Persistence of pathogens in liquid pig manure processed in manure tanks and biodigesters
Persistencia de patógenos en porcinaza líquida procesada en tanques estercoleros y biodigestores
Oscar Betancur H,1 Ph.D, Antonio Betancourt E,2 Ph.D, Julián Estrada A,3 Ph.D, Francisco Henao U,3* Ph.D.
1Elanco, Animal Health, Transversal 18 96-41, Bogotá D.C., Colombia.
2Corpoica, 140 street No 26 - 93, tower 2, Apartment 202, Floridablanca, Santander.
3Caldas University, Faculty of Agricultural Sciences, Doctorate in Agricultural Sciences, Headquarters 65 street N° 26-10, Manizales, Colombia.
Received: November 2014; Accepted: July 2015.
Objective. To evaluate the persistence of virus, bacteria, mold, yeast and parasites in liquid pig manure, processed in biodigesters and manure tanks in the central-western part of Colombia. Materials and methods. A directed observational study analyzed descriptively was carried out in three pig farms located where the manure tanks were assembled and its biodigesters were used. A sampling of liquid pig manure was taken to assess the presence of 26 pathogens at the beginning of the study and another one at the end of the process in manure tanks and biodigesters. For the manure tank, a 250 liters tank was filled with fresh pig manure and was analyzed after three days of storage. The biodigesters were of continuous flow and its effluents were analyzed, according to the specific hydraulic retention times. The diagnostic techniques were those recommended specifically for each microorganism and were carried out in certified labs by the Colombian Animal Health authority. Results. Of the 26 pathogens that were investigated, 15 appeared in the fresh pig manure used in pig manure tanks and 12 in the one used in biodigestors. In manure tanks, Porcine Circovirus type 2 (PCV2), mold, yeast, Salmonella spp., Balantidium coli and Strongylids did not persist. In biodigesters, PCV2, yeast, Strongylids, B. coli and Strongyloidesspp., did not persist. Conclusions. In both manure tanks and biodigesters, a variation could be seen in pathogen persistency, indicating that they act as transformation systems of pig manure for the removal of the latter, as long as the storage times are increased if the efficiency wants to be improved.
Key words: Bacteria, mould, parasite, virus, yeast (Source: CAB).
Objetivo. Evaluar la persistencia de virus, bacterias, mohos, levaduras, y parásitos en porcinaza líquida, procesada en biodigestores y tanques estercoleros en el centro–occidente de Colombia. Materiales y Métodos. Se realizó un estudio de observación dirigida analizada descriptivamente en tres explotaciones porcinas, donde se montaron tanques estercoleros y se aprovecharon sus biodigestores. Se realizó un muestreo de porcinaza fresca para analizar la presencia de 26 patógenos al comienzo del estudio y otro al final del proceso de estercoleros y biodigestores; los muestreos se repitieron en dos momentos en las tres granjas. Para el estercolero, se llenó un tanque de 250 litros con porcinaza fresca y se analizó después de tres días de almacenamiento. Los biodigestores fueron de flujo continuo, y se analizaron sus efluentes, según los tiempos de retención hidráulica específicos. Las técnicas diagnósticas fueron las recomendadas específicamente para cada microorganismo y se ejecutaron en laboratorios certificados por la autoridad sanitaria colombiana. Resultados. De los 26 patógenos investigados se detectaron 15 en la porcinaza fresca usada en estercoleros y 12 en la utilizada en biodigestores. En tanques estercoleros Circovirus Porcino tipo 2 (PCV2), mohos, levaduras, Salmonella spp., Balantidium coli y estrongilidos no persistieron. En biodigestores PCV2, levaduras, estrongilidos, B. coli y Strongyloidesspp., no persistieron. Conclusiones. Tanto en estercoleros como biodigestores se observó variación en la persistencia de agentes patógenos, indicando que funcionan como sistemas de transformación de la porcinaza para la remoción de éstos, siempre y cuando se aumenten los tiempos de almacenamiento si se quiere mejorar su eficiencia.
Palabras clave: Bacterias, levaduras, mohos, parásitos, virus (Fuente: CAB).
Pork is the most consumed animal protein in the world and the demand continues to grow. Its share of the world market is 37.4%, which makes pig farming a highly important agricultural activity in the world (1). According to the Colombian Association of Pig Farmers (2), in Colombia the domestic pork market reached 3,030,043 heads slaughtered in 2013, which represents approximately 254,000 tons of meat and indicates a growth of 2.2% compared to 2012. This growth is expected to continue in the coming years; per capita consumption in Colombia is 6.7 kg, which shows great future business opportunities. However, the large amount of pig manure that is produced daily limits the expansion of this industry, since it is associated with pollution and transmission of pathogens. Therefore, it is imperative to adequately dispose of and process pig manure so that it goes from being a source of pollution and health problems to a source of income for the producer. Proper use of pig manure may lead to it being used as a strategic bio-fertilizer or as raw material for animal feed.
Pig manure consists of feces, urine, bedding waste, rinse water, secretions (nose, throat, vagina, uterus, mammary gland, etc.), skin, hair, blood residue, and food waste. Organisms present in this mixture have the potential to persist in products derived from processing pig manure (3). To reduce the risk of contamination, pig manure should be treated using methods that ensure safety.
Diverse biological, physical and chemical processes to control pathogens in pig manure have been reported in literature, including: silage, composting, drying, earthworm composting, and the use of biodigesters, generally preceded by storage in manure tanks. For proper operation, with the exception of manure tanks, it is necessary to separate solid and liquid fractions of pig manure to aid in eliminating pathogens (4).
To process liquid pig manure in the pork industry in Colombia, it is common to use manure tanks and biodigesters. Manure tanks are designed to store manure (5); in the case of pig manure, the manure tank is used to store the complete mixture without prior separation. Normally storage time is three days, after which its contents are dumped in a fertilization field or an anaerobic fermentation system in a digester. So far, no formal reports have been done on the effectiveness of manure tanks in eliminating pathogens; Fongaro et al (6) reported the presence of Salmonella spp. and PCV2 in fresh liquid pig manure collected from a manure tank, without taking into consideration the time period it was retained in order to establish the capacity this system has to remove pathogens. The manure tank, therefore, is not strictly a mechanism to process pig manure.
It is important to clarify that in some farms tanks similar to manure tanks are used to store effluent from the biodigesters. Biodigesters are bio-reaction systems built to submit the liquid phase of agricultural waste, manure or industrial effluents to anaerobic fermentation in order to recover energy and materials (biogas, bio-fertilizer, and potentially useful substrates in various agricultural and agro-industrial processes) (7).
Notably, biodigesters also offer the possibility to control the spread of pathogens (8), main factors that influence pathogen reduction, which include: microbial competition, nutrient availability (9), temperature, pH, concentration of free ammonia and volatile fatty acids, biodigester design, and hydraulic retention time (HRT) or average time that the raw material is kept within the digester (10). To ensure an adequate biodigestion process, it is important that pig manure be sufficiently homogenized, and HRT should be sufficient to prevent endogenous bacteria from being expelled at a higher rate than that of reproduction (11). In this regard, Massé et al (12) reported significant reductions in the concentration of total and fecal coliforms, Escherichia coli, Salmonella spp., Campylobacter spp. and Yersinia enterocolitica when HRT was used for 7 to 14 days. In this sense, the Chen et al group (13) found that the rate of pathogen removal becomes more efficient as HRT increases from 11 to 25 days.
This study was done to help clarify real health risks from pig manure by evaluating the persistence of virus, bacteria, mold, yeast, and parasites in liquid pig manure processed in biodigesters and manure tanks in central-western Colombia.
MATERIALS AND METHODS
Type of study. A descriptively analyzed directed observational study was conducted.
Study site and population. The study was conducted in three full-cycle pig farms in central western Colombia, on a farm located in Pereira at 4°50’34” N; 75°46’02” W, on 800 sows (large farm), at an altitude of 1261 meters, average temperature of 25-30°C; another on 500 sows (median farm) in Villamaría at 5°01’46” N; 75°31’36” W, at 1850 meters, with an average temperature of 21-22°C; and 280 sows (small farm) in Santa Rosa de Cabal, at 4°50’34” N; 75°46’02” W, at 1450 meters, average temperature 22 to 26°C.
Treatment routes and sampling days. In 2013 fresh pig manure was sampled at baseline (June 4) and again when each route was processed (06, 07 and June 11); sampling was repeated on three farms (July 29 for fresh pig manure and July 31, August 01 and 05 for processed pig manure). On each farm a 250 plastic liter container was used as a manure tank, which was filled with 250 liters of fresh pig manure (38 kg solid and 212 L liquid parts) and was stored for three days. Continuous flow polyethylene tube biodigesters (14) were used in the study with the following HRT: 2.55 days on the small farm, 2.65 days on the median farm and 6.8 days on the large farm. Manure tanks were sampled at the end of the third day of storage and the biodigesters according to the respective HRT; sampling was conducted following the regulations of the Colombian Technical Standards NTC-ISO 5667-1 (15). Samples were transported in sterile sealable bags with 75g and 150g frozen Biothermics® gels according to manufacturer’s indications to preserve it at 4-8°C, and the samples arrived at the laboratory in a period of less than 24 hours.
Pathogens and diagnostic techniques. In fresh pig manure the presence of the following pathogens (Table 1) was analyzed:
Table 1. Studied microorganisms and diagnostic methods.
All the diagnostic tests were done in laboratories backed by the Colombian Agricultural Institute (ICA).
Selection criteria. The inclusion of the above pathogens in this study was done using the following criteria: PRRS, PCV2, PPV, L. intracellularis, A. pleuroneumoniae, for its high prevalence and economic impact. PCV1 was considered the control of PCV2. A. suum, E. coli, L. monocytogenes, Salmonella spp., Giardia spp., Leptospira spp., S. aureus due to the zoonotic risk involved. The virus for Aujesky’s disease because of the risk it represents despite not being officially reported in Colombia. Aerobic mesophilic bacteria, total coliforms, Clostridium sulfite reducers, molds and yeasts since over time they are indicators of microbial activity. The other bacteria and parasites (T. suis, B. coli, G. intestinalis, Strongyloidesspp., Metastrongylus spp., coccidia and strongylid) because of their economic impact, epidemiological importance and because they are potentially pathogenic to other species, including humans.
In this study the persistence of viruses, parasites, A. pleuropneumoniae, Leptospira spp., L. monocytogenes, L. intracellularis and Salmonella spp., was assessed from the presence of the organism, while for those diagnosed by counting (aerobic mesophilic bacteria, S. aureus, total coliforms, E. coli, Clostridium sulfite reducers, molds, yeasts), persistence depended on the final number of each agent in excess of the maximum permitted levels according to the reference values in table 2.
Table 2. Maximum counts adopted for ruminant feed.
Table 3 shows that of the 26 pathogens studied, only 15 were found in fresh pig manure which the manure tanks were filled with; after three days of storage 9 of them persisted (L. intracellularis, mesophilic aerobic, E. coli, total coliforms, S. aureus, Clostridium sulfite reducers, L. monocytogenes, Strongyloides spp., coccidia); therefore, the other 6 disappeared during this period. E. coli, total coliforms, and Clostridium sulfite reducers persisted on the 3 farms; aerobic mesophilic bacteria persisted in the 3 farms but only in the largest it persisted in 2 samples; S. aureus persisted on the 3 farms, and did not persist in one sampling of the large farm; L. intracellularis persisted in the largest farm, in the median one it appeared in only one of the samples and did not persist, and in the small one it appeared in two samples but only persisted in one. Strongyloides spp., persisted only in a sampling of the median farm. Coccidia persisted on the 3 farms, except in the second sampling of the small farm.
Table 3. Pathogens found in manure tanks on three farms in central western Colombia.
In data recorded in table 4, it can be seen that 12 of the 26 agents included in the study were initially diagnosed in liquid pig manure used to feed biodigesters, and 8 persisted (L. intracellularis, aerobic mesophilic bacteria, S. aureus, Clostridium sulfite reducers, total coliforms, E. coli, Salmonella spp., coccidia). E. coli persisted in the 3 farms; L. intracellularis persisted in the small farm, appeared in the median one in only one of the samples and persisted, and appeared in the large farm in the 2 samples but persisted in just one. Aerobic mesophilic bacteria persisted in the 3 farms but only in the first sampling. S. aureus persisted in median and on the small farm in one of the samples, while in the large farm it did not persist. In the case of Salmonella spp., these appeared only with the first sampling of the 3 farms and persisted in the median and small one, but in the large farm it did not persist. Clostridium sulfite reducers persisted in the medium and small farms, and the large one, in spite of appearing in the 2 samples, persisted in just one. Coccidia was observed in just one of the samples of the large and small farms, and persisted in both cases.
Table 4. Pathogens found in biodigesters on three farms in central western Colombia.
Pig manure is considered to have a leading role in the transmission of various pathogens (virus, bacteria, molds, yeasts and parasites), so its use in agricultural and agro-industrial processes is considerably limited. From previous experience we know that certain etiological agents can be removed completely or partially by processing pig manure using tanks, biodigesters, sun-drying, silage, worm composting and by manufacturing worm flour. The strategic use of pig manure, either in biofertilization or in animal feed, could be possible if preceded by similar processes, and its true role in the transmission of pathogens would be determined by persistence in the end product, or in other words, if these procedures are able to remove them.
Of the 26 pathogens initially investigated, 15 were found in fresh pig manure used in the manure tanks, and 12 in manure used in the biodigesters, perhaps because they were not present in the farms studied (negative farms), or due to differences in health and biosecurity conditions, or due to very low prevalence at the time of sampling. Also, the presence of antibiotic residues that modify the bacterial populations in pig manure should not be ruled out (23). Meanwhile, persistence could be due to short storage processing times, especially in the case of high amounts of pathogens. For this reason, some authors (13, 24) consider longer retention times than those used in this study to be necessary.
In the viruses evaluated in this research, only PCV2 was found in both fresh pig manure used for the process and biodigesters (Tables 3 and 4), and in both cases it was removed. This virus has been found by other authors in liquid pig manure and has persisted in this environment even after 30-40 days (6); these authors argue that PCV2, a DNA virus, is resistant to disinfection and is commonly excreted in pig feces and urine; for those reasons, they are considered bioindicators of environmental quality. Although in this study it was not possible to pinpoint what caused the removal of the viruses found in fresh pig manure, there is evidence that in liquid pig manure there are some proteolytic or ammonium ion compounds that have deleterious effects on these agents (23).
In the case of L. intracellularis, there was persistence both in manure tanks and biodigesters. Despite the clinical and economic importance of L. intracellularis, its efficiency has not been considered in studies of biodigesters or manure tanks to treat pig manure, possibly because it is a pathogen that does not affect other species of agricultural interest, it is not zoonotic, or due to difficulties in cultivating and identifying it. Lately, identifying it by PCR has been recommended; however, this technique may indicate an erroneous pattern in the persistence value found, given their ability to use DNA from dead cells (13).
In this study the persistence of aerobic mesophilic bacteria was found both in manure tanks and biodigesters. For manure tanks, it could be explained by the environmental conditions within the tanks, since during pig manure storage aerobic conditions are present on the surface, but 30 cm from the surface anaerobic conditions prevail (25). Biodigesters in this study showed persistence in only one sample. Chen et al (13) mention that different HRT lead to modifying the structure of the microbial community, which can have an effect on the survival of pathogenic bacteria.
S. aureus persisted both in the manure tanks and biodigesters. This is a facultative anaerobic bacteria, and this could favor its survival in both systems. Although the ammonium present in the liquid excreta has a bactericidal effect, given the lower cell permeability of gram positive cocci it is less susceptible than other bacteria (26). According to Poudel et al (27), it is only possible to eliminate S. aureus after a HRT of 10 days, and this study reinforces this approach, since the longest HRT was 6.8 days.
As for molds and yeasts, persistence was not found in manure tanks or biodigesters. There are few data on yeast and mold counts in animal manure (4). Greater amounts of bacteria than mold has been found in pig manure treated by anaerobic digestion because bacteria has a greater role in converting organic waste during this process (28). This study demonstrates the efficiency of treatment systems for liquid pig manure to remove these agents, given its susceptibility to ammonia and humic substances (28).
Meanwhile, Salmonella spp. was found to be persistent in biodigesters but not in manure tanks. Under the conditions of this study, this agent did not show persistence in the tanks. Possible reasons for this elimination could be high concentrations of ammonium (26) and the presence of native flora which directly competes due to the substrate (9). Other authors have found Salmonella spp. to persist in pig manure treated in anaerobic biodigesters even after 30-40 days, although the strains were not always pathogenic (6). Increased HRT contributes to inactivating Salmonella spp. (13). In this study, short retention times probably did not allow the complete removal of Salmonella spp., in biodigester effluent. Perhaps variations found among the authors consulted and the results of this study could be explained by the absence or presence technique, where 25g of sample was used to detect very low amounts of Salmonella spp., which would depend on the prevalence of the disease on each farm.
Clostridium sulfite reducers persist in manure tanks and biodigesters. This can be explained because it is an anaerobic spore form that can resist and multiply in low redox potentials, and it is therefore likely to survive the anaerobic digestion of manure (3). The persistence of this bacteria has been reported in psychrophilic anaerobic digestion processes of pig manure using sequential batch reactors (12).
Total coliforms and E. coli persisted both in manure tanks and biodigesters. Son et al (26) added 2% urea (ammonium) in manure tanks and after two weeks obtained <5 CFU/g values for total coliforms and E. coli due to the pH increase. Unlike this study, the manure tanks received no treatment. It can clearly be seen that retention times required for complete elimination of coliforms within a biodigester are greater than those in this study (maximum 6.8 days).
Anaerobic digestion has been highly efficient in removing E. coli with a HRT of 25 days (13). In this study E. coli persisted on all farms at all times; this can be explained by the short storage time. A difficulty that may arise when evaluating continuous flow anaerobic digesters is the lack of correlation between HRT and the reduction of pathogens such as E. coli, since a certain amount of pig manure passes quickly through the biodigester, since these biodigesters are continuously fed fresh pig manure. Low retention times could not only lead to increased survival of the pathogenic but also to increased problems with odors of effluents (29). Adding urea (ammonium) or increasing retention times are alternatives to be evaluated in further studies in order to minimize the risk posed by these microorganisms.
L. monocytogenes was observed and persisted in one sample manure tank. It is possible that the combination of an environment poor in organic matter, rich in salt, and an alkaline pH favored the survival of this pathogen that is known to withstand extreme stress conditions (30).
As for parasites in the manure, persistence of B. coli or Strongylid is not observed, while coccidia persisted. As Son et al (26) observed, conditions that favor high ammonium concentrations may inactivate parasites. As for Strongyloides spp., persistence was observed but it is known that free-living states of Strongyloides spp., have a short duration and are negatively affected by adverse environmental conditions. The persistence of coccidia may be explained by the liquid medium which favors the development of parasites and additionally due to its ability to form oocytes that are highly resistant to unfavorable environmental conditions. In nature, oocytes probably survive for weeks or months. In the case of biodigesters, persistence of Strongylid and Strongyloides spp., was not found, but coccidian was found. Biological considerations of these parasites described in pig manure tanks apply similarly to biodigesters. Cañon-Franco et al (24) evaluated the efficiency of anaerobic digesters in eliminating parasites after 15 days of HRT, finding that the time is insufficient for complete removal of Isospora suis oocytes and different species of Eimeria such as Strongyloides ransomi eggs. In this study the results are similar in terms of the persistence of coccidia, but not in terms of Strongyloidesspp. The persistence of coccidia on both routes could be explained by its cystic nature, which is very resistant to adverse environmental conditions. In this study, retention times were low compared to reports in literature, however, it was possible to eliminate some of the parasites. Very short HRT does not allow adequate removal of pathogens, particularly of parasite eggs, which have greater resistance to environmental stress as compared to viruses and bacteria (8).
In this study the results for manure tanks show that PCV2, molds, yeasts, Salmonella spp., B. coli and Strongylid did not persist, while L. intracellularis, aerobic mesophilic bacteria, S. aureus, Clostridium sulfite reducers, total coliforms, E. coli, L. monocytogenes, Strongyloidesspp., and coccidia did persist. On the other hand, in the biodigesters it can be seen that PCV2, yeast, strongylid, B. coli and Strongyloides spp. did not persist, while L. intracellularis, aerobic mesophilic bacteria, S. aureus, Clostridium sulfite reducers, total coliforms, E. coli, Salmonella spp., L. monocytogenes, and coccidia did.
Under the conditions of this study it was not possible to guarantee the safety of pig manure since some pathogens managed to persist in spite of various treatments. However, both manure tanks and biodigesters showed variations in persistence, indicating that they work as systems to transform pig manure to remove some pathogens, though it is necessary to increase storage times to improve efficiency, which, for the biodigesters, can be improved by increasing the size or by using less wash water in the facilities.
It is important to consider for further studies if the eggs or larvae of parasites, as well as viruses and bacteria, found to be persistent are viable or not. In the case of Salmonella spp., it is necessary to verify whether they are pathogenic or non-pathogenic strains, and for E. coli differentiate the type of adhesins, since these are specific to each species; for pigs K88, K99 for cattle.
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