Effect of water spinach and biochar on methane production in an in vitro system with substrate of Bauhinia acuminata or Bitter Neem (Azadirachta indica) leaves

Phonevilay Silivong, T R Preston¹, Nguyen Huu Van² and Duong Thanh Hai²

Souphanouvong University, Lao PDR
silivongpvl@yahoo.co.th
¹ Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV), Carrera 25 No 6-62 Cali, Colombia
² Faculty of Animal Sciences, Hue University, Vietnam

Abstract

The objective of this study was to determine the effect of water spinach as source of soluble protein, and of biochar as support for biofilms, on methane production in an in vitro incubation system using leaves of Bauhinia acuminata and Azadirachta indica as the substrate. For each source of leaves, the experimental design was a 2*2 factorial arrangement of 4 treatments with four replications. Measurements were made of total gas, percent methane in the gas and DM and N solubilized, at intervals of 6, 12, 18 and 24h.

Gas production and percent substrate solubilized were increased by water spinach on both Bauhinia and Bitter Neem. By contrast, water spinach did not affect methane production on Bauhinia but increased it on Bitter Neem. Biochar supplementation had no effect on total gas and methane production.

Keywords: fermentation, greenhouse gases, leaf meal, incubation, solubility

Introduction

In developing improved systems for feeding live stock, account must also be taken of the impacts on the environment. It is estimated that live stock presently account for some 18% of total greenhouses gases which contribute to global warming (Steinfeld et al 2006). Enteric methane from fermentative rumen digestion is the main source of these emissions. There is an urgent need to develop ways of reducing methane emissions from ruminants in order to meet future targets for mitigating global warming.

Previous research showed that methane production was less when fish meal rather than groundnut meal was the substrate (Preston et al 2013) and when Sesbania and cassava leaves were the substrate compared with water spinach and sweet potato foliage. The differences in methane production between the different protein sources appeared to be related to the solubility of the protein which was 17% in fish meal compared with 76% in groundnut meal and 33 and 35% in Sesbania and cassava compared with 71% for water spinach and sweet potato foliage.

Another positive approach to the problem of how to reduce methane emissions from live stock has been to incorporate a low level (1%) of biochar in the diet (Sangkhom et al 2012; Leng et al 2012a,b,c). Biochar is the product of incomplete carbonization of fibrous biomass at high temperatures (Lehmann and Joseph 2009). It is a highly porous material with a large surface area which gives it valuable properties as a support mechanism for biofilms that facilitate the adsorption of consortia of micro-organisms and nutrients.

The purpose of the present study was to measure the methane production from tree leaves with low protein solubility (Bauhinia acuminata and Azadirachta indica) and to determine if methane production would be increased by supplementation with water spinach, the protein in which is highly soluble.

Hypotheses

The hypotheses to be tested in an in vitro rumen incubation were:

• Gas production and dry matter fermented would be increased when water spinach was incorporated in the basal substrate of leaves from Bauhinia acuminata or Azadirachta indica (Bitter Neem).
  
  
• Biochar would reduce methane production

Materials and methods

Location and duration

For each substrate, the experimental design was a 2*2 factorial arrangement of 4 treatments with four replications.

The factors were:

•  With or without water spinach (WS and No-WS)

•  With or without biochar (BC and No-BC)

Preparation of substrate and the in vitro system

A simple in vitro system was used (Photo 1) with recycled plastic bottles as flasks for the incubation and gas collection.

• A and B bottles (1.5 liters capacity)
• C bottle (3 liters capacity)

The A bottle containing representative samples for fermentation was connected with the B bottle by a plastic tube (8mm of diameter). The B bottle was marked at 50ml interval before being suspended in the C bottle containing water. Clay was used to cover the stoppers of the plastic bottle and junction of stopper and plastic tube to prevent leakage of gas. Gas production was measured by water displacement. The leaves from Bauhinia and Bitter Neem, and leaves plus stems of water spinach, were chopped into small pieces (3-5mm) and dried at 65°C for 48h then ground with a coffee grinder, and mixed according to the proportions shown in Table 1. The mixtures (12g DM) were put in the incubation bottle with 960 ml of buffer solution (Table 2) and 240 ml of rumen fluid. The rumen fluid was taken at 3.00-4.00am from 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 taken to the laboratory, and stored until 5.00am, when the contents were filtered through a layer of cloth before being added to the incubation bottle. The remaining air in the flask was flushed out with carbon dioxide. The bottles were incubated at 38°C in a water bath for 24 h.

Photo 1. The in vitro fermentation system using recycled water bottles and water displacement to measure gas production

Data collection and measurements

Incubations were carried out for 6, 12, 18 and 24h. At the end of each incubation, the methane concentration in the gas was measured with a Crowcon infra-red analyser (Crowcon Instruments Ltd, UK; Photo 2). The residual DM in the incubation bottle was determined by filtering through cloth (Photo 3) and drying the residue (65°C for 72 h). N in the filtrate (N solubilized) was determined according to AOAC (1990) method. Solubility of the protein in the leaves was determined by shaking 3 g of dry leaf meal in 100 ml of M NaCl for 3h then filtering through Whatman No. 4 filter paper, and determining the N content of the filtrate (Whitelaw et al 1963).

Photo 2. Measurement of percentage of methane in the gas	Photo 3. The substrate residue filtered though cloth

Chemical analyses

The samples of foliage, water spinach and residual substrate were analysed for DM, ash and N according to AOAC (1990) methods.

Statistical analysis

The data from the experiment were analyzed by the general linear model option of the ANOVA program in the Minitab software (Minitab 2000). Sources of variation in the model were: water spinach, biochar, interaction water spinach*biochar and error.

Results and discussion

Chemical composition

Percentages of crude protein and ash were lower in the leaves of Bauhinia and Bitter Neem than in water spinach (Table 3). The protein solubility values for both Bauhinia and Bitter Neem were only one third of that in water spinach.

Values for the gas production, percent methane in the gas and methane produced per unit substrate solubilized increased with length of incubation time (Tables 4a and 4b; Figures 1-4a and 1-4b). Gas production and percent substrate solubilized were increased by water spinach on both Bauhinia and Bitter Neem. By contrast, water spinach did not affect methane production on Bauhinia but increased it on Bitter Neem.

Figure 1a. Effect of water spinach and biochar on gas production from Bauhinia

Figure 1b. Effect of water spinach and biochar on gas production from Bitter Neem

Figure 2a. Effect of water spinach and biochar on percent of methane in the gas from Bauhinia

Figure 2b. Effect of water spinach and biochar on percent of methane in the gas from Bitter Neem

Figure 3a. Effect of water spinach and biochar on percent DM solubilized from Bauhinia

Figure 3b. Effect of water spinach and biochar on percent DM solubilized from Bitter Neem

Figure 4a. Effect of water spinach and biochar on methane production per unit substrate solubilized from Bauhinia

Figure 4b. Effect of water spinach and biochar on methane production per unit substrate solubilized from  Bitter Neem

The increases in methane concentration in the gas and per unit DM solubilized, with fermentation time, are similar to the findings by several researchers (Inthapanya et al 2011, Binh Phuong et al 2011, Thanh et al 2011) who used a similar in vitro system but with different substrates). Marin et al (2014) reported a similar effect on methane production when the in vitro fermentation proceeded for 48h compared with 24h. Leng et al (2012a) proposed that the increase in methane with fermentation time reflected the change in substrate for methanogens from hydrogen to acetate as occurs in the biodigester. The increase in methane production on the Neem leaf substrate, due to inclusion of water spinach foliage, compared with absence of such an effect when water spinach was added to the Bauhinia, presumably reflected the decrease in the anti-microbial effects of the oils in the Neem leaves( http://en.wikipedia.org/wiki/Azadirachta_indica) as their concentration in the Neem substrate was decreased by addition of water spinach. In the in vitro rumen it is likely that the protozoal population was most affected by the oils in the Neem leaves, which in turn would reduce the population of methanogens in view of their close association with rumen protozoa (Williams 1986).

The effect of the water spinach on methane production ranged from no effect (with Bauhinia) to an increase in methane on the Neem substrate. Inthapanya and Preston (2014) reported that methane production was decreased when cassava leaves replaced water spinach in an in vitro fermentation of urea-treated rice straw. In this case, the decrease in methane could have been due to the effect of the tannins in cassava leaves reducing the activity of the methanogenic bacteria as reported by several researchers (Goel and Makkar 2012; Soltan et al 2012). Bauhinia leaves contain appreciable levels of tannins according to Queiroz Siqueira et al (2012) as do the leaves of Neem ( http://www.herbalextractsplus.com/neem-leaf.html), while water spinach has almost none (less than 0.01% in the report by Igwenji et al 2011).

Conclusions

• Increasing the length of the incubation in the in vitro rumen fermentation of leaves of Bauhinia acuminata and Azadirachta indica increased gas production, methane concentration in the gas and methane produced per unit substrate solubilized.
  
  
•  Supplementation with water spinach increased the rate of gas production and the percentage DM solubilized on both sources of leaves.
  
  
• Water spinach did not affect methane production from Bauhinia leaves but increased the methane production from Neem leaves.

Acknowledgements

This research was done by the senior author with support from SIDA MEKARN II program as part of the requirements for the PhD degree in Animal Production "Improving Livelihood and Food Security of the people in Lower Mekong Basin through Climate Change Mitigation". The authors acknowledge support for this research from the MEKARN II project financed by Sida. Special thanks to Mr Bountou and Mr Khamsay who provided valuable help in the laboratory.They also thank the Faculty of Agriculture and Forest Resource, Souphanouvong University for providing the facilities to carry out this research.

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Received 4 February 2015; Accepted 25 February 2015; Published 3 March 2015

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