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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.

Table 1. Changes in pH and chemical composition during production of the SRDBs

SRDB.F

SRDB.FB

SRDB.FD

pH  

After steaming

3.91

3.91

3.90

After fermenting 7 days

4.03

4.03

4.10

After processing

4.03

4.07

4.17

DM, %  

Before fermentation

27.2

27.2

27.2

After fermenting 7days

26.9

26.9

26.9

After processing

26.9

26.8

26.8

Ash, % in DM

Before fermentation

4.40

4.43

4.47

After fermenting 7days

4.25

4.24

4.23

After processing

4.24

4.20

4.17

Crude protein, % in DM

Before fermentation

5.64

5.64

5.64

After fermenting 7days

5.65

5.66

5.67

After processing

5.60

5.62

5.62

True protein, % in DM

Before fermentation

2.19

2.19

2.18

After fermenting 7days

4.68

4.67

4.67

After processing

4.57

4.62

4.62


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).

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

Sweet (or bitter) cassava leaves

CTL

SRDB.F

SRDB.FB

SRDB.FD

Ensiled cassava root

8.64

8.16

8.16

8.16

SRDB

-

0.48

0.48

0.48

Cassava leaves

3.00

3.00

3.00

3.00

Urea

0.24

0.24

0.24

0.24

Sulphur-rich minerals

0.12

0.12

0.12

0.12

Total

12.0

12.0

12.0

12.0

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.

Table 3. 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

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:

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 cassava leaf meal; (a*b)ij is the interaction between source of SRDB and cassava leaf meal and eijk 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.

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

Source of SRDB

SEM

p

Source of CLM

SEM

p

CTL

SRDB.FB

SRDB.FD

SRDB.F

Bitter

Sweet

Gas production, ml

0-6h

683

683

658

642

15.59

0.204

625

708

11.02

<0.001

6-12h

1200a

1083b

1042b

1017b

19.09

<0.001

1071

1100

13.50

0.150

12-18h

858a

758ab

717b

658b

33.59

0.005

688

808

23.75

0.002

18-24h

658a

583ab

567ab

533b

28.26

0.040

538

633

19.98

0.004

Methane, %

0-6h

11.7a

10.7a

9.33b

8.70b

0.2635

<0.001

9.67

10.5

0.186

0.006

6-12h

13.5a

12.0b

10.3c

9.67c

0.250

<0.001

10.8

11.9

0.177

0.001

12-18h

19.8a

17.8b

15.7c

14.8c

0.363

<0.001

16.9

17.2

0.257

0.501

18-24h

22.8a

21.2b

19.5c

18.3c

0.391

<0.001

19.8

21.1

0.276

0.006

Total gas, ml

3400a

3108b

2983bc

2850c

62.64

<0.001

2921

3250

44.29

<0.001

Methane, %

17.0a

15.4b

13.7c

12.9d

0.152

<0.001

14.3

15.2

0.107

<0.001

Total methane, ml

563a

462b

393c

350c

11.34

<0.001

402

481

8.018

<0.001

DM solubilized, %

71.0

69.2

67.5

65.9

2.049

0.357

67.5

69.3

1.449

0.398

Methane, ml/g DM solubilized

66.5a

55.8ab

48.6bc

44.5c

2.630

<0.001

50.0

57.7

1.860

0.010

CLM: cassava leaf meal; CTL: control; SRDB.F: fermented by yeast only; SRDB.FB: fermented by yeast, then boiled; SRDB.FD: fermented by yeast, then distilled

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


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

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

Hamdani H, Chami N, Oukhouia M, Jabeur I, Sennouni C and Remmal A 2019 Effect of a thymol-based additive on rumen fermentation, on methane emissions in eructed gas and on milk production in Holstein cows. Livestock Research for Rural Development. Volume 31, Article #107. http://www.lrrd.org/lrrd31/7/houdh31107.html

Inthapanya S, Preston T R and Leng R A 2016 Ensiled brewers’ grains increased feed intake, digestibility and N retention in cattle fed ensiled cassava root, urea and rice straw with fresh cassava foliage or water spinach as main source of protein. Livestock Research for Rural Development. Volume 28, Article #20. http://www.lrrd.org/lrrd28/2/sang28020.htm

Minitab 2010 Minitab Software Release 16.0

Phanthavong V, Preston T R, Viengsakoun N and Pattaya N 2016 Brewers' grain and cassava foliage (Manihot esculenta Cranz) as protein sources for local “Yellow” cattle fed cassava pulp-urea as basal diet. Livestock Research for Rural Development. Volume 28, Article #196. http://www.lrrd.org/lrrd28/11/phan28196.html

Phuong L T B, Preston T R, Van N H and Dung D V 2019 Effect of additives (brewer’s grains and biochar) and cassava variety (sweet versus bitter) on nitrogen retention, thiocyanate excretion and methane production by Bach Thao goats. Livestock Research for Rural Development. Volume 31, Article #1. http://www.lrrd.org/lrrd31/1/phuong31001.html

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, Binh P L T, Phung L D and Ngoan L D 2019 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. Livestock Research for Rural Development. Volume 31, Article #127. http://www.lrrd.org/lrrd31/8/sang31127.html

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

Silivong P, Preston T R, Van N H and Hai D T 2018 Brewers’ grains (5% of diet DM) increases the digestibility, nitrogen retention and growth performance of goats fed a basal diet of Bauhinia accuminata and foliage from cassava (Manihot esculenta Crantz) or water spinach (Ipomoea aquatica). Livestock Research for Rural Development. Volume 30, Article #55. http://www.lrrd.org/lrrd30/3/siliv30055.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


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

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