Contreras, R.

Oriol, E.

Raimbault, Maurice

Horizon / Pleins textes

I 
2-0- 
- 
- 
1- 
.- 
LIR. 
50- 
30- 
AI S 
ga- 
15- 
5 
Biotechnology Techniques V o l  1 No 2 79-84 (1987)  
Recel ved June 2 
USE OF MICROCALORIMETRY FOR MONITORING THE SOLID STATE 
CULTURE OF ASPERGILLUSNIGER 
Oriol, E.*, Contreras, R.l, Raimbault, M. 
ORSTOM, 213 rue Lafayette 75480 Paris-Cedex 10, FRANCE. 
Centro Nacional de Investigacion cientifica, P.O. Box 
6880, Cd. Habana, CUBA. -. 
*To whom all correspondance should be addressed: Present 
address: Departement de GCnie Biochimique et Alimentaire 
INSA, Av. de Rangueil 31077 Toulouse-Cedex, FRANCE. 
SUMMARY 
A modeì solid state culture of As~ersillus niser was 
monitored by microcalorimetry and a transient phase between 
germination and apical growth was characterized by an 
exothermic phenomenon whose Intensity could be related to 
culture conditlons. 
INTRODUCTION 
Microcalorimetry has been widely used for characterization 
and physiological studies of bacteria and yeasts (Belaich. 
1080; Lamprecht, 1980) but very few attempts have been made 
concerning growth and metaboiism of filamentous fungi. The 
critical conditions of agitation and aeration required to 
ensure mold growth have been difficult to provide in the 
microcalorimetry devices employed until now. Solid state 
culture (SSC)  conditions have been demonstrated to be 
i adequate in static reaction vessels (Cannel & Moo-Young, 
1980) and SSC might thus be a useful model to study the 
growth pattern of fungi (Contreras et al., 1986). i 
To provide a good oxygen transfer in the solid medium, a 
great interface between the liquid film and oxygen was 
necessary. This situation can be achieved by using a liquid 
synthetical medium inoculated with spores, absorbed on a 
porous matrix of low density. A lignocellulosic material, 
i.e. sugarcane bagasse has been chosen as  a suitable support 
material for the growth of Asperq i l l u s  niser. a strain with 
no cellulolytic action. 
MATERIAL AND METHODS 
I 
Nicroorqanism. Aspe rsi 1 lus nlqer var, hennebersi ( N o  10> 
described by Raimbault 8 klazard (1980) was used. I 
I 
79 
ORSKl&l Fonds Qocumentaire 
Preparation of spore Inoculum. Spore inoculum was obtained 
as d scribed by Raimbault 8 Alazard (1980) and was used at 3 
Cultlvation condltlons: Sugarcane bagasse, free of sugars, 
was obtained from a sugar factory In Zacatepec (Mesfco). The 
20-50 mesh fraction was used. 1 
Composition of the medium was: KH2P04, 5 mg; (NH4> S04,'9.8 
mg; Urea, 2.4 mg for 100 mg of glucose independant y of the 
glucose Concentration In the solution. 
15 mg of support were introduced in a 3 ml reaction vessel 
and sterilized apart from the liquid medium in an autoclave 
at 1 bar for 20 mn. The pH of the salts solution containing 
spores was adjusted to 2.7. The support was then moistened 
with 40 pl of the liquid glucose-salts medium inoculated 
with spores. A saturating oxygen flow was passed in the 
vessel for 1 mn before it was hermetically sealed. 
Microcalorimetry apparatus: A 4 channel LKB Bioactivity 
Monitor was used. Attenuation was fixed to 1000-3000 
microwatts and power-time curves (thermograms) were 
registered continuously. All experfments were duplicated and 
good reproductibility was observed. 
Petermination of water act i vi t u  Initial Water activity of 
the medium CAW) defined as the relative humidity of the 
gaseous atmosphere in equilibrium with the medlum was 
determined with a water activity detector (Humldat IC I I  
purchased from Novasinar>. Samples ( 3  to 4 g> were 
introduced in a relative humidity captor placed in a 
temperature regulation chamber at 30°C; the state of 
x 10 8 spores i reaction vessel unless otherwise speclfied. 
equilibrium was reached after 3 h. 
RESULTS 
Influence of the l i q u i d  phase slucose concentrat 
power-time curves 
Given standard inoculum conditions. four alucose 
concentrations ranging from 30 to 220 g/l-were used and the 
power-time curves (full line) are given in Fig. 1. The 
system enabled the detection of an activity as soon as 1 h 
after inoculation. Other monitoring devices such as exit gas 
analysis are not sensitive enough to give any signal during 
this period. The germination time increased with the glucose 
concentration and for the three higher glucose 
concentrations two peaks were observed while for the lower 
one, one only peak was detected. That first peak has not 
previously been reported in microcalorimetry experiments 
dealing with spore germination of Fusarium roseum CLjungholm 
et al., 1980). 
When an ideal curve (fig 1 : dashed lines) was drawn under 
the first peak, it became possible to measure the area of 
this peak. An increase in the glucose concentration of the 
liquid phase produced a decrease in the initial Aw of the 
medlum which could be correlated with the heat evolved in 
the first peak. as can be seen in Figure 2. For the three 
higher glucose concentrations, the heat corresponding t o  the 
first peak is about 5% the total heat produced during 
growth. 
80 
Figure 1: Power-tlme curves  observed during growth of 
A.niser on bagasse with different glucose concentrations in 
the absorbed 1 iquid medium. 
81 
95 .86 .97 -08 .99 
AW 
Figure 2: Heat evolved I n  the f i r s t  peak as a functlon of 
the water a c t i v i t y  CAW)  of the medium. 
Power 
[jlW) 
2x107~p.  
82 
I 
l 
A mlcroscuplc examlnatlon made at the maxlmum helght of the 
first peak showed germinated spores In an homogeneous phase 
wlth a germlnatlng tube approxlmately flve times larger than 
the spore. 
ce of the ampunt of spore Inoculum on the Power t h e  - 
!am!k3 
Given standard glucose concentration In the llquld phase (85, 
gil), three trlals were run wlth decreaslng amounts of 
spores In the reactlon vessel. As may be seen In Flg 3, the 
delayed appearance of the flrst peak as well as Its helght 
were affected by the slze of the Inoculum. Thls might 
slgnlfy that under SSC condltlons, the germlnatlon process 
occurs faster when the lnoculum 1s more concentrated, 
perhaps due to the presence of an auto-actlvator of 
germlnatlon (Sussman, 1976). 
Furthermore, the cultivation time Increased drastically when 
the inoculum was small, passing from 16 to 26 h for a 
25-fold decrease of the number of spores. 
DI SCUSS I ON 
The sensitivity of the apparatus allowed the detection of a 
signal corresponding to a transitional phase between 
germination and apical growth. Studies achieved in column 
incubators showed that this signal was not associated with 
respiration or biomass production. As the intensity of the 
peak seemed to be dependent on the water activity and thus 
the osmotical pressure of the medium and on the number of 
spores, two hypothesis might be established: 
- A large part of the spore germination i s  endogenous 
(Sussman, 1965) and the signal should thus be associated 
with a structural change such as a rearranging of the 
permease transport system at the beginning of the exogenous 
phase. 
- An accumulation of macromolecules (polyols) inside the 
germinating spore accompanied with a water expulsion for 
adapting to the osmotic conditions of the medium. 
As biomass is always difficult to determine precisely In 
SSC, the utilizatlon of the support model demonstrated that 
the mlcrocalorlmetry method is feasible for monitoring the 
growth of molds and for describing its variations to culture 
condi t ions. 
Acknowledgments 
This work was done as part of the cooperation agreement 
between the Comite Estatal de Cooperacion Economica (CECE, 
Cuba) and the Institut Francais de Recherche Scientifique 
pour le Developpement en Cooperation (ORSTOM, France) with a 
lspecific research .program agreed upon between the Centro 
Nacional de Investigacion Cientifica and ORSTOM. 
REFERENCES 
Belaich, J. P. (1980) In: Blologlcal Mlcrocalorlmetry, A .  
E. Breezer Ed., pp 1-42. London: Academic Press. 
83 
Cannel , E. and M~so-i'rsuny, M. (19HO) Proc. Blochem., 5 ,  
Contreras, O. R., Johnsrud, S. C. and Erlksson, K.E.  (1986) 
In Proceedlngs of "Blotechnology In the Pulp and Paper 
Industry" 3rd Int. Conf., Stockholm, June 16-19, 
Lamprecht, I. (1980) In: Blologlcal Mlcrocalorlmetry A. 
E.  Breezer E d . ,  pp 43-112, London: Academic Press. 
Ljungholm, K . ,  Noren, B.and Wadso, I. (1980) Olkos, 34, 
94-97. 
Raimbault, M. and Alazard, D. (1980) European J. Appl. 
Microbiol., 9, 199-209. 
Sussman, A. S. (1965) In: The Fungl Vol. JI: The Fungal 
Organism" G. S. Ainsworth and A. S. Sussman Eds., pp 
733-764, London: Academic Press. 
Function, D.J. Weber and W. M. Hess Eds., pp 101-132, 
New-York: Wiley Interscience. 
24-28. 
21 1-213. 
Sussman, A. S. (1976) In: The Fungal Spore: Form and 
84 
I 
T 
C 
P 
t 
W 
m 
i: 
I I  
Î Pl i 
I i hl 
i 
I 
S <  
Ci 
be 
YE 
de 
de 
t h  
W e  
hY 
P l  
Ba 
t h  
G e  
an 
se 


Use of microcalorimetry for monitoring the solid state culture of Aspergillus niger

Abstract

Similar works

Full text

Available Versions

Horizon / Pleins textes