Bioresource Technology 101 (2010) 7556–7562

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Bioresource Technology

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Direct microbial conversion of wheat straw into lipid by a cellulolytic fungusof Aspergillus oryzae A-4 in solid-state fermentation

Lin Hui a, Cheng Wan b, Ding Hai-tao a, Chen Xue-jiao a, Zhou Qi-fa c,*, Zhao Yu-hua a,**

a Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou 310058, Chinab College of Science, Jiangxi Agriculture University, Nanchang 330045, Chinac Institute of Plant Science, College of Life Sciences, Zhejiang University, Hangzhou 310058, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 15 January 2010Received in revised form 7 April 2010Accepted 9 April 2010Available online 4 May 2010

Keywords:Microbial lipidDirect microbial conversionAspergillus oryzae A-4CellulaseSolid-state fermentation

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.04.027

* Corresponding author.** Corresponding author. Tel.: +86 571 88208557; fa

E-mail addresses: zqifa2002@yahoo.com (Z. Qi-fa)(Z. Yu-hua).

Direct microbial conversion of wheat straw into lipid by a cellulolytic fungus of Aspergillus oryzae A-4 insolid-state fermentation (SSF) was investigated. In submerged fermentation, A. oryzae A-4 accumulatedlipid to 15–18.15% of biomass when pure cellulose was utilized as the sole substrate. In SSF of the wheatstraw and bran mixture, A. oryzae A-4 yielded lipid of 36.6 mg/g dry substrate (gds), and a cellulase activ-ity of 1.82 FPU/gds with 25.25% of holocellulose utilization in the substrates were detected on the 6thday. The lipid yield reached 62.87 mg/gds in SSF on the 6th day under the optimized conditions fromPlackett–Burman design (PBD). Cellulase secretion of A. oryzae A-4 was found to influence the lipid yield.Dilute acid pretreatment of the straw and addition of some agro-industrial wastes to the straw couldenhance lipid production of A. oryzae A-4.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Biodiesel, produced by transesterification reaction of triacyl-glycerol lipid, has received more and more attention as an alterna-tive diesel fuel (Vasudevan and Briggs, 2008). Microbial lipid,namely single cell oil (SCO), is considered as a promising feedstockfor biodiesel production due to some advantages such as short pro-ducing period, little labor required and easy to scale up (Li et al.,2008). Microbial lipid production utilizing glucose in submergedfermentation (SmF) has been intensively studied. However, thehigh costs of SmF system and glucose in microbial lipid productionhas become the major obstacle for the development and wideapplication of biodiesel production.

Exploration of inexpensive culture media and low-cost fermen-tation system could lower the cost of microbial lipid. Manyagro-industrial wastes (e.g. waste molasses, sludge sewage, mono-sodium glutamate wastewater, free-fatty acids, crude glycerol)have been reported to be used as raw materials for lipid production(Angerbauer et al., 2008; Fakas et al., 2008; Mlicková et al., 2004;Xue et al., 2008; Zhu et al., 2008). Some hydrolysates such as toma-to-waste hydrolysate and lignocellulose hydrolysate have alsobeen utilized for the production of SCO (Fakas et al., 2008; Huang

ll rights reserved.

x: +86 571 88206995., yhzhao1958@yahoo.com.cn

et al., 2009). Recently, there has been an increasing interest in uti-lizing cellulosic wastes such as straw as carbon source, althoughthe works related with microbial lipid production from cellulosicsubstrates are very limited (Singh, 1991, 1992).

Compared with SmF, solid-state fermentation (SSF) could di-rectly utilize insoluble wastes without physical–chemical or enzy-matic hydrolysis, therefore significantly reduces the processingcosts. SSF has been successfully used for the production of enzymesand secondary metabolites for a long time (Pandey et al., 2000).However, to the best of our knowledge, SSF of cellulosic wastesfor microbial lipid accumulation has been rarely studied and failedto work well without addition of expensive cellulase (Peng andChen, 2008). There are several important factors affecting lipidaccumulation in SSF processes. Among these factors, selection ofsuitable strains is crucial, and attempts have been made to explorethe possibilities of using endophytic fungi isolated from oleaginousplants for bioconversion of cellulosic wastes to lipid in SSF system(Pandey et al., 2000; Peng and Chen, 2007).

Aspergillus strains are natural fungal species frequently used inSSF processes, and have been successfully employed in the produc-tion of enzymes (e.g. b-glucosidase, xylanase, cellulase) and organ-ic acids (André et al., 2010; Nasuno, 1974; te Biesebeke et al.,2002). Several species of this genus like Aspergillus niger, Aspergillusnidulans and Aspergillus oryzae have also been proved to be able toaccumulate lipid up to 12–25% of biomass (Singh, 1991, 1992;Wynn and Ratledge, 1997; Yi and Zheng, 2006). Recently, twoA. niger strains (NRRL 364, LFMB 1), grown on glycerol medium,

L. Hui et al. / Bioresource Technology 101 (2010) 7556–7562 7557

have been reported to be able to accumulate up to 40% of lipids indry matter (André et al., 2010). In this study, we investigated thefeasibility of direct bioconversion of wheat straw into microbial li-pid by using a newly isolated cellulolytic fungus which has beenidentified as A. oryzae, a common member of the genus Aspergillus.

2. Methods

2.1. Microorganism and inoculum

A. oryzae A-4, previously isolated from the African jungle soilcollected in June, 2008 from Republic of Cameroon, was identifiedby Microbiology Lab, Zhejiang University and kept on Potato Dex-trose Agar (PDA) slants at 4 �C. The homogenized mycelia of A. ory-zae A-4 was used as the inoculum for SSF, which was prepared byhomogenizing the culture incubated in 100 ml of PDA at 30 �C,180 rpm for 48 h to well-distributed suspension.

2.2. Evaluation of lipid accumulation and cellulose degradation of A.oryzae A-4

Sudan dyes staining was performed for the observation of intra-cellular lipid globules in the mycelia of A. oryzae A-4 cultivated innitrogen-limited solid medium. The nitrogen-limited medium con-sisted of mineral salts solution (MS solution), 50 g/l glucose and1.0 g/l yeast extract. The MS solution contained (NH4)2SO4 1.7 g,KH2PO4 2.0 g, MgSO4�7H2O 0.5 g, CaCl�2H2O 0.2 g, FeSO4�7H2O0.01 g, ZnSO4�7H2O 0.01 g, MnSO4�4H2O 0.001 g, CuSO4�5H2O0.0005 g, 0.1% Tween-80 (w/v) and 1000 ml deionized water, withpH adjusted to 5.5. The cellulose degradation was determined byCongo red staining and filter paper decomposition.

2.3. Lipid accumulation in SmF of pure cellulose

Lipid accumulation of A. oryzae A-4 was carried out in 250 mlconical flasks at 30 �C with shaking at 180 rpm. 50 ml basal med-ium (Singh, 1991) containing 2% of microcrystalline cellulose wasinoculated with the homogenized mycelia. After fermentation,the mycelia were gathered for lipid and biomass assay, while resid-ual cellulose contents were determined by the methods of Updeg-raff (1969).

2.4. Substrate and SSF

Wheat straw was obtained from Anhui province in China, andadditive substrates such as wheat bran, orange peel, apple peel, su-gar cane bagasse were all collected from local market. Solid sub-strates were dried at 80 �C for 4 h before milled to 20–40 mesh.The fermentation media consisted of 3.6 g wheat straw, 0.4 gwheat bran, and 4 ml MS solution in Petri dishes (U = 9 cm). Aftersterilization, the media was cooled down and inoculated with0.5 ml of homogenized mycelia per dry substrate and static culti-vated at 30 �C, 50–80% humidity. Experiments were carried outwith three replications.

2.5. PBD for improvement of lipid production

PBD is a useful tool to screen a range of factors and is typicallyused as a preliminary optimization technique (Zhao et al., 2008).Ten numerical variables and one categorical variable were investi-gated in the study, including inoculum size, incubation tempera-ture, ratio of bran to straw, ratio of substrates to MS solution,fermentation volume, initial pH, concentration of salts in the MSsolution, and pretreatment strategy. Alkali pretreatment combinedwith microwave pretreatment (APCMP) was used for straw pre-

treatment (Zhao et al., 2010). The impact of each variable on lipidproduction was estimated based on comparison of the difference inthe mean between the high level (+1) and the low level (�1). Thelipid yield was expressed as the mean value of the three replica-tions. The significance of each variable in the PBD experimentwas determined by applying the Student’s t-test using MINITABstatistical software (Minitab 14). The statistics was performed bySPSS 16.0 (Chicago, IL).

2.6. Analytical methods

2.6.1. Lipid determinationCell lipid in SmF was extracted with methanol–chloroform mix-

ture (2:1, v/v) by the method of Bligh and Dyer (1959) and deter-mined gravimetrically. The total lipid in SSF was extracted by thesame method mentioned above with slight modification. The so-lid-state fermented residue was dried at 80 �C to constant weight.One gram of fermented product was subsequently washed andcentrifuged to remove soluble impurity on the surface. The samplewas soaked with 15 ml HCl (2 M) for 2 h, and then transferred intoboiling-water. After incubation for 10 min, the sample was cooledinstantly for cell disruption. In the later step, the sample was vor-texed with 3.75 ml methanol–chloroform mixture (1:1, v/v) for15 min, and then sucking filtered to obtain liquid phase containinglipid. The step was repeated twice for sufficient extraction, and li-quid phase from the same sample was collected together. Forremoving water-soluble impurity in the collection, 4 ml of deion-ized water was added to the collection and vortexed well. Finally,the collection was centrifuged for 5 min at room temperature togive a two-phase system, and bottom phase was carefully suckedout through the pipette. The solvent was removed by evaporationand the total lipid was measured gravimetrically. Fatty acids weremethylated by using trimethylsulphonium hydroxide (Peng andChen, 2008) and then analyzed by using GC–MS (Focus-GC-DSQII) equipped with DB-5MS column (30 � 0.25 mm, 0.25 m).

2.6.2. Enzyme extraction and determinationTo prepare the enzyme extract in SSF, 1 g of fermented residue

was mixed with 20 ml of citric acid buffer (pH 4.8). Then, the mix-ture was shaken at 37 �C for 2 h. The supernatant was obtained forfurther analysis. The activity of cellulase was measured by deter-mination of filter paper cellulase activity (FPA) according to US Na-tional Renewable Energy Laboratory (NREL) (Adney and Baker,1996). The reactions were all incubated at 50 �C for 50 min, andthe content of released reducing sugar was assayed using 3,5-dinitrosalicylic acid (DNS) method. One unit (U) of enzyme activitywas defined as the amount of enzyme required to release 1 lmol ofreducing sugar per minute under assay conditions. The enzymaticactivity was expressed as units per gram of dry substrate (FPU/gds).

2.6.3. Loss in dry matter (LDM)Loss in dry matter (LDM) was obtained using the following

equation:

%LDM ¼ initial dry weight of substrate� dry weight of spent substrateinitial dry weight

� 100%

2.6.4. Holocellulose utilizationCellulose and hemicellulose in the cellulosic wastes were deter-

mined according to USDA-ARS Agricultural Handbook (Goeringand Van Soest, 1971). The equation calculating holocellulose utili-zation was as follows:

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%Holocellulose utilization

¼ initial holocellulose� residual holocelluloseinitial holocellulose

� 100%holocellulose ¼ celluloseþ hemicellulose

2.6.5. Determination of biomassThe fungal biomass in SSF was determined by estimation of N-

acetyl glucosamine according to Roopesh et al. (2006), and the bio-mass was depicted as mg glucosamine per gram dry substrate (mg/gds).

3. Results and discussion

3.1. Evaluation of lipid accumulation and cellulose degradation of A.oryzae A-4

The mycelia of A. oryzae A-4 cultivated in the nitrogen-limitedsolid medium with glucose as carbon source presented a signifi-cant blue–black by Sudan black dye staining. Large, bright and or-ange lipid globules were observed by Sudan III dye staining underthe microscope. A lipid content of 15.4–22.1% with a maximumbiomass of 9.68 g/l was obtained when A. oryzae A-4 was cultivatedin liquid nitrogen-limited medium with glucose of 50 g/l as carbonsource after 6 days. Therefore, A. oryzae A-4 was an oleaginousmicroorganism. Congo red staining results showed that A. oryzaeA-4 formed a clearing zone of 32 mm diameter on carboxymethylcellulose plates within 2 days. Filter paper decomposition resultsshowed that Whatman No.1 Filter Paper (1 � 6 cm) was com-pletely disintegrated by A. oryzae A-4 in less than 2 weeks. Furtherstudy on lipid production from cellulose by A. oryzae A-4 (Table 1)showed that both the lipid content and the biomass increased withthe decrease of cellulose within 6 days, and a cellulose utilizationof 41.60%, lipid yield of 39.08 mg/g substrates, lipid content of18.15%, and biomass of 4.31 g/l were detected after 6 days. How-

Table 1Lipid production by A. oryzae A-4 cultivated on liquid medium with 20 g/l cellulose ascarbon source. Data are means of three replications.

Fermentationtime (d)

Biomass(g/l)

Celluloseutilization (%)

Lipidcontent (%)

Lipid yield(mg/g subs)

2 1.74 8.50 7.80 6.964 3.12 22.42 15.40 23.996 4.31 41.60 18.15 39.088 6.70 45.20 8.00 26.80

Fig. 1. The time course of lipid production (lipid yield,4), cellulase secretion (FPA, j), loswheat straw and bran mixture in SSF. Bars indicate standard errors (n = 3).

ever, lipid content decreased dramatically after the 6th day, whilethe biomass and cellulose utilization kept increasing, which mightbe attributed to the utilization of the storage lipids for biomassproduction. The lipid accumulation pattern of A. oryzae A-4 wasslightly different from that reported by Singh (1991), who showedthat the lipid content and the biomass of A. niger AS101 increasedcontinuously as cellulose decreased when cultivated on cellulosemedium. The results indicated that A. oryzae A-4 was capable ofconverting widely available cellulosic wastes into microbial lipid.

3.2. Lipid accumulation in SSF of the wheat straw and bran mixture byA. oryzae A-4

The lipid extracted from the wheat straw and bran mixture usedin the experiment was 21.6 mg/gds. Fig. 1 demonstrated lipid accu-mulation and cell growth under the SSF parameters as ratio of branto straw 1:9, ratio of substrates to MS solution 1:1 and tempera-ture 30 �C without addition of cellulase. At the early fermentationstage (within 5 days), FPA, LDM and the biomass increased withtime while the extracted lipid decreased slightly. That the ex-tracted lipid decreased could be attributed to the utilization ofthe original lipid in the substrate for anabolism, since the availablesubstrates hydrolyzed from cellulosic wastes were insufficient foranabolism due to the low cellulase secretion of A. oryzae A-4. Theextracted lipid increased abruptly to the maximum 36.6 mg/gdsfrom the 5th day to the 6th day, and decreased gradually andslightly after the 6th day. The FPA was also found to be maximum(1.82 FPU/gds) on the 6th day, and then a slight decline was ob-served. Assay of holocellulose in the substrate showed that25.25% of holocellulose was utilized on the 6th day. The LDMand the biomass tended to keep increased during the fermentation.The results suggested that A. oryzae A-4 could secrete cellulase tohydrolyze holocellulose in the cellulosic wastes into available car-bon and energy source. The direct utilization of holocellulose by A.oryzae A-4 could contribute to lowering the cost of lipid productionsince other oleaginous fungi previously reported utilized sugar-rich materials as the nutrient source rather than holocellulose(Conti et al., 2001; Stredansky et al., 2000).

3.3. Fatty acid compositions of total lipids from the solid-statefermented products

The GC–MS analysis results (Table 2) showed that the major fattyacid compositions of total lipids from the solid-state fermented

s in dry matter (LDM, s) and biomass estimation (d) by strain A. oryzae A-4 from the

Table 2Fatty acid compositions of total lipids produced by A. oryzae A-4 in SSF of wheat strawand bran mixture. Culture conditions: ratio of bran to straw 1:9, initial substrates toMS solution 1:1, incubation temperature 30 �C.

Fermentation time (day) Fatty acids (%, w/w)

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

0 28.15 13.52 1.96 6.816 32.95 9.96 22.64 27.74

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products were palmitic acid (C16:0, 32.95%), stearic acid (C18:0,9.96%), oleic acid (C18:1, 22.64%) and linoleic acid (C18:2, 27.74%).The fermentation resulted in a substantial increase in the contentof oleic and linoleic acids, which were 1.96% and 6.81%, respectively,in the solid substrates. The fatty acids profile obtained in this studyis similar to that of lard or palm olein, and is in line with earlier find-ings showing that Aspergillus strains produce lipids rich in oleic andlinoleic acids (André et al., 2010; Strayer et al., 2006; Wynn and Ratl-edge, 1997).

Table 4Factors included in the PBD and the importance ranking in lipid production.

Variable Level Coefficient t-Test P > |t|

�1 0 1

A 2 3 4 4.811 8.320 0.014a

B 25 28 30 �2.544 �4.400 0.048a

C 1:9 1.5:8.5 2:8 2.400 4.150 0.053D 4 5 6 1.944 3.360 0.078E 6 8 10 �1.456 �2.520 0.128F 0 0.0005 0.001 �0.156 �0.270 0.813G 0.01 0.02 0.03 �1.378 �2.380 0.140H 1 1.5 2 4.344 7.510 0.017a

I 0.1 0.3 0.5 �1.467 �2.540 0.127J 4:4 4:5 4:7 2.044 3.540 0.072K APCMP No

APCMPNoAPCMP

5.922 10.240 0.009a

A: inoculum size (ml/gds), B: temperature (�C), C: ratio of bran to straw, D: fer-mentation volume (g), E: pH, F: alcohol (g/l), G: ZnSO4 (g/l), H: (NH4)2SO4 (g/l), I:MgSO4 (g/l), J: ratio of substrates to MS solution and K: pretreatment strategies.

a Model terms are significant.

3.4. Optimization of lipid production in SSF

3.4.1. Lipid yield optimized by PBDThe coefficient R2 indicated that up to 95.6% of the data variabil-

ity could be explained by the model in the PBD. From the listeddata in Table 3, treatment 12 coded as A � B + C + D �E + F � G � H � I + J + K yielded the maximum lipid (62.87 mg/gds)on the 6th day under SSF conditions: inoculum size 1 ml/gds, ratioof bran to straw 2:8, fermentation volume 6 g, ratio of substrates toMS solution 4:7, temperature 25 �C, no pretreatment with wheatstraw, and concentration of salts in the MS solution was alcohol0 g/l; Zn2+ 0.01 g/l; NH4

2+ 1 g/l; Mg2+ 0.1 g/l. Lipid yield of treat-ment 12 increased by 71.87% as compared to the pre-optimizedtreatment (36.6 mg/gds as shown in Fig. 1), and was 49.52% higherthan that obtained from the endophytic fungi Microsphaeropsis sp.grown in the same condition (Peng and Chen, 2008). The FPAdetected in treatment 12 was more than four times of that in theMicrosphaeropsis sp. while the biomass of A. oryzae A-4(307.17 mg/gds) was 24 times of that of Microsphaeropsis sp. Thevigorous strain growth and the high FPA in Treatment 12 indicatedthat A. oryzae A-4 could be highly adaptable to the straw sub-strates, and thus yielded high lipid. The results also implied thatthe cellulase secretion of the strains have a considerable impact

Table 3The PBDa and the experimental results after SSF for 6 days.

Trials Coded value

A B C D E F G H

1 1 1 1 �1 1 1 �12 1 1 �1 1 1 �1 1 �3 �1 �1 �1 �1 �1 �1 �1 �4 1 1 �1 1 �1 �1 �15 �1 �1 �1 1 1 1 �16 0 0 0 0 0 0 07 �1 �1 1 1 1 �1 18 �1 1 �1 �1 �1 1 19 0 0 0 0 0 0 0

10 1 �1 �1 �1 1 1 1 �11 0 0 0 0 0 0 012 1 �1 1 1 �1 1 �1 �13 1 �1 1 �1 �1 �1 114 �1 1 1 �1 1 �1 �1 �15 �1 1 1 1 �1 1 1 �

A: inoculum size (ml/gds), B: temperature (�C), C: ratio of bran to straw, D: fermentationratio of substrates to MS solution and K: pretreatment strategies.

a |R| = 99.37%, R2 = 95.60%.

on lipid yield when cellulosic wastes are used as fermentationsubstrates.

3.4.2. Effects of SSF parameters on lipid yieldThe P-values in PBD, a tool for checking the significance of each

factor, showed that among the factors tested, inoculum size,(NH4)2SO4 concentration and pretreatment strategies had a signif-icant (P < 0.05) positive effect on lipid yield, while temperature hada significant (P < 0.05) negative effect (Table 4). It was observedthat lipid accumulation was inhibited by APCMP pretreatmentsince some reports showed that pretreatment would remove aconsiderable amount of free sugar, protein and oil, or caused endproduct inhibition (Zhao et al., 2010). However, search for a ra-tional design of pretreatment is necessary to improve enzymaticdigestibility since the holocellulose utilization was low in thisstudy. We also observed that low level of alcohol, Zn2+, Mg2+ wasbeneficial for lipid accumulation although their effects appearedto be insignificant (P > 0.05). The negative impact of the high levelof Mg2+ and Zn2+ on SCO production by Lipomyces starkeyi have alsobeen shown with the aid of PBD in the report of Zhao et al. (2008).These results are consistent with the findings made by Papaniko-laou et al. (2004), who showed that double (or multiple)-limitedmedia could repress storage lipid degradation.

Lipid yield (mg/gds) FPA (FPU/gds)

I J K

1 �1 �1 �1 43.73 ± 6.79 0.681 �1 �1 1 43.53 ± 6.22 1.551 �1 �1 �1 28.93 ± 3.20 0.601 1 1 �1 47.20 ± 8.20 0.481 1 �1 1 47.20 ± 0.98 1.890 0 0 0 45.27 ± 6.82 1.791 �1 1 �1 44.73 ± 3.74 1.171 �1 1 1 45.40 ± 4.10 1.840 0 0 0 49.07 ± 3.67 2.211 1 1 �1 33.73 ± 6.27 0.690 0 0 0 46.07 ± 2.55 2.151 �1 1 1 62.87 ± 3.25 1.691 1 �1 1 58.20 ± 3.46 2.001 1 1 1 38.73 ± 6.39 1.481 1 �1 �1 26.53 ± 1.47 0.78

volume (g), E: pH, F: alcohol (g/l), G: ZnSO4 (g/l), H: (NH4)2SO4 (g/l), I: MgSO4 (g/l), J:

Table 5Comparison of pretreatment strategies for lipid production (lipid yield) and cellulaseactivity (FPA) by A. oryzae A-4 after SSF of wheat straw and bran mixture for 6 days.

Pretreatment strategies Lipid yield (mg/gds) FPA (FPU/gds)

Un-pretreatment 50.87 ± 1.13 1.44 ± 0.018Dilute acid 59.40 ± 3.96 2.31 ± 0.034Alkaline peroxide 44.67 ± 4.03 1.31 ± 0.040Aqueous ammonia 43.73 ± 2.16 0.74 ± 0.040

7560 L. Hui et al. / Bioresource Technology 101 (2010) 7556–7562

3.4.3. Comparison of pretreatment strategiesDifferent pretreatment strategies were evaluated for lipid pro-

duction by cultivating A. oryzae A-4 for 6 days under the followingSSF conditions: inoculum size 0.5 ml/gds, ratio of bran to straw 2:8,fermentation volume 6 g, ratio of substrates to MS solution 4:7,temperature 25 �C and concentration of salts in the MS solutionwas alcohol 0 g/l; Zn2+ 0.01 g/l; NH4

2+ 1 g/l; Mg2+ 0.1 g/l. Three dif-ferent pretreatment strategies were used: dilute acid pretreatment(0.7% H2SO4, 121 �C, 1 h, ratio of straw to liquid 10%), alkaline per-oxide pretreatment (2.5% H2O2, pH 11.5, 37 �C, 24 h, ratio of strawto liquid 10%) and aqueous ammonia treatment (15% ammoniasolution, 90 �C, 24 h, ratio of straw to liquid 10%). The maximum li-pid yield (59.40 ± 3.96 mg/gds) and the maximum FPA(2.31 ± 0.034 FPU/gds) from straw and bran mixture in SSF was ob-tained by dilute acid pretreatment (Table 5), which yielded signif-icantly (P < 0.05) higher lipid than that the other pretreatmentstrategies.

3.4.4. Effects of different additive substrates on lipid yieldThe results presented in Fig. 2 showed that the lipid yield on the

6th day with addition of any of sugar cane bagasse (35.65 mg/gds),orange peel (33.84 mg/gds), banana peel (33.4 mg/gds) and wheatbran (37.3 mg/gds) to wheat straw were higher than that obtainedin wheat straw (28.5 mg/gds) without additive substrates. Branwas revealed as an excellent additive substrate for lipid productionsince it contains sufficient nutrients and remains free even underhigh moisture conditions. It could also be noted that maximumFPA (2.40 FPU/gds) and maximum LDM (17.25%) were achievedwhen orange peel was supplemented into the wheat straw, show-ing that addition of orange peel could not only improve lipid out-put but also yield high cellulase. Thus, an alternative approach forproducing lipid could be SSF of cellulosic wastes with carbon-rich

Fig. 2. Effects of different additive substrates on lipid production (mg/gds),cellulase secretion (FPA, FPU/gds) and loss in dry matter (LDM, %) by A. oryzae A-4 with the following SSF parameters: ratio of additive substrate to straw 1:9, initialsubstrates to MS solution 1:1, incubation temperature 30 �C and fermentation time6 days. Data are means of three replications. CK: no additive substrate, A: sugarcane bagasse, B: apple peel, C: orange peel, D: banana peel and E: wheat bran.

edible by-products, such as the orange peel produced in largequantities (Gema et al., 2002).

3.5. The relationship between cellulase secretion and lipid yield in SSF

To illuminate the relationship between cellulase secretion andlipid yield, we predicted the process of lipid accumulation by A.oryzae A-4 cultivated on the solid cellulosic medium as shown inFig. 3. At the early stage, the weight of the dry matter in the med-ium slowly decreased as the increase of cellulase activity (Fig. 1),since A. oryzae A-4 secreted enzymes (1) into the solid mediumto hydrolyze (2) holocellulose into available monomers in the SSFsystem. The released monomers were taken up by A. oryzae A-4for growth and lipid storage (3, 4), resulting in an increase of bio-mass and lipid yield (Fig. 1). It should be stressed that carbon up-take rate (carbon availability) is of crucial importance for theaccumulation of microbial lipid (Fakas et al., 2008). If the availablesubstrate level is too low, the strain can hardly accumulate lipidand will use the stored lipid (5) as a substrate (Holdsworth andRatledge, 1988). As the substrate is composed of insoluble poly-mers, microorganisms rely on extracellular enzymes to breakdown the insoluble polymers into available monomers for uptake.Thus, the decrease of cellulase would cause the limitation of avail-able substrate in the medium, finally leading to the storage lipidreadily converted to a new biomass. As shown in Fig. 1, the lipidyield decreased followed by a decline of cellulase activity, whilethe biomass kept increasing. The prediction in Fig. 3 illustratedthe important role of cellulase secretion during the process of lipidaccumulation with the cellulosic wastes as fermentation sub-strates. Similar findings leading to this conclusion have been re-vealed previously in Microsphaeropsis sp., Mortierella isabellinaand Cunninghamella echinulata growing on the complex mediacomposed of insoluble polymers like straw, pectin and starch(Papanikolaou et al., 2007; Peng and Chen, 2008).

Our results in Fig. 1 also showed that the lipid yield decreasedfollowed by a decline of FPA after the 6th day, and the lipid yieldwas significantly (r = 0.76, n = 7, P < 0.05) correlated with the FPA,which suggested that the decrease of cellulase led to the degrada-tion of storage lipid. This is confirmed by our further study, whichshowed that the lipid yield kept nearly stable after the 6th daywhen cellulase (10 FPU/gds) was added to the fermentation med-ium on the 6th day to suppress the lipid breakdown by breakingthe limitation of carbon source from the decrease of cellulase,while the lipid yield in the treatment without the addition of cel-lulase kept decreasing (Fig. 4). The results indicated that lipid deg-radation could be inhibited by the addition of cellulase. Inhibitionof the lipid degradation is of great importance in the industrial

Fig. 3. The schematic illustration of the process of lipid accumulation by strain A.oryzae A-4 using cellulosic wastes as substrates in the SSF.

Fig. 4. The lipid production (lipid yield, 4) and cellulase secretion (FPA, j) afteradditional cellulase was added on the 6th day. The SSF parameters were the same asthat in Fig. 1. Dash lines represent the time course without the additional cellulase.The arrow indicates the time for addition of cellulase.

L. Hui et al. / Bioresource Technology 101 (2010) 7556–7562 7561

processes for lipid production, since the lipid degradation occursregardless of the carbon source previously used for lipid produc-tion and begins very rapidly when the carbon uptake could notsaturate the metabolic requirement (André et al., 2010; Fakaset al., 2008). Previous works have shown that the employment ofrational strategies such as genetic engineering or fermentationtechnology could effectively suppress the lipid breakdown(Papanikolaou et al., 2004; Mlicková et al., 2004).

The optimal condition for lipid accumulation usually did notcorrespond to that of cellulase secretion (Fig. 2 and Table 1). InFig. 2, maximum lipid yield was observed in the treatment withthe addition of bran, while maximum FPA was obtained at thetreatment with the addition of orange peel. In Table 1, treatment12 presented the maximum lipid yield while treatment 9 pre-sented the maximum FPA. It might be owing to that lipid accumu-lation in microorganisms usually conducted in a condition with anexcess of carbon source and a limiting amount of nitrogen source(Ratledge, 2004), while synthesis of cellulase need sufficient nitro-gen source. Thus, to achieve significant levels of lipid production, itis essential to adjust the ratio of carbon and nitrogen in the med-ium for the balance of lipid accumulation and enzyme synthesis.Additionally, the illumination of genomics of A. oryzae could pro-vide potential to improve lipid accumulation and substrate utiliza-tion by further modification (Aachary and Prapulla, 2008;Courchesne et al., 2009), although the lipid yield in this studywas much lower than that in the nitrogen-limited culture mediumdue to insufficient free carbon source.

4. Conclusion

We isolated a cellulolytic fungus A. oryzae A-4, which was ableto directly convert the cellulosic wastes into lipid mainly com-posed of oleic and linoleic acids. The feasibility of efficient andlow-cost microbial lipid production by A. oryzae A-4 from wheatstraw was subsequently established under SSF. A. oryzae A-4 couldyield lipid as high as 62.87 mg/gds in SSF on the 6th day under theoptimized conditions. Our study also demonstrated that cellulasesecretion of A. oryzae A-4 has a crucial impact on lipid productionfrom cellulosic wastes.

Acknowledgements

The work was supported by the International CooperationProject in Science and Technology of Zhejiang Province, China

(No. 2008C14038), the National Hi-Tech Research and Develop-ment Program (863) of China (No. 2007AA06Z329), the Scienceand Technology Project of Zhejiang Province, China (No.2007C23036, 2008C13014-3).

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