TA-918CA (1s2024) Aula 4

Prévia do material em texto

Prof. Andreas K. Gombert
TA918C - Microbiologia 
e Fermentações
Aula 4 - 04/04/2024 
A CURVA DE CRESCIMENTO MICROBIANO
374 MONOD
phases may often be suppressed (see p. 388). The retardation
phase is frequently so short as to be imperceptible. The same is
sometimes true of the stationary phase. Conversely, more complex
growth cycles are not infrequently observed (see p. 389).
i
6
TIME
FzG. 1.--Phases o[ growth. Lower curve: log bacterial density. Upper curve:
variations of growth rate. Vertical dotted lines mark the limits of phases. Figures
refer to phases as defined in text (see p. 373).
GROWTH CONSTANTS
The growth of a bacterial culture can be largely, if not com-
pletely, characterized by three fundamental growth constants
which we shall define as follows:
Total growth: 2 difference between initial (xo) and maximum
(x~) bacterial density:
G -- Xm~-
Exponential growth rate: growth rate during the exponential
phase (R). It is given by the expression
log2x2- log2x2
R=
t2 -- tx
s "Croissanee totale," Monod 1941.
www.annualreviews.org/aronline
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Cul$vos 
em BIORREATORES
Cul$vos 
 em FRASCOS
Cinética de crescimento, consumo de 
substrato e formação de produto
Etanol: metabólito primário ou
produto catabólicoAntibiótico: metabólito secundário
Curva de crescimento típica
1
do livro Brock Biology of Microorganisms
Mas como são construídos 
esses gráficos?
🤔
Como medir 
concentração celular?
Meio líquido 
X
Meio sólido
© 2004 Pearson Education, Inc.
CAPÍTULO 5
NUTRIÇÃO, CULTIVO LABORATORIAL 
E METABOLISMO 
DOS MICRORGANISMOS
PARTE I 
 PRINCÍPIOS DE MICROBIOLOGIA
fissão binária 
(bactérias)[
uma duplicação ou uma geração
© 2004 Pearson Education, Inc.
Brotamento (leveduras)
uma duplicação ou uma geração
Fungos filamentosos ou 
bolores
zoom
Bolores em cultivo 
submerso
Crescimento de bolores 
 (fungos filamentosos): 
 
- alongamento e ramificação de hifas 
- esporulação 
- germinação de esporos 
http://www.youtube.com/watch?v=GQIi4KAN1QM
http://www.youtube.com/watch?v=sbaWbiFt_Go&feature=related
http://youtu.be/IRiwXMeKoGk
CÉLULAS ANIMAIS
Chinese Hamster Ovary (CHO)
cells
http://leelab.org/research/biomolecular
http://www.gembio.com/research-area-interest/
insect-cell-culture-products
de mamíferos
de insetos
Como medir 
concentração celular?
©!2004!Pearson!Education,!
Inc.
Contagem!direta!de!células
unidade: no. de células/volume
resposta rápida
funciona para seres unicelulares
não funciona para fungos filamentosos
impreciso para suspensões muito diluídas
permite uso de corantes para viáveis/não-viáveis
requer operadores muito bem treinados
©!2004!Pearson!Education,!
Inc.
Contagem!de!
células!viáveis!(I)
unidade: UFC*/volume
resposta demorada
trabalhoso
funciona para seres unicelulares
não funciona para fungos filamentosos
Vantagens:
- extremamente sensível
- mede somente células viáveis e capazes de formar colônias
*Unidades Formadoras de Colônia
Filtração!a!vácuo!
(massa!seca!de!céls.)
… ou centrifugação
vácuo
Medida de massa seca de células
unidade: massa seca de células/volume
resposta em alguns minutos
funciona para seres unicelulares
funciona para fungos filamentosos
não distingue células viáveis de não-viáveis
exige uma quantidade mínima de amostra e
não funciona para suspensões muito diluídas
Todos os nutrientes devem estar em solução
Extremamente reprodutível (erro < 2%)
©!2004!Pearson!Education,!
Inc.
Fissão!binária!em!procariotos
Para bolores, faz-se raciocínio análogo, com base na medida
da massa seca de células da população!
Duplicação do número de células da população
Métodos automáticos
medida rápida e automática
unidade: no. de partículas/volume
somente para unicelulares
desvantagem: alto preço
Microscopia
 “automatizada"Contador 
Coulter
Citômetro de
Fluxo
©!2004!Pearson!Education,!
Inc.
Medida!da!turbidez!
(absorbância)
Medida do Espalhamento de Luz
também conhecido como:
- Turbidimetria
- Medida da Densidade Óptica
(método indireto!)
Pode ser feita num 
espectrofotômetro simples!
C
al
ib
ra
çã
o:
!T
ur
bi
di
m
et
ria
!
(m
ét
od
o!
in
di
re
to
)!
X
!
C
on
ta
ge
m
!(
m
ét
od
o!
di
re
to
)
todos esses métodos 
são offline… :-(
voltando à curva de 
crescimento…
26
A value of k = 0.24 h-1 would describe most of the data, although it would slightly under-
estimate the initial growth rate. Another approach would be to take the log of the above
equation to give:
and to fit the data to this equation and estimate k from the intercept. In this case k would be
about 0.25 h-1.
b) The yields are estimated directly from the data as:
The above estimate of YX/S is only approximate, as maintenance effects and endogenous
metabolism have been neglected.
Y
g
g
Y
g
g
/
/
P S
X S
P
S
P
S
X
S
X
S
= - = - -
-
=
= - = - -
-
=
D
D
D
D
( )
( )
.
( . . )
( )
.
49 0
2 100
0 5
10 7 0 5
2 100
0 104
log log log
1
1 0
X
dX
dt
k X
X
= + -
Ê
ËÁ
ˆ
¯̃•
Dt (h)
–
X(g/L) k (h-1)
2 0.75 0.333 0.931 0.36
3 1.55 0.236 0.856 0.28
5 3.45 0.156 0.681 0.23
5 6.25 0.093 0.416 0.22
5 8.65 0.044 0.200 0.22
5 10.00 0.016 0.074 0.22
5 10.55 0.0057 0.023 0.25
1 -
Ê
ËÁ
ˆ
¯̃•
X
X
1 1
X
X tD D/ ( )h-
Figure 6.12. Logistic growth curve.
182 How Cells Grow Chap. 6
ch06 10/11/01 5:19 PM Page 182
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1	
2	
3	
4	
5	
6	
0.00	 2.00	 4.00	 6.00	 8.00	 10.00	 12.00	 14.00	
DO	1	
Cultivo da levedura Saccharomyces cerevisiae
Curva de crescimento típica
1
do livro "Brock Biology of Microorganisms”
374 MONOD
phases may often be suppressed (see p. 388). The retardation
phase is frequently so short as to be imperceptible. The same is
sometimes true of the stationary phase. Conversely, more complex
growth cycles are not infrequently observed (see p. 389).
i
6
TIME
FzG. 1.--Phases o[ growth. Lower curve: log bacterial density. Upper curve:
variations of growth rate. Vertical dotted lines mark the limits of phases. Figures
refer to phases as defined in text (see p. 373).
GROWTH CONSTANTS
The growth of a bacterial culture can be largely, if not com-
pletely, characterized by three fundamental growth constants
which we shall define as follows:
Total growth: 2 difference between initial (xo) and maximum
(x~) bacterial density:
G -- Xm~-
Exponential growth rate: growth rate during the exponential
phase (R). It is given by the expression
log2x2- log2x2
R=
t2 -- tx
s "Croissanee totale," Monod 1941.
www.annualreviews.org/aronline
Annual Reviews
A
nn
u.
 R
ev
. M
ic
ro
bi
ol
. 1
94
9.
3:
37
1-
39
4.
 D
ow
nl
oa
de
d 
fr
om
 a
rjo
ur
na
ls
.a
nn
ua
lre
vi
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s.o
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by
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or
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GROWTH OF BACTERIAL CULTURES 373
Although the two variables are not equivalent, it is convenient
to express growth rates in the same units (i.e., number of doublings
per hour) in both cases. When cell concentrations have been
estimated, it is equivalent to the true division rate. When bacterial
density is considered, it expresses the number of doublings of
bacterial density per unit time, or the division rate of cells postu-
lated to be of constant average size. In all that follows, unless
specified, we shall consider growth and growth rates in terms of
bacterial density.
These definitions involve the implicit assumption that in a
growing culture all the bacteria are viable, i.e., capable of division
or at least that only an insignificant fraction of the cells are not
capable of giving rise to a clone. This appears to be a fairly good
assumption, provided homogeneous populations only are con-
~idered. It has been challenged however [Wilson (4)] on the basisof comparisons of total and viable counts. But the cultures ex-
amined by Wilson were probably not homogeneous (see p. 378),
and the value of the viable count in determining the "absolute"
number of cells which should be considered viable under the
conditions of the culture is necessarily doubtful (see p. 378).
Direct observations by Kelly & Rahn (5) contradict these findings
and justify the assumption. [See also Lemon (42) and Topley 
Wilson (43).]
GROWTH PHASES
In the growth of a bacterial culture, a succession of phases,
characterized by variations of the growth rate, may be conveni-
ently distinguished. This is a classical conception, but the different
phases have not always been defined in the same way. The follow-
ing definitions illustrated in Fig. 1 will be adopted here:
1. lag phase: growth rate null;
2. acceleration phase: growth rate increases;
3. exponential phase: growth rate constant;
4. retardation phase: growth rate decreases;
5. stationary phase: growth rate null;
6. phase of decline: growth rate negative.
This is a generalized and rather composite picture of the growth
of a bacterial culture. Actually, any one or several of these phases
may be absent. Under suitable conditions, the lag and acceleration
www.annualreviews.org/aronline
Annual Reviews
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THE GROWTH OF BACTERIAL CULTURES
BY JACQUES MONOD
Pasteur Institute, Paris, France
INTRODUCTION
The study of the growth of bacterial cultures does not consti-
tute a specialized subject or branch of research: it is the basic
method of Microbiology. It would be a foolish enterprise, and
doomed to failure, to attempt reviewing briefly a "subject"
which covers actually our whole discipline. Unless, of course, we
considered the formal laws of growth for their own sake, an ap-
proach which has repeatedly proved sterile. In the present review
we shall consider bacterial growth as a method for the study of
bacterial physiology and biochemistry. More precisely, we shall
concern ourselves with the quantitative aspects of the method,
with the interpretation of quantitative data referring to bacterial
growth. Furthermore, we shall consider z exclusively the positive
phases of growth, since the study of bacterial "death," i.e., of the
negative phases of growth, involves distinct problems and meth-
ods. The discussion will be limited to populations considered
genetically homogeneous. The problems of mutation and selection
in growing cultures have been excellently dealt with in recent
review articles by Delbriick (1) and Luria (2).
No attempt is made at reviewing the literature on a subject
which, as we have just seen, is not really a subject at all. The
papers and results quoted have been selected as illustrations of the
points discussed.
DEFINITION OF GROWTH PHASES
AND GROWTH CONSTANTS
Division RATE AND GROWTH RATE
In all that follows, we shall define "cell concentration" as
the number of individual cells per unit volume of a culture and
"bacterial density" as the dry weight of cells per unit volume of a
culture.
Consider a unit volume of a growing culture containing at time
t, a certain number x, of cells. After a certain time has elapsed,
371
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Annual Reviews
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Cinética do crescimento 
limitado por nutrientes
�
µµµµ 8/µµµµ =1>
EF
GF H/EF
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H)%#')#G$,.%$%&'()*%'9#")-)'##(6#$9(+#)
I.1J&#:$K+%+6
5+0#"$7()L:$FMDNNobel Prize
 1965
Velocidade específica de crescimento ao longo do tempo
3 parâmetros principais:
- tempo de duração da fase lag
- tempo de duplicação na fase log 
(ou velocidade específica de 
crescimento)
- concentração celular final
2
C
ur
va
 d
e 
cr
es
ci
m
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to
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pi
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, c
om
 
se
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ra
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o 
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ai
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o 
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fa
se
s
Reta no gráfico
com escala 
Logarítmica = 
Fase exponencial
de crescimento 
Fase de adaptação (lag): metabolismo celular em adaptação a 
uma nova condição (eventualmente pode não existir!) [em que 
casos não existirá?] 
Fase exponencial do crescimento (log): tempo de duplicação 
constante e mínimo da população, velocidade específica de 
crescimento máxima, crescimento ilimitado. [o que quer dizer 
isto?]
Fase de redução de velocidade: entre o final do crescimento 
exponencial e a fase estacionária, inicia-se por alguma 
limitação nutricional e/ou por alguma inibição. 
Fase estacionária: concentração celular constante (depende do 
método de medida). 
Fase de declínio ou morte: diminuição da concentração celular 
(depende do método e pode não ser observada).
Fases que eu considero importantes:
32The lag phase occurs immediately after inoculation and is a period of adaptation of
cells to a new environment. Microorganisms reorganize their molecular constituents when
they are transferred to a new medium. Depending on the composition of nutrients, new
enzymes are synthesized, the synthesis of some other enzymes is repressed, and the inter-
nal machinery of cells is adapted to the new environmental conditions. These changes re-
flect the intracellular mechanisms for the regulation of the metabolic processes discussed
in Chapter 4. During this phase, cell mass may increase a little, without an increase in cell
number density. When the inoculum is small and has a low fraction of cells that are vi-
able, there may be a pseudolag phase, which is a result, not of adaptation, but of small in-
oculum size or poor condition of the inoculum. 
Low concentration of some nutrients and growth factors may also cause a long lag
phase. For example, the lag phase of Enterobacter aerogenes (formerly Aerobacter aero-
genes) grown in glucose and phosphate buffer medium increases as the concentration of
Mg2+, which is an activator of the enzyme phosphatase, is decreased. As another example,
even heterotrophic cells require CO2 fixation (to supplement intermediates removed from
key energy-producing metabolic cycles during rapid biosynthesis), and excessive sparging
can remove metabolically generated CO2 too rapidly for cellular restructuring to be ac-
complished efficiently, particularly with a small inoculum. 
Figure 6.3. Typical growth curve for a bacterial population. Note that the phase of
growth (shown here for cell number) depends on the parameter used to monitor growth.
Sec. 6.2 Batch Growth 161
ch06 10/11/01 5:19 PM Page 161
©!2004!Pearson!Education,!
Inc.
Crescimento!exponencial