Effects of nitrate and sulphur on in vitro methane production and dry matter degradation

Ho Quang Do, Tran Thi Thuy, Trinh Phuc Hao, T R Preston* and R A Leng**

Cantho University, Cantho, Vietnam
hqdo@ctu.edu.vn
* UTA-TOSOLY, AA 48 Socorro, Colombia
** University of New England, Armidale NSW 2351 Australia,
PO Box 361, Coolum Beach 4573, Queensland, Australia

Abstract

Three treatments: S0, S0.4 (0.4 % added sulphur) and S0.8 (0.8 % added sulphur) were compared in an in vitro incubation with 3 repetitions in a completely randomized design, using as substrate a basal diet of NaOH-treated rice straw, molasses and cottonseed meal. The forage components of the diets were dried and milled through a 1 mm screen and mixed with the other components of the diet. Representative samples of the substrate (20 g DM) were put in an incubation flask (2500 ml) to which were added 1.6 liters of buffer solution and 400ml of rumen fluid. The rumen fluid for each treatment was obtained from rumen-fistulated cattle that were on the same dietary treatments. The flasks were then incubated at 38⁰C in a water bath for 72h. During the incubation, each flask was connected to an aluminium bag for total collection of gas over the 72h incubation. At the end of the incubation the total gas volume was recorded and samples analyzed for the proportion of methane. The same treatments and the same methods were repeated to measure the loss of substrate during an incubation of 24h.

Methane production per unit substrate fermented was reduced linearly with increase in level of sulphur in the incubation medium.

Key words: Cotton seed meal, greenhouse gases, molasses, rice straw, urea

Introduction

This paper is part of a series of researches aimed at optimizing the use of nitrate salts as a strategy for reducing methane emissions from enteric fermentation in ruminants  in order to meet future targets for mitigating global warming (Leng 2008). Trinh Phuc Hao et al (2009) and Le Thi Ngoc Huyen et al (2010) showed that nitrate could be safely fed as the major source of fermentable N provided that the animals (goats and cattle, respectively) were adapted to the diet over a period of 2 weeks. The positive effect of nitrate salts in reducing methane production has been shown in both in vitro (Binh Phuong et al 2011; Inthapanya et al 2011; Outhen et al 2011; Thanh et al 2011) and in vivo (Anh Nguyen Ngoc et al 2010; Van Zijderveld et al 2010)The study of Sokolowski et al (1969) indicated that there was an apparent interaction between nitrate and sulphate with N retention and wool growth being enhanced when both were present in the diet, compared with either nitrate or sulphate given separately. The results reported by Van Zijderveld et al (2010) and Silivong et al (2011) also showed a synergistic effect on methane mitigation of feeding both nitrate and sulphate.

The objective of the present research was to study the effects of nitrate and sulphur on methane production and DM fermentation in an in vitro system using rice straw and molasses as the major  substrates.

Materials and methods

Location and experiment design

The experiment was conducted in the Laboratory of  the Department of Animal Science, College of Agriculture and Applied Biology, Can Tho Uinversity.

Treatments and design

The treatments in a completely randomized design with three repetitions were:

• S0: Basal diet with no added sulphur

• S0.4: Basal diet with addition of 0.4% sulphur

• S0.8: Basal diet with addition of  0.8% sulphur

The sulphur was in the form of the pure element. Sodium nitrate was included in all diets as the source of NPN (Table 1).

Preparation of substrates for the in vitro incubation

Rice straw was chopped to lengths of 3-4cm. A solution was prepared containing 3%  NaOH and 97% water. This solution was then sprayed on the rice straw at the rate of 30 litres solution to 100 g rice straw DM. The treated rice straw, grass and cottonseed meal were dried at 60°C and milled through a 1 mm screen.  A representative sample (20 g DM ) was put in  an incubation flask (2500ml) to which were added 1.6 liters of buffer solution and 400ml of rumen fluid, prior to filling each flask with carbon dioxide. A separate incubation was carried out with the same substrates to determine DM fermented over a 24h period. For this study, a representative sample of the substrates (0.5 g DM ) was put in an incubation flask (100ml) to which were added 40 ml of buffer solution and 10 ml of rumen fluid, prior to filling each flask with carbon dioxide. The rumen fluid for each treatment was obtained from rumen-fistulated cattle that were on the same dietary treatments. The flasks were incubated at 38^°C in a water bath for 72h for measuring methane and for 24h for measuring DM degradation.  During the incubation for methane production, each flask was connected to an aluminium bag for total collection of gas over the 72h period. At the end of the incubation the total gas volume was recorded and samples analyzed for the proportions of methane and carbon dioxide.

Measurements

Total gas volume was recorded with a "Ritter" gas flow meter (Calibrated Instrument Inc; Photo 1) and samples analyzed for the percentage of methane using a gas detector (Geotechnical Instruments GA 94; Photo 2). Feed samples were analyzed for DM, ash, NDF, ADF and N according to AOAC (1990). 

Photo 1. The gas flow meter	Photo 2. The gas meter for measuring methane

Statistical analysis

The results were analyzed by the General Linear Model option in the ANOVA program of the Minitab Software (version13.2). Sources of variation in the model were: level of added sulphur and error.

Results and Discussion

The chemical composition of the ingredients in the substrate is shown in Table 2.

Due to error, the measurement of substrate fermented was only determined after 24h of incubation, while gas measurements were made after 72h. There appear to be no data in this type of in vitro system for effect of incubation time on proportion of substrate fermented at times varying from 4h to 72h. However, in a recent report Inthapanya et al (2010) plotted substrate fermented against time over the period 4 to 24h using a substrate containing 67% alkali-treated rice straw and 30% cassava leaves (DM basis), with the NPN source being potassium nitrate or urea. The relationship of DM fermented with incubation time over the 24h period was linear with no difference between the slopes for urea and potassium nitrate. In the present experiment, the values for methane production per unit substrate fermented (calculated as methane production at 72h and substrate fermented at 20h) will not be the same as if both had been determined at the same incubation time (either 24 or 72h); however, it appears reasonable to assume that the relative differences due to source of additive (in this case sulphur) would be similar.

Figure 1. Relationship between substrate fermented and incubation time (substrate contained 67% rice straw and 30% fresh cassava leaf [DM basis] and either potassium nitrate or urea as NPN source; from Inthapanya et al 2011)

There was a tendency  (P=0.18) for the methane content of the gas after 72 h to be lower for the treatments with added sulphur (Table 3). The proportion of the substrate fermented after 24h was increased due to supplementation with sulphur. Methane production (at 72h) per unit of substrate fermented (at 24h) decreased linearly with sulphur supplementation (Figure 1).

Figure 2. Relationship between sulphur level and methane production at 72h per unit substrate fermented after 24h

The reduction in methane production with added sulphur is in agreement with the findings of Van Zijderveld et al (2010), Silivong et al (2011) and Binh Phuong et al (2011). The results of Binh Phuong et al (2011) are of interest as they indicate that the synergism between the NPN and sulphur on methane mitigation occurred only when the NPN source was nitrate and not urea.

Conclusions

• Methane production per unit substrate fermented was reduced linearly with increase in level of sulphur in an incubation medium that contained sodium nitrate.

References

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Received 31 August 2011; Accepted 2 October 2011; Published 10 October 2011

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