Effect of the endogenous phase duration on the maximum substrate removal rate in mixed cultures

;Vat. Res. Vol. 20, No. 12, pp. 1505-1509,1986 Printed in Great Britain

0043-1354/86$3.00+0.00 Pergamon Journals Ltd

EFFECT OF THE ENDOGENOUS PHASE DURATION ON THE MAXIMUM SUBSTRATE REMOVAL RATE IN MIXED CULTURES JAN CHUDOBA, PAVEL C H U D O B A and JAKUB S. {~ECH Department of Water Technology and Environmental Engineering, Prague Instituteof Chemical Technology, Suchb/ttarova 5, 166 28 Prague 6, Czechoslovakia (Received June 1985) Abstract--The maximum suhstrate removal rates, r~.~, were measured during the endogenous phase by means of a simple respirometric method. Excess biomass from a continuously-fed completely mixed (CM) and a semicontinuously-fed (SE) reactor was tested with ethylalcohol, glutamic acid, phenylalanine, acetic acid, phenol and glucose. It was found with the mixed culture from the CM system that during a 6-h endogenous period, the rx.,, values remained constant with all the substrates tested. With the mixed culture from the SE system, the rx.,~ values increased during first 8-12 h of the endogenous period and then decreased. The authors conclude that the respirometric method measuring r~.m values can be used to optimize the contact stabilization process. Key words--endogenous phase, mixed cultures, substrate removal rates, respirometric measurements, kinetic constants

INTRODUCTION

In the discussion o f the p a p e r " D e t e r m i n a t i o n o f kinetic c o n s t a n t s o f activated sludge microo r g a n i s m s " by (~ech et al. (1984), G r a d y a n d Philb r o o k (1984) pointed o u t t h a t a 1 h a e r a t i o n period w i t h o u t a n exogenous substrate involved before the start o f respirometric m e a s u r e m e n t s m i g h t considerably decrease the values o f the m a x i m u m substrate r e m o v a l rates, rx, m. Consequently, the first aim o f this p a p e r was to present o u r latest results o b t a i n e d in this field. T h e second aim was to show h o w the d u r a t i o n o f the e n d o g e n o u s p h a s e in the system involving sludge regeneration or r e a e r a t i o n (e.g. c o n t a c t stabilization process) influences the m a x i m u m substrate removal rate. DESCRIPTION OF EXPERIMENTS

Cultivation o f stock mixed culture The mixed culture used for kinetic measurements was cultivated in two units with the temperature maintained at 20°C. Technological parameters are summarized in Table 1. One unit was a continuously-fed, completely mixed reactor (CM) with a total working volume of 81. The other unit was operated semi-continuously (SE) on a once-a-day feeding schedule. This unit simulated a system with ideal plug flow. Simultaneously, this unit simulated a system with distinctly separated exogenous and endogenous phases (e.g. contact stabilization system). As shown by ~ech and Chudoba (1983), and as presented in Fig. l, the exogenous substrate in such a system is removed from solution during first 5-10 h (contact), whereupon a long endogenous period follows (regeneration). The composition of a multicomponent substrate dosed into both units slightly differed as shown in Table 2. The substrate solution was dosed into the CM unit by means of a peristaltic pump.

The excess biomass from both units was periodically used for respirometric measurements carried out with individual components. These measurements were started after a onemonth adaptation period. Respirometric measurements The respirometer described by (~ch et al. (1984), and further tested by Chudoba et al. (1985), was used for the kinetic measurements. The evaluation of respirograms was the same as described previously. The dependence of the maximum substrate removal rate, rx.m, and the half-velocity constant, K,, on the duration of the endogenous phase was measured with excess biomass from the CM system. The measurements were always carried out at zero, 3rd, 6th and 9th hour after the mixed culture being transferred from the aeration tank into the respirometric cell. During this 9-h period, also the endogenous respiration was recorded and its specific rate calculated (rob.e). Each substrate was tested at four initial concentrations in the range from 1 to 1 2 m g l -~ (e.g. 2, 4, 6, 10mgl-~). The testing lasted usually from 2 to 2.5 h. The dependence of the rx.m and Ks values on the duration of the endogenous phase was also measured with the mixed culture from the SE system. After the multicomponent substrate was dosed into the SE system, its volume made up to 81. and thoroughly mixed, 450 ml of the mixed liquor were immediately transferred into the respirometric cell, and aeration was started. An oxygen electrode measured the concentration of soluble oxygen, which was continuously recorded. When one component of the multi-component substrate had been removed, oxygen concentration always increased. In this way, it was possible to indicate a moment of removing all components from the solution. Moreover, occasionally courses of soluble COD and biomass concentration were followed in the SE system during one feeding cycle as shown in Fig. 1. It can be seen in Fig. 1 that approx. 90% of COD was removed during the first 6 h. Therefore, the first respirometric measurement with a given tested substrate was always started at the 6th hour following the dosing of the multicomponent substrate. One series of respirometric measurements did not last more than 2.5 h. Further measurements were carried out at every 4th

1505

JAN CHUDOBA et

1506 Table

al.

Technological parameters of activated sludge units

I.

Parameter

Completely mixed system (CM)

Semicontinuous system (SE)

8 1.5 1 48 3.5 1.62 550

8 -I 48 4.5 2.02 90

1.0 1.5

1.0 1.5

0.62 0.93

0.50 0.74

Aeration volume (I.) Volume of settler (1.) Recirculation ratio ( - ) HRT (h) SRT (day) MLSS (gl ~) SVI (mlg ]) Volumetric loading (kg m 3day ]) BOD basis COD basis Sludge loading (kg kg ] day i) BaD basis COD basis

Table 2. Composition of a multicomponent substrate used for feeding the activated sludge units. All components were dissolved in tap water Concentration (mg I- ') Component

Completely mixed system

Semi-continuous system

600 600 600 600 600 -160 100 2000 3000

600 600 600 600

Ethylalcohol (as COD) Glutamic acid (as COD) Phenylalanine (as COD) Acetic acid (as COD) Phenol (as COD) Glucosoe (as COD) NaHCO 3 KH2PO4 BOD 5 COD

hour, totally 24 h (i.e. at 10th, 14th, 18th, 22nd, 26th, and 30th hour, following the dosing o f the multicomponent substrate). Each substrate was tested at four initial concentrations as described above. The obtained values o f r x were plotted against the calculated values o f S, which could be expressed by the following relation: S

(1)

rx=rx.mK~+S"

Both constants, rx,m and K,, were calculated by m e a n s of a least square method from the following linearized form o f

1.4

L

Contact

_ I_ ~r

--

600 600 100 2000 3000

equation (1): rx

= r~.m - K, ~.

(2)

An example of the plots according to equations (1) and (2) is given in Fig. 2.

Analytical methods C O D was determined by the micromethod described by Jirka and biomass concentration was determined by means of m e m b r a n e filters (! .5 # m )

dichromate semiCarter (1975). The after being removed and dried at 105°C.

r~ /S

Regeneration

="

4 120

r

8 .

12

16

20

,

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l

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1 8

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1

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Time

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16

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20

I

24

(h)

Fig. 1. The courses of soluble C O D (l) and biomass concentration (2) during one feeding cycle in the SE unit.

S {mg

12

L-I)

Fig. 2. Typical relationships between r~ and S (equation 1) and between r x and rx/S (equation 2) as obtained with acetic acid during an adaptation period in the SE unit. rx,,~= 181 m g g - l h - ' , K , = 6 m g l '.

Endogenous phase duration Table 3. Values of K, and rx,,,, obtained during the endogenous phase with the mixed culture from the CM system Compound Ethylatcohol

Glutamic acid

Phenylalanine

Acetic acid

Phenol

Time* (h)

(mgl -t)

0 3 6 9 0 3 6 9 0 3 6 9 0 3 6 9 0 3 6 9

0.7 0.8 I.I 0.9 4.3 3.3 2.9 3.9 0.5 1.3 1.0 1.0 1.5 1.6 1.4 2.9 2.2 2.6 2.9 1.9

K,

20 "T

r x ,,1

(mgg

h- )

18 17 23 28 80 76 72 86 32 32 29 32 235 242 227 266 206 196 195 191

1507

16

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12 8 -

4

o

~

i

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120 S

100 80

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2

0

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i

1

2 3

t 4

I

t 5

t 6

t 7

t 8

t

0

I=

~ ~

910

Time (hi

Fig. 3. Results obtained with glutamic acid and mixed culture from the CM system, rox.,-----endogenous respiration rate.

• Time from the start of the endogenous phase.

RESULTS AND DISCUSSION o

The results are summarized in Tables 3 and 4 and some graphical examples are given in Figs 3-6. Figures 3 and 4 (CM system) show that without the exogenous substrate the values of rx.,, remained approximately constant during 6 h.

12 I-

~

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1

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o_-oJ I

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l

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l

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200

4

Table 4. Values of K, and rx.,, obtained during the endogenous phase with the mixed culture from the SE system Compound Ethylalcohol

Glutamic acid

Phenylalanine

Acetic acid

Glucose

Time* (h) 6 10 14 18 22 26 30 6 10 14 18 22 26 30 6 10 14 18 22 26 30 6 10 14 18 22 26 30 6 10 14 18 22 26 30

K,

rx m

(mgl -I)

( m g g -t h - )

1.3 1.9 1.4 1.8 1.4 1.5 2.1 3.7 6.1 6.6 5.8 3.8 6.6 5.7 3.7 4.1 3.3 6.5 4.9 5.3 4.2 1.8 2.2 3.4 4.4 3.1 2.7 2.8 8.3 12.6 15.7 13.7 7.1 5.7 8.1

*Time from the start of a oue-day feeding cycle.

36 45 42 54 49 48 53 58 75 80 85 65 77 79 58 66 62 88 77 81 73 120 130 150 186 145 142 141 84 144 210 192 125 118 124

1

0

I

t

I

I

1

2

3

I

I

I

I

4 .5 6 7 Time (h)

I

8

0

9 10

Fig. 4. Results obtained with acetic acid and mixed culture from the C M system.

,To~

16 12

E

8 , 0

i

I

I

I

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I

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I

200

~.~- 8

0

~

6

L, 4 o,

40

2_E

o

o ~" 6

10

14

18

22

26

30

Time (h) Fig. 5. R e s u l t s o b t a i n e d w i t h acetic a c i d a n d m i x e d c u l t u r e f r o m the S E system. T h e a r r o w i n d i c a t e s the e n d o f o n e f e e d i n g cycle.

1508

JAN CHUDOBAet al.

A

2O

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8 I

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200 150

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6

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14

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22

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30

Time (h)

Fig. 6. Results obtained with glucose and mixed culture from the SE system.

The rx.,, values remained constant despite the fact that the endogenous respiration rate was decreasing continuously during the first 3 h. The results have shown that the mixed cultures cultivated at relatively higher values of the SRT are able to keep their capability of substrate removing on a constant level for several hours. Moreover, for the r~.m values the results also indicate rather an increasing than a decreasing tendency during further aeration without the exogenous substrate. Grady and Philbrook (1984) cite the results of Law et al. (1976) showing evidence that bacteria rapidly lose some high affinity enzyme systems when removed from their source of the exogenous substrate. Our results have shown that this is not the case with the mixed cultures cultivated at relatively higher SRT as it is with those used in chemostats. The application of the results obtained in the chemostats (SRT usually in hours)to the activated sludge process (SRT in days) must be done very carefully. The SRT is a very important factor in species selection in mixed cultures. t~ech et al. (1984) have proposed to aerate mixed cultures without an exogenous substrate at least 1 h before using them. The results plotted in Figs 3 and 4 show that the mixed cultures should be aerated 3 h before testing rather than 1 h. This is because a constant endogenous respiration rate was not usually obtained sooner than after 3 h of endogenous metabolism. This period, however, will generally depend on the technological parameters of a given activated sludge system. Some results obtained in the SE system are graphically presented in Figs 5 and 6. The figures clearly show that in the system with distinctly separated exogenous and endogenous phases the rx.~ values increase during first8-12 h of the endogenous phase

and then decrease. The decrease was most probably due to the inactivation of transfer enzymes. The courses of rx.,, values indicate that from the kinetic point of view the SE system was not operated under optimum conditions because the maximum values of rx.,, were obtained after approx. 18 h of aeration. This means that an 18 h rather than a 24 h feeding cycle should have been used. It is evident that this method of measuring rx.,~values offers a simple and elegant way of optimizing the regenerator in the contact stabilization process. Although this process has been operated for about 60 years (Haseltine, 1961), there has been so far no objective method of determining a needful regeneration period. The respirometric determination of the kinetic constants, however, enables us to find this period, and thus to find an optimum volume of the regenerator. An increase of rx.m values during the endogenous phase in the SE system is believed to be mainly due to the accumulation capacity (AC) restoration. Consequently, the results obtained support the view on the role of the AC in the selection of non-filamentous microorganisms in a selector-type reactor (Chudoba, 1985). In agreement with Principle 5 (Chudoba, 1985) saying that filamentous microorganisms have low values of the AC, the r.~,m values remained constant during a 6 h endogenous phase with heavily filamentous culture from the CM system. Figures 5 and 6 also show that after a 24 h aeration period, the rx, mvalues remain approximately constant during further 6 h. This is in good agreement with the results obtained in the CM system (Figs 3 and 4). It also follows from Figs 5 and 6 that the higher rx,,, the higher K,, and vice versa. SUMMARY

The maximum substrate removal rates, rx.,,, were measured during the endogenous phase with mixed cultures. The mixed cultures were cultivated in two laboratory units, namely, a continuously-fed completely mixed and a semi-continuously-fed reactor. The latter was used to simulate the system with ideal plug flow and, simultaneously, the contact stabilization system. Both reactors were fed with a multicomponent substrate. The maximum substrate removal rates were determined by means of a simple respirometric method. Excess biomass from the completely mixed system was tested with ethylalcohol, glutamic acid, phenylalanine, acetic acid and phenol. It was found that during a 6 h endogenous period, the rx.,, values remained approximately constant with all the substrates tested. After the exogenous substrate in the semicontinuously-fed system being removed, the biomass was tested with ethylalcohol, glutamic acid, phenylalanine, acetic acid, and glucose. It was found that the values of rx.,, increased during the first 8-12 h of the endogenous period and then decreased. The

Endogenous phase duration results indicate that the semi-continuously-fed system was not operated under optimum conditions. The authors conclude that the respirometric method of determining rx.m values can be used to optimize the contact stabilization process. REFERENCES

(~ech J. S. and Chudoba J. (1983) Influence of accumulation capacity of activated sludge microorganisms on kinetics of glucose removal. War. Res. 17, 659-666. (~ech J. S., Chudoba J. and Grau P. (1984) Determination of kinetic constants of activated sludge microorganisms. War. Sci. Technol. 17, 259-272. Chudoba J. (1985) Control of activated sludge filamentous bulking--VI. Formulation of basic principles. Wat. Res. 19, 1017-1022.

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Chudoba J., ~ech J. S., Farka~ J. and Grau P. (1985) Control of activated sludge filamentous bulking--V. Experimental verification of a kinetic selection theory. Wat. Res. 19, 191-196. Grady C. P. L. and Philbrook D. M. (1984) Discussion of paper No. C21 "Determination of Kinetic Constants of Activated Sludge Microorganisms" by J. S. (~ech et al., Proc. 12th I A W P R C Conf., Amsterdam, Sept. 1984. Haseltine T. R. (1961) Sludge reaeration in the activated sludge process---a survey. J. War. Pollut. Control Fed. 33, 946-967. Jirka A. M. and Carter M. J. (1975) Micro semi-automated analysis of surface and wastewaters for chemical oxygen demand. Analyt. Chem. 47, 1397-1402. Law A. T., Robcrtson B. R., Dunker S. S. and Button D. K. (1976) On describing microbial growth kinetics from continuous culture data: some general considerations, observations, and concepts. Microbial Ecol. 2, 261-283.