Impact of effective microorganisms-treated wheat bran on nutritional parameters and methane production

E T Tadesse, G A Minneeneh, Y Y Mummed, M Dejene and E Kebreab

African Center of Excellence for Climate-Smart Agriculture and Biodiversity Conservation, Haramaya University, P.O. Box 138, Dire Dawa, Ethiopia
eden.tesfaye27@gmail.com

Abstract

The present study investigates the effects of Effective Microorganisms-treated wheat bran (EMWB) on organic matter digestibility (OMD), metabolizable energy (ME), short-chain fatty acids (SCFA) production, and in vitro gas and methane production in a mixed ruminant diet. The diet comprised 70% native pasture hay and 30% concentrate mix, with the latter including varying levels (0%, 33%, 66%, and 100%) of EMWB corresponding to EM0, EM33, EM66 and EM100, respectively. The total gas production at all incubation hours (3, 6, 12, 24, 48, 72 and 96 hr.) were higher (p<0.05) for EM100 and followed by EM66. The highest (p< 0.05) “a” fraction of gas production value was recorded for EM0 (1.56). The “b” fraction value was high (p<0.05) for EM100 (33.8) = EM66 (32.6) and low for EM33 (29.9) =EM0 (28.6). Gas production potential (a+b) value was greater (p<0.05) for EM100 (34.2) = EM66 (32.8) and lower for EM0 (30.2) = EM33 (30.4). The rate constant of gas production (c) was greatest (p<0.05) for EM100 (0.25) and lowest for EM0 (0.08) = EM33 (0.11). OMD and ME were in order of EM100 (56.6, 7.60) > EM66 (54.2, 7.14) > EM33 (50.1, 6.49) = EM0 (48.1, 6.21) respectively. The value of SCFA was in order of EM100 (0.77) =EM66 (0.72)>EM33 (0.60) =EM0 (0.57). Methane (CH₄) production at 24 hours showed a decreasing trend with increasing EMWB levels, underscoring the potential of EMWB as a sustainable feed supplement that not only enhances OMD and SCFA production but also contributes to reducing CH₄ emissions from livestock.

Keywords: in vitro gas, methane, EM-treated wheat bran

Introduction

The agricultural sector faces a significant challenge of feeding a growing population predicted to peak at 9.2 billion people by 2050 (FAO 2006). This challenge comes with the added responsibility of meeting social and environmental obligations, particularly the reduction of greenhouse gas (GHG) emissions especially methane (CH₄) which has 25 times higher global warming potential than carbon dioxide (CO₂) (IPCC 2007; 2014). Livestock, notably ruminants, represent the largest single source of CH₄ emissions from agriculture, contributing approximately 39% to global emissions related to human activities (32% from enteric fermentation and 7% from manure) (IPCC 2007). If enteric CH₄ emissions grow in tandem with increasing livestock numbers, global livestock-related CH₄ production will increase by 60% by 2030 (FAO 2009). However, in many developed countries, enteric CH₄ intensity (emission per unit of product) is declining due to improved productivity of dairy cows and a stable beef cattle population. Yet, developing countries particularly, Africa shows increases in enteric CH₄ emission (IPCC 2022; FAO 2023).

The GHG emission of livestock sector of Ethiopia is expected to increase from 65 Mt CO₂eq in 2010 to 125 Mt in 2030 due to an increase in CH₄ emissions from enteric fermentation, which accounts about 80% of the total emission from livestock (FDRE 2011). Enteric CH₄ emission from livestock sector of Ethiopia is increasing rapidly since 2005 mainly due to large number of livestock with low productivity (FAO 2023). However, Ethiopia aims to build a green economy by reducing the livestock number while boosting the productivity and efficiency of livestock sector. To achieve this aim, improving rumen ecology is one of the prioritized actions to reduce emission intensity from livestock (FDRE 2011).

Scientific studies (IPCC 2007, 2014, 2022), agree that significant reductions in GHG emissions are essential to meet the global goal of limiting global warming to no more than 2°C. Achieving this target necessitates a rapid acceleration of mitigation efforts (IPCC 2022). Reducing CH₄ emission is one of the strategies set by agriculture, forestry and other land use (AFOLU) sector both to achieve the climate goal and play central role to food security and sustainable development (IPCC 2007; 2022). To reduce enteric CH₄ emission, many novel mitigation strategies are being refined, such as use of supplements, feed additives, vaccines and probiotics (direct-fed microbials). However, there is still a major role for research and development to play in this area (IPCC 2014; 2022).

Direct-fed microbials (DFM) have been defined as naturally occurring microorganisms that enhance livestock health (Krehbiel et al 2003). They have been successfully used in ruminant production to increase productivity, prevent digestive disorders like acidosis and decrease pathogenic load in animals (Lettat et al 2012; McAllister et al 2011). They are also promising mitigation strategies of enteric CH₄ emission (Collado et al 2007; Jeyanathan et al 2014).

Effective microorganisms (EM), are a composite of selected microorganisms basically yeast (Saccharomyces cerevisiae) and lactic acid bacteria (Higa 1994). EM technology is widely used and accepted in a livestock sector due to its ability to increase productivity (Alexey et al 2019), improve digestibility (Yacout et al 2021; Tadesse et al 2024) and enhance nutritive value of feeds (Gulilat and Walelign 2017; Fikre et al 2019; Asmare et al 2020; Roba 2022; Eden et al 2023). Additionally, studies have shown effectiveness of EM in reducing GHG from livestock sector. Applications of EM in stored slurry and manure have significantly reduced total greenhouse gas emissions, including CH₄ and nitrous oxide (Amon et al 2004; 2005; Bastami et al 2016). Likewise, treating feed with EM increased butyrate and propionate production, improved gas production kinetics and reduced in-vitro CH₄ production (Yacout et al 2021). Moreover, using EM as a feed additive has reduced CH₄, ammonia and nitrous oxide emissions from pig's slurry, increased volatile fatty acids (VFA), decreased protozoan numbers, and significantly reduced enteric CH₄ emissions (Polyorach et al 2018).

However, despite these promising results, there is limited knowledge regarding the effects of EM-treated wheat bran on in-vitro gas and CH ₄ production. Therefore, the aim of this study was to evaluate the impact of EM-treated wheat bran in a mixed diet on organic matter digestibility (OMD), metabolizable energy, short-chain fatty acids (SCFA) production, and in-vitro gas and CH₄ production.

Materials and methods

Preparation of effective microorganisms-treated wheat bran

The preparation of EM-treated wheat bran involved obtaining a sufficient quantity of activated EM from Weljeji PLC (Bishoftu, Ethiopia) and molasses from the Ethiopian Sugar Corporation Wenji branch. The EM was diluted by mixing 1 liter of EM, 1 liter of molasses, and 18 liters of water in a 1:1:18 ratio. Subsequently, 20 liters of the diluted EM solution were poured gradually into 50 kg of wheat bran and thoroughly mixed. The mixture was placed in a concrete container to maintain anaerobic conditions and protect it from direct sunlight. It was left to ferment for a duration of 21 days, and it was considered ready for use once it emitted a sweet fermented smell.

Experimental treatments

In a companion feeding trial involving lactating dairy cows, a mixture consisting of 70% native pasture hay and 30% concentrate mixture was fed to the animals. The concentrate mixture comprised 35% wheat bran, 20% maize, 21% rice bran, 3% molasses, 4% niger seed cake, 11% sunflower cake, 3% salt, and 3% limestone, with the composition and proportions provided by the formulating company. To investigate the effects of EM-treated wheat bran on lactating dairy cow performance, the concentrate mixture was replaced with varying percentages of EM-treated wheat bran for different treatments (EM0, EM33, EM66, and EM100), corresponding to 0%, 33%, 66%, and 100% replacement, respectively. A mixed ration with proportions similar to the feeding trial was prepared and used to assess total gas production, ME, OMD and CH₄ production. For instance, a 100 g mixed ration was prepared for EM0 by combining 70 g of native pasture hay with 30 g of the concentrate mixture. Similar adjustments were made for EM33, EM66 and EM100. Samples of the treatment mixed feed were dried at 65°C for 48 hours and ground using a Wiley mill to pass through a 1 mm screen for subsequent measurements.

Experimental measurements

Chemical composition

The analysis of the chemical composition of the treatment samples, including dry matter (DM), ash, crude protein (CP), acid detergent fiber (ADF), acid detergent lignin (ADL) and neutral detergent fiber (NDF), followed the AOAC (1990) method. The organic matter (OM) content was calculated as 100% minus the ash content, while the CP content was calculated by multiplying the nitrogen content by a factor of 6.25. The determination of ADF, ADL and NDF in the samples was carried out using the method of Van Soest and Robertson (1985).

The chemical composition of the experimental feeds is given in Table 1.

In vitro gas production and gas production kinetics

In vitro gas production (GP) measurement was conducted according to the procedure outlined by Menke and Steingass (1988). For the in-vitro incubation of the mixed treatment rations, rumen fluid was obtained from three rumen fistulated Boran × Holstein Frisian cross-breed steers, which were fed a basal diet of natural pasture hay and supplemented with 2 kg of concentrate mixture before the morning feed. Pooled rumen fluid was prepared and transferred to a large glass beaker placed inside a 39°C water bath. The beaker was continuously purged with CO₂ and stirred, following the method recommended by Goering and Van Soest (1970). Media solutions, including buffer solution, macro and micro mineral solution, were prepared and utilized as described in Goering and Van Soest (1970).

Each treatment sample weighting about 200 mg, was placed into 100 ml calibrated glass syringe together with 30 ml rumen liquid and culture media solution on a 1:2 ratios in triplicate. Blank syringes with buffered rumen fluid with no treatment samples were also included in triplicate. All the syringes were incubated in a water bath maintained at 39°C. The syringes were shaken gently 30 min after the start of incubation and every hour for the first 10 hr. of incubation and volume was recorded at intervals of 3, 6, 12, 24, 48, 72, and 96 hours of incubation. Gas production (ml/200 mg) at different time points was calculated as:

Where: Gt = gas production value (ml/200 mg) at t hours, G0 = gas production of blank syringes (ml), V0 = volume in ml at beginning (0hr.), Vt = volume in ml at t hours, Ws = weight of dried sample in mg.

The kinetics of the gas production was estimated using the following exponential equation of Ørskov and McDonald (1979):

Y= a+b (1-e^-ct),

where Y = total gas production at time t; a = gas production from the immediately fermentable organic matter (OM) (the intercept of the gas production curve); b = gas production from fermentation of slowly but potentially fermentable OM; a+b = the potential gas production (the asymptote of the gas production curve); c = the rate constant for the gas production b, t = incubation time and e = base of natural logarithm (Blummel and Ørskov 1993).

The in vitro organic matter digestibility, metabolizable energy and short chain fatty acids measurements

The calculation of in-vitro OMD, ME and SCFA contents of the mixed treatment ration samples was based on the net 24-hour gas volume, CP and ash contents of treatment rations according to the following equations:

OMD (%) = 14.88 + 0. 889* GP24 + (0.45 *CP %) + (0.651*XA %) (Menke and Steingass 1988)

ME (KJ/kgDM) = 2.20 + 0.136*GP24 + 0.057*CP% (Menke and Steingass 1988)

SCFA (mmol) = 0.0239*GP24 - 0.0601 (Getachew et al 2002).

Where: GP24 = gas produce at 24-hr., CP = crude protein content of treatment ration samples, XA = ash content of treatment ration samples.

Measurement of methane production

Methane production after 24 hours of incubation was measured through the absorption of CO₂ with 40% of sodium hydroxide (NaOH) as described by Fievez et al (2005). Using this method, it was assumed that in-vitro gases primarily contained CH₄ and CO₂, while other gases produced during fermentation were relatively insignificant.

At the end of the 24-hour incubation period, after recording the final gas volume, the lower end of the syringe was connected to the lower end of another syringe containing 4ml of NaOH. The NaOH was introduced into the incubated treatment samples and blank syringe to prevent gas escape. Mixing of treatment sample with NaOH allowed absorption of CO₂, with the remaining gas (V2) in the syringe considered to be CH₄ and other insignificant gases (0.2%) (Fievez et al 2005). CH₄ production was calculated by subtracting the gas production of blank syringes after CO₂ removal from the remaining gas.

Statistical analysis

Data on total gas production at different incubation period, gas production kinetics, OMD, ME, SCFA and CH₄production at 24 hr. were analyzed using analysis of variance (ANOVA) following the general linear model (GLM) procedure of SAS statistical program (2010). Means were separated using LSD. The model used for analysis was Y_ij = μ + T_i + ß _j + e_ij,where; Y_ij = response variable; μ = overall mean; T_i = treatment effect; ß _j = replication effect; and e_ij = the random error.

Result

In vitro gas and methane production

In-vitro gas production of mixed diets containing different levels of EM-treated wheat bran is presented in Table 2.In-vitro gas production showed increasing trend as incubation period increased for all treatments. However, there was significant variation among treatments (p<0.05). The gas production pattern showed increasing trend as the level of EM-treated wheat bran increased in mixed diets. The greatest gas production value was recorded for EM100 under all incubation period and followed by EM66.

The greatest (p<0.05) CH₄ production at 24hr of incubation period was recorded from EM0 followed by EM33 and the lowest value was recorded from EM100.

Figure 1. Effect of EMWB on Methane Production

In-vitro gas production kinetics

The gas production values from immediately fermentable organic matter (OM) (a) were significantly higher for EM0 compared to the other treatments (EM33, EM66, EM100), while the values for EM33, EM66, and EM100 were similar. The gas production values from fermentation of slowly but potentially fermentable OM (b) exhibited an increasing trend as the level of EM-treated wheat bran in the mixed diets increased, with the order being EM100 = EM66 > EM33 = EM0. Furthermore, the rate constant for gas production (b) showed a significant increase as the level of EM-treated wheat bran increased in the mixed rations, with the order of EM100 > EM66 > EM33 = EM0. Similarly, the gas production potential (a+b) increased with the rising level of EM-treated wheat bran in mixed rations and the order of values was EM100 = EM66 > EM33 = EM0 (Table 3).

Organic matter digestibility, metabolizable energy and short chain fatty acids

Table 4 presents the calculated values of OMD, ME, and SCFA at the 24-hour incubation period. As shown in the table, all these parameters exhibited a linear increase with the higher levels of EM-treated wheat bran in the mixed diets. The EM100 had the highest (p<0.05) OMD and ME value, and the order of OMD values was EM100 > EM66 > EM33 = EM0. The SCFA values also reflected this trend, with EM100 and EM66 having the highest values.

Discussion

In vitro gas production

In vitro gas production is influenced by the fermentable carbohydrates present in the feed, and its volume and rate are dependent on the nature of these carbohydrates (Blummel et al 1997). Additionally, the composition of feed, particularly the cell-wall fractions and crude protein (CP) content, can impact feed degradability (Van Soest 1994). In this study, the consistently high gas production observed throughout the incubation period for EM100 and EM66 can be attributed to their low fiber content (NDF, ADF, and ADL) and high CP content, as indicated in Table 1.

This finding aligns with previous studies by Kumara et al (2009), Bezabih et al (2013) and Andualem et al (2016), which showed a positive correlation between CP content and in-vitro gas production, as well as a negative correlation between fiber content and in-vitro gas production. Furthermore, Maheri et al (2008) found that lower NDF and ADF contents in feedstuffs resulted in higher in-vitro gas production. Additionally, the inclusion of EM in mixed diets has been shown to improve in-vitro gas production and gas production kinetics (Yacout et al 2021).

Gas production kinetics

The parameters related to gas production kinetics exhibited variations among treatments, indicating differences in the rate and extent of fermentation characteristics in mixed diets. The extent of gas production potential (a+b) is known to correlate with dry matter intake and the nutritive value and degradability of feeds (Blummel and Ørskov 1993; Khazaal et al 1993; Getachew et al 2000). Consequently, the higher gas production potential values observed in EM100 and EM66 may be attributed to the improved nutritive values of these treatments, resulting from the increased level of EM-treated wheat bran in the mixed rations. As indicated in Table 1, the inclusion of EM in mixed diets enhanced the CP content and reduced structural carbohydrate content (NDF, ADF, and ADL).

Ndlovu and Nherera (1997) found that the gas production rate (c) was negatively correlated with NDF and ADF. Hence, the faster gas production rate (c) observed in EM100 and EM66 might be due to their lower NDF and ADF contents (Table 1). This suggests that rumen microbes were able to utilize the feed more effectively, possibly due to a higher content of fermentable nutrient.

Short chain fatty acids, metabolizable energy and organic matter digestibility

The greater values of OMD, ME and SCFA observed in EM100 and EM66 can be attributed to their highest gas production at the 24-hour incubation time, compared to the control. Gas production is positively correlated with SCFA content, OMD, and ME (Getachew et al 2000; Kumara et al 2009; Andualem et al 2016). Studies have shown that higher gas production can contribute significantly to energy supply through SCFA production (Maheri et al 2008). This may also be related to the higher CP content of EM100 and EM66 compared to the control, as positive correlations have been reported among CP, ME, and OMD values of feeds (Tolera et al 1997; Aderinboye et al 2016). In line with this finding, Yacout et al (2021) showed that the inclusion of EM in the feed significantly increased butyrate and propionate production, leading to a decrease in the acetate to propionate ratio.

Methane production

The reduction in CH₄ production observed in EM100 and EM66 may be associated with the presence of lactic acid bacteria and Saccharomyces cerevisiae in EM solution. Both lactic acid bacteria and Saccharomyces cerevisiae have the ability to individually and in combination reduce CH₄ production (Chung et al 2011; Jeyanathan et al 2014; Salem et al 2015).

Furthermore, the lower NDF, ADF, and ADL contents of EM100 and EM66 compared to the control may have played a role. Fermentation of NDF typically increases CH₄ production by shifting the SCFA proportion towards acetate, which produces more hydrogen (Kaitho et al 1997). Similarly, the study by Yacout et al (2021) showed a negative correlation between CH₄ production and propionate, which may be attributed to their competition for hydrogen. Moreover, Andualem et al (2016) indicated that CH₄ concentration is positively correlated with fiber fractions and negatively correlated with CP content in feed. Similarly, Junior et al (2017) showed a negative correlation between DM degradation and CH₄ production, suggesting that fiber-rich feeds exhibited higher CH₄ and lower CO₂ production, possibly due to increased methanogen activity resulting from a greater availability of CO₂ and H₂.

The reduction in CH₄ production in the current study is consistent with the findings of Polyorach et al (2018) and Amon et al (2005), where feeding EM as a feed additive significantly reduced enteric CH₄ emissions and CH₄ emissions from slurry, respectively. Additionally, the study by Yacout et al (2021) indicated that treating feed with EM reduced in vitro CH₄ emission. In agreement with these findings, Amon et al (2004), Amon et al (2005) and Bastami et al (2016) reported that EM was effective in reducing in-vitro CH₄ and nitrous oxide production when added to manure and slurry.

Conclusion

Reducing CH₄ production while maintaining feed quality and livestock production is of paramount importance. The use of EM-treated wheat bran as a feed additive offers a promising CH₄ mitigation strategy, particularly for developing countries like Ethiopia. This approach not only has the potential to enhance feed quality and improve livestock production but also contributes to a reduction in CH₄ emissions, thereby promotes climate smart livestock production. However, it is essential to emphasize that further in-vitro and in-vivo studies are required to validate and confirm these results.

Acknowledgements

We would like to express our gratitude for the PhD scholarship awarded to the first author by the World Bank through the African Center of Excellence for Climate Smart Agriculture and Biodiversity Conservation at Haramaya University. We also extend our appreciation to the Ethiopian Institute of Agricultural Research for their valuable feed support. Special thanks go to the laboratory of the School of Animal and Range Sciences at Hawassa University for their exceptional laboratory services. We are sincerely thankful to all individuals who played a part in the successful completion of this study.

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