Effect of added yeast fermented rice at differential levels on methane production in in vitro incubation using elephant grass and cassava leaves as basal substrate

L T B Phuong, L A Tuyet, D T M Linh, L T N Nguyen and T R Preston¹

Nong Lam University, Vietnam
phuong.lethuybinh@hcmuaf.edu.vn
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 used to determine the effect on fermentation parameters of adding yeast fermented rice (YFR) to a basal diet of elephant grass and cassava foliage. After 7 days of anaerobic fermentation, the YFR contained 16% of beta glucan and 32% water-soluble DM. It was then added to the basal diet at levels of 0, 1, 1.5, 3. 4.5 and 6% all on DM basis.

After 24 hours of incubation total gas production had increased by 30%. By contrast the proportion of methane in the gas had decreased by 35%.

There was a linear increase in the molar proportion of propionic acid and a decrease in the proportion of acetate, as the level of YFR in the fermentation medium was increased. But butyric gas the proportion in the VFA was no related to the YFR level. As the replacement of acetate by propionate increase the demand for hydrogen, the net response is less hydrogen available for methanogenesis and therefore lower emissions of methane as the level of YFR in the diet is increased. In the final balance must be added the economic advantage of included propionate from YFR instead of the alternative pathway of feeding 20-30% of starch-rich concentrates.

Key words: beta-glucan, lactic acid, propionic acid, methane

Introduction

Brewer's grain (BG) and rice distiller's byproduct (RDB) are two agricultural by-products that have long been used in ruminant diets, but their role as a nutritional intervention to reduce methane emissions in ruminants is a recent development (Preston 2023). Experiments by Phanthavong et al (2014, 2016a, 2016b) established a fattening diet for cattle consisting of cassava root pulp-urea, brewer’s grains and rice straw. When an attempt was made to replace the brewer’s grains and rice straw with fresh cassava foliage, it was observed that when the cassava was of a bitter variety, rich in HCN precursors, the cattle had a craving to eat brewer’s grains that were being fed to cattle in the adjoining pen. From this observation, the study of Binh et al (2017) demonstrated the benefits of adding 4% of BG to a diet containing 30% of bitter cassava foliage. The cattle supplemented with 4% BG gained weight and thiocyanate excretion through urine (products of HCN detoxification) was no longer observed. A follow-up from this study was the experiment by Sengsouly and Preston (2016), which demonstrated that a 4% supplement of rice distiller’s byproduct (RDB) was as effective as BG in enhancing the growth rate of cattle, with associated benefits of reducing methane production in the rumen. Sangkhom et al (2017) showed the beneficial effect of supplementing cattle with rice distillers' byproduct (RDB) on improving the growth rate and feed conversion of cattle by 40 and 20% respectively. In addition, supplementation of RDB increased propionic acid production in the rumen and reduced by 26% the ratio of methane: carbon dioxide in the mixed eructed gas and air.

Based on this series of results with 4% of rice distillers’ byproduct, a parallel series of experiments was carried out to simulate the procedure used by farmers to make rice wine but omitting the step in which the fermented rice is subjected to a distillation process to separate the alcohol component (the rice wine). These experiments were successful and are described in the paper by Preston et al (2022), giving rise to the term Yeast Fermented Rice (YFR) to distinguish the product from RDB.

The first experiment to evaluate the YFR supplement was a feeding trial to determine the response to YFR fed at levels of 0, 1.5. 3, 4.5 and 6% of a diet of ad libitum elephant grass and restricted protein supplement.

Growth rates of the cattle were improved on all levels of YFR above 1.5% but the response was variable (Figure 1) and the reduction in methane production was curvilinear with increases in methane at the highest level of YFR (Figure 2).

Figure 1. Effect of YFR on growth rate of cattle (Nguyen Van Thu et al, 2022)	Figure 2. Effect of YFR on ratio of methane:carbon dioxide ratio (Nguyen Van Thu et al, 2022)

Discussions of these experiments revolved around the assumption that the presence of beta-glucan in BG, RDB and YFR was responsible for changes in the rumen fermentation and that the production of lactic acid was an intermediate step in the conversion of glucose polymers to propionic acid.

The objective of the research described in this paper was to understand the contrasting results shown in Figure 1, in which production of methane was reduced at low levels of YFR and then increased when YFR was increased to 6% of the diet (Figure 2).

Materials and methods

Location

The experiment was conducted in Nong Lam University, Ho Chi Minh City, Vietnam.

Treatments and experimental design

The experiment was designed CRD with yeast fermented rice (YFR) adding levels as factors and 3 replications of each treatment. The treatments were:

Control: basal diet including elephant grass and cassava leaves as bypass protein at 25% of DM basis.

YFR1: basal diet + Yeast fermented rice (YFR) at 1 % of DM basis.

YFR1.5: basal diet + YFR at 1.5 % of DM basis.

YFR3: basal diet + YFR at 3% of DM basis.

YFR4.5: basal diet + YFR at 4.5 % of DM basis.

YFR6: basal diet + YFR at 6% of DM basis.

Material preparation

Fresh cassava leaves (sweet variety) with 4-5 months of regrowth and fresh elephant grass used in the experiment were planted on the University farm. Preparation of yeast fermented rice (YFR): 1kg of polished white rice was soaked in 1.5 liters of tap water for 5 hours, then milled and mixed with yeast (Saccharomyces cerevisiae) at 3% DM. This mixture was put into 02 kg plastic bags, sealed bags, and left to ferment for 07 days.

All ingredients would be analyzed for DM and N following the procedure of AOAC (1990) before being introduced into in vitro fermentation system.

In vitro incubation

A simple in vitro system was used based on the procedure reported by Inthapanya et al (2011).

Rumen fluid was taken from goat immediately after it was slaughtered at the local abattoir. Fluid in the rumen was filtered directly through 2 layers of cloth to reject the residual feed, filtered fluid was contained in thermal flask to keep warm, then moved quickly to the laboratory for mixing. The 12 grams DM of substrates (Table 1) were mixed with 0.24 liters of filtered rumen fluid and followed by 0.96 liters of buffer solution (Table 2). This mixture was contained in the fermentable bottle, gassed with carbon dioxide, and incubated in a water bath at 38°C for 24h.

Data collection and measurements

The gas volume was measured by water displacement from the receiving bottle suspended in water. The methane percentage in the gas after 24h fermentation was measured with a Crowcon meter (Crowcon Instruments Ltd, UK).

Residue DM and fluid of each in vitro bottle after 24h fermentation had separated in filtration using cloth and non-absorbent cotton wool. The DM residue was then dried to constant weight and the solubilized DM is calculated by subtracting the residue from the total. Sample of fluid were taken for determination of concentrations of individual volatile fatty acid and lactic acid (estimated by gas liquid chromatography following the method of Rowe et al 1979). pH value of fluid also was collected by using digital pH meter.

For measurement of YFR solubility in water: 3g DM of YFR were immerses in dissolved in 100ml NaCl solution (58g NaCl filled with distilled water up to 1000 ml). This mixture was then stirred for 2h, allowed to settle for 30 min, decant the water and collect the bottom insoluble matter to dry until getting constant weight. The YFR solubility was calculated by the difference between total YFR and insoluble YFR.

Beta-glucan measurements in YFR after 7 days fermentation in nylon bags was detected by β-Glucan Assay Kit (Yeast and Mushroom) Megazyme (K-YBGL 02/21). Total glucan was measured by solubilizing 1,3:1,6-β-D-Glucans, 1,3-β-D-glucans and a-glucans in ice cold 12M H2SO4 and then hydrolyzed to near completion in 2M H2SO4. Remaining glucan fragments are then quantitatively hydrolyzed to glucose using a mixture of highly purified exo-1,3-β-glucanase and β-glucosidase. Alpha-glucans and sucrose are specifically hydrolyzed to D-glucose and D-fructose and glucose is measured with amyloglucosidase and invertase using GOPOD reagent. Beta-glucan was calculated by difference of total glucan and alpha-glucan.

Lactic acid concentration in YFR after 7 days fermentation in nylon bags was estimated by gas liquid chromatography following the method of Rowe et al (1979).

Statistical analysis

The data were analysed with the general linear model (GLM) option in the ANOVA programme of the Minitab software (Minitab 18). Sources of variation were treatments and error.

Results and discussion

The activities in this experiment took place in two distinct steps.

Preparation of YFR

Step 1 was the addition of yeast to polished rice with the aim of releasing glucose polymers such as beta-glucan from the cell wall of the yeast. These compounds are water soluble and can be estimated by a simple test for DM soluble in water (Table 3).

In step 2, the yeast-fermented rice (YFR) is the source of nutrients for the in vitro rumen.

The in vitro fermentation

The increase in gas production as the level of supplementation with YFR was increased reflects the greater fermentability of the rice-based YFR compared with the basal diet which was elephant grass and cassava leaves (Table 4).

There was a linear increase in the molar proportion of propionic acid and a decrease in the proportion of acetate, as the level of YFR in the fermentation medium was increased (Table 5; Figures 3 and 4).

Figure 3. Effect of added YFR on volatile fatty acid (VFA) proportions

The ratio of Ac to Pr decreased from 4.34 to 2.44 as the content of YFR in the diet was increased from zero to 6% (Figure 4).

Figure 4. Effect of added YFR levels on ratio of acetic acid and propionic acid (Ac:Pr) production after 24h in vitro incubation

The proportion of HBu in the VFA showed no consistent response to increasing level of YFR (Figure 3). The overall effect was to increase the demand for hydrogen to form propionate, which resulted in a marked decrease in the production of methane in the fermentation (Figure 5).

Figure 5. Effect of YFR levels on methane in gas on the in vitro fermentation

The major impact of the increase in propionic acid was the linear decrease in the methane content of the rumen gas (Figure 6).

Figure 6. Correlation of methane and propionic acid production (HPr) among different levels of added YFR in in vitro rumen fermentation using elephant grass and cassava leaves as basal substrate

Water soluble DM (which reflecting level of glucose polymers in the YFR) was positively related to propionate production (Figure 7).

Figure 7. Correlation of propionic acid (HPr) production and soluble DM among different levels of added YFR in in vitro rumen fermentation using elephant grass and cassava leaves as basal substrate

The propionic acid production also showed a positive correlation with lactic acid content in the YFR (Figure 8).

Figure 8. Correlation of propionic acid (HPr) production in treatments after 24h in vitro fermentation and lactic acid content before fermentation (calculated by the amount added YFR in treatments)

Increasing the level of YFR in the in vitro fermentation resulted in an increase in water soluble DM (Figure 9)

Figure 9. Effect of added YFR levels on solubilized DM after 24h in in vitro fermentation using elephant grass and cassava leaves as basal substrate

Conclusions

• The results of this study indicate that the addition of yeast fermented polished rice to a basal diet of elephant grass and cassava leaves leads to the release of glucose polymers such as beta glucan, which in turn are a substrate for the production of propionic acid probably through the medium of lactic acid.
  
  
• As the replacement of acetic acid by propionic acid requires additional hydrogen there is less hydrogen for the reduction of caron dioxide to forma methane.
  
  
• The virtues of YFR are threefold: it is a means to reduce methane emissions from ruminant animals; it is a technology based on farmer experience (in production of rice wine) therefore easy to extend to oar me farmers.
  
  
• It is a means to make more efficient use of tropical pastures rather than supplementing then with high levels of concentrates.

Acknowledgments

The authors acknowledge support for this research from research funding of Nong Lam University, Ho Chi Minh City, Vietnam and equipment from Veterinary Bio-Science Department, Animal Sciences are acknowledged for providing the facilities to carry out this research.

References

AOAC 1990 Official methods of analysis.Association of Official Analytical Chemists, Arlington, Virginia, 15 ^th edition

Binh P L T, Preston T R, Duong K N and Leng R A 2017A 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. Retrieved July 27, 2023, from http://www.lrrd.org/lrrd29/5/phuo29104.html

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

Minitab 2018 Minitab reference manual release 18. 1.0. Minitab Inc.

Nguyen Van Thu, Preston T R and Leng R 2022 Supplementing the diet of growing cattle with yeast-fermented rice (YFR) increased the production of rumen propionate, decreased emissions of methane and improved growth and feed conversion. Livestock Research for Rural Development. Volume 34, Article #113. Retrieved July 24, 2023, from http://www.lrrd.org/lrrd34/12/34113thuv.html

Phanthavong V, Preston T R, Viengsakoun N and Pattaya N 2016b 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. Retrieved August 24, 2018.

Phanthavong V, Khamla S and Preston T R 2016a Fattening cattle in Lao PDR with cassava pulp. Livestock Research for Rural Development. Volume 28, Article #10. http://www.lrrd.org/lrrd28/1/phan28010.html

Phanthavong V, Viengsakoun N, Sangkhom I and Preston T R 2014 Cassava pulp as livestock feed; effects of storage in an open pit. Livestock Research for Rural Development. Volume 26, Article #169. http://www.lrrd.org/lrrd26/9/phan26169.htm

Preston T R 2023 Supplementing ruminant diets with yeast-fermented rice improves growth rate and feed conversion and reduces emissions of methane. Livestock Research for Rural Development. Volume 35, Article #66. http://www.lrrd.org/lrrd35/8/3566Pres.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.Retrieved October 11, 2017, from 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

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.