The Sanitation of Animal Waste Using Anaerobic Stabilization

Handling, storage, treatment and use of different forms of animals excrements entails two principle problems: epizootological or epidemiological and hygienical. Solid excrements contain high numbers of common intestinal microflora (E. coli, faecal streptococci, lactobacilli etc.), bacteria that are pathogenic also for man (salmonellae, mycobacteria, listeriae etc.), protozoa (Isospora spp., Balantidium coli) and eggs or larvae of enteronematodes (Ascaris suum, Oesophagostomum sp., Trichuris suis etc.) (Lauková et al., 2000; Krupicer et al., 2000).


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developmental stages (oocysts, eggs, larvae). Through faeces of infected dogs and cats the germs of parasitozoonoses spread into the environment. It is especially the case of cysts of intestinal parasitic protozoa -Entamoeba histolytica, Giardia intestinalis, Balantidium coli, Toxoplasma gondii, the eggs of tapeworms Dipylidium sp., Echinococcus sp. and parasitic nematodes (Antolová et al., 2004;Matsuo and Nakashio, 2005;Miterpáková et al., 2006). Regarding public health helminthozoonoses caused by Toxocara sp. (in dogs) and Ascaris sp. (in pigs) are very significant, especially due to their zoonotic character connected with the syndrome larva migrans.
Humans became infected usually orally (per os) by ingestion of substrates (soil, vegetables, etc.) with embryonated Toxocara eggs. Many symptoms are associated with this infection, including changes in blood cell counts and affection of various organs, as the ascaris larvae can migrate throughout the body. The symptoms of infection are often non-specific and may be mistaken with those of other infectious agents (common viral diseases, diarrhoea) or we may not observe any clinical signs. Toxocariasis manifests itself in two distinct forms: visceral, larva migrans visceralis, and ocular, larva migrans ocularis (Despommier, 2003).
A. suum infects pigs and is of major economical significance due to production losses linked to reduced feed conversion efficiency and losses to the mean industry associated with the condemnation of "milk-spot" livers . Ascaris infects over a quarter of the world´s human population (1.47 billion people worldwide) and clinically affects ~335 million people (Crompton, 1999).
The above-mentioned helminthozoonoses are classified among epidemiologically "low-risk" parasitozoonoses, because the propagative stages develop in the outdoor environment into the infectious stage and potentially secondarily contaminate the food chain. Therefore, a direct contact with infected animal, but also contaminated environment, or contaminated food chain (water, vegetables) are considered as a potential risk factor.
Attention has receantly been paid also to the problem of hygienical hazards in terms of the treatment and use of animal excrements and their application to soil as valuable nutrients for cultivated plants. The hazards are mainly connected with the quantity of continually produced solid and liquid wastes. The results is ecological disbalance, mainly with respect to environmental load with pathogenic microorgamism and nitrous organic substances. Animal organic wastes are also sources of greate amounts of gases releases. The most dangerous of them are ammonia and methane. Ammonia released into the atmosphere is irritating and toxic to the biotic component of the environment. On the other hand, animal excrements can supply essential plant nutrients and improve the fertility of soil by adding organic matter. Therefore, to prevent health risks (for human as well as for animals) and odour nuisance from animal wastes, different methods for a satisfactory utilisation and sanitation have been researched (Schwartzbrod et al., 1989, Tofant et al., 1999Juriš et al., 2000;Sasáková et al., 2005;Papajová and Juriš, 2009). There are big variations in the treatment of animal wastes (aerobic and anaerobic stabilization, composting etc.).
For the above-mentioned reasons our studies concentrated on anaerobic stabilization of liquid (slurry) and solid (manure, excrements) animal waste. The aims of our study were to www.intechopen.com The Sanitation of Animal Waste Using Anaerobic Stabilization 51 monitor the physical-chemical changes in pig slurry treated by ecologically acceptable and energetically beneficial anaerobic stabilisation, changes in the properties of anaerobically digested slurry stored in ground lagoon and the effect of anaerobically digested slurry stores in ground lagoon on the survival of parasitic germs. The impact of lime (two types) on the survival of parasitic organisms in anaerobic stored manure and dog excrements mixed with hay, was also studied.

Parasitological methods
To determine helminth eggs count in slurry (input and output samples from bioreactor and in lagoon samples -supernatant and sediment), 50 ml from each of the 1 l sample was taken and examined by a sedimentation-flotation mathod (Cherepanov, 1982).
A. suum eggs were isolated by dissection of a distal uterine part of female pig ascaris. The distal uterine ends were then removed to a glass homogenizer and processed. The water suspension of eggs was stored in an Erlenmayer flask in a refrigerator at 4°C.
We used the ''artificial contamination of lagoon and organic wastes'' approach to make sure that there is s sufficient number of positive samples in our observations. Model eggs were inoculated by a micropipette into polyurethane carriers, prepared according to Plachý and Juriš (1995), at a dose of 1 000 eggs per one carrier. A porous cellular plastic -soft expanded polyurethane, commercially known as a plastic foam, was used as a material for the carriers. It is an additive product of polyisocyanates and compounds with a high content of hydroxylic groups. It consists of a network of interconnected cells, resembling a honeycomb. Its polyurethane structure allows for a sufficient contact of helminth eggs with the environment, preventing them from release and consequently improving their recovery (Picture 1). For mechanical protection the carriers were placed to perforated plastic net (Picture 2) before introducing them into the organic wastes. Three samples were taken and analysed at each sampling interval. The eggs were re-isolated from the inoculated carriers as follows: the carriers were cut into small pieces and washed in a mortar with 3 · 5 ml portions of saline, thoroughly stirred and filtered through a sieve into test tubes. After centrifugation, sediments were transferred to Petri dishes.
The viability of exposed unembryonated A. suum eggs was determined by incubation up to the embryonated stage (Picture 6) in a thermostat at 26°C for 21 days. Petri dishes with A. suum eggs were aerated daily with micropipette. The developmental ability of A. suum eggs was compared with that of the control eggs which were kept in distilled water under aerobic conditions.

Physical and chemical methods
The following changes in physical and chemical properties of the solid and liquid wastes were monitored: pH, dry mater (DM), inorganic (IM) and organic (OM) matter, ammonium ions (NH 4 + ), total nitrogen (N t ), chemical oxygen demand (COD), soluble and insoluble substances and C:N ratio. Picture 1. Polyurethane carrier with adhered A. suum eggs Picture 2. Perforated plastic nets with the carriers The samples were examined for the pH (1:10 water extract) using a pH electrode (HACH Company, Loveland, Colorado, USA). Dry matter (drying at 105°C to a constant weight), residum-on-ignition (550°C for 4 h), and water soluble ammonium nitrogen (NH 4 + ) by titration (Mulvaney, 1996). Soluble and insoluble substances were determined by evaporation of the known amount of homogeneous sample filtrate on a water bath after absorption of insoluble substances on a filter, drying the evaporation residue at 105°C and determining its weight. COD was determined on the basis of organic substances oxidation in sample by potassium dichromate in sulfuric acid medium during 2-hour boiling in a COD reactor (HACH Company, Loveland, Colorado, USA). Portion of samples for N t determinations were digested using a HACH-Digesdahl apparatus (HACH Company, Loveland, Colorado, USA). N t was distilled with NaOH (40 %) (Bremner, 1996). The C content was calculated according to the content of OM by the method of Navarro et al. (1993) to obtain the C:N ratio.

Statistical analysis
The physical and chemical properties (pH, DM, IM, OM, NH 4 + , N t ,) of solid animal wastes, as well as the number of demaged eggs were expressed as mean values ± standard deviation ( x ±SD).
Significance of differences between experimental and control groups of parasites were determined using Student t-test, ANOVA and Dunnet Multiple Comparison test at the levels of significance 0.05; 0.01 and 0.001 (Statistica 6.0).

a) Anaerobic stabilisation of liquid animal wastes
Investigations were carried out under operating conditions of the large-capacity pig farm in Slovak Republic (Picture 3). Technological equipment for anaerobic treatment of pig slurry on the principle of methanogenesis with the production of biogas was built up on the farm (Picture 4). Pig slurry was treated in the bioreactor (2 500 m 3 ) manufactured by Mostáreň Brezno under the agreement with the firm BAUER Voitsberg. The stirring of the substrate in this reactor was done at the expense of energy of the generated biogas. Mean daily input of raw pig slurry in bioreactor of biogas plant varied between 78 and 144 m 3 . The volume of digested slurry after methanogenesis was equal to that of the input. Two lagoons were the part of the biogas plant. The volume of larger lagoon is 20 000 m 3 (Picture 5) and that of smaller lagoon is 5 000 m 3 . Both lagoons serve as reservoirs of digested slurry. Liquid fraction from the smaller lagoon was carried away and spread on fields. The presence and survival of parasite eggs were studied in the larger lagoon. Samples were taken from raw slurry collecting basin before the inlet in to bioreactor (input samples), from outlet of digested slurry after methanogenesis in bioreactor (output samples), from supernatant (liquid fraction) and from lagoon sludge (solid fraction -sediment). The slurry samples for parasitological and physical and chemical examination were collected monthly during 29 month.
Slurry from the pig farm stored in the collecting basin showed a considerable variability during the period of study (Table 1). Compared with mean pH value of 7.12 ± 0.26, pH raw slurry in the month 11, 17 and 21 was lower, ranging between 6.61 and 6.95. The most conspicuous differences were recorded in DM content, which is most likely associated with the amount of process water use. The DM content in raw slurry determined during the period studied ranged from 0.81 % to 5.30 %. The amount of NH 4 + in raw slurry was between 821 mg.l -1 and 1 774 mg.l -1 . Chemical oxygen demand (COD) for that period varied from 2 000 mg.l -1 to 22 530 mg.l -1 . The mean contents of N t , in slurry was 1 445 ± 420 mg.l -1 . This is caused by the projected input, reckoning on the 5 % of dry matter in raw pig slurry, but the mean dry matter content in raw slurry supplied to bioreactor was 1.96 % and therefor poultry excrements had to be regularly added (average DM content 22.27 %) to pig slurry prior to its supply into bioreactor. Stabilized slurry outlet of bioreactor contained as much as 3.23 ± 2.54 % DM on the average. Anaerobic digestion increased slurry pH which was ranging from 7.37 to 8.50. Compared with untreated slurry, anaerobic stabilization increased the content NH 4 + to 7.80 ± 0.29 mg.l -1 on average. Concentration of N t was increased twice.  Table 2. Physico-chemical properties of digested pig slurry (output sample of bioreactor) (COD -chemical oxygen demand, DM -dry matter, IM -inorganic mater, OM -organic matter; NH 4 + -ammonium ions, N t -total nitrogen, --not examined)

Storage
www.intechopen.com The Sanitation of Animal Waste Using Anaerobic Stabilization 57 Anaerobically stabilized slurry was pumped from bioreactors into slurry ground lagoon for further storage. A long-term storage of digested slurry in lagoon is the most effective way of treatment resulting in a elimination of helminth eggs (Schwartzbrod et al., 1989). At the same time there is an increase in biogenic elements, especially of nitrogen and phophorus which are transformed into the forms acceptable by plants. Results of the chemical analysis of liquid fraction (supernatant) are presented in Table 3 and those of solid fraction (sludge) of lagoon in Table 4. pH of supernatant has not changed much over the period studied.
Mean pH was 8.20 ± 0.11 %. Sediment pH decreased during the first period of the study (month 0-6) and than again increased. Ammonia content was about equal in both the fraction. The highest content of NH 4 + was detected in spring month with its decrease observed in the course of study. N t contained by supernatant samples varied between 882 mg.l -1 to 2 283 mg.l -1 (Table 3) and in sediment between 3 571 mg.l -1 to 57 831 mg.l -1 (Table 4). Sediment contained more DM and N t than supernatant (Tables 3, 4).  Table 3. Physico-chemical properties of supernatant from stabilized pig slurry stored in lagoon (COD -chemical oxygen demand, DM -dry matter, IM -inorganic mater, OM -organic matter; NH 4 + -ammonium ions, N t -total nitrogen, --not examined)

Storage
A. sum eggs and Oesophagostomum sp. eggs were rarely detected in slurry on the input and also on the output of bioreactor (Table 5). Similar results of helminths eggs occurrence in anaerobic slurry treatment were also presented by Juriš et al. (1996), No helminth eggs were found in the supernatant of digested slurry from the lagoon. A. suum eggs were found in sediment (Table 5).
High percentage of devitalised unembryonated A. suum eggs (47.46 ± 0.78 %) stored 11 months (from May -month 13 to March -month 23) in a ground slurry lagoon points to the impact of high concentration of NH 4 + (max. 5 358 mg.l -1 in sediment compared to 1 863 mg.l -1 in supernatant), which are releasing during a period of time from an open area of the ground lagoon, and nitrogen (max. 9 854 mg.l -1 in sediment compared to 2 283 mg.l -1 in supernatant) on devitalization of developmental stages of endoparasites. The number of devitalised A. suum eggs increased towards to the bottom of lagoon. In the control groups, only 19.60 ± 1.80 % of A. suum eggs were devitalized (Table 6).

Sediment (lagoon)
ND ND A-6 ND ND ND ND ND ND ND ND ND ND A-2 ND ND www.intechopen.com

b) Anaerobic stabilisation of solid animal wastes
The effect of anaerobic stabilisation of solid animal wastes (manure, dog excrements) with or without addition of lime on the survival of parasitic germs were studied under laboratory conditions. Two types of lime was used in the experiment: 1. quality dust lime and 2. dust rejects from lime production caught on the electrostatic precipitator. General characteristics of tested lime are given in Table 7. a. manure mixed with quality dust lime in a concentration of 20 g.kg -1 (ML20) b. manure mixed with dust rejects in a concentration of 20 g.kg -1 (M20) c. dog droppings mixed with dust rejects in a concentration of 20 g.kg -1 (D20), d. dog droppings mixed with dust rejects in a concentration of 70 g.kg -1 (D70).
The physical and chemical properties of treated manure and dog excrements are given in Tables 8 -13. Comparison of the changes in The physical and chemical properties of organic material during anaerobic stabilisation with or withou dust rejects is given in Fig. 1 Table 9. Physico-chemical properties of the pig manure mixed with dust rejects in a concentration of 20 g.kg -1 during anaerobic stabilization (M20) (DM -dry matter, IMinorganic mater, OM -organic matter; NH 4 + -ammonium ions, N t -total nitrogen)  Table 10. Physico-chemical properties of the pig manure mixed with quick lime in a concentration of 20 g.kg -1 during anaerobic stabilization (ML20) (DM -dry matter, IMinorganic mater, OM -organic matter; NH 4 + -ammonium ions, N t -total nitrogen, --not examined)    Table 13. Physico-chemical properties of the dog excrements mixed with dust rejects in a concentration of 20 g.kg -1 during anaerobic stabilization (D70) (DM -dry matter, IM -inorganic mater, OM -organic matter; NH 4 + -ammonium ions, N t -total nitrogen, --not examined)  For the sanitation of animal excrenemts, the use of dust rejects from lime production, at more affordable price than quality lime, is very suitable. An application of dust rejects to the mixed dogs' excrements at a concentration of 20 g.kg -1 of organic wastes, resulted in a devitalisation of 65.65 ± 2.84 % and at a concentration of 70 g.kg -1 77.05 ± 2.36 % of model unembryonated A. suum eggs within 24 hours (Table 15). A. suum eggs were totally devitalised as early as within 8 days in dogs' excrements after application of dust rejects at a concentration of 70 g.kg -1 and within 21 days after application of dust rejects at a concentration of 20 g.kg -1 due to the changes in physical and chemical properties of the stabilised materials (Tables 12, 13). 57.23±3.21 % of eggs were devitalised in the control without dust reject in the end of experiment (Table 15).

Storage (days) Demaged A. suum eggs (x%±SD)
CD P20 P70 0 12.62±1.14 12.62±1,14 12.62±1.14 1 Tab. 15. Survival of A. suum eggs during anaerobic stabilisation of manure with or without lime (* Significance at the level P<0.05; ** Significance at the level P<0.01; *** Significance at the level P<0.001) Our experiment showed that stabilisation of organic wastes with dust rejects result in complete devitalisation of A. suum eggs (Table 14, 15). The most important physico-chemical factors affecting viability of helminth eggs include pH and ammonia. We observed the highest pH and ammonia content especially in the organic wastes treated with tested types of lime. One of our previous studies (Ondrašovič et al., 2002) on the effect of ammonium hydroxide on A. suum eggs showed that at 10 % concentration of NH 4 OH, pH 12.16 and exposure time 180 min. approx. 94 % A. suum eggs were devitalised. Pescon and Nelson (2005) also reported that environmentally relevant concentrations of ammonia may significantly increase the rate of Ascaris eggs inactivation during alkaline stabilization.

Conclusion
Processes of slurry anaerobic stabilization represent an effective method in terms of energy, since the substantial portion of energy present in easily decomposable organic constituents of the substrate is acquired in the form of biogas. Non-decomposed organic matter is well stabilized from hygienic point of view. Anaerobic stabilization increases the proportion of biogenic element (especially nitrogen) converting stabilized excrements into quality fertilizer. Anaerobically stabilized pig slurry stored in lagoon significantly influence the quality and quantity of grasses, depending on the dose of slurry used and on weather conditions. From the nutritional point of view, the sludge (sediment) from ground lagoon is also important for plants (Valocká et al., 2000). The high amount of nitrogen is apparently the result of the decomposition process going on in lagoon.
Hazard of contamination of field fertilized with the lagoon effluent increases when raw slurry is used for fertilization of soil or pastures. When slurry is processed in a wastewater treatment plant, parasitic eggs concentrated in solid fraction. It is therefore necessary to pay a proper attention to slurry processing.
The anaerobic stabilization and the use of dust rejects from lime production, at more affordable price than quality lime dust, were demonstrated to be very suitable for the sanitation of organic wastes from animal production and dog excrements. This way of treatment is thus not associated with a risk of dissemination, survival and potential spread of developmental stages of endoparasites to the environment via stabilized organic wastes