Method for selective biocatalyst separation in high producing biocatalytic reactor systems

Abstract

Method for selective cell retention of a micro-organism comprising a (semi- )continuous fermentative production of an organic substance of interest, in a bioreactor system, where fluidly connected is present (a) a fermentation zone, wherein the organic substance is produced using a biocatalytic micro-organism, and (b) a separation zone, wherein a reaction mixture, comprising an aqueous fraction, containing the biocatalytic micro-organism, and the produced substance, is fed to the separation zone, or wherein at least the aqueous fraction containing the biocatalytic microbiological cells is fed to the separation zone, and wherein the reaction mixture is separated in two fraction, distinguished in solid content, as in the separation zone, the first liquid fraction is enriched in the biocatalytic micro-organism (relative to the second liquid fraction) and the second liquid fraction is enriched in a solid matter different from the biocatalytic micro-organism (relative to the first liquid fraction).

Description

P138981PC00Title: METHOD FOR SELECTIVE BIOCATALYST SEPARATION IN HIGH PRODUCING BIOCATALYTIC REACTOR SYSTEMSThe present disclosure relates to a method for fermentatively producing an organic substance. In particular, the present disclosure relates to a method, comprising a selective cell retention of a micro-organism used as a biocatalyst during a continuous or semi-continuous fermentative production of an organic substance.There is a strong world-wide effort to provide more sustainable production routes, e.g. transition from fossil based substances to bio-based substances, for production of various organic substances, e.g. alcohols, esters, amino acids, carbohydrates, ethers, lipids, ketones, aldehydes, organic acids, pyridines, and proteins. These substances can be used in a variety of applications for example as flavours, fragrances, cosmetic ingredients, food ingredients, nutraceutical products, bioinsecticides, specialty chemicals, commodity chemicals, or as biofuels. These substances can either provide a sustainable replacement of a known product or a novel more sustainable product.These more sustainable production routes include amongst others biocatalytic processes. It is known in the art to produce organic substances by biocatalytic processes such as fermentation and cell-free conversions. In a fermentation process, micro-organisms are used to convert a suitable substrate into an organic substance of interest. In a cell-free conversion, enzymes convert a substrate into an organic substance of interest. As is generally known in the art, various microorganisms and enzymes are known that can be used on an industrial scale for the production of a wide variety of organic substances. Generally known fermentative processes include the use of natural micro-organisms and / or genetically modified organisms, e.g. yeasts or bacteria, for the production of various organic substances, e.g. alcohols, esters, amino acids, carbohydrates, lipids, ketones, aldehydes, organic acids, ethers, pyridines, imines, proteins.Organisms may be genetically modified to increase the product titre of a naturally produced organic substance and / or to enable a microorganism to produce an organic substance that it does not produce naturally. E.g. a micro-organism maybe modified by incorporating genes from a plant responsible for the production of an organic substance, e.g. a terpene, a terpenoid, not naturally produced by the micro-organism.Current high producing continuous or semi-continuous fermentative biocatalytic reactor systems, most often chemostat, lack the ability for selective separation of biocatalytic micro-organisms (biocatalysts) from other solid matter. This lack of selective biocatalyst separation results in the following potential debits. (1) In any system, but especially for higher biocatalyst retention systems like air lift reactors and membrane bioreactors, (aged) biocatalyst and / or biocatalyst material with reduced to no activity builds up in the biocatalytic reaction medium leading to reduced biocatalytic reaction activity and efficiency as the run progresses. (2) In case of different populations of biocatalyst present inside the biocatalytic reaction system there is currently no selective way to control the size of each population other than trying to apply selection pressures onto the system to influence the population sizes. (3) In case of a solid organic product that precipitates in the biocatalytic medium there is currently no selective means of separation of the product from the biocatalytic micro-organisms and / or other microbial matter.Especially in the waste water treatment industry (WWTP) more extensive research has been done into biocatalyst retention and population shifts in continuous flow systems. However in waste water treatment the systems are focussed on converting matter and removing organic content and nitrogen, by activated sludge based systems. The reactor systems are not used for the production of end-products and are also mixed-culture (open) systems. Due to the diluted (feed) nature of the system, as water treatment is the objective, the liquid flowrates, and the corresponding hydraulic retention time of the system differs. Many reactor technologies and their impact on solid retention time (SRT) in combination with hydraulic retention time (HRT) and a specific conversion of components have been investigated. Reactor technologies tested comprise ‘closed digester tank’ (CDT, ), ‘Continuously stirrer tank reactor’ (CSTR,), Anaerobic sequencing batch reactor, stirred tank, modified ‘Anaerobic baffled reactor’ (ABR), ‘upflow anaerobic sludge blanket’ (UASB,), ‘Expanded granular sludge blanket’ (EGSB), ‘upflow anaerobic sludge fixed film reactor’ (UASFF), ‘Anaerobic fluidizedbed’ (AFB), 'membrane bioreactor’ (MBR), Anaerobic filter. These reactor systems operate at relatively low growth rates (mu) in the range of 0.023 h’1to 0.11 day-1, lower feed substrate concentrations than typical biocatalytic reaction systems (~0.3 to 19 wt%), relative low biocatalyst concentrations ‘mixed liquor suspended solids’ (MLSS) of 1.5-5 g / L for stirred tank kind of systems with similar SRT to HRT, but for airlift reactor systems as well and an MLSS of 10-12 g / L for systems with higher SRT (like a MBR) and relatively lower substrate conversion rates (-0.02 to 0.66 g / L / h) than typical biocatalytic reaction systems. Typical SRT ranged from 0.8 to 100 days and the HRT ranged from 0.3 to 16 days. It was found in this research that there is an optimum and even higher SRT isn’t always better. In all this is shown to illustrate that the application of solid and liquid residence time in WWTP covers a wide range and cannot be used to optimize a specific biocatalytic conversion step without undue burden.Especially relevant for assessment of more intensified systems in WWTP, the ‘Anammox’ processes is relevant; Also here solid retention time (and population distribution) control is desired but is limited, as shown by [Lackner et al, Full-scale partial nitridation / anammox experiences An application survey, water research 55 (2014) 292-303] and they surveyed authors stating: “Aside from growing and sustaining the slow growing anammox bacteria, balanced activity of aerobic ammonium oxidizing bacteria needs to be established in line with a suppression or out-selection of nitrite oxidizing bacteria. The growth rates of ammonium oxidizing bacteria are usually higher than those of nitrite oxidizing bacteria at elevated temperatures (> 30°C) which makes selective wash-out of nitrite oxidizing bacteria possible in suspended biomass systems for partial nitridation by adjusting the solids retention time (SRT) at a minimum level That concept, however, is not applicable for biofilm systems because biofilms can sustain microorganisms with very different growth kinetics due to the undefined SRT and their distinct substrate gradients; further also for combined PN / A SRT cannot be applied as sole selection criterion due to the significantly slower growth rate of the anammox biomass. The most practical approach to limit nitrite oxidation is considered to be reactor operation under oxygen limited conditions which favours growth of ammonium oxidizing bacteria versus nitrite oxidizing bacteria, as their oxygenaffinity is higher and combined with additionally competition for nitrite by the anammox bacteria.”Nicolella et al [wastewater treatment with particulate biofilm reactors, Journal of Biotechnology 80 (2000) 1-33] showed in Figure 2 that for Waste Water Treatment Systems, different reactor geometry and configurations, with their own intrinsic cell settling and retention properties, allow operation of waste water treatment, for a wide range of substrate concentrations and flowrates. However, high substrate concentrations in waste water treatment indicates the use of single cells (under high growth rate), as any dilution rate of the system is acceptable because wash-out is not limiting the operation in waste water treatment. For a fermentative production system this would lead to an undesirable product yield as substantial amount of substrate go to biomass growth.Apart from certain reactor configurations, current methods for increased biocatalyst retention include immobilization of the biocatalyst by fixating microbes or cell-free enzymes by for example a matrix, biofilm support structure, or preformed carrier. This does generally not allow selective separation of biocatalytically (highly) active living microbes from non-viable microbes that have at least substantially lost their activity. Further, fixation has drawbacks, such as the added complexity by having to immobilise the microbes, mass transfer limitation and a reduced biocatalytic activity, once fixated. Further, the support occupies inert space in the bioreactor system, which may also have an adverse effect on production capacity. Other methods include retention means of a membrane, which is generally not adequate either for selective retention of living cells relative to other solid matter. The use of membranes also adds to complexity plus brings typical challenges like risk of fouling of the membrane surface. Further, it has been proposed to separate biocatalyst from a liquid product by enhancing gravity of the biocatalyst itself by for example flocculation, like for example yeast flocculation for ethanol production in an ultra-low aerated biocatalytic reactor system (0.005 wm aeration, (Zhao et al, Yeast flocculation New story in fuel ethanol production, Biotechnology Advances 27 (2009) 849-856). Retention of the flocs is problematic due to the CO2 formation of the high producing system, in relation to the requirement of effluent withdrawal under the given conditions. For aerobic systemsan even higher gas input is required, due to oxygen now being required as substrate.Traditionally, fermentations and recovery of fermentatively produced substances were carried out using a batch process, but nowadays (semi-)continuous recovery processes and (semi-)continuous fermentative processes are also known in the art.A (semi-)continuous operation allows for control of residence time of certain fluid phases and / or substances in the system. Semi-continuous or Continuous fermentation in combination with continuous in-situ substance recovery, process sustainability and economic viability can be improved.US2017 / 0073710 Al relates to a method for recovering a specific group of products, namely liquid lipids or hydrocarbons, from a fermentation mixture, wherein first the lipid or hydrocarbon is fermentatively prepared in a fermentation reactor (or compartment thereof) and thereafter the fermentation reaction mixture is fed to a separator (a second vessel or a separate compartment). In the separator, phase-separation of the reaction mixture into an aqueous phase and a product phase is promoted by injecting a gas, thereby forming a product layer, which product layer is collected. Optionally, the process comprises a continuous operation. In particular, the working examples illustrate a continuous mode of operation for the product recovery stage. US2017 / 0073710 acknowledges in paragraph

[0088] that the presence of a specific part of the biomass, namely cell debris, has a negative effect on separating the product phase from the aqueous phase. However, it is not described or suggested to a separate a biocatalytically active microorganism from the cell debris, let alone that this would be desired.WO2021 / 010822 and WO2024 / 049295 disclose devices and methods allowing the continuous fermentative production and extraction of produced product in a single apparatus, using extractive overlay fermentation. Herein liquid-liquid phase separation by gravitational forces is applied as In Situ Product Recovery power (ISPR), which is integrated in the fermentation reactor system itself.Although the devices and methods of WO2021 / 010822 and WO2024 / 049295 thus allow for an additional degree of freedom in operation and allows matching of ISPR and separation capacity or requirement, it does not describe or suggest to selectively separate a desired active micro-organism, used as a biocatalyst for theproduction of the organic substance of interest, directly from other solid matter, like biocatalyst material with reduced to no activity, a different cell phenotype, other species of cells (contamination) and precipitated product in the bioreactor system, let alone how to achieve this.It is an object of the present disclosure to provide a novel method wherein an organic substance is fermentatively produced, in particular a method that addresses one or more drawbacks of known methods to produce an organic substance with the help of a biocatalyst, for instance one or more drawbacks known or mentioned for prior art methodology, as described herein.The inventors realised in particular that current technologies suffer from limitations like declining biocatalyst performance over time, reduced mass transfer, restrictions on other process operating parameters like aeration, high material cost, emulsion formation in the fermentation broth over time, fouling, pressure drop and the risk of contamination (introduced by external equipment for biocatalyst rich recycle streams like centrifuges).The inventors realised that it is possible to selectively retain biocatalytically active microbiological cells used for fermentative preparation of an organic substance of interest in a method comprising a (semi-)continuous operation of a bioreactor system. Moreover, they realised that this can be achieved by selectively separating biocatalytically active micro-organisms from other solid matter in a (semi-)continuously operated biocatalytic reactor system, and that this can be used to contribute to maintaining a satisfactory or even improved substrate conversion rate to a product of interest for prolonged run duration.Amongst others, the inventors realised that to enable optimisation on yield for fermentative productive systems more specific biomass retention and separation is required. Overall the flowrate of the substrate feed in, the substrate concentration and yield together give the productivity of the system. Product retention time is also a design parameter in production systems. This reinforces that the growth rate, the degree of cell retention (growth requirement) and ability to retain viable cell matter is an important requirement for reactor and process design. When combined more effectively this enables the development of processes with increased performance, specifically yield.Accordingly, the present disclosure relates to a method comprising a (semi-)continuous fermentative production of an organic substance of interest, in a bioreactor system comprising(a) a fermentation zone, wherein the organic substance is fermentatively produced using a biocatalytic micro-organism, and(b) a separation zone, wherein a reaction mixture, said reaction mixture comprising an aqueous fraction, containing the biocatalytic micro-organism, and the produced organic product, is (semi-)continuously fed to the separation zone, or wherein at least the aqueous fraction containing the biocatalytic microbiological cells is (semi-) continuously fed to the separation zone, and wherein the reaction mixture is separated into at least two fractions, distinguished in solid content, as in the separation zone, the first liquid fraction is enriched in the biocatalytic microorganism (relative to the second liquid fraction) and the second liquid fraction is enriched in a solid matter different from the biocatalytic micro-organism (relative to the first liquid fraction).Thus the present method allows the selective retention of the biocatalytically active cells in the bioreaction system. The retained cells (present in the first fraction) or a substantial part thereof, are typically returned to the fermentation zone.Typically, the method according to the present disclosure comprises a continuous or semi-continuous fermentative production of one or more organic substances in the bioreactor system in which conversion of substrate by the living micro-organism into at least one organic substance occurs in the fermentation zone, wherein the micro-organism is mobile in the reaction mixture. Thus, the biocatalytic micro-organism is (freely) distributed in the reaction mixture in the fermentation zone, at least during normal operation. The micro-organism is typically unbound to a support / carrier material. When freely distributed, typically at least a substantial part of the micro-organism is dispersed as individual cells, although it is also possible that biocatalytic cells form clusters essentially consisting of cells, in particular if the separation comprises some form of cluster formation, the clusters are returned to the fermentation zone, and the fermentation zone is operated under conditions at which the clusters are not fully disintegrated (such as when a relatively low stirring force is applied / relatively low turbulence)and the microbes are actively pushed to aggregate to form solid particulates in the desired size range due to the applied conditions.The reaction mixture is provided in the fermentation zone, which reaction mixture comprises the micro-organism and an aqueous phase, the aqueous phase comprising a substrate for the micro-organism. A substrate as such can be a liquid, a solid or a gas. A substrate is usually dissolved in the aqueous phase for efficient uptake by the micro-organism.The organic substance is fermentatively prepared from the substrate in the presence of the micro-organism in the fermentation zone. The micro-organism thus acts as a biocatalyst.A method according to the disclosure comprises a stage during which the fermentative production and a separation are simultaneously carried out, which stage may herein also be referred as the ‘simultaneous production and separation stage’, or ‘simultaneous stage’. During at least a (substantial) part of said simultaneous stage, process conditions are usually in an (essentially) steady state, in particular when operating under continuous process conditions.In accordance with a method of the present disclosure, the substrate is typically at least during a substantial part of said simultaneous production and separation stage, preferably during an essentially steady state production stage, continuously or semi-continuously fed into the fermentation zone.In accordance with a method of the present disclosure, the reaction mixture, comprising the micro-organism and the produced organic substance, or at least an aqueous fraction of the reaction mixture, comprising the micro-organism and optionally the produced organic substance, is typically at least during a substantial part of said simultaneous production and separation stage, preferably during an essentially steady state production stage, continuously or semi-continuously fed from the fermentation zone into the separation zone.In a method according to the disclosure, the reaction mixture, comprising the micro-organism and the produced organic substance, or at least an aqueous fraction of the reaction mixture, said aqueous fraction comprising the microorganism, fed into the separation zone, is subjected to a separation into at least two fractions, thereby forming a first fraction enriched in the micro-organism and a second fraction enriched in solid matter different from said micro-organism inwhich the first fraction is enriched. The separation typically comprises the formation of at least two liquid layers (an upper layer and a lower layer), wherein one of said two layers forms the first fraction (enriched in the biocatalytic microorganism) and the other of said two layers forms the second fraction (enriched in a different solid matter).At least during said simultaneous stage, in particular during an essentially steady state, at least a part of said second fraction from the bioreactor system is typically continuously or semi-continuously removed from the bioreactor system. The removed (part of the) second fraction may be further processed by any suitable, e.g. known, downstream processing e.g. as described in the prior art described herein. Such downstream processing can comprise (semi)continuous processing, batch processing or both.At least during said simultaneous stage, in particular during an essentially steady state, at least a part of said first fraction enriched in the micro-organism is returned from the separation zone to the fermentation zone.The present disclosure thus advantageously provides a method for selective microbial biomass separation in biocatalytic reactor systems in particular a method for selective microbial biomass separation in (semi-)continuous high producing fermentation system that overcomes one or more of the disadvantages of known microbial retention systems and methods. Advantageously the method according to the present disclosure allows for maintaining substrate conversion rates for prolonged run duration, by desired fraction of biomass, those with i.e. preferably the optimal conversion rate and product yield. This is especially beneficial for fermentation processes with slow growth. This method can be applied to both aerobic and anaerobic processes, in both general and precision fermentation processes.A method according to the present disclosure can be applied to any fermentation; it has specific benefits for processes that suffer from one or more of: product instability, product inhibition, an equilibrium limitation, a product yield limitation due to excess biomass formation, biocatalytic biomass activity loss over time.A major benefit of the present method is to enable or facilitate desired prolonged microbial activity (longer effective fermentation time), in particularenabling or facilitating maintaining substrate conversion rates for a prolonged run duration.The method of the present disclosure allows for process improvements like improved yield, reduced downtime, reduced resource requirements, reduced energy consumption. Leading to improved sustainability and improved economic feasibility of the process and therefore according to the present disclosure thus enables more fermentation processes to be competitive and successful in the current market.In particular effective is a method according to any of the claims 1-14.The method of the present disclosure can be beneficial for the upstream processing (biocatalytic reaction) as well as the downstream processing and / or purification.The disclosure mitigates one or more restrictions of known methods, like declining microbial performance over time, reduced mass transfer, restrictions on other process operating parameters like aeration, high material cost, fouling, pressure drop and the risk of contamination (introduced by external equipment for biocatalyst rich recycle streams like centrifuges.The selective retention of the biocatalytic micro-organism can be used to stimulate selection pressure for a slow- growing biocatalytic micro-organism with a favourable production rate or yield. This can be accomplished by biocatalytic microorganism retention in the fermentation zone (via returning said micro-organism in the first phase to the fermentation zone) in combination with removal of biomass with reduced or no activity towards the fermentative, or by population selection due to selective separation and removal of faster growing biocatalytic microorganisms.Terminology used for describing particular embodiments is not intended to be limiting of the invention. Unless defined otherwise herein, terms are as defined in WO2021 / 010822 and WO2024 / 049295. When not defined the terms are used in agreement with how they generally defined in the art.As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. The term “or” as used herein is defined as “and / or” unless specified otherwise.The term "and / or" includes any and all combinations of one or more of the associated listed items. It will be understood that the terms "comprises" and / or"comprising" specify the presence of stated features but do not preclude the presence or addition of one or more other features. It will be further understood that when a particular step of a method is referred to as subsequent to another step, it can directly follow said other step or one or more intermediate steps maybe carried out before carrying out the particular step, unless specified otherwise. Likewise it will be understood that when a connection between structures or components is described, this connection may be established directly or through intermediate structures or components unless specified otherwise.The term “(at least) substantial(ly)” or “essentially” is generally used herein to indicate that it has the general character or function of that which is specified. When referring to a quantifiable feature, this term is in particular used to indicate that it is at least 50 %, more in particular more than 75 %, even more in particular more than 90 % of the maximum that feature. The term ‘essentially free’ is generally used herein to indicate that a substance is not present (below the detection limit achievable with analytical technology as available on the effective filing date) or present in such a low amount that it does not significantly affect the property of the product that is essentially free of said substance. In practice, in quantitative terms, a product is usually considered essentially free of a substance, if the content of the substance is 0 - 1 wt.%, in particular 0 - 0.5 wt.%, more in particular 0 - 0.1 wt.%.In the context of this application, the term "about" means generally a deviation of 10 % or less from the given value, in particular a deviation of 5% or less, more in particular a deviation of 2% or less.As used herein “organic” refers to any organic substance (such as biocatalytically produced product that is chemically oxidisable, as can be determined by the Chemical Oxygen Demand (COD) test, as described in ISO 6060:1989.Terms like ‘biocatalytical(ly)’ and “biocatalyst” are generally known in the art to describe methods respectively biological material, wherein at least one reaction step in the method is catalysed by a biological material or moiety derived from a biological source, for instance an organism or a biomolecule derived there from. In the method of the present disclosure an organic substance is fermentatively produced, i.e. the biocatalyst is a living micro-organism. The micro-organismgenerally excretes the produced organic substance of interest into the reaction medium from which the organic substance can be recovered without having to lyse the micro-organism. Thus a method of the present disclosure is generally carried out without having to disrupt the biocatalyst in order to recover the product.Fermentative methods can be aerobic, oxygen-limited or anaerobic.For the purpose of clarity and a concise description, features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.The operation of the fermentation zone, including the feeding, choice of microorganism and substrate can be based on known methods, e.g. as described in the prior art cited herein, notably W02021 / 010822 or WO2024 / 049295 of which the contents are incorporated by reference.At least during an essentially steady state stage, the fermentation zone is typically operated under turbulent flow conditions, whilst the separation into said first fraction and said second fraction takes place under less turbulent or laminar conditions. The selective separation, resulting in said first fraction enriched in the biocatalytic micro-organism is enhanced by changing hydrodynamic conditions inside the biocatalytic reactor system. In particular a more laminar flow, resulting in more calm flow conditions enhances solid-solid, solid-liquid, liquid-liquid, gasliquid separation locally in the reactor system. The Reynolds number (Re) is a generally known dimensionless number defined as the ratio of inertial forces to viscous force (Re = density x fluid velocity x characteristic length I dynamic viscosity), that is used in the art to quantify the flow conditions. A low Re, typically of about 2300 or less is indicative of laminar flow, preferably of 2000 or less, e.g. 100-1800. A high Re, typically of about 2900 or more, e.g. 3500-10000, is indicative of turbulent flow. However, a person skilled in chemical engineering will also be able to determine visually whether flow conditions are essentially laminar (e.g. by injecting some ink and watching the flow pattern). Thus, the flow, at least in a part of the separation zone, used for settling of solid matter, usually has a Reynolds number (Re) of less than 3000, preferably of 2900 or less, more preferably of less than 2300. It is not necessary that there is a continuous flow though. Essentiallylaminar flow conditions and no-flow conditions can be alternated in the reaction zone.The changing of hydrodynamic flow conditions allows for increased relative movement of a certain phase, particle and / or droplet up and an increased relative movement of another certain phase, particle and / or droplet downwards. In case of large enough particles and / or droplets this phenomena takes place to a significant enough extent to allow for sufficient separation. In case there is a need for even further enhanced separation, a second liquid phase may be used and / or other operating conditions may be changed locally to further enhance separation. This second liquid phase is of particular benefit in case of smaller hydrophobic, a-polar material, like cell debris, precipitated product, extracellular polymeric substance (EPS), etc.. Thus, changing hydrodynamic flow conditions is of particular benefit in the case of using a difference in size between the solid matter for the first fraction (comprising the micro-organism to be retained) and the solid matter for the second fraction and / or droplet size of a liquid phase comprising fermentatively produced organic substance (e.g. when the method of the present disclosure is a method comprising an in-situ organic substance extraction with a liquid extraction phase, for instance in accordance with W02021 / 010822 or WO2024 / 049295).The separation into said first fraction and said second fraction generally makes use of at least one difference in a physical property between the microorganism for the first fraction and the solid matter for the second fraction.Advantageously, at least one physical property is used selected from the group of specific gravity (relative density), electrostatic forces, polarity / non-polarity, size, shape, magnetic properties and hydrophobicity. Carrying out such separation is, e.g., beneficial for yield of the produced organic substance of interest.In preferred embodiment the degree of hydrophobicity of a solid matter for the first fraction or the second fraction is influenced by changing one or more operating parameters, in particular one or more of pH, temperature, presence of ionic species in the biocatalytic aqueous liquid phase, changing the state of the product itself (e.g. in case of acids / bases change from dissociated to non- dissociated form / change from acid to its conjugated base or change from base to its conjugated acid), the cell membrane, folding state of protein (as a produced organic substance),aggregation state, material selection, charge state of the molecule itself, polarity of the molecule or group of molecules.In an advantageous embodiment, use is made of a difference in size, resulting in a difference in gravitational, buoyancy and / or drag forces, allowing for selective separation by settling.In an advantageous embodiment, the difference in the physical property, more specifically size, is enhanced by forming enlarged biomass particulates from the micro-organism used for the microbial conversion, e.g. by creating clusters of micro-organism cells, which forming comprises at least one of (1) aggregation, (2) flocculation, and (3) filamentation, and wherein said first fraction comprises said enlarged biomass particulates. In a further advantageous embodiment, at least one of aggregation, flocculation and filamentation is used to create (enlarged) particular of the solid matter for the second fraction. Such means of forming clusters are known in the art per se. The skilled person will be able to realise this in a method according to the present disclosure based on the present disclosure, the cited prior art and common general knowledge.The difference in the physical property, more specifically size, is enhanced by forming enlarged biomass particulates from the micro-organism used for the microbial conversion, e.g. by creating clusters of micro-organism cells, which forming comprises at least one of aggregation, flocculation, and filamentation, and wherein said first fraction comprises said enlarged biomass particulates.Dependent on the used biocatalytic micro-organism (or the nature of an undesired micro-organism of which the removal via the second fraction is to be accomplished) aggregation, flocculation, or filamentation can be naturally occurring and / or influenced by setting certain operating conditions optionally in combination with a certain reactor geometry (flow conditions, shear forces). The net sum will dictate the form and size of the flocs, aggregates or particles of filamented cells.Auto-flocculation or auto-aggregation can be influenced at biocatalyst level through microbe choice and selection, modifications and aggregate properties at cell level such as the surface properties and ability of cells to bind to each other, mass transfer properties, stability of the three-dimensional complex amongst other properties. Next to biocatalyst selection and modifications auto-flocculation or auto-aggregation can be influenced by process conditions, like amount of shear,power input, stirrer tip speed, flow conditions and velocities, presence of particulates. Flocculation can also be accomplished by addition of a flocculant. Various flocculation agents have been employed for the immobilisation or flocculation of biomass including inorganic, organic and microbial flocculants. Organic flocculants are often advantageous as they are fast acting and often do not require large dosages. Examples known in the art of organic flocculants include chitosan, polyacrylamide, polyethylene amine. Inorganic flocculants include salts of aluminium such as poly-aluminium chloride and aluminium sulphate as well as salts of iron including ferric chloride and ferric sulphate. Although salts of aluminium, iron and zinc can provide efficient flocculation of microalgae, these salts would often end up in the natural environment, causing detrimental environmental effects via soil and water pollution, or in the biomass, which may impact or limit applications.A cationic flocculant maybe used to attract negatively charged particles in the water, such as certain types of dirt, organic materials, or microorganisms. Chitosan is an example of an organic cationic flocculant. Calcium ions, aluminium ions and iron ions are examples of inorganic cationic flocculants. A flocculant can also be anionic, attracting particles with a positive charge. The choice of a cationic or anionic flocculant depends on the aqueous environment conditions, like alkalinity in combination with the electrical properties of the solids to be flocculated.A potential disadvantage associated with the use of natural or chemical flocculation for immobilisation is the relative weak strength of the microbial clusters and their fragmentation when exposed to shear stresses. Shear stresses are imposed through hydrodynamic forces in biocatalytic reactors by impeller mixing in the bulk fluid phase or fluid motions associated with the gas-liquid interface if gas-sparging is used. These shear stresses should be minimized to prevent fragmentation of the microbial flocs through weaker stirring or lower gas flow rates, possibly at the expense of decreased mass transfer rates and overall biocatalytic reactor performance, if the micro-organism is used in flocculated state in the fermentation zone.’Carriers for a biocatalyst often create large difference in retention and thus limit the type and geometry of the reactor for a suitable retention strategy. Thus itis a major advantage that the present method allows the selective retention of nonsupported biocatalytic micro-organisms.Microbes exist that can form filaments, thereby enlarging themselves. This can also be used to enhance selective separation in accordance with the disclosure, in particular when a filament forming micro-organism is used for the fermentative production. A well-known class that does this is the class of filamentous fungi. Typical for filamentous fungi is a high biomass density (~5-100 g / L), high titres, eukaryotic ‘post-translational modifications’ (PTM) and longer fermentation times. However the filamentous nature introduces some challenges as well, like increased broth viscosity. Preferred examples of filamentous fungi include Aspergillus, in particular A. niger, A. flavus, A terreus; Rhizopus arrhizus; Fusarium oxysporum; and Trichoderma reesei.In a preferred embodiment the selective separation is enhanced by using a second, liquid phase, as an auxiliary liquid. In a first preferred embodiment the auxiliary liquid is an organic liquid, forming a separate liquid phase. It is found that more hydrophobic, apolar material (than the microbiological cells) can be enriched at the interface between the two liquid phases, i.e. the aqueous phase of the reaction mixture and the auxiliary liquid phase. By selectively separating the auxiliary liquid phase from the aqueous phase the more hydrophobic, a-polar material is transported with the second auxiliary phase, as (part of) the second fraction, and selectively removed from the biocatalytic reactor system. In a further preferred embodiment, the auxiliary liquid forms a second aqueous phase, separate from the first aqueous fraction. This can be accomplished by forming emulsions / dispersions of the water-in-water type, in the presence of suitable aids, which can be polymeric or a salt. Advantageously, said second liquid phase comprising an aqueous phase with either a polymer and a salt or with two or more polymers. When using at least two or more polymers, these are chosen to be incompatible with each other, whilst individually dissolved in the aqueous phase. Thus at least two aqueous phases form. Suitable incompatible water-soluble polymer combinations are known in the art. Exemplary mutually incompatible polymers systems for water-in-water type emulsions are polyethylene glycols (PEG) and dextrans. An example of a combination of a salt and a polymer is PEG and potassium phosphate.Further, in an advantageous embodiment, the separation into said first and said second fraction is aided by the presence of a gas phase, said gas phase either being locally introduced and / or being the result of gas entraining in the aqueous fraction to the separation zone as result of hydrodynamics. This is in particular useful when employing flotation.In a preferred method of the present disclosure, the enrichment in the first fraction with biocatalytic micro-organism comprises the use of settling. In such method, the method is advantageously operated to provide an observed settling rate of 0.002 cm / min to 1.4 cm / min of particles of the micro-organism cells (such as aggregates, flocs, filamented micro-organism). This typically corresponding to a (spherical) particle size of about 7 micron to about 0.5 mm). More preferably, said particles have an observed settling rate of 0.01 cm / min to 0.8 cm / min (typically corresponding to a size of about 10 micron to about 0.3 mm). In a specific embodiment, said particles have an observed settling rate of 0.08 cm / min to 0.5 cm / min (typically corresponding to a size of about 50 micron to about 0.15 mm). The skilled person will be able to choose method conditions to effectuate such settling rate based on the information provided in the present disclosure, the cited prior art, and common general knowledge.In a preferred embodiment the selective separation into the first and second fraction is enhanced by the biocatalytic reactor geometry design, in combination with the degree of freedom and control of the process resulting from the selected reactor geometry. As is known in the field settling or sedimentation can be aided by angle of the reactor internal or wall, particular lamella type settling at 45°-70°, often 60° angle is typically applied. Reactor geometry can affect separation by influencing flow conditions, altering an angle in a channel between fermentation zone and separation zone (e.g. when using a system based on W02021 / 010822), location and number of outlets for the second fraction. E.g. use can be made of a principle described in WO2024 / 049295 in combination with the contents of the present disclosure. Geometries like slopes and angles to enhance, e.g. degassing, or optimize settling are known in the art and can be readily applied [e.g.: Zhao et al, Yeast flocculation: New story in fuel ethanol production, Biotechnology Advances 27 (2009) 849-856; Domingues et al. Contamination of a High -Cell-Density Continuous Bioreactor, Biotechnol. Bioeng. 68 (2000) 584-587; Zhang et al, A NewDeveloped Airlift Reactor Integrated Settling Process and Its Application for Simultaneous Nitrification and Denitrification Nitrogen Removal, The Scientific World Journal (2013), pl-7].Advantageously, in the method of the present disclosure,the retention time of said first fraction is significantly larger than the hydraulic retention time of the (main) aqueous phase in the bioreactor system;the retention time of said second fraction is equally or lower than the hydraulic retention time of the (main) aqueous phase in the bioreactor system;further, in case the separation is aided by the presence of a second liquid and / or gas phase the retention time of said second fraction is lower than the hydraulic retention time of the (main) aqueous phase in the bioreactor system.The produced organic substance of interest can be recovered from the bioreactor system as part of the second fraction or a third fraction. Besides using the present method to separate the organic substance as a solid, it is also possible to use (semi-continuous) in situ extraction, e.g. essentially as described in WO2021 / 010822 or WO2024 / 049295. When using in situ extraction, in case of a hydrophobic produced organic substance it can form its own phase. It is also possible to include a separate extraction phase (recovery liquid), which can be organic or inorganic. It is also possible to make use of the water-in-water emulsion principle, wherein one of the aqueous phases serves as a product recovery phase.A method according to the present disclosure allows fermentative production and recovery of organic substances for a prolonged time at a high liquid capacity for various types of product-recovery combinations, including for 1) organic substances that form their own liquid phase; 2) organic substances that are produced and extracted in situ (e.g. 2-phenyl ethanol (2-PE), butanol), using an extractant (product recovery phase); 3) product in aqueous phase of the reaction mixture (e.g. proteins). For illustrative purposes, the following table lists a typical product formation rate (g / L / h) and substrate yield (g / g) for exemplary products in each of these cases. The product retention time (PRT) follows from the liquid (orproduct recovery phase) retention time. The substrate used are concentrated (glucose) feed, 50 weight % solutions, unless specified otherwise.Case Product Substrate yield PRT formation rate [g / g] [h][g / l / h]1 self-formed liquid 2.5 0.25 6phase5 0.25 32 extraction (2-PE) 1 0.1 12 extraction (butanol) 5 0.3* 1-23 product in aqueous 1 0.1 100® phase (protein)**70 wt%# non-selective recovery via aqueous phase (reaction mixture)@ equal to HRTThe PRT is high in this example for case 3 (protein recovery in aqueous phase). The PRT (=HRT) can be reduced by diluting the feed, which requires more specific cell retention, by e.g. increasing the effective size of the micro-organism ‘particles’.Operation of fermentation system thus is dependent on actual production rates, feed concentration, liquid phase mass balances. The product residence time and the (aqueous) hydraulic residence time(s) often are at least an order of magnitude apart. Preferably, the (desired) solid(s) residence time is not depending on either the product residence time or the aqueous liquid residence time. For an optimal (desired) solid(s) residence time, and thereby overall biocatalytic process performance, it is important that desired solid(s) are retained, while undesired solid(s) / product(s) are removed. This reinforces the importance of being able to control the SRT of the different solid fractions that can occur in a fermentation process.If desired, the liquid phase comprising the product of interest can be subjected to further downstream processing. This can be done in a manner known per se, e.g. by one or more filtration steps, by back extraction to a lower boilingsolvent, and / or solvent or product evaporation as described in WO2021 / 010822 or in PCT / NL2024 / 050668, optionally followed by further purification.Separation of biocatalytic micro-organism and solid produced organic substanceA method according to the present disclosure is particularly suitable to separate cells of the micro-organism used to fermentatively produce an organic substance from solid matter comprising the produced organic substance. Such organic substance can be a substance that is liquid under the fermentation conditions or extracted from the reaction mixture by in situ extraction in the fermentation zone, yet subjected to solidification prior to separation in the separation zone (in particular by reducing the temperature to a temperature below the solidification temperature). The produced organic substance can also be an organic substance that is solid both under fermentation conditions and separation conditions.A substance can be present as a solid phase if present in a concentration is above the solubility of the substance in the aqueous broth (or the solubility of the substance in an 2nd liquid phase). A substance is solubilized if the concentration is within the solubility limits of the liquid phase(s).When the size of (particles of) the product (containing or consisting of the fermentatively produced organic substance of interest) is sufficiently smaller than the size of the biocatalytic micro-organism, it is usually possible to use flotation. The upward movement of the solid product particles can be stimulated with gas bubbles, and the product can then be recovered at the gas-liquid interface on top of the liquid in the separation zone, or by catching the product at another interface. I.e. solid is often preferentially present at the interface and can thus move upwards or downwards with a dispersed phase, being gas bubble, liquid droplet or solid particulate, depending on motion of the dispersed phase.Selective solid-solid separation is particularly beneficial in case of product and / or biocatalyst degradation over time as result of product stability by itself or as result of operational conditions (temperature, pH, shear force / power input, gradients within the reactor system, flow regime) or compounds (e.g. enzymes,biocatalytic species, oxygen, ionic / re active species) present in the biocatalytic aqueous liquid phase.In an embodiment comprising the separation of biocatalytic micro-organism and solid produced organic substance, preferably the solid matter for the second fraction comprises at least one organic substance (at least solid under separation conditions) selected from the group of proteins, fats, long chain alcohols, (typically CIO or more, preferably C12-C24), solid flavour molecules, vitamins, pigments and dyes. In particular, good results are achieved with a protein that is fermentatively produced and separated from the micro-organism. Such protein is advantageously selected from dairy proteins, meat proteins, egg proteins and enzymes. As the micro-organism used for the fermentative production, usually a genetically modified micro-organism is used, wherein the genetic modification allows the micro-organism to produce and excrete the protein. In principle any microorganism suitable for fermentative production of the organic substance of interest can be used. In particular, a micro-organism can be used as described elsewhere in the present disclosure or in the prior art cited herein.In a highly advantageous embodiment the micro-organism used for the production of the organic substance, preferably protein, that is separated as a solid from the micro-organism, is a fungus, preferably a yeast, more preferably Pichia; Aspergillus Niger or Trichoderma Reesei; the first fraction enriched in biomass is enriched in the yeast cells and the second fraction is enriched in the produced organic substance, preferably protein; the separation into said first fraction and said second fraction comprises the formation of aggregates comprising the fungus, and allowing said aggregates to settle in the separation zone, thereby forming the first fraction, enriched in the fungus cells, in a lower liquid layer in the separation zone and the second fraction enriched in the produced organic substance, preferably protein, in an upper layer. Selective settling of the fungus aggregates can further be enhanced by choosing a favourable flow channel geometry in the separation zone in combination with a selected liquid velocity (low Re), suppressing upward movement of the yeast aggregates. The inventors realised that principles described for upward movement of liquid recovery phase, containing produced organic substance, in WO2021 / 010822 or WO2024 / 049295, can also be translated not only to upward movement of droplets of low density, but also to downwardmovement of solid particles, such as yeast, aggregate more effectively. In particular, the configuration where separation zone is followed after the downcomer (W02021 / 01822), with suitable removal of gas phase, or lateral flow into a separation section (WO2024 / 049295), can be applied in combination with the present disclosure to enhance the retention possibilities of the micro-organism with which the first fraction is enriched.Separation of biocatalytic micro-organism and other solid biomassThe inventors further realised that it would be desirable to provide a method for removal of biomass material with reduced to no activity from the bioreactor system, whilst retaining active biocatalyst. Inventors found that by selectively removing biomass material with reduced to no activity the decline in biocatalytic activity over time in the fermentation zone is reduced, preferably to the point where substrate conversion rates can be maintained for prolonged run duration. This can advantageously provide one or more of benefits, like improved yield, reduced downtime, reduced resource requirements, reduced energy consumption, all leading to improved sustainability and improved economic feasibility of the process.Thus, in a further advantageous embodiment a method according to the present disclosure comprises a separation wherein the first fraction is enriched in the living micro-organism, having biocatalytic activity in the production of the microbiologically produced organic substance, and the second fraction is enriched in microbial material with reduced to no biocatalytic activity in the production of the microbiologically produced organic substance. Typically, said microbial material with which the second fraction is enriched comprises at least one matter selected from the group of cell debris, lysed cells, protein (cellular protein natural to the micro-organism, protein produced as the organic substance of interest or both), and the like. The first fraction or a substantial part thereof, including living micro-organism is returned to the fermentation zone, whilst second fraction can be discarded or taken from the bioreactor system and subjected to downstream processing, e.g. comprising recovery of the produced organic substance of interest if this is present in the second fraction. Hereby, the content of biocatalytically active micro-organism is selectively retained relative to biocatalyst material with no orreduced biocatalytic activity, and a high concentration of the biocatalytically active micro-organism can be maintained in the fermentation zone for a prolonged time.In particular, good results with respect to the separation of living microorganism and further microbial mass are achieved with an auxiliary liquid for which the microbial material (such as one or more of cell debris, lysed cells, cellular protein, fermentatively produced protein of interest) has an affinity. The auxiliary liquid is a liquid that forms a separate phase from the aqueous phase. Preferably the density of the auxiliary liquid is lower than the density of the aqueous phase, and auxiliary phase forms a layer on top of the aqueous phase in the separation zone. Such method advantageously comprises dispersing the auxiliary liquid into the reaction mixture or the aqueous phase of the reaction mixture, allowing the said microbial biomass to be enriched at the interface of dispersed auxiliary liquid and the aqueous phase, wherein in the separation zone the auxiliary liquid is allowed to form an upper liquid layer comprising the second fraction, enriched in microbial biomass having reduced to no biocatalytic activity in the production of the microbiologically produced organic substance, and a lower liquid layer, comprising the first fraction, enriched in the living organism having biocatalytic activity in the production of the microbiologically produced organic substance. The auxiliary liquid as a whole, enriched in the further solid biomass, may be taken as the second fraction, or - when in particular the interface between aqueous phase and auxiliary liquid phase is enriched - the volume close to the interface may be taken as the first fraction.It should be noted that at least in some embodiments, the biomass may potentially act as an emulsion stabiliser for the auxiliary liquid. In such case, similar to emulsion stabilization of dispersed droplets in aqueous system, where emulsion is avoided by control of the coalescence and break-up of the droplets (e.g. having a net outcome of a droplet diameter >20micron.), tailoring the biomass levels and specific interaction at the liquid-liquid interface is required. In the art surfactants, aggregating chemicals, ionic concentrations, temperature are examples known to impact this stabilization.Suitable auxiliary liquids include liquids that have interactions with the other solids, allowing enrichment of these solids into or on the interface of the auxiliary liquid and are dependent on the nature of the cell debris or protein orbiomass. Preferred are liquids that do not mix with water (are at least substantially insoluble) and that are non-toxic to the product producing microorganism. Preferred is a net repulsive interaction of the cell wall of the active microbial cells (or aggregates / filaments) with the auxiliary liquid’s liquid-surface.Advantageously, the first fraction contains solid particles formed of a plurality of the living micro-organism (clusters), which particles have a net downward impulse (orientation with the gravity force vector). The difference in the physical property, more specifically size, is enhanced by forming enlarged biomass particulates from the micro-organism used for the microbial conversion, e.g. by creating clusters of micro-organism cells, which forming comprises at least one of aggregation, flocculation, and filamentation, and wherein said first fraction comprises said enlarged biomass particulates. This leads to being able, depending on specific solid matter property to apply sedimentation, settling and / or flotation.In an embodiment, the produced organic substance of interest has a higher affinity for the auxiliary liquid than for the aqueous phase. In such case, at least in some embodiment, the auxiliary liquid may also be suitable as a product recovery phase, by using an in situ fermentative extraction, essentially as described in WO2021 / 010822 or WO2024 / 049295. In such case at least the bulk of the auxiliary liquid (remote from the interface with the aqueous phase) may be recovered as a third fraction. The auxiliary liquid does not have to extract the produced organic substance into the bulk of its phase in order to be useful in the product recovery. One may also make use of a hydrophobic interaction wherein the produced organic substance is enriched at / near the interface of the auxiliary liquid phase and the aqueous phase (forming the first fraction).In an embodiment the auxiliary liquid is also used to enrich produced protein and allow more enriched recovery, where the auxiliary liquid forms an aqueous two phase system with the aqueous phase of the reaction mixture (such as an emulsion of the water-in-water type).Separation of specific biocatalyst cells from other cells or microorganisms Additionally, the inventors realised that a method according to the present disclosure can be used to selectively separate the active micro-organism (as part ofthe first fraction), of which it is desired to return it to the fermentation zone and use it again for the fermentative production of interest from a different microorganism (as part of the second fraction).This embodiment is amongst others particularly advantageous for slow growing, low biomass yield processes and / or processes with initial growth phase, followed by production phase with less / no growth and higher product yield.Further, the method of the present disclosure may also be used advantageously for a specific microbial population enrichment in the system by using a difference in microbial (cell) size, a difference in cell membrane or a difference in density as basis for separation. This is particularly beneficial in case of a contamination that builds up over time, which in particular slow / not growing micro-organisms are sensitive to. Typically, a run needs to be terminated when the contamination build up reaches a certain level. By selectively removing the contaminating species a longer run duration and desired product formation can be maintained for longer periods of time.The different micro-organism can be of a different species (typically a contamination), a micro-organism of a different variety of the same species, or micro-organism of the same variety having a different phenotype. The different micro-organism (with which the second fraction is enriched) generally has an undesired or less desired property, such as a reduced activity or lack of activity with respect to the fermentative production of the organic substance of interest, compared to the desired active micro-organism.Separation into the first fraction and second fraction can generally be based on one or more of the principles described above; e.g. use can be made, for example, of a difference in size, resulting in a difference in gravitational, buoyancy and / or drag forces, allowing for selective separation by settling Other physical properties that influence selective solid separation: specific gravity, electrostatic forces, polarity / non-polarity, size, magnetic, hydrophobicity.Separation of biocatalytic cells form, inorganics, salt particles, side-products and the likeIn a further embodiment, the second phase is enriched in an inorganic solid, salt particle, solid side-product or the like. This can also be accomplished using atechnique of which the principle is described above, e.g. by selectively forming clusters of either the biocatalytic micro-organism or the solid matter that is to be separated from said micro-organism, thereby increasing the size of one of the solid matters relative to the other, and carrying separation on the basis of a difference in size.Micro-organism used for fermentationAdvantageously, a micro-organism is used for the fermentative production of the organic substance of interest selected from the group of bacteria, archaea and fungi, preferably selected from the genera Pseudomonas, Gluconobacter, Rhodobacter, Clostridium, Escherichia, Paracoccus, Methanococcus, Methanobacterium, Methanocaldococcus, Methanosarcina, Aspergillus, Penicillium, Saccharomyces, Kluyveromyces, Pichia, Komagataella, Candida, Hansenula, Bacillus, Corynebacterium, Blakeslea, Phaffia (Xanthophyllomyces), Yarrowia, Schizosaccharomyces, Zygosaccharomyces, Saccharopolyspora, Trichoderma and Zymomonas more preferably from the group of Corynebacterium glutamicum, Escherichia coli, Bacillus subtilis, Bacillus methanolicus, Pseudomonas aeruginosa, Pseudomonas putida, Rhodobacter capsulatus, Rhodobacter sphaeroides, Paracoccus carotinifaciens, Paracoccus zeaxanthinifaciens, Saccharomyces cerevisiae, Saccharomyces pastorianus, Schizosaccharomyces pombe, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Blakeslea trispora, Penicillium chrysogenum, Phaffia rhodozyma (Xanthophyllomyces dendrorhous), Pichia pastoris (also known as Komagataella phaffii), Yarrowia lipolytica, Saccharopolyspora spinosa, Trichoderma reesii and Zymomonas mobilis, in particular from the group of Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas putida, Saccharomyces cerevisiae, Saccharopolyspora spinosa, Trichoderma reesii, Pichia pastoris (Komagataella phaffii), Aspergillus niger and Zymomonas mobilis.A micro-organism selected from the above micro-organisms may in particular be used for the production of an organic substance disclosed herein. The skilled person will be able to choose a particularly useful micro-organism for the fermentative production of a specific organic substance of interest, based on the present disclosure and methodology known in the art.Fermentatively produced organic substances of interestA method according to the present disclosure can be used in the production of any organic substance that can be produced fermentatively. The produced organic can be solid, it can be dissolved in the liquid phase and / or a second liquid phase (auxiliary liquid or product recovery phase) or it can form its own phase. This product own phase can also be used for selective solid removal combining both product and second solid fraction removal.In a method wherein a microbiologically produced organic substance is separated from the first fraction as part of the second fraction (see also above), it can form a solid phase at least in the conditions used in the separation zone, in particular at least at a temperature of about 40 degrees C or less, preferably at a temperature in the range of between 0 degrees C and 35 degrees C, more preferably at a temperature in the range of between 4 and 30 degrees C.The fermentatively produced organic substance is excreted by the microorganism, such that it does not require lysis of the micro-organism.Typical process applications of a method according to the present disclosure include the production of biofuels / biofuel precursors, chemicals production, like biobased replacements / precursors for petrochemicals such as polymers for plastics; agricultural chemicals such as fertilizers, pesticides and herbicides, speciality chemicals and pharmaceuticals and food (additives), including proteins, amino acids, vitamins, microbial oils, flavours.Usually, the organic substance has at least 2 carbons, preferably at least 6 carbons, in particular at least 10 carbons, more in particular at least 12 carbons. The organic substance can have over 1000 carbons (such as in case of produced biopolymers. In an embodiment the organic substance has up to 500, up to 250, up to 100, up to 50, or up to 24 carbons.Advantageously, the method according to the invention is used to produce one or more organic substances selected from the group consisting of hydrocarbons, in particular monoterpenes, sesquiterpenes, aromatic hydrocarbons; isoprenoids (terpenoids); organic acids, in particular C5-C24 fatty acids, more in particular C12-C20 fatty acids; alcohols, in particular alcohols having at least 4 carbon atoms (in particular butanol or long chain alcohols); phenolic compounds, ketones, in particular ketones having at least 5 carbon atoms; aldehydes in particularaldehydes having at least 5 carbon atoms; cyclic carboxylic esters, in particular lactones; non-cyclic esters, in particular non-cyclic esters having at least 5 carbon atoms; lipids in particular glycerides; ethers; pyridines; imines; imides; amines; amino acids; and peptides. For further details of preferred organic substances that can in be produced in accordance with the invention, reference is made to the prior art cited herein, in particular W02021 / 010822 page 23, line 27 till page 27, line 10 of which the contents are incorporated herein by reference.Further particularly preferred is a method of the present disclosure wherein a protein is fermentatively produced as the organic substance of interest. Usually, the protein is produced by a genetically modified micro-organism, wherein one or more genes have been inserted or modified to allow expression of the protein (if the protein is not naturally produced by the wild-type micro-organism), to enhance expression, and / or to allow or enhance excretion by the micro-organism. The protein may e.g. serve as a food ingredient, a feed ingredient or pharmaceutical.The method of the present disclosure is in particular advantageous for the production of a dairy protein, such as at least one of Lactoferrin, Casein, Lactoglobulin, Lactalbumin; a meat protein, such as at least one of collagen-like / collagen, leghemoglobin, myoglobin; and egg protein, such as at least one of Ovomucoid and Ovalbumin.Modifying a micro-organism to make it suitable for the production of a protein can be done based on methodology generally known in the art. Further, specific means of modification are e.g. described in Domingues et al (Process Biochemistry, March 2005, Pages 1151-1154; https: / / doi. Org / 10.1016 / j.procbio.2004.04.016), describing Aspergillus niger B-galactosidase production; Robert et al (Biochemical Engineering Journal, Volume 147, 15 July 2019, Pages 39-47; https: / / doi. Org / 10.1016 / j.bej.2019.03.027) describing recombinant lipase B in Pichia pastoris; Aro et al (Food Research International Volume 163, January 2023, 11213; https: / / doi. Org / 10.1016 / j.foodres.2022.112131) describing bovine beta-lactoglobulin and hen egg ovalbumin by T reesei; Shao et al. (Bioresource Technology, Volume 363, November 2022, 127884; https: / / doi. Org / 10.1016 / j.biortech.2022.127884) describing leghemoglobin production in P. pastoris.When producing a protein, it is particularly preferred to use the method of the present disclosure to separate the produced protein as part of the second fraction from the first fraction. Separating the produced product from the reaction mixture already in the bioreactor system in the separation zone (semi-) continuously, instead of further downstream, allows separation at an early stage from components in the reaction mixture that may degrade or otherwise modify the protein. E.g. fermentation broths often contain proteases. The method according to the invention makes it possible to produce proteins effectively whilst having relatively low retention times in the reaction mixture, which addresses product instability problems. Low retention times also allows operation at a higher substrate concentration (less dilution), which increases production capacity, yield, and offers the possibility to make downstream processing easier.For the fermentative production of a protein in accordance with the present disclosure one may use bacteria. Gram negative bacteria are not preferred for food applications, due to the presence of endotoxins. Benefits of using bacteria are: fast growth, low complexity media, a broad product titre range. Examples of suitable bacteria include: E.coli and C. glutamicum.Good results are obtained in particular with a fungus, such as a yeast. Yeast species do have the eukaryotic post translation modifications (PTMs) to produce the proteins. Usually they offer by a medium titer. 1-22 g / L. Preferred examples are:, S. cerevisiae, K.marxianus and Pich ia pastoris (K. phaffi). Further, Filamentous fungi are particularly useful for the fermentative production of a protein or other organic substance in accordance with the present disclosure. Their filamentation can be used to facilitate separation in order to obtain the first and second fraction. They offer a high biomass density, high titers, eukaryotic PTMs, longer effective fermentation times. For filamentous based processes, Trichoderma, in particular T. resei, and Aspergillus, in particular, A. niger, are preferred filamentous fungi.Next, the present disclosure will be illustrated by a number of experimental examples.EXAMPLESEstablishing effective selective cell retention in a continuous or semi continuous biocatalytic process is a step forward compared to current chemostat systems, as it enhances overall process metrics, by preventing common problems that are known to hinder yield and (reactor) volumetric productivity, such as the build-up of dead or inactive biocatalyst, emulsification, product inhibition, lack of functional control of the size of different populations, and possible precipitation and accumulation of a solid organic product.The present disclosure provides some major ways to achieve this separation between a microorganism-enriched fraction and a solid matter fraction: using flow hydrodynamics and / or system geometry and / or biocatalyst particle size. A rationale behind controlling these factors is to effectively exploit the physical property differences that drive any separation. Logically, applying strategies to control settling rates and behaviour of biocatalysts in static or hydrodynamic conditions provides useful insights on how to control their separation from other fermentation elements in any conditions, including the system described in the present disclosure and earlier disclosures (such as US2017 / 0073710 Al W02021010822 and WO2024049295).As discussed in Govender et al., Applied and Environmental Microbiology 74,

[2008] (https: / / journals.asm.org / doi / 10.1128 / aem.00394-08) the industry standard is to actually remove flocculation genes or apply it only at the end of fermentation to aid clarification of the broth, as desired for instance when producing beer. As there are two conflicting demands from the microbe, as during fermentation, a high suspended yeast count is required to maintain a satisfactory rate of fermentation, while at completion, efficient settling is desired to enhance product clarification and recovery. Govender et al. discusses a need for a strain that does not flocculate during fermentation, but in which flocculation can be switched on at the end of fermentation to aid settling and clarification and avoid expensive and time consuming processes like filtration and centrifugation.This disclosure, however, makes use of means to control solid properties, like degree of flocculation, in order to enhance fermentation process performance during the fermentation. Primary considerations to come to the desired strategy tocontrol settling and settling rate, that can be applied in continuous flow systems to achieve in-situ selective solids separation, comprise factors that impact buoyancy, e.g. particle size distribution, shape, morphology, friction experienced, degree of turbulence in the surrounding liquid, density difference. Reactor geometry and flow conditions are also relevant factors and can be included in the strategy.Therefor the following examples provide factors intrinsic to the microorganism, such as the species or genetic drivers influencing flocculation, and factors related to the surrounding suspension conditions, such as pH, calcium and other solubilized ions, nutrient (e.g. phosphate) limitation, together with the scope to which different settling properties can be applied to control fermentation performance. The impact, when applying these factors in accordance with the present disclosure, is demonstrated for a range of yeasts and bacteria. Specifically the last examples address how these factors can be implemented within flow hydrodynamics and system geometry elements. These last examples clearly demonstrate the behaviour of particles in continuous fermentation systems and that the behaviour of particles can be manipulated in accordance with the present disclosure within design constraints of in-vivo systems to enhance overall fermentation process performance. Thus it can be understood that the effects shown in the examples can be further applied to actual process applications, such as protein production.One important consideration when referring to control of settling rate and particle size is that this control is not necessarily a maximization. The aim is to control the overall process performance by for example ensuring settling rates fall within a range that favours separation and respects constraints such as mass transfer limitations (hindering the diffusion of nutrients such as oxygen or glucose, or creating undesired gradients) that typically occur with larger floc sizes.Example 1: Benchmark settling of Saccharomyces cerevisiaeIt is well recognized that some wild- type strains of for example Saccharomyces cerevisiae can change their effective size (aggregation state) by innate flocculation properties. Flocculation is here driven by gene expression, which is possible to either enhance or suppress, thus creating strains with acontrolled flocculation capacity if so desired, which is well known in the field.Flocculation in S. cerevisiae is cell-cell aggregation (reversible clumping) thought to be driven by lectin-like proteins on the cell surface. These are encoded by the FLO gene family. Typically, in present day practice, Saccharomyces cerevisiae strains have been selected on lacking active gene / gene expression of the associated FLO genes to prevent flocculation. As (too much) flocculation can have negative mass transfer and therefore fermentation performance impacts or hinders or complicates e.g. the reproducibility of research.Example 1 was conducted to benchmark and validate the use of an optical method to quantify settling rate of, a strain lacking gene expression of FLO and so, non-flocculating Saccharomyces cerevisiae.2 mL cuvettes (with a 20 mm maximum height) were filled with different volumes (corresponding to column heights of 15, 17 and 19 mm) of a S. cerevisiae CEN. PK2-1C suspension with an optical density (OD, 600 nm) of 1.0. S. cerevisiae CEN. PK2-1C is a well-known genetically modified lab strain known for its usually non-flocculating behaviour under typical lab conditions. Spectrometer measurements of the samples were taken every minute during a span of 3 hours, at a fixed height of 8.5 mm. The settling rate was calculated as the time-averaged distance between liquid height and sensor height, and the OD monitoring informed on the fraction of the sample situated below the sensor height at any given moment.Results are shown in Figure 1. The results show a bell curve following a Gaussian distribution, showing typical normal distributed particle sizes. The observed range corresponds to the one reported in literature for non-flocculating yeast, of 0.008-0.015 cm / min (Eduardo V. Soares, ‘Quantification of yeast flocculation’ in J. Inst. Brew, March-April 1997).The normal variability of settling rates within a sample was confirmed to be proportional to different particle size I shape of yeast cells. Furthermore, within Figure 1, the values of mean are in line with the average settling rate of 0.01 cm / min, the natural settling rate achievable for yeast. Data results show there is no observed effect of sample column height in relation to applied sensor height on settling rate at this scale.The experimental optical setup indeed confirmed the settling rate of (genetically modified) non-flocculating yeast. The results confirm that an average spherical particle size can be used to describe a particle range distribution with an overall average settling velocity, as was done in claims 10-12. Using the single average settling rate value, corresponding to an approximately spherical particle, is a common approach in the field.Example 2: Settling of Saccharomyces cerevisiae with flocculationBeyond genetically determined properties, flocculation of microorganisms is known to be impacted by pH and calcium concentration (Soares, Biotechnology Leters 22: 1827-1832, 2000), amongst other factors. In these experiments calcium concentration, pH, EDTA concentration and biomass concentration are manipulated to control and obtain different settling rates of a flocculating strain, Saccharomyces cerevisiae CCUG (Culture Collection University of Gothenburg) 53310, to control settling within the desired rates and allow different fractions to be obtained, as per this disclosure.Example 2A: Calcium concentrationSaccharomyces cerevisiae CCUG 53310 cells were used. This strain (available from the Culture Collection University of Gothenburg) is a well known constitutively flocculating yeast under standard culture conditions. CCUG 53310 culture suspensions were washed twice with a 30 mM ethylenediaminetetraacetic acid solution (EDTA, a chelating agent that removes the cations in solution), to avoid interference from the cultivation medium, and twice more with demineralized water. The pellets were then resuspended in 7.5 mL of a solution comprising different volumes of a 50 mM phosphate buffer and a 100 g / L CaCl2solution, so as to achieve the final Ca2+concentrations in the mixture of 0, 2, 4, 8 and 32 mM. They were then incubated for 4 hours with horizontal shaking, mixed again and the settling rate was determined using the optical method as described in Example 1. The normalised optical density (OD) values in the first 9 minutes of settling and subsequent settling rates are shown in Figure 2 and Table 1.Table 1: Settling rate as function of calcium concentration and pH Ca2+[mM] 0 2 4 8 32 Settling rate [cm / min] 0.1 23.0 4.0 1.3 1.0 pH 6.9 6.7 6.5 5.9 5.3These results show that Ca2+concentration in solution can be applied to this Saccharomyces cerevisiae CCUG 53310 to manipulate the settling rate, increasing it from its natural settling rate to at least 23 cm / min at 2 mM Ca2+. Depending on floc size very large settling rates were achieved compared to natural settling rate of single cells.As shown in Example 1, the average settling rate of non-flocculating yeast is 0.01 cm / min. When combining the cells from this Example with the cells from Example 1 in the same fermentation selective separation of the two cell fractions can be achieved by settling with a upward liquid velocity higher than the settling rate of the non-flocculating yeast, but significantly lower than the settling rate of the Saccharomyces cerevisiae CCUG 53310.Example 2B: EDTA and pHThe Saccharomyces cerevisiae CCUG 53310 culture was grown in Verduyn mineral medium with the presence of calcium in trace amounts.This cultivation was performed without separate calcium additions. However, there are typically other sources of calcium in a fermentation medium, such as: preculture, vitamin solution, trace element solution and release of intracellular calcium, which can be stimulated by other nutrients like magnesium. As a chelating agent, EDTA can be used to control flocculation by decreasing the quantity of solubilized Ca2+ions. This effect is also dependent on media pH, since calcium tends to form more complexes with increasing alkalinity.Samples from the fermentation were subjected to EDTA solutions at different concentrations and adjusted to different pH values. Settling rate calculations were performed. Results are shown in Figure 3.Figures 4A-4D contain microscopic pictures of a sample without EDTA (4A), the same sample with 3.6 mM EDTA (4B), the same sample with 7.2 mM EDTA (4C) and the same sample with 7.2 mM EDTA and 7.2 mM Ca2+(4D). Figures 4A-4D show the impact on EDTA on the free calcium as with increasing concentration of EDTA (A versus B & C) the flocs become less pronounced and significantly smaller with a more open morphology. Addition of free calcium (Figure 4D) again increases the floc size, showing reversibility of the process, as is expected as the free calcium concentration impacts the degree of flocculation.These results confirm the inverse correlation between EDTA and settling rate, and demonstrate the significance of pH conditions. pH 6 and a dosing of 0.8 mM EDTA, and higher, result in settling rates in the desired range presented in claim 12 (0.08-0.50 cm / min). Literature, Soares et al. (2000), confirms the dependence of settling rate free calcium concentration. As it is known calcium tends to form more complexes with increasing alkalinity. EDTA is a chelating agent that can form a stable complex with Ca2+and have a similar impact as increasing alkalinity. Indeed this allows for control of settling rate by changing calcium concentration in combination with complex formation to either increase of decrease settling rate. So the separation efficiency and degree of selective separation between different solid fractions can be steered by these factors.Example 2C: Biomass concentration and pHBiomass concentration is known to have a negative impact on settling rate as increased biomass concentration or suboptimal growth leads to lighter flocs rather than dense, well-settling flocs, hindering the separation process, as is well understood in continuous operating waste water treatment plants.Using Saccharomyces cerevisiae CCUG 53310 suspensions similar to the Example 2A and 2B, the effect of biomass concentration on settling rate was determined for two pH values. The samples were diluted with culture medium (previously adjusted to the desired pH) to achieve five biomass concentrations, measured in cell dry weights, in the range of 5 to 25 g / kg. The samples were mixed and settling phenomena were observed in 15 mL test tubes, filled with 7.5 mL, and quantified via optical density measurements as in example 1. The results obtained, biomass settling rates determined in the first 30 seconds, are shown in Figure 5.Biomass concentrations were confirmed to enable control of settling rate in the range of about 0.1 to 5 cm / min. The effect of pH was a significant factor.Microscopic optical pictures were taken of the biomass after the settlingexperiments, of which Figure 6 is an example. Figure 6 shows the floc size corresponding with the measured 0.5 cm / min settling rate to be approximately 1200 x 800 pm, confirming the upper range of an ideal spherical particle around 0.5 mm to be possible. Biomass concentration increase was confirmed to decrease effective settling rate, although the relative change became less pronounced from 15 g / kg onwards. The desirable operating range for the settling of yeast, for example at pH 6 and CDW of 15-25 g / kg, was obtained. The settling rates achieved fall well within the range of claims 11 and 12, confirming control of settling rate at the desired level.So for the Saccharomyces cerevisiae a settling rate range of 0.01 - 5 cm / min was shown in example 1 and 2. This settling rate was confirmed to be controllable using genetic factors, degree of calcium complexation and free calcium concentration and biomass concentration and shape. This range of factors for settling rate control gives a degree of freedom to the fermentation process, as there are multiple ways to achieve the same result, and therefore improve control of selective solid separation.Example 3: Settling of Kluyveromyces marxianusExample 3A: pH / Ca2+ / biomass concentrationThe above-mentioned control strategies to achieve different settling rates are transversal to other species. K. marxianus DSM 5422 culture samples were collected from 3 L fermentations, washed with a 30 mM EDTA solution, resuspended in standard mineral growth media and screened for the impact of different variables on flocculating extent and thereby settling tendency. The experimental points and corresponding binary observations are compiled in Table 2.Table 2: Kluyveromyces marxianus flocculation behaviour for a range of pH values, a range of added Ca2+concentrations and a range of biomass concentration cell dry weight (CDW) values.Ca2+addition Biomass CDW [g / kg] (pH 5)pH (no added[mM] (with 200 mM (with 300 mM Ca2+)(pH 5) Ca2+) Ca2+)3 0 0 0 5 0 03.5 0 10 0 10 0 04 0 50 0 15 0 04.5 0 100 0 20 0 05 0 150 0 25 0 05.5 0 200 0 30 0 06 0 300 0 40 0 00: No settling observed; 0: Settling o ) servedFlocculation and settling of K. marxianus was not observed for any pH value within the growing range of this species (pH 3-6) without calcium addition.However for both pH 5 the addition of 200 mM Ca2+resulted in flocculation and thereby increased settling tendency. It was observed that the settling tendency was significantly more pronounced at low biomass concentration (5-10 g / kg). Similar observations were at 300 mM.The observations for different pH without added Ca2+are in line with expectations, as the pH has an impact on the degree of complexation of the Ca2+and thereby flocculation tendency of the microbe.As seen for the Saccharomyces cerevisiae, also the settling tendency of the Kluyveromyces marxianus is impacted by factors like calcium and biomass concentration. As settling can be controlled in a similar way with the Kluyveromyces marxianus, a food grade heat tolerant yeast, selective solid separation can thus be implemented in an industrial food grade application.Example 4: Settling of Komagataella phaffiiExample 4A: pHLiterature, Tanneberger et al. (‘A Novel Form of 6- Phospho fructokinase’ in Enzyme Catalysis and Regulation, August 2007)) and Sae-Tang et al. (Engineering Flocculation for Improved Tolerance and Production of d- Lactic Acid in Pichia pastoris. J. Fungi 2023, 9(4), 409), states that wild type strains of Komagataella phaffii, previously designated Pichia pastoris, have natural flocculation capability.Komagataella phaffii CBS2612 was cultivated in 2% yeast peptone extract (YPD) medium in shake-flasks, at 30 °C, for 51 h. 5.5 mL samples were taken at various fermentation time points, and the settling behaviour was observed in the test tubes. Figure 7A-C shows the observations 5 minutes after sampling from the shake-flask after 4 hours of cultivation (7A), 10 hours of cultivation (7B) and 51 hours of cultivation (7C).Full settling was observed in the lOh sample (Figure 7B), making it possible to approximate a 0.5 cm / min settling rate. Further, the pH was monitored along the fermentation and the lowest pH (5.3) was registered at 10 h as well. This differentiated performance could be, thus, either related to biomass concentration and / or pH. A pH screening was performed on the same sample, resulting in the observations in Figure 8. Figure 8 shows the samples after 5 minutes of settling at different pH values. From left to right, tube 1 to 7, the pH per tube is 7.0, 6.0, 5.0, 4.5, 4.0, 3.5 and 3.0, with a corresponding estimated settling rate / range of 0.004 cm / min for tube 1 and 2, 0.45 - 0.70 cm / min for tube 3, 4 and 5 and 0.90 cm / min for tube 6 and 7.These results show that flocculation and settling of Komagataella phaffii is highly dependent on pH and is favoured under acidic (pH 3.0-3.5) conditions. Thusin a preferred embodiment, selective solid separation is applied to protein production using the Komagataella phaffii, in which in a more preferred embodiment the retained fraction is the microbe and the second solid fraction is the protein product.Example 5: Settling of Escherichia coliBacterial cells, in this case Escherichia coli (E. coli), are smaller in size than yeast cells. In known methods, the settling rate of natural single bacterial cells, such as E. coli cells, is too slow for application of industrial settling. However, the inventors realised that, as for yeast, when aggregation of E. coli can be achieved, settling can be controlled in a continuous or semi-continuous fermentative production, further comprising a simultaneous recovery stage, in accordance with the present disclosure.Ojima et al, Applied and Environmental Microbiology September 2015 Volume 81 Number 17, describes genome overexpression can be used for Escherichia coli as a settling rate control factor, with the bcsB gene. Besides genetic approaches as described in previous examples also operating conditions and or chemicals can be used to enhance settling of Escherichia coli, in accordance with the present disclosure.Example5A #1, 2, 3, 4 and 5: chitosan concentration and pHRehn et al, Journal of Biotechnology 165 138 - 144 (2013) describes the flocculation of E. coli cell by the addition of chitosan. From the conclusion section of that paper: “Immobilization of E. coli cells by flocculation with chitosan may be implemented without need for advanced equipment or much additional efforts. The procedure is simple, effectively immobilizing a large amounts of cells using a small dosage of chitosan, which is a relatively low cost material.” Rehn et al. describes a clear method to flocculate E. coli with chitosan, although it focusses on immobilization. This method is used in Example 5A#1, #2, #3, #4 and #5.E. coli K12 was cultivated in Erlenmeyer flasks using standard media with glucose and K9-trace metals. Cultivation conditions were 37 °C and a starting pHof 7. Agitation of the shaker was at 200 rpm. Chitosan (~ 9 mg / g of stock solution or 1:200 chitosan:cells) was added in different phases of fermentation and at different pH values. The chitosan types used in the Examples were low molecular weight (LMW) (50-190kDa), Technical grade (TG) (medium to high) (190-375 kDa) and high molecular weight (HMW) (310-375kDa).The following Examples were generated: Example #1 - E. coli growth with chitosan; Example #2 -E. coli growth with chitosan and with pH correction to 7; Example #3 - reference growth of only E. coli; Example #4 - addition of chitosan after growth; Example #5 - chitosan addition after growth and pH correction to 7.The results (Figure 9) show very limited settling of E. coli without chitosan over time, while E. coli with chitosan addition shows almost full settling within 40 minutes.Figure 10 shows a histogram of % of cells settling grouped per settling rate interval. The bars are in order listed, with the example numbers being tl = example #1, t4 = example #4, t4 pH7 = example #5.The results (Figure 9 and Figure 10) show that chitosan can be used to promote aggregation and effectuate settling in minimal media. The growth rate of E. coli K12 is negatively impacted by the presence of chitosan and was observed to be about half. Addition of chitosan during growth, under the provided amount, enhanced the settling rate to a similar extent as addition after growth. An increased pH of 7 decreased the settling rate significantly, indicating a lower pH value is preferred for higher settling rates.So in this Example it is shown that chitosan addition has a significant impact on E. coli settling rate. Furthermore pH was identified as an additional control factor for settling. Addition of chitosan increases settling rate to >0.035 cm / min, which is near the lower limit of the preferred embodiment with regard to settling rate.Example 5B #1, 2, 3 and 4: Chitosan, temperature and pHRehn et al. (2013) describes the flocculation of E. coli cell by the addition of chitosan. The following Examples were generated by running a fed-batch fermentation in 3L Eppendorf Scivario reactors. E. coli K12 (wildtype) was used. The starting weight was 1 kg broth solution. Total fermentation time was 66 hours, (max) Glucose feed-rate applied was 3 g / kg starting weight of broth. Chitosan was added 40 hours after inoculation, at a dosing of 0.3 g / kg broth. Conditions were changed 18 hours after inoculation for Examples #2, #3, and #4, as shown in table 3 with the results of the E. coli settling. Addition of Chitosan was carried out by sterile addition, ~30ml solution in total). Media was M9 with 10g glucose / kg for the batch phase. N-source was added later to avoid precipitation.E.coli was cultivated in 3L STR with addition of chitosan at different time points during the fermentation. The calculated settling rate per experimental set point can be found in Table 3.Table 3: Setpoint (SP) temperature and pH at 18 h after inoculation and E. coli settling rate at end of fermentation (at 66 h).Example #1 #2 1 #3 #4Temperature SP 25 30 37 30pH SP 6.9 6.9 6.9 7.4Settling rate at 66 h; <0.003 0.006 0.023 0(cm / min)Temperature is observed to inversely effect biomass yield, as at the end of fermentation biomass yield for the 25°C setup is 0.4 g / g and 0.32 g / g for 37°C setup. Furthermore in these Eexamples the settling rate of E.coli is enhanced by Chitosan induced aggregation, depending temperature and pH. The observed effects of pH and temperature and their associated growth conditions are found to be significant.Similar to Example 5A a lower pH was found to enhance settling.Temperature is found to impact biomass yield and therefore concentration. As for yeast, biomass concentration is known to have an impact on settling. So for low effective growth rates, in continuous fermentation systems, in combination with the appropriate biomass concentration, subsequent settling rate and selective solid separation, yield to biomass in combination with fermentation product yield can be optimized.Example 5C #1 and #2: Chitosan grade, temperature and phosphate limitationGrowth and nutrient limitations are known to impact shape and morphology of microorganisms (Romano et.al, Phosphate Limitation Induces Drastic Physiological Changes, Applied and Environmental Microbiology, 2015, Volume 81 Number 10). Two examples were generated, varying chitosan grade, chitosan addition timing, exponential growth rate and phosphate limitation. Chitosan addition was 0.3 g / kg starting broth. The setpoint changes after 18h are shown in Table 4. The exponential feed was started after finishing the batch dosing of glucose and was triggered automatically based on a CO2 off-gas value change. The reactor in Example #1 was set at lower exponential feed in combination with phosphate limitation and gave a final biomass concentration of ~25 g / kg. The final biomass concentration of Example #2 was ~ 40 g / kg.Table 4: Experimental setup and results for example #1 and #2 (part of Example 5C)Example #1 Example #2 Chitosan Medium to High Mw High Mw Temperature (°C) start & 30 (30) 30 (37)(after 18 HAI)Start weight (kg) 1.0 1.2pH 6.9 6.9Timepoint addition chitosan 18 41Feed rate c-source [g / (kg.h)] 2.0 3.0Settling rate at 41 h(cm / min) 0.027 0.015Settling rate at 66 h[cm / min] 0.018 <0.005Phosphate limited yes noSettling rates were calculated to vary between 0.015-0.027 cm / min.Figure 11A-D show pictures of 4x diluted samples, after 30 min settling, of example #141h (11A) and 66h (11B) and example #241h (11C) and 66h (11D). Figure 12A and B show microscopic pictures of example #1 (12A) and example #2 (12B).Settling rates can indeed be varied and controlled within desired window, by addition of chitosan in combination with growth conditions.. E. coli under phosphate nutrient limitation experienced chain growth as expected, which further enhanced settling compared to normal growing E. coli. It was found that the phosphate limitation, in combination with growth rate and different grade chitosan addition severely impacts cell morphology (as can be seen in Figure 12A and Figure 12B).So as morphology can be steered, an increased settling rate can be achieved while maintaining sufficient accessible surface area for mass transfer. And therefore selective solids separation can be applied to E. coli cultivations without negatively impacting fermentation process performance.Example 6: Settling of Corynebacterium glutamicumRehn et al. (2013) describes a clear method to flocculate E. coli with chitosan. To further investigate the effect of chitosan on other relevant industrial hosts this same method will be used Corynebacterium glutamicum, a host typically used for amino acid production.Example 6A: chemical additive chitosanIn this Example settling behaviour of a wild-type Corynebacterium glutamicum with chitosan was demonstrated. The Corynebacterium glutamicum was cultivated in shake-flasks, using a LB media for the preculture and a CGXII media containing M9 trace metals for the growth step, both at 30°C. Subsequently settling experiments were performed using the cell culture material from the shake-flasks. In example #1 no chitosan was added and in example #2 10 g / kg TG chitosan in 0.1 M citric acid was added at tO of the growth step.Figure 13 shows the histogram of extent of settling expressed as settled cell fraction (%) of example #1 (A) and #2 (C) at timepoint t2 (5.5h) and Figure 14 shows the settling test of example #1 (left tube) and example #2 (right tube).As can be seen in Figure 13 and Figure 14, an adverse effect of chitosan was observed. The Corynebacterium glutamicum was found to be self- aggregating with a certain morphology (Figure 14) and the addition of chitosan is suspected to have changed this morphology resulting in an adverse effect on the settling rate of the aggregates. Figure 14 shows the settling experiments in which larger flocs are observed for Example 2 in combination with a decrease in effective settling rate. The physical properties of the flocs in example #2 are now such that settling is adversely impacted, meaning that there is an optimum in certain factors, like size, which is not necessarily the maximum value that leads to maximum settling.Example 6B #1,#2 and #3: chemical additive chitosan concentration, chitosan grade and biomass concentrationIn this Example settling behaviour of a wild-type Corynebacterium glutamicum with chitosan was demonstrated. The Corynebacterium glutamicum was cultivated in shake-flasks, using a LB media for the preculture and a CGXII media containing M9 trace metals for the growth step, both at 30°C. Subsequently settling experiments were performed using the cell culture material from the shake-flasks. Chitosan in 0.1 M citric acid was added at tO of the growth step.Example #1: Settling of Corynebacterium glutamicum (15 g / kg) with different grades of chitosan (HMW, TG & LMW) in two ratios (3 &5Figure 15 shows the settling test samples of example #1 with different chitosan grades and concentrations from left to right (after 61 minutes of settling): tube 1 = Chitosan HMW 3 mg / g cells, tube 2 = Chitosan HMW 5 mg / g cells, tube 3 = Chitosan TG 3 mg / g cells, tube 4 = Chitosan TG 5 mg / g cells, tube 5 = Chitosan LMW 3 mg / g cells, tube 6 = chitosan LMW 5 mg / g cells. From Figure 15 it can be observed that the samples with high molecular weight (HMW) chitosan have a higher settling rate than the samples with technical grade (TG) and low molecular weight (LMW) chitosan.Example #2: Settling of Corynebacterium glutamicum (15 g / kg) with high molecular weight (HMW) chitosan in different ratios (0-20Figure 16 shows the settling test samples of example #2 with different chitosan grades and concentrations from left to right (after 46 minutes of settling): tube 1: 20 mg HMW chitosan / g cells, tube 2: 13 mg HMW chitosan / g cells, tube 3: 10 mg HMW chitosan / g cells, tube 4: 5 mg HMW chitosan / g cells, tube 5: 3 mg HMW chitosan / g cells. From Figure 16 it can be observed that the samples with 20, 13 and 10 mg chitosan / g cells have a clear top layer, where 5 and 3 mg chitosan / g cells have a more cloudy top layer. The 20 mg chitosan / g cells sample has the fastest initial settling, followed by 13 and 10 mg chitosan / g cells. After 7 minutesthe sample with 5 mg chitosan / g cells showed significant settling. After 14 minutes the 3 mg chitosan / g cells sample has significant settling with a smaller settled fraction.It was observed that the top layer of the 5 and 3 mg chitosan / g cells concentration samples remains cloudy, indicating a wide particle size distribution. The lower the chitosan concentration the more smaller cell aggregates are present that do not settle within the given time frame. Also a settling rate tipping point was observed between the 10 mg chitosan / g cells and 5 mg chitosan / g cells concentration, as the 5 mg chitosan / g cells concentration experienced a higher settling rate of a certain fraction of the biomass, indicating a difference in morphology / size / shape between these two samples.Example #3: Settling of Corynebacterium glutamicum in different concentrations with HMW chitosan a ratio of 1Figure 17 shows the settling test samples of example #3 with the different biomass concentrations from left to right (after 20 minutes of settling): tube 1: 5 g / kg, tube 2: 10 g / kg, tube 3: 15 g / kg, tube 4: 20 g / kg. From Figure 17 it can be observed that the samples with more biomass settle more slowly.So from Example 6A and 6B it was observed that chitosan dosing needs to be carefully chosen and balanced, as there is an optimum in certain factors, like size, which is not necessarily the maximum value that leads to maximum settling. Different grades and concentrations of chitosan were observed to have different impacts on settling and settling rate ranges. When balanced correctly chitosan gives a settling rate distribution such that one fraction settles within minutes and the other fraction settles an order of magnitude slower, allowing selective separation between the two fractions.Example 7: Settling of Yarrowia lipolyticaExample 7A: chemical additive chitosanSimilar to Examples 5 and 6, the effect of chitosan on other relevant industrial hosts is demonstrated for Yarrowia lipolytica, Louhasakul et al, Renewable Energy 136 1311-1319 (2019). The experimental setup is given in Table 6. Yarrowia lipolytica is typically used for the production of lipids, biofuels and enzymes and can use unusual carbon sources as an substrate, allowing for high industrial process versatility.Table 6: Experimental setup4x Preculture 2x growth testShake-flask Shake-flaskStrain Y. lipolytica Y. lipolyticaMedia YPD VerduynTemp 30°C 30°CpH (start) 7 7Agitation 140 RPM 140 RPMTime 24h 24hSuspended biomass from the growth test was used to test a range of different chitosan grades in combination with different chitosan concentrations per amount of cells.Figure 18 shows the settling of Yarrowia lipolytica samples after 30 minutes (CDW: 15 g / kg) in Verduyn media at pH7, with different chitosan types. From left to right: tube #10.005g Chitosan high molecular weight (HMW) / gceiis; tube #2 0.013g Chitosan HMW / gceiis; tube #30.005g Chitosan technical grade (TG) / gceiis; tube #40.013g Chitosan (TG) / gceiis; tube #50.005g Chitosan low molecular weight (LMW) / gceiis; tube #60.013g Chitosan LMW / gceiis.The observed settling rate in Figure 18 is -0.002 cm / min. This is near the lower end of the desired settling rate window.Figure 19 shows the settling of Yarrowia lipolytica samples after 60 minutes settling, (CDW: 15 g / kg) in Verduyn media at pH7 with different chitosan types.From left to right: tube #10.005g Chitosan high molecular weight (HMW) / gceiis; tube #20.013g Chitosan HMW / gceiis; tube #30.005g Chitosan technical grade (TG) / gceiis; tube #40.013g Chitosan (TG) / gceiis; tube #50.005g Chitosan low molecular weight (LMW) / gceiis; tube #60.013g Chitosan LMW / gceiis.In both Figure 18 and 19 it can be observed that only the sample with chitosan type LMW at a concentration of 0.013 g / gceiis visibly settles slower than the rest of the samples. All other samples have about the same settling rate.In this Example it is observed that grade chitosan in combination with concentration can have an adverse effect on settling rate. Therefore application of factors impacting settling rate is not straightforward and the strategy to control settling and settling rate must be chosen carefully.Example 7B: pHAs other examples have shown an impact of pH on settling behaviour this has also been tested for Yarrowia lipolytica in a similar setup as for Example 7A.Figure 20 and 21 show the settling of Yarrowia lipolytica samples after respectively 30 minutes settling (Figure 20) and 60 minutes of settling Figure 21) (CDW: 15 g / kg) in Verduyn media at different pH without chitosan. From left to right pH of 3, 4, 5, 6 and 7.The observed settling rate (Figure 20) is -0.0015 cm / min. Again on the lower end of the desired settling rate window. In Figure 21 it is observed that samples with pH 6 & 7 settle slightly faster than the samples with lower pH.Overall, compared to Example 7A, there is almost no effect observed on settling rate over a large pH range. So industrial processes can be run at a wide range of pH without adversely effecting settling rate for selective solid separation.Example 8: Saccharomyces cerevisiae flocculationExample 8A: dynamic conditions, e.g. flow regimeYeast flocks break up under increased shear force and / or turbulent conditions such as those for example from a stirrer, agitator or pump. Under low shear and / orlow turbulence the break up is reduced and overall net flocculation is observed. Thus for a flocculating yeast flocculation is a reversible process..The example was generated using Saccharomyces cerevisiae CCUG 53310 in the a 3L fermentation system. During the fermentation stirring was stopped to demonstrate the effect of high versus low shear environment on the yeast flocculation state.In Figure 22A-D snapshots of the video are shown in which the stirring is stopped ((A) TO (0 sec); (B) T1 (10 sec); (C) T2 (20sec); (D) T3 (30 sec)). In time the degree of flocculation and settling changes significantly within a characteristic time of seconds to a minute. This characteristic time is within the typical residence time range of the continuous fermentation system (as described in example 9) more laminar flow separation zone. And therefore flocculation, subsequent very high settling rates, solid retention and selective separation of active biomass over cell debris or other solids is expected in the separation zone. Whereas break-up of the flocs is expected when returning from the separation zone into the fermentation zone, negating possible mass transfer limitations by large flocs in the fermentation zone. This phenomena is a preferred way to control settling rate, without negatively impacting fermentation process performance as result of mass transfer limitations.Example 9: Settling in continuous fermentation systemsExample 9A: Continuous fermentation system FASTMINIAs described in Example 8 yeast flocks break up under increased shear force and / or turbulent conditions such as those for example from a stirrer, agitator or pump. Under low shear and / or low turbulence the break up is reduced and overall net flocculation is observed. Thus for a flocculating yeast flocculation is a reversible process. This example is generated to demonstrate flocculation and settling in the fermentation zone versus the more laminar separation zone of a continuous fermentation system.A standard Verduyn medium was used, supplemented with the addition of 8 mM Ca2+to aid flocculation (Table 7). This was done in the form of Calciumsulphate dihydrate, as the chloride form could create pitting in the vessel’s metal parts. The optimal flocculating range for this embodiment is pH 3-5, therefore it was chosen to run at pH 4.A cultivation of the Saccharomyces cerevisiae CCUG 53310 was performed in a FASTMINI (device as disclosed in WO2024049295), similar to figure 3A, in which the fermentation zone is fluidly connected to a separation zone, which is in turn fluidly connected to a recirculation section. The recirculation zone returns the liquid broth to the fermentation zone. The fermentation zone is turbulent, while the separation zone has a more laminar flow pattern allowing gravity based phase separation of gas, liquid(s) and solid(s).Table 7: Experimental setupFASTminiStrain S. cerevisiae CCUG 53310Temp 30 degCpH 4Media Verduyn + 8 mM CalciumBatch 20 g / kg glucoseFeed 50 wt% glucoseFigure 23 shows a picture of FASTMINI fermentation, separation and recirculation zone during the experiment. As can be seen in Figure 23 larger flocs are present in the more laminar separation zone. This is line with the lower shear stress impact on the strain and the balance between flocculation and break up as observed in Example 8. Also settling of the flocs is observed in the separation zone. A higher floc density is observed towards the bottom of the separation zone versus the top, where a clear liquid layer can be seen. This clear liquid layer was continuously removed via the harvest dip tube (as seen in the picture in Figure 23).As discussed in Example 8, the flocculation, subsequent very high settling rates, solid retention and selective separation of active biomass over cell debris or other solids were expected in the separation zone and break-up of the flocs was expected when returning from the separation zone into the fermentation zone. This example confirms that indeed happens and this preferred way to control settlingrate, without negatively impacting fermentation process performance as result of mass transfer limitations, is possible in the continuous fermentation system FASTMINI. As larger scale FAST systems (as in described in W02021 / 010822 Al), like the FAST500 pilot reactor (in example 9B & 9C), and also at scale, have similar conditions in the fermentation and separation zones, this is expected not only to work for FASTMINI but to be widely applicable for any FAST system at any scale. Unlike the CIRCOX® type reactor, a non-producing continuous airlift fermentation system, used in wastewater treatment, as listed in WO2021 / 010822 Al, which uses granulated carrier material to retain biomass. As previously mentioned the applied residence time and turbulence significantly differ. The main method of retention in CIRCOX® is by the creation of (anaerobic) large biomass film layer covered granules. These granules have rapid settling, and can easily be applied, because diffusion limitations in waste water treatment are, due to the COD load / reactor productivity, much less constrictive, compared to industrial product producing reactors. Efficient fermentative production can not occur under such diffusion constraints.So selective separation can be applied in any continuous fermentation system with similar hydraulic zones and corresponding residence times at any scale. It is thus possible to separate active biomass from solid matter, different from said micro-organism, comprising one or more of the following: cell debris, lysed cells, protein, and the like, solid phase product, extracellular matter, different aggregation and / or flocculation state of the micro-organism(s) from said micro-organism.Example 9B: Continuous fermentation system FAST500 using Saccharomyces CerevisiaeIn a preferred embodiment the separation into said first and said second fraction is aided by the presence of an organic second liquid phase, said second liquid phase comprising either a (by)product forming its own liquid phase and / or auxiliary liquid phase supplied to the bioreactor system. It is known in the field that, typically more hydrophobic, solid material can adhere to the surface of such second (organic) liquid phase, depending on interactions.This example demonstrates the use of such second liquid phase and the resulting selective solid separation. Extractive production of 2-phenyl ethanol (2-PE) using oleyl alcohol as extractive phase with (non-flocculating) S. Cerevisiae (Hassing, E. J., et. al 2019. “Connecting Central Carbon and Aromatic Amino Acid Metabolisms to Improve De Novo 2- Phenylethanol Production in Saccharomyces cerevisiae.” Metabolic Engineering 56: 165-180) was carried out in a pilot scale reactor for 100+ hours using an experimental setup analogous to Brewster et al. (Biotechnology & Bioengineering 122:287-297, 2024). The reactor was configured as described in WO2021 / 010822 Al, see e.g. Figure 1 for a schematic view of the system.Similar as described in [WO2021 / 010822 Al] continuous addition and separation of extractive phase was applied during the production phase, at about ~20 kg / hr. Batch phase growth was directly followed by an exponential feed growth phase (together ~ 50 hours), going into a 50+ hours of a more steady-state production phase. The system had high internal liquid recirculation, from fermentation zone to separation zone and back via the recirculation zone, and allowed continuous removal of liquid matter from the separation zone via a harvest outlet..No accumulation of extractive phase in the fermentation zone was observed. The extractive phase was always below 5 v / v%. Figures 24 and 25 show samples of the harvest (left) and sample of the fermentation broth (right) from the FAST500 continuous flow fermentation system. The sample bottom layer is solids, middle layer is aqueous phase and top layer is extractive phase. The phase composition of the liquid removed from the separation zone via the harvest outlet was initially about 100% extractive phase (see Figure 24). Throughout the fermentation the harvest composition went to an average composition of 20 vol% aqueous phase and 80 vol% extractive phase. Cell dry weight (CDW) content of the fermentation broth was increasing during the production phase from 26 g / kg to an average steady state like value of 41 g / kg. Production of 2-PE reached a semi-steady state of 0.7 g / (L h), while the respiratory quotient, based on off-gas data, remained constant.The fermentation 2-PE production activity was maintained, while also the CDW was constant and thus the active cell level remained constant. However in 2-PE fed-batch fermentations the CDW was observed to be consistently far higher, namely 50 g / kg aqueous broth, at similar yield.This difference between lab cell dry weight (CDW) in the broth and CDW in the continuous flow system in combination with the effectuated 2-PE productivity shows that active biomass was selectively being retained in the system, as less active solid(s) were removed from the system, otherwise the CDW should have been the same. The solid fractions in Figure 24 and Figure 25 confirm indeed a higher solid fraction per amount of aqueous fraction in the harvest sample versus the fermentation broth sample.Example 9C: Continuous fermentation system FAST500 using E. coliIn a similar set up as the example 9B non-flocculating E.coli K12 was cultivated for 100h with ongoing addition and removal of oleyl alcohol as extractive phase, while the reactor was continuously dosed with geraniol (at levels that would have been inhibiting if the extractive phase was not present) and increased with each ramp of the extractive phase. The final geraniol dosing value corresponding to 1 g / (L h). The E.coli remained viable throughout the 100 hours and inhibition control was indeed achieved by the ongoing extraction and removal of geraniol. The extractive phase dosing was ramped and ranged ~3, ~6, ~10 kg / h. Again the extractive phase to water phase ratio in the harvest was about 80% / 20%. No buildup of extractive phase in the fermentation section occurred. The respiratory quotient remained almost constant throughout the fermentation at 0.8, which is the typical value for E. coli experiencing limited geraniol inhibition stress.Figure 26 shows a sample of harvest (left) and sample of fermentation broth (right) from the FAST500 continuous fermentation system. The sample bottom layer is solids, middle layer is aqueous phase and top layer is extractive phase. Note that the top layer is not significant enough to be visible in the tube on the right hand side.The amount of solid material in the aqueous phase fraction of the harvest is about 30% on volume basis, while the aqueous broth only has about 10% on volume basis. Significantly more solid mater was present in the harvest per amount of aqueous phase. So it is confirmed that selective solid separation has taken place, asthere is different amount of solids per amount of aqueous fraction in the harvest versus the fermentation broth.Compared to the difference in solid fraction the degree of selective separation for this example is much higher, so there must be a difference in solid physical properties and its distribution. This can either be explained by the properties of the E. coli versus the yeast in relation to the same extractive phase or the fact that more cell lysis with resulting cell debris was generated in this example. It was observed in the fermentation that, at a stable respiratory quotient, with the final ramp of the extractive phase the CDW remained the same, indicating that the extractive phase does not selectively pull the active biomass fraction.In summary, as demonstrated by the provided examples, cell settling can be controlled to occur at desired rates, by the carefully selecting relevant factors. While the cause-effect relations between these factors and floc size / settling rate alterations are well established. Balancing these factors is observed to be critical as they can also lead to adverse effects, either by combination or the wrong set point(s) / concentration(s). As literature focusses on settling or no settling, typically at end of fermentation, but do not aim to control the settling rate per se. The presented examples were generated for specific strains, and demonstrate industrial relevant operational windows for continuous fermentation systems.In this way, selective solid separation can help to overcome current limitations (e.g. mass transfer, wash out, product stability, emulsification) and can be used to improve the performance indicators of biocatalytic processes, such as yield, space time utilization, productivity, total product obtained from the system (i.e. titre), directly impacting the economic feasibility and sustainability.BRIEF DESCRIPTION OF THE FIGURESFigure 1 shows a distribution of sample cells by range of settling rates, for three sample column heights.Figure 2: shows normalised OD values in the first 9 minutes of settling, for five experimental conditions with different Ca2+concentrationsFigure 3 shows a setling rate of Saccharomyces cerevisiae suspensions, EDTA concentrations of 0.0-3.6 mM, and pH values of 4, 5 and 6.Figure 4: shows four photographs illustrating an effect of EDTA on flocculation: No EDTA (A), 3.6 mM EDTA (B), 7.2 mM EDTA (C), 7.2 mM EDTA + 7.2 mM Ca2+(D). Grid distance is 50 micron.Figure 5 shows biomass settling rates determined in the first 30 seconds of settling, which is an approximation of initial settling rate. The results are shown for variable cell dry weight (CDW) for pH 5 and 6.Figure 6 shows a microscopic observation of a Saccharomyces cerevisiae floc from a 3L fermentation at pH 6 (arrow is 200 micron).Figure 7 shows Komagataella phaffii fermentation samples 5 minutes after being taken from the shake flask at A) 4 h, B) 10 h and C) 51 hFigure 8 shows settling behaviour of a K.phaffii sample at different pH.Figure 9 shows E. coli K12 settling without chitosan (example #3) and with chitosan (example #1)Figure 10 shows a histogram of % of cells settling grouped per settling rate interval. The bars are in order listed, with the example numbers being tl = example #1, t4 = example #4, t4 pH7 = example #5.Figure 11 shows pictures of 4x diluted samples, after 30 min settling, of Example 5C#141h (A) and 66h (B) and example 5C#241h (C) and 66h (D)Figure 12 shows microscopic pictures of Example 5C#1 (A) and Example 5C#2 (B) Figure 13 shows histogram of extent of settling expressed as settled cell fraction (%) of Example 6#1 (A) and 6 #2 (C) at timepoint t2 (5.5h)Figure 14 shows settling test of Example 6A#1 (left tube) and Example 6A#2 (right tube)Figure 15 example 6B #1; Chitosan grade and concentration from left to right (after 61 minutes of settling): tube 1 = Chitosan HMW 3 mg / g cells, tube 2 = Chitosan HMW 5 mg / g cells, tube 3 = Chitosan TG 3 mg / g cells, tube 4 = Chitosan TG 5 mg / g cells, tube 5 = Chitosan LMW 3 mg / g cells, tube 6 = chitosan LMW 5 mg / g cells. Figure 16 shows example 6B #2 Chitosan grade and concentration from left to right (after 46 minutes of settling): tube 1: 20 mg HMW chitosan / g cells, tube 2: 13 mg HMW chitosan / g cells, tube 3: 10 mg HMW chitosan / g cells, tube 4: 5 mg HMW chitosan / g cells, tube 5: 3 mg HMW chitosan / g cellsFigure 17 shows Example 6B #3 Biomass concentration from left to right (after 20 minutes of settling): tube 1: 5 g / kg, tube 2: 10 g / kg, tube 3: 15 g / kg, tube 4: 20 g / kg Figure 18 shows settling of Yarrowia lipolytica samples (Example 7a), after 30 minutes settling, (CDW: 15 g / kg) in Verduyn media, pH7, with different chitosan types. From left to right: tube #1 0.005g Chitosan high molecular weight (HMW) / gcells; tube #2 0.013g Chitosan HMW / gcells; tube #3 0.005g Chitosan technical grade (TG) / gcells; tube #4 0.013g Chitosan (TG) / gcells; tube #5 0.005g Chitosan low molecular weight (LMW) / gcells; tube #6 0.013g Chitosan LMW / gcellsFigure 19 shows settling of Yarrowia lipolytica samples, after 60 minutes settling, (CDW: 15 g / kg) in SMG, pH7, with different chitosan types. From left to right: tube #1 0.005g Chitosan high molecular weight (HMW) / gcells; tube #2 0.013g Chitosan HMW / gcells; tube #3 0.005g Chitosan technical grade (TG) / gcells; tube #4 0.013g Chitosan (TG) / gcells; tube #5 0.005g Chitosan low molecular weight (LMW) / gcells; tube #6 0.013g Chitosan LMW / gcellsFigure 20 shows settling of Yarrowia lipolytica samples (Example 7B), after 30 minutes settling, (CDW: 15 g / kg) in Verduyn media, at different pH without chitosan. From left to right pH of 3, 4, 5, 6 and 7.Figure 21 shows settling of Yarrowia lipolytica samples, after 60 minutes settling, (CDW: 15 g / kg) in Verduyn media, at different pH without chitosan. From left to right pH of 3, 4, 5, 6 and 7.Figure 22 Picture of 3E fermenter at different times after stopping the stirring (a) TO (0 sec); (b) T1 (10 sec); (c) T2 (20sec); (d) T3 (30 sec)Figure 23 shows a picture of FASTMINI fermentation, separation and recirculation zone during the experiment.Figure 24 shows a sample of harvest (left) and sample of fermentation broth (right) from continuous fermentation system (Example 9B). Extractive phase fraction in harvest was -100%. Bottom layer is solids, middle layer is aqueous phase and top layer is extractive phase.Figure 25: shows a sample of harvest (left) and sample of fermentation broth (right) from continuous fermentation system. Extractive phase fraction in harvest was -80%. Bottom layer is solids, middle layer is aqueous phase and top layer is extractive phase.Figure 26 shows a sample of harvest (left) and sample of fermentation broth (right) from continuous fermentation system obtained in Example 9C.

Claims

Claims1. A method for selective cell retention of a micro-organism during a continuous or semi-continuous fermentative production of one or more organic substances in a bioreactor system, the bioreactor system comprising a fermentation zone, and a separation zone, which fermentation zone and separation zone are fluidly connected, the method comprising providing a reaction mixture in the fermentation zone, which reaction mixture comprises the micro-organism and an aqueous phase, the aqueous phase comprising a substrate for the micro-organism;the method comprising a stage during which the fermentative production and a separation are simultaneously carried out, whereinat least during a part of said simultaneous production and separation stage, substrate for the micro-organism is continuously or semi- continuously fed into the bioreactor;at least during a substantial part of said simultaneous production and separation stage, the reaction mixture, comprising the micro-organism and the produced organic substance, or at least an aqueous fraction of the reaction mixture, is continuously or semi-continuously fed from the fermentation zone into the separation zone,subjecting the reaction mixture, comprising the micro-organism and the produced organic substance, or at least an aqueous fraction of the reaction mixture, fed into the separation zone, to a separation into at least two fractions, thereby forming a first fraction enriched in the micro-organism and a second fraction enriched in solid matter different from said microorganism in which the first fraction is enriched, wherein the separation into said first fraction and said second fraction makes use of at least one difference in a physical property between said micro-organism in the first fraction and said solid matter in the second fraction, which physical property is selected from the group of specific gravity, electrostatic forces, polarity / non-polarity, size, shape, magnetic properties and hydrophobicity; at least during a substantial part of said simultaneous production and separation stage, at least a part of said second fraction is removed continuously or semi-continuously from the bioreactor system;at least during a substantial part of said simultaneous production and separation stage, at least part of the first fraction, enriched in the microorganism, is continuously or semi-continuously fed from the separation zone back into the fermentation zone.

2. The method according to claim 1, wherein said first fraction enriched in said micro-organism is retained in the bioreactor system by alignment of internal flow hydrodynamics, system geometry and effective microorganism ‘particle’ size, whereinsaid internal flow hydrodynamics are turbulent conditions (preferably a Re of more than 3000) in the fermentation zone and less turbulent (preferably Re of 2900 or less), essentially laminar conditions (Re < 2300), in the separation zone,wherein the solid matter different from said micro-organism, comprises one or more of the following: cell debris, lysed cells, protein, and the like, solid phase product, extracellular matter, different aggregation and / or flocculation state of the micro-organism(s) from said micro-organism in the first fraction,wherein said solid matter is separated from the first fraction by aqueous phase liquid withdrawal from the separation zone, and / or presence of a second liquid phase in the system, and / or presence of a gas phase in the separation zone.

3. The method according to claim 1 or 2, wherein the separation into said first and said second fraction is aided by the presence of an organic second liquid phase, said second liquid phase comprising either a (byproduct forming its own liquid phase and / or auxiliary liquid phase supplied to the bioreactor system.

4. The method according to any of the preceding claims, wherein the separation into said first and said second fraction is aided by the presence of an aqueous second liquid phase, said second liquid phase comprising an aqueous phase, containing one or more polymers and optionally a salt.

5. The method according to any of the preceding claims, wherein the separation into said first and said second fraction is aided by the presence of a gas phase, said gas phase either being locally introduced and / or beingthe result of gas entraining in the aqueous fraction to the separation zone as result of hydrodynamics.

6. The method according to any of the preceding claims, wherein- the retention time of said first fraction is significantly larger than the hydraulic retention time of the (main) aqueous phase in the bioreactor system;- the retention time of said second fraction is equally or lower than the hydraulic retention time of the (main) aqueous phase in the bioreactor system; and- in case the separation is aided by the presence of a second liquid and / or gas phase the retention time of said second fraction is significantly lower than the hydraulic retention time of the (main) aqueous phase in the bioreactor system.

7. The method according any of the preceding claims, wherein the solid matter by which said second fraction is enriched comprises the microbially produced organic substance, preferably a substance selected from the group consisting of proteins, fats, long chain alcohol, solid flavour molecules, vitamins, pigments and solid dyes.

8. The method according to claim 7, wherein the microbially produced organic substance comprises a protein, preferably a protein selected from dairy proteins, meat proteins, egg proteins and enzymes.

9. The method according to claim 7 or 8, whereinthe micro-organism used for the production of the organic substance, preferably protein, comprises Pichia cells;the first fraction enriched in biomass is enriched in the Pichia cells and the second fraction is enriched in the produced organic substance, preferably protein;the separation into said first fraction and said second fraction comprises the formation of aggregates comprising Pichia cells and allowing said aggregates to settle in the separation zone, thereby forming the first fraction, enriched in the Pichia cells in a lower liquid layer in the separation zone and the second fraction enriched in the produced organic substance, preferably protein, in an upper layer.

10. The method according to any of the preceding claims, wherein the first fraction comprises particles of micro-organism cells of the micro-organism with which the first fraction is enriched, such as aggregates, flocs, filamented micro-organism, said particles having an observed settling rate of 0.002 cm / min to 1.4 cm / min (typically corresponding to a ‘spherical’ particle size of about 7 micron to about 0.5 mm).

11. The method according to claim 10, wherein said particles have an observed settling rate of 0.01 cm / min to 0.8 cm / min (typically corresponding to a size of about 10 micron to about 0.3 mm).

12. The method according to claim 11, wherein said particles have an observed settling rate of 0.08 cm / min to 0.5 cm / min (typically corresponding to a size of about 50 micron to about 0.15 mm).

13. The method according to any of the preceding claims, wherein said simultaneous production and separation stage comprises an essentially steady state.

14. The method according to claim 13, wherein during said essentially steady state substrate is fed from the fermentation zone into the separation zone, said separation into said first fraction and said second fraction is carried out, at least a part of said second fraction is removed from the bioreactor system, and at least part of the first fraction, enriched in the microorganism, is fed from the separation zone back into the fermentation zone.

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