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

Citation of this paper

Mitigating methane emissions from ruminants; comparison of three nitrate salts as sources of NPN (and sinks for hydrogen) in an in vitro system using molasses and cassava leaf meal as substrates

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, UTA (Colombia)
AA#48, Socorro, Santander, Colombia
** University of New England, Armidale NSW, Australia

Abstract

This study aimed to evaluate the effect of three nitrate salts (calcium nitrate, potassium nitrate and sodium nitrate) compared with urea on methane production in an in vitro 24h incubation with molasses and cassava leaf meal as substrate.

Compared to urea treatment, all nitrate salts diminished total gas production, methane percentage in the gas and methane production per unit of substrate.

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


Introduction

It has been established that calcium nitrate acts as an alternative sink for hydrogen in in vitro incubations using molasses, sugar cane or cassava root meal as energy substrates and cassava leaf meal as the source of protein (Inthapanya Sangkhom et al 2011; Outhen et al 2011; Binh Phuong et al 2011; Du Thuy Thanh et al 2011). Potassium nitrate was used successfully by Trinh Phuc Hao et al (2009) in feeding trials with goats while sodium nitrate was studied by Ngoc Huyen et al (2010) with cattle.  However, there have been no comparisons of the different nitrate salts in the same experiment.

Calcium nitrate is marketed as Calcinit and contains a small percentage of ammonium ions. Ammonia under warm temperature reacts with sugars in molasses to form a toxic imidazole (Perdok and Leng 1987). In tropical conditions this could be factor in total mixed feeds, so it was decided to confirm that any nitrate salt would have the same effect on methane emissions before comparisons in the whole animal.

The objective of this study was to compare nitrate salts of calcium, sodium and potassium in an in vitro incubation using molasses as energy substrate and cassava leaf meal as the protein source. Urea served as the control treatment.


Materials and methods

Location and duration

The in vitro experiments were conducted in the laboratory of Nong Lam University, Ho Chi Minh city, Viet Nam, in July, 2011.

Experimental design

The four treatments in a completely randomized design (CRD) were iso-nitrogenous levels of urea and nitrates of calcium, potassium and sodium (Table 1). The substrates were molasses and cassava leaf meal.

Table 1. Individual treatments

 

Treatment

NPN sources

% N in NPN

% NPN in diet

CaN

Ca(NO3)2.4H2O

11.9

7.87

NaN

NaNO3

16.5

5.67

KN

KNO3

13.9

6.73

Urea

(NH2)2CO

46.7

2

 A simple in vitro system was used based on the procedure reported by Inthapanya Sangkhom et al (2011).

Material preparation and implementation of the method

Molasses was purchased in the market. Cassava leaves was 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 2. Representative samples of the mixtures (12g DM) were put in the incubation bottle to which was added 960ml of buffer solution (Table 4) and 240ml of cattle rumen fluid (taken immediately from a steer that was slaughtered at the local abattoir). The bottles with substrate were then incubated in a water bath at 39 C for 24h..

Table 2. Ingredients in the different  treatments (g)

     

Treatment

Molasses

Cassava leaf meal

NPN sources

Calcium nitrate

Sodium nitrate

Potassium nitrate

Urea

CaN

8.06

3

0.94

-

-

-

NaN

8.32

3

-

0.68

-

-

KN

8.19

3

-

-

0.81

-

Urea

8.76

3

-

-

-

0.24


Table 3. Ingredients of the buffer solution (adapted from Tilly and Terry 1963)

Ingredients

CaCl2

NaHPO4.12H2O

NaCl

KCl

MgSO4.7H2O

NaHCO3

Cysteine

(g/liter)

0.04

9.30

0.47

0.57

0.12

9.80

0.25

Measurements

The gas volume was measured by water displacement from the receiving bottle suspended in water. The bottle was calibrated at intervals of 50ml. The methane percentage in the gas was measured with a Crowcom meter (Crowcom Instruments Ltd, UK), and expressed as volume of methane per unit substrate.

The DM and crude protein contents of the substrates were determined according to AOAC (1990) methods. The sulphate in molasses was analyzed by Smart3 colorimeter. N solubility was determined by shaking 3g sample with 100ml NaCl 1M for 3 hours, filtering through Whatman No.4 filter paper and determining nitrogen in the filtrate.


Results and discussion

Chemical composition of ingredients in the substrate

Crude protein in the cassava leaf meal was high but very low solubility indicating that cassava leaf meal has potential bypass protein characteristic. Compared with cassava leaf meal, the crude protein in molasses is low, beside that it contains significant sulphate source. Thus, molasses is easily fermentable substrate for anaerobic incubation and is sulphate source providing for microorganism.  

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

 -

Gas and methane production

Compared with urea, all the nitrate treatments supported lower gas production (Figure 1). Percentages of methane in the gas were similar for all the nitrate salts, the values being some 25% less than with urea (Table 5; Figure 2). Expressed as volume of methane per  unit substrate, the reduction due to the nitrate salts was some 40% (Figure 3). This finding is similar to the report of Ngoc Huyen et al (2010), for sodium nitrate and ammonium nitrate as the sources of fermentable N compared with urea, and Lin et al (2011) who compared sodium nitrate and urea in an in vitro experiment.  

Table 5. Mean values for total gas production, methane volume, methane percentage and methane production per g substrate

 

CaN

KN

NaN

Urea

SE

P (nitrate)

SEM

P (NPN sources)

Gas production, ml

1593

1490

1535

1927

31.5

0.12

29.1

<0.001

Methane, ml

237

248

235

394

7.09

0.43

6.78

<0.001

Methane, %

14.9

16.6

15.3

20.4

0.30

0.007

0.27

<0.001

Methane, ml/g substrate

19.8

20.6

19.6

32.8

0.59

0.43

0.57

<0.001


The higher gas production on the urea treatment is partially due to the carbon dioxide liberated from hydrolysis of the urea. One mole of urea produces 1 mole of carbon dioxide which is 22.4 .litres of gas thus hydrolysis of  0.24 g of urea accounted for  90ml of the total gas production and would have been produced over a short time after initiation of the incubation. This would account for about 25%  of the difference in gas production (about 400 ml) between use of urea compared with the nitrate salts. These results are similar to the findings of Guo et al (2009), where addition of sodium nitrate in a 24h incubation lead to lower methane production and also less CO2 production, compared with urea as NPN source.

Figure 1. Effect of nitrate sources versus urea on gas production in
an in vitro system with molasses and cassava leaf meal as substrate
Figure 2. Effect of nitrate sources versus urea on methane percentage in
an in vitro system with molasses and cassava leaf meal as substrate

Figure 3. Effect of nitrate sources versus urea on methane production per unit substrate

Conclusions


Acknowledgements

We 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, which is part of the requirements for the degree of MScbe submitted by the Senior Author to Cantho University.


References

AOAC 1990 Official methods of analysis. Association of official Analysis (15th edition). Washington, D.C, USA.

Binh Phuong L T, Preston T R and Leng R A 2011  Mitigating methane production from ruminants; effect of supplementary sulphate and nitrate on methane production in an in vitro incubation using sugar cane stalk and cassava leaf meal as substrate. Livestock Research for Rural Development. Volume 23, Article #22. http://www.lrrd.org/lrrd23/2/phuo23022.htm

Du Thuy Thanh, Preston T R and Leng R A 2011  Effect on methane production of supplementing a basal substrate of molasses and cassava leaf meal with mangosteen peel (Garcinia mangostana) and urea or nitrate in an in vitro incubation. Livestock Research for Rural Development. Volume 23, Article #98. http://www.lrrd.org/lrrd23/4/than23098.htm

Inthapanya S, Preston T R and Leng R A 2011  Mitigating methane production from ruminants; effect of calcium nitrate as modifier of the fermentation in an in vitro incubation using cassava root as the energy source and leaves of cassava or Mimosa pigra as source of protein. Livestock Research for Rural Development. Volume 23, Article #21. http://www.lrrd.org/lrrd23/2/sang23021.htm

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Outhen P, Preston T R and Leng R A 2011  Effect of supplementation with urea or calcium nitrate and cassava leaf meal or fresh cassava leaf in an in vitro incubation using a basal substrate of sugar cane stalk. Livestock Research for Rural Development. Volume 23, Article #23.  http://www.lrrd.org/lrrd23/2/outh23023.htm

Perdok H B and Leng R A 1987 Hyper-excitability in cattle fed ammoniated roughages. Animal Feed Science and Technology, 17: 121-143.

Trinh Phuc Hao, Ho Quang Do, Preston T R and Leng R A 2009  Nitrate as a fermentable nitrogen supplement for goats fed forage based diets low in true protein.  Livestock Research for Rural Development. Volume 21, Article #10.  http://www.lrrd.org/lrrd21/1/trin21010.htm

Tilley J M A and Terry R  A  1963 A two stage technique for the in vitro digestion of forage crops. Journal of the British Grassland  Society 18 : 104.

Guo W S, Schaefer D M,  Guo L P,  Ren L P and  Meng Q X 2009 Use of Nitrate-nitrogen as a Sole Dietary Nitrogen Source to Inhibit Ruminal Methanogenesis and to Improve Microbial Nitrogen Synthesis In vitro. Asian-Australasian Journal of Animal Science  Volume 24, No. 4,  542 – 549.


Received 16 December 2011; Accepted 24 December 2011; Published 4 January 2012

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