Foam formation is a massive challenge in submerged aerated bioprocesses, e.g., in beer fermentation. While the use of antifoam may easily overcome foaming at laboratory scale, it is often an unattractive solution since the challenge remains in future upscaling, as reduced mass transfer and extra steps in product purification and analytics result in increased costs. Interestingly, the number of studies tackling this challenge is relatively low, although literature suggests a range of alternatives, from avoiding foaming to means of controlling or even using foaming as an in situ product removal. Here we give an overview of the topic in five subsections. (1) We argue that a sound understanding of the molecular origin of foaming can facilitate solutions for overcoming the challenge while introducing some long-known challenges (i.e., in beer fermentation). We then review in (2) the apparent avoidance of foam formation before we in (3) summarize possibilities to reduce and control foam after its formation. Subsequently, in (4), we discuss possible solutions that take advantage of foam formation, for example, via foam fractionation for in situ product removal. Finally, in (5), we provide an overview of microbial strain engineering approaches to cope with some aspects of foaming in fermentations. With this review, we would like to sensitize and inform the interested reader while offering an overview of the current literature for the expert, particularly with regard to the foam special issue in Discover Chemical Engineering.

Immobilization is an appropriate tool to ease the handling and recycling of enzymes in biocatalytic processes and to increase their stability. Most of the established immobilization methods require case-to-case optimization, which is laborious and time-consuming. Often, (chromatographic) enzyme purification is required and stable immobilization usually includes additional cross-linking or adsorption steps. We have previously shown in a few case studies that the molecular biological fusion of an aggregation-inducing tag to a target protein induces the intracellular formation of protein aggregates, so called inclusion bodies (IBs), which to a certain degree retain their (catalytic) function. This enables the combination of protein production and immobilization in one step. Hence, those biologically-produced immobilizates were named catalytically-active inclusion bodies (CatIBs) or, in case of proteins without catalytic activity, functional IBs (FIBs). While this strategy has been proven successful, the efficiency, the potential for optimization and important CatIB/FIB properties like yield, activity and morphology have not been investigated systematically. We here evaluated a CatIB/FIB toolbox of different enzymes and proteins. Different optimization strategies, like linker deletion, C- versus N-terminal fusion and the fusion of alternative aggregation-inducing tags were evaluated. The obtained CatIBs/FIBs varied with respect to formation efficiency, yield, composition and residual activity, which could be correlated to differences in their morphology; as revealed by (electron) microscopy. Last but not least, we demonstrate that the CatIB/FIB formation efficiency appears to be correlated to the solvent-accessible hydrophobic surface area of the target protein, providing a structure-based rationale for our strategy and opening up the possibility to predict its efficiency for any given target protein. We here provide evidence for the general applicability, predictability and flexibility of the CatIB/FIB immobilization strategy, highlighting the application potential of CatIB-based enzyme immobilizates for synthetic chemistry, biocatalysis and industry.

We have recently demonstrated that fusions of different target enzymes to the coiled-coil domain TDoT induced the formation of catalytically active inclusion bodies (CatIBs) in E. coli (doi:10.1016/j.jbiotec.2017.04.033). Here we show that the CatIB properties can be tailored to the requirements of different reaction systems using two different coiled-coil domains as fusion tags: TDoT and 3HAMP. As an example the benzaldehyde lyase from Pseudomonas fluorescens (PfBAL) was chosen, which catalyzes the formation of 2-hydroxy ketones and benzoins from aromatic donor aldehydes and aromatic or aliphatic acceptor aldehydes. Using these fusion tags two different kinds of PfBAL-CatIBs were successfully produced that differ in morphology, solubility during washing steps, initial rate activity, protein and lipid content. TDoT-PfBAL and 3HAMP-PfBAL CatIBs were studied relative to the soluble enzyme concerning their application in biocatalysis in continuous reaction mode and in batch using a mono- and biphasic solvent system. Both CatIBs outperformed the soluble enzyme with respect to stability under reaction conditions. In the buffer/DMSO system used for the continuous synthesis of (R)-2-hydroxy-1-phenylpropanone, TDoT-PfBAL was superior to the soluble enzyme. Besides, 3HAMP-PfBAL was more suitable in a biphasic reaction system for the synthesis of (R)-benzoins and revealed an up to 3-fold higher activity compared to the soluble enzyme.

Bioreactors are the operative backbone, for example, for the production of biopharmaceuticals, biomaterials in tissue engineering, and sustainable substitutes for chemicals. Still, the Achilles' heel of bioreactors nowadays is the aeration which is based on intense stirring and gas sparging, yielding inherent drawbacks such as shear stress, foaming, and sterility concerns. We present the synergistic combination of simulations and experiments toward a membrane stirrer for the efficient bubble‐free aeration of bioreactors. A digital twin of the bioreactor with an integrated membrane‐module stirrer (MemStir) was developed with computational fluid dynamics (CFD) studies addressing the determination of fluid mixing, shear rates, and local oxygen concentration. Usability of the MemStir is shown in a foam‐free recombinant production process of biosurfactants (rhamnolipids) from glucose with different strains of Pseudomonas putida KT2440 in a 3‐L vessel and benchmarked against a regular aerated process. The MemStir delivered a maximal oxygen transfer rate (OTR_max) of 175 mmol L⁻¹ h⁻¹ in completely foam‐free cultivations. With a high space‐time yield (STY) of 118 mg_RL L⁻¹ h⁻¹ during a fed‐batch fermentation, the effectiveness of the novel MemStir is demonstrated. Simulations show the generic value of the MemStir beyond biosurfactant production, for example, for animal cell cultivation.

Alcanivorax borkumensis is one of the most abundant marine bacteria found in areas of oil spills, where it degrades alkanes. The production of a glycine-glucolipid is considered an essential element for alkane degradation.

Polyol lipids (a.k.a. liamocins) produced by the polyextremotolerant, yeast-like fungus Aureobasidium pullulans are amphiphilic molecules with high potential to serve as biosurfactants. So far, cultivations of A. pullulans have been performed in media with complex components, which complicates further process optimization due to their undefined composition. In this study, we developed and optimized a minimal medium, focusing on biosurfactant production. Firstly, we replaced yeast extract and peptone in the best-performing polyol lipid production medium to date with a vitamin solution, a trace-element solution, and a nitrogen source. We employed a design of experiments approach with a factor screening using a two-level-factorial design, followed by a central composite design. The polyol lipid titer was increased by 56% to 48 g L⁻¹, and the space-time yield from 0.13 to 0.20 g L⁻¹ h⁻¹ in microtiter plate cultivations. This was followed by a successful transfer to a 1 L bioreactor, reaching a polyol lipid concentration of 41 g L⁻¹. The final minimal medium allows the investigation of alternative carbon sources and the metabolic pathways involved, to pinpoint targets for genetic modifications. The results are discussed in the context of the industrial applicability of this robust and versatile fungus.