THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY NGUYEN DUC HAI TOPIC TITLE: ISOLATION AND DETECTION OF BACTERIA IN SOIL ASH AND SEDIMENT WITH THE CONTAMINATION OF PARAQUAT AND GLYPHOSATE BACHELOR THESIS Study Mode : Full - time Major : Environmental Science and Management Faculty : International Training and Development Center Batch : 2010 - 2015 Thai Nguyen, 23/01/ 2015 n Thai Nguyen University of Agriculture and Forestry Degree Program Bachelor of Environmental Science and Management Student Name Nguyen Duc Hai Student ID DTN1053110068 Thesis Title “Isolation and Detection of bacteria in soil, ash and sediment with the contamination of Paraquat and Glyphosate” Supervisor (s) 1. Chiu-Chung Young, National Chung Hsing University - Taiwan 2. Nguyen Huu Tho Abstract: Paraquat and Glyphosate are well-known as two of the most widely used herbicides in agricultural activities and landscape turf management. However, their impacts are extremely numerous and serious on both human health and the environment, especially the uncontrolled usage of these herbicides with high levels causes soil contamination.
Currently, PCR is an useful and powerful technique, and it has been developed for various applications, particularly for DNA isolation and detection of microbial organisms, such as bacteria or viruses.Paraquat and Glyphosate were added to the soil, ash and sediment to make herbicide mixtures in this study. Bacteria lived in herbicide mixtures were isolated, and then were detected by a PCR detection method. In this study, there were 24 types of bacteria that were isolated and found out their names, in which 12 species of bacteria could grow well in the herbicide environment. The results indicated that bacteria are able to survive in the herbicide contaminated condition.
It also demonstrated that those bacteria have potentials to utilize herbicide as a sole carbon source for their survival, degrade herbicide toxic compounds Paraquat and Glyphosate, and reduce significant the level of Paraquat and Glyphosate toxicity in the soil, ash and sediment were polluted by the herbicides. Keywords Paraquat , Glyphosate, Microbial DNA isolation Herbicide, Biodegradation, Microbe Number of Pages 40 pages Date of 22/01/2015 Submission i n AKNOWLEDGEMENT This thesis was completed under international collaboration between the Thai Nguyen University of Agriculture and Forestry, Thai Nguyen - Viet Nam and the National Chung Hsing University – Taiwan in the graduate internship program academic year 2010 - 2015. During implementation and completion of the subject, I have received the help and support of Thai Nguyen University of Agriculture and Forestry and National Chung Hsing University, on this occasion I would like to thank you for the invaluable help. First of all I would like to express deep gratitude and respect to Professor Chiu Chung-Yong, who has direct supervision and guidance I made this topic, and I would like to thank Master Nguyen Huu Tho as a science instructor, he spent a lot of time and effort in helping me complete this research! This study can not complete without the encouragement and support of many colleagues, friends and family members, Finally, I would like to thank everyone who helped me complete this study! Thai Nguyen, January 15th, 2015 Supervisor Student Nguyen Huu Tho Nguyen Duc Hai ii n TABLE OF CONTENTS LIST OF FIGURES.1 LIST OF TABLES.2 LIST OF ABBREVIATIONS.
Soil, ash and sediment. Overview of Herbicide. Classification of herbicides. Effects of herbicide with environment and human.
Overview of soil, ash, sediment and soil pollution. Polymerase Chain Reaction ( PCR ). Process of PCR Method. Application of PCR.
Bushnell Haas basal medium. Preparing Herbicide Toxicity. Microbial DNA isolation. Preparing Nutrient Broth Agar medium and Bushnell Haas culture medium.
Preparing Herbicide Toxicity. Serial dilution of herbicide mixtures. Microbial DNA isolation. Polymerase chain reaction (PCR) detection of bacteria.
Imaging and Analysis. Preparing herbicide toxicity. Serial dilution of herbicide mixtures. Microbial DNA isolation.
Test the growth of bacteria in contamination environment. DISCUSSION AND CONCLUSION. 38 iv n LIST OF FIGURES Figure 2. The molecular structure of Paraquat.
The molecular structure of Glyphosate. The three main step of PCR process. PCR process for 16S rDNA .The color and density of bacteria in process of dilution for Ash-PRQ herbicide mixtures.The color and density of bacteria in process of dilution for Ash-GPT herbicide mixtures.The color and density of bacteria in process of dilution for .30 Sediment-PRQ herbicide mixtures .The color and density of bacteria in process of dilution for Sediment-GPT herbicide mixtures.The color and density of bacteria in process of dilution for Soil-PRQ herbicide mixtures.The color and density of bacteria in process of dilution for Soil-GPT herbicide mixtures.31 1 n LIST OF TABLES Table 2. Classification for the size of sediment (Holmes, 1978).1 Content of BH medium .2 Content of NA medium.
The content of PCR tube. Herbicide mixtures using in this experiment.U of bacteria per 1mL in each type of herbicide mixtures. Number of bacteria detected in six herbicide mixtures. The density of each type of bacteria in Ash – PRQherbicide mixture.
The density of each type of bacteria in Ash - GPTherbicide mixture. The density of bacteria in herbicide mixture. Bacteria isolated in ash, soil and sediment with contamination of Paraquat and Glyphosate. Ability for survival of bacteria in herbicide mixture.36 2 n LIST OF ABBREVIATIONS PRQ Paraquat GPT Glyphosate C.U Colony Forming Unit PCR Polymerase chain reaction 3 n PART I.
Research rationale At the end of the 20th century and the early of the 21st century, it is a period that marks the extremely strong development of science and technology in Vietnam and other nations in the world. As an agricultural country with a long wet rice culture, Vietnam primarily takes advantages of available resources. Nevertheless, by using outdated farming methods with the presence of plant protection products and the unscientific use of those ones, it causes contamination of the environment, especially in soil, water, sediments, etc. At the present, there are thousands of types of pesticides, herbicides, soil improvement products used.
Each type of those requests users must have certain knowledge about the dosage and usages to have the best efficiency and less impact on the environment as well. Nonetheless, in fact, almost of users measure the dose by the naked eyes and unscientific manners. They use pesticides, herbicides or other products arbitrarily, thus the efficiency is impacted and the environment is received adverse effects from that (Sribanditmongkol et al. Research’s objectives In this study, Paraquat and Glyphosate were added to the soil, ash and sediment for the enrichment and using a specific PCR detection method.
Bacteria that could grow in herbicide environment were detected. That were demonstrate that those bacteria could utilize herbicide as a sole carbon source for their survival. Therefore, this research could confirm the growth of bacteria in the that contaminated condition as well as found out the potential bacteria with a strong ability to remove Paraquat and Glyphosate compounds in the environment. Research questions Each bacterium has a different metabolic system to eliminate herbicide.
The application of bacteria in detected in this study was significant for bioremediation technology. Therefore, this research answers the following questions 1. How many kinds of bacteria have been isolated and detected in herbicide mixtures? 2. What kinds of bacteria have ability to survive and grow in the environment contaminated by these herbicides? 3.
How is the growth of bacteria in the herbicide contaminated environment? 1. Limitations This study concentrated on the ability of bacteria to reduce Paraquat and Glyphosate concentration and their growth within the contaminated environment in laboratory. This research brought many significances in scientific learning and research areas. In terms of this aspect, doing the proposed research required the application and promotion of academic knowledge, and the results are useful materials or references for future researches.
The potential bacteria, were able to biodegrade or reduce Paraquat and Glyphosate concentration effectively, could apply in soil, ash or sediments which were contaminated by these kinds of herbicide. Besides, this study was significant to bioremediation applications and treatment of the environmental issues. Herbicide Herbicide is defined as weed killers or a pesticide used to kill weeds or unwanted wild plants which makes crops grow poorly and impact to crop yield and quality of agricultural products (Kellogg et al. Soil, ash and sediment 1.
Soil Soil is a natural body, including solids (minerals and organic matter), liquids, and gases occurs on the surface, occupies space and is characterized by one or both of the following: leg sun, or the layer, which is distinguished from the original material as a result of the addition, losses, transfer, and transformation of energy and matter, or the ability to support rooted plants from a natural environment (Chesworth et al. Ash In analytical chemistry, ash is well-known as all remaining non-aqueousresidue after a combustion, which consists mostly of metal oxide. It is one of the components in the proximate analysis of biological materials, that mostly include salty and inorganic substances (IUPAC, 1997) Moreover, ash is defined as the white or grayish powder remaining after something has been thoroughly burned or a product or material of volcanic activity, that has a small particle size (Cristina, 2007) 1. Sediment Sediment is defined as solid material, which formed by natural processes of erosion and weathering, and moved from one place to another through water, ice, or wind (Prothero et al.
Overview of Herbicide 2. Classification of herbicides Herbicide is classified base on activity, use, chemical family, mode of action, or type of vegetation controlled (Andrew et al. - Herbicide selective and non-selective + Selective herbicide will only kill weeds without harming the crop + Non-selective herbicide be used where no cultivation, these herbicides are harmful to all species are present where spraying and herbicide exposure. - Herbicide spraying on the foliage and herbicides is spray, fertilizer or drenches on soil + Foliar spraying of herbicides is the only can penetrate the grass to harm the grass (herbicides are not able to penetrate into the root).
+ Herbicides is spray, fertilizer or drenches on soil can only penetrate into the weeds through the stem or roots of the grass. These herbicides have been used spray on kind of ground just finish plowing or finished sowing, when the grass has not appeared on the field. - Herbicide for pre-germination and for post-germination + Herbicide for pre-germination is used early, immediately after sowing, when the grass has not grown on the farm. + Herbicide for post-germination is used later, when the grass was growing or immature stage.
Effects of herbicide with environment and human Herbicides have widely variable toxicity, and the toxicity depends on occupational exposure levels. The influences of herbicide on human health are very serious. Several herbicides can cause health effects ranging from skin rashes to death. The pathway of attack can arise from intentional or unintentional direct consumption, improper application resulting in the herbicide coming into direct contact with people or wildlife, inhalation of aerial sprays, or food consumption prior to the labeled pre-harvest interval.
Under some conditions, certain herbicides can be transported via leaching or surface runoff to contaminate groundwater or distant surface water sources. Generally, the conditions that promote herbicide transport include intense storm events (particularly shortly after application) and soils with limited capacity to adsorb or retain the herbicides. Herbicide properties that increase likelihood of transport include persistence (resistance to degradation) and high water solubility (Havens et al. Moreover, the risk of Parkinson's disease has been shown to increase with occupational exposure to herbicides and pesticides (Gorell et al.
The herbicide paraquat is suspected to be one such factor (Dinis et al. In addition to health effects caused by herbicides themselves, commercial herbicide mixtures often contain other chemicals, including inactive ingredients, which have negative impacts on human health (Dinis et al. Paraquat Paraquat (or N,N′-dimethyl-4,4′-bipyridinium dichloride) is a toxic organic compound with the formula [(C5H4N)2]Cl2.,2012) It is a non-selective herbicide, which one of the most widely used herbicides. The molecular structure of Paraquat However, it is very toxic and active and can kill green plant tissue when exposure.