Methane production of fermented broken rice versus fermented cassava root in rumen in vitro fermentation using ammoniated rice straw as basal substrate

Phuong T B L¹, Tuyet A L¹ and Preston T R²

¹ Nong Lam University, Ho Chi Minh City, Vietnam
phuong.lethuybinh@hcmuaf.edu.vn
² Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV), Carrera 25 No 6-62 Cali, Colombia

Abstract

This study evaluated whether yeast-fermented rice (simulated by fermentation by-products such as brewer’s spent grain or rice wine distillers’ residues) differs from unfermented broken rice and cassava root in mitigating methane production in an in vitro system using ammoniated rice straw (ARS) as the basal substrate. The experiment was arranged as a completely randomized 2 × 2 factorial design with three replicates. The factors were: (i) Carb source at 4.5% DM (broken rice [BR] vs. cassava root [CR]) and (ii) Carb treatment at 4.5% DM (yeast-fermented vs. non-fermented), along with a negative control consisting of ARS alone.

Supplementation with 4.5% DM of starch-rich substrates increased total gas production and soluble dry matter (DM) after 24 h of fermentation compared with the control; however, no significant effects of Carb source or Carb treatment were observed. Methane production per unit of soluble DM did not differ significantly among treatments, suggested starch-rich supplementation showed potential to reduce methane output relative to control. Fermented BR/CR shows lower proportion of methane in total gas than their unfermented counterparts. No differences in methane production were detected between fermented BR and fermented CR, suggesting that cassava root may serve as a viable alternative to broken rice as a substrate for yeast fermentation in ARS-based diets. Furthermore, no interaction between Carb source and Carb treatment was observed.

Keywords: proportion of methane, soluble DM, starch-rich substrates, total gas

Introduction

The use of rice straw as a feed resource in ruminant diets represents an effective strategy for recycling agricultural by-products. However, its utilization is constrained by poor nutritional quality, particularly its low crude protein content and high structural fiber fraction. Rice straw is primarily composed of cellulose and hemicellulose, which provide substrates for fibrolytic bacteria, leading to increased acetate production relative to propionate and consequently higher enteric methane (CH₄) emissions (Ma et al., 2025). In addition, approximately 2–12% of gross feed energy intake may be lost as CH₄ during rumen fermentation. Therefore, improving the nutritional value of rice straw and mitigating methane emissions are important considerations for its efficient use in ruminant feeding systems.

Previous studies have demonstrated that supplementation with 4% yeast-fermented rice (YFR) or rice distillers’ by-products in cassava leaf source can reduce methane production during rumen fermentation (Inthapanya et al 2017, Sangkhom et al 2020). Similar effects have also been reported for diets supplemented with 4% urea-fermented cassava root. These responses were supported by the findings of Binh et al (2017) and Sangkhom and Preston (2016), who suggested that yeast cell wall components and fermented cereal substrates may act as prebiotic compounds, altering rumen fermentation patterns by enhancing propionate production relative to acetate production.

Nevertheless, the effects of 4% yeast-fermented substrates in high-fiber diets such as alkali-treated rice straw (ARS) remain unclear. Phuong et al (2024) reported that supplementation with 4% YFR in ARS-based diets did not consistently reduce methane production. Therefore, to further elucidate the effects of yeast-fermented substrates under high-fiber feeding conditions, the present study evaluated the effects of two carbohydrate sources, broken rice and fresh cassava root, together with their fermented forms, fermented broken rice and fermented cassava root, each supplemented at 4% of the diet, on methane production during in vitro rumen fermentation using ARS as the basal substrate.

Materials and methods

Experimental design

The experiment was arranged as a completely randomized 2*2 factorial design with 03 replicates. The two factors are: (i) Carb source (broken rice versus cassava root); (ii) Carb treatment (yeast fermentation versus non-fermentation) with ammoniated rice straw (ARS) as basal substrate. The experiment treatments are as follows:

ARS: ammoniated rice straw only as negative control

BR4.5: pure broken rice at 4.5 % of DM and 95.5% of ARS

FBR4.5: yeast fermented broken rice at 4.5 % of DM and 95.5% of ARS

CR4.5: fresh cassava root at 4.5 % of DM and 95.5% of ARS

FCR4.5: yeast fermented cassava root at 4.5 % of DM and 95.5% of ARS

Ingredients preparation

Fermentation of carbohydrate source: Broken rice (1 kg) was soaked in 1.5 L of tap water for 5 hours, then milled and inoculated with Saccharomyces cerevisiae at 0.5% of DM (1 × 10¹⁰ CFU/g). The mixture was packed into 2 kg nylon bags, sealed, and incubated for 7 days to allow fermentation. Fresh cassava roots were cut into small pieces, homogenized into a paste using a blender, and subsequently fermented following the procedure similar to broken rice described above.

Ammoniation procedure: Rice straw was chopped into lengths of approximately 5–6 cm prior to ammoniation. Chopped straw (500 g as fresh weight) was placed in 2 kg nylon bag and thoroughly mixed with an aqueous urea solution (3% urea of DM and dissolved in 500 ml of water) to achieve a final moisture content of approximately 40–45%, which is considered optimal for ammoniation. The treated rice straw was compressed in nylon bags to expel air, sealed, and stored at ambient temperature for 21 days. Bags were rotated weekly to ensure uniform distribution of urea throughout the material.

Samples removed from the bags should be processed immediately, undergoing chemical analysis and in vitro incubation without delay.

In vitro rumen incubation

A thermos flask was pre-warmed with warm water (approximately 40 °C) prior to rumen fluid collection. Rumen contents were obtained from beef cattle that had been fasted overnight before slaughter at a local abattoir. Immediately after slaughter, rumen contents were removed, thoroughly mixed, and strained through two layers of cheesecloth to remove residual feed particles. The strained rumen fluid was transferred to the pre-warmed thermos flask after remoning warm water, sealed, and transported to the laboratory within 30 min. In vitro incubation was conducted following the procedure described by Inthapanya et al (2011).

Twelve grams of substrate on a dry matter basis was accurately measured and combined with 0.24 L of filtered rumen fluid and 0.96 L of buffer solution (Table 1) for each incubation bottle. Following the addition of flushing carbon dioxide to create anaerobic conditions, then incubated in a water bath at 39 °C for 24 hours. In total, 18 in vitro incubation systems were prepared, including three blank systems (containing only rumen fluid and buffer solution without substrate) and 15 systems assigned to experimental treatments. Fermentation was stopped after 24 h by placing the bottles in an ice bath to inhibit further microbial activity.

Mesurements and data collection

Samples of ingredients including fermented cassava root, fresh cassava root, broken rice, fermented broken rice and ammoniated rice straw were analyzed for dry matter (DM) and crude protein (CP) according to AOAC (2011) procedures. For pH measurement, weigh 10 grams of the sample and blend it with 100 ml of distilled water for 30 seconds. Record the pH value after the reading has stabilized for 30 seconds using a pH meter (AD1020- Adwa Instruments, Hungary). Hydrogen cyanide (HCN) concentration was determined according to the method described in TCVN 10497:2015. Each sample was analyzed in duplicate, and each treatment analyzed two independent ammoniated batches.

Total gas production after 24 h of incubation was measured by water displacement using a graduated receiving bottle suspended in water. After gas volume measurement, gas samples were analyzed for methane and carbon dioxide concentrations (% vol) using a Crowcon gas analyzer equipped with an infrared sensor (Crowcon Instruments Ltd., UK).

Unfermented dry matter was recovered by filtering incubation contents through cloth and non-absorbent cotton wool. The residue was dried to constant weight, and fermented DM was calculated as the difference between the initial substrate (12 g DM) and the recovered residue. Gas and methane production per unit of soluble DM (ml/g) was calculated as gas and methane volume divided by fermented substrate DM.

Gas production, methane volume, and soluble dry matter (DM) for experimental treatments were calculated after correction for the blank values.

Statistical analysis

Data was analyzed using the General Linear Model (GLM) procedure of Minitab software version 19. The statistical model included all treatments (ARS, BR4.5, FBR4.5, CR4.5 and FCR4.5), Carb source (broken rice and cassava root), Carb treatments (yeast fermentation and non-fermentation), and the interaction between Carb source and Carb treatments. Mean comparisons were performed using Tukey’s test, and statistical significance was declared at p< 0.05.

Results and discussion

Chemical composition of ingredients

Yeast fermentation of fresh cassava root and broken rice decreased the pH from 6.1 and 6.3 to 4.5 and 4.3, respectively. The low pH also facilitated the processing of hydrocyanic acid (HCN) content in fresh cassava root, resulting in undetectable HCN levels in the fermented cassava root (FCR). Ammoniation of rice straw increased its pH and enhanced its crude protein content.

Gas and methane production

Supplementation with 4.5 % starch-rich substrates, such as broken rice and cassava root, in the ammoniated rice straw diet enhanced fermentation, as reflected by greater gas production relative to fiber-only control. This effect was further supported by higher soluble dry matter (DM) values in the carbohydrate-supplemented treatments compared with the control (Table 3). However, no significant differences in gas production were observed between carbohydrate sources (Carb source; e.g. broken rice group vs. cassava root group) or between carbohydrate treatments (Carb treatment; e.g., yeast-fermented vs. non-fermented).

The proportion of methane in total gas was lower in treatments supplemented with 4.5% DM of carbohydrate compared with the unsupplemented control (Figure 1). This likely reflects differences in biochemical fermentation pathways, whereby starch-rich substrates favor propionate formation, whereas fiber fermentation is associated with greater acetate production and, consequently, higher methane output. The lowest methane percentages were observed in the fermented broken rice and fermented cassava root treatments, which were significantly different from the control (Table 3; Figure 1).

Figure 1. Percentage of methane in gas of the treatments after 24 hours of in vitro rumen fermentation

However, when gas and methane production were expressed per unit of soluble DM, no significant differences were detected among treatments (Table 3). This is consistent with findings by Phuong et al (2024), who reported that the contribution of yeast-fermented rice in ammoniated rice straw (ARS) to methane mitigation was unclear at a 4% inclusion level, but did not evaluate the comparative effect relative to unfermented rice.

In the present study, we calculated the difference between starch-supplemented treatments and control. Figure 2 indicates that the type of carbohydrate source influences methane production per unit of soluble DM differently. Specifically, supplementation with 4.5% broken rice (BR) slightly increased methane production per unit soluble DM relative to the control. This may be attributed to enhanced DM solubility, leading to a slight increase in fermentation rate and, consequently, gas and methane production enhanced slightly (Table 3; Figure 2 and 3). In contrast, inclusion of 4.5% cassava root (CR) resulted in reduced soluble DM, gas production, and result in reducing methane output per soluble DM (Table 2). This effect may be associated with the presence of hydrocyanic acid (HCN) in fresh cassava root, as reported in previous studies (e.g. Vongkhamchanh et al 2015, Phuong et al 2012, Phuong et al 2015, Sangkhom et al 2019).

Figure 2. Differences in methane per unit soluble DM of carbohydrate supplement compared to control	Figure 3. Differences in gas per unit soluble DM of carbohydrate supplements compared to control

Nevertheless, at the inclusion low level of 4.5%, the reduction in methane production per unit soluble DM in the CR treatment was not statistically different from the control. Meanwhile, supplementation with 4.5% DM of fermented broken rice, fermented cassava root, or fresh cassava root tended to reduce methane production per unit of soluble DM. Yeast-fermented broken rice and cassava root showed a reduction compared with the control, more reduction that observed with unfermented broken rice (BR4.5) and even fresh cassava root (CR4.5). Although these differences were not statistically significant, they suggest a potential role of yeast fermentation in lowering methane production relative to non-fermented carbohydrate sources.

Previous studies (e.g., Binh et al 2017, Phanthavong et al 2016, Sangkhom et al 2019) reported that supplementation with approximately 4% DM of yeast-fermented cereal by-products, such as brewers’ spent grains and rice wine distillers’ residues, reduced methane production in both in vivo and in vitro systems. However, these studies were conducted using basal diets rich in soluble carbohydrates (e.g., cassava pulp–urea or cassava leaf meal), in contrast to the fiber-rich ammoniated rice straw used in the present study. Similarly, Phuong et al (2024) observed no significant effect at a comparable inclusion level of yeast-fermented rice in ARS substrate, which may be attributed to differences in substrate characteristics. The observed responses likely by there is a certain opposition between the fermentation of soluble carbohydrates (BR and CR) and structural carbohydrates (e.g., rice straw) in two issue (i) fermentation rate and (ii) methane production. Soluble carbohydrates generally increase fermentation rate, and in terms of mechanism, also increase methane production. In the present study, supplementation with 4.5% carbohydrate increased fermentation activity, as indicated by higher gas production and soluble DM compared with the control, yet did not significantly alter methane production per unit of soluble DM (Table 3). Overall, it could indicate that methane output was reduced relative to the control, with a more pronounced reduction observed in yeast-fermented treatments (FBR 4.5 and FCR 4.5) than in non-fermented counterparts (BR and CR), as reflected in the lower proportion of methane in total gas (Figure 4). Sangkawe et al (2025) reported that supplementation with Saccharomyces cerevisiae enhanced fiber degradation by stimulating the growth of key fibrolytic bacteria, including Ruminococcus albus, Ruminococcus flavefaciens, and Fibrobacter succinogenes . In this context, improved fermentation associated with reduced methane production may confer benefits for animal performance based on an ammoniated rice straw (ARS) basal diet.

Figure 4. Effect of Carb treatment (fermentation including FBR4.5 and FCR4.5; Non-fermentation including BR4.5 and CR4.5) on proportion of methane in total gas

There were no significant differences observed in gas or methane production per unit of soluble DM between carbohydrate sources (i.e., the broken rice group, including BR4.5 and FBR4.5, versus the cassava root group, including CR4.5 and FCR4.5) (Figure 5. Similarly, no differences were detected between FBR4.5 and FCR4.5 (Table 2), consistent with the findings of Sangkhom et al (2020). These results suggest that cassava root may serve as a viable alternative to broken rice as a yeast fermentation substrate in diets based on fiber-rich ARS.

Figure 5. Gas and methane production per unit soluble DM in Carb source (broken rice group including BR4.5 and FBR4.5; the cassava root group including CR4.5 and FCR4.5)

Furthermore, no interaction between carbohydrate source and carbohydrate treatment was identified. This may be explained by the contrasting effects of BR4.5 and CR4.5 on gas and methane production (Figures 2 and 3), potentially influenced by additional factors such as the presence of hydrocyanic acid (HCN) in fresh cassava root.

Conclusions

Overall, in a basal diet characterized by high fiber content from ammoniated rice straw, methane mitigation and fermentation efficiency, reflected in increased soluble dry matter (DM) and gas production, were improved by supplementation with yeast-fermented carbohydrate sources, such as fermented broken rice or fermented cassava root.

Supplementation with 4.5% DM of fresh cassava root in the ammoniated rice straw substrate slightly decreased methane production per unit of soluble DM compared with compared to the pure broken rice supplement at the same level; however, overall, there was no significant difference between them, nor was an interaction found between Carb source and Carb treatment.

Acknowledgments

The authors acknowledge support for this research from research funding of Nong Lam University, Ho Chi Minh City, Vietnam with proposal code No. CS-CB25-CNTY-04

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