ISOLATION AND CHARACTERIZATION OF SOIL BACTERIA CAPABLE OF DEGRADING METRIBUZIN IN SUGARCANE FARMS OF WESTERN KENYA

ABSTRACT
Nzoia River Drainage Basin is a major sugar production region in Kenya. Various pesticides are applied in this area to control weeds and boost sugar productivity. However, use of herbicides for weed control leads to increased chemical loads in the environment whose effects could be disastrous to the biotic component. The use of pesticide adapted microorganisms in the degradation and detoxification of many toxic xenobiotics, especially pesticides, is an efficient tool for the decontamination of polluted environments. The main objective of this work wasto isolate and identify soil bacteria capable of degrading metribuzin, a commonly used weed killer in sugarcane farms.Five farms with history of metribuzin application were purposively identified for soil sampling. Random soil sampling was used to obtain samples from a depth of 0 – 10cm in November 2013. A composite sample was used in isolation of the bacteria. Influence of temperature, pH, nitrogen and phosphorous on growth of the isolated bacteria was also tested. Experimental design was used to carry out the experiments in the laboratory. Mineral salts media containing metribuzin as the sole carbon source was used to culture and selectively isolate metribuzin degrading bacteria. Growth of the bacteria in the medium measured as absorbance at OD600nmfor various time intervals was an indication of tolerance to the herbicide and ability to utilize metribuzin as a carbon source. HPLC method was used to determine ability of the bacteria to degrade metribuzin and assess the metabolites after the 21 day incubation period. Molecular analysis was carried out by DNA extraction from each isolate and subjected to PCR using 16S primers. Sequences and blast results were compared to relevant data bases. ANOVA and separation of means using LSD at p ≤ 0.05 was used to analyse data. Seven different bacteria isolates with metribuzin degrading potential were coded NZ453A, NZ454B, NZ453C, NZ543A, NZ543B, NZ8070 and NZ1110. They were subjected to morphological, cultural, biochemical and molecular characterization. Results also revealed thattemperature, pH, nitrogen and phosphorous had different influence on the specific bacteria but generally, 35oC, pH 9, nitrogen and phosphorous concentrations of 7.5g/L recorded highest growth on most of the isolates. The isolates degraded more than 93% of metribuzin. The seven isolated bacteria were identified as Planococcus sp., Burkholderia cepacia, Pseudomonas sp., Bacillus sp., Arthrobacter sp., and Staphylococcus sp., all of which have been previously associated with degradation of recalcitrant compounds in the environment. This indicates that Nzoia sugarcane farms consist of different metribuzin degrading bacteria, which can grow in different physical chemical conditions. They can be multiplied and further developed for bioremediation or bioaugmentation of metribuzin contaminated sites.

CHAPTER ONE
INTRODUCTION
Background information
Sugarcane is an important cash crop in Kenya and it is the major economic activity undertaken in Western Kenya among other agricultural activities (KESREF, 2009). Weeds in sugarcane growing areas are a major challenge to cane production and can reduce sugar yields significantly as they compete for moisture, nutrients, and light during the growing season as well as acting as alternate hosts for disease and insect pests. Heavy weed infestations can also interfere with sugarcane harvest by adding unnecessary harvesting expenses and can act as source of seed bank replenishment and re-infestation in subsequent years (KESREF, 2009). Herbicides such as atrazine (as Gesapaxcombi), Metribuzin, hexazinone, diuron, actril DS (isoxynil + 2,4-D ester), ametryn and glyphosate are frequently used for controlling grasses and broad leaf weed in different crop fields (KESREF, 2009).

The excessive use of herbicides leads to accumulation of a huge amount of residues in the environment, thereby posing a substantial health hazard for the current and future generations due to uptake and accumulation of these toxic compounds in the food chain and drinking water (Monisha et al., 2014). Pesticides applied on land subsequently find their way into water bodies through runoff causing trans- media pollution. In deed residual pesticides have been detected in soil and water in the Nzoia River Drainage Basin (NRDB) in Kenya (Getenga et al., 2004, Ngigi et al., 2011).

Long exposure of the soils to triazine and chlorophenoxy-herbicides that are used in Kenyan sugarcane farms may have resulted in the adaption of the native soil microbes leading to enhanced degradation (mineralization) of the herbicides in the soil (Ramdas and Sims, 2011). Studies conducted in Kenya and other parts of the world show that adapted microbes indeed do exist in the soils with long exposure to the herbicides (Abdelhafid et al., 2000; Getenga et al., 2009; Ngigi et al., 2011). Microbes showing enhanced herbicide degradation abilities can be used in biogumentation experiments to stimulate degradation of herbicides in contaminated fields (Rousseaux et al., 2003; Aislabie et al., 2005).

Metribuzin is a herbicide in the group of triazinones and it is listed as a toxic release inventory (TRI) chemical (USEPA, 1996). It is moderately adsorbed on soils with high clay or organic content (HSDB, 2000) The half-life of metribuzin increases with decreasing soil moisture content, depth, temperature and pH (Malone et al., 2004). Microbial metabolism is the major pathway for the removal of metribuzin from soil. This study screened soils from sugarcane farms of Nzoia Sugar Company, in NRDB for bacteria with metribuzin degrading potential. The findings from this study may be useful for bioremediation of metribuzin contaminated soils in generating information that may be used in subsequent similar studies.

Statement of the problem
Pesticides have played an important role in the increase of agricultural production for food security to an ever increasing world population. The range of damage associated with the application of pesticides to the environment and different receptors is great. The major environmental impacts of pesticide use in agriculture include; loss of aquatic and terrestrial biodiversity, contamination of surface and ground water, agricultural produce and poisoning of agricultural workers among other potential negative impacts. Metribuzin is a widely preferred herbicide because of relatively low cost, unrestricted use and is considered for use more often, after banning of residual pesticides in most countries. It has been detected in both surface and ground water, in areas of use in different studies. It is a frequently used herbicide in sugarcane farms of Western Kenya.

Recent studies in Kenya have indicated the presence of pesticide residues in sugarcane cultivated soils and water from the drainage basins. As a result, there is growing concerns regarding the potential effects of pesticides on non-target organisms such as soil microbes, man, and livestock among others. Erosion of sugarcane soils and subsequent transport of sediment-bound contaminants in river runoffs into Lake Victoria is a growing concern as the sugar industry continues to expand. Lake Victoria is an important natural resource to the East African region and beyond in the fishing industry, farming, source of domestic water supply, transportation and source of river Nile, which is also the main source of livelihoods for North African countries. Available animal studies suggest metribuzin exposure causes low acute toxicity. Sub-chronic studies suggest that metribuzin could cause adverse effects in body weight gain, organ weight and haematological parameters.

Objectives
General objective
To assess the presence and physical chemical growth conditions of potential metribuzin degrading bacteria in Nzoia sugar belt farms, for probable use in bioremediation.

Specific objectives
1. To determine bacteria population distribution, isolate and characterize bacteria with potential of degrading metribuzin in Nzoia sugar company farms.

2. To determine the influence of temperature, pH, phosphorous and Nitrogen on growth of metribuzin degrading isolates.

3. To determine degradation potential and molecular identities of metribuzin degrading bacteria.

Hypotheses
1. There is no significant difference in soil bacteria population distribution and bacteria isolates with potential of degrading metribuzin in Nzoia sugar company farms.

2. There is no significant difference in growth of the isolates due to effects of temperature, pH, phosphorous and nitrogen.

3. There is no significant difference in degradation potential and molecular identities of metribuzin degrading bacteria.

Justification
In Kenya large amounts of metribuzin are used to control weeds in sugar belts of Nzoia, Mumias and Sony, as well as wheat and maize farming in Eldoret, tea farming and coffee plantations in the country. Despite the herbicide’s importance as a herbicide, it is known to have a half-life of between two and a half months to four months in soils and is a surface and ground water contaminant. It is also known to have negative health effects, for example internal organs enlargement and haematological conditions, in animals after ingestion. Microbial degradation is the principle route of removal of metribuzin and it is considered an environmental friendly approach in pollution management. Bioremediation which includes the gainful utilization of effective microorganism for the metabolism or biodegradation of target pollutants into safer and innocuous products is amongst the potent technologies that are being used globally for the restoration of contaminated sites. A contemporary approach in this regard is the utilization of microorganisms especially bacteria due to fast growth and metabolism. Bacteria are well known for their diversity and versatility with regard to both physical and chemical environments. Many bacteria that are able to degrade pesticides have been isolated from soil around the world. In Kenya, the presence and the role of metribuzin degrading bacteria in soil has not been demonstrated in agricultural systems, even though studies show metribuzin presence in natural waters where the herbicide is used. The growth conditions, bacteria distribution and influence of physico-chemical parameters on bacteria populations are also not known hence there is need to establish multiplication conditions of the bacteria for essential uses which include bioremediation. This study will contribute to acquisition of new knowledge in research, increased government database for use in policy formulations, in herbicide use and will create awareness among the nuclear estate managers about the effects of herbicides to the environment and bioremediation methods in restoration of contaminated sites.

Scope of the study
Among the many farms, soil samples were obtained purposively from five farms with history of metribuzin application within a period of two to three years at the time of sampling. It is within this duration that metribuzin was used frequently as per data from the Nzoia Sugar Company. The farms were part of Nzoia Sugar Company Nucleus estate farms with designated numbers and were representative of farms with metribuzin treatment. Among many other microorganisms known to degrade xenobiotic compounds, this study researched on bacteria in regard to their versatility and fast metabolism. This study was done in vitroasone of the most appropriate method of isolating and identifying specific degraders with limited resources for the study.Other herbicides were used in the study area but Metribuzin was studied because it has been detected as a residue in Nzoia River Drainage Basin, as reported in other studies (Ngigi et al., 2011). It is also frequently used as an alternate herbicideto other herbicides.

Assumptions
It was assumed that the genera of metribuzin degrading bacteria present were not affected by variations in weather, such that they were present in the soil in all seasons, although sampling was done during the rainy season. The bacteria were assumed to be adapted to degrading metribuzin insitu and their multiplication invitro could be successfully used for bioaugmentation of contaminated sites in the field.

Definition of terms Pesticide
Any chemical substance used for killing pests, as insects, weeds etc. (Anon, 1999)

Herbicide
Also commonly known as weed killers, are pesticides used to kill unwanted plants (Anon, 1999).

Xenobiotic compound
These are chemicals which are foreign to the biosphere.

Bioremediation
Bioremediation is a process that uses mainly microorganisms, plants, or microbial or plant enzymes to detoxify contaminants in the soil and other environments (Hance 1991).

Biodegradation
Biodegradation refers to the partial, and sometimes total, transformation or detoxification of contaminants by microorganisms and plants (Hance 1991).

Metribuzin degrading isolates
These refer to bacteria that were isolated from the soil samples, which utilized metribuzin as the sole carbon source, thus breaking down the metribuzin compound into metabolites.

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Item Type: Kenyan Material  |  Attribute: 51 pages  |  Chapters: 1-5
Format: MS Word  |  Price: KSh900  |  Delivery: Within 30Mins.
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