BIOREMEDIATION OF PETROLEUM HYDROCARBON CONTAMINATED SOIL BY INDIGENOUS BACTERIA IN ASSOCIATION WITH PLANT
Petroleum, the backbone of today's mechanized society, now became a threat to environment due to extraction and transportation. Due to transportation accidental spills occur regularly all over the world. Such spills have also occurred at many locations throughout Pakistan over time. Petroleum and its products contain carcinogenic and mutagenic compounds and therefore contamination of soil from petroleum hydrocarbons is a serious problem. Efforts are now focused on seeking potential remediation techniques for cleanup of petroleum contaminated soils in a cost effective and eco friendly way. Plant assisted bioremediation of petroleum contaminated soil is getting attention as compared to the alone use of either microorganism or plant. The challenging task for such efforts to be successful is not only the survival of microorganisms upon their inoculation into xenobiotic environment but also microbe's positives interaction with plants. Plant growth promoting bacteria (PGPB) having 1 aminocyclopropane 1 carboxylic acid deaminase (ACC deaminase) are considered to helpful for plants in stressed environment by reducing stress induced ethylene. Over three hundred bacterial isolates were cultured from collected petroleum contaminated soil samples. Bioremediation and ACCmetabolism assays screened out 27 from 301 bacterial isolates. These 27 bacterial isolates possessed medium to high bioremediation potential for PAHs and also high ACC deaminase activity. Compatibility of these 27 bacterial isolates with alfalfa, maize and canola was assayed in growth pouches and jar experiment under axenic conditions. Finally, on the basis of bioremediation, ACC deaminase and plant growth promotion activity, 8 bacterial isolates were screened out. Plant assisted bioremediation of artificially spiked coarse textured soil bearing 10,000 mg kg (w/w) crude oil concentration was carried out both under controlled and ambient conditions of light and temperature. Also growth performance of inoculated plants was compared with uninoculated plants for each crop. After 60 days, results revealed that four bacterial isolates Bacillus subtilis, Bacillus cereus and other Bacillus sp. significantly degraded petroleum hydrocarbons. The most efficient was Bacillus subtilis which reduced TPH contamination 47% 37% and 43% in combination with alfalfa, canola and maize, respectively. While the same bacterium when inoculated alone reduced 33% TPH contamination under controlled light and temperature while 31% under ambient light and temperature. Significant increase in growth attributes such as root length, shoot length and fresh biomass of all three crops was observed as compared to their corresponding un inoculated plant control. Comparatively, alfalfa with respect to germination and growth in petroleum contaminated soil performed better as compared to maize and canola. Study can be concluded that use of bacteria possessed with dual traits of bioremediation potential and ACC deaminase activity in combination with plant can be a good approach for remediation of petroleum hydrocarbons contaminated soil. Chapter 1: General Introduction 1 Chapter 1 General Introduction Petroleum derived from fossil fuels are important part of our today's mechanized society, however, an inevitable risk to environment is being posed due to extraction and transportation of these fuels. Contamination of terrestrial environment with petroleum hydrocarbons occurs due to both human and mechanical error. Accidental spills of petroleum hydrocarbons into various compartments of environment such as water and soil are increasing all over the world, however, the exact detail of extent of petroleum hydrocarbons contamination is difficult to bring into figures because the contamination is unintentional. Pakistan's coastal line near Karachi faced contamination of 28,000 tons of crude oil when in July, 2003 a Greek ship naming Tasman Spirit cracked into two pieces. The citizen of Korangi Town Karachi, Pakistan found oil in their streets, house yards and shops due to extraordinary rupture in the pipeline of Pak Arab Refinery Corporation (PARCO) during construction work by Sui Southern Gas Company (Alam, 2008) Another un intentional spill of 18 tankers of crude oil happened in September, 2009 in Sindh Province of Pakistan due to collision of two goods carrying trains (Khan, 2009) Most recently, another huge oil spill happened when two NATO oil tankers each bearing 60,000 liters gasoline toppled on Khojak Pass near Chaman, Pakistan. Further release of petroleum hydrocarbons into the environment occurs at well sites, petroleum refineries. Petroleum refining industries in Karachi, Rawalpindi, Multan, Qasba Gujrat Kot Adu are sources of contaminants such as phenols, sulphides and oily residues (National Environmental Policy of Pakistan, 1999) A significant contribution in hazardous waste level, according to Pakistan Environmental Protection Agency (2005) is from petroleum and petro chemical industries. Contamination of different compartments of the environment i.e. air, water and soil with petroleum hydrocarbons poses serious threat to health of human beings. For instance, among benzene, toluene, ethylbenzene and xylene commonly abbreviated as BTEX, benzene is carcinogenic, toluene damages central human nervous system, ethylbenze causes skin irritation and long exposure to xylene may lead to aplastic anemia (US EPA, 2011) BTEX are mono aromatic compounds and are important component of gasoline and constitute 40% of gasoline's composition (Ribeiro et al. 2012) Similarly, diesel fuel is composed of branched alkanes, cycloalkane, alkenes and recalcitrant aromatic Chapter 1: General Introduction 2 hydrocarbons (ATSDR 1996) Therefore, remediation measures of such pollutants in the environment especially in soil are of utmost importance. A number of remediation techniques have been applied for the cleanup of petroleum hydrocarbon contaminations including physical, chemical and biological techniques. Physical remediation measures such as incineration and thermal desorption and chemical remediation processes such as solvent extraction, encapsulation and oxidation reductions are not only expensive but also need substantial use of heavy machinery and energy. Moreover, bulk of soil has to be shifted to treatment facility which is ecologically disruptive (Huang et al. 2004) These limitations make biologically based remediation techniques such as phytoremediation and bioremediation more attractive. Phytoremediation is the use of plants for removal or containment of contaminants through various processes such as phytoextraction, phytostabilization, phytotransformation and phytovolatilization (Glick, 2003; 2010) Phytoremediation, no doubt, is an appealing strategy for decontamination of environmental xenobiotic compounds, however, in case of petroleum hydrocarbons it is evident from various studies that plant growth including root shoot biomass and elongation of root and shoot was reduced due to petroleum hydrocarbons stress (Masakorala et al. 2013; Peng et al. 2009; Germaine et al. 2009) Petroleum hydrocarbons are of various nature and have different characteristics especially solubility in water. For successful phytoremediation, contaminants must have solubility in water while petroleum hydrocarbons with octanol water partitioning co efficient (Kow) greater than 3 are not soluble in water thus not translocated to shoots. Due to less solubility in water, phytoremediation of such contaminants becomes difficult (Rojo, 2009) Another appealing strategy is the use of indigenous bacteria usually called as insitu bioremediation which is an established strategy for petroleum hydrocarbons decontamination. For successful application of this technique, it is necessary to isolate such bacteria that not only survive in the contaminated conditions but also capable of degrading the target contaminants (Malik and Ahmad, 2012) As petroleum hydrocarbons are complex mixture of different compounds having different structure such as n alkanes, polyaromatics and cycloalkanes, the in situ remediation of polyaromatics and cycloalkanes is sometimes difficult because of failure of bacteria in achieving contact with such contaminants (Sarma and Prasad, 2015) Moreover, high molecular weight compounds are not degraded at early stages of inoculation so persistent survival of Chapter 1: General Introduction 3 bacterial inoculants is necessary for degradation of such compounds (Mishra et al. 2001; Schwartz and Scow, 2001) Bioremediation assisted by plants is useful to overcome such constraints and thereby enhances degradation of recalcitrant soil contaminant like polyaromatic hydrocarbons (Kawasaki et al. 2012) Roots of higher plants play important role in assisting microbial population to degrade pollutants in many ways such as provision of molecular oxygen, inorganic nutrients through sloughed off cells, soluble exudates and lysates (Khan et al. 2013; Segura and Ramos, 2013; Phillips, 2008; Martin et al. 2014) Plants improve soil aeration by directly giving off oxygen to the root zone as well as allowing improved entry of oxygen into the soil by diffusion along old root channels (Nye and Tinker, 1977) If microbes do not have degradative pathways for petroleum hydrocarbons and cannot use them as carbon source, even then petroleum hydrocarbons can be mineralized through the process of co metabolism (Li et al. 2015) Cometabolism is mechanism by which plants assist microbes to co metabolize a contaminant in the soil using the root exudates as an energy source. Plant enzymes degrade the compounds, and then further degradation is carried out by microbes ultimately into carbon dioxide and water. Some structural analogs of polyaromatic hydrocarbons such as phenols, terpernes and flavonoids are released by some plant roots as exudates and thus promote the growth of petroleum hydrocarbons degrading bacteria (Khan et al. 2013;