A novel approach to single-cell protein (SCP) production via hydrogen-oxidizing bacteria (HOB) is introduced to minimize explosion risks in hydrogen-based bioprocesses while assessing its implications for renewable-energy-based protein production systems. An alternative process is proposed where hydrogen (H2) and oxygen (O2) are spatially separated into two compartments: bioreactor and regenerator. Hydrogen is combined with an alternative electron carrier in a bioreactor, while oxygen is directed to a second compartment where the electron carrier is regenerated and energy is recovered. A thermodynamic analysis was performed to estimate yields and energetic efficiencies with a 13% margin of biological variability. Alternative processes based on HNO3 as electron carrier showed the highest potential when reduced to N2, NO, or N2O at low pH. Energy demand for SCP production was estimated to be 5.26, 5.82, and 5.52 MJ/molprotein, respectively. This was comparable to the 5.47 MJ/molprotein when using O2 as electron acceptor. The estimated yields for HNO3 reduction to N2 were in accordance with empirical biological yields. This showed that HOB-SCP energetic efficiency could be competitive with that of the conventional single-compartment aerobic method, offering a comparable energy demand, and a more integrated process design while eliminating explosion risks. This perspective on energetic efficiency is crucial for feasibility and techno-economic analysis of hydrogen-based SCP as a renewable-energy-driven route for sustainable protein production.

Acoustofluidic techonolgies enable label-free manipulation and separaiton of particles and cells in microfluidic systems, offering strong potential for biological analysis and bioprocessing applications. However, fabrication of acoustofluidic chips remains costly, particularly for platforms that integrate external fields such as an acoustic field, which require additional components like transducers. To address these challenges, this study compares three rapid and cost-effective fabrication methods, namely 3D printing, laser cutting (LC), and Computer Numerical Control (CNC) milling, for producing acoustofluidic chips compatible with off-the-shelf 2.1 MHz piezoelectric transducers. The chip geometry and the simple fabrication workflow were optimised, along with post-processing strategies, to improve the performance and efficiency of bulk acoustophoresis. Experimental characterization of particle trajectories, supported by numerical simulations, elucidated acoustofluidic behaviour and reduced trial-and-error in chip design. Energy and resource requirements were further assessed to determine suitability for scalable applications. By combining inexpensive equipment with streamlined fabrication and optimization workflows, this approach lowers the entry barrier to acoustofluidic research while enabling rapid, low-cost chip development and facilitating translation toward practical applications. The results provide practical guidelines for selecting appropriate fabrication methods for acoustofluidic chips based on performance and manufacturability considerations.

Conventional alginate extraction from brown seaweed typically relies on harsh, non-recyclable chemicals, limiting process sustainability. This study presents temperature-responsive deep eutectic solvents (TRDES) as circular, recyclable extractants for alginate recovery. Using computational screening with COSMO-RS and experimental validation of TRDES affinity and alginate partitioning, TRDES1 (o-cresol: ethanolamine) was identified as the most promising combination, and was optimised and reused over eight cycles, yielding up to 55.6 +/- 14.4 mg/g DW. COSMO-RS modelling validated the observed increase in extraction efficiency over successive cycles, showing enhanced partition coefficients and reduced Gibbs free energy of transfer with reuse. The process enabled mild extraction of functional alginate with increasing efficiency over the cycles. The main solvent parameters for TRDES design found to govern extraction and recyclability were capacity (C), partition coefficient (K), and Gibbs free energy (Delta G). Optimal performance was achieved with moderate TRDES-water capacity (1.27 x 10(1) to 3.15 x 10(1)), low TRDES capacity (<1.2), and K > 1. This work establishes a theoretical framework with design rules for future TRDES development based on computational and experimental analysis and highlights the need for novel, biocompatible TRDES systems. As demonstrated, combining computational screening with these design principles enables the use of recyclable solvents. Incorporating natural compounds into TRDES design enhances both process efficiency and sustainability, facilitating the integration of DES technologies into circular biorefineries and supporting environmentally responsible biomass valorisation.

Microalgae are a promising source for proteins, lipids, and carbohydrates for the cosmetic, nutraceutical, chemical, food/feed, and biofuel industry. In comparison with soy and palm oil, microalgae can be produced in a more sustainable way. To make microalgae production economically feasible, all biomass ingredients need to be efficiently utilized, similar to petroleum refineries in which oil is fractionated in fuels and a variety of products with higher value. However severe conditions can affect the properties of some components in the biomass. To overcome this, focus needs to be put on biorefinery techniques which are mild and effective. Microalgal biorefinery is a linear process consisting of harvesting, cell disintegration, sequential extraction, and further fractionation. Among these steps, the cell disintegration often represents a bottleneck for the extraction of hydrophilic or hydrophobic components, due to the presence of a tough cell wall in many strains. State of the art knowledge on both novel and classical techniques for product extraction within cell disintegration is presented. Comparison is made on the basis of two main criteria: yield of disintegration and energy consumption. The current work gives also a comprehensive outlook on business cases for microalgae biorefinery.

The red seaweed Palmaria palmata is a promising source for alternative proteins. Development of mild and novel biorefinery approaches are needed for green processes yielding functional proteins. Process intensification using ultrasound-assisted unit operations can be an effective strategy to circumvent current seaweed biorefinery challenges, in particular limited protein extraction yields. In this research, first, effects of acoustic cavitation (AC) during ultrasound-assisted extraction (UAE) were studied using a multifactorial design of experiments for total protein and R-phycoerythrin (RPE) extraction from P. palmata. Secondly, novel enzyme-assisted high-frequency ultrasonic extraction (EAUE) was developed following the same approach and responses. Contrary to the traditional acoustic cavitation process, this intensification strategy allows a milder extraction of proteins from the recalcitrant P. palmata harnessing combined effects of acoustic irradiation and enzymes. Validation experiments showed the robustness of developed models. Maximum RPE yields were 2.6 mg/gdw for both approaches. However, maximum total protein extraction efficiencies were 13.6% when applying acoustic cavitation and increased to 36.0% under high-frequency acoustic irradiation together with the Viscozyme® L enzyme preparation. Moreover, intensification phenomena were observed during EAUE and an increased extraction yield in comparison to traditional enzymatic processes (EAE). We hypothesize the synergistic effect observed is due to acoustic fields improving cell wall disentanglement and enhancing enzyme-substrate interactions. The present study provides insights into the use of ultrasound for protein extraction of P. palmata and introduces, to the best of our knowledge, for the first time EAUE as process intensification strategy of seaweed biorefinery processes.

Ensuring an adequate supply of oxygen remains a significant challenge in the development of large engineered tissue constructs in the field of tissue engineering. To address this, novel strategies have recently been introduced, including the incorporation of photosynthetic microorganisms into engineered tissues. However, to take the full advantage of this co-culture approach, careful selection of photosynthetic microorganisms and a better understanding of their long-term interactions with mammalian cells are required. Here, we first examined the effects of continuous 28-day light exposure on the proliferation and biofunctionality of mammalian cells. We observed that articular cartilage-derived chondroprogenitor cells (ACPCs) did better withstand light exposure under chondrogenic conditions than mesenchymal stromal cells (MSCs). Next, four different photosynthetic microorganisms, capable of growing at 37°C, were co-cultured with cartilage cells. Among them, Leptolyngbya sp. (Leptolyngbya) and Synechococcus sp. (Synechococcus) did not compromise the morphology and chondrogenic capacity of mammalian cells in vitro over 28 days, whereas Chlorella sorokiniana (Chlorella) inhibited chondrogenesis. This inhibition might due to excessive oxygen release by Chlorella in chondrogenic culture medium, as Leptolyngbya and Synechococcus did not produce detectable oxygen under the same culture conditions. To further explore their potential for oxygen delivery to other tissue-derived cells, we also assessed the growth rate and oxygen production of these four microorganisms in different mammalian cell culture media. We found that the composition, especially the presence of trace elements in tissue medium, critically influenced oxygen production. The tested microorganisms were able to grow and release oxygen in different mammalian cell culture media typically used for the propagation of cardiac, cartilage and liver cells, highlighting their flexible metabolic pathways across the different environments. This study emphasizes the importance of carefully selecting photosynthetic microorganisms for different tissue types, ensuring a balance between oxygen production and the specific nutritional demands of mammalian cells.

Protein extraction from Palmaria palmata remains challenging due to structural features that restrict protein release. This study evaluated alkaline solvents, xylanase enzymes, and deep eutectic solvents (DES) to assess protein recovery and structural changes using chemical analysis and microscopy. Protein yield, sugar solubilization, tissue density, protein localization, and cell wall integrity were examined. Microscopy revealed that proteins are concentrated in densely packed cortical cells with minimal intercellular space, limiting solvent penetration. Alkaline extraction achieved the highest protein recovery (∼60%) with moderate structural disruption. Xylanase-assisted extraction caused greater cell wall breakdown but resulted in lower protein recovery (∼27%), while DES treatment caused minimal disruption and yielded the lowest recovery (∼14%). Carbohydrate solubilization did not correlate with protein recovery, indicating that cell wall degradation alone is insufficient. These findings identify dense cortical organization as a key constraint and highlight the need for combined mechanical and targeted biochemical strategies to improve extraction efficiency.

This study evaluates the impact of using deep eutectic solvents (DES), specifically betaine-urea, as a novel extraction technology for alginate from brown seaweed and its effect on the techno-functionality of the final product. Ten eutectic mixtures were assessed, with betaine-urea (BU) demonstrating the highest performance, yielding 14.9 +/- 2.0 % of the total amount of alginate in the first extraction step (BU-1). Although this yield was lower than the conventional acid-alkaline (AK) method (37.6 +/- 4.1 %), a sequential extraction combining water (BU-2) and alkaline conditions (BU-3) achieved higher overall yields for the BU method. The study highlights significant differences in the chemical and rheological properties of alginates extracted with BU compared to those obtained via traditional methods. BU-extracted alginates exhibited the characteristic shear-thinning behaviour of alginate solutions but with markedly higher apparent viscosity at low shear rates. BU-2 displayed gel-like behaviour (tan S = 0.1) with a consistency index (K) 62 times higher than the acid-alkaline extract. Chemical analysis revealed a higher fraction of high-molecular-weight alginates in BU-2 and BU-3, partially explaining their increased viscosity. However, the role of betaine-urea in shaping the alginate structure and function should be further studied. This research underscores the potential of DES technology in enhancing the functionality of alginate, offering a potentially more sustainable alternative to mineral acid-base methods.

The baculovirus expression vector system (BEVS) is a scalable platform used to produce recombinant adeno-associated virus vectors (rAAV) in insect cells. A major challenge in this system is reducing the formation of empty rAAV capsids, which do not contain vector DNA and lack therapeutic value. The proportion of empty capsids is influenced by the balance between the two baculovirus constructs that coinfect the producer cells: Bac-Rep-Cap, which supplies AAV replication and capsid proteins, and Bac-GOI-ITR, which delivers the therapeutic gene of interest. Digital holographic microscopy (DHM) is a label-free imaging technique that allows real-time monitoring of cell morphology in suspension cultures. Previous studies have used DHM to track cell density and baculovirus infection; however, its ability to evaluate different coinfections has not been explored. In this study, we combined DHM with machine learning to identify morphological patterns associated with various coinfections for rAAV production. Shaker-flask experiments with different Bac-Rep-Cap: Bac-GOI-ITR ratios created a dataset of cell morphologies to train a predictive classification model. When applied to real-time bioreactor measurements, the model revealed shiftsin the classification patterns related to the initial multiplicity of infection (MOI). The integration of DHM with the classification model has the potential to produce a qualitative "process fingerprint," where deviations in morphological patterns can serve as early indicators of suboptimal coinfection. Such early warning signs enable timely batch termination, reducing downstream processing of inconsistent material. Overall, DHM combined with machine learning offers a non-invasive, real-time tool for process benchmarking and quality assurance in rAAV manufacturing.

Polar microalgae possess unique mechanisms that enable them to thrive in extreme environments; however, their molecular responses remain poorly explored in biotechnological contexts. This study presents the physiological and transcriptomic responses of Chlamydomonas malina RCC2488 under two key environmental conditions: Thermal acclimation (8 vs 4 °C) and nitrogen deprivation (-N vs +N). Through de novo RNA-seq assembly (46,536 transcripts; 20,993 annotated), 1696 and 1296 differentially expressed genes were identified under thermal acclimation and nitrogen deprivation, respectively. Both conditions induced the accumulation of triacylglycerols (TAGs) enriched in polyunsaturated fatty acids (PUFAs) and repression of photosynthesis-related genes. However, the underlying regulatory mechanisms differed substantially. Thermal acclimation significantly reduced the growth rate (from 1.707 to 0.324 d⁻¹) and activated the de novo fatty acid and TAG biosynthesis pathway through upregulation of key genes including ACCase, KAS, KAR, HD, DGAT and G3PDH. In contrast, nitrogen deprivation halted cell division and triggered prioritized metabolic reprogramming characterized by the activation of critical lipogenic enzymes (PDH, ACCase, HD, G3PDH, and DGAT), transcriptional maintenance of nitrogen transporters and assimilation enzymes (NR, NRT2) in an alert state, and repression of non-essential growth-associated pathways such as amino acid and nucleotide metabolism, translation, cell division and protein degradation. The identification of shared differentially expressed genes between both conditions suggests the existence of a common transcriptomic core associated with cellular homeostasis and resource optimization. Collectively, these findings demonstrate that two distinct environmental perturbations converge toward the same lipid phenotype through differentiated regulatory programs, revealing an adaptive strategy oriented toward resource economy and energy storage in dynamic polar environments. This study positions Chlamydomonas malina RCC2488 as a relevant model for understanding metabolic plasticity in polar microalgae and as a promising platform for sustainable TAG and PUFA production under suboptimal conditions.

Conventional alginate extraction from brown seaweed relies on alkaline treatments that limit sustainability and solvent reuse. In this study, a novel deep eutectic solvent (DES)-salt aqueous two-phase system (ATPS) was developed for the integrated extraction and separation of alginate as a more sustainable alternative. By incorporating the precipitation principle of three-phase partitioning at elevated salt concentrations in an ATPS, alginate was selectively recovered in an intermediate phase between the DES and aqueous salt solution, enabling direct isolation while preserving the solvent for reuse. Salt selection significantly influenced phase formation, with K3PO4 exhibiting stronger phase-forming ability than K2HPO4 due to its enhanced salting-out effect, whereas alginate recovery was primarily governed by salt concentration. The highest recovery (76.4 +/- 0.1%) was achieved using betaine: urea (1,2) with 50 wt% K3PO4, with most alginate accumulating in the intermediate phase. The DES maintained stable recovery over six cycles (63-86%), while preserving alginate integrity. These results demonstrate the potential of DES-based ATPS as an efficient platform for simultaneous extraction, separation, and solvent recycling, providing a promising route toward more sustainable and integrated seaweed biorefinery processes.

Cultivation conditions are known to affect the structural organization of microbial cell walls. However, the impact of these structural variations on protein release following mild cell disruption remains largely unknown. Therefore, this study investigated the effects of cultivation conditions on the Saccharomyces cerevisiae cell wall. Its robustness and apparent protein permeability were assessed indirectly through, respectively, zymolyase susceptibility and intracellular protein release following hydrophobic deep eutectic solvent assisted membrane-permeabilization. More specifically, the influences of cultivation pH, glucose-limitation, growth phase and cultivation mode were evaluated. Shake-flask experiments demonstrated that cultivation conditions associated with different physiological states strongly affected cell wall structural organization, since a shift from glucose-limitation to glucose-abundance increased zymolyase susceptibility by 5-fold, and increased protein release by up to 3.8-fold. Therefore, the effects of growth phase and cultivation mode were further investigated in controlled bioreactors, resulting in more profound differences. Biomass harvested from exponentially growing batch cultures exhibited a 5.7-fold higher soluble protein release than low-rate fed-batch cultures, while zymolyase susceptibility increased by 19.4-fold. Moreover, strong correlations were found between physiological state, zymolyase susceptibility and protein permeation behaviour, suggesting that cultivation-dependent physiological states impact cell wall characteristics and intracellular protein release. Finally, apparent molecular size analysis demonstrated that the released material obtained from permeabilized biomass was enriched with components smaller than 100 kDa. Overall, this study demonstrates that cultivation-driven modulations in cell wall characteristics strongly affect mild disruption yields, thereby highlighting the importance of integrating upstream and downstream processes for efficient recovery of intracellular proteins.

The emergence of new viruses and the spread of existing pathogens necessitate efficient vaccine production methods. The baculovirus expression vector system (BEVS) is an efficient and scalable system for subunit and virus-like particle vaccine production and gene therapy vectors. However, current production processes are often limited to low cell concentrations (1-4 × 106 cells/mL) in fed-batch mode. To improve the volumetric productivity of the BEVS, a medium exchange strategy was investigated. Screening experiments were performed to test baculovirus (expressing green fluorescent protein; GFP) infection and productivity of insect cell cultures infected at high cell concentration (1-2 × 107 cells/mL), showing that infection at high cell concentrations was possible with medium exchange. Next, duplicate perfusion runs with baculovirus infection were performed using a cell concentration upon infection (CCI) of 1.2 × 107 cells/mL and a multiplicity of infection (MOI) of 0.01, reaching a maximum viable cell concentration of 2.8 × 107 cells/mL and a maximum GFP production of 263 mg/L. The volumetric productivity of these perfusion runs was 4.8 times higher than for reference batch processes with a CCI of 3 × 106 cells/mL and an MOI of 1. These results demonstrate that process intensification can be achieved for the BEVS by implementing perfusion, resulting in a higher volumetric productivity.

Microalgae possess diverse lipid classes as components of structural membranes and have adopted various lipid remodeling strategies involving phospholipids to cope with a phosphorus (P)-limited environment. Here, we report a unique adaptative strategy to P deficient conditions in two cold-adapted microalgae, Raphidonema monicae and Raphidonema nivale, involving the lipid class diacylglyceryl glucuronide (DGGA) and the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine. Lipidomic analyses showed that these two lipid classes were present only in trace amounts in nutrient replete conditions, whereas they significantly increased under P-starvation concomitant with a reduction in phospholipids, suggesting a physiological significance of these lipid classes to combat P-starvation. Additionally, we found two putative sulfoquinovosyldiacylglycerol (SQDG) synthases, known to be involved in DGGA synthesis in higher plants, in the draft genome of R. monicae, and compared it with SQDG synthases found in other organisms such as higher plants, Streptophyta, and Chlorophyta. DGGA has not been previously recognized in Chlorophyta, and our findings suggest that the lipid class may be present in other closely related green algae too. Thus, this study expands our knowledge on diverse lipid remodeling responses of Chlorophycean algae to adapt to low P environments.

Microalgae are considered a potential sustainable feedstock for producing ingredients used in food, feed and cosmetics. However, the rigid cell wall structure presents a significant challenge for the efficient extraction intracellular products. Enzymatic hydrolysis of cell wall polymers could offer a mild and environmentally sustainable cell disruption approach. Nevertheless, incomplete knowledge of the structural and architectural properties of the cell wall limits the commercial application of this method. In this study, a combination biochemical and microscopy techniques was used to decipher the composition, topography, ultrastructure and the architecture of the cell wall of Nannochloropsis oceanica IMET1. Acid hydrolysis of the extracted cell wall released neutral sugars, constituting 80 % of its total weight (w/w). Of these sugars, 64 % was glucose, which primarily constitutes the cellulose layer. This was further confirmed through calcofluor white fluorescence imaging using a scanning confocal microscope. Additionally, the presence of small amounts of arabinose, fucose, galactose, and mannose suggests the presence of polysaccharide networks connecting the outer layer, the cellulose-based layer and the plasma membrane. Scanning electron microscopy revealed a rugged cell surface covered with N-acetyl-glucosamine units as confirmed by fluorescent-lectin staining. Transmission electron microscopy revealed a double-layered cell wall structure. Hydrolysis of the extracted cell wall with lichenases and cellulases generated mostly glucose monomers alongside traces of other neutral sugars. Overall, this study provides insights into the complex structure of the cell wall of N. oceanica IMET1, which can enhance enzyme assisted extraction strategies in microalgae.

After more than four decades, the field of sponge biotechnology has finally overcome its most formidable obstacle: establishing a sponge cell line. Cells from the deep water marine sponge Geodia barretti divide rapidly and continuously in optimized nutrient media, clearing the path for new lines of research--whether to uncover how multicellular life evolved from unicellular organisms, study the origins of animal-microbe symbiosis, or develop production strains to industrialize new sponge-derived drugs and exploit the untapped biotechnological potential of the Porifera. Now is an exciting time in sponge biotechnology, so what's next?

Physiological, biochemical, and morphological responses of Nannochloropsis oceanica and Microchloropsis gaditana were investigated using a two-phase cultivation strategy, starting with nitrogen (N) replete conditions followed by N-starvation at different biomass-specific photon supply rates (BSPSRs). Cultures were grown in photobioreactors under a 16:8 hours light-dark cycle with a maximum light intensity of 1200 μmolphoton m-2 s-1. Subsequently, two BSPSRs, 9 and 18 μmolphoton gdw-1 s-1, were implemented at the onset of the N-starvation phase by adjusting biomass concentrations. Under N-starvation at the lower BSPSR of 9 μmolphoton gdw-1 s-1, N. oceanica exhibited substantially higher biomass and fatty acid productivities (0.62 and 0.35 g L-1 day-1, respectively), compared to M. gaditana's biomass and fatty acid productivities (0.22 and 0.085 g L-1 day-1, respectively), indicating the presence of a robust stress response mechanism in N. oceanica. While both species experienced decreased photosynthetic efficiency under N-starvation, M. gaditana maintained higher quantum yield values with (0.46 and 0.32 at low and high BSPSRs, respectively) compared to N. oceanica (0.42 and 0.15). Similarly, M. gaditana preserved higher EPA content (35.36 and 30.28 mgEPA gdw-1) than N. oceanica (31.34 and 28.98 mgEPA gdw-1) at low and high BSPSRs, suggesting distinct physiological adaptation strategies. Chlorophyll content substantially declined in both species under high BSPSR and N-starvation, with N. oceanica experiencing a more pronounced degradation. The morphological analysis aligned with these data, unveiling significant alterations in chloroplast structure and oil-body formation. Providing visual evidence of cellular adaptations to stress conditions and the differences between the two species, with N. oceanica formed larger oil bodies while experiencing more extensive chloroplast degradation than M. gaditana under N-starvation at the higher BSPSR. These findings demonstrate species-specific trade-offs between growth, stress tolerance, and biochemical composition, offering valuable insights for optimizing microalgal strains for targeted application, food and biofuel production with N. oceanica or EPA-rich biomass with M. gaditana.

Semi-hydrophobic eutectic solvents (ES) possess a great potential as lipid extraction solvent from untreated microalgae. However, the low vapor pressure of these solvents and the unknown effects on other biomolecules (e.g., proteins, carbohydrates) limit their application in microalgae biorefinery. In this work, recovery of the extracted lipids was performed by addition of antisolvents and the affecting parameters (i.e., antisolvent type, amount, temperature, ES imidazole content) were studied. The highest recovery was obtained with methanol addition to ES with 15 mol% imidazole at -20 °C, where lipid crystals were formed consisting mainly of saturated fatty acids. The remaining soluble lipids under the same condition were found to be fractions with mono- and poly-unsaturated fatty acids. Furthermore, based on the iterative extractions, the regenerated solvents could create sufficient driving force for lipid extraction despite the lipid accumulation. In addition, a scale-up study of lipid extraction and solvent recycling was performed (2 mL vs 500 mL), whereby the larger scale also showed a good performance. Finally, protein and carbohydrate isolation from the defatted biomass was feasible, but the proposed ES process was not sufficiently mild to maintain native proteins. On the other hand, opportunities are discussed to create new functionalities for proteins and carbohydrates so that a multiproduct biorefinery is feasible for this ES.

In biopharmaceutical manufacturing, continuous perfusion cultivation enables high space-time yields and increased plant utilization, which are critical targets for modern upstream process intensification. However, filter-based cell retention devices, utilized in these processes, have significant disadvantages: Significant sieving effects and the risk of filter blockage alongside the retention of harmful substances and non-viable cells, represent a major challenge and often reduce the viability of the culture. To enable the next-generation of continuous processes, novel cell retention strategies are required. Therefore, the aim of this study was to develop an approach for large-scale sorting of viable and non-viable cells and to investigate its applicability for novel continuous cultivation strategies. To remove non-viable cells and thus to enrich viable cells in the culture, a single-use fluidized bed centrifuge (FBC) was used, which is usually applied for concentration and washing of mammalian cells. A novel FBC method was introduced by overloading the centrifuge chambers that allows high throughput sorting depending on the culture´s viability. The impact of the sorting on the subsequent cultivation and productivity of the cells was investigated in a multi-parallel 15 mL bioreactor setup. Cell sorting after regular fed-batch cultivation showed +14% increase of viability, continued cell growth, and thus +13% higher titers. Thereafter, periodic cell sorting was tested on a 5-L scale bioreactor, combining the advantageous characteristics of fed-batch and perfusion cultivation. The feasibility was successfully demonstrated for 20 days, achieving a high average space-time yield of 0.75 g/L/d. In both cultivation trials, up to +38% higher cell specific antibody productivities were found after cell sorting. Overall, the FBC sorting method in combination with innovative cultivation concepts addresses current limitations and challenges of continuous biopharmaceutical manufacturing and has great potential to further advance modern process intensification.