EFFECT OF SEWAGE SLUDGE COMPOSTING PROCESS IN CYBERNETIC BIOREACTOR ON THE NUMBER OF SELECTED MICROORGANISMS GROUPS AND PROTEASES ACTIVITY

Agnieszka Wolna-Maruwka¹, Aleksander Jędruś^2
1 Department of Agricultural Microbiology, University of Life Sciences, Poznań, Poland
² Institute of Agricultural Engineering, University of Life Sciences, Poznań, Poland

ABSTRACT

Microbiological chatracteristics of sewage sludge from mechanical and biological sewage treatment plant composted in controlled conditions with straw and sawdust are presented. Prepared composts were placed in four bioreactors with air flow 4 l O₂ · min^-1. In bioreactor K1, K2 and K3 the composted mass consisted of 65 % sewage sludge (K1-sewage sludge 1, K2 – sludge 2, K3 – sludge 3) + 30 % sawdust + 5 % straw, while in bioreactor K4 the proportion was: 45 % sludge 2 + 50 % sawdust + 5 % straw. Compost samples were taken from all chambers at the same time depending on the actual temperature. Microbiological analyses consisted in the determination by plate method on selective medium the numbers of cellulolitic, proteolitic and solving phosphates microorganisms. Furthermore, in the experiment, the activity levels of proteases were determined using 1 % sodium caseinate as substratum. Studies have shown that the most intensive reproduction of cells of the studied groups of microorganisms were recorded after 20 hours of composting (term II), while the maximal level of proteolitic activity occurred between the 20th and 66th hour of the composting process. Furthermore, it was found that the composting process caused a decrease in the  number of the analysed microorganism groups in the majority of composts.

Key words: microorganisms, proteolitic activity, biowaste, sewage sludge.

INTRODUCTION

One of the effects of civilization development is the creation of different types of wastes including also the communal wastes [26]. An inseparable element accompanying the biological and chemical purification of sewages is the development of sewage sludge and its amount is the greater the higher the efficiency of the sewage treatment plant [3]. In Poland, about 359 thousand ton of dry matter of sewages are created. According to the prognoses of the Institute of Environmental Protection, in Poland, until the year 2010, the amount of communal sewage sludge will amount to 413–450 thous. ton of dry matter per year [20].

The increasing problem of a high amount of sewage sludges contributes to the search for the most rational and safe methods of their utilization and management [16]. Among the basic methods of municipal sewages utilization (among others: incineration, storage on dumping grounds,  throwing into the sea), increasingly more attention is devoted to the method of agricultural use of the sewages [10]. In spite of the fact, that agricultural utilization is a cheap method of sewage sludge management, accordidng to the data of the Main Statistical Office (year 2004), in Polish  agriculture, only as little as 13 % of communal waste mass is utilized and 37 % is still being stored. A rational management of the stable communal wastes is possible only after their processing into composts [5,13,30]. The composting process in comparizon with other methods of neutralization, has many advantages and the most important of them is a radical liquidation of wastes, minimalization of sanitary threat connected with the agglomeration of wastes,  return to the biological circulation of many plant nutrition components. Composting process permits also to include the communal wastes into the balance of organic fertilization [21].

Agricultural utilization of compost developed from sewage sludge includes among others the use of it for fertilization purposes because of a its abundant content of organic matter and nutritive components for plants, mainly oxygen (about 6 % d.m.) and phosphorus (about 3 % d.m.) which decide about their very  high utility [23]. A well prepared compost can have almost the same fertilization value for plants as manure. The decay of proteinaceous substance of plant and animal origin in composted sewage sludges is a complex and multistage process. Proteinaceous combinations in the sewage sludges are attacked by proteolitic microorganism groups producing proteases, specific extracellular enzymes secreted to the external environment [31]. Another group of microorganisms taking an active part in organic matter transformation in composts are cellulolitic microorganisms, which represent  a group which is able to utilize cellulose as a source of carbon and energetic substrate.

Activity and quantity of cellulolitic microorganisms are influenced by many factors. The main factor limiting the activity of cellulolitic microorganisms is a lack of available organic matter. Therefore, in agricultural practice, sewage sludges are frequently composted with different additions like e.g. straw, sawdust, which are characterized by a wide C:N proportion [6]. Also the content of phosphorus in sewage sludges is high and it oscillates between 1.5 and 6.0 % of dry matter [24]. It has been shown that the accessibility of mineral phosphorus compounds from not assimilable forms into forms available to plants and microorganisms takes place mainly through secretion by microorganisms of strong mineral and organic acids during their life processes [17]. The objective of the present studies was the determination of the effect of the composting process of sewage sludges with different additions, in controled conditions, on the number of three selected microorganism groups and the activity of proteases.

MATERIAL AND METHODS

Experiment was established in laboratory conditions in 2006. Material used in the studies consisted of sewage sludges originating from the  three, different Sewage Treatments Plant and consisted of wheat and rye straw and sawdust. The microbiological and chemical analyses are presented in Tables 1 and 2. Studies were carried out in four bioreactors of 125 dm³ capacity and equipped with electronic sensors for constant recording of some process parameters (temperature, carbon dioxide, methane, ammonia and oxygen). Materials for studies were thoroughly mixed in a container in weight proportion in relation to dry matter: 65 % of sewage sludge (bioreactor K1 – sludge 1, K2 – sludge 2, K3 – sludge 3) + 30 % of sawdust  + 5 % of straw in bioreactor K1, K2 and K3 while in bioreactor K4, the proportion was: 45 % of sewage sludge (sludge 2)+ 50 % of sawdust + 5 % of straw. The experiment was conducted with a constant air flow amounting to 4 L min^-1 in chambers. Material in bioreactors was composted for 34 days and18 hrs, while compost samples were taken from all chambers at the same time depending on the actual temperature of the composted material.

On microbiological selective medium, using plate method, the number of colony forming units (cfu) of cellulolitic, proteolitic and solving phosphates microorganisms were determined. Proteolitic bacteria were incubated on a selective medium according to Rodina [19] at 22°C for 48 hours. In order to increase the substrate contrast in relation to proteolitic bacteria (white colonies creating some clearings-up), for the determination of the quantity, the Frazier's reagent was applied. The quantity of cellulolitic microorganisms was determined on the medium according to Rodina [19]. Glass plates were incubated for 8 days at 28°C. Microorganisms solving  phosphates were determined on selective medium according to Rodina [19]. The plates were incubated for 7 days at 28°C. Furthermore, by spectrophotometric method, in the samples of composted material, the activity of proteases was determined using as substrate 1% sodium caseinate after one hour incubation at 50°C with wave length 578 nm. Enzyme activity was expressed in mg of tyrosine·kg^-1·h^-1 [14]. Statistical analyses applied in the experiment were carried out basing on Statistica 8.0 programm.

RESULTS AND DISCUSSION

A review of the subjective literature indicates that studies on the change in the quantities of proteolitic, cellulolitic and phosphates microorganisms in sewage sludges subject to composting process are not numerous, hence, in the performed experiment, attention was concentrated among others on the determination of the number of the above mentioned organisms.

Analisis of the obtained results (Tables 3, 5, 6) indicated that the main factor having an influence on changes in the number of the studied microorganism groups were the changes in the temperature values during the composting process. According to Ishii et al. [12], changes of temperature during composting are a condition for the microorganism succession understood as changes in the quantitative and qualitative composition of microorganisms. Species composition and the number of the particular populations depends not only on the type of organic matter subject to composting, but also on the composting technique. According to Taiwo and Oso [28], the type of the applied composting technique of organic matter has a significant influence on the number and activity of microorganisms and thereby on temperature changes in the compost. In turn, Daniel and Cowan [7] reported, that the reason of changes in the critical temperature of growth having an influence on the resistance of microorganisms to the action of high temperatures may be caused by the pH value of the environment and the chemical composition of the culture substrate.

Data illustrating the dynamics of changes in the number of cellulolitic microorganisms are shown in Table 3. Analysis of the number of cellulolitic microorganisms in the analysed composts revealed that their greatest number in the moment of of the experiment's establishment (term I) was in compost K2 and it amounted to 9693.05 cfu·10⁴·g^-1d.m. of compost. The 20-hrs composting process in controlled conditions caused an increase of temperature in the bioreactors by 12–23 °C, which contributed the increase of the number of the analysed microorganisms in all chambers by several times and even by dozens of times. Next to temperature which favoured the development of these microorganisms, also the pH value could have contributed to the creation of the optimal conditions for cellulolitic bacteria growth. The pH value was then 7.15–7.61 (Table 4). In the successive term of analyses (term III – analysis after 30 hrs) there followed a rapid drop of cellulolitic microorganisms proliferation most probably caused by a successive temperature increase (by 7 or even 12°C) in the composted materials. Starting with term III, the number of the discussed microorganisms was decreasing which depended on the high temperature was mainly in the composts reaching even 74°C. Such status was maintained until term VI (analysis after 5 days) with the exception of compost K3, where, with a slight drop of temperature from 64 to 61°C, an increase of the proliferation of the analysed microorganism group was recorded. Studies carried out by Chang and Hudson [4] indicate, that for cellulose degradation at the optimal temperature for thermophiles, fungi species Chaetomium thermophilum and Humicola insolens are responsible. Next to them, at 55–61°C, Aspergillus fumigatus developed which takes an active part in the degradation of cellulose (Strom 1985). According to Bergquist et al. [2], the decomposition of cellulose in high temperatures (optimum 70°C) is also carried out by Caldibacillus cellulovorans. After 20 days and 18 hrs from the moment of the experiment establishment (term VII), in all composts there followed an increase of cellulolitic microorganisms proliferation being the result of temperature drop in all chambers by 12–41°C.

In the last term of the microbiological analyses (term VIII – analysis after 34 days and 18 hrs), a decrease of the discussed microorganisms was observed in spite of the further drop of temperature. The above phenomenon could have been caused by the depletion of cellulose compounds, or by a strong development of actinomycetes with antibiotic properties [29]. On the basis of the obtaind results (Table 3), it can be showed that the greatest quantity of the analysed microorganism group was recorded in term III (analysis after 20 hrs), when the studied material subject to the composting process was still rich in the decomposable organic matter.The slow decrease of the number of cellulitic microorganisms during the experiment can  be a testimony that the available amount of substrates for that microorganism group decreased. Temperature was also a factor guarantying the occurrence of the studied group of microorganisms. Temperature increase above 45°C inactivated the proliferation of the discussed microorganisms.

Analogically as in case of cellulolitic microorganisms, changes in the number of phosphates solving microorganisms depended most probably on temperature changes in the composted materials (Table 5). Interpretation of study results shown in Table 5, permitted to state that the greatest number of microorgnisms in the moment of experiment establishment (term I) occurred in compost  K1 and it amounted to 944.57 cfu·10⁴·g^-1d.m. of compost. Temperature increase in bioreactors by  12–23°C during a 20-hour compostng process (term II) contributed to the increase of the number of the analysed microorganism group in compost K1 and K2 by 63–104 %, and a decrease of their proliferation in the composts K3 and K4 .Further increase of temperature after  30 hrs of composting (term III) cdontributed to the drop of the number of phosphates solving microorganisms in all chambers. The above situation was maintained until term VI in the composts K2 and K4. Analogical was the dynamics of the quantitative changes of microorganisms in composts K1 and K3, some deviation was recorded only in term IV (analysis after 41.5 hrs), when the proliferation of bacteria rapidly increased. repeated increase of the number of phosphates solving microorganisms was recorded as late as in term VII, when the temperature in the composted materials reached the values of 27–29 °C.

The main factors involved in the solving of the mineral compounds of phosphorus are mineral and organic acids. The above  situation was confirmed by Ishii et al. [12], who isolated from the communal wastes, mezophilic fermenting bacteria Leuconostoc paramesenteroides,  Pediococcus acidilactici, Staphylococcus piscifermentans which secrete into the compost organic acids, mainly lactic acid taking part in the liberation of mineral compounds of phosphorus. According to Shekhara et al. [22] proliferation and activity of phosphates solving bacteria depend on the C/N proportion, pH and temperature of the material. According to the authors, these processes take place in the most intensive way at 10 % substrate salination, pH = 12 and temperature  45 °C. This statement, however, is difficult to compare with our own studies, because during the performed experiment, the maximal values of pH obtained in the composted material was 9.7 (Table 4).

On the basis of the obtained results of microbiological analyses presented in Table 6, it was found that the main factor affecting the number of proteolitic microorganisms in the composted materials was most probably the temperature. Quantitative changes of proteolitic bacteria during the experiment were analogical as in case of cellulolitic microorganisms (Table 4). The analysis of proteolitic microorganisms proliferation after a 20-hour composting process (Term II) indicated that their number rapidly increased with the increase of temperature in the composted masses reaching the maximal values in the range of 1193.71–2748.92 cfu·104·g^-1s.m. of compost. In the successive terms of analyses, with the increase of temperature in the chambers, a decrease of proteolitic micoroorganisms was observed. According to Stroma (1985), in compost, in the thermophilous phase, in temperature range from 49 to 65°C, among the proteolitic bacteria, there dominate species belonging to Bacillus genus (over 85 %). In the range of temperatures from 55 to 75°C, optimum at 65·70°C, there mainly dominates  Bacillus schlegelli [1].

A repeated slight increase of the number of the discussed group of microorganisms was recorded as late as in the VII term (analysis after 20 days and 18 hrs), when  the temperature in  the composted materials dropped to the value of 27–49°C. From the data presented in Table 6, it follows that the greatest quantity of proteolitic microorganisms was recorded in the term II of analyses, in the range of temperatures favouring the development of mezophiles (25–45°C). The decrease of the proliferation of the studied group of microorganisms during our experiment may testify that the organic matter subject to decomposition had been exhousted, or there was no tolerance by this group of microorgaanisms to the thermophilic conditions.

Studies carried out by Hadas et al. [11] indicate that the greatest number of proteolitic microorganisms occurred on the 3rd day of the composting process of manure. In the successive days of analyses, with the increase of temperature in the composted material, a drop of the proliferation of microorganisms was recorded and this was also reflected in our own studies.

Biological transformation of nitrogen compounds is directly related with the proportion of carbon to nitrogen. If the proportion C/N is wide, then there comes to an increase of the number and mass of microorganisms [26]. This statement can be referred to our own studies, because the greatest quantity of the studied group of microorganisms was recorded in compost  K3, where the sewage sludge was a component of the composted mass. The C/N proportion in the above mentioned sludge was 19.68 and it was the widest one in comparison with the remaining sewage sludges applied in the experiment (Table 1). Analysis of study results revealed the occurrence of positive correlations (compost K1 and K4) and negative correlations (compost K2 and K3) between the number of proteolitic bacteria and the activity of proteases (Figs. 1–4).

Interpretation of data presented in Table 7 permit to notice that the greatest proteolitic  activity for compost  K1 occurred in term II of analyses and it amounted to 91.85 mg of tyrosine·kg^-1·h^-1 30-hour of  biowaste composting process contributing to the drop of its enzymatic activity. The above situation was maintained until the end of the experiment. Different was the course of changes in the activity of the discussed enzymes in the material composted in chamber K2, where an increased activity of proteases was recorded during the initial terms of analyses. The maximal activity was observed in term IV (after 41.5 hrs of composting) and it amounted at that time to 86.45 mg of tyrosine·kg^-1·h^-1, at the temperature of 72°C. Further increase of temperature inactivated the activity of proteases in the successive two terms of analyses (terms V and VI). As late as in the last term of studies, with a drop of temperature to 27 °C, a repeated increase of the proteolitic activity of compost was recorded. Also in the remaining two composts (K3 and K4), significnt oscillations in the level of proteolitic activity in the composted materials was recorded.

Enzymatic activity in compost K3 in term I of analyses amounted to 54.72 mg tyrosine·kg^-1·h^-1. 16-degree increase of temperature (term II) contributed to its decrease by about 47%. Further temperature increase by about 11 °C was observed in the composted mass and it caused a successive increase of proteolitic activity. In two successive terms, the activity of the studied enzymes alternately increased and decreased and first  at the beginning of the VI term (analysis after 5 days) it started to decrease and that state was maintained until the end of the experiment. Analysis of proteolitic activity changes in compost K4 showed that the increase of temperature during the composting process contributed to its increase in term II and III. In the successive terms of analyses, the activity in the studied material was decreasing. Only after the 5 days from the moment of experiment establishment, a small increase of activity was recorded in compost K4.

On the basis of the presented studies, one can state that the temperature value can be the only factor influencing the level of proteolitic activity. According to Margesin et al. [15], temperature increase to 55–60 °C inactiwates protease which was confirmed on the basis of enzymatic analyses. Carried out in our own studies in compost  K1 and K4 (Table 7). The above statement is also confirmed by the studies of McKiinley, Vestal [17], according to them, the maximal activity of microorganisms in the composting process of sewage sludges takes place in the temperature range from 25 to 45 °C, while in the range of 55 to 74 °C, a relatively lower activity was recorded. However, Finstein et al. [8] have a different opinion, they argue that the greatest activity of microorganisms was observed in the temperature optimal for the majority of thermophilic microorganisms which is close to 60 °C and this was confirmed in our own studies in composts  K2 and K3 (Table 7).

According to Hadas et al. [11], proteolitic activity depends on the availability of carbon. Results of experiment carried out by those authors indicate that  the highest preteolitic activity takes place during the first 7 days of composting. Studies carried out by Goyala et al. [9] reported that the highest  proteolitic activity was observed between the 10th and the 60th day of composting. The above presentation indicates that there is no explicit explanation of the causes of changes in the proteolitic activity of sewage sludge during composting. These differing observations  probably are the results of differentiated composition of the composted organic matter. It  may also result from differences in the applied method of composting, as well as from the selection of methods permitting to assess the activity and quantity of microorganisms. The obtained results do not authorize to draw definite conclusions, but they are  a successive contribution to the continued discussion referring to the changes in the quantity and activity of microorganisms in sewage sludge subject to the composting process.

CONCLUSIONS

1. Changes in the number of cellulolitic and phosphatic microorganisms and the proteolitic activity of composted sewage sludges with an addition of straw and sawdust were conditioned by the origin of sludges and by the temperature of the composting process.

2. The most intensive proliferation of the studied microorganism group cells were recorded after 20 hours of composting (term II). The maximal level of proteolitic activity occurred between the 20th and the 66th hours of the composting process.

3. The highest mean number of the analysed microorganism groups and the proteolitic activity were recorded in compost K3, which included sewage sludge characterized by C/N = 19.68.

4. Temperature increase during the composting process has shown to be a factor reducing the number of the studied microorganism groups and the proteolitic activity in the majority of composts.

5. Positive correlation between the number of proteolitic bacteria and proteolitic activity was recorded only in composts K1 and K4.

ACKNOWLEDGEMENTS

REFERENCES

Accepted for print: 10.12.2008

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