Growth and lipid production of Rhodotorula glutinis R4, in comparison to other oleaginous yeasts

Highlights

• Lipid production and growth by R. glutinis R4 and other oleaginous yeasts were compared.

• R. glutinis R4 showed remarkable capacity for lipid production and fast growth.

• Microbial oils produced by R. glutinis R4 are similar to vegetable oils.

• Fatty Acyl Methyl Esters (FAME) produced from R. glutinis R4 lipids are adequate for biodiesel synthesis.

• Biodiesel derived from R. glutinis R4 complies with international fuel standards.

Abstract

Some Rhodotorula spp. have been characterized as oleaginous yeasts. Under certain culture conditions they can accumulate neutral lipids, which are mainly triglycerides (TAG). Microbial TAG that can be used as raw material for biodiesel synthesis are attractive for the biofuel industry. In this study, the ability to synthesize lipids of Rhodotorula glutinis R4, isolated in Antarctica, was compared with eight strains belonging to the genera Rhodotorula and Yarrowia with the aim of proposing a novel source of oils for biodiesel synthesis. All strains were cultured under nitrogen (N) limiting conditions and an excess of carbon (C) in the culture medium. We found that yeasts accumulated between 9–48.9 % (w/w) of lipids. Among them, R. glutinis R4 showed the highest growth (14 g L⁻¹, μmax 0,092 h⁻¹) and lipid production (7 g L⁻¹; 47 % w/w). Microbial oils produced by R. glutinis R4 are similar to vegetable oils, with 61 % of oleic acid, indicating that it is adequate for biodiesel synthesis. Our results demonstrate that biodiesel derived from R. glutinis R4 complies with international fuel standards ASTM D6751 and EN 14214. Therefore, this work demonstrates that Rhodotorula glutinis R4 is a novel and valuable source of microbial oils for biodiesel synthesis.

Introduction

Because of the constant search for alternative energy sources to decrease dependence on petroleum-based fuel, biofuel production has emerged as a key player. Thereby, significant research is being performed on the production of biofuels from renewable sources, particularly in biodiesel production (Li et al., 2007). Currently, soy, palm, jatropha, rapeseed and sunflower seeds, among others, are the main source of oils used for biodiesel synthesis. This type of raw material has numerous disadvantages, the most important being high production costs, negative environmental impact, and direct competition with the production and availability of animal and human food. Additionally, vegetable oil production for biodiesel promotes deforestation and encourages monocultures. Moreover, large planting areas are required and yields are affected by weather conditions, among other limitations. Due to the above-mentioned reasons, social movements have campaigned against the use of this type of biofuels because of its negative effects on wild ecosystems (Elshout et al., 2019).

Nowadays there is great interest in finding new sources of lipids that can be used for biodiesel production (Beopoulos and Nicaud, 2012). A viable solution is the use of microbial oils. These oils are effectively synthesized by many microorganisms such as bacteria, yeasts, filamentous fungi and unicellular algae (Meng et al., 2009). Numerous studies conducted in this area have shown that, under certain conditions, some organisms have the ability to produce and accumulate lipids. When this ability to accumulate lipids is ≥20 % (w/w) with respect to their biomass, they are considered oleaginous microorganisms (Donot et al., 2014). In particular, these lipids accumulate in the cytoplasm, specifically within the lipid bodies (LB) (Park et al., 2017). Additionally, the production of these lipids starts at the end of the exponential growth phase and is more intense during the stationary phase.

Presently, oleaginous yeasts are being evaluated as oil factories due to the advantages they present compared to vegetable oils. They have a short life cycle and are less affected by seasons and climate, they require less labor and microbial oil production is easier to scale up. In addition, no extensive cultivation areas are required and microbial oil production is independent of geographic location (Liang and Jiang, 2013). In general, oleaginous yeasts belong to the genera Lipomyces, Cryptococcus, Trichosporon and Yarrowia as well as Rhodotorula (Rhodosporidium). The genus Rhodotorula, which includes red pigmented yeasts, belongs to the Basidiomycota division. It was found and isolated from different sources such as air, seawater, fruits and soil (Kurtzman et al., 2011). Today, this genus is being intensively studied since it has the natural ability to produce many different compounds of industrial interest besides lipids. Moreover, the above yeasts also synthesize carotenoids, enzymes and terpenes (Park et al., 2017). Up to now, several Rhodotorula spp. have been recognized as oleaginous yeasts, as they can accumulate between 40–70 % (w/w) of intracellular neutral lipids under nitrogen limitation and simultaneous excess of carbon source (Viñarta et al., 2016). However, it has been reported that nitrogen limitation is not the only condition that induces lipogenesis. Sulphate and phosphate limitation in the culture medium are also influencing factors that may affect lipid biosynthesis. All the above conditions may affect cell proliferation rate and could result in low biomass yield. In brief, there are numerous factors that affect lipid content and fatty acid composition, e.g. environmental factors, including aeration, pH, temperature, inoculum size, incubation period, medium composition and culture conditions. However, the most important factor is the microorganism used for lipid production (Subramaniam et al., 2010).

Due to the wide variety of factors that affect the production of lipids, different strategies for productivity optimization have been studied. In this regard, it has been demonstrated that the lipids synthetized by Rhodotorula spp. can be used as feedstock for biodiesel production, and that they can also be employed as a source of nutritionally valuable fatty acids (FA) (Sitepu et al., 2014).

During the last decade, interest has been focused on selecting new strains as promising lipid producers as well as developing new strategies to increase lipid production. These strategies include oxygenation conditions, temperature, pH and the optimization of the entire process itself. Progress has also been made in the development of molecular biology tools, which will increase the understanding of the metabolic pathways taking place in the control of lipid synthesis, accumulation and degradation. All this knowledge will provide the necessary tools to optimize the industrial process of lipid production by microorganisms (Lyman et al., 2019).

In addition, Rhodotorula species have proved to have a biotechnological advantage over other oleaginous yeasts because of their ability to grow on inexpensive substrates. They are capable of metabolizing many compounds used as carbon sources such as sugar (mono-, di-, or poly-saccharides), organic acids, glycerol, raw materials, industrial by-products and wastewaters (Park et al., 2017), which reduces total production costs. Microbial lipids, also known as single cell oils (SCO), are considered an important alternative source of lipids for several biotechnological applications, including biofuel and pharmaceutical industries. Among Rhodotorula spp., R. glutinis has been identified as an excellent lipid and carotenoid producer. Therefore in this work we decided to compare the ability for lipid biosynthesis of the cold-adapted oleaginous yeast Rhodotorula glutinis R4, isolated in Antarctica by our laboratory, with eight other yeast strains, including Rhodotorula spp. and Yarrowia spp. Our results suggest that Rhodotorula glutinis R4 could be a novel and valuable source of microbial oils for biodiesel synthesis.