Filaments in Bioprocesses

Filamentous fungi are widely used in the biotechnology industry for the production of industrial enzymes. Thus, considerable work has been done with the purpose of characterizing these processes. The ultimate goal of these efforts is to be able to control and predict fermentation performance on the basis of “standardized” measurements in terms of morphology, rheology, viscosity, mass transfer and productivity. However, because the variables are connected or dependent on each other, this task is not trivial. The aim of this review article is to gather available information in order to explain the interconnectivity between the different variables in submerged fermentations. An additional factor which makes the characterization of a fermentation broth even more challenging is that the data obtained are also dependent on the way they have been collected—meaning which technologies or probes have been used, and on the way the data is interpreted—i.e. which models were applied. The main filamentous fungi used in industrial fermentation are introduced, ranging from Trichoderma reesei to Aspergillus species. Due to the fact that secondary metabolites, like antibiotics, are not to be considered bulk products, organisms like e.g. Penicillium chrysogenum are just briefly touched upon for the description of some characterization techniques. The potential for development of different morphological phenotypes is discussed as well, also in view of what this could mean to productivity and—equally important—the collection of the data. An overview of the state of the art techniques for morphology characterization is provided, discussing methods that finally can be employed as the computational power has grown sufficiently in the recent years. Image analysis (IA) clearly benefits most but it also means that methods like near infrared measurement (NIR), capacitance and on-line viscosity now provide potential alternatives as powerful tools for characterizing morphology. These measuring techniques, and to some extent their combination, allow obtaining the data necessary for supporting the creation of mathematical models describing the fermentation process. An important part of this article will indeed focus on describing the different models, and on discussing their importance to fermentations of filamentous fungi in general. The main conclusion is that it has not yet been attempted to develop an overarching model that spans across strains and scales, as most studies indeed conclude that their respective results might be strain specific and not necessarily valid across scales.

Filamentous cultures, such as fungi and actinomycetes, contribute substantially to the pharmaceutical industry and to enzyme production, with an annual market of about 6 billion dollars. In mechanically stirred reactors, most frequently used in fermentation industry, microbial growth and metabolite productivity depend on complex interactions between hydrodynamics, oxygen transfer, and mycelial morphology. The dissipation of energy through mechanically stirring devices, either flasks or tanks, impacts both microbial growth through shearing forces on the cells and the transfer of mass and energy, improving the contact between phases (i.e., air bubbles and microorganisms) but also causing damage to the cells at high energy dissipation rates. Mechanical-induced signaling in the cells triggers the molecular responses to shear stress; however, the complete mechanism is not known. Volumetric power input and, more importantly, the energy dissipation/circulation function are the main parameters determining mycelial size, a phenomenon that can be explained by the interaction of mycelial aggregates and Kolmogorov eddies. The use of microparticles in fungal cultures is also a strategy to increase process productivity and reproducibility by controlling fungal morphology. In order to rigorously study the effects of hydrodynamics on the physiology of fungal microorganisms, it is necessary to rule out the possible associated effects of dissolved oxygen, something which has been reported scarcely. At the other hand, the processes of phase dispersion (including the suspended solid that is the filamentous biomass) are crucial in order to get an integral knowledge about biological and physicochemical interactions within the bioreactor. Digital image analysis is a powerful tool for getting relevant information in order to establish the mechanisms of mass transfer as well as to evaluate the viability of the mycelia. This review focuses on (a) the main characteristics of the two most common morphologies exhibited by filamentous microorganisms; (b) how hydrodynamic conditions affect morphology and physiology in filamentous cultures; and (c) techniques using digital image analysis to characterize the viability of filamentous microorganisms and mass transfer in multiphase dispersions. Representative case studies of fungi (Trichoderma harzianum and Pleurotus ostreatus) exhibiting different typical morphologies (disperse mycelia and pellets) are discussed.

Living with limits. Getting more from less. Producing commodities and high-value products from renewable resources including waste. What is the driving force and quintessence of bioeconomy outlines the lifestyle and product portfolio of Aspergillus, a saprophytic genus, to which some of the top-performing microbial cell factories belong: Aspergillus niger, Aspergillus oryzae and Aspergillus terreus. What makes them so interesting for exploitation in biotechnology and how can they help us to address key challenges of the twenty-first century? How can these strains become trimmed for better growth on second-generation feedstocks and how can we enlarge their product portfolio by genetic and metabolic engineering to get more from less? On the other hand, what makes it so challenging to deduce biological meaning from the wealth of Aspergillus -omics data? And which hurdles hinder us to model and engineer industrial strains for higher productivity and better rheological performance under industrial cultivation conditions? In this review, we will address these issues by highlighting most recent findings from the Aspergillus research with a focus on fungal growth, physiology, morphology and product formation. Indeed, the last years brought us many surprising insights into model and industrial strains. They clearly told us that similar is not the same: there are different ways to make a hypha, there are more protein secretion routes than anticipated and there are different molecular and physical mechanisms which control polar growth and the development of hyphal networks. We will discuss new conceptual frameworks derived from these insights and the future scientific advances necessary to create value from Aspergillus Big Data.

The aim of this work is to review bioprocess engineering aspects of lovastatin (antihypercholesterolemia drug) production by Aspergillus terreus in the submerged culture in the bioreactors of various scale presented in the scientific literature since the nineties of the twentieth century. The key factor influencing the cultivation of any filamentous species is fungal morphology and that is why this aspect was treated as the starting point for further considerations. Fungal morphology is known to have an impact on the following issues connected with the cultivation of A. terreus reviewed in this article. These are broth viscosity in conjunction with non-Newtonian behaviour of the cultivation broths, and multistage oxygen transfer processes: from gas phase (air) to liquid phase (broth) and diffusion in the fungal agglomerates. The latest achievements concerning the controlling A. terreus morphology during lovastatin biosynthesis with the use of morphological engineering techniques were also reviewed. Last but not least, some attention was paid to the type of a bioreactor, its operational mode and kinetic modelling of lovastatin production by A. terreus.

Solid-state fermentation (SSF) with filamentous fungi is a promising technique for the production of a range of biotechnological products and has the potential to play an important role in future biorefineries. The performance of such processes is intimately linked with the mycelial mode of growth of these fungi: Not only is the production of extracellular enzymes related to morphological characteristics, but also the mycelium can affect bed properties and, consequently, the efficiency of heat and mass transfer within the bed. A mathematical model that describes the development of the fungal mycelium in SSF systems at the particle scale would be a useful tool for investigating these phenomena, but, as yet, a sufficiently complete model has not been proposed. This review presents the biological and mass transfer phenomena that should be included in such a model and then evaluates how these phenomena have been modeled previously in the SSF and related literature. We conclude that a discrete lattice-based model that uses differential equations to describe the mass balances of the components within the system would be most appropriate and that mathematical expressions for describing the individual phenomena are available in the literature. It remains for these phenomena to be integrated into a complete model describing the development of fungal mycelia in SSF systems.

Filamentous fungi, especially basidiomycetes, produce a wide range of metabolites, many of which have potential biotechnological and industrial applications. Solid-state fermentation (SSF) is very suitable for the cultivation of basidiomycetes since it mimics the natural habitat of these fungi. Some of the major advantages of SSF are the robustness of the process, the use of low-cost residual materials as substrates, and the reduced usage of water. However, monitoring key variables is difficult, which makes process control a challenge. Specifically, it is very difficult to determine the biomass during SSF process involving basidiomycetes. This is problematic, as the biomass is normally a key variable in mass and energy balance equations. Further, the success of fungal SSF processes is often evaluated, in part, based on the growth of the fungus. Direct determination of the dry weight of biomass is impossible and indirect quantification techniques must be used. Over the years, various determination techniques have been developed for the quantification of fungal biomass in SSF processes. The current review gives an overview of various direct and indirect biomass determination methods, discussing their advantages and disadvantages.

The review presents a comprehensive overview on available solutions for the monitoring and modeling of various aspects of hairy root growth processes. Several online and offline measurement principles get explained exemplary and are being compared. It was found that no direct online measurement principle for hairy root biomass in submerged and solid-state culturing environment is available. However, certain indirect methods involving one or more measurement values have been developed for biomonitoring of hairy roots especially in bioreactors. In the field of modeling of hairy root growth processes, four independent architectures (continuous models, metabolic flux analysis, agent-based models, and artificial neural networks) are described and compared including literature references. The discussion is structured into microscopic model approaches, addressing only certain aspects of growth, and macroscopic model approaches, describing the hairy root network as a whole. An agent-based macroscopic model based on phenomenological data acquired with systematic imaging of hairy roots on culture dishes together with a 3D visualization of simulation results is presented in detail.

Plants synthesize a wide variety of natural products, which are traditionally termed secondary metabolites and, more recently, coined specialized metabolites. While these chemical compounds are employed by plants for interactions with their environment, humans have long since explored and exploited plant secondary metabolites for medicinal and practical uses. Due to the tissue-specific and low-abundance accumulation of these metabolites, alternative means of production in systems other than intact plants are sought after. To this end, hairy root culture presents an excellent platform for producing valuable secondary metabolites. This chapter will focus on several major groups of secondary metabolites that are manufactured by hairy roots established from different plant species. Additionally, the methods for preservations of hairy roots will also be reviewed.

White biotechnologies have several challenges to overcome in order to become a viable industrial process. Achieving highly concentrated lignocellulose materials and releasing fermentable substrates, with controlled kinetics in order to regulate micro-organism activity, present major technical and scientific bottlenecks. The degradation of the main polymeric fractions of lignocellulose into simpler molecules is a prerequisite for an integrated utilisation of this resource in a biorefinery concept. The characterisation methods and the observations developed for rheology, morphology, etc., that are reviewed here are strongly dependent on the fibrous nature of lignocellulose, are thus similar or constitute a good approach to filamentous culture broths. This review focuses on scientific works related to the study of the rheological behaviour of lignocellulose suspensions and their evolution during biocatalysis. In order to produce the targeted molecules (synthon), the lignocellulose substrates are converted by enzymatic degradation and are then metabolised by micro-organisms. The dynamics of the mechanisms is limited by coupled phenomena between flow, heat and mass transfers in regard to diffusion (within solid and liquid phases), convection (mixing, transfer coefficients, homogeneity) and specific inhibitors (concentration gradients). As lignocellulose suspensions consist of long entangled fibres for the matrix of industrial interest, they exhibit diverse and complex properties linked to this fibrous character (rheological, morphological, thermal, mechanical and biochemical parameters). Among the main variables to be studied, the rheological behaviour of such suspensions appears to be determinant for process efficiency. It is this behaviour that will determine the equipment to be used and the strategies applied (substrate and biocatalysis feed, mixing, etc.). This review provides an overview of (i) the rheological behaviour of fibrous materials in suspension, (ii) the methods and experimental conditions for their measurements, (iii) the main models used and (iv) their evolution during biocatalytic reactions with a focus on enzymatic hydrolysis.