Search Results (146)

Thraustochytrids are promising alternatives for the production of docosahexaenoic acid (DHA; C22:6 n-3), a long-chain polyunsaturated fatty acid with health benefits. For practical application of this oleaginous microorganism, an efficient cultivation method to enhance DHA production is required, which relies on several factors that support cell growth, lipid accumulation, and lipid turnover. In this study, the robust submerged fermentation of an acid- and high-temperature-tolerant strain of Aurantiochytrium limacinum was investigated. Under controlled temperature and acidic conditions (pH 4.5 and 30 °C), glucose and peptone were the best carbon and nitrogen sources for enhancing biomass and DHA production, respectively, with a glucose concentration of 60 g/L and a C/N ratio of 24 being optimal for DHA production. Applying an aeration rate of 2 vvm and an agitation speed of 300 rpm using a combination of a ring sparger and pitch-blade impeller in a stirred-tank bioreactor improved DHA production using intermittent fed-batch fermentation. The highest DHA titer was obtained at 3.01 g/L, and the DHA content in biomass was 10.69% (w/w) after intermittent feeding of cassava starch hydrolysate as the substrate. Full article

Palmitoleic acid (POA; 16:1 n-7 or cis-9 16:1) is a bioactive monounsaturated fatty acid (FA) with emerging metabolic and skin-health relevance, yet conventional botanical and animal sources provide limited and variable levels. Here we report on the development of a high-yield POA product platform in the heterotrophic microalga Prototheca moriformis through targeted genetic engineering. A Δ9-fatty acid desaturase from Macadamia integrifolia (MiSAD1618) was integrated using a phosphite-based selection system. Primary screening identified stable transformants producing up to 54% POA of total fatty acids, compared to 0.8% in the parental strain. In 1 L shake-flask cultivation, POA reached up to 58.2% of total fatty acids. In a 1 L fed-batch fermentation, the engineered strain accumulated 47.8 g/L of lipids with 43.5% POA after 96 h of fermentation, corresponding to 20.8 g/L of POA. GC–MS analysis of 4,4-dimethyloxazoline (DMOX) derivatives confirmed that the major 16:1 isomer was 16:1 n-7 (Δ9). Together, these results establish P. moriformis as a scalable fermentation platform for producing POA-rich oil and highlight its potential as an efficient alternative source of POA, providing a foundation for further strain and process optimization toward commercial production. Full article

Oleaginous yeasts are promising microbial platforms for lipid production from non-conventional carbon sources; however, acetate utilization is frequently constrained by physiological limitations associated with culture pH. In this study, acetate utilization, biomass formation, and lipid production by Lipomyces starkeyi were investigated under flask and fed-batch cultivation to evaluate the influence of culture pH and pH control strategy. Statistically supported flask-scale experiments demonstrated that acetate concentration and cultivation time significantly affected acetate consumption, biomass formation, volumetric lipid concentration, and culture pH, with excessive acetate loading resulting in culture alkalization, incomplete substrate utilization, and reduced process performance. Although volumetric lipid concentration increased with increasing acetate concentration, lipid content and fatty acid composition remained unchanged, indicating that enhanced lipid production was primarily attributable to increased biomass formation rather than to changes in lipid biosynthesis. Fed-batch cultivation under different pH-control strategies provided qualitative insights into the relationships among pH regulation, acetate availability, and lipid accumulation under controlled fermentation conditions. While lipid accumulation was observed under both HCl-based and acetic acid-based pH control, differences in pH stability and cumulative acetate availability were associated with distinct patterns of lipid production. Collectively, these results identify culture pH as a critical physiological parameter influencing acetate utilization and lipid accumulation in L. starkeyi and suggest that coordinated pH control and carbon feeding strategies may improve the robustness of acetate-based lipid production processes. Further replicated fed-batch studies will be required to quantitatively validate these trends and support industrial applications. Full article

Haemophilus influenzae b (Hib) is a pathogenic bacterium that causes meningitis worldwide, mainly in children less than two years old. The capsular polysaccharide b (PRP) is an essential antigen for vaccine formulation. This study aimed to develop a high-yield, technically accessible production strategy for PRP production to facilitate vaccine manufacturing in non-profit laboratories. Various fed-batch cultivation strategies were evaluated to address metabolic limitations and identify a robust, simplified process suitable for seamless scale-up to pilot scale. Glucose limitation strategies did not reduce inhibitory acetic acid accumulation due to deficiencies in Hib’s respiratory chain, whereas oxygen availability was identified as critical parameter. Increasing the specific air flow from 0.5 to 1.0 vvm in constant fed-batch (Cfb) resulted in a 33% yield increase, reaching 1706.40 mg PRP.L⁻¹. However, the highest PRP concentration was achieved using exponential fed-batch with cell recycling (EfbCR), resulting in 1879.28 mg PRP.L⁻¹. Although EfbCR offered high productivity, the Cfb strategy emerged to be the most technically feasible and robust solution and was successfully scaled up to an 80 L bioreactor, achieving 1885 mg PRP.L⁻¹. These results advance understanding of PRP production by Hib and provides valuable insight into an efficient and simplified strategy for producing this key/vital vaccine antigen. The findings support the potential for cost-effective local production in public health initiatives. Full article

Bovine chymosin is key for cheese production, yet its traditional sourcing is unsustainable. While microbial and plant-based alternatives exist, they often cause non-specific proteolysis, leading to bitter flavors in cheese. This study aims to develop a high-yield, methanol-independent platform for recombinant bovine chymosin production by engineering the expression system of Komagataella phaffii through multi-factorial optimization. Initially, the native bovine prochymosin gene (pcw) was codon-optimized (pcm14) and cloned, along with an mCherry-tag construct (clpcm14), into inducible vector pPIC9 for expression in Komagataella phaffii GS115. Screening identified the fusion-tagged strain clp2-91 as the highest producer. Subsequently, the inducible AOX1 promoter in the previously selected clp2-91 strain was replaced with a constitutive GAP promoter, yielding engineered strain GH1. Cultivated in a 3L fermenter, GH1 exhibited a volumetric productivity of 105.03 SU/(mL·h), twice that of inducible strain clp2-91 (53.59 SU/(mL·h)). The further optimization of fermentation conditions (pH 4.0, glucose as carbon source, fed-batch mode) boosted the enzyme activity of GH1 to 12,000 SU/mL. The recombinant chymosin exhibited enzymatic properties similar to those of the native enzyme and, importantly, demonstrated a broader pH stability (pH 2.0–6.0). This study demonstrates an efficient strategy for chymosin expression in K. phaffii, offering insights that may support the future development and optimization of heterologous protein production in this yeast. Full article

The increasing demand for recombinant proteins has driven innovation in bioprocessing strategies using Komagataella phaffii as a host organism. Conventional fed-batch cultivation with pure methanol induction remains widely used but presents challenges including high methanol consumption, extended downtime, and elevated operational costs. This study evaluates alternative strategies combining mixed induction (methanol/sorbitol) with continuous cultivation to enhance productivity, sustainability, and improved economic outcome. Using KEX2 protease as a model industrial recombinant protein, we compared four cultivation modes: fed-batch with methanol (benchmark), fed-batch with mixed induction, continuous with methanol, and continuous with mixed induction. Cell growth, volumetric yield, and specific productivity were evaluated at 5L scale and then modelled to simulate industrial scales (40 L and 400 L). Results demonstrate that continuous cultivation with mixed induction significantly improves yield up to 9-fold compared to conventional fed-batch and reduces methanol usage and oxygen demand. Techno-economic simulations reveal that a 40 L continuous process can match or exceed the output of two 400 L fed-batch runs, while lowering capital and operating costs and minimizing environmental footprint. This integrated strategy offers a scalable, low-cost, and safer method for recombinant protein production, supporting compact and sustainable manufacturing solutions. Full article

The recycling of fossil-based plastic waste remains a key challenge in reducing environmental pollution and greenhouse gas emissions. An innovative approach is the biotechnological conversion of the n-alkane mixture obtained from thermal pyrolysis of plastic waste. This study focuses on the use of the oleaginous yeast Yarrowia lipolytica for the valorization of polyethylene (PE)-derived pyrolysis oil. From a screening of 50 Y. lipolytica strains, the most promising candidate was selected, and its single-cell phenotype was stabilized by MHY1 deletion. In shake flask experiments, this strain grew similarly on 5–20 vol% of n-hexadecane, revealing no inhibitory effects. Subsequently, a high cell density fermentation was established in a 4 L bioreactor using a pulsed fed-batch approach, resulting in biomass concentrations of up to 145.6 g·L⁻¹, which contained 22.0% triacylglycerols. In addition, cultivation at pH 2.5, compared to pH 4.0, reduced citrate formation from 95.6 to 0.8 g·L⁻¹, while biomass and TAG titers remained similar. Overall, these results highlight the potential of integrating plastic waste-derived pyrolysis oil into future bioprocesses using Y. lipolytica as an effective platform for high cell density production. Full article

BslA (Biofilm surface layer protein A), a highly hydrophobic lipoprotein from Bacillus spp., self-assembles at fluid interfaces to form a crystalline film that reduces surface tension. In this study, we selected Pichia pastoris as a eukaryotic system for expressing recombinant BslA identified in Bacillus paralicheniformis BL-1. The secretory expression of recombinant BslA in the P. pastoris GS115 strain under the AOX1 promoter was confirmed in shake-flask cultivation. Next, two fed-batch fermentation strategies, constant dissolved oxygen strategy (DO-stat) and oxygen-limited fed-batch (OLFB) strategy, in a 5 L scale, were compared. The DO-stat process led to late-stage cell death and product degradation, limiting yields. Switching to the OLFB process by removing the glycerol feeding phase mitigated this issue, allowing extended fermentation and increasing the final recombinant BslA concentration to 657 mg/L. This study establishes P. pastoris with an OLFB strategy as an effective system for secreting recombinant BslA protein, providing a basis for future industrial-scale production. Full article

Zearalenone (ZEN) is a thermostable, lipophilic, non-steroidal estrogenic mycotoxin produced by Fusarium spp. that persistently contaminates food and feed. Its strong estrogenic activity and resistance to conventional detoxification strategies pose significant threats to food safety and human and animal health. Conventional physical and chemical degradation methods often compromise nutritional quality and leave toxic residues. Here we report the engineering of a novel Clonostachys rosea lactone hydrolase, Cr2zen, for efficient ZEN degradation in Pichia pastoris under mild conditions. Native Cr2zen exhibited a protein concentration of 0.076 mg/mL, achieving a degradation rate of approximately 17.9% within 30 min, with kinetic parameters of K_m 75.9 µM and V_max 0.482 µmol/L/s at 30 °C and pH 8.0. By integrating signal peptide screening and codon optimization, we identified Ser-Cr2 as the most effective variant, achieving a rapid 81.53% degradation of 10 ppm ZEN under mild conditions. Fed-batch cultivation in a 7.5 L bioreactor resulted in high cell densities of OD₆₀₀ 332.8 for Ser-Cr2 and 310.8 for Oser-Cr2, with extracellular protein concentrations of 0.62 and 0.79 g/L, respectively. The results demonstrate that signal peptide engineering and codon optimization substantially improved the production of lactone hydrolase in P. pastoris. This study establishes a scalable ZEN degradation under mild conditions in P. pastoris and outlines a strategy to integrate protein and process engineering for enhanced enzymatic mycotoxin degradation. Full article

Astaxanthin, a high-value keto-carotenoid with potent antioxidant and health-promoting properties, has gained global attention as a sustainable nutraceutical and biotechnological product. The green microalgae Haematococcus pluvialis and Chromochloris zofingiensis represent two promising natural producers, yet they differ markedly in physiology, productivity, and industrial scalability. This review provides a focused comparative analysis of these two species, emphasizing their quantitative performance differences. H. pluvialis can accumulate astaxanthin up to ~3–5% of dry biomass but typically reaches biomass densities of only 5–10 g L⁻¹, whereas C. zofingiensis achieves ultrahigh biomass concentrations of 100–220 g L⁻¹ under heterotrophic fed-batch fermentation, although its astaxanthin content is much lower (~0.1–0.5% DW). While H. pluvialis remains the benchmark for natural astaxanthin due to its exceptionally high cellular content, its thick cell wall, slow growth, and strict phototrophic requirements impose major cost and operational barriers. In contrast, C. zofingiensis exhibits rapid and flexible growth under heterotrophic, mixotrophic, or phototrophic conditions and can achieve ultrahigh biomass in fermentation, though its ketocarotenoid flux and astaxanthin accumulation remain comparatively limited. Meanwhile, a rapidly growing patent landscape demonstrates global technological competition, with major portfolios emerging in China, the United States, and Europe, spanning chemical synthesis, microbial fermentation, algal metabolic engineering, and high-density cultivation methods. These patents reveal clear innovation trends—ranging from solvent-free green synthesis routes to engineered microalgae and yeast chassis for enhanced astaxanthin production—which increasingly shape industrial development strategies. By synthesizing recent advances in metabolic engineering, two-stage cultivation, and green extraction technologies, this review identifies key knowledge gaps and outlines a practical roadmap for developing next-generation astaxanthin biorefineries, with an emphasis on scalable production and future integration into broader biorefinery frameworks. The findings aim to guide future research and provide actionable insights for scaling sustainable, cost-effective production of natural astaxanthin. Full article

The search for sustainable development has led several production processes to adopt biorefineries. We evaluated the cultivation of Spirulina platensis and Scenedesmus obliquus in consortium (50/50%), with the addition of effluent of the anaerobic digestion (AD) of cattle waste, in fed-batch mode, to obtain biomass in 10 L raceways. Subsequently, cultivation was carried out at pilot scale in a 100 L raceway. Zarrouk medium (20%) was used, with the addition of 10% (v/v) of effluent in the fed-batch process. The biomasses were characterized to evaluate their application. In 10 L raceways, higher biomass concentrations were obtained in the cultivation of Spirulina with the addition of effluent, or with the microalgae consortia without the addition of effluent (around 1 g/L). The addition of the effluent reduced the carbohydrate content and increased the protein content during the cultivation. Scale-up (100 L raceways) with Spirulina showed similar results to those obtained in the 10 L raceways, with removals of 48%, 88% and 11% for COD, nitrogen and total phosphorus, respectively. The cultivation of microalgae in consortium and Spirulina can be used in the post-treatment of effluent of AD, allowing the production of biomass for different applications. Full article

Model-based approaches provide increasingly advanced opportunities for optimizing and accelerating bioprocess development. However, to accurately capture the complexity of biotechnological processes, continuous refinement of suitable models remains essential. A crucial gap in this field has been the lack of suitable model for describing Escherichia coli growth in cultivation media containing yeast extract, while accounting for key bioprocess parameters such as biomass, substrate, acetate, and oxygen. To address this, a published mechanistic macro-kinetic model for E. coli was extended with a set of mathematical equations that describe key aspects of the uptake of yeast extract. The underlying macro-kinetic approach is based on the utilization of amino acids in E. coli, where growth is primarily influenced by two distinct classes of amino acids. Using fed-batch cultivation data from an E. coli K-12 strain supplemented with yeast extract, it was demonstrated that the proposed model extensions were essential for accurately representing the bioprocess. This approach was further validated through fitting the model on cultivation data from five different yeast extracts sourced from various manufacturers. Additionally, the model enabled reliable predictions of growth dynamics across a range of yeast extract concentrations up to 20 g L⁻¹. Further differentiation of the data into batch and fed-batch revealed that for less complex datasets, such as those obtained from a batch phase, a simplified model can be sufficient. Due to its modular structure, the developed model provides the necessary flexibility to serve as a tool for the development, optimization, and control of E. coli cultivations with and without yeast extract. Full article

Organic-waste-derived volatile fatty acids (VFAs) are promising substrates for fungal biomass cultivation, offering a nutrient-rich medium capable of meeting microbial growth requirements. However, the growth and biomass productivity are highly influenced by the VFAs’ composition and mode of operation. This study investigated the cultivation of Aspergillus oryzae fungal biomass using agro-industrial-derived VFA effluent, employing repeated-batch and fed-batch (stepwise and continuous-feeding) cultivation modes to evaluate fungal growth and biomass composition. The highest dry biomass yield of 0.41 dry biomass/gVFAs_fed (g/g) was achieved in fed-batch mode with continuous feeding, where the biomass exhibited pellet morphology, facilitating ease of harvesting. The crude protein content varied according to the cultivation strategy, reaching 45–53% in continuous-feeding fed-batch mode, while it was 34–42% in stepwise fed-batch mode. Additionally, the fungal biomass contained significant levels of essential macronutrients and trace elements, including Mg, Ca, K, Mn, and Fe, which are crucial if the biomass is intended to be used in animal feed formulations. This study highlights the effects of cultivation modes on biomass composition and the potential of VFA-derived fungal biomass as a sustainable feed ingredient. Full article

There is an increasing industrial demand for sustainable resources for lipid-based biofuels and platform chemical production. A promising, CO₂-efficient resource is autotrophically cultivated microalgae, either for direct single-cell oil (SCO) production or as a biomass substrate for fermentative SCO production via organisms like yeasts. Regarding the latter, chemical biomass hydrolysis typically results in high sugar yield and high salt concentrations due to the required neutralization prior to fermentation. In contrast, enzymatic hydrolysis is often lacking in mass efficiency. In this study, the enzymatic hydrolysis of both nutrient-replete and lipid-rich autotrophic Microchloropsis salina biomass was optimized, testing different pre-treatments and enzyme activities. Hereby, the protease treatment to weaken the cell wall integrity and the dosing of the Cellic CTec3 was identified to have the highest effect on hydrolysis efficiency. Sugar yields of 63% (nutrient-replete) and almost 100% (lipid-rich) could be achieved. The process was successfully scaled-up in mini bioreactors at a 250 mL scale. The resulting hydrolysate of the lipid-rich biomass was tested as a substrate of the oleaginous yeast Cutaneotrichosporon oleaginosus in a consumption-based acetic acid fed-batch setup. It outperformed both the model substrate and the glucose control, demonstrating the high potential of the hydrolysate as feedstock for yeast oil production. The presented sequential and circular SCO-producing value chain highlights the potential for mass- and space–time-efficient biofuel production, combining the autotrophic cultivation of oleaginous algae with decoupled yeast oil fermentation for the first time. Full article

The batch, repeated batch and fed-batch cultivation strategies, in stirred tank bioreactors, were evaluated to maximize biomass production and the cells’ desulfurization activity (CDA) of Rhodococcus qingshengii IGTS8. The batch culture reached 2.62 g DCW/L biomass, with a productivity of 0.03 g DCW·L⁻¹·h⁻¹ and only 26% glycerol consumption. The repeated batch strategy reduced cultivation time during the first cycle, increasing biomass production by 15%, with 30% glycerol consumed and productivity 2.3 times higher than the batch process; however, subsequent cycles showed no further improvement. CDA peaked early in both modes but declined to 12–13 U/mg DCW by the end of the exponential growth phase. Fed-batch cultivation achieved 8.35 g DCW/L with 87% glycerol consumption, resulting in a threefold increase in volumetric productivity and a 1.7-fold higher specific growth rate compared with the batch mode. CDA remained stable during the fed-batch process and was approximately 40% higher compared with the batch and repeated batch processes. The fed-batch culture was used directly in a two-phase bubble column bioreactor for the desulfurization of dibenzothiophene (DBT), 4-methyl-dibenzothiophene (4-MDBT) and their mixture. The complete desulfurization of 1.4 mM DBT was achieved at a rate of 21.6 mmol DBT/kg DCW/h, while 0.9 mM 4-MDBT was fully converted but at a 2.5-fold lower rate. The simultaneous conversion of the DBT/4-MDBT mixture showed reduced efficiencies of 59.6% and 41.2%, respectively. Full article