DYNAMICS OF THE MICROBIOLOGICAL INDEXES AT LIME TREATMENT OF SLUDGES FROM A PURIFICATION PLANT

Microbiological indexes have been studied in dynamics at lime treatment and independent planting of lavender (Lavandula angustifolia, variety “Hemus”) and sweet basil (Ocimum basilicum, variety “Genovese”) of fresh sludges from a purification plant, with concentrations of quicklime: 10%, 20% and 30% for a 40-day period of composting. The microbiological analysis includes determining of nonpathogenic (non-spore forming bacteria, bacilli, actynomycetes, micromycetes, bacteria, which assimilate mineral nitrogen) and pathogenic (Salmonella spp., Escherichia coli and coliforms, Enterococcus, Clostridium perfringens) microflora. Out of the beneficial microflora in the sludges the quantity of the non-spore forming bacteria is the biggest, and the lowest is of the actynomycetes. A basic share in the composition of the pathogenic microflora occupies Clostridium perfringens, followed by Escherichia coli and coliforms, and Enterococcus. Absence of Salmonella spp. was established in the studied sludges. The best results for the disinfection of the sludges displays the adding of 30% quicklime (followed by the variants with 20% and 30% lime), on 5-th day of the experiment setting. The pH decrease in the period of the study leads to a repeated development of pathogenic microflora. In the meantime the creation of an alkaline medium leads to decrease in the quantity of the beneficial microorganisms. The decrease of the moisture and the drying of the sludges for the study period display a weaker effect on the dynamics of the microflora in comparison with the pH increase impact. The planting of lavender and basil independently in the sludges samples from a purification plant has a beneficial effect (a little bit better with the lavender variants) for increase of the quantity of nonpathogenic microbes and pathogens frequency of occurrence decrease. The effect is intensified by increasing the plant development period.


Introduction
The sludges from the purification plants are substrata rich in organic substances, which could be used as organic fertilizers in agriculture. The problem for their usage as a fertilizer is the content of heavy metals in them, pathogenic microorganisms and parasites. The lime treatment leads to alkalization of the medium and respectively to sedimentation of the heavy metals in sludges from purification plants, as thus their solubility and mobility are decreased. When calcium oxide is added an exothermic reaction is obtained and the temperature of the sludges is increased, which helps for the destroying of the pathogenic types of microorganisms and parasites, consequently for acceleration of the processes of disinfection and deworming. The hydrated lime forms complexes with smelly sulphurous compounds, like hydrogen sulphide and organic mercaptans (end products from the microbes and parasites activity), as a result of which sediment with smell of calcium ions is obtained. Hence comes the problem with the deodorizing of the sludges, about the solution of which can be used the planting of essential oil crops, some of which also have antimicrobial properties.
According to a study by Marcinkowski (1985) the waste sludges from purification plants, deactivated by quicklime can be used for agricultural purposes. The processing by lime and the pasteurization of the sludges (50-day sanitation) are very efficient methods for disinfection and create safe sludges regarding content of faecal coliforms (Keller et al., 2004). Marinova et al. (2016) present methods for treatment of sludges from purification plants by lime materials. According to these au-

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thors the most perspective for a short period of time (around 1 month) is the disinfection of the sludges with adding 20% fine and 30% bigger fraction of quicklime. The treatment by quicklime according to them is especially appropriate for this purpose, since the lime is not dangerous from environmental point of view and in the same time it is effective. In the sludges are isolated pathogenic microorganisms like: Salmonella spp., Listeria spp., Escherichia coli, Campylobacter spp., Clostridium spp., Yersinia spp. and others (Arthurson, 2008;Dudley et al., 1980;Larsen and Munch, 1986;Straub et al., 1993;Strauch, 1991). According to a study by Dermendzhieva (2017) in the sludges from Bulgarian purification plants the number of the faecal (coliforms, Escherichia coli) and intestinal pathogenic (Enterobacteriaceae, Salmonella) microorganisms is over the admissible norms in the Regulations for the order and way of sludges utilization from the purification of waste waters by their usage in agriculture (State Gazette, issue 63/12.08.2016), which makes them unusable (but with potential) for fertilization in the agriculture without applying methods for their disinfection. These microorganisms have strong ability to constantly adapt to the changes in the environment for survival (Kearney et al., 1994) and they can be relatively resistant (especially the spore forming species, like Clostridium perfringens) to the frequently used methods for stabilization of sludges (Sahlström, 2003).
The plants synthesize flavonoids as a response of a microbial infection and it is established that these compounds are a mighty antimicrobial agent against a wide spectrum of pathogenic microorganisms (Górniak et al., 2019). In this relation the study of antimicrobial properties of plants at variants with sludges from purification plants, treated for disinfection and deodorizing deserves attention.
The purpose of the study is following the dynamics of microbiological indexes at lime treatment of fresh sludges from a purification plant (in a combination with independent planting of lavender and sweet basil), as a methods of their disinfection of pathogenic microorganisms.

Materials and methods
Fresh sludges from a purification plant have been studied as per physico-chemical and microbiological indexes, in dynamics, composted in greenhouse conditions, in plastic packings (containers), treated with quicklime in different concentrations and variants. Lavender (Lavandula angustifolia, variety "Hemus") and sweet basil (Ocimum basilicum, variety "Genovese") are additionally planted independently with three of the variants (table 1): Microbiological analyses are carried out as per the method of the limit dilutions and culture on growing medianonpathogenic microflora: Nutrient agar for non-spore forming bacteria and bacilli; MRS agar for lactobacilli; Actinomycete isolation agar for actynomycetes and bacteria, assimilating mineral nitrogen; Czapek-Dox agar for moulded fungi; pathogenic microflora: Desoxycholate citrate agar for Salmonella spp.; Endo agar for Escherichia coli and coliforms (oxidase confirming test); ChromoBio Enterococcus agarfor Enterococcus; Perfringens agar (TSC and Perfringens selective supplement) for Clostridium perfringens. The results are presented like colony-forming units (КОЕ) a unit substrate. A jar with reagent for generating anaerobic medium is used for the isolation of anaerobes.
For following the temperature of the sludges is used a temperature probe, for determining the moisturea moisture meter, and for registering pH (in water) -рН meter.

Results and discussion
The temperature of the studied sludges is increased to the highest degree on 40-th day from the beginning of the experiment, at adding 30% quicklime (with around 9 °С). By increasing the period of sludges disinfection the moisture decreases by around 1/3 in comparison with the beginning of the experiment (to the highest degree for the variants with 30% lime). The lime treatment leads to alkalization of the mediumto the highest degree on 5-th day after adding 30% lime. On 40-th day from the beginning of the experiment, the sludges values of рН are in the neutral zone again, a little bit higher than the sludges value of рН before the experiment beginning. These trends follow to a weaker degree for the variants with 20% lime and these with 10% lime (table 2). The microbiological analysis of the sludges from the purification plant has shown presence of specific and non-specific (pathogenic) microflora. The quantity of the nonpathogenic groups microorganisms is presented in the next table 3. The quantity of the non-spore forming bacteria and bacteria, assimilating mineral nitrogen is the highest, and the actynomycetes are presented the least (to complete absence with lime treatment). The lime treatment increases the pH values of the sludges (alkaline medium, to рН around 12), which leads to decrease of the quantity of the analyzed nonpathogenic groups microorganisms. On the other hand the decrease in the moisture of the sludges increases the quantity of the microorganisms. The moisture and the temperature of the substrata are of essential importance for the development of the microbes. The excessively high amount of moisture in the sludges (90%) in the beginning of the experiment limits in a certain degree the development of the microorganisms. The bacilli as spore-forming species and micromycetes as requiring higher moisture survive well in extreme conditionsas per amount in the content of the microflora they are after the non-spore forming bacteria and the bacteria, which assimilate mineral nitrogen.
The lime treatment renders stronger limiting impact over the development of the nonpathogenic groups of microorganisms in comparison with the decrease of the moisture (from 90% up to 30-38%) and the drying of the sludges by increase in the temperature with around 6 °С up to 9 °С in the end the experiment (the amount of the microbes with the variants without lime treatment is higher than the one with the variants with lime treatment). The results show that the adding of 30% quicklime decreases the amount of the microorganisms to the highest degreethe effect follows for the variants with 20% lime and least impact over the amount of the microbes renders the adding of 10% lime in the sludges. This trend regarding the dynamics in the amount of the microorganisms for the period of study, is preserved for the variants up to 5-th day from the setting of the experiment, until the sludges are with stronger alkaline medium (pH 11-12). The decrease of рН to a weaker alkaline medium (рН around 9,1-9,4) on 20-th day and a neutral medium (рН 7,2-7,4) on 40-th day increases the amount of all analyzed nonpathogenic groups of microorganisms. As a whole the amount of the microorganisms is a little bit higher with the variants with lavender in comparison with the variants with basil, followed by the ones without vegetation, regardless of the added amount of lime and the decrease of moisture in the period of analysis.
The lime treatment is a method which is used for disinfection (destroying of pathogenic microorganisms) of the sludges from purification plants. In the studied variants were established: Escherichia coli and coliforms, Enterococcus, Clostridium perfringens ( figure 1 up to figure 3). In the content of the pathogenic species in the sediment the frequency of occurrence of Clostridium perfringens (5060 cfu/g before beginning of the experiment) is the highest, followed by Escherichia coli and coliforms (3820 cfu/g before beginning of the experiment), and Enterococcus (3040 cfu/g before beginning of the experiment). Absence of Salmonella spp. is established. Clostridium perfringens is a spore forming species, which impede its destroying. The lime treatment leads to decrease in the quantity of the pathogenic microbes (at all variants with lime treatment) and their complete destroying until 5-th day from setting the experiment at the variants with 30% lime, regardless of the vegetation. Marinova et al. (2016) also establish best disinfection of sludges from purification plants at adding 20% and 30% quicklime. According to the study of these authors for 31 days after the mixing of the sludges with fine quicklime in the indicated concentrations, Clostridium perfringens is destroyed. The results of their studies confirm that for obtaining safe sediment from an epidemiological point of view, it should be processed for one month or a shorter period. The same authors have established that the massiveness of the quicklime also renders a different impact over the development of the microorganismsthe larger fraction homogenises more difficult with the sediment, because of which the usage of a higher norm is necessary. Therefore the creation of an alkaline medium is a method for the sediment purification of pathogenic microorganisms. The pH values decrease, however, (a weaker alkaline medium-20-th day and a neutral medium -40-th day) leads to a repeated development of pathogenic microorganismsto a higher degree for Clostridium perfringens. The adding of 30% lime and the planting of lavender display best results for the destroying of the pathogenic microorganisms. The variants with 30% lime and basil follow as per efficiency, after them the variants without vegetation. The decrease of the moisture and the drying of the sludges have significantly weaker effect than the lime treatment, which is indicative of the fact that at the variants without lime treatment the results are preserved close for the whole period of the study. The conditions of the medium, as the active reaction of the sludges, the salt content, the heavy metals content, temperature, can lead to a change in the progress of the microbial activity in the substrate and respectively to influence on the reproductive ability of the microorganisms (Dermendzhieva, 2017). The studied pathogenic microorganisms have strong ability to adapt constantly to the changes in the environment in order to survive (Kearney et al., 1994) and they can be relatively resistant (especially the spore forming species, like Clostridium perfringens) to the frequently used methods for stabilization of sludges (Sahl-  (Marcinkowski, 1985;Keller et al., 2004). On the one hand, for the complete destroying and prevention of repeated development of the pathogenic microflora is necessary an additional lime treatment after 5-th day for preservation of the alkaline medium, but on the other hand this would lead to decrease in the quantity of the useful microorganisms. Therefore, a combination of lime treatment with ash adding, microbial fertilizers, appropriate plant species and other methods are necessary in a process of repeated analyses and following the development of the microflora in the sludges in dynamics.

Conclusions
Fresh sludges from a purification plant have been studied after lime treatment and independent planting of lavender and basil for a period of 40 days regarding dynamics of nonpathogenic and pathogenic microorganisms. Non-spore forming bacteria, bacilli, including lactobacilli, actynomycetes, micromycetes, bacteria, assimilating mineral nitrogen, Salmonella spp., Escherichia coli and coliforms, Enterococcus, Clostridium perfringens have been analysed. Out of the beneficial microflora in the sludges the highest is the quantity of the non-spore forming bacteria, and the lowest of the actynomycetes. In the content of the pathogenic microflora a basic share occupy Clostridium perfringens, followed by Escherichia coli and coliforms, and Enterococcus. The absence of Salmonella spp. is established in the studied sludges.
The lime treatment renders a beneficial effect for the destroying of pathogenic microorganisms in samples with sludges from a purification plant. Best results for their disinfection has the adding of 30% quicklime (follow the variants with 20% and 10% lime), on 5-th day after setting of the experiment. In the same time, however, the creation of an alkaline medium leads to decrease in the quantity of the useful microorganisms. On the one hand, an additional lime treatment after 5-th day is necessary for a complete destroying and prevention of the pathogenic microbes' repeated development (especially for the spore forming species Clostridium perfringens), and on the other hand, probably this would lead to decrease of the beneficial microorganisms to a higher degree.
The temperature of the studied sludges is increased by around 6 °С up to 9 °С, and the moisture decreases by 1/3 until the 40-th day from beginning of the experiment, to the highest degree at adding 30% quicklime. The lime treatment leads to alkalization of the mediumto the highest degree on 5-th day after adding 30% lime. On 40-th day from the beginning of the experiment, the pH values of the sludges decrease to a neutral medium. These trends follow to a weaker degree for the variants with 20% lime and these with 10% lime. The decrease of the moisture and drying of the sludges for the period of study displays a weaker effect on the dynamics of the pathogenic, as well as of the nonpathogenic microorganisms in comparison with the impact of the pH increase.
The independent planting of lavender and basil in the samples of sludges from a purification plant has a favourable effect (a little bit better with the variants with lavender) for increase in the nonpathogenic microbes quantity and decrease of the frequency of occurrence of the pathogenic ones. The effect increases by increase in the period for development of the plants. The selection of these plant species is appropriate for disinfection and deodorizing of the sludges in a combination with lime treatment.
Continued studies and a multifactor approach are necessary for establishment of appropriate combined variants simultaneously for complete disinfection of the sludges and preservation of the beneficial microflora, with purpose their safe usage as an organic fertilizer in agriculture.