Development of a Methane Emission Reduction Technique for Dairy Cattle Using Soil Microorganisms: A Small-Scale Pilot Trial ()
1. Introduction
Among greenhouse gases contributing to global warming, methane (CH4) is second only to carbon dioxide (CO2) [1]-[3]. Methane has a global warming potential that is approximately 28 times that of CO2, making even small amounts highly impactful. Ruminants such as dairy cattle and sheep produce large quantities of methane during digestion, accounting for approximately 5% of total greenhouse gas emissions [4]-[7].
The rumen of dairy cattle harbors a diverse microbial community that facilitates feed decomposition [8] [9]. During this process, hydrogen (H2) and carbon dioxide (CO2) are produced as byproducts, which methanogenic archaea use to generate methane. This methane is released into the atmosphere through eructation, contributing to global warming [10]. Furthermore, it has been reported that this process results in an energy loss of approximately 2% - 15% of digestible feed [4].
Recent research has identified a novel bacterial strain in the rumen of dairy cows that is expected to help reduce methane emissions, suggesting that the rumen microbiome still holds untapped potential for methane mitigation. Moreover, previous studies have demonstrated that certain feed additives, such as garlic and seaweed, can effectively reduce methane emissions [11] [12], making them promising candidates for practical application. In addition, genetic selection and innovative feeding strategies have been explored to further decrease methane emissions in ruminants [4] [6] [13].
Livestock harbors a wide array of microorganisms, and we have focused on artificially maintaining a balanced microbial community. To date, we have administered various soil-derived bacterial formulations to livestock in an effort to enhance feeding efficiency and strengthen disease prevention systems. BX-1 is a feed additive produced by culturing a cocktail of multiple soil bacteria on a large scale, then inactivating (killing) them and converting them into powder (Kawashima Co., Ltd., Japan). A key advantage is that, as long as the original live bacteria are available, BX-1 can be mass-produced in virtually limitless quantities anywhere—even close to cattle facilities. In other words, BX-1 can be rapidly manufactured exactly where and when it is needed. This feature sets it apart from other additives, such as garlic or seaweed. Furthermore, because the soil bacteria that make up BX-1 are fully inactivated and powdered, it is easy to transport, resistant to both heat and cold, and can be stored long-term (about 2 years). It also does not proliferate in the environment or in cattle, ensuring safe use. In this study, we developed and tested BX-1 for its effectiveness and feasibility in mitigating methane emissions in dairy cattle.
2. Materials and Methods
2.1. Preparation and Administration of BX-1
BX-1 is a powdered feed additive composed of mass-cultured and inactivated soil bacteria that are commercially available as livestock feed supplements (Kawashima Co., Ltd., Japan). Bacterial species belonging to BX-1 are listed in Table 1.
Table 1. Micro-organisms in BX-1.
Taxon name |
Proportion (%) |
Staphylococcus saprophyticus group |
59.5524 |
Staphylococcus kloosii |
32.5953 |
Weissella paramesenteroides group |
2.0747 |
Staphylococcus aureus group |
1.3063 |
Kocuria koreensis |
1.1814 |
Staphylococcus pettenkoferi group |
0.7108 |
Bacillus cereus group |
0.5283 |
Leuconostoc pseudomesenteroides group |
0.3218 |
Pediococcus acidilactici group |
0.1153 |
Kocuria kristinae |
0.0912 |
Staphylococcus sciuri group |
0.0720 |
Corynebacterium variabile group |
0.0672 |
Enterococcus italicus group |
0.0672 |
Brachybacterium faecium group |
0.0528 |
Brevibacterium DL489154_s |
0.0480 |
Leuconostoc mesenteroides group |
0.0384 |
Corynebacterium nuruki group |
0.0336 |
Citricoccus muralis group |
0.0240 |
Corynebacterium flavescens |
0.0192 |
Corynebacterium minutissimum group |
0.0192 |
Enterococcus faecium group |
0.0192 |
Bacillus megaterium group |
0.0144 |
Lactobacillus acidipiscis |
0.0144 |
Lactococcus lactis group |
0.0144 |
Actinomyces slackii |
0.0096 |
Arthrobacter agilis group |
0.0096 |
Arthrobacter echini |
0.0096 |
Blautia wexlerae |
0.0096 |
Clavibacter michiganensis group |
0.0096 |
Kocuria rhizophila group |
0.0096 |
Neomicrococcus lactis |
0.0096 |
Ruminococcus faecis |
0.0096 |
Actinomyces provencensis group |
0.0048 |
Anaerostipes hadrus group |
0.0048 |
Bacillus carboniphilus group |
0.0048 |
Bacillus smithii |
0.0048 |
Bifidobacterium adolescentis group |
0.0048 |
Blautia faecis |
0.0048 |
Brevibacterium iodinum group |
0.0048 |
Caenibacillus caldisaponilyticus |
0.0048 |
Caldibacillus debilis |
0.0048 |
Corynebacterium tuberculostearicum |
0.0048 |
Corynebacterium xerosis group |
0.0048 |
Dorea formicigenerans |
0.0048 |
Enterococcus saccharolyticus group |
0.0048 |
Fusicatenibacter saccharivorans |
0.0048 |
Geobacillus stearothermophilus group |
0.0048 |
Geobacillus thermoleovorans group |
0.0048 |
Geobacillus toebii group |
0.0048 |
Kurthia zopfii group |
0.0048 |
Lactobacillus dextrinicus |
0.0048 |
Lactobacillus sakei group |
0.0048 |
Lactococcus taiwanensis |
0.0048 |
Listeria grayi |
0.0048 |
Dorea PAC000479_s |
0.0048 |
Sporobacter PAC001162_s |
0.0048 |
Sporobacter PAC001306_s |
0.0048 |
PAC001201_g PAC002029_s |
0.0048 |
Romboutsia timonensis |
0.0048 |
Rummeliibacillus pycnus |
0.0048 |
Staphylococcus succinus group |
0.0048 |
Weissella ghanensis group |
0.0048 |
2.2. Effect of BX-1 on Eructation Suppression and Methane
Emissions in Dairy Cattle
Five 12-month-old Holstein heifers were provided with drinking water containing either 1 g or 10 g of BX-1 per liter over three days. Eructation frequency was recorded for 40 min, starting at four hours post-feeding. The bar graphs represent the mean values of five cattle, with * indicating a significant reduction in comparison to the control group (Student’s t-test).
Additionally, the methane concentration in the exhaled breath was measured five times at four-minute intervals just from four hours post-feeding, and the average values were calculated. To measure methane concentrations, a methane gas sensor probe was positioned at approximately 10 cm from the cow’s mouth. The bar graphs represent the mean values of five cattle, with * indicating a significant reduction in comparison to the control group (Student’s t-test).
3. Results
Cattle typically burp once every 1 - 2 min. The number of burps in cattle measured in our study showed no significant difference from reports from other research groups [14]. However, we found that the oral administration of BX-1 drastically reduced this frequency.
The administration of BX-1 resulted in a dramatic reduction in both eructation frequency and methane concentrations in exhaled breath. Specifically, eructation frequency decreased markedly (Figure 1), whereas the methane concentration underwent an even more dramatic reduction (Figure 2). When dairy cattle were provided with free access to drinking water containing 10 g/L of BX-1, the number of eructations was reduced by approximately 77%, and the concentration of methane gas in exhaled air decreased by about 80%. Furthermore, veterinary examinations revealed no clinical symptoms. These results indicate that BX-1 is an effective methane reduction technology for dairy cattle in this small-scale pilot study.
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Figure 1. Suppression of eructation in dairy cattle by oral administration of BX-1. Five 12-month-old Holstein heifers were given ad libitum access to drinking water containing BX-1 powder (1 g or 10 g per liter) for three days. The number of eructation was recorded for 40 minutes starting four hours after feeding. Each bar in the graph shows the mean numbers of eructation from the five cattle. Asterisks indicate a significant decrease (P < 0.01, Student’s t-test) relative to the control group which received water alone.
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Figure 2. Reduction of methane emissions in dairy cattle by oral administration of BX-1. Five 12-month-old Holstein heifers were given ad libitum access to drinking water containing 1 g or 10 g of BX-1 per liter for three days. Starting four hours after feeding, methane concentrations in eructed gas were measured 5 times at four-minute intervals for each heifer, and the average value was calculated. Each bar in the graph shows the mean methane concentration of the five cattle. Asterisks indicate a significant decrease (P < 0.01, Student’s t-test) in comparison to the control group which received water alone.
4. Discussion
This study demonstrated that soil bacteria-based feed additive BX-1 effectively suppressed methane emissions and eructation in dairy cattle over a short period. The rumen of ruminants hosts a diverse microbial community, including methanogenic archaea responsible for methane production [8]-[10]. BX-1 is composed of inactivated bacterial cells that do not proliferate in the digestive tract. These results suggest that BX-1 may have altered the microbial balance in the rumen, potentially suppressing the metabolic activity of methanogens or exerting direct inhibitory effects on methanogenesis through bacterial enzymes or other components. Additionally, previous research has reported the efficacy of BX-1 for controlling Salmonella infection in hatched chickens and laying hens, as well as Newcastle disease in poultry [14]-[16]. These findings imply that BX-1 may have broader applicability beyond methane mitigation, potentially improving the health of various livestock. In this small-scale trial, a remarkable reduction in methane concentration was observed, which is a highly significant effect; however, validating these findings in more diverse and larger-scale settings is essential (at least 20 cows for each dosage of BX-1). We also plan to test BX-1 under various housing conditions, including indoor barns and open pastures. Future research should clarify the metabolic pathways through which BX-1 influences methanogens, identify any limitations or conditions under which its effect might diminish, and consider the impact of prolonged administration on rumen function, animal health, and feed efficiency. Although we used young dairy heifers in this study, we plan to administer BX-1 to lactating cows in future research. We will assess clinical signs, changes in body weight, and milk production to further evaluate the broader safety of BX-1. Cost-effectiveness and risk analysis, including safety for end consumers and environmental impact of large-scale production, will be crucial for real-world application. Moreover, dairy farming practices differ worldwide, so confirming the reproducibility of these results under various feed compositions, water availability, and housing systems is critical. It will also be necessary to investigate how BX-1 can be integrated into different feeding regimens—whether mixed in total mixed rations or provided via drinking water—and to determine optimal dosage levels and treatment durations for sustained methane reductions. Since BX-1 is a bacterial-based product, it can potentially be mass-produced in many regions, reducing logistical barriers and highlighting its high potential for global scalability. In conclusion, while this small-scale pilot trial provides encouraging data on the efficacy and safety of BX-1 in reducing methane emissions in dairy cattle, further investigations that address the mechanisms of action, limitations, cost-effectiveness, and broader applicability are needed to establish a robust evidence base for its integration into global livestock practices aimed at mitigating methane emissions.
NOTES
*Equally contributed.
#Corresponding author.