Rhizopus microsporus var. Oligosporus

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Classification

Higher Order Taxa

Eukaryote (Domain); Fungi (Kingdom); Zygomycota (Division); Mucormycotina (Class); Mucorales (Order); Mucoraceae (Family); Rhizopus (Genus); Microsporus (Species); Oligosporus (Variety)  

Description and Significance

Tempeh.

Rhizopus oligosporus is most commonly known for its role in the production of tempeh, a food staple in southeast Asia [1]. R. oligosporus is the crucial driver of the fermentation of soybeans into tempeh, which is becoming an increasingly popular meat substitute worldwide [1]. Although best known for its role in fermented food production, R. oligosporus is also able to produce the enzyme phytase, which is commonly used as an additive to animal feed in order to increase its nutritional value [2]. In addition to its key role in tempeh production, Rhizopus oligosporus is also important for the production of industrial enzymes and the treatment of waste [1] [3].

Rhizopus oligosporus is considered a domesticated strain of Rhizopus microspores and is not found in nature. Because of its domestication, it does not produce rhizonins and rhizoxins as secondary metabolites,, unlike other strains of R. microsporus [1]. Though domesticated, R. oligosporus maintains a highly specific morphology related to its large and irregular spores, giving it an exotic appearance.  

Genome structure

The genome of R. oligosporus has not been sequenced completely; neither has it been studied beyond the specific isolation of a gene coding for chitinase I. Researchers examined antifungal activity of chitinase I through plasmid transformation from R. oligosporus inserted in tobacco plants [4]. The isolation of chitinase I, a hydrolytic enzyme which targets the chitin found in the cell walls of many fungal species, indicates that R. oligosporus may have some antimicrobial activity [4]. Other variants of the Rhizopus microspores species often produce toxic metabolites, but R. oligosporus strains do not possess any genes for these metabolic toxins [rhizonins and rhizoxins), having apparently lost the genes for toxic secondary metabolites present in R. microspores during domestication [1].  

Cell structure

The major components of the cell wall of R. oligosporus are chitin and chitosan, which give strength and structure to the cell [5]. R. oligosporus needs to produce chitinase in order to breakdown the chitin in its cell wall. The breakdown of this cell wall allows for the germination of the visible spores produced by R. oligosporus [5].

R. oligosporus forms large spores that are sub-globulose to globulose. Its spores have non-parallel ridges and valleys and the plateaus of the spores are often granular. R. oligosporus also has a high proportion of irregular spores and defects in spore formation [3].  

Metabolic processes

R. oligosporus is a heterotroph, which is able to utilize a diverse range of food sources to best support its growth, most notably common sugars, such as xylose, glucose, fructose, galactose, maltose, and mannitol, and various vegetable oils, such as cottonseed, soybean, peanut, and grapeseed oils [6]. R. oligosporus is able to utilize both organic and inorganic nitrogen sources. One study revealed that R. oligosporus is insensitive to experimental changes between organic and inorganic nitrogen sources; arginine was used as the organic nitrogen source, while sodium nitrates and ammonium chloride were used as inorganic nitrogen sources [6].  

Ecology

As R. oligosporus is a widely used starter culture for tempeh production, many studies have been directed towards its growth in the context of industrial application. In particular, many early research centered on determining R. oligosporus’s optimal growth conditions, alongside those of other industrially-used microbes, and investigated factors such as pH, temperature, media type, etc., for [9] [7] [8] [6]. Researchers know little about R. oligosporus’ ecology beyond its optimal growth conditions in an industrial-/research-type environment.

The optimal growth conditions of R. oligosporus are at temperatures between 35-40⁰C, with water activity at Aw = 0.995, and atmospheric composition of normal air [7] [8]. High concentrations of carbon dioxide or oxygen can slow the growth of R. oligosporus [8]. High water activity at otherwise optimal conditions can cause growth sensitivity to changes in temperature in R. oligosporus, as can other other abiotic environmental fluctuations. In anaerobic conditions of 99.5% N2 and 0.5% O2, R. oligosporus’ growth at a certain colony diameter is much less in terms of biomass than in aerobic conditions, indicating more resources directed to hyphae growth for scavenging oxygen from the atmosphere rather than growth in mycelial density [8]. The biomass growth rate of R. oligosporus on solid medium has also been determined in a mathematical model [8].

Compared to R. microsporus and Actinomucor elegans, a bacteria used in sufu (a Chinese fermented soybean food) production, R. oligosporus grows at a faster rate than R. microsporus in terms of biomass but grows at the same rate in terms of radial growth [8]. When compared to A. elegans, R. oligosporus grows faster in terms of biomass than A. elegans at temperatures between 35-40⁰C [7].  

Pathology

R. oligosporus is a domesticated strain of Rhizopus microsporus and appears to have lost the toxic secondary metabolites rhizoxin and rhizonins A and B present in R. microsporus [1]. However, R. oligosporus can be used for antifungal purposes. In one study, the Chitinase I gene from R. oligosporus was inserted into tobacco plant leaves and the increased production of Chitinase I increased the plant’s antifungal activity [4]. Genetically modifying tobacco plants and other agricultural plants with Chitinase I may be a possible solution to protect agricultural plants from fungal infections. R. oligosporus can also produce an antibacterial protein that inhibits the growth of some Gram-positive bacteria, thus leading to fewer intestinal infections in consumers of tempeh [10].  

Current Research

Current research focuses on characterizing R. oligosporus itself and other members of the Rhizopus genus, in a way that is not directly related to industrial application. One curiosity studied in R. oligosporus is its ability to produce antibiotic and antifungal compounds [10, 4]. In addition, another recent study showed that R. oligosporus, along with the related species R. chinensis, produced no secondary metabolites such as pharmaceutical rhizoxins and highly toxic rhizonins, whereas R. microsporus produced rhizoxins and some strains highly-toxic rhizonins [1]. In another recent study, R. oligosporus was morphologically identified by its high spore irregularity and its sporangiospores’ distinct ornamental patterns via low temperature scanning electron microscopy (SEM) and light microscopy [3]. Morphological features of twenty-seven other members of the R. microsporus and R. oryzae species were examined in this study as well [3].  

References

[1] Jennessen, J., Nielsen, K.F., Houbraken, J., Lyhne, E.K., Schnürer, J., Frisvad, J.C., and Samson, R.A. 2005. Secondary metabolite and mycotoxin production by the Rhizopus microsporus group. Journal of Agricultural and Food Chemistry 53:1833-1840.

[2] Sabu, A., Sarita, S., Pandey, A., Bogar, B., Szakacs, G., & Soccol, C. R. 2002. Solid-state fermentation for production of phytase by Rhizopus oligosporus. Applied Biochemistry and Biotechnology 102(1-6):251-260.

[3] Jennessen, J., Schnürer, J., Samson, Robert A., and Dijksterhuis, J. 2008. Morphological characteristics of sporangiospores of the tempe fungus Rhizopus oligosporus differentiate it from other taxa of the R. microscopus group. Mycological Research 112: 547–562.

[4] Terakawa, T., N. Takaya, H. Horiuchi, M. Koike, and M. Takagi. 1997. A Fungal Chitinase Gene From Rhizopus Oligosporus Confers Antifungal Activity to Transgenic Tobacco. Plant Cell Reports 16(7) 439-43.

[5] Yanai, K., Takaya, N., Kojima, N., Horiuchi, H., Ohta, A., & Takagi, M. 1992. Purification of two chitinases from Rhizopus oligosporus and isolation and sequencing of the encoding genes. Journal of bacteriology 174(22):7398-7406.

[6] Sorenson, W. G., and C. W. Hesseltine. 1966. Carbon and Nitrogen Utilization by Rhizopus Oligosporus. Mycologia 58(5):681.

[7] Han, B.-Z.; Ma, Y. 2003. Effects of temperature and relative humidity on growth and enzyme production by Actinomucor elegans and Rhizopus oligosporus during sufu pehtze preparation. Food Chem 81:27-34.

[8] Han, B.-Z., & Nout, M. J. R. 2000. Effects of temperature, water activity and gas atmosphere on mycelial growth of tempeh fungi Rhizopus microsporus var. microsporus and R. microsporus var. oligosporus. World Journal of Microbiology & Biotechnology 16: 853–858.

[9] Breeuwer, Pieter, et al. 1997. Nonanoic Acid, a Fungal Self-Inhibitor, Prevents Germination of Rhizopus oligosporus Sporangiospores by Dissipation of the pH Gradient. Applied and environmental microbiology 63(1):178-185.

[10] Kobayasi, S.Y., Naoto O., and Takuya K. 1992. Purification and Characterization of an Antibiotic Substance Produced from Rhizopus oligosporus IFO 8631. Biosci. Biotech. Biochem 56(1):94-98.  

 
Edited by [Harim Kim, Yu-Ching Chang, Rae Lynch, Bronte Escobar], student of Jennifer Talbot for BI 311 General Microbiology, 2016, Boston University.