Potential of biochar in enhancing the effectiveness of probiotics Bacilli and Lactobacilli on in vitro microbial populations, hydrolytic enzymes, and ruminal fermentation in sheep

Introduction: Probiotics accelerate and improve rumen development, stability, and balance of beneficial microbes in the digestive system and decrease microflora disruption, which will increase the activity of desirable enzymes. This action of probiotics increases digestibility, feed efficiency, livestock performance, and general defense including antioxidant power. However, the response of different animals to the specific probiotics is not uniform and similar. Therefore, it is necessary to provide factors that improve the conditions for the establishment and functioning of microbial additives in the rumen or to use compounds that have a synergistic effect with probiotics to receive a uniform and reassuring response from the herd. One of the potentially useful materials to achieve this goal is biochar. In the present study, it was hypothesized that if biochar (as a favorable habitat for microorganisms) is added to probiotic-containing diets, the conditions for establishing these microbial products in the fermentation environment may be improved and probiotics can act more efficiently. Moreover, a probable synergy between probiotics and biochar may increase the efficiency of these additives compared to their separate usage. However, there is no special information available in this regard, especially about the effect of including biochar in probiotic-containing diets on different microbial populations and rumen hydrolytic enzymes. Therefore, this study aimed to investigate the potential of biochar in enhancing the effectiveness of the probiotic sources (Bacilli and/or Lactobacilli) on microbial populations, hydrolytic enzyme activity, digestibility, antioxidant capacity, and fermentation products in the sheep rumen, in vitro. Materials and methods: The experimental treatments were: 1. A basal diet without probiotics and biochar (control), 2. Basal diet containing probiotic Bacilli (B. coagulans, B. subtilis, and B. licheniformis, at the ratio of 2×1011, 5×109, and 5×109 CFU/g, respectively), 3. A basal diet containing probiotic Lactobacilli (L. plantarum, L. rhamnosus, and Enterococcus faecium, at the ratio of 2×109, 2×1010, and 2×1010 CFU/g, respectively), 4. A basal diet containing biochar, 5. A basal diet containing Bacilli-biochar, 6. A basal diet containing Lactobacilli-biochar, and 7. A basal diet containing Bacilli-Lactobacilli-biochar. Diet digestibility was determined by the two-stage Tilley and Terry method. In addition, the 24 and 72-h in vitro gas production techniques were conducted. At the end of each incubation, cellulolytic and proteolytic bacteria, as well as protozoa population, enzymatic activity (carboxymethyl cellulose, microcrystalline cellulose, filter paper degrading, and α-amylase), truly digestible substrate (TDS), partitioning factor (PF), microbial biomass production (MBP), methane release, antioxidant capacity, pH, ammonia-N (NH3-N), and volatile fatty acids (VFA) were determined by the standard methods. All in vitro tests were done in three replicates and two batches (runs) in different weeks. Data were analyzed in a completely randomized design using the GLM procedure of SAS. Results and discussion: Separate inclusion of probiotic Bacilli and biochar in the diet increased the cellulolytic bacteria population and activity of fibrolytic enzymes compared to the control (P<0.05), but these variables were not affected by Lactobacilli. The highest values of these variables were observed with the inclusion of biochar in probiotic Bacilli-containing diets (i.e., Bacilli-biochar and Bacilli-Lactobacilli-biochar treatments). These results were due to probiotics' ability to provide superior growth conditions for useful ruminal microbes, as well as the positive influence of the biochar structure, as a desirable habitat, on establishing, attaching, and developing rumen microorganisms. The protozoa population was not affected by biochar, but it was decreased (P<0.05) in the probiotic-containing diets (Bacilli and Lactobacilli treatments without or with biochar) in comparison to the control. The number of proteolytic bacteria and protease activity were not affected by the experimental treatments. Alpha-amylase activity in 24-h incubation in Basilli and Lactobacilli treatments was higher than the control (P<0.05) but was not affected by biochar. The activity of this enzyme in 72-incubation was lower in the control than in probiotic or biochar treatments (P<0.05). The alpha-amylase activity was the highest in the Lactobacilli-biochar and Bacilli-Lactobacilli-biochar groups. Separate inclusion of probiotics and biochar in the diet increased the diet digestibility, degraded substrate, and microbial biomass production (P<0.05). The maximum values of these parameters were detected in the probiotics-biochar diets. The reason for these increases can be related to the improvement of the cellulolytic bacteria population, alpha-amylase, and fibrolytic enzyme activity in the probiotic or biochar groups. The 24-h total antioxidant activity was not affected by the treatments. In 72-h incubation, the probiotics did not affect this variable, but a tendency to increase in antioxidant capacity was observed in diets containing biochar (without or with probiotics). The improving effect of biochar on the antioxidant power could be owing to its activity in trapping pollutants, poisons, and adverse factors in the incubation medium. Methane release, NH3-N, and acetate to propionate ratio were decreased, but total VFA was increased by separate use of the probiotics or biochar in the diet, compared to the control (P<0.05). More importantly, the inclusion of biochar in the diets containing the probiotic (i.e., Bacilli-biochar, Lactobacilli-biochar, and Bacilli-Lactobacilli-biochar) resulted in the greatest total VFA and the lowest NH3-N and methane release (P<0.05). One reason for the increased methane and ammonia was the lower protozoa population in the additives groups. The methane decline may also be related to the decreasing effect of the additives on methanogens, and their increasing effect on methanotrophs. Moreover, the improved bacterial biomass production could be considered as another reason for the decreased ammonia; i.e., more ammonia was assimilated into the bacterial protein. The increased VFA production was due to the higher digestibility and degraded substrate in the probiotic and/or biochar groups. Regarding the valuable characteristics of probiotics and biochar and their probable synergistic effects, their simultaneous application resulted in the highest improvement of fermentation variables. Conclusions: Separate use of probiotics Bacilli and Lactobacilli or biochar in the diet improved in vitro ruminal microbial and enzymatic activity and reduced energy (as methane) and nitrogen (as ammonia) losses. More importantly, the addition of biochar to the probiotics-containing diets was a suitable strategy for enhancing the effectiveness of the probiotic sources on digestibility and microbial biomass and reduction of methane and ammonia. However, these results need to be confirmed by further experiments.Materials and methods: The experimental treatments were: 1- basal diet without probiotic and biochar (control), 2 - basal diet containing probiotic Bacilli (B. coagulans, B. subtilis, and B. licheniformis, at the ratio of 2×1011, 5×109, and 5×109 CFU/g, respectively), 3- basal diet containing probiotic Lactobacilli (L. plantarum, L. rhamnosus, and Enterococcus faecium, at the ratio of 2×109, 2×1010 and 2×1010 CFU/g, respectively), 4- basal diet containing biochar, 5- basal diet containing Bacilli-biochar, 6- basal diet containing Lactobacilli-biochar, and 7- basal diet containing Bacilli-Lactobacilli-biochar. Diet digestibility was determined by the two-stage Tilley and Terry method. In addition, the 24 and 72-h in vitro gas production techniques were conducted. At the end of each incubation, cellulolytic and proteolytic bacteria, as well as protozoa population, enzymatic activity (carboxymethyl cellulose, microcrystalline cellulose, filter paper degrading, and α-amylase), truly digestible substrate (TDS), partitioning factor (PF), microbial biomass production (MBP), methane release, antioxidant capacity, pH, ammonia-N (NH3-N) and volatile fatty acids (VFA) were determined by the standard methods. All in vitro tests were done in three replicates and two batches (runs) in different weeks. Data were analyzed in a completely randomized design using the PROC GLM of SAS.Results and discussion: Separate including probiotic Bacilli and biochar in the diet increased the cellulolytic bacteria population and activity of fibrolytic enzymes compared to the control (P<0.05), but these variables were not affected by Lactobacilli. The highest values of these variables were observed with the inclusion of biochar in probiotic Bacilli-containing diets (i.e., Bacilli-biochar and Bacilli-Lactobacilli-biochar treatments). These results were due to probiotics' ability to provide superior growth conditions for useful ruminal microbes, as well as the positive influence of the biochar structure, as a desirable habitat, on establishing, attaching, and developing rumen microorganisms. The protozoa population was not affected by biochar, but it was decreased (P<0.05) in the probiotics-containing diets (Bacilli and Lactobacilli treatments without or with biochar) in comparison to the control. The number of proteolytic bacteria and protease activity were not affected by the experimental treatments. Alpha-amylase activity in 24-h incubation in Basilli and Lactobacilli treatments was higher than the control (P<0.05) but was not affected by biochar. The activity of this enzyme in 72-incubation was lower in the control than in probiotic or biochar treatments (P<0.05). The alpha-amylase activity was the highest in Lactobacilli-biochar and Bacilli-Lactobacilli-biochar groups. Separate inclusion of probiotics and biochar in the diet increased the diet digestibility, degraded substrate, and microbial biomass production (P<0.05). The maximum values of these parameters were detected in the probiotics-biochar diets. The reason for these increases can be related to the improvement of the cellulolytic bacteria population, alpha-amylase, and fibrolytic enzyme activity in the probiotic or biochar groups. The 24-h total antioxidant activity was not affected by the treatments. In 72-hour incubation, the probiotics had no effect on this variable, but a tendency to increase in antioxidant capacity was observed in diets containing biochar (without or with probiotics). The improving effect of biochar on the antioxidant power could be owing to its activity in trapping pollutants, poisons, and adverse factors in the incubation medium. Methane release, NH3-N, and acetate to propionate ratio were decreased, but total VFA was increased by separate use of the probiotics or biochar in the diet, compared to the control (P<0.05). More importantly, the inclusion of biochar in the diets containing the probiotic (i.e., Bacilli-biochar, Lactobacilli-biochar, and Bacilli-Lactobacilli-biochar) resulted in the greatest total VFA and the lowest NH3-N and methane release (P<0.05). One reason for the increased methane and ammonia was the lower protozoa population in the additives groups. The methane decline may also be related to the decreasing effect of the additives on methanogens, and their increasing effect on methanotrophs. Moreover, the improved bacterial biomass production could be considered as another reason for the decreased ammonia; i.e., more ammonia was assimilated into the bacterial protein. The increased VFA production was due to the higher digestibility and degraded substrate in the probiotic and/or biochar groups. Regarding the valuable characteristics of probiotics and biochar and their probable synergistic effects, their simultaneous application resulted in the highest improvement of fermentation variables.Conclusions: Separate use of probiotics Bacilli and Lactobacilli or biochar in the diet improved in vitro ruminal microbial and enzymatic activity and reduced energy (as methane) and nitrogen (as ammonia) losses. More importantly, the addition of biochar to the probiotics-containing diets was a suitable strategy for enhancing the effectiveness of the probiotic sources on digestibility and microbial biomass and reduction of methane and ammonia. However, these results need to be confirmed by further experiments.