taxonomic characterization of an acidophilic bacterium

To understand the roles of microorganisms in this operator, we have studied bacterial community structure and isolated one strain that was first identified as the genus Nitrobacter reide

Taxonomic characterization of an acidophilic bacterium isolated from the

acidophilic nitrifying process

Taxonomic characterization of an acidophilic bacterium isolated from the

acidophilic nitrifying process

Abstract

Nitrification and denitrification processes in which ammonia in municipal and industrial wastewater is converted into nitrogen by nitrifying and denitrifying microorganisms, are the centre of nitrogen cycle Nitrification and denitrification at acidophilic condition is considered as a prospective solution for treatment wastewater containing amonia Toward this purpose, our laboratory has designed an operator ANSBR (Acidophilic Nitrifying Sequencing Batch Reactor) to treatment wastewater at acidophilic condition with sludge as seed To understand the roles of microorganisms in this operator, we have studied bacterial community structure and isolated one strain that was first identified as the genus

Nitrobacter (reidentified to the genera Dyella) In this study, a new strain isolated from

ANSBR (Acidophilic Nitrifying sequencing Batch Reactor) were characterized taxonomically The strain were characterized using phenotypic analysis and molecular based methods Physiological test and quinone analysis were carried out as well as 16S rRNA sequence Using 16S rRNA gene sequence analysis and phylogenetic tree showed

this strain was similar to the genus Dyella, Frateuria and Rhodanobacter These

Frateuria strains are generally known as acidophilic bacteria and also found in other

acidophilic wastewater treatment systems (Kevin et al., 2005) As the result, our strain is Gram-negative, motile, could grow at pH 4.0 – 8.0 and optimum pH was 5.0 This strain grew slowly at 10 or 55oC, and optimum temperature for for growth was 25oC This strain could grow at R2A medium, and very well at NA medium but could not grow at the

medium that used for Frateuria, Acetobacter and Glucenobacter Therefore, based on medium test datas, we found this strain was not the genus Frateuria that can produce a

water-soluble brown pigment on glucose-yeast extract-CaCO3, and use AE broth – the medium

was used to identified the genus Frateuria (Swings et al., 1984) In conclusion, we suggest this strain belongs to the genus Dyella as an acidophilic bacterium This study with the aim is to

provide the first general characteristics of the strain isolated from activated sludge for the further intensive research, particurlarly G/C content and fatty acid analysis, two powerful tools to identified strains at species level

Content

Chapter 1 Introduction about acidophilic nitrifying process and microorganism population in this process

1.1 General introduction about nitrifying process 5

1.2 The invention of acidophilic nitrifying process 6

1.3 The genus Dyella, Frateuria and Rhodanobacter 8

1.4 Purpose of our study 9

Chapter 2 Phenotypic characterization of an isolated strain 2.1 Introduction 10

2.2 Materials and methods 12

2.2.1 Test strain 12

2.2.2 Test growth medium 12

2.2.3 Cell morphology and cultural characteristic 12

2.2.4 pH test 12

2.2.5 Temperature test 13

2.2.6 NaCl tolerance test 13

2.2.7 Growth measurement 13

2.2.8 Quinone analysis 14

2.3 Results 16

2.3.1 Test strain and cultivation 16

2.3.2 Test growth medium 17

2.3.3 Cell morphology and cultural characteristic 18

2.3.4 pH test 19

2.3.5 Temperature test 20

2.3.6 NaCl tolerance test 21

2.3.7 Growth measurement 22

2.3.8 Quninone analysis 23

2.4 Discussion 23

Chapter 3 Molecular characteristic of the isolated strains 3.1 Introduction 24

3.2 Materials and methods 27

3.2.1 16S rRNA analysis 27

3.2.2 Phylogenetic tree 32

3.3 Results 32

3.4 Discussion 32

Chapter 4 Concluding remarks 33

Annex 34

Index 36

References 44

Chapter 1 : Introduction about acidophilic nitrifying process and microorganism population in this process

1.1.General introduction about nitrifying process

Nitrification is the biological oxidation of ammonia with oxygen into nitrite followed by the oxidation of these nitrites into nitrates Degradation of ammonia to nitrite is usually the rate limiting step of nitrification Nitrification is an important step in the nitrogen cycle in soil This process was discovered by the Russian microbiologist Sergei Winogradskyi The oxidation of ammonium into nitrite is performed by two groups of organisms, ammonia oxidizing bacteria and ammonia oxidizing archaea Ammonia oxidizing bacteria can be found among the β – proteobacteria and γ – proteobacteria In soils the most studied

ammonia oxidizing bacteria belong to the genera Nitrosomonas and Nitrosococcus

Although in soils ammonia oxidation occurs by both bacteria and archaea, archaeal ammonia oxidizers dominate in both soils and marine environments, suggesting that

Crenarchaeota may be greater contributors to ammonia oxidation in these environments

The second step (oxidation of nitrite into nitrate) is mainly done by bacteria of the genus

in aeration (bringing oxygen in the reactor) and the addition of an external carbon source (e.g methanol) for the denitrification In most environments, both organisms are found together, yielding nitrate as the final product It is possible however to design systems in which selectively nitrite is formed (the Sharon process) Together with ammonification, nitrification forms a mineralization process which refers to the complete decomposition of organic material, with the release of available nitrogen compounds This replenishes the nitrogen cycle

1.2.The invention of acidophilic nitrifying bioreactor

Chemolithoautrophic nitrifying bacteria, i.e., ammonia-oxidizing bacteria (AOB), catalyzing the first oxidation step of ammonia to nitrite and nitrite-oxidizing bacteria (NOB) completing the oxidation of the intermediate nitrite to nitrate are known to be sensitive to low pHs Optimum growth occurs under neutral to moderately alkaline condition (pH 7.5 to 8.0) In liquid pure culture, growth is usually restricted to a low pH of 5.8 (AOB) or 6.5 (NOB) (Watson et al., 1989) and activity ceases typically below pH 5.5 (Hankinson et al., 1988, Jiang et al., 1999) The failure of AOB to cope with acidic conditions is thought to be primarily based on the unavailability of a substrate : with decreasing pHs, ammonia, the substrate of AOB (Suzuki et al., 1974), is increasing protonated Nitrite, the substrate of NOB, undergoes protonation to nitric acid, which disproportionates to nitrate and gaseous nitric oxide at low pHs (Bock et al., 2001) Furthermore, when present at elevated concentrations under low pHs, free nitric acid negatively affects the growth and activity of nitrifying bacteria (Anthonisen et al., 1976) Despite these limitations, autotrophic nitrifying bacteria have been isolated from, or nitrifying activity has been demonstrated in, acidic environments, such as soils, activated sludge, and biofilms Numerous nitrifying isolates have been obtained from soild with pHs around 4 (deBoer et al., 2001) even as low as 2.5 (Prosser, J.I 1989) However, the majority

of such isolates do not show nitrifying activity in acidic mineral medium (deBoer et al., 2001) In contrast, autotrophic nitrifying activity in soil sample could be maintained at a

pH as low as 4 (deBoer et al., 2001)

Toward this trend, our laboratory has developed an acidophilic nitrifying sequencing batch reactor called ANSBR to treatment wastewater at acidophilic condition with sludge as seed Two graduated students in our lab, Kuroki and Matsuba were successful to prove that the addition of yeast extract has affected to keep sludge and the high performance of nitrification in the reactor To reveal change of the microbial population by the addition of yeast extract, succcessions of the bacterial populations in the acidophilic nitrification sequence batch reactor were evaluated by PCR-DGGE analyses In addition of yeast extract, predominant bacteria in the reactor were novel microbes affiated with a candidate phylum TM7 that they are functionally unknown Moreover, a member of our group, Nguyen Minh Giang has isolated a bacterium that first of all, identified to belong to the

genus Nitrobacter (similar 98% to Nitrobacter winogradskyi (ATCC 25301) by sequencing

a short distance gene (665 bp) The discovery of this genus from sludge at acidophilic condition seems a significant finding From this view, we take on responsibility to study this strain Reidentified full gene of this strain by 16S rRNA sequence analysis and

phylogenetic tree shows that this strain is closely related to the genus Dyella japonica

XD53T (AB110498)

Besides, similar >95% to the genus Frateuria and Rhodanobacter These genus belong to the family Xanthomonadaeae, class Gammaproteobacteria

Fig 1-1 The phylogenetic tree of a subset of the γ-Proteobacteria , based on 16S rRNA gene

sequence comparision, determined by neighbour-joining Escherichia coli was used as the

outgroup Bootstrap percentages of 1000 replicates are indicated at nodes

1.3 The genera Dyella, Frateuria, Rhodanobacter

Our strain is the most closely related to the genus Dyella In literature about Dyella, there are seven genus : Dyella ginsengisoli (2009), Dyella japonica (2005), Dyella koreensis (2005),

Dyella marensis (2009), Dyella soli (2009), Dyella terrae (2009), Dyella yeojuensis (2006) Dyella japonica came from Japan, Dyella ginsengisoli – one of two strains came from China,

others came from Korea Most of these strains were isolated from soil, mesophile and neutrality

bacteria, unknown function except Dyella koreensis,Dyella japonica RB28, and Dyella

ginsengisoli Dyella koreensis is identified as β-glucosidase-producing bacterium More specially, Dyella japonica RB28 is reported to cause human infection Dyella gingengisoli is used to

biodegradation of biphenyl and isolated from activated sludge Our strain is similar 97% to

Dyella japonica XD22, XD10, RB28; 96% to Dyella ginsengisoli LA-4, LA-3 and 97% to Dyella sp CHNCT14, CHNCT13, CHNCT15 The information of all strains of Dyella sp is

unpublic About Dyella ginsengisoli, two strain, as we have said above, are used to biodegradation of biphenyl and isolated from activated sludge About Dyella japonica, only

Dyella japonica RB28 is known as an damage bacterium to human

The genus Frateuria was validly established by Swings et al in 1980 for bacteria that are polarly

flagellated, produce brown pigment, have branched cellular fatty acids, and are Q – 8 equipped

Gram-negative, incorporating “Acetobacter aurantius” Frateuria aurantia is type species and only a species of this genus The genus Frateuria is recognized as Pseudo-acetic acid bacteria

because this strain showed intermediate characteristics to acetic acid bacteria Up to now, four

case of the isolation of Frateuria aurantius, all from flowers and fruits, there are three in Japan,

and other in tropical region (Indonesia) Three general characteristics of this genus are acidophilic bacteria, can produce a water-soluble brown pigment on glucose-yeast extract-CaCO3, and use AE broth According to the phylogenetic tree, our strain is closely related to the

genus Frateuria sp (DQ419968) and Frateuria WJ69 (AY495959) Both of these strains that are quite different from those above metioned Frateuria, are isolated from wastewater systems that contain heavy metals The paper of Frateuria sp (DQ419968) is unpublic, but the paper of

Frateuria WJ69 was public in 2005, but this isolate is uncharacteristic In general, the genus Frateuria can be isolated from either flowers and fruits or systems that contain heavy metals

As the genus Dyella, all strains of the genus Rhodanobacter are also not acidophilic bacteria Only the strain of Dyella koreensis of the genus Dyella is non-motile On the opposite, all of genus Rhodanobacter are non-motile except Rhodanobacter fulvus These strains were isolated

from soil, only Rhodanobacter thiooxydans sp was isolated from a biofilm on sulfur particles

used in an autotrophic denitrification process In this process, this strain reduced nitrate, but not nitrite

In summary, three above genus belong to the family Xanthomonadaea, Gammaproteobacteria

Therefore, they are relative to some characteristics, the most differences are G/C content, composition of fatty acid In conclusion, to identified one strain exactly, we need to carry out these experiments

1.4 Purpose of our study

In this study, a strain was isolated from acidophilic nitrifying sequencing reactor was studied taxonomically Phylogenetic tree indicates this strain is closely related to the genus

Frateuria, Dyella and Rhodanobacter Therefore, this study aims to identified and

characteristic of this strain to understand their taxonomic position at physiologically and biochemically characteristics as well as DNA-based molecular properties

Chapter 2 Phenotypic characterization of an isolated strain

2.1 Introduction

Taxonomy is the science of classification and consists of two major subdisciplines,

identification and nomenclature Although the goal of identification is merely to provide

the name of an isolate, most identification systems depend on first determining a number

of morphological, biochemical, cultural, antigenic, and other phenotypic characteristics of the isolate before the name can be assigned Phenotypic analyses have traditionally played

an important role in bacterial identification and classification Characteristics of taxonomic value that are widely used include various aspects of morphology, motility, nutrition and physiology, biochemistry

The morphology of cells normally includes cell shape, cell size, the gram stain reaction In culture the morphology indicated the status of the cells, both in terms of the cells and in the case of primary isolates the differentiation state may be critical Cell shape is generally characteristic of a given bacterial species, but can vary depending on growth conditions Typical examples include coccus (spherical), bacillus (rod-like), spirillum (spiral) and filamentous Bacteria generally form distinctive cell morphologies when examined by light microscopy and distinct colony morphologies when grown on petri plates Gram staining is used to differentiate bacterial species into large groups (Gram possitive and Gram negative) based on the physical properties of their cell walls

Bacterial motility styles include motile by flagella, motile by gliding, motile by gas vessels, and nonmotile Motile by gliding can be observed directly from examination of the tubes following incubation Growth spreads out from the line of inoculation of the organism is motile Flagella are readily seen with the electron microscope In extremely large prokaryote, tufts of flagella can also be observed by phase contrast microscopy

Nutrition test is the test for mechanism of energy conservation (phototroph, chemooraganotroph, chemolithotroph) Phototrophic bacteria – green and purple bacteria use energy from sunlight and carbon from dioxide or organic carbon Chemoauthotrophic bacteria obtains its nourishment through the oxidation of inorganic chemical compounds as opposed to photosynthesis Chemolithotrophy is defined as the production of metabolically useful energy by the oxidation of inorganic compounds

Physiological bacteria performs by relationship between bacteria and environment such as oxygen, temperature, pH, and salt tolerances, ability to use various carbon, nitrogen, and

sulfur sources.Temperature is one of the most environmental factors affecting growth and survival of microorganisms Classification of bacteria based on temperature includes four groups : psychrophile, mesophile, thermophile, and hyperthermophile Psychrophile can live from -10 to 20oC and optimum temperature is 4oC Mesophile can live from 10 to

45oC and optimum is 39oC Thermophile can live from 40 to 75oC and optimum is 60oC Hyperthermophile can live from 65 to 120oC and optimum is 90 to 100oC Each organism has a pH range within which growth is possible and usually has a well – defined pH optimum Organisms that grow best at low pH are types of extremophiles called acidophiles The most critical factor for obligate acidophily is the stability of the cytoplasmic membrane When the pH is raised to neutrality, the cytoplasmic membrane of strongly acidophilic bacteria actually dissolves and the cells lyse, suggesting that high concentrations of hydrogen ions are required for membrane stability In a batch culture, pH can change during growth as the result of metabolic reactions that consume or produce acidic or basic substances Thus, chemical called buffers are frequently added to microbial culture media to keep the pH relatively constant Microorganisms vary in their need for oxygen In fact, microorganisms can be divided into several groups depending on the effect

of oxygen

Biochemical tests for taxonomy oftenly are fatty acid and quinone Some fatty acid are unique to a group of bacteria For exanple the iso and anteiso saturated fatty acid are widely found in gram positive bacteria Gram negative bacteria very often contain straight fatty acids with one double bond Such characteristic features in the alkyl chain of fatty acids are used as a guide in the classification of bacteria Isoprenoid quinone are liquid molecules in all species of respiratory and photosynthetic microorganisms and exhibit marked structural variations depending upon the microbial taxon Taking advantage of this, quinones have been used not only as chemotaxonomic markers in microbial systematics but also as good measures of microbial populations in the environment in terms of quantity, quality, and activity The quinone profile method allows good measurement of both fundamental and applied aspects of ecological and environmental microbiology In

Proteobacteria, Q-10 is found mostly in the α subclass, Q-8 in the β and γ subclass, Q-9 in

the γ subclass

2.2 Materials and methods

2.2.1 Test strain

This strain was isolated from Nguyen Minh Giang, a member of bioreactor group, and

store at 10oC for 2 month in AOB medium, pH 4.0 This strain was first identified similar

to the genus Nitrobacter after sequencing a short distance gene of this strain (665bp)

Therefore we decided to use NOB medium, pH 4.0 for next cultivation Before transfering this strain to new medium – NOB, we did multiple passages of this type of colonies grown

on ANSBR medium gellangum into intermediate medium gellangum (50% AOB medium + 50% NOB medium) This strain was cultivated at 25oC, 140 rpm/min Purification was test by observation with phase-contrast microscope

2.2.2 Test growth medium

Growth was test on AOB, NOB medium , YMG agar, nutrient agar – NA (Difco) and R2A agar (Difco), AE broth , and Glucose – CaCO3 – yeast extract (two last medium were used

to identified the genus Frateuria, ) The isolate was incubated at 30oC, 140 rpm/min (if use liquid medium) Daily observation were made and recorded for up to 14 days We used buffer (50 mM KH2PO4, 50 mM K2HPO4, pH 7.0) to keep pH stable

2.2.3 Cell morphology and cultural characteristic

Cells, on above medium, will be test morphology Pick up one colony from solid medium (incubate for 24h) to a tube 2 ml containing milliQ Centrifuge 12,000 rpm for 5 mins Remove supernatant then Use loop to remove the strain from tube to a lame (loop is heated until red hot and cooled in air briefly) Cover by lamme General cell morphology was studied using an Olympus model BX-50 epifluorescence microscope at phase contrast

to view individual shape and size Cells are also observed after Gram reaction Test was test griding motile by semi – liquid of the best medium

2.2.4 pH test

After growth medium test, we chose the medium that is the best for growthing of bacteria

to continue pH test Colonies were picked up from petri plate to liquid medium Incubate

200 ml culture for 24 h After that, 1ml culture dilutes with 9 ml medium (initial OD is 1.0)

We used buffer (50 mM KH2PO4, 50 mM K2HPO4, pH 7.0) to keep pH stable Growth was test at pH 3.0 – 8.0 with intervals of 1.0 pH unit Incubate samples at 25oC, 140 rpm/min Cell growth was monitored spectrophotometrically by measuring the optimal density at

660 nm (OD660) every 2 hours until OD constant (stationary phase)

Based on OD660, the growth rate at each pH is calculated as follow :

µ =

)12(

)1log2(log

*303.2

t t

OD OD

Based on OD660, the growth rate at each temperature is calculated as follow :

µ =

)12(

)1log2(log

*303.2

t t

OD OD

2.2.6 NaCl tolerance test

To do this experiment, we used best medium with NaCl 0%, 2%, 4%, 6% at optimum pH Growth was incubated at optimum temperature for 24h Daily observations were made and recorded

2.2.7 Growth measurement

Growth was cultivated at the best nutrient, optimum pH, temperature and %NaCl for growth measurement Cell growth was monitored spectrophotometrically by measuring the optimal density at 660 nm (OD660) every 2 hours until OD constant (stationary phase) The number of cells in the culture was calculated based on the standard line

The standard line was made by the follow method : Growth was cultivated at the best medium, optimum pH, temperature and %NaCl for growth measurement for 24h Prepare 9ml pure medium (the best medium, optimum pH and %NaCl).Transfer 1 ml culture from this medium to 9 ml pure medium to have 10-1 dilution Continue transfer 1ml culture of 10-1 dilution to 9 ml pure medium to have 10-2 dilution Density of cells at 10-1,

10-2 dilution are measured by spectrophotometer at 660 nm Repeat this step until 10-n

dilution (n dilution time, ODn = 0) 10-n dilution are measured total cell count by SYBR Green I staining

Number of cells at 10-y dilution = number of cells at 10-n x 10n-y (0 <y < n)

Total cell count by SYBR Green I staining : Volume 1 ml of the sample and

dissolved in 9 ml of filtered PBS buffer in a 50 ml conical tube Sonicate the sample with ultrasonic disintegrator for sec and settle it for 10 minutes Take appropriate dilution (the 10-5 to 10-7 dilutions for activate sludge sample) then pipette 5 ml (50 – 200 cells per vision) and add 1/ 10.000 SYBR GREEN I and incubate for 5 minutes in the dark Filtered with ISOPORE 0.2 µm GTBP membrane filter Put one drop of antifade solution, CITI FLUOR (Agar SCIENTIFIC), then observe the trapped cells with epifluorescence microscope Chose the right one that gives you 50 – 200 cells per vision Count at least 10 screens and take average to calculate the amounts of cells per ml in your sample Turn the mercurry ramp on at least 30 minutes before turn it off or vise versa Cells are count by software WinROOF

Number of cells per 1 ml =

6-10

x 210

x 170

x CellsAVERAGE

7.176

x10-x

With : x : dilution times

Area of view = 170 µm x 210 µm (when magnification of 400x is used)

Usable area of filter = 176.7 µm2

2.2.8 Quinone analysis

Strain was incubated at the best medium, optimum pH and temperature for 24h Quinone was extracted and purified as follow :

Extraction and purification

1 During the process of extraction, the sample were covered with black paper to avoid exposure to light

2 50 ml of the sample was allowed to settle in a measuring cylinder for 30 minutes

3 The upper layer was decanted and the residue was transferred to a new corning tube

of 50 ml capacity

4 The tubes were centrifuged at 6000 rpm, 4oC for 10 mins

5 Supernatant was discarded and 20 ml of 50 mM phosphate buffer containing 1 mM Potassium ferricyanide was added

6 The tubes were shaken vigorously for the complete re – suspension of the pellets and centrifuged again

7 The washing was repeated twice same way, and finally the pellets were suspended in 5-7 ml of same buffer

re-8 Three volumes of a mixture of chloroform and methanol (2:1 vv-1) was added and the samples were shaken vigorously

9 The clumps were dispersed by sonication at 100 W, 90 sec

10 The samples were centrifuged at 6000 rpm for 10 mins at 4oC

11 The uppermost layer of the water was discarded using Pasteur pipette

12 The bottom layers of chloroform and methanol was filtered into an oval flask using no.2 whattman filter

13 The mixture of chloroform and methanol was evaporated at 37oC using a evapotator with a water bath

rotatory-14 To remove any fraction of water left 2-3 ml of hexane and 1 ml of aceton was again added

15 The contents were transferred to a corning tube and little water was also added

16 The tubes were kept for a while to separate water phase from hexane and after the separation hexane layer was transferred to an oval flask with a Pasteur pipette

17 The hexane was evaporated using ratatory evaporator

18 The lipid extract was re-suspended in 1-2 ml of hexane

19 Quinone were eluted through Sep-pack cartridges using a mixture of hexane and 10% diethyl ether into 50 ml oval flasks

20 The eluted quinones were dried under rotatory evaporator and re-suspended in 50

µl of aceton

HPLC analysis

Quinone components were separated by reverse-phase partion HPLC with a mixture of methanol-Diisopropyl ether (9:2) as mobile phase at a flow rate of 1.5 ml h-1 and column temperature of 30oC

An injection volume of 20 µl was used

Elution was monitored with a UV spectrophometric detector at 270 nm (menaquinones) and 275 nm (Ubiquinones)

Quinone species were identified by their specific elution time

Where xik, xjk ≥ 0.01 and x jk x ik 100、xik and xjk indicates the mole % of quinone

homolog k in samples i and j The D values can be interpreted to reveal the extent of

differences in microbial community structures among samples

Another parameter of phylogenetic importance is Microbial divergence index MDq, which

can be explained as follows

Where xk ≥ 0.001 and xk indicates the molar ratio of quinone homolog k to the total

quinone contents as 1 If all quinone types detected constitute equal molar proportions, the

MDq value becomes equal to the number of Quinone types detected

2.3 Results

2.3.1 Test strain

We failed to transfer this strain from ANSBR medium to NOB medium directly, so

we used intermediate medium including 50% AOB medium (1mM NH4+) and 50% NOB medium (1mM NO2-) In AOB and NOB medium, this strain grew slowly The colonies were tiny and white After two months, the color changed to brown yellow

Fig 2.1 Strain was incubate at AOB, intermediate medium, NOB medium

(From left to right), pH 4.0 after 6 days

By observation with phase – contrast microscope, we saw a pure culture This bacterium is rod, thin , length of a single cell = 1.5 - 2.0 µm

Fig 2.2 Morphology of this strain at AOB and NOB medium by microscope at phase

contrast Bar = 20 µm

2.3.2 Test growth medium

Cells could grow at NA, R2A, AOB, NOB, but could not grow at AE, Glucose – Yeast extract – CaCO3, YMG And strain grew very well at NA medium after 24h incubation

In NA , the strain grew better than R2A The streak of strain on the surface of agar are yellow in color, could reach 2.5- 4.0 mm in width after 3 days incubation (Fig A) The colonies on NA medium were fairly circle, white in color in color , flat after 2 days incubation (Fig B) We saw the difference in color between streak (yellow) and colonies (white) of strain When we used a loop to cover strain (moving as circular ), we got a viscous liquid (Fig C)

In AOB and NOB, strain growth slowly Colonies are tiny, white and become dark yellow after 2 month (Fig A)

In YMG medium, colonies are quite not detected Also, in AE, Glucose – Yeast

extract medium – the medium for Frateuria, colonies are quite not detected too

NOB YMG

A

2.3.3 Cell morphology and cultural characteristic

In both NA and R2A medium, strain is straight rod, single, double chain or long chain, 5.0 – 7.0 µm in length This size is bigger 3 – 4 times comparing to the strain in AOB and NOB medium Gram-negative (Fig.).In semi-solid culture, growth spreads out from the line of inoculation of the organism Therefore, this strain is motile Phase-contrast microscope shows many visible small pigments inside of intracellular These pigments could not identified, and will be investigate in the next study

Fig 2.3 (A) Strain grew at 30oC after 14 days (B) Strain grew at NA at 30o after

3 days (C) The statement of strain after incubate at solid NA at 30oC for 2

weeks, then remove by loop to liquid NA

2.3.4 pH test

From the result of medium test, we cultivate this strain at NA medium, 25oC, 140 rpm/min The relationship between growth rate (µ) and pH is shown in the Fig From the chart, we saw this strain coulg grow at pH 4.0 – 8.0 and optimum pH is 5.0 From the fig, we can see the culture at pH 5.0 is more turbidity than others At pH 3.0, we can not detect the growth

of this strain in liquid medium by spectrophotometre (Fig.)

Fig 2.5 Phase-contrast micrographs showing many visible small pigments –

bold green inside of intracelluar (from NA medium), pH 6.890 Bar = 20µm

Small pigment

0 0.05 0.1 0.15 0.2 0.25 0.3

2.3.5 Temperature test

The isolate could grow at temperature 10 – 55oC, and optimum temperature is 25oC Strain grew limit at 10 or 55oC

Fig 2.6 (A) The relationship between growth rate - µ (1/h) and pH 3.0 – 7.0 (intervals of 1

unit) (B) Strain was incubated in NA, pH 6.890, 25oC, 140 rpm/min after 72h From left to

right, pH 3, 4, 5, 6, 7, 8

10

20 25

37

55

0 0.05 0.1 0.15 0.2 0.25

2.3.6 NaCl tolerance test

This strain was not tolerant to NaCl In medium supplied NaCl, we could not detect growth

Fig 2.8 Strain was incubated at NA medium, pH 5.0, 140 rpm/min after 72h

From left to right, temperature is 10, 20, 25, 37, 55oC

Fig 2.9 Strain was incubated at NA medium, pH 5.0, 25oC after 72h

NaCl 0% NaCl 2%

NaCl 6% NaCl 4%

2.3.7 Growth measurement

14.6 14.8 15 15.2 15.4 15.6 15.8 16

Fig 2.10 The standard line shows the relationship between OD660nm

and total number of cells (cells/ml)

Fig 2.11 Rate growth of this strain when it was incubated at NA, pH5.0, 25oC, 140

rpm/min,

2.3.8 Quninone analysis

The quinone profile for this strain showed Q-8 as the unit detected components (data is

given in Index) In the theory, Q-8 and Q-9 present γ-proteobacteria

2.4 Discussion

The discription of the isolate for phenotypic characteristics :

Cells are straight rod (5.0 – 7.0 µm) single, double or long chain Gram-negative Motile Colonies grow well on nutrient agar and R2A They do not grow in AE broth, YMG and Glucose-Yeast extract-CaCO3 The condition for growth are10 – 55oC, pH 4.0 – 8.0 and optimum is 25oC, pH 5.0 Not tolerant to NaCl Q-8 is the major component of the quinone system

From phenotypic analysis, we made sure that this strain didn’t belong to the genus

Nitrobacter.The datas of medium tests supported our conclusion by this strain could

develop well in the medium that is not used to identify the genus Nitrobacter (Fig 2.3A) In

addition the change of size cells when this strain grew up from NOB medium to NA, R2A medium clearly showed the same point (Fig 2.2 and 2.4) More over, quinone data showed

this strain belonged to Gammabacteria On the other hand, Nitrobacter belongs to

Alphaproteobacteria The reason why this strain is not Nitrobacter but can grow in AOB

and NOB medium may be explained by the characteristic of reduction of ammonia and nitrite In literature, the scientists are used to do API test for ammonia and nitrite reduction

experiment We will test these characteristics so far to prove our hypothesis

Medium test also give us other conlusions that our strain is also not the genus Frateuria

The test showed our strain grew well on NA, R2A but could not grow at Glucose-Yeast extract-CaCO3 medium used to identify the genus Frateuria More over, this strain also

could not grow in AE and YMG medium According to the composition of growth medium (innex),we give a hypothesis that this strain cannot reduct glucose that is composed of YMG, AE, Glucose-Yeast extract-CaCO3 medium This hyphothesis also must be supported by API test

The phylogenetic tree showes the isolate related to Rhodanobacter fulvus, Rhodanobacter

lindaniclasticus and Rhodanobacter spathiphylli In the genus Rhodanobacter, the motile

characteristic only appear with Rhodanobacter fulvus

In general, the phenotypic datas identify the isolate relate to the genus Dyella and

Rhodanobacter fuvus

Chapter 3 Molecular characteristic of the isolated strains

3.1 Introduction

Because bacteria are so small and contain relatively few structural clues to their evolutionary roots, the phylogeny of prokaryotes has only emerged from genotypic analyses Determination of genomic DNA G + C content, and chemotaxonomic methods such as analysis of cell wall and lipid composition, in many cases proved superior to classical methods based upon morphological and physiological traits

One system that has proven extremely useful is automated cellular fatty acid (CFA) analysis (Onderdonk and Saaer, 1995) The system depends on saponifying the fatty acids with sodium hydroxide, converting them to their volatile methyl esters, and then separating and quantifying each fatty acid by gas-liquid chromatography A computer compares the resulting fatty acid profile with thousands of others in a huge database and calculates the best match or matches for the isolate The computer can also indicate that the isolate does not closely match any other fatty acid profile, which can lead to discovery of new genera or species The entire procedure is simple and takes about 2h, and numerous specimens can

be analyzed rapidly each day One drawback is that the isolate must be cultured under highly standardized conditions of media and temperature in order to provide a valid basis

of comparison woth other fatty acid profiles

A second universal system, and the one of choice at present, is one in which all or most of the nucleotide sequence of the 16S rRNA gene of an unknown isolate is determined The basis of this is the fact that approximately 70% of the 16S rRNA genes (i.e., 16S rDNA) of all procaryotes is highly conserved (identical in sequence) whereas other regions are unique to particular genera or species DNA is isolated from the strain and then universal primers are used to amplify the 16S rDNA by the polymerase chain reaction (PCR) The sequence of the PCR product is compared with other sequences stored in an enormous database One such database is used in the Ribosomal Database Project-II (RDP-II), which

is a cooperative effort by scientists at Michigan State University and the Lawrence Berkeley National Laboratory 16S sequencing is rapidly enough to handle a large number

of isolates in a short time; this service is provided by a number of institutions for medical and other types of isolates Given a well-equipped sequencing lab, 16S sequences can be obtained and analyzed within 48 hours The main drawback with sequence-based identification is that of the need for sophisticated equipment, which is present in relatively

few microbiology labs Another drawback is that although sequence-based identification is very effective for assigning as isolate to its most likely genus, it may not be able to identify

an isolate to the species level if the sequence for two or more related to the species level if the sequences for two or more related species have greater than 97% similarity

Genomic G+C (guanine plus cytosine) content is the percentage of nitrogenous bases on a DNA molecule which are either guanine or cytosine (from a possibility of four different ones, also including adenine and thymine) This may refer to a specific fragment of DNA

or RNA, or that of the whole genome G+C ratios within a genome are markedly variable Initially, overall base compositions of DNAs (mol% G + C values) were used to compare procaryotic genomes, and organisms for which mol% G + C values differed markedly were obviously not of the same species If, however, two organisms had the same mol% G + C value, they might or not belong to the same species, and thus a much more precise method

of comparison was needed The development of DNA – DNA hybridization techniques fulfilled this need The continue that often blurred the separation between groups defined

by phenotypic characteristics did not usually occur with DNA – DNA hydridization Organisms tended to be either closely related or not, because DNA – DNA duplex formation did not even occur if base pair mismatches exceeded 10 – 20% Thus DNA – DNA hydridization solved many of the problems that had long plagued bacterial taxonomy

at the species level of classification

With improvement in molecular sequencing techniques, the idea of Zuckerkandl and Pauling (1965) to deduce the phylogenetic history of organisms by comparing the primary structures of macromolecules became applicable The first molecules to be analyzed for this purpose were cytochromes and ferredoxins (Fitch and Margoliash, 1967) Subsequently, Carl Woese and coworkers demonstrated the usefulness of small subunit (SSU) rRNA as a universal phylogenetic marker (Fox et al., 1977) The critical initial step

of sequence-based phylogenetic analyses is undoubtedly the alignment of primary structures Alignment is necessary because only changes at positions with a common ancestry can be used to infer phylogenetic conclusions These homologous positions have

to be recognized and arranged in common columns to create an alignment, which then provides the basis for subsequent calculations and conclusions Sequences such as SSU rRNA that contain a number of conserved sequence positions and stretches can be aligned using multiple sequence alignment software such as CLUSTAL W (Swofford et al., 1996)