Biocatalysis and Agricultural Biotechnology 2 (2013) 339–343

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Biocatalysis and Agricultural Biotechnology

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Original Research Paper

Influence of culture conditions on lipid production by Candidasp. LEB-M3 using glycerol from biodiesel synthesis

Susan Hartwig Duarte a,n, Gislaine Ghiselli b, Francisco Maugeri a

a Laboratory of Bioprocess Engineering, Faculty of Food Engineering—UNICAMP, Campinas, Brazilb Instrumental and Chemical Analysis Centre—EMBRAPA AGROENERGY, Brasília, Brazil

a r t i c l e i n f o

Article history:Received 27 May 2013Received in revised form4 July 2013Accepted 16 July 2013Available online 24 July 2013

Keywords:Fatty acidsBiodieselRaw glycerolLipidLinolenic acid

81/$ - see front matter & 2013 Elsevier Ltd. Ax.doi.org/10.1016/j.bcab.2013.07.001

espondence to: Department of Food Engineerinty of Campinas—UNICAMP, Rua Monteiro Loba083-862 Campinas, SP, Brazil. Tel.: +55 19 3521ail addresses: susanduarte@hotmail.com,ea.unicamp.br (S.H. Duarte).

a b s t r a c t

The goal of this work was to use glycerol produced from biodiesel synthesis to grow Candida sp. LEB-M3and select significant variables of culture conditions on lipid production. This yeast was isolated from aBrazilian biome which showed capacity to accumulate up to 55% lipids (w/w) and convert about 43% ofglycerol into lipids. Production and lipid profile at different growth temperatures were studied. Theexperimental design showed that glycerol, FeCl3 �6H2O, yeast hydrolyzate and temperature weresignificant on the lipid production. Cultivation at 23 1C promoted the highest concentration of lipidsabout 9.9 g/L. However, the lipid profiles for the different growth temperatures were similar, with highconcentrations of linoleic acid (C18:2) (∼45–55%) and smaller amount of gamma linolenic acid (C18:3)(∼2–5%), both essential fatty acids, leading to the conclusion that lipid produced by Candida sp. LEB-M3has potential to be used both as a feedstock for biodiesel production or as a source for essentialfatty acids.

& 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Biodiesel has received considerable attention in recentyears because it is a biodegradable, renewable and non-toxicfuel, contributing to the environment by emitting less pollut-ing gases in the atmosphere than regular diesel (Antolin et al.,2002). Brazil is a large producer and consumer of biodieselbesides the conditions for cultivation of oil plants, raw materialfor biodiesel production, are favorable in several areas (Silvaet al., 2009).

The traditional production of biodiesel using vegetable oils haseconomic impacts due to their high costs and the fact that they arealso used for food, thus it is a raw material of low viability(Marchetti et al., 2008). Moreover, biodiesel produced from vege-table oils generates about 10% glycerol as the main by-product,whose generation in excess may represent an environmentalproblem, since currently the market does not absorb the entireproduction (Silva et al., 2009). According to ANP (Agência Nacionaldo Petróleo, Gás Natural e Biocombustíveis) in 2011 Brazilianmarket produced 2.6 million m³ of biodiesel, which resulted overthan 273 million m³ of glycerin. Therefore, there is an increased

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g, Faculty of Food Engineering,to 80, Barão Geraldo,4052.

interest in exploring alternatives for the production of lipids toproduce biofuel and also to use glycerol as a carbon source (Chiet al., 2007; Easterling et al., 2009; Kaur et al., 2012; Papanikolaouet al., 2008).

Oleaginous microorganisms are able to accumulate 20% ormore of their biomass in lipids, mainly in the form of triacylgly-cerol (TAG) (Ratledge, 2005), which can be used to producebiodiesel by transesterification process, where ester bonds inTAG are broken leading to two products: fatty acid methyl estersand glycerol. These microorganisms present great industrialpotential because of their ability to store lipids with propertiesand composition often similar to those of animal and vegetableproducts (Papanikolaou et al., 2008).

When it comes to use of the residual glycerol from biodiesel inculture media, without prior purification, advantages over thetraditional use of pure glycerol as a substrate are observed, mainlywith respect to lower cost and higher lipid production. Howeverrelatively few studies have reported the use of this substrate as thesole carbon source (Papanikolaou et al., 2008).

In order to obtain an alternative for the use of glycerolgenerated in the synthesis of biodiesel, as well as to study theproduction of microbial lipids as an alternative for biodieselproduction, this study sought to select significant variables inthe production of lipids and in the growth of Candida sp. LEB-M3on glycerol from biodiesel, including medium components andcultivation conditions, and also to study the influence of tempera-ture on fatty acid composition.

S.H. Duarte et al. / Biocatalysis and Agricultural Biotechnology 2 (2013) 339–343340

2. Materials and methods

2.1. Microorganism

The culture was isolated from flowers found in the PantanalbyMaugeri and Hernalsteens (2007) and screened as a oleaginousyeast strain identified of Candida sp. LEB-M3 (Laboratory ofBioprocess Engineering, UNICAMP, Brazil) in previous work(Duarte et al., 2013). The yeast was maintained on GYMP (yeast,malt, glucose, peptone) agar slant at 5 1C, as stock cultures. Beforeeach culture, colonies were reactivated on GYMP slants composedof 20.0 g/L glucose, 5.0 g/L yeast extract, 10.0 g/L malt extract,2.0 g/L KH2PO4 and 20.0 g/L agar, pH 5.5 and incubated at 30 1C for48 h.

2.2. Preparation of inoculum

Two tubes of the microbial culture on slant GYMP were scrapedwith 10 mL of 0.1% peptone water for removal of the microorgan-ism cells and transferred into Erlenmeyer flasks containing 180 mLof culture medium composed of: 30.0 g/L glycerol, 7.0 g/L KH2PO4,2.5 g/L Na2HPO4, 1.5 g/L MgSO4 �7H2O, 0.15 g/L CaCl2, 0.15 g/LFeCl3 �6H2O, 0.02 g/L ZnSO4 �7H2O, 0.06 g/L MnSO4 �H2O, 0.5 g/L(NH4)2SO4 and 0.5 g/L yeast extract, pH 6.0 (Papanikolaou andAggelis, 2002). The inoculum was cultivated at 28 1C in shakenflasks (New Brunswick Scientific model Innova 4430) at 185 rpm.The cell concentration was monitored by counting in a Neubauerchamber until reaching approximately 1�108 cells/mL (Zhang et al.,2005).

2.3. Flask cultures

For selection of the significant variables in the production oflipids, a Plackett–Burman design was performed (Rodrigues andIemma 2012). Eight independent variables were studied: concen-trations of crude glycerol (carbon source) (20–40 g/L), FeCl3 �6H2O(0–0.2 g/L), MnSO4 �H2O (0–0.06 g/L), MgSO4 �7H2O (0–1.0 g/L),(NH4)2SO4 (inorganic salts) (0.2–0.6 g/L), yeast hydrolyzate ProdexLacs (Prodesa S.A., Campinas, Brazil) (1.0–3.0 g/L), initial pH (5.5–6.5) and cultivation temperature (25–35 1C).

The flasks were inoculated with 10% (v/v) of the inoculum andmaintained in the incubator at the determined temperature foreach test, and samples were taken at pre-set intervals. Two typesof glycerol were used in the experiments: commercial glycerol, forinoculum, and crude glycerol (from the synthesis of biodiesel),containing 42.4% (w/v) glycerol, free fatty acids, methanol, saltsand other impurities, obtained by transesterification of soybean oilwith methanol without any previous treatment, which was kindlysupplied by SP-Bio, Sumaré, Brazil. The amount of crude glyceroladded to the culture medium was determined by considering thedesired concentration of the carbon source substrate.

Different experimental levels of the Plackett–Burman designwere carried out, totaling 12 trials and three replications at thecentral point. The dependent variables studied were the lipidcontent (g lipid/100 g of biomass), lipid concentrations (g/L offermented medium) and lipid yield (g lipid/g of glycerol�100).

2.4. Influence of temperature on lipid production

According of the results from the Placket–Burman design,values of each variable were chosen to study the influence oftemperature. Cultures were grown in Erlenmeyer flasks containing180 mL of culture medium composed of: 30.0 g/L crude glycerol,7.0 g/L KH2PO4, 2.5 g/L Na2HPO4, 0.15 g/L CaCl2, 0.02 g/LZnSO4 �7H2O, 0.4 g/L (NH4)2SO4 and 3.0 g/L yeast hydrolyzate, atpH 6.0. The inoculum used was the same as that in the tests

carried out in the Plackett–Burman design. Flasks were inoculatedwith 10% (v/v) of the inoculum at the desired temperature, and185 rpm.

2.5. Analysis

2.5.1. Cell growthSamples were collected and centrifuged at 785g (Dupont

Sorvall centrifuge model RC 26 Plus). After removing the super-natant, the cells were washed once with distilled water, centri-fuged again and re-suspended in a known volume of water.Absorbance was then measured at 600 nm. The absorbance valueswere converted to cell concentration (g/L) using a biomassstandard curve (Easterling et al., 2009; Duarte et al., 2013).

2.5.2. Glycerol concentrationSamples of culture medium were first diluted and filtered

through 0.22 mm filters. The analysis of glycerol was performedin HPLC (Varian 9095), using an HPX-87H column, a mobile phasecomposed of 0.005 N H2SO4, pH 2.6 at a flow of 0.6 mL/min, and aRI detector. Concentrations of glycerol (retention time about15 min) were calculated based on calibration curves constructedfor this compound using external standards.

2.5.3. Lipids extractionThe dried biomass was treated with a 2 M HCl solution to

rupture the cell wall then lipid concentration was determinedusing the Bligh and Dyer method (Bligh and Dyer, 1959), followedby re-extraction (Manirakiza et al., 2001). The chloroform phase,containing the lipids, was evaporated and lipids were measured bydry weight.

2.5.4. Determination of fatty acidsDetermination of fatty acids was performed after the lipid

fraction was esterified to obtain the fatty acid methyl esters(Metcalfe et al., 1966). The identification and quantification offatty acids was performed on a Varian 3800 GC, gas chromato-graph with 1 mL sample manual injection, a Carbowax (30 m�0.25 mm�0.25 mm) chromatographic column and a flame ioniza-tion detector (FID). Analysis conditions were: injector temperature230 1C, detector 250 1C, initial column temperature 140 1C for20 min, 2.5 1C/min to 220 1C and 10 min at 220 1C; 1.6 mL/mingas flow (N2), split ratio 1:100, gas flow in the detector: 30/30/300N2/H2/synthetic ar. Fatty acids were identified by direct com-parison of retention times with standards obtained from Sigma-Aldrich and quantified by normalization of areas.

2.6. Statistical methods

For the Plackett–Burman design the level of significance wasdetermined by the Student's-test and to evaluate the results a 90%confidence interval (p≤0.1) was used. Tests of the influence oftemperature on lipid production were performed in triplicate; datawas treated by the ANOVA and Tukey test to determine significantdifferences between the results at the different temperaturesstudied, at 95% confidence (p≤0.05). The software Statistica7.0 was used to analyze the results.

3. Results and discussion

3.1. Selection of variables for the production of lipids

The coded values of the independent variables and the resultsof the Plackett–Burman design are presented in Table 1. Thehighest lipid accumulation was observed in assay 10, where the

S.H. Duarte et al. / Biocatalysis and Agricultural Biotechnology 2 (2013) 339–343 341

lipid content was 55.02%, lipid concentration of 13.14 g/L and yieldof 38.69%, in which the variables glycerol, (NH4)2SO4, yeasthydrolyzate and initial pH were at the highest studied levels andMgSO4 �7H2O, FeCl3 �6H2O, MnSO4 �H2O and temperature at thelowest levels. However, the best conversion occurred in assay 7,where the variables glycerol, (NH4)2SO4 concentrations and tem-perature were encountered at the lowest level (�1) and theremaining variables at the highest level (+1).

Considering the effect of estimates for the three responses inTable 2, it can be seen that only the variable glycerol had asignificant effect (p≤0.1) on the lipid content response at 90%confidence. For the concentration of lipids, the variablesFeCl3 �6H2O and temperature showed a significant negative effect,while glycerol and yeast hydrolyzate concentrations and initial pHshowed a significant positive effect. For the yield of lipids,temperature and yeast hydrolyzate had significant effects at 90%confidence, indicating that organic nitrogen sources are morebeneficial to lipid production of Candida sp. LEB-M3 than inorganicnitrogen sources, which was also observed with Trichosporonfermentans (Zhu et al., 2008). Curvature was a significant effectdecreasing the standard error and preventing that effects of thevariables were masked (Papanikolaou and Aggelis, 2002).

The compounds MgSO4 �7H2O, MnSO4 �H2O and FeCl3 �6H2Opresented insignificant effects or significant and negative effects(Table 2), which means that be removed from the medium.Concerning glycerol, whose effect is significant and positive for

Table 1Plackett–Burman design (coded values) and responses in lipid content (%) lipidconcentration (g/L) and yield (%) at the end of cultivation.

Assays Independent variablesa Lipids

X1 X2 X3 X4 X5 X6 X7 X8Content

(%)Concentration

(g/L)Yield(%)

1 + – + – – – + + 47.68 5.53 16.902 + + – + – – – + 53.18 6.28 17.273 – + + – + – – – 28.10 3.83 20.384 + – + + – + – – 41.16 7.70 23.135 + + – + + – + – 48.85 10.09 28.756 + + + – + + – + 51.37 8.43 22.017 – + + + – + + – 37.84 7.64 43.248 – – + + + – + + 31.18 4.34 21.029 – – – + + + – + 40.37 6.05 32.19

10 + – – – + + + – 55.02 13.14 38.6911 – + – – – + + + 33.61 5.62 25.9212 – – – – – – – – 47.70 6.35 32.3413 0 0 0 0 0 0 0 0 21.74 3.48 10.9114 0 0 0 0 0 0 0 0 22.81 3.86 12.1615 0 0 0 0 0 0 0 0 19.59 3.14 9.62

a X1: glycerol, X2: MgSO4 �7H2O, X3: FeCl3 �6H2O, X4: MgSO4 �7H2O, X5: (NH4)2SO4,X6: yeast hydrolyzate, X7: initial pH, X8: temperature.

Table 2Effect estimates for the dependents variables (significant at 90% confidence).

Factor Lipid content (%) Lipid concentr

Effect (%) St error t (5) p-value Effect (g/L)

Mean 43.00 1.84 23.24 o0.001 7.08Curvature �43.25 8.27 �5.22 0.003 �7.17Glycerol 13.07 3.69 3.53 0.016 2.89MgSO4 �7H2O �1.69 3.69 �0.45 0.666 �0.20FeCl3 �6H2O �6.90 3.69 �1.86 0.121 �1.67MnSO4 �H2O �1.81 3.69 �0.49 0.644 �0.13(NH4)2SO4 �1.04 3.69 �0.28 0.788 1.12Yeast hydrolyzate 0.44 3.69 0.12 0.908 2.02Initial pH �1.28 3.69 �0.34 0.742 1.28Temperature �0.21 3.69 �0.05 0.956 �2.08

lipid content and lipid concentration, it should be kept at thecentral level, because at higher concentrations there are consider-able glycerol residues at the end of fermentations which isundesirable, as shown by Fig. 1. Furthermore it is possible toreduce the cultivation time when the goal is total consumption ofglycerol.

For yeast hydrolyzate concentrations, whose effect is significantand positive for lipid concentration and lipid yield, it is maintainedat the highest level (+1). Regarding pH, since it is significant onlyfor the lipid concentration, it will be kept at the central level forthe next step, as well as (NH4)2SO4, which is necessary for cellgrowth as an inorganic nitrogen source.

Temperature is an important variable, showing a negativeeffect for all responses and should be studied in specific experi-ments at lower temperatures. Moreover there are studies report-ing in the literature that temperature can be cause influence onfatty acid profile formation and saturation, and then it is animportant variable to study separately on profile lipids andaccumulation of this (Ruangudom and Punpeng, 2011; Leathersand Scragg, 1989).

3.2. Influence of temperature on the production of lipids

As shown by Fig. 2, the growth of Candida sp. LEB-M3 onmedium containing glycerol from biodiesel synthesis is slightlydifferent according to the fermentation temperature. It can be seenthat at 23 1C growth reached the stationary phase after 192 h ofculture, where biomass reached the highest concentration(19.771.07 g/L), while at 25 1C and 27 1C the stationary phase isnot reached even after 240 h of cultivation.

Lipid content showed no significant difference between thetemperatures, as shown in Table 3. However, the concentration oflipids at 23 1C is significantly different from the others, reaching9.9070.87 g/L. This result is due to the fact that for the determi-nation of lipid concentration, the amount of biomass produced istaken into account for the concentration of lipids, which washigher at 23 1C as shown in Fig. 2. Lipid yield was highest at 23 and25 1C according to the above results.

Ruangudom and Punpeng (2011) studied the influence oftemperature on lipid production by the yeast Rhodosporidiumtoruloides TISTR 5123 from sugar cane juice; they found thattemperature was important variable and with the cultivation at20 1C reached a maximum value of lipid content about 55%. Also,the microbial lipid profile, quantity, productivity and conversionefficiency are influenced by various process conditions (Beopouloset al., 2009). However, in this present work temperature wassignificant only for lipid content, as previously stated (Table 3).

ation (g/L) Lipid yield (%)

St error t (5) p-value Effect (%) St error t (5) p-value

0.30 23.13 o0.001 26.82 1.61 16.65 o0.0011.36 �5.23 0.003 �31.84 7.20 �4.42 0.0060.61 4.71 0.005 �4.72 3.22 �1.46 0.2020.61 �0.33 0.753 �1.11 3.22 �0.34 0.7430.61 �2.73 0.040 �4.74 3.22 �1.47 0.2000.61 �0.21 0.836 1.55 3.22 0.48 0.6480.61 1.84 0.125 0.70 3.22 0.21 0.8350.61 3.30 0.021 8.08 3.22 2.51 0.0530.61 2.10 0.089 4.53 3.22 1.40 0.2180.61 �3.40 0.019 �8.53 3.22 �2.65 0.045

0 24 48 72 96 120 144 168 192 216 240

Time (h)

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Gly

cero

l (g/

L)Fig. 1. Time courses for biomass growth and glycerol consumption at three different initial glycerol concentrations, (a) and (c) for the assays: 1 (○), 2 (□), 3 (◊), 4 (Δ), 5 (●), 6(■), 7 (♦), 8 (▲), and 9 (+); (b) and (d) for the assays 10 (○), 11 (□), 12 (◊), 13 (Δ), 14 (●), and 15 (■).

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Fig. 2. Time course of Candida sp. LEB-M3 growth and glycerol consumption at different temperatures: (o) 23 1C, (□) 25 1C e and (◊) 27 1C.

Table 3Mean and standard deviations for lipid content (%), lipid concentration (g/L) andlipid yield (%) according to Tukey's test of statistical analysisa.

Temperature (1C) Lipid content (%) Lipid concentration (g/L) Lipid yield (%)

23 50.1872.75a 9.9070.87a 32.0773.39a

25 47.6573.7a 7.6570.44b 28.5372.81a,b

27 44.1471.63a 7.7070.84b 22.6770.36b

a Same letters in a column means no significant differences between values(po0.05).

Table 4Fatty acid composition (% w/w of total lipid) in the biomass of Candida sp. LEB-M3grown in culture media containing biodiesel glycerol at different temperatures andcomparison with the profile of other vegetable oils [25].

Fatty acid 23 1C 25 1C 27 1C Soybean Sunflower Palm

Saturated Fatty acidC16:0 12.9 9.8 14.2 11.4 7.1 42.6C18:0 3.1 3.5 1.4 4.4 4.7 4.4

Monounsaturated fatty acidsC16:1 0.6 0.4 0.51 — — 0.3C18:1 34.0 25.5 27.4 20.8 25.5 40.5

Polyunsaturated fatty acidsC18:2 44.6 54.9 52.3 53.8 62.4 10.1γ C18:3 2.1 5.1 3.6 9.3 — 0.2

S.H. Duarte et al. / Biocatalysis and Agricultural Biotechnology 2 (2013) 339–343342

3.3. Fatty acid profile

In the cells cultivated at 23 1C (Table 4), the fatty acid profilewas 16.06% saturated fatty acids, especially palmitic acid (C16:0),and 35.32% monounsaturated fatty acids, with emphasis on oleicacid (C18:1). However the amount of polyunsaturated fatty acidswas higher, being 46.73% of the total, with prominence of theessential linoleic acid (C18:2), making up 44.6% and gammalinolenic acid (C18:3) (GLA) composing 2.13%.

In the cells cultivated at 25 1C, the percentage of saturated fattyacids was 13.26%, composed primarily of C16:0, while of the26.78% monounsaturated fatty acids, C18:1 stood out. Polyunsatu-rated fatty acids accounted for 60.0% in this case, of which C18:2made up 54.9% and C18:3 5.10%. In cells cultivated at 27 1C, thepercentage of saturated fatty acids was 15.66%, with prominence

of the C16:0, while oleic acid C18:1 was prominent amongmonounsaturated fatty acids. The polyunsaturated fatty acidsreach a total of 55.94%, with 52.32% of C18:2 and 3.62% of C18:3.

The current results are in agreement with other studies, whereit was observed that the oleaginous yeast strain Cryptococcuscurvatus NRRLY-1511 produced lipids consisting mainly of 30.68%(C18:2), 22.66% (C18:1) and 16.74% (C16:0) (El Fadaly et al., 2009).Papanikolaou et al. (2004) also reported the microbial productionof lipids containing 3.5% of GLA, corresponding to 16–19 mg of GLAper gram of dry biomass. In the present study the amount of GLA

S.H. Duarte et al. / Biocatalysis and Agricultural Biotechnology 2 (2013) 339–343 343

produced by Candida sp. LEB-M3 ranged between 10.65 and24.3 mg/g of biomass (dry weight basis), depending on thefermentation temperature, which represents significant produc-tion of this essential fatty acid when using residual glycerol frombiodiesel as a substrate.

Evans and Ratledge (1983) reported the lipid production byCandida curvata in medium containing xylose as the carbonsource, where the fatty acid profile consisted of 15% stearic acid(18:0) and 4% of linoleic acid (18:2). Li et al. (2010), when studyingthe production of lipids by the yeast Rhodotorula mucilaginosaTJY15a using cassava starch as the substrate, obtained a fatty acidprofile composed of 63.5% C18:1 and 22.3% C16:0, and 5.7% C18:2,the only polyunsaturated fatty acid.

When comparing the fatty acid profiles produced by Candidasp. LEB-M3 for the different temperatures, it can be found that at25 1C there was a higher polyunsaturated fatty acid production,followed by the temperature of 27 1C, which showed a similarprofile, and finally at 23 1C was the lowest production of essentialfatty acids. It seems that temperatures above 23 1C are less suitablefor increased production of these acids.

Single-cell oils, those obtained from microorganisms, are nowaccepted as biotechnological products playing key roles in thesupply of major polyunsaturated fatty acids (PUFA), such as thegamma linolenic acid produced in this work, which are known tobe essential for human nutrition and development (Ratledge,2005).

The lipids produced by Candida sp. LEB-M3 at all temperaturescan be used as feedstock for biodiesel production because thecomposition on fatty acids is similar to the composition ofvegetable oils commonly used for biodiesel production (Akohet al., 2007) mainly soybean oil, and still can be used as a sourceof essential fatty acids for use in foods.

4. Conclusion

The Plackett–Burman screening experimental design enabledthe establishment of important variables for lipid production bythe yeast Candida sp. LEB-M3 from biodiesel based glycerol, whichincluded the glycerol concentration, yeast hydrolyzate concentra-tion, pH and temperature. The yeast was able to accumulate up to55% (dry weigh basis) lipids and convert about 43% of glycerolinto lipids. The fatty acid profile showed similarity at the threetemperatures, with a predominance of C18:2, but during cultiva-tion at 23 1C there was increased production of C18:1 and lowerproduction of linoleic acid when compared to the other tempera-tures. Lipids produced by Candida sp. LEB-M3 represent a sig-nificant source of this essential fatty acid when using residualglycerol from biodiesel as a substrate.

Acknowledgments

The authors would like to thank the Laboratory of Ecology andBiotechnology of Yeasts (Federal University of Minas Gerais) andCAPES for their financial support.

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