Livestock Research for Rural Development 24 (1) 2012 Guide for preparation of papers LRRD Newsletter

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Mitigating methane production from ruminants; effect of supplementary sulphate and nitrate on methane production in an in vitro incubation using molasses and cassava leaf meal as substrate

Le Thuy Binh Phuong, Duong Nguyen Khang, T R Preston* and R A Leng**

Nong Lam University, Viet Nam
binhphuongty27@yahoo.com
* Finca Ecologica TOSOLY, AA#48, Socorro, Santander, Colombia
** University of New England, Armidale NSW, Australia

Abstract

This in vitro incubation was arranged as a 2*3 factorial in a completely randomized block design with 6 treatments and 4 repetitions. The first factor was the source of fermentable N (potassium nitrate or urea); the second factor was the level of added sulphur (0, 0.4 and 0.8% of substrate DM, as sodium sulphate.  The substrate was molasses and cassava leaf meal. Incubations were carried out on successive days for 6, 12, 18, 24 and 48h.  At the end of each incubation, volume of gas and  percentage methane were recorded, and the residual substrate filtered to determine the amount of substrate solubilized. 

Gas production, percentage methane in the gas and methane production  were reduced by replacing urea by nitrate at incubation times from 6 to 48h, and were increased with length of incubation. Added  sulphur  increased methane emissions in the presence of nitrate over the early incubation periods indicating a greater fermentation rate in that period, but sulphur was additive in decreasing methane in longer incubations, indicating nitrate had been fully reduced and sulphur reduction commenced The reduction in methane production after 48h of incubation, when  sulphur and nitrate were combined, was greater than when nitrate was used alone

Key words: climate change, fermentation, gas production, greenhouse gases, potassium nitrate, urea


Introduction

Livestock are a significant source of global methane (CH4) emissions, producing some 80 tonnes  annually which represents about 28% of anthropogenic methane emissions (Steinfeld et al 2006).  Enteric methane from ruminant livestock is also an energy cost reducing available feed energy.  Blaxter and Clapperton (1965) indicated that  8-12% of the gross feed energy was lost in the form of methane resulting from the ruminant digestion process.

In the absence of hydrogen sinks other then carbon dioxide in the rumen, methane production is indispensable as it maintains molecular hydrogen levels low at the sites in the biofilm (MacAllister et al 1994) close to hydrolytic breakdown of polymers, such as cellulose and other structural carbohydrates, and fermentative degradation of the solubilized  organic matter to VFA. This process is a critical requirement for continuous fermentation because of the low partial pressure of hydrogen that is required to re-oxidise reduced cofactors produced in the pathways of metabolism (Cheng et al 1980).

Leng (2008) suggested  the more energetically favourable nitrate and sulphate as potential high affinity electron acceptors, competing with carbon dioxide for hydrogen and thus maintaining the availability of  oxidized cofactors generated in fermentative degradation of carbohydrates, while at the same time lowering methane production. The products of nitrate and sulphate reduction are then ammonia and sulphide, respectively.

There are several potential benefits from using nitrate as an alternative electron acceptor in the diets of ruminants. These include potentially higher microbial growth efficiency as ATP is generated  in the reduction of nitrate to ammonia and the nitrate can substitute fermentable N (e.g. urea) in a low protein diet. The conditions which appear to favor the use of nitrate as a fermentable N source,  that also mitigates methane production,  are a source of readily fermentable carbohydrate, an adequate level of sulphur, and a protein supplement low in content of soluble protein and high in “bypass” protein  (Leng 2008).  A source of supplementary sulphur may have an important interaction as sulphide availability in the rumen may be  a critical issue in conversion of nitrate to ammonia without releasing nitrite into the medium (Leng and Preston 2010).

Molasses contains approximately 50% of sugars (Cleasby 1963) and may have 3-7g  sulphur/kg dry matter (Suttle 2010), depending on the process used for clarification of the sugar-rich juice before centrifugation of the sugar. It is therefore convenient to use molasses as the source of sulphur and  fermentable carbohydrate.

Cassava forage has been shown to be a good source of bypass protein in diets based on molasses-urea (Ffoulkes and Preston 1978). Keo Sath et al (2008) and Tham et al (2008) reported that cassava leaf meal improved growth performance and feed conversion in cattle diets based on urea-supplemented rice straw.  However, the presence of cyanogenic glucosides in cassava leaves may increase the requirement for sulphur to detoxify the cyanide produced in the rumen.  Blakeley and Coop (1949) concluded that approximately 1.2g of  sulphur was required to detoxify 1.0g of HCN. Wheeler et al (1975) showed that the supply of  sulphur licks to ruminants effectively protected them against chronic cyanide toxicity.

The research reported here examines the role of supplementary  sulphur in the synergistic interaction between sulphate and nitrate in lowering methane production in an in vitro fermentation of a substrate based on molasses and cassava leaf meal.


Materials and methods

Location and duration

The experiment was conducted in the laboratory of Nong Lam University, Ho Chi Minh city, Viet Nam, from July to August, 2011.

Experimental design

The experiment was arranged as a 2*3 factorial in completely randomized block design (CRBD) with 6 treatments and 4 repetitions (Table 1). The first factor was the source of fermentable N (potassium nitrate [6% of substrate DM] or urea [1.8% of substrate DM);  the second factor was the level of added sulphur (0, 0.4 and 0.8% of diet DM, as sodium sulphate).  The substrates were molasses and cassava leaf meal.

Table 1. Individual treatments (as % of substrate DM)

Treatment

NPN levels

Sulphur levels

KN0S

6.0

0

U0S

1.8

0

KN0.4S

6.0

0.4%

U0.4S

1.8

0.4%

KN0.8S

6.0

0.8%

U0.8S

1.8

0.8%

The experiment was conducted using 24 incubation flasks every day (equivalent to 6 treatments and 4 repetitions) for 5 days corresponding to the lengths of each incubation, which were for 6, 12, 18, 24 and 48h. The in vitro procedure was that described by Sangkhom Inthapanya et al (2011).

Table 2. Plan of the incubations

Day

Number of flasks

Length of incubation, h

1

24

6

2

24

12

3

24

18

4

24

24

5

24

48

Material preparation and implementation of the method

Molasses was purchased in the market. Cassava leaves (from cassava plants managed for foliage production) were sun-dried and ground through a 1 mm sieve. The ingredients in the substrate (molasses, cassava leaf meal, source of NPN) were mixed according to the proportions shown in Table 3. Representative samples of the mixtures (12g DM) were put in the incubation bottle to which was added 960ml of buffer solution (Tilly and Terry 1963) and 240ml of cattle rumen fluid. The rumen fluid was taken immediately from a steer that was slaughtered at the local abattoir, and held in a thermos flask for about 1hr  until placed in the incubation bottles. The bottles with substrate were then incubated in a water bath at 39 C for the different lengths of incubation (6, 12, 18, 24 and 48h).

Table 3 . Ingredients in the treatments (g DM)

 

KN

U

KN-0.4S

U-0.4S

KN-0.8S

U-0.8S

Molasses

8.28

8.784

8.06

8.57

7.85

8.35

Cassava leaf meal

3

3

3

3

3

3

Urea

0

0.22

0

0.22

0

0.22

KNO3

0.72

0

0.72

0

0.72

0

Na2SO4

0

0

0.21

0.21

0.42

0.42

Measurements

The gas volume was measured by water displacement from the receiving bottle suspended in water which was calibrated at intervals of 50ml. Methane percentage was measured by Crowcom meter (Crowcom Instruments Ltd, UK). At the end of each incubation period, residual substrate was filtered through 2 layers of cloth and absorbent cotton, followed by drying of the residue at 100C for 48h to determine the DM residue.  

The DM and N in the molasses and cassava leaf meal, and in the residue after incubation, were determined according to AOAC (1990) methods. The sulphate in molasses was measured with a " Smart 3" colorimeter. N solubility was determined by shaking 3g sample with 100ml 1M NaCl  for 3 hours, filtering through Whatman No.4 filter paper and determining nitrogen in the filtrate.

Statistic analysis

The data were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab Softwar (Minitab 2000). Sources of variation in the model were: levels of  sulphur, NPN source, interaction  sulphur*NPN source and error


Results and Discussion

Chemical composition of ingredients in the substrate

Crude protein in the cassava leaf meal was high but of very low solubility indicating that cassava leaf meal has potential bypass protein characteristics (Table 4). At the levels used,  molasses contributed an additional 0.18% sulphur to the substrates.

Table 4. Chemical composition of ingredients \in the substrate

 

 

DM,%

CP in DM, %

N solubility, %

Sulphur, g/kg DM

Molasses

57.2

5.77

-

2.53

Cassava leaf meal

88.0

22.6

24.3

 -

In all incubations, gas production, percentage methane in the gas and methane production, were: (i) reduced by replacing urea by nitrate at incubation times from 6 to 48h; and (ii) were increased with length of incubation (Table 5). There were no effects of added sulphur (Figures 1 and 2) with incubation times of 6 and 12h but for incubations of 18, 24 and 48h, methane was reduced by both levels of added  sulphur where urea provided the fermentable N source  In the presence of nitrate sulphur appeared to increase methane production over the short incubation period possibly indicating an increased fermentation of the substrate but in the longer incubation periods reversed this effect indicating that sulphur reduction increased with time, possibly through depletion of the energetically more favourable nitrate reduction. The lowering  in methane production after 48h of incubation, when  sulphur and nitrate were combined, was greater than when nitrate was used alone (Figure 3). These results are similar to our earlier report (Binh Phuong et al 2011) when added sulphur had no effect on methane production over 8h of fermentation, but decreased methane when the incubation was for 48h, with more pronounced effects when nitrate was the NPN source. After 48 hr incubation all the soluble sugar from the added molasses was obviously utilized and the overall effect of nitrate v urea, and their interactions can be calculated according to the amount of total dry matter included in the flasks that was solubilized (this was not done for the earlier incubations since soluble sugars could lead to error). These results clearly indicate the lowered methane production per g of digestible carbohydrate (Figure 3) owing to nitrate and sulphate additions, relative to urea.

Table 5. Mean value for gas production, methane production, substrate fermented and methane production per unit substrate fermented for lengths of incubation of 6, 12, 18, 24 and 48h

  

K-nitrate

Urea

SE

P (NPN)

0S

0.4S

0.8S

SEM

P ( sulphur)

P (NPN*S)

6h

 

 

 

 

 

 

 

 

 

 

Gas production, ml

852

960

14.8

<0.001

970

874

874

18.2

0.002

0.10

Methane, ml

101

134

3.84

<0.001

129

104

119

4.70

<0.001

0.01

Methane, %

11.8

13.8

0.25

<0.001

13.1

11.9

13.5

0.31

0.004

0.009

Methane, ml/g substrate

17.3

21.1

0.73

<0.001

20.5

17.0

20.1

0.89

0.02

0.01

12h

 

 

 

 

 

 

 

 

 

 

Gas production, ml

1204

1457

20.6

 <0.001

1280

1366

1345

25.2

0.06

0.05

Methane, ml

156

272

4.38

 <0.001

200

219

222

5.36

0.02

0.15

Methane, %

12.9

18.7

0.24

 <0.001

15.3

15.7

16.4

0.30

0.04

0.68

Methane, ml/g substrate

23.2

36.9

0.63

 <0.001

27.3

30.9

32.1

0.77

0.001

0.08

18h

 

 

 

 

 

 

 

 

 

 

Gas production, ml

1879

2077

17.6

 <0.001

2048

1954

1933

21.58

0.003

0.06

Methane, ml

277

466

8.15

 <0.001

398

364

352.

9.98

0.01

0.56

Methane, %

14.7

22.4

0.37

 <0.001

19.2

18.5

17.9

0.46

0.21

0.70

methane, ml/g substrate

38.7

61.4

1.33

 <0.001

52.5

48.3

49.4

1.63

0.18

0.22

24h

 

 

 

 

 

 

 

 

 

 

Gas production, ml

1796

2074

21.3

 <0.001

2023

1905

1878

26.1

0.002

0.53

Methane, ml

381

505

4.40

 <0.001

479.84

429.45

420

5.39

 <0.001

0.78

Methane, %

21.2

24.4

0.20

 <0.001

23.7

22.4

22.2

0.24

0.001

0.90

Methane,, ml/g substrate

52.9

65.5

0.98

 <0.001

60.9

57.6

59.1

1.20

0.18

0.34

48h

 

 

 

 

 

 

 

 

 

 

Gas production, ml

1889

2219

27.4

 <0.001

2217

1987

1957

33.5

 <0.001

0.40

Methane, ml

411

572

5.67

 <0.001

554

470

448

6.94

 <0.001

0.05

Methane, %

21.7

25.7

0.19

 <0.001

24.9

23.4

22.7

0.23

 <0.001

0.02

methane, ml/g substrate

50.5

65.9

0.96

 <0.001

64.6

55.6

54.6

1.17

 <0.001

0.02

DM solubilized, %

73.5

74.8

0.61

0.145

74.3

74.4

73.7

0.74

0.76

0.34


Figure 1. Effect of added sulphur on methane production for different lengths of incubation and substrates with potassium nitrate as NPN source Figure 2. Effect of added sulphur on methane production for different lengths of incubation and substrates with urea as NPN source

Figure 3. Effect of added sulphur on methane production in the presence of urea or sulphate after 48h incubation

The results of these studies indicate that nitrate-reducing organisms (NRB) are present in the rumen of cattle un-adapted to nitrate, as methane emissions were decreased with nitrate addition compared to that when urea was added. Apparently nitrate reduction takes precedence over sulphur reduction but the apparent effect of added sulphur in the late stage of the fermentation possibly indicates that nitrate N had been fully converted to ammonia and then sulphur reduction commences indicating the presence of sulphur-reducing bacteria (SRB) in the medium. The lowering of methane production is consistent with higher affinity  for hydrogen (or greater Gibbs Free energy change) from reduction of  nitrate and sulphur in the rumen as compared to reduction of carbon dioxide. The Gibbs Free energy change of the reduction is greater for nitrate than sulphate which is greater than for carbon dioxide.  SRB activity is likely to reflect both sulphur and nitrate reduction as these organisms have a wide range of substrates that they can use in their energy metabolism (Moura et al 2007).

The stoichiometry of nitrate reduction to ammonia indicates that the reduction of 1 mole of nitrate  results in the lowering of methane production  by 1 mole. From the lowering of methane release measured it appears that the efficiency of nitrate reduction approaches 100% after 12-18h incubation period and then sulphide reduction  over the next 30hours contributes further lowering  methane production  by about 10% over the next 12h.


Conclusions


Acknowledgements

This research was done by the senior author with support from NUFU as part of the requirements for the MSc degree in Animal Production "Specialized in Response to Climate Change and Depletion of Non-renewable Resources" of Cantho University, Vietnam. The authors wish to thank SIDA-SAREC for funding this research through the regional MEKARN project. We also acknowledge the administration at Nong Lam University for support in providing the facilities to carry out the research.


References

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Received 15 December 2011; Accepted 30 December 2011; Published 4 January 2012

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