Appl Microbiol Biotechnol (1991) 35:662-668 017575989100199U

@pried Afwrobiology Biotechnology © Springer-Verlag 1991

Biodegradation of 4-chlorophenol by adsorptive immobilized Alcaligenes sp. A 7-2 in soil Jiirgen Balfanz and Hans-Jiirgen Rehm Institut fiir Mikrobiologie, Universitat Miinster, Corrensstrasse 3, W-4400 Miinster, Federal Republic of Germany Received 23 January 1991/Accepted 26 April 1991

Summary. Alcaligenes sp. A 7-2 immobilized on granular clay has been applied in a percolator to degrade 4chlorophenol in sandy soil. G o o d adsorption rates on granular clay were achieved using cell suspensions with high titres and media at p H 8.0. The influence o f various parameters such as aeration rate, p H , temperature, concentration o f 4-chlorophenol and size of inoculum on the degradation rate were investigated. During fedbatch fermentations under optimal culture conditions, concentrations o f 4-chlorophenol up to 1 6 0 m g . 1 - 1 could be degraded. Semicontinuous culture experiments demonstrated that the degradation potential in soil could be well established and enhanced by the addition of immobilized bacteria. Continuous fermentation was p e r f o r m e d with varying 4-chlorophenol concentrations in the feed and different input levels. The m a x i m u m degradation rate was 1.64 g. 1- 1. d a y - 1.

1985, 1989; Westmeier and R e h m 1986; Keweloh et al. 1989). The strain Alcaligenes sp. A 7-2 has been shown to degrade m o n o c h l o r o p h e n o l s (Westmeier and R e h m 1985, 1986, 1987). During our investigations we examined Alcaligenes sp. A 7-2 for degrading 4-chlorophenol in sandy soil. By determination o f various parameters a model system for biodegradation of xenobiotics in soil was established.

Materials and methods Microorganism Pure cultures of.41caligenes sp. A 7-2 (Schwien and Schmidt 1982) were used throughout the experiments.

Media

Introduction Haloaromatics are widely used as intermediates in chemical syntheses, as insulators, pesticides, plasticizers and solvents (Leisinger and Brunner 1986). A m o n g these, chlorophenols are mainly used as efficient fungicides. I n a d e q u a t e handling of chlorinated phenols has led to their release into the environment. Because o f their toxicity and persistence they represent a serious pollution problem, especially if these xenobiotics m o v e through the soil by washing out and contaminate ground water aquifers (Apajalahti and Salkinoja-Salonen 1986). The advantages of immobilized bacteria for biodegradation of phenols have been demonstrated in earlier investigations. By using immobilized microorganisms, biomass is protected against washing out and often increased resistance to xenobiotics such as phenol or 4chlorophenol can be observed (Ehrhardt and R e h m

Offprint requests to: H.-J. Rehm

The basis of all media was a mineral medium containing (per 1): Na2HPO4.2H20, 3.5 g; KH2PO4, 1.0g; (NH4)2SO4, 1.0g; MgSO4- 7 H20, 0.2 g; Ca(NO3)2.4 H20, 0.05 g; Fe(III)-ammonium citrate, 0.01 g; 1 ml of a trace element solution (Pfennig and Lippert 1966).

Precultivation medium. 3-Chlorobenzoate was added as the sodium salt (3 mM) to the mineral salts medium. The pH was adjusted to 7.0. Fermentation medium. 4-Chlorophenol was added to the mineral salts medium after sterilization as the only source of energy and carbon. The pH was set to 7.0. For fed-batch, semicontinuous and continuous fermentations in soil columns the medium was alkalized by changing the phosphate buffer: Na2HPO4.2H20, 4.5 g.l-1; KH2PO4, 0.2 g-1-1. The final pH wa~ set to 8.0.

Carrier materials and soil Granular clay (Lecaton, Hutzel Hydrokulturen, Bad Iburg, FRG) with a diameter of 2.5-5 mm was used as the carrier. The Lecaton particles were cooked in 10 -3 N HC1 for 30 min and washed in distilled H20 before use. Aquarium sand served as model soil. A sieve analysis showed that about 90% of the sand particles were

663 0.63-1.12 mm in diameter (Omar and Rehm 1988). The density was 1.57 g . c m -3 and the surface was about 70 cmZ.g -1 sand. Before use the sand was heated at 800°C for 5 h in a muffle furnace.

and continuous culture the cells were suspended in 20 mM phosphate buffer, pH 8.0, and recycled for 48 h.

Culture conditions Immobilization

Batch cultures of free bacteria were grown on a gyratory shaker at 30°C. Five millilitres of a 1-day-old preculture was inoculated into 15 ml fermentation medium (or 3 ml of a 3-day-old preculture into 150 ml fermentation medium). Fermentations in soil columns were carried out with a packedbed fermentor (50 mm diameter, 800 ml total volume) filled with 50 g granular clay and 675 g sand (Fig. 1).

Immobilization experiments. Alcaligenes sp. A 7-2 was grown on agar plates (3 mM 3-chlorobenzoate) for 2 days at 28 ° C. Precultivation medium (100 ml in a 500-ml erlenmeyer flask) was inoculated and incubated on a rotary shaker for 2 days at 30°C and 125 rpm. Cells were centrifuged (20 min, 10,000g) and resuspended in the medium used for adsorption experiments (0.9% NaC1 and 20 mM phosphate buffer, respectively, at different pH values. Adsorption was carded out by pumping the cell suspension through a cylindrical container (made of high-grade steel wire netting) filled with 20 g granular clay in a glass column (21 cm length x 4 cm diameter) by means of a peristaltic pump at a flow rate of 2.41. h - 1 for 24 h. The amount of cells immobilized was determined by counting the cells of the suspension before and during immobilization in a Thoma counting chamber.

Fed-batch fermentation. The liquid phase of the fermentor was 350 ml mineral medium and about 30 ml capillary medium of granular clay. For aeration experiments and fermentations with different 4-chlorophenol concentrations, immobilization was carded out with a 1 : 10 diluted cell suspension of a preculture. Fermentations were incubated at room temperature. The aeration rate was generally set at 0.5 1-min -1. When 4-chlorophenol had been totally degraded it was supplied twice more.

Fermentation in soil columns. Cell suspension (300ml) was pumped through-five containers each filled with 10 g granular clay. The cells were suspended in 0.9% NaCl. For semicontinuous

Semicontinuous fermentations. One fermentor was filled with 875 g sand and 220 ml fermentation medium. It was inoculated with 10 ml cell suspension used for immobilization. "When 4-chlo-

24

m ~

--1

3

2

I

14 • 14

5

13

11

10 9

8 7

Fig. 1. Schematic view of the fermentation equipment used for biodegradation of 4-chlorophenol in soil with adsorbed cells of Alcaligenes sp. A 7-2: 1, thermostatted packed-bed fermentor; 2, pH electrode; 3, pH meter; 4, 02 electrode; 5, O2-measuring instrument; 6, recorder; 7, air source; 8, stop valve; 9, air-flow meter; 10, sterile filter; 11, air wetting; 12, aeration frit; 13, outlet valve; 14, peristaltic pump; 15, mixing desk; 16, fermentation medium; 17, mineral medium; 18, wire netting; 19, sand; 20, granular clay; 21, percolation loop; 22, percolation loop (only for semicontinuous cultivation); 23, septum for sampling medium; 24, reflux condenser

664

80 \

70

~ - - 4 ~ .

_

~

_

6o

70 =

=

=

60

_

5o

r

o

;

4O

~ 3o

r.-

? 2o

2o

10 0

10 0

10

20

30

-

i

40 80 120 160

t (h)

0

20

40

i

,

I

60

80

,

i

i

I

i

100 120 140 160

Fig. 2. Biodegradation of different 4-chlorophenol concentrations

t (h) Fig. 3. Biodegradation of different 4-chlorophenol concentrations

(mg/l) in shake cultures inoculated with 1.5. l0 s cells.m1-1. @, 30; O, 40; @ , 50; O, 60; I , 70

in a shake culture inoculated with 1.5.107 cells, m l - ' (for symbols see Fig. 2 legend)

rophenol had been degraded the medium was run off and the fermentor was washed with 200 ml mineral medium and filled with 200 ml fermentation medium (40 mg.l -I of 4-chlorophenol). By this method the titre of free cells was diminished by a factor of ten. A second fermentor was packed as described before.

Continuousfermentation. The fermentation was started as a fedbatch culture. Five doses of 4-chlorophenol (40 mg-1-1) were added to the fermentor. When 4-chlorophenol had been degraded, 160 ml medium was run off and the continuous fermentation was started. There was backwater only at the bottom of the column.

~o 12 v-4 x

10

~

8

c

2

\

1

f

f

_-------0 U

Analytical methods 4-Chlorophenol was determined quantitatively by the photometric method of Martin (1949) using 4-aminoantipyrene as the colour reagent. At the end of fermentation the total number of cells adsorbed on sand and granular clay was determined by protein measurement (Lowry et al. 1951; Hanson and Phillips 1981) with reference to a calibration curve constructed with defined dilutions of a cell suspension. The degradation rate was defined as the concentration of 4chlorophenol divided by the total time needed for degradation (mg.1-1.h-1).

0

,

,

,

I

4

,

~

,

I

8

,

,

0

,

I

,

,

,

12

I

,

,

,

I

16

20

,

,

,

I

24

t (h)

%

2,0

v-i

x 1,6 "0 1,2

b

"

~ o

"~ 0,8

~ 0,4 if)

Results

Biodegradation of 4-chlorophenol in shake cultures Batch fermentations in shake cultures with Alcaligenes sp. A 7-2 s h o w e d a significant i n t e r d e p e n d e n c e between the time n e e d e d for degradation, 4 - c h l o r o p h e n o l c o n c e n t r a t i o n a n d size o f inoculum. Cultures with high cell c o n c e n t r a t i o n s (1.0.109 cells/ml) at the beginning (Fig. 2) were able to d e g r a d e 4 - c h l o r o p h e n o l f r o m 30 a n d 60 rag. 1-~ with nearly the same d e g r a d a t i o n rate (average rate: 2.2 m g . 1-1. h - 1). The capability o f deg r a d a t i o n was restricted to a c o n c e n t r a t i o n o f 7 0 m g . 1 - 1 . I f the inocula were r e d u c e d to a tenth (1.0.10 7 cells, m l - 1 ) the d e g r a d a t i o n rate diminished b y 60% (30 a n d 40 m g . l - ' ) c o m p a r e d to cultures with a high i n o c u l u m (Fig. 3). C o n c e n t r a t i o n s o f m o r e t h a n 50 mg- 1-1 were not degraded.

u

0 0

4

8

12

16

20

24

t (h)

Fig. 4. a Adsorption of Alcaligenes sp. A 7-2 on granular clay in relation to the cell concentration. Cell suspensions used (cells/ ml): O, 1.21.107; 0 , 5.49.107; I"1, 1.16.108; I , 6.45.108. b Adsorption ofAlcaligenes sp. A 7-2 on granular clay in relation to the pH of the cell suspension used: O, pH 5; 0 , pH 6, [3, pH 7; I , pH 8

Adsorption of Alcaligenes sp. A 7-2 onto granular clay Figure 4a a n d b d e m o n s t r a t e that m o s t cells were ads o r b e d d u r i n g the first 5 h o f immobilization. With increasing cell concentrations the percentage o f a d s o r b e d cells to the total a m o u n t o f cells s u s p e n d e d in 100 ml m e d i u m diminishd (Table 1). The p H in w h i c h i m m o -

665 Table 1. Adsorption ofAlcaligenes sp. A 7-2 in relation to the titre of the cell suspension used

Cell titre (1. ml-1)

Cell adsorption per g granular clay after 24 h

Total amount of cells adsorbed (%)

1.21.107 5.49.107 1.16.108 6.45.10 a

4.22.107 1.27.108 2.48.108 1.36.109

69.6 46.2 42.8 42.2

t(h) F

A

B [l

so b

I-I

t

II

40

/

40rng/I ]

so r /I I

E 60

I

30 20 10

Table 2. Adsorption ofAlcaligenes sp. A 7-2 in relation to the pH of the cell suspension used

pH and cell titre (1. ml- 1)

Cell adsorption per g granular clay after 24 h

Total amount of cells adsorbed (%)

5/6.23.107 6/6.56.107 7/6.58-107 8/7.24-107

9.72.107 1.47.10 s 1.37.10 s 1.95-108

31.2 44.8 42.2 53.8

0

0,1 0,5 1,0 2,0

I/min

2.

3.

Fig. 5. A Biodegradationof 40 mg. 1- i of 4-chlorophenol at different aeration rates. B Biodegradationof 4-chlorophenol in relation to the concentrationused, added at three successivetimes (1., 2., 3.)

t (h) bilization was performed also modified the adsorption of bacteria (Table 2). At pH 8.0 cells adsorbed twofold better than at pH 5.0.

1.

addition of 4-chlorophenol

A

B

30 25 20

Biodegradation of 4-chlorophenol in soil Degradation of 4-chlorophenol in soil columns. The following experiments were carded out to build up an optimized system for biodegradation of haloaromatics by adsorptive immobilized bacteria in soil (Balfanz 1988). Various aeration rates were tested during the mineralization of 40 mg. 1- ~ 4-chlorophenol (Fig. 5a). No interdependence of aeration rate and degradation rate was observed. Oxygen saturation did not reach values below 60%. High aeration rates (1.0 and 2.01.min -1) caused strong turbulence in the fermentor and cell material was sheared off the granular clay and sand. Therefore further experiments were carded out at an aeration rate of 0.5 1. min -1. Fed-batch fermentations at different concentrations of 4-chlorophenol (Fig. 5b) showed that repeated use of immobilized cells increased the degradation rate and decreased the degradation time. After the third addition of 4-chlorophenol at concentrations of 40 and 50 rag. 1-1, the degradation rate increased by a factor of four compared with those rates after the first addition. In the course of fermentation with 60 mg. 1-1 of 4-chlorophenol the degradation rate was only doubled. For further experiments the concentration of 4-chlorophenol was set to 40 mg. 1-1. Figure 6a demonstrates that the mineralization of 4chlorophenol could be significantly enhanced by a high inoculaum. This effect was essentially limited to the first degradation phase, whereas after the second and third addition of 4-chlorophenol there were no more distinct differences. Fermentations at different pH values (Fig. 6b) showed that media with an initial pH of

15 10 5 0

1.

2.

3.

L 1.

2.

3.

addition of 4-chlorophenol

Fig. 6. A Biodegradationof 40 mg. l - ~of 4-chlorophenol in relation to the cell loading of the carder. The symbols indicate the titres of the cell suspensions used for immobilizationin m1-1. B Biodegradationof 40 mg. 1- ~of 4-chlorophenol in relation to the initial pH value

7.7 reduced the degradation time for the first addition of 4-chlorophenol (17 h at pH 6.1; 14.5 h at pH 7.7). Experiments at different fermentation temperatures (Fig. 7) showed that temperature effects were also mainly restricted to the first degradation phase. The degradation time was reduced from 42h (15°C) to 11 h (32.5 ° C). The best degradation results were achieved at 30 ° C even after the third addition of 4-chlorophenol.

Degradation of increasing concentrations of 4-chlorophenol in fed-batch culture. The toxicity limit, i.e. the maximal degradable concentration of 4-chlorophenol, was determined using fed-batch cultures (Fig. 8). The maximal degradation rate (14.1 m g . l - l . h -1) could be detected at 70.3 mg-l-1 of 4-chlorophenol. Higher concentrations up to 132.3 mg. 1-1 reduced the degradation rate continuously to 3.5 mg.1-1-h -1. At 157.1 mg.1-1 biodegradation slowed down to 0.7 m g . l - 1 , h-1. Concentrations of more than 105 mg.1-1 seemed to be very

666 t(h)

F

]

,.,5,0

401-1

2. 17,s

tl

/

3. 20,o

4. 2 2 , 5

301-11 l II 251-11

5. 25,o ° c 6. 27,5 7 30,0 8. 32,5

20

~ ....

hi

60

/

351-1

_

/

/ / /

/

--

"-5 cO-

50

lo

4-0

,el

30 0

107 ~=

20 I ,,¢

10

10 0

I

,

,

t

1 23456789

1.

1 23456789

1 23456789

7O

2. 3. addition of 4-chlorophenol

6o

Fig. 7. Biodegradation of 40 mg. 1-1 of 4-chlorophenol in relation to the fermentation temperature

1801/

p 30

1109

150

50 ~, ¢0 c-

60

~

,

,

,

10

I

. . . .

20

I

. . . .

I

30

t (h)

4-0

J:J

b) free and irrrnobilized cells

10a

0

10z "

--e 2o

?

"¢ 10 0 0

c-"

I

~

0

5

0

10 9

a) free cells

70

10z ~:

10

20

4-0

30

t (h)

Fig. 9. Biodegradation of 40 mg.l- ~ of 4-chlorophenol by free cells (a) or free and immobilized cells (b) in a semicontinuous cultivation: O, 4-chlorophenol; O, titre of free cells

U

~-

30 0

106

0

1£

80 t (h)

Fig. 8. Biodegradation of 4-chlorophenol in fed-batch culture. After 161 h, 10 ml of a concentrated mineral medium was added: Q, 4-chlorophenol; ©, titre of free cells

80

¢1

$2

$3

$

4

7O

~ 5o

i

4o toxic to the system. Bacterial growth of free cells stopped and the titre o f free cells diminished. After fermentation, 1.109 cells, g - 1 granular clay were immobilized in each zone of the fermentor and 1.6.10 s cells-g -1 sand were adsorbed at the top and 3.5.108 cells.g -a sand at the bottom of the column, respectively.

Degradation of 4-chlorophenol in semicontinuous culture. Semicontinuous culture experiments should compare the degradation rates of two different inoculated soils: the first soil was inoculated only with free cells (Fig. 9a) and the second one was inoculated with free and clayadsorbed cells (Fig. 9b). After total degradation o f the first doses of 4-chlorophenol, the titre of free cells was nearly equal in both experiments (3.5.108 cells-ml-]). During fermentation with free cells the degradation rate hardly intensified (first addition, 2.4 mg. 1-1- h - 1 third addition, 2.6 mg. 1-1. h - 1). By using immobilized cells, the degradation rate continuously increased despite a very low titre of free cells (first addition, 3.1 mg.1-1 . h - l ; sixth addition, 10.0 m g . 1 - 1 - h - 1 ) . During the fermentation a gradient of cell material adsorbed onto granular clay had been built up in the

_~ 3o ? 2o o

.......... .,4 ,

0

,

,

,

[

100

,

,

,

,

[

,

,

,

200

,

....... I

300

,

,

i

" i

|

i

i

400

t (h)

Fig. 10. Biodegradation of 4-chlorophenoi in continuous cultivation. The arrows indicate a change of 4-chlorophenol concentration in the feed: 1, from 40 to 20mg.l-1; 2, 40mg.l-I; 3, 80mg.l-1; 4, 100mg.l-]; solid line, 4-chlorophenol input in mg. 1-1. h- ]; dotted line, 4-chlorophenol efflux in mg- 1-1

soil column (at the top, 4.6.108 c e l l s . g - l ; at the bottom, 2.3.108 cells.g-I). In both experiments 5.107 cells, g - ] sand were immobilized. No gradient could be detected.

Degradation of 4-chlorophenol in continuous culture. To test the maximum degradation capacity a continuous fermentation was established (Fig. 10). Like the upper layers of soil outdoors, the soil was only wet by the capillary water of sand and granular clay at the top of the

667

soil column during continuous cultivation. At the end of the fed-batch phase the titre of free cells was 1.8- 10 9 cells.m1-1. The degradation rate increased from 3 . 0 m g . l - l . h -1 to 32.1 m g . l - l . h - k During the first phase (0-45 h) the titre of free cells diminished to 3 . 5 . 1 0 7 by washing out. At an input of 25 mg.1-1 -h -1 of 4-chlorophenol (40 mg. 1-1 in the feed) degradation was incomplete and 4-chlorophenol accumulated in the medium. In the course of continuous fermentation, the degradation rate increased to 68.3 m g . l - l . h - 1 by changing stepwise the input and concentration of 4chlorophenol in the feed. At that time the input was 73.5 m g . l - l - h -1 (100 mg.1-1 in the feed).

Discussion

Westmeier and Rehm (1985, 1986) found good degradation capacities of immobilized Alcalioenes sp. A 7-2 for 4-chlorophenol in a packed-bed fermentor with granular clay as the carrier material, especially during continuous culture experiments. The present work indicates that the application of immobilized Alcalioenes sp. A 7-2 to decontaminate soil can be advantageous under certain conditions. All degradation kinetics for 4-chlorophenol could be divided into a lag phase and a degradation phase. This effect is probably due to induction processes of the enzyme systems needed for degradation. Precultures were grown on 3-chlorobenzoate. Therefore the lag phase might be explained by a lack of induced phenolhydroxylase (Dorn and Knackmuss 1978; Westmeier and Rehm 1985). The extended lag phases at the beginning of 4-chlorophenol degradation with diluted and 3-dayold cell suspensions can be traced back to a general loss of enzyme activity and to the small inoculum size. These results correspond to those of Westmeier and Rehm (1985). They found a rapid decrease in degradation activity after a starvation period of 3 days during semicontinuous fermentations of Alcaligenes sp. A 7-2 at different pH values. The ability to degrade higher concentrations of 4-chlorophenol is consequently based on the status of enzyme induction and the use of high cell titres to lower toxic concentrations to ones tolerated well. Phenolic compounds are toxic to microorganisms, even to those with biodegradation potential (M6rsen and Rehm 1990). If cells are exposed too long to high amounts of chlorophenols cell damage can occur. Therefore we particularly directed our attention to the lag phase in order to find optimal parameters that would minimize this phase. Best degradation results were achieved using optimal parameters, especially temperature and high biomass concentration. Concentrations of up to 160 mg-1-1 could be degraded. These results correspond with those of Molin and Nilsson (1985) and to Crawford and Mohn (1985). Using high biomass concentrations they found increasing degradation activities during the degradation of phenol and pentachlorophenol.

In the course of our investigations it became obvious that mainly the induction period could be effected by different parameters. As soon as the enzyme systems had been induced and a particular cell concentration in soil had been exceeded, degradation of 4-chlorophenol was independent of biomass concentration, due to low substrate levels, and temperature (in the range 20-

30oc).

Granular clay proved to be a carrier material with satisfactory adsorption capacities (Omar and Rehm 1988). At the beginning of each experiment nearly 75% of adsorbed cells were washed off the carrier. The same effect had been observed by Westmeier and Rehm (1985). Among others, this effect can be traced back to the different surface energies of cell and carrier material. Formation of solid biofilms was made easier by formation of surface film of either bound water or organic material (Gerson and Zajic 1979). Depending on the type of fermentation biofilm formation resulted from resorption of free cells and cell growth on the carrier. Biofilms on sand particles were only available after long fermentation periods. This effect is probably due to the relatively small colonizable surface area compared to the porosity of granular clay (Li and DiGiano 1983). Acknowledgements. This work was supported by a grant of the Bergbauforschung Essen/DMT/BMFT (1460521) and DECHEMA/AIF.

References Apajalahti JHA, Salkinoja-Salonen MS (1986) Degradation of polychlorinated phenols by Rhodococcus chlorophenolicus. Appl Microbiol Biotechnol 25:62-67 Balfanz J (1988) Abbau von 4-C1-Phenol durch immobilisierte Bakterien im Modellboden. Diploma-Thesis, University of MOnster Crawford RL, Mohn WW (1985) Microbiological removal of pentachlorophenol from soil using a Flavobacterium. Enzyme Microb Technol 7:617-620 Dorn E, Knackmuss HJ (1978) Chemical structure and biodegradability of halogenated aromatic compounds. Two catechol 1,2dioxigenases from a 3-chlorobenzoate-grown pseudomonad. Biochem J 174:73-84 Ehrhardt HM, Rehm HJ (1985) Phenol degradation by microorganisms adsorbed on activated carbon. Appl Microbiol Biotechnol 21:32-36 Ehrhardt HM, Rehm HJ (1989) Semicontinuous and continuous degradation of phenol by Pseudomonas putida P 8 adsorbed on activated carbon. Appl Microbiol Biotechnol 30:312-317 Gerson DF, Zajic JE (1979) The biophysics of cellular adhesion. In: Venkatasubramanian K (ed) Immobilized microbial cells. ACS Symp Ser 106:29-57 Hanson RS, Phillips JA (1981) Chemical composition. In: Gerhardt P, Murray RGE, Costilow RN, Nester EW, Wood WA, Krieg NR, Phillips GB (eds) Manual of methods for general bacteriology. Am Soc Microbiol, Washington DC, pp 358359 Keweloh H, Heipieper H J, Rehm HJ (1989) Protection of bacteria against toxicity of phenol by immobilization in calcium algihate. Appl Microbiol Biotechnol 31:383-389 Leisinger T, Brunner W (1986) Poorly degradable substances. In: Rehm H J, Reed G, Sch6nborn W (eds) Biotechnology, vol 8. Microbial degradations. VCH, Weinheim, pp 475-513

668 Li AYL, DiGiano FA (1983) Availability of sorbed substrate for microbial degradation on granular activated carbon. J WPCF 55 (4):392-399 Lowry OH, Rosenbrough NJ, Lewis Farr A, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275 Martin RW (1949) Rapid colodmetdc estimation of phenol. Anal Chem 21 : 1419 Molin G, Nilsson I (1985) Degradation of phenol by Pseudomonas putida ATCC 11 172 in continuous culture as different ratios of biofilm surface to culture volume. Appl Environ Microbiol 50:946-950 M6rsen A, Rehm HJ (1990) Degradation of phenol by a defined mixed culture immobilized by adsorption on activated carbon and sintered glass. Appl Microbiol Biotechnol 33:206-212 Omar SH, Rehm HJ (1988) Degradation of alkanes by Candida parapsilosis and Penicilliumfrequentans immobilized on granu-

lar clay and aquifer sand. Appl Microbiol Biotechnol 28:103108 Pfennig N, Lippert KD (1966) 0ber alas Vitamin B12-Bediiffnis phototropher Schwefelbakterien. Arch Microbiol 55:245-256 Schwien U, Scbmidt E (1982) Improved degradation of monochlorophenols by a constructed strain. Appl Microbiol Biotechnol 44:33-39 Westmeier F, Rehm HJ (1985) Biodegradation of 4-chlorophenol by entrapped Alcaligenes sp. A 7-2. Appl Microbiol Biotechnol 22:301-305 Westmeier F, Rehm HJ (1986) Einsatz von Immobilisationsverfabren zum biologischen Abbau chlorierter Phenole. Chem Ind 3:158-160 Westmeier F, Rehm HJ (1987) Degradation of 4-chlorophenol in municipal wastewater by adsorptive immobilized Alcaligenes sp. A 7-2. Appl Microbiol Biotechnol 26:78-87