Isolation and Characterization of Yeasts Able to...

Research ArticleIsolation and Characterization of Yeasts Able toAssimilate Sugarcane Bagasse Hemicellulosic Hydrolysate andProduce Xylitol Associated with Veturius transversus(Passalidae Coleoptera and Insecta)

Italo Thiago Silveira Rocha Matos Enedina Nogueira Assunccedilatildeo Edson Junior do CarmoVerena Makaren Soares and Spartaco Astolfi-Filho

Departamento de Genetica Universidade Federal do Amazonas Av Gal Rodrigo Octavio 3000 Manaus AM Brazil

Correspondence should be addressed to Italo Thiago Silveira Rocha Matos italo_matosufamedubr

Received 6 April 2017 Accepted 10 May 2017 Published 6 June 2017

Academic Editor Giuseppe Comi

Copyright copy 2017 Italo Thiago Silveira Rocha Matos et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Yeasts are an important component of insect gut microbial content playing roles such as degradation of polymers and toxiccompounds biological control and hormone vitamin and digestive enzyme production The xylophagous beetle gut is ahyperdiverse habitat and a potential source of new specieswith industrial abilities such as enzymeproduction pentose fermentationand biodetoxification In this work samples of Veturius transversus (Passalidae Coleoptera and Insecta) were collected fromthe Central Amazon Rainforest Their guts were dissected and a total of 20 microbial colonies were isolated using sugarcanebagasse hemicellulosic hydrolysate They were identified as having 10 distinct biochemical profiles and genetic analysis allowedidentification as three clades in the genera Candida Williopsis and Geotrichum All colonies were able to assimilate D-xyloseand 18 were able to produce xylitol especially a strain of Geotrichum with a maximum yield of 0502 gsdotgminus1 These results agreewith a previous prediction that the microbial community associated with xylophagous insects is a promising source of species ofbiotechnological interest

1 Introduction

Yeasts are microorganisms of the Fungi kingdom distributedin the phylaAscomycota Basidiomycota andDeuteromycota[1] On the other hand beetles are themost abundant order ofinsects (Coleoptera Insecta Arthropoda andMetazoa) withmore than 400000 species currently described [2]

The association of yeasts with beetles was decisive in theevolutionary success of these insects the microbiota beingindispensable to them playing fundamental roles such asin the synthesis of amino acids lipids pheromones anddigestive enzymes and in biodetoxification [3 4] Accordingto Suh et al [5] the microbial content of a xylophagousbeetlersquos gut is a hyperdiverse source of undescribed species

Xylitol is a promising polyol of five carbons with medicalapplications inmiddle ear otitis [6] andobesity prevention [7]

It is obtained from D-xylose reduction performed by micro-bial fermentation or a chemical process [8] As this latterreleases a high number of by-products demanding severalsteps for purification sampling efforts aiming to isolatemicrobes with ability to produce xylitol keep being necessary

Recent yeast and yeast-like fungi sampling efforts haveincreased the number of known species and strains ableto produce vitamins enzymes and other products fromfermentation of sugars such as ethanol and xylitol [9] In thisway isolation and characterization of wild-type yeasts andyeast-like fungi remains an important approach

The aim of this work was to isolate and characterize yeastsassociated with the xylophagous beetle Veturius transversus(Passalidae Coleoptera and Insecta) able to assimilate sug-arcane bagasse hemicellulosic hydrolysate (SBHH) as solecarbon source and produce xylitol by D-xylose fermentation

HindawiInternational Journal of MicrobiologyVolume 2017 Article ID 5346741 6 pageshttpsdoiorg10115520175346741

2 International Journal of Microbiology

2 Material and Methods

Under authorization (protocol number 34652-1) of theInstituto Chico Mendes de Conservacao da Biodiversidade(Brazilian authority for biodiversity access) 15 beetles werecollected from the Central Amazon Rainforest (3∘061015840052010158401015840S 59∘581015840231410158401015840 W) They were identified as V transversusa highly representative passalid beetle in this region Threesampleswere deposited in the Entomological collectionPauloBurnheim (UFAM Brazil)

The beetles were washed in 70 ethanol for 1min theelytra were removed and the gut was dissected Fragmentsof intestine of about 1 cm were incubated for 48 h (120 rpm28∘C) in tubes with 10mL of SBHH prepared as previouslydescribed [10 11] After this time 100 120583L of this suspensionwas spread on SBHH added to agar Yeasts and yeast-likecolonies were isolated in Petri dishes containing Sabouraudagar (yeast extract 10 gL glucose 40 gL agar 20 gL)

To evaluate their ability to ferment D-xylose a loopfulof each isolate was cultured in tubes containing 10mL ofYNBX medium (yeast nitrogen base without amino acids67 gL D-xylose 40 gL) After 7 days of incubation at28∘C and 120 rpm the medium content was centrifugedand the supernatant was analysed by an HPLC systemusing a Rezex RPM monosaccharide column (300 times 78mmPb2+ 8 Phenomenex) The D-xylose consumption rate ()was calculated according to the final and initial D-xyloseconcentrations Xylitol yield (gsdotgminus1) was calculated by theratio xylitol producedD-xylose consumed

For taxonomic identification biochemical characterizationwas performed using kit ID32C (BioMerieux) according tothemanufacturerrsquos instructionsThe resultswere plotted in theonline applicationApiWeb (httpsapiwebbiomerieuxcom)for physiological similarity identification

Furthermore the isolates were evaluated by genomicinternal transcribed spacer (ITS) and ribosomal genenucleotide sequences The DNA was extracted according toHarju et al [12] and amplified by PCR using primers ITS1(51015840 TCC GTA GGT GAA CCT GCC 31015840) and ITS4 (51015840 TCCTCC GCT TAT TGA TAT GC 31015840) The PCR products wereused to perform a sequencing reaction using a BigDyekit (Applied Biosystems) and nucleotide sequences wereobtained in an Applied Biosystems 3130 Genetic Analyzerautomatic sequencer

The obtained sequences were compared to the NCBIdatabase (httpswwwncbinlmnihgov) using BLAST(Basic Local Alignment Search Tool) and deposited inGenBank For phylogenetic relationship analysis nucleotidesequences were aligned using Clustal-W and analysed byneighbour-joining (bootstrap 2000 replicas) provided byMEGA 60 [13] Nucleotide sequences from the genomic ITSregion of Meyerozyma guilliermondii (GenBank JN974905)Trichosporon mycotoxinivorans (GenBank JX891097) andScheffersomyces stipitis (GenBank GU256745) were includedin the phylogenetic tree as reference groups this last beingthe external group

3 Results

A total of 20 colonies were isolated and evaluated corre-spondent to 10 species of four genera Candida (12 isolates)

Cryptococcus (five isolates) Debaryomyces (one isolate) andGeotrichum (two isolates) their biochemical profiles aredescribed in Table 1

Nucleotide sequence BLAST results identified three dif-ferent groups one close to Candida tropicalis another com-posed of members of the speciesWilliopsis saturnus and thethird composed of the genus Geotrichum sp The fragmentlength was about 550 bp for C tropicalis and W saturnuscontaining 18S rDNA (partial) ITS1 (complete) 58S rDNA(complete) ITS2 (complete) and 28S rDNA (partial) ForGeotrichum sp fragment length was on average 250 bpcontaining ITS1 (partial) 58S rDNA (complete) and ITS2(partial)

For isolates 07 16 and 18 identity greater than or equalto 99 allows us to conclude that these are members of thespeciesW saturnus Isolates 03 08 09 10 11 and 20 presentedidentity varying from 97 to 99 with C tropicalis makingit admissible that they are closely related to this specieshere named clade C tropicalis The other isolates presentedidentity varying from 92 to 96 with Geotrichum sp orGalactomyces spp

Considering the short length of the fragment whichallows classification only at genus level and the synonymybetween Geotrichum and Galactomyces they were classifiedas clade Geotrichum sp The complete BLAST results andGenBank accession number of sequences are presented inTable 2

Neighbour-joining phylogenetic analysis endorsed theconclusion about the three groups that clade W saturnusis close to Meyerozyma guilliermondii and Scheffersomycesstipitis whereas C tropicalis and Geotrichum sp are closelyrelated to Trichosporon mycotoxinivorans The phylogenetictree is presented in Figure 1

Fermentation tests indicated that none of the isolatesproduces ethanol using xylose as carbon source This resultwas expected because xylose fermentation to ethanol is anuncommon feature being presented by less than 1 of knownyeast species [14] However most of them were able toproduce xylitol only isolates 12 and 13 (Geotrichum sp) beingunable to do this The highest yield was observed in isolate01 (Geotrichum sp) reaching 0502 gsdotgminus1 and consuming926 of the D-xylose The complete results are presented inTable 3

4 Discussion

All isolates were able to assimilate D-xylose a commonfeature in yeasts able to metabolize SBHH because this isthe most abundant monosaccharide in hemicellulose [15]Candida is the most representative but that occurs becausethere are a great number of asexual phase (anamorph) speciesclassified in this genus which is a polyphyletic group [5 16]

The other genera Cryptococcus and Debaryomyces haveremarkable biotechnological potential in incorporation oflipids in their biomass being reported as oleaginous yeasts[17 18] Suh andBlackwell [19] describe the genusGeotrichumas dimorphic fungi being anamorphs of the genera Dipodas-cus andGalactomyces and growing being yeast-like accordingto environmental conditions

International Journal of Microbiology 3

Table1Biochemicalprofi

leso

fisolates

from

Vtra

nsversusidentified

accordingto

ApiW

eb(BioMerieux

)Isolate

GAL

ACT

SAC

NAG

LAT

ARA

CEL

RAF

MAL

TER

2KG

MDG

SOR

XYL

RIB

GLY

RHA

PLE

ERY

MEL

GRT

MLZ

GNT

LVT

MAN

LAC

INO

GLU

SBE

GLN

ESC

Species

Similarity(

)

01+

++

++

++

++

++

++

++

++

++

++

++

++minusminus

+minus

++

Cryptococcus

humico

la984

02+minus

++

++

++

++

++

++

++

++minusminusminusminusminusminus

++

++minusminusminus

Cryptococcus

curvatus

mdash

03+minus

++minusminusminusminus

++

++

++minus

+minus

+minusminusminus

+minusminus

+minusminus

+minusminusminus

Debaryomyces

etchellsii

791

04+minus

++

++

++

++

++

++

++

++

++minusminus

+minus

+minusminus

+minusminusminus

Cand

ida

mem

branifa

-cie

nsmdash

05+minus

++minusminus

+minus

++

+minus

++

+minus

++minusminus

++

+minusminusminusminus

+minus

+minus

Cand

ida

interm

edia

mdash

06+

++


++

++

++minus

+minus

+minusminusminus

+minus

++

++

++

+minus

Cand

ida

parapsilosis

mdash

07+minus

++

++

++

++

++

++

++

++

++

++

++

++

++

++

+Cr

yptococcus

humico

la992

08+minus

++

++

++

++

++

++

++

++

++

++

++

++

++minus

+minus

Cryptococcus

humico

la997

09+minus

++

++

++

++

++

++

+minusminus

+minusminusminus

++

++minusminus

+minus

+minus

Cand

ida

tropicalis

536

10+minus

++

++

++

++

++

++

++

++

++

++

++

++minus

++

+minus

Cryptococcus

humico

la995

11+

++

++

+minusminus

++

+minus

++minus

+minus

++minusminus

+minusminus

+minusminus

+minusminus

+Ca

ndida

famata

mdash

12minus

+minusminusminusminusminusminusminusminusminusminusminus

+minus


+minusminusminus

Geotrichum

capitatum

977

13minus


+minus


+minusminus

+Geotrichum

capitatum

977

14+

++

++minusminusminus

++

++

++minus

+minus

+minusminusminus

+minusminus

+minusminus

+minusminus

+Ca

ndidasake

995

15+

++

++minusminus

++

++minus

++minus

+minus

+minusminusminus

+minusminusminusminusminus

++

+minus

Cand

idasake

990

16+

++

++

+minusminus

++

++

++minus

+minus

+minusminusminus

++minus

+minusminus

+minusminus

+Ca

ndida

parapsilosis

831

17+

++

++minus

+minus

++

++

++

++minus

+minusminusminus

+minusminus

+minus

++minus

+minus

Cand

ida

tropicalis

mdash

18+

++


++

++

++minus

+minus

+minusminusminus

+minus

++

++

++

+minus

Cand

idasake

954

19+

++

++minusminusminus

++

++

++minus

+minus

+minusminusminus

+minusminus

++minus

+minus

++

Cand

ida

tropicalis

944

20+minus

++minus

++minus

++

+minus

++

+minusminus

+minusminus

++

+minus

+minusminus

++

++

Cand

ida

interm

edia

957

GAL

galactoseAC

Tcycloh

exim

ideSA

CsucroseNAG

N-acetylglucosamineLA

TlacticacidA

RAarabino

seC

ELcellobioseRA

Fraffino

seM

AL

maltoseT

REtrehalose2KG

2keto-gluconateM

DG

120572-m

ethyl-g

lucopyrano

sideSO

Rsorbito

lXY

LxyloseR

IBriboseGLY

glycerolRH

Arhamno

seP

LEpalatinoseER

Yerythrito

lMEL

melibioseG

RTsod

ium

glucuron

ateMLZ

melezito

seG

NT

potassium

glucon

ateLV

Tlevulin

icacidM

ANm

annitolLA

ClactoseIN

Oino

sitolG

LUglucoseSBE

sorbo

seG

LNglucosamineES

Cesculin

ironcitrate


Table 2 Identification of isolates according to BLAST result and nucleotide sequence GenBank accession number

Isolate Species Max identity () Query coverage () 119890-value GenBank accession number01 Geotrichum sp 93 92 1119890 minus 119 KP27664402 Geotrichum sp 95 93 3119890 minus 131 KP27663603 Candida tropicalis 99 98 00 KP27664504 Geotrichum sp 94 100 1119890 minus 121 KP27663705 Galactomyces candidum 96 97 1119890 minus 131 KP27663806 Geotrichum sp 96 99 1119890 minus 126 KP27663907 Williopsis saturnus 99 100 00 KP25757508 Candida tropicalis 98 99 00 KP27664609 Candida tropicalis 99 99 00 KP27664710 Candida tropicalis 98 99 00 KP27664811 Candida tropicalis 98 99 00 KP27664912 Galactomyces candidum 96 98 1119890 minus 136 KP27664013 Galactomyces geotrichum 96 98 1119890 minus 136 KP27664114 Geotrichum sp 95 100 1119890 minus 125 KP27664215 Geotrichum sp 94 100 3119890 minus 102 KP28848816 Williopsis saturnus 99 100 00 KP25757417 Geotrichum sp 93 99 7119890 minus 129 KP28848718 Williopsis saturnus 99 100 00 KP25757319 Geotrichum sp 94 99 5119890 minus 134 KP27664320 Candida tropicalis 97 99 00 KP276650

Table 3 D-xylose consumption rate () and xylitol yield of eachisolate

Isolate D-xylose consumption rate () Xylitol yield (gsdotgminus1)01 926 050202 299 021003 334 021404 362 025505 307 018606 356 022407 310 016908 332 018009 340 017010 307 010011 1000 033912 275 0013 187 0014 1000 031515 1000 032616 1000 030417 414 030418 1000 034119 1000 037020 1000 0347

All biochemical profile results presented similarity withspecies able to perform pentose fermentation andor anotherprocess with biotechnological potential However according

to Barnett [20] biochemical profiles may be used as comple-mentary information but cannot be conclusive for taxonomicidentification because they can present high variation withit being recommended to evaluate genomics data Accordingto Hou-Rui et al [21] up to 1 of nucleotide substitutionin a ribosomal domain is permitted for strains of a singlebiological species rDNA sequence analysis being a simpleand reliable tool for taxonomic identification

The phylogenetic analysis endorses that predicted by Bar-nett [20] noticeable because clade W saturnus is composedof isolates with three different biochemical profiles whereasclade C tropicalis is composed of five different biochemicalprofiles (one of those C tropicalis) and clade Geotrichum haseight different biochemical profiles Furthermore there weresome isolates with the same biochemical profile distributedin all clades strengthening that hypothesis

The maximum theoretical yield for xylitol productionfrom D-xylose fermentation is 10 gsdotgminus1 Despite this asmicrobes produce xylitol as a compatible solute it is excretedin osmotic stress conditions and then consumed as themedium becomes less harsh [22] common yields range from40 to 70 [23] The highest yield value for microbialfermentation is reported byGranstrom et al [24] forCandidasp at 085 gsdotgminus1

With Geotrichum sp (isolate 01) being a wild-type strainwith the capability to produce xylitol like some industrialstrains it can be considered a promising xylitol-producingyeast This is the first work to report xylitol productionby wild-type yeast strains associated with beetles from theCentral Amazon Rainforest


Clade Geotrichum sp

Clade Candida tropicalis

Clade Williopsis saturnus

62

32

34

43

29

7

3

4

10

100

84

54

99

100

100

58

46

11

VT06

VT19

VT04

VT14

VT01

VT15

VT17

VT13

VT12

VT02

VT05

VT20

VT11

VT03

VT08

VT09

VT1017

33

VT07

VT16

VT18

Meyerozyma guilliermondii

P stipitis ATCC62970 (GU256745)

Trichosporon mycotoxinivorans

Figure 1 Neighbour-joining phylogenetic tree endorsing the distribution of the isolates into three groups

5 Conclusion

The yeast community associated with V transversus gut isrich in D-xylose-assimilating and xylitol-producing speciessome of which present potential close to industrial strainsGeotrichum is a highly representative group in this commu-nity

Geotrichum sp (isolate 01) presents high xylitol yieldreaching about 50 of the maximum theoretical yield andis a promising xylitol-producing strain

Subsequent efforts must be concentrated on developingbioprocesses using these isolates

Conflicts of Interest

The authors declare that there are no conflicts of interest

Acknowledgments

Special thanks are due to Coordenacao de Aperfeicoamentode Pessoal de Nıvel Superior (CAPES) Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) andFundacao de Amparo a Pesquisa do Estado do Amazonas(FAPEAM) The authors are also grateful to Dra NairOtaviano Aguiar for beetle identification

References

[1] C J ALexopoulos C W Mims and M Blackwell IntroductoryMycology JohnWiley amp Sons New York NY USA 4th edition1996

[2] A S Lelej and S Y Storozhenko ldquoInsect taxonomic diversity inthe Russian Far Eastrdquo Entomological Review vol 90 no 3 pp372ndash386 2010


[3] T S Davis ldquoThe Ecology of Yeasts in the Bark Beetle HolobiontA Century of Research RevisitedrdquoMicrobial Ecology vol 69 no4 pp 723ndash732 2015

[4] S-O Suh C J Marshall J V McHugh and M BlackwellldquoWood ingestion by passalid beetles in the presence of xylose-fermenting gut yeastsrdquo Molecular Ecology vol 12 no 11 pp3137ndash3145 2003

[5] S-O Suh N H Nguyen andM Blackwell ldquoNine new Candidaspecies near C membranifaciens isolated from insectsrdquo Myco-logical Research vol 109 no 9 pp 1045ndash1056 2005

[6] A Azarpazhooh H Limeback H P Lawrence and P S ShahldquoXylitol for preventing acute otitis media in children up to 12years of agerdquo Cochrane database of systematic reviews no 11Article ID CD007095 2011

[7] J C Parajo H Domınguez and J M Domınguez ldquoBiotech-nological production of xylitol Part 1 Interest of xylitol andfundamentals of its biosynthesisrdquo Bioresource Technology vol65 no 3 pp 191ndash201 1998

[8] Z D V L Mayerhoff I C Roberto and S S Silva ldquoXylitolproduction from rice straw hemicellulose hydrolysate usingdifferent yeast strainsrdquo Biotechnology Letters vol 19 no 5 pp407ndash409 1997

[9] H Urbina J Schuster and M Blackwell ldquoThe gut ofGuatemalan passalid beetles A habitat colonized bycellobiose-and xylose-fermenting yeastsrdquo Fungal Ecology vol 6 no 5 pp339ndash355 2013

[10] I T S R Matos L A Cassa-Barbosa R S Medeiros GalvaoC G Nunes-Silva and S Astolfi Filho ldquoIsolation taxonomicidentification and investigation of the biotechnological poten-tial of wild-type Meyerozyma guilliermondii associated withAmazonian termites able to ferment D-xyloserdquo BioscienceJournal vol 30 no 1 pp 260ndash266 2014

[11] I T S R Matos L A Cassa-Barbosa P Q Costa Neto and SAstolfi Filho ldquoCultivation of Trichosporon mycotoxinivoransin sugarcane bagasse hemicellulosic hydrolyzaterdquo ElectronicJournal of Biotechnology vol 15 no 1 2012

[12] S Harju H Fedosyuk and K R Peterson ldquoRapid isolation ofyeast genomic DNA Bust nrsquo Grabrdquo BMC Biotechnology vol 4article no 8 2004

[13] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 Molecular Evolutionary Genetics Analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013

[14] B Hahn-Hagerdal H B K Karhumaa C Fonseca I Spencer-Martins and M F Gorwa-Grauslund ldquoTowards industrialpentose-fermenting yeast strainsrdquo Applied Microbiology andBiotechnology vol 74 no 5 pp 937ndash953 2007

[15] B C Saha ldquoHemicellulose bioconversionrdquo Journal of IndustrialMicrobiology and Biotechnology vol 30 no 5 pp 279ndash2912003

[16] F Schauer and R Hanschke ldquoTaxonomy and ecology of thegenus CandidardquoMycoses vol 42 no S1 pp 12ndash21 1999

[17] J M Ageitos J A Vallejo P Veiga-Crespo and T G VillaldquoOily yeasts as oleaginous cell factoriesrdquo Applied Microbiologyand Biotechnology vol 90 no 4 pp 1219ndash1227 2011

[18] E A Johnson ldquoBiotechnology of non-Saccharomyces yeasts -The ascomycetesrdquo Applied Microbiology and Biotechnology vol97 no 2 pp 503ndash517 2013

[19] S-O Suh and M Blackwell ldquoThree new asexual arthroconidialyeasts Geotrichum carabidarum sp nov Geotrichum histeri-darum sp nov andGeotrichumcucujoidarum sp nov isolated

from the gut of insectsrdquoMycological Research vol 110 no 2 pp220ndash228 2006

[20] J A Barnett ldquoA history of research on yeasts 8 TaxonomyrdquoYeast vol 21 no 14 pp 1141ndash1193 2004

[21] Z Hou-Rui Q Xiang-Xiang S S Silva et al ldquoNovel isolates forbiological detoxification of lignocellulosic hydrolysaterdquo AppliedBiochemistry and Biotechnology vol 152 no 2 pp 199ndash2122009

[22] G M Walker Yeast Physiology and Biotechnology John Wileyamp Sons New York NY USA 1998

[23] J S Aranda-Barradas C Garibay-Orijel J A Badillo-Coronaand E Salgado-Manjarrez ldquoA stoichiometric analysis of biologi-cal xylitol productionrdquo Biochemical Engineering Journal vol 50no 1-2 pp 1ndash9 2010

[24] T B Granstrom K Izumori andM Leisola ldquoA rare sugar xyli-tol Part II Biotechnological production and future applicationsof xylitolrdquo Applied Microbiology and Biotechnology vol 74 no2 pp 273ndash276 2007

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Microbiology


2 Material and Methods

Under authorization (protocol number 34652-1) of theInstituto Chico Mendes de Conservacao da Biodiversidade(Brazilian authority for biodiversity access) 15 beetles werecollected from the Central Amazon Rainforest (3∘061015840052010158401015840S 59∘581015840231410158401015840 W) They were identified as V transversusa highly representative passalid beetle in this region Threesampleswere deposited in the Entomological collectionPauloBurnheim (UFAM Brazil)

The beetles were washed in 70 ethanol for 1min theelytra were removed and the gut was dissected Fragmentsof intestine of about 1 cm were incubated for 48 h (120 rpm28∘C) in tubes with 10mL of SBHH prepared as previouslydescribed [10 11] After this time 100 120583L of this suspensionwas spread on SBHH added to agar Yeasts and yeast-likecolonies were isolated in Petri dishes containing Sabouraudagar (yeast extract 10 gL glucose 40 gL agar 20 gL)

To evaluate their ability to ferment D-xylose a loopfulof each isolate was cultured in tubes containing 10mL ofYNBX medium (yeast nitrogen base without amino acids67 gL D-xylose 40 gL) After 7 days of incubation at28∘C and 120 rpm the medium content was centrifugedand the supernatant was analysed by an HPLC systemusing a Rezex RPM monosaccharide column (300 times 78mmPb2+ 8 Phenomenex) The D-xylose consumption rate ()was calculated according to the final and initial D-xyloseconcentrations Xylitol yield (gsdotgminus1) was calculated by theratio xylitol producedD-xylose consumed

For taxonomic identification biochemical characterizationwas performed using kit ID32C (BioMerieux) according tothemanufacturerrsquos instructionsThe resultswere plotted in theonline applicationApiWeb (httpsapiwebbiomerieuxcom)for physiological similarity identification

Furthermore the isolates were evaluated by genomicinternal transcribed spacer (ITS) and ribosomal genenucleotide sequences The DNA was extracted according toHarju et al [12] and amplified by PCR using primers ITS1(51015840 TCC GTA GGT GAA CCT GCC 31015840) and ITS4 (51015840 TCCTCC GCT TAT TGA TAT GC 31015840) The PCR products wereused to perform a sequencing reaction using a BigDyekit (Applied Biosystems) and nucleotide sequences wereobtained in an Applied Biosystems 3130 Genetic Analyzerautomatic sequencer

The obtained sequences were compared to the NCBIdatabase (httpswwwncbinlmnihgov) using BLAST(Basic Local Alignment Search Tool) and deposited inGenBank For phylogenetic relationship analysis nucleotidesequences were aligned using Clustal-W and analysed byneighbour-joining (bootstrap 2000 replicas) provided byMEGA 60 [13] Nucleotide sequences from the genomic ITSregion of Meyerozyma guilliermondii (GenBank JN974905)Trichosporon mycotoxinivorans (GenBank JX891097) andScheffersomyces stipitis (GenBank GU256745) were includedin the phylogenetic tree as reference groups this last beingthe external group

3 Results

A total of 20 colonies were isolated and evaluated corre-spondent to 10 species of four genera Candida (12 isolates)

Cryptococcus (five isolates) Debaryomyces (one isolate) andGeotrichum (two isolates) their biochemical profiles aredescribed in Table 1

Nucleotide sequence BLAST results identified three dif-ferent groups one close to Candida tropicalis another com-posed of members of the speciesWilliopsis saturnus and thethird composed of the genus Geotrichum sp The fragmentlength was about 550 bp for C tropicalis and W saturnuscontaining 18S rDNA (partial) ITS1 (complete) 58S rDNA(complete) ITS2 (complete) and 28S rDNA (partial) ForGeotrichum sp fragment length was on average 250 bpcontaining ITS1 (partial) 58S rDNA (complete) and ITS2(partial)

For isolates 07 16 and 18 identity greater than or equalto 99 allows us to conclude that these are members of thespeciesW saturnus Isolates 03 08 09 10 11 and 20 presentedidentity varying from 97 to 99 with C tropicalis makingit admissible that they are closely related to this specieshere named clade C tropicalis The other isolates presentedidentity varying from 92 to 96 with Geotrichum sp orGalactomyces spp

Considering the short length of the fragment whichallows classification only at genus level and the synonymybetween Geotrichum and Galactomyces they were classifiedas clade Geotrichum sp The complete BLAST results andGenBank accession number of sequences are presented inTable 2

Neighbour-joining phylogenetic analysis endorsed theconclusion about the three groups that clade W saturnusis close to Meyerozyma guilliermondii and Scheffersomycesstipitis whereas C tropicalis and Geotrichum sp are closelyrelated to Trichosporon mycotoxinivorans The phylogenetictree is presented in Figure 1

Fermentation tests indicated that none of the isolatesproduces ethanol using xylose as carbon source This resultwas expected because xylose fermentation to ethanol is anuncommon feature being presented by less than 1 of knownyeast species [14] However most of them were able toproduce xylitol only isolates 12 and 13 (Geotrichum sp) beingunable to do this The highest yield was observed in isolate01 (Geotrichum sp) reaching 0502 gsdotgminus1 and consuming926 of the D-xylose The complete results are presented inTable 3

4 Discussion

All isolates were able to assimilate D-xylose a commonfeature in yeasts able to metabolize SBHH because this isthe most abundant monosaccharide in hemicellulose [15]Candida is the most representative but that occurs becausethere are a great number of asexual phase (anamorph) speciesclassified in this genus which is a polyphyletic group [5 16]

The other genera Cryptococcus and Debaryomyces haveremarkable biotechnological potential in incorporation oflipids in their biomass being reported as oleaginous yeasts[17 18] Suh andBlackwell [19] describe the genusGeotrichumas dimorphic fungi being anamorphs of the genera Dipodas-cus andGalactomyces and growing being yeast-like accordingto environmental conditions



leso

fisolates

from

Vtra

nsversusidentified

accordingto

ApiW

eb(BioMerieux

)Isolate

GAL

ACT

SAC

NAG

LAT

ARA

CEL

RAF

MAL

TER

2KG

MDG

SOR

XYL

RIB

GLY

RHA

PLE

ERY

MEL

GRT

MLZ

GNT

LVT

MAN

LAC

INO

GLU

SBE

GLN

ESC

Species

Similarity(

)

01+

++

++

++

++

++

++

++

++

++

++

++

++minusminus

+minus

++

Cryptococcus

humico

la984

02+minus

++

++

++

++

++

++

++


++

++minusminusminus

Cryptococcus

curvatus

mdash

03+minus


++

++

++minus

+minus

+minusminusminus

+minusminus

+minusminus

+minusminusminus

Debaryomyces

etchellsii

791

04+minus

++

++

++

++

++

++

++

++

++minusminus

+minus

+minusminus

+minusminusminus

Cand

ida

mem

branifa

-cie

nsmdash

05+minus

++minusminus

+minus

++

+minus

++

+minus

++minusminus

++


+minus

+minus

Cand

ida

interm

edia

mdash

06+

++


++

++

++minus

+minus

+minusminusminus

+minus

++

++

++

+minus

Cand

ida

parapsilosis

mdash

07+minus

++

++

++

++

++

++

++

++

++

++

++

++

++

++

+Cr

yptococcus

humico

la992

08+minus

++

++

++

++

++

++

++

++

++

++

++

++

++minus

+minus

Cryptococcus

humico

la997

09+minus

++

++

++

++

++

++

+minusminus

+minusminusminus

++

++minusminus

+minus

+minus

Cand

ida

tropicalis

536

10+minus

++

++

++

++

++

++

++

++

++

++

++

++minus

++

+minus

Cryptococcus

humico

la995

11+

++

++

+minusminus

++

+minus

++minus

+minus

++minusminus

+minusminus

+minusminus

+minusminus

+Ca

ndida

famata

mdash

12minus


+minus


+minusminusminus

Geotrichum

capitatum

977

13minus


+minus


+minusminus

+Geotrichum

capitatum

977

14+

++

++minusminusminus

++

++

++minus

+minus

+minusminusminus

+minusminus

+minusminus

+minusminus

+Ca

ndidasake

995

15+

++

++minusminus

++

++minus

++minus

+minus

+minusminusminus


++

+minus

Cand

idasake

990

16+

++

++

+minusminus

++

++

++minus

+minus

+minusminusminus

++minus

+minusminus

+minusminus

+Ca

ndida

parapsilosis

831

17+

++

++minus

+minus

++

++

++

++minus

+minusminusminus

+minusminus

+minus

++minus

+minus

Cand

ida

tropicalis

mdash

18+

++


++

++

++minus

+minus

+minusminusminus

+minus

++

++

++

+minus

Cand

idasake

954

19+

++

++minusminusminus

++

++

++minus

+minus

+minusminusminus

+minusminus

++minus

+minus

++

Cand

ida

tropicalis

944

20+minus

++minus

++minus

++

+minus

++

+minusminus

+minusminus

++

+minus

+minusminus

++

++

Cand

ida

interm

edia

957

GAL

galactoseAC

Tcycloh

exim

ideSA

CsucroseNAG


TlacticacidA

RAarabino

seC

ELcellobioseRA

Fraffino

seM

AL

maltoseT

REtrehalose2KG

2keto-gluconateM

DG

120572-m

ethyl-g

lucopyrano

sideSO

Rsorbito

lXY

LxyloseR

IBriboseGLY

glycerolRH

Arhamno

seP

LEpalatinoseER

Yerythrito

lMEL

melibioseG

RTsod

ium

glucuron

ateMLZ

melezito

seG

NT

potassium

glucon

ateLV

Tlevulin

icacidM

ANm

annitolLA

ClactoseIN

Oino

sitolG

LUglucoseSBE

sorbo

seG

LNglucosamineES

Cesculin

ironcitrate












Clade Geotrichum sp



62

32

34

43

29

7

3

4

10

100

84

54

99

100

100

58

46

11

VT06

VT19

VT04

VT14

VT01

VT15

VT17

VT13

VT12

VT02

VT05

VT20

VT11

VT03

VT08

VT09

VT1017

33

VT07

VT16

VT18





5 Conclusion






Acknowledgments


References


































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



leso

fisolates

from

Vtra

nsversusidentified

accordingto

ApiW

eb(BioMerieux

)Isolate

GAL

ACT

SAC

NAG

LAT

ARA

CEL

RAF

MAL

TER

2KG

MDG

SOR

XYL

RIB

GLY

RHA

PLE

ERY

MEL

GRT

MLZ

GNT

LVT

MAN

LAC

INO

GLU

SBE

GLN

ESC

Species

Similarity(

)

01+

++

++

++

++

++

++

++

++

++

++

++

++minusminus

+minus

++

Cryptococcus

humico

la984

02+minus

++

++

++

++

++

++

++


++

++minusminusminus

Cryptococcus

curvatus

mdash

03+minus


++

++

++minus

+minus

+minusminusminus

+minusminus

+minusminus

+minusminusminus

Debaryomyces

etchellsii

791

04+minus

++

++

++

++

++

++

++

++

++minusminus

+minus

+minusminus

+minusminusminus

Cand

ida

mem

branifa

-cie

nsmdash

05+minus

++minusminus

+minus

++

+minus

++

+minus

++minusminus

++


+minus

+minus

Cand

ida

interm

edia

mdash

06+

++


++

++

++minus

+minus

+minusminusminus

+minus

++

++

++

+minus

Cand

ida

parapsilosis

mdash

07+minus

++

++

++

++

++

++

++

++

++

++

++

++

++

++

+Cr

yptococcus

humico

la992

08+minus

++

++

++

++

++

++

++

++

++

++

++

++

++minus

+minus

Cryptococcus

humico

la997

09+minus

++

++

++

++

++

++

+minusminus

+minusminusminus

++

++minusminus

+minus

+minus

Cand

ida

tropicalis

536

10+minus

++

++

++

++

++

++

++

++

++

++

++

++minus

++

+minus

Cryptococcus

humico

la995

11+

++

++

+minusminus

++

+minus

++minus

+minus

++minusminus

+minusminus

+minusminus

+minusminus

+Ca

ndida

famata

mdash

12minus


+minus


+minusminusminus

Geotrichum

capitatum

977

13minus


+minus


+minusminus

+Geotrichum

capitatum

977

14+

++

++minusminusminus

++

++

++minus

+minus

+minusminusminus

+minusminus

+minusminus

+minusminus

+Ca

ndidasake

995

15+

++

++minusminus

++

++minus

++minus

+minus

+minusminusminus


++

+minus

Cand

idasake

990

16+

++

++

+minusminus

++

++

++minus

+minus

+minusminusminus

++minus

+minusminus

+minusminus

+Ca

ndida

parapsilosis

831

17+

++

++minus

+minus

++

++

++

++minus

+minusminusminus

+minusminus

+minus

++minus

+minus

Cand

ida

tropicalis

mdash

18+

++


++

++

++minus

+minus

+minusminusminus

+minus

++

++

++

+minus

Cand

idasake

954

19+

++

++minusminusminus

++

++

++minus

+minus

+minusminusminus

+minusminus

++minus

+minus

++

Cand

ida

tropicalis

944

20+minus

++minus

++minus

++

+minus

++

+minusminus

+minusminus

++

+minus

+minusminus

++

++

Cand

ida

interm

edia

957

GAL

galactoseAC

Tcycloh

exim

ideSA

CsucroseNAG


TlacticacidA

RAarabino

seC

ELcellobioseRA

Fraffino

seM

AL

maltoseT

REtrehalose2KG

2keto-gluconateM

DG

120572-m

ethyl-g

lucopyrano

sideSO

Rsorbito

lXY

LxyloseR

IBriboseGLY

glycerolRH

Arhamno

seP

LEpalatinoseER

Yerythrito

lMEL

melibioseG

RTsod

ium

glucuron

ateMLZ

melezito

seG

NT

potassium

glucon

ateLV

Tlevulin

icacidM

ANm

annitolLA

ClactoseIN

Oino

sitolG

LUglucoseSBE

sorbo

seG

LNglucosamineES

Cesculin

ironcitrate












Clade Geotrichum sp



62

32

34

43

29

7

3

4

10

100

84

54

99

100

100

58

46

11

VT06

VT19

VT04

VT14

VT01

VT15

VT17

VT13

VT12

VT02

VT05

VT20

VT11

VT03

VT08

VT09

VT1017

33

VT07

VT16

VT18





5 Conclusion






Acknowledgments


References


































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












Clade Geotrichum sp



62

32

34

43

29

7

3

4

10

100

84

54

99

100

100

58

46

11

VT06

VT19

VT04

VT14

VT01

VT15

VT17

VT13

VT12

VT02

VT05

VT20

VT11

VT03

VT08

VT09

VT1017

33

VT07

VT16

VT18





5 Conclusion






Acknowledgments


References


































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Isolation and Characterization of Yeasts Able to...

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