Bioresource Technology 110 (2012) 711–714

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Bioresource Technology

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Short Communication

Oil production by the yeast Trichosporon dermatis cultured in enzymatichydrolysates of corncobs

Chao Huang a,b, Xue-fang Chen b,c, Lian Xiong a,b, Xin-de Chen a,b,⇑, Long-long Ma a,b

a Key Laboratory of Renewable Energy and Gas Hydrate, Chinese Academy of Sciences, Guangzhou 510640, PR Chinab Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 510640, PR Chinac Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China

a r t i c l e i n f o

Article history:Received 28 September 2011Received in revised form 10 January 2012Accepted 16 January 2012Available online 24 January 2012

Keywords:Trichosporon dermatisMicrobial oilCorncob enzymatic hydrolysate

0960-8524/$ - see front matter � 2012 Elsevier Ltd. Adoi:10.1016/j.biortech.2012.01.077

⇑ Corresponding author at: No. 2, Nengyuan Road510640, PR China. Tel./fax: +86 20 37213916.

E-mail address: cxd_cxd@hotmail.com (X.-d. Chen

a b s t r a c t

Corncob was hydrolyzed with Trichoderma reesei cellulase and used as substrate for growth by the oleag-inous yeast Trichosporon dermatis without detoxification or addition of a nitrogen source or trace ele-ments. A total biomass of 24.4 g/L with a lipid content of 40.1% (corresponding to a lipid yield of 9.8 g/L), and a high lipid coefficient (lipid yield per mass of sugar, % g/g) of 16.7 could be achieved after culti-vation for 7 days. Therefore, T. dermatis is a promising strain for microbial oil production from lignocel-lulosic biomass.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Microbial oils, namely single cell oils (SCO), can be used as sub-stitutes for value-added lipids such as cocoa–butter (Adamczaket al., 2009; Papanikolaou and Aggelis, 2011), but also as feedstockfor biodiesel production because their fatty acid composition issimilar to that of vegetable oils (Zhu et al., 2008; Huang et al.,2009; Papanikolaou and Aggelis, 2011).

Various fermentation substrates have been explored to lowerproduction costs of microbial oils. These materials include not onlyglycerol (Fakas et al., 2009), oils and fats (Papanikolaou and Agge-lis, 2003), wastewater (Xue et al., 2008), and wastes from foodindustry (Zhu et al., 2008), but also lignocellulosic biomass suchas rice straw (Huang et al., 2009), wheat straw (Yu et al., 2011),and bagasse (Tsigie et al., 2011). Corncob is another abundant agri-cultural residue that could be utilized for SCO production.

Although acid-hydrolysis is most often used to produce ligno-cellulosic hydrolysates for SCO production (Huang et al., 2009; Tsi-gie et al., 2011; Yu et al., 2011), it generates inhibitory substancesrequiring a detoxification step prior to fermentation (Rubin, 2008;Huang et al., 2011). Enzymatic hydrolysis of lignocellulosic materi-als would avoid such problems. To date, only a few oleaginousmicroorganisms such as Trichosporon fermentans (Huang et al.,2009), Yarrowia lipolytica (Tsigie et al., 2011), Cryptococcus curvatus(Yu et al., 2011) have been examined for microbial oil production

ll rights reserved.

, Tianhe District, Guangzhou

).

on lignocellulosic hydrolysates. Hence, screening new strains forSCO production on lignocellulosic hydrolysates would be beneficialfor the development of microbial oil production. In the presentstudy, Trichosporon dermatis, an oleaginous yeast was investigatedfor its capability to produce oil when grown on corncob hydroly-sate generated with Tricoderma reesei.

2. Methods

2.1. Microorganisms and raw materials

Trichoderma reesei CICC 13052 was used for cellulase productionand Trichosporon dermatis CH007 (Laboratory of Energy and Bio-chemical Engineering, Guangzhou Institute of Energy Conversion,Chinese Academy of Sciences) was used for SCO production. Corn-cobs pretreated with organic solvents (Teramoto et al., 2008) werekindly provided by ZHONGKE New Energy Co., LTD (Yin-kou,China).

2.2. Enzyme production and enzymatic hydrolysis of corncob

T. reesei was cultured in medium containing (g/L) glucose, 10;peptone, 1; citric acid, 0.5; Vogel’s Medium (Vogel, 1964), 20;Tween 80, 0.15, pH 5.0 at 30 �C and 150 rpm for 36 h. A 10% inoc-ulum of this culture was transferred to a culture medium contain-ing (g/L): glucose, 1; citric acid, 0.5; (NH4)2SO4, 2; Vogel’s Medium,20; Tween 80, 0.15; wheat bran, 10; rice straw, 20, pH 5.0. Cultiva-tion was performed in a 250-mL conical flask containing 100 mLmedium in a rotary shaker at 28 �C and 150 rpm. After

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fermentation for 5 days, the solid fraction was separated by vac-uum filtration and the fermentation broth was used for hydrolysis.The pretreated corncob was mixed with tap water and the liquidenzyme generated by T. reesei (1:1 v/v) to give a mixture with a so-lid loading of 10% (w/v). Hydrolysis was carried out at 50 �C for3 days. The resulted hydrolysate was recovered by vacuum filtra-tion using a No. 102 Qualitative Filter (Whatman-Xinhua Co.,LTD, China) and stored at 4 �C prior to use.

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2.3. Microbial oil production on corncob enzymatic hydrolysate

Sterilized by autoclaving at 115 �C for 20 min, the corncobenzymatic hydrolysate (initial pH 7.0) was used without detoxifi-cation and nutrient addition as medium for oil production byT. dermatis. The yeast was cultured first on medium containing(g/L) glucose, 20; peptone, 10; yeast extract, 10; at 28 �C and150 rpm for 24 h. Then, 5% seed culture was inoculated into thecorncob hydrolysate. Cultivation was performed in a 250-mLconical flask containing 50 mL of hydrolysate in a rotary shakerat 28 �C and 150 rpm.

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Fig. 1. Production of microbial oil on corncob enzymatic hydrolysate medium by T.dermatis. (A)Time course of cell growth and lipid accumulation. (4) Biomass; (h)lipid yield; (s) lipid content; (B)Time course of sugar and ammonium nitrogenutilization.

2.4. Analytical methods

Biomass was harvested by centrifugation at 8000 g for 10 minand the dry cell weight was determined (Xue et al., 2008). Lipidcontent and lipid yield were measured as descried by Zhu et al.(2008). Lipid coefficient (%, g/g) refers to the lipid yield (g/L) on su-gar consumption (g/L). The fatty acid profile was determined byconversion of fatty acids to fatty acid methyl esters and gas chro-matography (GC-2010, Shimadzu Corporation, Japan) with ioniza-tion detector and an Rtx�-Wax capillary column (RestekCorporation, USA). The column temperature was maintained at195 �C for 12 min and upgraded from 195 �C to 230 �C at a rateof 10 �C min�1 and kept for 15 min. Nitrogen was used as the car-rier gas at 1.0 mL/min. Split ratio was 1:30 (v/v). The injector andthe detector temperatures were set at 250 �C and 280 �C, respec-tively. Sugar concentrations (D-glucose, D-xylose, and D-cellobi-ose) in corncob hydrolysate were analyzed by HPLC (Waters2685 systems, Waters Corp., USA), with a RI detector (Waters2414), and on Shodex Sugar SH-1011 column using 5 mM H2SO4

solution at a flow rate of 0.5 mL/min at 50 �C. Ammonium nitrogenwas evaluated by a Hach DR2700 Water Quality Analyzer (HachCompany, USA). All reported data were averages of experimentsperformed in triplicate.

Table 1Lipid composition of T. dermatis during cultivation on the corncob enzymatichydrolysate.

Fermentation time (days) Lipid composition (%)

C16:0 C18:0 C18:1 C18:2 Othersa

1 21.6 12.8 41.6 14.9 9.22 30.0 15.6 36.9 11.4 6.03 30.3 15.6 37.9 10.4 5.74 29.3 15.6 39.5 9.9 5.75 28.8 14.4 41.3 9.9 5.56 26.6 14.5 40.0 10.0 8.87 27.5 15.0 42.5 9.3 5.78 29.1 13.4 40.4 9.6 7.59 27.1 14.3 40.9 9.9 7.87b 26.1 15.0 43.8 9.4 5.77c 26.7 13.2 43.7 11.1 5.37d 27.7 13.6 43.4 10.0 5.3

a Others were C8:0, C10:0, C12:0, C14:0, C16:1, C18:3, C20:0 C20:1, C20:2, C22:0,and C24:0.

b Fermentation on the hydrolysate with initial sugar concentration of 70 g/L.c Fermentation on the hydrolysate with initial sugar concentration of 80 g/L.d Fermentation on the hydrolysate with initial sugar concentration of 90 g/L.

3. Results and discussion

In contrast to previous studies using lignocellulosic acid hydrol-ysates for microbial oil production (Huang et al., 2009; Tsigie et al.,2011; Yu et al., 2011), corncob hydrolysate was obtained by enzy-matic means in this study. The hydrolytic enzymes were producedwith low-cost substrates (wheat bran or rice straw). After enzy-matic hydrolysis for three days, the corncob hydrolysate contained35.6 g/L glucose, 8.0 g/L cellobiose, and 16.5 g/L xylose (measuredby HPLC). Interestingly, besides glucose, the corncob enzymatichydrolysate also contained xylose, suggesting that the xylan incorncob could also be hydrolyzed by the enzymes generated byT. reesei. A low concentration of cellobiose (8.0 g/L) was detectedin the corncob hydrolysate, indicating that glucosidase might alsobe generated by T. reesei. Lipid production by oleaginous yeasts re-quires nitrogen sources and trace elements (Zhu et al., 2008), but inthis study, no additional nutrients were added to the corncobhydrolysate. Also, detoxification processes such as overliming orabsorption were not necessary.

Growth and lipid accumulation by T. dermatis on corncobhydrolysate are shown in Fig. 1A. Sugar and ammonium nitrogenutilization by T. dermatis are illustrated in Fig. 1B. No obvious lagphase was observed at the beginning of fermentation (after oneday’s fermentation, the biomass of T. dermatis was about 6.5 g/L).The lipid content of T. dermatis (7.6%) was relatively low afterone day’s fermentation. It is also possible that the nitrogen levelswere still too high at that time since lipid accumulation usually be-gins when nitrogen becomes limited (Papanikolaou and Aggelis,2011). The initial ammonium nitrogen presented in the corncob

Table 2Lipid production on different carbon sources by various microorganisms.

Strain Carbon source Biomass (g/L) Lipid content (%) Lipid yield (g/L) Reference

Y. lipolytica Industrial fats 8.7 44.0 3.8 (Papanikolaou et al., 2001)M. isabellina Glycerol 7.8 25.6 2.0 (Fakas et al., 2009)C. echinulata Glycerol 6.2 53.2 3.3 (Fakas et al., 2009)L.starkeyi Sewage sludge 9.4 68.0 6.4 (Angerbauer et al., 2008)R. glutinis Monosodium glutamate wastewater 25.0 20.0 5.0 (Xue et al., 2008)Mucor sp. RRL001 Tapioca starch 28.0 17.8 5.0 (Ahmed et al., 2006)Aspergillus sp. Waste cooking oils 18.0 64.0 11.5 (Papanikolaou et al., 2011)T. dermatis Corncob enzymatic hydrolysate 24.4 40.1 9.8 This work

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hydrolysate was about 110 mg/L. After 1 day’s fermentation, mostof the ammonium nitrogen was utilized and only about 12 mg/Lammonium nitrogen still existed in the medium (Fig 1B), and thelipid content of T. dermatis increased. It is worth noting that lipidaccumulation by T. dermatis was associated with its growth. Inter-estingly, as shown in Fig. 1B , T. dermatis utilized glucose, xyloseand cellobiose simultaneously. This pattern is different from thatof Trichosporon fermentans which used xylose after the exhaustionof glucose on rice straw hydrolysate (Huang et al., 2009). When theglucose was almost used up, the consumption of xylose increasedand finally, glucose, xylose, and cellobiose were depleted by T. der-matis. Also, the ability of using cellobiose for SCO production showsthe potential of T. dermatis to use simultaneous saccharificationand fermentation process to produce microbial oil which wouldfurther reduce the cost of SCO production. After 7 days’ fermenta-tion, the lipid content and lipid yield of T. dermatis were at theirhighest points of 40.1% and 9.8 g/L, respectively.

In some cases, changes in the fatty acids composition with theage of the culture have been observed (Fakas et al., 2009). In thepresent study, as shown in Table 1, the percentage of unsaturatedfatty acids (58.9%) in the fatty acid methyl esters on day 1 washigher than that on day 2–9. From the 2nd day to the 9th day,the lipid composition of T. dermatis changed during the fermenta-tion, suggesting that the fermentation time influenced lipid com-position of T. dermatis. Overall, the lipid extracted from T.dermatis mainly contained palmitic acid, stearic acid, oleic acidand linoleic acid, and the unsaturated fatty acids amounted toabout 52.2%. This composition of the lipid is similar to that of palmoils, and so the lipid is a promising feedstock for bio diesel produc-tion. After 7 days’ fermentation, a clear decline in the lipid contentof T. dermatis was observed due to the use of the accumulated lipidfor cell proliferation (Papanikolaou et al., 2001; Papanikolaou andAggelis, 2003). Changes in fatty acid composition could have re-sulted from selective utilization of oleic and stearic acids.

The initial sugar concentration (60 g/L) in the corncob enzy-matic hydrolysate was increased with time and solid–liquid ratioduring hydrolysis. The higher initial sugar concentration (70, 80and 90 g/L) in the corncob enzymatic hydrolysate resulted in high-er biomass (27.2, 30.1, and 30.7 g/L, respectively) and lipid content(40.3%, 41.0%, and 42.6%, respectively) of T. dermatis after 7 days0

fermentation in spite that its lipid coefficients (16.2%, 15.7%, and15.3%, respectively) were a little lower. A higher initial sugar con-centration increased the ratio of unsaturated fatty acids in the lipidcomposition of T. dermatis (Table 1) and the highest ratio of unsat-urated fatty acids was achieved on the hydrolysate with an initialsugar concentration of 80 g/L. Interestingly, the fermentation pHchanged little throughout the fermentation process and rangedfrom 6.7 to 7.2, indicating that the corncob enzymatic hydrolysatehad a certain buffer capacity which could maintain the growth andlipid accumulation of T. dermatis at a suitable pH.

Cultivation of C. curvatus on the wheat straw hydrolysate (Yuet al., 2011) and Y. lipolytica on the bagasse hydrolysate (Tsigieet al., 2011) had resulted in lipid yields of 5.8 and 6.7 g/L,

respectively. These values are lower than that obtained in the cur-rent study (9.8 g/L). The lipid yield of T. dermatis was lower thanthat of T. fermentans (11.5 g/L) on the rice straw hydrolysate(Huang et al., 2009), but the lipid coefficient for T. dermatis(16.7% g/g) on the corncob enzymatic hydrolysate was higher thanthat for T. fermentans (11.9% g/g) grown on rice straw hydrolysate.Comparison of lipid production by oleaginous microorganisms onvarious low-cost mediums is provided in Table 2. Apparently, theability of utilizing different agro-industrial residues varied withmicrobial strains. Although L. starkeyi and C. echinulata had thehigher lipid content (68.0% and 53.2%) when cultivated on sewagesludge and glycerol, a higher lipid yield was obtained by T. dermatison corncob enzymatic hydrolysate. The comparison shows the po-tential of using lignocellulosic hydrolysates for SCO production byT. dermatis.

4. Conclusions

Trichosporon dermatis was able to utilize corncob hydrolyzed byan enzyme extract from Trichoderma reesei for oil production with-out detoxification of the hydrolysate or the addition of a nitrogensource or trace elements. The lipid yield of T. dermatis suggeststhe potential of economic production of microbial oil from thislow-cost feedstock. More research on efficient pretreatment of lig-nocellulosic biomass and genetic modification of both T. dermatisand T. reesei need to be done to make this bioconversion commer-cially viable.

Acknowledgements

The authors acknowledge the financial support of industrializa-tion project of high-new technology of Guangdong province(2009B011200008), the important project of knowledge innovationengineering of Chinese Academy of Sciences (KSCX2-YW-G-063)and the support plan project of national science and technology(2012BAD32B07).

References

Adamczak, M., Bornscheuer, U.T., Bednarski, W., 2009. The application ofbiotechnological methods for the synthesis of bio-diesel. Eur. J. Lipid Sci.Technol. 111 (8), 800–813.

Ahmed, S.U., Singh, S.K., Pandey, A., Kanjilal, S., Prasad, R.B.N., 2006. Effects ofvarious process parameters on the production of gamma-linolenic acid insubmerged fermentation. Food Technol. Biotechnol. 44 (2), 283–287.

Angerbauer, C., Siebenhofer, M., Mittelbach, M., Guebitz, G.M., 2008. Conversion ofsewage sludge into lipids by Lipomyces starkeyi for biodiesel production.Bioresour. Technol. 99 (8), 3051–3056.

Fakas, S., Papanikolaou, S., Batsos, A., Galiotou-Panayotou, M., Mallouchos, A.,Aggelis, G., 2009. Evaluating renewable carbon sources as substrates for singlecell oil production by Cunninghamella echinulata and Mortierella isabellina.Biomass Bioenerg. 33 (4), 573–580.

Huang, C., Zong, M.H., Wu, H., Liu, Q.P., 2009. Microbial oil production from ricestraw hydrolysate by Trichosporon fermentans. Bioresour. Technol. 100 (19),4535–4538.

714 C. Huang et al. / Bioresource Technology 110 (2012) 711–714

Huang, C., Wu, H., Liu, Q.P., Li, Y.Y., Zong, M.H., 2011. Effects of aldehydes on thegrowth and lipid accumulation of oleaginous yeast Trichosporon fermentans. J.Agric. Food Chem. 59 (9), 4606–4613.

Papanikolaou, S., Aggelis, G., 2003. Modeling lipid accumulation and degradationin Yarrowia lipolytica cultivated on industrial fats. Curr. Microbiol. 46 (6),398–402.

Papanikolaou, S., Chevalot, I., Komaitis, M., Aggelis, G., Marc, I., 2001. Kinetic profileof the cellular lipid composition in an oleaginous Yarrowia lipolytica capable ofproducing a cocoa-butter substitute from industrial fats. Anton. Leeuw. Int. J.G.80 (3-4), 215–224.

Papanikolaou, S., Aggelis, G., 2011. Lipids of oleaginous yeasts. Part II: Technologyand potential applications. Eur. J. Lipid Sci. Technol. 113 (8), 1052–1073.

Papanikolaou, S., Dimou, A., Fakas, S., Diamantopoulou, P., Philippoussis, A.,Galiotou-Panayotou, M., Aggelis, G., 2011. Biotechnological conversion ofwaste cooking olive oil into lipid-rich biomass using Aspergillus andPenicillium strains. J. Appl. Microbiol. 110 (5), 1138–1150.

Rubin, E., 2008. Genomics of cellulosic bio fuels. Nature 454 (7206), 841–845.

Teramoto, Y., Lee, S.H., Endo, T., 2008. Pretreatment of woody and herbaceousbiomass for enzymatic saccharification using sulfuric acid-free ethanol cooking.Bioresour. Technol. 99 (18), 8856–8863.

Tsigie, Y.A., Wang, C.Y., Truong, C.T., Ju, Y.H., 2011. Lipid production from Yarrowialipolytica Po1g grown in sugarcane bagasse hydrolysate. Bioresour. Technol. 102(19), 9216–9222.

Vogel, H.J., 1964. Distribution of lysine pathways among fungi: evolutionaryimplications. Am. Nat. 435–446.

Xue, F.Y., Miao, J.X., Zhang, X., Luo, H., Tan, T.W., 2008. Studies on lipid productionby Rhodotorula glutinis fermentation using monosodium glutamate wastewateras culture medium. Bioresour. Technol. 99 (13), 5923–5927.

Yu, X., Zheng, Y., Dorgan, K.M., Chen, S., 2011. Oil production by oleaginous yeastsusing the hydrolysate from pretreatment of wheat straw with dilute sulfuricacid. Bioresour. Technol. 102 (10), 6134–6140.

Zhu, L.Y., Zong, M.H., Wu, H., 2008. Efficient lipid production with Trichosporonfermentans and its use for bio diesel preparation. Bioresour. Technol. 99 (16),7881–7885.