Methane production in an in vitro rumen fermentation is reduced when the carbohydrate substrate is fresh rather than ensiled or dried cassava root, and when biochar is added to the substrate

Bounthavy Vongkhamchanh, Sangkhom Inthapanya¹ and T R Preston²

Faculty of Agriculture and Forestry, Champasack University, Champasak, Lao PDR
vongkhamchanhd@yahoo.com
¹ Faculty of Agriculture and Forest Resource, Souphanouvong University, Lao PDR
² Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV), Carrera 25 No 6-62 Cali, Colombia

Abstract

This study aimed to evaluate the effect of biochar on methane production in an in vitro rumen incubation in which dried, ensiled or fresh cassava roots were used as energy substrate. Urea and cassava leaf meal were used as sources of NPN and protein. Gas production and methanein the gas were measured over successive 6h intervals for 24h, after which the residaul dry matter (DM) in the substrate was determined by filtration.

There were no differences in gas production among the cassava root treatments during any of the fermentation intervals. By contrast, methane percent in the gas was lowest in the fresh root and highest in the dried root, with intermediate values for the ensiled root. The total DM mineralized after 24h fermentation was not affected by root processing. Gas production tended to be reduced (p=0.07) by biochar for the 18-24h interval but was not affected at the earlier intervals. The methane concentration in the gas was reduced by biochar in the 18-24h fermentation interval, but there was no effect for fermentation intervals of 0-6, 6-12 and 12-18h. Biochar did not affect the proportion of DM mineralized after 24h, but reduced the production of methane per unit DM mineralized.

Key words: greenhouse gases, HCN, processing

Introduction

As most greenhouse gases (GHG), with the exception of methane, have a half-life of over a hundred years, global GHG will have to peak by 2020 and drop by 75-80 per cent in the period to 2050 to limit global warming to two degrees (The Climate Group 2008). The total GHG emissions in 2010 were estimated to have increased by more than 6 per cent, and for 2011 were estimated to have increased by 3.2 per cent (The Guardian 2011; IEA 2011).

Agriculture emissions of methane (CH₄) and nitrous oxide (N₂O), which account for 90 per cent of total agricultural GHG emissions, grew by 17 per cent in the period 1990-2005, roughly proportional to the increase in global cereal production volume, but about three times as fast as the productivity increase in global cereal production (IPCC 2007a). These GHG emissions were predicted to rise by 35-60 per cent by 2030 in response to population growth and changing diets in developing countries, especially in response to greater consumption of ruminant meat and dairy products, as well as the future spread of industrial and factory farming, particularly in developing countries (IPCC 2007b).

The methane emissions from enteric fermentation in herbivorous animals, especially ruminants, are considered a major source of greenhouse gases (Stavi and Lal 2013). Therefore, much research has been designed to investigate the mitigation of methane emissions. In an earlier report (Leng et al 2012) it was shown that biochar derived from rice husks reduced methane production in an in vitro incubation with rumen fluid and a substrate of cassava root meal and cassava leaf meal supplemented with urea or potassium nitrate as the major fermentable N source.

The purpose of this study was to test the effect of biochar on methane production in an in vitro incubation with rumen fluid, in which dried, ensiled and fresh cassava roots were used as energy substrate.

Materials and Methods

Location and duration

The experiment was done in the laboratory of the Faculty of Science, Champasack University, Champasak province, Lao PDR in March, 2015.

Treatments and experimental design

The experiment was designed as a 2*3 factorial in a completely randomized design (CRD) with 4 replications. The factors were:

Source of biochar

• BC: Biochar 1% of the DM in the substrate
• NBC: No biochar

Energy substrate

• FC: Fresh cassava root
• EC: Ensiled cassava root
• DC: Dried cassava root

The in vitro system

Gas production was measured in an in vitro system using recycled PEP water bottles (1 liter) for the fermentation, and gas collection by water displacement (Inthapanya et al 2012).

Experimental procedure

Samples of cassava root and cassava leaves were chopped into small pieces around 1-2 cm in length and dried in the sun for three days then ground to pass through a 1mm sieve. For the ensiled cassava root, it was ground then ensiled for four days. Rice husks were carbonized in an “updraft” stove (Olivier 2010) to produce biochar, which was used as a supplement in the in vitro incubation.

Representative samples of the substrates (12 g DM) 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 cattle in the slaughter house), prior to filling each bottle with carbon dioxide. The bottles were incubated at 38°C in a water bath for 24 h.

Data collection and measurements

During the incubation the gas volume was recorded at 6, 12, 18 and 24h. After each time interval, the methane concentration in the gas was measured with a Crowcon infra-red analyser (Crowcon Instruments Ltd, UK). At the end of the incubation, the residual solids in the incubation bottle were separated by filtering through cloth, to determine mineralization of the substrate.

Chemical analyses

The samples of fresh, dried and ensiled cassava root and dried cassava leaves were analysed for DM and total N according to AOAC (1990) methods.

Statistical analysis

The data were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab Software (version 14.0). Sources of variation in the model were: Cassava root (CR), biochar (BIO), interaction CR*BIO and error.

Results and discussion

There were no differences in gas production among the cassava root treatments at any of the fermentation intervals (Table 1). By contrast, methane percent in the gas was lowest in the fresh root and highest in the dried root, with intermediate values for the ensiled root (Figure 1). The total DM mineralized after 24h fermentation was not affected by root processing.

Gas production tended to be reduced (p=0.07) by biochar for the 18-24h incubation interval but was not affected at the earlier intervals. The methane concentration in the gas was reduced by biochar in the 18-24h fermentation interval, but there was no effect for fermentation intervals of 0-6, 6-12 and 12-18h. Biochar did not affect the proportion of DM mineralized after 24h, but reduced the production of methane per unit DM mineralized (Figure 2).

The effect of cassava root processing on methane production would appear to be related to the levels of hydrocyanic acid (HCN) precursors, which are known to be reduced by ensiling and to a greater extent by drying (Bui Huy Nhu Phuc et al 2001). Phuong et al (2012, 2015) showed that HCN precursors in cassava leaves were reduced by drying and that this effect was subsequently manifested in reduced production of methane, apparently due to the toxic effect of HCN on methanogens (Smith et al 1985: Rojas et al 1999).

The reduction in methane production due to biochar was less than was reported by Leng et al (2012) when biochar was added to an in vitro rumen incubation with cassava root meal as substrate, and nitrate salts or urea as sources of NPN. The reason may be that the biochar used in our experiment had been exposed to lower temperatures during carbonization, which would have reduced the surface area per unit weight. This would have resulted in  a lower capacity to adsorb nutrients and form habitat for syntrophic microbial communities in the biofilm that is believed to be a determining factor in facilitating microbial fermentation of feed organic matter (Leng 2014).

0-6h	6-12h
12-18h	18-24h
Figure 1.  Methane concentrationm in the gas was reduced when the energy substrate was fresh rather than ensiled or dried cassava root

Figure 2. The effect of biochar on methane per unit substrate mineralized from dried, ensiled and fresh cassava root

Conclusions

• In an in vitro rumen fermentation methane percent in the gas was lowest when the energy susbstrate was fresh cassava oot and highest for dried root, with intermediate values for the ensiled root.
  
  
• Biochar reduced methane production: (i)  expressed as a function of the substrate mineralised; and (ii) in the final 18-24h of incubation but with no effect in the earlier intervals of the fermentation.
  
  
• The effect of cassava root processing on methane production would appear to be related to the levels of hydrocyanic acid (HCN) precursors, which are known to be reduced by ensiling and to a greater extent by drying.

Acknowledgements

This research is part of the requirement by the senior author for the degree of PhD at Nong Lam University.  The support from the MEKARN II project, financed by Sida, is gratefully acknowledged, as is the that from the Faculty of Natural Science, Champasack University for providing laboratory facilities to carry out this research.

References

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Received 28 August 2015; Accepted 30 August 2015; Published 1 October 2015

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