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γ-Linolenic acid production with Thamnidium elegans by solid-state fermentation on apple pomace
Bioresource Technology 73 (2000) 41±45
c-Linolenic acid production with Thamnidium elegans by solid-state fermentation on apple pomace Miroslav Stredansky*, Elena Conti, Silvia Stredanska, Flavio Zanetti POLYtech S.C. ar. l., Area Science Park, Padriciano 99, I-34012 Trieste, Italy Received 14 June 1999; received in revised form 20 August 1999; accepted 26 August 1999
Abstract Apple pomace (AP) and spent malt grains (SMG) were used as the major substrate components for the production of a highvalue fungal oil containing up to 11.43% biologically active c-linolenic acid (GLA). The solid-state fermentation (SSF) process developed for GLA production was aected by substrate composition and moisture, agitation and aeration. Under optimised conditions GLA yields as high as 3.50 g/kg moist substrate were obtained from Thamnidium elegans CCF 1456, grown on a substrate consisting of a mixture of AP and SMG impregnated with peanut oil and a nutrient solution, after 8 days of incubation. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: c-Linolenic acid; Solid-state fermentation; Mucorales
1. Introduction c-Linolenic acid (GLA, cis,cis,cis-6,9,12-octadecatrienoic acid), a precursor of a variety of biologically active eicosanoids, is found in the seed oil of evening primrose, borage and black currants and is incorporated into pharmaceutical preparations which claim bene®cial eects in the treatment of eczema, diabetes, cancers and other diseases (Horrobin, 1992). In addition to the pharmaceutical applications, the forti®cation of food with polyunsaturated fatty acids, coupled with an increasing public awareness of ``healthy'' foods, has brought these compounds to the attention of consumers (Gill and Valivety, 1997). Moulds of the Zygomycetes, especially the Mucorales, are known to accumulate GLA in the mycelium. Currently the only industrial plant for the production of GLA by fungal fermentation is in Japan (Ratledge, 1993; Hiruda et al., 1996). Cheaper substrates than the commonly used glucose have been assayed for the fungal production of GLA, such as starch (Chen and Chang, 1996), acetic acid (Immelman et al., 1997), ethanol (Emelyanova, 1997), in order to make this process competitive with the traditional extraction from plant
sources. Solid-state fermentation (SSF) on cereal substrate was used for the production of GLA (Emelyanova, 1996) and some other polyunsaturated fatty acids (Stredanska et al., 1993). SSF is a process in which micro-organisms grow on a moist solid substrate in the absence of free water and which allows utilization of cheap raw materials and residues of agro- and food-industries as fermentation substrates (Pandey, 1992). Apple pomace, a solid residue obtained after crushing and pressing apples during the manufacture of applejuice, is a cumbersome waste product often considered of little value as animal foodstu or for pectin production. SSF on apple pomace has been performed for the production of ethanol (Ngadi and Correia, 1992) and citric acid (Hang and Woodams, 1987). In this work, we evaluated the feasibility of GLA production by SSF on apple pomace with a number of Mucorales strains. The fermentation process was then optimised using the producing strain Thamnidium elegans CCF 1456. 2. Methods 2.1. Micro-organism maintenance and spore inoculum preparation
*
Corresponding author. Tel.: +39-40-375-6621; fax: +39-40-9220016. E-mail address: [email protected] (M. Stredansky).
The fungal strains, listed in Table 1, were obtained from the Culture Collection of Fungi (Charles Univer-
0960-8524/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 1 3 2 - 7
42
M. Stredansky et al. / Bioresource Technology 73 (2000) 41±45
Table 1 Fungal oil and GLA production with Mucorales strains on solid substrates consisting of 15 g AP, 5 g SMG and 30 ml of nutrient solution (8 days cultivation at 24°C)
a b
Strain
Conversion of AP into fungal oil (% w/w)b
Oil content in dry fermented mass (% w/w)
GLA in TFAa
GLA yield in g/kg of
(% w/w)
Wet substrate
Dry substrate
Mortierella isabelina CCF14 Cunninghamella elegans CCF1318 Cunninghamella echinulata CCF103 Thamnidium elegans CCF 1456
15.7 12.5 14.1
16.8 16.6 15.0
4.23 7.43 5.78
1.40 1.95 1.70
3.49 4.89 4.26
13.1
17.2
7.53
2.07
5.17
TFA: Total fatty acids. In grams of oil produced per 100 g of AP.
sity, Prague, Czech Republic) and maintained on Czapek-Dox agar slants at 4°C. The spore inoculum was prepared by washing the mycelium grown on Sabouraud agar for 4 days at 28°C, with an aqueous solution of 0.1% (w/v) Tween 80. The so-obtained spore suspension, containing ca. 1 ´ 105 spores/ml, was used for inoculating the sterilised substrate. 2.2. Solid substrate preparation and cultivation Flask cultivation. 250 ml Erlenmeyer ¯asks were ®lled each with 15 g of dry crushed apple pomace (AP obtained from ERSA, Pozzuolo del Friuli, Italy) impregnated with 30 ml of a nutrient solution containing (g/l): NaNO3 , 4; K2 HPO4 , 2; MgSO4 7H2 O, 0.5; and yeast extract, 1. Then 5 g of dry spent malt grains (SMG, obtained from the brewery plant Moretti, S.Giorgio di Nogaro, Italy) were added and mixed with the moistened AP, so as to increase substrate porosity. In some experiments a dierent ratio of AP to SMG was used, as speci®ed in Section 3. Static cultivation was performed at 24°C in a humidi®ed (near to 100%) atmosphere for 8 days, after inoculating 2 ml of spore suspension into each ¯ask. Cultivation in rotating bottles. 500 ml bottles (75 mm diameter, 165 mm length) were ®lled each with 45 g of AP impregnated with 90 ml of the nutrient solution and mixed with 15 g of SMG. Bottles were placed in a roller culture apparatus (Wheaton Instruments) set at a rotation rate of 0.5 rpm. Cultivation was performed at 24°C in a humidi®ed atmosphere for 8 days, after inoculating 6 ml of spore suspension into each bottle. Bag cultivation. Autoclavable bags (500 300 mm2 , cultivation area 600 cm2 ) were ®lled each with 90 g AP impregnated with 180 ml of nutrient solution and mixed with 30 g of SMG. The aperture of the bag was sealed with a plug bearing air inlet and outlet openings, those provided with sterile ®lters. During cultivation (8 days, 24°C), humidi®ed sterile air was supplied at a constant rate of 100 ml/min. Each bag was inoculated with 12 ml spore suspension.
In experiments with enriched substrates glucose or glycerol were added to the nutrient solution, while oil was applied to the impregnated substrate. The fermentation vessels containing the solid substrate were sterilised in the autoclave at 121°C before inoculating. Triplicate cultures were set up for each condition tested. 2.3. Analytical procedures Extraction of lipid and determination of fatty acids. The whole fermented mass (biomass plus residual substrate) was dried in the oven, weighed and milled with a Waring blender. The lipid fraction was extracted with chloroform/methanol (2:1) according to Folch et al. (1957). Lipid extracts were derivatised to methyl esters by treatment with 0.5 M NaOH methanolic solution, followed by heating at 80°C for 5 min and addition of BF3 /methanol complex. The mixture was boiled for 2 min and extracted with heptane. Fatty acid methyl esters were analysed by gas chromatography with a HewlettPackard 5890A gas-chromatography apparatus, equipped with an SP-2330 Supelco fused silica capillary column, using heptadecanoic acid (Sigma) as internal standard.
3. Results and discussion 3.1. Screening of strains Twenty-three strains of the genera Mortierella, Cunninghamella, Rhizopus, Mucor and Thamnidium were screened for GLA production on solid substrates containing AP. Fungal oil production and GLA yields obtained from the best strains screened are reported in Table 1. All the tested strains of the genera Mortierella, Thamnidium and Cunninghamella showed good growth, with the mycelium penetrating inside the substrate particles. Mucor and Rhizopus strains exhibited poor or no growth on AP, due to the acidic pH (3.5) of the sub-
M. Stredansky et al. / Bioresource Technology 73 (2000) 41±45
Fig. 1. Time course of oil and GLA production by T. elegans CCF 1456 in ¯ask cultivations at 24°C. Symbols: n, % fungal oil in the fermented mass (w/w); d, yield of GLA in g/kg of wet substrate; m, % GLA in total fatty acids (w/w).
strate. In fact, when the AP was neutralised with KOH before inoculating, a satisfactory growth was observed with these strains. However, GLA production was lower than that found with the strains listed in Table 1. Thamnidium elegans was chosen for further work. The time-course of the process with T. elegans is shown in Fig. 1. It is interesting to note that while the ®nal yield of GLA reached a plateau after 7 days of fermentation, the percentage of GLA in the oil extracted increased steadily until day 10 of fermentation. Thus, prolonged fermentation might allow of obtaining a product with a GLA content near to that found in plant seed oil (evening primrose oil from 8% to 12%). 3.2. Eect of solid substrate type and composition The AP provides a good source of assimilable nutrients for micro-organisms, such as sugars (glucose and fructose account for up to 50% of the dry matter), pectin, organic acids, etc. However, the addition of the amount of water required for fungal growth produces a slurry matter which is unsuitable for the fermentation process. SMG consisting of a high portion of cellulose and other insoluble polysaccharides, were found to prevent fermentation-mass packing (Stredansky and Conti, 1999) and were mixed with the impregnated AP so as to provide a porous solid substrate, also allowing a higher oxygen availability to the growing organisms. As shown in Fig. 2, a ratio of AP to SMG of 3 to 1 proved optimal for fungal oil and GLA production. Increasing the proportion of AP in the substrate resulted in the formation of a more compact mass, in which substrate utilization was incomplete, and less lipid, with a lower content in GLA, was accumulated in the mycelium. Conversely, if a greater portion of SMG was used, a poorer oil production was recorded, although it was richer in GLA, as a consequence of the reduced availability of assimilable compounds in the substrate.
43
Fig. 2. Fungal oil and GLA production by T. elegans CCF 1456 with increasing ratio of AP to SMG in the substrate. 8 days cultivation at 24°C. Symbols: n, % fungal oil in the fermented mass (w/w); m, % GLA in total fatty acids (w/w); d, yield of GLA in g/kg of wet substrate.
As an alternative to AP and SMG, citrus peel and crushed corn cobs were used to form the solid substrate. However, when citrus peel was used instead of AP, the GLA production by T. elegans and C. elegans decreased to ca. 40% of that obtained from AP. Similarly, when corn cobs were used in substitution for SMG, the GLA yields decreased to ca. 60%. 3.3. Eect of the water content in the substrate The degree of moisture in the solid substrate strongly aects the performance of SSF processes (Pandey, 1992). Fig. 3 shows that increasing the amount of water used to impregnate the substrate led to an increment of both the GLA content in the fungal oil and the GLA yield, as referred to the dry substrate, giving an indication of a superior substrate utilization under higher moisture conditions. On the other hand, the GLA yield, as referred to the wet substrate, decreased with in-
Fig. 3. Eect of substrate moisture on GLA production. 8 days cultivation at 24°C. Symbols: n, % GLA in total fatty acids (w/w); d, yield of GLA in g/kg of dry substrate; m, yield of GLA in g/kg of wet substrate.
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M. Stredansky et al. / Bioresource Technology 73 (2000) 41±45
It was surprising to ®nd that, despite the high sugar content in the AP + MSG mix, the most signi®cant eect on the lipid yield was achieved when the substrate was supplemented with additional C compounds (Table 2). The highest GLA yield was achieved with the substrate enriched with peanut oil (not containing GLA), which serves as a direct precursor for the formation of fungal oil. Plant oils are often supplied to fungal cultivations to increase the PUFA yield (Shinmen et al., 1989).
creasing water content, which indicated a drop in the process productivity. We used the intermediate value of 60% (w/w) water in the substrate in the following experiments, which fell within the optimal range for SSF processes with fungi (Hang and Woodams, 1987), while it was unfavourable for bacterial fermentation (Stredansky and Conti, 1999). The low moisture content combined with the acidic pH of the AP-based substrate may signi®cantly decrease the risk of bacterial contamination in fungal fermentation systems. The oil content in the fermented mass was not affected by the substrate moisture (data not shown).
3.5. Eect of agitation and aeration Agitation and aeration facilitate the maintenance of homogeneous conditions within the SSF bioreactor, especially with respect to the temperature and the gaseous environment. These are especially important in view of a process scaling-up (Mitchell et al., 1989). The eect of agitation was tested using the rotating bottle. The results obtained with rotating culture were very similar to those with static culture (Table 3), although a dierent mode of growth was observed. In fact, spherical aggregates of substrate and mycelial growth of ca. 1 cm diameter formed during cultivation due to the rotating movement which resulted in a higher density of the fermented mass as compared to the static culture. The results obtained indicated that process scale-up might be feasible with the use of a rotating drum-type bioreactor. The eect of aeration was investigated in cultures growing in sterile plastic bags with the same substrate layer thickness as used in the Erlenmeyer ¯asks. The
3.4. Enrichment of the substrate In spite of the satisfactory results obtained using AP as the main nutrient source for the production of GLA enriched oil by T. elegans, the unbalanced content of C compounds with respect to N compounds, the latter being found at a very low concentration as compared to the former, could pose a limitation to the optimal process productivity. Thus, the C/N ratio of the substrate was adjusted to more favourable values through the addition of exogenous N compounds. NaNO3 proved a suitable N source in screening experiments, and was used at a concentration of 4 g/l in the impregnating solution. Also the addition of yeast extract, potassium phosphate and magnesium sulphate up to concentrations of 1, 2 and 0.5 g/l, respectively, showed a positive eect on the lipid and GLA production, and they were always included in the nutrient solution (see Section 2).
Table 2 Fungal oil and GLA production with Thamnidium elegans CCF 1456 on solid substrates consisting of 15 g AP, 5 g SMG and 30 ml of nutrient solution enriched with 2 g of C-source (8 days cultivation at 24°C)
a
Substrate supplement
Oil content in dry fermented mass (% w/w)
GLA in TFAa
GLA yield in g/kg of
(% w/w)
Wet substrate
Dry substrate
± Glucose Glycerol Peanut oil
17.0 16.8 19.0 21.0
7.72 8.75 7.58 7.61
2.11 2.61 2.58 3.04
5.27 6.17 6.10 7.16
TFA: Total fatty acids.
Table 3 Eect of substrate supplementation with C-sources on GLA production by Thamnidium elegans under either agitation (rotation rate of 0.5 rpm) or forced aeration (air constant rate of 100 ml/min) conditions (8 days cultivation at 24°C)
a
GLA yield in g/kg of
Cultivation mode
Substrate supplement
Oil content in dry fermented mass (% w/w)
GLA in TFAa (% w/w)
Wet substrate
Dry substrate
Agitation Agitation Agitation Aeration Aeration Aeration
± Glucose Peanut oil ± Glucose Peanut oil
18.0 17.9 20.8 17.6 18.2 21.2
7.22 8.51 7.38 9.39 11.43 9.11
2.15 2.49 2.98 2.57 3.16 3.50
5.37 6.23 7.45 6.43 7.90 8.75
TFA: Total fatty acids.
M. Stredansky et al. / Bioresource Technology 73 (2000) 41±45
application of forced aeration had a positive eect on the process performance (Table 3). While the variation of fungal oil yield was negligible, the proportion of GLA to total fatty acids increased, so as to result in a higher GLA yield. Since GLA is synthesised from stearic acid via oleic and linoleic acids through three consecutive desaturation steps, these results suggest that the availability of excess oxygen through forced aeration directly aected fatty acids desaturation. It was previously shown that micro-organisms require molecular oxygen for the desaturation steps occurring in the PUFA biosynthetic pathway, and that oxygen availability determines the degree of unsaturation of the fatty acids produced (Yongmanitchai and Ward, 1989). The highest yield was achieved on the substrate enriched with peanut oil, giving 3.50 g GLA per kg of initial wet substrate, or 4.54 g/kg of wet fermented mass. The product with the highest concentration of GLA (11.4% of total fatty acids) was achieved from cultivation on the substrate supplemented with glucose, when the yields were 3.16 g/kg of initial wet substrate and 4.15 g/kg of wet fermented mass. Other major fatty acids were found in the following percentages: palmitic 17.5%; stearic 5.8%; oleic 42.8%; linolenic 18.9%. These results are not easily comparable with published data on the same subject. Emelyanova (1996) reported on the GLA production by Cunninghamella japonica in SSF, but data on yields, as referred to the substrate weight and on substrate conversion into the fungal oil, are ambiguous or missing. Furthermore, it is impossible to correlate results from SSF to those obtained in conventional submerged fermentation. Reported yields of GLA in submerged cultivation are: 0.96 g/l (Cheng and Chang, 1996) from Cunninghamella echinulata cultivated on 10% soluble starch; 0.51 g/l achieved by Immelman et al. (1997) cultivating Mucor circinelloides on acetate; and 5.61 g/l obtained by Hiruda et al. (1996) from Mortierella ramanniana on 30% glucose in a 600 l pilot fermentor. GLA yields from submerged cultivation of the strain T. elegans CCF 1456 used in this study never exceeded 1 g/l (our unpublished results). In conclusion, the SSF technique was successfully applied to produce GLA-enriched fungal oil. As compared to submerged fermentation, this technique oers the additional advantages of a substantial reduction of wastewater from the fermentation process and the possibility of using wastes from the agro-food industries as
45
substrate components, thus making the overall process more favourable to the environment.
References Chen, H.-C., Chang, C.-C., 1996. Production of c-linolenic acid by the fungus Cunninghamella echinulata CCRC31840. Biotechnol. Prog. 12, 338±341. Emelyanova, E.V., 1996. c-Linolenic acid production by Cunninghamella japonica in solid state fermentation. Process Biochem. 31, 431± 434. Emelyanova, E.V., 1997. Lipid and c-linolenic acid production by Mucor inaquisporus. Process Biochem. 32, 173±177. Folch, J., Lees, M., Sloane-Stanley, G.H., 1957. A simple method for isolation and puri®cation of total lipids from animal tissues. J. Biol. Chem. 226, 450±497. Gill, I., Valivety, R., 1997. Polyunsaturated fatty acids, part 1: Occurrence, biological activities and applications. Trends Biotechnol. 15, 401±409. Hang, Y.D., Woodams, E.E., 1987. Eect of substrate moisture content on fungal production of citric acid in a solid state fermentation system. Biotechnol. Lett. 9, 183±186. Hiruda, O., Futamura, T., Takebe, H., Satoh, A., Kamisaka, Y., Yokochi, T., Nakahara, T., Suzuki, O., 1996. Optimization and scale up of c-linolenic acid production by Mortierella ramanniana MM 15-1, a high c-linolenic acid producing mutant. J. Ferment. Bioeng. 82, 366±370. Horrobin, D.F., 1992. Nutritional and medical importance of clinolenic acid. Prog. Lipid Res. 31, 163±194. Immelman, M., du Preez, J.C., Kilian, S.G., 1997. Eect of C:N ratio on c-linolenic acid production by Mucor circinelloides grown on acetic acid. System. Appl. Microbiol. 20, 158±164. Mitchell, D.A., Lonsane, B.K., Durand, A., Renaud, R., Almanza, S., Maratray, J., Desgranges, C., Crooke, P.S., Hong, K., Tanner, R.D., Malaney, G.W., 1989. General principles for bioreactor design and operation for SSC. In: Doelle, H.W., Mitchell, D.A., Rolz, C.E. (Eds.), Solid Substrate Cultivation. Elsevier, London, pp. 115±140. Ngadi, Y.D., Correia, L.R., 1992. Solid-state ethanol fermentation of apple pomace is aected by moisture and bioreactor mixing speed. J. Food Sci. 57, 667±670. Pandey, A., 1992. Recent process developments in solid state fermentations. Process Biochem. 27, 109±117. Ratledge, C., 1993. Single cell oils ± have they a biotechnological future?. Trends Biotechnol. 11, 278±284. Shinmen, Y., Shimidzu, S., Akimoto, K., Kawashima, H., yamada, H., 1989. Production of arachidonic acid by Mortierella fungi. Appl. Microbiol. Biotechnol. 31, 11±16. Stredanska, S., Slugen, D., Stredansky, M., Grego, J., 1993. Arachidonic acid production by Mortierella alpina grown on solid substrate. World J. Microbial. Biotechnol. 9, 511±513. Stredansky, M., Conti, E., 1999. Xanthan production by solid state fermentation. Process Biochem., in press. Yongmanitchai, W., Ward, O.P., 1989. Omega-3 fatty acids: alternative sources of production. Process Biochem. 24, 117±125.
c-Linolenic acid production with Thamnidium elegans by solid-state fermentation on apple pomace Miroslav Stredansky*, Elena Conti, Silvia Stredanska, Flavio Zanetti POLYtech S.C. ar. l., Area Science Park, Padriciano 99, I-34012 Trieste, Italy Received 14 June 1999; received in revised form 20 August 1999; accepted 26 August 1999
Abstract Apple pomace (AP) and spent malt grains (SMG) were used as the major substrate components for the production of a highvalue fungal oil containing up to 11.43% biologically active c-linolenic acid (GLA). The solid-state fermentation (SSF) process developed for GLA production was aected by substrate composition and moisture, agitation and aeration. Under optimised conditions GLA yields as high as 3.50 g/kg moist substrate were obtained from Thamnidium elegans CCF 1456, grown on a substrate consisting of a mixture of AP and SMG impregnated with peanut oil and a nutrient solution, after 8 days of incubation. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: c-Linolenic acid; Solid-state fermentation; Mucorales
1. Introduction c-Linolenic acid (GLA, cis,cis,cis-6,9,12-octadecatrienoic acid), a precursor of a variety of biologically active eicosanoids, is found in the seed oil of evening primrose, borage and black currants and is incorporated into pharmaceutical preparations which claim bene®cial eects in the treatment of eczema, diabetes, cancers and other diseases (Horrobin, 1992). In addition to the pharmaceutical applications, the forti®cation of food with polyunsaturated fatty acids, coupled with an increasing public awareness of ``healthy'' foods, has brought these compounds to the attention of consumers (Gill and Valivety, 1997). Moulds of the Zygomycetes, especially the Mucorales, are known to accumulate GLA in the mycelium. Currently the only industrial plant for the production of GLA by fungal fermentation is in Japan (Ratledge, 1993; Hiruda et al., 1996). Cheaper substrates than the commonly used glucose have been assayed for the fungal production of GLA, such as starch (Chen and Chang, 1996), acetic acid (Immelman et al., 1997), ethanol (Emelyanova, 1997), in order to make this process competitive with the traditional extraction from plant
sources. Solid-state fermentation (SSF) on cereal substrate was used for the production of GLA (Emelyanova, 1996) and some other polyunsaturated fatty acids (Stredanska et al., 1993). SSF is a process in which micro-organisms grow on a moist solid substrate in the absence of free water and which allows utilization of cheap raw materials and residues of agro- and food-industries as fermentation substrates (Pandey, 1992). Apple pomace, a solid residue obtained after crushing and pressing apples during the manufacture of applejuice, is a cumbersome waste product often considered of little value as animal foodstu or for pectin production. SSF on apple pomace has been performed for the production of ethanol (Ngadi and Correia, 1992) and citric acid (Hang and Woodams, 1987). In this work, we evaluated the feasibility of GLA production by SSF on apple pomace with a number of Mucorales strains. The fermentation process was then optimised using the producing strain Thamnidium elegans CCF 1456. 2. Methods 2.1. Micro-organism maintenance and spore inoculum preparation
*
Corresponding author. Tel.: +39-40-375-6621; fax: +39-40-9220016. E-mail address: [email protected] (M. Stredansky).
The fungal strains, listed in Table 1, were obtained from the Culture Collection of Fungi (Charles Univer-
0960-8524/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 1 3 2 - 7
42
M. Stredansky et al. / Bioresource Technology 73 (2000) 41±45
Table 1 Fungal oil and GLA production with Mucorales strains on solid substrates consisting of 15 g AP, 5 g SMG and 30 ml of nutrient solution (8 days cultivation at 24°C)
a b
Strain
Conversion of AP into fungal oil (% w/w)b
Oil content in dry fermented mass (% w/w)
GLA in TFAa
GLA yield in g/kg of
(% w/w)
Wet substrate
Dry substrate
Mortierella isabelina CCF14 Cunninghamella elegans CCF1318 Cunninghamella echinulata CCF103 Thamnidium elegans CCF 1456
15.7 12.5 14.1
16.8 16.6 15.0
4.23 7.43 5.78
1.40 1.95 1.70
3.49 4.89 4.26
13.1
17.2
7.53
2.07
5.17
TFA: Total fatty acids. In grams of oil produced per 100 g of AP.
sity, Prague, Czech Republic) and maintained on Czapek-Dox agar slants at 4°C. The spore inoculum was prepared by washing the mycelium grown on Sabouraud agar for 4 days at 28°C, with an aqueous solution of 0.1% (w/v) Tween 80. The so-obtained spore suspension, containing ca. 1 ´ 105 spores/ml, was used for inoculating the sterilised substrate. 2.2. Solid substrate preparation and cultivation Flask cultivation. 250 ml Erlenmeyer ¯asks were ®lled each with 15 g of dry crushed apple pomace (AP obtained from ERSA, Pozzuolo del Friuli, Italy) impregnated with 30 ml of a nutrient solution containing (g/l): NaNO3 , 4; K2 HPO4 , 2; MgSO4 7H2 O, 0.5; and yeast extract, 1. Then 5 g of dry spent malt grains (SMG, obtained from the brewery plant Moretti, S.Giorgio di Nogaro, Italy) were added and mixed with the moistened AP, so as to increase substrate porosity. In some experiments a dierent ratio of AP to SMG was used, as speci®ed in Section 3. Static cultivation was performed at 24°C in a humidi®ed (near to 100%) atmosphere for 8 days, after inoculating 2 ml of spore suspension into each ¯ask. Cultivation in rotating bottles. 500 ml bottles (75 mm diameter, 165 mm length) were ®lled each with 45 g of AP impregnated with 90 ml of the nutrient solution and mixed with 15 g of SMG. Bottles were placed in a roller culture apparatus (Wheaton Instruments) set at a rotation rate of 0.5 rpm. Cultivation was performed at 24°C in a humidi®ed atmosphere for 8 days, after inoculating 6 ml of spore suspension into each bottle. Bag cultivation. Autoclavable bags (500 300 mm2 , cultivation area 600 cm2 ) were ®lled each with 90 g AP impregnated with 180 ml of nutrient solution and mixed with 30 g of SMG. The aperture of the bag was sealed with a plug bearing air inlet and outlet openings, those provided with sterile ®lters. During cultivation (8 days, 24°C), humidi®ed sterile air was supplied at a constant rate of 100 ml/min. Each bag was inoculated with 12 ml spore suspension.
In experiments with enriched substrates glucose or glycerol were added to the nutrient solution, while oil was applied to the impregnated substrate. The fermentation vessels containing the solid substrate were sterilised in the autoclave at 121°C before inoculating. Triplicate cultures were set up for each condition tested. 2.3. Analytical procedures Extraction of lipid and determination of fatty acids. The whole fermented mass (biomass plus residual substrate) was dried in the oven, weighed and milled with a Waring blender. The lipid fraction was extracted with chloroform/methanol (2:1) according to Folch et al. (1957). Lipid extracts were derivatised to methyl esters by treatment with 0.5 M NaOH methanolic solution, followed by heating at 80°C for 5 min and addition of BF3 /methanol complex. The mixture was boiled for 2 min and extracted with heptane. Fatty acid methyl esters were analysed by gas chromatography with a HewlettPackard 5890A gas-chromatography apparatus, equipped with an SP-2330 Supelco fused silica capillary column, using heptadecanoic acid (Sigma) as internal standard.
3. Results and discussion 3.1. Screening of strains Twenty-three strains of the genera Mortierella, Cunninghamella, Rhizopus, Mucor and Thamnidium were screened for GLA production on solid substrates containing AP. Fungal oil production and GLA yields obtained from the best strains screened are reported in Table 1. All the tested strains of the genera Mortierella, Thamnidium and Cunninghamella showed good growth, with the mycelium penetrating inside the substrate particles. Mucor and Rhizopus strains exhibited poor or no growth on AP, due to the acidic pH (3.5) of the sub-
M. Stredansky et al. / Bioresource Technology 73 (2000) 41±45
Fig. 1. Time course of oil and GLA production by T. elegans CCF 1456 in ¯ask cultivations at 24°C. Symbols: n, % fungal oil in the fermented mass (w/w); d, yield of GLA in g/kg of wet substrate; m, % GLA in total fatty acids (w/w).
strate. In fact, when the AP was neutralised with KOH before inoculating, a satisfactory growth was observed with these strains. However, GLA production was lower than that found with the strains listed in Table 1. Thamnidium elegans was chosen for further work. The time-course of the process with T. elegans is shown in Fig. 1. It is interesting to note that while the ®nal yield of GLA reached a plateau after 7 days of fermentation, the percentage of GLA in the oil extracted increased steadily until day 10 of fermentation. Thus, prolonged fermentation might allow of obtaining a product with a GLA content near to that found in plant seed oil (evening primrose oil from 8% to 12%). 3.2. Eect of solid substrate type and composition The AP provides a good source of assimilable nutrients for micro-organisms, such as sugars (glucose and fructose account for up to 50% of the dry matter), pectin, organic acids, etc. However, the addition of the amount of water required for fungal growth produces a slurry matter which is unsuitable for the fermentation process. SMG consisting of a high portion of cellulose and other insoluble polysaccharides, were found to prevent fermentation-mass packing (Stredansky and Conti, 1999) and were mixed with the impregnated AP so as to provide a porous solid substrate, also allowing a higher oxygen availability to the growing organisms. As shown in Fig. 2, a ratio of AP to SMG of 3 to 1 proved optimal for fungal oil and GLA production. Increasing the proportion of AP in the substrate resulted in the formation of a more compact mass, in which substrate utilization was incomplete, and less lipid, with a lower content in GLA, was accumulated in the mycelium. Conversely, if a greater portion of SMG was used, a poorer oil production was recorded, although it was richer in GLA, as a consequence of the reduced availability of assimilable compounds in the substrate.
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Fig. 2. Fungal oil and GLA production by T. elegans CCF 1456 with increasing ratio of AP to SMG in the substrate. 8 days cultivation at 24°C. Symbols: n, % fungal oil in the fermented mass (w/w); m, % GLA in total fatty acids (w/w); d, yield of GLA in g/kg of wet substrate.
As an alternative to AP and SMG, citrus peel and crushed corn cobs were used to form the solid substrate. However, when citrus peel was used instead of AP, the GLA production by T. elegans and C. elegans decreased to ca. 40% of that obtained from AP. Similarly, when corn cobs were used in substitution for SMG, the GLA yields decreased to ca. 60%. 3.3. Eect of the water content in the substrate The degree of moisture in the solid substrate strongly aects the performance of SSF processes (Pandey, 1992). Fig. 3 shows that increasing the amount of water used to impregnate the substrate led to an increment of both the GLA content in the fungal oil and the GLA yield, as referred to the dry substrate, giving an indication of a superior substrate utilization under higher moisture conditions. On the other hand, the GLA yield, as referred to the wet substrate, decreased with in-
Fig. 3. Eect of substrate moisture on GLA production. 8 days cultivation at 24°C. Symbols: n, % GLA in total fatty acids (w/w); d, yield of GLA in g/kg of dry substrate; m, yield of GLA in g/kg of wet substrate.
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M. Stredansky et al. / Bioresource Technology 73 (2000) 41±45
It was surprising to ®nd that, despite the high sugar content in the AP + MSG mix, the most signi®cant eect on the lipid yield was achieved when the substrate was supplemented with additional C compounds (Table 2). The highest GLA yield was achieved with the substrate enriched with peanut oil (not containing GLA), which serves as a direct precursor for the formation of fungal oil. Plant oils are often supplied to fungal cultivations to increase the PUFA yield (Shinmen et al., 1989).
creasing water content, which indicated a drop in the process productivity. We used the intermediate value of 60% (w/w) water in the substrate in the following experiments, which fell within the optimal range for SSF processes with fungi (Hang and Woodams, 1987), while it was unfavourable for bacterial fermentation (Stredansky and Conti, 1999). The low moisture content combined with the acidic pH of the AP-based substrate may signi®cantly decrease the risk of bacterial contamination in fungal fermentation systems. The oil content in the fermented mass was not affected by the substrate moisture (data not shown).
3.5. Eect of agitation and aeration Agitation and aeration facilitate the maintenance of homogeneous conditions within the SSF bioreactor, especially with respect to the temperature and the gaseous environment. These are especially important in view of a process scaling-up (Mitchell et al., 1989). The eect of agitation was tested using the rotating bottle. The results obtained with rotating culture were very similar to those with static culture (Table 3), although a dierent mode of growth was observed. In fact, spherical aggregates of substrate and mycelial growth of ca. 1 cm diameter formed during cultivation due to the rotating movement which resulted in a higher density of the fermented mass as compared to the static culture. The results obtained indicated that process scale-up might be feasible with the use of a rotating drum-type bioreactor. The eect of aeration was investigated in cultures growing in sterile plastic bags with the same substrate layer thickness as used in the Erlenmeyer ¯asks. The
3.4. Enrichment of the substrate In spite of the satisfactory results obtained using AP as the main nutrient source for the production of GLA enriched oil by T. elegans, the unbalanced content of C compounds with respect to N compounds, the latter being found at a very low concentration as compared to the former, could pose a limitation to the optimal process productivity. Thus, the C/N ratio of the substrate was adjusted to more favourable values through the addition of exogenous N compounds. NaNO3 proved a suitable N source in screening experiments, and was used at a concentration of 4 g/l in the impregnating solution. Also the addition of yeast extract, potassium phosphate and magnesium sulphate up to concentrations of 1, 2 and 0.5 g/l, respectively, showed a positive eect on the lipid and GLA production, and they were always included in the nutrient solution (see Section 2).
Table 2 Fungal oil and GLA production with Thamnidium elegans CCF 1456 on solid substrates consisting of 15 g AP, 5 g SMG and 30 ml of nutrient solution enriched with 2 g of C-source (8 days cultivation at 24°C)
a
Substrate supplement
Oil content in dry fermented mass (% w/w)
GLA in TFAa
GLA yield in g/kg of
(% w/w)
Wet substrate
Dry substrate
± Glucose Glycerol Peanut oil
17.0 16.8 19.0 21.0
7.72 8.75 7.58 7.61
2.11 2.61 2.58 3.04
5.27 6.17 6.10 7.16
TFA: Total fatty acids.
Table 3 Eect of substrate supplementation with C-sources on GLA production by Thamnidium elegans under either agitation (rotation rate of 0.5 rpm) or forced aeration (air constant rate of 100 ml/min) conditions (8 days cultivation at 24°C)
a
GLA yield in g/kg of
Cultivation mode
Substrate supplement
Oil content in dry fermented mass (% w/w)
GLA in TFAa (% w/w)
Wet substrate
Dry substrate
Agitation Agitation Agitation Aeration Aeration Aeration
± Glucose Peanut oil ± Glucose Peanut oil
18.0 17.9 20.8 17.6 18.2 21.2
7.22 8.51 7.38 9.39 11.43 9.11
2.15 2.49 2.98 2.57 3.16 3.50
5.37 6.23 7.45 6.43 7.90 8.75
TFA: Total fatty acids.
M. Stredansky et al. / Bioresource Technology 73 (2000) 41±45
application of forced aeration had a positive eect on the process performance (Table 3). While the variation of fungal oil yield was negligible, the proportion of GLA to total fatty acids increased, so as to result in a higher GLA yield. Since GLA is synthesised from stearic acid via oleic and linoleic acids through three consecutive desaturation steps, these results suggest that the availability of excess oxygen through forced aeration directly aected fatty acids desaturation. It was previously shown that micro-organisms require molecular oxygen for the desaturation steps occurring in the PUFA biosynthetic pathway, and that oxygen availability determines the degree of unsaturation of the fatty acids produced (Yongmanitchai and Ward, 1989). The highest yield was achieved on the substrate enriched with peanut oil, giving 3.50 g GLA per kg of initial wet substrate, or 4.54 g/kg of wet fermented mass. The product with the highest concentration of GLA (11.4% of total fatty acids) was achieved from cultivation on the substrate supplemented with glucose, when the yields were 3.16 g/kg of initial wet substrate and 4.15 g/kg of wet fermented mass. Other major fatty acids were found in the following percentages: palmitic 17.5%; stearic 5.8%; oleic 42.8%; linolenic 18.9%. These results are not easily comparable with published data on the same subject. Emelyanova (1996) reported on the GLA production by Cunninghamella japonica in SSF, but data on yields, as referred to the substrate weight and on substrate conversion into the fungal oil, are ambiguous or missing. Furthermore, it is impossible to correlate results from SSF to those obtained in conventional submerged fermentation. Reported yields of GLA in submerged cultivation are: 0.96 g/l (Cheng and Chang, 1996) from Cunninghamella echinulata cultivated on 10% soluble starch; 0.51 g/l achieved by Immelman et al. (1997) cultivating Mucor circinelloides on acetate; and 5.61 g/l obtained by Hiruda et al. (1996) from Mortierella ramanniana on 30% glucose in a 600 l pilot fermentor. GLA yields from submerged cultivation of the strain T. elegans CCF 1456 used in this study never exceeded 1 g/l (our unpublished results). In conclusion, the SSF technique was successfully applied to produce GLA-enriched fungal oil. As compared to submerged fermentation, this technique oers the additional advantages of a substantial reduction of wastewater from the fermentation process and the possibility of using wastes from the agro-food industries as
45
substrate components, thus making the overall process more favourable to the environment.
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