Comprehensive Exploration of the Growth and Lipid Synthesis Phases of T. oleaginosus Cultures Implementing Design of Experiments and Response Surface Methodology.

Trichosporon oleaginosus is an unconventional oleaginous yeast distinguished by its remarkable capacity to accumulate lipids in excess of 70% of its dry weight, particularly when cultivated in nitrogen-restricted conditions with ample carbon sources. A pivotal question that arises pertains to the nutrient dynamics in the culture medium, which give rise to both the excessive lipid content and corresponding lipid concentration. While previous research has predominantly focused on evaluating the impact of the initial carbon-to-nitrogen (C/N) ratio on lipid production, the precise critical thresholds of glucose and ammonium sulfate ((NH4)2SO4) at which growth and intracellular lipid production are either stimulated or impeded remain inadequately defined. This study employs an experimental design and response surface methodology to investigate the complex mechanism of lipid accumulation and its interaction with cellular growth. Application of the aforementioned methodologies resulted in the production of 10.6 g/L of microbial oil in batch cultures under conditions that correspond to a C/N ratio of 76. However, the primary objective is to generate knowledge to facilitate the development of efficient fed-batch cultivation strategies that optimize lipid production exclusively employing inorganic nitrogen sources by finely adjusting carbon and nitrogen levels. The intricate interaction between these levels is comprehensively addressed in the present study, while it is additionally revealed that as glucose levels rise within a non-inhibitory range, lipid-free biomass production decreases while lipid accumulation simultaneously increases. These findings set the stage for further exploration and the potential development of two-stage cultivation approaches, aiming to fully decouple growth and lipid production. This advancement holds the promise of bringing microbial oil production closer to commercial viability.

Enzymatic hydrolysis for the systematic production of second-generation glucose from the dual polysaccharide reserves of an anti-pollutant plant.

In this study, the anti-pollutant macrophyte Typha domingensis is exploited for the production of highly concentrated second-generation glucose. A two-stage starch and cellulose enzymatic hydrolysis process is compared for the first time with a single-stage simultaneous starch and cellulose hydrolysis approach, with the former achieving enhanced glucose production, making it more promising for large-scale deployment. The proposed two-stage process is optimized via the Box-Behnken response surface methodology achieving glucose yield values of 74.4% and 71.7% with respect to the starch and cellulose fraction, respectively. Elevated shaking rates are shown to exert a positive effect on both starch and cellulose enzymatic hydrolysis only under high initial substrate concentrations and high initial enzyme to substrate ratios, indicating the importance of accounting for the synergies between key process variables when aiming to increase glucose production. The findings of the presented experimental framework aspire to support future scale-up studies and techno-economic assessments.

Model-based intensification of a fed-batch microbial process for the maximization of polyhydroxybutyrate (PHB) production rate.

An integrated metabolic-polymerization-macroscopic model, describing the microbial production of polyhydroxybutyrate (PHB) in Azohydromonas lata bacteria, was developed and validated using a comprehensive series of experimental measurements. The model accounted for biomass growth, biopolymer accumulation, carbon and nitrogen sources utilization, oxygen mass transfer and uptake rates and average molecular weights of the accumulated PHB, produced under batch and fed-batch cultivation conditions. Model predictions were in excellent agreement with experimental measurements. The validated model was subsequently utilized to calculate optimal operating conditions and feeding policies for maximizing PHB productivity for desired PHB molecular properties. More specifically, two optimal fed-batch strategies were calculated and experimentally tested: (1) a nitrogen-limited fed-batch policy and (2) a nitrogen sufficient one. The calculated optimal operating policies resulted in a maximum PHB content (94% g/g) in the cultivated bacteria and a biopolymer productivity of 4.2 g/(l h), respectively. Moreover, it was demonstrated that different PHB grades with weight average molecular weights of up to 1513 kg/mol could be produced via the optimal selection of bioprocess operating conditions.

Microbial production of polyhydroxybutyrate with tailor-made properties: an integrated modelling approach and experimental validation.

The microbial production of polyhydroxybutyrate (PHB) is a complex process in which the final quantity and quality of the PHB depend on a large number of process operating variables. Consequently, the design and optimal dynamic operation of a microbial process for the efficient production of PHB with tailor-made molecular properties is an extremely interesting problem. The present study investigates how key process operating variables (i.e., nutritional and aeration conditions) affect the biomass production rate and the PHB accumulation in the cells and its associated molecular weight distribution. A combined metabolic/polymerization/macroscopic modelling approach, relating the process performance and product quality with the process variables, was developed and validated using an extensive series of experiments and measurements. The model predicts the dynamic evolution of the biomass growth, the polymer accumulation, the consumption of carbon and nitrogen sources and the average molecular weights of the PHB in a bioreactor, under batch and fed-batch operating conditions. The proposed integrated model was used for the model-based optimization of the production of PHB with tailor-made molecular properties in Azohydromonas lata bacteria. The process optimization led to a high intracellular PHB accumulation (up to 95% g of PHB per g of DCW) and the production of different grades (i.e., different molecular weight distributions) of PHB.

A combined metabolic/polymerization kinetic model on the microbial production of poly(3-hydroxybutyrate).

In the present work, an integrated dynamic metabolic/polymerization kinetic model is developed for the prediction of the intracellular accumulation profile and the molecular weight distribution of poly(3-hydroxybutyrate) (P(3HB) or PHB) produced in microbial cultures. The model integrates two different length/time scales by combining a polymerization kinetic model with a metabolic one. The bridging point between the two models is the concentration of the monomer unit (i.e. 3-hydroxybutyryl-CoA) produced during the central aerobic carbon metabolism. The predictive capabilities of the proposed model are assessed by the comparison of the calculated biopolymer concentration and number average molecular weight with available experimental data obtained from batch and fed-batch cultures of Alcaligenes eutrophus and Alcaligenes latus. The accuracy of the proposed model was found to be satisfactory, setting this model a valuable tool for the design of the process operating profile for the production of different polymer grades with desired molecular properties.