Index

Introduction
Materials and Methods
Results
Discussions
Conclusion
References

Abstract

Lignocellulosic substrates are wastes in the environment whose reducing sugars are not readily available for use. Biological pretreatment is the use of microorganisms and/or their metabolites to break down substrates to obtain simple sugars which is also cheap compared with other pretreatment techniques. This work is aimed at degrading lignocellulosic substrates with higher mushrooms to obtain simple sugars that could be used as raw materials for other industrial processes. The two mushrooms [Pleurotus ostreatus (PO) and Lentinus squarrosulus (LS)] with the ability to produce cellulase, xylanase, and lignase were used for degradation of lignocellulosic substrates [groundnut shell (GS), maize cob (MC), maize straw (MS), rice straw (RS) and sugarcane bagasse (SB)]. The residual extractives, cellulose, hemicellulose, lignin, and reducing sugar contents were determined every 7 days. Least extractives (1.12 %), hemicellulose (15.09 %), lignin (17.60 %), and cellulose (5.60 %) were recorded in PO-degraded MS, POLS-degraded GS, LS-degraded GS, and PO-degraded MS at 28, 35, 49 and 42 days of degradation, respectively. The highest reducing sugar contents (mg/g) obtained in GS (11.83), MS (27.03), SB (28.70), and RS (37.96) were recorded when degraded by PO for 49, 14, 7, and 49 days, respectively while that of MC (13.32) was recorded when degraded by LS for 42 days. Reducing sugar obtained was higher from sole degradation with PO compared with LS and POLS. Degraded MS, RS, and SB had better yield of reducing sugar than GS and MC. The amount of reducing sugar released varied with substrates, organisms, and degradation time.

Keywords: Biological pretreatment, Cellulose, Degradation, Extractives, Hemicellulose, Lentinus squarrosulus, Lignin, Lignocellulosic substrates, Pleurotus ostreatus, Reducing sugar.

Received: 2 February 2023 / Revised: 6 April 2023/ Accepted: 20 April 2023/ Published: 8 May 2023

Contribution/ Originality

We have analyzed nutritional recovery status in hospitalized children with malnutrition.

1. INTRODUCTION

Second-generation biofuels are produced from non-edible agricultural wastes however, reducing sugars needed for the generation of biofuel in these wastes are not readily released from them [1-3]. Therefore, it is necessary to pretreat non-edible agricultural wastes to obtain simple sugars needed for the production of biofuel. Non-edible agricultural wastes are lignocellulosic substrates that contain mainly cellulose, hemicellulose, and lignin [4]. The cellulose and hemicellulose when broken down contain fermentable sugar but lignin does not contain any sugar [5, 6]. Biological, chemical and physical, and physicochemical methods of pretreatment are available [7, 8]. The biological pretreatment method is the cheapest among different pretreatment techniques and it is also environmentally friendly [9]. Organisms or their metabolites are used to break down lignocellulosic substrates in the biological pretreatment method. Lignocelluloses are broken down by soft rot, brown rot, and white rot fungi [10]. There are two kinds of white rot fungi such as selective and non-selective. Hemicellulose and lignin portions of lignocellulose are degraded by selective white rot fungi while the cellulose part is essentially not degraded but all parts of lignocellulose are equally degraded by non-selective white rot fungi [10-12]. Some of the white rot fungi that have been utilized to degrade lignocellulose are Obba rivulosa Ceriporia lacerate, Ceriporiopsis subvermispora, Cyathus stercolerus, Pycnoporus cinnarbariunus, Lentinus squarrosulus, Pleurotus ostreatus, Pleurotus tuber-regium, Gelatoporia subvermispora and Phanerochaete chrysoporium [1-3, 9, 13, 14].

Lignocellulosic wastes in the environment could include groundnut shell, maize straw, rice straw, sugar cane bagasse, maize cob, and others. Utilization of these non-edible wastes will reduce waste in the environment. Degradation of these lignocellulosic substrates with fungi and obtaining a good yield of reducing sugar required for fermentation of biofuel and other industrial processes would make this technique economically viable. This work aimed at degrading lignocellulosic substrates with fungi to obtain fermentable sugars.

2. MATERIALS AND METHODS

2.1. Collection of Lignocellulosic Wastes

All lignocellulosic biomass were collected from Oyo state (8.1196° N, 3.4196° E). Groundnut shell was collected from Saki, maize cob from Ajegunle, Oyo town while maize straw was collected from Okunlola’s farm, Ilora. Sugar cane bagasse was obtained from Akunlemu, Oyo town and rice straw was collected from the International Institute of Tropical Agriculture (IITA), Ibadan. All the samples were dried and milled with a milling machine in Oyo town and taken to the Laboratory of the Department of Biological Sciences, Ajayi Crowther University, Oyo town.

2.2. Collection of White Rot Fungi

Two white rot fungi (Lentinus squarrosulus and Pleurotus ostreatus) with the potential of producing cellulases, hemicellulases, and lignin-modifying enzymes were collected from the Department of Botany, University of Ibadan, Ibadan.

2.3. Pretreatment of Lignocellulose with White Rot Fungi

Five lignocellulosic wastes (maize cob, maize straw, rice straw, groundnut shell, and sugar cane bagasse) were pretreated with Pleurotus ostreatus and Lentinus squarrosulus singly and in consortium. One hundred (100) grams of each lignocellulosic material were weighed into separate glass jars and 300 mL of distilled water was added and mixed. They were sterilized at 121 °C and 1.05kg cm-2 for 15 minutes and allowed to cool. Each cooled biomass was inoculated separately with eight agar plugs (7mm in diameter) of Pleurotus ostreatus and Lentinus squarrosulus separately and consortium (4 agar plugs each of Pleurotus ostreatus and Lentinus squarrosulus) [2]. They were incubated at 28 ±2°C for 70 days. Samples were taken from degrading substrates every 7 days and were analyzed for cellulose, hemicellulose, lignin, extractives, and reducing sugar contents.

2.4. Determination of Extractives, Hemicellulose, Lignin, and Cellulose

The extractive in each biomass was determined using solvent extraction. Sixty millilitres of acetone was added to a dried biomass sample (1 g). The temperature was maintained at 56 ℃ for 2 hours. The samples were then dried at 105 ℃ until they reached a constant weight. The amount of extractives in the biomass is the weight difference between the sample before and after extraction [15]. Extractive-free dried biomass (0.4 g) was added to four millilitres of 0.5 mol/L NaOH, and the mixture was maintained at 80 ℃ for three and a half hours. The sample was washed with distilled water until the solution's pH reached 7, and it was then dried in a hot air oven at 105 ℃ until a constant weight was observed. The amount of hemicellulose is the weight difference between the sample's initial and final weights [15]. Extractive-free dried biomass (0.4g) was mixed with 12 millilitres (98% sulphuric acid) and left at room temperature for 24 hours. The sample was diluted with 80 mL of distilled water after 24 hours, and it was then heated for 1 hour at 100 °C. The mixture was allowed to cool, filtered, and the residue was washed with distilled water until there were no sulphate ions left in the filtrate. The residue was dried until it reached a constant weight. The lignin content is the weight of the dried residue [15].

The difference was used to compute the amount of cellulose, with the assumption that each biomass consisted solely of cellulose, lignin, hemicellulose, and extractive [15].

Weight of the total biomass - (lignin + hemicellulose + extractive) content equals the content of cellulose

2.5. Determination of Reducing Sugar Content

Extraction of reducing sugar from the degraded substrate was done by adding 1 g of the degraded substrate to 20 mL of distilled water and shaking every 15 minutes for 2 hours. It was filtered, and the obtained filtrate was utilized to determine the amount of reducing sugar. The Dinitrosalicylic acid method was used to determine the amount of reducing sugar [16].

2.6. Statistical Analysis

The level of significance was set at P≤0.05 for the analysis of variance, which was performed on the experimental data to estimate the means, using SPSS version 23.

3. RESULTS

The component (extractive, hemicellulose, lignin, and cellulose) of groundnut shell pretreated with Pleurotus ostreatus and Lentinus squarrosulus is shown in Table 1. Extractives of groundnut shell degraded by Pleurotus ostreatus (PO), Lentinus squarrosulus (LS), and a consortium of Pleurotus ostreatus and Lentinus squarrosulus (POLS) ranged from 2.09 – 9.99 %; 2.02 – 9.99 % and 2.31 – 9.99 % with the least recorded at 21, 28 and 28 days of degradation, respectively and their highest extractives were recorded before degradation. The highest hemicellulose contents of groundnut shell degraded by PO (30.39 %), LS (26.31 %), and POLS (26.07 %) were obtained on 14 days of degradation, and the least (15.09 %) was obtained when degraded by POLS for 35 days. The least lignin contents (33.43, 17.60, and 18.78 %) of all treatments (PO, LS, and POLS) were observed in the 49-day degraded sample, respectively. The cellulose content of groundnut shell degraded by PO, LS, and POLS ranged from 18.55 – 37.68 %, 23.44 – 52.61 %, and 21.00 – 52.69 %, respectively. The least and highest cellulose content by the degrading fungi were obtained at 14 and 49 days of degradation, respectively. Statistical analysis revealed that the extractives, hemicellulose, lignin, and cellulose contents of degraded groundnut shell by Pleurotus ostreatus, Lentinus squarrosulus, and a consortium of the two were significantly different (P≤0.5) with days of degradation. The component of degraded maize cob by all the treatments was in the order of hemicellulose > lignin > cellulose > extractives Table 2. The highest extractives of PO (8.78 %), LS (8.59 %), and POLS (10.33 %) were recorded in 7-day degraded maize cob. The highest hemicellulose contents of 40.70 %, 43.92 %, and 44.88 % were recorded when maize cob was degraded by PO, LS and POLS at 14, 63, and 14 days of degradation, respectively. The lignin content of maize cob degraded by PO, LS, and POLS ranged from 24.29 – 35.66 %, 26.45 – 35.37 %, and 22.65 – 35.85 %, respectively. The initial cellulose content (29.08 %) of undegraded maize cob was higher than those degraded except for maize cob degraded by PO for 49 days (29.13 %) which was not significantly different (P>0.05) from the undegraded. The least cellulose content was observed at 14 days of degradation by PO (21.34 %) and POLS (15.07%). Maize cob degraded by LS had least cellulose content (16.22 %) at 49 days of degradation which was not significantly different (P>0.05) from cellulose content (16.68 %) obtained at 14 days of degradation.

Table 1. Component of groundnut shell pretreated with Pleurotus ostreatus and Lentinus squarrosulus singly and combined.

Days

Extractives (%)

Hemicellulose (%)

Lignin (%)

Cellulose (%)

POLS

9.99e

9.99d

15.59a

40.79c

33.64f

33.64g

33.64f

8.19d

9.55d

7.09bc

28.41e

26.18g

24.44g

43.06de

35.38b

37.13b

20.34b

28.90de

31.35de

3.01a

2.90a

3.13a

30.39e

26.31g

26.07h

48.05f

47.35f

49.80f

18.55a

23.44a

21.00a

2.09a

3.47a

3.41a

21.40c

20.91ef

20.75de

50.24g

48.79f

49.41f

26.27c

26.83bc

26.43b

2.45a

2.02a

2.31a

16.81a

16.81ab

17.25b

49.21fg

51.57g

50.20f

31.52e

29.59de

30.24d

5.74b

6.31b

18.88b

18.89cd

15.09a

41.64cd

43.27de

41.53cd

33.74f

31.53f

37.07g

6.28bc

7.62bc

6.38b

18.58b

18.01bc

19.60cd

48.87fg

48.50f

47.84e

26.27c

25.87b

26.18b

7.95d

7.58bc

7.71bc

20.94c

22.21f

20.81de

33.43a

17.60a

18.78a

37.68g

52.61h

52.69h

7.68cd

8.54cd

8.23c

23.92d

21.02ef

22.70f

42.24cde

42.33d

40.94c

26.16c

28.10cd

28.13c

6.28bc

7.14bc

7.16bc

24.43d

19.84de

18.34bc

43.60e

43.88e

42.58d

25.69c

29.15de

31.93e

7.19bcd

7.89c

7.47bc

24.86d

21.19ef

21.25e

38.74b

40.71c

41.05c

29.21d

30.21ef

30.24d

Note:

Mean values with different alphabetical superscripts along the column are significantly different (P≤0.05).
PO:                Pleurotus ostreatus.
LS:                 Lentinus squarrosulus.
POLS:           Consortium of Pleurotus ostreatus and Lentinus squarrosulus.

Table 2. Component of maize cob pretreated with Pleurotus ostreatus and Lentinus squarrosulus singly and combined.

Days

Extractives (%)

Hemicellulose (%)

Lignin (%)

Cellulose (%)

POLS

8.26bc

8.26c

35.27ab

35.27a

27.38b

27.38ab

27.38b

29.08e

29.08f

29.08e

8.78c

8.59c

10.33d

37.37cde

42.80de

39.73cd

27.77b

28.52b

28.66bc

26.08c

20.09b

21.28b

3.59a

5.10b

4.20ab

40.70g

42.84de

44.88f

34.37de

35.37e

35.85h

21.34a

16.68a

15.07a

3.20a

3.88b

5.38b

39.43fg

40.02c

39.54cd

35.66e

32.99d

34.82gh

21.72ab

23.11d

20.27b

3.24a

2.33a

2.92a

34.02a

39.01c

37.22b

34.55de

33.91de

33.79fg

28.20de

24.74e

26.07d

8.62c

7.39c

7.40c

36.39bc

42.86de

35.73a

32.13c

28.00b

32.01e

22.86b

21.75cd

24.86d

6.93b

8.12c

7.34c

36.71bc

36.91b

38.45bc

33.37cd

33.74d

33.02ef

22.99b

21.22bc

21.19b

7.72bc

7.64c

7.79c

38.86ef

41.75d

41.49e

24.29a

34.39de

29.39cd

29.13e

16.22a

21.33b

7.90bc

8.55c

8.60c

38.54def

38.91c

42.05e

26.72b

31.23c

28.29bc

26.83cd

21.31bc

21.07b

6.95b

7.61c

38.22def

43.92e

38.98c

26.68b

26.45a

30.57d

28.15de

22.02cd

22.84c

7.03b

7.56c

7.87c

37.22cd

36.91b

40.91de

27.29b

27.60ab

22.65a

28.46e

27.93f

28.57e

Note:

Mean values with different alphabetical superscripts along the column are significantly different (P≤0.05)
PO:              Pleurotus ostreatus.
LS:               Lentinus squarrosulus.
POLS:         Consortium of Pleurotus ostreatus and Lentinus squarrosulus.

Table 3. Component of maize straw pretreated with Pleurotus ostreatus and Lentinus squarrosulus singly and combined.

Days

Extractives (%)

Hemicellulose (%)

Lignin (%)

Cellulose (%)

POLS

10.72g

10.72f

10.72e

42.05a

42.05ab

42.05def

29.52e

29.52b

29.52c

17.71g

17.71ef

17.71a

2.87b

3.18a

4.98a

48.29cd

42.73bc

39.35c

26.18b

30.53b

18.42a

22.66h

23.56g

37.24e

4.59c

5.22b

5.30ab

47.55bc

46.47f

36.14b

31.00f

35.99d

33.44d

16.86fg

12.33ab

25.12d

7.39de

6.66bc

6.40abc

46.34b

43.41bcd

40.77d

32.81g

38.99f

35.23e

13.46d

10.94a

17.60a

1.12a

2.00a

13.97f

49.78e

45.11ef

33.40a

37.42h

37.59e

35.13e

11.68c

15.30d

17.50a

9.05f

8.67e

8.05d

46.57b

41.14a

43.77g

29.12de

27.14a

25.70b

15.27e

23.05g

22.48c

7.06de

6.93cd

6.65bcd

50.66ef

43.55cd

41.40de

36.69h

36.06d

35.76e

5.60a

13.46bc

16.19a

7.65ef

8.34de

7.00cd

51.62f

43.70cde

42.78efg

30.98f

33.42c

33.68d

9.75b

14.55cd

16.54a

7.62ef

7.32cde

7.47cd

49.5de

44.34de

42.97fg

27.77cd

29.51b

29.73c

15.12e

18.84f

19.83b

6.03d

6.30bc

6.62bcd

53.33g

46.35f

43.54fg

27.05bc

30.65b

26.41b

13.59d

16.70e

23.43c

8.29ef

7.01cd

6.94cd

54.16g

43.59cd

42.75efg

21.84a

30.97b

30.16c

15.71ef

18.43f

20.15b

Note:

Mean values with different alphabetical superscripts along the column are significantly different (P≤0.05)
PO:                Pleurotus ostreatus.
LS:                 Lentinus squarrosulus.
POLS:           Consortium of Pleurotus ostreatus and Lentinus squarrosulus.

Table 4. Component of sugarcane bagasse pretreated with Pleurotus ostreatus and Lentinus squarrosulus singly and combined.

Days

Extractives (%)

Hemicellulose (%)

Lignin (%)

Cellulose (%)

POLS

11.12f

11.12e

11.12g

36.15a

29.71b

29.71c

29.71e

23.01def

23.01c

23.01d

4.45ab

3.52ab

4.48b

35.44a

38.33b

39.34b

27.27a

27.49b

26.10bc

32.85h

30.66f

30.08g

3.67ab

2.35a

2.43a

35.21a

39.46bc

42.17cd

33.35c

34.70e

35.77g

27.78g

23.48c

19.62c

4.83bc

4.39b

5.38bc

40.51c

40.36cd

45.03f

32.86c

30.00c

34.63g

21.80cd

25.25d

14.95a

3.01a

3.27ab

2.19a

39.02b

42.25e

41.12c

33.64c

32.10d

32.46f

24.33f

22.38c

24.23de

8.73e

7.17cd

4.80b

41.84cd

39.67bcd

43.66ef

25.88a

29.88c

27.91d

23.55ef

23.29c

23.63de

6.72d

7.08cd

6.31cd

40.63c

40.50cd

41.55c

34.23c

32.21d

33.07f

18.41b

20.21b

19.07c

9.26e

7.96d

7.81ef

40.56c

41.21de

43.84ef

33.35c

33.01d

31.62f

16.83a

17.83a

16.73b

9.98ef

11.17e

8.92f

42.57de

40.98cde

41.43c

26.88a

26.89b

24.95b

20.57c

20.97b

24.70e

6.00cd

6.00c

7.40de

42.52de

44.17f

41.32c

29.01b

26.41b

27.16cd

22.47de

23.42c

24.12de

6.77d

6.87cd

7.35de

43.66e

40.69cd

43.06de

26.98a

23.91a

23.21a

22.58de

28.53e

26.38g

Note:

Mean values with different alphabetical superscripts along the column are significantly different (P≤0.05)
PO:                Pleurotus ostreatus.
LS:                 Lentinus squarrosulus.
POLS:            Consortium of Pleurotus ostreatus and Lentinus squarrosulus.

The Extractives, hemicellulose, lignin, and cellulose contents of degraded maize straw is as shown in Table 3. The highest extractive (10.72 %) was recorded in undegraded maize straw and the lowest extractives of 1.12 %, 2.00 %, and 4.98 % were observed in PO, LS, and POLS-degraded maize straw at 28, 28, and 7 days of degradation, respectively. Hemicellulose content ranged from 42.05 – 54.16 %, 41.14 – 46.47 %, and 33.40 – 43.77 % in PO, LS, and POLS degraded maize straw, respectively. The hemicellulose content of PO-degraded maize straw was higher than LS and POLS-degraded maize straw throughout the period of degradation. The observed highest lignin contents by maize straw degraded by PO (37.42 %), LS (38.99 %), and POLS (35.76%) were recorded at 28, 21, and 42 days of degradation, respectively. It was observed that maize straw degraded for 7 days had highest cellulose content for PO (22.66 %), LS (23.56 %), and POLS (37.24 %). Days of degradation had a significant effect (P≤0.05) on extractives, hemicellulose, lignin, and cellulose of degraded maize straw.

The component of pretreated sugarcane bagasse is shown in Table 4. From the table, undegraded sugarcane bagasse had the highest extractives when compared with those degraded by selected white rot fungi. The least extractive observed in sugarcane bagasse degraded by PO, LS, and POLS were 3.01 %, 2.35 %, and 2.19 % at 28, 14, and 28 days of degradation, respectively. Hemicellulose contents of 35.21 – 43.66 %, 36.15 – 44.17 %, and 36.15 – 45.03 % were observed in PO, LS, and POLS-degraded sugarcane bagasse with their highest content at 70, 63, and 21 days of degradation, respectively. While the highest lignin content of PO-degraded maize straw (34.23 %) was observed at 42 days of degradation, the highest lignin contents of LS (34.70 %) and POLS (35.77%) -degraded sugarcane bagasse were recorded at 14 days of degradation. The least cellulose content obtained when sugar cane bagasse was degraded by PO, LS, and POLSwere 16.83 %, 17.83 %, and 14.95 % at 49, 49, and 21 days of degradation, respectively. There were significant differences (P≤0.05) in the values of extractives, hemicellulose, lignin, and cellulose with the day of degradation.

Table 5 shows the extractives, hemicellulose, lignin, and cellulose contents of PO, LS, and POLS-degraded rice straw. The extractives of rice straw degraded by PO, LS, and POLS ranged from 3.20 – 8.86 %, 3.84 – 8.86 %, and 3.07 – 8.86 %, respectively. Hemicellulose content of 39.61 – 56.70 %, 39.61 – 50.38 %, and 36.95 – 51.66 % were recorded in PO, LS, and POLS-degraded rice straw, respectively. During degradation, ranges of 20.91 – 31.54 %, 24.91 – 34.85 %, and 24.37 – 35.19 % of lignin content were obtained in PO, LS, and POLS-degraded rice straw, respectively. The cellulose content of rice straw degraded by PO ranged from 12.16 to 27.97 % with the least and highest at 70 and 7 days of degradation, respectively. Cellulose content range of 8.06 – 25.25 % and 15.31 – 30.46 % were recorded in LS and POLS degraded rice straw, respectively. Statistical analysis revealed values of extractives, hemicellulose, lignin, and cellulose of degraded rice are significantly different (P≤0.05) with days of degradation.

The reducing sugar content of lignocellulosic samples (groundnut shell, maize cob, maize straw, sugar cane bagasse, and rice straw) determined every 7 days during 70 days of degradation is shown in Table 6. Reducing sugar of PO-pretreated groundnut shell ranged from 2.61 to 11.23 mg/g with the least and highest at 56 and 49 days of degradation, respectively. The least reducing sugar (0.78 mg/g) of groundnut shell degraded by LS was obtained at 70 days of degradation and the highest (11.83 mg/g) at 21 days of degradation. The reducing sugar content of POLS-degraded groundnut shell ranged from 2.90 to 8.01 mg/g with the least and highest at 7 and 49 days of degradation, respectively, and were not significantly different (P>0.05). Reducing sugar of maize cob degraded by PO, LS, and POLS ranged from 0.37 – 8.16 mg/g, 3.64 – 13.32 mg/g, and 3.76 – 13.25 mg/g, respectively. The highest reducing sugar contents of PO, LS, and POLS-degraded maize straw were 27.03 mg/g, 20.41 mg/g, and 19.70 mg/g at 14, 35, and 0 days, respectively. The reducing sugar content of PO-degraded maize straw was generally higher than the reducing sugar of LS, and POLS-degraded maize straw. Sugarcane bagasse degraded by PO, LS, and POLS have reducing sugar in the range of 9.08 – 28.70 mg/g, 11.75 – 25.58 mg/g, and 10.28 – 31.94 mg/g with their highest content at 7, 7, and 49 days, respectively. The lowest reducing sugar (6.88 mg/g) was recorded in rice straw before degradation.

Table 5. Component of rice straw pretreated with Pleurotus ostreatus and Lentinus squarrosulus singly and combined.

Days

Extractives (%)

Hemicellulose (%)

Lignin (%)

Cellulose (%)

POLS

8.86d

8.86c

39.61a

39.61b

26.95f

26.95c

26.95b

24.59f

24.59fg

24.59d

4.26a

3.84a

3.58a

46.87c

39.97a

36.95a

20.91a

30.99d

29.01c

27.97g

25.20g

30.46e

3.31a

6.71bc

3.40a

51.37d

50.38f

43.23cd

31.54h

34.85e

35.19f

13.79b

8.06a

18.18b

6.53b

4.86a

4.46a

45.29b

48.96ef

48.18f

25.61ef

31.54d

31.19d

22.57e

14.64c

16.17a

3.20a

4.53a

3.07a

52.40de

49.44ef

45.91e

30.16gh

34.79e

32.93e

14.24b

11.24b

18.09b

8.19cd

7.85bcd

7.60bc

51.37d

48.52e

48.29f

23.68cd

26.72bc

28.79c

16.76c

16.91d

15.31a

8.21cd

6.94bc

7.97bc

45.36b

45.33d

44.17d

29.41g

34.13e

32.17de

17.03c

13.61c

15.70a

7.16bc

6.50b

7.22b

47.86c

41.41b

42.08c

24.88de

30.27d

30.75d

20.11d

21.82e

19.95c

8.17cd

8.18cd

7.77bc

53.66e

42.80bc

46.18e

24.96de

27.17c

26.90b

13.21ab

21.85e

19.15bc

7.98bcd

6.73bc

8.34bc

53.65e

42.62b

48.77f

22.08ab

25.40ab

26.23b

16.29c

25.25g

16.66a

7.97bcd

7.21bc

8.11bc

56.70f

44.14cd

51.66g

23.17bc

24.91a

24.37a

12.16a

23.73f

15.86a

Note:

Mean values with different alphabetical superscripts along the column are significantly different (P≤0.05).
PO:                 Pleurotus ostreatus.
LS:                  Lentinus squarrosulus.
POLS:            Consortium of Pleurotus ostreatus and Lentinus squarrosulus.

Table 6. Reducing sugar content (mg/g) of lignocellulosic substrates degraded by Lentinus squarrosulus and Pleurotus ostreatus singly and combined.

Period
(Days)

POLS

3.99ab

3.99a

3.76abcd

3.76a

19.70a

19.70b

16.88abc

16.88ab

6.88a

6.37abc

4.54ab

2.90a

7.06cd

13.26b

10.67a

14.76a

11.64ab

12.39ab

28.70d

25.58c

23.68abc

17.80bc

15.68a

15.74ab

4.20abc

5.16ab

3.12a

3.32abcd

8.45ab

6.68a

27.03a

15.16ab

9.14a

22.66cd

14.53ab

10.28a

19.04bcd

11.19a

15.80ab

7.14bc

11.83c

6.36a

4.58abcd

6.49ab

8.59a

22.26a

18.81ab

13.29ab

16.23abc

22.66bc

23.13abc

31.84ef

11.57a

20.70ab

3.42ab

8.56bc

7.88a

4.45abcd

3.85a

5.82a

18.24a

15.91ab

14.12ab

11.52ab

22.17bc

12.58ab

25.88cde

10.27a

15.17ab

8.08cd

3.44a

7.03a

4.74abcd

9.34ab

9.66a

24.94a

20.41b

18.93b

19.51bc

18.90abc

17.68abc

30.44ef

13.46a

19.27ab

5.67abc

1.71a

4.98a

0.37a

13.32b

10.73a

18.14a

16.29ab

14.27ab

9.61a

17.59abc

13.03ab

11.93ab

17.01a

17.92ab

11.23d

4.08ab

8.01a

5.76bcd

7.37ab

8.92a

11.93a

16.76ab

11.80ab

16.00abc

19.88abc

31.94c

37.96f

12.55a

11.38ab

2.61a

5.64ab

7.99a

2.89abc

4.92ab

5.20a

15.54a

16.70ab

11.86ab

11.08ab

20.26abc

21.73abc

19.85bcd

14.00a

17.45ab

3.55ab

1.51a

7.26a

8.16d

7.62ab

13.25a

17.48a

13.72ab

11.25ab

18.60abc

11.75a

26.37bc

27.47de

13.37a

28.74b

5.50abc

0.78a

6.25a

0.76ab

3.64a

7.12a

16.96a

9.66a

12.58ab

9.08a

15.01ab

20.80abc

18.79bcd

8.34a

18.14ab

Note:

Mean values with different alphabetical superscripts along the column are significantly different (P≤0.05).
GS:              Groundnut shell.
MC:             Maize cob.
MS:             Maize straw.
SB:               Sugarcane bagasse.
RS:              Rice straw.
PO:              Pleurotus ostreatus.
LS:               Lentinus squarrosulus.
POLS:         Consortium of Pleurotus ostreatus and Lentinus squarrosulus.

The highest reducing sugar of rice straw degraded by PO (37.96 mg/g), LS (17.01 mg/g), and POLS (28.74 mg/g) were recorded at 49, 42, and 63 days, respectively. Statistical analysis revealed that there was no significant difference (P>0.05) in the reducing sugar content of rice straw that was degraded by Lentinus squarrosulus with days of degradation.

4. DISCUSSIONS

Changes in chemical composition observed when different lignocellulolytic substrates were degraded with Pleurotus ostreatus and Lentinus squarrosulus through solid-state fermentation could be due to the metabolites (cellulase, xylanase, lignase/laccase, etc.) produced by these organisms, which can degrade different parts of lignocellulose. This observation corroborates the work of Issaka, et al. [17] and Wuanor and Ayoade [18] who degraded groundnut shell with Pleurotus species and reported changes in the chemical composition of groundnut shell. Costa-Silva, et al. [19] also observed changes in the composition of grape stalks degraded by some white rot fungi.Lower extractives recorded in most of the degraded substrates than in non-degraded ones might probably be due to the utilization of the extractives as nutrients during degradation by these mushrooms [20]. The values of extractives vary from biomass to biomass and between different parts of the same plant [20, 21].

Higher hemicellulose content observed in degraded lignocellulolytic substrates compared with non-degraded might be a result of low required nutrients needed for the production of hemicellulases (xylanase and others) on the substrates that could have converted hemicellulose to glucose and xylose. This is contrary to the findings of Issaka, et al. [17] and Wuanor and Ayoade [18] who recorded a decrease in hemicellulose content after degrading groundnut shell with Pleurotus species. The percentage composition of lignocellulolytic substrates differs from one another based on the class of the substrate which is softwood or hardwood [20]. Generally, lower hemicellulose content was observed when degraded by co-culture of Pleurotus ostreatus and Lentinus squarrosulus than when degraded singly. This might be due to the synergistic relationship between Pleurotus ostreatus and Lentinus squarrosulus in the utilization of hemicellulose. There have been reports that organisms performed differently when in consortium from when used singly [22]. The decrease in lignin content of groundnut shell observed after 49 days of degradation by Pleurotus ostreatus, Lentinus squarrosulus and consortium of Pleurotus ostreatus and Lentinus squarrosulus showed that these mushrooms can remove lignin bonds that prevent holocellulose from being broken down to simple and fermentable sugar. This observation has been reported to be due to the production of lignin-degrading enzymes by these organisms [23-26]. A similar observation of a decrease in lignin content of degraded groundnut shell by Pleurotus ostreatus for 5 weeks [17] and 30 days [18] has also been reported.

Conversion of cellulose to simple sugars by cellulase-producing mushrooms selected for degradation in this work could be responsible for a decrease in cellulose content observed at most sampling times in all selected lignocellulolytic substrates. The cellulose part of lignocellulosic substrates would have been extensively utilized and converted to hexoses by selected mushrooms leading to a decrease in cellulose after degradation as reported by some researchers [27]. A similar observation of a decrease in cellulose content after degradation with Pleurotus species was reported by Akinfemi [28] and Huang, et al. [29] from maize cob and crop straw respectively.

The higher reducing sugar released from groundnut shell degraded by the monoculture of Pleurotus ostreatus and Lentinus squarrosulus than co-culture of the two might be due to the high utilization of the released reducing sugar as carbon and energy sources by the co-culture than monoculture [30, 31] or the organisms might be having an antagonistic effect on each other leading to decrease in released reducing sugar when grown together. While the observed higher reducing sugar released in Pleurotus ostreatus and Lentinus squarrosulus-degraded maize cob than non-degraded one was probably because of the interaction between hydrolytic and oxidative enzymes released by these organisms when degrading maize cob, breaking down cellulose and hemicellulose to simple sugar [32, 33]. A similar observation of increased reducing sugar content of maize cob, when degraded by Pleurotus ostreatus, was reported by Adamafio, et al. [32]. The increase in reducing sugar content observed in degraded maize straw could be due to the breaking down of different components of maize straw to reducing sugar by the enzymes produced by the organisms which could be influenced by both genetic makeup and environmental conditions [29, 34, 35]. Higher reducing sugar content observed in degraded sugarcane bagasse could be due to the ability of Pleurotus ostreatus and Lentinus squarrosulus to produce cellulase and xylanase which could have broken the holocellulose content of sugarcane bagasse to reducing sugar Ravichandran, et al. [26]; Jonathan and Akinfemi [36]; Dong, et al. [37]; Shankarappa, et al. [38]; Gani, et al. [39]. Gani, et al. [39] reportedhigh reducing sugar when sugarcane bagasse was pretreated with alkaline and acid. The ability of Pleurotus ostreatus and Lentinus squarrosulus to produce lignocellulolytic enzymes that can breakdown cellulose, hemicellulose, and lignin to simple sugar could be responsible for high amount of reducing sugar recorded in degraded rice straw Jonathan and Akinfemi [36]; Belal [40]; Nurika, et al. [41]. Belal [40] reported high reducing sugar in rice straw degraded with Trichoderma reesei for 14 days while Nurika, et al. [41] observed a higher amount of reducing sugar after 21 days of degradation of rice straw with Serpula lacrymans.

5. CONCLUSION

The ability of Pleurotus ostreatus and Lentinus squarrosulus to degrade lignocellulosic substrates to simple sugars shows that these organisms could be employed in second-generation biofuel production where simple sugars released from lignocellulose would be used for ethanol production. Highest reducing sugar content (37.96 mg/g) was obtained by degrading rice straw by Pleurotus ostreatus for 49 days. Sole degradation with Pleurotus ostreatus had a better yield of reducing sugar than Lentinus squarrosulus and co-cultured. The amount of reducing sugar released varied with substrates, organisms, and degradation time.

Funding: This study received no specific financial support.

Competing Interests: The authors declare that they have no competing interests.

Authors’ Contributions: All authors contributed equally to the conception and design of the study.

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