The present invention relates to a method for cultivating a microorganism in fed-batch culture. According to a further aspect, the present invention relates to a fermentation plant comprising more than one bioreactor for cultivating a microorganism in fed-batch culture.

Background

Industrial fermentation plants can be mono-plants where many repeated batches of a single product are executed in multiple bioreactors, also known as fermentation vessels. When these industrial fermentation processes are executed in conventional fed-batch mode, they are characterized by a low initial and high final bioreactor filling. By consequence the average degree of filling of these bioreactors often is low in comparison to processes run in batch and chemostat mode. Thus, while many fermentation plants are operated at maximum capacity, a significant part of the available fermentation capacity is filled with air rather than fermentation broth. This is a waste of capital invested. This problem is particularly present for large bioreactors, typically ranging from 30 to 500 m ³ .

WO2016/189203 discloses a method for producing a biosynthetic product in a cascade of bioreactors comprising a biomass production reactor and a product formation reactor, wherein at least part of the microorganism culture from the biomass production reaction is fed to the product formation reactor which contains nutrient depleted medium. The method can be carried out in fed-batch manner. WO2026/189203 does not relate to filling capacity of a fermentation plant. There is a need in the art for improved methods for industrial fermentation processes.

Detailed description

The present invention relates to a method for cultivating a microorganism, comprising the steps of:

(i) introducing into at least one first bioreactor a medium and an inoculum of the microorganism, and cultivating the microorganism in fed-batch culture until the degree of filling of the at least one first bioreactor is at least 80%, wherein cultivating the microorganism comprises producing a fermentation broth;

(ii) transporting a first part of the fermentation broth from the at least one first bioreactor to a second bioreactor; and

(iii) cultivating the microorganism in the at least one first and at least one second bioreactor in fed-batch culture until the degree of filling of the at least one first and at least one second bioreactor is at least 80%, wherein cultivating the microorganism in step (i) and (iii) comprises adding a feed to the at least one first bioreactor and / or the at least one second bioreactor. We found that a degree of filling of at least 80% of the first bioreactor and then transporting a part of the fermentation broth to a second bioreactor, preferably to an empty second bioreactor, and continuing the fed-batch fermentation in both bioreactors delivers a higher productivity as compared to a method for cultivating microorganisms in fed-batch culture in at least two bioreactors, wherein each bioreactor is emptied and cleaned after each fed-batch culture has been completed.

The term ‘degree of filling’ of a bioreactor as used herein refers to the part of the gross volume of the bioreactor that is occupied by gassed fermentation broth.

The term ‘first’, ‘second’ and ‘third’ bioreactors as used in the present context means that the first, second and third bioreactors are separate bioreactors, which can be of identical size or of different size. The term includes the scenario where there are more than one first, second or third bioreactors.

The term ‘first’ and ‘second’ part of the fermentation broth as used herein mean that the first part is of a younger fermentation age than the second part. The amount of fermentation broth in the present first and second part can be the same or can be different. The term ‘age of the broth’ as used herein means the time that has elapsed since the moment the first bioreactor was inoculated and the moment at which the age of the broth is specified.

As used herein, the first part and / or the second part of the fermentation broth from the at least one first bioreactor is not being concentrated before transporting the fermentation broth to at least one second and / or to at least one third bioreactor. Accordingly, the fermentation broth is transported as such from the at least one first bioreactor to at least one second and / or at least one third bioreactor.

In the present invention, only the at least one first bioreactor is filled with medium and inoculum to start up the cultivation of the microorganisms. Hence, in a preferred embodiment, the at least one second and/or the at least one third bioreactor do not comprise a step of introducing a medium and/or inoculum of the microorganism into the second and/or third bioreactor. Preferably, the at least one second bioreactor is empty and/or sterile before receiving a first part of the fermentation broth from the first bioreactor. Preferably, the at least one third bioreactor is empty and/or sterile before receiving a second part of the fermentation broth from the first and second bioreactor.

Preferably, cultivating the microorganism in the at least one first bioreactor in step (i) until the degree of filling of the at least one first and/or at least one second bioreactor is larger than, or at least 80%, comprises cultivating the microorganism in the at least one first bioreactor until the degree of filling of the first is larger than, or at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%.

Preferably, cultivating the microorganism in the at least one first and at least one second bioreactor in step (iii) until the degree of filling of the at least one first and/or at least one second bioreactor is larger than, or at least 80%, comprises cultivating the microorganism in the at least one first and at least one second bioreactor until the degree of filling of the first and/or second bioreactor is larger than, or at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%.

Cultivating the microorganism in the second bioreactor comprises producing a fermentation broth.

Cultivating the microorganism in step (i) and (iii) in the method according to the present invention comprises adding a feed to the bioreactor. The feed in the method ofthe invention, such as in step (i) and step (iii) and / or any other step in the method of the invention, comprises one or more suitable nutrients for cultivating the microorganism known to a person skilled in the art, for instance a carbon source such a glucose, fructose, maltose or sucrose. Usually the feed in a fed batch culture comprises a nutrient that is not present in the medium that is added into bioreactor. Adding a feed to a bioreactor is performed under sterile conditions known to a person skilled in the art.

In a preferred embodiment, the first part of the fermentation broth in step (ii) is an amount that results in a similar degree of filling of the at least one first and at least one second bioreactors after step (ii). A similar degree of filling is preferably a percentage within the range of plus or minus 10% or 5% of the averaged degree of filling of the bioreactors. For example with an averaged degree of filling of 40% the degree of filling in the bioreactors is within 30% to 50%.

Alternatively the first part of the fermentation broth in step (ii) is an amount within the range of 30% wtto 70% wt ofthe fermentation broth, such as 40% wtto 60% wt ofthe fermentation broth at the end of step (i). More preferably, the first part of fermentation broth is an amount within the range of 45% wt to 55% wt, or most preferably around 50% wt of the fermentation broth at the end of step (i). In a fermentation plant wherein multiple bioreactors of equal volume are used for the fed-batch fermentation, it is advantageous to equally divide the fermentation broth at the end step (i) in the first bioreactor amongst the first and second bioreactor. This equal division provides the maximum bioreactor capacity for further growth of the microorganisms in the fermentation broth. In a fermentation plant wherein multiple bioreactors of different volume are used for the fed-batch fermentation, it is advantageous to divide the fermentation broth at the end of step (i) in the first bioreactor amongst the first and second bioreactor in proportion to their respective volumes. For example, if the first bioreactor has a volume of 200 m ³ and the second bioreactor has a volume of 150 m ³ then the fraction of fermentation broth transferred to the second bioreactor in step (ii) would be 150/(200 +150) and the fraction remaining in the first bioreactor in step (ii) would be 200/(200 + 150).

Cultivating a microorganism is performed in fed-batch culture in the at least one first, second and / or third bioreactor. In the method according to the present invention, cultivating a microorganism is performed in fed-batch culture in each first, second and / or third bioreactor.

A fed-batch culture is known to a person skilled in the art, and generally can be defined as a process wherein one or more nutrients or substrates are fed to a bioreactor during fermentation or cultivation and in which products remain in the bioreactor until the end of a culture.

In some conventional fed-batch fermentation processes the degree of filling of the bioreactor at the start of fermentation is within the range of 20% to 30% of the bioreactor, and the fermentation is ended when the bioreactor is full. In the present invention however, the present medium and inoculum are preferably introduced to the at least one first bioreactor until a degree of filling is reached of more than 30% of the at least one first bioreactor, preferably more than 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% or even more than 75% of the at least one first bioreactor. Preferably, the present medium and inoculum are preferably introduced to the at least one first bioreactor until a degree of filling is reached within the range of 40 to 80% of the first bioreactor, more preferably 50 to 75% of the at least one first bioreactor. In comparison with conventional fed-batch fermentation processes the initial volume of medium plus inoculum is higher, in order to benefit from the increased capacity for the fast growth phase of the microorganisms in the fermentation broth.

The present inventors found that by increasing by a factor of more than one, for example two, the amount of medium of the first bioreactor and preferably also increasing the size of the inoculum of this bioreactor, and by transporting a part of the fermentation broth to a second bioreactor once the first bioreactor is full or nearly full, the average degree of filling of the bioreactors increases significantly, and thus improves the return on investment of the fermentation plant.

In a preferred embodiment, the present method comprises repeating the steps (i) to (iii) and/or wherein the number of first bioreactors relative to the number of second bioreactors is 2:1 or is 3:2 or is 4:3. In this way the present method can advantageously be used in fermentation plants having for example three, five or seven bioreactors, or a multiple of three, five or seven bioreactors.

To improve the efficiency in fermentation plants having several bioreactors, it is advantageous to repeat the present steps (i) to (iii) for all or nearly all first bioreactors that are newly inoculated. One can always use the same bioreactors in the plant as first bioreactor and as second bioreactors, but one can also vary the use of bioreactors as first and as second bioreactor.

The advantage of the present method is that the degree of filling of the bioreactors is higher than in conventional fermentation processes. A conventional time-averaged degree of filling is lower than 60%, excluding idle time of the bioreactor during ‘turn-around’ of the bioreactor in between two consecutive batches. Preferably, the time-averaged degree of filling in the present first and second bioreactors from the beginning of step (i) towards the end of step (iii) is higher than 60%, such as higher than 65% or even higher than 70%.

To improve the efficiency of a fermentation plant, the number of bioreactors used as first bioreactors (for carrying out steps (i) and (iii)) is always larger than the number of bioreactors used as second bioreactors (for carrying out step (iii)). Preferably, the number of first bioreactors to the number of second bioreactors is 2:1 or 3:2 or 4:3.

In a further preferred embodiment, the present method further comprises the steps of:

(iv) transporting a second part of the fermentation broth from the at least one first bioreactor to at least one third bioreactor; (v) transporting a part of the fermentation broth from the at least one second bioreactor to at least one third bioreactor; and

(vi) cultivating the microorganism in the first, second and third bioreactor until the degree of filling of the first, second and/or third bioreactor at least 80%, wherein cultivating the microorganism comprises adding a feed to the bioreactor.

The concept of the present invention can be used to further improve the efficiency of a fermentation plant by following the steps (iv) to (vi). The concept of dividing fermentation broth can advantageously be repeated to even further increase efficient use of the capacity of a fermentation plant.

A part of the fermentation broth from the at least one second bioreactor in step (v) is a first or a second part of the fermentation broth from the second bioreactor, preferably a second part of the fermentation broth from the second bioreactor.

Preferably, the second part of the fermentation broth is not being concentrated before transporting from the first and second bioreactor into the third bioreactor.

Preferably, present step (vi) of cultivating the microorganism in the at least one first, second and/or third bioreactor until the degree of filling of the at least one first and/or second bioreactor is larger than or at least 80%, comprises cultivating the microorganism in the at least one first and at least one second bioreactor until the degree of filling of the at least one first and/or at least one second bioreactor is larger than or at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%.

In a preferred embodiment, the present second part of the fermentation broth in step (iv) and in step (v) is an amount that results in a similar degree of filling of the at least one first and second and third bioreactors after step (v). A similar degree of filing is preferably a percentage within the range of plus or minus 10% or 5% of the averaged degree of filling of the bioreactors. For example with an averaged degree of filling of 40% the degree of filling in the bioreactors is within 30% to 50%.

Alternatively, the second part of the fermentation broth in step (iv) and in step (v) is an amount within the range of 25% wt to 40% wt of the fermentation broth at the end of step (iii). More preferably, the second part is an amount within the range of 30% wt to 35% wt, or most preferably around 33% wt of the fermentation broth at the end of step (iii). In a fermentation plant wherein multiple bioreactors of equal volume are used for the fed-batch fermentation, it is advantageous to equally divide the fermentation broth at the end of step (iii) in the first and second bioreactor(s) amongst the at least one first, second and third bioreactor(s). This equal division of the fermentation broth amongst three bioreactors provides the maximum bioreactor capacity for further cultivation of the microorganisms in the fermentation broth. In a fermentation plant wherein multiple bioreactors of different volume are used forthe fed-batch fermentation, it is advantageous to divide the fermentation broth at the end of step (iii) in the at least one first and second bioreactors amongst the at least one first and second and third bioreactor in proportion to their respective volumes. For example, if the at least one first bioreactor has a volume of 200 m ³ and the at least one second bioreactor has a volume of 150 m ³ and the at least one third bioreactor has a volume of 100 m ³ then the fraction of fermentation broth transferred from the at least one first to the at least third bioreactor in step (iv) would be 100/(200 + 150 + 100), the fraction remaining in the at least first bioreactor in step (iv) would be (200 + 150)/(200 + 150 + 100), the fraction of fermentation broth transferred from the second to the third bioreactor in step (v) would be 100/(200 + 150 + 100), the fraction remaining in the second bioreactor in step (v) would be (200 + 150)/(200 + 150 + 100).

The use of the present invention can be even pushed further to equally divide the fermentation broth at the end of step (vi) of the at least one first, second and third bioreactor(s) amongst four bioreactors, followed by cultivating the microorganism in the at least first, second, third and fourth bioreactors.

In a preferred embodiment, in present step (i) the medium and inoculum are introduced to the at least one first bioreactor until a degree of filling is reached of more than 60% of the at least one first bioreactor. This high amount of initial filling allows a further division of fermentation broth over an at least one first, second and third bioreactor, and hence improves the average degree of filling of the bioreactors.

In a preferred embodiment, the present method comprises repeating the steps (i) to (vi) and/or wherein the number of first bioreactors relative to the number of second bioreactors relative to the number of third reactors is 3:2:1. To improve the efficiency in fermentation plants having several bioreactors, it is advantageous to repeat the present steps (i) to (vi) for all or nearly all first bioreactors that are newly inoculated. One can always use the same bioreactors in the plant as first bioreactor and as second bioreactors and as third bioreactors, but one can also vary the use of bioreactors as first and as second bioreactor and as third bioreactors.

To improve the efficiency of a fermentation plant, the number of bioreactors used as first bioreactors (for carrying out steps (i) and (iii) and (vi)) is preferably larger than the number of bioreactors used as second bioreactors (for carrying out steps (iii) and (vi)), and is larger than the number of bioreactors used as third bioreactors (for carrying out step (vi)). Preferably, the number of first reactors to the number of second reactors to the number of third reactors is 3:2:1. In this way the present method can advantageously be used in fermentation plants having for example six bioreactors, or a multiple of six bioreactors.

In a preferred embodiment, the present bioreactors have an equal volume, or a volume within the same volume range. To benefit most from the present invention, it is advantageous if the bioreactors have an equal volume. An equal volume as used herein is defined as a volume that deviates not more than 5% from the averaged bioreactor volume. For example, if the averaged bioreactor volume of the present first, second and/or third bioreactors is 500 m ³ , the volume of the bioreactors is within the range of 475 m ³ to 525 m ³ .

The method for cultivating a microorganism as disclosed herein is preferably carried out on an industrial scale. Industrial scale as used herein comprises cultivating a microorganism in bioreactors having a volume of 1 to 1000 m ³ , or 10 to 800 m ³ , such as 30 to 500 m ³ , preferably a volume of 50 to 450 m ³ , more preferably a volume of 100 to 400 m ³ In a preferred embodiment, the at least one first, second and / or third bioreactors each have a volume within the range of 1 to 1000 m ³ , or 10 to 800 m ³ , or 30 to 500 m ³ , preferably within the range of 50 to 450 m ³ , more preferably within the range of 100 to 400 m ³ .

The advantage of the present method is that the degree of filling of the bioreactors is higher than in conventional fermentation processes. A conventional degree of filling is smaller than 60%. Preferably, the degree of filling in the present first, second and third bioreactors from the beginning of step (i) towards the end of step (vi) is higher than 70%, such as higher than 75%.

In a preferred embodiment, the present microorganism is chosen from the group consisting of yeast, filamentous fungi, bacteria and algae.

A yeast cell is preferably a yeast belonging to the genus of Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia. More preferably, the present yeast is Kluyveromyces lactis, Saccharomyces cerevisiae, Hansenula polymorpha, Yarrowia lipolytica or Pichia pastoris.

Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et ai., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. Filamentous fungal strains include, but are not limited to, strains of Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Rasamsonia, Thermoascus, Thielavia, Tolypocladium, and Trichoderma.

Preferred filamentous fungi belong to a species of an Acremonium, Aspergillus, Chrysosporium, Myceliophthora, Penicillium, Talaromyces, Rasamsonia, Thielavia, Fusarium or Trichoderma genus, and most preferably a species of Aspergillus niger, Acremonium alabamense, Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae, Aspergillus fumigatus, Talaromyces emersonii, Rasamsonia emersonii, Aspergillus oryzae, Chrysosporium iucknowense, Fusarium oxysporum, Myceliophthora thermophila, Trichoderma reesei, Thielavia terrestris or Penicillium chrysogenum. A more preferred filamentous fungi belongs to the genus Aspergillus, more preferably the filamentous fungi belongs to the species Aspergillus niger or is Aspergillus niger.

The term “bacteria” includes both Gram-negative and Gram-positive microorganisms. Suitable bacteria may be selected from e.g. Escherichia, Anabaena, Caulobactert, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus, Bacillus, Brevibacterium, Corynebacterium, Rhizobium ( Sinorhizobium ), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Staphylococcus, Streptomyces, Actinomycetes, Xanthomonas or Sphingomonas. Preferably, the bacterial cell is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus, G. oxydans, Caulobactert crescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides, Pseudomonas zeaxanthinifaciens, Paracoccus denitrificans, E. coli, C. glutamicum, Staphylococcus carnosus, Streptomyces lividans, Sinorhizobium melioti and Rhizobium radiobacter.

The present algae are preferably chosen from the group consisting of glaucophytes, rhodoplasts and chloroplasts. More preferably the present algae are heterotrophic algae, more preferably heterotrophic algae like Chlorella, Nannochloropsys, Nitzschia, Thraustochythum, Aurantiochytrium, or Schizochytrium.

A method for cultivating a microorganism as disclosed herein comprises producing a fermentation broth. Producing a fermentation broth may comprise producing a compound of interest by the microorganism.

In a preferred embodiment, the microorganism in a method of the inventioncomprises at least one polynucleotide coding for a compound of interest or at least one polynucleotide coding for a compound involved in the production of a compound of interest by the microbial cell.

The compound of interest can be any biological compound. The biological compound may be biomass or a biopolymer or metabolite. The biological compound may be encoded by a single polynucleotide or a series of polynucleotides composing a biosynthetic or metabolic pathway or may be the direct result of the product of a single polynucleotide or products of a series of polynucleotides. The biological compound may be native to the host cell or heterologous.

The term "heterologous biological compound" is defined herein as a biological compound which is not native to the cell; or a native biological compound in which structural modifications have been made to alter the native biological compound.

The term "biopolymer" is defined herein as a chain (or polymer) of identical, similar, or dissimilar subunits (monomers). The biopolymer may be any biopolymer. The biopolymer may for example be, but is not limited to, a nucleic acid, polyamine, polyol, polypeptide (or polyamide), or polysaccharide.

The biopolymer may be a polypeptide. The polypeptide may be any polypeptide having a biological activity of interest. The term "polypeptide" is not meant herein to refer to a specific length of the encoded product and, therefore, encompasses peptides, oligopeptides, and proteins. Polypeptides further include naturally occurring allelic and engineered variations of the above- mentioned polypeptides and hybrid polypeptides. The polypeptide may be native or may be heterologous to the host cell. The polypeptide may be a collagen or gelatin, or a variant or hybrid thereof. The polypeptide may be an antibody or parts thereof, an antigen, a clotting factor, an enzyme, a hormone ora hormone variant, a receptor or parts thereof, a regulatory protein, a structural protein, a reporter, or a transport protein, protein involved in secretion process, protein involved in folding process, chaperone, peptide amino acid transporter, glycosylation factor, transcription factor, synthetic peptide or oligopeptide, intracellular protein. The intracellular protein may be an enzyme such as, a protease, ceramidases, epoxide hydrolase, aminopeptidase, acylases, aldolase, hydroxylase, aminopeptidase, lipase. The polypeptide may also be an enzyme secreted extracellularly. Such enzymes may belong to the groups of oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, catalase, cellulase, chitinase, cutinase, deoxyribonuclease, dextranase, esterase. The enzyme may be a carbohydrase, e.g. cellulases such as endoglucanases, b- glucanases, cellobiohydrolases or b-glucosidases, hemicellulases or pectinolytic enzymes such as xylanases, xylosidases, mannanases, galactanases, galactosidases, pectin methyl esterases, pectin lyases, pectate lyases, endo polygalacturonases, exopolygalacturonases rhamnogalacturonases, arabanases, arabinofuranosidases, arabinoxylan hydrolases, galacturonases, lyases, or amylolytic enzymes; hydrolase, isomerase, or ligase, phosphatases such as phytases, esterases such as lipases, proteolytic enzymes, oxidoreductases such as oxidases,, transferases, or isomerases. The enzyme may be a phytase. The enzyme may be an aminopeptidase, asparaginase, amylase, a maltogenic amylase, carbohydrase, carboxy peptidase, endo-protease, metallo-protease, serine- protease catalase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta- glucosidase, haloperoxidase, protein deaminase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, galactolipase, chlorophyllase, polyphenoloxidase, ribonuclease, transglutaminase, or glucose oxidase, hexose oxidase or monooxygenase.

The compound of interest may be a heterologous product. The compound of interest can be a glucose oxidase, such as a heterologous glucose oxidase. Alternatively the compound of interest is a lipolytic enzyme, e.g. a lipolytic enzyme having one or more of the activities selected from the group consisting of: lipase (triacyl glycerol lipase), phospholipase (e.g phospholipase A1 and/or phospholipase A2 and/or phospholipase B and/or phospholipase C), galactolipase.

A polypeptide or enzyme can also be a product as described in W02010/102982.A polypeptide can also be a fused or hybrid polypeptide to which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding one polypeptide to a nucleic acid sequence (or a portion thereof) encoding another polypeptide.

Techniques for producing fusion polypeptides are known in the art, and include, ligating the coding sequences encoding the polypeptides so that they are in frame and expression of the fused polypeptide is under control of the same promoter (s) and terminator. The hybrid polypeptides may comprise a combination of partial or complete polypeptide sequences obtained from at least two different polypeptides wherein one or more may be heterologous to the host cell. Example of fusion polypeptides and signal sequence fusions are for example as described in W02010/121933.

The biopolymer may also be a polysaccharide. The polysaccharide may be any polysaccharide, including, but not limited to, a mucopolysaccharide (e. g., heparin and hyaluronic acid) and nitrogen-containing polysaccharide (eg., chitin). In a more preferred option, the polysaccharide is hyaluronic acid. In another preferred option, the polysaccharide is a hydrocolloid, e.g. xanthan, gellan, pectin, welan or another polysaccharide.

The polynucleotide coding for the compound of interest or coding for a compound involved in the production of the compound of interest according to the invention may encode an enzyme involved in the synthesis of a primary or secondary metabolite, such as organic acids, alcohols, lipids, carotenoids, beta-lactam, antibiotics, and vitamins. Such metabolite may be considered as a biological compound according to the present invention. Preferably, the present compound of interest is beta-lactam. Preferably, the present compound of interest is ethanol.

The term "metabolite" encompasses both primary and secondary metabolites; the metabolite may be any metabolite. Preferred metabolites are citric acid, gluconic acid, adipic acid, fumaric acid, itaconic acid and succinic acid.

The metabolite may be encoded by one or more genes, such as in a biosynthetic or metabolic pathway. Primary metabolites are products of primary or general metabolism of a cell, which are concerned with energy metabolism, growth, and structure. Secondary metabolites are products of secondary metabolism (see, for example, R. B. Herbert, The Biosynthesis of Secondary Metabolites, Chapman and Hall, New York, 1981).

The primary metabolite may be, but is not limited to, an amino acid, fatty acid, triacy Ig lycerol , nucleoside, nucleotide, sugar, triglyceride, or vitamin. For example, vitamin A, B2, C, D or E.

The secondary metabolite may be, but is not limited to, an alkaloid, coumarin, flavonoid, polyketide, quinine, steroid, peptide, or terpene. The secondary metabolite may be an antibiotic, antifeedant, attractant, bacteriocide, fungicide, hormone, insecticide, or rodenticide. Preferred antibiotics are cephalosporins and beta-lactams. Other preferred metabolites are exo-metabolites. Examples of exo-metabolites are Aurasperone B, Funalenone, Kotanin, Nigragillin, Orlandin, Other naphtho-y-pyrones, Pyranonigrin A, Tensidol B, Fumonisin B2 and Ochratoxin A.

The biological compound may also be the product of a selectable marker. A selectable marker is a product of a polynucleotide of interest which product provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Selectable markers include, but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricinacetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), ble (phleomycin resistance protein), hyg (hygromycin), NAT or NTC (Nourseothricin) as well as equivalents thereof.

Given the advantage of the present method for fermentation plants, the present invention relates, according to another aspect, to a fermentation plant comprising at least one first bioreactor and at least one second bioreactor, , wherein preferably the bioreactor is a production bioreactor, wherein the bioreactors, preferably the production bioreactors, have an equal volume and wherein the bioreactors, preferably the production bioreactors, are connected with means for transporting fermentation broth between the bioreactors or production bioreactors. Preferably, wherein all bioreactors, or production bioreactors, are connected with means for transporting fermentation broth between the bioreactors to all other bioreactors or production bioreactors. Preferably, the means for transporting fermentation broth between the bioreactors or production bioreactors are conduits. More preferably, the present means for transporting fermentation broth between the bioreactors, or conduits, are situated in such a way that the inlet and/or outlet of the means for transporting fermentation broth allows the transport of fermentation broth between the bioreactors or production bioreactors. More preferably, the inlet of the means for transporting fermentation broth is situated at the underside or bottom of the bioreactor.

A fermentation plant as disclosed herein is suitable for carrying out the method according to the present invention.

The invention is further illustrated in the non-limiting examples below, wherein reference is made to the figures 1 to 6.

Figure 1 shows the time-averaged degree of filling in a bioreactor following a conventional fermentation process as shown in Example 1 .

Figure 2 shows a repetitive schedule of batches following the conventional fermentation process as shown in Example 1.

Figure 3 shows the time-averaged degree of filling in a first bioreactor following the method of Example 2.

Figure 4 shows a repetitive schedule of batches following the method of Example 2.

Figure 5 shows the time-averaged degree of filling in a first bioreactor following the method of Example 3.

Figure 6 shows a repetitive schedule of batches following the method of Example 3.

Figure 7 shows the time-averaged degree of filling in a first bioreactor following a method of Example 4.

Figure 8 shows a repetitive schedule of batches following the method of Example 4.

EXAMPLES

Reference Example 1 Base case

The following base case which is representative for an aerobic, large scale industrial fermentation is calculated: a fed-batch processes executed in six 236 m ³ bioreactors, a gas holdup of 15%, a liquid density near 1 ton/m ³ so a maximum liquid amount of 200 tons, an inoculum of 20 tons and a medium batch weight of 30 tons. The time to reach end of fermentation is 72h per bioreactor. For the base case, the time-averaged degree of filling can be calculated to be 58%, see Figure 1. This number excludes idle time of the fermenter during emptying, cleaning, sterilization and refilling (‘turn-around time’ or TAT), which further lowers the time-averaged filling.

Applying the base case in a plant with six 236 m ³ bioreactors, calculating with a TAT of 12h, and year-round production, this plant executes 626 batches per year producing a total of 125 kton of broth. The scheduling of this plant is shown in Figure 2. Example 2 Double filling case

In the double filling case, a first bioreactor is started up with a double inoculum and a double batch medium weight and the feed rate is double compared to the base case. Now, the degree of filling is higher, and the bioreactor is full after 32h. At the moment the bioreactor is full, half of its contents is transferred to a clean, empty second bioreactor and feeding of both bioreactors is continued at the original (base case) rate. The degree of filling over time is shown in Figure 3. The time averaged filling of the first bioreactor is 74%. The second bioreactor has a time-averaged filing of 75%. Again, both numbers exclude TAT.

Applying the double filling case in the same plant having six bioreactors of 236m ³ , the fed-batches can be scheduled as shown in Figure 4. Two out of six bioreactors serve as first bioreactors. These are inoculated and cultivated until end of fermentation, and the remaining two bioreactors serve as second bioreactors and are only filled with fermentation broth from the other four first bioreactors. The plant now produces 796 batches per year, each resulting in 200 tons of broth. The total annual broth production of the plant is 159 kton. This is an increase of 27% compared with the base case of Example 1 .

Example 3 Triple filling case

In the triple filling case, a first bioreactor is started up with a triple inoculum and a triple batch medium weight and that the feed rate is triple compared to the base case. Now, the degree of filling is higher, and the bioreactor is full after 14h. At the moment the first bioreactor is full, half of its contents is transferred to a clean, empty second bioreactor and feeding of both bioreactors is continued at the triple rate until both the first and second bioreactors are full again after 43h. Thereafter, 1/3 of the first bioreactor content and 1/3 of the second bioreactor content is transferred to a clean, empty third bioreactor and feeding of all three bioreactors is continued at the triple rate until end of fermentation. The degree of filling over time for the first bioreactor is shown in Figure 5. The time averaged filling of the first bioreactor is 79%. The second bioreactor has a time-averaged filing of 77%. The third bioreactor has a time-averaged filling of 83%. Again, both numbers exclude TAT.

Applying the triple filling case in the same plant having six bioreactors of 236m ³ , the fed- batches can be scheduled as shown in Figure 6. Now three first bioreactors are inoculated and cultivated until end of fermentation, two second bioreactors are only filled with fermentation broth from these three first bioreactors, and one third bioreactor is filled with fermentation broth from the two first and the two second bioreactors. The plant now produces 822 batches per year, each resulting in 200 tons of broth. The total annual broth production of the plant is 164 kton. This is an increase of 31% compared with the base case of Example 1 and in increase of 3% compared with the double filling case of Example 2. Example 4

Double filling case with lower filling

In an alternative double filling case, a first bioreactor is started up with a double inoculum and a double batch medium weight and the feed rate is double compared to the base case. Now, the degree of filling is higher, and the bioreactor is filled to 80% after 22h. At the moment the first bioreactor has reached a 80% filling, half of its contents is transferred to a clean, empty second bioreactor and feeding of both bioreactors is continued at the original (base case) rate. The degree of filling over time is shown in Figure 7. The time averaged filling of the first bioreactor is 67%. The second bioreactor has an average filling of 70%.

Applying this double filling case with a broth transfer from the first to the second bioreactor when the first reactor is filled to 80% can only be scheduled in a plant having (a multifold of) five bioreactors of 236m ³ . The scheduling is shown in Figure 8. Three out of the five bioreactors serve as first bioreactors. These are inoculated and cultivated until end of fermentation, and the remaining two bioreactors serve as second bioreactors and are only filled with fermentation broth from the other three first bioreactors. The plant now produces 598 batches per year with five bioreactors, each resulting in 200 tons of broth. The total annual broth production of the plant is 120 kton. This is an increase of 15% compared with the base case of Example 1 , when correcting for the fact that the plant has five rather than six bioreactors.