Effect of yeast-fermented cassava root compared with yeast-fermented rice 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

An experiment was designed to respond to the hypothesis that whole rice grain and fresh cassava root when fermented with yeast would have similar “prebiotic” qualities as yeast fermented polished rice in their capacity to reduce methane formation in an in vitro rumen incubation of ensiled cassava root supplemented with urea and cassava foliage. Two sources of cassava foliage (from sweet and bitter varieties) were also tested. The rationale for this idea was that cassava root was less competitive with polished rice as a staple food in human nutrition.

At all stages of the 24h in vitro incubation, the gas production and the methane content of the gas were reduced when the additive was yeast-fermented polished rice or yeast-fermented cassava root, compared with yeast-fermented whole rice grain. The methane content of the gas was increased linearly with the length of the incubation and represented a higher percentage of the gas as the rate of gas production increased. It is hypothesized that reducing the rate of rumen fermentation of a substrate rich in precursors of glucose (starch) and amino-acids (leaf protein) would reduce enteric methane production ,and facilitate the escape of nutrients for more efficient utilization by intestinal enzymes and acetic acid-producing microorganisms in the cecum.

Keywords: amino acids, global warming, glucose precursors, greenhouse gas, prebiotics, ruminants

Introduction

In previous papers we showed that polished rice fermented with yeast apparently acted as a prebiotic as reflected in reduced production of methane when 4% (DM basis) was added to an in vitro rumen incubation of ensiled cassava root supplemented with urea and cassava leaf meal (Inthapanya et al 2020). It was hypothesized that the action of the yeast-fermented rice was mediated by its effect in creating “habitat” for biofilms supporting synergistic activities between rumen microorganisms and their substrate (Leng 2017). In this respect the yeast-fermented rice appeared to have similar properties to rice distillers’ byproduct (RDB), the residue after yeast-fermentation of polished rice followed by distillation of the ethanol to produce rice wine In the experiment reported by Inthapanya et al (2020), yeast-fermented rice and RDB were almost equally effective in reducing methane production (by 21 and 16%, respectively). An important finding was that when the rice was supplemented with urea and diammonium phosphate the product after yeast-fermentation did not reduce rumen in vitro methane production (Inthapanya et al 2019). It appeared that the products/byproducts from growth of yeast differed markedly from those resulting from those produced when the yeast was in the ethanol-producing mode.

The following experiment was designed to respond to the hypothesis that yeast fermentation of whole rice grain and fresh cassava root would also produce a product with similar “prebiotic” qualities as yeast fermentation of polished rice. The rationale for this idea was that cassava root was less competitive with polished rice as a staple food in human nutrition.

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 June to July 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 prebiotic:

· GRG: Ground whole rice grain

. GCR: Ground cassava root

· PR: Polished rice

Source of cassava leaves:

· 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).

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

Feed ingredients

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 80ºC for 24hours before grinding. The cassava root was chopped into small pieces, ground and then ensiled for 7 days in closed plastic bags.

The preparation of the prebiotics

Rice grain (with husk) and rice grain (without husk) were weighed (1 kg), wet-milled in a liquidizer, then soaked in 1.5 liters of water for 5 hours prior to mixing with yeast (Saccharomyces cerevisiae) at 3% DM basis. The mixtures were 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).

The in vitro incubation

Amounts of the substrates (Table 1), equivalent to 12g DM, were put in the incubation bottles, 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 24hours.

Data collection and measurements

During the incubation the gas volume was recorded at 6h intervals (0-6, 6-12, 12-18 and 18-24hours). 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 100^oC to determine the DM mineralized (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 (2010) software (version 16.0). In the model the sources of variation were prebiotic, cassava leaf, additive*leaf interaction and error. The statistical model used was:

Y_ijk = µ +a_i +b_j + (a*b)_ij + e_ijk

Where: Y_ijk is dependent variable; µ is overall mean; a _i is the effect of prebiotic source; b_j is the effect of cassava leaf source; (a*b)_ij is the interaction between source of prebiotic and cassava and e_ijk is random error.

Results and discussion

Chemical composition of substrate

In the absence of any added source of nitrogen there were no changes in the crude protein content of the substrates during the 7-day fermentation and therefore no growth of the yeast. The pH of the selected raw materials was low before and after the yeast fermentation (Table 3).

Gas production and methane content of the gas

At all stages of the incubation the gas production, and the methane content of the gas, were greater when yeast-fermented whole rice grain was the additive as compared with fermented polished rice or fermented cassava root which did not differ (Table 4; Figure 1). Leaves from the bitter cassava variety produced less gas, with lower methane content, than leaves from the sweet variety. As a result, the same trends were seen in total (24h) gas production and percent methane in the gas (Figures 1 and 2). The methane content of the gas increased as the fermentation progressed (Figure 3).

Figure 1. Gas production in 24h according to source of prebiotic and source of cassava leaves (bitter or sweet)	Figure 2. Methane % in the gas in 24h according to source of prebiotic and source of cassava leaves (bitter or sweet)

DM mineralized (digested)

This was similar for all three additives with the result that the quantities of methane per unit DM mineralized were highest for fermented whole rice grain followed by fermented cassava root with the lowest value for fermented, polished rice (Table 4).

Relationships between length of the incubation and methane content of the gas

There was a positive linear relationship between the percent methane in the gas and the length of the incubation (Figure 3), confirming earlier findings (Sangkhom et al 2019).

Figure 3. Effect of incubation length on the methane content of the gas

Relationship between methane content in the gas and (i) total gas production and (ii) the proportion of the substrate DM mineralized

There was a close relationship (Figure 4) between methane content of the gas and total gas production. The relationship between methane content of the gas and the proportion of the substrate mineralized was positive but less pronounced (Figure 5).

Figure 4. Relationship between total gas production and methane percent in the gas	Figure 5. Methane percent in the gas does not increase with percent of substrate mineralized

Sweet and bitter cassava

The comparisons between leaves from sweet and bitter cassava varieties, as sources of protein in the incubation, consistently demonstrated the reduced gas production and lower methane content in the gas, for the bitter variety (Figures 1 and 2).

Discussion

Recent researches with the in vitro rumen incubation system (Binh et al 2015; Inthapanya et al 2020) have shown that methane production in a simulated ruminant feeding system (ensiled cassava root or root-pulp, urea and cassava foliage) is reduced by up to 35% when the substrate is supplemented with 4% (as DM) of byproducts of yeast fermentation of cereal grains (brewers’ spent grains, rice wine distillers’ byproduct and yeast-fermented polished rice).

In the present experiment, the treatments of yeast-fermented whole rice grain (RG) and yeast-fermented cassava root (GCR) have to be compared with those of yeast-fermented polished rice (PR) which represents the positive control in terms of the “expected “results. In this respect, it is clear that the fermented whole rice grain did not possess the characteristics needed to reduce rumen methane production, while yeast-fermented cassava root gave results comparable with those from fermented polished rice (PR).

Other findings, namely the direct relationship between in vitro rumen fermentability (gas production) and the percentage of methane in the gas, confirm previous findings. It has been hypothesized (Inthapanya et al 2019) that these findings can be interpretated as indicative of the advantages of reducing the degree of rumen fermentation (and hence encouraging rumen escape) of nutrients with potential to be digested enzymically in the small intestine (protein and starch) and by fermentation in the cecum-colon (fibrous particles). Under these circumstances the reduced rumen fermentation (and resultant nutrient escape) would result in associated joint benefits of reduced enteric methane emissions with increased productivity in milk (Hamdai et al 2019) and beef production (Phanthavong et al 2016).

Conclusions

• At all stages of the 24h in vitro incubation, the gas production and the methane content of the gas were reduced when the additive was yeast-fermented polished rice or yeast-fermented cassava root, compared with yeast-fermented whole rice grain.
  
  
• Methane content of the gas increased linearly with the length of the incubation.
  
  
• It is hypothesized that reducing the rate of rumen fermentation of a substrate rich in precursors of glucose (starch) and amino-acids (leaf protein) will reduce enteric methane production ,and facilate the escape of nutrients for more efficient utilization by intestinal enzymes and acetic acid producing microorganisms in the cecum.

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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