Bioresource Technology 101 (2010) 6262–6264

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Bioresource Technology

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Short Communication

Two-stage utilization of corn straw by Rhizopus oryzae for fumaric acid production

Qing Xu, Shuang Li, Yongqian Fu, Chao Tai, He Huang *

State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, No. 5 Xinmofan Road,Nanjing 210009, People’s Republic of China

a r t i c l e i n f o

Article history:Received 5 December 2009Received in revised form 20 February 2010Accepted 22 February 2010Available online 16 March 2010

Keywords:BiomassFumaric acidCorn strawRhizopus oryzaeXylose

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.02.086

* Corresponding author. Tel./fax: +86 25 83172094E-mail address: biotech@njut.edu.cn (H. Huang).

a b s t r a c t

Due to the abundant, low price characteristic, lots of efforts have been put into producing bulk chemicalfrom lignocellulose biomass, but the low utility of xylose, which is the second main component in ligno-cellulose, becomes a bottleneck for efficient lignocellulose utilization. This study investigated a noveltwo-stage corn straw utilization strategy for fumaric acid production by Rhizopus oryzae. Fungal growthwas approached in hydrolysates from acid hydrolysis of corn straw, contained 30 g/l xylose; and fumaricacid production was occurred in hydrolysates from enzymatic hydrolysis of the residue corn straw afteracid hydrolysis, contained 80 g/l glucose. Under the optimal condition using this two-stage corn strawutilization strategy, the fumaric acid production, was up to 27.79 g/l, with the yield of 0.35 g/g, produc-tivity of 0.33 g/l/h.

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1. Introduction

With the depletion of global conventional oil, the world is indesperate need of new resources to address vital economic andenvironment problems. Due to the volume and environmentalattributes, lignocellulosic biomass is considered to be the mostpromising option. Large amounts of waste lignocellulosic biomassare generated from forestry or agriculture, and the biomass couldbe hydrolyzed to oligosaccharide, further converted to various va-lue-added products by microorganisms (Howard et al., 2003).

Fumaric acid is a four-carbon unsaturated dicarboxylic acid thatis widely used in food, chemical and pharmaceutical industry. Thezygomycetes fungus Rhizopus oryzae is a well-known producer offumaric acid (Carol et al., 2008). Many studies have beenconducted on the potential of utilizing lignocellulosic biomass.Woiciechowski investigated fumaric acid production from woodchips hydrolysate, and only 5.085 g/l fumaric acid could beobtained from 56.55 g/l sugar (Woiciechowski et al., 2001). Liaoindicated that the fumarate concentration from dairy manurehydrolysates by acid pretreatment was only 4.9 g/l with a yieldof 0.15 g/g (Liao et al., 2008). A more effective way was requiredfor lignocellulosic biomass utilization.

Depending on the pretreatment means, xylose and glucose con-tained in lignocellulosic biomass could be mostly separated (Charleset al., 2005). In the present study, a two-stage corn straw utilizationstrategy for fumaric acid production was developed. Corn straw wasfirst pretreated by dilute acid to obtain a xylose-rich liquid that could

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be used as a carbon source to grow fungal biomass, further the resi-due of corn straw was digested by enzyme to obtain a glucose-richliquid that could be used for fumaric acid production.

2. Methods

2.1. Material and preparation

Corn straw were collected and milled to a particle size no largerthan 1 mm. Acid hydrolysis of corn straw was carried out in a med-ium containing 2% H2SO4 with a liquid/solid ratio of 10:1 (w/w) at100 �C for 2 h (Bai et al., 2008, with little modified). The remainingsolid was removed from aqueous solution by filtration and forenzymatic hydrolysis. Enzymatic treatment was performed basedon our previous study (Yan et al., 2009): the reaction conditionswere 50 �C, pH 4.8 (0.05 M sodium citrate buffer) for 72 h withenzyme loading of 60 FPU/g of glucan was used. The hydrolysatesfrom acid treatment consisted of 23.6 g/l xylose and 2.3 g/l glucose,the enzymatic hydrolysate consisted of 31.2 g/l glucose and 1.8 g/lxylose. All of the hydrolysates were concentrated by vacuum evap-oration at 40 �C till the concentration of xylose reached 50 g/l foracid hydrolysate, the concentration of glucose reached 120 g/l forenzymatic hydrolysate. The concentrated hydrolysates were storedat 4 �C and diluted to desirable concentration before use.

2.2. Microorganism and medium

R. oryzae ME-F12, as the mutant of R. oryzae ATCC 20344, weregrown on potato-dextrose agar slants at 35 �C for 6 days, the fungalspores in the slants were suspended in sterilized water maintained

Fig. 1. Fungal growth with pure xylose or glucose. Dash lines are for sugarconsumption, solid lines are for biomass accumulation, triangle is for xylose, squareis for glucose.

Q. Xu et al. / Bioresource Technology 101 (2010) 6262–6264 6263

at 4 �C. Pre-culture medium (g/l): xylose or glucose 50, urea 2.0,KH2PO4 0.6, MgSO4�7H2O 0.5, ZnSO4�7H2O 0.11, FeSO4�7H2O0.0088, pH 3.0. Acid production medium (g/l): glucose 80, urea0.2, CaCO3 50, with other ingredients similar as the pre-culturemedium. To evaluate the effects of corn straw hydrolysates on fun-gal growth and fumaric acid production, different concentratedhydrolysates from acid pretreatment (contained 20–50 g/l xylose)were employed as carbon source in pre-culture medium andhydrolysates from enzymatic hydrolysis (contained 60–120 g/lglucose) were employed as carbon source in fumaric acid produc-tion medium. The initial pH for pre-culture medium with hydroly-sate was 4.0.

2.3. Culture method

Pre-culture and acid production was carried out in a 250 mlflask with 50 ml medium at 200 rpm, 35 �C. For pre-culture, 1 mlspore suspension was inoculated into the flask. For acid productionexperiments, 10% (v/v) of pre-cultured R. oryzae were employed.

2.4. Analytical methods

Fumaric acid was quantified by HPLC as Zhou et al. (2000) re-ported: the final culture broth was diluted by the addition of water,and HCl was added to neutralize the excess CaCO3. The broth washeated at 80 �C until clear, the supernatant was collected for anal-ysis. A Dionex HPLC was employed equipped with a refractive in-dex detector, the mobile phase was 5 mM H2SO4 at a flow rate of0.8 ml min�1 through a Bio-Rad Aminex HPX-87H column at 60 �C.

Sugars were determined and quantified by HPLC as reported:xylose and glucose were analyzed by HPLC (Dionex) equipped witha Bio-Rad Aminex HPX-87P column and a refractive index detector.The mobile phase was water at a flow rate 0.6 ml/min and columntemperature was 85 �C (Yan et al., 2009).

Biomass was measured by washing the mycelia twice with dis-tilled water, drying at 70 �C until constant weight was achieved.

2.5. Calculations

The yield of fumaric acid (g/g) is expressed as amount of prod-uct synthesized (g) divided by the amount of sugar consumed (g).The productivity of fumaric acid (g/l/h) is expressed as grams offumaric acid produced per liter per hour. Biomass yield (g/g) is cal-culated as amount of biomass (g dry weight) divided by theamount of sugar consumed (g).

Fig. 2. The effect of different xylose concentration in acid hydrolysates on R. oryzaegrowth. (1) A, 20 g/l xylose; B, 30 g/l xylose; C, 40 g/l xylose; D, 50 g/l xylose. (2)Dash lines are for xylose consumption, solid lines are for fungal biomass.

3. Results and discussion

3.1. Effect of xylose on fungal growth

Xylose (50 g/l) or glucose (50 g/l) was used as carbon source forthe pre-culture of fungal, respectively. Different carbon sourceshad a significantly influence on R. oryzae cultivation (Fig. 1). Com-pared to glucose, the consumption of xylose by R. oryzae was muchslower, which was consisted with earlier studies that most of nat-ural microorganisms utilize hexose faster than other forms of sug-ars (Mass et al., 2006). After 48 h pre-culture, the biomass forxylose medium was 8.84 g/l and the residual xylose was 18.3 g/l;the biomass for glucose medium was 7.59 g/l and the residual glu-cose was 3.0 g/l. More biomass and higher biomass yield (0.28 g/g)could be reached with xylose as carbon source. It is probably due tothe specific metabolic pathways, in most eukaryotic organisms, xy-lose is first converted to xylulose, further transformed to xylulose-5-phosphate and then converted to glyceraldehyde-3-phosphatevia the pentose phosphate pathway (Mass et al., 2008), following

biomacromolecules synthesis, such as RNA, DNA, protein, resultin an increase of biomass.

There were several reports about the utilization of xylose byRhizopus sp. for acid production, showed a common problem ofxylose conversion. Mass et al. (2006) described only 40 g/l ofxylose could be converted to lactic acid by R. oryzae no matterwhat concentration of initial xylose was exposed. Mass et al.(2006) used immobilized R. arrhizus with polyurethane foam toferment xylose, only 16.4 g/l fumaric acid could be obtained witha productivity of 0.087 g/l/h. Thus, xylose is a suitable carbon re-source for fungal growth but not for acid production, it is moreprofitable for the two-stage utilization of sugars which containedin lignocellulose.

3.2. Effect of corn straw acid hydrolysates on fungal growth

Fungal growth using different concentrated hydrolysates fromacid hydrolysis of corn straw were demonstrated in Fig. 2. The fun-gal biomass profiles were similar during initial culture time withdifferent concentrated of xylose, and biomass increased from4.84 to 8.86 g/l following an increase of xylose concentration from20 to 50 g/l in hydrolysates. Acid hydrolysis of lignocellulose, espe-cially concentrated acid hydrolysis, usually contained inhibitors

Table 1The effect of different pre-cultured seeds on fumaric acid production with pureglucose (80 g/l).a

Pre-culturemedium withxylose(g/l)

Fermentation with pure glucose

Fumaric acidconcentration(g/l)

Fumaricacid yield(g/g)

Fermentationtime (h)

Fumaricacidproductivity (g/l/h)

20 20.80 ± 0.29 0.26 84 0.2530 30.74 ± 0.31 0.38 84 0.3740 30.64 ± 0.24 0.38 96 0.3250 30.67 ± 0.17 0.38 108 0.28

a Data with sign ‘‘ ± ” are the average of triplicates with standard deviation(n = 3).

Fig. 3. Effect of different glucose concentration in enzymatic hydrolysis on fumaricacid production. (1) A, 60 g/l glucose; B, 80 g/l glucose; C, 100 g/l glucose; D, 120 g/lglucose. (2) Dash lines are for glucose consumption, solid lines are for fumaric acidproduction.

6264 Q. Xu et al. / Bioresource Technology 101 (2010) 6262–6264

such as formic acid, phenol and furfural. These substrates couldhave some inhibition effects on microorganism, but the datashowed that R. oryzae could well resist the inhibitors inhydrolysates.

Different pre-cultured R. oryzae with acid hydrolysates was cul-tivated in acid production media with 80 g/l pure glucose (Table 1).The highest fumaric acid production, yield, productivity reached upto 30.74 g/l, 0.38 g/g, 0.37 g/l/h, respectively by fungal pre-culturedwith acid hydrolysates contained 30 g/l xylose. Higher concen-trated hydrolysates could not significantly improve fumaric acidproduction and yield, but dramatically decrease the productivity.Thus hydrolysates contained 30 g/l xylose was utilized for the fun-gal pre-culture in the following experiments.

3.3. Effect of corn straw enzymatic hydrolysates on fumaric acidproduction

To study the effect of glucose concentration of hydrolysates onfumaric acid production, the optimal pre-cultured fungal in acidhydrolysates were cultivated in fermentation medium with enzy-matic hydrolysates contained 60, 80, 100 and 120 g/l glucose(Fig. 3). The fumaric acid production increased from 15.30 to28.92 g/l corresponding to concentrated hydrolysates with60–100 g/l glucose after 60–96 h fermentation. Higher glucoseconcentration (120 g/l) resulted in a significant decrease in fumaricacid production. The highest fumaric acid yield and productivitywere 0.35 g/g and 0.33 g/l/h with a production of 27.79 g/l at a res-idence time of 84 h with hydrolysates contained 80 g/l glucose,which were close to the results attained in the earlier study with80 g/l pure glucose (Table 1), indicated that the hydrolysates withan enzymatic treatment had no inhibitory effect on fumaric acidaccumulation. Glucose concentration in medium of hydrolysatesincreased during the initial culture time. It is probably because asmall amount of cellobiose may be leaved in the hydrolysate afterpretreatment, and degraded into glucose by R. oryzae duringfermentation, leading to the increase of glucose concentration.

4. Conclusions

As xylose and glucose were the main components of corn straw,a novel strategy was developed for the efficient utilization of sug-ars for fumaric acid production from corn straw. Fungal growthwas approached in xylose-rich hydrolysates from acid hydrolysisof corn straw; and fumaric acid production was occurred in glu-cose-rich hydrolysates from enzymatic hydrolysis of the residuecorn straw after acid hydrolysis. Under the optimal condition,

fumaric acid production was achieved at 27.79 g/l, with the fuma-ric acid yield, productivity of 0.35 g/g, 0.33 g/l/h, which were thebest result of fumaric acid production from lignocellulosic biomass.

Acknowledgements

This work was financially supported by the National Basic Re-search Program of China (No. 2007CB707805), the Key Programof the National Natural Science Foundation of China (No.20936002), the National Natural Science Foundation of China(No. 20706031), and the United Foundation of NSFC and Guang-dong Province (No. U0733001).

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