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

Phuong L T Binh, T R Preston¹, Van Huu Nguyen² and Dinh Van Dung²

Faculty of Animal Sciences and Veterinary Medicine, Nong Lam University, Vietnam
binhphuonglt@gmail.com
¹ Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV), Carrera 25 No 6-62 Cali, Colombia,
² Hue University of Agriculture and Forestry, Hue University, Hue City, Vietnam

Abstract

Total gas and methane production were determined in an in vitro rumen incubation of cassava-pulp-urea supplemented with a range of additives. The experimental design was 3 replications of two factors: (i) the source of supplementary cassava foliage (Gon [sweet] and KM94 [bitter]); ND (ii) no additive (CTL), brewers’ grain (BG), rice wine starter culture (RWS), yeast fermented cassava pulp (YFCP) and cassava pulp fermented with yeast, urea and di-ammonium phosphate (YFCP-U-DAP).

Gas production was increased and methane content in the gas was reduced when the protein supplement was from leaves of the bitter cassava variety. There was an interaction between the source of the leaves and the effect of the brewers’ grain on methane in the gas which was reduced when the cassava leaf source was a sweet variety but there was no effect of the brewers’ grain when the cassava leaf was from a bitter variety. Additives based on cassava pulp fermented with, or without, addition of urea and diammonium phosphate during the incubation, had no apparent effect on gas production or methane content of the gas. By contrast, adding 1% of a traditional rice wine starter culture (containing yeast and molds) reduced gas production but did not affect the methane content in the gas compared with the control of no additive.

Key words: alcohol, beta-glucan, HCN, live yeast, prebiotic

Introduction

Recent studies have shown: (i) that adding small amounts (4% of diet DM) of brewers’ grain to a diet of cassava-pulp-urea-bitter cassava foliage improved DM intake and liveweight gain of cattle, and reduced excretion of urine thiocyanate (Binh et al 2017); and (ii) a 4% addition of rice distillers’ byproduct to ensiled cassava root, urea and cassava foliage, had similar effects with growth rate of cattle increased by 37% and feed conversion by 21% (Sengsouly et al 2016).

There are similarities in the production of brewers’ grains and rice distillers’ byproduct. In each case the original substrate (barley grain or polished rice) is fermented with yeast to produce ethanol, which is then separated by distillation. The resultant residues are: brewers’ grains and rice distillers’ byproduct. Both these byproducts will have undergone acid hydrolysis during the distillation process which may have led to release of the prebiotic β-glucan from the yeast cell walls (reference) with resultant positive effects on animal performance and wellbeing (eg: reduction of HCN toxicity from the cyanogenic glucosides in cassava).

Brewers’ grains and rice distillers’ byproduct are not readily available in all rural areas. Thus, the objective of the present study was to evaluate possible alternatives to these two byproducts that could potentially act as prebiotics in ruminant diets based on cassava root pulp and cassava foliage of the sweet and bitter varieties.

Materials and methods

Location and duration

The experiments were conducted in the laboratory of Nong Lam University, Ho Chi Minh city, Viet Nam, from November to December 2017.

Treatments and design

Two factors were studied in an in vitro rumen incubation. The design was a 2*5 factorial with 3 replications. The factors were:

Source of cassava foliage: Sweet (Gon)  or Bitter (KM94) variety

Potential source of prebiotic: No supplement (CTL), Brewers’ grain (BG), Rice wine starter culture (RWS), Cassava pulp fermented with RWS (YFCP) and Cassava pulp fermented with RWS, urea and di-ammonium phosphate (YFCP-U-DAP)

The basal substrate (DM basis) was 73% ensiled cassava pulp, 2% urea and 25% cassava leaves (sweet or bitter variety). The additives were incorporated in the substrate at levels (DM basis) of 4%, except for the RWS which was added at 2%.

Materials

The brewer’s grains were taken directly, in the fresh state, from the brewing process at the beer factory in Ho Chi Minh City. YFCP-U-DAP was made by fermenting fresh cassava pulp for 7 days with 2% rice wine starter culture (RWS), 1% sodium chloride, 3% urea and 1% di-ammonium phosphate (DAP) (all on DM basis). It was stored 7 days in closed polyethylene bags. FCP was made by the same procedure but without urea and DAP.

In vitro incubation

The in vitro procedure was the same as that described by Inthapanya et al (2011). Representative samples of the mixtures (12g DM) were put in the incubation bottles to which were added 960ml of buffer solution (Table 1) and 240ml of goat rumen fluid (taken immediately from a goat that was slaughtered at the local abattoir). The bottles with substrate were then incubated in a water bath at 38 °C for 24h.

Data collection

Samples of the additives were analysed prior to, and after 7 days of fermentation for: DM and crude protein (CP) according to methods of AOAC (1990); pH and Brix values were made using a digital pH meter and hand-refractometer.

For microbiological analysis: 5 g fresh sample was diluted in Wilkin medium in different concentrations. MRS medium was used to isolate lactic acid bacteria (LAB) at 37⁰C .To 1 liter MRS medium was added 2g natamycin dissolved in 40ml sterile water to prevent the development of fungi (Dung et al 2007). The Sabouraud medium was used to isolate Saccharomyces at room temperature. Colonies were counted and the results expressed as colonies per gram of sample (CFU/g).

The volume of gas produced in the in vitro incubation was recorded by water displacement at 7, 12 and 24h. The methane percentage in the gas at each of these times was measured by infra-red meter (Crowcon Ltd, UK).

Statistical analysis

The data were analyzed with the general linear model (GLM) option in the ANOVA program of the Minitab software (Minitab 2000). Sources of variation were: source of cassava foliage, additive, interaction forage source*additive and error.

Results and discussion

Composition of the additives

The low crude protein concentration in fresh cassava pulp is the reason for the need to add urea to support the rumen fermentation (Table 2). As was to be expected, the supplementation of both urea and di-ammonium phosphate in the yeast-fermented cassava pulp resulted in higher pH and crude protein than without them.

The increase in the brix values (Measures % soluble sugars) after fermentation reflects the effect of the yeast enzymes in causing partial hydrolysis of starch (in the cassava pulp) to soluble sugars. Addition of urea-DAP to the substrate appeared to accelerate this process.

Methane content of the gas

The methane content of the gas increased progressively as the fermentation advanced over the 24h of the incubation (Table 2a; Figure 1).

Figure 1. Effect of stage of the fermentation on the methane content of the gas

Gas production was higher when the protein source was leaf from bitter as opposed to the sweet cassava variety; and was higher when 4% brewers’ grains were added to the substrate (Tables 3a, 3b; Figure 2). The exception was in the case of the rice wine starter additive (RWS). We have no explanation for this anomaly.

Figure 2. Effect of additives, and source of cassava leaf (bitter or sweet variety) on gas production after 24h fermentation

When the cassava leaf was from the sweet variety, brewers’ grains reduced the methane in the gas, but there was no effect of the other additives (Figure 3). With the bitter variety of cassava, the results were more variable.

Figure 3. Effect of additives, and source of cassava leaf (bitter or sweet variety) on methane content of the gas after 24h fermentation

By restricting the comparison to the control and brewers’ grains additive, the effects are clearer (Figure 4). There was an interaction between the effects of the brewers’ grains additive and the source of the cassava leaf meal on the percentage of methane in the gas. When the cassava leaf was from the bitter variety, there were no benefits from supplementation with brewers’ grains; in contrast, with the sweet cassava variety, brewers’ grains reduced methane percentage by 31% (Figures 3 an 4. Sangkhom et al (2017) also found that supplementing 4% of brewers’ grain, to an in vitro incubation of cassava root-urea, reduced methane production when the protein source was leaf from sweet cassava.

Figure 4. Interaction between source of cassava leaf meal and supplementation with brewers’ grains on methane content in the gas

The decrease in methane production when bitter, rather than sweet, cassava leaves were the protein source, is consistent with results from previous studies (Phuong et al 2012ab, 2015; Phanthavong et al 2015). The toxicity of HCN to methanogens has been documented (Smith et al 1995; Cuzin and Labat 1992).

The brewer’s grain used in our experiment was taken immediately after distillation of the ethanol in the beer production process. There were no viable yeast cells in the brewers’ grain but high populations in the two yeast-fermented additives (Figure 5). There were traces of lactobacilli in the brewers’ grains but much greater quantities in the yeast-fermented additives (Figure 6). It is evident that the yeast was no longer viable in the brewers’ grains. By contrast, in the treatments YFCP-U-DAP and RWS the fermented substrate had not been submitted to “heat” treatment and thus the yeast in these additives was still “viable”.

Figure 5. Viable yeast cells in brewers’ grain and in cassava pulp fermented only with yeast (YFCP) or with yeast and urea YFCP-U-DAP) after 07 days of fermentation	Figure 6. Lactobacilli in brewers’ grain and in cassava pulp fermented only with yeast (YFCP) or with yeast and urea YFCP-U-DAP) after 07 days of fermentation

The beneficial role of brewers’ grain and rice wine distillers’ byproduct in animal feeding is thought to be due to the prebiotic effect of β-glucan, that had been released from the cell walls of the cereal grain (brewers’ grain) and/or the yeast (rice distillers’ byproduct and brewers’ grain) (Binh et al 2017; Sivilai et al 2018). It is hypothesized that heating under acid conditions (pH about 4), as in the final stage of distillation of the alcohol in brewers’ grains and rice distillers’ byproduct, submits the residue to “acid hydrolysis”, which is a necessary step in solubilizing the β-glucan (Sang Hoon Lee et al 2015) and hence facilitating its role as a “prebiotic” (Lam Ka-Lung and Chi-Keung Cheung 2013).

The research reported in this paper may help to explain why the byproducts of ethanol fermentation and distillation are effective as prebiotics. The byproducts provide the source of the β-glucan (the cell walls in barley/rice and in yeast); the distillation of the ethanol from the fermented acid substrate (pH<4.0) creates the condition for the necessary treatment (acid-hydrolysis) to make the β-glucan soluble and therefore available to the host species.

Conclusions

• In an in vitro rumen incubation of a substrate of cassava-pulp-urea with cassava leaves and a range of addditives, the methane content of the gas increased progressively as the fermentation advanced over the 24h of the incubation.
  
  
• Gas production increased when the protein supplement was from leaves of a bitter compared with sweet variety of cassava and when 4% brewers’ grains were added to the incubation.
  
  
• There was an interaction between the source of the leaves and the effect of the brewers’ grain on methane in the gas which was reduced when the cassava leaf source was the sweet variety but there was no effect of the brewers’ grain when the cassava leaf was from a bitter variety.
  
  
• Additives based on cassava pulp fermented with, or without, addition of urea and diammonium phosphate during the incubation, had no apparent effect on gas production or methane content of the gas. By contrast, adding 1% of a traditional rice wine starter culture (containing yeast and moulds) reduced gas production but did not affect the methane content in the gas compared with the control of no additive.

Acknowledgements

This research is part of the requirement for the PhD of the senior author in the doctoral program of Hue University of Agriculture and Forestry, Vietnam. Financial support from the Sida-financed project, MEKARN II, is gratefully acknowledged.

References

AOAC 1990 Official Methods of Analysis: Association of Official Analytical Chemists. 15^th Edition. Washington, DC. UAD Arlington pp 1230

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

Cuzin N and Labat M 1992 Reduction of cyanide levels during anaerobic digestion of cassava. International Journal of Food Science 27 329-326

Dung N T P, Rombouts F M and Nout M J R 2007 Characteristics of some traditional Vietnamese starch-based rice wine fermentation starters (men). LWT - Food Science and Technology 40(1): 130-135.

Inthapanya S, 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

Lam Ka-Lung and Chi-Keung Cheung P 2013 Digestible long chain beta-glucans as novel prebiotics. Bioactive Carbohydrates and Dietary Fibre. Volume 2, pp 45-64 https://doi.org/10.1016/j.bcdf.2013.09.001

Minitab 2000 Minitab Software Release 16.0

Phanthavong V, Viengsakoun N, Sangkhom I and Preston T R 2015 Effect of biochar and leaves from sweet or bitter cassava on gas and methane production in an in vitro rumen incubation using cassava root pulp as source of energy. Livestock Research for Rural Development. Volume 27, Article #72. Retrieved January 11, 2018, from http://www.lrrd.org/lrrd27/4/phan27072.html

Phuong L T B, Khang D N and Preston T R 2012a Effect of NPN source, level of added sulphur and source of cassava leaves on growth performance and methane emissions in cattle fed a basal diet of molasses. Livestock Research for Rural Development. Volume 24, Article #70. http://www.lrrd.org/lrrd24/4/phuong24070.htm

Phuong L T B, Preston T R and Leng R A 2012b Effect of foliage from “sweet” and “bitter” cassava varieties on methane production in in vitro incubation with molasses supplemented with potassium nitrate or urea. Livestock Research for Rural Development. Volume 24, Article #189. http://www.lrrd.org/lrrd24/10/phuo24189.htm

Phuong L T B, Khang D N and Preston T R 2015 Methane production in an in vitro fermentation of cassava pulp with urea was reduced by supplementation with leaves from bitter, as opposed to sweet, varieties of cassava. Livestock Research for Rural Development. Volume 27, Article #162. http://www.lrrd.org/lrrd27/8/phuo27162.html

Sang Hoon Lee, Gwi Yeong Jang, In Guk Hwang, Hyun Young Kim, Koan Sik Woo, Kee Jong Kim, Mi Ja Lee, Tae Jip Kim, Junsoo Lee and Heon Sang Jeong 2015 Physicochemical properties of β-glucan from acid hydrolyzed barley. Preventative Nutrition and Food Science 20(2): 110–118 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4500513/

Sangkhom I and Preston T R 2016 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. Livestock Research for Rural Development. Volume 28, Article #194. RetrievedFebruary 6, 2018, from http://www.lrrd.org/lrrd28/11/sang28194.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. Retrieved February 8, 2018, from http://www.lrrd.org/lrrd28/10/seng28178.html

Sivilai B, Preston T R, Hang D T and Linh N Q 2018 Effect of a 4% dietary concentration of rice distillers’ byproduct, or of brewers' grains, on growth rate and feed conversion during pregnancy and lactation of native Moo Lath gilts and their progeny. Livestock Research for Rural Development. Volume 30, Article #20. Retrieved February 6, 2018, from http://www.lrrd.org/lrrd30/1/lert30020.html

Smith M R, Lequerica J L and Hart M R 1985 Inhibition of methanogenesis and carbon metabolism in Methanosarcina sp. by cyanide, Journal of Bacteriology, 162, 67-71.

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 1 March 2018; Accepted 21 March 2018; Published 1 April 2018

Go to top