Regulation of odor gas emission and performance by probiotic Bacillus in livestock industry

Livestock operations have transitioned over time from small farms to industrial facilities. Industrialized farms have improved the efficiency of animal management. However, there are problems with these large-scale operations, such as infectious disease and waste disposal [1]. Waste disposal can cause environmental issues, including soil erosion and the production of global greenhouse gases and air pollutants [2,3].


Introduction
Livestock operations have transitioned over time from small farms to industrial facilities. Industrialized farms have improved the efficiency of animal management. However, there are problems with these large-scale operations, such as infectious disease and waste disposal [1]. Waste disposal can cause environmental issues, including soil erosion and the production of global greenhouse gases and air pollutants [2,3].
In terms of air pollutants, various emissions such as ammonia (NH 3 ), hydrogen sulfide (H 2 S), methane (CH 4 ), nitrous oxide (N 2 O), volatile organic compounds (VOCs), and other odors are released from livestock production facilities [4]. These emissions are not only a nuisance to people living in nearby residential areas [5] but can also result in health problems for farm workers. Ammonia and H 2 S have shown critical negative effects on farm workers, including chronic or acute pulmonary disorders, as well as on domestic animals like swine and poultry.
Ammonia is generated from livestock barns, open feedlots, and manure storage facilities on farms, as well as during manure handling, treatment, and spreading. Ammonia dissolves readily in water (e.g., swine urine and drinking water) where it ionizes to form an ammonium ion. The atmospheric pressure and temperature affect ammonia solubility in water from dissolved or suspended materials [6]. On the other hand, ammonia produced in poultry facilities is created by urea and uric acid degradation [7].
Another source of odor in livestock production is H 2 S, which has been recognized as harmful to humans, and animals in deeppit production systems [8,9]. Hydrogen sulfide is formed under anaerobic conditions by bacteria reducing sulfate to sulfide;

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sulfide then combines with hydrogen ions to form hydrogen sulfide [10]. Pigs are affected by different levels of hydrogen sulfide. Severe distress, eye irritation, and drooling can be caused by concentrations of 100 ppm. Pigs exposed to 250 ppm of H 2 S may exhibit cyanosis, convulsions, and death [11].
Farm workers are also affected negatively by hydrogen sulfide exposure. Humans can detect a smell like rotten eggs when exposed to 0.1 to 5 ppm of H 2 S, even though these levels are not toxic. Eye and respiratory irritation in humans can occur at H 2 S levels of 100 ppm. High levels of H 2 S, (e.g., 150 to 200 ppm) cannot be detected by humans due to olfactory paralysis. At levels >200 ppm, H 2 S affect the nervous system and levels >1,000 ppm result in immediate collapse and respiratory paralysis [12].
There are several possible solutions to mitigate the environmental pollution from animal housing. Excretion of nitrogen and phosphorus can be reduced by formulating diets that improve nutrient digestibility [13]. Feed utilization and dry matter intake can be improved by fine grinding and pelleting, which reduce the size and increase the surface area of grains, thereby increasing the potential for interaction with digestive enzymes [13].
Enzymes can also be used to increase nutrient availability in animal feed. Enzymes can supplement the host's endogenous enzyme production, increasing the availability of nutrients, improving the digestibility of fibrous material, and decreasing any anti-nutritional factors present in feed ingredients [14]. For example, protease can degrade protein sources such as soybean meal and improve protein digestibility [13]. Other indirect contributors to improving swine house environments include antibiotics, probiotics, and organic acids. Low crude protein formulations using synthetic amino acids can also be used to reduce N excretion.
Probiotics can protect young animals against enteropathogenic disorders and improve growth performance [15]. Studies have shown that probiotics can create a gastrointestinal tract environment that is unfavorable to pathogenic growth [16]. Probiotics can decrease intestinal microbial catabolism and have a protein sparing effect, leading to reduced nitrogen flows [17].
A number of Bacillus strains could be used for feed additive in livestock industry. Bacillus are aerobic or facultative anaerobic, gram-positive, rod-shaped, and spore-forming bacteria. The spore-forming habit of Bacillus is highly beneficial for long term storage without a loss of activity, compared with non-sporeforming bacteria. Spores also have the ability to survive low pH, harsh environments, meaning their probiotic properties can benefit the small intestine [18].
Bacillus in swine can help to improve gut health and immunity for piglets and reduce environmental pollutants such as odor gas emissions from pig manure [19]. Upadhaya et al. [20] proposed that the reduction of fecal NH 3 emissions was observed when Bacillus-including feed was supplied to pigs, suggesting the improvement of nutrient digestibility by probiotics. However, Wang et al. [21] reported that it has no influence to enhance nutrient digestibility but indicates the effectiveness for the reduction of slurry NH 3 emissions.

Reduction of ammonia (NH 3 ) and hydrogen sulfide (H 2 S) excretion
Ammonia and hydrogen sulfide are negative substances on farm workers as well as animals in swine production and cause environment pollution [26]. Nguyen et al. [27] found that the supplementation of Bacillus-containing feed showed an advantageous effect to decrease NH 3 emissions but have no effect on the reduction of other gases (H 2 S and mercaptan). A recent study [27], showed that the addition of the increase in Lactobacillus inhibited pathogenic microorganisms and improved nutrient digestibility, resulting in reduced fecal NH 3 emissions.
Growing pigs fed diets with Bacillus licheniformis and Bacillus subtilis for 15 weeks, showed improved performance and reduced gas emissions due to increased fecal Lactobacillus counts and improved utilization of sulfur-containing amino acids [28]. It was concluded that the increase in Lactobacillus reduced intestinal pH through the production of organic acids, and that the bacitracin (bacteriocin) secreted by B. licheniformis inhibited the microbes that produce urease, thereby reducing NH 3 gas emissions. These results are supported by our research data, which showed that pigs fed diets with B. subtilis complex probiotics produced lower NH 3 and H 2 S emissions after a three-week growing period (unpublished data). These results suggest that three weeks of feeding is needed for probiotic adherence in the gut to have positive effects in swine.
According to Balasubramanian et al. [29], when probiotics containing Bacillus coagulans, B. licheniformis, and B. subtilis were fed to growing and finishing pigs over 16 weeks, no reduction in fecal noxious gas (NH 3 , H 2 S) emissions was observed. Yan et al. [30] found that increased nutrient digestibility reduced the substrate for microbial fermentation in the large intestine, which resulted in a decrease in fecal gas emissions. Chen et al. [31] showed that dietary Bacillus supplementation decreased NH 3 emissions, however, other odor substances such as H 2 S and mercaptan did not decrease.

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Bacillus spp. as probiotics can also affect the production of malodorous substances such as skatole. Skatole is a malodorous compound in meat and fecal that causes an off-flavor, so called "boar taint" [32]. Sheng et al. [33] demonstrated that dietary B. subtilis natto and B. coagulans supplementation decreased the skatole content of meat and feces. Doerner et al. [34] found that the reduced number of Clostridium in the feces of pigs fed Bacillus spp. was consistent with a lower skatole concentration in the meat and feces; Clostridium in feces is involved in the conversion of tryptophan to skatole.

Growth Performance in swine
Nguyen et al. [27] reported that dietary supplementation with probiotics-based Bacillus in weaning pigs linearly improved average daily gain (ADG) and average daily feed intake (ADFI) on days 0 to 7 of the experiment, as well as ADG and feed conversion ratio (FCR) on days 8 to 21. According to research by Lan et al. Kim et al. [28], dietary supplementation with B. licheniformis and B. subtilis complex probiotics (Bioplus YC) in growing pigs for 15 weeks resulted in improved growth performance in some periods but there was no significant difference from the control over the study period as a whole. The reasons for improved performance may be explained by changes in intestinal microorganisms and an increase in the secretion of digestive enzymes.
Hu et al. [35] observed that the ADG and FCR of piglets were improved and diarrhea occurrence was reduced when weaning pigs were fed B. subtilis KN-42 for 26 days. Greater bacterial diversity in the intestinal environment indicated an increase in the relative number of Lactobacillus and reduction in the relative number of E. coli in the feces. Wang et al.
[21] also reported that ADG tended to increase linearly and ADFI increased as the levels of probiotic (Bioplus 2B ® ) increased, however, no linear or quadratic effects were observed in FCR.
In growing and finishing pigs, dietary direct-fed microbial (DFM) supplementation has been shown to have negative effects on growth performance. Growing and finishing pigs have better digestibility, improved immunity, and increased resistance to intestinal disorders [36]. Balasubramanian et al. [29] reported that dietary supplementation of three probiotic Bacillus strains (B. coagulans, B. licheniformis, and B. subtilis) did not show a positive effect on the ADG and FCR without affecting ADFI in growing and finishing pigs. Upadhaya et al. [37] reported that there were significantly effective to ADG and ADFI, when two probiotic complexes (B. licheniformis and B. subtilis) was supplied to growing and finishing pigs as feed additive during the experimental period.

Effects of other beneficial microbials
Growing pigs fed a diet with 10% palm kernel meal (PKM) and added probiotics (B. subtilis and Saccharomyces cerevisiae), showed a reduction in fecal NH 3 , total mercaptans, and H 2 S content [42]; pigs fed a diet without PKM produced less mercaptans than pigs fed diets with PKM. The addition of probiotics to a non-PKM diet had a significant effect on ADG and FCR, but the addition of probiotics to a diet with PKM did not have a positive effect on performance. These results may be due to the presence of nonstarch polysaccharides in PKM creating a viscous environment in the gut.
Chen et al. [43] found the dietary supplementation of three probiotic complex (Lactobacillus acidophilus, S. cerevisiae, and B. subtilis) enhance ADG, when it was provided to the growing pigs for six weeks. In addition, fecal NH 3 -N excretion was reduced when pigs were fed a probiotic complex, however, there was no effect on volatile fatty acid (VFA) production. Chen et al. [31] reported that dietary supplementation probiotics combination (B. subtilis, B. coagulans, and L. acidophilus) in finishing pigs reduced fecal NH 3 -N production and improved ADG, however, there was no effect on ADFI or FCR. In their study, digestibility of N was not increased, therefore, the reduction in fecal NH 3 -N may not have resulted from nutrient digestibility but rather changes in intestinal microflora.

Reduction in ammonia (NH 3 ) and hydrogen sulfide (H 2 S) excretion
In the poultry industry, Bacillus spp. probiotics are widely used. A reduction in NH 3 gas emissions from excreta was observed for poultry fed metabolic energy (ME)-and crude protein (CP)reduced diets [44]. Poultry fed probiotic-supplemented diets also showed reduced NH 3 gas emissions compared with those fed diets

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without probiotics. Decreasing the CP content of the feed reduces the amount of synthetic amino acids supplied, thereby reducing the amount of N that is excreted by the poultry [45]. In addition, the feeding of probiotics can lead to increased nutrient utilization and changes in the balance of intestinal microorganisms, which can reduce NH 3 gas emissions.
According to research by Jeong et al. and Kim et al. [25], broilers fed a diet supplemented with B. subtilis C-3102 for five weeks, showed a reduction in NH 3 due to an increase in the number of Lactobacillus and reduction in the number of pathogenic bacteria. However, there were no effects on H 2 S, mercaptan, or acetic acid production.
Ahmed et al. [46] reported that the supplementation of feed containing Bacillus amyloliquefaciens showed the effect of NH 3 reduction in feces during raising term. The observed reduction in NH 3 emissions from broiler excreta may be due to increased nutrient utilization and changes in intestinal microbiota. Another reason is that B. amyloliquefaciens reduced the pH of the feces. A reduced concentration of E. coli and improved utilization of sulfur amino acids in the intestine could reduce the conversion of fecal ammonium to volatile ammonia.
Tang et al. [47] indicated that inclusion of B. amyloliquefaciens product in laying hens reduced NH 3 production in a six-week feeding trial; the number of cecal Lactobacillus was increased, but the number of E. coli and Salmonella bacteria and NH 3 gas emission was reduced.

Performance in Poultry
The use of antibiotics in the poultry industry to control pathogenic infections, such as necrotic enteritis, has been banned in some places due to concerns about consumer safety. In such cases, Bacillus spp. have been used to improve performance through positive changes in intestinal microbiota.
Bacillus subtilis was added to a ME-and CP-reduced diet to evaluate the effects of probiotic supplementation related to energy and protein [44]. Poultry fed diets with reduced energy and protein content showed a decreased in ADG and FCR. However, animals fed diets with probiotics showed significant improvements in ADG and FCR in the growing and finishing periods. These performance improvements did not appear immediately; three weeks were required for normal enzyme production that produced effects.
A recent study by Jeong et al. & Kim et al. [25] found that broilers fed a diet with B. subtilis C-3102 showed improved ADG and FCR, however, there was no effect on meat quality. In this study, Lactobacillus counts in the cecum, ileum, and excreta were significantly increased, and E. coli counts in the cecum and excreta were decreased with dietary B. subtilis supplementation.
Ahmed et al. [46] reported that ADG, ADFI, and FCR were improved when broilers were fed a diet with a B. amyloliquefaciens probiotic; serum IgG and IgA were also increased. Tang et al. [47] reported that laying hens fed a diet with B. amyloliquefaciens commercial product for six weeks had better egg production, eggshell strength, and eggshell thickness than hens that received a non-supplemented diet.
Bacillus amyloliquefaciens has the ability to produce extracellular enzymes, such as cellulose, α-amylases, protease, and metalloproteases. Those enzymes can help to increase the efficiency of digestion and absorption of nutrients [48]. Bacillus amyloliquefaciens also produce bacteriocins, such as subtilin, which have antibacterial effects against pathogenic microorganisms [49].

Effects with other beneficial microbial
Probiotics have been used to reduce NH 3 emissions, improve performance, and maintain livestock product safety in the poultry industry, most commonly with a Bacillus spp. complex. In one study, a combination of Pichia guilliermondii, B. subtilis, and Lactobacillus plantarum, at a ratio of 1:2:1, reduced NH 3 gas emissions by 46% in vitro test [50]. This probiotic complex significantly decreased crude protein digestibility, pH, NH 3 -N, urease, and uricase activity. Furthermore, the number of microorganisms responsible for fermenting carbohydrates to produce short chain fatty acids was increased.

Conclusion
In conclusion, dietary Bacillus spp. probiotic supplementation in monogastric animals can reduce NH 3 and H 2 S production depending on the conditions. In terms of performance, there were various effects of supplementation level, viability, and composition of probiotic species, diet formulation, age of animals, livestock house environment, and so on. Nutrient digestibility can be improved by the enzymes or bacteriocin produced by Bacillus spp.
In addition, supplementation with Bacillus spp. can help reduce fecal odor production, gas emission, and improve the performance of monogastric animals. Additional studies of complex probiotics that satisfy both odor reduction and performance requirements for monogastric animals are recommended.