The article is devoted to the problems of sorption purifying of the water environments polluted  by heavy metals ions. The influence of microorganisms on sorption properties of aluminosilicate sorbents is examined. It was studied sorption of Fe 2 + , Cu 2 + ions with bacterial cell in the free state immobilized on a surface of aluminosilicate sorbents and also at formation of biofilms. It is shown that  the presence of the immobilized microorganisms increases the limiting capacity of heavy metals  sorption by aluminosilicate sorbent. The formation of biofilms on the surfaces of sorbent increases  heavy metals resistance of microorganisms and their sorption efficiency in comparison with the aluminosilicate sorbent

Ignatenko, A. V.

Sazanovets, M. A.

The article is devoted to the problems of sorption purifying of the water environments polluted by heavy metals ions. The influence of microorganisms on sorption properties of aluminosilicate sorbents is examined. It was studied sorption of Fe 2 + , Cu 2 + ions with bacterial cell in the free state immobilized on a surface of aluminosilicate sorbents and also at formation of biofilms. It is shown that the presence of the immobilized microorganisms increases the limiting capacity of heavy metals sorption by aluminosilicate sorbent. The formation of biofilms on the surfaces of sorbent increases heavy metals resistance of microorganisms and their sorption efficiency in comparison with the aluminosilicate sorbent

Belarusian State Technological University

Biotechnology 189УДК 628.112+574.582  M. A. Sazanovets, PhD student (BSTU);  A. V. Ignatenko, PhD (Biology), assistant professor (BSTU) ANALYSIS OF INFLUENCE OF MICROORGANISMS  ON HEAVY METALS ADSORPTION BY ALUMINOSILICATE SORBENTS The article is devoted to the problems of sorption purifying of the water environments polluted by heavy metals ions. The influence of microorganisms on sorption properties of aluminosilicate sor-bents is examined. It was studied sorption of Fe2 +, Cu2 + ions with bacterial cell in the free state im-mobilized on a surface of aluminosilicate sorbents and also at formation of biofilms. It is shown that the presence of the immobilized microorganisms increases the limiting capacity of heavy metals sorption by aluminosilicate sorbent. The formation of biofilms on the surfaces of sorbent increases heavy metals resistance of microorganisms and their sorption efficiency in comparison with the alu-minosilicate sorbent. Introduction. Environmental pollution by heavy metals is one of actual environmental problems. Ion-exchange pitches, carbon and alumosilicate sorbents are most widely used for cleaning of wastewater from heavy metals. Sorbents with well developed internal surface of micropores reaching 1000 m2/g, possess the greatest efficiency and sorption capacity for binding of ions of heavy met-als (HM) in the range of 100–580 mg/g [1, 2].  One of shortcomings of microporous sorbents is pollution of their surface by organic, inorganic pollutants, and also microorganisms. Fast devel-opment on a surface of sorbents of bacteria and other microorganisms leads to change of sorption properties of such sorbents. Main part. The purpose of the real work – the analysis of binding of bacteria on a surface of alu-minosilicate sorbents and their influence on sorp-tion of heavy metals. As an object of the research we used alumi-nosilicate sorbent on TU 2163–001–01115840–94 with a specific surface of 30 m2/g. Sorbent was fractioned with the help of sieves selecting gran-ules with the size of (0.9 ± 0.1) mm. Pure cultures of bacteria from the collection of the department of biotechnology and bioecology: Bacillus sp., E. coli, of Pseudomonas sp. and also the strains allocated from sewage were used. Collection cultures of bacteria were sowed in a loop on the slanted nutritious agar (NA) and culti-vated during 3 days. Then washouts of cultures in the nutritious broth (NB) cultivated during 2 h for transferring cells to the exponential growth phase. After it cells have been sediment by centrifugation at 8000 rev./min. within 10 min., washed three times in physiological solution from the nutrient medium under the same conditions of centrifuga-tion.  Assessment of concentration of microorgan-isms in water was carried out by the turbidimetric method [3]. For this purpose calibration curve of light intensity changing at 600 nm (D600) from concentration of cells in suspension were built.  A total number of microorganisms in water media were determined by their cultivation in nu-tritious agar as N = a · 10f / V,                         (1) where a – average number of the grown colonies on the plates; f – dilution degree of suspension of cells; V – suspension volume placed on agar sur-face. Concentration of microorganisms in water media varied in the range of 106–108 CFU/ml. The maintenance of Fe2+, Cu2+ ions in water media was determined by a chelatometric method based on optical density testing of heavy metals complexes with EDTA (EthyleneDiamineTetraace-tic Acid) at 370 nm and 750 nm, respectively. For this purpose calibration curves of optical density changes from concentration of HM previously were built. The process of HM sorption was studied in a static mode. Stationary sorption of HM was carried out in flasks V = 50 cm3 containing 2 g of a sor-bent and 20 cm3 of HM solution of the correspond-ing concentration. Flasks were stirred up in a shaker during 1 h selecting on 2 cm3 of the sample within 10 min., 2 cm3 of a complexon of EDTA were added (1:1) and then optical density of the received complexes HM–EDTA was measured.  A value of HM sorption by a sorbent was de-termined by expression  0( ) ,pC C VAm− ⋅=                       (2) where Co, Cp – initial and equilibrium HM concentra-tions; V – solution volume; m – dry mass of sorbent.  Correspondence of НM sorption to the equa-tion I. Langmuir [4] was checked: ,( 1)A K CAK C∞⋅ ⋅=⋅ +                         (3) where A∞ – the limiting capacity of HM sorption; K – constant of Langmuir; C – equilibrium concen-tration of HM. ISSN 1683-0377. Proceedings of BSTU. 2013. No. 4. Chemistry, Organic Substances Technology and Biotechnology 190 Value A∞ was found by transforming the ex-pression (3) in the return coordinates 1/A from 1/C.  Sorption of microorganisms at the surface of aluminosilicate sorbent was registered by tur-bidimetric method as the change of concentration of сells in water after sorbents particles sedi-mentation. Dry biomass was determined by its weighting on analytical scales after centrifugation of сells and their drying up to the constant weight at 105oC. For studying of influence of microorganisms on sorption properties of a sorbent daily culture of cells washed by centrifugation in physiological solution were passed through a column with alu-minosilicate granules up to the full saturation of a sorbent. Exit of microorganisms from a column was registered by the changes of optical densities at 280 nm and 600 nm. Columns with the cells of bacteria immobilized on a sorbent were used fur-ther for building of biofilms. For this purpose glu-cose solution (10–4 M) was passed through the col-umns during 3 days.  After adsorption of microorganisms on the alu-minosilicate granules and formations of biofilms solutions of HM were passed through the received samples under the same conditions, as well as in the case of an initial aluminosilicate sorbent. Assessment of viability of microorganisms in presence of HM was carried out by reductase method in accordance with State standard 23454–79 by means of redox-dye methylene blue (MB) [5], and also by the method of diffusion of sub-stances into agar.  Concentration of MB in solution before and af-ter connection with sorbent was measured at    680 nm. Knowing the surface area occupied by a mole-cule of MB, and the concentration of the connected dye, the size of a specific surface of a sorbent was accounted as it is described in [6]. The obtained data were processed statistically, using Microsoft Excel software. Comparative study of sorption properties of an aluminosilicate sorbent based on analysis of iso-therms of HM sorption was made before and after microorganism’s immobilization and after forma-tion of a biofilm.  Time of sorption of HM was chosen equal 2 h as a record of kinetic curves of sorption showed that the equilibrium state in the analyzed case was established during 1–2 h and further didn't change. At first A∞ value of HM sorption by an alumi-nosilicate sorbent was estimated. The results of the analysis of the received isotherms of HM sorption are given in fig. 1.  Apparently from Fig. 1, Fe2+ binding is well described by the equation of an isotherm of monomolecular sorption of I. Langmuir. Similar dependence is observed and in the case of Cu2 +. The calculated values A∞ are given in the table. At the second stage sorption of bacteria on a surface of an aluminosilicate sorbent was studied (Fig. 2).  As seen from the table a specific capacity of binding of cells on a surface of a sorbent varies in the range of 0.45–0.75 mg/g for the studied micro-organisms. Unlike an aluminosilicate sorbent the isotherm of HM sorption on the immobilized microorga-nisms has more complicated character (Fig. 3).  The area of low concentration of HM in which sorption submits to Langmuir's equation, and the area of high concentration described by depend-ence of G. Freundlich is allocated [3]. It is proba-bly connected with penetration of HM at high con-centration inside the cells and their death. The calculated values of Fe2+, Cu2+ sorption on aluminosilicate sorbent and biosorbents in the free and immobilized states are given in the table at 20oC.   00,050,10,150,20,250,30,350,40 50 100 150ΔC, мг/лCравн, мг/л   02468101214160 0,1 0,2 0,31/ΔC1/Cравн  Fig. 1. Isotherm of Fe2+ sorption on aluminosilicate sorbent  in straight and return coordinatesΔС, mg/l l/ΔС  Сp, mg/l l/Сp 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0  0                   0.1                 0.2                  0.3 Biotechnology 191Table Sorption capacity of sorbents and biosorbents Sorbents Sorbated object A∞, mg/g Aluminosilicate gra-nules  Fe2+ 2.2 ± 0.1 Aluminosilicate gra-nules Cu2+ 2.9 ± 0.4 Aluminosilicate gra-nules E. сoli 0.45 ± 0.05 Aluminosilicate gra-nules Pseudomonas sp. 0.75 ± 0.10 E. сoli  Fe2+ 3.5 ± 0.3 Pseudomonas sp Fe2+ 27.6 ± 0.5 E. сoli on alumino-silicate sorbent  Cu2+ 6.5 ± 0.4 Pseudomonas sp bio-film on aluminosili-cate sorbent Fe2+ 120.1 ± 0.8  As it is seen from the table, a sorption capac-ity of aluminosilicate sorbent in the presence of   the immobilized microorganisms of E. сoli in-creases by 2–3 times in comparison with the ini-tial sorbent. In actual practice the majority of microorgan-isms immobilized on a surface of sorbents, can form biofilms. In that case their stability to HM can be changed.  Fig. 1 shows the reductase activity of Bacillus sp. in suspension as well as in biofilm (Fig. 4). According to the received data cells of micro-organisms are 3–4 times steadier against cop-per ions being a part of a biofilm, than in a  free state. It was checked also how the formation of a biofilm influences the sorption properties of an aluminosilicate sorbent (Table).  In the case of sorption of Pseudomonas sp. cells, steady against HM and formed a biofilm on a surface of aluminosilicate sorbent, a value of A∞ for Fe2+ increased at 50–60 times in comparison with an aluminosilicate granules.  00,010,020,030,040,050,060,070,080 0,05 0,1 0,15 0,2 0,25ΔN·10−8 , кл/млNравн·10−8, кл/мл  0501001502002500 20 40 601/ΔN·10−8 , мл/кл1/Nравн·10−8, мл/кл  Fig. 2. Isotherm of E. сoli sorption on aluminosilicate sorbent in straight and return coordinates 00,020,040,060,080,10,120,140,160,180 0,1 0,2 0,3 0,4ΔC, мг/млCравн, мг/мл 0 2 46-10123456789101/ΔC1/Cравн  Fig. 3. Isotherm of Cu2+ sorption on E. сoli in straight and return coordinates Сp, mg/m1 Np ⋅ 10–8, cells/ml l/Np ⋅ 108  ΔN ⋅ 10–8, cells/ml l/ΔN ⋅ 10–8 , ml/kl ΔС, mg/ml l/ΔС –1/Сp 0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0  0              0.1            0.2            0.3            0.4                        2                    4                  6  0         0.05       0.1       0.15       0.2       0.25 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0  ISSN 1683-0377. Proceedings of BSTU. 2013. No. 4. Chemistry, Organic Substances Technology and Biotechnology 192 2100,20,40,60,810 1 2 3Время культивирования, дни Fig. 4. Reductase activity of Bacillus sp. in the presence of Cu2+ (10–3 M): 1 – cells in NB; 2 – cells in a biofilm  Conclusion. The obtained data show that mi-croorganisms are well attached to a surface of an aluminosilicate sorbent and form biofilms. Steady to HM cultures of cells poses a higher sorption capacity of HM than usual cells. Bacteria cells show the increased resistance to HM in the immobilized state and keep the viability at higher concentration of HM in comparison with free microorganisms. Formation of a biofilm by the steady cultures of cells increases their sorption capacity to  HM on 1–2 orders in comparison with the initial aluminosilicate sorbent and also increases stabil-ity of cells to HM in comparison with cells in a free state. References 1. Смирнов, А. Д. Сорбционная очистка воды / А. Д. Смирнов. – Л.: Химия, 1982. – 168 с. 2. Илялетдинов, А. Н. Микробиологическая очистка воды от тяжелых металлов / А. Н. Иля-летдинов // Водные ресурсы. – 1980. – № 2. –  С. 158–169. 3. Фридрихсберг, Д. А. Курс коллоид- ной химии / Д. А. Фридрихсберг. – Л.: Химия, 1984. – 368 с. 4. Грег, С. Адсорбция, удельная поверхность, пористость / С. Грег, К. Синг. – 2-е изд. – М.: Мир, 1984. – 306 с. 5. Методы определения ингибирующих ве-ществ: ГОСТ 23454–79. – Введ. 01.01.80. – М.: Стандарты, 1989. – 6 с. 6. Савицкая, Т. А. Коллоидная химия: лаб. практикум: в 2 ч. / Т. А. Савицкая, Д. А. Коти-ков. – Минск: БГУ, 2004. – Ч. 1. – 104 с. Received 28.02.2013                           Relative time  of discolouring of MB Cultivation time, days 1 0.8 0.6 0.4 0.2 0 

Analysis of influence of microorganisms on heavy metals adsorption by aluminosilicate sorbents

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Analysis of influence of microorganisms on heavy metals adsorption by aluminosilicate sorbents

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