In the present study, the yeast Rhodotorula glutinis has been assessed with the aim of producing microbial lipids from glycerol under different aeration conditions. For such a purpose, experiments were carried out in shake flasks, under different conditions of agitation (150 to 250 rpm) and aeration (2.5 to 5.0 of flask volume-to-medium volume ratio). Furthermore, their influence on fermentative parameters (lipid and cell concentration, biomass yield; lipid yield; and lipid volumetric productivity) has been investigated using a 2 2 full factorial design. The statistical analysis has revealed a strong influence of both variables on substrate consumption, lipid accumulation, cell growth and lipid productivity. As a whole, results suggest that higher aeration levels provide greater cell and lipid concentrations, and lipid volumetric productivity. The best results (4.5 g/L of lipids and Q P = 0.95 g/L ⋅day) were achieved at the highest aeration (5.0 flask volume-to-medium volume ratio) and agitation (250 rpm) levels. Their fatty acid profile showed that oleic acid was produced in greater quantity (53.5%), followed by linoleic acid (18.7%), palmitic acid (6.8%) and stearic acid (9.9%). The microbial oil presented viscosity of 39.3 cP at 50°C and free fatty acid content of 1.93% ± 0.08%. These are significant results and contribute to establishing operational conditions that maximize single-cell oil production from glycerol by Rhodotorula glutinis, i.e. an alternative source as renewable raw material for lipid-based biorefineries.
Renewable raw material sources have been sought in order to replace petroleum in various industrial sectors for quite some time. In this context, the concept of biorefineries arises, i.e. industrial plants capable of processing renewable raw materials on a large scale in order to obtain products of interest to various sectors. Lipid-based biorefineries can use oleaginous raw materials that can be mainly obtained from vegetable oils [
An extensive use of vegetable oils as raw material requires large agricultural areas for planting oil crops and harvest periods of over 60 days for processing such crops [
In addition to using vegetable raw materials, studies have demonstrated that oleaginous microorganisms have great potential for such a purpose. Heterotrophic oleaginous microorganisms, such as yeasts, are able to accumulate lipids rapidly at greater proportions; furthermore, they do not depend on climate, region and are not affected by seasonality, unlike vegetable crops. Such characteristics make these microorganisms potentially competitive as lipid source [
According to Rawat et al. [
Oxygen availability by culturing oleaginous yeast is a parameter that exerts great influence on microbial growth, lipid productivity and composition. Oxygen availability in the culture medium generally has a positive correlation with biomass concentration and lipid productivity of yeast [
In this context, the present study aimed to assess the influence of oxygen availability on lipid production by Rhodotorula glutinis NRRL Y-12905 from glycerol. Although several pieces of research have been published about microbial oil production by yeast, the present work demonstrates a systematic study on using statistical tools for evaluating and determining optimal process conditions for obtaining single cell oil.
The yeast Rhodotorula glutinis NRRL Y-12905 has been used as microorganism in experiments. It was provided by the USDA (United States Department of Agriculture), Peoria, Illinois, and kept on malt extract agar slant at 4˚C. The inoculum was prepared by transferring cells from malt extract agar to 250 mL Erlenmeyer flasks containing 50 mL of a culture medium composed of (g/L) glycerol (30.0), yeast extract (3.0), MgSO4∙7H2O (1.0) and (NH4)2HPO4 (3.0). The flasks were incubated for 24 hours at 30˚C and 200 rpm. Afterwards, the cells were recovered by centrifugation (2000 xg for 10 minutes) and resuspended in sterile distilled water and transferred to the culture medium so as to reach an initial cells concentration of about 1 g/L.
The influence of oxygen availability on lipid production from glycerol by Rhodotorula glutinis has been evaluated through the design of experiments and response surface methodology. Fermentation assays were carried out according to a face-centered 22 full factorial design with center points in triplicate. In this study, agitation and aeration effects on substrate consumption, cell growth, lipid accumulation, biomass yield (YX/S―biomass yield on glycerol consumed; g/g) and lipid yield (YP/S―lipid yield on glycerol consumed; g/g) and lipid volumetric productivity were assessed. Statistica 13 (license: JKK510H198630AR-B) was used in the statistical analysis.
Fermentation assays were performed in 250-mL Erlenmeyer flasks containing 50, 67 or 100 mL of a fermentation solution medium composed of (g/L) glycerol (70.0), yeast extract (3.0), MgSO4∙7H2O (1.0), (NH4)2HPO4 (3.0) and KH2PO4 (20.0) inoculated with 1 g/L cells. The inoculated flasks were incubated for 120 h in a rotary shaker at 30˚C. The different conditions of agitation (150 to 250 rpm) and aeration (2.5 to 5.0 of flask volume-to-medium volume ratio) employed in essay were varied according to a factorial design. Throughout the essay, samples were taken at every 24 h for assessing lipid and glycerol concentrations, and cell growth determinations.
Cell concentration was determined by measuring the fermentation broth UV-spectrophotometric absorbance at 600 nm, which was correlated to a calibration curve (dry weight by optical density).
Glycerol concentration was determined by High Performance Liquid Chromatography (HPLC) in an Agilent chromatograph equipped with a Bio-Rad Aminex HPX-87H column (300 × 7.8 mm) and a refractive index detector. Operation conditions were 45˚C of temperature, 0.005 mol/L of sulfuric acid as eluent at flow rate of 0.6 mL/min and sample volume of 20 µL.
Total lipids were extracted from cells by the Bligh & Dyer method [
Lipid was extracted from cells by the Bligh & Dyer method to analyze fatty acid profile, absolute viscosity and acidity index [
FAMEs were prepared by methyl transesterification of lipids [
The absolute viscosity of microbial oil was determined with a Brookfield Model LVDVII viscometer (Brookfield Viscometers Ltd., England) using the CP 52 cone. Measurements were made at 50˚C in triplicate. To verify the Newtonian fluid behavior, the obtained data (viscosity, strain rate and shear stress) were adjusted to Equation (6), where: where: K is the consistency index, γ is strain rate, n is the angular coefficient and τ is shear stress).
τ = K ⋅ γ n (6)
Acidity index was determined according to the AOCS methodology [
AI ( mg KOH / g ) = Alkali volume ( mL ) × Alkali molarity ( mol / L ) × ( 56.1 g / mol ) Sample weight ( g ) (7)
FFA % = 100 × Alkali volume ( mL ) × Alkali molarity ( mol / L ) × ( 282 g / mol ) Sample weight ( g ) (8)
Assay | Variables | Responses | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
Coded values | Real values | |||||||||
X1 | X2 | Agitation (rpm) | Aeration* | Cell (g/L) | Lipid (g/L) | Consumption of glycerol (%) | YP/S (g/g) | Yx/S (g/g) | QP (g/L∙day) | |
1 | −1 | −1 | 150 | 2.5 | 10.5 | 0.89 | 33.3 | 0.038 | 0.45 | 0.13 |
2 | 1 | −1 | 250 | 2.5 | 24.2 | 2.32 | 74.4 | 0.044 | 0.47 | 0.47 |
3 | −1 | 1 | 150 | 5.0 | 18.0 | 1.65 | 64.7 | 0.037 | 0.40 | 0.22 |
4 | 1 | 1 | 250 | 5.0 | 39.4 | 4.45 | 87.6 | 0.073 | 0.64 | 0.95 |
5 | −1 | 0 | 150 | 3.75 | 16.8 | 1.24 | 54.1 | 0.033 | 0.44 | 0.21 |
6 | 1 | 0 | 250 | 3.75 | 30.9 | 4.18 | 81.0 | 0.074 | 0.54 | 0.80 |
7 | 0 | −1 | 200 | 2.5 | 14.9 | 1.54 | 46.3 | 0.048 | 0.46 | 0.30 |
8 | 0 | 1 | 200 | 5.0 | 27.6 | 3.62 | 73.1 | 0.071 | 0.54 | 0.65 |
9 | 0 | 0 | 200 | 3.75 | 21.5 | 1.60 | 68.4 | 0.033 | 0.45 | 0.27 |
10 | 0 | 0 | 200 | 3.75 | 18.9 | 1.21 | 69.6 | 0.025 | 0.39 | 0.23 |
11 | 0 | 0 | 200 | 3.75 | 19.6 | 1.06 | 58.3 | 0.026 | 0.48 | 0.19 |
*Aeration is defined as the ratio between the volume of the Erlenmeyer flask and the volume of fermentation medium used in the experiments.
production by yeast Rhodotorula glutinis. The following response variables were assessed: cell and lipid concentration; substrate consumption; lipid yield; biomass yield; and lipid productivity. The yeast was able to accumulate lipids during the observed culture period in all conditions, but there was a great variation in the final lipid and cell concentrations, thus demonstrating the influence of aeration on microbial oil production. A maximum lipid concentration of 4.45 g/L was obtained under higher oxygen availability conditions (level +1 of both variables, X1 and X2). On the other hand, under lower oxygen availability conditions (level −1 of both variables), there were limitations in cell growth and in lipid accumulation if compared to higher aeration conditions. Increased oxygen availability conditions provided a lipid accumulation about five times greater than that accumulated by yeast under lesser aeration conditions. These data indicate a positive effect of increased aeration on microbial oil production and cell growth.
Agitation and aeration effects have been assessed more carefully through statistical tools using the response surface methodology. The Pareto charts shown in
The Pareto chart in
By analyzing the Pareto chart as regards response variables of lipid accumulation (
As for the lipid productivity response variable (
For both response variables, i.e. biomass yield (YX/S) and lipid yield (YP/S), the Pareto chart (
that none of the variables were significant at 95% confidence interval. Such behavior may be due to the fact that substrate consumption, cell growth and lipid accumulation varied together, so that, within the variation range observed, lipid yield or biomass yield presented similar values, regardless of aeration condition.
With respect to terms considered as significant by the Pareto chart analysis, mathematical models capable of describing the behavior of response variables of cell concentration, lipid accumulation, substrate consumption and lipid productivity (Equations (1)-(4), respectively) were developed as a function of Agitation and Aeration variables within the studied region. The analysis of variance of models (ANOVA) is presented in
ANOVA analysis parameters | Responses of models | |||
---|---|---|---|---|
Cell* (g/L) | Lipid* (g/L) | Consumption of glycerol* (%) | QP* (g/L∙day) | |
Model P-value | <0.0005 | 0.004 | <0.0005 | 0.005 |
Lack of fit P-value | 0.551 | 0.104 | 0.681 | 0.076 |
R2 | 0.9834 | 0.7458 | 0.9015 | 0.8926 |
*Significant at 95% confidence; P < 0.05.
75%, 90% and 89% for cell concentration, lipid concentration, substrate consumption and lipid productivity response variables, respectively. Based on these mathematical models, response surface charts shown in
Cell ( g / L ) = 20.5 + 8.2 X 1 + 5.9 X 2 + 2.8 X 1 2 + 1.9 X 1 X 2 (1)
Lipid ( g / L ) = 2.16 + 1.19 X 1 + 0.83 X 2 (2)
Glycerol consumption ( % ) = 64.6 + 15.2 X 1 + 11.9 X 2 (3)
Q P ( g / L ⋅ day ) = 0.33 + 0.28 X 1 + 0.15 X 2 + 0.13 X 1 2 + 0.10 X 1 X 2 (4)
was used to assess oxygen availability to the yeast. Thus, these authors report that, for batch cultures, an increase in aeration from 0 to 2 vvm led to higher cell concentration and accumulated lipid concentration. The authors also reported that maximum lipid concentration reached in batch cultures was 4.32 g/L, i.e. a very similar result to that found herein.
As a whole, the results suggest that higher cell and lipid concentrations, as well as higher volumetric lipid productivity, could be achieved by providing the culture medium with greater aeration levels. It was also observed that, within the studied range, there were no significant variations in biomass yield (YX/S) and lipid yield (YP/S). Notwithstanding, it was found that lipid production can be favored by higher aeration conditions, cultures in shaken flasks did not enable tests with greater agitation (maximum possible agitation at 250 rpm). The tendency observed in the analysis of results suggests that an improvement in lipid production can be achieved under higher aeration conditions. Thus, yeast cultures performed on bioreactors which could provide higher aeration levels, or even the use of oxygen enriched air, are potentially promising alternatives to make important improvements in the process productivity. In fact, higher aeration levels have been reported as favorable conditions for lipid production. Machado Junior [
Magdouli et al. [
By analyzing the microbial oil produced by the yeast Rhodotorula glutinis, it was found fatty acids with chain size ranging from ten to eighteen carbons with different saturation degrees (
Fatty acid | Present study | Sitepu (2013) [ | Kot et al.; (2016) [ | Zhang et al.; (2014) [ | |
---|---|---|---|---|---|
Caprylic Acid | C8:0 | n.d. | 0.0 | n.r. | n.r. |
Capric Acid | C10:0 | 0.02 ± 0.01 | 0.0 | n.r. | n.r. |
Lauric Acid | C12:0 | 0.07 ± 0.01 | 0.0 | 0.0 | n.r. |
Myristic Acid | C14:0 | 0.87 ± 0.02 | 1.7 | 0.4 | 0.7 |
Palmitic Acid | C16:0 | 16.83 ± 0.06 | 12.0 | 24.3 | 16.5 |
Palmitoleic acid | C16:1 | n.d. | n.r. | 0.2 | 0.4 |
Stearic Acid | C18:0 | 9.97 ± 0.05 | 5.5 | 10.1 | 3.7 |
Oleic Acid | C18:1 | 53.54 ± 0.02 | 54.7 | 53.2 | 51.3 |
Linoleic Acid | C18:2 | 18.73 ± 0.03 | 17.1 | 6.8 | 21.6 |
Arachidonic acid | C20:0 | n.d. | n.r. | 0.3 | n.r. |
n.d.―not detected; n.r.―not reported.
unsaturated fatty acids, which correspond to 72%. Four fatty acids (palmitic acid, stearic acid, oleic acid and linoleic acid) constitute about 99% of lipid constituents. Among which, oleic acid (C18: 1) was the most commonly found fatty acid, i.e. about 53.5%, followed by linoleic (C18: 2), palmitic (C16: 0) and stearic (C18: 0) with 18.7%, 16.8%, 9.9%, respectively. Traces of capric (C10: 0) and lauric (C12: 0) acids were also detected, 0.02% and 0.07%, respectively. These results are in agreement with previously reported data in literature for the yeast Rhodotorula glutinis, as described by Kot et al. [
According to the rheological analysis of the microbial oil obtained from Rhodotorula glutinis, a Newtonian fluid behavior was observed with constant viscosity at deformation rate ranging between 100 and 360 s−1. This behavior is in agreement with literature data, since oils and fats commonly present Newtonian fluid behavior [
Acidity index is an important parameter for monitoring the quality of oils and fats. It reveals the degree of free fatty acids in the material, which is calculated in milligrams of potassium hydroxide to neutralize the free fatty acids present in one gram of sample. The oil produced by the yeast Rhodotorula glutinis NRRL Y-12905 achieved acidity index of 5.8 ± 0.2 mg KOH/g oil, which corresponds to 1.93% ± 0.08% FFA. According to Porphy and Farid [
Rhodotorula glutinis NRRL Y-12905 was able to produce and accumulate lipids from glycerol in all cultivation conditions evaluated herein; however, aeration and agitation proved to be important variables, since they were, in fact, capable of affecting the process efficiency.
As regards fermentation performance, the level of aeration has had a significant influence on substrate consumption, cell growth, lipid concentration and volumetric productivity. In general, cell growth, lipid accumulation and lipid volumetric productivity were favored by greater oxygen availability. Agitation and aeration at 250 rpm and 5.0 (flask volume-to-medium volume ratio), respectively, were the optimal operational conditions in the evaluated range of values, due to promoting high cell and microbial oil concentrations (39.4 g/L and 4.5 g/L, respectively) and QP (0.95 g/L∙day). These results demonstrate that the establishment of a suitable aeration condition is of paramount importance to improve the process of microbial oil production from glycerol by R. glutinis. In addition, glycerol bioconversion processes can provide a way of integrating with current biodiesel production, contributing to the development of lipid-based biorefinery.
The authors gratefully acknowledge the financial support from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo―Process Number 2016/06683-0) and the CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico-Process Number 455260/2014-1). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior―Brasil (CAPES)―Finance Code 001.
The authors declare that they have no conflict of interest.
Bento, T.F.S.R., Viana, V.F.M., Carneiro, L.M. and Silva, J.P.A. (2019) Influence of Agitation and Aeration on Single Cell Oil Production by Rhodotorula glutinis from Glycerol. Journal of Sustainable Bioenergy Systems, 9, 29-43. https://doi.org/10.4236/jsbs.2019.92003