DEGRADATION OF BRANCHED CHAIN ALIPHATIC AND AROMATIC PETROLEUM HYDROCARBONS BY MICROORGANISMS

I n a u g u r a l d i s s e r t a t i o n

zur

Erlangung des akademischen Grades

doctor rerum naturalium (Dr. rer. nat.)

an der Mathematisch-Naturwissenschaftlichen Fakultät

der

Ernst-Moritz-Arndt-Universität Greifswald

vorgelegt von Le Thi Nhi Cong geboren am 18/02/1980 in Thanhhoa, Vietnam

Greifswald, September 2008

Dekan: Prof. Dr. Klaus Fesser Mathematisch-Naturwissenschaftlichen Fakultät Ernst-Moritz-Arndt-Universität Greifswald 1. Gutachter: Prof. Dr. Frieder Schauer Institut für Mikrobiologie Ernst-Moritz-Arndt-Universität Greifswald 2. Gutachter: Prof. Dr. Ulrich Klinner Biologie Fakultät IV (Mikrobiologie und Genetiks) Universität Aachen

Tag der Promotion: 14 November 2008

Acknowledgements

The work described here was carried out between July 2005 and September 2008, in the Institute for Microbiology, Ernst-Moritz-Arndt-University of Greifswald (EMAU) - Germany in partial fulfilment of the requirements for the Degree of Ph. D. I wish to express my deepest gratitude to my supervisor Prof. Dr. Frieder Schauer for suggesting the topic of this thesis as well as for his professional guidance and constructive criticism. I am also greatly indebted to Dr. Annett Mikolasch, whose worthful advice, excellent suggestions and permanent motivating support were in valuable. Sincere thanks are also due her for her knowledgeable correction of this work and for her encouragement to me in hard times. My sincerest thanks go to Prof. Dr. Robert Jack, Institute for Immunology, for his careful correction in English writing. My thanks also go to Dr. Peter-Hans Klenk, DSMZ - German Collection of Microorganisms and Cell Cultures, for his help in bacterial identification. I wish to express my honest gratitude to Ms. Anne Rheinhard, Ms. Brigite Fricke for providing microorganisms and medium throughout this work and for their kindness. My thanks are also due to Susanne Awe for her help with protocols and her guidance with all experimental techniques as well as her experience in HPLC and GC/MS analyses. For the friendship, helpful comments, professional advice and pleasant working environment, I wish to thank Dr. Rabea Sietmann, Doreen Waldau, Veronica Hahn, Jahn Kabisch and other members of Prof. Schauer´s group. I would like to thank all students working in this group for sharing with me late evening and weekend work. My deepest thanks are also extended to Assoc. Prof. Dr. Lai Thuy Hien, head of Petroleum Microbiology laboratory, Institute of Biotechnology, Vietnam for advice and encouragement to me during the preparation of this work. I gratefully thank Vietnamese Ministry of Education and Training (MOET) and German Academic Exchange Service (DAAD) as well as Overseas Organization Office, Greifswald for financial support. I would like to thank the organizers of the Joint Graduate Education Program (JGEP) for giving me the opportunity to be a Ph. D. student in EMAU Greifswald, Germany. I am grateful to my friends in Germany as well as in Vietnam and other lands for being near me and for encouraging me. I wish to extend my sincere thanks to everybody who helped me and promoted me during this study. Last but not least, I would like to take this opportunity to express my deeply gratitude to my mother and my family for loving me and supporting me endlessly.

CONTENTS

Contents

1 INTRODUCTION ...... 1 1.1 Biodegradation and bio-remediation...... 1 1.2 Degradation of branched chain alkanes...... 2 1.3 Degradation of pristane ...... 3 1.4 Degradation of alkylbenzene ...... 5 2 MATERIAL AND METHODS ...... 8 2.1 Microorganisms ...... 8 2.2 Culture media ...... 8 2.3 Culture conditions ...... 11 2.3.1 Strain storage ...... 11 2.3.2 Determination of biomass...... 11 2.3.3 Culture condition of pristane-degrading bacteria ...... 12 2.3.3.1 Selection and isolation of pristane-degrading bacteria ...... 12 2.3.3.2 Toxicity tests...... 12 2.3.3.3 Growth kinetics and degradation of pristane by bacteria...... 13 2.3.4 Culture conditions of aromatic-degrading microorganisms...... 13 2.3.4.1 Preculture ...... 13 2.3.4.2 Toxicity tests...... 14 2.3.4.3 Biotransformation experiments for aromatic compounds...... 14 2.4 Extraction and derivatization methods ...... 15 2.5 Chemical analyze and measurement procedure...... 16 2.6 Chemicals ...... 18 3 RESULTS...... 20 3.1 Pristane-degrading microorganisms...... 20 3.1.1 Selection of pristane-degrading microorganisms...... 20 3.1.2 Identification ...... 22 3.1.3 Pristane degradation...... 25 3.1.3.1 Kinetics of pristane degradation ...... 25 3.1.3.2 Detection of possible degradation products...... 27 3.2 Transformation of alkyl-substituted aromatics...... 32 3.2.1 Toxicity of aromatic compounds on microorganisms ...... 32

CONTENTS

3.2.2 Screening microorganisms transforming aromatics...... 35 3.2.3 Biotransformation of iso -pentylbenzene...... 37 3.2.3.1 Identification of biotransformation metabolites of iso -pentylbenzene by HPLC and GC/MS ...... 37 3.2.3.1.1 iso -Pentylbenzene biotransformation metabolites by M. neoaurum 109 ...... 37 3.2.3.1.2 iso -Pentylbenzene biotransformation metabolites by N. cyriacigeorgica 1472.....42 3.2.3.1.3 iso -Pentylbenzene biotransformation metabolites by R. ruber SBUG 82...... 44 3.2.3.1.4 iso -Pentylbenzene biotransformation metabolites by T. mucoides SBUG-Y 801..46 3.2.3.2 Kinetics of the iso -pentylbenzene transformation metabolites...... 48 3.2.3.3 Transformation of transformation metabolites ...... 50 3.2.4 Biotransformation of sec -octylbenzene...... 52 3.2.4.1 Biotransformation of sec-octylbenzene by N. cyriacigeorgica SBUG 1472.....52 3.2.4.2 Kinetics of the sec-octylbenzene transformation by N.cyriacigeorgica 1472...57 3.2.4.3 Transformation of N. cyriacigeorgica 1472 with intermediate products ...... 58 3.2.4.3.1 Biotransformation of 3-phenylbutyric acid ...... 58 3.2.4.3.2 Biotransformation of ß methylcinnamic acid...... 60 3.2.4.3.3 Biotransformation of acetophenone ...... 61 3.2.4.4 Biotransformation of sec-octylbenzene by M. neoaurum 109...... 62 3.2.4.5 Kinetics of the sec-octylbenzene transformation by M. neoaurum 109 ...... 64 3.2.4.6 Transformation of M. neoaurum 109 with intermediate products...... 65 3.2.4.6.1 Biotransformation of 3-phenylbutyric acid ...... 65 3.2.4.6.2 Biotransformation of ß-methylcinnamic acid...... 67 3.2.4.6.3 Biotransformation of 2-phenylpropionic acid ...... 67 3.2.4.6.4 Biotransformation of acetophenone ...... 68 3.2.4.6.5 Biotransformation of benzoic acid ...... 68 3.2.4.7 Biotransformation of sec-octylbenzene by R. ruber 82...... 69 3.2.4.8 Kinetics of the sec-octylbenzene transformation by R. ruber 82...... 72 3.2.4.9 Transformation of R. ruber 82 with intermediate products ...... 73 3.2.4.9.1 Biotransformation of 3–phenylbutyric acid...... 73 3.2.4.9.2 Biotransformation of ß–methylcinnamic acid...... 75 3.2.4.9.3 Biotransformation of 2-phenylpropionic acid ...... 76 3.2.4.9.4 Biotransformation of acetophenone ...... 77 3.2.4.9.5 Biotransformation of benzoic acid ...... 79 3.2.4.10 Biotransformation of sec-octylbenzene by T. mucoides SBUG-Y 801 ...... 80 4 DISCUSSION ...... 81

CONTENTS

4.1 Pristane degradation ...... 81 4.2 Degradation of aromatic compounds...... 86 4.2.1. Biotransformation of iso-pentylbenzene...... 87 4.2.2. Biotransformation of sec -octylbenzene...... 92 5 REFERENCES ...... 101 6 APPENDIX ...... 109

SUMMARY

Summary

The overall aim of the work was to investigate the ability of several Gram-positive bacteria including Mycocbacterium neoaurum SBUG 109, Nocardia cyriacigeorgica SBUG 1472 and Rhodococcus ruber SBUG 82 and the yeast Trichosporon mucoides SBUG-Y 801 to degrade and transform branched chain hydrocarbons which occur in petroleum and its fraction products such as gasoline or gas oil and which are known as important and recalcitrant environmental pollutants. Pristane, iso -pentylbenzene and sec - octylbenzene were used in this work as model compounds. These compounds represent significant groups of petroleum constituents (branched chain alkanes and aromatic hydrocarbons).

Three bacteria and the yeast T. mucoides SBUG-Y 801 were selected in a screen of 16 hydrocarbon-utilizing strains in the SBUG collection and from 21 isolated hydrocarbon- utilizing strains from oil-contaminated habitats of Saudi Arabian Desert and of Vietnam. The bacteria were identified in cooperation with DSZM (Deutsche Sammlung von Mikroorganismen und Zellkulturen) as M. neoaurum SBUG 109, N. cyriacigeorgica SBUG 1472, R. ruber SBUG 82. These bacterial and yeast strains were shown to possess high potential for degrading and transforming pristane, iso -pentylbenzene and sec - octylbenzene.

The intermediates produced by these bacteria during incubation with pristane were analyzed by GC and GC/MS. The products 4-methyl pentanoic acid; methyl butanedioic acid; 2-methyl pentadioic acid; methyl propanedioic acid; 4-methyl heptanedioic acid and 2,6,10,14–tetramethyl-pentadecan–3–one were detected in M. neoaurum cultures . In R. ruber , methyl butanedioic acid; 2-methyl pentadioic acid; 4,8-dimethylnonanoic acid, 4- methyl heptanedioic acid; 2,6,10–trimethylundecanoic acid; 3,7-dimethyl decanedioic acid and 2,6,10,14–tetramethyl–pentadecan–3-one were identified. In N. cyriacigeorgica , 2- methylpentanedioic acid; 4,8-dimethylnonanedioic acid; 2,6-dimethylheptanedioic acid and pristanic acid were found.

The detection of 11 intermediates during pristane degradation by the three Gram- positive bacteria provided sufficient information to elucidate in detail three degradative pathways of pristane involving mono-, di- and sub-terminal oxidations. The sub-terminal oxidation by M. neoaurum and R. ruber was demonstrated for the first time. This

SUMMARY

occurence of a sub-terminal oxidation in these strains was strengthened by further results of aromatic compounds transformation (see below). During this pathway, ketone mono- oxygenation reactions seem to be involved. Because of this it will be of interest to look more closely at the catalytic processes involved and their possible extension to the bio- degradation of other branched chain hydrocarbons. Since in the present study 59 %, 51 % and 84 % of pristane were degraded in 3 weeks by M. neoaurum , R. ruber and N. cyriacigeorgica , this illustrated that the degradation rates of this isoprenoid alkane were high.

The bacteria we studied were not only effective degraders of multiple branched chain alkane but also useful transformers of aromatic hydrocarbons. The intermediates produced were analyzed by comparing the retention times and UV/Vis spectra of the HPLC elution profile as well as the retention times and mass spectra of the GC/MS with those of available standards. Using iso -pentylbenzene as a substrate, 8 metabolites were generated by M. neoaurum transformation including product A (phenylacetic acid), B (acetophenone), D ( iso -valerophenone), E (succinic acid), F (benzoic acid), G [(2-hydroxy- phenyl)-acetic acid] and H (2-methyl-4-phenyl-butyric acid). We additionally identified an alkyl hydroxylated iso -pentylbenzene derivative as 2-methyl-4-phenyl-butan-2-ol or 2- methyl-4-phenyl-butan-1-ol. Two metabolites (C and D) were detected by N. cyriacigeorgica transformation and three metabolites (A, D and F) were identified by R. ruber transformation which led to the complete biotransformation of this substance. iso - Pentylbenzene transformation by M. neoaurum was initiated by attack on the alkyl side chain followed by ring cleavage. The appearance of iso -valeorophenone confirmed the occurrence of a sub-terminal oxidation mechanism in M. neoaurum and R. ruber .

In addition to products A, C, D and G, the identification X-(3–methyl–butyl)-phenol (X means that position of the hydroxy group on the aromatic ring system, such as 2, 3 or 4 remained unclear) in T. mucoides cultivation demonstrated for the first time the capacity of alkyl side chain attack by this organism which was hitherto known only for its ability of ring cleavage.

The detection of 15 degradation products of sec -octylbenzene (including 2- phenylpropionic acid, 3-phenylbutyric acid, ß-methylcinnamic acid, 5-phenylhexanoic acid, acetophenone, 2-hydroxy-acetophenone, 2,3-dihydroxy-benzoic acid, succinic acid, 7-phenyloctan-2-one, benzoic acid, phenylacetic acid, 7-phenyl-octan-2-ol, hydroxy- phenylacetic acid and 2-hydroxybenzoic acid), in the studied bacteria pointed to an

SUMMARY

effective sec -octylbenzene degradation pathway in which dehydrogenation of 3- phenylbutyric acid to form ß-methylcinnamic acid is a newly described option. The identification of 2-phenylpropionic acid and 3-phenylbutyric acid in sec -octylbenzene transformation experiments by T. mucoides confirmed the possibility of alkyl side chain attack by this yeast. Summarizing the results, we describe for the first time in detail the biotransformation of sec -octylbenzene by M. neoaurum , N. cyriacigeorgica , R. ruber and T. mucoides .

Our results suggest that these microorganisms may be useful as potential strains for hydrocarbon degradation and it may be of interest to investigate their suitability to solve specific environmental pollutant problems associated with branched chain aliphatic and alkyl-branched compounds which contribute to the persistence of hydrocarbon fractions in the environment.

INTRODUCTION 1

1 Introduction

Humanity requires petroleum hydrocarbons for power generation, heating and for the operation of vehicles, aircraft, and ships. As a collateral consequence environmental contamination with petroleum products has occurred due accidental spills and to past disposal practices. Given that petroleum contains numerous compounds of varying structural complexity, there is considerable resistance to biodegradation of the residual mixture. Nevertheless, bio-remediation techniques have shown potential for broad application in the treatment and removal of oil pollutants from aquatic and terrestrial ecosystems. Certain naturally occurring microorganisms have the ability to degrade petroleum and they play an important role in the natural attenuation of petroleum pollutants. The process of bio-remediation, defined as the use of microorganisms (especially bacteria) to detoxify or remove pollutants, is an emerging environment-friendly technology, based on the fact that biological degradation causes minimal ecological side effects (Moran et al. 2000). This technology is an evolving method for the removal and destruction of many environmental pollutants, including those produced by the petroleum industry. There are two approaches used for bio-remediation: the first relies upon the metabolic capacities of the indigenous microbial populations. In the second, exogenous microbial populations, selected for their hydrocarbon-degradation activities, are added (Atlas 1995b).

1.1 Biodegradation and bio-remediation

Biodegradation is the partial breakdown or complete destruction of the molecular structure of an environmental pollutant by physiological reactions catalyzed mainly by microorganisms (Atlas 1995b). Hydrocarbon degraders, typically bacteria, yeasts and filamentous fungi are widely distributed in the environment and their numbers are usually enhanced following hydrocarbon spillage. Many microorganisms are capable of degrading alkanes and converting them to easily metabolizable substances (Wentzel et al. 2007). It is generally considered that the most important hydrocarbon-degrading bacteria in the soil environment are members of the genera Mycobacterium (Solano-Serena et al. 2000), Nocardia (Zeinali et al. 2008), Corynebacterium (McKenna and Kallio 1971), Brevibacterium (Pirnik et al. 1974), and Rhodococcus (Takei et al. 2008) and n-alkane enrichment generally selects for these bacteria. Less frequently occurring genera include Pseudomonas (Schauer 1981; Weide and Schauer 1981), Acinetobacter (Lal and Khanna

INTRODUCTION 2

1996) and Achromobacter (Stephens 1987). In the marine environment Alcanivorax (Engel 2007) and another Rhodococcus (Sharma and Pant 2000) tend to dominate. A better understanding of these organisms might well lead to further industrial applications. Bio-remediation is the intentional use of biodegradation processes to eliminate environmental pollutants from sites where they have been released either intentionally or inadvertently. Bio-remediation technologies use the physiological potential of microorganisms, as assessed most readily in laboratory assays, to eliminate or reduce the concentration of environmental pollutants in field sites (Schauver 1993). Most oil components were reported to be readily degradable. Thus, for example, the biodegradability of benzene, toluene, ethylbenzene and xylene have all been demonstrated using complex microflorae (Mallakin and Ward 1996; Matteau and Ramsay 1997). Some polyalkylated benzene (Lang 1996) and some linear and branched alkanes (Schaeffer et al. 1979) were also shown to be degraded. However, branched alkanes and polyalkylated benzenes are still significant environmental pollutants problems because of their resistance to degradation. Studies on biodegradaion rates of hydrocarbons indicate a hierarchy of ease of biodegradation in which n-alkane > branched alkanes > low molecular weight aromatics > cycloalkanes (Leahy and Colwell 1990). Therefore, branched alkanes and aromatic compounds which contain branched side chain were focused on in this study.

1.2 Degradation of branched chain alkanes

Aliphatic hydrocarbons are suitable substrates for several bacteria and fungi and therefore they can be cleared from hydrocarbon polluted areas in a relatively short period. However, a minor fraction of crude oil, gas oil and petroleum are relatively resistant against microbial attack and these fractions, which contain a high amount of branched chain hydrocarbons, are scarcely degraded by most microorganisms and hence accumulate during bio-remediation. The reason for this phenonmenon may be either that the alkyl branches hinder the uptake of the hydrocarbons into the cell or that the branches are not susceptible to the enzymes of the ß-oxidation pathway (Schaeffer et al. 1979). Nevertheless, a few bacteria are known to be able to oxidize these persistent substrates. Up till now, only a few bacterial species have been described which are able to degrade branched alkanes. Certain alkyl branched compounds are recalcitrant to degradation by most bacteria and thus accumulate in the biosphere (Alexander 1973). Using an isooctane-degrading microflora, Marchal and co-workers isolated a pure strain, identified as Mycobacterium austroafricanum , which had a metabolic pathway allowing

INTRODUCTION 3

for ß-oxidation and which grew on anteiso-methyl alkanes (Marchal et al. 2003). In addition, the Gram-negative bacterium Pseudomonas aeruginosa was reported to transform isoalkanes such as 2-methylhexane, 3-methylhexane and 2,4-dimethylhexane (Schauer 2001) while another P. aeruginosa strain (W51D) was shown to be have citronellol oxidizing capacity and to be able to grow with citronellic and geranic acids as substrate (García and Chávez 2000). A denitrifying bacterium ( Marinobacter sp. Strain CAB) isolated from marine sediments was shown to metabolize 6,10,14-trimethylpentadecan-2- one under aerobic and anaerobic conditions (Rontani et al. 1997). Mycobacterium fortuitum NF4 and Mycobacterium ratisbonense SD4 isolated from a sewage treatment plant were described to be able to utilize the multiply branched hydrocarbon squalane and its analogous unsaturated hydrocarbon squalene as the sole carbon source for growth (Berekaa and Steinbuchel 2000). A Rhodococcus sp. was shown to have the potential to degrade and utilize phytane (2,6,10,14-tetramethylhexadecane), norpristane (2,6,10- trimethylpentadecane) and farnesane (2,6, 0-trimethyldodecane) as sole sources of carbon and energy (Nakajima et al. 1985). In general, branched chain alkanes tend to be less readily degraded than n-alkanes and the study of branched chain alkane biodegradation properties is thus an interesting and necessary work. However, because of the lack of branched chain standard substrates, pristane often serves as a suitable and commercially available substrate.

1.3 Degradation of pristane

Pristane has been detected in bacteria, algae, higher plants, various tissues of fish and mammals, and in sediments, coal, and mineral oil. Pristane has been taken as an internal marker in environmental hydrocarbon analyses since it is viewed as being highly persistent during the degradation of crude oil and petroleum products (Atlas 1981; Bartha and Atlas 1977). However, this assumption is now known to be invalid as research on the bio-remediation of hydrocarbon pollutants in Prince William Sound, has shown that pristane and phytane are degraded at similar rates to straight-chain alkane. Because of this, alternative internal standards, such as hopanes, are required (Atlas 1995a). Some bacterial strains have been isolated that have the ability to utilize pristane as sole carbon and energy source. McKenna and Kallio (1971) investigated the degradation of pristane by a Corynebacterium sp. and, based on the isolation and identification of 4,8,12- trimethyltridecanoic acid and α-methylglutaric acid, proposed a mono- and di-terminal oxidation pathway operating in this bacterial strain. Using a Brevibacterium erythrogenes

INTRODUCTION 4

strain isolated from a seawater sample by selective enrichment culture on crude petroleum, Pirnik et al . (1974) investigated the mechanism and regulation of pristane catabolism in the absence of n-alkanes. The soil bacterium Rhodococcus sp. BPM 1613 (Nakajima and Sato 1983) was found to produce the mono-terminal oxidation metabolites pristanol, pristanic acid, pristyl pristanate, pristyl aldehyde, and other pristane-derived products, which are further biodegraded through ω- or ß- oxidation. A new Mycobacterium sp. strain P101 was isolated from a soil sample and shown to have the capacity to grow on methyl-branched alkanes (pristane, phytane, and squalane) and was suitable to study the role of ß- methylacyl CoA racemase in the degradation of methyl-branched hydrocarbons (Sakai et al. 2004). The degradation pathways of pristane have been established based on analyses of the metabolic intermediates formed from pristane by using Brevibacterium erythrogenes (Pirnik et al. 1974), Nocardia globerula (Alvarez et al. 2001) and several strains such as Corynebacterium , Mycobacterium , and Nocardia (Beguin 2003; McKenna and Kallio 1971; Pirnik et al. 1974) and are illustrated in Figure 1.1. Furthermore, two isolated strains of Rhodococcus TMP2 and T12 were reported to degrade pristane at moderately low temperature (Kunihiro et al. 2005). β - oxidation COOH COOH

COOH

β - oxidation

HOOC COOH HOOC COOH

Figure 1.1 Metabolic pathway of pristane degradation by microorganisms (Beguin 2003; McKenna and Kallio 1971; Pirnik et al. 1974). This pathway serves as a model for the degradation of isoprenoid aliphatic hydrocarbons in bacteria.

In addition to the degradation of pristane by ω- and ß-oxidation, Rontani et al (1986), using a mixed bacterial population, described a third mechanism which starts with an attack at the third carbon atom. However, little is known about the detailed steps of this third mechanism and about the species of bacteria involved. In general, despite the fact that the degradation of pristane by mono- and di-terminal oxidation has been elucidated and

INTRODUCTION 5

reviewed by Pirnik (1977), very little investigation has been done to explain the precise reaction mechanism of the pristane sub-terminal oxidation.

1.4 Degradation of alkylbenzene

Alkylbenzenes are major constituents of oily wastes and pesticides as well as of many synthetic detergents, and the biodegradation of such molecules by sewage organisms is therefore of environmental significance (Amund and Higgins 1985). Although a number of publications are available on the biodegradation of alkylaromatic compounds, most of the studies are related to alkylaromatics with short linear alkyl chains (Amund and Higgins 1985; Baggi et al. 1972; Bains and Boulanger 2008; Bartha and Atlas 1977; Chuchev and Belbruno 2007; Zylstra and Kim 1997). It has been reported that aromatic hydrocarbons with branches and/or condensed ring structures, are more recalcitrant to biodegradation, meaning that fewer microorganisms can degrade those structures and the rates of biodegradation are lower than the rates of the simpler hydrocarbon structures found in petroleum. The greater the complexity of the hydrocarbon structure, for instance the higher the number of methyl branched substituents or condensed aromatic rings, the slower the rates of degradation and the greater the likelihood of accumulating partially-oxidized intermediary metabolites (Atlas 1995b). The degradation of alkylbenzene may proceed via three different routes: oxidation of the aromatic ring followed by ring cleavage without prior attack of the alkyl side chain; initial oxidation of the alkyl side chain; or a combination of both. Aromatic rings generally are hydroxylated to form diols; the rings are primarily oxidized to substituted catechols which are ring-cleavaged via meta -, or ortho - cleavages (Figure 1.2). When the alkyl chain length exceeds C7 the preferred route seems to be by attack on the alkyl chain via ω- and ß-oxidation (Figure 1.3). Generally, the terminal methyl group of a long n-alkyl side chain is initially oxidized to a carboxylic group, which is followed by classical ß-oxidation to form the carboxylic or actetic acid derivative, depending on whether there is an odd or even number of carbons in the alkyl side chain (Awe et al. 2008).

INTRODUCTION 6

ortho -cleavage R O OH 2 R OH R 1 O COOH 2 COOH OH O OH

meta -cleavage R R OH O COOH COOH COOH COOH R O R R OH 2 OH OH

3 2 OH COOH CO 2 OH O CHO R H O O

HCOOH COOH

Figure 1.2 Ring-cleavage of alkylbenzenes via ortho - or meta - cleavage; R, alkyl side chain.

(a ) HOOC ω− / β − R oxidation

ω− / β − oxidation HOOC (b ) R = Alkyl chain with odd (a) or even (b) number of carbon atoms

Figure 1.3 Initial alkyl side chain attack via ω-/ß- oxidation. Depending on the number of carbon atoms (odd or even), benzoic acid or phenyl acetic acid (respectively) is the degradation product.

Pseudomonas sp. NCIB 10643 was reported to catabolise a series of branched side chain benzene structures including iso -propylbenzene, tert -butylbenzene via ring attack rather than side chain attack (Smith and Ratledge 1989). Using linear alkyl benzenes (LAB) as substrates 2-, 3-, and 4-phenylbutyric acids were isolated as major metabolites indicating that initial shortening of alkyl side chain by ω- and ß-oxidation takes place without cleavage of the benzene ring in the degradation of these phenyl position isomers of LAB (Bhatia and Singh 1996). Utilization of 1-phenylalkanes by Nocardia isolates (Sariaslani et al. 1974) and Acinetobacter lwoffi (Amund and Higgins 1985) was shown that 1-phenyl alkanes were degraded by a combination of ω-, ß-, and α-oxidations of the alkyl side chains.

INTRODUCTION 7

Aim of the dissertation

The general aim of the work presented in this thesis is to investigate the microbial degradation of recalcitrant compounds, especially of hydrocarbons with a branched alkane chain and aromatic alkanes with branched side chains. In order to study the processes of biodegradation of these hydrocarbons in the laboratory, pristane, iso -pentylbenzene and sec -octylbenzene were selected to represent branched alkanes and aromatics with branched side chains. Special emphasis was directed towards the degradation or transformation of these substrates by Mycobacterium neoaurum SBUG 109, Rhodococcus ruber SBUG 82, Nocardia cyriacigeorgica SBUG 1472 and Trichosporon mucoides SBUG-Y801 which are all alkane-utilizing microorganisms. These investigations may be important to develop a new approach to deal with specific bioaccumulation problems associated with the alkyl- branched compounds which confer molecular recalcitrance. For these reasons the dissertation is focused on the following main tasks: I. Investigation of the biodegradation of pristane by selected bacteria
Selection of pristane-degrading bacteria.
Analyses of metabolites produced during the incubation of selected bacteria with pristane by GC/MS.
Study of the degradation pathways of pristane based on these analyzed metabolites. II. Studying the biotransformation of aromatic compounds with branched side chain by selected organisms
Screening of aromatic hydrocarbon-transforming microorganisms.
Determining the intermediate products during the biotransformation of iso -pentylbenzene by these organisms by HPLC and GC/MS.
Detecting the intermediate products during the biotransformation of sec - octylbenzene by these organisms by HPLC and GC/MS.
Elucidating the transformation routes of these compounds based on the detected intermediates.

MATERIAL AND METHODS 8

2 Material and methods

2.1 Microorganisms

In this study, 13 n-alkane-utilizing bacteria, 13 yeasts and 11 fungi from the strain collection (Stammsammlung des Fachbereiches Biologie der Ernst-Moritz-Arndt- Universität Greifswald am Institut für Mikrobiologie - SBUG) which were isolated from hydrocarbon-contaminated soils in the north of Germany and oil-contaminated soil samples collected in Saudi Arabian Desert and Vietnam (as described in the next part 2.3.3.1) were used (Table 2.1).

Table 2.1 Microorganisms used for screening experiments of hydrocarbon-utilization.

Bacteria Yeasts Filamentous fungi

Gordonia rubropentincta SBUG 105 Candida maltosa SBUG-Y 700 B1 Gordonia terrae SBUG 252 Trichosporon mucoides SBUG-Y 801 D1 Mycobacterium vaccae SBUG 109 Trichosporon asahii SBUG-Y 833 G2 Mycobacterium sp. SBUG 246 Trichosporon domesticum SBUGY 752 H2 Nocardia sp. SBUG 82 B2 L1 Norcadia sp . SBUG 175 D2 HK1 Nocardiform rods SBUG 510 G1 HK2 Rhodococcus erythropolis SBUG 271 H1 HK3 Rhodococcus fascians SBUG 635 HL 10a HK4 Rhodococcus globerulus SBUG 1390 HL 4a HK5 Rhodococcus rhodochrous SBUG 189 L2 HK6 Rhodococcus sp. SBUG 179 L3 Strain Halah 3b SBUG 1472 L4

2.2 Culture media

Malt agar (MAg) for storage and cultivation of yeasts on solid medium.

Agar-agar 18 g Bio malt 25 g Distilled water add to 1000 ml

The pH value was regulated to 6.8 with NaOH 25 % (weight/volume – wt/v) solution.

MATERIAL AND METHODS 9

Nutrient agar II for storage and cultivation of bacteria on solid medium. Peptone from meat 2.5 g Peptone from gelatine 2.5 g Peptone from casein 1.75 g Casein hydrolysate 1.75 g Yeast extract 1.5 g Sodium chloride 5.0 g Agar-agar 20 g

25 g of nutrient agar were suspended in 1 litre distilled water and were brought to the boil to dissolve completely. The solution was sterilised by autoclaving 15 minutes at 121 oC and cooled down to 45 ÷ 50 oC. The pH value was regulated to 7.2 ± 0.2 with NaOH 25 % (wt/v) solution.

Nutrient broth (NB II) medium Peptone from casein 3.5 g Peptone from meat 2.5 g Peptone from gelatine 2.5 g Yeast extract 1.5 g Sodium chloride 5.0 g

15 g of NB II were dissolved in 1 litre distilled water and were sterilized by autoclaving at 121 oC for 15 minutes. The pH value was regulated to 7.2 ± 0.2 with NaOH 25 % (wt/v) solution.

Mineral salts agar For incubation of cells with different kinds of substrates in mineral salts medium (according to Hornei et al. 1972 – see below) 18 g litre -1 agar-agar were added. The pH value was regulated to 6.8 with NaOH 25 % (wt/v) solution. Mineral salts medium – MSM For cultivation and incubation of bacteria and yeasts in liquid culture two different types of mineral salts medium were used - mineral salts medium for bacteria MSM-Ba and mineral salts medium for yeasts MSM-Y.

MATERIAL AND METHODS 10

For bacteria (MSM-Ba) (Schauer, 1981) For yeast (MSM-Y) (Hornei et al. 1972)

NH 4H2PO 4 5.0 g NH 4H2PO 4 5.0 g

K2HPO 4 2.5 g KH 2PO 4 2.5 g

MgSO 4 x 7 H 2O 0.5 g MgSO 4 x 7 H 2O 1 g

NaCl 0.5 g Ca(NO 3)2 x 4 H 2O 0.02 g

K2SO 4 0.46 g FeCl 3 x 6 H 2O 2 mg

CaCl 2 0.07 g H3BO 3 0.5 mg

FeCl 3 x 6 H 2O 2 mg MnSO 4 x 5 H 2O 0.4 mg

H3BO 3 0.5 mg ZnSO 4 x 7 H 2O 0.4 mg

MnSO4 x 5 H 2O 0.4 mg Na 2MoO 4 0.2 mg

ZnSO 4 x 7 H 2O 0.4 mg CoCl 2 0.1 mg

Na 2MoO 4 0.2 mg CuSO 4 x 5 H 2O 0.1 mg

CoCl 2 0.1 mg KJ 0.1 mg

CuSO 4 x 5 H 2O 0.1 mg KJ 0.1 mg pH 6.3 pH 5.4 Vitamin solution 10 ml litre -1

Distilled water was added to 1000 ml. Distilled water was added to 1000 ml.

Vitamin solution (van der Walt and van Kerken 1961) The vitamin solution was sterilized with the sterile filter (Rotrand FP 030/3, 0.2 µm, Schleicher & Schuell) and was used for the yeast mineral salt medium with the concentration of 10 ml litre -1.

p-Aminobenzoic acid 20 mg Pantothenic acid, Ca-salt 40 mg Biotin 0.2 mg Pyridoxinhydrochloride 40 mg Folic acid 0.2 mg Riboflavin 20 mg myo-Inositol 200 mg Thiaminhydrochloride 100 mg Nicotinic acid 40 mg Distilled water added to 1000 ml

MATERIAL AND METHODS 11

Phosphate buffer for cultivation bacteria and yeast in the transformation experiments (Sörensen et al. 2002).

KH 2PO 4 6.806 g per 1 l bidistilled water 99.65 ml

Na 2HPO 4 x 12 H 2O 17.907 g per 1 l bidistilled water 0.35 ml pH 5.5 All media and buffers (except vitamin solution) were autoclaved at 121 oC for 20 minutes.

2.3 Culture conditions

2.3.1 Strain storage

An aliquot of 100 µl of liquid culture or diluted sample was spread on MAg and/or NAg plates using a Drigalski spatula. In other cases a sample from paraffin storage was streaked by means of an inoculation needle. The plates were incubated at 30 °C for 1-3 days. The single strains were stored on NAg or MAg slants below a layer of paraffin at 4 °C.

2.3.2 Determination of biomass

Dry weight measurement To analyze the growth curve of bacteria it was necessary to determine the dry weigh, because the intergraded bacteria had a hydrophobic cell surface (especially with hydrocarbons as substrate) and grow as pellet in the water phase. For determination of biomass concentration as dry weight, the bacteria were precultured on agar plates and then brought as inoculum into shaking flasks. During incubation samples of the culture broth were filtered through membrane filters (NC 45 Schleicher & Schuell, Dassel, 0.45 µm, Ø 24 mm). The filters with the retained biomass were dried at 100 oC for 2 h and then cooled in an exsiccator with Silica-Gel at room temperature and weighted. In control experiments the filters were dried, cooled and weighted before using for filtration. The difference between the first and second weight was used to calculate the dry weight of biomass as g litre -1.

Optical density For biotransformation experiments with yeasts, the cells were suspended in the medium up to homogeneity; therefore the cell concentration could be determined with the

optical density. The optical density was measured at a wavelength of 600 nm (OD 600nm ) in plastic micro-cuvettes with 1 cm deepth in a photospectrometer (Pharmacia LKB

MATERIAL AND METHODS 12

Biochrom, England). If the OD 600nm was more than 0.8, the cell suspension was diluted with buffer or MSM.

2.3.3 Culture condition of pristane-degrading bacteria

2.3.3.1 Selection and isolation of pristane-degrading bacteria Selection 12 hydrocarbon degrading strains from the strain collection of the Department of Biology at the University of Greifswald (SBUG) were incubated on mineral salts agar plates. The agar plates were supplemented with pristane evaporated from a sterile filter sheet in the plate cover as carbon and energy source for 2 - 6 days at 30 oC to allow the cells to grow. The well growing bacteria were selected for further experiments. Isolation In order to isolate additional new strains 1 gram of several petroleum-contaminated soil samples from Saudi Arabia was incubated for enrichment with 10 ml litre -1 pristane as the only source of carbon and energy in 500-ml flasks containing 100 ml of mineral salt medium. After appropriate times (3 to 7 days) of shaking at 30 oC and 180 rpm, 5 ml were transferred to 95 ml of fresh medium and incubated under the same conditions. Identical transfers were performed twice. Afterwards, microorganisms were obtained by plating 0.1 ml of cultures on mineral salt agar medium with the pristane on the filter sheet as described above. 2.3.3.2 Toxicity tests For the degradation and transformation experiments, it was necessary to carry out toxicity tests to identify the concentration of the substrate which inhibits on the growth of microorganisms. The cultivation of cells was performed in 500-ml Erlenmeyer flasks containing 100 ml of NB II medium supplemented with various concentration of the substrates (0.01 %, 0.05 %, 0.1 %, 0.5 % and 1 %, volume/volume – v/v). Cultures were incubated for 48 h at 30 oC with 300 rpm rotary shaking so that the chemicals could reach saturation in the liquid medium. After each 2 h, a whole culture flask was removed to determine the cell growth with the biomass weight. Inoculated flasks without substrates were used as controls. From the measured data the growth kinetics in the presence of the pristane as substrate were estimated to show at which concentration the cells died or were inhibited.

MATERIAL AND METHODS 13

2.3.3.3 Growth kinetics and degradation of pristane by bacteria Cultures were shake-incubated in 500-ml Erlemeyer flasks with 50 ml MSM-Ba medium and 0.5 % (v/v) of pristane at 30 oC and 300 rpm. As additional controls, flasks with autoclaved cells and pristane were used. At each sampling period, the shaking experiment of one whole 500-ml Erlemeyer flask was stopped and the same volume of tetradecane (0.5 %) as internal standard was added then the whole mixture (whole cells and supernatant). The mixture was extracted three times with diethyl ether. Extracts were dried over anhydrous sodium sulfate and concentrated by rotary evaporation. The residues obtained were dissolved in 1 ml hexane and analyzed by gas chromatography (GC). The cell pellet was determined with the dry weight biomass method and the pH value was measured with a pH meter. After GC analyses, the kinetics of degradation and growth were estimated by calculating of the peak area of pristane and the dry weight values of cells.

2.3.4 Culture conditions of aromatic-degrading microorganisms

2.3.4.1 Preculture Bacteria To start the aromatics-degrading experiments, the bacteria were cultivated on MSM-Ba agar plates with n-alkanes as carbon and energy sources. In the agar plates (Petri plates) one filter paper was placed into the cover and sterilised 2h at 170 °C, then the plates were poured with 10 ml sterilized MSM-Ba agar. The filter paper was supplemented with

0.3 ml of n-alkanes (C 10 to C 20 , mostly n-tetradecane) slowly evaporating from the sterile filter sheet as vapours (Kreisel and Schauer 1987) and used as carbon source for 2 to 7 days at 30 °C to allow growth of cells.

In case of using n-alkanes with shorter chain lengths (C 6 to C 9), the inoculated plates were put in a larger glass with evaporating n-alkane (closed by a lid) according to method F 10 of Kreisel and Schauer (1987). The biomass was used for the following experiments. Yeasts For yeasts different compounds were used for precultivation, such as glucose, yeast extract, dodecane or phenol. Experiments were carried out in 500–ml-Erlenmayer flasks. 100 ml of MSM-Y medium were inoculated with pour colonies picked from MAg plates and supplemented with 2 % of yeast extract or 1 % glucose or 1 % dodecane (v/v). After 24 hours of incubation at 300 rpm on a large shaking machine (Infors AG, Bottmingen,

MATERIAL AND METHODS 14

Suisse) at 30 °C, the cells were harvested by centrifugation for 10 minutes at 4 °C and 6000 x g (Sorvall ® RC-53 Refrigerated Superspeed Centrifuge, DuPont Instruments) and washed three times in sterile buffer (phosphate buffer – pH 5.5) to remove the residues of yeast extract or glucose or dodecane. The cell pellet was resuspended in 10 ml buffer to reach a homogenous cell suspension which was used to adjust an optical density at 600 nm -1 -1 (OD 600nm ) of 1. For transformation tests 0.1 ml litre and 0.025 ml litre of the sterile filtered substrates were added. For phenol-preculture, 100 ml of MSM-Y medium in 500–ml-Erlenmayer flasks were inoculated with pure colonies picked from MAg plates and supplemented with 0.05 % phenol, 1 % glucose and 0.4 % yeast extract (v/v) at 300 rpm and 30 °C. After 24 hours, 10 ml of this incubation were brought in 100 ml MSM-Y with 0.05 % phenol and 0.4 % yeast extract (v/v). After 16 hours 0.05 % phenol were added and then, after 2 hours the cells were harvested by centrifugation as described above. 2.3.4.2 Toxicity tests As described in part 2.3.3.2, toxicity tests were carried out with a variety of concentrations of aromatic compounds including 0.005 %, 0.01 %, 0.025 %, 0.05 % and 0.1 % (v/v). Cultures were incubated for 48 h at 30 oC with 300 rpm rotary shaking, and after each 6h, the cultures were estimated by biomass dried weight (bacteria) or OD (yeast) methods. Inoculated flasks without substrates were used as controls. From the obtained data thegrowth kinetics in the presence of the respective (more or less toxic) aromatic substrate was estimated to show at which concentration the cells died or were inhibited. 2.3.4.3 Biotransformation experiments for aromatic compounds Bacteria For bacteria biotransformation experiments the cells were precultured on tetradecane or pristane as described above. Bacterial biomass from agar plates was transferred to 500-ml flasks with 100 ml of sterilized mineral salts medium supplemented with trace elements (see above), and sec -octylbenzene, sec -hexylbenzene or iso - pentylbenzene (0.1 ml and 0.25 ml liter −1 ) were added. For controls we used (i) cells in mineral salts medium without the compound that was to be transformed and (ii) sec - octylbenzene, sec -hexylbenze or iso -pentylbenze in mineral salts medium without cells. After incubation for 3 days at 30°C and 300 rpm on a rotary shaker, formation of metabolites was followed by analyzing 0.1 ml of the culture supernatant by high- performance liquid chromatography (HPLC). For kinetic studies the formation of

MATERIAL AND METHODS 15

metabolites was analyzed by HPLC from time to time during the course of the incubation period. At the end of the experiment, the supernatants of all samples and controls were filtered and extracted as described below (part 2.4) The residues were dried under a nitrogene stream and disolved in methanol or n-hexane. These extraction samples were analyzed by gas chromatography-mass spectrometry (GC/MS) and HPLC. Yeasts To carry out the biotransformation experiments of Candida maltosa SBUG 700, Trichosporon mucoides SBUG 801, Trichosporon domesticum SBUG 830, Trichosporon asahii SBUG 833 with sec -octylbenzene, sec -hexylbenzene and iso -pentylbenzene, and the cells were precultivated with different carbon sources as described above. The transformation experiments were carried out in 500-ml shaking flasks with 100 ml of phosphate buffer and shaked at 300 rpm and 30 oC. Buffer without substrate and without cells were used as controls. After various times, the samples were taken. For the detection and quantification of metabolites in the aqueous supernatant at different intervals during the incubation, 1 ml samples were centrifuged at 4 oC with 6000 x g for 5 minutes to remove cells and then 100 µl of the supernatant were analyzed by HPLC. At the end of the experiment, the centrifuged supernatants of all samples and controls were extracted as described below (part 2.4). The residues were dried under a nitrogen stream and dissolved in methanol or n- hexane. These extraction samples were analyzed by GC/MS and HPLC.

2.4 Extraction and derivatization methods

Extraction To identify the transformation or degradation products by GC/MS, the supernatants were extracted. For experiments with bacteria, the reaction mixture was filtered as described above to obtain the supernatant. For yeast, the incubation mixture was centrifuged at 4 oC with 8000 rpm in 10 minutes. After that, the pH values of the supernatants were brought to pH 9 with 25 % (wt/v) NaOH solution and then the supernatants were extracted three times with an equal amount of ethyl acetate (or diethyl ether). For this purpose the mixture of supernatant and solvent was filled into the funnel flask and shaked for 5 minutes. Then the shaking wasstopped and it was necessary to wait until two clearly separated phases appeared, one is the aqueous phase and the other is the organic phase. After removing th organic phase, the aqueous phase was used for further

MATERIAL AND METHODS 16

extraction and was extracted again in the same manner after acidification to pH 2 with 6 N HCl. The two organic extracts (pH 9 and pH 2) were dried over anhydrous sodium sulfate and concentrated by rotary evaporation. The residues obtained were dissolved in methanol and analyzed by HPLC and GC/MS analyses for detection and characterization of the transformation products.

Derivatisation

The evaporation volatility temperature of the transformation products which contained carboxyl, amino or hydroxyl groups is usually too high; therefore, it was required to derivatize such metabolites to decrease their boiling points for the GC-analyses.

Methylation with diazomethane

The extracts containing hydroxylated or acid products were methylated with diazomethane. For this reason, 2 ml 50 % (wt/v) KOH, 1 ml diethyl ether and 1 ml carbitol [2-(2-ethoxyethoxy) ethanol] were filled in one special closed apparatus (Aldrich, Steinheim, Germany) by glass Pasteur pipettes. The reaction started when 0.5 mg diazald (N-methyl-N-nitroso-p-toluensulfonamid) was added. Another clean glass Pasteur pipette was used to carry the diazomethane gas directly into the extracts to methylate. If the sample become yellow, the methylation reaction was finished. After methylation, the samples were stored at room temperature under a fume hood for 24 h and then samples were analyzed by GC/MS.

2.5 Chemical analyze and measurement procedure

To analyze substrates and identify intermediate metabolites GC/MS and HPLC were used.

Combined gas chromatography/mass spectrometric analyses (GC/MS)

After convesion of metabolites to their methyl derivatives by diazomethane, the products for MS were separated on a GC coupled to a mass spectrometer. The analyses was carried out by the Company of Fisons Instruments (Mainz-Kastell) with the following configuration:

MATERIAL AND METHODS 17

Gas chromatograph GC8000 (Fisons Instruments, Mainz, Germany) Mass spectrometer MD800 (Fisons Instruments) Column BPX5; MS (J&W Scientific, Folsom, USA) 0.21 mm x 30 m Adhesion-preventing film 95 % dimethyl-polysiloxane, 5 % phenylsiloxane Carrier gas Helium, pressure 20 psi Volume injection 1 µl Temperature program 10 min in 60 oC, 60 – 290 oC, 8 o C min -1 10 min in 40 oC, 40 – 290 oC, 8 oC min -1 Ionisation 70 eV Interface temperature 240 0C Source temperature 250 0C The sample was analyzed over a scan range of m/z (mass-to-charge ratio) 35-600 Da (Dalton).

High-performance liquid chromatography (HPLC)

To determine substrates and metabolites in the culture, 1 ml of the culture fluid was filtered or centrifuged to remove biomass. 100 µl of the supernatant were injected into the HPLC. The HPLC was performed on a Hewlett Packard (Bad Homburg, Germany) with the following configuration: Pump HP 1050 series pump system Detector HP 1040 M series II diode array detector Integrator HP HPLC Chem Station Column LiChroCART 125-4 RP-18 end-capped, 5 µl (Merck, Darmstadt) Flow rate 1 ml min -1

-1 An aqueous solution of methanol and 1 g litre H 3PO 4 in bidestilled water was used as the mobile phase with a linear gradient from 30 to 100 % methanol within 14 min at a flow rate of 1 ml min -1.

MATERIAL AND METHODS 18

2.6 Chemicals

Table 2.2 All chemicals used have the highest purity.

Chemicals Purity Sources CAS-Nr. Benzoic acid 99 % Aldrich 65-85-0 Biomalt (1) Decane purum Fluka 124-18-5 Dodecane purum Fluka 112-40-3 Diazald 99 % Aldrich 80-11-5 (N-Methyl-N-nitroso-toluol- sulfonamid) Diethylether 99 % Aldrich 60-29-7 2,3-Diehydroxybenzoic acid 99 % Aldrich 303-38-8 3,4-Dihydroxybenzoic acid > 97 % Fluka 99-50-3 Dimethyl pimelate > 99 % Aldrich 1732-08-7 Dimethyl succinate > 98 % Aldrich 106-65-0 4,8-Dimethylnonanoic acid ND (2) 7540-70-7 Hexadecane > 99 % Merck 544-76-3 Hexane 99.9 % Merck 110-54-3 Heptadecane 99 % Fluka 629-78-7 Heptane 99 % Fluka 142-82-5 4-Hydroxybenzoic acid > 98 % Fluka 99-96-7 2-Hydroxyphenylacetic acid > 99 % Aldrich 614-75-5 3-Hydroxyphenylacetic acid > 99 % Aldrich 621-37-4 2-iso -Butyl-heptanedioic acid ND (2) 937617-70-4 iso -Pentylbenzene > 97 % Fluka 2049-94-7 iso -Valerophenone > 98 % Aldrich 582-62-7 D,L-Malic acid > 99 % Aldrich 6915-15-7 D,L-Mandelic acid > 98 % Fluka 90-64-2 2-Methoxy-benzenpropanoic acid- ND ND 55001-09-7 methylester Methyl-4-hydroxyphenylacetate > 99 % Aldrich 14199-15-6 ß-Methylcinnamic acid Aldrich 1199-20-8

MATERIAL AND METHODS 19

2-Methylglutaric acid 98 % Aldrich 18069-17-5 3-Methylglutaric acid > 98 % Fluka 626-51-7 2-Methylhexane > 97 % Fluka 591-76-4 3-Methylhexane > 97 % Fluka 589-34-4 Methylmalonic acid 99 % Aldrich 516-05-2 2-Methyl-4-phenyl-butyric acid > 97 % Aldrich 1949-41-3 2-Methyl-4-phenyl-butanol 97 % Aldrich 103-05-9 Methylsuccinic acid > 97 % Fluka 498-21-5 4-Methylvaleric acid 99 % Aldrich 646-07-1 Nonane > 98 % Merck 111-84-2 Nutrient agar II (3) 2011-06-30 Nutrient broth (NB II) (4) 2011-03 Octane Rein Sigma-Aldrich 111-65-9 Pentadecane 99 % Fluka 629-62-9 Phenylacetic acid > 99 % Merck 103-82-2 3-Phenylbutyric acid 98 % Aldrich 4593-90-2 2-Phenylpropionic acid > 97 % Aldrich 492-37-5 3-Phenylpropionic acid > 98 % Fluka 501-52-0 Phosphoric acid (crystal) 99 % Merck 7664-38-2 Pristane > 98% Sigma 1921-70-6 Pristanic acid > 98 % Sigma 1189-37-3 sec-Hexylbenzene > 99 % Labortest OHG ND sec-Octylbenzene > 99 % Labortest OHG ND Tetradecane > 99 % Fluka 629-59-4 Tetrahydrogeranic acid ND (2) 5698-27-1 Tridecane 99 % Fluka 629-50-5 Undecane 99 % Fluka 1120-21-4 Vanillic acid > 97 % Aldrich 121-34-6

CAS Nr., Chemical Abstracts Registry Number; 1, Hergestell für Gesund produckte GmbH, Kirn, Germany; 2, Institute of Applied Synthetic Chemistry; 3 Becton, Dickinson & Company, Difco TM ; 4, SIFIN, Institut für Immunpräparate & Nährmedien; ND, not determined

RESULTS 20

3 Results

3.1 Pristane-degrading microorganisms

3.1.1 Selection of pristane-degrading microorganisms

Microorganisms are able to carry out a wide variety of metabolic biotransformations on alkanes which range from linear ( n-alkanes), circular ( cyclo -alkanes) and branched ( iso -alkanes) to aromatic hydrocarbons. The effectivity and suitability of a selected microbial strain to degrade or transform various substrates is determined by

incubating with different hydrocarbon sources such as n-alkanes (from C 6 to C 16 ), ethylcyclodecane, pristane and phenyldecane Table 3.1. All used strains were isolated from hydrocarbon-contaminated soils in the north of Germany, Vietnam or Saudi Arabia by enrichment cultivation.

Table 3.1 Growth of different bacteria on alkanes.

Strain Chain leghth of n-alkane Bacteria C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 a b c Gordonia - - + + + + ++ ++ ++ ++ ++ + - - rubropentincta SBUG 105 Gordonia terrae - - - - - + ++ ++ ++ ++ ++ + - - SBUG 252 Mycobacterium ------++ ++ ++ ++ ++ ++ - - vaccae SBUG 109 Mycobacterium sp. - - - - - + ++ ++ ++ ++ ++ + + - SBUG 246 Nocardia sp. ------++ ++ ++ ++ ++ ++ + + SBUG 82 Nocardiform rods - - - + + + ++ ++ ++ ++ ++ + - - SBUG 510 Nocardia sp . 175 - - - - + + ++ ++ ++ ++ ++ + - - Rhodococcus - - - - - + + ++ ++ ++ ++ + - - erythropolis SBUG 271 Rhodococcus - - - - - + + ++ ++ ++ ++ + + + fascians SBUG 635 Rhodococcus - - - - - + + ++ ++ ++ ++ - - - globerulus SBUG 1390

RESULTS 21

Rhodococcus - - - - - + ++ ++ ++ ++ ++ - - - rhodochrous SBUG 189 Rhodococcus sp. - - - - - + ++ ++ ++ ++ ++ + + - SBUG 179 Strain Halah3b ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + SBUG 1472 Yeasts B2 ------+ + - - - Candida maltosa - - - - - + + + ++ ++ ++ - - + SBUG-Y 700 D2 ------+ + + + + - + G1 ------+ + - - + H1 ------+ + + + - - + HL 10a - - - - + ++ + + ++ ++ ++ - - - HL 4a ------L2 ------+ + + + + - - L3 ------+ + + + - - - L4 ------+ + + + - - + Trichosporon ------+ + ++ ++ ++ - - - mucoides SBUG- Y 801 Trichosporon - - - - + + + + ++ ++ ++ - - - asahii SBUG- Y 833 Trichosporon - - - - - + + + + ++ ++ - - - domesticum SBUG-Y 752 Filamentous fungi B1 - - - - + + + + ++ ++ ++ + + - D1 - - - - + + + + ++ ++ ++ + - + G2 ------+ + + - - H2 - - - - + + + + ++ ++ ++ + + + L1 ------+ + + - - + HK1 ------+ + + - - - HK2 ------+ + + + - - HK3 ------+ + + + - - - HK4 - - - - + + + + ++ ++ ++ + + + HK5 ------+ + ++ ++ ++ + + + HK6 ------+ + - - - -: no growth; +: uncertain growth; + +: growth; +++: good growth

C6 to C 16 : the subscripts mean the number of carbon atoms containing in the n-alkane chain; a: pristane, b: ethylcyclodecane, c: phenyldecane.

RESULTS 22

From 37 hydrocarbon utilizing strains, we could select 3 strongly pristane degrading bacteria including M. vaccae 109, Nocardia sp. 82 and Halah 3b. Specific emphasis was therefore directed to the branched chain alkane - pristane and its degradation pathway by analyzing secreted products and intermediates with GC/MS and to the transformation of aromatic compounds consisting of a benzene ring with a branched side chain such as iso-pentylbenzene and sec -octylbenzene analyzed by HPLC and GC/MS.

3.1.2 Identification

Because of the preliminary on incomplete identification of the three selected bacterial strains showing good growth on pristane, these strains were further investigated in detail to prove their exact and recent taxonomic position. All three bacteria were determined, therefore, by morphological, physiological and chemical properties (described in the Table 3.2) in cooperation with Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). The experiments were completed by 16 S rDNA sequence analyses (see page 23 and 24)

Table 3.2 Physiological and biochemical characteristics of the strains M. vaccae 109, Nocardia 82 and Halah 3b 1472.

Characteristics M.vaccae Nocardia Halah3b SBUG 109 SBUG 82 SBUG 1472 Color of colonies yellow red Pearch-white

Growth on sole carbon source

Glucarate + + + Gluconate + + + L-Valine + + + Acetamide + - + p-Nitrophenyl-ß-D-xyloside + + + Caprate - - + Succinate - - + 3-Hydroxybenzoate - - + 2-Hydroxyvalerate + - - L-Alanine + - - L-Leucine + + - N-Acetyl-D-glucoseamine - - -

RESULTS 23

D-Galactose - - - D-Glusosaminic acid - - - L-Rhamnose - - - D-Ribose - - - D-Sucrose - - - D-Turanose - - - D-Arabitol - - - i-Inositol - - - Citrate - - - 2-Oxoglutarate - - - Pimelate - - - 4-Hydroxybenzoate - - - L-Aspartate - - - L-Proline - - - L-Serine - - - Putrescine - - - Tyroamine - - - Benzoate - - - 4-Aminobutyrate - - - Phenylacetate - - - Quinate - - - p-Nitroph.phosphorylcholine - - - 2-Desoxythymidine-5-p-nitrophenyl - - - –phospate Mycolic acid one, long chain one, long chain one, long chain Fatty acid composition S, U S, U S, U +, positive; -, negative; S, saturated; U, unsaturated.

For determination purpose the morphology of the strains was first analyzed under a light microscope. Polar lipids were extracted and purified using the small-scale integrated procedure of Minnikin (Minnikin et al. 1984) . Dried preparations were dissolved in 200 µl 2-propanol and 1-10 µl amounts were separated by HPLC without further purification and analyzed as described previously (Kroppenstedt 1985) . Polar lipids were extracted, examined by two-dimensional TLC (Stanek and Roberts 1974) and identified using published procedures (Minnikin et al. 1977). Fatty acid methyl-esters and mycolic acid trimethyl-silylesters were prepared and analyzed as previously described (Klatte et al.

RESULTS 24

1994) using the standard Microbial Identification System (MIDI Inc. Delaware) for automated gas chromatographic analyzes (Sasser 1990). Numerical analyses of fatty acids pattern was performed according to Kämpfer and Kroppenstedt (Kämpfer and Kroppenstedt 1996). Physiological characterization of both strains was performed as previously described by Kämpfer (Kämpfer et al. 1990). 16S rRNA genes were amplified with standard PCR primers, sequenced on a Beckman Capillary sequencer and compared with the reference sequences in the ribosmal database projects (Maidak et al. 1996).

Conform with the description of the genus Mycobacterium (M.D. and Keddie 1987), colonies of SBUG 109 were non-motile, non-sporulating, saffon- (RAL 1017) to chrome-yellow (RAL 1007), growing scotochromogenic as rods of 2 µl length and 1 µl diameter. Both, SBUG 109 as well as reference strain DSM 44074 were catalase positive (while only DSM 44074 could grow at 42 °C), but otherwise differed in many of the tested substrates (data not shown). Determination of the length of myolic acids failed, however, α-mycolic acids and mycolic acid wax esters were clearly identified by TLC (data not shown). The pattern of unbranched, saturated and unsaturated fatty acids as well as tuberculostearic acid is diagnostic for Mycobacteria and closely related genera. Qualitative and qualtitative analyses of the fatty acid pattern allowed the identification of SBUG 109 as a member of the species Mycobacterium neoaurum (type strain DSM 44074), although with a higher degree of similarity to M. aurum . 100 % sequence similarity of a 500 base pairs partial 16S rRNA sequence with M. neoaurum and 3 % sequence difference to M. aurum as well as 4.7 % sequence difference to M. vaccae , finally allowed the identification of SBUG 109 as a strain of the species Mycobacterium neoaurum .

In agreement with the description of the genus Rhodococcus (Goodfellow and Lechevalier 1987), colonies of SBUG 82 were matt and winkled, salmon colored (RAL 3024), growing in short, branched hypha which decomposed to rods and cocci in aging cultures. Only minor differences could be observed in its substrate usage, as compared to tested typus and reference strains (Klatte et al. 1994). Mycolic acids had a length of 42-48 C-atoms, as typical for other R. ruber strains. The pattern of unbranched, saturated and unsaturated fatty acids as well as tuberculostearic acid is diagnostic for Rhodococci and closely related genera. Qualitative and qualtitative analyses of the fatty acid pattern allowed the identification of SBUG 82 as a member of the Rhodococci (data not shown), but only the 100 % sequence similarity of a 500 base pairs partial 16S rRNA sequence finally allowed the identification of SBUG 82 as a strain of the species Rhodococcus ruber .

RESULTS 25

Corresponding with the description of the genus Nocardia (Yassin et al. 2001), colonies of the strain Halah 3b SBUG 1472 showed a pearl white aerial mycel (RL 1013) and an ivory colored (RAL 1014) substrate mycel. Only minor differences could be observed in its substrate usage, as compared to the typus strain (data not shown), these differences can be explained by the different natural habitats (environmental habitat vs . clinical isolate). Mycolic acids had a length of 50 – 58 carbon atoms, with a pattern very similar to the one known from N. otitidiscaviarum (DSM 43010), but clearly different from

the C 46 – C 54 pattern known from N. cyriacigeorgica . Different pathways and precursors in the sythesis of mycolic acids in the oil-containing habitats of SBUG 1472 and the clinical environment of DSM 44484 might explain these striking differences between the two closely related strains. The pattern of unbranched, saturated and unsaturated fatty acids as well as tuberculo stearic acid is diagnostic for Nocardia and close relatives, Mycobacteria , Rhodococci and Gordonia . Qualitative and quantitative analyses of the fatty acid pattern allowed the identification of SBUG 472 as a member of the Nocardia (data not shown), but only the 100 % sequence similarity of a 500 base pairs partial 16S rRNA sequence finally allowed the identification of SBUG 1472 as a strain of the species Nocardia cyriacigeorgica .

3.1.3 Pristane degradation

3.1.3.1 Kinetics of pristane degradation In order to estimate the quantitative uptake and decomposition of pristane by M. neoaurum , R. ruber and N. cyriacigeorgica, media from growing culture of bacteria supplemented with either 0.01 % or 0.5 % (v/v) of pristane were extracted with diethyl ether at different times and analyzed by GC method with tetradecane used as an internal standard. When 0.01 % (v/v) pristane was used as a substrate, after 8 hours at 30 0C, pristane degradation of M. neoaurum, R. ruber and N. cyriacigeorgica were recorded at the rate of 96 %, 93 % and 90 %, respectively. Figure 3.1 depicts that when 0.5 % (v/v) pristane was used, M. neoaurum , R. ruber and N. cyriacigeorgica degraded 59 %, 51 % and 84 % in 3 weeks, respectively. The amount of pristane evaporated during the incubation was not significant (2 %). By the end of experiments using 0.5 % (v/v) of pristane, the pH of the media decreased from 6.3 to 4.6 in all strain cultures. This decrease could be explained by the presence of produced intermediate acids during oxidation of pristane. The curves of growth kinetics and substrate degradation showed that all strains have utilized pristane as a carbon and energy source for growth.

RESULTS 26

(a) 4.5 1.2 77

4.5 ) )

4 -1 -1 66 ) 1 -1 3.5 3.5 55 3 0.8 2.5 44 2.5 0.6 2 33 pH value value pH pristane (g/l) pristane 1.51.5 0.4 22 1 0.2 (g/l) weight mass dry 1 pristane (gram litre litre (gram pristane 1 dry weight dry (gram litre weight dry (gram litre 0.50.5 weight dry (gram litre weight dry (gram litre 0 0 00 0 5 10 15 20 25 (b) Incubation time (days) 4.54.5 1.8 7

) -1 ) 4 1.6 -1 6 3.53.5 1.4 5 3 1.2 2.5 2.5 1 4

2 0.8 1.5 3 1.5 0.6 value pH pristane (g/l) pristane 2 1 0.4 pristane (gram litre litre (gram pristane 0.5 (g/l) weight mass dry dry weight dry (gram litre 0.5 0.2 weight dry (gram litre 1 0 0 0 0 5 10 15 20 25

Incubation time(c) (days)

4.5 1.6 7

) ) 4 1.4 -1 6 -1 3.5 1.2 5 3 1 2.5 4 0.8 2 3

0.6 pH value 1.5 2 1 0.4

pristane (gram litre 0.5 0.2 1 dry weight (gram litre 0 0 0 0 5 10 15 20 25

Incubation time (days)

Figure 3.1 Degradation of 0.5 % (v/v) pristane (x), kinetic growth ( ) and pH value decreasing (●) of M. neoaurum (a), R. ruber (b) and N. cyriacigeorgica (c)

RESULTS 27

3.1.3.2 Detection of possible degradation products M. neoaurum , R. ruber and N. cyriacigeorgica have been shown to possess high abilities in degrading pristane. To determine potential metabolites accumulated in the culture medium supplemented with pristane, the culture supernatants were extracted with diethyl ether at pH 9 and pH 2 and analyzed by GC/MS. To analyze acid-extractable metabolites, the compounds in the extracts from the acidified supernatants were derivatized by methylation with diazomethane in a micro apparatus. In order to avoid the co-eluting of expected metabolites with the solvent during running with GC/MS, two different gradients were created, one with starting point temperature at 40 oC and the other at 60 oC. By analytical chemical methods and in comparison with reference MS spectra of the National Institute of Standard Technology (NIST) libraries, 6 metabolites have been identified in M. neoaurum SBUG 109; 7 metabolites in R. ruber SBUG 82, and 4 metabolites in N. cyriacigeorgica SBUG 1472 (Table 3.3).

Table 3.3 Metabolites analyzed by GC/MS during growth on pristane by M. neoaurum SBUG 109, R. ruber SBUG 82 and N. cyriacigeorgica SBUG 1472.

Product name Product structure M R N (metabolite number) (as methylester)

4-Methylpentanoic acid (1) O + O O O Methylbutanedioic acid (2) O + + O

2-Methylpentanedioic acid (3) O O + + + O O

Methylmalonic acid (4) O O + O O

4,8-Dimethylnonanedioic acid (5) O + + O

4-Methylheptanedioic acid (6) O O + + O O

2,6-Dimethylheptanedioic acid (7) O + O

RESULTS 28

2,6,10-Trimethylundecanoic acid (8) O + O O O 3,7-Dimethyldecanedioic acid (9) O + O 2,6,10,14-Tetramethyl pentadecan– + + 3–one (10) O

Pristanoic acid (11) O + O ., M. neoaurum SBUG 109; R, R. ruber SBUG 82 and N, N. cyriacigeorgica SBUG 1472 ; +, metabolite detectable.

To confirm these observed products and their structures, standard acids were purchased and analyzed with GC/MS. According to the comparison of the retention times and mass spectra of these products with those of synthesized compounds they were determined (Table 3.4, and see also in Appendix - Figure 6.1 to Figure 6.11)

Table 3.4. Mass spectrometric data of compounds isolated during growth on pristane by M. neoaurum SBUG 109, R. ruber SBUG 82 and N. cyriacigeorgica SBUG 1472. The extracted acids were transformed for analytical purposes by methylation to the corresponding methylesters.

No tR Metabolites m/z (%, 70 eV) (min) Analyzing with GCMS starting temperature 40 oC 1 7.6 M 99 (10.66), 87 (40.13), 74 (87.77), 73 (13.04), 59 (19.12), 57 (45.45), 55

(39.50), 43 (100.00), 41 (33.54), 39 (18.81) O

8.0 st O 99 (14.11), 87 (47.83), 74 (100.00), 73 (13.04), 59 (19.92), 57 (44.46), 55 (41.67), 43 (87.28), 41 (38.15), 39 (17.31) 2a 15.9 R 129 (29.72), 100 (8.53), 59 (100.00), O 58 (60.60), 44 (7.83), 42 (39.63) O O 15.9st O 129 (29.08), 100 (20.76), 59 (100.00), 58 (60.60), 42 (12.03), 41 (22.65)

RESULTS 29

3a 18.5 R 143 (30.57), 142 (11.42), 115 (36.36), 114 (81.50), 101 (11.00), 99 (29.84), 88 (28.58), 87 (13.57), 83 (32.81), 74 (8.34), 73 (64.88), 69 (11.94), 59 (69.80), 57 (9.76), 56 (24.18), 55 (100.00), 45 (10.51), 43 (27.53), 42 (16.32), 41 (29.05), 39 (16.34) O O O O 18.5 st 143 (33.57), 142 (12.64), 115 (40.16), 114 (88.73), 101 (11.90), 99 (32.75), 88 (32.01), 87 (14.50), 83 (34.69), 74 (8.94), 73 (65.45), 69 (10.57), 59 (71.17), 57 (10.34), 56 (25.56), 55 (100.00), 45 (8.81), 43 (21.17), 42 (16.58), 41 (27.82), 39 (14.94) Analyzing with GCMS starting temperature 60 oC 4 5.6 M 115 (33.06), 114 (9.74), 87 (11.56), 72 (7.30), 59 (100.00), 57 (33.67), 56 M (12.78), 55 (20.49) ( ) O O

O O 5.6 st 115 (41.45), 114 (18.82), 87 (19.70), 72 (15.43), 59 (100.00), 57 (31.20), 56 (17.24), 55 (25.35) ( st ) 2b 11.2 M 129 (19.23), 128 (11.12), 101 (11.74), 100 (12.18), 87 (9.15), 69 (12.30), 59 M O (100.00), 41 (21.08), 39 (10.26) ( ) O O

O 11.2 st 129 (27.04), 128 (17.16), 101 (14.45), 100 (19.34), 87 (11.46), 69 (14.85), 59 (100.00), 41 (22.25), 39 (9.97) ( st ) 3b 15.3 M , N 143 (22.84), 142 (7.80), 115 (32.81), 114 (68.60), 99 (29.19), 88 (25.63), 87 O O (10.98), 83 (28.04), 73 (52.41), 59 (70.81), 57 (10.86), 56 (28.64), 55 O O (100.00), 43 (22.89), 42 (18.66), 41

(28.14), 39 (18.41) ( M,N)

RESULTS 30

15.2st 143 (35.13), 142 (13.53), 115 (39.08), 114 (92.98), 99 (33.61), 88 (32.23), 87 14.48), 83 (35.37), 73 (64.93), 59 (71.65), 57 (10.20), 56 (24.82), 55 (100.00), 43 (21.12), 42 (15.75), 41 (27.83), 39 (14.97) ( st ) 5 19.2 R, N 157 (4.29), 143 (6.34), 127 (8.59), 95 (6.37), 88 (10.50), 87 (100.00), 85

(14.71), 83 (16.69), 74 (68.09), 71 (37.77), 69 (25.20), 59 (14.84), 57

(40.91), 56 (11.19), 55 (45.92), 43 (51.88), 41 (40.25) ( R,N) O

19.1 st O 157 (6.32), 143 (14.63), 127 (8.54), 95 (14.63), 88 (19.50), 87 (100.00), 85 (23.17), 83 (6.69), 74 (85.37), 71 (26.83), 69 (23.17), 59 (18.29), 57 (39.02), 56 (10.21), 55 (35.37), 43 (60.98.88), 41 (20.73) ( st ) 6 20.4 M, R 171 (16.54), 142 (20.04), 139( 29.74), 129 (68.90), 115 (13.65), 87 (22.92), 83 (43.04), 74 (46.97), 69 (75.63), 59 O O (47.71), 55 (100), 43 (34.35), 41 M R O O (44.21) ( , ) 171, 142, 139, 129, 115, 87, 83, 74, 69, 59, 43, 41 standard from the library NIST 7 20.4 N 129 (25.84), 101 (7.65), 97 (25.30), 88 (100.00), 69 (31.57), 59 (40.83), 57

O (19.07), 56 (17.93), 55 (44.48), 41 (31.67) ( N) O 129, 101, 97, 88, 69, 59, 57, 56, 55, 41 standard from the library NIST 8 22.5 R 169 (10.18), 141 (6.82), 129 (9.16), 113 (11.51), 99 (71.89), 88 (100), 71 O (34.34), 59 (34.57), 43 (41.11) ( R) O 169, 141, 129, 113, 99, 88, 71, 59, 43 standard from the library NIST

RESULTS 31

9 22.9 R 185 (33.19), 152 (72.63), 143 (27.00), 124 (23.79), 111 (46.49), 103 (18.90), 87 (28.65), 74 (77.15), 69 (36.12), 59 O O (57.23), 55 (100), 43 (50.81), 41 O R O (48.06) ( ) 185, 152, 143, 124, 111, 103, 87, 74, 69, 59, 55, 43, 41 standard from the library NIST 10 25.7 M, R 238 (11.88), 226 (7.60), 212 (12.83), 197 (6.41), 185 (24.23), 167 (26.84), 155 (12.35), 125 (4.75), 113 (12.59), 85 (34.92), 71 (11.16), 69 (20.90), 58 (35.63), 43 (100.00) ( M,R) O 238, 226, 212, 197, 185, 167, 155, 125, 113, 85, 71, 69, 59, 43 st from the library NIST 11 29.7 N 101 (40.93), 97 (13.03), 88 (100.00), 71 (12.83), 69 (17.56), 57 (27.44), 55 N (24.91), 43 (31.58), 41 (20.30) ( )

O 29.2 st O 101 (37.56), 97 (12.11), 88 (100.00), 71 (13.97), 69 (20.03), 57 (32.86), 56 (10.22), 55 (30.00), 43 (38.47), 41 (24.97) ( st ) Substrate 113 (12.26), 99 (10.20), 85 (28.75), 71 pristane (77.45), 69 (11.63), 57 (100.00) 56 (20.72), 55 (20.89), 43 (65.60), 41 tR 25.4 (30.99)

M: isolated metabolites produced by M. neoaurum SBUG 109; R: isolated metabolites produced by R. ruber SBUG 82; N: isolated metabolites produced by N. cyriacigeorgica SBUG 1472; st: purchased standard; standards from the library NIST only showed the fragments but the intensity data were not shown.

RESULTS 32

3.2 Transformation of alkyl-substituted aromatics

As well as branched alkanes, alkyl-substituted aromatic hydrocarbons which are ubiquitous in nature are significant environmental pollutants. Among the aromatic compounds occuring in crude oil, alkyl substituted benzenes are dominating. Members of bacteria and yeasts are known for its capacity to degrade aromatic hydrocarbons. Therefore, the present work reports on biodegradation of sec -octylbenzene and iso - pentylbenzene by the bacteria M. neoaurum , R. ruber and N. cyriacigeorgica and the yeasts C. maltosa , T. asahii , T. mucoides , and T. domesticum .

3.2.1 Toxicity of aromatic compounds on microorganisms

Prior to biotransformation experiments, the toxicities of the named aromatic compounds affecting the growth of microorganisms were tested. Due to the similarity of sec -octylbenzene and iso -pentylbenzene and their effects on the microbial growth, this section describes mainly in details the influence of iso -pentylbenzene.

iso -Pentylbenzene affected the growth of bacteria cultured on nutrient broth in presence of variable concentrations of this aromatic compound (Figure 3.2). For three bacteria including M. neoaurum , R. ruber and N. cyriacigeorgica , biomass weight measuring method was used. Each bacterium was cultivated in 100 ml of nutrient broth medium supplemented with different concentration of iso -pentylbenzene such as 0.005 %, 0.01 %, 0.025 %, 0.05 % and 0.10 % in parallels. At different period of culturing, one parallel was stopped, filtered through the glass fibre filter, then the filter was dried at 100 oC in 2 hours and weight measured before and after filtering. Cells growing in nutrient broth without the presence of aromatic compound were used as control. All bacteria showed similar results. The control grew well on the nutrient broth medium; while with cells cultured in presence of higher concentration of iso -pentylbenzene - 0.05 % and 0.1 % - no growth occurs (Figure 3.2). The growth of all bacteria was reduced in the cultures supplemented with 0.01 % and 0.025 %.

The influence of iso -pentylbenzene on growth of yeasts cultured with different concentrations of the aromatic compound was shown in Figure 3.3.

RESULTS 33

2.4 M. neoaurum ) 1 - 2

1.6

1.2

0.8 Dry weight (g/l) weight Dry 0.4

Dried weight (g litre (g weight Dried 0 0 10 20 30 40 50

time (h)

2.4 R. ruber ) 1 - 2

1.6

1.2

0.8 Dry weight (g/l) weight Dry 0.4 Dried weight (g litre (g weight Dried 0 0 10 20 30 40 50 time (h)

2.4 N. cyriacigeorgica ) 1 - 2

1.6

1.2

0.8 Dry weight (g/l) weight Dry 0.4 Dried weight (g litre (g weight Dried 0 0 10 20 30 40 50 time (h)

control (0 %) 0.005% 0.01% 0.025% 0.05% time0.10% (h)

Figure 3.2 Toxicity test for M. neoaurum SBUG 109, R. ruber SBUG 82 and N. cyriacigeorgica SBUG 1472 in nutrient broth supplemented with different concentrations of iso -pentylbenzene (data are shown as the means ± SD of three independent cultures). Measurement as dry weight of biomass of the culture.

RESULTS 34

4.5 C. maltosa 4 3.5 3 2.5 2

OD value OD 1.5

OD value 1 0.5 0 0 10 20 30 40 50

4.5 T. asahii 4 3.5 3 2.5 2

OD value OD 1.5

OD value 1 0.5 0 0 10 20 30 40 50

4.5 T. mucoides 4 3.5 3 2.5 2

OD value OD 1.5 1 OD value 0.5 0 0 10 20 30 40 50

4.5 T. domesticum 4 3.5 3 2.5 2

OD value OD 1.5 OD value 1 0.5 0 0 10 20 30 40 50 Time (h)

control (0 %) 0.01% 0.025%time (h) 0.05% 0.1% 0.2%

Figure 3.3 Toxicity test for C. maltosa SBUG-Y 700, T. asahii SBUG-Y 833, T. mucoides SBUG-Y 801, and T. domesticum SBUG-Y 752 in mineral salts medium for yeasts supplemented with 1 % of glucose and different concentrations of iso -pentylbenzene (data are shown as the means ± SD of three independent cultures). Measurement as optical density of the culture.

RESULTS 35

For C. maltosa , T. asahii , T. mucoides , and T. domesticum the optical density

measuring method was used ( A600 ). Cells were cultured in mineral salts medium for yeast with 1 % of vitamin solution and 1 % of glucose, and supplemented with various concentration of iso-pentylbenzene such as 0.01 %, 0.025 %, 0.05 %, 0.1 % and 0.2 %. Controls consisted of cells grown with vitamin solution and glucose but without the aromatic compound. After 4 h, 8 h, 12 h, 28 h and 48 h of cultivation, the optical densities

of all cultures and of their controls were measured at 600 nm ( A600 ). The growth of all yeast was reduced in the cultures supplemented with 0.01 % and higher concentration (Figure 3.3). Concentrations of iso -pentylbenzene of 0.05 % and higher inhibits growth completely. The yeasts showed similar effects as estimated for bacteria but with 0.025 % of iso -pentylbenzene a somewhat stronger inhibition effect was observed. In conclusion, all tested microorganisms presented the capacity of growing with the concentration of iso -pentylbenzene lower than 0.05 %. In test with sec -octylbenzene similar results were obtained. Therefore, for further experiments, the concentrations of 0.01 % and 0.025 % (v/v) were used.

3.2.2 Screening microorganisms transforming aromatics

M. neoaurum SBUG 109, R. ruber SBUG 82, C. maltosa SBUG-Y 700, T. asahii SBUG-Y 833, T. mucoides SBUG-Y 801, and T. domesticum SBUG-Y 752 have been reported to have abilities of biotransformation of aromatic substances including mono- and di-chlorophenols, di-phenylether, biphenyl and alkylphenol (Hammer 1996; Mikolasch et al. 2003; Schauer et al. 1995; Sietmann et al. 2001). Therefore, in this study, these strains which were precultured on different carbon sources such as dodecane, glucose, nutrient agar, phenol, pristane, tetradecane and yeast extract were used to transform several aromatic hydrocarbons with a branched-chain side including iso -pentylbenzene and sec - octylbenzene. For yeasts, the experiments were performed with different celll densities (OD). According to Schauer (1981), the optimal OD for transformation of these yeasts are OD 2 and OD 6, therefore these ODs were used to transform aromatic compounds. To determine the pathway of iso -pentylbenzene and sec -octylbenzene up to cleavage of the aromatic ring system and to determine the intermediates via which the transformation of aromatics occurs at different time intervals during the incubation, 1 ml of cell suspension was removed aseptically and centrifuged to remove the biomass. 100 µl aliquots of supernatant were then analyzed by HPLC. After 8 h, 24 h, 48 h the supernatants were collected and extracted three times with an equal volume of ethyl acetate after

RESULTS 36

alkalization to pH 9 and acidification to pH 2 as described above (part 2.4 page 15). The residues obtained after reducing the extract volume by rotary evaporation were dissolved in methanol and analyzed by HPLC and GC/MS. The different strains of microorganisms had various capacities for biotransformation of iso -pentylbenzene and sec -octylbenzene dependent from the conditions of cultivation (Table 3.5).

Table 3.5 Biotransformation capacities of microorganims on aromatic compounds analyzing by HPLC and GC/MS.

iso -pentylbenzene sec -octylbenzene Strain Precultivation 0.01 % 0.025 % 0.01 % 0.025 % M .neoaurum Nutrient agar + + + + Tetradecane + ++ + ++ Pristane + + + + R. ruber Nutrient agar - + + + Tetradecane - ++ + ++ Pristane - + + + N. cyriacigeorgica Nutrient agar - + + + Tetradecane - ++ + ++ Pristane - + + + C. maltosa Glucose OD 2 - - - - OD 6 - - - - Yeast extract OD 2 - - - - OD 6 - - - - T. mucoides Phenol OD 2 - - - - OD 6 + ++ + ++ Yeast extract OD 2 - - - - OD 6 + + + + T. domesticum Glucose OD 2 - - - - OD 6 - - - - Yeast extract OD 2 - - - - OD 6 - - - - T. asahii Dodecane OD 2 - - - - OD 6 - - - - Yeast extract OD 2 - - - - OD 6 - - - - -, negative results; +, detected metabolites obtained by GC/MS analyses or in a trace amount of in HPLC; ++, detected metabolites by HPLC and GC/MS analyses.

RESULTS 37

According to these above presented results, it was focused on the experiments with M. neoaurum SBUG 109, R. ruber SBUG 82 and N. cyriacigeorgica SBUG 1472 precultured on nutrient broth, pristane as well as tetradecane; and with T.mucoides SBUG- Y 801 precultured on phenol to transform iso -pentybenzene and sec -octylbenzene with a concentration of 0.25 ml litre -1 (0.025 %, v/v).

3.2.3 Biotransformation of iso -pentylbenzene

3.2.3.1 Identification of biotransformation metabolites of iso -pentylbenzene by HPLC and GC/MS 3.2.3.1.1 iso -Pentylbenzene biotransformation metabolites by M. neoaurum 109 On the basic of the results shown in Table 3.5, M. neoaurum 109 was described as a good candidate for biotransformation of iso -pentylbenzene . Interestingly, it also was reported by Mikolasch et al. (2003) that M. neoaurum 109 (previously name was M. vaccae SBUG 109) acted as a well hydrocarbon transforming bacterium. These results were an encouragement to use this bacterium precultivated at different conditions for transformation experiments, resulting in several metabolites which called as products A, B, C and D with the retention time 6.2 min, 6.7 min, 10.4 min and 11.4 min, respectively as described in Figure 3.4.

Product A Product B Product C Product D Substrate (*)

Figure 3.4 HPLC elution profile of the aqueous culture supernatant after incubation of M. neoaurum SBUG 109 with iso-pentylbenzene (0.25 ml liter -1; 24 h). mAU, mili absorbance units; *, cellular excreted product.

RESULTS 38

Detailed GC/MS analyses gave hints for further metabolites named as E, F, G, H and formed from iso -pentylbenzene. MVEX04 (a) Scan EI+ 21.051 TIC 100 4.98e6 RT

18.433 % 12.381

11.965 14.099 0 rt 5.000 7.500 10.000 12.500 15.000 17.500 20.000 22.500 25.000 27.500 Sample ID: M.vaccae pre. on pristane, +0,01% isopentylbenzene, extracted, 24h, pH2 Acquired on 09-Nov-2006 at 19:56:10

MVEX10 (b) Scan EI+ 15.316 TIC 100 3.39e6 RT

20.834 %

21.184

8.814 22.084 25.469 28.770 30.920 31.487 0 rt 8.000 10.000 12.000 14.000 16.000 18.000 20.000 22.000 24.000 26.000 28.000 30.000 32.000

Figure 3.5 GC/MS elution profiles of extracts after alkaline (a) and acidic (b) extractions after incubation of M. neoaurum SBUG 109 with iso -pentylbenzene (0.25 ml liter -1; 24 h).

All of these products were identified by comparison of retention times and UV/Vis spectra of the HPLC elution profiles as well as retention times and mass spectra of the GC/MS to those of veritable standards (Table 3.6, see also in Appendix - Figure 6.12 to Figure 6.15).

RESULTS 39

Table 3.6 Retention time and UV/Vis-spectrum of the identified metabolites produced by M. neoaurum SBUG 109 cultured with 0.25 ml litre -1 iso -pentylbenzene in comparison with standard substances.

1 2 tR tR Met UV/Vis-spectrum m/z (%, 70 eV) (min) (min)

mAU A 6.2 20 15.1 150 (28.4), 92 (7.8), 91 (100.0), 65 17.5 COOH 15 (15.8), 39 (8.1) 12.5 10 7.5 6.1 st 5 15.1 st 150 (29.1), 92 (8.1), 91 (100.0), 65 2.5 0 (14.7), 39 (6.1) st 300 400 500 nm

mAU B 6.7 500 11.0 120 (19.3), 105 (100.0), 77 (92.3), 51 O 400 (28.6), 50 (11.0), 43 (15.4)

300 6.5 st 200 11.0 st 120 (22.7), 105 (100.0), 77 (90.3), 51 100

0 (30.0), 50 (13.7), 43 (15.1) st 300 400 500 nm C 10.4 18.38 146 (43.1), 131 (62.3), 91 (97.6), 78 mAU OH (11.6), 77 (20.4), 65 (14.7), 59 (100.0), 14 12 51 (10.8), 43 (15.5), 41 (8.7), 39 (9.5) 10 or 8 OH 10.4 st 6 18.35 st 146 (33.2), 131 (47.4), 91 (78.9), 78 4 2 (8.9), 77 (10.5), 65 (14.1), 59 (100.0), (*) (*) 0 51 (7.3), 43 (14.8), 41 (8.3), 39 (8.5) st -2 400 nm

mAU D 11.4 17.5 18.43 162 (9.3), 120 (28.9), 105 (100.0), 77 O 15 (46.6), 51 (15.3), 41 (7.3) 12.5 10 7.5 11.4 st 5 18.43 st 162 (9.0), 120 (29.4), 105 (100.0), 77 2.5 (55.2), 51 (17.8), 41 (8.5) st 0 300 400 500 nm Sub 14.0 st mAU 14.1 st 148 (14.2), 105 (10.3), 93 (7.42), 92 350 300 (100.0), 91 (71.2), 77 (7.4), 65 (11.8), 250 200 57 (11.7), 41 (14.6), 39 (8.5) st 150 100 50 0 300 400 500 nm 1 2 Met: metabolite; t R : Retention time in HPLC; t R : Retention time in GC/MS; Sub: substrate; st: purchased standard; (*), 2-methyl-4-phenyl-butan-2-ol.

RESULTS 40

Amongst observed major metabolites by HPLC, product A has a UV/Vis-spectrum characteristic for an acidic aromatic compound and has a retention time shorter than the substrate. By matching with the available library and purchased standard, this acidic metabolite was determined as phenylacetic acid because of the same retention time and UV/Vis-spectra similarity. To confirm the identification, the cultivation supernatant without cell was extracted and the residues were analyzed by GC/MS. According to computer matching of the mass spectra with the library spectra using NIST database and comparing with an authentic standard, the fragmentation pattern of this compound were corresponding to the standard, only the intensities of the fragment ions little varied (Table 3.6). Therefore, this metabolite was confirmed to be phenylacetic acid.

The other major metabolites (B and D) with retention times 6.7 min and 11.4 min had UV/Vis-spectra corresponding to that of ketone compounds. Analyzing by HPLC acetophenone as a standard, it was shown that this standard has also a retention time at 6.7 min and the same UV-spectrum as metabolite B. Thus, it was suggested that, this

metabolite was acetophenone. The other metabolite (t R at 11.4 min) contained a ketone group with a longer branched chain length than acetophenone. Analyzing by HPLC and GC/MS the purchased standard iso -valerophenone, product D has confirmed as iso - valerophenone (Table 3.6).

Product C had a similar UV/Vis-spectrum and HPLC retention time (10.4 min) with both 2–methyl–4-phenylbutyric acid and 2-methyl-4-phenyl-butan-2-ol. Analyzing by GC/MS product C was identified as 2-methyl-4-phenyl-butan-2-ol in the alkaline extract at

the same tR 18.4 min, meanwhile the tR of 2–methyl–4-phenyl butyric acid was 20.8 min. However, 2-methyl-4-phenyl-butan-1-ol has the same molecule weight with 2-methyl-4- phenyl-butan-2-ol, they differ only on the position of the hydroxy group. Because of non availability of 2-methyl-4-phenyl-butan-1-ol for analyzing by GC/MS and HPLC, there was no complete confirmation of structure for product C. Because of the limited sensitivity of analyzing aqueous samples by HPLC, the culture supernatant without cells were extracted at pH 9, 7 and 2 and concentrated. In addition to the four upper products (A to D), the acidic extract analyses by HPLC and GC/MS pointed to one previously undetected compound, called as product E, and two others, called as F and G. By matching with the available standards (Figure 3.6 and in

Appendix Table 6.1), they were estimated as succinic acid (tR 9.1 min), benzoic acid (tR

12.5 min) and 2-hydroxy-phenylacetic acid (tR 20.1 min), respectively.

RESULTS 41

Reverse fit factor [REV]: 981 MVEX08→ 278 (9.131) Cm (275:282-(269:272+295:322)) COOH 4.57e4 HOOC 115 100 Product E

55 59 % 114 87 45 57 60 116 41 42 72 85 88 0 R:981 CONG 33: DIMETHYL SUCCINATE Hit 1 115 100 Dimethyl 55

. 59 succinic acid % 114 87 56 42 45 60 88 116 41 85 101 0 m/z 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100105 110115120 125130Reverse 13fit5 factor 140 145 [REV]: 150 947 MVEX08 482 (12.532) Cm (482:486-(478+495:504)) 8.06e3 105 COOH 100 Product F 77 % 51 136 43 39 41 56 106 57 69 70 78 129 135 137 37 45 53 68 83 84 92 97 0 R:947 CONG 26: BENZOIC ACID, METHYLATED Hit 1 105 100 Benzoic acid 77

% 51 136 50 106 52 78 137 38 39 74 91 92 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

MVIPB102 930 (20.001) Cm (928:933-(909:925+940:965)) COOH 556 91 121 Product G 100 OH

% 180 78 51 65 77 37 52 80 90 93 99 110 122 129 135 148 189 40 44 61 107 152 159 170 176 185 203 205 0 R:779 PHALK 85: 2-OH-PHENYLESSIGSÄURE 2FACH METHYLIERT Hit 1 91 100 2-hydroxy phenylacetic a acid 121

%

180 65 78 93 51 59 77 122 39 52 66 89 120 148 181 38 50 79 105 107 133 149 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 Figure 3.6 Mass spectra of metabolite peaks with GC retention times of 9.1 min (product E), 12.5 min (product F) and 20.1 min (product G) are illustrated in comparison with reference mass spectra of succinic acid (product E), benzoic acid (product F) and 2-hydroxy- phenylacetic acid (product G), respectively. All acidic analyses were methylated.

RESULTS 42

Interestingly, analyzing by GC/MS, 2-methyl-4-phenyl butyric acid was detected in acidic extract at retention time of 20.8 min, called product H (Figure 3.7). This products consists of main peak at m/z 192 (5.2 %), 161 (6.6 %), 160 (7.4 %), 105 (10.0 %), 104 (12.9 %), 91 (52.2 %), 88 (100.0 %), 65 (14.6 %), 57 (20.8 %), 51 (4.5 %), 39 (6.2 %) in comparison with standard which comprised main fragments at m/z 192 (5.1 %), 161 (4.6 %), 160 (6.4 %), 105 (10.0 %), 104 (11.8 %), 91 (52.8 %), 88 (100.0 %), 65 (14.8 %), 57 (23.1 %), 51 (3.9 %), 39 (6.3 %). The high similarity of the fragment ions and retention times confirmed the presence of this acid in the extract.

Reverse fit factor [REV]: 992 MVEX10 980 (20.834) Cm (979:981-(971:975+986:992)) 3.35e5 88 Product H 100 COOH

91 %

57 65 104 39 41 79 87 92 105 115 160 161 51 66 77 132 133 192 0 R:992 CONG 49: 2-METHYL-4-PHENYL-BUTYRIC ACID, METHYLATED Hit 1 88 100 2-methyl-4-phenylbutyric acid

91 %

57 65 104 39 59 77 92 105 115 160 41 51 66 78 132 133 161 192 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210

Figure 3.7 Mass spectra of metabolite H was detected by GC/MS at retention time 20.8 min in comparison with the purchased standard (st) and confirmed the structure as 2-methyl-4- phenyl-butyric acid. All metabolites and standard were methylated.

3.2.3.1.2 iso -Pentylbenzene biotransformation metabolites by N. cyriacigeorgica 1472 Members of Nocardia are well-known for their capacities to degrade hydrocarbons. N. cyriacigeorgica (formerly named Nocardia cyriacigeorgici ) is a newly recognized species within the genus, however, it is known for its epidemiology but not for its hydrocarbons degradation (Elsayed et al. 2006; Fux et al. 2003; Yassin et al. 2001; Fux C. 2003). iso -Pentylbenzene biotransformation experiments had also done with this organism. However, the number of products was smaller than that of M. neoaurum 109. HPLC elution profiles of the aqueous culture supernatant were summarized in Figure 3.8.

RESULTS 43

mAU mAU 3.639 14 300 12 mAU 10 Product C 17.5 8 15 250 Cellular 6 12.5 4 Product D 10 excreted 2 7.5 200 0 product 5 -2 400 nm 2.5 0

150 16.695 300 400 500 nm

100

50 10.437 10.938 13.905 1.400 13.077 12.780 11.392 6.927 7.697

0 6.269

2 4 6 8 10 12 14 16 18 min

Figure 3.8 HPLC elution profiles of the aqueous culture supernatant after incubation of N. cyriacigeorgica SBUG 1472 with iso -pentylbenzene (0.25 ml liter -1; 24 h) and UV/Vis spectra of metabolites. mAU, mili absorbance units.

The elution profiles of N. cyriacigeorgica compared with the available standards and the identified metabolites of other investigated strains showed that product D could be identified as iso -valerophenone, and product C was remained questionable of being 2- methyl-4-phenyl-butan-2-ol or 2-methyl-4-phenyl-butan-1-ol. Computer matching of the mass spectra with the library spectra using NIST database and comparing with authentic standards (iso-valerophenone and 2-methyl-4-phenyl-butan-2-ol), the fragmentation pattern of this compound were corresponding to the standard, only the intensities of the fragment ions little varied (Table 3.7 see also in Appendix - Figure 6.16 and Figure 6.17). Therefore, the metabolite was confirmed as iso -valerophenone (product D); but there was still questionable whether 2-methyl-4-phenyl-butan-2-ol or 2-methyl-4-phenyl-butan-1-ol was product C.

RESULTS 44

Table 3.7 Retention times and UV/Vis-spectra of the identified metabolites produced by N. cyriacigeorgica SBUG 1472 cultured with 0.25 ml litre -1 iso -pentylbenzene in comparison with standard substances.

t 1 t 2 Met R UV/Vis-spectrum R m/z (%, 70 eV) (min) (min) C 10.4 mAU 18.36 146 (36.6), 131 (52.0), 91 (89.6 ), 78 14 12 (12.0), 77 (32.0), 65 (14.7), 59 (100.0), 51 10 8 (12.3), 43 (24.1), 41 (10.6), 39 (11.3) 6 4 10.4 st 2 18.35st 0 146 (33.2), 131 (47.4), 91 (78.9), 78 (8.9), -2 77 (10.5), 65 (14.1), 59 (100.0), 51 (7.3), 400 nm (*) 43 (14.8), 41 (8.3), 39 (8.5) st mAU D 11.4 17.5 18.43 162 (10.3), 120 (29.9), 105 (100.0), 77 15 O (50.6), 51 (15.3), 41 (8.3) 12.5 10 7.5 11.4 st 5 18.43st 162 (9.0), 120 (29.4), 105 (100.0), 77 2.5 0 (55.2), 51 (17.8), 41 (8.5) st 300 400 500 nm 1 2 Met: metabolite; tR : Retention time in HPLC; tR : Retention time in GC/MS; st: purchased standard; (*), 2-methyl-4-phenyl-butan-2-ol. 3.2.3.1.3 iso -Pentylbenzene biotransformation metabolites by R. ruber SBUG 82 R. ruber were reported to grow on different hydrocarbon substrates such as n- alkanes, iso -alkanes, aromatic compounds, or their oxidation products; which are commonly introduced into gasoline formulations (Sayavedra-Soto et al. 2006; Schumacher and Fakoussa 1999; Sivan et al. 2006; Zhukov et al. 2007) Using R. ruber SBUG 82 to transform iso -pentylbenzene three metabolites were obtained (Figure 3.9). mAU 140 120 Substrate 100

80 Product D 60 Product A 14.930 Product F

40 11.015

20 6 .3 6 11.468

6 .1 5

0 7 .7 4 3

-20

2 4 6 8 10 12 14 16 18 min

Figure 3.9 HPLC elution profile of the aqueous culture supernatant after incubation R. ruber SBUG 82 with iso -pentylbenzene (0.25 ml liter-1; 24 h). mAU, mili absorbance units.

RESULTS 45

The elution profiles of R. ruber SBUG 82 included product A – phenylacetic acid, product D – iso -valerophenone and product F – benzoic acid. GC/MS analyses also confirmed these metabolites including product A with retention time at 15.2 min; product D at retention time 18.42 min and product F at 12.4 min (Table 3.8, see also Appendix - Figure 6.18 to Figure 6.20).

Table 3.8 Retention times and UV/Vis-spectra of the identified metabolites produced by R. ruber SBUG 82 cultured with 0.25 (ml litre -1) iso -pentylbenzene in comparison with standard substances.

1 2 tR tR Met UV/Vis-spectrum m/z (%, 70 eV) (min) (min)

mAU A 6.2 20 15.2 150 (26.4), 92 (8.8), 91 (100.0), 65 17.5 COOH 15 (15.2), 39 (7.1) 12.5 10 7.5 6.1 st 5 15.1 st 150 (29.1), 92 (8.1), 91 (100.0), 65 2.5 0 (14.7), 39 (6.1) st 300 400 500 nm

mAU D 11.4 17.5 18.42 162 (8.5), 120 (28.6), 105 (100.0), 77 15 O (49.6), 51 (16.3), 41 (8.3) 12.5 10 7.5 11.4 st 5 18.43st 162 (9.0), 120 (29.4), 105 (100.0), 77 2.5 0 (55.2), 51 (17.8), 41 (8.5) st 300 400 500 nm F 6.3 mAU 12.4 136 (31.9), 106 (6.4), 105 (100.0), 77 200 COOH (75.6), 51 (26.8), 50 (1397) 150 100 6.3 st 50 12.3 st 136 (33.5), 106 (6.3), 105 (100.0), 77 0 300 400 500 nm (74.6), 51 (28.9), 50 (14.5)

1 2 Met: metabolite; t R : Retention time in HPLC; t R : Retention time in GC/MS; st: purchased standard.

RESULTS 46

3.2.3.1.4 iso -Pentylbenzene biotransformation metabolites by T. mucoides SBUG-Y 801 T. mucoides SBUG-Y 801 was known as one of the yeasts which can grow on either glucose or phenol and has capacity of transforming biphenyl into a variety of mono-, di-, and trihydroxylated derivatives hydroxylated on one or both aromatic rings (Sietmann et al. 2000; Sietmann et al. 2001). Using T. mucoides SBUG-Y 801 to transform iso - pentylbenzene, the observed results were described in Figure 3.10. mAU 2.722 400

350 Cellular excreted

300 products

250 Product C Product D 200

150

100 1.395 10.427 5.753 9.867 1.166 3.591 16.699

50 13 .8 84 2.329 11.381 7.16 3 3.884 10 .7 32 6.043 4.363 6.91 0 0

2 4 6 8 10 12 14 16 18 min

Figure 3.10 HPLC elution profile of the aqueous culture supernatant after incubation of T. mucoides SBUG-Y 801 with iso -pentylbenzene (0.25 ml liter-1; 24 h). mAU, mili absorbance units.

The elution profiles of T. mucoides included product C and product D. Product D was identified as iso -valerophenone by HPLC. GC/MS analyses also confirmed this result. However, product C was compared with 2-methyl-4-phenyl-butan-2-ol and gave similar result, but with 2-methyl-4-phenyl-butan-1-ol was unavailable. Therefore, the exact structure of product C remains unknown. Furthermore, product A – phenylacetic acid was identified in the acid extracts of T. mucoides ; and product G – 2–hydroxy-phenylacetic acid was found in T. mucoides acid extracts (Table 3.9, see also in Appendix – Figure 6.21 to Figure 6.23).

RESULTS 47

Table 3.9 Retention time of the identified metabolites produced by T. mucoides SBUG-Y 801 cultured with 0.25 ml litre -1 iso -pentylbenzene in comparison with standard substances.

Met Structure tR (min) m/z (%, 70 eV)

A COOH 15.2 150 (26.4), 92 (8.8), 91 (100.0), 65 (15.2), 39 (7.1)

15.1 st 150 (29.1), 92 (8.1), 91 (100.0), 65 (14.7), 39 (6.1) st C 18.36 146 (32.6), 131 (42.0), 91 (79.6 ), 78 (11.3), 77 (22.4),

OH 65 (13.1), 59 (100.0), 51 (11.1), 43 (13.6), 41 (9.2), 39

(7.3) or 18.35 st 146 (33.2), 131 (47.4), 91 (78.9), 78 (8.9), 77 (10.5), OH (*) 65 (14.1), 59 (100.0), 51 (7.3), 43 (14.8), 41 (8.3), 39 (8.5) st D O 18.42 162 (8.6), 120 (24.8), 105 (100.0), 77 (59.6), 51 (12.3),

41 (6.4) 18.43 st 162 (9.0), 120 (29.4), 105 (100.0), 77 (55.2), 51 (17.8), 41 (8.5) st G 20.9 180 (25.5), 124 (7.1), 121 (95.3), 93 (9.4), 91 (100.0), COOH 78 (18.9), 77 (13.4), 65 (18.4), 52 (6.2), 51 (11.5) OH 20.0 st 180 (22.9), 124 (7.4), 121 (95.7), 93 (11.4), 91 (100.0), 78 (12.3), 77 (11.2), 65 (19.5), 52 (6.7), 51 (10.9)

Met: metabolite; t R: Retention time in GC/MS; st: purchased standard; (*), 2-methyl-4-phenyl- butan-2-ol. By GC/MS analyses, addition to other similar metabolites with M. neoaurum SBUG 109, one more metabolite was distinguished in alkaline extract of T. mucoides , designated as product K with the retention time of 18.3 min. Mass spectra analyses

revealed a molecular weight of 164. Further fragment ions at m/z 121 [M–CH(CH 3)2], 107

[M–CH 2CH(CH 3)2], 93 [M–(CH 2)2CH(CH 3)2], 77 [M–(CH 2)2CH(CH 3)2– OH], and 43 [-

CH(CH 3)2] corresponding with a structure bearing a branched alkane side with 5 carbon atoms, a phenyl ring and one hydroxyl group on the ring, estimating that the structure of product K is X-(3–methyl–butyl)-phenol (X means that position of the hydroxy group at the aromatic ring system, such as 2, 3 or 4 remained unclear) (Figure 3.11).

92 100 - 93 - 107 - 121

91

% 107

OH + 71 + 57 + 43 79

55 73 77 41 43 65 103 93 51 121 59 108 131 45 63 66 80 89 94 146 149 164 37 85 129 132 157 165 180 190 0 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190

Figure 3.11 Mass spectrum of metabolite K – X-(3-methyl-butyl)-phenol or X-hydroxy iso- pentylbenzene , X means that position of the hydroxy group at the aromatic ring system.

RESULTS 48

Summarizing the results, during iso -pentylbenzene biotransformation, by HPLC and GC/MS analyses, 8 metabolites (from products named as A to H) were detected in M. neoaurum ; whereas 2 metabolites (product C and D) were found in N. cyriacigeorgica SBUG 1472, 3 metabolites (product A, D and F) in R. ruber SBUG 82; and 5 products (A, C, D, G and K) in T. mucoides SBUG-Y 801.

3.2.3.2 Kinetics of the iso -pentylbenzene transformation metabolites The kinetics of the iso -pentylbenzene transformation was depicted in Figure 3.12. During the biotransformation of iso -pentylbenzene, the number of metabolites, which could observed in HPLC chromatograms without GC/MS data, produced by M. neoaurum SBUG 109 is 4; by N. cyriacigeorgica SBUG 1472 is 2; by R. ruber SBUG 82 is 3 and by T. mucoides SBUG-Y 801 is 2. However, 2-methyl-4-phenyl-butan-2-ol (product C) and 2- methyl-4-phenyl-butyric acid (product H) were detectable at the same retention time (10.4 min) in the HPLC profiles. Therefore, it was proposed that in the culture of M. neoaurum with iso -pentylbenzene supplemented as the substrate, in addition to phenylacetic acid (product A), acetophenone (product B) and iso -valerophenone (product D) a mixture of product C and H was observed by HPLC analyses. The substrate was reduced with the highest amount by N. cyriacigeorgica , about 50 %. Acetophenone was found only in the reaction mixture of M. neoaurum after 24 h and it was presented over the whole cultivation period. Benzoic acid (product F) was detectable in R. ruber after 24 h in a trace amount but after 30 h it could not be observed. iso-Valerophenone and 2-methyl-4-phenyl-butan-2-ol were detected in all experiments. In the supernatant cultures of N. cyriacigeorgica and T. mucoides , 2-methyl-4-phenyl-butan-2-ol was quickly identified after 2 h and maintained with a remarkable amount, (more than 0.05 µM and 0.03 µM), from 4 h and 8 h to the end of the experiment, respectively. Also in both of these strains and in M. neoaurum , iso - valerophenone was determined with a trace amount, but in R. ruber the compound was prolonged detectable until the end of experiments with sum of about 0.016 µM. Phenylacetic acid (product A) was excreted into the culture of M. neoaurum after 8 h with a content increasing from 0.01 µM to 0.016 µM.

RESULTS 49

0,018 M. neoaurum 0,08 0,016 0,07 0,014 0,06 0,012 0,05 0,01 0,04 0,008 0,03 0,006 Products (µM) Products 0,004 0,02 (µM) Substrate 0,002 0,01 0 0 0 5 10 15 20 25 30 35 40 45 50

0.07 N. cyriacigeogica 0.07 0.06 0.06 0.05 0.05 0.04 0.04 0.03 0.03 0.02 0.02 Products Products (µM) Substrate (µM) Substrate 0.01 0.01 0 0 0 5 10 15 20 25 30 35 40 45 50

0,02 R. ruber 0,08 0,07 0,015 0,06 0,05 0,01 0,04 0,03 Products (µM) 0,005 0,02 Substrate (µM) 0,01 0 0 0 5 10 15 20 25 30 35 40 45 50

T.mucoides 0.035 0.07 0.03 0.06 0.025 0.05 0.02 0.04 0.015 0.03 0.01 0.02 Products Products (µM) 0.005 0.01 (µM) Substrate 0 0 0 5 10 15 20 25 30 35 40 45 50

Time (h) product A product B mixture of C and H product D product F substrate product C

Figure 3.12 Kinetics of substrate uptake and product formation during the transformation of iso -pentylbenzene; (data are shown as the means ± SD of three independent cultures).

RESULTS 50

Summarizing the results from the Figure 3.12 (data are shown as the means ± SD of three independent cultures) the biotransformation of 0.025 % (v/v) iso -pentylbenzene determined by HPLC; phenylacetic acid (product A); acetophenone (product B); 2-methyl- 4-phenyl-butan-2-ol or 2-methyl-4-phenyl-butan-1-ol (product C); iso -valerophenone (product D); benzoic acid (product F); 2-methyl-4-phenyl-butyric acid (product H). In case of M. neoaurum the products C and H could not be separated by HPLC. 3.2.3.3 Transformation of transformation metabolites 0.005 % (v/v) of phenylacetic acid (product A), 2-methyl-4-phenyl-butan-2-ol (assumed as product C because 2-methyl-4-phenyl-butan-1-ol was not available), iso - valerophenone (product D) and 2-methyl-4-phenyl-butyric acid (product H) were used as substrates for biotransformation by M. neoaurum , R. ruber, N. cyriacigeorgica and T. mucoides . The summarized results were presented in Table 3.10.

Table 3.10 Detected metabolites by HPLC analyses in the cultures of M. neoaurum , R. ruber , N. cyriacigeorgica and T. mucoides with product A, C, D and H as substrates.

Substrate Formed metabolites M. n. R. r. N. c. T. m. from product A-H Product A n.d. n.d. n.d. n.d. n.d. Product C n.d. n.d. n.d. n.d. n.d. Product D Benzoic acid + n.d. n.d. n.d. Product H Phenylacetic acid ++ ++ n.d. n.d. Benzoic acid + n.d. n.d. n.d. 2-hydroxy benzoic acid + n.d. n.d. n.d. M.n., M. neoaurum , R.r., R. ruber , N. c., N. cyriacigeorgica; T.m., T. mucoides; ++, main metabolite; +, trace metabolite; n.d., not detectable.

Kinetics of the 2-methyl-4-phenyl-butyric acid biotransformation metabolites were described in the Figure 3.13. Phenylacetic acid was the main intermediate of this transformation. It was observed with a remarkable amount after 2 h of cultivation by M. neoaurum with 2-methyl-4-phenyl-butyric acid. Benzoic acid was obtained with a trace amount. Though in the cultivation of R. ruber , the transformation was occurred somewhat later then in M. neoaurum, after 48 h of incubation all of the substrate was transformed.

RESULTS 51

0.09 M. neoaurum 0.09

0.08 0.08

0.07 0.07

0.06 0.06

0.05 0.05

0.04 0.04 Product (µM) Product Substrate (µM) Substrate 0.03 0.03

0.02 0.02

0.01 0.01

0 0 0 10 20 30 40 50 60 Time (h)

R. ruber 0.09 0.09

0.08 0.08

0.07 0.07 0.06 0.06 0.05 0.05 0.04 0.04 0.03 Product (µM) Product Substrate (µM) Substrate 0.03 0.02 0.02 0.01

0.01 0

0 -0.01 0 10 20 30 40 50 60 Time (h)

substrate phenylacetic acid benzoic acid

Figure 3.13 Kinetics of the transformation of 2-methyl-4-phenyl-butyric acid by M. neoaurum and R. ruber ; ♦, substrate; , phenylacetic acid; , benzoic acid; (data are shown as the means ± SD of three independent cultures).

RESULTS 52

3.2.4 Biotransformation of sec -octylbenzene

The numbers of detectable metabolites were very different. The highest number of metabolites, which were detected by HPLC, was formed by N. cyriacigeorgica . Therefore, the following presentationof results started with this strain. 3.2.4.1 Biotransformation of sec-octylbenzene by N. cyriacigeorgica SBUG 1472 N. cyriacigeorgica has high capacities to degrade n-alkanes and branched-alkanes. Therefore, this strain precultured on tetradecane or pristane was used to transform several aromatic hydrocarbons with a branched-chain side such as iso-pentylbenzene, sec - hexylbenzene or sec -octylbenzene. It was found that sec -octylbenzene was transformed by tetradecane- as well as pristane-precultivated cells. The metabolites formed from sec - octylbenzene were characterized by using aqueous samples of the supernatant to analyze them with HPLC. At separation times during the incubation, 1 ml of cell suspension was taken and removed the cell biomass by filtration. After that, 100µl of aqueous culture was analyzed by HPLC. Transformation of sec -octylbenzene resulted in a series of acid metabolites. Identification of these metabolites has shown that the strain accumulated four oxidation products, designated as product I, II, III and IV which had the retention times of 7.9, 9.1, 9.6 min and 11.4 min, respectively (Figure 3.14).

mAU 180 9.113 Product II 160

140 Product III

120

100 9.623

1.264 80 Product I Product IV

60

40 7.935 13.436

1.064 11.423 10.095 20 5.404 0 2 4 6 8 10 12 14 16 min

Figure 3.14 HPLC elution profiles of the aqueous culture supernatant after incubation N. cyriacigeorgica SBUG 1472 with sec -octylbenzene (0.25 ml liter -1; 72 h) and UV/Vis absorption spectra of products. mAU, mili absorbance units.

RESULTS 53

All of these products were identified by comparison of retention time and UV/Vis spectra of the HPLC elution profiles as well as retention time and mass spectra of the GC/MS analyses to those of authentic standards (Table 3.11, see also in Appendix – Figure

6.24 to Figure 6.27). The HPLC retention time (t R) and mass spectrum of product I (tR 7.9 min) suggested that this compound could be an acid product with shorter branched-chain side than the parent compound. The mass spectrum, obtained by GC/MS analyses, indicated a molecular ion peak at m/z 164. The main fragment ions at m/z 105 [M-

COOCH 3] and 77 [C6H5] correspond with a structure containing one carboxyl function, an n-propyl chain and a phenyl residue. In comparison of the retention time, mass spectrum and UV/Vis absorption spectrum of the available standard product I was identified as 2- phenylpropionic acid.

Table 3.11 Retention time and UV/Vis-spectrum of the identified metabolites in cultures of N. cyriacigeorgica with sec -octylbenzene in comparison with standard substances.

1 2 tR tR Met UV/Vis-spectrum m/z (%, 70 eV) (min) (min)

mAU I 7.9 60 16.2 164 (22.4), 105 (100.0), 79 (11.1), 78 (5.9), 50 77 (17.4), 51 (7.2), 39 (4.4)

40 30 COOH 7.9 st 20 16.2 st 164 (22.4), 105 (100.0), 79 (12.9), 78 (5.6), 10 77 (17.6), 51 (7.5), 39 (3.8) 0 300 400 500 nm II 9.1 18.6 178 (19.0), 121 (12.4), 119 (6.0), 118

mAU (81.6), 106 (6.5), 105 (100.0), 103 (17.6),

1000 91 (15.5), 79 (9.1), 78 (9.2), 77 (19.5), 51 800 (11.6), 41 (9.4), 39 (11.3) 600 COOH 400 9.1 st 200 18.5 178 (11.1), 121 (16.2), 119 (7.1), 118 0 (81.6), 106 (6.5), 105 (100.0), 103 (17.6), 300 400 500 nm 91 (15.5), 79 (9.1), 78 (9.2), 77 (19.5), 51 (11.6), 41 (9.4), 39 (11.3) III 9.6 20.9 176 (56.0), 175 (44.8), 145 (80.5), 144

mAU (36.5), 117 (39.4), 116 (27.7), 115 (100.0), 160 140 102 (12.5), 91 (34.8), 89 (10.9), 77 (13.2), 120 100 65 (12.0), 63 (11.0), 51 (19.0), 50 (10.5), 39 80 (28.0) 60 COOH 40 20 20.9 st 176 (58.2), 175 (47.3), 145 (89.9), 144 9.6st 0 300 400 500 nm (45.3), 117 (46.9), 116 (29.8), 115 (100.0), 102 (11.0), 91 (34.8), 89 (10.0), 77 (13.0), 65 (12.7), 63 (10.6), 51 (18.2), 50 (8.6), 39 (20.7)

RESULTS 54

mAU

IV 11.4 40 27.7 206 (15.1), 178 (38.8), 131 (22.0), 130 (100.0), 118 (21.1), 117 (26.4), 115 (20.1), 30 105 (84.1), 92 (9.5), 91 (90.3) 83 (9.5), 77 20 COOH (13.4), 74 (22.0), 71 (9.8), 59 (10.8), 55 10 (15.4), 20.7), 43 (35.4), 41 (27.6), 39 (10.3)

0 300 400 500 nm mAU Sub 15.4 50 20.7 190 (11.6), 106 (12.8), 105 (100.0), 91 (13.3), 79 (5.6), 77 (6.0), 41 (4.1) 40 30 20 10 0 300 400 500 nm 1 2 Met: metabolite; tR : Retention time in HPLC; tR : Retention time in GC/MS; Sub: substrate; st: purchased standard.

Product II was isolated from the supernatant extraction for further analyzes with HPLC and GC/MS. MS analyses of this product revealed a molecular weight of 178. The difference of 14 in comparison with the molecular weight of product A (M+ 164) was considered with an additional methyl group in this molecule. Comparing HPLC and GC/MS analyses of the available standard, product II was confirmed as 3-phenylbutyric acid. Analyzing the isolated product III from the supernatant extract by HPLC and GC/MS suggested that this compound could have an unsaturated branched-chain side. The mass spectrum showed a molecular weight at m/z 176 resulted from the loss of two atoms

of hydrogen from the 3-phenylbutyric acid. Further major fragment ions at m/z 145 [C 6H5

+CH 3C(CH)CO], 117 [C 6H5 +CH 3C(CH)/+H], 115 [CH 3C(CH)CH 2COOCH 3], 91 [C 6H5 +

C/ + 2H] and 77 [C 6H5] correspond with a structure containing one carboxyl function, an n-butyl chain with a double bond and a phenyl residue, suggesting the structure ß- methylcinnamic acid for product III. Also comparing HPLC and GC/MS analyses of the available standard, product III was confirmed as ß-methylcinnamic acid. The longer HPLC retention time of product IV (11.4 min) and the similar UV spectrum with product I and II suggested that this compound is a phenylalkanoic acid with a longer branched side chain than the side of product I and II. The mass spectrum, obtained by GC/MS analyses, indicated a molecular weight of m/z 206. The main fragment ion are

at 174 [C 6H5 +CH 3CH(CH 2)3CO/-H], 130 [-CH 3CH(CH 2)3COOCH 3/+H], 117 [C 6H5

+CH 3CHCH 2/-2H], 104 [C 6H5 +CH 3C-], 91 [C 6H5 +CH 2], 87 [-CH 2CH 2COOCH 3], 77

[C 6H5], 74 [CH 3COOCH 3], 59 [-COOCH 3] and 45 [COCH 3/+2H]. UV spectrum, retention

RESULTS 55

time in HPLC, retention time in GC/MS, and mass spectrum led to the suggestion that product IV can be identified as 5-phenylhexanoic acid. Because of the confined sensitivity of analyzing aqueous samples by HPLC, the culture supernatant without cells were extracted at pH 9; 7 and 2 and concentrated. Besides four of the upper products, the neutral and alkaline extract analyses by HPLC and GC/MS pointed out one previously undetectable compound, called as product V, furthermore by GC/MS analyses, another product, designated as product VI was identified, and the acidic extract determined two others, called as VII and VIII (Table 6.2). Product V found in neutral and alkaline extracts suggested a metabolite containing a keto or an alcohol group. The absolute similarity of the UV spectrum and retention time in analyses by HPLC between product V and available standard suggested product V is acetophenone. Moreover, the mass spectrum showed a base peak ion at m/z 120, with

fragment ions at m/z 105 [C 6H5+CCH 3/+H] or 105 [C 6H5+CO], and 77 [C 6H5]. Comparing mass spectra and retention times by GC/MS analyzes, acetophenone was confirmed for product V (Figure 3.15 and Figure 6.28).

mAU mAU 700 O 300 Rt 6.7 min 250 600 200 Product V 150

500 100 50 0 400 300 400 500 nm

300

200 3.671

100 6.671 1.381 13.018 13.396 12.754 9.254 1.705 10.644 9.010 8.797 1.891 0 3.302

2 4 6 8 10 12

Figure 3.15 HPLC elution profiles of the alkaline extract after incubation N. cyriacigeorgica SBUG 1472 with sec -octylbenzene (0.25 ml liter -1; 72 h) and UV/Vis absorption spectra of products. mAU, mili absorbance units.

Metabolite VI was found at a retention time of 17.2 min, matching with the chemicals in library NIST, it was suggested as α-hydroxyacetophenone. Analyzing the available standard, the retention time was observed at 16.9 min. By comparing the mass

RESULTS 56

spectrum fragments, this metabolite was confirmed as α-hydroxyacetophenone (Figure 3.16).

O 2OHACEPH 743 (16.883) Scan EI+ 105 3.73e5 100 Product VI OH 77 OH

% O

51

50 78 106 52 58 65 86 89 133 147 152 39 42 43 62 63 66 74 91 92 98 102 119 120 121 136 137 145 0 3BSC8-04 762 (17.199) Cm (761:766-(744:755+770:775)) Scan EI+ 105 2.98e3 100 α -hydroxyacetophenone

77 %

91 51 78 106 120 148 50 52 119 128 133 145 150 38 43 58 61 65 66 72 75 86 89 9396 101 115 122 130 0 m/z 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105110 115 120 125 130 135140 145 150

Figure 3.16 Mass spectrum of product VI with a GC retention time of 17.2 min, in comparison with a mass spectrum of reference standard of α-hydroxyacetophenone.

Metabolite VII was found in very small quantities and was not detected by HPLC analyses, but by GC/MS analyses. The retention time of this metabolite was at 21.6 min. The mass spectrum of this compound revealed a molecular ion peak at m/z 196 and contained the characteristic fragment ions m/z 163, m/z 149, m/z 135, m/z 122, m/z 107, and m/z 77. Matching with the retention time and the mass spectrum of the available standard, compound VII was confirmed as 2,3-dihydroxybenzoic acid (Figure 3.17, see also in Appendix - Table 6.2) Product VIII was found also in trace amounts and was only detectable by GC/MS. The retention value and fragmentation pattern of the compound was very similar to succinic acid (methylated), thus these obtained data led to the identification of this compound as succinic acid (as described in Appendix- Table 6.2).

RESULTS 57

COOH Product VII Reverse fit factor [REV]: 952 3BAP182 1028 (21.634) Cm (1027:1030-(1016:1025+1032:1041)) OH 5.94e3 163 100 OH 165

166 196 107 % 77 122 197 45 51 79 135 149 53 65 121 167 39 76 92 95 108 123 150 80 136 198 38 69 82 97 181 0 R:952 PHALK 42: 2,3-DIMETHOXY-BENZOESÄURE-METHYLESTER 2,3-dihydroxybenzoic acid Hit 1 163 100 165 196

% 107 77 122 149 45 51 79 135 167 65 53 76 92 121 123 136 150 197 39 91 93 181 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220

Figure 3.17 Mass spectrum of product VI with a GC retention time of 21.6 min, in comparison with a mass spectrum of reference standard. All samples were methylated.

3.2.4.2 Kinetics of the sec-octylbenzene transformation by N.cyriacigeorgica 1472 The substrate sec -octylbenzene is insoluble in water, therefore, in aqueous culture supernatant analyses by HPLC it could not be observed. The main products were formed rapidly after 4 h incubation and represented phenylalkanoic acid with shorter branched side chain, which is soluble in water and then detectable in aqueous supernatants by HPLC (Figure 3.18).

0.16 0.14 0.12 0.1 0.08 0.06

products (µM) products 0.04 0.02 0 0 10 20 30 40 50 60 70 80

time (h)

Figure 3.18 Kinetics of the transformation of sec -octylbenzene (0.025 %) by N. cyriacigeorgica SBUG 1472 as determined by HPLC; , 3–phenylbutyric acid; x, 2– phenylpropionic acid; o, ß–methylcinnamic acid; ♦, 5–phenylhexanoic acid (data are shown as the means ± SD of three independent cultures).

RESULTS 58

The quantity of 3-phenylbutyric acid from the beginning to the end was always higher than that of other acids. Three other products (V, VI and VII) were not detectable in the aqueous supernatant by HPLC analyses, but can be identified by GC/MS analyses of the extracts. It could be that these products took a long time to be created and in case of excretion in very little amounts.

3.2.4.3 Transformation of N. cyriacigeorgica 1472 with intermediate products

2-phenylpropionic acid, 3-phenylbutyric acid, ß-methylcinnamic acid and acetophenone were used as substrates in the biotransformation experiments of tetradecane- precultivated cells of N.cyriacigeorgica to investigate the ring cleave products and to complete the pathway of sec -octylbenzene transformation. The intermediates were analyzed by comparing the retention time and UV of the HPLC elution profile as well as the retention time and mass spectra of the GC/MS analyses with those of available standards. However, N.cyriacigeorgica could not transform 2-phenylpropionic acid.

3.2.4.3.1 Biotransformation of 3-phenylbutyric acid

Using 0.005 % (v/v) of 3-phenylbutyric acid as the substrate, after various times, aqueous sample of the supernatant were analyzed by HPLC, the supernatant was also alkalified and acidified then extracted with equal volumes of ethyl acetate as describe in method 2.4 for GC/MS analyses. It was determined that ß-methylcinnamic acid was the main metabolite of the 3-phenylbutyric acid transformation (Table 3.12 and Figure 3.19).

Table 3.12 Retention time and MS spectra of the identified metabolites produced by N. cyriacigeorgica SBUG 1472 cultured with 0.05 ml litre -1 3-phenylbutyric acid in comparison with standard substances.

Compound tR (min) m/z (%, 70 eV) 20.9 176 (54.9), 175 (45.2), 145 (87.5), 144 (44.5), 117 (49.1), 116 (28.1), 115 (100.0), 102 (11.5), 91 (34.1), 89 (10.1), 77 (13.3),

65 (12.1), 63 (11.1), 51 (18.0), 50 (9.5), 39 (23.1)

COOH 20.9 st 176 (58.2), 175 (47.3), 145 (89.9), 144 (45.3), 117 (46.9), 116

(29.8), 115 (100.0), 102 (11.0), 91 (34.8), 89 (10.0), 77 (13.0), 65 (12.7), 63 (10.6), 51 (18.2), 50 (8.6), 39 (20.7) O 15.1 120 (19.3), 105 (100.0), 77 (92.3), 51 (28.6), 50 (11.0), 43

(15.4) 15.1 st 120 (22.7), 105 (100.0), 77 (90.3), 51 (30.0), 50 (13.7), 43 (15.1) st tR: Retention time in GC/MS; st: purchased standard.

RESULTS 59

mAU 9.503 mAU 140 mAU 600 200 500 120 Substrate 150 400 9.003 COOH 300 100 100 200 50 100 0 0 80 300 400 500 300 400 500 nm nm

60

40 20 1.234 0

-20 2 4 6 8 10 12 14 16 18 min

Figure 3.19 HPLC elution profiles of the aqueous supernatant after incubation N. cyriacigeorgica SBUG 1472 with 3–phenyl butyric acid (0.05 ml liter -1; 72 h) and UV/Vis absorption spectra of substrate and formed products. mAU, mili absorbance units.

Also a small amount of acetophenone was found in the alkaline supernatant extract by HPLC and GC/MS analyses (Table 3.12). The 3-phenylbutyric acid was dehydrogenized after 2 h incubation to produce ß-methylcinnamic acid and after 96 h 34 % of the substrate was transformed to ß-methylcinnamic acid (Figure 3.20).

0.3 0.08

0.07 0.25 0.06 0.2 0.05

0.15 0.04 Product (µM)

0.03

Substrate (µM) 0.1 0.02 0.05 0.01

0 0 0 10 20 30 40 50 60 70 80 90 100 Time (h)

Figure 3.20 Kinetics of the transformation of 3–phenyl butyric acid (0.005 %) by N. cyriacigeorgica SBUG 1472 as determined by HPLC; o, substrate; ♦, ß–methylcinnamic acid (data are shown as the means ± SD of three independent cultures).

RESULTS 60

3.2.4.3.2 Biotransformation of ß methylcinnamic acid By using ß-methylcinnamic acid as a substrate, acetophenone was gained as the major metabolite (Figure 3.31), and 2,3-dihydroxybenzoic acid was detected in the organic extract The detailed results of the metabolite profiles were presented in Table 3.13.

mAU

70 9.415

Cellular mAU 60 20 excretion 15 Substrate 50 10 O 40 product 5 0 30 300 400 500 nm

3.841 20

10 6.815

0

-10

2 4 6 8 10 12

Figure 3.21 HPLC elution profiles of the alkaliphatic extract after incubation N. cyriacigeorgica SBUG 1472 with ß–methylcinnamic acid (0.05 ml liter -1; 72 h) and UV/Vis absorption spectra of products. AU, absorbance units.

Table 3.13 Retention times and UV/Vis-spectra of the identified metabolites produced by N. cyriacigeorgica SBUG 1472 cultured with 0.05 ml litre -1 ß-methylcinnamic acid in comparison with standard substances.

Compound tR (min) m/z (%, 70 eV) 15.1 120 (19.3), 105 (100.0), 77 (92.3), 51 (28.6), 50 (11.0), 43 O (15.4)

15.1st 120 (22.7), 105 (100.0), 77 (90.3), 51 (30.0), 50 (13.7), 43 (15.1) st 21.6 196 (60.1), 167 (15.2), 166 (71.8), 165 (71.2), 163 (100.0), 150 (11.3); 149 (22.4); 135 (25.4), 125 (10.9),

123 (11.2), 122 (37.6), 121 (19.1), 119 (10.3), 108 (10.5),

COOH 107 (42.8), 92 (16.5), 79 (22.7), 77 (41.6), 76 (11.0), 65 (22.1), 53 (11.7), 51 (33.1), 50 (13.3), 45 (30.3)

OH OH 21.7 st 196 (62.4), 167 (16.6), 166 (80.3), 165 (75.2), 163 (100.0), 150 (11.5); 149 (30.0); 135 (27.6), 125 (9.6), 123 (11.3), 122 (36.0), 121 (17.1), 119 (10.9), 108 (10.3), 107 (46.1), 92 (16.2), 79 (20.2), 77 (40.7), 76 (10.7), 65 (20.6), 53 (14.5), 51 (32.2), 50 (14.8), 45 (30.8) tR: Retention time in GC/MS; st: purchased standard.

RESULTS 61

3.2.4.3.3 Biotransformation of acetophenone When acetophenone was used as substrate in addition to 2,3-dihydroxybenzoic acid, succinic acid were detected by GC/MS analyses. The results of the metabolite profiles were described in details in Table 3.14.

Table 3.14 Retention time and MS-spectra of the identified metabolites produced by N. cyriacigeorgica SBUG 1472 cultured with 0.05 ml litre -1 acetophenone in comparison with standard substances.

Compound tR (min) m/z (%, 70 eV) 21.6 196 (60.1), 167 (17.1), 166 (85.8), 165 (72.3), 163 (100.0), 150 (13.1); 149 (27.4); 135 (25.4), 125 (10.1), 123 (12.0),

122 (36.7), 121 (16.9), 119 (10.1), 108 (12.0), 107 (52.2),

COOH 92 (16.5), 79 (21.2), 77 (40.6), 76 (10.1), 65 (20.1), 53 (13.7), 51 (31.9), 50 (14.3), 45 (30.2)

OH OH 21.7 st 196 (62.4), 167 (16.6), 166 (80.3), 165 (75.2), 163 (100.0), 150 (11.5); 149 (30.0); 135 (27.6), 125 (9.6), 123 (11.3), 122 (36.0), 121 (17.1), 119 (10.9), 108 (10.3), 107 (46.1), 92 (16.2), 79 (20.2), 77 (40.7), 76 (10.7), 65 (20.6), 53 (14.5), 51 (32.2), 50 (14.8), 45 (30.8) 9.2 115 (100.0), 114 (34.9), 87 (20.0), 59 (55.7), 57 (7.2), 56 (7.2), 55 (78.2), 45 (2.5), 42 (4.4), 41 (4.7) COOH HOOC 9.2 st 115 (100.0), 114 (30.2), 87 (19.3), 59 (58.2), 57 (7.2), 56 (7.2), 55 (72.7), 45 (12.5), 42 (5.9), 41 (5.2)

tR: Retention time in GC/MS; st: purchased standard.

RESULTS 62

3.2.4.4 Biotransformation of sec-octylbenzene by M. neoaurum 109 The same protocol was used to analyze formed metabolites during the biotransformation of sec -octylbenzene by M. neoaurum 109. It was designated the metabolite names using the same system as the metabolites detected in N. cyriacigeorgica 1472. By HPLC and GC/MS analyses, product II, III and V (3–phenylbutyric acid, ß– methylcinnamic acid and acetophenone, respectively) were confirmed as the major metabolites (Table 3.15, see also in Appendix – Figure 6.29 to Figure 6.31).

Table 3.15 Retention times and UV/Vis-spectra of identified metabolites after incubation of M. neoaurum with 0.25 ml litre -1 sec -octybenzene in comparison with standard substances by HPLC and GC/MS analyses.

1 2 tR tR Met UV/Vis-spectrum m/z (%, 70 eV) (min) (min)

mAU II 9.1 1000 18.6 178 (10.8), 121 (13.3), 119 (6.0), 118

800 (69.7), 106 (6.6), 105 (100.0), 103 (15.9),

600 91 (15.7), 79 (10.8), 78 (9.5), 77 (20.2), 51 COOH 400 (9.96), 41 (7.5), 39 (8.1) 200

0 9.1 st 300 400 500 nm 18.5 178 (11.1), 121 (16.2), 119 (7.1), 118 (81.6), 106 (6.5), 105 (100.0), 103 (17.6), 91 (15.5), 79 (9.1), 78 (9.2), 77 (19.5), 51 (11.6), 41 (9.4), 39 (11.3) mAU III 9.6 160 20.8 176 (64.4), 175 (51.7), 145 (93.7), 144 140 (43.5), 117 (44.1), 116 (30.5), 115 (100.0), 120 100 102 (11.1), 91 (33.9), 89 (9.5), 77 (12.9), 65 80 60 COOH (11.8), 63 (9.7), 51 (15.8), 50 (7.9), 39 40 (19.3) 20 0 300 400 500 nm 9.6 st 20.9 st 176 (58.2), 175 (47.3), 145 (89.9), 144 (45.3), 117 (46.9), 116 (29.8), 115 (100.0), 102 (11.0), 91 (34.8), 89 (10.0), 77 (13.0), 65 (12.7), 63 (10.6), 51 (18.2), 50 (8.6), 39 (20.7) mAU V 6.7 500 O 11.0 120 (14.4), 105 (100.0), 77 (91.2), 51 400 (23.2), 50 (9.8), 300

200 6.5 st 100 11.0st 120 (22.7), 105 (100.0), 77 (90.3), 51 0 (30.0), 50 (13.7), 43 (15.1) 300 400 500 nm 1 2 Met: metabolite; tR : Retention time in HPLC; tR : Retention time in GC/MS; st: purchased standard.

RESULTS 63

Product I and VIII were identified by GC/MS analyses (Table 3.16, see also Appendix – Figure 6.32 and Figure 6.33).

Table 3.16 Retention times and MS-spectra of identified metabolites after incubation of M. neoaurum with sec -octybenzene in comparison with standard substances by GC/MS analyses.

Met Structure tR (min) m/z (%, 70 eV) I 16.1 164 (13.0), 119 (15.1), 105 (100.0), 79 (10.4), 78 (4.1), 77 (14.9), 51 (5.9), 39 (5.1)

164 (22.4), 105 (100.0), 79 (12.9), 78 (5.6), 77 COOH 16.2 st (17.6), 51 (7.5), 39 (3.8) VIII 9.2 115 (100.0), 114 (24.9), 87 (10.0), 59 (53.7), 57 (7.2), 56 (7.2), 55 (72.2), 45 (20.5), 42 (4.7), 41

COOH (4.7) HOOC 9.2 st 115 (100.0), 114 (30.2), 87 (19.3), 59 (58.2), 57 (7.2), 56 (7.2), 55 (72.7), 45 (12.5), 42 (5.9), 41 (5.2) Met: metabolite; t R: Retention time in GC/MS; st: purchased standard. In addition to these described upper metabolites, analyzing alkaline extract by GC/MS at retention time 23.7 min, another important product was observed and called as product IX, a ketone compound. Mass spectra of the compound were reported in Figure 3.22.

43 1.11e3 100

O 161 57

+71 +57 +43 +99 +85 -105 -119 -- 133 -147 -161 105 % 71

41 85

143 144 77 162 55 79 106 37 97 99 113 142 44 69 76 118 134 186 189 204 52 62 91 148 154 171 177 198 0 m/z 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200

Figure 3.22 Mass spectra of product IX named 7-phenyl-octan-2-one identified in cultivation of M. neoaurum with sec-octybenzene.

RESULTS 64

The mass spectra of this compound showed a molecular ion peak at m/z 204. The

main fragment ion at m/z 161 (M-CH 3CO), 105 [M-(CH 2)4C(O)CH 3], 85 [-

(CH 2)3C(O)CH 3], 77 (C 6H5), 71 [-(CH 2)2C(O)CH 3], 57 [-CH 2C(O)CH 3], and 43 (-CH 3CO) correlated with a structure that have a ketone group, an n-octyl chain and a phenyl ring. In comparison with the mass data of the substrate (data not shown), product IX was defined as 7-phenyl-octan-2-one.

3.2.4.5 Kinetics of the sec-octylbenzene transformation by M. neoaurum 109 According to HPLC data, 3 metabolites were found to include 3–phenylbutyric acid, 5–phenylbutyric acid and acetophenone ( Figure 3.23 ). They were all occured after incubating bacteria 8 h under growing conditions, the amount of these metabolitse kept raising up to 48h. The sarturation occurred after 48h. Among these metabolites, 3– phenylbutyric acid was detected at the highest concentration of 0.12 µM during the cultivation; the others were about 0.05 µM.

0.14 0.12 0.1

0.08 0.06 0.04 Products (µM) 0.02

0 0 10 20 30 40 50 60 70 80

Time (h)

Figure 3.23 Kinetics of the transformation of sec -octylbenzene (0.025 ml litre -1) by M. neoaurum SBUG 109 as determined by HPLC; , 3–phenylbutyric acid; x, 5–phenylhexanoic acid; , acetophenone (data are shown as the means ± SD of three independent cultures).

RESULTS 65

3.2.4.6 Transformation of M. neoaurum 109 with intermediate products 3.2.4.6.1 Biotransformation of 3-phenylbutyric acid When 3-phenylbutyric acid was used as the substrate, based on the molar yield, only 2 % of this substance was transformed. Analyzing with HPLC, a small amount of ß- methylcinnamic acid (product III) and benzoic acid was detected with retention times at 9.4 min and 6.4 min, respectively (Figure 3.24 and Table 3.17). GC/MS data of the acid extracts confirmed these results. Benzoic acid was named as product X (Appendix Figure 6.34).

Table 3.17 Retention times and UV/Vis-spectra of product III and X during inbation of M. neoaurum 109 with 0.05 ml litre -1 of 3-phenylbutyric acid.

1 2 Met tR UV/Vis-spectrum tR m/z (%, 70 eV) (min) (min) III 9.3 20.8 176 (54.4), 175 (47.7), 145 (91.7), 144 (41.9),

mAU 117 (44.3), 116 (30.1), 115 (100.0), 102 160 140 (12.1), 91 (32.8), 89 (9.5), 77 (12.9), 65 120 (11.8), 63 (9.1), 51 (18.5), 50 (7.9), 39 (19.7) 100 80 60 9.6st COOH 20.9 st 176 (58.2), 175 (47.3), 145 (89.9), 144 (45.3), 40 20 117 (46.9), 116 (29.8), 115 (100.0), 102 0 (11.0), 91 (34.8), 89 (10.0), 77 (13.0), 65 300 400 500 nm (12.7), 63 (10.6), 51 (18.2), 50 (8.6), 39 (20.7) X 6.3 mAU 12.5 136 (32.9), 106 (7.4), 105 (100.0), 77 (69.6), 200 COOH 51 (27.8), 50 (12.7) 150

100

6.3 st 50 12.3 st 136 (33.5), 106 (6.3), 105 (100.0), 77 (74.6), 0 51 (28.9), 50 (14.5) 300 400 500 nm 1 2 Met: metabolite; t R : Retention time in HPLC; t R : Retention time in GC/MS; st: purchased standard.

RESULTS 66

mAU

120 mAU 8.938 Substrate mAU 200 15 100 150 10

100 COOH Substrate 5 80 COOH 50 0 0 60 300 400 500 nm 300 400 500 nm

40

20 9.392

0 6.398

-20

2 4 6 8 10 12 14 16 18 min

Figure 3.24 HPLC elution profiles of the aqueous supernatant after incubation M. neoaurum 109 with 3-phenylbutyric acid (0.05 ml liter -1; 72 h) and UV/Vis absorption spectra of products. mAU, mili absorbance units.

In comparison with the mass data of other standard chemicals, in addition to succinic acid (product XIII) which was also detected in the acid phase, acetophenone (product V) was found in alkaline phase. Furthermore, 3,4–dihydroxybenzoic acid was estimated as the product XI and had the same retention time at 23.5 min as the available standard. The mass spectra showed the molecular weight at m/z 196 (100.0 %) and contained the base peak ion at m/z 165 (98.5 %), and fragment ions occurred at m/z 125 (14.5 %), 97 (10.6 %), 83 (14.3 %), 79 (32.2 %), 77 (12.0 %), 51 (15.5 %), 50 (11.6 %), 43 (43.0 %), 41 (38.6 %), 39 (18.2). The standard had also a molecular weight of m/z 196 (84.0 %) and contained the base peak ion at m/z 165 (100.0 %), and fragment ion at m/z 125 (11.8 %), 97 (10.6 %), 83 (10.3 %), 79 (31.2 %), 77 (18.1 %), 51 (18.9 %), 50 (8.8 %), 43 (43.0 %), 41 (28.6 %), 39 (15.2). The fragmentation pattern of the compound and standard was identical but the intensities of the fragment ions were different. By comparing with product VII (2,3-dihydroxybenzoic acid), the compound had the same molecular weight but appeared 2 minutes after product VII (retention time 21.6 min in GC/MS analyses) and fragments were different. Therefore, product XI was confirmed as 3,4– dihydroxybenzoic acid (Appendix-Figure 6.35).

RESULTS 67

3.2.4.6.2 Biotransformation of ß-methylcinnamic acid Analyzing HPLC and GC/MS data for the extracts of supernatant without cell acetophenone was determined with all characteristics as distinguished in as a metabolite (Table 3.18).

Table 3.18 Retention time and UV/Vis-spectrum of the identified metabolites in culture of M. neoaurum with 0.05 ml litre -1 ß-methylcinnamic acid in comparison with standard substance.

1 2 tR tR Met UV-spectrum m/z (%, 70 eV) (min) (min)

mAU V 6.7 500 O 10.9 120 (21.9), 105 (100.0), 77 (90.5), 51 400 (30.2), 50 (15.7), 43 (16.7)

300 6.5 st 200 11.0st 120 (22.7), 105 (100.0), 77 (90.3), 51 100 (30.0), 50 (13.7), 43 (15.1) 0 300 400 500 nm 1 2 Met: metabolite; t R : Retention time in HPLC; t R : Retention time in GC/MS; st: purchased standard.

3.2.4.6.3 Biotransformation of 2-phenylpropionic acid Alternatively to N. cyriacigeorgica 1472, by analyzing with HPLC and GC/MS, it was observed that M. neoaurum 109 could transform a little amount of 2–phenylpropionic acid into acetophenone and benzoic acid. UV/Vis–spectra and mass spectra as well as retention times were summarized in Table 3.19.

RESULTS 68

Table 3.19 Retention times and UV/Vis-spectra of the identified metabolites during incubation of M. neoaurum with 0.05 ml litre -1 2-phenylpropionic acid in comparison with standard substances.

1 2 tR tR Met UV/Vis-spectrum m/z (%, 70 eV) (min) (min)

mAU V 6.7 500 O 10.9 120 (20.9), 105 (100.0), 77 (93.5), 51 400 (32.2), 50 (14.7), 43 (17.6)

300 6.5 st 200 11.0st 120 (22.7), 105 (100.0), 77 (90.3), 51 100 (30.0), 50 (13.7), 43 (15.1) 0 300 400 500 nm X 6.3 mAU 12.3 136 (31.2), 106 (5.4), 105 (100.0), 77 COOH 200 (71.3), 51 (29.4), 50 (13.2) 150

6.3 st 100 12.3st 136 (33.5), 106 (6.3), 105 (100.0), 77 50 (74.6), 51 (28.9), 50 (14.5) 0 300 400 500 nm 1 2 Met: metabolite; tR : Retention time in HPLC; t R : Retention time in GC/MS; st: standard. 3.2.4.6.4 Biotransformation of acetophenone As the result of biotransformation of acetophenone, in addition to benzoic acid ditermined by HPLC and GC/MS analyses, 3, 4 – dihydroxybenzoic acid was identified by GC/MS analyses by comparing with available standards (Table 3.20).

Table 3.20 Retention time and MS-spectra of the identified metabolites in cultivation of M. neoaurum with 0.05 ml litre -1 acetophenone in comparison with standard substances.

Met Structure tR (min) m/z (%, 70 eV) X 12.3 136 (32.1), 106 (4.4), 105 (100.0), 77 (73.1), 51 COOH (28.4), 50 (12.3) 12.3 st 136 (33.5), 106 (6.3), 105 (100.0), 77 (74.6), 51 (28.9), 50 (14.5) V 23.6 196 (87.4), 165 (100.0), 125 (12.3), 97 (15.6), OH 83 820.3), 79 (28.0), 77 (17.0), 69 (23.3), 57

OH (18.3), 56 (13.0), 51 (16.6), 43 (37.5), 41 (32.0), 39 (11.8) 23.6 st 196 (84.1), 165 (100.0), 125 (11.8 ), 97 (10.6 ), COOH 83 (10.3 ), 79 (31.2 ), 77 (18.1 ), 51 (18.9 ), 50 (8.8 ), 43 (43.0 ), 41 (28.6 ), 39 (15.2) Met: metabolite; t R: Retention time in GC/MS; st: purchased standard. 3.2.4.6.5 Biotransformation of benzoic acid 0.05 g litre -1 of benzoic acid was used as substrate, but no metabolite was detected and after 48 h, this compound still maintained in the culture supernatant.

RESULTS 69

3.2.4.7 Biotransformation of sec-octylbenzene by R. ruber 82 Detection by HPLC showed that product II (3–phenylbutyric acid) and III (ß– methylcinnamic acid) were the main metabolites in the aqueous supernatant of R. ruber 82 incubated with 0.25 ml litre -1 of sec -octylbenzene. Analyzing the extracts of cell-free supernatants by HPLC after 24 h, 48 h, 72 h and 96 h incubation, in addition to product I

(t R 7.9 min), another product, called as product XII, was found at a retention time of 6.0 min (Figure 3.25 and Table 3.21). By comparing UV/Vis-spectra and mass spectra as well as retention in both HPLC and GC/MS this compound was defined as phenylacetic acid (at retention time 15.4 min in GC/MS analyses) with the major fragment ions at m/z 150 [M],

91 [M-COOCH 3] and 77 [M-CH 2COOCH 3] (Appendix – Figure 6.36 to Figure 6.39).

mAU Cellular 9.382 600 Product III excretion

500

2.670 product Product II 400

300 Product I

200 Product XII

100 16.700 8.893 1.382 5.997 7.78 13.844 12.695 3.534

3.859 0

2 4 6 8 10 12 14 16 18 min

Figure 3.25 HPLC elution profiles of the acid extract after incubation of R. ruber SBUG 82 with sec -octylbenzene (0.25 ml liter -1; 72 h). mAU, mili absorbance units.

Moreover, after 8 h of incubation, phenylacetic acid (product XII) and after 54 h, benzoic acid (product X) appeared as trace metabolites in aqueous supernatant (Table 3.21 and Appendix-Figure 6.40). However, after 72 h, in aqueous samples, no more benzoic acid was observed.

RESULTS 70

Table 3.21 Retention times and UV/Vis-spectra of the identified metabolites during incubation of R. ruber with 0.25 ml litre -1 sec -octybenzene in comparison with standard substances by HPLC and GC/MS analyses. 1 2 tR tR Met UV/Vis-spectrum m/z (%, 70 eV) (min) (min)

mAU I 7.9 60 164 (21.5), 105 (100.0), 79 (12.1), 78 (5.1), 16.2 50 77 (16.4), 51 (7.2), 39 (4.0)

40 30 COOH 20 164 (22.4), 105 (100.0), 79 (12.9), 78 (5.6), 7.9 st 10 77 (17.6), 51 (7.5), 39 (3.8) 0 16.2 st 300 400 500 nm II 9.1 18.6 178 (11.8), 121 (12.3), 119 (7.0), 118 (79.7), 106 (6.1), 105 (100.0), 103 (16.9), mAU 1000 91 (15.1), 79 (10.1), 78 (8.5), 77 (20.2), 51 800 (9.9), 41 (8.5), 39 (8.1)

600 COOH 400 9.1 st 18.5 178 (11.1), 121 (16.2), 119 (7.1), 118 200 (81.6), 106 (6.5), 105 (100.0), 103 (17.6), 0 300 400 500 nm 91 (15.5), 79 (9.1), 78 (9.2), 77 (19.5), 51 (11.6), 41 (9.4), 39 (11.3) III 9.6 20.8 176 (54.4), 175 (41.7), 145 (91.2), 144

mAU (43.1), 117 (45.1), 116 (29.5), 115 (100.0), 160 140 102 (11.1), 91 (32.9), 89 (9.5), 77 (12.9), 120 65 (11.9), 63 (9.7), 51 (15.8), 50 (8.0), 39 100 80 (19.3) COOH 60 20.9 st 9.6st 40 20 176 (58.2), 175 (47.3), 145 (89.9), 144 0 (45.3), 117 (46.9), 116 (29.8), 115 (100.0), 300 400 500 nm 102 (11.0), 91 (34.8), 89 (10.0), 77 (13.0), 65 (12.7), 63 (10.6), 51 (18.2), 50 (8.6), 39 (20.7) X 6.3 mAU 12.3 136 (30.2), 106 (6.4), 105 (100.0), 77 COOH 200 (75.3), 51 (29.1), 50 (11.2)

150 6.3 st 100 12.3 st 136 (33.5), 106 (6.3), 105 (100.0), 77 50 (74.6), 51 (28.9), 50 (14.5) 0 300 400 500 nm

mAU XII 6.0 20 15.4 150 (27.4), 92 (8.1), 91 (100.0), 65 (14.2), 17.5 COOH 39 (5.1) 15 12.5 10 6.1 st 7.5 15.1 150 (29.1), 92 (8.1), 91 (100.0), 65 (14.7), 5 39 (6.1) st 2.5 0 300 400 500 nm 1 2 Met: metabolite; t R : Retention time in HPLC; t R : Retention time in GC/MS; st: purchased standard.

RESULTS 71

By GC/MS analyses of the acid extracts, benzoic acid, succinic acid and 2,3– dihydroxybenzoic acid were characterized with the reference data described in Table 3.22 (see also Appendix - Figure 6.40 and Figure 6.41).

Table 3.22 Retention times and MS-spectra of the identified metabolites produced by R. ruber SBUG 82 cultured with 0.25 ml litre -1 sec -octybenzene in comparison with standards.

Compound tR (min) m/z (%, 70 eV) 21.6 196 (61.1), 167 (16.1), 166 (81.8), 165 (78.3), 163 (100.0), 150 (12.1); 149 (22.4); 135 (24.1), 125 (10.4), 123 (12.9), 122

(36.2), 121 (17.9), 119 (10.5), 108 (13.0), 107 (42.2), 92 COOH (16.1), 79 (21.2), 77 (40.6), 76 (10.1), 65 (20.1), 53 (12.7), 51

(31.9), 50 (14.4), 45 (30.4) OH 21.7 st 196 (62.4), 167 (16.6), 166 (80.3), 165 (75.2), 163 (100.0), 150 OH (11.5); 149 (30.0); 135 (27.6), 125 (9.6), 123 (11.3), 122 (36.0), 121 (17.1), 119 (10.9), 108 (10.3), 107 (46.1), 92 (16.2), 79 (20.2), 77 (40.7), 76 (10.7), 65 (20.6), 53 (14.5), 51 (32.2), 50 (14.8), 45 (30.8) 9.2 115 (100.0), 114 (31.2), 87 (20.0), 59 (57.5), 57 (7.2), 56 (7.2),

COOH 55 (78.2), 45 (10.0), 42 (3.5), 41 (4.7) HOOC 9.2 st 115 (100.0), 114 (30.2), 87 (19.3), 59 (58.2), 57 (7.2), 56 (7.2), 55 (72.7), 45 (12.5), 42 (5.9), 41 (5.2) tR: Retention time in GC/MS; st: purchased standard. GC/MS analyses of the alkaline extract revealed a phenylalkanol metabolite at a retention time of 22.2 min, designated as product XIII. Mass spectra of this metabolite resulted in a molecular weight of 206 and contained of significant fragment ions at m/z 191

(M-CH 3), 161 [M-CH(OH)CH 3], 147 [M-CH 2CH(OH)CH 3], 133 [M-(CH 2)2CH(OH)CH 3],

119 [M-(CH2)3CH(OH)CH 3], 105 [M-(CH 2)4CH(OH)CH 3], 91 [C 6H5CH/ +H], and 77

[C 6H5] representing a structure consisting of an n-alkanol chain with 8 carbon atoms and a phenyl ring, indicating that the structure of product XIII was 7–phenyl–octan–2–ol (Figure 22.15071487 3.26). NOSEC119 1059 (22.151) Cm (1059:1061-(1047:1053+106m/z4:1066)) 206 Scan EI+ 161 3.18e4 100

OH

119 147 161 191 105 133 191 % 119 57 91 41 117

43 105 192 115 162 77 39 149 103 163 206 65 74 79 107 120 131 51 55 63 73 92 147 193 89 133 150 164 175 200 207 214 50 88 95 176 183 0 m/z 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 Figure 3.26 Mass spectrum of product XIII identified 7–phenyl–octan–2–ol after incubation of R. ruber SBUG 82 with sec -octylbenzene (0.25 ml liter -1; 72 h) by GC/MS.

RESULTS 72

3.2.4.8 Kinetics of the sec-octylbenzene transformation by R. ruber 82 During transformation experiments of each sampling period, 100 µl of supernatant was analyzed by HPLC. The metabolites were determined in comparison with the UV/Vis- spectra and retention time of the available standards. According to the relation between the amount of the standard and its peak area, the concentration of metabolites was calculated and their kinetics were presented in Figure 3.27.

0.03

0.025

0.02

0.015

0.01 Products (µM) Products

0.005

0 0 20 40 60 80 100

Time (h)

Figure 3.27 Kinetics of product formation during transformation of sec -octylbenzene (0.25 ml litre -1) by R. ruber SBUG 82 as determined by HPLC; , 3–phenylbutyric acid; ▲, 5– phenylhexanoic acid; x, phenylacetic acid; ○, benzoic acid (data are shown as the means ± SD of three independent cultures).

It was shown that 5–phenylhexanoic acid occuring in the incubation fluid with a high molarity of 0.025 µM; 3–phenylbutyric acid was detected after 4 h of cultivation and slightly reduced after 24 h. Also phenylacetic acid and benzoic acid appeared in minor amounts in the culture.

RESULTS 73

3.2.4.9 Transformation of R. ruber 82 with intermediate products 3.2.4.9.1 Biotransformation of 3–phenylbutyric acid Based on the molar yield, 3–phenylbutyric acid was estimated to decrease 25 % after 96 h of biotransformation. ß–methylcinnamic acid was the major product of the 3– phenylbutyric acid transformation (Figure 3.28).

mAU

9 .3 8 7 1750 Substrate mAU 1500 1250 1500 mAU 1000 100 750 COOH 1250 80 COOH 500 60 250 40 0 1000 20 300 400 500 nm 0 750 300 400 500 nm

500 8 .8 5 7

250 2 .6 7 7

1 .3 7 9 3 .7 6 9 16 .700 6 .0 0 5 3 .5 3 0 13.84 9 12.70 6 0 1 .7 3 6 2 4 6 8 10 12 14 16 18 min

Figure 3.28 HPLC elution profiles of the acidic extract after incubation R. ruber SBUG 82 with 3–phenyl butyric acid (0.05 g liter -1; 72 h) and UV/Vis absorption spectra of products. mAU, mili absorbance units. It was determined after 4 h of cultivation and maintained with a large amount, 0.1 µM (Figure 3.29).

0.3 0.12

0.25 0.1

0.2 0.08

0.15 0.06

0.1 0.04 (µM) Product Substrate (µM) Substrate

0.05 0.02

0 0 0 10 20 30 40 50 60 70 80 90 100

Time (h)

Figure 3.29 Kinetics of substrate oxidation and product formation of 3–phenyl butyric acid (0.05 g litre -1) by R. ruber SBUG 82 as determined by HPLC; o, substrate; ♦, ß– methylcinnamic acid (data are shown as the means ± SD of three independent cultures).

RESULTS 74

In aqueous supernatants analyses by HPLC, phenylacetic acid was not detected. However, analyses of the acid extract by HPLC and GC/MS showed the presence of this acid. Additionally, 2–phenylpropionic acid was found in the acid extract if analyzed by GC/MS (Table 3.23).

Table 3.23 Retention time and UV/Vis-spectra of the identified metabolites in cultivation of R. ruber with 0.025 g liter -1 3–phenyl butyric acid in comparison with standard substances by HPLC and GC/MS analyses.

UV/Vis- Met t 1 (min) t 2 (min) m/z (%, 70 eV) R spectrum R I 16.2 164 (20.1), 105 (100.0), 79 (12.5), 78

(5.1), 77 (16.1), 51 (7.3), 39 (4.0) 16.2 st 164 (22.4), 105 (100.0), 79 (12.9), 78 COOH (5.6), 77 (17.6), 51 (7.5), 39 (3.8) III 9.6 20.8 176 (56.4), 175 (45.7), 145 (90.2), 144 mAU 160 (42.1), 117 (44.9), 116 (25.5), 115 140 120 (100.0), 102 (11.1), 91 (32.4), 89 (9.9),

100 77 (12.9), 65 (11.9), 63 (9.7), 51 (16.8), 80 9.6st COOH 50 (8.1), 39 (19.3) 60 40 20.9 st 176 (58.2), 175 (47.3), 145 (89.9), 144 20 0 (45.3), 117 (46.9), 116 (29.8), 115 300 400 500 nm (100.0), 102 (11.0), 91 (34.8), 89 (10.0), 77 (13.0), 65 (12.7), 63 (10.6), 51 (18.2), 50 (8.6), 39 (20.7) mAU XII 6.0 20 15.3 150 (27.4), 92 (8.1), 91 (100.0), 65 17.5 COOH (14.2), 39 (5.1) 15 12.5

10 150 (29.1), 92 (8.1), 91 (100.0), 65 6.1 st 7.5 15.1 5 (14.7), 39 (6.1) st 2.5 0 300 400 500 nm 1 2 Met: metabolite; t R : Retention time in HPLC; t R : Retention time in GC/MS; st: purchased standard.

RESULTS 75

3.2.4.9.2 Biotransformation of ß–methylcinnamic acid Using 0.025 g litre -1 of ß–methylcinnamic acid as substrate for the transformation, after 24 h of incubation a trace amount of benzoic acid and phenylacetic acid was detected at retention times of 6.4 min and 6.0 min, respectively (Figure 3.30 and Table 3.24).

mAU

1600 1 .2 8 4

1400

1200 Substrate 1000 Benzoic acid 800 Phenylacetic acid 600 9 .4 0 6 400 1 .1 0 6

200 6 .0 2 4

4 .2 2 3 0 6 .4 5 5

2 4 6 8 10 12 14 16 18 min

Figure 3.30 HPLC elution profiles of the aqueous supernatant after incubation R. ruber SBUG 82 with ß–methylcinnamic acid (0.025 g liter -1; 24 h). mAU, mili absorbance units.

Table 3.24 Retention times and UV/Vis-spectra of the identified metabolites during incubation of R. ruber with 0.025 g liter -1 ß–methylcinnamic acid in comparison with standard substances by HPLC and GC/MS analyses.

1 2 tR tR Met UV/Vis-spectrum m/z (%, 70 eV) (min) (min) X 6.4 mAU 12.3 136 (32.2), 106 (6.1), 105 (100.0), 77 200 COOH (74.3), 51 (29.0), 50 (12.2)

150

6.3 st 100 12.3 st 136 (33.5), 106 (6.3), 105 (100.0), 77 50 (74.6), 51 (28.9), 50 (14.5) 0 300 400 500 nm

mAU XII 6.0 20 15.4 150 (28.4), 92 (8.1), 91 (100.0), 65 (14.5), 17.5 COOH 39 (5.4) 15 12.5 10 6.1 st 7.5 15.1 150 (29.1), 92 (8.1), 91 (100.0), 65 (14.7), 5 39 (6.1) st 2.5 0 300 400 500 nm 1 2 Met: metabolite; t R : Retention time in HPLC; t R : Retention time in GC/MS; st: purchased standard.

RESULTS 76

3.2.4.9.3 Biotransformation of 2-phenylpropionic acid Although in the biotransformation of sec -octylbenzene, 2–phenypropionic acid was not detectable as a metabolite, the biotransformation of 3–phenylbutyric acid resulted in the formation of 2–phenypropionic acid that was detected by GC/MS. Therefore, the compound was also used as substrate with the concentration of 0.005 % (v/v). Unlike to the biotransformation of 2–phenylpropionic acid by N. cyriacigeorgica 1472 where no metabolite was formed, in the transformation of 2–phenylpropionic acid by R. ruber 82, during HPLC and GC/MS analyses 4 metabolites were detectable and identified as acetophenone, phenylacetic acid (by HPLC and GC/MS – presented in Table 3.25), 2,3-dihydroxybenzoic acid and succinic acid (by GC/MS – presented in Table 3.26). Amongst these metabolites only acetophenone was found during biotransformation by M. neoaurum 109.

Table 3.25 Retention times and UV/Vis-spectra of the identified metabolites during incubation of R. ruber with 0.05 ml litre -1 2-phenylpropionic acid in comparison with standard substances by HPLC and GC/MS analyses.

1 2 tR tR Met UV/Vis-spectrum m/z (%, 70 eV) (min) (min)

mAU XII 6.2 20 15.2 150 (28.6), 92 (8.5), 91 (100.0), 65 17.5 COOH 15 (14.1), 39 (5.5) 12.5 10 6.1 st 7.5 15.1 150 (29.1), 92 (8.1), 91 (100.0), 65 5 2.5 st (14.7), 39 (6.1) st 0 300 400 500 nm mAU V 6.7 500 10.9 120 (21.7), 105 (100.0), 77 (90.1), 51 400 O (30.1), 50 (13.7), 43 (17.1)

300 6.5 st 200 11.0 120 (22.7), 105 (100.0), 77 (90.3), 51

100 (30.0), 50 (13.7), 43 (15.1) 0 st 300 400 500 nm 1 2 Met: metabolite; t R : Retention time in HPLC; t R : Retention time in GC/MS; st: purchased standard.

RESULTS 77

Table 3.26 Retention times and MS-spectra of the identified metabolites produced by R. ruber SBUG 82 cultured with 0.05 ml litre -1 2-phenylpropionic acid in comparison with standards.

Compound tR (min) m/z (%, 70 eV) 21.6 196 (62.1), 167 (16.2), 166 (80.8), 165 (78.2), 163 (100.0), 150 (12.6); 149 (22.2); 135 (22.1), 125 (11.4),

123 (12.9), 122 (36.6), 121 (17.9), 119 (10.2), 108 (13.0),

COOH 107 (42.2), 92 (16.1), 79 (21.4), 77 (40.6), 76 (10.1), 65 (20.1), 53 (12.7), 51 (31.9), 50 (14.1), 45 (30.3)

OH OH 21.7 st 196 (62.4), 167 (16.6), 166 (80.3), 165 (75.2), 163 (100.0), 150 (11.5); 149 (30.0); 135 (27.6), 125 (9.6), 123 (11.3), 122 (36.0), 121 (17.1), 119 (10.9), 108 (10.3), 107 (46.1), 92 (16.2), 79 (20.2), 77 (40.7), 76 (10.7), 65 (20.6), 53 (14.5), 51 (32.2), 50 (14.8), 45 (30.8) 9.2 115 (100.0), 114 (30.1), 87 (21.0), 59 (58.5), 57 (7.2), 56 (7.1), 55 (73.2), 45 (11.6), 42 (3.5), 41 (5.7) COOH HOOC 9.2 st 115 (100.0), 114 (30.2), 87 (19.3), 59 (58.2), 57 (7.2), 56 (7.2), 55 (72.7), 45 (12.5), 42 (5.9), 41 (5.2)

tR: Retention time in GC/MS; st: purchased standard.

3.2.4.9.4 Biotransformation of acetophenone When using acetophenone as a substrate, 2,3-dihydroxybenzoic acid and succinic acid were detected in the culture supernatant of N. cyriacigeorgica 1472; benzoic acid and 3,4–dihydroxybenzoic acid were found with M. neoaurum 109. In contrast, in culture of R. ruber SBUG 82 with acetophenone, by comparing GC/MS data with the available standards, in addition to 3,4–dihydroxybenzoic acid and succinic acid (Table 3.27), D,L– mandelic acid was detected at retention time 18.0 min (Figure 3.31 and Table 3.27).

RESULTS 78

Table 3.27 Retention times and MS-spectra of the identified metabolites in cultivation of R. ruber SBUG 82 with 0.05 ml litre -1 acetophenone comparing with standard substances.

Met Structure tR (min) m/z (%, 70 eV)

V OH 23.6 196 (87.4), 165 (100.0), 125 (12.3), 97 (15.6), 83 OH 820.3), 79 (28.0), 77 (17.0), 69 (23.3), 57 (18.3), 56 (13.0), 51 (16.6), 43 (37.5), 41 (32.0), 39 (11.8) 23.6 st 196 (84.1), 165 (100.0), 125 (11.8 ), 97 (10.6 ), 83 COOH (10.3 ), 79 (31.2 ), 77 (18.1 ), 51 (18.9 ), 50 (8.8 ), 43 (43.0 ), 41 (28.6 ), 39 (15.2) XIV 18.0 166 (6.0), 108 (8.6), 107 (100.0), 105 (12.1), 79 (81.3), 78 (8.8), 77 (62.8), 51 (18.1), 50 (6.8) 39 (8.1)

HOOC OH 17.7 st 166 (6.0), 108 (10.1), 107 (100.0), 105 (12.2), 79 (83.1), 78 (8.2), 77 (60.8), 51 (16.9), 50 (5.9) 39 (7.1) VIII 9.2 115 (100.0), 114 (29.9), 87 (19.0), 59 (53.7), 57 (7.2), 56 (7.0), 55 (74.2), 45 (10.5), 42 (4.7), 41

COOH (4.4) HOOC 9.2 st 115 (100.0), 114 (30.2), 87 (19.3), 59 (58.2), 57 (7.2), 56 (7.2), 55 (72.7), 45 (12.5), 42 (5.9), 41 (5.2)

Met, metabolite; t R, retention time in GC/MS; st, purchased standard. Reverse fit factor [REV]: 949 NOAP112 810 (17.999) Cm (809:815-(799:806+824:830)) 5.26e3 107 100 Product XIV 79 77 HOOC OH %

51 105 59 80 111 39 41 50 52 63 115 166 38 69 74 89 125 139 0 R:949D,L-mandelic PHALK 57: D,L-MANDELSÄURE-METHYLESTER acid Hit 1 107 100

79

77 %

51 105 108 50 80 38 39 52 63 74 89 90 166 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 Figure 3.31 Mass spectra of product XIV identified as D,L–mandelic acid during biotransformation of 0.05 ml litre -1 of acetophenone by R. ruber in comparison with a standard. All samples were methylated.

RESULTS 79

3.2.4.9.5 Biotransformation of benzoic acid 0.025 g litre -1 of benzoic acid was used as the substrate to transformation experiments by R. ruber 82. After incubating 4h, the substrate was consumed. The cell-free supernatants were extracted at acidic and alkaline conditions followed by HPLC analyses. Phenylacetic acid and trace of salicylic acid were found at retention times of 5.8 min and 7.1 min, respectively, and compared with UV/Vis–spectra with available standards (Figure 3.32).

mAU mAU 20 16.601 17.5 COOH 100 15 12.5 mAU COOH 10 250 80 7.5 200 5 150 OH 2.498 2.5 0 100 60 300 400 500 nm 50 0 40 300 400 500 nm 5.810

20 13.782 0 3.303 7.123 -20

2 4 6 8 10 12 14 16 18 min

Figure 3.32 HPLC elution profiles of the aqueous supernatant after incubation R. ruber SBUG 82 with benzoic acid (0.025 g liter -1; 24 h) and UV/Vis absorption spectra of product. mAU, mili absorbance units.

RESULTS 80

3.2.4.10 Biotransformation of sec-octylbenzene by T. mucoides SBUG-Y 801 The biotransformation of sec –octylbenzene by T. mucoides SBUG-Y 801 was examined by HPLC and GC/MS analyses. However, only 3–phenylbutyric acid and 2– phenylpropionic acid could be found in a trace amounts even in the extract samples (Table 3.28, see also Appendix - Figure 6.42 and Figure 6.43). To prove the capacity of T. mucoides SBUG-Y 801, to transform other compounds including 3–phenylbutyric acid, ß–methylcinnamic acid, 2–phenylpropionic acid and acetophenone, similar experimental procedures were applied. Analyzed by HPLC and GC/MS, only ß–methylcinnamic acid was detected as the major metabolite of 3- phenylbutyric acid transformation by T. mucoides SBUG-Y 801 (Table 3.28 and Figure 6.44).

Table 3.28 Retention times and UV/Vis-spectra of identified metabolites during incubation of T. mucoides SBUG-Y 801 with 0.25 ml litre -1 sec -octybenzene in comparison with standard substances by HPLC and GC/MS analyses.

1 2 tR tR Met UV-spectrum m/z (%) (70 eV) (min) (min)

mAU I 7.9 60 16.2 164 (22.5), 105 (100.0), 79 (11.1), 78 (5.1), 50 77 (15.4), 51 (7.9), 39 (3.0)

40 30 COOH 7.9 st 20 16.2 st 164 (22.4), 105 (100.0), 79 (12.9), 78 (5.6), 10 77 (17.6), 51 (7.5), 39 (3.8) 0 300 400 500 nm II 9.1 18.6 178 (11.2), 121 (13.3), 119 (7.4), 118 (79.7), 106 (6.4), 105 (100.0), 103 (18.9), mAU 1000 91 (17.1), 79 (10.0), 78 (8.9), 77 (20.1), 51 800 (9.5), 41 (8.5), 39 (9.9) 600 COOH 9.1 st 400 18.5 178 (11.1), 121 (16.2), 119 (7.1), 118 200 (81.6), 106 (6.5), 105 (100.0), 103 (17.6), 0 91 (15.5), 79 (9.1), 78 (9.2), 77 (19.5), 51 300 400 500 nm (11.6), 41 (9.4), 39 (11.3) III 20.9 176 (54.4), 175 (41.9), 145 (91.2), 144 mAU 160 (41.3), 117 (41.9), 116 (29.5), 115 (100.0), 140 120 102 (12.1), 91 (34.4), 89 (9.9), 77 (12.1), 100 65 (11.1), 63 (9.7), 51 (16.8), 50 (8.1), 39 80 COOH (19.1) 60 40 20.9 st 176 (58.2), 175 (47.3), 145 (89.9), 144 20 (45.3), 117 (46.9), 116 (29.8), 115 (100.0), 0 300 400 500 nm 102 (11.0), 91 (34.8), 89 (10.0), 77 (13.0), 65 (12.7), 63 (10.6), 51 (18.2), 50 (8.6), 39 (20.7) 1 2 Met: metabolite; t R : Retention time in HPLC; t R : Retention time in GC/MS; st: standard.

DISCUSSION 81

4 Discussion

To screen for microorganisms which are able to degrade aliphatic and aromatic hydrocarbons, especially pristane, iso -pentylbenzene and sec -octylbenzene, 16 hydrocarbon utilizing strains from the strain collection of the Department of Biology of the University Greifswald (Stammsammlung des Fachbereiches Biologie der Ernst-Moritz- Arndt-Universität Greifswald – SBUG) were incubated on the different hydrocarbon sources. Additionally, 21 hydrocarbon utilizing strains were isolated from oil-contaminated habitats of the Saudi Arabian Desert and from Vietnam. Three very effective pristane– utilizing bacterial strains were identified as Mycobacterium neoaurum SBUG 109, Nocardia cyriacigeorgica SBUG 1472, and Rhodococcus ruber SBUG 82. These strains were used for degradation experiments and for determining the catabolic pathways of pristane. In addition, all of these strains and the phenol-utilizing yeast Trichosporon mucoides SBUG-Y 801 were investigated for their ability to transform iso -pentylbenzene and sec -octylbenzene.

4.1 Pristane degradation

Growth experiments with pristane as substrate show that M. neoaurum SBUG 109, N. cyriacigeorgica SBUG 1472, and R. ruber SBUG 82 utilized pristane as a carbon and energy source. About 2.5 g litre -1 pristane were oxidized by these strains during 15 to 20 days. M. neoaurum SBUG 109, N. cyriacigeorgica SBUG 1472 and R. ruber SBUG 82 are aerobic actinomyceteous bacteria which possess mycolic acids at the external surface of the cell. These compounds are unusual long–chain hydroxylated and branched fatty acids, esterified to the peptidoglycan of the cell wall. Probably, these lipophilic cell structures are significant for the affinity of these bacteria to lipophilic pollutants. It was suggested that the ability of Mycobacterium , Nocardia and Rhodococcus species to produce mycolic acids, nocardols, and nocardone thus give rise to a number of features important or potentially important in n–alkane utilization (Stephens 1987).

Pristane has been reported to be degraded mainly via mono- and di-terminal oxidation. In this study, we show that it was degraded not only via these pathways but also via sub-terminal oxidation (Figure 4.1). Four of the identified metabolites, 2,6,10,14– tetramethylpentadecanoic acid (pristanic acid, metabolite 12), 2,6,10–trimethylundecanoic acid (metabolite 8), 4,8–dimethylnonanoic acid (metabolite 5), 2,6–dimethylheptanoic acid

DISCUSSION 82

(metabolite 7), and 4–methylpentanoic acid (metabolite 1) appeared to derive from the mono-terminal oxidation pathway of pristane followed by ß–oxidation of pristanic acid (Figure 4.1, pathway I). This pathway was previously shown to be operating in several microorganisms for instance in Brevibacterium erythrogenes (Pirnik et al. 1974) , Corynebacterium sp. (McKenna and Kallio 1971) and Rhodococcus sp. (Beguin 2003). A special feature of the oxidation of branched chain hydrocarbons of the pristane type is that, because of the branch points, not only acetyl coenzyme A but also propionyl coenzyme A is produced during ß–oxidation. Finally, mono-terminal oxidation of pristane followed by ß–oxidation results in 2–methyl propionyl coenzyme A as end product (Britton 1984).

The second significant branch of pristane biodegradation involves the di-terminal oxidation pathway (Margesin and Schinner 2001; Pirnik 1977). The series of identified dicarboxylic acids have been attributed to an oxidation via dicarboxylic acid pathway (Figure 4.1, pathway II). After the formation of the initial terminal oxidation products, the degradative pathway branches into mono- and di-terminal oxidation. Whether the second oxidation at the ω–position starts at pristanol, pristanal, pristanic acid or at other mono- terminal ß–oxidation products, remains to be established. The first detectable di-terminal degradation product of the strains investigated is 4–methylheptanedioic acid (metabolite 3). However, because of the missing terminal branched points the ω–oxidation of the

mono-terminal pathway intermediates must start before - at the latest at the methylated C 9 chain (4,8–dimethylnonanoic acid, metabolite 5). The dicarboxylic acid oxidation continues until 2–methylpentanedioic acid (metabolite 4) is formed from propionyl–CoA and acetyl–CoA primary di-terminal oxidation products. The end product of di-terminal oxidation is believed to be α-methylmalonyl–CoA, though we were only able to show this for M. neoaurum SBUG 109. This product can then be isomerised to succinyl–CoA (Britton 1984).

Only a few microorganisms are able to utilize branched–chain hydrocarbons because for most organisms these structures are not suitable substrates either for the initial hydroxylation nor for the ß-oxidation. There are three conditions necessary for aerobic degradation of isoprenoid hydrocarbons in nature; first, the bacteria have to possess genes and enzymes for alkane mono-oxygenation (cytochrome P450 is involved in Gram positive bacteria); second, the ß-oxidation enzymes must also be able to interact with branched chain fatty acids; and third, unusual end products of ß-oxidation must be integrated into intermediate metabolism (Beguin 2003; Britton 1984) Among the microorganisms, which

DISCUSSION 83

fulfil these criteria, actinomycetes have a very high capacity to degrade branched-chain hydrocarbons (Stephens 1987). These bacteria are versatile degraders of many organic compounds and their ß-oxidation pathway serves not only as a catabolic pathway but also as a source of fatty acids for the production of a large variety of lipids of different complexity and functions (Alvarez et al. 2001).

CH2OH CH2OH OH (M)(R) (N)

COOH HOOC COOH met. 11 met. 10 O

COOH COOH HOOC COOH

(R) COOH COOH HOOC COOH HOOC met. 8 (R) N R ( )( ) COOH HOOC COOH HOOC COOH met. 9 met. 5

(N) (M)(R) COOH HOOC COOH HOOC COOH met. 6 met. 7

M N R COOH HOOC COOH ( )( )( ) COOH (M) HOOC met. 3 met. 1 (M)(R) COOH M HOOC COOH ( ) HOOC COOH met. 4 met. 2

Pathway I Pathway II Pathway III

Figure 4.1 Proposed pathway for the degradation of pristane by R.ruber SBUG 82 (R), M. neoaurum SBUG 109 ( M) and N. cyriacigeorgica SBUG 1472 (N) based on the identification of 11 pristane derived catabolites; met, metabolite.

DISCUSSION 84

Mycobacterium sp. and Rhodococcus sp . were reported to grow on different hydrocarbon types including n–alkanes, isoalkanes, aromatic compounds, or alicyclic hydrocarbons and some of their derivatives; which are commonly introduced into gasoline formulations (Berekaa and Steinbuchel 2000; Bicca et al. 1999; Burback and Perry 1993; Khan et al. 2002; Kostichka et al. 2001). Studying nine bacteria including Rhodococcus , Norcadia , Gordonia and Dietzia genera, Alvarez H.M. (2003) concluded that the mono- terminal and ß-oxidation pathways play the main role in hydrocarbon degradation by actinomycetes. Though most Mycobacterium sp. were assumed to oxidize pristane through mono-terminal oxidation (Sakai et al. 2004). Berekaa and Steinbüchel (2000) showed that Mycobacterium sp. could also degrade squalane via the dioic acid pathway. Similarly, a Rhodococcus strain was able to degrade pristane via mono- and di-carboxylic acids (Takei et al. 2008). These two degradation pathways have been confirmed to proceed also in the three pristane-utilizing bacteria M. neoaurum , R. ruber and N. cyriacigeorgica which we used. For N. cyriacigeorgica the capacity to utilize aliphatic and branched chain hydrocarbons was detected for the first time.

In M. neoaurum and R. ruber , in addition to mono-terminal and di-terminal products, other intermediates were formed which cannot be integrated into these degradation pathways (pathway I and pathway II in Figure 4.1). For example, the intermediate 2,6,10,14-tetramethylpentadecan-3-one (metabolite 10) was found in the alkaline extracts. This metabolite 10 suggests that pristane was attacked on carbon 3. This kind of attack has been proposed by Rontani et al. (1986) using a marine mixed bacterial population, though the available data concerning bacterial degradation of isoprenoids by this pathway are still very limited. Rontani et al. (1986) proposed that the last compound of this sub-terminal oxidation process is then hydrolyzed into propan-2-ol and 4,8,12– trimethyltridecanoic acid (Figure 4.2, pathway III B). The acid was further degraded via mono-terminal oxidation (pathway I). The results of our work, however, suggest that after attack on the pristane at the third carbon atom and the formation of 2,6,10,14– tetramethylpentadecan-3-one (metabolite 10), a long chain aliphatic primary alcohol and 2- methylpropanoic acid are formed, and that the primary alcohol is then oxidized to 3,7,11– trimethyldodecanoic acid (Figure 4.2, pathway III A). Indeed the formation of further metabolites found in the culture supernatants can only be understood in terms of pathway III A. Furthermore, the production of the acids 2 and 9, requires a combination of a sub- terminal (pathway III A) and a di-terminal oxidation. The 3,7,11–trimethyldodecanoic acid

DISCUSSION 85

thus formed can be oxidized to 2,6,10–trimethyldodecandioic acid which is subsequently metabolized via alternative ß-oxidation to form 3,7–dimethyldecandioic acid (metabolite 9), and 2–methylbutandioic acid (metabolite 2).

In the present study, we have shown conclusively that 2,6,10,14– tetramethylpentadecan-3-one (metabolite 10), 3,7–dimethyldecandioic acid (metabolite 9) and 2–methylbutandioate (metabolite 2) are metabolites of sub-terminal degradation. These metabolites may only be derived from the sub-terminal oxidative pathway which generates a series of metabolites with even chain length. This is the first report describing in detail the third degradation pathway of pristane for the isolated and characterized bacteria R.ruber SBUG 82 and M. neoaurum SBUG 109.

Using 1-phytanyl-1-octadecanyl-ether (phytanyl is 3,7,11,15- tertramethylhexadecyl), Jenish et al (1999) confirmed that the R. ruber degraded the substrate via an “internal oxidative pathway” by the biological Baeyer-Villiger-Oxidation. The chemical oxidation of a ketone to an ester by treatment with peroxy acid or hydrogen peroxide is well known as Baeyer-Villiger-Oxidation and can be used as model to explain the sub-terminal oxidation in alkane and cycloalkane catabolism, which is referred to as biological Baeyer-Villiger-Oxidation. It has been shown that in R. ruber CD4 this mechanism is operative during cyclododecane degradation in which Baeyer-Villinger monooxygenase (BVMO) catalyzes the substrate by inserting an oxygen atom next to the alicyclic keto group (Schumacher and Fakoussa 1999). Given that chemical methods for Baeyer-Villiger oxidation are still somewhat unsatisfactory with respect to stereo specificity, process safety and ecology, the bio-catabolic approach is, in many cases, the tool of choice to optimize the reaction. Additionally, it has been reported that there are seven BVMO genes in Mycobacterium tubercolosis (Fraaije 2002) so that it is likely that BVMO also occurs in M. neoaurum SBUG 109 and that the sub-terminal degradation pathway of pristane by M. neoaurm and R. ruber may be explained by such a ketone mono-oxygenase catalyzed Baeyer-Villiger mechanism.

DISCUSSION 86

O

O O O O

O + HO + OH OH HO O 3, 7, 11- trimethyldodecan-1-ol 2 - methylpropanoic acid 4, 8, 12- trimethyltridecanoic acid propan-2-ol

O

OH

3, 7, 11- trimethyldodecanoic acid

Pathway III A Pathway III B

Figure 4.2 Proposed sequence of metabolite production by sub-terminal oxidation of pristane via 2,6,10,14-tetramethylpentadecan-3-one as intermediate followed by two different monooxidation reactions by ketone monooxygenase. The esters formed can be split by esterase action to yield different products. In R . ruber SBUG 82 and M. neoaurum SBUG 109 pathway III A seems to be involved. Pathway III B is described for a marine bacterial mixed population (Rontani 1986).

In conclusion, the results described in this study show that N. cyriacigeorgica, M. neoaurum and R. ruber are able to degrade branched chain hydrocarbons efficiently and that the degradation of pristane by M. neoaurum and R. ruber also proceeds via a sub- terminal oxidation mechanism. During this pathway, ketone mono-oxygenation reactions seem to be involved. Because of this it will be of interest to look more closely at the catalytic processes involved and their possible extension to the bio-degradation of other branched chain hydrocarbons.

4.2 Degradation of aromatic compounds

In this study, iso -pentylbenzene and sec -octylbenzene have been chosen as model compounds to investigate the biotransformation of branched chain alkylbenzene compounds by several alkane-utilizing microorganisms. Aromatic hydrocarbons, such as benzene, toluene, ethylbenzene, xylene, naphthalene, and branched chain alkylbenzenes

DISCUSSION 87

belong to the large volume petrochemicals, widely used as fuels and industrial solvents (Dong et al. 1993; Dutta and Harayama 2001; Ellis et al. 1996). As a consequence of their widespread use and subsequent introduction into the environment many of these compounds are considered to be priority pollutants (Keit and Telliard 1979). Furthermore, alkylbenzenes containing long alkyl chains (more than 7 carbon atoms) generally display low volatility and water solubility (Hoeppel 1991). It is known that hydrophobic compounds are often recalcitrant to biodegradation because of their low bioavailability. In particular, compounds with branched alkyl side chain are difficult to biodegrade, though a few biotransformation pathways have been documented (Baggi et al. 1972; Sariaslani et al. 1974; Sariaslani et al. 1982; Simoni et al. 1996; Tanghe et al. 1999). Typically 3-methyl- branched and quaternary substituted alkyl chains can result in considerable environmental recalcitrance (Pirnik 1977; Rontani et al. 1997).

M. neoaurum SBUG 109, N. cyriacigeorgica SBUG 1472 and R. ruber SBUG 82 have all been shown to possess high capacities of effective biodegradation of iso - pentylbenzene and sec-octylbenzene. Additionally, T. mucoides SBUG-Y 801, known as a phenol utilizing and biphenyl-biotransforming yeast (Sietmann et al. 2000), was able to transform these aromatic compounds effectively. The physiological studies presented here were designed to further analyze the pathways of microorganisms by which alkylated aromatic structures could be degraded and to identify the structures of the metabolites formed. These studies used iso -pentylbenzene and sec -octylbenzene as model substrates which were used to answer the questions as to whether the primary attack occurs at the branched alkyl side chain or at the aromatic ring and whether ring fission was performed via ortho- or meta- cleavage.

4.2.1. Biotransformation of iso -pentylbenzene

The metabolites identified by HPLC and GC/MS analyses of the studied microorganisms are summarized in the Table 4.1. The key reaction of the iso - pentylbenzene transformation is that the alkyl side chain was oxidatively attacked and then shortened. This mode of catabolism has been previously demonstrated in other bacteria and yeasts e.g. Alcanivorax sp. MBIC 4326 (Dutta and Harayama 2001), N. salmonicolor (Sariaslani et al. 1974) and Candida maltosa (Awe et al. 2008). However, the alkylbenzenes used in these studies were linear n-alkylbenzenes without branched side chain, whereas iso -pentylbenzene has one branching point at the end of the alkyl chain.

DISCUSSION 88

Table 4.1 Detected metabolites analyzed by HPLC and/ or GC/MS formed during iso - pentylbenzene transformation by selected microorganisms.

M. neoaurum N. cyriacigeorgica R. ruber T. mucoides Met Structure SBUG 109 SBUG 1472 SBUG 82 SBUG-Y 801

A COOH + + +

B O +

C + + +

O D + + + +

E + COOH HOOC F COOH + +

G COOH + +

OH

H COOH +

K + OH

Because of the non-availability of a 2-methyl-4-phenyl-butan-1-ol standard and the impossibility of isolating product C by HPLC for NMR analysis, the exact structure of this product remains unconfimed. Theoretically, after formation of 2-methyl-4-phenyl-butan-1- ol, this alcohol is carboxylated to 2-methyl-4-phenyl-butyric acid followed by other shorter acid products. Therefore, we propose that product C is 2-methyl-4-phenyl-butan-1-ol. On the basis of HPLC and GC/MS analysis, iso -pentylbenzene was proposed to be transformed by M. neoaurum via two different pathways. In the first, terminal oxidation of the methyl group of iso -pentylbenzene and further oxidation to form 2-methyl-4-phenyl- butan-1-ol is followed by the formation of 2-methyl-4-phenyl-butyric acid, which is degraded via ß-oxidation to form phenylacetic acid. Further degradation of phenylacetic acid probably proceeds in two directions, one continues oxidation and decarboxylation to form benzoic acid, and the other involves hydroxylation of the aromatic ring to form (2- hydroxy-phenyl)-acetic acid. Benzoic acid and (2-hydroxy-phenyl)-acetic acid are

DISCUSSION 89

probably transformed by ortho - or meta -cleavage to succinic acid. In the second pathway, iso -valerophenone is formed from the corresponding secondary alcohol as a result of attack at carbon 4 of the branched alkyl side chain of iso -pentylbenzene. iso -Valerophenone probably underwent further biological Bayer-Villiger-Oxidation to produce an ester intermediate (not detectable) which has been hydrolyzed to benzoic acid. The benzoic acid could then be cleaved to form succinic acid. Both of these pathways start with the initial attack of the alkyl side chain, whereas iso -propylbenzene and iso -butylbenzene were reported to undergo primarily ring-fission as described for Pseudomonas sp. (Jigami et al. 1975). Degradation of iso -pentylbenzene through 2-methyl-4-phenyl butyric acid, phenylacetic acid and benzoic acid suggested the involvement of a ß-oxidation mechanism operating on the branched alkyl side chain and a decarboxylation process of phenylacetic acid to yield benzoic acid. Investigating the mechanism of decarboxylation of ortho - substituted benzoic acids, Chuchev and Belbruno (2007) concluded that aromatic decarboxylation processes play a significant role in biochemical reactions (Chuchev and Belbruno 2007). A similar mechanism has been reported for Pseudomonas sp. by Sariaslani et al. (1982) during studies on the degradation of 3-phenylbutyric acid which was detected as an intermediate resulting from ω-oxidation and subsequent decarboxylation. 3-Phenylpropionic acid was then α-oxidized to produce phenylacetic acid followed by a further decarboxylation step leading to benzoic acid. When 2-methyl-4- phenyl butyric acid was used as substrate for biotransformation experiments, M. neoaurum produced phenylacetic acid as the major intermediate. Thus, mono-terminal oxidation, followed by ß-oxidation of the alkyl side chain to phenylacetic acid, has been confirmed in M. neoaurum . The presence of (2-hydroxy-phenyl)-acetic acid and succinic acid indicate that the proposed the ring cleavage via ortho - or meta -pathway was operative. The formation of small amount of benzoic acid from 2-methyl-4-phenyl-butyric acid may be explained by an additionally integrated decarboxylation step. Detection of phenylacetic acid (product A) and benzoic acid (product F) in the incubation of R. ruber with iso -pentylbenzene suggests that the same mechanism was operating here too. Identification of iso -valerophenone (product D) and benzoic acid (product F) in the cultures of M. neoaurum and R. ruber gave hints for an ability of these organisms to degrade branched alkyl side chain also via sub-terminal oxidation , and this has been confirmed in pristane degradation by these bacteria. Several microorganisms such as

DISCUSSION 90

Marinobacter sp. (Rontani et al. 1997), Rhodococcus ruber (Kostichka et al. 2001) and Arthrobacter sp. (Cripps et al. 1978) can metabolize ketones by a sequence of reactions initiated by the insertion of an oxygen atom between the carbon atom of the oxo group and an adjacent carbon atom to form either an ester or a lactone (Cripps 1975). Other transformation steps of ketone metabolism have, however, been proposed. With butan-2-one, three transformation steps of different metabolic pathways have been suggested: first, the ketone was converted into ethyl acetate by Nocardia sp., but further transformation was not described; second, the ketone was metabolized to propionic acid by M. vaccae , but further transformation was also not described; third, the ketone was catalyzed to 3-hydroxybutan-2-one by Pseudomonas sp., and further metabolism of this

latter product proceeded via butan-2,3-dione followed by cleavage into two C 2 units (Cripps 1975 and reference therein). These three types of reaction are summarized in Figure 4.3.

O a O ethyl acetate

b O + C H 3O H O O propionic acid c O OH O O + OH OH O O 3-hydroxybutan-2-one

Figure 4.3 Proposed transformation steps of butan-2-one metabolism by Nocardia sp. (a); by M. vaccae (b) and Pseudomonas sp. (c).Modified after Cripps (1975).

Therefore, it was reasonable to assume that iso -valerophenone was degraded by the initial Bayer-Villiger-Oxidation which can be performed in two different ways resulting in phenol or benzoic acid as products (Figure 4.4 The second pathway has been proposed for M. neoaurum and R. ruber . The sub-terminal mechanism can be carried out by R. ruber as shown during the degradation of pristane (Nhi-Cong et al. 2008a) and for another R. ruber strain with cyclododecane as substrate (Schumacher and Fakoussa 1999). Previous studies on the metabolism of propane by another Mycobacterium ( M. vaccae JOB-5) indicated that the major assimilatory pathway involves an initial sub-terminal oxidation (Johnson et al. 2004). However, hitherto there has no information about the degradation of branched alkyl side chains of aromatics via sub-terminal mechanism by either M. neoaurum nor R. ruber .

DISCUSSION 91

O OH HO + O O O

O O OH HO O +

Figure 4.4 Proposed pathways of iso -valerophenone metabolism by M. neoaurum and R. ruber

In contrast to both of these strains, in which several metabolites of different pathways of iso-pentylbenzene degradation were detected, N. cyriacigeorgica 1472 yielded only two detectable metabolites - product D and product C – which also indicate an attack on the alkyl side chain of iso-pentylbenzene. Species of the genus Nocardia are well known for their capability to catabolize phenylalkanes and recalcitrant branched hydrocarbons (Alvarez et al. 2001; Sariaslani et al. 1974; Smith 1990). For example, the degradation of 3-phenyldodecane by Nocardia sp. was initiated with an oxidation of the alkyl side chain (Baggi et al. 1972). Webley (1954) and Webley et al. (1955, 1956) showed also that in Nocardia opaca n-alkylbenzenes were degraded by ß-oxidation of the side chain. N. cyriacigeorgica 1472 it has been shown to have a high capacity to degrade a variety of n-alkanes and pristane (up to 84 % after 15 to 20 days) (Nhi-Cong et al. 2008b). These results indicate that the transformation of iso -pentylbenzene by this bacterium also starts at the alkyl side chain. The fact that T. mucoides 801 can grow on phenol as the sole carbon source may suggest that the degradation of iso -pentylbenzene starts at the ring of the molecule. Furthermore, it has been reported by Sietmann et al. (2000) that this yeast can transform 40 % of biphenyl within the first 24h of incubation by hydroxylation. The identification of product K - X -(3-methyl-butyl)-phenol which contains an OH-group on the ring of iso - pentylbenzene gives support for this proposal. Moreover, the appearance of phenylacetic acid (product A), 2-methyl-4-phenyl-butan-1-ol (product C), iso -valerophenone (product D) and benzoic acid (product F) demonstrated the attack on the branched side chain. Another yeast, Candida maltosa SBUG 700, was also reported to have high capacity of degradation of phenylalkanes on the alkyl side chain (Awe et al. 2008). In conclusion, under aerobic conditions, iso -pentylbenzene was degraded on the branched side chain via both mono- and sub-terminal oxidation Figure 4.5. It is of particular interest in this respect that the benzene ring was cleaved by M. neoaurum SBUG

DISCUSSION 92

109. These features could be potentially interesting for the biotransformation of other recalcitrant aromatic compounds.

(M, N, T) (M, N, R) O T ( ) OH

? Product K Product C Product D OH O (M) COOH ? O Product H

(M, R, T) COOH Product A

CO 2 O M T ( , ) (M, R, T) COOH OH HO + OH Product G Product F

Ring cleavage

(M) COOH HOOC Product E Figure 4.5 Proposed pathways for the degradation of iso -pentylbenzene by M. neoaurum (M), N. cyriacigeorgica (N), R. ruber ( R) and T. mucoides (T); [], undetectected metabolite; , not proven by experiments; , proven by experiments.

4.2.2. Biotransformation of sec -octylbenzene

The identification of numerous phenylalkanoic acids containing shorter branched side chains and/or shorter unsaturated side chains than sec -octylbenzene during biotransformation by N. cyriacigeorgica, M. neoaurum , and R. ruber suggested that the degradation of sec -octylbenzene started primarily from the side chain. Most investigations of benzene with a branched alkyl side chain shows that ring cleavage is preferred to a branched side chain attack, in line with the fact that branched alkyl groups are considered to be degradation recalcitrant. The key reaction of Pseudomonas sp. NCIB 10643 growing

DISCUSSION 93

on a range of mono-substituted alkylbenzenes (C2-C7), including both normal and branched chain species, was the extra-diol cleavage between carbon atom 1 and 2, also known as meta -ring cleavage (Smith and Ratledge 1989). P. acidovorans was also concluded to catabolize 2-phenylbutane, 3-phenylpentane and 4-phenylpentane by a ring cleavage pathway (Baggi et al. 1972), whereas Smith (1990) and Sariaslani et al . (1974) reported the degradation of n-alkylbenzenes by an initial side chain attack. The appearance of several ring hydroxylated and ring opened metabolites (including 3-phenylbutyric acid, 2-phenylpropionic acid, ß-methylcinnamic acid, acetophenone and benzoic acid) during the biotransformation by the microorganisms studied in this work confirmed the occurrence of ring cleavage after primary oxidation of the side chain (Table 4.2).

Table 4.2 Detected metabolites by HPLC and/or GC/MS of sec -octylbenzene transformation by studied microorganisms.

Substrate Product Structure N.c M. n R. r T. m sec -Octylbenzene

I COOH + + + +

II COOH + + + +

III COOH + + + -

- IV COOH + - -

O V + + - -

O VI + + - - OH COOH VII + - + - OH OH

COOH VIII HOOC - + + -

IX - + - - O

COOH X - - + -

COOH

DISCUSSION 94

XII - - + -

XIII OH - - + -

3-Phenylbutyric acid (IV) I COOH - - + +

III COOH + + + +

O V + - - -

COOH X - + - -

COOH

XI HO - + - - OH

XII COOH - - + -

ß-Methylcinnamic O acid (III) V + + - -

COOH X - - + -

XII COOH - - + -

2-Phenylpropionic O acid (I) V - + + -

COOH VII - - + - OH OH COOH X - + + -

COOH VIII HOOC - - + -

XII COOH - - + -

Acetophenone (V) VIII COOH + - + - HOOC

DISCUSSION 95

Acetophenone (V) COOH

VII OH + - - - OH

OH

XIV COOH - - + -

COOH X - + - -

COOH

XI HO - + - - OH Benzoic acid (X) XII COOH - - + -

COOH XV - - + - OH N. c, Nocardia cyriacigeorgica; M. n, Mycobacterium neoaurum; R. r, Rhodococcus ruber; T. m, Trichosporon mucoides . The degradation of sec -octylbenzene by all studied microorganisms seems to start from the alkyl side chain by terminal oxidation , which is the preferred pathway for many

microorganisms when the alkyl chain length exceeds C 7 (Smith 1990). Sariaslani et al . (1974) reported on the degradation of n-dodecyl- and n-nonylbenzenes by initial side chain attack via ω- and ß–oxidation. The detection of 5-phenylhexanoic acid and 3-phenylbutyric acid as the major products indicated that initial shortening of alkyl side chain by ß- oxidation takes place before cleavage of the benzene ring as in the case of 1-phenyl alkane degradation (Webley 1956). Bhatia et al. , (1996) when studying the biodegradation of linear alkyl benzenes by Nocardia amarae, detected 2-, 3-, and 4-phenylbutyric acid as the major metabolites without cleavage of the benzene ring (Bhatia and Singh 1996). Two other Nocardia strains capable of utilizing 3-phenyldodecane as the sole carbon and energy source were isolated and 2-phenylbutyric acid, 3-phenylvaleric acid and 4-phenylhexanoic acid were detected in the cultures, though neither diol or semialdehyde-related compounds were formed (Baggi et al. 1972). Because of this it was proposed that sec -octylbenzene was transformed via a monooxygenation pathway to 2-phenyloctanoic acid, and that this product was ß-oxidized to 2-phenylhexanoic acid and then to 3-phenylbutyric acid. The identification of 7-phenyl-octan-2-ol, 7-phenyloctan-2-one and 2- phenylpropionic acid indicated the occurrence of sub-terminal oxidation in which 7- phenyloctan-2-one (metabolite IX) was formed from the corresponding secondary alcohol (Figure 4.6). Further oxidation of the ketone may occur in two diverse directions, on one

DISCUSSION 96

hand, the ketone may be transformed to 6-phenylheptanoic acid which was later oxidized to 4-phenylpentanoic acid and 2-phenylpropionic acid (metabolite I), while on the other hand, this ketone may degraded to the 5-phenylhexanoic acid (metabolite IV) which was metabolized to 3-phenylbutyric acid via ß-oxidation.

O OH CH OH O O + 3

6-phenyl-heptanoic acid

OH O

Product XIII Product IX O HO O OH + O

O

OH

Product V 5-phenyl-hexanoic acid

Figure 4.6 Putative oxidation of sec -octylbenzene via formation of product XIII, IX and IV could be explained by two different routes to produce 6-phenylheptanoic acid, which can lead to the formation of 2-phenylpropionic acid, and 5-phenylhexanoic acid.

Because of the methyl group on the alkyl side chain of the 3-phenylbutyric acid , this acid could not be oxidized to phenylacetic acid as can normal linear even chain phenylalkanoic acids. In this investigation, it was shown that 3-phenylbutyric acid was dehydrogenated to ß-methylcinnamic acid leading the formation of acetophenone (Figure 4.7). Normally the dehydrogenation step and the following hydrolysis happens very quickly but in this case ß-methylcinnamic acid was relatively stable because of the steric hindrance of the branch point. Using Rhodococcus rhodochrous PB1 cultured with racemic 3-phenylbutyric acid, Simoni et al . (1996) reported the conversion of (R)-3-phenylbutyric acid to 3-phenylpropionic acid followed by its further metabolism via meta cleavage; whereas (S)-3-phenylbutyric acid was co-metabolically oxidized to (S)-3-(2,3- dihydroxyphenyl)butyric acid for which no further degradation steps were described (Simoni et al. 1996). Sariaslani et al . (1982) proposed the degradation of 3-phenylbutyric acid in Pseudomonas sp. via two different routes, the dioxygenation of 3-phenylbutyric acid to form 3-(2,3-dihydroxyphenyl)-butyric acid accompanied by meta ring cleavage and the ω-oxidation of the alkyl side chain with subsequent decarboxylation to generate 3- phenylpropionic acid which in turn yielded phenylacetic acid and benzoic acid (Sariaslani

DISCUSSION 97

et al. 1982). The occurrence of phenylacetic acid and benzoic acid in this investigation, therefore, was elucidated (Figure 4.7).

COOH COOH

Product I Product X

COOH COOH

Product XII Product II

COOH O

Product V Product III

Figure 4.7 Proposed formation of benzoic acid, phenylacetic acid and acetophenone via 3- phenylbutyric acid . , not proven by experiments; , proven by experiments.

The appearance of phenylacetic acid in the series of sec -octylbenzene, 3- phenylbutyric acid, ß-methylcinnamic acid, 2-phenylpropionic acid and benzoic acid biotransformations by R. ruber SBUG 82 is not easily to explain and may be also related to an unclear phenomenon of excreted cellular product when the cells were brought in contact with any aromatic compounds. This phenomenon of excreting aromatic acids was also observed when T. mucoides or other microorganisms were incubated with any aromatic hydrocarbons (Hammer 1996). Using acetophenone as a substrate, metabolites such as α-hydroxyacetophenone (product VI), hydroxyl-phenylacetic acid (product XIV), benzoic acid (product X), 2,3- dihydroxy-benzoic acid (product VII), and 3,4-dihydroxy-benzoic acid (product XI), and succinic acid (product VIII) were detected. 2-Hydroxyacetophenone (product VI) and hydroxyl-phenylacetic acid (product XIV) can be generated by ring hydroxylation and by oxidation of the side chain. The further degradation of these intermediates is quite unclear at the moment, though it is conceivable that ring cleavage occurs. The appearance of benzoic acid as an intermediate of acetophenone degradation can be explained by two possibilities . Strains like M. neoaurum and R. ruber with the capacity for sub-terminal degradation can perform the biological Bayer-Villiger-Oxidation on acetophenone to form ester intermediates which can be hydrolyzed to benzoic acid and methanol (Figure 4.8). Though, N. cyriacigeorgica has not been described as strain with the ability to carry out

DISCUSSION 98

sub-terminal degradation, 2,3-dihydroxy-benzoic acid was nevertheless detected as a product of acetophenone. An oxidative demethylation or conversion step could be possible (Figure 4.8) as described by Sariaslani et al . (1982) and Simoni et al . (1996) to explain demethylation steps on branched alkyl side changes. Under aerobic condition, benzoic acid was degraded via known – ortho - or meta -cleavage pathways after ring hydroxylation. In N. cyriacigeorgica SBUG 1472, the hydroxylation positions were on carbon atoms number 2 and 3; whereas in M. neoaurum SBUG 109 at carbon atoms number 3 and 4. Succinic acid was considered as a result of these dihydroxylated benzoic acids.

O

Ring cleavage ? OH Product VI

O OH Ring cleavage COOH ? COOH Product V Product XIV HO Ring cleavage OH Product XI COOH COOH COOH HOOC Product VIII OH Product X Product XV Ring cleavage COOH

OH OH Product VII

Figure 4.8 Presumed oxidation pathways of acetophenone via different routes, one was the attack on the ring, the others were attacks on the side chain followed by the ring cleavage.

Benzoic acid transformation experiments with 0.025 g litre -1 were performed for all studied microorganisms. After 4 h of incubation all of the substrate was degraded by R. ruber , and salicylic acid was the main product of the biotransformation. In the case of N. cyriacigeorgica all of the substrate was degraded after 8 h incubation but no metabolite was observed, suggesting that in this strain benzoic acid was used completely as the energy and carbon sources for the cells. M. neoaurum and T. mucoides were not able to transform benzoic acid. In summary, based on the combination of metabolites detected in the sec - octylbenzene transformation process, the degradative pathways shown in Figure 4.9 were

DISCUSSION 99

postulated. sec -Octylbenzene was demonstrated to be degraded via attack on the side chain by mono- and sub-terminal oxidation. Benzene ring cleavage was considered to be carried out by N. cyriacigeorgica , M. neoaurum and R. ruber resulting in the occurrence of succinic acid.

(R) OH

COOH (M) O

(N) COOH COOH

(N,M,R,T) COOH COOH

(N,M,R,T) COOH

R COOH ( ) N M R (N,M,R) ( , , ) COOH O

COOH (N,M) (M,R) (R) O COOH OH OH

(N,R) (R) (M) COOH COOH COOH Ring cleavage Ring cleavage OH OH HO OH OH Ring cleavage

(M,R) COOH HOOC

Figure 4.9 Postulated degradation pathways of sec -octylbenzene. The accumulated and detected products were marked N, M, R, and T for N. cyriacigeorgica , M. neoaurum , R. ruber and T. mucoides , respectively. , not proven by experiments; , proven by experiments.

DISCUSSION 100

Conclusions

1. Three bacteria and the yeast Trichosporon mucoides SBUG-Y 801 which possess high potential for degrading and transforming pristane, iso -pentylbenzene and sec - octylbenzene.were selected. 2. The bacteria were identified in cooperation with DSZM as Mycocbacterium neoaurum SBUG 109, Nocardia cyriacigeorgica SBUG 1472 and Rhodococcus ruber SBUG 82. 3. 11 intermediates produced by these bacteria during incubation with pristane were analyzed by GC and GC/MS. This detection provided sufficient information to elucidate in detail three degradative pathways of pristane involving mono-, di- and sub-terminal oxidations. 4. The identification of iso -pentylbenzene transformation products by HPLC and GC/MS analyses led to the complete biotransformation of this substance. 5. The occurrence of a sub-terminal oxidation mechanism in M. neoaurum and R. ruber was confirmed. 6. By detecting 15 degradation products of sec -octylbenzene, we describe for the first time in detail the biotransformation of this substrate. This detection also confirmed the possibility of alkyl side chain attack by these bacteria and this yeast. 7. In addition, in the studied bacteria pointed to an effective sec -octylbenzene degradation pathway in which dehydrogenation of 3-phenylbutyric acid to form ß- methylcinnamic acid is a newly described option.

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APPENDIX 109

6 Appendix

Table 6.1 Retention times and MS-spectra of the identified metabolites produced by M. neoaurum SBUG 109 cultured with 0.25 (ml litre -1) iso -pentylbenzene in comparison with standards

Met Structure tR (min) m/z (%, 70 eV) E 9.1 115 (100.0), 114 (31.4), 87 (22.5), 59 (58.3), 57 (7.1), 56 (7.1), 55 (79.3), 45 (10.2), 42 (5.9), 41

COOH (4.6)

HOOC 9.3 st 115 (100.0), 114 (30.2), 87 (19.3), 59 (58.2), 57 (7.2), 56 (7.2), 55 (72.7), 45 (12.5), 42 (5.9), 41 (5.2) F 12.5 136 (29.9), 105 (100.0), 77 (65.6), 56 (9.0), 51 COOH (27.8), 50 (12.7)

12.3 st 136 (53.3), 105 (100.0), 77 (74.6), 56 (8.0), 51 (28.9), 50 (14.5) G 20.9 180 (29.2), 124 (6.1), 121 (95.6), 93 (6.9), 91 (100.0), 78 (15.4), 77 (15.5), 65 (16.6), 52 (5.4),

COOH 51 (10.6)

OH 20.0 st 180 (22.9), 124 (7.4), 121 (95.7), 93 (11.4), 91 (100.0), 78 (12.3), 77 (11.2), 65 (19.5), 52 (6.7), 51 (10.9) Met: metabolite; t R:Retention time in GC/MS; st: purchased standard

APPENDIX 110

Table 6.2 Retention times and MS-spectra of the identified metabolites produced by N. cyriacigeorgica SBUG 1472 cultured with 0.25 ml litre -1 sec -octylbenzene in comparison with standards

Met Structure tR (min) m/z (%) (70 eV) VI 17.2 120 (9.8), 105 (100.0), 91 (24.0), 77 (56.8), 51 O (27.8)

OH 16.9st 120 (7.5), 105 (100.0), 91 (20.1), 77 (72.5), 51 (23.2) VII 21.6 196 (61.6), 167 (18.2), 166 (51.8), 165 (79.2), 163 (100.0), 150 (10.3); 149 (28.4); 135 (24.4), 125

(10.3), 123 (12.2), 122 (37.6), 121 (16.1), 119 (10.2), 108 (12.5), 107 (52.8), 92 (16.0), 79 (26.7), 77 (42.6), 76 (11.9), 65 (22.6), 53 (19.7), 51 (33.9), COOH 50 (16.3), 45 (30.1)

OH OH 21.7 st 196 (62.4), 167 (16.6), 166 (80.3), 165 (75.2), 163 (100.0), 150 (11.5); 149 (30.0); 135 (27.6), 125 (9.6), 123 (11.3), 122 (36.0), 121 (17.1), 119 (10.9), 108 (10.3), 107 (46.1), 92 (16.2), 79 (20.2), 77 (40.7), 76 (10.7), 65 (20.6), 53 (14.5), 51 (32.2), 50 (14.8), 45 (30.8) VIII 9.1 115 (100.0), 114 (26.3), 87 (22.8), 59 (59.5), 57 (7.1), 56 (7.1), 55 (78.2), 45 (10.2), 42 (5.9), 41

COOH (5.9)

HOOC 9.3 st 115 (100.0), 114 (30.2), 87 (19.3), 59 (58.2), 57 (7.2), 56 (7.2), 55 (72.7), 45 (12.5), 42 (5.9), 41 (5.2)

Met: metabolite; t R:Retention time in GC/MS; st: purchased standard

APPENDIX 111

Reverse fit factor [REV]: 855 MV-01 178 (7.463) Cm (177:185-(162:166+200:208)) (a) 311 43 100 74

% 57 41 55 87 39 59 53 73 81 99 101 108 37 65 124 139 147 162 172 186 200 205 224 238 240 256 261 0 R:855 CONG 16: 4-METHYLVALERIC ACID, METHYLATED Hit 1 74 100 (b) 43

87 % 41 55 57

39 59 73 81 88 99 45 115 124 155 69 101 137 148 167 170 205 224 238 246 255 264 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 Figure 6.1 Mass spectra of metabolite 1 (a) produced by M. neoaurum SBUG 109 in comparison with the purchased standard 4-methylvaleric acid (b). All compounds were methylated. Reverse fit factor [REV]: 516 NO-04 682 (15.866) Cm (682:685-(665:679+686:694)) 254 59 100 (a)

%

125 129 101 36 47 49 65 81 90 102 108 118 140 154 54 62 75 79 86 93 100 136 150 0 Reverse fit factor [REV]: 904 EPRIMV12 405 (11.248) Cm (402:408-(386:395+415:419)) 4.70e3 59 (b) 100

%

41 129 39 42 60 69 87 100 101 128 45 56 74 0 R:904 CONG 48: METHYLSUCCINIC ACID, METHYLATED Hit 1 59 100 (c)

%

129 41 100 42 69 101 128 39 60 87 45 55 74 85 130 0 m/z 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140

Figure 6.2 Mass spectra of metabolite 2a (a) produced by R. ruber SBUG 82 and 2b (b) produced by M. neoaurum SBUG 109 in comparison with the purchased standard methylsuccinic acid (c). All compounds were methylated.

APPENDIX 112

NO-01 840 (18.500) Cm (839:841-(824:831+846:854)) 2.68e4 55 100 (a) 114 59 73

% 115 83 143 41 43 88 99 39 74 82 101 142 45 51 53 60 69 91 116 65 150 0 E-PRIS26 652 (15.366) Scan EI+ 55 2.84e3 100 (b)

59 114 73 % 41 43 115 99 83 88 39 91 143 45 69 74 101 38 50 53 60 82 95 105 142 144 150 65 77 85 97 123 130 158166 171 174 0 m/z 40 50 60 70 80 90 100 110 120 130 140 150 160 170 EPRIMV12 648 (15.299) Cm (646:650-(635:640+656:666)) 1.92e4 55 100 (c)

59 114 73 % 115 41 83 99 39 88 42 143 54 74 82 101 51 60 69 84 107 142 144 38 91 122 0 R:983 CONG 11: 2-METHYL GLUTARIC ACID, METHYLATED Hit 1 55 100 (d) 114 59 73

% 115 83 88 99 143 41 43 56 39 82 101 142 69 74 45 51 53 60 89 116 144 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160

Figure 6.3 Mass spectra of metabolite 3a produced by R. ruber SBUG 82 (a) and 3b produced by N. cyriacigeorgica SBUG 1472 (b) and M. neoaurum SBUG 109 (c) in comparison with the purchased standard 2-methylglutaric acid (d). All compounds were methylated.

APPENDIX 113

55 115 1.11e3 100 (a) 59

% 43 69 87 114

53 71 85 90 109 116 152 189 232 65 125 161 181 219 223 252 268 272 299 309 321 339 347 0 m/z 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 5.64627409 CH3MALO3 69 (5.646) Cm (63:70-(56:60+73:78)) (b) Scan EI+ 59 1.63e5 100

% 115 57

87 114 43 72 88 53 60 101 116 281 292 307 83 142 146 165 179191 206 208 233 237 256300 322 333 336 348 0

Figure 6.4 Mass spectra of metabolite 4 produced by M. neoaurum SBUG 109 (a) in comparison with the purchased standard methylmalonic acid (b). All compounds were methylated.

APPENDIX 114

E-PRIS26 881 (19.184) Rf (5,3.000) 87 100 (a)

43 69 74

% 59

146 97 113 44 141 157

0 40 60 80 100 120 140 160 EPRINO02 882 (19.200) Cm (882:883-(879:881+883:885)) 87 100 (b) 74

123 153 % 43 84 56 145 89 107 50 73 132

0 40 60 80 100 120 140 160

19.13418579 48DIMEC9 878 (19.134) 87 (c) 100

74

43 % 55 57 69 83 88 115 53 127 143 157 0

Figure 6.5 Mass spectra of metabolite 5 produced by R. ruber SBUG 82 (a) and produced by N. cyriacigeorgica SBUG 1472 (b) in comparison with the purchased standard 4,8- dimethylnonanoic acid (c). All compounds were methylated.

APPENDIX 115

E04-N07B 954 (20.401) Cm (953:955-(946:950+961:971)) 4.40e5 55 (a) 100

69 97 129 59 % 41 74 83 43 139 82 87 142 39 53 96 110 115 125 171 45 65 67 98 130 155 79 93 109 143 170 172 0 MVPRI16 957 (20.450) Cm (955:963-(945:953+989:995)) 9.11e3 55 100 (b) 69 97 129 59 74 % 41 83 43 139 39 82 87 53 67 96 110 115 125 142 171 98 130 155 143 170 172 0 R:871 NIST 23286: HEPTANEDIOIC ACID, 4-METHYL-, DIMETHYL ESTER Hit 1 97 100 (c) 129

55 % 69 74 83 139 171 41 43 87 142 59 82 96 110 111 115 125 29 39 44 130 143 155 170 172 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210

Figure 6.6 Mass spectra of metabolite 6 produced by R. ruber SBUG 82 (a) and produced by M. neoaurum SBUG 109 (b) in comparison with the purchased standard 4- methylheptanedioic acid (c). All compounds were methylated.

APPENDIX 116

E-PRIS26 959 (20.484) Cm (958:961-(956+966)) (a) 3.40e3 88 100

55 % 97 129 39 139 257 302 182 329 0 R:734 NIST 15787: HEPTANOIC ACID, 2,6-DIMETHYL-, METHYL ESTER Hit 4 88 100 (b)

% 101 41 57 129 141 0 m/z 100 200 300

Figure 6.7 Mass spectra of metabolite 7 produced by N. cyriacigeorgica SBUG 1472 (a) in comparison with the standard 2,6-dimethylheptanoic acid in NIST library (b). All compounds were methylated. (a) E04-N07B 1084 (22.568) Cm (1083:1085-(1075+1093)) 7.67e4 88 100

99

43 % 41 59 71 55 39 69 67 87 101 113 72 79 81 89 95 125 129 140 141 169 172 45 53 139 162 163 0 R:7882,6,10-trimethylundecanoic NIST 15787: HEPTANOIC acid, -methylated ACID, 2,6-DIMETHYL-, METHYL ESTER Hit 1 88 (b) 100

%

43 55 101 41 57 59 87 39 69 71 83 89 102 73 97 115 129 141 157 0 m/z 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190

Figure 6.8 Mass spectra of metabolite 8 produced by R. ruber SBUG 82 (a) in comparison with the standard 2,6-dimethylheptanoic acid in NIST library (b). All compounds were methylated.

APPENDIX 117

7.62e3 55 100 (a) 74 152 83 43 % 111 185 39 97 153 186 0 R:746 NIST 35509: DECANEDIOIC ACID, 3,7-DIMETHYL-, DIMETHYL ESTER Hit 13 55 100 41 69 (b) 74 185 % 83 153 112 227 115 186 228 0 m/z 50 100 150 200

Figure 6.9 Mass spectra of metabolite 9 produced by R. ruber SBUG 82 (a) in comparison with the standard 3,7-dimethyldecanedioic acid in NIST library (b). All compounds were methylated.

461 43 100 (a)

% 58 41 85 167 185 69 125 39 111 0

E-PRIS01 1500 (29.504) Cm (1497:1503-(1479:1483+1531:1535)) 2.93e4 69 100 43 (b) % 55 71 85 129 149 167 0 R:8462,6,10,14-tetramethyl NIST 25894:pentadecan–3–one 2,10-UNDECANEDIONE, 6,6-DIMETHYL- Hit 1 43 100 (c) 69 109 137 % 71 85 55 97 121 153 197 0 m/z 50 100 150 200

Figure 6.10 Mass spectra of metabolite 10 produced by R. ruber SBUG 82 (a) and produced by M. neoaurum SBUG 109 (b) in comparison with the standard 2,6,10,14- tetramethylpentadecan-3-one in NIST library (c).

APPENDIX 118

H3BPRI14 1514 (29.737) Cm (1513:1517-(1488:1494+1524:1533)) 4.41e4 88 100 (a)

% 101

43 57

69 157 222 312 0 R:983 CONG 4: PRISTANIC ACID Hit 1 88 100 (b)

% 101 43 57

69 157 222 0 m/z 50 100 150 200 250 300

Figure 6.11 Mass spectra of metabolite 7 produced by N. cyriacigeorgica SBUG 1472 (a) in comparison with the purchased standard pristanoic acid (b). All compounds were methylated.

Sample ID: M.vaccae pre. on pristane, +0,01% isopentylbenzene, extracted, 24h, pH2 Reverse fit factor [REV]: 990 MVEX10 648 (15.299) Cm (647:648-(640:642+659:662)) 1.25e6 91.1 (a) 100

% 150.1

59.1 65.1 92.1 39.1 89.1 151.1 77.1 119.1 0 R:990 PHALK 9: PHENYLESSIGSÄURE-METHYLESTER Hit 1 91.0 100 (b)

%

150.0 65.0 59.0 89.0 92.0 39.0 105.0 118.0 151.0 77.0 0 m/z 40.0 60.0 80.0 100.0 120.0 140.0 160.0 180.0 Figure 6.12 Mass spectra of m etabolite A produced by M. neoaurum SBUG 109 (a) in comparison with the purchased standard phenylacetic acid (b). All compounds were methylated.

APPENDIX 119

Reverse fit factor [REV]: 921 MVIPB102 388 (10.965) Cm (386:396-(353:364+411:434)) (a) 1.41e3 105 100 77

% 51 120 43 49 52 78 37 38 54 63 75 91 109 140 174 188 0 R:921 CONG 17: ACETOPHENONE Hit 1 105 100 77 (b)

% 51 120 43 50 78 106 52 65 121 38 39 62 63 74 91 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 Figure 6.13 Mass spectra of m etabolite B produced by M. neoaurum SBUG 109 (a) in comparison with the purchased standard acetophenone (b).

Sample ID: M.vaccae pre. Pristane + 0,01%isopentylbenzen, 24h, pH9, 081106 Reverse fit factor [REV]: 971 MVEX04 833 (18.383) (a) 2.81e5 59 100 91

131

% 146 105 77 43 65 78 92 106 132 147 39 51 58 63 103 120 129 145 50 68 75 89 115 162 0 R:971 CONG 50: ALPHA, ALPHA-DIMETHYL-3-PHENYLPROPANOL Hit 1 59 100 (b) 91

% 131 146

43 65 92 39 51 60 75 77 78 105 129 132 50 52 68 89 103 106 115 116 149 0 m/z 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 Figure 6.14 Mass spectra of m etabolite C produced by M. neoaurum SBUG 109 (a) in comparison with the purchased standard α,α-dimethyl-3-phenylpropanol (b). All compounds were methylated.

APPENDIX 120

Reverse fit factor [REV]: 960 MVEX01 835 (18.417) Cm (834:837-(827+848)) 1.39e5 105.1 (a) 100 O

% 77.1 120.1 51.1 78.1 147.1 162.2 0 R:960 NIST 13002: 1-BUTANONE, 3-METHYL-1-PHENYL- Hit 1 105.0 100 (b)

% 77.0

120.0 51.0 78.0 147.0 162.0 0 m/z 50.0 100.0 150.0 200.0 250.0 300.0 350.0

Figure 6.15 Mass spectra of m etabolite D produced by M. neoaurum SBUG 109 (a) in comparison with the purchased standard iso -valerophenone (b).

(a) Reverse fit factor [REV]: 932 3B-IPB01 825 (18.250) 3.04e4 59 100 91

131 % 105 146 77 43 78 39 51 60 65 92 103 106 45 120 129 132 55 69 74 89 115 135 149 162 0 R:932 CONG 50: ALPHA, ALPHA-DIMETHYL-3-PHENYLPROPANOL Hit 1 59 100 (b) 91

% 131 146

43 65 77 92 39 51 60 75 78 105 129 132 50 52 68 89 103 106 115 116 149 0 m/z 40 50 60 70 80 90 100 110 120 130 140 150 160 170 Figure 6.16 Mass spectra of m etabolite C produced by N. cyriacigeorgica SBUG 1472 (a) in comparison with the purchased standard α,α-dimethyl-3-phenylpropanol (b).

APPENDIX 121

1.39e5 105.1 (a) 100 O

% 77.1

120.1 51.1 78.1 147.1 162.2 0 R:960 NIST 13002: 1-BUTANONE, 3-METHYL-1-PHENYL- Hit 1 105.0 100 (b)

% 77.0

120.0 51.0 78.0 147.0 162.0 0 m/z 50.0 100.0 150.0 200.0 250.0 300.0 350.0

Figure 6.17 Mass spectra of m etabolite D produced by N. cyriacigeorgica SBUG 1472 (a) in comparison with the purchased standard iso -valerophenone (b).

NOSEC113 647 (15.282) Cm (644:648-(629:641+653:666)) 2.33e5 91 (a) 100

%

65 150 92 39 41 59 63 89 151 38 50 51 52 77 105 118 119 0 R:975 PHALK 9: PHENYLESSIGSÄURE-METHYLESTER Hit 1 91 100 (b)

%

150 65 92 39 41 59 89 151 38 46 51 52 77 105 118 119 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170

Figure 6.18 Mass spectra of metabolite A produced by R. ruber SBUG 82 (a) in comparison with the purchased standard phenylacetic acid (b). All compounds were methylated.

APPENDIX 122

1.39e5 105.1 100 (a) O

% 77.1

120.1 51.1 78.1 147.1 162.2 0 R:960 NIST 13002: 1-BUTANONE, 3-METHYL-1-PHENYL- Hit 1 105.0 100 (b)

% 77.0

120.0 51.0 78.0 147.0 162.0 0 m/z 50.0 100.0 150.0 200.0 250.0 300.0 350.0

Figure 6.19 Mass spectra of metabolite D produced by R. ruber SBUG 82 (a) in comparison with the purchased standard iso -valerophenone (b).

NOSEC152 470 (12.331) Cm (470:475-(449:455+475:484)) (a) 1.62e3 105 100

77 109

%

51 136 140 50 52 78 95 110 115 120 38 49 66 73 91 92 145 150 160 0 R:899 CONG 26: BENZOIC ACID, METHYLATED Hit 1 105 100 (b) 77

% 51 136 50 106 52 78 137 38 39 74 91 92 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160 Figure 6.20 Mass spectra of metabolite F produced by R. ruber SBUG 82 (a) in comparison with the purchased standard benzoic acid (b). All compounds were methylated.

APPENDIX 123

Reverse fit factor [REV]: 952 TMIP0993 827 (18.284) Cm (820:828-(814:817+838:845)) 1.80e5 59 (a) 100 91

131 % 105 146 77 43 65 78 92 39 51 63 103 106 120 129 132 50 55 74 75 89 115 149 162 0 R:952 CONG 50: ALPHA, ALPHA-DIMETHYL-3-PHENYLPROPANOL Hit 1 59 100 (b) 91

% 131 146

43 65 92 39 51 60 75 77 78 105 129 132 50 52 68 89 103 106 115 116 149 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

Figure 6.21 Mass spectra of metabolite C produced by T. mucoides SBUG-Y 801 (a) in comparison with the purchased standard α,α-dimethyl-3-phenylpropanol (a).

Reverse fit factor [REV]: 962 TMIPB119 828 (18.301) (a) 6.06e5 105 100

77 % 120

39 51 78 91 106 162 41 57 59 121 131 146 50 65 69 74 92 144 147 163 0 R:962 CONG 51: ISOVALEROPHENONE Hit 1 105 100 (b)

77 % 120 51 78 106 39 41 63 121 144 162 50 57 65 69 74 91 147 163 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190

Figure 6.22 Mass spectra of metabolite D produced by T. mucoides SBUG-Y 801 (a) in comparison with the purchased standard iso -valerophenone (b).

APPENDIX 124

556 91 121 100 (a)

% 180

65 77 78 51 52 80 90 93 99 110 122 129 135 148 189 37 40 44 61 107 152 159 170 176 185 203 205 0 R:779 PHALK 85: 2-OH-PHENYLESSIGSÄURE 2FACH METHYLIERT Hit 1 91 100 (b) 121

%

180 65 78 93 51 59 77 122 39 52 66 89 120 148 181 38 50 79 105 107 133 149 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200

Figure 6.23 Mass spectra of metabolite G produced by T. mucoides SBUG-Y 801 (a) in comparison with the purchased standard 2-hydroxy-phenylacetic acid (b). All compounds were methylated. Sample ID: 3b+0,025%sec-octybenzene, methylated Reverse fit factor [REV]: 986 3BPH2ME1 701 (16.182) Cm (696:703-(686:694+706:717)) 2.23e5 105 (a) 100

%

77 164 106 51 165 0 R:986 CONG 13: 2-PHENYLPROPIONIC ACID-METHYLATED Hit 1 105 100 (b)

%

77 106 164 51 165 0 m/z 50 100 150 200 Figure 6.24 Mass spectra of metabolite I produced by N. cyriacigeorgica SBUG 1472 (a) in comparison with the purchased standard 2-phenylpropionic acid (b). All compounds were methylated.

APPENDIX 125

Sample ID: 3b+0,025%sec-octybenzene, methylated Reverse fit factor [REV]: 983 3BPH2ME1 845 (18.583) Cm (838:846-(831:834+849:853)) (a) 5.73e5 105 100 118

%

39 77 121 178 51 179 0 R:983 CONG 14: 3-PHENYLBUTYRIC ACID - METHYLATED Hit 1 105 100 (b) 118

%

77 121 39 51 178 0 m/z 50 100 150 200 Figure 6.25 Mass spectra of metabolite II produced by N. cyriacigeorgica SBUG 1472 (a) in comparison with the purchased standard 3-phenylbutyric acid (b). All compounds were methylated.

3BPH2ME1 981 (20.850) Cm (978:982-(965:976+985:995)) Scan EI+ 115 4.38e4 100 (a) 145

176 175

% 117 144 91 39 51 58 63 65 77 102 105 146 177 50 76 78 89 92 118 161 37 41 67 99 131 133 159 162 189 192 198 0 BCH3CIN 983 (20.885) Cm (979:984-(962:977+988:1000)) Scan EI+ 115 2.82e5 100 145 (b)

176 117 % 144 175 91

39 51 58 65 77 102 105 118 146 177 50 52 76 78 89 92 161 37 41 67 99 131 133 147 162 181 192 202 0 m/z 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 Figure 6.26 Mass spectra of metabolite III produced by N. cyriacigeorgica SBUG 1472 (a) in comparison with the purchased standard ß-methyl cinnamic acid (b). All compounds were methylated.

APPENDIX 126

3B-SEC01 1233 (25.051) Cm (1232:1235-(1212:1222+1235:1240)) Scan EI+ 91 3.18e3 100

% 119

173

121 41 43 59 115 77 131 55 92 145 174 202 78 104 129 205 234 51 65 155 185 199 74 97 110 143 158 214 223 246 0 m/z 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 Figure 6.27 Mass spectra of metabolite IV produced by N. cyriacigeorgica SBUG 1472 5-phenylhexanoic acid (b). Compound was methylated.

ACETOPHE 386 (10.931) Scan EI+ 105 2.13e5 100 77 (a)

%

51 120 50 43 52 78 106 39 44 65 74 86 89 121 163 62 63 71 91 92 98 102 113 118 129134 135 143 148 156 161 166 0 3BSC8-04 384 (10.898) Scan EI+ 105 1.88e5 100 77 (b)

%

51 120 43 50 52 78 106 39 44 65 69 74 85 89 112 121 125 151 162 62 63 91 92 98 135141147 165 166 0 m/z 40 50 60 70 80 90 100 110 120 130 140 150 160 Figure 6.28 Mass spectra of metabolite V produced by N. cyriacigeorgica SBUG 1472 (b) in comparison with the purchased standard acetophenone (a).

APPENDIX 127

Sample ID: M.vaccae 109 +0,025% sec.-octylbenzene, 96h, pH2, methylated Acquired on 01-Jul-2008 at 12:02:14 Reverse fit factor [REV]: 999 MVSEC102 840 (18.500) Cm (837:840-(827:832+848:857)) 7.39e5 105 100 (a) 118

%

103 77 79 91 51 106 121 178 39 41 53 59 74 65 89 92 115 131 146 147 179 0 R:999 CONG 14: 3-PHENYLBUTYRIC ACID - METHYLATED Hit 1 105 100 (b)

118

%

77 103 79 91 51 106 121 178 39 41 59 74 65 89 92 115 131 146 147 179 0 m/z 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 Figure 6.29 Mass spectra of metabolite II produced by M. neoaurum SBUG 109 (a) in comparison with the purchased standard 3-phenylbutyric acid (b). All compounds were methylated. 115 4.38e4 100 145 (a)

176 175

% 117 144 91 39 51 58 63 65 77 89 102 105 118 146 177 50 76 78 92 161 37 41 67 99 131 133 159 162 189 192 198 0 BCH3CIN 983 (20.885) Cm (979:984-(962:977+988:1000)) Scan EI+ 115 2.82e5 100 145 (b)

176 117 % 144 175 91

39 51 58 65 77 102 105 118 146 177 50 52 76 78 89 92 161 37 41 67 99 131 133 147 162 181 192 202 0 m/z 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 Figure 6.30 Mass spectra of metabolite III produced by M. neoaurum SBUG 109 (a) in comparison with the purchased standard ß-methyl cinnamic acid (b). All compounds were methylated.

APPENDIX 128

MV3PB8-9 380 (10.831) Cm (376:384-(356:368+400:411)) 8.62e3 105 100 77 (a)

% 51 43 50 120 78 106 39 52 65 38 63 74 91 0 R:968 CONG 17: ACETOPHENONE Hit 1 105 100 77 (b)

% 51 120 43 50 78 106 52 65 121 38 39 62 63 74 91 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150

Figure 6.31 Mass spectra of metabolite V produced by M. neoaurum SBUG 109 (a) in comparison with the purchased standard acetophenone (b). Reverse fit factor [REV]: 847 MVSEC102 697 (16.116) Cm (695:698-(683:688+713:717)) 3.54e3 105 100 (a)

%

79 119 77 103 106 164 39 41 51 56 59 69 88 91 50 65 73 128 150 0 R:847 CONG 13: 2-PHENYLPROPIONIC ACID-METHYLATED Hit 1 105 100 (b)

%

77 79 103 106 164 51 65 91 39 50 52 59 63 89 165 0 m/z 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

Figure 6.32 Mass spectra of metabolite I produced by M. neoaurum SBUG 109 (a) in comparison with the purchased standard 2-phenylpropionic acid (b). All compounds were methylated.

APPENDIX 129

9.24708557 DICH3SUC 285 (9.247) (a) Scan EI+ 115 1.73e5 100

55

59 % 114 87 45 116 41 42 57 60 74 83 88 91 127 47 53 68 71 72 75 85 99 101 102 106 131 133 145 151156 160 0 MVSEC102 282 (9.197) Cm (278:289-(258:262+298:312)) Scan EI+ 115 451 100 (b) 55

59 %

114 87 42 45 57 67 137 155 41 47 54 65 73 77 82 91 94 96 101 110 117 122 132 135 146 152 159 0 m/z 40 50 60 70 80 90 100 110 120 130 140 150 160 Figure 6.33 Mass spectra of metabolite VIII produced by M. neoaurum SBUG 109 (b) in comparison with the purchased standard succinic acid (a). All compounds were methylated.

Reverse fit factor [REV]: 928 MVIPB102 471 (12.348) Cm (469:477-(442:447+498:511)) 3.59e3 105 100 (a)

77

109 % 51 136 140 50 53 78 82 110 38 39 74 91 92 0 R:928 CONG 26: BENZOIC ACID, METHYLATED Hit 1 105 100 (b)

77

% 136 51 50 106 52 78 137 38 39 74 91 92 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160 Figure 6.34 Mass spectra of metabolite X produced by M. neoaurum SBUG 109 (a) in comparison with the purchased standard benzoic acid (b). All compounds were methylated.

APPENDIX 130

23.58426476 VANILLIC 1145 (23.584) Scan EI+ (a) 165 4.21e5 100 196

% 79 51 77 121 166 197 50 59 63 94 107 125 137 181 39 76 80 92 95 149 153 218 109 126 167 182 199 207 223 229 0 MV3PB8-2 1141 (23.518) Cm (1139:1143-(1112:1133+1154:1173)) Scan EI+ 196 527 100 (b) 165

%

79 43 39 51 55 77 122 125 50 71 83 149 153 197 62 85 97 107 137 171 181 190 212 224 231 0 m/z 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 Figure 6.35 Mass spectra of metabolite XI produced by M. neoaurum SBUG 109 (b) in comparison with the purchased standard 3,4–dihydroxybenzoic acid (a). All compounds were methylated.

Reverse fit factor [REV]: 948 NOSEC113 699 (16.149) Cm (698:703-(682:693+707:719)) 1.30e4 105 100 (a)

% 77 103 51 79 106 164 39 50 53 59 65 91 63 74 89 165 0 R:948 CONG 13: 2-PHENYLPROPIONIC ACID-METHYLATED Hit 1 105 100 (b)

%

77 79 103 106 164 51 65 91 39 50 52 63 89 165 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 Figure 6.36 Mass spectra of metabolite I produced by R. ruber SBUG 82 (a) in comparison with the purchased standard 2-phenylpropionic acid (b). All compounds were methylated.

APPENDIX 131

Reverse fit factor [REV]: 993 NOSEC113 844 (18.566) Cm (839:844-(826:833+848:864)) 5.10e5 105 100 (a)

118

%

77 103 79 91 117 121 51 178 39 41 59 74 65 89 92 131 146 147 179 0 R:993 CONG 14: 3-PHENYLBUTYRIC ACID - METHYLATED Hit 1 105 100 (b)

118

%

103 77 79 91 121 106 178 39 41 51 53 74 59 65 92 115 131 146 147 179 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210

Figure 6.37 Mass spectra of metabolite II produced by R. ruber SBUG 82 (a) in comparison with the purchased standard 3-phenylbutyric acid (b). All compounds were methylated. 115 4.38e4 100 (a) 145

176 175

% 117 144 91 39 51 58 63 65 77 89 102 105 118 146 177 50 76 78 92 161 37 41 67 99 131 133 159 162 189 192 198 0 BCH3CIN 983 (20.885) Cm (979:984-(962:977+988:1000)) Scan EI+ 115 2.82e5 100 145 (b)

176 117 % 144 175 91

39 51 58 65 77 102 105 118 146 177 50 52 76 78 89 92 161 37 41 67 99 131 133 147 162 181 192 202 0 m/z 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200

Figure 6.38 Mass spectra of metabolite III produced by R. ruber SBUG 82 (a) in comparison with the purchased standard ß-methyl cinnamic acid (b). All compounds were methylated.

APPENDIX 132

NO2PP112 654 (15.399) Cm (635:676-(500:559+873:927)) 3.44e4 91 100 (a)

%

150 65 92 39 41 59 89 151 51 77 98 118 119 0 R:967 PHALK 9: PHENYLESSIGSÄURE-METHYLESTER Hit 1 91 100 (b)

%

150 65 92 39 59 151 38 46 51 77 89 105 118 119 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190

Figure 6.39 Mass spectra of metabolite XII produced by R. ruber SBUG 82 (a) in comparison with the purchased standard phenylacetic acid (b). All compounds were methylated. Reverse fit factor [REV]: 899 NOSEC152 470 (12.331) Cm (470:475-(449:455+475:484)) (a) 1.62e3 105 100

77 109

%

51 136 140 50 52 78 95 110 115 120 38 49 66 73 91 92 145 150 160 0 R:899 CONG 26: BENZOIC ACID, METHYLATED Hit 1 105 100 (b)

77

% 136 51 50 106 52 78 137 38 39 74 91 92 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160

Figure 6.40 Mass spectra of metabolite X produced by R. ruber SBUG 82 (a) in comparison with the purchased standard benzoic acid (b). All compounds were methylated.

APPENDIX 133

NOSEC152 1032 (21.700) Cm (1030:1032-(1024:1026+1035:1041)) (a) 292 163 100 165

196 % 107 166 122 149 197 111 167 190 36 38 45 54 64 73 76 91 137 141 150 178 181 202 62 82 94 97 123 209 0 R:537 PHALK 42: 2,3-DIMETHOXY-BENZOESÄURE-METHYLESTER Hit 1 163 100 (b) 165 196

% 107 77 122 149 45 51 79 135 167 65 121 123 150 53 59 76 92 105 136 197 39 66 91 93 118 181 0 m/z 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 Figure 6.41 Mass spectra of metabolite VII produced by R. ruber SBUG 82 (a) in comparison with the purchased standard 2,3-dihydroxyl-benzoic acid (b). All compounds were methylated.

TMSEC092 702 (16.200) Cm (699:706-(681:692+714:723)) 623 105 100 (a)

%

77 79 103 39 51 53 72 82 98 112 116 148 164 45 5962 69 88 93 125 139 140 141 157 169 0 R:781 CONG 13: 2-PHENYLPROPIONIC ACID-METHYLATED Hit 1 105 100 (b)

%

77 79 103 106 164 51 65 91 39 50 52 59 63 89 165 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 Figure 6.42 Mass spectra of metabolite I produced by T. mucoides SBUG 801 (a) in comparison with the purchased standard 2-phenylpropionic acid (b). All compounds were methylated.

APPENDIX 134

Reverse fit factor [REV]: 995 TMSEC092 838 (18.467) Cm (837:840-(825:831+846:855)) 5.98e4 105 100 (a)

118

%

77 79 103 41 91 106 121 39 51 74 178 43 59 65 89 115 131 147 0 R:995 CONG 14: 3-PHENYLBUTYRIC ACID - METHYLATED Hit 1 105 100 (b)

118

%

77 79 103 41 91 106 121 39 51 53 74 178 43 59 65 89 92 115 131 146 147 179 0 m/z 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 Figure 6.43 Mass spectra of metabolite II produced by T. mucoides SBUG-Y 801 (a) in comparison with the purchased standard 3-phenylbutyric acid (b). All compounds were methylated.

TM3PB092 983 (20.885) Cm (980:984-(967:976+985:998)) (a) 714 115 100 145

176 % 117 144 175 91 39 51 65 89 102 118 45 57 63 77 78 105 114 146 157 173 177 41 76 97 122 133 140 161 186 188 0 R:885 CONG 45: BETA-METHYLCINNAMIC ACID, METHYLATED Hit 1 115 (b) 100 145

176 117 144 % 175 91

39 51 58 65 77 105 118 146 50 67 76 78 87 89 92 102 161 177 41 52 131 132 0 m/z 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190

Figure 6.44 Mass spectra of metabolite III produced by T. mucoides SBUG 801 (a) in comparison with the purchased standard ß-methyl cinnamic acid (b). All compounds were methylated.

CURRICULUM VITAE Personal Name: Le Thi Nhi Cong Gender: Female Date of birth: 18 th February 1980 Place of birth: Thanhhoa, Vietnam Nationality: Vietnamese Marital status: Single

E-mail: [email protected] or [email protected]

Education 1987 - 1995 Primary school 1995 - 1998 Secondary school Third prize in Biology National Examination, level 12 Grade : Good 1998 - 2002 Studied in Faculty of Biology, Hanoi University of Science Major subject: Microbiology Grade : Very Good 2002 - 2004 Studied for Master Degree in Faculty of Biology, Hanoi University of Science Major subject: Microbiology Grade : Very Good 2003 – 10/2004 Studied in Joint Graduated Education Program Hanoi-Greifswald for Diploma Equivalent (DE) and won “Ministry of Education and Training (MOET) scholarship” in Improve Human Resources Project (Project 322) of Vietnamese Government. July 2005 - now Ph. D. Student of Ernst-Moritz-Arndt-University of Greifswald Project: Degradation of branched-chain alkanes by microorganisms

Work experience 2002 – now: Worked as a researcher in Department of Petroleum Microbiology, Institute of Biotechnology, Vietnam. Fields of research: - Microflora in gas oil wells and petroleum products. - Diversity of microorganisms in marine. - Application of microorganism in industry and environmental treatment. - Study on biosurfactant-producing microorganisms.

Skills Good English skills German skills Good office computer skills

Personalities Patient and willing to learn

Interests Going on tourist, Reading books and newspapers, Listening classical music, Watching films

List of publications and other scientific achievements

Lai Thuy Hien, Do Thu Phuong, Hoang Hai, Pham Thi Hang, Le Thi Nhi Cong , Kieu Huu Anh (2003). Selection of biosurfactant-producing microorganisms for application in gas-oil industry and environmental treatment. Journal of Biotechnology 1 (1): 119-129.

Tran Dinh Man, Vuong Thi Nga, Pham Thi Hang, Le Thi Nhi Cong , Lai Thuy Hien, Le Tran Binh (2004). Distribution of useful and harmful microorgamisms in shrimp aquaculture water in Tien Hai coastal of Thai binh province. The 2 nd seminar on environmental science and technology issue related to the urban and coastal zones development. 234-238.

Nhi-Cong, Le Thi ; Mikolasch, Annett; Klenk, Hans-Peter; Schauer, Frieder. Degradation of the multiple branched alkane 2,6,10,14-tetramethylpentadecane (pristane) in Rhodococcus ruber and Mycobacterium neoaurum. International Biodeterioration and Biodegradation ( Accepted )

Le Thi Nhi-Cong , Annett Mikolasch, Susanne Awe, Halah Sheikhany, Hans-Peter Klenk, Frider Schauer. Oxidation of aliphatic, branched chain, and aromatic hydrocarbons by Nocardia cyriacigeorgica isolated from oil-polluted sand samples collected in Saudi Arabian Desert. (Submitted )

Erklärung

Hiermit erkläre ich, dass diese Arbeit bisher von mir weder an der Mathematisch- Naturwissenschaftlichen Fakultät der Ernst-Moritz-Anrdt-Universität Greifswald noch einer anderen wissenschaftlichen Einrichtung zum Zwecke der Promotion eingereicht wurde. Ferner erkläre ich, dass diese Arbeit selbständig verfaßt und keine anderen als die darin angegebenen Hilfsmittel benutzt habe.

Le Thi Nhi Cong