Abstract Background The co-cultivation of filamentous fungi and actinobacteria is challenging due to their complex growth interactions. This study investigates how key parameters, such as inoculation strategy, glutamic acid concentration, hydrodynamic stress, and dissolved oxygen, influence the growth dynamics between Aspergillus niger and Streptomyces coelicolor in shake flask co-cultures. Recognizing the crucial role of macromorphology in filamentous microorganisms, an automated image analysis pipeline was developed to quantitatively assess the heterogeneity and reproducibility of each population. Results Simultaneous growth was achieved when both microorganisms were inoculated in pelleted form, whereas spore inoculation led to complete A. niger dominance. At 1:2 and higher inoculation ratios (fungus to bacteria), S. coelicolor could compete effectively. While A. niger growth-maintained dominance at 136 and 250 rpm (1:1), S. coelicolor growth outcompeted the fungus at 60 rpm, a shift attributed to a reduced oxygen transfer rate. Notably, only the highest shear forces (250 rpm) produced homogeneous, reproducible fungal pellet populations. Overall, bottom-baffled flasks enhanced reproducibility compared to non-baffled flasks. Conclusion It is possible to regulate the growth of S. coelicolor and A. niger in a co-culture by the aforementioned parameters. Among these, the inoculation ratio is most important to achieve different dynamics. A quantitative analysis of morphology development while optimising inoculation strategies provides a foundation for designing co-culture experiments that achieve balanced and reproducible growth.

Abstract The filamentous fungus Aspergillus niger is a well-established cell factory in biotechnology. Its productivity depends on macromorphological development which remains difficult to control, partly because the relationship between seed culture and reactor-specific shear force conditions has not been systematically investigated. This study examined how high or low shear forces affect pellet development at both micro- and macromorphological levels in stirred-tank reactors (STR, high shear regime) and rocking-motion bioreactors (RMB, low shear regime). A. niger seed cultures with initially either large or small pellets were used to inoculate batch STR or RMB. Comparable cultivation conditions were applied so that fermentations differed mainly in shear force regime. Growth characteristics and pellet macromorphologies were analysed using 2D and 3D image analyses, enabling us to classify pellets according to three different classes based on their inner pellet architecture. The distribution of these classes depended on both the macromorphologies of the seed culture and the reactor type. Under high shear forces in the STR, pellets underwent breakage shortly after stirrer activation, were limited in their size to an average diameter of 500–600 µm, and formed a homogeneous population. In addition, broken pellets occurred predominantly under STR conditions. In contrast, cultivations in RMB preserved the initial pellet architecture, allowed the formation of larger pellets (median diameter ~ 800 µm) and supported pellet fusion, thus resulting in a more heterogeneous macromorphological population. Notably, glucose uptake rate correlated with the surface-to-volume ratio of the pellet populations, i.e., glucose became faster consumed under STR conditions accompanied with lower biomass yields and higher protein secretion. Citric acid production, however, was detectable in both STR and RMB only when reactors were inoculated with seed cultures characterised by a loose pellet morphology. Overall, our study demonstrates how shear regime and seed culture morphology jointly shape pellet architecture, population heterogeneity and productivity in scale-up processes. Such a comprehensive understanding of morphological developments is instrumental for optimising bioprocesses and future predictive modelling approaches. Key points • 2D/3D analysis of defined seed cultures in different shear-induced environments • High shear restricts and homogenises pellets, while low shear maintains heterogeneity • Highest citric acid and total protein levels were found in smaller, compact pellets.

Phosphorus-containing fertilisers are essential for global food production but depend on finite phosphate rock reserves. Recovery of phosphorus from waste streams, such as those in wastewater treatment plants, will be required to follow future regulations. Certain Acinetobacter spp. can accumulate 24–30

Direct combustion (DC) and anaerobic digestion (AD) are widely recognized technologies for converting agro-industrial residues into energy. Although numerous studies have evaluated the technical and economic feasibility of biomass-based energy alternatives, existing financial risk assessments are mainly based on stochastic methods focused on sensitivity analysis, expected values, or the probability of financial losses, while overlooking the influence of uncertainty on adverse and extreme risk scenarios. To address this gap, this study conducts a financial risk comparison between DC and AD for a sugarcane mill, evaluating extreme scenarios of potential financial losses and feedstock requirements. Uncertainty in key technical and economic variables is modeled using Monte Carlo simulation, and extreme risk outcomes are assessed through tail-risk measures. Results indicated that DC remains as a financially viable option under current market conditions, while AD exhibits a higher dependence on policy incentives and greater exposure to adverse risk scenarios. Outcomes are also critically discussed under the consideration of sustainability measures and regulatory frameworks.The study provides a transferable risk-based evaluation framework for assessing the economic feasibility of biomass-based energy projects under uncertainty, thereby contributing to informed decision-making in the utilization or residual biomass.

The marine protist Schizochytrium limacinum SR21 is a promising producer of docosahexaenoic acid (DHA) from short-chain carboxylic acids (SCCAs). Dark fermentation effluent (DFE) is an SCCA- and nutrient-rich substrate derived from biogenic residues and has previously been evaluated for microbial lipid production. However, its low carbon concentration limits volumetric productivity and causes substantial culture dilution when larger feed volumes are required. Membrane-based cell retention is an established process intensification strategy for low-concentration feed solutions, as it decouples hydraulic residence time from biomass residence time. However, the combination of DFE utilisation and cell retention for DHA production by S. limacinum has received little attention. In this study, repeated fed-batch and cell retention strategies were evaluated to overcome dilution limitations and enable intensified biomass and lipid production from DFE. Additional cultivations with defined SCCA mixtures showed preferential uptake of acetate and butyrate over propionate and lactate, which was also observed during DFE conversion. In repeated fed-batch operation, biomass output reached 7.5 g after 95 h. In contrast, a final biomass output of 33 g was achieved in cell retention mode, representing a 4.4-fold increase. This improvement was not associated with enhanced substrate conversion efficiency but resulted from retaining biomass while increasing DFE throughput. While applying cell retention, the specific palmitic acid concentration increased to 116 mg g⁻¹, while DHA remained comparatively stable at 57 mg g⁻¹. This resulted in a DHA titre of 1.9 g L⁻¹ compared with 0.24 g L⁻¹ in repeated fed-batch. Nitrogen and phosphate removal reached 58

Sustainability has become a central challenge for the biopharmaceutical industry, which is characterized by resource-intensive manufacturing processes, complex supply chains, and increasing regulatory expectations for environmental transparency. This review examines the role of single-use technologies (SUTs) and sustainability by design (SbD) approaches in improving the environmental performance of modern biomanufacturing systems. While SUTs offer operational advantages such as increased flexibility, faster facility turnaround, and reduced water and energy consumption compared with stainless steel systems, their widespread adoption also raises concerns regarding plastic waste generation and end-of-life management. Survey results from industry stakeholders further indicate increasing awareness of sustainability challenges, but also reveal gaps in knowledge, governance structures, and practical implementation across the value chain. The paper aims to summarize recent developments in environmental assessment, process engineering, and digital bioprocessing to evaluate pathways toward more sustainable production and, at the same time, to identify knowledge gaps that remain. Emphasis is placed on the integration of life cycle assessment (LCA) and quantitative sustainability metrics into early process development, enabling more informed technology selection and design decisions now and in the future. In addition, emerging technological strategies are discussed, including process intensification, continuous and hybrid manufacturing configurations, and advanced process analytical technologies (PATs) that support real-time monitoring and resource-efficient operation. Overall, the findings highlight that an integrated systems perspective is required that combines technological innovation, standardized environmental metrics, and collaborative industry frameworks. Embedding SbD principles early in product and process development can significantly reduce environmental impacts while maintaining regulatory compliance and economic feasibility.

Yeast and bacteria co-cultures can be found in nature and have multiple advantages that can be exploited, nowadays also in a controlled bioproduction environment. Various types of co-cultivation have been used for food applications such as production of flavor compounds in dairy products and alcoholic beverages. Co-cultures can broaden the substrate spectrum for microbial food and feed production, they can increase productivity and efficiency, and the nutritional value. Workflows have been developed from plate to bioreactor scale to increase reproducibility and optimize benefits of individual co-cultivation strategies. Nonetheless, certain limitations need to be overcome for industrial application. Many interactions of microbes, in particular in suspension cultures, are not sufficiently understood or even explored. While more possibilities arose from on-line monitoring of individual populations or even single cells, off-line measurement techniques are still typically applied in order to assess growth and product formation. Promising advances have been achieved, however, by methods for single-cell at-line and on-line analysis in co-cultures which are accounted for to emphasize the current opportunities and challenges in monitoring and controlling co-cultures. This review aims to summarize the recent advances with a particular focus on cultivation procedures and process analysis in bacteria, yeast and bacteria-yeast co-cultures. The implementation of suitable monitoring methods to enable (remote) control and contribute to quality assurance will accelerate the development and optimization of industrial co-culture bioprocesses. This will support transferability and process standardization across world regions adding to the advancement of bioproduction. The applicability of some relevant technology is, however, in its infancy.

Polyhydroxyalkanoate (PHA) is an important bioplastic, its production has been commercialized, and an increase of production capacities is expected. As with many other basic chemicals, PHA production requires a currently unavailable amount of renewable carbon if bioplastic production is ever to compete with plastic production from petroleum. This extensive demand for raw materials poses challenges in terms of costs, logistics, and land use. The application of biogenic residues is therefore one of the prerequisites for any economically significant and environmentally friendly PHA production. Against this background, recent findings on the possibilities of using biogenic residues from food production and consumption to produce PHA are summarized. Waste animal fats, waste cooking oil, but also mixed food waste, either from food production or consumer food waste represent the most abundant food-related residues. They are explored for their potential to serve as substrate for PHA production. While waste animal fat and waste cooking oil can be fed directly into suspension cultures, mixed food waste can be converted into short-chain carboxylic acids from microbial hydrolysis and acidogenesis in dark fermentation before being fed. Titers and productivity of the several feedstock options are compared. The potential for economically viable and sustainable production and integration into local material cycles is highlighted, although there are still several challenges to overcome. • Waste cooking oil enables low-cost and scalable PHA production • Thermally liquefied animal fats are a suitable feed for emulsifier-free PHA production • Coupling dark fermentation and PHA production is economically feasible • The impact of carboxylic acid composition on PHA synthesis is explored

The transition to a sustainable bioeconomy requires effective transformation of biomass, including biogenic residues, into high-value products. Dark fermentation effluent is a promising feedstock for microbial lipid production, offering a renewable and cost-effective alternative to conventional carbohydrate substrates. Rich in short-chain carboxylic acids, this effluent provides essential carbon sources for microbial growth and supports circular biorefinery strategies, particularly when produced from locally available biogenic waste streams. This review assesses the potential of microbial lipid production from dark fermentation effluent, focusing on three key microbial groups: oleaginous yeasts, marine protists, and microalgae. Among these, yeasts and protists exhibit significant advantages over microalgae, including faster growth rates, higher lipid yields, and superior adaptability to the complex composition of dark fermentation effluent. To enhance utilization, two-stage operation allow for the independent optimization of biomass production and lipid accumulation, with controlled feeding in dissolved oxygen or pH-auxostat modes. However, challenges remain, especially with respect to solid-liquid separation, sterilization, and the overall integration of two distinctly different biotechnological processes, which require suitable monitoring and control strategies.

Digital holographic microscopy (DHM) is a label-free analytical technique for the determination of the cells' volume and their cytosolic refractive index. Here, we demonstrate the suitability of DHM for the quantification of total lipid accumulation in the oleaginous yeast Yarrowia lipolytica. Presently, microbial lipids are gaining increasing attention due to their nutritional value in feed and food applications. Their microbiological synthesis in algae and yeast is subject to optimization studies, which necessitates rapid quantification of total lipids for faster progress and the possibility of process control. So far, quantification of the total intracellular long-chain fatty acid concentration in yeast cells is time-consuming though when common chromatography for a volumetric analysis or staining and flow cytometry for a single-cell based analysis are used. This study, however, demonstrates that 3D-DHM facilitates a quasi-real-time measurement that allows for a rapid quantification of total intracellular lipid accumulation on a single-cell level without cell staining. Data from wild-type and lipid overproducing Y. lipolytica strains with specific yields of long-chain fatty acids in a range between 70 and 360 mg/gCDW show a good correlation with the optical volume determined by DHM, as the total lipid accumulation in the cell is typically well correlated with the long-chain fatty acid concentration. The results further correlate with data obtained from gas chromatography and flow cytometry of Nile Red-stained cells, which proves the reliability of DHM for lipid quantification in Y. lipolytica.

This study investigates a two-stage anaerobic digestion (AD) process to enhance methane production from lignocellulosic biomass, in this case bedding straw, co-fed with maize silage. The system combines phase separation and double thin-slurry recirculation under mesophilic conditions. The first hydrolysis-acidogenesis stage produced over 10 g L-1 short-chain carboxylic acids (SCCA) at an organic loading rate of 1.3-3.0 g (L d)-1 when fed with a content of 50 wt% straw. The subsequent methanogenesis stage achieved a maximum methane yield of 260 mL CH4 gCOD-1, with 50 % variation in methane production within 24 h. Thin-slurry recirculation was shown to improve the methane content by 20 %. Cell activity and quick recovery of gas production was proven after fasting periods of several weeks. The approach demonstrates the potential for flexible, efficient processing of bedding straw in AD without further treatment beyond cutting.

Dissolved hydrogen (dH2) is an important parameter in anaerobic digestion (AD) processes, in particular in a two-stage operation mode encompassing separate hydrolytic/acidogenic and methanogenic stages. Then, monitoring of dH2 as a substrate of hydrogenotrophic methanogenesis is essential to avoid feast and famine conditions. Despite its significance, there is currently a lack of established monitoring systems which are capable of reliably measuring dH2 concentrations in culture broth. To address this challenge, we propose a novel measurement system, which is based on the membrane-free extraction of hydrogen in an extraction chamber and the subsequent analysis by a metal-oxide (MOX) gas sensor. The response time of the MOX sensor lies in the range of seconds, while the entire measurement process completes within a total processing time of about 70 min). This study explores the measurement performance of the dH2 sensor in the hydrolytic/acidogenic and methanogenic stage during lab-scale anaerobic digestion. The measurement principle was consistently applied for over three months. During this period, the methanogenic stage was partly sparged with gaseous hydrogen to monitor the dH2 response afterwards. The dH2 sensor responded reliably to these and other dynamic changes. Depending on the process conditions, concentrations between < 10 and > 4,000 Pa were detected, corresponding to dH2 concentrations of < 0.074 to > 30 µmol L−1. The findings demonstrated the importance of dH2 monitoring and show that it facilitates the control of H2 addition, thereby preventing both under- and oversupply during methanogenesis.

Abstract Background. Short-chain carboxylic acids are interesting building blocks that can be synthesized from biogenic residues with the so-called dark fermentation. One challenge though is the hydrolysis of lignocellulosic residues to make them accessible for whole cell biotransformation. Accessibility can be achieved through conversion of lignocellulose through microbial exoenzymes. In this study, bacteria and fungi were isolated from different lignocellulose-containing feedstock. A workflow of an automated screening with the isolated strains for their ability to accumulate short-chain carboxylic acids in fermentation was elaborated. The screening was performed with and without addition of fungal enzymes. Results. Results show a three-fold increase in acetic acid concentration and nearly a six-fold increase in succinic acid concentration if the feedstock was pre-treated in comparison to untreated feedstock. When adding the enzyme-containing supernatant to a continuous dark fermentation with wood barks as feedstock, the acid concentration increased remarkably from 1 to 2.6 g⋅L− 1. Conclusion. The methodology, thus, is suitable to identify strains and enzyme mixtures for improving hydrolysis of complex lignocellulosic feedstock.

Lycium schweinfurthii, a wild shrub of the Solanaceae family, has received increasing attention in the last decade for its therapeutic potential in traditional medicine due to its diverse array of secondary metabolites, including phenolic substances and terpenoids. The aim of this study was to investigate the accumulation of phenolics, flavonoids, and the terpenoid lupeol in L. schweinfurthii cell suspension shake flask cultures and a single-use 2-dimensional rocking motion bioreactor. Three different media formulations were compared for in vitro cell cultures. Various parameters, such as biomass accumulation, settled cell volume, cell viability (assessed via a 2,3,5-triphenyl tetrazolium chloride assay), and sucrose consumption were determined as indicators of cell activity and growth. Total phenolic and flavonoid contents were estimated spectrophotometrically, lupeol was quantified via High-Performance Thin Layer Chromatography (HPTLC). Although a higher fresh biomass concentration of 464 g L− 1 was obtained in MS medium supplemented with a combination of each, 1 mg L− 1 of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 1-Naphthaleneacetic acid (NAA), the rocking-motion bioreactor cultivation was performed with 2 mg L− 1 NAA due to its superior reproducibility in viability, productivity, and content of bioactive compounds (e.g., phenolics, flavonoids, lupeol). A final fresh biomass concentration of 185 g L− 1 was achieved in a 16 L cultivation scale with a notable increase in the concentration of phenolics (1.4-fold) and flavonoids (1.7-fold). Most importantly, the concentration of lupeol, a pentacyclic triterpenoid known for its anti-inflammatory, antibacterial, and anti-atherogenic properties, exhibited a remarkable 5.5-fold increase in the bioreactor cultivation (585 µg g− 1) compared to shake flask cultivations (106 µg g− 1). The current study demonstrated the profound impact of media composition and non-limited fed-batch conditions in a rocking-motion bioreactor on the accumulation of bioactive compounds. The findings are also relevant to other plant cell cultures. Scaling-up of Lycium schweinfurthii suspension culture is possible in a rocking motion bioreactor for production of phenolics and lupeol.

Hydrolysis at changing hydraulic retention time, recirculation, bedding straw content in the feed, bioaugmentation and the impact of those changes on gradient formation in the liquid phase in plug-flow reactors (PFRs) was examined. The pH-value, conductivity and oxidation–reduction potential (ORP) were monitored at three spots along the PFRs to study potential correlations to process performance during a total process time of 123 weeks. The on-line monitoring showed good correlations to acidogenesis: namely, the pH and ORP to the acidification, to butyric (and lactic) acid concentration and to the acid yield. The ORP (measured at the inlet) showed the most stable correlation to acidogenesis under dynamic operation, while the conductivity (at the outlet) correlated to the acid concentration in dependence on the feedstock. Multiple measurement spots as used in this study allow to gain more information about acidogenic fermentation than a single spot, simplifying process control and automation attempts with recalcitrant feedstock.

In the present study, a tailor-made plug-flow reactor with 3D-printed parts and a multi-position monitoring of the pH-value, conductivity and ORP at the inlet, center and outlet part is introduced and applied in anaerobic digestion in order to investigate whether i) the formation of gradients can be detected, ii) ideal single or multiple spots for the location of sensors during an acidification process can be identified, and iii) the spatial gradient acquisition allows for an indirect monitoring of process performance measures, e.g. the accumulation of shortchain carboxylic acids.During the acidification of a full digestion and in dark fermentation with maize and grass silage, the multiposition monitoring of the liquid phase revealed that the local monitoring of conductivity at the inlet and center, the ORP at the center and outlet and the pH-value at the outlet are relevant to gain information of the process performance, while the spatial gradient monitoring of the conductivity between the inlet and center and of the ORP between the center and outlet are relevant for the early detection of process disturbances.

The filamentous fungus Aspergillus niger is an important production host in biotechnology. Shear force regimes are one of the key factors that affect macromorphology and product yield. While morphology changes under intensive agitation have been widely investigated, studies at a low shear force regime independently from oxygen limitation has remained a challenge. Therefore, in this study, a 2-dimensional rocking-motion bioreactor is used as an alternative platform for studying the macromorphology under a low shear force regime, but sufficient supply of dissolved oxygen. Talcum macroparticles were added at different concentrations to control the development of a certain macromorphology. Results showed that 0.25% and 1% (ww-1) of talcum led to a mixture of dispersed mycelia and loose clumps, similar to what is obtained in lab-scale stirred tank reactors. At lower talcum concentrations, distinct pellet formation was observed. Quantitative analysis of pellets showed that with 0.05% of talcum, 95% of the pellets had a diameter smaller than 850 mu m after 36 h. In case of 0.1% of talcum, 94% ( +/- 5.0%) of the pellets had a diameter below 650 mu m.The presented approach makes it possible to achieve a certain morphology as, for example, observed in large scale cultivations to study the consequences for product synthesis.

Since natural resources for the bioproduction of commodity chemicals are scarce, waste animal fats (WAF) are an interesting alternative biogenic residual feedstock. They appear as by-product from meat production, but several challenges are related to their application: first, the high melting points (up to 60 °C); and second, the insolubility in the polar water phase of cultivations. This leads to film and clump formation in shake flasks and microwell plates, which inhibits microbial consumption. In this study, different flask and well designs were investigated to identify the most suitable experimental set-up and further to create an appropriate workflow to achieve the required reproducibility of growth and product synthesis. The dissolved oxygen concentration was measured in-line throughout experiments. It became obvious that the gas mass transfer differed strongly among the shake flask design variants in cultivations with the polyhydroxyalkanoate (PHA) accumulating organism Ralstonia eutropha. A high reproducibility was achieved for certain flask or well plate design variants together with tailored cultivation conditions. Best results were achieved with bottom baffled glass and bottom baffled single-use shake flasks with flat membranes, namely, >6 g L-1 of cell dry weight (CDW) with >80 wt

Abstract Hydrolysis at changing hydraulic retention time (HRT), recirculation, bedding straw content in the feed, bioaugmentation and the impact of those changes on gradient formation in the liquid phase in plug-flow reactors (PFRs) was examined. The pH, conductivity and oxidation-reduction-potential (ORP) were monitored at three spots along the PFRs to study potential correlations of gradient formation to process performance during a total process time of 123 weeks. Local on-line monitoring showed good correlations to acidogenesis: namely the pH and ORP to the acidification, to butyric (and lactic) acid concentration and to the acid yield. The ORP (inlet) showed the most stable correlation to acidogenesis under dynamic operation, while the conductivity (outlet) correlated to the acid concentration in dependance on the feedstock. Multiple measurement spots as used in this study allow to gain more information about acidogenic fermentation than a single measurement spot, simplifying process control and automation attempts with recalcitrant feedstock.

Two parallel plug-flow reactors were successfully applied as a hydrolysis stage for the anaerobic pre-digestion of maize silage and recalcitrant bedding straw (30