Livestock Research for Rural Development 31 (8) 2019 Guide for preparation of papers LRRD Newsletter

Citation of this paper

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:

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:

The individual treatments were:

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.

Table 2. The quantities of ingredients in the substrates (DM basis)

DAP+U+Y
boiled

DAP+U+Y
not boiled

Yeast
boiled

Yeast
not boiled

Ensiled cassava root

8.16

8.16

8.16

8.16

Bitter cassava leaf meal

3.00

3.00

3.00

3.00

Urea

0.24

0.24

0.24

0.24

Minerals

0.12

0.12

0.12

0.12

SRDB

0.48

0.48

0.48

0.48

Total

12.0

12.0

12.0

12.0



Table 3. Chemical composition of ensiled cassava root and cassava leaf meal

 

DM,
%

CP

Ash

Insoluble CP,
% of total CP

As, % of DM

Ensiled cassava root

32.0

2.48

0.85

9.20

Bitter cassava leaf meal

90.0

19.1

6.12

31.1

CP: crude protein; DM: dry matter

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 80şC 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.

Table 4. Ingredients of the buffer solution

Ingredients

CaCl2

NaHPO4.12H2O

NaCl

KCl

MgSO4.7H2O

NaHCO3

Cysteine

(g/liter)

0.04

9.30

0.47

0.57

0.12

9.80

0.25

Source: Tilley and Terry (1963)

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:

Yijk = µ +ai +bj +(a*b)ij + eijk

Where: Yijk is dependent variable; µ is overall mean; a i is the effect of SRDB source; bj 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).

Table 5. Mean values for gas production, methane in the gas, digestibility and methane per unit substrate solublidized

Additive

p

Boiling

p

SEM

DAP+U+Y

Yeast

Boiled

Not Boiled

Gas production, ml

0-3h

469

431

0.262

444

456

0.702

22.5

3-6h

656

619

1.00

656

619

1.00

14.9

6-12h

1131

1013

0.004

1119

1025

0.017

24.1

12-18h

931

806

<0.001

906

831

0.010

17.31

18-24h

656

594

0.166

656

594

0.166

29.9

Methane, %

0-3h

8.13

7.38

0.007

8.13

7.38

0.007

0.161

3-6h

11.3

9.63

0.002

10.8

10.1

0.146

0.284

6-12h

14.1

12.5

0.002

13.8

12.9

0.050

0.284

12-18h

19.1

17.0

<0.001

18.6

17.5

0.042

0.350

18-24h

21.8

19.1

<0.001

21.1

19.8

0.004

0.275

0-24h

Gas, ml

3844

3463

<0.001

3781

3525

<0.01

60.4

Methane, %

15.4

13.5

<0.001

15.0

13.9

<0.001

0.133

Methane, ml

593

469

<0.001

570

492

<0.001

8.77

DM solubilized, %

74.6

67.0

0.004

72.1

69.5

0.258

1.53

Methane, ml/g DM solubilized

68.2

60.3

<0.001

67.8

60.7

0.003

1.33

DAP: di-ammonium phosphate; U: urea; Y: yeast; p: probability; SEM: standard error of the mean



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


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

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

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