EFFECTS OF BIOCHAR PRODUCED FROM TROPICAL RICE HUSK AND PEANUT SHELL AT DIFFERENT PROCESSING TEMPERATURES ON IN VITRO RUMEN FERMENTATION AND METHANE PRODUCTION

D. V. Dung¹, L. D. Phung^1*, L. D. Ngoan¹, N. H. Quan¹, T. T. T. Tra¹, V. T. M. Tam¹, N. X. Ba¹, L. D. Thao¹ and H. Roubík²

¹Faculty of Animal Sciences and Veterinary Medicine, University of Agriculture and Forestry, Hue University, Hue city, Vietnam.

²Department of Sustainable Technologies, Faculty of Tropical AgriSciences, Czech University of Life Sciences Prague, Kamýcká 129, 165 00 Prague, Czech Republic.

^*Correspondence author’s Email: ldphung@hueuni.edu.vn

ABSTRACT

 The aim of this study was to investigate the effects of biochar produced from tropical biomass resources at different processing temperatures on methane production and rumen fermentation in vitro. Two available tropical biomass resources of rice husk and peanut shell were used for pyrolysis at three temperature levels of 300, 500 and 700^oC. Biochar was supplemented at 3% in diets of dry matter basis. In vitro fermentation characteristics and methane production were measured at 4, 24 and 48h after incubation. Results showed that there were no significant differences in terms of (i) gas and methane production, (ii) dry matter and organic matter digestibility (iii) pH and NH₃-N concentration between diets supplemented either rice husk or peanut shell drived biochar in an in vitro system (P>0.05). Whereas, different processing temperatures affected on total gas, production of methane and NH₃-N concentration (P<0.05), increasing processing temperature decreased methane production. There were no interactions between biomass resources and processing temperature on in vitro rumen fermentation and methane production. These results implicate that rice husk and peanut shell derived biochar produced at 700^oC can be used to mitigate methane emission from cattle production, further in vivo studies are required to confirm practical parameters.

Keyword: Rice husk, Peanut shell, Biochar, In vitro fermentation, Methane production

http://doi.org/10.36899/JAPS.2022.3.0463
Published first online October 19. 2021

INTRODUCTION

Methane produced from cattle production by enteric fermentation process up to 85-90% and by excretion of feces (Ribeiro et al., 2015). Bell et al. (2011) reported that 15% of global warming is due to enteric methane production. In addition, Johnson and Johnson (1995) concluded that, methane production is responsible for a loss of 2 to 12% of gross energy of dietary for the ruminants, which could be available used for growth or other performance. Therefore, reduction of methane emission from ruminants could bring about both environmental and economic benefits, with improving sustainable and energy efficient ruminant production, and in addition offer a potential way for global warming slowing (Cabeza et al., 2018).

Biochar is a byproduct produced from the pyrolysis of cellulose-rich biomass (Lehmann and Joseph, 2009), many previous studies reported that, the use of the biochar has the potential way to reduce methane production in cattle (Leng et al., 2012; Hansen et al., 2012; Saleem et al., 2018; Zhang et al., 2019). However, the mode of action of biochar is not fully understood (Winders et al., 2019). The reason is that the previous studies provided little or no detailed characteristics of the biochar (Cabeza et al., 2018). Some authors suggested that the gas adsorption by biochar in the rumen resulted in decreased methane belching, or the porous characteristic nature of biochar increases inert surface area in the rumen acknowledge for improved habitat of microbial, or change the community of microbial (Saleem et al., 2018; Leng, 2014). Furthermore, according to Sonoki et al. (2013) and Feng et al. (2012) when biochar is applied to the soil, the balance between methanotrophic and methanogenic organisms group was changed favorably toward decreasing the methanogen to methanotrophs ratio in paddy soil, and in the rumen this process may also occur (Winders et al., 2019). These functions of biochar are dependent on the characteristics of biochar. However, the characteristics of biochar are dependent on both the pyrolysis temperature and the sources of biomass from which it was produced (Tomczyk et al., 2020).

Vietnam is a tropical country with abundant crop byproducts, in which rice husk and peanut shell are dominant and there are potential biomass resources for biochar production. An estimated annual production of rice husk and peanut shell are 8.6 and 0.12 million ton, respectively (GSO, 2019). These byproducts were mainly used as fuel for cooking or burning on the field. This creates environmental pollution. However, so far, there have been few studies on processing and using biochar for ruminant production in Vietnam and in Asia as well. This study was conducted to determine effects of biochar produced from available tropical biomass resources of rice husk and peanut shell, and prepared at different processing temperatures on rumen fermentation characteristics and methane emissions in vitro.

MATERIALS AND METHODS

Materials: The experiment was carried out in the Lab Center of Animal Sciences and Veterinary Medicine Faculty, University of Agriculture and Forestry, Hue University, Hue city, Thua Thien Hue province, Vietnam. Two tropical biomass resources were used for producing biochar including rice husk and peanut shell. Biochar was produced at three processing temperatures of 300, 500 and 700⁰C. Biochar was produced as described by Nguyen et al. (2018).

Experimental design: A 2 (biomass resources) x 3 (processing temperature) factorial design was used to study effects of biochar produced from rice husk and peanut shell, and prepared at different processing temperatures on characteristics of rumen fermentation and methane production in vitro. Biochar biomass resources included rice husk and peanut shell and biochar processing temperature included 300, 500 and 700⁰C. Gas and methane production,dry matter (DM) and organic matter (OM) digestibility, and rumen fermentation characteristics (NH₃-N concentration and pH) were measured in vitro. These parameters were measured at three time points after the incubation (4, 24 and 48h). Total 90 bottles (2 biochar resources x 3 processing temperatures x 5 bottles/treatment combination x 3 time points) and 5 bottles for 5 bank samples were used for incubation.

Rumen inoculum: Ruminal fluid was collected from 4 fistulated beef cattle before the morning feeding in the farm of the Animal Sciences and Veterinary Medicine Faculty of Hue University of Agriculture and Forestry, Hue University. Cattle were fed diets consisting of forage (50%) and concentrate (50%). After being collected, the rumen fluids of 4 cattle were mixed and putted in a warmed thermos flask, then were quickly transferred to the lab where rumen fluid filtered through 4 layers of cheese and then carefully used buffer mineral solution to mix with rumen fluid with a ratio of of 1 part of remen fluid and 4 parts of buffer solution. All processes were made with anaerobic condition by flushing with CO₂ gas. Buffer solution as described by Theodorou et al. (1994) and was put in a water bath at 39⁰C and used CO₂ gas to purged continuously for 30 minutes.

Substrates and chemical analyses: The feed substrate consisted of forage (50%) and concentrate (50%). Biochar was added to the feed substrate at 3% diet in DM basic. Using a hammer mill (Pullerisette 19, Fritsch GmbH, Laborgeratebau, Germany) to ground the substrates samples to pass a 1 mm sieve and then substrates was analysed chemical composition (DM, OM, CP, NDF).

In vitro fermentation and fermentation attributes analyses: 250 mg of (the air-dried basis) substrate were incubated in the bottle (120mL) which contained 25 mL of mixed rumen fluid and buffer mineral solution. The total gas production was determined at 4, 24 and 48h after incubation by using a manual pressure transducer combined with a syringe. Methane production was determined at the same time by Gas chromatography (Model 8610C Gas Chromatograph, SRI instruments Europe GmbH, USA).

Digestibilities of DM and OM, pH and concentration of NH₃-N were measured at 4, 24, and 48h after incubation. At each time point, pH value was determined immediately by pH meter (Hana, Germany), and then collection approximately 10 mL of end liquids and mixed with 0.2M HCl for analyses NH₃ -N concentration later. Centrifuge the rest of end liquids in each bottle at 10,000xg for 5 min, remove supernatant and dried at 105⁰C for 12 h and burned at 550⁰C for 4 h to measure DM and ash. DM digestibility and OM digestibility were determined as difference weight between after and before incubation, accurated by a blank which consisted of five bottles containing only rumen fluid and buffer solution. NH₃-N concentration was measured by method of AOAC (1990).

Statistical analysis: The effects of biomass resources and processing temperatures on gas and methane productions, in vitro digestibility of DM, OM, and invitro rumen fermentation characteristics (pH and NH₃-N concentration) were analyzed using ANOVA of SPSS 16.0 with a statistical model following:

Y_ijk = µ + B_i + T_j + B_i*T_j+ e_ijk

Where Y_ij is the independent factor; µ is the overall mean; B_i is the effect of biomass resources; T_j is the effect of processing temperature; B_i*T_j is interaction between biomass resources and processing temperature; e_ij is the residual effect. Tukey test was used for pairwise comparison between two treatments when the P value of F test <0.05. In all the analyses, statistically significant differences were declared at P<0.05.

RESULTS

Chemical composition of biochar and the substrates: The chemical composition of biochar is presented in Table 1. Biochar produced at higher temperature had a larger surface area and higher water holding capacity. When processing temperature increased from 300 to 500 and 700 ^oC, biochar surface area increased from 2.0, to 3.9 and 103.2 and from 2.3 to 3.1 and 101.0 m²/g, respectively for rice husk and peanut shell; water holding capacity was increased from 3.1 to 3.6 and 5.2 and from 3.9 to 4.1 and 4.8, respectively. Rice husk derived biochar produced at different temperatures had higher ash concentration than peanut shell, from 20.7 to 24.7% compared to from 4.3 to 5.2%, respectively. In contrary, peanut shell derived biochar produced at different temperatures had higher organic matter concentration than rice husk, from 94.8 to 95.7% compared to 75.3 t0 79.3%, respectively (Table 1). The chemical composition of substrates when supplemented with 3% biochar is presented in Table 2. It can be seen from the table that substrates supplemented with 3% biochar had similar chemical compositions of DM, ash, OM, CP, EE and NDF.

Table 1. Chemical composition of biochar produced from tropical rice husk and peanut shell at different processing temparatures

DM: Dry matter; OM: Organic matter; WC: Water capacity

Table 2. Chemical composition of substrates when supplemented with 3% biochar

DM: Dry matter; OM: Organic matter; CP: Crude protein; EE: Ether extracts; NDF: Neutral detergent fibre

Total gas and methane production: The total gas and methane production at 4, 24 and 48h after incubation are presented in Table 3. The biochar produced from rice husk, peanut shell had no significant effects on total gas and methane emission (P>0.05). Whereas, different processing temperatures had significant effects on total gas and methane emission (P<0.05), increasing processing temperature decreased methane production. There were no interactions between biomass resources and processing temperature on total gas and methane emission (P>0.05).

Digestibility of DM and OM, pH value and NH₃-N concentration: The biochar produced from rice husk and peanut shell had no effects on in vitro digestibility of DM and OM, pH and concentration of NH₃-N (P>0.05). Whereas, different processing temperatures had a significant effect on concentration of NH₃-N (P<0.05). Similar to total gas or methane emission, there were no interactions between biomass resources and processing temperature on in vitro digestibility, pH value and NH₃-N concentration at 4, 24 and 48h after incubation (Table 4).

Table 3. In vitro gas and methane production at 4, 24 and 48h after incubation

RH: Rice husk; PH: Peanut shell; B: Biomass; T: Temperature; BxT: Interaction between biomass resource and processing temperature; RSD: Residual standard deviation; ^a,b Means within rows within each factor missing a common superscript letter are different at P < 0.05.

Table 4. In vitro digestibility, pH and N-NH₃ concentration

RH: Rice husk; PH: Peanut shell; B: Biomass; T: Temperature; BxT: Interaction between biomass resource and processing temperature; RSD: Residual standard deviation; ^a,b,c Means within rows within each factor missing a common superscript letter are different at P < 0.05.

DISCUSSION

Effects of biochar on methane and total gas production in an in vitro system: Both rice husk and peanut shell are available agro-byproducts in tropical countries such as Vietnam. However, currently very few rice husk or peanut shell are used for any beneficial purposes, they are mainly burned and this process causes environmental pollution. Methane production from livestock should be mitigated to ensure sustainable development. Biochar has been declared for their potential of methane production reduction both in vivo and vitro studies (Leng et al., 2012; Winders et al., 2019). Therefore, using rice husk or peanut shell to produce the biochar should be considered. Biochar is not only used for reducing methane emission in livestock production (Cabeza et al., 2018) but also as a potential strategy to mitigate of greenhouse gas when applied to soils (Gurwick et al., 2013).

The present results indicate that the biochar produced from different tropical available biomass types of rice husk and peanut shell had no significant effects on methane and total gas production. This confirms findings of Cabeza et al. (2018); Hansen et al. (2012); McFarlane et al. (2017). Effects of biochar on gas and methane production depend on their physical and chemical characteristics (Hansen et al., 2012). The similar characteristics of biochar produced from rice husk and peanut huks (Table 1) could be the reason for this observation.

In this study, the effects of biochar produced from different processing temperatures on total gas or methane production were found. Cabeza et al. (2018) found that methane production decrease was higher with biochar produced at 550⁰C than 700⁰C. While, Calvelo Pereira et al. (2014) documented that there is no difference in methane production reduction between biochar produced at 350⁰C or 550⁰C. In the present study, methane production was decreased when biochar was produced at higher temperature. However, in general, no differences (P>0.05) were found between biochar prepared at 500⁰C and 700⁰C. The results of Cabeza et al. (2018), Calvelo Pereira et al. (2014) and this study showed that effects of biochar processed at different pyrolysis temperatures on methane production reduction were not consistent among studies. In current study, higher reduced methane production in biochar prepared at 500⁰C and 700⁰C compared with biochar prepared at 300⁰C could be explained by different surface areas of biochar (Table 1), especially biochar prepared at 700⁰C. High surface areas of biochar could help biochar adsorbed gases and/or methane emission (Hansen et al., 2012).

Inclusion of biochar has been shown to reduce methane production in cattle in both in vitro and in vivo condition (Leng et al., 2012; Hansen et al., 2012; Calvelo Pereira et al., 2014; Saleem et al., 2018; Cabeza et al., 2018; Winders et al., 2019). However, the mechanisms of their effects on methane production have not been explored clearly enough, although biochar has been shown as a potential methane mitigation strategy (Winders et al., 2019). The biochar used in these researches were prepared from different resources of biomass and at different processing temperatures, however their characteristics such as pH, surface area were not indicated (Calvelo Pereira et al., 2014; Leng et al., 2012; Hansen et al., 2012). Saquing et al. (2016) recommended that the acts of biochar as a redox-active mediator of electron that takes up electrons from microbial oxidation reaction and supply the electron at a certain distance from the center of microbial reaction, this process could reduce hydrogen in rumen. Previous studies recommended that biochar was prepared from rice husk at high temperatures with a high conductivity of electrical and capacity of electron buffering of fodder decomposing redox reactions (Yu et al., 2015; Sun et al., 2017). Sun et al. (2017) reported that different biochar has differences in conductivity of electrical and in capacity of electron buffering which depends on the biomass sources and pyrolysis temperature. Based on the above literature, the mitigation in methane was higher with biochar produced at 700⁰C, or 500⁰C than 300⁰C in present study could be explained. First, higher surface area of biochar could adsorb more methane production. Second, biochar produced from high pyrolysis temperature has high electrical conductivity and electron buffering capacity of fodder decomposing redox reactions (Sun et al., 2017). Furthermore, according to Feng et al. (2012) and Sonoki et al. (2013) when biochar application in the soil, the balance between methanotrophic and methanogenic organisms group was changed unfavourably toward methanogenesis rather than methanotrophism. However, there is no evidence to prove the effectiveness of this process by different biochar types.

Effects of biochar on DM, OM digestibility, pH and NH₃-N concentration in an in vitro system: The biochar produced from different tropical available biomass resources had no different effects on in vitro DM and OM digestibility, pH and NH₃-N concentration. Similarly, biochar prepared at different pyrolysis temperature had no significant effects on in vitro DM and OM digestibility and pH value, however, had a significant effect on NH₃-N concentration. There were no interactions between biomass resources and processing temperature on in vitro DM, OM digestibility, pH and NH₃-N concentration. These results are similar with McFarlane et al. (2017) who reported that, in vitro digestibility was not differred by biochar resources. The pH value is an important parameter to monitor rumen fermentation (Kumar et al., 2013; Zhang et al., 2019). The pH value in this study is no differences among biochar resources, and ranged from 6.61 to 6.92, these pH values were higher than the 5.0 to 5.5 concluded by Hoover (1986) which activity of microbial in rumen was negatively affected. Zhang et al. (2019) documented that biochar supplementation leads to increased pH value due to the alkaline nature of the biochar. This means that the buffering capacity of biochar improved the suitable range for microbial activity. The effects of different biochar resources on NH₃-N concentration were not consistent in literature. Cabeza et al. (2018) reported that both processing temperature and biomass resource affected NH₃-N concentration. NH₃-N concentration were higher for biochar produced at 700⁰C than 550⁰C with biomass sources of rice husk, oil seed rape straw, Miscanthus straw and wheat straw. However, the concentration was lower for biochar prepared at 700⁰C than 550⁰C for the biomass of soft wood. They are in contrast to our present study in which concentrations of NH₃-N were higher for biochar prepared at 700⁰C than 550⁰C or 300⁰C, that is in agreement with the findings of Cabeza et al. (2018). The effect of biochar on concentration of NH₃-N could be explained that biochar adsorb NH₃-N production (Cabeza et al., 2018), while Gai et al. (2014) and Winnin (2014) reported that the efficacy of adsorbing NH₃-N by biochar was no improved by the pyrolysis temperature, increasing processing temperature decrease cation exchange capacity. This may be the reason why NH₃-N concentration is higher for biochar produced at 700⁰C than 550⁰C or 300⁰C in the present study. The NH₃-N concentrations in this experiment were higher 5 mg/100 mL, which is required to improve the growth rate of bacteria in rumen for the optimal feed fermentation and digestion of DM and OM in the rumen (McDonald et al., 1995). 

Conclusions: The biochar produced from rice husk and peanut shell had no significant effects on gas, methane production, in vitro digestibility of DM and OM, pH and concentration of NH₃-N. Whereas total gas, methane production and NH₃-N concentration were significantly affected by different processing temperatures, increasing processing temperature decreased methane production and concentration of NH₃-N. There were no interactions between biomass resources and processing temperature on total gas and methane production. These results highlight the advantages and need for more research in animals (in vivo condition) to understand and confirm the practical parameters.

Acknowledgements: The authors wish to thank The National Foundation for Science and Technology Development (NAFOSTED), Vietnam for their support and funding of this project (Code 106.05-2019.22).

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