Methane production in an in vitro rumen fermentation of molasses-urea was reduced by supplementation with fresh rather than dried cassava (Manihot esculenta, Crantz) leaves and by biochar

Phonethep Porsavathdy, Sengsouly Phongphanith¹, T R Preston² and R A Leng³

Faculty of Agriculture and Environment, Savannakhet University, Laos PDR
pphonetheb@gmail.com
¹ Faculty of Agriculture and Forest Resource, Souphanouvong University, Laos PDR
² Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV), Carrera 25 No 6-62 Cali, Colombia
³ University of New England, Armidale NSW, Australia

Abstract

The aim of this study was to evaluate the effect of fresh and sun-dried cassava leaves together with biochar on methane production in an in vitro rumen incubation of molasses-urea as basal substrate. The design was a 2*2 factorial in CRD with 4 treatments and 4 replications. The factors were processing of cassava leaves: fresh and sun-dried; and with or without biochar (1% of substrate DM). Total gas and percent methane in the gas were measured at intervals of 12 and 24h.

Methane as a proportion of the gas and as a function of substrate fermented was reduced when fresh rather than dried cassava leaf was the protein source and when biochar was added to the substrate.

Keywords: cyanide, greenhouse gas, HCN, methanogenesis

Introduction

Ruminant animals are a major source of total anthropogenic methane emissions producing an estimated 80 million tonnes of methane annually accounting for 33% of anthropogenic emissions of this greenhouse gas (Beauchemin et al 2008).

Successful mitigation of ruminant GHG emission is challenging technically but is made even more difficult because of the rising demand for milk and meat (Steinfeld et al. 2006). Thus it is important to study ways of maintaining good animal performance but with reduced levels of methane. The challenge is to increase the production of meat and milk from ruminants without increasing, and preferably reducing, methane emissions.

The foliage of cassava (Manihot esculenta, Crantz) has been shown to be an effective source of bypass protein for stimulating growth rates of fattening cattle (Ffoulkes and Preston 1978; Sath et al 2008) and sheep (Ho Quang Do et al 2002) and growth and milk production of goats (Thanh et al 2013; Dung et al 2010). It is widely cultivated in all tropical counties and is thus a logical forage to provide the additional protein required in diets high in fermentable carbohydrate and urea. Cassava leaves are known to contain variable levels of condensed tannins; about 3% in DM according to Netpana et al (2001) and Bui Phan Thu Hang and Ledin (2005), which are reported to decrease methane production and increase the efficiency of microbial protein synthesis (Makkar et al 1995; Grainger et al 2009). Reductions of methane production by 13 to 16% were reported by Carulla et al (2005), Waghorn et al (2002) and Woodward et al (2004), apparently through a direct toxic effect on methanogens.

According to MAF (2013) the total planted area of cassava in Lao PDR is 43,975 ha, with an average root yield of 24tonne/ha, which in 2013 increased to 45,185 ha and an average yield of 28 tonne/ha (Tin Maung Aye 2015). Fresh foliage yields of cassava managed as a semi-perennial forage crop in Cambodia with repeated harvests at 50 to 70 day intervals and fertilized with biodigester effluent were 20 tonnes/ha (Preston et al 2000).

Biochar is the residue when fibrous biomass is carbonized at high temperatures. It has been shown to play an active role in systems involving microbial fermentation (Lehmann and Joseph 2009). It is believed that the biochar, which is highly porous, may also act as “habitat” for biofilms that facilitate the functioning of consortia of micro-organisms and their nutrients (Leng et al 2012a).

The purpose of the present study was to determine the effects on methane production in an in vitro rumen fermentation when biochar was combined with either fresh or sun-dried cassava leaves provided as supplements to a basal substrate of molasses-urea.

Hypotheses

Methane production would be reduced when a basal substrate of molasses-urea in an in vitro rumen incubation was supplemented with biochar as a source of biofilm, and by fresh compared with sun-dried cassava leaves as a source of bypass protein.

Materials and methods

Location and duration

The experiment was conducted in the laboratory of the Faculty of Agriculture and Forest Resource, Souphanouvong University, Luang Prabang province, Lao PDR, from November to December 2015.

Treatments and experimental design

Four treatments were arranged as a 2*2 factorial in completely randomize design with 4 replications. The factors were:

· Fresh (FC) or dried (DC) cassava leaves

· Biochar at 0 (NBio) or 1% (Bio) of the substrate DM

The basal substrate was molasses with 3% urea in DM (Table 1).

The in vitro system

Recycled water bottles (capacity 1500 ml) were used for the fermentation and collection of the gas (Photo 1). A hole was made in the lid of each of the bottles, which were inter-connected with a plastic tube (id 4mm). The bottle receiving the gas had the bottom removed and was suspended in a larger bottle (2 liter capacity) partially filled with water, so as to collect the gas by water displacement. The bottle that was suspended in water was calibrated at 50 ml intervals to indicate the volume of gas.

Photo 1. The in vitro system

Preparation of diets and rumen fluid

The cassava leaves were chopped into small pieces and milled in a coffee grinder either fresh or after drying in an oven at 105 °C for 24 h,. They were then mixed with urea. A representative sample of the mixtures (12 g DM) (Table 1) was put in the fermentation bottle to which was added 0.96 liters of buffer solution (Table 2) and 240 ml of rumen fluid (obtained from a newly slaughtered buffalo in the town abattoir), prior to displacing the air with carbon dioxide. Each junction of the connecting tube with the bottles was covered by "plasticine" (modelling clay) to ensure a gas-tight seal (Photo 2). The bottles with substrate were then incubated at 38°C in a water bath for 24h.

Data collection and measurements

Gas production was estimated by water displacement. The percentage of methane in the gas was measured by infra-red sensor (Crowcom Instruments Ltd, UK), after 12 and 24h of fermentation. At the end of the incubation the total gas volume and total methane production were calculated. Residual insoluble DM from the substrate was determined by filtration through cloth (Photo 2) and drying the residues at 100°C for 24h.

Photo 2.The substrate residue filtered through cloth

Statistical 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: cassava leaf, biochar, interaction cassava leaf*biochar and error.

Results

Gas production was lower, as was percent methane in the gas, when fresh rather than dried cassava leaves was the protein source; and when biochar was added to the substrate (Table 3; Figures 1-2).

DM mineralized was reduced when fresh rather than dried cassava leaves were the protein source, and tended (p = 0.055) to be reduced when biochar was added to the substrate (Table 3; Figures 3 and 4).

Methane production per unit substrate DM mineralized was reduced by fresh compared with dried cassava leaves and by biochar (Figures 5 and 6).

Figure 1. Effect of drying cassava leaves on gas production (0-24h)	Figure 2. Effect of biochar on gas production (0-24h)

Figure 3. DM mineralized was lower when fresh rather than dried cassava leaves were the protein source	Figure 4. DM mineralized was lower when biochar was added to the substrate.

Figure 5. Methane produced per unit substrate mineralized was lower for fresh rather than dried cassava leaves	Figure 6 . Methane produced per unit substrate mineralized was lower when biochar was added to the substrate.

Discussion

The reduced production of methane in an in vitro rumen incubation when fresh leaves replaced dried leaves is in agreement with a previous study by Sangkhom et al (2012) when cassava root was the carbohydrate substrate and cassava leaves were used fresh or after sun- or oven-drying. The mechanism for the reduction in methane with fresh leaves is that: (i) drying the cassava leaves reduces the liberation of HCN (Phuc et al 1996; Phoung et al 2012a,b); and (ii) it is the liberated HCN which inhibits methanogenesis (Smith et al 1985; Gijzen et al 2000).

The effect of biochar in reducing methane production has been reported in in vitro rumen incubation systems (Leng et al 2012a; Seengsouly and Preston 2016a), and in growing cattle (Leng et al 2012b; Sengsouly and Preston 2016b). Leng (2012a) ascribes this effect of the biochar to its role as support for biofilms which facilitate rumen microbial ecosystems including those that reduce methanogenesis.

Conclusions

The percent methane in the gas and methane produced per unit substrate mineralized was: lower for fresh than for sun-dried cassava leave; and lower with added biochar.

Acknowledgements

This research was done by the senior author as part of the requirements for the MSc degree in Animal Production "Improving Livelihood and Food Security of the people in Lower Mekong Basin through Climate Change Mitigation" in Cantho University, Vietnam. The authors acknowledge support for this research from the MEKARN II project financed by Sida.

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Received 21 November 2016; Accepted 5 February 2017; Published 1 March 2017

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