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

Le Thuy Binh Phuong, 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

The aim of this study was to evaluate the effect of replacing urea by calcium nitrate  with or without supplementary sulfur (0, 0.4, 0.8% on DM basis) on methane production in an in vitro incubation medium inoculated with rumen fluid and  using sugar cane stalk and cassava leaf meal as substrate.  The design was a 3*2 factorial arrangement of the treatments with 4 replications.

Compared with urea, calcium nitrate reduced methane production The effect was consistent over successive periods in the 48h incubation. Adding 0.4% sulfur, in the form of sodium sulphate, increased methane production, while 0.8% sulphur reduced methane production. When 0.8% sulfur was combined with nitrate the effects on methane reduction were additive. Methane production  increased linearly with the length of the incubation on all treatments

Key word: Calcium nitrate, climate change, fermentation, gas production, greenhouse gases, urea, substrate

Introduction

Emissions of greenhouse gases (GHG) from anthropogenic sources( man made) are considered to be the main cause of global warming (IPCC 2007). Live stock production is a major contributor to GHG levels, accounting for about 18% of the anthropogenic GHG emissions (Steinfield et al 2006).  Ruminants produce globally about 80 tonnes of methane gas annually which represents some 28% of anthropogenic methane emissions (Leng 2008).

Methane  is produced in the fore-stomach of ruminants as a result of microbial fermentation of feed components In the process of digestion of organic matter  reduced co factors are generated and a requirement for continuous fermentation is that theses co factors are re oxidised. The oxidation of theses reduced cofactors is coupled to high affinity electron acceptors mainly sulphur and nitrate However if these are in low concentrations or absent, which is the case under normal feeding conditions,  carbon dioxide is reduced to methane

Recently, Leng (2008) has emphasised that nitrate as a feed component replacing urea has a dual role as an electronic sink for hydrogen produced by fermentation ,and the  ammonia  produced is the preferred source of fermentable nitrogen in diets low in crude protein. The dietary conditions which favour utilization of nitrate to lower the production of methane are a source of easily fermentable carbohydrate, a low content of soluble protein,  an adequate level of sulfur and a source of bypass protein (Leng 2008). Sugar cane contains 50% of sugars on DM basis and almost no protein. It is thus logical  to test whether   nitrate can be used to lower enteric methane production  and at the same time maintain or  promote microbial growth in the rumen. A diet of sugar cane with 3% urea (in DM) and with rice polishing as a bypass protein source has been shown to support growth rates of 800 g/day in fattening cattle (Preston et al 1976).  Leng and Preston (2010) have emphasized that the reduction of both nitrate and nitrite could be stimulated by a source of ruminal sulphide produced locally. As most organisms that reduce sulphate are also capable of reducing nitrate or nitrite, the feeding of nitrate and sulphate is likely to have important interactions. Recently, Van Zijderveld et al 2010) demonstrated an added effect on lowering of methane production by feeding a combination of nitrates and sulphate to sheep.

Hypothesis

Supplementary sulphate will have a synergistic effect in reducing methane production in a basal diet of sugar cane in which the source of non-protein nitrogen (NPN) is nitrate rather than urea.

Objectives

To study the effect of sulphate and nitrate on methane production in an in vitro system inoculated with rumen fluid using sugar cane as the basal substrate and cassava leaf meal as the source of protein.

Materials and methods

Location and duration

The experiment was conducted in the laboratory of An Giang University from September to November, 2010.

Experimental design

Three levels of sulfur and two sources of NPN were compared in an in vitro fermentation system. The design was a 3*2 factorial arrangement with 4 replications. The levels of sulfur (as sodium sulphate) were 0, 0.4 and 0.8% (in DM); the NPN sources were urea (2% in DM) and calcium nitrate (3.8% in DM).

The individual treatments were:

• CaN: Calcium nitrate

• U: Urea

• CaN-0.4S; CaN + 0.4% sulfur as sodium sulphate

• U-0.4S: Urea + 0.4% sulfur as sodium sulphate

• CaN-0.8S; CaN + 0.8% sulfur as sodium sulphate

• U-0.8S: Urea + 0.8% sulfur as sodium sulphate

Material preparation and implementation of the method

Fresh sugar cane stalk was purchased in the market. The outer rind was removed with a knife and the central part (the sugar-rich pith) chopped into small pieces (about 2mm section). Cassava leaves were sun-dried and ground through a 4 mm sieve. These two components (cassava leaf meal and sugar cane stalk) were mixed with the sources of N (urea or nitrate) and sulphate (according to the proportions shown in Table 1). Representative samples of the mixtures (12g DM) were put in the incubation bottle to which were added 960ml of buffer solution (Table 2) and 240ml of rumen fluid obtained from a buffalo, prior to filling each bottle with carbon dioxide. The rumen fluid was taken at 10-11pm in the slaughter-house from a buffalo immediately after the animal was killed. A representative sample of the rumen contents (including feed residues) was put in a vacuum flask and stored until 8-9am  the following morning when the contents were filtered through one layer of cloth before being added to the incubation bottle. The gas space inside the bottle was then flushed with carbon dioxide prior to the incubation at 38°C for 48h. A simple in vitro system was used (Photo 1) based on the procedure reported by Sangkhom et al (2011).

Measurements

The gas volume was measured at 8, 24, and 48h by water displacement from the receiving bottle suspended in water which was calibrated at intervals of 50ml. On each occasion, after measuring the volume,  the gas was ejected from the receiving bottle though a tube attached to a Crowcom meter (Crowcom Instruments Ltd, UK) fitted with an infra-red sensor to measure methane (Photo 2).  Undigested substrate at the end of the incubation after 48h was filtered by cloth and was then dried at 105⁰C for 24h to determine residual DM. The DM and crude protein contents of the substrates were determined according to AOAC (1990) methods.

Statistic analysis

The data were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab Software (version13.2) (Minitab 2000). Sources of variation in the model were: levels of sulfur, NPN source, interaction sulfur*NPN source and error

Results and Discussion

At each stage of the fermentation and overall, gas production, per cent of methane in the gas, was less when nitrate rather than when urea, was the source of NPN (Table 3). Increasing the level of sulfur reduced gas production but the effect on methane production was variable. Methane production increased with sulfur level in samples taken after 8 and 24h. Only the last samples (25-48h) and overall values showed reduction in methane with level of sulfur. There was an interaction between NPN source and level of sulfur, such that when nitrate was the supplementary source of N, the relative reduction in methane with added 0.8% sulfur was greater than when 0.8% sulfur was given with urea as the fermentable N source, indicating suppression of sulphur-reducing bacteria by nitrate.

The substrate DM fermented in 48h incubation was less with nitrate than with urea; and was reduced with added sulfur.

Methane production per unit substrate fermented was lowered by some 30% when nitrate was the NPN source but there was no difference due to added sulfur. On all levels of added sulfur, the nitrate consistently reduced methane production (Figure 1), with the greatest reduction being observed when 0.8% sulfur was added to the substrate.

Figure 1. Effect on methane production of replacing urea by calcium nitrate as NPN source in an in vitro fermentation of sugar cane stalk and cassava leaf meal with different levels of added sulfur	Figure 2. Reduction in methane due to addition of 0.8% sulfur, 3.8% calcium nitrate or 3.8% nitrate plus 0.8% sulfur

The interaction between nitrate and sulfur is apparent in the incubations when methane production rate was compared on the treatments with zero and 0.8% sulfur with urea or nitrate as NPN source. Sulfur alone appeared to increase methane production whereas in combination with nitrate the lowering in methane production was greater than on nitrate alone (Figure 2). The effects with 0.8% sulfur are similar to those reported by Van Zijderveld et al (2010) and Silivong et al (2011) when 0.8% sulfur and nitrate had additive effects in lowering methane production.  A low level of sulphur appears to stimulate fermentation rate as it actually increased methane output  indicating that the incubation medium on the 0% sulphur treatment was actually deficient in S . At higher sulphate additions undoubtedly the extra S was reduced  through the production of hydrogen sulphide. As the electron acceptors in the medium are used up there will be a swing with time from nitrate conversion to ammonia to sulphate reduction to hydrogen sulphide and when sulphate is exhausted it appears that methane will be generated from reduction of carbon dioxide. These are governed by, and in accord with, the Gibbs free energy change of the reactions.

The curvilinear increase in methane production with duration of incubation (Figures 3 and 4), indicative of the transition to a secondary fermentation of the VFA to methane,  supports the  findings of Sangkhom et al (2011), who used cassava leaves or mimosa foliage as supplements to cassava root meal, and Outhen et al (2011) who employed fresh or dried cassava leaves with sugar cane stalk, as substrate in a similar in vitro system.

Figure 3. Effect of nitrate vs urea on methane production at intervals during the incubation	Figure 4. Effect of level of added sulfur on methane production at intervals during the incubation

The lowering of methane production when nitrate replaced urea is in accord with its high affinity for electron capture but following the depletion of nitrate would see the reversion to methane generation  by reduction of hydrogen to methane  Moreover, the transition from a primary fermentation to  a secondary fermentation where VFA are degraded is more likely to produce relatively higher increments of methane per unit of organic acid degraded.   However, with higher sulfur level in treatment 0.8%S, the effect of sulfur in lowering  methane production  may be more prolonged, while nitrate is depleted  Nitrate is known to inhibit sulphur reducing bacteria (see Hubert and Voordouw 2007). As nitrate is depleted by dissimulatory conversion to ammonia, sulphur reduction will return and hydrogen sulphide production may dilute  the methane percentage in the gas.(see Bracht and Kung 1997).

Conclusions

• Compared with urea, calcium nitrate reduced methane percentage in the gas produced in an in vitro system with sugar cane stalk and cassava leaf meal as substrate. The effect was consistent over successive periods in the 48h incubation.

  
  

• Adding 0.4% sulfur, in the form of sodium sulphate, increased methane production indicating S deficiency in the medium; however, at the 0.8% level there appeared to be an additive  effect on methane reduction when combined with nitrate.

  
  

• Both nitrate compared with urea and increasing level of sulfur decreased rate of gas production and  the rate of fermentation of the substrate.

  
  

• The methane production increased linearly with the length of the incubation.

Acknowledgements

This research was carried out in the laboratory of the Faculty of Agriculture, An Giang University, An Giang province, Vietnam. We wish to thank SIDA-SAREC for funding this research as part of the MSc course at Cantho University through the regional MEKARN project. Special thanks to Ms. Le Thi Thuy Hang and Mr. Ho Xuan Nghiep who helped us in the laboratory.  We thank the administration at An Giang university laboratory for support in providing the facilities to carry out the research

References

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Received 10 January 2011; Accepted 25 January 2011; Published 1 February 2011

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