Sustainable Production Of Lipids Research Articles

Sustainable production of lipids from cocoa fatty acid distillate fermentation driven by adaptive evolution in Yarrowia lipolytica

Single cell oil production using oleaginous yeasts is a promising alternative to animal and plant-derived lipids. But substrate costs for microbial fermentation are a major bottleneck. Using side streams as alternative to substrates like glucose, for growing yeast, is a potential cost-effective solution. By combining a previously reported process of growing yeasts on a solid cocoa fatty acid distillate side stream with adaptive evolution techniques, the growth of oleaginous yeast Yarrowia lipolytica was improved by 2-fold. The lipid titre was also boosted by more than 3-fold. Using transcriptomics, key genes were identified that are possibly involved in tailoring of lipid composition, side stream utilisation and enhancement of lipid titres. Candidate genes were also identified that might enable efficient growth and utilization of fatty acids and triacylglycerides found in cocoa fatty acid distillate. In summary, this research has improved the understanding of side stream utilisation for lipid production in oleaginous yeast.

Elucidation of sterol biosynthesis pathway and its co-regulation with fatty acid biosynthesis in the oleaginous marine protist Schizochytrium sp.

Sterols constitute vital structural and regulatory components of eukaryotic cells. In the oleaginous microorganism Schizochytrium sp. S31, the sterol biosynthetic pathway primarily produces cholesterol, stigmasterol, lanosterol, and cycloartenol. However, the sterol biosynthesis pathway and its functional roles in Schizochytrium remain unidentified. Through Schizochytrium genomic data mining and a chemical biology approach, we first in silico elucidated the mevalonate and sterol biosynthesis pathways of Schizochytrium. The results showed that owing to the lack of plastids in Schizochytrium, it is likely to use the mevalonate pathway as the terpenoid backbone pathway to supply isopentenyl diphosphate for the synthesis of sterols, similar to that in fungi and animals. In addition, our analysis revealed a chimeric organization of the Schizochytrium sterol biosynthesis pathway, which possesses features of both algae and animal pathways. Temporal tracking of sterol profiles reveals that sterols play important roles in Schizochytrium growth, carotenoid synthesis, and fatty acid synthesis. Furthermore, the dynamics of fatty acid and transcription levels of genes involved in fatty acid upon chemical inhibitor-induced sterol inhibition reveal possible co-regulation of sterol synthesis and fatty acid synthesis, as the inhibition of sterol synthesis could promote the accumulation of fatty acid in Schizochytrium. Sterol and carotenoid metabolisms are also found possibly co-regulated, as the inhibition of sterols led to decreased carotenoid synthesis through down-regulating the gene HMGR and crtIBY in Schizochytrium. Together, elucidation of the Schizochytrium sterol biosynthesis pathway and its co-regulation with fatty acid synthesis lay the essential foundation for engineering Schizochytrium for the sustainable production of lipids and high-value chemicals.

Production of single cell oil by two novel nonconventional yeast strains of Curvibasidium sp. isolated from medicinal lichen.

Oleaginous yeasts utilize renewable resources to produce lipids, which benefits sustainable development, and it is of great interest to screen robust lipid producers. Curvibasidium sp. belongs to nonconventional yeast that are very limitedly studied. Here, two cold-adaptive strains of Curvibasidium sp., namely, Y230 and Y231, isolated from the medicinal lichen Usnea diffracta were investigated for their potential in lipid production. Genome mining of Curvibasidium sp. Y231 was performed, and the special features related to fatty acid biosynthesis were revealed. Glucose, xylose, and glycerol were tested as sole carbon sources for yeast cell growth and lipid production. The total lipid contents of Curvibasidium sp. Y230 and Y231 range from 38.43% to 54.62% of the cell dry cell weight at 20°C, and glucose is the optimal carbon source. These results indicate that the Curvibasidium sp. strains are promising for sustainable lipid production. Our study provides basis for exploration of lichen-derived strains for biotechnological applications, and also benefits utilization of other nonconventional yeasts for sustainable production based on genome-based studies.

Key role of fluorescence quantum yield in Nile Red staining method for determining intracellular lipids in yeast strains

BackgroundMicrobial lipids are found to be an interesting green alternative to expand available oil sources for the chemical industry. Yeasts are considered a promising platform for sustainable lipid production. Remarkably, some oleaginous yeasts have even shown the ability to grow and accumulate lipids using unusual carbon sources derived from organic wastes, such as volatile fatty acids. Recent research efforts have been focused on developing rapid and accurate fluorometric methods for the quantification of intracellular yeast lipids. Nevertheless, the current methods are often tedious and/or exhibit low reproducibility.ResultsThis work evaluated the reliability of different fluorescence measurements (fluorescence intensity, total area and fluorescence quantum yield) using Nile Red as lipid dye in two yeast strains (Yarrowia lipolytica ACA-DC 50109 and Cutaneotrichosporon curvatum NRRL-Y-1511). Different standard curves were obtained for each yeast specie. Fermentation tests were carried with 6-month difference to evaluate the effect of the fluorometer lamp lifetime on lipid quantification.ConclusionsFluorescence quantum yield presented the most consistent measurements along time and the closer estimations when compared with lipids obtained by conventional methods (extraction and gravimetrical determination). The need of using fluorescence quantum yield to estimate intracellular lipids, which is not the common trend in studies focused on microbial lipid production, was stressed. The information here provided will surely enable more accurate results comparison.

Isolation, identification, and statistical optimization of a psychrotolerant Mucor racemosus for sustainable lipid production

Lipid accumulating fungi are promising tools as alternative lipid source with different applications. In this study, seven oleaginous fungal strains were isolated from dung samples from the Egyptian ecosystem which later investigated for lipid accumulation, and Mucor racemosus AH1 represented the highest one. Statistical optimization of M. racemosus AH1 for lipid production was carried out using Taguchi design. Accordingly, dry biomass, total lipids, and lipid content were 3.72 gL−1, 1.21 gL−1, and 32.4%, respectively. Fatty acid profile of the produced lipids at different temperatures from 5 to 35 °C was investigated using gas chromatography mass spectroscopy GC–MS. Results revealed that the best temperature range for unsaturated fatty acids production particularly polyunsaturated fatty acids (PUFAs) was between 10 and 20 °C in which unsaturated fatty acids (USFAs) were higher than saturated fatty acids (SFAs); 54.47% and 43.67%, respectively. In conclusion, a promising lipid accumulating and cold-adapted M. racemosus MG547571 are considered as hopeful source of USFAs particularly oleic and linoleic acids which can be recruited for pharmaceutical applications, additionally, the high lipid yield could be exploited for biodiesel production.

Assessing the potential of Schizochytrium sp. HX-308 for microbial lipids production from corn stover hydrolysate.

Schizochytrium sp. has received increasing attention as promising commercial resource for the sustainable production of lipids, due to their fast growth rate and high lipid content. However, the price of glucose represents a significant proportion of the total substrate cost. Therefore, in this study, the lignocellulosic hydrolysate of corn stover hydrolysate (CSH) was used as low-cost culture medium to replace glucose in Schizochytrium sp. fermentation. When Schizochytrium sp. HX-308 was fermented with 20% glucose from CSH and 80% of glucose from pure glucose, the lipid production reached 21.2gL-1 , which is lower than that of using 100% of pure glucose. However, the shifts of fatty acid composition indicated that CSH has great potential to enhance the percentage of polyunsaturated fatty acids (PUFAs) in total lipids. However, as the second largest carbon source in CSH, xylose was not utilized by the Schizochytrium sp. HX-308, and further analysis showed that probably because it does not possess a functional xylulose kinase. In addition, the degradation products in lignocellulosic hydrolysate have a strong inhibitory effect on cell growth, so it is necessary to investigate the tolerance of Schizochytrium sp. HX-308 to degradation products. Here, the effects of five typical degradation products on the growth and lipid synthesis were further investigated. Schizochytrium sp. HX-308 showed good tolerance to furan derivatives and organic acids, but low tolerance to phenolic compounds. Furthermore, in order to improve the lipid accumulation using CSH, the two-stage fermentation strategy was developed, resulting in a 54.8% increase compared to that of the one-stage strategy. In summary, this study provides a reference for further fermentation engineering with cheap lignocellulosic biomass as substrate.

Highly Valuable Polyunsaturated Fatty Acids from Microalgae: Strategies to Improve Their Yields and Their Potential Exploitation in Aquaculture.

Microalgae have a great potential for the production of healthy food and feed supplements. Their ability to convert carbon into high-value compounds and to be cultured in large scale without interfering with crop cultivation makes these photosynthetic microorganisms promising for the sustainable production of lipids. In particular, microalgae represent an alternative source of polyunsaturated fatty acids (PUFAs), whose consumption is related to various health benefits for humans and animals. In recent years, several strategies to improve PUFAs’ production in microalgae have been investigated. Such strategies include selecting the best performing species and strains and the optimization of culturing conditions, with special emphasis on the different cultivation systems and the effect of different abiotic factors on PUFAs’ accumulation in microalgae. Moreover, developments and results obtained through the most modern genetic and metabolic engineering techniques are described, focusing on the strategies that lead to an increased lipid production or an altered PUFAs’ profile. Additionally, we provide an overview of biotechnological applications of PUFAs derived from microalgae as safe and sustainable organisms, such as aquafeed and food ingredients, and of the main techniques (and their related issues) for PUFAs’ extraction and purification from microalgal biomass.

The Effect of Soybean Oil on Lipid Metabolism in Mucor circinelloides WJ11 by Metabolomic Analysis

Oleaginous fungus Mucor circinelloides utilizes different carbon sources associated with sustainable production of lipids, using soybean oil as carbon source, the fungus accumulated more intracellular lipids. Nonetheless, the metabolic changes in M. circinelloides upon vegetable oil as main carbon source have not been yet reported. Therefore, this study was conducted to investigate the metabolomics of M. circinelloides WJ11 cultivated in mixed glucose and soybean oil as carbon source and to reveal its effects on lipid metabolism. M. circinelloides strain WJ11 was cultured under optimized conditions in a fermenter and the biomass samples were collected after 24 h for lipid metabolite investigation. The frozen biomass samples were subjected to various chromatographic analyses like liquid chromatography mass spectrometry analysis and high resolution-mass spectrometry for metabolite profiling. A total of 438 differential metabolites were identified, of which 48 were up-regulated and 33 were down-regulated. Among them, lyso-phosphatidic acid and monoglyceride were up-regulated whereas phosphatidylglycerol and lyso-phosphatidylglycerol were down-regulated in the experimental group (soybean oil and glucose as mixed carbon sources) compared with the control group (glucose as single carbon source). Significant changes in metabolite levels correlated to lipid synthesis were identified. This study showed that the addition of soybean oil to the medium favors the triacylglycerol synthesis in M. circinelloides. Our results can also be applied to the investigation of other microorganisms and would contribute to further genetic engineering for higher lipid accumulation in fungi.

Symbiotic Associations: Key Factors That Determine Physiology and Lipid Accumulation in Oleaginous Microorganisms.

Symbiosis naturally provides an opportunity for microorganisms to live together by mutual or one-way benefit. In symbiotic relationships, the microorganisms usually overcome the limitations of being free-living. Understanding the symbiotic relationships of oleaginous microorganisms provides potential route for the sustainable production of microbial-based alternative fuels. So far, several studies have been conducted in oleaginous microorganisms for the production of alternative fuels. However, some oleaginous microorganisms require high quantity of nutrients for their growth, and high level of energy and chemicals for harvest and separation of lipid bodies. Symbiotic associations can successfully be applied to address these issues. Of symbiotic associations, lichens and selective species of oleaginous endosymbiotic mucoromycotina have received substantial interest as better models to study the evolutionary relationships as well as single-cell oil production. Construction of artificial lichen system composed of cyanobacteria and oleaginous yeast has been achieved for sustainable production of lipids with minimum energy demand. Recently, endosymbiotic mucoromycotina species have been recognized as potential sources for biofuels. Studies found that endohyphal bacterium influences lipid profiling in endosymbiotic mucoromycotina species. Studies on the genetic factors related to oleaginous characteristics of endosymbiotic mucoromycotina species are scarce. In this regard, this review summarizes the different forms of symbiotic associations of oleaginous microorganisms and how symbiotic relationships are impacting the lipid formation in microorganisms. Further, the review also highlights the importance of evolutionary relationships and benefits of co-culturing (artificial symbiosis) approaches for sustainable production of biofuels.

Optimization of high-throughput lipid screening of the microalga Nannochloropsis oceanica using BODIPY 505/515

BackgroundMicroalgae are considered a promising platform for sustainable lipid production. Despite this, productivities need to be improved to facilitate an economically viable production process. This can be achieved through strain improvement, for instance by genetic engineering. Strain improvement strategies often deploy high-throughput screening platforms i.a. involving single-cell methodologies such as fluorescence-activated cell sorting (FACS) for the identification and isolation of better-performing strains. The heterokont microalga Nannochloropsis is a prospective candidate for the industrial production of lipids. Previous studies have reported the isolation of high lipid-producing Nannochloropsis strains by combining qualitative staining of lipid bodies using the fluorophoric dye BODIPY with FACS methodology. However, it has never been investigated how cellular physiology and different staining conditions hamper the reproducibility of this method as a quantitative screening procedure. ResultsHere we report the development of an optimized single cell lipid screening procedure for Nannochloropsis oceanica. Systematic assessment of different staining conditions revealed that treatment with 6% DMSO and 1.2 μg ml−1 BODIPY for 15 min is ideal for staining neutral lipids in an exponentially growing culture of N. oceanica. Cultures that are overproducing lipids, for example after exposure to external stimuli such as nutrient deprivation stress, require treatment with 10% DMSO and 1.2–1.6 μg ml−1 BODIPY for 36 min to facilitate complete staining of lipid bodies. We verify that DMSO is required to permeabilize the particularly tough cell barrier of Nannochloropsis and we show that exposure to 10% DMSO does not affect cell viability. Increasing concentrations of BODIPY, however, correlated with a decrease in viability when screening stressed cultures. Using the optimized protocol, reproductive viabilities can be expected to be ~91% and 83–82% for non-stressed and stressed cultures respectively. The optimized procedure allows for a quantitative prediction of cellular neutral lipids (R2= 0.981), as determined by comparison to results obtained through a reference procedure.

Conversion of sugar beet residues into lipids by Lipomyces starkeyi for biodiesel production

BackgroundLipids from oleaginous yeasts emerged as a sustainable alternative to vegetable oils and animal fat to produce biodiesel, the biodegradable and environmentally friendly counterpart of petro-diesel fuel. To develop economically viable microbial processes, the use of residual feedstocks as growth and production substrates is required.ResultsIn this work we investigated sugar beet pulp (SBP) and molasses, the main residues of sugar beet processing, as sustainable substrates for the growth and lipid accumulation by the oleaginous yeast Lipomyces starkeyi. We observed that in hydrolysed SBP the yeast cultures reached a limited biomass, cellular lipid content, lipid production and yield (2.5 g/L, 19.2%, 0.5 g/L and 0.08 g/g, respectively). To increase the initial sugar availability, cells were grown in SBP blended with molasses. Under batch cultivation, the cellular lipid content was more than doubled (47.2%) in the presence of 6% molasses. Under pulsed-feeding cultivation, final biomass, cellular lipid content, lipid production and lipid yield were further improved, reaching respectively 20.5 g/L, 49.2%, 9.7 g/L and 0.178 g/g. Finally, we observed that SBP can be used instead of ammonium sulphate to fulfil yeasts nitrogen requirement in molasses-based media for microbial oil production.ConclusionsThis study demonstrates for the first time that SBP and molasses can be blended to create a feedstock for the sustainable production of lipids by L. starkeyi. The data obtained pave the way to further improve lipid production by designing a fed-batch process in bioreactor.Graphical abstract

Isolation and functional analyses of PvFAD2 and PvFAD3 involved in the biosynthesis of polyunsaturated fatty acids from Sacha Inchi (Plukenetia volubilis)

The development of ω-3 fatty acid-rich vegetable oils is essential to enrich the production of functional foods. Sacha Inchi (Plukenetia volubilis L.) is a unique oilseed crop with much potential. Its seeds contain rich polyunsaturated fatty acids (PUFAs), especially linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3). Endoplasmic reticulum -located ω-6 and ω-3 fatty acid desaturases (FAD) are responsible for the biosynthesis of LA and ALA, respectively, in plant seeds. Here, we isolated two full-length FAD genes from Sacha Inchi, named PvFAD2 and PvFAD3, which encoded predicted amino acid residues of 384 and 379 in protein, respectively. Protein sequence and subcellular localization analysis revealed that they were located in the endoplasmic reticulum (ER). Heterologous expression in Saccharomyces cerevisiae confirmed that PvFAD2 and PvFAD3 could catalyze LA and ALA synthesis, respectively. The stability and catalytic efficiency of the PvFAD3 protein may be closely related to temperature. In transgenic tobacco, using seed-specific expression promoters, PvFAD2 and PvFAD3 significantly promotes the production of LA (from 68% to 70.5%) and ALA (from 0.7% to 3.1%) in seed oil. These results show that PvFAD2 and PvFAD3 do, indeed, function as crucial enzymes for PUFAs biosynthesis, and provide a key gene source for the sustainable production of lipids with tailored fatty acid compositions via genetic engineering in other oil crops.

Benchtop flow NMR spectroscopy as an online device for the in vivo monitoring of lipid accumulation in microalgae

The sustainable production of lipids by microalgae is widely developed among the bioprocess community targeting various applications such as feed, food, health or bioenergy. The cultivation of microalgae needs dedicated systems with the optimal illumination geometry. Performing non-invasive online analyses on these bioprocesses is limited to few analytical techniques, often based on optical properties and can however rarely be related to intracellular products. The real-time knowledge of the lipids accumulation in microalgae is –in this case– not possible. In this article, the proof-of-concept that the recent benchtop NMR spectroscopy device can be used for the non-invasive and selective detection of lipids inside microalgae cells is carried out. Three cultures of Nannochloropsis gaditana were analyzed in flow conditions. The relative quantitative feature is confirmed by the correlation with a reference technique classically used for lipid analysis, i.e. the FAME (Fatty Acid Methyl Ester) profiling by gas chromatography.

Biomimetic light dilution using side-emitting optical fiber for enhancing the productivity of microalgae reactors

Photoautotrophic microbes present vast opportunities for sustainable lipid production, CO2 storage and green chemistry, for example, using microalgae beds to generate biofuels. A major challenge of microalgae cultivation and other photochemical reactors is the efficiency of light delivery. In order to break even on large scale, dedicated photon management will be required across all levels of reactor hierarchy – from the harvesting of light and its efficient injection and distribution inside of the reactor to the design of optical antenna and pathways of energy transfer on molecular scale. Here, we discuss a biomimetic approach for light dilution which enables homogeneous illumination of large reactor volumes with high optical density. We show that the immersion of side-emitting optical fiber within the reactor can enhance the fraction of illuminated volume by more than two orders of magnitude already at cell densities as low as ~5 104 ml−1. Using the green algae Haematococcus pluvialis as a model system, we demonstrate an increase in the rate of reproduction by up to 93%. Beyond micoralgae, the versatile properties of side-emitting fiber enable the injection and dilution of light with tailored spectral and temporal characteristics into virtually any reactor containment.

Oleaginous yeasts for sustainable lipid production-from biodiesel to surf boards, a wide range of "green" applications.

A growing world population and a growing number of applications for vegetable oils are generating an increasing demand for these oils, causing serious environmental problems. A sustainable lipid production is then fundamental to address these problems. Oleaginous yeasts are a promising solution for sustainable lipid production, but, with the current knowledge and technology, they are still not a serious alternative in the market. In this review, the potential of these yeasts is highlighted and a discussion is made mainly focused on the economics of the oleaginous yeast oil production and identification of the key points to be improved to achieve lower production costs and higher income. Three main stages of the production process, where costs are higher, were identified. To render economically feasible the production of oils using oleaginous yeasts, a reduction in production costs must occur in all stages, lipid yields and productivities must be improved, and production must be targeted to high-value product applications.

Characterization of newly isolated oleaginous yeasts - Cryptococcus podzolicus, Trichosporon porosum and Pichia segobiensis

The yeast strains Cryptococcus podzolicus, Trichosporon porosum and Pichia segobiensis were isolated from soil samples and identified as oleaginous yeast strains beneficial for the establishment of microbial production processes for sustainable lipid production suitable for several industrial applications. When cultured in bioreactors with glucose as the sole carbon source C. podzolicus yielded 31.8% lipid per dry biomass at 20°C, while T. porosum yielded 34.1% at 25°C and P. segobiensis 24.6% at 25°C. These amounts correspond to lipid concentrations of 17.97 g/L, 17.02 g/L and 12.7 g/L and volumetric productivities of 0.09 g/Lh, 0.1 g/Lh and 0.07 g/Lh, respectively. During the culture of C. podzolicus 30 g/l gluconic acid was detected as by-product in the culture broth and 12 g/L gluconic acid in T. porosum culture. The production of gluconic acid was eliminated for both strains when glucose was substituted by xylose as the carbon source. Using xylose lipid yields were 11.1 g/L and 13.9 g/L, corresponding to 26.8% and 33.4% lipid per dry biomass and a volumetric productivity of 0.07 g/Lh and 0.09 g/Lh, for C. podzolicus and T. porosum respectively. The fatty acid profile analysis showed that oleic acid was the main component (39.6 to 59.4%) in all three strains and could be applicable for biodiesel production. Palmitic acid (18.4 to 21.1%) and linolenic acid (7.5 to 18.7%) are valuable for cosmetic applications. P. segobiensis had a considerable amount of palmitoleic acid (16% content) and may be suitable for medical applications.

Biocrude production by activated sludge microbial cultures using pulp and paper wastewaters as fermentation substrate

Municipal wastewater activated sludge contains a mixed microbial community, which can be manipulated to produce biocrude, a lipid feedstock for biodiesel production. In this study, the potential of biocrude production by activated sludge microorganisms grown in three different types of pulp and paper mill wastewaters was investigated. A 20% (v/v) activated sludge was inoculated into pulp and paper wastewater, supplemented with glucose (60 g/L) and nutrients (nitrogen and phosphorus) to obtain a high carbon to nitrogen ratio (70:1). The culture was incubated aerobically for seven days. The results showed that the activated sludge microorganisms were able to grow and accumulate lipids when cultivated in amended wastewaters. Microorganisms growing in anaerobic settling pond effluent water showed the highest lipid accumulation of up to 40.6% cell dry weight (CDW) after five days of cultivation compared with pulp wash wastewater (PuWW) (11.7% CDW) and mixed wastewater (MWW) (8.2% CDW) after seven days of cultivation. The lipids mostly contained C16‒C18 fatty acids groups with oleic acid and palmitic acid being the dominant fatty acids. The maximum biodiesel yield was about 6–8% CDW for all the wastewaters. The results showed the potential of utilizing pulp and paper mill effluents and other waste streams, such as activated sludge for the sustainable production of lipids for biofuel production.