Search

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.

Agricultural activities produce large quantities of organic byproducts and waste rich in lignocellulosic materials, which are not sufficiently utilized. In this study, alternative agricultural waste products, namely, spent mushroom substrate (SMS) from the cultivation of edible Pleurotus ostreatus mushrooms and the roots of leafy vegetables from hydroponic cultivation (HRL), were evaluated for their potential to be used as substrates for the cultivation of Pleurotus citrinopileatus and their effects on the quality, the nutritional value, the chemical properties (lipid, protein, carbohydrate, ash, fatty acid and carbohydrate composition) and the bioactive content (total phenolic compounds and antioxidant activity) of produced mushrooms. SMS and HRL (in different ratios with and without additives) and wheat straw with additives (WS-control) were used. During incubation, the linear growth rate of the mycelium (Kr, mm/day) was measured and used for screening. Mushroom cultivation took place in bags, where several characteristics were examined: earliness (duration between the day of substrate inoculation and the day of first harvest) and biological efficiency (B.E. %, the ratio of the weight of fresh mushrooms produced per dry weight of the substrate × 100). Furthermore, this study aimed to investigate the effect of the protein extract (PE) and carbohydrate extract (CE) of P. citrinopileatus after in vitro digestion (fraction less than 3kDa: PE-DP-3; digestate fraction: CE-D, respectively) on the expression of antioxidant-related genes in the THP-1 cell line. The results showed that mushrooms grown on SMS 50%-HRL 40% had the fastest growth (6.1 mm/d) and the highest protein and lipid contents (34.7% d.w.; 5.1% d.w.). The highest B.E. (73.5%), total carbohydrate (65.7%) and total phenolic compound (60.2 mg GAE/g d.w.) values were recorded on the control substrate. Antioxidant activity was observed in all extracts; the total flavonoid content was low in the samples, and the maximum total triterpene value was detected in SMS 80%-HRL 20% (9.8 mg UA/g d.w.). In all mushrooms, linoleic acid (C18:2) was the main fatty acid (above 60%), and fructose was the dominant individual saccharide. In the investigation of the regulation pathway, NFE2L2 gene expression was upregulated only in the SMS 60%-HRL 40% intervention during incubation with CE-D samples. Additionally, the transcription levels of antioxidant-related genes, SOD1, CAT, HMOX1 and GSR, were increased in the SMS 60-30% intervention. Compared to WS, the alternative substrates are observed to trigger a pathway concerning CE that may resist oxidative stress. This study supports the utilization of agricultural byproducts through sustainable and environmentally friendly practices while simultaneously producing high-value-added products such as mushrooms. Therefore, alternative substrates, particularly those containing HRL, could serve as natural sources of antioxidant potential.

The present work examined the production of single-cell protein (SCP) by a newly isolated strain of Kluyveromyces marxianus EXF-5288 under increased lactose concentration of deproteinized cheese whey (DCW) and different temperatures (in °C: 20.0, 25.0, 30.0 and 35.0). To the best of the authors' knowledge, this is the first report examining the ability of Kluyveromyces marxianus species to produce SCP at T = 20.0 °C. Different culture temperatures led to significant differences in the strain's growth, while maximum biomass and SCP production (14.24 ± 0.70 and 6.14 ± 0.66 g/L, respectively) were observed in the cultivation of K. marxianus strain EXF-5288 in shake-flask cultures at T = 20.0 °C. Increased DCW lactose concentrations (35.0-100.0 g/L) led to increased ethanol production (Eth max = 35.5 ± 0.2 g/L), suggesting that K. marxianus strain EXF-5288 is "Crabtree-positive". Batch-bioreactor trials shifted the strain's metabolism to alcoholic fermentation, favoring ethanol production. Surprisingly, K. marxianus strain EXF-5288 was able to catabolize the produced ethanol under limited carbon presence in the medium. The dominant amino acids in SCP were glutamate (15.5 mg/g), aspartic acid (12.0 mg/g) and valine (9.5 mg/g), representing a balanced nutritional profile.

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., Competing Interests: The authors declare no conflict of interest.