Master Thesis: Bioremediation of waste gas and soil by ...€¦ · Master Thesis . submitted by...
Transcript of Master Thesis: Bioremediation of waste gas and soil by ...€¦ · Master Thesis . submitted by...
Universität für Bodenkultur Wien
University of Natural Resources and Life Sciences, Vienna
Department of Biotechnology Institute of Applied Microbiology
ACBR
Bioremediation of waste gas and soil by black extremotolerant fungi
Master Thesis
submitted by Caroline Poyntner, Bakk. techn.
Vienna, 2014
Supervisor: Katja Sterflinger-Gleixner, Assoc. Prof. Dr.
Acknowledgement
I would like to thank my supervisor, Prof. Sterflinger-Gleixner, for giving me the opportunity, support and trust to conduct this thesis in her group and in the collaborating group in Spain. Thanks for always having an open door and advice during the last years. I also want to thank the group members of the ACBR, who helped to create a good atmosphere in the lab and who always tried to help and became friends. Also thankful thoughts go to my supervisor in Spain, Prof. Prenafeta-Boldú, and his colleagues Miriam and Laura, who were great support in a new (Catalonian) environment. I would also like to thank the institute of biotechnology of the University of Natural Resources and Applied Life Sciences Vienna and the FWF for supporting this project Of course, I also want to thank my parents and my brother Lukas, who not only gave me financial backing but also the freedom to find my own way even if this meant that I would be out of town another time and an end of my education further out of reach. Supporters in different kind of ways were my friends who had time for me when I was worried, unhappy or unsatisfied during my studies, spent their precious free time with correcting my thesis, or distracted my with climbing and snowboarding. Thank you, Dani, Vicky, LFs, Clara and Gabriel.
What you do
makes a difference,
and you have to decide
what kind of difference you want to make.
Jane Goodall
Abstract Around the world there are a lot of polluted sites which need treatment because they are dangerous to human and environmental health. A possible treatment is bioremediation, where the focus lies on engineered microorganism, mostly bacteria. As remediation with bacteria has some disadvantages, this study focuses on finding fungal strains for bioremediation. Next to bioremediation, the usage of the strains on biofilters is a target. Biofilters could be used for treatment of waste gases in industry. In this study, three different pollutants of the group of hydrocarbons were tested. Hydrocarbons were chosen, because it is already known that some fungal strains are able to degrade them. Another factor is that the chemicals, toluene, hexadecane and polychlorinated biphenyl 126, are often found in polluted sites, as they are very abundant chemicals in petrol and oils. Therefore treatment is necessary. In this study, screenings were conducted to see which fungal strains are able to live in the presence of the three hydrocarbons and furthermore are able to degrade them. The focus lied on fungal strains from extreme environment, especially the group of black fungi, as they are known to be very stress resistant. Therefore they are good candidates for bioremediation. In this study, 163 different strains from the Centraalbureau voor Schimmelcultures (CBS) were screened. The screening was performed in two steps. First, a general microtiter plate screening with the help of a Tecan reader was carried out. The Tecan reader observed growth through measuring the change of absorption during the test period. Second step was a detailed screening to detect the detailed degradation. This was done with gas chromatography, which measured concentrations of the hydrocarbons and the production of CO2. The first screening was done at the ACBR in Austria and the second one at IRTA in Spain. The screening methods for the studies were successfully developed and we were able to find two strains which showed a good degradation performance for toluene, growing on this compound as the sole source of carbon and energy and, thus, converting it into water and carbon dioxide. Therefore they are promising candidates for bioremediations applications. Keywords: Bioremediation, biofilters, extremotolerant fungi, black fungi, hydrocarbons, microtiter plate screening, Gas Chromatography Screening
Zusammenfassung
Weltweit gibt es eine große Anzahl an Altlasten und Deponien, die einer Sanierung bedürfen, da sie eine Bedrohung für die Gesundheit des Menschen und der Umwelt darstellen. Bei der Sanierung wird in vielen Fällen zum Mittel der biologischen Altlastensanierung gegriffen. Hierbei wird meist mit Hilfe modifizierter Bakterien die Verschmutzung abgebaut. Da jedoch die Methode mit Bakterien auch Nachteile hat, wird oftmals nach Alternativen gesucht. In dieser Studie wurden daher verschiedene Pilzstämme untersucht, um sie als alternative Kandidaten in der biologischen Altlastensanierung einzusetzen. Eine andere Anwendung wäre der Einsatz von Pilzstämmen in sogenannten Biofiltern, die zur Reinigung von Abgasen eingesetzt werden könnten. In der Arbeit wurde der Fokus auf extremotolerante Pilzstämme gerichtet, da diese unter sehr viel Stress und extremen Umwelteinflüssen wachsen zu können. Vor allem Vertreter der Gruppe schwarze Pilze wurden untersucht, da von ihnen bereits einige bekannt sind, die Kohlenwasserstoffe abbauen können. Als Kontaminationsstoffe wurde Kohlenwasserstoffe gewählt, da diese sehr oft als langlebige Schadstoffe in Altlasten zu finden sind. 163 Pilzstämme des Centraalbureau voor Schimmelcultures (CBS) wurden mit drei verschiedenen Kohlenwasserstoffen, Toluol, Hexadekan und polychloriertes Biphenyl 126 in zwei verschiedenen Screenings auf ihre Abbaueigenschaften getestet. Im ersten Screening wurde das Wachstum der Stämme in Anwesenheit der Kohlenwasserstoffe mit Hilfe von Mikrotiterplatten getestet. Das Wachstum wurde durch Absorptionsmessungen mit einem Tecan Reader analysiert und am ACBR in Österreich durchgeführt. Im nachfolgenden Screening am IRTA in Spanien wurde der Abbau der Kohlenwasserstoffe mit Gaschromatographiemessung gemessen. Mit der Chromatographiemethode konnten genaue Konzentration der Kohlenwasserstoffe und CO2 Konzentrationen gemessen werden. Die Screening Methoden wurden erfolgreich entwickelt und am Ende konnten zwei Stämme gefunden werden, die gute Abbauergebnisse von Toluol erzielten. Sie nutzten diesen Kohlenwasserstoff als einzige Kohlenstoff und Energiequelle und konnten es zu Kohlendioxid und Wasser abbauen. Daher sind die Stämme vielversprechende Kandidaten für Bodensanierung. Schlagwörter: Bodensanierung, Biofilter, Kohlenwasserstoffe, extremotolerante Pilze, schwarze Pilze, Mikrotiterplatten Screening, Gas Chromatographie
List of contents
1 Introduction ..................................................................................................................................... 9 1.1 Aim of the thesis ......................................................................................................................9 1.2 Bioremediation ........................................................................................................................9 1.3 Choice of microorganism: fungi or bacteria?........................................................................ 10 1.4 Black fungi ............................................................................................................................. 11 1.5 Species .................................................................................................................................. 13 1.6 Pathways ............................................................................................................................... 13 1.7 Hydrocarbons ........................................................................................................................ 14
1.7.1 Toluene .............................................................................................................................. 15 1.7.2 Hexadecane ....................................................................................................................... 15 1.7.3 Polychlorinated biphenyl 126 ............................................................................................ 16
2 Materials and Methods ................................................................................................................. 17 2.1 Materials ............................................................................................................................... 17
2.1.1 Equipment ......................................................................................................................... 17 2.1.2 Media Micotiter Plates ...................................................................................................... 18 2.1.3 Media Screening Teflon coated bottles ............................................................................ 19 2.1.4 Media for cultivation of fungi ............................................................................................ 20 2.1.5 Hydrocarbons .................................................................................................................... 21 2.1.6 Microorganisms ................................................................................................................. 21
2.2 Methods ................................................................................................................................ 22 2.2.1 Pre-tests ............................................................................................................................ 22
4.2.1.1 Cultivation of strains __________________________________________________ 22 4.2.1.2 Microtiter plates Pre-tests _____________________________________________ 22
a. Hexadecane ____________________________________________________ 22 b. PCB 126 ________________________________________________________ 22 c. Toluene ________________________________________________________ 22
2.2.1.3 Inoculum _________________________________________________________ 23 2.2.1.4 TTC ______________________________________________________________ 23
2.2.2 Microtiterplate screening .................................................................................................. 23 4.2.2.1 Cultivation of strains __________________________________________________ 23 2.2.2.2 Preparation of microtiter plates _______________________________________ 24
2.2.3 Screening with Gas-Chromatography (GC) ....................................................................... 24 2.2.3.1 Teflon bottles _____________________________________________________ 24
Negative Control_________________________________________________ 25
Positive Control _________________________________________________ 25
Hydrocarbon ____________________________________________________ 25 2.2.3.2 GC-measurements __________________________________________________ 25 2.2.3.3 GC-FID ___________________________________________________________ 25 4.2.3.4 GC-TCD_____________________________________________________________ 26
3 Results ........................................................................................................................................... 27 3.1 Pre-tests ................................................................................................................................ 27 3.2 Microtiter plate screening .................................................................................................... 27 3.3 Results of GC - screening ...................................................................................................... 30
3.3.1 Negative results ................................................................................................................. 31 3.3.2 Positive results for toluene degradation ........................................................................... 32
3.3.2.1 Cladophialophora immunda __________________________________________ 32 3.3.2.2 Exophiala mesophila ________________________________________________ 34
3.3.3 Toxicity .............................................................................................................................. 36 4 Discussion ...................................................................................................................................... 39 5 Conclusion and Outlook ................................................................................................................ 43
6 References ..................................................................................................................................... 44 7 Appendix: ...................................................................................................................................... 48
7.1 Diagrams microtiter plate pre-tests ...................................................................................... 48 7.2 Diagrams microtiter plate screening .................................................................................... 50
7.2.1 PCB 126 .............................................................................................................................. 50 7.2.2 Toluene .............................................................................................................................. 69 7.2.3 Hexadecane ....................................................................................................................... 88
7.3 Diagrams GC screening ....................................................................................................... 107
List of tables Table 1: Equipment ACBR, University of Natural Resources and Applied Life Sciences, Austria ...... 17 Table 2: Equipment IRTA, Spain ........................................................................................................... 17 Table 3: Gas Chromatographs (GC) ...................................................................................................... 18 Table 4: Trace element solution ........................................................................................................... 18 Table 5: Vitamin solution ..................................................................................................................... 19 Table 6: Growth medium ...................................................................................................................... 19 Table 7: Glucose vitamin/trace element media .................................................................................. 19 Table 8: Glucose medium ..................................................................................................................... 19 Table 9: Ingrediens vitamin solution .................................................................................................... 20 Table 10: Solution A .............................................................................................................................. 20 Table 11: Solution B .............................................................................................................................. 20 Table 12: Malt extract agar .................................................................................................................. 20 Table 13: Malt-Pepton Solution / Glycerin Solution ........................................................................... 20 Table 14: Hydrocarbon used for Screening .......................................................................................... 21 Table 15: Strains pre-test ..................................................................................................................... 22 Table 16: Results Microtiter plate screening: ...................................................................................... 28
List of figures
Figure 1: Phylogenetic tree based on 1700 positions of small subunit ribosomal DNA .................... 12 Figure 2: Three different black yeast species: Exophiala sideris, Exophiala spinifera, Cladophialophora minourae(from left to right) .................................................................................... 13 Figure 3: Different metabolic pathways starting form toluene, styrene, ethylbenzene and benzene. ............................................................................................................................................................... 14 Figure 4: Various ways how hydrocarbons can penetrate soil (Young & Cerniglia 1995, p. 83). ....... 14 Figure 5: Chemical formula for toluene (National Institutes of Health, 2012). ................................... 15 Figure 6: Chemical formula of hexadecane (National Institutes of Health, 2012). ............................. 15 Figure 7: Chemical formula of PCB 126 (Royal Society of Chemistry, 2014). ....................................... 16 Figure 8 Lyophilized vials, tubes with tilted agar ................................................................................ 23 Figure 9: Toluene Plates in desiccator, Tecan Reader. ........................................................................ 24 Figure 10 GC-FID and GC-TCD ............................................................................................................... 26 Figure 11: A, B, C: Microtiter plate screening of strain 114, Pseudallescheria boydii, with the hydrocarbons PCB126 (A), toluene (B) and hexadecane (C). .............................................................. 31 Figure 12: Teflon coated bottle: ........................................................................................................... 32 Figure 13: GC-results ............................................................................................................................. 32 Figure 14 A, B, C: Microtiter plate screening of strain 17, Cladophialophora immunda, with the hydrocarbons PCB126 (A), toluene (B) and hexadecane (C). OD 700 plotted against the days of measurement. ....................................................................................................................................... 33 Figure 15: Teflon coated bottles: ......................................................................................................... 33 Figure 16: GC-results: ............................................................................................................................ 34 Figure 17 A, B, C: Microtiter plate screening of strain 64, Exophiala mesophila, with the hydrocarbons PCB126 (A), toluene (B) and hexadecane (C).OD 700 plotted against the days of measurement. ....................................................................................................................................... 35 Figure 18: Teflon coated bottles: ......................................................................................................... 35 Figure 19: GC-results: ............................................................................................................................ 36 Figure 20 A, B, C: Microtiter plate screening of strain 25, Exophiala jeanselmei, with the hydrocarbons PCB126 (A), toluene (B) and hexadecane (C).OD 700 plotted against the days of measurement. ....................................................................................................................................... 37 Figure 21: GC-results: ............................................................................................................................ 38 Figure 22: Two different microtiter plates with different fungal strains; .......................................... 40
1 Introduction
1.1 Aim of the thesis
A series of fungal strains were screened for their ability to degrade hydrocarbons. Screenings were carried out at the ACBR, Vienna and at IRTA, Caldes. The aim of the study was to find promising fungal strains, which could be used for bioremediation purposes in soil, water or in biofilters. Hydrocarbons are regulated as pollutants in the EU as they are toxic to humans and environment. Strategies to treat those pollutions are searched strongly. One of them is remediation. Hydrocarbons are classified in different groups, one of them are aromatic hydrocarbons. Aromatic hydrocarbons like benzene, toluene, ethyl benzene and the xylene isomers (collectively known as BTEX) are among the most abundant components from the water soluble fraction of crude oil and refined fuels(Prenafeta-Boldú et al., 2001a). Due to their toxicity and persistence, aromatic hydrocarbons are regarded as one of the major environmental pollutant group and have therefore been subjected to stringent environmental regulations (Mehlman et al. 1992). Treating those toxic compounds is challenging, because of wide dispersal and concentrations that cannot be treated chemically or physically due to high expenses. Hydrocarbon pollution can result from leaking gasoline tanks, gasoline accidents and other sources. BTEX and additive methyl-tert-butyl ether (MTBE) are the most water-soluble components of gasoline. Therefore these compounds predominate groundwater contaminant plumes from recent gasoline spills (Prenafeta-Boldú et al., 2004). Existing bioremediation techniques are mostly using bacteria as degradation organisms, while using fungi is a rather new approach. Bacteria have a high metabolic diversity and can assimilate a great variety of organic chemicals. Their disadvantage is to have a low physiological flexibility. If environmental conditions like pH, water availability or temperature change, they have problems to adapt.
Fungal strains mostly metabolize xenobiotics by co-metabolism which leads to low degradation rates and the need of co-substrates but they can grow on under more stringent conditions. Therefore they are able to grow on solid matrixes, extreme pH, low water content, low/high temperatures and can adapt if conditions change. The chosen organisms for this study, extremotolerant fungi, can live in extreme conditions and are therefore good candidates for bioremediation applications. The extremotolerant fungi used for the screening carried out in the frame of this thesis, mostly belong to the group of black yeasts-also called microcolonial fungi (MCF) which are among the most stress-tolerant organism on the Earth (de Hoog and Grube M., 2008). In the experiments of the thesis it was screened for strains which are able to degrade the hydrocarbons completely to carbondioxide and water and therefore no additional inputs of co-substrates in the bioremediation application is needed. The application in bioremediation would be either as living strains or in well-known genetically modified host organisms, where their attributes are expressed.
1.2 Bioremediation
In Austria, currently 65.586 hazardous sites are registered and only 3-9% treated (Granzin and Valtl, 2013; Skala et al., 2007) either by excavation, chemical, physical, biological cleaning methods or a combination of these. As this is not only the case for Austria but a problem all around the world, finding good waste treatment solutions is urgent. A big focus lies on remediation technologies, which are currently already working in some sites and different novel approaches are searched. Remediation technologies serve to immobilize contaminants, separate them from the soil, or destroy those (Caliman et al., 2010). They can be divided into in situ and ex situ methods. The U.S. Environmental Protection Agency (Engineering Forum, 2006) differentiates the in situ techniques into
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biological-treatments
thermal-treatments
physical/chemical treatments
The aim in this study belongs to the so called bioremediation, a remediation with the use of microorganisms. This treatment is defined as biological treatment. Biological treatments can be further differentiated into:
bio-augmentation: a bioremediation option for hydrocarbon-contaminated, oily-sludge restoration (Ayotamuno et al., 2007)
biosparging: pressurized air is injected under the water table and therefore enhances the rate of biological degradation of contaminant by naturally occurring bacteria (Gavrilescu, 2005; Kao et al., 2008)
bioventing: oxygen is enriched through a system of extraction and injection wells, which lasts from several months to years (Caliman et al., 2010)
composting
and several other less frequently applied methods;
The method aimed for this study is bioaugmentation. Bioaugmentation is not an easy technique, as lot of criteria have to be taken in account when working with microbes. Critical points in bioremediation are the bioavailability of the contaminant for the microorganisms, the degradation to less toxic compounds and the opportunity for optimization of biological activity (Crawford et al., 2004; Dua et al., 2002; Gavrilescu, 2005; Ward and Sing, 2004).
1.3 Choice of microorganism: fungi or bacteria?
The well-known bioremediation techniques which are using engineered bacteria have the disadvantages to show slow dispersal, long processes and are less effective if conditions change. Also nitrogen as well as phosphor are limiting factors. Contaminated soil is often lacking microorganisms with the ability to degrade the pollutants. Therefore in usage of bioaugmention additional microbial colonies are added to contaminated soil. Bacteria are often too slow to fill the open spaces in the soil and as a consequence, less effective microorganisms take over the place. This leads to very long processes in bioremediation, where conditions change until bacteria colonies are not able to adapt anymore. Fungi, on the other hand, can survive changing environmental conditions. This is an important factor in industrial spills. They can survive low amount of oxygen, changes in pH, water availability and temperature as well as limited access to nitrogen and phosphor. Fungal enzymes are more abundant than bacterial enzymes. Furthermore, fungal strains which live in extreme environments can produce so called extremozymes, for example laccases. Laccases can be a contribution to biodegradation for a wide range of aromatic pollutants (Hölker et al., 2004). All this listed advantages lead to the conclusion that fungal strains could be good candidates for bioremediation. Nevertheless, there are some disadvantages. A big disadvantage of fungal strains is, that most of them are not able to completely degrade contaminants. In partial degradations, they produce co-metabolites, which need to be studied in advance to know if they are toxic. Some strains are just able to degrade the toxic contaminant in presence of another substance, which means that additional inputs can be necessary. This leads to lower metabolic rates and biomagnification and therefore makes the process more difficult. In this thesis, two screenings were conducted with 163 fungal strains. The mentioned disadvantages are the reason why so many different isolates were tested. A lot of species adapt to their environment and differ between their isolation sources. In the first screening, the strains were tested on their ability to grow in the presence of hydrocarbons while in the second screening the detailed metabolic process was examined.
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1.4 Black fungi
Fungi represent the greatest eukaryotic diversity on earth and they are among the primary decomposers in ecosystems (Tsui et al., 2011). They share a long history with us, being useful producers of antibiotics or being a threat to our health in form of mycosis (Galagan et al., 2005). There is a huge amount of different species and groups differentiating themselves through different attributes like morphologies, genetics and reproduction. The group of black fungi was chosen for the purpose of bioremediation, as they are found in extreme environments and are therefore resistant to stress conditions. On one hand numerous species exhibit a significant human-pathogenic potential (de Hoog et al., 2003; Zeng et al., 2007), which can affect skin, lungs and nervous systems of humans and on the other hand, they have the unique ability to thrive in environments enriched with toxic hydrocarbons (benzene and xylene; Prenafeta-Boldú et al. 2006). For example, three different species of Exophiala have been isolated from sites polluted with hydrocarbons or with the aid of alkyl benzene enrichment (Zhao et al., 2010). A study of Badali (Badali et al., 2011) suggests that the two attributes of being pathogen and of being able to live on hydrocarbons are not necessarily combined in the same species. The stress resistance is the major argument for using them in bioremediation. Recent experiments showed that the stress resistance of MCF against solar radiation, radioactivity, desiccation and oligothophic conditions even allow them to survive space and Martian conditions (Zakharova et al., 2013). “Black yeast” is a terminus technicus subscribing a group of fungi that is quite heterogeneous from the taxonomic and phylogenetic point of view. They have a melanized cell wall in common and the formation of daughter cells by yeast-like multilateral and polar budding (Sterflinger, 2005). The umbrella term is used to indicate heterogeneous lineages of Chaetothyriomycetidae and Dothideomycetidae. The different orders can be seen in the phylogenetic tree in Figure 1. “Meristematic fungi” is another term to describe fungi with melanized cell walls, which grow and reproduce in isodiametric division. MCF describes a growth pattern, by both kinds – black yeasts and meristematic fungi. They characteristically grow in small, clump-like colonies which can survive stress like desiccation, pH difference, solar radiation and even radioactivity. They are also known to be hydrocarbonoclastic. The surface-volume-ratio is kept small to save water and survive UV-radiation. Some are even able to do micro- and macropitting on the rocks and stones they are living, to absorb minerals, which is a problem in cultural heritage protection. It is known that on rock surfaces, where microbial interactions occur, there are constant changes in atmospheric conditions (Zakharova et al., 2013). In this sense, as assumed by Gorbushina (Gorbushina et al., 2008), ubiquitous subaerial biofilms are bioindicators that are continually subjected to climate change. Therefore we can determine climate change by monitoring biofilms of fungi and other microorganisms on rocks. Another term for the attributes of black yeasts is “poikilo tolerant”. The term poikilo-tolerant (resistant to variable stress; from poikilos: variegated) has been used to describe the behaviour of living organisms in environments, where tolerance to multiply and variable parameters is essential for survival (Gorbushina, 2007). Living on filter membranes or as soil inoculation, they could degrade pollutants without getting disturbed by the environment easily.
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Figure 1: Phylogenetic tree based on 1700 positions of small subunit ribosomal DNA (tree modified from Sterflinger 2005).MCF; growing on rock are highlighted. Bootstrap values were generated from 1000 trees using the Felsenstein method (Sterflinger et al., 2012)
1.5 Species
163 strains from the Centraalbureau voor Schimmelcultures (CBS, Netherlands Royal Academy of Science, www.cbs.knaw.nl) were chosen for the screening in the thesis. Recent studies (Prenafeta-boldu et al. 2001a; Prenafeta-Boldú et al. 2004; Prenafeta-Boldú et al. 2006; Isola et al. 2013) showed that a high diversity of fungi have degrading abilities, most of them belonging to the group of black yeasts. Therefore also for the screening as seen in Figure 2, the majority of strains belonged to that group. The groups which were used belonged to black yeast-like organisms (order Chaeotrhyriales) and Scedosporium species (order: Microascales). Scedosporium species are associated with alkane-contaminated sites and degrade linear aliphatic compounds (April et al., 1998; Claussen and Schmidt, 1998; Janda-Ulfig et al., 2008; Onodera et al., 1990). For both orders various publications show that they are able to live in polluted sites. Belonging to the black yeast-like group, they have melanin as a protection attribute, which helps them to survive in stressful environments. Next to their habitats in extreme niches, they are also found as human pathogens, forming life threatening mycoses.
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Figure 2: Three different black yeast species: Exophiala sideris, Exophiala spinifera, Cladophialophora minourae(from left to right)
1.6 Pathways
While degradation of polyaromatic compounds by fungi is well studied, the metabolism of monoaromatic hydrocarbons is still poorly understood and studies on this are rare (Prenafeta-Boldú et al. 2001b). Two enzymatic systems are suggested to take part in the oxidation of aromatic hydrocarbons. These are the enzymatic systems of the detoxifying cytochrome P450 monooxygenases, laccases and the lignin-decomposing peroxidase (Luykx et al., 2003). In Figure 3, the different mentioned pathways (Luykx et al. 2003; Rustler et al. 2008) are described. For benzene, the assimilatory pathway is not known yet.
Figure 3: Different metabolic pathways starting form toluene, styrene, ethylbenzene and benzene.
1.7 Hydrocarbons
For the screening, three different hydrocarbons were applied. They were used as representatives for the groups of alkenes, aromatic hydrocarbons and polychlorinated biphenyls. Hydrocarbons are substances in mineral oil, which next to heavy metals is suspected to be the most frequent soil contamination at investigated sites. Mineral oil and chlorinated hydrocarbons are the most frequent contaminants found in groundwater (EEA 2007). Distribution ways for the example of oil pollution in soil are shown in Figure 4.
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Figure 4: Various ways how hydrocarbons can penetrate soil (Young & Cerniglia 1995, p. 83).
1.7.1 Toluene
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Figure 5: Chemical formula for toluene (National Institutes of Health, 2012).
Toluene is a chemical compound, which can be found in natural crude oil. It is used as a solvent found in aviation gasoline, spray and wall paints, paint thinner, medicine, dyes, explosives, detergents, fingernail polish, spot removers , lacquers, adhesives, rubber and antifreeze (Tox Town, 2002). It can affect health, depending on exposure levels to the chemical. While the inhalation of high levels of toluene cause unconsciousness, low levels lead to headache, dizziness, fatigue, nausea and other short term affects. Long-time exposure can affect kidneys, nervous system, liver, brain and the heart. Any gasoline pollution can contain toluene and is therefore a substance, which should be removed from the environment. It is already known that fungal species are able to live with toluene as sole carbon source. For instance Weber et al., 1995, were able to find Cladosporium sphaerospermum, which is able to grow on toluene. Later it was reclassified as Cladophialophora saturnica through molecularbiological techniques (Badali et al., 2009), while before it was classified morphologically.
1.7.2 Hexadecane
Figure 6: Chemical formula of hexadecane (National Institutes of Health, 2012).
As toluene, hexadecane can be found in gasoline and diesel, rubber, shale oil production, coal combustion, biomass and refuse combustion and tobacco smoke (National Institutes of Health, 2012). Not only exposure due to inhalation is problematic, but also pollution of water, which leads to a hexadecane intake of fishes as well as seafood and furthermore to human intake through food.
1.7.3 Polychlorinated biphenyl 126
Figure 7: Chemical formula of PCB 126 (Royal Society of Chemistry, 2014). Polychlorinated biphenyls are proved to be toxic in animals(National Institutes of Health, 2012). Due to their widespread, uncontrolled industrial applications, PCB's have become a ubiquitous contaminant in the environment (National Institutes of Health, 2012). Especially in foods coming from animal sources, a lot of PCBs can be detected and are therefore a critical health risks. They can affect the immune, reproductive, nervous and endocrine systems and have been linked to low intelligence quotients in children (Van Emon et al., 2013). Although banned in many countries, PCB is very persistent and can be found in air, soil, dusts and sediments. In-situ bioremediation is an attractive alternative for the treatment of PCB-contaminated soils and sediments (Ruiz-Aguilar et al., 2002), but only aerobic bioremediation with bacteria has been studied extensively (Fiebig et al., 1993; Quensen et al., 1990; Rojas-Avelizapa et al., 1999). Some fungi were found to be able to degrade PCB (Ruiz-Aguilar et al., 2002).
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2 Materials and Methods
2.1 Materials
2.1.1 Equipment
The Equipment used at ACBR, University of Natural Resources and Applied Life Science, Austria and at GIRO, IRTA, Spain is listed in Table 1 and Table 2. The parameters used for the gas chromatography are listed in Table 3. Table 1: Equipment ACBR, University of Natural Resources and Applied Life Sciences, Austria
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device supplier Tecan reader TECAN reader infinite® M1000 reader, Tecan group Ltd. (switzerland) laminar flow bench Thermo scientifc MSC advantage pipettes/ multichannel pipettes Eppendorf / VWR microtiter plate shaker Stuart SSL5 microtiter plate shaker, Bibby Scientific Limited desiccator Glaswerk-Wertheim microtiter plates Cellstar, Greiner-Bio-One, 655180 sterile syringe filter Rotilab ®, CARL ROTH GMBH + CO. KG ribolyzer MP Fast Prep 24 autoclave CertoClav sterilizer GmbH scale Santorius AG
Table 2: Equipment IRTA, Spain device supplier 250 mL boston flask with Teflon Waddinxveen, the Netherlands mininert valves
hydrocarbon chamber Captair by Erlab bio safety cabinet Telstar Bio IIA Burdinola sterile syringe Thermo Scientific, USA pipettes Eppendorf microsyringes 1705-1710 (Luer-Lock), Hamilton Bonaduz AG Needle P/N 7729-07/00.
autoclave JP SELECTA S.A. incubator Memmert GmbH scale Scaltec SBC 51
Table 3: Gas Chromatographs (GC)
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device supplier / properties name Trace 2000 series Thermo Quest CE Instruments column TRB624 N° series NF8261 gas carrier He (2 mL/min) septum Diskobolus/Teknokroma Septa
Marathon 12 mm TR-D033064 slope isothermal initial temperature 180 °C (toluene)
220 °C (hexadecane) end temperature 180 °C (toluene) 220 °C (hexadecane) injector temperature 250 °C
temperature detector 250 °C injection split/ split less split 1:50 (toluene)
1:5 (hexadecane) injection volume 100 µL hold time 2.00 min (toluene)
6.00 min (hexadecane) split flow 100 mL/min gas saver flow 20 mL/min
Varian CP-3800 Varian Inc. column Haysep Q 80-100 method CO2measurement Front_2013, Software Galaxie N° series 320056731-17 gas carrier He (45 mL/min) septum DISKOLBOLUS Green Septa 9mm TR-
D033032 slope isothermal initial temperature 90 °C end temperature 90 °C injector temperature 180 °C temperature detector 180 °C injection on column pressure 26.7 kPa-psi injection volume 200 µL hold time 1 min
2.1.2 Media Micotiter Plates
For the microtiter plate screening, two solutions were needed as described in Table 4 and Table 5, a vitamin solution and a trace element solution respectively. The vitamin solution was sterile filtrated and then stored at 4°C while the trace element solution was kept at room temperature. Those two basic solutions were needed to prepare the three working solutions: growth medium (Table 6), glucose vitamin/trace element medium (Table 7) and glucose medium (Table 8). All three solutions were sterilized at 120 °C for 15 minutes and the vitamin solution was added in sterile conditions after cooling down of the medium. PH was adjusted to 5.5 with potassium hydroxide. The three media were kept at room temperature. Table 4: Trace element solution ingredients concentration (per L) supplier magnesium chloride hexahydrate 40 g Merck GesmbH calcium chloride 10 g Merck GesmbH sodium chloride 10 g AppliChem GmbH iron(III) chloride hexahydrate 200 mg Merck GesmbH hydrogen borate 50mg Sigma Aldrich GmbH copper sulphate 10 mg Merck GesmbH manganese chloride 40mg Merck GesmbH potassium iodide 10mg AppliChem GmbH sodium molybdate 20mg Alfa Aesar GmbH zinc sulphate 40mg Merck GesmbH hydrogen chloride for dissolving Merck GesmbH
Table 5: Vitamin solution
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ingredients concentration (per 0.1L) supplier biotin 2mg Merck GesmbH calcium - panthothenate 200 mg Merck GesmbH folic acid 0.2 mg Merck GesmbH myo-inositol 1 g Merck GesmbH niacin 40 mg Merck GesmbH 4-aminobenzoic acid 20 mg AppliChem GmbH pyridoxine hydrochloride 40 mg AppliChem GmbH riboflavin 20 mg Merck GesmbH thiamine hydrochloride 40 mg Merck GesmbH
Table 6: Growth medium ingredients concentration (per L) supplier potassium dihydrogen phosphate (KH2PO4) 9.5 g Merck GesmbH dipottasium hydrogen phosphate (K2HPO4) 0.5 g AppliChem GmbH magnesium sulphate heptahydrate (MgSO4*7H2O) 0.1 g Merck GesmbH ammonium chloride (NH4Cl) 2 g AppliChem GmbH trace element solution 10 mL vitamin solution 1 mL
Table 7: Glucose vitamin/trace element media ingredients concentration (per L) supplier KH2PO4 9.5 g Merck GesmbH K2HPO4 0.5 g AppliChem GmbH MgSO4*7H2O 0.1 g Merck GesmbH NH4Cl 2 g AppliChem GmbH glucose 2 % AppliChem GmbH trace element solution 10mL vitamin solution 1 mL
Table 8: Glucose medium ingredients concentration (per L) supplier KH2PO4 9.5 g Merck GesmbH K2HPO4 0.5 g AppliChem GmbH MgSO4*7H2O 0.1 g Merck GesmbH NH4Cl 2 g AppliChem GmbH glucose 2 % AppliChem GmbH
Next to those three media a tetrazolium chloride (TTC) solution (0.2 %, Serva Chemicals) was prepared and sterilized through a filter (Roth, 0.22 µm). It was kept shielded from light at room temperature.
2.1.3 Media Screening Teflon coated bottles
In the Teflon coated bottles, a mineral medium (Hartmans and Tramper 1991) and a solution of vitamins (2 mL/L, Table Table 9) were used. The mineral medium was composed of solution A and B (see Table 10: Solution A and Table 11: Solution B respectively), which were mixed in a proportion of A: 10 mL and B: 25 mL in one litre. Solution A and B were autoclaved, afterwards the vitamin solution was added. The sterile filtration was done with a sterile filter (Roth, 0, 22 µm) under sterile conditions.
Table 9: Ingredients vitamin solution
20
ingredients concentration (per L) supplier
nicotinic acid 100 mg Sigma Aldrich GmbH calcium Pantothenate 200 mg Scharlau cyanocobalamin 25 mg Scharlau inositol 100 mg Sigma Aldrich GmbH p-aminobenzoate 20 mg Sigma Aldrich GmbH thiamine 50 mg Sigma Aldrich GmbH pyridoxine 25 mg Scharlau biotin 10 mg Scharlau riboflavin 10 mg Scharlau folic acid 10 mg Sigma Aldrich GmbH thioctic acid 10 mg Sigma Aldrich GmbH
Table 10: Solution A ingredients concentration (per L) supplier ammonium sulphate 200 g Sigma Aldrich GmbH magnesia chloride hexahydrate 10 g Fluka Chemicals ethylenediaminetetraacetic acid 1 g Sigma Aldrich GmbH zinc sulphate heptahydrate 0.2 g Sigma Aldrich GmbH calcium chloride dehydrate 0.1 g Scharlau iron sulphate heptahydrate 0.5 g Scharlau sodium molybdate Dihydrate 0.02 g Sigma Aldrich GmbH copper sulphate 0.02 g Sigma Aldrich GmbH cobalt chloride hexahydrate 0.04 g Sigma Aldrich GmbH manganese chloride 0.1 g Sigma Aldrich GmbH
Table 11: Solution B ingredients concentration (per L) supplier potassium phosphate 155 g Sigma Aldrich GmbH sodium dihydrogen Phosphate 85 g Fluka Chemicals
.
2.1.4 Media for cultivation of fungi
The different strains were grown on Petridishes filled with Malt extract agar (MEA, Table 12). The agar was sterilized at 120°C and then poured into the Petri dishes under sterile conditions. Table 12: Malt extract agar ingredients concentration (per L) supplier maltextract 20 g/L AppliChem GmbH glucose 20 g AppliChem GmbH agar agar 15 g AppliChem GmbH bacto pepton 0,1 % Difco
The freeze-dried cultures were homogenised in malt-peptone solution as described in Table 13. The malt-peptone solution was sterilized at 120°C for 15min. Glycerine (70 %, Table 13) was autoclaved and used for freezing of the cultures.
Table 13: Malt-Pepton Solution / Glycerine Solution ingredients concentration supplier malt extract 2% AppliChem GmbH bacto peptone 0,1% Difco glycerine 70% Sigma Aldrich GmbH
2.1.5 Hydrocarbons
The hydrocarbons (Table 14) used for the screenings were 99 % analytical grade. Polychlorinated biphenyl (PCB) 126 was also 99 % analytical grade but 10ng dissolved in Isooctane. Table 14: Hydrocarbon used for Screening
21
hydrocarbon used for supplier toluene Teflon coated bottle screening Scharlau hexadecane Teflon coated bottle screening Scharlau toluene microtiterplate screening Merck KGaA hexadecane microtiterplate screening Alfa Aesar – A Johnson
Matthey Company PCB 126 microtiterplate screening Dr.Ehrenstorfer GmbH
2.1.6 Microorganisms
The fungal strains tested in this study were distributed by the Centraalbureau voor Schimmelcultures (CBS), Utrecht, Netherlands). Each fungal strain was marked with a CBS number and their growth conditions. All strains came from different isolates and made up 163 different species out of 9 genera. Most of them were so-called “black yeasts”.
Order: Chaetothyriales
• Exophiala
• Cladophialophora
• Pseudallescheria
• Phialophora
• Rhinocladiella
• Selenophoma
Order: Dothideales • Aureobasidium
Order: Microascales • Scedosporium
• Graphium
2.2 Methods
2.2.1 Pre-tests
The first microtiter plate-tests performed were used as pre-tests to develop the method. For this, two strains (Table 15) of the ACBR culture collection were tested for the growth in presence of hexadecane, PCB 126 or toluene. The designed method was based on two publications: (Wrenn and Venosa, 1996) and Strong-Gunderson and Palumbo, 1994 and experiments done before at the ACBR (Sterflinger, personal communication).
4.2.1.1 Cultivation of strains
The cultivation was done through three point inoculation of pre-existing plates. Depending on the strain, different temperatures were chosen for incubation as indicated in Table 15. Table 15: Strains pre-test
22
Strain CBS/MA Nr. temperature/humidity Exophiala xenobiotica MA 2883 25°C Exophiala dermatididis CBS 525.76 38°C, high humidity
4.2.1.2 Microtiter plates Pre-tests
The approach for PCB 126 and hexadecane was to fill the wells with 150 µL of the growth media and 50µL of the hydrocarbon solution. For toluene 200µL of media were used due to the volatility of toluene.
a. Hexadecane
Into each well 150 µL of growth medium and 50 µL of hexadecane were pipetted. As positive control 200 µL of glucose media with and without vitamin + trace element solution were used. Plates were kept at room temperature on a shaker (Stuart SSL5, 250 rpm), sealed with parafilm. Additionally, one row was added with TTC and one in which TTC was added at a later time point.
b. PCB 126
Similar to the experimental setup of the hexadecane plates, the PCB 126 plates were prepared: 150 µL of growth medium, 50 µL PCB 126 solution (10 ng/ µL PCB 126 in isooctane), positive Control: 200µL of glucose media with and without vitamin + trace element solution. Also two rows for TTC were done. Together with the hexadecane plates, they were kept on a shaker at room temperature, sealed with parafilm.
c. Toluene
The setup for toluene differs from the other two hydrocarbon plates: in case of toluene 200 µL of growth medium, glucose media with and without vitamin + trace element solution was used. The plates were kept in a desiccator as toluene is a volatile chemical. A saturated solution of toluene should reach a value of 515 ppm in a closed environment. As the environment should be the same for all the plates it was not possible to have a positive control as in the other hydrocarbon trials. The
volatile toluene reaches all wells including the positive control (growth control with only glucose as carbon source). For this reason the wells for positive control in the other hydrocarbon plates were used as growth control for the toluene experiment.
2.2.1.3 Inoculum
For the inoculum, one cm2 biomass was taken from the Petridishes and added in a 2mL Eppendorf tube, where 0.5 mL of glass beads and 1 mL of NaCl were added before. The eppendorf tube and the glass beads were sterilized before. The biomass was ribolyzed (4.0 m/s for 5 sec) in order to get a homogenic distribution of cells in the NaCl solution which could then be used for inoculation of the microtiter plates. For each strain the homogenisation step had to be optimized and was checked under the light microscope. Afterwards, 20 µL of the homogenic cell suspension were added into the wells of the microtiter plates.
2.2.1.4 TTC
One row was created where 50 µL of TTC (0.2 %) where added to the normal setup of different media and a second additional row where TTC was added at the end of the test.
2.2.2 Microtiterplate screening
4.2.2.1 Cultivation of strains
The strains were sent from the CBS as freeze-dried cultures in glass ampoules. Those ampoules were handled according to the CBS instruction for reviving freeze-dried-cultures. The lyophilized vials were cleaned with ethanol, cut on the top with a glass cutter near the Bunsen flame and poured into 2 ml of malt peptone solution. This suspension was kept at room temperature for at least 5 hours. One mL was then poured on two malt extract agar plates and was plated with single-use spatulas. To the remaining one mL of suspension, one mL of 70 % glycerine was added and frozen at 4 °C. The cultures which were delivered in tubes with tilted agar were seeded on malt extract agar-plates. Each strain was cultivated in duplicate. All plates were inoculated at room temperature and sealed with parafilm except for Cladophialophora chaetospira, which was incubated with access to sunlight due to the need of UV light for growth. A visible growth of the different strains was achieved after 1 to 3 weeks, which varied within the different strains. Once there was a significant amount of biomass, they were treated further.
Figure 8 Lyophilized vials, tubes with tilted agar
23
2.2.2.2 Preparation of microtiter plates
On each measured plate three blanks were prepared. Two wells with growth medium, one well with glucose and vitamin+ trace element solution and in one well the glucose solution was appended. Concerning the samples, for each strain two wells with media and hydrocarbon, one with Glucose and Vitamin + Trace element solution and one with Glucose solution alone were done. The two different glucose wells were done to see if vitamins or trace elements do inhibit growth. To each well 20 µl of inoculum were added, 150 µl of medium and 50 µl of hydrocarbon. As in the pre-test, the amounts for toluene were different (200 µl medium for all three, 20 µl inoculum). Plates with hexadecane and PCB 126 were sealed with parafilm and placed on a shaker (Stuart SSL5, 250 rpm). The measurement was done every second day at 700 nm with the Tecan reader a total period of 40 days under the following conditions: 24 °C, shaking: 1 sec, 2 mm amplitude, linear, multiple reads, 25 flashes, settle time: 10 ms. By measuring the OD 700 the growth of the cultures was detected. Due to condensation, evaporation due to heat and growth of the fungi, there was a loss of media during the measured time. In those cases, the wells were refilled with sterilized water. For toluene plates (see Figure 9), the desiccator was cleaned carefully with ethanol to avoid any contaminations. The silica gel was dried in an incubator at 120°C for at least one hour until it had changed colour from red to blue. The toluene was poured into a beaker (250 ml) and placed next to the opened microtiter plates. In those wells that were desiccated, water was added. For the OD measurement, lids were placed on the toluene plates and the plates were measured under the same conditions as the hexadecane and PCB plates.
Figure 9: Toluene Plates in desiccator, Tecan Reader.
2.2.3 Screening with Gas-Chromatography (GC)
This second screening was done at IRTA, Caldes, Spain, where the positive strains of the microtiter plate screening were used.
2.2.3.1 Teflon bottles
To determine the growth kinetics, 250 mL Boston flasks were used. Those bottles were sealed with Teflon Miniert valves (Phase Separations, Waddinxveen, The Netherlands) to have a closed system. The bottles where filled with 25 mL of buffered mineral media (Hartmans & Tramper 1991) with a pH of 7. The bottles were cleaned and rinsed with desalinated water, then the media was added and the bottles were autoclaved at 120 °C. The sterile filtrated vitamin solution was added with a pipette afterwards under sterile conditions. Toluene was added based on its toxicity level for black yeast and the known water/air partition coefficient (Amoore & Hautala 1983). The concentration of 6.6x10-8 mol was added with a Hamilton microsyringe. The injection was done in Captair by Erlab to capture the volatile hydrocarbons. The volume of hexadecane containing the same amount of carbon was calculated (1.2x10-5 mol) and added to through the injection valve.
24
For the first ten strains three types of bottle contents where used:
Negative Control
25 mL mineral media + 0.3 mL of inoculum
Positive Control
25 mL mineral media + 0.3 % glucose + hydrocarbons + 0.3 mL inoculum
Hydrocarbon
25 mL mineral media + hydrocarbons + 0.3 mL inoculum A spore solution of the fungal strains was taken as inoculum which was done by taking one cm2 biomass from the MEA plate, soling it in sterile water, vortex it and inject it with a sterile needle under sterile conditions. The inoculation was done after the bottle substrates reached equilibrium. The bottles were kept at 25 °C in the incubator. Another 15 strains were treated, where just the hydrocarbon was used as substrate and neither a positive nor a negative control was performed. Due to the fact that Cladiophialophora immunda is known from literature (Prenafeta-Boldú et al. 2001a) to be able to degrade toluene, it was taken as positive control for the whole experiment.
2.2.3.2 GC-measurements
The growth was monitored by visual observation and GC measurements of the headspace. For the GC measurements, the consumption of hydrocarbons in the headspace was measured with GC –FID (Trace 2000 series, Thermo Quest CE Instruments). The production of CO2 was measured with GC-TCD. Measurement was done over 30 days where starting point for hydrocarbon measurements was day 0. The CO2 measurement was started when hydrocarbon depletion was measured or growth could be seen optically.
2.2.3.3 GC-FID
Two methods were elaborated: Toluene: Oven temperature: 180 °C hold Time: 2.00 min, Split: 50, Peak around 1.45 min Hexadecane: Oven Temperature: 220 °C, Hold Time: 6.00min, Split: 5.1 min 100 µL of the headspace was injected with a Hamilton microsyringe into the column. To calculate the amount of toluene/hexadecane, a calibration curve was done beforehand. The calibration curve was performed with following concentrations: Toluene: 2, 4, 6, 8 µL of toluene in 25 µL of desalinated water, Hexadecane: 2, 10 µL of hexadecane in 25 mL of desalinated water. To define the method, the different bottles of the calibration curve were measured and the different parameters of the GC were changed until an optimal peak was received. For correcting the daily variations of the instrument, two standards with known amounts of hydrocarbon were measured before and after the whole measurement. Those results were used for calculations afterwards. Additionally, the column was cleaned with injection of a sample volume of air in between measurements.
25
For hexadecane, the pure substance was injected in liquid form and diluted in pentane (1ml pentane, 10µL hexadecane) to see the expected retention time. Having a low vapor pressure, hexadecane signal peaks were rather small and hard to detect. In the used mineral medium, hexadecane could not dissolve properly and built droplets. Therefore it was hardly detectable it in the headspace. To develop the best method, different injection volumes (100 – 500 µl) and heating the samples up to 70°C before measuring were checked. Although heating up the sample would increase detectability, this treatment was not possible as heating up growing fungi to higher temperatures results in a growth disturbance. Finally the method used was to inject 100 µL at 220 °C oven temperature. The amount of hexadecane could not be measured quantitatively because only a small peak was detectable in this sequence. For this reason, the small peak was taken as a qualitative indicator for the presence of hexadecane. Moreover a stable amount of toluene in combination with an increase of CO2 would have been a second proof for the degradation of hexadecane.
4.2.3.4 GC-TCD
For measurement of CO2 production the GC-TCD (Varian CP-3800) was used. The method was already defined, named Front 2013. This method uses the column at 90°C and the detector and injector temperature was set as 180°C. For injecting the sample volume of 200 µL a Hamilton SGE was used. Furthermore, two standards were measured to determine the performance of the instrument. One standard contained a known content of N2, air, CH4 and H2 and the second one CO2 and N2. The standards were injected with different volumes and analyzed with the same method.
Figure 10 GC-FID and GC-TCD
26
3 Results
3.1 Pre-tests
The pre-tests were performed to receive the best method for the microtiter screening of 163 fungal strains of the CBS. For the screening, conditions remained the same as in the pre-test, apart from TTC. Those rows did not show a noticeable colour change caused redox-reduction of TTC due to fungal growth which made the method not useful. The screening was therefore performed as described in chapter2.2.2.
3.2 Microtiter plate screening
The plates were measured for a time period of 40 days and data were analysed in Excel. For calculating the change in OD 700, the blanks were subtracted from the OD 700 values of all 163 strains. Growth curves were plotted in diagrams, resulting in 9 diagrams per strain (3 hydrocarbons, plate 1, 2 and the average of the two plates). In the analysed data set, 114 strains out of 163 showed growth in the microtiter plate screening (see Table 16). Some fungal strains were able to grow up to levels comparable to the positive controls, while others just showed a slight rise in OD 700 from the beginning to the end of the measurements. In Table 16 the results were divided into + for growth, ~ for little growth and – for no growth and different hydrocarbons. There was found one strain which was able show growth in presence of all the three hydrocarbons, 14 with two hydrocarbons and 19 with one hydrocarbon (marked with + in Table 16). The two positive controls, glucose with vitamins and trace elements and glucose without vitamins and trace elements, conducted themselves in most cases similar. Therefore the vitamins and trace elements did not inhibit growth of the strains, although sometimes growth was slower or less. Just in very few cases it inhibited growth. Optically, it was possible to check for contaminations, for example if there was growth outside of the wells or on the plate cover. The results for the toluene curves showed unexpected variations which may be a result of the toxic impact of toluene for some fungal species. Furthermore, deviations were also seen due to desiccation of the wells and a refill with sterile water. The different diagrams of the plates can be seen in chapter 3.3 and the appendix. Bold marked strains in Table 16 were used to do further screening at IRTA. They were chosen by their performance in the microtiter plate screening and by their isolation source. Some strains were not able to grow after reviving them from the freeze dried culture. These are also mentioned in the table below but their hydrocarbon cells are empty.
27
Table 16: Results Microtiter plate screening: strains ordered by name, +=growth, ~= little growth, x= no growth, strains written bold are the ones, which were used for the screening at IRTA, strains with empty cells for Hexadecane (Hex), Toluene (Tol) and PCB 126 (PCB) were not able to grow after the freeze dried culture
28
Nr Name CBS N° Hex Tol PCB
1 Exophiala dermatidtidis 122239 x + X 2 Exophiala xenobiotica 102455 ~ + X 3 Pseudallescheria agusta 254.72 x ~ X 4 Exophiala sideris 121838 ~ + X 5 Cladophialophora carrionii 260.83 x x X 6 Cladophialophora saturnica 118724 x + X 7 Exophiala castellanii 109812 ~ ~ ~ 8 Phialophora verrucosa 138.67 x x X 9 Exophiala exophialae 668.76 x x X 10 Selenophoma mahoniae 388.92 x + X 10 Exophiala sideris 121819 x ~ X 11 Exophiala oligosperma 109807 x + X 12 Exophiala alcalophila 122256 x x X 13 Scedosporium apiospermum 117407 + ~ ~ 14 Pseudallescheria ellipsoidea 219.85 + + ~ 15 Rhinocladiella basitona 101460 16 Cladophialophora arxii 409.96 x ~ X 17 Cladophialophora immunda 110551 x + ~ 18 Pseudallescheria boydii 116899 ~ + X 19 Graphium eumorphum 987.73 + ~ + 20 Exophiala bergeri 119100 x x X 21 Exophiala lecanii-corni 102400 x x X 22 Cladophialophora yegresii 114407 ~ ~ ~ 23 Cladophialophora samoёnsis 259.83 x x X 24 Cladophialophora minourae 987.96 x x X 25 Exophiala jeanselmei 507.90 + x + 26 Pseudallescheria fusoidea 106.53 + ~ X 27 Cladiophialophora boppii 110029 x + ~ 28 Aureobasidium pullulans var. pullulans 100524 x x X 29 Cladophialophora subtilis 122642 ~ ~ ~ 30 Phialophora americana 840.69 x x X 31 Exophiala spinifera 899.68 x + + 32 Rhinocladiella similis 116299 + x + 33 Exophiala spinifera 110628 x ~ ~ 34 Exophiala heteromorpha 648.76A x ~ X 35 Pseudallescheria minutispora 116911 + x ~ 36 Cladophialophora australiensis 112793 x x X 37 Cladophialophora emmonsii 979.96 x ~ X 38 Exophiala mesophila 121964 39 Cladophialophora mycetomatis 454.82 x x X 40 Exophiala alcalophila 520.82 x x X 41 Exophiala sideris 121834 x x X 42 Pseudallescheria ellipsoidea 332.75 + + X 43 Exophiala oligosperma 265.49 + x X 44 Pseudallescheria boydii 316.54 45 Exophiala spinifera 425.92 ~ x X 46 Aureobasidium pullulans 110374 x x X 47 Exophiala dermatitidis 115663 x ~ ~ 48 Phialophora verrucosa 286.47 x x X 49 Cladophialophora carrionii 114392 x x X 50 Exophiala jeanselmei 117.86 51 Exophiala xenobiotica 118157 x ~ ~ 52 Exophiala bergeri 121846 x ~ X 53 Exophiala mesophila 836.95 x x X 54 Exophiala dermatitidis 748.88 55 Cladophialophora immunda 122255 x ~ X 56 Exophiala lecanii-corni 232.39 x ~ X 57 Cladophialophora boppii 126.86 x + ~
29
Nr Name CBS N° Hex Tol PCB
58 Cladophialophora immunda 834.96 x + X 59 Cladophialophora yegresii 114406 x x X 60 Exophiala oligosperma 725.88 + x X 61 Pseudallescheria boydii 119709 + x X 62 Cladophialophora arxii 306.94 x + X 63 Exophiala dermatitidis 149.90 x x ~ 64 Exophiala mesophila 120910 + ~ ~ 65 Exophiala heteromorpha 633.69 x x X 66 Pseudallescheria boydii 117405 + + X 67 Cladophialophora minourae 556.83 x x X 68 Exophiala sideris 121813 x x X 69 Cladophialophora emmonsii 640.96 70 Exophiala mesophila 121509 x + X 71 Pseudallescheria boydii 101720 + + ~ 72 Pseudallescheria angusta 116914 + ~ X 73 Exophiala castellanii 581.76 x x ~ 74 Rhinocladiella similis 111763 + ~ X 75 Pseudallescheria boydii 115829 + + x 76 Cladophialophora immunda 122257 x + x 77 Pseudallescheria boydii 375.77 78 Aureobasidium pullans 584.75 x ~ + 79 Exophiala xenobiotica 117672 x ~ x 80 Pseudalleschria boydii 119696 + + ~ 81 Pseudalleschria boydii 116894 + x x 82 Pseudalleschria boydii 593.73 + x x 83 Exophiala jeanselmei 122339 x x x 84 Cladophialophora immunda 109797 x + ~ 85 Exophiala jeanselmei 109635 x x ~ 86 Pseudalleschria boydii 101721 + ~ + 87 Exophila oligosperma 537.76 + ~ x 88 Pseudallescheria boydii 116658 + ~ x 89 Exophiala dermatitidis 116.97 x x x 90 Pseudallescheria boydii 116421 + + x 91 Cladophialophora immunda 122636 x ~ x 92 Pseudalleschria boydii 115.59 + + + 93 Aureobasidium pullulans 110373 x x x 94 Pseudalleschria ellipsoidea 301.79 + + x 95 Exophiala sideris 121818 x x ~ 96 Exophiala heteromorpha 232.33 ~ ~ ~ 97 Exophiala oligosperma 115966 + ~ x 98 Cladophialophora carrionii 160.54 x ~ ~ 99 Aureobasidium pullulans var. Subglaciale 123388 100 Aureobasidium pullulans var. Pullulans 701.76 ~ ~ ~ 102 Pseudallescheria boydii 108.54 + ~ x 103 Exophiala dermatitidis 150.90 x x ~ 104 Exophiala spinifera 269.28 x ~ x 105 Pseudallescheria boydii 117387 + + x 106 Cladophialophora carrionii 114398 x ~ ~ 107 Pseudallescheria boydii 116595 + ~ x 108 Exophiala xenobiotica 117647 + + x 109 Pseudalleschria boydii 329.93 + ~ ~ 110 Pseudalleschria boydii 117395 + ~ x 111 Exophiala mesophila 120907 x x x 112 Exophialophora alcalophila 118723 113 Exophiala bergeri 102241 ~ x ~ 114 Pseudalleschria boydii 117404 + + x 115 Exophiala spinifera 194.61 + x x 116 Cladophialophora chaetospira 491.70 + x x 117 Cladophialophora potuelntorum 114772 x x x 118 Cladophialophora chaetospira 114747 x ~ x 119 Exophiala castellanii 110025 x x x 120 Pseudallescheria boydii 101723 + + x 121 Aureobasidium pullulans 122385 x ~ ~ 122 Pseudallescheria desertorum 489.72 + ~ ~ 123 Exophiala moniliae 520.76 x x x
30
Nr Name CBS N° Hex Tol PCB
124 Cladophialophora potulentorum 112222 ~ ~ ~ 125 Cladophialophora potulentroum 115144 x x x 126 Exophiala lecanii-corni 122266 x ~ x 127 Fonsecaea 109628 x + ~ 128 Pseudallescheria boydii 116594 + x ~ 129 Exophiala alcalophila 118722 x x x 130 Exophiala alcalophila 521.82 x x x 131 Pseudallescheria boydii 116410 + ~ ~ 132 Pseudallescheria boydii 322.51 + x x 133 Pseudallescheria boydii 117393 ~ + x 134 Exophiala bergeri 119094 x ~ x 135 Pseudallescheria boydii 116898 + ~ x 136 Exophiala sideris 121832 x + x 137 Pseudallescheria boydii 116897 x + ~ 138 Cladophialophora mycetomatis 122637 x ~ x 139 Pseudallescheria boydii 101.22 + ~ ~ 140 Pseudallescheria boydii 117417 + ~ x 141 Exophiala dermatitidis 109148 x ~ ~ 142 Exophiala bergeri 353.52 x x x 143 Pseudallescheria boydii 117408 + + x 144 Exophiala mesophila 121497 ~ ~ x 145 Pseudallescheria ellipsoidea 418.73 + + x 146 Fonsecaea 109630 x + x 147 Pseudallescheria boydii 117415 + x x 148 Exophiala jeanselmei 528.76 x x x 149 Exophiala xenobiotica 117754 x ~ x 150 Cladophialophora 102230 x + ~ 151 Pseudallescheria boydii 116403 + + ~ 152 Exophiala jeanselmei 677.76 x ~ ~ 153 Exophiala spinifera 667.76 x ~ x 154 Exophiala dermatitidis 120479 x ~ x 155 Pseudallescheria boydii 100396 + x ~ 156 Exophiala mesophila 121507 x x x 157 Pseudallescheria boydii 330.93 + ~ x 158 Pseudallescheria boydii 499.90 + ~ x 159 Pseudallescheria boydii 117403 ~ ~ x 160 Exophiala sideris 121820 x ~ ~ 161 Exophiala dermatitidis 116726 x x x 162 Exophiala heteromorpha 102696 x x x 163 Wangiella 110555 x x x
3.3 Results of GC - screening
For the GC-screening at IRTA, Spain, 25 strains were chosen from the fungal strains listed in Table 16. They were chosen by their performance in the first screening and by their isolate source. One strain, Cladophialophora immunda, 17, served as positive control as it was known from literature that this strain can degrade toluene (Prenafeta-Boldu et al., 2001 a). After a few days it was possible to see strong growth in all glucose controls, which could be compared to the bottles with sole hydrocarbon source. Additionally, a comparison with the negative controls was useful, as some of the fungal strains were able to sporulate without any carbon source. Three different cases were observed: negative results (shown in chapter 3.3.1), positive results for toluene degradation (shown in chapter 3.3.2) and toxicity (shown in chapter3.3.3). Results were analysed in Excel and were corrected by a daily factor. This factor was calculated by measuring two standards before and after the daily measurements. The difference to the actual concentration of toluene in the standards was calculated. The measurements were then corrected.
3.3.1 Negative results
Negative results were expressed by missing CO2 production and hydrocarbon degradation. This applied to the strains with following numbers: 26, 19, 14, 31, 71, 32, 25, 110, 114, 158, 94, 139, 75, 159, 143, 97, 90, 105, 107, 86, 92 and 81 (presented in Table 16). As an example the strain 114, Pseudalleschria boydii, exhibited an increase in OD in the presence of hexadecane (Figure 11 C) and toluene (Figure 11 B) in the microtiter plate screening. In the GC – screening, no growth was detected (Figure 12). The GC - FID measurements showed a slight decrease in toluene concentration. CO2 values (presented in Figure 13) did not increase but showed slight variation due to equipment variations.
A B
C Figure 11: A, B, C: Microtiter plate screening of strain 114, Pseudallescheria boydii, with the hydrocarbons PCB126 (A), toluene (B) and hexadecane (C). OD 700 plotted against the days of measurement. The graphs consist of the average data points of duplicates. They show 4 curves, medium plus hydrocarbon (M), medium plus glucose (G), medium plus glucose, vitamins and trace elements (GVT). M curves are, as expected, quite similar In Figure A, a big difference between G, GVT curves and M curve is seen. The values of the M curves were not increasing. The strain was not able to grow in presence of the PCB126. In the graphs B and C, there were increasing values of OD 700. The strain was able to grow in presence of the hydrocarbons.
M; day 0; 0,15457
M; day 2; 0,147145
M; day 4; 0,169235
M; day 7; 0,251935
M; day 9; 0,26473
M; day 11;
0,431725
M; day 14;
0,411855
M; day 16;
0,37814
M; day 18;
0,34843
M; day 21;
0,33322
M; day 23;
0,315985
M; day 25;
0,299025
M; day 28;
0,31373
M; day 30;
0,30855
M; day 32;
0,31232
M; day 35;
0,30697
M; day 37;
0,30901
M; day 39;
0,324105
M; day 43;
0,40534
M; day 44;
0,414995
M; day 46;
0,40863 M; day 0; 0,112885
M; day 2; 0,098245
M; day 4; 0,11782
M; day 7; 0,19966
M; day 9; 0,207765
M; day 11;
0,208075
M; day 14;
0,207115
M; day 16;
0,18897
M; day 18;
0,183455
M; day 21;
0,192475
M; day 23;
0,19196
M; day 25;
0,188545
M; day 28;
0,223035
M; day 30;
0,209165
M; day 32;
0,206245
M; day 35;
0,20453
M; day 37;
0,20442
M; day 39;
0,23531
M; day 43;
0,37075
M; day 44;
0,3799
M; day 46;
0,378685 G; day 0; 0,184315
G; day 2; 0,216835
G; day 4; 0,37081
G; day 7; 0,97743
G; day 9; 1,041025
G; day 11;
0,98427
G; day 14;
0,96181
G; day 16;
0,976155
G; day 18;
0,988935
G; day 21;
1,019875
G; day 23;
1,018905
G; day 25;
1,011155
G; day 28;
1,00815
G; day 30;
0,99647
G; day 32;
1,00043
G; day 35;
1,175415
G; day 37;
1,34074
G; day 39;
1,491925
G; day 43;
1,571695
G; day 44;
1,55189
G; day 46;
1,546785
GVT; day 0;
0,14467
GVT; day 2;
0,18028
GVT; day 4;
0,312605
GVT; day 7; 0,4371
GVT; day 9;
0,488665
GVT; day 11;
0,53715
GVT; day 14;
0,590605
GVT; day 16;
0,64497
GVT; day 18;
0,698765
GVT; day 21;
0,76212
GVT; day 23;
0,790395
GVT; day 25;
0,816805
GVT; day 28;
0,86374
GVT; day 30;
0,874745
GVT; day 32;
0,8968
GVT; day 35;
0,92827
GVT; day 37; 0,952
GVT; day 39;
0,97079
GVT; day 43;
1,01577
GVT; day 44;
1,028265
GVT; day 46;
1,03877
M
M
G
GVT
M; day 0;
0,108515
M; day 2;
0,117375
M; day 4;
0,135255
M; day 7;
0,18489
M; day 9;
0,20609
M; day 11;
0,205135
M; day 14;
0,230905
M; day 16;
0,248275
M; day 18;
0,24943
M; day 21;
0,28307
M; day 23;
0,301235
M; day 25;
0,32858
M; day 28;
0,367385
M; day 30;
0,37851
M; day 32;
0,37531
M; day 35;
0,455775
M; day 37;
0,428745
M; day 39;
0,433045
M; day 43;
0,531675
M; day 44;
0,505675
M; day 46;
0,48674
M; day 0;
0,062035
M; day 2;
0,067415
M; day 4;
0,08224
M; day 7;
0,119645
M; day 9;
0,13642
M; day 11;
0,125415
M; day 14;
0,15331
M; day 16;
0,166255
M; day 18;
0,16568
M; day 21;
0,190465
M; day 23;
0,20707
M; day 25;
0,233565
M; day 28;
0,26714
M; day 30;
0,277755
M; day 32;
0,285885
M; day 35;
0,37579
M; day 37;
0,35032
M; day 39;
0,352345
M; day 43;
0,44739
M; day 44;
0,414455
M; day 46;
0,39764
G; day 0; 0,0903
G; day 2; 0,10702
G; day 4; 0,16283
5
G; day 7; 0,34927
5
G; day 9; 0,46936
5
G; day 11;
0,61191
G; day 14;
0,672345
G; day 16;
0,71691
G; day 18;
0,73366
G; day 21;
0,76356
G; day 23;
0,76892
G; day 25;
0,780905
G; day 28;
0,78786
G; day 30;
0,79948
G; day 32;
0,77908
G; day 35;
0,79536
G; day 37;
0,792565
G; day 39;
0,79182
G; day 43;
0,825165
G; day 44;
0,813995
G; day 46;
0,772805
GVT; day 0;
0,061875
GVT; day 2;
0,064435
GVT; day 4;
0,102245
GVT; day 7;
0,26606
GVT; day 9;
0,333085
GVT; day 11; 0,39652
GVT; day 14; 0,46683
GVT; day 16; 0,49363
5
GVT; day 18; 0,49104
5
GVT; day 21; 0,52631
GVT; day 23; 0,54722
5
GVT; day 25; 0,56957
5
GVT; day 28; 0,59145
5
GVT; day 30; 0,62025
5
GVT; day 32; 0,612
GVT; day 35; 0,64338
5
GVT; day 37; 0,64583
GVT; day 39; 0,66338
5
GVT; day 43; 0,70482
5
GVT; day 44; 0,70020
5
GVT; day 46; 0,67488
5
M
M
G
GVT
M; day 0; 0,148275 M; day 2; 0,142835 M; day 4; 0,17841
M; day 7; 0,3995
M; day 9; 0,61329
M; day 11; 0,78686
M; day 14; 0,90021 M; day 16; 0,94927
M; day 18; 0,988295 M; day 21; 1,01549 M; day 23; 1,03849 M; day 25; 1,0333 M; day 28; 1,04847 M; day 30; 1,044305 M; day 32; 1,05824 M; day 35; 1,05115 M; day 37; 1,05097 M; day 39; 1,03697 M; day 43; 1,029385 M; day 44; 1,023585 M; day 46; 1,023875
M; day 0; 0,130945 M; day 2; 0,13937 M; day 4; 0,163375
M; day 7; 0,375895
M; day 9; 0,586845
M; day 11; 0,783435
M; day 14; 0,90185 M; day 16; 0,96199
M; day 18; 1,008685 M; day 21; 1,061185 M; day 23; 1,084255 M; day 25; 1,091885 M; day 28; 1,106975 M; day 30; 1,11327 M; day 32; 1,12521 M; day 35; 1,12463 M; day 37; 1,118685 M; day 39; 1,118925 M; day 43; 1,123915 M; day 44; 1,12151 M; day 46; 1,12383
G; day 0; 0,124955 G; day 2; 0,16534
G; day 4; 0,291415
G; day 7; 0,70829 G; day 9; 0,77756 G; day 11; 0,78301 G; day 14; 0,78049 G; day 16; 0,7991215
G; day 18; 0,750215 G; day 21; 0,727085 G; day 23; 0,71548
G; day 25; 0,8007255 G; day 28; 0,8272455 G; day 30; 0,84408 G; day 32; 0,889935 G; day 35; 0,863485 G; day 37; 0,864405 G; day 39; 0,87479
G; day 43; 0,9156245 G; day 44; 0,9429895 G; day 46; 0,890855
GVT; day 0; 0,135945 GVT; day 2; 0,15523
GVT; day 4; 0,28257
GVT; day 7; 0,421395
GVT; day 9; 0,496075 GVT; day 11; 0,55954
GVT; day 14; 0,658795
GVT; day 16; 0,734258 GVT; day 18; 0,742455 GVT; day 21; 0,80053
GVT; day 23; 0,83196 GVT; day 25; 0,834875 GVT; day 28; 0,87095
GVT; day 30; 0,912165 GVT; day 32; 0,972795 GVT; day 35; 0,986383 GVT; day 37; 0,999355 GVT; day 39; 1,01345
GVT; day 43; 1,079457 GVT; day 44; 1,118384 GVT; day 46; 1,089675
M
M
G
GVT
31
32
Figure 12: Teflon coated bottle: No visible growth could be detected in the medium.
T; 0,00; 3,89087E
-09
T; 4,00; 4,11327E
-09
T; 5,01; 3,75858E
-09
T; 9,00; 3,88915E
-09 T; 12,00; 2,97341E
-09
T; 15,01; 3,35744E
-09
T; 17,00; 3,17596E
-09
T; 18,01; 3,10307E
-09
T CO2; 5,00;
8,53E-11
T CO2; 9,01;
2,96E-10
T CO2; 11,00;
2,96E-10
T CO2; 15,01;
5,43E-10
T CO2; 17,01;
2,86E-10
T CO2; 18,00;
1,96E-10
T
T CO2
Figure 13: GC-results Toluene (T) degradation and CO2 (T CO2) production by Pseudallescheria boydii, 114. Carbon equivalence [mol] of the two molecules plotted against runtime [days]. The toluene was decreasing slightly while CO2 remained stable.
3.3.2 Positive results for toluene degradation
Two strains, Cladophialophora immunda, 17, (presented in chapter 3.3.2.1) and Exophiala mesophila, 64, (presented in chapter Fehler! Verweisquelle konnte nicht gefunden werden.), showed the ability to grow on toluene as the sole carbon and energy source. This was confirmed by an increase of CO2and a toluene decrease. Cladophialophora immunda, was already known to be able to assimilate toluene (Prenafeta-Boldú et al., 2001a) and therefore served as positive control for the whole experiment. Some strains seemed to be optically growing, but measurements showed, that those strains were not able to produce CO2. The optically seen growth was sporulation.
3.3.2.1 Cladophialophora immunda
For this strain, the results of the microtiter plate screening were negative for hexadecane and PCB 126. The OD 700 for toluene varied in both plates but showed an increasing trend. Optical detection also showed more growth in the bottle with sole hydrocarbon source than in the negative control (see Figure 15). The graph of the GC results shows a decrease in toluene and increase in CO2. At the point where toluene is degraded completely, CO2 also reached maxima and stayed stable for the next measuring points. 70% of the toluene was recovered in CO2. In figure 20 it can be seen, that the bottle with hydrocarbons as carbon source behaved similar to the positive control.
A B
M; day 0;
0,0426015
M; day 1;
0,05077
M; day 2;
0,032595
M; day 4;
0,107055
M; day 11;
0,136955
M; day 14;
0,126055
M; day 17;
0,126055
M; day 20,;
0,113305
M; day 22;
0,092055
M; day 24;
0,07314
M; day 29;
0,09568
M; day 31;
0,12065
M; day 33;
0,14763
M; day 36;
0,14542
M; day 0;
0,04641
M; day 1;
0,1187125
M; day 2;
0,074405
M; day 4;
0,19057
M; day 11;
0,44591
M; day 14;
0,416985
M; day 17;
0,416985
M; day 20,;
0,437825
M; day 22;
0,41806
M; day 24;
0,458655
M; day 29;
0,39149
M; day 31;
0,28472
M; day 33;
0,405395
M; day 36;
0,39565 G; day 0; 0,02587
G; day 1; 0,14981
5
G; day 2; 0,08009
5
G; day 4;
0,368745
G; day 11;
2,14423
G; day 14;
2,27213
G; day 17;
2,27213
G; day 20,;
2,13063
G; day 22;
2,13403
G; day 24;
2,02967
G; day 29;
1,83336
G; day 31;
1,810645
G; day 33;
1,64435
G; day 36;
1,5578
GVT; day 0;
0,00979
GVT; day 1;
0,109285
GVT; day 2;
0,090315
GVT; day 4;
0,278035
GVT; day 11; 0,5247
GVT; day 14; 0,56566
5
GVT; day 17; 0,56566
5
GVT; day 20,; 0,58713
5
GVT; day 22; 0,57295
5
GVT; day 24; 0,60978
5
GVT; day 29; 0,61938
5
GVT; day 31; 0,68426
5
GVT; day 33; 0,58722
5
GVT; day 36; 0,69865
5
M
M
G
GVT
M; day 0;
0,04203
M; day 1;
0,068825
M; day 2;
0,055665
M; day 7;
0,061565
M; day 10;
0,096345
M; day 11;
0,068695
M; day 14;
0,10008
M; day 17;
0,1105235
M; day 18;
0,103935
M; day 19;
0,09831
M; day 21;
0,09482
M; day 28;
0,1166745
M; day 31;
0,1204015
M; day 34;
0,2099035
M; day 37;
0,15417
M; day 39;
0,16207
M; day 41;
0,21176
M; day 0;
0,0292
M; day 1;
0,041355
M; day 2;
0,033525
M; day 7;
0,027855
M; day 10;
0,06844
M; day 11;
0,045065
M; day 14;
0,07593
M; day 17;
0,098205
M; day 18;
0,069255
M; day 19;
0,06899
M; day 21;
0,069715
M; day 28;
0,093501
M; day 31;
0,0965225
M; day 34;
0,11961
M; day 37;
0,129095
M; day 39;
0,13607
M; day 41;
0,18067
G; day 0;
0,014475
G; day 1;
0,02416
G; day 2;
0,019715
G; day 7;
0,017235
G; day 10;
0,046205
G; day 11;
0,017685
G; day 14;
0,02512
G; day 17;
0,04624
G; day 18;
0,0398
G; day 19;
0,036265
G; day 21;
0,041525
G; day 28;
0,046675
G; day 31;
0,04692
G; day 34;
0,02199
G; day 37;
0,057955
G; day 39;
0,07451
G; day 41;
0,10817 GVT; day 0;
0,042785
GVT; day 1;
0,05202
GVT; day 2;
0,049595
GVT; day 7;
0,044395
GVT; day 10; 0,0672
GVT; day 11; 0,05880
5
GVT; day 14; 0,06829
GVT; day 17; 0,08428
5
GVT; day 18; 0,08401
GVT; day 19; 0,08229
5
GVT; day 21; 0,08064
GVT; day 28; 0,09144
5
GVT; day 31; 0,08871 GVT; day 34; 0,01737
5
GVT; day 37; 0,07316
GVT; day 39; 0,09119
5
GVT; day 41; 0,12845
M
M
G
GVT
M; day 0;
0,05367
M; day 1;
0,050715
M; day 2;
0,064865
M; day 7;
0,126715
M; day 10;
0,11336
M; day 11;
0,14128
M; day 14;
0,147005
M; day 17;
0,17895
M; day 18;
0,1839
M; day 19;
0,196245
M; day 21;
0,154165
M; day 28;
0,144555
M; day 31;
0,10973
M; day 34;
0,14667
M; day 37;
0,12284
M; day 39;
0,19806
M; day 41;
0,14946
M; day 0;
0,077215
M; day 1;
0,075315
M; day 2;
0,09738
M; day 7;
0,15113
M; day 10;
0,17376
M; day 11;
0,14857
M; day 14;
0,15789
M; day 17;
0,180745
M; day 18;
0,184295
M; day 19;
0,191185
M; day 21;
0,15887
M; day 28;
0,15387
M; day 31;
0,11589
M; day 34;
0,155695
M; day 37;
0,10227
M; day 39;
0,191835
M; day 41;
0,15396
G; day 0;
0,00596
G; day 1;
0,03701
G; day 2;
0,042885
G; day 7;
0,891255
G; day 10;
1,552215
G; day 11;
1,736885
G; day 14;
2,161945
G; day 17;
2,430795
G; day 18;
2,476935
G; day 19;
2,50972
G; day 21;
2,59997
G; day 28;
2,59188
G; day 31;
2,574705
G; day 34;
2,553415
G; day 37;
2,512115
G; day 39;
2,131935
G; day 41;
2,523745
GVT; day 0;
0,03426
GVT; day 1;
0,07292
GVT; day 2;
0,11053
GVT; day 7;
0,676875
GVT; day 10; 0,84945
5
GVT; day 11; 0,91408
GVT; day 14; 1,04284
GVT; day 17; 1,18482
5
GVT; day 18; 1,20517
5
GVT; day 19; 1,21617
GVT; day 21; 1,27484
GVT; day 28; 1,39087
5
GVT; day 31; 1,47441
GVT; day 34; 1,53255
5
GVT; day 37; 1,6215
GVT; day 39; 1,52990
5
GVT; day 41; 1,65765
M
M
G
GVT
C Figure 14 A, B, C: Microtiter plate screening of strain 17, Cladophialophora immunda, with the hydrocarbons PCB126 (A), toluene (B) and hexadecane (C). OD 700 plotted against the days of measurement. The graphs consist of the average data points of duplicates. They show 4 curves, medium plus hydrocarbon (M), medium plus glucose (G), medium plus glucose, vitamins and trace elements (GVT). M curves are, as expected, quite similar. In figure A, a big difference between the G curve and M curve is seen. There was still a slight increase of the OD 700 values from the beginning. All the curves in figure B increased drastically during the 41 days of the measurement. The strain was able to grow in presence of toluene. Figure C shows growth with hexadecane. The values for the M curve stayed around 0 as result of no growth with hexadecane.
33
Figure 15: Teflon coated bottles: negative control, positive control, bottle with hydrocarbons (from left to right). Higher growth than in the negative control was shown in positive control and hydrocarbon bottle.
Toluene + Glucose ;
0,00; 4,3912E-09
Toluene + Glucose ;
13,00; 3,51925E-
09
Toluene + Glucose ;
14,00; 2,8234E-09
Toluene + Glucose ;
15,01; 2,52602E-
09
Toluene + Glucose ;
19,00; 1,74633E-
09
Toluene + Glucose ;
21,00; 1,90599E-
09
Toluene; 0,00;
4,3146E-09
Toluene; 13,00;
2,60646E-09
Toluene; 14,00;
2,17209E-09
Toluene; 15,01;
1,79783E-09
Toluene; 19,00;
4,3196E-11
Toluene; 25,01; -
2,8601E-10
G+T CO2; 7,00;
2,40818E-10
G+T CO2; 8,00;
7,99866E-10
G+T CO2; 13,01;
6,08205E-10
G+T CO2; 14,01;
7,36696E-10
G+T CO2; 15,00;
1,62794E-09
G+T CO2; 19,01;
1,26712E-09
G+T CO2; 20,00;
1,26E-09
G+T CO2; 25,00;
2,30E-09
G+T CO2; 27,01;
2,20E-09
G+T CO2; 28,00;
2,11E-09 G+T CO2; 33,00;
1,41E-09
T CO2; 8,00; -
2,67853E-10
T CO2; 13,01;
3,6562E-10
T CO2; 14,01;
5,71652E-10
T CO2; 15,00;
1,02761E-09
T CO2; 19,01;
1,3044E-09
T CO2; 20,00;
1,51E-09
T CO2; 25,00;
2,07E-09
T CO2; 27,01;
1,82E-09
T CO2; 28,00;
1,77E-09
T CO2; 33,00;
1,40853E-09
Toluene + Glucose
Toluene
G+T CO2
T CO2
34
Figure 16: GC-results: Toluene and CO2 values by Cladophialophora immunda, 17. Carbon equivalence [mol] of the two molecules was plotted against runtime [days]. There were 4 different growth curves:
Toluene + Glucose: toluene of the positive control
G+T CO2: CO2 of the positive control
Toluene: toluene of bottles with hexadecane and toluene as sole carbon source
T CO2: CO2 of bottles with hexadecane and toluene as sole carbon source For the positive control, there can be seen a harsh decline in toluene and an increase in CO2. The trend of the curves of both bottles was quite similar. The CO2 curves of both bottles reached a maximum, when no more toluene could be degraded.
3.3.2.2 Exophiala mesophila
In the microtiter plate screening, the strain Exophiala mesophila, 64, exhibited an obvious growth in case of toluene and hexadecane while no growth could be detected in presence of PCB 126 (Figure 17). Optically, it was possible to see growth in the Teflon coated bottles (see Figure 15). The GC analysis of the headspace (see Figure 16) showed, that the strain was able to degrade toluene; 40% of the hydrocarbon was recovered in CO2.
A B
M; day 0;
0,1902665
M; day 1;
0,25193
M; day 2;
0,21972
M; day 4;
0,251365
M; day 11;
0,30848
M; day 14;
0,2215
M; day 17;
0,2215
M; day 20,;
0,280755
M; day 22;
0,17603
M; day 24;
0,20477
M; day 29;
0,16603
M; day 31;
0,14902
M; day 33;
0,178175
M; day 36;
0,200725
M; day 0;
0,18114
M; day 1;
0,2336625
M; day 2;
0,20347
M; day 4;
0,24896
M; day 11;
0,2741
M; day 14;
0,20199
M; day 17;
0,20199
M; day 20,;
0,26549
M; day 22;
0,2668
M; day 24;
0,22262
M; day 29;
0,157995
M; day 31;
0,133405
M; day 33;
0,14114
M; day 36;
0,13649
G; day 0;
0,14329
G; day 1;
0,26747
G; day 2;
0,313945
G; day 4;
0,58231
G; day 11;
1,56583
G; day 14;
1,46528
G; day 17;
1,46528
G; day 20,;
1,29793
G; day 22;
1,33813
G; day 24;
1,23812
G; day 29;
1,09011
G; day 31;
1,049195
G; day 33;
0,89565
G; day 36;
0,8336
GVT; day 0;
0,15037
GVT; day 1; 0,2718
GVT; day 2;
0,33201
GVT; day 4;
0,43496
GVT; day 11; 0,55977
GVT; day 14; 0,52142
GVT; day 17; 0,52142
GVT; day 20,; 0,53063
5
GVT; day 22; 0,49662
5
GVT; day 24; 0,53798
5
GVT; day 29; 0,55688
5
GVT; day 31; 0,53421
5
GVT; day 33; 0,58767
5
GVT; day 36; 0,57110
5
M
M
G
GVT
M; day 0;
0,206415
M; day 1;
0,186415
M; day 2;
0,20785
M; day 7;
0,278965
M; day 10;
0,26403
M; day 11;
0,25512
M; day 14;
0,251765
M; day 17;
0,2593385
M; day 18;
0,25923
M; day 19;
0,260095
M; day 21;
0,263055
M; day 28;
0,3572295
M; day 31;
0,3177065
M; day 34;
0,4781535
M; day 37;
0,3256
M; day 39;
0,295415
M; day 41;
0,29816
M; day 0;
0,199225
M; day 1;
0,17976
M; day 2;
0,20091
M; day 7;
0,24669
M; day 10;
0,241785
M; day 11;
0,23266
M; day 14;
0,230835
M; day 17;
0,22954
M; day 18;
0,2326
M; day 19;
0,237855
M; day 21;
0,250295
M; day 28;
0,308061
M; day 31;
0,2837275
M; day 34;
0,412735
M; day 37;
0,37283
M; day 39;
0,35218
M; day 41;
0,33899 G; day 0;
0,20065
G; day 1;
0,22534
G; day 2;
0,3046
G; day 7;
0,46393
G; day 10;
0,49435
G; day 11;
0,492825
G; day 14;
0,500475
G; day 17;
0,500105
G; day 18;
0,49536
G; day 19;
0,49554
G; day 21;
0,509395
G; day 28;
0,586255
G; day 31;
0,595075
G; day 34;
0,63911
G; day 37;
0,667565
G; day 39;
0,682975
G; day 41;
0,677645
GVT; day 0;
0,22849
GVT; day 1;
0,24562
GVT; day 2;
0,317925
GVT; day 7;
0,42359
GVT; day 10; 0,43818
GVT; day 11; 0,43957
GVT; day 14; 0,45531
GVT; day 17; 0,45198
GVT; day 18; 0,4502
GVT; day 19; 0,45093
GVT; day 21; 0,45543
GVT; day 28; 0,50783
GVT; day 31; 0,51226
5
GVT; day 34; 0,49609
GVT; day 37; 0,60667
GVT; day 39; 0,61669
5
GVT; day 41; 0,61108
M
M
G
GVT
M; day 0;
0,26142
M; day 1;
0,28733
M; day 2;
0,29546
M; day 7;
0,285355
M; day 10;
0,25918
M; day 11;
0,29999
M; day 14;
0,302415
M; day 17;
0,3132
M; day 18;
0,37266
M; day 19;
0,385525
M; day 21;
0,360795
M; day 28;
0,42202
M; day 31;
0,42125
M; day 34;
0,47475
M; day 37;
0,521555
M; day 39;
0,48902
M; day 41;
0,62839
M; day 0;
0,318385
M; day 1;
0,297965
M; day 2;
0,298915
M; day 7;
0,27197
M; day 10;
0,2747
M; day 11;
0,27724
M; day 14;
0,295955
M; day 17;
0,323995
M; day 18;
0,37136
M; day 19;
0,384085
M; day 21;
0,38811
M; day 28;
0,48502
M; day 31;
0,509615
M; day 34;
0,61157
M; day 37;
0,643475
M; day 39;
0,694555
M; day 41;
0,78883 G; day 0; 0,22276
G; day 1;
0,285645
G; day 2;
0,365595
G; day 7;
1,74426
G; day 10;
2,109915
G; day 11;
2,235635
G; day 14;
2,342895
G; day 17;
2,378895
G; day 18;
2,362485
G; day 19;
2,34282
G; day 21;
2,47547
G; day 28;
2,49693
G; day 31;
2,484305
G; day 34;
2,477265
G; day 37;
2,387765
G; day 39;
2,108185
G; day 41;
2,435645
GVT; day 0;
0,229475
GVT; day 1;
0,280315
GVT; day 2;
0,355535
GVT; day 7;
0,66723
GVT; day 10; 0,76155
5
GVT; day 11; 0,79344
GVT; day 14; 0,85957
GVT; day 17; 0,92237
5
GVT; day 18; 0,92037
5
GVT; day 19; 0,92307
GVT; day 21; 0,95739
GVT; day 28; 1,05012
5
GVT; day 31; 1,08761
GVT; day 34; 1,12460
5
GVT; day 37; 1,15605
GVT; day 39; 1,16315
5
GVT; day 41; 1,1906
M
M
G
GVT
C Figure 17 A, B, C: Microtiter plate screening of strain 64, Exophiala mesophila, with the hydrocarbons PCB126 (A), toluene (B) and hexadecane (C).OD 700 plotted against the days of measurement. Those graphs consist of the average data points of duplicates. They show 4 curves, medium plus hydrocarbon (M), medium plus glucose (G), medium plus glucose, vitamins and trace elements (GVT). M curves are, as expected, quite similar in in all figures. In figure A, the M curves varied around 0.2 OD 700 and did not increase during the period of measurement while the G and GVT curves increased. This strain was not able to grow with PCB 126. In figure B, an increase in the M curve is seen. For the hexadecane (C), an increase of the M values can be seen.
Figure 18: Teflon coated bottles: negative control, positive control, bottle with hydrocarbons (from left to right). Higher growth was seen in positive control and hydrocarbon bottle than in the negative control. The medium seemed blurred in the two bottles with growth.
35
G+T; 0,00; 5,17712E-
09 G+T; 13,00; 3,95012E-
09
G+T; 14,00; 3,44108E-
09
G+T; 15,01; 3,12963E-
09 G+T; 19,00; 1,80301E-
09 G+T; 22,00; 1,2353E-09
T; 0,00; 4,8935E-09
T; 8,00; 2,63481E-
09
T; 13,00; 2,16954E-
09
T; 14,00; 2,0919E-09
T; 15,01; 7,0367E-10
T; 19,00; 4,81711E-
10
G+T CO2; 7,00;
1,63474E-09
G+T CO2; 8,00; 1,5E-
09
G+T CO2; 13,01;
3,54855E-09
G+T CO2; 14,01;
3,54855E-09
G+T CO2; 15,00;
8,31996E-09
G+T CO2; 19,01;
6,95492E-09
G+T CO2; 21,00;
5,69E-09
T CO2; 8,00;
3,33E-10
T CO2; 13,01;
9,54861E-10
T CO2; 14,01;
7,04492E-10
T CO2; 15,00;
1,48356E-09
T CO2; 19,01;
2,43275E-09
T CO2; 21,00;
1,43E-09
G+T
T
G+T CO2
T CO2
Figure 19: GC-results: Toluene and CO2 values by Exophiala mesophila, 64.Carbon equivalence [mol] of the two molecules was plotted against runtime [days]. There are 4 different growth curves:
G+T: toluene of the positive control
G+T CO2: CO2 of the positive control
T: toluene of bottles with hexadecane and toluene as sole carbon source
T CO2: CO2 of bottles with hexadecane and toluene as sole carbon source The curve for toluene decreased very quickly in both bottles while CO2 production differed. For the positive control the curve reached higher levels of C equivalence than gas samples of the sole hydrocarbon bottle.
3.3.3 Toxicity
One strain, Exophiala jeanselmei, 25, showed an unexpected curve during the GC-screening as concentration of toluene was toxic for this strain. It showed neither an increase of CO2in the sole hydrocarbon bottle nor in the positive control bottle (Figure 21). During the microtiter plate screening, the strain was able to grow in presence of hexadecane and PCB 126 (Figure 20 A, C). In the case of toluene (Figure 20B), the strain did not show growth in wells with toluene as sole carbon source nor in the wells with additional glucose. Also optically, no growth was seen.
36
A B
C Figure 20 A, B, C: Microtiter plate screening of strain 25, Exophiala jeanselmei, with the hydrocarbons PCB126 (A), toluene (B) and hexadecane (C).OD 700 plotted against the days of measurement. The graphs consist of the average data points of duplicates. They show 4 curves, medium plus hydrocarbon (M), medium plus glucose (G), medium plus glucose, vitamins and trace elements (GVT). M curves are, as expected, quite similar in in all figures. In figure A all curves were plotted in the same area and did not differ a lot. Exophiala jeanselmei was able to grow in wells with glucose as in well with PCB 126. The G curve showed minus values in the end of the measurement. Figure B shows no OD 700 increase in all wells. The fungal strain was not able to grow in presence of toluene, also if glucose was available as additional carbon source. In figure C wells without glucose showed an increase in OD 700 after a few days of incubation time. The strain was able to grow in presence of hexadecane.
M; day 0 ; -
0,049305
M; day 3;
0,121275
M; day 4;
0,142895
M; day 5;
0,2347
M; day 6;
0,55525
M; day 7;
0,6281
M; day 12;
0,652135
M; day 15;
0,61858
M; day 16;
0,64297
M; day 19;
0,708705
M; day 22;
0,708705
M; day 23;
0,716255
M; day 24;
0,720285
M; day 26;
0,6893
M; day 33;
0,6268
M; day 36;
0,50878
M; day 40;
0,572965
M; day 43;
0,56357
M; day 45;
0,524285
M; day 0 ;
0,136595
M; day 3;
0,12777
M; day 4;
0,152245
M; day 5;
0,191355
M; day 6;
0,57572
M; day 7;
0,64689
M; day 12;
0,719075
M; day 15;
0,720205
M; day 16;
0,73437
M; day 19;
0,85623
M; day 22;
0,85623
M; day 23;
0,884545
M; day 24;
0,893795
M; day 26;
0,8517
M; day 33;
0,821915
M; day 36;
0,66842
M; day 40;
0,769095
M; day 43;
0,773835
M; day 45;
0,737675
G; day 0 ;
0,155505
G; day 3; 0,20897
G; day 4; 0,36849
G; day 5; 0,52747
G; day 6; 1,01492
5
G; day 7; 1,11706
5
G; day 12;
0,92684
G; day 15;
0,7941
G; day 16;
0,730455
G; day 19;
0,644855
G; day 22;
0,644855
G; day 23;
0,539935
G; day 24;
0,498035
G; day 26;
0,44784
G; day 33; -
0,77715
G; day 36; -
0,748
G; day 40; -
0,93655
G; day 43; -
0,93755
G; day 45; -
0,9987
GVT; day 0 ;
0,21494
GVT; day 3;
0,116835
GVT; day 4;
0,11615
GVT; day 5;
0,15327
GVT; day 6;
0,29766
GVT; day 7;
0,3161
GVT; day 12;
0,686825
GVT; day 15;
0,759675
GVT; day 16;
0,720585
GVT; day 19;
0,732495
GVT; day 22;
0,732495
GVT; day 23;
0,96011
GVT; day 24;
1,008855
GVT; day 26;
1,116785
GVT; day 33;
0,95108
GVT; day 36;
0,831815
GVT; day 40;
0,86777
GVT; day 43;
0,830795
GVT; day 45;
0,810925
M
M
G
GVT
M; day 0; -
0,049305
M; day 03; -
0,008315
M; day 4; 0,2347
M; day 05; -
0,007095
M; day 06; -
0,006375
M; day 07; -
0,00627
M; day 12; -
0,008655
M; day 15; -
0,00062
M; day 16;
0,008155
M; day 19; -
0,006975
M; day 22;
0,00768
M; day 23;
0,000325
M; day 24; -
0,003065
M; day 26;
0,000575
M; day 33;
0,00963
M; day 36; -
0,00534
M; day 40; -
0,009715
M; day 43; -
0,01529
M; day 45; -
0,018145
M; day 0; 0,136595
M; day 03; -
0,00121
M; day 4; 0,191355
M; day 05; -
0,00272
M; day 06; -
0,00308
M; day 07; -
0,002805
M; day 12; -
0,006165
M; day 15;
0,002485
M; day 16;
0,01961
M; day 19; -
0,00148
M; day 22;
0,00813
M; day 23;
0,00425
M; day 24; -
0,009365
M; day 26;
0,003295
M; day 33;
0,011105
M; day 36;
0,000235
M; day 40; -
0,008535
M; day 43; -
0,01539
M; day 45; -
0,02203
G; day 0; 0,155505
G; day 03;
0,001795
G; day 4; 0,52747
G; day 05; -
0,002905
G; day 06; -
0,001985
G; day 07; -
0,001435
G; day 12; -
0,003745
G; day 15;
0,00434
G; day 16;
0,008405
G; day 19; -
0,00649
G; day 22; -
0,0022
G; day 23; -
0,01187
G; day 24; -
0,022615
G; day 26; -
0,01592
G; day 33; -
0,000145
G; day 36; -
0,01015
G; day 40; -
0,018825
G; day 43; -
0,01982
G; day 45; -
0,020095
GVT; day 0;
0,21494
GVT; day 03; -
0,001095
GVT; day 4;
0,15327 GVT; day 05; -
0,003785
GVT; day 06; 8E-05
GVT; day 07; -
0,00409
GVT; day 12; -
0,01005
GVT; day 15;
0,01087
GVT; day 16;
0,01375
GVT; day 19; -
0,012905
GVT; day 22; -
0,008915
GVT; day 23; -
0,016525
GVT; day 24; -
0,023975
GVT; day 26; -
0,018905
GVT; day 33;
0,00155
GVT; day 36; -
0,00782
GVT; day 40; -
0,017955
GVT; day 43; -
0,02227
GVT; day 45; -
0,01946
M
M
G
GVT
M; day 0; -
0,003315
M; day 03;
0,02759
M; day 04; -
0,014645
M; day 05;
0,043935
M; day 06;
0,052385
M; day 07;
0,066605
M; day 15;
0,40489
M; day 16;
0,50126
M; day 19;
0,68935
M; day 22;
0,766265
M; day 23;
0,720795
M; day 24;
0,773495
M; day 26;
0,786035
M; day 33;
0,78393
M; day 36;
0,773495
M; day 40;
0,7605
M; day 43;
0,787425
M; day 45;
0,72309
M; day 0;
0,02118
M; day 03;
0,03161
M; day 04;
0,023785
M; day 05;
0,04205
M; day 06;
0,05326
M; day 07;
0,06912
M; day 15;
0,44069
M; day 16;
0,529795
M; day 19;
0,67614
M; day 22;
0,72883
M; day 23;
0,705285
M; day 24;
0,73316
M; day 26;
0,729875
M; day 33;
0,73731
M; day 36;
0,73316
M; day 40;
0,72916
M; day 43;
0,749607
M; day 45;
0,652435
G; day 0; 0,00326
5
G; day 03;
0,097465
G; day 04;
0,247335
G; day 05;
0,50534
G; day 06;
0,6504
G; day 07;
0,637785
G; day 15;
0,602275
G; day 16;
0,591135
G; day 19;
0,540205
G; day 22;
0,53068
G; day 23;
0,51627
G; day 24;
0,51766
G; day 26;
0,49168
G; day 33;
0,45465
G; day 36;
0,51766
G; day 40;
0,4867315
G; day 43;
0,4205 G; day
45; 0,29647
GVT; day 0; -0,005
GVT; day 03;
0,07122
GVT; day 04;
0,19263
GVT; day 05;
0,258005
GVT; day 06;
0,39555
GVT; day 07;
0,3644
GVT; day 15;
0,610115
GVT; day 16;
0,61429
GVT; day 19;
0,500935
GVT; day 22;
0,47628
GVT; day 23;
0,46666
GVT; day 24;
0,4668
GVT; day 26;
0,449265
GVT; day 33;
0,31201
GVT; day 36;
0,4668
GVT; day 40;
0,3318515
GVT; day 43;
0,274164
GVT; day 45;
0,2818475
M
M
G
GVT
37
G+T; 0,00; 4,18771E-
09
G+T; 13,00;
3,84693E-09
G+T; 14,00;
3,38323E-09
G+T; 15,01;
2,85383E-09
T; 0,00; 4,20721E-
09
T; 13,00; 4,1817E-
09
T; 14,00; 4,01142E-
09
T; 15,01; 3,83632E-
09
G+T CO2; 7,00;
1,91E-10
G+T CO2; 8,00;
1,78948E-10
G+T CO2; 13,01;
5,23923E-10
G+T CO2; 14,01;
1,89964E-10
G+T CO2; 15,00;
2,13128E-10
T CO2; 8,00;
1,84812E-10
T CO2; 13,01;
2,10981E-10
T CO2; 14,01;
2,18875E-10
T CO2; 15,00;
1,21E-10
G+T
T
G+T CO2
T CO2
38
Figure 21: GC-results: Toluene and CO2 values by Exophiala jeanselmei, 25.Carbon equivalence [mol] of the two molecules was plotted against runtime [days]. There are 4 different growth curves:
G+T: toluene of the positive control
G+T CO2: CO2 of the positive control
T: toluene of bottles with hexadecane and toluene as sole carbon source
T CO2: CO2 of bottles with hexadecane and toluene as sole carbon source Toluene values were decreasing slightly in both bottles. The speciality of this strain expressed itself in the stable value of CO2. It was not increasing in both bottles, not even in the positive control although having glucose as carbon source.
4 Discussion
We were able to successfully develop two screening methods to gain knowledge about the different strains and their degradation and growth abilities. The microtiter plate screening was newly developed and gave answers about the growth of the strains in presence of the hydrocarbons. 114 out of the 163 fungal strains showed growth in the microtiter plate screening on at least one of the hydrocarbon sources supplied. Finally, in the subsequent assimilation tests in liquid closed batches, we successfully selected two strains – Cladophialophora immunda and Exophiala mesophila- who were able to degrade toluene. Cladophialophora immunda comes from a hydrocarbon rich isolation source, a gasoline station. Therefore, we can state that it adapted to the environment and is able to degrade toluene. Exophiala mesophila, on the other hand, was isolated from a human, from a chronic sinusitis. This strain is able to degrade toluene although not having the hydrocarbon in his environment. This means that strains don’t need a hydrocarbon rich environment to have the features to degrade them although the isolate from the hydrocarbon rich source showed better degradation performance. The method of the microtiterplate screening provided a lot of data points by the Tecan reader. With data from the microtiter plate screening, growth curves were created with the help of Excel to demonstrate if the strains exhibited growth under certain conditions. In some cases growth of the positive control with vitamins and trace elements was slower or less but just in very few cases it inhibited growth. If there was inhibition by the vitamins and trace elements, the fungal strain could also not grow in presence of the hydrocarbon. The microtiter plate screening was a basic tool, to gain a general answer if the strains are able to grow in presence of the different hydrocarbons, not if the strain can degrade it. Therefore the number of positive strains was rather high. There are several reasons for a follow up screening in a second phase of investigation:
Graph interpretation Interpretation of the graphs was rather difficult as some showed a slight increase in the OD values compared to the positive control, while others were on the same level as the positive control. Therefore, the differentiation between + and was done in the result table (see Table 16).
Growth curve variations Curves varied a lot and did not always show a clear trend (see figures in chapter 3.3. and in the appendix). On one hand this occurred due to residual growth of fungi. Although being small, the wells of the microtiter plates have different options where the fungi can grow. Some of them preferred to grow on the surface, while others grew only on the walls or in some other region, where detection with the help of the Tecan reader was not possible. On the other hand desiccation of the wells and the need to refill wells with water affected growth of some fungi and as a consequence resulted in graph variations. The test method endured long and although sealing the plates with parafilm, the wells dried out after one week on the shaker. The growth differences in the wells are presented in Figure 22. Another reason for variations was the toxic effect of toluene to some strains. The chemical was poured in a beaker, which was then put in the desiccator. Therefore, in the desiccator the gaseous phase of the substance was saturated. For some fungal strains this was toxic, which could also be seen in the more detailed screening at IRTA. Due to this toxicity some strains were only able to grow slightly or stopped growth abruptly which resulted in graph variations. Also the concentration change of volatile toluene due to opening of the desiccator for measurements could have resulted in growth variations.
39
Figure 22: Two different microtiter plates with different fungal strains; Variations are seen between the 3rd and 4th, 7th and 8th and 11th and 12th well in each row although containing the same strain, growth was therefore different in the wells.
Homogenisation Homgenisation of the fungal strain from the agar plate in NaCl was a difficult task. While some fungal strains homogenised easily after a short time in the ribolyzer, some were hard to homogenise and therefore a difference appeared in the amount of cells in the inoculum between the different wells. As the screening was meant to generate a general yes or no answer to growth, this was seen as unproblematic.
Tecan Reader The results of the Tecan reader varied from day to day. A daily factor could have been introduced to correct the results. As data were corrected by a daily measured blank, the correction by a daily factor was considered as not necessary. The equipment delivered just little variation in the results, which was considered sufficient for the basic screening.
Oligotrophic strains Strains for the screening were mainly oligotrophic. Oligotrophic means growing and metabolizing slowly but steadily at low concentrations of energy sources, nutrients and water (Gostinčar et al., 2012). This is also exhibited in their potential to survive in extreme and stressful environments. This fact means that they can also survive using carbon sources other than hydrocarbons. For example they could metabolize ingredients of the microtiter plates or from any other sources. Although the plates were generally closed in general with lids, they were opened from time to time for refilling or other reasons. Hence, other nutrients could have entered. Some fungal strains sporulated and this optical density could still be measured with the Tecan reader.
In general, the microtiter plate screening provided the expected information. The results exhibited a general idea on the growth behaviour of the strains. Also a high throughput in a small time window was reached, as it is difficult to handle 163 strains in the same time and under same conditions. The data gave basic ideas of which strains could be good degrading candidates and therefore were used in the GC screening. In the GC screening the main research goal was to define if the fugal strain can use hydrocarbons as sole carbon source and therefore degrade those. For analytical detection, the GC-measurements were used. As equipment Teflon coated bottles were chosen due to the fact that hydrocarbons are not able to penetrate through. As mentioned in 3.3 the results were divided in three possible cases:
40
Negative results: no increase in CO2 and stable amount of toluene in the sample bottle, in glucose control increase in CO2 and decrease of toluene
Positive results: increase in CO2 and decrease of toluene, in glucose control increase in CO2 and decrease of toluene
Toxicity: neither in the sample nor in the glucose control increase of CO2 or decrease of toluene
Another case would have been the degradation of hexadecane. Therefore, the hexadecane concentration in the sample bottles would have decreased, while toluene concentration would have stayed stable and CO2 would have increased. This case didn’t appear in any of the bottles therefore it seems to be very difficult to degrade hexadecane for fungal strains. The PCB degradation abilities have not been tested in this screening and will be tested in further tests. The results of the GC screening and the microtiter plate screening results differed. The microtiter plate results were more often interpreted as positive for living in presence of hydrocarbons, while the GC screening showed that they were not able to degrade the hydrocarbon. This can be explained by sporulation and their being oligotrophic (see Figure 22). In all bottles, the graphs showed slight variations which happened due to equipment instability. There was a daily factor introduced for the correlation, but still there were slight variations. Additionally, toluene decreased slightly in bottles of non-degraders. The reason for this fact can be due to leakages in the bottles. The Teflon coated bottles were inherent stable for hydrocarbons but the connection to the injection valve and the valve itself can have minor leakages. Also while retrieving the injection volume, small amounts of toluene could have escaped. As the GC measurement is precise, it is not easy to find sample equipment in the same precise range which also fulfils all other requirements needed (i.e. sterility, volume, usage for microbes). Compared to the concentrations of the degrading strains it is a minor loss which does not disturb interpretation of the results. The glucose bottles exhibited that the fungal strains are able to degrade toluene in presence of glucose. Two candidates were found to degrade hydrocarbons: Cladophialophora immunda and Exophiala mesophila are two strains which were able to use toluene as sole carbon source. The negative controls were very important, as all of the strains were able to sporulate without any carbon source. Therefore, an optical comparison was possible to recognize the difference between sporulation and real growth. CO2 variations can be a result of a change in pH. The media were buffered and therefore no pH change should happen due to growth. However no measurement of pH was possible, as the sterility of the media was given more importance than the pH. Still this could have been a trigger for variations in CO2 levels. One big point in the results is the CO2 recovery. In the case of Cladophialophora immunda, 70% of toluene was recovered in CO2. For Exophiala mesophila around 40% of toluene was recovered. Taking the bioremediation goal in account, those percentages are very important as it is not known yet what happened to the rest of the toluene. Other unwanted toxic compounds could be produced in their degradation process or toluene degradation products could go into the biomass. This could be tested further through drying and weighing of the biomass. Especially for the bioremediation goal it is important to be sure that no toxic compounds are produced in the degrading process to make sure that not more dangers are introduced for environment and animals through applying the method. Another critical point for the Exophiala strain is that toluene is not degraded completely. Further tests would be necessary if with more inoculum all toluene can be degraded or if there will always remain some hydrocarbon.
41
Next to the toxicity of the intermediates the H2 level of the strains should not be forgotten. The strains can be human pathogenic and therefore the metabolic pathways of the fungal strains, their changes during the degradation and their pathogenic features have to be studied in detail. Cladophialophora immunda was isolated near a petrol station. Therefore it was already in hydrocarbon rich environment and adapted. A transcriptome study would be a good tool, to know in detail how this strain is able to survive in such an environment and how the strain can use toluene as a carbon source. Another interesting point is that although black fungi are known to survive high levels of stress, some of them were not able to survive low levels of toluene. They were toxic for them as seen for Exophiala jeanselmei (see 3.3.3). Therefore, not all abilities of black fungi are known and in some parts they are quite extraordinary while in other parts too high expectations are made regarding their stress tolerance.
42
5 Conclusion and Outlook
Both screening methods met the intended goals to define fungal strains with biodegradative abilities. The microtiter plate method was successfully developed and fulfilled the need of high throughput in a small time period in constant conditions was met. Although having some limitations caused by the inhomogeneous growth of fungi, the microtiter plate method turned out to be appropriate to answer the main questions. In the microtiter plate screening 114 fungal strains were found, which were able to grow in media with hydrocarbon as sole carbon source. Those were taken to the second screening, where the most promising candidates were screened for hydrocarbon degradation. In the second screening, two strains were proven to be able to degrade and assimilate toluene, while hexadecane was not degraded by any strain. PCB-126 still needs to be observed in the GC method. PCB-126 must be added into the Teflon bottles and afterwards measured in the GC-FID for degradation. The reasons for the differences between the results of the microtiter plate screening and the GC-screening can be seen in the preciseness of the methods and also on the two different targets. Further investigations for the knowledge were the rest of the degraded toluene goes (into biomass and/or into co-metabolites) and what kinds of co-metabolites are produced, are necessary before using them in bioremediation or on biofilters. It must be sure that no toxic or pathogenic compounds are produced during degradation. Moreover, the biochemical pathways should be studied on the basis of proteomic and trascriptomic (RNA sequence, gene expression) analysis before using the strain in field experiments or applications. The techniques also have to be developed from a lab scale to one which can be applied for large scale bioremediation. Being aware of possible risks and studying those before applying anything into nature is one of the major challenges of further studies. Anyhow, the strains found in the screening are useful candidates and are worth being tested further. Having a lot of pollutions around us, any possible approach for a solution should be tested. Additionally, the group of black yeast is not known completely and they have a lot of surprising attributes which could be used in a lot of approaches. For example the mechanisms which help them to survive in such extreme conditions are not fully understood. There are always new technologies invented and further developed, especially in the field of proteomics, metabolomics and genomics which could help us to understand all the processes better and use them in the field of biotechnology. Therefore research in this field is highly promising.
43
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7 Appendix:
7.1 Diagrams microtiter plate pre-tests
Test plates, different concentrations and equipment parameters were tested
-1
-0,5
0
0,5
TAG 0(18.04.13)
TAG 20 TAG 40 TAG 64
Average Hexadecane
Hexa
GlucVT
Gluc
-0,3
-0,2
-0,1
0
0,1
0,2
0,3
TAG
0
TAG
5
TAG
8
TAG
14
TAG
20
TAG
26
TAG
33
TAG
36
TAG
41
TAG
49
TAG
57
TAG
62
TAG
67
TAG
70
TAG
76
Average Tol
Tol
Tol2
GlucVT
Gluc
-0,2
-0,1
0
0,1
0,2
TAG
0
TAG
5
TAG
9
TAG
16
TAG
21
TAG
27A
bs
n Hexadecan Average
Hex1
Hex2
GlucVT
Gluc
-0,4
-0,3
-0,2
-0,1
0
0,1
TAG0
TAG5
TAG9
TAG16
TAG21
TAG27
Average Toluol
Tol
Tol2
GlucVT
Gluc
0
0,1
0,2
0,3
0,4
0,5
day0
day9
day17
day29
day39
day46
day53
Average Hexadecan
Hex 1
Hex 2
GlucVT
Gluc0
0,1
0,2
0,3
0,4
0,5
1 4 7 10 13 16 19 22
Average Hex with TTC Hex 1
Hex 2
GlucVT
Gluc
48
0
0,05
0,1
0,15
day0
day9
day17
day29
day39
day46
day53
Tol average
Tol 1
tol 2
GlucVT
Gluc0
0,1
0,2
0,3
1 4 7 10 13 16 19 22
Tol average with TTC Tol 1
tol 2
GlucVT
Gluc
-0,1
0
0,1
0,2
0,3
0,4
0,5
day0
day9
day17
day29
day39
day46
day53
PCB Average
pcb 1
pcb 2
GlucVT
Gluc 0
0,2
0,4
0,6
day0
day6
day15
day26
day30
day36
day43
Average PCB
pcb 1
pcb 2
GlucVT
Gluc
49
7.2 Diagrams microtiter plate screening
Runtime in days against absorbtion M = medium plus hydrocarbon G = medium plus glucose GVT = medium plus glucose, vitamins and trace elements
7.2.1 PCB 126
-2
0
2
day
0
day
4
day
6
day…
day…
day…
day…
day…
day…
day…
10 Selenophoma mahoniae / Average
PCB
M
M
G-2
-1
0
1
day
0
day
4
day
6
day
12
day
16
day
22
day
24
day
33
day
40
day
45
13 Scedosporium apiospermum / Average
PCB
M
M
G
GVT
-2
0
2
day 0day 3day 4day 5day 6day 7day 12day 15day 16day 19day 22day 23day 24day 26day 33day 36day 40day 43day 45
12 Exophiala alcalophila / Average
PCB
M
M
G-2
0
2
day 0day 3day 4day 5day 6day 7day 12day 15day 16day 19day 22day 23day 24day 26day 33day 36day 40day 43day 45
11 Exophiala oligosperma / Average PCB
M
M
G
-1
0
1
2
day
0
day
4
day
6
day
12
day
16
day
22
day
24
day
33
day
40
day
45
7 Exophiala castellanii / Average PCB
M
M
G
GVT-2
0
2
day 0day 3day 4day 5day 6day 7day 12day 15day 16day 19day 22day 23day 24day 26day 33day 36day 40day 43day 45
9 Exophiala exophialae / Average
PCB
M
M
G
50
-2
0
2
day
0
day
4
day
6
day…
day…
day…
day…
day…
day…
day…
2 Exohiala xenobiotica / Average
PCB
M
M
G -1
0
1
2
day
0
day
4
day
6
day
12
day
16
day
22
day
24
day
33
day
40
day
45
4 Exophiala sideris / Average PCB
M
M
G
GVT
-1
0
1
2
day
0
day
4
day
6
day
12
day
16
day
22
day
24
day
33
day
40
day
45
1 Exophiala dermatitidis / Average
PCB
M
M
G
GVT-0,5
0
0,5
1
1,5
day
0
day
5
day
12
day
19
day
24
day
36
day
45
24 Cladophialophora minourae / Average PCB
M
M
G
GVT
-2
-1
0
1
day
0
day
4
day
6
day
12
day
16
day
22
day
24
day
33
day
40
day
45
28 Aureobasidium pullulans var. pullulans / Average PCB
M
M
G
GVT
-2
0
2
day
0
day
4
day
6
day
12
day
16
day
22
day
24
day
33
day
40
day
45
19 graphium eumorphum / Average
PCB
M
M
G
GVT
-1,5
-1
-0,5
0
0,5
1
day
0
day
5
day
12
day
19
day
24
day
36
day
45
35 Pseudallescheria minutispora / Average PCB
M
M
G
GVT -1
0
1
2
day
0
day
4
day
6
day
12
day
16
day
22
day
24
day
33
day
40
day
45
36 Cladophialophora australiensis / Average
PCB
M
M
G
GVT
51
-2
-1
0
1
2
day
0
day
4
day
6
day
12
day
16
day
22
day
24
day
33
day
40
day
45
33 Exophiala spinifera / Average PCB
M
M
G
GVT-2
0
2
day 0day 3day 4day 5day 6day 7day 12day 15day 16day 19day 22day 23day 24day 26day 33day 36day 40day 43day 45
34 Exophiala heteromorpha /
Average PCB
M
M
G
-2
-1
0
1
day
0
day
4
day
6
day
12
day
16
day
22
day
24
day
33
day
40
day
45
32 Rhinocladiella similis / Average PCB
M
M
G
GVT-1
0
1
day
0
day
4
day
6
day
12
day
16
day
22
day
24
day
33
day
40
day
45
31 Exophiala spinifera / Average PCB
M
M
G
GVT
-2
-1
0
1
day
0
day
4
day
6
day
12
day
16
day
22
day
24
day
33
day
40
day
45
29 Cladophialophora subtilis / Average PCB
M
M
G
GVT -1
0
1
2
day
3
day
5
day
7
day
15
day
19
day
23
day
26
day
36
day
43
30 Phialophora americana / Average
PCB
M
M
G
GVT
-1
0
1
2
3
day
0
day
2
day
11
day
17
day
22
day
29
day
33
day
38
day
43
37 Cladophialophora emmonsii/ Average PCB
M
-2
0
2
day
0
day
2
day
11
day
17
day
22
day
29
day
33
day
38
day
43
66 Pseudallescheria boydii/ Average PCB
52
-2
0
2
day
0
day
2
day…
day…
day…
day…
day…
day…
day…
26 Pseudallescheria fusoidea/ Average
PCB
-1
0
1
2
day
0
day
2
day
11
day
17
day
22
day
29
day
33
day
38
day
43
20 Exophiala bergeri/ Average PCB
-2
0
2
day
0
day
2
day…
day…
day…
day…
day…
day…
day…
78 Aureobasidium pullulans/ Average
PCB M
-2
0
2
4d
ay 0
day
2
day…
day…
day…
day…
day…
day…
day…
5 Cladophialophora carrionii/ Average
PCB M
-1
0
1
2
day
0
day
2
day
11
day
17
day
22
day
29
day
33
day
38
day
43
21 Exophiala lecanii-corni/ Average PCB
-2
-1
0
1
2
day
0
day
2
day
11
day
17
day
22
day
29
day
33
day
38
day
43
27 Cladophialophora boppii/ Average PCB
M
-2
0
2
day
0
day
2
day…
day…
day…
day…
day…
day…
day…
14 Pseudallescheria ellipsoidea / Average
PCB
0
1
2
3
day0
day2
day11
day17
day22
day29
day33
17 Cladophialophora immunda/ Average PCB
M
53
-2
0
2
day
0
day
2
day…
day…
day…
day…
day…
day…
day…
63 Exophiala dermatitidis/ Average
PCB M
-2
0
2
day0
day2
day11
day20,
day24
day31
day36
day40
46 Aureobasidium pullulans/ Average
PCB M
-2
-1
0
1
2
day0
day2
day11
day17
day22
day31
day36
day40
3 Pseudallescheria agusta/ Average PCB
M
-1
0
1
2
day0
day2
day11
day17
day22
day31
day36
day40
16 Cladophialophora arxii / Average PCB
M
0
2
day 0day 2day 11day 17day 22day 31day 36day 40
53 Exophiala mesophila/ Average
PCB M
-5
0
5
day 0day 2day 14day 20,day 24day 31day 36day 40
70 Exophiala mesophila/ Average
PCB M
-1
0
1
2
3
day0
day2
day11
day17
day22
day31
day36
day40
74 Rhinocladiella similis/ Average PCB
-2
0
2
day0
day2
day11
day17
day22
day31
day36
day40
51 Exophiala xenobiotica/ Average
PCB
54
-5
0
5
day 0day 2day 11day 17day 22day 31day 36day 40
39 Cladophialophora mycetomatis/ Average PCB
-2
0
2
day0
day2
day11
day17
day22
day31
day36
day40
65 Exohiala heteromorpha/
Average PCB M
-5
0
5
day 0day 2day 11day 17day 22day 31day 36day 40
22 Cladophialophroa yegresii / Average
PCB M
0
2
day 0day 10day 17day 21day 26day 30day 35day 40
40 Exophiala alcalophila/ Average
PCB
M
M
G
0
1
2
day 7day 14day 19day 23day 28day 33day 37
68 Exophiala sideris /
Average PCB
M
M
G
GVT-1
0
1
2
3
day 0day 10day 17day 21day 26day 30day 35day 40
49 Cladophialophora carrionii/ Average PCB
M
0
0,5
1
1,5
2
day 0day 10day 17day 21day 26day 30day 35day 40
59 Cladophialophora yegresii/ Average PCB
M
0
1
2
3
day 0day 10day 17day 21day 26day 30day 35day 40
41 Exophiala sideris/ Average PCB
M
M
G
GVT
55
-0,5
0
0,5
1
1,5
day 0day 10day 17day 21day 26day 30day 35day 40
42 Pseudallescheria ellipsoidea/ Average PCB
M
-1
0
1
2
day 0day 10day 17day 21day 26day 30day 35day 40
47 Exophiala dermatitidis/ Average
PCB
M
M
G
-1
0
1
2
day 0day 10day 17day 21day 26day 30day 35day 40
45 Exophiala spinifera / Average PCB
M
-2
0
2
4
day 0day 10day 17day 21day 26day 30day 35day 40
8 Phialophora verrucosa/ Average
PCB M
0
2
4
day 0day 10day 17day 21day 26day 30day 35day 40
43 Exophiala oligosperma/ Average
PCB
M
M
G-0,5
0
0,5
1
1,5
day 0day 10day 17day 21day 26day 30day 35day 40
73 Exophiala castellanii/ Average PCB
M
-1
0
1
2
day 0day 10day 17day 21day 26day 30day 35day 40
72 Pseudallescheria angusta/ Average PCB
M
M
G
GVT-2
0
2
4
day 0day 10day 17day 21day 26day 30day 35day 40
60 Exophiala oligosperma/ Average
PCB M
56
-1
0
1
2
day 0day 10day 17day 21day 26day 30day 35day 40
61 Pseudallescheria boydii/ Average PCB
M
-2
0
2
4
day 0day 10day 17day 21day 26day 30day 35day 40
48 Phialophora verrucosa/ Average
PCB
M
M
G
-5
0
5
day 0day 10day 17day 21day 26day 30day 35day 40
67 Cladophialophora minourae/ Average
PCB
M
M
G-1
0
1
2
day 0day 10day 17day 21day 26day 30day 35day 40
23 Cladophialophora samoёnsis/ Average PCB
M
M
G
GVT
-2
0
2
4
day 0day 10day 17day 21day 26day 30day 35day 40
6 Cladophialophora saturnica / Average PCB
-0,5
0
0,5
1
1,5
day 0day 10day 17day 21day 26day 30day 35day 40
59 Cladophialophora yegresii / Average PCB
M
M
G
GVT
-1
0
1
2
3
day 0day 10day 17day 21day 26day 30day 35day 40
62 Cladophialophora arxii / Average PCB
M
-0,5
0
0,5
1
day 0day 10day 17day 21day 26day 30day 35day 40
71 Pseudallescheria boydii/ / Average PCB
M
M
G
GVT
57
0
0,5
1
1,5
2
day0
day4
day9
day14
day18
day23
day28
day32
day37
57 Cladophialophora boppii / Average PCB
0
0,5
1
1,5
day0
day4
day9
day14
day18
day23
day28
day32
day37
104 Exophiala spinifera / Average PCB
-1
0
1
2
3
4
day0
day4
day9
day14
day18
day23
day28
day32
day37
76 Cladophialophora immunda / Average PCB
-0,5
0
0,5
1
1,5
2
day0
day4
day9
day14
day18
day23
day28
day32
day37
52 Exophiala bergeri / Average PCB
0
1
2
3
4
day 0day 4day 9day 14day 18day 23day 28day 32day 37
75 Pseudallescheria boydii / Average PCB
-1
0
1
2
3
day0
day4
day9
day14
day18
day23
day28
day32
day37
58 Cladophialophora immunda / Plate 2 PCB
-1
0
1
2
3
day0
day4
day9
day14
day18
day23
day28
day32
day37
55 Cladophialophora immunda / Average PCB
-1
0
1
2
3
day0
day4
day9
day14
day18
day23
day28
day32
day37
56 Exophiala lecanii-corni / Average PCB
58
-0,5
0
0,5
1
1,5
day0
day4
day9
day14
day18
day23
day28
day32
day37
105 Pseudallescheria boydii / Average PCB
-0,5
0
0,5
1
1,5
2
day0
day4
day9
day14
day18
day23
day28
day32
day37
102 Pseudallescheria boydii / Average PCB
-2
0
2
4
day0
day4
day9
day14
day18
day23
day28
day32
day37
108 Exophiala xenobiotica / Average
PCB
-1
0
1
2
3
4
day0
day4
day9
day14
day18
day23
day28
day32
day37
91 Cladophialophora immunda / Average PCB
0
0,5
1
1,5
day0
day4
day9
day14
day18
day23
day28
day32
day37
103 Exophiala dermatitidis / Average
PCB
0
0,5
1
1,5
day0
day4
day9
day14
day18
day23
day28
day32
day37
101 Exophiala sideris / Average PCB
0
0,5
1
1,5
day0
day4
day9
day14
day18
day23
day28
day32
day37
93 Aureobasidium pullulans / Average PCB
0
0,5
1
1,5
2
day0
day4
day9
day14
day18
day23
day28
day32
day37
95 Exophiala sideris / Average PCB
59
0
0,5
1
day0
day4
day9
day14
day18
day23
day28
day32
day37
80 Pseudallescheria boydii / Average PCB
0
0,5
1
1,5
day0
day4
day9
day14
day18
day23
day28
day32
day37
113 Exophiala bergeri / Average PCB
0
1
2
3
day0
day4
day9
day14
day18
day23
day28
day32
day39
115 Exophiala spinifera / Average PCB
0
0,5
1
1,5
2
day0
day4
day9
day14
day18
day23
day28
day32
day37
106 Cladophialophora carrionii / Average PCB
0
1
2
3
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
117 Cladophialophora potulentorum / Average
PCB
-2
0
2
4
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
118 Cladophialophora chaetospira / Average
PCB
0
0,5
1
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
110 Pseudallescheria boydii / Average PCB
0
1
2
3
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
111 Exophiala mesophila / Average
PCB
60
0
0,5
1
1,5
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
109 Pseudallescheria boydii / Average PCB
0
0,5
1
1,5
2
2,5
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
84 Cladophialophora immunda / Average PCB
0
0,5
1
1,5
2
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
83 Exophiala jeanselmei / Average PCB
-2
0
2
4
day 0day 6 day13
day20
day27
day34
day42
97 Exophiala oligosperma / Average PCB
0
1
2
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
124 Cladophialophorum potulentora / Average
PCB
-1
0
1
2
3
4
day 0day 6 day13
day20
day27
day34
day42
123 Exophiala moniliae / Average PCB
0
0,5
1
1,5
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
107 Pseudallescheria boydii / Average PCB
0
0,5
1
1,5
2
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
92 Pseudallescheria boydii / Average PCB
61
-0,5
0
0,5
1
1,5
day 0day 6 day13
day20
day27
day34
day42
120 Pseudallescheria boydii / Average PCB
-0,5
0
0,5
1
day 0day 6 day13
day20
day27
day34
day42
119 Exophiala castellanii / Average PCB
0
0,5
1
day 0day 6 day13
day20
day27
day34
day42
121 Aureobasidium pullulans / Average PCB
-0,5
0
0,5
1
1,5
day 0day 6 day13
day20
day27
day34
day42
122 Pseudallescheria desertorum / Average
PCB
0
1
2
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
94 Pseudallescheria ellipsoidea / Average
PCB
-2
0
2
4
day 0day 6 day13
day20
day27
day34
day42
96 Exophiala heteromorpha / Average
PCB
-0,5
0
0,5
1
1,5
2
2,5
day 0day 6 day13
day20
day27
day34
day42
98 Cladophialophora carrionii / Average PCB
-0,2
0
0,2
0,4
0,6
0,8
1
day 0day 6 day13
day20
day27
day34
day42
90 Pseudallescheria boydii / Average PCB
62
-0,5
0
0,5
1
day 0day 6 day13
day20
day27
day34
day42
100 Aureobasidium pullulans var. pullulans /
Average PCB
0
1
2
3
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
125 Cladophialophora potulentorum / Average
PCB
-1
0
1
2
3
day 0day 6 day13
day20
day27
day34
day42
116 Cladophialophora chaetospira / Average
PCB
-0,5
0
0,5
1
1,5
2
2,5
day 0day 6 day13
day20
day27
day34
day42
85 Exophiala jeanselmei / Average PCB
0
0,5
1
1,5
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
88 Pseudallescheria boydii /Average PCB
-1
0
1
2
3
day 0day 6 day13
day20
day27
day34
day42
87 Exophiala oligosperma / Average
PCB
0
1
2
3
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
79 Exophiala xenobiotica / Plate 2
PCB
-0,5
0
0,5
1
1,5
day 0day 6 day13
day20
day27
day34
day42
82 Pseudallescheria boydii / Average PCB
63
-0,5
0
0,5
1
1,5
day 0day 6 day13
day20
day27
day34
day42
81 Pseudallescheria boydii / Average PCB
0
1
2
3
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
149 Exophiala xenobiotica / Average
PCB
0
1
2
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
154 Exophiala dermatitidis / Average
PCB
0
0,5
1
1,5
2
2,5
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
136 Exophiala sideris / Average PCB
0
0,2
0,4
0,6
0,8
1
day 0day 6 day13
day20
day27
day34
day42
139 Pseudallescheria boydii / Average PCB
0
0,5
1
1,5
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
141 Exophiala dermatitidis / Average
PCB
0
1
2
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
129 Exophiala alcalophila / Average
PCB
-0,5
0
0,5
1
day 0day 6 day13
day20
day27
day34
day42
159 Pseudallescheria boydii / Average PCB
64
0
1
2
3
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
161 Exophiala dermatitidis / Average
PCB
0
1
2
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
162 Exophiala heteromorpha / Average
PCB
0
2
4
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
163 Wangiella / Average PCB
-2
0
2
4
day 0day 6 day13
day20
day27
day34
day42
89 Exophiala dermatitidis / Average
PCB
-0,5
0
0,5
1
1,5
day 0day 6 day13
day20
day27
day34
day42
86 Pseudallescheria boydii / Average PCB
-2
0
2
4
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
148 Exophiala jeanselmei / Average
PCB
0
1
2
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
144 Exophiala mesophila / Average
PCB
0
1
2
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
130 Exophiala alcalophila / Average
PCB
65
0
0,5
1
1,5
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
132 Pseudallescheria boydii / Average PCB
-0,5
0
0,5
1
1,5
2
2,5
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
146 Fonsecaea / Average PCB
0
0,2
0,4
0,6
0,8
1
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
143 Pseudallescheria boydii / Average PCB
-0,5
0
0,5
1
1,5
2
2,5
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
150 Cladophialophora sp. / Average PCB
0
0,5
1
1,5
2
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
153 Exophiala spinifera / Average PCB
-0,5
0
0,5
1
1,5
day 0day 7 day14
day21
day28
day35
day42
151 Pseudallescheria boydii / Average PCB
0
0,2
0,4
0,6
0,8
1
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
128 Pseudallescheria boydii / Average PCB
-0,5
0
0,5
1
1,5
2
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
126 Exophiala lecanii-corni / Average PCB
66
0
0,5
1
1,5
2
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
160 Exophiala sideris / Average PCB
-0,5
0
0,5
1
1,5
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
158 Pseudallescheria boydii / Average PCB
-1
0
1
2
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
134 Exophiala bergeri / Average PCB
0
0,5
1
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
133 Pseudallescheria boydii / Average PCB
0
1
2
3
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
142 Exophiala bergeri / Average PCB
-0,5
0
0,5
1
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
157 Pseudallescheria boydii / Average PCB
0
2
4
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
156 Exophiala mesophila / Average
PCB
-0,5
0
0,5
1
1,5
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
155 Pseudallescheria boydii / Average PCB
67
-2
0
2
4
day 0day 7 day14
day21
day28
day35
day42
152 Exophiala jeanselmei / Average
PCB
-0,5
0
0,5
1
1,5
2
day 0day 2day 5day 7day 9day 12day 14day 16day 19day 21day 23day 26day 28day 30day 33day 35day 37day 41day 42day 44day 47
137 Pseudallescheria boydii / Average PCB
0
0,2
0,4
0,6
0,8
day 0 day 2 day 5 day 7 day 9 day12
day14
135 Pseudallescheria boydii / Average PCB
-0,5
0
0,5
1
day0
day2
day5
day7
day9
day12
day14
145 Pseudallescheria ellipsoidea / Average
PCB
0
1
2
3
4
day 0 day 2 day 5 day 7 day 9 day12
day14
18 Pseudallescheria boydii / Average PCB
-0,5
0
0,5
1
1,5
day 0day 2day 5day 7day 9 day12
day14
138 Cladophialophora mycetomatis / Average
PCB
-0,5
0
0,5
1
1,5
day0
day2
day5
day7
day9
day12
day14
140 Pseudallescheria boydii / Average PCB
-0,5
0
0,5
1
1,5
2
day 0day 2day 5day 7day 9 day12
day14
127 Fonsecaea / Plate 2 PCB
68
7.2.2 Toluene
-0,2
0
0,2
0,4
0,6
day0
day2
day5
day7
day9
day12
day14
131 Pseudallescheria boydii / Average PCB
0
0,5
1
1,5
day 0day 2day 5day 7day 9 day12
day14
147 Pseudallescheria boydii / Average PCB
-0,1
0
0,1
0,2
0,3
day
0
day
05
day
12
day
19
day
24
day
36
day
45
24 Cladophialophora minourae/ Average Tol
M
M
G
GVT -0,2
0
0,2
0,4
0,6
day
0
day
05
day
12
day
19
day
24
day
36
day
45
28 Aureobasidium pullulans var. pullulans/ Average Tol
M
M
G
GVT
-0,5
0
0,5
1
day0
day05
day12
day19
day24
day36
day45
19 Graphium eumorphum /
Average Tol
M
M
G -0,5
0
0,5
1
day
0
day
05
day
12
day
19
day
24
day
36
day
45
Ach
sen
tite
l
35 Pseudallescheria minutispora/ Average
Tol
M
M
G
GVT
-0,5
0
0,5
day
0
day
05
day
12
day
19
day
24
day
36
day
45
Ach
sen
tite
l
36 Cladophialophora australiensis / Plate 2
Tol
M
M
G
GVT -0,5
0
0,5
1
day0
day05
day12
day19
day24
day36
day45
33 Exophiala spinifera/ Average Tol
M
M
G
GVT
69
-0,5
0
0,5
1
day0
day05
day12
day19
day24
day36
day45
34 Exophiala heteromorpha / Average
Tol M
M
G
GVT
0
1
2
3
day
0
day
4
day
06
day
12
day
16
day
22
day
24
day
33
day
40
day
45
32 Rhinocladiella similis/ Average Tol
M
M
G
GVT
-0,5
0
0,5
1
day0
day05
day12
day19
day24
day36
day45
31 Exophiala spinifera/ Average Tol
M
M
G
GVT -0,2
0
0,2
0,4
0,6
day0
day05
day12
day19
day24
day36
day45
29 Cladophialophora subtilis/ Average Tol
M
M
G
GVT
-0,5
0
0,5
1
day0
day05
day12
day19
day24
day36
day45
30 Phialophora americana / AverageTol
M
M
G
00,5
1
day0
day05
day12
day19
day24
day36
day45
10 Selenophoma mahoniae / AverageTol
M
M
G
-0,5
0
0,5
1
day0
day05
day12
day19
day24
day36
day45
13 Scedosporium apiospermium/ Average
Tol
M
M
G
GVT
00,5
1
day0
day05
day12
day19
day24
day36
day45
12 Exophiala alcalophila / Average
Tol
M
M
G
70
0
1
2
day
0
day
4
day…
day…
day…
day…
day…
day…
day…
day…
11 Exophiala oligosperma /
Average Tol
M
M
G -0,5
0
0,5
1
1,5
day0
day05
day12
day19
day24
day36
day45
7 Exophiala castellanii / Average Tol
M
M
G
GVT
00,5
1
day0
day05
day12
day19
day24
day36
day45
9 Exophiala exophialae / Average
Tol
M
M
G -0,5
0
0,5
1
day0
day05
day12
day19
day24
day36
day45
2 Exophiala xenobiotica /
AverageTol
M
M
G
0
0,5
1
day0
day05
day12
day19
day24
day36
day45
4 Exophiala sideris / Average Tol
M
M
G
GVT
0
1
2
day
0
day
4
day…
day…
day…
day…
day…
day…
day…
day…
1 Exophiala dermatitidis /
AverageTol
M
M
G
0
0,5
1
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
27 Cladiophialophora boppii/ Average Tol
M
M
G
GVT -0,5
0
0,5
1
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
26 Pseudallescheria fusoidea/ Average Tol
M
71
-0,1
-0,05
0
0,05
0,1
0,15
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
22 Cladophialophora yegresii/ Average Tol
M
M
G
GVT-0,5
0
0,5
1
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
3 Pseudallescheria agusta/ Average Tol
M
0
0,5
1
1,5
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
74 Rhinocladiella similis/ Average Tol
M
M
G
GVT-0,2
0
0,2
0,4
0,6
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
14 Pseudallescheria ellipsoidea/ AverageTol
M
-0,2
-0,1
0
0,1
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
5 Cladophialophora carrionii/ Average Tol
M
00,10,20,30,4
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
16 Cladophialophora arxii / Average Tol
M
00,20,40,60,8
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
78 Aureobasidium pullans/ Average Tol
M
-0,2
-0,1
0
0,1
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
21 Exophiala lecanii-corni/ Average Tol
72
0
0,1
0,2
0,3
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
17 Cladophialophora immunda/ Average Tol
M
00,5
1
day
0
day
2 day…
day…
day…
day…
day…
day…
day…
70 Exophiala mesophila/ Average
Tol
M
M
G
0
0,2
0,4
0,6
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
46 Aureobasidium pullulans/ AverageTol
-0,050
0,050,1
0,150,2
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
37 Cladophialophora emmonsii/Average Tol
M
-0,4
-0,2
0
0,2
0,4
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
39 Cladophialophora mycetomatis/ Average Tol
M
-0,1
0
0,1
0,2
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
20 Exophiala bergeri/ Average Tol
M
0
1
2
day
0
day
2 day…
day…
day…
day…
day…
day…
day…
65 Exophiala heteromorpha/
AverageTol M
-0,5
0
0,5
1
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
66 Pseudallescheria boydii/ Plate 2 Tol
73
-0,5
0
0,5
day
0
day
2 day…
day…
day…
day…
day…
day…
day…
63 Exophiala dermatitidis/ Average
Tol
00,5
1
day
0
day
2 day…
day…
day…
day…
day…
day…
day…
51 Exophiala xenobiotica/ Average
Tol M
00,5
1
day
0
day
2 day…
day…
day…
day…
day…
day…
day…
53 Exophiala mesophila/ Average
Tol
0
1
2
day 0day 10day 17day 21day 26day 30day 35day 40
40 Exophiala alcalophila/ Average
Tol
0
0,5
1
day 7day 14day 19day 23day 28day 33day 37
68 Exophiala sideris /
Average Tol
-0,1
0
0,1
day 0day 10day 17day 21day 26day 30day 35day 40
49 Cladophialophora
carrionii/ Average Tol
0
0,5
1
day 0day 10day 17day 21day 26day 30day 35day 40
59 Cladophialophora yegresii/Average Tol
0
1
2
day 0day 10day 17day 21day 26day 30day 35day 40
41 Exophiala sideris/ Average Tol
74
-0,5
0
0,5
1
day 0day 10day 17day 21day 26day 30day 35day 40
42 Pseudallescheria ellipsoidea/ Average Tol
0
0,5
day 0day 10day 17day 21day 26day 30day 35day 40
47 Exophiala dermatitidis/ Average
Tol
-0,5
0
0,5
1
day 0day 10day 17day 21day 26day 30day 35day 40
45 Exophiala spinifera / Average Tol
-0,5
0
0,5
1
day 0day 10day 17day 21day 26day 30day 35day 40
8 Phialophora verrucosa/ Average
Tol
0
1
2
day 0day 10day 17day 21day 26day 30day 35day 40
43 Exophiala oligosperma/ Average
Tol
0
0,5
1
day 0day 10day 17day 21day 26day 30day 35day 40
73 Exophiala castellanii/
Average Tol
-0,5
0
0,5
1
day0
day10
day17
day21
day26
day30
day35
day40
Ach
sen
tite
l
72 Pseudallescheria angusta/ Average Tol
-0,5
0
0,5
day0
day10
day17
day21
day26
day30
day35
day40
60 Exophiala oligosperma/ Average
Tol
75
-0,5
0
0,5
1
day 0day 10day 17day 21day 26day 30day 35day 40
61 Pseudallescheria boydii/ Average Tol
-1
0
1
2
day 7day 14day 19day 23day 28day 33day 37
48 Phialophora verrucosa/ Average
Tol
-0,1
0
0,1
0,2
day 0day 10day 17day 21day 26day 30day 35day 40
67 Cladophialophora minourae/ Average Tol
-0,2
0
0,2
0,4
0,6
day 0day 10day 17day 21day 26day 30day 35day 40
23 Cladophialophora samoёnsis/ Average Tol
-0,05
0
0,05
0,1
day
0
day
10
day
17
day
21
day
26
day
30
day
35
day
40
6 Cladophialophora saturnica / Average Tol
-0,5
0
0,5
1
day 0day 10day 17day 21day 26day 30day 35day 40
59 Cladophialophora yegresii / Average Tol
-0,1
-0,05
0
0,05
0,1
day0
day10
day17
day21
day26
day30
day35
day40
62 Cladophialophora arxii / Average Tol
-0,2
0
0,2
0,4
0,6
day 0day 10day 17day 21day 26day 30day 35day 40
71 Pseudallescheria boydii/ / Average Tol
76
0
0,05
0,1
0,15
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
57 Cladophialophora boppii / Average Tol
0
0,1
0,2
0,3
0,4
0,5
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
104 Exophiala spinifera / Average Tol
-0,2
0
0,2
0,4
0,6
0,8
1
day 0day 7 day14
day21
day28
day35
day43
76 Cladophialophora immunda / Average Tol
-0,04
-0,02
0
0,02
0,04
day 0day 7 day14
day21
day28
day35
day43
52 Exophiala bergeri / Average Tol
-0,5
0
0,5
1
1,5
2
day 0day 7 day14
day21
day28
day35
day43
75 Pseudallescheria boydii / Average Tol
0
0,05
0,1
0,15
0,2
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
58 Cladophialophora immunda / Average Tol
-0,2
0
0,2
0,4
0,6
0,8
day 0day 7 day14
day21
day28
day35
day43
55 Cladophialophora immunda / Average Tol
0
0,5
1
1,5
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
56 Exophiala lecanii-corni / Average Tol
77
-0,2
0
0,2
0,4
0,6
0,8
day 0day 7 day14
day21
day28
day35
day43
105 Pseudallescheria boydii / Average Tol
-0,2
0
0,2
0,4
0,6
0,8
1
day 0day 7 day14
day21
day28
day35
day43
102 Pseudallescheria boydii / Average Tol
-0,5
0
0,5
1
day 0day 7 day14
day21
day28
day35
day43
108 Exophiala xenobiotica / Average
Tol
-0,05
0
0,05
0,1
0,15
day 0day 7 day14
day21
day28
day35
day43
91 Cladophialophora immunda / Average Tol
0
1
2
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
103 Exophiala dermatitidis / Average
Tol
0
0,5
1
1,5
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
101 Exophiala sideris / Average Tol
0
0,5
1
1,5
day 0day 7 day14
day21
day28
day35
day43
93 Aureobasidium pullulans / Average Tol
0
0,5
1
1,5
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
95 Exophiala sideris / Average Tol
78
0
0,2
0,4
0,6
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
80 Pseudallescheria boydii / Average Tol
0
1
2
3
day 0day 7 day14
day21
day28
day35
day43
113 Exophiala bergeri / Average Tol
0
0,5
1
1,5
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
115 Exophiala spinifera / Average Tol
0
0,2
0,4
0,6
0,8
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
106 Cladophialophora carrionii / Average Tol
0
0,5
1
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
117 Cladophialophora potulentorum / Average
Tol
-0,05
0
0,05
0,1
day 0day 6 day13
day20
day27
day34
day42
118 Cladophialophora chaetospira / Average
Tol
0
0,5
1
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
110 Pseudallescheria boydii / Average Tol
0
0,5
1
1,5
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
111 Exophiala mesophila / Average Tol
79
0
0,2
0,4
0,6
0,8
1
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
109 Pseudalleschria boydii / Average Tol
0
0,2
0,4
0,6
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
84 Cladophialophora immunda / Plate 2 Tol
0
0,5
1
1,5
2
2,5
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
83 Exophiala jeanselmei / Average Tol
-0,5
0
0,5
1
1,5
day 0day 6 day13
day20
day27
day34
day42
97 Exophiala oligosperma / Average
Tol
0
0,1
0,2
0,3
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
124 Cladophialophorum potulentora / Average
Tol
0
0,02
0,04
0,06
0,08
0,1
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
123 Exophiala moniliae / Average Tol
0
0,2
0,4
0,6
0,8
1
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
107 Pseudallescheria boydii / Average Tol
0
0,5
1
1,5
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
92 Pseudallescheria boydii / Average Tol
80
-0,2
0
0,2
0,4
0,6
0,8
day 0day 6 day13
day20
day27
day34
day42
120 Pseudallescheria boydii / Average Tol
-0,2
0
0,2
0,4
0,6
day 0day 6 day13
day20
day27
day34
day42
119 Exophiala castellanii / Average Tol
0
0,5
1
day 0day 6 day13
day20
day27
day34
day42
121 Aurepbasidium pullulans / Average Tol
0
0,5
1
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
122 Pseudallescheria desertorum / Average
Tol
0
0,2
0,4
0,6
0,8
1
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
94 Pseudallescheria ellipsoidea / Average Tol
-0,1
0
0,1
0,2
day 0day 6 day13
day20
day27
day34
day42
96 Exophiala heteromorpha / Average
Tol
-0,02
0
0,02
0,04
0,06
0,08
0,1
day0
day6
day13
day20
day27
day34
day42
98 Cladophialophora carrionii / Average Tol
-0,5
0
0,5
1
1,5
day 0day 6 day13
day20
day27
day34
day42
90 Pseudallescheria boydii / Average Tol
81
-0,05
0
0,05
0,1
0,15
day0
day6
day13
day20
day27
day34
day42
100 Aureobasidium pullulans var. pullulans/
Average Tol
0
0,5
1
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
125 Cladophialophora potulentronum /
Average Tol
0
1
2
day1
day6
day10
day15
day20
day24
day29
day34
day38
day43
116 Cladiophialophora
chaetospira / …
0
1
2
3
day0
day6
day13
day20
day27
day34
day42
85 Exophiala jeanselmei / Average Tol
0
0,5
1
day0
day6
day13
day20
day27
day34
day42
88 Pseudallescheria boydii / Plate 1 Tol
-1
0
1
2
day0
day6
day13
day20
day27
day34
day42
87 Exophiala oligosperma /
Average Tol
0
0,5
1
1,5
day0
day6
day13
day20
day27
day34
day42
79 Exophiala xenobiotica / Plate 2 Tol
-0,5
0
0,5
1
1,5
day0
day6
day13
day20
day27
day34
day42
82 Pseudallescheria boydii / Average Tol
82
-0,5
0
0,5
1
day0
day6
day13
day20
day27
day34
day42
81 Pseudallescheria boydii / Average Tol
0
1
2
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
149 Exophiala xenobiotica / Average
Tol
0
0,5
1
1,5
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
154 Exophiala dermatitidis / Average
Tol
0
0,2
0,4
0,6
0,8
day0
day6
day13
day20
day27
day34
day42
136 Exophiala sideris / Average Tol
0
0,5
1
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
139 Pseudallescheria boydii / Average Tol
0
1
2
day0
day6
day13
day20
day27
day34
day42
141 Exophiala dermatitidis / Average
Tol
012
day0
day6
day13
day20
day27
day34
day42
129 Exohiala alcalophila / Average
Tol
-1
0
1
2
day1
day6
day10
day15
day20
day24
day29
day34
day38
day43
159 Pseudallescheria boydii / Average Tol
83
0
2
4
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
161 Exophiala dermatitidis /
Average Tol
0
1
2
day0
day6
day13
day20
day27
day34
day42
162 Exophiala heteromorpha / Average
Tol
0
1
2
day0
day6
day13
day20
day27
day34
day42
163 Wangiella / Average Tol
0
1
2
day0
day6
day13
day20
day27
day34
day42
89 Exophiala dermatitidis / Average
Tol
-0,5
0
0,5
1
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
86 Pseudallescheria boydii / Average Tol
-1
0
1
2
day0
day7
day14
day21
day28
day35
day42
148 Exophiala jeanselmei / Average
Tol
0
1
2
day
2
day
7
day…
day…
day…
day…
day…
day…
day…
day…
144 Exophiala mesophila / Average
Tol
0
0,5
1
1,5
day0
day7
day14
day21
day28
day35
day42
130 Exophiala alcalophila / Average Tol
84
0
0,5
1
day0
day7
day14
day21
day28
day35
day42
132 Pseudallescheria boydii /Average Tol
-0,5
0
0,5
1
1,5
day0
day7
day14
day21
day28
day35
day42
146 Fonsecaea / Average Tol
0
0,2
0,4
0,6
0,8
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
143 Pseudallescheria boydii / Average Tol
-0,5
0
0,5
1
1,5
day0
day7
day14
day21
day28
day35
day42
150 Cladophialophora / Average Tol
012
day0
day7
day14
day21
day28
day35
day42
153 Exophiala spinifera / Average
Tol
-0,5
0
0,5
1
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
151 Pseudallescheria boydii / Average Tol
0
0,5
1
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
128 Pseudallescheria boydii / Average Tol
-0,5
0
0,5
1
day0
day7
day14
day21
day28
day35
day42
126 Exophiala lecanii-corni / Average Tol
85
0
0,5
1
1,5
day0
day7
day14
day21
day28
day35
day42
160 Exophiala sideris / Average Tol
0
0,2
0,4
0,6
day0
day7
day14
day21
day28
day35
day42
158 Pseudallescheria boydii / Average Tol
-0,1
0
0,1
0,2
day0
day7
day14
day21
day28
day35
day42
134 Exophiala bergeri / Average Tol
0
0,5
1
day2
day7
day12
day16
day21
day26
day30
day35
day41
day44
133 Pseudallescheria boydii / Average Tol
0
1
2
day0
day7
day14
day21
day28
day35
day42
142 Exophiala bergeri / Average Tol
0
0,5
1
day0
day7
day14
day21
day28
day35
day42
157 Pseudallescheria boydii / Average Tol
0
1
2
day0
day7
day14
day21
day28
day35
day42
156 Exophiala mesophila / Average Tol
-0,2
0
0,2
0,4
0,6
0,8
day0
day7
day14
day21
day28
day35
day42
155 Pseudallescheria boydii / Average Tol
86
-0,05
0
0,05
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
152 Exophiala jeanselmei / Average
Tol
-0,05
0
0,05
0,1
1 2 3 4 5 6 7 8 9101112131415161718192021
137 Pseudallescheria boydii / Average Tol
0
0,5
1
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
135 Pseudallescheria boydii/ Average Tol
0
0,5
1
day2
day7
day12
day16
day21
day26
day30
day35
day41
145 Pseudallescheria ellipsoidea / Average
Tol
0
0,1
0,2
0,3
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
18 Pseudallescheria boydii/ Average Tol
-0,05
0
0,05
0,1
day0
day7
day14
day21
day28
day35
day42
138 Cladophialophora mycetomatis /Average
Tol
0
0,5
1
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
140 Pseudallescheria boydii / Average Tol
-0,5
0
0,5
1
1,5
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
127 Fonsecaea / Average Tol
87
7.2.3 Hexadecane
-0,5
0
0,5
1
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
131 Pseudallescheria boydii / Average Tol
0
0,5
1
1,5
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
147 Pseudallescheria boydii / Average Tol
0
2
4
day
0
day
04
day
06
day
15
day
19
day
23
day
26
day
36
day
43
24 Cladophialophora minourae/
Average Hex
M
M
G
GVT-0,5
00,5
11,5
day
0
day
04
day
06
day
15
day
19
day
23
day
26
day
36
day
43
28 Aureobasidium pullulans var. pullulans/ Average Hex
M
M
G
GVT
0
1
2
day
0
day
04
day
06
day
15
day
19
day
23
day
26
day
36
day
43
19 Gaphium eumorphum/ Average
Hex
M
M
G
GVT-0,5
0
0,5
1
day
0
day
04
day
06
day
15
day
19
day
23
day
26
day
36
day
43
35 Exophiala jeanselmei/ Average
Hex
M
M
G
GVT
0
2
4
day
0
day
04
day
06
day
15
day
19
day
23
day
26
day
36
day
43
36 Cladophialophora australiensis / Average
Hex
M
M
G
GVT
0
1
2
day
0
day…
day…
day…
day…
day…
day…
day…
day…
33 Exophiala spinifera/ Average
Hex
M
M
G
88
0
2
4
day
0
day
04
day
06
day
15
day
19
day
23
day
26
day
36
day
43
34 Exophiala heteromorpha/ Average
Hex
M
M
G
GVT
0
1
2
3
day
0
day
04
day
06
day
15
day
19
day
23
day
26
day
36
day
43
32 Rhinocladiella similis / Average Hex
M
M
G
GVT
0
1
2
3
day
0
day
04
day
06
day
15
day
19
day
23
day
26
day
36
day
43
31 Exophiala spinifera / Average Hex
M
M
G
GVT
0
0,5
1
1,5
2
day
0
day
04
day
06
day
15
day
19
day
23
day
26
day
36
day
43
29 Cladophialophora subtilis / Average Hex
M
M
G
GVT
0
0,5
1
1,5
2
day
0
day
04
day
06
day
15
day
19
day
23
day
26
day
36
day
43
30 Phialophora americana/ Average Hex
M
M
G
GVT
0
0,5
1
1,5
day
0
day
04
day
06
day
15
day
19
day
23
day
26
day
36
day
43
10 Selenohoma mahoniae/ average Hex
M
M
G
GVT
0
0,5
1
day
0
day
04
day
06
day
15
day
19
day
23
day
26
day
36
day
43
13 Scedosporium apiosermum/ Aveage hex
M
M
G
GVT
0
0,5
1
1,5
2
day
0
day
04
day
06
day
15
day
19
day
23
day
26
day
36
day
43
12 Exophiala alcalohila/ Average Hex
M
M
G
GVT
89
0
1
2
day
0
day
04
day
06
day
15
day
19
day
23
day
26
day
36
day
43
11 Exophiala oligosperma / Average
Hex
M
M
G
GVT
0
0,5
1
1,5
2
day
0
day
04
day
06
day
15
day
19
day
23
day
26
day
36
day
43
7 Exophiala castellanii/ Average Hex
M
M
G
GVT
0
1
2
3
day
0
day
04
day
06
day
15
day
19
day
23
day
26
day
36
day
43
9 Exophiala exophialae/ Average Hex
M
M
G
GVT
0
1
2
3
day
0
day
04
day
06
day
15
day
19
day
23
day
26
day
36
day
43
2 Exophiala xenobiotica / Average Hex
M
M
G
GVT
0
0,5
1
1,5
2
day
0
day
04
day
06
day
15
day
19
day
23
day
26
day
36
day
43
4 Exophiala sideris/ Average Hex
M
M
G
GVT
0
0,5
1
1,5
2
day
0
day
04
day
06
day
15
day
19
day
23
day
26
day
36
day
43
1 Exophiala dermatitidis/ Average Hex
M
M
G
GVT
0
1
2
3
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
27 Cladiophialophora boppii/ Average Hex
M
M
G
GVT
0
0,5
1
1,5
2
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
22 Cladophialophora yegressii/ Average Hex
M
M
G
GVT
90
-0,5
0
0,5
1
1,5
2
day0
day2
day10
day14
day18
day21
day31
day37
day41
3 Pseudallescheria agusta/Average Hex
M
M
G
GVT
0
1
2
3
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
74 Rhinocladiella similis/ Average Hex
M
M
G
GVT
-1
0
1
2
3
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
14 Pseudallescheria ellipsoidea/ Plate 2 Hex
M
M
G
GVT -0,5
0
0,5
1
1,5
2
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
5 Cladophialophora carrionii/ Average Hex
M
M
G
GVT
0
1
2
3
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
16 Cladophialophora arxii/ Average Hex
M
M
G
GVT
0
0,2
0,4
0,6
0,8
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
78 Aureobasidium pullans/ Average Hex
M
M
G
GVT
-1
0
1
2
3
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
21 Exophiala lecanii-corni/ Average Hex
M
M
G
GVT0
1
2
3
day0
day7
day14
day19
day31
day39
Ach
sen
tite
l
70 Exophiala mesophila / Average Hex
M
M
G
GVT
91
0
0,5
1
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
46 Aureobasidium / Average Hex
M
M
G
GVT -0,5
0
0,5
1
1,5
2
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
37 Cladophilalophora emmonsii/ Average Hex
M
M
G
GVT
0
1
2
3
day 1day 7day 11day 17day 19day 28day 34day 39
39 Cladophialophora mycetomatis / Average Hex
M
M
G
GVT
0
1
2
3
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
20 Exophiala bergeri/ Average Hex
M
M
G
GVT
0
1
2
3
4
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
65 Exophiala heteromorpha / Average Hex
M
M
G
GVT-0,5
0
0,5
1
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
66 Pseudallescheria boydii/ Average Hex
00,5
11,5
2
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
63 Exohiala dermatitidis/ Average Hex
M
M
G
GVT
0
1
2
3
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
51 Exohiala xenobiotica/ Average Hex
M
M
G
GVT
92
0
1
2
3
day
0
day
2
day
10
day
14
day
18
day
21
day
31
day
37
day
41
53 Exophiala mesophila/ Average Hex
M
M
G
GVT0
0,5
1
1,5
2
day0
day10
day17
day21
day26
day30
day35
day40
40 Exophiala alcalophila/ Average Hex
0
1
2
3
day0
day10
day17
day21
day26
day30
day35
day40
68 Exophiala sideris /
Average Hex
-1
0
1
2
3
day0
day10
day17
day21
day26
day30
day35
day40
49 Cladophialophora carrionii/ Average Hex
M
0
1
2
3
day 0day 10day 17day 21day 26day 30day 35day 40
59 Cladophialophora yegresii/Average Hex
0
1
2
3
day0
day10
day17
day21
day26
day30
day35
day40
41 Exophiala sideris/ Average Hex
M
0
0,5
1
1,5
day 0day 10day 17day 21day 26day 30day 35day 40
42 Pseudallescheria ellipsoidea/ Plate 2 Hex
M
M
G
GVT
0
0,5
1
1,5
2
day0
day10
day17
day21
day26
day30
day35
day40
47 Exophiala dermatitidis/
Average Hex
M
M
G
GVT
93
0
0,5
1
1,5
2
day0
day10
day17
day21
day26
day30
day35
day40
45 Exophiala spinifera / Average Hex
M
M
G
GVT -1
0
1
2
3
4
day0
day10
day17
day21
day26
day30
day35
day40
8 Phialophora verrucosa/ / Average Hex
M
0
1
2
3
day0
day10
day17
day21
day26
day30
day35
day40
43 Exophiala oligosperma/ Average Hex
M
0
0,5
1
1,5
day0
day10
day17
day21
day26
day30
day35
day40
73 Exophiala castellanii/ Average Hex
M
0
0,5
1
1,5
day0
day10
day17
day21
day26
day30
day35
day40
72 Pseudallescheria angusta/ Average Hex
M
0
1
2
3
day0
day10
day17
day21
day26
day30
day35
day40
60 Exophiala oligosperma/ Average Hex
M
-0,5
0
0,5
1
1,5
day0
day10
day17
day21
day26
day30
day35
day40
61 Pseudallescheria boydii/ Average Hex
M
-1
0
1
2
3
4
day7
day14
day19
day23
day28
day33
day37
48 Phialophora verrucosa/ Average Hex
M
94
-1
0
1
2
3
day0
day10
day17
day21
day26
day30
day35
day40
67 Cladophialophora minourae/ Average Hex
M
-1
0
1
2
3
day0
day10
day17
day21
day26
day30
day35
day40
23 Cladophialophora samoёnsis/ Average Hex
-1
0
1
2
3
day0
day10
day17
day21
day26
day30
day35
day40
6 Cladophialophora saturnica / Average Hex
-0,5
0
0,5
1
1,5
day0
day10
day17
day21
day26
day30
day35
day40
59 Cladophialophora yegresii / Average Hex
M
M
G
GVT
-1
0
1
2
3
4
day0
day10
day17
day21
day26
day30
day35
day40
62 Cladophialophora arxii / Average Hex
M
0
0,5
1
1,5
day0
day10
day17
day21
day26
day30
day35
day40
71 Pseudallescheria boydii/Average Hex
M
M
G
GVT
0
0,5
1
1,5
2
2,5
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
57 Cladophialophora boppii / Average Hex
0
0,2
0,4
0,6
day 0day 7 day14
day21
day28
day35
day43
104 Exophiala spinifera / Average Hex
95
-1
0
1
2
3
day 0day 7 day14
day21
day28
day35
day43
76 Cladophialophora immunda / Average Hex
-0,5
0
0,5
1
1,5
2
day 0day 7 day14
day21
day28
day35
day43
52 Exophiala bergeri / Average Hex
-1
0
1
2
3
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
75 Pseudallescheria boydii / Average Hex
0
0,5
1
1,5
2
2,5
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
58 Cladophialophora immunda / Average Hex
-1
0
1
2
3
4
day 0day 7 day14
day21
day28
day35
day43
55 Cladophialophora immunda / Average Hex
0
1
2
3
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
56 Exophiala lecanii-corni / Average Hex
-0,5
0
0,5
1
1,5
day 0day 7 day14
day21
day28
day35
day43
105 Pseudallescheria boydii / Average Hex
-0,5
0
0,5
1
1,5
day 0day 7 day14
day21
day28
day35
day43
102 Pseudallescheria boydii / Average Hex
96
-1
0
1
2
3
day 0day 7 day14
day21
day28
day35
day43
108 Exophiala xenobiotica / Average
Hex
-1
0
1
2
3
4
day0
day7
day14
day21
day28
day35
day43
91 Cladophialophora immunda / Average Hex
0
1
2
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
103 Exophiala dermatitidis / Average
Hex
0
1
2
3
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
101 Exophiala sideris / Average Hex
0
1
2
day 0day 7 day14
day21
day28
day35
day43
93 Aureobasidium pullulans / Average Hex
0
0,5
1
1,5
2
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
95 Exophiala sideris / Average Hex
0
0,5
1
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
80 Pseudallescheria boydii / Average Hex
0
1
2
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
113 Exophiala bergeri / Average Hex
97
0
0,5
1
1,5
2
2,5
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
115 Exophiala spinifera / Average Hex
0
0,5
1
1,5
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
114 Pseudallescheria boydii / Average Hex
0
0,5
1
1,5
2
2,5
day0
day4
day9
day14
day18
day23
day28
day32
day37
day43
day46
106 Cladophialophora carrionii / Average Hex
0
1
2
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
117 Cladophialophora potulentorum / Average
Hex
0
1
2
3
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
118 Cladophialophora chaetospira / Average
Hex
0
0,5
1
1,5
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
110 Pseudallescheria boydii / Average Hex
0
1
2
3
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
111 Exophiala mesophila / Average
Hex
0
0,5
1
1,5
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
109 Pseudallescheria boydii / Average Hex
98
0
1
2
3
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
84 Cladophialophora immunda / Average Hex
0
0,5
1
1,5
2
2,5
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
83 Exophiala jeanselmei / Average Hex
-2
0
2
4
day 0day 6 day13
day20
day27
day34
day42
97 Exophiala oligosperma / Average
Hex
0
1
2
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
124 Cladophialophora potulentorum/ Average
Hex
0
1
2
3
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
123 Exophiala moniliae / Average Hex
0
0,5
1
1,5
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
107 Pseudallescheria boydii / Average Hex
0
0,5
1
1,5
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
92 Pseudallescheria boydii / Average Hex
-0,5
0
0,5
1
1,5
day 0day 6 day13
day20
day27
day34
day42
120 Pseudallescheria boydii / Average Hex
99
-0,5
0
0,5
1
1,5
day 0day 6 day13
day20
day27
day34
day42
119 Exophiala castellanii / Average Hex
0
0,5
1
day 0day 6 day13
day20
day27
day34
day42
121 Aurepbasidium pullulans / Average Hex
0
0,5
1
1,5
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
122 Pseudallescheria desertorum / Average
Hex
0
1
2
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
94 Pseudallescheria ellipsoidea / Average
Hex
-2
0
2
4
day 0day 6 day13
day20
day27
day34
day42
96 Exophiala heteromorpha / Average
Hex
0
1
2
3
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
98 Cladophialophora carrionii / Average Hex
-0,5
0
0,5
1
1,5
day 0day 6 day13
day20
day27
day34
day42
90 Pseudallescheria boydii / Average Hex
-0,5
0
0,5
1
day 0day 6 day13
day20
day27
day34
day42
100 Aureobasidium pullulans var. Pullulans /
Average Hex
100
0
2
4
day 0day 1day 3day 6day 8day 10day 13day 15day 17day 20day 22day 24day 27day 29day 31day 34day 36day 38day 42day 43day 45
125 Cladophialophora potulentorum /
Average Hex
0
1
2
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
116 Cladophialophora chaetospira / Average
Hex
0
0,5
1
1,5
2
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
85 Exophiala jeanselmei / Average Hex
0
0,5
1
1,5
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
88 Pseudallescheria boydii / Average Hex
-2
0
2
4
day 0day 6 day13
day20
day27
day34
day42
87 Exophiala oligosperma / Average
Hex
0
1
2
3
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
79 Exophiala xenobiotica / Plate 2 Hex
-0,5
0
0,5
1
1,5
day 0day 6 day13
day20
day27
day34
day42
82 Pseudallescheria boydii / Average Hex
-0,5
0
0,5
1
1,5
day 0day 6 day13
day20
day27
day34
day42
81 Pseudallescheria boydii / Average Hex
101
0
1
2
3
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
149 Exophiala xenobiotica / Average
Hex
0
1
2
3
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
154 Exophiala dermatitidis / Average
Hex
0
0,5
1
1,5
2
2,5
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
136 Exophiala sideris / Average Hex
0
0,5
1
1,5
day 0day 6 day13
day20
day27
day34
day42
139 Pseudallescheria boydii / Average Hex
0
0,5
1
1,5
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
141 Exophiala dermatitidis / Average
Hex
0
1
2
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
129 Exophiala alcalophila / Average
Hex
-0,5
0
0,5
1
day 0day 6 day13
day20
day27
day34
day42
159 Pseudallescheria boydii / Average Hex
0
1
2
3
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
161 Exophiala dermatitidis / Average
Hex
102
0
1
2
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
162 Exophiala heteromorpha / Average
Hex
0
2
4
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
163 Wangiella / Average Hex
0
1
2
3
day0
day3
day8
day13
day17
day22
day27
day31
day36
day42
day45
89 Exophiala dermatitidis / Average
Hex
-0,5
0
0,5
1
1,5
day 0day 6 day13
day20
day27
day34
day42
86 Pseudallescheria boydii / Average Hex
-2
0
2
4
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
148 Exophiala jeanselmei / Average
Hex
0
1
2
3
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
144 Exophiala mesophila / Average
Hex
0
1
2
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
130 Exophiala alcalophila / Average
Hex
0
0,5
1
1,5
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
132 Pseudallescheria boydii /Average Hex
103
0
0,5
1
1,5
2
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
146 Fonsecaea / Average Hex
0
0,5
1
1,5
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
143 Pseudallescheria boydii / Average Hex
-0,5
0
0,5
1
1,5
2
2,5
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
150 Cladophialophora sp. / Average Hex
0
0,5
1
1,5
2
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
153 Exophiala spinifera / Average Hex
-0,5
0
0,5
1
1,5
day 0day 7 day14
day21
day28
day35
day42
151 Pseudallescheria boydii / Average Hex
0
0,5
1
1,5
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
128 Pseudallescheria boydii / Average Hex
-0,5
0
0,5
1
1,5
2
2,5
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
126 Exophiala lecanii-corni / Average Hex
0
0,5
1
1,5
2
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
160 Exophiala sideris / Average Hex
104
0
0,5
1
1,5
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
158 Pseudallescheria boydii / Average Hex
-1
0
1
2
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
134 Exophiala bergeri / Average Hex
0
0,5
1
1,5
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
133 Pseudallescheria boydii / Average Hex
0
0,5
1
1,5
2
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
142 Exophiala bergeri / Average Hex
0
0,5
1
1,5
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
157 Pseudallescheria boydii / Average Hex
0
2
4
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
156 Exophiala mesophila / Average
Hex
0
0,5
1
1,5
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
155 Pseudallescheria boydii / Average Hex
-2
0
2
4
day 0day 7 day14
day21
day28
day35
day42
152 Exophiala jeanselmei / Average
Hex
105
0
1
2
day 0day 2day 5day 7day 9day 12day 14day 16day 19day 21day 23day 26day 28day 30day 33day 35day 37day 41day 42day 44day 47
137 Pseudallescheria boydii / Average Hex
-0,2
0
0,2
0,4
0,6
0,8
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
135 Pseudallescheria boydii / Average Hex
0
0,5
1
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
145 Pseudallescheria ellipsoidea / Average
Hex
0
1
2
3
4
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
18 Pseudallescheria boydii / Average Hex
0
1
2
3
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
138 Cladophialophora mycetomatis /Average
Hex
0
0,5
1
1,5
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
140 Pseudallescheria boydii / Average Hex
-0,5
0
0,5
1
1,5
2
2,5
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
127 Fonsecaea / Average Hex
0
0,2
0,4
0,6
0,8
1
day 0day 7 day14
day21
day28
day35
day42
131 Pseudallescheria boydii / Average Hex
106
7.3 Diagrams GC screening
Carbon equivalence [mol] of the Toluene and CO2 was plotted against runtime [days]. different curves:
G+T: toluene of the positive control
G+T CO2: CO2 of the positive control
T: toluene of bottles with hexadecane and toluene as sole carbon source
T CO2: CO2 of bottles with hexadecane and toluene as sole carbon source
0
0,5
1
1,5
day0
day5
day9
day14
day19
day23
day28
day33
day37
day42
day47
147 Pseudallescheria boydii / Average Hex
-5E-09
0
5E-09
1E-08
0,00 10,00 20,00
C e
q
Runtime
26 Pseudallescheria fusoidea
G+T
T
G+T CO2
T CO2
0
5E-09
0,00 10,00 20,00
C e
q
Runtime
14 Pseudallescheria ellipsoidea
G+T
T
G+T CO2
T CO2
0
2E-09
4E-09
6E-09
8E-09
0,00 10,00 20,00
C e
q
Runtime
31 Exophiala spinifera
G+T
T
G+T CO2
T CO2
0
2E-09
4E-09
0,00 10,00 20,00
C e
q
Runtime
19 Graphium emorphum
G+T
T
G+T
T
107
0
5E-09
0,00 10,00 20,00
C e
q
Runtime
71 Pseudallescheria boydii
G+T
T
G+T CO2
T CO2
0
5E-09
1E-08
0,00 10,00 20,00
C e
q
Runtime
32 Rhinocladiella similis
G+T
T
G+T CO2
T CO2
0
5E-09
1E-08
0,00 10,00 20,00
C e
q
Runtime
27 Cladiophialophora boppii
G+T
T
G+T CO2
T CO2
0
5E-09
0,00 10,00 20,00
C e
q
Runtime
110 Pseudallescheria boydii
T
T CO2
0
5E-09
0,00 5,00 10,0015,00
C e
q
Runtime
158 Pseudallescheria boydii
T
T CO20
5E-09
0,00 2,00 4,00 6,00
C e
q
Runtime
94 Pseudallescheria ellipsoidea
T
T CO2
0
5E-09
0,00 2,00 4,00 6,00
C e
q
Runtime
139 Pseudallescheria boydii
T
T CO20
2E-09
4E-09
0,00 5,00 10,0015,00
C e
q
Runtime
75 Pseudallescheria boydii
T
T CO2
108
0
5E-09
0,00 2,00 4,00 6,00
C e
q
Runtime
159 Pseudallescheria boydii
T
T CO20
5E-09
0,00 2,00 4,00 6,00
C e
q
Runtime
143 Pseudallescheria boydii
T
T CO2
0
5E-09
0,00 5,00 10,0015,00
C e
q
Runtime
97 Exophiala oligosperma
T
T CO20
5E-09
0,00 5,00 10,0015,00
C e
q
Runtime
90 Pseudallescheria boydii
T
T CO2
0
5E-09
0,00 5,00 10,0015,00
C e
q
Runtime
105 Pseudallescheria boydii
T
T CO20
5E-09
0,00 5,00 10,0015,00
C e
q
Runtime
107 Pseudallescheria boydii
T
T CO2
0
5E-09
0,00 2,00 4,00 6,00
C e
q
Runtime
86 Pseudallescheria boydii
T
T CO20
5E-09
0,00 2,00 4,00 6,00
C e
q
Runtime
92 Pseudallescheria boydii
T
T CO2
109
0
5E-09
0,00 2,00 4,00 6,00
C e
q
Runtime
81 Pseudallescheria boydii
T
T CO2
110
STATUTORY DECLARATION I declare that I have authored this thesis independently, that I have not used other than the declared sources/resources, and that I have explicitly marked all material which has been quoted either literally or by content from the used sources.
Vienna, April 2014
Caroline Poyntner
EIDESSTATTLICHE ERKLÄRUNG Ich erkläre an Eides statt, dass ich die vorliegende Arbeit selbstständig verfasst, andere als die angegebenen Quellen/Hilfsmittel nicht benutzt, und die den benutzten Quellen wörtlich und inhaltlich entnommene Stellen als solche kenntlich gemacht habe.
Wien, April 2014 Caroline Poyntner
111