Oleaginous yeasts- substrate preference and lipid productivity: a view on the performance of microbial lipid producers

Background Oleaginous yeasts are promising microbial platforms for sustainable, bio-based production of biofuels and oleochemical building blocks. Bio-based residues provide sustainable and cost-effective carbon sources for fermentative yeast oil production without land-use change. Considering the regional abundancy of different waste streams, we chose complex biomass residue streams of marine origin; macroalgae hydrolysate, and terrestrial origin; wheat straw hydrolysate in the presence, and absence of corn steep liquor as a complex nitrogen source. We investigated the biomass and lipid yields of an array of well-described oleaginous yeasts; R. glutinis, T. asahii, R. mucilaginosa, R. toruloides, C. oleaginosus growing on these hydrolysates. Furthermore, their sugar utilization, fatty acid profile, and inhibitory effect of the hydrolysates on yeast growth were compared. For correlative reference, we initially performed comparative growth experiments for the strains on individual monomeric sugars separately. Each of these monomeric sugars was a dominant carbon source in the complex biomass hydrolysates evaluated in this study. In addition, we evaluated N-acetylglucosamine, the monomeric building block of chitin, as a low-cost nitrogen and carbon source in yeast fermentation. Results C. oleaginosus provided the highest biomass and lipid yields. In the wheat straw and brown algae hydrolysates, this yeast strain gained 7.5 g/L and 3.8 g/L lipids, respectively. Cultivation in algae hydrolysate resulted in a higher level of unsaturated fatty acids in the lipids accumulated by all yeast strains. R. toruloides and C. oleaginosus were able to effectively co-utilize mannitol, glucose, and xylose. Growth rates on wheat straw hydrolysate were enhanced in presence of corn steep liquor. Conclusions Among the yeast strains investigated in this study, C. oleaginosus proved to be the most versatile strain in terms of substrate utilization, productivity, and tolerance in the complex media. Various fatty acid profiles obtained on each substrate encourage the manipulation of culture conditions to achieve the desired fatty acid composition for each application. This could be accomplished by combining the element of carbon source with other formerly studied factors such as temperature and oxygen. Moreover, corn steep liquor showed promise for enhancement of growth in the oleaginous strains provided that carbon substrate is available.

Wood-feeding termite gut symbionts as an obscure yet promising source of novel manganese peroxidase-producing oleaginous yeasts intended for azo dye decolorization and biodiesel production

Background The ability of oxidative enzyme-producing micro-organisms to efficiently valorize organic pollutants is critical in this context. Yeasts are promising enzyme producers with potential applications in waste management, while lipid accumulation offers significant bioenergy production opportunities. The aim of this study was to explore manganese peroxidase-producing oleaginous yeasts inhabiting the guts of wood-feeding termites for azo dye decolorization, tolerating lignocellulose degradation inhibitors, and biodiesel production. Results Out of 38 yeast isolates screened from wood-feeding termite gut symbionts, nine isolates exhibited high levels of extracellular manganese peroxidase (MnP) activity ranged between 23 and 27 U/mL after 5 days of incubation in an optimal substrate. Of these MnP-producing yeasts, four strains had lipid accumulation greater than 20% (oleaginous nature), with Meyerozyma caribbica SSA1654 having the highest lipid content (47.25%, w/w). In terms of tolerance to lignocellulose degradation inhibitors, the four MnP-producing oleaginous yeast strains could grow in the presence of furfural, 5-hydroxymethyl furfural, acetic acid, vanillin, and formic acid in the tested range. M. caribbica SSA1654 showed the highest tolerance to furfural (1.0 g/L), 5-hydroxymethyl furfural (2.5 g/L) and vanillin (2.0 g/L). Furthermore, M. caribbica SSA1654 could grow in the presence of 2.5 g/L acetic acid but grew moderately. Furfural and formic acid had a significant inhibitory effect on lipid accumulation by M. caribbica SSA1654, compared to the other lignocellulose degradation inhibitors tested. On the other hand, a new MnP-producing oleaginous yeast consortium designated as NYC-1 was constructed. This consortium demonstrated effective decolorization of all individual azo dyes tested within 24 h, up to a dye concentration of 250 mg/L. The NYC-1 consortium's decolorization performance against Acid Orange 7 (AO7) was investigated under the influence of several parameters, such as temperature, pH, salt concentration, and co-substrates (e.g., carbon, nitrogen, or agricultural wastes). The main physicochemical properties of biodiesel produced by AO7-degraded NYC-1 consortium were estimated and the results were compared to those obtained from international standards. Conclusion The findings of this study open up a new avenue for using peroxidase-producing oleaginous yeasts inhabiting wood-feeding termite gut symbionts, which hold great promise for the remediation of recalcitrant azo dye wastewater and lignocellulosic biomass for biofuel production. Graphical Abstract

The history, state of the art and future prospects for oleaginous yeast research

Lipid-based biofuels, such as biodiesel and hydroprocessed esters, are a central part of the global initiative to reduce the environmental impact of the transport sector. The vast majority of production is currently from first-generation feedstocks, such as rapeseed oil, and waste cooking oils. However, the increased exploitation of soybean oil and palm oil has led to vast deforestation, smog emissions and heavily impacted on biodiversity in tropical regions. One promising alternative, potentially capable of meeting future demand sustainably, are oleaginous yeasts. Despite being known about for 143 years, there has been an increasing effort in the last decade to develop a viable industrial system, with currently around 100 research papers published annually. In the academic literature, approximately 160 native yeasts have been reported to produce over 20% of their dry weight in a glyceride-rich oil. The most intensively studied oleaginous yeast have been Cutaneotrichosporon oleaginosus (20% of publications), Rhodotorula toruloides (19%) and Yarrowia lipolytica (19%). Oleaginous yeasts have been primarily grown on single saccharides (60%), hydrolysates (26%) or glycerol (19%), and mainly on the mL scale (66%). Process development and genetic modification (7%) have been applied to alter yeast performance and the lipids, towards the production of biofuels (77%), food/supplements (24%), oleochemicals (19%) or animal feed (3%). Despite over a century of research and the recent application of advanced genetic engineering techniques, the industrial production of an economically viable commodity oil substitute remains elusive. This is mainly due to the estimated high production cost, however, over the course of the twenty-first century where climate change will drastically change global food supply networks and direct governmental action will likely be levied at more destructive crops, yeast lipids offer a flexible platform for localised, sustainable lipid production. Based on data from the large majority of oleaginous yeast academic publications, this review is a guide through the history of oleaginous yeast research, an assessment of the best growth and lipid production achieved to date, the various strategies employed towards industrial production and importantly, a critical discussion about what needs to be built on this huge body of work to make producing a yeast-derived, more sustainable, glyceride oil a commercial reality.

Recycling Industrial Food Wastes for Lipids Production by Oleaginous Yeasts Rhodosporidiobolus Azoricus and Cutaneotrichosporon Oleaginosum.

BackgroundMicrobial lipids have been emerging as a sustainable alternative to vegetable oils and animal fat to produce biodiesel and industrial relevant chemicals. The use of wastes for microbial processes can represent a way for upgrading low value feedstock to high value products, addressing one of the main goals of circular economy, the reduction of wastes by recycling. Two oleaginous yeasts, Rhodosporidiobolus azoricus and Cutaneotrichosporon oleaginosum, were used in this study to demonstrate the feasibility of the proposed approach. ResultsIn this study wastes from industrial food processing, as pumpkin peels and syrup from candied fruits manufacture, were used for yeast cultivation and for lipids production. Evaluation of growth and sugar consumption revealed marked differences between the yeasts in capacity to utilize sucrose and glucose, the main sugars present in the feedstock. In particular, we observed an unexpected limitation in glucose metabolism on mineral media by R. azoricus. Both species showed ability to grow and accumulate lipids on media exclusively composed by undiluted pumpkin peels hydrolysate, and R. azoricus was the best performing. By a two-stage process carried out in bioreactor, this species reached a biomass concentration of 45 g/L (dry weight) containing 55% of lipids, corresponding to a lipid concentration of 24 g/L, with a productivity of 0.26 g/L/h and yield of 0.29 g lipids per g of utilized sugar. These values are close to the highest reported so far from organic wastes. ConclusionsWastes from industrial food processing were sufficient to completely support yeast growth and to induce lipid accumulation. This study provides strong evidence that the concept of valorisation through the production of lipids from the complete metabolism of nutrients present in agro-industrial wastes by oleaginous yeasts is promising for implementation of biotechnological processes in a circular economy contest.

Whole conversion of agro-industrial wastes rich in galactose-based carbohydrates into lipid using oleaginous yeast Aureobasidium namibiae

Background Raw materials composed of easily assimilated monosaccharides have been employed as carbon source for production of microbial lipids. Nevertheless, agro-industrial wastes rich in galactose-based carbohydrates have not been introduced as feedstocks for oleaginous yeasts. Results In this study, Aureobasidium namibiae A12 was found to efficiently accumulate lipid from soy molasses and whey powder containing galactose-based carbohydrates, with lipid productions of 5.30 g/L and 5.23 g/L, respectively. Over 80% of the fatty acids was C16:0, C18:0, C18:1, and C18:2. All kinds of single sugar components in the two byproducts were readily converted into lipids, with yields ranging between 0.116 g/g and 0.138 g/g. Three α-galactosidases and five β-galactosidases in the strain were cloned and analyzed. Changes of transcriptional levels indicated GalB and GalC were key α-galactosidases, and GalG was key β-galactosidase. In 10 L fermentor, lipid production from SM and WP achieved 6.45 g/L and 6.13 g/L, respectively. β-galactosidase was responsible for lactose hydrolysis; sucrase and α-galactosidase both contributed to the efficient hydrolysis of raffinose and stachyose in a cooperation manner. Conclusions This is a new way to produce lipids from raw materials containing galactose-based carbohydrates. This finding revealed the significance of sucrase in the direct hydrolysis of galactose-based carbohydrates in raw materials for the first time and facilitated the understanding of the efficient utilization of galactose-based carbohydrates to manufacture lipid or other chemicals in bioprocess. Graphic abstract

Engineering of Saccharomyces cerevisiae for the accumulation of high amounts of triacylglycerol

Background Fatty acid-based substances play an important role in many products, from food supplements to pharmaceutical products and biofuels. The production of fatty acids, mainly in their esterified form as triacylglycerol (TAG), has been intensively studied in oleaginous yeasts, whereas much less effort has been invested into non-oleaginous species. In the present work, we engineered the model yeast Saccharomyces cerevisiae, which is commonly regarded as non-oleaginous, for the storage of high amounts of TAG, comparable to the contents achieved in oleaginous yeasts. Results We investigated the effects of several mutations with regard to increased TAG accumulation and identified six of them as important for this phenotype: a point mutation in the acetyl-CoA carboxylase Acc1p, overexpression of the diacylglycerol acyltransferase Dga1p, deletions of genes coding for enzymes involved in the competing pathways glycogen and steryl ester synthesis and TAG hydrolysis, and a deletion of CKB1, the gene coding for one of the regulatory subunits of casein kinase 2. With the combination of these mutations in a S. cerevisiae strain with a relatively high neutral lipid level already in the non-engineered state, we achieved a TAG content of 65% in the dry biomass. High TAG levels were not only obtained under conditions that favor lipid accumulation, but also in defined standard carbon-limited media. Conclusions Baker's yeast, which is usually regarded as inefficient in the storage of TAG, can be converted into a highly oleaginous strain that could be useful in processes aiming at the synthesis of fatty acid-based products. This work emphasizes the importance of strain selection in combination with metabolic engineering to obtain high product levels.

Lipid yields from oleaginous yeasts isolated from the north Peruvian Andes by culture media non-limiting nitrogen

Oleochemicals can be obtained from oily yeasts due to their ability to produce a high lipid content. This research aimed to isolate them from the North Peruvian Andes with a lipid content greater than 20%. They were identified by sequencing internal transcribed spacer regions ITS of conserved ribosomal DNA (rDNA), evaluate their growth kinetics, biomass and lipid yields, using culture media with C/N 100:1+xylose (MS-1-7) and 2:1+glucose (MS-2-7). Growth kinetics up to the maximum stationary phase was evaluated using the parameterized Gompertz type II model. Rhodotorula glutinis, R. mucilaginosa, and R. kratochvilovae were selected. The C/N ratio in the culture medium influenced growth kinetics, biomass and lipids yields. With MS-1-7, a high specific growth rate (?max) was obtained, reaching the stationary phase between 6 to 9 h and the highest lipid accumulation between 23.1% and 31.5%. With the MS-2-7 medium, maximum biomass value obtained in the stationary phase between 37 and 51 h, which generated the highest biomass yields at the end of the entire process and lipid yield of 4.65, 5.59, and 8.80 g L-1 in the strains mentioned. There is potential to obtain high lipid yields using a culture media non-limiting nitrogen, examining not only the C/N ratio. But also, the quantities, nature of the components, and type of oleaginous yeasts taking care to avoid a high carbon concentration to prevent the Cabtree effect.

Oleaginous yeasts respond differently to carbon sources present in lignocellulose hydrolysate

Background Microbial oils, generated from lignocellulosic material, have great potential as renewable and sustainable alternatives to fossil-based fuels and chemicals. By unravelling the diversity of lipid accumulation physiology in different oleaginous yeasts grown on the various carbon sources present in lignocellulose hydrolysate (LH), new targets for optimisation of lipid accumulation can be identified. Monitoring lipid formation over time is essential for understanding lipid accumulation physiology. This study investigated lipid accumulation in a variety of oleaginous ascomycetous and basidiomycetous strains grown in glucose and xylose and followed lipid formation kinetics of selected strains in wheat straw hydrolysate (WSH). Results Twenty-nine oleaginous yeast strains were tested for their ability to utilise glucose and xylose, the main sugars present in WSH. Evaluation of sugar consumption and lipid accumulation revealed marked differences in xylose utilisation capacity between the yeast strains, even between those belonging to the same species. Five different promising strains, belonging to the species Lipomyces starkeyi, Rhodotorula glutinis, Rhodotorula babjevae and Rhodotorula toruloides, were grown on undiluted wheat straw hydrolysate and lipid accumulation was followed over time, using Fourier transform-infrared (FTIR) spectroscopy. All five strains were able to grow on undiluted WSH and to accumulate lipids, but to different extents and with different productivities. R. babjevae DVBPG 8058 was the best-performing strain, accumulating 64.8% of cell dry weight (CDW) as lipids. It reached a culture density of 28 g/L CDW in batch cultivation, resulting in a lipid content of 18.1 g/L and yield of 0.24 g lipids per g carbon source. This strain formed lipids from the major carbon sources in hydrolysate, glucose, acetate and xylose. R. glutinis CBS 2367 also consumed these carbon sources, but when assimilating xylose it consumed intracellular lipids simultaneously. Rhodotorula strains contained a higher proportion of polyunsaturated fatty acids than the two tested Lipomyces starkeyi strains. Conclusions There is considerable metabolic diversity among oleaginous yeasts, even between closely related species and strains, especially when converting xylose to biomass and lipids. Monitoring the kinetics of lipid accumulation and identifying the molecular basis of this diversity are keys to selecting suitable strains for high lipid production from lignocellulose.