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Revista de Microbiologia

Print version ISSN 0001-3714

Rev. Microbiol. vol. 29 n. 4 São Paulo Oct./Dec. 1998

https://doi.org/10.1590/S0001-37141998000400008 

CHARACTERIZATION OF SELECTED STRAINS OF MUCORALES USING FATTY ACID PROFILES

 

Manuela da Silva1*, Gilson Paulo Manfio2, Vanderlei Perez Canhos1
1Departamento de Ciência de Alimentos, Faculdade de Engenharia de Alimentos, UNICAMP, Campinas, SP, Brasil; 2Fundação Tropical de Pesquisas e Tecnologia "André Tosello", Campinas, SP, Brazil

Submitted: September 16, 1997; Returned to authors for corrections: April 09, 1998;
Approved: September 19, 1998

 

 


ABSTRACT

The fatty acid profiles of several fungi of the order Mucorales (Zygomycetes), including Backusella lamprospora (Lendner) Benny and R.K. Benj., Benjaminiella youngii P.M. Kirk, Circinella simplex van Tieghem, Cunninghamella blakesleeana Lendner, Mortierella ramanniana (Möller) Linnem., Mucor circinelloides f. janssenii (Lendner) Schipper, Mycotypha microspora Fenner, Rhizomucor miehei (Cooney and R. Emerson) Schipper and Rhizomucor pusillus (Lindt) Schipper, and of Volutella sp. Fr., from the class Ascomycetes, were qualitatively analysed by gas-liquid chromatography in order to determine the taxonomic value of these chemotaxonomic markers. The fatty acids present in all strains were palmitic (16:0), oleic (18:1), linoleic (18:2) and g-linolenic (18:3) acid, with the exception that the latter was not found in Volutella sp. Chemotaxonomic markers for some species and genera were obtained, including a non-identified fatty acid, FAME8 (minimum and maximum retention times of 27.92 and 28.28 minutes) for Rhizomucor miehei CCT 2236 and Rhizomucor pusillus CCT 4133, and FAME3 (minimum and maximum of 16.53 and 16.61 minutes) for Benjaminiella youngii CCT 4121. The chemotaxonomic marker of the order Mucorales was the fatty acid 18:3w6, confirming previous data from literature. The results of the present study suggest that qualitative fatty acid analysis can be an important chemotaxonomic tool for the classification of fungi assigned to the order Mucorales (Zygomycetes).

Key words: taxonomy, chemotaxonomic marker, fatty acids, Mucorales


 

 

INTRODUCTION

The order Mucorales, class Zygomycetes, is comprised mainly by saprobic fungi with a cosmopolitan distribution, including some opportunistic parasites of plants or animals (8), and spoilage molds, such as Rhizopus spp. The Mucorales have important economical applications, including the production of microbial rennet by Rhizomucor miehei, and lipases by Rhizomucor miehei and R. pusillus, both for dairy industry; fumaric acid for paper industry by Rhizopus spp. (7), and fatty acids for pharmaceutical (precursors of prostaglandins) and food industries by Mortierella spp. and Mucor spp. (18).

The classification and identification of fungi are traditionally based on morphological criteria. However, the study of closely related, non-sporulating fungi and of those with unknown life cycle is limited by scarce differential morphological features (2). Biochemical and molecular methods, such as fatty acid profiles, protein and nucleic acid sequencing have been of great value to overcome these limitations (3,23).

Fatty acids are useful taxonomical markers which have been widely used in the classification of several groups of microorganisms (27). However, fatty acid composition has been applied to the characterization of relatively few fungal taxa, including Ganoderma australe (15), Mortierella spp. (1), phytopathogenic (10,11,12) and endomycorrhizal fungi (6,19).

Reproducible fatty acids profiles can be obtained under rigorous control of growth temperature, incubation time and culture media formulation (4, 14, 18). The early studies applying fatty acids analysis as a taxonomic tool for filamentous fungi were accomplished in the 60´s by Shaw (21, 22, 23, 24). This author investigated the presence of g-linolenic fatty acid in fungi from different classes, delimiting two taxonomic groups on that basis (21); one group included representatives from the class Oomycetes, Zygomycetes and Chytridiomycetes, and the other representatives from the class Ascomycetes and Basidiomycetes. Similar results were observed in later studies (30), where Zygomycetes were characterized by the presence of w6 series of C18 polyunsaturated fatty acids, in particular g-linolenic acid.

The classical characterization of Mucorales includes morphological characteristics and cultural features (1,2,6). The objective of this study was to assess the potential of fatty acid profiles in the differentiation of selected species belonging to the order Mucorales (Zygomycetes), focusing mainly on economically important taxa (Mortierella spp., Mucor spp. and Rhizomucor spp.).

 

MATERIALS AND METHODS

Fungi. Backusella lamprospora (CCT 3480; collected in Belém PA, Brazil), Benjaminiella youngii (CCT 4121, originally IMI 336111), Circinella simplex (CCT 4260; collected from roots of Cariniana sp. in the Atlantic forest in Estação Ecológica Juréia-Itatins, Peruíbe SP, Brazil), Cunninghamella blakesleeana (CCT 4123, originally IMI 200337), Mortierella ramanniana (CCT 4428; collected from Araucaria angustifolia root in São Francisco de Paula RS, Brazil), Mucor circinelloides f. janssenii (CCT 4396), Mycotypha microspora (CCT 4126, originally IMI 282443; CCT 4127, originally IMI 108621), Rhizomucor miehei (CCT 2236, originally NRRL 3420), Rhizomucor pusillus (CCT 4133; isolated from shoyu (soybean sauce) in Campinas SP, Brazil), Volutella sp. (CCT 2995; collected from soil under the Atlantic forest in Estação Ecológica Juréia-Itatins, Peruíbe SP, Brazil) were preserved by freeze drying at the Culture Collection Tropical (CCT), Fundação Tropical de Pesquisas e Tecnologia "André Tosello", Campinas SP, Brazil.

Culture methods. Fungal strains were cultivated on Potato Carrot Agar medium (25) for 7 to 10 days at 28°C. Biomass for fatty acid analysis was grown in 250 ml Erlenmeyer flasks containing 100 ml of Saboraud Dextrose Broth (20), seeded with inocula from solid cultures, and incubated at 28°C on a rotatory shaker at 150 rpm for 3 to 4 days. Biomass was harvested by filtration using 8 µm cellulose acetate membranes (Millipore) and lyophilized.

Extraction and analysis of fungal fatty acids. Fatty acids were extracted and derivitised as recommended in the protocols of the MIDI chromatographic system (20), with minor modifications. The extraction procedure included a saponification step using 1.0 ml of NaOH 3,75 M - methanol 50% (1:1, v/v) and 100 mg of powdered dry biomass. The tubes were mixed by vortexing for 5 to 10 s, and heated in a boiling water bath for exactly 5 min. Tubes were further vortexed for 5 to 10 s, returned to the boiling water bath for an additional 25 min, then cooled to room temperature. The saponificate was acidified and methylated by addition of 2.0 ml of HCl 6.0 N (titrated) - 100% methanol (13:11, v/v), followed by vortexing for 5 to 10 s, heating for 10 ±1 min at 80°C, and rapid cooling. The fatty acid methyl esters (FAMEs) were extracted by adding 1.25 ml of hexane - methyl tert-butyl ether (1:1, v/v) and gentle mixing by continuous inversion for 10 min. The top phase containing the organic solvent with the extracted FAMEs was removed with a Pasteur pipette and transferred to a new set of tubes. A final washing step was performed by addition of 3.0 ml of NaOH 0.3 M and gentle mixing by continuous inversion for 5 min. Approximately 2/3 of the organic phase were transferred to chromatographic vials, sealed and stored at -20°C until analysis.

Prior to FAMEs analyses, the samples were evaporated under nitrogen and resuspended in 50 µl of hexane. The analyses were carried out in triplicate in a Shimadzu GC-14A gas chromatograph equipped with a flame ionization detector and a split/splitless injector using a 30 m x 0.32 mm, 0.25 µm internal diameter, polar OmegawaxTM 320 capillary column (Supelco). The carrier gas was helium at a flow rate of 20 cm3 s-1 and a split-ratio of 40/1. The temperature of the injector was 250°C and that of the flame ionization detector was 250°C. The oven temperature after sample injection (2µl) was 20 min at 160°C, increasing to 200°C at 5°C/min and held at this temperature for 15 min.

The chromatograms were recorded in a Shimadzu Model C-R4A Chromatopac integrator. The identification of individual FAMEs was based on the retention times relative to a mixture of FAMEs standards (Matreya, Inc. n° 4232), including lauric acid (12:0), myristic acid (14:0), pentadecanoic acid (15:0), palmitic acid (16:0), palmitoleic acid (16:1), margaric acid (17:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), g-linolenic acid (18:3 w6), nonadecanoic acid (19:0), arachidic acid (20:0), gadoleic acid (20:1), eicosadienoic acid (20:2), dihomo-g-linolenic acid (20:3), arachidonic acid (20:4), behenic acid (22:0). The identified and non-identified fatty acids which reproductively amounted up to at least 1% of the total peak area in the samples were used for taxonomic characterization of the strains in a tabular format.

 

RESULTS AND DISCUSSION

The fatty acid profiles of ten representative strains belonging to the order Mucorales (Zygomycetes), and one asexual representative of the class Ascomycetes, Volutella sp., were analysed. One of the Mucorales strains, Cunninghamella blakesleeana CCT 4123, was analysed in duplicate in order to evaluate the fatty acid profile variability present in different independent cultures of the same organism. The analyses were carried out in triplicate and the resulting profiles were qualitatively identical, with some variation in the amounts of individual compounds. These results suggest that the standardization of the growth conditions used are limited to the generation of qualitative fatty acid data. Quantitative analysis of fatty acid data would require the analysis of a larger number of replicates and the use of classical and non-parametrical statistics in the analysis of the data.

Out of twenty fatty acids present in the fungi studied, ten could not be identified by comparison with the fatty acid standard mixture used; these were named FAME1 to FAME10 according to their retention times (Table 1). The qualitative fatty acid profiles, including identified and non-identified fatty acids, are presented in Table 2. Palmitic acid (16:0), oleic acid (18:1), linoleic acid (18:2) and g-linolenic acid (18:3) were present in all strains analysed, with the exception that the latter was not present in Volutella sp. Pentadecanoic acid (15:0), margaric acid (17:0), nonadecanoic acid (19:0), arachidic acid (20:0), gadoleic acid (20:1), dihomo-g-linolenic acid (20:3) and arachidonic acid (20:4) were absent in all strains analysed. The remaining fatty acids were discontinuosly distribuited among the strains analysed (Table 2).

 

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The fatty acid profiles obtained were, in most of the cases, comparable to data derived from the literature. The fatty acid profile of Mucor circinelloides f. janssenii CCT 4396 was identical to that of Mucor circinelloides CBS 478.70, identified by Westhuizen et al. (30), except for the presence of 20:2 in strain CCT 4396. Strains of Rhizomucor miehei CCT 2236 showed the same fatty acid profile as the strains analysed by Sumner and Morgan (26) and Westhuizen et al. (30). The 16:0, 16:1, 18:1, 18:2 and 18:3w6. The 16:0, 16:1, 18:0, 18:1, 18:2 and 18:3w6 fatty acids present in Rhizomucor pusillus CCT 4133 had already been verified in other Rhizomucor pusillus strains by Sumner and Morgan (26), Mumma et al. (17) and Westhuizen et al. (30).

Rhizomucor pusillus CCT 4133 and Rhizomucor miehei CCT 2236 could be differentiated on the basis of their fatty acid profiles by the absence of 18:0 in the latter. The non-identified FAME8 was restricted to these two species only, and thus could be considered as a chemotaxonomic marker for the genus Rhizomucor.

Backusella lamprospora CCT 3480 was characterized by a fatty acid profile including all the identified fatty acids present in the other fungi. The presence of 22:0 was restricted to this fungus and to the strains of Rhizomucor analysed.

Amano et al. (1) differentiated representatives of the subgenus Micromucor and Mortierella by the occurrence of polyunsaturated C20 fatty acids in the latter group. The C20 polyunsaturated fatty acids were characteristically absent in the strains of Mortierella ramanniana var. ramanniana, which belong to the subgenus Micromucor. The fatty acid profile of Mortierella ramanniana var. ramanniana was composed by 16:0, 18:0, 18:1, 18:2 and 18:3w6 (1). In a later study, Westhuizen et al. (30) identified 14:0, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3w6, 20:0, 20:1, 20:2 and 20:4 in Mortierella ramanniana var. ramanniana CBS 243.58. Nevertheless, the presence of fatty acids from the C20 series in strain CBS 243.58 may have been influenced by growth temperatures used in the study. CBS 243.58 was grown at 21°C, whereas strains analysed by Amano et al. (1) were grown at 28°C. Mortierella ramanniana CCT 4428 was also grown at 28°C and presented a similar composition of identified fatty acids to the CBS 243.58 strain, except by the occurrence of small amounts of 20:2 (approx. 3.2% of relative proportional average) in the former strain. The discrepant results from the different studies indicate that synthesis of C20 fatty acids by Mortierella ramanniana is temperature dependent.

A smaller number of fatty acids were detected in Benjaminiella youngii CCT 4121. FAME3 (Table 2) was found exclusively in this strain, suggesting that this fatty acid may be a potential chemotaxonomic marker for Benjaminiella youngii.

The fatty acid profile of Mycotypha microspora CCT 4126 (A) was identical to that of Mycotypha microspora CCT 4127 (B), showing that there are no differences between strains of the same species. Similarly, the fatty acid profiles of Cunninghamella blakesleeana CCT 4123, derived from duplicate cultures, showed no significant difference, demonstrating the reproducibility of the method. The presence of the fatty acids 16:0, 18:1, 18:2 and 18:3 in the Cunninghamella blakesleeana CCT 4123 was previously observed by Shaw (22) in Cunninghamella blakesleeana IMI 6387.

One species of the genus Volutella, included in this study as a reference organism different from the studied Zygomycetes, showed a profile with the smallest number of fatty acids. This organism was the only one that did not present the fatty acid 18:3(w6), g-linolenic acid, characteristic of the Zygomycetes fungi, in accordance with earlier reports (14,16,30).

In general, the major Ascomycetes and Basidiomycetes fatty acids are 16:0, 18:1 and 18:2(w6) (9,10,14,15). Ascomycetes and their asexual representatives can be characterized by the presence of 18:3(w3), which does not occur in Zygomycetes, or by a profile composed of up to 18:2(w6) fatty acid (13,23,30). From the identified fatty acids of Volutella sp. CCT 2995 it can be observed that, as a member of Ascomycetes, this strain is characterized by the absence of 18:3(w6) and the presence of 18:2(w6), corroborating the previous investigations.

According to Amano et al. (1), the fatty acids can provide valuable information to establish an accurate taxonomic system for fungi. However, the fatty acid profiles, as any other taxonomic character, should not be considered isolatedly. In combination with taxonomic information from different sources, including morphological, molecular and biochemical data, the analysis of fatty acid profiles can be considered a valuable tool for the polyphasic taxonomy of fungi (5,13,28,29).

The results of the present study suggest that qualitative fatty acid analysis can be an important chemotaxonomic tool for the classification of fungi assigned to the order Mucorales (Zygomycetes). Fatty acid profiles allowed the differentiation of closely related species assigned to the same genus (Rhizomucor miehei CCT 2236 and Rhizomucor pusillus CCT 4133) whereas strains from the same species (Mycotypha microspora CCT 4126 and 4127), as well as duplicate cultures of the same organism (Cunninghamella blakesleeana CCT 4123), showed identical profiles.

Chemotaxonomic markers that could be potentially used for the identification at species and genus were also found. A non-identified fatty acid, FAME8, was only detected in the strains of the genus Rhizomucor, whereas non-identified fatty acid FAME3 was characteristic for Benjaminiella youngii. The fatty acid 18:3w6 was confirmed as chemotaxonomic marker for strains belonging to the order Mucorales studied in the present investigation.

 

ACKNOWLEDGMENT

MS was supported by a M.Sc. grant from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). We would like to thank Dr. Tania Akiko Anazawa, from the Food Science Department of FEA/UNICAMP, for valuable assistance with chromatographic analyses. The authors are also indebted to Dr. Ludwig H. Pfenning for suggestions and guidance.

 

 


RESUMO

Caracterização de linhagens de Mucorales através do perfil de ácidos graxos

O perfil de ácidos graxos de Backusella lamprospora (Lendner) Benny e R.K. Benj., Benjaminiella youngii P.M. Kirk, Circinella simplex van Tieghem, Cunninghamella blakesleeana Lendner, Mortierella ramanniana (Möller) Linnem., Mucor circinelloides f. janssenii (Lendner) Schipper, Mycotypha microspora Fenner, Rhizomucor miehei (Cooney e R. Emerson) Schipper e Rhizomucor pusillus (Lindt) Schipper, da ordem Mucorales (Zygomycetes), e Volutella sp. Fr., da classe Ascomycetes, foram analisados qualitativamente por cromatografia gás-líquida, tendo como objetivo determinar o valor taxonômico destes marcadores quimiotaxonômicos. Os ácidos palmítico (16:0), oléico (18:1), linoléico (18:2) e g-linolênico (18:3) foram encontrados em todas as linhagens, com exceção do último, o qual não foi encontrado na linhagem de Volutella analisada. Foram obtidos marcadores quimiotaxonômicos para algumas espécies e gêneros estudados, incluindo um ácido graxo não-identificado, FAME8 (tempos de retenção mínimo e máximo de 27,92 e 28,28 minutos) para Rhizomucor miehei CCT 2236 e Rhizomucor pusillus CCT 4133 e FAME3 (tempos de retenção mínimo e máximo de 16,53 e 16,61 minutos) para Benjaminiella youngii CCT 4121. Para a ordem Mucorales, o marcador quimiotaxonômico obtido foi o ácido graxo 18:3w6, confirmando dados da literatura. Os resultados do presente estudo sugerem que a análise qualitativa do perfil de ácidos graxos pode ser uma ferramenta importante na classificação de fungos da ordem Mucorales (Zygomycetes).

Palavras-chave: taxonomia, marcador quimiotaxonômico, ácidos graxos, Mucorales.


 

 

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* Corresponding authors: Mailing address: Departamento de Ciência de Alimentos, Faculdade de Engenharia de Alimentos, UNICAMP, CEP 13083-010, Campinas, SP, Brasil. Fax: (+5519) 242-7827. E-mail: manuela@bdt.org.br

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