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Effect of yeast-fermented rice and 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 varieties of 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

The effect of an additive (yeast-fermented rice) on methane production was evaluated in a rumen in vitro incubation of ensiled cassava root supplemented with urea and leaf meal from sweet or bitter cassava. The experiment was arranged as a 3*2 factorial in a completely randomized design with 4 replications of the treatments which were: source of additive (CTL: no supplement; FR: yeast-fermented rice; or RDB: rice distillers’ byproduct) and source of bypass protein (cassava leaf meal from sweet or bitter variety). The quantity of substrate in each fermentation bottle was 12g DM to which were added 240 ml of rumen fluid (from a slaughtered cow in a nearby abattoir) and 960 ml of buffer solution. Measurements of gas production and methane percentage in the gas were made over intervals of: 0-6, 6-12, 12-18 and 18-24h. Residual substrate DM was measured at the end of the 24h incubation.

The methane content of the gas was reduced by 21% when yeast-fermented rice was the source of prebiotic and by 16% when rice distillers’ byproduct was the additive. The overall effect of the combination of “fermented rice” and ”bitter” cassava leaves was to reduce the methane content of the gas by 32%. The proportion of the substrate solubilized was not affected by the additives but was reduced when bitter cassava leaves replaced sweet cassava leaves as the source of bypass protein.

Key words: ensiling, incubation, methane, prebiotic


Introduction

In developing the cattle fattening system based on cassava root pulp, urea and cassava foliage (Phanthavong et al 2014, 2016a,b) it was observed that when the animals 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 of these observations 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 were ameliorated by the addition of 5% of brewers’ grains to the diet). Further confirmation for the role of brewers’ grains as a potential prebiotic was the research of Binh et al (2017), which demonstrated a direct relationship between a supplement of brewers’ grains, growth enhancement of cattle and reduced excretion in the urine of thiocyanate - the product of the detoxification of HCN in the rumen.

The follow-on from this research was the demonstration that a dietary level of 4% of rice distillers’ by-product, 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 distillers’ by-product were confirmed in a follow-on study with cattle (Sangkhom et al 2017).

The logical sequence in this research was to develop a simple method to reproduce the rice distillers’ by-product but without the associated production of rice wine (Inthapanya et al 2019). Sticky and normal rice were steamed (farmer system), or not steamed, fermented with yeast under anaerobic conditions for 7 days and then boiled or distilled (to simulate the farmer method) or not heated. There were no benefits, as measured by effects of the simulated RDB on methane production in the in vitro system (Sangkhom et al 2020), from steaming the rice or from boiling or distilling the rice after fermentation.

On the basis of these findings it was hypothesized that the critical step in the development of the RDB was probably the initial fermentation with the yeast and that the “steaming” and use of “sticky rice” were elements that facilitated the production of rice wine but had no carry-over effect on the RDB in its role as a potential prebiotic.

The following experiment was designed to compare the product from the simple fermentation of rice with the classical RDB as an additive in the in vitro rumen simulation of the cattle fattening system based on ensiled cassava root, urea and cassava foliage. Leaves from sweet and bitter cassava varieties were compared as the source of bypass protein to create conditions in which the effect of the fermented rice would be more clearly manifested.


Materials and methods

Location and duration

The experiment was carried out in the laboratory of the Faculty of Agriculture and Forest Resource, Souphanouvong University, Lao PDR, from December 2019 to January 2020.

Treatments and experimental design

The experiment was arranged as a 3*2 factorial in a completely randomized design (CRD) with 4 replications of each treatment. The factors were:

Source of additivc:

· CTL: no supplement

· FR: fermented rice

. RDB: rice distillers’ byproduct (from farmer)

Source of cassava:

· SW: sweet cassava leaf meal

· BT: bitter cassava leaf meal

Ensiled cassava root, urea, and sulphur-rich minerals were added to all the substrates.

Table 1. The quantities of ingredients in the substrates (DM basis)

No

Items

Sweet cassava leaf meal

Bitter cassava leaf meal

CTL

FR

RDB

CTL

FR

RDB

1

Ensiled cassava root

8.04

7.56

7.56

8.04

7.56

7.56

2

Fermented rice

0.48

0.48

3

Rice distillers’ byproducts

0.48

0.48

4

Urea

0.24

0.24

0.24

0.24

0.24

0.24

5

Sulphur-rich minerals

0.12

0.12

0.12

0.12

0.12

0.12

6

Bitter cassava leaf meal

3.60

3.60

3.60

7

Sweet cassava leaf meal

3.60

3.60

3.60

Total

12.0

12.0

12.0

12.0

12.0

12.0

CTL: no supplement; FR: fermented rice; RDB: rice distillers’ byproduct

In vitro system

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

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

The cassava root and cassava leaves (bitter and sweet) were collected from the farmer area in Phouxangkham village, Luang Prabang province. The leaves were chopped into small pieces of 1-2 cm, and then dried at 80şC for 24h before grinding. The cassava root was chopped into small pieces, ground and then ensiled for 7 days in closed plastic bags.

Fermented rice (FR)

Rice grain (without husk) was weighed (1 kg), wet-milled in a liquidizer, then soaked in 1.5 litres of water for 5h prior to mixing with yeast ( Saccharomyces cerevisiae) at 3% DM basis. The mixture was then put in closed plastic bags and allowed to ferment for 7 days before being evaluated in the in vitro rumen incubation following the procedure developed by Sangkhom et al (2011).

Rice distillers’ byproduct (RDB)

The RDB was collected from a farmer accustomed to making alcohol (rice wine).

The in vitro incubation

Amounts of the substrates (Table 1), equivalent to 12g DM, 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 24h.

Table 2. 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: Tilly and Terry (1963)

Data collection and measurements

During the incubation the gas volume was recorded at 6h intervals (0-6, 6-12, 12-18 and 18-24h). After each interval, the methane concentration in the gas 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 100oC 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.

Statistical analysis

The data were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab software (version 16.0). In the model the sources of variation were source of additive, source of cassava leaf, additive*leaf interaction and 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 additive; bj is the effect of cassava leaf source; (a*b)ij is the interaction between source of additive and cassava leaf source and eijk is random error.


Results and discussion

Chemical composition of diets

Fermentation of the rice with yeast did not affect the protein content of the rice, presumably because of lack of available nitrogen-rich substrate needed for the yeast to grow. The much higher protein content in the rice distillers’ byproduct would appear to be the result of the yeast “working” instead of “growing”, fermenting the rice starch to ethanol, which was subsequently removed by distillation, thus “concentrating” the effective protein content of the residue (RDB).

Table 3. Chemical composition of substrate ingredients (% in DM except for DM which is on fresh basis)

DM

CP

Ash

Ensiled cassava root

29

2.48

0.85

Bitter cassava leaf meal

90

19.1

6.12

Sweet cassava leaf meal

90

19.3

6.04

Fermented rice

7.5

5.63

4.32

Rice distillers' byproduct

7.9

22.5

5.43

Gas production

Gas production over the 24h incubation was not affected by the additives but was reduced when “bitter” cassava leaf replaced “sweet” cassava leaf as the protein source (Table 4; Figure 1). The methane content of the gas increased during successive stages in the fermentation, a finding common to all the in vitro incubations conducted in our laboratory, beginning with Sangkhom et al (2011).

DM solubilized

The proportion of the substrate solubilized was not affected by the additives but was reduced when bitter cassava leaves replaced sweet cassava leaves as the source of bypass protein.

Methane content of the gas

The methane content of the gas in the overall 24h incubation was reduced by 21% when fermented rice was additive to the substrate and by 16% when RDB was the additive (Figure 2). Methane produced per unit substrate solubilized showed similar tendencies (Figure 3). The overall effect of the combination of “fermented rice” and ”bitter” cassava leaves was to reduce the methane content of the gas by 32% (Table 5; Figure 4).

Table 4. Mean values for gas production, methane in the gas, DM solubilized and methane per unit of substrate solubilized

Additive

SEM

p

Cassava

SEM

p

CTL

FR

RDB

BT

SW

Gas production, ml

0-6h

606

613

594

19.32

0.786

567

642

15.77

0.003

6-12h

1038

1006

1031

15.87

0.359

971

1079

12.95

<0.001

12-18h

781

725

750

22.34

0.231

688

817

18.24

<0.001

18-24h

569

563

494

25.52

0.097

500

583

20.83

0.011

Methane, %

0-6h

12.0

9.75

10.1

0.232

<0.001

9.83

11.4

0.189

<0.001

6-12h

17.3

12.6

13.5

0.253

<0.001

13.5

15.4

0.207

<0.001

12-18h

20.9

16.3

17.4

0.317

<0.001

16.8

19.5

0.259

<0.001

18-24h

23.1

19.1

20.3

0.344

<0.001

19.7

22.0

0.281

<0.001

Total gas, ml

2994

2906

2869

50.56

0.227

2725

3121

41.28

<0.001

Methane, %

18.3

14.4

15.3

0.184

<0.001

15.0

17.1

0.150

<0.001

Total methane, ml

548

414

433

9.211

<0.001

402

528

7.521

<0.001

DM solubilized, %

73.3

69.9

72.0

1.086

0.113

69.7

73.8

0.887

0.004

Methane, ml/g DM solubilized

62.1

49.2

49.9

1.052

<0.001

48.0

59.5

0.859

<0.001

BT: bitter cassava leaf meal; CTL: no supplement; FR: fermented rice; P: probability; RDB: rice distiller’s by-product; SW: sweet cassava leaf meal; SEM: standard error of the mean


Figure 1. Effect of additives (fermented rice and rice distillers' byproduct)
and of source of cassava leaf on gas production after 24h (CTL: no
supplement; FR: fermented rice; RDB: rice distiller’s by-product)
Figure 2. Effect of additives (fermented rice and rice distillers' byproduct) and source of
cassava leaf on the methane content in the gas during the 24h incubation (CTL: no
supplement; FR: fermented rice; RDB: rice distiller’s by-product)


Figure 3. Effect of Effect of additives (fermented rice and rice distillers' byproduct) and source
of cassava leaf on the methane production per unit of substrate DM solubilized
(CTL: no supplement; FR: fermented rice; RDB: rice distiller’s by-product)


Table 5. Mean values for percent methane in the gas after 24h incubation in vitro

Cassava leaf

Additive

% methane

Sweet

None

19.2

Bitter

None

17.3

Sweet

RDB

16.2

Sweet

FR

15.3

Bitter

RDB

13.7

Bitter

FR

13.1

SEM

0.291



Figure 4. Interaction between cassava leaf variety and source of additives on methane content of the gas in a 24h
in vitro incubation (CTL: no supplement; RDB: rice distillers' byproduct; FR: fermented rice)


Discussion

Reduced production of methane, when leaves or foliage from the bitter variety of cassava, rather than the sweet variety, were the source of bypass protein, has been demonstrated on several occasions both in vitro (Phuong et al 2012; Binh et al 2018; Phanthavong et al 2018) and in vivo with cattle (Binh et al 2017) and with goats (Phuong et al 2019). The positive effect of RDB in reducing enteric methane in cattle has been shown by Sengsouly and Preston (2016) and Sangkhom et al (2017).

The results presented in this paper are the outcome of a series of experiments that aimed to develop a simple additive, building on the experience obtained with brewers’ grains (Binh et al 2017) and rice distillers’ byproduct (RDB) (Sengsouly and Preston 2016). The initial attempts using rice grain fermented with yeast, diammonium phosphate and urea, were not successful (Inthapanya et al 2019); there were also no advantages from steaming of the rice prior to fermentation nor from boiling or distilling off the ethanol (Sangkhom et al 2020).

Anaerobic fermentation of milled rice with yeast over a period of 7 days is a simple operation easily done under farm conditions. The next step is to test this system with cattle and goats to confirm: (i) the reduction in enteric rumen methane: and (ii) the expected improvement in growth rate and feed conversion that should result from the net energy gained from the expected shift in fermentation end-products from methane to propionate (Figure 5).

 
Figure 5. Linear relationship between the methane: carbon dioxide ratio in expired breath and molar
propionic:acetic acid ratio in the rumen of cattle fed ensiled cassava root, urea, brewers’
grains and rice straw supplemented with glycerol (Phanthavong et al 2017)


Conclusions


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 31 December 2019; Accepted 7 February 2020; Published 2 March 2020

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