Biotechnological potential of red yeast isolated from birch forests in Poland.

OBJECTIVES: This study aimed to isolate red yeast from sap, bark and slime exudates collected from Polish birch forests and then assessment of their biotechnological potential. RESULTS: 24 strains of red yeast were isolated from the bark, sap and spring slime fluxes of birch (Betula pendula). Strains belonging to Rhodotorula mucilaginosa (6), Rhodosporidiobolus colostri (4), Cystrofilobasidium capitaum (3), Phaffia rhodozyma (3) and Cystobasidium psychroaquaticum (3) were dominant. The highest efficiency of carotenoid biosynthesis (5.04 mg L-1) was obtained by R. mucilaginosa CMIFS 004, while lipids were most efficiently produced by two strains of P. rhodozyma (5.40 and 5.33 g L-1). The highest amount of exopolysaccharides (3.75 g L-1) was produced by the R. glutinis CMIFS 103. Eleven strains showed lipolytic activity, nine amylolytic activity, and only two proteolytic activity. The presence of biosurfactants was not found. The growth of most species of pathogenic moulds was best inhibited by Rhodotorula yeasts. CONCLUSION: Silver birch is a good natural source for the isolation of new strains of red yeast with wide biotechnological potential.

Carotenoids production by Rhodosporidium paludigenum yeasts: Characterization of chemical composition, antioxidant and antimicrobial properties.

The high market potential imposed by natural carotenoids has turned the scientific interest in search for new strains, capable of synthesizing a wide spectrum of these pigments. In this study, Rhodosporidium paludigenum NCYC 2663 and 2664 were investigated for carotenoids production and lipid accumulation utilizing different carbon sources (glucose, fructose, sucrose, mixture of glucose: galactose). Strain R. paludigenum 2663 produced the highest total carotenoids titer (2.21 mg/L) when cultivated on sucrose, together with 4 g/L lipids (30% w/w content) and 7 g/L exopolysaccharides. In the case of R. paludigenum 2664, glucose favored the production of 2.93 mg/L total carotenoids and 1.57 g/L lipids (31.8% w/w content). Analysis of the chemical profile during fermentation revealed that ß-carotene was the prominent carotenoid. Strain 2663 co-produced γ-carotene, torulene and torularhodin in lower amounts, whereas 2664 synthesized almost exclusively ß-carotene. The produced lipids from strain 2663 were rich in oleic acid, while the presence of linoleic acid was also detected in the lipoic fraction from strain 2664. The obtained carotenoid extracts exhibited antioxidant (IC50 0.14 mg/mL) and high antimicrobial activity, against common bacterial and fungal pathogenic strains. The results of this study are promising for the utilization of biotechnologically produced carotenoids in food applications.

Challenges and prospects of yeast-based microbial oil production within a biorefinery concept.

Biodiesel, unlike to its fossil-based homologue (diesel), is renewable. Its use contributes to greater sustainability in the energy sector, mainly by reducing greenhouse gas emissions. Current biodiesel production relies on plant- and animal-related feedstocks, resulting in high final costs to the prices of those raw materials. In addition, the production of those materials competes for arable land and has provoked a heated debate involving their use food vs. fuel. As an alternative, single-cell oils (SCOs) obtained from oleaginous microorganisms are attractive sources as a biofuel precursor due to their high lipid content, and composition similar to vegetable oils and animal fats. To make SCOs competitive from an economic point of view, the use of readily available low-cost substrates becomes essential. This work reviews the most recent advances in microbial oil production from non-synthetic sugar-rich media, particularly sugars from lignocellulosic wastes, highlighting the main challenges and prospects for deploying this technology fully in the framework of a Biorefinery concept.

From straw to salmon: a technical design and energy balance for production of yeast oil for fish feed from wheat straw.

BACKGROUND: Aquaculture is a major user of plant-derived feed ingredients, such as vegetable oil. Production of vegetable oil and protein is generally more energy-intensive than production of the marine ingredients they replace, so increasing inclusion of vegetable ingredients increases the energy demand of the feed. Microbial oils, such as yeast oil made by fermentation of lignocellulosic hydrolysate, have been proposed as a complement to plant oils, but energy assessments of microbial oil production are needed. This study presents a mass and energy balance for a biorefinery producing yeast oil through conversion of wheat straw hydrolysate, with co-production of biomethane and power. RESULTS: The results showed that 1 tonne of yeast oil (37 GJ) would require 9.2 tonnes of straw, 14.7 GJ in fossil primary energy demand, 14.6 GJ of process electricity and 13.3 GJ of process heat, while 21.5 GJ of biomethane (430 kg) and 6 GJ of excess power would be generated simultaneously. By applying economic allocation, the fossil primary energy demand was estimated to 11.9 GJ per tonne oil. CONCLUSIONS: Fossil primary energy demand for yeast oil in the four scenarios studied was estimated to be 10-38% lower than for the commonly used rapeseed oil and process energy demand could be met by parallel combustion of lignin residues. Therefore, feed oil can be produced from existing non-food biomass without causing agricultural expansion.

Shotgun-Based Mass Spectrometry Analysis of Phospholipid and Triacylglycerol Molecular Species and Eicosanoids in Salmon Muscle Tissue on Feeding Microbial Oil.

The continuous growth of aquaculture places a growing demand on alternative sources of fish oil (FO). Certain microorganisms provide a sustainable replacement for FO due to their content of EPA and DHA, which are essential for fish health. Appreciable evidence shows that changes in feeding sources may alter the nutritional components of salmon; however, the influence of diets on lipid species remains unclear. In this study, the identification and semi-quantification of lipid molecular species in salmon muscle during feeding with a microbial oil (MO) were carried out by focusing on triacylglycerol (TAG) and diacyl-phospholipid using shotgun-based mass spectrometry analysis. DHA in the MO diet was efficiently incorporated into phospholipid structures on feeding, followed by accumulation in salmon muscle. The MO diet elevated the level of certain EPA-containing TAGs, such as TAG C52:5 (16:0_16:0_20:5) and TAG C54:6 (16:0_18:1_20:5), indicating that the MO diet may be an excellent source for enhancement of the abundance of ω3 lipids. Further, prostaglandins (PGs) PGE2 and PGF3α were identified and quantified for the first time in salmonid tissue.

Food Toxicology and Food Safety: Report of the 3rd International Electronic Conference on Foods: Food, Microbiome, and Health-A Celebration of the 10th Anniversary of Foods' Impact on Our Wellbeing.

The purpose of the conference session summarized in this article was to bring together international experts on food toxicology and food safety and share the current scientific knowledge on these topics. The presentations covered a wide range of interdisciplinary issues, including (i) the impact of diet on body weight and health outcomes including results from animal models of carcinogenesis, (ii) methods for microbial oil extraction, (iii) food processing and its impact on food safety and health, (iv) novel compounds to avoid mycotoxin contamination of agricultural products, and (v) the safety of cannabidiol in food supplements based on Cannabis sativa extracts. Some of the conclusions of the presentations included that correct food choices may impact on the risk of non-communicable diseases such as cancer, that food processing may have an influence on health, by either reducing or increasing risks, and that research regarding novel compounds is important, which may have preventive but also detrimental effects on health.

Predicting performance of in-situ microbial enhanced oil recovery process and screening of suitable microbe-nutrient combination from limited experimental data using physics informed machine learning approach.

Screening of suitable microbe-nutrient combination and prediction of oil recovery at the initial stage is essential for the success of Microbial Enhanced Oil Recovery (MEOR) technique. However, experimental and physics-based modelling approaches are expensive and time-consuming. In this study, Physics Informed Machine Learning (PIML) framework was developed to screen and predict oil recovery at a relatively lesser time and cost with limited experimental data. The screening was done by quantifying the influence of parameters on oil recovery from correlation and feature importance studies. Results revealed that microbial kinetic, operational and reservoir parameters influenced the oil recovery by 50%, 32.6% and 17.4%, respectively. Higher oil recovery is attained by selecting a microbe-nutrient combination having a higher ratio of value between biosurfactant yield and microbial yield parameters, as they combinedly influence the oil recovery by 27%. Neural Network is the best ML model for MEOR application to predict oil recovery (R2≈0.99).

Agroindustrial byproduct-based media in the production of microbial oil rich in oleic acid and carotenoids.

This study focuses on the potential of Rhodotorula mucilaginosa CCT 7688 in simultaneous production of lipids and carotenoids in agroindustrial byproduct-based media and specially aims at establishing a process condition that guarantees high concentrations of both bioproducts, i.e., a carotenoid-rich microbial oil with potential economic value and health benefits attributed to carotenoids and fatty acids. Four different combinations of cultivation modes (batch and fed-batch) and alternative substrates (crude glycerol, sugarcane molasses and corn steep liquor) were tested. The M2-B assay, which comprises the use of an agroindustrial byproduct-based medium without any supplementation (70 g L-1 sugarcane molasses and 3.4 g L-1 corn steep liquor) and batch mode, was selected as the most promising one to produce both compounds. Total carotenoid production and total lipid content were 1794.2 µg L-1 and 43.2% (w/w), respectively, after 144 h of cultivation. The fatty acid profile showed predominance of oleic acid (69.9%) and palmitic acid (23.2%). Thus, R. mucilaginosa CCT 7688 may be used in simultaneous production of lipids and carotenoids successfully; its fatty acid profile is similar to that found in olive oil. Both compounds are economically interesting and have great possibility of future commercial applications.

Potential of food waste hydrolysate as an alternative carbon source for microbial oil synthesis.

Volatile fatty acids (VFAs) have great potential as cheap raw materials in microbial oil synthesis and reducing the cost of substrates is essential for the development of microbial oil biosynthesis. In this study, the food waste hydrolysate and synthetic VFAs media were both used as substrate to synthesis microbial oil. The optimal short-chain VFAs ratio for microbial oil synthesis is 20:5:5 and increasing the proportion of propionic acid is the key to obtaining odd fatty acids. The hydrolysate obtained from food waste under the total solid condition of 2:1 and pH 5 is the most suitable medium for microbial oil synthesis. The biological products obtained from food waste hydrolysate were comparable to synthetic VFAs media, obtaining a 34.02% of lipid content. Results prove that food waste hydrolysate has great potential as the available feedstock for microbial oil synthesis and a promising application value in food waste recycling.

Schizochytrium sp. (T18) Oil as a Fish Oil Replacement in Diets for Juvenile Rainbow Trout (Oncorhynchus mykiss): Effects on Growth Performance, Tissue Fatty Acid Content, and Lipid-Related Transcript Expression.

In this study, we evaluated whether oil extracted from the marine microbe, Schizochytrium sp. (strain T18), with high levels of docosahexaenoic acid (DHA), could replace fish oil (FO) in diets for rainbow trout (Oncorhynchus mykiss). Three experimental diets were tested: (1) a control diet with fish oil (FO diet), (2) a microbial oil (MO) diet with a blend of camelina oil (CO) referred to as MO/CO diet, and (3) a MO diet (at a higher inclusion level). Rainbow trout (18.8 ± 2.9 g fish-1 initial weight ± SD) were fed for 8 weeks and evaluated for growth performance, fatty acid content and transcript expression of lipid-related genes in liver and muscle. There were no differences in growth performance measurements among treatments. In liver and muscle, eicosapentaenoic acid (EPA) was highest in trout fed the FO diet compared to the MO/CO and MO diets. Liver DHA was highest in trout fed the MO/CO diet compared to the FO and MO diets. Muscle DHA was highest in trout fed the MO and MO/CO diets compared to the FO diet. In trout fed the MO/CO diet, compared to the MO diet, fadsd6b was higher in both liver and muscle. In trout fed the FO or MO/CO diets, compared to the MO diet, cox1a was higher in both liver and muscle, cpt1b1a was higher in liver and cpt1a1a, cpt1a1b and cpt1a2a were higher in muscle. Schizochytrium sp. (T18) oil was an effective source of DHA for rainbow trout.

Obtaining hemicellulosic hydrolysate from sugarcane bagasse for microbial oil production by Lipomyces starkeyi.

OBJECTIVE: The extraction of the hemicellulose fraction of sugarcane bagasse (SCB) by acid hydrolysis was evaluated in an autoclave and a Parr reactor aiming the application of the hydrolysate as a carbon source for lipid production by Lipomyces starkeyi. RESULTS: The hydrolysis that resulted in the highest sugar concentration was obtained by treatment in the Parr reactor (HHR) at 1.5% (m/v) H2SO4 and 120 °C for 20 min, reaching a hemicellulose conversion of approximately 82%. The adaptation of the yeast to the hydrolysate provided good fermentability and no lag phase. The fermentation of hemicellulose-derived sugars (HHR) by L. starkeyi resulted in a 27.8% (w/w) lipid content and YP/S of 0.16 g/l.h. Increasing the inoculum size increased the lipid content by approximately 61%, reaching 44.8% (w/w). CONCLUSION: The hemicellulose hydrolysate from SCB is a potential substrate for L. starkeyi to produce lipids for biodiesel synthesis based on the biorefinery concept.

Bioprocess development for the production of novel oleogels from soybean and microbial oils.

This study presents the production of novel oleogels via circular valorisation of food industry side streams. Sugarcane molasses and soybean processing side streams (i.e. soybean cake) were employed as fermentation feedstocks for the production of microbial oil. Fed-batch bioreactor fermentations carried out by the oleaginous yeast Rhodosporidium toruloides led to the production of 36.9 g/L total dry weight with an intracellular oil content of 49.8% (w/w) and 89.4 µg/g carotenoids. The carotenoid-rich microbial oil and soybean oil were evaluated as base oils for the production of wax-based oleogels. The wax esters, used as oleogelators, were produced via enzymatic catalysis, using microbial oil or soybean fatty acid distillate as raw materials. All oleogels presented a gel-like behaviour (G' > G″). However, the highest G' was determined for the oleogel produced from soybean oil and microbial oil-wax esters, which indicated a stronger network. Thermal analysis showed that this oleogel had a melting temperature profile up to 35 °C, which is favorable for applications in the confectionery industry. Also, texture analysis demonstrated that soybean oil-microbial oil wax oleogel was stable (1.9-2.2 N) within 30-days storage period. This study showed the potential of novel oleogels production through the development of bioprocesses based on the valorisation of various renewable resources.

Using 1-propanol to significantly enhance the production of valuable odd-chain fatty acids by Rhodococcus opacus PD630.

Odd-chain fatty acids (OCFAs) have been reported to possess pharmacological activity and have been used in the manufacture of agricultural and industrial chemicals. We here provided a new method to increase the OCFAs content in oil produced by Rhodococcus opacus PD630 through addition of 1-propanol to the fermentation media. The OCFAs in oil of R. opacus PD630 are primarily pentadecanoic acid (C15:0), heptadecanoic acid (C17:0) and heptadecenoic acid (C17:1). After adding 0.5-1.5% (v/v) 1-propanol, the production of oil increased from 1.27 g/L to 1.31-1.61 g/L, and the OCFAs content in oil increased by 46.7-55.1%. Metabolic intermediates determination and transcriptome analysis revealed that R. opacus assimilated 1-propanol through methylmalonyl-CoA pathway. When the nitrogen source was limited, propionyl-CoA was converted to propionyl-acyl carrier protein (ACP) which could be used as primer during the elongation of fatty acid synthesis. Then OCFAs were produced when odd number of propionyl-ACP was incorporated in the cycles of fatty acid synthesis.

Development of a Circular Oriented Bioprocess for Microbial Oil Production Using Diversified Mixed Confectionery Side-Streams.

Diversified mixed confectionery waste streams were utilized in a two-stage bioprocess to formulate a nutrient-rich fermentation media for microbial oil production. Solid-state fermentation was conducted for the production of crude enzyme consortia to be subsequently applied in hydrolytic reactions to break down starch, disaccharides, and proteins into monosaccharides, amino acids, and peptides. Crude hydrolysates were evaluated in bioconversion processes using the red yeast Rhodosporidium toruloides DSM 4444 both in batch and fed-batch mode. Under nitrogen-limiting conditions, during fed-batch cultures, the concentration of microbial lipids reached 16.6-17 g·L-1 with the intracellular content being more than 40% (w/w) in both hydrolysates applied. R. toruloides was able to metabolize mixed carbon sources without catabolite repression. The fatty acid profile of the produced lipids was altered based on the substrate employed in the bioconversion process. Microbial lipids were rich in polyunsaturated fatty acids, with oleic acid being the major fatty acid (61.7%, w/w). This study showed that mixed food side-streams could be valorized for the production of microbial oil with high unsaturation degree, pointing towards the potential to produce tailor-made lipids for specific food applications. Likewise, the proposed process conforms unequivocally to the principles of the circular economy, as the entire quantity of confectionery by-products are implemented to generate added-value compounds that will find applications in the same original industry, thus closing the loop.

Bioconversion of Poplar Wood Hemicellulose Prehydrolysate to Microbial Oil Using Cryptococcus curvatus.

Poplar wood hemicellulose prehydrolysate was used for microbial oil production using an oleaginous microorganism Cryptococcus curvatus. Initially, the effect of substrate concentration and nitrogen content was investigated on synthetic media. Then poplar wood prehydrolysate without detoxification was used as substrate in the fermentation. The result showed that this strain is capable of consuming both hexose and pentose sugars, a challenge in fermentation of hemicellulosic streams. It was able to accumulate 36.98% of lipid and the fermentation resulted in 13.78 g/L of biomass and 5.13 g/L of lipid under optimum conditions after 164 h of fermentation. The lipid product obtained was characterized in terms of their fatty acid profiles. Overall, this study shows that it is possible to produce microbial oil from a sustainable renewable feedstock like poplar wood hemicellulose prehydrolysate. This robust strain used has the ability to grow on industrially produced hemicellulose which can help in the development of an integrated biorefinery, where all the three components of lignocellulosic biomass are utilized.

Microscale direct transesterification of microbial biomass with ethanol for screening of microorganisms by its fatty acid content

Abstract We present an improved method of direct transesterification suitable for the quantitative analysis of multiple dry samples for its fatty acid content, using a minimal amount of biomass and reactants. The method features an acid-catalyzed direct alcoholysis of microgram samples of dry biomass; the rationale behind the solvent and reagent proportions chosen is discussed. The method was validated using seven microbial strains with diverse lipid content (Saccharomyces cerevisiae, Saccharomyces boulardii, Candida tropicalis, Haematococcus pluvialis, Chlorella vulgaris, Spirulina platensis and Schizochytrium limacinum), and compared with a macroscale direct transesterification method, and with gravimetric analysis of lipids extracted with solvents. The microscale method showed a conversion of 98.06 ± 0.87% of the lipids, using approximately 3 mg of dry biomass, 1mL of 0.2M H2SO4 dissolved in anhydrous ethanol (the acid is the catalyzer and ethanol the reactant)). The mixture was maintained at 70 °C for 20 h with periodic mixing, and then extracted with 2mL n-heptane and analyzed by GC-FID. The lipid content was then calculated considering dilution and sample mass. This method is effective, reliable, and technically attractive for analytical and comparative purposes.

Refined soybean oil wastewater treatment and its utilization for lipid production by the oleaginous yeast Trichosporon fermentans.

BACKGROUND: The release of refined soybean oil wastewater (RSOW) with a high chemical oxygen demand (COD) and oil content burdens the environment. The conversion of RSOW into lipids by oleaginous yeasts may be a good way to turn this waste into usable products. RESULTS: The oleaginous yeast Trichosporon fermentans was used for treating the RSOW without sterilization, dilution, or nutrient supplementation. It was found that the COD and oil content of the RSOW were removed effectively; microbial oil was abundantly produced in 48 h; and the phospholipids in the RSOW tended to contribute to a higher biomass and microbial lipid content. With Plackett-Burman design and response surface design experiments, the optimal wastewater treatment conditions were determined: temperature 28.3 °C, amount of inoculum 5.9% (v/v), and initial pH 6.1. The optimized conditions were used in a 5-L bioreactor to treat the RSOW. The maximum COD degradation of 94.7% was obtained within 40 h, and the removal of the oil content was 89.9%. The biomass was 7.9 g/L, the lipid concentration was 3.4 g/L, and the lipid content was 43% (w/w). The microbial oil obtained, with a main component of unsaturated fatty acids, was similar to vegetable oils and was suggested as a potential raw material for biodiesel production. CONCLUSION: Trichosporon fermentans can be effectively used for RSOW treatment, and lipid production and can complete pretreatment and biochemical treatment simultaneously, allowing the utilization of RSOW, which both solves an environmental problem and positively impacts the use of resources. These results provide valuable information for developing and designing more efficient waste-into-lipid bioprocesses.

Bioprocess development for biolubricant production using microbial oil derived via fermentation from confectionery industry wastes.

Microbial oil produced from confectionery and wheat milling side streams has been evaluated as novel feedstock for biolubricant production. Nutrient-rich fermentation media were produced by a two-step bioprocess involving crude enzyme production by solid state fermentation followed by enzymatic hydrolysis of confectionery industry waste. Among 5 yeast strains and 2 fungal strains cultivated on the crude hydrolysate, Rhodosporidium toruloides and Cryptococcus curvatus were selected for further evaluation for biolubricant production based on fermentation efficiency and fatty acid composition. The extracted microbial oils were enzymatically hydrolysed and the free fatty acids were esterified by Lipomod 34-MDP in a solvent-free system with trimethylolpropane (TMP) and neopentyl glycol (NPG). The highest conversion yields were 88% and 82.7% for NPG esters of R. toruloides and C. curvatus, respectively. This study also demonstrates that NPG esters produced from microbial oil have promising physicochemical properties for bio-based lubricant formulations that could substitute for conventional lubricants.

Bioprospecting microbes for single-cell oil production from starchy wastes.

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

Production of wax esters via microbial oil synthesis from food industry waste and by-product streams.

The production of wax esters using microbial oils was demonstrated in this study. Microbial oils produced from food waste and by-product streams by three oleaginous yeasts were converted into wax esters via enzymatic catalysis. Palm oil was initially used to evaluate the influence of temperature and enzyme activity on wax ester synthesis catalysed by Novozyme 435 and Lipozyme lipases using cetyl, oleyl and behenyl alcohols. The highest conversion yields (up to 79.6%) were achieved using 4U/g of Novozyme 435 at 70°C. Transesterification of microbial oils to behenyl and cetyl esters was achieved at conversion yields up to 87.3% and 69.1%, respectively. Novozyme 435 was efficiently reused for six and three cycles during palm esters and microbial esters synthesis, respectively. The physicochemical properties of microbial oil derived behenyl esters were comparable to natural waxes. Wax esters from microbial oils have potential applications in cosmetics, chemical and food industries.