Effect of sweet or bitter cassava leaves and biochar on methane production in an in vitro incubation with substrates of Bauhinia acuminata and water spinach (Ipomoea aquatica)

Phonevilay Silivong¹, T R Preston², Nguyen Huu Van³ and Duong Thanh Hai³

¹ Animal Science Department, Faculty of Agriculture and Forest Resource, Souphanouvong University, Lao PDR
silivongpvl@yahoo.com
² Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV), Carrera 25 No 6-62 Cali, Colombia
³ Faculty of Animal Sciences, Hue University of Agriculture and Forestry, Hue University, Vietnam

Abstract

The aim of this study was to determine the effect of biochar and leaf meal from sweet and bitter cassava leaves on methane production in an in vitro incubation of Bauhinia acuminata and water spinach as basal substrate. The experimental design was a 2*2 factorial arrangement of 4 treatments (leaf meal of sweet or bitter cassava; with and without biochar) with 4 replications. Measurements were made of gas production, percent methane in the gas and DM digested after incubation periods of 6, 12, 18 and 24h.

Increasing the length of the incubation increased methane concentration in the gas and methane produced per unit substrate digested. The methane content of the gas was reduced when leaves of bitter cassava replaced leaves of sweet cassava as protein source and when 1% of biochar was added to the substrate. The magnitude of the differences was relatively small but consistent for all incubation times. The proportion of the substrate DM that was digested during the incubation was increased when biochar was included in the substrate and reduced when the protein supplement was leaf meal from bitter compared with sweet cassava.

It is concluded that the higher levels of cyanogenic glucosides (precursors of hydrocyanic acid HCN) in the leaves from bitter compared with sweet cassava were the reasons for the reduction in digestibility of the substrate and in the production of methane during the in vitro incubations

Key words: cyanogenic glucosides, fermentation, greenhouse gas, HCN, incubation

Introduction

Reducing GHG emissions from agriculture, especially from livestock, is a priority in order to reduce global warming (Sejian et al 2010). Ruminants - cattle, buffalo, sheep and goats - are the major contributors of methane emissions from the agriculture sector (Lassey 2007). There is therefore a need to develop feeding systems for ruminants that will result in reduced emissions of methane gas from the enteric fermentation in these animals. Promising ways to do this are: (i) by the feeding of biochar (Leng et al 2012) derived from the carbonization of fibrous wastes (Orosco et al 2018); and (ii) using as protein supplement the foliage from bitter as opposed to sweet cassava (Phuong et al 2012).

The objective of the research described in this paper was to combine both of these factors as a means to reduce methane production in goats fed foliage from the legume tree Bauhinia acuminata supplemented with foliage from water spinach.

Materials and methods

Location and duration

The experiment was carried out from May to June 2018 at the Animal Science Laboratory of the Faculty of Agriculture and Forest Resources, Souphanouvong University, Luang Prabang Province, Lao PDR.

Treatments and experimental design

The experimental design was a 2*2 factorial arrangement of 4 treatments with four replications.

The factors were:

Source of cassava leaves: Sweet (SC) or bitter (BC) variety

Biochar: With (Bio) or without (NoBio) biochar

Preparation of substrate and the in vitro system

The in vitro system used recycled “PET” plastic bottles as flasks for the incubation and gas collection (Diagram 1).

Diagram 1. A schematic view of the in vitro system to measure gas production in an in vitro incubation (Inthapanya et al 2017)

The B bottle containing the substrate for fermentation was connected to the D bottle by a plastic tube (4mm diameter). The D bottle was marked at 50ml intervals 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, cassava and water spinach were chopped into small pieces (3-5mm) and dried at 65°C for 48h then ground with a coffee grinder. The biochar was produced by burning rice husks in a top-lit updraft (TLUD) gasifier stove (Photos 1-3).

Photos 1-3. Rice husks, gasifier stove and biochar derived from the rice husks

The biochar was ground to a particle size that passed through a 1 mm sieve and mixed with the other ingredients according to the proportions shown in Table 1. The mixtures (12g DM) were put in the incubation bottle with 960ml of buffer solution (Table 2) and 240ml of rumen fluid. The rumen fluid was taken at 3.00-4.00am from a buffalo immediately after the animal was slaughtered in the Luang Prabang abattoir. A representative sample of the rumen contents (including feed residues) was put in a vacuum flask and stored in the laboratory. At 5.00am the contents were filtered through a layer of cloth and 240ml of the filtrate added to each of the incubation bottles. The remaining air in the bottles was flushed out with carbon dioxide. The bottles were incubated at 38°C in a water bath for periods of 6, 12, 18 and 24h, with separate incubations for each time.

Data collection and measurements

At the end of each incubation, the volume of gas was measured, and the methane concentration recorded by passing the gas through a Crowcon infra-red analyser (Crowcon Instruments Ltd, UK). The residual DM in the incubation bottle was determined by filtering through cloth and drying the residue (65°C for 72 h). Solubility of the protein in the cassava leaves was determined by shaking 3g 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). The leaves of cassava, bauhinia, water spinach and residual substrate were analysed for DM, ash and N according to AOAC (1990) methods="_Toc315720164"

Statistical analysis

The data were analyzed by the general linear model option of the ANOVA program in the Minitab software (Minitab 2014). In the model the sources of variation were: treatments, replicates and error. The statistical model used was:

Y_ijk = μ + P_i + A_j + P_i*A _j+ e_ijk

μ = Overall mean

P_i = Source of sweet or bitter of cassava leaves supplementation effect

A_j = with or without biochar supplementation effect

P_i*A_j = Interaction between source of cassava leaves * with or without biochar

e_ijk = random error

Results and discussion

Chemical composition

Protein solubility was lowest in Bauhinia, was lower in bitter than in sweet cassava leaves and highest in water spinach leaves (Table 3).

The linear increase in methane concentration in the gas with duration of the incubation (Table 4; Figures 1 and 3) is similar to the trends observed by Outhen et al (2011), Binh Phuong et al (2011), Inthapanya et al (2011) and Silivong and Preston (2015) who used the same in vitro fermentation model. These results indicate that there is a lag time either in the growth of organisms that ferment carbohydrate to VFA and hydrogen, and/or in the growth of those that convert hydrogen to methane.

The methane content of the gas was reduced when leaves of bitter cassava replaced leaves of sweet cassava as protein source (Figure 1) and when 1% of biochar was added to the substrate (Figure 2). The magnitude of the differences was relatively small but consistent for all incubation times.

Figure 1. Effect of sweet or bitter cassava on % methane in the gas	Figure 2. Effect of biochar on % methane in the gas

The proportion of the substrate DM that was digested during the incubation was increased when biochar was included in the substrate (Figure 3) and reduced when the protein supplement was from bitter compared with sweet cassava (Figure 4).

Figure 3. Effect of supplementation with biochar on proportion of substrate digested	Figure 4. Effect of bitter versus sweet cassava on proportion of substrate digested

Addition of biochar reduced the production of methane per unit substrate digested (Figure 5). However, there was no consistent trend for effects of bitter versus sweet cassava (Figure 6). As was to be expected the production of methane per unit substrate digested increased linearly with duration of the incubation.

Figure 5. Effect of supplementation with biochar on production of methane per unit substrate digested	Figure 6. Effect of bitter versus sweet cassava on production of methane per unit substrate digested

Discussion

The reduction in production of methane when leaves of bitter cassava replaced those from sweet cassava has been reported in several in vitro incubations (Phuong et al 2012; Binh et al 2018) and appears to be a direct effect of the higher HCN levels in the bitter varieties being toxic to methanogens (Smith et al 1985). However, the major reduction (18%) in digestibility in the 24h incubation, when leaves from bitter cassava replaced those from sweet cassava, has not previously been reported; in fact, the opposite effect was observed by Phuong et al (2012). The logical assumption is that the reduced digestibility reflected a more general HCN toxicity on the activity of all rumen microbes.

The reduction in methane production due to biochar was much less than has been observed in previous studies. A reduction of 13% was recorded by Phuong et al 2012; of 8% by Vongkhamchanh et al 2015; and 12% by Binh et al 2018). The implication is that the sample of biochar used in this experiment may have been of inferior quality in terms of its relative surface area: weight ratio (and hence its capacity to support microbial communities in biofilms [Leng 2017]). This stresses the need for a simpler quality test than the standard “BETS” ratio (for which the measuring equipment is not available in laboratories in Lao PDR).

Conclusions

• Increasing the length of the incubation from 6 to 24h in an in vitro rumen fermentation increased methane concentration in the gas and methane produced per unit substrate DM digested.
  
  
• The methane content of the gas was reduced when leaves of bitter cassava replaced leaves of sweet cassava as protein source and when 1% of biochar was added to the substrate. The magnitude of the reduction due to biochar was relatively small but consistent for all incubation times.
  
  
• The proportion of the substrate DM that was digested during the incubation was increased when biochar was included in the substrate and reduced when the protein supplement was leaf meal from a bitter compared with a sweet cassava variety.

Acknowledgements

This research was done by the snior author with support from SIDA MEKARN II program "Improving Livelihood and Food Security of the people in Lower Mekong Basin through Climate Change Mitigation" as part of the requirements for the PhD degree in Animal Production. The authors acknowledge support for this research from the MEKARN II project financed by Sida. Special thanks to Mr. Singkham and Mr. Syphone 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 10 August 2018; Accepted 14 August 2018; Published 3 September 2018

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