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Citation of this paper

Effect of supplements of yeast (Saccharomyces cerevisiae), rice distillers’ by-product and fermented cassava root on methane production in an in vitro rumen incubation of ensiled cassava root, urea and cassava leaf meal

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

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

An in vitro rumen incubation was carried out to determine effects on methane production of supplementing ensiled cassava root, urea and cassava leaf meal with rice distillers’ byproduct, fermented cassava root and yeast (Saccharomyces cerevisiae). The four treatments in a completely randomized design were: CTL: No supplement; RDB: 4% (in DM) rice distillers’ byproduct; FCR: 4% (in DM) urea-fermented cassava root; Yeast: 1% (in DM) commercial yeast. The quantity of substrate in each fermentation bottle was 12g DM to which were added 240 ml of rumen fluid (from slaughtered cattle) and 960 ml of buffer solution. The incubations were done using one-liter “PEP” bottles with gas collection by water displacement. Measurements of total gas production and methane percentage in the gas were made at intervals of 0-3, 3-6, 6-12, 12-18 and 18-24h.

The hourly rate of gas production increased to a maximum in the 3-6h of incubation interval and then decreased linearly. In contrast, the proportion of methane in the gas increased linearly with incubation interval from the beginning until the final 18-24h interval. Total gas production was highest for the fermented cassava root additive, followed by the rice distillers’ byproduct with lowest values for the control and yeast treatments. The methane content of the gas was highest for the control treatment, followed by the fermented cassava root and yeast with the lowest value for rice distillers’ byproduct, for which the overall reduction in methane was of the order of 25%.

 Methane production per unit DM digested showed a similar trend as the methane percentage in the gas. It is suggested that the benefits from brewers’ grains and rice distillers’ byproduct, in reducing methane production in the rumen fermentation, both in vitro and in vivo, are the indirect effects of these additives increasing the proportions of propionic acid in the rumen VFA.

Key words: β-glucan, greenhouse gas, hydrolysis, propionate, urea


Introduction

Agriculture is an important source of greenhouse gas mainly through emissions of methane from enteric fermentation in ruminants and decomposition of manure (Gerber et al 2013). Ruminants are estimated to produce up to 95 million tonnes of methane annually, mainly from enteric fermentation and to a lesser extend from decomposition of manure (O’Mara 2011; Patra 2014).

Strategies to reduce these emissions should first address the need to increase productivity of ruminant livestock. This will reduce methane emissions per unit of livestock product – meat and milk (Figure 1); secondly, the feeding systems that lead to increased ruminant productivity are those that lead to increased proportions of propionic acid; and thirdly, the escape of protein from the rumen contributes amino acids directly to the animal through enzymic digestion of protein in the intestines. The additional advantage of this process is that fibrous feed particles that escape the rumen attached to the protein will still be fermented to useful end products but in the cecum-colon section of the ruminant digestive tract in which the fermentation process does not produce methane (Demeyer 1991).

Figure 1. Relationship between growth rate of cattle (kg/d) and the production of
methane (g/kg live weight gain) (Klieve and Ouwerkerk 2007)

In countries located in temperate latitudes, cereal crops such as maize and barley are the choice of feeds for intensifying ruminant production. Maize is grown in tropical latitudes but yields do not compete with those in temperate climates. By contrast, cassava (Manihot esculenta Crantz) is a crop that originated in the tropics (in the Caribbean) and is now grown in over 90 countries world-wide (Lebot 2009). Of importance in a warming world is that it appears that cassava is potentially highly resilient to future climatic changes and according to Jarvis et al (2012) “could provide Africa with options for adaptation whilst other major food staples face challenges”.

Cassava has become a major crop in Lao PDR mainly because of the export of starch that is extracted from the cassava root. There are five cassava starch factories with a total planted area of 43,975ha, giving an average yield of fresh roots of 24 tonnes/ha. Annual production is of the order of 1,000,000 tonnes (Lao PDR Department of Agriculture 2012).

The increasing popularity of cassava for industrial purposes has focussed attention on its potential as animal feed, partly because of the availability of the by-products (some 20% of the original weight of roots remains as high moisture pulp) and partly through the development of high yield varieties.

These developments have been the driving force for a series of researches directed at optimising the use of both the pulp and the fresh cassava root as the basis of intensive systems of livestock production, especially the fattening of local cattle (Phanthavong et al 2014, 2015, 2016, 2017; Inthapanya et al 2016). An additional advantage of cassava over cereal crops such as maize is that the foliage has proved to be a a valuable source of bypass protein such that the cassava plant becomes a source both of highly digestive carbohydrate (from the root) as well as protein (from the foliage. The only additional features needed are a source of fermentable nitrogen (available locally as fertilizer grade urea) and minerals.

The development of cattle feeding systems based on cassava has stimulated an important outcome, namely how to manage the potential toxicity linked with the presence throughout the plant of cyanogenic glucosides that give rise to hydrocyanic acid when exposed to favorable conditions (eg: appropriate enzymes) in the plant itself or within the digestive of animals that consume it. Recent research, much of it in the laboratory of the senior author of this paper, has shown that the potentially toxic cyanogenic glucosides in cassava can be neutralized by supplementing the animal diet with small quantities of locally available byproducts from fermentation industries, specifically from the production of beer which gives rice to “brewer’ grains”; and the artisan distillation of fermented rice to make an alcoholic wine, which produces a byproduct known in Lao PDR as “Quilao”, in Vietnam as “Hem” and in Cambodia as “Bar Ran”. Brewers’ grains have been shown to aid directly in the detoxification of HCN in cases where forage of a “bitter” (high HCN potential variety) was fed (Binh et a 2017). Both brewers’ grains and rice distillers’ byproduct, fed at less than 5% of the diet, have resulted in reduced production of rumen methane and improved growth rates in cattle fed ensiled cassava pulp-urea of ensiled cassava root-urea as basal diet (Keopaseuth et al 2016; Sengsouly and Preston 2016; Inthapanya et al 2017).

Access to brewers’ grains is limited to farmers living in close proximity to the beer factory. Rice distillers’ byproduct is more widely available in rural areas, but supplies are limited. For these reasons, research to identify alternatives to both brewers’ grains and rice distillers’ by-product are considered to be of high priority. As both brewers’ gains and rice distillers’ by-product are products of fermentation by yeast (specifically Saccharomyces cerevisiae), it was decided to evaluate: (i) a commercial source of yeast commonly available in local markets; and (ii) cassava root enriched by yeast fermentation with additional sources of nitrogen (urea) and phosphorus (diammonium phosphate – DAP).

The purpose of the present study was to determine effects on methane production in an in vitro rumen fermentation when yeast, rice distillers’ byproduct, or fermented cassava root, were added in small amounts (4% DM basis) to a basal substrate of ensiled cassava root supplemented with urea and cassava leaf meal.


Materials and methods

Location and duration

The experiment was conducted in the laboratory of the Department of Animal Science, Faculty of Agriculture and Forest Resources, Souphanouvong University, Luang Prabang province, Lao PDR.

Treatments and experimental design

The experimental design was completely randomized (CRD) with 4 treatments and 5 replicates of each treatment.

· CTL: Ensiled cassava root

· RBD: CTL + rice distillers’ byproduct at 4% of DM

· FCR: CTL+ fermented cassava root at 4% of DM

· Yeast: CTL+ yeast at 1% of DM

All the treatments included urea (2% of root DM), cassava leaf meal from a bitter variety (25% of substrate DM) and S-rich minerals (1% of DM substrate).

In vitro rumen fermentation system

The in vitro rumen fermentation system (Diagram 1) was the same as that used by Sangkhom et al (2011). Recycled “PEP”water bottles (capacity 1500ml) were used for the fermentation and collection of the gas. These were connected by plastic tube (id 4mm) to a similar bottle which received the gas (the bottom of which had been removed) and which was suspended in a larger bottle (5liter capacity) partially filled with water, so as to collect the gas by water displacement. The bottle that was suspended in water was calibrated at 50ml intervals to indicate the volume of gas.

Diagram 1. A schematic view of an apparatus to measure gas production in an in vitro fermentation
Experimental procedure

The cassava roots and leaves were collected from the Souphanouvong University farm, The roots were chopped into pieces around 1-2 cm of length, ground in a liquidizer, and then stored in a plastic bag for ensiling over 7 days. Cassava leaves were chopped into small pieces around 1-2 cm in length, then dried in the oven at 80ºC for 24 h before grinding. The rice distillers’ byproduct was collected from a farmer accustomed to produce “rice wine alcohol”. The procedure for producing “fermented cassava root” was as follows: the roots were chopped and steamed for 30 minutes, allowed to cool for 15mintutes and then mixed with di-ammonium phosphate (DAP) at 0.8%, 3% of yeast (Saccharomyces cerevisiae) and 2% of urea (all on DM basis) prior to being fermented in a closed plastic bag for 7 days. Representative samples (12g DM) of the substrates was put in the incubation bottle to which was added 0.96 liters of buffer solution (Table 1) and 240 ml of rumen fluid (obtained from a slaughtered cow) prior to filling each bottle with carbon dioxide. The bottles were incubated at 38 0C in a water bath for 24h.

Table 1. 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 0-3h, 3-6h, 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 digested.

Chemical analyses

Samples were analyzed for dry matter (DM), ash, crude protein (CP) and crude fiber (CF) according to AOAC (2010) methods. The solubility of the protein in diet ingredients was determined by extraction with M NaCl according to the method outlined in Whitelaw and Preston (1963).

Statistical analysis

The data were subjected to ANOVA analyses in the Minitab software (Mintab 2010).  Tukey’s pair-wise comparison was used to determine the differences between treatments at a probability of p<0.05.


Results and discussion

Chemical composition

The values for DM, crude protein and solubility of the protein in the rice distillers’ byproduct (Table 2) were similar to those reported for the same product by Luu Huu Manh et al (2009) and Sangkhom et al (2017). The cassava root after fermentation with yeast, urea and DAP had a similar level of crude protein as reported for the same procedure by Vanhnasin et al (2016) and Manivanh et al (2016).

Table 2. Chemical composition of substrate components


DM
%

CP

CF

Ash

Soluble CP
% of total CP

As % of DM

Ensiled cassava root

32.1

2.51

1.12

0.88

9.22

Fermented cassava root

39.9

10.4

1.13

0.87

11.6

Bitter cassava leaf meal

90.3

19.0

15.2

6.14

31.3

Rice distillers’ byproduct

7.46

24.2

2.29

4.45

37.9

Yeast


45.0



32.4

Effect of incubation interval

The rate of gas production increased to a maximum in the interval 3-6h of incubation then decreased linearly (Table 3; Figure 2). In contrast, the proportion of methane in the gas increased linearly with incubation interval (Figure 3).

Table 3. Mean values for percent methane in the gas, and gas production per hour, during successive intervals of the fermentation

Interval, h

SEM

p

0-3

3-6

6-12

12-18

18-24

Methane, %

7.25a

9.35b

16.0c

20.0d

22.2e

0.203

<0.0001

Gas, ml/h

128b

193d

150c

125b

91a

3.8

<0.0001



Figure 2. Effect of fermentation interval on rate of gas production in an in vitro fermentation of ensiled
cassava root supplemented with urea and cassava leaf meal and different additives


Figure 3. Effect of fermentation interval on the methane content of the gas in an in vitro fermentation
of ensiled cassava root supplemented with urea and cassava leaf meal
Effect of additives

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

Gas production, ml

CTL

FCR

Yeast

RDB

SEM

p

0-3h

360a

420b

370a

380a

11.7

0.012

3-6h

510a

700b

520a

580a

36.7

0.008

6-12h

760a

1070b

820a

950c

36.7

<0.001

12-18h

640a

940b

690ac

720c

20.6

<0.001

18-24h

470a

630b

480a

600b

19.0

<0.001

Methane, %

0-3h

8.4a

7.8a

6.4b

6.4b

0.28

0.001

3-6h

11.4a

10b

8.4c

7.6c

0.35

<0.001

6-12h

18.6a

17.6a

15c

12.6b

0.70

<0.001

12-18h

22a

21.2a

19.6c

17b

0.39

<0.001

18-24h

24.2a

23.6a

21.2c

19b

0.37

<0.001

Total gas, ml

2740a

3760b

2880a

3230c

62.7

<0.001

Total methane, ml

484b

637c

427a

426a

11.5

<0.001

Methane, % total gas

17.7d

16.9 c

14.8b

13.2a

0.227

<0.001

DM digestibility, %

59.2a

69.4b

61.1a

65.2c

0.83

<0.001

CH4, ml/ g DM digested

68.4b

76.5c

58.2a

54.4a

1.76

<0.001

a,b,c values on the same row with different superscripts differ (p<0.05) CTL= Ensiled cassava root; RBD= CTL + rice distillers’ byproduct at 4% of DM; FCR= CTL+ fermented cassava root at 4% of DM; Yeast=CTL+ yeast at 1% of DM

Total gas production was highest for fermented cassava root, followed by RDB with lowest values for the control and yeast treatments (Table 4; Figure 4). The methane content of the gas (Figure 5) differed among treatments with highest values for the control treatment, followed by the fermented cassava root, yeast and RDB. On the RDB treatment the overall reduction in percent methane was of the order of 25%.  Methane production per unit DM digestibility (Figure 6) showed a similar trend as the methane percent in the gas.

Figure 4. Effect of addition (% in DM) of fermented cassava root (4%) = FCR, yeast (1%) =yeast, rice distillers’
byproduct (4%) = RDB, on the gas production in 24h in an in vitro fermentation of
ensiled cassava root supplemented with urea and cassava leaf meal = CTL


Figure 5. Effect of addition (% in DM) of fermented cassava root (4%) = FCR, yeast (1%) =yeast, rice distillers’
byproduct (4%) = RDB, on percent methane in the gas in 24h in an in vitro fermentation of
ensiled cassava root supplemented with urea and cassava leaf meal = CTL


Figure 6. Effect of addition (% in DM) of fermented cassava root (4%) = FCR, yeast (1%) = Yeast, or rice distillers’
byproduct (4%) = RDB, on the methane produced per unit substrate DM digested = CTL


Discussion

The increases in methane production in the gas with duration of fermentation time, indicative of the transition to a secondary fermentation of the VFA to methane, supports earlier findings using a similar in vitro incubation system but with different substrates (Le Thy Binh Phuong et al 2011; Outhen et al 2011; Sangkhom et al 2011; Thanh et al 2011).

The beneficial effect of the small quantity (4% of substrate DM) of rice distillers’ byproduct in reducing methane production from rumen fermentation is similar to the response reported by Sangkhom and Preston (2016) when brewers’ grains and rice distillers’ byproduct were added to in vitro incubations of ensiled and fermented cassava roots. Addition of yeast at 1% of the substrate also reduced methane production but was slightly less effective than the rice distillers’ byproduct. By contrast, an attempt to simulate some of the features of RDB by fermenting cassava root with yeast, urea and diammonium phosphate had no effect on methane production in the in vitro fermentation.

It has been postulated that the benefits of yeast-based additives in improving human health and growth rates of animals are related to the β-glucan present in the yeast cell wall and their effect in stimulating the immune system (eg: Dritz et al 1995; Hanh et al 2006; Novak and Vetvicka 2008; Waszkiewicz -Robak 2013). Increases in growth rate of weanling pigs and improved resistance to E coli infections were reported by Thuy and Hanh (2017) when pure β-glucan, isolated from spent brewers’ yeast was included in the diet.

In ruminant systems it is probable that their effects are modulated through effects on microbial ecosystems in the rumen and/or lower down the digestive tract. Thus alleviation of hydrocyanic acid toxicity, in cattle fed cassava foliage from a variety rich in HCN-precursors, has been suggested as being due to the βglucans in brewers’ grains supporting biofilm-based fermentations in the rumen that favored detoxification of the HCN (Inthapanya et al 2017).

A shift in the microbial fermentation towards propionate at the expense of acetate production increases the overall yield of Metabolisable energy. It also increases availability of glucogenic substrate which is often critically low in some feeds (Preston and Leng 1987), Hydrogen produced when acetate is the end product of organic matter fermentation is converted to methane and thus reduces overall Metabolisable energy. The magnitude of the inverse relationship is illustrated in Figure 7 which shows the concentration of propionic acid and that of methane in the rumen of cattle that was achieved by varying the levels of rumen protozoa (Whitelaw et al 1984).

Figure 7. Relationship between energy in eructed methane and the proportion of propionic acid in the rumen VFA of cattle,
resulting from changes in the rumen population of protozoa. Closed symbols are data from faunated
animals; open symbols are from ciliate-free animals (Whitelaw et al 1984)

A similar relationship between rumen levels of methane and propionic acid was observed by Inthapanya et al (2017).

Improvements in growth rate and feed conversion in cattle supplemented with brewers’ grains or rice distillers’ product are thus to be expected from the combined benefits of increased availability of both glucose precursors from propionic acid and increased total Metabolisable energy as a result of decreased methane production in the rumen.

Improvements in growth rate and feed conversion in cattle supplemented with brewers’ grains or rice distillers’ product are thus to be expected from the combined benefits of increasing the availability of glucose precursors from propionic acid and the direct saving in Metabolizable energy as a result of decreased methane production in the rumen.

We therefore suggest that the consistently reported benefits of brewers’ grains and rice distillers’ byproduct in reducing methane production in rumen fermentations, both in vitro (Sangkhom and Preston 2016) and in vivo (Sengsouly and Preston 2016; Sangkhom et al 2017; Binh et al 2017; are the indirect effects of these additives in increasing the proportions of propionic acid in the rumen VFA.

The explanation of why small proportions in the diet of brewers’ grains, or rice distillers’ byproduct, result in more propionic acid in the rumen VFA is less obvious. Common to both brewers’ grains and rice distillers’ byproduct is the presence of yeast which has been subjected to heating (100°C) under acid conditions during the process of distilling off the ethanol as is standard practice in production of beer from barley (brewers’ grains) and from polished/broken rice (rice “wine”). It is then assumed that it is the β-glucan derived from the cell walls of the yeast (and of the barley), which modifies microbial activities in the rumen biofilms (Leng 2014) favoring higher proportions of propionic acid in the rumen VFA. β-glucans are present in the cell wall of barley (Havrlentová and Kraic 2006) and yeast (Waszkiewicz-Robak 2013).

An important issue is the need, or otherwise, to isolate the β-glucan which, as described by Nguyen Thi Thuy and Nguyen Cong Hanh (2016), required high pressure homogenization to break the yeast cell wall followed by acid, then alkaline hydrolysis. The results from our experiment, in which there were no effects on methane by supplementing the substrate with fermented cassava root, partially support the hypothesis that breakage of the yeast/barley cell walls, followed by acid hydrolysis, are necessary first steps in facilitating the action of the β-glucan. The positive effect of the commercial yeast “starter” in decreasing methane production implies that some of the β-glucan in this product may have been in the free state. However, the greater impact of the rice distillers’ byproduct in reducing methane production would probably have been facilitated by the degree of hydrolysis of the yeast cells that were likely to have occurred in the process of distilling the ethanol.

The positive effect of rice distillers’ byproduct in reducing methane production in vitro mirrors the results obtained in vivo when local cattle were fed basal diets of ensiled or fermented cassava root supplemented with urea and fresh cassava foliage (Sengsouly et al 2016; Sangkhom et al 2016). In both these experiments the reduction in methane emissions were directly linked with improved growth rates and better feed conversion.


Conclusions


Acknowledgments

This research is part of the requirement by the senior author for the degree of PhD at Hue University of Agriculture and Forestry, Hue University, Vietnam. The authors acknowledge support for this research from the MEKARN II project financed by Sida and the help from Mr. Bounthan at laboratory of Animal Science. The Faculty of Agriculture and Forest Resource (FAF), Souphanouvong University (SU) is acknowledged for providing the facilities to carry out this research.


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Received 12 October 2017; Accepted 12 November 2017; Published 1 December 2017

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