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Biodegradation of Aliphatic Chlorinated Hydrocarbon (PCE, TCE and DCE) in Contaminated Soil. TIBUI Aloysius Cho
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Biodegradation of Aliphatic Chlorinated Hydrocarbon (PCE, TCE and DCE) in Contaminated Soil.

TIBUI Aloysius Cho

Rapporttyp Report category Licentiatavhandling Examensarbete AB-uppsats C-uppsats D-uppsats Övrig rapport ________________

Språk Language Svenska/Swedish Engelska/English ________________

Titel Title: Biodegradation of Aliphatic Chlorinated Hydrocarbon (PCE, TCE and DCE) in Contaminated Soil. Författare Author :TIBUI Aloysius Cho

Sammanfattning Abstract Soil bottles and soil slurry experiments were conducted to investigate the effect of some additives on the aerobic and anaerobic biodegradation of chlorinated aliphatic hydrocarbons; tetrachloroethylene (PCE), trichloroethylene (TCE) and dichloroethylene (DCE) in a contaminated soil from Startvätten AB Linköping Sweden. For the aerobic degradation study the soil sample was divided into two groups, one was fertilised. The two groups of soil in the experimental bottles were treated to varying amount of methane in pairs. DCE and TCE were added to all samples while PCE was found in the contaminated soil. Both aerobic and anaerobic experiments were conducted. For aerobic study air was added to all bottles to serve as electron acceptor (oxygen). It was observed that all the samples showed a very small amount of methane consumption while the fertilised soil samples showed more oxygen consumption. For the chlorinated compounds the expected degradation could not be ascertained since the control and experimental set up were more or less the same. For the anaerobic biodegradation study soil slurry was made with different media i.e. basic mineral medium (BM), BM and an organic compound (lactate), water and sulphide, phosphate buffer and sulphide and phosphate buffer, sulphide and ammonia. To assure anaerobic conditions, the headspace in the experimental bottles was changed to N2/CO2. As for the aerobic study all the samples were added DCE and TCE while PCE was found in the contaminated soil. The sample without the soil i.e. the control was also given PCE. It was observed that there was no clear decrease in the GC peak area of the pollutants in the different media. The decrease in GC peak area of the pollutants could not be seen, this may be so because more susceptible microorganisms are required, stringent addition of nutrients and to lower the risk of the high concentration of PCE and petroleum products in the soil from Startvätten AB.

ISBN _____________________________________________________ ISRN LIU-TEMA/ES-D--06/05--SE _________________________________________________________________

ISSN _________________________________________________________________ Serietitel och serienummer Title of series, numbering Handledare Tutor

Nyckelord Keywords: Aerobic biodegradation, Anaerobic biodegradation, Chlorinated compound, Tetrachloroethene (PCE), Trichloroethene (TCE), Dichloroethene DCE), Micro-organisms, Methane, Basis mineral medium, Gas Chromatography (GC), Inorganic fertilizer

Datum Date . 2006/12/11

URL för elektronisk version http://www.ep.liu.se/index.sv.html

Institution, Avdelning Department, Division Insitutionen för tema Miljövetenskap The Tema Institute Environmental Science

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Table of content -Abstract……………………………………………………………………………..2 Introduction……………………………………………………………………...…3 -Aim and objectives…………………………………………………………………5 -Principles used……………………………………………………………………...6 -Research question…………………………………………………………………..6 State of the art………………………………………………………………………7 Materials and methods -Preparation of soil samples………………………………………………………...10 -Determination of water content of soil sample…………………………………….11 -Determination of organic content of the soil……………………………………….11 -Determination of water holding capacity of the soil……………………………….11 -Determination of oxygen and methane consumption……………………………....11 - Preparation of experimental solution solutions (BM, NH4 and lactate)………...…12 -Calibrations (split and splitless)…………………………………………………....13 Results -Results for aerobic degradation studies…………………………………………….14 -Results for anaerobic degradation studies………………………………………….17 Discussion -Discussion for the aerobic study……………………………………………………20 -Discussion for the anaerobic study…………………………………………………21 Conclusions -Conclusions………………………………………………………………………...22 -Reference…………………………………………………………………………...23 -Appendix…………………………………………………………………………...25

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Biodegradation of Aliphatic Chlorinated Hydrocarbon (PCE, TCE and DCE) in Contaminated Soil.

Abstract: Soil bottles and soil slurry experiments were conducted to investigate the effect of some additives on the aerobic and anaerobic biodegradation of chlorinated aliphatic hydrocarbons; tetrachloroethylene (PCE), trichloroethylene (TCE) and dichloroethylene (DCE) in a contaminated soil from Startvätten AB Linköping Sweden. For the aerobic degradation study the soil sample was divided into two groups, one was fertilised. The two groups of soil in the experimental bottles were treated to varying amount of methane in pairs. DCE and TCE were added to all samples while PCE was found in the contaminated soil. Both aerobic and anaerobic experiments were conducted. For aerobic study air was added to all bottles to serve as electron acceptor (oxygen). It was observed that all the samples showed a very small amount of methane consumption while the fertilised soil samples showed more oxygen consumption. For the chlorinated compounds the expected degradation could not be ascertained since the control and experimental set up were more or less the same. For the anaerobic biodegradation study soil slurry was made with different media i.e. basic mineral medium (BM), BM and an organic compound (lactate), water and sulphide, phosphate buffer and sulphide and phosphate buffer, sulphide and ammonia. To assure anaerobic conditions, the headspace in the experimental bottles was changed to N2/CO2. As for the aerobic study all the samples were added DCE and TCE while PCE was found in the contaminated soil. The sample without the soil i.e. the control was also given PCE. It was observed that there was no clear decrease in the GC peak area of the pollutants in the different media. The decrease in GC peak area of the pollutants could not be seen, this may be so because more susceptible microorganisms are required, stringent addition of nutrients and to lower the risk of the high concentration of PCE and petroleum products in the soil from Startvätten AB.

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Introduction: In many parts of the world today the term pollution has become a household word meaning the emission into the environment of unwanted substances, which are harmful to man, animals, plants and the environment as a whole. In broad terms pollution can be divided into land, air and water pollution. The Oxford Dictionary of Ecology in Earth and Environmental Science define the environment as: ‘the complete range of external condition, physical and biological in which an organism lives (air, land or water). The environment includes social, cultural and (for humans) economical and political considerations’. According to Adriano et al. (1998) the U.S. Environmental Protection Agency (USEPA) estimates that, more than one million tons of toxic chemicals have been released on land or into air and water, by factories, refineries and chemical plants. These unwanted substances have caused tremendous damage to our environment. For example vast land areas that would have been put into better use have been left abandoned due to risks they pose to the ecological system and human health (Adriano et al. 1998). Beside other types of pollutants, some of these chlorinated compounds, like chloroform, trichloroethene (TCE) and tetrachloroethene (PCE), have also been seen to pose a risk for drinking water sources as they can be transported to groundwater (Laturnus, 2003). These chlorinated pollutants in groundwater are suspected carcinogenic in mice (Beeman, 1994). Recently it is acknowledged that there are many compounds ubiquitous in our environment having a disrupting effect on our reproductive system (decrease in sperm counts and increase in male genital track disorder), endocrine system (breast and blood tissue cancer has been linked to high concentrations of PCBs) and the immune system. About half of these compounds are chlorinated compounds and the undesirable effects are due to their dioxins (Eder et al. 1995). The water also suffers from unwanted materials such as excess amounts of nutrients from farm land runoffs, industrial waste, municipal waste etc, leading to eutrophication (Boorman et al. 1999) and (Howarth, 2000). Eutrophication renders the surface water unfit for drinking e.g. due to the bloom of toxic algae, it renders aquatic habitats undesirable to some aquatic species such as in the Baltic Sea (Laturnus, 2003). About 7 million different types of pollutants have been identified. Of this huge amount approximately 330,000 are said to be anthropogenic (man-made) which are practically used by man, while the rest are biogenic (natural occurring) compounds (van Agteren et al. 1998). It has also been seen that the production of most chemicals is often associated with the formation of unwanted hazardous by-products, which usually ends up being dumped into the environment (Adriano et al. 1998). ). Due to the increase demand of certain goods and services in our communities, there has been increase use of chlorofluorohydrocarbons (CFCs) and other volatile organohalogens in our industries, thus, leading to a large annual release of these compounds into the environment (Laturnus, 2003). Focusing this study on the following chlorinated aliphatic solvents: tetrachloroethene (PCE) trichloroethene (TCE) and dichloroethene (DCE), it is now known that they are

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often found in groundwater as they are widely used as solvents (Aulenta et al. 2002). It has also been shown that most of our daily goods and services contain these pollutants such as petroleum products, detergents, fire extinguishers, aerosol, pesticides etc. (Solomon, 2004). This means more of such pollutants will be dumped into the environment. It is for this reason that better methods of cleaning the environment is being investigated into everyday. Many techniques are currently being employed for the degradation of these chlorinated compounds. They are generally grouped in two main types, the biological treatments and non-biological methods. The non-biological could be by a physical or a chemical means. In recent times it has been seen that biological treatments are the most preferable, for the reasons that biotreatments are usually less destructive and at times less costly to carry out as compared to the non-biological means like incineration methods, which require much energy and leaves behind residues that has to be taken care of (Adriano et al. 1998). More to this is that, with the biological means, the soil quality is maintained, while with the incineration method the soil is e.g. depleted of its organic fraction and structure. The biological treatment is a naturally occurring process with the bacteria already doing the degradation, but mostly at a low rate (van Agteren et al. 1998). Biodegradation from an environmental standpoint is the use of biological means (living organisms) to remove completely or transform unwanted harmful substances in the environment to less harmful ones such as the conversion of PCE to ethylene and Cl- (Adriano et al. 1998). Depending on how the normal natural state of the environment is altered, there are several types of bioremediation techniques available. They are: natural attenuation, bioaugmentation and accelerated bioremediation. Natural attenuation is when both biotic and abiotic degradation processes are taking place in a contaminated site without the influence of man. Bioaugmentation, this is the inoculation of specific microorganisms with a set of genes enabling them to degrade target pollutants in a contaminated site. Accelerated bioremediation, is the addition of selected additives into an affected area to stimulate the indigenous microorganisms to grow and degrade certain pollutants (Deweerd et al. 1998). Different types of organisms have been used in biodegradation, such as plants, fungi, algae and bacteria (Keppler et al. 2000). Biodegradation can be divided into aerobic and anaerobic reactions, i.e. in the presence or absent of oxygen respectively. For the complete degradation of PCE both the aerobic and anaerobic processes are needed. The degradation of PCE to TCE and TCE to DCE is favourable in the absence of oxygen while the conversion DCE to vinyl chloride (VC) and finally VC to ethane and CO2 can only take place in the presence of oxygen (Adriano et al. 1998). Naturally in the environment some of these pollutants are being degraded by microorganisms. But these microorganisms poorly degrade most of these synthetic products. This is because there are few microorganisms that produce the enzymes that can recognize and transform these pollutants and also for the fact that the degradation involves many steps which are controlled by different enzymes (Adriano et al. 1998). It is

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also known that most organic pollutants of synthetic origin are often toxic chemicals that need to be removed to low levels in treatment system (van Agteren et al. 1998). Several reaction pathways have now been investigated intensively and an increasing number of reaction rate constants can now be estimated quite accurately using a variety of Quantitative Structure Activity Relationships (QSARs) (Peijnenburg and Damborsky, 1996). It is said that among organic chemicals, biodegradation is the dominant pathway, contributing significantly to the attenuation of their environmental concentrations’ (Peijnenburg and Damborsky, 1996). Biodegradation frequently, although not necessary, leads to the complete conversion of the complex molecules to inorganic products that is mineralization (van Agteren et al. 1998). Based on the ability of microorganisms to degrade organic pollutants, the pollutants have been classified as biodegradable, persistent or recalcitrant (van Agteren et al. 1998). Some biodegradation and biotransformation processes are promoted by specific microorganisms attacking only a given site of a contaminant molecule. For a single species of organisms to be able to completely degrade a complex pollutant compound it must contain many different biocatalysts (enzymes) thus a consortium of microorganisms will work together in a treatment train (Wilson and Clarke, 1994). The metabolic pathways are often dictated by specific environmental conditions such as the presence or absence of molecular oxygen, pH, temperature, or nutritional conditions and other additives (Suarez and Rafai, 1999). The results of field experiments are much more reliable than those of laboratory experiments, as they give the insight of the actual effect of the heterogeneity of the complex environment. However, often laboratory measurements are needed in order to gain a representative picture (Otten et al. 1997). Research efforts in bioremediation technology have expanded rapidly during the past years, but basic research is still needed to better understand the biological chemical and the physical factors affecting biotransformation processes (Adriano et al. 1998). -Aim and objectives The essence of this research is to see how certain additives (methane, inorganic fertilizer and microbial growth media) can be manipulated to contribute significantly to the alteration of biodegradation of aliphatic chlorinated hydrocarbon in a contaminated soil, i.e. PCE, TCE and DCE. Carrying out such a work in the laboratory gives a good ground to evaluate and predict the feasibility for cleaning up such an area by bioremediation and consequently maintaining the quality of the soil. The degradation is going to be investigated under aerobic and anaerobic conditions. Under aerobic condition it will be in order to cover the effects of oxygen, inorganic fertilizer and different levels of methane while under the anaerobic conditions it will uncover the different redox levels by the use of different types of microbial growth media such as basic mineral medium (BM), BM plus organic compounds, effect of sulphide (which will affect the redox condition) phosphate and ammonia in the growth medium.

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The aerobic incubation will include supply of methane and fertilizer additions to soil samples to possibly enhance the growth and metabolism of the microorganisms, which will likely lead to a more rapid degradation of the pollutants. Although this is a laboratory study, it is intended to give good grounds for effective and efficient basis for an in situ remediation. -Principles involve: Biodegradation of chlorinated compounds can occur through two pathways or principles, they are: - The chlorinated compound can be used as an electron acceptor (reductive

dechlorination). This can only occur under anaerobic conditions. This will only take place in a redox system, where the chlorinated compounds are reduced by an electron donor. In this case our possible electron donors will be ammonia (NH4), an organic compound (lactate). That is here the chlorine atoms (Cl) in PCE are replaced by hydrogen from NH4 to give TCE and further replacement of Cl from TCE will give DCE (Otten et al. 1997) and finally to VC

- They can be used as electron donor by microorganisms in oxidative dechlorination.

This is in an aerobic condition, where oxygen is used as the final electron acceptor. The chlorinated hydrocarbons i.e. TCE, DCE and VC are used as co-metabolites, while methane and ethanol have been shown to be used as energy and carbon sources for the microorganisms performing such co-metabolic processes (Van Agteren et al. 1998).

The two principles will be exploited in this research. In general biodegradation of chlorinated solvent is an electron-donor-limited process (Adriano et al. 1998). Generally speaking, biodegradation rates are greater under aerobic, oxidative conditions, than under anaerobic conditions. However, there are many situations, where anaerobic metabolism provides the sole means for biodegradation (Aulenta et al. 2005) Adriano et al. (1998) say that the supply of laboratory propagated microorganisms to a contaminated environment are less effective than naturally occurring microbes already present in the specific environment. It is for this reason that the source of microorganisms in this investigation will be those present in the soil at the time of sampling. -Research Questions: The main question here is how those certain additives specifically nutrients (phosphorus, ammonia, sulphide, lactate and methane) may affect the natural process of biodegradation in the environment? Is the degradation caused by microorganisms? What is the fate of the methane added to the soil? What is the effect of the organic matter content of the soil? Does biodegradation occur in nature? How do the oxygenated and anoxic conditions affect the biodegradation of these compounds? Are inorganic nutrients required for an increase of the rate of biodegradation? .

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State of the arts: As to the state or the arts of this research area, much has been done, much is currently going on and much is still to be done. In situ and laboratory works have been done in this area, and careful laboratory estimations and predictions have about 80% successes in field situations (U.S. E.P.A., 1991). Biodegradations of chlorinated solvents (PCE, TCE, cisDCE, transDCE, VC etc.) mostly occur via reductive dechlorination. This is a process where the chlorinated aliphatic hydrocarbons act as electron acceptors and where an adequate supply of electron donor such as natural organic carbon, fuel, hydrocarbon, landfill leachates etc is needed for a complete degradation (U.S. E.P.A., 1991). Chang and Alvarez (1996) studied microbial degradation of chlorinated and non-chlorinated methane, ethane and ethene by a mixed culture grown under chemostate and batch conditions. The activity by the mixed culture was evaluated and compared with that of two pure methanotrophic strains CACI (isolated from the mixed culture) and Methylosus trichosporium OB3b. He saw that none of the cultures were able to degrade the fully chlorinated aliphatics such as PCE and carbon tetrachloride (CT). Of the four cultures tested, the chemostate-grown mixed culture exhibited the highest transformation capacity for TCE, cis-1, 2-DCE, tetrachloroethane, 1,1,1-trichloroethane and 1,2-dichloroethane. Likely the different bacteria in the mixed culture favoured the degradation of the intermediate products of PCE by separately producing the different enzymes needed to degrade the assembly of intermediate products. Generally highly substituted chlorinated aliphatics are rapidly dechlorinated under anaerobic conditions. However reductive dechlorination reaction proceeds progressively more slowly as the number of chlorine or other halogen groups attached to carbon atom decreases (Wilson and Clarke. 1994). He also mentions that in contrast aerobic biodegradation usually progresses more rapidly as the number of chlorine constituent declines. Under anaerobic conditions, chloroethene act as an electron acceptor that is sequential replacement of chlorine by hydrogen, that is PCE is reductively dechlorinated via TCE, 1,2-DCE and 1,1-DCE to vinyl chloride (VC) (Van Agteren et al. 1998). The conversion of VC to ethene and to ethane is the slowest step in the anaerobic pathway. VC generally accumulates and pollutes the reaction medium (Van Agteren et al. 1998) and (Coleman et al. 2002). Studies were conducted to examine the biodegradation of 14C-labelled VC in samples taken from a shallow aquifer. Under aerobic conditions, vinyl chloride was readily degraded, with greater than 99% of the labelled material being degraded after 108 days and approximately 65% being mineralized to 14CO2 (Davis and Carpenter 1990). Lower chlorinated compounds such as cisDCE and VC often accumulate in chloroethene-contaminated aquifers due to incomplete reductive dechlorination of higher chlorinated compounds (Sing et al. 2004). A highly enriched aerobic culture that degrades VC as a growth substrate was obtained from a chloroethene-contaminated aquifer material. The culture rapidly degraded 50-250 µmoles aqueous VC to below GC detection limit with a

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first-order rate constant of 0.2 day-1 (Sing et al. 2004). Besides VC, the culture also degraded ethene as the sole carbon source. In addition, the culture degraded cisDCE, but only in the presence of VC. However, no degradation of trans-DCE or TCE occurred either in the presence or absence of VC (Sing et al. 2004). Experiments examining the effect of oxygen concentrations on VC degradation showed that the culture was able to metabolize VC efficiently at extremely low concentrations of dissolved oxygen (DO) (Sing et al. 2004). Complete removal of 150µmoles of VC occurred in the presence of only 0.2µmol of oxygen (1.8 mg/L DO) and this is important since most groundwater environments contain low DO (1-2 mg/L) (Sing et al. 2004). This study showed that the culture was able to withstand long periods of oxygen starvation for example; the culture was able to assimilate VC with minimal lag time even after 5 months of starvation (Sing et al. 2004). This is impressive from the point of its sustenance under field conditions. Overall the culture is robust and degrades VC to below the detection limit rendering this culture suitable for field application (Sing et al. 2004). The enzyme system, methane monooxygenase (MMO) functions intracellularly either attached to the cell membrane (particulate MMO) or free in the cellular fluid (soluble MMO) (Wilson and Clarke, 1994). Recent studies have shown that TCE and other chlorinated hydrocarbon are degraded primarily by soluble MMO, a form of enzyme that occurs mainly when copper becomes depleted in the medium (Wilson and Clarke, 1994). In excess of copper, and some other nutrients some methanotrophs promote the development of particulate MMO and a decline in the more bioactive soluble form of MMO (Wilson and Clarke, 1994). Methane or methanol induces the MMO synthesis in methanotrophs. But high concentrations of these can cause competitive inhibition of the dechlorination process. In this situation the inducing power must be maintained by the presence of an electron donor, thus formaldehyde and format can serve as external electron donors for MMO and their addition to the treatment has increase the TCE oxidation rate (Wilson and Clarke, 1994). Cometabolic degradation of TCE yields toxic intermediates. These toxic intermediates limit the practical application of cometabolic process in bioremediation (Van Agteren et al. 1998). Intermediates formed during the conversion of TCE by M. trichosporium OB3b attach the monooxygenase and other cell components, resulting in a rapid decrease in viability and a slower decrease in activity of the soluble MMO (Van Agteren et al. 1998). The loss of viability at high TCE loading is much less severe than in non methanotrophic bacteria such as Burkholderia cepacia G4, which is resistant to TCE as well as it’s degradation products but its capacity to degrade TCE is due to the monooxygenase involved in the degradation of aromatic compounds such as toluene, phenol, etc thus the aromatics have been used as an inducing co substrate (Van Agteren et al. 1998). Reductive dechlorination by anaerobic bacteria is important because some highly chlorinated compounds, such as CT and PCE, are not known to be degraded under aerobic conditions (Wilson and Clarke, 1994). The decease in reductive dechlorination in an anaerobic condition can be seen in the light that VC, a potential carcinogen, has been observe to accumulate, probably because it is not rapidly dechlorinated further under

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anaerobic conditions (Wilson and Clarke, 1994). However, recent data have shown that VC does not always accumulate in the environment, but the anaerobic degradation rate and degradation products of VC were found to depend on the type and quantity of nutrients added to the growth medium (Wilson and Clarke, 1994). In the presence of methane, methanol, ammonia, phosphate and phenol the main biotransformation products were non- chlorinated compounds instead compounds such as methane and ethane (Wilson and Clarke, 1994). If partially oxidized nutrients such as acetate and citate were added, VC will be oxidized primarily to CO2 (Wilson and Clarke, 1994). Complete and rapid biodegradation for many contaminants may not require specific environmental conditions but perhaps changing conditions to satisfy the need of other microbial consortia (Wilson and Clarke, 1994). This is true with the case of PCE and PCA where reductive dechlorination, anaerobic condition is required initially to remove the first chlorine groups and then followed by aerobic condition to degrade the less chlorinated compounds that tend to accumulate and inhibit the reaction process.

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Materials and methods: The experiment was conducted in two parts. The first part was under aerobic condition in which the effect of methane and inorganic fertilizer was investigated while the second part was in anoxic conditions in which the effect of different redox levels and different types of basic growth medial was investigated in soil slurry. Both set ups had the same source of soil, collected at the same time and place. All the equipment used was kept under the same conditions and place. The first part was done for 10 weeks with splitless injection while the second part was done for 4 weeks using split injection. The soil sample used in this study was obtained from a laundry company Startvätten AB, in Linköping a town found in Sweden. This company area is know to have harboured a dry cleaning facility since the mid fifty’s. From past investigations it is now known that the area is contaminated with high levels of PCE and its degradation products. This is as a result of improper and inadequate disposal of the chemicals, which are used by this company as detergents and bleaching agents. The soil is grey moist silt/fine sand mostly covered by buildings or asphalt, thus not likely to supporting any microflora (Surma et al. 2004).The contaminated soil labelled ECO 405 (borehole number 405) was collected at the depths of 3.4 to 3.7 m in May 2005 and was stored at 4˚C until analysis in October 20th, 2005 The choice of inorganic nutrients was the common Farmer’s fertiliser containing 12% of nitrate N, 5% of phosphate P and 14% of potassium K was due to the fact that these nutrients were likely to be at low levels in the soil investigated and also readily available. The addition of methane was done to supply a source of carbon and energy for the growth of the microbial population which will in turn hasten the consumption and the cleaning up of the soil from the chlorinated organics, since it has been shown that methane is a good source of electrons donor while the organochlorine is transformed as a co-metabolite. (van Agteren et al. 1998). -Preparation of sample In this first part sample preparation, 800 g of the soil was sieved (mesh 2 mm) and divided in two lots of which one was supplied with 40 mg of an inorganic fertilizer (common farmers’ fertilizer NPK) which was thoroughly mixed in a beaker. 30 g of soil with and without fertilizer was weighed into eleven different 118 ml serum bottles each. They were closed by butyl rubber stopper and capped with aluminium crimps. Three series of triplicates were given 0.9, 1.5, and 3 ml of methane, (99.995% air liquid, Malmö) by the use of sterilised disposable syringes (BD plastipakTM). The last two from each series served as controls. All the 22 bottles were given 4 µl of DCE (99%, Aldrich-chemie D.7924 Steinheim) and 5 µl of TCE (99.5% E. Merck, Darmstadt Germany) using separate 5 µl glass syringes i.e. 53 µM and 56 µM, respectively. All the bottles were injected with 20 ml air that served both as over pressure and oxygen supply. They were incubated at 15˚C in the dark for 20 day. The samples with fertilizers were known

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as methane fertilised soils (MF) while those without fertilizers were known as methane non-fertilised (MNF). Control one is no methane with fertilised (NMF) and control 2 no methane no fertilizer (NMNF). The fates of the compounds were followed by withdrawing 0.3 ml from the head space by a syringe. The procedure was as follows in order to secure that the amount aimed at was captured although the over pressure in the bottles: after filling the syringe with 0.3 ml of headspace, its needle was drawn back into the stopper, which sealed the tip. The plunger was then further withdrawn to the 0.9 ml mark to create an underpressure, before the sample was finally transferred for injection into a gas chromatograph equipped with an electron captured detector (GC ECD Hewlett Packard Gas Chromatograph series 5880). Chromatographic peaks were produced with areas that depicted the amount of the corresponding compounds injected from the headspace. The injections were repeated for ten weeks making one injection each week from all the bottles. The Gas chromatography was done with nitrogen as the carrier gas at 32psi and the split flow was at 10psi and separated on a 25 m x 0.53 mm poraplot Q column (df = 20 µm) run at temperature programme: Initially 150˚C for 3 min. and at increasing temperature of 10˚C/min. Until a final level of 200˚C, this completed the analysis after 10 min. Post temp = 210˚C Post time = 5 min -Determination of water content Three empty porcelain cups were weighed separately and labelled 1, 2, and 3. The soil was sieved in 2 mm mesh size, and 10 g of the soil sample was put in each cup and were taken to the oven at 150˚C, for 20 hours. After 20 hours, the soil in each cup was weighed together with the cup. From this the water content was calculated as shown in appendix table 1. -Determination of organic content The dry soil in their respective porcelain cups were further taken to another oven for 5 hours and at 450˚C. The soil and the cups were weighed separately after cooling in an exicator at room temperature. The organic content of the soil was obtained as shown in table 2. -Determination of oxygen (O2) and methane consumption After fourteen days of the set up of the experiment for aerobic biodegradation, the amount of oxygen in each of the 118 ml bottles was determined. This was done by injecting 0.3 ml of headspace into a GC with a thermoconductivity detector (TCD) using a 1ml syringe. Air was used as a standard by injecting 0.3ml (air contain 20% O2, but here the 20% is considered to be100%) into the GC TCD. For methane a calibration curve was also made. The result obtained is seen in table 3. -Preparation of the experimental solutions for the second part of the study: -Basal Mineral Medium (BM): 15 ml of W1 (0.2M KH2PO4) was measured into a 1000 ml flask followed by the addition 900 ml of milli-Q water. A further 15 ml of W2 (0.2M

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Na2HPO4) was added. The flask was shaken and then boiled for 30 minutes. It was then cooled under N2 gas and the head space changed to N2 gas. When 45 ml of the boiled solution (W1+W2) plus 2.5 ml of C1 (macro and micronutrients and vitamins) and 2.5 ml of C2 (1M NaCO3 x 9H2O) are mixed together, it forms the BM. In this study the BM was without the C2 since the C2 was used as a source of Sulphide. For further details on this composition see Ejlertsson et al. (1996). -NH4

2.4 g of NH4NO3 was dissolved in 100 ml of boiled milli-Q water and 2.5 ml of it will be added into each of three 118 ml bottle -Lactate 2.5 g of sodium lactate was dissolved in 100 ml of boiled milli-Q water. 0.5 ml of this solution will be added to three bottles as indicated in the procedure below. -Preparation of soil slurry Eighteen 118 ml serum bottles were washed clean in preparation of the experiment. 200 g of the experimental soil was sieve with a 2 mm mesh size. 10 g of the sieved soil was weight into fifteen of the 118 ml bottles. The gas phase of all the bottles was then changed to N2 gas. Of the 15 bottles with soil, three of them were added 47.5 ml of boiled milli-Q water under N2 gas. The gas phase of these three bottles was then changed to N2/CO2 and 2.5 ml of C2 was added to each of these bottles. They were then labelled water with soil and sulphide (W+S). With the remaining 15 bottles, 45 ml of W1 +W2 solution was added to each of these bottles under N2 gas. Their gas phase was then changed to N2/CO2 gas, and 2.5 ml of C1 was added to each under N2/CO2 gas. Of these 15 bottles, the three without soil were added 2.5 ml of boiled milli-Q water and were then labelled BM without soil (BM-S). Of the remaining 12 bottles all with soil, in three 2.5 ml of boiled milli-Q water was added and labelled BM with soil (BMS). The next three bottles with soil out of the remaining 9 were added 2.5 ml boiled milli-Q water and 0.5 ml lactate solution. The next three were added 2.5 ml boiled milli-Q water and 2.5 ml of NH4 solution, and the last three were added 2.5 ml of C2. They were labelled BM with soil and organic compound (BM+O), BM with soil and NH4 (BM+NH4) and BM with soil and sulphide (BM+S) respectively. This was done under N2/CO2 and closed with butyl rubber stopper. The Head space was changed to N2/CO2 in order to prevent the phosphate buffer (W1+W2) from reacting with C1 and C2 to produce CO2 which will affect the pH of the medium. In the three bottles without soil, 5 µl of PCE (99.5% E. Merck, Darmstadt Germany) was injected by slipping the needle of the syringe between the stopper and the bottle, while in all 18 bottles 5 µl of TCE and DCE were also added using separate micro syringes. The stoppers were then capped with aluminium crimps while maintaining an overpressure of one atmosphere of N2/CO2. The bottles were then transferred to the climate room at 15oC

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-GC analysis At 9:30 am the next day 0.3 ml of the head space of each of the bottles were injected in a G C ECD machine split at 118 split gas flow rate. The injection procedure was the same as in the first part. -Calibration for the split injection The Calibration bottles were prepared by labelling three of the 118 ml serum bottles with the following quantities; 1 µl, 2.5 µl, and 5 µl respectively. Each of these bottles was then covered with a butyl rubber stopper. In the first bottle labelled 1 µl, 1 µl of PCE was injected into it by slipping the needle syringe in between the rubber stopper and the bottleneck. In that same bottle, 1 µl TCE and DCE were also injected in the same manner. The bottle was now firmly covered with the rubber stopper and capped with aluminium crimps. The same was done with second and the third bottle but using 2.5 µl and 5 µl of the compounds respectively. The three bottles were given of 10 ml of air as overpressure using a 10 ml syringe. The three bottles were then taken to the oven at 100˚C for 10 minutes. This was to allow the liquid compounds injected to evaporate to the gaseous phase. They are then allowed to cool down to ambient (18˚C). At this point, four bottles of size 9 ml were sealed with butyl rubber stoppers and capped with aluminium crimps. 0.3 ml headspace is taken from each of the 118 ml sampled bottles into a separate 9 ml sealed bottle and labelled accordingly. That is making a 30 times dilution. 0.3 ml of headspace was split injected into the GC ECD equipment. The data generated was registered in excel and a calibration curve made. The calibration equation was computed and the line trend was also obtained. Calculations are seen in the appendix table 6, figure 19a. -Calibration for the splitless injection 0.3 ml headspace was taken from the 1 ml 9 ml bottles with a 1 ml syringe and injected to the GC ECD equipment (splitless) and the areas of the peaks of the 3 compounds under investigation were recorded. The same was repeated for the 2.5 µl and the 5 µl 30 time dilution bottles. The data generated was registered in excel and a calibration curve made. The calibration equation was computed and the line trend was also obtained. See appendix table 6, figure 19b.

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Results: The result is presented in two sections: one for the aerobic degradation study and the other for the anaerobic degradation study. Methane consumption was noticed with the highest occurring in the samples with 3.0 ml methane followed by those with 1.5 ml methane and lastly those with 0.9 ml methane in both the fertilised and the non-fertilises samples while oxygen consumption was as high as 52% of that present in the air supplied in the samples containing the fertilizer and 19% in the soil samples without t fertilizers, see appendix table 4. It was observed that the organic content of the soil was too low while the water content was average or accepted for most soil as concerns microbial activities. It is measured with a ± standard deviation. This soil properties need to be suitable for biological activities to prevail in that medium see table 5 below. Table 5 Soil properties Water content, ml/g 0.14(±0.2) Organic content, %/dry wt. 0.5(±0.01)

For the aerobic and the anaerobic biodegradation studies measurements are taken in terms of GC peak areas. The different peaks correspond to the different compounds present in the headspace injected into the GC. The peaks are produced at different intervals which correspond to the retention time of the different pollutants present in the headspace. The size peak areas correspond to the amount of the pollutant present in the injected headspace. A decrease is considered when the numbers of points below the first point of analyses on the graph are more than the number of points above and the reverse is increase. The graphs are produced by computing the median value of GC peak area of a pollutant for each triplicate set up against time in days. Results of the aerobic section Graphical representations of results per treatment for the aerobic degradation of the three chlorinated compounds are shown in figures 1A - 2C. From the graphical presentation of the results of the aerobic degradation, it can be seen that there is no trend in the amount of the compounds under investigation. The variation among the triplicates is too wide to allow any claim of differences in results within the dates. Furthermore, due to the mistakes of not calibrating the GC-system on the dates of measurement, the trends indicated maybe due to the variation in the detector response in the different dates of head space measurement. The results of the aerobic studies are presented in two parts that is the fertilised and the non-fertilised parts. The first 20 days were considered as incubation period thus the results will be from day 21 to day 60. Results of the fertilised part Analysing the graphical results presented in figure 1A to C it could be seen that from day 21 day 60 there was some decrease in the DCE of the following methane fertilised soil

15

(MF) treatments; 1.5 and 3.0 ml methane while there was no apparent decrease in the amount of DCE in the 0.9 ml methane treatment. No decrease was also seen with the DCE of control 1, and variation between all the triplicates. a

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Fig. 1 DCE (a), TCE (b) and PCE (c) GC peak area with time for different amounts of added MF soil. Results of the non-fertilised part Analysing the result obtained from the methane non-fertilised (MNF) soil set up, it was observed that there was noticeable decrease in the DCE of the 0.9ml MNF set up while the was no noticeable decrease in the DCE of the 1.5 ml and 3.0 ml MNF soil treatment and also in control 2 that is non methane non-fertilised (NMNF) treatment there was also no decrease in the DCE (see Figure 2a). For the TCE as shown in figure 2b there was decrease in the 0.9 ml MNF soil and control 2 while there was no reduction in the amount of TCE in the 1.5 ml and 3.0 ml MNF soil set up. For the PCE in figure 2c, a decrease in amount was noticed in the 0.9 ml MNF and the control 2 while the was decrease in the 1.5 ml and 3.0 ml MNF treatment a

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Fig. 1 DCE (a), TCE (b) and PCE (c) GC peak area with time for different amounts of added MNF soil. Results of anaerobic biodegradation From figure 3A – 4C the graphs are presenting the change in the amount of the compounds under investigation. The results are those from day 10 to day 30 for the reason that the first three dates of analysis has been considered as incubation period. They are presented as GC peak area against time in days. The result is in two parts of three set ups each. Part one, results of the BM without Soil (BM-S), BM with Soil (BMS), BM with Soil and Organic compound (BM+O) set ups and part two, results of the Water with Soil and Sulphide (W+S), BM with Soil and Sulphide (BM+S) and BM with Soil and NH4 (BM+NH4). Part one: From figure 3a, it was seen that there was a slight decrease in the amount of DCE in the BMS set up while no decrease was seen in the DCE of the BM–S and the BMO set ups. For TCE as shown in figure 3b, there was a slight decrease in GC peak area observed in the BMS set up and no change was seen in the BM-S and the BM+O set ups. For the PCE, the same result was obtained as in the case of DCE and TCE shown by figure 3c

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Figure 3 DCE (a), TCE (b) and PCE (c) GC peak area with time in the BM–S, BMS and BM+O set ups.

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Part two: For the DCE as shown by figure 4a there was a decrease in GC peak areas in all three set ups. The same result was also seen in the TCE and PCE of these three set-ups. a

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Discussion Aerobic study From the result no trend could be established due to the variation in the data sets over time. This makes it difficult to say with any degree of certainty if the additives could promote the biodegradation of the pollutants in question. In this case comparing the extent of decrease in the pollutants as per the set-ups will be out of place for the reason that, the decrease in a set-up is not significant from a statistical point of view. Chang and Alvarez (1996) showed that a mixed culture of microorganisms will cause the degradation of PCE and its intermediate compounds than a single culture. From this analogy one can deduce that the microorganisms in this experiment have been there for a long time and due to the toxic nature of the pollutant (van Agteren et al. 1998) very few species of bacteria would withstand this soil medium. This thus eliminates certain types of microorganisms that would have provided the soil medium with the different types of enzymes needed for the biodegradation of the pollutants and its intermediate products (Adriano et al. 1998). Due to this insignificant difference the result has been presented in GC peak area without any particular units. As such our interest is to know if there is any degradation with such additives. However there was a fundamental problem of calibration in the methodology which made the result very difficult to interpret. That is there was the absence of calibration preceding each day of sample analysis, thus, there was lack of proper optimisation and compensation of the GC column sensitivity due to continuous injection of samples headspace. The calibration made at the end of the experiment was helpful in calculating the amount of the compounds injected into the various set-ups at the start of the experiment. One other point concerning the methodology is the manner in which the pollutants where added to the set-up. They were injected into the serum sampling bottles through the thick butyl rubber stopper with a micro-syringe which was very difficult to penetrate due to much friction thus some delay and lose of the volatile pollutants before delivery. The result presented was from day 21 to day 60 in the sense that the early days was considered to be pre incubation period, since the pollutants were injected in the liquid phase, thus, some time was needed for it to go into the gas phase and equilibrate with the headspace. The oxygen consumption showed in the fertilized set-ups was more than 50% of that supplied in the air; this indicated more biological activity in the fertilized treatments. This is also an indication that microorganisms are limited by the amount of nutrient available for their metabolism and growth (Wilson and Clarke, 1994). For methane consumption, it was not so much influenced by the presence or absence of the fertilizer but the amount of methane at the start influence the amount consumed thus the more the methane the more that will likely be used by methanotrophs, while concomitant cometabolic degradation may take place (Chang and Alvarez, 1996). However Chang and Alvarez (1996) showed that methane is an electron donor in the reductive dechlorination of PCE. Anaerobic study In this study, the different growth media used seemed to have a positive influence on the rate of biodegradation of the chlorinated compounds under investigation. Beeman et al. (1994) showed that ammonia is a good electron donor for the reductive dechlorination

21

and DeWeerd et al. (1998) said that sulphide is a preferred electron donor for PCE and TCE and not for DCE. However, due to the lack of a substantial difference in the decrease of the amount of the pollutants between the different set-ups as in the first case, no accurate comparison could be made between the set-ups. From the results one can say that the additives and the experimental set-up caused the degradation of the pollutants to an extent. However in this situation I will rather advocate for a further research in order to confirm these indicative trends. Also the results obtained can not be used to assess biodegradation of the pollutants with certainty due to the fundamental experimental error of not making a calibration at the start of each day of a GC analysis and also after the change of a septum. It was also noticed that the decrease in GC peak area was more pronounced for TCE than for DCE, this could be explained from the point that during reductive dechlorination TCE is first converted to DCE thus adding DCE amount while DCE is converted to VC. Unfortunately, the amount of VC produced was below the detection limit for the GC ECD. The decrease in peak area found in BM with soil, sulphide and NH4 could be explained from the point that the ammonia and the sulphide added more electrons in system (Otten et al. 1997) thus more PCE and TCE were needed as electron acceptor and were consequently believed to degrade. Well not much analysis on how much and why the reduction in the GC peak area of the pollutant due to the problems encountered but will rather comment on why and how to go about it. The expected biodegradation in this research was not reached may be for the following reasons which has to be taken into consideration for subsequent work on this site. The site of soil collection shows that it is barren due to the fact that it is void of vegetation and at a dept which is more than 3 m from the surface. These aspects would lead to the soil incapable of supporting microbial activities leading to very little or no microorganisms in this soil. This is as a result of lack of enough nutrients as shown by the organic content of the soil (Table 5) and other suitable conditions. In addition to this, the soil has high levels of PCE and petroleum products which could also cause the inability of the microorganisms to thrive well. One other reason could be for the fact that the experiment was conducted for a short period of time. This then suggest that for in situ bioremediation to take place on this site more stringent measures should be taken to improve the soil nutrients, more active additives and other conditions for microbial growth and metabolisms and even the inoculation of more vibrant strains of microorganisms with broad spectrum of substrate and strains that could withstand high levels of these pollutants.

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Conclusion: From the above results one can not categorically ascertain any point for conclusion. From table 4 it is observed that the samples with fertilizer had consumed more oxygen than the samples without. This shows that the rate of metabolism and growth was higher in the samples containing the fertilizer. Thus it goes without saying that aerobic degradation will obviously be higher in the fertilised samples. That is inorganic nutrients are necessary for aerobic biodegradation. The amount of organic matter in the soil is also an aspect to check for degradation both in the aerobic and anaerobic condition since they will serve as carbon source. In the anaerobic situation little or no change took place in the amount of DCE and same with the PCE in the aerobic condition. No definitive conclusion could be draw about the degradation of these compounds since this experiment had some lapses that should be taken into consideration. At this point one can conclude that this research has just paved way for further investigation taking particular attention with the methodology and other shortcomings involved in scientific investigations.

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References Adriano, D.C. Bollag, J.M. Frankenberger, J.M. and Sims, R.C. eds. (1998). Bioremediation of contaminated soil. Wisconsin USA, American society of agronomy, inc., Crop science society of America, inc. and Soil science society of America, inc. van Agteren, M.H., Keuning, S. and Janssen, D.B. (1998). Handbook of biodegradation and biological treatment of hazardous organic compounds. Volume 2. Dordrecht, Kluwer academic. Aulenta, F. Majone, M. Verbo, P. and Tandoi, V. (2002). Complete dechlorination of tetrachloroethene to ethane in the presence of methanogenesis and acetogenesis by an anaerobic sediment microcosm. Biodegradation. 13 (6), pp. 411- 424 Aulenta, F. Potalvivo, M. Majone, M. Papini, M.P. and Tandoi, V. (2005). Anaerobic bioremediation of groundwater containing a mixture of 1,1,2,2-tetrachloroethane and chloroethenes. Biodegradation. 17, pp.193 - 206 Beeman, R.E. (1994). In situ Biodegradation of groundwater Contaminants. U.S. Patent No.5,277,815. Boorman, G.A. Dellarco, V. Dunnick, J.K. Chapin, R.E. Hunter, S. Hauchman, F. Gardner, H., Cox, M. and Sills, R. C. (1999). Drinking water disinfection byproducts: review and approach to toxicity evaluation. Environmental health perspectives. 107 (1), pp. 207-217. Chang, H.L. and Alvarez-Cohen, L. (1996). Biodegradation of individual and multiple chlorinated aliphatic hydrocarbons by methane-oxidizing culture. Applied Environmental and Microbiology. 62 (9), pp. 3371-3377. Coleman N. V., Mattes T.E.,Gossett J.M. and Spain J.C. (2002). Phylogenetic and kinetic diversity of vinyl chloride-assimilating bacteria from contaminated sites. Applied and Environmental Microbiology. 68, pp. 6162-6171. Davis, J. W. and Carpenter, C.L. (1990). Aerobic biodegradation of Vinyl chloride in groundwater sample. Applied and Environmental Microbiology. 56 (12), pp. 3878-3880 Deweerd, A.K. Flanagan, P.W. Brennan, J.M., Principe, J.M. and Spivack, L.J. (1998). Biodegradation of trichloroethylene and dichloromethane in contaminated soil and groundwater. Research Report/General Electric Corporate Research and Development. Schenectady, NY 12301-0008 Eder, T. and Schhmidt, W. (1995). Organochlorine contamination in The Great Lakes: the risks are real and demand action. Ecological society of America. 5 (2), pp. 298-301.

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Ejlertsson, J., Johansson, E., Karlsson, A., Meyerson, U. and Svensson, B.H. (1996). Anaerobic degradation of xenobiotics by organisms from municipal solid waste under landfilling conditions. Antonie van Leeuwenhoek. 69, pp. 67-74 Howarth, R.W. (2000). Clean coastal waters; understanding and reducing the effect of nutrient pollution. Washington DC, National academy press. Keppler, F. Eiden R. Niedan, V. Pracht, J. and Schoele, H. F. (2000) Halocarbon produced by natural oxidation processes during degradation of organic matter. Nature 403, pp. 298-301. Laturnus, F. (2003). Natural sources of volatile organohalogens: Impact on the biosphere. Ph.D. thesis, University of Copenhagen Denmark. . National Chrysanthemum Society USA. (2003). Soil composition and management [internet], Advanced Grower's Handbook. Available form: <http://www.mums.org/journal/articles/soil_management.htm> [Accessed 18 December 2005]. Otten, A. Alphenaar, A. Pijls, C. Spuij, F. and Han de Wit. (1997). In situ soil remediation. Dordrecht. Kluwer Academic. Sing, H. Loffler, F.E. and Fathepure, B. Z. (2004) Aerobic biodegradation of vinyl chloride by a highly enriched mixed culture. Biodegradation. 15 (3), pp.178 - 204 Solomon, S. (2004). The hole truth: what’s news (and what’s not) about the ozone hole. Nature. 427, pp. 289-291. Suarez, P.M. and RAfai, S.H. (1999) Biodegradation rate for fuel hydrocarbons and chlorinated solvents in groundwater. Civil and Environmental Engineering University of Houston. Tx 77204 - 4791 Surma, R. B. Liska K. S. and Hydrogeotechnike. (2004).Innovative process for the on-site decontamination of soil. Research report/Ecosoil. COOP-CT-2004-508442. Peijnenburg, J.C.W. and Damborsky, J. (1996): Biodegradability Prediction. Dordrecht, Kluwer Academic. U.S. Environmental Protection Agency. (1991). Innovation treatment technologies, seminal status report. EPA/540/2-91/001, No.2.U.S. Office of solid waste and emergency response. Washington DC. Wilson, D.J. and Clarke, A.N. (1994). Hazardous waste site soil remediation: Theory and application of innovative technology. New York, Marcel Dekker.

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Appendix Graphical presentation of raw data as area of GC peaks against time in days. Aerobic: a b c Figure 5a – c show the result of DCE, TCE and PCE for the 0.9 ml methane fertilised set-up respectively. a b c Figure 6a – c show the result of DCE, TCE and PCE for the 1.5 ml methane fertilised set-up respectively. a b c Figure 7a – c show the result of DCE, TCE and PCE for the 3.0 ml methane fertilised set-up respectively. a b c Figure 8a – c show the result of DCE, TCE and PCE for the non-methane fertilised set-up respectively (Control 1).

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a b c Figure 9a – c show the result of DCE, TCE and PCE for the 0.9 ml methane non-fertilised set-up respectively. a b c Figure 10a – c show the result of DCE, TCE and PCE for the 1.5 ml methane non-fertilised set-up respectively. a b c Figure 11a – c show the result of DCE, TCE and PCE for the 3.0 ml methane non-fertilised set-up respectively. a b c Figure 12a – c show the result of DCE, TCE and PCE for the non-methane non-fertilised set-up respectively (control 2).

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Anaerobic Study: a b c Figure 13a – c show the result of DCE, TCE and PCE for BM – Soil set-up respectively. a b c Figure 14a – c show the result of DCE, TCE and PCE for BM + Soil set-up respectively. a b c Figure 15a – c show the result of DCE, TCE and PCE for BM soil + Organic compound set-up respectively. a b c Figure 13a – c show the result of DCE, TCE and PCE for Water soil + Sulphide set-up respectively.

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2000

2500

9 14 19 24 29 34

Time (Days)

are

a

PCE BM +Soil

Series2

0

50

100

150

200

9 14 19 24 29 34

Time (Days)

area

DCEBM Soil+OrgSeries

01000200030004000

50006000700080009000

9 14 19 24 29 34

Time (Days)

are

a

TCEBM Soil+OrgSeries2

0

500

1000

1500

2000

2500

9 14 19 24 29 34

Time (Days)

area

PCEBM Soil+OrgS i 2

0

50

100

150

200

9 14 19 24 29 34

T i me ( Days)

area

DCEwater+sulphideSeries2

0

2 0 0

4 0 0

6 0 0

8 0 0

10 0 0

12 0 0

9 14 19 2 4 2 9 3 4

T i me ( D a y s )

area

TCEwater+sulphideSeries2

0

500

1000

1500

2000

2500

9 14 19 24 29 34

T i me ( D a y s )

area

PCEwater+sulphideSeries2

28

a b c Figure 17a – c show the result of DCE, TCE and PCE for BM soil + Sulphide set-up respectively. a b c Figure 18a – c show the result of DCE, TCE and PCE for BM soil sulphide + NH4 set-up respectively. Composition of the Fertilizer used: N = 12% P = 5% K = 14% Mg = 1.2% S = 8% Zn = 0.01% Mo = 0.0005% B = 0.02% Soil Composition: Nutrient part per million in soils Nitrates = 25 - 60ppm Phosphate (PO4

3-) = 4 -5ppm i.e. 500g of soil contain 2-2.5mg PO43-

Potash = 25 -50ppm Calcium = 150 - 250ppm (National Chrysanthemum Society USA. 2003). Calculation of the amount of fertilizer (PO4

3-) 1kg of soil contain 5mg of PO4

3- →400g of soil = 2mg of PO4

3- But 5g of PO4

3- contain 100g of fertilizer →2mg of PO4

3-= 40mg of fertilizer Thus 40mg of fertilizer was added to 400g of soil.

0

50

100

150

200

9 14 19 24 29 34

T ime ( D ays)

are

a

DCEwater+sulphidelSeries2

0

200

400

600

800

1000

1200

9 14 19 24 29 34

T i me ( Days)

area TCE

water+sulphidelSeries2

0

500

1000

1500

2000

2500

9 14 19 24 29 34

T i me ( D a y s )

area

PCEwater+sulSeries2

0

20

40

60

80

100

120

140

160

9 14 19 24 29 34

Time (Days)

area

DCE BMSulphide+HN4Series2

0100200300400500600700800900

1000

9 14 19 24 29 34

Time (Days)

are

a

TCE BMSulphide+HN4

Series2

0

500

1000

1500

2000

2500

9 14 19 24 29 34

Time (Days)

are

a

PCE BMSulphide+HN4

Series2

29

Table1. Weight measurements (g) for the determination of the water content of the soil

Porcelain cups

Wt of cups (Wc)

Wt of soil (Ws)

Dry wt of soil & cup (Wsc)

Water content (WC)

1 30.42 10 39.08 1.34 2 30.43 10 39.06 1.37 3 31.46 10 39.82 1.34

Table2. Weight measurements (g) for the determination of the organic content of the soil (S = soil & C = cup) Porcelain cups

Dry wt of SC at 105(Dw105)

Dry of SC at 450(Dw 450)

Organic content

1 39.08 39.04 0.04 2 39.06 39.02 0.04 3 39.82 39.78 0.06 Table3. Weight measurements (g) for the determination of water holding capacity (WHC)

Test tubes (Tb)

Dry Wt. of wet Tb A

Wt of wet Tb B

Wt. of soil(S) C

Wt of immersed S&Tb. D

WHC D-C-B

1 8.62 11.54 27.74 41.40 2.12 2 8.99 13.16 27.65 44.67 3.41 3 8.85 12.66 27.90 43.57 3.01

Table4. Percentage of oxygen and methane consumed per treatment group within the first month of the experiment. Type of treatment

Amount of O2 present (Area) Triplicates

Standard in Air (Calibration)

O2 % Consumed (Mean value)

CH4 % Consumed (Mean value)

0.9ml CH4- fertilised soil

74,74,70 134 (100%) 50 (±2) 0.7 (±0.1)

1.5ml CH4 fertilised soil

79,75,83 134 46 (±3) 1.1 (±0.2)

3.0ml CH4- fertilised soil

52,31,69 134 65 (±13) 1.3 (±0.5)

30

0.9ml CH4- non fertilised soil

127,119,123 134 15 (±3) 0.6 (±0.1)

1.5ml CH4-non fertilised soil

130,103,125 134 18 (±10) 0.9 (±0.2)

3.0ml CH4- fertilised soil

102,124,110 134 23 (±8) 1.6 (±0.1)

No CH4-fertilised soil

89,73 134 45 (±8) 00

No CH4-non fertilised soil

127,112 134 18 (±7) 00

Table 6: Raw data for Calibration of DCE, TCE and PCE Calibration 30 times

dilution split

A 0 4908 4591 6429 D 0 1460 8217 20201 T 0 10690 144767 562727 P 0 14312 345186 1451070

splitless A 0 32171 19927 32760 D 0 20022 33725 79471 T 0 478512 102223

01497820

P 0 1461310

2016130

2394872

31

a

30X dilution split

PCEy = 302.04x - 189.34R2 = 0.9208

TCEy = 117.05x - 68.978R2 = 0.9306

DCEy = 4.185x - 1.6432R2 = 0.9754

-5000

500100015002000

0 2 4 6Vol. (µl)

Peak

are

a

Fig.19a shows a 30 times dilution calibration curve for the split injection (anaerobic study) with the linear equation and the R2 b

30x dilution splitless

DCEy = 15.322x + 0.6916R2 = 0.9869

TCEy = 292.59x + 128R2 = 0.9568

PCEy = 422.77x + 569.62R2 = 0.765

0

1000

2000

3000

0 2 4 6

Vol (µl)

peak

are

a

Fig.19b shows a 30 times dilution calibration curve for the splitless injection (aerobic study) with the linear equation and the R2

32

Calculation of the amount of pollutants injected into each treatment system Aerobic study DCE 4µl was injected in all treatment bottles DCE density is 1265g/l Mwt (C2H2Cl2) = {(12x2)+(1x2)+(35.45x2)}=96.9 →1l = 1265g →mass = 4 x 10-6l = 1265 x 4 x 10-6

= 5.06 x 10-3g Mole = mass/Mwt → Mole = 5.06 x 10-3/96.9 = 5.22 x 10-5 = 52µmoles was used. TCE 5µl was injected in all the treatment bottles TCE density is 1462g/l Mwt. (C2HCl3) = 131.4 →1l = 1462g →mass = 5 x 10-6l = 1462 x 5 x 10-6g = 7.31 x10-3g Mole = mass/Mwt. → Mole =7.31 x 10-3/131.4 = 5.56 x 10-5 = 56µmoles was used. Anaerobic study DCE 5µl was injected in all treatment bottles But DCE density is 1265g / l RMwt = 96.9 →1l = 1265g →mass = 5 x 10-6l = 1265 x 5 x 10-6 = 6.325 x10-3g Mole = 6.325 x10-3g / 96.9 That is 65µmoles was used TCE Same 5µl thus 56µmoles was used Equations for the calculations: Water content = Wc + Ws - Wsc Dry weight = Wet soil - water content Organic content =(Dw105 – Dw450)*100/Dw105. Water holding capacity = D-C-B

33

Table7. Shows the mean and standard deviations of some of the soil compositions Item/samples 1 2 3 mean std Water content (ml) 1.34 1.37 1.34 1.35 0.017321 Dry weight of soil (g) 8.62 8.99 8.85 8.82 0.186815 Organic content (%) 0.04 0.04 0.06 0.046667 0.011547 WHC (ml) 2.12 3.41 3.01 2.846667 0.660328

Table8. Shows the mean and the standard deviations (%) of the methane consumption Replicates 1 2 3 mean std MF 0.9 0.7 0.7 0.6 0.666667 0.057735 MF 1.5 0.9 1.2 1.1 1.066667 0.152753 MF 3.0 1.7 0.8 1.5 1.333333 0.472582 MNF 0.9 0.6 0.6 0.7 0.633333 0.057735 MNF 1.5 0.8 0.8 1.1 0.9 0.173205 MNF 3.0 1.5 1.6 1.7 1.6 0.1

Table9. Shows the mean and the standard deviations (%) of the oxygen consumption Replicates 1 2 3 Mean std MF 0.9 47.5 49 52 49.5 2.291288 MF 1.5 44 46 49 46.33333 2.516611 MF 3.0 53 79 64 65.33333 13.05118 MNF 0.9 13 19 15.9 15.96667 3.000556 MNF 1.5 11.3 29.8 14.7 18.6 9.847335 MNF 3.0 30.5 15.3 24.5 23.43333 7.655935 NMF 39.5 50.5 45 7.778175 NMNF 13.6 24.1 18.85 7.424621


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