INFLUENCE OF DIFFERENT CONDITIONS FOR BIOFILM FORMATION ON THE OXIDATION ACTIVITY OF ACIDITHIOBACILLUS FERROOXIDANS V. Mamatarkova Sofia University St. Kliment Ohridski, Faculty of Biology, Department of Biotechnology, Sofia, Bulgaria Correspondence to: Vyara Mamatarkova E-mail: vmamatarkova@yahoo.com ABSTRACT The dynamics of formation and functioning of biofilm of Acidithiobacillus ferrooxidans JCM 3863 have been investigated in batch in two quite different types of biofilm reactors design shake flasks (SFBFR) and bubble columns (BCBFR). However the packed bed has been consisted of the same elements - long thin expended polystyrene parallelepipeds. The bioreactors have been very different by the hydrodynamic conditions ensured and their mode of aeration. It has been observed that the biofilms formed in SFBFR and BCBFR under the same conditions (temperature, ph, ferrous and ferric ions concentrations) had different surface structure and thickness. The influence of the bioreactor design and their capacity to ensure conditions for ferrous ion oxidation has been investigated by moving the biofilms, formed and functioning in one type of biofilm reactor to the another type of bioreactor design. Under such conditions, the average specific surface oxidation rates of ferrous to ferric ions have been about five times higher in SFBFR that those in BCBFR. The comparison of the experimental results obtained in the two types of biofilm reactors also showed that these rates obtained with biofilm formed in SFBFR have not been influenced significantly by the change of the type of bioreactor in which the biofilm functions, then it has been of crucial importance for the biofilm of BCBFR. Keywords: Acidithiobacillus ferrooxidans, biofilm, bioreactors Introduction Bacteria Acidithiobacillus ferrooxidans oxidize ferrous to ferric ions. The solutions of ferric ions are used in biohydrometallurgy (18), for purification of waste gases containing sulfur compounds and in acid mine drainage treatment (1). These bacteria are capable to form biofilms on solid supports with cell concentration in the biofilm of about 1 11 cells/cm 3 (19), which make them interesting for use in high performance biofilm reactors. The biofilms themselves formed by these bacteria possess some interesting properties, different from those, formed by other types of microorganisms. They are solid, consisted mainly of inorganic compound named jarosite (3,4) and small amount of extracellular polysaccharides. Their mechanical strength allows easy manipulations, without any damages of their entities. This makes them convenient for setting up special experiment for elucidating some of the most important phenomena in biofilm reactors. The most interesting amongst them are the investigations on the influence of the mechanical conditions on the biofilm properties. Especially it is important for biofilm reactors with inverse fluidized bed (IFBBFR) (11,12) and fixed bed in its annulus (FBBFR) (15) based on the airlift principle. In these reactors there are two different compartments in the reaction zone - one in the airlift upriser with intensive hydrodynamics and other - in the downcomer with quiet mode of liquid phase movement. In IFBBFR the high intensity of the hydrodynamics is used for biofilm thickness control, when in FBBFR the biofilm grows without control. The maintenance of the biofilm thickness in IFBBFR is carried out by means of different mechanisms (9,11,17) occurring simultaneously in the same space. This makes difficult the evaluation of their contribution in the biofilm control, which induces necessity of additional experimentations. The preliminary observations showed that it was possible to model the compartment with intensive hydrodynamics by bubble column biofilm reactor (BCBFR). As to the compartment with quiet liquid flow, it was modeled by shake flasks biofilm reactor (SFBFR) (8). The aim of this work is to investigate the different hydrodynamic conditions on surface structure and oxidation activity of biofilm of Acidithiobacillus ferrooxidans JCM 3863 using these two 99 12 YEARS OF ACADEMIC EDUCATION IN BIOLOGY
types of biofilm reactors. Materials and methods Bacteria. The experiments were carried out with the strain Acidithiobacillus ferrooxidans JCM 3863 kindly submitted by Japan Collection of Microorganisms. Media. The liquid medium used for cultivation of suspended culture of bacteria and forming and functioning of biofilm of Acidithiobacillus ferrooxidans JCM 3863 contained the following components (1,2): (NH 4 ) 2 SO 4.4 g/l, MgSO 4.7H 2 O.4 g/l, K 2 HPO 4.4 g/l, FeSO 4.7H 2 O 5 g/l. ph was corrected by 1 N H 2 SO 4 maintaining its value to 1.9±1. Bioreactors and conditions. The experiments were carried out in batch in two types of laboratory heterogeneous bioreactors with biofilm of Acidithiobacillus ferrooxidans JCM 3863: shake flasks biofilm reactor (SFBFR) with working volume 35 ml and bubble column biofilm reactor (BCBFR) with working volume 1.65 l (8). As solid support were used parallelepipeds from expended polystyrene 45 1 8 mm for SFBFR and 29 8 8 mm for BCBFR. In SFBFR liquid volume was.25 l and total support surface.78 m 2. In BCBFR liquid volume was 1.35 l and total support surface.22 m 2. The aeration rate in BCBFR was maintained at 1.3 vvm, when for SFBFR the oxidation occurred at 21 rpm. These stirring conditions were accepted as suitable for providing oxygen supply in excess and for ensuring the ideal mixing in the reaction zones. The temperature was 28±1 C and biofilm thickness was about 7 µm in all experiments. Formation of biofilm. In both types of bioreactors the biofilm of Acidithiobacillus ferrooxidans JCM 3863 was formed in feed-batch regime using a method especially developed for these microorganisms (14,15,16). Analytical methods. The concentration of ferrous and ferric ions and total iron were determined by spectrophotometric analysis (7). The biofilm thickness was determined on the base of its dry weight and specific density 237 kg/m 3 (11). This procedure is described in details previously (6,8). Dynamics of oxidation using biofilms. Originally ferrous ions oxidation was investigated using the biofilms formed in the each of biofilm reactors. Later the biofilm from SFBFR has been moved in BCBFR, whereas the biofilm of BCBFR has been installed in the SFBFR. The volumetric and surface rates of oxidation were calculated at degree of oxidation.85-.9 as follows: r ( S S ) o f vol = - volumetric rate (g/l.h), where S and S f t are initial and final concentration of Fe 2+ ions (g/l) respectively, t duration of cycle (h) rsv rsurf = - surface rate (g/m 2.h), where V is the volume of F liquid phase (l), F total biofilm surface (m 2 ), calculated on the base of total surfaces of solid support. S S f η = - degree of oxidation of Fe 2+ (-). S Results and Discussion Surface structure and thickness of biofilm formed in bioreactors The biofilms are showed on Fig. 1. Fig. 1. Surface structure of 4 days aged biofilms: A inert solid support without biofilm; B formed in SFBFR; C formed in BCBFR. The visible difference in surface structures is due to different hydrodynamics of two bioreactors. In SFBFR the bioprocess occurred only in two phases liquid medium and solid support with biofilm under the conditions of low shear stress. Thеre, the conditions were soft and due to this reason no erasing of biofilm was going on and its thickness increased. The biofilm surface was rough with developed surface near-by to the liquid phase. In the BCBFR reaction zone there were three phases liquid medium, solid support with biofilm and gas phase in the form of air bubbles. There, the level of shear stress was high, which provoked erosion of surface layers of biofilm. This led to smooth biofilm surface. It is important to mention that the color of the biofilms in both reactors was the same and close to this obtained in the biodisk reactors [13]. Similar results were obtained during 91 12 YEARS OF ACADEMIC EDUCATION IN BIOLOGY
investigations on influence of conditions for formation of biofilm from Acidithiobacillus ferrooxidans on its properties in biodisk reactors, packed bed and fluidized bed biofilm reactors [5,1,11,13]. It was found in these works that the nature of the solid supports, the type of bioreactor [5], initial concentrations of substrate Fe 2+ [13] play important role on the properties of biofilm such as color, density, porosity [1,13] etc. In our case the type of solid support and initial concentration of ferrous ions especially were selected to be the same in the both bioreactors. This gave possibility to observe the differences in connection with the type of biofilm reactor used and mainly with the intensity of hydrodynamics in their reaction zones. Obviously, they determine the appearance of the biofilm surface structure. Along with differences in the biofilm surface structure, a different in the dynamics of biofilm formation and growth of each of bioreactors was defined (Fig. 2). δ [μm] 32 3 28 26 24 22 2 18 16 14 12 1 8 6 4 2 5 1 15 2 25 3 35 4 45 t [days] Fig. 2. Dynamics of biofilm formation in SFBFR; in BCBFR. The soft conditions of aeration in SFBFR promoted faster biofilm growth (fig.2). The presence of air bubbles in the BCBFR provoked the biofilm erosion and in such a way hindered the biofilm thickness growth. For equal period (for example 2 days) the biofilm in SFBFR reached to about 1 µm, then in BCBFR it was of about 66 µm. These results showed that the main reason for effective biofilm thickness control in the upriser of IFBBFR probably is the high level of the shear stress in there. Oxidation activity of biofilms The activity of biofilm was investigated in repeated batch regime. Each of cycles in both type bioreactors was carried out under the same conditions. r vol [g/l.h] 2,6 A 18 B,5,4,3,2,1, 1 2 3 4 5 6 cycle number r surf [g/m 2.h] 16 14 12 1 8 6 4 2 1 2 3 4 5 6 cycle number Fig. 3. Oxidation rates - A volumetric oxidation rate, r vol; B surface oxidation rate, r surf - in SFBFR; in BCBFR. The biofilms in the separate biofilm reactors were formed in feed-batch regimes. After reaching the biofilm thickness of about 6 μm the essential experimental series were switched over to batch regimes. Fig. 3 represents the oxidation rates in the both types of bioreactors after the change the modes of functioning to the batch regimes. The duration of these experiments was rather short and for this reason it was supposed that the biofilm thickness was not changed significantly remaining in the intervals 6-7 μm. It can be seen, that during the first two cycles the volumetric rates in SFBFR and BCBFR were quite different. At the third cycle they became equal. In the following two cycles in BCBFR the volumetric rate remained almost the same, when in the SFBFR increased slowly. In the fifth cycle in SFBFR and BCBFR remained nearly equal.58 and.54 g/l.h respectively. This behavior was due to the ratio of volume of liquid phase to the surface of biofilm V/F 32.5 l/m 2 in SFBFR and 6.14 l/m 2 in BCBFR. The activity of biofilms expressed as specific surface rates in both of bioreactors was quite different 18.1 and 3.27 g/m 2.h respectively. In batch regime in the liquid phase of bioreactors there was a good quantity of swimming cells of Acidithiobacillus ferrooxidans (1 7-1 8 cells/ml) [8], removed from the biofilm by erosion to the liquid phase. They also took part in the oxidation process. Thus, the ferrous ions oxidation occurred through heterogeneous homogeneous mechanism and the total rate of oxidation was determined by the overall activity of microbial cells in biofilm and those in the liquid phase. But it is quite possible that this mechanism is not only responsible for these differences in overall oxidation activity. We made 911 12 YEARS OF ACADEMIC EDUCATION IN BIOLOGY
series of experiments combining the conditions of biofilm formation with the mode of biofilm reactor functioning. The biofilm formed in SFBFR was installed in the BCBFR and inversely the biofilm formed in BCBCR to SFBFR in fresh nutrient media. In these experiments the ratio V/F for the both bioreactors was the same and equal to 38.5 l/m 2. As a base of comparison there were used biofilms produced and functioning in same SFBFR and BCBFR with the same ratio of V/F 38.5 l/m 2 (Table 1). TABLE 1 Surface rates of oxidation: bioreactors 1 and 3 controls; bioreactors 2 and 4 combinations of biofilm and type bioreactor Bioreactors 1. SFBFR with biofilm from SFBFR 2. BCBFR with biofilm from SFBFR 3. BCBFR with biofilm from BCBFR 4. SFBFR with biofilm from BCBFR r surf [g/m 2.h] 14.4 12.6 8.41 4.36 It can see that the biofilm formed in SFBFR keeps approximately the same activity in bioreactors 1 and 2 the difference is about 12 %. Contrary to this result the biofilm formed in BCBFR and functioning in SFBFR (bioreactor 4) demonstrates almost two times lower surface rate compared to this in control bioreactor 3. It can be supposed that this effect is due to higher real biofilm surface when its superficial layers are rough with highly developed specific surface. Thus, the ratio V/F reflects only the geometrical relationship, which is apparent but not realistic. Conclusions The results obtained show the significant role of the conditions under which the biofilms are formed. Their influence on the biofilm superficial layers structure is evident. For the future it will be very interesting to investigate these differences in the depth of biofilms formed in different bioreactors under various conditions. The experience of the present work will be useful as the base for setting up a new type of biofilm investigations. As to the differences in oxidation activity it is clear that the role of biofilm formation and functioning are also extremely important. The experiments show that the higher oxidation activity can be improved if the hydrodynamic conditions are successfully chosen. This reveals again the crucial role of the combination of bioreactor design, the process and regime of functioning. Acknowledgment The author would like to express her gratefulness to Assoc. Prof. Ludmil Nikolov, Biological Faculty of Sofia University, Bulgaria, and Prof. Dimitre Karamanev, Department of Chemical and Biochemical Engineering, University of Western Ontario in London, Canadа, for their fruitful discussions. REFERENCES 1. Ahonen L, Tuovinen O.H. (1991) Appl. Environ. Microbiol., 57, 138-145. 2. Ahonen L, Tuovinen O.H. (1991) Appl. Environ. Microbiol., 58, 6-66. 3. Daoud J., Karamanev D. (26) Miner. Eng., 19, 96-967. 4. Grishin S.I., Bigham J.M., Tuovinen O.H. (1988) Appl. Environ. Microbiol., 54, 311-316. 5. Grishin S.I., Tuovinen O.H. (1989) Appl. Microbiol. Biot., 31, 55-511. 6. Karamanev D., Nikolov L. (1988) Biotechnol. Bioeng., 31, 295-299. 7. Karamanev D., Nikolov L., Mamatarkova V. (22) Miner. Eng., 15, 341-346. 8. Mamatarkova V. (22) Ph.D. Thesis, Sofia University, Bulgaria, 61-63. 9. Mamatarkova V., Nikolov L., Karamanev D. (1994) Proceedings of the International Workshop and Young Scientist School, Sofia, Bulgaria, 132-135. 1. Nikolov L. (1992) In: Biofilms: Science and Technology, Kluwer Academic Publishers (eds. L.F.Melo et al.), NATO ASI Series, Series E: Applied Science, 223, 511-521. 11. Nikolov L., Karamanev D. (199) J. Ferment. Bioeng., 69, 265-267. 12. Nikolov L., Karamanev D., Elenkov D. (1981) Bioreactor with Moving Bed, Bulg. Invention, 3291/9.1.1981. 912 12 YEARS OF ACADEMIC EDUCATION IN BIOLOGY
13. Nikolov L., Karamanev D., Mamatarkova V., Mehochev D., Dimitrov D. (22) Biochem. Eng. J., 12, 43-48. 14. Nikolov L., Mehochev D., Dimitrov D. (1986) Biotechnol. Lett., 8, 77-71. 15. Nikolov L., Penev Tz., Popova B., Ivanov A., Kuzmanov L. (1986) Biotechnol. Biotech., 2, 14-16. 16. Nikolov L., Valkova-Valchanova M., Mehochev D. (1988) J. Biotechnol., 7, 87-94. 17. Rittmann B.E. (1989) In: Structure and Function of Biofilms (eds. Characklis W.G. and Wilderer P.A.), Wiley, 49-58. 18. Torma A.E. (1977) Adv. Biochem. Eng., 6, 1-37. 19. Vulkova-Vulchanova M., Nikolov L., Penev Ts. (1987) CR Acad. Bulg. Sci., 4, 69-72. 913 12 YEARS OF ACADEMIC EDUCATION IN BIOLOGY