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Production of reducing sugar from acid pretreated leaf litter using crude enzyme from Aspergillus niger strain AK10.


Leaf litters are considered a kind of plant waste materials that needed to be managed appropriately. Mostly, they are used for making silage or animal feed (Lattimore and Griffiths, 2006). However, there are quite a large numbers plant leaves that are not utilized and become leaf litters lying around. When wet, they can be good source for production of mold, spores and many airborne pollutants that lead to respiratory problems in human and animals. In many areas, leaf litters are removed by burning which, in fact, creates even worst health problems (Sannigrahi, 2009). Moreover, leaf burning could lead to soil nutrients depletion since they are taken up by plant and parts of them are stored in plant leaves.

The major component of plant cell wall is cellulose which is a polymer of glucose subunits. Hydrolysis of plant cell wall releases glucose molecules available for many microbial fermentation processes including ethanol production. The residues remained after hydrolysis can be further used as fertilizer or as animal feed composition which allow fully usage of all the nutrients present in plant cells (Sannigrahi, 2009).

In this study, a process of combined acid-enzymatic hydrolysis of leaf litter using sulfuric acid and crude enzyme extract from rice bran culture of Aspergillus niger AK10 (Cha et al., 2010) was developed. A potential application of the reducing sugar produce in cellulosic ethanol production has also been shown.


Fungal cultivation of crude enzyme production

Stock culture of Aspergillus niger AK10 (AK10) was maintained on Potato Dextrose Agar slant (PDA; Oxoid, USA). Solid state fermentation was done in a 100-ml bottle containing 10 gm of rice bran with 10 ml mineral solution containing 0.1% [K.sub.2]HP[O.sub.4], 0.02% MgS[O.sub.4], 0.01% Ca[Cl.sub.2] and 0.25% [(N[H.sub.4]).sub.2]S[O.sub.4]. After the media were autoclaved, they were inoculated with 1 ml of spore suspension in 0.05% Tween-80. Incubation was done at 30[degrees]C for 96 hours prior to crude enzyme extraction.

Enzyme extraction

The 96-hour culture of AK10 grown on rice bran solid medium was added with 40 ml of distilled water and left agitated at room temperature for 1 hr. The liquid part was isolated by pressing through cheese-cloth. This suspension was centrifuged to separate the remained particles from the supernatant. The clear supernatant was collected as the crude enzyme extract, simply called "AK10ase".

Cellulase Assay

Cellulase activity was assayed by measuring the amount of reducing sugars released from carboxymethyl cellulose (Sigma, USA) which interacts with 3,5-dinitrosalicylic acid or DNS (Miller, 1959). The reaction mixture containing 0.9 ml of 1% (w/v) carboxymethyl cellulose in 50mM phosphate buffer, pH 6.8 and 0.1 ml of enzyme extract. The enzyme reaction was carried out at 50[degrees]C for 30 min, then, 1 ml of DNS was added to stop the reactions. The solution was incubated in a boiling water bath for 5 min for color development and the absorbance was measured at 540 nm against the enzyme blank. One unit of cellulase was defined as the amount of enzyme required to release 1 [micro]mol reducing sugar or glucose equivalent per min under the above assay conditions.

Xylanase Assay

Xylanase activity was assayed using 1% oat spelt xylan solution as the substrate, as described by Bailey et al.(1992) and the amount of reducing sugars released was determined by the method of Miller (1959) using DNS reagent. One unit of enzyme activity was defined as 1 [micro]mol of xylose equivalent produced per minute under the assay conditions.

Digestion of leaf litter using crude AK10ase

The basic digestion process was done by mixing 10 gm of shredded plant leaves in a reaction mixture containing 40 ml of 0.05 M acetate buffer, pH 4.0, and 5 ml of crude AK10ase. The reaction was incubated at 37[degrees]C for 1 hour. The content of each component will be optimized in the study. The increase in the amount of metabolizable reducing sugar was determined by DNS assay (Miller, 1959).

Pretreatment of shredded leaves with sulfuric acid

The acid pretreatment was done by mixing 10 gm of shredded leaves with 20 ml of acid solutions at different concentrations and incubated at 95[degrees]C for 1 hour.

DNS Assay for Reducing Sugar concentration

This was done following the method of Miller (1959)

Alcohol fermentation

Stock culture of the alcohol producing yeast, Saccharomyces cerevisiae strain Yy (Yy) was maintained on PDA slant. Minimal medium for yeast cultivation (MMY) was composed of 0.7% of [K.sub.2]HP[O.sub.4], 0.2% of K[H.sub.2]P[O.sub.4], 0.01% of MgS[O.sub.4], 0.1% of [(N[H.sub.4]).sub.2]S[O.sub.4], and 0.03% of Ca[Cl.sub.2]. When needed, glucose was added as carbon source at 1% concentration. The fermentation was carried out at 28[degrees]C.

Alcohol content measurement

This was done using alcohol refractometer (gauging percentage of alcohol in water) (Trans Instruments (S) PTE LTD, Singapore)


Enzymatic digestion of shredded leaf litter using crude enzyme AK10ase

AK10ase was obtained from the 96-hr solid culture of AK10 grown on rice bran medium. The crude AK10ase contained 66.8 U/ml of cellulase activity and 324.5 U/ml of xylanase activity. Reactions for leaf litter digestion were set up at 3 different pH conditions; i.e. pH2, pH4 and pH6. The amount of reducing sugar released from the 3 reactions were measured at various periods and compared. It was found that the highest amount of reducing sugar was released from the reaction under pH4 for 2 days (Figure 1). Leaving the reaction mixture proceed longer than 2 days showed rapid decrease in the amount of reducing sugar release. This could be due to the denaturation of the enzyme protein due to a too long storage at 37[degrees]C. Alternatively, there could be some microbial contamination into the mixture since the reactions were carried out at permissive temperature. This could also explain the reason for low reducing sugar release under the condition of pH6 which allowed active growth of any microbes in the atmosphere.


However, the reducing sugar obtained at Day 1 was approximately 16% less than that obtained from the 2-day digestion period. Thus, one-day period was considered enough for enzymatic hydrolysis which produced approximately 6.7 mg/gm of shredded leaves.

The optimum concentrations of enzyme and buffer for effective leaf litter digestion

The suitable proportion of enzyme to substrate was needed to be verified in order to obtain an efficient degradation. The suitable amount of the 0.05M acetate buffer used in the reaction mixture was also determined. It was found that the leaf litter digestion gave the highest reducing sugar product when the reaction mixture was composed of 10 gm of shredded leaves, 40 ml of 0.05 M acetate buffer, pH4.0 and 5 ml of AK10ase (Table 1). This was proportionally equivalent to the standard reaction mixture for amylase assay based on the enzyme activity on hydrolyzing gelatinized starch (Bernfeld, 1955).

The suitable sulfuric acid concentration for pretreatment of the shredded leaf litter

In order to improve the enzymatic reaction on shredded leaves, pretreatment of the cellulosic waste materials was tested. Among many methods of pretreatment that had been reported (Sun and Cheng, 2002; Rajoka et al., 2005; Hendriks and Zeeman 2009), acid hydrolysis seemed to be more often used since it could efficiently solubilize the hemicelluloses leading to a better enzymatic hydrolysable substrate (Gregg and Saddler, 1996; Hendriks and Zeeman, 2009). Thus, the optimum concentration of sulfuric acid for pretreatment process was determined. In this experiment, sulfuric acid solutions were prepared in 20 ml volume for each treatment of 10 gm shredded leaves. The acid hydrolysis was carried out at 95[degrees]C for 1 hour. The result (Table 2) showed that the 3% acid concentration gave the highest sugar release from the treatment. However, the sugar obtained from the pretreatment with 1% acid was only 15.9% less. Thus, pretreatment with 1% acid solution was, instead, considered as the process of choice.

Degradation of shredded leaves using combined acid-enzyme hydrolysis

The combined process of leaf litter digestion was carried out as two separate sets of acid-pretreatment; one was done with 0.5% sulfuric acid and the other was with 1.0% concentration. After 1 hour, both sets were separately adjusted to pH4.0 prior to enzymatic digestion with AK10ase. The combined process that started with 0.5% acid pretreatment gave 3.5 mg/ml reducing sugar while that with 1.0% acid gave 4.5 mg/ml (Figure 2).


However, pretreatment with higher concentration of acid affected the reaction rate of the subsequent enzymatic digestion; i.e. for the batch with 1% acid pretreatment, the rate of reducing sugar production was 21.7 mg/ml/min while that with 0.5% acid pretreatment, the rate was 58.6 mg/ml/min.

Thus, the process of the fermentable sugar production was summarized as diagram shown in Figure 3. The reducing sugar product was conveniently called "Leaf Litter Sugar" or "LLS". This process included pretreatment of the shredded leaves in 0.5% sulfuric acid at 95[degrees]C for 1 hour. Then, the pH was adjusted to pH4.0. The enzymatic hydrolysis was carried by adding 35 ml of 0.05 M acetate buffer plus 5 ml of crude AK10ase and incubated at 37[degrees]C for 30 min. The resulted LLS solution contained 0.3% reducing sugar.


This process was considered to be very environmental friendly as compared to many other processes which the acid hydrolysis were carried out at higher than 100[degrees]C, using chemicals at higher concentration (Woiciechowski et al., 2002; Lu et al., 2007; Hendriks and Zeeman, 2009; Tippayawong and Chanhom, 2011). However, moderate temperature treatment could lead to low saccharification due to sugar decomposition (McMillan, 1994; Sun and Cheng, 2002). In the case of the acid concentration, 0.5% sulfuric acid was about the lowest concentration to be used for effective acid pretreatment of cellulosic materials (Lu et al., 2007; Wang et al., 2011). Although acid hydrolysis using 0.4% acid was reported, the treatment was held at a higher temperature of 130[degrees]C (Tippayawong and Chanhom, 2011). Moreover, the enzymes used in other reported processes were commercial purified or partial purified enzymes which would increase the processing costs (Woiciechowski et al., 2002; Lu et al., 2007; Hendriks and Zeeman, 2009; Tippayawong and Chanhom, 2011).

Potential application of LLS for alcohol fermentation.

In this study, LLS was tried using as nutrient source for cultivation of S.cerevisiae strain Yy (Yy). The LLS solution containing 0.3% reducing sugar was concentrated 10 times by evaporation. The concentrated LLS (conc.LLS) containing 3% reducing sugar was added, as a carbon source, to the minimal medium, MMY, to give a final concentration of 1% reducing sugar. Alcohol production from Yy was determined and compared to those obtained from the other two conditions; i.e. one was in MMY plus 1% glucose, the other was in non-concentrated LLS without any supplements. The result (Table 3) showed that Yy could use the reducing sugar present in conc.LLS to produce alcohol. Moreover, Yy could produce alcohol in MMY plus conc.LLS more efficient that in MMY plus glucose. Non-concentrated LLS, by itself, did not contain all essential nutrients for supporting the alcohol production from Yy. Nonetheless, the cell number in sole LLS solution did not decrease indicating that there were enough nutrients to maintain the survival of the cells and there was no antimicrobial substance in LLS. The cell number remained on the Day-11 of fermentation in MMY plus conc.LLS indicated that Yy could tolerate alcohol as high as 28% concentration.

Therefore, this indicated that the process of combined acid-AK10ase enzyme hydrolysis of leaf litter could be effectively used for producing fermentable sugar. The sugar produced showed high potential for application in alcohol production, at least from S.cerevisiae strain Yy used in this study.


This research was supported by the Mahidol University Research Grant Targeted on the Environmental Management, Mahidol University, Thailand.


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Saovanee Chancharoensin *, Thanyaporn Srimahaeak, Punyawee Dulyayangkul and Sudaporn Luechai.

Science Division, Mahidol University International College, Mahidol University, Nakhon Pathom 73170, Thailand

* Corresponding Authors: Saovanee Chancharoensin Science Division, Mahidol University International College, Mahidol University, Salaya, Nakhonpathom 73170, Thailand

Table 1 Leaf litter digestion in various reaction mixtures.

Amount of shredded Volume of Volume of Reducing sugar
leaves (gm) enzyme (ml) buffer (ml) (mg/gm

10 5 40 6.7
10 10 40 2.6
10 5 20 1.6
10 10 20 1.7

Table 2 Pretreatment of shredded leaves with sulfuric
acid at various concentrations

Concentrations of sulfuric acid (%) Reducing sugar released from
 the pretreatment (mg/ml)

0 0.8
0.5 2.5
1.0 3.7
1.5 4.0
3.0 4.4

Table 3. The cell number and alcohol production in the 11-day
culture of Saccharomyces cerevisiae (Yy) grown in various media.

Media Cell Number Alcohol production
 (CFU/ml) (%)

MMY + 1% glucose 7.6x[10.sup.6] 3
LLS 5.7x[10.sup.5] 8
MMY + 1% reducing sugar 2.9x[10.sup.7] 28
 from conc. LLS
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Author:Chancharoensin, Saovanee; Srimahaeak, Thanyaporn; Dulyayangkul, Punyawee; Luechai, Sudaporn
Publication:International Journal of Biotechnology & Biochemistry
Date:Jul 1, 2012
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