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Journal of Environmental Quality 31:1502-1508 (2002)
© 2002 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America

TECHNICAL REPORTS
Atmospheric Pollutants and Trace Gases

Evaluation of Leachate Recirculation on Nitrous Oxide Production in the Likang Landfill, China

Chun Man Leea, Xue Rui Linb, Chong Yu Lanb, Samuel Chun Lap Loa and Gilbert Yuk Sing Chan*,a

a Dep. of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Hong Kong SAR, China
b School of Life Sciences, Zhongshan University/National Key Laboratory of Biocontrol, Guangzhou, China

* Corresponding author (bcyschan{at}polyu.edu.hk)

Received for publication July 9, 2001.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Landfill leachate recirculation is efficient in reducing the leachate quantity handled by a leachate treatment plant. However, after land application of leachate, nitrification and denitrification of the ammoniacal N becomes possible and the greenhouse gas nitrous oxide (N2O) is produced. Lack of information on the effects of leachate recirculation on N2O production led to a field study being conducted in the Likang Landfill (Guangzhou, China) where leachate recirculation had been practiced for 8 yr. Monthly productions and fluxes of N2O from leachate and soil were studied from June to November 2000. Environmental and chemical factors regulating N2O production were also accessed. An impermeable top liner was not used at this site; municipal solid waste was simply covered by inert soil and compacted by bulldozers. A high N2O emission rate (113 mg m-2 h-1) was detected from a leachate pond purposely formed on topsoil within the landfill boundary after leachate irrigation. A high N2O level (1.09 µg L-1) was detected in a gas sample emitted from topsoil 1 m from the leachate pond. Nitrous oxide production from denitrification in leachate-contaminated soil was at least 20 times higher than that from nitrification based on laboratory incubation studies. The N2O levels emitted from leachate ponds were compared with figures reported for different ecosystems and showed that the results of the present study were 68.7 to 88.6 times higher. Leachate recirculation can be a cost-effective operation in reducing the volume of leachate to be treated in landfill. However, to reduce N2O flux, leachate should be applied to underground soil rather than being irrigated and allowed to flow on topsoil.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
NITROUS OXIDE is an important infrared-absorbing trace gas that contributes 5 to 6% of the anthropogenic forcing of the global energy balance. Because of its persistent half-life of 150 yr in the atmosphere (Hao et al., 1987), it can be transported to the stratosphere where it is photochemically oxidized to NO and responsible for O3 depletion (Rodhe, 1990). Although the concentration of the N2O in ambient air is about 1000 times less than that of CO2 (Watson et al., 1992), it is about 200 times stronger than that of CO2 in terms of radioactive forcing. Since pre-industrial times, the atmospheric concentration of N2O has increased from about 270 µg L-1 to a recent measure of 315 µg L-1 (Department of the Environment, Transport and the Regions, 1998; Leuenberger and Siegenthaler, 1992). Soils and oceans are the primary natural sources of N2O (Seiler and Conard, 1987). The planetary sum of N2O sources was estimated to be about 15 Tg N yr-1, of which more than one-half results from soil microbial activity (McElroy and Wofsy, 1986; Davidson, 1991).

In developing countries such as China, urban refuse disposal is often in open dumps, and in most cases the N2O produced is released into the atmosphere. Composting and landfilling operations have been identified as possible significant sources of N2O (Christensen et al., 1996; Rettenberger and Schreier, 1996). Borjesson and Svensson (1997) conducted a study on three Swedish landfill sites where soil N2O levels reached more than 18 000 mg L-1 in a landfill covered with pure sewage sludge. However, there are no reported studies on the fluxes of N2O due to storage and recirculation of landfill leachate.

In landfill topsoil where oxygen is readily available from ambient air, irrigation or flowing of leachate, having a high level of ammoniacal N, is expected to result in high levels of N2O production from ammonium oxidation or nitrate reduction. According to Ludvigsen et al. (1998), denitrification was the main process responsible for the production of 0.3 to 57 mol N2O d-1 kg-1 (dry wt.) of soil from an anaerobic aquifer contaminated with landfill leachate. The rates of denitrification and nitrification can be affected by a number of factors, such as temperature, oxygen availability, soil moisture content, soil pH, soil total organic carbon, and nitrogen contents. However, there is a lack of information concerning N2O flux in relation to leachate recirculation practice. In the present study, N2O flux from Likang Landfill, Guangzhou, China, which practiced leachate recirculation for 8 yr, was monitored monthly from June to November 2000. The contributions of nitrification and denitrification to N2O production were determined by laboratory incubation studies. The relationships between climatic factors and physico–chemical properties of landfill topsoil and leachate to N2O production were determined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Study Site
Likang Landfill is one of only two ongoing landfill sites in Guangzhou, China. The site has been used for waste disposal since 1992. It is surrounded by hills, except on the northeast side. The landfill consists of an area of about 32 ha and 2870000 m3 capacity. Recent daily loadings of municipal solid waste have been 1400 to 1700 Mg (Zhou et al., 1999). Waste is spread in compacted layers with a bulldozer without regard for bulk density or percent saturation. The waste is covered with soil to reduce odor, prevent waste from blowing away, and prevent animals such as dogs from carrying waste from the site. Triplicate covering topsoil samples were collected in terms of distance from the leachate pond boundary (1 and 10 m) for chemical analysis and laboratory incubations. Synthetic impermeable top liners were not used in this site for landfill gas emission control.

To reduce loading to the leachate treatment plant, leachate rich in ammoniacal N from a collection pond at the lower site level was pumped via surface pipes at a rate of 400 m3 h-1 and discharged onto the topsoil at the southern and uppermost level of the site. Leachate was then allowed to flow by gravity as surface runoff (Fig. 1) . Under this leachate recirculation practice, leachate ponds of various sizes (2–105 m2) were formed in the uneven contour of the site.



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  		Fig. 1. Diagrammatic sketch of the sampling site (not to scale).

 
Site Measurement
The humidity and temperature of ambient air at the field site were measured by a wet-bulb hydrometer (Model DO706585; YSI, Yellow Springs, OH). To study the profile and cause of N2O elevation in ambient air, gas samples were collected in triplicate in 10-mL glass pre-evacuated tubes 1.5 m above ground at four locations: 1 m away from the leachate collection pond at the lower site level and 1, 10, and 150 m from a leachate pond of 27 to 105 m2 in size formed after leachate recirculation. The in situ dissolved oxygen level and temperature of leachate samples were measured by an oxygen probe of a dissolved oxygen meter (Model 54ARC; YSI).

To determine the dissolved N2O level in leachate, a 50-mL sample was collected in triplicate from the same leachate pond with a 100-mL syringe. An equal volume of headspace was filled by ambient air and shaken vigorously for 10 min. The volume expanded in the headspace after degassing was recorded and a 10-mL gas sample was collected from the headspace via a three-way valve with a 10-mL pre-evacuated sampling tube. All gas samples were stored in the dark before N2O quantification in the laboratory.

Gas bubbles were observed continuously escaping from the pond. To estimate gas emission rate and N2O level in the bubbles, gas samples were collected by a 225-cm3 sampler modified from a plastic vial. The sampler was immersed into the leachate, fully filled with leachate, and then inverted (upside down) so that gas bubbles were trapped. A three-way valve was connected to the bottom of the sampler and gas samples were collected by suction and passed to a 10-mL pre-evacuated sampling tube. Plastic tubing was connected between a landfill gas analyzer (Model LFG10-15597; ADC, Leesburg, VA) and the sampler output; the methane and carbon dioxide contents in gas samples were thus determined. The time required to fill the whole sampler was recorded and the gas emission rate from the pond in the form of bubbles was estimated.

Topsoil on the field site was highly contaminated by leachate, especially in the zones where fresh leachate was continuously flowing through. Nitrous oxide flux from leachate-contaminated topsoil was determined by collecting gas samples 1 and 2 m away from the leachate pond. Holes of 1 cm in diameter and 10 cm in depth were made by inserting an iron pipe into the soil at distances 1 and 2 m from the leachate pond boundary. Plastic boxes were inserted in triplicate into the soil at each distance and equilibrated for at least 0.5 h. Gas samples were collected with 10-mL pre-evacuated sampling tubes. Temperature of landfill topsoil was measured by inserting a thermometer to a 5-cm depth. The soil moisture content was the total weight loss of a soil sample after it has been dried at 105°C until a constant weight was obtained (Allen, 1989).

Physico–Chemical Analysis
Extractable soil NH4–N, NO3–N, and NO2–N were determined by shaking 10-g soil samples in 100 mL 2 M KCl solution. Ammoniacal N was determined by the Indophenolblue method (Allen, 1989), while NO3–N and NO2–N were determined with ion selective probes (Models 9307BN and C9346BN, respectively; Orion, Beverly, MA). Soil pH was determined from suspensions of air-dried soil in deionized water by an ISE meter (Model 720A, Orion). Total organic matter was determined by the loss in weight after igniting at 450°C for 16 h (Allen, 1989). Inorganic N sources in leachate were also measured by the same methods described above while the extraction step was omitted and dilution factors were applied if necessary.

Laboratory Incubations
Nitrification and Denitrification Nitrous Oxide Production
An incubation study was conducted to study the proportion of N2O produced in a landfill of leachate-contaminated soil due to autotrophic nitrification and denitrification. Soil samples were taken in triplicate at depths of 2 to 7 cm, at 1 and 10 m away from the leachate pond boundary. All soil samples were chilled at their field moisture condition and brought back to the laboratory and stored at 4°C. All incubations were completed within 5 d. Triplicate 10-g samples were incubated at 25°C in 50-mL serum bottles under four different conditions: (i) ambient air; (ii) 10 Pa of C2H2 (5 mL of 0.1% C2H2 per bottle) to inhibit autotrophic nitrification (Berg et al., 1982); (iii) 10 kPa of C2H2 (5 mL of 100% C2H2 per bottle) to inhibit denitrification N2O reduction (Ambus, 1998); and (iv) pure N2 (flushing with N2 for 3 min) to simulate an anaerobic environment. The N2O in headspace was measured after 2, 12, and 24 h. The nitrification N2O production was calculated as N2O0Pa - N2O10Pa. The relative denitrification N2O production was calculated as [N2O10Pa/N2O10kPa] x 100% (Ambus, 1998).

Nitrification and Denitrification Potential Assays
Since N2O can be produced by both nitrifiers and denitrifies, the potential for N2O production in landfill soils under natural conditions is related to the rates and extent of nitrification and denitrification. Thus, nitrification potential assay was conducted to estimate the activity of the nitrifier population in the soil, which is basically a static analysis (Schmidt and Belser, 1982). Triplicate 20-g samples of field-moist soil were mixed with 100 mL substrate (1 mM phosphate buffer, pH 7.1–7.4 and 50 mg NH4Cl L-1) in 500-mL wide-mouth Erlenmeyer flasks and incubated at 25°C in the dark and shaken at 150 rpm horizontally to maintain equilibrium between the dissolved N2O and that in the headspace. All flasks were covered with aluminum foil to reduce evaporation, but with pinholes made to allow free gas flow. Twelve-milliliter slurry samples were taken at 0, 5, and 24 h and centrifuged at 14 000 rpm for 15 min. The concentrations of NO3 + NO2 were determined from the supernatant. The nitrifying activity was determined by subtraction with initial and final values of (NO3 + NO2) kg-1 of soil over the 24 h.

The principle of the denitrification potential assay is to determine the relative quantity of denitrifying enzymes present in landfill soil samples. Triplicate 10-g field-moist portions of soil were mixed with 20 mL substrate (1.2 g KNO3 L-1, 5 g glucose L-1) in 50-mL serum bottles and 1 g L-1 chloramphenicol was added to inhibit enzyme synthesis during incubations (Ambus, 1998; Smith and Tiedje, 1979). The headspace was made anaerobic by flushing N2 for 3 min to remove O2 inhibition and diffusion limitations for substrates and products. Acetylene was added to 10 kPa and then N2O levels in the sample headspace were determined after 0, 30, and 60 min. The denitrification potential was calculated as the amount of N2O produced kg-1 soil h-1.

Gas Chromatography
All gas samples collected in the pre-evacuated tubes from the landfill and incubation studies were kept at 4°C and in the dark until quantitative analysis. Nitrous oxide contents were determined by injecting a 1-mL gas sample to a gas chromatograph (Model HP 4890; Hewlett–Packard, Wilmington, DE), equipped with Porapak Q column (80/100 mesh; 350 cm in length x 1/8 in. stainless steel; Alltech Associates, Deerfield, IL) (Upstill-Goddard et al., 1996). The column temperature was maintained at 70°C. Nitrogen was used as carrier gas at 15 mL min-1 and the temperature for the electron capture detector was set to 350°C. The N2O concentration was calculated by comparing the peak areas of N2O standards (Model Scotty II, Analyzed Gases 072263; Orion, Deerfield, IL).

Statistics
Differences between treatment groups and control groups were tested by Student's t test following Bhattacharya and Johnson (1977) and Daniel (1987) and the r values were reported.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
Field Study
Climatic information, including topsoil and leachate temperatures (0.5 m from pond surface), are shown in Table 1. The highest ambient temperature of 38°C was detected in September, while the highest pond leachate temperature of 42°C was detected on the sampling day in August. The lowest ambient temperature was recorded in November (24°C). Nitrous oxide emission from the pond showed a positive correlation to pond temperature (r = 0.703). The relative humidity ranged from 44 to 66%. The China Meteorological Administration reported no precipitation on any of the sampling days except in July.


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  		Table 1. Climatic information on sampling days and physico–chemical properties of leachate samples collected from leachate ponds from June to November 2000.

 
The physico–chemical parameters of leachate including inorganic N contents and pH are listed in Table 1. The dissolved oxygen detected in the pond was low (0.6–0.7 mg L-1). The concentration of NH4–N in the leachate samples was high (1353–4220 mg L-1) (Table 2).


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  		Table 2. Physico–chemical properties of topsoil samples collected near the leachate pond from July to November 2000.

 
Landfill soil samples collected near the leachate pond had a relatively high concentration of NH4+–N (33.7 to 1735 mg kg-1). The soil samples were generally alkaline and the total organic carbon content was about 3 to 14%. Moisture contents of the soil samples collected 1 m from the leachate pond were all significantly higher (P < 0.01) than soil samples collected 2 m from the leachate pond, indicating that the topsoil adjacent to the pond was highly contaminated and flooded with leachate.

The gas bubbled in the leachate pond was mostly landfill gas produced after anaerobic degradation of municipal solid waste, as it generally had a high methane content (12 to 37%) and high CO2 content (8.5 to 17%) (Table 3). The gas also had a high level of N2O, 0.73 to 1.56 µg N L-3. The dissolved N2O in the water sample collected in the leachate pond was 0.35 to 0.80 µg N L-3.


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  		Table 3. Nitrous oxide, CH4, and CO2 levels in gas bubbled out from the leachate pond, and dissolved N2O level in pond water sample from June to November 2000.

 
Nitrous oxide emitted from contaminated topsoil near the leachate pond ranged from 0.6 to 1.1 µg L-1 in the period from September to November (Table 4). Compared with uncontaminated landfill-covering soil (0.6 ± 0.32 µg L-1) the concentrations were 0 to 0.9 times higher (P < 0.05 or P < 0.01) for 1- and 2-m samples. The N2O contents in gas samples emitted from the topsoil 2 m from the leachate pond were significantly correlated with the soil NO3–N level (r = 0.719). Similarly, the N2O contents (far from the leachate pond boundary) were also highly correlated with soil NH4–N levels (r = 0.820 and 0.739 for samples collected 1 and 2 m from the pond, respectively).


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  		Table 4. Nitrous oxide concentration in gas samples emitted from topsoil near the leachate pond from July to November 2000.

 
Contributions of Nitrification and Denitrification on Nitrous Oxide Production
Production of N2O in soil samples collected from September to November was not affected by 10 Pa of C2H2, indicating that no N2O was produced by autotrophic nitrification (Table 5). Relative net N2O production from denitrification ranged from 26.4% to more than 100%.


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  		Table 5. Nitrification N2O production and the relative N2O production by denitrification in the Likang Landfill site from September to November 2000.

 
Denitrification and Nitrification Potentials
The denitrification potential was observed to be higher at 1 m from the pond than 10 m from the pond, except in the soil samples collected in September (Table 6). There was no consistent relation between nitrification potential and topographic position. The greatest value of denitrification and nitrification potential was observed in the soil samples collected in October (1.35 µg N kg-1 h-1) and November (420 µg N kg-1 h-1), respectively.


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  		Table 6. Nitrification and denitrification potentials of the Likang Landfill soil samples from September to November 2000.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
Field measurements were conducted to quantify N2O flux from Likang Landfill after leachate recirculation. The relative importance of biological nitrification and denitrification for N2O production was compared.

The ambient and pond leachate temperatures increased gradually from June 2000, reaching a climax in the summer (ambient 38°C, September; leachate 42°C, August) and then declined. The leachate was probably heated by strong solar irradiation, as it is a subtropical landfill, and also by the landfill gas generated after degradation of waste and bubbled from the pond bottom. Gas samples bubbled from the leachate pond had high N2O levels, ranging from 0.7 to 1.6 µg N L-1, and exhibited a positive correlation with pond temperature (r = 0.703). A doubling of N2O emission from nitrogenous soil for each 10°C rise in temperature within the range of 15 to 35°C has been reported (Blackmer et al., 1982). However, in the present study, soil temperature (28 to 37°C) showed a loose negative correlation with the topsoil N2O emission (r = -0.201). The decline in N2O emission at temperatures higher than 35°C was probably due to the reduction in autotrophic nitrifier activity. Soil desiccation after solar irradiation might also cause a reduction in the mobility of available nutrients for microbes (Focht and Verstraete, 1977).

Both nitrification and denitrification activities are substrate limited. Therefore, N transformation and N2O emission are expected to be highly related to soil NH4 or soil NO3 levels. Our field survey results indicated a significant correlation between the soil NH4–N level and concentration of N2O (0.6 to 1.1 µg L-1) detected in the topsoil gas samples (1 and 2 m away from the leachate pond) (r = 0.820 and 0.739, respectively). Similar correlations between NH4 levels and N2O fluxes were reported by Mosier et al. (1982)(1983) in cropped soils and Hutchinson et al. (1993) in fertilized soils. Furthermore, the soil NO3–N level also had a similar positive correlation with the N2O concentration in topsoil gas samples collected at 2 m from the leachate pond (r = 0.719). However, a low correlation (r = 0.260) between soil NO3–N level and N2O production was noted; leachate in the pond kept wetting the nearby soil and thus supplied an unlimited NO3–N source for N2O production.

In this study, the dissolved oxygen content in Likang leachate pond was maintained at a low level of 0.7 to 0.8 mg L-1 (3 to 5 cm from surface), and the N2O emission rates ranged from 8.45 to 113.11 mg N m-2 h-1. Under low O2 conditions, Poth and Focht (1985) suggested that the production of N2O by NH4 oxidizers resulted from a reductive process in which the bacteria use NO2 as an electron acceptor. This mechanism not only allows the organisms to conserve limited O2 but also avoids the potential for accumulation of toxic levels of NO2–N. Tortoso and Hutchinson (1990) suggested that the NO to N2O ratio of nitrification products, which normally ranges between 10 and 20 in fully aerobic environments, decreased along with O2 partial pressure. Despite the effect on nitrification, O2 also inhibits denitrifying enzyme activity and represses synthesis of new denitrifying enzymes (Payne, 1973). Soil denitrification rates have been shown to increase with added water or reduced aeration (Ardakani et al., 1977; Bremner and Shaw, 1958).

Landfill topsoil usually has a low O2 partial pressure if a superior landfill top liner is not provided. To determine the correlation between O2 partial pressure and N2O flux, a laboratory test was conducted by incubating soil samples at low O2 partial pressure. The results indicated that anaerobiosis (pure N2) increased N2O production in samples by up to 2.5 orders of magnitude. Since the production of N2O was mainly derived from denitrification, limiting O2 increased its rate of production. Moreover, N2O evolved during NH4–N oxidation arose from the interactions of hydroxylamine oxidoreductase and nitrate reductase, which was in turn influenced by the conditions of aerobiosis (Firestone, 1982). Hydroxylamine oxidoreductase forms the presumed intermediate (NOH) or its dimer hyponitrite, which may dismutate chemically under reduced O2 tensions to N2O (Nicholas, 1978).

The results of laboratory incubations of leachate-contaminated soil samples indicated that N2O was mainly produced through the denitrification process, whereas nitrification was less responsible for biological production of the gas; less than 3% of N2O was produced by nitrification. However, since C2H2 was used as an inhibitor for autotrophic nitrification, heterotrophic nitrification might not be completely eliminated in the assay (Anderson et al., 1993). The results were contradictory to some reports on forest ecosystems where nitrification was interpreted as an important source of N2O (Martikainen and De Boer, 1993; Robertson and Tiedje, 1987; Sitaula and Bakken, 1993). Heterotrophs responsible for denitrification have higher tolerance to elevated temperature (i.e., broad range of 15–75°C), and also tolerate low O2 tension (Focht and Verstraete, 1977). The continuous supply of organic carbon (energy source for denitrifiers) in the site might increase the proportion of N2O production noted at the Likang Landfill site.

There were significant differences (P < 0.05 or 0.01) between the N2O levels in air at different distances from the pond (Table 7). The N2O levels determined were all 0.1 to 1.4 times higher than that in the ambient air outside the landfill site. Borjesson and Svensson (1997) conducted a similar study in Swedish landfill sites, and the maximum N2O emission rates ranged from 3.9 x 10-2 to 1.31 g m-2 d-1 from the landfill cover consisting of pure sewage sludge. Bogner et al. (1999) also conducted temporal studies from July to January which indicated that peak production occurred in August in an Illinois (USA) landfill, reaching an emission rate of 8.55 x 10-3 g m-2 d-1 from the landfill surface. In our case study, Likang Landfill, with the practice of leachate recirculation, showed high emission rates of N2O ranging from 24.83 to 45.86 x 10-3 g m-2 d-1 from landfill topsoil. Moreover, N2O produced from leachate pond was about 1.12 to 1.43 times higher than that produced from landfill topsoil (µg N L-1) and the emission rate of N2O (ng N cm-2 h-1) from the leachate pond was 61.8 to 79.7 times higher than that detected from different ecosystems (Table 8). Due to the practice of leachate recirculation in Likang landfill, accumulation of leachate on soil would provide abundant inorganic N sources for the nitrifying and denitrifying bacteria. Moreover, leachate covering the topsoil would also decrease the O2 level in the soil underneath and thus increase the N2O production through nitrification and denitrification, especially the latter.


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  		Table 7. Nitrous oxide levels presented in ambient air at four different locations on the site from June to November 2000.

 

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  		Table 8. Literature comparison of the N2O fluxes from different ecosystem types.

 

   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
Although leachate recirculation can reduce the cost of running a leachate treatment plant and enhance biodegradation, the free flow and uneven contour of leachate through landfill topsoil forms leachate ponds with extremely low partial pressure of oxygen. Low O2 conditions in leachate favor N2O production. To fully benefit from leachate recirculation while avoiding excessive formation of N2O, which is a greenhouse gas, underground pipes should be installed to evenly distribute leachate and an impermeable top liner should be used to control gas emission.


   ACKNOWLEDGMENTS
 
Support from the Hong Kong Polytechnic University Central Research Grant (G-V885, G-T449, and G-YC11) and Zhongshan University (NSFC, 39970144 and GDNFSF, 990248) are greatly acknowledged.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 




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