16 Magdalena Błaszak and Andrzej Nowak Material and methods Soil for analyses was collected from the humus layer (0 10 cm) of a cultivated field of Na

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1 ECOLOGICAL CHEMISTRY AND ENGINEERING A Vol. 15, No Magdalena BŁASZAK and Andrzej NOWAK* COPPER IONS AS A FACTOR DETERMINING THE ENZYMATIC ACTIVITY OF SOIL BACTERIA JONY MIEDZI JAKO CZYNNIK KSZTAŁTUJĄCY AKTYWNOŚĆ ENZYMATYCZNĄ BAKTERII GLEBOWYCH Summary: The experiment has been aimed at evaluating the way and the range of bacterial activity due to copper ions. Copper(II) chloride dihydrate was introduced into the soil at two rates to achieve the total copper content corresponding to weak and strong soil pollution by the element. After two-month soil incubation several hundreds of bacterial strains were isolated and ranges of proteolytic, amylolytic, and lipolytic activities were determined for every strain. Microorganisms were cultured on solid medium containing casein, tributyrin, or starch. Achieved results were compared with the same activity of microorganisms isolated from control soil. Contamination of soil with copper ions changed bacterial hydrolytic activity to a different degree, but it was not always statistically significant. In most cases, increase in hydrolytically inactive mic- roorganism number and decrease in extremely active strains (from several to several dozens of per cents) was observed after copper chloride application. Higher differences in relation to control were recorded after weak pollution of soil with copper for amylolytic and lipolytic activities. Keywords: copper ions, soil bacteria, hydrolytic activity Due to particularly significant role of soil microorganisms in mineralization of organic matter and, in consequence, affecting the soil fertility, determination of bacterial hydrolytic activity under conditions of weak and strong soil pollution with copper ions has been undertaken. When content of components harmful for the natural environment reaches high level, only microorganisms resistant to xenobiotics may form biological activity of a soil. The accumulation of heavy metals in soil leads to the decrease of biodiversity of soil microorganisms. An attempt was made to answer the question of how particular heavy metals affect the whole group of bacteria with respect to hydrolytic capabilities. The experiment has been aimed at evaluating the way and the range of bacterial activity shaping by copper, as well as directions in which the so-called soil metabolic profile in reference to selected substrates can be changed. * Department of Microbiology and Environmental Biotechnology, Agricultural University of Szczecin, ul. J. Słowackiego 17, Szczecin, mblaszak@agro.ar.szczecin.pl

2 16 Magdalena Błaszak and Andrzej Nowak Material and methods Soil for analyses was collected from the humus layer (0 10 cm) of a cultivated field of National Allotment Gardens at Kasprzak street in Gorzów Wielkopolski. The soil was characterized by the mechanical composition of strong loamy sand (58 % of sand, 25 % of dust and 17 % floatable fraction), ph H2 O = 6.5, and organic matter content of 1.5 %. Natural copper concentration in collected material was 6.1 mg kg 1. The soil was dried and sieved through the sieve of 2 mm mesh to remove mechanical contaminations. Maximum water absorption capacity of the soil was determined and then it was adjusted to 50 %. Copper (II) chloride dihydrate was introduced into the soil at two doses to achieve total copper content corresponding to weak and strong soil pollution by the element [1]. The first dose was set up as 80 mg kg 1, the second as 500 mg kg 1. After two-month soil incubation at 20 C (control soil, soil with the 1 st and 2 nd copper doses), several hundreds of bacterial strains were isolated with Bunt-Rovir s method by means of soil dilution inoculation [2]. Isolated bacteria were inoculated onto subsequent solid mediums containing organic substrates. Hydrolytic activity of inoculated bacteria was estimated on the same mediums that contained: starch (1 %) [3], tributyrin (1 %) [4], casein (1.4 %) [5]. After seven-day incubation, the loss of substrates in mediums was observed. The range of hydrolytic activity towards starch, tributyrin, and casein was determined for every strain. The activity was expressed as index of activity (IA), ie the ratio of hydrolysis zone diameter around the colony to diameter of the colony itself (mm). Microbial parameters: 1. Average hydrolytic activity index. In order to evaluate the activity index (proteolytic, lipolytic, or amylolytic IA) for every strain, the ratio of clarification zone diameter (due to casein, tributyrin or starch loss) around the colony to the diameter of colony itself was measured. Mean IA value for a given carbon and/or energy source was calculated on a base of results for the entire group of bacteria isolated from the soil (200 strains examined in three replications). 2. Groups of hydrolytic activity. Within the proteolytic, lipolytic or amylolytic activity, microorganisms were divided into 4 groups with varied hydrolytic activities: microorganisms showing no activity (IA = 1); microorganisms with moderate activity (1.6 IA > 1); microorganisms with high activity (3 IA > 1.6); microorganisms showing extreme activity (IA > 3). To evaluate statistically significant differences of hydrolytic activity of microorganisms originating from soils of different levels of copper contamination, variance analysis at 0.05 significance level was carried out. Bacterial hydrolytic activities were compared among soils by means of Duncan s multiple range test. Results and discussion The influence of copper ions on bacterial hydrolytic activity was found. After xenobiotic introduced to a soil, the increase in hydrolytically inactive or moderately ac-

3 Copper Ions as a Factor Determining the Enzymatic Activity of Soil Bacteria 17 Fig. 1. Amylolytic activity of bacteria isolated from the control, moderately copper contaminated (I) and strongly copper contaminated soil (II)

4 18 Magdalena Błaszak and Andrzej Nowak Fig. 2. Proteolytic activity of bacteria isolated from the control, moderately copper contaminated (I) and strongly copper contaminated soil (II)

5 Copper Ions as a Factor Determining the Enzymatic Activity of Soil Bacteria 19 Fig. 3. Lipolytic activity of bacteria isolated from the control, moderately copper contaminated (I) and strongly copper contaminated soil (II)

6 20 Magdalena Błaszak and Andrzej Nowak tive bacteria share occurred, number of extremely active bacteria decreased (Fig. 3). The percentage of lipolytically inactive bacteria increased after weak and strong soil contamination with copper ions (by about 5 % and 10 %, respectively). Share of inten- sively hydrolizing tributyrin (high and extreme activity) was decreased in weakly and strongly contaminated soil (by over 60 % and 80 %, respectively) (Fig. 3B). Mean IA for microorganisms from both soil types decreased by 0.3 (Fig. 3A). Other authors when studying the copper influence on selected groups of microorganisms, also observed the decrease in their number or activity [6 10]. Low copper doses introduced into the soil may stimulate the development and enzymatic activity of microorganisms contained [11 13]. That effect can result from a relatively weak influence of copper ions as compared with other heavy metals [14] as well as disorganizing interaction of high copper rates on metabolic processes, which manifests itself as short metabolic shock [15]. Also in present experiment, the activation of microorganisms extremely able to hydrolyze starch (increase in bacteria number by about 10 %) occurred due to weak pollution of soil with copper, along with the decrease of moderately active microorganisms number (by about 20 %) (Fig. 1B). Average index of activity (IA) for that soil was 2.1. Strong copper contamination did not reveal con- siderable changes in reference to amylolytic activity of control group bacteria. Share of extremely active bacteria decreased by several per cents, and content of inactive strains increased by about 20 % (Fig. 1B). However, differences between activities of bacteria from control and strongly contaminated group were not statistically significant with mean IA for strongly polluted soil bacteria amounting to 1.4 and 1.5 for control soil. Experiments of Nowak et al. [11] revealed that amylolytic microorganisms appeared to be the most sensitive to copper; complete inhibition of amylolytic activity occurred after copper application at 5 mm dm 3 rate. No statistically significant influence of weak copper contamination on bacterial proteolytic activity was recorded. Average IA for control and weakly polluted soils was 2.6 (Fig. 2A). Majority of studied microorganisms showed high or extreme activity (about 70 %); proteolytically inactive and moderately active strains made up about 30 %. Addition of copper to the soil at the amounts leading to strong pollution caused changes in particular activity groups percentage. Share of bacteria extremely active in protein hydrolysis decreased by about 20 %. Content of high activity bacteria increased by similar percentage (Fig. 2B). Mean IA for that soil bacteria was 2.1 (Fig. 2A). Conclusions 1. Copper appeared to be factor that determines bacterial hydrolytic activity. Both in weakly and strongly polluted soil, microorganisms showed varied range and level of activities. 2. The largest changes related to lipolytic microorganisms; bacteria manifested di- minished ability to lipids hydrolysis in copper contaminated soil. Amylolytic bacteria from weakly polluted soil were in majority amylolytically inactive or extremely active; the strongly contaminated soil was settled by bacteria with amylolytic activity profile similar to that in control soil.

7 Copper Ions as a Factor Determining the Enzymatic Activity of Soil Bacteria Decrease in the number of bacteria with extremely proteolytic activity and the increase in moderately and high activities was observed along with the increasing soil contamination with copper ions. References [1] Kabata-Pendias A. and Pendias H.: Biogeochemia pierwiastków śladowych. PWN, Warszawa [2] Bunt J.S. and Rowir A.D.: Microbiological studies of some susubantarctic soil.. J. Soil Sci. 1955, 6(1), [3] Cooney D.G. and Emerson R.: Thermophilic fungi. An Account of Their Biology, Activities and Clas- sification. W.H. Freeman and Company, San Francisco, London 1964, pp.188. [4] Kosewska L.: Analiza mikrobiologiczna w przemyśle spożywczym. PWSZ, Warszawa [5] Kędzia W. and Konar H.: Diagnostyka mikrobiologiczna. PZWL, Warszawa [6] Lugauskas A., Levinskaitë L., Peèiulytë D., Repeèkienë J., Motuzas A., Vaisvalavièius R. and Prosyèe- vas I.: Effect of copper, zinc and lead acetates on microorganisms in soil.. Ekologija 2005, 1,, [7] Wyszkowska J. and Kucharski J.: Właściwości biochemiczne i fizykochemiczne gleby zanieczyszczonej metalami ciężkimi.. Zesz. Probl. Post. Nauk Rol. 2003, (492), [8] Kucharski J., Hłasko A., Wyszkowska J. and Jastrzębska E.: Reakcja drobnoustrojów i bobiku na zanieczyszczenie gleby miedzią.. Zesz. Probl. Post. Nauk Rol. 2000, (427), [9] Roane T.M.: Lead Resistance in Two Bacterial Isolates from Heavy Metal Contaminated Soils.. Mi- crob. Ecol. 1999, 37, [10] Hemida S.K., Omar S.A. and Abel-Mallek A.Y.: Microbial populations and enzyme activity in soil treat- ed with heavy metals.. Water, Air, Soil Pollut. 1997, 95,, [11] Nowak A., Przybulewska K., Szopa E. and Stacewicz A.: Wpływ metali ciężkich (Hg, Cd, Cu, Pb) na wzrost i aktywność enzymatyczną bakterii glebowych.. Folia Univ. Agric. Stetin., Agric. 2001, 221, 88, [12] Przybulewska K., Nowak A. and Smolińska M.: Wpływ metali ciężkich na wybrane elementy cyklu przemian węgla.. Zesz. Probl. Post. Nauk Rol. 2003, (492), [13] Rajapaksha R.M.C.P., Tobor-Kapłon M.A. and Bååth E.: Metal toxicity affects and bacterial activities in soil differently. Appl. Environ. Microbiol. 2004, 70(5), [14] Barna J.: Badanie wpływu kadmu, miedzi, ołowiu i rtęci na zmiany ilości biomasy żywych mikroorganiz- mów w glebie. Praca magisterska. AR, Szczecin [15] Nowak A., Szopa E. and Błaszak M.: Wpływ metali ciężkich (Cd, Cu, Pb, Hg) na ilość biomasy żywych mikroorganizmów w glebie.. Acta Agr. Silv., Ser. Agr. 2004, 42,, JONY MIEDZI JAKO CZYNNIK KSZTAŁTUJĄCY AKTYWNOŚĆ ENZYMATYCZNĄ BAKTERII GLEBOWYCH Streszczenie z e e Celem doświadczenia było ustalenie, czy jony miedzi mogą zmieniać zakres aktywności enzymatycznej populacji bakterii glebowych. Do gleby wprowadzono dihydrat chlorku miedzi(ii) w dwóch dawkach, tak by całkowita zawartość miedzi odpowiadała słabemu i silnemu zanieczyszczeniu gleby tym pierwiastkiem. Po dwumiesięcznej inkubacji gleby, izolowano z niej szczepy bakteryjne (po kilkaset) i ustalano zakres ich aktywności proteolitycznej, amylolitycznej i lipolitycznej. Mikroorganizmy hodowano na podłożu stałym z ka- zeiną, tributyryną lub skrobią. Otrzymane wyniki porównywano do tej samej aktywności mikroorganizmów wyizolowanych z gleby kontrolnej. Zanieczyszczenie gleby jonami miedzi zmieniało w różnym zakresie ak- tywność hydrolityczną bakterii, jednak nie zawsze w sposób statystycznie istotny. W większości przypadków, po wprowadzeniu do gleby chlorku miedzi, zanotowano zwiększanie się ilości mikroorganizmów nieaktyw- nych hydrolitycznie oraz zmniejszenie udziału szczepów wybitnie aktywnych (od kilku do kilkudziesięciu procentów). W przypadku aktywności amylolitycznej i lipolitycznej większe różnice wobec kontroli odnoto- wano po słabym, a nie silnym zanieczyszczeniu gleby miedzią. Słowa kluczowe: jony miedzi, bakterie glebowe, aktywność hydrolityczna