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ISSN 1330–9862
original scientific paper
(FTB-1503)
Production and Recovery of Aroma Compounds
Produced
by Solid-State Fermentation Using
Different Adsorbents
Adriane B. P. Medeiros
1
, Ashok Pandey
2
, Luciana P. S. Vandenberghe
1
,
Gláucia M. Pastore
3
and Carlos R. Soccol
1
*
1
Bioprocess Engineering and Biotechnology Division, Chemical
Engineering Department, Federal
University of Paraná, CEP 81531–970, PR–Curitiba,
Brazil
2
Biotechnology Division, Regional Research Laboratory, CSIR,
695019–Trivandrum, India
3
Food
Engineering Faculty, FEA, State University of
Campinas, Unicamp, CP 6121,
CEP 13083–862, SP–Campinas, Brazil
Received: April 27, 2005
Accepted: October 24, 2005
Summary
Volatile compounds with fruity characteristics were produced by
Ceratocystis fimbriata
in two different bioreactors: columns (laboratory scale) and
horizontal drum (semi-pilot scale). Coffee husk was used as substrate for the
production of volatile compounds by so- lid-state fermentation. The
production of volatile compounds was significantly higher when horizontal
drum bioreactor was used than when column bioreactors were used. These
results showed that this model of bioreactor presents good perspectives for
scale-up and application in an industrial production. Headspace analysis of
the solid-state culture detected
twelve compounds, among them: ethanol, acetaldehyde, ethyl acetate, ethyl
pro- pionate, and isoamyl acetate. Ethyl acetate was the predominant product
in the headspace (28.55
mmol/L/g of initial dry matter). Activated carbon, Tenax-TA, and
Amberlite XAD-2
were tested to perform the recovery of the compounds. The adsorbent
columns were con- nected to the column-type bioreactor. All compounds present
in the headspace of the co- lumns were adsorbed in Amberlite XAD-2. With
Tenax-TA, acetaldehyde was adsorbed in higher concentrations. However, the
recovery found by using the activated carbon was very
low.
Agro-industrial residues have been used as
efficient
substrates in several bioprocesses such as the produc- tion of
organic acids (1), production of enzymes and bio- logical detoxification of
coffee husks (2). The application of agro-industrial residues not only
provides alternative substrates for solid-state fermentation (SSF), but
it also helps to solve pollution problems (3). Cassava bagasse, sugar cane
bagasse, apple pomace, giant palm bran, and
coffee husk have been used as substrates for aroma pro- duction in
SSF (4–7). It is estimated that around 100 aro- ma compounds are produced
industrially by microbial fermentation
(8).
Strains of the fungi Ceratocystis have been
identified
as aroma
producers. Christen et al. (9) studied the pro- duction of aroma compounds by
employing different substrates (wheat bran, cassava bagasse, and sugar
cane bagasse complemented with a synthetic medium). It was concluded that
the type of aroma
depended on two
47
A.B.P. MEDEIROS et al.: Aroma Compounds Produced
by Fermentation,
Food Technol.
Biotechnol. 44 (1) 47–51 (2006)
different sources (carbon and nitrogen). Fruity aroma was detected in
the cultures of Ceratocystis fimbriata us- ing coffee husk as substrate.
Soares et al. (5) found that the odour detected in the headspace of the
culture de- pended on the amount of added glucose. High levels of the
addition of glucose decreased aroma intensity.
Ac- cording to the authors it seems that glucose concentra- tion had a
direct influence on the metabolic pathways and thus on the nature of the
volatile compounds. Among the compounds produced ethanol and ethyl acetate
were the most abundant.
Product recovery is often a difficult step in
biopro-
cesses, especially for flavour compounds because of
their volatility and low solubility. It is also necessary to keep the
concentration of volatile compounds in the fermen- tation medium below a
certain level due to its inhibitory effect on microbial growth. There are
many on- and off- -line technologies that can make the extraction and
con- centration of flavour compounds (10).
One of the most used methods to remove organic
compounds from fermentation medium
involves solvent extraction, separation on specific membranes and
adsor- ption on activated carbon and porous hydrophobic po- lymers. The
two last ones have been used for the con- centration of aroma
compounds.
There are numerous reports on the adsorption of
fla-
vour compounds. Sorption on activated carbon and po- rous
hydrophobic polymers is a suitable method to ex- tract and concentrate
volatile compounds from aqueous medium. Solid sorbents were used
in an on-line extrac- tion of
g-decalactone during a bioconversion process (11).
The sorbents tested were: activated carbon and three po- rous
polystyrene-type polymers (Porapak Q, Chromo- sorb 105 and Resin SM4).
Sporidiobolus salmonicolor was cultivated on fermentation broth.
Adsorbents were add- ed to culture medium at 20 and 30
g/L.
g-decalactone
was extracted from the adsorbents using hexane. The presence of
adsorbents in the bioconversion medium al- lowed a very low concentration of
the lactones in liquid medium, as a consequence, it limited the toxicity of
the flavour compound to the yeast.
In
this work, the volatile compounds produced by
Ceratocystis fimbriata with coffee husks as substrate by solid-state fermentation in two
different bioreactor ty- pes, column bioreactor (laboratory scale) and
horizontal drum bioreactor (semi-pilot scale), were evaluated. Vol- atile
compounds were recovered by trapping them in different adsorbents. Activated
carbon and two porous polymers (Amberlite XAD-2 and Tenax-TA) were
tested as adsorbents. The objective of the study was to com- pare the
production of volatile compounds in different scales and to test the
efficiency of porous polymers and carbon as adsorbents to recover these
aromas.
Material and Methods
Microorganism and inoculum
Ceratocystis fimbriata (CBS 374.83) was used
during
this work. The strain was maintained on potato dextrose agar (PDA)
and stored at 4 °C in agar slants. Inoculum was prepared after 5 days of
growth at 30 °C into 250-mL Erlenmeyer flasks with 50 mL of potato dextrose
agar.
Spores were collected with sterile distilled water contai- ning a
few drops of Tween 80 and small glass beads by agitation in shaker (120 rpm,
15 min, 25 °C). The spore suspensions contained 10
8
spores/mL, which were pre-
pared by dilution with sterile distilled water and count- ed with
the Neubauer’s chamber.
Preparation of the substrate
Coffee husks were dried at 60 °C in an air oven
for
24 h. The dried substrate was milled and sieved to ob- tain
particles of 0.8–2.0 mm size. The material was steril- ized in an autoclave
at 121 °C for 15 min and enriched with glucose. The initial pH of the medium
was adjus- ted to 6.0 and moisture to 65 %. The medium was sub- sequently
inoculated using 1·10
7
spores/g initial dry
matter (IDM).
Fermentation
procedure
SSF was carried out in two different bioreactors:
col-
umns (Fig. 1) and horizontal drum (Fig. 2) connected with an air
distributor.
The glass columns (diameter 4 cm, length 20 cm)
were
filled with 20 g of coffee husks inoculated with spore suspension.
Fig. 1 shows the schematic set-up of this fermentation system. The
temperature of the water bath was maintained at 30 °C and the columns were
connec- ted with an air distributor. Initial moisture content and pH of
the substrate were 65 % and 6.0, respectively. The substrate (coffee husk)
was supplemented with 20 % mass fraction of glucose dissolved in water, which
was used to humidify the initial substrate. The aeration rate was fixed at
0.6 L/h/column. Fermentation was
carried out for 192 h, or until reducing sugars reached low
levels. Reducing sugars were measured by Somogyi and Nel- son
(12,13).
The production of volatile compounds by Ceratocy-
stis fimbriata in a stainless steel horizontal drum bioreac- tor
(Fig. 2) was also studied. An air compressor suppli- ed the air required by
the growth of fungi inside the bioreactor. Experiments were carried out using
1.5 kg of coffee husks as substrate. The same conditions as used in the
column experiments were applied.
48
A.B.P. MEDEIROS et al.: Aroma Compounds Produced
by Fermentation,
Food Technol.
Biotechnol. 44 (1) 47–51 (2006)
Fig. 1.
Schematic set-up of column bioreactor system. 1
air
pump, 2 air filter, 3 air moisturizing unit, 4 air distributor, 5
co- lumns in water bath, 6 CaCl
2
columns, 7 adsorbent columns
Volatile compounds recovery
Volatile metabolites were collected in adsorbent
col-
umns, which were connected at the outlet of the column bioreactor
during fermentation. To avoid
water interfer- ence, pre-columns with CaCl
2
were connected before the
adsorbent columns. The adsorbent columns were made of glass
(length 10 cm, internal diameter 6 mm) and were packed with 320 mg of
granular activated carbon (Ultraporous FBC, mesh size 6–8 mm). The same
packed quantity of polymeric resins Tenax-TA (60–80 Supelco) and Amberlite
XAD-2 were also tested. The columns were fitted between glass wool. The
adsorbed volatile compounds were continuously eluted three times with
a small volume of solvent (3 mL/adsorbent column). Di- chloromethane was
the solvent used for activated car- bon columns and methanol for two other
sorbents (Te- nax-TA and Amberlite XAD-2). Column apparatus
was constructed in order to desorb the volatile compounds from the
adsorption columns according to Janssens et al. (14). Thus, a concentrated
solution of compounds was obtained. A volume of 1
mL of this extract was injected
into the capillary column and analyzed by gas chroma- tography. The
conditions used for the GC are given be- low.
Analytical procedures
Aroma
compounds were determined by gas chro-
matography. A volume of 1 mL of the headspace of the culture was
injected directly into a Shimadzu gas chromatograph GC17A, equipped with a
flame ioniza- tion detector at 230 °C and HP-DB5 capillary column (30 m x
0.32 mm). The temperature program employed was set to start at 40 °C, hold
for 5 min, gradually increasing to 150 °C at 20 °C/min rate and holding at
150 °C for 5 min. The injector temperature was maintained at 230 °C under
split mode of 1:5 rate. In order to quantify all the compounds, a standard
curve of ethanol (Merck) was con- structed. Total and individual volatiles
were expressed as
mmol per liter of headspace.
Results and Discussion
Although the same conditions were applied in col-
umn and horizontal drum bioreactor experiments, the results with
the horizontal drum bioreactor reached high productivity. The total volatile
production was 6 times higher than that obtained in column bioreactors. All
the volatile compounds were present in the headspace of both types of
bioreactors (columns and horizontal drum).
Fig. 3 presents the evolution of different
parameters
in the production of volatile compounds in both col- umns (A) and
horizontal drum bioreactor (B). The re- sults show that in glass column
bioreactors the maximal production of volatile compounds per g of IDM was
23 mmol/L after 72 h of fermentation. In this
case, total pro- ductivity was found to be 0.251
mmol/(L·g·h). The hori-
zontal drum bioreactor showed a better performance, which could be
observed in the higher concentration of volatiles obtained
(144
mmol/(L·g)) after 72 h and total
productivity of 1.52
mmol/(L·g·h). These results showed
great and promising perspectives for the scale-up of the process
for aroma
production by solid-state fermenta- tion
with agro-industrial residues as fermentation
sub- strates.
A
total of twelve compounds were produced, out of
which ethyl acetate, ethanol and acetaldehyde were the major
compounds. Other compounds, including ethyl
49
A.B.P. MEDEIROS et al.: Aroma Compounds Produced
by Fermentation,
Food Technol.
Biotechnol. 44 (1) 47–51 (2006)
1
2
3
8
5
6
7
Fig. 2.
Schematic set-up of a horizontal drum bioreactor system for aroma production by SSF.
1 air pump, 2 air filter, 3 air moisturi-
zing unit, 4 bioreactor, 5 rotatory agitator, 6 motor, 7 rpm
controller, 8 gas outlet
0
50
100
150
0
48
96
144
192
t/h
0
4
8
12
16
w ( reducin
g su g ars
) /%
Total volatile
Reducing sugars
B
c ( tota
l vo
l at il e )/
mo
l/( Lg
))
( m
×
0
5
10
15
20
25
0
48
96
144
192
t/h
c ( tota
l vo
l at il e )/
mo
l/( Lg
))
( m
×
Total volatile
Reducing sugars
A
240
0
4
8
12
16
w (reducing
s ugars)/%
Fig. 3.
Evolution of aroma production and
reducing sugars
during fermentation in column
bioreactors (A) and horizontal drum bioreactor
(B)
propionate, propyl acetate, ethyl isobutyrate, butyl ace- tate,
were also produced during fermentation. Four
com- pounds remained unidentified. Table 1 shows the con- centration of
each individual compound produced by
C.
fimbriata, each of them was accumulated in the head- space at their
maximum concentration, which was ob- served on the 3rd day of fermentation. The
presence of fruity aroma produced by the
culture was attributed to the production of esters. As it is known, alcohols
do not contribute to any flavour, although together with other compounds
they affect overall flavour quality. Esters of low molecular mass are
responsible for fruity odours and consist of acids and their derived
compounds such as acetates, propionates, and butyrates. Some examples are
ethyl butyrate and isoamyl acetate, which are found in strawberry and banana
flavours (
15).
It
is possible to say that the concentration of volatile
compounds in the headspace of the culture is generally affected by
several factors, including the nature and concentration of the fermentation medium and
its vapor partial pressure. There is the possibility that the com- pounds,
which are less volatile in nature, might not be accurately
measured.
Table 2 gives the quantities of the volatile com-
pounds which were recovered by solvent elution of acti- vated
carbon, Tenax and Amberlite XAD-2 columns. The recovery of the volatile
compounds on activated carbon column was not efficient, probably due to the
hydrophi- lic nature of that support. Only the compounds at higher amounts
in the headspace such as acetaldehyde (2.36
mmol), ethanol (24.47 mmol), and ethyl acetate
(108.68
mmol) were recovered by solvent elution of the
activated carbon column. Porous resins (Tenax and Am- berlite
XAD-2) showed the best results for the recovery of volatile compounds. In the
case of Tenax, acetalde- hyde was recovered in significant amounts
(649.7
mmol).
This compound was easily adsorbed demonstrating
that
there is an affinity of the resins for acetaldehyde. Sup- posing
that the volatile concentrations in the headspace were constant during a day,
the total volatile quantity produced could be calculated multiplying the
concentra- tion by total flow. Thus, the efficiency of the recovery on
each adsorbent can be calculated. For example, ethyl acetate was recovered in
XAD-2 column with 9 % effi- ciency. Amberlite XAD-2 and Tenax demonstrate to
be more efficient in trapping volatile compounds than acti- vated carbon.
Amberlite XAD-2 could adsorb ten of the twelve compounds produced
by
Ceratocystis fimbriata us-
ing coffee husk as substrate. Adsorption on activated carbon of
aroma compounds in
wastewaters from aro- matic plants distillation was considered excellent
(
³90
%) to moderate (44–77 %) by Edris
et al. (16). The au-
thors verified that some components were more selec- tively
adsorbed. They also observed a moderate recov- ery (
»70 %) of aroma adsorbed on carbon
with diethyl
ether. This could explain why few components (ethyl acetate,
acetaldehyde and ethanol) were found in the ex- tract of the activated carbon
using dichloromethane to recover the volatile compounds. The desorption of
the volatile compounds from the adsorbent columns should be
improved.
Conclusion
The production of volatile compounds was signifi-
cantly higher in a horizontal drum bioreactor, showing good
prospects for the process scale-up. Twelve com- pounds were separated by GC
headspace analysis of the culture. The predominant compounds were ethyl
aceta- te, ethanol and acetaldehyde. Comparing different types of
adsorbents used to recover the aroma compounds, the
resin Amberlite XAD-2 adsorbed almost all com- pounds present in the
headspace of the culture when compared with Tenax and activated carbon. The
results obtained from the adsorption experiments showed the possibility of
using porous resins to recover microbial volatile compounds produced by SSF
processes.
50
A.B.P. MEDEIROS
et al.: Aroma Compounds Produced
by Fermentation,
Food Technol.
Biotechnol. 44 (1) 47–51 (2006)
Table 1. Volatile compounds present in the »headspace«
of
C.
fimbriata solid-state cultures in aerated
columns (maximum concentration)
Compound
c/(mmol/L)
Acetaldehyde
12.25
A*
8.75
B*
4.25
Ethanol
62.50
Ethyl acetate
472.50
Isopropyl acetate
0
Ethyl propionate
6.50
Ethyl isobutyrate
3.00
Isobutyl acetate
5.25
Ethyl butyrate
2.75
Isoamyl acetate
1.50
D*
0.18
*not identified
Table 2. Volatile compounds recovered from different
materials during fermentation of coffee
husk by C
eratocystis fimbriata
Compound
n(adsorbed compounds)/mmol
Activated carbon Amberlite XAD-2
Tenax
Acetaldehyde
2.36
519.09
649.70
Ethanol
24.47
107.32
69.37
Ethyl acetate
108.68
610.76
128.19
Propyl acetate
–
0.85
0.06
Ethyl isobutyrate
–
2.38
0.27
Isobutyl acetate
–
3.90
1.47
Ethyl butyrate
–
1.76
–
C*
–
0.31
–
Isoamyl acetate
–
0.06
–
D*
–
0.25
–
*not identified
Acknowledgements
Adriane B.P. Medeiros and Carlos R. Soccol thank
CAPES and CNPq, respectively, for financial
support.
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Soccol, Coffee residues as substrates for aroma production by
Ceratocystis fimbriata in solid-state fermentation, Braz.
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Vandamme,
N.M. Schamp, Production of flavours by microorganisms, Process
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Proizvodnja i izdvajanje aromatskih spojeva
dobivenih
fermentacijom ~vrstog supstrata koriste}i razne
adsorbente
Sa`etak
Hlapljivi spojevi s vo}nom aromom dobiveni su s pomo}u plijesni
Ceratosystis fimbria-
ta u dva razli~ita bioreaktora, i to u kolonskom bioreaktoru (u
laboratoriju) i horizontal- nom bubnju (poluindustrijski). Kao ~vrsti
supstrat za fermentaciju upotrijebljena je lupina kave. Proizvodnja
hlapljivih spojeva bila je kudikamo ve}a u horizontalnom bubanjskom reaktoru.
Stoga ovaj model bioreaktora ima sve preduvjete za uve}anje i primjenu u
indu- strijskoj proizvodnji. Kromatografskom analizom plinske faze iznad
fermentiranog sup- strata prona|eno je 12 hlapljivih spojeva, a me|u njima
etanol, acetaldehid, etilni acetat, etilni propionat i izoamilni acetat.
Etilni acetat bio je glavni hlapljivi proizvod u plinskoj fazi
(28,55
mmol/L/g po~etne suhe tvari). Za izdvajanje hlapljivih spojeva
ispitani su ak-
tivni ugljen, Tenax-TA i Amberlite XAD-2. Kolone za adsorpciju bile su
povezane s kolon- skim bioreaktorom. Sve hlapljive spojeve iz plinskog
prostora bioreaktora adsorbirao je Amberlite XAD-2. Tenax-TA adsorbirao je
vi{e acetaldehida, a izdvajanje hlapljivih spoje- va na aktivnom ugljenu bilo
je vrlo slabo.
51
A.B.P. MEDEIROS et al.: Aroma Compounds Produced
by Fermentation,
Food Technol.
Biotechnol. 44 (1) 47–51 (2006)
