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Optimization of culture conditions of Rhodotorula graminis S1/2R to obtain saponifiable lipids for the production of second-generation biodiesel

  • Adalgisa Martinez-Silveira
  • Virginia Pereyra
  • Gabriela Garmendia
  • Caterina Rufo
  • Silvana VeroEmail author
Original Article
  • 130 Downloads

Abstract

The use of biodiesel has generated a diversification of the energy matrix, which has led to a lower dependence on fossil fuels and a concomitant reduction of the adverse effects on the environment generated by their use. Currently, biodiesel is mainly produced by transesterification with short-chain alcohols of oils from different sources, in particular from oil crops, such as soybean and canola. Since the use of crops for the generation of fuels competes with their use as food, it seems appropriate to explore new oil sources for the production of biodiesel, such as the intracellular triglycerides accumulated by oleaginous yeasts. In this case, to achieve a competitive process, yeast production should be carried out using low-cost fermentable substrates such as industrial wastes or by-products. In this work we optimized the production of intracellular lipids by a previously selected oleaginous yeast strain in a fed-batch culture in presence of crude glycerol as a source of carbon and energy. In the optimized conditions an important improvement of lipid concentration was achieved, obtaining 12.2 g L−1 of fatty acid methyl esters (FAMEs) and 26.6 g L−1 of dry biomass in 360 h of culture.

Keywords

Oleaginous yeast Biodiesel Lipid production Crude glycerol 

Introduction

The demand for fossil fuels as sources of energy is ever increasing (Mosarof et al. 2015). However, the possibility of their depletion is forcing the fuel industry to look for alternative and renewable energy sources (Tai and Stephanopoulos 2013). Biodiesel generated by transesterification of triglycerides with short chain alcohols, constitutes one of the options. This biofuel is currently produced in many countries and it is mainly obtained from triglycerides from oil crops, such as soybeans and canola seeds (Sharma and Singh 2009). However, the overall production costs are still quite high, and so biodiesel is still an expensive product when compared to fossil fuels (Pan et al. 2009). The price of oil feedstock, which represents about 70–90% of the total cost of biodiesel (Meng et al. 2009; Vicente et al. 2009) has been recognized as the main reason for high prices. Moreover, concerns have been raised about the risk of diverting agricultural land and food crops for biofuel production to the detriment of the food supply (Kraisintu et al. 2010). Consequently, biofuel production may move food production to previously non-agricultural land, such as forests, increasing the net greenhouse gases emission. In that sense, the European Union has encouraged research on the development of new biofuels that do not compete with food crops (EU Directive 2015).

The search of novel raw materials for biodiesel production seems to be a challenge. Triglycerides accumulated in lipid bodies inside the cells of oleaginous microorganisms seem to be a feasible alternative. Microorganisms that, in certain culture conditions, are able to accumulate intracellular lipids exceeding the 20% of their dry weight, are categorized as oleaginous (Ratledge 1979). Strains of microalgae, yeast, fungi and bacteria of different species have been classified in that category. In many cases in adequate culture conditions, intracellular lipids could represent more than 40% of the dry microbial biomass (Sitepu et al. 2014). However, it should be considered that not all lipids obtained from microbial biomass are suitable to produce biodiesel. Only lipids with fatty acid ester linkages (also referred to as saponifiable lipids) and free fatty acids can be converted into alkyl esters and therefore can be used as raw material for biodiesel (Saenge et al. 2011). In the case of oleaginous yeasts, intracellular lipids are mainly composed by triacylglicerides (Subramaniam et al. 2010), and therefore are considered promising candidates as sustainable biodiesel precursors. Biodiesel, regardless of its origin, must meet certain requirements to be used as a fuel. Some of these requirements are based on the fatty acid profile of the alkylesters that constitute the fuel. In Uruguay, standards for biodiesel indicate that cetane number must be higher than 45, and no more than 12% of linolenic acid esters or 1% of esters from polyunsaturated acids (more than 4 double bonds) is admitted (UNIT 1100:2014 2014). In general, the fatty acid profile of methyl esters obtained from lipids accumulated by oleaginous yeasts chemically resemble vegetable oils, with oleic acid (C18:1) as the main component (Patel et al. 2017). However, every microorganism will produce a unique fatty acids profile depending on its nature and on the growth conditions, which must be determined in each individual case.

Oleaginous yeasts have several advantages to be used in biodiesel production. They can store high amount of lipids, they have short life cycles, are easy to manipulate, and have the ability to use a broad variety of carbon sources (Sitepu et al. 2014). Yeast lipids are a promising alternative feedstock to produce biodiesel; however, the implementation of cost-effective production processes is mandatory. A possible strategy to reduce production costs could be the use of waste materials or industrial by-products, such as biodiesel-derived crude glycerol, as the base for the culture medium to grow oleaginous yeasts. Crude glycerol is constituted by a mixture of glycerol (65–85%, w/w), methanol, and soap and is obtained as a by-product of biodiesel production in a 1:10 ratio to biodiesel (Liang et al. 2010a). Since biodiesel production has increased, the amount of crude glycerol available has also raised. A fraction of crude glycerol is refined for cosmetic and pharmaceutical uses through an expensive process, not feasible for all the producers, especially for small and medium-sized ones (Liang et al. 2010b). Moreover, the current supply of crude glycerol exceeds the demand exerted by both industries. Crude glycerol is also being used as an ingredient in animal feeds (Yang et al. 2012). However, according to Satpathy et al. (2014) a considerable fraction of crude glycerol is being disposed as waste with the concomitant detrimental effect on the environment. Thus, the search of alternative uses seems to be imperative.

In a previous work (Pereyra et al. 2014), our group selected an oleaginous yeast identified as Rhodotorula graminis. This yeast could accumulate saponifiable lipids corresponding to more than 40% of its dry weight, when it was cultivated in batches in a medium with a high carbon/nitrogen (C/N) ratio using glucose as a carbon source. In addition, it was determined that it could also grow and accumulate saponifiable lipids in presence of crude glycerol as the only carbon source. The mixture of fatty acid methyl esters (FAMEs) obtained from the transesterification of those lipids was characterized and it was determined that it met the requirements to be used as fuel in Uruguay (UNIT 1100:2014 2014). The aim of this work is to optimize the production conditions of the selected strain, in the presence of crude glycerol as carbon and energy source in order to maximize the accumulation of intracellular lipids to be transformed into biodiesel.

Materials and methods

Microorganism

Rhodotorula graminis S1/2R isolated and characterized as oleaginous yeast by Pereyra et al. (2014) was used in the present study. It was maintained on Potato Dextrose Agar (PDA, Oxoid) at 4 °C and on Brain Heart Infusion broth (BHI, Difco™) amended with 20% glycerol at − 80 °C.

Analytical procedures

Yeast biomass was determined by cell dry weight. Wet yeast cells obtained from culture medium were centrifuged at 7000 rpm for 5 min, washed twice with distilled water and then dried at 60 °C to constant weight. Glycerol concentration was determined by high performance liquid chromatography (HPLC) in a system with of a pump Waters 510 (Waters, Millipore Corporation), an injection valve equipped with a 20-µL loop, an Aminex HPX-87C column (250 mm × 4 mm, BioRad Laboratories) and a Shimadzu RID-10A refractive index detector. Data were processed using Peak Simple version 4.26 software. The analysis was performed at 67 °C with a flow rate of 0.5 mL/min using isocratic elution with water as a mobile phase.

The content of intracellular saponifiable lipids was determined as the amount of FAMEs obtained from dry yeast biomass by the in situ transesterification process described by Martinez-Silveira et al. (2018). FAMEs analysis was conducted by gas chromatography (GC) in an Agilent Technologies 7820A GC equipped with a DB-225 (20 m, 0.100 mm ID, 0.10 µm) column and a flame ionization detector. N2 at 0.2 mL/min was used as carrier gas. Temperature of injection port and detector was set in 250 °C. The temperature program applied was the following: 40 °C for 0.5 min, increase at 25 °C/min to 195 °C, increase at 3 °C/min to 205 °C, increase at 8 °C/min to 230 °C, and hold at 230 °C for 10 min. Methyl nonadecanoate (74208-1G methyl nonadecanoate analytical standard Fluka Analytical Sigma-Aldrich 1891) (1.5 mg/mL) was used as an internal standard. FAMEs identity was determined by comparison of retention times with those obtained with standards from Supelco 37 Component FAME Mix, Sigma-Aldrich 1891 separated on the same GC conditions. Total FAMEs were calculated as the sum of each individual peak area related to the area of the internal standard. Saponifiable lipid concentration was expressed as gram of methyl esters obtained from yeast biomass per liter of culture medium.

Growth medium and culture conditions for intracellular lipid accumulation

Rhodotorula graminis S1/2R was cultivated in 250 mL Erlenmeyer flasks, containing 50 mL of liquid medium, in an orbital shaker at 150 rpm at 28 °C for 5 days, according to conditions previously described by Pereyra et al. (2014). In the present study, the medium defined by Thakur et al. (1989) was used as a base, replacing glucose with crude glycerol which was previously treated according to Pyle et al. (2008) with modifications. Briefly, the crude glycerol was diluted 1:2 (v/v) and then, acidified to pH 1 with 3 N HCl, in order to release the fatty acids which were subsequently separated by centrifugation (5000 rpm, 15 min). The supernatant was then adjusted to pH 5 with 1 M NaOH and glycerol concentration was determined by HPLC, as described above. The obtained solution was added to the basal medium in order to reach a glycerol concentration of 40 g L−1, which was previously determined as the maximum concentration that did not affect the growth of the yeast strain (Pereyra et al. 2014). The concentration (g L−1) of the remaining components of the medium were: KH2PO4, 0.75; yeast extract (YE), 1.5; NH4NO3, 0.285; CaCl2·2H20, 0.4; MgSO4·7H2O, 0.4. Three biological replicates were carried out. After 5 days of culture, biomass, glycerol and the amount of FAMEs obtained through a direct transesterification process from yeast biomass was determined as described above.

Experimental design to determine variables that affect lipid accumulation

A fractional factorial design (FFD) of 25−1 (resolution V) was carried out to study the influence of pH, C/N ratio, CaCl2, MgSO4 and YE concentration, on saponifiable lipid accumulation in yeast biomass. In all cases, glycerol concentration was maintained at 40 g L−1, and the concentration of KH2PO4 was set at 0.75 g L−1 to regulate the pH. Each factor was examined at two levels with three replicated runs at the central point to give an estimate of the experimental error. The levels assayed for each factor were 5 and 7 for pH, 40 and 120 for C/N ratio, 0.0 and 1.5 g L−1 for YE concentration and 0.0 and 0.4 g L−1 for both CaCl2 and MgSO4 concentration. For the central point treatments all factors were assayed at an intermediate level corresponding to the mean value of each interval. The total number of trials, including the 3 central points, were 19. Each flask containing 50 mL of the medium was inoculated with 10% (v/v) pre-inoculum of the yeast from a 24 h old culture obtained in the same medium. Flasks were incubated as described above and after 5 days biomass and FAMEs corresponding to intracellular saponifiable lipids were determined as described before. The concentration of FAMEs corresponding to intracellular saponifiable lipids of the yeast biomass (as grams per liter of culture medium) was used as the response.

The experimental design and statistical analysis were performed with the aid of Design Expert, Version 10 software (STAT-EASE Inc., Minneapolis, USA). Since a two-level factorial design was used results were fitted to a first order model. Analysis of variance (ANOVA) was performed to further evaluate the model and to determine the factors that had a significant effect (95% level of confidence) on lipid accumulation.

Optimization of the composition of the medium to maximize lipid accumulation

Response surface methodology (RSM) based on central composite design (CCD) was used to optimize the levels of the significant variables in order to maximize lipid production. Design-Expert, Version 10 (STAT-EASE Inc., Minneapolis, USA) was used to perform the experimental designs and statistical analysis of the results.

Three variables C/N ratio and YE and MgSO4 concentrations at five concentration levels were included in the design. The levels assayed for C/N ratio were 59.77, 70, 85, 100 and 110.23; 0.33, 0.50, 0.7, 1.00 and 1.17 g L−1 for YE concentration and 0.10, 0.15, 0.23, 0.30 and 0.35 g L−1 for MgSO4. Seventeen experiments were conducted including three replicates at the central point. The concentration of FAMEs corresponding to intracellular saponifiable lipids of the yeast biomass was used as the response. An empirical model was obtained relating the response with the variables of the experiment. Using this model, the optimal values of significant variables were determined, in order to maximize the concentration of saponifiable lipids. The model was validated experimentally in triplicate. The theoretical maximum concentration of saponifiable lipids obtained by the model was compared with the experimental value obtained in such conditions. That experimental value was also compared with the concentration of saponifiable lipids obtained previously in the basal medium.

Batch and fed-batch processes in a 3 L fermenter

Batch and fed-batch fermentations were carried out in a 3 L jar fermenter (Fermenter BLBIO-3GJ, ShanghaiBailunBio-Technology.co, Ltd) with a 2 L working volume. In both cases the culture medium was the one defined as optimal by the CCD, whose composition (g L−1) was: glycerol, 40; KH2PO4, 0.75; YE, 1.12; NH4NO3, 0.744; MgSO4·7H2O, 0.11. Additionally, 0.5 mL of antifoam (Sigma-A6426) was added to the medium before being inoculated with a 24 h old culture of the yeast obtained in the same medium. Cultures were maintained at 28 °C, aerated with 2 vvm air and agitated at 250 rpm. Temperature was monitored with a platinum probe (Pt100), and pH and dissolved O2 (% DO) were measured and recorded continuously with a pH meter 405-DPAS-SC-K8S (Mettler, Toledo) and an oxygen probe InPro 6800 (Mettler, Toledo), respectively. Samples were taken every 24 h. Biomass, FAMEs corresponding to intracellular saponifiable lipids and glycerol concentration was determined in each sample as described above. Batch culture was finished after glycerol depletion when a stop in the increase of biomass and lipids was registered. In fed-batch culture, three feedings with 200 mL of a 10 × concentrated medium were made when % DO reached 80%.

Results

Growth and intracellular lipid accumulation in presence of glycerol

To promote the intracellular accumulation of lipids, R. graminis S1/2R was cultivated in presence of pretreated crude glycerol as carbon and energy source, in agitated flasks at 28 °C. After 5 days the concentration of glycerol in the culture medium was almost depleted (0.44 ± 0.19 g L−1), the concentration of biomass was 13.22 ± 0.33 g L−1 and the accumulation of intracellular saponifiable lipids was 2.90 ± 0.13 g L−1, determined as FAMEs obtained by direct transesterification from yeast biomass.

Determination of variables that influence the concentration of intracellular lipids

Table 1 shows the 19 experimental runs and response values obtained in the screening of significant variables on the concentration of FAMEs (g L−1 of culture) from intracellular lipids accumulated by R. graminis S1/2R. The results were evaluated through ANOVA and a model involving significant factors was generated. These results are shown in Table 2. The C/N ratio and the concentrations of YE and MgSO4 were the only variables with a significant effect (p = 0.0117, p < 0.0001 and p = 0.0058, respectively). A significant curvature was observed, with p < 0.0099, which means that in the center of the design space, the behavior of the response was not linear.
Table 1

Fractional factorial design matrix and FAMEs concentration after 5 days of culture

Trial

Independent variables

Dependent variable

C/N ratio

YE (g L−1)

pH

CaCl2 (g L−1)

MgSO4 (g L−1)

FAMEs (g L−1)

1

120

1.50

5

0.4

0.0

3.18

2

120

0.00

7

0.4

0.0

0.10

3

120

0.00

7

0.0

0.4

1.73

4

40

1.50

7

0.4

0.0

2.68

5

80

0.75

6

0.2

0.2

3.07

6

40

0.00

7

0.4

0.4

1.91

7

40

1.50

7

0.0

0.4

3.33

8

120

0.00

5

0.4

0.4

1.44

9

80

0.75

6

0.2

0.2

3.16

10

40

0.00

7

0.0

0.0

0.03

11

120

0.00

5

0.0

0.0

0.09

12

40

1.50

5

0.0

0.0

0.48

13

80

0.75

6

0.2

0.2

3.28

14

40

0.00

5

0.4

0.0

0.09

15

40

0.00

5

0.0

0.4

0.31

16

120

1.50

7

0.0

0.0

2.76

17

120

1.50

5

0.0

0.4

3.27

18

120

1.50

7

0.4

0.4

3.09

19

40

1.50

5

0.4

0.4

2.36

Table 2

ANOVA of the fractional factorial design showing the effect of factors on FAMEs concentrations

Source

Sum of squares

Degrees of freedom

Mean square

F value

p-value prob > F

Model

1744.34

5

348.87

11.42

0.0003

A-C/N

269.68

1

269.68

8.83

0.0117

B-YE

1089.42

1

1089.42

35.65

< 0.0001

C-pH

40.94

1

40.94

1.34

0.2696

D-CaCl2

1.52

1

1.52

0.05

0.827

E-MgSO4

342.78

1

342.78

11.22

0.0058

Curvature

286.29

1

286.29

9.37

0.0099

Optimization of the composition of the medium to maximize the accumulation of lipids

Based on the previous factorial design, RSM using CCD was applied to determine the optimal levels of the three variables (C/N ratio and YE and MgSO4 concentrations) that significantly influenced the lipids accumulated by R. graminis S1/2R. Since the variation in the concentration of CaCl2 and pH had no significant effect on the concentration of FAMEs, both factors remained constant in the CCD, in values within the range studied previously. In this way, pH of 6 was set for all cases and CaCl2 was not included in the medium to reduce the cost. Table 3 presents the design and the concentration of FAMEs obtained from the yeast biomass in each case.
Table 3

Central composite design matrix and FAMEs concentrations after 5 days of culture

Trial

C/N ratio

YE (g L−1)

MgSO4 (g L−1)

FAMEs (g L1)

1

85

0.75

0.23

2.90

2

85

0.75

0.1

3.22

3

100

0.5

0.15

2.47

4

70

0.5

0.15

3.00

5

85

0.75

0.23

3.32

6

100

1

0.15

3.02

7

70

0.5

0.3

2.86

8

110.23

0.75

0.23

2.52

9

70

1

0.15

3.45

10

100

1

0.3

2.97

11

85

0.75

0.35

3.10

12

85

0.33

0.23

2.50

13

100

0.5

0.3

2.62

14

59.77

0.75

0.23

3.42

15

85

1.17

0.23

3.47

16

85

0.75

0.23

3.08

17

70

1

0.3

3.70

The experimental results were analyzed by ANOVA (Table 4). In the assayed range the YE concentration and the C/N ratio had a significant effect on the concentration of FAMEs in the culture (p < 0.0001), but the concentration of MgSO4 was not significant (p = 0.9742). The CCD was fitted with the following first order equation, in which only the significant variables were considered:
Table 4

ANOVA of the central composite design showing the effect of factors on FAMEs concentration

Source

Sum of squares

Degrees of freedom

Mean square

F value

p-value prob > F

Model

5.35

3

1.78

35.28

< 0.0001

A-YE

2.95

1

2.95

58.28

< 0.0001

B-MgSO4

5.51E−05

1

5.51E−05

1.09E−03

9.74E−01

C–C/N

2.4

1

2.4

47.49

< 0.0001

FAMEs (g L−1) = 1.12 YE − 0.02 C/N + 3.63.

The fit of the model was checked by R2, which was calculated to be 0.89 indicating that 89% of the variability in the response could be explained by the model. The statistical significance of the model equation was also assessed by ANOVA (p = 0.0001).

The response surface curve (Fig. 1) shows the effect of the two significant variables on FAMEs concentration. As the concentration of YE increased, higher concentrations of FAMEs were obtained, whereas this relationship was the reverse in the case of the C/N ratio. The model predicted that the optimal values of the two significant variables were YE = 1.00 g L−1 and C/N ratio = 70, and the maximum predicted FAMEs concentration was 3.52 g L−1.
Fig. 1

Response surface plot, of the combined effects of YE and C/N ratio on FAMEs concentration obtained with the central composite design

In order to confirm the optimized culture conditions, three additional experiments in shake flasks were performed using the predicted optimal culture conditions. The concentrations of the components of the medium (g L−1) were: glycerol in the form of pretreated crude glycerol, 40; KH2PO4, 0.75; MgSO4, 0.10; YE, 1.00. Additionally, ammonium nitrate was added to reach a C/N ratio = 70 and initial pH was set at 6. The concentration of FAMEs obtained in those conditions was 3.79 ± 0.20 g L−1, which differed only 7% from the value predicted by the model. Although optimal values of variables were in the extreme of the intervals assayed in the CCD, higher values of YE and lower values of C/N ratio were not evaluated based on the results of the previous factorial design. The curvature effect found in that analysis showed that optimum values of significant variables would be near to the central point (C/N ratio = 85 and YE concentration = 0.75 g L−1) and not in the extremes of the interval. Therefore, they would not be beyond the lowest and the highest values assayed for C/N ratio (40) and YE concentration (1.5 g L−1) in the first experimental design. The results of the CCD confirmed those presumptions since the maximum concentration of FAMEs obtained (3.79 g L−1) was higher than the highest value obtained in the factorial assay (3.33 g L−1).

Table 5 presents a comparison between the optimized and the original medium used in this work. After optimization, the concentration of FAMEs obtained from the yeast S1/2R increased 30.6%. At the same time, in the optimized medium of the concentration of some of the components (YE, MgSO4 and CaCl2) were lower than in the original medium which could contribute to reduce production costs.
Table 5

Composition of the optimized culture medium compared to the medium defined by Thakur et al. (1989) and concentrations of biomass and FAMEs obtained in each case

 

Optimized medium

Medium defined by Thakur et al. (1989)

Glycerol (crude glycerol) (gL−1)

40

40

YE (g L−1)

1.12

1.50

MgSO4 (g L−1)

0.11

0.40

CaCl2 (g L−1)

0.40

NH4NO3 (g L−1)

0.774

0.285

KH2PO4 (g L−1)

0.75

0.75

C/N Ratio

69.41

82

pH

6

5

Biomass (g L−1)

13.60

13.22

FAMEs (g L−1)

3.79

2.90

Batch cultivation in 3 L fermentor

The growth of R. graminis S1/2R and the accumulation of intracellular saponifiable lipids in the optimized medium was evaluated in batch culture in a 3 L fermenter. The culture was monitored daily for 8 days, during which, the concentration of biomass, FAMEs and glycerol were analyzed. Dissolved oxygen was monitored daily over 8 days (Fig. 2).
Fig. 2

Time course of DO (%) measured continuously (primary vertical axis), biomass (g L−1) (black diamond), lipid production (g L−1) (black circle) and residual glycerol levels (g L−1) (black triangle) measured at periodic intervals (secondary vertical axis) during the batch culture of the strain S1/2R in the optimized medium

When glycerol was completely consumed after 120 h a marked increase in the dissolved oxygen concentration was assessed. At the same time, both yeast growth and lipids accumulation were stopped. The maximum amount of FAMEs obtained from the biomass in these conditions in the fermenter was 3.6 g L−1 at the fifth day of culture, which was very similar to that obtained in batch cultures in Erlenmeyer flasks.

Fed-batch cultivation

A fed-batch fermentation was performed in order to increase the amount of FAMEs obtained from the biomass. The concentration of glycerol, biomass, FAMEs and dissolved oxygen were monitored daily (Fig. 3). At start, glycerol concentration in the medium was 40 g L−1. Feedings with concentrated fresh medium to reach a glycerol concentration of 40 g L−1 were performed each time that the % DO increased up to 80% and glycerol was completely exhausted. Two feedings were performed, one after 118 h and the other after 247 h. Yeast growth, observed by an increment in biomass and a reduction of % DO, could be achieved after the first feeding (Fig. 3). However, after the second feeding, growth at the same rate could not be restored and so the culture was stopped at 450 h. At 360 h, the highest concentrations of FAMEs and dry biomass were obtained (12.2 g L−1 and 26.6 g L−1, respectively). At that time, glycerol concentration was 7.30 g L−1 meaning that 112 g L−1 were consumed by the yeast. In those conditions, lipid productivity was estimated to be 0.81 g L−1 day−1 and a lipid yield of 0.11 g/g of consumed glycerol was obtained.
Fig. 3

Time course of DO (%) measured continuously (primary vertical axis), biomass (g L−1) (black diamond), lipid production (g L−1) (black circle) and residual glycerol levels (g L−1) (black triangle) measured at periodic intervals (secondary vertical axis) during the fed- batch culture of the strain S1/2R in the optimized medium

The percentage composition of the mixture of fatty acids obtained from R. graminis S1/2R intracellular oils at the end of the fed-batch culture was the following: 0.5% lauric acid (14:0), 12.2% of palmitic acid (16:0), 4.8% palmitoloeic acid (16:1), 1.2% stearic acid (18:0), 73.7% of oleic acid (18:1), 6.0% linoleic acid (18:2), 0.8% linolenic acid (18:3), 0.3% behenic acid (20:0) and 0.5% lignoceric acid (24:0), Oleic and palmitic acid were the main components. No polyunsaturated fatty acids were detected and linolenic acid was below 1%. The cetane number obtained for the mixture, calculated according to Azam et al. (2005) was 59.

Discussion

In the present study the oleaginous yeast R. graminis S1/2R, previously selected for its ability to store saponifiable lipids (Pereyra et al. 2014) was grown in a crude glycerol based medium, to produce a mixture of FAMEs that fulfills the composition requirements for biodiesel. When the selected yeast was grown in batch at 150 rpm in a glycerol based medium, 2.90 g L−1 of FAMEs were obtained in 5 days at 28 °C. After optimizing the composition of the medium by means of a CCD, 3.79 g L−1 of FAMEs were achieved, which represented an improvement of 31% in the lipid yield. Moreover, the optimized medium resulted simpler and of lower cost than the initial one, due to a reduction in the concentration of YE, magnesium and calcium salts. Since the economic feasibility of biodiesel production from single cell oils is mainly determined by the cost of microbial production process (Ageitos et al. 2011), the use of a low cost culture medium is indispensable. In that sense, the carbon and energy source, which is the main component of a culture medium to grow heterotrophic microorganisms, like yeasts, should be selected among byproducts or even wastes from nearby industries, to minimize production and transportation costs. In this work crude glycerol, a byproduct of biodiesel production, was used as the main component of the culture medium. Even though after a costly purification process, glycerol could be sold to pharmaceutical or cosmetics industries, the increasing of biodiesel production makes essential the search of new fates for that byproduct. Its use as a component of culture media to produce biomass, extracellular or intracellular microbial products such as single cell oils seems to be a feasible alternative.

Previous works have used glycerol-based media, to achieve lipid accumulation in different oleaginous yeasts, including Rhodotorula species. In that sense, Galafassi et al. (2012) obtained a concentration of 2.86 g L−1 of total lipids from a R. graminis strain, while Saenge et al. (2011) obtained a maximum lipid yield of 2.5 g L−1 from a R. glutinis strain in agitated batch culture with glycerol as carbon and energy source. In both cases lipid yield resulted lower than the obtained in our work in similar conditions (3.79 g L−1). However, in case of Saenge et al. (2011) an important improvement (almost 75%) was obtained when the yeast was cultivated in a stirred and aerated bioreactor (aeration rate 2 vvm), reaching a concentration of 4.33 g L−1 of total lipids. In our case no improvement was achieved when R. graminis S1/2R was cultivated in stirred and aerated conditions, showing that dissolved oxygen was not the limiting factor in our cultures, as it seemed to be in Saenge et al. (2011). In most studies, lipid yield was expressed as total lipid content of microbial cells. However, in our work, results were expressed as FAMEs yields, which were obtained only from the saponifiable lipid fraction of yeast biomass. These data represent the actual amount of biodiesel that could be obtained following the process (production and extraction) described in the present work, which undoubtedly is lower than the total lipid content of yeast biomass.

In this work, an important improvement of lipid concentration was achieved in fed batch culture. More than 12 g L−1 of FAMEs were obtained from R. graminis S1/2R after two feedings with concentrated fresh medium. That yield represented an enhancement up to threefold compared with the yield obtained in batch culture. In those conditions, approximately 112 g of glycerol per liter of culture were consumed by the yeast, which represented a yield of 0.11 g of biodiesel per gram of glycerol. Although the maximum theoretical yield of triacylglicerides on glycerol is around 0.30 g/g, the experimental values obtained in most cases are lower, around 0.10 gg−1 (Papanikolaou and Aggelis 2011), which are in accordance with the values obtained in this study.

In the present work, the implementation of a fed batch strategy was necessary to overcome yeast growth inhibition by substrate since the selected yeast could not grow if glycerol concentration was higher than 40 g L−1 (Pereyra et al. 2014). In such conditions, considering a yield of 0.11 g/g of consumed glycerol, the expected FAMEs concentration in a batch culture could not be higher than 4.4 g L−1. Thus, successive feedings were necessary to obtain higher amounts of FAMEs.

After the third feeding, yeast growth could not be restored. Since a high concentration of biomass (more than 25 g L−1) was present in the bioreactor, a problem with oxygen transfer was postulated as the cause of the stop of yeast growth. According to those results, fermentation could be stopped at 360 h, after two feedings, achieving 12 g of biodiesel per liter of culture medium. With such yield approximately 50,000 L of culture would be necessary to produce a biodiesel amount equivalent to that obtained from one hectare of soybean in a year (Gui et al. 2008). In those conditions second generation biodiesel produced from yeast biomass seems to be a reliable and sustainable alternative. Yeast production does not involve the use of cropland, is not affected by seasons or climate and is environmentally friendly since no pesticide applications are needed. On the other hand, concerns about environmental sustainability of using farmland to grow crops for biofuels production have risen. It has been demonstrated that greenhouse gases emissions have increased when forest and grassland are converted into new croplands for biodiesel feedstock production or to replace the grain diverted to biofuel (Kazamia and Smith 2014).

The composition of biodiesel is highly influenced by feedstock and production conditions. National and international standards namely UNIT 1100:2014 (2014) (Uruguay), ASTM D6751-12 (2012) (USA), and EN 14214 (2003) (Europe) have been set up to monitor the quality of biodiesel. Among the characteristics related to composition of methyl esters mixture, biodiesel in Uruguay must have a cetane number higher than 45 and contain no more than 12% of linolenic acid esters or 1% of esters of polyunsaturated acids (more than 4 double bonds). Similar requirements can be found in international standards with slight variations in the minimum cetane number which, in case of EN 14214 and ASTM D6751, must be higher than 51 and 47, respectively (Patel et al. 2017). Saponifiable lipids accumulated by R. graminis S1/2R in the culture conditions described in this work fulfilled those requirements and therefore represent a promising alternative as raw material for second generation biodiesel production.

Conclusion

The present report describes the use of the oleaginous yeast R. graminis S1/2R, for the production of lipid and the conversion of yeast oil into biodiesel. Optimization of the process and scaling up in a 3 L fermenter using crude glycerol as carbon source resulted in the intracellular accumulation of saponifiable lipids representing almost 46% of the oleaginous yeast dry weight. The use of a crude glycerol-based medium seems to be a sustainable way of reducing biodiesel production costs and represents a convenient fate for this waste by-product.

Notes

Acknowledgements

This work was supported by Agencia Nacional de Investigación e Innovación (ANII) (Grant no. FSE144810), Comisión Sectorial de Investigación Científica (CSIC Uruguay) and Programa de Desarrollo de las Ciencias Básicas (PDECIBA).

Compliance with ethical standards

Conflict of interest

The authors state that they have no conflict of interest.

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Copyright information

© Society for Environmental Sustainability 2019

Authors and Affiliations

  1. 1.Área de Microbiologia, Departamento de Biociencias, Facultad de QuímicaUniversidad de la RepúblicaMontevideoUruguay
  2. 2.Instituto Polo Tecnológico, Facultad de QuímicaUniversidad de la RepúblicaPandoUruguay

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