Single Cell Oil Production Research Articles

Potential of Mortierellaceae for polyunsaturated fatty acids production: mini review.

The health benefits of polyunsaturated fatty acids (PUFAs) have encouraged the search for rich sources of these compounds. However, the supply chain of PUFAs from animals and plants presents environmental concerns, such as water pollution, deforestation, animal exploitation and interference in the trophic chain. In this way, a viable alternative has been found in microbial sources, mainly in single cell oil (SCO) production by yeast and filamentous fungi. Mortierellaceae is a filamentous fungal family world-renowned for PUFA-producing strains. For example, Mortierella alpina can be highlighted due to be industrially applied to produce arachidonic acid (20:4 n6), an important component of infant supplement formulas. Thus, the state of the art of strategies to increase PUFAs production by Mortierellaceae strains is presented in this review. Firstly, we have discussed main phylogenetic and biochemical characteristics of these strains for lipid production. Next, strategies based on physiological manipulation, using different carbon and nitrogen sources, temperature, pH and cultivation methods, which can increase PUFA production by optimizing process parameters are presented. Furthermore, it is possible to use metabolic engineering tools, controlling the supply of NADPH and co-factors, and directing the activity of desaturases and elongase to the target PUFA. Thus, this review aims to discuss the functionality and applicability of each of these strategies, in order to support future research for PUFA production by Mortierellaceae species.

Recent Advances in using Lipomyces starkeyi for the Production of Single-Cell Oil

The clean energy demand and limited fossil fuel reserves require an alternate source that is sustainable and eco-friendly. This demand for clean energy steered the introduction of biofuels such as bioethanol and biodiesel. The third-generation biodiesel is promising as it surpasses the difficulties associated with food security and land usage. The third-generation biodiesel comprises biodiesel derived from oil produced by oleaginous microbes. The term oleaginous refers to microbes with the ability to accumulate lipids to about 20% of the biomass and is found in the form of triacylglycerols. Yeasts can be grown easily on a commercial scale and are amenable to modifications to increase single-cell oil (SCO) productivity. The oleaginous yeast L. starkeyi is a potential lipid producer that can accumulate up to 70% of SCO of its cell dry weight under optimum conditions. Compared to other oleaginous organisms, it can be grown on a wide range of feedstock and a good part of the lipid produced can be converted to biodiesel. This review presents the recent advances in single-cell oil production from L starkeyi and strategies to increase lipid production are analyzed.

Pilot Scale Production of Single Cell Oil by Apiotrichum brassicae and Pichia kudriavzevii from Acetic Acid and Propionic Acid

Volatile fatty acids can be used as a cheap carbon source for biotechnological lipid production with oleaginous yeasts, but one factor limiting their large-scale use is their inherent cytotoxicity. Developing a suitable cultivation strategy can help mitigate the adverse effect volatile fatty acids have, since these effects are strongly dependent on concentration and pH. This work shows that, by employing a pH-stat fed-batch approach for the cultivation of Apiotrichum brassicae V134 and Pichia kudriavzevii V194, lipid contents above 56 g/100 g dry cell weight and dry cell weight concentrations above 30 g/L can be reached. Furthermore, volumetric lipid productivities up to 0.29 g/Lh could be achieved using acetic and propionic acid as a sole carbon source. It was also demonstrated that the developed process is robust and scalable. Scale-up to the 500 L scale resulted in a similar lipid yield, dry cell weight (31–37 g/L), and single cell oil content (56 g/100 g dry cell weight–58 g/100 g dry cell weight). The main fatty acid present in the produced lipids was oleic acid (36–43%), but also odd-numbered fatty acids, especially heptadecanoic acid (7–15%), were present. Additionally, different methods for the pretreatment of biomass prior to lipid extraction were assessed, and the iodine value (48), peroxide value (7.3), and acid value (4.3) of the extracted single cell oil were determined.

Novel evolved Yarrowia lipolytica strains for enhanced growth and lipid content under high concentrations of crude glycerol

BackgroundYarrowia lipolytica is a well-studied oleaginous yeast known for its ability to accumulate and store intracellular lipids, while growing on diverse, non-conventional substrates. Amongst them, crude glycerol, a low-cost by-product of the biodiesel industry, appears to be an interesting option for scaling up a sustainable single-cell oil production process. Adaptive laboratory evolution (ALE) is a powerful tool to force metabolic adaptations endowing tolerance to stressful environmental conditions, generating superior phenotypes with industrial relevance.ResultsY. lipolytica MUCL 28849 underwent ALE in a synthetic medium with increasing concentration of pure or crude glycerol as a stressing factor (9–20% v/v) for 520 generations. In one case of pure glycerol, chemical mutagenesis with ethyl methanesulfonate (EMS) was applied prior to ALE. Growth profile, biomass production and lipid content of 660 evolved strains (EVS), revealed 5 superior isolates; exhibiting from 1.9 to 3.6-fold increase of dry biomass and from 1.1 to 1.6-fold increase of lipid concentration compared to the parental strain, when grown in 15% v/v crude glycerol. NGS for differential gene expression analysis, showed induced expression in all EVS affecting nucleosomal structure and regulation of transcription. As strains differentiated, further changes accumulated in membrane transport and protein transport processes. Genes involved in glycerol catabolism and triacylglycerol biosynthesis were overexpressed in two EVS. Mismatches and gaps in the expressed sequences identified altered splicing and mutations in the EVS, with most of them, affecting different components of septin ring formation in the budding process. The selected YLE155 EVS, used for scale-up cultivation in a 3L benchtop bioreactor with 20% v/v crude glycerol, achieved extended exponential phase, twofold increase of dry biomass and lipid yields at 48 h, while citric acid secretion and glycerol consumption rates were 40% and 50% lower, respectively, compared to the parental strain, after 24 h of cultivation.ConclusionALE and EMS-ALE under increasing concentrations of pure or crude glycerol generated novel Y. lipolytica strains with enhanced biomass and lipid content. Differential gene expression analysis and scale-up of YLE155, illustrated the potential of the evolved strains to serve as suitable “chassis” for rational engineering approaches towards both increased lipid accumulation, and production of high-added value compounds, through efficient utilization of crude glycerol.

Bioconversion of non-food corn biomass to polyol esters of fatty acid and single-cell oils

BackgroundLignocellulose is a valuable carbon source for the production of biofuels and biochemicals, thus having the potential to substitute fossil resources. Consolidated bio-saccharification (CBS) is a whole-cell-based catalytic technology previously developed to produce fermentable sugars from lignocellulosic agricultural wastes. The deep-sea yeast strain Rhodotorulapaludigena P4R5 can produce extracellular polyol esters of fatty acids (PEFA) and intracellular single-cell oils (SCO) simultaneously. Therefore, the integration of CBS and P4R5 fermentation processes would achieve high-value-added conversion of lignocellulosic biomass.ResultsThe strain P4R5 could co-utilize glucose and xylose, the main monosaccharides from lignocellulose, and also use fructose and arabinose for PEFA and SCO production at high levels. By regulating the sugar metabolism pathways for different monosaccharides, the strain could produce PEFA with a single type of polyol head. The potential use of PEFA as functional micelles was also determined. Most importantly, when sugar-rich CBS hydrolysates derived from corn stover or corncob residues were used to replace grain-derived pure sugars for P4R5 fermentation, similar PEFA and SCO productions were obtained, indicating the robust conversion of non-food corn plant wastes to high-value-added glycolipids and lipids. Since the produced PEFA could be easily collected from the culture via short-time standing, we further developed a semi-continuous process for PEFA production from corncob residue-derived CBS hydrolysate, and the PEFA titer and productivity were enhanced up to 41.1 g/L and 8.22 g/L/day, respectively.ConclusionsHere, we integrated the CBS process and the P4R5 fermentation for the robust production of high-value-added PEFA and SCO from non-food corn plant wastes. Therefore, this study suggests a feasible way for lignocellulosic agro-waste utilization and the potential application of P4R5 in industrial PEFA production.

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.

Sustainable production of single-cell oil and protein from wastepaper hydrolysate: identification and optimization of a Rhodotorula mucilaginosa strain as a promising yeast.

This study investigated the potential of wastepaper hydrolysate as a sustainable and low-cost carbon source for single-cell oil and protein production, attending to the growing need for alternative feedstocks and waste management strategies. Wastepaper, characterized by its high carbohydrate content, was subjected to enzymatic and chemo-enzymatic treatments for carbohydrate release. The chemo-enzymatic treatment performed better, yielding 65.3g l-1 of fermentable sugars. A total of 62 yeast strains were screened for single-cell oil accumulation, identifying Rhodotorula mucilaginosa M1K4 as the most advantageous oleaginous yeast. M1K4 lipid production was optimized in liquid culture, and its fatty acid profile was analyzed, showing a high content of industrially valuable fatty acids, particularly palmitic (28%) and oleic (51%). Batch-culture of M1K4 in a 3-l reactor demonstrated the strain's ability to utilize wastepaper hydrolysate as a carbon source, with dry cell weight, total lipid and protein production of 17.7g l-1, 4.5g l-1, and 2.1g l-1, respectively. Wastepaper as a substrate provides a sustainable solution for waste management and bioproduction. This research highlights the potential of R. mucilaginosa for lipid and protein production from wastepaper hydrolysate.

Production of Single Cell Oils from Bacterial Sources

Single Cell Oils (SCO) is of profound interest for a variety of purposes ranging from biofuels to nutritional adjuvants, pharmaceutical applications and biotransformation for valuable products. A number of microorganisms have been shown to produce and accumulate SCO. In the present study a methodical attempt was made to isolate potential SCO producers from Indian water sources. Saltwater samples from the Arabian Sea and freshwater samples from an Indian cold-water river (Pindhari River, Uttarakhand) were collected and studied for the occurrence of lipid producing microbes. Of the several isolates shortlisted as lipid producers three isolates from this study were identified as potential SCO producers based on their lipid producing abilities. The types of fatty acids comprising the SCO from selected isolates were studied by Gas Chromatography (GC) and confirmed by Gas Chromatography/Mass Spectrometry (GC/MS). Lipid profiles from the GC analysis showed that the isolates in this study produced economically and nutritionally valuable Monounsaturated fatty acids (MUFA) like Palmitoleic acid and Oleic acid. Also, two isolates from the Arabian Sea were seen to produce a valuable omega-3 Polyunsaturated Fatty Acid (PUFA) like Eicosapentanoic acid. While a freshwater isolate produced Linoleic acid an omega-6 PUFA. Selected isolates were characterized for their biochemical characteristics and identified molecularly by 16S rRNA sequencing. Ornithinibacillus sp. Marseille-P3601 strain isolated in our study from the cold-water River Pindhari, Uttarakhand is found capable of producing PUFA.

Water footprint and wastewater quality assessment of yeast single cell oil production: Gate to gate approach for industrial water sustainability

Effective water resource utilization and sustainability for industrial operations is a growing concern. With increased industrial water demand, abstraction and water quality changes are rising. In India, distilleries generate more than 40.4 billion litres of effluent daily within the fermentation industry. Water, a public good with market and opportunity costs, needs effective mapping and management. Emerging distillery processes such as yeast lipid fermentation, if developed along with water sustainability, could aid in advancing water resource management. In the scope of this idea, the present study focuses on assessing the water footprint and water quality mapping for Rhodotorula mucilaginosa IIPL32 lipid production using crude glycerol, a by-product of the biodiesel industry. The assessment was based on primary data generated during the 500 L plant scale operation. The process's blue water footprint was assessed by applying a chain-summation approach, and the grey water requirement was determined by measuring water quality parameters for the effluent streams. The process's net blue and grey water footprint were estimated to be 3.87 and 23.66 m3 water/kg of lipid, respectively. Water quality index ratings were identified for all the respective water streams within the processing system, and human risk factors were estimated. The results suggested proper treatment of the spent broth, whereas the secondary effluent stream from cleaning operations could be reutilized within the system. Quality mapping also suggested that the effluent's high organic and mineral load can be processed for water and material recovery, which may significantly reduce the process's grey water and pollution load.

Growth Potential of Selected Yeast Strains Cultivated on Xylose-Based Media Mimicking Lignocellulosic Wastewater Streams: High Production of Microbial Lipids by Rhodosporidium toruloides

The potential of Rhodosporidium toruloides, Candida oleophila, Metschnikowia pulcherima, and Cryptococcus curvatus species to produce single-cell-oil (SCO) and other valuable metabolites on low-cost media, based on commercial-type xylose, was investigated. Rhodosporidium strains were further evaluated in shake-flasks using different lignosulphonate (LS) concentrations, in media mimicking waste streams derived from the paper and pulp industry. Increasing the LS concentration up to 40 g/L resulted in enhanced dry cell weight (DCW) while SCO production increased up to ~5.0 g/L when R. toruloides NRRL Y-27012 and DSM 4444 were employed. The intra-cellular polysaccharide production ranged from 0.9 to 2.3 g/L in all fermentations. Subsequent fed-batch bioreactor experiments with R. toruloides NRRL Y-27012 using 20 g/L of LS and xylose, led to SCO production of 17.0 g/L with maximum lipids in DCW (YL/X) = 57.0% w/w. The fatty acid (FA) profile in cellular lipids showed that oleic (50.3–63.4% w/w) and palmitic acid (23.9–31.0%) were the major FAs. Only SCO from batch trials of R. toruloides strains contained α-linolenic acid. Media that was supplemented with various LS concentrations enhanced the unsaturation profile of SCO from R. toruloides NRRL Y-27012. SCO from R. toruloides strains could replace plant-based commodity oils in oleochemical-operations and/or it could be micro- and nano-encapsulated into novel food-based formulas offering healthier food-products.

Optimized conversion of wheat straw into single cell oils by Yarrowia lipolytica and Lipomyces tetrasporus and synthesis of advanced biofuels

This paper deals with the optimized conversion of undetoxified wheat straw hydrolysates into microbial lipids by two oleaginous yeasts, Yarrowia lipolytica and Lipomyces tetrasporus. Wheat straw were pretreated by steam explosion at 203 °C for 300 s and hydrolysed at 20% solid-to-liquid ratio by using an enzymatic loading of 15 FPU/g substrate. The mixed wheat straw hydrolysates (WHS) contained 86 gL-1 glucose and 22 gL-1 xylose, 2.3 gL-1 acetic acid, 0.9 gL-1 furanic compounds. The fermentation process was optimized in terms of the inoculum age and density, medium composition, and bioreactor feeding strategy. In particular, the different capacity of the two yeasts to overcome the toxic effect of the biomass degradation by-products, in different inoculum ages, was deeply investigated. Two hydrolysates concentration were tested: WSH containing 86 gL-1 glucose and 22 gL-1 xylose and the diluted medium containing 40 gL-1 glucose and 22 gL-1 xylose. The results indicated that both yeasts were able to detoxify WSH and grow on undetoxified hydrolysates as effect of the intrinsic capacity to metabolize the furan derivatives. Y. lipolytica was able to detoxify the medium in all the investigated set-ups, while L. tetrasporus was able to detoxify the medium only if inoculated in the stationary phase of growth. After the process optimization in shaken flasks, the production of Single Cell Oils (SCOs) by L. tetrasporus was carried out in a medium-scale bioreactor of 10L obtaining lipid yield and cell content of 21% and 62%, respectively. The extracted SCOs, with high oleic and palmitic acid content, were converted into biodiesel displaying overall features in accordance with international biodiesel standards, namely ASTM and EN 14214.

Screening and Molecular Characterization of Oleaginous Yeasts and Their Potentiality for Single Cell Oil Production

The study aimed to isolate oleaginous yeasts from different sources (rotten fruits, fruit juices, milk, fish and air) and to identify and to study the possibility of production of single cell oil (SCO) from wheat straw and sugarcane molasses media. Thirty samples of yeasts were isolated using different methods according to the source of isolates. The isolated yeasts were characterized using microscopic appearance, colony morphology, physiological tests, assimilation tests and molecular identification. The effectiveness of oleaginous yeast to produce the single cell oil was studied, by growing the yeast on two types of wheat straw media (detoxified liquid hydrolysate (DLH) and non-detoxified liquid hydrolysate (NDLH)), and molasse media. Single cell oil was extracted by hexane. Eighteen isolates were found to be yeast, and the profile of ten yeast samples was identified as Saccharomyces cerevisiae, while, one sample was identified as Pichia guilliermondii. DLH wheat straw gave higher productivity of oil than NDLH wheat straw. Generally, S. cerevisiae gave higher oil productivity (84 %), compared to P. guilliermondii (52 %).

Biotechnological Conversions of Mizithra Second Cheese Whey by Wild-Type Non-Conventional Yeast Strains: Production of Yeast Cell Biomass, Single-Cell Oil and Polysaccharides

The cultivation of oleaginous yeasts on various agro-industrial residues and the subsequent production of microbial lipids (single-cell oils), which can be used as starting materials for the synthesis of both “second generation” biodiesel and various types of oleochemicals, is considered as one of the most important approaches of the Industrial Biotechnology, in terms of circular economy and green and sustainable development. In this study, seven wild-type non-conventional yeast strains were evaluated for their growth on a semi-defined medium with cheese whey lactose as a sole carbon source. Five of these strains were further batch-cultivated into the complex substrate that contained second cheese whey, which is the main by-product of Mizithra whey cheese manufacture, after centrifugation and filtration without any extra mineral salts, carbon, or nitrogen source addition. All these five strains grown in second cheese whey produced mainly yeast biomass and to lesser extent microbial lipids and other interesting metabolites, such as polysaccharides. The strain Cryptococcus curvatus ATCC 20509 produced the highest total dry weight (TDW) amount (22.4 g/L), that contained 3.4 g/L of intra-cellular lipids, followed by C. curvatus NRRL Y-1511, which produced 20.6 g/L of TDW and 3.2 g/L lipids. A novel, non-previously systematically studied strain, namely Papiliotrema laurentii NRRL Y-2536, produced significant quantities of TDW (22.0 g/L) and, interestingly, secreted quantities of exopolysaccharides. Fed-batch shake-flask cultivation of C. curvatus ATCC 20509 in pretreated second cheese whey, pulse-supplemented with condensed cheese whey-derived lactose, led to the significant TDW quantity of 38.1 g/L that contained c. 57% w/w of total lipids (lipids at a concentration 21.7 g/L were produced). Cellular lipids of all microorganisms, mainly stored as triacylglycerols, contained in variable quantities the fatty acids Δ9C18:1, C16:0, Δ9,12C18:2 and C18:0, constituting perfect candidates for the synthesis of “second generation” biodiesel.

Lipid production from lignocellulosic biomass using an engineered Yarrowia lipolytica strain

BackgroundThe utilization of industrial wastes as feedstock in microbial-based processes is a one of the high-potential approach for the development of sustainable, environmentally beneficial and valuable bioproduction, inter alia, lipids. Rye straw hydrolysate, a possible renewable carbon source for bioconversion, contains a large amount of xylose, inaccessible to the wild-type Yarrowia lipolytica strains. Although these oleaginous yeasts possesses all crucial genes for xylose utilization, it is necessary to induce their metabolic pathway for efficient growth on xylose and mixed sugars from agricultural wastes. Either way, biotechnological production of single cell oils (SCO) from lignocellulosic hydrolysate requires yeast genome modification or adaptation to a suboptimal environment.ResultsThe presented Y. lipolytica strain was developed using minimal genome modification—overexpression of endogenous xylitol dehydrogenase (XDH) and xylulose kinase (XK) genes was sufficient to allow yeast to grow on xylose as a sole carbon source. Diacylglycerol acyltransferase (DGA1) expression remained stable and provided lipid overproduction. Obtained an engineered Y.lipolytica strain produced 5.51 g/L biomass and 2.19 g/L lipids from nitrogen-supplemented rye straw hydrolysate, which represents an increase of 64% and an almost 10 times higher level, respectively, compared to the wild type (WT) strain. Glucose and xylose were depleted after 120 h of fermentation. No increase in byproducts such as xylitol was observed.ConclusionsXylose-rich rye straw hydrolysate was exploited efficiently for the benefit of production of lipids. This study indicates that it is possible to fine-tune a newly strain with as minimally genetic changes as possible by adjusting to an unfavorable environment, thus limiting multi-level genome modification. It is documented here the use of Y. lipolytica as a microbial cell factory for lipid synthesis from rye straw hydrolysate as a low-cost feedstock.

Oleaginous yeast Rhodotorula toruloides biomass effect on the metabolism of Arctic char (Salvelinus alpinus).

Sustainability issues arise when using fish oil and vegetable oils in fish feed production for aquaculture purposes. Microbial production of single cell oil is a potential alternative as a lipid ingredient in the production of fish feed. In this study, we replaced the vegetable oils with the oleaginous yeast R. toruloides biomass in the diet of Arctic char (S. alpinus) and investigated the effects on health and composition. Measurement of fish growth parameters showed a higher liver weight and hepatosomatic index in the experimental group of fish fed partly with yeast biomass compared to a control group fed a diet with vegetable oils. No significant differences in the lipid content of muscle and liver tissues were found. The fatty acid profiles in the muscle of both fish groups were similar while the experimental fish group had a higher amount of monounsaturated fatty acids in the liver. Histology of livers showed no significant difference in the number of lipid droplets. The size of hepatic lipid droplets seemed to be related to liver fat content. Quantification of metabolites in the liver revealed no differences between the fish groups while plasma metabolites involved in energy pathways such as alanine, 3-hydroxybutyrate, creatinine, serine, betaine, and choline were significantly higher in the experimental fish group.

Use of Pseudomonas fluorescens in the production of single-cell oil and estimate the oil content of fatty acids

The study aimed at producing single-cell oil using Pseudomonas fluorescens bacteria and conducting solubility and solubility testing to confirm oil production and estimate the content of oil produced from fatty acids, corn coalts were used as a carbon source and applied optimal conditions forthe production obtained from the previous study, as used amid liquid corn coalts in the production experiment and oil was extracted with the saxolite device (Soxhletlet) ) We obtained the highest oil productivity by 55.47%, the oil produced was fully soluble in chloroform and dissolved to a lesser degree in ethanol and the inability of the oil produced to melt in water, and the oil produced contained the highest percentage of linoleic acid Unsaturated at 69,214%, while stearyk acid was 23.62%, palmetic acid 2.92%, propionic acid 1.6% and butaric acid 0.146%.

Impact of brewery wastewater inhibitors in pure and mixed cultures of the yeast Rhodosporidium toruloides NCYC 921 and the microalga Tetradesmus obliquus ACOI 204/07

Brewery wastewater (BWW) is an appealing low-cost substrate for the production of single cell oils by oleaginous microorganisms. However, it may contain inhibitor compounds that may affect the microbial metabolism. This work investigated, for the first time, the presence of potential inhibitor compounds in primary brewery wastewater (PBWW) and secondary brewery wastewater (SBWW) for the pure and mixed cultivation of the yeast Rhodosporidium toruloides NCYC 921 and the microalga Tetradesmus obliquus ACOI 204/07. Three organic acids (OrgAc) were identified in the brewery effluents (acetic, propionic and butyric acids). Yeast and microalga pure and mixed cultivations were performed in PBWW and SBWW in order to understand the behaviour of the microorganisms, individually and together. Flow cytometry (FC) was used to monitor each microbial population during the mixed cultivations, and to study the yeast and microalga cell viability throughout all cultivations. The yeast cells in pure cultures grown in both effluents were severely affected by the OrgAc presence confirmed by the cell stress results obtained by FC. However, in the mixed cultures, the yeast cells were able to develop, and the levels of stress conditions were considerably lower. Only in microalga pure and mixed cultures efficient OrgAc removal was observed.

Microbes: A Hidden Treasure of Polyunsaturated Fatty Acids.

Microbes have gained a lot of attention for their potential in producing polyunsaturated fatty acids (PUFAs). PUFAs are gaining scientific interest due to their important health-promoting effects on higher organisms including humans. The current sources of PUFAs (animal and plant) have associated limitations that have led to increased interest in microbial PUFAs as most reliable alternative source. The focus is on increasing the product value of existing oleaginous microbes or discovering new microbes by implementing new biotechnological strategies in order to compete with other sources. The multidisciplinary approaches, including metabolic engineering, high-throughput screening, tapping new microbial sources, genome-mining as well as co-culturing and elicitation for the production of PUFAs, have been considered and discussed in this review. The usage of agro-industrial wastes as alternative low-cost substrates in fermentation for high-value single-cell oil production has also been discussed. Multidisciplinary approaches combined with new technologies may help to uncover new microbial PUFA sources that may have nutraceutical and biotechnological importance.

Production of single cell oil by Yarrowia lipolytica JCM 2320 using detoxified desiccated coconut residue hydrolysate.

Nowadays, the replacement of petro-diesel with biodiesel has raised the concern among the community for the utilization of improper feedstocks and the cost involved. However, these issues can be solved by producing single cell oil (SCO) from lignocellulosic biomass hydrolysates by oleaginous microorganisms. This study introduced Yarrowia lipolytica JCM 2320 with a desiccated coconut residue (DCR) hydrolysate (obtained from the 2% dilute sulphuric acid pretreatment) as a carbon source in generating SCO. However, common inhibitors formed during acid pretreatment of biomass such as five-hydroxymethylfurfural (HMF), furfural, acetic acid and levulinic acid resulting from the sugar degradations may have detrimental effects towards the fermentation process. To visualize the effect of inhibitors on Y. lipolytica, an inhibitory study was conducted by adding 0.5–5.0 g/L of potential inhibitors to the YPD (yeast, peptone and D-glucose) medium. It was found that the presence of furfural at 0.5 g/L would increase the lag phase, which beyond that was detrimental to Y. lipolytica. Furthermore, increasing the five-hydroxymethylfurfural (HMF) concentration would increase the lag phase of Y. lipolytica, whereas, for acetic acid and levulinic acid, it showed a negligible effect. Detoxification was hence conducted to remove the potential inhibitors from the DCR hydrolysate prior its utilization in the fermentation. To examine the possibility of using adsorption resins for the detoxification of DCR hydrolysate, five different resins were tested (Amberlite® XAD-4, Amberlite® XAD-7, Amberlite® IR 120, Amberlite® IRA 96 and Amberlite® IRA 402) with five different concentrations of 1%, 3%, 5%, 10% and 15% (w/v), respectively. At resin concentration of 10%, Amberlite® XAD-4 recorded the highest SCO yield, 2.90 ± 0.02 g/L, whereas the control and the conventional overliming detoxification method, recorded only 1.29 ± 0.01 g/L and 1.27 ± 0.02 g/L SCO accumulation, respectively. Moreover, the fatty acid profile of the oil produced was rich in oleic acid (33.60%), linoleic acid (9.90%), and palmitic acid (14.90%), which indicates the potential as a good biodiesel raw material.

The influence of different carbon sources on growth and single cell oil production in oleaginous yeasts Apiotrichum brassicae and Pichia kudriavzevii

Oleaginous yeasts offer an interesting possibility for renewable lipid production, since the single cell oil accumulated can be based on a wide range of cheap, waste-derived carbon sources. Here, several short chain carboxylic acids and sugars commonly found in these substrates were assessed as carbon sources for Apiotrichum brassicae and Pichia kudriavzevii. While both strains were able to utilize all carbon sources employed, high volumetric lipid productivities (0.4 g/Lh) and lipid contents (68%) could be reached particularly with acetic acid as carbon source. Odd-numbered volatile fatty acids led to lower productivities and lipid contents, but the lipids contained unusually high proportions of odd-numbered fatty acids (up to 80% of total fatty acids). These fatty acids are rather uncommon in nature and might offer the possibility for various high value applications. In conclusion both strains are able to utilize a wide range of substrates potentially present in waste-derived substrates. Lipid content and volumetric lipid productivity strongly depend on the carbon source, with even-numbered volatile fatty acids resulting in the highest values. For volatile fatty acids in particular, the carbon source also strongly influences the composition of the lipids produced by the yeast strains.