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

Abstract

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.

Keywords: C/N ratio parameterization; Cryptococcus curvatus; design of experiments; inorganic nitrogen source; lipogenesis; media composition optimization; microbial lipids; oleaginous yeast; response surface methodology (RSM).

Figure 1

Main effect plots of the initial glucose concentration on the (a) total biomass and (b) lipid content for the studied (NH₄)₂SO₄ concentrations after 96 h of cultivation.

Figure 2

The response surface plot of the (a) biomass concentration, (b) lipid content, and (c) lipid free-biomass concentration as a function of glucose and (NH₄)₂SO₄. The red bullets represent the experimentally measured process performance under the model-derived optimal conditions (text boxes refer to the coordinates of the experimental points, where the x-axis represents the glucose concentration and y-axis represents the (NH₄)₂SO₄ concentration in the culture medium).

Figure 3

Comparison of the key performance indices between the batch cultures of T. oleaginosus grown on glucose and (NH₄)₂SO₄ after 96 h of cultivation. Table entries refer to the initial medium compositions in terms of glucose and (NH₄)₂SO₄ in g/L and the corresponding C/N ratio.

Figure 4

Correlation between the (a) biomass concentration, (b), lipids concentration, and (c) lipid-free biomass concentration and glucose in cultures of T. oleaginosus grown on organic (0.26 g/L) and inorganic (0.21 g/L) nitrogen sources. Data from organic nitrogen cultures were obtained from [24].

Figure 5

Comparison of the time profiles of the (a) biomass concentration, (b) lipids concentration, (c) lipid free-biomass concentration, (d) lipid content, (e) NH₄⁺ concentration, and (f) glucose concentration during the batch fermentation of T. oleaginosus in a lab-scale bioreactor using two culture media, C/N 38 and C/N 85.