Biochar lowers net methane production from rumen fluid in vitro

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

Souphanouvong University, Luang Prabang Province, Lao PDR
inthapanyasangkhom@yahoo.com

*University of New England, Armidale NSW, Australia
 **Finca Ecológica TOSOLY, AA#48, Socorro, Colombia

Abstract

Three experiments were carried out to evaluate the effect of biochar on methane production from buffered ruminal fluid in an in vitro system using cassava root meal as substrate with either potassium nitrate or urea as the NPN source.

Experiment 1: The treatments in 2*2 factorial arrangements with four replications of each treatment were: urea or potassium nitrate as NPN source; and presence or absence of 5% biochar. The quantity of substrate was 12 g DM to which was added 240 ml rumen fluid (from slaughtered buffalo) and 960 ml of buffer solution. The incubation was for 24 and 48hours with measurements of gas production, percent methane, substrate solubilized and methane produced per unit substrate solubilized. 

Gas production, methane percentage in the gas, substrate solubilized and methane produced per unit substrate solubilized were all lowered when nitrate replaced urea as the fermentable  N source at either 24 or 48 hours of the incubation. Addition of biochar did not affect gas production but increased the percentage DM solubilized. Methane produced and methane produced per unit substrate solubilized was lowered by 14% due to addition of biochar when urea was the NPN source but was not affected when nitrate was the source of NPN.

Experiment 2: The treatments in a 2*6 factorial with three replications were: (i) concentration of biochar (0, 1, 2, 3, 4 and 5% on DM basis); (ii) washing or no washing of the biochar. The substrate was cassava root meal and urea. The general procedure and analyses were similar to those in experiment 1.

Methane produced was reduced by 11-13% by adding 1% biochar but there were no further benefits from increasing the biochar level to between 2 and 5%. Methane production and per unit substrate DM solubilized were reduced by about 5% by washed compared with unwashed biochar.

Experiment 3: The design was a completely randomized comparison of: No biochar with urea, 0.5% biochar with urea, 1.0% biochar with urea, 1% biochar with 50% urea and 50% potassium nitrate and 1% biochar with 100% potassium nitrate (at 6% of diet DM).  

Biochar at 0.5% reduced methane by 10% and at 1% reduced it by 12.7%. With 50% nitrate N and 50% urea N, plus biochar at 1%,  the reduction in methane was 40.5% and with 100% nitrate N plus biochar  at 1%,  it was 49%.

Keywords: climate change, fermentation, gas production, greenhouse gas    

Introduction

Methane emissions from biological sources are a balance between production by methanogenic Archae and oxidation by methanotrophic micro-organisms. Methane oxidation has been reported in both aerobic and anaerobic environments, which restricts the flux of methane entering the atmosphere (Hanson and Hanson 1996). Measurements in flooded rice fields indicated that a high proportion (up to 80%) of the methane produced was oxidized at the soil surface (Hanson and Hanson 1996). Both aerobic and anaerobic methanotrophic bacteria are unique in their ability to utilize methane as a sole carbon and energy source.

Stocks and McCleskey (1964) isolated methane-utilizing bacteria from the rumen of steers that were similar to methanotrophs isolated from soil and water and Mitsumori et al (2002) demonstrated methanotrophs were present in both rumen fluid and attached to the rumen wall. However, a study using an artificial rumen indicated that only 0.3% of methane flux was oxidized (Kajikawa and Newbold 2000). Further studies by Kajikawa et al (2003) indicated that 0.2 to 0.5 of the methane flux was anaerobically oxidized by reversal of methanogenesis with sulphate as the terminal electron acceptor.

Recent studies have demonstrated that the application of biochar in paddy soils lowered methane release (Liu et al 2011) although other studies under different conditions report the opposite (Zhanga et al 2010). It appears that amounts of methane emissions will depend on the soil type, the chemical properties of the biochar, and on the fertilization and water management regimes (Cai et al 1997). However the decrease in methane emissions under biochar amendment in the research of Liu et al (2011) was not the result of inhibition of the growth of methanogenic archaea but to increased methanotrophic proteobacterial abundances with greatly increased ratios of methanotrophic to methanogenic abundances in the paddy soils as measured by real-time polymerase chain reaction (qPCR) and PCR–DGGE (denaturing gradient gel electrophoresis) (Feng et al 2012). The possibility of increasing methanotrophic activity in the rumen led us to examine the effect of biochar amendment on ruminal fluid methane production in vitro as a preliminary to whole animal research.

It is now well established that  nitrate can replace urea as a major source of fermentable N (rumen ammonia) in rations fed to ruminants (see Trinh Phuc Hao et al 2009) and at the same time it  lowers  methane production because of it’s higher affinity for  hydrogen as compared to carbon dioxide (see Leng 2008). In preliminary studies the methane mitigating effect of biochar was seen and it  was therefore decided to examine the possibility of an additive effect of nitrate and biochar.

Hypothesis

The hypothesis to be tested was:

• Inclusion of biochar in the diet of ruminants would lead to a reduction in enteric methane emissions

Objectives

• To test the effect of biochar on methane production from buffered ruminal fluid in an in vitro system using cassava root meal as substrate and with either potassium nitrate or urea as the NPN source.

Materials and methods

Location and duration

Three in vitro incubation experiments were conducted in the laboratory of the Faculty of Agriculture and Forest Resources, Souphanouvong University, Luang Prabang province, Lao PDR, from April to May 2012. 

Experiment 1

Treatments and experimental design 

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

• U: Urea at 2% of substrate DM

• U-BC: Urea with added biochar at 5% of substrate DM

• KN: Potassium nitrate at 6% of substrate DM

• KN-BC: 6% Potassium nitrate with added biochar at 5% of substrate DM

Cassava root meal was the substrate added to provide the energy source in all incubations (Table 1).

The equipment and procedure was that used by Sangkhom Inthapanya et al (2011).

Newly harvested cassava root was 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 and passed 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. The biochar was mixed with the cassava root meal and either potassium nitrate or urea (Table 1) prior to adding to flasks containing 1.2 liters of diluted rumen fluid (240 ml of rumen fluid plus 960 ml of buffer solution made according to Tilly and Terry 1963). Rumen fluid was collected from a newly slaughtered buffalo in the village abattoir into an insulated flask and used immediately or within 30 min of sampling. The buffalo had been grazing local grasses and had been fasted overnight.

The substrate was put in the incubation flask containing the diluted rumen fluid which was then gassed with carbon dioxide and the flasks were incubated at 38 ⁰C in a water bath for 24 and 48 hours.

Data collection and measurements

The gas volume was read from the collection bottles directly after 24 and 48 hours and the percentage of methane in the gas  was measured using a Crowcon infra-red analyser (Crowcon Instruments Ltd, UK) for the separate incubations. Gas from the collection bottle was drawn into   the measuring apparatus. Three samples were measured from each collection bottle. At the end of each incubation time 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: Biochar, NPN source, interaction Biochar*NPN and error.

Experiment 2

Objectives and experimental design

The objectives were to examine the effect of the concentration of biochar in the fermentation medium over the range of 0 to 5% (DM basis).

The design was a 2*6 factorial with three replications. The factors were:

• Washed or unwashed biochar

• Concentration of biochar (0, 1, 2, 3, 4 or 5% on DM basis)

The general procedure and analyses were similar to those in experiment 1.

Biochar was used either as an untreated powder or after washing. Biochar was washed by adding 100ml of distilled water to 50g of biochar, stirring and the flask allowed to stand for 10 minutes before pouring off the water phase. This was repeated two times and the biochar dried before adding  to the incubation flasks containing buffer,  rumen fluid and substrate.

Statistical analysis

The data were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab (2000) Software. Two sets of data were analysed. The first set included all the data to test effect of levels of biochar. The sources of variation were: levels of biochar and error. In the second set the data for the control treatment were omitted so as to test the effect of washing or not washing the biochar. Sources of variation in the model for set 2 were: Level of biochar, washed or not washed, interaction level of biochar*washing and error.

Experiment 3

Objectives and experimental design

The objectives were to examine a lower concentration of biochar (0.5% in substrate DM) and the interaction with different proportions of potassium nitrate and urea as NPN source in diluted rumen fluid incubated with cassava root meal as described above .

The design was a completely randomized comparison of:

• No biochar with urea (2% in DM)

• 0.5% biochar with urea (2% in DM)

• 1.0% biochar with urea(2% in DM)

• 1.0% biochar with 50% urea (1% in DM) and 50% nitrate (3% in DM)

• 1.0% biochar with 100% nitrate (6% nitrate in DM)

There were 4 replicates of each treatment. The general procedure and analyses were similar to those in experiment 1.

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: Treatments and error. 

Results

Experiment 1

Effects of NPN source

Gas production, methane percentage in the gas, substrate solubilized and methane produced per unit substrate solubilized were all lowered when nitrate replaced urea as the fermentable nitrogen source at either 24 or 48hrs of the incubation (Table 1; Figures 1 to 4).  These results are similar to those reported by Guo et al (2009) and Lin et al (2011), and in the many studies from the MEKARN  project (http://www.mekarn.org) (Binh Phuong et al 2011; Inthapanya et al 2011; Outhen et al 2011; Phuong et al 2012a,b; Quang Do et al 2011; Silivong et al 2012; Sophea Iv and Preston T R 2011 ; Thanh et al 2011, 2012).

Effects of Biochar

Addition of biochar did not affect gas production but increased the percentage DM solubilized. Methane produced and methane produced per unit substrate solubilized were lowered by added  biochar by 11.5 and 12.8% at 24 and 48h when urea was the NPN source but appeared to be less affected (interactions were P=0.16 and P=0.23 at 24h and P=0.052 and P=0.069 at 48h) when nitrate was the source of NPN (8.3 and 11.5%; Figures 1 to 4). These effects (for lowering of methane production) were thus similar for incubation periods of 24 and 48 h with urea, but more pronounced for the longer period of fermentation (Table 2).

Experiment 2

Effects of level of biochar

Methane produced in 24h was reduced by adding 1% biochar (by 12%) but there was no further effects from raising biochar level to between 2 and 5% (Table 3; Figure 5). The same effect was observed when methane production was expressed as ml per unit substrate DM solubilized (Figure 7).

Effect of washing the biochar

Methane production in 24h and per unit substrate DM solubilized was reduced by about 5% by washing the biochar (Table 3; Figures 6 and 8).

Experiment 3

Biochar at 0.5% of the substrate reduced methane by 10% and at 1% reduced it by 12.7% (Table 4; Figures 9 and 10). With a 50% mix of nitrate and  urea N, plus biochar at 1%, the reduction in methane was 40.5% and with 100% nitrate N plus biochar at 1%, it was 49%.

Discussion

As has been observed in numerous studies nitrate lowers methane production from rumen fluid indicating the presence of nitrate reducing bacteria that use nitrate as a terminal electron acceptor and outcompete methanogens for hydrogen produced in fermentation. However this is the first study to show that biochar may also have a role in reducing rumen methanogenesis. Adding 5% biochar to the substrate used in the incubation flasks apparently lowered net methane production by 14% and by 13% when this was calculated on an, as is or, per unit of dry matter apparently fermented basis, respectively. In the presence of nitrate there was a significant reduction of methane production by 34 %. The presence of biochar appeared to further lower methane production in the presence of nitrate but the lowered production was quantitatively small but still 9% of the methane produced when nitrate was incubated without biochar.

In the second study increasing levels of biochar added to the incubation medium demonstrated that the optimum biochar level for maximum mitigation in vitro was less than 1% of the substrate added.   Even though the major reduction in methane production by biochar was similar to that in the first study, at every level of inclusion of unwashed biochar in the incubation medium there was both a higher total methane production and  methane production per unit of substrate apparently digested.So washing the biochar appears to increase it’s methane mitigating benefits.Biochar’s overall mechanism  for lowering methane production appeared to be mostly associated with its insoluble components.

This research was initiated because of the demonstration that biochar may encourage methanotrophic microbes to proliferate in anoxic soils associated with rice growing (Feng et al 2012). However it is recognized that biochar does not always decrease methane release from amended soils (Cai et al 1997) and there are multiple interactions that come into play in any anoxic ecosystem that may affect the results. Similarly it will be necessary to study different biochars with differing sources of rumen fluid to clarify the potential mitigation possibilities for enteric methane.

It appears unlikely that biochar lowers methane production by anaerobic oxidation  as in natural anaerobic environments methanotrophs grow slowly limited by the energy availability. The  short turn over rate of rumen fluid appears to negate the substantial growth of these organisms.  However methanotrophs depend on methane oxidation and they are also sulphur-reducing bacteria (SRB) because sulphur is the terminal electron acceptor in anaerobic methane oxidation according to the equation CH₄+SO₄²⁻ ->HCO₃⁻ +HS⁻+H₂O. Other studies have suggested that SRB did not carry out anaerobic oxidation directly, but rather a consortium with unknown organisms and SRB was involved (Smemo and Yavitt 2011). However,  it appears  that anaerobic methanotrophs are present in  rumen fluid (Stock and McCleskey 1964; Kajikawa et al 2003) and  aerobic methanotrophs are attached to rumen epithelium (Mitsumori et al 2002). To be effective in capturing methane, anaerobic methanotrophs would have to be spatially distributed close to the site of methane production which is likely to be close to the methanogenic Archae that probably colonize  the outer layers of the biofilm consortia attached to feed particles (McAllister and Cheng 1996; Leng 2011) where the partial pressure of methane will be the highest.

The spatial distribution of organisms relative to their preferred substrate  is important as illustrated  in in vitro incubations of marine  sediments containing high numbers of microbial consortia, consisting of organisms that affiliate with methanogenic archaea and with sulphate-reducing bacteria, where an increase in partial pressure of the methane from 0.1 MPa (approximately 1 atm) to 1.1 MPa (approximately 11 atm) resulted in a four to fivefold increase of the sulphide production rate and therefore methane oxidation (Nauhaus et al 2002), Thus the spatial distribution of  methane oxidizing organisms or consortia close to the site of methanogenesis appears to be a critical issue in stimulating the overall reactions. The question raised here is “does biochar with its relatively large surface area (http://en.wikipedia.org/wiki/BET_theory)  and highly porous structure (Photo 1) provide  a favourable habitat for the  organisms involved in a methanogenic methanotrophic interaction increasing the potential for anaerobic methane oxidation”.  This then leads to ecological studies of how best to increase the efficiency of these associations. The BET surface area is a measure of the ability of a material to absorb gases. Biochars often have BET surface areas of 2-4 m² /g biochar  but much greater surface areas maybe produced by particular production technologies. As shown in the photo the potential to create habitat for biofilm residing microbes is substantial where gases could be adsorbed on to the surfaces of the biochar.

There are also other potential explanations for the net decrease in methane release from rumen fluid including a change in surface ion exchange capacity for microbial biofilm formation or a direct effect of chemicals not soluble in water on fermentation pathways and the end products produced. A direct toxic effect on methanogens seems unlikely as the rate of fermentation of the substrate appeared to be unchanged by biochar addition and the amounts of biochar were exceedingly small. It is also unlikely that the biochar washed or unwashed could supply high affinity electron accepting substrate, particularly at the lowest level of inclusion (1% of the total substrate). However hydrogen uptake could be reduced in some way as nitrate  out-competes most other electron acceptors (sulphate and carbon dioxide) and the effect of biochar appears to be reduced when nitrate replaced urea as the major fermentable N source. Nitrate appears to inhibit anaerobic methane oxidation (Hanson and Hanson 1996). Since the depression in methane production was observed in vitro there can be no involvement of the rumen wall associated methanotrophs, which are probably aerobic bacteria dependant  on diffusion of oxygen across the rumen epithelium as the terminal electron acceptor (Mitsumori et al 2002). 

The lack of, or small,  response of methane production to biochar inclusion in the substrate when nitrate provided the fermentable N source may be a result of nitrate inhibition of methanotrophs through competition with these sulphur reducing bacteria for available electrons. 

The preliminary and speculative nature of the present report is acknowledged but the importance of this observation to atmospheric methane accumulation if repeatable in other situations is so immense that   bringing the finding to the attention of other research scientists is warranted and this early publication is to put the information in the public domain as it is felt that any attempt to patent a process using biochar to mitigate enteric methane production from all animals is not in the interests of people in general. If these results are repeated when animals are fed biochar in their diet, then there would be good reason to suggest that enteric methane production maybe lowered from all animals including humans by extremely small amounts of dietary biochar whether fermentation sacs precede or follow digestion in the intestines. The exception will be where acetogenesis replaces methanogenesis in the fermentation areas of the tract as in the kangaroo that produce little methane (Kempton et al 1976). A major point here is that if the results are applicable to animals under domesticated conditions the small amounts likely to be needed would probably indicate this could be the least expensive methodology for mitigating methane production.

Conclusions

• Methane production by rumen fluid was decreased when nitrate replaced urea as NPN source at both 24 and 48 h of fermentation time.  

• There was no effect of added biochar on  gas production but the percentage of DM solubilized was increased.

• Methane production and methane per unit substrate solubilized was reduced by added biochar with urea as NPN source

• Overall, the methane production by rumen fluid was reduced by 12% by adding 1% of biochar and there were no further benefits from increasing biochar content .

• Washing the biochar appeared to improve its methane mitigation benefits.

• With 3% KN and 1% urea, plus biochar at 1%, the reduction in methane was 40.5% and with 6% KN plus biochar at 1%, it was 49%.

Acknowledgements

The authors acknowledge support for this research from the MEKARN project financed by Sida. Special thanks to Mr Sengsouly Phongphanith who provided valuable help in the laboratory and preparation of the samples. Thanks also to the Department of Animal Science laboratory, Faculty of Agriculture and Forest Resource, Souphanouvong University for providing the facilities to carry out this research. 

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