Methane production is reduced in an in vitro incubation when the rumen fluid is taken from cattle that previously received biochar in their diet

R A Leng*, Sangkhom Inthapanya and T R Preston**

Faculty of Agriculture and Forest Resource, Souphanouvong University, Lao PDR
inthapanyasangkhom@yahoo.com
* University of New England, Armidale NSW, Australia
** Finca Ecológica TOSOLY, AA 48 Socorro, Colombia

Abstract

In an in vitro incubation the treatments in a 2*2 factorial arrangement were: rumen fluid from cattle previously fed biochar (BA), rumen fluid from cattle previously fed biochar +  biochar added to the substrate (BA+BC), rumen fluid from cattle not previously fed biochar (NBA) and rumen fluid from cattle not previously fed biochar +  biochar added to the substrate (NBA+BC). There were 4 replications of each treatment. The substrate contained  (DM basis): 70% cassava root meal, 26.5-28% cassava leaf meal and 2% urea. In treatments BA+BC and NBA+BC the biochar was added at 1.5% of the substrate DM. These ingredients were mixed together in the incubation flask to which was added 480 ml of buffer solution and 120 ml of strained rumen fluid taken by stomach tube from cattle fed cassava root, cassava foliage and urea and either biochar (0.62% of diet DM) (for BA treatments) or no biochar (treatments NBA).

Gas production and percentage substrate DM solubilized were increased, and percent methane in the gas was reduced, when: (i) the rumen fluid in the incubation flask was taken from cattle adapted to 0.62% biochar in their diet (DM basis) over a 4 month period; and (ii) when biochar was added to the incubation medium at 1.5% of DM. There were additive effects on methane reduction when rumen fluid from adapted cattle was combined with biochar added to the incubation medium.

Key words: biofilm, climate change, consortia, global warming,  greenhouse gases

Introduction

In a previous report from our laboratory (Leng et al 2012), we showed that addition of 0.62% biochar to the cassava root and cassava leaf meal diet, fed to local “Yellow” cattle, resulted in decreased production of methane in eructated gases. In the present experiment we tested the hypothesis that the rumen fluid from the cattle that had been fed biochar would retain properties conducive to decreasing methane production when added as inoculum to an in vitro incubation with cassava root and cassava leaf meal supplemented with urea as the substrate.

Materials and methods

Location and duration

The in vitro incubation experiment was conducted in the laboratory of the Faculty of Agriculture and Forest Resource, Souphanouvong University, Luang Prabang province, Lao PDR, during October 2012.  

Treatments and experimental design 

The experimental design was a 2*2 factorial arrangement with four replications of each treatment.  Individual treatments were:

• BA: Rumen fluid from cattle previously fed biochar
• BA+BC:  Rumen fluid from cattle previously fed biochar +  biochar added to the substrate
• NBA: Rumen fluid from cattle not previously fed biochar
• NBA+BC: Rumen fluid from cattle not previously fed biochar +  biochar added to the substrate

The substrate contained cassava root meal, cassava leaf meal and urea (Table 1). 

The equipment and procedures were those used in previous experiments in our laborataory (Inthapanya et al 2011).

Newly harvested cassava roots and leaves were chopped into small pieces of around 1-2 cm of length and dried in the oven for 24 h at 80°C and then ground through a 1 mm sieve. The biochar was produced locally by burning rice husks in a top lit updraft (TLUD) gasifier stove (Olivier 2010). The biochar had a particle size that passed through a 1 mm sieve and was produced at a temperature of 900-1000^oC (Olivier 2010). The biochar in treatments BA+BC and NBA+BC was mixed with the cassava root and leaf meal and urea (Table 1) prior to adding to flasks containing 120 ml of rumen fluid and 480 ml of buffer solution made according to Tilly and Terry (1963).  The incubation flasks were then gassed with carbon dioxide and incubated in a water bath at 38 ^°C for 24 hours. The rumen fluid was collected by stomach tube from the cattle in the experiment reported by Leng et al (2012).

Data collection and measurements

The gas volume was read from the collection bottles directly after 24  hours and the percentage of methane in the gas measured using a Crowcon infra-red analyser (Crowcon Instruments Ltd, UK).   Three samples were measured from each collection bottle. At the end of the incubation the residual insoluble substrate in the incubation bottle was determined by filtering the contents through several layers of cloth that retained particle sizes to at least 0.1mm and then this was dried (100 °C for 24 hours) and weighed.

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: Replictates, Biochar, Rumen fluid source, Interaction Biochar*Rumen fluid source and error.

Results and discussion

Gas production and percentage DM solubilized were increased, and percent methane in the gas was reduced, when: (i) the rumen fluid in the incubation was taken from cattle adapted to 0.62% biochar in their diet (DM basis) over a 4 month period; and (ii) when biochar was added to the incubation medium at 1.5% of DM (Table 2 and Figures 1 and 2). There were additive effects on methane reduction when rumen fluid from adapted cattle was combined with biochar added to the incubation medium (Figure 3).   

Figure 1. Effect of rumen fluid (from cattle fed or not fed biochar during previous 4 months) and  biochar (with or without) in the substrate in an in vitro incubation with cassava root meal, cassava leaf meal and urea.	Figure 2. Effect of biochar (with or without) in the substrate in an in vitro incubation with cassava root meal, cassava leaf meal and urea and with rumen fluid from cattle adapted or not to biochar in the diet

Figure 3. Effect of adaptation to biochar in the diet, and of addition of biochar to the substrate, on percent reduction in methane production (ml methane/g DM solubilized) in an in vitro incubation of cassava root meal with cassava leaf meal and urea

Discussion

The studies now presented confirm that biochar produced from rice husk at high temperature when added to an anaerobic fermentative system using rumen fluid lowers the net production of methane. The mechanisms for this net reduction are not explained by any of the studies so far undertaken and we provide below some speculative suggestions of how this might be accomplished in order to stimulate discussions in this area, particularly by young scientists.

Biochar provides  a large surface area for mixed microbial communities  and undoubtedly, in the presence of organic matter, biochars become impregnated with microorganisms (Anonymous 2012).

In these studies a mixture of high starch and high fibre feed has been used as substrate for microbes in or from the rumen. Digestion of complex substrates in the rumen requires the coordinated activities of a number of different microbial species and  is most efficient when  these microbes are components of communities, self-organised within a structured biofilm (Wang and Chen 2009). Association and attachment  to feed particles by ruminal organisms is rapid (Cheng et al 1980) occurring within five minutes of feed entering into rumen fluid (Bonhomme 1990).  Ruminal organisms attach to plant derived feed particles containing mostly structural carbohydrates, and usually adhere through an extensive glycocalyx which encloses the bacterial colonies. These commence structural plant degradation by secreting  enzymes, which then hydrolyse the exposed structural components of fibrous plant parts (see Cheng et al 1980).  The hydrolytic products of these initiating bacteria attract other species with particular substrate requirements and in turn produce an endogenous extra-cellular polymeric substance which  forms the matrix for biofilm formation to which microbial populations are attracted and grow  in a structured or layered biofilm (Costerton 2007). These multi species biofilms are either positioned on the external surface  of the plant tissue,  or certainly with cereal  grain particles, develop internally as substrate is solubilised and utilised (McAllister et al 1994) . In these layered structures sequential degradation of both complex structural carbohydrates and more readily fermentable starches/sugars occurs  through a train of microbes that complement one another in that the end products of the energy metabolism of one group provide substrate for other closely closely associated microbial colonies. As an example of this the cellulolytic organism Fibrobacter succinogenese has the capacity to hydrolyse  a number of complex carbohydrates but only uses cellulose and its hydrolytic  products in its energy metabolism (Suen et al 2011).  In doing so it strips other recalcitrant structural carbohydrates to soluble components (mainly soluble sugars) providing enzymic access to the cellulose fibres they surround . The soluble mono and disaccharides not used by F succinogneses  then appear to  diffuse from the surface of the plant materials and are  likely to be degraded further by other microbes in the outer components of the biofilm matrix with the production of VFA  and ATP and reduction of cofactors . Overall  the rate of the fermentative process depends on the rate of regeneration of reduced cofactors and the release of hydrogen.  However,  a requisite for continuing fermentation is a low partial pressure of hydrogen since hydrogen build up would inhibit hydrogenase activity  resulting in lowered levels of NAD, NADP and FAD which slows or inhibits fermentation (see Wolin 1979; McAllister and Newbold 2008) and this is normally controlled by  the rate of methanogenesis.

Ruminal microorganisms can be functionally described as four sub populations: (i) those associated with ruminal fluid; (ii) those loosely attached to the  feed particles probably in the outer layer of the biofilm; (iii) those tightly bound to the particle surface:  and (iv) those that remain in the biofilm within feed particles (see Wang and McAllister 2002). The rumen fluid used in the incubation procedures is likely to have a population of microbes different to the mixed digesta but the rapidity of attachment and biofilm growth (Cheng et al 1980) suggests that these will be a source of inoculating organisms and will reflect therefore the population densities in the rumen. Thus it is useful to study the actions of rumen fluid from animals adapted to biochar and unadapted as the differences in rumen conditions should be reflected in the changes particularly of net  methane production.

In these studies we are interested in seeing whether there is benefit in mitigating enteric methane production  by increasing the  inert surface areas where microbes may come together for mutual feeding benefits.

The hypothesis developed from the companion paper (Leng et al 2012) is that solubilised feed materials and hydrogen diffusing from the particles being digested are partially or totally taken up as  they diffuse  towards the bulk fluid in the rumen  and that this will be aided by inert materials that provide surfaces either closely associated with the feed particle and biochar particles or by biochar suspended in the fluid.

Rumen fluid obtained in these studies is not representative of rumen digesta being lower in particles where it could be expected most of the effects of biochar will be exerted  However, we also anticipated that since the colonisation of feed is extremely rapid, that if biochar increased the population density of microbes in the digesta,  that the levels of these would be amplified in the liquor/small particle mix used in the incubation medium.

The results overall indicate that adapted rumen fluid  reduced net methane production and increased the rate of substrate solubilisation perhaps consistent with a larger population in rumen digesta of the consortia that oxidise methane. The additive effect of biochar in vitro with rumen fluid from animals adapted to biochar can also be hypothesised to be due to an initial higher population of these same organisms in the rumen fluid from adapted animals. The response to added biochar to rumen fluid from non adapted sources  which is higher than that from adapted rumen fluid without biochar suggests that the population dynamics of these were lower and that the activity had been lower in the absence of the biochar.

It is clear that biochar in a diet/substrate alters the population mix of microbes in the digesting medium. Its ability to be impregnated with microbes is probably the major association here and it is tentatively suggested that the biochar creates a house (habitat) for the association of microbes that either more efficiently ferment feed materials or possibly where methane oxidation is facilitated by bringing together methanogenic Archae and a methanotrophic consortia  (Knittel and  Boetius 2009). Such an association could result in an increased oxidation of methane by a bolstered population density of methanotrophs in biochar adapted rumens, where normally  these populations are  extremely small  (Kajikawa and Newbold  2003). It appears that the anaerobic methane oxidation is carried out by an as yet unknown consortia of Archae /bacteria and it is possible  that the lowering of methane by biochar addition to the rumen is solely a result of  the increase in potential habitat for this consortium. However, in the biodigester with added biochar, the biochar has been observed after 4  weeks to become impregnated with, in particular, methanosarcina-like species (Anonymous 2012) so also providing habitat for both methanogenic and methanotrophic organisms in very close association. To complete these speculations we put forward the concept that reduced methane production could be a result of  carbon and sulfur back flux during anaerobic microbial oxidation of methane and coupled sulfate reduction that has been recently described  by Holler et al (2012) where the methanogenic Archae were responsible for both production of methane and some oxidation of methane by reversal of methanogenesis.

Conclusions

• Gas production and percentage substrate DM solubilized were increased, and percent methane in the gas was reduced, when: (i) the rumen fluid in the incubation flask was taken from cattle adapted to 0.62% biochar in their diet (DM basis) over a 4 month period; and (ii) when biochar was added to the incubation medium at 1.5% of DM.
  
  
• There were additive effects on methane reduction when rumen fluid from adapted cattle was combined with biochar added to the incubation medium.

Acknowledgements

The authors acknowledge support for this research from the MEKARN project financed by Sida. Special thanks to Mr Sengsouly Phongphanith, Mr Khamphout Thammavong and Mr Touvieu Xaiker, who provided valuable help in the farm. We also thank the Department of Animal Science, Faculty of Agriculture and Forest Resources, Souphanouvong University for providing infrastructure support to carry out this research.

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Received 12 October 2012; Accepted 31 October 2012; Published 6 November 2012

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