Scum actinomycetes in sewage treatment plants—Part 1

War. Res. Vol. 22, No. 6, pp. 755-759, 1988 Printed in Great Britain. All fights reserved

0043-1354/88 $3.00 + 0.00 Copyright © 1988 Pergamon Press plc

SCUM A C T I N O M Y C E T E S IN SEWAGE TREATMENT PLANTS--PART 1 GROWTH

KINETICS

OF

N O C A R D I A A M A R A E IN C H E M O S T A T CULTURE

MICHAELA BAUMANN,l HILDE LEMMER1 and HARALD RIES2 ~Bayerische Landesanstalt fiir Wasserforschung, Kaulbachstrasse 37, D-8000 Munich 22 and 2University of Munich, Sektion Physik, Amalienstrasse 54, D-8000 Munich 22, F.R.G. (Received September 1987; accepted December 1987)

Abstract--The scum actinomycete Nocardia amarae was grown in batch culture and in chemostat culture on a fructose-peptone-yeast extract substrate. Chemostat growth is modeled with a two-substrate Monod equation incorporating 6 parameters which include maximum growth, kinetic constants for substrate and oxygen, cell yield and wall growth. These parameters were identified by numerically optimizing the fit. Among these parameters/z,~ x = 0.087 + 0.0075 h- ~and Ks = 675 + 124 mg 1- *, a value much higher than found for other activated sludge bacteria. The results are compared with findings of other researches for other sludge bacteria. Different growth strategies of these organisms are discussed. Key words--actinomycetes, Nocardia amarae, scum, wastewater treatment plant, growth kinetics, Monod parameters, chemostat modeling, activated sludge

INTRODUCTION Proper sludge settling in the secondary clarifier o f wastewater treatment plants is often disturbed by sludge scumming due to an abundant growth of nocardioform actinomycetes. These organisms are versatile in the utilization of different substrates. They are able to degrade even hydrophobic substrates such as hydrocarbons which are considered refractory. Previous investigations (Lemmer, 1985) have shown that hydrophobic substrates are selectively available to actinomycetes and allow them to compete successfully in activated sludge. Living on these substrates actinomycetes can grow only with long generation times. Therefore it was maintained that abundant actinomycete growth in wastewater treatment plants is connected with high sludge ages. However, in many plants a rapid increase of actinomycete biomass, leading to scum production, is observed in spite of sludge ages as short as I-2 days. Previous work indicated that scum actinomycetes grow faster on readily degradable food than on hydrophobic substrates (Lemmer, 1985). However, in actinomycete batch cultures with readily degradable food in concentrations about 100 times higher than those c o m m o n l y found in sewage treatment plants. characteristic growth times---defined here as the reciprocal of the growth r a t e - - o f 6-11 h were observed depending on the genus (Segerer, 1984; Lemmer, 1985). This contrasts with the findings mentioned for municipal scumming plants which imply shorter growth times. Thus the aim of this investigation was 755 W.R. 22/6--G

to determine the generation times of actinomycetes in a continuous flow system and to see under which conditions they agree with the observed rapid growth in sewage treatment plants.

MATERIALS AND METHODS

The test strain was Nocardia amarae isolated from a municipal wastewater treatment plant, where 80% of the wastewater originates in a papermill. First doubling times of N. amarae were determined in batch cultures as described by Segerer (1984). The actinomycete was grown at 28°C with primary nutrient broth. The biomass was monitored both photometrically at 560 nm and by measuring dry weight after filtration. The factor for converting optical density into dry weight was determined to be 8.54mgl -t dry wt for an optical density of I cm -~. Thereafter chemostat cultures with N. amarae were started. Conventional chemostats are usually run with a strong turbulent aeration. In such systems the cultivation of actinomycetes is troublesome because of excessive foam production and because the hydrophobic organisms tend to agglomerate and attach to the glass walls within the foam fraction. This prevents proper sampling. To avoid these problems the chemostat was aerated by an air flow above the liquid in the culture vessel (200h-~). The culture was vigorously stirred, which also prevented the hyphae from agglomerating. Chemostats were run at different, but constant, concentrations and dilution rates in a range of 0.01-0.4 h -I. The primary nutrient broth consisted of 1 g 1-~ fructose, 10 g 1- ~ peptone and 5 g 1- ~ yeast extract totalling 16 g lcarbon source. For substrate limitation tests this primary broth was diluted I" 10 [biological oxygen demand (BOD) about 1200mgl -~ O,], 1:30, 1:50 and 1:100 (BOD about 120 mg 1-I 02). 7 g I-i NaCI and traces of thiamine were added to all dilutions. The pH was adjusted to 7.2. BOD was measured in a sapromat (Voith BI2).

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The culture vessel contained 350 ml of nutrient broth and was kept at 28~C. At the beginning of the test it was inoculated with 5 ml of an 8 10 day batch culture, which was previously homogenized for 4 m i n with ultrasonic. N. amarae was allowed to grow up to an optical density of 0.4~1.0 cm ~before the continuous flow regime was started. Biomass concentration was monitored daily until it reached a steady state. This could take as long as 10 days. Thereafter the dilution rate was increased and the procedure repeated. Biomass concentration was determined photometrically at 560 nm. Additionally, metabolic activity was measured as the dehydrogenase activity after INT-staining (iodonitrotetrazolium chloride, Serva 26840) at 490 n m ( K o o p m a n et aL, 1984). Both values were required to reach a steady state. For INT-determination ultrasonic or ultraturrax homogenization of the samples was avoided because it kills some of the cells and thereby affects dehydrogenase activity. A close inspection of the measured data (which consisted of 30 triplets of substrate S fed into the chemostat, dilution D and biomass x) suggested that a simple M o n o d model with constant cell yield, where the actual steady state growth rate is assumed to be equal to the dilution, would not suffice (Pirt, 1985, pp. 39~10 and 107 109). Hence we used a generalized M o n o d equation which includes the effect of both substrate concentration Si and oxygen concentration O x within the chemostat: K , / S i + K o / O x - #max~It + 1 = 0

(la)

We also tried as an alternative generalization of the M o n o d equation: K~/Si + K o / O x + (K~Ko)/(SiOx) - p,n~,'It + I = (I

(lb/

Here K~ and Ko are the associated growth constants, lq,,,~ is the m a x i m u m growth rate if both oxygen and substrate are abundant and /~ is the actual growth rate. An equation equivalent to equation (la) is used to describe enzyme kinetics for two substrate reactions of the double displacement (ping-pong) type. Equation (lb) is equivalent to that describing ordered single displacement enzyme reactions (Lehninger, t975, pp. 204---205). An equation equivalent to equation (lb) was used by Lau et al. (1984). Si, O x and/z are not measured and therefore have to be inferred. Assuming constant cell yield Y, the substrate concentration in the chemostat Si can be calculated from the substrate concentration fed into the chemostat S and the biomass x: Si = S

x ; Y.

(2)

Analogously the oxygen concentration O x can be calculated assuming an imput proportional to the difference in oxygen tension between air and chemostat and a constant oxygen demand described by L: O x = Os -. x D L

(3)

where Os is the saturation concentration of oxygen. If the biomass is uniformly distributed within the chemostat the steady state growth rate must be equal to the dilution rate D. This is not true if a quantity W of biomass grows attached to the walls of the chemostat. Balancing the washout with the combined growth rates of suspended and attached biomass yields: p = xD,'(x + /4).

(4)

Substituting equations (2). (3) and (4) into equation (1) yields an equation which contains the parameter vector P (p ..... K0` K,, Y, L, W ) to be identified together with the measured variables S, D and x. Generally because the number of measurements exceeds the number of parameters and also because measurements are not free of error, there is no set of parameter values for which the system of equations (1) (4) holds true for all measured data. Assuming that errors are restricted to x we solve equation (1) for x. The left-hand side of equation (1)

has 3 poles at # = 0, Si = 0 and O x = 0 which correspond to x-values of 0, S Y and 1 / D L , respectively. Equation (1) has 3 solutions. The lowest one, situated between the first two poles, corresponds to positive values of Si and O x . The other two are unphysical. Let Xmod,~(S, D, P) represent this solution for given parameter vector P, substrate S and dilution D. It is the biomass predicted by the model. The parameters are identified with those values, for which agreement between model prediction and measured biomass is best. As merit function M we used the adjusted mean square deviation: [

M(P) = ~k (Xmodel[S(i), D ( i ) , P] - x .............di))2/(N - p )

(51

The summation extends over the measured data. p denotes the number of parameters. For optimization we used a downhill simplex method (Press, 1986, pp. 289-292). In order to avoid local minima we also incorporated simulated annealing (Press, 1986, pp. 326 328) with an annealing schedule devised by Brian Rogers (personal communication). The statistical uncertainty of the estimated parameter values was calculated by inverting the estimated Hessian (Jacobian) matrix of the function of merit (Kennedy and Gentle, 1980, pp. 475--478). The correlation matrix of the parameters was also calculated. The measurement consisted of 30 points. An optimization of the model described by equations (1)-.{4) for the complete data set yielded an o p t i m u m fit which was unacceptable because the mean deviation estimated by x / M was too high (0.6mgl). Even several more sophisticated models which included the oxygen for maintainance and substrate dependent cell yield could not model the complete data set much better. Accordingly the statistical uncertainties of the parameters were very high. The data include 5 measurements at a very low dilution rate of 0.01 h ~. Although the fit was not particularly bad for these points, we excluded these measurements for biological reasons: they were the first measurements in each series and the organisms m a y not yet have overcome the lag phase; equilibrium is approached slowly (time constant up to 100 h) and therefore is difficult to detect at this dilution: many other effects, such as cell respiration, introduce errors which are highest at low dilution rates. For reduced data set optimization yielded significantly better fits with a mean deviation of 0.14 m g l - J dry wt.

RESULTS AND DISCUSSION The batch culture of N. amarae with primary nutrient broth (carbon source= 16gl-~; BOD= 1 2 g l -~ 0 2 ) yielded a m a x i m u m g r o w t h rate o f 0.14 h ~. T h i s a g r e e s w i t h r e s u l t s o b t a i n e d b y Segerer (1984). F o r t h e c h e m o s t a t m e a s u r e m e n t s b o t h m o d e l s [ e q u a t i o n ( l a ) a n d (lb)] yield t h e s a m e r e s u l t s a n d t h e s a m e m e a n d e v i a t i o n o f t h e fit. F i g u r e 1 s h o w s t h e m e a s u r e d b i o m a s s as a f u n c t i o n o f t h e d i l u t i o n for different s u b s t r a t e c o n c e n t r a t i o n s . T h e c o n t i n u o u s c u r v e s r e p r e s e n t t h e p r e d i c t i o n o f t h e m o d e l des c r i b e d by e q u a t i o n s ( l a ) - ( 4 ) w i t h t h e o p t i m a l v a l u e s o f t h e p a r a m e t e r s . T h e s e v a l u e s a r e g i v e n in T a b l e 1. T h e m a x i m u m c a p a c i t y o f o u r a e r a t i o n c a n be c a l c u l a t e d as O S Y / L = 0.35 m g 0 2 h J. W i t h exception o f K0, w h i c h is n o t well c o n d i t i o n e d , t h e o t h e r p a r a m e t e r s c a n be identified w i t h r e a s o n a b l e a c c u racy. T h e h i g h e s t e l e m e n t o f t h e c o r r e l a t i o n m a t r i x

Growth kinetics of N. amarae

757

will be the organisms with the highest ratio of Pmax/Ks. This can be seen by approximating Monods equation as a first order Taylor series for low substrate:

0.4,

0.~

I~(S) ~ S(dl~/dS)s~o

~O.r,

for S<
(6)

where

o

d p / d S = (l~maxKs)/(Ks + S) ~ = #max~Ks for S = 0. (7)

0.1 C3

0

0

Y 0

0.1

0.2

0.4

0.3

D( h-11 Fig. l. Chemostat growth of N.

amarae.

Steady state

biomass is plotted as a function of dilution for substrate concentrations of 16gl -I, C); 1.6gl -t, A; 0.533 gl -I, I--1; 0.32 g 1-~, x ; 0.16 g 1-~, O. Continuous curves represent the best fit of the model defined by equations (la) and (4). The adjusted mean deviation between model prediction and measured points amounts to 0.14 mg 1-~ dry wt. The corresponding parameter values are given in Table 1. Points at dilution D = 0.01 h -~ are omitted.

(by absolute value) is the correlation between ~max and W (-0.89). In the beginning we hoped to find higher maximal growth in chemostat culture basically because even for dilution rates as high as 0.3 h-~ N. amarae has not been washed out. However, the detailed evaluation indicates a/~m~x-value which is somewhat lower, but still is in agreement with the values found in batch culture. Even a small wall growth can prevent N. amarae from being washed out at dilution rates higher than its growth rate. For our chemostat volume, the value of W implies that only 13#gl -~ dry wt of cells grew attached to the wall. Yet this was enough to yield substantial biomass concentrations in the chemostat of 0.4 mg I ~at dilutions twice as high as the maximum growth rate and to prevent the organisms from being washed out even at a dilution 4 times as high as the maximum growth rate. This mechanism might be effective also in sewage treatment plants allowing actinomycetes to increase their biomass without a growth rate that exceeds the dilution. CONCLUSIONS

In chemostat culture those microorganisms which have the highest growth rate under the given circumstances will successfully compete. At high substrate concentrations these will be the organisms with the highest t~m~" At low substrate concentrations these Table I. G r o w t h kinetic parameters for N . a m a r a e determined for chemostat growth on readily degradable substrate /~m~ = 0.087 _+ 0.0075 h-~; t d = 7.97 h Ks = 675 _+ 124mg 1-I substrate K0=l/~gl IO2-1mgl IO 2 Y = 0.0018 _+ 0.0002 (mg dry wt)/(mg substrate) L = 54.7 _+ 2.3 (mg O2)/(mg h i dry wt) W = 0.367 _+ 0.094 m g I- i dry wt

Thus at low Ks-values those microorganisms with higher ratios of #max/K, will be successful. However, for low oxygen concentrations an analogous approximation can be made. Equation (6) fails to account for maintainance requirements. Linking the success of competition to the growth rate does not apply if microorganisms of the same biocenosis are subject to different dilution rates. If microorganisms avoid dilution by attaching themselves to interfaces (walls, water surface, gas bubbles), they can gain an advantage over other microorganisms. Several theories concerning growth strategies of the different activated sludge microorganisms have been presented, especially for filamentous bacteria which cause sludge bulking and for actinomycetes which cause sludge scumming. Chudoba et al. (1973) proposed that floc forming bacteria are/~max-strategists. They can grow fast (high /~max), but need also high substrate concentrations (high Ks). On the other hand, filamentous organisms are K-strategists. They can grow even on diluted substrate (low Ks), but only slowly (low #max)' However, because the ratio Pmax/Ks is higher for these organisms, they grow faster at low substrate concentrations. The more detailed theory of Chiesa and Irvine (1985) includes two additional types of microorganisms. It distinguishes floc formers, which can grow at low oxygen concentrations, from others, which need relatively high oxygen concentrations. Moreover these authors describe "fast growing starvation susceptible filaments" which have higher maintainance substrate demands. For scum causing actinomycetes it was proposed that they can switch between a pmax-strategy when growing on readily degradable substrate and K-strategy when growing on refractory substrates (Lemmer, 1986). The experimental data on the growth kinetic of floc formers and filamentous microorganisms are not as rich. Table 2 summarizes results of other researchers with the growth parameters determined in this work for N. amarae growing on readily degradable substrate. The ratio of Pm~x/K~ was also calculated and listed in Table 2. The results are not strictly comparable, because one must assume that both growth parameters #m~ and Ks are not only specific for a microorganism but also for a given substrate. Especially when growing on readily as compared to refractory substrate microorganisms can be expected to show strongly differing growth parameters. Moreover some of the results in Table 2 refer to chemostat measurements whereas others are derived from batch

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MICHAELA BAUMANN et al.

Table 2. Growth kinetic parameters determined in this work for N. amarae in comparison with data for filamentous organisms and floc formers from activated sludge found by other researchers Sludge scumming Sludge bulking N. amarae FlocformS. natans HaliscAN HaliscAZ S. natans Typel701 Flocform 021N(2) 021N(7) Reference Lau et aL (1984) van Veen et al. (1982) Richard et aL (1985) Richard et al. (1984) p~a~(h-~) 0.087 + 0.0075 0.379 0.271 0.050 0.090 0.0271 0.108 0.179 0.158 0.058 ta(h) 8.0 1.8 2.6 13.9 7.7 2.6 6.3 3.9 4.4 12 K~(mg I ~) 675 + 124" 5* 10" 5t .... 10 2 <1 <1 Ko(mg I i) 0.001-1 0.15 0.01 ....... 0.033 0.014 0.073 --Y[(gdry wt)/ 0.0018+ 0.0002 0.55 0.53 0.59 0.42 0.53 0.44 0.71 0.56 (g substrate)] td(h)~ 5-7 1.9-2.0 3.8-5.8 19.8 3 8 . 5 . . . . . . . . . #ma~/Ks 0.13 76 27 10 -27 54 " >158 >58 [1 (g h) ~] *Reference to carbon source, the other data are measured with glucose. tFrom Krul (1977). :~Found in batch culture.

cultures. It is generally observed that batch cultures tend to yield lower #m~x-values t h a n c o r r e s p o n d i n g c h e m o s t a t measurements. The reasons are not fully understood. The c h e m o s t a t d e t e r m i n a t i o n of #~x in this work, however, agrees fairly well with earlier batch culture measurements. The data presented in Table 2 shed some light u p o n the different growth strategies of sludge bacteria. The bulking sludge causing filament type 021N (strain 2, Richard et al., 1984) appears to be a Kstrategist as c o m p a r e d to the floc former investigated by Lau et al. (1984). Its Ks-value < 1 m g I i a n d the #max-value 0.158 h - ~are lower t h a n the c o r r e s p o n d i n g values o f the floc former, however, the ratio #max/K, [ > 1 5 8 1 ( g h ) -~] is a factor of 2 higher t h a n the corresponding ratio for the floc former. F o r the strain 021N(7), however, this ratio is rather low. O n the other h a n d , Sphaerotilus natans appears to be a #max-strategist having relatively high # . . . . but a low ratio #max/Ks. The filamentous bacteria type 1701 and H a l i s c o m e n o b a c t e r hydrossis b o t h have low #max a n d a relatively low ratio #max~Ks [54 1 (g h) ~ and 101 ( g h ) ~, respectively]. Therefore their successful competition m u s t be based on o t h e r features. The relatively low K0-value for type 1701 ( 0 . 0 1 4 m g t 02) which gives this organism a n a d v a n t a g e at low oxygen conditions, as discussed by Richard et al. (1985), is notable. The features u p o n which H a l i s c o m e n o b a c t e r hydrossis bases its successful competition in some plants are not yet fully u n d e r s t o o d (Krul, 1977). The scum actinomycete N o c a r d i a a m a r a e growing on the substrate described a b o v e shows a r a t h e r low #m~x (0.087 _+ 0.075 h - l ) , the ratio of #max/Ks being very low [0.13 1 (g h ) - 1], because the Ks-value is nearly 100 times higher t h a n for other sludge bacteria. Therefore N. a m a r a e c a n n o t be a K-strategist w h e n growing on such readily degradable substrate. These findings agree with earlier conjectures (Lemmer, 1986). On the o t h e r h a n d , this organism is not a successful #,,~x-strategist, because, for example, the floc former, S. natans a n d type 1701 have c o m p a r a b l e or even higher maximal growth rates. Moreover, for substantial actinomycete growth available substrate

c o n c e n t r a t i o n s should exceed 700 mg I ~which is not c o m m o n in the p r i m a r y effluent o f sewage t r e a t m e n t plants. Therefore excessive actinomycete growth c a n n o t be traced to simple growth kinetics. O t h e r features such as wall growth or adherence to interfaces which can cause e n h a n c e m e n t of substrate availability a n d / o r m e a n cell residence time a p p e a r to be i m p o r t a n t for competitional success. It is k n o w n that N. a m a r a e can grow on such refractory substrate as h y d r o c a r b o n s , a r o m a t i c substances, pesticides etc. which only few o t h e r organisms can utilize. It would be interesting to investigate actinomycete g r o w t h strategies o n such substrates which might give these organisms a selective advantage. Acknowledgements--The authors wish to thank B. Rogers and W. Spirkl for the permission to use their optimization program. We thank B. Knabe for technical assistance. This work was supported by the Kuratorium fiir Wasserwirtschaft, Grant No. C00-1.07/83.

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Chiesa S. C. and Irvine R. L. (1985) Growth and control of filamentous microbes in activated sludge: an integrated hypothesis. Wat. Res. 19, 471-479. Chudoba J., Grau P. and Ottova V. (1973) Control of activated sludge filamentous bulking--lI. Selection of microorganisms by means of a selector. Wat. Res. 7, 1389- 1406. Kennedy W. J. and Gentle J. E. (1980) Statistical Computing, Chap. 10.3, pp. 475-478. Dekker, New York. Koopman B., Bitton G., Logue C., Bossart J. M. and Lopez J. M. (1984) Validity of tetrazolium reduction assays for assessing toxic inhibition of filamentous bacteria in activated sludge. In Toxicity Screening Procedures Using Bacterial Systems (Edited by Liu D. and Dutka J.), pp. 147-162. Dekker, New York. Krul J. M. (1977) Experiments with Haliscomenobacter hydrossis in continuous culture without and with Zoogloea ramigera. Wat. Res. 11, 197-204. Lau A. O., Strom P. F. and Jenkins D. (1984) Growth kinetics of Sphaerotilus natans and a floc former in pure and dual continous culture. J. War. Pollut. Control Fed. 56, 41-51. Lehninger A. L. (1975) Biochemistry, 2nd edition. North Publishers.

Growth kinetics of N. amarae l.eramer H. (1985) Mikrobiologische Untersuchungen zur Bildung von Schwimmschlamm auf Kl,~ranlagen, Ph.D. thesis, Technische Universitit, Munich, F.R.G. Lemmer H. (1986) The ecology of scum causing actinomycetes in sewage treatment plants. Wat. Res. 20, 531-535. Pirt S. J. (1985) Principles of Microbe and Cell Cultivation. Blackwell Scientific Publications, Oxford. Press W. H., Flannery B. P., Teukolsky S. A. and Vetterling W. T. (1986) Numerical Recipes. University Press, Cambridge. Richard M. G., Shimizu G. P. and Jenkins D. (1984) The growth physiology of the filamentous organism type 02IN and its significance to activated sludge bulking. 57th

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A. Conf. Wat. Pollut. Control Fed. New Orleans, La, U.S.A. Richard M. G., Hao O. and Jenkins D. (1985) Growth kinetics of Sphaerotilus species and their significance in activated sludge bulking. J. War. Pollut. Control Fed. 57, 68-81. Segerer M. (1984) Untersuchungen zur Schwimmschlammbildung in Kl/iranlagen durch Actinomyceten. Korr. Abw. 31, 1973-1076. van Veen W. L., Krul J. M. and Bulder C. J. E. A. (1982) Some growth parameters of Haliscomenobacter hydrossis (syn. Streptothrix hyalina), a bacterium occurring in bulking activated sludge. War. Res. 16, 531-534.