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`September-; 1985
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`Russian Original Vol. 54, No. 2, March-April, 1985
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`ISSN 0026-2617
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`MIBLAO 54 (2) 137-268 (1985)
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`'. M IC'ROBIDLOGY
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`1MHKP06HOJ10rHR I MIKROBIOLOGIYA
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`BEQ 1014
`Page 1
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`Microb_lology . is a ·translation of Mikrol5iologiya, a pub I ication
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`- Institute of Microbi~logy
`Aca d!!my of Sciences of the USSR
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`BEQ 1014
`Page 2
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`

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`INfLUENCE OF ACETATE ON THE GROHTH OF Escherichia coli
`uNDER AEROBIC AND ANAEROBIC CONDITIONS
`
`G. V. Smirnova and 0 . N. Oktyabr'skii
`
`UDC 579.842.11-24.04
`
`A study of the dyna~ics of the accumulation of acetate in batch cultures of Esche(cid:173)
`richia coli K-12 with an addition of a substrate showed that at pH 7. 0 stoppage of
`growth under aerobic conditions occurs at an acetate concentration of an average
`of 150 mM, while under anaerobic conditions it occurs at a concentration of 35 mM.
`Experiments with artificial introduction of acetate suggest that acetic acid plays
`the main role in the inhibition of growth of a culture of E. coli by acid products
`of metabolism. The authors discuss a possible mechanism of the inhibiting action
`of acetate.
`
`Op timization of the conditions and methods of culturing of microorganisms in modern fer(cid:173)
`menters permits yields of biomass up to 125-150 g of dry cells/liter to be obtained [3, 4].
`The production of higher yields is limited by the accumulation of products of metabolism,
`the inhibiting action of which becomes the deciding factor limiting growth under conditions
`when limitation with respect to all components of the nutrient medium and oxygen is removed,
`In the opinion of a number of authors [3 , 4, 7], for Esche(cid:173)
`and the pH level is controlled .
`riahia coli organic acids are such inhibiting metabolites.
`The purpose of this work was to study the dynamics of the accumulation of acid products
`of metabolism in batch cultures of E. coli with an addition of the substrate, as well as the
`influence of acetate as one of the main metabolites on the growth of the bacterial population
`under aerobic and anaerobic conditions.
`MATERIALS AND METHODS OF INVESTIGATION
`In the experiments we used the strain E. coli K- 12 (S) . Culturing was conducted in a
`batch system with an addition of substrate in ANKUM-2 apparatuses (working volume of the
`fe~enter 1 liter) with continuous recording of the -optical density, pH, Eh, and p0 2 at 37°C .
`The bacteria were cultured on a synthP.tic medium of the following composition ( in g/liter
`of distilled water): NaHP0 4•12H 20- 15,13; KH2P04 -3; NH4Cl- 5; CaCl2- 0 .022; MgS04•7H20
`0.246 ; FeS0 4•7H 20 - 0 . 014 ; MnS0 4•4H 20 - 0 , 004 ; ZnCl2- 0 , 004 ; CuS04•SH20- 0 . 0008 ; CoCl2•
`6H20 - 0 , 0008. The initial glucose concentration in the medium was 0 .5 g/liter, with in(cid:173)
`creasing density of the culture, the portion of glucose added at the moment of stoppage of
`growth in the case of exhaustion of . the substrate gradually increased. Supply of the culture
`with a nitrogen source and a constant pH level of 7.0 were maintained by automatic titration
`of the medium with NH40H, With increasing biomass in the fermenter, portions of a concen(cid:173)
`In experiments studying the influence of
`trated solution of MgS0 4 were periodically added.
`sodium acetate, the necessary concentrations of it were introduced into the initial medium
`together wtth the other components.
`An anaerobic system was created by continuous purging of the medium with argon,
`case of an aerobic system, throughout the entire period of culturing a high level of
`the order of 80%) was maintained by constantly increasing the rate of pumping of air
`number of revolutions of the mixer as the density of the cultures increased .
`The weight of the dry cells was determined on Synpor membrane filters (diameter of pores
`0,3, , The total content of acids formed in the process of culturing was found by titration
`W~th alkali and expressed in milliequivalents/liter . Acetate was determined by the method
`of gas~liquid chromatography on a Tsvet 101 chromatograph with a .stainless steel column (3 m x
`·2,5 mm) filled with 25% neopentyl glycol succinate on chromaton NAW DMCS (0.200- 0.2501).
`
`In the
`p02 (on
`and the
`
`!n~titute of the Ecology of Plants and Animals, Urals Science Center, Academy of Sciences
`of the USSR, Perm'. Translated from Mikrobiologiya, Vol. 54, No. 2, pp. 252-256, March- April
`1985 , Original article submitted August 30, 1983.
`
`0026-2 617/85/5402-0205$09.50 © 1985 Plenum Publishing Corporation
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`BEQ 1014
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`Fig. 1. Batch cul t ure of E. coli K-12 with an addition of the substrate
`under anaerobic (a) and aerobic (b) conditions, pH 7.0. 1) Biomass; 2)
`specific rate of growth; 3) acetate; 4) acid products.
`Statistical treatment of the data and calculation of the regression lines were performed
`on a Mir-2 computer.
`
`RESULTS AND DISCUSSION
`In the culturing of E. coli K-12 under anaerobic conditions (Fig. la), exponential
`to 0 .28 h- 1 continues for a period of 5 h. Subsequently the specific
`growth with ~max equal
`Tate of growth gradually fell, reaching zero at a level of biomass on the order of 0.9 g of
`ary cell~/liter, Growth of the culture was accompanied by the accumulation of acid products,
`A third of which consisted of acetate. By the time the growth stopped, the acetate content
`in the medium had reached 35 mM. Growth of the bacteria did not resume after the addition
`of all the nutrient components of the medium; this is evidence that stoppage of growth was
`not a~s~ciated with their exhaustion, and inhibition of growth was the result of the influence
`ot metabolites. The glucose consumption and, correspondingly, accumulation of acid products
`(not shown on the figure), which continue after growth ceases, are most likely consequences
`of uncoupling of glycolysis and constructiv-e metabolism. The inhibiting action of metabolites
`under anaerobic conditions was reversible; growth of the culture resumed when it was changed
`over to a system of aerobic culturing.
`Under aerobic conditions (Fig. lb), growth continued at a maximum specific rate for 7 h.
`A further dr op in ~ was accompanied by a rapid increase in the production of acid metabolites,
`including acetate, the concentration of which in the culture medium by the time of stoppage of
`gro\Y'th \Y'as 150 mM, more than four times the value characteristic of the anaerobic system.
`The yield of biomass reached 35 g of dry cells /l iter, which is a maxim for batch cultures of
`·E, coli with an addition of substrate with aeration with air in the absence of dialysis [3,
`4J, The cessation of growth is probably associated with an uncoupling of the energy and
`constructive metabolism, since the consumption of glucose and the excretion of acid metabolites
`continue even after complete stoppage of growth of the culture . The addition of nutrient
`components did not lead to a resumption of growth, i.e., under aerobic conditions also, stop(cid:173)
`page of growth was a consequence of the accumulation of inhibiting metabolites and not of an
`exhaustion of nutrient resources.
`E, coU metabolizes glucose with the formation of a rather wide spectrum of organic acids,
`including acetate, lactate, succinate, formate, pyruvate, etc. Our preliminary experiments
`ahowed that in E, coli~ of all the enumerated acids, the most pronounced inhibiting effect is
`In view of this, on the basis of experiments on the culturing of E.
`exhibited by acetate.
`coli under anaerobic and aerobic conditions, graphs of the dependence of the specific rate of
`growth on the concentration of acetate accumulated in the medium with increasing density of
`the culture were constructed, Culturing under anaerobic conditions (Fig. 2a, No. 1) was char(cid:173)
`acterized by a smooth decrease in ~ as acetate was accumulated; the inhibition constant,
`equal to the concentration of the products at ~ = ~max/2, · was 11.5 mM. The calcula ted acetate
`concentration at which growth stopped entirely was equal to 36 mM.
`Tn the case of aerobic growth, the dependence of ~ on the concentration of acetate was
`~f a somewhat different nature:
`an initially sharp drop in ~ gave way to a more gradual,
`almost linear fall (Fig . 2b, No. 1); Keq in this case was 23 mM, and the calculated acetate
`concentrAtion inhibiting growth by 100% was 150 mM.
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`206
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`4
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`40 0
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`60
`Acetate, mM
`Fig. 2. Dependence of the specific rate of growth of E. coli on the acetate
`concentration in the culture fluid (1, 2) and index of inhibition (3, 4) un(cid:173)
`der anaerobic (a) and aerobic (b) conditions. 1, 3) The curves were calcu(cid:173)
`lated according to the data of Fig . 1; 2, 4) the curves were obtained ac(cid:173)
`cording to the data of acute experiments.
`The following stage of the work was a study of the influence of acetate on the growth
`of E. coli by artificial addition of CH 3 COONa to t he culture medium. Acetate was introduced
`into the nutrient medium in amounts of 5, 10, 15, 20, and 25 mM in the case of the anaerobic
`system and 25, SO, 75, 100 , 125, and 150 mM under aerobic conditions. The investigations
`were conducted according t o the method of acute experiments, when the concentration of cells
`is negligible (under aerobic conditions no more than 1 g of dry cells/liter, under anaerobic
`conditions no more than 0,3)J the physicochemical parameters of the medium and the concentra(cid:173)
`tion of nutrient components were optimal. Under these conditions ~ = ~max and depends only
`on the concentration of the inhibitor. Chromatographic analysis showed that with such a lev(cid:173)
`el of accumulation of biomass there was no appreciable increase in the acetate concentration
`~n account of glucose metabolism, The curves of the dependence of the specific rate of growth
`on the concentration of CH 3 C00Na are presented in Fig. 2a, b, No. 2. We should mention that
`in all cases the detected dependences cannot be described by the equation of noncompetitive
`inhibition of enzymatic reactions [1]. Statistical treatment of the data shows that there
`is no sign~ficant difference between the regression- lines reflecting the dependence of the
`~pec~fic rate of growth on the acetate concentration in the case of its natural accumulation
`and art~fic:l.al introduction into the medium. Thus, it can be concluded that in a medium with
`a neutral pH acetate evidently makes the main contribution to the inhibition of the growth
`of E, coli by acid products of metabolism. The increase in the ionic strength of the solu(cid:173)
`tion and the influence of Na+ ions in the case of addition of sodium acetate to the medium
`can be neglected, since special experiments that we conducted showed that when equimolar
`amounts of NaCl are introduced into themedium instead of CH 3 COONa, the specific growth rate
`of E, coli is unchanged,
`The ability of fatty acids, including acetate, to have an inhibiting effect on the
`growth of bacteria has been noted repeatedly in the literature [6, 8].
`It is extremely
`probable that acetate in high concentrations inhibits the growth of E. coli by means of vari(cid:173)
`ous mechanisms: nonspecific inhibition of enzymatic reactions as a result of acidification
`of the cytoplasm, inhibition by acetate of the end products, etc. The authors, however,
`believe that the deciding factor is the influence of acetate on the energetics of the bacterial
`cells and primarily on such an important component of it as the electrochemical proton gra(cid:173)
`dient A~+. This effect is associated with the properties of acetate as a penetrating weak
`ac;l.d, Compounds of this class ( for example, benzoate, salicylate, propionate) possess the
`ability to remove ApH when they pass through the bacterial membrane in protonated form. Ac(cid:173)
`tually, the introduction of acetate into the culture medium f or E. coli under aerobic condi(cid:173)
`tions leads to a decrease or complete removal of ApH, its conversion to A~ against a back(cid:173)
`ground of general decrease in A~ [5]. Since it is now well known that A~+ and its com(cid:173)
`ponents are the energy base for a large number of physiological processes in bacteria (syn(cid:173)
`theeb of ATP, transport of substances, osmotic work, chemotaxis, etc.), it c. an be assumed
`that the inhibiting influence of acetate on the growth of the bac teria is realized through
`its influence on the components of A~+.
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`207
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`Page 5
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`pH
`7,0
`
`5,9
`
`5,8
`
`._
`0
`
`'>$i 0,5~ ~ G,LI
`~ oo; O,Z
`..... {)
`0
`
`I:Q
`
`I
`
`Fig. 3. Action of acetate on an anerobic
`culture of E. coli and the transition pro(cid:173)
`cess in the case of delivery of air. 1, 2)
`Biomass; 3, 4) pH.
`a) Anaerobiosis; b)
`aeration. Solid lines: acetate (25 mM) was
`added to the medium. Dashed lines: control,
`without the addition of acetate.
`Kashket has shown [2] that under anaerobic conditions in E. coli ~UH+ is represented
`only by the gradient of the chemical potential of protons (i.e., ~~H+ +~pH) . We should have
`expected that acetate, present in large amounts, should act as a protonophore under such
`conditions, removing ~pH against a background of continuing consumption of the energy sub(cid:173)
`strate ~~~+ = 6pH = 0), Actually, in our experiments at an acetate concentration of 25 rnM
`under anaerobic conditions, there was no growth of the culture, although the consumption of
`glucose and the corresponding acidification of the medium occurred at approximately the same
`rate as in the control (Fig. 3).
`According to Kashket's data, in E. coli ~pH under aerobic conditions is significantly
`higher than under anaerobic conditions; therefore the generation of ~pH and an increase in
`its value should be accompanied by a redi~tribution of acetate between the medium and the
`cells, in this case by supplementary introduction of it into the cell. The increase in the
`pH of the medium during the transition process, accompanying the turnon of aeration, can be
`explained in precisely this way (Fig. 3).
`The resumption of growth observed in this case is probably the main result of the ap(cid:173)
`pearance of ~~+ as a result of generation of ~~ and ~pH during the work of the respiratory
`chain,
`In aerobic culturing, the inhibiting effect of acetate on E. coli is probably realized
`more complexly,
`In this case, thanks to the conversion of ~pH to ~W under the influence of
`acetate· f5J, the value of ~~H+ = ~W remains rather high to provide for the physiological
`processes that dependon ~~H+. Probably this is the main cause of the substantial increase
`in the inhibiting acetate concentration in the case of its influence on the growth of E. coli
`under aerobic conditions. Another cause was mentioned earlier - under aerobic conditions
`the value of ApH is significantly higher than under anaerobic conditions; correspondingly,
`a higher acetate concentration is required for removal of ~pH.
`We should keep in mind that acetate has a far more pronounced ability to remove ApH than
`do other penetrating acids; therefore its influence on ~~+ should be manifested in the
`presence of a more substantial acetate content in the medium, which is observed in culture
`with an addition of substrate at a high biomass density.
`Thus, an analysis of the data obtained suggests that in batch culturing of E. coli with
`an addition of the substrate at the optimum pH, the main factor inhibiting growth of the cul(cid:173)
`ture becomes the accumulation of acid products of metabolism in the medium and primarily acetic
`acid, The uncoupling of the energy and constructive metabolism by acetate, the reversibilitY
`of the inhibition of growth by acetate after delivery of air to an anaerobic culture , and
`the difference of the inhibiting concentrations of acetate for aerobic and anaerobic condi(cid:173)
`tions permit the authors to suggest that the mechanism of its inhibition action may be as(cid:173)
`sociated with an influence on the proton motive force and the cellular processes coupled with
`;it,
`
`208
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`BEQ 1014
`Page 6
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`

`
`LITERATURE CITED
`N. F. Ierusalimskii and N. M. Neronova, Dokl . Akad. Nauk SSSR, 161, No. 6, 1437 (1965) .
`E. R. Kashket~ J. Bacteriol., 146, No. 1, 345 (1981) .
`P. Landwall and T. Holme, J. Gen. Microbiol., 103, 345 (1977) .
`H. Mori, T. Yano, T. Kobayashi, and S. Shimizu, J. Chern. Eng. Jpn., 12, No. 4, 313
`(1979) .
`D. R. Repaske and J. Adler, J. Bacteriol ., 145, No. 3, 1196 (1981) .
`C. W. Sheu and E. Freese, J. Bacteriol., ll~No. 2, 516 (1972 ) .
`N. A. Sinclair and J. L. Stokes, J . Bacteriol., 83 , No. 5, 1147 (1962) .
`N. Weiner and P. Draskoczy, Pharm. Exp. Ther., 132, 299 (1961) .
`
`1.
`2.
`3.
`4.
`
`5.
`6.
`7.
`8.
`
`KINETIC PRINCIPLES OF THE GROWTH OF Methanosaraina vaauoZata
`ON METHANOL
`
`S. V. Ka1yuzhnyi, A. N. Nozhevnikova,
`and ~. D. Varfolomeev
`
`UDC 579 .851.12-24.017.8
`
`The kinetics of the decomposition of methanol by growing cultures of Methano(cid:173)
`saraina vaauoZata was studied, It was shown that the reaction products are carbon
`dioxide and methane in a 1:3 ratio. It was established with an accuracy within
`10% that the metabolism of the microorganism does not contain other components ex(cid:173)
`cretable into the culture fluid or gas phase. A differential method of analysis
`of the kinetic growth curves in batch culturing is proposed in two modifications:
`analysis of the initial portions of the growth curve (10-20% consumption of the sub(cid:173)
`strate ) and analysis of the complete kinetic growth curve. The proposed method is
`illustrated in the analysis of the growth curves of a culture of M. vaauoZata.
`For t his culture the most important kinetic parameters were determined:
`the maxi(cid:173)
`mum specific rate of growth 11m and the constant of "affinity" of the microorganism
`for the substrate K8 • The proposed method of determining the kinetic parameters
`-makes it possible to give clear recommendations for the intensification and opti(cid:173)
`mization of microbiological processes.
`
`Methane bacteria carry out the final stage of the destruction of organic matter under
`anaerobic conditions.
`The genus Methanosaraina includes obligate-anaerobic coccoid microorganisms, which form
`methane from a hydrogen-carbon dioxide mixture, acetate, methanol, and methylamines , With
`reg~rd to the assortment of utilizable substrates, these are the most universal of the methane
`forming bacteria, combining all three pathways of methane formation and occupying a central
`position in many methanogenic communities . Morphologically Methanosaraina species are cocci
`of ~arious degrees of aggregation, possessing a characteristic mode of division -
`fragmenta(cid:173)
`tion ll~3J, A morphological peculiarity of Methanosaraina vaauoZata is the presence of gas
`~esicles, collected into packets- vacuoles [1- 4]. The most preferred substrate for Methano(cid:173)
`sdraina is methanol, since the largest amount of energy is liberated in its decomposition in
`comparison with other substrates of methane formation [5]. Representatives of the genus
`Methcmosa'r'aina in a pure culture convert methanol directly to methane wi··.:hout the formation
`o£ intermediate products, in contrast to the complex of MethanobaaiZZus kuzneaeovii, where a
`clostridial organism decomposes methane to molecular hydrogen and ca·,:·hc•L1 dioxide, while the
`~ethane~£orming bacterium Methanobaaterium the~oautotrophiaum synthesizes methane from them
`14 , 6, 7J,
`The purpose of the present work was to investigate the kinetic principles of the growth
`o~ ~ culture of M, vaauoZata under stationary conditions.
`
`}{, V, Lomonosov Mosco~q State University,
`Institute of Microbiology, Academy of Sciences
`of the USSR, Translated from Mikrobiologiya, Vol, 54, No. 2, pp. 2~7-262, March-April, 1985.
`Original article submitted March 22, 1984,
`
`0026 ~ 2617/85/5402~0209$9.50 © 1985 Plenum Publishing Corporation
`
`209
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`BEQ 1014
`Page 7