Chemical composition

Figure 3.2 In vitro gas production after 72h (IVGP) of incubation in rumen fluid of substrates treated with four different fungi for 0, 2, 4, 8 and 12 weeks ((▲) untreated autoclaved material, (●) Pleurotus ostreatus, (■) Pleurotus eryngii, (○) Ganoderma lucidum, (□) Lentinula edodes). Error bars indicate standard deviations, n=3.

3.3.2 IVGP

Untreated, autoclaved wheat straw showed an IVGP of 252.8 ml/g OM, IVGP of untreated autoclaved miscanthus, wood chips and rice straw was 184.2, 54.0, and 274.9 ml/g OM, respectively. There was a significant (P < 0.01) interaction between fungus and time for all substrates, indicating that the different fungi had different effects over time. While none of the fungi increased IVGP of wheat straw and wood chips during 4 weeks of treatment, IVGP of miscanthus was improved between 2 and 4 weeks treatment with G. lucidum (P = 0.0018) and L. edodes (P = 0.0111) (Figure 3.2). All fungal species tested here increased (P < 0.05) IVGP of wood chips after 8 weeks. Eight weeks of G.

lucidum treatment of miscanthus resulted in a maximum in IVGP (Figure 3.2). Twelve weeks of L. edodes treatment resulted in an increase in IVGP of 58.5 ml/g OM (23.1%) for wheat straw (P < 0.05), 80.5 ml/g OM (43.7%) for miscanthus (P = 0.0196) and 123.4 ml/g OM (228.8%) for wood chips (P < 0.0001) compared to untreated, autoclaved material (Figure 3.2). Furthermore, 12 weeks of treatment of wood chips with P. eryngii increased (P < 0.0001) IVGP by 76.1 g/ml OM (141.1% increase) (Figure 3.2). The IVGP of rice straw was not improved by fungal treatment (Figure 3.2).

3.3.3 Chemical composition

L. edodes treatment resulted in a significant improvement in IVGP of wheat straw and even in the greatest improvement in IVGP of miscanthus and wood chips as shown in Figure 3.2. Therefore, chemical composition of L. edodes treated material was measured

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after 2, 4, 8 and 12 weeks (Table 3.2), while for the other fungi chemical composition was measured only after 12 weeks (Table 3.1). Furthermore, as no improvement in IVGP of rice straw compared to the untreated autoclaved control was observed, no chemical analyses of the material were done. L. edodes caused a decrease (P < 0.05) in hemicellulose and ADL concentration of wheat straw from 2 weeks on. Cellulose concentration was increased (P < 0.05) in miscanthus from 2 weeks on (Table 3.2).

Treatment of wheat straw with L. edodes for 8 weeks or longer resulted in an increased cellulose concentration (Table 3.2). L. edodes did decrease (P < 0.0001) ADL concentration of not only wheat straw, but also of wood chips and miscanthus during 8 weeks of treatment (Table 3.2). However, the absolute amount of ADL in miscanthus started decreasing from 2 weeks on, as did the absolute amount of hemicellulose (P <

0.01) (Table 3.2). After 8 weeks of treatment, cellulose in miscanthus started to be degraded (P < 0.01) by L. edodes (Table 3.2). Although all fungi decreased the ADL and hemicellulose concentration of wheat straw and did not decrease the cellulose concentration, the IVGP of wheat straw was only increased after 12 weeks of treatment with L. edodes. This fungus caused a higher loss of ADL (65.6 g/kg DM) in wheat straw compared to the other fungi, but also a high loss of hemicellulose of 172.9 g/kg DM (Table 3.1). L. edodes also caused the highest loss (164.4 g/kg DM) of hemicellulose in miscanthus. Besides L. edodes, G. lucidum degraded (P < 0.0001) ADL in miscanthus, and P. eryngii (P = 0.0072) and P. ostreatus (P = 0.0017) caused a decreased ADL concentration in wood chips after 12 weeks treatment (Table 3.2). L. edodes decreased (P < 0.0001) the hemicellulose concentration most in miscanthus and was the only fungus which degraded (P < 0.05) hemicellulose at the same rate compared to other components in wood chips (Table 3.1). After 12 weeks of colonization, all fungi increased the cellulose concentration in wheat straw and miscanthus (P < 0.05) (Table 3.1). It is important to note that cellulose in miscanthus was degraded by all fungi except P. eryngii varying from 3 up to7.5 g (which corresponds to 7 up to 20%) of the absolute amount of cellulose present in the original substrate. The absolute amount of cellulose in wheat straw, decreased only after treatment with P. ostreatus (17.2 g) for 12 weeks (Table 3.1). The latter is in line with the highest DM loss in wheat straw caused by this fungus. The fact that G. lucidum degraded cellulose after 12 weeks treatment of miscanthus suggests that in the current experiment, 12 weeks of G. lucidum treatment of miscanthus is too long to result in maximum rumen fermentation. None of the fungi degraded cellulose of wood chips significantly, both when expressed as concentration or absolute amounts, whereas the absolute amount of ADL was decreased (P < 0.05) by L.

edodes and P. ostreatus treatment for 12 weeks (Table 3.2). Throughout the fungal treatment, substrates are enriched in terms of ash and crude protein concentration (Tables 3.1 and 3.2).

(%)

Substrate Treatment HC Cell ADL Ash CP HC Cell ADL Ash CP

Wheat straw

Control 260.1^a 479.6^a 81.1^a 24.1^a 26.6^a 24.2^a 44.6^a 7.5^a 2.2 2.5 0^a

G. lucidum 185.2^b 525.8^bc 56.7^b 27.7^ab 31.3^a 14.4^b 40.9^a 4.4^b 2.2 2.4 16^b

L. edodes 87.2^c 544.2^b 15.5^c 36.4^c 42.6^b 5.7^c 35.6^a 1.0^c 2.4 2.8 30^b

P. eryngii 171.3^bd 507.4^c 43.9^d 31.2^b 34.7^c 13.7^b 40.6^a 3.5^d 2.5 2.8 14^ab

P. ostreatus 165.5^d 477.6^d 46.7^d 44.9^d 51.8^d 8.3^c 24.0^b 2.4^e 2.3 2.6 46^c

SEM 6.97 7.29 0.85 0.91 0.61 0.85 1.71 0.13 0.12 0.10 0.03

P-value <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.41 0.09 <0.01

Miscanthus

Control 285.2^a 471.2^a 101.4^a 9.4^a 19.1^a 22.5^a 37.2^a 8.0^a 0.7^a 1.5 0^a

G. lucidum 194.2^b 524.0^b 72.3^b 12.3^b 22.0^b 12.7^b 34.2^b 4.7^b 0.8^b 1.4 17^b

L. edodes 120.8^c 582.3^c 36.8^c 14.4^c 27.4^c 6.2^c 29.7^c 1.9^c 0.7^ab 1.4 35^c

P. eryngii 253.4^d 508.3^b 96.2^a 10.4^a 19.1^a 19.5^d 39.2^d 7.4^a 0.8^b 1.5 3^a

P. ostreatus 239.2^d 516.9^b 96.3^a 13.0^b 23.1^b 14.6^b 31.6^c 5.9^d 0.8^b 1.4 23^d

SEM 5.28 2.81 1.31 0.24 0.47 0.47 0.41 0.12 0.01 0.03 0.02

P-value <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.08 <0.01

Wood chips

Control 198.2^a 453.0 198.2^a 96.6^a 29.8^a 11.0^ab 35.3 15.4^a 7.5 2.3 0^a

G. lucidum 151.3^ab 448.4 161.6^ab 96.8^ab 29.5^ab 11.3^ab 33.7 12.2^ab 7.3 2.2 4^ab

L. edodes 105.6^c 442.9 107.0^c 131.3^b 40.1^c 6.8^b 28.5 6.9^c 8.5 2.6 17^c

P. eryngii 164.6^abd 465.0 146.9^b 103.6^ab 33.2^abcd 15.0^a 41.9 13.1^ab 9.7 3.0 3^ab

P. ostreatus 143.0^abcd 421.3 135.2^bc 128.3^ab 40.9^cd 8.9^ab 26.2 8.4^bc 7.8 2.5 21^c

SEM 9.06 11.19 7.96 11.27 1.87 1.58 3.37 1.04 1.44 0.22 0.03

P-value <0.01 0.26 <0.01 0.04 <0.01 0.02 0.06 <0.01 0.49 0.19 <0.01

Values with different superscripts within column are significantly (P<0.05) different. ADL, acid detergent lignin; HC, hemicellulose; Cell, cellulose; CP, crude protein; DM loss, dry matter loss.

Substrate

Duration of treatment (weeks)

DM Loss (%)

HC Cell ADL Ash CP HC Cell ADL Ash CP

Wheat straw

Control 260.1^a 479.6^bc 81.1^a 24.1^c 26.6^d 24.2^a 44.6^a 7.5^a 2.2 2.5 0^c

2 202.7^b 461.2^c 74.6^b 28.6^b 30.8^c 17.4^b 39.5^ab 6.4^b 2.5 2.6 8^c

4 148.1^c 496.3^b 57.0^c 30.1^b 32.9^c 12.2^c 41.0^ab 4.7^c 2.5 2.7 11^bc

8 94.6^d 537.3^a 33.2^d 34.4^a 37.5^b 6.9^d 39.1^ab 2.4^d 2.5 2.7 22^ab

12 87.2^d 544.2^a 15.5^e 36.4^a 42.6^a 5.7^d 35.6^b 1.0^e 2.4 2.8 30^a

SEM 6.63 6.81 1.28 0.61 0.53 0.70 1.85 0.17 0.09 0.11 0.03

P-value <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.07 <0.01 0.24 0.39 <0.01

Miscanthus

Control 285.2^a 464.7^d 101.4^b 9.4^c 19.1^c 22.5^a 37.2^a 8.0^a 0.7^b 1.5 0^c

2 241.3^b 442.4^e 139.4^a 9.9^c 19.8^c 18.7^b 34.3^ab 10.8^b 0.8^a 1.5 2^c

4 185.6^c 501.1^c 97.5^b 10.6^c 21.3^c 12.6^c 34.1^ab 6.6^c 0.7^b 1.4 14^bc

8 149.1^d 549.7^b 73.6^c 12.1^b 24.6^b 8.8^d 32.2^bc 4.3^d 0.7^b 1.4 26^ab

12 120.8^e 575.8^a 36.8^d 14.4^a 27.4^a 6.2^e 29.7^c 1.9^e 0.7^ab 1.4 35^a

SEM 4.76 3.43 1.87 0.29 0.48 0.50 0.75 0.17 0.01 0.03 0.03

P-value <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.05 <0.01

Wood chips

Control 140.8^a 445.5^c 198.2^a 96.6 29.8^c 11.0^a 35.3 15.4^a 7.5 2.3 0

2 96.3^ab 486.2^b 187.3^ab 105.5 30.3^bc 7.8^ab 39.2 15.1^a 8.5 2.4 0

4 103.6^ab 497.4^ab 163.8^b 86.6 32.8^bc 6.8^ab 31.1 10.1^b 5.4 2.1 20

8 79.9^b 520.6^a 126.6^c 104.8 35.4^ab 5.6^b 36.1 8.8^b 7.2 2.5 11

12 105.6^ab 436.5^c 107.0^c 131.3 40.1^a 6.8^ab 28.5 6.9^b 8.5 2.6 17

SEM 9.80 6.69 5.48 10.33 1.14 1.04 2.61 0.83 0.80 0.18 0.07

P-value 0.02 <0.01 <0.01 0.10 <0.01 0.04 0.10 <0.01 0.11 0.44 0.16

Values with different superscripts within column are significantly (P<0.05) different. ADL, acid detergent lignin; HC, hemicellulose; Cell, cellulose; CP, crude protein;

DM loss, dry matter loss.

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