Effect of a simulated rice distillers’ byproduct on methane production in an in vitro rumen incubation of ensiled cassava root supplemented with urea and leaf meal from sweet or bitter cassava

Sangkhom Inthapanya, T R Preston¹, Le Duc Ngoan² and Le Dinh Phung²

Animal Science Department, Faculty of Agriculture and Forest Resource Souphanouvong University Lao PDR
inthapanyasangkhom@gmail.com
¹ Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV), Carrera 25 No 6-62 Cali, Colombia
² Faculty of Animal Sciences, Hue University of Agriculture and Forestry, Hue University, Vietnam

Abstract

Two experiments were carried out to study different ways of preparing a “simulated rice distillers’ byproduct (SRDB)”; and to determine the effects of the SRDB on methane production in an in vitro incubation of ensiled cassava root and urea supplemented with leaf meal from sweet or bitter cassava. Sticky rice was steamed then fermented with yeast (Saccharomyces cerevisiae) in closed plastic bags for 7 days. The fermented rice was then submitted to three treatments: boiling for 3h (SRDB-FB), distillation for 3h (SRDB-FD), or neither boiling nor distillation (SRDB-F). The SRDBs were tested for their effects on methane production in an in vitro rumen incubation of ensiled cassava root, urea and leaf meal from sweet or bitter cassava.

The SRDB-F reduced by 40% the methane production when added at 4% (DM basis) to an in vitro incubation of ensiled cassava root and urea supplemented with bitter cassava leaf meal. Boiling (SRDB-FB) or distilling (SRDB-FD) the yeast-fermented sticky rice reduced the effectiveness of the resultant product to reduce methane production. Leaf meal from a bitter cassava variety was 16% more effective than meal from a sweet variety in reducing methane production. The combined effect of the SRDB-F and bitter cassava leaf was a 50% reduction in methane production.

Keywords: β-glucan,brewers’ grains, HCN, prebiotic

Introduction

The rationale for this experiment was predicated on the observation by Phanthavong et al (2016) that when cattle were fed foliage from bitter cassava, rich in HCN precursors, they had a craving to eat brewers’ grains. It was hypothesized that the brewers’ grains were acting as a “prebiotic” providing habitat enabling the evolution of rumen microbial communities capable of detoxifying the HCN when the cassava foliage was consumed by the cattle. To test this hypothesis, fresh brewers’ grains were supplemented at 5% of the diet DM of local Yellow cattle fed ensiled cassava root and urea and given either sweet cassava foliage or fresh water spinach (Inthapanya et al 2016). The 33% increase in N retention when the cattle were fed the low level of brewers’ grains was considered to be evidence that the brewers’ grains were having a positive “prebiotic” effect on overall animal wellbeing rather than being simply an additional source of “bypass” protein. It was notable that the effect of the brewers’ grains was more pronounced when cassava foliage was the source of dietary protein rather than water spinach. The implication was that the cassava foliage was a superior source of bypass protein (solubility of the protein was 30% for cassava compared with 67% for water spinach) but this potential advantage was constrained by the negative effect of the HCN precursors (which was ameliorated by the addition of 5% of brewers’ grains to the diet). Further confirmation for the role of brewers’ grains as a prebiotic was the research of Binh et al (2017), which demonstrated a direct relationship between feeding brewers’ grains, growth enhancement of cattle and reduced excretion in the urine of thiocyanate - the product of the detoxification of HCN.

The follow-on from this research was the demonstration that a dietary level of 4% of rice distillers’ byproduct - the residue from home-distilled rice “wine” - was equally effective as brewers’ grains in enhancing growth rate of cattle, with the related benefit of reduced production of enteric methane (Sengsouly and Preston 2016). These benefits from low level supplementation of rice distiller’s byproduct have been confirmed in further studies with cattle (Sangkhom et al 2017) and with goats (Silivong et al 2018; Phuong et al 2019).

The purpose of the present study was: (i) to develop a simple method to reproduce the rice distillers’ byproduct but without the associated production of rice wine; and (ii) to test the effectiveness of the simulated rice distillers’ byproducts (SRDB) to reduce rumen methane production in an in vitro rumen incubation of cassava root-urea. This basal substrate was supplemented with leaves of either sweet or bitter cassava to create conditions in which the “prebiotic” effect of the SRDBs would be more clearly manifested.

The experiments were conducted in the laboratory of the Department of Animal Science, Faculty of Agriculture and Forest Resources, Souphanouvong University, Luang Prabang province, Lao PDR, from August to September 2019.

Experiment 1: Simulation of rice distillers’ byproduct (SRDB)

Materials and methods

“Sticky” rice was bought from the market in Luang Prabang capital. The steps in the processing to simulate rice distillers’ byproduct (SRDB) were: the sticky rice (1 kg) was wet-milled in a liquidizer, then soaked in 1.5 liters of water for 5 hours and then steamed for 30 minutes. It was then cooled for 30 minutes and mixed with yeast ( Saccharomyces cerevisiae) at 3% DM basis. The mixtures were put in closed plastic bags and allowed to ferment for 7 days after which the fermented rice was divided in three portions which were submitted to: (i) boiling for 3h (SRDB-FB); (ii) distillation for 3 h (SRDB-FD); or (iii) no further treatment (SRDB-F).

The samples of sticky rice before and after fermentation and after boiling/distilling were analyzed for pH, DM, ash, crude protein and true protein according to AOAC (1990) methods.

Results and discussion

The pH of the sticky rice after steaming was 3.9, increasing only slightly (4.1) after yeast fermentation and remaining at this level through boiling or distillation (Table 1). After steaming the DM content of the sticky rice was reduced to 27% and remained at this level through fermentation and boiling/distillation. Fermentation increased the true protein (TP) from 2.2% (39% of the crude protein) to 4.7% (61% of the crude protein). Part of this increase was the contribution from the added yeast (0.15% units of TP) but the greater part (2.45% units of TP) presumably was from growth of the yeast using the NPN produced by steaming as the N source for yeast growth. Neither boiling nor distillation affected the level of true protein beyond what was achieved by fermentation.

Experiment 2: Effect of the SRDB on the production of methane in an in vitro rumen incubation

Materials and methods

Experimental design

The experiment was arranged as a completely randomized 4*2 factorial design with 3 replicates. The factors were:

Source of SRDB

CTL: no SRDB

SRDB-F: fermented by yeast

SRDB-FB: fermented by yeast then boiled for 3h

SRDB-FD: fermented by yeast then distilled for 3h

Source of cassava leaf meal:

SW-CLM: leaf meal from sweet cassava

BT-CLM: leaf meal from bitter cassava

The SRDBs were added (DM basis) at 4% of the substrate which was: ensiled cassava root (72%), urea (2%), cassava leaf meal (25%) and 1% sulphur-rich minerals (Table 2).

Experimental procedure

The in vitro incubation procedure (Diagram 1) was the same as was developed by Sangkhom et al (2011).

Diagram 1. A schematic view of the rumen in vitro incubation system

The cassava root and cassava leaves (sweet and bitter) were collected from the Souphanouvong University farm. The leaves were chopped into small pieces of 1-2 cm, and then dried at 80ºC for 24h before grinding. After chopping into small pieces, the root was ground and then ensiled in closed plastic bags for 7 days.

Representative samples (12g DM) of the substrates (Table 2) were put in the incubation bottles followed by 0.96 liters of buffer solution (Table 3), 240 ml of rumen fluid (obtained from a slaughtered cow) and carbon dioxide (to replace the residual air in each bottle). The bottles were incubated at 38 ⁰C in a water bath for 24h.

Data collection and measurements

The gas volume was recorded over intervals of 0-6h, 6-12h, 12-18h and 18-24h. The methane concentration in the gas collected over each interval was measured with a Crowcon infra-red analyser (Crowcon Instruments Ltd, UK). At the end of the incubation, the remaining substrate was filtered through cloth and the solid residue dried at 100°C to determine the DM solubilized.

Statistical analysis

The data were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab software (Minitab 2010). In the model the sources of variation were treatments, treatment interaction and random error. The statistical model used was:

Y_ijk = µ +a_i +b_j +(a*b)_ij + e_ijk

Where: Y_ijk is dependent variable; µ is overall mean; a _i is the effect of SRDB source; b_j is the effect of cassava leaf meal; (a*b)_ij is the interaction between source of SRDB and cassava leaf meal and e_ijk is random error.

Results

Gas and methane production

At all incubation intervals, after the first 6h, the gas production, % methane in the gas, DM solubilized and methane per unit of DM solubilized were lower for all SRDB treatments compared with the control with no SRDB (Table 4). The most effective treatment for reducing methane was SRDB.F followed by SRDB.FD then SRDB.FB.

The proportion of methane in the gas, on all treatments, increased linearly with the length of the incubation (Figure 1).

Figure 1. The methane percentage in the gas decreased according to SRDB treatment and increased with the length of the incubation

There was a positive linear relationship between total gas production and the proportion of methane in the gas (Figure 2).

Figure 2. Relationship between total gas production and the proportion of the gas in the form of methane

Irrespective of the source of SRDB, the gas production (Figure 3), and the proportion of methane in the gas (Figure 4), were reduced when the protein supplement was from bitter as opposed to sweet cassava leaves.

Figure 3. Effect of method of producing the SRDB and the source of cassava leaf meal on gas production

Figure 4. Percent methane in the gas; effect of method of producing the SRDB and the source of cassava leaf meal

There was a dramatic reduction in methane production per unit substrate DM solubilized according to the method used for preparing the SRDB (Figure 5). The maximum reduction (40%) was recorded when the SRDB was produced by yeast fermentation alone. Boiling or distillation after fermentation decreased the efficacy of the SRDB to reduce the percent methane in the gas. The methane produced per unit substrate solubilized, was always less when the cassava leaves were from the bitter as opposed to the sweet variety.

Figure 5. Methane per unit DM solubilized ; effect of method of producing the SRDB and the source of cassava leaf meal

General discussion

Preparation of the SRDB

The increase in true protein of the sticky rice after fermentation with yeast confirms the observation made in the previous experiment (Sangkhom et al 2019). The only explanation for this effect is that the process of steaming the sticky rice resulted in the hydrolysis of some of the protein to peptides and/or amino acids which were subsequently used as substrate for growth of yeast.

Effectiveness of the SRDB to reduce methane production in an in vitro rumen incubation

The differences in the degree of effectiveness among the SRDBs in their capacity to reduce methane production in the in vitro rumen have no obvious explanation other than a deleterious effect of temperature on the β-glucan that presumably is responsible for the action of the SRDBs in facilitating the reactions responsible for the decline in methane production. The fact that there were consistent benefits (reduction of methane) when the cassava leaf meal was from a bitter rather than a sweet variety confirms that the mechanism for methane reduction was through reduced activity of methanogens (or the microbial flora in general). The latter explanation is supported by the research of Hamdani et al (2019) that a thymol-based additive: (i) reduced gas production in an in vitro rumen incubation: (ii) reduced the methane content of eructed gas when fed to dairy cows ; and (iii) increased the cows’ milk production.

It is relevant to emphasize the magnitude of the reduction in methane per unit substrate solubilized which was close to 50% from the effects of the SRDB supplement produced by yeast fermentation of sticky rice allied with the bitter cassava leaf meal as the protein supplement. Such a reduction in methane losses would translate into a major increase in the metabolizable energy content of the diet and a resultant increase in productivity of the host ruminant animal.

Conclusions

• The product of the fermentation of sticky rice by yeast (SRDB-F) reduced by 40% the methane production when added at 4% (DM basis) to an in vitro incubation of ensiled cassava root and urea supplemented with cassava leaf meal.
  
  
• Boiling (SRDB-FB) or distilling (SRDB-FD) the yeast-fermented sticky rice reduced the effectiveness of the resultant product to reduce methane production.
  
  
• Leaf meal from a bitter cassava variety was 16% more effective than meal from a sweet variety in reducing methane production.
  
  
• The combined effect of the SRDB-F and bitter cassava leaf was a 50% reduction in methane production.

Acknowledgements

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

References

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Received 9 September 2019; Accepted 19 September 2019; Published 2 October 2019

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