Microflora in swine manure and odor indicators

94 J. Zhu Agriculture, Ecosystems and Environment 78 2000 93–106 rely on the microbial properties in the swine manure. Since the malodor originates from microbial activi- ties involving a variety of microbes, understanding the characteristics of the microflora present in swine ma- nure is essential for developing effective odor control techniques. This paper reviews the available information in the literature related to the types of bacteria in swine ma- nure, the potential odorous compounds associated with different bacterial genera, and the corresponding tech- niques used to control odor based on microbiological principles. Areas that need further research are also recommended.

2. Microflora in swine manure and odor indicators

2.1. Bacterial genera indigenous to swine manure Several studies have revealed the types of bacte- ria that can be isolated from fresh intestinal or fe- cal material of swine. Rall et al. 1970 reported that there were six groups of swine fecal bacteria according to metabolic functions: lactose fermenters; nonlactose fermenters; Clostridium sp.; Lactobacillus sp.; Ente- rococci; and Staphylococcus sp.. The population sizes for the identified bacterial groups in descending order are lactose fermenters, Lactobacillus sp., Clostridium sp., nonlactose fermenters, and Staphylococcus sp.. The classifications of bacterial groups based on func- tions instead of genera or species may not be of much help in determining the contributions of each single species to manure odor generation. On the other hand, it is usually difficult to clearly classify the bacterial groups only according to their metabolic characteris- tics. For example, Enterococci in the above discussion can also be grouped into lactose fermenters Orvin, 1986. Nuru et al. 1972 found that the fecal bacteria of pigs mainly consisted of gram-positive cocci Strep- tococcus, Peptostreptococcus, and Staphylococcus, Lactobacillus, Escherichia, and Bacillus. By using strictly anaerobic culturing methods, Salanitro et al. 1977 concluded that the predominant fecal mi- croflora isolated from adult swine comprised several bacterial groups; namely, fecal streptococci, Eubac- terium sp., Clostridium sp., and Propionibacterium acnes. Similar results were also reported from another study conducted by Russell 1979 which showed that the gram-positive cocci were the predominant organisms identified in swine manure, containing Streptococcus, Peptococcus, Peptostreptococcus, and Megasphaera. The reason that Russell 1979 classi- fied Megasphaera as gram-positive was because most of strains in this group stained gram-positive in his study. According to the above researchers, the swine fecal bacterial genera found can be listed in order of quantity from high to low as: Gram-positive cocci ca. 39, Eubacterium ca. 27, Lactobacillus ca. 20, Gram-negative rods Escherichia, ca. 8, Clostrid- ium ca. 4, and some other minor groups such as Propionibacterium acnes and Bacteroides 2. Among these bacterial genera, Clostridium sp., Lac- tobacillus sp., Peptostreptococcus, Eubacterium, Pep- tococcus, Propionibacterium acnes, Bacteroides, and Megasphaera are anaerobes; Streptococcus, Staphy- lococcus, and Bacillus are facultative anaerobes; Escherichia are aerobes or facultative anaerobes. Ob- viously, the anaerobic or facultative anaerobic bacteria account for the major portion of the bacterial species found in swine manure due to the anaerobic environ- ment in the intestinal tract of pigs. The level of aero- bic bacteria if Escherichia can be counted as one is low. Identification of the bacterial genera in swine manure is important to reveal the microbial consor- tium, but it is not enough to explain the complex odor generation processes taking place in manure storage systems. In order to investigate the poten- tial of producing odorous compounds by the bacte- ria from different groups, a further examination on each individual genus of the indigenous bacteria is needed. 2.2. The characteristics of indigenous bacterial species 2.2.1. Streptococcus Streptococcus is a group of chemoorganotrophs with fermentative metabolism microbes that use reduced and preformed organic compounds as sources of energy, hydrogen, electrons, and car- bon for biosynthesis. Most of the species are facultatively anaerobic, but some require addi- J. Zhu Agriculture, Ecosystems and Environment 78 2000 93–106 95 tional CO 2 for growth and some may be strictly anaerobic. Optimum growth temperature is usu- ally 37 ◦ C but maximum and minimum tempera- tures vary among species. Neutral or near neu- tral pH will favor the growth and low 4.0 or high 9.6 pH will inhibit the growth. All strep- tococci ferment carbohydrates, producing predom- inantly lactic acid; minor amounts of acetic and formic acids, ethanol, and CO 2 may also be pro- duced. Many species in this genus produce ammo- nia. A total of five species were found in swine manure Russell, 1979. However, no information regarding the identification of these species was given. 2.2.2. Peptostreptococcus Peptostreptococcus is a group of anaerobic chemoorganotrophs that metabolize peptone and amino acids to acetic, formic, propionic, caproic, iso-butyric, butyric, iso-valeric, and iso-caproic acids. Volatile amines and various alcohols may also be produced. The pH for growth ranges from 6.0 to 8.0 with the optimum being 7.0–7.5, and the temperature ranges generally from 25 to 45 ◦ C with optimum being 35–37 ◦ C. There is a total of three species identified in swine manure in this genus Russell, 1979. They are P. asaccharolyticus, P. magnus, and P. productus. 2.2.3. Eubacteria Eubacteria is a group of obligately anaerobic chemoorganotrophs that produce mixtures of organic acids from carbohydrates or peptone. Growth usually is most rapid at 37 ◦ C and pH near 7. Most of them of- ten produce large amounts of butyric, acetic, formic, and lactic acids Moore and Holdeman, 1986. Available literature regarding the number of species in this genus identified in swine excreta is limited. According to Russell 1979, a total of five species in this genus has been identified so far E. aerofaciens, E. rectale, E. tenue, E. ventriosum, and E. lentum. There were another seven species in this genus that could not be identified in his study. Since many species in this genus produce volatile fatty acids and indole and more than half of the species have not been identified, the effect of this bacterial group in general, and of individual species in particular, on swine manure odor generation should receive further research. 2.2.4. Lactobacillus Lactobacilli are strictly fermentative, aero-tolerant or anaerobic, and aciduric or acidophilic bacte- ria. With glucose as a carbon source, lactobacilli may be either homofermentative, producing 85 lactic acid, or heterofermentative, producing lactic acid, CO 2 , ethanol, andor acetic acid in equimolar amounts. Lactobacilli grow best in slightly acidic media with pH of 4.5–6.4. Optimal pH for growth usually ranges from 5.5 to 6.2. Growth ceases when pH 3.6–4.0 is reached, depending upon the species and strains. The growth rate is often reduced when the environment becomes neutral or alkaline. Growth temperature ranges from 2 to 53 ◦ C with an optimum range of 30–40 ◦ C. There were nine species in this group found in swine manure and six of them were identified Russell, 1979. Since the major product of this genus is lac- tic acid only a small amount of acetic acid produced by some of the species under heterofermentative con- dition, it could be assumed that the contributions to odor offensiveness by the biological activities of this bacterial group is not significant. 2.2.5. Escherichia The typical species included in this genus is Es- cherichia coli which is aerobic or facultatively anaer- obic, having both respiratory and fermentative types of metabolism. Glucose and other carbohydrates are fermented by the bacteria with the production of pyru- vate, which is further converted into lactic, acetic, and formic acids. The optimum growth temperature is 37 ◦ C. Most of the strains in this species 90–100 produce indole which is very odorous. 2.2.6. Clostridium Most species in genus Clostridium are obligately anaerobic, although tolerance to oxygen varies widely and some species will grow but not sporulate in the presence of air at atmospheric pressure. For most species, growth is most rapid at pH 6.5–7 and at temperatures 30–37 ◦ C; the range of temperature for growth is 15–69 ◦ C Cato et al., 1986. Clostridium often can ferment amino acids to pro- duce energy by oxidizing one amino acid and using another as an electron acceptor in a process called 96 J. Zhu Agriculture, Ecosystems and Environment 78 2000 93–106 ‘the Stickland reaction’ Prescott et al., 1996. This generates ammonia, hydrogen sulfide, fatty acids, and amines during the anaerobic decomposition of pro- teins. The volatile fatty acids produced by different species in this genus in order of quantities from high to low are acetic, butyric, caproic, lactic, formic, pro- pionic, succinic, valeric, iso-butyric, iso-caproic, and iso-valeric acids. Some of the species also produce indoles and phenols. There were four species in this genus found in the swine excreta Russell, 1979, but none of them were identified. The complexity of the odorous products produced by Clostridium shows a great potential that the species within this genus may be major contributors to swine manure odor. Therefore, to better understand the involvement of the bacteria in this group in producing different odorous compounds, further research is needed that should focus on the identification of the species found in swine manure. 2.2.7. Propionibacterium Propionibacterium contains genera which are ei- ther obligately anaerobic or aerotolerant. Bacteria included in this group can produce propionic acid and acetic acids and lesser amounts of iso-valeric, formic, succinic, and lactic acids. The optimum growth temperature is between 30 and 37 ◦ C and pH near neutral. There are only two species found in swine manure, i.e., Propionibacterium acnes and Propionibacterium granulosum. Both species have the general characters as discussed above; however, only P. acnes has the capability of producing indole. 2.2.8. Bacteroides Bacteroides are obligately anaerobic chemoorgan- otrophs metabolizing carbohydrates or peptone. Fer- mentation products include a combination of suc- cinic, lactic, acetic, formic or propionic, and butyric acids. When n-butyric acid is produced, iso-butyric and iso-valeric acids are also present. Growth usually progresses most rapidly at 37 ◦ C and pH near 7. Two species in this genus have been found in swine manure and only one of them has been identified B. ruminicola. B. ruminicola has an optimum growth temperature of 37 ◦ C with a growth range between 25 and 45 ◦ C Holdeman and Moore, 1974. 2.2.9. Megasphaera There are only two species M. elsdenii and M. cerevisiae in this genus listed in Bergey’s Manual of Determinative Bacteriology Holt et al., 1994. Bac- teria in this genus are anaerobic chemoheterotrophs that ferment lactate to produce acetate, propionate, and volatile fatty acids with carbon numbers from 2 to 6 including 4-carbon straight- and branched-chain acids. Sulfur containing compounds are also produced by this genus. The growth temperature ranges from 25 to 40 ◦ C and the optimal pH for growth is slightly above neutral pH ≈ 7.4. The bacterial profile discussed above may not com- pletely cover all the bacterial species in swine manure e.g., methanogens were not reported in any of the above studies, while it is a common bacterial genus in the gastrointestinal tract of all mammals; how- ever, it does provide information on most of the in- digenous genera in the swine manure, especially those producing odorous compounds. To relate these genera to malodor production requires a basic knowledge of the major odorous compounds in swine manure. Thus, a thorough review of the past research regarding the odorous compounds in swine manure appears essen- tial to determine the relationship between the bacterial genera and these compounds. 2.3. Odorous compounds in swine manure A considerable amount of research has been con- ducted in determining odorous compounds in swine manure Merkel et al., 1969; Barth and Polkowski, 1974; van Gemert and Nettenbreijer, 1977; Schaefer, 1977; Lunn and van De Vyver, 1977; Spoelstra, 1977; Spoelstra, 1980; Yasuhara and Fuwa, 1980; Williams, 1984; Yasuhara et al., 1984; O’Neill and Phillips, 1992. Generally speaking, the odorous compounds are produced and accumulated in the storage systems where the mixture of feces and urine is decomposed by bacteria under the prevailing anaerobic conditions. These compounds can be divided into four different chemical classes Mackie, 1994. 2.3.1. Volatile fatty acids VFAs Typical acids in this group consist of acetic, propi- onic, butyric, iso-butyric, valeric, iso-valeric, caproic, and capric acids. The VFAs can be produced from J. Zhu Agriculture, Ecosystems and Environment 78 2000 93–106 97 Table 1 The indigenous bacterial genera in swine manure and their odorous compounds Bacterial genera Potential odorous compounds Streptococcus formic, acetic, propionic, butyric acids, ammonia and volatile amines Peptostreptococcus formic, acetic, propionic, and butyric acids, iso-butyric, valeric, caproic, iso-valeric, ammonia and volatile amines, and iso-caproic acids Eubacterium formic, acetic, propionic, and butyric acids, iso-butyric, valeric, caproic, iso-valeric, iso-caproic acids, indoles and phenols Lactobacilli formic, acetic, propionic, and butyric acids Escherichia formic, acetic, propionic, and butyric acids Clostridium formic, acetic, propionic, and butyric acids, iso-butyric, valeric, caproic, iso-valeric, iso-caproic acids, indoles and phenols Propionibacterium formic, acetic, propionic, and butyric acids, iso-butyric, valeric, caproic, iso-valeric, iso-caproic acids, indoles and phenols Bacteroides formic, acetic, propionic, and butyric acids, iso-butyric, valeric, caproic, iso-valeric, iso-caproic acids, ammonia and volatile amines Megasphaera formic, acetic, propionic, and butyric acids, iso-butyric, valeric, caproic, iso-valeric, iso-caproic acids, volatile sulfur-containing compounds the deamination of amino acids that are produced dur- ing the process of protein degradation and breakdown of carbohydrates. In the gastrointestinal tract, a neu- tral pH 6–7 normally prevails. Under this condi- tion, deamination is the major route for metabolism of amino acids, which results in the production of VFAs, CO 2 , H 2 , as well as ammonia. Bacterial gen- era involved in this activity normally include Eubac- teria, Peptostreptococcus, Bacteroides, Streptococcus, Escherichia, Megasphaera, Propionibacterium, Lac- tobacilli, and Clostridium. 2.3.2. Indoles and phenols Indole, skatole, cresol, and 4-ethylphenol appear to be the major components included in this group of compounds. Phenolic compounds such as phenols and p-cresols are produced from the microbial degra- dation of tyrosine and phenylalanine in the intestinal tract of animals Ishaque et al., 1985. Metabolism of tryptophan can result in the production of indoleac- etate which is subsequently converted into skatole 3-methylindole and indole by a different group of bacteria Mackie, 1994. Bacterial genera involved in these processes include Propionibacterium, Es- cherichia, Eubacteria, and Clostridia. 2.3.3. Ammonia and volatile amines Volatile amines include putrescine, cadaverine, methylamine, and ethylamine. Usually, aliphatic amines methyl- and ethyl-amine are present at low concentrations. During the storage of fresh manure, amino acids can most likely undergo decarboxylation to produce putrescine, cadaverine, and ammonia. Bac- terial genera involved in this activity include Strepto- coccus, Peptostreptococcus, and Bacteroides. Another big source of ammonia is from urea and nitrates in addition to amino acid deamination Spoelstra, 1980. 2.3.4. Volatile sulfur-containing compounds Included in this group are sulfides as well as methyl- and ethyl-mercaptans. The sulfur-containing compounds are produced by bacteria through two processes, i.e., reduction of sulfate and metabolism of sulfur-containing amino acids. Sulfate reduction proceeds via either assimilatory or dissimilatory path- ways. In the first process, bacteria produce enough reduced sulfur for cell biosynthesis, while in the sec- ond process sulfate is utilized as terminal electron acceptor and large quantities of sulfide are produced Hao et al., 1996. Bacterial genera involved in this activity include Megasphaera. The odorous compounds as well as the bacterial species associated with these compounds are summa- rized in Table 1. It appears that all the listed bacte- rial genera found in swine manure are more or less related to different types of odorous compounds. For odor control only, it may not be realistic to deal with all the genera unless the manure needs to be steril- ized. Since one bacterial species may produce one or 98 J. Zhu Agriculture, Ecosystems and Environment 78 2000 93–106 more odorous compounds and different compounds may contribute differently to the overall odor offen- siveness of the manure, it seems helpful and worth- while to find the major odorous compounds as well as the bacterial species associated with them. Once the bacterial species producing the major odorous com- pounds are determined, effective strategies and tech- niques for controlling these bacterial activities may be developed. 2.4. Major odor indicators and the related bacterial genera Research on the major indicators for malodors of swine manure has been carried out for many years. Merkel et al. 1969 found alcohols were unimportant in determining the nature of swine confinement ma- nure odors. Barth and Polkowski 1974 reported that the volatile organic acids correlated best with the odor intensity. Ammonia was thought to be useful as an in- dicator for malodor, but in spite of the relatively high concentrations and the easy determination ammonia was proved to be a poor parameter in evaluating odor intensities Lunn and van De Vyver, 1977. A study conducted by Spoelstra 1977 showed that indole and skatole could not be recommended as indicators for malodor because the concentrations of these two com- pounds might decline during storage. Later, Spoelstra 1980 reported in another study that both ammonia and hydrogen sulfide were not suitable indicators for the smell. The most pungent and the greatest variety of obnoxious smelling compounds originate from the de- composition of proteins. Ammonia does not reflect this degradation kinetics of the manure because the major part of ammonia in the manure originates from urea hydrolysis. Moreover, ammonia remains unchanged by methanogenesis and shows a retarded reaction to aerobic treatment compared with organic volatiles. Hydrogen sulfide formation also does not reflect ma- nure degradation kinetics because a relatively large part is derived from sulfate reduction. He concluded that the VFAs seemed to be useful indicators to test whether an effect has occurred in all odor-abatement methods. The VFAs show typical reactions for the group of accumulated volatile compounds when en- vironmental changes are made in swine manure to diminish odor. Williams 1984 found that the most widely applicable indicator was supernatant biochem- ical oxygen demand BOD both during aerobic treat- ment and post-treatment storage; VFAs, total organic acids, indoles and phenols can indicate acceptable and unacceptable limits of offensiveness during aero- bic treatment and post-treatment storage; sulfide is a misleading indicator during aerobic treatment but is a useful indicator during post-treatment storage; and ammonia is of no value as an indicator. A study con- ducted by Zahn et al. 1997 reported that the volatile organic acids with carbon numbers from 2 to 9 specif- ically demonstrated the greatest potential for the de- creased air quality. Therefore, according to the above researchers, it appears that VFAs could be used as a suitable odor indicator for swine manure. However, in recent years, it has been shown by a few studies that different VFAs will have differ- ent contribution to the odor generation. Thus, a fur- ther classification within this group is needed to deter- mine the potential of producing malodors by different VFAs so the specific bacterial species involved can be studied. The odorous nature of VFAs progresses from pun- gent odors of formic and acetic acids to the distinctly unpleasant and offensive odors of valeric and caproic acids Morrison, 1987. Although the short chain acids are present in much higher concentrations and have higher volatility, the VFAs with higher carbon num- bers have lower odor detection threshold thus are more offensive in nature Mackie, 1994. Therefore, the high concentration of VFAs in swine manure may not nec- essarily cause high intensity of malodor as a large por- tion of the VFAs could be composed of short chain acids with less odor potential. A study conducted by Zhu et al. 1997 appeared to provide evidence sup- porting this argument. They evaluated five commer- cial pit additive products and found that some products could reduce odor threshold without significantly re- ducing the total amount of VFAs. The offensive odor potential was not directly associated with the total con- centration of VFAs in the manure. It was depending upon the types and characteristics of certain acids not necessarily existing in high concentrations in the ma- nure. Therefore, the purview of VFAs responsible for odor generation can be narrowed down to those with long carbon chains C 10 or branchings. The acids in this group include iso-butyric, valeric, iso-valeric, caproic, and iso-caproic acids. The bacterial genera J. Zhu Agriculture, Ecosystems and Environment 78 2000 93–106 99 Table 2 pH and temperature ranges for growth of the related bacteria Bacterial genera pH a Temperature ◦ C a Oxygen tolerance Peptostreptococcus 6–8 25–45 35–37 No Eubacteria 6.5–7.5 20–45 37 No Clostridium 6.5–7 15–69 30–37 No for most strains Propionibacterium 6.5–7.5 30–37 35 No to aerotolerant Bacterioides 5–8.5 25–45 37 No Megasphaera 7.4–8.0 25–40 30 No a Numbers in parenthesis are optimum conditions. producing these compounds include Peptostreptococ- cus, Clostridium, Bacteroides, Eubacteria, Propioni- bacterium, and Megasphaera Table 1. The bacterial genera in Table 1 are normally active in the specific environment, e.g., in the gastrointesti- nal tract of animals, where the bacteria can effectively perform their metabolic activities for growth. That en- vironment may change or even no longer exist once the feces are excreted. Since swine manure contains sufficient nutrients for bacterial growth, the limiting factors that could affect the growth of these bacterial genera are most likely pH and temperature. The suit- able ranges of these two parameters for the growth of different bacterial genera are presented in Table 2. De- viations in pH and temperature from the values listed in Table 2 may not completely stop the bacterial activ- ities in producing volatile fatty acids, but may affect the productivity of different bacterial genera in vary- ing degrees. According to Table 2, genus Clostridium has the widest temperature range for growth among the bac- terial genera. Thus, as compared with other bacterial genera, Clostridium could be more active in producing odorous acids in the real manure storage environment, especially at low temperatures. Since little appears to have been made in systematically studying the mi- crobes at the species level in swine manure under the storage environment, experimental data that estimate the contributions of this genus to the VFA produc- tion on a quantitative basis are not available. However, there is plenty of evidence showing that Clostridium, if the environment favors the growth of this group of bacteria, is a major genus that is responsible for pro- ducing all types of VFAs through amino acids fer- mentation Gunsalus and Stanier, 1961; Mead, 1971; Gottschalk, 1985; Hill, 1986. In operating lagoons or earthen basins, the storage temperature of wastes usually ranges from 10 to 20 ◦ C, depending upon the season Spoelstra, 1980. Local lagoon waste temper- ature variation has also been reported by several other researchers Ohio, 2–27 ◦ C White et al., 1977; Okla- homa, 0–30 ◦ C Rice, 1977; Georgia, 3–25 ◦ C Smith and Franco, 1985. The temperature range for ma- nure stored in the pits is between 2 and 18 ◦ C Don- ham et al., 1985. The pH range for swine manure is between 6.5 and 7.5 Cooper and Cornforth, 1978. Comparing these situations to the parameters listed in Table 2, plus the anaerobic environment that always exists in the liquid manure storage systems where aer- ation is not available and the availability of nutri- ents, would suggest that Clostridium very likely is a genus that can play a major role in producing odorous VFAs. Genus Eubacterium could also make significant contributions to generating odorous acids. Although it has a narrower temperature range than Clostridium, it has the largest population among the genera listed in Table 2 and most of the strains produce long-chain fatty acids. So it is reasonable to assume that, under the manure storage environment, the major portion of the odorous VFAs could be produced by these two genera. A substantial increase in various VFAs after manure stored anaerobically for 24 h were observed by Williams 1981, which could be due mainly to the active growth of these bacteria. However, this hypothesis needs further study. It is also reasonable to assume that controlling the bacterial growth in these two groups may help reduce malodor genera- tion. Unfortunately, a lack of experimental data to verify this postulate and the incomplete identifica- tion of the species within these two groups make it difficult to draw specific conclusions in terms of the odor-related acids production with regard to different bacterial species. Therefore, further research on these 100 J. Zhu Agriculture, Ecosystems and Environment 78 2000 93–106 genera seems of significance in determining the types and quantities of odorous compounds produced by different species in these two groups. As mentioned above, pH could be a factor that af- fects bacterial growth. It can be seen from Table 2 that all the bacterial genera have a neutral or near neutral pH for their growth. This offers an opportunity to reg- ulate bacterial growth by adjusting the pH in manure liquid. This can be achieved more easily than control- ling temperature that is neither practical nor effective at the farm level. There have been several studies in which alkaline materials were added into manure to increase the manure pH Hammond and Day, 1968; Veenhuizen and Qi, 1993; Vincini et al., 1994; Bundy and Greene, 1995. These studies demonstrated odor reductions in varying degrees when manure pH was raised to a range of 8–11. However, none of these studies presented an explanation for the mechanisms. Based on previous discussions, it could be concluded that one major reason that the raised pH could reduce odor is that it inhibits the growth of those odor-causing bacteria indigenous to swine manure. Another mech- anism of reducing odor by alkaline materials is due to the precipitation of volatile fatty acids by forma- tion of salts. At high levels of pH, the formed salts will not be converted back to acids; thus, both the lev- els and volatility of the odorous acids will be reduced Rainville and Morin, 1985; Morrison, 1987. One problem associated with the pH adjustment is the emission of large quantities of either ammonia at the raised pH or hydrogen sulfide at the lowered pH from the treated swine manure. The emission of these two gases may cause severe problems to the environment and losses of animals and human lives under certain conditions. Therefore, to avoid the po- tential damage caused by the emission of these two gases during the pH adjustment, it would be better to treat fresh manure instead of aged manure. In fresh manure, the bacterial activity of decomposing organic substances to form ammonia and hydrogen sulfide has not fully developed, so the volatile portion of the gases is relatively low. Accordingly, these two gases may not reach a threatening level on both the environment and the properties in a short time period. The problem with this treatment is that continuous adjustment of pH has to be conducted to maintain the adjusted pH. Other- wise, due to the biological activities, pH will change and odor may return during the manure storage time.

3. Odor control techniques