Isoprene degradation by soil bacteria associated with wild himalayan cherry in tropical forests thailand

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY NGUYEN KIM LUYEN ISOPRENE DEGRADATION BY SOIL BACTERIA ASSOCIATED WITH WILD HIMALAYAN CHERRY IN TROPICAL FORESTS – THAILAN

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

NGUYEN KIM LUYEN

ISOPRENE DEGRADATION BY SOIL BACTERIA ASSOCIATED WITH WILD HIMALAYAN CHERRY IN TROPICAL FORESTS – THAILAND

BACHELOR THESIS

Study mode : Full-time Major : Environmental Science and Management Faculty : Advanced Education Program Office Batch : 2014 - 2018

Thai Nguyen, 20/09/2018

DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University of Agriculture and Forestry

Degree Program Bachelor of Environmental Science and Management

Student name Nguyen Kim Luyen

Thesis Title Isoprene Degradation by Soil Bacteria Associated with

Wild Himalayan Cherry in Tropical Forests-Thailand

Supervisors Dr Tho Huu Nguyen

Assist Prof Thararat Chitov Supervisor’s

Signature

Abstract:

Isoprene is a Biogenic Volatile Organic Compound (BVOC), which represents nearly one-third of total global hydrocarbon released in the atmosphere Isoprene can easily react with oxides of nitrogen to form tropospheric ozone that is harmful for life Broadleaf trees and shrubs are the greatest source of isoprene emission Certain proportion of isoprene is absorbed in soil, which can be reduced by some soil bacteria The aims of this research work are to measure isoprene degradation in the terrestrial environment, with a particular focus on soil associated with Wild Himalayan Cherry trees, one of the framework tree species used in forest restoration, and to isolate bacteria responsible for isoprene degradation Soil samples taken from underneath 6 Wild Himalayan Cherry trees in Mae-Sa Mai restoration area were examined for their properties and rates of isoprene consumption in soil incubated with isoprene was measured using Gas Chromatography-Flame Ionization Detector (GC-FID) Bacteria responsible for isoprene consumption were isolated from soil, using minimal medium (agar and broth) supplemented with isoprene as the sole carbon source The bacterial isolates were characterised based on the features of colony and cell morphology and some biochemical tests It was found that the soil samples had the average pH of 4.4, moisture content of 40.8%, and total viable microorganisms of 5.3106 CFU/g

Consumption of isoprene by soil microorganisms was observed in the combined soil sample; which occurred at a constant rate during the first 4 days of incubation of the soil sample with isoprene and remained unchanged afterwards Some of the soil bacterial isolates associated with Himalayan Cherry were capable of utilising isoprene as the sole carbon source Morphological and biochemical characterisation revealed these bacterial isolates to be of different types These bacterial isolates will be subjected to further identification and characterisation of their role in isoprene degradation

Key words isoprene degradation, soil bacteria, Wild Himalayan Cherry Number of pages 49

Date of

Submission:

24/09/2018

ACKNOWLEDGEMENT

First of all, I would like to thank International College and The ASEAN International Mobility for Student Program for providing opportunity for me to doing

my undergraduate research in Chiang Mai University

I would also like to thank Microbiology Division, Department of Biology, Faculty of Science, Chiang Mai University for their technical, financial, and scientific support

I am extremely grateful to my project supervisor Assistant Professor Dr Thararat Chitov at Chiang Mai University for her excellent advice and support throughout this study Being a member of your research group has been a privilege and this work would not have been possible without your immense knowledge and true guidance

I am also very grateful to my internship supervisor Dr Tho Huu Nguyen at Thai Nguyen University of Agriculture and Forestry for his insightful advice, discussions and comments which brought an added value to this research work

I am deeply grateful to the staff of FORRU attached to the Biology Department, Faculty of Science, Chiang Mai University for supporting me to conduct my research

I thank all members of SCB 2806 laboratory especially Ms Tuangporn Uttarothai for being around to give me advice and assistance in the laboratory Thank you all for your support, friendship, and encouragement and for making the time I spent at Microbiology Division very memorable

Finally, a special thank goes to my family and friends for their unequivocal support and faith in me

Author

Nguyen Kim Luyen

TABLE OF CONTENT

DOCUMENTATION PAGE WITH ABSTRACT i

ACKNOWLEDGEMENT iii

TABLE OF CONTENT iv

LIST OF TABLES vi

LIST OF FIGURES vii

LIST OF ABBREVIATIONS viii

PART I INTRODUCTION 1

1.1 Research rationale 1

1.2 Research’s objectives 2

1.3 Research questions and hypothesis 2

1.3.1 Research questions 2

1.3.2 Hypothesis 2

1.4 Limitations 2

1.5 Definitions 3

PART II LITERATURE REVIEW 4

2.1 Significance and Production of Isoprene 4

2.2 Significance of Isoprene 6

2.3 Isoprene’s Biological Roles 7

2.4 Isoprene Emission by Plants 7

2.5 Soil as a sink for isoprene: the effects of location, season, and isoprene degradation 8

2.6 Isoprene Degradation by Soil Bacteria 9

2.7 Isoprene-degrading Bacteria and Forest Restoration 10

PART 3 MATERIALS AND METHODS 13

3.1 Equipment and Materials 13

3.1.1 Equipment 13

3.1.2 Microbiological Media, Reagents, and Chemicals 13

3.2 Methods 14

3.2.1 Soil Sampling 14

3.2.2 Determination of soil characteristics 14

3.2.3 Determination of isoprene degradation by microorganisms in

soil associated with Wild Himalayan Cherry trees 15

3.2.4 Isolation of soil bacteria capable of degrading isoprene 15

PART IV RESULTS 18

4.1 Physical and Chemical Characteristics of Soil Samples 18

4.2 Microbiological characteristics of soil samples 18

4.3 Isoprene degradation by soil microorganisms 19

4.4 Isolation of bacteria capable degrading isoprene 21

4.5 Characterisation of isoprene-degrading bacterial isolates 24

PART V DISCUSSION 27

5.1 Physical and Chemical characteristics of Soil Samples Beneath

Wild Himalayan Cherry Trees 27

5.2 Microbiological Characteristics of Soil Samples 28

5.3 Investigation of Isoprene Degradation by Soil Microorganisms 29

5.4 Characterisation of Isoprene-Degrading Bacterial Isolates 29

PART VI CONCLUSION 31

REFERENCES 32

Appendix A 39

Appendix B 43

Selected Details of Analytical Procedures and results 43

Standard graph of isoprene measuring by GC-FID 43

Appendix C 48

LIST OF TABLES

Table 4.1 Location and basic characteristics of soil samples 18

Table 4.2 Count of total viable microorganisms in soil 19

Table 4.3 Isoprene consumption by soil microorganisms 20

Table 4.4 Characteristics of bacterial colony morphology 21

Table 4.5 Cell morphological characteristics of isoprene-degrading bacterial isolates 25 Table 4.6 Biochemical characteristics of isoprene-degrading bacterial isolates 26

LIST OF FIGURES

Figure 2.1 The chemical structure of Isoprene (CH2=C(CH2) CH=CH2)

(Source: Polymer Science Learning Center, 2000) 4

Figure 2.2 Isoprene cycle by sources and sinks (Source: McGenity, 2018) 5

Figure 2.3 Wild Himalayan Cherry (Source A: Chaiudom, 2018; B, C, D: Thakur, 2015) 12

Figure 4.1 The decrease of isoprene due to consumption by soil microorganisms 20

Figure 4.2 Bacteria isolates grew on MM agar plate 22

Figure 4.3 The isolates were more turbid after 1st cultured in MM broth 23

Figure 4.4 The isolates were more turbid after 2nd cultured in MM broth 24

LIST OF ABBREVIATIONS

T

MC CFU

MM G+

Negative microlitre millilitre litre millimetre peak area teragram

PART I INTRODUCTION 1.1 Research rationale

Atmospheric pollution is currently one of the major threats to human health and the environment Due to this, it is important to gain a thorough understanding of the cycling of atmospheric pollutants Some of the atmospheric pollutants are formed from the reactions between non-pollutant compounds, such as Biogenic Volatile Organic Compounds (BVOCs) Because of their existence in large amounts, BVOCs play an important role in atmospheric chemistry, climate conditions, and air quality Isoprene (2-methyl-1,3-butadiene [C5H8]), one of the BVOCs, represents approximately one-third

of total global hydrocarbon released into the atmosphere It is the second-most abundant volatile organic compound in the atmosphere after methane In areas with elevated levels

of oxides of nitrogen, the oxidation of isoprene leads to the formation of tropospheric ozone, which is harmful to health and ecosystems Ozone is a major pollutant and

greenhouse gas (Chameides et al., 1992), which also contribute to global warming

Other effects of atmospheric isoprene oxidation include the formation of tropospheric carbon monoxide, global transport of nitrogenous compounds, extended residence times

of other atmospheric trace gases, and the formation of secondary organic aerosols (Monson and Holland, 2001) The sources of atmospheric isoprene have been relatively well-studied, with terrestrial plants accounting for 90% of isoprene emissions to the

atmosphere (Pacifico et al., 2009) Besides, Pegoraro (2005) reports that soil acts as a

significant atmospheric sink of isoprene (3% of global emissions)

Although the proportion of isoprene in soil is small, compared with that in the atmosphere, the amount is significant Since soil surface is the interface between the absorbed isoprene in soil and atmospheric isoprene, it is hope that reduction of isoprene

in soil would affect the total amount of isoprene in the atmosphere Previous research works have demonstrated that some soil microorganisms possess the ability to degrade isoprene, and these organisms have association with certain types of plants Since the isoprene flux between soil and atmosphere and the interactions between plant and the associated isoprene-degrading microbes are not yet well understood, this research work was designed to address these questions Since this work was carried out in the North of

Thailand where deforestation is an issue of concern, one type of plant is chosen as a subject of this study: Wild Himalayan Cherry This is a tree significant in forest restoration and it has additional values in tourism and medicine It is expected, through this approach of study, that more clarity would be gained in the understanding of isoprene flux and isoprene degradation in soil by soil microorganisms Moreover, it is hoped that plant-microbe interactions, especially between Wild Himalayan Cherry and its rhizospheric bacteria capable of degrading isoprene would be more clearly understood

1.2 Research’s objectives

 Determine isoprene-degradation rates by soil microorganisms associated with

Wild Himalayan Cherry trees

 Isolate and characterize soil bacteria responsible for isoprene-degradation

1.3 Research questions and hypothesis

to the lower quantity of bacteria collected Secondly, limited time is the main culprit minimizing the range of scope of this research and hindering further study about identification of bacteria

1.5 Definitions

Isoprene is one of the Biogenic Volatile Organic Compounds, represents

approximately one-third of total global hydrocarbon released into the atmosphere

Soil bacteria is the microorganisms live in soil

PART II LITERATURE REVIEW

2.1 Significance and Production of Isoprene

Biogenic Volatile Organic Compounds (BVOCs) play an important role in atmospheric chemistry and the carbon cycle Around 1,150 Tg of the hydrocarbons in

the atmosphere are BVOCs (Guenther et al., 1995) Non-Methane Hydrocarbons

(NMHC) and short chain (<10 carbon atoms) volatile carbon-based molecules make up most of these BVOCs (Shaw, 2001) Isoprene is one of these NMHCs and represents

approximately one third of total global hydrocarbon release (Guenther et al., 2012)

Isoprene is also known as 2-methyl-1,3-butadiene (C5H8) (Figure 1.1) with the boiling point of 33°C and a mass of 68.0626 g/mol (Seinfeld and Pandis, 2012) It is a non-polar compound, and therefore has low water solubility (Sharkey, 1996a) Isoprene is denser than air, uncharged, and highly reactive in the atmosphere due to its two carbon-carbon double bonds (Seinfeld and Pandis, 2012) This means that it has important effects on

atmospheric chemistry (Pacifico et al., 2009)

(Source: Polymer Science Learning Center, 2000)

Isoprene emissions are estimated to be 600 million tons per year (Guenther et al.,

2006) The vast majority of isoprene is produced by trees and shrubs, with broadleaf trees accounting for 51% of total production, and shrubs for 46% (Figure 1.2) (Guenther

et al., 2006) In addition to plants, many animals also produce isoprene (Sharkey,

1996a) However, no global data is available for isoprene emission from soils, animals

or bacteria, although it seems unlikely that these have a significant impact (Guenther et

al., 2006)

Figure 2.2 Isoprene cycle by sources and sinks (Source: McGenity, 2018)

Isoprene emission from human is estimated to be 17 mg/day on average Human activity can affect isoprene production For example, changing in land use due to increasing demand for biofuel crops, such as poplar or oil palm, can increase isoprene emission in the atmosphere An example for this is seen in the increase of oil palm plantation in Malaysia In 2009, Malaysia had 13% oil palm plantation area, which increased from 1% in 1974 By 2014, the oil palm plantation took up to approximately 16% of land mass, which was about at 5.2 million hectares (Armstrong, 2013) The increase of isoprene emission by human’s activities combined with the warming climate’s effect on isoprene production by plants is predicted to contribute to a

substantial increase in isoprene emissions in the future (Sanderson et al., 2003a)

Atmospheric isoprene concentrations vary depending on the tree species, tree coverage in the area, and the air temperature (Wagner and Kuttler, 2014) Higher isoprene concentrations are found in hot climates, such as tropical rainforests and

deciduous forests, than in the regions with more temperate climates (David et al., 1991)

For example, in Amazonia in 1998, isoprene concentrations ranged from 4 to 10 ppb,

and in a forest in Tennessee (USA), from 3 to 10 ppb (Baldocchi et al., 1995); whereas

in Essen, Germany, isoprene concentrations are typically between 0.13 and 0.17 ppb during the day, and around 0.01 ppb at night time (Wagner and Kuttler, 2014) In

Shanghai (China), a warmer urban environment near large forests, isoprene concentrations ranged from 1 to 6 ppb, and in Venezuela’ isoprene concentrations

reached 3 ppb in a number of locations (Donoso et al., 1996) Low isoprene

concentrations can be found near marine environments, which were between 0.001 and

0.01 ppb (Lewis et al., 1997)

2.2 Significance of Isoprene

As isoprene is highly reactive and is produced in significant quantities, it is regarded as a very important biogenic hydrocarbon (Seinfeld and Pandis, 2012) Isoprene reacts in the atmosphere in a number of ways, however, the main reactions are through the reactions with hydroxyl radicals (OH) and nitrogen oxides (NOx) One of the major problems with isoprene is that its reactivity with nitrogen oxides produces tropospheric (low level) ozone In the upper atmosphere (stratosphere), ozone provides protection from UV light; but in the lower atmosphere (troposphere), ozone is

undesirable as it harms human and plant health and is a greenhouse gas (Sanderson et

al., 2003b)

The safe limit of ozone recommended by the World Health Organisation (WHO)

is 60 ppb (WHO, 2000), a figure that is sometimes exceeded, particularly in eastern USA

and China (Sanderson et al., 2003b) At 80 ppb ozone level, there is a significant

decrease in pulmonary function and increase in respiratory problems, including inflammation and changes in responsiveness Longer term exposure to ozone levels between 120 and 250 ppb has been shown to cause changes in the epithelium cells in the airways and connective tissues in the lung, often resulting in fibrotic changes (WHO, 2000) An estimate of 1365 deaths was predicted caused by ozone formed from

increased isoprene reacting with NOx (Ashworth et al., 2013) In addition to effects on

human, ozone can cause significant damage to ecological systems, microbial

populations in the environment, and crop losses (Ashworth et al., 2013)

Isoprene can also react with hydroxyl radicals, resulting in fewer radicals that would otherwise react with methane, extending its residence time, thus enhancing global

warming (Collins et al., 2002) Isoprene also causes the formation of secondary organic

aerosols, although effects resulting from the aerosols are not yet clear Hydrocarbons, particularly isoprene and other terpenes, can be oxidised to compounds with a lower

volatility and condense to form particulate matter (aerosols) The aerosols can absorb or scatter solar radiation, changing the distribution of energy in the atmosphere and reflecting radiation from the planet, which causes a cooling effect Secondary organic

aerosols can also change cloud precipitation characteristics (Sharkey et al., 2008) The

yield of aerosol per molecule is much lower for isoprene than for monoterpenes and larger molecules However, there are much more isoprene entering the atmosphere than other molecules, thus isoprene could be a significant factor in aerosol formation (Olcese

et al., 2007)

2.3 Isoprene’s Biological Roles

Isoprene emission is believed to be a protective mechanism from abiotic stresses Isoprene protects plants against heat stress by reducing heat-induced cell-membrane damage, enhances tolerance of reactive oxygen species and may affect plant-insect

interactions (Sharkey et al., 2008) The amount of isoprene released from

isoprene-emitting vegetation depends on leaf mass, leaf area, light (particularly photosynthetic

photon flux density, or PPFD) and leaf temperature (Alves et al., 2014) Daytime

emissions of isoprene are expected to be substantial during hot and sunny days, up to 25 μg/g (dry-leaf-weight)/hour in many oak species Isoprene emissions by plants are expected to be low during the night (Hester and Harrison, 2007)

2.4 Isoprene Emission by Plants

Most researchers find that hydrocarbon in the atmosphere emitted by plants was much more than that coming from human activities, especially during extended warm weather (Monson and Holland, 2001) Isoprene emission is the predominant biogenic source of hydrocarbons in the atmosphere, which is roughly equivalent to methane’s

global emission from all sources (Guenther et al., 2006) The global distribution of

isoprene emitting plants is unequal, with the majority (80%) of isoprene being produced

in tropical forests, mid-level emissions from temperate forests, and low emissions from

boreal forests and agricultural areas (Pacifico et al., 2009) The reason that isoprene

emissions is higher in the tropics is thought to be due to limits on transpiration rates, caused by the relative humidity outside the plant As water loss is one of the other primary mechanisms for plants to reduce their temperatures, isoprene production

becomes more important (Sharkey and Yeh, 2001)

Peter et al (1999) reported that while most plants are not isoprene emitters, still

a significant fraction of species has this capability In general, most isoprene emitters are woody species, although some ferns, vines, mosses, and other herbaceous species can also emit isoprene (Niinemets and Monson, 2013) In plant families that contain isoprene-emitting species, not all species are emitters A good illustration of this point

is in the case of oak trees (genus Quercus) All the North American oaks are high

isoprene emitters, but many European oak species are nonisoprene emitters (Csiky and

Seufert, 1999) High isoprene emitters include species of Casuarina (ironwood),

Eucalyptus, Populus (poplars and aspen), and Quercus (oaks) (Hewitt, 1998)

2.5 Soil as a sink for isoprene: the effects of location, season, and isoprene degradation

Most isoprene that are being emitted is degraded in the atmosphere by reactions with other atmospheric components However, it is understood that a proportion of atmospheric isoprene ends up degraded biologically in soils (Cleveland and Yavitt,

1997, 1998) The initial attempts to characterise biological isoprene degradation rates led to a global estimate of 20.4 MTy-1, or around 3% to 4% of isoprene production being degraded by biological processes in soils (Cleveland and Yavitt, 1997), indicating that soils represent a potentially important sink of isoprene The vast majority of isoprene

emission comes from plants (Guenther et al., 2006), and the foliage is expected to be

the main source of isoprene emission in soils Plant roots do not produce any isoprene,

and containing very little isoprene synthase (Cinege et al., 2009) With the effect of

production source on the degradative capacity of soil, and as isoprene has a very short atmospheric lifespan (Atkinson & Arey, 2003), it is expected that soils near to isoprene-producing trees would contain bacterial communities capable to degrade isoprene

As with most biological processes, there are the multitude of factors influencing isoprene degradation rates in soils So far, works on these have been focused on forest type, temperature, pH, moisture content, depth, and CO2 (Pegoraro et al., 2005)

Besides, seasons, or factors related to seasons are also significant factors that can affect soil isoprene degradation It is reported that isoprene emissions were related to seasons, which were low in spring and autumn, compared to summer, and this fact is closely

related to temperature (Harley et al., 1996) Therefore, soil microbial communities

would be expected to metabolise isoprene better in summer months, both because their metabolic activity should be higher in warmer soils and more isoprene is available

The estimates for global isoprene biological degradation had been based on rates determined in the laboratory, which were usually tested at high concentrations, and may not reflect the actual atmospheric concentrations (i.e 3 ppbv) (Cleveland and Yavitt, 1997) A more recent work, which was an in-situ test, using the atmospheric isoprene present at the time of sampling has demonstrated an isoprene-degradation rate of 2.0 nmol m-2 h-1 (Gray et al., 2014), which is significantly lower than previous estimates It

is notable that the isoprene-degradation rates in soils increase as the concentrations of isoprene increase (Cleveland and Yavitt, 1997, 1998) The ability of soil microbes to degrade isoprene at higher rates in the set-up conditions than those in the atmospheric conditions may indicate that isoprene is also produced in soil by other factors besides

vegetation, such as soil bacteria (Kuzma et al., 1995), fungi (Back et al., 2010), and household waste/compost (Mayrhofer et al., 2006)

2.6 Isoprene Degradation by Soil Bacteria

The soil layer (< 8 m deep) contains large numbers of bacteria and is an important

part of matter transformation and atmospheric gas exchange (Whitman et al., 1998) One

gram of soil can contain up to 1010 bacteria, and between 6400 and 38000 species, with many functional potentials Some soil bacteria have been known to play a role in BVOC

cycling (Ramirez et al., 2009), and some are known to be able to utilise isoprene, one of

the BVOCs, as a growth substrate (Cleveland and Yavitt, 1997) The bacteria that are

capable to degrade isoprene include Nocardia (Van Ginkel et al., 1987), Alcaligenes

denitrifcans and Rhodococcus erythropolis (Ewers et al., 1990) Some Methanotrophs

and an Arthrobacter isolate have also been known to epoxidise isoprene, but not utilise isoprene as a sole carbon source (Hou et al 1981) None of these terrestrial isolates was

extensively characterised The best documented isoprene degrader to date is

Rhodococcus sp AD45, a Gram-positive actinobacterium isolated nearly 20 years ago

from fresh water sediment by the group of Dick Janssen (Van Hylckama Vlieg et al.,

1999) This strain was first isolated under a condition in which isoprene was the sole carbon and energy source The gene cluster responsible for isoprene degradation has been cloned, which encodes a multicomponent monooxygenase, the enzyme that plays

an important role in isoprene metabolism The molecular factors associated with the enzyme complex have also been identified Since then, isoprene degradation has been

demonstrated in cultures of Actinobacteria: Gordonia, Leifsonia, Rhodanobacter,

Dyadobacter and Shinella, showing that isoprene-degradation is phylogenetically

widespread (Johnston, 2014) A recent bioreactor study has shown Pseudomonas,

Alcaligenes and Klebsiella to have high efficiency in isoprene degradation (Srivastva et al., 2015)

2.7 Isoprene-degrading Bacteria and Forest Restoration

In Thailand, as in most tropical countries, deforestation is widely recognised as major threats to environmental stability, economic prosperity and social welfare, particularly amongst rural communities Remaining forest has become fragmented and are less supportive to viable populations of many species (Lynam, 1999) In the northern highlands, which constitute the country’s most important watershed, large areas of degraded forestland require urgent reforestation Reforestation can come in the form of natural succession or economic forestry with monoculture plantations One method of natural forest restoration that has been proved successful to restore degraded agricultural sites is “The framework tree species method.” Framework tree species are indigenous forest tree species, planted to complement and accelerate natural regeneration of forest ecosystems and encourage biodiversity recovery, on degraded sites This method involves planting of 20-30 native tree species that have some essential characteristics such as high field performance (high survival rate), and high growth rates in open degraded sites; fast spreading, dense crowns that shade out herbaceous weeds and provision shelter and food for wildlife at an early age (Elliott and Kuaraksa, 2008) Since fire is a serious annual threat establishment of tropical forests in seasonally dry climates,

an ability to resist or recover after fire is therefore another important characteristic of framework species Furthermore, rapid germination and growth of seedlings to a plantable size (50–60 cm) in less than 1 year (FORRU, 1998) is desirable

The purpose of planting framework tree species as pioneer trees is so that dispersing mammals and birds, attracted by the planted trees, will help to restore species diversity of the vegetation, thus improving wildlife habitats Some of the indigenous species that have been proved to be suitable as framework species in deforested areas in

seed-Doi Suthep-Pui National Park, Thailand, are Spondias axillaris Roxb., Prunus

cerasoides D Don and Ficus altissima Bl., which had more than 80% of survival rates

after being planted for 3 years (Elliott et al., 2003) Since this method is one of the most

efficient approach to forest restoration and will potentially be applied in a more extended area, it is also important to consider the framework tree species in terms of being potentially beneficial to climates and the environments Previous studies have shown that some of the rhizosphere microbes associated with some framework tree species were able to degrade isoprene (Uttarothai, 2018 (personal communication)) One of

these tree species is Wild Himalayan cherry (Prunus cerasoides), a deciduous cherry

tree which grows up to 30 metres (98 ft) in height When the tree is not in flower, it is characterised by glossy, ringed bark and long, dentate stipules Flowers are hermaphroditic and are pinkish white in color (Figure 2.3) It has ovoid yellow fruit that turns red as it ripens This plant is popularly grown in Northern Thailand, not only for forest restoration but also for tourist’s attraction because of its beauty, bringing high economic efficiency for local people Furthermore, it has a range of uses including edible fruit, seed and gum, various medicinal applications, a timber, dyestuff, source of tannins, and beads

This research work, therefore, was designed to investigate isoprene degrading activity in soil associated with Wild Himalayan Cherry trees and to isolate and characterise soil bacteria that are responsible for this activity It is expected that the output from this research would add clarity to the effect of soils on the atmospheric isoprene flux and to the role of soil bacteria in isoprene-degradation

Figure 2.3 Wild Himalayan Cherry (Source A: Chaiudom, 2018; B, C, D: Thakur, 2015)

A

D

PART 3 MATERIALS AND METHODS

3.1 Equipment and Materials

3.1.2.2 Reagents and Chemicals

Nessler’s reagent Sulfanilic acid solution α-Naphthylamine solution Gram’s Stain Solutions Hydrogen peroxide Zinc powder

3.2 Methods

3.2.1 Soil Sampling

Soil samples were taken from beneath 6 Wild Himalayan Cherry trees in Mae Sa

Mai forest plantation area (18°53′44″ N, 98°56′27” E) Each soil sample was collected from 3 spots around the stump of the tree and these three subsamples were combined Soil temperature was measured at the sampling site using a thermometer

3.2.2 Determination of soil characteristics

3.2.2.1 Physical and Chemical characteristics

Measurement of soil pH

To measure soil pH, soil sample was added in 0.01 M CaCl2 solution (with soil: CaCl2 ratio of 1:5) Then, the soil suspension was centrifuged and mixed on the shaker After that, the pH of soil was measured using a PH meter

Measurement of moisture contents of soil samples

Soil samples’ moisture content were measured after drying soil in the hot air oven

at the temperature between 105°C and 110°C for 6 days The moisture content of the soil in percentage was calculated from the dry soil weight using the following formula:

MC% = 𝑊2−𝑊3

Where: W1 = Weight of tin (g)

W2 = Weight of moist soil + tin (g)

W3 = Weight of dried soil + tin (g)

3.2.2.2 Microbiological characteristics

Soil microbiological characteristics by means of colony count was performed after 6 days of incubation using a colony counter Counts were performed from the plates which contained approximately 30-300 colonies were grown The colony-forming units

(CFU per gram of soil) were calculated based on the average colony counts from 3 plates

using this formula:

a rubber cap After that, 100 µl of gas inside the vial was drawn and analysed for isoprene concentration using for GC-FID The FID signal (in pA; peak area) was read after 3 minutes The remaining isoprene was analysed every day for 5 days (measurement was performed) and the isoprene degradation rates were calculated 3.2.4 Isolation of soil bacteria capable of degrading isoprene

3.2.4.1 Isolation of soil bacteria using spread plating

A 10-gram portion of soil was added into 90 ml of peptone water and homogenised in Stomacher for 30 seconds (10-1 diluted solution) Then, 1 ml of 10-1diluted solution was transferred to a test tube containing 9 ml peptone water, making a

10-2 dilution This process was repeated until 10-5 diluted solution was achieved After that, 100 µl of the suspension from each dilution was spread on the surface of Minimum Medium (MM) Agar plate (3 plates for each dilution) The agar plates were incubated

in an anaerobic jar containing 5 ml of isoprene inside under room temperature

3.2.4.2 Culturing bacteria in Minimum Medium Agar plate

The representative colonies from 18 selected spread plates were re-isolated on

MM agar plate using streak plating All the streaked plates were placed in an anaerobic jar and incubated with 5 ml isoprene at room temperature After 4 days, the well-grown colonies were re-streaked on MM agar plate in order to isolate single colonies After

4-day incubation under isoprene supplement as the sole carbon source, colony characteristics, such as size, colour, form, margin, elevation and surface were observed

3.2.4.3 Culturing bacteria in Minimum Medium Broth

Selected colonies grown on MM agar incubated with isoprene were cultured in

agar slants before being transferred into 5 ml MM broth A positive control, Gordonia

polyisoprenivorans, was also cultured in order to provide a comparison for the level of

turbidity, which indicated growth in the MM broth Ten microlitres of isoprene were injected in each vial, which was then placed in an anaerobic jar and incubated under shaking condition at room temperature (~25°C) for 3 days

3.2.5 Characterisation of isoprene-degrading bacterial isolates

All the bacterial isolates were re-streaked on new plates, then incubated at 37°C for 2-3 days Gram’s staining, catalase test, ammonification test, nitrate reduction test, and urease test were carried out

3.2.5.1 Gram’s staining test

One loopful of an isolate was smeared on a glass slide and mixed with a drop of water, air-dried and fixed over the flame Then, it was stained with Crystal violet (1 minute), followed by Iodine solution (1 minute), decolorized using Decoloriser, and counter stained with Safranin O (30 seconds) Gram reaction and cell morphology of the

isolate was examined under the microscope

3.2.5.2 Catalase test

One loopful of an isolate was smeared on the glass slide in H2O2

Catalase-positive reaction is indicated by the appearance of gas bubbles

3.2.5.3 Ammonification test

A 200 µl portion of an overnight culture from Peptone Broth was transferred in

a depression of a spot plate After that, 1 drop of Nessler’s reagent was added in the depression to test for ammonia production, which would cause the change in colour from orange to brown due to the precipitate

3.2.5.4 Nitrate reduction test

A 200 µl portion of culture from Nitrate Broth was transferred in the depression

of a spot plate After that, 1 drop of Sulfanilic acid and 1 drop of α-Naphthylamine were

added in order to check for the presence of nitrite as indicated by the red colour If no nitrite (NO2-) was detected, Zn powder was added to detect the remaining nitrate (NO3-),

which, if present, would result in the development of a red coloured substance

3.2.5.5 Urease test

Urease production was performed by spotting the test bacteria in the middle of Urea agar plate and incubated at 37°C The results were observed after 24 hours Urease positive reaction was indicated by the agar turning to bright pink colour

PART IV RESULTS

4.1 Physical and Chemical Characteristics of Soil Samples

Six soil samples were taken from beneath 6 Wild Himalayan Cherry trees at 6

locations in Mae Sa Mai, Thailand The samples were designated A, B, C, D, E, F,

respectively Soil temperatures were measured at the sampling sites The pH of the samples was measured in the laboratory within 24 h after sampling Moisture contents

of soil were also analysed The results are shown in Table 4.1

Table 4.1 Location and basic characteristics of soil samples

* T: Soil temperature, MC: Moisture content of soil

Table 4.1 shows the location and basic characteristics of soil samples which were taken

in Mae Sa Mai village (18°53′44″ N, 98°56′27” E), Pong Yang subdistrict, Mae Rim district, Chiang Mai province, Thailand As can be seen, the highest temperature of soil belonged to sample D at 19.7°C while the lowest one was that of sample C and E at 19°C In addition, the average pH of soil found in this study was 4.4 and the average of soil moisture content was found to be 40.83% with the highest belonged to sample F (43.39%) and the lowest was 38.02% belonged to sample C

4.2 Microbiological characteristics of soil samples

Microbiological characteristics of soil were analysed using dilution/spread plate techniques and the results are shown in table 4.2

Table 4.2 Count of total viable microorganisms in soil

Sample

No of colonies in

spread plate 1

No of colonies in spread plate 2

No of colonies in spread plate 3

Average

No of colonies

CFU/g

Average colony count (CFU/g)

* CFU: Colony Forming Unit

Table 4.2 shows the total viable microorganisms in each soil sample with the average

colony count was 5.3106 CFU/g The highest colony-forming units per gram of soil was seen in sample B (10-4) at 8.4106 CFU/g, followed by that of sample D (10-4) and

C (10-4) (roughly 6.7106 CFU/g and 6.6106 CFU/g respectively), and the lowest was that of sample E (10-3) at 1.7106 CFU/g

4.3 Isoprene degradation by soil microorganisms

Soil was incubated with isoprene for 5 days and isoprene consumption was measured Isoprene degradation was observed, particularly during the first 4 days of incubation (Table 4.3 and Fig 4.1)

1950 2050 2150 2250 2350

Isoprene degradation rates

Table 4.3 Isoprene consumption by soil microorganisms

Day

Remaining isoprene (pA) reduction Average

per day (pA)

Total reduction

in 5 days (pA) [µl]

Total % reduction (pA)

y: peak area in unit of pA x: the amount of isoprene in unit of µl

Table 4.3 shows the isoprene consumption by soil microorganisms By the end of 5-day

incubation, microorganisms in the soil consumed an average of 15.32% of the isoprene injected The total reduction of isoprene in 5 days was 358.11 pA which was equivalent

to 22.12 µl As can be seen, the isoprene reduction was highest in day 3 (at 126.97 pA) and lowest in day 5 (at 7.15 pA)

Figure 4.1 The decrease of isoprene due to consumption by soil microorganisms

(Day) (pA)