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Introduction
The distribution of molecules and their union within the cell wall of tropical forages affect the metabolic action of microorganisms. Tropical forages have high contents of hemicellulose, cellulose, pectin and lignin in the cell walls, accounting for 35 to 80% of its lignocellulosic biomass, which provides structural integrity to the forage (Trejo-López et al, 2018). This reduces its digestibility by ruminants and limits animal productivity in the tropics. The enzymatic complex of β-1-4 cellulases hydrolyzes cell walls and determines digestibility of tropical forages by ruminants. It should be noted that 10 to 35% of the energy consumed is absorbed as net energy since 20 to 70% of the cellulose is not digested. Few studies aimed at increasing the efficiency of fiber utilization in tropical forages have been reported (Barahona and Sánchez, 2005).
Ruminal anaerobic environment and its microorganisms are responsible for the digestion of structural carbohydrates (Cai et al, 2010; Sattar et al, 2018; Azizi et al, 2020) by degrading fiber through enzymatic digestion (Berny et al, 2019; Gudeta and Krishna, 2019; Liu et al, 2019). The potential cellulolytic bacteria in the rumen are Bacteroides succinogenes, Clostridium, Trichonympha, Actinomycetes, Butyrivibrio fibrisolvens, Ruminococcus albus and Methanobrevibacter ruminantium (Gudeta and Krishna, 2019). However, their cellulolytic potential varies with the species present and what the host eats (Qian et al, 2019; Gudeta and Krishna, 2019).
Several chemical, physical, and biological methods have been used to improve fiber digestibility in ruminant diets (Azizi et al, 2020). A biological method tested uses bacteria capable of degrading plant cell wall components (Harsini et al, 2019). Fiber digestibility in the rumen is not due to the enzymatic activity of individual bacteria, but rather to their interaction with other microorganisms (Sattar et al, 2018). Its efficiency depends on the diversity and density of microorganisms, including bacteria, protozoa, fungi, and archaea (Qian et al, 2019). Cellulolytic microorganisms can be used as probiotics in ruminant diets to improve digestion of fibrous components (Gudeta and Krishna, 2019). Therefore, the objective of this study was to estimate the in vitro production of biogas, methane (CH4) and fermentative characteristics of cobra grass inoculated with ruminal bacteria (RB) in coculture with cellulolytic bacteria isolated (ICB) either from bovine or water buffalo.
Materials and methods
Ethical considerations
All the procedures involving animals were in accordance with the ethical standards of Universidad Autónoma de Guerrero (Mexico) and were performed according to the protocols of the Federal Animal Health Law and NOM-062-ZOO-1999.
Isolated cellulolytic bacteria
This study is a sequel of previously published work on cellulolytic bacterial consortia (CBC) obtained from water buffalo and Swiss-bu cow (Herrera-Pérez et al, 2018; Torres-Salado et al, 2019), from which cellulolytic bacteria evaluated in the present study were isolated. The culture medium based on ruminal fluid (MRF) was described by Hungate (1950) and modified by Torres-Salado et al (2020). To isolate cellulolytic bacteria, a sterile solid culture medium [MRF + 0.2% carboxymethylcellulose (Sigma-Aldrich®, St Louis, MO, USA) + 2% agar (Sigma-Aldrich®, St Louis, MO, USA)] was prepared in sterile Petri dishes. The CBC was inoculated by the streak plate seeding method, and plates were placed in an anaerobic jar with GasPakTM (BD Bioxon®, Oaxaca, Oaxaca, Mexico). The anaerobic jar was placed in an incubator (Ecoshel 9082, Ciudad de Mexico, Mexico) for 72 h at 39 °C for development of colonies.
In sterile test tubes (18X150 mm), 9 mL of sterile MRF with cellobiose (0.2%; Sigma-Aldrich®, St Louis, MO, USA) (MFRC) were added under anaerobic conditions with CO2. Colonies with good definition and isolation were transferred to a tube containing medium and incubated for 24 h at 39 °C. After incubation, a sample was observed under a microscope (BX31, Olympus®, Allentown, Pennsylvania, USA) to identify bacterial morphologies. The process was repeated until a single morphology of ICB of water buffalo (ICBbuf) and Swiss-bu cow (ICBbov) was obtained. The ICB was morphologically characterized according to Ramírez (2015), and Gram staining was conducted.
Substrate
Cobra grass (Brachiaria hibrido) was harvested at 56 d of regrowth and dehydrated at 60 °C until constant weight in an oven (Felisa® FE-293A, San Juan de Ocotán Zapopan, Jalisco, Mexico). The grass was then ground to pass 1 mm sieve in a Thomas-Wiley Mill (Thomas Scientific®, Swedesboro, NJ, USA). Bromatological composition of cobra grass was 7.5% crude protein, 69.05% neutral detergent fiber (NDF), 47.96% acid detergent fiber and 87.85% organic matter.
Inocula
1) RB = 5 mL ruminal bacteria from Swiss-bu cow ruminal fluid, centrifuged for 3 min at 1,157 g (Dehority et al, 1960). 2) ICBbov = 5 mL ICBbov incubated in MRF with cellobiose (0.2%) for 48 h. 3) ICBbuf = 5 mL ICBbuf incubated in MRF with cellobiose (0.2%) for 48 h. 4) Coculturebov = 5 mL RB and 5 mL ICBbov; 5) Coculturebuf = 5 mL RB and 5 mL ICBbuf.
Test of in vitro gas production
In serological vials (120 mL), 0.5 g cobra grass and 45 mL MFR medium were added. All the vials were maintained under anaerobic conditions with CO2. They were hermetically sealed with a neoprene stopper (20 mm diameter) and an aluminum ring, then sterilized 15 min in an autoclave (All American® 1941X, Madison, Wisconsin, USA) at 121 °C and 15 psi. The vials were then inoculated and incubated for 72 h at 39 °C. Biogas production and CH4 was measured following Menke and Steigass (1988) and modifications by Torres-Salado et al (2019).
Fermentative characteristics
Variables measured after incubation were pH (Herrera-Pérez et al, 2018), total bacterial count (Harrigan and McCance, 1979; Sánchez-Santillán et al, 2016), ammoniacal nitrogen (NH3-N; McCullough (1967); DMD (Getachew et al, 2004; Hernández-Morales et al, 2018), NDFD (Sánchez-Santillán et al, 2015), and volatile fatty acids (VFA) as described by Cobos et al (2007).
Results
Morphology and Gram staining of ICB from water buffalo and Swiss-bu cow rumen CBC (Herrera-Pérez et al, 2018; Torres-Salado et al, 2019) indicated that they were Gram positive cocci. These cocci showed formation of diplococci and, occasionally, chains of three or more cocci.
Biogas production accumulated at 72 h by ICBbov and ICBbuf represented on average 42.11% of that produced by RB (p<0.05). Coculturebov produced 14.24% more biogas accumulated at 72 h than RB (p<0.05). The ICBbov and ICBbuf were not different (p>0.05) from RB in partial biogas production at 24 h, while Coculturebov produced 41.38% more biogas than RB (p<0.05). The ICBbov and ICBbuf produced 14.07 and 26.30%, respectively, of the partial biogas produced by RB at 48 and 72 h (p<0.05). Moreover, neither of the cocultures was different from RB (p>0.05). The accumulated and partial production (48 and 72 h) of CH4 showed that ICBbov and ICBbuf produced less CH4 than RB, Coculturebov or Coculturebuf (p<0.05). However, partial production of CH4 at 24 h by RB, ICBbov and ICBbuf was not different (p>0.05; Table 1).
Table 1 Effect of adding isolated cellulolytic bacteria to ruminal bacteria on biogas and methane production (mL g-1 DM) using cobra grass as substrate.
| Variable | ICBbov | ICBbuf | RB | Coculturebov | Coculturebuf | SEM |
|---|---|---|---|---|---|---|
| Biogas24 1 | 80.44c | 80.11c | 99.42bc | 140.56a | 119.21ab | 5.35 |
| Biogas 48 2 | 16.74b | 15.53b | 114.57a | 110.01a | 92.53a | 9.40 |
| Biogas72 3 | 8.17b | 8.18b | 31.1a | 29.42a | 25.31a | 2.26 |
| Biogas4 | 102.58c | 103.82c | 245.08b | 279.99a | 237.06b | 15.83 |
| Methane24 1 | 11.03b | 12.67b | 15.1ab | 18.38a | 17.15a | 0.69 |
| Methane48 2 | 7.96b | 6.65b | 25.71a | 26.56a | 27.76a | 2.00 |
| Methane72 3 | 5.11b | 5.11b | 10.21a | 11.44a | 11.84a | 0.66 |
| Methane4 | 24.10b | 23.52b | 51.54a | 57.19a | 56.74a | 3.47 |
Means with different superscript letters (a, b, c) within rows indicate significant difference (p<0.05).
ICBbov = isolated bovine cellulolytic bacteria (6.90x108 cell mL-1); ICBbuf = isolated buffalo cellulolytic bacteria (8.40x108 cell mL-1); RB = ruminal bacteria (1.39x109 cell mL-1); Coculturebov = ICBbov and RB; Coculturebuf = ICBbuf and RB; SEM = Standard error of the mean; 1partial production with 24 h incubation; 2partial production with 24 to 48 h of incubation; 3partial production with 48 to 72 h of incubation; 4cumulative production.
The DMD and NDFD were not different in RB, Coculturebov and Coculturebuf (p>0.05). However, ICBbov degraded 37.10 and 96.34% more DMD and NDFD than ICBbuf (p<0.05). The total bacterial count was not different in RB, Coculturebov and Coculturebuf; and ICBbov was not different from RB (p>0.05). The NH3-N content of the culture medium was not different among inocula (p>0.05). The pH of the culture medium was different among inocula, which had pH within the range for RB (Table 2).
The concentration of VFA, acetate, propionate and butyrate were similar in RB, Coculturebov and Coculturebuf (p>0.05). The average VFA of these inocula was 82.62% more than the VFA produced by ICBbov and ICBbuf (p<0.05). Acetate and propionate production showed no difference between ICBbov and ICBbuf (p>0.05). The mean values of acetate and propionate of ICBbov and ICBbuf were 59.08 and 39.71%, respectively, of the mean production of RB, Coculturebov and Coculturebuf (p<0.05). The ICBbuf produced 24.06% more butyrate than ICBbov (p<0.05; Table 2).
Table 2 Effect of the addition of isolated cellulolytic bacteria to ruminal bacteria on in vitro fermentative characteristics of cobra grass substrate.
| Variable | ICBbov | ICBbuf | RB | Coculturebov | Coculturebuf | SEM |
|---|---|---|---|---|---|---|
| DMD (%) | 30.45b | 22.21c | 68.20a | 68.99a | 70.90a | 4.41 |
| NDFD (%) | 15.21b | 1.91c | 70.46a | 69.58a | 72.06a | 6.37 |
| Bacteria (109 cell mL-1) | 0.80bc | 0.50c | 0.97ab | 1.04ab | 1.14a | 0.06 |
| pH | 6.85b | 6.88a | 6.61d | 6.63cd | 6.65c | 0.02 |
| NH3-N (mg dL-1) | 26.22 | 24.73 | 23.55 | 23.03 | 22.13 | 0.51 |
| VFA (mM L-1) | 37.27b | 41.67b | 71.62a | 72.77a | 71.85a | 4.35 |
| Acetate (mM L-1) | 18.20b | 21.18b | 35.43a | 33.00a | 31.57a | 1.21 |
| Propionate (mM L-1) | 9.22b | 12.55b | 24.71a | 29.50a | 27.99a | 2.98 |
| Butyrate (mM L-1) | 7.94d | 9.85c | 11.49ab | 10.27bc | 12.29a | 0.42 |
Means with different superscript letters (a, b, c, d) within rows indicate significant difference (p<0.05). ICBbov = isolated bovine cellulolytic bacteria (6.90x108 cell mL-1); ICBbuf = isolated buffalo cellulolytic bacteria (8.40x108 cell mL-1); RB = ruminal bacteria (1.39x109 cell mL-1); Coculturebov = ICBbov and RB; Coculturebuf = ICBbuf and RB; SEM = Standard error of the mean; DMD = dry matter degradation; NDFD= neutral detergent fiber degradation; NH3-N = ammoniacal nitrogen; VFA = volatile fatty acids.
Discussion
Studies involving isolation of cellulolytic bacteria are based on genomic identification or metabolic tests (Qian et al, 2019; Xie et al, 2018; Hyung et al, 2018), but there is little research (Azizi et al, 2020; Gang et al, 2020) on the coculture of ICB with RB. In the present study, the ability of both ICB in coculture to increase fermentation characteristics and in vitro gas production was evaluated to determine whether they can be used as probiotics for ruminants. Although we are aware of the limitations of the technique used, it can be a useful method for determining its functionality because it describes the kinetics of microbial activity in response to the substrate and measures the effect of the inocula used (Williams, 2000; Harsini et al, 2019).
The ICBbov and ICBbuf are strict anaerobic cocci that require fermentable carbohydrates for their growth (carboxymethylcellulose, cellobiose, fiber from cobra grass) producing acetate as a product of fermentation (Table 2). Based on the characteristics described and Bergey’s Manual® of Systematic Bacteriology (Ezaki, 2015), ICBbov and ICBbuf are classified within the genus Ruminococcus.
Biogas production of CH4 (Table 1), DMD and NDFD (Table 2) did not show that ICBA potentiates DMD or NDFD of RB (coculture). Its use as a probiotic did not improve these variables; that is, fermentation and degradation of cobra grass did not improve. Azizi et al (2020) published similar results in in vitro tests with wheat straw inoculated with a coculture of RB and ICB from termite intestine; they found it was not different from RB alone.
Partial production of biogas makes it possible to infer the type of carbohydrates fermented during the incubation period. The production of biogas and CH4 produced at 24 h was not different between RB and ICB because the cell content, that is, non-structural carbohydrates (Texta et al, 2019) and a certain protein fraction (Rodríguez et al, 2010) of cobra grass was fermented. After 48 h, differences in biogas production among inocula occurred because structural carbohydrates fermented, suggesting the capacity of cellulolytic bacteria to use these carbohydrates (González et al, 2011; Texta et al, 2019) and to interact with other cellulolytic bacteria. Increased biogas production is assumed to be the result of increased population of cellulolytic bacteria and fermentation of structural carbohydrates such as cellulose. Cellulose in grass produces acetate, 2 molecules of CO2 and 8 of H+ as fermentation products (Hungate, 1966), and it is the only carbon source, reflected in a higher production of biogas. Anaerobic fermentation of cobra grass requires a complex interaction of microorganisms (Deng et al, 2017; Torres-Salado et al, 2019) and we intended to manipulate it by adding ICBA to RB. In vitro biogas production values lower than those found in our study were reported in wheat straw inoculated with RB in coculture with ICB from termite intestine (Azzi et al, 2020) or ICB from Arabian horses (Harsini et al, 2019). In contrast, in vitro fermentation of corn silage inoculated with RB in coculture with Lactobacillus plantarum, Enterococcus mundtii, or Enterococcus faecalis (Gang et al, 2020) produced more biogas than the cocultures used in the present study.
Bacterial consortia are diverse communities that interact with each other and their environment to carry out interdependent physiological processes (Davey and O’Toole, 2000; Bader et al, 2010; Zuroff et al, 2013; Torres-Salado et al, 2019). When comparing the ICB of our study with CBC of bovine or buffalo origin (Torres-Salado et al, 2019; Herrera-Pérez et al, 2018) in the production of biogas from cobra grass, the results were similar. Thus, we infer that ICB require interaction with other cellulolytic bacteria for heterofermentative activity due to food interdependence and cross-feeding (Sánchez-Santillán and Cobos-Peralta, 2016) because it includes hydrolysis, acidogenesis, syntrophic acetogenesis of volatile fatty acids and methanogenesis (Deng et al, 2017; Torres-Salado et al, 2019).
Ruminal CH4 production involves energy losses (Liu et al, 2019; Gang et al, 2020) between 2 and 12% (Liu et al, 2019). The factors that determine CH4 production are the bromatological characteristics of the substrate and the fermentation products of cellulolytic bacteria (Venegas et al, 2017; Torres-Salado et al, 2019). The different values in partial and accumulated production of CH4 among inocula (Table 1) are due to NDF content of cobra grass, bacterial conformation of the inocula and production pattern of VFA. Acetate, CO2 and H2 are fermentation products of cellulolytic bacteria (Gang et al, 2020) generating a syntrophic relationship with methanogenic archaea (Liu et al, 2019; Torres-Salado et al, 2019), which use CO2 and H2 as a metabolic strategy and produce CH4 (Torres-Salado et al, 2019). This is a consequence of increasing the fermentation of structural carbohydrates since their fermentation by cellulolytic bacteria will always produce CO2 and H2 that the archaea will use. However, the present study focused on improving the fermentation of these carbohydrates by manipulating the ruminal population. Values similar to our results were reported by Herrera-Pérez et al (2018) and Torres-Salado et al (2019) during cobra grass fermentation inoculated with RB in coculture with CBC.
The use of ICB in coculture with RB did not improve cobra grass DMD or NDFD (Table 2). These results agree with Azizi et al (2020), who mention that inoculation of fibrolytic bacteria in the rumen did not improve fiber digestion. This contradicts the study by Gang et al (2020), who reported that an increase in cellulolytic bacteria increases NDFD. The above can be attributed to the origin of the inoculum (RB and ICB), type of inoculum and conformation of the microorganism population (Abad-Guaman et al, 2015; Torres-Salado et al, 2019). Azizi et al (2020) reported an average of 38.36% DMD and 33.4% NDFD in wheat straw inoculated with RB in coculture with 3 ICB from termite intestine, while Harsini et al (2019) reported 41.30% DMD and 40.16% NDFD in wheat straw inoculated with RB in coculture with 3 ICB isolated from horses. These values are lower than the results of the present study. Torres-Salado et al (2019) reported 61.80 and 65.73% DMD, as well as 55.41 and 59.42% NDFD with bovine and buffalo CBC, respectively; these values are higher than those obtained with the ICB in our study. This supports the idea that ICB needs to interact with other bacteria to improve fiber degradation (Sánchez-Santillán and Cobos-Peralta, 2016).
Coculturebov, coculturebuf and RB showed lower pH levels than ICBs (Table 2) due to higher production of organic acids by hydrolysis of acetyl groups (Du et al, 2019). However, these pH values did not affect the enzymatic activity of cellulolytic bacteria, since values lower than 6.0 are required for their inhibition (Nagaraja, 2016). In total bacterial counts (Table 2), the lower values of IBSs compared to Cocultures are assumed to be attributed to a catabolic repression of IBSs due to the presence of glucose or other compounds in the medium that inhibited their enzymatic activity (Texta et al, 2019). In contrast, in RB and Cocultures, cross-feeding was present (Texta et al, 2019), reflected in the bacterial population for each type of inoculum. In contrast, other inocula interact by cross feeding (Texta et al, 2019). The inocula did not show differences in NH3-N, which is the result of degradation of nitrogen compounds (Du et al, 2019), and in our study the population of cellulolytic bacteria was modified. Azizi et al (2020) reported 8.97 log10 total bacteria g-1, pH 6.43 and 13.87 mg dL-1 of NH3-N in culture medium with wheat straw substrate inoculated with RB in coculture with ICBs from termite intestine. These values are higher for total bacteria and lower in pH and NH3-N than those of the present study.
The VFA are positively correlated with DMD and NDFD (Sánchez-Santillán and Cobos-Peralta, 2016). The VFA of cocultures and RB were higher than those of the ICBs because the DMD and NDFD were higher in cocultures and in RB (Table 2). The average production rate of acetate was 80.88% higher than that of propionate in the ICBs, while for the other inoculum the acetate production rate was 21.64% higher than that of propionate, confirming that cellulolytic bacteria mainly produce acetate during their metabolic path (Sánchez-Santillán et al, 2016). Gang et al (2020) reported 73.06, 24.07, 11.17, and 115 mM L-1 of acetate, propionate, butyrate and VFA in corn silage inoculated with RB in coculture with ICB from horses, and higher values in acetate and VFA, as well as values in propionate and butyrate similar to those in Coculturebuf in our study.
We conclude that the use of ICB from bovine or water buffalo in coculture with RB does not improve production of biogas, DMD or NDFD with respect to RB. The ICBs do not produce a synergistic effect under the conditions of the present study. The ICBs do not have potential for use as a probiotic to enhance cobra grass degradation.
