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Evaluation of Chemo-Micobial Methods for Cellulose Hydrolysis in Sugarcane Bagasse and Saw Dust

SJA_37_3_1009-1015

Research Article

Evaluation of Chemo-Micobial Methods for Cellulose Hydrolysis in Sugarcane Bagasse and Saw Dust

Samra Aftab1, Saleem Ullah1 and Farida Anjum2*

1Department of Agricultural Chemistry, Faculty of Nutrition Sciences, the University of Agriculture Peshawar, Khyber Pakhtunkhwa, Pakistan; 2Department of Soil and Plant Nutrition, Agriculture Research Institute, Tarnab, Peshawar, Khyber Pakhtunkhwa, Pakistan.

Abstract | Saw dust and sugarcane bagasse were treated with acids/bases, microbial cultures and their enzyme extracts to check their effect on cellulose hydrolysis. Microbes were isolated from soil by using serial dilution method and were selected on efficiency basis (screening of microbes on the basis of their activities as cellulose degrading agents). The cellulose and related constituent of saw dust and sugarcane bagasse showed that cellulose (49 %), hemicelluloses (23.5 %) and ash (1.8 %) were higher in sugarcane bagasse, while lignin (7.9 %) was higher in saw dust. The data regarding microbe isolation showed highest colonies of Aspergillus sp. (8 CFU × 104g-1) followed by Penicillium sp. and Trichoderma sp. (7 CFU × 104g-1 and 5 CFU × 104g-1), while the lowest colonies were noted for Candida ablican and Saccharomyces cerevisiae. The screening test of these microbes for cellulose degradation showed higher degradation for Trichoderma sp. (6 % and 8 %) followed by Penicillium sp. (4 % and 4.5 %) in both of the samples (saw dust and sugarcane bagasse) respectively. Similarly the combined effect of acids, bases and microbes showed higher result of NaOH, Trichoderma sp. (29.5 % and 41 %) in both samples (saw dust and sugarcane bagasse). The optimization of incubation time and temperature showed 72 hrs of incubation time and tempreture of 50 °C was best for cellulose degradation. From the data, it was concluded that Trichoderma sp. among microbes, HCl in acid and NaOH in bases were best for cellulose hydrolysis in samples while, incubation of 72 hrs at 50 °C showed higher cellulose degradation rate.


Received | August 04, 2018; Accepted | May 11, 2021; Published | July 15, 2021

*Correspondence | Farida Anjum, Department of Soil and Plant Nutrition, Agriculture Research Institute, Tarnab, Peshawar, Khyber

Pakhtunkhwa, Pakistan; Email: faragric@gmail.com

Citation | Aftab, S., S. Ullah and F. Anjum. 2021. Evaluation of chemo-micobial methods for cellulose hydrolysis in sugarcane bagasse and saw dust. Sarhad Journal of Agriculture, 37(3): 1009-1015.

DOI | https://dx.doi.org/10.17582/journal.sja/2021/37.3.1009.1015

Keywords | Cellulose, Sugarcane, Bagasse, Saw dust, Enzymes



Introduction

Plants cell wall has cellulose as its major component which serves to maintain their structure. Cellulose is also present in bacteria, fungi, algae, and even in animals (Mayer and Staples, 2002). Cellulose content was worked out by French chemist named Anselm Payne during 1838. He extracted the cellulose from green plants. Cellulose due to its digestibility in to fermentable sugars, capture the attention of energy scientist in the world. These sugars can easily be converted in to biofuels (Bansal et al., 2012). Cellulose is also important for its uses in pharmaceutical industry. The derivatives of cellulose are used as a film coating agent on tablets, which reduces its vulnerability to environmental attack and help in transportation (Florencio et al., 2007). The cellulose is also present in agricultural residues which can be used in biofuel production, the world scientists tend to use agricultural residues in different valuable biomass energy products such as oil, generating electricity and other biodegrading products at the same time (Wiselogel et al., 1996). The fermentation of cellulose is carried out in two steps that are pretreatment and hydrolysis. In nature many microorganisms for example bacteria, fungi and actinomycetes produces enzymes (hydrolytic and oxidative) that degrade the lignocellulosic material. These enzymes are important in agricultural and industrial use (Perez et al., 2002). The objective of this study is the digestion of cellulose which is complicated and still need to be standardized for economical use in various industries, especially in biofuel technology.

Materials and Methods

The present study is related to cellulose digestion of two samples viz. saw dust and sugar cane bagasse. First microbes were isolated from soil samples and were screened for their cellulose digestion efficiency in the form of whole cells and aqueous enzymatic extracts. Isolated species were tested for cellulolytic enzyme production on basal medium. The efficiency of microbial enzymes was checked in the form of reducing sugar in the samples which was determined by dinitrosalicylic acid (DNS) method using glucose as standard.

Secondly the samples of saw dust and sugar cane baggasse were treated with 1 normal solution of various acids (HCl, H2SO4, H3PO4) and bases (NaOH, Ca(OH) 2, NH4OH) and they then were subjected to enzymatic digestion with crude extract of microbes selected for their high cellulose digestion efficiency screening. microbial isolation was carried out by using PDA media. For preparation of PDA media 200gm of potatoes were washed and peeled properly and then boiled in 300 ml distilled water until it become soft. Then it was filtered through muslin cloth. After this 20 gm of agar and dextrose were added and volume was made to 1 liter by adding distilled water. Then media was sterilized in an autoclave at 121°C for 15 min. Saw dust was collected from saw machines and was brought in polythene bags to the laboratory. Dust was passed through 1mm sieve and used for further analysis. Sugarcane bagasse was collected from cane juice extractors, selling cane juice in local markets. The sample was squeezed to remove the sweet liquid and soften the fiber for further treatment. Then the pulp sample after squeezing was soaked overnight and washed thoroughly in distilled water. After washing, sample was dried in the lab oven first at 70°C and then at temperature of 100°C for complete dryness. After drying the pulp samples was milled with lab grinder and was passed through a sieve of about 1 mm pore size. The samples were stored in polythene bags. The fine powder was used for subsequent analysis.

Cellulose and hemicelluloses in samples were determined as acid detergent fiber (ADF). Neutral detergent fiber (NDF) were used as a source of lignin and ash. Method by Goering and Van Soest (1970) with some modification was used for the analysis of Acid detergent fiber (ligno-cellulose) and Neutral detergent fiber in the representative samples of sugar cane bagasse and saw dust.

Chemical treatment

The samples were subjected to chemical treatment of acid and bases in order to make accessible to microbial activity by the procedure of Detroy et al. (1981) and Lynd et al. (2002) with some modifications.

Acid treatment

Sawdust and sugarcane baggasse of 20 g was taken in conical flasks. Each sample was dissolved in 200 ml of 1N HCl, H2SO4, H3PO4 and were kept at 85 °C for 1 hour. After acid treatment samples was washed with distilled water and placed in an oven at 70 °C for complete dryness.

Base treatment

Sawdust and sugarcane baggasse of 20 g was taken. Dissolved each sample in 200 ml of 1N NaOH, Ca (OH)2, NH4OH and was kept at 85 °C for 1 hour. After base treatment samples was washed with distilled water and placed in an oven at 70 °C for complete dryness.

After acid and base treatment, all treated and non-treated samples were placed in an autoclave 121 °C for 15 min. These treated samples were further used for enzyme hydrolysis.

Statistical analysis

Data were subjected to the Completely Randomized Design (CRD) by Gomez and Gomez (1984) with LSD Test at 5% probability level (Steel and Torrie, 1997).

Results and Discussion

Cellulose is useful constituent in agricultural raw material specially those which go into waste. For this purpose, two samples of cellulosic raw materials, saw dust and sugar cane baggase were subjected to microbial degradation alone where microbial species isolated from soil were screened for their hydrolyzability on the samples mentioned above. The effect was observed in form of cellulose degradation and reducing sugar.

Results in Table 1 showed that saw dust contained 1.3 % ash, 41 % cellulose, 19 % hemicellulose and 24 % lignin. Sugar cane bagasse contained 1.8 % ash, 49 % cellulose, 23.5 % hemicellulose and 7.9 % lignin. The study of Chinedu et al. (2008) also supported the present data who found ash content in sawdust and sugarcane pulp that were 1.1 % and 1.8 % respectively. These results reflect the fact that saw dust is a good source of cellulose.

 

Table 1: Cellulose content in sample of sawdust and sugar cane bagasses.

Samples

Cellulose (%)

Hemicellulose (%)

Lignin (%)

Ash (%)

Sawdust

41.0

19.0

24.0

1.3.0

Sugarcane bagasses

49.0

23.5

7.9.0

1.8.0

 

The data presented in Table 2 showed that maximum colonies were observed for Aspergillus sp. (8.0 CFU × 103g) followed by Penicillium sp. (7.0 CFU × 104g), while the colonies of Candida ablican and Saccharomyce cerevisiae were same in amount and the lowest one. The study of Sharma et al. (2004) could be related to the present study who isolated fungal species from soil and examined maximum species that of Aspergillus group.

 

Table 2: Isolated microbes (CFU × 104g-1) from soil.

S/No

Species

CFU × 104g-1

1

Alternaria

2.0

2

Aspergillus

8.0

3

Penicillium

7.0

4

Saccharomyces cerevisia

1.0

5

Candida ablican

1.0

6

Fusarium

4.0

7

Absidia

1.5

8

Trichoderma

5.0

 

Screening of microbial species for cellulose hydrolysis in saw dust and sugar cane bagasse

Table 3 showed that maximum degradation in saw dust was observed in sample treated with Trichoderma sp. (up to 35 %), while degradation in saw dust samples treated with Saccharomyces cerevisiae, Absidia, Candida ablican and alternaria was negligible. Similarly, the highest degradation in sugarcane bagasse was observed in sample treated with Trichoderma sp. (up to 41 %) this might be due to the fact that Trichoderma sp. is enzyme common use for hydrolysis of lignocellulosic biomass, while sugarcane bagasse samples treated with Saccharomyces cerevisiae, Absidia, Candida ablican and alternaria showed negligible cellulose degradation. The present work is closely related with the findings of Lennox et al. (2010) who worked on degradation of cellulose content of saw dust and observed maximum degradation by Trichoderma sp., Penicillium sp., Aspergillus sp., while lowest degradation by Absidia.

 

Table 3: Cellulose content (%) in saw dust and sugarcane baggase incubated with fungal species.

Microbial isolates

% Saw dust*

% Sugarcane bagasse**

Alternaria

40.9

48.8

Aspergillus sp.

37.5

44.0

Penicillium sp.

37.0

44.5

Saccharomyces cerevisiae

40.5

48.6

Candida ablican

40.9

48.9

Fusarium sp.

40.0

47.5

Absidia sp.

40.9

48.9

Trichoderma sp.

35.0

41.0

* Cellulose content of saw dust (41 %); ** Cellulose content of sugarcane baggase (49 %).

 

Acid hydrolysis

For acid hydrolysis saw dust and sugar cane bagasse was treated with 1 N HCl, 1 N H2SO4 and 1 N H3PO4. These acid treated samples were further digested with microbial species. Data presented in Table 4 showed the highest amount of reducing sugars (32.90 %) for sugar cane bagasse treated with 1 N HCl followed by 1 N H2SO4 (26 %) by Trichoderma sp., while the lowest hydrolysis (1.1 % and 1.5 %) for sugar cane bagasse was observed in non treated sample and treated sample with H3PO4 by Fusarium sp. That might be due to saw dust contain high level of lignin content which could reduce the efficiency of enzymatic hydrolysis. Similarly, the highest amount of reducing sugars (29 %) for saw dust was found in sample treated with 1 N HCl by Trichoderma sp. while the lowest (0.98 % and 1.2 %) amount of reducing sugars for saw dust was observed in non treated sample followed by treated sample with H3PO4 by Fusarium sp. Laopaiboon et al. (2010) was in line with previous study who tested different acids for hydrolysis of sugarcane bagasse and found best results with HCl. generally, acid is useful in hydrolysis of saw dust and sugarcane biomass at an optimum condition of pH and temperature.

 

Table 4: Percent reducing sugar of saw dust and sugarcane baggase samples hydrolyzed with acids and microbial species.

Sample

Treatment

Species

Mean

Penicillium

Fusarium

Trichoderma

Aspergillus

Saw dust

Control

1.10op

0.98p

5.20k

03.50mn

2.70h

H2SO4

12.80g

3.50mn

29.00b

12.10g

7.58e

HCl

11.20h

3.90lm

1.90o

12.20g

14.08c

H3PO4

3.30mn

1.20op

10.10i

5.20k

4.95f

Sugarcane baggase

Control

2.90n

1.10op

7.30j

4.10lm

3.85g

H2SO4

14.50f

4.70kl

26.00c

24.20d

17.35b

HCl

17.90e

4.50kl

32.90a

23.90d

19.80a

H3PO4

7.10j

1.50op

14.30f

14.30f

9.30d

Sample x Species

Saw dust

7.10f

2.40h

11.55c

8.25e

7.32b

Sugarcane baggase

10.60d

2.95g

20.125a

16.625b

12.58a

Treatment x Species

Control

2.00j

1.04k

6.25g

3.80i

3.27d

H2SO4

13.65d

4.10i

13.95cd

18.15b

12.64b

HCl

14.55c

4.20i

30.95a

18.05b

16.99a

H3PO4

5.20h

1.35k

12.20e

9.75f

7.13c

Mean

8.85c

2.67d

15.84a

12.44b

Values within columns sharing a common letter are not significantly different at P<0.01. *Incubation time=72 hrs; *Incubation temperature=50°C

 

Table 5: Percent reducing sugar of saw dust and sugarcane baggase samples hydrolyzed with bases and microbial species.

Sample

Treatment

Species

Mean

Penicillium

Fusarium

Trichoderma

Aspergillus

Saw dust

Control

1.10rs

0.98s

5.20l

3.50no

2.70h

NaOH

15.00f

4.85lm

29.50b

18.90e

17.06 b

Ca(OH)2

9.00j

2.50pq

18.10e

10.10i

9.93d

NH40H

5.08l

1.30rs

9.20j

4.76lm

5.09f

Sugarcane baggase

Control

2.90op

1.10rs

7.30k

4.10lm

3.85g

NaOH

15.00f

5.10l

41.00a

27.90c

23.75a

Ca(OH)2

11.40h

3.76n

21.30d

15.20f

12.92c

NH40H

5.38l

1.90qr

13.50g

7.45k

7.06e

Sample x Species

Saw dust

7.55f

2.41h

15.50b

9.32e

8.69b

Sugarcane baggase

10.17d

2.97g

20.78a

13.67c

11.89a

Treatment x Species

Control

2.00l

1.04m

6.25h

3.80j

3.27d

NaOH

18.00d

4.98i

35.25a

23.40b

20.41a

Ca(OH)2

10.20g

3.13k

19.70c

12.65e

11.42b

NH40H

5.23i

1.60lm

11.35f

06.11h

6.07c

Mean

8.86c

2.69d

18.14a

11.49b

Values within columns sharing a common letter are not significantly different at P< 0.01. *Incubation time=72 hrs; *Incubation temperature=50°C

 

Alkali hydrolysis

Maximum amount of reducing sugars (41 %) for sugarcane bagasse was showed by sample treated with 1N NaOH by Trichoderma sp. (Table 5). while the lowest amount of reducing sugar (1.1 % and 1.9 %) for sugar cane bagasse was observed in non treated sample followed by treated sample with 1 N NH4OH by fusarium sp. Similarly, for saw dust reducing sugar (29.5 %) was higher in sample treated with 1 N NaOH by Trichoderma sp. while the lowest amount of reducing sugar (0.98 % and 1.3%) for saw dust was observed in non treated sample followed by treated sample with 1N NH4OH by fusarium sp. The work of Sharma et al. (2004) also supported the present study who treated the sample of sunflower hulls with 0.5 % (w/v) sodium hydroxide autoclaved at 121 °C and showed maximum saccharification of 59.8 % by hydrolysis enzyme of Trichoderma reesei.

The effect of different temperature (control, 30°C, 40°C, 50°C and 60°C) for an incubation period of 72 hrs is shown in Table 6. The data presented in table 6 showed the highest amount of reducing sugar (45%) for sugarcane bagasse was produced by crude enzyme extract of Trichoderma sp. at 50 °C while the lowest amount of reducing sugar (1.5 % and 3.5 %) for sugarcane bagasse was observed in control sample followed by incubated sample at 30°C by Fusarium sp. Similarly, the highest amount of reducing sugar (29.9%) for sawdust was observed in sample incubated at 50°C by Trichoderma sp. while the lowest amount of reducing sugar (0.99 % and 1.9 %) for saw dust was observed in control sample followed by incubated sample at 30 °C by Fusarium sp. The present work is in agreement with Rajesh et al. (2012) who determined the effect of temperature on enzymatic activity by using Trichoderma reesei in solid state fermentation process.

The data presented in Table 7 showed the highest amount of reducing sugar (46.2 %) for sugarcane bagasse was produced in sample incubated for 72 hrs at 50°C by crude enzyme extract of Trichoderma sp., while the lowest amount of reducing sugar (0.98 % and 1.30 %) for sugarcane bagasse was observed in control sample followed by sample, incubated

 

Table 6: Percent reducing sugar of saw dust and sugarcane baggase samples hydrolyzed with NaOH and microbial species at different incubation temperature.

Sample

Treatment

Species

Mean

Penicillium

Fusarium

Trichoderma

Aspergillus

Saw dust

control

2.90rs

0.99t

3.900opqr

3.50pqr

02.84i

30 oC

6.90m

1.90st

9.20l

5.30n

05.83g

40 oC

19.30h

4.20nopqr

18.10e

19.10h

17.23d

50 oC

16.10j

4.47nopq

29.90e

21.23g

17.93c

60 oC

7.20m

3.10rs

18.10hi

11.30k

09.93e

Sugarcane baggase

control

5.15no

1.50t

4.20nopqr

3.90opqr

03.69h

30 oC

6.70m

3.50qr

12.00k

07.00m

07.30f

40 oC

17.10ij

4.90nop

38.40b

26.50f

21.73b

50 oC

21.50g

5.30n

45.00a

31.50d

25.83a

60 oC

11.16k

3.30qr

33.80c

18.90h

16.79d

Sample x Species

Saw dust

10.48d

2.93f

17.48b

12.10c

10.75b

Sugarcane baggase

12.32c

3.70e

26.68a

17.56b

15.07a

Treatment x Species

Control

4.02jkl

01.25n

04.05jkl

03.73kl

03.26e

30 oC

6.80i

02.70m

10.60g

06.15i

06.56d

40 oC

18.20e

04.55jk

32.35b

22.80d

19.48b

50 oC

18.80e

04.88j

37.45a

26.37c

21.87a

60 oC

9.18h

03.20lm

25.95c

15.10f

13.36c

Mean

11.40c

3.32d

22.08a

14.83b

Values within columns sharing a common letter are not significantly different at P < 0.01. *Incubation time=72 hrs.

 

Table 7: Percent reducing sugar of saw dust and sugarcane baggase samples hydrolyzed with base (NaOH) and microbial species at different incubation time.

Sample

Treatment

Species

Mean

Penicillium

Fusarium

Trichoderma

Aspergillus

Saw dust

Control

1.10q

0.55q

1.50q

1.35q

1.12g

24 hrs

4.10p

1.10q

9.40mn

04.12p

4.68f

48 hrs

9.80lmn

1.10q

24.90ef

18.10j

13.47d

72 hrs

21.10h

4.30p

31.00d

23.20fg

19.90b

96 hrs

9.10n

1.10q

23.10g

20.30hi

13.40d

Sugarcane Baggase

Control

3.71p

0.98q

1.90qr

1.30q

1.97g

24 hrs

4.80op

1.10q

11.00lm

06.30o

5.85e

48 hrs

16.20k

1.90q

33.40c

19.20ij

17.67c

72 hrs

25.10e

5.10op

46.20a

30.50d

26.72a

96 hrs

11.47l

1.80q

35.80b

19.80hij

17.21c

Sample x Species

Saw Dust

9.04f

1.63g

17.98b

13.41d

10.52 b

Sugarcane Baggase

12.25e

2.21g

25.66a

15.42c

13.89 a

Treatment x Species

Control

2.40j

0.76k

1.70jk

1.32jk

1.549d

24 hrs

4.45i

1.20k

10.20h

5.21i

5.265c

48 hrs

13.00g

1.50jk

29.15b

18.65f

15.575b

72 hrs

23.10d

4.70i

38.60a

26.85c

23.313a

96 hrs

10.28h

1.45jk

29.45b

20.05e

15.31b

Mean

10.649c

1.923d

21.82a

14.42b

Values within columns sharing a common letter are not significantly different at P < 0.01. *Incubation temperature=50 °C

 

for 24 hrs at 50°C by Fusarium sp. Similarly the highest amount of reducing sugar (31.0%) for sawdust was observed in sample incubated for 72 hrs at 50°C by Trichoderma sp., while the lowest amount of reducing sugar (0.55% and 1.10%) for saw dust was observed in control sample followed by sample, incubated for 24 hrs at 50°C by Fusarium sp. Iqbal et al. (2010) studied that beyond the optimum incubation time (72 hrs) resulted decrease in reaction activity that might be due to depletion of nutrients or accumulation of some inhibitory compound in media. Results showed that Aspergillus sp. and Penicillium sp. also showed maximum hydrolysis for saw dust and sugarcane bagasse after 72 hrs of incubation period, while lowest amount of reducing sugar was obtained at control and 24 hrs of incubation time by Fusarium sp. followed by Penicillium sp. for saw dust and sugarcane bagasse. The present study agreed to Juliet et al. (2013) who observed that Trichoderma sp. was found with highest enzyme activity as compared to other isolated species and reported maximum hydrolysis of 35 % after 72 hrs of incubation time at 6.5 pH, respectively.

Conclusions and Recommendations

We concluded that cellulosic content in sugar cane bagasse was hydrolyzed more efficiently than saw dust during various treatments. Among different acids hydrochloric acid was more efficient in hydrolysis of cellulose content, while among bases sodium hydroxide was efficient. Combine effect of chemical and fungi (pretreated sample) was more efficient than individual effect of fungi (non-pretreated sample). In fermentation process, incubation time of 72 hrs and temperature at 50 °C were best for cellulose hydrolysis.

Novelty Statement

Digestion of cellulose using economically low-cost procedure for biofuel industries been specially given consideration during this research article.

Author’s Contribution

Samra Aftab: Conducted experiment, did data analysis and compilation.

Saleem Ullah: Supervised all the activities.

Farida Anjum: Original draft writing and data analysis.

Conflict of interest

The authors have declared no conflict of interest.

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Sarhad Journal of Agriculture

September

Vol. 37, Iss. 3, Pages 714-1097

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