tioki

otience

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18,

yeast in Antarctic soil. Moreover, we previously reported that

of East Antarctica were Mrakia spp. [12]. These reports suggestedthat Mrakia spp. are the dominant culturable fungi in East Antarc-tica and the most adaptive to the Skarvenes ice-free area. Mrakiablollopis SK-4 was isolated from Naga-ike Lake (6929.20S,3935.90E) in the Skarvsnes ice-free area, East Antarctica. This yeast

glucose, cellulaseen yeast is mixedrmed by cellulaseat a low cyeast. Thi

nique is therefore expected to improve saccharication and

on mechanochemically treated Eucalyptus and Japanese cedarwood meals using the DEF technique with the non-ionic surfactantTween 80, theoretical ethanol yields from these biomasses were51.2% and 65.1%, respectively. Since these fermentation rates didnot exceed 70%, DEF from lignocellulosic biomass with M. blollopisSK-4 is required to improve fermentation efciency. In this study,we tried to improve DEF by using the non-ionic surfactant, Tween80, and lipase at 10 C.

Corresponding authors. Fax: +81 82 420 8291.E-mail addresses: spindletuber@gmail.com, masaharu-tsuji@aist.go.jp (M. Tsuji),

tamotsu.hoshino@aist.go.jp (T. Hoshino).

Cryobiology 68 (2014) 303305

Contents lists availab

io

.eabout 35% of culturable fungi isolated from lake sediment and soil ethanol fermentation rates. However, when SK-4 was fermentedfor these yeasts in health, agriculture and industry [6].The cryophilic yeast [9] Mrakia spp. and Mrakiella spp. have

been found in the Arctic, Siberia, Alaska, Central-Russia, the Alps,Apennines, Patagonia and Antarctica [11]. di Menna [3] reportedthat the genus Mrakia accounted for about 24% of the culturable

time. In the presence of a high concentration ofactivity is considerably depressed. However, whwith an enzymatic hydrolysis reactor, glucose, fohydrolysis of cellulolytic biomass, is maintainedtration and is rapidly converted to ethanol by thehttp://dx.doi.org/10.1016/j.cryobiol.2013.12.0080011-2240/ 2014 Elsevier Inc. All rights reserved.oncen-s tech-The biosphere of this planet is principally cold, and many mi-crobes can be found inhabiting polar, deep sea and alpine regions[6]. Since microbes that have adapted to such cold environmentsare able to grow at temperatures below 0 C, it is expected thatthey can be utilized in de novo bioprocesses involving cold-adaptedenzymes [2]. Although there have been few studies on cryophilicyeasts from polar regions, these studies have revealed applications

strain showed high nucleotide sequence homologies (>99.6%) toM.blollopis CBS8921T in the ITS and D1/D2 sequences, and was shownto have fermentation ability for typical sugars such as glucose, su-crose, maltose, rafnose and fructose at cold temperatures [15].

Direct ethanol fermentation (DEF) from cellulosic biomass wasrst reported by Takagi et al. [10]. In this technique, enzymatichydrolysis and ethanol fermentation are carried out at the samea r t i c l e i n f o

Article history:Received 10 October 2013Accepted 26 December 2013Available online 2 January 2014

Keywords:Cryophilic yeastAntarcticaEthanol fermentationDirect ethanol fermentationWoody biomassa b s t r a c t

The Antarctic basidiomycetous yeast Mrakia blollopis SK-4 can quite uniquely ferment various sugarsunder low temperature conditions. When strain SK-4 fermented lignocellulosic biomass using the directethanol fermentation (DEF) technique, approximately 30% to 65% of the theoretical ethanol yield wasobtained without and with the addition of the non-ionic surfactant Tween 80, respectively. Therefore,DEF from lignocellulosic biomass with M. blollopis SK-4 requires the addition of a non-ionic surfactantto improve fermentation efciency. DEF with lipase converted Eucalyptus and Japanese cedar to12.6 g/l, and 14.6 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80 and 5 U/g-dry substratelipase, ethanol concentration increased about 1.4- to 2.4-fold compared to that without Tween 80 andlipase. We therefore consider that the combination ofM. blollopis SK-4 and DEF with Tween 80 and lipasehas good potential for ethanol fermentation in cold environments.

2014 Elsevier Inc. All rights reserved.dGraduate School of Life Science, Hokkaido University, N10W8 Kita-ku, Sapporo, Hokkaido 060-0810, JapanBrief Communication

Improvement of direct ethanol fermentathe Antarctic basidiomycetous yeast, Mratemperature condition

Masaharu Tsuji a,, Yuji Yokota b, Sakae Kudoh c, TamaBiomass Renery Research Center (BRRC), National Institute of Advanced Industrial ScHiroshima 739-0046, JapanbBio-production Research Institute, National Institute of Advanced Industrial Science anHokkaido 062-8517, JapancNational Institute of Polar Research (NIPR), 10-3 Midori-cho, Tachikawa, Tokyo 190-85

Cryob

journal homepage: wwwn from woody biomasses bya blollopis, under a low

su Hoshino a,d,and Technology (AIST), 3-11-32 Kagamiyama, Higashihiroshima,

chnology (AIST), 2-17-2-1 Tsukisamu-higashi, Toyohira-ku, Sapporo,

Japan

le at ScienceDirect

logy

lsevier .com/locate /ycryo

Ten ll (2.0 108 cells) of M. blollopis SK-4 was inoculated into400 ml YPD liquid medium (10 g/l yeast extract, 20 g/l peptone,and 20 g/l glucose) and cultured at 120 rpm for 120 h at 10 C, then400ml of the culture was collected by centrifugation at 3500g for10 min at 4 C. The pellet was suspended in 50 ml of distilled water.The initial cell density in the DEF mixture was adjusted toOD600 = 7.5, which corresponds to approximately 6.0 107 cells/ml.

Mechanochemically treated Eucalyptus wood meal [4] and Jap-anese cedar wood meal were kindly provided by Dr. TomoakiMinowa (BRRC, AIST). Eucalyptus wood meal was composed of40.0% glucan, 10.4% xylan, and 28.8% lignin, and Japanese cedarwood meal was composed of 38.2% glucan, 6.0% xylan and 34.6%lignin. These woody biomasses were used as substrates for DEF.

DEF experiments were carried out in 28-ml vials. Each basal DEFmixture consisted of 10% (w/v) woody biomass, 5 g/l yeast extract,5 g/l Bacto peptone, 2 g/l NH4Cl, 1 g/l KH2PO4 and 0.3 g/l MgSO47H2Oin 20 mM citrate buffer (pH 5.0), 50 FPU/g-dry substrate Acremo-zyme (Meiji Co., Ltd, Tokyo, Japan) and 20 ll/g-dry substrateOptimash BG (Genencor International, Rochester, NY, USA). Themedium was inoculated with 50 ml/l of inoculum. All of the DEFexperiments were carried out at 180 rpm for 144 h at 10 C.

DEF with lipase was performed under the same conditions asthose described for the basal DEF mixtures except for the additionof 5 U/g-dry substrate Aspergillus niger lipase (Wako Pure ChemicalIndustries, Osaka, Japan) to the basal reaction mixtures. DEF withlipase samples were collected every 24 h and centrifuged at

12100g for 5 min at 4 C. The supernatant was used as DEF withlipase solutions, and residual glucose and ethanol concentrationsin the solutions were measured.

DEF with Tween 80 and lipase was performed under the sameconditions as those described for the basal DEF mixtures exceptfor the addition of 1% (v/v) Tween 80 and 5 U/g-dry substrateA. niger lipase to the reaction mixtures. DEF with Tween 80 andlipase samples were collected every 24 h and centrifuged at12100g for 5 min at 4 C. The supernatant was used as DEF withTween 80 and lipase solutions, and residual glucose and ethanolconcentrations in the solutions were measured.

Glucose and ethanol concentrations in the ethanol fermentationand DEF solutions without/with lipase, or without/with Tween 80and lipase, were measured by high-performance liquid chromatog-raphy (HPLC). All samples were analyzed by HPLC with an AminexHP87 cation exchange column using both UV and RI detection at aow rate of 0.6 ml/min at 65 C or 80 C. The methods used foranalysis were described previously in detail by Goshima et al. [7].

Table 1Summary of DEF assay from Eucalyptus and Japanese cedar with/without Tween 80,lipase, and Tween 80 and lipase by Mrakia blollopis.

EtOHM (g/l) YE/G (g/g) YE/ET (%) Reference

EucalyptusDEF 7.1 (0.25) 0.16 (0.01) 31.4 (1.10) [13]T 11.6 (0.14) 0.26 (0.02) 51.2 (0.49) [13]L 12.6 (1.25) 0.28 (0.03) 55.6 (3.11) This studyT+L 17.3 (0.54) 0.39 (0.02) 76.4 (1.72) This study

Japanese cedarDEF 12.5 (0.76) 0.29 (0.01) 57.8 (1.50) [13]

304 M. Tsuji et al. / Cryobiology 68 (2014) 303305T 14.1 (0.19) 0.33 (0.04) 65.2 (0.83) [13]L 14.6 (0.96) 0.34 (0.03) 67.5 (2.27) This studyT+L 17.5 (0.54) 0.41 (0.01) 81.0 (1.46) This study

DEF, DEF without 1% (v/v) Tween 80 and lipase; T, DEF with 1% (v/v) Tween 80; L,DEF with 5 U/g-dry substrate lipase; T+L, DEF with 1% (v/v) Tween 80 and 5 U/g-drysubstrate lipase; EtOHM, maximum ethanol concentration after 144 h of DEF; YE/G,ethanol yield based on total glucose content in the DEF mixture; YE/ET, theoreticalyield of ethanol (0.51 g ethanol/g glucose). Upon hydrolysis, 1 g cellulose produces1.11 g glucose.Fig. 1. DEF from Eucalyptus wood meal with lipase or Tween 80 and lipase. (A) DEF wsubstrate lipase. Closed circles denote ethanol concentrations and open circles indicatecarried out in triplicate and error bars denote standard deviation values.The ethanol yields for DEF from Eucalyptus and Japanese cedarwere each calculated as a percentage of the maximum theoreticalethanol yield of 0.51 g ethanol per gram of glucose. The maximumethanol concentration (EtOHM), ethanol yield based on total glu-cose content in the ethanol fermentation mixture (YE/G), and theo-retical ethanol yield (YE/EY) are summarized in Table 1.

As a result of DEF of Eucalyptus and Japanese cedar with 5 U/g-drysubstrate lipase, 13.8 g/l and 18.6 g/l glucose were formed, respec-tively, after 24 h of fermentation. Finally, 12.6 g/l ethanol from Euca-lyptus and 14.6 g/l ethanol from Japanese cedar were produced after144 h of fermentation (Figs. 1 and 2A). When Eucalyptus was mixedwith 1% (v/v) Tween 80 and 5 U/g-dry substrate lipase, 10.3 g/l glu-cose was rapidly released from the biomass and was continuouslyconverted to ethanol for up to 120 h of fermentation (Fig. 1B). In thecase of DEF from Japanese cedar with 1% (v/v) Tween 80 and 5 U/g-drysubstrate lipase, 6.1 g/l glucose was formed within 24 h and nally17.5 g/l ethanol was produced after 144 h of fermentation (Fig. 2B).

Ethanol fermentation under cold temperature conditions is gener-ally used to produce wine and sake [1,16]. Little is known about thefermentation ability of basidiomycetous yeast. Eight species ofMrakiahave so far been reported: M. frigida, M. gelida, M. stokesii, M. nivalis,M. curviuscula, M. psychrophila, M. robertii and M. blollopis [11]. How-ever, species in this basidiomycetous yeast genus are known for theirability to ferment sugars. Actually, 7 of the 8 species are able toferment glucose, with only M. curviuscula lacking this ability.

M. blollopis SK-4, isolated from Naga-ike lake in the Skarvsnesice-free area, could convert 120 g/l of glucose to 48.7 g/l ethanolith 5 U/g-dry substrate lipase and (B) DEF with 1% (v/v) Tween 80 and 5 U/g-dryglucose concentrations. All DEFs were carried out at 10 C. All experiments were

5 Ue con

ologyat 10 C, while other Mrakia spp. could not produce more than 2%(v/v) ethanol [11,13]. Moreover, strain SK-4 could completely con-vert glucose to ethanol at pH 4.010.0 at 10 C [14]. We previouslyreported DEF from lignocellulosic biomass using SK-4 at 10 C.When DEF was carried out with 1% (v/v) Tween 80, the yield of eth-anol was about 1.1- to 1.6-fold higher than that when DEF was car-ried out without Tween 80 [13]. However, in the case of DEF with1% (v/v) Tween 80, ethanol conversion rates were only 51.2% and65.1% the theoretical maximum from Eucalyptus and Japanese ce-dar cellulose, respectively (Table 1). Therefore, DEF from lignocel-lulosic biomasses with M. blollopis SK-4 is required to improvefermentation efciency.

Eriksson et al. [5] reported that enzymatic hydrolysis was im-proved by the addition of the non-ionic surfactants, Tween 80 andTween 20, compared to the glucose concentration obtained by enzy-matic hydrolysis without a non-ionic surfactant. This improvement inhydrolysis reaction was thought to be caused by prevention of theadsorption of cellulase on the biomass. Moreover, Eucalyptus andJapanese cedar trees are known to maximally contain about 4% oilcontent [8]. The cell structures of these biomasses were completelydestroyed by mechanochemical treatment [4]. We speculate thatthe oil covers the surfaces of Eucalyptus and Japanese cedar woodmeals and that contact of cellulase with the biomass is impeded bythe oil. Since it is thought that oil can be decomposed by the additionof lipase, and since it is thought that adsorption of cellulase on bio-

Fig. 2. DEF from Japanese cedar wood meal with Tween 80 and lipase. (A) DEF withlipase. Closed circles denote ethanol concentrations and open circles indicate glucostriplicate and error bars denote standard deviation values.

M. Tsuji et al. / Cryobimass can be prevented by the addition of Tween 80, we conductedDEF in the presence of the non-ionic surfactant, Tween 80, and lipase.

When DEF was carried out with 5 U/g-dry substrate lipase, theyield of ethanol was approximately 1.2- to 1.8-fold higher thanthat when DEF was carried out without lipase. When DEF was car-ried out with 1% (v/v) Tween 80 and 5 U/g-dry substrate, the yieldof ethanol was about 1.4- to 2.4-fold higher than that when DEFwas carried out without Tween 80 and lipase. Theoretical ethanolyields were approximately 76.5% and 81.0% from Eucalyptus andJapanese cedar, respectively.

We therefore believe that the use of DEF with Tween 80 and lipaseis a good technique for the production of ethanol from woody bio-mass under cold temperature conditions. Moreover, if it becomespossible for cryophilic basidiomycetous yeasts to produce usefulmaterials by genetic engineering, the combination of SK-4 and DEFwith Tween 80 and lipase should be useful for the production of thesematerials from woody biomass under cold temperature conditions.

Funding statement

NoneAcknowledgments

We are grateful to Dr. T. Minowa, (AIST, Japan) for providingmechanochemically treated Eucalyptus and Japanese cedar woodmeals. This work is part of the Science Program of JARE-48. Itwas supported by NIPR under MEXT.

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Improvement of direct ethanol fermentation from woody biomasses by the Antarctic basidiomycetous yeast, Mrakia blollopis, under a low temperature conditionFunding statement AcknowledgmentsReferences