XIVth International Society for Biological Calorimetry Conference, 2006

Microcalorimetric Monitoring
of Microbial Growth in Solid-
State Fermentations

Menert, A., Kazarjan, A., Stulova, I., Lee,
C.C., Vilu, R.
Tallinn University of Technology

Why calorimetry?
Calorimetry is an extremely appropriate method for
studying microbiological processes.

Thermal power-time curves are influenced by the
metabolic activity and can be related to the
different physiological states of bacteria (Kemp and
(
Lamprecht, 2000).

From microcalorimetric data the thermodynamic
(∆H) as well as kinetic (µ=dX/(X·dt)) parameters of
a process can be calculated.

XIVth International Society for Biological Calorimetry
Conference June 2 - 6, 2006 Sopot, Poland

Ice calorimeter of Lavoisier-Laplace

The quantities of heat that are
produced or absorbed are proportional
to the extent of the processes
involved.


Isothermal
microcalorimeter
2277 TAM


General advantages of calorimetry

low specificity
good reproducibility
non-destructive analysis
continuous registration of processes
possibility to analyze turbid or coloured samples
high throughput of samples


Multichannel calorimeters

TAM III System


Reproducibility of data on TAM III
Lab Assistant Results Report
Ampoule (5-25-06)-lactic acid bacteria.rslt

Summary
Name: Ampoule (5-25-06)-lactic acid bacteria.rslt
Start time: May 25, 2006 21:10:59
End time: Jun 01, 2006 12:44:15
Operator: AM
Results file path: C:Documents and SettingsAnneMy DocumentsTAM III
experimentsAmpoule (5-25-06)-lactic acid bacteria.rslt

General Experiment Info 150
Bath temperature: 25 °C

Sample - Ch 3:1
Sample - Ch 3:2
Name: Lactic acid bacteria in MRS
100

Hea t flo w (µ W)
µmax1= 0.2658 h-1
µmax2= 0.2580 h-1 50

Qtot1= 29,408 J
Qtot2= 29,514 J 0

May 27 May 29 Jun 01


Introduction
Solid-phase fermentations are of great
interest in:
Cheese ripening
Spoiling of meat products
etc.

Solid matrix forces the cells to grow in
colonies, not free flowing

Solid-phase fermentations have been
less studied


Structure of agar

β-(1-3)-D and α-(1-4)-L bonded galactose

Scheme of agar gelatination

Repeating unit in agar structure


Bacterial growth curve

1 – Lag- phase
2 – Exponential phase
3 – Declining growth phase
4 – Stationary phase
5 – Lysis phase

Usually more attention is paid to phases 1-3, as biomass growth takes place there
which is essential in biotechnological industry. The most important is phase 2
(exponential growth) where productivity is the greatest.


Bacterial growth curve

Lag- phase
Exponential phase
Stationary phase

Van Impe et al., 2005

Bacterial growth in colonies
Every colony starts to grow from one
single cell: the radius of colony Rcol
increases in time by addition of new
cells

Lag-phase – acclimatization phase
Rcol
Exponential phase – the radius of
colony increases
Stationary phase – the increase of
the radius of colony has stopped


Parameters used to describe the growth of
colonies (Malakar et al, 2002)

Rboundery –
boundery of living
space
Rcol
Rcol
Rcol – radius of
growing colony
Rboundery

dX dX dRcol
= µX =c
dt dt dt
Malakar, P. K., Martens, D.E., Van Breukelen, W., Boom, R. M., Zwietering, M. H., Van ,t Riet, K. Appl. Environ.
Microbiol., 2002, 3432-3441


Every colony starts to grow from one single cell

Morphology of colonies is dependant
on many factors; the simpliest
geometrical shapes are sphere and
ellpisoid


Determination of bacterial growth in solid state
dependant on the concentration of glucose and agar

104 cfu/flask

Bacteria studied

Lactobacillus paracasei S1R1 – lactic acid
bacterium isolated from estonian type cheese,
belonging to NSLAB (non-starter lactic acid
bacteria)
Lactococcus lactis – typical lactic acid bacterium


The parameters determined
Outgrowth percentage (%)
how many of the cells inoculated will form the colonies
duration of lag-phase (t – hours)
Growth rate (µ – h-1)
The dependance of these parameters on glucose (2-50 g/L) and agar
concentration (1, 3, 5%) was measured

The growth of individual colonies was monitored in the
experiments
The deep inoculation was made with low inoculation rate
The aim was to achieve long distances between
the colonies, to guarantee the independant
growth of individual colonies
Constant incubation temperature 31oC was kept

Rakkude koguse kasv
3.50E+10
17 h
3.00E+10

2.50E+10
Change of bacterial
number in time
kogus 1 cm3

2.00E+10

1.50E+10

1.00E+10

5.00E+09

0.00E+00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
17 h
aeg OD muutus ajas
1.7
1.6
1.5
1.4
Change of bacterial 1.3

OD in time 1.2
OD (540 nm)

1.1

1
0.9
0.8
0.7

XIVth International Society for Biological Calorimetry 0.6
0.5
Conference June 2 - 6, 2006 Sopot, Poland 0.4

0.3
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Tunnid

The concentration of colonies in volumetric unit of
sample depending on agar and glucose concentrations
1800

1600

1400

1200 1 % agar
col/mL

1000
3% agar
800
5 % agar
600

400

200

0
0 10 20 30 40 50 60
glucose g/L


Outgrowth percentage of lactic acid bacteria at various
glucose and agar concentrations
Glucose, g/ L 2 5 10 15 20 30 40 50
Agar, %
1 2,60 3,40 3,12 2,72 6,29 0,05 0,05 0,03
3 1,03 2,63 2,45 1,77 4,15 0,04 0,05 0,03
5 0,71 0,53 0,41 0,47 0,55 0,04 0,04 0,02

Glucose, g/ L 2 5 10 15 20 22 24 26 28 30 40 50
Agar, %
3 0,10 0,17 0,58 1,46 1,44 1,44 1,66 1,60 1,50 0,04 0,05 0,03

1,80

1,60

Outgrowth percentage, % 1,40

1,20

1,00

0,80

0,60

0,40

0,20

0,00
0 5 10 15 20 25 30 35 40 45 50 55
Glucose, g/L

Dependance of lag-phase length on glucose concentration
Lag- faas i pik k us e rine vate l glük oos i k onts . 3% agar
(in 3% agar)
100

90

80

70
Lag- faasi pikkus, t

60

50

40

30

20

10

0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54
glük oos , g/l


Determination of growth rate of Lactobacillus paracasei S1R1 in solid-
state fermentations at various glucose concentrations (3% agar)

0,04

0,03
-1
Growth rate, h

0,02

0,01

0,00
0 5 10 15 20 25 30 35 40 45 50 55

Concentration of glucose, g/L


Outgrowth percentage, duration of lag phase
dependant on glucose and agar concentration

It was shown that the outgrowth percentage of bacteria studied
increases with the increase of glucose concentration 2-15g/L, the
outgrowth percentage is maximal and practically the same at
limiting substrate concentrations 15-30g/L, but it is dependant on
the concentration of agar. The maximum outgrowth percentage
was measured in 1% agar at 20g/L glucose -– 6%. At Gucose
concentrations > 30g/l the outgrowth percentage decreased
dramatically. At the same glucose concentrations the outgrowth
percentage decreased with increasing the agar concentration.
Almost in the same manner behaved the duration of lag-phase. The
measured minimum lag-phase duration was 20 hours.


Preparation of inocula with different concentration of
bacteria


Thermal Activity Monitor TAM 2277

Combination measuring unit

Growth curves at high colony number and low colony
number are different
180

160 Lb paracasei 7 kol
Lb. paracasei 7 kol
L. lactis 6 kol
140
L. lactis 40 kol
L. lactis 45 kol
120 Lb. paracasei 40 kol

100
dQ/dt

80

60

40

20

0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
XIVth International Society for Biological Calorimetry Time, h

Growth at large number of colonies can be
approximated to the growth in liquid culture
180

160 L. lactis 40 col
L. lactis 45 col
Lb. paracasei 40 col
140 Lb. paracasei 45 col

120

100
dQ/dt

80

60

40

20

0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
Time, h

Growth in large number of colonies can be
approximated to the growth in liquid culture
180

160 L. lactis 40 col
L. lactis 45 col
Lb. paracasei 40 col
140 Lb. paracasei 45 col

120

150
100
dQ/dt

80 100

Hea t flo w (µW)
60
50

40

0
20
May 27 May 29 Jun 01

0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
Time, h

Biomass growth rate is proportional to the heat
production rate (in exponential phase)

Specific growth rate of X
Ansorbance
microorganisms µ Cellcount
Biomass ln X
dX/dt = µX µ=(lnXt-lnX0)/t
µ=1/X*dX/dt
Xt=X0*eµt lnXt =lnX0 + µt Time
q
dQ/ Qs1 my1
Q µ
µW/mL µJ/mL 1/h
dt 2.5e+06 0.50
150
6 5

5 ln dQ/dt = 0.648 + 0.272 t
µmax = 0.272 h-1 3
120 2e+06 0.40 4
3
1
90 1.5e+06 0.30 2

ln dQ/dt
1

ln Q
0 5 10 15 20
-1
0
60 1e+06 0.20
0 5 10 15 20
-1
ln Q = - 3.382 + 0.254 t -3
-2 µmax = 0.254 h-1
30 500000 0.10
-3 -5
hours -4
0 0 0
0 4 8 12 16 20 -5 -7

time Time / h

Region for calculation of maximum
specific growth rate

ln (dQ/dt) = ln (dQ/dt)t=0 + µt

Calculation of specific growth rate µ
• In exponential growth phase dX/dt = µX (1)
• If the stoichiometry of biomass growth does not change during the growth

(dX/dt) is proportional to dQ/dt and
(X-X0) is proportional to Q.
• The rate of biomass increase is proportional to the rate of increase in the heat
production (where YQ is the proportionality factor):
dX/dt = YQ * dQ/dt (2)
• From definition of specific growth rate (Eq. 1) and replacing it into Eq. 2 we get the
relationship between µ and dQ/dt:
µX = YQ * dQ/dt (3)
• The increase of biomass in the exponential growth phase is an exponential
function: X = X0 * eµt (4)
• Replacing X from Eq. (4) into Eq. (3) µ * X0 * eµt = YQ * dQ/dt (5)
dQ/dt = 1/YQ * µ * X0 * eµt (6)

• After integrating :ln (dQ/dt) = ln (dQ/dt)t=0 + µt (7)
where ln (dQ/dt)t=0 = ln (1/YQ * µ * X0 * eµ).

Specific growth rates and heat production of Lb. paracasei
and L. lactis in agarose
Experiment 101005
-1
Channel Inoculum µmax, h Time, h dQ/dt max, µW/ml τ1, d Q1, J τ2, d Q2, J Q1+Q2, J Qtot, J Colonies
1
sm1 Lb.paracasei S1R1 10 0,3128 36,15 213,59 1,5063 10,581 2,2967 19,809 30,390 31,981 40
1
2
2

Experiment 171005
Channel Inoculum µmax, h-1 Time, h dQ/dt max, µW/ml τ1, d Q1, J τ2, d Q2, J Q1+Q2, J Qtot, J Colonies
0
sm1 Lb.paracasei S1R1 10 0,1501 52,52 96,13 2,1882 10,800 39,330 2
0
1
1

Experiment 091105
-1
Channel Inoculum µmax, h Time, h dQ/dt max, µW/ml τ1, d Q1, J τ2, d Q2, J Q1+Q2, J Qtot, J Colonies
1
sm1 L. lactis S1R1 10 0,1943 52,58 96,07 2,1910 12,402 41,741 6
sm2 L. lactis S1R1 102 0,4243 38,27 165,76 1,5944 12,902 2,6637 23,709 36,611 40,178 45
1
2

Experiment 151105
Channel Inoculum µmax, h-1 Time, h dQ/dt max, µW/ml τ1, d Q1, J τ2, d Q2, J Q1+Q2, J Qtot, J Colonies
1
2
sm2 Lb.paracasei S1R1 10 - 190,07 8,08 7,9194 - 0
1
sm3 L. lactis S1R1 10 0,3015 37,70 170,86 1,5708 11,672 2,5363 22,085 33,757 43,237 50
2
sm4 L. lactis S1R1 10 0,3961 39,50 150,76 1,6458 8,261 2,5534 19,591 27,851 31,623 40

The difference in growth curve is
dependent on

growth limitation by diffusion
screening effect – in large colonies bacteria
grow only on the outer layer
growth limitation by the production of lactic acid


At high colony number (40 - 50)
Growth curves in liquid medium were
similar to those with higher number of
colonies (~40) in solid state. Initially,
after the lag-phase the bacteria grow
with maximum growth rate µmax= 0.30-
0.40 h-1. After that limitation of
substrate (glucose) starts retarding the
growth. The growth is finally hindered
by the production of lactic acid.

The limiting factor at large number of
colonies or in liquid culture is
production of lactic acid


Cultivation of Lb.casei at high inoculation rate (103 – 107)
8 10 300
8 10 200
B
7
9 A 100
7
9 200

100
8
8

-1
6

dQ/dt, µW mL
-1

-1
log cfu mL

6 0 0

log cfu mL
-1
dQ/dt, µW mL
7
7
pH

pH
-100
5 -100
5 6
6

-200
5 5
4 -200 4
-1 -1
dQ/dt, µW mL dQ/dt, µW mL -300
-1 -1
log cfu mL log cfu mL
4 pH 4 pH
3 -300 3 -400
0 10 20 30 40 50 0 10 20 30 40 50
Tim e, h
Time, h
8 10 300

9
C
7 200

Bacterial count (♦), thermal power (--) and pH ( ) for
8
100 inoculum size
dQ/dt, µW mL-1
6
log cfu mL-1

7 A - 103 cfu mL-1 (MRS + agarose)
pH

0 B - 105 cfu mL-1 (MRS + agarose)
5 6
C - 107 cfu mL-1 (MRS + agarose)
-100
5
4
-1
dQ/dt, µW -1
mL
-200 log cfu mL
4 pH
3
Conference June 2 10 6, 2006 Sopot, Poland
0 - 20 30 40 50
Time, h

At low colony number (< 10)
Maximum specific growth rate µmax is
lower (0.15-0.30 h-1)
Provided that inhibition of growth is limited
by diffusion the growth is retarded mainly
due to substrate (glucose) defficiency
If the number of colonies is small (~7), the
measured growth rate µ< µmax as the
growth rate is determined by the diffusion
rate as well as (possibly) by the screening
effect of outer layer cells. The growth is
limited by diffusion of glucose until the
end, when it stops as a result of ending
the glucose supply and/or inhibition by
formation of lactic acid


Colony growth limitation by diffusion


Heat production during lag-phase and exponential phase is
independant of the number of colonies in the ampoule;
Q1 = const Q1=9-12 J / per ampoule
P,µW Pin[1](t) Pin[2](t) Pin[3](t) Pin[4](t) P,µW Pin[1](t) Pin[2](t) Pin[3](t) Pin[4](t)

10.581 J 9.0436 J

150

45 colonies 7 colonies
400

100

200
30.390 J
Q3 50

Q1 Q2
Q1 Q2 31.981 J 31.875 J

0 0

0 20 40 60 Time,hour 0 2 4 Time,day

Qtot= Q1 + Q2 + Q3

Total heat production in ampoule is constant, Qtot= const
Qtot=30-40 J / per ampole

Conclusions (outgrowth percentage)
The outgrowth percentage dependance on glucose
concntration has “three phases” –
2-15 g/L: with increasing the glucose concentration
the outgrowth percentage increases;
15-28 g/L: the region with with maximum outgrowth
percentage
30-50 g/L: high glucose concentrations inhibit
outgrowth
With increasing the concentration of agar the outgrowth
percentage decreases.


Conclusions (microcalorimetric data)

At high number of colonies (40-50) in a closed volume bacterial
growth in solid state is similar to the growth in liquid medium. The
growth is mainly limited by the production of lactic acid.

At low number of colonies (<10) the growth rate is dependant on the
number of colonies in the closed volume. The growth is mainly
limited by hindered diffusion of glucose.

Heat production during lag-phase and exponential phase is
independant of the number of colonies in the ampoule.

Total heat production in a closed volume (ampoule) is constant and
independant of the number of colonies.


Special thanks...
Vallo Kõrgmaa
Romi Mankin
Glafira Shkaperina
Natalja Kabanova
Signe Adamberg


XIVth International Society for Biological Calorimetry Conference, 2006

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