METHOD FOR ESTIMATING THE BIOMASS OF HAIRY ROOTS IN A MULTI-PHASIC CULTURE

Abstract

A method for estimating the hairy root biomass concentration X.sub.(t) of a multi-phasic culture of hairy roots in a culture medium, the culture medium including at least one source of carbon being at least one sugar, and at least one source of nitrogen. The biomass yield surprisingly correlates linearly with the consumed concentration of nitrogen and/or sugars over the entire time course of the multi-phasic culture of hairy roots. The method for estimating the hairy root biomass concentration X.sub.(t) according to the invention is also a useful tool to estimate the amount of one or more molecule(s) of interest synthesized by the hairy roots.

Claims

1-15. (canceled)

16. A method for estimating the hairy root biomass concentration X.sub.(t) of a multi-phasic culture of hairy roots in a culture medium, the culture medium comprising at least one source of carbon being at least one sugar, and one source of nitrogen, the method comprising: a) measuring, at time t.sub.0, the concentration C.sub.0 in the culture medium of at least one compound selected from the group consisting of at least one sugar and at least one nitrogen source; b) measuring, at a time t of any one of the culture phases, the concentration C.sub.(t) in the culture medium of the at least one compound; c) calculating the differential concentration C.sub.0-C.sub.(t) of the at least one compound; d) estimating the hairy root biomass concentration X.sub.(t) at the time t of any one of the culture phases by means of the following equation: $X_{\left(t\right)}=\left[Y_{X/i}\left(C_0-C_{\left(t\right)}\right)\right]+X_0,$ wherein: X.sub.0 is the hairy root biomass concentration at time t.sub.0, and Y.sub.X/i is the biomass yield empirically pre-determined by means of the following equation: $Y_{X/i}=\frac{X_1-X_0}{C_0-C_1}$ wherein X.sub.1 is the hairy root biomass concentration at a predetermined time t.sub.1; C.sub.1 is the concentration of the at least one compound at a predetermined time t.sub.1; X.sub.0 is the hairy root biomass concentration at time t.sub.0; C.sub.0 is the concentration of the at least one compound at time t.sub.0; and i is S for sugar(s) or N for nitrogen.

17. The method according to claim 16, wherein the at least one sugar is selected from the group consisting of glucose, fructose, sucrose, and any combinations thereof.

18. The method according to claim 16, wherein the at least one sugar is selected from the group consisting of a combination of glucose, fructose and sucrose.

19. The method according to claim 16, wherein each phase of the multi-phasic culture is characterized by its own biomass growth rate.

20. The method according to claim 16, wherein the multi-phasic culture is a bi-phasic culture.

21. The method according to claim 16, wherein the multi-phasic culture comprises: a) a first phase of culturing hairy roots dedicated to biomass growth; and b) one or more further phase(s) of culturing hairy roots dedicated to the production of one or more molecule(s) of interest.

22. The method according to claim 16, wherein the multi-phasic culture of hairy roots is aimed at producing fresh biomass and/or one or more molecule(s) of interest.

23. The method according to claim 16, wherein the one or more molecule(s) of interest is/are selected from the group consisting of recombinant proteins, metabolites, non-peptidic hormones, structured associations of recombinant proteins, virus-like particles and viruses.

24. The method according to claim 23, wherein the recombinant protein is selected from the group consisting of allergens; vaccines; viral proteins; enzymes; enzyme inhibitors; antibodies; antibody fragments; antigens, toxins; anti-microbial peptides; peptidic hormones; growth factors; blood proteins, in particular albumin, coagulation factors, transferrin; receptors and/or signaling proteins; protein components of biomedical standards; protein components of cell culture media; fusion and/or tagged proteins; cysteine (disulfide bridges)-rich peptides and proteins; and plant proteins, in particular lectins, papain.

25. The method according to claim 23, wherein the metabolite is selected from the group consisting of polyphenols; alkaloids; cannabinoids; terpenoids and steroids; flavonoids; and tannins.

26. The method according to claim 16, wherein the hairy root is selected from the group of families consisting of the Brassicaceae family; the Solanaceae family; the Cannabaceae family; the Caryophyllaceae family; the Saponaria family; and the Vitaceae family.

27. The method according to claim 16, wherein the hairy root is selected from the group of species consisting of Brassica rapa rapa, Brassica napus, Salvia Milthiorrhiza, Panax Ginseng, Armoracia rusticana, Trigonella foenumgraceum, Lippia dulcis, Lithospermum erythrorhizon, Ophiorrhiza pumila, and Echinacea purpurea, Echinacea Angustifolia, Puerariaphaseoloides, Harpagophytum Procumbens, Morinda Citrifolia, Hypericum Perforatum, Derris trifolia, Salvia miltiorrhiza, Salvia prevalzkii, Echinacea pallida, Cistanche tubulosa, Glycyrrhiza glabra, Sophora flavescens, Rhodiola Rosea, Polygonum cuspidatum, Fallopia multiflora, Lepidium peruvianum, Whitania Somnifera, Astragalus Membranaceous, Berberis Vulgaris, Sanguinaria canadensis, Eleutherococcus Senticosus, Cannabis sativa, Hydrastis Canadensis, Arctium Majus, Piper methysticium, Pueraria lobata, Glycyrrhiza uralensis, Ptychopetalum olacoides, Dioscorea Vollosa, Yucca shidigera, Panax quinquifolium, Azadirachta indica, Catharanthus trichophyllus, Calystegia sepium, Atropa belladonna, Hyoscyamus muticus, Artemisia annua, Datura stramonium, Arabidopsis thaliana, Stizolobium, Hassjoo, Ipomea aquatica, Perilla fruitescnens, Catharanthus roseus, Taxus brevifolia, Gloriosa Superba, Saponaria officinalis, Solanum tuberosum, Nicotiana tabacum, Nicotiana benthamiana, Cannabis sativa, Vitis vinifera, Duboisia leichhardtii, Ducoisia myoporoides and Cinchosa Pubescens.

28. The method according to claim 21, wherein the one or more further phase(s) of culturing the hairy roots is/are performed in the presence of a chemical and/or physical and/or biological inductor of the production of the one or more molecule(s) of interest.

29. The method according to claim 16, wherein when the at least one compound is at least one sugar, the biomass yield Y.sub.X/S ranges from about 0.25 to about 2.50.

30. The method according to claim 16, wherein when the at least one compound is a nitrogen source, the biomass yield Y.sub.X/N is ranges from about 5 to about 40.

31. The method according to claim 16, wherein the multi-phasic culture is performed in a bioreactor, in a volume of culture medium of at least about 20 L.

32. The method according to claim 31, wherein the multi-phasic culture is performed in a bioreactor, in a volume of culture medium of at least about 350 L.

33. The method according to claim 31, wherein the multi-phasic culture is performed in a bioreactor, in a volume of culture medium of at least about 500 L.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0215] FIGS. 1A-1D are a set of plots showing: (FIG. 1A) the biomass concentration (g.sub.DW.Math.L.sup.1) (closed circles) and protein of interest production (relative productivity, in %) (closed squares); (FIG. 1B) the conductivity (mS) (closed squares) and pH (open circles) of the extracellular culture medium; (FIG. 1C) the extracellular sugar concentrations (g.sub.C.Math.L.sup.1), including glucose (closed squares), sucrose (closed circles), total sugars IC (sucrose, fructose and glucose concentrations measured by ionic chromatography (IC); crosses), fructose (closed triangles) and total sugars (sucrose, fructose and glucose concentrations; open diamonds); and (FIG. 1D) the evolution of the amounts of the main nutrients (mg.Math.L.sup.1), including sulfate (closed diamonds), ammonium (closed triangles), potassium (crosses), phosphate (closed circles) and nitrate (closed squares); during the growth kinetics of Brassica rapa rapa hairy roots in Erlenmeyer flasks. Dotted vertical line indicates the culture medium renewal and addition of 2,4D.

[0216] FIGS. 2A-2C are a set of plots showing the biomass produced in function of the evolution of conductivity decrease (FIG. 2A), the total sugar uptake (FIG. 2B) and the total nitrogen consumption (FIG. 2C). The dotted square represents the growth phase (from day 0 to day 14) and error bars are standard deviations.

[0217] FIGS. 3A-3B are a set of histograms showing the biomass measurement and estimations using the correlations given in Table 1 at the end of the growth phase (FIG. 3A) and during the production phase at day 26 (FIG. 3B). The dotted horizontal lines correspond to 10% of the measured biomass concentration (experimental data). Error bars are standard deviations.

[0218] FIGS. 4A-4C are a combination of plots showing the evolution of conductivity (closed squares) and pH (open circles) (FIG. 4A); extracellular sugar concentrations (g.sub.C.Math.L.sup.1), including glucose (closed squares), sucrose (closed circles), total sugars IC (sucrose, fructose and glucose concentrations measured by ionic chromatography (IC); crosses), fructose (closed triangles) and total sugars (sucrose, fructose and glucose concentrations; open diamonds) (FIG. 4B); and main nutrient concentrations, including sulfate (closed diamonds), ammonium (closed triangles), potassium (crosses), phosphate (closed circles) and nitrate (closed squares) (FIG. 4C); in the culture medium during the growth phase of Brassica rapa rapa hairy roots in a large-scale bioreactor.

[0219] FIG. 5 is a histogram showing the biomass measurement and estimations using correlations determined in small-scale culture conditions (Table 1) at the end of the growth phase of a culture of Brassica rapa rapa hairy roots in a large-scale bioreactor. Dotted horizontal lines represent10% of the actual biomass concentration measured at the end of the culture. Error bars are standard deviations.

[0220] FIGS. 6A-6B are a set of plots showing the biomass concentration (g.sub.DW.Math.L.sup.1) over time (in days) during a bi-phasic culture of Brassica napus Westar 8 (FIG. 6A) and Cannabis sativa Santhica 10-70 (FIG. 6B) hairy roots in Erlenmeyer flasks.

[0221] FIGS. 7A-7B are a set of plots showing the biomass (g.sub.DW.Math.L.sup.1) produced in function of the evolution of conductivity (mS) during a bi-phasic culture of Brassica napus Westar 8 (FIG. 7A) and Cannabis sativa Santhica 10-70 (FIG. 7B) hairy roots in Erlenmeyer flasks.

[0222] FIGS. 8A-8B are a set of plots showing the dry weight biomass (g.sub.DW.Math.L.sup.1) produced in function of the evolution of the total sugar uptake (g.sub.S.Math.L.sup.1) during a bi-phasic culture of Brassica napus Westar 8 (FIG. 8A) and Cannabis sativa Santhica 10-70 (FIG. 8B) hairy roots in Erlenmeyer flasks.

[0223] FIGS. 9A-9B are a set of plots showing the dry weight biomass (g.sub.DW.Math.L.sup.1) produced in function of the evolution of the nitrogen uptake (g.sub.N.Math.L.sup.1) during a bi-phasic culture of Brassica napus Westar 8 (FIG. 9A) and Cannabis sativa Santhica 10-70 (FIG. 9B) hairy roots in Erlenmeyer flasks.

EXAMPLES

[0224] The present invention and disclosure are further illustrated by the following examples.

Example 1

Materials and Methods

1. Biological Materials

[0225] Hairy roots of a transformed Brassica rapa rapa clone producing recombinant -L-iduronidase (rIDUA; as described in Cardon et al., Plant Biotechnol J. 2019 Feb; 17 (2): 505-516) were grown at 100 rpm and 25 C. in 200 mL of Gamborg's B5 medium, supplemented with 30 g.Math.L.sup.1 of sucrose. For their preservation, hairy roots were sub-cultured each two weeks.

2. Hairy Root Culture

[0226] The experiments of growth monitoring in shake flasks and in a bioreactor were carried following the same protocol. Culture systems were seeded with 10 g.sub.FW.Math.L.sup.1 (gram of fresh weight (FW) per liter) of 11 days old hairy roots of Brassica rapa rapa. After 14 days of growth, roots were transferred into fresh medium with addition of 2,4D (2,4-dichlorophenoxyacetic acid) for the induction of rhizocals which allows to increase the production of recombinant protein, as described in Ekouna et al. (Plant Cell Tiss Organ Cult 131, 601-610 (2017)), and kept for 25 more days.

3. Growth Kinetics Experiments in Shake Flasks

[0227] The experiments of growth monitoring in shake flasks were made in 250 mL Erlenmeyer flasks filled with 100 mL of Brassica growth medium (BGM) medium (proprietary culture medium, specifically developed for the culture of Brassicaceae hairy root). The flasks were inoculated with 10 g.sub.FW.Math.L.sup.1 of 11 days old hairy roots and maintained at 100 rpm and 25 C. To follow the evolution of the culture, hairy roots were harvested in triplicates at regular time intervals. The harvested roots were washed with ultrapure water and dried at 70 C. for 24 hours to measure the dry weight (DW) of hairy roots. The pH and conductivity of the culture medium were also measured using a HI 5221 pH meter (Hanna Instruments, Woonsocket, RI, US) and samples were taken and stored at 20 C. for further analysis of sugar and nutrient concentrations. Three different experiments at different dates were done for the growth phase (the first 14 days) and only one for the whole culture process (14 days of growth and 25 days in rhizocals). For the presentation of the growth kinetics in Erlenmeyer flasks, means were calculated based on the three growth phase experiments, with the associated standard deviations.

4. Characterization in Large Scale Bioreactor

[0228] The characterization of the cultures in large scale was carried out in a 25 liters air-lift bioreactor, filled with 25 L of BGM medium. The bioreactor was seeded with 10 g.sub.FW.Math.L.sup.1 of 11 days old hairy roots, aerated and mixed by air injection at 2 L.Math.min.sup.1 and maintained in a thermostatically controlled chamber at 25 C. At regular time intervals, samples of culture medium were taken in sterile conditions and stored at 20 C. for further analysis of sugar and nutrient concentrations, and pH (Steamline, SI Analytics, Weilheim Germany) and conductivity (Sentek K10S7, Stepney, Australia) were measured. At the end of the culture, the hairy roots were harvested from the bioreactor, washed with ultrapure water and dried at 70 C. during 48 h to measure the dry weight.

5. Culture Analysis

5.1. Biomass

[0229] The biomass concentration X (in gram of dry weight (g.sub.DW) per liter or g.sub.DW.Math.L.sup.1) was obtained from the dry weight measurement, dividing the mass of dried roots (in g.sub.DW) by the total volume of culture (0.1 L and 25 L for cultures in flasks and bioreactor, respectively).

5.2. Sugars and Nutrients

[0230] The concentrations of anions (nitrate, sulfate and phosphate), cations (ammonium and potassium) and sugars (sucrose, glucose and fructose) in the culture medium were determined by ionic chromatography (Dionex ICS-3000; Thermo Fisher Scientific). Anions were quantified using an IonPAC AS11-HC (4250 mm) ion exchange column (Thermo Fisher Scientific) thermostatically at 30 C., linked to an ASRS 300 (4 mm) suppressor (at 248 mV) and a conductivity detector. The elution gradient was composed of the following steps: 98% of H.sub.2O and 2% of NaOH 100 mM from 0 to 5 min, 94% of H.sub.2O and 6% of NaOH 100 mM from 5 to 30 min, 60% of H.sub.2O and 40% of NaOH 100 mM from 30 to 45 min, 98% of H.sub.2O and 2% of NaOH 100 mM from 45 to 55 min, at a flowrate of 1 mL.Math.min.sup.1. Before injection, samples were filtered on 0.45 m cellulose membrane. Standard ranges were made with standard anion solutions (CPAchem). Cations were quantified using an IonPAC CS19 (4250 mm) ion exchange column (Thermo Fisher Scientific) thermostatically at 30 C., linked to an CSRS 300 (4 mm) suppressor (at 147 mV) and a conductivity detector. The elution gradient was the same as the anions' one replacing the NaOH solution by a 50 mM methanesulfonic acid solution. Sugars were measured with a ProPac PAI (4250 mm) ion exchange column (Thermo Fisher Scientific) and an amperometric detector. The elution gradient was composed of the following steps: 70% of H.sub.2O and 30% of NaOH 100 mM from 0 to 8.4 min, 40% of H.sub.2O and 60% of NaOH 100 mM from 8.4 to 10 min, 100% of NaOH 100 mM from 20 to 30 min, 100% of sodium acetate 1M and NaOH 100 mM from 30 to 35 min, 70% of H.sub.2O and 30% of NaOH 100 mM from 35 to 45 min, at a flowrate of 1 mL.Math.min.sup.1. Before injection, samples were filtered on 0.45 m cellulose membrane. Standard ranges were made with HPLC grade sucrose, glucose and fructose. Monitoring and peak areas determination were done by the software Chromeleon 7.0. For better comparison, all sugars were expressed in gram of carbon per liter dividing the concentration in g.Math.L.sup.1 by the molar mass of the concerned sugar (M.sub.Glc=M.sub.Fru=180 g/mol and M.sub.Suc=342 g/mol) and multiplying it by the number of carbon atoms and the molar mass of carbon (M.sub.C=12 g/mol).

5.3. Total Sugars

[0231] The concentration of total sugars was determined using the phenol-sulfuric method (Dubois et al., Analytical Chemistry 1956 28 (3), 350-356). For the dosage of total sugars in the culture medium, 0.2 mL of a 5% aqueous phenol solution (Sigma Aldrich, ref #: P4557) was added to 0.4 mL of carbohydrate sample. 1 mL of 95-98% sulfuric acid (Sigma Aldrich, ref #: 258105) was then quickly added to the mixture. After 10 minutes at room temperature, samples were vortexed for 15 sec and incubated in a water bath at room temperature for 20 min, in order to stop the reaction. Calibration samples consisted in glucose (Amresco, ref #: 0188) solutions at 0, 0.02, 0.04, 0.06, 0.08 and 0.1 g/L prepared in ultrapure water. Absorbance of samples was then measured at 490 nm (maximal intensity of the glucose peak) and 750 nm (to measure the turbidity). The concentration of total sugars in gram of carbon per liter (g.sub.C.Math.L.sup.1) was then calculated based on the calibration curve.

5.4. Estimation of Growth Rate and Apparent Biomass Yields

[0232] For the characterization experiments in flasks, the biomass growth was represented as exponential and the growth rate () was estimated from the regression of the exponential growth phase, according the following equation (1):

[00012]$X_{\left(t\right)}=X_0{e}^{{}_{}t}$

wherein X.sub.(t) represents the time dependent biomass concentration in g.sub.DW.Math.L.sup.1, X.sub.0 represents the initial biomass concentration, represents the growth rate in d.sup.1 and t represents the time in days.

[0233] The apparent biomass yield coefficient Y.sub.X/i represents the amount of biomass produced for a quantity of substrate (i.e., at least one compound comprised within the culture medium) degraded over time (e.g., carbon (such as sugars), nitrogen, sulfate, etc.), according to the following equation (2):

[00013]$Y_{X/i}=\frac{X_1-X_0}{C_0-C_1}$

wherein X.sub.1 is the hairy root biomass concentration at a predetermined time t.sub.1; C.sub.1 is the concentration of the at least one compound (i.e., substrate) at a predetermined time t.sub.1; X.sub.0 is the hairy root biomass concentration at time t.sub.0; C.sub.0 is the concentration of the at least one compound (i.e., substrate) at time t.sub.0; and i is for the at least one compound (i.e., substrate), for example, S for sugar(s) or N for nitrogen.

[0234] The biomass yield can be therefore estimated from the linear phase of the evolution of the quantity of biomass produced X.sub.1-X.sub.0 in function of the amount of substrate consumed C.sub.0-C.sub.1. Even if it is not considered as a substrate, the conductivity indirectly represents the concentration of nutrients in the culture medium and therefore a ratio between the biomass produced and the decrease in conductivity can also be calculated on the same basis as the biomass yield presented above.

5.5. Measurement of Recombinant Protein Production

[0235] The activity of rIDUA was measured with a fluorimetry assay by using the fluorogenic substrate sodium 4-methylumbelliferyl--L-Iduronide (4MU-I) (Santa Cruz Biotechnology, Dallas, US) as described in Ou et al. (Mol Genet Metab. 2014 Feb; 111 (2): 113-5). Before drying, hairy roots were washed in a saline solution to recover the protein of interest. 25 L of substrate (400 M 4MU-I prepared in 0.4M Sodium formate, pH 3.5, as assay buffer) were added to 25 L of protein samples and were incubated at 37 C. during 30 min. 200 L of glycine carbonate buffer pH 9.8 were added to the mixture to stop the reaction. 4-methylumbelliferone (4MU) (Sigma Aldrich, Saint-Louis, US) was used to prepare the standard calibration curve. Fluorescence was measured using a plate reader (TECAN Infinite M1000, Mnnedorf, Switzerland) with excitation at 355 nm and emission at 460 nm. Enzyme activity was first measured in mol of product formed per minute and per sample volume. The productivity was then calculated by dividing the enzyme activity by the corresponding biomass concentration and the culture time and was then expressed in % of the maximum productivity.

Results

1. Growth Characterization of a Transgenic Clone of Brassica rapa Rapa Hairy Roots in Shake Flasks

[0236] The growth of Brassica rapa rapa hairy roots during 39 days in 100 mL of BGM medium in Erlenmeyer flasks, corresponding to 14 days of biomass growth followed by 25 days of production through rhizocal culture, is presented in FIG. 1A. From day 2 to day 7, the growth of hairy roots was exponential with a maximum growth rate .sub.max=0.340.02 d.sup.1, corresponding to a doubling time of 2.040.12 d. The biomass concentration reached 6.990.63 g.sub.DW.Math.L.sup.1 before the culture medium renewal at the 14.sup.th day of culture. The standard deviation of U max was less than 10% despite the mean calculated from three different experiments carried out at three different dates, which shows the strong reproducibility and repeatability of the process. After the culture medium renewal at day 14 and the addition of 2,4D for the induction of rhizocals, the hairy roots growth was strongly slowed down, with a maximum growth rate of 0.040.01 d.sup.1 (doubling time of 17.335.77 d) and reached a final biomass concentration of 22.590.09 g.sub.DW.Math.L.sup.1. FIG. 1A also shows the relative production of recombinant protein (rIDUA) after the culture medium renewal. A progressive increase of the protein production was observed, which confirms the capacity of the clone to produce recombinant protein in liquid cultures.

[0237] The growth of the hairy roots was linked to a decrease of the conductivity of the culture medium, from 3.65 to 1.65 mS during the growth phase and from 3.65 to 2.48 mS during the rhizocal phase, due to the consumption of the ions needed for the biomass growth (FIG. 1B).

[0238] The evolution of the different sugars (sucrose, glucose, fructose and total sugars) was followed during the whole culture (FIG. 1C). The concentration of sucrose progressively decreased from the beginning of the culture. The decrease was concomitant with an increase of the concentrations of glucose and fructose in equimolar amounts. This was characteristic of the hydrolysis of sucrose which was complete at day 13. As indicated by the total sugar analysis, the carbon uptake was very low during the seven first days with only 1.54 g.sub.C.Math.L.sup.1 consumed (about 7.3% of the initial amount). A more important decrease of the total sugar concentration occurred after day 7 (3.2 g.sub.C.Math.L.sup.1), which corresponded to the beginning of glucose uptake. After the culture medium change at day 14, sucrose was completely hydrolyzed in 6 days. Glucose uptake began 3 days after the culture medium renewal and was progressively consumed until the end of the production phase. When the glucose concentration became lower than 2 g.sub.C.Math.L.sup.1, fructose started to be consumed from day 31 to the end of the culture. The total sugars uptake was 4.75 g.sub.C.Math.L.sup.1 at the end of the growth phase and 14.61 g.sub.C.Math.L.sup.1 at the end of the production phase.

[0239] The nutrient composition of the culture medium was also analyzed during the culture by following the concentrations of the main cations and of the main anions (FIG. 1D). Ammonium was quickly and fully consumed during the first five days of the culture and 90% of the nitrate amount was consumed during the 14 days of the growth phase. During the same period, an uptake of 81% of the phosphate source was observed and seemed to stabilize after 7 days of growth around 20 mg.Math.L.sup.1. Potassium and sulfate sources were also consumed but to a lesser extent (51% and 36% respectively). After the culture medium renewal and the induction of rhizocals, the nutrient uptake confirmed that nitrogen and phosphate were the main consumed compounds. Indeed, ammonium was completely consumed in only 3 days and the whole initial amount of nitrate was consumed at the 31.sup.th day. Phosphate uptake followed the same kinetics during the production phase as during the growth phase, with 66% of the initial phosphate amount consumed until reaching a steady concentration around 30 mg.Math.L.sup.1.

[0240] Nutrient monitoring shows that hairy roots of Brassica rapa rapa are able to assimilate both ammonium (NH.sub.4.sup.+) and nitrate (NO.sub.3.sup.) as nitrogen source for amino acids production, as most plant species. In the culture described herein, NH.sub.4.sup.+ was preferably consumed over NO.sub.3.sup. between day 0 and 3 corresponding to the beginning of the exponential growth phase and a decrease of the culture medium pH. The uptake of phosphate during the growth phase occurred from the beginning of the culture until day 7, which corresponds to the exponential growth phase and the moment where the pH is maximum.

[0241] Thus, in the culture conditions described above, nitrogen was the main limiting compound. As the main carbon source, glucose which was completely consumed at the end of the production phase, could also be a potential limitation, even if fructose took over as the secondary carbon source.

1.2. Sugars, Nitrogen and Conductivity Correlations for the Estimation of the Biomass Concentration

[0242] Thanks to the different measurements performed for each sampling point, the biomass concentration evolution was correlated to the uptake of total sugars, total nitrogen, potassium and sulfate, and to the evolution of conductivity, in order to better understand and characterize the culture kinetics of Brassica rapa rapa hairy roots in shake flasks. FIGS. 2A-2C represents the evolution of the biomass produced in function of the conductivity decrease (FIG. 2A), the total sugars uptake (FIG. 2B) and the total nitrogen consumption (FIG. 2C). As expressed with Equation 2, this representation allows to obtain the apparent yield Y.sub.X/i of the biomass X in function of a substrate S, provided that the evolution is linear.

[0243] Even if the conductivity of the culture medium is not a substrate, strictly speaking, it directly represents the concentration of ions in the culture medium and therefore the nutrient concentration. As presented in FIG. 2A, the conductivity decrease is linearly correlated to the biomass production, both during the growth phase (first phase of the culture of hairy roots) and the rhizocal phase (second phase of the culture of hairy roots), with apparent biomass yields of 3.901.11 g.sub.DW.Math.L.sup.1.Math.ms.sup.1 and 12.123.30 g.sub.DW.Math.L.sup.1.Math.ms.sup.1 respectively, regression coefficients r.sup.2 of 0.965 and 0.970 respectively and a correlation coefficient r=0.985 for both phases. Moreover, taking into account the standard deviations, all the experimental points are fitted with the linear regression.

[0244] By representing the evolution of the biomass production in function of the total sugar uptake (i.e., from sulfuric-phenol method and expressed in g.sub.C.Math.L.sup.1), a good linear correlation can be highlighted between these two parameters with an apparent biomass yield Y.sub.X/S=1.210.23 g.sub.DW.Math.g.sub.C.sup.1, a correlation coefficient r=0.994 and a regression coefficient r.sup.2=0.978 (FIG. 2B). Strikingly, the same correlation and apparent biomass yield Y.sub.X/S apply during both the growth phase (first phase) and the production phase (second phase), which means that the carbon requirement for the hairy roots is constant during the entire bi-phasic culture. As well as for conductivity, all the experimental data are well fitted with the linear regression, taking into account the standard deviations.

[0245] FIG. 2C represents the evolution of biomass production in function of the total nitrogen uptake (addition of ammonium and nitrate uptake, both expressed in gram of nitrogen (g) per liter). It shows that, when nitrogen is not limiting, the biomass production is linearly correlated with the nitrogen uptake with a biomass yield of 24.052,44 g.sub.DW.Math.g.sub.N.sup.1 both during the growth (first phase) and the production (second phase) phases, a correlation coefficient r=0.995 and a regression coefficient r.sup.2=0.988. Therefore, the nitrogen requirement for the biomass is constant during the entire bi-phasic culture, as is the case for the sugar requirement.

[0246] The correlations for the 5 parameters are given in Table 1 below. FIGS. 3A-3B compare the biomass actually measured (experimental data) and the biomass estimations obtained with the 5 correlations, at the end of the growth phase (first phase) (FIG. 3A) and during the production phase (second phase) (FIG. 3B).

TABLE-US-00001 TABLE 1 Correlations derived from conductivity (), total sugars (S), total nitrogen (N), potassium (K) and sulfate (Su) measurements during a bi- phasic culture in shake flasks, for estimation of the biomass concentration X.sub.(t) in function of time t. Subscripts 0 refer to initial time. Apparent biomass yield Growth phase Production phase (first phase) (second phase) Conductivity () Y.sub.X/ = 3.90 1.11 (r = 0.985) Y.sub.X/ = 12.20 3.30 (r = 0.985) Total sugars (S) .sup.Y.sub.X/S = 1.21 0.23 (r = 0.994) Total nitrogen (N) Y.sub.X/N = 24.05 2.44 (r = 0.995) Potassium (K) Y.sub.X/K = 11.44 1.44 (r = 0.991) .sup.Y.sub.X/K = 47.75 14.54 (r = 0.891) Sulfate (Su) Y.sub.X/Su = 99.10 33.33 (r = 0.989).sup. Y.sub.X/Su = 229.97 68.27 (r = 0.975)

[0247] The biomass concentrations estimated at the end of the growth phase (first phase) are all within the 10% range of the experimental data but only sugars and nitrogen estimations lead to a standard deviation below 10% (FIG. 3A). For the production phase (second phase), all the correlations gave estimated biomass concentrations within the 10% range of the experimental data, considering the standard deviations of the correlations (FIG. 3B). However, the average value of the estimated biomass from potassium and sulfate correlations are outside the 10% range of the experimental data, with a large standard deviation which indicates an underestimated prediction.

[0248] These correlations are particularly interesting for the estimation of the biomass concentration during a large-scale production where the biomass cannot be measured before the end of the production. So, the on-line and direct measurement of the conductivity of the culture medium with a conductivity probe (Sentek K10S7 for example) could be a good growth marker to control and estimate the biomass concentration during the culture, using the corresponding apparent biomass yield Y.sub.X/. However, when performing a multi-phasic culture, comprising for example a growth phase (first phase) and a production phase (second phase), it should be noted that biomass yields Y.sub.X/ must be pre-determined for each phase of the culture. Indeed, as illustrated in FIG. 2A and Table 1, with conductivity, a single biomass yield Y.sub.X/ does not apply in the different phases of a multi-phasic culture.

[0249] A regular sampling of culture medium to measure the total nitrogen evolution (easily measured using colorimetric kits (Merck, ref #114763, for example) or by liquid chromatography) or total sugar evolution (with the phenol-acid method for example) can thus constitute an appropriate and more convenient way to estimate the biomass evolution during a large scale production, in particular for a multi-phasic culture.

1.3. Growth of Hairy Roots of Brassica rapa Rapa in Large Scale Bioreactor

[0250] A culture of Brassica rapa rapa hairy roots was carried out in a pilot-scale bioreactor (25 L of useful volume), with the same method as in small scale, i.e., seeded with 10 g.sub.FW.Math.L.sup.1 of 11 days old hairy roots, with a growth phase of 14 days.

[0251] At the end of the growth phase, hairy roots were harvested, dried and weight to measure the biomass produced which was 3.24 g.sub.DW.Math.L.sup.1. The Brassica rapa rapa hairy roots culture monitoring is presented in FIGS. 4A-4C, with the evolution of conductivity (FIG. 4A), the evolution of extracellular sugars (FIG. 4B) and the evolution of the main nutrients used for the growth (FIG. 4C). As the biomass concentration cannot be measured during the process, but only after harvest at the end of the culture, only the conductivity and the pH of the culture medium were monitored (FIG. 4A).

[0252] During the first 3 days of the culture, a very small decrease of the culture medium conductivity from 3.59 to 3.46 mS was observed, concomitant with a decrease from 5.86 to 5.25 of the pH. An important decrease of the conductivity was then measured from 3.46 to 2.39 mS between day 3 and day 14, concomitant with an increase of the pH from 5.25 to 7.56. Regarding the sugar evolution (FIG. 4B), a progressive decrease in sucrose was observed from the beginning of the culture to the end, mostly due to hydrolysis into glucose and fructose, even if a direct uptake of the sucrose could be possible during the first 3 days, considering the small decrease in total sucrose during this period (from 19.6 to 16.8 g.sub.C.Math.L.sup.1, according to the ionic chromatography measurement). The total sugar concentrations remained almost stable from day 3 to day 10, which attests that only hydrolysis of sucrose happened during this period without any extracellular carbon fixation. After the 10.sup.th day, a slow-down in glucose accumulation and a small decrease of the total sugar concentration were observed, showing the start of glucose uptake.

[0253] FIG. 4C shows the evolution of the main ions during the growth phase in a large-scale bioreactor. Ammonium was completely consumed during the first 3 days and nitrate level was steady until day 2 before it progressively decreased until the end of the growth phase (first phase) from 1,421 to 619 mg.Math.L.sup.1 (42% of the initial nitrate level still remained at the end of the growth phase). An important uptake of phosphate also occurred between day 1 and day 8 with almost 80% of the initial amount consumed, which then stabilized at a basal level around 15 mg.Math.L.sup.1 until the end of the culture. Sulfate and potassium were less consumed with respectively 25% and 29% of initial amounts.

[0254] Compared with the cultures in Erlenmeyer flasks (FIG. 1), the culture in large scale bioreactors led to a longer lag time at the beginning of the growth phase (first phase). Indeed, although it was not possible to directly measure the biomass concentration during the process, several markers showed this delay. First, the nutrient consumption was slower to start: this was clearly reflected in the conductivity evolution whose main decrease began on day 3 while it started from the first day in small scale cultures. Nitrate consumption also showed this delay as it only began between day 2 and day 3 versus day 1 for the culture in Erlenmeyer flasks. Second, the hydrolysis of sucrose, which is correlated to invertases and sucrose synthase concentrations and therefore biomass concentration, was much slower than in Erlenmeyer flasks (6 g.sub.C.Math.L.sup.1 vs 21 g.sub.C.Math.L.sup.1 in 14 days).

[0255] Despite the delay observed, the growth of Brassica rapa rapa hairy roots in a large-scale bioreactor remained similar to the growth in small scale cultures. Indeed, the decrease of the culture medium conductivity from 3.65 mS to 2.7 mS during the growth phase showed a consumption of the nutrients which was due to the biomass production, and the evolution of pH was similar to that observed in small scale cultures with a decrease from 5.8 to 5.0 at the beginning of the culture followed by an increase to reach a pH of 6.7 at the end of the growth phase (FIG. 4A). However, the biomass concentration reached at the end of the growth phase was lower than in small scale cultures (3.24 g.sub.DW.Math.L.sup.1 in large bioreactor and 6.99 g.sub.DW.Math.L.sup.1 in Erlenmeyer flasks). This could be caused by the lag time observed in the large bioreactor which would have reduced the time dedicated to the exponential growth phase. The dynamics of sugars was similar to that observed in small scale cultures with hydrolysis of sucrose into fructose and glucose in equimolar amounts from the beginning of the culture, and a consumption of glucose before fructose. Evolution of nutrients was also similar to what was observed in small scale cultures, with a large uptake of nitrogen, first characterized by a quick and total uptake of ammonium and followed by nitrate consumption. Even if nitrogen was the most and main consumed nutrient, uptake of phosphate was also important and reached an equivalent basal concentration to that observed in small scale cultures (around 15-20 mg.Math.L.sup.1) and at the same culture period (between 7 and 8 days of growth).

1.4. Estimation of the Biomass Concentration with the Small-Scale Correlations

[0256] As it is not possible to directly measure the biomass amount inside the large scale bioreactor during the culture, the correlations for the biomass estimation established in small scale cultures (Table 1) were used to see if it is possible to obtain an accurate estimation of the biomass concentration in a large culture (FIG. 5). The actual biomass concentration measured after harvesting the culture was 3.24 g.sub.DW.Math.L.sup.1. Considering a 10% range around the experimental biomass value, the estimations of the biomass concentration at the end of the growth phase are statistically identical when obtained with sugar correlation. The conductivity correlation over-estimated the biomass concentration.

[0257] Strikingly, similarly to the observations made in small-scale conditions, it is possible to estimate accurately the biomass concentration (within the 10%-margin of error) in a large-scale culture of hairy roots via the measurement of sugar concentrations in the culture medium.

Example 2

Materials and Methods

[0258] Brassica rapa rapa hairy roots were grown as in Example 1.

[0259] Wild type Brassica napus hairy roots were grown at 100 rpm and 25 C. in 200 mL of Gamborg's B5 medium, supplemented with 30 g.Math.L.sup.1 of sucrose. For their preservation, hairy roots were sub-cultured every two weeks.

[0260] Cannabis sativa hairy roots were grown at 70 rpm and 25 C. in 200 mL of MS medium, supplemented with 30 g.Math.L.sup.1 of sucrose. For their preservation, hairy roots were sub-cultured every two weeks.

[0261] Culture analysis with respect to biomass, sugars and nutrients uptakes, estimation of growth rate and apparent biomass yields, and measurement of recombinant protein production were performed as in Example 1.

Results

[0262] As shown in FIGS. 6A-6B, the growth of Brassica napus and Cannabis sativa hairy roots species during 39 days in 100 mL of BGM medium in Erlenmeyer flasks, consisting of 14 days of biomass growth (first phase) and then 25 days of production through rhizocal culture (second phase), followed the same trend as the growth of Brassica rapa rapa hairy roots in identical conditions (see FIG. 1A for comparison). However, the growth rate was lower for both Brassica napus and Cannabis sativa hairy roots species, as compared to the growth rate of Brassica rapa rapa hairy roots, as seen in Table 2.

TABLE-US-00002 TABLE 2 Maximal biomass growth rate and doubling time in multi-phasic cultures of three Brassica species. Maximal biomass growth rate (d.sup.1) Doubling time t.sub.D (d) B. rapa rapa B. napus C. sativa = 0.40 d.sup.1 = 0.26 d.sup.1 = 0.19 d.sup.1 t.sub.D = 1.73 d t.sub.D = 2.66 d t.sub.D = 3.65 d

[0263] Similarly to Brassica rapa rapa hairy roots (see FIG. 2A), the measured conductivity (mS) for both Brassica napus and Cannabis sativa hairy roots is linearly correlated to the biomass production, both during the growth phase (first phase) and the rhizocal phase (second phase or production phase), with distinct apparent biomass yields for each phase (FIGS. 7A-7B). For Brassica napus hairy roots the biomass yields for conductivity are 2.570.48 g.sub.DW/L/mS and 8.482.58 g.sub.DW/L/mS, for the first phase of biomass production and for the second phase of rhizocal induction, respectively (see Table 3); whereas the biomass yields for conductivity during the first phase of biomass production and the second phase of rhizocal induction are respectively 2.110.42 g.sub.DW/L/mS and 4.493.42 g.sub.DW/L/mS, for Cannabis sativa hairy roots (see Table 3).

TABLE-US-00003 TABLE 3 Biomass growth yield for conductivity in multi-phasic cultures of three Brassica species. Biomass growth yield for conductivity (g.sub.DW/L/mS) B. rapa rapa B. napus C. sativa Growth phase 3.90 1.11 2.57 0.48 2.11 0.42 (first phase) Production phase 12.20 3.30 8.48 2.58 4.49 3.42 (second phase)

[0264] Again, similarly to Brassica rapa rapa, the biomass growth yield for total sugars and for total nitrogen for both Brassica napus and Cannabis sativa hairy roots are identical when growth phase and production phase are compared see FIGS. 8A-8B and Table 4; FIGS. 9A-9B and Table 5; respectively.

TABLE-US-00004 TABLE 4 Biomass growth yield for total sugars in multi-phasic cultures of three Brassica species. Biomass growth yield for total sugars (g.sub.DW/g.sub.S) B. rapa rapa B. napus C. sativa Growth phase 1.21 0.23 0.49 0.01 1.08 0.27 (first phase) Production phase 1.21 0.23 0.49 0.01 1.08 0.27 (second phase)

TABLE-US-00005 TABLE 5 Biomass growth yield for total nitrogen in multi-phasic cultures of three Brassica species. Biomass growth yield for total nitrogen (g.sub.DW/g.sub.N) B. rapa rapa B. napus C. sativa Growth phase 24.05 2.44 16.91 0.73 8.35 1.24 (first phase) Production phase 24.05 2.44 16.91 0.73 8.35 1.24 (second phase)

[0265] Consequently, the total sugar and/or total nitrogen consumption provide a useful biomarker to assess the biomass production of hairy roots, at any time of the phase of a multi-phasic culture of the hairy roots in a culture medium, i.e., either at the growth phase (first phase) or the production phase (second phase).