Open Access

Improved yield of α-L-arabinofuranosidase by newly isolated Aspergillus niger ADH-11 and synergistic effect of crude enzyme on saccharification of maize stover

  • Harshvadan Patel1,
  • Digantkumar Chapla2,
  • Jyoti Divecha3 and
  • Amita Shah1Email author
Bioresources and Bioprocessing20152:11

DOI: 10.1186/s40643-015-0039-7

Received: 5 December 2014

Accepted: 6 February 2015

Published: 10 March 2015

Abstract

Background

In the view of depleting resources and ever-increasing price of crude oil, there is an urge for the development of alternative sources to solve the issue of fuel in the coming years. Lignocellulosic biomass is considered to be the most potential alternative resources for fossil fuel. Bioconversion of cellulosic and hemicellulosic components into fermentable sugars is the key step in producing fuel ethanol from lignocellulose. The enzymatic hydrolysis of lignocellulosic biomass needs a highly balanced composition of cellulases and hemicellulases. Commercial enzymes are usually poor in accessory hemicellulolytic enzymes like α-L-arabinofuranosidase. The addition of such accessory enzymes in combination with cellulase or hemicellulase plays a vital role in improving the total yield of fuel ethanol by enhancing the saccharification yield.

Results

The newly isolated fungal strain Aspergillus niger ADH-11 produced a maximum of 22.14 U/g of α-L-arabinofuranosidase under solid-state fermentation using wheat bran as the substrate and modified Mandels-Weber medium at 30°C after 180 h of incubation. The optimization of various fermentation parameters was performed by response surface methodology employing Plackett-Burman design followed by Box-Behnken design. The yield of α-L-arabinofuranosidase was enhanced by 2.34-fold after executing statistical optimization of various fermentative parameters. Crude α-L-arabinofuranosidase was found to be highly stable for 3 h at its optimum temperature (55°C) and pH (4.0). The assessment of the crude enzyme extract in saccharification of alkali-treated maize stover revealed that the supplementation of crude α-L-arabinofuranosidase to commercial cellulase and crude xylanase mixture increased the saccharification yield up to 730 mg/g of maize stover.

Conclusions

The newly isolated A. niger ADH-11 was found to be a potential producer of α-L-arabinofuranosidase. The crude enzyme was active at low pH and high temperature which makes it suitable for various industrial applications such as enzymatic saccharification of lignocellulosic biomass. The supplementation of α-L-arabinofuranosidase enzyme to commercial cellulases and hemicellulases improves the bioconversion of lignocellulosic biomass to a greater extent.

Keywords

Aspergillus niger ADH-11 α-L-Arabinofuranosidase Solid-state fermentation Response surface methodology Maize stover

Background

Hemicelluloses are the second most abundant renewable source on the earth after celluloses representing up to 30 to 35% of the total dry weight of the carbohydrate polymers, out of which xylan is the major constituent [1,2]. Xylan-rich agroindustrial wastes are among the most important biomass sources in the world, representing an annual generation of 40 million tons causing considerable damage to economic activities in the agroindustrial sector and environment as well [3]. As xylan is the complex structure, its complete degradation requires the action of accessory hemicellulolytic enzymes out of which α-L-arabinofuranosidase is one of the most important enzymes [2,4].

α-L-Arabinofuranosidase is an exo-type enzyme that generally catalyzes the cleavage of the terminal α-L-arabinofuranosyl residues of arabinoxylan, arabinan, and arabinogalactan present in the hemicellulose structure. The arabinose side chains on hemicelluloses participate in cross-linking within the plant cell wall structure. L-Arabinose substitutions on xylan strongly inhibit the action of xylan-degrading enzymes, thus preventing the complete degradation of the polymer to its basic xylose units [5]. It is now well established that the action of α-L-arabinofuranosidase alone or in the combination with other hemicellulolytic enzymes is inevitable for enzymatic bioconversion of lignocellulosic biomass in to sugars which can be further fermented to ethanol. This enzyme is also finds a wide range of applications in food, feed, paper pulp, and pharmaceutical industries.

The production of α-L-arabinofuranosidase is widespread among bacteria and fungi. However, filamentous fungi are more attractive than bacteria as potential producers of these enzymes as fungi secrete higher enzyme levels in the culture medium than bacteria [6]. α-L-Arabinofuranosidase production has been reported by many fungal strains, such as Trichoderma reseei (142 nkat/mg), Aspergillus awamori (22.0 U/mg), Aspergillus nidulans (30.0 mIU/ml), Aspergillus terreus (6.0 nkat/ml), Penicillium purpurogenum (1.0 U/ml), Aspergillus oryzae (0.02 U/mg), and Penicillium chrysogenum (0.34 U/mg) [7-13].

Solid-state fermentation (SSF) is a microbial process in which solid material is used as the substrate which may provide necessary nutrients and serves as an anchorage for the cell. SSF can be of special interest in those processes where the crude fermented product may be used directly as the enzyme source [14]. Fungi grow well on moist substrates in the absence of free-flowing water [15] and produce large amount of extracellular enzymes like cellulases, hemicellulases, and ligninases. The production of enzymes using fungi under SSF is many a times higher than submerged fermentation (SmF), since SSF processes reproduce the natural living conditions of such fungi [16]. The use of SSF for production of enzymes has many economic advantages over SmF like no need for complex and sophisticated machinery, easy product recovery, simple and inexpensive substrate for the fermentation, low energy demand, high volumetric productivity, and often a high yield of product [17]. Response surface methodology has been extensively used in optimizing various fermentative parameters for enzyme production using solid-state fermentation [18-21].

Considering the potential and future prospects of α-L-arabinofuranosidase, the present study was aimed at production of α-L-arabinofuranosidase under SSF by a newly isolated fungal strain of Aspergillus niger ADH-11 employing response surface methodology. The properties of crude α-L-arabinofuranosidase were also evaluated to predict its end application, and the crude enzyme was supplemented to cocktails containing cellulase and xylanase to check synergistic action during saccharification of maize stover.

Methods

Materials

All the reagents, media, and chemicals used under study were of analytical grade purchased from Qualigens Fine Chemicals Pvt Ltd, HiMedia Laboratories Pvt Ltd, Merck & Co., and Loba Chemie Pvt Ltd from Mumbai, Maharashtra, India. p-Nitrophenyl-α-L-arabinofuranoside was obtained from Sigma-Aldrich, St. Louis, MO, USA. Wheat straw, rice straw, corn cobs, and maize bran were provided by a local farmer, whereas wheat bran and rice bran were procured from local market. The raw materials were washed thoroughly with water, dried at 80°C, and stored at room temperature in air tight plastic bags until use. The commercial cellulase used in this study was provided by MAPs Enzyme Limited (Ahmedabad, Gujarat, India).

Microbial strain

The α-L-arabinofuranosidase-producing fungal strain was isolated from decaying custard apple and it was identified as A. niger ADH-11 by the Agharkar Research Institute (ARI) Pune, India on the basis of its molecular characteristics. The sequence of the strain was submitted to NCBI gene bank with accession no. KF026012. The culture was grown on potato dextrose agar (PDA) slant and store at 4°C. It was subcultured every 15 days.

α-L-Arabinofuranosidase production under SSF using different agro residues by A. niger ADH-11

The α-L-arabinofuranosidase production was carried out in 250-ml Erlenmeyer flasks containing 5 g of washed dried agro residues (wheat bran, wheat straw, rice bran, rice straw, corn cobs, and maize bran) moistened with 10 ml of modified Mandels-Weber medium containing the following (g/l): urea, 0.3; ammonium sulfate, 1.4; KH2PO4, 0.3; CaCl2, 0.3; MgSO4.7H2O, 0.3; proteose peptone. 1.0 and (mg/l) MnSO4.7H2O. 1.6; ZnSO4.7H2O, 1.4; CoCl2, 2; Tween 80, 0.1%; and initial pH 5.3. The medium and the substrate were sterilized separately at 121°C for 15 min at 15 lbs. The inoculum was prepared using 7 days old A. niger ADH-11 slants. The spore suspension was prepared by adding 5 ml of 1% Tween 80 to each slant. The spore count was carried out using Neuber's chamber. The medium and the substrate were mixed at the time of inoculation at 108 spore/ml and incubated at 30°C under static condition. The contents of the flasks were mixed intermittently (twice a day), and the crude enzyme was extracted from each flask at regular interval of time (at every 24 h).

Enzyme extraction

The contents from each flask were extracted using a minimum quantity (30 ml) of 50 mM sodium citrate buffer (pH 5.3) along with 0.2 ml of Tween 80. The flasks were kept at 30°C with 150 rpm for 30 min for thorough mixing of the contents, followed by filtration through a wet muslin cloth with thorough squeezing, and the filtrate was centrifuged at 8,000 rpm for 30 min. The clear supernatant thus obtained was used as a crude enzyme for further studies.

Enzyme assays

The α-L-arabinofuranosidase activity was determined according to the method reported by Yanai and Sato [22] with some modifications. The reaction mixture consisting of 1 mM p-nitrophenyl-α-L-arabinofuranoside in 50 mM sodium citrate buffer (pH 5.3) was incubated with enzyme at 50°C for 10 min in total volume of 0.5 ml. The reaction was terminated by adding 1 ml of 2 M sodium carbonate solution. The amount of p-nitrophenol released was determined by measuring absorbance at 410 nm. One unit of α-L-arabinofuranosidase activity is defined as amount of enzyme required to release 1 μmol of p-nitrophenol per minute under assay condition. The xylanase activity was measured using 1% birch wood xylan solution as reported earlier [23]. The substrate was prepared in 50 mM sodium citrate buffer of pH 5.3. The enzyme reaction was carried out at 50°C for 10 min. After incubation, the reaction was terminated by adding 1 ml of DNS reagent. The enzyme activity was determined by measuring the release of reducing sugar using xylose as standard [24]. One unit of xylanase activity is defined as the amount of enzyme releasing 1 μmol of xylose per minute under assay condition. The filter paper activity was measured according to IUPAC recommendation employing filter paper (Whatman no. 1) as a substrate [25]. The release of reducing sugars in 60 min at 55°C and pH 4.8 (50 mM sodium citrate buffer) was measured as glucose equivalent using DNS method. One unit of filter paper activity is defined as amount of enzyme releasing 1 μmol of glucose per minute under assay condition.

Protein estimation

The soluble protein was estimated by Folin's method using bovine serum albumin as a standard [26].

Optimization of α-L-arabinofuranosidase production using response surface methodology

The optimization of the physicochemical parameters for α-L-arabinofuranosidase production was performed in two stages. Initially, ten variables were considered for screening using Plakett-Burman design (PBD) to identify the variables, which significantly influenced α-L-arabinofuranosidase production, and in the second stage the significant variables screened from PBD were further optimized using a Box-Behnken design (BBD).

Screening of parameters affecting α-L-arabinofuranosidase production by PBD

In this study, urea, ammonium sulfate, proteose peptone, yeast extract, KH2PO4, CaCl2, MgSO4, pH, moisture ratio, and fermentation time were selected as the independent variables. Each variable was set at two levels, higher and lower (Table 1). The experimental design is given in Table 2. The significance of regression coefficients was tested by T test for α-L-arabinofuranosidase production (Table 3).
Table 1

Actual and coded level of variables tested with Plakett-Burman design

 

Code

−1

1

Urea

X1

0.1

5

MgSO4

X2

0.1

3

CaCl2

X3

0.1

3

pH

X4

3

8

Ammonium sulfate

X5

1

10

Fermentation time

X6

48

216

Protease peptone

X7

1

5

Yeast extract

X8

1

5

KH2PO4

X9

0.1

3

Moisture ratio

X10

1:1

1:3

Table 2

PBD matrix for the screening of variables influencing α-L-arabinofuranosidase production

Run

X1

X2

X3

X4

X5

X5

X7

X8

X9

X10

α-L-Arabinofuranosidase (U/g)

1

0.1

0.1

0.1

3

1

48

1

1

0.1

1

8.75

2

5

0.1

0.1

8

10

216

1

5

0.1

3

35.41

3

5

0.1

3

3

1

216

5

5

0.1

1

35.01

4

0.1

3

3

3

10

48

5

5

0.1

3

9.4

5

0.1

3

3

8

1

48

1

5

3

3

4.97

6

5

0.1

0.1

3

10

48

1

5

3

1

12.35

7

0.1

3

3

8

10

216

1

1

0.1

1

19.85

8

0.1

0.1

0.1

8

1

216

5

5

3

1

35.08

9

0.1

0.1

0.1

3

10

216

5

1

3

3

30.41

10

5

3

3

3

1

216

1

1

3

3

32.15

11

5

3

3

8

10

48

5

1

3

1

10.79

12

5

0.1

0.1

8

1

48

5

1

0.1

3

11.81

Table 3

Regression coefficient for α-L-arabinofuranosidase production

Term

Effect

Coefficient

Standard error Coefficient

T value

P value

Constant

 

26.02

1.579

16.48

0.039

Urea

15.88

7.94

1.579

5.03

0.025

Ammonium sulfate

−12.63

−6.32

1.579

−4.00

0.050

Protease peptone

4.21

2.10

1.579

1.33

0.410

Yeast extract

4.12

2.06

1.579

1.30

0.417

KH2PO4

−0.12

−0.06

1.579

−0.04

0.059

CaCl2

−28.81

−14.40

4.737

−3.04

0.002

MgSO4

36.24

18.12

5.470

3.31

0.047

pH

−12.73

−6.37

1.579

−4.03

0.050

Moisture Ratio

−0.65

−0.33

1.579

−0.21

0.870

Fermentation time

32.68

16.34

1.579

10.35

0.047

Optimization of significant parameters for α-L-arabinofuranosidase production by BBD

BBD involves full factorial search by observing simultaneous, systematic, and efficient variation of important components on the fermentation process. Urea, MgSO4, CaCl2, pH, ammonium sulfate, and fermentation time were selected as individual variable for the production of α-L-arabinofuranosidase. BBD in six factors having three center runs (with a total of 54 experimental runs) was used for the optimization of α-L-arabinofuranosidase production.

The design allowed to evaluate the main and interactive effects of urea (X1), MgSO4 (X2), CaCl2 (X3), pH (X4), ammonium sulfate (X5), and fermentation time (X6) for α-L-arabinofuranosidase yield (U/g). The α-L-arabinofuranosidase yield (U/g) corresponding to the combined effects of six variables was studied in their specific range as shown in Table 4. The temperature was kept constant at 30°C throughout the experiment. All flasks were analyzed for α-L-arabinofuranosidase yield at specific time intervals as planned in BBD. The plan of BBD in the coded levels of six independent variables is shown in Table 5.
Table 4

Actual and coded level of variables tested with Box-Behnken design for α-L-arabinofuranosidase production

Process variable

Coded level of variable

−1

0

+1

Urea (X1)

0.1

2.55

5

MgSO4 (X2)

0.1

1.55

3

CaCl2 (X3)

0.1

1.55

3

pH (X4)

3

5.5

8

Ammonium sulfate (X5)

1

5.5

10

Fermentation time (X6)

48

132

216

Table 5

Full factorial BBD matrix for α-L-arabinofuranosidase production by Aspergillus niger ADH-11

 

Urea

MgSO 4

CaCl 2

pH

Ammonium sulfate

Fermentation time

α-L-Arabinofuranosidase

Run

X1

X2

X3

X4

X5

X6

Predicted activity (U/g)

Experimental activity (U/g)

1

−1

−1

0

−1

0

0

24.803

23.750

2

1

−1

0

−1

0

0

23.506

21.940

3

−1

1

0

−1

0

0

21.359

24.070

4

1

1

0

−1

0

0

22.517

20.990

5

−1

−1

0

1

0

0

21.465

22.510

6

1

−1

0

1

0

0

22.089

19.860

7

−1

1

0

1

0

0

20.756

21.840

8

1

1

0

1

0

0

23.835

25.370

9

0

−1

−1

0

−1

0

18.686

20.850

10

0

1

−1

0

−1

0

18.814

20.140

11

0

−1

1

0

−1

0

19.025

19.720

12

0

1

1

0

−1

0

14.183

12.240

13

0

−1

−1

0

1

0

16.684

17.410

14

0

1

−1

0

1

0

19.828

20.350

15

0

−1

1

0

1

0

21.293

18.750

16

0

1

1

0

1

0

19.467

18.520

17

0

0

−1

−1

0

−1

20.493

17.850

18

0

0

1

−1

0

−1

17.673

19.390

19

0

0

−1

1

0

−1

12.448

13.680

20

0

0

1

1

0

−1

16.328

15.430

21

0

0

−1

−1

0

1

22.543

23.930

22

0

0

1

−1

0

1

18.641

16.920

23

0

0

−1

1

0

1

21.868

20.640

24

0

0

1

1

0

1

24.666

26.820

25

−1

0

0

−1

−1

0

16.175

16.160

26

1

0

0

−1

−1

0

17.414

17.340

27

−1

0

0

1

−1

0

14.703

12.370

28

1

0

0

1

−1

0

17.861

17.410

29

−1

0

0

−1

1

0

19.621

19.590

30

1

0

0

−1

1

0

18.245

21.060

31

−1

0

0

1

1

0

17.154

17.710

32

1

0

0

1

1

0

17.697

17.230

33

0

−1

0

0

−1

−1

15.256

15.060

34

0

1

0

0

−1

−1

15.484

14.150

35

0

−1

0

0

1

−1

15.089

14.820

36

0

1

0

0

1

−1

18.333

16.440

37

0

−1

0

0

−1

1

22.735

23.410

38

0

1

0

0

−1

1

17.793

19.280

39

0

−1

0

0

1

1

23.168

25.720

40

0

1

0

0

1

1

21.242

20.220

41

−1

0

−1

0

0

−1

16.569

15.160

42

1

0

−1

0

0

−1

16.174

18.470

43

−1

0

1

0

0

−1

13.391

15.960

44

1

0

1

0

0

−1

20.412

21.240

45

−1

0

−1

0

0

1

24.726

23.410

46

1

0

−1

0

0

1

19.487

16.430

47

−1

0

1

0

0

1

20.467

18.660

48

1

0

1

0

0

1

22.643

24.540

49

0

0

0

0

0

0

18.998

21.490

50

0

0

0

0

0

0

18.998

16.630

51

0

0

0

0

0

0

18.998

20.180

52

0

0

0

0

0

0

18.998

17.460

53

0

0

0

0

0

0

18.998

18.670

54

0

0

0

0

0

0

18.998

19.560

Wheat bran was used as the substrate at 30°C under SSF.

For statistical calculation the independent variables were coded as follows:
$$ {x}_i = \left({X}_i-{X}_{\mathrm{o}}\right)/\delta {X}_i $$
(1)

Where X i is the experimental value of the variable; X o is the midpoint of X i , \( \delta \) X i is the step change in X i , and x i is the coded value for X i , i = 1 − 6.

The α-L-arabinofuranosidase yield was fitted using response surface methodology applying Equation 2 and was analyzed using MINITAB 16.0 version:
$$ Y = {\beta}_{\mathrm{o}} + {\beta}_i{x}_i + \sum\ {\beta}_{\mathrm{ii}}{x_i}^2 + \sum\ {\beta}_{ij}{x}_i{x}_i $$
(2)
where Y is the predicted response variable, β o, β i , β ii , β ij are fixed regression coefficients of the model, x i , x j (i = 1, 2, 3, 4, 5 and 6, i ≠ j, i < j = 1, 2, 3, 4, 5, 6) represent independent variables in the form of original values.

Interpretation and data analysis

The results of the experimental design were analyzed and interpreted using Minitab 16 (Minitab Inc, State College, PA, USA) statistical software. The prediction of fermentation parameters and generation of response contour plot by the model were also done by the same software. Analysis of variance (ANOVA) was used to establish the significance of the model parameters.

Effect of temperature and pH on α-L-arabinofuranosidase activity

The optimum temperature for α-L-arabinofuranosidase was determined by assaying relative activity at different temperatures ranging from 40°C to 75°C. The optimum pH for α-L-arabinofuranosidase was determined by assaying relative activity at different pH (3 to 7) using 50 mM sodium citrate buffer for pH 3, 4, 5, and 50 mM sodium phosphate buffer for pH 6 and 7.

Temperature and pH stability of α-L-arabinofuranosidase

To determine the thermal stability of α-L-arabinofuranosidase, the enzyme solution was treated at different temperatures (45°C, 55°C, and 65°C) in 50 mM sodium citrate buffer (pH 4.0) in a temperature-controlled water bath and the residual activity was measured at different time intervals (60 min) up to 3 h. To determine the pH stability of α-L-arabinofuranosidase, the enzyme solution was appropriately diluted at different pH buffers (4, 5, 6 and 7) and left to room temperature and the residual activity was measured at different time intervals (60 min) up to 3 h.

Pretreatments of maize stover

The substrate (maize stover) was washed, dried, and sieved to get uniform particle size before its use. The maize stover was given two separate pretreatments. In one case, pretreatment was given by preparing 15% slurry of maize stover in 4% NaOH solution and was incubated at 30°C for 12 h. The second pretreatment was given by 15% ammonia solution in the ratio of (1:4.5 (w/v)) to prepare the slurry. The slurry was autoclaved at 121°C and 15 lbs for 1 h. The substrate was washed completely with distilled water until neutral and dried in oven at 80°C until moisture was evaporated.

Enzymatic hydrolysis of maize stover

The enzymatic saccharification of pretreated and untreated maize stover was carried out using crude α-L-arabinofuranosidase from A. niger ADH-11, crude xylanase from Aspergillus foetidus MTCC 4898 [18], and commercial cellulase individually. The crude enzyme from A. niger ADH-11, crude enzyme from A. foetidus MTCC 4898, and commercial cellulase also have other cellulolytic and xylanolytic enzymes. Crude α-L-arabinofuranosidase was used at 4.0 U/g, crude xylanase was used at 300 U/g, while commercial cellulase was used at 5.0 FPU/g during saccharification of maize stover. The enzymatic hydrolysis was performed in 150-ml screw cap Erlenmeyer flasks containing 2.5% pretreated and untreated maize stover and diluted enzyme, as mentioned earlier, in 50 mM sodium citrate buffer (pH 4.8) containing 0.1% Tween 80 in a final volume of 40 ml. Controls were kept for each reaction in which the active enzyme was replaced with heat-inactivated enzyme. The reaction system was fortified with 10 mg% sodium azide. The reaction was carried out at 50°C in water bath with mild shaking. The samples were withdrawn every 4 h and incubated in boiling water bath (100°C) to inactivate the enzyme; the reaction mixture was then filtered through wet muslin cloth by thorough squeezing and centrifuged to collect the clear supernatant. This supernatant was used for further analysis to estimate total reducing sugar by DNS method.

Enzymatic hydrolysis of pretreated maize stover using commercial cellulase supplemented with crude xylanase and crude α-L-arabinofuranosidase

The enzymatic hydrolysis of alkali (NaOH)-treated maize stover was performed using commercial cellulase (5.0 FPU/g), crude xylanase from A. foetidus MTCC 4898 (300 U/g), and crude α-L-arabinofuranosidase from A. niger ADH-11 (4.0 U/g) individually and cocktail of all. The reaction system was same as described in ‘Enzymatic hydrolysis of maize stover’ section. The hydrolysed products were analyzed by high-performance liquid chromatography (HPLC; Phenomenex, Rezex ROA-organic acid H+, column; Phenomenex Inc, Torrance, CA, USA). The degree of synergy or synergy is defined as ‘the ratio of the yield of product released by enzymes when used together to the sum of yield of these products when the enzymes are used separately in the same amounts as they were employed in the mixture’.

Results and discussion

α-L-Arabinofuranosidase production using different agro residues under SSF

The production of α-L-arabinofuranosidase was carried out using various agro residues like wheat bran, wheat straw, rice bran, rice straw, corn cobs, and maize bran as a sole carbon source and Mandels-Weber medium as a moistening agent at 30°C under SSF. It was observed that all six substrates supported production of α-L-arabinofuranosidase but wheat bran was found to be the most appropriate substrate under SSF, yielding a maximum yield of α-L-arabinofuranosidase up to 9.45 U/g after 192 h of incubation (Figure 1); hence, wheat bran was selected as the substrate for production of α-L-arabinofuranosidase. The biochemical composition of wheat bran indicates that it contains predominantly non-starch carbohydrate polymers (approximately 58%), starch (approximately 19%), and crude protein (approximately 18%). The non-starch carbohydrate polymers are being primarily approximately 70% arabinoxylans, approximately 24% cellulose, and approximately 6% β-(1,3) (1,4)-glucan [27]. The presence of higher amount of arabinoxylans in wheat bran may have induced α-L-arabinofuranosidase production. Relatively very few attempts have been reported on the production of these enzymes under SSF. Khandeparker et al. [28] reported wheat bran was the best substrate for α-L-arabinofuranosidase production by Arthrobacter sp. MTCC 5214 under SSF.
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Figure 1

α-L-Arabinofuranosidase production using agro-residues under solid-state fermentation at 30°C.

Optimization of α-L-arabinofuranosidase production under SSF using response surface methodology

Response surface methodology has been successfully applied for the optimization of fermentation medium components, conditions for enzymatic hydrolysis, and fermentation processes [29-32]. It predicts the maximum enzyme production among the selected range of variables and also studies the interaction among independent variables.

In the present study, the effect of urea, ammonium sulfate, proteose peptone, yeast extract, KH2PO4, CaCl2, MgSO4, pH, moisture ratio, and fermentation time was evaluated on the basis of α-L-arabinofuranosidase production using PBD (Table 1). Among these variables, urea, ammonium sulfate, KH2PO4, CaCl2, MgSO4, pH, and fermentation time were identified as the most significant and contributing variables (Table 3) for α-L-arabinofuranosidase production (Figure 2a). These parameters were further analyzed as variables using BBD for better production of α-L-arabinofuranosidase.
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Figure 2

Pareto chart and contour plot. (a) Pareto chart showing effect of media components on α-L-arabinofuranosidase production. (b) Contour plot showing interaction of MgSO4 and incubation time on α-L-arabinofuranosidase production at hold values of urea, CaCl2, ammonium sulfate and pH.

The result of 54 run BBD in six variables urea, ammonium sulfate, KH2PO4, CaCl2, MgSO4, pH, and fermentation time for the optimization of α-L-arabinofuranosidase production is shown in Table 5.

The experimental results suggest that the variables selected for the fermentation process had a strong effect on α-L-arabinofuranosidase production. On the basis of these experimental values, statistical testing was carried out using Minitab 16.0. The analysis of the model was tested by Fisher's F test and Student's T test. ANOVA of α-L-arabinofuranosidase production showed that the response surface model was significant (P = 0.002) (Table 6) due to the square portion of the regression model. A P value below 0.05 indicates that the test parameters are significant. In common, the larger the magnitude of T and smaller the value of P, more significant is the consequent coefficient term [33]. The fitted second-order response surface model as specified by Equation 2 for α-L-arabinofuranosidase yield in coded process variables is as follows:
Table 6

ANOVA for the response surface quadratic model for α-L-arabinofuranosidase production

Source

Degree of freedom

Sequential sums of squares

Adjusted sums of squares

Adjusted mean squares

F value

P value

Regression

27

464.93

464.93

17.220

3.12

0.002

Linear

6

193.24

193.24

32.207

5.84

0.001

Square

6

123.23

123.23

20.539

3.73

0.008

Interaction

15

148.46

148.46

9.897

1.80

0.092

Residual error

26

143.28

143.28

5.511

  

Lack-of-fit

21

127.27

127.27

6.061

1.89

0.249

Pure error

5

16.00

16.00

3.201

  

Total

53

608.21

    
$$ \begin{array}{l}Y = 18.9983 + 0.4454{x}_1\hbox{--}\ 0.4446{x}_2\hbox{--}\ 0.0054{x}_3\hbox{--}\ 0.5050{x}_4 + 0.8204{x}_5 + 2.5971{x}_6 + 0.5010{x_1}^2\hfill \\ {}\kern4em + 2.4422{x_2}^2\hbox{--}\ 0.2028{x_3}^2 + 5997{x_4}^2\hbox{--}\ 2.7403{x_5}^2\hbox{--}\ 0.0628{x_6}^2 + 0.6138{x}_1{x}_2 + 1.8538{x}_1{x}_3\hfill \\ {}\kern4em \hbox{--}\ 1.2113{x}_1{x}_6 + 0.7537{x}_2{x}_5\hbox{--}\ 1.2925{x}_2{x}_6 + 0.6838{x}_3{x}_4 + 1.0675{x}_3{x}_5\hbox{--}\ 0.2487{x}_4{x}_6 + 0.1500{x}_5{x}_6\hfill \\ {}\hfill \end{array} $$
(3)
where Y is α-L-arabinofuranosidase yield square root of the predicted response, and x 1, x 2, x 3, x 4, x 5, and x 6 are the coded values of urea, MgSO4, CaCl2, pH, ammonium sulfate, and fermentation time, respectively. A comparison of the experimentally obtained values of the enzyme with the predicted values indicated that these data are in reasonable agreement with predictive values (Table 5).
The corresponding P value showed that the independent variable MgSO4 (X2) and fermentation time (X6) had significant effect (0.003, 0.000) on α-L-arabinofuranosidase yield (Table 7). A significant interaction was also observed between urea (X1) and CaCl2 (X3) and between pH (X4) and ammonium sulfate (X5) (0.034, 0.035) contributing to the response at significant level for α-L-arabinofuranoside production (Table 7). The R 2 value provides a measure of variability in the observed response value that can be explained by the experimental factors and their interactions. The coefficient R 2 of α-L-arabinofuranoside was 76.4%.
Table 7

Regression coefficient for α-L-arabinofuranosidase production

Term

Coefficient

Standard error coefficient

T value

P value

Constant

18.9983

0.9583

19.824

0.000

Urea

0.4454

0.4792

0.930

0.361

MgSO4

−0.4246

0.4792

−0.886

0.384

CaCl2

−0.0054

0.4792

−0.011

0.991

pH

−0.5050

0.4792

−1.054

0.302

Ammonium sulfate

0.8204

0.4792

1.712

0.099

Fermentation time

2.5997

0.4792

5.420

0.000

Urea × Urea

0.5010

0.7319

0.684

0.500

MgSO4 × MgSO4

2.4422

0.7319

3.337

0.003

CaCl2 × CaCl2

−0.2028

0.7319

−0.277

0.784

pH × pH

0.5997

0.7319

0.819

0.420

Ammonium sulfate × Ammonium sulfate

−2.7403

0.7319

−3.744

0.001

Fermentation time × Fermentation time

−0.0628

0.7319

−0.086

0.932

Urea × MgSO4

0.6138

0.8300

0.739

0.466

Urea × CaCl2

1.8538

0.8300

2.234

0.034

Urea × pH

0.4800

0.5869

0.818

0.421

Urea × Ammonium sulfate

−0.6538

0.8300

−0.788

0.438

Urea × Fermentation time

−1.2113

0.8300

−1.459

0.156

MgSO4 × CaCl2

−1.2425

0.8300

−1.497

0.146

MgSO4 × pH

0.6838

0.8300

0.824

0.418

MgSO4 × Ammonium sulfate

0.7537

0.5869

1.284

0.210

MgSO4 × Fermentation time

−1.2925

0.8300

−1.557

0.131

CaCl2 × pH

1.6750

0.8300

2.018

0.054

Ammonium sulfate × Fermentation time

1.0675

0.8300

1.286

0.210

CaCl2 × Ammonium sulfate

−0.2706

0.5869

−0.461

0.649

CaCl2 × Fermentation time

−0.2487

0.8300

−0.300

0.767

pH × Ammonium sulfate

1.8425

0.8300

2.220

0.035

pH × Fermentation time

0.1500

0.8300

0.181

0.858

The fitted response for the above regression model was plotted in contour plot (Figure 2b) for the pairs of interactive variables while keeping other variables at their predicted optimum level. Among the variables, MgSO4 (X2), CaCl2 (X3), pH (X4), and fermentation time (X6) were the most significantly linear and had a positive effect on enzyme production. The significant interaction was observed between MgSO4 (X2) and fermentation time (X6) for α-L-arabinofuranosidase production. Increasing the fermentation time (X6) up to certain level may lead to an increase in α-L-arabinofuranosidase yield, while increasing the MgSO4 (X2) up to 2.55 g/l may lead to a drastic decrease in the α-L-arabinofuranosidase yield. MgSO4 is considered as a good stimulator of mycelial growth which decreases the dormancy of the spores and can affect the enzyme yields [34]. Guerfali et al. [35] performed PBD followed by CCD and found that MgSO4 played an important role in α-L-arabinofuranosidase production.

The application of RSM with BBD predicted that the maximum α-L-arabinofuranosidase production will be up to 20.30 U/g at decoded values of process parameters as MgSO4 1.59, urea 2.55, CaCl2 1.55, and ammonium sulfate 5 g/l with initial pH 5.5 after incubation 180 h of fermentation time.

Validation of the experimental model

A repeat fermentation for α-L-arabinofuranosidase production was carried out under optimal conditions to validate the parameters predicted by the model. The α-L-arabinofuranosidase production under optimized parameters viz. MgSO4 1.59, urea 2.55, CaCl2 1.55, and ammonium sulfate 5 g/l at pH 5.5 and fermentation time of 180 h yielded α-L-arabinofuranosidase activity of 22.14 U/g (3.16 U/ml). This was significantly higher than the predicted value (20.30) by the model. Crude enzyme extract also contained 140 U/g (20.0 U/ml) endo-xylanase, 70.0 U/g (10.0 U/ml) β-xylosidase, and 0.14 U/g (0.02 U/ml) filter paper activity. Thus, it was proved that the statistical optimization increased α-L-arabinofuranosidase production by 2.34-fold as compared to their initial production which was 9.45 U/g (1.35 U/ml) before statistical optimization. Khandeparker [28] reported a maximum 3 U/g of α-L-arabinofuranosidase production after 120 h of incubation by Arthrobacter sp. MTCC 5214 under SSF. Guerfali et al. [35] reported a maximum 0.6 U/ml of α-L-arabinofuranosidase production after 120 h of incubation by Talaromyces thermophilus using response surface methodology under submerged fermentation. Hence, newly isolated A. niger ADH-11 was found to be a strong producer of α-L-arabinofuranosidase; however, the fermentation time was higher as compared to other reports.

Effect of temperature and pH on α-L-arabinofuranosidase activity

The influence of temperature on α-L-arabinofuranosidase activity was evaluated in the range of 40°C to 75°C. The results revealed that the optimum temperature for α-L-arabinofuranosidase activity was 55°C (Figure 3a). α-L-Arabinofuranosidase activity was reduced by only 13% and 44% at 50°C and 60°C, respectively. The result was similar to many α-L-arabinofuranosidases from different strains of fungi [36]. Guerfali et al. [35] also reported that the optimum temperature of α-L-arabinofuranosidase from Talaromyces thermophilus was 55°C.
https://static-content.springer.com/image/art%3A10.1186%2Fs40643-015-0039-7/MediaObjects/40643_2015_39_Fig3_HTML.gif
Figure 3

Effect of temperature (a) and pH (b) on α-L-arabinofuranosidase activity.

The α-L-arabinofuranosidase activity at various pH (3 to 7) was measured using p-nitrophenyl-α-L-arabinofuranoside as a substrate at 55°C. The optimum pH for α-L-arabinofuranosidase activity was found to be at 4 (Figure 3b). The enzyme was remarkably active even at pH 3.5 and 4.5, with loss of only 11% and 17% activities. Kaneko et al. [37] also found the optimum pH as 4.0 for α-L-arabinofuranosidase from A. niger 5-16. Most of the fungal α-L-arabinofuranosidases exhibited acidic pH in the range of 4.0 to 5.0. The enzyme activity is markedly affected by variation in pH outside its optimum pH. This may be due to substrate binding and catalysis, which are often affected by charge distribution on both substrate and enzyme molecules [38].

Temperature and pH stability of α-L-arabinofuranosidase

The α-L-arabinofuranosidase from A. niger ADH-11 was found to be highly stable in the range of 45°C to 55°C (Figure 4a). At 55°C the enzyme retained 82.38% of its activity after 3 h. Yan et al. [39] reported that α-L-arabinofuranosidase from Chaetomium sp. was stable at 55°C for 30 min. Guais et al. [40] found that α-L-arabinofuranosidase activity was dropped by 50% at 60°C after 12 min. The α-L-arabinofuranosidase could retain its activity up to 87.37% at pH 4 after incubation of 3 h (Figure 4b). The activity of α-L-arabinofuranosidase was gradually reduced to 30.21% and 23.07% at pH 5 and 6 after 3 h, respectively, while only 6.59% activity was retained at pH 7 after 3 h. Filho et al. [41] reported that α-L-arabinofuranosidases I and II from Penicillium capsulatum retained 50% activity at pH 4.0 after 9 and 17.5 min, respectively. Yan et al. [39] reported that α-L-arabinofuranosidase from Chaetomium sp. retained 80% activity at pH 4.0 after 30 min. In comparison to the above reports, the crude α-L-arabinofuranosidase from A. niger ADH-11 was found to be more stable at high temperature and low pH, so it can be suitable for saccharification of lignocellulosic biomass along with commercial cellulases.
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Figure 4

Thermal stability (a) and pH stability (b) of α-L-arabinofuranosidase.

Enzymatic hydrolysis of maize stover

The major conversion steps in biochemical production of cellulosic biofuels are pretreatment and enzymatic hydrolysis. Together, these steps work to breakdown the cellulose and hemicelluloses found in the cell walls of plants (biomass) to simple sugars which can then can be fermented to ethanol. Maize stover is currently the largest waste biomass in the India, consisting of more than one-third of the total solid waste produced. Maize stover typically contains 70% cellulose and hemicellulose and 15% to 20% lignin. Ammonia and NaOH-pretreated maize stover were used to test the ability of the crude α-L-arabinofuranosidase, crude xylanase, and commercial cellulase for the production of fermentable sugar by saccharification. Maximum reducing sugars of 203.6 mg/g were produced using crude α-L-arabinofuranosidase, while crude xylanase and commercial cellulase yielded 190.7 and 400.0 mg/g respectively using NaOH-pretreated maize stover (Figure 5a,b,c). A higher yield of reducing sugars was achieved when the substrate was pretreated with NaOH as compared to ammonia.
https://static-content.springer.com/image/art%3A10.1186%2Fs40643-015-0039-7/MediaObjects/40643_2015_39_Fig5_HTML.gif
Figure 5

Enzymatic hydrolysis of untreated and alkali (NaOH)- and ammonia treated maize stover using different enzymes. (a) Enzymatic hydrolysis of untreated and alkali- and ammonia-treated maize stover using 4.06 U/g crude α-L-arabinofuranosidase at 50°C. (b) Enzymatic hydrolysis of untreated and alkali-, and ammonia-treated maize stover using 300 U/g crude xylanase at 50°C. (c) Enzymatic hydrolysis of untreated and alkali- and ammonia-treated maize stover using 5.0 FPU/g commercial cellulase at 50°C.

The maximum bioconversion of lignocellulosic biomass requires action of not only efficient cellulolytic enzymes but also main chain depolymerizing and debranching hemicellulolytic enzymes at appropriate levels. It is evident from Table 8 that the commercial enzyme used in this study was deficient in β-xylosidase, α-L-arabinofuranosidase, and feruloyl esterase and hence, the indigenously produced crude α-L-arabinofuranosidase was supplemented to form a balanced enzyme cocktail for saccharification of alkali-pretreated maize stover. There was a remarkable increase in β-xylosidase, α-L-arabinofuranosidase, feruloyl esterase, endo-xylanase, and β-glucosidase loading upon supplementation of crude α-L-arabinofuranosidase to commercial cellulase-crude xylanase cocktail (Table 9). As shown in Figure 6, the cocktail produced maximum 730.1 mg/g of reducing sugar after 24 h which was higher as compared to each enzyme used alone (crude α-L-arabinofuranosidase 203.6 mg/g, crude xylanase 134.4 mg/g, and commercial cellulase 294.1 mg/g, respectively) for saccharification. The co-action of crude α-L-arabinofuranosidase with commercial cellulase-crude xylanase produced a superior saccharification yield. The HPLC analysis of enzymatic hydrolysates revealed synergistic rise in glucose, xylose, and arabinose (degree of synergy 1.21, 1.00, and 1.33, respectively). The levels of glucose, xylose, and arabinose were 5.57, 4.99, and 0.64 mg/ml when the cocktail was used (Table 10). Thus, the supplementation of crude α-L-arabinofuranosidase (4.0 U/g) with commercial cellulase and crude xylanase increased the glucose yield up to 176.8% and xylose yield up to 166.8% (Table 10). Although supplementation of crude xylanase with commercial cellulase increased the loading of endoglucanase, β-glucosidase, and endoxylanase (Table 9), the yield of glucose, xylose, and arabinose was lesser compared to the cocktail of crude α-L-arabinofuranosidase (4.0 U/g) with commercial cellulase (5 FPU/g) and crude xylanase (300 U/g) cocktail. These results clearly suggest that the higher yield of reducing sugars obtained by this cocktail was due to higher amount of α-L-arabinofuranosidase and β-xylosidase enzymes which were contributed by the crude α-L-arabinofuranosidase produced by A. niger ADH-11. The presence of α-L-arabinofuranosidase plays an important role by removing arabinose from the side chains of xylan, and β-xylosidase plays a crucial role in reducing the end product inhibition of xylanases by hydrolyzing xylobiose and hence facilitates better bioconversion of maize stover. A similar observation was reported by Delabona et al. [42]. Their studies on sugarcane bagasse strongly suggested that the supplementation of α-L-arabinofuranosidase with commercial cellulase resulted in greater sugar release. The present study clearly demonstrated that the crude α-L-arabinofuranosidase-rich enzyme obtained from A. niger ADH-11 can be used to enhance the efficiency of commercial cellulase for saccharification of biomass.
Table 8

Composition of enzymes in each cocktail

Enzyme

β-Xylosidase (U/ml)

α-L-Arabinofuranosidase (U/ml)

Feruloyl esterase (U/ml)

Xylanase (U/ml)

Filter paper activity (U/ml)

Endoglucanase (U/ml)

β-Glucosidase (U/ml)

Crude α-L-arabinofuranosidasea

10.0

3.16

1.0

20.0

0.02

5.56

0.18

Crude xylanaseb

0.2

ND

ND

375.0

0.12

4.5

0.20

Commercial cellulasec

ND

ND

ND

300.0

43.9

2,798.0

43.0

aCrude α-L-arabinofuranosidase from A. niger ADH-11; bCrude xylanase from A. foetidus MTCC 4898; cCommercial cellulase MAPs 450. ND, not detected.

Table 9

Enzyme loading for saccharification of pretreated maize stover

Enzyme

β-Xylosidase (U/g)

α-L-Arabinofuranosidase (U/g)

Feruloyl esterase (U/g)

Xylanase (U/g)

Filter paper activity (U/g)

Endoglucanase (U/g)

β-Glucosidase (U/g)

Crude α-L-arabinofuranosidasea

12.7

4.0

1.27

25.4

0.025

7.06

0.22

Crude xylanaseb

0.16

ND

ND

300

0.09

3.6

0.16

Commercial cellulasec

ND

ND

ND

33.0

5.0

307.7

4.78

aCrude α-L-arabinofuranosidase from A. niger ADH-11; bCrude xylanase from A. foetidus MTCC 4898; cCommercial cellulase MAPs 450. ND, not detected.

https://static-content.springer.com/image/art%3A10.1186%2Fs40643-015-0039-7/MediaObjects/40643_2015_39_Fig6_HTML.gif
Figure 6

Enzymatic hydrolysis of alkali-treated maize stover using individual and cocktail of different enzymes. Enzymatic hydrolysis of alkali-treated maize stover using individual and cocktails of crude α-L-arabinofuranosidase, crude xylanase, and commercial cellulases at 50°C.

Table 10

Monomer sugar composition of each cocktail used for enzymatic saccharification of NaOH-treated maize stover

Enzyme cocktails

Glucose (mg/ml)

Xylose (mg/ml)

Arabinose (mg/ml)

Commercial cellulase (5 FPU/g)

2.98

1.69

ND

Crude α-L-arabinofuranosidase (4.0 U/g)

0.69

1.59

0.48

Crude xylanase (300 U/g)

0.93

1.70

ND

Commercial cellulase (5 FPU/g) + Crude xylanase (300 U/g)

3.15

2.99

ND

Commercial cellulase (5 FPU/g) + Crude α-L-arabinofuranosidase (4.0 U/g) + Crude xylanase (300 U/g)

5.57

4.99

0.64

ND, not detected.

Conclusions

The present investigation showed that newly isolated A. niger ADH-11 is a potential α-L-arabinofuranosidase producer. The statistical optimization for α-L-arabinofuranosidase production by SSF was highly advantageous as enzyme production was enhanced by 2.34-fold. To the best of our knowledge, this report describes the highest level of α-L-arabinofuranosidase production under SSF. The crude enzyme was highly active and stable at 55°C and pH 4.0, which makes it suitable for application in enzymatic saccharification of biomass. Moreover, it was evident from the present investigation that the supplementation of crude α-L-arabinofuranosidase can play significant role in the enzymatic hydrolysis of maize stover.

Declarations

Acknowledgements

The authors gratefully acknowledge Gujarat State Biotechnology Mission (GSBTM), Gandhinagar, Gujarat, India for providing the research grants to support this work. The authors are also thankful to MAPs Enzyme Limited, India for providing the cellulase enzyme.

Authors’ Affiliations

(1)
BRD School of Biosciences, Sardar Patel Maidan Satellite Campus, Sardar Patel University
(2)
Department of Microbiology, Shree Ramkrishna Institute of Computer Education and Applied Sciences - MTB College Campus
(3)
Department of Statistics, Sardar Patel University

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© Patel et al.; licensee Springer. 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.

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