Growth and biodegradation by Sporidiobolales yeasts in ...hig.diva-portal.org/smash/get/diva2:1119832/FULLTEXT01.pdf · Vanillin (4-hydroxy-3-methoxybenzaldehyde), is the major component

AKADEMIN FÖR HÄLSA OCH ARBETSLIV Avdelningen för arbets- och folkhälsovetenskap

Growth and biodegradation by Sporidiobolales yeasts in vanillin-supplemented medium

Natalia González Gaarslev

2017

Examensarbete, G2E, 15 hp Biologi

Examensarbete i Biologi

Handledare: Sandra A. I. Wright

Examinator: Mikael Lönn

Summary

Studies of biodegradation in lignins by basidiomycetes yeasts show the conversion of lignin in various

degradation products among which vanillin, a valuable substance, suggested to be a strong inhibitor of

both fermentation and growth of yeasts, stands. Sporidiobolales yeasts used in these experiments were

aimed to be identified by their highly conserved ITS region as well as studied in vanillin-

supplemented medium through, vanillin-supplemented plates, TLC and Neubauer’s chamber to find

out which, among the several isolates tested, were the most resistant ones, understand how they take

up vanillin and how their growth is affected by the presence of the phenolic compound. Two strains

were identified as Rhodotorula babjevae. One of them, L4, together with LS22, Rhodosporidium

kratochvilovae, could withstand and biodegrade high concentrations of vanillin, producing

biodegradation products with Rf values similar to the ones know for vanillic acid and vanillyl alcohol.

Better growth in medium supplemented with small doses of vanillin was found, as well as disparity

among the same species and their metabolic features, therefore, herbicides resistance was suggested as

a reason for strains divergence. Further morphological-species comparison could also describe if there

exist a relation between them.

Resumen Estudios sobre la biodegradación de ligninas por levaduras basidiomicetes muestran la conversión de

lignina en distintos productos de degradación, entre los cuales se encuentra la vainillina, un fuerte

inhibidor de la fermentación y el crecimiento de levaduras. Las levaduras Sporidiobolales utilizadas en

estos experimentos han intentado ser identificadas a través de la región ETI, muy conservada, además

de estudiadas en medios suplementados con vainillina mediante placas suplementadas con vainillina,

CCF y cámara de Neubauer para averiguar cuáles son las cepas más resistentes, entender cómo

metabolizan la vainillina y cómo su crecimiento se ve afectado por la presencia de dicho compuesto.

Dos cepas fueron identificadas como Rhodotorula babjevae. Una de ellas, L4, junto con con la cepa

LS22, Rhodosporidium kratochvilovae, pudieron soportar y biodegradar elevadas concentraciones de

vainillina, originando productos de biodegradación con valores de Rf similares a los del ácido vanílico

y alcohol vanílico previamente conocidos. Se encontró un crecimiento mejor en medios

suplementados con pequeñas dosis de vainillina además de una disparidad entre mismas especies y sus

características metabólicas, así, herbicidas han sido sugeridos como una posible causa en dicha

divergencia. Una futura comparación morfología-especie podrá describir si existe relación entre

ambos.

Key words/Palabras clave: Sporobolomyces/Sporobolomyces, Vanillin/Vainillina,

Internal Transcribed Spacer (ITS)/Espaciador Transcribible Interno (ETI), Thin layer

chromatography (TLC)/Cromatografia en capa fina (CCF), Neubauer’s chamber/Cámara de

Neubauer.

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Contents

Summary ........................................................................................................................... 2

1. Introduction .................................................................................................................. 2

1.1. Background ............................................................................................................ 2 1.1.1. Lignin, an abundant organic polymer in Earth ............................................... 2 1.1.2. Vanillin as a biodegradation product from lignin ........................................... 2 1.1.3. Biodegradation of vanillin by microorganisms .............................................. 3

1.1.4. Sporidiobolales yeasts .................................................................................... 3 1.1.5. Ecological uses of Sporidiobolales yeasts ...................................................... 4 1.1.6. Methods of identification of fungi .................................................................. 4

1.2. Aims, objectives .................................................................................................... 4

2. Materials and methods .................................................................................................. 5

2.1. Culture media ........................................................................................................ 5 2.1.1. Yeast extract-peptone-dextrose (YEPD) ........................................................ 5 2.1.2. Lilly-Barnet, LiBa (Lilly VG, 1951) .............................................................. 5

2.1.3. LiBa-Vanillin (VA) stock solution 0.1 M ...................................................... 5 2.2. Yeast collection ..................................................................................................... 5

2.3. Identification experiments ..................................................................................... 7 2.3.1. Genomic DNA extraction ............................................................................... 8 2.3.2. Polymerase chain reaction (PCR), amplification of ITS regions ................. 10

2.3.3. PCR product classification ........................................................................... 11 2.3.4. Purification of the PCR product gel bands ................................................... 11

2.3.5. Verification of an adequate amount of DNA in the samples ........................ 12 2.3.6. Preparation of sequencing samples .............................................................. 12

2.3.7. Sequencing ................................................................................................... 12 2.3.8. Identification ................................................................................................. 13

2.4. Vanillin resistance experiments ........................................................................... 13 2.4.1. Selection test of the most resistant isolates .................................................. 13

2.4.2. Vanillin resistance and degradation in liquid cultures ................................. 14 2.5. Yeast growth in vanillin supplemented medium ................................................. 15

2.5.1. Cell count in Neubauer’s chamber ............................................................... 16

3. Results ........................................................................................................................ 16

3.1. Identification experiments ................................................................................... 16 3.1.1. DNA extraction ............................................................................................ 16

3.1.2. PCR product ................................................................................................. 16 3.1.3. Gel elution and recovery of purified ITS fragments .................................... 18 3.1.4. Verification of required minimum concentration of template DNA ............ 18 3.1.5. Identification results ..................................................................................... 18

3.2. Vanillin experiment: selection of most vanillin resistant isolates ....................... 19 3.3. Vanillin biodegradation in liquid cultures, TLC results ...................................... 20 3.4. Vanillin resistance: Neubauer’s chamber results................................................. 22

4. Conclusions and discussion ........................................................................................ 23

5. References .................................................................................................................. 24

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1. Introduction

1.1. Background

1.1.1. Lignin, an abundant organic polymer in Earth

Lignin is a high molecular weight three-dimensional macromolecule (Hedges & Mann,

1979), a complex heteropolymer (Campbell & Sederoff, 1996) synthesized in vascular

plants through the dehydrogenative polymerization of three cinnamyl alcohols: 4-

hydroxycinnamyl alcohol, coniferyl alcohol and synapyl alcohol (Boudet, et al., 1995) .

Lignin is the main structural component in plants secondary wall (Guo, et al., 2001)

being deposited in wall cells at the last stages of their differentiation (Boudet et al.,

1995). Besides mechanical support, lignin plays a significant role in both water

transport and defence in vascular plants (Campbell & Sederoff, 1996).

Vascular plants, mainly constituted of lignin, cellulose and hemicellulose (Demain, et

al., 2005) when dead, remain in the environment as lignocellulosic biomass, considered

a natural renewable resource due to its abundance (Demain et al., 2005). Lignin,

therefore, is one of the most abundant polymers in nature (Fengel & Wegener, 1984)

and due to its non-water solubility and optical inactivity, the study of its degradation is

very complicated (Fengel & Wegener, 1984).

Lignin is known to be very resistant to microbial degradation (Higuchi, 1990). Studies

of biodegradation in decayed wood lignins by whiterot basidiomycetes groups (Hata,

1966; Kirk & Chang, 1974) show the conversion of lignin in several degradation

products among which vanillin, considered a valuable substance, stands (Henderson,

1961; Higuchi, 1981; Zimmermann, 1990).

1.1.2. Vanillin as a biodegradation product from lignin

Vanillin (4-hydroxy-3-methoxybenzaldehyde), is the major component of natural

vanilla (Walton, et al., 2003), widely used for flavouring food, aromatise perfumes and

it can also intervene in pharmaceuticals synthesis (Voitl & Rohr, 2009). Considered a

valuable substance, vanillin can be synthetically produced from lignin, obtained from

the black liquor produced in Kraft processes in pulp and paper industry (da Silva et al.,

2009) and its scale production began in 1936 in the United States (Hocking, 1997).

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1.1.1.1 Vanillin toxicity

Vegetal substances used as flavouring agents, like vanillin, are known to possess

antimicrobial activity, being suitable in products conservation (Beuchat & Golden,

1989; Jay & Rivers, 1984). Vanillin is an antimycotic substance (Beuchat & Golden,

1989) which can used in food preservation, reducing thus the addition of non-natural

preservatives.

Vanillin is known as a strong inhibitor of fermentation and growth of yeasts as it has

been studied in the fermentation of lignocellulose hydrolysates, in bioethanol

fermentation, and it acts in very low concentrations (Endo, et al., 2008).

1.1.3. Biodegradation of vanillin by microorganisms

Biodegradation of vanillin, among several wood hydrolysates, has been studied with the

basidiomycete fungus Trametes versicolor (Jönsson, et al., 1998) as well as with the

yeast Saccharomyces cerevisiae, a good resistant to inhibitory compounds (Palmqvist,

et al., 1998).

Clostridium methoxybenzovorans sp., an anaerobic, spore-forming bacterium has also

shown to be a good degrader of methoxylated aromatic compounds (Mechichi et al.,

1999).

The tolerance of vanillin by the yeast Rhodosporidium toruloides, a Sporidiobolales

yeast, has been studied, providing novel mutant strains to optimize biofuel production

from lignocellulosic biomass (Qi et al., 2014).

In this project, the behaviour in vanillin-supplemented medium of red coloured yeasts

belonging to the class Uredinomycetes, order Sporidiobolales is studied.

1.1.4. Sporidiobolales yeasts

Sporidiobolales order belongs to Puccinimycotina subphylum, in Basidiomycota

division and pertains to Uredinomycetes class, including the genera Rhodotorula p.p.,

Sporidiobolus, Sporobolomyces p.p. and Rhodosporidium (Sampaio, et al, 2003).

Sporidiobolales yeasts or “red yeasts”, named due to their production of carotenoid

pigments, which provide them of a characteristic colour (Kurtzman, et al., 2011), can be

easily cultivated and are worldwide distributed (Aime et al., 2006).

Thirty-nine strains of basidiomycetes red yeasts in their asexual unicellular state

(Sampaio et al., 2003), belonging to a larger collection created at the Department of

Plant Pathology at the University of Molise in 2008 (Palmgren, L., 2009), are used to

carry out this project. The strains had undergone preliminary investigations based on its

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morphological characterization as well as their study as biocontrol agents of fungal

pathogens of plants and fruits (Castoria, et al., 2003; Castoria et al., 2005; Lima, et al.,

1998).

1.1.5. Ecological uses of Sporidiobolales yeasts

Sporidobolales yeasts have a wide range of usages such as: studies in tarball

(weathering of crude oil in marine environments) degradation (Shinde, et al, 2017), their

usage as biocontrol agents of fungal pathogens of plants and fruits (Castoria et al., 2003;

Castoria et al., 2005; Lima et al., 1998), carotenoid production (Yurkov, et al., 2008) for

colorants in animal feed, ubiquinone q10 production for antioxidants (Yurkov et al.,

2008) and vanillin biodegradation studies (Qi et al., 2014) for environmental cleaning.

1.1.6. Methods of identification of fungi

Fungi, even though they are the second largest kingdom of eukaryotic life (Blackwell,

2011) have not a primary DNA barcode marker. The internal transcribed spacer (ITS)

region seems to be the DNA region with the highest probability of success in

identification in the broadest range of (Schoch et al., 2012).

Internal transcribed spacer (ITS) region is has two variable non-coding regions nested

within the rRNA repeat between the 5.8S subunit and genes of the large rRNA subunit

(Gardes & Bruns, 1993). Features such as the amount of base pairs, between 600 and

800 when using primers ITS5 and ITS4 (White, et al., 1990), or the fact that it is a very

conserved sequence within species (Scorzetti, et al., 2002) make them a perfect target to

be amplified with “universal primers” (White et al., 1990). ITS regions are a

convenient region for molecular identification of fungi (White et al., 1990). Several

studies have demonstrated the variability of ITS regions according to morphological

distinction in fungal species (Gardes, et al., 1991; Gardes & Bruns, 1993).

1.2. Aims, objectives

This project has two main objectives. The first one is to test 39 different strains of

yeasts belonging to the order Sporidiobolales searching for the most resistant ones

growing in increasing concentrations of vanillin. The ones that grow better are selected

to study how they take up vanillin during their development, both through the yeast

growth and vanillin biodegradation pathway. The second objective aims accomplish and

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optimize several genomic experiments to identify the tested yeasts which can be

reproduced to identify more Sporidiobolales yeasts.

2. Materials and methods

2.1. Culture media

2.1.1. Yeast extract-peptone-dextrose (YEPD)

YEPD is a rich medium for yeast growth. Two different variants have been used, liquid

YEPD with glycerol, to prepare samples for a frozen backup, liquid YEPD to grow the

strains before the DNA extraction process and solid YEPD, with agar in a concentration

of the 2%, for petri dishes. To prepare one litre the solution contains 3.0 g Yeast extract,

10 g peptone of casein, 10 g D-glucose and 20 g bacteriological agar.

2.1.2. Lilly-Barnet, LiBa (Lilly VG, 1951)

LiBa medium is a minimum growth medium developed to study fungi strains features

such as carbon nutrition and physiology, concluding that structural variation and

configuration of carbon molecules compounds affect to the way in which fungi utilize

them (Lilly VG, 1951). In the present study, the amount of the different compounds

integrating the medium vary from the original formula as follows, containing in one

litre: 10.32 g of D-glucose, 2.0 g of L-asparagine, 1.0 g of KH₂PO₄, 0.5 g MgSO₄ x

7H₂O, 2 x 10-3 g of FeSO₄ x 7H₂O, 8,7 mg ZnSO₄ x 7 H2O, 4x10-6 mg MnSO₄ x H₂O,

4x10⁴ mg biotin and 4x10-4 mg thiamine

LiBa medium was used as liquid LiBa but also as semisolid LiBa (2% agar) on petri

dishes.

2.1.3. LiBa-Vanillin (VA) stock solution 0.1 M

A stock of LiBa-VA 0.1 M was used in all the vanillin-supplemented experiments to

adjust concentrations of LiBa-VA 1 mM, LiBa-VA 5 mM and LiBa-VA 10 mM. To

prepare 10 ml of stock solution, 0.512 g of vanillin were necessary.

2.2. Yeast collection

Concrete strains (Table 1) belonging to the collection created in 2008 in of University

of Molise (Palmgren, L., 2009) were selected due to their not reliable identification in a

first attempt. For this reason, some of them have a species name between quotation

6

marks (“ ”). The execution and optimization of this experiments will lead to a

forthcoming identification of the isolates.

The yeast strains used in this project were sampled from different vegetal species in

different climatic zones in central-southern Italy: Larino, Isole Tremiti, Campomarino,

Petacciato, Castel San Vincenzo and Portocannone (Curtis F, et al., 2009; Curtis, et al.,

2009; de Curtis et al., 2010). All the isolates were conserved in 40% glycerol YEPD at -

80 ºC.

Table1: Collection and reference strains: locations and plants where the different strains were sampled strain numbers, collection

number designated in Italy and the number assigned to the tubes in the frozen backup in Sweden. The table cells highlighted with a

light grey background indicate the reference strains.

Italian number Strain number Plant of origin Place isolated Frozen number

1 L2 Unknown Unknown F37


13 L105 Malus domestica Larino F3

36 L151 Ficus carica Larino F1

37 L153 Olea europea Larino F32





47a L166A Malus domestica Larino F7

52 L177 Malus domestica Larino F39



62 L209 Prunus persica Isole Tremiti F9

67 L215 Prunus persica Isole Tremiti F10


79 L241 Rosmarinus

officinalis

Molise coastal F12

114 L384 Ficus carica Isole Tremiti F36





196 L411 Olea europea Isole Tremiti F19

83”70” L249 Prunus armeniaca Portocannone F20

87 L257 Pistacia sp. Molise coastal F21

88 L268 Euphorbia paralias Campomarino F22

89 L269 Euphorbia paralias Campomarino F23

98 L359 Crithmum maritimum Isole Tremiti F24

116 L386 Ficus carica Isole Tremiti F34

123 “Rhodotorula sp.” Unknown Unknown F25

124 “Erytrhobasidium

hasegawianum”

Asparagus acutifolia Isole tremiti F26

“104” L393 Unknown Unknown F27

“105” L394 Asparagus acutifolia Isole tremiti F28

156 L452 Malus domestica Castel San Vincenzo F29

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185 L-76-2* Olea europea Larino F33

187 LS11* F30

188 LS22* Solanum tuberosum Venfaro F14

189 LS28* F15

190 Sporobolomyces sp. * F16

All the strains indicated with an asterisk (“*”) were studied before and some of them

took part in published experiments. They were used as reference strains.

Sporobolomyces roseus / Sporidiobolus sp*. is the isolate L-76-2* in the collection of

the Dipartimento A.A.A. dell‟Università degli Studi del Molise”, it was isolated from

potato surface (Wright, S. A. I., personal communication).

Rhodosporidium kratochvilovae is the isolate LS11* in the collection of the

Dipartimento A.A.A. dell‟Università degli Studi del Molise”, it was isolated from olive

leaves (Lima et al., 1998), and it is an efficient biocontrol agent against common post-

harvest pathogenic fungi (Castoria et al., 2003; Castoria et al., 2005; Lima et al., 1998)

and an in vitro degrader of the mycotoxin patulin (Castoria et al., 2005). Studies of the

phenotypic and genetic heterogeneity of the isolate R. kratochvilovae, have led to a

reclassification of the isolate LS11 from Rhodotorula glutinis to R. kratochvilovae

(Castoria et al., 2011).

Rhodosporidium kratochvilovae is the isolate LS22* in the collection of the

Dipartimento A.A.A. dell‟Università degli Studi del Molise”, it was isolated from olive

leaves (Wright, S. A. I., personal communication).

Cryptococcus laurentii is the isolate LS28* in the collection of the Dipartimento A.A.A.

dell‟Università degli Studi del Molise”, it was isolated from Annurca apples (Lima et

al., 1998) and it is an efficient biocontrol agent with antagonist activity against B.

cinerea and P. expansum in injured apples stored at 3ºC and 20ºC (Lima et al., 1998;

Lima, de Curtis, Castoria, & De Cicco, 2003).

The strain called Sporobolomyces sp.* in the table is strain IAM 13481 (Valerio, et al.,

2008).

2.3. Identification experiments

DNA barcoding is a rapid developing alternative in species identification, being

nowadays a critical tool in fungal taxonomy (Harrington et al., 2014). The target for the

DNA barcoding was the internal transcriber sequence (ITS) region. The results were

supported by GenBank database of the US National Centre for Biotechnology

8

Information (NCBI) using the Basic Local Alignment Search Tool (BLAST). Barcoding

projects are of high value for documenting fungal diversity.

To identify the isolates, several experiments, such as DNA extraction, Polymerase

Chain Reaction and agarose gel electrophoresis and their optimization were carried out

in these pigmented yeasts, which we know on beforehand belong to order

Sporidiobolales of the class Urediniomycetes (Gadanho & Sampaio, 2005).

2.3.1. Genomic DNA extraction

To study molecular systematics of any organism, high quality DNA is required

(Aljanabi & Martinez, 1997). A genomic extraction was completed according to the

protocol developed by Hoffman, C. in 2001 (Hoffman, 2001) with specific

modifications. One loopful of a single colony of each strain (grown for 24h in a YEPD

petri dish) was grown in 5 ml liquid YEPD in a 50 ml falcon tube in a horizontal shaker

overnight, at room temperature and 2300 rpm. The shaking allows cells survival

enabling them to reach the surface and take the O2 from the environment, fundamental

for them to grow. After 24 h, the tube was centrifuged 5 minutes at 4000 revolutions per

minute (rpm), the supernatant discarded, and the pellet transferred to an 1,5 ml

Eppendorf tube. The tube was frozen for 30 min at -80 ºC, in this step, the cell

membranes break due to the mechanical stress caused by the congelation. Subsequently

the cells were resuspended in 0,5 mL TENTS buffer (extraction buffer 10 mM Tris HCl

pH 7,5, 1 mM EDTA pH 8, 100 mM NaCl, 2% Triton X-100 and 1% SDS) to recover

the cell conditions avoiding DNA damage. All buffers containing a detergent, in this

case, Sodium-Dodecyl-Sulphate (SDS) lysate the nucleus releasing the DNA.

The mixture was transferred to a new autoclaved Eppendorf tube containing the

equivalent of 0.2 ml of acid washed glass beads. Later 0.5 ml of phenol-chloroform-

amylalcohol (25:24:1) were added to the mixture which was afterwards bead-beated for

15 min in a tabletop bead shaker. Phenol-chloroform-amylalcohol solutions denature the

proteins, this, together with the mechanical stress caused by the glass beads and the

shaking will divide the solution in an aquose phase (in the upper part of the Eppendorf)

containing the DNA, and an organic phase, containing the phenolic and cell residua. On

the next step of the DNA extraction cells were frozen for 30 min at -80 ºC, bead-beated

other 30 min in a tabletop bead shaker and finally spun at 13000 rpm at room

temperature (RT) for 10 min.

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The aqueous phase (which contains the DNA) was transferred to new autoclaved

Eppendorf tubes and 0.4 ml of isopropanol were added. Isopropanol helps to precipitate

the DNA re-establishing its three-dimensional structure. The DNA was precipitated by

rocking and spin at 4 ºC at 13000 rpm for 20 minutes and subsequently washed with

EtOH 70% (which also precipitates the DNA). Finally, the DNA pellet was dissolved in

0.2 ml double distilled MilliQ water (ddH20 MilliQ) and stored at -20 ºC.

2.3.1.1. RNAse treatment

For the procurement of a purer DNA sample, an RNAse treatment was included adding

30 µl of 1 mg/ml DNase-free RNase, which after mixture with the sample was

incubated 5 minutes at 37 ºC. Later, 10 µl of 4 M ammonium acetate and 1 ml of 100%

ethanol were included in the mixture and mixed by inversion to reprecipitate the nucleic

acid. Finally, the tubes were micro-centrifuged 3 minutes at high speed and RT, the

supernatant discarded and after the DNA pellet had dryed, this one was resuspended in

100 µl Tris-EDTA (TE) buffer.

2.3.1.2. Verification of DNA’s extraction succeed

All DNA samples had to be checked after their setting up to find out if the DNA

extraction was successful. Agarose electrophoresis can accomplish this commitment,

enabling to see the migration of a charged molecule with high resolution (Lehrach, et

al., under the influence of an electric field (Lima Pastrana, 2011).

The prepared agarose gels, after sample loading, were submerged in a tampon with pH

8, knowing that DNA molecules have negative charge in mediums with a pH higher

than 5, and that therefore they will migrate to the positive pole (Lima Pastrana, 2011).

Each gel contained a concentration of 0,7% in agarose and was prepared mixing 0.35

grams of agarose with 50 ml of 100X Tris-Acetate-EDTA (TAE) buffer. To dissolve the

agarose in the buffer, short periods of microwaving were necessary (around 30

seconds). After dissolving, the solution was poured in the electrophoresis tray, already

prepared with the wells comb. It takes about 30 minutes to cool down, then, the comb is

removed and the gel tray is ready to be introduced in the electrophoresis bucket. The

bucket was filled with 100 X TAE buffer covering the wells before the samples were

pipetted, avoiding so the removal of the samples from the wells.

Each sample had a volume of 20 µl, consisting of 16 µl autoclaved MilliQ H2O, 1 µl of

DNA sample (the one obtained after the DNA extraction) and 3 µl of a loading buffer,

to make the mixture heavier enabling the samples migration to the positive pole in the

gel. A molecular weight ladder containing fragments of DNA with a known number of

10

base pairs (bp) and known concentration was included for the interpretations of the

results. After closing the bucket, the electrical current was turned on for two hours at 70

mV.

GelGreen™, a safer alternative to Ethidium Bromide and SYBR dyes was used to stain

the gel dissolving 30 µl of the staining solution in 100 ml of MilliQ H2O in a bucket.

One hour after the submergence of the gel in the staining solution, its content was

revealed in a UV light lamp.

2.3.2. Polymerase chain reaction (PCR), amplification of ITS regions

Polymerase chain reaction, also known as PCR, is a method consisting on the

exponential amplification through a three-step cycling process (Schochetman, et al.,

1988) of a nucleic acid, or nucleotide sequences, in vitro. When amplifying a sequence,

the ends must be known in sufficient detail to synthesize oligonucleotides that can

hybridize it and initiate the reaction (Mullis & Faloona, 1987). Two synthetic oligos are

necessary to work as primers, annealing at either end of the targeted nucleotide

sequence to amplify in opposite directions orientation. After a proper annealing of the

primers, the functioning of a DNA polymerase in multiple rounds of denaturation

(opening the double helix), annealing and 3’ extension leads to an exponential

amplification of the target sequence (Ho, Hunt, Horton, Pullen, & Pease, 1989). Primers

ITS5 and ITS4 were used and their corresponding sequences are: ITS 5:

GGAAGTAAAAGTCGTAACAAGG, ITS 5: GGAAGTAAAAGTCGTAACAAGG

(White et al., 1990).

2.3.2.1. Reagents

Polymerase chain reactions were carried out using the Platinum™ Taq Green Hot Start

DNA Polymerase (Invitrogen™) with several modifications in the manufacturer

specifications (Perkin Elmer Cetus). 100 µl of PCR reaction sample were prepared for

each strain as follows: first, a master mix was prepared with 12.5 µl of 10 X Green PCR

buffer, which contains among other substances Tris, that regulates the reaction pH,

affecting the activity and fidelity of the enzyme and KCl, which increments the enzyme

activity (Johnson, 2000; Schochetman et al., 1988), 2.5 µl MgCl2, 2.5 µl of 10 mM

dNTP mix (dNTPs need Mg2+ to bind) and 0.5 µl of Platinum™ Taq DNA Polymerase.

An insufficient amount of Mg2+ affects the DNA polymerase by stopping its reaction

while its excess causes non-specific reactions (Johnson, 2000). Afterwards, 2.5 µl of

both ITS5 and ITS4 primers were added in a concentration of 10 mM (they were firstly

11

diluted from 100 mM), mixed together with the master mix ,72 µl of autoclaved and

filter sterilized MilliQ H20 and 5 µl of the DNA sample previously prepared in the DNA

extraction.

The 100 µl were divided in two small autoclaved Eppendorf tubes, having so each strain

two PCR products.

2.3.2.2. Generation of PCR products

50 µl PCR samples were introduced in a thermal cycler to incubate the reactions. Even

though the company had specifications, modifications on the times and temperatures of

the cycles were necessary for the circumstances of the modified mixture. The thermal

cycler was programmed for 35 cycles. First an initial denaturation at 94 ºC for 5 minutes

was necessary, then, in each cycle a denature stage occurred at 94 ºC for 1 minute (due

to de length of the sequence aimed to be replicated, this is, around 700bp), after, the

anneal phase was carried out at 61.5 ºC (being this the optimal temperature for the

primers to operate) for 30 secs and the extension stage took place at 72º for 50 seconds.

Finally, the sample was hold at 4º indefinitely.

2.3.3. PCR product classification

Agarose gel electrophoresis can clear up the size (in bp) of the ITS fragments obtained

in the PCR, determining if the PCR has been successful and has replicated the target

fragment of the DNA molecule. The size of the bands belonging to the PCR samples

can be defined by the proportional relation of the mobility of the sample in the gel and

the empirical logarithm of its size (Lehrach et al., 1977). A ladder with standards of

concentration and fragments size was necessary to set a calibration line in which the

band migration of each strain PCR ITS fragment was extrapolated, thus, determining

the fragment size. In this experiment, the ladder used was ZR 100 bp DNA Marker™

aiming to find the same or very similar fragment sizes in the PRC products as the PCR

is expected to replicate the same sequence considered to length between 600-700 bp

(White et al., 1990).

2.3.4. Purification of the PCR product gel bands

When the PCR product was considered to have the appropriate concentration, the next

step was to purify the bands in the gel containing our target sequence, cleaning possible

remaining PCR components. GenElute™ Gel Extraction Kit was the kit used and the

fragment of interest was extracted from the agarose gel in a slice, deposited inside an

12

Eppendorf tube containing a silica binding column and dissolved and cleaned with

products from the kit through several centrifugations. Our target, the ITS fragment, was

expected to get stuck in the silica binding column and it only be released in the last step

of the protocol, when releasing the clean sequence from the silica column.

2.3.5. Verification of an adequate amount of DNA in the samples

Before shipping the samples to the sequencing firm, it was important to know

approximately the concentration of the eluted samples to fulfil the sequencing company

requirements.

To the achievement of this task, new agarose gels were prepared and several

measurements were made in photographs taken from these gels to estimate the

concentration of the ITS region in the purified tubes. In the photographs, the luminance

of the bands was measured. To estimate the concentration of a band, a luminance value

is given to the molecular weight markers with known concentration, 600 bp, 26 ng and

700 pb, 24 ng. An average of both luminance and concentration values of the two ladder

bands was used to calculate the concentration of the sample bands because the expected

ITS regions were expected to appear between 600 bp and 700 bp. The calculation

procedure was a simple proportion of luminance-concentration. For making a more

accurate experiment, each luminance value was an average of 3 luminance values

measured along the gel band.

2.3.6. Preparation of sequencing samples

After measuring the concentrations of the purified ITS region, the samples were

prepared to be sent to LIGHTRUN tube, a sequencing PCR fragments company which

uses Sanger light-speed methodology. The sample requirements were related to the

concentration of the DNA template, between 20 ng and 80 ng, and its amount, minimum

5 µl, as well as the primer’s amount and concentration, 5 µl in concentrations of 5 µM

for each primer, ITS4 and ITS5.

2.3.7. Sequencing

Sequencing samples arrived three working days after the shipping. They came with the

analysed sequence in three different kind of files: .seq (the sequence), .ab1 (the

electropherogram) and .fas (for a FASTA DNA-program).

13

2.3.8. Identification

The sequences were analysed and edited by using the package DNAStar®, making

alignments and visually correcting the sequences by using the software MegAlign™.

For the identification, the obtained sequences were compared with those known of all

yeast species available at the GenBank database of the US National Centre for

Biotechnology Information (NCBI). For this task, the Basic Local Alignment Search

Tool (BLAST) available at http:// www.ncbi.nlm.nih.gov was used.

2.4. Vanillin resistance experiments

Several Sporidiobolales strains were tested in diverse minimum medium to study their

reaction to different concentrations of vanillin. The growth of a strain in a medium with

a higher concentration of this phenolic compound would make the strain to be

considered as good degrader of vanillin and therefore, object of study in liquid culture

experiments, studying its capacity for biodegrading vanillin aiming to understand a bit

better its biodegradation pathway.

2.4.1. Selection test of the most resistant isolates

In a first screening experiment, the 39 isolates were tested together with an isolate

belonging to another yeast collection, FMYD002 (Nehvonen, C., 2017) already used for

some vanillin tests. A total of 40 strains were imprinted and transferred from YEPD-

agar petri dishes to several LiBa-agar petri dishes (20 colonies per plate) with different

concentrations with replica prong. The experiment had two variants, one consisted on

transferring the colonies grown for 48h in YEPD-agar plates to LiBa-agar plates with

concentrations of LiBa-VA 0 mM, LiBa-VA 1 mM , LiBa-VA 5 mM and LiBa-VA 10

mM all at the same time. The second variant of this experiment, consists on the

transference of the colonies in order of increasing concentration from one plate to

another only after their growth was evident. Thus, the not vanillin resistant strains were

ruled out leading to the permanence of the best candidates withstanding high

concentrations of vanillin.

2.4.1.1. Solid culture media preparation

LiBa-agar plates are prepared mixing a stock of LiBa with autoclaved MilliQ H2O

containing agar up to a 2 %. Different amounts of vanillin stock solution (0.1 M) were

added to the flask containing LiBa-agar after pouring the plates. The flask contained

320 ml and each plate 20 ml of the medium, starting from LiBa-agar, and continuing in

14

crescent concentrations of vanillin order (1 mM, 5 mM and 10 mM). From each

concentration four plates were prepared making a total of sixteen plates.

Single colonies of the strains were cultivated with toothpicks in regular YEPD-agar

plates following the pattern of the replica prong to make the transference easier.

2.4.2. Vanillin resistance and degradation in liquid cultures

The strains that showed more resistance in the solid cultures were selected to grow in

the same vanillin concentrations (0 mM, 1 mM, 5 mM and 10 mM) in liquid LiBa

cultures, extracting samples in certain timepoints.

2.4.2.1. Experiment setup

Before starting the proper experiment, a preculture to set the concentration of the main

experiment was necessary. A loopful of the selected strain was grown in 40 ml of liquid

LiBa for 24 h. Afterwards, the number of cells was measured and each flask of the

experiment set at a concentration of 6 x 106 CFU/ml. The experiment consisted of eight

flasks, four flasks containing 60 ml of LiBa and concentrations of 0 mM, 1 mM, 5 mM

and 10 mM of vanillin and a cell concentration of 6 x 106 CFU/ml of the selected yeast

strain taken from the preculture, and other four containing the same concentrations of

vanillin but without the yeast, this is, controls. The flasks stayed 192 hours in a circular

shaker at 230 rpm and 25 ºC (in a sterile environment) and six timepoints were

established at 0, 12, 24, 36, 48 and 196 h to collect samples aiming to see how the yeast

degrade the supplemented vanillin in each medium.

2.4.2.2. Sample collection

At each timepoint, two millilitres of each flask were extracted and transferred to

Eppendorf tubes, spin at 4000rpm for 5 minutes and filter sterilized with a syringe. Each

sample was kept in the freezer at -20 ºC and defrosted to prepare a thin layer

chromatography

2.4.2.3. Thin layer chromatography (TLC)

Thin layer chromatography is a separation technique used in qualitative analysis of

compounds. TLC enables to monitor the progress of a reaction, the number of

components in a mixture, and it also gives an idea of the sample’s purity (Hess, 2007).

This technique has two phases, a stationary phase consisting on a silica slab coated on

aluminium and a glass chamber in which the back of the slab lays, and a mobile phase,

the eluent, made of a mixture of toluene, ethyl acetate and formic acid (4:5:1).

Capillarity is the principle of this methodology allowing the migration of the eluent and

15

the compounds upwards in the silica slab (Hahn-Deinstrop, 2007). Each compound has

a different affinity and polarity which affects the migration speed and the separation of

the compounds. Less polar compounds migrate further upwards while polar compounds

stick the polar silica slab, reducing the migrating speed. Usually TLC experiments take

80 min and the elution must be stopped before reaching the top of the silica slab. After,

the TLC can be studied under UV light or MBTH staining.

2.4.2.3.1. Sample preparation

Each sample for the TLC was taken from the frozen samples that were collected from

the main culture flasks. The samples were transferred to glass tubes and adjusted to pH

2 with HCl 1 M. For 2 ml, 800 µl of ethyl acetate were added dividing the mixture in

two phases, an organic phase constituted by the ethyl acetate and an inorganic phase

consisting of the LiBa medium. The samples were centrifuged for 5 min at 5000 rpm

and during the spin the compounds stick to the ethyl acetate. 500 µl of the organic phase

were extracted, transferred to small glass tubes, and dried with gaseous N2 (this step

takes around 15 min). Once the samples dried, 20 µl of ethyl acetate were added to the

tube resuspending the compounds. The 20 µl samples and 10 μl standards of vanillin 10

X (5 mM), vanillyl alcohol 10 X (5 mM) and vanillic acid 5 X (10 mM) were pippeted

in the silica slab in small doses, using a hair dryer to dry each drop before adding the

next. After the TLC plate was introduced in the chamber with the eluent. The standards

were chosen to help identifying the vanillin degradation sub-products providing

guidance in the staining contrast and travel distance of the stationary phases on the TLC

plates.

2.4.2.3.2. Treatment of the data

When revealed, the chromatography showed the compounds at different heights, this

migration distance is a signature of every compound found in the sample (Hahn-

Deinstrop, 2007). The data were treated using the retention factor (Rf) values of each

sub-product, consisting on the distance travelled by the sample over the distance

travelled by the eluent (solvent front).

2.5. Yeast growth in vanillin supplemented medium

The isolates were expected to struggle growing as the vanillin concentration increases

due to the known fact that vanillin is an inhibitor in yeasts growth at small

concentrations (Endo et al., 2008).

16

2.5.1. Cell count in Neubauer’s chamber

Haemocytometers enable cells counting. Neubauer’s chamber consist of a thick crystal

plaque divided in three sections. In the central section, a quadrangular grid is engraved

containing two counting areas, each of them divided in nine squares of 1 mm square

each. To prepare the samples, 20µl of each flask were extracted at the same timepoints

as the TLC samples, diluted in 180µl of LiBa, and 10 µl were added to 10 µl of

methylene blue. 10 µl of each sample were pippeted in the chamber, covered with a

glass slide and observed at 40x in the microscope. Neubauer’s chamber formula is:

𝑐𝑒𝑙𝑙 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 =𝑐𝑒𝑙𝑙𝑠 𝑛𝑢𝑚𝑏𝑒𝑟 𝑥 10000

(𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛)

Where “10000” is the area of each big square:

• 0,1cm x 0,1cm = 0,01cm2 (area of a square)

• 0,01cm2 x 0,01cm (depth) = 0,0001cm3

Neubauer’s chamber is also used to measure the cell concentration from the preculture.

3. Results

3.1. Identification experiments

The main aim was to optimize and find out the correct proportions of chemicals in the

different steps of the identification of yeast strains. The experiments were successful

and can be replicated.

3.1.1. DNA extraction

A first attempt of DNA extraction was done, unfortunately, with an outdated phenol. In

a second try all the samples succeeded in the DNA extraction. Nevertheless, the samples

were not as clean as aimed, therefore an RNAse treatment was included in the protocol.

Due to the immense size of DNA molecules, the samples did not migrate much down in

the gel. All in all, the gels showed a successful DNA extraction, and therefore the

experiments of DNA fingerprinting were carried out.

3.1.2. PCR product

After all the cycles in the thermal cycler were completed, the PCR product was checked

through an agarose gel. The gel results proved the PCR to be successful. An analysis of

the band sizes and migrations was performed to assure that all the ITS fragments

17

replicated in the PCR had a similar base pairs (bp) amount, this is, that all the fragments

have an approximate size (Table2-3).

Table 2-3: sizes of PCR products and migration distance after amplification of genomic DNA with primers ITS4 and ITS5.

A) Strains of the culture collection

strain L2 L4 L105 L151 L153 L154 L155 L156 L160 L166A L177 L200

Migration (mm) 30 30 30 30 29 29 30 29 29 29 30 30

Fragments size (bp) 625 625 625 625 675 675 625 675 675 675 625 625

L206 L209 L215 L234 L241 L384 L388 L389 “E.hasegawianum” L393

30 30 30 29 31 30 30 30 30 31

625 625 625 675 600 625 625 625 625 600

L212 L400 L411 L249 L257 L268 L269 L359 L386 “Rhodotorula sp.” L452 L394

30 30 30 30 30 30 31 30 31 31 30 30

625 625 625 625 625 625 600 625 600 600 625 625

B) Reference strains

strain LS11 L-76-2 LS22 LS28 “Sporobolomyces sp.

Migration (mm) 30 30 31 33 24

Fragments size (bp) 625 625 600 500 1000

Ladder fragments size (bp) 19 22 24 25 26 28 31 33 35 38

Migration (mm) 1500 1200 1000 900 800 700 600 500 400 300

Figure 1: PCR product: analysis of the proportional relation between the sample migration through the gel and the size of the

fragments.

20

22

24

26

28

30

32

34

450

550

650

750

850

950

1050

L2

L4

L10

5L

15

1L

15

3L

15

4L

15

5L

15

6L

16

0L

16

6A

L17

7L

20

0L

20

6L

20

9L

21

5L

23

4L

24

1L

38

4L

38

8L

38

9L

-76

-2L

S22

LS

28

“S. R

ose

us”

L21

2L

40

0L

41

1L

24

9L

25

7L

26

8L

26

9L

35

9L

38

6“R

ho

do

toru

la s

p.”

“Ery

trh

ob

asid

iu…

L39

3L

39

4L

45

2L

S11

Mig

rati

on

(m

m)

Fra

gm

ents

siz

e (b

p)

PCR product analysis

Fragments size (bp) Migration (mm)

18

The analysis of the band sizes and band migration (Figure 1, Table 2-3), showed that

all the culture collection strains have ITS fragments of the expected amount of bp (600-

800bp).

3.1.3. Gel elution and recovery of purified ITS fragments

After the purification and elution of the ITS regions extracted from gel slices, the

concentration of the template DNA vas determined through luminance measurements of

the bands to fulfil the requirements (20-80 ng/µl) of the sequencing company

(LightRun). Due to a lack of time, not all the samples reached this stage in the path to

sequencing.

3.1.4. Verification of required minimum concentration of template DNA

The ITS regions of strains L4 and L105 showed the highest concentration in PCR

product and therefore were selected to be shipped to the sequence company. During the

gel elution process, DNA losses could reach a 50%, so the concentration of the samples

was checked after the elution. Three luminance measurements were made for each band

and an average value (Table 4) was be used to relate luminance with concentration.

Table 4: Luminance values (Nits) average of the template DNA measured in gels run after the gel elution

Luminance measures 1st 2nd 3rd Average

L4 155 155 155 155

L105 155 156 155 155,33

L151 112 113 112 112,33

Ladder 700 146 145 146 145,67

Ladder 600 145 145 145 145

Table 5: Luminance-concentration relation. The concentration of the fixed average ladder was 25 ng/µl and the luminance 145,33

Nits and the results come from the direct proportion existing between luminance and onncentration.

Strains and ladders Luminance (Nits) Concentration (ng/µl)

L4 155 26,66

L105 155,3333333 26,72

L151 112,33 19,32

600-700 ladder 145,33 25

Luminance and concentration are directly proportional values (Table 5). The estimated

value for the samples was higher than the minimum required and therefore the samples

were prepared and sent to the company.

3.1.5. Identification results

Positive sequencing results were obtained from the company for both strains. Among

the diverse results found in a preliminary analysis it was possible to confirm that both

strains belong to the species Rhodotorula babjevae as their closest match was the strain

19

CBS-322 in the CBS culture collection, designated as Rhodotorula babjevae (Vu et al.,

2016).

3.2. Vanillin experiment: selection of most vanillin resistant isolates

As shown in Figure 2, in the experiment in which the strains were transferred with a

replica prong from YEPD plates simultaneously to the different concentrations of LiBa-

VA and grown for 48 hours, all the strains show growth in LiBa and LiBa-VA 1 mM

plates, while no growth is noticed in the LiBa-VA 5 mM and LiBa-VA10 mM plates.

The gradual transference experiment showed more hopeful results (Figure 2) as in

LiBa-VA 5 mM plates, after many hours, Rhodotorula babjevae (strain L4) and

Rhodosporidium kratochvilovae (strain LS22) grew. In LiBa 10 mM, only strain L4

grew. The two strains, due to their capacity to withstand higher concentrations of

vanillin were chosen to be studied in liquid LiBa-VA experiments, the biodegradation

pathway of vanillin and how the strains growth was affected in its different

concentrations.

48 h cultures Growth after gradual transfer to increasing

concentrations of vanillin (VA)

A

B

B

C

D

D

7 days growth

5 days growth

10 days growth

20

Figure 2: growth of red yeasts on Lilly Barnet (LiBa) medium supplemented with: (A) LiBa without VA; (B) LiBa + 1 mM VA;

LiBa + 5 mM VA; and (D) LiBa + 10 mM VA. The plates in the left column refer to a simultaneous transference of the yeasts grown in YEPD for 24 hours directly to all the plates with the different concentrations and grown for 48 h. In the right column, the

colonies were only transferred to an increasing concentration after they grew in the plate. Numbers in the plates refer to the strain

names (see Table 1).

3.3. Vanillin biodegradation in liquid cultures, TLC results

Vanillin biodegradation was studied through TLC plates running each strain samples

collected at 0, 12, 24, 36, 48 and 192 hours from the 4 different flasks containing the

liquid cultures with LiBa, LiBa-VA 1 mM, LiBa-VA 5 mM and LiBa-VA 10 mM and

the Rf of the revealed compounds was measured (Table 6), finding eight different

intermediaries in this cultivation process.

Table 6: Compounds revealed in TLC plates and their corresponding Rf value for Rhodotorula babjevae and Rhodosporidium

kratochvilovae..

Vanillin Comp. Y Comp. A Comp. X Comp. B Comp. C Comp. D Comp. E

Rf 0,8 0,74 0,67 0,65 0,64 0,62 0,58 0,49

For both, Rhodotorula babjevae and Rhodosporidium kratochvilovae the TLC results

showed degradation products of LiBa medium which were obviated (compounds A, B,

C, D and E).

Rhodotorula babjevae, (Figure 3) at concentrations of LiBa-VA 1 mM showed the

consumption of all the vanillin by the yeast in the first 12 hours. At time 0 had already

started degrading vanillin in compound X, which has the same Rf value as the standard

of vanillyl alcohol used. Once the vanillin disappeared the remaining compounds,

compound X and compound Y (with a Rf value similar to the one obtained in the

vanillic acid standard), were stable until 48 hours, and after 192 hours only compound

X remained. At concentrations of vanillin 5 mM, and 10 mM vanillin and compound X

were present during the 192 hours, in LiBa-VA 5 mM, compound Y appeared at 48

hours and remained even after 192 hours while in LiBa-VA 10 mM, compound X

appeared at 48 hours and at 192 hours it was already gone.

5 days growth

10 days growth

D 16 days growth

21

Figure 3: Biodegradation products of vanillin by Rhodotorula babjevae in LiBa, LiBa-VA 1 mM, LiBa-VA 5 mM and LiBa-VA 10

mM

In the TLC results for strain Rhodosporidium kratochvilovae, Figure 4, it was assumed

that at concentrations of vanillin 1 mM, all the medium was consumed by the yeast

during the first 24 hours due to the lack of products after 36 hours. At time 0, only

vanillin appeared and after 12 hours it had already been transformed in compound Y

and compound X, which disappeared after 24 hours. At concentrations of vanillin 5

mM, and 10 mM, vanillin stayed during the 192 hours, compound X disappeared after

192 hours and compound Y, formed at 36 hours, remained until the end of the

experiment in vanillin 5 mM flask while in vanillin 10 mM it was already consumed at

192 hours.

Figure 4: Biodegradation products of vanillin by Rhodosporidium kratochvilovae in LiBa, LiBa-VA 1 mM, LiBa-VA 5 mM and

LiBa-VA 10 mM

0

0,2

0,4

0,6

0,8

1

0 L

iBa-

VA

1 m

M

12

LiB

a-V

A 1

mM

24

LiB

a 1

mM

36

LiB

a-V

A 1

mM

48

LiB

a-V

A 1

mM

19

2 L

iBa-

VA

1 m

M

0 L

iBa-

VA

5 m

M

12

LiB

a-V

A 5

mM

24

LiB

a-V

A 5

mM

36

LiB

a-V

A 5

mM

48

LiB

a-V

A 5

mM

19

2 L

iBa-

VA

5 m

M

0 L

iBa-

VA

10 m

M

12

LiB

a-V

A 1

0 m

M

24

LiB

a-V

A 1

0 m

M

36

LiB

a-V

A 1

0 m

M

48

LiB

a-V

A 1

0 m

M

19

2 L

iBa-

VA

10

mM

Rf

Time (h)

Rhodotorula babjevae: vanillin biodegradation products

Vanillin Compound Y Compound X

0

0,2

0,4

0,6

0,8

1

0 L

iBa-

VA

1 m

M

12

LiB

a-V

A 1

mM

24

LiB

a 1

mM

36

LiB

a-V

A 1

mM

48

LiB

a-V

A 1

mM

19

2 L

iBa-

VA

1 m

M

0 L

iBa-

VA

5 m

M

12

LiB

a-V

A 5

mM

24

LiB

a-V

A 5

mM

36

LiB

a-V

A 5

mM

48

LiB

a-V

A 5

mM

19

2 L

iBa-

VA

5 m

M

0 L

iBa-

VA

10 m

M

12

LiB

a-V

A 1

0 m

M

24

LiB

a-V

A 1

0 m

M

36

LiB

a-V

A 1

0 m

M

48

LiB

a-V

A 1

0 m

M

19

2 L

iBa-

VA

10

mM

Rf

Time (h)

Rhodosporidium kratochvilovae: vanillin biodegradation products

Vanillin Compound Y Compound X

22

3.4. Vanillin resistance: Neubauer’s chamber results

To measure the resistance of the yeast to different concentrations of vanillin, the cell

growth was measured in six timepoints (0, 12, 24, 36, 48 and 192 hours).

Figure 5: growth of Rhodotorula babjevae in cultures with different concentrations of VA for 192 hours

As shown in Figure 5, Rhodotorula babjevae showed a better average growth in a LiBa

medium supplemented with 1 mM vanillin than it did in LiBa. This indicates that

probably small doses of vanillin can nutritive for the strain, enhancing its growth. Even

though, at 36 hours the growth was excellent, afterwards it started to decrease until 192

hours (unlike in LiBa medium in which the strain kept growing at 192 hours). In the

beginning the strain seemed to struggle to grow in the medium until it could benefit

from it. In concentrations of vanillin 5 mM the growth was very poor and after 48 hours

the cells started their apoptosis and in concentrations of vanillin 10 mM the strain didn’t

grow until 36 hours and after it died.

Figure 6: growth of Rhodosporidium kratochvilovae in cultures with different concentrations of VA for 192 hours

Rhodosporidium kratochvilovae showed different results in its growth (Figure 6). In

LiBa medium, the strain grew until 36 hours and after that the cells concentration

0

10

20

30

40

0 1 2 2 4 3 6 4 8 1 9 2CF

U/m

l (i

n x

10

6sc

ale)

Time (h)

Growth of Rhodotorula babjevae in different VA

concentrat ions

LiBa LiBa-Va 1 mM LiBa-VA 5 mM LiBa-VA 10 mM

0

10

20

30

40

50

60

70

0 1 2 2 4 3 6 4 8 1 9 2CF

U/m

l (i

n x

10

6sc

ale)

Time (h)

Growth of Rhodosporidium kratochvi lovae in different

VA concentrat ions

LiBa LiBa-Va 1 mM LiBa-VA 5 mM LiBa-VA 10 mM

23

started to decrease unlike it happened with Rhodotorula babjevae. In vanillin 1 mM

supplemented medium the strain reached its peak of growth after 2 hours and after that

it seemed like it had consumed all the medium due to its vast decrease in cell

concentration. At concentrations of vanillin 5 and 10 mM the strain progressively

reduces the number of cells, as it happens with strain L4.

4. Conclusions and discussion

The results obtained in the identification study show that the performed and optimized

experiments work for obtaining a clean ITS sequence, this means that the rest of the

strains which were selected for these experiments and that have not been identified yet,

can be identified reproducing these experiments.

The fact that the two identified strains belong to the same species (Rhodotorula

babjevae) and one withstands high concentrations of vanillin while the other one does

not opens a question mark for the rest of the strains, does the location of collection of

the sample has an influence in the species variation regarding vanillin biodegradation? It

seems like a possibility as strain LS11, Rhodosporidium kratochvilovae, a good

biodegrador of patulin (Castoria et al., 2005) is not resistant to vanillin, while, LS22,

also Rhodosporidium kratochvilovae, sampled from potatos does biodegrade vanillin.

This opens a second question mark, does the resistance to vanillin can be a consequence

of the usage of herbicides? Also, morphological studies can take place to find out if a

genomic identification would match with a morphological characterization distribution

of the isolates.

The strain Rhodosporidium dibovatum has been studied and documented as capable of

growing in the presence of vanillin (Luque et al., 2016) also, it was one of the strains

with the most similar ITS region to the one in Rhodotorula babjevae when the sequence

was introduced in the NCBI database. A futher study could consist on a classification of

ITS regions similarity and the capacity of the microorganisms withstandig vanillin.

Even though, exceptions were found such as the disparity between the ITS region length

found in Sprobolomyces sp. and L-76-2 which belong to the same species. It could be

due to inespecific amplifications during the PCR.

It can be concluded that strains L4, Rhodotorula babjevae, and LS22, Rhodosporidium

kratochvilovae were the two best strains growing in LiBa medium supplemented with

increasing concentrations of vanillin. Strain L4 in liquid cultures of LiBa-VA 1 mM,

consumed the medium more slowly than strain LS22, and that all in all, both of them

24

soon or later produce the same biodegradation products from vanillin, with Rf values

similar to the ones for vanillic acid and vanillyl alcohol.

It is also remarkable the influence of small doses of vanillin in the growth of yeasts,

concretely in strain LS22 once the vanillin is already degraded (1 mM VA) not only the

subproducts are consumed, what is more, if the results of Figure 6 are contrasted to the

results in Figure 4, it is possible to appreciate that the time in which the cells start to die

correspond to the time in which the biodegradation products are consumed. Studies

performed with strain FMYD002 (Nehvonen, C., 2017) also showed better growth

patterns supplementing the medium with 1 mM VA than only growing the yeast with

LiBa.

5. References

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M., Hibbett, D. (2006). An overview of the higher level classification of

pucciniomycotina based on combined analyses of nuclear large and small subunit

rDNA sequences. Mycologia, 98(6), 896-905.

Aljanabi, S. M., & Martinez, I. (1997). Universal and rapid salt-extraction of high

quality genomic DNA for PCR-based techniques. Nucleic Acids Research, 25(22),

Beuchat, L. R., & Golden, D. A. (1989). Antimicrobials occurring naturally in foods.

Food Technology (USA),

Blackwell, M. (2011). The fungi: 1, 2, 3 ... 5.1 million species? American Journal of

Botany, 98(3), 426-438. doi:10.3732/ajb.1000298 [doi]

Boudet, A., Lapierre, C., & Grima‐Pettenati, J. (1995). Biochemistry and molecular

biology of lignification. New Phytologist, 129(2), 203-236.

Campbell, M. M., & Sederoff, R. R. (1996). Variation in lignin content and composition

Plant Physiology, 110(1), 3-13. doi:110/1/3 [pii]

Castoria, R., Caputo, L., De Curtis, F., & De Cicco, V. (2003). Resistance of

postharvest biocontrol yeasts to oxidative stress: A possible new mechanism of

action. Phytopathology, 93(5), 564-572.

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Growth and biodegradation by Sporidiobolales yeasts in ...hig.diva-portal.org/smash/get/diva2:1119832/FULLTEXT01.pdf · Vanillin (4-hydroxy-3-methoxybenzaldehyde), is the major component