Effect of brewers’ grains and rice distillers’ byproduct on methane production in an in vitro rumen fermentation using ensiled or fermented cassava root (Manihot esculenta, Cranz) as energy substrate

Sangkhom Inthapanya and T R Preston¹

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

Abstract

An in vitro rumen incubation was carried out to study effects on methane production when: (i) cassava root was fermented with yeast, urea and di-ammonium phosphate (DAP) compared with first ensiling the root and then adding the supplements; and (ii) brewers’ grains or rice-wine distillers' byproduct were added to the substrate at low levels (4% of the substrate DM). The treatments were arranged in a 2*3 factorial with 4 replications. All treatments contained cassava leaf meal (30% of substrate DM) and sulphur-rich minerals (1%). Gas production and proportion of methane in the gas were recorded at intervals over the 48h incubation.

Fermenting cassava root with yeast, urea and DAP increased the true protein content from 1.8 to 7.6% in DM. Gas production was lower for fermented than ensiled cassava root but was increased by supplementation with brewers’ grains and rice distillers’ byproduct. The concentration of methane in the gas increased with the duration of the incubation and was lower for the fermented rather than the ensiled cassava root. The DM mineralized during the incubation was less for the fermented than the ensiled cassava root but was not affected by supplementation with brewers’ grains or rice distillers’ byproduct. Methane production per unit substrate DM fermented was less for the fermented compared with the ensiled root and was reduced by supplementation with brewers’ grains and rice distillers’ byproduct.

Keywords: acetogenesis, di-ammonium phosphate, DAP, mineralization, urea, yeast

Introduction

Global warming, caused by increasing atmospheric concentrations of greenhouse gases, is major worldwide environmental, economic, and social threat, and it is well documented that livestock production contributes to this problem (O’Mara 2011). Ruminants are estimated to produce up to 95 million tonnes of methane (CH₄) annually and are implicated as a major source of greenhouse gas production (Patra 2014).

Enteric CH₄ emissions are often predicted from the chemical analysis of the diets (Hristov et al 2013; Moraes et al 2014); however, these methods do not seem sufficiently accurate and appropriate for all feeding situations. Kebreab et al (2008) showed that CH₄ emissions inventories were more accurately estimated through diet-specific mechanistic models. Chagunda et al (2010) indicated that, in order to mitigate CH₄ emissions in a way acceptable for both the environment and animal welfare, it was important to quantify the effects of different diets on methane emissions. Other results suggest that a major difference is needed in dietary starch concentration in order to alter ruminal methanogenesis (Hassanat et al 2013).

Brewers’ spent grains (BG) are the major by-product of the brewing industry, representing around 85% of the total by-products generated (Mussato et al 2006. It is a lignocellulosic material available in large quantities throughout the year. It is considered to be a good source of bypass protein (Promkot and Wanapat 2003).

Wine is probably one of the main fermented beverages for which the recognition of the “territoriality” is fundamental for its appreciation. Many factors are known to affect the microbial ecology of wine production and yeasts, as both lactic acid and bacteria are involved in different ways in winemaking. Several studies showed the positive effects of spontaneous fermentations on the organoleptic complexity of wine as a consequence of the growth of different species and/or strains together at high levels (Le Jeune et al 2006; Wondra and Boveric 2001). Rice distillers’ by-product is the residue from production of “rice wine” which is an alcoholic drink made from sticky rice, maize, sweet potato, cassava or bananas (Oosterwijk and Vongthilath 2003). Studies in Vietnam by Luu Huu Manh et al (2000) and Luu Huu Manh et al (2009) reported 23% of high quality protein in the DM. These authors suggested that this by-product was appropriate for supplementing feeds of lower nutritional density such as rice bran and forages. More recently, Taysayavong et al (2010) in a study in Lao PDR reported increased growth rates from the feeding of rice distillers’ by-product to pigs.

Cassava (Manihot esculenta Crantz) is an annual crop grown widely in the tropical and subtropical regions. It is currently the third most important crop in Lao PDR, after rice and maize. It is widely grown throughout the country by upland farmers but in small areas using local varieties and with very few inputs (CIAT 2001). Roots of cassava have high levels of energy (75 to 85% of soluble carbohydrate) and minimal levels of crude protein (2 to 3% CP); they have been used as a source of readily-fermentable energy in cattle diets (Kang et al 2015; Polyorach et al 2013).

Cassava foliage is an agricultural by-product, considered to be a good source of bypass protein for ruminants (Ffoulkes and Preston 1978; Wanapat et al 2001; Promkot and Wanapat 2003; Keo Sath et al 2008). It has been fed successfully to improve performance of sheep (Hue et al 2008), goats (Do et al 2001; Phengvichith and Ledin 2007) and cattle (Wanapat et al 2000; Thang et al 2010) in fresh, wilted or dried form. Cassava leaves are known to contain variable levels of condensed tannins; about 3% in DM according to Netpana et al (2001) and Bui Phan Thu Hang and Ledin (2005). Cassava contains cyanogenic glucosides, mainly linamarin, which release hydrogen cyanide after hydrolysis in the rumen by an endogenous linamarase (Butler et al 1965). Anaerobic digestion can be inhibited by cyanide, because of the high sensitivity of methanogenic bacteria to this compound (Eikmanns and Thauer 1984; Smith et al 1985). Cuzin and Labat (1992) showed that additions of 5, 10, and 25 mg/litre of cyanide (HCN or linamarin) temporarily inhibited methanogenic bacteria.

A recent development in Lao PDR is the industrial production of starch for export using cassava roots as the feedstock. There are presently 5 factories in operation with a yearly demand of 200,000 tonnes of cassava roots. The extracted starch accounts for some 60% the root DM, the remainder being a byproduct known as cassava pulp. Recent research has demonstrated that the pulp is of similar nutritive value to the original cassava root (Phanthavong et al 2015) and can be used as he basis of an intensive system of cattle fattening.

This development has led to increased interest in the use of the whole cassava roots as the basis of a feeding system for cattle. The roots contain 60 to 70% moisture and are usually sun-dried or ensiled for long-term storage. The fresh roots can also be fermented (Malavanh and Preston 2016) with the objective of enhancing the protein content which in the fresh roots is only of the order of 2-3% in DM.

The purpose of the present study was to compare ensiled versus fermented cassava roots as the energy substrate in an in vitro rumen fermentation and to investigate the effect of incorporating small quantities of brewers’ grains based on the observations of Phanthavong and Preston (2016) that the former appeared to have “probiotic” effects in counteracting the apparent toxicity caused by feeding cattle with fresh cassava foliage when this was derived from a bitter variety with known high levels of cyanogenic glucosides. Rice distillers‘ byproduct was included as an additional treatment in view of its similarity to brewers’ grains in terms of being a byproduct from the fermentation of a cereal grain (rice) to make rice “wine”.

Materials and methods

Location and duration

The experiment was conducted in the laboratory of Animal Science department, Faculty of Agriculture and Forest Resource, Souphanouvong University, Lao PDR, from April to May 2016.

Treatments and experimental design

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

Source of cassava root:

• ECR: Ensiled cassava root
• FCR: Fermented cassava root

Source of supplement:

• No: No supplement
• BG: Brewers’ grains
• RD: Rice distiller’s byproduct

Cassava leaf meal, urea, biochar and sulphur-rich minerals were added to all the substrates.

Experimental procedure

The in vitro system was the same as described by Inthapanya et al (2011).

The cassava root was chopped into small pieces around 1-2 cm of length and was ground in a liquidizer, and then stored in a plastic bag for ensiling over 7 days. For the process of fermenting the cassava root, the additives (% DM basis) were yeast (3%), di-ammonium phosphate (DAP) (1%), urea (3%) and sulphur-rich minerals (1%) (Table 2). These were mixed with the ground cassava root and the mixture stored anaerobically in a sealed plastic bag for fermenting over 7 days.

The cassava leaves was collected from farm of the Faculty of Agriculture and forest resource, Souphanouvong University. They were chopped into small pieces around 1-2 cm in length, then dried in the oven at 80ºC for 24 hours before grinding. Biochar was made in a gasifier stove (Olivier 2010) using rice husks as the feedstock.

Brewers’ grains were bought from Lao beer factory in Vientiane; rice-wine distiller’s byproduct was collected from farmers who make “Lao Kao” wine in Luang Prabang.

Amounts of the substrates equivalent to 12g DM were put in the incubation bottle, followed by 0.96 liters of buffer solution (Table 3) 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 48 hours.

Data collection and measurements

During the incubation the gas volume was recorded at 3, 6, 12, 24 and 48 hours. After each time 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 contents of the incubation bottle were filtered through cloth to determine the residual DM.

Chemical analyses

The samples of ensiled cassava root, fermented cassava root, brewers’ grains, rice distiller’s byproduct, cassava leaf meal and biochar were analyzed for DM, crude protein and ash (AOAC 2010). Soluble protein was determined by extraction with M NaCl according to the method outlined in Whitelaw et al (1961) True protein in the ensiled cassava root and fermented cassava root was measured after precipitation with Trichlor-acetic acid (AOAC 2010).

Statistical analysis

The data were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab (2000) software (version 16.0). Sources of variation in the model were: replicates, source of cassava root, source of supplement, interaction between cassava root*supplement and error.

Results and discussion

Chemical composition

Fermenting the cassava root with yeast, urea and DAP increased the true protein content from 1.8 to 7.6% in DM, a value similar to that reported for the same procedure by Vanhnasin and Preston (2016) and Manivan and Preston (2016).

The protein content and the solubility of the protein were similar in the brewers’ grains and in the rice distillers’ byproduct (Table 4). However, as the proportions in the substrate were low (4% of the DM) their role as sources of protein would be small as compared with the protein derived from cassava leaf meal.

Gas production

Gas production was higher for ensiled than fermented cassava root in the first 12h and for the total incubation over 48h. Both brewers’ grains and rice distillers’ byproduct increased the gas production compared with the control that was not supplemented (Table 5; Figure 1).

Figure 1. Total gas production from sources of cassava root (fermented or ensiled) and different supplements

On all the treatments, the concentration of methane in the gas increased with the duration of the incubation (Table 5), lower values being recorded for the fermented rather than the ensiled cassava root (Figures 2, 3). The DM mineralized during the incubation was less for the fermented than the ensiled cassava root but was not affected by supplementation with brewers’ grains or rice distillers’ byproduct (Figure 4). Methane production per unit substrate was less for fermented than for ensiled cassava rooot and was reduced by supplementation with brewers’ grains and rice distillers’ byproduct (Figures 5 and 6).

Figure 2. Effect of ensiled or fermented cassava root in the methane in the gas	Figure 3. Effect of brewers’ grains and rice distiller’s byproduct in the methane in the gas

The increases in methane concentration in the gas with fermentation time are similar to the findings by several researchers (Inthapanya et al 2011, Binh Phuong et al 2011, Thanh et al 2011, Outhen et al 2011) who used a similar in vitro system but with different substrates

Figure 4. DM mineralized after 48h from cassava root (fermented or ensiled) and different supplements	Figure 5. Methane per unit substrate from ensiled or fermented cassava root supplemented with brewers’ grains or rice distillers’ byproduct

Methane production per unit substrate

Figure 6. Methane per unit substrate from ensiled or fermented cassava root with addition of brewers’ grains, rice distillers’ byproduct or no supplemen

The lower values for DM mineralized with fermented compared with ensiled cassava root are in line with the values for gas production which was lower for fermented than ensiled cassava root. This can be explained the fact that yeast fermentation results in part of the carbohydrate being converted to protein. Yeast protein is of low solubility and thus will be fermented to only a small extent in the in vitro rumen, the overall effect being to decrease the gas production and percentage DM mineralized. The effect of fermentation is thus to change the balance of the site of digestion with less nutrients being fermented in the rumen relative to those digested in the intestines and fermented in the cecum-colon. As disposal of hydrogen in fermentative degradation in the cecum-colon appears to be dominated by acetogenesis (see Demeyer 1991; Immig 1996; Popova et al 2013; Leng 2016) this would account for the decreased methane production for the fermented compared with the ensiled cassava root.

The reduction in methane production due to supplementation with brewers’ grains and rice distillers’ solubles is supported by research in growing goats fed cassava foliage when 4% brewers’ grains in the diet led to decreased production of methane (Vor Sina 2016, personal communication). A similar response was reported by Sengsouly and Preston (2016) in cattle fed on ensiled cassava root, when methane production was decreased when the diet was supplemented with 4% of rice distillers’ byproduct.

Conclusions

• Fermenting cassava root with yeast, urea and DAP increased the true protein content from 1.8 to 7.6% in DM.
  
  
• Gas production was lower for fermented than ensiled cassava root but was increased by supplementation with brewers’ grains and rice distillers’ byproduct.
  
  
• The concentration of methane in the gas increased with the duration of the incubation and was lower for the fermented rather than the ensiled cassava root.
  
  
• The DM mineralized during the incubation was less for the fermented than the ensiled cassava root but was not affected by supplementation with brewers’ grains or rice distillers’ byproduct.
  
  
• Methane production per unit substrate was less for the fermented compared with the ensiled root and was reduced by supplementation with brewers’ grains and rice distillers’ byproduct.

Acknowledgements

This research is part of the requirement by the senior author for the degree of PhD at Nong Lam University. The support from the MEKARN II project, financed by Sida, is gratefully acknowledged, as is the help received from the Animal Science Department, Faculty of Agriculture and Forest Resource, Souphanouvong University, Lao PDR.

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Received 24 September 2016; Accepted 25 September 2016; Published 1 November 2016

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