequent crop growth. University of Ghana http://ugspace.ug.edu.gh 6 CHAPTER TWO LITERATURE REVIEW 2.1 What is biochar? Biochar is an extremely complex stable form of carbon produced by the controlled heating of plant and/or animal material (biomass feedstock) at high temperatures (350 – 600 o C) in a low oxygen environment (Jenkins and Jenkinson, 2009). This definition includes chars and charcoal, and excludes fossil fuel products or geogenic carbon (Lehmann et al., 2006).The technique of heating in a low oxygen environment is called pyrolysis. Biochar‘s complex chemical structure is defined by the feedstock it is made from and the temperature conditions used in its manufacture. Biochar is a form of charcoal but is different in that, biochar is produced in controlled conditions so that most of the carbon is converted to usable products (Jenkins and Jenkinson, 2009). Charcoal usually has a total carbon content of over 75% whilst biochar often has much less total carbon (often 40-75%) but it has a higher mineral content, containing minerals such as Calcium (Ca), Potassium (K), Phosphorus (P) and Nitrogen (N) (Jenkins and Jenkinson, 2009). The characteristic of any biochar is a function of the material from which it is made and the temperature conditions used to make it. The range of biochars available could be considerable, representing a wide range of feedstock, temperature, residence times and heating rates used in their creation (Jenkins and Jenkinson, 2009). Incorporation of biochar into soil is shown to affect the preexisting soil properties in ways attributed to the physical and chemical properties of biochar. University of Ghana http://ugspace.ug.edu.gh 7 2.1.1 Physical properties of biochar Unlike the structure of graphite which consists of aromatic rings arranged in perfectly stacked and aligned sheets, biochar is made of irregular arrangements of C containing O and H and, in some cases, minerals depending upon feedstock (Lehmann and Joseph, 2009). Charred biomass consists of recalcitrant aromatic rings as well as more easily degradable aliphatic and oxidized carbon structures (Lehmann, 2007). Key physical features of most biochars are their highly porous structure and large surface area which can provide refuge for beneficial soil micro- organisms, such as mycorrhizae and bacteria, and influences the binding of important nutritive cations and anions (Atkinson et al., 2010). Biochar is often macro porous in nature which reflects cellular structures in the feedstock from which it is produced, which is potentially important for water holding and adsorption of soil (Sohi et al., 2010). When added to soil, biochar appears to divide rapidly into particles of silt size or less due to abrasion, shrink-swell, and other physical weathering processes (Brodowski et al., 2007). Process temperature is the main factor governing surface area, increasing in one study from 120 m 2 g -1 at 400 °C to 460 m 2 g -1 at 900 °C (Day et al., 2005). Low temperature biochar is stronger than high temperature products with regards to adsorptive properties, but it is more brittle and prone to abrading into finer fractions once incorporated into soil (Sohi et al., 2010). 2.1.2 Chemical properties of biochar Much research has produced unequivocal proof that biochar is not only more stable than any other amendment to soil and increases nutrient availability beyond a fertilizer effect, but its stability and nutrient retention properties make it more effective than any other organic material in soil (Lehmann and Joseph 2009). Chemical and physical properties such as high charge University of Ghana http://ugspace.ug.edu.gh 8 density and its particulate nature along with specific chemical structure, and high microbial and chemical stability, all contribute to greater nutrient retention and resistance to microbial decay than other organic matter (Atkinson et al., 2010). Baldock and Smernik (2002) determined that thermal treatment of organic materials at temperatures > 200°C induces significant variations in their chemical composition. Changes in chemical composition, as measured by 13 C nuclear magnetic resonance (NMR) indicated that changes with increased pyrolysis temperature included a conversion of O-alkyl C to aryl and O- aryl furan-like structures, which are more chemically active oxygen-containing carbon ring (Baldock and Smernik, 2002). Research suggests that biochar created at low temperatures may be suitable for controlling the release of fertilizer nutrients while high temperatures would lead to a material similar to activated carbon (Sohi et al., 2010). 2.1.3 Structural composition of biochar Thermal degradation of cellulose between 250 and 350ºC results in considerable mass loss in the form of volatiles, leaving behind a rigid amorphous C matrix (Baldock and Smernik, 2002). As the pyrolysis temperature increases, so does the proportion of aromatic carbon in the biochar, due to the relative increase in the loss of volatile matter (initially water, followed by hydrocarbons, tarry vapours, H2, CO and CO2), and the conversion of alkyl and O-alkyl C to aryl C (Baldock and Smernik, 2002; Demirbas 2004). Around 330ºC, polyaromatic graphene sheets begin to grow laterally, at the expense of the amorphous C phase, and eventually coalesce (Demirbas 2004; Baldock and Smernik, 2002). Above 600ºC, carbonization becomes the dominant process. Carbonization is marked by the removal of most of the remaining non-C atoms and consequent University of Ghana http://ugspace.ug.edu.gh 9 relative increase of the C content, which can be up to 90% (by weight) in biochars from woody feedstocks. (Antal and Gronli, 2003; Demirbas, 2004). Fig. 2.1 Putative structure of charcoal (adopted from Bourke et al., 2007). University of Ghana http://ugspace.ug.edu.gh 10 It is commonly accepted that each biochar particle comprises of two main structural fractions: stacked crystalline graphene sheets and randomly ordered amorphous aromatic structures (Fig. 2.1). Hydrogen (H), Oxygen (O), Nitrogen (N), Phosphorus (P) and Sulphur (S) are found predominantly incorporated within the aromatic rings as heteroatoms (Bourke et al., 2007). The presence of heteroatoms is thought to be a great contribution to the highly heterogeneous surface chemistry and reactivity of biochar. 2.1.4 Chemical composition and surface chemistry of biochar Biochar is produced from biomass and is predominantly composed of recalcitrant organic C with contents of plant micro and macro-nutrients retained from the starting feedstock. It is known from research on wildfire occurrence and the development of Anthrosols (e.g. Terra Preta soils) in the Amazon that charcoal can remain in the soil for hundreds to thousands of years (Agee 1996; Lehmann and Rondon 2006). Consequently, biochar can rapidly increase the recalcitrant soil C fraction of soil. The C in biochar is held in aromatic form which is resistant to decomposition when added as a soil amendment (Amonette and Joseph 2009), making it a C sequestration tool. However, composition varies by feedstock type and conditions of pyrolysis (Downie 2009). Actual C contents can range between 172 g kg -1 and 905 g kg -1 . Nitrogen content ranges from 1.8 g kg -1 to 56.4g kg -1 , total P from 2.7g kg -1 to 480g kg -1 and total potassium (K) from 1.0g kg -1 to 58g/ kg (Chan et al., 2007; Lehmann et al. 2002, Lima and Marshall 2005). Biochar also contains varying concentrations of other elements such as Oxygen (O), Hydrogen (H), Nitrogen (N), Sulphur (S), Phosphorus (P), base cations, and heavy metals (Goldberg 1985; Preston and Schmidt, 2006). The outer surfaces contain various O and H functional groups and the graphene sheets may contain O groups and free radicals (Bourke et al., 2007). Additionally, University of Ghana http://ugspace.ug.edu.gh 11 biochar has been produced with a range of pH values between 4 and 12, dependent upon the starting feedstock and operating conditions (Lehmann 2007). Generally, low pyrolysis temperatures (< 400° C) yield acidic biochar, while increasing pyrolysis temperatures produce alkaline biochar. Once incorporated into the soil, surface oxidation occurs due to reactions of water, Oxygen (O2) and various soil agents (Cheng et al., 2006; Lehmann, 2007). The cation exchange capacity (CEC) of fresh biochar is typically very low, but increases with time as the biochar ages in the presence of O2 and water (Cheng et al. 2008; Cheng et al., 2006; Liang et al., 2006). Biochar composition is highly heterogeneous, containing both stable and labile components (Sohi et al., 2009). Carbon, volatile matter, mineral matter (ash) and moisture are generally regarded as its major constituents (Antal and Gronli, 2003) and Table 2.1 summarizes ranges their relative proportion ranges in biochar as commonly found for a variety of source materials and pyrolysis conditions (Antal and Gronli, 2003; Brown, 2009). Table 2.1 Ranges of the relative proportion range of the four main components of biochar (weight percentage) as commonly found for a variety of source materials and pyrolysis conditions (adapted from Brown, 2009; Antal and Gronli, 2003) Component Proportion (w w -1 ) Fixed carbon 50-90 Volatile matter (e.g. tars) 0-40 Moisture 1-15 The relative proportion of biochar components determines the chemical and physical behaviour and function of biochar as a whole (Brown, 2009), which in turn determines its suitability for a site specific application, as well as transport and fate in the environment (Downie, 2009). For University of Ghana http://ugspace.ug.edu.gh 12 example, coarser and more resistant biochars are generated by pyrolysis of wood-based feedstocks (Winsley, 2007). In contrast, biochars produced from crop residues (e.g. rye, maize), manures and seaweed are generally finer and less robust (lower mechanical strength). The latter are also nutrient-rich, and therefore, more readily degradable by microbial communities in the environment (Sohi et al., 2009). The ash content of biochar is dependent on the ash content of the biomass feedstock. Grass, grain husks, straw residues and manures generally produce biochar with high ash contents, in contrast to that from woody feedstocks (Demirbas 2004). For instance, manure (e.g. chicken litter) biochars can contain 45% (by weight) as ash (Amonette and Joseph, 2009). Moisture is another critical component of biochar (Antal and Gronli, 2003), as higher moisture contents increase the costs of biochar production and transportation for unit of biochar produced. Keeping the moisture content up to 10% (by weight) appears to be desirable (Collison et al., 2009). In order for this to be achieved, pre-drying the biomass feedstock may be a necessity, which can be a challange in biochar production. Breaking and rearrangement of the chemical bonds in the biomass during processing results in the formation of numerous functional groups (e.g. hydroxyl -OH, amino-NH2, ketone -OR, ester -(C=O)OR, nitro - NO2, aldehyde -(C=O)H, carboxyl -(C=O)OH) occurring predominantly on the outer surface of the graphene sheets (e.g. Harris, 1997; Harris and Tsang, 1997) and surfaces of pores (van Zwieten et al., 2009). Some of these groups act as electron donors, while others as electron acceptors, resulting on coexisting areas which properties can range from acidic to basic and from hydrophilic to hydrophobic (Amonette and Joseph 2009). Some functional groups also contain other elements, such as N and S, particularly in biochars from manures, sewage sludge and rendering wastes. University of Ghana http://ugspace.ug.edu.gh 13 2.1.5 Pore size distribution and connectivity of biochar Biomass feedstock and the processing conditions are the main factors determining pore size distribution in biochar, and therefore its total surface area (Downie et al., 2009). During thermal decomposition of biomass, mass loss occurs mostly in the form of organic volatiles, leaving behind voids, which form an extensive pore network. Biochar pores are classified in this review into three categories (Downie et al., 2009), according to their internal diameters (ID): macropores (ID >50 nm), mesopores (2 nm< ID <50 nm) and micropores (ID <2 nm). These categories are orders of magnitude different to the standard categories for pore sizes in soil science. The elementary porosity and structure of the biomass feedstock is retained in the biochar product formed (Downie et al., 2009). The vascular structure of the original plant material, for example, is likely to contribute for the occurrence of macropores in biochar, as demonstrated for activated carbon from coal and wood precursors (Wildman and Derbyshire, 1991). In contrast, micropores are mainly formed during processing of the parent material. While macropores have been identified as a ‗feeder‘ to smaller pores (Martinez et al., 2006), micropores effectively account for the characteristically large surface area in charcoals (Brown, 2009). The development of microporosity in biochar, which is linked to an increase in structural and organizational order, has been shown to be favoured by higher pyrolysis temperature and retention times, as previously demonstrated for activated carbon (e.g. Lua et al., 2004). For example, increasing pyrolysis temperature from 250 to 500 o C enhanced the development of micropores in chars derived from pistachio-nut shells, due to increased evolution of volatiles. Similarly, heating rate and pressure during processing have also been found to influence the mass transfer of volatiles produced at any given temperature range, and are therefore regarded as key contributing parameters influencing pore size distribution (Antal and Grønli, 2003). University of Ghana http://ugspace.ug.edu.gh 14 2.1.6 Feedstock and its influence on biochar characteristics Feedstock is the term conventionally used for the type of biomass that is pyrolysed and turned into biochar. Feedstock is, along with pyrolysis conditions, the most important factor controlling the properties of the resulting biochar. Firstly, the chemical and structural composition of the biomass feedstock relates to the chemical and structural composition of the resulting biochar and, therefore, is reflected in its behaviour, function and fate in soils. Secondly, the extent of the physical and chemical alterations undergone by the biomass during pyrolysis (e.g. attrition, cracking, microstructural rearrangements) is dependent on the processing conditions (mainly temperature and residence times). Pyrolysis of wood-based feedstocks generates coarser and more resistant biochars with carbon contents of up to 80%, as the rigid ligninolytic nature of the source material is retained in the biochar residue (Winsley, 2007). Biomass with high lignin contents (e.g. olive husks) have shown to produce some of the highest biochar yields, given the stability of lignin to thermal degradation, as demonstrated by Demirbas (2004). Whereas woody feedstock generally contains low proportions (< 1% by weight) of ash, biomass with high mineral contents such as grass, grain husks and straw residues generally produce ash- rich biochar (Demirbas, 2004). These latter feedstocks may contain ash up to 24% or even 41% by weight, such as rice husk (Amonette and Joseph, 2009) and rice hulls (Antal and Grønly, 2003), respectively. The mineral content of the feedstock is largely retained in the resulting biochar, where it concentrates due to the gradual loss of carbon (C), hydrogen (H) and oxygen (O) during processing (Demirbas, 2004). The mineral ash content of the feedstock can vary widely and evidence seems to suggest a relationship between mineral ash and biochar yield University of Ghana http://ugspace.ug.edu.gh 15 (Amonette and Joseph, 2009). Many different materials have been proposed as biomass feedstocks for biochar, including wood, grain husks, nut shells, manure and crop residues, while those with the highest carbon contents (e.g. wood, nut shells), abundance and lower associated costs are currently used for the production of activated carbon (e.g. Lua et al., 2004; Martinez et al., 2006; Gonzaléz et al., 2009). Crystalline silica has also been found to occur in some biochars. Rice husk and rice straw contain unusually high levels of silica (220 and 170 g kg -1 ) compared to that in other major crops (van Zwieten et al., 2007). High concentrations of calcium carbonate (CaCO3) can be found in pulp and paper sludge (van Zwieten et al., 2007) and are retained in the ash fraction of some biochars. Regarding the characteristics of some plant feedstocks, Collison et al. (2009) go further, suggesting that even within a biomass feedstock type, different compositions may arise from distinct growing environmental conditions (e.g. soil type, temperature and moisture content) and those relating to the time of harvest. In general, wood biochars had higher total C, lower ash content, lower contents of total nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), aluminum (Al), sodium (Na), sulfur(S), and copper(Cu), and lower potential CEC and exchangeable cations than poultry litter biochars, whereas tree leaf biochars were generally intermediate (Singh et al., 2010). Much of the mineral content of the feedstock remains in the resulting biochar, where it is concentrated due to the loss of C, H and O during pyrolysis (Amonette and Joseph, 2009). There is considerable variation in the content of many elements especially N and P due to feedstock characteristics and range of production temperatures. Feedstocks typically high in N, P, K and S are sewage sludge, animal manures and biosolids. University of Ghana http://ugspace.ug.edu.gh 16 2.1.7 Temperature and its influence on biochar characteristics High temperature biochar pyrolyzed at 700 ˚C has recalcitrant characteristics and is advantageous when the chief objective is to remove atmospheric CO2 and sequester C in soil for millennia (Keiluweit et al., 2010). However, synchrotron-based near edge X-ray absorption fine structure (NEXAFS) spectra have revealed that biochars produced at high temperatures are typically poorly crystalline (Keiluweit et al., 2010). This implies that some metals in the C lattice may possibly be volatilized, and that the mineral fraction will be less (Bridgwater and Boocock, 2006). Therefore, these biochars would consequently have lesser reactivity in soils than lower temperature biochars, which tend to have a better impact on soil fertility (Steinbeiss et al., 2009). A study carried out by Gaskin et al. (2008) showed that biochars produced at 500 ˚C concentrated their most essential plant nutrients; namely P, K, Ca, and Mg. This subsequently led to considerably higher quantities in the final biochar product. Consequently, biochar that is produced with the key role of being a soil fertility amendment needs to be specifically aimed at carbonizing the biomass material under moist conditions and at low temperatures (Novak et al., 2009). Investigations conducted on the effect of different pyrolytic temperatures on pine chars showed that there was a reduction in the organic content with increasing pyrolytic temperature in the range of 300 to 700 °C. These studies also showed that the weight loss of chars declined from 37 % to 24 % when the biomass was pyrolyzed at 500 °C during different time intervals comprising 10 to 300 minutes. Therefore, it was suggested that pyrolytic temperatures play a more important role than pyrolytic time to carbonize pine wood (Zhou et al., 2009). Other studies revealed that the pyrolysis temperature has an effect on the yield of biofuel and biochar. An increase in temperature resulted in a reduction in the recovery of biochar, while the concentration of carbon increased (Daud et al., 2001; Demirbas, 2004). University of Ghana http://ugspace.ug.edu.gh 17 In a recent study, Cao and Harris (2010) investigated the effect that different heating temperatures have on the physical, chemical, and mineralogical properties of dairy-manure derived biochar. The untreated air dried biochar was dried at a room temperature of 25 °C and 500 °C respectivelyand used for comparative purposes. It was found that the following properties increased as a result of increased temperature during pyrolysis; specific surface area (SSA), ash content, pH and concentrations of P, Ca, and Mg. The SSA increased exponentially between 200 and 500 °C. The increase in ash content was due to the high presence of calcite and quartz minerals in the manure. At a temperature of 500 °C, the biochar produced more than 95 % ash, thus indicating the complete combustion of C. The pH increase was dependent on the heating temperature. Initially, the untreated manure at room temperature was alkaline at pH 7.5-8.0. At 200 °C, the pH declined to neutrality at about pH 7 (Cao and Harris 2010). At temperatures of 300 °C and above, the C began to ash, and subsequently increased the biochar pH to above 10.0, where it became constant (Cao and Harris 2010). In addition, the mean total P, Ca, and Mg concentrations increased from 0.91 %, 3.23 %, and 1.11 %, respectively at 100 °C to 2.66 %, 9.75 %, and 3.02 % at 500 °C. The total P, Ca, and Mg increases were attributed to increasing temperature. 2.2 Biochar’s Impact on Soil Performance  Improve nutrients availability, storage and CEC  Increase soil carbon content  Increase Water Holding Capacity  Increase soil pH University of Ghana http://ugspace.ug.edu.gh 18  Decrease Aluminum toxicity  Decrease tensile strength  Change microbiology of the soil  Decrease greenhouse gases emissions from the soil (N2O and CH4 )  Improve soil conditions for earthworm populations  Improve fertilizer use efficiency.  Increase soil water infiltration and permeability  Sorption of organic and other chemical substances 2.2.1 Improvement of NutrientAvailability, Storage and CEC 2.2.1.1 Biochar effect on soil N Lehmann et al. (2006) have suggested that biochar can adsorb both NH4 + and NH3 - from the soil solution thus reducing solution inorganic N at least temporarily, but perhaps concentrating it for microbial use. Biochar is an N depleted material having a uniquely high C/N ratio (839). It is also possible that some amount of decomposition might have occurred when fresh biochar is added to soil (Schneour, 1966; Liang et al, 2006), which could induce net immobilization of inorganic N already present in the soil solution. Gundale and DeLuca (2006) reported that biochar addition to soil caused reduction in ammonification compared to the control due to adsorption and reduced the potential for NH3 volatilization. The reduction could be due to high C/N ratio of biochar and greater potential for N immobilization (Lehmann et al., 2006). It should be noted however that, immobilization potential associated with biochar additions to soil would be greatly limited by the recalcitrant nature of biochar (DeLuca and Aplet, 2007). University of Ghana http://ugspace.ug.edu.gh 19 Biochar has the potential to catalyze the reduction of N2O to N2; potentially reducing the emission of this important greenhouse gas to the atmosphere, and thus biochar could directly or indirectly influence denitrification. The process of denitrification requires the presence of substrate (available C) and a terminal electron acceptor, such as NO3 - (Stevenson and Cole, 1999). 2.2.1.2 Biochar effect on P availability Fig. 2.2 Effect of biochar on soil (Cowie A. et al., 2006) Soils found in tropical regions are particularly poor in plant available phosphorus resulting in P deficient environments. These soils contain sesquioxides that have the ability to strongly sorb phosphate (Turner et al. 2007), and thereby creating a sink on the availability of inorganic The effect of biochar on P Indirect Direct Reduction of P fixation (pH, CEC, Microbial activities Water-soluble P University of Ghana http://ugspace.ug.edu.gh 20 phosphorus for plants (Oberson et al., 2006). Sandy textured soils give biochar the potential to ameliorate P leaching in soils, therefore, it is expected that P will increase with increasing levels of biochar additions (Novak et al., 2009). Addition of black charcoal to the previously mentioned Ferralsol and Anthrosol was correlated with increased phosphorus nutrition and plant uptake. Higher crop growth observed in this Anthrosol compared to the Ferralsol was largely an effect of elevated phosphorus and other nutrient availability along with comparatively low nutrient leaching (Lehmann et al., 2003). The availability and, subsequently, the adsorption of phosphorus is highly pH dependent. Increases in soil pH are likely to influence P availability, with available forms most common between pH of 4 to 8.5 (Atkinson et al., 2010). The availability of some elements toxic to plant growth, particularly at low pH, such as Al, Cu and Mn, can be reduced by biochar incorporation while the availability of other elements can increase, with biochar induced increases in soil pH enhancing solubility of phosphorus as well as N, Ca, Mg and Mo (Atkinson et al., 2010). The increasingly investigated characteristics of biochar uphold a reputation for it to help ameliorate problems of poorly fertile soils. Additions were investigated to determine if biochar could contribute to improving fertility of a sandy, acidic soil (Novak et al., 2009). Phosphorus concentration in leachate was found to decrease with increasing biochar application (Novak et al., 2009). The decrease was attributed to a combination of reactions such as retention of o-PO4 3- through ligand exchange reactions involving oxygen-containing functional groups on the biochar surface, adsorption of o-PO4 3- by Fe and Al oxides and hydroxides, and by adsorption and precipitation by Ca, Mg-phosphates (Bohn et al., 1979). Additions of biochar to soils result in alterations with effects that are beneficial to phosphate solubilizing bacteria (PSB), whose activities increase soil P (Lehmann et al., 2007). University of Ghana http://ugspace.ug.edu.gh 21 2.2.1.3 Biochar effect on other nutrients Incorporation of biochar into acid soils increased soil cation exchange capacity (CEC) and adsorption capacity of the soils for selected nutrients (Steiner et al., 2008; Novak et al., 2009; Sohi et al., 2010), especially in tropical and subtropical regions. Many studies have analyzed biochar‘s effect on nutrient availability and leaching, and have shown that it clearly has an influence on nutrient transformations. The extent of this influence depends highly on the ion of interest and the properties of biochar obtained from the feedstock and soil environment. The sources of organic matter used as biochar feedstocks are shown to alter the availability of key macronutrients such as N and P, and some metal ions such as Ca and Mg, when incorporated into the soil (Atkinson et al., 2010). Both increasing and decreasing nutrient uptake and biomass productivity have been reported following biochar additions to soil and the effect of biochar additions on nutrient availability is not yet entirely clear (Lehmann et al., 2003). Large proportions of black carbon in an Anthrosol of the Amazon basin was found to have significantly higher availability of P, Ca, Mn, and Zn than a nearby Ferrasol, minimal nutrient leaching, and increased plant uptake of P, K, Ca, Zn, and Cu(Lehmann et al., 2003). 2.2.1.4 Biochar effect on CEC Biochar was shown to increase the cation exchange capacity (Lehmann et al., 2003). Evidence suggests the cation exchange capacity (CEC) of biochar is consistently higher than that of the whole soil, clay minerals, or soil organic matter (Sohi et al., 2010). Soil CEC increases are due to carboxylate groups on the surfaces of the biochar itself and exposed carboxylate groups of organic acids sorbed by the biochar, both of which contribute negative surface charge to biochar University of Ghana http://ugspace.ug.edu.gh 22 particles (Novak et al., 2009). Simultaneously, increases in charge density per unit surface of organic matter develop, which equate with a greater degree of oxidation, or increases in surface area for cation adsorption, or a combination of both (Atkinson et al., 2010). 2.2.2 Biochar effect on soil organic carbon A change in microbial abundance and community structure may affect not only biochar mineralization itself, but also mineralization of other soil C (Busscher et al., 2010). The commonly observed greater microbial biomass has been presented as a reason for a greater decomposition of soil C (also called priming) in the presence of biochar (Wardle et al., 2008). The fact that this has generally not been observed beyond an initial greater mineralization after fresh biochar additions (Hamer et al., 2004; Wardle et al., 2008; Zimmerman et al., 2011) suggests different explanations for the C loss observed in these studies that may instead be related to physical export of C, changes in nutrient contents or pH (Lehmann and Sohi, 2008). Also, labile substances in biochars (such as condensable volatiles as found in smoke) may stimulate microbial activity shortly after biochar application to soil (Fischer and Bienkowski, 1999; Uvarov, 2000; Das et al., 2008; Steiner et al., 2008a), but these are mineralized within a relatively short period of time (Cheng et al., 2006). Longer incubations (beyond one year) and field trials have shown that biochars decrease mineralization of other soil C (Kuzyakov et al., 2009; Kimetu and Lehmann, 2010; Zimmerman et al., 2011). However, the conundrum of greater microbial biomass yet lower soil C respiration still warrants closer examination. Interestingly, similar observations of greater microbial biomass yet lower metabolism have been made in waste water treatment, where biofilms on sand showed greater removal and University of Ghana http://ugspace.ug.edu.gh 23 mineralization rates of dissolved aromatic C than biofilms on activated carbons (Koch et al., 1991) that typically have large surface areas (Downie et al., 2009). It is possible that CO2 precipitates as carbonates on biochar surfaces that have high pH and abundant alkaline metals, which would explain reduced detection of CO2 evolved, despite measured increases in microbial biomass. For example, lipases have been shown to sorb well to activated carbon matrices with long life and high activity (Quirós et al., 2011). So called ―immobilization‖ of enzymes on materials such as biochar is by now used in many industrial processes that allow stable conditions for optimum enzyme activity (Novick and Rozzell, 2005). A dominance of certain groups of microorganisms, such as coenocytic fungi degrading simple C compounds (e.g., Zygomycota) was observed when corn biochar was added to a temperate Alfisol, whereas, abundance of septate fungi (such as Basidiomycota, known lignin degraders) and Ascomycota) decreased (Jin, 2010). An increase in fungi that metabolize simpler sugars would be in accordance with greater microbial biomass and sorption of labile C compounds on biochar surfaces, rather than the inaccessibility of sorbed organic matter (Jin, 2010). 2.2.3 Biochars’ effect on water holding capacity of the soil Biochar incorporation into a soil can have widespread impacts on the intrinsic properties of a soil. Water holding capacity is influenced by both the mineral and organic components of a soil (Glaser et al., 2002). Higher levels of organic matter are associated with higher water holding capacity and Glaser et al. (2002) found water retention to be 18% higher in terra preta than in adjacent soils, a difference believed to be attributed to the higher biochar content and higher levels of organic matter associated with charcoal in these soils. The high stability of biochar, due to the extensive structure of aromatic carbons, offers potential for providing long-term University of Ghana http://ugspace.ug.edu.gh 24 modification to soil water holding capacity through its generally macro porous nature (Sohi et al., 2010). It is found that the long-term effect of biochar on available moisture will be positive in sandy soils dominated by larger pores, than in neutral or in medium-textured soils, and potentially detrimental in clay soils (Sohi et al., 2010). Gaskin et al. (2007) determined moisture release curves for a loamy sand field soil to which different amounts of biochar were added. The highest application rate was determined to have a significant effect on volumetric water content, double that of the control soil containing no biochar (Gaskin et al., 2007). Tryon (1948) studied the effect of charcoal on thepercentage of available moisture in soils of different textures and found different responsesamong soils. In sandy soil, the addition of charcoal increased available moisture by 18%after adding 45% biochar by volume, while no changes were observed in loamy soil, and soilavailable moisture decreased in the clayey soil. The high surface area of biochar can lead toincreased water retention, although the effect seems to depend on the initial texture of thesoil. Improved water holding capacity with biochar additions is most commonly observed in coarse-textured or sandy soils (Gaskin et al., 2007; Glaser et al., 2002). The impact of biochar additions on moisture content may be due to increased surface area relative to that found in coarse-textured soils (Glaser et al., 2002). Therefore, improvements in soil water retention by biochar additions may only be expected in coarse-textured soils or soils with large amounts of macro pores. Additionally, a large amount of biochar may need to be applied to the soil before it increases water retention. 2.2.4 Biochar effect on pH and aluminum toxicity Hydrogen (H + ) and aluminum (Al 3+ ) ion dominance in the soil exchangeable complex causes acidity which limits crop yield and utilization of many essential nutrients by plants and microorganisms (Black, 1993; Chintala et al., 2012a). Liming to remediate acidic soils has a University of Ghana http://ugspace.ug.edu.gh 25 longer history than the use of any other forms of soil amendments (McLean, 1971). Biochar contains some alkaline materials and has relatively high pH (Steiner et al., 2007; Gaskin et al., 2008) and, thus, can neutralize soil acidity and increase the pH of acid soils (Chan et al., 2008; Novak et al., 2009). Effects of biochar incorporation on pH and exchangeable acidity of acid soils have been reported (Chan et al., 2007; 2008; van Zwieten et al., 2010). Biochars can have pH values of below 4 or above 12, depending on feedstock type and pyrolysis temperature (Lehmann, 2007a; Chan and Xu, 2009). Similar to nutrient and C changes, the effects of pH changes induced by biochar will largely depend on the pre-existing soil pH, the direction and magnitude of change (Novak et al., 2009). Higher pyrolytic temperature (>400°C) was observed to produce biochars with alkaline pH (Novak et al., 2009). Before applying these biochars to acidic soils as amendment, it will be necessary to analyze their composition and liming potential. The physical and chemical characteristics of any amendment determine its effectiveness as liming agent (Barber, 1984). The liming effect of any amendment can be determined by studying soil indices such as soil pH and exchangeable acidity (Wong and Swift, 1995; Wang et al., 2009). Liming potential of a material can also be predicted by its properties such as calcium carbonate equivalence (Mokolobate and, Haynes 2002) and ash alkalinity (Noble et al., 1996). The ameliorating effect of biochars on acidic soil was assumed to be consistent with their composition and properties which depend on biomass feedstock type and pyrolytic conditions (Noble et al., 1996). Soil pH has the potential to undergo a change when either the biochar or a cation in the biochar reacts with the soluble monomeric Al species, or alternatively displaces it from the exchange sites of clay or soil organic matter (Sparks, 2003). Depending on the biochar biomass used, basic University of Ghana http://ugspace.ug.edu.gh 26 cations such as Ca, K, Mg, and silicon (Si) can form alkaline oxides or carbonates during the pyrolysis process (Noble et al., 1996). Following the release of these oxides into the environment, they can react with the H + and monomeric Al species, raise the soil pH, and decrease exchangeable acidity (Novak et al., 2009). Furthermore, research conducted by Novak et al. (2009) on pecan shell derived biochar revealed that there was a high concentration of calcium oxide (CaO) in the biochar, which neutralizes soil acidity as follows: 2Al – soil + 3CaO + 3H2O → 3Ca – soil + 2Al (OH)3 The reaction describes the reduction in exchangeable acidity whereby Ca replaces the monomeric Al species on the soil exchangeable sites and generates alkalinity. Subsequently, there is an increase in soil solution pH as a result of the reduction of the readily hydrolysable monomeric Al and the subsequent formation of the neutral [Al(OH)3]0 species (Sparks, 2003). 2.2.5 Biochar effect on soil biology The soil biota is vital to the functioning of soils and provides many essential ecosystem services. Understanding the interactions between biochar when it is used as a soil amendment, and the soil biota is therefore vital. It is largely through interactions with the soil biota, such as promoting arbuscular mychorrizal fungi (AMF) as well as influencing on water holding capacity, which lead to the reported effects of biochar on yields(Steiner et al., 2008; Kolb et al., 2009). Soil is a highly complex and dynamic habitat for organisms, containing many different niches due to its incredibly high levels of heterogeneity at all scales. On the micro scale, soil is often an aquatic habitat, as micro pores in soil are full of water at all times, apart from during very extreme drought, due to the high water tension which exists there (Steiner et al., 2008; Kolb et al., 2009). This is vital for the survival of many microbial species which require the presence of water for mobility as well as to function (Steiner et al., 2008; Kolb et al., 2009). Indeed, many soil University of Ghana http://ugspace.ug.edu.gh 27 organisms, specifically nematodes and microorganisms such as protozoa enter a state of cryptobiosis, whereby they enter a protective cyst form and all metabolism stops in the absence of water (Steiner et al., 2008; Kolb et al., 2009). When biochar application leads to an increased water retention of soils, it seems likely therefore that this will have a positive effect on soil organism activity, which may well lead to concurrent increases in soil functioning and the ecosystem services which it provides. Organisms in the soil form complex communities and food webs and engage in many different techniques for survival and to avoid becoming prey, ranging from hiding in safe refuges, through to conducting forms of chemical ‗warfare‘ (Zackrisson et al., 1996). Biochar, due to its highly porous nature, has been shown to provide increased levels of refugia where smaller organisms can live in small spaces which larger organisms cannot enter to prey on them. Microorganisms within these micro pores are likely to be restricted in growth rate due to relying on diffusion to bring necessary nutrients and gases, but as this occurs in micro pores within the soil, this demonstrates that microorganisms utilizing these refugia almost certainly would not be reliant of decomposition of the biochar for an energy source. This is likely to be one of the mechanisms for the demonstrated increases in microbial biomass (Steiner et al., 2008; Kolb et al., 2009), and combined with the increased water holding potentials of soil is a possible mechanisms for the increased observed basal microbial activity (Steiner et al., 2008; Kolb et al., 2009). However, due to the complexities of the soil system and its biota, it is probable that many more mechanisms are at work. For example Kolb et al. (2009) demonstrated that while charcoal additions affected microbial biomass and microbial activity, as well as nutrient availability, differences in the magnitude of the microbial response was dependent on the differences in base nutrient availability in the soils studied. However, they noted that the influences of biochar on the soil microbiota acted in a relatively similar way in the soils they studied, albeit at different University of Ghana http://ugspace.ug.edu.gh 28 levels of magnitude, and so suggested that there is considerable predictability in the response of the soil biota to biochar application. There is some evidence that the positive effects of biochar on plant production may be attributable to increased mycorrhizal associations (Nisho and Okano, 1991). The majority of studies concerning biochar effects on mycorrhiza show that there is a strong positive effect on mycorrhizal abundance associated with biochar in soil (Harvey et al., 1976; Ishii and Kadoya, 1994). The possible mechanisms were hypothesised by Warnock et al. (2007) to include (in decreasing order of currently available experimental evidence) a) Alteration of soil physico-chemical properties b) Indirect effects on mycorrhizae through effects on other soil microbes c) Plant–fungus signaling interference and detoxification of allelochemicals on biochar d) Provision of refugia from fungal grazers 2.2.6 Biochar effect on climate change Incorporation of biochar into soils increases the locking of atmospheric carbon dioxide (CO2) through a C-negative process (Glaser et al., 2009) and thus reduces emission of greenhouse gases such as CO2, methane (CH4), and nitrous oxide (N2O) compared with its feedstock (Lehmann et al., 2006; Spokas and Reicosky, 2009). Biochar is primarily composed of both single and condensed ring aromatic C, and subsequently has a mutual high surface area per unit mass and a high surface charge density (Lehmann 2007a). The biochars largely composed of single-ring aromatic and aliphatic C mineralize more rapidly in comparison to those composed of condensed aromatic C (Lehmann, 2007a; Novotny et al., University of Ghana http://ugspace.ug.edu.gh 29 2007). Spectra using NEXAFS reveal that aromatic and quinonic compounds are more common when aliphatic groups are lost at 400 ˚C (Keiluweit et al., 2010). Lehmann (2007a) reported that biochar may be an alternative to renewable energy because it is not carbon neutral, but rather carbon negative. This implies that because biochar is formed by a carbon negative process, it may serve as a long-term terrestrial sink of carbon. The carbon negative process means that the feedstock parent material used to manufacture biochar initially withdraws organic carbon from photosynthesis and decomposition carbon cycle pathway (Lehmann, 2007b). This process is then followed by storing this organic carbon in the soil, thus causing it to accumulate over time (Glaser, 2007). Relative to merely using fresh material to store C, because biochar decomposes over a long period of time, it is able to create the slow release of CO2 into the atmosphere over an extended period, and thus reduces CO2 emissions (Gaunt and Lehmann, 2008). Therefore, because biochar is able to gain CO2 from the atmosphere, it would circumvent from the contribution of climate change, and hence aid in reducing global warming (Lehmann, 2007a). It is generally accepted that reducing atmospheric concentrations of CO2 by permanently sequestering C in the soil could reduce the impact of climate-related damage. Increasing soil organic carbon (SOC) storage by conventional soil management practices such as conservation tillage, no-till, and perennial cropping systems can take many years and there is uncertainty about the C sequestration potential of these systems (Baker et. al., 2007; Denman et al., 2007). By contrast, application of biochar to agricultural soils is an immediate and easily quantifiable means of sequestering C and is rapidly emerging as a new management option that may merit high value C credits (McHenry, 2008; Glaser at al., 2009; Tenenbaum, 2009; Steinbeiss et. al., 2009). University of Ghana http://ugspace.ug.edu.gh 30 In many studies where biochar has been shown to reduce N2O fluxes, a number of mechanisms have been proposed based mainly on prior knowledge of the requirements of nitrifiers and denitrifiers. These include: (i) enhanced soil aeration (reduced soil moisture) inhibiting denitrification due to more oxygen being present; (ii) labile C in the biochar promoting complete denitrification i.e., dinitrogen (N2)formation; (iii) the elevated pH of the biochar creating an environment where N2O reductase activity is enhanced thus promoting N2 formation and higher N2/N2O ratios; and (iv) a reduction in the inorganic-N pool available for the nitrifiers and/or denitrifiers that produce N2O, as a result of NH4 + and/or NO3 - adsorption, greater plant growth, NH3 volatilisation loss, or immobilisation of N. Increases in N2O fluxes have been attributed to: (i) the release of biochar embodied-N or priming effects on SOM following biochar addition; (ii) biochar increasing soil water content and improving conditions for denitrification; and (iii) biochar providing inorganic-N and/or carbon substrate for microbes(Knoblauch et al., 2011; Scheer et al., 2011; Taghizadeh-Toosi et al., 2011; Clough et al., 2010; Singh et al., 2010; Van Zwieten et al., 2010b; Zhang et al., 2010 ; Spokas and Reicosky, 2009; Yanai et al., 2007). 2.2.7 Biochar effect on sorption of hydrophobic organic compounds (HOCS) and others The sorption of anthropogenic hydrophobic organic compounds (HOC) (e.g. PAHs, polychlorinated biphenyl - PCBs, pesticides and herbicides) in soils and sediments, is generally described based on two coexisting simultaneous processes: absorption into natural (amorphous) organic matter (NOM) and adsorption onto occurring charcoal materials (Cornelissen et al., 2005; Koelmans et al., 2006). Comparatively to that of NOM, charcoals (including soot) generally hold up to 10 to1000 times‘ higher sorption affinities towards such compounds (Chiou and Kile, 1998; Bucheli and Gustafsson, 2000, 2003). It has been estimated that black carbon (BC) can account for as much as 80 to 90% of total uptake of trace HOC in soils and sediments University of Ghana http://ugspace.ug.edu.gh 31 (Cornelissen et al., 2005), and that it applies to a much broader range of chemical species than previously thought (Bucheli and Gustafsson, 2003; Cornelissen et al., 2004). Biochar application is therefore expected to improve the overall sorption capacity of soils (Chiou 1998), and consequently, influence toxicity, transport and fate of trace contaminants, which may be already present or are to be added to soils. Enhanced sorption capacity of a silt loam for diuron (Yang and Sheng, 2006) and other anionic (Hiller et al., 2007) and cationic (Sheng et al., 2005) herbicides has previously been reported following the incorporation of biochar ash from crop (wheat and rice) residues. The relative importance of these latter studies is justified by the fact that charring of crop residues is a widespread agricultural practice (Hiller et al., 2007). Previous studies have convincingly demonstrated that adsorption to charcoals is mainly influenced by the structural and chemical properties of the contaminant (i.e. molecular weight, hydrophobicity, planarity) (Cornelissen et al., 2004, 2005; Zhu and Pignatello, 2005; Zhu et al., 2005; Wang et al., 2006), as well as pore size distribution, surface area and functionality of the charcoal (e.g. Wang et al., 2006; Chen et al., 2007). For example, sorption of tri- and tetra- substituted-benzenes (such as trichlorobenzene, trinitrotoluene and tetramethylbenzene) to maple wood charcoal (400°C) was sterically restricted, when compared to that of the lower size benzene and toluene (Zhu and Pignatello, 2005). In fact, experimental evidence has recently demonstrated that organic structures in the form of BC (including biochar) or NOM, which are equipped with strong aromatic π-donor and -acceptor components, are capable of strongly adsorbing to other aromatic moieties through specific sorptive forces other than hydrophobic interactions (Keiluweit and Kleber, 2009). Although a large body of evidence is available on the way the characteristics of HOC influence sorption to biochars, the contribution of the char‘s properties to that process has been far less evaluated. It is University of Ghana http://ugspace.ug.edu.gh 32 generally accepted that mechanisms leading to an increase in surface area and/or hydrophobicity of the char, reflected in an enhanced sorption affinity and capacity towards trace contaminants, as demonstrated for other forms of BC (Jonker and Koelmans, 2002). The influence of pyrolysis temperatures mostly in the 340-400°C range (James et al., 2005; Zhu et al., 2005) and feedstock type (Pastor-Villegas et al., 2006) on such phenomena has been recently evaluated for various wood chars by a number of authors. Interestingly, sorption to high-temperature chars appears to be exclusively by surface adsorption, while that to low-temperature chars derive from both surface adsorption and (at a smaller scale) absorption to residual organic matter (Chun et al., 2004). The influence of micro pore distribution on sorption to biochars has been clearly demonstrated by Wang et al. (2006). Diminished O functionality on the edges of biochar‘s graphene sheets due to heat treatment (e.g. further charring), resulted in enhanced hydrophobicity and affinity for both polar and non-polar compounds, by reducing competitive adsorption by water molecules (Zhu et al., 2005; Wang et al., 2013). The treated char also revealed a consistent increase in micropore volume and pore surface area, resulting in better accessibility of solute molecules and an increase in sorption sites (Wang et al., 2013). The underlying sorption mechanism, including the way it is influenced by a wide range of factors inherent to the contaminant, to the char material and to the environment, remains far from being fully understood (Fernandes and Brooks, 2003). In this context, it is vital to comprehensively assess the environmental risk associated to these species in biochar-enriched soils, while re- evaluating both the use of generic OC-water distribution coefficients (Jonker et al., 2005) and of remediation endpoints (Cornelissen et al., 2005). For instance, remediation endpoints (undetectable, non-toxic or environmentally acceptable concentrations, as set by regulatory University of Ghana http://ugspace.ug.edu.gh 33 agencies) for common environmental contaminants in biochar-enriched soils would need to be assessed based on dissolved (bioavailable) concentrations rather than on total concentrations (Cornelissen et al., 2005). In order to achieve that, prior careful experimental evaluation of the contaminant distribution, mobility and availability in the presence of biochar is paramount. 2.2.8 Impact of biochar on crop productivity Positive yield effects from biochar addition were reported by Kimetu et al. (2008), who were able to establish that the impact was in part due to non-nutrient improvement to soil function. Improved fertilizer use efficiency was pin-pointed as an explanation for biochar maintaining crop yields after forest clearance in Amazonia, in essentially a recreation of terra preta (Steiner et al., 2008a). Biochar-amended plots receiving NPK sustained higher crop yield compared to control plots where yield declined rapidly. Results from semi-arid soils in Australia have shown positive response of rice to biochar in combination with fertilizer in pot trials (Chan et al., 2007), and in Indonesia maize and peanut yields were enhanced where bark charcoal was applied in combination with N fertilizer in the field (Yamato et al., 2006). The view that nutrient management and pre-existing soil nutrient status determine crop response to biochar was supported by a study on rice (Asai et al., 2009), where statistically higher first-season yield was observed only when biochar (at a low rate) was applied together with fertilizer N and in a low- yielding crop variety. Yield was lower than the control in an equivalent treatment using a high- yielding (and thus N-demanding) variety. However, some studies show no significant yield response, for example at low rates of application in an Australian study in wheat (Blackwell et al., 2007). A pot study of cowpea showed higher biological nitrogen fixation with biochar addition due to nutrient effects (Rondon et al., 2005); higher yield and N uptake reported in pot trials using radish (Chan et al., 2007, 2008). A key consideration highlighted in several studies is University of Ghana http://ugspace.ug.edu.gh 34 the potential for biochar to immobilize previously plant available N. This could be from the mineralization of labile, high C–to–N fractions of biochar drawing N into microbial biomass, sorption of ammonium, or sequestration of soil solution into fine pores. The increase in crop yield with biochar application has been reported elsewhere for crops such as cowpea (Yamato et al., 2006), soybean (Tagoe et al., 2008), maize (Yamato et al., 2006; Rodríguez et al., 2009), and upland rice (Asai et al., 2009). Haefele (2007) and Haefele et al. (2008) discussed the possibility of biochar applications for rice-based cropping systems. Reichenauer et al. (2009) applied biochar in tsunami-affected paddy fields in Sri Lanka, and the experimental results showed that the application of 2 t rice-husk-charcoal ha -1 increased the grain yield from less than 4 t ha -1 for the control treatment to more than 5 t ha -1 for the biochar treatment. 2.3 Petroleum and its’ products 2.3.1 Origin and formation of petroleum oil. Even though disagreement exists about the origin of petroleum oil, years of research by geologists has resulted in a reasonably clear understanding of how crude oil forms in the earth‘scrust, its composition, and how it occurs. Ideas about the origin of oil follow two different lines of thinking: organic theories and inorganic theories (Hedberg, 1969). One of the earliest inorganic theories originated with Arab philosophers who, in about 850 A.D., suggested that water and air combined with fire to produce sulfur and mercury. The sulfur and mercury then combined with ―earth‖ and, at great subterranean temperatures, yielded ―naft‖ (naphtha) and ―qir‖ (asphalt) (Hedberg, 1969). Two nineteenth-century scientists, Louis Joseph University of Ghana http://ugspace.ug.edu.gh 35 Gay- Lussac (1850) and Alexander von Humboldt (1859) proposed that oil formed as a result of impregnation of marine sediments by subaqueous hot springs. Another nineteenth-century idea was that oil formed when hot alkalis combined with carbon dioxide deep in the earth‘s interior (Hunt, 1996). A Russian chemist, Dimitri Mendeleev (Mendeleev was also the ―inventor‖ of the Periodic Table), believed that percolating water encountered iron carbide deep in the earth, generating hydrocarbons. Other scientists, noting that methane occurs in trace amounts in volcanic gases and in fluid inclusions in igneous rocks, assumed that it was ―sweated‖ out of the earth‘s interior throughout geologic time, rose in the crust, changed into heavier hydrocarbons, and finally accumulated into the petroleum deposits we use today (Hedberg, 1969). Hypotheses suggesting an organic origin for oil is also old. Oil and coal were linked by some naturalists as early as the sixteenth century. Abundant imprints of leaves, stems, and other evidence of vegetation left little doubt as to the origin of coal (Rogers, 1863). Chemists discovered that small amounts of oil could be distilled from coal in the laboratory and postulated that this occurred in nature as well. Geologists had problems with this idea though, because the primary oil-producing strata lacked associated coals, and naturally-occurring oils were chemically different from the oils derived from the distillation of coal. Other nineteenth-century workers believed that oil was derived from terrestrial vegetation, which was washed into the sea and deposited with the sediments containing the petroleum. Problems with this idea include the fact that some oil is produced from rocks containing only marine fossils, and also the high temperatures needed to convert wood into liquid organic matter are not geologically reasonable. By the late 1800‘s and early 1900‘s, the prevailing view was that crude oil represents an accumulation of hydrocarbons that were originally produced by living organisms, both plants and animals and coal came from the accumulations of dead plants (Tissot and Welte, 1984). University of Ghana http://ugspace.ug.edu.gh 36 Other scientists tried to explain the origin of oil in other ways. The occurrence of hydrocarbons in meteorites has been well known to scientists since the mid-1800‘s (Tissot and Welte, 1984). In the early 1930‘s, astronomers learned that methane is a major component of the large outer planets – Jupiter, Saturn, Uranus, and Neptune. Because it was believed that all the planets in our solar system were closely related in origin, some researchers concluded that the raw materials for hydrocarbons must have been present in the substances from which the primordial earth accreted 4.6 billion years ago. By the 1950‘s, such reasoning led astronomer Fred Hoyle to argue that the deep earth must contain vast untapped reserves of oil just awaiting our technological ability to find and exploit them (Tissot and Welte, 1984). This idea is still favored by a small group of scientists. It is now accepted today by most geologists that oil was formed millions of years ago from a combination of hydrocarbons synthesized by living organisms and hydrocarbons formed by thermal alteration of organic matter in sedimentary rocks (Carter, 2004). Ten to twenty percent of the oil in the earth‘s crust is thought to form from living organisms, whereas 80 to 90 percent is formed by thermal alteration (Edwards, 1997). Marine planktons are the major components in both methods of natural crude oil formation (Edwards, 1997). Several lines of evidence support this contemporary view of the origin of petroleum: 1. Oil is rarely found in rocks that formed before life developed on the earth; 2. Oil contains compounds derived from the pigments of living organisms; 3. The ratio of carbon isotopes in oil is similar to that in organic matter; 4. Hydrocarbon compounds found in oil affect polarized light in the same way that hydrocarbons and other compounds synthesized by living organisms affect polarized light; 5. The structures of many oil compounds are similar to those of fats and waxes found in living organisms and, therefore, could be formed from them. University of Ghana http://ugspace.ug.edu.gh 37 When organisms die, bacteria attack their remains. These bacteria require oxygen, and if oxygen is plentiful, destruction of the organic remains is complete. Abundant remains of marine plankton, however, sometimes accumulate along with mud in stagnant underwater environments. The aerobic bacteria use up any dissolved oxygen quickly. Anaerobic bacteria, which obtain their oxygen from dissolved sulfur compounds and hydroxides in the pore waters of the mud, then take over. These bacteria consume most of the easily decomposable compounds in the organic matter, such as carbohydrates and proteins. As the muds are buried by an increasingly thicker cover of sediment, physical and low temperature chemical reactions continue to alter the chemical structure and composition of much of the organic matter. At even deeper burial depths, rising temperatures and pressures cause the organic debris to decompose further to form crude oil (Edwards, 19