JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 69, No. 4, 265-267. 1990

The Inverse Fluidized Bed Biofilm Reactor: A New Laboratory Scale Apparatus for Biofilm Research L U D M I L N. NIKOLOV* AND D I M I T A R G. K A R A M A N E V

Center o f Biotechnology, Sofia University, 8 Dragan Tsankov Str., 1421 Sofia, Bulgaria

Received 6 April 1989/Accepted 8 February 1990

A new bioreactor, the inverse fluidized bed biofiim, reactor, is proposed as a laboratory apparatus for kinetic and diffnsional studies of biofilm. Characteristics of the "ideal" laboratory biofilm reactor are established. Comparison between published in the literature types of bioreactors and the inverse fluidized bed biofilm reactor is made on the basis of these characteristics.

Heterogeneous bioreactors with fixed or immobil ized microorganisms are among the most promising appa- ratuses in b iotechnology now. Great progress was made in appl icat ion of these reactors to different processes in the last few years (Adler, I. , Proc. 4th Eur. Cong. Biotechnol. , Amste rdam, The Netherlands, p. 9, 1987). However , there are only a few types o f specialized labora tory equipment for investigation of b iof i lms- - the ma jo r representative of the heterogeneous biocatalysts .

Kornegay and Andrews (1) and Bakke et al. (2) have used a vertical tubular biofilm reactor for measurement of biofilm kinetics. Hoehn and Ray (3) did their experiments for measurement of some kinetic and diffusional pa- rameters of biofilm, using a horizontal drum as a sup- port . Harr is and Hans fo rd (4) and Onuma et al. (5) have used a flat biofilm carrier. Mulcahy et al. (6) p roposed a single-disk appara tus for the study of kinetics and diffu- sion in a biofilm of denitr ifying bacteria. Another bioreac- tor is called the thin-layer film fermentor (7). Wichlacz and Unz (8, 9) and Nakamura et al. (10) have measured some kinetic characteristics of ferrous iron oxidat ion by a biofilm of Thiobacillus ferrooxidans in a biodisk reactor. A review of the reactor confgura t ions for biofilm process research has been done by Moser (11).

The main aim of this study is to define the main characteristics of the "ideal" reactor for biofilm investiga- t ions, mainly of kinetics and diffusion, and to propose on this basis a new bioreactor , the inverse fluidized bed biofilm reactor, which could be used as a laboratory-scale appara tus for biofilm process research.

A schematic of the inverse fluidized bed biofilm reactor (Nikolov et al., Bulgarian Pat . no. 32910, 1981) is given in Fig. 1. It has been described in detail (12, 13). The liquid circulates in the reactor due to the airlift principle. A bed of particles with a density smaller than that of the liquid is placed in the annulus. The liquid, moving downwards , ex- pands the bed, thus forming the "inverse" fluidized bed. In- itially the lower level of the bed is well above the lower draf t tube opening. The biofilm growth on the surface of particles increases the biopart ic le (support particle with biofilm) density. Due to this, the inverse fluidized bed ex- pands, and the lower bed level moves slowly down, and at a certain biofilm thickness it reaches the lower draft tube opening. The biofilm of the particles, entering the draf t tube, is par t ia l ly s tr ipped off due to the intensive

* Corresponding author.

hydrodynamics . There is a three-phase fluidized bed to in- crease the shear stress. This is the way to control the biofilm thickness. The bioreactor used had a working volume of 5.4•. Its external diameter was 100mm, the draf t tube diameter was 4 0 m m , and the height was 500 mm. S tyrofoam spheres (2.5 l volume) with diameters between 1.8 and 2.2 mm and a density of 250 k g / m 3 were used as support particles.

The bacterial culture of T. ferrooxidans (strain G-15 of the collection of the Biological Facul ty of Sofia University) was originally isolated from drainage waters of a copper mine. The kinetics of ferrous iron oxidat ion by this bacter ium in a free submerged state and in biofilm has been examined (13; Nikolov, L. and Karamanev, D., 34th Canadian Chem. Eng. Conf . , Canada, p. 295, 1984).

The biofilm thickness was measured indirectly by the dry biofilm weight. A minimum of 100 bioparticles were dried at 80°C, and then the biofilm thickness was calculated by the following equation:

= [mbp/(47rrp 2) -- rppp/3]/Pbf ( 1 )

where d is the biofilm thickness; Pb~ is the biofilm density (equal to 2000 kg /m 3 (13)); mbp is the average biopart icle weight; and rp and pp a re the support particle radius and density, respectively.

The oxygen concentrat ion profiles a round and within the biofilm were calculated using a mathemat ical model of the inverse fluidized bed biofilm reactor (14).

The main condit ions that have to be realized in an ideal l abora tory heterogeneous catalytic reactor for process research are: (i) Gradientless fluid, i.e. complete mixing. This is achieved usually by intensive recirculation of the liquid through the bed of catalyst. The reactor volume must be as small as possible. (ii) No effect of external mass transfer (from the bulk of the liquid to the catalyst sur- face). Intensive hydrodynamics should be provided. (iii) No internal mass transfer l imitat ion, i.e. catalyst size has to be so small that the concentrat ion drop due to diffu- sional resistances inside the catalyst particle have no effect on the overall b ioreact ion rate. The condit ions specific for the biofilm (biocatalyst) are: (iv) Maintaining of a constant biofilm thickness at a value according to condi t ion 3. The biofilm grows due to cell reproduct ion. (v) The rat io be- tween the volume of biofilm and that of the liquid phase has to be as big as possible. When this ratio is low, the dilu- t ion rate of the substrate has to be low, and free submerged cells will not be washed out of the reactor.

265

266 NIKOLOV AND KARAMANEV

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FIG. 1.

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These condit ions could serve as a basis for analysis of the published types of biofilm reactors. The first condi- t i o n - n o gradients of the physical and physicochemical properties of liquid media in the reactor-- is fulfilled in almost all of the bioreactors cited above• The only excep- tions are the plate reactors, when the liquid velocity is low. This also results in a low external mass transfer rate. The biofilm thickness is small at the starting period in all the types of reactors, by which the third condit ion is satisfied• However, after a certain period, biofilm thickness in- creases, and the problem of internal diffusion limitation arises• Effective biofilm thickness control is not realized in most types of bioreactors. Partially biofilm thickness is controlled only in the thinlayer fermentors (7) and in the vertical tubular reactors (1, 2). The fifth condit ion con- tradicts the third one. According to condition 3, the biofilm thickness has to be small• In the same time, it is necessary to maintain a large amount of fixed cells• The cell quanti ty is proport ional to the biofilm volume, and at constant biofilm thickness, to the support area (condition 5). Since the specific support area is relatively small in many of the above-mentioned bioreactors, except partially the horizontal drum, biodisc, and vertical cylinder, it is difficult to satisfy simultaneously both requirements 3 and 5. An analysis of the published types of apparatuses is sum-

J . F E R M E N T . B I O E N G . ,

TABLE 1. Characteristics of the biofilm reactors for kinetic studies

No• Type of bioreactor Ref. Condition number ~'

l 2 3 4 5

1 Vertical cylinder 1, 2 : - ~ / * / - / 2 Horizontal drum 3 - - / +,' 3 Flat support 4, 5 + / - , 4 Single disk 6 ~ ~ - / 5 Thin-layer 7 - ~ + /' ! / 6 Biodisk 8, 9, 10 - +/" ~/

7 Inverse fluidized bed This stud3,' + / + • + -

(+), The condition is realized; ( ! / ), realized in part; ( ), not realized.

" Condition number: see text.

marized in Table 1 (rows 1 to 6). Summarizing all the data, the conclusion can be drawn that there are two main pro- blems which have not been solved in general in the existing laboratory biofilm reactors• First, effective control of the biofilm thickness was not reached• The second unsolved problem is the low specific support area•

Analysis of the inverse fluidized bed biofilm reactor pro- posed in this paper was also done on the basis of the characteristics of the ideal laboratory biofilm reactor• It was previously found (12) that the liquid in this reactor is completely mixed (Peclet number 0•001-0.006) under the hydrodynamic conditions typical for cultivation of most types of microorganisms• However, in the case of ferrous iron oxidation by the bacterium T. f e r roox idans , a gra- dient of the dissolved oxygen concentration with reactor height was observed (13)• It did not influence the kinetics of the process, because even the lowest oxygen concentra- tion (3.5rag//) was higher than the limiting one - - 0.64 m g / l (Nikolov, L. and Karamanev, D., 34th Cana- dian Chem• Eng. Conf. , Canada, p. 295, 1984)• Never- theless, a bigger gradient could be expected in the case of microorganisms with more intensive oxygen consumption. In this case, the oxygen concentration drop could be decreased by a decrease of the reactor height, or by using Lighter support particles, which require higher liquid recir- culation, but not high enough to cause biofilm detach- ment.

The profile of the oxygen concentration within the biofilm was calculated by the mathematical model of the inverse fluidized bed biofilm reactor, which included Fick's equation in radial coordinates:

1 1 d d C r,

where Do2 is the diffusivity of oxygen in water, r is the radial coordinate, r~ is the substrate usage rate, and C is the substrate concentration• The values of the constants in the model were the same as that in reference 14 except for the bioreactor design parameters, mentioned above• It was calculated that the concentration drop of oxygen due to the liquid-solid mass transfer resistance is less than 5% of the concentration in the bulk of liquid (Fig. 2).

The biofilm thickness regulation in the inverse fluidized bed biofilm reactor satisfies requirements for work in the kinetic regime• Biofilm thickness can be kept constant for more than two months (13). The experimental profile of the biofilm thickness by reactor height is given in Fig. 3. It can be seen that the biofilm thickness is uniform, except about 15 cm in the top of the inverse fluidized bed, where it is lower• This gradient of the biofilm thickness can be

VOL. 69, 1990 NOTES 267

~ 5 = ¢J

10 support

0

biofilm liquid

1

2

3

0 20 40 60 80 ,pm FIG. 2. Oxygen concentration profile in biofilm. Oxygen concen-

tration in bulk of liquid: 1-8.1 mg/l (at top of the inverse ftuidized bed), 2-5.5 mg/l (at middle of the bed); 3-3.5 mg/l (at the lower bed boundary).

decreased, using proper mater ial for the three-phase fluid- ized bed in the draf t tube. When the biofilm thickness in the reactor is set at a value lower than the critical one (characterized by substrate diffusion into the film), then both condit ions 3 and 4 are satisfied. For the case of biofilm thickness of 85/am, as that in Fig. 3, the profile of the oxygen concentrat ion by the biofilm radial coordinate is shown in Fig. 2. It was calculated by the mathemat ical model (14), including Eq. 2. It can be seen that even when the oxygen concentrat ion in liquid is as low as 3.5 mg/l (concentrat ion in the lower part of the inverse fluidized bed), there is almost no oxygen deplet ion in the biofilm.

The large biofilm suppor t area, usually up to 3000 m2/ m 3 (in this s tudy 1000 m2/m~), allows the mainta inance of a high rat io between the volumes of biofilm and liquid media even at small biofilm thicknesses. Because o f the large biofilm volume, the inverse fluidized bed bioreactor works at high di lut ion rates. In the case of ferrous iron ox- idat ion by T. ferrooxidans, the di lut ion rate was 1.1 h -~, while free submerged bacteria are completely washed out at di lut ion rates over 0.1 h l (data not shown).

It was found out that shear stress caused by particle- part icle interact ion does not disturb biofilm format ion and growth. Previously (Karamanev, D. et al., Proc. 4th Eur. Cong. Biotechnol. Amste rdam, The Netherlands, p. 328- 331, 1987) it was shown that the biofilm growth rate in this reactor is almost the same as that in a biodisk reactor.

Another useful p roper ty of the bioreactor is that the biofilm itself can be studied easily. It is possible to take biopart icles out of the reactor for detailed study of the physical and biological propert ies of the biofilm.

150

100

E

5O /

I I I I 0 !0 20 30 40 50

h, c m

FIG. 3. Profile of the biofilm thickness by reactor height, h =0 at top of the bed.

REFERENCES

1. Kornegay, B . H . and Andrews, J .F . : Kinetics of fixed film biological reactors. J. Water Poll. Control Fed., 40, 460-468 (1968).

2. Bakke, R., Trulear, M.G. , Robinson, $. A., and Charaeklis, W.J. : Activity of Pseudomonas aeruginosa in biofilms: steady state. Biotechnol. Bioeng., 26, 1418-1424 (1984).

3. I-Ioehn, R. C. and Ray, A. D.: Effect of thickness on bacterial film. J. Water Poll. Control Fed., 45, 2302-2320 (1973).

4. Harris, N. P. and I-Iansford, G. S.: A study of substrate removal in microbial film reactor. Water Res., 10, 935-943 (1976).

5. Onuma, M., Omura, T., Umita, T., and Aizawa, J.: Diffusion coefficient and its dependence on some biochemical factors. Biotechnol. Bioeng., 19, 1533-1539 (1985).

6. Muleahy, L. T., Shieh, W. K., and LaMotta, E. $.: Experimental determination of intrinsic denitrification kinetic constants. Biotechnol. Bioeng., 23, 2403-2406 (1981).

7. Moser, A.: Bioreactors with thin-layer characteristics. Bio- technol. Lett., 4, 281-286 (1982).

8. Wichlaez, P .L. and Unz, R.F. : Growth kinetics of attached iron-oxidizing bacteria. Appl. Environ. Microbiol., 50, 460-467 (1985).

9. Wiehlacz, P. L. and Unz, R. F.: Fixed film biokinetics of ferrous iron oxidation. Biotechnol. Bioeng. Symp. Ser., 11, 493-506 (1977).

10. Nakamura, K., Noike, T., and Matsumoto, J.: Effect of opera- tional conditions of biological Fe 2~ oxidation with rotating biological contactors. Water Res., 20, 73-77 (1986).

11. Moser, A.: Biotechnology, p. 311-347. In Rehm, H.-J. and Reed, G. (ed.), WCH Press, Weinheim (1985).

12. Nikolov, L. and Karamanev, D.: Experimental study of the in- verse fluidized bed biofilm reactor. Can. J. Chem. Eng., 65,214- 217 (1987).

13. Karamanev, D. and Nikolov, L.: Influence of some physicochemical parameters on bacterial activity of biofilm. Fer- rous iron oxidation by Thiobacillus ferrooxidans. Biotechnol. Bioeng., 31, 295-299 (1988).

14. Chavarie, C., Karamanev, D., Nikolov, L., and Champagne, J.: Simulation d'un r6acteur ~. biofilm h fluidisation invers6e, p. 254- 263. La Fluidisation, Toulouse, France (1985).