Folia Microbiol.

42 (5), 505-508 (1997)

Performance of Immobilized Cells for Dihexyl Sulfosuccinate B iotransformation*

J. HUSKAa**, I. Z,/~VADSKA a, D. TOTHa and P. GEMEINER b
alnstitute of Microbiology, Slovak Academy of Sciences, 949 01 Nitra, Slovakia blnstitute of Chemistry, Slovak Academy of Sciences, 842 38 Bratislava, Slovakia Received April 2, 1997

ABSTRACT. Possibilitiesof using immobilizedbacterial cells for waste water treatment in a continuous process was determined. Cells of Comamonas terrigena strainN3H immobilizedin calcium alginate beads were successfulby used in packed bead-typereactor for continuous biotransformation of the anion-active surfactant dihexyl sulfosuccinate. Absence of calcium ions from the treated medium led to the disruption of alginate beads within 8 d of usage. When the medium was supplementedwith Ca2+ ions the beads were stable for at least one month in the continuous process. During the whole time period the transformationeffectivitywas in the range of 80-100 % even at the highest flow rate of 14 mL/min.

Versatile usage of surfactants (surface-active agents) in almost all human activity resulted in this group of xenobiotics being one of the most abundant in the aqueous environment. Because of their negative impact on aquatic microbiota (Swisher 1987), and operation of waste water treatment plants (Bla~ej et al. 1981) there is an urgent need for their effective removal. Introduction of the bacterial biodegradation potential provides an advantage especially from the economical and environmental viewpoint. Exploration of bacterial specialist-degraders, especially in combination with immobilization procedures is a very promising step in waste-water technology (Scott 1987). Immobilization of bacterial cell offers several advantages, such as the prevention of cell losses occurring in continuous processes, and permits one to work with high cell densities. Furthermore, immobilized microorganisms are more resistant to adverse effects occurring during the degradation processes, such as changes of cultivation parameters, including pH, temperature, fluctuations of substrate concentration, presence of toxic substances, etc. (Woodward 1988; Dawson et al. 1981). Alginate is most widely used as a carder matrix for immobilization of living cells. Entrapment of cells within beads of Ca2+-alginate gels ensures a very mild immobilization procedure and, moreover, this system secures the minimalization of activity losses. Immobilization by entrapment methods also makes it possible to work with high cell densities in comparison with attachment methods (Smidsrod and Skjak-Brrk 1990). Dihexyl sulfosuccinate (DHSS) used here, as a secondary sulfonate belongs to the group of anionic surfactants. This group represents 65 % of the total world surfactant production. Because of excellent wetting, foaming, emulsifying and dispersing properties these surfactants play an important commercial role (Linfield 1976). Therefore some knowledge about their impact and/or fate in the environment and their biodegradation is required. The aim of the present study was to investigate the possibility of immobilization of the DHSS specialist degrader Comamonas terrigena strain N3H in calcium alginate beads for surfactant biotransformation and testing of this system under continuous conditions.

M A T E R I A L AND M E T H O D S
Microorganism. N3H isolate from a site with elevated levels of surfactant pollution identified as Comamonas terrigena (Gram-negative rod bacterium) was obtained during a screening process for surfac-

tant biodegradation. The isolate was stored on MPA no. 1 (meat-peptone agar) slants in a refi'igerator (4 ~ Cultivation and media. To obtain sufficient biomass, bacteria were grown overnight at 30 ~ in 1-L Erlenmeyer flasks filled with 500 mL YEP (yeast extract peptone) broth under static conditions. Overnight cultures (A575 ,~ 0.35) were centrifuged (3000 g, 20 min), the supernatant was discharged and bacteria were resuspended in a minimum amount of sterile distilled water and used for experiments. All experiments were performed in a modified Tris-HCI-DMM (phosphate-flee Davis mineral medium) at pH 7.4. In the case of continuous biotransformation in a small bioreactor the medium was supplemented with 50 mmol/L CaC12. *Presented at the 4th Mini-Symposiumon Biosorptionand MicrobialDegradation, Prague, Czech Republic,November26-29, 1996. **Correspondenceauthor.

506 J. HI]SKA et al.

Vol. 42

Preparation o f immobilized cells. Methods for immobilization of bacterial cells and thereafter for the hardening of alginate gel is described in Hflska et al. (1996). Also the methods for the fitting of optimum biotransformation conditions are described in the same paper. MBAS assay. To determine the surfactant concentration a modified method previously described by Hayashi (1975) was used. Continuous biotransformation. Glass columnar reactor equipped with four outlets was used for continuous biotransformation. Two different dimensions of reactors were applied (data for the smaller are shown in parentheses): total height 530 mm (400 ram); height filled with immobilized biocatalyst 430 mm (220 mm); distance between two outlets 100 mm (70 mm); inner diameter 30 mm (25 mm); volume 300 mL (100 mL). Direction of medium flow was from bottom up. Outlets 1st to 3rd (the small reactor was equipped only with three outlets) were used only for sample taking. To prevent choking of outlets in their neck by alginate beads they were filled with glass cotton. The stock solution of medium left above the reactor and the flow was achieved by hydrostatic pressure. The stock solution of the medium was stirred with a magnetic stirrer for achieving the homogeneous distribution of DHSS (surfactants have a tendency to accumulate at the interfaces) and for aeration. The experiments were carried out at room temperature (24 ~ For the large reactor cells harvested from 7 L of YEP broth were used. In the case of the small reactor cells were obtained from 5.5 L of YEP broth. Free volume represents the amount of medium for the filling of the reactor after packing with beads. Retention time (rain) was calculated as the ratio of free volume to flow rate. Chemicals. Anionactive surfactant - dihexyl sulfosuccinate (DHSS, Merck) as sodium salt - was used; surfactant concentration was 100 mg/L. Powdered sodium alginate (Protanal LF 20/60, Drammen, Norway) was used for immobilization. All other chemicals were of analytical grade.

RESULTS AND DISCUSSION Running of continuous biotransformation of DHSS in packed bead type of bioreactor was based on preliminary results from batch or semicontinuous experiments (Hflska et al. 1996a). The data are summarized in Table I. One of the main disadvantages of alginate Table I. Optimal data for DHSS biotransformation by Comagel is its low stability in the presence of phosmonas terrigena immobilizedin calcium alginate gela phate or citrate ions due to the liberation of cross-linking Ca2+ ions from the gel structure leading to destabilization of the beads (Smidsrod Parameter Value and Skjak-Brdk 1990). In our previous work Adsorption of DHSSto carrier none (Hfiska et al. 1996b) we reported the advantage Temperature 25 ~ of hardening of alginate beads with polyethylepH 7.4 t;eimine and glutaraldehyde on the stability of Biomass loading 120 mg wet mass beads over nonhardened beads. In semicontinuper mL alginate ous batch DHSS transformation experiments the Bead diameter 1.2 mma DHSS tolerance at least 1000mg/L cells immobilized in hardened alginate beads Stabilityb - nonhardenedbeads 6 days were stable for over two months, in comparison - hardened beads 2 months with 6 d for nonhardened beads. After these preliminary results the cells of C. terrigena immoaThe lowest diameter we can produce but an additional increbilized in Ca2+-alginate gel were introduced into ase of biotransformationeffectivityshould be expected below this value. the bioreactor for continual biotransformation of bStability of alginate beads was obtained from semicontinuous DHSS. Fig. 1 shows the course of DHSS transexperiments. formation when the bioreactor was fed with a medium without calcium ions. It is evident that around 70 % of the initial DHSS amount was transformed within the first fourth of the reactor. Table II shows the influence of the initial DHSS concentration on DHSS transformation. It is evident that with a higher concentration (up to 1000 mg/L) the total transformed amount of DHSS is higher in a given time unit. Probably the same concentration gradient was formed in the bioreactor. During the whole running period the flow rate was adjusted to 9 mL/min. Problems arise on the 6th day of operation, when the alginate beads start to swell and prevent liquid flow. Therefore we refilled the bioreactor with the same beads and used it again. Problems recurred and on the 8th day all beads were disrupted and the system ceased to function. The total amount of treated medium was 65 L. Hence the pressure to wash out the calcium ions from alginate structure was over 75 times higher than in a batch experiment. For this reason the addition of calcium ions to the medium seems to be essential for prolonging the life time of alginate beads.

1997

D1HEXYL SULFOSUCCINATE BIOTRANSFORMATION

507

Fig. 2 shows the results from the small bioreactor where the medium was supplemented with 50 mmol/L CaC12. During the 30 d of operation no changes on alginate beads were observed and the cells retained their biotransformation capability throughout this period. To increase of the biotransformation rate with time clearly confirms our previous results t~om semicontinuous biotransformation of DHSS. During the operation of the reactor the DHSS concentration never dropped by more than 20 % of the initial concentration. The total treated amount in this case was 450 L.
100 %
, ,

Table II. Amount of transformed DHSS (mg) in time dependence with initial concentration of DHSS

lnitialconcentration ofDHSS, mg/L Time h 0 2 5 50 19 40 48 100 34 69 89 200 71 122 154 300 135 202 223 500 185 298 333 1000 352 574 648

90

80

70

____2
I
2

s0

I
4

I
6

.....

8 d

Fig. 1. Continuous DHSS biotransformation (%) in a large reactor where the medium contained no calcium ions; I - first outlet V1, 2 - outlet V2, 3 - outlet V3, 4 - last outlet V4; flow rate 9 mL/min.

At the end of the experiment no visual changes in the bead structure were observed. The present results show the possibilities of exploitation of C. terrigena in an immobilized form for continuous treatment of water containing DHSS. However, application of alginate gels, or other kinds of natural polymers in open systems, such as waste-water treatment plants is not suitable for their low stability and because of the abundance of alginate degrading bacteria (Gacesa 1992). Anyway, alginate represents the best laboratory model for testing the behavior of immobilized cells and the results obtained with alginate are in close correlation with many other synthetic hydrogels such as polyvinylalcohol (Hertzberg et al. 1995).

This work was partly supported by

8 10

14

13 12

10

PECO grant no. ERBCIPACTT923020 and by VEGA grant no. 2/3040/96. The authors wish to
thank Mrs. Smogrovi~ov/l and Mr. D6m6ny from the Department of Biochemical Technology, Slovak Technical University in Bratislava for their assistance with the reactor.

% 80

60 REFERENCES BLA~EJ A., TOLGYESSYJ., HAEAMAD., BATORAV., ROSWAL L., RAK J.: Chemical Aspects in Environment. (In Slovak) Alfa, Bratislava 1981. DAWSON M.P., HUMPHREY B., MARSHAL K.C.: Adhesion: a tactic in the survival strategy of a marine vibrio during starvation. Curr.Microbiol. 6, 195-201 (1981). GACESA P.: Enzymic degradation of alginates. Int. J.Biochem. 24, 545-552 (1992). HAVASHi K. : A rapid determination of sodium dodecyl sulphate with methylene blue. Anal.Biochem 67, 503-506 (1987).

20

I
8

I
16

I
2r d 32

Fig. 2. Continuous DHSS biotransformation (%) in a small reactor where the medium was supplemented with Ca2+ ions; 1 - first outlet V1, 2 - outlet V2, ,.3- last outlet V3. The arrows indicate the changes of flow rate (mL/min).

508

J. HUSKA et al.

Vol. 42

HERTZBERGS., MOEN E., VOGELSANG C., OSTGAARD K.: Mixed photo-crosslinked polyvinylalcohol and Ca-alginate gels for cell entrapment. AppLMicrobioLBiotechnol. 1, 10-17 (1994). HUSKAJ., Z,/~VADSKA I., Tt3THD., DOBROTOVA M., GEMEINERP.: Immobilization of suffactant degrading bacteria in alginate gel. Biologia 51,279-283 (1996a). HUSKAJ., Z,~VADSKA I., DOBROTOV,/~ M., TOTHD., GEMEINERP., VRBANOV,kA., AUGUST~IJ.: Anionactive surfactant degradation by immobilized cells, pp. 731-738 in Immobilized Cells: Basics and Applications; Progress m Biotechnology, Vol. 11 (R.H. W ijffels, R.M. Buitelaar, C. Bucke, J. Tramper, Eds). Elsevier, Amsterdam-Lausanne-New York- Oxford--Shannon-Tokyo 1996b. LINFIELDW.M.: Sulfopolycarboxylic acid derivatives in anionic surfactants, pp. 405--433 in Surfactant Science Series (W.M. Linfield, Ed.), Vol. 7, Part 1I. Marcel Dekker, New York 1976. SCOTT C.D.: Techniques for producing monodisperzed biocatalists beads for use in columnar bioreactors. Ann.N.Y.Acad, Sci. 501, 487-493 (1987). SMIDSRODO,, SKJAK-BRCK G,: Alginate as immobilization matrix for cells. Trends Biotechnol. 8, 71-78 (1990). SWISHERR.D.: Surfactant biodegradation. Surfactant Science Series, Vol. 18, 2nd ed. Marcel Dekker, New York 1987. WOODWARDJ.: Methods of immobilization of microbial cells, d.Microbiol.Meth. 8, 91-102 (1988).