Isolation and detection of bacteria in soil, ash and sediment with the contamination of paraquat and glyphosate

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 PARA

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

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 Prof Chiu-Chung Young, National Chung Hsing

Keywords Paraquat , Glyphosate, Microbial DNA isolation

Herbicide, Biodegradation, Microbe Number of Pages 40 pages

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 15 th , 2015

TABLE OF CONTENTS

LIST OF FIGURES 1

LIST OF TABLES 2

LIST OF ABBREVIATIONS 3

PART I INTRODUCTION 4

1.1 Research rationale 4

1.2 Research’s objectives 4

1.3 Research questions 5

1.4 Limitations 5

1.5 Definitions 5

1.5.1 Herbicide 5

1.5.2 Soil, ash and sediment 6

PART II LITERATURE REVIEW 7

2.1 Overview of Herbicide 7

2.1.1 Classification of herbicides 7

2.1.2 Effects of herbicide with environment and human 8

2.1.3 Paraquat 8

2.1.4 Glyphosate 9

2.2 Overview of soil, ash, sediment and soil pollution 10

2.2.1 Soil 10

2.2.2 Ash 11

2.2.3 Sediment 11

2.2.4 Soil Contamination 12

2.3 Polymerase Chain Reaction ( PCR ) 14

2.3.1 Definition 14

2.3.2 Process of PCR Method 14

2.3.3 Application of PCR 16

2.4 Related study 17

2.4.2 Domestic research 18

PART III METHODS 19

3.1 Materials 19

3.1.1 Bushnell Haas basal medium 19

3.1.2 NA medium 19

3.1.3 Preparing Herbicide Toxicity 20

3.1.4 Microbial DNA isolation 20

3.1.5 Equipment 20

3.2 Methods 21

3.2.1 Preparing Nutrient Broth Agar medium and Bushnell Haas culture medium 21

3.2.2 Preparing Herbicide Toxicity 21

3.2.3 Serial dilution of herbicide mixtures 22

3.2.4 Streaking Plates 22

3.2.5 Microbial DNA isolation 22

3.2.6 Polymerase chain reaction (PCR) detection of bacteria 23

3.2.7 Imaging and Analysis 25

PART IV RESULTS 26

4.1 Preparing herbicide toxicity 26

4.2 Serial dilution of herbicide mixtures 26

4.3 Microbial DNA isolation 28

4.4 Test the growth of bacteria in contamination environment 34

PART V DISCUSSION AND CONCLUSION 37

5.1 DISCUSSION 37

5.2 CONCLUSION 37

REFERENCES 38

LIST OF FIGURES

Figure 2.1 The molecular structure of Paraquat 9

Figure 2.2 The molecular structure of Glyphosate 9

Figure 2.3 The three main step of PCR process 16

Figure 3.1 PCR process for 16S rDNA 24

Figure 4.1.The color and density of bacteria in process of dilution for Ash-PRQ herbicide mixtures .29

Figure 4.2.The color and density of bacteria in process of dilution for Ash-GPT herbicide mixtures .29

Figure 4.3.The color and density of bacteria in process of dilution for 30

Sediment-PRQ herbicide mixtures 30

Figure 4.4.The color and density of bacteria in process of dilution for Sediment-GPT herbicide mixtures .30

Figure 4.5.The color and density of bacteria in process of dilution for Soil-PRQ herbicide mixtures .31

Figure 4.6.The color and density of bacteria in process of dilution for Soil-GPT herbicide mixtures .31

LIST OF TABLES

Table 2.1 Classification for the size of sediment (Holmes, 1978) 12

Table 3.1 Content of BH medium 19

Table 3.2 Content of NA medium 19

Table 3.3 The content of PCR tube 24

Table 4.1 Herbicide mixtures using in this experiment 26

Table 4.2 The C.F.U of bacteria per 1mL in each type of herbicide mixtures .27

Table 4.3 Number of bacteria detected in six herbicide mixtures .28

Table 4.4 The density of each type of bacteria in Ash – PRQherbicide mixture .32

Table 4.5 The density of each type of bacteria in Ash - GPTherbicide mixture .32

Table 4.6 The density of bacteria in herbicide mixture 33

Table 4.7 Bacteria isolated in ash, soil and sediment with contamination of Paraquat and Glyphosate 34

Table 4.8 Ability for survival of bacteria in herbicide mixture .36

LIST OF ABBREVIATIONS

PART I INTRODUCTION 1.1 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 in Vietnam

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., 2012)

1.2 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

1.3 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?

1.4 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

1.5 Definitions

1.5.1 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

1.5.2 Soil, ash and sediment

1.5.2.1 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., 2008)

1.5.2.2 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,

1.5.2.3 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., 1996)

PART II LITERATURE REVIEW 2.1 Overview of Herbicide

2.1.1 Classification of herbicides

Herbicide is classified base on activity, use, chemical family, mode of action, or type of vegetation controlled (Andrew et al., 2011)

- 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

2.1.2 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., 1995)

Moreover, the risk of Parkinson's disease has been shown to increase with occupational exposure to herbicides and pesticides (Gorell et al., 1998) The herbicide paraquat is suspected to be one such factor (Dinis et al., 2006)

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., 2006)

2.1.3 Paraquat

Paraquat (or N,N′-dimethyl-4,4′-bipyridinium dichloride) is a toxic organic

compound with the formula [(C5H4N)2]Cl2 (Paraquat dichloride ICSC: 0005.,2012) It is a non-selective herbicide, which one of the most widely used herbicides

Figure 2.1 The molecular structure of Paraquat

However, it is very toxic and active and can kill green plant tissue when exposure Today, many countries have removed this herbicide from the list of use, but

in Vietnam it has been popular, approximately 85% of farmers use Paraquat for agricultural productions (Buzik et al., 1997) It is very harmful for human health, Paraquat effected on respiratory system, lung, eye, esophageal, digestive, kidney with many health factor like mouth ulcers, respiratory failure, liver failure, renal insufficiency, collapse circuit, esophageal perforation etc Paraquat can cause death from 3-5 days, and has no cure

Glyphosate is a common herbicide widely used by farmers, because it help to kill weeds without killing their crops In 2007, glyphosate was the most used herbicide

in the United States agricultural sector Nevertheless, its effects on humans and the environment persist are numerous This herbicide impacts on eye, skin, respiratory system, cardiovascular system, kidneys with very serious consequences eye, skin irritation, fingers acute, sore throat, nausea, vomiting, diarrhea, respiratory, etc (Sribanditmongkol et al., 2012)

Furthermore, Glyphosate adsorbs strongly to soil and is not expected to move vertically below the six-inch soil layer; residues are expected to be immobile in soil Glyphosate is readily degraded by soil microbes to aminomethylphosphonic acid (AMPA) and carbon dioxide Glyphosate and AMPA are not likely to move to ground water due to their strong adsorptive characteristics However, glyphosate does have the potential to contaminate surface waters due to its aquatic use patterns and through erosion, as it adsorbs to soil particles suspended in runoff If glyphosate reaches surface water, it would not be broken down readily by water or sunlight (R.E.D.,1993)

2.2 Overview of soil, ash, sediment and soil pollution

2.2.1 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., 2008)

2.2.2 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,

2.2.3 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.,1996) Sediment can enrich soil with nutrients The areas which

have sedimentary soil is usually better forfarming Deltas and river banks, where much

sediment is deposited, are often the most fertile agricultural areas in a region

Sediments are often transported by waterfluvial processes, wind (Aeolian processes) and glacier (Holmes, 1978) For example, water wash sediment from gravel

or pebbles, then sediment is flowed down from a stream into a river, and eventually to

that river's delta; or from river to sea Glaciers can deposit sediment and then the

sediment is brought to other regions following to flows of glaciers when they are being

melted

The size of sediment ismeasured on a log base 2 scale, and is classified from

"colloid" to "boulder"

Table 2.1 Classification for the size of sediment (Holmes, 1978)

Size range Size range Aggregate class

φ scale

Other names

-5 to -6 32–64 mm 1.26–2.5 in Very coarse gravel Pebble -4 to -5 16–32 mm 0.63–1.26 in Coarse gravel Pebble -3 to -4 8–16 mm 0.31–0.63 in Medium gravel Pebble

-1 to -2 2–4 mm 0.079–0.157 in Very fine gravel Granule

0 to -1 1–2 mm 0.039–0.079 in Very coarse sand

Soil pollution is caused by agricultural chemicals (such as pesticide, herbicide or

are known as a causing agent which pollutes the soil environment, for example heavy metals, petroleum hydrocarbons, polynuclear aromatic hydrocarbons, solvents, herbicides and pesticides are commonly toxic products used

Heath Effects

Contaminated soil directly impacts on human and other organisms health Toxic chemicals can hurt plants when the plants grow in the polluted soil, they will absorb hazardous substances via their root Animals and humans eat those plants, they can be impacted by contamination And then humans may be affected, when they ingest the contaminated animals, or expose to contaminated soil when they work in that soil or inhale small soil particles or dust evaporated in the air Moreover, threats are greater

by the infiltration of soil contamination into groundwater aquifers used for human consumption

Damages for health from soil contamination vary seriously, which depend on types of pollutant and pathway of exposure and vulnerability of people Long term exposure to chromium, lead and other metals, petroleum, solvents, and many pesticide and herbicide can cause skin inflammation, cancers, congenital disorders, or more severe is other chronic health conditions Agricultural products such as herbicides, pesticides, insecticides, etc have also been identified as health hazards in soil and groundwater

Environment Effects

Soil contaminantion can bring numerous consequences for ecosystems and the environment.There are radical soil chemistry changes which can occur with many toxic substances even at low concentration The metabolism, food chain and habitat of

Moreover, soil that has been contaminated cannot grow food well or become impossible to grow due to depending on the level of pollution, because the chemicals can leech into the food and harm people who ingest it If contaminated soil is used to grow food, farmers will usually gain lower yields of products And the untreated soil pollution over the long term will have less healthy food, plants and animals Soil pollution can possibly affect drinking water or underground water sources, as well This will be more serious if there is a lack of plants on the soil, it will cause more erosion, spreading pollutants over other land

In addition, soil pollutants will change texture or structure of the soil and the types of microorganisms that live inside it If certain organisms in one area will be die and reduced, the predator will lose their food supply Therefore, soil pollution can probably change the ecosystems system

2.3 Polymerase Chain Reaction ( PCR )

2.3.1 Definition

Polymerase Chain Reaction ( PCR ) is one of the most powerful method

based on using the ability of DNA polymerase to synthesize new strand of DNA

complementary to the offered template strand and then amplify thousands to millions

of copies of a particular DNA sequence (Bartlett et al.,2003)

PCR was developed in 1983 by Kary-B-Mullis, an American biochemist who won the Nobel Prize for Chemistry in 1993 for his invention.(Bartlett et al,2003)

Nowadays, PCR is an useful and indispensable technique in molecular biology or medical area with various applications (Saiki et al., 1988)

2.3.2 Process of PCR Method

A basic PCR set up requires some following components and reagents

- DNA template that consists of the DNA region (target) to be synthesized

- Two primer that are complementarity to the 3’ (three primer) ends of each of the sense and anti-sense strand of the DNA template

- Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C

- Deoxynucleoside triphosphates (dNTPs) or called as nucleotide containing

triphosphate groups is assigned as the building-blocks from which the DNA polymerase synthesizes a new DNA strand

- Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase

- Bivalent cations , magnesium or manganese ions; generally Mg2+ is used, but

Mn2+ can be utilized for PCR-mediated DNA-mutagenesis, as higher

Mn2+ concentration increases the error rate during DNA synthesis

- Monovalent cation potassium ions

The PCR is commonly carried out in a reaction volume of 10–200 µl in small reaction tubes (0.2–0.5 ml volumes) in athermal cycler (Bartlett and Stirling., 2003) PCR is a three-step process that is carried out in repeated cycles These three steps are repeated for 30 or 40 cycles The cycles are done on an automated cycler, a device which rapidly heats and cools the test tubes containing the reaction mixture

Firstly, Denaturation: occur at 94 oC-96 oC, the double-stranded DNA is melted and opened into two pieces of single-stranded DNA

Secondly, Annealing: At medium temperatures, around 54 oC (129.2 F), the primers pair up (anneal) with the single-stranded "template" The DNA polymerase

attaches on the small length of double-stranded DNA (the joined primer and template), and starts to copy the template

Thirdly, Extension or Elongation: At 72 oC, the DNA polymerase works best, and DNA building blocks complementary to the template are coupled to the primer, a double stranded DNA molecule is created

For one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA for amplification in the next cycle Finally, the number of copies of the DNA template is increased exponentially (Bartlett and Stirling., 2003)

Figure 2.3 The three main step of PCR process ( Source: http://en.wikipedia.org/wiki/Polymerase_chain_reaction)

2.3.3 Application of PCR

At the present, PCR is very valuable in a number of newly emerging laboratories and clinical techniques, include DNA cloning for sequencing, detection of bacteria or viruses (particularly AIDS), functional analysis of genes; the diagnosis of

hereditary diseases or genetic disorders; the identification of genetic fingerprints; and the detection and diagnosis of hazardous diseases etc (Bartlett and Stirling, 2003)

Particularly, PCR is commonly used in selective DNA isolation and detection

of bacteria or viruses PCR allows isolation of DNA fragments from genomic DNA

by selective amplification of a specific region of DNA PCR supplies these techniques with high amounts of pure DNA to analyze DNA samples even from very small amounts of initial material

Other applications of PCR include DNA sequencing to determine unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to expedite recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid or the genetic material of another organism Bacterial colonies (E coli) can be rapidly screened by PCR for correct DNA vector constructs By comparing experimental DNAs combined with different methods, PCR may also be used for genetic fingerprinting; a forensic technique used to identify a person, genetic relationships between individuals or organisms (Pavlov