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

BACKGROUND: Sustainable 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. RESULTS: The 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. CONCLUSION: This 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.

Isolation of Hexavalent chromium tolerant fungal species from urban vegetable farm soil and effluent waste in Addis Ababa& Rift valley, Ethiopia.

Hexavalent chromium is resistant to degradation and harmful toxic substance to environment and community health. Physicochemical treatment methods are demanding high cost, used large quantities of chemicals & energy, release large amount of secondary toxic degradants. Mycoremediation is an eco-friendly alternative treatment method. The main objective of this research is to isolate and characterize chrome (VI) tolerant fungi from farm soil & industry effluent for mycoremedation purpose. The screening and isolation of yeast was carried out on potato dextrose agar media. PDA and broth assay test for fungi tolerance to hexavalent chromium at different concentration, temperature and pH was evaluated. Fungi species was identified biochemically using Biolog Microstation depending on carbon utilization and chemical sensitivity test. The result revealed that 10 yeast species was identified with full ID from effluent waste and farm soil based on their probability ≥  75% and similarity index ≥  0.5 as well as their Cr (VI) tolerance ability up to 2500 ppm. These are Yarrowia lipolytica (100%, 0.7), Cryptococcus luteolus(100%, 0.64), Rhodotorula aurantiaca A(100%, 0.62), Ustilago maydis(100%, 0.58) Trichosporon beigelii B (100%, 0.51), Cryptococcus terreus A (100%, 0.62), Zygosaccharomyces bailii (98%, 0.65), Nadsoniafulvenscens (90%, 0.62), Schizoblastosporonstarkeyihenricii (89%, 0.56), Endomycopsis vivi (84%, 0.62), Rhodotorula pustula (Sim, 0.59). Two yeast species Yarrowia lipolytica and Nadsoniafulvenscens show the highest growth mean Optical density (OD) measure 0.74 ± 0.2 & 0.60 ± 0.2 respectively at pH 7 & 25 °C. The highest tolerance index (mm) was recorded by Schizoblastosporon starkey henricii 0.3067 ± 0.152. Cr (VI)-tolerance ability of these yeast strains used in the development of chromium-bioremediation technologies provide an alternative option for chromium sequestration after HPLC analysis& molecular characterization.

Turnover of Benzoxazinoids during the Aerobic Deterioration of Maize Silage (Zea mays).

While plant-specialized metabolites can affect mammal health, their fate during the aerobic deterioration of crop silage remains poorly understood. In this study, we investigated the metabolization of benzoxazinoids (BXs) in silages of two maize genotypes (W22 wild type and bx1 mutant line) during aerobic deterioration. In W22 plants, concentrations of the aglucone BXs DIMBOA and HMBOA in silage decreased over time upon air exposure, while concentrations of MBOA and BOA increased. Mutant plants had low levels of BXs, which did not significantly vary over time. Aerobic stability was BX-dependent, as pH and counts of yeasts and molds were higher in W22 compared to that in bx1 silage. The nutrient composition was not affected by BXs. These preliminary results may be used to estimate the amounts of BXs provided to farm animals via silage feeding. However, further research is warranted under different harvest and storage conditions.

Examining How the Fermentation Medium Influences Thiol Expression and Its Perceived Aroma in Commercial Brewing Yeast Strains.

In Saccharomyces, the IRC7 gene encodes for a cysteine S-conjugate ß-lyase enzyme which can release polyfunctional thiols from their cysteinylated precursor forms, thereby promoting thiol aroma in beer. This study examined the thiol production of 10 commercial yeast strains in two different media, a hopped yeast extract-peptone-dextrose (YPD) medium and a 100% barley malt wort to explore how differences in yeast strain and medium conditions influence the release of polyfunctional thiols. 3-Sulfanylhexan-1-ol was most affected by medium conditions, and its concentrations were highest in wort fermentations. The higher nitrogen content and pH of the YPD medium relative to the wort fermentations were notable differences, and significant correlations between these variables and the extent of free thiol production were observed. A strong association existed between polyfunctional thiol concentrations and the fermentation-derived, malt, and hop-derived compounds 2-phenylethanol, ß-damascenone, and ß-ionone. The sensory impressions of thiol character in beer were influenced by the presence of other aromatic compounds such as esters and terpene alcohols, and aroma attributes such as "tropical" were not the most suitable for describing beers brewed with yeasts that fully express homozygous IRC7F. Sensory attributes "sweaty", "vegetal", and "overripe fruit" were more strongly associated with these strains.

Characterization of selected species of Pichia and Candida for their growth capacity in apple and grape must and their biofilm parameters.

Pichia and Candida species include biofilm-forming yeasts able to spoil foods and beverages. Strains belonging to 10 Pichia and Candida species isolated from apples, grape musts, and wines were analysed. They were subjected to molecular typing and characterized for their ability to grow and ferment must for cider and wine production, and for their biofilm properties. All strains grew similarly in apple and grape must. Glucose-fermenting strains displayed differentiated fermentation performances. Great variation in SO2 and ethanol sensitivity was observed among the strains. Pichia manshurica strains showed high tolerance to both molecules. Eleven and five surface-spreading biofilm (MAT) phenotypes were identified in solid and liquid media, respectively. Strains produced biofilms with variable thicknesses and widths in culture tubes. Cell adherence and aqueous-hydrocarbon biphasic hydrophobicity assays were carried out. Some Pichia manshurica and P. membranifaciens strains exhibited a high capacity to form a thick biofilm and had high cell adherence and hydrophobicity values. These strains could be more likely to colonize the internal surfaces of tanks. This study evidenced that some Pichia and Candida strains can proliferate during apple and grape must fermentation and may be detrimental the beverage quality, due to their specific biofilm properties.

Microbial cell factories based on filamentous bacteria, yeasts, and fungi.

BACKGROUND: Advanced DNA synthesis, biosensor assembly, and genetic circuit development in synthetic biology and metabolic engineering have reinforced the application of filamentous bacteria, yeasts, and fungi as promising chassis cells for chemical production, but their industrial application remains a major challenge that needs to be solved. RESULTS: As important chassis strains, filamentous microorganisms can synthesize important enzymes, chemicals, and niche pharmaceutical products through microbial fermentation. With the aid of metabolic engineering and synthetic biology, filamentous bacteria, yeasts, and fungi can be developed into efficient microbial cell factories through genome engineering, pathway engineering, tolerance engineering, and microbial engineering. Mutant screening and metabolic engineering can be used in filamentous bacteria, filamentous yeasts (Candida glabrata, Candida utilis), and filamentous fungi (Aspergillus sp., Rhizopus sp.) to greatly increase their capacity for chemical production. This review highlights the potential of using biotechnology to further develop filamentous bacteria, yeasts, and fungi as alternative chassis strains. CONCLUSIONS: In this review, we recapitulate the recent progress in the application of filamentous bacteria, yeasts, and fungi as microbial cell factories. Furthermore, emphasis on metabolic engineering strategies involved in cellular tolerance, metabolic engineering, and screening are discussed. Finally, we offer an outlook on advanced techniques for the engineering of filamentous bacteria, yeasts, and fungi.

Attenuating the triacylglycerol catabolism enhanced lipid production of Rhodotorula strain U13N3.

Enhancing the lipid production of oleaginous yeasts is conducive to cutting the cost of feedstock for biodiesel. To increase the lipid productivity of Rhodotorula sp. U13N3, genes involving lipid degradation were knocked out and fermentation conditions were investigated. Results of transcription analysis demonstrated that genes encoding the ATG15-like lipase (ATG15) and peroxisomal acyl-CoA oxidase (ACOX2) were upregulated significantly at the lipogenesis stage. When ATG15 and ACOX2 were knocked out separately from the genome by the CRISPR/Cas9 method, both ΔATG15 and ΔACOX2 mutants showed better lipid production ability than the parent strain. Flow cytometry and confocal microscopic analyses indicated that simultaneous the knockout of ATG15 and ACOX2 did not impact the cell viability, whereas the lipid production was enhanced markedly as the lipid yield increased by 67.03% in shake flasks. Afterward, the ΔATG15ΔACOX2 transformant (TO2) was cultivated in shake flasks in the fed-batch mode; the highest biomass and lipid yield reached 45.76 g/L and 27.14 g/L at 216 h, respectively. Better performance was achieved when TO2 was cultivated in the 1-L bioreactor. At the end of fermentation (180 h), lipid content, yield, yield coefficient, and productivity reached 65.53%, 27.35 g/L, 0.277 g/g glycerol, and 0.152 g/L/h, respectively. These values were at the high level in comparison with Rhodotorula strains cultivated in glycerol media. Besides, fermentation modes did not affect the fatty acid composition of TO2 significantly. In conclusion, blocking the lipid degradation was an applicable strategy to increase the lipid production of Rhodotorula strains without compromising their cell viability. KEY POINTS: • ATG15-like lipase and acyl-CoA oxidase (ACOX2) participated in lipid degradation. • Knockout of ATG15 and ACOX2 increased lipid productivity, and lipid yield coefficient. • Cell viability maintained at high level in the knockout mutants during fermentation.

Effect of commercial yeasts (Saccharomyces cerevisiae) on fermentation metabolites, phenolic compounds, and bioaccessibility of Brazilian fermented oranges.

Brazil is the largest producer of oranges worldwide, as well as one of the largest producers of orange juice. Alcoholic fermented beverages have been considered a marketable alternative for oranges. In this study, four S. cerevisiae commercial yeasts were evaluated for metabolites generated during orange juice (cv. 'Pêra D9') fermentation. Alcohols, sugars, and organic acids were evaluated by HPLC-DAD-RID during fermentation, and phenolic compounds were analyzed in fermented orange. Orange juice and fermented oranges were also subjected to digestion simulations. The yeasts presented an adequate fermentation activity, based on sugar consumption, and high ethanol (>10.5%) and glycerol (4.8-5.5 g/L) contents. The yeast strains T-58 and US-05 produced high levels of lactic acid. Phenolic compounds and antioxidant activity did not differ amongst yeasts, presenting hesperidin levels between 115 and 127 mg/L, respectively. The fermented orange showed a >70% bioaccessibility, compared to juice, especially for catechin, epigallocatechin-gallate, procyanidin-B2, rutin, and procyanidin-B1.

Strategy to reduce acetic acid in sugarcane bagasse hemicellulose hydrolysate concomitantly with xylitol production by the promising yeast Cyberlindnera xylosilytica in a bioreactor.

The yeast Cyberlindnera xylosilytica UFMG-CM-Y309 has been identified as a promising new xylitol producer from sugarcane bagasse hemicellulosic hydrolysate (SCHH). However, SCHH pretreatment process generates byproducts, which are toxic to cell metabolism, including furans, phenolic compounds, and carboxylic acids, such as acetic acid, typically released at high concentrations. This research aims to reduce acetic acid in sugarcane hemicellulose hydrolysate concomitantly with xylitol production by yeast strain Cy. xylosilytica UFMG-CM-Y309 in a bioreactor by strategically evaluating the influence of volumetric oxygen transfer coefficient (kLa) (21 and 35 h-1). Experiments were conducted on a bench bioreactor (2 L volumetric capacity) at different initial kLa values (21 and 35 h-1). SCHH medium was supplemented with rice bran extract (10 g L-1) and yeast extract (1 g L-1). Cy. xylosilytica showed high xylitol production performance (19.56 g L-1), xylitol yield (0.56 g g-1) and, maximum xylitol-specific production rate (µpmáx 0.20 gxylitol·g-1 h-1) at kLa value of 21 h-1, concomitantly slowing the rate of acetic acid consumption. A faster acetic acid consumption (100%) by Cy. xylosilytica was observed at kLa of 35 h-1, concomitantly with an increase in maximum cellular growth (14.60 g L-1) and reduction in maximum xylitol production (14.56 g L-1 and Yp/s 0.34 g g-1). This study contributes to pioneering research regarding this yeast performance in bioreactors, emphasizing culture medium detoxification and xylitol production.

Dynamic Transcriptome Analysis Reveals Transcription Factors Involved in the Synthesis of Ethyl Acetate in Aroma-Producing Yeast.

Ethyl acetate is an important flavor element that is a vital component of baijiu. To date, the transcription factors that can help identify the molecular mechanisms involved in the synthesis of ethyl acetate have not been studied. In the present study, we sequenced and assembled the Wickerhamomyces anomalus strain YF1503 transcriptomes to identify transcription factors. We identified 307 transcription factors in YF1503 using high-throughput RNA sequencing. Some transcription factors, such as C2H2, bHLH, MYB, and bZIP, were up-regulated, and these might play a role in ethyl acetate synthesis. According to the trend of ethyl acetate content, heat map results and STEM, twelve genes were selected for verification of expression levels using quantitative real-time PCR. This dynamic transcriptome analysis presents fundamental information on the transcription factors and pathways that are involved in the synthesis of ethyl acetate in aroma-producing yeast. Of significant interest is the discovery of the roles of various transcription factor genes in the synthesis of ethyl acetate.

Biosynthesis of bacterial cellulose nanofibrils in black tea media by a symbiotic culture of bacteria and yeast isolated from commercial kombucha beverage.

Bacterial cellulose has drawn the attention for its unique properties and applications including; medicine, pharmacy, food, agricultural, textile and electronics. The present study focused on the production of bacterial cellulose nanofibrils (BCNF) from black tea as cost effective alternative medium in addition to study the effect of gamma radiation on BCNF properties. A symbiotic culture of bacteria and yeast (SCOBY) were isolated from commercial Kombucha beverage and were identified as Acinetobacter lowffii and Candida krusei, respectively. The symbiotic culture was used for production of BCNF on Hestrin-Schramm (HS), black tea (BT) and modified BT media. BCNF was purified (0.5 N NaOH) and quantified by dry weight, yield and productivity determination. Characterization and effect of gamma radiation (5-25 kGy) on BCNF were studied using Scanning Electron Microscope (SEM), Fourier transform infrared (FTIR) and X-Ray Diffraction (XRD). The highest BCNF production was achieved using BT medium with 0.2% tea and 6.0% commercial sugar (with dry weight 4.77-4.61 g/l and productivity 68.14% and 65.85%, respectively). Supplementation of BT medium with 1% ethanol, 0.27% Na2HPO4 and 0.5% yeast extract individually, enhanced the BCNF production (7.85, 6.84 and 5.73 g/l), respectively. FTIR spectrum of BCNF from sugared water (SW), HS and BT showed similar structure with high purity. As a conclusion, gamma irradiation has no effect on the BCNF structure while showed different effects on its crystallinity index and size with the different doses. The changes in CrI were ranged between (17 and 23.5%), while the crystallinity size (Cs) was affected by gamma irradiation in a positive relationship where the crystalline size was decreased (33%) by exposure to 5 kGy then increased by increasing the dose of radiation reaching 25.7% at 25 kGy. SEM graphs showed the morphology of microbial culture and its symbiotic relationship in addition to the ultrafine structure of non-irradiated and irradiated BCNF.

Plant growth-promoting properties of the phosphate-solubilizing red yeast Rhodosporidium paludigenum.

Phosphorus (P) is one of the essential elements that are necessary for plant development and growth. However, the availability of soluble forms of P for plants in the soils is limited, because a large proportion of it is bound to soil constituents. Thus, the concentration of P available to plants at any time is very low and, moreover, its availability depends on the soil pH. As a solution, phosphate-solubilizing microorganisms (PSMs) are employed that render inorganic P available to plants in soluble form. Thus far, research into PSMs has been insufficient, and only few such organisms have been considered for exploitation as microbial fertilizer strains. The characteristics of plant growth promotion with the plant-PSMs coculture system remain to be elucidated. In the current study, we report on the isolate Rhodosporidium paludigenum JYC100 that exhibits good performance for solubilizing calcium phosphate. We found that it can be regulated by the amount of soluble phosphate. Furthermore, R. paludigenum JYC100 promotes plant growth under specific conditions (P deficiency, but with insoluble phosphate) in different media and soil pots. In contrast, the yeast Aureobasidium pullulans JYC104 exhibited weak phosphate-solubilizing capacities and no plant growth-promoting ability. Compared to control plants, the biomass, shoot height, and cellular inorganic P content of plants increased in plants cocultivated with R. paludigenum JYC100. In addition, histochemical GUS and qRT-PCR assays of phosphate starvation-induced (PSI) genes showed that the transcript levels of these PSI genes are decreased in the plants cocultured with R. paludigenum JYC100. These findings reflect the unique ability of R. paludigenum JYC100 to convert insoluble P compounds to plant-available P, thereby leading to growth promotion. Our study results highlight the use of yeasts as potential substitutes for inorganic phosphate fertilizers to meet the P demands of plants, which may eventually improve yields in sustainable agricultures.

Enhancement production of lipid and unsaturation of fatty acids in Cryptococcus humicola via addition of calcium ion.

Lipids synthesized by oleaginous yeasts are considered to be the best candidates for biodiesel production. Cryptococcus humicola as an oleaginous yeast accumulated lipid in cells. In order to optimize the conditions for lipid production, different carbon and nitrogen sources were used and metals were added into the medium. Ca2+ addition increased the lipid production greatly. Xylose and peptone were optimal carbon source and nitrogen source, respectively for lipid accumulation. Response surface experiment results revealed that the accumulation of lipid could be maximized when the xylose, peptone and Ca2+ concentration was 61 g/L, 4.31 g/L, 0.67 mmol/L. C16 and C18 fatty acid account for about 91% of the total fatty acids. The most abundant fatty acid was oleic acid (42.68%), followed by palmitic acid (29.7%) and stearic acid (13.87%). The addition of Ca2+ increased the content of unsaturated fatty acids (such as C16:1 and C18:1) and improved the unsaturation of fatty acids. Quantitative real time PCR analysis revealed that expression of genes related to lipid biosynthesis showed up-regulated by Ca2+ treatment. This study provided a strategy for increase in lipid production and content of unsaturated fatty acids.

Chemical characterization and biological effect of exopolysaccharides synthesized by Antarctic yeasts Cystobasidium ongulense AL101 and Leucosporidium yakuticum AL102 on murine innate immune cells.

The current study aimed to investigate exopolysaccharides (EPSs) produced by two Antarctic yeasts isolated from soil and penguin feathers samples collected on Livingston Island (Antarctica). The strains were identified as belonging to the species Leucosporidium yakuticum (LY) and Cystobasidium ongulense (CO) based on molecular genetic analysis. The EPS production was investigated using submerged cultivation. Different chemical, chromatographic, and spectral analyses were employed to characterize EPSs. LY accumulated 5.5 g/L biomass and 4.0 g/L EPS after 120 h of cultivation, while CO synthesized 2.1 g/L EPS at the end of cultivation, and the biomass amount reached 5.5 g/L. LY-EPS was characterized by a higher total carbohydrate content (80%) and a lower protein content (18%) by comparison with CO-EPS (62%, 30%). The LY-EPS mainly consisted of mannose (90 mol%), whereas CO-EPS had also glucose, galactose, and small amounts of uronic acids (8-5 mol%). Spectral analyses (FT-IR and 1D, 2D NMR) revealed that LY-EPS comprised a typical ß-(1 → 4)-mannan. Branched (hetero)mannan, together with ß/α-glucans constituted the majority of CO-EPS. Unlike LY-EPS, which had a high percentage of high molecular weight populations, CO-EPS displayed a large quantity of lower molecular weight fractions and a higher degree of heterogeneity. LY-EPS (100 ng/mL) elevated significantly interferon gamma (IFN-γ) production in splenic murine macrophages and natural killer (NK) cells. The results indicated that newly identified EPSs might affect IFN-γ signaling and in turn, might enhance anti-infectious responses. The data obtained also revealed the potential of EPSs and yeasts for practical application in biochemical engineering and biotechnology.

Assessment of Cosmetic and Dermatological Properties and Safety of Use of Model Skin Tonics with Kombucha-Fermented Red Berry Extracts.

Kombucha is a health-promoting beverage that is produced by fermenting sweetened tea using symbiotic cultures of bacteria belonging to the genus Acetobacter, Gluconobacter, and yeast of the genus Saccharomyces. This study compared the cosmetic and dermatological properties of the extracts of the following redberries: R. rubrum, F. vesca, and R. idaeus, and their ferments, which were obtained by fermentation for 10 and 20 days using tea fungus. For this purpose, the fermented and non-fermented extracts were compared in terms of their chemical composition using the HPLC/ESI-MS chromatographic method, demonstrating the high content of biologically active compounds that were present in the ferments. The antioxidant activity of the tested samples was evaluated using DPPH and ABTS tests, as well as by evaluating the scavenging of the external and intracellular free radicals. The cytotoxicity of the extracts and the ferments, as well as the cosmetic formulations, were also determined by conducting Alamar Blue and Neutral Red tests assessing the cell viability and metabolism using skin cell lines: fibroblasts and keratinocytes. In addition, application tests were conducted showing the positive effects of the model cosmetic tonics on the TEWL, the skin hydration, and the skin pH. The results indicate that both the extracts and the ferments that were obtained from kombucha can be valuable ingredients in cosmetic products.

CRZ1 transcription factor is involved in cell survival, stress tolerance, and virulence in fungi.

The calcineurin-CRZ1 signaling pathway is conserved from yeasts to humans, and is involved in survival, tolerance to biotic and abiotic stress conditions, virulence, and drug resistance. The primary target of calcineurin is Crz1p in yeasts and nuclear factor of activated T-cells (NFAT) in mammals. Calcineurin regulates the expression of various genes involved in cell wall regeneration, ionic homeostasis, virulence, and other cellular processes. Another protein called regulator of calcineurin (RCN) also regulates calcineurin functions. This review discusses the structure, functions, and regulations of CRZ1 in fungi and related organisms. We have also discussed the role of CRZ1 in pathogenicity and virulence in fungi, plants, and animals.

Oleaginous yeasts: Time to rethink the definition?

Oleaginous yeasts are typically defined as those able to accumulate more than 20% of their cell dry weight as lipids or triacylglycerides. Research on these yeasts has increased lately fuelled by an interest to use biotechnology to produce lipids and oleochemicals that can substitute those coming from fossil fuels or offer sustainable alternatives to traditional extractions (e.g., palm oil). Some oleaginous yeasts are attracting attention both in research and industry, with Yarrowia lipolytica one of the best-known and studied ones. Oleaginous yeasts can be found across several clades and different metabolic adaptations have been found, affecting not only fatty acid and neutral lipid synthesis, but also lipid particle stability and degradation. Recently, many novel oleaginous yeasts are being discovered, including oleaginous strains of the traditionally considered non-oleaginous Saccharomyces cerevisiae. In the face of this boom, a closer analysis of the definition of "oleaginous yeast" reveals that this term has instrumental value for biotechnology, while it does not give information about distinct types of yeasts. Having this perspective in mind, we propose to expand the term "oleaginous yeast" to those able to produce either intracellular or extracellular lipids, not limited to triacylglycerides, in at least one growth condition (including ex novo lipid synthesis). Finally, a critical look at Y. lipolytica as a model for oleaginous yeasts shows that the term "oleaginous" should be reserved only for strains and not species and that in the case of Y. lipolytica, it is necessary to distinguish clearly between the lipophilic and oleaginous phenotype.

The Role of Microorganisms and Carbon-to-Nitrogen Ratios for Microbial Protein Production from Bioethanol.

With industrial agriculture increasingly challenging our ecological limits, alternative food production routes such as microbial protein (MP) production are receiving renewed interest. Among the multiple substrates so far evaluated for MP production, renewable bioethanol (EtOH) is still underexplored. Therefore, the present study investigated the cultivation of five microorganisms (2 bacteria, 3 yeasts) under carbon (C), nitrogen (N), and dual C-N-limiting conditions (molar C/N ratios of 5, 60, and 20, respectively) to evaluate the production (specific growth rate, protein and biomass yield, production cost) as well as the nutritional characteristics (protein and carbohydrate content, amino acid [AA] profile) of MP production from bioethanol. Under C-limiting conditions, all the selected microorganisms showed a favorable AA profile for human nutrition (average AA score of 1.5 or higher), with a negative correlation between protein content and growth rate. Maximal biomass yields were achieved under conditions where no extracellular acetate was produced. Cyberlindnera saturnus and Wickerhamomyces anomalus displayed remarkably high biomass yields (0.40 to 0.82 g cell dry weight [CDW]/g EtOHconsumed), which was reflected in the lowest estimated biomass production costs when cultivated with a C/N ratio of 20. Finally, when the production cost was evaluated on a protein basis, Corynebacterium glutamicum grown under C-limiting conditions showed the most promising economic outlook. IMPORTANCE The global protein demand is rapidly increasing at rates that cannot be sustained, with projections showing 78% increased global protein needs by 2050 (361 compared to 202 million tonprotein/year in 2017). In the absence of dedicated mitigation strategies, the environmental effects of our current food production system (relying on agriculture) are expected to surpass the planetary boundaries-the safe operating space for humanity-by 2050. Here, we illustrate the potential of bioethanol-renewable ethanol produced from side streams-as a main resource for the production of microbial protein, a radically different food production strategy in comparison to traditional agriculture, with the potential to be more sustainable. This study unravels the kinetic, productive, and nutritional potential for microbial protein production from bioethanol using the bacteria Methylorubrum extorquens and Corynebacterium glutamicum and the yeasts Wickerhamomyces anomalus, Cyberlindnera saturnus, and Metschnikowia pulcherrima, setting the scene for microbial protein production from renewable ethanol.

Isolation and test of novel yeast strains with lignin usage capability and phenolic compound resistance.

Five yeast fungi strains (i.e., two Cryptococcus albidus, one Candida guillermondii, and two Candida tropicalis) were isolated from sugarcane and tested for their use of lignin as sole carbon source and their potential to grow in the presence of phenol and phenol derivatives (i.e., pentachlorophenol and p-nitrophenol). The full set of isolated yeasts showed ligninolytic activity, achieving at least 36% lignin degradation after 25 days. The C. albidus JS-B1 strain had the highest ligninolytic activity, achieving 27% lignin degradation within 4 days. This increased activity was associated with the production of ligninolytic laccase enzymes. All the tested yeast fungi strains showed growth in the presence of high concentrations of phenolic compounds (i.e., 900 mg/L phenol, 200 mg/L p-nitrophenol, 50 mg/L pentachlorophenol) and showed significant potential for lignin and lignin by-product degradation. Each of these five strains has the potential to be used in biological treatment processes for contaminated effluents from paper pulping and bleaching or phenol and phenol-derivative biodegradation processes for other industrial wastewater effluents.

Antifungal cultures and metabolites of lactic acid bacteria for use in dairy fermentations.

Fungal spoilage limits the shelf life of fermented dairy products. To address the problem, this study explores the potential of lactic acid bacteria as antifungal adjunct cultures in dairy matrices. Strains of lactic acid bacteria (113) representing 19 species were screened for their activity against Penicillium caseifulvum, Aspergillus clavatus and Mucor racemosus in modified MRS medium, milk, and yogurt. Strains of Lactiplantibacillus plantarum, Furfurilactobacillus milii, and Lentilactobacillus parabuchneri inhibited the growth of mycelial fungi. The inhibitory effects of lactic acid bacteria against yeasts were also determined in yogurt with Candida sake, Saccharomyces bayanus, and Torulaspora delbrueckii as challenge strains. The inhibition of yeasts by lactic acid bacteria was strain-specific and unrelated to the activity towards mycelial fungi. Organic acids and hydroxy fatty acids were quantified by liquid chromatograph coupled with refractive index detector and tandem mass spectrometry, respectively. Principal component analysis indicated 10-OH 18: 1 fatty acids and acetate are the main antifungal metabolites and explained over 50 % of the antifungal activity. The correlation analysis of metabolites and mold-free shelf life of milk and yogurt confirmed the role of these compounds. The genomic study analysed genes related to the production of major antifungal metabolites and predicted the formation of 1,2-propanediol and acetate but not of hydroxy unsaturated fatty acids. The findings provide new perspectives on the selection of antifungal strains, the characterization of antifungal metabolites and the exploration of antifungal mechanisms among different species.