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effect of substrate availability on bacterial growth kinetics. F. Volkering, A. M. ... have been shown to be mutagenic and/or carcinogenic. (Lavoie et al. 1979).

Appl Microbiol Biotechnol (1992) 36:548-552

Applied Microbiology Biotechnology © Springer-Verlag 1992

Microbial degradation of polycyclic aromatic hydrocarbons: effect of substrate availability on bacterial growth kinetics F. Volkering, A. M. Breure, A. Sterkenburg, and J. G. van Andel National Institute of Public Health and Environmental Protection, Laboratory for Waste Materials and Emissions, P.O. Box 1, 3720 BA Bilthoven, The Netherlands Received 22 July 1991/Accepted 13 September 1991 Summary. It is demonstrated that bacterial growth on crystalline or adsorbed polycyclic aromatic hydrocarbons can result in a linear increase in biomass concentration. A simple mathematical approach is presented, showing that under these circumstances mass transfer from the solid phase to the liquid phase is rate-limiting for growth.

Introduction Polycyclic aromatic hydrocarbons (PAH) are hazardous compounds originating from oil, tar, wood-preserving creosote, or from incomplete combustion of fossil fuels. The contamination of air (Daisey et al. 1979), soil (Bossert and Bartha 1984) and water (Andelman and Snodgrass 1974) by PAH has been reported and several PAH have been shown to be mutagenic and/or carcinogenic (Lavoie et al. 1979). Because of their hydrophobicity PAH occur in the environment mainly attached to particles. It has been shown that PAH are biodegradable compounds (e.g. Davies and Evans 1964; Evans et al. 1965) and therefore biotechnological techniques might be applicable for remediation of polluted soil. However, application of such techniques for remediation of polluted soils has demonstrated that PAH of higher molecular mass are degraded very slowly, and that the residual concentration of PAH is too high to permit unrestricted application of the treated soil according to Netherlands governmental guidelines (Ministry of Housing, Physical Planning and the Environment 1987; Socz6 and Staps 1988; Staps 1990). It has been shown that pure cultures of bacteria can use naphthalene and phenanthrene in the dissolved state only (Wodzinski and Bertolini 1972; Wodzinski and Coyle 1974) and therefore the dissolution of solid PAH is a prerequisite for growth. This implies that even in systems without intraparticle mass transfer limitaOffprint requests to: F. Volkering

tion, such as shaking cultures, mass transfer from the solid phase to the aqueous phase might be rate-limiting. This can explain the often-observed linear growth of bacteria and yeasts on slightly soluble substrates (e.g. McLee and Davies 1972; Prokop et al. 1971; Stucki and Alexander 1987; Thomas et al. 1986). This study has been performed to investigate the kinetics of PAH degradation in relation to the solubilization rate of PAH. Insight into these kinetics may lead to better understanding of the factors involved in the incomplete removal of PAH in biotechnological soil remediation processes. Theory The rates of biomass formation and substrate uptake are related according to the equation (Tempest 1970): -

dXt -

~

_

Y. dS,

- -

dt

(1)

dt

where Xt is the biomass concentration (kg-m-3), St is the total amount of substrate per unit volume (kg-m-3), Y is the yield constant (kg biomass formed.kg substrate used -1) and t is the time (h). When the substrate is present in a solid and a liquid phase, the change in total substrate concentration is: dSt dt

~

1 V

-

-

,

dQ, dCt + dt dt

(2)

where V is the volume (m3), Q, is the amount of solid or adsorbed substrate (kg) and C, is the concentration of substrate in solution (kg.m-3). Consequently, Eq. 1 can be written as: dXt

d-~- =

- Y.

(3)

.--+

dt

-di-/

The rate of mass transfer from the solid phase to the aqueous phase can be described as (Perry et al. 1963): dQ, dt

KI. A-(Ceq- C,)

(4)

549 w h e r e K~ is a c o n s t a n t ( m . h - ~), A is t h e c o n t a c t s u r f a c e (m 2) a n d C~q is the e q u i l i b r i u m c o n c e n t r a t i o n o f substrate in s o l u t i o n ( k g . m - 3 ) . A t l o w e r cell d e n s i t i e s ( w h e n C, >>0) m a s s t r a n s f e r is n o t l i m i t i n g a n d e x p o n e n t i a l g r o w t h c a n occur. A t h i g h cell d e n s i t i e s in g r o w i n g cultures, h o w e v e r , o n e m a y a s s u m e t h a t C, is n e g l i g i b l e c o m p a r e d to Ceq and constant \dt

= 0

)

and, therefore, that the mass

..................................................................... x~ ............. . . . . . . . . . . . . . . .

~

~x~\

transfer approaches the maximal rate:

\

i

:......................

\\

i

\ i

0

- ~ - ] m~ = K , . A . Ceq

(5)

I n this s i t u a t i o n , t h e rate o f u p t a k e o f s u b s t r a t e b y t h e b i o m a s s c a n n o t e x c e e d t h e d i s s o l u t i o n rate a n d , b y c o n s e q u e n c e , t h e b i o m a s s f o r m a t i o n rate is l i m i t e d to t h e m a s s t r a n s f e r rate a c c o r d i n g t o :

dX, Y ~ = - ~

[dQ, I ~/~.x

-

Y . K , . A . C~q v

(6)

Equation 6 describes the linear increase with time of the biomass concentration when the surface and the y i e l d are c o n s i d e r e d c o n s t a n t . W h e n t h e s u b s t r a t e is a d d e d in c r y s t a l l i n e f o r m , t h e m a s s t r a n s f e r r a t e - ~d Q , ~s . e q u a l to t h e d i s s o l u t i o n dt rate J a n d t h e e q u i l i b r i u m c o n c e n t r a t i o n C~q c a n b e replaced by the saturation concentration, C~. ~e equat i o n for l i n e a r g r o w t h in this s i t u a t i o n is:

dXt ~t

~en the

Y

Y. K~ . A . C ~

= v " Jm.~ =

V

(7)

t h e i n c r e a s e in b i o m a s s c o n c e n t r a t i o n is l i n e a r , specific growth

rate

(

~t = Xt

dt ] is c o n t i n -

uously decreasing. To complete the model describing b a t c h g r o w t h o n s l i g h t l y s o l u b l e s u b s t r a t e s the M o n o d e q u a t i o n is u s e d to y i e l d the r e l a t i o n s h i p o f ~ a n d C,: ~, = ~ a ~ "

~

C~

(8)

Ct + K~

w h e r e ~ is t h e a c t u a l s p e c i f i c g r o w t h rate ( h - 1), ~max is t h e m a x i m a l s p e c i f i c g r o w t h rate (h -~) a n d K , is t h e s a t u r a t i o n c o n s t a n t ( k g . m - 3 ) . C~ is c a l c u l a t e d b y integ r a t i o n o f Eq. 4 y i e l d i n g :

c,=co+

o dt

V

o dt

(9)

w h e r e Co a n d C~ a r e t h e d i s s o l v e d s u b s t r a t e c o n c e n t r a t i o n s at t i m e t = 0 a n d t = t respectively. An example of a graphical representation of the m o d e l is g i v e n in Fig. 1.

Materials

and methods

Bacterial cultures. Mixed bacterial populations that were capable of growth on naphthalene, phenanthrene, anthracene or fluoranthene as single sources of carbon and energy were isolated by se-

\

time

Fig. 1. Graphical representation of the model for batch growth on solid substrate: - - , biomass concentration (X,) calculated according to the equation X, = Xo e ' ' ' ; - - - , substrate concentration (Ct) calculated according to Eq. 9 (see text); . . . . , specific growth rate (~tt) calculated according to Eq. 8 (see text) lective enrichment from a domestic waste-water treatment plant in Dordrecht (The Netherlands). From the mixed populations thus obtained a strain, 8909N, was isolated using naphthalene as the selective substrate. It grew also on phenanthrene, anthracene and fluoranthene. This strain was a Gram-negative motile rod and was identified as Pseudomonas sp. Organisms used in experiments for growth on mixtures of PAH adsorbed on activated carbon were isolated from inoculation material obtained from a plant where polluted soil was remediated by an extraction technique (HWZ, Amsterdam, The Netherlands).

Growth conditions. Organisms were grown at 30 ° C in mineral media, essentially as described by Evans et al. (1970), with 1 mM EDTA as chelating agent and the concentrations of other medium components being one-half of those described. When used for batch culture the medium was buffered at pH 7.0 with 50 mM phosphate. Pure cultures were maintained on agar slants containing 0.1% (w/v) of the PAH required (storage at 4 ° C). Mixed cultures degrading singly dosed PAH were maintained in sequential batch cultures in 300-ml erlenmeyer flasks supplied with 100 ml medium. Crystalline PAH were added as sole sources of carbon and energy. Mixed cultures growing on mixtures of PAH adsorbed to activated carbon (see below) were frozen in 15% glycerol at - 196 ° C and maintained at - 30 ° C. Strain 8909N was grown in naphthalene-limited, continuous culture using a stirred (1000 rpm) fermentor (Applicon, Schiedam, The Netherlands) with a working volume of 1.2 1 at a dilution rate of 0.2 h -1. The mineral medium used was the same as described above. The pH was maintained at 7.0+0.1 with 0.5 M NaOH and the temperature at 30 ° C. Aeration and substrate supply were accomplished by leading naphthalene-saturated air (1801.h -1, = 4.84.10 - 4 mol naphthalene, h - 1) through the culture. Batch growth experiments with mixed cultures on naphthalene and phenanthrene were carried out at 30°C in 300-ml conical flasks containing 100 ml mineral medium. Substrate was added in crystalline form. Samples were taken for measurement of the optical density at 540 nm (OD54o). Batch growth experiments with strain 8909N on naphthalene were carried out with shaking (150 rpm) in 500-ml conical flasks with 200 ml mineral medium to which 0.4 g of a sieved fraction of naphthalene had been added. The experiments were started by inoculation with 5 ml of a washed suspension of chemostat-grown cells of strain 8909N. The optical density at 540 nm was measured every 30 min. In some experiments 1-ml samples were taken to determine the naphthalene concentration.

550

Adsorption of PAH to activated carbon. PAH originating from extraction sludge, the main stream of waste resulting from the extraction procedure of soil remediation, were concentrated tenfold onto activated carbon. In order to achieve this, 200 g sludge was added to 500 ml acetone, and the suspension was mixed thoroughly for 10 min. Thereafter, 1 1 hexane was added. After thorough mixing for another 10 min the suspension was centrifuged (20 rain, 5000g). The supernatant organic phase was washed three times with demineralized water. Granular activated carbon (20 g, ROW 0.8, Norit, Amersfoort, The Netherlands) was added and the PAH were allowed to adsorb by mixing for 10 min under lowered pressure (2 kPa) and subsequently removing the organic solvent by distillation in a rotavapor apparatus (2 kPa, 40° C). By this procedure a load amounting to 71.0 I~g total PAH.mg -~ dry weight of activated carbon was achieved. Analytical procedures. Protein was assayed after alkali treatment of cells (Herbert et al. 1971) by the method of Lowry et al. (1951) using bovine serum albumin as a standard. Bacterial growth was determined by OD54omeasurement with the samples being diluted to an OD < 1.0. For determination of dissolved naphthalene in the culture fluid, 200 p.l aliquots of supernatant samples were diluted with 800 pJ acetonitrile and analysed subsequently by HPLC using a ChromSpher C18 (PAH) column (Chrompack, Middelburg, The Netherlands). The eluent was an 80/20 mixture of acetonitrile/ water. Peaks were detected by use of a fluorescence detector (Shimadzu, Kyoto, Japan). The wavelengths of excitation and emission were 278 nm and 324 nm, respectively.

Table 1. Growth characteristics of mixed cultures grown in mineral salts media at pH 7.0 and 30° C Substrate

Cmax (mg.l-')

Growth rate (h-')

Naphthalene Phenanthrene Anthracene

32 1.3 0.075

0.3 0.03 0.0003

C.... substrate saturation concentration

1.0

During the enrichment procedures by which mixed bacterial cultures were obtained that grew on naphthalene, anthracene or phenanthrene as the sole sources of carbon and energy, it was observed that growth occurred with a constant increase in biomass with time. Figure 2 shows the increase in biomass observed with growth on crystalline naphthalene or phenanthrene. The linear relationship in this figure demonstrates that under such conditions with high cell density, bat.chwise growth did not proceed according to the exponential kinetics by which growth occurred at low cell density.



,,

4 E 0 ,~to

.'"

0

- ~ " ~

. .1~"" @--"" .~" .~'" /." ..~"

..~.i ; " "" ..:;."~ 0

2

,

,

,

,

,

,

4

6

8

10

12

14

time

(days)

16

Fig. 2. Batch growth of a mixed bacterial culture on naphthalene (~,) and phenanthrene (Q) added in crystalline form: OD, optical density

-~

30

~

25 20

~ t~ .~. ~" O)

15

o

10

t_O) _ t~ ¢-

0.8

-=

0.6

t-

0.4 ,,;,s~. , ,- ,,~,: ?~ ;,

0

to

cO

0

0

0.2

'

I

;

5

II ~,%, ,,~~, •

0



0.0 0

Results and discussion

35

7

0 14

21

time

(h)

28

0

m ¢-

35

Fig. 3. Batch growth of strain 8909N on naphthalene: , OD at 540 n m ; . , concentration of dissolved naphthalene; . . . . , specific growth rate

Table 1 shows the maximal specific g r o w t h rate found during the exponential phase of growth and relates the value of this growth parameter to solubility of the substrates. For the three PAH studied, the growth rate decreased remarkably in parallel with a decrease in solubility of the substrates. From this observation one might assume that even during the exponential phase low substrate solubilities determine the kinetics of microbial growth. However, in order to quantify this phen o m e n o n in terms of Ks and ~max further studies are needed that are harassed experimentally by the low biomass concentrations that are required and the extremely low growth rates that may occur. For this reason, and for reasons of quantifying the linear growth p h e n o m e n o n demonstrated in Fig. 2, experiments were carried out with pure cultures at higher cell densities with naphthalene as the substrate. A typical curve for growth on naphthalene is given in Fig. 3, showing a good similarity to the theoretical curve in Fig. 1. At low biomass concentrations in the initial stages of the experiment a constant growth rate was found. After the naphthalene concentration in solution (C,) had fallen to below 250 lxg. 1-1, the growth rate decreased rapidly. As solid naphthalene was still visible in the conical flask where the batch experiment was performed, this decrease in naphthalene concentration was caused by a naphthalene uptake rate exceed-

551 2.50

2.00 E 0 ,~

1.50

i~

1.00

0 0.50

0.00 0

10

20

30

40

50

60

70

time (h)

Fig. 4. Batch growth of strain 8909N on sieved fractions of naphthalene with different diameters: A, 600-10001xm; ~ , 10002400 ~tm; O, 2400-3350 Ixm; V, >3350 ~tm 60 v

E

50

+ /

t--

.£ .,-,

/

40 /

k. .~

"

/ /

30

/

~_..+~

~-

~. / ;

O

~

20

~ /

¢._

"~ O

~,~ •

~.~.~

/

10

~

/

-

/

/

~_ EL

/

/

0 0

4

f i

i

i

8

12

16

time

20

(days)

In the latter case, desorption of substrate from the surface m a y limit growth in a way similar to that by which dissolution of P A H affects growth. In order to investigate any effect of the rate of desorption of P A H on the rate of increase in biomass, a mixture of P A H that originated from polluted soil was adsorbed to activated carbon. Batch cultures with 250 and 500 mg activated carbon in 100 ml mineral m e d i u m were grown and the increase in biomass was followed by determination of the cell protein. Figure 5 shows that a linear increase in biomass was found also with P A H adsorbed to activated carbon, and that also for adsorbed substrate growth was proportional to the a m o u n t of P A H on activated carbon added, i.e., the total desorbing surface. In the experiments described in Figs. 3, 4 and 5, the hypothesis was confirmed that the rate of substrate dissolution or desorption m a y restrict bacterial growth. This implies that estimation of specific bacterial growth rates on poorly soluble substrates in b a t c h culture is often not very reliable. The observation that the specific growth rate increases with increasing amounts of (solid) substrate (Keuth and R e h m 1991) can be explained by the m e c h a n i s m p r o p o s e d in this paper. It is often recognized that the results of soil remediation plants, when expressed in terms of a residual concentration of polluting components, in casu PAH, do not match those of laboratory experiments. The studies presented here indicate that the rate by which pollutants m a y dissolve or desorb, and thereby b e c o m e available for microbial degradation, m a y play a crucial part in the success of biotechnological soil sanitation programmes. Our investigations are now focused on increase in bioavailability by changing physical and biological conditions to optimize biodegradation rates.

Acknowledgements. This work was supported by the Netherlands Integrated Soil Research Program (grant no. 8977).

Fig. 5. Batch growth of a mixed bacterial culture on a mixture of polycyclic aromatic hydrocarbons, added to a batch adsorbed to activated carbon: @, 250 mg loaded carbon added; +, 500 mg loaded carbon added. For preparation of the loaded activated carbon see Materials and methods

ing the dissolution rate, J . . . . At the end of the growth curve the naphthalene concentration measured was 7 ~tg. 1-1 and the Ks estimated from the course of lxt and Ct was 40 ktg. 1-1. The m a x i m a l dissolution rate o f a component, J . . . . depends on the total crystal surface, A. F r o m the hypothesis that the increase in biomass is proportional to J . . . . it was inferred that it is, by consequence, also proportional to A. This was tested by inoculation o f strain 8909N in batch cultures with equal amounts of naphthalene with different particle diameters, after which the biomass concentration was followed in time. Figure 4 shows that the slope in the curves thus obtained increased with decreasing diameter of the substrate particles, and thereby d e p e n d e d on the available crystal surface area. P A H present in waste streams and polluted soil occur mostly in crystalline form or adsorbed to surfaces.

References Andelman JB, Snodgrass JE (1974) Incidence and significance of polynuclear aromatic hydrocarbons in the water environment. Crit Rev Environ Control 5:69-83 Bossert I, Bartha R (1984) The fate of petroleum in soil ecosystems. In: Atlas RM (ed) Petroleum microbiology. Macmillan, New York, pp 434-476 Daisey JM, Leyko MA, Kneip TJ (1979) Source identification and allocation of polynuclear aromatic hydrocarbon compounds in the New York City aerosol: methods and applications. In: Jones PW, Leber P (eds) Polynuclear aromatic hydrocarbons. Ann Arbor Science Publishers, Ann Arbor, Mich., pp 201215 Davies JI, Evans WC (1964) Oxidative metabolism of naphthalene by soil pseudomonads: the ring-fission mechanism. Biochem J 91:251-261 Evans CGT, Herbert D, Tempest DW (1970) The continuous cultivation of microorganisms. 2. Construction of a chemostat. Methods Microbiol 2:277-327 Evans WC, Fernley HN, Griffith E (1965) Oxidative metabolism of phenanthrene and anthracene by soil pseudomonads: the ring-fission mechanism. Biochem J 95:819-831 Herbert D, Phipps PJ, Strange RE (1971) Chemical analysis of microbial cells. Methods Microbiol 5B:209-343

552 Keuth S, Rehm H-J (1991) Biodegradation of phenanthrene by Arthrobacter polychromogenes isolated from a contaminated soil. Appl Microbiol Biotechnol 34:804-808 Lavoie EJ, Bendenko V, Hirota N, Hecht SS, Hoffmann D (1979) A comparison of the mutagenicity, tumor-initiating activity and complete carcinogenicity of polynuclear aromatic hydrocarbons. In: Jones RW, Leber P (eds) Polynuclear aromatic hydrocarbons. Ann Arbor Science Publishers, Ann Arbor, pp 705-721 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275 McLee AG, Davies SL (1972) Linear growth o f a Torulopsis sp. on n-alkanes. Can J Microbiol 18:315-319 Ministry of Housing Physical Planning and Environment (1987) Environmental Program of the Netherlands 1988-1991. Staatsuitgeverij, The Hague Perry RH, Chilton CH, Kirkpatrick SD (eds) (1963) Chemical engineers' handbook. McGraw-Hill, New York Prokop A, Erickson LE, Paredes-Lopez O (1971) Growth models of cultures with two liquid phases. V. Substrate dissolved in disperse phase-experimental observations. Biotechnol Bioeng 13:241-256

Socz6 ER, Staps JJM (1988) Review of biological soil treatment techniques in The Netherlands. In: Wolf K, Brink WJ van den, Colon FJ (eds) Contaminated soil '88. Kluwer Academic Publishers, Dordrecht, pp 663-670 Staps JJM (1990) International evaluation of in-situ biorestauration of contaminated soil and groundwater. National Institute of Public Health and Environmental. Protection (RIVM), Bilt! hoven Report no. 738708006 Stucki G, Alexander M (1987) Role Of dissolution rate and solubility in biodegradation of aromatic compounds. Appl Environ Microbiol 53:292-297 Tempest DW (1970) The continuous cultivation of micro-organisms. 1. Theory of a chemostat. Methods Microbiol 2:259276 Thomas JM, Yordy JR, Amador JA, Alexander M (1986) Rates of dissolution and biodegradation of water-insoluble organic compounds. Appl Environ Microbiol 52:290-296 Wodzinski RS, Bertolini D (1972) Physical state in which naphthalene and bibenzyl are utilized by bacteria. Appl Microbiol 23:1077-1081 Wodzinski RS, Coyle JE (1974) Physical state of phenanthrene for utilization by bacteria. Appl Microbiol 27:1081-1084

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