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Fermented sugarcane bagasse with the fungus Pleurotus eryngii reduced in vitro methane production

Nguyen Thi Huyen, Nguyen Thi Tuyet Le and Bui Quang Tuan

Department of Animal Nutrition and Feed Technology, Faculty of Animal Science, Vietnam National University of Agriculture, Trau Quy, Hanoi, Vietnam
nthuyencnts@gmail.com

Abstract

The objective of this experiment was to determine the nutrients value and methane production of sugarcane bagasse (SB) after fermented with the fungus Pleurotus eryngii. In experiment 1, the sugarcane bagasse was treated with P. eryngii at the concentrations of 2.5% of wet weight of substrate for 4 weeks with 3 replicates. Results showed that fungal treatment increased the CP content and reduced NDF, ADF and ADL of the SB. In experiment 2, the SB and fungal treated SB were incubated for 24h in the buffer rumen fluid mixture. Results showed that total gas accumulation after 24h incubation was higher in the fungal treated SB while the methane production was lower in the fungal treated SB. Based on the results mentioned above, it could be concluded that Pleurotus eryngii can be used to improve nutrients value and digestibility of sugarcane bagasse and the fungal treated SB can be used in ruminants diet to reduce methane production.

Keywords: nutrients value, methane production, Pleurotus eryngii, sugarcane bagasse, wood-rot fungi


Introduction

The feed cost for ruminants is increasing and during dry season feed resources is limited. Therefore searching for alternative feed resources is becoming increasingly essential. Sugarcane bagasse (SB) is a by-product in the process of sugar production. Approximately 300 kg of this by-product is produced from every ton of raw sugarcane. In 2019-2020, approximate 914 thousand tons of sugar was produced in Vietnam. Besides, the primary source, 117 thousand tons of sugarcane bagasse was produced (estimated). The SB has low nutritive value with crude protein content (less than 3% DM) and lignin content (16% DM). However, the moisture content of the SB is 40 - 45%, which makes it easy to rot and mold. This is a cause for environmental pollution and waste the renewable source of roughage for ruminants.

Chemical treatment of sugarcane bagasse to increase the intake and digestibility by ruminants studied for years ago Nirawan et al (2016). However, Tuyen et al (2012) stated that physical and chemical treatments can be expensive, harmful to users or unfriendly to the environment. Biological methods using white-rot fungi may be a more viable alternative to improve the nutritional value of rice straw. This method is environmentally friendly and potentially economical (Tuyen et al 2012).

Using white-rot fungi to treat sugarcane bagasse has been studied by Mahmood Molaei Kermani et al. (2019) and Kamra et al (1993). The results of Huyen et al (2019, 2021) indicated that the nutritional value of rice straw, dried maize cobs were improved when incubated for 4 weeks with Pleurotus eryngii. The following experiment was conducted to confirm Pleurotus eryngii also can be used to improve nutrients value, digestibility and reduce methane production sugarcane bagasse of in Vietnam.


Materials and methods

Fungi treatments of sugarcane bagasse for 4 weeks

The experiment was conducted at Department of Animal Nutrition and Feed Technology, Vietnam National University of Agriculture, Hanoi, Vietnam. The sugarcane bagasse (SB) was collected in Yen Chau and Mai Son, Son La province, Vietnam. One kilograms (fresh basis) of sugarcane bagasse was packed in polyethylene bags (40 cm length and 20 cm diameter and 2.54 mm thickness), that was immediately tied up with a little cotton on the top of bag by nylon rope. The bags were autoclaved for 1 h at 121 C. The autoclaved SB bags were cooled at 20 C and then were inoculated with spawn at 2.5% of rice straw (fresh weight basis). The SB was fermented with Pleurotus eryngii (P. eryngii; strain MES 03757) according to the procedure developed by Tuyen et al (2012). All the bags were transferred to the fermentation room, which was maintained at 30 C and the relative humidity of the room was maintained at 75 % for 4 weeks. Then all bags were removed from the fermentation room and the fungal treated SB was oven-dried at 65 C for 3 days. The SB and fungal treated SB were ground in a cross-beater mill to pass through a 1mm sieve, then was stored at 4C before analysis of DM, ash, N, NDF, ADF and ADL and using for gas production experiment.

Table 1. Chemical composition of sugarcane bagasse in raw and after autoclaved

Items

Sugarcane
bagasse (Raw)

Sugarcane bagasse
(Autoclaved)

DM

531.5 3.6

542.5 2.1

Chemical composition (g/kg DM)

OM

928.6 1.0

930.0 1.8

CP

29.0 0.7

26.3 0.6

NDF

791.4 11.8

803.1 11.0

ADF

405.5 5.0

410.8 3.7

ADL

148.6 8.2

156.7 7.9

In vitro gas production experiment

The gas production was conducted according to the method described by Menke et al (1979). In summary, a mixture of rumen fluid was collected before feeding time in the morning from three different rumen fistulated lactating Holstein-Friesian dairy cows. These cows were fed a grass and maize silage mixture and concentrate according to their requirements 2 times per day. The rumen fluid was filtered through four layers of cheesecloth into pre-warmed thermo flasks. A strict anaerobic condition was maintained during rumen fluid collection. Buffer solution was made as described in the method of Cone et al (1996). The rumen fluid was mixed with a buffer solution in a 1:2 (v/v) ratio under a continuous flux of CO2. Approximately 500 mg of the oven-dried SB (control) and fungal treated SB substrates were weighted triplicates into 100-mL calibrated glass syringes. The grass syringes were pre-warmed at 39C before adding with 60 mL of the buffer rumen fluid mixture then they were incubated in a water bath at 39C. Three blank glass syringes only contained 60 mL of the buffer rumen fluid mixture. The gas production was manually recorded at 0, 3, 6, 12, 18 and 24h. The gas production was calculated by subtracting the mean of gas production from three blank syringes and expressed on an OM basis. After 12h and 24h of incubation, the fermentation fluid from each glass syringe was collected for volatile fatty acid (VFA) analysis.

The in vitro degradability of dry matter at 24 h of incubation was performed by pouring the samples from each glass syringe into crucible (pre-weighed and recorded the weight) and emptying the glass syringe with distilled water, then the crucible were brought to oven-drying at 103 C for 24 hours and weighed. The data of residue DM after subtracted the residue DM of blank were used for in vitro DM degradability (IVDMD).

Measurements sampling and analytical procedures

The SB (raw and autoclaved) and fungal treated SB samples were analysed for DM, ash and nitrogen according to AOAC (2005) methods. Neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) were determined according to Van Soest et al (1991). The VFA concentration was analysed by gas chromatography following the procedures of Pellikaan et al (2011).

Methane (CH4) production was estimate following the equation of Moss et al. (2000):

Methane (CH4) production = 0.45 [Acetic acid] - 0.275 [Propionic acid] + 0.4 [Butyric acid], expressed as mmol.

Statistical analysis

The different of chemical composition, gas production and methane production of the SB and fungal treated SB were analysed by ANOVA using the MIXED procedure of SAS (SAS, 2010). The model was:

Y = μ + Ti + εij (1)

where Y = the dependent variable, μ = the overall mean, Tj = the effect of treatment (i=1 to 3) and εij=the residual error term. The results are presented as the least squares means and standard error of the means. Differences among main effects were analysed using Tukey-Kramer’s multiple comparison procedure in the LSMEANS statement of SAS (SAS 2010) with effects considered significant at p≤0.05 and a trend at 0.05<p.


Results and discussion

Fungal treatment increased the CP content of the SB (Table 2 and Table 3). Similar results were reported by Mahmood Molaei Kermani et al. (2019) who reported that CP content of sugarcane bagasse was increased when the SB was treated with oyster mushroom (Pleurotus florida) for 30 days. Huyen et al (2019, 2021) found that the CP content improved by Pleurotus eryngii treated rice straw and dried maize cobs for 28 days.

The concentrations of ADF, NDF and ADL were reduced by fungal treatment. Similar results were reported by Kamra et al (1993), Zuo et al (2019), Akinfemi (2010), Vorlaphim et al. (2018) and Huyen et al (2019, 2021). The fungi require substrates such as cellulose, hemicellulose or other carbon sources for their growth, the end products being fungal protein and carbon dioxide, the latter accounting for the overall 15.5% loss of substrate DM during the fermentation (Table 3). Kamra et al (1993), reported 11.5-13.3% DM loss when sugarcane bagasse was treated with Pleurotus fungi.

Table 2. Chemical composition of sugarcane bagasse (autoclaved) and fungal-treated sugarcane bagasse after 4 weeks

Items

Control

4 weeks

SEM

p

DM

542.5a

458.3b

3.07

<.0001

Chemical composition, g/kg DM

OM

930.0a

792.8b

1.01

<.0001

CP

26.3a

57.9b

0.36

<.0001

NDF

803.1a

693.2b

5.80

0.0002

ADF

410.8a

366.1b

2.47

0.0002

ADL

156.7a

109.2b

3.58

0.0007

ab Row means with different superscripts differ at p<0.05



Table 3. The loss of elements from sugarcane bagasse after incubation with fungi for 4 weeks

Items

Sugarcane
bagasse, g

Fungal-treated
sugarcane bagasse, g

Loss/gain,%

DM

100 0.0

84.5 1.6

-15.5 1.6

OM

93.0 0.2

79.3 0.17

-14.7 0.3

CP

2.6 0.1

5.7 0.1

+120.1 6.6

NDF

80.3 1.1

69.3 0.9

-13.7 2.2

ADF

41.1 0.4

36.6 0.5

-10.9 0.9

ADL

15.7 0.8

10.9 0.4

-30.3 1.5

Total gas accumulation and in vitro DM degradability (IVDMD) after 24h incubation was higher when the SB was treated with Pleurotus fungi for 28 days (Table 4). These findings are supported by in vitro studies of Kamra et al (1993) who found the IVDMD increased 4.1 - 10.4% when the SB was treated with Pleurotus fungi. Mahmood Molaei Kermani et al (2019) who reported that the digestibility increased when sugarcane bagasse was treated with oyster mushroom (Pleurotus florida) for 30 days. The higher gas production in the fungal treated samples could be related to the higher CP content and lower content of NDF, ADF and ADL compared to the control samples (Osuga et al 2006). The lower content of fibre in the fungal treated samples can facilitate the colonization of the feed by the rumen microbial population, which in turn might increase the fermentation rate, therefore improving digestibility. Methane production in the fungal-treated SB was lower compared with the control. Hook et al. (2011) reported that the more fibre in ruminants diet produces more acetate and butyrate proportion which increases hydrogen availability and activity of rumen methanogens. In this experiment, the proportion of acetate and butyrate at 24h in the fungal-treated SB and the SB are 70 and 75%, respectively. The lower content of NDF in the fungal-treated SB could be a reason for the reduction of methane production.

Table 4. Total gas and methane production of sugarcane bagasse and fungal-treated sugarcane bagasse after 4 weeks

Items

Control

4 weeks

SEM

p

Gas production (ml/0.5g OM substrate)

0-6h

14.3a

19.8b

0.15

<.0001

6-12h

18.2a

23.1b

0.10

<.0001

12-18h

14.6a

17.5b

0.25

0.0011

18-24h

11.8a

14.6b

0.23

0.0010

Total gas accumulation

58.9a

75.0b

0.36

<.0001

IVDMD, %

35.6a

50.2b

0.77

0.0002

Volatile fatty acids (mmol/ 0.5 g OM substrates) after 12h incubation

Acetic acid

25.1a

27.1b

0.15

0.0006

Propionic acid

7.4a

9.9b

0.05

<.0001

Butyric acid

5.8a

4.5b

0.02

<.0001

Total VFAs

41.1a

45.2b

0.24

0.0003

Volatile fatty acids (mmol/ 0.5 g OM substrates) after 24h incubation

Acetic acid

40.4a

43.2b

0.25

0.0015

Propionic acid

11.9a

15.8b

0.09

<.0001

Butyric acid

9.3a

7.2b

0.04

<.0001

Total VFAs

66.2a

71.9b

0.42

0.0007

Methane production (mmol/ 0.5g OM substrates)

CH4 at 12h

11.56a

11.28b

0.061

0.0324

CH4 at 24h

18.60a

17.95b

0.106

0.0125

ab Row means with different superscripts differ at p<0.05 Methane (CH4) production = 0.45 [Acetic acid] - 0.275 [Propionic acid] + 0.4 [Butyric acid], expressed as mmol.


Conclusions


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