Selection and Characterization Of Bacterial Isolated For Monocyclic Aromatic Degradation

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SELECTION AND CHARACTERIZATION OF BACTERIAL

ISOLATES FORMONOCYCLIC AROMATIC DEGRADATION

BY

DWI

SURYANTO

GRADUATE PROGRAM

(119)

To my dearest children:

(120)

ABSTRACT

A number of anoxygenic photosynthetic bacteria (APB) were isolated from Moluccas, Central Kalimantan, West Kalimantan, West Java, and Yogyakana. Approximately, 65.8% of the APB isolates were able to utilize benzoate as their sole carbon source when grow photosynthetically. The ability of these bacteria to grow in gentisate was firstly reported. No growth was demonstrated in the presence of phenol, cathecol and salysilate as sole carbon sources. In addition, we also isolated five aerobic bacterial isolates that could grow at least in three different monocyclic aromatic compounds. One of the aerobic isolates could grow in atrazine.

The three APB, DS-1, DS-4 and Cas-13 as well as aerobic isolate DS-8 were Gram negative, motile, non-halophilic, non alkali- and acidophilic. The APB were rod-shape cells with swollen terminal. DS-8 was ovale-rod cell, has excellent swarming activity and produce extracellular protease and chitinase. Growth of the selected APB isolates were optimum at NaCl concentration of 0.5% (w/v) and initial p H of 7.5, respectively, while optimum NaCl concentration and initial pH for DS-8 growth, one of the aerobic isolates, were 1-1.5% (w/v) and 7-8.5, respectively. Each of the isolates could grow in benzoate up to 10 rnM.

APB isolates could grow and utilize cassamino acid and dextrin as their sole carbon sources. However, DS-8 could utilize either cassamino acid, glutamate, glucose, acetate, potato starch, or ethanol as the only carbon sources. Macrorestriction Fragment Length Polymorphisms (MFLP) employing Pulsed-Field Gel Electrophoresis (PFGE) of the APB isolates indicated that DS-1 was closely related to Cas-13 rather than to DS-4. Physiological or metabolic identification using Microbact (Medvet Science Pty. Ltd., Adelaide, Australia) indicated that DS-8 was closely related to Klebsiella ozaenae (49.85%) and Smatiu lzquefaciens (24.42%). Sequence analysis of 16s rRNA genes showed that the APB isolates were strains of R h o d o p ~ m o n u s p c t l ~ , while DS-8 was S. marcexens. A complete 16s rRNA sequence analysis, however, is needed to give more definitive information about the taxonomic position of the isolates.

(121)

STATEMENT O F RESEARCH ORIGINALITY

This is to verify that the dissertation entitled:

SELECTION AND CHARACTERIZATION O F BACTERIAL ISOLATES FOR MONOCYCLIC AROMATIC DEGRADATION

is my own work and has never previously been published. All of the incorporated

data and information are validated and stated clearly.

Bogor, 10 October 2001

(122)

SELECTION AND CHARACTERIZATION OF BACTERIAL

ISOLATES FOR MONOCYCLIC AROMATIC DEGRADATION

BY

DWI SURYANTO

A DISSERTATION

Submitted to the Bogor Agricultural University in partial fulfillment of the requirements for

the Doctorate Degree (Dr.)

GRADUATE PROGRAM

BOGOR AGRICULTURAL UNIVERSITY

200 1

(123)

This is to certify that the dissertation

Title SELECTION AND CHARACTERIZATION OF

BACTERIAL ISOLATES FOR MONOCYCLIC AROMATIC DEGRADATION

Name DWI SURYANTO

Student number 965080 BIO

Study Program/ Biology/Microbiology Sub Program

has been accepted toward fulfillment of the requirements for Doctorate degree in Biology/Microbiology

1. Committee members

Dr. Antonius Suwanto Dr. Ania Meryandini

Chairman

Prof. Dr. Bibiana W. Lay

J '

w

Prof. Dr. Muhammad Sri Saeni

2. Head of Study Program

4

Dr. Dede Setiadi

(124)

BIOGRAPHY

Dwi Suryanto. Born in Sungailiat, April 9, 1964. The second son of

father, Muhammad Sahuri and mother, Sujatmi. Graduated from Elementary

School (SD UPTB Pemali), Junior High School (SMP UPTB Pemali), and Senior

High School (SMA Negeri Sungailiat) in 1976, 1979, and 1982, respectively.

Continuing education at Faculty of Biology, Gadjah Mada University, Yogyakarta

in 1982, and obtaining bachelor degree majoring in Ecology in 1987. In 1991,

master degree w a s started in Department of Entomology, College of Natural

Sciences, Michigan State University, East Lansing, USA, under a USAID

scholarship program, and granted in 1993. In 1996, w a s admitted as a doctorate

student at Study Program of Biology, Sub-program of Microbiology, Bogor

Agricultural University, Bogor.

Appointed as a lecturer at Department of Biology, Faculty of Mathematics

and Natural Sciences, North Sumatra University, Medan. Married in 1992, and

have two dearest children, Muhammad Aditya Haryawan and Nindya Laksita

(125)

AKNOWLEDGMENTS

I would like to express my deepest appreciation and sincere thanks to my

major advisor, Dr. Antonius Suwanto, for his guidance, patience and support

throughout my doctorate degree, and for giving an opportunity to learn more

about the art of microbiology.

I would also like to express my gratitude to other committee members,

Dr. Anja Meryandini, Prof. Dr. Maggy T. Suhartono, Prof. Dr. Bibiana W. Lay,

and Prof. Dr. Muhammad Sri Saeni for their unfailing support, encouragement,

and guidance during the course of my work.

I have enjoyed the interaction and aid from the marvelous students,

friends, and technicians in Laboratory of Microbiology and Biochemistry of

Research Center for Biotechnology, Artini Pangastuti, Dr. Budiasih Wahyuntari,

Diana E. Waturangi, Etty Pratiwi, Irawan Tan, Munti Yuhana, Dr. Nisa

Rachmania, Nurhaemi Haris, Rina Martini, Temmy Desiliyarni, Dr. Wibowo

Mangunwardoyo, Widanarni, Witri Djasmasari, Dr. Yusminah Hala, Stefani

Adijuwana, Eni Sumartini and Ika Malikah, and the many others too numerous to

be named, but will remain dear to my heart.

To my dearest children, Muhammad Aditya Haryawan and Nindya Laksita

Laras, thank you both for being the light of my life.

Special thanks must go to Center for Microbial Diversity, FMIPA, IPB for

funding this research. Finally, I would also like to thank to my government, the

North Sumatra University, and PT Timah Tbk. for their support and confidence.

(126)

TABLES

CHAPTER 2.

MATERIALS

AND

METHODS

Table 1. Bacterial strains and plasmid used in this study

...

2 1

CHAPTER 3.

[image:128.511.31.464.33.743.2]

SELECTION AND ISOLATION OF BACTERIA FOR BENZOATE DEGRADATION

Table 1. The ability of anoxygenic photosynthetic bacteria to grow in

[image:128.511.37.461.217.736.2]

monocyclic aromatic compounds in anaerobic condition illuminated

...

with 40 W tungsten at a distance of 30 cm from cultures 30 Table 2. The ability of bacterium to grow in monocyclic aromatic compounds

in aerobic condition

...

34

CHAPTER 4.

ISOLATION AND CHARACTERIZATION OF A NOVEL BENZOATE-UTILIZING Serratia marcescens

Table 1. The appearance of DS-8 colonies when grown on different media

...

42 Table 2. Physiological and biochemical characterization of DS-8

...

45 Table 3. Comparison of 16s rRNA sequence similarity of DS-8 and

a number of closely related bacteria

...

45

CHAPTER 5.

CHARACTERIZATION OF BENZOATE DEGRADING ANOXYGENIC PHOTOSYNTHETIC BACTERIA ISOLATED

FROM ENVIRONMENT

Table 1. Observation on cell morphology

...

56 Table 2. Peaks (nrn) and absorbances of cell extract of

Rb.

sphaerozdes, DS-1,

...

DS-4, and Cas-13 59

(129)

LIST OF

FIGURES

CHAPTER 1.

[image:129.511.40.467.40.667.2]

GENERAL INTRODUCTION

Figure 1. General pathway of aerobic catabolism of aromatic compounds

...

6

Figure 2. General pathway of anaerobic catabohm of aromatic compounds

..

8

*

CHAPTER 3.

SELECTION AND ISOLATION OF BACTERIA FOR

BENZOATE DEGRADATION

Figure 1. Photograph of growth of anoxygenic photosynthetic bacteria in modified Sistrom with 5 rnM benzoate as C source supplemented with vitamins

...

29 Figure 2. Growth of anoxygenic photosynthetic bacteria in modified Sistrom with 5 mM benzoate as sole C source supplemented with vitamins

..

32

Figure 3. Growth of aerobic bacteria in modified salt medium with 5 mM

...

benzoate as sole C source supplemented with vitamins 34

CHAPTER 4.

ISOLATION

AND

CHARACTERIZATION OF A NOVEL

BENZOATE-UTILIZING Serratia rnarcescens

Figure 1. Photograph of DS-8 cells ... 40

Figure 2. Photograph of colony expansion of DS-8 by its swarming activity after 12 hours of incubation time..

...

43

Figure 3. Colony appearance of DS-8 on LB agar containing ampicillin,

...

streptomycin and spectinomycin, and gentamycin 43

Figure 4. Colony appearance of DS-8 on chitin agar (A) and skim milk agar (B)

...

43

Figure 5. Dendrogram of DS-8 and its relatives

...

46

~ i & r e 6 . ~ u h r e o f DS-8 on modified salt medium supplemented with 5 mM benzoate (A) without vitamin supplementation, (B) with vitamin supplementation

...

46

Figure 7. Growth of DS-8 in benzoate supplemented with vitamins and without vitamins

...

47

Figure 8. Profiles of growth and benzoate degradation of DS-8 in different benzoate concentration (A), NaCl concentration . . (B), . . and different initial pH (C)

...

49

Figure 9. Growth of DS-8 in different C-sources

...

51

CHAPTER 5.

CIlARACTERIZATION OF BENZOATE DEGRADING ANOXYGENIC PHOTOSYNTHETIC BACTERIA ISOLATED

FROM ENVIRONMENT

(130)

Figure 2. Colony appearance of (A) DS-1, (B) DS-4, and

(C)

cas-13 on [image:130.515.42.469.0.738.2]

modified Sistrom with 5 mM succinate as their C-source

...

.. .

. .

57 Figure 3. Colony appearance of (A) DS-1, (B) DS-4, and (C) Cas-13 on

modified Sistrom with 5 mM benzoate as their C-source

...

... . . .

58

Figure 4. Absorption spectrum of cell extract of DS-1, DS-4, Cas-13, and

[image:130.515.41.467.44.569.2]

Rb. sphaero2des 2.4.1.

. . .

.

. . .

.

. . .

.

. . .

.

. .

.

. . .

. .

. . . .

.

59 Figure 5.

MFLP

profiles of total genome digested with A d . .

.. .. .

...

.

.. ...

.. .

.

60 Figure 6. Phylogenic tree of MFLP profiles after digestion with A d . .

. .

. . .

60 Figure 7. Phylogenic tree of 165 rRNA gene sequences

...

. .. ...

...

. . .

.. .

61 Figure 8. Growth of DS-1, DS4, and Cas-13 in 5 mM benzoate with

vitamins (A) and with no vitamins (B)

... .. . . .. . .

...

.

.. . . .

.

. . .

63 Figure 9. Colony appearance of (A) DS-1, (B) DS4, and (C) Cas-13 on

modified Sistrom with 5 mM gentisate as their C-source

...

.

.. .

..

.

. .

64 Figure 10. Histogram of cell density and benzoate utilization of DS-1.

.

. . . .

..

65 Figure 1 1. Histogram of cell density

and

benzoate utilization of DS-4

. . . ..

66 Figure 12. Histogram of cell density and benzoate utilization of Cas-13..

. . . .

67 Figure 13. Growth of DS-1, DS-4, Cas-13, and Rb. sphaerozdes 2.4.1. in 5 m M succinate(A) and acetate

(B)

with vitamins..

. . .

. .

. . .

. . . .

. .

. . .

.

..

69 Figure 14. Histogram of cell density of other C-source utilization of DS-1,

DS4, andCas-13

... ...

...

... ...

... ... ... ...

...

... ...

...

... ...

...

... ...

70

CHAPTER

6.

IDENTIFICATION OF RESPONSIBLE GENE INVOLVED IN

BENZOATE DEGRADATION OF

Swratia marcescens DS-8 AND Rhodopseudomonas palustris DS-4 Figure 1. Southern blot analysis of total cellular DNA of DS-8 and

-

(131)

CHAPTER

1.

GENERAL

INTRODUCTION

In the last few decades, many hydrocarbon compounds especially

aromatic hydrocarbons have been introduced in large quantity and accumulated in

soil, aquatic environment, anaerobic sediment, or even in deep-ground water

(Mohn and Kennedy, 1992; Kuo and Genthner, 1996; Laine and Laine and

Jergensen, 1996; Semple and Cain, 1996; Werwath et al., 1998). They become a

serious problem since they are toxic and carcinogenic (Shimao et al., 1989; Leahy

and Colwell, 1990; Dong et al., 1992; Valenzuela et al., 1997).

The persistence of the aromatic hydrocarbon compounds in environment

depends on the structure and the complexity of the compounds. Haloaromatic

and polycydic aromatic hydrocarbon in general are relatively recalcitrant (Leahy

and Colwell, 1990; Valenzuela et al., 1997). It is known that mineralization rate

(degradation of the compounds to C02 and H20) of higher-molecular-weight

complex aromatic hydrocarbon, such as resin, and asphalten is much slower than

degradation of lower-molecule-weight aromatic hydrocarbon such as monocydic

aromatic compounds (Leahy and Colwell, 1990). However, previous studies

showed that complex hydrocarbons were rapidly degraded in optimum condition

(Leahy and Colwell, 1990).

One of the important monocyclic aromatic introduced to environment is

benzoate. It is introduced through herbicide application or other industrial

practices (Werwath et al., 1998). It is also encountered as an important

intermediate in metabolic pathway of many aromatic hydrocarbon compounds

(132)

(Harwood and Gibson, 1988; Grund et al., 1990; Powlowski and Shingler, 1994;

Arendorf et al., 1995).

Metabolism of aromatic hydrocarbon compounds as well as other

hydrocarbons in nature depends on the catabolic reaction of microorganisms

(Semple and Cain, 1996). Degradation rate of the compound is affected by its

natural propeny, its concentration, and the microbial community in the

environment (Leahy and Colwell, 1990; Nicholson et al., 1992).

Biodegradation process are versatile and can be utilized at various stages

of treatment (Porcier, 1991). However, introduced microorganisms may

eventually not worked as they are supposed to (Nicholson et al., 1992). Lower

resistance, predation, competition, inhibition

by

other toxic chemicals or much

toxic intermediate, and other microbial contaminants are responsible for

uncompleted biodegradation of organic compounds (Harwood et al., 1990;

Hiepieper et al., 1992; Miethling and Karlson, 1996; Blasco et al., 1997). Since

biotransformation products are not necessary safe, the complete mineralization of

toxic organic substances to C02 and H20 is the most desirable goal (Laine and

Jerrgensen, 1996; Blasco et a/., 1997).

Utilization of microorganisms as bioremediation agents of hydrocarbon

compounds has been plentifully reported (Blasco and Castillo, 1992; Lobos et al.,

1992; Nicholson et al., 1992; Blasco et al., 1997). Furthermore, industrial devoted

to the bioremediation of toxic organic pollutants are growing rapidly (Wyndham

et al., 1994). Biotechnological approach in biodegradation process of complex

hydrocarbons was established by choosing proper microorganisms or engineering

(133)

essential to assess risks at contaminated sites, implement biological treatment

processes, or design effective bioremediation strategies (Nicholson et al., 1992).

Selection and characterization of new prospecting aromatic-utilizing

microorganisms are still in need, while many workers are trying to optimize the

utilization of the available strains. For the former groups, many restricted their

works only on the ability of the bacteria to utilize the aromatic compounds. A

knowledge of genetic, physiological, and ecological characterization in the

screening and selection of bacteria are crucial in term of providing comprehensive

information of the strains in order to determine and to establish proper

technology for bioremediation (Leahy and Colwell, 1990; Nicholson etal., 1992;

Wyndham et al., 1994).

Aerobic Degradation of Monocyclic Aromatic Hydrocarbon Compounds

Study on biodegradation of aromatic hydrocarbon compounds has

primarily conducted for aerobic microorganisms (Genthner etal., 1989). Of these

microorganisms, bacteria like Achmonas, Acinetobacter, Akaligenes, Ahobacter,

3acillus, P d m w , and B u r k h o k spp. (ShLaao

etd,

1989; Leahy and Colwell,

1990; Shen and Wang, 1995; Kuo and Genthner, 1996; Blasco et al., 1997), and

the fungi (Bugos et al., 1988; Bumpus, 1989; Spadaro et al., 1992; Gemble et al.,

1996) are degradation organisms that have been reported most successful in

catabolizing the aromatic compounds. Several other microbes (Grund etal., 1990;

Lenke and Knackmuss, 1992; Lenke etal., 1992; Lobos etal., 1992; Schmidt etal.,

1992; Behki et al., 1993; Allen et al., 1997; Miehling andKarlson, 1996) includmg

algae (Semple and Cain, 1996) have also shown their ability to aerobically

catabolize aromatic hydrocarbons.

(134)

Although a new oxidation pathway has been described via benzoyl-

Coenzyme A and 3 - h y d r o ~ y ~ ~ l - C o e n z ~ m e A in a denitnfylnglkabnzm sp.

(Altenschmidt et al., 1993), the most common routes for aerobic degradation of

these compounds are usually through destabhtion of the aromatic ring to form

an intermediate catechol(1,2 dhydroxybenzene) (Grund et al., 1990; Blasco etal.,

1997). Protocatechuat (3,4 dihydroxybenzoate) and gentisate (2,5

dihydroxybenzoate) as intermediates were also postulated (Crowford, 1976;

Fuenmayor et al., 1998).

The conversion of the monocyclic aromatic to intermediate catechol

involves ring-dioxygenases. However, in some bacteria ring-monooxygenases are

the common enzymes for the conversion (Powlowski and Shingler, 1994;

Williams and Sayers, 1994; Shield etal., 1995). The catechols are substrates for the

second stage of catabolism which is performed by the actions of ring-cleavage

dioxygenases that break one of the carbon-carbon bonds of the ring by addition

of molecular oxygen. This reaction produces an unsaturated aliphatic acid

(William and Sayers, 1994). The ring cleavage usually occurs through ortho

cleavage (intradiol) which produced cis,cis muconic acid (or a derivative) and

through meta cleavage (extradiol) which produces Zhydro~~muconic

semialdehyde (or a derivative) (Williams and Sayers, 1994; Laine and Laine and

Jerrgensen, 1996; Blasco et al., 1997). The enzyme systems resemble each other,

even though many different metabolic pathways have been identified (Williams

and Sayers, 1994; Kudo et al., 1998).

The biochemistry of the two reaction sequences appears to be conserved

(Figure 1) in all eubacteria in which they are found. Therefore, aerobic aromatic

(135)

catabolism consists of a variety of pathways that converge on a common

intermediate (catechols) which are further assidated by a common pathway

(Williams

and Sayers, 1994). The pathway itself has undoubtedly been in existence

for a considerable period of evolutionary time (Williams and Sayer, 1994).

Phenol, benzoate, and their derivatives have generally been a subject of

intensive study on aerobic biodegradation of monocyclic aromatic

(Gurujeyalakshmi and Oriel, 1989; William and Sayer, 1994; Shen and Wang,

1995; Semple and Cain, 1996; Valenzuela et al., 1996). Phenol is found as natural

phenolic compound in plant materials as well as in the effluents of oil refineries,

petrochemical plants, pesticide application, and other industrial processes

(Gurujeyalakshmi and Oriel, 1989; Lenke et al., 1992; Werwath et al., 1998). It is

also found as an important intermediate in the anaerobic degradation of many

complex and simple aromatic compounds (Zhang and Wiegel, 1990). Like

phenol, benzoate and its derivatives are often encountered as intermediate of

complex aromatic hydrocarbon catabolism including biphenyl (Williams and

Sayers, 1994; Arendorf etal., 1995), chlorophenol (Williams and Sayers, 1994),

cinnamate, mandelate, 5-phenylvalerate, 3-phenyl propionate, and benzoylformate

(Harwood and Gibson, 1988). Thus, the factors that influence the rate and extent

of benzoate degradation may also influence biodegradation of other aromatic

compounds (Hopkins et al, 1995; Warikoo et al., 1996). It was also introduced

(136)

c i s , c i s

MUCONATE

ACETYLCoA

+

o r t: h o or IntradLol Pathway. SUCCINULCOA

ACE'I'YL-CoA

[image:136.753.29.700.40.489.2]

+ la c c a ox XxEradiol Pathway. P Y R U V ~

(137)

Anaerobic Catabolism of Monocyclic Aromatic Hydrocarbons

Anaerobic degradation pathway of aromatic hydrocarbon (Figure 2) has

not been fully understood (Coschigano and Young, 1997; Harwood and Gibson,

1988; Pelletier and Harwood, 1998), but the process has been reported (Genthner

et al., 1989; Madsen and Licht, 1992; Nicholson et al., 1992; H o p b et al., 1995).

Madsen and Licht (1992) isolated and characterized an anaerobic

bacterium from municipal sludge. The bacterium, related to Closdum, was able

to dechlorinate chlorophenol. Anaerobic biodegradation of atrazine by the

facultatively anaerobic bacterium M9 1-3 has been studied (Crawford et al., 1998).

The isolate was capable to utilize atrazine as its sole

C

and N source under

aerobic as well as anaerobic conditions (Radosevich et al., 1995). Nicholson et al.

(1992) showed that pentachlorophenol-acclimated methanogenic consortium

dechlorinated pentachlorophenol to ~chlorophenol, although not all routes

produce this intermediate. Kuo and Genthner (1996) isolated a new bacterium,

strain SB, that degrades benzoate only when coculture with an H2 or formate-

(138)

[image:138.511.39.467.40.711.2]

(139)

The effect of electron acceptors and electron donors availability in

anaerobic degradation of aromatic compounds might differ. Mohn and Kennedy

(1992) reported that addition of electron donors such as sucrose and some

potential products of sucrose fermentation, H2, formate, acetate and propionate

in a reactor had negligible effect on dehalogenation of chlorophenol. Added

elemental S, sulfate, nitrate, and ferric ions inhibited dehalogenation. However, Heindriksen et al. (1992) demonstrated that the addition of glucose in a glucose-

amended reactor stimulated dechlorination rate of pentachlorophenol. This might

be due to a higher concentration of the biomass. Hfggblom etal. (1993) showed

that the process depended on the availability of electron acceptor, and on the

position of the chlorine substituent. In anoxic sediments, nitrate, sulfate, or

carbonate may serve as terminal electron acceptors (Kohring et al., 1989;

Haggblom et al., 1993). When sulfate concentration tend to be low, such as in

anaerobic freshwater environment, carbonate reduction to methane serves as the

predominant electron sink. On the other hand, in marine system, sulfate

concentration tend to be high, and sulfate reduction or sulfidogenesis serves as

the major electron accepting process (Haggblom et al., 1993).

The effect of heavy metal ions like C d o ,

Cue,

C r o , or H g o on

biodegradation of 2-chlorophenol, 3-chlorobenzoate, phenol, and benzoate in

anaerobic bacterial consortia has been examinated. Although the effect of the

ions was different in different aromatic compounds, increasing degradation rate

was observed in benzoate with 0.01 ppm C r o , Cd(II) and Cu(II), in phenol

with 0.01 ppm C r o , and in 2-chlorophenol and 3-chlorobenzoate with 1.0 to

2.0 ppm Hg@) after an extended acclimation period (Kuo and Genthner, 1996).

(140)

Although previous works showed anaerobic degradation of many

monocyclic aromatic (Genthner etal., 1989; Madsen and Licht, 1992; Mohn and

Kennedy, 1992; Kuo and Genthner, 1996; Crawford et al., 1998), anaerobic

catabolism of benzoate has gotten more attention. Haggblom et al. (1993),

Hopkin et al. (1995), Kuo and Genthner (1996), and Warikoo et al. (1996)

observed anaerobic benzoate degradation in various bacteria. Harwood and

Gibson (1988), Kamal and Wyndham (1990), Wright and Madigan (1991), Blasco

and Castillo (1992), Gibson and Gibson (1992), Sasikala et al. (1994, and Shoreit

and Shaheb (1994) saw that anoxygenic phototrophic bacteria photo-

anaerobically catabolize benzoate and its derivatives or homologs. Since the

anoxygenic photosynthetic bacteria demonstrate biochemical versatility, they are

relatively easier to study rather than any other obligately anaerobic bacteria. A

complete pathway of the anaerobic degradation of aromatic compound has been

postulated from anoxygenic photosynthetic bacteria (Figure 2) (Pelletier and

Harwood, 1998).

Genetic and Biochemistry of Aromatic Hydrocarbon Catabolism

Genetic and biochemical analysis of aerobic degradation has been done

primarily in Psardornonas (Altenschdt et al., 1993; Dunaway-Mario and Bab bin,

1994; Powlowslu and Shingler, 1994; William and Sayers, 1994; Shield etal., 1991;

de Souza et al., 1995; Blasco et al., 1997; Fuenmayor etal., 1998). Degradation of

aromatic compound is encoded in plasmids or chromosome (Harayama et al.,

1991; Jeffrey eta/., 1992; Bremer eta/., 1993). Some transposable elements such as

Tn4651 and Tn4653, the toluene transposons, and Tn4655, the naphthalene

transposon also carry the degradative genes (Wyndham et al., 1994). Shield et al.

(141)

(1995) found that TOM plasmid, a 108 kb degradative plasmid, are responsible

for toluene and phenol catabolism. This plasmid possesses genes coding for

toluene ortho monooxygenase and catechol 2,3-dioxygenase. Large plasmid

collectively called the TOL plasmids carrying xyl gene for toluene/xylene has

been a subject of intensive study (Assinder and Williams, 1990). Several other

degradative genes have also been identified. These include bph, dmp, nab and tod

(Williams and Sayers, 1994), gtd (Werwath et al., 1998),

ben

(Jeffrey et al., 1992), and

nag (Fuenmayor et a!., 1998).

Several study on homology of the degradative genes has been carried out.

Kim et al. (1996) has conducted homology study of degradative genes in

Sphingomonas. Harayama et al. (1991) observed that

xylXYZ

of Pdmonusputzdz

and benABC of Acimbacter calcoaceticus shared a common ancestry. Bundy et al.

(1998) saw the similarity between

the

anul BC-encoded anthranilate dioxygenase

and the benABC-encoded benzoate dioxygenase of Acinetobacter sp. strain ADPI.

Substitution of antC of Acinetobacter mutants by benC during growing in

anthranilate suggesting relatively broad substrate specificity of the BenC

reductase. In contrast, the bem4 B genes did not substitute for anulB (Bundy et al.,

1998) indicating a narrow substrate specificity (Hara~arna et al., 1991; Bundy et al.,

1998). The genes responsible in conversion of naphthalene to gentisate, nag, from

Pseudomonas sp. strain

U2

isolated from oil-contaminated soil have been

sequenced. Sequence comparisons suggested that the novel genes represented the

archetype for naphthalene strains which use the gentisate pathway rather than the

(142)

Comparative study on enzyme responsible for degradation of aromatic

compound were conducted by Dong et al., (1992) and Neidle et al. (1991).

Catechol2,3 dioxygenase of

B.

rtearothennqphilus was functionally the same as the

enzyme encoded by xylE in

P.

putida, although their thermostability and

homology between the two genes were rather

different

(Dong etal., 1992). Neide

et al. (1991) demonstrated that the comparison of the deduced amino acid

sequences of BenABC of A. calcoaceticus with relative sequences including those

for the multicomponent toluate, toluene, benzene, and naphthalene 1,2

dioxygenase indicated that the similar size of the hydroxylase component sub-

units were derived from a common ancestor.

Study on genetic of anaerobic catabolism of aromatic compounds was

almost limited on anoxygenic photosynthetic bacteria. Anaerobic catabolism of

benzoate by anoxygenic photosynthetic bacteria involves

bad

genes. For the ring-

cleavage of benzoate,

badl

that codes for Bad, a 2-ketocyclohexanecarboxyl

Coenzyme-A hydrolase, are seemingly responsible (Palletier and Harwood, 1998).

Biochemical analysis of the anaerobic monocyclic aromatic hydrocarbon

catabolism showed the possible pathways (Pelletier and Harwood, 1998) with

cyclohex-1,s-diene-1 carboxyl-CoA and 3-hydro~~pimelil Co-A as a common

intermediate before separating to their specific pathway and entering TCA cycle,

respectively.

Cloning of degradative genes has been reported. Kim and Oriel (1995)

successfully clonedpheA andpheB from B. stearothemzophilus BR.219 to Eschaachiu

coli. The genes are coding for the conversion phenol to catechol and catechol to

2-hydroxymuconic semialdehyde, respectively. Cloning and mapping of phenol

(143)

degradative genes for me& pathway from B. stearothermophilus

FDTP-3

to E. coli

was also carried out by Dong et al. (1992). Springael et al. (1994) reported a

transfer of degradative genes into heavy metal resistant Alraligenes eutrophus strains.

Goyal and Zylstra (1996) cloned degradative genes that distinct from the classical

gene nah from Cornamonus testosteroni GZ39, capable of degradation of polycy&c

aromatic hydrocarbon. Cloning and parrial sequence of atrazine degradative gene

from Pseudomonas sp. strain ADP have been conducted (de Souza et al., 1995).

They observed that the gene was wide spread in nature and contribute to the

formation of hydroxyztrazine in soil.

Only limited studies on degradative genes of anaerobic degradation of

aromatic compounds has been conducted. Coschigano and Young (1997) carried

out cloning and sequencing of tut genes which involved in anaerobic toluene

degradation pathway of a denitrdying bacterium.

The General Objectives of The Research

The objective of t h s study is to select and characterize bacterial isolates

capable of utilizing monocyclic aromatic hydrocarbon as their C-source. The

study of examination of anaerobic degradation of aromatic compounds was

restricted only on anoxygenic photosynthetic bacteria. To achieve this aim, we

utilized a number molecular techniques including analysis of 16s-rRNA genes of

selected isolates, Macro Restricted Fragment Length Polymorphism (MFLP) for

DNA ~rofiling analysis and spectral analysis for the anoxygenic photosynthetic

bacteria, transposition and mutation by transconjugation, and Southern

hybridization. Bacterial idendication for selected aerobic bacteria was performed

using Microbact kit (Medvet Science PTY Ltd., Adelaide, Australia). Physiological

(144)

properties were determined by growing the isolates in different monocyclic

aromatic compounds, different concentrations of benzoate and NaC1, and

different initial pH of the medium as well as non-aromatic C-sources. Microscope

observation was employed to examine morphological properties includmg cell

(145)

CHAPTER 2.

MATERIAL AND METHODS

Isolation and Screening of Benzoate-utilizing Bacteria

Screening of anoxygenic photosynthetic bacteria (APB) was carried out by

growing the isolates of Laboratory of Microbiology and Biochemistry, Research

Center for Biotechnology, Bogor Agricultural University and of environment

samples from Central Kalimantan, West Kalimantan, West Java, Molucca and

Yogyakarta

(DIY)

in Sistrom modified medium in 10 ml completely filled screw

cap culture tubes. The medium contains 5.44 g K.iPo4,0.39 g WCl, 1 g NaCl,

0.6 g MgS04.7H20, 0.0884 g CaCl2.2H20, 0.004 g FeS04.7H20, 40pl

N H -

molybdate 1%,0.2 ml trace element

O

solution (0.1765 mgA EDTA, 0.1540

mg/l MnSO4.2H20,0.5 mg/l ZnSO4.7H20,0.0392 mg/l CuSO4.5H20,0.0248

mg/l Co(N03)2.6H20, and 0.01 1 mg/ml H3BO3) and 0.2 ml vitamins (1 pg/ml

nicotinic acid, 0.5 pg/ml thiamin, and 0.01 pg/ml biotin) in 2000 ml, with 1 m M

Na-benzoate, 0.1 mM phenol, 5 mM Na-salysilate (2-hydroxibenzoate), and 0.5 mM

catechol as C-source.

Screening of aerobic bacteria was done by growing the isolate of the

environment from Central Kalimantan and West Java in modified salt medtum. The

medium contains 0.5 g K2HPO4,5 g NaC1,l g NH4C1,l g MgS04.7H20,20 pl

NH4-molybdate l0/o, 0.2 ml

TE

solution, and 0.05 ml vitamins in 1000 ml, with 5

(146)

Aromatic Hydrocarbon Utilization Test

In order to obtain single colony, one oose of selected bacterial solution

was stricken into modified salt medium agar or modified Sistrom agar with 5 mM

benzoate as C source and incubated in appropriate growth condition. The ability

of aerobic isolates to grow in 5 mM Nagentisate, 1 mM phenol, 5 mM Na-

sal~silate, and 1 mM atrazine (2-chloro-4-ethylamino-6-is~pro~ilamino-l,3,5-

triazine (9040 purity) was examined by striking single colony in modified salt

medium solidified with 1.5% agar with tested aromatic compounds. Similar test

was done for APB with 5 rnM gentisate. For 0.1 mM phenol, 5 mM Na-salysilate

(2-hydroxibenzoate), and 0.5 m M catechol as C-source, liquid medium was used.

Benzoate and Other C-Source Utilization Test

Three isolates of anoxygenic photosynthetic bacteria, DS-1, DS-4 and

Cas-13, and one aerobic bacteria, DS-8, were chosen for further study. The two

formers of APB strains were isolated from Java, and the last was isolated from

Molucca. DS-8 was isolated from sewage in Bogor.

Benzoate utilization of DS-8 was determined by growing the isolate in a

modified salt medium supplemented with or without vitamins with 5 m M Na-

benzoate as a carbon source. Escherichia coli TOP10 was used as a control. For

testing DS-1, DS-4, and Cas-13 growth on aromatic compounds and the ability to

degrade aromatic compounds, the isolates were grown in modified Sistrom

by

omitting a l l carbon sources including nitrilo-triacetic acid, with 5 mM benzoate as

C-source supplemented with or without vitamins in 100 ml completely filled

(147)

supplemented with vitamins with 5 mM succinate and 5 mM acetate as their

carbon sources.

To examine degrading ability of three isolates of anoxygenic

photosynthetic bacteria, DS-1, DS-4 and Cas-13, and one aerobic bacteria, DS-8,

in different conditions, the isolates were grown either in modified salt or modified

Sistrom medium with different NaCl concentrations (0.5,1,1.5,2,2.5, and 3O/0),

benzoate concentrations (2.5,5,7.5,10 mM) and 5 mM benzoate without vitamin

supplementation, and at different initial pH (4,4.5,5,5.5,6,6.5,7,7.5,8,8.5, and

9).

Growth in other C sources was performed in modified salt medium

supplemented with vitamins with either 5 mM succinate, 5 m M glucose, 190

cassamino acid, 5 mM citrate, 5 mM glutarnate, 5 mM acetate, 3% ethanol, or 1%

potato starch as carbon sources for DS-8, and in modified Sistrom with either 5

mM mannitol, 5 mM glucose, 5 mM glutarnate, 3% cassamino acid, 5 mM Na-

tartrate, 1% dextrin, 3% ethanol, 3% glycerol added with 1°/o CaCO3, and 5 mM Na-

citrate as sole carbon sources for the APB.

Growth Condition, Measurement of Growth, and Quantitation of Benzoate Utilization

Test of utilization of benzoate by aerobic bacteria was carried out in 250

ml flask filled with 50

ml

salt medium with 5 mM benzoate. The flask were

shaken with 200 rpm in temperature of 30°C. Anaerobic test of the isolate was

done in modified salt agar medium supplemented with vitamins with 5 mM

benzoate as carbon source in anaerobic jar with BBL GasPak Plus (Becton

(148)

Similar test for APB was done in 1M)

ml

completely filled tube with Sistrom

medium with 5 mM benzoate. All cultures of APB were illuminated with 40 W of

tungsten bulb in a distance of 30 cm. Growth were turbidimetrically measured

every 24 hours but 12 hours for 5 mM succinate and 5 mM acetate at 660 nm.

The ability to grow in benzoate with different condition was measured at

120 hours of incubation time. Utilization of other C-sources was measured at 72

hours of inoculation.

For all inoculation, the seed cultures were taken from 2-days old culture of

aerobic bacteria or 3days old culture of APB of 5 mM Na-benzoate medium.

Other C-source inoculations were taken from 2 days old culture. The cultures

were grown with the initial cell concentration of approximately 5xlV cell/ml.

All

aromatic compounds, but phenol were filter-sterilized.

Cell density was measured as absorbance at 660 n m (Harwood and

Gibson, 1988; Kamal and Wyndham, 1990) using Novaspec 11 (Pharmacia,

Uppsala, Sweden) spectrophotometer. Benzoate concentration was measured at

its absorption maximum of 276 nm using Hitachi Model U-2010 UV/Vis

spenrophotometer Wtachi Intnunent, Inc. Japan) following the establtshrnent of

standard curve relating benzoate concentration to W absorbance (Shoreit and

Shaheb, 1994).

Unless it mentioned otherwise, all media was adjusted to pH 7.2.

Examination of Cell Morphology and Physiological Properties

Cell shape, motility, and Gram staining were evaluated using a Nikon YS2-

T

microscope. Physiological characteristics of DS-8 were analyzed using

Microbact

kit

test wedvet Science Pty. Ltd., Adelaide, Australia). Test of

(149)

production of extracellular protease and chitinase were monitored on salt medtum

agar with 2% colloidal chitin and Luria Bertani (LB) agar with 2% skim milk. The

salt medium contains 0.1 g K m 0 4 , 0.1 g NaC1, 0.7 g W4)2S04, 0.01 g

MgS04.7H20, and 0.05 g yeast extract in 100

d.

Eosin methylene blue

(EMB)

agar was used for preliminary screening for Enterobacteriaceae isolates. The appearance of the colonies were observed in LB

agar supplemented with either 50 p g / d ampicillin, 50 pg/ml spectinomicin and

streptomicin, 10 pg/ml trimethoprim, 10 p g / d tetracyclin, 20 p g / d

gentamicin, 5 rnM benzoate, 5 mM salysilate, 5 mM gentisate, or 5 mM phenol.

The ability to degrade catechol was conducted by spraying 0.03% catechol on 1-

day old colony on LB agar. Test of growth in different temperature was

monitored on LB agar.

Total Genomic DNA Preparation

Modified phenol-chloroform-isoamyIalcoho1 treatment and ethanol

precipitation were used to extract the genomic DNA. A 5-ml overnight culture

was centrifuged at 5000 rpm for 5 minutes, washed with 1 ml0.85% NaC1, and

resuspended with 500 pl IxTE. Solution was added with 100 pl lisoz~me (50

mg/ml) and incubated at 37°C for 1 h. Freeze-thaw was carried out 2-3 times.

Incubating by shakmg for 1 hour at 65°C was carried out following an addition of

100 ~ 1 1 0 % SDS into the solution. A 10 p1 Proteinase-K (10mg/ml) was added

into the microtube followed by incubation for 1 hour at 37°C. After treated with

100 pl NaCl and 100 p1 pre-heated CTAB/NaCl (65"C), the solution was

incubated at 65°C for 20 minuts. A 0.5 ml phenol: chloroform: isoamylalcohol

(150)

(25:24:1) was added and mixed gently by inverting the tube. The tube was

centrhged for 10 minutes at room temperature. Aqueous phase was transferred

by pipetting and precipitated with 0.6 volume of cold isopropanol at room

temperature for 30 minutes. The aqueous phase was discarded following a

centrifugation of the tube for 10 minutes. Pellet was washed with 70% ethanol,

and discard the ethanol. Air dry the pellet to remove residual ethanol. Add 25 pl

nuclease-free water or TE

M

buffer. If desired, the samples may be stored at

-

20°C (Sambrook et

al.,

1989).

Total Genomic Analysis

Gel inserts for total genomic analysis have been prepared as Smith and

Cantor (1987). DNA restriction used method as describe previously (Suwanto

and Kaplan, 1989). Pulsed-field gel electrophoresis to obtain Macro Restricted

Fragment Length Polymorphism profiles was utilized for DNA separation using

CHEF-DR@

II

(Bio-Rad, Richmond, CA).

Spectral Analysis

Cell of DS-1, DS-4, Cas-13, and

Rb.

sphaemrdes

2.4.1 were harvested from 7

days old anaerobphototrophic culture of modified Sistrom supplemented with

vitamins with 5 rnM succinate as sole carbon source and suspended in ICM buffer

(10 rnM phosphate buffer pH 7.0 and 1 rnM Na-EDTA pH 7.0). Sonication were

carried out using Soniprep 150 (MSE, UK) at amplitude of 2 1 for 2 minutes three

time with time interval of 1 minutes. Cell extractions were

centrifuged

at 3.000 rpm

(151)

protein concentration off 100 pg/ml. Protein concentration were determined

by

Pierce BCA* Protein Assay

Kit

(Rockford,

El,

USA).

Amplification and Sequencing of Part of 16s rRNA Genes

The 16s-rRNA genes were PCR-amplified using specific primers of 63f

and 1387r from genomic DNA (200 ng) using Ready-To-GO PCR Beads

(Pharmacia-Biotech, Uppsala, Sweden). These primers were successfully to work

with a broad range environmental samples (Marchesi et al., 1998). Phenol-

chloroform-isoamylalcohol(25:24:1) treatment, ethanol precipitation, and agarose

gel electrophoresis were used to purdy the genomic DNA. Total volume of PCR

reaction (25 pl) consisted of 1.5 U Taq DNA Polymerase, lOmM Tris-HC1 (pH 9

at room temperature), 50 mM KC1,l.S mM MgC12,200 ph4 of each dNTl?s and

stabilizer including BSA. The reaction was incubated in a Gene Amp PCR System

2,400 Thermocycler perkin-Elmer Cetus, Norwalk, Conn).

Part of 16s-rRNA gene was sequenced to infer the closest related

organism from Ribosomal Database Project (RDP) maintained in the University of

Illinois, Urbana-Champaign. The sequencing reactions were done by using the Big

Dye Ready Reaction Dye Deoxy Terminator kit, purify with ethanol-sodium

acetate ~recipitation. The reactions were run on an ABI PRISM 377 DNA

Sequencer (PE Applied Biosystems, Foster City, CA.).

Construction of Phylogenic Tree

Cluster analysis of 16s-rRNA gene was done by computer program from

European Bioinformatics Institute (http://www.ebi.ac.uk). Treecon computer

program (Yves Van de Peer of Department of Biochemistry, University of

(152)

their nucleotide sequences and MFLP profiles obtained from pulsed-field gel

electrophoresis.

Strains and Plasmids

Escherichia coli S17-1 was used to promote a transfer of plasmid pJFF350

(Omegon-Km) to DS-8. E. coli JM109 (pTnMod-OGm) and E. coli HBlOl

(pRK2013) were used for triparental mating of DS-4. E. coli DH5a used to

propagate and transfer DNA, was d o n n e d by the calcium chloride technique

[image:152.540.45.473.27.774.2]

(Sambrook et al., 1989). Bacterial strain and plasmids are listed in Table 1.

Table 1. Bacterial strains and plasmids used in t h study.

Di-

and Triparental Matings.

S17-1 (pJFF350) and DS-8 were grown in LB-kanamycin and LB-

ampicillin overnight, respectively. A 1-rnl sample of DS-8 was mixed with a 200-

pl sample of S17-1 (pJFF350) and centrduged for 5000 rpm for 5 minutes. Pellet

was washed once with 1 ml of 0.85% NaCl solution, resuspended with 40 pl LB Bacterial strains

or plasmid Strains El. coli JM109

E. coli S17-1

E. coli DHSa E. coli HBlOl DS-4 DS8 Plasmids pRK2013 pJFF350 PTnMod-Ogm

broth, and spotted into a microtube containing 500 p1

LB

agar. After 1-day

incubation at 30°C, culture were resuspended with 400 p l of 0.85% NaCl solution Relevant genotype/phenotype

recA 1 endA 1 gytA% thi-1 hsa'Rl7 (m- m ~ + ) supE44 & 1 k A(k-pAB) {F' traD36pmAB+ k I q ZAMlS)

r e d thi pro hdrR4 (K m ~ + ) (R.4-2Tc-Mu-Km-Tn7) Tpr Smr

supE44 AlacU169 (080 kZAM15) h d 1 7 r d l endA 1 gyrA96 thi-1 relA 1 supE44 hsdS20 (m-m~-) red13 ara-14poA2 lacYl galK2 rpsL20 xyl-5 mtl-1 wildtype Kmr Sp/Smr

wildtype Amp'

Col-El replicon Kmr Mob+ Tra+ helper plasmid ~ m r

pMBl replicon Tn5 tnp RP4 oriT Gmr

(153)

incubation, single transconjugant colonies were isolated on the same medium.

Negative selection were done on modified salt medium unsupplemented with

vitamins with 5 mM benzoate as its C source.

Similar technique were used for tripvental mating in which E. coli JM109

(pTnMod-OGm), E. coli HBlOl @RK2013), and DS-4 were mixed together, but

LB agar containing gentamicin, and streptomicin and spectinomicin was used

instead of LB-kanamycin and LB-ampicillin.

Transformation of Flanking DNA.

Since no mutant was obtained from D M , the study was restricted only on

DS-8 mutants.

Suspected colony of knocked down genes by transposition were grown in

LB kanamycin and ampicillin broth overnight in 30°C at 200 rpm. Modified

phenolchloroform-isoamyIalcoho1 treatment, and ethanol precipitation were used

to extract the genomic DNA as described previously. The DNA were digested

with Kpnl'and transformed to DH5a using method as described by Sambrook et

al. (1989).

A 1-ml overnight culture of DH5a was subcultured in LB broth for 3 h.

The culture was harvested by centrdugation at 5000 rpm for 2 minutes at 4.C.

The supernatant was discharge. Pellet was resuspended in 200 ml of ice-cold 50

mM CaC12

+

50 mM Tris and incubated on ice for 20 minutes; The cells were

pelleted by centrdugation at 5000 rpm for 2 minutes at 40C. The supernatant was

discharged. Pellet was resuspended in 250 ml of ice-cold 0.1 M CaC12 and

reincubated on ice for 10 minutes. KpnI-digested DNA was put into the

(154)

seconds. The tubes was rapidly placed on ice to cool for 60 minutes. The cells

were transferred into 2 rnl of SOC broth. The culture was incubated for 45-60

minutes at 370C to allow the cell to recover. A 50-100

ml

of the transformation

mix were plated onto LB-kanamycin agar and incubated overnight.

Plasmid Preparation

In general, DNA plasmid minipreparation was done with Quantum

Prep" Plasmid Miniprep Kit (Bio-Rad, Hercules, CA). The preparation was

done as specified by the manufacturer.

Southern Hybridization

Total bacterial DNA was extracted as previously described (Sambrook et

al. 1989). After digested with KpnI, DNA were fractionated on 1.5O/0 agarose gel

in Ix TAE buffer. The gel was stained with EtBr and photographed under UV

illumination. DNA was denaturated by soaking the gel into denaturation solution

(1.5 N NaCl and 0.5 N NaOH) for 30 minutes at room temperature with

constant, gentle agitation and then rinsed briefly in deionized water.

Neutralization was done by soaking the gel for 15 minutes 2 times into the

neutralization solution p H 7.5 (1 M Tris and 1.5 N NaCl) at room temperature

with constant, gentle agitation.

DNA was transferred in 20x SSC to a nylon Zeta-Probe (Bio-Rad

Laboratories, CA) following NEBlot Phototope Kit protocol (New England

Biolabs, Inc. MA).

Hybridization of biotynilation labeled probes using to the blot was

performed as described in Phototope Detection Kit protocol. Random

biotynilated octamers were used to prime DNA synthesis in vitro from

(155)

denaturated double-stranded template DNA as described by NEBlot Phototope

Probe Labelling protocol.

Amplification Using

benA

Primer

Primers for amplification of suspected benA gene from

S.

marcescens DS-8

were designed based on available sequences of benA of Alcaligenes calcoaceticus and

Pseudornonasputzda obtained from GenBank database. Technique for ampldication

was described previously.

Genomic DNA (200 ng) of

S.

marcescens DS-8 were PCR-amplified using

b e d primers forward (5'-GTGCACTGGAACTA) and reverse (5'-

TCCTTCGTCTTC) using Ready-To-GO PCR Beads (Pharmacia-Biotech)

.

Agarose gel electrophoresis were used to purrfy the genomic DNA. Total volume

of PCR reaction (25 pl) consisted of 1.5 U

Taq

DNA Polymerase, 10mM Tris-

HCl (pH 9 at room temperature), 50 mM KCI, 1.5 rnM MgCL, 200 pM of each

dNTPs and stabilizer including BSA. The reaction was incubated in a Gene Amp

(156)

CHAPTER

3.

SELECTION AND ISOLATION OF BACTERIA

FOR

BENZOATE DEGRADATION

ABSTRACT

Thuzy-four anoxygenic photosynthetic bacteria and 7 other environmental isolates from West Java and DXY were examined for their ability to grow anaerobically in light in monocydic aromatic compound, including benzoate, salysilate (2-hydro~~benzoate), phenol, gentisate (2,5-dihydroxybenzoate), and catechol (1,2-benzenediol). Five aerobic bacteria were tested for aerobic utilization of the monocyclic aromatic compounds. Twenty-seven out of 41 of isolated anoxygenic photosynthetic bacteria (65.8%) were able to grow in 5 rnM

benzoate. DS-1, DS-4, and Cas-13 of the purple non-sulfur bacteria and DS-8, MR1.2, and PG9.1 of the aerobic bacteria showed relatively short generation time. The ability of purple non-sulfur bacteria to grow in 5 rnM gentisate was firstly reported. MR1.2 was the only Gram positive isolate of the aerobic bacteria capable of growing in 1 mM atrazine (2chloro-4-ethylamino-6-isopropylamino-

1,3,5-triazine).

(157)

INTRODUCTION

Various bacteria show their ability to aerobically catabolize aromatic

hydrocarbon compounds (Shimao et al., 1989; Lenke et al., 1992; Miethling and

Karlson, 1996; Valenzuela et al., 1997), however Pseudomoms and Bacillus are the

most common bacteria that have gotten much attention for intensive study in

their aromatic degradation ability (Brenner et al., 1993; Shen and Wang 1995;

Molina et a/., 1998) and genetic properties (Orser and Lange, 1994; Powlowski

and Shingler, 1994; Williams and Sayers, 1994; Shields et al., 1995).

Unlike aerobic degradation of aromatic compounds, relatively less

information in anaerobic degradation of those compounds are available

(Genthner, 1989). However, previous works showed excellent ability of various

bacteria to anaerobically degrade the compounds. Anaerobic degradation of

benzoate and its derivates has mainly been concerned in anoxygenic photosynthetic

bacteria (APB) (Harwood and Gibson, 1988; Kamal and Wyndham, 1990; Wright

and Madigan, 199 1; Shoreit and Shabeb, 1994). The ability to utilize other aromatic

compounds has also been demonstrated. Blasco and Castillo (1992) observed that

Rhodobacter capsulatus E l F l degraded mononitrophenol and dinitrophenol with

acetate as

C

source. Rhodopseudomonaspalus~ was able to utilize various phenolic

compounds, hydroxylated and methoxylated aromatic, aromatic aldehyde, and

hydroaromatic acid (Harwood and Gibson, 1988). Rdp. palustris also showed the

ability to catabolism pyridine and pyrazine (Sasikala etal., 1994). The ability of APB

to grow in monocyclic aromatic both aerobically and anaerobically is one of the

(158)

(Harwood and Gibson, 1988; Kamal and Wyndham, 1990; Wright and Madigan,

1991; Shoreit and Shabeb, 1994).

The aim of this study is to screen and to isolate bacteria capable of uulivng

benzoate as their sole C-source aerobically and phototrophic anaerobically. Other

aromatic compounds includmg phenol, salysilate, and gentisate were also chosen as

substrates for their growth.

RESULTS

AND

DISCUSSION

The result on ability of APB to grow in 5 rnM benzoate as their sole C

source showed that 15 isolates (36.6%) well grew, 12 isolates (29.3%) grew poorly,

and 14 isolates (34.1%) did not grow (Figure 1). Although some isolates grew

poorly, the number of exarninated isolates including environmental isolates that

capable of utilizing benzoate were moderately high (65.896) (Table 1). Thu

showed that the degradation of benzoate within the group were common in APB.

Degradative genes for the anaerobic benzoate utilization might be involved (Pelletier

and Harwood, 1998). Unlike the aerobic degradation of monocyclic aromatic

compound that yields catechol or its derivates as common intermediates before ring

cleavage (Williams and Sayers, 1994), the anaerobic degradation of this compound

produces 2-ketocyclohexane-lcarboxyl-CoA immediately before ring cleavage

(Pelletier and Harwood, 1998). Of the APB,

Rs*,

palustris were the most common

member capable of u h g benzoate as its C source (Harwood and Gibson, 1988;

(159)

(160)

[image:160.772.43.672.85.483.2]

(161)

+ + + + excessive grow + + + well grow

+ + moderately grow

+

less grow not grow Ut untested

? grow very slow, GT not calculated

(162)

[image:162.776.38.597.39.495.2]

0 48 % 144 192 240 Tim (lntus)

Figure 2. Growth of anoxygenic photosynthetic bacteria in modified Sistrom with 5 mM

Selection and Characterization Of Bacterial Isolated For Monocyclic Aromatic Degradation

SELECTION AND CHARACTERIZATION OF BACTERIAL

STATEMENT O F RESEARCH ORIGINALITY

SELECTION AND CHARACTERIZATION O F BACTERIAL ISOLATES FOR MONOCYCLIC AROMATIC DEGRADATION

SELECTION AND CHARACTERIZATION OF BACTERIAL

Submitted to the Bogor Agricultural University in partial fulfillment of the requirements for

GRADUATE PROGRAM

Dr. Antonius Suwanto Dr. Ania Meryandini

BIOGRAPHY

AKNOWLEDGMENTS

TABLE OF CONTENTS

ABSTRACT

CHAPTER 3

FROM ENVIRONMENT

TABLES

CHAPTER 2.

CHAPTER 4.

LIST OF

CHAPTER 1.

CHAPTER 4.

CHAPTER 5.

CHAPTER

IDENTIFICATION OF RESPONSIBLE GENE INVOLVED IN

CHAPTER

Aerobic Degradation of Monocyclic Aromatic Hydrocarbon Compounds

(Williams

MUCONATE

ACETYLCoA

xylXYZ

different

CHAPTER 2.

Growth Condition, Measurement of Growth, and Quantitation of Benzoate Utilization

Examination of Cell Morphology and Physiological Properties

Total Genomic DNA Preparation

Total Genomic Analysis

CHEF-DR@

Spectral Analysis

sphaemrdes

centrifuged

Amplification and Sequencing of Part of 16s rRNA Genes

Construction of Phylogenic Tree

Strains and Plasmids

Transformation of Flanking DNA.

Amplification Using

CHAPTER

INTRODUCTION

1991; Shoreit and Shabeb, 1994).

RESULTS