Glycerol-treated rice straw releases less methane than untreated straw in an in vitro rumen incubation

Sangkhom Inthapanya, T R Preston¹ and R A Leng²

Animal Science Department, Faculty of Agriculture and Forest Resource. Souphanouvong University Lao PDR
inthapanyasangkhom@gmail.com
¹ Investigador Emérito, Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV), Carrera 25 No 6-62 Cali, Colombia
² University of New England, Armidale NSW, Australia

Abstracts

The purpose of the present study was to assess the effect on methane production in an in vitro rumen incubation of rice straw which had been prior reacted over a 14 day period with increasing levels of crude glycerol.

Gas production and percent of substrate DM fermented, after a 24h in vitro fermentation, was increased by prior incubation of rice straw with increasing levels of crude glycerol of up to 20% in the straw DM. These effects appeared to be due to the presence of the glycerol per se and that there had been no reaction between the glycerol and the structural components of the rice straw. The methane content of the gas increased linearly as the fermentation progressed and, at all fermentation intervals, was linearly decreased by prior incubation of the rice straw with glycerol.

Keywords: biodiesel, byproducts, fermentation, global warming, palm oil

Introduction

Energy is the most fundamental requirement for human existence and activities. Thus the demand of energy has increased rapidly with growing of world population. However, the main sources of energy are fossil fuels – the burning of which contributes directly to global warming. By 2050, all fossil fuels should remain in the ground if global warming and resultant climate change is to be halted (IPCC 2014).

The other major contributor to global warming is methane (CH₄) with a global warming potential substantially larger than that of carbon dioxide (CO₂) (IPCC 2014). Ruminants are estimated to produce up to 95 million tonnes of methane (CH₄) annually and are implicated as a major source of greenhouse gas production (Patra 2014). There is therefore an urgent need to develop ways of reducing methane production from ruminant animal production systems.

Amongst the various alternate fuels being developed, biodiesel has received special attention because it can be produced from renewable sources (vegetable oils and animal fats). There are several sources which are being used as feed stock for biodiesel production such as soybean, sunflower, oil palm, rape seed, cotton seed, Jatropha and soybean oil (Singh and Singh 2009).

The oil palm (Elaeis guineensis) is a perennial crop which has the highest yield in terms of oil production per hectare of plantation (Ong et al 2011). During the trans esterification process in the manufacture of biodiesel, the triglyceride (oil or fat) is reacted with methanol or ethanol to produce biodiesel as the main product and glycerol as a by-product (Silva et al 2010, Imahara 2009).

The cultivation of oil palms is a recent activity in Lao PDR and has given rise to local production of biodiesel and with it the availability of crude “glycerol”. Ongoing research has shown that local “Yellow” cattle, fattened on cassava pulp-based diets (Phantavong et al 2016), respond with increased growth rate and a major reduction in methane emissions when 11% of crude glycerol was included in the diet (Vongsamphanh et al 2017).

The purpose of the present study was to assess the effect on methane emissions in an in vitro rumen incubation of rice straw which had been reacted over a 14 day period with crude glycerol.

Materials and methods

Location and duration

The experiment was conducted in the laboratory of the Animal Science Department, Faculty of Agriculture and Forest Resource, Souphanouvong University, Lao PDR, from January to February 2016.

Treatments and experimental design

The five treatments, arranged in a completely randomized block design with 4 replications, were levels of crude glycerol of 0, 5, 10, 15 and 20% (DM basis) added to rice straw and incubated anaerobically for 14 days. The treated straw, supplemented urea and water spinach, were used as the substrate in an in vitro rumen fermentation.

Experimental procedure

Rice straw was chopped into small pieces around 1-2 cm of length, ground (1mm sieve) and then mixed with crude glycerol at 0, 5, 10, 15 and 20% DM. The treated straw was then incubated anaerobically in sealed polyethylene bags for 14 days. Water spinach was collected from the farm of Souphanouvong University. It was chopped into small pieces and then dried in the oven at 80ºC for 24 hours before grinding (1mm sieve).

The in vitro system was the same as described by Sangkhom Inthapanya et al (2011). Amounts of the substrates equivalent to 12g DM (Table 1) were put in the incubation bottle, followed by 0.96 liters of buffer solution (Table 2) and 240 ml of rumen fluid obtained from a cow immediately after being slaughtered. The bottles were then filled with carbon dioxide and incubated at 38 ⁰C in a water bath for 24 hours.

Data collection and measurements

During the incubation the production of gas, and the concentration of methane, was recorded for successive 6h intervals over 24h. Methane was measured with an infra-red analyser (Crowcon Instruments Ltd, UK). At the end of the incubation, the contents of the incubation bottle were filtered through cloth to determine the residual DM.

Statistical analysis

The data were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab (2000) software (version 16.0). Sources of variation in the model were: treatments, replicates and error. Relationships among variables were analyzed by regression.

Results

Gas production

Gas production was highest over the 6-12h fermentation interval and lowest over the 18-24h interval, with intermediate values for 0-6h and 12-18h (Table 3; Figure 1).

Figure 1. Effect of incubating rice straw with increasing levels of glycerol (0, 5, 10, 15, 20%) on gas production during the in vitro fermentation.

Over the 24h incubation the gas production increased linearly, with increasing levels of glycerol incubated with the straw (Figure 2).

Figure 2. Production of gas over the 24 h incubation from rice straw incubated with increasing levels of glycerol

DM digestibility

The digestibility of the substrate increased when the rice straw was incubated with increasing levels of glycerol (Table 3; Figure 3). However, if it is assumed that the glycerol was 100% digested then subtracting it from the rest of the substrate results in a decline in the “corrected” digestibility as the glycerol level in the incubation was increased.

Figure 3. Effect on digestibility of the substrate as the rice straw component of the substrate was incubated with increasing levels of glycerol (corrected values were derived by assuming all the added glycerol was 100% digestible)

Methane production

The methane concentration in the gas increased as the incubation proceeded up to 24h (Figure 4) and was reduced linearly as the rice straw was incubated with increasing levels of glycerol (Figure 5).

Figure 4. Increases in methane concentration in the gas in succeeding 6h intervals (6=0-6h, 12=6-12h, 18=12-18h, 24=18-24h) during the incubation

Figure 5. The methane content in the gas increased with each 6h fermentation interval and decreased with increasing level of glycerol (G0 to G20) added to the rice straw

Methane produced per unit substrate digested decreased linearly as the rice straw component of the substrate was incubated with increasing levels of glycerol (Figure 6).

Figure 6. Methane produced per unit substrate digested decreased linearly with as the rice straw component of the substrate was incubated with increasing levels of glycerol

Discussion

The linear increase in methane production with increasing length of incubation in in vitro rumen systems has been a constant finding in a wide range of experiments (eg: Inthapanya et al 2011; Binh Phuong et al 2011; Thanh et al 2011; Outhen et al 2011; Sangkhom and Preston 2016; Duy et al 2016; Sengsouly and Preston 2016). The explanation (Inthapanya et al 2011) for this shift in the proportion of methane in the fermentation end-products with increase in residence time in the in vitro system is that it reflects the likely order of use of fermentation substrates. Initially any soluble sugars, starches and proteins will be degraded by the microbial consortium with a much slower hydrolysis of structural carbohydrates such as cellulose and hemicelluloses and insoluble protein, all of which are also fermented to VFA. As the more digestible components of the substrate are solubilized and converted into VFA there will be a change in the microbial population to those organisms that utilize VFA as an energy source (similar to the reactions that occur in a biodigestor) with the production of methane and carbon dioxide gas. The implication of this relationship is that when in vitro systems are used to estimate substrate digestibility and/or composition of fermentation end products, then the residence time in the in vitro system probably should not exceed 24h.

Addition of 20% glycerol to the substrate increased total gas production by 10% over 24h. However, the effect on the proportion of DM digested was more variable with the maximum increase being only some 4-5%. Furthermore, if digestibility values are corrected for an assumed 100% digestibility of the glycerol, then it would appear that incubating rice straw with glycerol had no effect on straw digestibility.

The linear decreases in methane production from rice straw incubated with increasing levels of glycerol from 0 to 20% in straw DM are in accordance with the results reported by Syahniara et al (2016) from 13 in vitro experiments with glycerol additions to a variety of substrates. In the functional rumen there is opportunity for supplementary glycerol to be absorbed through the rumen epithelium (by as much as 60% according to Omazic 2013). This obviously was not possible in the in vitro system used in our experiment and in those reported by Syahniara et al (2016).

Conclusions

• Gas production, after a 24h in vitro fermentation, was increased linearly (R² = 0.99) by prior incubation of rice straw with increasing levels of crude glycerol of up to 20% in the straw DM.
  
  
• The methane content of the gas increased linearly as the fermentation progressed and, at all fermentation intervals, was linearly decreased by prior incubation of the rice straw with glycerol.

Acknowledgements

This research was supported by the MEKARN II project, financed by Sida. The help received from the Animal Science Department, Faculty of Agriculture and Forest Resource, Souphanouvong University, Lao PDR is gratefully acknowledged.

References

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Received 6 January 2017; Accepted 26 May 2017; Published 2 July 2017

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