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Effects of Different Additives and Lignin Peroxidase Enzyme on in vitro Gas Production Kinetics and Methane Production of some Straws




Effects of Different Additives and Lignin Peroxidase Enzyme on in vitro Gas Production Kinetics and Methane Production of some Straws

Unal Kilic1*, Abdiwali Mohamoud Abdi1 and Deniz Ekinci2

1Department of Animal Science, Ondokuz Mayis University, Turkey

2Department of Agricultural Biotechnology, Ondokuz Mayis University, Turkey


This study was aimed to determine the effects of different additives and lignin peroxidase (LiP) enzyme on the in vitro gas production and methane production of some straws. The study involved 8 groups for each forage, 3 different straws (wheat straw, soybean straw, sorghum straw), which comprise of 2 different LiP enzyme treatments (Available –Not Available) and 4 different treatment groups (control, 4% urea, 10% molasses and 14% urea+molasses (4% urea, 10% molasses). Hohenheim gas test was used in in vitro gas production measurements while infrared methane analyzer was used to determine methane production. The experiments were conducted as per the randomized factorial design in randomized complete blocks. In terms of crude protein content, the highest CP was obtained from the urea+molasses treatment for wheat, sorghum and soybean straws, while control groups were found to have the lowest CP content. The lowest NDF, ADF and lignin contents were found in sorghum straws (P<0.001). The highest gas production value was obtained from sorghum straws for 24-hours incubation process (P<0.001). Treatments did not cause any effects on 24-hours gas production for straws without enzyme addition (P>0.05). LiP enzyme was found to increase in vitro gas production and methane production for all straws and treatments (P<0.001). As a result, sorghum straws were proven to offer a higher feed value and additional in vivo and in vitro studies are required with the aim of determining the possibilities of using LiP enzyme in forages.

Article Information

Received 14 March 2017

Revised 11 May 2018

Accepted 10 April 2019

Available online 13 August 2019

Authors’ Contribution

UK contributed in all experiment design, in vitro analysis of samples, acquisition of data, statistically evaluated the data and drafting of the article. AMA collected the data, did chemical analysis of samples and statistically evaluated the data. DE performed experiments and revised the article.

Key words

In vitro gas production, Lignin peroxidase, Methane, Straw


* Corresponding author:

0030-9923/2019/0006-2057 $ 9.00/0

Copyright 2019 Zoological Society of Pakistan


The most common issue in ruminant nutrition is the insufficiency of the forages available in winter months. As the fodder crops are not produced in sufficient amounts, manufacturers commonly have to consider straws. As straws offer low protein, energy and mineral contents, while offering higher cellulose content, their nutritive values are relatively lower (Kalkan and Filya, 2011). Commonly preferred for their cost-efficiency, straws are subjected to physical (chopping, grounding, boiling, etc.), chemical (urea and some alkali treatments) and biological methods in order to increase their nutritive values. Furthermore, it is also known that it is possible to increase the forage quality of straws using a number of additives. Scientific research has recently took an interest in biological methods (treatment with enzymes such as (cellulase, hemicellulase, pectinase and xylanase, etc.) and it is aimed to increase the nutritive value of straws with enzyme addition (Rodrigues et al., 2001, 2008; Hossain and Anantharaman, 2008; Kalkan and Filya, 2011; Pinto et al., 2012; Selcuk et al., 2016).

Straws offer a low digestibility with their high cellulose content and lignin content at an average between 10% and 15% (Sarnklong et al., 2010). It is reported that there is a direct relationship between digestibility of straw and the digestion of their lignin content (Moyson and Verachtert, 1991) and that forage digestibility is degraded in ruminants fed with poor quality forages as rumen does not have an enzyme to digest lignin. Crosby-Galváne et al. (2018) reported that residues of chihua pumpkin improved dry matter digestibility and reduced gas production in the in vitro study. In this context, use of fungi is common in order to break down the compounds including lignin (Arora et al., 2002; Hossain and Anantharaman, 2008; Wulandari et al., 2013). However, it was reported that the extended period required for the production of enzymes by fungi may lead to losses in nutrition (Ramos et al., 2004). Therefore, research suggests that the direct use of LiP enzyme is more practical and that LiP enzyme breaks down non-phenolic compounds including lignin (Wan and Li, 2011). Wulandari et al. (2013) reported that addition of ligninase enzyme in rice straw decreases the cellulose content and that 66.3% of lignin is broken down. However, literature review showed that the studies using LiP enzyme were only interested in determining the enzyme activity (Khazaal et al., 1990; Arora et al., 2002) and no studies were found in which enzyme is added to the rumen fluid directly. Kalkan and Filya (2011) reported that the direct addition of cellulase enzyme into forages improves the nutritive value, however, they have recommended further research on the effects of enzymes in the rumen environment. Ruminants produce approximately 16% of the methane available in the world. Even larger amounts of methane is produced especially when poor quality forages such as straws are consumed by ruminants. Therefore, consumption of straws without additives also have significant problems with ecological relevance.

This study aims to determine the effects of different additives (urea, molasses and urea+molasses) on the nutritive content, in vitro gas production, methane production, energy contents and organic matter digestibility of straws. Moreover, it was aimed to define the additives which will offer the best rumen conditions for straws with respect to ecology. it is known that forages have different lignocellulosic structures and that soybean straw consists of two different types of lignin (guaiacyl and syringyl) (Xu et al., 2007). Another purpose of this study is to determine the effects of LiP enzyme on the in vitro gas production and methane production of legume (soybean) and graminaes (wheat and sorghum) straws. The hypothesis of this study is LiP enzyme reduces the enteric methane production as it offers the properties suitable for the breaking down of peroxidases forming in rumen, in other words, it uses the hydrogens available in the rumen.



Establishment of treatment groups and ensiling straws

In this study, three different straws (wheat straw, soybean straw and sorghum straw) with 4 different treatment groups (Control, urea (4%), molasses (10%) and urea + molasses (14%)) were used. Total of 12 treatment groups were established with 4 groups for each straw. The straws were ensiled into PVC laboratory silos (2.5-L capacity, 5 cm radius × 30.0 cm height) using pressing tool. A total of 45 laboratory silos (Three treatments × three straws × five replicate for each treatments) were made and stored at ambient temperature (27-34 °C).The straws included in the control group (100% straw) were silaged and preserved under laboratory conditions until other silos were opened.

As water soluble carbohydrate sources prevent lignin peroxidase (Sigma EC# = LiP) activity (Khazaal et al., 1993), no enzymes were added to the straw silage during the fermentation process. Moreover, Rehman et al. (2014) reported no significant differences for the straw silages with enzyme addition. Therefore, LiP enzyme was added in the process of in vitro gas production determination. In in vitro gas production tecnique, 2 different LiP enzyme applications (with or without) were used in the study and total of 24 treatment groups were established with 8 groups for each straw. The urea and molasses used in this study was defined in accordance with the literature review conducted (Sarwar et al., 2011; Rehman et al., 2014; Polyorach and Wanapat, 2015) . The percentages used in this study are: straw, 70%; urea 4%, molasses 10%; urea + molasses 14% and water for the remaining part.

Chemical analyses

All the silages were dried in a forced air oven at 55 °C for 72 h. Then, samples were milled in a hammer mill through a 1 mm sieve for determination of chemical compositions and IVGP’s assays. The samples were analyzed for dry matter (DM), ash and crude protein (CP) contents were analysed according to AOAC (1998). CP was calculated by multiplying N by 6.25. The crude fiber (CF), acid detergent fiber (ADF), neutral detergent fiber (NDF) and acid detergent lignin (ADL) analysis were done according to the method of Van Soest et al. (1991) using AnkomA2000 automated fiber analyser. The ether extract (EE) content was determined with Ankom XT15 analyzer (AOCS, 2005). The contents of cellulose (Cel=ADF-ADL), hemicellulose (HCel = NDF-ADF), organic matters (OM = DM-ash)) and nitrogen free extract (NFE=DM-(CP+ash+EE+CF)) were determined by calculation.

In order to identify the optimum LiP enzyme dosage, LiP enzyme was tried in different buffers (pH=3.5 and pH=6.0), in different dosages (0, 10µl, 100µl and 1000µ), and using hydrogen peroxide (H2O2) and veratryl alcohol with wheat straw and the treatments were cultured for 48 h. Then the LiP enzyme activity was read with a spectrophotometer at 310nm. One enzyme unit was defined as the amount of enzyme necessary to induce 1µmol veratryl alcohol into veratraldehite in 1 min. According to the results of preliminary study in which the remaining amount of ADL was investigated, it was found that buffer with a pH of 3.5 performs better than the buffer with a pH of 6.0 with respect to the LiP enzyme. Moreover, in spite of the positive outcomes observed at some pH=3.5 levels, it was decided to consider the pH level similar to the rumen’s (5.5-7.0), therefore, as also reported by Khazaal et al. (1990), it was decided that the use of 1 enzyme unit, i.e. 100µl (0.1 unit), at a pH of 6.0 and a treatment without the use of veratryl alcohol and hydrogene peroxide would be suitable.

Determining in vitro gas production of samples (Hohenheim gas test)

Approximately 200 mg dry weight of samples were weighed into 100 ml calibrated glass syringes following procedures of Menke and Steingass (1988). Rumen fluid used in in vitro gas technique collected from three Brown Swiss x Native Cattle bulls (Average 24-30 months age and 400-500 kg Live Weight) slaughtered at a slaughterhouse. The bulls fed twice daily with a diet containing alfalfa hay (55%) and barley grain (45%). Rumen fluid was brought to the laboratory within 15-20 min in thermos (38-40°C). Then, rumen fluid mixed and it was taken under CO2 atmosphere and were filtered through two layers of cheesecloth. The syringes were warmed at 39ºC before the injection of 30 ml rumen fluid-buffer mixture (1:2) into each syringe and incubated in a water bath at 39ºC. Gas volumes were recorded at 0, 3, 6, 9, 12, 24, 48, 72 and 96 h of incubation. Five repetitions of each sample were used in in vitro gas production experiment. 0.1 unit LiP enzyme was added to each 1 gram of the rumen fluid-buffer mix used in in vitro gas production technique, while control groups were introduced only with the rumen fluid-buffer mix without enzyme addition.

Net gas productions of samples were determined at 24 h after incubation and corrected for hay standard of University of Hohenheim and blank (without enzyme and with enzyme). Cumulative gas production data were fitted to the model of Ørskov and McDonald (1979) by the NEWAY computer package programme: y = a + b (1-exp-ct) where: a, gas production from the immediately soluble fraction (ml), b, gas production from the insoluble fraction (ml), a + b, potential gas production (ml), c, gas production rate constant for the insoluble fraction (ml/h), t, incubation time (h), y, gas produced at time t. Organic matter digestibility, ME and NEL contents of samples were estimated using equations given below:

OMD, % = 14.88+ 0.8893 GP + 0.651 ash + 0.448 CP (Menke et al. 1979)

ME, MJ/kg DM = 2.20+0.136GP + 0.002859 EE2 + 0.057CP (Menke et al. 1979)

NEL, MJ/kg DM = 0.101GP + 0.11 EE + 0.051CP (Menke and Steingass, 1988)

Where; GP: 24 h gas production (ml/200mg DM), EE: Ether extract (%), CP: Crude protein (%)

Determination of methane production of samples

Methane contents (%) of total gas produced at 24 h fermentation of samples were measured using a methane analyzer (Sensor Europe GmbH, Germany) according to Goel et al. (2008). After measuring gas produced at 24 h incubation, 25-40 mL gas samples was transferred into inlet of the methane analyzer. Methane production (mL) was calculated as follows:

Methane production (mL) = The percent of methane (%) X Total gas production (mL)

Determination of rumen fluid pH, volatile fatty acids and ammonia-N contents

The pH value of rumen fluid was determined using Hanna 1332 digital pH meter with 3 replications. The volatile fatty acids analysis of rumen fluid were done using a gas chromatography (Agilent Technologies 6890N gas chromatography, Cat. 11023, Stabilwax-DA, 30 m, 0.25 mm ID, 0.25 um df. Maximum temperature: 260°C.) according to Wiedmeier et al. (1987). Rumen fluid amonnia-N analysis were done using Kjeldahl methods according to Blümmel et al. (1997) in 3 replicates.

Statistical analysis

One Sample Kolmogorov Smirnov and normality hypothesis tests were used in order to test the compliance of the data for variance analysis and it was found that the data had a normal distribution (P>0.05). Levene Homogeneity of Variances test was used to test the homogeneity of the variances and it was found that the variances were homogeneous (P>0.05). The data obtained from the study were analysed with randomized factorial design. All analyses of the data were performed by using the SPSS Program (Windows Version of SPSS, release 20.0). Duncan’s multiple range test was used for the comparison of mean values.



Nutrient compositions of the straws tested in the experiment are shown in Table I. According to the Table I, among all three straws the highest DM content was found in the control groups. With respect to the crude protein content, the highest CP was obtained from the urea+molasses treatment for wheat, sorghum and soybean straws (P<0.001). It was found that control groups offer the lowest CP content (P<0.001) and the treatment increases the CP content of the straws (P<0.001).

The highest NFE content was found in control group and the group treated with molasses (P<0.001). However, urea and urea+molasses addition decreased the NFE content of the straws (P<0.001). The lowest NDF, ADF and lignin contents were found in sorghum straws in this study (P<0.001). The treatments involving molasses as additive were found to have lower NDF and ADF contents. The highest NDF content was found in control group and urea treatment of wheat straw (P<0.001). A decrease trend in NDF content was identified for the groups treated with molasses. When compared to the control group of soybean straw, the group with urea+molasses addition was found to have a lower NDF value (P<0.001). The lowest ADF content was found in the urea+molasses treatment (P<0.001). There was no significant effect of


Table I.- Nutrient compositions of the straws used in the study (as DM%).














69.64 ± 0.91c

89.01 ± 0.69bcd

7.23 ±


1.35 ± 0.2c

42.67 ± 0.31d

10.99 ±0.69bcd

37.76 ± 1.19d

76.78 ± 0.61a

6.09 ± 0.21c

27.34 ±0.32bc

43.34 ± 0.72a


75.32 ± 1.04b

87.7 ± 0.85de

4.27 ±


1.11 ± 0.23c

38.34 ± 0.8e

12.3 ± 0.85ab

43.98 ± 0.34b

71.31 ± 0.63b

5.63 ± 0.34c

26.71 ±0.17bcd

38.97 ± 0.86c


76.55 ± 0.54b

87.92 ± 0.45cde

10.21 ± 0.5c

1.32 ± 0.09c

37.71 ± 0.36e

12.08 ±0.45abc

38.68 ± 0.45d

69.89 ± 1.02b

5.48 ± 0.28c

26.2 ± 1.25bcd

38.22 ± 0.31c


91.81 ± 0.1a

90.2 ± 0.17ab

2.93 ±


1.31 ± 0.11c

41.9 ± 1.36d

9.8 ± 0.17de

44.06 ± 1.55b

78.89 ± 1.1a

6 ±


31.36 ± 0.82a

41.53 ± 0.53ab


66.22 ± 0.18d

91.14 ± 0.1a

10.57 ± 0.05c

1.24 ± 0.19c

34.44 ± 0.16f

8.86 ± 0.1e

44.89 ± 0.11b

63.16 ± 0.22c

3.11 ± 0.91d

25.67 ±0.45cd

34.38 ± 0.98d


75.99 ± 0.1b

90.5 ± 0.03ab

5.29 ±


3.02 ± 0.21a

31.49 ± 0.8g

9.5 ± 0.03de

50.71 ± 0.72a

59.07 ± 0.22d

1.96 ± 0.11d

24.91 ± 0.23de

32.2 ± 0.23d


74.29 ±


91.11 ± 0.05a

17.62 ± 0.05a

1.6 ± 0.13bc

30.67 ± 0.65g

8.89 ± 0.05e

41.22 ± 0.65c

54.89 ± 0.42e

1.82 ± 0.22d

23.41 ± 0.26e

29.66 ± 0.66e


90.09 ±


91.33 ± 0.03a

3.28 ± 0.09hi

2.26 ± 0.37ab

34.68 ± 0.94f

8.67 ± 0.03e

51.12 ± 1.24a

64.7 ± 0.22c

2.74 ± 0.47d

27.71 ± 0.29b

34.24 ± 0.04d


69.2 ±


87.47 ± 0.43e

9.32 ±


1.6 ± 0.21bc

50.2 ± 0.41b

12.53 ± 0.43a

26.36 ± 0.46f

69.2 ± 1.24b

13.15 ± 0.37a

13.85 ± 0.35fg

42.2 ± 1.27a


74.5 ±


89.36 ± 0.3bc

4.66 ±


0.91 ± 0.16c

50.12 ± 0.25b

10.64 ± 0.3cd

33.66 ± 0.59e

69.21 ± 0.56b

12.89 ± 0.31ab

14.29 ± 0.66fg

42.03 ± 0.27a


75.5 ±


86.83 ± 0.46e

12.61 ± 0.26b

1.53 ± 0.43bc

45.36 ± 0.21c

13.17 ± 0.46a

27.34 ± 1.09f

63.81 ± 0.58c

11.52 ± 0.08b

12.68 ± 0.4g

39.61 ± 0.29bc


90.12 ±


90.42 ± 0.26ab

3.64 ±


1.41 ± 0.77bc

53.1 ± 0.43a

9.58 ± 0.26de

32.27 ± 0.08e

71.46 ± 0.28b

12.64 ± 0.3ab

14.98 ± 0.23f

43.84 ± 0.81a













WSU, wheat straw urea; WSM, wheat straw molasses; WSU+M, wheat straw urea+molasses; WSCont, wheat straw control; SSU,sorghum straw urea; SSM, sorghum straw molasses; SSU+M, sorghum straw urea+molasses; SSCont, sorghum straw control; SySU, soybean straw urea; SySM, soybean straw molasses; SySU+M, soybean straw urea+molasses; SySCont, soybean straw control. P<0.001;a,b…, Means with different supercripts in the same column are significantly different.


urea and molasses additions to soybean straw separately on ADF content. Sorghum straw gave the lowest lignin (ADL) content among others while wheat straws followed (P<0.001). Nevertheless, there was no effect of treatments on the lignin contents of sorghum and wheat straw (P>0.05). However, it was shown that among the soybean straws, the lignin (ADL) content of urea+molasses treatment was lower than mere urea treatment (P<0.001).

The values obtained from the rumen fluid used in the in vitro gas production (IVGP) technique are as follows: pH, 5.35 (5.30-5.39); total volatile fatty acids content, 96.35±0.54 mmol/L ; acetic acid, 50.60±0.71 mmol/L; propionic acid, 23.69±0.21 mmol/L; butyric acid, 17.75±0.54 mmol/L; isobutyric acid, 2.12±0.25 mmol/L; valeric acid, 1.19±0.07 mmol/L; and isovaleric acid content, 1.01±0.02 mmol/L. Amonnia nitrogen content of the rumen fluid was found to be 28.85±1.05 mg/100 ml (between 221.7 and 371.0 mg/l).

Table II shows the in vitro gas production of the straws with and without LiP enzyme According to the table, it was found that LiP enzyme addition increases in vitro gas production upon each and every treatment of the straws incubated for 3 h up to 96 h. The highest gas production value was obtained from sorghum straws for 24-h incubation process (P<0.001). Wheat straw control group gave the lowest IVGP value among the straws without enzyme treatment (P<0.001). Treatments were not found to have any effects on 24-h gas production for wheat straws, sorghum straws, and soybean straws without enzyme addition (P>0.05).

Table III shows the in vitro gas production parameters, OMD, ME, NEL and methane productions of straws. LiP enzyme addition increased methane production significantly for the straws (P<0.001).


Table II.- In vitro gas production of the straws and pH values for 96-hours incubation.


3 h

6 h

9 h

12 h

24 h

48 h

72 h

96 h

96.h pH



4.16± 0.70fgh



6.08± 0.95efg



22.02± 3.74cd

32.02± 1.79def

34.95± 1.09cd

36.38± 1.26def

6.41± 0.03bc


18.64± 0.78de

21.02± 0.63cd

27.58± 0.91bcd

33.85± 0.9bc

57.86± 1.35ab

71.12± 1.35abc

75.16± 2.06ab

75.3± 3.02abc

6.04± 0.01jk



2.02± 0.21gh

2.35± 0.39efg

4.26± 0.58efg



15.04± 0.82cd

23.55± 2.07def

26.67± 3.09df

29.37± 2.73d-g

6.42± 0.06bc


21.28± 0.52b-e

23.53± 1.03a-d

28.62± 1.83a-d

34.31± 2.22bc

56.8± 1.58ab

70.44± 1.91abc

74.63± 2.1ab

76.13± 2.07abc

6.05± 0.02jk



1.58± 0.56gh

1.35± 0.72fg

2.47± 1.05fg

4.73± 1.24de

13.86± 1.3cd

23.11± 2.03def

25.02± 2.02df

26.61± 1.9fg

6.39± 0.01cd


20.73± 0.51cde

23.66± 2.1a-d

30.87± 3.02a-d

32.47± 8.66bc

55.01± 11.88ab

71.01± 13.95abc

76.19± 15.99ab

76.42± 15.32abc

6.27± 0.02fg



0.45± 0.21h



1.12± 0.37g



9.98± 0.78d

20.62± 0.84f



29.02± 0.76efg

6.30± 0.01ef


16.58± 2.48e

17.78± 3.54d

22.71± 4.57d

31.67± 5.51c

54.37± 5.17ab

65.73± 9.12bc

69.02± 10.72b

70.96± 10.39bc

6.14± 0.04hi



2.93± 0.62fgh

3.49± 0.58efg

5.29± 0.62efg

8.89± 0.55de

16.55± 0.48cd

27.24± 0.45def

32.76± 1.37cde

33.43± 0.34d-g

6.33± 0.01def


22.75± 3.53a-d

24.85± 4.6abc

31.44± 6.03a-d

38.17± 7.34abc

61.83± 8.78ab

80.69± 9.32ab

85.63± 9.57a

87.28± 9.77ab

6.11± 0.02ijk



7.17± 0.62f



12.37± 1.04e

15.95± 1.14d

25.55± 1.33c

37.34± 1.69de

42.66± 2.01c

45.55± 2.04de

6.32± 0.01def


27.64± 5.17a



36.09± 8.9ab

46.06± 12.18a

61.89± 13.46ab

78.01± 15.29abc

80.77± 14.62ab

82.78± 17.21abc

6.03± 0.01k



2.97± 0.22fgh

3.54± 0.32efg

5.02± 0.39efg

7.65± 0.49de

14.74± 0.76cd

24.67± 1.12def

30.27± 1.27cde

33.47± 1.45defg

6.38± 0.01cde


26.17± 1.72ab

28.9± 2.18ab

36.41± 2.9ab

42.78± 3.39abc

65.31± 5.09a

82.61± 6.54a

88.29± 7.24a

89.89± 7.03a





6.21± 1.35fg

8.09± 1.83ef

11.31± 2.2ef

14.53± 2.35d

24.51± 3.55c

37.71± 5.22d

43.16± 5.12c

46.27± 5.06d

6.31± 0.02def


25.67± 3.00abc

28.03± 3.74ab

34.38± 4.52abc

40.57± 5.58abc

64.82± 4.09ab

82.67± 5.4a

86.08± 5.29a

89.46± 5.25a





3.10± 0.54fgh

4.23± 0.98efg

5.9± 1.43efg

9.6± 1.88de

15.36± 2.02cd



18.79± 1.26df

19.46± 1.46fg




19.16± 1.20de

22.3± 1.62bcd

26.49± 3.33cd

32.18± 3.23bc

51.64± 3.52b

63.62± 5.19c

67.51± 5.32b

67.96± 5.48c

6.29± 0.02f



3.46± 0.42fgh

5.14± 0.48efg

8.49± 0.57efg

10.95± 0.62de

15.76± 0.51cd

17.32± 0.67f

17.77± 0.66f

18.56± 0.59g

6.51± 0.02a


25.51± 1.00abc

29.99± 1.25a

36.85± 1.27a

41.93± 1.07abc

59.53± 2.27ab

72.52± 3.4abc

76.69± 3.74ab

77.28± 4.08abc

6.21± 0.01gh



3.97± 0.30fgh

6.13± 0.58efg

8.86± 0.79efg

13.73± 1.04de

17.59± 1.3cd

21.78± 1.2ef

22.81± 1.13df

23.49± 1.02fg

6.53± 0.01a


21.49± 0.34bcd

23.98± 0.33a-d

26.69± 0.32cd

33.25± 0.06bc

55.4± 3.56ab

65.59± 2.38bc

68.53± 2.14b

68.98± 2.14c

6.12± 0.01ij



3.40± 0.50fgh

5.09± 0.7efg

7.7± 0.53efg

10.76± 0.77de

17.1± 0.77cd

20.61± 0.97f

21.18± 1.47df

21.86± 1.88fg

6.48± 0.04ab


23.27± 0.78a-d

28.34± 1.93ab

36.25± 2.89ab

43.55± 2.85ab

63.83± 3.35ab

77.56± 3.74abc

82.18± 4ab

83.22± 4abc

6.21± 0.03g











For abbreviations, see Table I.


Table III.- In vitro gas production parameters, OMD, ME, NEL and methane productions of straws.


LİP Enzyme

c, ml/h

a+b, ml

OMD, %



Methane, ml




39.83± 1.68cd

44.86± 3.33c

5.61± 0.51c

2.74± 0.38c

3.59± 0.53c


0.047± 0.003de

77.61± 2.85ab

76.73± 1.2ab

10.49± 0.18ab

6.36± 0.14b

9.92± 0.14ab



0.028± 0.005ghı

31.95± 3.68def


4.49± 0.11cd

1.86± 0.08cd

2.45± 0.14c


0.043± 0.003def

78.22± 2.09ab

75.31± 1.4ab

10.17± 0.21b

6.08± 0.16b

9.13± 0.48ab



0.030± 0.001f-ı

29.14± 2.06def


4.67± 0.18cd

2.07± 0.13cd

2.00± 0.29c


0.035± 0.005e-h

79.7± 15.1ab


10.27± 1.62b

6.22± 1.20b

9.29± 2.86ab



0.02± 0.001ı


31.44± 0.69d

3.73± 0.1d

1.30± 0.08d

1.09± 0.18c


0.047± 0.007de


70.92± 4.6b

9.76± 0.7b

5.78± 0.52b

8.79± 2.22ab



0.030± 0.001f-ı


40.1± 0.43cd

5.06± 0.07cd

2.35± 0.05cd

2.22± 0.10c



90.43± 9.15a

80.37± 7.81ab

11.22± 1.19ab

6.92± 0.89ab

10.57± 2.29ab



0.030± 0.001f-ı

48.65± 2.23c

46.15± 1.19c

6.0± 0.18c

3.18± 0.13c

4.03± 0.22c


0.043± 0.003def

84.16± 15.7ab

78.47± 11.97ab

10.94± 1.83ab

6.85± 1.36ab

11.07± 3.28ab



0.020± 0.001ı

39.97± 1.73cd

41.67± 0.67cd

5.22± 0.1cd

2.56± 0.08cd

2.39± 0.17c



92.34± 7.31a

86.64± 4.52a

12.1± 0.7a

7.67± 0.51a

10.43± 2.44ab



0.025± 0.003

50.42± 5.03c

43.79± 3.15c

5.74± 0.48c

2.89± 0.36c

4.02± 1.04c


0.04± 0.001d-g

91.63± 5.14a

79.64± 3.64ab

11.22± 0.56ab

6.96± 0.41ab

11.87± 1.46a



0.063± 0.015b

19.75± 1.5f

40.87± 1.8cd

4.83± 0.28cd

2.20± 0.20cd

2.65± 0.50c


0.047± 0.003de

69.87± 5.46b

73.14± 3.14b

9.76± 0.48b

5.87± 0.36b

7.9± 1.26b



0.08± 0.001a

18.17± 0.62f

37.91± 0.45cd

4.61± 0.07cd

1.93± 0.05cd

2.64± 0.31c


0.047± 0.003de

78.68± 4.25ab

76.84± 2.02ab

10.56± 0.31ab

6.35± 0.23b

10.38± 0.71ab



0.063± 0.003bc

23.22± 1.14ef

44.74± 1.16c

5.32± 0.18cd

2.59± 0.13cd

3.07± 0.25c


0.045± 0.005de

70.95± 2.05b

78.37± 3.17ab

10.46± 0.48ab

6.41± 0.36ab

10.08± 0.82ab



0.063± 0.006bc

21.98± 1.61ef

37.95± 0.68cd

4.74± 0.10cd

2.07± 0.08cd

2.59± 0.16c


0.05± 0.001cd

84.24± 3.78ab

79.52± 2.98ab

11.09± 0.46ab

6.79± 0.34ab

11.99± 0.64a








For abbreviations, see Table I.


While no statistically significant differences were found for the straws without enzyme addition with respect to the methane production, urea addition to soybean straw with enzyme addition proved to have a lower methane production level (P<0.001) and the treatments did not have significant effects on the other straws with enzyme addition (P>0.05). It was observed that LiP enzyme addition to straws increases the total gas production (a+b value), OMD, ME and NEL content at a significant level for all straws and all treatments (P<0.001).



Chemical compositions of straws

The reason behind the decreasing DM content with ensiling process found in this study was the addition of water and molasses during the ensiling process. The reason behind the lower CP contents found for treatments with only urea when compared to treatments with molasses+urea was the added protein provided by molasses. It is known that different dosages (4%, 5% and 6% urea) of urea applied to wheat and sorghum straws increase the CP content (Kraidees, 2005; Mattoni et al., 2007). These findings were similar to the findings of this study and the values agree with the literature reports.

The CP content of sorghum straw was reported between 3.3% and 5.3% in a number of studies (Mattoni et al., 2007;

Hamed and Elimam, 2009; Jonathan et al., 2012;

ElObied and Ali, 2013) and the CP content found in this study (3.28%) was in this reported range. The CP content of soybean straw was reported between 5.0% and 7.88% in the literature (Stanton and LeValley, 2006; Mule et al., 2008) and the CP content found in our study (3.64%) was lower than the reported range. The CP content of wheat straw was found to be 2.93%. This content was reported by several authors (Can et al., 2004; Dhali et al., 2005; Stanton and LeValley, 2006; Hassan et al., 2011) at a range between 2.9% and 4.0%. Accordingly, the CP content range reported in the literature for wheat straw was close to the one found in our study. It is believed that several factors such as forage type, soil structure, fertilization, harvest time, ratio of stalks and seeds in the hay, ratio of impurities, etc. may account for the differences between the studies. Furthermore, it was shown by El-Shatnawi and Mohawesh (2000) that CP needs of the sheep for maintenance (7-9%) and lactation (10-12%) can be compared with urea or urea+molasses treatment of the straws.

The reason behind the decreasing NDF content in straws with molasses addition is that molasses is a water soluble carbohydrate source. This fact was the reason behind the proportionately decreasing NDF content of straw. Having found to have the lowest NDF content among other straws, sorghum straws are believed to increase the feed consumption of animals when molasses addition treatments are preferred. In addition, the NDF content found in this study for sorghum straw (64.7%) was similar to the reports of Mattoni et al. (2007) (64.6%) and Jonathan et al. (2012) (69.3%). With respect to soybean straw, Fluharty (2009) reported an NDF content of 70.0% while Stanton and LeValley (2006) reported 54.0%. In this study, the NDF content of soybean straw was 71.46% which is similar to the report of Fluharty (2009). The NDF content of wheat straws was reported by a number of authors (Can et al., 2004; Stanton and LeValley, 2006; Fluharty, 2009) within the range between 54.4% and 73.0%. It can be seen that the value obtained in this study (78.89%) is higher than the literature reports. In a study which used 6% urea with wheat straw, it was found that the NDF content was similar to the one obtained from control group (78.8% vs. 78.3%). These results are in agreement with the findings of our study. The assessment with respect to NDF and ADF recommends the use of sorghum straws with the lowest values and urea+molasses treatment for their high digestibility and feed intake.

Treatments (urea, molasses and urea+molasses treatments) had no effect on the lignin content of neither sorghum straws nor wheat straws. The fact that the lignin content of soybean straws with urea+molasses addition was lower than the one with only urea addition may be explained with the use of molasses, a water soluble carbohydrate source during fermentation. Moreover, it may be that urea is broken down during silage fermentation, which may lead to lower lignin content in the treatment with molasses+urea addition as the urea is not extracted in the form of ammonia. The ADL content of sorghum straw was found to be 2.74% in this study; Serna-Saldivar et al. (2012) and Cardoso et al. (2013) have reported similar results (7.0% and 7.52%, respectively) while Jonathan et al. (2012) who stated that they have used sorghum stalks reported an ADL content of 28.2%. According to these results, the ADL data found in this study was quite lower than the literature reports. The ADL content of soybean straw was found to be 12.64%. This value is similar to the report of Maheri-Sis et al. (2011) (13.0%) while lower than the report of Fluharty (2009) (16.0%).The lignin content of wheat straw was found to be 6% while it was lower than the report of Fluharty (2009). Factors such as forage type, soil structure, fertilization, harvest time, ratio of stalks and seeds in the hay, treatments applied on forages before analysis (grounding size), differences in silage fermentation may account for the differences found

(Kilic and Saricicek, 2006).

In vitro gas productions and methane productions of straws

The pH value measured from the rumen fluid remaining after 96 h incubation shows whether the buffer used is consumed by microorganisms or not in terms of in vitro gas production. If the pH is acidic then buffer may be consumed. In this context, it was observed that the LiP addition decreases the pH value obtained after 96 h, however, it does not acidify the environment and that it has no effect on the results. Kalkan and Filya (2011) reported that cellulose enzyme addition increases the in vitro gas production of wheat straw. Authors reported a 24-h gas production at 12.46 ml for the control group of wheat straw, while this value reached up to 20.19 ml with the addition of cellulase. In this study, 24-h gas production was found at 9.98 ml for the control group and the same value reached up to 54.37 ml with LiP addition. Rodrigues et al. (2008) reported higher gas production in groups with enzymes they have extracted from white rot fungi and stated that it improves the digestibility of forages. We have found a similar effect in this study. Nevertheless, Rodrigues et al. (2001) reported that the addition of enzymes to break down cell membrane in perennial rye grass silage had no effect on gas production. This result may be accounted for factors such as intrarumen conditions, processing of forages and differences in the application, dosage differences, reduced enzyme activity in the process, etc. Kalkan and Filya (2011) reported that OMD and DMD is significantly affected by the processing conditions and that there are highly positive correlations between in vitro gas production and OMD.

Denek et al. (2014) reported 24-h gas production of 38.44ml methane production of 11.31% and ME of 7.68 MJ/kg DM for wheat straw. It can be seen that the values reported in this study are lower than the ones reported by Denek et al. (2014). It is believed that several factors such as forage variety, soil structure, fertilization, chemical composition, harvest time, ratio of stalks and seeds in the hay and differences in the in vitro gas production technique used may account for the differences found (Kilic and Saricicek, 2006). Lopez et al. (2005) reported “a value” (59.6-50.8 ml), “c value” (0.036-0.046 ml/h) and ME values (5.66-7.38 MJ/kg DM) legume and poaceae straws. Among these reported values, the “a value” and ME values were higher when compared to the wheat straw control and soybean straw control groups in this study. Gas production rate (c value), on the other hand, was higher than WS but lower than SS. In addition, LiP enzyme addition led to higher values in terms of a value, c value and ME values and it was suggested that LiP enzyme addition increases the forage value.

Methane, as the final product of rumen fermentation, is known to cause 8-10% energy loss. Particularly difficult to biodegrade, lignin available in straw reduces the activity of cellulose and hemicellulase enzymes provided by rumen microorganisms. Consequently, straw leaves rumen with a quite low rumen fermentation rate and cannot be utilized by ruminants which leads to methane production (Khan and Mubeen, 2012). Johnson and Johnson (1995) reported that non-fibrous carbohydrate fermentation leads to lower methane production when compared to carbohydrate fermentation of high cellulose content and noted that feeding animals with forages containing high soluble carbohydrate content will lead to reduced methane production. This is a result of the fact that poor quality forages have a lower rate of digestible parts. Accordingly, LiP addition to poor quality forages will reduce methane formation having broken down compounds with lignin, therefore increasing the nutritional value.

Dias et al. (2010) reported that enzyme addition increases the cellulose/lignin ration of wheat straw and Pinto et al. (2012) identified lignin saccharification in wheat straw exposed to lignin peoxidase enzyme. These results are in agreement with the findings of our study. Selcuk et al. (2016) reported that cellulase enzyme addition increases the digestibility of rice straw and Tang et al. (2008) reported that fibrolytic enzyme preparation (containing cellulase and xylanase) increases the cumulative gas production, IVDMD and IVOMD of poor quality forages. In this study, it was found that in vitro gas production and methane production increases significantly along with OMD value with the LiP enzyme use for all the straws included in the study. As it would be expected, breaking down of lignin also increases the digestibility of organic matter.

Wan and Li (2011) reported that fungal pretreatment (enzyme production) has a less significant effect in soybean straws in terms of lignoselulosic break down when compared to wheat straws and that soybean straws are more resilient to biodegradation. Wan et al. (2011) reported that the cellulose digestibility of soybean straw increases significantly with the pretreatment of hot water. Although soybean straw had a higher lignin content when compared to other straws experimented in this study, it was found that LiP enzyme addition increases the OMD value of all the straws.

Kalkan and Filya (2011) reported that cellulose addition increases the ME content of wheat straw. Similarly, in this study LiP enzyme addition also increased the ME content of wheat straw. Nevertheless, it is believed that the reason behind the increase in in vitro gas production, methane production and digestibility of straws is the fact that the lignin structure available in the forage breaks down (acetic acid production).

Kamalak (2005) reported the in vitro gas productions of wheat and barley straws which are rich in NDF (75.56%, 72.73% respectively) and ADF (54.33%, 53.23%, respectively) and poor in CP content (3.14%, 4.22%, respectively) are 13.50-45.33 ml and 13.33-47.00 ml, respectively for the time range between 0th to 96th h. In this study, a range between 0.45 and 29.02 ml was found for wheat straw for the gas productions between 0th to 96th h. However, these values increased up to 16.58-70.96 ml with the addition of LiP enzyme. Accordingly, it was found that LiP enzyme has an important effect on increasing in vitro gas production and nutritive values of wheat straw.

It was reported for the ammonia treated soybean straw that cellulase enzyme addition decreases the lignin content to 30.16% (that it increases the breaking down of lignin) (Xu et al., 2007). In this study, it was found that LiP enzyme addition increases the gas production and methane production which means that lignocelulosic structure is broken down. However, it is believed that the differences found accounts for the different lignocellulosic structures straws have.



A general assessment will show that sorghum straw offers the best forage value among other straws. In addition, it is recommended that in lands where drought is common, sorghum straw should be enriched with additives and used in animal nutrition. Sorghum straw offers a great forage potential in this respect. It was found that treatments applied in this study increased the CP content of straws when compared to control groups and that the highest crude protein contents were possible with the urea+molasses treatment. Moreover, it was shown that LiP enzyme addition increases the gas production and methane production significantly for all the treatments. When there was no enzyme addition to any of these three straws it was observed that methane production was not affected by the treatments while enzyme addition increased methane production in all straws and treatments. Nevertheless, it was found that urea addition to soybean straw led to a lower methane production when compared to the control group. Increased methane production in rumen is not a favorable situation. Feeding strategies aimed at reducing the methane production which is increased by the use of LiP enzyme. Accordingly, it is necessary to use the energy more efficiently with urea addition to straws or its combined use with forages rich in protein. In conclusion, all the treatments applied in this experiment were proved to be useful in increasing the nutritional value of poor quality forages and straws. Thus, it will be possible to minimize the methane production caused by the use of poor quality forages. In addition to its use in animal nutrition, it is also possible to use LiP enzyme in biogas systems where methane production is desired for better efficiency of the system. However, in order to do this, the enzymes are required to be economical and to operate at an advanced level. Indeed, white-rot fungi have been studied due to their potential to produce ligninolytic enzymes for delignification of some lignocellulosic materials. In addition, it should be considered that treatment of straws with LiP enzyme before they are fed to the animals and in vitro and in vivo studies are recommended for the use of LiP enzyme in poor quality forages.



This work was supported by Turkish Scientific and Technological Research Council, TUBITAK (TOVAG 216O606), Turkey.


Conflict of interest statement

We declare that we have no conflict of interest.



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