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Extraction of benzaldehyde from fermentation broth by pervaporation
Process Biochemistry, Vol. 31, No. 6, pp. 533-542, 1996
Copyright© 1996Elsevier ScienceLtd Printed in Great Britain. All rights reserved 0032-9592/96$15.00+ 0.00
FLSEVIER
0032-9592(95)00098-4
Extraction of Benzaldehyde from Fermentation Broth by Pervaporation T. Lamer, a H. E. S p i n n l e r , b I. S o u c h o n a & A. ~,oilley a "Laboratoire de G6nie des Proc6d6s Alimentaires et Biotechnologiques, ENS.BANA, 1 esplanade Erasme, C.U.M., 21000 Dijon, France ~'Laboratoire de recherche sur les ar6mes, INRA, rue Sully, 21000 Dijon, France (Received 24 May 1995; revised manuscript received 27 October 1995 and accepted 19 November 1995)
The application of pervaporation to extract benzaldehyde produced by microorganisms is considered. A model system was used to study the influence of different parameters and attempts to understand better the mass transfer of this flavour compound. Benzaldehyde was removed from a culture medium from which it was produced by Bjerkandera adusta. The performance of these processes is compared.
INTRODUCTION
ethanol. During the past 10 years, several new applications of pervaporation have been proposed in biotechnology and the food industry.3-5 Pervaporation consists of a partial vaporization of the liquid solution through a dense membrane. In this membrane process, solutes are dissolved in the polymer material at the upstream face of the membrane and then diffuse into the polymeric network and are finally evaporated in the downstream gas phase. This evaporation occurs because of a continuous pumping (vacuum pervaporation) or a continuous sweeping gas which brings a low partial pressure in the downstream compartment. Due to the selectivity of the polymeric material, the substances pass through the membrane according to their affinity for the membrane. Therefore, water and hydrophilic compound fluxes are greater in hydrophilic membranes (polyvinylalcohol, cellulose acetate) whereas hydrophobic fluxes are greater in hydrophobic membranes (polydimethylsiloxane, polyetherblockamide). The pervaporation process allows both extraction and concentration and is very
Several works concerning the production of flavours by microorganisms have shown that the production rate decreases sharply when the product concentration exceeds a toxicity threshold beyond which growth and/or production are inhibited. 1"2 In such cases, a continuous extraction of the product is necessary to improve the productivity of the fermentation process. However, continuous extractions are not easy to perform using classical techniques like distillation because, quite often, operational conditions are not compatible with cell viability. Other systems such as vacuum fermentation are not selective and are very costly with dilute broth. Other means, such as in situ extraction with oils may be quite selective, but the concentration factor is low.2 Membrane processes like pervaporation seem to have a lot of advantages since continuous recovery under non-stressful conditions is possible. Pervaporation is a recent process used to separate liquid mixtures; its main application being to dehydrate organic solvents such as 533
534
T. Lamer et al.
attractive for biotechnological separations especially when product concentrations are very low. A first attempt to couple pervaporation and a fermentation was made by Groot et al. in 19846 to extract butanol. More recently, other compounds have been studied but these were essentially alcohols. The first references on the extraction of aroma compounds by pervaporation are seven years old. 4'7 Dettwiller et al. 8 attempted to remove butanediol and acetoin from a Bacillus subtilis fermentation. Poor fluxes and selectivities were obtained because of the hydrophilic nature of these organic compounds. The present work deals with the extraction of benzaldehyde. This compound is involved in bitter almond and sherry aroma and was produced by a filamentous fungus, Bjerkandera adusta. It is a basidiomycete known for its ability to produce benzaldehyde from phenylalanine.9 The extraction of this substance is necessary since inhibition of production growth is observed at concentrations over 300 ppm. 2 Moreover, the last reaction of the metabolic pathway is an equilibrium between benzyl alcohol and benzaldehyde. Selective extraction would continuously shift the equilibrium to the production of the benzaldehyde and as a consequence may improve the bioconversion yield.2 The extraction of benzaldehyde and benzyl alcohol in a simplified model medium was investigated in order to understand the mass transfer of this compound by pervaporation and the coupling between the fermentation and the extraction by pervaporation.
Table 1.
MATERIALS AND METHODS Properties of the aroma compounds The principle physico-chemical properties of benzaldehyde and benzyl alcohol given by the suppliers are presented in Table 1. Some other properties of these compounds have been determined as:
• saturated vapour pressures have been estimated by the Lee-Kesler's correlation, 1° • water solubility at 25°C of both aroma compounds was determined by the mutual solubility technique, 1~ • diffusion coefficients in water have been measured at 25°C with a porous diaphragm cell (Stoke's ceU). 12 Membranes Two kinds of membrane were used to extract the organic compounds from the aqueous solutions.
• A composite membrane from GFT-Le Carbone Lorraine (Paris, France). This composite membrane consists of an active top layer of 10/~m made of polydimethylsiloxane (PDMS) on a support of polyacrylonitrile and non-woven polyester. The total thickness of the membrane is 200 #m. • An homogeneous PDMS membrane prepared with a preparation kit traded by Dow Coming Corp. (Midland, USA). Two reagents (9 g of silicone and 1 g of curing agent) were mixed and dissolved in chloroform. The gas was removed into a vacuum
Physico-chemicalproperties of benzaldehyde and benzyl alcohol
Compound
Benzaldehyde
Benzyl alcohol
Supplier Purity (%) Formula
Merck-Schuchardt, BRD 98 (~O40
E. Merck Darmstadt, BRD > 99 ( ~ Oqt,Ofl
Molecular weight (g mol l) Density (g m1-1) 20°C Boiling temperature (°C) Saturated vapour pressure (mbar) Odour
106'13 1.042-1-045 179.5 1"33 (26°C) Bitter almond
108.14 1-045-1.046 205.2 0-09 (25°C) Fruity
Extraction of benzaldehyde by pervaporation
535
pounds in the pervaporate to that in the feed).
flask for a few minutes. The mixture was laid out on a glass support then reticulation was conducted at 100°C for a period of 90 min. Different membrane thickness were produced in the range 50-130/~m. The silicone density of the Dow Coming membrane was measured and found to be equal to 1140 kg m -3.
These pervaporation data were obtained using two kinds of experimental apparatus. The first set-up was used to study pervaporation of benzaldehyde and benzyl alcohol in a model aqueous solution whereas the second one was used when pervaporation was coupled with fermentation (Fig. 1). The materials used were glass, stainless steel and PTFE to prevent sorption of aroma on the wall of the system. For both experimental apparatus, the same pervaporation cell was used. The characteristics of this cell are presented in Fig. 2. The membrane with an effective area of 50 x 10 -4 m 2 was lying on a porous stainless steel support. A laminar flow rate was maintained at the upstream side of the membrane. The flow rate of the liquid was 1.2 litre min-1 corresponding to a Reynolds number (Re) of 600. All the surfaces in contact with the liquid were smooth. The permeate was removed from the downstream side of the membrane by a continuous pumping and collected in either of the parallel cold traps at -196°C. In order to study the influence of the downstream pressure on mass transfer, pervaporation experiments were performed with different downstream pressures in the range 35-700 Pa controlled by a valve at the inlet of the pump, and measured by a pressure gauge (BP101, Alcatel, France).
Sorption measurements Sorption measurements allow solubility and diffusion coefficients to be obtained. The former coefficient is deduced from the equilibrium by plotting the sorption isotherm, whereas the latter is obtained from the non-steady-state of sorption, kinetics. The experimental procedure to determine the solubility and diffusion coefficients of aroma compounds in polydimethylsiloxane (PDMS) was described in a previous study by Lamer et al. 13
Pervaporation experiments Each pervaporation experiment allowed the determination of two data: • the flux of component (defined as the mass of water and/or organic compound pervaporating through a membrane per square meter and per hour), • the selectivity fl, or enrichment factor, (defined as the ratio mass fraction of corn-
9 "4
8
,1
l0
V"
Fig. 1. System for continuous extraction of benzaldehyde by pervaporation: 1, feed tank; 2, stirrer; 3, nylon sifter; 4, air injection; 5, PTFE pump; 6, buffer; 7, flow meter; 8, pervaporation cell; 9, glass valves; 10, cold traps; 11, pressure gauge; 12, dessicant; 13, vacuum pump.
536
T. Lamer et al.
The main differences between the two experimental systems were the nature of the medium in the feed tank. With the model solution the apparatus used was simple; elements 3-6 of the apparatus were not present. However, in the case of the in situ extraction of a fermentation broth, a preliminary filtration of the medium was necessary because of the destruction of the mycelium in the pump and of the possible growth of the fungus in the pervaporation cell. This filtration was performed using a nylon sifter. Regular injections of compressed sterile air into the upstream line (1 s every 30 s) was applied to prevent the sifter from plugging. High liquid flow rates (up to 60 litres h -~) in the pervaporation cell could be maintained for a period of several hours. In order to maintain a constant flow throughout the pervaporation cell, a second pump and a 2 litre buffer tank were added. During pervaporation experiments,
...............
L.,, !~
.....
,~
the volume of culture medium immobilized in the buffer tank and in the tubes was about 500 ml. The details of the pervaporation experiments and the determination of fluxes are given in a previous paper, a4 In the case of model system experiments, it was verified that a constant flux of aroma compound was reached. This steady state was obtained 30 min of pervaporation. Strain and growth conditions Ej~r/~nder~ ad~st~ (Wild ex Fr.) Karst CBS 595.79 was maintained on Potato Dextrose Agar (PDA) slants at 4°C. The composition of the media was as follows: L,u-lecithin from soya bean (Sigma, St Louis, MO) (10 g), phenylalanine (2 g), yeast extract (0"5 g), K H 2 P O 4 (0"2 g), M g S O 4
(0"2 g), C u S O 4
(5 mg), distilled water (1 litre). The medium was adjusted to pH 5-5 with (1 N) H2504, dis-
265 100 ,.._l ~...... ~ .........o~ . . ~
!~ t ~;~ ~ .!~i ~ ~
r
~
1
A1
(2)~
r
r
~
r
(1)
B2
B1
(6)
Fig. 2. Pervaporation cell. A1 and A2: view from the inside (A1) and the side (A2) of the upper part of the cell, B1 and B2: view from the inside (B1) and the side (B2) of the bottom part of the cell. (1) Inlet of the liquid, (2) outlet of the liquid, (3) PTFE joint, (4) membrane lain on the support, (5) stainless steel microporous support, (6) outlet of the pervaporate.
Extraction of benzaldehyde by pervaporation
pensed in 500 ml Erlenmeyer flasks, sterilized for 20min at 121°C and then inoculated to obtain a final concentration of 105 spores m l Flasks were shaken (200 rev min -1, 3.6cm diameter) at a growth temperature of 25°C. Glass fermenters (7 litre): (Applikon, Schiedam, NL) were filled with 5 litres of medium and sterilized at 121°C for 40 min. The condenser at the air outlet was cooled with glycolated water at - 4 ° C to prevent stripping of volatiles. The medium was inoculated with three 5-day-old flask cultures (corresponding to 1.5 g of dry biomass). Extraction and measurements of substrates and aroma
Each culture was filtered through cheesecloth. The mycelium was retained and dried in an oven (70°C) to constant weight and the growth rate calculated. Phenylalanine and products were measured using HPLC. The apparatus included two pumps, a gradient former and a UV detector (Gilson, Villiers Le Bel, F). A C18 #Bondapak column (250 × 4.6 mm) (Waters, Milford, MA) was also used. Solvents were water (A) and methanol (B). The solvent gradient was 45% B to 40% B in 10 min and then isocratic for 15 min. In both cases the flow rate was 0.6 ml min -1. The compounds were detected at 254 nm wavelength. Before injection into the HPLC system, samples were diluted twice in acetonitrile to dissolve lecithin, and then centrifuged at 600g for 15 min. Twenty microlitre samples of the supernatant were injected. The pervaporates and feed compositions (for model assays) were measured using a 427A gas-liquid chromatograph with a column, 3 m length x 3 mm diameter, packed with Chromosorb W-AW 100-200 mesh and 10% Carbowax
537
20 M and N2 as carrier gas (flow rate 20 ml min-1). An integrator (HP 3396A) performed the acquisition and treatment of the data given by the flame ionization detector (FID). Temperatures for analysis were: FID (200°C); injector (200°C); oven (1500C). A calibration curve was made for both chromatographic methods and used for quantification. No differences between the quantifications with HPLC and GC were recorded. Mass spectrometry: mass spectra were obtained with a Nermag R10-10 coupled with a Girdel 31 GC. Column and GC conditions were as described previously.15 Electron impact was recorded with an ion source energy of 70 eV. The scanning rate was 0.8s from 25 to 300 a.m.u.
RESULTS AND DISCUSSION Model system To understand better the behaviour of benzaldehyde and benzyl alcohol during pervaporation, it was necessary to examine the physico-chemical characteristics of both compounds. A summary of the physical characterised properties is given in Table 2, with the comparison of the fluxes and selectivities for both aroma compounds through the composite membrane (GFT). The solubility of benzyl alcohol in water is thus 5.5 times higher than that of benzaldehyde. The solubilities in water can be related to hydrophobicity constants (log P) calculated by the Rekker method. 16 Diffusion coefficients in water of both compounds are almost the same. The solubility coefficient of benzaldehyde in PDMS is more than three times higher than
Table 2. Physico-chemical characteristics at 25°C of benzaldehyde and benzyl alcohol (in water and in PDMS) and comparison of mass transfer of both aroma compounds (concentration in water: 100 ppm, composite membrane GFT, Reynolds number: 600, downstream pressure: 60 Pa)
Solubility in water (g litre-1) Diffusion coefficient in water x 1011 (m 2 s-1) Hydrophobicity constant (log P) Solubility coefficient in PDMS Diffusion coefficient in PDMS x 1011 (m 2 s-1) Flux of pervaporation (g h - 1 m-E) Selectivity fl
Benzaldehyde
Benzyl alcohol
6"95 103 1-48 7"8 2"6 1-04 272
38"14 100 0"92 2"4 0-02 2"5
538
T. Lamer et al.
that of benzyl alcohol. This coefficient is the partition coefficient of the aroma compound between water and the membrane and quantifies the affinity of the compounds for the membrane. Lamer (1993) 17 found that for linear molecules with eight atoms and close hydrophobicities, the sequence for sorption is: alcohol < aldehyde, which is the same sequence found here for benzyl alcohol and benzaldehyde. The diffusion and solubility coefficients are closely related. 18'19 The more the compounds sorb into the membrane, the weaker is the diffusion coefficient. The benzaldehyde flux was 50 times higher than for benzyl alcohol for the same concentration on the upstream side of the membrane. The sequence in fluxes is identical to that found for the sorption of the aroma compounds to the polymer. Lamer et al. (1994) 13 showed that a strong relationship exists between the observed fluxes in the steady state of transfer of pervaporation and the sorption characteristics in the membrane. A selectivity equal to 272 means that for a 100 ppm benzaldehyde solution, the benzaldehyde concentration in the cold trap is 27200 ppm. In fact, the water solubility of benzaldehyde is equal to 5500 ppm and two phases were obtained in the trap: a liquid phase saturated with the aldehyde and an organic phase corresponding to the benzaldehyde. This phenomenon was obviously observed with higher selectivities and during the whole study. Such a separation did not occur with benzyl alcohol, a selectivity equal to only 2 being obtained.
Influence of the liquid concentration During fermentation, the concentration of aroma compounds in the culture medium is not constant. For this reason the effect of benzaldehyde and benzyl alcohol concentration in the liquid has been studied at 25°C with the composite membrane (GFT). Figure 3 shows that when the concentration was increased from 50 to 300 ppm, the flux of benzaldehyde increased from 0.5 to 3 g m -2 h -~, whereas the flux of benzyl alcohol increased from 0.008 to 0-05 g m - 2 h - . 1 These increases result from the increase in the driving force. Linear developments of the fluxes with the concentration are thus observed. The fluxes for benzaldehyde are 50 times higher than for benzyl alcohol, for the same concentration at the upstream side of the membrane.
3.0 2.5
y
2.0 ~ . ~ . _ . ~1.5 ' 1.0' 0.5"
0.C0
B~,~
__
~
"~ , Concentration(mg/litra) Fig. 3. Changein flux of benzaldehydeandbenzy|alcohol with concentration in solution (T=25°C, membrane GFF, Re=600, downstream pressure---60 Pa).
In the range of concentrations studied, the selectivities of both aroma compounds were constant. As in the case of the fluxes observed, the selectivity values for benzyl alcohol is lower than that of benzaldehyde: the selectivities being 250 for benzaldehyde and 2.5 for benzyl alcohol. This is consistent with earlier reports that aldehydes have a higher enrichment factor than alcohols and a lower one than esters. 5' 13 As a consequence of these differences, it is possible to extract preferentially benzaldehyde from benzyl alcohol. The flux variations of the benzyl alcohol with the operational parameters were negligible, therefore, the following discussions focuses mostly on the behaviour of benzaldehyde. In all cases, the flux of water remained constant and equal to the flux observed with pure water whatever the flux of aroma compounds. This observation confirmed the absence of plasticization of the membrane by very dilute solutions of organic compounds. That was also mentioned by Nguyen and Nobe 2° for highly diluted solutions of chloroform, dichloromethane and bromoethane.
Influence of the membrane Several silicone based membranes were tested at 25°C and a benzaldehyde concentration equal to 100 ppm (Table 3). The higher flux was obtained with thin membranes, but in the case of the composite membrane, for which the dense layer thickness was very low (about 10 pm), flux was not at its highest. This may be due to the influence of the microporous support limiting the evaporation
Extraction of benzaldehyde by pervaporation Table 3. Pervaporation fluxes and selectivities of benzal-
dehyde at T=250C (model system, concentration in water: 100 ppm, Reynolds number: 600, downstream pressure: 60
Pa)
70
~
539
-
....
Thickness of Flux Selectivity PDMS (g m -2 h - t ) fl (Itm)
zs ~%
60 ',,
\
/
/
2.0~ W -r
Q.
',\ m
1.5 .~
\, m.
10a
Composite membrane GFT Homogeneous membrane
1-04
30 60 130
1.75 1.48 0.91
272 477 495 558
step. Homogeneous membranes presented high fluxes and selectivities when the thickness was small (30 and 70/~m). However, the 30#m thick membrane had very poor mechanical properties and was not selected for this work. Selectivities of homogeneous membranes were higher than those of composite membranes due to their thickness which also reduced the water flux.
Change in downstreampressure When the saturated vapour pressure of the component was low, the downstream pressure may influence the mass transfer. The effect of pressure was studied between 35 and 700 Pa with a benzaldehyde concentration equal to 100 ppm (Fig. 4). In this range, flux decreased with higher pressures. Other authors have observed a similar change in flux with downstream pressure. 21 Studies on other aroma compounds revealed that below saturated vapour pressure, the aroma flux was constant at the maximum
1.sli
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~o
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eoo
20
1.0 "i ®
o.s
PHE
• ! /
5O
100
150
0.0 200
T i m e (h)
Fig. 5. Change in the concentrations of benzaldehyde and phenylalanine during fermentation (T=25°C).
"Thickness of the active top layer.
0.9
3.0
~o
looo
Fig. 4. Change in benzaldehyde flux with downstream pressure (T=25°C, homogeneous PDMS membrane 130 pro, concentration=0.1 g litre -1, Re=600).
value. ]4 This was not observed here. Measurements of flux with lower pressure may be useful to show this phenomenon.
Fermentation/pervaporation coupling experiments Two extractions were performed in this study and they differ from each other by the use of different membranes for the pervaporation. For the first trial, the production of benzaldehyde reached a concentration of about 63.4mg litre -~ after 4 days (Fig. 5). In contrast, the phenylalanine concentration (precursor of benzaldehyde) decreased sharply since it was consumed by Bjerkandera adusta. Pervaporation was performed using GFT composite membranes. Four periods of coupling have been made at 1, 2, 4 and 5 days during brief periods of a few hours. The flux increased when benzaldehyde concentration in the culture broth increased, as observed in the model systems (water + aroma)• However, the fluxes were lower than those previously measured, for example with 63.4mg aldehyde litre-1, the flux was about two times less than in the model medium (0"36 g m-2 h-1 instead of 0.70g m -2 h - l ) . This decrease resulted from the presence of the microorganism and from a lower flow rate. To improve fluxes and selectivities, a second trial of coupling was performed using a homogeneous PDMS membrane with a thickness of 100/~m (Table 4). The ratio of the concentration of benzaldehyde and benzyl alcohol in the fermenter was inverted in the pervaporate, benzaldehyde was concentrated by pervaporation by a factor of 24-89. When considering the coupling of pervapora-
T. Lamer et al.
540
Table 4. Change in concentration of benzaldehyde and benzylalcohol in fermenter and in pervaporate during the coupling of fermentation with pervaporation (T=25°C, homogeneous membrane PDMS, thickness=100gm, downstream pressure=200 Pa) Time (h)
Concentration in fermenter (mg litre - 1)
Benzaldehyde 102 132 139 214 228
78 69 80 112 132
Concentration in pervaporate (mg litre - 1)
Benzyl alcohol 120 737 790 490 170
3700 6200 4800 5300 3200
tion with fermentation, it is clear that pervaporation permits a selective extraction of benzaldehyde from the medium by a favourable shift of the equilibrium benzaldehyde~benzyl alcohol. The pervaporate was analyzed by GC/MS to determine its composition and to identify the different volatile compounds (Table 5). This pervaporate was rich in benzaldehyde (about 68%) and it is clear that the pervaporation was appropriate to remove this aldehyde selectively from the mixture. The quantity of the benzyl alcohol was low in spite of its concentration in the culture medium which was equal to several hundred mg litre-1. As the concentration of the hydrophobic solutes in the pervaporate was much higher than that in the broth, it was possible to identify metabolites in the pervaporate which were not detected in the broth. Their structures including
Table 5. Compounds in the pervaporate (mass spectrometry analysis) Compounds
Proportion (%)
Benzaldehyde
68.5
Benzyl alcohol
4.5
~,-Decalactone 1-Phenyl ethanone 3-Chloro-p-anisaldehyde Ethyl benzoate Phenyl acetaldehyde Styrene Phenyl acetonitrile Toluene Cinnamaldehyde Benzyl formate
4.4 1.5 1.4 1.0 0.9 0.9 0-6 0.9 0.3 0.3
Hydrocarbon (unknown) Anisaldehyde
Benzaldehyde
9.6 5"0
Selectivity of benzaldehyde
Benzyl alcohol 900 3100 1900 2800 1100
47 89 60 47 24
a benzene ring suggest a relationship between these compounds and the catabolic pathway from phenylalanine to benzaldehyde (Fig. 6). Most of the phenylalanine catabolites are hydrophobic which may be the reason why some of the compounds which were not detected in the culture broth were detected in the pervaporate. This is the case especially for 1-phenyl ethanone, phenyl acetaldehyde, styrene, phenyl acetonitrile and cinnamaldehyde. It has to be emphasized that the pervaporation process may be a means of showing the different intermediary metabolites of the bioconversion. Some other compounds detected in the pervaporate including 3-chloro-p-anisaldehyde~22' 15 and y-decalactone 9 are normal products of Bjerkandera adusta.
CONCLUSIONS Two kinds of silicone membranes can be selected to perform the extraction in culture medium since fluxes were similar. However, the homogeneous membrane is more selective. There is a large influence of downstream pressure on the flux of benzaldehyde, and so for further experiments, this parameter needs to be at a minimum. Previous reports have shown the influence of fluid velocity on pervaporation of aroma compounds 17 and so, flow rate must be maximal. This may be the main problem to solve. Because of the presence of biomass, a preliminary separation step is necessary to maintain biomass activity and to prevent membrane fouling. Some practical problems need to be solved before an industrial application may be considered but it is clear that pervaporation is a.downstream process which is well adapted
541
Extraction of benzaldehyde by pervaporation
to the extraction of flavour compounds. These results showed selectivity which brought about a concentration factor of 100 for benzaldehyde,
O~
OH
o~
H2N~
the highest published for such a diluted broth. Furthermore, good flux could be obtained with a weak vacuum, which is quite inexpensive to
OH
H2N
Phenylalanine OH Tyrosine
\ c~
V
O
Anisaldehyde t
r° Styrene(1)
Cinnamaldehyde(1) s
"'~,
0
o\ CH3 3-Chloro Anisaldehyde
l-Phenvl~ethanone(1)
Phenylacetaldehyde(1) /I
\ i
~'
\
\
",~ N
II BenzylAlcohol
Benzaldehyde O
Phenyl~
Rrile (l)
OH
BenzylFormate(1) Ethyl Benzoate(l) Benzoic Acid(1) Fig. 6. Hypothetical pathway of benzaldehyde production by Bjerkandera adusta. (1) Compounds not detected in the culture broth but detected in the pervaporate.
542
T. Lamer et al.
produce. At the moment the limiting step of the process is the productivity of the fungus used. 10.
ACKNOWLEDGEMENTS
11.
We thank G. Mauvais for technical assistance and E. S~mon for GC-MS analysis. 12.
REFERENCES 1. Spinnler, H. E., Dufoss6, L., Souchon, I., Latrasse, A., Piffaut, C., Voilley, A. & Delest, P., Production of gamma-decalactone by bioconversion. Patent number FR-93.06673 (1994). 2. Spinnler, H. E., Roche, N. & Mauvais, G., Production of benzaldehyde concentrates by Bjerkandera adusta. (1995) (submitted). 3. Escudier, J. L. & Le Bouar, M., Applications and evaluation of pervaporation for the production of low alcohol wines. In Proceedings of the Third International Conference on Pervaporation Processes in the Chemical Industry, Bakish Materials Corp., Englewood, NJ, 1988, pp. 387-97. 4. VoiUey, A., Schmidt, B., Simatos, D. & Baudron, S., Extraction of aroma compounds by the pervaporation technique, In Proceedings of the Third International Conference on Pervaporation Processes in the Chemical Industry, Bakish Materials Corp., Englewood, NJ, 1988, pp. 429-38. 5. Bengtsson, E., Tr~gardh, G. & HallstrSm, B., Concentration of apple juice aroma from evaporator condensate using pervaporation. Lebensm. Wiss. Technol., 25 (1992) 29-34. 6. Groot, W. J., Van den Oever, C. E. & Kossen, N. W. F., Increase of substrate conversion by pervaporation in the continuous butanol fermentation. Biotechnol. Lea., 11 (1984) 709-14. 7. Bengtsson, E., Tfiigardh, G. & Hallstr6m, B., Recovery and concentration of apple juice aroma compounds by pervaporation. J. Food Eng., 10 (1989) 65-71. 8. Dettwiler, B., Dunn, I. J. & Prenosil, J. E., Bioproduction of acetoin and butanediol: product recovery by pervaporation. In Proceedings of the Fifth International Conference on Pervaporation Processes in the Chemical Industry, Bakish Materials Corp., Englewood, NJ, 1991, pp. 308-18. 9. Berger, R. G., Neuhauser, K. & Drawert, F., Characterization of the odor principles of some
13.
14.
15.
16. 17.
18.
19. 20. 21. 22.
basidiomycetes: Bjerkandera adusta, Poria aurea. Tyromyces sambuceus. Flavour Fragrance J., (1986) 1, 181-5. Reid, R. C., Prausnitz, J. M. & Poling, B. E., The Properties of Gasses and Liquids, 4th edn., McGrawHill, New York, 1987. Le Thanh, M., Lamer, T., Voilley, A. & Jose, J., D6termination des coefficients de partage vapeurliquide de compos6s d'ar6me h partir de leurs caract6ristiques physico-chimiques. J. Chim. Phys., 90 (1993) 545-60. Cussler, E. L., Diffusion, Mass Transfer in Fluid Systems. Cambridge University Press, Cambridge, 1984. Lamer, T., Rohart, M. S., Voilley, A. & Baussart, H., Influence of sorption and diffusion of aroma compounds in silicone rubber on their extraction by pervaporation. J. Membr. Sci., 90 (1994) 251-63. Lamer, T. & Voilley, A., Influence of different parameters on the pervaporation of aroma compounds. In Proceedings of the Fifth International Conference on Pervaporation Processes in the Chemical Industry, Bakish Materials Corp., Englewood, NJ, 1991, pp. 110-22. Spinnler, H. E., De Jong, E., Mauvais, G., Semon, E. & Le Quere, J. L., Production of halogenated compounds by Bjerkandera adusta. Appl. Microbiol. Biotechnol., 42 (1994) 212-21. Rekker, R. F., The Hydrophobic Fragmental Constant, eds W. Th. Nauta & R. F. Rekker. Elsevier, Amsterdam, 1977. Lamer, T., Extraction de compos6s d'ar6me par pervaporation: Relation entre les propri~t6s physico-chimiques des substances d'ar6me et leurs transferts h travers des membranes ~ base de polydim6thylsiloxane. PhD Thesis, Dijon, Universit6 de Bourgogne, 1993. Heintz, A., Funke, H. & Lichtenthaler, R. N., Sorption and diffusion in pervaporation membranes. In Pervaporation Membrane Separation Processes, ed. R. Y. M. Huang. Elsevier, Amsterdam, 1991, pp. 279-319. N6el, J., Introduction to pervaporation. In Pervaporation Membrane Separation Processes, ed. R. Y. M. Huang. Elsevier, Amsterdam, 1991, pp. 1-109. Nguyen, Q. T. & Nobe, K., Extraction of organic contaminants in aqueous solutions by pervaporation. J. Membr. Sci., 30 (1987) 11-22. B6ddeker, K. W., Bengston, G. & Bode, E., Pervaporation of low volatility aromatics from water. J. Membr. Sci., 53 (1990) 143-58. De Jong, E., Field, J. A., Spinnler, H. E., Wijnberg, J. B. P. A. & de Bont, J. A. M., Significant biogenesis of chlorinated aromatics by fungi in natural environments. Appl. Environm. Microbiol., 60 (1994) 264-70.
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FLSEVIER
0032-9592(95)00098-4
Extraction of Benzaldehyde from Fermentation Broth by Pervaporation T. Lamer, a H. E. S p i n n l e r , b I. S o u c h o n a & A. ~,oilley a "Laboratoire de G6nie des Proc6d6s Alimentaires et Biotechnologiques, ENS.BANA, 1 esplanade Erasme, C.U.M., 21000 Dijon, France ~'Laboratoire de recherche sur les ar6mes, INRA, rue Sully, 21000 Dijon, France (Received 24 May 1995; revised manuscript received 27 October 1995 and accepted 19 November 1995)
The application of pervaporation to extract benzaldehyde produced by microorganisms is considered. A model system was used to study the influence of different parameters and attempts to understand better the mass transfer of this flavour compound. Benzaldehyde was removed from a culture medium from which it was produced by Bjerkandera adusta. The performance of these processes is compared.
INTRODUCTION
ethanol. During the past 10 years, several new applications of pervaporation have been proposed in biotechnology and the food industry.3-5 Pervaporation consists of a partial vaporization of the liquid solution through a dense membrane. In this membrane process, solutes are dissolved in the polymer material at the upstream face of the membrane and then diffuse into the polymeric network and are finally evaporated in the downstream gas phase. This evaporation occurs because of a continuous pumping (vacuum pervaporation) or a continuous sweeping gas which brings a low partial pressure in the downstream compartment. Due to the selectivity of the polymeric material, the substances pass through the membrane according to their affinity for the membrane. Therefore, water and hydrophilic compound fluxes are greater in hydrophilic membranes (polyvinylalcohol, cellulose acetate) whereas hydrophobic fluxes are greater in hydrophobic membranes (polydimethylsiloxane, polyetherblockamide). The pervaporation process allows both extraction and concentration and is very
Several works concerning the production of flavours by microorganisms have shown that the production rate decreases sharply when the product concentration exceeds a toxicity threshold beyond which growth and/or production are inhibited. 1"2 In such cases, a continuous extraction of the product is necessary to improve the productivity of the fermentation process. However, continuous extractions are not easy to perform using classical techniques like distillation because, quite often, operational conditions are not compatible with cell viability. Other systems such as vacuum fermentation are not selective and are very costly with dilute broth. Other means, such as in situ extraction with oils may be quite selective, but the concentration factor is low.2 Membrane processes like pervaporation seem to have a lot of advantages since continuous recovery under non-stressful conditions is possible. Pervaporation is a recent process used to separate liquid mixtures; its main application being to dehydrate organic solvents such as 533
534
T. Lamer et al.
attractive for biotechnological separations especially when product concentrations are very low. A first attempt to couple pervaporation and a fermentation was made by Groot et al. in 19846 to extract butanol. More recently, other compounds have been studied but these were essentially alcohols. The first references on the extraction of aroma compounds by pervaporation are seven years old. 4'7 Dettwiller et al. 8 attempted to remove butanediol and acetoin from a Bacillus subtilis fermentation. Poor fluxes and selectivities were obtained because of the hydrophilic nature of these organic compounds. The present work deals with the extraction of benzaldehyde. This compound is involved in bitter almond and sherry aroma and was produced by a filamentous fungus, Bjerkandera adusta. It is a basidiomycete known for its ability to produce benzaldehyde from phenylalanine.9 The extraction of this substance is necessary since inhibition of production growth is observed at concentrations over 300 ppm. 2 Moreover, the last reaction of the metabolic pathway is an equilibrium between benzyl alcohol and benzaldehyde. Selective extraction would continuously shift the equilibrium to the production of the benzaldehyde and as a consequence may improve the bioconversion yield.2 The extraction of benzaldehyde and benzyl alcohol in a simplified model medium was investigated in order to understand the mass transfer of this compound by pervaporation and the coupling between the fermentation and the extraction by pervaporation.
Table 1.
MATERIALS AND METHODS Properties of the aroma compounds The principle physico-chemical properties of benzaldehyde and benzyl alcohol given by the suppliers are presented in Table 1. Some other properties of these compounds have been determined as:
• saturated vapour pressures have been estimated by the Lee-Kesler's correlation, 1° • water solubility at 25°C of both aroma compounds was determined by the mutual solubility technique, 1~ • diffusion coefficients in water have been measured at 25°C with a porous diaphragm cell (Stoke's ceU). 12 Membranes Two kinds of membrane were used to extract the organic compounds from the aqueous solutions.
• A composite membrane from GFT-Le Carbone Lorraine (Paris, France). This composite membrane consists of an active top layer of 10/~m made of polydimethylsiloxane (PDMS) on a support of polyacrylonitrile and non-woven polyester. The total thickness of the membrane is 200 #m. • An homogeneous PDMS membrane prepared with a preparation kit traded by Dow Coming Corp. (Midland, USA). Two reagents (9 g of silicone and 1 g of curing agent) were mixed and dissolved in chloroform. The gas was removed into a vacuum
Physico-chemicalproperties of benzaldehyde and benzyl alcohol
Compound
Benzaldehyde
Benzyl alcohol
Supplier Purity (%) Formula
Merck-Schuchardt, BRD 98 (~O40
E. Merck Darmstadt, BRD > 99 ( ~ Oqt,Ofl
Molecular weight (g mol l) Density (g m1-1) 20°C Boiling temperature (°C) Saturated vapour pressure (mbar) Odour
106'13 1.042-1-045 179.5 1"33 (26°C) Bitter almond
108.14 1-045-1.046 205.2 0-09 (25°C) Fruity
Extraction of benzaldehyde by pervaporation
535
pounds in the pervaporate to that in the feed).
flask for a few minutes. The mixture was laid out on a glass support then reticulation was conducted at 100°C for a period of 90 min. Different membrane thickness were produced in the range 50-130/~m. The silicone density of the Dow Coming membrane was measured and found to be equal to 1140 kg m -3.
These pervaporation data were obtained using two kinds of experimental apparatus. The first set-up was used to study pervaporation of benzaldehyde and benzyl alcohol in a model aqueous solution whereas the second one was used when pervaporation was coupled with fermentation (Fig. 1). The materials used were glass, stainless steel and PTFE to prevent sorption of aroma on the wall of the system. For both experimental apparatus, the same pervaporation cell was used. The characteristics of this cell are presented in Fig. 2. The membrane with an effective area of 50 x 10 -4 m 2 was lying on a porous stainless steel support. A laminar flow rate was maintained at the upstream side of the membrane. The flow rate of the liquid was 1.2 litre min-1 corresponding to a Reynolds number (Re) of 600. All the surfaces in contact with the liquid were smooth. The permeate was removed from the downstream side of the membrane by a continuous pumping and collected in either of the parallel cold traps at -196°C. In order to study the influence of the downstream pressure on mass transfer, pervaporation experiments were performed with different downstream pressures in the range 35-700 Pa controlled by a valve at the inlet of the pump, and measured by a pressure gauge (BP101, Alcatel, France).
Sorption measurements Sorption measurements allow solubility and diffusion coefficients to be obtained. The former coefficient is deduced from the equilibrium by plotting the sorption isotherm, whereas the latter is obtained from the non-steady-state of sorption, kinetics. The experimental procedure to determine the solubility and diffusion coefficients of aroma compounds in polydimethylsiloxane (PDMS) was described in a previous study by Lamer et al. 13
Pervaporation experiments Each pervaporation experiment allowed the determination of two data: • the flux of component (defined as the mass of water and/or organic compound pervaporating through a membrane per square meter and per hour), • the selectivity fl, or enrichment factor, (defined as the ratio mass fraction of corn-
9 "4
8
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Fig. 1. System for continuous extraction of benzaldehyde by pervaporation: 1, feed tank; 2, stirrer; 3, nylon sifter; 4, air injection; 5, PTFE pump; 6, buffer; 7, flow meter; 8, pervaporation cell; 9, glass valves; 10, cold traps; 11, pressure gauge; 12, dessicant; 13, vacuum pump.
536
T. Lamer et al.
The main differences between the two experimental systems were the nature of the medium in the feed tank. With the model solution the apparatus used was simple; elements 3-6 of the apparatus were not present. However, in the case of the in situ extraction of a fermentation broth, a preliminary filtration of the medium was necessary because of the destruction of the mycelium in the pump and of the possible growth of the fungus in the pervaporation cell. This filtration was performed using a nylon sifter. Regular injections of compressed sterile air into the upstream line (1 s every 30 s) was applied to prevent the sifter from plugging. High liquid flow rates (up to 60 litres h -~) in the pervaporation cell could be maintained for a period of several hours. In order to maintain a constant flow throughout the pervaporation cell, a second pump and a 2 litre buffer tank were added. During pervaporation experiments,
...............
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the volume of culture medium immobilized in the buffer tank and in the tubes was about 500 ml. The details of the pervaporation experiments and the determination of fluxes are given in a previous paper, a4 In the case of model system experiments, it was verified that a constant flux of aroma compound was reached. This steady state was obtained 30 min of pervaporation. Strain and growth conditions Ej~r/~nder~ ad~st~ (Wild ex Fr.) Karst CBS 595.79 was maintained on Potato Dextrose Agar (PDA) slants at 4°C. The composition of the media was as follows: L,u-lecithin from soya bean (Sigma, St Louis, MO) (10 g), phenylalanine (2 g), yeast extract (0"5 g), K H 2 P O 4 (0"2 g), M g S O 4
(0"2 g), C u S O 4
(5 mg), distilled water (1 litre). The medium was adjusted to pH 5-5 with (1 N) H2504, dis-
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Fig. 2. Pervaporation cell. A1 and A2: view from the inside (A1) and the side (A2) of the upper part of the cell, B1 and B2: view from the inside (B1) and the side (B2) of the bottom part of the cell. (1) Inlet of the liquid, (2) outlet of the liquid, (3) PTFE joint, (4) membrane lain on the support, (5) stainless steel microporous support, (6) outlet of the pervaporate.
Extraction of benzaldehyde by pervaporation
pensed in 500 ml Erlenmeyer flasks, sterilized for 20min at 121°C and then inoculated to obtain a final concentration of 105 spores m l Flasks were shaken (200 rev min -1, 3.6cm diameter) at a growth temperature of 25°C. Glass fermenters (7 litre): (Applikon, Schiedam, NL) were filled with 5 litres of medium and sterilized at 121°C for 40 min. The condenser at the air outlet was cooled with glycolated water at - 4 ° C to prevent stripping of volatiles. The medium was inoculated with three 5-day-old flask cultures (corresponding to 1.5 g of dry biomass). Extraction and measurements of substrates and aroma
Each culture was filtered through cheesecloth. The mycelium was retained and dried in an oven (70°C) to constant weight and the growth rate calculated. Phenylalanine and products were measured using HPLC. The apparatus included two pumps, a gradient former and a UV detector (Gilson, Villiers Le Bel, F). A C18 #Bondapak column (250 × 4.6 mm) (Waters, Milford, MA) was also used. Solvents were water (A) and methanol (B). The solvent gradient was 45% B to 40% B in 10 min and then isocratic for 15 min. In both cases the flow rate was 0.6 ml min -1. The compounds were detected at 254 nm wavelength. Before injection into the HPLC system, samples were diluted twice in acetonitrile to dissolve lecithin, and then centrifuged at 600g for 15 min. Twenty microlitre samples of the supernatant were injected. The pervaporates and feed compositions (for model assays) were measured using a 427A gas-liquid chromatograph with a column, 3 m length x 3 mm diameter, packed with Chromosorb W-AW 100-200 mesh and 10% Carbowax
537
20 M and N2 as carrier gas (flow rate 20 ml min-1). An integrator (HP 3396A) performed the acquisition and treatment of the data given by the flame ionization detector (FID). Temperatures for analysis were: FID (200°C); injector (200°C); oven (1500C). A calibration curve was made for both chromatographic methods and used for quantification. No differences between the quantifications with HPLC and GC were recorded. Mass spectrometry: mass spectra were obtained with a Nermag R10-10 coupled with a Girdel 31 GC. Column and GC conditions were as described previously.15 Electron impact was recorded with an ion source energy of 70 eV. The scanning rate was 0.8s from 25 to 300 a.m.u.
RESULTS AND DISCUSSION Model system To understand better the behaviour of benzaldehyde and benzyl alcohol during pervaporation, it was necessary to examine the physico-chemical characteristics of both compounds. A summary of the physical characterised properties is given in Table 2, with the comparison of the fluxes and selectivities for both aroma compounds through the composite membrane (GFT). The solubility of benzyl alcohol in water is thus 5.5 times higher than that of benzaldehyde. The solubilities in water can be related to hydrophobicity constants (log P) calculated by the Rekker method. 16 Diffusion coefficients in water of both compounds are almost the same. The solubility coefficient of benzaldehyde in PDMS is more than three times higher than
Table 2. Physico-chemical characteristics at 25°C of benzaldehyde and benzyl alcohol (in water and in PDMS) and comparison of mass transfer of both aroma compounds (concentration in water: 100 ppm, composite membrane GFT, Reynolds number: 600, downstream pressure: 60 Pa)
Solubility in water (g litre-1) Diffusion coefficient in water x 1011 (m 2 s-1) Hydrophobicity constant (log P) Solubility coefficient in PDMS Diffusion coefficient in PDMS x 1011 (m 2 s-1) Flux of pervaporation (g h - 1 m-E) Selectivity fl
Benzaldehyde
Benzyl alcohol
6"95 103 1-48 7"8 2"6 1-04 272
38"14 100 0"92 2"4 0-02 2"5
538
T. Lamer et al.
that of benzyl alcohol. This coefficient is the partition coefficient of the aroma compound between water and the membrane and quantifies the affinity of the compounds for the membrane. Lamer (1993) 17 found that for linear molecules with eight atoms and close hydrophobicities, the sequence for sorption is: alcohol < aldehyde, which is the same sequence found here for benzyl alcohol and benzaldehyde. The diffusion and solubility coefficients are closely related. 18'19 The more the compounds sorb into the membrane, the weaker is the diffusion coefficient. The benzaldehyde flux was 50 times higher than for benzyl alcohol for the same concentration on the upstream side of the membrane. The sequence in fluxes is identical to that found for the sorption of the aroma compounds to the polymer. Lamer et al. (1994) 13 showed that a strong relationship exists between the observed fluxes in the steady state of transfer of pervaporation and the sorption characteristics in the membrane. A selectivity equal to 272 means that for a 100 ppm benzaldehyde solution, the benzaldehyde concentration in the cold trap is 27200 ppm. In fact, the water solubility of benzaldehyde is equal to 5500 ppm and two phases were obtained in the trap: a liquid phase saturated with the aldehyde and an organic phase corresponding to the benzaldehyde. This phenomenon was obviously observed with higher selectivities and during the whole study. Such a separation did not occur with benzyl alcohol, a selectivity equal to only 2 being obtained.
Influence of the liquid concentration During fermentation, the concentration of aroma compounds in the culture medium is not constant. For this reason the effect of benzaldehyde and benzyl alcohol concentration in the liquid has been studied at 25°C with the composite membrane (GFT). Figure 3 shows that when the concentration was increased from 50 to 300 ppm, the flux of benzaldehyde increased from 0.5 to 3 g m -2 h -~, whereas the flux of benzyl alcohol increased from 0.008 to 0-05 g m - 2 h - . 1 These increases result from the increase in the driving force. Linear developments of the fluxes with the concentration are thus observed. The fluxes for benzaldehyde are 50 times higher than for benzyl alcohol, for the same concentration at the upstream side of the membrane.
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"~ , Concentration(mg/litra) Fig. 3. Changein flux of benzaldehydeandbenzy|alcohol with concentration in solution (T=25°C, membrane GFF, Re=600, downstream pressure---60 Pa).
In the range of concentrations studied, the selectivities of both aroma compounds were constant. As in the case of the fluxes observed, the selectivity values for benzyl alcohol is lower than that of benzaldehyde: the selectivities being 250 for benzaldehyde and 2.5 for benzyl alcohol. This is consistent with earlier reports that aldehydes have a higher enrichment factor than alcohols and a lower one than esters. 5' 13 As a consequence of these differences, it is possible to extract preferentially benzaldehyde from benzyl alcohol. The flux variations of the benzyl alcohol with the operational parameters were negligible, therefore, the following discussions focuses mostly on the behaviour of benzaldehyde. In all cases, the flux of water remained constant and equal to the flux observed with pure water whatever the flux of aroma compounds. This observation confirmed the absence of plasticization of the membrane by very dilute solutions of organic compounds. That was also mentioned by Nguyen and Nobe 2° for highly diluted solutions of chloroform, dichloromethane and bromoethane.
Influence of the membrane Several silicone based membranes were tested at 25°C and a benzaldehyde concentration equal to 100 ppm (Table 3). The higher flux was obtained with thin membranes, but in the case of the composite membrane, for which the dense layer thickness was very low (about 10 pm), flux was not at its highest. This may be due to the influence of the microporous support limiting the evaporation
Extraction of benzaldehyde by pervaporation Table 3. Pervaporation fluxes and selectivities of benzal-
dehyde at T=250C (model system, concentration in water: 100 ppm, Reynolds number: 600, downstream pressure: 60
Pa)
70
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Thickness of Flux Selectivity PDMS (g m -2 h - t ) fl (Itm)
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Composite membrane GFT Homogeneous membrane
1-04
30 60 130
1.75 1.48 0.91
272 477 495 558
step. Homogeneous membranes presented high fluxes and selectivities when the thickness was small (30 and 70/~m). However, the 30#m thick membrane had very poor mechanical properties and was not selected for this work. Selectivities of homogeneous membranes were higher than those of composite membranes due to their thickness which also reduced the water flux.
Change in downstreampressure When the saturated vapour pressure of the component was low, the downstream pressure may influence the mass transfer. The effect of pressure was studied between 35 and 700 Pa with a benzaldehyde concentration equal to 100 ppm (Fig. 4). In this range, flux decreased with higher pressures. Other authors have observed a similar change in flux with downstream pressure. 21 Studies on other aroma compounds revealed that below saturated vapour pressure, the aroma flux was constant at the maximum
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Fig. 5. Change in the concentrations of benzaldehyde and phenylalanine during fermentation (T=25°C).
"Thickness of the active top layer.
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Fig. 4. Change in benzaldehyde flux with downstream pressure (T=25°C, homogeneous PDMS membrane 130 pro, concentration=0.1 g litre -1, Re=600).
value. ]4 This was not observed here. Measurements of flux with lower pressure may be useful to show this phenomenon.
Fermentation/pervaporation coupling experiments Two extractions were performed in this study and they differ from each other by the use of different membranes for the pervaporation. For the first trial, the production of benzaldehyde reached a concentration of about 63.4mg litre -~ after 4 days (Fig. 5). In contrast, the phenylalanine concentration (precursor of benzaldehyde) decreased sharply since it was consumed by Bjerkandera adusta. Pervaporation was performed using GFT composite membranes. Four periods of coupling have been made at 1, 2, 4 and 5 days during brief periods of a few hours. The flux increased when benzaldehyde concentration in the culture broth increased, as observed in the model systems (water + aroma)• However, the fluxes were lower than those previously measured, for example with 63.4mg aldehyde litre-1, the flux was about two times less than in the model medium (0"36 g m-2 h-1 instead of 0.70g m -2 h - l ) . This decrease resulted from the presence of the microorganism and from a lower flow rate. To improve fluxes and selectivities, a second trial of coupling was performed using a homogeneous PDMS membrane with a thickness of 100/~m (Table 4). The ratio of the concentration of benzaldehyde and benzyl alcohol in the fermenter was inverted in the pervaporate, benzaldehyde was concentrated by pervaporation by a factor of 24-89. When considering the coupling of pervapora-
T. Lamer et al.
540
Table 4. Change in concentration of benzaldehyde and benzylalcohol in fermenter and in pervaporate during the coupling of fermentation with pervaporation (T=25°C, homogeneous membrane PDMS, thickness=100gm, downstream pressure=200 Pa) Time (h)
Concentration in fermenter (mg litre - 1)
Benzaldehyde 102 132 139 214 228
78 69 80 112 132
Concentration in pervaporate (mg litre - 1)
Benzyl alcohol 120 737 790 490 170
3700 6200 4800 5300 3200
tion with fermentation, it is clear that pervaporation permits a selective extraction of benzaldehyde from the medium by a favourable shift of the equilibrium benzaldehyde~benzyl alcohol. The pervaporate was analyzed by GC/MS to determine its composition and to identify the different volatile compounds (Table 5). This pervaporate was rich in benzaldehyde (about 68%) and it is clear that the pervaporation was appropriate to remove this aldehyde selectively from the mixture. The quantity of the benzyl alcohol was low in spite of its concentration in the culture medium which was equal to several hundred mg litre-1. As the concentration of the hydrophobic solutes in the pervaporate was much higher than that in the broth, it was possible to identify metabolites in the pervaporate which were not detected in the broth. Their structures including
Table 5. Compounds in the pervaporate (mass spectrometry analysis) Compounds
Proportion (%)
Benzaldehyde
68.5
Benzyl alcohol
4.5
~,-Decalactone 1-Phenyl ethanone 3-Chloro-p-anisaldehyde Ethyl benzoate Phenyl acetaldehyde Styrene Phenyl acetonitrile Toluene Cinnamaldehyde Benzyl formate
4.4 1.5 1.4 1.0 0.9 0.9 0-6 0.9 0.3 0.3
Hydrocarbon (unknown) Anisaldehyde
Benzaldehyde
9.6 5"0
Selectivity of benzaldehyde
Benzyl alcohol 900 3100 1900 2800 1100
47 89 60 47 24
a benzene ring suggest a relationship between these compounds and the catabolic pathway from phenylalanine to benzaldehyde (Fig. 6). Most of the phenylalanine catabolites are hydrophobic which may be the reason why some of the compounds which were not detected in the culture broth were detected in the pervaporate. This is the case especially for 1-phenyl ethanone, phenyl acetaldehyde, styrene, phenyl acetonitrile and cinnamaldehyde. It has to be emphasized that the pervaporation process may be a means of showing the different intermediary metabolites of the bioconversion. Some other compounds detected in the pervaporate including 3-chloro-p-anisaldehyde~22' 15 and y-decalactone 9 are normal products of Bjerkandera adusta.
CONCLUSIONS Two kinds of silicone membranes can be selected to perform the extraction in culture medium since fluxes were similar. However, the homogeneous membrane is more selective. There is a large influence of downstream pressure on the flux of benzaldehyde, and so for further experiments, this parameter needs to be at a minimum. Previous reports have shown the influence of fluid velocity on pervaporation of aroma compounds 17 and so, flow rate must be maximal. This may be the main problem to solve. Because of the presence of biomass, a preliminary separation step is necessary to maintain biomass activity and to prevent membrane fouling. Some practical problems need to be solved before an industrial application may be considered but it is clear that pervaporation is a.downstream process which is well adapted
541
Extraction of benzaldehyde by pervaporation
to the extraction of flavour compounds. These results showed selectivity which brought about a concentration factor of 100 for benzaldehyde,
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OH
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the highest published for such a diluted broth. Furthermore, good flux could be obtained with a weak vacuum, which is quite inexpensive to
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r° Styrene(1)
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II BenzylAlcohol
Benzaldehyde O
Phenyl~
Rrile (l)
OH
BenzylFormate(1) Ethyl Benzoate(l) Benzoic Acid(1) Fig. 6. Hypothetical pathway of benzaldehyde production by Bjerkandera adusta. (1) Compounds not detected in the culture broth but detected in the pervaporate.
542
T. Lamer et al.
produce. At the moment the limiting step of the process is the productivity of the fungus used. 10.
ACKNOWLEDGEMENTS
11.
We thank G. Mauvais for technical assistance and E. S~mon for GC-MS analysis. 12.
REFERENCES 1. Spinnler, H. E., Dufoss6, L., Souchon, I., Latrasse, A., Piffaut, C., Voilley, A. & Delest, P., Production of gamma-decalactone by bioconversion. Patent number FR-93.06673 (1994). 2. Spinnler, H. E., Roche, N. & Mauvais, G., Production of benzaldehyde concentrates by Bjerkandera adusta. (1995) (submitted). 3. Escudier, J. L. & Le Bouar, M., Applications and evaluation of pervaporation for the production of low alcohol wines. In Proceedings of the Third International Conference on Pervaporation Processes in the Chemical Industry, Bakish Materials Corp., Englewood, NJ, 1988, pp. 387-97. 4. VoiUey, A., Schmidt, B., Simatos, D. & Baudron, S., Extraction of aroma compounds by the pervaporation technique, In Proceedings of the Third International Conference on Pervaporation Processes in the Chemical Industry, Bakish Materials Corp., Englewood, NJ, 1988, pp. 429-38. 5. Bengtsson, E., Tr~gardh, G. & HallstrSm, B., Concentration of apple juice aroma from evaporator condensate using pervaporation. Lebensm. Wiss. Technol., 25 (1992) 29-34. 6. Groot, W. J., Van den Oever, C. E. & Kossen, N. W. F., Increase of substrate conversion by pervaporation in the continuous butanol fermentation. Biotechnol. Lea., 11 (1984) 709-14. 7. Bengtsson, E., Tfiigardh, G. & Hallstr6m, B., Recovery and concentration of apple juice aroma compounds by pervaporation. J. Food Eng., 10 (1989) 65-71. 8. Dettwiler, B., Dunn, I. J. & Prenosil, J. E., Bioproduction of acetoin and butanediol: product recovery by pervaporation. In Proceedings of the Fifth International Conference on Pervaporation Processes in the Chemical Industry, Bakish Materials Corp., Englewood, NJ, 1991, pp. 308-18. 9. Berger, R. G., Neuhauser, K. & Drawert, F., Characterization of the odor principles of some
13.
14.
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16. 17.
18.
19. 20. 21. 22.
basidiomycetes: Bjerkandera adusta, Poria aurea. Tyromyces sambuceus. Flavour Fragrance J., (1986) 1, 181-5. Reid, R. C., Prausnitz, J. M. & Poling, B. E., The Properties of Gasses and Liquids, 4th edn., McGrawHill, New York, 1987. Le Thanh, M., Lamer, T., Voilley, A. & Jose, J., D6termination des coefficients de partage vapeurliquide de compos6s d'ar6me h partir de leurs caract6ristiques physico-chimiques. J. Chim. Phys., 90 (1993) 545-60. Cussler, E. L., Diffusion, Mass Transfer in Fluid Systems. Cambridge University Press, Cambridge, 1984. Lamer, T., Rohart, M. S., Voilley, A. & Baussart, H., Influence of sorption and diffusion of aroma compounds in silicone rubber on their extraction by pervaporation. J. Membr. Sci., 90 (1994) 251-63. Lamer, T. & Voilley, A., Influence of different parameters on the pervaporation of aroma compounds. In Proceedings of the Fifth International Conference on Pervaporation Processes in the Chemical Industry, Bakish Materials Corp., Englewood, NJ, 1991, pp. 110-22. Spinnler, H. E., De Jong, E., Mauvais, G., Semon, E. & Le Quere, J. L., Production of halogenated compounds by Bjerkandera adusta. Appl. Microbiol. Biotechnol., 42 (1994) 212-21. Rekker, R. F., The Hydrophobic Fragmental Constant, eds W. Th. Nauta & R. F. Rekker. Elsevier, Amsterdam, 1977. Lamer, T., Extraction de compos6s d'ar6me par pervaporation: Relation entre les propri~t6s physico-chimiques des substances d'ar6me et leurs transferts h travers des membranes ~ base de polydim6thylsiloxane. PhD Thesis, Dijon, Universit6 de Bourgogne, 1993. Heintz, A., Funke, H. & Lichtenthaler, R. N., Sorption and diffusion in pervaporation membranes. In Pervaporation Membrane Separation Processes, ed. R. Y. M. Huang. Elsevier, Amsterdam, 1991, pp. 279-319. N6el, J., Introduction to pervaporation. In Pervaporation Membrane Separation Processes, ed. R. Y. M. Huang. Elsevier, Amsterdam, 1991, pp. 1-109. Nguyen, Q. T. & Nobe, K., Extraction of organic contaminants in aqueous solutions by pervaporation. J. Membr. Sci., 30 (1987) 11-22. B6ddeker, K. W., Bengston, G. & Bode, E., Pervaporation of low volatility aromatics from water. J. Membr. Sci., 53 (1990) 143-58. De Jong, E., Field, J. A., Spinnler, H. E., Wijnberg, J. B. P. A. & de Bont, J. A. M., Significant biogenesis of chlorinated aromatics by fungi in natural environments. Appl. Environm. Microbiol., 60 (1994) 264-70.