Bioprospecting microbes for single-cell oil production from starchy wastes

Production of lipid from oleaginous yeast using starch as a carbon source is not a common practice; therefore, the purpose of this investigation was to explore the capability of starch assimilating microbes to produce oil, which was determined in terms of biomass weight, productivity, and lipid yield. Saccharomyces pastorianus, Rhodotorula mucilaginosa, Rhodotorula glutinis, and fungal isolate Ganoderma wiiroense were screened for the key parameters. The optimization was also performed by one-factor-at-a-time approach. Considering the specific yield of lipid and cell dry weight yield, R. glutinis and R. mucilaginosa showed superiority over other strains. G. wiiroense, a new isolate, would also be a promising strain for starch waste utilization in terms of extracellular and intracellular specific yield of lipids. Extracellular specific yield of lipid was highest in R. glutinis culture (0.025?g?g^?1 of biomass) followed by R. mucilaginosa (0.022?g?g^?1 of biomass) and G. wiiroense (0.020?g?g^?1 of biomass). Intracellular lipid was again highest in R. glutinis (0.048?g?g^?1 of biomass). The most prominent fatty acid methyl esters among the lipid as detected by GC-MS were saturated lipids mainly octadecanoic acid, tetradecanoate, and hexadecanoate. Extracellular lipid produced on starch substrate waste would be a cost-effective alternative for energy-intensive extraction process in biodiesel industry.     

Overexpression of malic enzyme (ME) of Mucor circinelloides improved lipid accumulation in engineered Rhodotorula glutinis

The oleaginous yeast Rhodotorula glutinis has been known to be a potential feedstock for lipid production. In the present study, we investigated the enhancement of expression of malic enzyme (ME; NADP⁺ dependent; EC 1.1.1.40) from Mucor circinelloides as a strategy to improve lipid content inside the yeast cells. The 26S rDNA and 5.8S rDNA gene fragments isolated from Rhodotorula glutinis were used for homologous integration of ME gene into R. glutinis chromosome under the control of the constitutively highly expressed gene phosphoglycerate kinase 1 to achieve stable expression. We demonstrated that by increasing the expression of the foreign ME gene in R. glutinis, we successfully improved the lipid content by more than twofold. At the end of lipid accumulation phrase (96 h) in the transformants, activity of ME was increased by twofold and lipid content of the yeast cells was increased from 18.74 % of the biomass to 39.35 %. Simultaneously, there were no significant differences in fatty acid profiles between the wild-type strain and the recombinant strain. Over 94 % of total fatty acids were C_16:0, C_18:0, C_16:1, C_18:1, and C_18:2. Our results indicated that heterologous expression of NADP⁺-dependent ME involved in fatty acid biosynthesis indeed increased the lipid accumulation in the oleaginous yeast R. glutinis.     

Oily yeasts as oleaginous cell factories

Oily yeasts have been described to be able to accumulate lipids up to 20% of their cellular dry weight. These yeasts represent a minor proportion of the total yeast population, and only 5% of them have been reported as able to accumulate more than 25% of lipids. The oily yeast genera include Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces. More specifically, examples of oleaginous yeasts include the species: Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, and Yarrowia lipolytica. Yeast do exhibit advantages for lipid production over other microbial sources, namely, their duplication times are usually lower than 1 h, are much less affected than plants by season or climate conditions, and their cultures are more easily scaled up than those of microalgae. Additionally, some oily yeasts have been reported to accumulate oil up to 80% of their dry weight and can indeed generate different lipids from different carbon sources or from lipids present in the culture media. Thus, they can vary their lipid composition by replacing the fatty acids present in their triglycerides. Due to the diversity of microorganisms and growth conditions, oily yeasts can be useful for the production of triglycerides, surfactants, or polyunsaturated fatty acids.     

Fatty acids from oleaginous yeasts and yeast-like fungi and their potential applications

Purpose: Oleaginous yeasts, fatty acids biosynthesis and regulation in the oleaginous yeasts and the fatty acids from the oleaginous yeasts and their applications are reviewed in this article.

Results: Oleaginous yeasts such as Rhodosporidium toruloides, Yarrowia lipolytica, Rhodotorula mucilaginosa, and Aureobasidium melanogenum, which can accumulate over 50% lipid of their cell dry weight, have many advantages over other oleaginous microorganisms. The fatty acids from the oleaginous yeasts have many potential applications. Many oleaginous yeasts have now been genetically modified to over-produce fatty acids and their derivatives. The most important features of the oleaginous yeasts are that they have special enzymatic systems for enhanced biosynthesis and regulation of fatty acids in their lipid particles. Recently, some oleaginous yeasts such as R. toruloides have been found to have a unique fatty acids synthetase and other oleaginous yeasts such as A. melanogenum have a unique highly reducing polyketide synthase (HR-PKS) involved in the biosynthesis of hydroxyl fatty acids.

Conclusions: It is necessary to further enhance lipid biosynthesis using metabolic engineering and explore new applications of fatty acids in biotechnology.     

Kinetics of growth and fatty acid production of Rhodotorula glutinis

Summary The effect of culture conditions, especially nitrogen/carbon (N/C) ratio and temperature, on acyl lipid profiles in the oleaginous yeast Rhodotorula glutinis NRRL Y 1091 have been investigated. Cultivation of the microorganism under N-limited conditions (N/C < 0.14 g/g) resulted in enhanced fatty acid (FA) cell content but a reduced relative amount of polyunsaturated fatty acids (PUFA). The maximal FA productivity was obtained for a 0.025 N/C ratio resulting from the arrangement between the specific rate of FA synthesis and the concentration of lipid-free biomass. Under nitrogen-non-limiting conditions, cells grown at lower temperatures had a higher PUFA content and the maximal productivity of -linolenic acid was obtained by shifting the temperature of the culture from 30° C to 25° C. Offprint requests to: A. Pareilleux     

Determining intracellular lipid content of different oleaginous yeasts by one simple and accurate Nile Red fluorescent method

Abstract

A simple and accurate Nile Red fluorescent method was built to evaluate the lipid content of three different oleaginous yeasts by one standard curve. The staining of cells can be observed clearly by laser scanning confocal microscope, showing that Nile Red can enter into the cells of oleaginous yeasts easily. A series of conditions such as pretreating temperature, cell suspension concentration (OD₆₀₀), staining time, Nile Red concentration and the type of suspension solvent were learnt systematically to obtain the optimal process parameters for Nile Red staining. After optimization, the fitting curve of Nile Red fluorescent method was established under suitable conditions (pretreating temperature: 50?°C, OD₆₀₀: 1.0; staining time: 5?mins; Nile Red concentration: 1.0?μg/mL; suspension solvent: PBS) and it had a suitable correlation coefficient (R² = 0.95) for lipid content measurement of different oleaginous yeasts. By this study, the possibility of lipid content determination of different oleaginous yeasts by one fitting curve can be proven and this will improve the efficiency of researches related to microbial lipid production.     

Lipid accumulation inRhodotorula glutinis on sugar cane molasses in single-stage continuous culture

Microbial lipids produced byRhodotorula glutinis grown in continuous culture with molasses under nitrogen-limiting conditions were evaluated and the effects of growth rate on fatty acid composition were studied. As the growth rate decreased, cell biomass, lipid content and lipid yield gradually increased. The maximum lipid content recorded was 39% (w/w) of dry cell biomass at a dilution rate of 0.04 h^–1. The growth rate also affected fatty acid composition: oleic acid decreased with decreasing growth rate while stearic acid increased.     

Effect of various nutrient limitations on fatty acid production by Rhodotorula glutinis

The effects of nitrogen (N), phosphorus (P), zinc (Zn) or iron (Fe) limitations on lipid and especially -linolenic acid (ALA) synthesis in the oleaginous yeast Rhodotorula glutinis have been investigated. Exhaustion of the limiting element in the medium resulted in an enhancement of both the fatty acid cell content (FA/X) and the corresponding productivity. Except for Fe-limited media, lipid accumulation was always coupled with an increase in the yield of ALA synthesis, whereas in all the limiting conditions the productivity of ALA declined. Kinetic investigations showed that this decline resulted from a sharp decrease in the specific rates of ALA synthesis associated with slowing down of growth. A comparative study showed that the highest yields and specific rates of fatty acid and ALA synthesis were induced by P-limitation early in the lipid accumulation phase. However, above a FA/X of 15%, N-limiting conditions become more attractive for producing lipids. During P-limited growth a down-shift of temperature from 30° C to 25° C was shown ao reduce the range of FA/X values at which the specific rate of fatty acid synthesis was maximal but without any other effect on fatty acid formation. In contrast, the decreased temperature resulted in enhanced ALA production by maintaining the specific rate of synthesis near to the maximal value of 3.6 mg g _X ^–1 h^–1, where X^* is free fatty acid biomass, and increased the corresponding yield by a factor of up to three. Correspondence to: A. Pareilleux     

Accumulation of lipid byRhodotorula glutinis in continuous culture

Summary Rhodotorula glutinis accumulated 35% (w/w) lipid when grown nitrogen-limited in a chemostat at a dilution rate (D) of 0.02^–1 . At D = 0.10 h^–1, the lipid content was only 15% (w/w). Dual limitation of nitrogen and phosphate increased neither the amount of lipid produced nor the lipid yield (14g lipid per 100g glucose consumed). The fatty acid composition was unchanged by the growth rate.     

Comparative Proteomics Profile of Lipid-Cumulating Oleaginous Yeast: An iTRAQ-Coupled 2-D LC-MS/MS Analysis

Accumulation of intracellular lipid in oleaginous yeast cells has been studied for providing an alternative supply for energy, biofuel. Numerous studies have been conducted on increasing lipid content in oleaginous yeasts. However, few explore the mechanism of the high lipid accumulation ability of oleaginous yeast strains at the proteomics level. In this study, a time-course comparative proteomics analysis was introduced to compare the non-oleaginous yeast Saccharomyces cerevisiae, with two oleaginous yeast strains, Cryptococcus albidus and Rhodosporidium toruloides at different lipid accumulation stages. Two dimensional LC-MS/MS approach has been applied for protein profiling together with isobaric tag for relative and absolute quantitation (iTRAQ) labelling method. 132 proteins were identified when three yeast strains were all at early lipid accumulation stage; 122 and 116 proteins were found respectively within cells of three strains collected at middle and late lipid accumulation stages. Significantly up-regulation or down-regulation of proteins were experienced among comparison. Essential proteins correlated to lipid synthesis and regulation were detected. Our approach provides valuable indication and better understanding for lipid accumulation mechanism from proteomics level and would further contribute to genetic engineering of oleaginous yeasts.     

Optimization of pineapple pulp residue hydrolysis for lipid production by Rhodotorula glutinis TISTR5159 using as biodiesel feedstock

The higher lipid productivity of Rhodotorula glutinis TISTR5159 was achieved by optimizing the pineapple pulp hydrolysis for releasing the high sugars content. The sequential simplex method operated by varied; solid-to-liquid ratio, sulfuric acid concentration, temperature, and hydrolysis time were successfully applied and the highest sugar content (83.2 g/L) evaluated at a solid-to-liquid ratio of 1:10.8, 3.2% sulfuric acid, 105 °C for 13.9 min. Moreover, the (NH₄)₂SO₄ supplement enhanced the lipid productivity and gave the maximum yields of biomass and lipid of 15.2 g/L and 9.15 g/L (60.2%), respectively. The C16 and C18 fatty acids were found as main components included oleic acid (55.8%), palmitic acid (16.6%), linoleic acid (11.9%), and stearic acid (7.8%). These results present the possibility to convert the sugars in pineapple pulp hydrolysate to lipids. The fatty acid profile was also similar to vegetable oils. Thus, it could be used as potential feedstock for biodiesel production.     

Lipid formation by Cryptococcus albidus in nitrogen-limited and in carbon-limited chemostat cultures

Summary Cryptococcus albidus var. Albidus CBS 4517 was grown in nitrogen-limited and in carbon-limited chemostat cultures. The effect of growth rate and limiting nutrient on lipid accumulation and fatty acid composition was investigated.The maximum lipid content in the biomass was, in both cultivation systems, observed at the lowest dilution rate (growth rate) tested. At this dilution rate, D=0.31 h^-1, cells from the nitrogen-limited culture contained 41% (w/w) lipid and cells from the carbon-limited culture 37%. These results indicate the ability of C. albidus, unlike other oleaginous yeasts, to accumulate lipid also in carbon-limited chemostats.The yield of lipid from carbon source was about the same at D=0.031 h^-1 in nitrogen-limited (Y _L/S=0.16 g/g) as in carbon-limited (Y _L/S=0.17 g/g) cultures and decreased with increasing growth rates. In the nitrogen-limited culture, the lipid productivity was about constant at low growth rates (0.031–0.056 h^-1) and a slight decrease was observed at D=0.08 h^-1, while the specific lipid productivity, q _L, increased to 27.5 mg/g per hour. In the carbon-limited culture, however, lipid productivity increased with increasing growth rates and reached its maximum value near _max, whereas q _L was about constant at 20 mg/g per hour.The fatty acid composition was influenced by the specific growth rate in nitrogen-limited as well as in carbon-limited cultures, although the changes were more pronounced during carbonlimitation. A decrease in the degree of unsaturation (/mole) was also observed with increasing lipid content in the cells.     

Lipids of Cunninghamella echinulata with emphasis to γ-linolenic acid distribution among lipid classes

Changes in lipid composition of the oleaginous fungus Cunninghamella echinulata were monitored during growth. Lipid fractions and individual lipid classes varied in amount, relative proportions, and fatty acid profile depending on the developmental stage. Neutral lipids (N), comprised mainly of triacylglycerol, were accumulated in the fungal mycelium during both the late exponential and the stationary growth phases with a concomitant decrease in the amount of polar lipids. While fatty acid composition of N fraction remained almost constant, individual N classes showed a noticeable alteration in γ-linolenic acid (GLA) concentration. The glycolipid plus sphingolipid (G+S) fraction consisted mainly of monoglycosylglycerol and diglycosylglycerol. The sugar composition of G+S fraction was analyzed and showed a partial replacement of galactose for glucose as growth proceeded. Phospholipid (P) major classes were phosphatidylcholine (PC) and phosphatidylethanolamine, followed by phosphatidylinositol, phosphatidylserine, and diphosphatidylglycerol. P fatty acid composition showed significant changes with time, resulting in a considerable drop in the unsaturation index of this fraction. While in mid exponential growth phase, all P classes contained more than 20% w/w GLA of total fatty acids, and their concentration decreased to 12–17% w/w, except for the PC class where GLA concentration remained at high levels (e.g., more than 20% w/w). The constant level of GLA in PC at all growth phases suggests that PC was the major source of GLA. Sterol analysis showed that their concentration increased during growth, whereas ergosterol was the major component.     

Fatty acid composition in lipid fractions lengthwise the mycelium of Mortierella isabellina and lipid production by solid state fermentation

This paper investigates the correlation between mycelial age and fatty acid biosynthesis. The correlation was investigated by analyzing the lipid composition lengthwise the mycelium of the oleaginous fungus Mortierella isabellina, a potential producer of γ-linolenic acid (GLA). Young mycelia were rich in polar lipids (glycolipids plus sphingolipids and phospholipids), while neutral lipid content increased in aged mycelia. In young mycelia, each polar lipid fraction contained almost 40% (w/w) polyunsaturated fatty acids (PUFAs), but this content decreased to less than 30% (w/w) in aged mycelia. On the other hand, PUFA content in neutral lipids fluctuated slightly with age. These results indicate that PUFA biosynthesis is favored in young, fast growing mycelia, while it decreases significantly in aged mycelia. This trend was also observed when we grew M. isabellina on pear pomace, an agro-industrial waste. Pear pomace cultures yielded significant amounts of lipid, which reached 12% (w/w) in dry fermented mass. The produced lipid was rich in GLA and the maximum GLA content in dry fermented mass was 2.9 mg/g.     

Characterization of the mitochondrial NAD+ -dependent isocitrate dehydrogenase of the oleaginous yeast Rhodosporidium toruloides

Early biochemical studies have demonstrated that lipid accumulation by oleaginous yeasts is linked to the activity of the NAD⁺-dependent isocitrate dehydrogenase (Idh). However, molecular study of Idh of oleaginous microorganisms remains limited. Here, we present the cloning of a mitochondrial NAD⁺-specific Idh from Rhodosporidium toruloides (RtIdh), an excellent microbial lipid producer that uses carbohydrates as the carbon source. The evolutionary relationship analyses among RtIdhs and other yeast Idhs revealed that RtIdh had a closer relationship with the Idhs of Ustilago maydis and Schizophyllum commune. We expressed the RtIDH gene in the yeast Saccharomyces cerevisiae idhΔ mutant. Under the nitrogen-limited condition, the intracellular lipid content and extracellular citrate concentration of the culture of the S. cerevisiae idhΔ carrying the RtIDH gene increased as the carbon/nitrogen molar ratio of the media increased, while the wild-type S. cerevisiae strain showed no correlation. Our data provided valuable information for elucidating the molecular mechanism of microbial oleaginicity and for engineering microorganisms to produce metabolites of fatty acid pathway.     

Transformation of vegetable oils by an oleaginous yeast:Rhodotorula glutinis IIP-30

Summary A locally isolated oleaginous yeastRhodotorula glutinis IIP-30 was grown on vegetable oils obtained from coconut, ground nut and til. The fatty acid composition of yeast oil was quite similar to that of the substrate oil in case of ground nut and til, while it was different with coconut oil. Utilization of C₁₂ and C₁₄ fatty acids of coconut oil to yield higher proportions of C₁₈₁ and C₁₈₂ fatty acids was observed.     

Carbon accumulation in <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis> induced by nitrogen limitation

Background

Oleaginous microorganisms, such as bacterium, yeast and algal species, can represent an alternative oil source for biodiesel production. The composition of their accumulated lipid is similar to the lipid of an oleaginous plant with a predominance of unsaturated fatty acid. Moreover this alternative to conventional biodiesel production does not create competition for land use between food and oleo-chemical industry supplies. Despite this promising potential, development of microbial production processes are at an early stage. Nutritional limited conditions, such as nitrogen limitation, with an excess of carbon substrate is commonly used to induce lipid accumulation metabolism. Nitrogen limitation implies modification of the carbon-to-nitrogen ratio in culture medium, which impacts on carbon flow distribution in the metabolic network.

Results

The goal of the present study is to improve our knowledge of carbon flow distribution in oleaginous yeast metabolism by focusing carbon distribution between carbohydrate and lipid pools in order to optimize microbial lipid production. The dynamic effects of limiting nitrogen consumption flux according to carbon flow were studied to trigger lipid accumulation in the oleaginous yeast Rhodotorula glutinis. With a decrease of the specific nitrogen consumption rate from 0.052 Nmol.Cmol_X^?1.h^?1 to 0.003 Nmol.Cmol_X^?1.h^?1, a short and transitory intracellular carbohydrate accumulation occurred before the lipid accumulation phase. This phenomenon was studied in fed-batch culture under optimal operating conditions, with a mineral medium and using glucose as carbon source. Two different strategies of decreasing nitrogen flow on carbohydrate accumulation were investigated: an instantaneous decrease and a progressive decrease of nitrogen flow.

Conclusions

Lipid production performance in these fed-batch culture strategies with R. glutinis were higher than those reported in the previous literature; the catalytic specific lipid production rate was 0.07 Cmol_lip.Cmol_X*^?1.h^?1. Experimental results suggested that carbohydrate accumulation was an intrinsic phenomenon connected to the limitation of growth by nitrogen when the nitrogen-to-carbon ratio in the feed flow was lower than 0.045 Nmol.Cmol^?1. Carbohydrate accumulation corresponded to a 440% increase of carbohydrate content. These results suggest that microbial lipid production can be optimized by culture strategy and that carbohydrate accumulation must be taken account for process design.

     

Oleaginous yeasts: Biodiversity and cultivation

Microbial lipids produced by oleaginous microorganisms, also called microbial oils and single cell oils (SCOs), are very promising sources for several oil industries. The exploration of efficient oleaginous yeast strains, meant to produce both high-quantity and high-quality lipids for the production of biodiesel, oleochemicals, and the other high value lipid products, have gained much attention. At present, the number of oleaginous yeast species that have been discovered is 8.2% of the total number of known yeast species, most of which have been isolated from their natural habitats. To explore high lipid producing yeasts, different methods, including high-throughput screening methods using colorimetric or fluorometric measures, have been developed. Understanding of the fatty acid composition profiles of lipids produced by oleaginous yeasts would help to define target lipid-related products. For lipid production, the employment of low-cost substrates suitable for yeast growth and lipid accumulation, and efficient cultivation processes are key factors for successfully increasing the amount of the accumulated lipid yield while decreasing the cost of production.     

Modeling lipid accumulation in oleaginous fungi in chemostat cultures. II: Validation of the chemostat model using yeast culture data from literature

A model that predicts cell growth, lipid accumulation and substrate consumption of oleaginous fungi in chemostat cultures (Meeuwse et al. in Bioproc Biosyst Eng. doi:, 2011) was validated using 12 published data sets for chemostat cultures of oleaginous yeasts and one published data set for a poly-hydroxyalkanoate accumulating bacterial species. The model could describe all data sets well with only minor modifications that do not affect the key assumptions, i.e. (1) oleaginous yeasts and fungi give the highest priority to C-source utilization for maintenance, second priority to growth and third priority to lipid accumulation, and (2) oleaginous yeasts and fungi have a growth rate independent maximum specific lipid production rate. The analysis of all data showed that the maximum specific lipid production rate is in most cases very close to the specific production rate of membrane and other functional lipids for cells growing at their maximum specific growth rate. The limiting factor suggested by Ykema et al. (in Biotechnol Bioeng 34:1268–1276, 1989), i.e. the maximum glucose uptake rate, did not give good predictions of the maximum lipid production rate.     

The expression of the Cuphea palustris thioesterase CpFatB2 in Yarrowia lipolytica triggers oleic acid accumulation

The conversion of industrial by‐products into high‐value added compounds is a challenging issue. Crude glycerol, a by‐product of the biodiesel production chain, could represent an alternative carbon source for the cultivation of oleaginous yeasts. Here, we developed five minimal synthetic glycerol‐based media, with different C/N ratios, and we analyzed the production of biomass and fatty acids by Yarrowia lipolytica Po1g strain. We identified two media at the expense of which Y. lipolytica was able to accumulate ～5 g L^?1 of biomass and 0.8 g L^?1 of fatty acids (0.16 g of fatty acids per g of dry weight). These optimized media contained 0.5 g L^?1 of urea or ammonium sulfate and 20 g L^?1 of glycerol, and were devoid of yeast extract. Moreover, Y. lipolytica was engineered by inserting the FatB2 gene, coding for the CpFatB2 thioesterase from Cuphea palustris, in order to modify the fatty acid composition towards the accumulation of medium‐chain fatty acids. Contrary to the expected, the expression of the heterologous gene increased the production of oleic acid, and concomitantly decreased the level of saturated fatty acids. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:26–35, 2016