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

Valorization of lignocellulosic biomass, such as Spent Mushroom Substrate (SMS), as an alternative substrate for biogas production could meet the increasing demand for energy. In view of this, the present study aimed at the biotechnological valorization of SMS for biogas production. In the first part of the study, two SMS chemical pretreatment processes were investigated and subsequently combined with thermal treatment of the mentioned waste streams. The acidic chemical hydrolysate derived from the hydrothermal treatment, which yielded in the highest concentration of free sugars (≈36 g/100 g dry SMS, hydrolysis yield ≈75% w/w of holocellulose), was used as a potential feedstock for biomethane production in a laboratory bench-scale improvised digester, and 52 L biogas/kg of volatile solids (VS) containing 65% methane were produced in a 15-day trial of anaerobic digestion. As regards the alkaline hydrolysate, it was like a pulp due to the lignocellulosic matrix disruption, without releasing additional sugars, and the biogas production was delayed for several days. The biogas yield value was 37 L/kg VS, and the methane content was 62%. Based on these results, it can be concluded that SMS can be valorized as an alternative medium employed for anaerobic digestion when pretreated with both chemical and hydrothermal hydrolysis.

Microalgae have gained significant attention as sustainable sources of high value compounds, such as bioactive polysaccharides that are usually rich in sulfated groups and exhibit antioxidant properties. Here, 14 new microalgae strains of the genera Tetraselmis, Dunaliella, and Nannochloropsis, isolated from Greek coastal lagoons were analyzed to quantify and characterize their polysaccharide content. Heterogeneity was observed regarding the content of their total sugars (5.5–40.9 g/100 g dry biomass). The strains with a total sugar content above 20% were analyzed concerning the content of total, α- and β-glucans. Tetraselmis verrucosa f. rubens PLA1-2 and T. suecica T3-1 were rich in β-glucans (11%, and 8.1%, respectively). The polysaccharides of the two Tetraselmis strains were isolated and they were mainly composed of glucose and galactose. The isolated polysaccharides were fractionated using ion-exchange-chromatography. The anionic fraction from T. verrucosa f. rubens PLA1-2 was rich in sulfated polysaccharides, had antioxidant capacity, and exhibited healing properties. The anionic polysaccharides from the two Tetraselmis strains did not negatively influence the viability of human cells, while exhibiting antiviral properties against the replication of Hepatitis C Virus (HCV), with median efficient concentrations (EC50) at a range of 210–258 μg/mL.

A study on the ability of new microbial strains to assimilate biodiesel-derived glycerol at low purity (75% w/w) and produce extra-cellular platform chemical compounds of major interest was carried out. After screening several bacterial strains under different fermentation conditions (e.g., pH, O₂ availability, glycerol purity), three of the screened strains stood out for their high potential to produce valued-added products such as 2,3-butanediol (BDO), 1,3-propanediol (PDO) and ethanol (EtOH). The results indicate that under aerobic conditions, Klebsiella oxytoca ACA-DC 1581 produced BDO in high yield (Y_BDO/Gly = 0.46 g/g, corresponding to 94% of the maximum theoretical yield; Y_mt) and titer, while under anaerobic conditions, Citrobacter freundii NRRL-B 2645 and Enterobacter ludwigii FMCC-204 produced PDO (Y_PDO/Gly = 0.56 g/g, 93% of Y_mt) and EtOH (Y_EtOH/Gly = 0.44 g/g, 88% of Y_mt), respectively. In the case of C. freundii, the regulation of pH proved to be mandatory, due to lactic acid production and a subsequent drop of pH that resulted in fermentation ceasing. In the fed-batch culture of K. oxytoca, the BDO maximum titer reached almost 70 g/L, the Y_BDO/Gly and the mean productivity value (Pr_BDO) were 0.47 g/g and 0.4 g/L/h, respectively, while no optimization was imposed. The final BDO production obtained by this wild strain (K. oxytoca) is among the highest in the international literature, although the bioprocess requires optimization in terms of productivity and total cost. In addition, for the first time in the literature, a strain from the species Hafnia alvei (viz., Hafnia alvei ACA-DC 1196) was reported as a potential BDO producer. The strains as well as the methodology proposed in this study can contribute to the development of a biorefinery that complements the manufacture of biofuels with high-value biobased chemicals.

Microbial cultures repurposing organic industrial residues for value-added metabolite production is pivotal for sustainable resource use. Highlighting polyunsaturated fatty acids (PUFAs), particularly gamma-linolenic acid (GLA), renowned for their nutritional and therapeutic value. Notably, Zygomycetes’ filamentous fungi harbor abundant GLA-rich lipid content, furthering their relevance in this approach. In this study, the strain C. elegans NRRL Y-1392 was evaluated for its capability to metabolize glycerol and produce lipids rich in GLA under different culture conditions. Various carbon-to-nitrogen ratios (C/N = 11.0, 110.0, and 220.0 mol/mol) were tested in batch-flask cultivations. The highest GLA production of 224.0 mg/L (productivity equal to 2.0 mg/L/h) was observed under nitrogen excess conditions, while low nitrogen content promoted lipid accumulation (0.59 g of lipids per g of dry biomass) without yielding more PUFAs and GLA. After improving the C/N ratio at 18.3 mol/mol, even higher PUFA (600 mg/L) and GLA (243 mg/L) production values were recorded. GLA content increased when the fungus was cultivated at 12 °C (15.5% w/w compared to 12.8% w/w at 28 °C), but productivity values decreased significantly due to prolonged cultivation duration. An attempt to improve productivity by increasing the initial spore population did not yield the expected results. The successful scale-up of fungal cultivations is evidenced by achieving consistent results (compared to flask experiments under corresponding conditions) in both laboratory-scale (Working Volume—Vw = 1.8 L; C/N = 18.3 mol/mol) and semi-pilot-scale (Vw = 15.0 L; C/N = 110.0 mol/mol) bioreactor experiments. To the best of our knowledge, cultivation of the fungus Cunninghamella elegans in glycerol-based substrates, especially in 20 L bioreactor experiments, has never been previously reported in the international literature. The successful scale-up of the process in a semi-pilot-scale bioreactor illustrates the potential for industrializing the bioprocess.