Microbial Lipid Research Articles

Dark fermentation of expired fruit juices for biohydrogen production followed by treatment and biotechnological exploitation of effluents towards bioplastics and microbial lipids

The treatment and biotechnological exploitation of expired fruit juices (EFJ) towards biohydrogen, polyhydroxyalkanoates (PHAs) and lipids production was investigated. The EFJ were initially fermented via an acidogenic consortium, during which the effect of the organic loading rate (OLR, 20–40 g sugars/L.d) on the sugars uptake and the yields of metabolites i.e. hydrogen, volatile fatty acids (VFAs) and lactate, was studied. The increase of the OLR resulted to lower bioconversion of sugars and metabolic shift to lactate production, whereas the highest hydrogen yield, 10.83 ± 0.41 L H2/m3 EFJ, was obtained for the OLR 30 g sugars/L.d. The acidogenic effluents were then utilized for the production of PHAs and microbial lipids, via bacterial heterotrophic and cyanobacterial consortia, respectively. The PHAs produced were in all cases copolymers, and the highest yield, 20.69 ± 1.01 kg PHAs/m3 EFJ was achieved from the effluent with the highest VFAs content. Microbial lipids were also successfully produced from all effluents. The results revealed that low VFAs and high sugars concentration, allowed cyanobacterial growth of up to 373.5 mg /L.d and maximum lipid content of up to 11.7 % d.w. (5.47 ± 0.49 kg biolipids/m3 EFJ), as well as significant substrate degradation (dissolved oxygen demand - d-COD, total nitrogen-ΤΝ, orthophosphates-PO43-, total sugars up to about 93 %, 95 %, 67 % and 96 %, respectively).

Improvement in oxidative stability and quality characteristics of functional chicken meat product supplemented with aqueous coriander extract

ABSTRACT The aim of the current study was to explore the preservative effect of aqueous coriander extract and butylated hydroxytoluene (BHT) on the shelf life of refrigerated chicken patties for 9 days. For this purpose, BHT (100 ppm) and aqueous coriander extract (1%) was applied to improve the storage stability. The patties were evaluated for their physiochemical characterization including total phenolic content (TPC), thiobarbituric acid reactive substances (TBARS), peroxide value (PV), total carbonyl, metmyoglobin, and instrumental color, microbial analysis including (total plate count), and sensory attributes including (color and odor). The results showed that coriander extract has a significant (P < .05) effect in terms of delaying microbial growth, lipid and protein oxidation compared to BHT application. Likewise, higher phenolic contents were observed in patties treated with coriander extract. Conclusively, coriander extract did not impose any undesirable effect of physical attributes (instrumental color and color/odor) of cooked chicken patties. It is concluded that aqueous coriander extract is a plenteous source of various antioxidants and should be used as a functional food additive in meat products at 1% concentration without affecting the product’s quality attributes.

Microbial methane cycling in sediments of Arctic thermokarst lagoons.

Thermokarst lagoons represent the transition state from a freshwater lacustrine to a marine environment, and receive little attention regarding their role for greenhouse gas production and release in Arctic permafrost landscapes. We studied the fate of methane (CH4 ) in sediments of a thermokarst lagoon in comparison to two thermokarst lakes on the Bykovsky Peninsula in northeastern Siberia through the analysis of sediment CH4 concentrations and isotopic signature, methane-cycling microbial taxa, sediment geochemistry, lipid biomarkers, and network analysis. We assessed how differences in geochemistry between thermokarst lakes and thermokarst lagoons, caused by the infiltration of sulfate-rich marine water, altered the microbial methane-cycling community. Anaerobic sulfate-reducing ANME-2a/2b methanotrophs dominated the sulfate-rich sediments of the lagoon despite its known seasonal alternation between brackish and freshwater inflow and low sulfate concentrations compared to the usual marine ANME habitat. Non-competitive methylotrophic methanogens dominated the methanogenic community of the lakes and the lagoon, independent of differences in porewater chemistry and depth. This potentially contributed to the high CH4 concentrations observed in all sulfate-poor sediments. CH4 concentrations in the freshwater-influenced sediments averaged 1.34 ± 0.98 μmol g-1 , with highly depleted δ13 C-CH4 values ranging from -89‰ to -70‰. In contrast, the sulfate-affected upper 300 cm of the lagoon exhibited low average CH4 concentrations of 0.011 ± 0.005 μmol g-1 with comparatively enriched δ13 C-CH4 values of -54‰ to -37‰ pointing to substantial methane oxidation. Our study shows that lagoon formation specifically supports methane oxidizers and methane oxidation through changes in pore water chemistry, especially sulfate, while methanogens are similar to lake conditions.

Biotreatment of discarded fruit juices and simultaneous lipid production using Leptolyngbya sp.

AbstractBACKGROUNDFruit juices are an exceptional source of nutrients; however, due to their short shelf‐life and vulnerability to microbial degradation, large volumes of juice are regularly discarded worldwide. As a wastewater, discarded fruit juices (DFJ) possess high organic loads with significant amounts of nitrogen and phosphorus, making them a suitable substrate for microbial growth and to produce natural bioactive compounds.RESULTSDiscarded fruit juices (DFJ) were treated and simultaneously valorized (producing microbial lipids) using a microbial consortium dominated by the photosynthetic cyanobacterium Leptolyngbya sp. The effects of N:P and C:N ratios on growth and lipid production were examined, along with the ability of the consortium to biotreat DFJ in laboratory‐scale (LS) reactors. Using the optimum nutrient ratios (N:P 5:1, C:N 50.2:1), scaled‐up reactors (aquariums) were then tested under suspended and attached growth conditions. The biomass produced was further exploited for lipid production and the results revealed that Leptolyngbya sp. was able to produce up to 19% d.w. lipids and simultaneously efficiently treat DFJ. High dissolved‐chemical oxygen demand (d‐COD), total nitrogen, and PO43− removal rates (85.0% and 93.9%, 93.4% and 95.2%, 84.6% and 83.9%, respectively) were achieved in both the suspended and attached growth systems, along with high biomass productivities (316.3, 340.0 mg L−1 day−1, respectively).CONCLUSIONThe biological approach used in this study demonstrated that is possible to exploit DFJ and achieve satisfactory lipid contents (up to 19% d.w.) using a cyanobacterial‐based consortium. © 2023 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley &amp; Sons Ltd on behalf of Society of Chemical Industry (SCI).

Incorporation of betel leaf extract provides oxidative stability and improves phytochemical, textural, sensory and antimicrobial activities of buffalo meat sausages

The antioxidant effect of betel leaf extract (BLE) on lipid and protein oxidation, microbial count and physicochemical attributes was investigated in meat sausages during refrigerated storage at 4 ± 1 °C. Buffalo meat sausages were developed after incorporating 0, 250, 500 and 750 mg kg−1 of BLE (BLE0, BLE1, BLE2 and BLE3) respectively. The sausages showed no changes in proximate composition due to BLE inclusion, but there was an improvement in microbial quality, color score, textural properties and lipid and protein oxidative stability. Further, higher sensory scores were observed for the BLE-incorporated samples. The images from scanning electron microscopy (SEM) revealed a reduction in surface roughness and unevenness showing microstructure modification in BLE treated sausages compared to the control sausages. Hence, to improve the storage stability and impede the rate of lipid oxidation in sausages, BLE incorporation proved to be an effective strategy.

Co‐production of lipids and carotenoids by Rhodosporidium toruloides from cane molasses using temperature and pH shifting strategies

AbstractThe industrialized production of microbial lipids and carotenoids is facing various challenges including difficulties with microbial strains, fermentation processes, and downstream processing. This study aimed to optimize the fermentation conditions for the efficient co‐production of lipids and carotenoids using Rhodosporidium toruloides. By using temperature and pH fermentation shift strategies, the lipid content was increased by 0.57 times (78.6%), and the carotenoid content was increased by 2.5 times (2.71 mg g−1) in comparison with a control. To reduce the fermentation cost, an inexpensive carbon source, cane molasses, was used to replace nitrogen, phosphorus, and carbon sources. This enabled a maximum lipid content of 68.9% and 2.85 mg g−1 of carotenoids to be obtained. These results provided an efficient method for lipid and carotenoid co‐production, which could provide a basis for the industrial production of microbial lipids and carotenoids.

Economic and environmental evaluation for a closed loop of crude glycerol bioconversion to biodiesel

Crude glycerol, a byproduct of biodiesel production, was utilized as a carbon source to produce microbial lipids by the oleaginous yeast Rhodotorula toruloides in this study. The maximum lipid production and lipid content were 10.56 g/L and 49.52%, respectively, by optimizing fermentation conditions. The obtained biodiesel met the standards of China, the United States, and the European Union. The economic value of biodiesel produced from crude glycerol increased by 48% compared with the sale of crude glycerol. In addition, biodiesel production from crude glycerol could reduce 11,928 tons of carbon dioxide emissions and 55 tons of sulfur dioxide emissions. This study provides a strategy for a closed loop of crude glycerol to biofuel and ensures sustainable and stable development of the biodiesel industries.

Rapid remodeling of the soil lipidome in response to a drying-rewetting event

BackgroundMicrobiomes contribute to multiple ecosystem services by transforming organic matter in the soil. Extreme shifts in the environment, such as drying-rewetting cycles during drought, can impact the microbial metabolism of organic matter by altering microbial physiology and function. These physiological responses are mediated in part by lipids that are responsible for regulating interactions between cells and the environment. Despite this critical role in regulating the microbial response to stress, little is known about microbial lipids and metabolites in the soil or how they influence phenotypes that are expressed under drying-rewetting cycles. To address this knowledge gap, we conducted a soil incubation experiment to simulate soil drying during a summer drought of an arid grassland, then measured the response of the soil lipidome and metabolome during the first 3 h after wet-up.ResultsReduced nutrient access during soil drying incurred a replacement of membrane phospholipids, resulting in a diminished abundance of multiple phosphorus-rich membrane lipids. The hot and dry conditions increased the prevalence of sphingolipids and lipids containing long-chain polyunsaturated fatty acids, both of which are associated with heat and osmotic stress-mitigating properties in fungi. This novel finding suggests that lipids commonly present in eukaryotes such as fungi may play a significant role in supporting community resilience displayed by arid land soil microbiomes during drought. As early as 10 min after rewetting dry soil, distinct changes were observed in several lipids that had bacterial signatures including a rapid increase in the abundance of glycerophospholipids with saturated and short fatty acid chains, prototypical of bacterial membrane lipids. Polar metabolites including disaccharides, nucleic acids, organic acids, inositols, and amino acids also increased in abundance upon rewetting. This rapid metabolic reactivation and growth after rewetting coincided with an increase in the relative abundance of firmicutes, suggesting that members of this phylum were positively impacted by rewetting.ConclusionsOur study revealed specific changes in lipids and metabolites that are indicative of stress adaptation, substrate use, and cellular recovery during soil drying and subsequent rewetting. The drought-induced nutrient limitation was reflected in the lipidome and polar metabolome, both of which rapidly shifted (within hours) upon rewet. Reduced nutrient access in dry soil caused the replacement of glycerophospholipids with phosphorus-free lipids and impeded resource-expensive osmolyte accumulation. Elevated levels of ceramides and lipids with long-chain polyunsaturated fatty acids in dry soil suggest that lipids likely play an important role in the drought tolerance of microbial taxa capable of synthesizing these lipids. An increasing abundance of bacterial glycerophospholipids and triacylglycerols with fatty acids typical of bacteria and polar metabolites suggest a metabolic recovery in representative bacteria once the environmental conditions are conducive for growth. These results underscore the importance of the soil lipidome as a robust indicator of microbial community responses, especially at the short time scales of cell-environment reactions.A8GGwc64vbJf6mXPFnxxi9Video

Dynamics of the Lipidome in a Colon Simulator

Current evidence suggests that gut microbiome-derived lipids play a crucial role in the regulation of host lipid metabolism. However, not much is known about the dynamics of gut microbial lipids within the distinct gut biogeographic. Here we applied targeted and untargeted lipidomics to in vitro-derived feces. Simulated intestinal chyme was collected from in vitro gut vessels (V1-V4), representing proximal to distal parts of the colon after 24 and 48 h with/without polydextrose treatment. In total, 44 simulated chyme samples were collected from the in vitro colon simulator. Factor analysis showed that vessel and time had the strongest impact on the simulated intestinal chyme lipid profiles. We found that levels of phosphatidylcholines, sphingomyelins, triacylglycerols, and endocannabinoids were altered in at least one vessel (V1-V4) during simulation. We also found that concentrations of triacylglycerols, diacylglycerols, and endocannabinoids changed with time (24 vs. 48 h of simulation). Together, we found that the simulated intestinal chyme revealed a wide range of lipids that remained altered in different compartments of the human colon model over time.

Integrative analysis of genomic and metabolomic data reveals key metabolic pathways involved in lipid and carotenoid biosynthesis in oleaginous red yeast Rhodosporidiobolus odoratus XQR

Rhodosporidiobolus odoratus, one of the oleaginous red yeasts, is gaining biotechnological importance for their ability to produce microbial lipids and carotenoids. However, to date, the genomic resource underling lipid and carotenoid biosynthesis in R. odoratus has not been reported. Here, we reported the first genome assembly of R. odoratus using a combination of PacBio and Illumina techniques. The final genome assembly is 23.74 Mb in size, containing 52 scaffolds with a N50 length of 1200,460 bp and a GC content of 56.90%. Benchmarking Universal Single-Copy Orthologs (BUSCO) assessment showed that our assembly contains 94.23% of Basidiomycota universal single-copy orthologs. The genome was predicted to contain 4986 protein-coding genes, 4967 of which were functionally annotated. Metabolomic profiling identified 574 lipids, 3 carotenoids, and 208 volatile organic compounds synthesized by R. odoratus. Integrative analysis of genomics and metabolomics provides insights into the biosynthesis of lipid, carotenoid, and other bioactive compounds in R. odoratus. Collectively, the results presented herein greatly enhance our understanding of R. odoratus in lipids and carotenoids biosynthesis, and thus further accelerate its fundamental molecular investigations and biotechnological applications.

Exploration of the evolution and production of volatile compounds in grouper (Epinephelus coioides) during cold storage

Microbial growth, protein degradation and lipid oxidation played an important role in the flavor of grouper during cold storage. In order to study the relationship between volatile compounds (VOCs) and microorganisms, proteins and lipids in grouper during cold storage, headspace-solid phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) was used to analyze the VOCs of grouper during cold storage. The total viable counts (TVC), Pseudomonas count, total sulfhydryl (SH) content, carbonyl content, TBARs, total free fatty acids (FFAs) content and free amino acids (FAAs) were measured. The results showed that the indicators related to microorganisms, proteins and lipids deteriorated to different degrees during cold storage. A total of 67 VOCs were identified in the grouper. After screening, 10 key VOCs were found, 8 of which had a negative odor and were highly correlated with the deterioration of grouper quality. Through further correlation analysis, the source of key VOCs was basically determined, and the main factors affecting VOCs production were determined to be microbial growth, followed by protein degradation and lipid oxidation. It laid a foundation for regulating the flavor formation of grouper during cold storage from the source in the future.

Effectiveness of Terminalia bellerica based bioactive edible film in controlling the microbial growth and lipid oxidation of cheese during storage

A novel bioactive film was developed using Terminalia bellerica as a bioactive ingredient for improved lipid-oxidative stability and microbial quality of cheese using kalari, a Himalayan cheese, as a food-model system. The carrageenan-based film with optimum characteristics was developed using 0.5% T. bellerica extract and characterized for various physicochemical and oxidative properties. The cheese samples were packaged within the films (T1 = control film, T2 = film containing 0.5% T. bellerica) and stored for four weeks at 4±1°C along with control samples. The addition of T. bellerica significantly increased the thickness and density and decreased the moisture content, transparency, solubility and water-vapour transmission rate of the film. It also increased (P<0.05) the antioxidant potential (total-phenolic and flavonoid contents, DPPH and ABTS radical scavenging activities) of the film. A significant impact of the film was observed on lipid stability and microbiological quality and the samples stored within the T2 film showed significantly lower values for thiobarbituric acid reactive substances, free-fatty acids, peroxide values and microbial counts on days 14 and 28. The T2 film showed a significant positive effect on the sensory quality of the stored product indicating the potential of the film for controlling the quality loss during storage.

Automatic Fed-Batch Cultivation Enhances Microbial Lipid Production from Volatile Fatty Acids

Organic waste is generated worldwide, and its disposal and recycling are becoming a challenge. Due to its high carbon content, however, it may be converted into valuable products. Carbon neutrality is essential, and unstable international oil prices stress the increasing importance of biofuels significantly. Volatile fatty acids (VFA) derived from organic waste can be converted to microbial lipids by oleaginous yeast using it as a carbon source. When VFA is consumed by oleaginous yeast, the pH of the medium rises; hence, acidic agents have to be added to the medium to maintain the broth’s pH. In this study, we enhanced microbial lipid productivity by automatic fed-batch cultivation using VFA as an acidic agent, and the modified cultivation showed 48.9% and 69.0% higher biomass and lipid productivity than manual multi-fed culture. At a VFA concentration of 5 g/L and pH 7.0, a lipid yield of 0.25 g/g alongside lipid productivity of 0.11 g/L/h was obtained from an automatic fed-batch system. Oleic acid accounted for the largest proportion of microbial lipids, and the fatty acid composition was suitable for biodiesel production.

Alternative microbial-based functional ingredient source for lycopene, beta-carotene, and polyunsaturated fatty acids

The acquisition of carotenoids and polyunsaturated fatty acids (PUFAs) from plants and animals for use as functional ingredients raises concerns regarding productivity and cost; utilization of microorganisms as alternative sources is an option. We proposed to evaluate the production of carotenoids and PUFAs by Rhodopseudomonas faecalis PA2 using different vegetable oils (rice bran oil, palm oil, coconut oil, and soybean oil) as carbon source, different concentrations of yeast extract as nitrogen source at different cultivation time to ensure the best production. Cultivation with soybean oil as source of carbon led to the most significant changes in the fatty acid profile. Compared to the initial condition, the strain cultivated in the optimal conditions (4% soybean oil, 0.35% yeast extract, and 14 days of incubation) showed an increase in μmax, biomass, carotenoid productivity, and microbial lipids by 102.5%, 52.7%, 33.82%, and 34.78%, respectively. The unsaturated fatty acids content was raised with additional types of PUFAs; omega-3 [alpha-linolenic acid and eicosapentaenoic acid] and omega-6 [linoleic acid and eicosatrienoic acid] fatty acids were identified. The results of ultra high-performance liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry (UHPLC-ESI-QTOF-MS/MS) indicated the molecular formula and mass of bacterial metabolites were identical to those of lycopene and beta-carotene. The untargeted metabolomics revealed functional lipids and several physiologically bioactive compounds. The outcome provides scientific reference regarding carotenoids, PUFAs, and useful metabolites that have not yet been reported in the species Rhodopseudomonas faecalis for further use as a microbial-based functional ingredient.

Optimization of Wheat Straw Conversion into Microbial Lipids by Lipomyces tetrasporus DSM 70314 from Bench to Pilot Scale

Microbial lipids are renewable platforms for several applications including biofuels, green chemicals, and nutraceuticals that can be produced from several residual carbon sources. Lignocellulosic biomasses are abundant raw materials for the production of second-generation sugars with conversion yields depending on the quality of the hydrolysates and the metabolic efficiency of the microorganisms. In the present work, wheat straw pre-treated by steam explosion and enzymatically hydrolysed was converted into microbial lipids by Lipomyces tetrasporus DSM 70314. The preliminary optimization of the enzymatic hydrolysis was performed at the bench scale through the response surface methodology (RSM). The fermentation medium and set-up were optimized in terms of the nitrogen (N) source and carbon-to-nitrogen (C/N) ratio yielding to the selection of soy flour as a N source and C/N ratio of 160. The bench scale settings were scaled-up and further optimized at the 10 L-scale and finally at the 50 L pilot scale bioreactor. Process optimization also included oxygen supply strategies. Under optimized conditions, a lipid concentration of 14.8 gL−1 was achieved corresponding to a 23.1% w/w lipid yield and 67.4% w/w lipid cell content. Oleic acid was the most abundant fatty acid with a percentage of 57%. The overall process mass balance was assessed for the production of biodiesel from wheat straw.

Effect of sage seed gum film incorporating Zataria multiflora Boiss essential oil on the storage quality and biogenic amine content of refrigerated Otolithes ruber fillets

The effect of an edible film based on sage seed gum (SSG) incorporating 3 % Zataria multiflora Boiss essential oil (ZEO) was investigated on the storage quality and shelf life of tiger-tooth croaker (Otolithes ruber) fillets during storage at 4 ± 1 °C compared to the control film (SSG film without ZEO) and the Cellophane. The SSG-ZEO film significantly decelerated microbial growth (evaluated by total viable count, total psychrotrophic count, pH, TVBN) and lipid oxidation (evaluated by TBARS) compared to the others (P ˂ 0.05). The antimicrobial activity of ZEO was the highest and the lowest on E. aerogenes (MIC: 0.196 μL/mL) and P. mirabilis (MIC: 0.977 μL/mL), respectively. E. aerogenes was identified as an indicator biogenic amine-producer in O. ruber fish at refrigerated temperature. The active film significantly lowered biogenic amine accumulation in the samples inoculated with E. aerogenes. A clear relationship was observed between the release of ZEO's phenolic compounds from the active film to the headspace and the reduction of microbial growth, lipid oxidation, and biogenic amine production in the samples. Consequently, SSG film containing 3 % ZEO is proposed as a biodegradable antimicrobial-antioxidant packaging to extend the shelf life and decrease the biogenic amine production in refrigerated seafood.

Combination of a Push-Pull-Block Strategy with a Heterologous Xylose Assimilation Pathway toward Lipid Overproduction from Lignocellulose in Yarrowia lipolytica.

The production of biodiesel using microbial lipids derived from renewable lignocellulosic biomass is considered a promising strategy to reduce environmental pressure and promote the green energy transition. The hydrolysates of lignocellulosic biomass are rich in glucose and xylose, which makes it crucial to efficiently utilize both sugars. Here, we combined metabolic engineering and adaptive laboratory evolution (ALE) to construct an engineered Yarrowia lipolytica strain that can efficiently produce lipids from glucose and xylose. First, the "Push-Pull-Block" strategy was adopted to increase lipid content to 73.42% of the dry cell weight (DCW). Then, a heterologous xylose-utilization pathway was integrated into the engineered strain, which was subjected to ALE. The final evolved strain could accumulate 53.64% DCW of lipids from xylose, and the lipid titer reached 16.25 g/L. This work sheds light on the potential of microbial lipid overproduction from lignocellulose using engineered Y.lipolytica.

Quality impact of sustainable ma-packaging options for emulsion-type sausage: A German case study

The industrial packaging of meat products is an important process factor to preserve high quality and prolong shelf life by reducing microbial growth, lipid oxidation, and enzymatic autolysis. Currently, legislation and regulations are forcing companies to eliminate or reduce the use of (conventional fossil-based plastic) packaging.This case study in the German meat industry was designed to ascertain the effectiveness of four innovative packaging materials, differing in foil thickness and composition, to preserve the quality of ma-packed emulsion-type sausages during its shelf life compared to a conventional control packaging (multi-layer APET/PE 300µ/62µ). During storage, different product parameters and the atmosphere inside the packages were investigated to analyze the effect on product quality and shelf life.Except for one plastic-reduced multi-layer APET/PE 250µ/47µ, all materials showed significantly higher values for defect packages and gas permeability compared to the control. The decline in product quality varied between the materials showing no correlation between microbial spoilage and a decrease in product quality for emulsion-type sausages in the tested materials in comparison to the control. Besides the control, the multi-layer APET/PE 230µ/62µ was most effective in keeping the quality of ma-packed emulsion-type sausages and preserved an acceptable product quality during its shelf life.

Maximising biotransformation of pine needles to microbial lipids using Lipomyces starkeyi MTCC 1400T

Pine needles are relatively less explored lignocellulosic forest waste which can be saccharified to fermentable sugars and be used in subsequent conversion to biofuels. Microbial lipids were produced from pine needles hydrolysate and recovered lipid liquor. Ultrasound assisted pretreatment increased the cellulose content by 20.679%, 27.17%, 32.21%, and 35.61% when treated for 60 min at varying NaOH concentrations (1–4%). The 4-feed batch sachharification was performed to investigate the effect of increasing substrate loadings and fixed initial concentration (5% w/v) and yielded maximum increase of 70.23% in reducing sugars. The pine needle hydrolysate yielded 19.581 g/L total lipids, 35.888 g/L biomass concentration and lipid concentration at 67.46% g/g. Simultaneously, at equimolar ratios of HCl and liquor resulted in highest yields for total lipids (6.92 g/L) and traces of inhibitors. The fatty acids distribution and biodiesel properties revealed a similar biodiesel quality to that of palm oil. Findings from this study facilitate the complete utilisation of pine needles for microbial lipid production to resolve forest fires, and in keeping view of climate, fuels and inorganic resources towards developing a circular bioeconomy.

Mastering targeted genome engineering of GC-rich oleaginous yeast for tailored plant oil alternatives for the food and chemical sector

BackgroundSustainable production of triglycerides for various applications is a major focus of microbial factories. Oleaginous yeast species have been targeted for commercial production of microbial oils. Among all the oleaginous yeasts examined in a previous comparative study, Cutaneotrichosporon oleaginosus showed the highest lipid productivity. Moreover, a new lipid production process for C. oleaginosus with minimal waste generation and energy consumption resulted in the highest lipid productivity in the history of oleaginous yeasts. However, productivity and product diversity are restricted because of the genetic intractability of this yeast. To date, successful targeted genetic engineering of C. oleaginosus has not yet been reported.ResultsThe targeted gene editing was successfully carried out in C. oleaginosus using CRISPR/Cas system. A tailored enzyme system isolated to degrade the C. oleaginosus cell wall enabled the isolation of viable spheroplasts that are amenable to in-cell delivery of nucleic acids and proteins. The employment of both Cas9 protein and Cas mRNA was effective in obtaining strains with URA5 knockout that did not exhibit growth in the absence of uracil. Subsequently, we successfully created several strains with enhanced lipid yield (54% increase compared to that in wild type) or modified fatty acid profiles comparable with those of cocoa butter or sunflower oil compositions.ConclusionThis study establishes the first targeted engineering technique for C. oleaginosus using the CRISPR/Cas system. The current study creates the foundation for flexible and targeted strain optimizations towards building a robust platform for sustainable microbial lipid production. Moreover, the genetic transformation of eukaryotic microbial cells using Cas9 mRNA was successfully achieved.