The Effect of Essential Oils on Rumen Microbiota: Analysis of the Correlation Between Antibacterial Activity and Fermentation Modulation In Vitro

Abstract

This study aimed to quantitatively determine the composition of 25 essential oils (EOs) using gas chromatography–mass spectrometry (GC-MS) to assess their minimum inhibitory concentrations (MICs) against selected rumen microorganisms and to confirm their effects in in vitro tests on volatile fatty acid (VFA) formation. GC-MS analysis identified over 80 compounds across the tested oils. The MICs were determined for Butyrivibrio fibrisolvens, Prevotella albensis, Lactobacillus delbrueckii ssp. lactis, and Streptococcus bovis, revealing diverse sensitivities. The rumen bacteria’s sensitivity to essential oils varied by strain, with some microorganisms inhibited at low concentrations while others required higher doses, highlighting the potential for targeted modulation of the rumen microbiota. Amyris balsamifera and Zingiber officinale demonstrated strong inhibitory effects at low concentrations and simultaneously enhanced VFA production. In contrast, Lavandula officinalis showed inhibitory effects on VFAs. Amyris balsamifera and L. officinalis also exhibited methane reduction. These findings demonstrate that selected essential oils can modulate rumen microbiota and fermentation by either inhibiting or stimulating specific bacterial groups, highlighting their potential as natural modulators to improve rumen function and animal health.

1. Introduction

In recent years, there has been growing interest in natural bioactive compounds, including essential oils (EOs), as a promising alternative to antibiotic growth promoters and synthetic feed additives [1,2,3,4,5]. Essential oils are complex mixtures of volatile secondary metabolites of plant origin, characterized by antibacterial, antioxidant, and anti-inflammatory properties [6,7,8]. The largest chemical group of essential oils is terpenes (monoterpenes and sesquiterpenes), often accompanied by phenols, alcohols, aldehydes, ketones, esters, and other oxygenated hydrocarbon derivatives [9,10,11,12]. At present, there are relatively few studies simultaneously evaluating the chemical composition of numerous essential oils obtained from different plant materials.

The activity of EOs against Gram-positive and Gram-negative bacteria has been widely described, but the effects of individual oils on the rumen microbiota remain poorly understood [11,13,14]. Importantly, essential oils may have a bidirectional effect, inhibiting the growth of some microorganisms while stimulating others, which suggests their potential as selective modulators of rumen fermentation rather than merely broad-spectrum antimicrobial agents [4,15,16,17]. Furthermore, as shown by a recent review, there is a lack of research on the appropriate doses and mechanisms through which essential oils affect methane emissions in ruminants [18]. It is crucial to understand the relationship between the dose of these metabolites, the rumen microbiome, and methanogenesis to maximize the effectiveness of supplementation [15].

The rumen microbiota plays a key role in digestive processes and the overall health of ruminants, determining feed fermentation efficiency, nutrient availability, and methane emissions [4,5,15]. Therefore, maintaining a stable and functionally diverse community of microorganisms in the rumen is essential for ensuring optimal animal productivity and sustainable animal production. On the other hand, disturbances in the microbiological balance of the rumen, caused by changes in nutrition, environmental factors, or infections, can lead to impaired fermentation processes and reduced animal performance. It has been shown that supplementation with an appropriate dose of essential oil alters the composition of the rumen microbiota and affects digestive enzymes as well as short-chain fatty acid profiles [19]. In one of the most recent studies, essential oils from Rosmarinus officinalis and Zingiber officinale were shown to influence metabolic parameters in dairy cows, including the fatty acid profile of milk [20]. In many studies, essential oil doses were determined based on the literature. However, to achieve such goals, it is crucial to develop more standardized methods for essential oil extraction and, above all, to establish a safe dosage for supplementation in animal diets [21].

Current research increasingly focuses on the possibility of precisely shaping the composition of rumen microbiota in order to increase fermentation efficiency and reduce energy losses in the form of methane [22]. Despite numerous studies describing the qualitative impact of essential oils on fermentation processes in the rumen, the quantitative relationships between their chemical composition, antibacterial activity, and ability to modulate the microbiota in vitro remain unclear. Essential oils, which are complex mixtures of monoterpenes and phenols, have been shown to modulate rumen microbial activity while affecting volatile fatty acid (VFA) ratios and methane production [4,5,13,23,24]. However, despite promising results, there are some discrepancies between in vitro and in vivo studies, and the standardization of methods (for determining the MICs of volatile substances) remains a methodological challenge [8]. Properly selected EO mixtures can have synergistic effects in reducing CH₄ emissions without significantly inhibiting fermentation [4]. Although recent studies indicate that 3-nitrooxypropanol (3-NOP) is an effective inhibitor of methane emissions in cows, the findings are somewhat controversial [25]. Natural compounds appear to be an attractive alternative to antibiotic growth promoters and coccidiostats such as monensin or 3-NOP because they have the ability to modulate rumen microbiota and inhibit methanogens, which translates into reduced methane emissions in in vitro models and some in vivo studies [2,26,27,28]. EO blends have also been reported to lower methane and ammonia formation in vitro and, in some cases, in vivo—supporting their potential as scalable “natural” methane-mitigation tools [29]. Despite numerous studies on the qualitative effects of essential oils on rumen fermentation, there remains a lack of systematic and quantitative data linking their chemical composition, strain-specific antibacterial activity (MIC), and effects on rumen fermentation parameters under standardized in vitro conditions. Therefore, the present study addresses this knowledge gap by integrating GC–MS analysis, MIC determination, and in vitro fermentation assays to provide a rational basis for the targeted use of essential oils in sustainable ruminant nutrition.

This study aimed to quantitatively determine the composition of 25 essential oils using gas chromatography coupled with mass spectrometry (GC-MS), to determine the minimum inhibitory concentrations (MICs) for selected microorganisms in the rumen of cows, and to confirm these effects on the formation of VFA profiles in in vitro tests. We hypothesize that selected essential oils have the potential to modulate the rumen ecosystem, which may be crucial in fermentation processes. This research is important for the development of sustainable feeding practices and reducing the environmental impact of ruminant farming by reducing methane emissions.

2. Materials and Methods

2.1. Chromatographic Analysis of Essential Oils

The essential oils used in this study were purchased from a reputable commercial supplier (Herbiness Sp. z o.o., Chomiec, Poland, and Avicenna Oil, Wrocław, Poland). The oils were selected from a group of the most widely available oils at relatively low prices and based on preliminary literature data concerning their actual biological activity. For analysis, samples were prepared by adding 20 µL of essential oil to 980 µL of dichloromethane (GC-MS grade, Sigma-Aldrich, Darmstadt, Germany), after which the solutions were transferred into chromatographic vials. Their chemical composition was analyzed using a gas chromatograph coupled with a mass spectrometer (GC-MS; Shimadzu GC-MS QP 2020, Shimadzu, Kyoto, Japan) and GC-MS Bruker Scion 8900 (Bruker Daltonics, Billerica, MA, USA). The volatile compounds were separated using a Zebron ZB-5 MSi capillary column (30 m × 0.25 mm × 0.25 µm; Phenomenex, Torrance, CA, USA).

The GC-MS operating conditions were set as follows: a mass spectrometer scan range of 50–350 m/z at a rate of 0.3 scans·s⁻¹, with helium as the carrier gas at a flow of 1.08 mL·min⁻¹ and a split ratio of 1:49. The oven temperature program started at 70 °C and increased to 200 °C at 5 °C·min⁻¹, and then increased to 280 °C at 20 °C·min⁻¹, followed by an isothermal hold for 3 min, resulting in a total run time of 34.0 min. A 1 µL aliquot of each sample was injected at 260 °C.

The identification of volatile compounds was based on the combined evaluation of several criteria applied during data analysis. Mass spectra obtained for each peak were compared with reference spectra from the NIST 23 (National Institute of Standards and Technology) and FFNSC (Flavors and Fragrances of Natural and Synthetic Compounds) libraries. For this comparison, we used the AMDIS (v. 2.73) and GCMS solution (v. 4.20; Shimadzu, Kyoto, Japan). In parallel, linear retention indices (LRIs) were calculated using a retention index calculator, with the values verified against those reported in the NIST 23 and FFNSC databases. The relevant spreadsheet is attached as Supplementary Table S1. Additionally, the retention times were compared with those of authentic reference standards to support compound identification. To facilitate a clear comparison of differences among essential oil chemotypes, four representative oils—Lavandula officinalis, Amyris balsamifera, Z. officinale, and Citrus sinensis—are presented in the main body of the manuscript in Section 3.1, as these oils were subsequently evaluated in vitro in rumen fluid.

2.2. Cultures of Rumen Microorganisms

The effects of EOs on the growth of ruminal bacterial strains were measured by inoculating a single colony from a solid medium into liquid Schaedler’s broth. The bacterial strains used in this study are listed in

Table 1. Bacterial strains used in the study.

Strain	Source
Butyrivibrio fibrisolvens 35459T	CCUG
Prevotella albensis 51935T	CCUG
Streptococcus bovis 2092	PCM
Lactobacillus delbrueckii lactis 2611	PCM

CCUG—Culture Collection University of Gothenburg; PCM—Polish Collection of Microorganisms.

. After 48 h of incubation under anaerobic conditions at 39 °C, the starter cultures were added to serial twofold dilutions of essential oils, ensuring that the final optical density corresponded to 0.5 on the McFarland scale, in accordance with CLSI guidelines [30]. The essential oils were prepared in DMSO (1:1) and added aseptically after autoclaving the medium to achieve final concentrations ranging from 25 to 6400 ppm. The 96-well plates were incubated for 24 h at 39 °C for Lactobacillus delbrucki lactis and Streptococcus bovis and for 48 h at 39 °C for Precotella albensis and Butyrivibrio fibrolesnsis, anaerobically, in a GasPak EZ anaerobe pouch system (BD). Bacterial growth was assessed by measuring the optical density at 650 nm [Spark, Tecan, Männedorf, Switzerland]. All the bacterial procedures were performed in a glove box (Coy Anaerobic Chamber) under anaerobic conditions (oxygen concentration below 0.5%). Using this approach, the MIC (minimum inhibitory concentration) and IC₅₀ (half maximal inhibitory concentration) values were determined. The inhibition percentage of each oil concentration was calculated in relation to the growth control. The MIC was considered the lowest concentration that inhibited 90% of the bacterial growth, while the IC₅₀ represents the EO concentration at which the optical density was half that measured in the absence of EO.

2.3. Animals and Rumen Fluid Collection

Rumen fluid was obtained via cannulas from non-lactating Polish Holstein–Friesian cows with a body weight of approximately 600 ± 30 kg. The animals received daily grass silage, hay, rapeseed meal, and a mineral–vitamin premix. Fresh water was available to the cows at all times. The cow cannulation was approved by the Local Ethics Committee on Animal Experiments (Protocol No. 053/2019). Rumen contents were collected through the fistulas of rumen-cannulated cows, with approximately 3 L obtained 3 h after the morning feeding. The fluid was immediately transferred to insulated thermoses maintained at an internal temperature of 39 °C. Before use, the rumen fluid was filtered through a triple layer of cheesecloth.

2.4. In Vitro Fermentation and Experimental Design

Fermentation trials were conducted using an Ankom RF gas production system (ANKOM Technology, Macedon, NY, USA). Each sample was homogenized and combined with a pre-warmed 39 °C buffer solution in a 1:4 ratio (75 mL of rumen fluid to 300 mL of buffer), after which the mixtures were placed into preheated 500 mL glass fermentation bottles. Each jar was loaded with Ankom bags containing 1 g of feed, and additional bags enriched with the selected essential oils were included in the experimental jars. The inoculum was flushed with carbon dioxide and transferred to a shaking water bath set at 39 °C. All incubation steps were conducted under strictly controlled anaerobic conditions at 39 °C to maintain consistent microbial activity.

The EOs used in the incubation trials were selected based on the results obtained in the preceding in vitro experiments on rumen microbial cultures described in Section 2.2. The fermentation assays included a control group (without essential oils) and treatments supplemented with selected EOs at defined volumes. The following essential oils were tested—ginger (Z. officinale), amyris (A. balsamifera), orange (C. sinensis), and lavender (L. officinalis)—in volumes of 10 and 20 μL. These oils were selected based on their different compositions and MIC values. Two control groups were created: CON (incubation of contents with buffer) and MON (positive control with the addition of 10 mg of monensin per bottle). Monenzine (purity > 98%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The dose of monensin for the positive control group was determined based on the literature [31]. The appropriate volume of each essential oil was added to the designated Ankom bags before incubation.

2.5. Sample Collection and Ammonia Analysis

The fluid collected after 4 h and 24 h of incubation was divided into 10 mL Falcon tubes (15 mL) and stabilized by adding 0.5 mL of concentrated sulfuric acid (H₂SO₄), which prevented the secondary fermentation and degradation of volatile acids. For the analysis, 1 mL of the acidified sample was centrifuged at 14,500× g for 4 min to remove particulate matter, and the resulting supernatant was diluted (1:9) with deionized water to fit the linear range of the photometer. The analysis was performed using a portable colorimeter (Hanna Instruments, Smithfield, RI, USA, model HI-97733) and dedicated reagents according to the manufacturer’s protocol based on the Nessler method ASTM D1426-92 [32]. The ammonia concentration was automatically calculated by the device using its built-in calibration curve.

2.6. Volatile Fatty Acid Analysis

For VFA analysis, 1 mL of each sample from incubation trials was taken and transferred to Eppendorf tubes (2 mL). Then, an internal standard—heptanoic acid (C₇) in an amount corresponding to 0.5 mg per sample—was added. A 0.8 mL volume of diethyl ether was added to each sample, after which the contents were mixed vigorously on a vortex mixer for 30 s and centrifuged for 4 min at 14,000 rpm in a tabletop centrifuge. After centrifugation, the organic phase was separated and purified by filtration on a layer of celite. The remaining aqueous phase was re-extracted with 0.8 mL of diethyl ether. After re-mixing (30 s, vortex) and centrifugation (4 min, 14,000 rpm), the extract was filtered in the same way. The combined ether extracts were transferred to chromatography vials and analyzed by gas chromatography coupled with mass spectrometry (GC-MS).

Volatile fatty acids were analyzed using the same GC-MS system as described for the essential oil analyses (Shimadzu GCMS QP 2020, Shimadzu, Kyoto, Japan). Separation was carried out on a Zebron ZB-FAME capillary column (60 m × 0.25 mm × 0.20 μm; Phenomenex, Torrance, CA, USA). The oven temperature program started at 80 °C with a 1 min hold, followed by an increase at 7 °C·min⁻¹ to a final temperature of 200 °C with a 2 min hold. The mass spectrometer scan range was 42–300 m/z, the rate was 1 scans·s⁻¹, and helium was the carrier gas at a flow of 1.80 mL·min⁻¹. The injector temperature was set at 260 °C, and the split ratio was 50. Individual VFAs were quantified using calibration curves prepared from authentic reference standards.

2.7. Statistical Analysis

Statistical analysis was conducted using the R environment (version 2025.09.2+418, RStudio, Boston, MA, USA, 2025) and the packages dplyr, tidyr, car, DescTools, rstatix, openxlsx, and ggplot2. Prior to the main analysis of variance (ANOVA), key assumptions were verified using the residuals from the full factorial model. The normality of residuals was tested using the Shapiro–Wilk test. The homogeneity of the variances across all the treatment–time interaction groups was checked using Levene’s test (centered at the mean). For each in vitro fermentation parameter, a two-way analysis of variance (two-way ANOVA) was performed using the type III sum of squares method. A conditional post hoc analysis was subsequently applied to each time point based on the outcome of the Levene’s test: Dunnett’s test or Student’s t-tests with Welch’s correction. Statistical significance was declared at p < 0.05. When applicable, significance levels are reported as p < 0.05 (a), p < 0.01 (b), and p < 0.001 (c).

3. Results

3.1. Chromatographic Analysis of Essential Oils

In the present study, 25 essential oils representing diverse botanical origins and distinct phytochemical profiles were subjected to GC–MS chromatographic analysis. In total, nearly 150 individual volatile compounds were identified across the analyzed samples, encompassing a broad range of chemical classes, including monoterpene hydrocarbons, oxygenated monoterpenoids, sesquiterpenes, esters, aromatic aldehydes, and phenylpropanoids. The resulting chromatographic profiles exhibited substantial heterogeneity: some essential oils were characterized by only a few dominant constituents, whereas others displayed high compositional complexity, comprising several dozen compounds present at varying relative abundances. Comprehensive analytical data, including retention index values, peak identification, and the relative percentage contribution of individual components, are provided in Supplementary Table S1.

The essential oil of L. officinalis was characterized by a profile clearly dominated by linalyl acetate (39.11%) and linalool (36.60%), which together accounted for more than 75% of the total volatile fraction. In addition, smaller amounts of terpinen-4-ol (4.83%), lavandulyl acetate (3.81%), and β-cis-ocimene (3.81%) were detected. This composition is indicative of a classical lavender chemotype, in which the predominance of monoterpene esters and alcohols determines both the aromatic properties and the biological activity of the oil.

In contrast, the essential oil of A. balsamifera exhibited a markedly different chemical profile, with a pronounced dominance of oxygenated sesquiterpenes. The major constituents were valerianol (38.43%), 7-epi-α-eudesmol (14.52%), epi-γ-eudesmol (11.06%), and elemol (10.89%). In addition, moderate levels of γ-eudesmol (5.84%) and β-sesquiphellandrene (4.98%) were identified. The prevalence of oxygenated sesquiterpenes clearly distinguishes A. balsamifera oil from monoterpene-rich samples and reflects its comparatively heavier terpenoid profile.

The essential oil of Z. officinale also displayed a complex aromatic composition, dominated primarily by sesquiterpenes, including α-zingiberene (36.67%), β-sesquiphellandrene (13.79%), α-curcumene (9.77%), and β-bisabolene (7.85%). Among monoterpenes, camphene represented the most abundant component (7.31%). Aldehydic compounds such as geranial (2.73%) and neral (2.27%) were also present, further underscoring the phytochemical complexity of this oil.

Finally, the essential oil of C. sinensis, listed as the last entry in

Table 2. Chemical composition of selected essential oils determined by GC–MS, expressed as relative peak area (%).

No.	Peak Name	RI Exp. 1	RI Lit. 2	Lavandula officinalis	Amyris balsamifera	Zingiber officinale	Citrus sinensis
				Area (%) 3
1	α-Pinene	932	937	1.47	-	-	0.87
2	Camphene	958	952	-	-	7.31	-
3	Sabinene	971	974	-	-	-	0.51
4	β-Myrcene	990	991	1.43	-	0.55	2.14
5	Octanal	1006	1003	-	-	-	0.23
6	3-Carene	1012	1011	-	-	-	0.24
7	Limonene	1034	1030	-	-	-	95.41
8	β-Phellandrene	1035	1031	-	-	4.78	-
9	Eucalyptol	1036	1032	-	-	3.15	-
10	β-cis-Ocimene	1038	1038	3.81	-	-	-
11	trans-β-Ocimene	1049	1049	1.49	-	-	-
12	Linalool	1103	1099	36.60	-	-	0.35
13	Lavandulol	1166	1170	1.09	-	-	-
14	Borneol	1178	1172	2.42	-	0.84	-
15	Terpinen-4-ol	1183	1177	4.83	-	-	-
16	α-Terpineol	1199	1198	-	-	0.48	-
17	Decanal	1209	1206	-	-	-	0.25
18	Neral	1240	1240	-	-	2.27	-
19	Linalyl acetate	1255	1257	39.11	-	-	-
20	Geranial	1269	1270	-	-	2.73	-
21	Lavandulol acetate	1288	1290	3.81	-	-	-
22	Neryl acetate	1358	1364	1.62	-	-	-
23	Copaene	1376	1375	-	-	0.48	-
24	Geranyl acetate	1381	1382	-	-	0.46	-
25	β-Elemene	1397	1390	-	-	0.76	-
26	(E)-β-Farnesene	1455	1457	2.33	-	0.49	-
27	α-Neocallitropsene	1469	1480	-	1.17		-
28	α-Curcumene	1482	1480	-	1.2	9.77	-
29	γ-Curcumene	1488	1482	-	1.03		-
30	Valencene	1490	1491	-	-	0.46	-
31	cis-β-Guaiene	1493	1498	-	0.34		-
32	α-Zingiberene	1501	1499	-	3.57	36.67	-
33	α-Selinene	1502	1501	-	-	1.24	-
34	α-Farnesene	1504	1508	-	-	3.08	-
35	β-Bisabolene	1510	1509	-	0.55	7.85	-
36	β-Dihydroagarofuran	1512	1510	-	1.5	-	-
37	γ-Cadinene	1519	1520	-	-	2.41	-
38	β-Sesquiphellandrene	1530	1523	-	4.98	13.79	-
39	Selina-4(15),7(11)-diene	1541	1540	-	0.39	-	-
40	Selina-3,7(11)-diene	1543	1546	-	2.09	-	-
41	α-Elemol	1558	1547	-	10.89	-	-
42	Germacrene B	1559	1557	-	-	0.43	-
43	Rosifoliol	1614	1609	-	0.53	-	-
44	5-epi-7-epi-α-Eudesmol	1617	1610	-	0.83	-	-
45	epi-γ-Eudesmol	1623	1624	-	11.06	-	-
46	Eremoligenol	1624	1627	-	1.08	-	-
47	γ-Eudesmol	1643	1632	-	5.84	-	-
48	Valerianol	1655	1657	-	38.43	-	-
49	7-epi-α-Eudesmol	1670	1666	-	14.52	-	-

¹ Experimentally calculated linear retention index. ² Literature-derived retention index according to the NIST 23 Mass Spectral Library. ³ Calculated by peak area normalization.

, was distinguished from the remaining samples by an exceptionally high limonene content (95.41%), which overwhelmingly dominated its chromatographic profile.

3.2. Effects of Essential Oils on Cultures of Rumen Microorganisms

In order to apply the optimal concentration of plant-derived EOs, the inhibition of the growth of Butyrovibrio fibrosolvens, Prevotella albensis, Lactobacillus delbrueckii ssp. Lactis and Streptoccoccus bovis was observed and is presented in

Table 3. Inhibition effects of EOs on rumen microorganisms. MIC: the lowest concentration that inhibited 90% of the bacterial growth. IC₅₀ is the concentration of essential oils that led to a 50% decrease in cell density at 24 h of incubation. The results shown are means of three culture tubes at each concentration.

	Strain
Essential Oil	Butyrovibrio fibrosolvens		Prevotella albensis		Lactobacillus delbrueckii ssp. Lsactis		Streptoccoccus bovis
	MIC (ppm)	IC50 (ppm)	MIC (ppm)	IC50 (ppm)	MIC (ppm)	IC50 (ppm)	MIC (ppm)	IC50 (ppm)
Lavandula officinalis	1600	1200	1600	800	1600	50	1600	800
Amyris balsamifera	200	150	200	50	200	50	1600	100
Thuja occidentalis	800	400	800	400	1600	800	1600	400
Matricaria chamomilla	400	200	800	600	3200	2400	3200	200
Acorus calamus	200	150	3200	800	1600	800	3200	800
Anethum graveolens	400	300	800	200	1600	1200	1600	800
Helichrysum italicum	3200	400	3200	300	3200	2400	3200	2400
Rosmarinus officinalis	1600	800	1600	800	1600	100	3200	50
Cannabis indica	4000	1300	3200	800	1600	800	1600	800
Mentha piperita	1600	800	3200	1200	3200	600	3200	800
Litsea cubeba	3200	1100	3200	1200	3200	800	3200	1600
Humulus lupulus	200	100	100	no	3200	800	3200	400
Zingiber officinale	3200	1600	1600	1000	800	400	6400	1600
Citrus limon	1600	400	1600	400	400	200	no	no
Citrus sinensis	1600	800	1600	400	1600	200	3200	1600
Citrus reticulata	400	200	200	150	400	200	1600	800
Limonka Citrus	800	200	200	100	200	100	1600	800
Piper nigrum	1600	200	800	200	1600	200	1600	400
Origanum majorana	1600	800	1600	100	800	400	1600	800
Melissa officinalis	400	100	200	25	400	200	800	400
Cannabis sativa	3200	400	1600	200	1600	200	1600	800
Cannabis indica (1)	400	100	400	100	400	100	800	400
Commiphora myrrha	1600	200	1600	400	400	100	3200	1600
Valeriana officinalis	1600	400	1600	400	1600	200	3200	1600
Cymbopogon citratus	200	50	100	50	1600	400	200	100

MIC (minimum inhibitory concentration); IC₅₀ (half maximal inhibitory concentration).

. The analysis included 25 distinct essential oils tested at 14 concentrations, ranging from 25 to 4000 parts per million (ppm). The use of higher concentrations would not be commercially or applicationally justified. An inhibitory effect for B. fibrosolvens was observed, with an MIC of 200 ppm for A. balsamifera, Acorus calamus, Humulus lupulus, and Cymbopogon citratus. For this strain, the weakest inhibitory effect was found for Helichrysum italicum, Cannabis indica, Z. officinale, Litsea cubeba, and Canabis sativa (3200, 4000, 3200, 3200, and 3200 ppm, respectively). Similarly to the fibrolytic B. fibrisolvens, the amylolytic P. albensis showed high sensitivity to A. balsamifera (200 ppm), H. lupulus (100 ppm), and C. citratus (100 ppm), as well as to citrus essential oils such as Citrus reticulata (200 ppm), Citrus limon (200 ppm), and Melissa officinalis (200 ppm). This strain showed the weakest sensitivity (3200 ppm) for A. calamus, H. italicum, C. indica, Mentha piperita, and L. cubeba. Interestingly, the same MIC values were observed for all strains when treated with L. officinalis and A. balsamifera. The least sensitive strain proved to be S. bovis. As many as 11 of the tested EOs (Matricaria chamomilla, A. calamus, H. italicum, R. officinalis, M. piperita, L. cubeba, H. lupulus, Z. officinale, and C. sinensis) showed inhibitory concentrations of 3200 ppm or higher. S. bovis was most sensitive to C. citratus (200 ppm). For Lactobacillus delbrueckii ssp. lactis, the highest inhibitory concentrations were recorded for essential oils from M.chamomilla, H. italicum, M. piperita, and L. cubeba (3200 ppm), while the lowest inhibitory concentrations were observed for A. balsamifera and C. limon (200 ppm).

An increase in the optical density of the tested strains was observed under anaerobic conditions compared to the control without essential oils. The heatmap (Figure 1) illustrates the percentage change in the growth of four bacterial strains relative to the untreated control. Prevotella albensis exhibited moderate sensitivity to the essential oils tested. Strong inhibitory activity at concentrations of 800 ppm (Figure 1A, dark blue) was observed for oils such as Commiphora myrrha and Origanum majorana. A similar sensitivity pattern was observed for Butyrivibrio fibrisolvens. Among the examined bacteria, Lactobacillus delbrueckii proved to be the most resistant to inhibition by essential oils. In contrast, Streptococcus bovis displayed the highest overall sensitivity. Nearly all the tested oils had minimum inhibitory concentrations (MICs) of 800 ppm or above (Table 3, Figure 1D, dark blue). Several oils, particularly those belonging to the Cannabis and Citrus groups, exhibited strong inhibitory effects at much lower concentrations, beginning at approximately 150–200 ppm. Based on the culture results obtained for individual strains, four essential oils were selected for in vivo testing in the artificial rumen. Selection was based on their potential to promote the growth of fibrolytic and amylolytic bacteria, or their lack of effect on these groups, and their commercially applicable concentrations.

Table 4. Effects of Zingiber officinale, Amyris balsamifera, Citrus sinensis, and Lavandula officinalis essential oils on in vitro fermentation characteristics.

Time	Treatments										SEM	p-Value
	CON	MON	Zingiber officinale		Amyris balsamifera		Citrus sinensis		Lavandula officinalis
		10 mg	25 ppm	50 ppm	25 ppm	50 ppm 20 μL	25 ppm 10 μL	50 ppm 20 μL	25 ppm 10 μL	50 ppm 20 μL
pH
4 h	6.84	6.68 **	6.81	6.73	6.85	6.67 **	6.71 *	6.76	6.88	6.96	0.15	0.08
24 h	6.57	6.59	6.60	6.66	6.66	6.71 **	6.57	6.58	6.59	6.58	0.19	0.12
NH3-N, mg × L−1
4 h	166.6	159.6	147.6	145.3	175.0	120.0 *	157.0	163.0	129.3	157.3	5.82	0.42
24 h	147.6	142.0	142.6	165.3	180.3	153.0	184.6	147.0	206.6 **	222.3 ***	14.52	0.37
NH3, mg × L −1
4 h	202.6	208.0	175.6	174.6	198.0	150.6 **	190.6	198.3	159.0	192.3	14.23	0.21
24 h	179.6	171.0	164.0	206.0	205.6	186.0	219.3	181.3	261.0 **	271.3 ***	7.43	0.37
NH4+, mg × L −1
4 h	214.3	213.3	184.3	180.6	217.6	149.3 *	201.6	210	164.6	205.6	11.2	0.24
24 h	190	188.0	174.0	210.6	225.3	193.3	236.0	188.6	270.3 **	286.0 ***	8.46	0.33
Total VFA, mg × mL −1
4 h	1.14	1.16	1.49 ***	1.41 ***	1.53 *	1.36 ***	1.16	1.21	1.14	1.29	0.09	0.08
24 h	1.30	1.46	1.60 ***	1.59 ***	1.66 ***	1.67 ***	1.12	1.16	1.01 ***	0.99 ***	0.14	0.12
Individual VFA, mg × mL −1
Acetate
4 h	0.52	0.53	0.79 ***	0.73 ***	0.82 ***	0.68 ***	0.52	0.55	0.55	0.63	0.11	0.12
24 h	0.59	0.66	0.85 ***	0.87 ***	0.89 ***	0.91 ***	0.47	0.50	0.43 **	0.41 ***	0.19	0.18
Propionate
4 h	0.35	0.36	0.42 ***	0.41 ***	0.43	0.40 ***	0.36	0.37	0.33	0.37	0.09	0.08
24 h	0.42	0.49	0.46 ***	0.43 ***	0.47 ***	0.46 ***	0.35	0.36	0.32 ***	0.32 ***	0.13	0.06
Butyrate
4 h	0.27	0.27	0.28	0.27	0.28	0.26	0.28	0.29	0.26	0.29	0.20	0.13
24 h	0.29	0.31	0.29	0.29	0.30	0.30	0.30	0.30	0.26	0.26	0.34	0.18
Acetate to propionate ratio
4 h	1.43	1.47	1.84 ***	1.78 ***	1.73 ***	1.80 ***	1.44	1.49	1.36	1.48	0.15	0.07
24 h	1.50	1.34 ***	1.84 ***	1.78 ***	1.95 ***	1.88 ***	1.34 **	1.38	1.34 **	1.28 ***	0.24	0.11
Methane, mL × L −1
4 h	3.68	2.40	2.63	2.60	2.50	2.29	3.05	3.08	2.25	2.90	0.23	0.56
24 h	18.08	12.50 ***	17.60	15.6 ***	15.17 ***	13.66 ***	17.26	13.2 ***	14.22 ***	13.60 ***	0.34	0.89

Values represent means; differences between treatment groups and control group (p < 0.05 *, p < 0.01 **, p < 0.001 ***).

presents the effects of the additives on the rumen pH and NH₃-N, NH₃/NH₄⁺, and VFA concentrations. A statistically significant decrease in pH was observed after 4 h for monensin (p < 0.03) and for the application of 50 ppm of A. balsamifera essential oil (EO) after 4 h (p < 0.001), persisting up to 24 h. A 25 ppm concentration of C. sinensis reduced the pH up to 4 h post-application (p < 0.03). A significant decrease in NH₃-N was observed after 4 h for 50 ppm of A. balsamifera EO (p < 0.03), which returned to a level close to the control after 24 h. Conversely, after 24 h, the amount of ammoniacal nitrogen increased for the L. officinalis essential oil used (at both concentrations) (p < 0.01). Similar dependencies were observed for all the analyzed protein metabolism parameters in the fermentation chamber. A decrease was observed for 50 ppm of A. balsamifera (NH₃ p < 0.04; NH₄+ p < 0.01) and an increase for both concentrations of L. officinalis (25 ppmL: NH₃ p < 0.01; NH₄+ p < 0.01; 20 μL: NH₃ p < 0.01; NH₄+ p < 0.01). A statistically significant decrease in methane production compared to the control was observed at both tested concentrations for ginger essential oil and A. balsamifera essential oil (p < 0.01).

The total VFA increased after the application of all essential oils, although these changes were not statistically significant. The EOs from Z. officinale and A. balsamifera induced an increase in total VFAs as early as 4 h post-administration, and this effect persisted up to 24 h (p < 0.01). The rise in total VFAs resulted from increased acetate and propionate concentrations, as a significant elevation of both fatty acids is observed after applying A. balsamifera and Z. officinale at both tested concentrations (p < 0.01). No significant changes in butyrate levels are noted. In contrast, lavender essential oil led to a decrease in acetate and propionate 24 h after administration, accompanied by a reduction in total VFAs at the same time point.

4. Discussion

A detailed chemical characterization was conducted to determine the qualitative and quantitative composition of the volatile fractions, which may underlie the observed biological effects. The potential of EOs as microbiome regulators results from their broad spectrum of action on various types of microorganisms, not only pathogens but also the microflora of the digestive tract [24]. The antimicrobial activity of essential oils is strongly dependent on their chemical composition. Owing to the complexity of these mixtures, it is not possible to unambiguously attribute the observed biological effects to individual constituents; instead, synergistic interactions among multiple compounds are suggested, as well as the simultaneous modulation of ruminal bacterial cell membranes by several components acting in concert [33]. The present study indicates that selected essential oils have the potential to modulate rumen microbial populations rather than acting solely as broad-spectrum inhibitors. The determination of MIC and IC₅₀ values allowed the identification of concentration ranges that may influence microbial activity without severely disrupting the rumen ecosystem. Some essential oils may transiently stimulate microbial growth or metabolic activity (Figure 1). However, given that microbial growth was assessed using optical density rather than viable cell counts, this effect should be interpreted with caution and may reflect increased metabolic activity rather than enhanced bacterial survival. This selective mode of action has also been reported by other authors, who demonstrated that essential oils preferentially affect hyper-ammonia-producing bacteria and methanogenic archaea while sparing fibrolytic populations critical for fiber degradation [4,13].

Nevertheless, our team’s observations indicate that certain essential oils, such as ginger and A. balsamifera, have the potential to modulate in vitro fermentation processes. Some studies emphasize that it is necessary to evaluate more essential oils in order to determine their optimal dosage [20]. Our own research corresponds with these postulates. In vitro reports indicate that the activity of EOs arises primarily from the presence of terpenes (e.g., limonene, menthol, 1,8-cineole), phenolic terpenoids (thymol, carvacrol), aldehydes (citral), and sesquiterpenes (β-caryophyllene, humulene) [1,10,13,24]. These compounds may inhibit the growth of proteolytic and deaminating bacteria, modulate cellulolytic bacterial populations, and consequently alter volatile fatty acid (VFA) production while reducing methanogenesis [4,15,17,22,24]. Recent studies confirm that essential oils such as rosemary, peppermint, ginger, lemon, calamus, and lemon balm exhibit strong activity against both Gram-positive and Gram-negative bacteria, whereas hop and hemp oils additionally reduce the abundance of certain methanogenic archaea, thereby decreasing methane emissions under in vitro conditions [34,35]. Earlier studies conducted by our group on the application of C. sativa and C. indica indicated that these oils exerted a greater effect on Lactobacillus spp. and Butyrivibrio spp. than monensin did. Moreover, the application of cannabis-derived EOs resulted in increased total VFA concentrations, particularly of acetate and propionate, after 6 h of incubation [36].

In a study by Joch et al., a series of EOs with comparable compositions (eugenol, carvacrol, citral, limonene, 1,4-cineole, p-cymene, linalool, bornyl acetate, α-pinene, and β-pinene) were evaluated in vitro. Among these compounds, only limonene, 1,4-cineole, bornyl acetate, and α-pinene did not inhibit VFA production, while only bornyl acetate reduced methane formation [37]. It should be noted, however, that a very high dose was applied (1000 μL × L⁻¹ of incubated rumen fluid).

The observed effects of essential oils in this study support the concept that their activity in the rumen is primarily dose-dependent and selective rather than broadly antimicrobial. Similar dose-dependent and selective effects of essential oils on rumen microorganisms have been widely reported, whereas higher doses may suppress overall fermentation activity [4,22,24]. The determination of MIC and IC₅₀ values enabled the identification of concentration ranges that may modulate microbial activity without causing extensive disruption of the rumen ecosystem. Such an approach is consistent with in vitro screening strategies recommended for essential oils, which emphasize the importance of defining sub-inhibitory concentrations to avoid detrimental effects on rumen fermentation and microbial efficiency [4,8]. In lavender oil, the dominance of two compounds (linalyl acetate and linalool) was associated with a reduction in total VFA production in vitro, accompanied by lower acetate and propionate concentrations. The greatest increase in total VFA was observed following the application of A. balsamifera, at both lower and higher doses, with a marked increase in acetate and propionate. A similar, albeit slightly weaker, effect was observed for Z. officinale. Consistent with previous reports, p-coumaric and ferulic acids have also been shown to effectively modulate ruminal fermentation by influencing nitrogen metabolism and the relative proportions of individual VFAs [38,39].

Some essential oils may adversely affect the stability of the rumen microbiota, leading to disturbances in fermentation processes, reduced VFA production, and, consequently, decreased milk yield [13]. In addition, the intense or organoleptically unacceptable sensory characteristics of certain EOs may limit feed intake, resulting in reduced energy and nutrient consumption and, ultimately, lower milk production [40]. It has been demonstrated that citronella, clove, and anise oils reduce methane production but simultaneously exert negative effects on ruminal microbial fermentation. Similar observations were made in the present study with respect to lavender oil [41].

Reducing enteric methane emissions in ruminants requires the strategic application of validated interventions, among which dietary management has received the greatest attention. The effectiveness of additives such as garlic, tannins, saponins, and essential oils largely depends on their matrix, dosage, ruminant species, and overall diet composition [28,42,43]. Recent research increasingly focuses on the targeted manipulation of the rumen microbiota to enhance fermentation efficiency and reduce energy losses in the form of methane. Castañeda-Correa et al. demonstrated that thymol and carvacrol, applied individually or in combination, can significantly modulate rumen microbial diversity, affecting fermentation kinetics and VFA profiles [33]. More recent microbiome studies based on metagenomic and metabolomic approaches further support the multidirectional effects of these compounds [44,45].

An important finding of the present study was the consistent reduction in CH₄ production following in vitro incubation with all four essential oils, particularly at the higher dose. The magnitude of methane reduction was dose-dependent. Citrus sinensis oil at higher doses effectively reduced methane emissions despite not exerting a beneficial effect on individual VFA production. In contrast, A. balsamifera and L. officinalis were effective in reducing methane production, regardless of the dose. Notably, A. balsamifera limited undesirable fermentative processes (methanogenesis) while simultaneously optimizing beneficial energy-related processes by increasing VFA production. Our results suggest synergistic interactions among the major constituents of A. balsamifera (valerianol, 7-epi-α-eudesmol, epi-γ-eudesmol, and elemol). Similar relationships have been reported in studies demonstrating synergistic anti-methanogenic mechanisms following the in vitro application of garlic derivatives and rumen buffers, resulting in substantial CH₄ reductions and improved VFA profiles [2]. In this context, in vivo evidence indicates that essential oils such as ginger and rosemary can be incorporated into dairy cow diets without compromising productivity while still modulating ruminal fermentation patterns associated with methane mitigation [20]. These findings highlight the high efficacy of natural compounds, including EO blends, in hydrogen sequestration within the rumen. Although the methane-mitigating feed additive Bovaer^® (3-nitrooxypropanol; 3-NOP) is well supported mechanistically and can deliver substantial reductions in enteric CH₄ under controlled conditions [25], its real-world rollout has recently faced acceptance and implementation headwinds. In some markets, consumer backlash and online misinformation have created reputational pressure, while farm-level concerns and policy/operational uncertainty have contributed to cases where parts of the sector have paused trials or stepped back from routine use. Against this background, the search for natural, diet-compatible alternatives or complements is scientifically justified—especially compounds that can be integrated into existing feeding strategies while maintaining animal performance and product quality. One promising direction involves phytogenic preparations, including essential oils (EOs), which can act as rumen microbiome modulators [33,34,37,40].

Previous studies also indicate that phenolic compounds may inhibit methanogenesis by modifying hydrogen utilization by rumen microorganisms and promoting propionate formation at the expense of acetate [39,43]. Increased propionate production relative to acetate may be associated with higher CO₂ production in the rumen [46]. Numerous studies have reported effective methane mitigation following the application of natural compounds [42]. For example, cashew nut shell extract resulted in lower methane production compared with a monensin-treated control group [47]. In the present study, monensin exhibited the greatest overall efficacy. Finally, it should be noted that discrepancies between in vitro and in vivo studies may also arise from differences in retention time, which is typically longer in vitro than the residence time of the same feed or supplement in the rumen of a cow [48].

The application of essential oils aligns with the global trend toward natural and sustainable strategies to improve the efficiency of animal production. The observed relationships between the chemical composition of essential oils and their antimicrobial activity suggest that these substances may serve as promising natural modulators of ruminal fermentation. Future studies will help determine the feasibility of dietary supplementation with the EOs investigated here in the dairy sector, which should ultimately confer benefits first to the animals and subsequently to consumers.

5. Conclusions

Rumen bacteria’s sensitivity to essential oils varies by strain, with some, such as B. fibrosolvens and P. albensis, inhibited at low concentrations, while others, including S. bovis and L. delbrueckii ssp. lactis, require higher doses after application of a particular EO. These observations highlight the potential for targeted modulation of the rumen microbiota. Certain essential oils, including A. balsamifera and Z. officinale, exhibited strong inhibitory effects at low concentrations (25 ppm) while simultaneously enhancing VFA production under in vitro conditions. Application of A. balsamifera and Z. officinale resulted in significant increases in acetate and propionate, whereas L. officinalis inhibited these VFAs. Amyris balsamifera and L. officinalis essential oils showed the highest potential for methane reduction. These findings suggest that appropriately selected essential oils can modulate both the composition and activity of the rumen microbiota, influencing key metabolic parameters and fermentation processes. The data obtained may contribute to the rationalization of the use of essential oils in ruminant nutrition, while improving animal health and fermentation efficiency. The results obtained in this study will enable the future composition of mixtures that modulate fermentation processes in the rumen with concentrations of active substances in formulations that have been pre-selected on the basis of the MIC values determined. In addition, the MIC values we have determined for Butyrivibrio fibrosolvens will enable the selection of EO mixtures that will have a potentially beneficial effect not only on milk yield but also on the functional characteristics of cow’s milk.