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Biodegradation of azaarenes by
Novosphingobium Subartica (sp. LH128)
12-01-2004Master Project 2003
Gijs Manneveld, 9900411Supervisor: John Parsons
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Biodegradation of Azaarenes
by
Novosphingobium Subartica (sp. LH128)
Gijs Manneveld
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Contents
1 Summary.........................................................................................................................................4
1.1 Introduction..............................................................................................................................4
1.2 Degradation kinetics................................................................................................................4
1.3 Metabolic pathways .................................................................................................................5
2 Introduction....................................................................................................................................6
2.1 Degradation kinetics................................................................................................................7
2.2 Metabolic pathways .................................................................................................................9
3 Materials and methods ................................................................................................................11
3.1 Chemicals...............................................................................................................................11
3.2 Equipment ..............................................................................................................................12
3.3 Bacterial strain ......................................................................................................................13
3.4 Extraction test ........................................................................................................................13
3.5 Recovery determination of the concentration step.................................................................13
3.6 Abiotic degradation ...............................................................................................................13
3.7 Continuous culture.................................................................................................................14 3.7.1 Continuous culture; co-substrate concentration 15% in relation to the carbon source concentration..................................................................................................................................15 3.7.2 Continuous culture; co-substrate concentration 60% in relation to the carbon source concentration..................................................................................................................................15 3.7.3 Measurement of optical density .....................................................................................15 3.7.4 Determination glucose concentration ............................................................................16 3.7.5 Determination total protein concentration .....................................................................16 3.7.6 Selective plating.............................................................................................................16 3.7.7 HPLC analysis ...............................................................................................................16
3.8 Metabolite analysis ................................................................................................................17 3.8.1 HPLC analysis ...............................................................................................................17
3.8.1.1 HPLC/LC-MS............................................................................................................17 3.8.2 LC-MS analysis .............................................................................................................17 3.8.3 Co-substrates used for the metabolite analysis ..............................................................18
4 Results ...........................................................................................................................................19
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4.1 Extraction test ........................................................................................................................19
4.2 Recovery determination of the concentration step.................................................................20
4.3 Abiotic degradation ...............................................................................................................21
4.4 Continuous culture: co-substrate concentration 15% in relation to the carbon-source .......22 4.4.1 Optical density (OD) and total protein measurements:..................................................22 4.4.2 HPLC .............................................................................................................................24
4.4.2.1 Culture I and II: with glucose as carbon source.........................................................24 4.4.2.2 Cultures III and IV: phenanthrene as carbon source..................................................25 4.4.2.3 Phenanthridine degradation .......................................................................................26 4.4.2.4 Acridine degradation..................................................................................................28
4.4.3 Calculation of kinetic degradation constants .................................................................30
4.5 Continuous culture: co-substrate concentration 60% of carbon source ...............................31 4.5.1 Optical density (OD) and total protein measurements...................................................32 4.5.2 HPLC .............................................................................................................................33 4.5.3 Calculation of kinetic degradation constants .................................................................34
4.6 Metabolite analysis ................................................................................................................35 4.6.1 Carbazole metabolites ....................................................................................................36
5 Discussion......................................................................................................................................40
5.1 Continuous culture.................................................................................................................40
5.2 Metabolite analysis ................................................................................................................41
6 Appendix A; Wick-medium ........................................................................................................43
7 Reference ......................................................................................................................................44
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1 Summary
1.1 Introduction Polycyclic aromatic hydrocarbons (PAH’s) contain two or more fused rings made of
carbon and hydrogen atoms. If one carbon is replaced with nitrogen, they are called nitrogen polycyclic hydrocarbons (NPAH’s), or more general azaarenes. Azaarenes are usually found around land polluted with coal tar or crude oil. They are also found around wood preserving facilities. The toxicity of the azaarenes is normally based on genotoxic, teratogenic and other developmental effects. Because they resemble biological active molecules they have the tendency to disrupt biological systems like DNA-replication.
In order to predict the degradation of the azaarenes in the environment different parameters need to be found. An important parameter is the biological fate of the azaarenes. The biological fate can be divided in two parameters, the degradation kinetics and the metabolic pathways of azaarenes. From earlier studies is known that azaarenes can be degraded by Novosphingobium Subartica sp. LH128, but only in the presence of a growth substrate, which is called co-metabolism.
1.2 Degradation kinetics
In a continuous culture the degradation constants in the Michaelis-Menten kinetics were determined, but due to high efficiency degradation of the azaarenes by LH128 those constants could not be determined. In a chemostat a culture of LH128 was started growing on either glucose or phenanthrene in the presence of the azaarenes acridine and phenanthridine. The concentrations of the azaarenes were followed by HPLC-FLU.
In the first experiments the azaarenes could not be determined anymore, degradation
efficiency was >99.9%. After some adjustments, bacterial density down a 5-fold and azaarene concentration >60% in comparison with the carbon source concentration, the degradation was still >99% and the constants could still not be determined. Also the low growth rate of the culture led to instability in the chemostat, so further experiments were stopped. For future experiments another bacterial strain would be best to use at higher bacterial density. A bacterial strain that can handle high concentrations of azaarenes in order to make sure the concentrations stay above the detection limit of the HPLC-FLU.
The results from the above experiments showed that different enzymes were probably
used in the degradation of the azaarenes than used for the utilisation of phenanthrene. Therefore batch cultures were started to find the metabolic pathway of the azaarenes within LH128.
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1.3 Metabolic pathways
The batch cultures were pre-grown on n-nonadecane to make sure that during the analysis similar metabolites of phenanthrene were not detected along metabolites of the azaarenes. But it seems that LH128 was not able to degrade the azaarenes anymore. Also some cultures did not grow at all, with exception of carbazole, due to the toxicity of the azaarenes. Extra cultures were started with lower azaarene concentration, after a considerable density was reached; the cultures were extracted, concentrated and measured on the LC-MS. Only for carbazole probable metabolites could be detected, but due to low-resolution mass spectra no structure elucidation could be done. Probably growing on n-nonadecane did not trigger the induction of enzymes necessary for the degradation of the azaarenes, or the induction time for the degradation enzymes is longer than the time the cultures were followed
For future experiments it is advisable to use phenanthrene as carbon source instead, in
order to make sure the azaarenes are degraded. And to be sure that there is degradation, the culture and azaarenes concentration should be. But to counteract any low concentration problems several cultures should be started and every day one culture should be extracted and measured on the LC-MS. This way you will have a high enough concentration for the LC-MS and can you follow the culture in time.
It is advisable to use the LC-MS for the pathway elucidation, because with the LC-MS
there is also the possibility of structure elucidation, which cannot be done with HPLC-FLU or HPLC-UV. If the LC-MS is used, the different parts of the system should be better optimised for the metabolites and the parent compound. For example optimising the cone-voltage for better fragmentation and the source voltage for a better ionisation.
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2 Introduction
Azaarenes are nitrogen heterocyclic polyaromatic hydrocarbons (NPAH’s), derivatives of polycyclic aromatic hydrocarbons (PAH’s), which contain two or more fused rings made of carbon and hydrogen atoms. Azaarenes are found naturally in a mixture together with PAH’s and other organic compounds in crude oils and tars, but are also found in synthetic oils and various pesticide formulations. They were even found in aroma of tea, coffee and tobacco (i). NPAH’s are usually present in much smaller quantities than PAH’s.
Many azaarenes are able to disrupt cellular processes like DNA replication and can cause cancer. The azaarenes form the basic structure for several alkaloids (i) and are also similar to molecules with biological functions including co-enzymes, vitamins, and nucleic acids and can take their place in different biological systems. The general toxicity of the azaarenes is similar to PAH’s, they are for example both able to induce narcosis. Also other toxic effects such as genotoxic, teratogenic and other developmental effects have been found. (ii)
Industrial effluents, oil spills and incomplete combustion of for example vehicle exhaust, diesel, kerosene, but also domestic residential cooking/heating and wood burning are the primary entry routes of azaarenes into the environment. (iii) Because the nitrogen-substituted compounds are more water-soluble than their corresponding PAH’s they tend to remain in the aqueous phase rather than adsorbing to organic matter. It makes the compounds environmentally more dangerous than PAH’s, because they can spread more easily in the environment, while PAH’s are more likely to stay around the source. Due to their high mobility and toxicity they can have a larger environmental impact that PAH’s (iv). In order to predict the fate of the azaarenes in the environment different parameters need to be found.
An important parameter is the biological fate of the azaarenes. In nature the azaarenes are the subjected to degradation through a variety of processes. In the aquatic environment biological transformations are of primary importance (iv). Bacteria usually transform the azaarenes into more polar metabolites in order to make their excretion and transport in the aquatic environment easier, biotransformation. (see figure 1). This also gives another problem, because the more polar compounds are usually more toxic than their parent compounds.
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N N
OH
OH
Figure 1: example of co-metabolism/biotransformation, partially degradation of phenanthridine into the more polar phenanthridine-2,3-diol.
The azaarenes can be degraded aerobically by several bacterial strains. Novosphingobium Subartica sp. LH128 is know to be able to degrade azaarenes to some extend in the presence of phenanthrene or glucose (viii). The azaarenes cannot be utilised as the sole carbon- and energy source by LH128, they will only be degraded in the presence of another compound that acts as the carbon and energy source, for example a homologue PAH; phenanthrene. This is called co-metabolism, which is defined as the transformation of a non-growth substrate by growing cells in the presence of a growth substrate. Co-metabolism usually results from the lack of specificity of enzymes and co-factors. (v)
2.1 Degradation kinetics
One of the biological parameters to be determined are the degradation constants, the kinetics of the degradation of azaarenes. In earlier studies the kinetics were already determined in batch cultures of Novosphingobium Subartica sp. LH128 (viii). To get more representative constants, they are also determined in a in a chemostat. In a chemostat the culture will be a continuous culture, which will give a more nature like environment, (see 3.7). A continuous culture has a constant flux of the necessary nutrients in and out of the culture. By fixing the dilution rate and limiting the carbon source the growth rate of the bacteria can be controlled. The ability to fix all the parameters of the culture in a continuous culture, for example growth rate, temperature and pH, will lead to a high degree of control on the culture. (see figure 2). An advantage is the longer periods in which the culture can be used. Also there is a more nature like competition for the necessary compounds than in a batch culture.
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ratedilutionVFD
rategrowthSK
S
steady
state
s
µ
µµ
==
+= max
Figure 2: equations for the growth rate and dilution rate in a continuous culture, by fixing the dilution rate of the chemostat the growth rate becomes equal to the dilution rate. In the equations S is the substrate concentration, Ks is the affinity constant for the substrate, µ(max) is the (maximal) growth rate, D is the dilution rate, F is the flow rate of the medium, and V is the volume of the chemostat.
The kinetic constants of the degradation can be determined with help of Michaelis-Menten kinetics. (see figure 3). Two different parameters can be determined, the Km, an affinity constant (the affinity of the enzyme for the co-substrate) and Vmax, the maximum degradation rate. Both parameters will give a tool for determining the degradation modes of the different azaarenes.
)(][
][max
SSDVSK
SVV
R
m
−=+
=
Figure 3: equation to determine the maximum degradation rate and affinity constants of the different azaarenes in a continuous culture. Where the first equation the Michealis-Menten kinetics determines, V(max) is the (maximum) degradation rate, Km is the affinity constant, S is the substrate concentration in the chemostat, D is the dilution rate and SR is the substrate concentration in the reservoir.
In the above equations D and SR are known and S can be measured. Changing one of the pre-determined parameters D or SR, the constants Vmax and Km can be determined. In this case the substrate concentrations are changed. With a plot of the different V against the different S the constants can be determined (see figure 4). Ignoring the fact that Michaelis-Menten is only for enzymes and assuming that the enzyme catalysis/transformation step is the rate-limiting step in the degradation and this method works only if the degradation of the co-substrate reaches a steady state and the concentration of the substrate stays constant over longer periods of time.
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0
0,5
1
1,5
2
2,5
3
3,5
0 0,15 0,3 0,45 0,6 0,75 0,9 1,05 1,2S
V
Vmax
1/2 Vmax
Km
Figure 4: example of the determination of the kinetic constants Km and Vmax by plotting S against V of the continuous culture.
2.2 Metabolic pathways
The metabolic pathways of the azaarenes within LH128 play an important role in the determination of the effect of azaarenes in the environment. Microbial degradation of azaarenes is one of the most important factors in reducing their concentrations, therefore it is of special interest to study the degradation of such compounds and gain a better understanding of the fate of these compounds in the environment (vi). Especially the fate of the metabolites can have a profound influence on the impact of the azaarenes on the environment. More polar metabolites will have a higher distribution among the environment and the toxic compounds will be more spread out than before the transformation. If the polar compounds are more toxic this could be a large threat to the environment. But it is also possible that LH128 can degrade the azaarenes completely so the azaarenes form after the degradation no threat anymore.
Little is known of the biodegradation pathways of these compounds within the species LH128, some first degradation steps are known, for example the transformation of acridine into acridinone and phenanthridine into phenanthridinone (viii). The azaarenes are probably degraded further than the aldehyde/ketone form, but how exactly is not known. Probably the pathways are similar to those of phenanthrene and other analogue PAH’s which has been determined (vii). In earlier studies (viii) it was assumed that azaarenes were degraded with the help of the same enzymes as being used in the utilisation of phenanthrene as carbon and energy source by LH128, so it can expected that the azaarenes have a similar metabolic pathway.
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Different cultures are followed, with different azaarenes present, in time with the HPLC-FLU, HPLC-UV and also extracting whole cultures after longer period of exposure, for detection with the LC-MS. In this way there is going to be searched for the formation of possible metabolites of azaarenes by LH128 and the possible elucidation of metabolic pathways of the azaarenes. The LC-MS can be used to elucidate the structure of the different metabolites. By changing the cone voltage different fragmentation spectra can be obtained. In figure 31 a representation is given of the interface of the LC-MS used in the metabolite analysis.
Cone, entrance to the MS, by changing the cone voltage fragmentation can be controlled
Ionization and evaporation area
Source voltage, determines the ionisation grade of the molecules
LC -> MS
Figure 31: a photograph of an LC-MS interface in the ESI-mode.
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3 Materials and methods
3.1 Chemicals The following chemicals were used with the experiments described below: Acridine, 97%, Sigma-Aldrich, Steinheim, Germany n-Nonadecane, 99%, Acros Organics, Geel, Belgium 7,8-Benzoquinoline, 97%, Sigma-Aldrich, Steinheim, Germany Isoquinoline, 97%, Aldrich Chemie, Steinheim, Germany 9-Methylacridine, 95%, Aldrich Chemical Co., Millwaukee, WI (USA) Quinoline, 98% (GC), Sigma-Aldrich, Steinheim, Germany Phenanthridine, 98% (GC,T), Sigma-Aldrich, Steinheim, Germany Dibenz[c,h]acridine, no. 0015, Commission of the European Communities, Community
bureau of reference (BCR) Alpha-D(+)-Glucose, anhydrous, 99+%, Acros Organics, Geel, Belgium Phenanthrene, 97%, Acros Organics, Geel Belgium Fluoranthene, 97%, Acros Organics, Geel, Belgium Bacteriological Agar, for molecular biology, Sigma-Aldrich Co., St. Louis, MO (USA) Glucose (Trinder) 100, Sigma-Diagnostics Inc., St. Louis, mo (USA) Bio-Rad Dc protein assay, reagents A, B and S, Bio-Rad Laboratories, Hercules, CA
(USA) Acetic acid, glacial 100%, p.a., Merck, Darmstadt, Germany Acetonitrile, HPLC-grade, Rathburn Chemicals Limited, Walkerburn, Scotland Dichloromethane, stabilized c.p., Acros Organics, Geel, Belgium Methanol, HPLC-grade, Rathburn Chemicals Limited, Walkerburn, Scotland Ethyl acetate, 99.5+%, Acros Organics, Geel, Belgium Acetone, p.a., Acros Organics, Geel, Belgium Pentane, p.a., Acros Organics, Geel, Belgium Sulfuric Acid, 4M
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3.2 Equipment The following equipment was used during experiments described below: For the measurement of the optical density:
Vitatron Universal photometer, λabsorbance=540 nm For the chemostat the following filters were used to filter incoming air into the growth medium:
Acrodisc 25 mm, Syringe filter 0.2 µm, HT Tuffryn membrane, Pall Corp. Ann Harbor, MI (USA)
(were also used for filtering proteins from LC-MS samples) The HPLC-system consisted of:
Waters 717plus Autosampler Waters 484 Tunable Absorbance detector Waters 600 Controller Waters 474 Fluorescence detector column; 125-2.00 mm 3 micron Lichrospher 3u RP18, Phenomenex Data-analysis; Chromstar 4.0
For the measurement of the protein and glucose concentration:
UV500, UV-Visible photometer, Unicam Data-analysis; Vision 32-bit version 1.24, Thermospectronic
The LC-MS-system consists of:
Spectrasystem SN4000 Spectrasystem P4000 (pump) Spectrasystem AS300 (autosampler) Spectrasystem FL3000 (fluorescence detector) Navigator LC/MS, Thermoquest, Finnigan Copr., quadropole Column: 125x2.00 mm 3 micron Lichrosphere 4u, RP18, phenomenex Data-analysis; Xcalibur, Home Page version 1.2, Finnigna Corp.
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3.3 Bacterial strain
For the degradation experiments performed in this project, Novosphingobium Subartica sp. LH128 was chosen, because of its capability to co-metabolise azaarenes and its ability to utilize phenanthrene as a sole source of carbon and energy (viii). The species were discovered in a coal tar polluted landfill, where these species were also isolated from. The strain was first determined as a part of the Sphingomonas family, but later re-determined as Novosphingobium Subartica sp. LH128 (ix). The strain used in the experiments for the continuous cultures is a laboratory strain pre-grown on Wick-medium (see appendix A) and phenanthrene as a sole source for carbon and energy. For the metabolism analysis the same strain of LH128 was pre-grown on Wick-medium and n-nonadecane as a sole source for carbon and energy. (x)
3.4 Extraction test
To 11 ml of distilled water were added 800 µl of184 µg/ml phenanthrene (in methanol) and 800 µl of 31 µg/ml phenanthridine (in methanol). The solution was respectively extracted 5 times with 4 ml pentane and 4 ml dichloromethane at a pH of 7 and 3.1 ml sample of the water phase was after every extraction measured directly into the HPLC. (see 3.7.7.)
3.5 Recovery determination of the concentration step
To 15 ml of dichloromethane were added 100 µl of 25 µg/ml fluoranthene, 50 µl of 184 µg/ml phenanthrene, 100 µl of 31 µg/ml phenanthridine and 100 µl of 42.1 µg/ml acridine (all in methanol). The dichloromethane was blown to dryness under a nitrogen flow, the residue was dissolved in 1 ml of acetonitril and directly measured on the HPLC. (see 3.7.7.)
3.6 Abiotic degradation
In order to see if phenanthrene, acridine and phenanthridine weren’t degraded in sunlight, a degradation test was done. For the abiotic degradation test two erlenmeyers with 20 ml destilled water and with 600 µl 184 µg/ml phenanthrene (in methanol) and respectively 600 µl 41.6 µg/ml acridine and 31 µg/ml phenanthridine (in methanol) were left on the lab table in sunlight and daily for 9 days 1 ml samples (in duplicate) were taken and measured on the HPLC. (see 3.7.7.)
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3.7 Continuous culture
For the determination of the kinetic constants of the degradation of azaarenes in a continuous culture, a culture of Novosphingobium Subartica sp. LH128 was grown and maintained in a chemostat (see figure 5). LH128 was pre-grown on phenanthrene and Wick-medium (see appendix A) in a batch culture. The chemostat was filled with 400 ml Wick-medium (see appendix A), closed and sterilised for 45 min at 5 kPa.
Figure 5: representation of the chemostat system used with the continuous culture experiments
~12 g/l glucose under sterile conditions was added to the chemostat as carbon source, after which 2 ml of pre-grown (phenanthrene) LH128 was added to the culture. The chemostats were thermostated at 25 °C and sterile air was blown into the culture at ~19 ml/min. After a considerable density has been reached, the chemostat was connected to the media with a constant dilution rate, D, which is determined by the ratio of the flow rate, F, over the volume, V, of the chemostat. After a steady state has been reached, control by measurement of the optical density, glucose concentration and protein concentration of the culture, the co-substrates were added to the media and the concentrations were followed in time with HPLC.
For the HPLC, 10 ml samples were taken (with glass syringes) and acidified with 1 ml of 4M sulfuric acid and 200 µl 25 µg/ml fluoranthene was added as the internal standard. The samples were stored in the fridge at 4 °C in brown flasks with 4 ml dichloromethane. Before injection the samples were three times extracted with 4 ml of dichloromethane, dried under reflux with the help of a vigreux and the last dichloromethane under a nitrogen flow. The residue was dissolved in methanol and directly injected on the HPLC (see 3.7.7.)
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The steady state of the culture is followed in time with the measurements of the optical density (see 3.7.3), glucose concentration (see 3.7.4) and the protein concentration (see 3.7.5.) Selective plating checked the purity of the culture (see 3.7.6.)
3.7.1 Continuous culture; co-substrate concentration 15% in relation to the carbon source concentration
Four chemostats were used, two with glucose as carbon and energy source for the
bacteria and respectively acridine and phenanthridine as co-substrate and two with phenanthrene as carbon and energy source and respectively acridine and phenanthridine as co-substrate. The concentrations of the carbon sources were 0.12 g/l for glucose at ~0.26 ml/min and 3 g/l phenanthrene (in methanol) at a dilution rate of ~0.010 ml/min, which resulted in a concentration in the culture of respectively 0.03 mg/min glucose and 0.03 mg/min phenanthrene. The co-substrate concentration was 15% of the phenanthrene concentration. The co-substrates were also added in methanol at a dilution rate of ~0.010 ml/min.
3.7.2 Continuous culture; co-substrate concentration 60% in relation to the carbon source concentration
Three chemostats were used, two with phenanthrene as carbon source and one with
glucose as carbon source. One chemostat culture served as control and was only growing on phenanthrene. The other cultures had phenanthridine as co-substrate in a concentration 60% of the carbon source concentration. The concentrations of the carbon source were respectively 0.024 g/l glucose at ~0.26 ml/min and 0.6 g/l phenanthrene (in methanol) at ~0.010 ml/min, which resulted in a concentration in the culture of respectively 0.006 mg/min glucose and 0.006 mg/min phenanthrene. Also phenanthridine was also added in methanol to the culture at ~0.010 ml/min.
3.7.3 Measurement of optical density
2 ml culture has been sampled under sterile conditions and the absorbance at λ=540 nm was measured.
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3.7.4 Determination glucose concentration
2 ml culture has been sampled under sterile conditions and centrifuged down for 6 minutes at 14,000 rpm. 500 µl of the supernatant was mixed with 1ml Trinder reagent and vortexed. The solution was placed in a waterbath of 37°C for 15 minutes. The absorbance at λ=515 nm was measured and the data has been compared with a calibration line.
3.7.5 Determination total protein concentration
2 ml culture has been sampled under sterile conditions and centrifuged down for 6 minutes at 14.000 rpm. The pellet has been mixed with 25 µl of 2% (w/v) SDS, 25 µl of 0.6 M KOH, 125 µl of reagent A (with 1% reagent S) (BioRad) and 1 ml of reagent B (BioRad), after every step the solution was vortexed. The solution was incubated for 15 minutes at room temperature and the absorbance at λ=750 nm was measured against a blank (distilled water). The data has been compared with a calibration line of bovine serum gamma-globulin.
3.7.6 Selective plating 2 ml culture has been sampled under sterile conditions and a dilution range of 10 steps was made under sterile conditions. 10 µl drops of the different dilution steps were placed on a glucose agar plate and a glucose agar plate with 200 µg/ml streptomycine. The plates were incubated at 30 °C for a couple of days until colonies could be counted. The plates were made from Wick-medium (see appendix A), 20 g/l Agar and 2 g/l glucose (carbon/energy source). Sterilised at 2 kPa for 20 min. After sterilisation, if necessary, the streptomycin was added.
3.7.7 HPLC analysis
The samples were measured on the HPLC with a mobile phase of 60% acetonitrile, 40% 4 g/l sodium acetate (aq) pH=4, 0.2 ml/min and an injection volume of 1 µl. The fluorescence detection of phenanthrene and phenanthridine at λexcitation=254, λemission=364 nm, of acridine at λexcitation =250, λemission =400 nm and of fluoranthene at λexcitation =288, λemission =462 nm. (xi) If necessary the data were correlated to the concentration of fluoranthene on a calibration line.
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3.8 Metabolite analysis
3.8.1 HPLC analysis
In the first experiments an erlenmeyer with 100 ml Wick –medium was sterilised at 5kPa for 45 min. Under sterile conditions 4 g/l n-nonadecane (carbon/energy source) and 0.5 g/l co-substrate (see table 1) were added dissolved in acetone. Overnight the acetone was evaporated at 25 °C shaking the erlenmeyer at 200 rpm. After a night shaking, 1 ml of LH128, pre-grown on n-nonadecane was added under sterile conditions to the medium in the erlenmeyer. The erlenmeyer was put away shaking at 200 rpm and at 25 °C. Every 24h 1.5 ml sample was taken under sterile conditions. 1 ml was used for the determination of the optical density (see 3.7.3), to 0.5 ml sample 0.5 ml 4M sulphuric acid was added and the solution was centrifuged at 14.000 rpm for 6 min. The supernatant was directly measured at the HPLC and later also on the LC-MS
In later experiments the n-nonadecane and co-substrate (see table 1) were not added in acetone anymore and the temperature was raised to 34 °C, two degrees above the melting point of n-nonadecane.
3.8.1.1 HPLC/LC-MS
The samples were measured three times on the HPLC, with a mobile phase of 60% acetonitrile, 40% 4 g/l sodium acetate (aq) pH=4, 0.2 ml/min and an injection volume of 1 µl. The detection of the compounds and metabolites was done with UV-absorbance at 254, 200 and 300 nm and with fluorescence detection at λexcitation= 254 nm, λemission=364, 400, 464 nm. The samples were also measured on the LC-MS, with a mobile phase starting with 80% water/methanol (3:1) with 0.1 mM sodium acetate and 20 % methanol, stepwise changing in three minutes to a mobile phase consisting of 30% water/methanol (3:1) with 0.1 mM sodium acetate and 70% methanol, with a flow of 0.25 ml/min and an injection volume of 100 µl. The detection was done with a cone voltage of 4kV and a fragmentation voltage of, ±25 eV, ±100 eV, full scan for m/z between 50 and 300.
3.8.2 LC-MS analysis
For the LC-MS analysis of metabolites an erlenmeyer with 100 ml Wick –medium was sterilised at 5kPa for 45 min. Under sterile conditions 4 g/l n-nonadecane (carbon/energy source) and a small amount of co-substrate (see table x) were added. After 48 hours of stabilizing at 34 °C and 200 rpm 1 ml of LH128, pre-grown on Wick-medium and n-nonadecane, were added to the medium. The culture was left shaking until a considerable density was reached; this was checked under the microscope. After the density was high enough, the culture was centrifuged down at 8000 rpm for 30 minutes. The supernatant was
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extracted 3 times with ethylacetate, half the volume of the supernatant; the ethylacetate was evaporated at 60°C under a nitrogen flow. The residue was rinsed with 2 ml methanol and filtered through a 0.2 µm filter to remove the proteins and after filtering the sample was directly measured on the LC-MS, with a mobile phase consisting of 40% water/methanol (3:1) with 0.1 mM sodium acetate and 60% methanol, with a flow of 0.25 ml/min and an injection volume of 10 µl. The detection was done with a cone voltage of 4kV and a fragmentation voltage of, ±25 eV, ±50 eV, ±75 eV, ±100 eV, full scan for m/z between 50 and 350.
3.8.3 Co-substrates used for the metabolite analysis Co-substrate HPLC/LC-MS LC-MS Acridine No Yes Phenanthridine Yes Yes Carbazole Yes Yes Benzo(h)quinoline No Yes Quinoline No Yes Isoquinoline Yes Yes 9-methylacridine No Yes Dibenzacridine No Yes
Table 1: the co-substrates used in the analysis of metabolites
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4 Results
4.1 Extraction test
Using dichloromethane under acid and base conditions, most of the compounds are removed from the water phase after one extraction, as can be seen in figure 6 for an extraction with dichloromethane under acidic conditions, pH~3.
Figure 6: two chromatograms of the extraction test done with dichloromethane under acidic conditions, 14ma0007 is the water phase before the extraction and 14ma0008 after one extraction with dichloromethane
The results of the extraction with ethylacetate under acid conditions can be seen in figure 7. After two extractions with acid conditions there are still azaarenes in the water phase. The same results are seen with basic conditions.
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Figure 7: HPLC-chromatogram of the extraction of phenanthridine, phenanthrene and fluoranthene with ethylacetate under acidic conditions, where 18ma0002 before the extraction is and 18ma0003 after 2 times extracting with ethylacetate.
For the extraction of azaarenes out of the water phase the best organic solvent used is dichloromethane. As the experiments were qualitative, no recoveries are calculated for the extraction.
4.2 Recovery determination of the concentration step
In order to determine the recoveries of the concentration step of the extraction samples under a nitrogen flow, standard solutions were concentrated under a nitrogen flow and measured on the HPLC and compared with the concentration at the start of the concentration step. As can be seen in table 2, the drying under a nitrogen flow will not give large deviations and is thereby a suitable method to concentrate the samples. compound %-recovery phenanthridine 96.6 phenanthrene 104.3 acridine 100.7
Table 2: The recovery of the concentration step after blowing a standard solution with phenanthreen, phenanthridine and acridine in dichloromethane to dryness under a nitrogen flow at 40°C.
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4.3 Abiotic degradation
In order to see if phenanthridine, phenanthrene and acridine were degraded in sunlight a degradation test was done. As can be seen in figure 8, there seems to be no abiotic degradation of phenanthridine, acridine and phenanthridine in daylight. The degradation of phenanthridine, phenanthrene and acridine takes only place biotic in the presence of LH128.
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
0 1 2 3 4 5 6 7 8 9 10
days
peakarea
Figure 8: abiotic degradation test of phenanthrene, acridine, phenanthridine in water in daylight (duplicate HPLC-measurements). The purple squares represent the phenanthrene concentration, the red triangles represent the phenanthridine concentration and the dark blue diamonds represent the acridine concentration.
21
4.4 Continuous culture: co-substrate concentration 15% in relation to the carbon-source
The first experiments were started with a co-substrate concentration of 15% of the
carbon source. The cultures were followed in time by optical density, total protein concentration and the HPLC (see figures 9 t/m 11, 4.4.1 and 4.4.2).
4.4.1 Optical density (OD) and total protein measurements:
0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.070
0.080
0.090
0 2 4 6 8 10 12 14 16
days
ng/m
l pro
tein
e
Cultures III and IV, azaarenes added
Cultures I and II azaarenes added
Figure 9: total protein measurements of cultures of LH128 in a chemostat; I: glucose and phenanthridine, II: glucose and acridine, III: phenanthrene and phenanthridine and IV: phenanthrene and acridine before and after the azaarenes were added. The dark blue diamonds represent culture I, the purple squares culture II, the green triangles culture III and the orange diamonds represent culture IV.
22
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0 2 4 6 8 10 12 14 16
days
A54
0
Cultures III and IV, azaarenes added
Cultures I and II azaarenes added
Figure 10: Optical density of cultures of LH128 in a chemostat at 540 nm; I: glucose and phenanthridine, II: glucose and acridine, III: phenanthrene and phenanthridine and IV: phenanthrene and acridine before and after the azaarenes were added. The dark blue diamonds represent culture II, the purple squares culture I, the red triangles culture III and the orange diamonds represent culture IV.
The cultures growing on phenanthrene were at a relative stable steady state, before and after the azaarenes were added. The cultures growing on glucose were relative stable before the azaarenes were added, but had some more problems maintaining a steady growth rate after the azaarenes were added, it seems that the cultures need a longer lag phase in order to react onto the possible toxicity of the azaarenes. This is also according to van Herwijnen et al (viii), who found that a culture of LH128 grown on glucose in a batch needed a longer lag phase before it could properly co-metabolise the azaarenes. The bacteria need that time to induce the enzymes needed for the metabolism of the azaarenes. Because the azaarenes resembles phenanthrene in structure, the bacteria growing on phenanthrene probably do not need to induce the enzymes needed for degradation of the co-substrate.
23
4.4.2 HPLC
4.4.2.1 Culture I and II: with glucose as carbon source
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
0 1 2 3 4 5 6 7 8
days
co-s
ubst
rate
(µg/
ml)
Figure 11: concentration of the co-substrate in cultures I and II (in duplicate), phenanthridine and acridine respectively, followed in time with glucose as growth substrate. The dark blue diamonds represent culture I and the purple squares represent culture II. (after 5 days no more measurements were done on culture I)
The HPLC-results of the cultures growing on glucose show that the degradation efficiency of the co-substrates are high in the beginning, but decreases after 3 days. After which the degradation efficiency of acridine slowly increases, it seems that bacteria need some time to induce the proper enzymes or that the bacteria need some time to adjust to a ‘toxic’ metabolite, which the concentration, see figure 11, increases and decreases equally with acridine. The degradation efficiency of phenanthridine seems to decrease even further, but the metabolite concentration seems to decrease after a few days (see figure 12). This could be caused by a higher affinity of the bacteria for the metabolite than for phenanthridine.
24
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
0 1 2 3 4 5 6 7 8
days
rel a
rea
(/FA
)
Figure 12: relative area's of the metabolites of phenanthridine and acridine against time in the cultures with glucose as carbon source. The purple squares represent the relative area of the metabolite of acridine and the dark blue diamonds represent the metabolite of phenanthridine
4.4.2.2 Cultures III and IV: phenanthrene as carbon source
0.000
0.020
0.040
0.060
0.080
0.100
0.120
0.140
0 1 2 3 4 5 6 7 8 9
days
phen
anth
rene
(µg/
ml)
azaarenes added
Figure 13: The phenanthrene concentration (carbon source, duplicate) in time of cultures III en IV, respectively phenanthridine and acridine as co-substrate. The purple squares represents culture IV and the dark blue diamonds culture III. (after 5 days no more measurements were done on culture III)
25
After the azaarenes were added the degradation of phenanthrene in combination with acridine seems to be more efficient, for phenanthridine there seems to be inhibition.(see figure 13) of the degradation of phenanthrene, but more measurements could give a more detailed answer.
The concentrations of acridine and phenanthridine are below the detection level, so nothing can be said about the degradation efficiency in combination with phenanthrene as carbon source, except it is almost completely.
4.4.2.3 Phenanthridine degradation
0,00 5,00 10,00 15,00 20,00 25,00
Retention time (min)
Metabolite of phenathridine
phenanthridine
Internal standard
phenanthrene
phenanthrene-metabolite
Figure 14: HPLC Chromatograms of a culture with glucose as carbon source and phenanthridine as co-substrate represented with the dark green line, and two cultures grown on phenanthrene with phenanthridine as co-substrate and without, represented respectively by the red and dark blue lines.
In the above displayed chromatograms a peak representing a possible metabolite of phenanthridine and a peak representing phenanthridine can be seen by the culture grown on glucose. In the culture grown on phenanthrene, without phenanthridine, there is a peak representing a possible metabolite of phenanthrene, but in the culture with phenanthridine those metabolites are gone. In the same culture the phenanthridine-metabolite, as well as phenanthridine, inside the culture with glucose as carbon source, is not detectable anymore and the phenanthrene-metabolite concentration decreases in time (see figure 15). The metabolites of phenanthridine and phenanthrene are probably utilised in the metabolic pathways of LH128 and are further degraded. Possibly the enzymes for the degradation of phenanthrene are not specific for phenanthrene alone and can degrade more compounds that are similar to phenanthrene.
26
In a glucose grown culture there seems to be some degradation of the metabolites
(see figure 12), but the bacteria aren’t as efficient as the phenanthrene-grown LH128, which could indicate that the enzymes induced by the glucose grown bacteria for the degradation of phenanthridine are not the same as the one induced in the phenanthrene grown bacteria. But it could also indicate that LH128 grown on phenanthrene induces more enzymes to counteract the larger amount of similar substrates available for the bacteria.
0.000
0.020
0.040
0.060
0.080
0.100
0.120
0.140
0.160
0.180
0.200
0 1 2 3 4 5 6 7
days
rel a
rea
(/FA
)
phenanthridine added
Figure 15: relative area of the phenanthrene metabolite detected by HPLC in time in the culture with phenanthridine as co-subtrate.
27
4.4.2.4 Acridine degradation
0.00 5.00 10.00 15.00 20.00 25.00
retention time (min)
acridinone, metabolite of acridine Internal standard
acridine
phenanthrene-metabolite
phenanthrene
Figure 16: HPLC chromatograms of the culture grown on glucose with acridine as co-substrate, represented by the dark blue line, and the cultures grown on phenanthrene with and without acridine as co-substrate, represented respectively by the orange and purple line.
In a culture with phenanthrene as carbon source, without a co-substrate, there is a peak representing a metabolite of phenanthrene, which decreases in time. (see figures 14 and 15) After acridine is added, the phenanthrene-metabolite, as well as acridine, are completely degraded. In both cultures (glucose and phenanthrene as carbon-source) one of the acridine metabolites is probably acridinone (tested with a commercial available acridinone on the HPLC), which also decreases in time more rapidly in the phenanthrene-grown culture than in the glucose grown culture. In the latter culture the metabolite concentration first increases and after several days it decreases again (see fig. 18), probably this is caused by the time the bacteria need to induce the enzymes for the degradation of acridinone as well as acridine.
After adding the acridine, the phenanthrene concentration also decreases in time and the metabolite of phenanthrene is not detectable anymore. If acridine makes the degradation of phenanthrene more efficient acridine also triggers the induction of more enzymes to degrade acridine and phenanthrene, which will make the degradation of both compounds more efficient.
28
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
0 1 2 3 4 5 6 7 8 9
days
rel a
rea
(/FA
)
acridine added
Fig 17: relative areas (divided by the internal standard, fluoranthene) of the metabolite of phenanthrene and acridine (acridinone) in time in a culture of LH128 grown on phenathrene, where the dark blue diamonds represent the phenanthrene metabolite and the red squares acridinone.
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
0 1 2 3 4 5 6 7 8
days
rel a
rea
(/FA
)
Fig 18: relative area in time (/internal standard) of acridinone, formed as a degradation product in the degradation of acridine in a glucose grown culture of LH128.
29
4.4.3 Calculation of kinetic degradation constants
Due to the fact that the co-substrates are completely degraded in the cultures with phenanthrene as growth substrate and almost completely degraded in the cultures grown on glucose, no kinetic degradation constants could be calculated for the azaarenes in this experiment. However the degradation rates of the culture with glucose as carbon source, could be determined. For the cultures grown on phenanthrene, the degradation rates of phenanthrene are determined. (see table 3) The degradation rates are calculated with the help of the equation in figure 3. (see introduction). Table 3: degradation rates of the different substrates degraded in a continuous culture of LH128 Co-substrate Carbon source V (µg/min) V (µg/h) Phenanthridine Glucose 4.5 0.075 Acridine Glucose 4.5 0.075 Phenanthridine Phenanthrene 30* 0.50* Acridine Phenanthrene 30* 0.50* *) degradation rates for the degradation of phenanthrene, the degradation of the co-substrates was 100%
30
4.5 Continuous culture: co-substrate concentration 60% of carbon source
With a 15% co-substrate concentration of the carbon source the co-substrates were
(almost) completely degraded. In order to calculate the degradation efficiency more information is needed. To test a higher substrate concentration for this purpose only phenanthridine is used in the following experiments and the density of the culture was lowered by a factor of 5 by lowering the carbon source concentration. This factor was chosen by figure 19, where the determination limit of the protein analysis is determined by diluting a culture of LH128 from a concentration known from previous continuous cultures to the determination limit. Three cultures were followed in time by optical density, total protein determination and the HPLC.
0,000
0,020
0,040
0,060
0,080
0,100
0,120
0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1
dilution (1= original protein conc. of previous cultures)
prot
ein
conc
. (ng
/ml)
Chosen dilution
Figure 19: determination maximum dilution for the determination limit of the protein analysis. 1 is the original protein concentration in the first continuous cultures. The dotted lines represent the determination limit.
31
4.5.1 Optical density (OD) and total protein measurements
0
0,01
0,02
0,03
0,04
0,05
0,06
0,07
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
days
A54
0
phenanthridine added culture 2
phenanthridine added
Fig 20: Optical density of cultures of LH128 in a chemostat at 540 nm; 1: glucose and phenanthridine , 2: phenanthrene and phenanthridine and 4: phenanthrene before and after the azaarenes were added. The purple squares represent culture 1, the red triangles culture 2 and the dark blue diamonds culture 4.
As can be seen in figure 20, the cultures were relatively stable before and after the phenanthridine is added. The protein concentration (see figure 21) does not show the same trend. Due to a too low density of bacteria the protein concentration cannot be measured accurately and there are a lot of fluctuations in the protein concentration in time. The dilution was determined by figure 19, as can be seen the dilution of the culture in relation to the first continuous cultures, should lead to a detectable protein concentration.
32
0,000
0,005
0,010
0,015
0,020
0,025
0,030
0,035
0 2 4 6 8 10 12 14 16 18
days
prot
ein
conc
. ng/
ml
Phenanthridine added to culture II
Phenanthridine added to culture I
Figure 21: total protein concentration of the cultures 1, 2 en 4 in time, 1: glucose and phenanthridine, 2: phenanthrene and phenanthridine before and after the azaarenes were added. Culture 4 is only phenanthrene. The red triangles represents culture 1, the dark blue diamonds represents culture 2 and the purple squares represents culture 4.
4.5.2 HPLC
The cultures are also followed in time by HPLC-FLU, but still with a lower bacterial density and a higher percentage of co-substrate compared with the carbon source the degradation efficiencies are near the 100% degradation. See for example figure 22 en 23:
0,00
0,10
0,20
0,30
0,40
0,50
0,60
0,70
0,80
0 2 4 6 8 10
days
Co-
subs
trat
e (µ
g/m
l)
0,998
0,9982
0,9984
0,9986
0,9988
0,999
0,9992
degr
adat
ion
effic
ienc
y
Figure 22: degradation efficiency and co-substrate concentration (phenanthridine) in time followed in a culture of LH128 with phenanthrene as carbon and energy source. The purple squares represent the degradation efficiencies and the red triangles represent the co-substrate concentration
33
0
0,5
1
1,5
2
2,5
3
3,5
4
0 2 4 6 8 10
days
co-s
ubst
rate
(µg/
ml)
0,988
0,99
0,992
0,994
0,996
0,998
1
degr
adat
ion
effic
ienc
y
Figure 23: degradation efficiency and co-substrate concentration (phenanthridine) in time followed in a culture of LH128 with glucose as carbon and energy source. The purple squares represent the degradation efficiencies and the red diamonds represent the co-substrate concentration
As can be seen in figure 22, the degradation is so efficient that no conclusion can be drawn if there is any inhibition or increasing efficiency. This is also the case with the culture grown on glucose (see figure 23).
4.5.3 Calculation of kinetic degradation constants Also with >60% co-substrate concentration compared with the carbon source, no
degradation kinetic constants can be determined. But the degradation rates can be calculated with the data available. (see table 4) As can be seen in table 4, the degradation rate for phenanthridine is the same for both growth substrates, which indicates that the efficiency of the degradation by LH128 in a continuous culture is the same at higher co-substrate concentrations for respectively glucose and phenanthrene as the carbon source. Table 4: degradation rates for phenanthridine for the degradation in a continuous culture of LH128 at different carbon sources Co-substrate Carbon source V (µg/min) V (µg/h)phenanthridine glucose 3.6 0.060 phenanthridine phenanthrene 3.6 0.060
34
4.6 Metabolite analysis
The analysis of metabolites of the degradation of azaarenes by LH128 was first done with the HPLC and later on also with the LC-MS. With the first method water phase samples were measured with 3 fluorescence programmes, only carbazole has shown some degradation and the forming of metabolites. (see figure 24)
Figure 24: 3d graph, of time against retention time (R-time) and intensity of the water phase of a culture of LH128 grown on n-nonadecane as carbon source and carbazole as growth substrate
The other cultures showed hardly any growth or degradation of the azaarenes. Probably the concentrations used of the azaarenes had a toxic level for LH128. To counteract that problem new cultures were started, with less amount of azaarenes and growing at an elevated temperature to increase the growth rate. The cultures with phenanthridine, benzo(h)quinoline, carbazole, acridine and dibenzacridine showed growth, but only carbazole was degraded and metabolites could be found with the LC-MS. Isoquinoline, quinoline and 9-methylacridine still did not show any growth. The concentrations were probably still too toxic. See also table 5.
35
Co-substrate HPLC LC-MS growth metabolites growth metabolitesAcridine Yes No Phenanthridine No No Yes No Carbazole Yes Yes Yes Yes Benzo(h)quinoline Yes No Quinoline No No Isoquinoline no No No No 9-methylacridine No No Dibenzacridine Yes No Table 5: summary of growth and metabolite detection with the different methods used in the metabolite search
4.6.1 Carbazole metabolites The culture growing on carbazole and n-nonadecane showed some degradation of
carbazole and metabolites are probably found with the help of the LC-MS.. The spectra obtained with a cone voltage of 50, 75 and 100 eV are not of much use in the determination of the structure of the metabolite, because the obtained fragmentation spectra at those cone voltages are not more than background spectra. From known metabolites (xii,xiii) the masses and the masses of probable adducts are compared with the largest mass peak in the spectra. In figure 25 the metabolites that are probably detected are summarized.
NH
OH
NH
OH
OH
OH
O
O
O
O
H
H
H
H
H
H
carbazol-y-ol 1,1'-biphenyl-y-ol
carbazole-x,y-diol (2E,4E)-hexa-2,4-dienedioic acid Figure 25: possible metabolites of carbazole, detected in the water phase of a culture of LH128 growing on n-nonadecane and carbazole.
The above structures were found by comparing the several molecular ions, with or without acetate adducts, in the spectra obtained with the LC-MS, with known metabolites and expected metabolites of carbazole. Only the above compounds showed a similarity in mass with the literature metabolites. In the following figures the different mass spectra are
36
shown of which there is a probability that they are the metabolites described in figure 25, also the spectra of the parent compound, carbazole, and the carbon source, n-nonadecane, are shown.
CH3CH3
n-nonadecane
Figure 26: mass spectra and chromatogram (SIM m/z = 189) of the extracted water phase of a culture of LH128 grown on n-nonadecane and carbazole. The spectrum shows the carbonsource n-nonadecane (fragments)
37
NH
OH
m/z = 183, acetate adduct = 242
Figure 27: mass spectra and chromatogram (SIM m/z = 242) of the extracted water phase of a culture of LH128 grown on n-nonadecane and carbazole. The spectrum shows probably the acetate adduct (m/z = 242) of a probable metabolite of carbazole
O
O
O
O
H
H
H
H
H
H
m/z = 201
Figure 28: mass spectra and chromatogram (SIM m/z = 201) of the extracted water phase of a culture of LH128 grown on n-nonadecane and carbazole. The spectrum shows a probable metabolite (m/z = 201) of carbazole
38
NH
Carbazole
Figure 29: mass spectra and chromatogram (SIM m/z = 166) of the extracted water phase of a culture of LH128 grown on n-nonadecane and carbazole. The spectrum shows the acetate adduct (m/z = 226) of carbazole and carbozole itself. (m/z = 166)
OH
m/z = 170, acetate adduct m/z = 229
Figure 30: mass spectra and chromatogram (SIM m/z = 229) of the extracted water phase of a culture of LH128 grown on n-nonadecane and carbazole. The spectrum shows probably an acetate adduct of a probable metabolite (m/z = 229) of carbazole
39
5 Discussion
5.1 Continuous culture
As could be seen in the results, the original plan to determine the kinetic constants of the degradation of azaarenes failed. The azaarenes were degraded too efficiently by LH128 to determine the Michaelis-Menten constants. However the degradation rates could be calculated for phenathridine and acridine in a culture grown on glucose and for phenanthrene. By lessening the density of the culture by a 5-fold, the protein concentration was too low to measure any stability in the culture or correlate the degradation kinetics to the protein concentration of the culture. Also the azaarenes were degraded very efficient again, >99% of the original azaarene was degraded. However the degradation rate for phenanthridine in both cultures could be calculated. As can be seen in tables 4 and 5, the degradation rate of phenanthridine lowers from 0.075 µg/h to 0.060 µg/h, which indicates that at higher concentrations the degradation efficiency is lowered for the degradation of phenanthridine. Phenanthrene on the other hand is degraded more efficient with higher concentrations of phenanthridine, 0.5 µg/h at 15% phenanthridine concentrations to a 100% degradation. More enzymes are induced by LH128 in the presence of phenanthridine, but the enzymes are probably more efficient in the degradation of phenanthrene than they are in the degradation of phenanthridine.
In order to make sure there is still enough azaarenes so it is above the detection limit, a
higher concentration can be used, with a high bacterial density, the only problem is the toxicity of the azaarenes for LH128 at higher concentrations. It is advisable to use a continuous culture for the determining of the kinetic constants because a continuous culture will be a better representation of nature, but for the determination of the kinetic constants of the azaarenes in a continuous culture another bacterial strain would be better to use, a strain that will be able to grow longer in a steady state than LH128.
The use of glucose as carbon source cannot be advised either, because of the easy and badly controlled contamination with other bacterial strains. Badly controlled, because a good method to check the purity of the culture is not available. The purity of the culture was checked by putting a sample of the culture onto an agar plate with glucose as carbon source and streptomycin as an antibiotic; only members of the Sphingomonas family are able to grow on the plate (also the re-determined species, see 3.3). In earlier experiments LH128 was able to grow on these plates, but randomly. Probably they were more sensitive to the antibiotics than the rest of the bacterial family.
A better method to test, is a plate with phenanthrene as carbon source, but spraying a
probably carcinogenic compound on a plate cannot be advised. And due to the low stability of phenanthrene at higher temperatures, the phenanthrene cannot be added before or just
40
after the sterilisation. But also this method cannot distinguish LH128 from other phenanthrene degrading species.
The degradation of azaarenes in a continuous culture seems to be done with the help of different enzymes than for phenanthrene is used (see 4.4.2.3 and 4.4.2.4). In the presence of azaarenes the degradation of phenanthrene seems to go more efficient and in the case of acridine; phenanthrene metabolites are degraded more efficient. If the bacteria were using the same enzyme you could expect inhibition and a less efficient degradation of phenanthrene.
The bacteria react probably on the toxicity of the azaarenes by inducing other enzymes that are also able to degrade phenanthrene and it metabolites. If the cultures are in a steady state, all concentrations, also the metabolites, should stay constant. If with the introduction of another compound there seems to be extra losses of the metabolites of the azaarenes and the carbon source, phenanthrene, there must be a different degradation system for the azaarenes. Therefore batch cultures were started to find the metabolic pathway of the azaarenes within LH128.
5.2 Metabolite analysis
The batch cultures were pre-grown on n-nonadecane to make sure that during the analysis similar metabolites of phenanthrene were not detected along metabolites of the azaarenes. The first experiments gave the impression that the used bacterial strain of LH128 was not able to degrade the azaarenes anymore, but the strain was able to grow on phenanthrene and with some certainty can be concluded that the strain still is LH128. Several probable causes could have been the reason why LH128 was not able to degrade the azaarenes (with exception of carbazole). Growing on n-nonadecane did not trigger the induction of enzymes necessary for the degradation of the azaarenes, but it is strange why they do grow on glucose and are able to degrade the azaarenes in the presence of glucose (after a longer lag phase). Also possible is that the time to induce the enzymes for the degradation of the azaarenes is longer than the time the cultures were followed. The bacteria had enough problems growing on n-nonadecane they needed longer time and effort to induce the proper enzymes.
For future experiments it is advisable to use phenanthrene as carbon source instead, in order to make sure the azaarenes are degraded. In order to counteract the problems with similar metabolites, a blank with only phenanthrene available as carbon source should be started next to the degradation experiments. But no quantitative measurements on the metabolites can be done in this setting.
To be sure that there is degradation, the culture should be followed in time and also the co-substrate concentration should be measured in time. The problem with direct water phase measurements is the low concentration of the co-substrates due the low solubility of the
41
azaarenes in the water phase and the toxicity of the azaarenes at higher concentrations. Also the concentrations were too low for measurements on the LC-MS. Larger volumes could be taken from the cultures, but with batch cultures taking large samples can influence the culture and its environment, so no reproducible results can be obtained. For good results several cultures should be incubated with phenanthrene and grown to a fixed density, if every culture has the same optical density or protein concentration the azaarenes could be added and every day a culture could be centrifuged, the water phase extracted and concentrated and measured on the LC-MS. This way you will have a high enough concentration for the LC-MS and can you follow the culture in time. In order to make sure every culture has the same density the culture could be diluted.
It is advisable to use the LC-MS for the pathway elucidation, because with the LC-MS there is also the possibility of structure elucidation, which cannot be done with HPLC-FLU or HPLC-UV. If the LC-MS is used, the different parts of the system should be better optimised for the metabolites and the parent compound. By optimising the system, better resolution can be obtained and structures could be elucidated. For example optimising the cone-voltage for better fragmentation and the source voltage for a better ionisation, can give better resolution and higher yields in the mass spectrometer. (see figure 31) So more time should be invested in the improvement of the MS-system, so you will have good fragmentation spectra for the elucidation of the structure. Also a better baseline separation of the different compounds should improve the resolution of the MS and better results should be obtained.
42
6 Appendix A; Wick-medium The medium is prepared with distilled water All ingredients are of P.A. quality Buffer Stock-Solution
(dilute 1:40)(25ml/L) 70.4 g/L Na2HPO4*2 H2O (m=358.14) 4 g/L KH2PO4 (m=136.09) Mineral Stock-Solution
(dilute 1:10)(100ml/L) 10 g/L (NH4)2SO4 (m=132.14) 2 g/L MgCl2 * 6 H2O (m=203.31) 1 g/L Ca(NO3)2 * 4 H2O (m=236.15) Trace Element Stock-Solution
(dilute 1:200)(5ml/L) 800 mg/L Na-EDTA * 2 H2O (m=372.23) 300 mg/L FeCl2 *4 H2O (m=198.81) 10 mg/L MnCl2 * 4 H2O (m=197.91) 4 mg/L CoCl2 * 6 H2O (m=237.93) 1.56 mg/L CuSO4 * 5 H2O (m=249.68) 3 mg/L Na2MoO4 * 2 H2O (m=241.95) 2 mg/L ZnCl2 (m=136.28) 0.5 mg/L LiCl (m=42.39) 0.5 mg/L SnCl2 * 2 H2O (m=225.63) 1 mg/L H3BO3 (m=61.83) 2 mg/L KBr (m=119.01) 2 mg/L KI (m=166.01) 0.59 mg/L BaCl2 *2H2O (m=244.28)
43
7 Reference (i) Pollutional potentiality and biodegrability of some of heterocyclic nitrogenous bases in environment, S. Sandhya, R.A. Pandey, J. Microb. Biotechnology,1991, 6 (1): 15-30 (ii) Toxic and genotoxic effects of Azaarenes: Isomers and Metabolites, E.A.J. Bleeker et al., Polycyclic Aromatic Compounds, 1999, 13: 191-203 (iii) Fate and movement of Azaarenes and their anaerobic biotransformation products in an aquifer contaminated by wood-treatment chemicals, Wilfred E. Pereira et al., Environmental Toxicoligy and Chemistry, 1987, 6:163-176 (iv) Toxcity of Azaarenes, Eric A.J. Bleeker et al., Rev. Environ. Contam. Toxicol., 2002, 173: 39-38 (v) The Kinetics of Cometabolism, Craig S. Criddle, Biotechnology and Bioengineering, 1993, 41: 1048-1056 (vi) Microbial degradation of azaarene related compounds, Tishiaki Kimura, Toshio Omori, Life Chemistry reports, 1995, 12: 207-225 (vii) Elucidation of the metabolic pathway of fluorene and cometabolic pathway of fluorine and cometabolic pathways of phenanthrene, fluoranthene, anthracene and dibenzothiophene by Sphingomonas sp. LB126, René van Herwijnen et al., Research in Microbiology, 2003, 154 (3): 199-206 (viii) Biotransformation of three-ring azaarenes by the phenanthrene degrading strain Sphingomonas sp. LH128, René van Herwijnen, Chris de Graaf, Harrie A.J. Govers, John R. Parsons, Environ. Toxicol. Chem., 2004 23, No. 2. (ix) Proposal of the genus Sphingomonas sensu stricto and three new genera, Sphingobium, Novosphingobium and Sphingopyxis, on the basis of phylogenetic and chemotaxonomic analyses, M Takeuchi, K Hamana, and A Hiraishi, Int J Syst Evol Microbiol 2001 51: 1405-1417. (x) Karolien Vanbroekhoven, Ph-D-thesis, table II.7, page 59 (xi) Determination of basic nitrogen-containing polynuclear aromatic hydrocarbons formed during thermal degradation of polymers by high-performance liquid chromatography-fluorescence detection, M. Wilhelm et al., Journal of Chromatography A, 2000, 878: 171-181 (xii) Metabolisme van Azaarenen, Olaf Oudendijk, Masters thesis 1997 (xiii) Environmental Organic Chemistry, 2ed, Rene P. Schwarzenbach, Philip M. Geschwend, Dieter M. Imboden, 2003, John Wiley & Sons Inc., USA, chapter17: 687-774
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