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Enzyme and Microbial Technology 52 (2013) 105– 110

Contents lists available at SciVerse ScienceDirect

Enzyme and Microbial Technology

jou rn al h om epage: www.elsev ier .com/ locate /emt

nhanced ethanol production from wheat straw by integrated storage andre-treatment (ISP)

olkmar Passotha,∗, Muhammad Rizwan Tabassuma,1, Harikrishnan A.S. Naira,2, Matilda Olstorpea,evgeniia Tiukovaa, Jerry Ståhlbergb

Swedish University of Agricultural Sciences, Department of Microbiology, Box 7025, SE-75007 Uppsala, SwedenSwedish University of Agricultural Sciences, Department of Molecular Biology, Box 590, SE-751 24 Uppsala, Sweden

r t i c l e i n f o

rticle history:eceived 14 September 2012eceived in revised form 5 November 2012ccepted 6 November 2012

eywords:

a b s t r a c t

Integrated storage and pre-treatment (ISP) combines biopreservation of moist material under airtightconditions and pre-treatment. Moist wheat straw was inoculated with the biocontrol yeast Wicker-hamomyces anomalus, the xylan degrading yeast Scheffersomyces stipitis or a co-culture of both. Thesamples and non-inoculated controls were stored at 4 or 15 ◦C. The non-inoculated controls were heavilycontaminated with moulds, in contrast to the samples inoculated with W. anomalus or S. stipitis. These

heat strawio-preservationre-treatmentthanol

two yeasts were able to grow on wheat straw as sole source of nutrients. When ethanol was producedfrom moist wheat straw stored for four weeks at 4 ◦C with S. stipitis, an up to 40% enhanced yield (finalyield 0.15 g ethanol per g straw dry weight) was obtained compared to a dry sample (0.107 g/g). In allother moist samples, stored for four weeks at 4 ◦C or 15 ◦C, 6–35% higher yields were obtained. Thus,energy efficient bio-preservation can improve the pre-treatment efficiency for lignocellulose biomass,which is a critical bottleneck in its conversion to biofuels.

. Introduction

Plant biomass is a valuable renewable resource that can be usedo produce biofuels, feedstock for human and animal nutrition anduilding blocks for chemicals. Lignocellulose is the most abun-ant biomass, and its use for biofuel production will be essentialo obtain a long-term sustainable supply of energy and chemicals,ince lignocellulose usually does not compete with food produc-ion [1]. Lignocellulose consists of the polysaccharides cellulosend hemicellulose and the polyphenolic compound lignin. It car-ies the structure of plants and is evolved to resist degradation.ince ethanol production yeasts can only ferment hexose mono-nd few disaccharides to ethanol, degradation of the polysac-haride chains is essential for ethanol generation. This can onlye achieved by harsh thermochemical pre-treatment followed

y enzymatic digestion. Thermochemical pre-treatment is energyemanding and results in the release of inhibitors of subsequentrocesses. Enzyme treatments are expensive, accounting for about

∗ Corresponding author. Tel.: +46 18 673380; fax: +46 18 673393.E-mail address: Volkmar.Passoth@slu.se (V. Passoth).

1 Present address: Department of Bioinformatics and Biotechnology, Governmentollege University, Faisalabad, Pakistan.2 Present address: Singapore Centre on Environmental Life Sciences Engineering,anyang Technological University, 60 Nanyang View 637551, Singapore.

141-0229/$ – see front matter © 2012 Elsevier Inc. All rights reserved.ttp://dx.doi.org/10.1016/j.enzmictec.2012.11.003

© 2012 Elsevier Inc. All rights reserved.

10% of the total production costs of lignocellulosic ethanol [2].Decreasing the input of costs and energy for pre-treatment is crucialfor establishing a commercial ethanol production process based onlignocellulose substrate.

Plant biomass production is seasonal, while fuels, chemicals,feed and food are continuously consumed. This implies thatharvested biomass must be stored until processing. Insufficientconservation of biomass not only results in substantial losses ofmaterial, but also in the growth of moulds and bacteria that canbe pathogenic, produce toxins persisting in the production chainor spores causing allergic reactions. Efficient conservation requiresthe input of energy and/or chemicals. To prevent growth of undesir-able microorganisms during storage, conservation measures haveto be taken, in many cases drying being the most efficient method.Especially in regions with a temperate climate, like Scandinaviaplant biomass can have a very high moisture content at harvest,making drying an extremely energy demanding process. In somecases more than half of the total process energy for biomass pro-duction is spent on drying [3]. During later process steps, frequentlywater has to be added again. Water removal during storage mightnot be necessary for biofuel production, as recently demonstratedfor moist cereal grain where the presence of moisture during stor-

age made polymeric sugars better accessible to subsequent enzymetreatment, finally resulting in an increase in ethanol yield of morethan 10% [4]. Better accessibility of polysaccharides to enzymedegradation has also been demonstrated for ensiled sugar beet

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ulp [5]. Moist storage requires new measures of conservation, anddding a biocontrol yeast has been proven to be one of the mostfficient ways of preservation [3].

Attempts have been performed to introduce a bio-pre-reatment of lignocellulose material, where lignin degrading fungiere cultivated on the lignocellulose biomass, which resulted inp to 33% increased ethanol yields. However, in these experimentshe fungal activity was purely regarded as a pre-treatment. Storageas not regarded, the experiments have been performed in ster-

le systems, and were time consuming, which is not applicable forarge-scale processes [6,7].

Our study aimed to establish an integrated storage and pre-reatment (ISP) process, using yeasts that can prevent microbialontamination of the biomass and at the same time soften the struc-ure of the material. An ISP should be adapted to the conditionsf the region where the biomass is processed. Under Scandinavianonditions, biomass is usually harvested before the cold season, andhen stored at low temperatures. Thus, organisms should be usedhat are active at low temperatures. To prove the concept of ISP weompared ethanol production from moist wheat straw preservedith the known biocontrol yeast Wickerhamomyces anomalus [3]

syn. Hansenula, Pichia anomala) and the xylan-degrading yeastcheffersomyces stipitis [8] with that of non-inoculated moist wheattraw and dry material in laboratory scale experiments.

. Materials and methods

.1. Wheat straw

Dry wheat straw was obtained from the experimental farm Kungsängen of thewedish University of Agricultural Sciences (SLU).

.2. Microorganisms and growth media

The strains W. anomalus J121 (CBS100487) and S. stipitis CBS5774 were used forSP. Test ethanol fermentations were performed with the industrial Saccharomyceserevisiae isolate J672 [9]. The strains were conserved in freeze cultures at −70 ◦C asescribed [10]. Before the experiments, strains from the freeze stock were cultivatedn solid YPD (yeast extract 10 g/l, peptone 20 g/l and 16 g/l agar for solidification)ver night at 30 ◦C. Strains for ISP were pre-cultivated in minimal medium, contain-ng 6.7 g/l yeast nitrogen base (Difco Laboratories, Detroit, USA) and 20 g/l glucose,n 100 ml shake flask cultures in 300 ml Erlenmeyer flasks at 200 rpm and 25 ◦C for4 h. The fermentation strain S. cerevisiae J672 was pre-cultivated in 100 ml liquidPD over night at 30 ◦C. Before starting the fermentation, the cells were centrifuged,ashed with saline (NaCl, 9 g/l) and cells were counted or the optical density at

00 nm (OD600) was determined (see below). Wheat straw agar plates contained00 g/l milled wheat straw and 16 g/l agar.

.3. Storage experiments

To be able to handle wheat straw in a laboratory system for testing storage it wasilled (Ultra centrifugal mill ZM 1000; Retsch Germany) into fine powder (particle

ize < 0.5 mm) and kept airtight in a plastic bag at 2 ◦C until use. The moisture contentf the wheat straw was 5.78% corresponding to a water activity of 0.223. The waterctivity of the wheat straw was adjusted to 0.973 (moisture content 30%, materialurther designated as “moist wheat straw”). The moist wheat straw was mixed in

blender (Electrolux) with cultures of the ISP-microbes (105 yeast cells per g (dryeight) of straw). Non-inoculated control samples of moist wheat straw of the sameater activity and dry wheat straw were also kept along with the test samples for

omparison. Six grams (wet weight) of the moist samples were filled into 50 mlalcon tubes and kept at 4 ◦C and 15 ◦C for four weeks storage, if not otherwisetated. Examples of storage tubes are shown in Fig. 1. The caps of all tubes werelosed tightly and a needle was introduced in the cap of each tube to simulate aireakage. Experiments were performed in duplicates.

.4. Isolation and identification of microorganisms

Microbes from storage tubes were isolated by mixing a complete content of tube with 44 ml peptone water (2 g/l bacteriological peptone, 0.2 g/l tween 80),

lled into a Stomacher bag and extracted as described earlier [11]. One ml of thebtained suspension was further diluted and spread on YPD-medium containing.1 g/l chloramphenicol. Yeasts were identified by sequencing the D1D2-region ofhe 26S rRNA-gene of the ribosomal large subunit as described earlier [10]. Mouldsere identified to the genus level after morphological characteristics according to

l Technology 52 (2013) 105– 110

the procedures and taxonomic keys of Samson et al. [12]. Microbial analyses weredone from three parallel tubes of each storage approach.

2.5. Thermochemical pre-treatment

Thermochemical pre-treatment (TCP) was performed in 100 ml Schott flasks,which lids had a hole with a diameter of about 1 cm, which was closed with rubberstoppers. This rubber stopper was perforated with a syringe needle to enable gasexchange. Stored samples and empty flasks were weighed and 2.73 g wet weight(corresponding to 1.91 g dry weight) were transferred into the flasks. The moisturecontent of dry samples was adjusted to 30% by adding sterile water before trans-ferring to the flasks. The wheat straw was suspended in 19.8 ml of 0.75% H2SO4

(0.135 M) and the samples were autoclaved (CertoClav Sterilizer GmbH, Traun,Austria) for 120 min at 121 ◦C, whereafter pressure was released manually, untilatmospheric pressure was reached inside the autoclave.

2.6. Simultaneous saccharification and fermentation (SSF)

After TCP, the pH in each bioreactor was adjusted under sterile conditions to 5,using 10 M NaOH. Sodium citrate buffer, pH 5.0 was added to 0.1 M final concentra-tion and enzyme solution (Accellerase DUET enzyme, Genencor, Copenhagen) wasintroduced into the flasks through the needles at a concentration of 0.1 g/g of wheatstraw (dry matter). Accellerase DUET is a cocktail of several enzymes including,xylanase (3600 ABX/g), cellulases (2200–2500 CMCU/g) and �-glucosidase activi-ties (400 U/g). Finally, S. cerevisiae cells were added (final OD 1) and water to obtaina total reaction weight of 35 g (corresponding to 35 ml, dry weight content 54.8 g/l).Air from the flask headspace was removed by nitrogen flushing for two minutes (flowrate two litres per minute) through an introduced second sterile needle through therubber stopper. All flasks were incubated in a shaker at 35 ◦C for 4 days at 120 rpm.Samples were withdrawn through a sterile needle after every 24 h during the SSF andwere centrifuged, filter sterilized (pore size 0.2 �m) and stored for HPLC analysis.

2.7. Analytical methods

Moisture content (MC) was determined using a Sartorius Moisture analyzer MA-45 (Göttingen, Germany) and water activity by using an Aqua Lab CX-2 (DecagonDevices Inc. Washington DC, USA). Yeast cells were quantified using a Hemocy-tometer (Scherf, Burker, Germany) under an Olympus BH2 Research Microscope(Olympus America Inc.), and appropriate dilutions were generated with normalsaline (9 g/l NaCl). Optical density was measured in an Ultrospec 1100 pro, Biochrom(Agilent, Germany) spectrophotometer. Normal saline was used as blank for ODmeasurements and for diluting cell suspension to get an OD range from 0.1 to 0.4for an accurate reading at 600 nm. Glucose, xylose and ethanol concentrations weredetermined by high performance liquid chromatography (HPLC) as described earlier[13].

2.8. Statistical analysis

Results of the fermentations were compared to each other by means of Student’st-test, using Microsoft Office Excel 2007. Differences were regarded as significant atp < 0.05.

3. Results

S. stipitis CBS 5774 and W. anomalus J121 were tested for growon wheat straw agar and both strains showed clear growth afterthree days of incubation. The pH of the material was 7.5 at start ofthe storage and was not significantly changed after ISP.

Cultures of W. anomalus, S. stipitis or co-cultures of both wereinoculated to moist straw (moisture content 30%, cell titre 105

cells/g (dry weight) wheat straw) and stored for four weeks. Duringincubation, no mould growth was observed in the samples inocu-lated with yeasts, in contrast to the non-inoculated controls, whichwere overgrown by black or green moulds (Fig. 1). However, thetotal weight of the samples was similar, with no detectable dryweight losses in the 4 ◦C storages and 1.5–2% dry weight lossesin the 15 ◦C storages. Several samples were stored for more thanone year and also in these samples no mould growth was observedin storages inoculated with W. anomalus or S. stipitis. No furtherweight losses were observed in these long-term storages.

Yeast and mould growth in the tubes was further investigated

by extracting cells from the material and spreading them on agarplates. In cultures inoculated with W. anomalus or S. stipitis morethan 99% of the colonies were formed by yeasts, with almost equalcolony forming units (cfu) numbers of 1–2 × 107 per g (dry weight)

V. Passoth et al. / Enzyme and Microbial Technology 52 (2013) 105– 110 107

Fig. 1. Storage of moist wheat straw with restricted access of air. (a) Storage tubes containing moist wheat straw (30% moisture content). PA- inoculated with W. anomalusJ c- now

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121, PS- inoculated with S. stipitis CBS 5774, CO- inoculated with both organisms, Uater extracts from non-inoculated control tubes.

heat straw for both yeasts and temperatures. Some colonies (fiveer parallel) were identified by sequencing the D1D2 regions, andhe isolates belonged to S. stipitis or W. anomalus, respectively.lates obtained from the non-inoculated controls were mainlyvergrown with moulds, which made it impossible to perform anccurate quantification (Fig. 1), cfu numbers were about 106 per

wheat straw (dry weight). All identified moulds belonged to theenus Penicillium. From dry wheat straw isolated moulds reachedbout 102 cfu per g wheat straw, indicating that some mould sporesere present on the dry material.

For thermochemical pre-treatment a simplified dilute acidreatment was performed, where the samples were mixed withilute sulphuric acid (0.75%) and then autoclaved for 2 h. The mois-ure content of the dry controls was adjusted to 30% just beforehe treatment. After autoclaving, the pH was adjusted to 5.0, andermentation was performed by simultaneous saccharification andermentation (SSF) for 96 h. In pre-experiments it was found that inome cases the fermentation yeast started consuming the formedthanol between 72 and 96 h. Therefore the headspaces of the cul-ures were flushed with nitrogen at the beginning of the cultivationnd after 48 h, which prevented ethanol consumption.

The ethanols yields (calculated on the weight at the beginning oftorage) from moist straw were higher than from the dry material,hich yielded 0.106 g/g wheat straw (Table 1, Fig. S1). The differ-

nce to the dry sample was proven to be significant for both S.

n-inoculated control. (b) YPD plates inoculated with different dilutions of peptone

stipitis inoculated treatments, the S. stipitis–W. anomalus co-culturestored at 4 ◦C and the non-inoculated control stored at 4 ◦C. Fromthe co-culture and the non-inoculated control stored at 15 ◦C signif-icance of increased yields could not be proven (p = 0.057 and 0.085,respectively). The highest yield (0.149 g/g) was obtained from thematerial stored at 4 ◦C with S. stipitis as ISP organism. The yieldsobtained from W. anomalus at 4 ◦C treated wheat straw were onlyabout 6% above those obtained from dry wheat straw, and lowerthan those obtained from the non-inoculated control (differencesnot significant except to the non-inoculated control stored at 4 ◦C).From the W. anomala sample stored at 15 ◦C slightly higher yieldswere obtained than from the 4 ◦C sample, but these differenceswere not significant. However, the non-inoculated controls wereheavily infected by moulds, which may be problematic in commer-cial processes. Co-cultures of S. stipitis and W. anomalus gave yieldsthat were above the non-inoculated controls but below the S. stipitisISP.

Most of the ethanol was already produced during the first 24 hreaching from 0.067 g/g wheat straw in the fermentation of thedry material to 0.112 in the fermentation of the material storedwith S. stipitis at 4 ◦C (Table 1, Fig. S1). No glucose was detected

in the SSF-process, indicating that all glucose released by theenzymes was consumed immediately. Xylose, which represents themajor monosaccharide of the hemicellulose cannot be degradedby normal bakers’ yeast [14], and may thus be a measure of the

108 V. Passoth et al. / Enzyme and Microbia

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l Technology 52 (2013) 105– 110

polysaccharide degradation in the process. In most fermentations,the maximum concentrations were reached during the first 24 hor at the latest at the second sampling at 48 h (Table 2, Fig. S2).The concentrations remained almost constant at about 13 g/l duringthe fermentations, representing yields of more than 0.23 g/g wheatstraw (dry weight). However, in our method the amount of xylosewas most probably overestimated, since other sugars present in thehydrolysates had very similar retention times, resulting in occa-sional co-integration of the peak areas. Nevertheless, at highestxylose release the ethanol yield also reached maximum values, i.e.in the S. stipitis 4 ◦C ISP samples (Table 2, Fig. S2).

Storage temperature also had an impact on the ethanol yieldsince the yields were higher at 4 ◦C than in the 15 ◦C for both the S.stipitis ISP samples and the non-inoculated control. In W. anomalustreated material an opposite effect was observed, i.e. more ethanolwas produced from the 15 ◦C stored material.

4. Discussion

By applying biopreservation of moist lignocellulose we couldobtain an up to 40% increased yield compared to dry samples.Thus, the storage process also acted at as a pre-treatment. Similarto earlier results with cereal grain [4] more ethanol was obtainedfrom moist stored samples, which also included the non-inoculatedcontrols. Thus, hydration during storage is most probably a majorfactor in degrading polysaccharides to mono- and oligomers insub-sequent treatments. This, anyway, provides a new quality ofutilizing straw as raw material for biofuel production: up to nowhigh moisture content is regarded a major obstacle in utilizing itstechnical production potential (i.e. the maximum amount that canbe generated from a certain area) for energy production, especiallyin regions with a humid climate [15]. Conserving moist materialusing low energy input biopreservation can thus increase the rawmaterial basis for bioenergy production and even turn the disad-vantage of high moisture content to an advantage since it improvesthe pre-treatment efficiency. Similar results have been obtained forsugar beet pulp, where ensiling improved both enzymatic degra-dation and ethanol yield after storage [5,16].

When using moist stored cereal grain for ethanol production, noimpact of the conservation organism on the final yield was seen,as the ethanol yield was the same as in non-inoculated controls,as long the samples were not infected by moulds [4]. However, onwheat straw in the samples treated with S. stipitis during storage aneffect of the inoculated organism became obvious, as most ethanolwas formed from this material. Also the fact that an intermediateamount of ethanol was formed from co-cultivations of both yeastsis an indication that S. stipitis was involved in a de-stabilizationof the material. Production of xylanases has been documented forS. stipitis [8], while in W. anomalus, no genes encoding for hemi-cellulase or cellulase activity have been detected in the genome[17]. Interestingly, from S. stipitis treated samples most ethanolwas produced and most xylose was released. This indicates thatsome degradation of the hemicelluloses was obtained by the pres-ence of S. stipitis, although the yeast obviously did not assimilatesubstantial amounts of xylose during storage.

Wheat straw consists of about 0.75 g/g polysaccharides [7,18],including about 0.49–0.54 g/g glucose monomers [18,19]. Whenfermenting the material with S. cerevisiae that can only fermentthe glucose, the theoretical ethanol yield would be 0.24–0.27 gethanol per g wheat straw. However, by dilute acid treatmentonly 25% of the glucose was recovered [18]. Thus, by using a non-

optimized thermochemical treatment, simple autoclaving in thepresence of dilute sulphuric acid, we obtained a comparativelyhigh ethanol yield from the moist stored material. Moreover, sincethe pre-treatment was far less intensive than the usually applied

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V. Passoth et al. / Enzyme and Mi

reatment methods, most probably lower amounts of inhibitorsere produced. Consequently there was no demand for diluting

he sample before fermentation (in contrast to a recent studyhere we fermented steam exploded lignocellulose material [20])

nd most of the ethanol was produced during the first 24 h. Weave indications that further decreasing the pre-treatment inten-ity (decreasing the autoclaving time to 30 min) does not negativelympact the ethanol yield (unpublished results). Thermochemi-al pre-treatment is one of the major bottlenecks for obtainingconomically viable bioethanol production from lignocellulose,ecause it is energy demanding and co-generates inhibitors of theermentation process. The presence of inhibitors limits the con-entration of dry matter in the fermentation process, keeping thenal ethanol concentration low. This in turn increases the energyemand for distillation, which stands for the major energy inputuring ethanol production [2]. Thus, ISP can represent a major stepowards sustainable lignocellulose ethanol production.

Biological pre-treatment of lignocellulose material, using lignin-egrading fungi has been described before. However, all thesetudies have been performed in sterile systems [6,7], which is notustainable when generating a bulk product like ethanol. Our studyhows that similar to cereal grain [4], mould contamination is aeneral problem during storage of moist wheat straw. During owntudies on bio-pre-treatment with lignin degrading fungi or theryophilic yeasts Holtermanniella takashimae or Cryptococcus cere-lis [21,22] the organisms were outcompeted by the moulds presentn the material (unpublished results). Although no negative impactn ethanol yields was observed in this study, moulds, beside ofeteriorating the material may have other negative impacts due tohe potential production of mycotoxins and allergenic spores. Thus,he identification of organisms viable on wheat straw and exposingnti-mould activity is essential for developing the ISP-system.

W. anomalus and S. stipitis obviously inhibited moulds on theaterial. W. anomalus is a known biocontrol yeast with anti-mould

nd antibacterial activities on a variety of substrates, however, thiss to our knowledge the first report about anti-mould activity of thiseast on wheat straw [23–25]. Moreover, our results demonstrateor the first time an anti-mould activity for S. stipitis. The mecha-ism of mould inhibition is not clear yet. For W. anomalus, severalotential metabolites with antifungal activity have been identified,

ncluding ethyl acetate and ethanol [26,27], and the production ofeta-glucanases and killer proteins [28,29]. However, it still needso be proven, whether one of these mechanisms is active in thetraw storage. Nothing is known about any antimould activities of S.tipitis. Several glucanases are present in the genome [30] but theirelevance for antifungal activity needs to be proven. Both yeastsre respiratory [13,31], with a high activity of respiration even atestricted access to air. It is possible that these yeasts efficientlyonsume the oxygen present in the airtight system, which wouldnhibit the aerobic moulds. However, other mechanisms may bective as well, since some air was available through the syringeeedle in the lid of the storage tube. Both W. anomalus and S. stipi-is can use a broad spectrum of carbon and nitrogen sources, whichncludes a variety of sugars, amino acids (both as carbon and nitro-en sources), ammonium and nitrate [14,32,33]. This may enablehem to use even trace amounts of nutrients, removing resourcesor potential contaminants.

This study opens up a new perspective on handling lignocellu-ose biomass by regarding storage as a pre-treatment method, butt also indicates a demand for characterizing lignocellulose storageystems. Physiology, growth kinetics and mechanism of antifungalctivity of the ISP organisms inside the system are largely unknown,

nd thus factors for optimizing ISP still need to be identified. Identi-ying limits of biopreservation by challenging the system with mostommon contaminants will be required, and it will be necessary todentify further ISP-organisms to manage varying conditions like

[

l Technology 52 (2013) 105– 110 109

temperature fluctuations or different lignocellulose materials likeforest residues. Anyway, this study provides a first proof of conceptthat storage and pre-treatment can be integrated to decrease thetotal energy input during processing of lignocellulose for biofuelproduction.

Acknowledgements

We thank Majid Haddad Momeni at the Department of Molec-ular Biology, SLU, for help with preparation of enzyme solution.This work was supported by the thematic research programmeMicrodrive (http://microdrive.slu.se) at the Swedish University ofAgricultural Sciences, Uppsala and The Swedish Research Councilfor Environment, Agricultural Sciences and Spatial Planning (For-mas).

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.enzmictec.2012.11.003.

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