Fermentation

Ricotta cheese whey (RCW) is a by-product with nutritional potential, but its use in the human diet is limited due to its high salinity. Chlorella vulgaris can use RCW as a substrate to enhance biomass productivity. The aim of this work was to evaluate different conditions for C. vulgaris growth in RCW, during scaling-up analysis. After preliminary assays to select growth conditions, two systems were prepared as follows: 500 mL Erlenmeyer flasks (control-system) and a 3 L Bioreactor. Microfiltrated RCW was used as a substrate for C. vulgaris LPMA39 production. Biomass was measured and productivity at 96 h, cell growth kinetics behaviour, biomass biochemical characterisation, and the efficiency of nutrient removal were determined. Both systems presented the same biomass concentration at 96 h (2.2–2.8 g·L⁻¹) and productivity (0.021–0.027 g·L⁻¹·h⁻¹). Nevertheless, 11 h lag-period for cell adaptation to the 3 L Bioreactor was required; thereafter, cells grew faster (µ_max: 0.32 ± 0.08 h⁻¹) than control-system. Finally, slight but significantly lower C_max: 2.14 ± 0.08 was obtained when comparing it to control-system. Lipids, proteins, and pigment contents decreased by the scaling-up; meanwhile, higher reduction in chemical oxygen demand (COD), total phosphorus, and total nitrogen were recorded in the 3 L Bioreactor. Identifying the operating conditions that improve C. vulgaris performance in non-diluted RCW remains a challenge from a sustainability standpoint.

In this study, nine different Propionibacterium freudenreichii strains were isolated from Kars Gravyer produced by traditional methods in Turkey and identified by sequencing the 16S–23S intergenic region using species-specific primers. The isolated strains were examined in vitro for the presence of the β-galactosidase enzyme, autoaggregation ability, sensitivity against eight selected antibiotics and survivability under harsh conditions in order to determine their potential probiotic properties. After probiotic potentials were evaluated, an experimental design was made to optimize the production of vitamin B12 in a 3 L glass bioreactor P. freudenreichii NUV774. While all strains showed similar resistance (92–98%) to gastric juice (0.3% pepsin, pH 3.0), they showed resistance to intestinal fluid (0.1% pancreatin, 0.3% bile salt, pH 8.0) between 60% and 92%. It was determined that the viability after 3 and 6 h of incubation in 0.5% and 1% bile salt differed between strains. All isolates exhibited resistance to ciprofloxacin, ampicillin, and trimethoprim–sulphamethoxazole; however, most were sensitive to ofloxacin. Overall, P. freudenreichii strains showed resistance to the gastrointestinal tract, tolerance to pH 3.0, and high tolerance to bile salts. As a result of optimization, maximum vitamin B12 production was found to be 156.8 mg/L. The optimum operating conditions were calculated as temperature = 36.9 °C, aeration = 2.430 vvm, and agitation = 159.120 rpm. Hence, P. freudenreichii, as future probiotic strain candidates, will offer an alternative source to Lactobacillus, Bifidobacterium and some Bacillus spp. In addition, this study denoted that the alteration of the production of active vitamin B12 by P. freudenreichii occurs in a strain-dependent manner.

High organic loading is known to destabilize anaerobic digestion (AD). This study compared bioaugmentation and pH adjustment under increasing organic loading rate (OLR: 2.0, 4.0 and 6.0 gVS L⁻¹ d⁻¹), focusing on the responses of microbial structure, metabolic pathways, and energy metabolism. Results demonstrated that bioaugmentation maintained stable methane production of 400.54 ± 10.08 and 374.15 ± 24.32 mL·g-VS⁻¹ at 4.0 and 6.0 gVS L⁻¹ d⁻¹, respectively, whereas control and pH-adjusted reactors failed at 4.0 gVS L⁻¹ d⁻¹. The acidified system restored methane yield from 86.30 to 382.13 mL·g-VS⁻¹ after bioaugmentation, whereas pH adjustment and feeding cessation were ineffective, failing to produce methane within 25 days. Microbial analysis showed bioaugmentation enriched Methanosarcina, enhanced hydrogenotrophic/methylotrophic methanogenesis, and strengthened syntrophy with syntrophic propionate-oxidizing bacteria (SPOB), reducing volatile fatty acid accumulation via reinforced syntrophic propionate/butyrate oxidation. Upregulation of osmoregulatory (nha, kdp, proP) and energy metabolism genes (eha, mvh, hdr) maintained osmotic balance and energy supply under high load. In contrast, pH adjustment downregulated SPOB and propionate oxidation genes, causing persistent acid inhibition. This study elucidated the distinct regulatory effects of bioaugmentation and pH adjustment on high-load AD systems, providing actionable strategies for both maintaining operational stability in high-load reactors and recovering methanogenesis in acid-inhibited systems.