Simulating rice distillers’ by-product with fermented sticky rice; effects on methane production in an in vitro rumen fermentation of ensiled cassava root, cassava foliage and urea

Sangkhom Inthapanya, T R Preston¹, Le Thuy Binh Phuong², Le Dinh Phung³ and Le Duc Ngoan³

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 Nong Lam University, Vietnam
³ Faculty of Animal Sciences, Hue University of Agriculture and Forestry, Hue University, Vietnam

Abstract

Experiments were carried out: (i) to produce a simulated “rice distillers’ byproduct (SRDB)” which would act as a prebiotic in ruminant diets based on cassava root and cassava foliage; and (ii) to test the efficacy of the SRDB to reduce methane production in an in vitro rumen incubation of ensiled cassava root, cassava leaf meal and urea. In experiment 1, sticky rice was steamed then supplemented with either yeast alone (Y) or yeast in combination with urea and di-ammonium phosphate (Y+U+DAP). After a 7/day anaerobic fermentation the products were boiled for 3h or not boiled. In the rumen in vitro incubation (experiment 2), using 1 liter recycled plastic “Pet” water bottles, with gas collection by water displacement, the substrate (12 g DM) was ensiled cassava root (72%), bitter cassava leaf meal (25%), urea (2%) and S-rich minerals (1%), all on DM basis. Buffer solution (960 ml) and rumen fluid (240ml) from a slaughtered cow were added and air in the gas space was displaced with carbon dioxide. The fermentation treatments (SRDBs) in a 2*2 factorial design with 4 replications were: DAP+U+Y with boiling for 3h after fermentation; DAP+U+Y no boiling; Yeast with boiling after fermentation; Yeast no boiling. The SRDBs were added at 4% (DM basis) to the substrate in the rumen in vitro incubation. Measurements were made of total gas production and methane percentage in the gas at intervals of 0-3, 3-6, 6-12, 12-18 and 18-24h.

The SDRB was more effective in reducing methane production in the in vitro rumen incubation when the sticky rice was fermented only with yeast rather than with the combination of yeast, DAP and urea. Boiling the SRDBs after fermentation reduced their capacity to reduce rumen methane in the in vitro incubation.

Key words: bitter cassava, byproduct, gas, HCN, Prebiotic, sticky rice

Introduction

The purpose of this study was to develop and test a feed supplement with “prebiotic” properties that can be produced by the farmer. The research was based on earlier findings that the products of fermentation of cereal grains (brewers’ grains and rice distillers’ byproduct) have “prebiotic” properties as reflected in improved animal performance and wellbeing when added to cassava-based diets containing elements such as cyanogenic glucosides that can give rise to toxic HCN in the animals’ digestive system (Sengsouly et al 2016; Binh et al 2017; Sangkhom et al 2017; Binh et al 2018).

Hypotheses:

• It is the boiling (distillation) of the fermented rice in the making of rice wine that brings about the acid hydrolysis, needed to release β-glucan from cereal grains and yeast, and that this gives “prebiotic” properties to brewers’ grains and rice distillers’ byproduct.
  
  
• Adding urea and diammonium phosphate (DAP) to the yeast in the fermentation of sticky rice will prevent formation of ethanol but will not have a negative effect on the release of β-glucan.

As our laboratory in not equipped to analyze directly the presence of β-glucan in the end products of cereal grain fermentation, the presence of “prebiotic” properties will be measured indirectly by the potential of the supplement to reduce methane production in rumen in vitro incubations of substrates based on roots and leaves of the cassava plant.

The experiments were carried out in the laboratory of the Department of Animal Science, Faculty of Agriculture and Forest Resources, Souphanouvong University, Luang Prabang province, Lao PDR, from January to February 2018.

Experiment 1: Simulation of rice distillers’ by-product (SRDB)

Materials and methods

The “sticky” rice was bought from the market in Luang Prabang capital. The steps in the processing to simulate rice distillers’ byproduct were: the sticky rice was weighed (1 kg) and wet-milled in a liquidizer, then soaked in 1.5 litres of water for 5h then steamed for 30 minutes. It was then cooled for 30 minutes and mixed with yeast ( Saccharomyces cerevisiae) alone (3% DM basis) or yeast, urea and dicalcium phosphate (DAP) (3, 2 and 0.8%, respectively on DM basis). The mixtures were then put in closed plastic bags and allowed to ferment for 7 days after which each treatment was divided into two portions, one of which was boiled for 3h. The four products (SRDBs) from this process (Table 1) were then evaluated as potential “prebiotics” in rumen in vitro incubations following the procedure developed by Sangkhom et al (2011).

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

Table 1. Chemical composition and pH values of the SRDBs
	Boiled		Not boiled
	DAP+U+Y	Yeast	DAP+U+Y	Yeast
DM, %
Before fermentation	27.7	27.6	27.7	27.6
After fermenting 7d	26.1	27.6	25.9	25.9
Then after boiling 3h	7.83	7.59
Ash, % in DM
Before	4.54	4.59	4.54	4.43
After fermenting 7d	4.11	4.11	3.97	4.01
Then after boiling 3h	4.09	3.99	-	-
Crude protein, % in DM
Before	11.6	5.50	11.5	5.45
After fermenting 7d	11.4	4.89	11.2	4.88
Then after boiling 3h	11.3	5.23	-	-
True protein, % in DM
Before fermentation	2.05	1.80	2.00	1.75
After fermenting 7d	7.55	4.40	7.45	4.25
After boiling 3h	5.60	2.80	-	-
pH
Before fermentation	3.91	3.71	3.86	3.66
After fermenting 7d	5.42	4.00	5.38	3.75
Then after boiling 3h	6.10	4.10

Results

The pH of the SRDBs after fermentation was increased (to 5.42 and 5.38) when DAP and urea were added prior to the fermentation as compared with adding only yeast (4.00 and 3.75) (Table 1). The effect of boiling was to increase the pH further (from 5.42 to 6.1) in the case of the DAP+U+Y treatment but had negligible effect on the yeast only treatment (4.0 to 4.1).

As expected, the values for crude protein before and after fermentation and after boiling reflected the addition of urea, DAP and yeast and were not affected by the process of fermentation nor of boiling. In contrast, the true protein content of the SRDBs increased from 2% in DM (derived originally from the sticky rice) to 7.45-7.83% in DM for the DAP+U+Y treatment and to 4.25-4.4% when only yeast was added. Boiling the SRDB decreased the true protein by about 2 units. The greater increases in true protein after fermentation with the DAP+U+Y treatment reflect the effect of the nitrogen and phosphorus from urea and DAP in providing nutrients for growth of the yeast. We have no explanation for the reduction in true protein caused by boiling.

Experiment 2: Effect of simulated rice distillers’ by-product (SRDB) on methane production in a rumen in vitro incubation of ensiled cassava root, urea and cassava leaf meal

Experimental design

The experiment was arranged as a completely randomized 2*2 factorial design with 4 replicates. The two factors (from Experiment 1) were:

• Source of fermentation additives
• Boiling

The individual treatments were:

• DAP+U+Y with boiling after fermentation
• DAP+U+Y no boiling
• Yeast with boiling after fermentation
• Yeast no boiling

The SRDBs were added at 4% (DM basis) to the substrate (Table 2) in the rumen in vitro incubation, which was composed (DM basis) of 72% ensiled cassava roots, 2% urea, 25% leaf meal from a bitter cassava variety and 1% sulphur-rich minerals. The composition of the ingredients in the substrates is in Table 3.

In vitro rumen fermentation system

The in vitro rumen fermentation system (Diagram 1) followed the model described by Sangkhom et al (2011).

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

Experimental procedure

The bitter cassava leaves were collected from the Souphanouvong University farm. They were chopped into small pieces of 1-2 cm, then dried at 80ºC for 24h before grinding. Representative samples (12g DM) of the substrates (Table 2) were put in the incubation bottle followed by 0.96 liters of buffer solution (Table 4), 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 100C to determine the DM solubilized (digested) during the incubation.

Chemical analyses

Samples were analyzed for DM, ash, and crude protein according to AOAC (1990) methods. The solubility of the protein in the SRDB and the cassava leaf meal was determined by extraction with M NaCl according to the method outlined in Whitelaw and Preston (1963).

Statistical analysis

The data were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab software (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 boiling; (a*b)_ij is the interaction between source of SrDB and boiling and e _ijk is random error.

Results

Gas and methane production

At all incubation times after 6h, and over the full 24h period, the gas production was reduced when the SRDBs were derived from sticky rice fermented only with yeast compared with yeast plus urea and DAP (DAP+U+Y) (Table 5; Figure 1). Gas production was increased when the SRDBs were boiled following fermentation (Figure 2).

The percentage of methane in the gas was reduced when yeast was the only additive used in the production of the SRDB (Figure 3); however, it was increased when the SRDBs were boiled after fermentation (and when the additives included yeast, DAP and urea); but not affected when only yeast was the additive (Figure 4).

The DM solubilized during the incubation was decreased when the SRDBs were fermented only with yeast rather than “mixed yeast, DAP and urea” (Figure 5). By contrast, DM solubilized was increased when the SRDBs had been boiled after mixed yeast, DAP and urea were added in the fermentation but with no effect when only yeast was added (Figure 6).

Methane produced per unit DM solubilized was reduced when the SRDBs had been fermented with yeast as compared with mixed yeast, DAP and urea (Figure 7); and increased when the SRDBs had been boiled after the fermentation (Figure 8).

Figure 1. Total gas production; effect of adding DAP+U+Y or Yeast alone	Figure 2. Total gas production; effect of boiling

Figure 3. Percent methane in the gas; effect of adding DAP+U+Y or Yeast alone	Figure 4. Percent methane in the gas; effect of boiling

Figure 5. DM solubilized; effect of adding DAP+U+Y or Yeast alone	Figure 6. DM digested; effect of boiling

Figure 7. Methane per unit DM solubilized; effect of adding DAP+U+Y or Yeast alone	Figure 8. Methane per unit DM solubilized; effect of boiling

As expected, there was a direct linear relationship between total gas production and the DM solubilized during the incubation (Figure 9). However, the methane “content” in the gas was not constant but increased linearly as total gas production (and DM solubilized) was increased (Figure 10). In other words: the gas from the treatments that produced most rumen gas was also richer in methane.

Figure 9. Gas production and percent DM solubilized during the incubation are directly elated	Figure 10. The methane content of the gas increased linearly as gas production increased

Discussion

In hindsight there was a mistake in the design of the two experiments, in not including as a control treatment, the traditional byproduct from rice wine production (RDB) with proven properties as a “prebiotic” in diets based on ensiled cassava root-urea-cassava foliage (Sengsouly et al 2017: Sangkhom et al 2017). Thus, it could not be ascertained why heating (boiling) the yeast-fermented sticky rice had a negative effect on the “simulated RDB compared with “distilling” the yeast-fermented sticky rice as in traditional rice wine production

There are three issues that arise from this research, assuming that the benefits of rice distillers’ byproduct in reducing enteric methane, and increasing animal productivity, are due to the action of β-glucan released from cell wall carbohydrate by yeast fermentation of sticky rice (as in the traditional method of producing rice wine): (i) β -glucan is not produced when additional nutrients are added to encourage the yeast to multiply as opposed to producing alcohol (as in the traditional way to produce rice wine); (ii) that boiling the fermented substrate (as opposed to traditional distillation) negates the “prebiotic” effect achieved by fermentation alone; (iii) although it was shown that the simulated SRDB produced by simple yeast fermentation of sticky rice, without distillation or boiling of the fermented substrate, was effective in reducing methane production in an in vitro incubation of cassava pulp-urea and cassava leaf meal, it is still to be proved that the simulated SRDB will improve growth and feed conversion in growing cattle as has been demonstrated with the traditional rice distillers’ byproduct (RDB) (Sengsouly et al 2016 ; Sangkom et al 2017).

Conclusions

• A simulated rice distillers’ byproduct (SRDB) made by fermenting sticky rice with yeast was more effective in reducing methane production in an in vitro rumen incubation than when it was made by fermenting sticky rice with a combination of yeast, di-ammonium phosphate and urea.
  
  
• Boiling the SRDBs after fermentation negated their capacity to reduce rumen methane in the in vitro incubation.

Acknowledgements

This research is part of the requirement by the senior author for the degree of PhD at Hue University of Agriculture and Forestry, in Vietnam. 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

AOAC 1990 Official methods of analysis.15th ed. AOAC, Washington, D.C (935-955)

Binh P L T, Preston T R, Duong K N and Leng R A 2017 A low concentration (4% in diet dry matter) of brewers’ grains improves the growth rate and reduces thiocyanate excretion of cattle fed cassava pulp-urea and “bitter” cassava foliage. Livestock Research for Rural Development. Volume 29, Article #104. http://www.lrrd.org/lrrd29/5/phuo29104.html

Binh P L T, Preston T R, Van H N and Dinh V D 2018 Methane production in an in vitro rumen incubation of cassava pulp-urea with additives of brewers’ grain, rice wine yeast culture, yeast-fermented cassava pulp and leaves of sweet or bitter cassava variety. Livestock Research for Rural Development. Volume 30, Article #77. http://www.lrrd.org/lrrd30/4/binh30077.html

Minitab 2010 Minitab Software Release 16.0 Minitab Inc., Pennsylvania, USA.

Sangkhom I, Preston T R and Leng R A 2011 Mitigating methane production from ruminants; effect of calcium nitrate as modifier of the fermentation in an in vitro incubation using cassava root as the energy source and leaves of cassava or Mimosa pigra as source of protein. Livestock Research for Rural Development. Volume 23, Article #21. http://www.lrrd.org/lrrd23/2/sang23021.htm

Sangkhom I, Preston T R, Leng R A, Ngoan L D and Phung L D 2017 Rice distillers’ byproduct improved growth performance and reduced enteric methane from “Yellow” cattle fed a fattening diet based on cassava root and foliage (Manihot esculenta Cranz). Livestock Research for Rural Development. Volume 29, Article #131. http://www.lrrd.org/lrrd29/7/sang29131.html

Sengsouly P and Preston T R 2016 Effect of rice-wine distillers’ byproduct and biochar on growth performance and methane emissions in local “Yellow” cattle fed ensiled cassava root, urea, cassava foliage and rice straw. Livestock Research for Rural Development. Volume 28, Article #178. http://www.lrrd.org/lrrd28/10/seng28178.html

Tilley J M A and Terry R A 1963 A two stage technique for the in vitro digestion of forage crops. Journal of the British Grassland Society. 18 : 104

Whitelaw F G and Preston T R 1963 The nutrition of the early-weaned calf. III. Protein solubility and amino acid composition as factors affecting protein utilization. Animal Production. Volume 5 pp 131-145.

Received 28 February 2019; Accepted 20 July 2019; Published 1 August 2019

Go to top