Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.researchgate.net/publication/283329257

Exceptionalhexose-fermentingabilityofthexylitol-producingyeastCandidaguilliermondiiFTI20037

ArticleinJournalofBioscienceandBioengineering·May2016

DOI:10.1016/j.jbiosc.2015.10.011

CITATIONS

2

READS

62

8authors,including:

NajjapakSooksawat

5PUBLICATIONS27CITATIONS

SEEPROFILE

NicoleKrystynaHarner

UniversityofGuelph

7PUBLICATIONS64CITATIONS

SEEPROFILE

ParamjitBajwa

UniversityofGuelph

22PUBLICATIONS758CITATIONS

SEEPROFILE

HungLee

UniversityofGuelph

259PUBLICATIONS9,654CITATIONS

SEEPROFILE

AllcontentfollowingthispagewasuploadedbyNajjapakSooksawaton04January2017.

Theuserhasrequestedenhancementofthedownloadedfile.Allin-textreferencesunderlinedinblueareaddedtotheoriginaldocumentandarelinkedtopublicationsonResearchGate,lettingyouaccessandreadthemimmediately.

www.elsevier.com/locate/jbiosc

Journal of Bioscience and BioengineeringVOL. 121 No. 6, 631e637, 2016

Exceptional hexose-fermenting ability of the xylitol-producing yeastCandida guilliermondii FTI 20037

Xin Wen,1 Sukhdeep Sidhu,1 Spencer K.C. Horemans,1 Najjapak Sooksawat,1,2,x Nicole K. Harner,1

Paramjit K. Bajwa,1 Zhirun Yuan,3 and Hung Lee1,*

School of Environmental Sciences, University of Guelph, Guelph, ON N1G 2W1, Canada,1 Department of Biology, Mahidol University, Bangkok 10400, Thailand,2 and FPInnovations,570 Boulevard St-Jean, Pointe-Claire, QC H9R 3J9, Canada3

Received 8 June 2015; accepted 20 October 2015Available online 18 November 2015

* CorrespondE-mail add

x Current addAgricultural an

1389-1723/$http://dx.doi

The yeast Candida guilliermondii FTI 20037 is well-known for its ability to produce xylitol from xylose. Recently, thisstrain was found to produce greater than 5% (w/v) ethanol from glucose. This level of ethanol is typically not exceeded bywild-type strains of other native pentose-fermenting yeasts. This prompted the current study to examine the ability ofC. guilliermondii FTI 20037 to utilize and ferment high concentrations of each of the hexoses commonly found inlignocellulosic hydrolysates. In defined media, FTI 20037 fermented 14.4%e25.9% (w/v) of glucose, mannose or galactoseindividually to ethanol in concentrations ranging from 6% to 9.3% (w/v). Fermentation was completed within 36 h (forglucose) to 100 h (for galactose). In 25.9% (w/v) glucose, FTI 20037 produced 9.3% (w/v) ethanol within 40 h. FTI 20037produced xylitol exclusively when xylose was given as the sole carbon source. The strain utilized arabinose poorly. Underthe same fermentation conditions, an industrial Saccharomyces cerevisiae strain produced slightly higher levels ofethanol [9.9% (w/v)] from 25.0% (w/v) glucose. Another pentose-fermenting yeast Pachysolen tannophilus also fermentedhigh concentrations of glucose and mannose to produce relatively high peak ethanol concentrations; however, this yeastrequired considerably longer to completely consume these hexoses. The ability of FTI 20037 to produce high level ofethanol rapidly from glucose is remarkable. To our knowledge, this is the first known instance of a non-modified nativexylose-fermenting yeast strain able to produce such high levels of ethanol from glucose as rapidly as S. cerevisiae in adefined medium.

� 2015 The Society for Biotechnology, Japan. All rights reserved.

[Key words: Ethanol; Fermentation; Hexose; Xylitol; Xylose; Yeast]

Xylose is an aldopentose that occurs primarily in polymer formas xylan in the hemicellulose portion of plant cell walls. Next toglucose, xylose is the second most abundant renewable sugar innature, forming up to 25% of the total dry weight of some forestryand agricultural residues. The efficient utilization of pentose sugarsis, therefore, important in the overall bioconversion of plantbiomass for the production of liquid fuels and chemicals. However,xylose is not as readily fermented as glucose by yeasts. Nativestrains of the fermentative yeast Saccharomyces cerevisiae are un-able to utilize xylose as a sole carbon source (1). In this connection,the discovery of xylose-fermenting yeasts in the early 1980s (2e4)was considered a milestone as it lends hope that xylose can also befermented (5). Since this initial discovery, numerous xylose-fermenting yeasts have been reported. Among the better xylose-fermenting yeasts, three categories are known: (i) those such asScheffersomyces (Candida) shehatae and Scheffersomyces (Pichia)stipitis that ferment xylose almost exclusively to ethanol (5); (ii)those such as Candida guilliermondii (6,7) and some Candida tropi-calis strains (8) that produce mostly xylitol from xylose; and (iii)

ing author. Tel.: þ1 519 824 4120x53828; fax: þ1 519 837 0442.ress: hlee@uoguelph.ca (H. Lee).ress: Rajamangala University of Technology Tawan-ok, Faculty ofd Natural Resources, Chonburi, Thailand.

e see front matter � 2015 The Society for Biotechnology, Japan..org/10.1016/j.jbiosc.2015.10.011

Pachysolen tannophilus that produces a mixture of ethanol andxylitol (7). The reason(s) for the preferential production of ethanolor xylitol by these yeasts is not known.

C. guilliermondii FTI 20037 is a naturally occurring yeast strainthat was maintained at the culture collection of the Foundation forIndustrial Technology (FTI), São Paulo, Brazil. The strain was firstdeposited at the National Research Council Canada (NRCC) CultureCollection in 1985 and designated as NRC 5578. In the early 1990s,one of us (H. Lee) deposited this strain in the American Type CultureCollection (ATCC) where it is currently designated as C. guillier-mondii ATCC 201935 and is available to researchers worldwide.

In a yeast screening study carried out at NRCC, strain FTI 20037was found to be an excellent xylitol producer from xylose (6). Sincethis discovery in the late 1980s, many papers have been publishedon various aspects of xylose-to-xylitol fermentation by FTI 20037 inboth defined media and xylose-rich lignocellulosic hydrolysates. Insome studies, researchers have noted that low amounts of ethanolwere produced by C. guilliermondii, likely from the small amount ofhexoses present (9e12). However, given the almost exclusive focuson xylitol production by C. guilliermondii FTI 20037, its ability toproduce a small amount of ethanol was not considered significantand typically not investigated further.

Recently, we assessed the fermentative performance ofC. guilliermondii FTI 20037 in several lignocellulosic hydrolysates.

All rights reserved.

FIG. 1. Sugar fermentation by C. guilliermondii FTI 20037. (A) Glucose fermentation. Theinitial concentrations were 15.3% (round dotted line), 20.1% (dash line) and 25.9% (w/v)(solid line). (B) Mannose fermentation. The initial concentrations were 15.6% (dashline) and 20.3% (w/v) (solid line). (C) Galactose fermentation. The initial concentrationswere 14.4% (dash line) and 19.6% (w/v) (solid line). (D) Xylose fermentation. The initialconcentration was 10.5% (w/v). Values shown are means� SEM of three independent

FIG. 2. Sugar fermentation by S. cerevisiae T2. (A) Glucose fermentation. The initialconcentrations were 14.4% (round dotted line), 18.3% (dash line) and 25.0% (w/v) (solidline). (B) Mannose fermentation. The initial concentrations were 15.4% (dash line) and20.6% (w/v) (solid line). (C) Galactose fermentation. The initial concentrations were17.7% (dash line) and 22.6% (w/v) (solid line). Values shown are means� SEM of threeindependent experiments. Crosses indicate sugar utilization trend; closed circlesindicate ethanol production.

632 WEN ET AL. J. BIOSCI. BIOENG.,

One of the hydrolysates contained considerably higher hexosecontent than pentoses. Interestingly, greater than 5% (w/v) ethanolwas produced rapidly by FTI 20037 in this hydrolysate (data notshown). This level is considered to be near the high end of peakethanol concentrations reported to be produced by some well-known native pentose-fermenting yeasts such as S. stipitis,S. shehatae (13,14) and P. tannophilus (15). This led to a series ofexperiments described below to specifically examine the ability of

experiments. Crosses indicate sugar utilization trend; closed circles indicate ethanolproduction and open triangles indicate xylitol production (dotted line).

FIG. 3. Sugar fermentation by P. tannophilus NRRL Y-2460. (A) Glucose fermentation.The initial concentrations were 15.9% (round dotted line), 20.5% (dash line) and 26.6%(w/v) (solid line). (B) Mannose fermentation. The initial concentrations were 15.0%(dash line) and 20.5% (w/v) (solid line). (C) Galactose fermentation. The initial

VOL. 121, 2016 HEXOSE FERMENTATION BY C. GUILLIERMONDII 633

FTI 20037 to ferment high concentrations of three hexoses (glucose,mannose and galactose) in defined liquid medium. These threehexoses are commonly found in lignocellulosic hydrolysates. Ourresults established the remarkable ability of strain FTI 20037 toproduce high concentrations of ethanol from each of these hexosesugars. S. cerevisiae strain T2, used for fermentation of spent sulfiteliquor (SSL) at the Tembec Alcohol Plant, as well as three othernative pentose-fermenting yeasts, P. tannophilus NRRL Y-2460,S. shehatae NRC 2886 and S. stipitis NRRL Y-7124, were tested underthe same conditions for comparison.

MATERIALS AND METHODS

Microorganisms and media C. guilliermondii FTI 20037 (ATCC 201935, NRC5578), S. shehatae NRC 2886 (ATCC 34887; NRRLY-12858) and S. stipitis NRRL Y-7124(NRC 2548; ATCC 58376) were obtained from the NRCC Culture Collection (Ottawa,ON, Canada). P. tannophilusNRRLY-2460 (ATCC 32691) was kindly provided by CletusKurtzman (USDA, Peoria, IL, USA). A SSL-adapted S. cerevisiae T2 strain used in SSLfermentation at the Tembec Alcohol Plant (16), was kindly provided by JurajStrmen (formerly of Tembec, Témiscaming, QC, Canada). All sugars were purchasedfrom SigmaeAldrich (Oakville, ON, Canada). All culture media components werepurchased from Thermo Fisher Scientific Company (Ottawa, ON, Canada).

Culture maintenance and inoculum preparation Yeast strains weremaintained individually on YEPD (yeast extract, peptone, dextrose) agar plates at4 �C and subcultured periodically. For inoculum preparation, a loopful of cells from adistinct colony grown on the YEPD plate was used to inoculate 100 mL of definedmedia containing 0.67% (w/v) yeast nitrogen base (YNB) without amino acid orammonium sulphate supplemented with 0.225% (w/v) urea and 2% (w/v) of eitherxylose for C. guilliermondii and other pentose-fermenting yeasts or glucose forS. cerevisiae in 250-mL Erlenmeyer flasks. The cultures were incubated at28� 2 �C for 48 h with gyratory shaking at 180 rpm as described by Bajwa et al.(17). The OD600 values of these cultures, measured using an Ultraspec 3100 ProUV/Visible spectrophotometer (Biochrom Ltd., UK), were about 7e8 (1.7e1.9 g drycell weight/L).

Fermentation experiments The methods used for fermentation studieswere as described by Bajwa et al. (17,18). Briefly, cells from the 48-h inoculumculture were centrifuged at 3700 �g for 15 min at 21�2 �C in a Sorvall centrifuge(Thermo Fisher Scientific). The cell pellet was washed with sterile deionized waterand re-suspended in 100 mL of defined media supplemented with varyingconcentrations of one of the test sugars (glucose, mannose, galactose, arabinose orxylose) in a 250-mL Erlenmeyer flask. The sugar concentrations tested rangedfrom 10% to 25.9% (w/v). Xylose fermentation was performed as a control toconfirm the ability of strain FTI 20037 and other pentose-fermenting yeasts toconvert xylose to ethanol and/or xylitol. The flasks were incubated at 28� 2 �Cwith gyratory shaking at 180 rpm. Samples (1.75 mL) were withdrawnperiodically for sugar and ethanol analysis. Samples were centrifuged at 17,000 �gfor 5 min; the supernatant was collected and stored at �20 �C before analysis.Each fermentation experiment was done at least three times using independentlygrown inocula. The mean and SEM were calculated and shown as values witherror bars in figures.

Analytical methods Sugar and ethanol concentrations were analyzed usingan Agilent-1200 HPLC system equipped with a BioRad Aminex HPX-87H(300 � 7.8 mm) column with 5 mM sulphuric acid as the mobile phase running at0.6 mL/min at 40 �C as previously described (19). Sugar samples were dilutedbefore HPLC analysis when the initial sugar concentration was above 10% (w/v).

RESULTS AND DISCUSSION

Native xylose-fermenting yeasts can ferment the dominanthexose and pentose sugars in lignocellulosic hydrolysates individ-ually to ethanol. However, the rate and yield of ethanol producedfrom pentoses are considerably lower compared to hexosefermentation by native strains of the fermentative yeastS. cerevisiae (5). Moreover, some well-known native xylose-fermenting yeasts exhibit low ethanol tolerance (19) which canlimit the efficient fermentation of sugars in lignocellulosic

concentrations were 15.7% (dash line) and 20.8% (w/v) (solid line). (D) Xylosefermentation. The initial concentration was 10.3% (w/v). Values shown are mean-s� SEM of three independent experiments. Crosses indicate sugar utilization trend;closed circles indicate ethanol production and open triangles indicate xylitol produc-tion (dotted line).

FIG. 4. Sugar fermentation by S. shehatae NRC 2886. (A) Glucose fermentation. Theinitial concentrations were 15.7% (round dotted line), 20.0% (dash line) and 25.6% (w/v)(solid line). (B) Mannose fermentation. The initial concentrations were 15.2% (dashline) and 21.1% (w/v) (solid line). (C) Galactose fermentation. The initial concentrations

634 WEN ET AL. J. BIOSCI. BIOENG.,

hydrolysates to ethanol. As an example, S. stipitis NRRL Y-7124 wasreported to produce a peak ethanol concentration of 5.7% (w/v)from 20% (w/v) xylose in a yeast extract-containing medium, whileseveral other pentose-fermenting yeast strains such as S. shehataeNRRL Y-12856 and NRRL Y-12857 and S. stipitis NRRL Y-11545 couldnot completely consume the sugar at this high initial concentrationand produced lower peak ethanol levels of 1.6%e2.6% (w/v) (20).Furthermore, fermentation by some strains of S. shehatae andS. stipitis was inhibited by concentrations of ethanol as low as 4%(w/v) (14). P. tannophilus NRRL Y-2460 was reported to produce5.5% (w/v) ethanol from 12% (w/v) glucose (15). In another study,the peak ethanol concentrations produced by Spathaspora passali-darum from 10% (w/v) xylose or 10% (w/v) glucose in a definedmedium were reported to be 3.74% and 3.14% (w/v), respectively(21). Thus, it is generally thought that themaximum concentrationsof ethanol produced from any sugars by native pentose-fermentingyeasts do not exceed 6% (w/v).

Glucose fermentation C. guilliermondii FTI 20037 is knownto produce high levels of xylitol from xylose. To our knowledge, itsability to ferment high concentrations of hexoses has not beenexamined. In this study, we specifically examined the ability of FTI20037 to ferment high concentrations of three hexoses (glucose,mannose and galactose) in defined liquid medium.

In defined medium, glucose was readily consumed by FTI 20037and fermented to high levels of ethanol. The strain completelyconsumed 15.3% (w/v) glucose in 21 h, producing a peak ethanolconcentration of 5.3% (w/v) (Fig. 1A). Higher concentrations ofglucose [20.1% and 25.9% (w/v)] were also completely utilized in 27and 36 h, respectively, and high maximum ethanol concentrations[7.3% and 9.3% (w/v), respectively] were produced. The yields were0.35, 0.36 and 0.36 g ethanol/g glucose consumed for 15.3%, 20.1%and 25.9% (w/v) glucose, respectively.

Interestingly, the remarkable glucose-fermenting ability ofC. guilliermondii was comparable to that of S. cerevisiae T2, withrespect to ethanol production from the highest glucose concen-trations tested in this study. Glucose at an initial concentration of24.9% (w/v) was completely utilized by S. cerevisiae T2 within 24 h(Fig. 2A). A maximum ethanol concentration of 9.9% (w/v) wasobtained from 24.9% (w/v) of glucose; and this corresponded to ayield of 0.40 g/g.

Three other well-known pentose-fermenting yeasts,P. tannophilus NRRL Y-2460, S. shehatae NRC 2886 and S. stipitisNRRL Y-7124, were also tested for fermentation of high concen-trations of glucose under similar conditions. P. tannophiluscompletely consumed 26.6% (w/v) glucose in 120 h, producing apeak ethanol concentration of 8.9% (w/v) (Fig. 3A). However,S. shehatae (Fig. 4A) and S. stipitis (Fig. 5A) were unable tocompletely utilize 25.6% and 25.7% (w/v) glucose, respectively, over144 h of incubation. The peak ethanol concentrations produced bythe two yeasts were less than 6% (w/v). Amongst the pentose-fermenting yeasts tested, C. guilliermondii was the best at fer-menting glucose based on the peak ethanol concentration [9.3% (w/v)] achieved, shortest time required for glucose utilization (36 h)and complete consumption of glucose at the highest concentration[26.9% (w/v)] tested. The glucose fermentation performance pa-rameters for these yeasts are summarized in Table 1 forcomparison.

Mannose fermentation Mannose is another hexose sugarfound in lignocellulosic biomass andmay represent up to 15% of the

were 15.0% (dash line) and 20.3% (w/v) (solid line). (D) Xylose fermentation. The initialconcentration was 10.1% (w/v). Values shown are means� SEM of three independentexperiments. Crosses indicate sugar utilization trend; closed circles indicate ethanolproduction and open triangles indicate xylitol production (dotted line).

FIG. 5. Sugar fermentation by S. stipitis NRRL Y-7124. (A) Glucose fermentation. Theinitial concentrations were 16.0% (round dotted line), 20.8% (dash line) and 25.7% (w/v)(solid line). (B) Mannose fermentation. The initial concentrations were 14.9% (dash

TABLE 1. Fermentation performance parameters of selected yeasts in highconcentrations of glucose in defined media.

Yeast [Glucose]i(% w/v)

[EtOH]max

(% w/v)YE1 (g/g)(% of Yt)

YE2 (g/g) t (h)

C. guilliermondii15.3� 0.2 5.3� 0.1 0.35 (68%) 0.35 21 � 020.1� 0.4 7.3� 0.3 0.36 (71%) 0.36 27 � 125.9� 0.9 9.3� 0.3 0.36 (71%) 0.36 36 � 0

P. tannophilus15.9� 0.4 5.9� 0.2 0.37 (72%) 0.37 36 � 320.5� 0.8 7.8� 0.1 0.38 (74%) 0.38 66 � 226.6� 0.7 8.9� 0.3 0.35 (68%) 0.33 120 � 0

S. shehatae15.7� 0.9 4.5� 0.3 0.29 (56%) 0.29 48 � 920.0� 0.2 4.9� 0.0 0.26 (51%) 0.25 INC25.6� 0.9 5.7� 0.6 0.25 (49%) 0.22 INC

S. stipitis16.0� 0.7 4.6� 0.0 0.29 (57%) 0.29 72 � 1320.8� 0.5 4.3� 0.1 0.27 (53%) 0.21 INC25.7� 1.5 4.3� 0.2 0.26 (52%) 0.17 INC

S. cerevisiae14.4� 0.5 6.3� 0.0 0.44 (86%) 0.44 18 � 318.3� 0.1 8.5� 0.1 0.46 (91%) 0.46 18 � 324.9� 0.1 9.9� 0.4 0.40 (78%) 0.40 24 � 4

[ ]i, initial sugar concentration; [EtOH]max, peak ethanol concentration; YE1, ethanolyield based on the amount of sugar consumed, YE2, ethanol yield based on theamount of sugar supplied; Yt, theoretical yield of ethanol is 0.51 g/g; t, time requiredfor complete sugar utilization; INC, incomplete sugar utilization.

VOL. 121, 2016 HEXOSE FERMENTATION BY C. GUILLIERMONDII 635

total sugars in some softwood hydrolysates (22). In definedmedium, mannose fermentation took slightly longer than glucoseby all the yeast strains tested. Mannose at 15.6% (w/v) wascompletely utilized by C. guilliermondii FTI 20037 in 21 h (Fig. 1B).The maximum ethanol concentration produced was 5.3% (w/v),with a corresponding yield of 0.35 g/g. C. guilliermondii utilized20.3% (w/v) mannose in 54 h, producing a peak ethanolconcentration of 6.7% (w/v) with an ethanol yield of 0.33 g/g(Table 2).

Mannose at 15.4% and 20.6% (w/v) was completely utilizedwithin 18 and 24 h, respectively, by S. cerevisiae T2 (Fig. 2B). Themaximum ethanol concentrations producedwere 5.9% and 7.7% (w/v), respectively. These corresponded to an ethanol yield of 0.38 g/gat both mannose concentrations. The results showed thatS. cerevisiae fermented mannose slightly better thanC. guilliermondii at the lower concentration, but considerably fasterat the higher concentration.

Interestingly, P. tannophilus fermented 15.0% and 20.5% (w/v)mannose effectively to produce peak ethanol concentrations of5.8% and 7.8% (w/v), respectively (Fig. 3B), with correspondingethanol yields of 0.39 and 0.38 g/g, respectively. These levels aresimilar to those produced by S. cerevisiae and better than thoseproduced by C. guilliermondii. However, P. tannophilus took 78 h tocompletely consume 20.5% (w/v) mannose; this time frame wasconsiderably longer than either S. cerevisiae or C. guilliermondii.

S. shehatae (Fig. 4B) and S. stipitis (Fig. 5B) completely utilized15.2% and 14.9% (w/v) mannose, respectively, within 72 h. However,these yeasts were unable to completely utilize higher concentra-tions of mannose. From the initial 21.1% and 19.8% (w/v) mannose,1.6% and 4.6% (w/v) mannose remained unused by S. shehatae andS. stipitis, respectively, after 144 h of incubation. The peak ethanolconcentrations produced by the two yeasts were under 5% (w/v) forall mannose concentrations tested.

line) and 19.8% (w/v) (solid line). (C) Galactose fermentation. The initial concentrationswere 15.1% (dash line) and 20.2% (w/v) (solid line). (D) Xylose fermentation. The initialconcentration was 10.1% (w/v). Values shown are means� SEM of three independentexperiments. Crosses indicate sugar utilization trend; closed circles indicate ethanolproduction and open triangles indicate xylitol production (dotted line).

TABLE 2. Fermentation performance parameters of selected yeasts in highconcentrations of mannose in defined media.

Yeast [Mannose]i(% w/v)

[EtOH]max

(% w/v)YE (g/g)

(% of Yt)YE2 (g/g) t (h)

C. guilliermondii15.6� 0.1 5.3� 0.1 0.35 (69%) 0.34 24� 020.3� 0.1 6.7� 0.0 0.33 (65%) 0.33 78� 0

P. tannophilus15.0� 0.1 5.8� 0.2 0.39 (76%) 0.39 48� 320.5� 0.2 7.8� 0.2 0.38 (75%) 0.38 78� 5

S. shehatae15.2� 0.2 4.3� 0.1 0.28 (55%) 0.28 60� 721.1� 0.9 4.6� 0.1 0.24 (46%) 0.22 INC

S. stipitis14.9� 0.3 4.9� 0.1 0.33 (65%) 0.33 72� 319.8� 0.8 4.3� 0.1 0.28 (55%) 0.22 INC

S. cerevisiae15.4� 0.2 5.9� 0.0 0.38 (75%) 0.38 18� 320.6� 0.0 7.7� 0.0 0.38 (75%) 0.38 24� 6

All abbreviations are as in Table 1.

636 WEN ET AL. J. BIOSCI. BIOENG.,

Galactose fermentation Compared to glucose and mannose,galactose constitutes a smaller proportion of the overall sugarcomposition ranging from 0% to 4% in agricultural and softwoodbiomass and less than 2% of the total carbohydrate found in hard-wood (23). Galactose was not as readily or well fermented asglucose or mannose by the yeasts tested in this study. This wasseen by the longer times required by the yeasts to consume thissugar and the lower ethanol yields produced.

Galactose at 14.4% (w/v) was completely consumed in 72 h byC. guilliermondii FTI 20037 to produce a peak ethanol concentrationof 4.7% (w/v) (yield¼ 0.32 g/g) (Fig. 1C). However, C. guilliermondiicould not completely utilize 19.6% (w/v) galactose over 144 h ofincubation. C. guilliermondii consumed approximately 84% of 19.6%(w/v) galactose to produce a peak ethanol concentration of 4.4% (w/v), representing a yield of 0.27 g/g (Table 3).

S. cerevisiae T2 was slightly better at fermenting galactose,requiring 60 and 108 h to completely utilize 17.7% and 22.6% (w/v)galactose, respectively (Fig. 2C). The maximum ethanol concen-trations produced were 5.3% and 7.3% (w/v), respectively, and thesecorresponded to ethanol yields of 0.30 g and 0.32 g/g, respectively(Table 3).

Similar to C. guilliermondii, 20% (w/v) galactose was notcompletely consumed by S. stipitis and S. shehatae (Figs. 4C and 5C).The peak ethanol concentrations produced from galactose were3.2% and 3.9% (w/v) by S. shehatae and S. stipitis, respectively. Ofparticular note was the very slow galactose utilization byP. tannophilus. This yeast consumed about 59% and 55% of the initial15.7% and 20.8% (w/v) galactose, respectively, after 144 h of

TABLE 3. Fermentation performance parameters of selected yeasts in highconcentrations of galactose in defined media.

Yeast [Galactose]i(% w/v)

[EtOH]max

(% w/v)YE (g/g)

(% of Yt)YE2 (g/g) t (h)

C. guilliermondii14.4� 0.1 4.7� 0.1 0.32 (63%) 0.32 72� 319.6� 0.1 4.4� 0.1 0.27 (53%) 0.22 INC

P. tannophilus15.7� 0.3 0.2� 0.0 0.01 (2%) 0.01 INC20.8� 0.8 0.1� 0.0 0.01 (2%) 0.01 INC

S. shehatae15.0� 0.1 3.4� 0.3 0.23 (45%) 0.23 96� 1420.3� 1.6 3.2� 0.2 0.18 (35%) 0.16 INC

S. stipitis15.1� 0.3 4.7� 0.1 0.31 (61%) 0.31 78� 020.2� 0.6 3.9� 0.2 0.29 (57%) 0.19 INC

S. cerevisiae17.7� 1.6 5.3� 0.1 0.30 (59%) 0.30 60� 522.6� 1.6 7.3� 0.1 0.32 (63%) 0.32 108� 6

All abbreviations are as in Table 1.

incubation and produced negligible amount of ethanol [about 0.1%(w/v)] (Fig. 3C).

Overall, C. guilliermondii FTI 20037 exhibited excellent glucose-fermenting ability which was the best amongst the pentose-fermenting yeasts and comparable to that of S. cerevisiae T2.However, mannose and galactose were not fermented as efficientlyby C. guilliermondii as S. cerevisiae. Of the other pentose-fermentingyeasts tested, P. tannophilus also fermented glucose and mannosewell to yield high peak ethanol concentrations which rivalled thoseproduced by C. guilliermondii and S. cerevisiae. However, this straintook considerably longer to ferment these two hexoses. Moreover,it utilized galactose poorly and did not produce any ethanol fromthis sugar. S. shehatae and S. stipitis were relatively poor at fer-menting the hexose sugars.

Pentose fermentation Xylose fermentation was conductedas control experiments to ascertain the ability of pentose-fermenting yeasts to produce ethanol and/or xylitol from thissugar. C. guilliermondii FTI 20037 efficiently fermented 10.5% (w/v) xylose to produce 6.44% (w/v) xylitol in 72 h (Fig. 1D).Arabinose was poorly utilized by C. guilliermondii FTI 20037, andno ethanol was produced during 114 h of fermentation (data notshown). The other pentose-fermenting yeasts produced mostlyethanol [S. shehatae (Fig. 4D) and S. stipitis (Fig. 5D)] or bothethanol and xylitol [P. tannophilus (Fig. 3D)] from xylose. This isconsistent with their known xylose-fermenting abilities.

As expected, S. cerevisiae was unable to utilize xylose or arabi-nose over 144 h of incubation and neither ethanol nor xylitol wasproduced (data not shown).

In summary, C. guilliermondii FTI 20037 was found to fermenthigh concentrations of glucose rapidly in a definedmedium to yieldvery high peak ethanol concentrations. To our knowledge, the un-characteristically high peak ethanol concentrations achieved (9.3%,w/v) has not been seen in any other unmodified native pentose-fermenting yeasts. This remarkable glucose-fermenting abilitywas similar to that of an industrial S. cerevisiae T2 strain.C. guilliermondii also fermented mannose and galactose to ethanol,although less efficiently than glucose. P. tannophilus fermented highconcentrations of glucose to ethanol; however this yeast tookconsiderably longer compared to C. guilliermondii or S. cerevisiae. Incomparison, S. shehatae and S. stipitiswere relatively poor at hexosefermentation. The results are of considerable interest in thatC. guilliermondii is known to produce high levels of xylitol fromxylose, but its exceptional hexose-fermenting ability has not beenreported to date.

ACKNOWLEDGMENTS

Our research on native pentose-fermenting yeasts was sup-ported by the NSERC Bioconversion Network was funded by NSERCunder grant no. NETGP350246-07, BioFuelNet Canada and OntarioMinistry of Agriculture, Food and Rural Affairs. We thank JurajStrmen (formerly of Tembec, Témiscaming, QC, Canada) forproviding S. cerevisiae T2 strain and Cletus Kurtzmann for providingPachysolen tannophilus NRRL Y-2460.

References

1. Batt, C. A., Carvallo, S., Easson, D. D., Akedo, M., and Sinskey, A. J.: Directevidence for a xylose metabolic pathway in Saccharomyces cerevisiae, Bio-technol. Bioeng., 28, 549e553 (1986).

2. Jeffries, T. W.: Conversion of xylose to ethanol under aerobic conditions byCandida tropicalis, Biotechnol. Lett., 3, 213e218 (1981).

3. Schneider, H., Wang, P. Y., Chan, Y. K., and Maleszka, R.: Conversion of D-xylose into ethanol by the yeast Pachysolen tannophilus, Biotechnol. Lett., 3,89e92 (1981).

VOL. 121, 2016 HEXOSE FERMENTATION BY C. GUILLIERMONDII 637

4. Slininger, P. J., Bothast, R. J., van Cauwenberge, J. E., and Kurtzman, C. P.:Conversion of D-xylose to ethanol by the yeast Pachysolen tannophilus, Bio-technol. Bioeng., 24, 371e384 (1982).

5. Harner, N. K., Wen, X., Bajwa, P. K., Austin, G. D., Ho, C. Y., Habash, M. B.,Trevors, J. T., and Lee, H.: Genetic improvement of native xylose-fermentingyeasts for ethanol production, J. Ind. Microbiol. Biotechnol., 42, 1e20 (2015).

6. Barbosa, M. F. S., Medeiros, M. B., Mancilha, I. M., Schneider, H., and Lee, H.:Screening of yeasts for production of xylitol from D-xylose and some factorswhich affect xylitol yield in Candida guilliermondii, J. Ind. Microbiol., 3, 241e251(1988).

7. Lee, H., Atkin, A. L., Barbosa, M. F. S., Dorscheid, D. R., and Schneider, H.:Effect of biotin limitation on the conversion of xylose to ethanol and xylitol byPachysolen tannophilus and Candida guilliermondii, Enzyme Microb. Technol.,10, 81e84 (1988).

8. Silva, S. S. and Afschar, A. S.: Microbial production of xylitol from D-xyloseusing Candida tropicalis, Bioprocess Eng., 11, 129e134 (1994).

9. Bura, R., Vajzovic, A., and Doty, S. L.: Novel endophytic yeast Rhodotorulamucilaginosa PTD3 I: production of xylitol and ethanol, J. Ind. Microbiol. Bio-technol., 39, 1003e1011 (2012).

10. Mateo, S., Puentes, J. G., Roberto, I. C., Sanchez, S., and Moya, A. J.: Optimi-zation of acid hydrolysis of olive tree pruning residue. Fermentation withCandida guilliermondii, Biomass Bioenergy, 69, 39e46 (2014).

11. Sene, L., Converti, A., Zilli, M., Felipe, M. G. A., and Silva, S. S.: Metabolic studyof the adaptation of the yeast Candida guilliermondii to sugarcane bagassehydrolysate, Appl. Microbiol. Biotechnol., 57, 738e743 (2001).

12. Villarreal, M. L. M., Prata, A. M. R., Felipe, M. G. A., and Silva, J. B. A. E.:Detoxification procedures of eucalyptus hemicellulose hydrolysate for xylitolproduction by Candida guilliermondii, EnzymeMicrob. Technol., 40, 17e24 (2006).

13. Delgenes, J. P., Moletta, R., and Navarro, J. M.: The ethanol tolerance of Pichiastipitis Y-7124 grown on a D-xylose, D-glucose and L-arabinose mixture,J. Ferment. Technol., 66, 417e422 (1988).

14. du Preez, J. C., Driessel, B., and Prior, B. A.: Ethanol tolerance of Pichia stipitisand Candida shehatae strains in fed-batch cultures at controlled low dissolvedoxygen levels, Appl. Microbiol. Biotechnol., 30, 53e58 (1989).

View publication statsView publication stats

15. Jeffries, T. W., Fady, J. H., and Lightfoot, E. N.: Effect of glucose supplementson the fermentation of xylose by Pachysolen tannophilus, Biotechnol. Bioeng.,27, 171e176 (1985).

16. Bajwa, P. K., Ho, C. Y., Chan, C. K., Martin, V. J. J., Trevors, J. T., and Lee, H.:Transcriptional profiling of Saccharomyces cerevisiae T2 cells upon exposure tohardwood spent sulphite liquor: comparison to acetic acid, furfural and hydrox-ymethylfurfural, Antonie Leeuwenhoek, 103, 1281e1295 (2013).

17. Bajwa, P. K., Shireen, T., D’Aoust, F., Pinel, D., Martin, V. J. J., Trevors, J. T.,and Lee, H.: Mutants of the pentose-fermenting yeast Pichia stipitis withimproved tolerance to inhibitors in hardwood spent sulfite liquor, Biotechnol.Bioeng., 104, 892e900 (2009).

18. Bajwa, P. K., Pinel, D., Martin, V. J. J., Trevors, J. T., and Lee, H.: Strainimprovement of the pentose-fermenting yeast Pichia stipitis by genome shuf-fling, J. Microbiol. Methods, 81, 179e186 (2010).

19. Barbosa, M. F. S., Lee, H., and Collins-Thompson, D. L.: Additive effects ofalcohols, their acidic by-products, and temperature on the yeast Pachysolentannophilus, Appl. Environ. Microbiol., 56, 545e550 (1990).

20. Slininger, P. J., Bothast, R. J., Okos, M. R., and Ladisch, M. R.: Comparativeevaluation of ethanol production by xylose-fermenting yeasts presented highxylose concentrations, Biotechnol. Lett., 7, 431e436 (1985).

21. Long, T. M., Su, Y. K., Headman, J., Higbee, A., Willis, L. B., and Jeffries, T. W.:Co-fermentation of glucose, xylose, and cellobiose by the beetle-associatedyeast Spathaspora passalidarum, Appl. Environ. Microbiol., 78, 5492e5500(2012).

22. Gírio, F. M., Fonseca, C., Carvalheiro, F., Duarte, L. C., Marques, S., and Bogel-qukasik, R.: Hemicelluloses for fuel ethanol: a review, Bioresour. Technol., 101,4775e4800 (2010).

23. van Maris, A. A., Abbott, D., Bellissimi, E., Brink, J., Kuyper, M., Luttik, M. H.,Wisselink, H. W., Scheffers, W. A., Dijken, J., and Pronk, J.: Alcoholicfermentation of carbon sources in biomass hydrolysates by Saccharomycescerevisiae: current status, Antonie Leeuwenhoek, 90, 391e418 (2006).