Livestock Research for Rural Development 25 (6) 2013 Guide for preparation of papers LRRD Newsletter

Citation of this paper

All biochars are not equal in lowering methane production in in vitro rumen incubations

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

Faculty of Agriculture and Forest Resource, Souphanouvong University, Lao PDR
* University of New England, Armidale NSW, Australia
rleng@ozemail.com.au
** Center for Research and Technology Transfer
Nong Lam University, Ho Chi Minh City, Vietnam

Abstract

Two experiments were carried out to measure effects of different sources of biochar and bentonite on methane production when they were incubated with cassava root, cassava leaf and urea. The treatments were:  bentonite clay powder from Australia (Ben-A) and Vietnam (Ben-V); two commercial samples of biochar from Australia (Bio-AL and Bio-AM),  two biochar samples from Lao PDR (Bio-LP1 and Bio-LP2) produced by carbonization of rice husks in an updraft gasifier stove and a control (No-bio) treatment with no additive. The incubation was for 24 h in experiment 1 and 48 h in experiment 2.

The relative effect of the various additives in decreasing methane production was improved (from 8 to 14%) as the fermentation time (and the production of methane) increased. The general tendency appeared to be for a reduction in methane production with incorporation of either bentonite or biochar in the incubation medium when the incubation time was extended to 48 h.

Keywords: biofilms, carbonization, clay, consortia, fermentation, gasifier stove


Introduction

In any anaerobic ecosystem the availability of  suitable habitat for microbes is paramount in determining the rate and extent  at which organic molecules are converted to their end products (Costerton 2007). Anaerobic degradation of organic matter requires numerous species of microorganisms that progressively convert large complex molecules to soluble organic acids and eventually to complete mineralization to carbon dioxide and water. Efficient fermentative degradation is only achieved when the various organisms are in structured consortia where the distance between end product producers are close to end product users particularly for inter-species transfer of hydrogen (Cheng et al 1995). Anaerobic ecosystems such as waste water treatment or biodigesters have long turnover times (often days or weeks) and complete mineralization of organic matter can be achieved ( eg: in wastewater treatment plants). This is achieved by the microbial consortia organizing their layered structures in self-produced biofilms consisting of extracellular polymeric substances (see for review Davey and O’Tool 2000). In the rumen the turnover time rarely exceeds 17-20hrs and  the biofilm mode of degradation of complex polysaccharides (eg: cellulose, hemicellulose, starch) extends to the production of organic acids with hydrogenotrophic methane production. It is  clear that digestion of complex organic matter is achieved by organisms that attach to the surface of feed particles and hydrolytically convert  these to sugars which can  then be progressively degraded to VFA and methane by consortia of microbes organized in  biofilms (Cheng et al  1980: McAllister et al 1994;  Leng 20101). There is limited information, from isotope studies using specifically labeled VFA (Leng and Brett 1967),  indicating direct oxidation (secondary fermentation) of small amounts of butyrate and propionate to acetate (see Sharp et al 1982 ) in the rumen.

We hypothesized that these ruminal microbial consortia would be enhanced by providing solid surface areas where the microbes could readily form organized transfer of substrate between different species of microbes. As closeness to feed source is related to efficiency of the reactions this should increase productivity (see de Bok et al 2004). We were mainly interested in the possibility of increasing microbial growth efficiency and potentially increasing the activity of methanotrophic microbes that are present in small populations in the rumen (Kajikawa and Newbold 2003; Kajikawa et al 2003) and which may lead to a decrease in net methane production.

Earlier studies in our laboratory (Leng et al 2012a,b) showed that incorporation of biochar,prepared by carbonization of rice husks in a gasifier stove reduced methane production both  in vitro and  in vivo (Leng 2012c). We hypothesized that the action of biochar in the rumen resulted from it's potential to ct as an improved location for biofilm microbial consortia and that  this would facilitate microbial activity, including oxidation of methane by methanotrophic  organisms . The idea that biochar could  act as a functional site for improved biofilm formation is based on the  large surface to weight ratio (>30m2/g and up to 500m2/g), creating opportunities for adsorption of both micro-organisms, nutrients and gases..

Bentonite clays are also characterized by their high adsorptive capacity (Kaufhold et al 2010) and have been demonstrated to improve  microbial protein availability in sheep (Fenn and Leng 1989; Ivan et al  1992).  There are some large differences in the porosity of both biochar (depends very much on heat used in preparation and source of the original substrate )  and bentonite which has different associated cations and a smaller surface to weight ratio. Recent studies have also indicated the possibility of promoting direct interspecies electron transfer with activated charcoal through a high conductivity of biochar providing better electrical connections for inter-species electron transfer than those forged in the biofilm on feed particles (Liu et al  2012).

The objectives of the studies reported here were to obtain some preliminary information on the relative effects of bentonite clays compared with known (and unknown) sources of biochar on methane production in a rumen in vitro system with a substrate of cassava root and leaf meal.


Materials and Methods

Location and duration

Two experiments were conducted in the laboratory of the Department of Animal Science, Faculty of Agriculture and Forest Resource, Souphanouvong University, Luang Prabang province, Lao PDR, from January to February 2013.

Treatments and experimental design 

In each experiment, the design was a completely randomized block with eight treatments and 4 replicates.

In experiment1, fermentation was over a total period of 24 h with measurements of gas production and content of methane in the gas at 3, 6, 12 and 24 h. At the end of the 24 h  fermentation the DM mineralized was determined by filtering the contents of the fermentation bottle through several layers of cloth that retained particle sizes to at least 0.1mm and then this was dried (100C for 24 hours) and weighed.

In experiment 2, the fermentation was for 48 h with the same measurements as in Experiment 1.

The treatments in each experiment were:

Bentonite clay powder from Australia (BEN-A) and Vietnam (BEN-V)

Two commercial samples of biochar from Australia (Bio-AL and Bio-AM) and two from Lao PDR (Bio-LP1 and Bio-LP2) that were prepared by carbonization of rice husks in a gasifier stove

One commercial sample of activated charcoal from Colombia (AC)

Control with no additive (No-bio)

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

Table 1. The ingredients in the substrate (g DM)

 

Bio-AL

Bio-AM

Bio-LP1

Bio-LP2

BEN-A

BEN-V

AC

No-bio

Cassava root meal

9.6

9.6

9.6

9.6

9.6

9.6

9.6

9.6

Cassava leaf meal

2.04

2.04

2.04

2.04

2.04

2.04

2.04

2.16

Urea

0.24

0.24

0.24

0.24

0.24

0.24

0.24

0.24

Additive

0.12

0.12

0.12

0.12

0.12

0.12

0.12

 

Total

12

12

12

12

12

12

12

12

The in vitro system was that used by 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 80C and then ground through a 1 mm sieve. The biochar samples from Lao PDR (LP-1 and LP-2) were  produced by carbonizing rice husks in a top lit updraft (TLUD) gasifier stove (Olivier 2010) at estimated temperatures in the range 700-900 C

Photo 1: Biochar

Photo 2: Bentonite

The biochar / bentonite was mixed with the cassava root and leaf meal and 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 at Phosi 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 C in a water bath for 48 hours.

Data collection and measurements

The gas volume was read from the collection bottles. 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. At the end of 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 (100C 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: replicates, source of biochar / bentonite and error.


Results and Discussion

Experiment 1

Gas production from the Bio-AL treatment was greater than from the control (Table 2). The methane percentage in the gas was reduced on all treatments compared with the control.

Table 2. Mean values for gas production, methane in the gas, DM mineralized during the incubation and methane production per unit DM mineralized in an in vitro rumen incubation over 24 h, with cassava root meal, cassava leaf meal and urea as substrate and with addition of biochar or bentonite

 

AC

BEN-A

BEN-V

Bio-AM

Bio-AL

Bio-LPN

Bio-LPO

No-bio

SEM

p

Total gas, ml

2630a

2648ab

2668ab

2750ab

2825b

2773ab

2738ab

2575a

37.9

0.002

Total CH4, ml

660

676

682

685

683

684

679

702

8.51

0.13

Methane, % in the gas

25.1 b

25.5 b

25.6 b

24.9 b

24.2 b

24.7 b

24.8b

27.3 a

0.23

0.001

DM disappea5rance, %

70.1

70.0

69.4

70.4

71.0

70.2

69.8

69.9

0.64

0.73

Methane, ml/g DM mineralized

80.1

82.1

83.5

82.8

81.8

82.8

82.8

85.5

1.01

0.26

ab Means without common subscript differ at P<0.05

The percentage methane in the gas increased with a curvilinear trend as the fermentation time was increased (Figures 1 and 2). The relative effect of the various additives in decreasing methane production appeared to improve as the fermentation time (and the production of methane) increased.

Figure 1. Effect of incubation time and sources of biochar and bentonite on methane content of the gas
in a rumen in vitro system with substrate of cassava root, cassava leaf and urea


Figure 2. Effect of incubation time on the lowering of the methane content of the gas
in a rumen in vitro system with substrate of cassava root, cassava leaf and urea

Experiment 2

Production of methane was reduced when commercial biochar samples from Australia were included in the incubation medium (Table 3). A similar effect was observed on methane production per unit substrate mineralized, with the biochar from Lao PDR (LPN) responding similarly to Bio-AL and Bio-AM. The general tendency appeared to be for a lowering in methane production with incorporation of either bentonite and biochar in the incubation medium (Figures 3 and 4).

Table 3. Mean values for gas production, methane in the gas, DM mineralized during the incubation and methane production per unit DM mineralized in an in vitro rumen incubation over 48 h, with cassava root meal, cassava leaf meal and urea as substrate and with addition of biochar or bentonite

 

 

No-bio

Bio-LPO

BEN-V

AC

BEN-A

Bio-LPN

Bio-L

Bio-M

SEM

Prob.

Gas volume, ml

 

1508

1413

1488

1503

1480

1400

1438

1375

45.5

0.325

Methane, %

 

35.5a

34.8 a

32.5a

32.0b

32.2a

33.3a

32.5b

32.8b

0.60

0.004

Methane, ml

 

535a

490a

483a

479a

478a

465b

466b

450b

14.4

0.022

Digestibility, %

 

69.8a

70.3a

70.a

70.4a

70.5a

70.1a

71.3b

71.3b

0.33

0.025

Methane,  ml/g DM mineralized

65.2a

59.4a

58.6a

57.9a

57.7 a

56.5b

55.7b

53.6b

1.80

0.011

ab Means without common superscript differ at P<0.05


Figure 3. Effect of sources of biochar and bentonite on methane content of the gas in a rumen
in vitro 48 h incubation with substrate of cassava root, cassava leaf and urea


Figure 4. Effect of sources of biochar and bentonite on methane produced per unit substrate mineralized in a  rumen in vitro 48 h incubation with substrate of cassava root, cassava leaf and urea


The overall degree of mitigation  of methane  was small  but significant effects were found with some biochars. The use of biochar as a feed ingredient will be followed up since there are obviously differences between these biochars. It will be interesting to examine the in vivo effects of biochar in practical production systems as the biochar will be present in excreta and may have considerable influence on the performance of biochar in affecting methane release from manure and soil. Further work is on-going in this area.

 

A recent publication (Hansen et al 2013) has provided supporting evidence for the effect of biochar in mitigating methane production from rumen fluid in vitro, supporting our original work (Leng et al 2012a,b,c). This evidence from a different laboratory, together with our own studies,  suggests that the research priority should be to identify the “best ”biochars for this purpose and to follow the potential benefits from the rumen through the caecum into feces and then the effects this could have in anaerobic biodigesters and /or carbon storage and methane emissions from soil fertilised with excreta from biochar-supplemented animals.


Conclusions


Acknowledgements

The authors acknowledge support for this research from the MEKARN project financed by Sida. Special thanks are given to Mr Sengsouly Phongphanith, who provided valuable help in the laboratory. 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 13 February 2013; Accepted 12 May 2013; Published 2 June 2013

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