Continuous culture of bacteria with biomass retention

Trends in Biotechnology, Vol. 2, No. 1, 1984

Continuous culture of bacteria with biomass retention Henk W. wan Verseveld, Michael Arbige and William R. Chesbro Bacterial cells have three phases of growth which are characterized respectively by: (1) balanced growth with a high yield of biomass; (2) balanced growth with lower biomnm yield; and (3) unbalanced growth with lowest biomass yield. Phases 2 and 3 are associated with elevated concentrations of the regulatory nudeotides centered on guauosine~'diphosphate~'-diphosphate. Maintenance of the correct growth phase is important in optlmiT~ industrial product formation by bacterial populations. All microbial processes have three basic ingredients: substrate (nutrient); cells (biomass); and an environment in which the first two interact. In bioteclmology the main focus is on the third of these ingredients, the process reactor. A reactor's configuration determines the rates at which nutrients, biomass and catabolic products transfer into and out of the microbes' environment. It is now possible to design reactors that are open or closed to the entry or exit of any combination of these materials. This design flexibility has resulted in a variety of devices that can be characterized as belonging primarily to one or the other of the four types listed in Table 1: substrate open-biomass open (SoBo), substrate open-biomass closed (SoBc), substrate closed-biomass open (ScBo), and substratc closed-biomass closed (ScBc). There are some perfect or pure examples of these reactor types: it is difficult to operate a chemostat except SoBo; and an ScBc process occurs whenever a nutrient mixture is seeded with an appropriate microorganism, tightly sealed and incubated. Imperfect modes of operation are, however, probably more common. Even with the. existence of mixed modes, it usually requires little beyond simple inspection to see how mass transfers into and out of a given reactor type take place. Much less obvious are the mass transfers from nutrients into

biomass by assimilation and into catabolic products by dissimilation. Fig. 1 shows examples of changes in biomass concentration, Specific growth rate (~; h -]) and mass doubling time (tD; h) in a batch culture (ScBc mode), a chemostat (SoBo mode) and a recycling fermenter (SoBc mode). In the log phase of a hatch culture the specific growth rate is generally constant and maximal, and in the late log phase it decreases due to the exhaustion of a limiting substrate. Although the most widely used equation to describe this dependence is that deduced by Monod ~(see Table 2), there are at least six other equations that will produce curves fitting a plot of/~ against the concentration of the limiting substrate. This embarassing availability of suitable equations is essentially due to the phenomenological nature of simple mass transfer analysis 2 (i.e. the analyses are descriptive and do not postulate specific mechanisms). Whatever the best mathematical description of the dependence of/a on substrate concentration may be, the existence of s u c h ' a dependence is confirmed by the observed behavior of la in the chemostat, where it becomes

stabilized at a value that depends on the dilution rate of fresh, limiting substrate entering the reactor. When this steady state is attained, growth is balanced and the molar growth yield, Y, for any limiting anabolic substrate is constant. In the cell, there are two broad classes of ATP-consuming processes. There is a class of reactions in which ATP is consumed to drive the net synthesis of biomass from anabolic substrates; and there is a second class of reactions whose ATP consumption does not result in formation of more biomass, but whose roles in the cell are less easily characterized. The reactions of the latter class have been termed maintenance reactions, emphasizing that they are related to cell survival, and they have also been called, leakage and uncoupling reactions 2's, to emphasize the diversion of energy from the processes of assimilation. The first term, 'maintenance reactions', ignores those reactions which consume energy without producing biomass or contributing to cell maintenance, such as random hydrolysis of ATP. The latter terms, 'leakage', and to a lesser extent 'uncoupling', carry the implication that reactions such as the synthesis of regulatory nucleotides like cAMP, which consume A T P without biomass synthesis, do not contribute to cell survival. No commonly used term seems wholly satisfactory, so we will refer to two broad kinds of ATP usage in the cell simply as classes 1 (increasing biomass) and 2 (not increasing biomass). The distribution of catabolic substrate consumption between the support of class 1 and class 2 reactions has been estimated by use of equations developed by Pirt 4 (see Table 2). A coefficient, m, was defined as being independent of ta. More recently, a

Table I. Classification of some biological process reactors by mass transfer characteristics.

Open (Bo)

Biomass Transfer Closed or retained (Be)

Continuous reactors, including: chemostats, turbidostats and some types of mixed tower reactors

Henk van Verseveld is at the Microbiology Depm-tmem of the Biology Laboratory of the Free University, A n m t e r a - ~ , The Netherlands; Mldmel Arbige is a senior microbiologist in fermemmtion development for C~-neeor, Inc., South San ~.c~ "~ Some types of dialysis culture F n m e h c o , CA, USA; and Willimn Chesbro Some types ofsolid substrate reactors is ]Professor o f Microbiology in the Microbiology Department of the Univendty of ~o New l-lmnlmhire, Durham, Nil, USA. © l~4, EIscvkn'Scitt~PubliahcxsB.V.,Amstcrd~a 0166-9430/84/$02.00

Some types of dialysis culture Recycling reactors Fed-batch reactors Fixed-film reactors, including trickling tower or trickling filter types Expanded bed reactors Immobilized (viable) cell reactors Stratified tower reactors, including sedimentation and sludge blanket types Stirred tank (batch) reactors Stratified (batch) reactors, including the special case ofthe plug, or pipe, flow reactor

9

Trends in Biotechnology, Fol. 2, No. 1, 1984

A. BATCH Biomass (g dry weighf/t )

B. CONTINUOUS

8

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Fig. I. Changes in biomass concentration, specific growthrate, and mass doubling time in: (A) a batch culture;(B) a chemostat; and ( C) a recycling f~'menter.

growth-rate dependent coefficients, being a linear function of 14 has been introduced. These formulae permit m in a chemostat to be readily estimated and thus make it possible to calculate the fraction of catabolic substrate consumed for class 2 reactions. The fraction supporting class 1 reactions is then obtained by subtraction and is represented as the maximum yield of biomass, Y~=, that would be obtained per mole catabolic substrate in the absence of class 2 reactions. The Pirt formulae have formed the basis for predicting the biomass increase in a dialysis culture (SoBc mode)6. As the biomass grows, application of the maintenance coefficient, m, requires that a continuously increasing fraction of the incoming energy substrate be used to support class 2 reactions. As this fraction increases to 1.0, all the energy substrate is needed to support class 2 reactions, leaving none for growth, and /~ fails to 0. This analysis established a feature that must be a general characteristic of growth rate behavior in SoBc reactors. In this type of reactor, a continuously falling growth rate develops as a result of the progressively dwindling supply of energy per cell. Although the recycling reactor is closely analogous to a dialysis culture 7

as a mass transfer system, the growth pattern actually observed fits the Pitt formulae only approximately. The population size does not approach the maximum asymptotically, as these formulae predict, but instead passes through three successive, sharply defined phases: a first phase* of balanced exponential growth, a second phase of balanced linear growth and a third phase, indefinitely long, of unbalanced linear growth (Fig. 1C). The specific growth rate falls (and the mass doubling time, tD, lengthens) along two successive trajectories in the second and third phases. Although /~ is a term that is convenient to manipulate mathematically in making calculations for exponentially growing cultures in any type of reactor, it is misleading when considering slowly growing cultures. About 98°7o of the positive values o f g can be studied reliably in a chemostat 8, from 2.07 to 0.05 h -1. In terms of to, however, this is 0.33 to 13.8 h, and that represents only about 7% of the tD range examined when atD of 200 h has been reached in a recycling reactor. Consequently, using to to describe growth rates when they are below a/~ of * The terms 'domain; "state ' or "zone' are sometimes used instead of 'phase'.

0.05 h-' gives a direct appreciation of the range being considered. At least two conclusions can be drawn from the growth behavior observed in.a recycling reactor. First, that class 2 energy costs cannot be cap culated using the specific maintenance coefficient, m, when the tD is longer than 15 h. Second, that the relationship between either/~ or Yand the substrate input rate shows two discontinuities, one when the tD is about 15 h and the other when it is about 55 h 9. This latter outcome is not predicted by the Pirt, Monod or equivalent mass transfer formulae, since all of these use continuous functions to relate these terms. Regulatory nucleotides in slowgrowing bacteria Discontinuities in the relationship between g and (tD) or Y and substrate input rates could have been confidently predicted by at least 19781° from the understanding reached by then about the connection between nutrient concentrations and the synthesis and degradation of the family of regulatory nucleotides centered on guanosine-5'diphosphate-3'-diphosphate (ppGpp). The collective regulatory effects of these nucleotides are generally called the stringent response, the most striking manifestation of which is a

Trends in Biotechnology, VoL 2, No. 1, 1984

10

simultaneous inhibition of the transcription ofrRNA, tRNA, and the mRNA for ribosomal protein, thus halting the synthesis of ribosomes. " or PPPfiPP pppA -~{ow rates of carbon This family ofnucleotides affects such / nitrogenassimitafion Pi a range of cell functions t°, however, that their activities compose the I inhibifion broadest regulatory system yet found in PPi +ppl3 • •* ppl3pp bacteria. ' inhibifion(?) Synthesis of ppGpp occurs via two tow rare of energi / distinct pathways (Fig. 2). Along one pppA ----_~.oduc~ion in ceil i path, its rate of synthesis is stimulated by uncharged tRNA. Hence ppGpp is synthesized in cells which are suffering pA ~ ~ sfimutafionn from amino acid shortages caused, for example, by either nitrogen or carbon Fig. 2. Nutrient-dependent synthesis and degradation of regu/atory guanosine pol~hosphates. starvation. Along the other path, the Pi inorganic phosphate; PPi = inorganic pyrophos,phate; pA = adenosine-5'-monophosphate; rate of synthesis is stimulated by in- pppA = adenosine-5'-triphosphate; ppG = guanosine-5'-diphosphate; ~ = guanosine-5'creasing energy shortage in the cell. At triOhosf~ote; ppGpp = guanosine-5'-diphosphatc~3'-diphosphate; pppC,pp = guanosine-5'the same time, ppGpp degradation is triphosphate- 3 '-diphosphate. progressively slowed. Working in con#) falls low enough to increase the con- ducible as the ceils enter the first phase cert, these reactions function to meter centration of ppGpp to a level which of linear growth (Fig. ~ 1C). Concurthe rate of nutrient uptake. The cell stops ribosome synthesis,/a and Y will rently, the cellular concentration of thus meters its own growth rate. When drop relatively abruptly. ppGpp starts to rise above the basal the nutrient uptake rate (and therefore The discontinuity caused by this level of the exponential growth phase. Table 2. Formulae and glossary physiological mechanism occurs at atD The production of ppGpp and cAMP Equatiom Symbols Descriptionand of 55-65 h (the second discontinuity, are coordinate 15, and the activities of dimensions Fig. 1C) in species of Escherichid'" products of genes under cAMP conMonad Bacillus 12, Paracoccus9 and Rhizo- trol are elevated 20-70-fold by the Relation of specific Specific mass growth bium'. Apparently evolutionary selec- simultaneous presence ofppGpp in the rate (h-l) growth rate to concentration of a tion pressure has acted to make the t D at cell. limiting gtbstrate: The tD values (or g values) at which which the stringent response is invoked gnm Maximum g=~(~ common among this broad range of the two discontinuities occur divide attainable in the S+K~ growth system bacteria. growth rates into 3 phases (Table 3), S Substrate The other break in the plot of tD each with different dominant features concentration (M) against substrate uptake rate is at about of metabolism and physiology. The P/n Relation of molar K m Saturation constant 12-15 h a. The physiological mechan- limitation to this characterization of catabolic sulmtrate fm) ism underlying this break is not exactly metabolism and physiology by growth growth yield to known, but since ppGpp decreases the rate phases is that it is partly based on specific growth rate frequency of translational errors", it is the invocation of stringent regulation 1 =ra+ 1 suggested that this discontinuity is by low rates of nutrient uptake, and so partly caused by an increase in the far the synthesis of ppGpp has been Relation of specific Y Apparent molar energy cost of protein synthesis, result- demonstrated only in eubacteria. catabolic sulmtrate growthyield(g ing in a lower Yvahie (see Fig. 3). Since Whether or not it is also synthesized by consumption rate biomsss/mol to specific growth limiting sul~trate these changes depend only on the regu- archaebacteria, such as the methanorate utilized) latory physiology of the microbial cell, gens, has not been established. It is not Ymax Molar growth yield q f A-(u)+~ corrected for and not reactor configuration, they will synthesized by eukaryotic cells generalYm~x substrate utilization take place in any of the reactors listed in ly, although yeast may make it in not leading to response to heat shock. ~+ biomass increase (g Table 1 when the appropriate nutrient bioma~mol In addition, cAMP is not synthesized uptake rates and corresponding tD limiting su~trate by the industrially important genus values are reached. utilized for In addition to the increased con- Bacillus ~7. The growth of Bacillus biomass) m Maintenance centration o f p p G p p in cells growing at polymyxa in a recycling reactor ~2, coefficient (tool tD longer than 14 h (Refs 9 and 11), shows the three phases typical of euenergy substrate operons affected by cAMP can be in- bacterial growth but it remains to be utilized without producing biomass duced. When E. coli grows anaerobic- determined how the expression of increase (h- z g ally in a recycling reactor with glucose adaptive functions that would be biomass- ]) as the carbon-energy source and mediated by cAMP in other eubacterial tD .MmS$doubling time lactose or L-tryptophan are present at genera changes with growth rate phase q Specific substrate concentrations sufficient to induce/3- in Bacillus. In fact, Bacillus generally consumption rate galactosidase or tryptophanase respect- deal with the problems of nutritional (mol h - ~ biomass-1) ively, the two enzymes become in- impoverishment and growth at slow

pA ~

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11

Trends in Biotechnology, Vol. 2, No. 1, 198#

Table 3. Growth rate phases of the eubacteria,

Feature t D range

1: Exploitation approx 15 h and shorter y observed Maximum ApParent energy demand Lowest by class 2 reactions ppGpp effects Basal level

Phase 2: Adaptation approx 33 to 55 h

50- 75% of maximum Higher

3: Maintenance approx I00 h and longer 30-50% of maximum Highest

cAMP effects when

Basal level

Effects other than Full stringent response inhibition of ribosome including inhibition of ribosome synthesis synthesis may be present Elevated Elevated

growthrate is limited by catabolicsubstrate Endogenouscellular

Accumulated

May be consumed

reseT~ Cell morphology

'Normal' shape; size Shape may change; varies with growth size need not be growth rate related to growth rate

rates by sporulation. Therefore, phases 2 and 3 growth rates are likely to be reached only when sporulation is inhibited or in asporogenous strains]~.

Applications of the growth rate phase concept Because Y will be highest in phase 1 for catabolicaUy limited growth (Fig. 3), this is the desired phase for operations aimed at maximum production of biomass, and SoBo reactors are the best for this purpose. Continuous reactors, of course, are generally understood to be a superior choice for biomass production. SoBc reactors, on the other hand, minimize biomass yield and maximize production of eatabolites because they develop slowly growing, rapidly catabolizing phase 2 and 3 populations. Again the low biomass yield high catabolic rates which are characteristic of SoBc reactors is widely recognized, and such reactor designs are commonly used in waste stream treatment to give maximum mineralization of substrate with minimum accumulation of biomass, reducing the problem that the accumulated biomass (sludge) poses in its further disposal. What the phase concept implies for waste treatments is that, for the eubacterial populations involved, biomass yield will be essentially constant anywhere within domains 2 and 3. That is, at any of the tD values in domain 2, Y will be about 70o7o that of domain 1. Consequently, from the standpoint of maximizing this population's catabolic activity while minimizing its anabolic syntheses, only the domain in which the reactor's operation keeps the population is

Consumed Shape may change; size need not be related to growth rate

important, not where in that domain. For similar reasons, SoBc reactors are to be preferred for the production of fermentation products by bacteria. In addition, the rapid process rates made possible by the high cell densities obtainable in the reactor favour fermentation yields. In either SoBc or SoBo reactors, anabolic limitation of growth can be accompanied by high, uncoupled rates of catabolism, so that recovery cost can be lowered by raising the concentration of input catabolic substrate which correspondingly raises the concentrations of fermentation products in the output. In an SoBo reactor, however, the cell density in the reactor is determined by Y for the particular anabolic substrate limiting growth, so

%Y 100-

-

-

that the steady state population density, and consequently the steady state process rate of the catabolic substrate (which depends on the population density), will be limited in proportion to the severity of the anabolic limitation. In an SoBc reactor, the population density is limited not by Y, but by the effective limit of the technique used to keep the cells in the reactor. Another advantage of the SoBc reactor for production of fermentation end products comes from the fact that high end product concentrations in a reactor usually negatively affect /a before they affect the specific catabolic substrate consumption rate, q. That is, they act to increase class 2 energy costs, thereby withdrawing energy from class 1 reactions, and growth. Limitation of growth rate by class 2 energy demand that arises from conditions other than nutrient limitation is termed growth energy diversion, to distinguish it from catabolic or anabolic growth limitations. In an SoBo reactor, growth energy diversion by the effects of high end product concentrations aggravates the problem of achieving high population densities because neither added anabolic nor catabolic substrate will raise cell production, and its limitation on/a means that the maximum throughput rate of the process (D, in chemostat terms) is limited. In an SoBc reactor, growth energy diversion like nutrient limitation only restricts the rate at which a suitable population density is

\ \ |

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40-

20-

o

16o

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2&o L#

Fig, 3. Percentage o/Y for a catabolic, grov/th-limiting substrate as a function of ~ in a ppGpp ~

synthesizing bacterium.

12 achieved, and because the population is not being washed out of the reactor, it does not limit the throughput rate of the process. Manipulation of class 2 energy metabolism, and thus the growth energy diversion it produces, provides a useful method of controlling microbial processes TM, primarily in SoBc reactors. Increasing the class 2 energy demand by adverse thermal, pH, ionic strength, or oxidation-reduction conditions, or by other stresses, can be used directly to depress biomass production and increase catabolic product yield, and it can be used to shift a population from one domain to another. By lowering g (increasing tD), increased stress moves a population in the direction of phases 2 and 3; decreased stress has the opposite effect. This makes growth energy diversion a technique that is complementary to nutrient up- or downshifts, that produce transitions between phases through their effects on catabolic or anabolic limitations. One o f the consequences of intermittent fed batch operation is that the intermittent feeding can upshiti a bacterial population's growth rate back across one or more of the phase boundaries, changing metabolism concomitantly to that o f the higher growth rate domain reached. A function o f the stringent response, in fact, is to preserve anabolic integrity so that the cell can take rapid advantage of any nutrient change (or stress change) that permits a growth rate upshift '°. The production of secondary metabolites in SoBc reactors is particularly interesting when viewed using the phase concept. Secondary metabolites have been defined as molecules which are not essential for the growth of the organism that produces them 19. Antibiotics exemplify this kind of metabolite, and it has been pointed out that although they may be produced at a maximum rate in stationary phase cultures, their production can also occur in exponentially growing cultures, i.e. their status as secondary metabolites does not depend on the growth phase in which they are produced. Nonetheless, their maximum production can occur in late log-stationary phase cultures: phases 2 and 3. It should thus be

Trends in Biotechnology, VoL 2, No. I, 1984

interesting to examine how antibiotic production by eubacteria is affected by the metabolic regimes of these phases. The same considerations apply to adaptive enzymes under the control of cAMP and ppGpp (modified for the case o f Bacillus), and probably to toxins and adhesins where these substances are produced adaptively. We started with the premises that all microbial processes have only three basic components, and that the spatial circumstances in which the processes take place control microbial growth rates by controlling mass transfer into and out of the system. Since the ingredients of microbial processes were made general, the scheme for their classification in Table 1 is general and applies whether the process reactor is manufactured or natural. The scheme becomes limited when applied to natural (and many manmade) process reactors where there is uncertainty about the kinds of nutrients being transferred to the microbial population, the rates and routes o f their transfer, and by the taxonomic complexity o f the population. The case of a root nodule enclosing a population of Rhizobium bacteroids, an example of a natural process reactor, would seem to be an SoBc reactor. But the kinds of nutrients reaching the Rhizobium bacteroids from the plant tissue as well as the routes and rates of their transfer are extremely difficult to identify and quantitate. As an example of the complications raised by mixed types of bacteria being present in the reactor, the human intestinal tract contains both adherent and non-adherent bacterial populations, so that it might be a pulsed SoBc reactor for the former at the same time it is being a plug flow ScBc reactor for the latter. However, the phase concept may fred its broadest utility in the analysis of situations just such as these, involving mixed microbial populations and complex, hard-to-quantify mass transfers. The eubacterial cell by adjusting its content of regulatory nucleotides to its tD, and by shifting its metabolic behavior from patterns typical of one growth rate range to those typical of another at specific tD values, provides

signals about the rate at which it is obtaining nutrients, about the pathways of catabolism it is employing, about what biomass yield coefficient is in force, and generally whether it is displaying exploitive, adaptive, or conservative features of its survival potential. Learning to estimate the concentrations o f these nucleotides in a particular bacterial population directly, or indirectly through measurement of one o f the aspects of cell metabolism they control, so that the signals can be interpreted, would open the possibility of letting the bacterial cell itself analyse these complex process situations for us. References

1 Monod,J. (1949) Ann. Rev. Microbiol. 3, 371-394 2 Westerhof,H. V., Lolkema,J. S., Otto, R. and Hellingwerf, K. J. (1982) Biochim. Biophys. Acta 683, 181-220 3 Hellingwerf,K. J., Lolkema,J. S., Otto, R., Neijssel, O. M.,Stouthamer, A. H., Harder, W., van Dam, K. and Westerhof, H. V. (1982) FEMS Micro. Lett. 15, 7-17. 4 Pirt, S. J. (1965) Pro~ Roy. Soc. Lond. (Biol.) 163, 224--231 5 Pitt, S. J. (1982) Arch. Microbiol. 133, 300-302 6 Schultz, J. S. and Gerhardt, P. (1969) Bacterw/. Rev. 33, 1-47 7 Chesbro, W., Evans, T. and Eifert, R. (1979) J. Bacteriol. 139, 625-638 8 Hansford, G. S. and Humphrey, A. E. (1966) Biotechnol. Bhang. 8, 85-96 9 van Verseveld,H., Chesbro, W., Braster, M. and Stouthamer, A. H. Arch. Microbial. in press. 10 Gallant,J. A. (1979)Ann. Rev. Genet. 13, 300-302 11 Arbige,M. and Chesbro, W. (1982)J. Cre~ Microbiol. 128, 693-703 12 Arbige,M. and Chesbro, W. (1982)Arch. Microbial. 132, 338-344 13 Stare, H., van Verseveld, H. and Stouthamer, A. H. (1983)Arch. Microbial. 135, 199-204 14 Gerhart,E., Wagner, H., Ehrenberg, M. and Kttrland, C. G. (1982) Mol. Gen. Genet. 185, 269-274 15 Braedt, G. and Gallant, J. (1977) J. Bacteriol. 129, 564-566 16 Silverman, R. H. and Atherly, A. G. (1979) Microbiol. Rev. 43, 27-53 17 Botsford,J. L. (1981) Microbiol. Rev. 45, 620-642 18 Chesbro, W. R., Eifert, R. and Evans, T. (1979) U.S. Patent 4, 167, 450 19 Demain, A. L., Kennel, Y. M. and Aharonowitz, Y. (1979) in: Microbial Technology: Current State, Future Prospects

(A. T. Bull, D. C. Ellwood, C. Ratledge, eds), pp. 163-185, CambridgeUniversity Press, Cambridge.