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Characterization of volatile compounds produced by Rhizopus strains grown on agro-industrial solid wastes
Bioresource Technology 71 (2000) 211±215
Characterization of volatile compounds produced by Rhizopus strains grown on agro-industrial solid wastes P. Christena,b,*, A. Bramorskib,c, S. Revaha, C.R. Soccolc b
a Department of Chemical Engineering, Universidad Aut onoma Metropolitana ± Iztapalapa, AP 55534, CP 09340 Mexico, DF, Mexico IRD Microbiology Laboratory, CESB/ESIL, Universit e de Provence, Case 925, 163 Avenue de Luminy, 13288 Marseille, Cedex 9, France c Biotechnological Processes Laboratory, Universidade Federal do Paran a, Caixa Postal 19001, CEP 81531-970, Curitiba, PR, Brazil
Received 26 September 1998; received in revised form 10 May 1999; accepted 24 May 1999
Abstract Four edible Rhizopus strains were cultivated on eight combinations of solid agro-industrial wastes (cassava bagasse, apple pomace), soyabean, amaranth grain and soyabean oil. Signi®cant dierences in growth were observed among strains on the dierent media studied. The medium containing cassava bagasse with soyabean (5:5 w/w) gave the highest CO2 production, while Rhizopus oryzae ATCC 34612 was the best producer of volatiles. The aromas of the cultures were light and rather pleasant. The amaranth medium with mineral salts solution produced the highest amount of volatile compounds (VC), demonstrating that the aroma of fermented solid substrates can be improved. The VC production was very rapid, attaining, in most of the cases, its maximum around the ®rst day of culture. These maxima markedly varied according to the medium used. Ó 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction The processing of agro-industrial raw materials such as cassava or apples produces large amounts of waste whose accumulation leads to important problems of environmental pollution. Such wastes, as they contain high amounts of starch or reducing sugars, could be adequate for growing edible fungi. Most of the research in this ®eld has dealt with enzyme or biomass production (Pandey, 1992); few papers have reported the production of ¯avouring compounds. However, some recent reviews pointed out the great potential of microorganisms, especially fungi, for this purpose (Bigelis, 1992; Janssens et al., 1992; Berger, 1995). Rhizopus strains have been traditionally used to ferment solid substrates such as soyabean, e.g. in the fabrication of tempeh in Indonesia (Hachmeister and Fung, 1993). During this process, a pleasant odour is developed but the volatile compounds (VC) generated have not been identi®ed. Rhizopus species were also reported to be used for the biotransformation of raw cassava by
*
Corresponding author. Address: IRD Microbiology Laboratory, CESB/ESIL, Universite de Provence, Case 925, 163 Avenue de Luminy, 13288 Marseille, Cedex 9, France.
protein enrichment (Raimbault et al., 1985) and the production of L -lactic acid and fumaric acid (Soccol et al., 1994). The objective of this work was to study the bioconversion by edible fungi of the Rhizopus genus of agroindustrial solid wastes or substrates into (i) biomass and/ or (ii) volatile metabolites of interest, such as ¯avouring alcohols or esters. Four Rhizopus strains were grown on eight mixtures of solid substrates in order to select one strain and one medium for further optimization of the process. The 32 combinations were evaluated in terms of CO2 and total VC produced. This study also identi®ed some of the VC and established the dynamics of total VC detected in the headspace of the cultures. 2. Methods 2.1. Microorganisms and maintenance A previous screening (Bramorski, 1997) allowed selection of four strains among 20 tested for the production of pleasant aromas when grown on cassava bagasse: R. oryzae ATCC 34612 (Ro1), R. oryzae NRRL 395 (Ro2), R. oryzae MUCL 28627 (Ro3) and R. sp. NRRL 25975 (Rsp). Strains were maintained on Potato
0960-8524/00/$ ± see front matter Ó 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 0 8 4 - X
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P. Christen et al. / Bioresource Technology 71 (2000) 211±215
Dextrose Agar (PDA) slants at 30°C for 5 days and were then stored at 4°C for periods not exceeding 3 months. 2.2. Spore production Spores were produced on PDA after 5 days of culture at 30°C. They were collected in sterile water containing 0.1% Tween 80 and small glass beads. The suspension provided 1 ´ 107 spores per gram of initial dry matter (IDM) as inoculum. 2.3. Comparison of substrates As reported previously, VC production by fungi can be in¯uenced by the type of substrate used (Christen et al., 1994, 1997) and the presence of precursors (Christen et al., 1997). In order to take advantage of the variability in the composition of the materials, whose representative analysis is given in Table 1, they were mixed in various proportions to obtain a broad range of C/N ratios (Table 2). As some Rhizopus species can utilize long chain fatty acids as sole carbon source (Graham et al., 1995), we decided to add soyabean oil to test the ability of the strains to use its fatty acids. Amaranth was tested alone as no data were available about its use in solid-state fermentation. As we observed that Rhizopus could grow on amaranth and produce some interesting VC, we tried to improve this production by adding a mineral salt solution to the solid substrate. Soyabean was previously dried and ground in order to eliminate the hull. Amaranth was used in a popped form to improve its nutrient availability. It was then ground, as were cassava bagasse and apple pomace. The four raw materials were then oven dried at 85°C for 12 h, sieved through a 0±0.8 mm screen and autoclaved at 121°C for 15 min. 2.4. Fermentation conditions Static solid fermentations were performed in 250 ml Erlenmeyer ¯asks covered with 6 layers of gauze and
containing 15 g of dry matter. Initial water contents of the media were calculated according to the maximum absorption capacity of each substrate as determined according to Hernandez Jimenez (1990) (Table 1). Experiments involving the 32 combinations were conducted in duplicate for four days. Fermentation conditions were: initial pH, 6; temperature, 30°C, and aeration by passive diusion through the gauze. 2.5. Analytical methods C/N molar ratio was determined using a CHNS/O analyzer Series II, model 2400 (Perkin Elmer, USA). Growth was evaluated from the dynamics of headspace CO2 concentration in the ¯ask (Mitchell, 1992; Auria et al., 1995), which is the balance between CO2 generation by the microorganism and its loss by passive diusion through the gauze layers. This parameter must only be considered as an indicator of growth, and the values given are only meaningful if compared between themselves. CO2 was measured by gas chromatography (GC) according to Christen et al. (1993) and reported as % (v/v). Results are the average of two determinations. The odours of the cultures were evaluated by sensorial evaluation with an untrained panel of six members, with no restriction in descriptive terms. The VC produced were characterized from the headspace of the cultures by GC with a Hewlett-Packard 5890 apparatus equipped with a 5 m Megabore HP1 column and a ¯ame ionization detector. The operating conditions were: temperatures, injector and detector: 210°C, oven held at 40°C during 2 min and then programmed at 10°C/min increments up to 150°C. The nitrogen gas ¯ow was 1.5 ml/min and split ratio was 1:32. Total VC produced were expressed as lmol ethanol equivalent per liter of air (lmol eq. ethanol/l). Some compounds were also determined individually and their concentration in the headspace was expressed from standard curves as lmol/l. Due to the high volatility of these compounds, the results are largely underestimated
Table 1 Composition of the wastes and substrates Substratea
Cassava bagasse
Soyabean
Apple pomace
Amaranth grain
Protein Starch Reducing sugars Fat Ashes Water saturationb Reference
2.3 63.6 0.24 0.65 0.83 65 Cereda (1994)
40.0 5.6 7.0 18.0 4.6 40 Pyler (1973)
5.1 0.0 57.0 4.2 2.8 75 Hang (1987)
15.6 62 Not given 6.3 3.7 65 Teutonico and Knorr (1985)
a Cassava bagasse was from Lorenz S.A. (Paran a, Brazil). Soyabean was from Ceval S.A. (Sta Catarina, Brazil). Apple pomace was from Tecnovin, Industria de sucos S.A. (Sta Catarina, Brazil). Amaranth was from Tulyehualco (Mexico, DF, Mexico). Proportions are given on a dry matter basis and expressed as % (w/w). b Determined in our laboratory, expressed in % (w/w).
P. Christen et al. / Bioresource Technology 71 (2000) 211±215 Table 2 Composition of the dierent solid media studied Medium
Substrate
C/Na
A B C D
Cassava bagasse + soyabean (8:2 w/w) Cassava bagasse + soyabean (2:8 w/w) Cassava bagasse + soyabean (5:5 w/w) Cassava bagasse + soyabean + soyabean oilb (4.5:4.5:1 w/w/v) Apple pomace + cassava bagasse + soyabean (8:1:1 w/w/w) Apple pomace + cassava bagasse + soyabean (3:3:4 w/w/w) Amaranth grain Amaranth grain + mineral salt solutionc
29.3 10.8 20.5 20.8
E F G G0
38.6 18.0 29.4 24.8
a
Molar ratio. Soyabean oil was from Ceval S.A. (Sta Catarina, Brazil). Proportions are given on a dry matter basis. c The salt solution was the one optimized by Christen and Raimbault (1991). b
and can be used only for relative comparison of the treatments. 2.6. Statistical treatment of the results Carbon dioxide and VC production data were analysed by one-way ANOVA and Tukey's studentized range (TSR) test, using StatView software (Abacus Concepts, version 1.02, Berkeley, CA). 3. Results and discussion 3.1. Growth characterization Variance analysis of CO2 production showed signi®cant eects (p < 0:0001) of the media, strains and interaction on growth. The TSR test allowed the
213
classi®cation of media and strains for CO2 production and growth (Table 3). The best growth was obtained with media C, D, B and F with proportions of substrates leading to a C/N ratio between 10 and 21. The addition of soyabean oil had no eect on growth (D medium). Lower CO2 production was observed with unbalanced media, particularly for E medium (with a high-reducing sugar content and, consequently, a lower initial water activity) and G medium (amaranth). However, in the case of amaranth, the addition of a mineral salt solution (G0 medium) greatly improved CO2 production. The A medium with a high starch content (and high C/N ratio) gave intermediate results. The range of CO2 production was larger among media (3.4±16.3%) than among strains (10.0±12.1%). 3.2. VC production The odours detected were rather pleasant with a slight alcoholic note and no dierences were reported by the panel between the 32 combinations. This diers from the fruity notes detected in cultures of Ceratocystis ®mbriata grown in the same conditions on cassava bagasse (Christen et al., 1997). Signi®cant dierences were observed in VC production between media (p < 0:0001), but not between strains. This indicates that VC production depends mostly on the medium used. The highest VC production was obtained with amaranth (Table 3), and was greatly enhanced by the addition of the mineral salts solution (G0 medium). Medium E gave an intermediate result. The VC production was rather poor with the other media. The addition of soyabean oil (D medium) neither increased the VC production nor promoted speci®c odours, although some species of this genus are known to produce esterases. For all media used, no relation was
Table 3 Classi®cation of media and strains for CO2 and total VC production by a TSR test CO2 production
*
Total VC production
Tukey groups by medium
Mean value (% v/v)
Medium
Tukey groups by medium
Mean value (lmol eq. ethanol/l)
Medium
1 2 3 3 4 5 6 7
16.35 14.70 13.58 13.40 11.98 10.37 5.42 3.42
C D B F A G0 G E
1 2 3 4 5 5 5 6
209.2 71.9 49.1 31.8 21.8 15.5 14.4 8.0
G0 G E F C B D A
Tukey groups by strain
Mean value (% v/v)
Strain
Tukey groups by strain
Mean value (lmol eq. ethanol/l)
Strain
1 2 3 4
12.10 11.62 10.93 9.96
Ro3 Ro1 R. sp. Ro2
1 2 2 2
63.2 52.0 48.4 47.3
Ro1 Ro2 R. sp. Ro3
Signi®cant with p < 0:05.
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P. Christen et al. / Bioresource Technology 71 (2000) 211±215
Table 4 Compounds identi®ed in the headspace of cultures of R. oryzae 1 Medium A B C D E F G G0
Acetaldehyde a
+ + + ++d + + + ++
Ethanol b
+++ +++ +++ +++ +++ +++ +++ +++
1-propanol + + + + + + + +
Ethyl acetate + + + + + + + ++
Ethyl propionate c
± ± ± ± ± + ± +
3-MB + + + + + + + ++
a
<1 lmol/l. >10 lmol/l. c None. d 1±10 lmol/l. b
observed between CO2 and total VC production. These results oppose those obtained for C. ®mbriata grown on various solid substrates (Meza et al., 1998). The four Rhizopus strains showed few dierences in the metabolites produced. The case of Ro1 is presented in Table 4. The number of compounds detected varied between 6 and 9 according to the media, among them acetaldehyde, ethanol, 1-propanol, ethyl acetate, ethyl propionate, and 3-methyl butanol (3-MB) were identi®ed. Some of these compounds were previously reported as growth indicators of moulds, especially 3-MB (B orjesson et al., 1990). Lanciotti and Guerzoni (1993) observed that metabolic vapours of R. arrhizus containing ethanol and 3-MB displayed an antifungal activity. Ethanol was always the most abundant volatile compound ( >80%). Its production was favoured by the high C/N ratio. Also low aeration, associated with static cultivation, promoted the fermentative activity. Rhizopus oryzae 1, which produced the highest quantity of VC, was used to study the time course of total VC production on the eight media (Fig. 1). In media A±F, total VC did not exceed 40 lmol eq. ethanol/l. Their production was increased when amaranth was used as substrate, and attained 283 lmol eq. ethanol/l when mineral salt solution was added to amaranth. For media A, B, C and D, after a short lag phase, maxima were attained within 20 h (as for CO2 ), while 40 h were needed on media F and G0 . The lag phase was much longer on medium E as observed with CO2 production. 4. Conclusion This study showed that agro-industrial substrates could be rapidly transformed by Rhizopus strains to produce VC. Large dierences were found, in terms of VC and CO2 productions, depending on the medium used. Dierences were not statistically signi®cant among
Fig. 1. Total volatile evolution in the headspace of cultures of R. oryzae 1 on eight dierent solid media.
strains for VC production. Study with strain Ro1 showed that ethanol was the major VC produced. Acetaldehyde, ethyl acetate, 1-propanol, and 3-MB were always present, at low or moderate concentrations. The odours of the cultures were pleasant but no relationship was established between the sensorial analysis and the amount of VC produced. VC production increased three times and CO2 production twice when a mineral salt solution was added to the amaranth medium. The system of open cultures used, adequate for this screening, did not allow VC production at levels satisfactory from an economic point of view. This could be improved by using forced aeration, a closed system and an adequate recovery set-up (e.g. with Tenax resin or activated carbon).
P. Christen et al. / Bioresource Technology 71 (2000) 211±215
Acknowledgements The authors wish to thank P. Torres (UAM, Mexico) for his help in statistical analysis, Dr. S. Huerta (UAM, Mexico) for the C/N analysis and Dr. P. Roger (IRD, France) for his helpful comments on the manuscript. This project was partly funded by IRD (France), CNPq (Brazil) and CONACyT (Mexico). A. Bramorski received a grant from the International Relation Service of IRD during her stay in Mexico.
References Auria, R., Ortiz, I., Villegas, E., Revah, S., 1995. In¯uence of growth and high mould concentration on the pressure drop in solid state fermentations. Process Biochem. 30, 751±756. Berger, R.G., 1995. Aroma Biotechnology. Springer, Berlin. Bigelis, R., 1992. Flavor metabolites and enzymes from ®lamentous fungi. Food Technol. 46, 151±161. B orjesson, T., St ollman, U., Schn urer, J., 1990. Volatile metabolites and other indicators of Penicillium aurantiogriseum growth on dierent substrates. Appl. Environ. Microbiol. 56, 3705±3710. ao de Bramorski, A., 1997. Caracterizacß~ ao do crescimento e producß~ metabolitos volateis por fungos ®lamentosos cultivados sobre substratos agroindustriais. M.Sc. Thesis. Faculty of Technology, Federal University of Paran a, Brazil. Cereda, M.P., 1994. Caracterizacß~ ao dos residuos da industrializacß~ ao da mandioca. In: Cereda, M.P. (Ed.), Industrializacß~ ao da mandioca no Brasil, Pauliceia, S~ ao Paulo, pp. 11±50. Christen, P., Auria, R., Vega, C., Villegas, E., Revah, S., 1993. Growth of Candida utilis in solid state fermentation. Biotechnol. Adv. 11, 549±557. Christen, P., Villegas, E., Revah, S., 1994. Growth and aroma production by Ceratocystis ®mbriata in various fermentation media. Biotechnol. Lett. 16, 1183±1188. Christen, P., Meza, J.C., Revah, S., 1997. Fruity aroma production in solid state fermentation by Ceratocystis ®mbriata: in¯uence of the
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substrate type and the presence of precursors. Mycol. Res. 101, 911±919. Christen, P., Raimbault, M., 1991. Optimization of culture medium for aroma production by Ceratocystis ®mbriata. Biotechnol. Lett. 13, 521±526. Graham, A.J., Gordon, M., H, , Westby, A., Owens, J.D., 1995. Nutrition of tempe moulds. Lett. Appl. Microbiol. 21, 223± 227. Hachmeister, K.A., Fung, D.Y.C., 1993. Tempeh: a mold-modi®ed indigenous fermented food made from soyabean and/or cereal grains. Crit. Rev. Microbiol. 19, 137±188. Hang, Y.D., 1987. Production of fuels and chemicals from apple pomace. Food Technol. 41, 115±117. Hernandez Jimenez, S., 1990. Caracterizaci on y evaluaci on fõsicobiol ogica de soportes inertes para fermentaci on s olida. Reportes de Biotecnologõa (UAM, Mexico) 21, 29±31. Janssens, L., Pooter, H.L., Schamp, N.M., Vandamme, E.J., 1992. Production of ¯avours by microorganisms. Process Biochem. 27, 195±215. Lanciotti, R., Guerzoni, M.E., 1993. Competitive inhibition of Aspergillus ¯avus by volatile metabolites of Rhizopus arrhizus. Food Microbiol. 10, 367±377. Meza, J.C., Christen, P., Revah, S., 1998. Eect of added amino acids on the production of a fruity aroma by Ceratocystis ®mbriata. Sci. Alim. 18, 627±636. Mitchell, D.A., 1992. Biomass determination in solid-state cultivation. In: Doelle, H.W., Mitchell, D.A., Rolz, C.E. (Eds.), Solid Substrate Cultivation. Elsevier, London, pp. 53±63. Pandey, A., 1992. Recent process developments in solid-state fermentation. Process Biochem. 27, 109±117. Pyler, E.J., 1973. Baking Science and Technology. Vol. 1, Siebel Pub. Comp., Chicago, IL. Raimbault, M., Revah, S., Pi~ na, F., Villalobos, P., 1985. Protein enrichment of cassava using molds isolated from traditional foods. J. Ferment. Technol. 63, 395±399. Soccol, C.R., Stonoga, V.I., Raimbault, M., 1994. Production of L -lactic acid by Rhizopus species. World J. Microbiol. Biotechnol. 10, 433±435. Teutonico, R.A., Knorr, D., 1985. Amaranth: composition, properties and application of a rediscovered food crop. Food Technol. 39, 49±60.
Characterization of volatile compounds produced by Rhizopus strains grown on agro-industrial solid wastes P. Christena,b,*, A. Bramorskib,c, S. Revaha, C.R. Soccolc b
a Department of Chemical Engineering, Universidad Aut onoma Metropolitana ± Iztapalapa, AP 55534, CP 09340 Mexico, DF, Mexico IRD Microbiology Laboratory, CESB/ESIL, Universit e de Provence, Case 925, 163 Avenue de Luminy, 13288 Marseille, Cedex 9, France c Biotechnological Processes Laboratory, Universidade Federal do Paran a, Caixa Postal 19001, CEP 81531-970, Curitiba, PR, Brazil
Received 26 September 1998; received in revised form 10 May 1999; accepted 24 May 1999
Abstract Four edible Rhizopus strains were cultivated on eight combinations of solid agro-industrial wastes (cassava bagasse, apple pomace), soyabean, amaranth grain and soyabean oil. Signi®cant dierences in growth were observed among strains on the dierent media studied. The medium containing cassava bagasse with soyabean (5:5 w/w) gave the highest CO2 production, while Rhizopus oryzae ATCC 34612 was the best producer of volatiles. The aromas of the cultures were light and rather pleasant. The amaranth medium with mineral salts solution produced the highest amount of volatile compounds (VC), demonstrating that the aroma of fermented solid substrates can be improved. The VC production was very rapid, attaining, in most of the cases, its maximum around the ®rst day of culture. These maxima markedly varied according to the medium used. Ó 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction The processing of agro-industrial raw materials such as cassava or apples produces large amounts of waste whose accumulation leads to important problems of environmental pollution. Such wastes, as they contain high amounts of starch or reducing sugars, could be adequate for growing edible fungi. Most of the research in this ®eld has dealt with enzyme or biomass production (Pandey, 1992); few papers have reported the production of ¯avouring compounds. However, some recent reviews pointed out the great potential of microorganisms, especially fungi, for this purpose (Bigelis, 1992; Janssens et al., 1992; Berger, 1995). Rhizopus strains have been traditionally used to ferment solid substrates such as soyabean, e.g. in the fabrication of tempeh in Indonesia (Hachmeister and Fung, 1993). During this process, a pleasant odour is developed but the volatile compounds (VC) generated have not been identi®ed. Rhizopus species were also reported to be used for the biotransformation of raw cassava by
*
Corresponding author. Address: IRD Microbiology Laboratory, CESB/ESIL, Universite de Provence, Case 925, 163 Avenue de Luminy, 13288 Marseille, Cedex 9, France.
protein enrichment (Raimbault et al., 1985) and the production of L -lactic acid and fumaric acid (Soccol et al., 1994). The objective of this work was to study the bioconversion by edible fungi of the Rhizopus genus of agroindustrial solid wastes or substrates into (i) biomass and/ or (ii) volatile metabolites of interest, such as ¯avouring alcohols or esters. Four Rhizopus strains were grown on eight mixtures of solid substrates in order to select one strain and one medium for further optimization of the process. The 32 combinations were evaluated in terms of CO2 and total VC produced. This study also identi®ed some of the VC and established the dynamics of total VC detected in the headspace of the cultures. 2. Methods 2.1. Microorganisms and maintenance A previous screening (Bramorski, 1997) allowed selection of four strains among 20 tested for the production of pleasant aromas when grown on cassava bagasse: R. oryzae ATCC 34612 (Ro1), R. oryzae NRRL 395 (Ro2), R. oryzae MUCL 28627 (Ro3) and R. sp. NRRL 25975 (Rsp). Strains were maintained on Potato
0960-8524/00/$ ± see front matter Ó 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 0 8 4 - X
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P. Christen et al. / Bioresource Technology 71 (2000) 211±215
Dextrose Agar (PDA) slants at 30°C for 5 days and were then stored at 4°C for periods not exceeding 3 months. 2.2. Spore production Spores were produced on PDA after 5 days of culture at 30°C. They were collected in sterile water containing 0.1% Tween 80 and small glass beads. The suspension provided 1 ´ 107 spores per gram of initial dry matter (IDM) as inoculum. 2.3. Comparison of substrates As reported previously, VC production by fungi can be in¯uenced by the type of substrate used (Christen et al., 1994, 1997) and the presence of precursors (Christen et al., 1997). In order to take advantage of the variability in the composition of the materials, whose representative analysis is given in Table 1, they were mixed in various proportions to obtain a broad range of C/N ratios (Table 2). As some Rhizopus species can utilize long chain fatty acids as sole carbon source (Graham et al., 1995), we decided to add soyabean oil to test the ability of the strains to use its fatty acids. Amaranth was tested alone as no data were available about its use in solid-state fermentation. As we observed that Rhizopus could grow on amaranth and produce some interesting VC, we tried to improve this production by adding a mineral salt solution to the solid substrate. Soyabean was previously dried and ground in order to eliminate the hull. Amaranth was used in a popped form to improve its nutrient availability. It was then ground, as were cassava bagasse and apple pomace. The four raw materials were then oven dried at 85°C for 12 h, sieved through a 0±0.8 mm screen and autoclaved at 121°C for 15 min. 2.4. Fermentation conditions Static solid fermentations were performed in 250 ml Erlenmeyer ¯asks covered with 6 layers of gauze and
containing 15 g of dry matter. Initial water contents of the media were calculated according to the maximum absorption capacity of each substrate as determined according to Hernandez Jimenez (1990) (Table 1). Experiments involving the 32 combinations were conducted in duplicate for four days. Fermentation conditions were: initial pH, 6; temperature, 30°C, and aeration by passive diusion through the gauze. 2.5. Analytical methods C/N molar ratio was determined using a CHNS/O analyzer Series II, model 2400 (Perkin Elmer, USA). Growth was evaluated from the dynamics of headspace CO2 concentration in the ¯ask (Mitchell, 1992; Auria et al., 1995), which is the balance between CO2 generation by the microorganism and its loss by passive diusion through the gauze layers. This parameter must only be considered as an indicator of growth, and the values given are only meaningful if compared between themselves. CO2 was measured by gas chromatography (GC) according to Christen et al. (1993) and reported as % (v/v). Results are the average of two determinations. The odours of the cultures were evaluated by sensorial evaluation with an untrained panel of six members, with no restriction in descriptive terms. The VC produced were characterized from the headspace of the cultures by GC with a Hewlett-Packard 5890 apparatus equipped with a 5 m Megabore HP1 column and a ¯ame ionization detector. The operating conditions were: temperatures, injector and detector: 210°C, oven held at 40°C during 2 min and then programmed at 10°C/min increments up to 150°C. The nitrogen gas ¯ow was 1.5 ml/min and split ratio was 1:32. Total VC produced were expressed as lmol ethanol equivalent per liter of air (lmol eq. ethanol/l). Some compounds were also determined individually and their concentration in the headspace was expressed from standard curves as lmol/l. Due to the high volatility of these compounds, the results are largely underestimated
Table 1 Composition of the wastes and substrates Substratea
Cassava bagasse
Soyabean
Apple pomace
Amaranth grain
Protein Starch Reducing sugars Fat Ashes Water saturationb Reference
2.3 63.6 0.24 0.65 0.83 65 Cereda (1994)
40.0 5.6 7.0 18.0 4.6 40 Pyler (1973)
5.1 0.0 57.0 4.2 2.8 75 Hang (1987)
15.6 62 Not given 6.3 3.7 65 Teutonico and Knorr (1985)
a Cassava bagasse was from Lorenz S.A. (Paran a, Brazil). Soyabean was from Ceval S.A. (Sta Catarina, Brazil). Apple pomace was from Tecnovin, Industria de sucos S.A. (Sta Catarina, Brazil). Amaranth was from Tulyehualco (Mexico, DF, Mexico). Proportions are given on a dry matter basis and expressed as % (w/w). b Determined in our laboratory, expressed in % (w/w).
P. Christen et al. / Bioresource Technology 71 (2000) 211±215 Table 2 Composition of the dierent solid media studied Medium
Substrate
C/Na
A B C D
Cassava bagasse + soyabean (8:2 w/w) Cassava bagasse + soyabean (2:8 w/w) Cassava bagasse + soyabean (5:5 w/w) Cassava bagasse + soyabean + soyabean oilb (4.5:4.5:1 w/w/v) Apple pomace + cassava bagasse + soyabean (8:1:1 w/w/w) Apple pomace + cassava bagasse + soyabean (3:3:4 w/w/w) Amaranth grain Amaranth grain + mineral salt solutionc
29.3 10.8 20.5 20.8
E F G G0
38.6 18.0 29.4 24.8
a
Molar ratio. Soyabean oil was from Ceval S.A. (Sta Catarina, Brazil). Proportions are given on a dry matter basis. c The salt solution was the one optimized by Christen and Raimbault (1991). b
and can be used only for relative comparison of the treatments. 2.6. Statistical treatment of the results Carbon dioxide and VC production data were analysed by one-way ANOVA and Tukey's studentized range (TSR) test, using StatView software (Abacus Concepts, version 1.02, Berkeley, CA). 3. Results and discussion 3.1. Growth characterization Variance analysis of CO2 production showed signi®cant eects (p < 0:0001) of the media, strains and interaction on growth. The TSR test allowed the
213
classi®cation of media and strains for CO2 production and growth (Table 3). The best growth was obtained with media C, D, B and F with proportions of substrates leading to a C/N ratio between 10 and 21. The addition of soyabean oil had no eect on growth (D medium). Lower CO2 production was observed with unbalanced media, particularly for E medium (with a high-reducing sugar content and, consequently, a lower initial water activity) and G medium (amaranth). However, in the case of amaranth, the addition of a mineral salt solution (G0 medium) greatly improved CO2 production. The A medium with a high starch content (and high C/N ratio) gave intermediate results. The range of CO2 production was larger among media (3.4±16.3%) than among strains (10.0±12.1%). 3.2. VC production The odours detected were rather pleasant with a slight alcoholic note and no dierences were reported by the panel between the 32 combinations. This diers from the fruity notes detected in cultures of Ceratocystis ®mbriata grown in the same conditions on cassava bagasse (Christen et al., 1997). Signi®cant dierences were observed in VC production between media (p < 0:0001), but not between strains. This indicates that VC production depends mostly on the medium used. The highest VC production was obtained with amaranth (Table 3), and was greatly enhanced by the addition of the mineral salts solution (G0 medium). Medium E gave an intermediate result. The VC production was rather poor with the other media. The addition of soyabean oil (D medium) neither increased the VC production nor promoted speci®c odours, although some species of this genus are known to produce esterases. For all media used, no relation was
Table 3 Classi®cation of media and strains for CO2 and total VC production by a TSR test CO2 production
*
Total VC production
Tukey groups by medium
Mean value (% v/v)
Medium
Tukey groups by medium
Mean value (lmol eq. ethanol/l)
Medium
1 2 3 3 4 5 6 7
16.35 14.70 13.58 13.40 11.98 10.37 5.42 3.42
C D B F A G0 G E
1 2 3 4 5 5 5 6
209.2 71.9 49.1 31.8 21.8 15.5 14.4 8.0
G0 G E F C B D A
Tukey groups by strain
Mean value (% v/v)
Strain
Tukey groups by strain
Mean value (lmol eq. ethanol/l)
Strain
1 2 3 4
12.10 11.62 10.93 9.96
Ro3 Ro1 R. sp. Ro2
1 2 2 2
63.2 52.0 48.4 47.3
Ro1 Ro2 R. sp. Ro3
Signi®cant with p < 0:05.
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P. Christen et al. / Bioresource Technology 71 (2000) 211±215
Table 4 Compounds identi®ed in the headspace of cultures of R. oryzae 1 Medium A B C D E F G G0
Acetaldehyde a
+ + + ++d + + + ++
Ethanol b
+++ +++ +++ +++ +++ +++ +++ +++
1-propanol + + + + + + + +
Ethyl acetate + + + + + + + ++
Ethyl propionate c
± ± ± ± ± + ± +
3-MB + + + + + + + ++
a
<1 lmol/l. >10 lmol/l. c None. d 1±10 lmol/l. b
observed between CO2 and total VC production. These results oppose those obtained for C. ®mbriata grown on various solid substrates (Meza et al., 1998). The four Rhizopus strains showed few dierences in the metabolites produced. The case of Ro1 is presented in Table 4. The number of compounds detected varied between 6 and 9 according to the media, among them acetaldehyde, ethanol, 1-propanol, ethyl acetate, ethyl propionate, and 3-methyl butanol (3-MB) were identi®ed. Some of these compounds were previously reported as growth indicators of moulds, especially 3-MB (B orjesson et al., 1990). Lanciotti and Guerzoni (1993) observed that metabolic vapours of R. arrhizus containing ethanol and 3-MB displayed an antifungal activity. Ethanol was always the most abundant volatile compound ( >80%). Its production was favoured by the high C/N ratio. Also low aeration, associated with static cultivation, promoted the fermentative activity. Rhizopus oryzae 1, which produced the highest quantity of VC, was used to study the time course of total VC production on the eight media (Fig. 1). In media A±F, total VC did not exceed 40 lmol eq. ethanol/l. Their production was increased when amaranth was used as substrate, and attained 283 lmol eq. ethanol/l when mineral salt solution was added to amaranth. For media A, B, C and D, after a short lag phase, maxima were attained within 20 h (as for CO2 ), while 40 h were needed on media F and G0 . The lag phase was much longer on medium E as observed with CO2 production. 4. Conclusion This study showed that agro-industrial substrates could be rapidly transformed by Rhizopus strains to produce VC. Large dierences were found, in terms of VC and CO2 productions, depending on the medium used. Dierences were not statistically signi®cant among
Fig. 1. Total volatile evolution in the headspace of cultures of R. oryzae 1 on eight dierent solid media.
strains for VC production. Study with strain Ro1 showed that ethanol was the major VC produced. Acetaldehyde, ethyl acetate, 1-propanol, and 3-MB were always present, at low or moderate concentrations. The odours of the cultures were pleasant but no relationship was established between the sensorial analysis and the amount of VC produced. VC production increased three times and CO2 production twice when a mineral salt solution was added to the amaranth medium. The system of open cultures used, adequate for this screening, did not allow VC production at levels satisfactory from an economic point of view. This could be improved by using forced aeration, a closed system and an adequate recovery set-up (e.g. with Tenax resin or activated carbon).
P. Christen et al. / Bioresource Technology 71 (2000) 211±215
Acknowledgements The authors wish to thank P. Torres (UAM, Mexico) for his help in statistical analysis, Dr. S. Huerta (UAM, Mexico) for the C/N analysis and Dr. P. Roger (IRD, France) for his helpful comments on the manuscript. This project was partly funded by IRD (France), CNPq (Brazil) and CONACyT (Mexico). A. Bramorski received a grant from the International Relation Service of IRD during her stay in Mexico.
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