Effects of L-tryptophan, Fructan, and Casein on Reducing Ammonia, Hydrogen Sulfide, and Skatole in Fermented Swine Manure

The effects of daily dietary Bacillus subtilis (Bs), and adding L-tryptophan, fructan, or casein to fecal fermentation broths were investigated as means to reduce the production of noxious gas during manure fermentation caused by ammonia, hydrogen sulfide (H2S), and 3-methylindole (skatole). Eighty swine (50.0±0.5 kg) were equally apportioned to an experimental group given Bs in daily feed, or a control group without Bs. After 6 weeks, fresh manure was collected from both groups for fermentation studies using a 3×3 orthogonal array, in which tryptophan, casein, and fructan were added at various concentrations. After fermentation, the ammonia, H2S, L-tryptophan, skatole, and microflora were measured. In both groups, L-tryptophan was the principle additive increasing skatole production, with significant correlation (r = 0.9992). L-tryptophan had no effect on the production of ammonia, H2S, or skatole in animals fed Bs. In both groups, fructan was the principle additive that reduced H2S production (r = 0.9981). Fructan and Bs significantly interacted in H2S production (p = 0.014). Casein was the principle additive affecting the concentration of ammonia, only in the control group. Casein and Bs significantly interacted in ammonia production (p = 0.039). The predominant bacteria were Bacillus spp. CWBI B1434 (26%) in the control group, and Streptococcus alactolyticus AF201899 (36%) in the experimental group. In summary, daily dietary Bs reduced ammonia production during fecal fermentation. Lessening L-tryptophan and increasing fructan in the fermentation broth reduced skatole and H2S.


INTRODUCTION
Environmental contamination caused by swine manure has provoked much public concern. More than 160 noxious substances have been discovered in swine waste (Le et al., 2005). Ammonia and hydrogen sulfide (H 2 S) are routinely measured, and 3-methylindole (skatole), a byproduct of tryptophan degradation, also increases fecal odor. Deposition of skatole in adipose tissues can taint the meat, with harmful consequences for the beef and pork industries.
In efforts to reduce the production of ammonia, H 2 S, and skatole, dietary additives have been investigated. These have included probiotics (Wutzke et al., 2010), fructan (Xu et al., 2002), and enzymes or amino acids (Vhile et al., 2012). Castration and vaccination have also been used to control the production of noxious gases. Among the probiotics, effective microorganisms, Bacillus subtilis (Bs), and lactic acid bacteria reduce the production of ammonia by modifying the intestinal microflora (Jeon et al., 1996). Dietary fructan has been shown to selectively promote Bifidobacterium spp. in the gut, thereby reducing the production of skatole (Li, 2009), and there is evidence for the prebiotic effects of inulin-derived fructans in humans (Salazar et al., 2014). Amino acid-balanced diets (such as those containing tryptophan) were shown to reduce the protein ratio in daily feedings, which led to less ammonia production in swine manure (Powers et al., 2007). The purpose of the above methods was to reduce the production of foul-smelling gas from within the bodies of live swine. To our knowledge, there have been no investigations to reduce the unpleasant odor of fecal matter once outside the body, i.e., conducted in vitro.
The present study evaluated the effects of externally applied tryptophan, fructan, or casein on manure fermentation, and noted the interactions among these additives and dietary Bs. Our results should provide a solid reference for studies aimed at reducing the odor of fecal waste outside the live swine body.

MATERIALS AND METHODS
The Animal Welfare Committee of Shandong Academy of Agricultural Sciences reviewed and approved the study protocol.

Experimental design
The 80 pigs were randomly divided into an experimental group and a control group (n = 40, each), constituting four replicates in each group and ten pigs in each replicate. Both groups were raised on a concrete floor. Pigs in the experimental group (Bs + ) were given a cornsoybean basal diet supplemented with 0.2% Bs. The control group (Bs -) received the same diet, but without the bacteria. The basal diet was prepared in accordance with the China feeding standard for swine (NY/T65-2004).
After 6 weeks, fresh manure was quickly collected, before feeding in the morning, from 2 randomly selected pigs in each replicate for subsequent studies (Yokoyama and Carlsonet, 1974). In brief, 280 g of each fecal sample was first suspended in 2.8 L anaerobic mineral buffer (5.0 g/L NaHCO 3 , 0.9 g/L NaCl, 0.9 g/L (NH 4 ) 2 SO 4 , 0.45 g/L KH 2 PO 4 , 0.45 g/L K 2 HPO 4 ·3H 2 O, 0.03 g/L CaC1 2 ·2H 2 O, 0.02 g/L MgCl 2 , 0.01 g/L MnSO 4 ·4H 2 O, 0.01 g/L CoCl 2 ·6H 2 O, 0.01 g/L FeSO 4 ·7H 2 O, 0.1 g/L cysteine). The suspensions were subsequently transferred to sterile plastic bags filled with CO 2 , thoroughly shaken for 5 min, and filtered through 6 layers of gauze to remove small particles. The filtrate from each suspension was distributed into 27 aliquots with 100 mL in each flask, and divided into 9 groups based on the amounts of L-tryptophan, fructan, or casein added to each flask (Table 1). The broths were incubated anaerobically for 24 h at 38°C. A 20-mL sample was taken from each flask to measure the gas composition. One milliliter of clear fermented broth was separately taken from both the Bs + and Bssamples (the blank, containing no added L-tryptophan, fructan, or casein, broth 1 in Table  1), mixed, and stored at -20°C for microflora isolation and identification.

Measurement of substances in the noxious gas
The fermented broth samples were measured after filtration through a 0.45-μm organic membrane. Ammonia was measured by the Nessler reagent colorimetric method. H 2 S was quantified via sulfate-iodometric titration. Ltryptophan and skatole were measured using highperformance liquid chromatography, with reference to Li (2009), and conducted at the Center for Food Quality Supervision and Testing Ministry of Agriculture (Jinan, China).

Isolation and identification of fecal bacteria in the fermentation broth
Genomic DNA of fecal bacteria was purified using an OMEGA Stool DNA kit (Omega Bio-Tec, Norcross, GA, USA). Using V3-340F (5'-FAM-TCCTACGGGAGGCA GCAGT-3') and V3-532R (5'-TCCTACGGGAGGCAGC AGT-3') as universal primers, the V3 region of 16S rDNA was amplified through fluorescence-based polymerase chain reaction-single-strand conformation polymorphismfragment length polymorphism (F-PCR-SSCP-FLP) methods, as described by Zhang et al. (2000). The 195-bp PCR products were analyzed with an Applied Biosystems 3730xl DNA Analyzer (Applied Biosystems, Carlsbad, CA, USA, 96-capillary array), in which the amount of each specific bacteria was quantified by the area size of their corresponding peaks. The 16S rDNA clone library (target fragment size 1.6 kb) was constructed by amplifying the 16S rDNA region of each bacteria using the V3 region fluorescent-labeled primers. The PCR products were purified and sequenced at the Biotechnology Research Center, Shandong Academy of Agricultural Sciences. All the sequences were BLASTed in the NCBI database to identify the bacterial species, and quantified using Mega6 software (http://www.megasoftware.net).

Statistical analyses
The amounts of ammonia, H 2 S, and skatole of the Bs + and Bsgroups were compared using one-way analysis of variance and Student's t-test. A probability (p) value less than 0.05 was the standard for significance. The generalized linear model procedure (PROC GLM) was used in the 3×3 orthogonal experimental design, to analyze the average k values and the residual R values. Duncan's method was used for multi-comparisons (p = 0.05). SAS (V9.1) was used for statistical analyses. Data are presented as means± standard error of the mean.

Effect of different additives on ammonia, H 2 S, and skatole production
The amounts of ammonia, H 2 S, tryptophan, and skatole produced in the fermentation broths prepared from fecal samples from pigs given Bs (Bs + group) were significantly lower than that of pigs of the control group (Bs -). This suggests that the addition of Bs in the diet was associated with reduced ammonia, H 2 S, tryptophan, and skatole production in the fermentation broths ( Table 2).

Effect of different added substances on the residual Rvalue of noxious gas
Casein showed the largest R-value to the ammonia in the fermentation broth in the Bs + group, and L-tryptophan resulted in the largest R in the Bsgroup (Table 3). This indicated that the main experimental additives affecting the Bs + and Bsgroups were different. In the production of H 2 S, fructan resulted in the largest R-value in both groups, which indicated that fructan was the main substance that affected the production of H 2 S. In the result regarding tryptophan, Ltryptophan showed biggest R-value in both groups, indicating that external added tryptophan was the main cause for the tryptophan in the fermentation broth. For skatole, external L-tryptophan showed the biggest R-value, which indicated that external L-tryptophan was the main affecting element.
Regarding the effect of adding casein to the fermentation broth on ammonia production, there was no correlation (r = -0.1551) between Bs + group (k1>k2>k3) and the Bsgroup (k1>k3>k2; Table 4). This shows that the effects of casein at different concentrations were different in the Bs+ group and the Bsgroup. Fructan at different   concentrations showed similar effects on H 2 S, since the two effects (both k1>k3>k2) were highly correlated (r = 0.9981); k1 was higher than k3 in both groups (p<0.05), which indicated that the 1.5% added fructan reduced the H 2 S levels in both the Bs + and Bsgroups. Tryptophan showed similar effects on skatole (both k2>k3>k1, r = 0.9992), and k1 was lower than k3 in both groups (p<0.05). This indicated that the 0.1% added tryptophan increased the concentrations of skatole in both the Bs + and Bsgroups.

Correlation between externally added substances and Bacillus subtilis
In the Bs + group, there were no associated changes between externally added L-tryptophan and ammonia, H 2 S, tryptophan, or skatole production (p>0.05; Table 5). There was significant correlation between casein and Bs in the production of ammonia (p = 0.0395), as well as between fructan and Bs on the production of H 2 S (p = 0.0141).

DISCUSSION
The ammonia, H 2 S, and skatole produced during pig raising not only adversely affect the growth of pigs, but also contaminate the environment and harm human health. In this study, we found that incorporating Bs in the pigs' daily feed significantly reduced the concentrations of ammonia, H 2 S, and skatole in the manure fermentation broth. Moreover, adding external L-tryptophan increased the production of skatole in the manure fermentation broths. Fructan inhibited the production of H 2 S. The effect of casein to the ammonia production depended on the presence of Bs. These discoveries provide an important reference for the control of noxious gases.

Effects of Bacillus subtilis on the production of ammonia, H 2 S and skatole in the fermented broth
Bs is routinely added to the daily swine diet to reduce the production of noxious gas. In the present study, ammonia production from fermentation broths prepared from the fecal matter of pigs given Bs was also reduced compared with that of pigs of the control group, which is consistent with the results of Kim et al. (2005). This may be because Bs secretes proteases that degrade proteins which otherwise transition to ammonia (Zhang et al., 2009). It is also possible that Bs promotes the presence of Lactobacillus amylovorus in the microflora (Su et al., 2006), which produces amylase (Eom et al., 2009) that facilitate the breakdown of ammonia.
Consistent with the discovery of Lee et al. (2009), in the present study dietary Bs was also associated with less H 2 S production from the fermentation broths, compared with the  control group. This reduction may be caused by metabolites of Bs that contain catalase and oxidase, which decrease the production of H 2 S (Yumoto et al., 2004).
In the present study, dietary Bs was also associated with less skatole produced by the fermentation broths, compared with the control group. It was reported previously that bacteria could affect the pathway of skatole formation by way of tryptophan metabolism (Li, 2009;Vhile et al., 2012), although the microorganisms tested were not Bs. The identity of the microorganisms responsible for the conversion of tryptophan to skatole has not been established. However, bacteria known to facilitate the degradation of skatole are: lactic acid bacteria 11201 (Yokoyama et al., 1983), Clostridium drakei and C. scatologenes (Whitehead et al., 2008), C. disporicum (Li, 2009), and C. scatologenes ATCC 25775 (Doerner et al., 2009).
The predominant bacteria discovered in the manure broth in this study were Streptococcus alactolyticus AF201899 and Lactobacillus amylovorus. No reports regarding the function of Streptococcus alactolyticus AF201899 have been published previously. Rinkinen et al. (2004) demonstrated that S. alactolyticus was the predominant lactic acid bacteria in the empty intestine and feces. S. alactolyticus could secret β-glycoside hydrolase, αgalactosidase, amylase, urease, acidic galactose, and galactosidase.
It has also been reported that lactic acid in the daily diet could reduce skatole production in the gut (Nowak and Libudzisz, 2009). Investigated as a probiotic, L. amylovorus secreted lactic acid and amylase (Eom et al., 2009) and promoted the growth of lactobacilli and bifidobacteria when galactooligosaccharides and Bifidobacterium were added to an in vitro model of the large intestine (Martinez et al., 2013). L. amylovorus was not found to degrade skatole directly. Skatole could be degraded in vitro by Streptococcus 6020 (Li, 2011). The effect of S. alactolyticus AF201899, L. amylovorus, and Bacillus MA001 need further investigation.
In this study, the effect of Bs in reducing ammonia, H 2 S, and skatole occurred in the swine gut, in the in vitro manure fermentation process, or both. Suárez-Estrella et al. (2013) reported that Bs could reduce changes in bacterial species during vegetable anaerobic composting, but there are no reports on the amount of ammonia, H 2 S, or skatole. In the present study, some isolated bacteria could not be grown in culture, which is consistent with the general knowledge that there are many undiscovered bacteria in the swine intestine. Further investigations are needed to elucidate the effect of Bs on the production of ammonia, H 2 S, and skatole.

Effect of added L-tryptophan, casein, and fructan on ammonia, H 2 S and skatole production
In the present study, tryptophan as an additive was the main variant that affected ammonia production in the Bs + group, but not the Bsgroup. No significant interaction was observed between tryptophan and Bs, indicating that added tryptophan might affect the nitrogen level in feces through other bacteria. Pierce et al. (1931) discovered that yeast might enhance nitrogen and indole production; added tryptophan, cysteine, and phenylalanine inhibited ammonia production in feces. There has been no report regarding whether added tryptophan could affect the production of H 2 S in swine manure. In our study, added tryptophan was not the main element affecting the production of H 2 S. We found that added tryptophan was the main variant affecting the amount of tryptophan and skatole produced in the fermented manure broth. This is consistent with the previous discovery that skatole in swine blood could be increased by injecting tryptophan into the swine appendix (Jensen, 2006). However, it was also found that addition of tryptophan into the daily diet had no significant effect on the level of skatole (Wesoly and Weiler, 2012). The differences might be caused by how the tryptophan was acquired by the swine or how it interacted with the manure. Wesoly and Weiler (2012) indicated that the insignificance of dietary tryptophan might be because tryptophan might be absorbed by the small intestine, instead of being utilized by bacteria in the colon. In the present study, added Ltryptophan showed no significant effect on skatole in either the Bs + or Bsgroup (same k value, r = 0.9992), indicating that the fermentation of Bs in the intestine might not be simulated by the fermentation of tryptophan in vitro.
In this study, casein was the main factor affecting ammonia production in the control group, but was not significant for H 2 S production, which is consistent with the reports of Powers et al. (2007). They showed that a diet balanced in amino acids with low crude proteins helped to reduce the ammonia level in feces, but exhibited no effect on H 2 S. We found that the combination of dietary Bs with casein added to the fermentation broth was significantly associated with a reduction in ammonia (p = 0.0395), but not H 2 S (p = 0.9505) produced from the fermentation broth. This was in accord with the various effects of different concentrations of casein on ammonia production in both the Bs + and Bsgroups (r = -0.1551). The effect of casein on ammonia production may be related to the protease secreted by Bs. Casein was not the main additive to affect skatole production. This is consistent with the conclusion of Lin et al. (1992) that daily diet had no effect on the production of skatole. Lundstrma et al. (1994) reported that an increase in the concentration of skatole did not correlate with an increase of protein. Casein or dietary proteins could result in changes in the microbiota in the intestine and therefore affect the fermentations .
Fructan, as a probiotic, modifies the microflora by increasing the amount of beneficial bacteria (Salazar et al., 2014). In the present study, the addition of fructan to fermentation broths was associated with a reduction in H 2 S, which correlated with the addition of dietary Bs. This is similar to a previous study (Zhao et al., 2013), which reported that a 1% addition of fructan had no effect on fertility, but lactic acid bacteria dramatically increased, with a significant decrease in E. coli (p<0.001); the amount of ammonia, H 2 S, and organosulfur significantly decreased (p<0.05). Fructan had similar effects on H 2 S in both the Bs + and Bsgroups of the present study (r = 0.9981), indicating that Bs did not affect the amount of H 2 S. However, fructan did not significantly affect skatole, which is consistent with the conclusion drawn by Xu et al. (2002). They observed that 0.5% to 1% fructan increased the amount of indole, but not skatole, perhaps because the pH change resulted in a difference in the microbiota. In our research, although the microbiota of the Bs + and Bsgroups differed, we regret that the pH was not measured.
In this study, Bs was added to the diet, and not directly to the fecal fermentation broths. In fact, Bs can also be added directly to the broths. The production of crude proteins in fecal broth depended on the amount of casein added. Further study is required to determine the optimal means of introducing Bs to fermentations broths, and its interactions with different protein, tryptophan, and fructan concentrations.

CONCLUSION
Adding L-tryptophan to fecal fermentation broths increased production of skatole. Adding fructan to the broths reduced the production of H 2 S. The effect of casein on ammonia depended on the addition of Bs in the daily diet. Daily dietary Bs reduced the production of ammonia, H 2 S, and skatole in fecal fermentation broths.