Professional Documents
Culture Documents
2
Copyright 1971 American Society for Microbiology Printed in U.S.A.
One of the major uses of mixed microbial popu- studies because it is readily used by many organ-
lations is in processes for the biological treatment isms and probably permits the maximum hetero-
of organic wastes. It has become increasingly im- geneity of species which can be obtained in a
portant that the kinetic formulas used in the minimal medium. The studies reported herein
design of biological waste treatment facilities and were undertaken to extend the usefulness of the
in prediction of their operational characteristics previous work by testing the applicability of the
be based upon controlled studies of microbial Monod equation to heterogeneous populations
behavior rather than upon empirical rules. An growing on a variety of substrates. As the most
equation which is of considerable importance in severe possible test of the utility of the equation
such applications is one describing the relation- in biological waste treatment, municipal sewage
ship between the logarithmic growth rate constant was concentrated and the soluble portion was
of the population and the substrate concentration. used as substrate.
For pure cultures, the following equation was
proposed by Monod (6) and was further MATERIALS AND METHODS
developed and tested by Herbert et al. (4): A = Microbial populations were developed on each
nm(s)1/ks + s where a is the logarithmic growth carbon source from initial inocula of sewage obtained
rate constant, s is the substrate concentration, from the municipal treatment plant at Stillwater, Okla.
,Unl is the maximum logarithmic growth rate con- Five milliters of primary clarifier effluent were added
stant for the culture under the conditions used, to 45 ml of synthetic medium containing a specific
and k, is the substrate concentration at which carbon source (1,000 mg/liter of final concentration,
see Table 1) and aerated on a shaker apparatus (250-
A = i,/2. Several modifications of this equation ml Erlenmeyer flasks, 90 oscillations per min, tem-
have been suggested [for review, see (5)]. Appli- perature 23 + 2 C). Daily, 5 ml of the cell suspension
cations of the original and modified equations to was transferred into 45 ml of fresh medium. After
heterogeneous populations or waste treatment seven such transfers, a portion of the mixed culture
processes have been proposed [for review, see (2) ], was employed in growth rate studies. Using the re-
but many design formulations in present use mainder of the culture, the daily transfer procedure
assume a linear relation between ,u and s rather was continued for several weeks, and the growth rate
than the hyperbolic one described by the Monod experiment was repeated.
equation. In the growth rate experiments, 2.5 ml of the cell
We have previously reported that the Monod suspension was placed in each of the growth flasks
(250 ml, Erlenmeyer), and the volume was made up to
relationship best describes the growth of hetero- 50 ml with growth medium containing various con-
geneous populations in a synthetic waste contain- centrations of the carbon source under study. The in-
ing glucose as the sole carbon source and limiting organic salt concentrations were those given in Table
nutrient (3, 7). Glucose was employed in these 1, except that for substrate concentrations above 1,000
253
254 PEIL AND GAUDY APPL. MICROBIOL..
TABLE 1. Composition of growth medium observed in the two experiments. Because of the
Constituents Concn (per liter) heterogeneity of the population and the attendant
opportunities for changes in predominance in
Carbon sourcea ................... 1,000 mg such systems, a usable range rather than a pre-
Ammonium sulfate, (NH4)2S04 .... 500 mg cise value must be expected for the constants in
Magnesium sulfate, MgSO4 7H20.. 100 mg any kinetic model which is applied to depict
Ferric chloride, FeC13 6H20 .0.50 mg growth of heterogeneous populations.
Manganous sulfate, MnS04-H200 10.0 mg The values of growth constants obtained for all
Calcium chloride, CaCl2............ 7.5 mg experiments are given in Table 2. In general,
1.0 M potassium phosphate buffer, hyperbolic curves were obtained in plots of ,u
pH 7.0 10.0 ml
versus s for all experiments. In 7 of the 27 experi-
Tap water 1........................I00.0 ml ments the hyperbolic fit was questionable, but in
Distilled water ............ to volume
each of these cases a continuous hyperbolic curve
a The following compounds were employed provided a better fit of the data than a straight line
individually as sole carbon source: glucose, (see Fig. 1). With most of the substrates used,
lactose, sucrose, sorbitol, alanine, glutamic acid, different values for i,m and k. were obtained in
serine, histidine, phenylalanine, cysteine, acetic experiments carried out at different times. In most
acid, and propionic acid.
data than does a straight line intersecting a hori- FIG. 1. Relation between logarithmic growth rate
zontal line through i,m (see dotted lines). Only at constant, ,u, and substrate concentration,
s, for hetero-
very low substrate concentrations could a straight geneous microbial populations growing on glutamic
line be fitted through the data. Significant differ- acid as sole source ofcarbon. (a) Monodplot, ,u versus s.
ences in the values of the growth constants were (b) Lineweaver-Burk plot, s/,u versus s.
VOL. 21, 1971 AEROBIC GROWTH OF MICROBIAL POPULATIONS 255
TABLE 2. Values of Mm anid k8 for heterogenzeous studies, it was necessary to concentrate the settled
populationis growing on various substratesa sewage. The substrates used in the two growth
rate experiments on sewage consisted of a series of
Substrate Expt. kC
no. /Am (hr-1) (mg/liter) dilutions of the soluble (filterable) fraction of
settled sewage which had been concentrated to
Glucose 0.49 29 its original volume. Portions of 500 ml each
Glucose 2 0.38 11 were evaporated under vacuum to a final volume
Lactose 0.53 55 of 25 ml in an evaporator (Buchler Instrument
Lactose 2 0.44 37 Co.) Evaporation temperatures of 65 and 55 C
Lactose 3l 0.20 were employed (substrates for experiments 1 and
Lactose 4 0.43 33 2, respectively). There was a significant loss of
Sucrose 0.55 17 COD during concentration. Determinations of
Sucrose 0.28 6
Sorbitol 0.60 18 the COD of the settled sewage before concentra-
Sorbitol 2 0.44 13 tion and at concentrations of 5-, 10-, 15-, and
Alanine 0.33 27 20-fold indicated that, at all concentration ratios,
Alanine 0.18 15 approximately 35% of the sewage COD was
Glutamic acid 0.78 47 volatilized and swept out of the system when
Glutamic acid 2 0.59 95
Serine 0.43 50 SEWAGE
Serine 2 0.54 30 PJM= 0.46 hr-
Histidine 0.50 17 0
0.50 0ks 55 mg/i _ _
Histidine 2 0.67 50
Phenylalanine 1 0.33 41
Phenylalanine 0.33 54 0.40 _ -
Cysteine 0.16 23
Acetic acid 2
2
0.36 41
Acetic acid 0.29 47 0.30
Propionic acid 0.38 6
Propionic acid 2 0.37 17
Sewage 1 0.49 41 020
Sewage 0.43 62
0.10 X
-Value of AMm and k5 were obtained from plots
of s/M versus s.
0
concentration was conducted at 65 C. At 55 C, little doubt concerning the validity of the Monod
20 to 25% of the sewage COD was volatilized relationship for this complex "substrate." Values
during concentration. The COD removed from of pm are approximately the same as those ob-
the evaporating flask was not recovered in the served in the many experiments reported pre-
condensation flask, indicating that the material viously using glucose as carbon source (3, 7), and
was quite volatile (strippable). The condensate they are in general agreement with the other
was analyzed for COD and never contained more values obtained in the present study (see Table 2).
than 30 mg/liter COD. Check runs were made in The agreement between values obtained with
which a known concentration of glucose solution sewage and with some pure compounds, e.g.,
was placed in the apparatus at 65 C and 100%/ of glucose, as substrates is perhaps not unpredict-
the glucose was retained in the evaporation flask. able since a simple medium may often be con-
Also, a solution of acetic acid was concentrated. verted to a "complex" one by the accumulation of
Complete recovery of this compound was at- partially metabolized products (2).
tained; 30%O remained in the evaporation flask In general, based upon the massive amounts of
and 70% was found in the condensation flask. data previously reported (3, 7) for heterogeneous
Thus, it appears that the apparatus functioned populations using glucose as substrate, the experi-
properly and that the material stripped from the mental results presented here in which various
settled sewage under the conditions of evapora- single carbon sources were employed and, of even
tion was of such volatility that it could not be greater importance, those in which sewage
condensed in the cold flask and was forced from medium was employed, it is concluded that the
the system. Concentration studies at lower tem- hyperbolic function can be employed to describe
peratures were conducted, but the time required the relation between growth kinetics and sub-
to concentrate a 500-ml portion to 25 ml was strate concentration for systems which may be
sufficient to allow microbial growth and it was heterogeneous with respect to both species of
undesirable to add microbial inhibitors. Each 25 microbe and carbon sources. Clearly, the ki-
ml of concentrated sewage was filtered imme- netic constants g,,, and k. cannot be considered
diately through membrane filters, 0.45-/.m pore as precise values for systems in which species pre-
size, and the filtrates were pooled and stored at domination is subject to fluctuations and a usable
0 C for use in a growth rate experiment. Thus, the range of values must be employed in depicting the
sewage substrate consisted, not of all the organic behavior of the system.
components of sewage, but of the soluble organic
portion minus the highly volatile material in the ACKNOWLEDGMENTS
sewage. In any event, there should be no doubt This work was supported by research grant WP 00 325 and
about the complexity and heterogeneity of this by training grant WP-19, both from the Federal Water Quality
substrate. The concentrate exhibited the charac- Administration, U.S. Department of Interior.
teristic odor of fresh sewage and the appearance
of urine. It was slightly alkaline, pH 8.5. This LITERATURE CITED
material diluted to various concentrations (ex- 1. American Public Health Association. 1965. Standard methods
pressed as COD) in distilled water comprised the for the examination of water and waste water, 12th ed.
Amer. Pub. Health Ass. Inc., New York.
sole growth medium (i.e., no mineral salts or 2. Gaudy, A. F., Jr., and Elizabeth T. Gaudy. 1966. Microbiology
buffer was added). The pH remained at 8.5 during of waste waters. Annu. Rev. Microbiol. 20:319-336.
the growth experiments. The heterogeneous popu- 3. Gaudy, A. F., Jr., M. Ramanathan, and B. S. Rao. 1967.
lations used as initial inocula in the growth ex- Kinetic behavior of heterogeneous populations in completely
periments consisted of cells grown from random mixed systems. Biotechnol. Bioeng. 9:387-411.
sewage samples through 3 to 5 serial, 24-hr trans- 4. Herbert, D., R. Elsworth, and R. C. Telling. 1956. The con-
tinuous culture of bacteria: a theoretical and experimental
fers in the sewage medium. study. J. Gen. Microbiol. 14:601-622.
Plots of ,u versus s for experiments run on 5. James, T. W. 1961. Continuous culture of microorganisms.
sewage concentrated at 65 and 55 C are shown in Annu. Rev. Microbiol. 15:27-46.
Fig. 2. The decrease in growth rate constant with 6. Monod, J. 1949. The growth of bacterial cultures. Annu. Rev.
high substrate concentrations (1,000 and 1,300 Microbiol. 3:371-394.
7. Ramanathan, M., and A. F. Gaudy, Jr. 1969. Effzct of high
mg/liter COD, Fig. 2b) suggests that an inhibitory substrate concentration and cell feedback on kinetic behavior
compound may have been removed from the of heterogeneous populations in completely mixed systems.
sewage at 65 but not at 55 C. The results leave Biotechnol. Bioeng. 11:207-237.