Effect of potassium nitrate as modifier of the fermentation in an in vitro incubation using as substrate NaOH and/or lime treated straw supplemented with fresh cassava leaves

Sangkhom Inthapanya, Duong Nguyen Khang*, R A Leng*** and T R Preston**

Souphanouvong University, Lao PDR
inthapanyasangkhom@yahoo.com
* Department of Animal Physiology and Biochemistry, Nong Lam University,
Ho Chi Minh City, Vietnam
** Finca Ecológica TOSOLY, AA#48, Socorro, Colombia
*** University of New England, Armidale NSW 2351 Australia, PO Box 361, Coolum Beach 4573, Queensland, Australia

Abstract

The aim of this study was to evaluate the effect of combining alkali treatment of rice straw with non-protein nitrogen (NPN) from nitrate in reducing methane production in an in vitro incubation system. The treatments in a split-plot 2*6 arrangement with four replications were: alkali treated straw with NaOH (0; 1; 2; 3; 4%) plus lime (Ca(OH)₂) (4; 3; 2; 1; 0%) or untreated straw, and as NPN source  potassium nitrate or urea. All treatments had fresh cassava leaf as protein source.  The quantity of substrate was 12g to which were added 240 ml rumen fluids (from slaughtered buffalo) and 960 ml of buffer solution. The incubation was for 24h with measurements of total gas production,  methane percentage at intervals of 4; 8; 12; 16 and 24 hours and determination of residual unfermented substrate at the end.

The proportion of substrate fermented after 24h was increased by alkali  treatment and tended to increase as lime replaced NaOH. Total gas production and methane percentage  increased with  incubation time  and  were reduced when nitrate replaced urea  at all  fermentation stages. After 24h, the methane production per unit of fermented substrate was less when nitrate replaced urea. It is concluded that  lime ) can replace NaOH as a means of increasing the fermentability of rice straw and that methane production is decreased with potassium nitrate  as NPN source.

Keywords: Alkali, climate change, gas production, greenhouse gases, urea

Introduction

The green house gases (GHG) emissions from the agriculture sector account for about 25.5% of total global radiative forcing and over 60% of anthropogenic sources (FAO 2009). Animal husbandry accounts for 18% of GHG emissions. Emission of methane (CH4) is responsible for nearly as much radiative forcing as all other non-CO2 GHG gases combined (Beauchemin and McGinn 2005). While atmospheric concentrations of GHGs have risen by about 39% since pre-industrial era, CH4 concentration has more than doubled during this period (WHO 2009). Reducing GHG emissions from agriculture, especially from livestock, should therefore be a top priority since it could curb global warming fairly rapidly (Sejian et al 2010).

Ruminants,  such as cattle, buffalo, sheep and goats, are  the major contributors  of total methane agricultural emissions(Leng 1993; Lassey 2007; Chhabra et al 2009).. In ruminants, the H2 produced in rumen fermentation is normally removed by the reduction of CO2 to  methane. However, nitrate has a higher affinity for H2 than CO­2 and is first used to  reduce of NO3 to NO2 and then NO2­ to NH­3, thereby reducing methane  production from CO2. Renewed recognition that nitrate supplements in ruminants  compete successfully for H2 ,and thus decrease methane production, is a promising development (Leng 2008).

The use of nitrate as a source of rumen fermentable nitrogen had previously been discouraged due to the possible formation of toxic  nitrite  under some circumstances  during  nitrate reduction of nitrate to ammonia  (Leng and Preston 2010). However, recent research has shown  that if the adaptation to nitrate  is done  gradually, toxicity is not a problem (Hao Trinh Phuc et al 2009).

The key to improving the use of crop residues for ruminants is to overcome the inherent barriers to rumen microbial fermentation, which in rice straw is the lignification of the cellulose and hemicellulose cell wall components, and to provide supplements of fermentable nitrogen, vitamins and minerals. Among the strategies to improve the straw fermentation, alkaline substances have been the most widely investigated and accepted  for practical on-farm application (Chenost and Kayouli, 1997). The most commonly used alkaline agents are sodium hydroxide (NaOH), ammonia (NH₃), lime (Ca[OH]₂) and urea. Chemical treatments appear to be the most practical for on-farm use since  inexpensive machinery is required,  chemicals are relatively cheap and the procedures  are relatively simple. Hao Trinh Phuc et al. (2009) showed that in goat production studies  given a low-protein rice straw diet, similar improvements in growth rate and N retention were obtained irrespective of whether the animals were supplemented with nitrate or urea. In a similar cattle study, Le Thi Ngoc Huyen et al (2010) compared sodium nitrate and urea as iso-nitrogenous sources of supplementary N to NaOH-treated rice straw, molasses and cottonseed meal. They found that methane production was reduced by nitrate supplementation while feed intake, digestibility and growth rate did not differ between treatments.

The protein in cassava (Manihot esculenta, Crant) leaves is considered to be a good source of bypass protein (Ffoulkes and Preston 1978; Wanapat et al 1997, Sath et al 2008). It is widely cultivated in all tropical counties and is thus a logical forage to provide the additional protein required in diets high in non-protein nitrogen.

The purpose of the present study was to determine if lime (Ca[OH]₂) could replace sodium hydroxide as an alkaline source to improve straw digestibility, and how this might affect methane production in an in vitro incubation, in which the NPN source was either potassium nitrate or urea.

Hypothesis

The hypotheses to be tested were:

• Lime (Ca[OH]2) could replace NaOH to treat rice straw to improve the digestibility in an in vitro incubation.

• Providing the NPN source as potassium nitrate rather than urea will reduce the methane production irrespective of the method of treating the straw.

Materials and methods

Location and duration

An in vitro incubation was conducted in the laboratory of the Faculty of Agriculture and Forest resources, Souphanouvong University, Luang Prabang province, Lao PDR, from May to June 2011. 

Treatments and experimental design 

The experimental design was arranged as a split-plot 2*6 factorial arrangement with four replications of the following treatments:

Source of NPN:

• Urea

• Potassium nitrate

Alkali treatment:

• NaOH 0%  plus  Ca(OH)2 4%

• NaOH 1%  plus  Ca(OH)2 3%

• NaOH 2%  plus  Ca(OH)2 2%

• NaOH 3%  plus  Ca(OH)2 1%

• NaOH 4%  plus  Ca(OH)2 0%

• Untreated

The main plots (one run with 4 flasks) were the NPN sources; the split plots were the alkali treatments. 

 The in vitro system

The equipment and procedure was that used by Sangkhom Inthapanya et al (2011) (Photos 1 and 2).

Photo 1. The in vitro system	Photo 2. Gas production after fermentation

Experimental procedure

The rice straw was chopped into small pieces of around 1-2 cm of length, then ground (1mm sieve). A sample of 500g was taken to treat with combinations of NaOH (% in straw DM: 0, 1, 2, 3, 4) and lime (% in straw DM: 4, 3, 2, 1, 0). The alkalis were dissolved / suspended in water (50% solution) and applied to the straw, which was then stored in plastic bags at room temperature for 14 days. The treated straw was mixed with 30% (DM basis) of fresh cassava leaf before  the incubation, and with either potassium nitrate (6% of substrate DM) or urea (2% of substrate  DM) as source of NPN.

Representative samples of the substrates (12 g DM which included 8 g straw, 3.73g fresh cassava leaf, 0.72 g potassium-nitrate or 0.22 g urea) were put in the incubation bottle to which were added 0.96 liters of buffer solution (Table 2) and 240 ml of rumen fluid (obtained from a recently slaughtered buffalo at the village abattoir), prior to filling each bottle with carbon dioxide. The bottles were incubated at 38 ⁰C in a water bath for 4, 8, 12, 16 and 24 h.  

Data collection and measurements

The gas volume and the percentage of methane in the gas  were recorded at 4, 8, 12, 16 and 24h with a Crowcon infra-red analyser (Crowcon Instruments Ltd, UK, photo 3).  At  24h  the residual DM in the incubation bottle was determined by filtering through cloth and drying (100 ⁰C for 24h) the residue (Photo 4).

Photo 3. Measurement of methane with the Crowcom meter	Photo 4. The substrate residue filtered though cloth

Chemical analyses

Samples of straw and fresh cassava leaf were analysed for DM, ash and N according to methods outlined in Ly and Nguyen Van Lai (1997).

Statistical analysis

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

Results and discussion

Chemical composition

The DM content of rice straw treated with lime or NaOH was lower than in untreated straw (Table 3). Straw treated with lime had higher ash content than straw treated with NaOH. The relatively low solubility of the N in the fresh cassava leaf was indicative of potential bypass protein characteristics.   

Effects of alkali treatment and NPN sources on total gas and methane production

The proportion of substrate fermented after 24h was increased by NaOH-lime treatment and tended to increase as lime replaced NaOH (Table 4; Figures 1 and 2). The total gas production and percentage of methane in the gas increased with time of incubation (Figures 3 and 4). The gas production and the percentage of methane in the gas were reduced when nitrate replaced urea  all stages of the fermentation. After 24h, the percentage methane in the gas and the methane production per unit of fermented substrate was less when nitrate replaced urea as the source of NPN (Figures 5 and 6). There was no consistent effect of alkali treatment of the straw on methane percentage in the gas nor on methane production per unit substrate fermented (Figures 5 and 6). 

Figure 1. Effect on percentage of substrate fermented of combinations of lime (L) and NaOH (N)	Figure 2. Effect on percentage of substrate fermented of replacing NaOH with lime treatment
Figure 3. Effect of incubation time on gas production with KNO3 or urea as NON source	Figure 4. Effect of incubation time on methane content of the gas with KNO3 or urea added to the substrate
Figure 5. Effect of alkali treatment and proportion of lime (L) and NaOH (N) in the alkali medium on percentage methane in the gas	Figure 6. Effect of alkali treatment and proportion of lime (L) and NaOH (N) in the alkali medium on methane produced per unit substrate fermented

The beneficial effects in reduction of methane production when nitrate replaced urea as  NPN source in an in vitro incubation are similar to those reported for more fermentable substrates by Phommasack Outhen et al (2011) for sugar cane, Sangkhom Inthapanya et al (2011) for cassava root, Le Thuy Binh Phuong et al (2011) for sugar cane and Thanh et al (2011) and Phonevilay  et al (2011) for molasses. 

Conclusions

• The total gas production and percentage of methane in the gas increased with  incubation time.

• The gas production and the percentage of methane in the gas were reduced when nitrate replaced urea as the NPN source at all stages of the fermentation.

• After 24h, the methane production per unit of fermented substrate was less when nitrate replaced urea as  NPN source.

• The proportion of substrate fermented after 24h was increased by NaOH-lime treatment and tended to increase as lime replaced NaOH.

• There was no consistent effect of alkali treatment on methane percentage in the gas nor on methane production per unit substrate

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". The authors acknowledge support for this research from the MEKARN project financed by Sida. Special thanks to Mr Nouyang and Mr Jator who provided valuable help in the laboratory.  Thanks also to the staff of Department of Animal Science laboratory, Faculty of Agriculture and Forest Resource, Souphanouvong University for providing the facilities to carry out this research.

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Received 16 August 2011; Accepted 28 September 2011; Published 10 October 2011

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