Lysinibacillus fusiformis: a novel fibrolytic native strain from the rumen microbiome that increases in vitro digestibility of central agricultural residues

ABSTRACT

Increasing the digestibility of agricultural residues is one goal of sustainable livestock production, and the role of rumen microbiome-feed interaction is critical for efficient digestion. This study was aimed at isolating and evaluating in vitro the effect on dry-matter digestibility (IVDMD), relative feed values (RFV), volatile fatty acid (VFA) synthesis, pH changes, and gas production (GP) by the inclusion of native rumen strains in mixtures of corn, sorghum, and oat agricultural residues with ruminal fluid. The results show that an inoculum of 1 × 10¹⁰ colony-forming unit per millilitre (CFU/mL) of the aero-tolerant native rumen strain Lysinibacillus fusiformis increases the IVDMD, RFV, and VFA values of all mixtures. During agricultural residue fermentation, the pH was stable, and propionic acid was the primary fatty acid synthesized, indicating increased energy availability for efficient cattle growth performance while limiting molecular hydrogen (H₂) synthesis for conversion to methane (CH₄). These results suggest that L. fusiformis could be used as a direct-feed microbial to promote sustainable livestock production. To the best of our knowledge, this is the first report to link the fermentation of fibrous agricultural residues in ruminal fluid inoculated with a fibrolytic native strain and digestibility in favour of increasing the efficiency of livestock production.

KEYWORDS:

• Direct-fed microbial
• crop residues
• effective utilization
• cattle nutrition
• sustainable livestock production

Introduction

The major obstacle to efficient livestock production is their adequate nutrition or feeding (Mahesh and Mohini 2014). When cattle feed on low-quality feed, such as some central agricultural residues, the consequences can be low feed intake and poor performance (Mahesh and Mohini 2014; Yanti and Yayota 2017). Central agricultural residues, such as corn, oats, and sorghum, are characterized by low-protein and high-fibre content, representing a significant source of potential energy for ruminants (Mahesh and Mohini 2014; Kılıc and Gulecyuz 2017). Nevertheless, the efficient digestion of fibre polysaccharides cellulose and hemicellulose is limited by lignin content because it hinders the efficacy of the fibrolytic enzymes from native microbiota (Kılıc and Gulecyuz 2017). Despite this obstacle, agricultural residues constitute an important feed for ruminants during the long dry season. As a matter of fact, they are the main feed source in developing countries due to their high accessibility and low cost (Kılıc and Gulecyuz 2017; Yanti and Yayota 2017). Various methods have been suggested to enhance the digestibility of agricultural waste, such as physical, chemical, and biological treatments. Among them, a biological treatment that involves the use of silage and exogenous fibrolytic enzymes has shown promising outcomes (Yanti and Yayota 2017; Kılıc and Gulecyuz 2017). However, if silage is not made correctly, it will not be accepted by animals, and wastage will be high. Adding exogenous fibrolytic enzymes to ruminant diets can potentially enhance the digestion of plant cell walls, leading to improved feed efficiency (Gado et al. 2009; Salem et al. 2013; Kondratovich et al. 2019). However, the results are inconsistent because the enzyme activity depends on several factors, such as the origin, the proper mixture of fibrolytic enzymes cellulose and xylanase according to the diet composition, livestock race, physicochemical properties of the rumen among others (Tirado-González et al. 2018). Successful results have been obtained using a mixture of fibrotic enzymes obtained from anaerobic ruminal bacteria consisting of cellulose, xylanase, α-amylase, and protease (called ZADO®) in a proportion of 7.1, 2.3, 61.5, and 29.2 units/g respectively (Gado et al. 2009).

In recent years, feed additives, including antimicrobial growth promoters, prebiotics, and probiotics, have been used as supplements to animal diets to improve animal production and health (Ban and Guan 2021). These results and conclusions indicate that modifying the rumen microbiome composition could significantly impact the host animal's fermentation, physiology, and productivity. In this context, several studies have been developed using selected direct-fed microbials (DFM) (Elghandour et al. 2015). However, the response to DFM is not constant, and the outcomes have been fleeting or unsuccessful. According to Weimer et al. (2017), the use of DFM only results in a temporary increase in the microbiome, which may be due to microbial instability in the ruminal system. Hence, using native strains like DFM can help mitigate this issue (Seo et al. 2010).

Previous works, including some of our own, have shown that natural fermentation could be directed to increase the process yield or provide specific characteristics to the final product by incorporating selected native strains. It has also been displayed that the success of the process is based on the natural adaptation of the microorganisms in system fermentation, including substrate characteristics and interactions with other native strains (Navarrete-Bolaños 2012; Navarrete-Bolaños and Serrato-Joya 2023). Therefore, increasing the composition of the rumen microbiome by adding fibrolytic native strains could increase agricultural residue digestibility and evolve from low to high livestock production with sustainability features. However, to our knowledge, strategies to modify the native microbial population by incorporating selected fibrolytic native strains have not succeeded, despite the vast number of references that mention the existence of three fibrolytic rumen native strains that are highly active, identified as Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens (Seo et al. 2010; Henderson et al. 2015; Yeoman et al. 2021). Perhaps because they are obligately anaerobic microorganisms, and the massive biomass production in free oxygen environments is not easy to perform, as well as any subsequent conditioning process to prolong the shelf life to supply as a feed supplement. Although some representative ecological and functional properties of the cultured fibrolytic strains have been demonstrated, the molecular studies have revealed that currently recognized fibrolytic species represent only a small proportion of the total fibrolytic population (Koike and Kobayashi 2009). Hence, the microbial ecology studies and rumen fibrolytic strain function should be extended.

Based on the above, in this research, rumen microbial ecology was studied to select fibrolytic native strains to increase the digestibility of the agricultural residues from corn, sorghum, and oats, the central cultures in Mexico and other parts of the world which have a low nutritional value due to their high fibre content. The goal is to find fibrolytic native strains that can be supplied as a supplement along with cattle feeds to enhance feed digestibility and increase the efficiency of livestock production.

Materials and methods

Raw materials. Dry corn, sorghum, and oat agricultural residues were obtained from three locations near Celaya, Guanajuato, Mexico (100° 48′ 55″ W, 20° 31′ 40″ N). The samples were milled in a hammer mill and screened using a set of 8,10, and 12 mesh sieves to obtain a particle size of 2 mm. These samples were used for the digestibility assays.

Ruminal fluid. Two ruminal samples were collected to obtain ruminal fluid: one was collected directly from fistulated livestock housed at the National Center for Disciplinary Research in Animal Physiology and Improvement in Queretaro City, Mx. The other was collected directly from the stomachs of cows slaughtered in the local slaughterhouse in Celaya Gto. Mx. Each sample was homogenized and squeezed, and the liquid was collected in an anaerobic environment using an anaerobic chamber (model 818-GB, Plas-Labs, Lansing, MI, USA).

Native strain isolation. Aliquots from each collected ruminal fluid were added into Petri dishes containing the culture medium proposed by Bryant and Burkey (1953) for cellulolytic bacteria isolation and physiological studies, changing the primary substrate according to the strains to be isolated (Table 1). In all inoculated Petri dishes, classic plate − casting processes were used for inoculation according to Stanier et al. (1986), and incubated at 39 (±1)°C for 24, 48, and 72 h in an anaerobic incubator (CO₂ incubator, model NU-5510, Nuaire, Plymouth, MN, USA). Colonies developed with different morphologies were collected, transferred to new Petri dishes containing fresh agar media, and incubated again. The procedure was repeated until pure cultures were obtained. To assure the purity of the isolated colonies, they were analysed by microscope (DMRAX2, Leica Microsystems, Wetzlar, Germany) using staining techniques.

Table 1. Culture media composition used for culture and isolation of rumen bacteria.

Ingredients	CM1	CM2	CM3	CM4
Ruminal Fluid (mL)	200	200	200	200
Mineral solution 1 (mL)	150	150	150	150
Mineral solution 2 (mL)	150	150	150	150
Cysteine hydrochloride 3% (mL)	16.5	16.5	16.5	16.5
Sodium carbonate 6% (mL)	66.5	66.5	66.5	66.5
trypticase (g)	15	15	15	15
Yeast extract (g)	5	5	5	5
Glucose (g)	13.3	13.3	13.3	13.3
Bacteriological Agar (g)	15	15	15	15
cellulose (g)	5	–	–	–
Corn Straw (g)	–	5	–	–
Sorghum Straw (g)	–	–	5	–
Oat Straw (g)	–	–	–	5

Mineral solution 1: K₂HPO₄ to 3%.

Mineral solution 2: KH₂PO₄ to 0.3%, (NH₄)₂SO₄ to 0.6%, NaC1 to 0.6%, MgSO₄ to 0.06%, and CaCl₂ to 0.06%.

Enzymatic extracts production. Samples from each axenic culture were collected and transferred to 50 mL Falcon tubes containing 40 mL of fortified culture medium without agar and 5 mL of mineral oil under anaerobic conditions. The inoculated tubes were incubated at 39°C for 48 h at 100 rpm on a rotary shaker (model 4520, Forma Scientific, Marietta, Ohio, USA) for biomass propagation. The product of the propagation was centrifuged at 10,000 g (model Z383, Hermle Labortechnik, Wehingen, Germany), and the phases separated. The phase cell-free is the enzymatic extract, which evaluated the presence of fibrolytic enzymes.

Strain selection. The strain selection was based on analysis of the content of the fibrolytic enzymes laccase, lignin peroxidases and cellulases in each enzymatic extract. The laccase and lignin peroxidases were quantified using the method described by Ander and Messner (1998), and the cellulases by the method described by Navarrete-Bolaños et al. (2003).

Strain identification. Once the best fibrolytic strain had been selected, molecular identification was performed, including DNA extraction using the method described by Reilly and Attwood (1998), and PCR amplification and nucleotide sequencing of the almost complete 16S rRNA gene, using primers 27F [5’-AGAGTTTGATCCTGGCTCAG−3’] and 1492R (5’-GGTTACCTTGTTACGACTT−3’). The PCR reactions were carried out in a T1 thermocycler (Biometra, Göttingen, Germany), using a cycling program that included an initial cycle of 94°C for 3 min, 30 cycles of 94°C for 1 min, 50°C for 1 min, 72°C for 1.5 min, and a final extension at 72°C for 10 min. Sequences of the primers 27F and 1492R were obtained (Macrogen, Seoul, South Korea) and compared with data in the GenBank database (National Center for Biotechnology Information [NCBI]). The sequences were aligned, and for phylogenetic identification of the strains, alignments were made with CLUSTAL X version 2.0 (Larkin et al. 2007), using the default settings. The evolutionary history was inferred by using the maximum likelihood (ML) and Bayesian inference methods. To select a nucleotide substitution model, the Bayesian information criterion was implemented, and ML analysis (MEGA version 6.0; Tamura et al. 2013), was performed. Initial tree/s for the heuristic search were obtained by applying the neighbour − joining method to a matrix of pairwise distances, estimated using the maximum composite likelihood approach. The trees were bootstrapped, with 1,000 replicates of each sequence, to evaluate the reliability of the tree topologies. Bayesian phylogenetic reconstructions were composed with the program MrBayes version 3.2 (Ronquist et al. 2012).

Specific culture medium design. Once Lysinibacillus fusiformis was selected, an experimental strategy to design the specific culture medium to maximize biomass growth was developed based on the strategy proposed by Navarrete-Bolaños et al. (2017) for the ingredients listed in Table 1. The strategy began with constructing and performing a screening folded Plackett − Burman design for 12 variables or ingredients (Table 3), taking those shown in Table 1 as reference values and defining the frontier values as shown in Table 2. This was followed by the construction and performance of a central composite design for five variables (Table 4) to obtain a culture medium that allows significant biomass growth.

Table 2. Culture medium components analysis for L. fusiformis growth.

Component	Reference composition (g/L)	Lower frontier value (g/L)	Upper frontier value (g/L)
W1: KH2PO4	10	7	13
W2: K2HPO4	1	0.5	1.5
W3: (NH4)2SO4	2	1	3
W4: NaCl	2	1	3
W5: MgSO4	0.2	0	0.4
W6: CaCl2	0.2	0	0.4
W7: Cysteine	1.1	0.6	1.6
W8: Carbonate	9	6	12
W9: Tryptone	4.5	3	6
W10: Yeats Ext	1.5	1	2
W11: Glucose	4.5	3	6
W12: Cellulose	3	4.5	6

Table 3. a screening folded Plackett-Burman for 12 variables or ingredients (212×5/512, for N=40 assays). The value of the ingredients is expressed in g/L and the output function as UFC/mL.

Assay	W1	W2	W3	W4	W5	W6	W7	W8	W9	W10	W11	W12	Y
1	13	0.5	3	3	0	0	1.5	12	3	2	6	3	1.88E+07
1	7	1.5	3	1	0.4	0	1.5	6	3	2	6	6	4.04E+07
1	7	0.5	1	8.25	0.18	0	1.5	12	3	2	3	3	1.24E+07
1	13	1.5	1	8.25	0	0	0.5	6	6	2	3	6	2.00E+07
1	7	1.5	3	2.75	0	0	1.5	12	3	1	3	6	1.44E+07
1	10	1	2	2	0.2	0.2	1	9	4.5	1.5	4.5	4.5	9.65E+06
1	7	0.5	1	2.75	0	0	1.5	6	6	1.5	6	3	2.57E+07
1	13	1.5	1	8.25	0.4	0	1.5	6	3	1.5	3	3	2.25E+07
1	7	0.5	3	8.25	0	0	0.5	6	3	2	6	6	9.50E+06
1	7	0.5	1	8.25	0	0.4	1.5	6	6	1.5	3	6	1.29E+08
1	13	0.5	1	2.75	0.4	0	1.5	12	6	1.5	3	6	1.40E+07
1	7	1.5	1	2.75	0.4	0.2	0.5	6	6	1.5	3	6	2.25E+07
1	13	1.5	3	2.75	0	0.2	1.5	6	3	2	3	3	3.44E+07
1	13	0.5	1	8.25	0	0.2	1.5	12	3	1.5	6	6	2.60E+08
1	10	1	2	5.5	0.2	0.4	10	9	4.5	1	4.5	4.5	1.83E+07
1	13	0.5	3	8.25	0.4	0	0.5	6	3	1.5	3	6	1.63E+08
1	7	1.5	1	8.25	0	0	1.5	12	6	2	3	6	4.03E+07
1	7	0.5	1	2.75	0	0	0.5	6	3	1.5	3	3	2.22E+08
1	13	0.5	1	8.25	0	0.2	0.5	12	6	1.5	3	3	1.79E+07
1	7	1.5	1	8.25	0.4	0.2	1.5	6	3	1.5	6	6	2.31E+07
1	13	0.5	1	8.25	0.4	0.2	0.5	6	6	2	6	3	2.28E+08
1	13	1.5	1	2.75	0	0	0.5	12	3	1.5	6	6	7.20E+06
1	13	1.5	3	8.25	0	0	1.5	6	6	1.5	6	3	4.51E+07
1	7	1.5	1	2.75	0	0.2	1.5	12	6	2	6	3	2.99E+07
1	7	0.5	1	2.75	0.4	0.2	0.5	12	3	2	3	6	2.30E+07
1	7	1.5	3	8.25	0	0.2	0.5	12	3	1.5	3	3	2.78E+07
1	7	0.5	3	8.25	0.4	0.2	1.5	6	6	2	3	3	1.93E+07
1	13	0.5	1	2.75	0	0.2	1.5	6	3	2	3	6	2.44E+07
1	13	0.5	3	2.75	0.4	0.2	0.5	6	3	1.5	6	3	3.08E+07
1	7	0.5	3	2.75	0.4	0.2	1.5	12	3	1.5	6	3	4.10E+06
1	13	1.5	3	8.25	0.4	0.2	0.5	12	3	2	3	6	5.95E+06
1	7	0.5	1	8.25	0.4	0	0.5	12	6	1.5	6	6	2.30E+07
1	13	1.5	1	2.75	0.4	0	0.5	12	3	2	6	3	1.86E+07
1	13	0.5	3	2.75	0	0	0.5	12	6	2	3	3	2.47E+07
1	7	1.5	1	8.25	0	0.2	0.5	6	3	2	6	3	1.55E+07
1	13	0.5	1	2.75	0.4	0	1.5	6	6	2	6	6	2.41E+08
1	13	1.5	1	2.75	0.4	0.2	1.5	12	6	1.5	3	3	1.51E+08
1	7	1.5	3	2.75	0.4	0	0.5	6	6	2	3	3	2.70E+07
1	7	1.5	3	8.25	0.4	0	0.5	12	6	1.5	6	3	1.24E+07
1	13	1.5	3	8.25	0.4	0.2	1.5	12	6	2	6	6	2.74E+07

Table 4. Central composite design to define a culture medium for F. fusiformis biomass production. The value of the ingredients is expressed in g/L and the output function as UFC/mL

Assay	W1	W5	W6	W9	W11	Y
1	13	0.6	1.2	6.4	5.2	445000000
2	13	0.6	1.2	6.4	5.2	217000000
3	10	0.4	1	4.9	6.7	245000000
4	16	0.4	1.4	3.4	3.7	128000000
5	10	0.4	1	4.9	3.7	198000000
6	16	0.8	1.4	3.4	6.7	140000000
7	16	0.4	1	4.9	6.7	170000000
8	13	1.07568	1.2	6.4	5.2	135000000
9	10	0.8	1	3.4	0.6	50000000
10	13	0.6	1.2	6.4	8.76762	215000000
11	13	0.6	1.2	8.46762	5.2	315000000
12	16	0.8	1	3.4	3.7	178000000
13	13	0.124317	1.2	6.4	5.2	143000000
14	16	0.4	1.4	3.4	6.7	163000000
15	16	0.6	1.67568	6.4	5.2	100000000
16	16	0.8	1.4	3.4	3.7	235000000
17	10	0.8	1	4.9	3.7	153000000
18	10	0.8	1.4	4.9	6.7	100000000
19	10	0.8	1.4	3.4	6.7	100000000
20	16	0.8	1	3.4	6.7	170000000
21	20.1352	0.6	1.2	6.4	5.2	355000000
22	16	0.8	1.4	4.9	6.7	85000000
23	13	0.6	1.2	1.33238	5.2	165000000
24	10	0.4	1.4	3.4	6.7	175000000
25	16	0.4	1.4	4.9	6.7	703000000
26	10	0.8	1.4	3.4	3.7	233000000
27	10	0.4	1	3.4	3.7	100000000
28	16	0.4	1	4.9	3.7	95000000
29	13	0.6	1.2	6.4	1.63238	155000000
30	10	0.4	1.4	3.4	3.7	140000000
31	16	0.4	1	3.4	3.7	198000000
32	10	0.8	1.4	4.9	3.7	113000000
33	5.86476	0.6	1.2	6.4	5.2	110000000
34	16	0.8	1	4.9	6.7	345000000
35	10	0.4	1.4	4.9	3.7	365000000
36	16	0.4	1.4	4.9	3.7	60000000
37	13	0.6	0.724317	6.4	5.2	188000000
38	10	0.8	1	4.9	6.7	148000000
39	16	0.8	1.4	4.9	3.7	313000000
40	16	0.8	1	4.9	3.7	52500000
41	10	0.4	1	3.4	6.7	70000000
42	10	0.4	1.4	4.9	6.7	90000000
43	10	0.8	1	3.4	6.7	668000000
44	16	0.4	1	3.4	6.7	193000000

Optimizing process variables for biomass production in a stirred-tank bioreactor. Once defined a culture medium that allows obtaining significant biomass quantitative of fibrolytic strain selected, an experimental strategy to maximize biomass production at level bioreactor was constructed and performed, taking into consideration the variables agitation (Z₁), aeration (Z₂), acidity (expressed as pH value) (Z₃), which were singled out because of their relationship with the enzyme activity and cell metabolism (pH value), maintain homogeneity in the system (agitation), and provide oxygen for cell metabolic activities (aeration). Based on a preliminary screening design, the feasible ranges for these variables were defined as 1–200 rpm for Z₁, 0–1 VVM for Z₂, and 5.5–7.5 pH values for Z₃. A central composite design was constructed (Table 5) as described by Montgomery (2005). In all experimental assays, the temperature was maintained constant at 39 (±1) °C which is the average temperature of in vivo cattle rumen system, the medium culture composition, based on the previous results (specific culture medium design section), and the inoculum quantitative (1 × 10⁶ CFU/mL) in a ratio of 1:10.

Table 5. Central composite design to optimizing variables process to maximizes F. fusiformis biomass.

Assay	Z1	Z2	Z3	UFC/mL
1	100	0	6.5	2.08E+07
2	200	1	5.5	3.00E+06
3	0	0	7.5	1.92E+07
4	200	0	5.5	1.09E+07
5	200	0	7.5	3.89E+07
6	0	1	7.5	1.80E+07
7	200	1	7.5	1.46E+07
8	100	0.5	8.18179	1.47E+07
9	−68.1793	0.5	6.5	2.69E+07
10	100	0.5	6.5	1.01E+08
11	0	1	5.5	1.23E+07
12	0	0	5.5	5.00E+07
13	268.179	0.5	6.5	1.60E+07
14	100	1.5	6.5	1.83E+07
15	100	0.5	6.5	2.51E+07
16	100	0.5	4.81821	7.80E+06

In vitro agricultural residues fermentation. Three groups were created as a result of the mixtures among three different Agricultural residues (corn, sorghum, and oats) with ruminal fluid and inoculated with three different cell densities of L. fusiformis (1 × 10⁸, 1 × 10¹⁰, and 1 × 10¹² CFU/mL) (Table 6). During fermentation, the native microbial population of the ruminal fluid digested the sample, along with L. fusiformis, as usually occurs in vivo in the rumen. After each incubation period, samples were collected from each group and analysed to determine acid detergent fibre (ADF) and neutral detergent fibre (NDF) according to the methods of Van Soest and Wine (1967).

Table 6. Effects of inoculum density in mixtures of corn, sorghum and oat agricultural residues on relative feed values (RFV), and in vitro true digestibility (IVDMD).

Assay		NDF				ADF				RFV				IVDMD
AR	ID	Y0	Y12	Y24	%(-)	Y0	Y12	Y24	%(-)	Y0	Y12	Y24	%(+)	Y0	Y12	Y24	%(+)
CC	0	97,23	83,04	76,75	21,06	50,12	42,45	39,90	20,39	47,70	62,54	70,08	46,91	49,86	55,83	57,82	15,97
C1	8	97,23	86,15	67,48	30,60	50,12	34,49	27,96	44,21	47,70	66,98	92,53	93,98	49,86	62,03	67,12	34,62
C2	10	97,23	84,92	60,43	37,85	50,12	34,57	26,66	46,81	47,70	67,88	104,88	119,88	49,86	61,97	68,13	36,66
C3	12	97,23	80,89	59,28	39,03	50,12	35,51	30,93	38,29	47,70	70,42	101,69	113,20	49,86	61,24	64,81	29,98
SC	0	91,58	82,34	67,40	26,40	80,28	38,33	33,27	58,56	26,78	66,70	86,93	224,63	26,36	59,04	62,98	138,92
S4	8	91,58	76,61	76,72	16,23	80,28	29,81	29,17	63,66	26,78	79,75	80,24	199,65	26,36	65,68	66,18	151,03
S5	10	91,58	65,71	55,35	39,56	80,28	26,85	21,66	73,02	26,78	96,24	121,05	352,07	26,36	67,98	72,03	173,22
S6	12	91,58	68,95	62,89	31,33	80,28	32,65	27,96	65,17	26,78	85,62	99,28	270,76	26,36	63,47	67,12	154,61
OC	0	85,77	79,32	66,82	22,09	63,80	39,56	41,17	35,47	42,51	68,12	79,11	86,09	39,20	58,08	56,83	44,97
O7	8	85,77	60,83	64,67	24,60	63,80	39,58	35,17	44,87	42,51	88,80	88,47	108,09	39,20	58,07	61,50	56,90
O8	10	85,77	63,83	53,12	38,07	63,80	32,23	31,66	50,38	42,51	92,97	112,49	164,59	39,20	63,79	64,24	63,87
O9	12	85,77	66,25	65,48	23,66	63,80	32,42	26,65	58,23	42,51	89,37	96,80	127,69	39,20	63,64	68,14	73,83

AR: agricultural residues, C=Corn, S=Sorghum, O=Oat; CC, SC, OC: Controls; ID: Inoculum Density; 0= without inoculum, 8=1.0 × 10⁸, 10=1.0 × 10¹⁰, 12=1.0 × 10¹² UFC/mL respectively; NDF: Neutral fibre detergent; ADF: Acid Fiber Detergent; %(+): increase; %(-): decrease.

Dry-matter digestibility, dry-matter intake, and relative feed value. The results from ADF and NDF were used to estimate the in vitro dry-matter digestibility (IVDMD), in vitro dry matter intake (IVDMI), and relative feed values (RFV) for each agricultural residue fermented according to Rohweder et al. (1978): $\mathrm{RFV}=\frac{\mathrm{IVDMD}\times \mathrm{IVDMI}}{1.29}$ $\mathrm{IVDMD}=88.9-\left(0.779\times \mathrm{ADF}\right)$ $\mathrm{IVDMI}=\frac{120}{\mathrm{NDF}}$

Volatile fatty acid (VFA) measurement. Fermented samples from each assay were centrifuged at 10,000 g and the phases separated. Then 1 mL of the light phase (supernatant) was mixed with 250 uL of metaphosphoric acid (25% w/v). The reaction mixtures were transferred to GC vials and analysed using a gas chromatographer (Perkin − Elmer, Model Clarus 500) at a split ratio of 3:23, and at 100–150 °C at 10 °C m⁻¹, with the temperature of the injector set at 200 °C and the detector at 250 °C using a Stabilwax–DA column (30 mL x 0.25 mm ID). Peak integration was performed using the internal software, and their identification was made based on a standard external solution containing acetate, propionate and butyric acid.

Gas production. Gas production was performed using a digital manometer (D1005PS, Ashcroft, Stratford, CT, USA) coupled to a 0.6 mm hypodermic needle to measure the pressure of the gas in the headspace during the fermentation. Each fermenter was incubated at 39°C, and the headspace gas pressure in each was adjusted to ambient pressure. Measurements were taken at 2, 4, 6, 8, 10, 12, 18, 24, 30, 36, and 48 h. After each measurement, the headspace gas pressure was adjusted to ambient pressure. The gas pressure of the reaction system, expressed in kg/cm², was converted to gas volume (mL/g substrate) using the equation V = p/0.019, as proposed by Sandoval-Gonzalez et al. (2016).

pH measurement. The pH values of the fermented samples were determined using an Ultra Basic pH Meter UB-10 (Denver Instrument, Bohemia, NY, USA). Measurements were taken at 12, 24, 36, and 48 h during in vitro digestibility to analyse pH evolution.

Statistical analysis. Statistical analysis was performed using Statgraphics Centurion software (Statgraphics Technologies, The Plains, VA, USA). Variables were analysed using ANOVA procedures and significant differences were accepted for a 95% confidence interval (P ≤ 0.05). Experiments were carried out in a completely randomized statistical design of response surface type to optimize the independent variables for the culture medium design and biomass production, a 3 × 3 Latin Square was used for inoculum densities and straw types of analysis, and Tukey's Multiple-Range Test was used to compare means among treatments. All experimental assays were performed three times and the analyses of the digestibility parameters also were performed three times for each treatment.

Results

Microbial ecology studies. Forty (X₁, X₂, X₃, … , X₄₀) axenic cultures were isolated from the ruminal fluid samples. Seventeen (X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇, and X₁₈) of them were satisfactorily propagated in vitro, four (X₁, X₂, X₄, X₁₅) showed the capacity to synthesize fibrolytic enzymes, and X₂ was selected as the best strain due to its ability to most efficiently synthesize the enzymatic complex made up of both oxidative (laccase and lignin peroxidase) and hydrolytic enzymes (cellulases) that increase degradability of agricultural residues.

Strain identification. The entire dataset for the phylogenetic analysis totalled 11 nucleotide sequences of various bacilli isolated from rumens. The ML and Bayesian analyses yielded trees with similar topologies, each resolving six well-supported clades relative to the out group. These phylogenetic analyses indicated that the sequence obtained from the selected isolate was clustered with the reference to bacilli with 100% bootstrap support. L. fusiformis (X₂) was identified according to the blastx algorithm of the NCBI database, with 100% identity and 0% expectancy. The 5’−29R sequence of the selected L. fusiformis was deposited in GenBank under accession number MH03289.

Culture medium for L. fusiformis growth. The results of the Plackett − Burman design allowed a screening to be performed of the original ingredients, reducing the number from twelve to seven with two of them in constant value (NaCl at 0.368, and cellulose at 0.411) and the other five analysed under a central composite design (Table 4). The results (last column of Table 4) were analysed by analysis of variance (data not shown), which showed, for a confidence interval of 0.05, that no individual ingredient or interaction between them exhibited a significant effect within their defined limits. Hence, the experimental results were used to construct a second-order mathematical model using the least squares method to find the values of the variables that maximize output function, resulting in the following expression: $\begin{array}{rl}Y& =-2.744\times {10}^9-3.677\times {10}^7{W}_1+3.357\times {10}^9{W}_5\\ & +2.852\times {10}^9{W}_6+1.363\times {10}^7{W}_9+1.979\\ & \times {10}^8{W}_{11}-2.097\times {10}^6{W}_1^2-3.317\times {10}^7{W}_1{W}_5\\ & +4.368\times {10}^7{W}_1{W}_6+7.995\times {10}^6{W}_1{W}_9+4.806\\ & \times {10}^6{W}_1{W}_{11}-8.578\times {10}^8{W}_5^2-9.874\times {10}^8{W}_5{W}_6\\ & -1.074\times {10}^8{W}_5{W}_9-3.365\times {10}^7{W}_5{W}_{11}-1.047\\ & \times {10}^9{W}_6^2+9.437\times {10}^6{W}_6{W}_9-8.218\times {10}^7{W}_6{W}_{11}\\ & -6.107\times {10}^6{W}_9^2+769023W_9{W}_{11}-1.267\times {10}^7{W}_{11}^2\end{array}$The model solution is based on solving a system linear equation obtained via the derivation of the second-order model as follows: ${\displaystyle \frac{\mathrm{dY}}{d{W}_1}=3.677\times {10}^7-\left(2\right)\times 2.097\times {10}^6{W}_1+3.317\times {10}^7{W}_5+4.368\times {10}^7{W}_6+7.995\times {10}^6{W}_9+1.979\times {10}^8{W}_{11}}$ ${\displaystyle \frac{\mathrm{dY}}{d{W}_5}=3.357\times {10}^9-1.455\times {10}^6{W}_1+\left(2\right)\left(8.578\times {10}^8\right)W_5+9.874\times {10}^8{W}_6+1.074\times {10}^8{W}_9+3.365\times {10}^7{W}_{11}}$ ${\displaystyle \frac{\mathrm{dY}}{d{W}_6}=2.852\times {10}^9+4.368\times {10}^7{W}_1-9.874\times {10}^8{W}_5-\left(2\right)\left(1.047\times {10}^9\right)W_6+9.437\times {10}^6{W}_9-8.218\times {10}^7{W}_{11}}$

${\displaystyle \frac{\mathrm{dY}}{d{W}_9}=1.363\times {10}^7+7.995\times {10}^6{W}_1-1.074\times {10}^8{W}_5+9.437\times {10}^6{W}_6-\left(2\right)\left(6.107\times {10}^6\right)W_9+769023W_{11}}$ ${\displaystyle \frac{\mathrm{dY}}{d{W}_{11}}=1.979\times {10}^8+4.806\times {10}^6{W}_1-3.365\times {10}^7{W}_5-8.218\times {10}^7{W}_6+769023\times {10}^6{W}_9+\left(2\right)\left(1.267\times {10}^7\right)W_{11}}$

The system solution was solved using the Levenberg–Marquardt method (Gill et al. 1997), obtaining the following results: W₁= 20.135, W₅= 0.159, W₆= 1.515, W₉= 8.468, and W₁₁= 6.497. The strategy performed to design a specific culture medium for L. fusiformis growth indicates that it must contain 20.135 g/L of KH₂PO₄, 0.159 g/L of MgSO₄, 1.515 g/L of CaCl₂, 8.468 g/L of tryptone, 6.497 g/L of glucose, 0.368 g/L of NaCl and 0.411 of cellulose, which allow a theoretical maximum yield of 6.784 × 10⁸ CFU/mL. Confirmation assays, using the optimal process conditions were performed, with the results yielding 8.89 × 10⁸ CFU/mL, which compares favourably against the predicted model value.

Biomass production of L. fusiformis in bioreactor. Once performed the central composite design assays to maximize L. fusiformis biomass production, the results (last column of Table 5) analysed by analysis of variance (data not shown), which showed that, for a confidence interval of 0.05, no individual variable or interaction between them exhibited a significant effect within their defined limits. This analysis suggested that the optimal values that maximized the output function were among the defined limits. Hence, the experimental results were used to construct a second-order mathematical model using the least squares method to find the values of the variables that maximized output function, resulting in the following expression: $\begin{array}{rl}& Y=-5.0106\times {10}^8-358938Z_1-1.0951\times {10}^7{Z}_2\\ & +1.76027\times {10}^8{Z}_3-1060.26Z_1^2+16750Z_1{Z}_2\\ & +80875Z_1{Z}_3-3.1514\times {10}^7{Z}_2^2+5.025\times {10}^6{Z}_2{Z}_3\\ & -1.42089\times {10}^7{Z}_3^2\end{array}$The model solution is based on solving a system linear equation obtained via the derivation of the second − order model as follows: ${\displaystyle \frac{\mathrm{dY}}{d{Z}_1}=-358938-\left(2\right)\times 1060.26Z_1+16750Z_2+80875Z_3}$ ${\displaystyle \frac{\mathrm{dY}}{d{Z}_2}=-1.0951\times {10}^7+16750Z_1-\left(2\right)\times 3.1514\times {10}^7{Z}_2+5.025\times {10}^6{Z}_3}$ ${\displaystyle \frac{\mathrm{dY}}{d{Z}_3}=1.76027\times {10}^8+80875Z_1+5.025\times {10}^6{Z}_2-\left(2\right)\times 1.42089\times {10}^7{Z}_3}$

The system solution was solved using the Levenberg–Marquardt method (Gill et al. 1997), obtaining the following results: Z₁= 81.137, Z₂= 0.365, and Z₃= 6.489. This procedure indicated that the parameter values for L. fusiformis biomass propagation are 81.137 rpm of agitation, 0.365 VVM of aeration level, 6.489 of pH value for a theoretical maximum yield of 5.356 × 10⁷ CFU/mL. Confirmation assays, using the optimal process conditions were performed, with the results yielding 8.89 × 10⁷ CFU/mL, which compares favourably against the predicted model value. The fermented product at optimal condition was filtered and the biomass collected, which was used for in vitro digestibility studies.

In vitro digestibility and the nutritional value of agricultural residues. In vitro digestibility assays were proposed to estimate agricultural residues in in vivo digestibility as a function of the structural changes due to the metabolic activity of L. fusiformis, shown in Tables 6 and 7. Qualitative data analysis shows increased yield values of in vitro dry-matter digestibility (IVDMD), relative feed values (RFV), and volatile fatty acid (VFA) in the agricultural residues of corn, oats, and sorghum as the fermentation time increased, independent of the inoculum density. Quantitative data analysis based on Tukey’s multiple-range test revealed that the three inoculums (1 × 10⁸, 1 × 10¹⁰, and 1 × 10¹² CFU/mL) increase IVDMD [F_(3,8)= 12458.77, p < 0.001; F_(3,8)= 9.58, p < 0.005; and F_(3,8)= 3328.71, p < 0.001], RFV [F_(3,8) = 12458.77, p < 0.001; F_(3,8)= 9.58, p < 0.005; and F_(3,8)= 3328071, p < 0.001], and VFA − T [F_(3,8)= 4625.53, p < 0.001; F_(3,8)= 212.53, p < 0.001; and F_(3,8)= 120864.00, p < 0.001] in corn, sorghum and oat straw. It also shows that the IVDMD, RVF, and VFA − T yield values differ for each agricultural residue depending on the inoculum density. The Latin square design data analysis revealed that the effect of the substrates is significant and the inoculum density effect is almost significant (F_(2,8)= 8.65, p < 0.001 and F_(2,8)= 4.46, p < 0.06 respectively). The data analysis based on 3² response surface design revealed that an inoculate containing 1 × 10¹⁰ CFU/mL allows obtaining of the maximum values of RVF, IVDMD, and VFA-T, and that the best agricultural residue is sorghum, followed by oats, and finally, corn.

Table 7. Effects of inoculum density in mixtures of corn, sorghum and oat agricultural residues on volatile fatty acids (VFA) synthesis.

	Volatile Fatty Acid				Percentual contribution
Assay	Acetic	Propionic	Butyric	Total	Acetic	Propionic	Butyric
CC	1646,81	1092,02	827,93	3566,76	46,17	30,62	23,21
C1	1878,88	2503,46	927,71	5310,05	35,38	47,15	17,47
C2	3160,88	3844,43	1589,01	8594,32	36,78	44,73	18,49
C3	2109,50	2947,59	762,35	5819,44	36,25	50,65	13,10
SC	1317,95	900,85	697,85	2916,65	45,19	30,89	23,93
S4	3015,92	1992,31	932,49	5940,72	50,77	33,54	15,70
S5	3340,20	1420,07	1334,77	6095,04	54,80	23,30	21,90
S6	4513,27	2939,97	1621,72	9074,96	49,73	32,40	17,87
OC	2036,60	742,13	572,78	3351,51	60,77	22,14	17,09
O7	2083,28	1909,34	625,68	4618,30	45,11	41,34	13,55
O8	2569,25	2797,81	948,97	6316,03	40,68	44,30	15,02
O9	4472,27	3027,33	458,44	7958,04	56,20	38,04	5,76

Gas production. Qualitative data analysis shows an increase in gas production directly correlated to in vitro fermentation time for the three agricultural residues, independent of the inoculum density. Quantitative data analysis based on Tukey's multiple − range test revealed no significant differences in gas production at different inoculum densities [F_(3,8) = 12458.77, p < 0.001; F_(3,8)= 9.58, p < 0.005; and F_(3,8)= 3328071, p < 0.001] during in vitro digestibility for the same substrate. It also revelated that there are significant differences in gas production for different substrates [F(3,8) = 12458.77, p < 0.001; F(3,8) = 9.58, p < 0.005; and F(3,8) = 3328071, p < 0.001], independent of inoculum density.

pH value changes. Qualitative data analysis shows no significant differences in pH values during in vitro digestibility. Qualitative data analysis shows significant differences in pH evolution during in vitro digestibility, beginning with a value of 6.2 (±0.1), increasing until 7.4 (±0.2) after 36 h of assay time, and ending with a value of 6.5 (±0.3). Quantitative data analysis based on Tukey's multiple-range test revealed significant differences in pH evolution for the different substrates [F(3,8) = 12458.77, p < 0.001; F(3,8) = 9.58, p < 0.005; and F(3,8) = 3328071, p < 0.001] and different inoculum density [F(3,8) = 12458.77, p < 0.001; F(3,8) = 9.58, p < 0.005; and F(3,8) = 3328071, p < 0.001].

Discussion

Rumen microbiome studies and the native strain Lysinibacillus fusiformis. Considering the importance of rumen microbiome − feed interaction in livestock nutrition, the use of agricultural residues as feed, and their low − quality nutritional value due to their high fibre content were fundamental to looking at a native strain that increases its digestibility and, livestock production. Rumen microbiome studies for fibrolytic strains show that L. fusiformis can efficiently synthesize an enzymatic complex comprised of the oxidative fibrolytic enzymes laccase and lignin peroxidase, which are the key enzymes directly involved in the depolymerization of lignin of the natural lignocellulosic substrates (Jing 2010), in addition to cellulase, which hydrolyses β−1,4 linkages in cellulose chains and liberates hexoses that are subsequently metabolized to pyruvate and later on to volatile fatty acids (VFAs).

L. fusiformis is a gram-positive, endospore-forming bacterium recognized as a ubiquitous environmental bacterium of pathogenic nature – not yet thoroughly characterized. It has received immense attention in recent years for its biotechnological potential, in particular for its ability to produce relevant enzymes (especially esterases, peptidases, and xilases) with potential industrial application (Mechri et al. 2017; Jabeur et al. 2020; Omisore et al. 2022), and now laccase, lignin peroxidase, and cellulase. Maybe this vast array of enzymes synthesized by L fusiformis is required to degrade the complex arrangements of structural carbohydrates in plant cell walls, as was described by Morgavi et al. (2013) and Koike and Kobayashi (2009), among others.

Microbial biomass production of L. fusiformis. The culture medium composition and process parameter values significantly affect microbial metabolism. The analysis of the components of the culture medium used for ruminal bacteria isolation allowed a new feasible culture medium for F. fusiformis biomass propagation. The components analysis of the new culture medium showed similitude with a general − purpose liquid medium supporting from undemanding microorganisms to fastidious organisms’ growth, as well as culture media recommended for the control of industrial fermentations. Specifically, tryptone provides nitrogen, vitamins, minerals, and amino acids essential for growth. Glucose is a fermentable carbohydrate that provides carbon and energy. Monopotassium phosphate is an efficient buffer. Potassium, calcium, and iron chlorides provide essential ions for osmotic equilibrium. Magnesium sulphate is a source of divalent cations. Cellulose seemingly is an enzyme inductor. Related to process variables, the technologies developed for massive aero-tolerant microbial biomass production based on submerged fermentation – a simplistic process that provides an adequate process variable control – make viable the disponibility of F. fusiformis in large quantities.

Based on the above, F. fusiformis can be mass-produced and subsequently conditioned to prolong the shelf life and characteristics that facilitate their incorporation in cattle feed as a direct-feed microbial supplement. Moreover, because F. fusiformis is rumen native, the stability and viability within the gastrointestinal tract, the interactions with the endogenous microbiota, and the host health should not be at risk. Therefore, supplementing the diet of cattle with L. fusiformis will increase the native rumen microbiota, acting synergistically with native digesters Fibrobacter succinogenes, Ruminobacter amylophilus, Selenomonas ruminantium, and others native strains to improve the digestibility of agricultural residues and increase livestock performance.

Enzymatic hydrolysis of the fibre in agricultural residues. The quantitative effect of the enzymatic complex synthesized by F. Fusiformis on in vitro fermentation assays in mixtures of rumen fluid, L. fusiformis, and agricultural residues of corn, oat, or sorghum is detailed in Table 6 and 7. The results indicate a diminution of values of the neutral detergent fibre (NDF) and acid detergent fibre (ADF). The NDF fraction is related to lignin content, and the amount of lignin is negatively associated with the feed digestibility, according to Mendoza et al. (2014). Therefore, a low NDF value implies an increase in the intake and digestibility of feed. However, ADF measures the plant components in forages that are the least digestible by livestock, including cellulose and lignin. Therefore, as ADF values increase, digestibility decreases, so forage with high ADF concentrations is typically lower in energy availability. Hence, low NDF and ADF values in livestock feed are essential because they imply improved cattle digestion, energy levels, performance, and productivity. These results are consistent with those reported by Kılıc and Gulecyuz (2017), who suggested that the lower the NDF and ADF contents of the feed, the higher the RFV index and quality of the forage. Also, with the reports by Gado et al. (2009) and Salem et al. (2013) who found that adding the exogenous fibre-degrading enzymes ZADO® (mix of anaerobic bacteria and their enzymes of cellulases, xylanases, alpha amylase, and protease) enhanced degradation of NDF, ADF and DM, which improves feed utilization and animal performance.

In vitro digestibility (IVD). Digestibility is the factor that expresses the potential of an ingredient to be digested, absorbed, and processed by the host-digestive system, whether in terms of nutrients or energy. Their value has an inverse relationship with the NDF and ADF values, the same relationship displayed in the results in Table 6. Overall, in vitro fermentation increases the digestibility of corn, oat, and sorghum agricultural residues. In particular, sorghum has the highest value of digestibility, followed by oats and, finally, corn, which is consistent with the average fibre content present in these agricultural residues. These results suggested that adding L. fusiformis to the diet of cattle enhances the nutritional value of the three agricultural residues studied due to an increase in the degradation of dietary fibre, which are consistent with those reported by Kondratovich et al. (2019)

who suggested that fibrolytic enzymes positively affected the digestion of multiple roughage sources commonly fed to cattle and in beef cattle growing diets stimulated intake and generated positive effects on ruminal fermentation.

Relative feed value (RFV). RFV is a parameter related to the quality of feed for animals, which depends to a great extent on the concentration of structural carbohydrates in the feed, as measured by the ADF and NDF fractions, which represent the more indigestible parts of the plant. As a result, digestibility and energy obtained through fermentation increase RFV (see Table 3). According to Rohweder et al. (1978), RFV combines the digestibility value and feed intake. Hence, increased RFV values indicate increased digestibility and feed intake by cattle. The data analysis in Table 6 shows that the agricultural residues of sorghum were found to have a higher RFV than those of oats and corn. The increased RFV values in the three agricultural residues fermented indicate that these would be classed as high quality for cattle nutrition. Therefore, the increased RFV values on corn, oat, or sorghum agricultural residues fermented by native rumen microbiome augmented with L. fusiformis evolve from low to high nutritional feed.

Volatile fatty acid (VFA) production. In the rumen system, insoluble carbohydrates contained in the cattle feed are broken down into soluble sugars, which then undergo anaerobic fermentation to yield volatile fatty acids, including acetate, propionate, and butyrate. Hence, the presence and augmentation of the VFA concentration in the fermented agricultural residues indicates a fibre degradation, which facilitates the access of digestive enzymes to structural carbohydrates for hydrolysis. Therefore, a direct relationship exists between VFA content in the ruminal fluid and rumen feed digestibility by the rumen microbiome, which is consistent with the results shown in Table 7. In addition, data analysis indicates that the fermentation of the agricultural residues of sorghum results in a higher VFA concentration than in oats and corn. Furthermore, Table 7 also indicates a low acetic to propionic acid ratio (average 1.24) in all treatments, which are consistent with the findings of Wang et al. (2020), who suggested that fibre-degrading bacteria produced acetate and insoluble carbohydrate-decomposing bacteria produced propionate, with Arriola et al. (2011), who observed that a decrease in the acetate: propionate ratio implies an increase in the total volatile fatty acid concentration, and with Eun et al. (2007), who showed that enhancement in fibre degradation was often accompanied by a decrease in acetate: propionate.

This allows us to hypothesize that fermented agricultural residue will significantly affect animal performance toward efficient productivity. As propionate is glycogenic, a lower acetic to propionic acid ratio indicates increased production and availability of energy for nutrient utilization, growth, milk production/lactation and foetal development in pregnant animals, depending on the physiological state of the animal. Conversely, a higher acetic to propionic acid ratio has some negative implications for the performance of animals. For instance, dairy animals will tend to produce higher butterfat than milk yields. In animal fattening, energy use is less efficient because it is lost in methane synthesis.

pH value changes. Under normal conditions, the pH of the ruminal fluid varies according to the composition of the food consumed by cattle in the range of 5.5 − 7. In rations high in carbohydrates of easy fermentation, the pH value is reduced to a range between 5 and 5.5. In rations high in fibre, the pH value is close to 7.0, a desirable value but at the expense of consuming food with low nutritional value. Hence, a well-formulated balanced ration between grains and straw is the best way to achieve a stable pH of around 6.5 or by providing low carbohydrate rations during the day to avoid ruminal acidosis (Zhang et al. 2017). In this work, the pH value recorded in the in vitro fermentation assays ranged from 6.2 to 7.4 (6.8 average), which is a relatively desirable pH because of gradual fibre degradation by fibrolytic enzyme action and the synergistic effect with other hydrolytic enzymes synthesized by the native microbiome. In the first phase of fibre digestibility, the pH value increased from 6.2 to 7.4 as a result of fibre degradation, and in the second phase, it decreased due to the synthesis of fatty acids. Therefore, pH can be considered an indicator parameter for adequate fibre degradation.

Gas production (GP). GP increased as in vitro digestibility time increased in all assays for the three agricultural residues studied (see Table 6). GP results from the fermentation of carbohydrates into short − chain, volatile fatty acids, namely acetate, propionate and butyrate, which cattle use for obtaining energy and for protein synthesis. This fermentation process also leads to the generation of carbon dioxide (CO₂) and molecular hydrogen (H₂), which a group of archaea that naturally occur in the rumen use as substrates to produce methane (CH₄), a major source of greenhouse gases (GHG) worldwide. Hence, increasing livestock production also increases gas production, particularly enteric CH₄ (Knapp et al. 2014; Yanti and Yayota 2017; Mizrahi et al. 2021). Therefore, reducing the GHG intensity of ruminant production is a considerable challenge for the research community. Among the proposed strategies to reduce gas emissions are dietary supplementation with antimethanogenic compounds, suppressing methanogenesis substrates, and enhancing the nutritional quality of the feed for livestock (Haque 2018). Therefore, dietary supplementation with F. fusiformis may reduce methane production by enhancing the nutritional quality of the feed for livestock. Also, because F. fusiformis induces major acid propionic synthesis compared to acetic and butyric acid synthesis, and the propionate fermentation pathway is distinguished from the pathways leading to acetate and butyrate by not liberating hydrogen, which is necessary to reduce CO₂ to CH₄ (Knapp et al. 2014; Shabat et al. 2016; Chen et al. 2020).

Conclusion

Studies for increasing the digestibility of ruminant feed remain at the forefront of research today as the digestibility of agricultural residues is a priority due to the enormous amount of residue left after harvesting. These residues are not in direct competition with human food, so, if put to good use, will reduce the cost of production associated with the cost of feeding, thereby decreasing the negative environmental impact caused by dumping or burning residues and improving livestock production.

L. fusiformis, a fibrolytic native strain from the rumen microbiome, synthesizes the enzymatic complex that modifies the lignin structure and, in synergistic interaction with other fibrolytic and cellulolytic native rumen strains, degrades fibre from agricultural residues of corn, oats, and sorghum. The saccharification of cellulose to fermentable carbohydrates and subsequent volatile fatty acids synthesis indicates an evolution of agricultural residue from low- to high-quality feed. Propionic acid is the primary fatty acid synthesized, which increases energy availability for efficient cattle growth performance, and their metabolism limits molecular hydrogen synthesis and enteric methane.

This study's results suggest that supplementing cattle's diet with L. fusiformis can improve feed utilization, animal performance, and livestock production while enhancing fibre degradation without significant enteric methane production.

The aero-tolerant capacity of F. fusiformis helped us to study the physicochemical effectors associated with the growth and definition of a culture media and the process parameters in a stirred-tank bioreactor that generated significant biomass production.

Based on the ability to synthesize a wide range of digestive enzymes, be it a rumen native strain, aero-tolerant, and some critical physicochemical effectors to significant biomass production, F. fusiformis must be an attractive alternative for use as a direct-feed microbial because it is viable during preparation, stable until intake by animals, and able to survive in the digestive environment.

As far as we can determine, a direct-feed fibrolytic strain has not been proposed to increase the digestibility of high-fibre content feeds, which is due, among other factors, to the lack of a stable and efficient strain under normal conditions of ruminal fermentation, and that it is available in sufficient quantity to cover the market demand, and can now be addressed.

Declaration of interest

There is no known actual or potential conflict of interest including any financial, personal, or other relationship with other people or organizations associated with this work.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by Consejo Nacional de Ciencia y Tecnología [grant number: ITC-1-2015-028-221208]; Tecnológico Nacional de México [grant number: 5275.14P].