Production of polyhydroxyalkanoates using a free fatty acids containing substrate and a nutrient-containing co-substrate in presence of microorganisms

Abstract

Processes for producing PHA using PHA-producing microorganisms are provided. The process can include using a culture medium comprising a substrate mixture that comprises an FFA-containing substrate and a nutrient-containing substrate. The FFA-containing substrate has a carbon content accounting for between about 50 wt% to 100 wt% of a total carbon content of the substrate mixture. The FFA-containing substrate can have a weight ratio of unsaturated fat to saturated fat between 3 and 10, and / or a viscosity of between 40 and 60 CP. The process can include monitoring in real time at least one fermentation parameter comprising at least one of an optical density parameter, a suspended solids parameter or a PHA concentration parameter, using measured fluorescent spectrum data being correlated to the at least one fermentation parameter.

Description

PRODUCTION OF POLYHYDROXYALKANOATES USING A FREE FATTY ACIDS CONTAINING SUBSTRATE AND A NUTRIENT-CONTAINING CO-SUBSTRATE IN PRESENCE OF MICROORGANISMSTECHNICAL FIELDThe present invention generally relates to the production of polyhydroxyalkanoates (PHA) via PHA producing microorganisms, and more particularly to a process for producing PHA including supplying free fatty acids as a main carbon source in a culture medium comprising PHA producing microorganisms.BACKGROUND

[0001] The PHAs are the family of bioplastics which can be produced by microorganisms. The major problem with the production of PHAs is the cost of production and numerous works have been done using different waste sources to overcome the cost problems associated with the PHA production. However, the availability and composition of waste sources (crude glycerol, waste cooking oil, etc.) is always limited which impacts the efficiency of developed PHA process using waste sources with bacteria culture.

[0002] There is still a need for a technology that overcomes at least some of the drawbacks of what is known in the field.SUMMARY

[0003] The presently proposed techniques involve the use of free fatty acids (FFAs) as a main carbon source to sustain PHA production by PHA-production microorganisms, along with nutrient source(s). The free-fatty acids are provided in a substrate mixture having an optimized carbon content and optimized nutrient content.

[0004] For example, there is provided the substrate mixture including an FFA- containing substrate, a micronutrient-containing substrate and a macro-nutrient substrate, wherein between about 50 wt% and 100 wt% of a total required optimized carbon content of the substrate mixture is provided by the FFA-containing substrate. TheFFA-containing substrate is thus qualified as the main carbon source. The composition of the substrate mixture and the relative proportions of the used substrates are adjustable depending on the nature of the used substrates.

[0005] A method to prepare such substrate mixture having the optimized carbon content and optimized nutrient content is also provided. The substrate mixture can be used as a production media, prior to fermentation, and can be inoculated with an inoculum comprising the PHA-producing microorganisms prior to PHA-production via fermentation. Optionally, water can be further added to the substrate mixture to form the production media, in accordance with a volume of the culture medium required.

[0006] There is further provided a process for producing PHA including real time monitoring of substrate (carbon / nutrient) consumption to optimize PHA production. The process for producing PHA comprises supplying the substrate mixture as described herein as a production media to PHA-producing microorganisms. For example, the PHA- producing microorganisms are provided in an inoculum, such that the substrate mixture can be inoculated with the inoculum at a start-up stage of the process to form the culture medium. The process further includes fermenting the PHA microorganisms in the culture medium by maintaining a temperature between 25°C and 35 °C. The process further includes recovering PHA. The process further includes real-time monitoring of the carbon content and / or nutrient content to react to or prevent a change in the carbon / nutrient content of the culture medium by supplying carbon / macronutrient(s) to the culture medium via an additional adjusted amount of the FFA-containing substrate and / or macronutrient-containing substrate to the culture medium. The supplying can be performed continuously or at a specific fermentation duration.

[0007] In some implementations, the process can include monitoring / determining the total carbon content of the production media / culture medium prior to fermentation and / or during fermentation.

[0008] In some implementations, the process can further include monitoring / determining the total fatty acids content of the production media / culture medium prior to fermentation and / or during fermentation.

[0009] In some implementations, the process can further include monitoring / determining the total nutrient content of the production media / culture medium prior to fermentation and / or during fermentation.

[0010] The developed process allows for efficient PHA production (e.g., approximately 60-80 g / L of biomass with 70-90 % w / w of PHA). The developed process also advantageously includes the use of sludge solids as a co-substrate and a source of micronutrients.

[0011] There is further provided a use of a wavelength range between about 360 nm and about 460 nm, optionally between about 420 nm and about 450 nm to radiate / excite the culture medium for in situ measurement of at least one of the carbon content and the nutrient content of the culture medium. Optionally, additional physico-chemical fermentation parameters can be monitored in real-time based on this specific wavelength excitation. For example, the real-time monitoring can be performed by probing the culture medium using a bioptic probe.

[0012] In certain aspects, there is provided a process for producing PHA including providing a culture medium comprising a substrate mixture and PHA-producing microorganisms; subjecting the culture medium to fermentation conditions to sustain biomass growth and to trigger production of PHA by the PHA-producing microorganisms; and recovering PHA from the culture medium. The substrate mixture comprises an FFA- containing substrate and a nutrient-containing substrate. The FFA-containing substrate has a carbon content accounting for between about 50 wt% to 100 wt% of a total carbon content / demand of the culture medium.

[0013] For example, the FFA-containing substrate has a weight ratio of unsaturated fat to saturated fat between 3 and 10. For example, the FFA-containing substrate has a weight ratio of unsaturated FFA to saturated FFA between 20 and 200. For example, the FFA-containing substrate has a saturated fat content between 10 wt% and 20 wt% with respect to a total weight of the FFA-containing substrate. For example, the FFA- containing substrate has a trans fat content of at most 0.05 wt% with respect to the total weight of the FFA-containing substrate. For example, the FFA-containing substrate has at least one of a C16:0 saturated FFA content of at most 50 g / kg of FFA-containing substrate; a C16:1 unsaturated FFA content between 5 g / kg and 200 g / kg of FFA-containing substrate; a C18:3 unsaturated FFA content between 150 g / kg and 400 g / kg of FFA-containing substrate; and a C18:0 saturated FFA content of at most 20 g / kg of FFA-containing substrate. For example, the FFA-containing substrate has a viscosity of between 40 and 60 CP as measured with ASTM D445 standard at 40°C.

[0014] In some embodiments, the nutrient-containing substrate comprises at least one of a micronutrient-containing substrate and a macronutrient-containing substrate. The micronutrient-containing substrate provides between 30 wt% and 100 wt% of a total micronutrient content / demand of the culture medium.

[0015] In some embodiments, providing the culture medium comprises providing the substrate mixture as at least part of a production media; and inoculating the production media with an inoculum comprising the PHA-producing microorganisms to produce the culture medium.

[0016] In some embodiments, the process further comprises pre-treating the substrate mixture prior to being provided as part of the culture medium to prevent contamination of the microorganisms.

[0017] In some embodiments, the process further comprises measuring and adjusting a pH of the culture medium prior to or during fermentation to maintain the pH between 6 and 8.

[0018] In some embodiments, the process further comprises monitoring at least one of a carbon content, a nutrient content, a biomass growth, and a cell mass of the culture medium during fermentation; and adding at least one of the FFA-containing substrate and the nutrient-containing substrate in accordance with monitored information.

[0019] For example, the at least one of the FFA-containing substrate and the nutrientcontaining substrate is further continuously added in an adjusted amount in accordance with the monitored information to maintain PHA concentration at at least about 60 wt% of the total weight of suspended solids in the culture medium.

[0020] In some embodiments, the monitoring is performed in real time in the culture medium during fermentation.

[0021] In some embodiments, the monitoring includes:measuring at least one fermentation parameter of the culture medium during fermentation, the at least one fermentation parameter comprising an optical density, total suspended solids, PHA concentration or any combinations thereof; and based on the at least one fermentation parameter, determining at least one of the carbon content, the nutrient content, the biomass growth, and the cell mass of the culture medium.

[0022] For example, measuring the at least one fermentation parameter can include probing the culture medium for in situ spectroscopic measurement to collect fluorescent spectrum data that is correlated to the at least one fermentation parameter.

[0023] In some embodiments, the process includes adding the FFA-containing substrate to the culture medium during fermentation to maintain a total carbon content in the culture medium at the optimal total carbon content of the culture medium.

[0024] For example, the optimal total carbon content of the culture medium is between 3 g / L and 12 g / L of the culture medium.

[0025] In certain aspects, there is also provided a use of an FFA-containing substrate for the production of PHA as part of a culture medium, the FFA-containing substrate having at least one feature as defined herein.

[0026] In some embodiments, the FFA-containing substrate is used as a production media prior to fermentation and / or as a continuous feed during fermentation.

[0027] In some embodiments, the substrate mixture is used to provide between 50 wt% and 100 wt% of the total carbon content consumed by the PHA-producing microorganisms to yield at least 60 wt% of PHA with respect to a total weight of the suspended solids.

[0028] In certain aspects, there is provided a method for optical monitoring and analysis of a PHA production process in a culture medium comprising microorganisms, the method comprising: exciting the culture medium with excitation light;detecting fluorescent light emitted by the culture medium in response to the excitation light; and analyzing the detected fluorescent light using a response model relating fluorescent spectrum data with fermentation parameter data to derive at least one fermentation parameter of the culture medium; wherein the at least one fermentation parameter comprises at least one of an optical density parameter, a suspended solids parameter, or a PHA concentration parameter.

[0029] In some embodiments, the process further comprises controlling the PHA production process based on the analysis to adjust or maintain the at least one fermentation parameter. For example, controlling the PHA production process comprises adding at least one of a nutrient source or a carbon source to the culture medium based on the at least one fermentation parameter.

[0030] In some embodiments, the excitation light lies within a wavelength range of 280 nm to 490 nm.

[0031] In some embodiments, the fluorescent light is detected within a wavelength range of 250 nm to 595 nm.

[0032] In some embodiments, the at least one fermentation parameter comprises the optical density parameter, and the method further comprises determining cell growth information from the optical density parameter. For example, the optical density parameter is determined from fluorescent light detected within a wavelength range of 360 nm to 595 nm in response to excitation light within a wavelength range of 340 nm to 490 nm.

[0033] In some embodiments, the at least one fermentation parameter comprises the suspended solids parameter, and the method further comprises determining cell mass information from the suspended solids parameter. For example, the suspended solids parameter is determined from fluorescent light detected within a wavelength range of 305 nm to 595 nm in response to excitation light within a wavelength range of 280 nm to 490 nm.

[0034] In some embodiments, the at least one fermentation parameter comprises the PHA concentration parameter, the method further comprising determining nutrient and / or carbon content information about the culture medium from the PHA concentration parameter. For example, the PHA concentration parameter is determined from fluorescent light detected within a wavelength range of 480 nm to 595 nm in response to excitation light within a wavelength range of 450 nm to 490 nm.

[0035] In some embodiments, the response model is a partial least squares model.

[0036] In some embodiments, excitation and detection are performed using a light source and a spectral detector contained within a single probe.

[0037] In certain aspects, there is also provided a PHA production process in a culture medium comprising PHA-producing microorganisms comprising monitoring in real time at least one fermentation parameter comprising at least one of an optical density parameter, a suspended solids parameter or a PHA concentration parameter. The monitoring comprises using the method for optical monitoring and analysis of the PHA production process as defined herein.

[0038] While aspects of the invention will be described in conjunction with example embodiments, it will be understood that it is not intended to limit the scope of these aspects to such embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included as defined by the present description. The objects, advantages and other features of the present techniques will become more apparent and be better understood upon reading of the following non-restrictive description of the invention, given with reference to the accompanying drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Implementations of the PHA producing process and related use of free fatty acids as a main carbon source are represented in and will be further understood in connection with the following figures.

[0040] Figure 1 is a three-dimensional graph of detected values at different detection wavelengths (in nm) along with fermentation time (in days and hours) for a culturemedium being excited at an excitation wavelength of 420 nm and being continuously fed with free fatty acids as the main carbon source (Bosk-02).

[0041] Figure 2 is a three-dimensional graph of detected values at different detection wavelengths (in nm) along with fermentation time (in days and hours) for a culture medium being excited at an excitation wavelength of 450 nm and being continuously fed with free fatty acids as the main carbon source (Bosk-02).

[0042] Figure 3 is a three-dimensional graph of detected values at different detection wavelengths (in nm) along with fermentation time (in days and hours) for a culture medium being excited at an excitation wavelength of 450 nm and being continuously fed with free fatty acids as the main carbon source (Bosk-02).

[0043] Figure 4 is a three-dimensional graph of detected values at different detection wavelengths (in nm) along with fermentation time (in days and hours) for a culture medium being excited at an excitation wavelength of 450 nm and being continuously fed with waste cooking oil as the main carbon source (Bosk-01).

[0044] Figure 5 includes four graphs showing fluorescence-correlated optical density based on a model developed using external optical density measurements at 600 nm.

[0045] Figure 6 is four graphs showing fluorescence-correlated total suspended solids based on another model developed using external total suspended solids measurement using an external analytical scale and a drying method.

[0046] Figure 7 includes four graphs showing fluorescence-correlated PHA concentration based on another model developed using external PHA concentration measurement using gas chromatography-flame ionization detector (GC-FID).DETAILED DESCRIPTION

[0047] The techniques described herein involve the production of PHA using a substrate mixture including FFAs and nutrients, in presence of PHA-producing microorganisms. The FFAs are provided by an FFA-containing substrate. The FFAs are used herein as a main carbon source which can represent a main portion of or the total carbon being required for bacterial growth and PHA production. The nutrients of the substrate mixture include micronutrients and macronutrients which are also required,along with carbon, bacterial growth and PHA production. The micronutrients are provided by a micronutrient containing substrate that can be activated sludge. The macronutrients are provided by a macro-nutrient containing substrate. It is noted that micronutrients and macronutrients can be combined and provided as a nutrientcontaining (co-)substrate.

[0048] In one general aspect, there is provided the substrate mixture including the FFA-containing substrate as described herein, the micronutrient-containing substrate as described herein and the macro-nutrient substrate described herein, wherein between about 50 wt% and 100 wt% of the total optimized carbon content of the substrate mixture is provided by the FFA-containing substrate. The FFA-containing substrate is thus qualified as the main carbon source. The composition of the substrate mixture and the relative proportions of the used substrates are adjustable depending on the nature of the used substrates.

[0049] The FFAs as referred to herein are derived by the action of lipase on ester bonds from trigacylgycerols, diacylglycerols, or monoacylglycerol in presence of moisture and high temperatures. FFAs are carboxylic acids that exist in an unbound form in oils and fats. During PHA synthesis, bacteria use FFAs as a primary carbon source to produce PHA. FFAs are preferred as they are consumed faster by microorganisms as compared to glycerides.

[0050] The FFAs provided by the FFA-containing substrate can include saturated fatty acids, unsaturated fatty acids or a combination thereof. They can be characterized by a lipid number having a C:D form, with C being the number of carbon atoms in the fatty acid and D being the number of double bonds in the fatty acid. When referring to the FFA-containing substrate as including an FFA, one should understand that the FFA- containing substrate includes at least one FFA (non-esterified form). The FFA-containing substrate can for example include at least one FFA resulting from the degradation of a fat / lipid (e.g., triglyceride). The FFA-containing substrate can for example further include byproducts of fat / lipid degradation. The processes encompassed herein can thus include conditioning of a source of fat / lipid, for example a waste source, to produce the FFA- containing substrate having an enhanced FFA content with respect to the source of fat / lipid, for example by degradation of the source of fat / lipid. The FFA-containing substrate can be referred to as an FFA source.

[0051] For example, the FFA-containing substrate can include a combination of at least one saturated FFA and at least one unsaturated FFA. For example, the FFA- containing substrate can include at least one saturated fat and at least one unsaturated fat. For example, the FFA-containing substrate can include at least one fat and at least one FFA (naturally occurring). For example, the FFA-containing substrate can include a triglyceride and at least one FFA, such as palmitoleic acid having a lipid number of 18:3.

[0052] The FFA-containing substrate as encompassed herein is used as a production media that sustains bacterial growth upon inoculation by microorganisms, thereby forming a culture medium. The FFA-containing substrate can also be used as a carbon- containing feed to the culture medium to sustain PHA production during fermentation of the grown bacteria. The FFA-containing substrate is thus selected to provide a sufficient and adequate carbon source to the microorganisms for sustaining bacterial growth and further PHA production.

[0053] The FFA-containing substrate is characterized by a weight ratio of unsaturated fat to saturated fat between 3 and 10, optionally between 3 and 9, further optionally between 4 and 8, yet further optionally between 4 and 7. It has been found that a specific range of ratios of unsaturated FFAs to saturated FFAs favors PHA production as the FFA-containing substrate is optimally consumed by the microorganisms. It is understood that the techniques described herein including selection of the FFA- containing substrate as a main carbon source allow producing PHA at a concentration of at least 60 wt% with respect to a total weight of suspended solids in the culture medium.

[0054] The FFA-containing substrate has a fat profile that can be determined using GC-FID (Gas Chromatography Flame Ionization Detection). The fat profile is the composition in fats of the FFA-containing substrate, which allows determining ratios of unsaturated fats to saturated fats, and ratio of unsaturated FFAs to saturated FFAs that are available to the microorganisms. The following Table 1 exemplifies four industrial byproducts that can be used as the FFA-containing substrate as defined herein, with Table 1 showing their fat profile as determined using GC-FID.Table 1

[0055] In some embodiments, the FFA-containing substrate can be characterized by at least one of the following:- a weight ratio of unsaturated fat to saturated fat between 3 and 10, optionally between 4 and 7;- a weight ratio of unsaturated FFA to saturated FFA between 20 and 200, further optionally between 25 and 160;- a saturated fat content between 10 wt% and 20 wt% with respect to a total weight of the FFA-containing substrate, optionally between 16 and 19 wt%;- a trans fat content of at most 0.05 wt% with respect to the total weight of the FFA- containing substrate;- a C16:0 saturated FFA content of at most 50 g / kg of FFA-containing substrate;- a C16:1 unsaturated FFA content between 5 g / kg and 200 g / kg of FFA- containing substrate;- a C18:3 unsaturated FFA content between 150 g / kg and 400 g / kg of FFA- containing substrate;- a C18:0 saturated FFA content of at most 20 g / kg of FFA-containing substrate; and- a viscosity between 40 and 60 CP as measured with ASTM D445 (Brookfield Viscosimeter) standard at 40°C.

[0056] In some embodiments, the FFA-containing substrate has a weight ratio of unsaturated fat to saturated fat between 3 and 10, and a viscosity between 40 and 60 CP as measured with ASTM D445 (Brookfield Viscosimeter) standard at 40°C. The FFA- containing substrate can further include a C16:0 saturated FFA content of at most 50 g / kg of FFA-containing substrate; a C16:1 unsaturated FFA content between 5 g / kg and 200 g / kg of FFA-containing substrate; a C18:3 unsaturated FFA content between 150 g / kg and 400 g / kg of FFA-containing substrate; and a C18:0 saturated FFA content of at most 20 g / kg of FFA-containing substrate.

[0057] It is noted that the viscosity of the FFA-containing substrate is selected to facilitate mixing of the FFA-containing substrate with other fermentation media components (such as nutrient containing substrate and / or water) and flowing of the production media, thereby favoring carbon availability to bacteria during fermentation. For example, acid oil has a viscosity of 41.4 CP which is suitable for preparation of theproduction media. However, soap stock and deodorizer distillate have a viscosity of 25.8 CP and 35.5 CP, respectively, which makes it difficult for mixing with other fermentation media components and reduce availability of the carbon to bacteria during fermentation.

[0058] It is further noted that the use of the FFA-containing substrate as described herein as a main carbon source in the PHA-producing process as described herein allows production of at least 50 wt% with respect to a total weight of suspended solids in the culture medium, for example, between 60 wt% and 100 wt% with respect to the total weight of suspended solids in the culture medium.

[0059] In some embodiments, the FFA-containing substrate includes at least one FFA and has an FFA content between about 30 wt% and 100 wt%, optionally between 80 wt% and 100 wt% with respect to a total weight of the FFA-containing substrate. For example, the FFA-containing substrate can include one or more FFA(s) as provided in below Table 2. For example, the FFA-containing substrate can be industrially produced, e.g., from hydrolysis of triglycerides, or can be derived from a natural source of FFAs, e.g., animal fats and / or vegetable fats.Table 2 showing the FFA content (in g / kg) in FFA-containing different substrates

[0060] FFAs can be derived from food and agro-industries as high FFA content in oils / fats leads to non-desirable odors and taste of the edible oils / fats. Therefore, a major portion of FFAs can be industrially removed and treated as waste. For example, the FFA-containing substrate can include or be a waste stream such as waste cooking oil, soap stocks, spent bleaching oil, deodorizer distillates, waste of triglyceride-based feeds of biodiesel industry, and any combinations thereof.

[0061] In some embodiments, the carbon content of the FFA-containing substrate can be calculated based on the GC-FID values, which allows to determine any initial amount of FFA-containing substrate and / or any additional amount of FFA-containing substrate that is necessary to maintain the available carbon content in the culture medium at an optimal carbon content (that is predetermined for optimization of fermentation parameters for the process). For example, the optimized carbon content can be selected based on highest biomass (growth) and highest PHA produced (PHA production).

[0062] The FFA-containing substrate that can include one or more FFA(s) is part of the substrate mixture as the main carbon source selected for the PHA producing bacteria / microorganisms used in process for PHA production. Main carbon source should be understood herein as providing between 50 wt% and 100 wt% of the total carbon content that is needed by the bacteria during fermentation for optimized growth and PHA production.

[0063] In some embodiments, the substrate mixture comprises at least one micronutrient that is present in the culture medium in an amount of at most 0.05 g per L of the culture medium, and the substrate mixture comprises at least one macronutrient that is present in an amount of at least 8 g per L of the culture medium. Macronutrients are present in larger quantities than micronutrients for efficient production of PHA by promoting cell multiplication. Micronutrients can be present in the substrate mixture as trace or in smaller amounts as they act as precursors in various metabolic pathways.

[0064] The micronutrient-containing substrate of the substrate mixture is provided to account for between 30 wt% and 100 wt%, optionally between 40 % and 80 wt%, of the total micronutrient content of the substrate mixture. The micronutrient-containing substrate can include one or more micronutrient(s) being ionic chloride, iron, calcium, calcium chloride (CaCh), sulphates, copper sulphate (CuSC t), copper (Cu), chromiumchloride (CrCh), nickel chloride, cobalt chloride (C0CI2), nickel (Ni), chromium (Cr), cobalt (Co), H3BO3, AIK(SO4)2.12H2O, MnCI2, ZnSO4'7H2O, ZnSO4, NaMoO4-2H2O, NaMoO4, C0CI2 6H2C), CUSO45H2O, MnCI2-4H2O, nitroacetic acid, NaCI, and FeSO4.7H2O.

[0065] For example, the micronutrient-containing substrate can include wastewater and / or activated sludge. Activated sludge is to be understood as a suspended aerobic sludge obtained after a wastewater treatment. For example, the wastewater and / or the activated sludge can be from the pulp and paper mill industry, cheese industry, sugar and molasses industry, starch industry, lignocellulose industry, other food industries, agro-industries or a combination thereof. For example, the micronutrient-containing substrate can include pulp and paper mill activated sludge (PPMAS), pulp and paper mill wastewater, or a combination thereof. For example, the micronutrient-containing substrate can be pulp and paper mill activated sludge (PPMAS) having a typical composition and characterization as provided in below Table 3.Table 3: Physical and chemical characterization of activated sludge containing nutrients (PPMAS) - provide per liter of sludge (without centrifugation)*VSS: volatile suspended solids*TS: Total solids and *TSS: Total suspended solids

[0066] It should be noted that the micronutrient-containing substrate, when being or comprising activated sludge for example, can include components being a source of carbon, thereby contributing to the total carbon content of the substrate mixture, in addition to the FFA-containing substrate. For example, the micronutrient-containing substrate can account for between about 0 wt% and 50 wt% of the total carbon content of the substrate mixture.

[0067] It should further be noted that the activated sludge that can be used for PHA production thus eliminates addition of micronutrients into the process, because the necessary micronutrients are already contained in the activated sludge. The activated sludge further includes macronutrients as exemplified in Table 3. However, there is still a need for additional macronutrients which can be added separately via the macronutrientcontaining substrate for efficient PHA production.

[0068] The macronutrient-containing substrate of the substrate mixture can include one or more macronutrient(s) to enhance the growth of the PHA-producing microorganisms and production of PHA during fermentation. The one or more macronutrients include phosphate, sodium, nitrogen, magnesium, potassium, urea, ammonium ferric citrate, nitriloacetic acid, or any combinations thereof. For example, the macronutrient-containing substrate can include or be Na2HPO4, KH2PO4, MgSCU,Na2HPO4.12H2O, Na2HPO4.7H2O, MgSO4.7H2O, (NH4)2SO4, CO(NH2)2and any combinations thereof.

[0069] The techniques described herein are developed for production of PHA by and growth of PHA-producing microorganisms being provided in a culture medium. The PHA- producing microorganisms comprise Bacillus megaterium, Comamonas testosteroni, Cupriavidus necator 11599, Cupriavidus necator H16, Pseudomonas guezennei biovar. Tikehau, R. eutropha, E. coli, engineered E. coli, Alcaligenes latus, Sphingobacterium sp. ATM, Plasticicumulans acidivorans, Bacillus tequilensis, Haloferax mediterranei, H. mediterranei, Pseudomonas fluorescens A2a5, Ralstonia eutropha H16 and any combinations thereof. In some implementations, the culture medium can be a pure culture medium comprising a single type of bacteria at a time.

[0070] There is further provided a method to prepare and provide the substrate mixture to be added to a culture medium including the PHA-producing microorganisms in view of PHA production upon fermentation. The preparation of the substrate mixture includes determining a carbon content of at least one of the FFA-containing substrate and the micronutrient-containing substrate, and adjusting the relative amount of these two substrates to be supplied to the microorganisms based on the determined carbon content to achieve a predetermined optimal total carbon content for optimal biomass growth and PHA production. For example, the method can include determining the carbon content of the micronutrient-containing substrate, and providing an adjusted amount of the FFA-containing substrate based on said determined carbon content to achieve the predetermined optimal total carbon content in the culture medium. For example, the optimized total carbon content can be between about 3 g / L and 12 g / L, optionally between about 4 g / L and 8 g / L of the culture medium .

[0071] Providing the adjusted amount of the FFA-containing substrate based on said determined carbon content can include adding the adjusted amount of the FFA- containing substrate to the nutrient-containing substrate, with the FFA-containing substrate being the main source of carbon for the substrate mixture / production media.

[0072] In some implementations, the micronutrient-containing substrate can be an activated sludge, for example an industrial activated sludge that can include lignocellulosic waste. The method can include characterization / determination of thecarbon content of the activated sludge. Such characterization can include centrifugating a sample of the activated sludge to recover a sludge solid component including organic matter and determining a carbon content of the sludge solid component. For example, the carbon content of the activated sludge can be determined using a Total Organic Carbon Analyzer, for example from Shimadzu. The adjusted amount of the FFA- containing substrate is then determined in terms of equivalent carbon. The method further includes providing the micronutrient-containing substrate with an adjusted solids concentration. For example, the sludge solid component can be provided as the micronutrient-containing substrate with a solids concentration between about 5 g / L and 50 g / L. For example, the sludge solid component can be resuspended in water to achieve a solids concentration between about 5 g / L and 50 g / L to form a suspension that is used as the micronutrient-containing substrate.

[0073] In some implementations, the method can include determining at least one of the total fatty acids content (e.g., via titration) and the total carbon content of the substrate mixture (or of the combination of the micronutrient-containing substrate and of the FFA-containing substrate) prior to fermentation.

[0074] The preparation of the substrate mixture can also include determining a nutrient content of the micronutrient-containing substrate and providing an adjusted amount of the macronutrient-containing substrate based on said determined nutrient content to achieve an optimized nutrient content in the substrate mixture / production media. For example, the quantity of macronutrients to be added via the macronutrient-containing substrate can be tailored based on the nutrient content present in sludge solid component (example acceptable range is given in Table 4). This adjusted mixture of macronutrients is defined as a “NUTRIMIX” (macronutrient-containing substrate). The concentration of macronutrient changes in the nutrimix with each type of industrial activated sludge waste. For example, the nutrient content can be determined using liquid chromatography mass spectrometry (LC-MS) analysis.Table 4: Example macronutrients used for PH A production

[0075] It is noted that the inoculated production media can be referred to as the culture medium at an initial stage of the fermentation (TO). The culture medium being exposed to fermentation conditions after TO can be referred to as a fermenting / fermented culture medium having an evolving cell mass, carbon content and PHA content.

[0076] The method for preparing the substrate mixture can further include at least one pre-treatment step of one or more of the substrates to avoid contamination that could derive from the use of wastes and to allow the growth of bacteria inside the media for the production of PHA. For example, the at least one pre-treatment step can be based on the nature of the waste stream(s) that are used for the FFA-containing substrate and / or micronutrient-containing substrate. In general, a heat-treatment enabling sterilization of the waste-derived substrate (that can include the FFA-containing substrate and / or the micronutrient-containing substrate) can be performed. For example, the at least one pretreatment step can be selected based on the industry type of the activated sludge that is used as the micronutrient-containing substrate. For example, when the activated sludge is or includes whey waste, the at least pre-treatment step of the method for preparing the substrate mixture can include catalysis treatment. For example, when the activated sludge is or includes pulp and paper mill activated sludge (PPMAS), the at least pretreatment step of the method for preparing the substrate mixture can include heat treatment of the PPMAS with addition of calcium containing source. For example, the heat-treatment can be sterilization via autoclave.

[0077] In some implementations, the method for preparing the substrate mixture / production media can include adjusting a pH thereof between 6 and 8, optionally between 6.4 and 7.2.

[0078] It should be noted that although the substrate mixture is referred to as a “mixture”, one or more of the substrate(s) of the substrate mixture can be provided to the PHA-producing microorganisms separately from the other(s), thereby forming the production media to be inoculated with the inoculum to initiate / maintain PHA production via fermentation. For example, the macronutrient-containing substrate can be added to the culture medium separately from and after the FFA-containing substrate and micronutrient-containing substrate (combined or not). The mixture should thus be understood as a combination or assembly of substrates that can be supplied independently or together to the PHA-producing microorganisms to form the culture medium.

[0079] In another general aspect, there is provided a process for producing PHA comprising supplying the substrate mixture as described herein as a production media to PHA-producing microorganisms. For example, the PHA-producing microorganisms are provided in an inoculum, such that the substrate mixture can be inoculated with the inoculum at a start-up stage of the process to form the culture medium. The process further includes fermenting the PHA microorganisms in the culture medium by maintaining a temperature between 25°C and 35 °C. The process further includes recovering PHA. It should be noted that fermentation and PHA recovery can be performed according to various techniques as readily known and available in the art.

[0080] In some implementations, prior to supplying the substrate mixture (including the FFA-containing substrate, the micronutrient-containing substrate and the macronutrientcontaining substrate) to the inoculum at an initial stage of the process, the process can include preparation of the substrate mixture according to at least one of the method steps / features as described hereinabove.

[0081] In some implementations, the process can include adjusting a pH of the culture medium prior to and / or during fermentation. For example, addition of at least one of acetic acid and phosphoric acid can be performed to adjust the pH.

[0082] Optionally, the process can include operating fermentation for 70 hours to 160 hours.

[0083] It should be noted that the carbon content stemming from the micronutrientcontaining substrate (e.g., activated sludge) is accounted for prior to fermentation, and if the PHA-producing microorganisms require more carbon upon fermentation, the FFA- containing substrate is added as the sole carbon source during fermentation. It should further be noted that the nutrient content stemming from the micronutrient-containing substrate (e.g., activated sludge) is accounted for prior to fermentation, and if the PHA- producing microorganisms require more nutrients upon fermentation, the macronutrientcontaining substrate is added as the sole nutrient source during fermentation.

[0084] In some implementations, the process can further include monitoring of the carbon content of the culture medium and comparing a monitored value of the carbon content to a standard value or range of the carbon content that is pre-determined to ensure optimal bacteria growth and PHA production. The process can then include supplying carbon to the culture medium by further continuously adding an adjusted amount of the FFA-containing substrate to maintain the carbon content at the optimized value or within the optimized range for the carbon content for optimal PHA production / concentration as defined herein. Alternatively, the process can then include supplying carbon to the culture medium by further adding an adjusted amount of the FFA-containing substrate when the monitored value of the carbon content during fermentation (at a specific fermentation duration) is different from the optimized value or optimized range for the carbon content for optimal PHA production / concentration as defined herein.

[0085] For example, the process can include monitoring the carbon content of the culture medium and maintaining the carbon content of the culture medium between 4 g / L and 12 g / L during fermentation. For example, maintaining the carbon content of the culture medium within the optimized range is performed by adding the FFA-containing substrate in the adjusted amount necessary to bring the carbon content back into the standard range. In another example, maintaining the carbon content of the culture medium within the optimized range is performed by periodically adding a given amount of the FFA-containing substrate during fermentation. In another example, maintaining the carbon content of the culture medium within the standard range is performed bycontinuously adding a given amount of the FFA-containing substrate during fermentation. Optionally, the carbon content can be maintained between 3 g / L and 12 g / L of the culture medium, optionally between 4 g / L and 8 g / L of the culture medium, for example, by supplying between about 6 g and 18 g of the FFA-containing substrate to the culture medium.

[0086] Optionally, the monitoring of the carbon content can be performed in real-time so as to quickly react to a change in the carbon content of the culture medium by supplying carbon to the culture medium via the additional adjusted amount of the FFA- containing substrate to the culture medium continuously or at a specific fermentation duration. For example, real-time monitoring of the carbon content can include performing in situ spectroscopic measurement at an excitation wavelength range between about 360 nm and about 460 nm, optionally between about 420 nm and about 450 nm.

[0087] In some implementations, the process can include monitoring / determining the total carbon content of the production media / culture medium prior to fermentation and / or during fermentation.

[0088] In some implementations, the process can further include monitoring / determining the total fatty acids content of the production media / culture medium prior to fermentation and / or during fermentation.

[0089] In some implementations, the process can further include monitoring / determining the total nutrient content of the production media / culture medium prior to fermentation and / or during fermentation.

[0090] In some implementations, the process can further include monitoring of the nutrient content of the culture medium and comparing a monitored value of the nutrient content to an optimized value or range of the nutrient content that is pre-determined to ensure optimal bacteria growth and PHA production. The monitored nutrient content includes at least one of magnesium, sodium, iron, calcium, potassium, phosphate, and ammonia content. The process can then include supplying macronutrient(s) to the culture medium by further adding an adjusted amount of the macronutrient-containing substrate continuously or at a specific fermentation duration when the monitored value of the macronutrient content during fermentation is different from the optimized value or optimized range for the nutrient content.

[0091] For example, the process can include monitoring the nutrient content of the culture medium and maintaining the nutrient content of the culture medium according to the ranges provided in Table 3 during fermentation. For example, maintaining the nutrient content of the culture medium within the optimized range is performed by adding the macronutrient-containing substrate (that can be a mixture of macronutrients, each provided in an adjusted amount - cf nutrimix) in the adjusted amount necessary to bring the nutrient content back into the optimized range. In another example, maintaining the nutrient content of the culture medium within the optimized range is performed by periodically adding a given amount of the macronutrient-containing substrate during fermentation. In another example, maintaining the nutrient content of the culture medium within the optimized range is performed by continuously adding a given amount of the macronutrient-containing substrate during fermentation.

[0092] Optionally, the monitoring of the carbon content and / or nutrient content can be performed in real-time so as to quickly react to a change in the carbon / nutrient content of the culture medium by supplying carbon / macronutrient(s) to the culture medium via the additional adjusted amount of the FFA-containing substrate and / or macronutrientcontaining substrate to the culture medium at a specific fermentation duration. For example, real-time monitoring can include performing in situ wet chemical photometric, ion-selective electrodes and / or turbidity measurement(s) of the culture medium at an excitation wavelength range between about 360 nm and about 460 nm, optionally between about 420 nm and about 450 nm.

[0093] In another aspect, the present disclosure relates to the use of a wavelength range between about 360 nm and about 460 nm, optionally between about 420 nm and about 450 nm to radiate the culture medium for in situ measurement of at least one fermentation parameter being correlated to at least one of the carbon content and the nutrient content of the culture medium. Optionally, additional physico-chemical parameters can be monitored / correlated to in real-time based on this specific wavelength excitation range. For example, the real-time monitoring can be performed by probing the culture medium using a bioptic probe. For example, the real-time monitoring can be based on measuring a fluorescence response / signal of the culture medium.

[0094] For example, the carbon and / or nutrient content of the culture medium can be monitored in real-time to enable quick adjustments in response to changes. Theseadjustments can be made by supplying additional carbon and / or macronutrients to the culture medium via the additional adjusted amount of the FFA-containing substrate and / or macronutrient-containing substrate to the culture medium at a specific fermentation stage. For example, real-time monitoring of at least one of the carbon content and the nutrient content can be performed using in situ measurement such as wet chemical photometric analysis, ion-selective electrodes, and / or turbidity measurement on the culture medium, with excitation wavelengths ranging from about 360 nm to about 460 nm, optionally between about 420 nm and about 450 nm. Optionally, additional physico-chemical parameters can also be monitored in real-time within this specific excitation wavelength range.

[0095] In certain aspects, the present disclosure relates to a method for optical monitoring of a PHA production process in a culture medium. The method can include a step of exciting the culture medium with excitation light, and a step of detecting fluorescent light emitted by the culture medium in response to the excitation light. The method can also include a step of analyzing the detected fluorescent light using a response model relating fluorescent spectrum data with fermentation parameter data to derive at least one fermentation parameter of the culture medium. The fermentation parameters determined through this analysis can include at least one of an optical density parameter, a suspended solids parameter (e.g., in g / L), or a PHA concentration parameter (e.g., in wt%).

[0096] In some embodiments, the excitation and detection steps are performed using a single fluorescent probe — also referred to below as a bioptic probe — containing both an excitation light source and a spectral detector. However, the use of a single probe is not required. The bioptic probe may be used to measure metabolic activity in the culture medium using fluorescent signals to analyze processing efficiency, based on parameters such as optical density, suspended solids, and PHA concentration.

[0097] Upon excitation of the culture medium with light in a specific excitation wavelength range (e.g., 280 nm to 490 nm, though other ranges may be used), the culture medium emits fluorescent light in a corresponding detection wavelength range (e.g., 250 nm to 590 nm, though other ranges are possible). The spectral characteristics of this detected fluorescent light can vary based on the parameters (e.g., composition) of the culture medium, including its contents in nutrients and metabolic molecules. Asdifferent culture medium parameters are expected to interact differently with excitation light, it is possible to correlate changes in measured fluorescence spectra with changes in fermentation parameters of the culture medium, such as optical density, suspended solids, and PHA concentration.

[0098] Probing the culture medium in the context of fluorescence-based monitoring can refer herein to the use of a physical probe (hardware / instrument) operatively positioned with respect to the culture medium to deliver excitation light to the medium and collect resulting fluorescent light emitted from the excited medium. Typically, the probe includes a light emitter that provides the excitation light and a spectrometer or spectral detector that detects the emitted fluorescence. For example, the probe may be positioned to deliver excitation light through a glass wall of a bioreactor containing the culture medium. In another example, the probe may be located in situ, i.e., within the culture medium inside the bioreactor. Various other arrangements are also possible. In some instances, the light emitter and spectral detector may be provided in separate devices, functioning together as a combined probe.

[0099] Additionally, probing the culture medium in fluorescence-based monitoring can also refer herein to the addition of chemical probes, which are specific fluorescent molecules or compounds (e.g., dyes, tags, or indicators) that are incorporated into the culture medium to enable or enhance fluorescence measurements. Both physical probes and chemical probes can be used in certain PHA production monitoring applications, where a physical instrument probes the fluorescence response of a culture medium in which specific chemical fluorescent probes are added to generate or enhance the fluorescence signal.

[0100] In some embodiments, the bioptic probe includes or is connected to a processing unit (e.g., a computing system with processors and memories) configured to receive, possibly in real-time, fluorescent spectrum data measured by the probe. This processing unit may apply a response model, which can be developed using external data obtained through independent analytical methods (e.g., sample analysis via external equipment). The correlation between fluorescence measurements from the probe and fermentation parameters may be determined using a mathematical model, such as a partial least squares model, as represented by the following equation:

[0101] Using this approach, models can be developed to analyze (i) optical density, for example, to monitor cell growth during fermentation; (ii) suspended solids, to monitor cell mass of the culture medium during fermentation; and (iii) PHA concentration (e.g., from PHA content in suspended solids during fermentation), to monitor PHA production in real-time.(i) Model for Optical Density

[0102] Using an external spectrophotometer, a model has been developed to analyze the increase in cell number with optical density values at 600 nm. The graphs shown in Figure 5 demonstrates the increase in cell number based on a model developed using external optical density measurements at 600 nm.(ii) Model for Suspended Solids

[0103] Using an external analytical scale and a drying method, a model has been developed for suspended solids measurements as shown in graphs of Figure 6. The bioptic probe allows for real-time measurement of total suspended solids based on fluorescence data.(iii) Model for PHA concentration

[0104] Using an external gas chromatography-flame ionization detector (GC-FID), a model has been developed to determine the percentage of PHA in suspended solids as shown in graphs of Figure 7. The bioptic probe allows for real-time measurement of PHA production based on fluorescence data.

[0105] Examples ranges of excitation and detection wavelengths used for the models developed for optical density, suspended solids, and PHA content in suspended solids (SS) are summarized in the table below.

[0106] The probe can continuously emit excitation light at various excitation wavelengths and detect fluorescence signals at different wavelengths throughout the fermentation. This generates a dataset of excitation-emission signal pairs, which can be stored in a database and analyzed using software to establish correlations with multiple physico-chemical parameters. The software can detect relationships between different excitation-emission signal pairs and fermentation parameters. Once calibrated, the probe can detect and quantify various components by analyzing variations in within one or among multiple excitation-emission signal pairs.

[0107] In some instances, the target molecule itself does not necessarily need to be fluorescent. Instead, the probe could detect fluorescence changes in one or multiple molecules that are produced (or degraded) during the production of a biomolecule that emit a correlating signal that can be detected by the probe. The software may perform a principal component analysis to determine whether the variation of a given molecule is related to one, two, three or ten excitation-emission signal pairs. The software may further employ a mathematical model to calculate a quantitative relationship between this variation and fluorescence signal variations.

[0108] Once calibrated for a specific process, the bioptic probe can replace multiple probes to simultaneously correlate at least one excitation-emission signal pair to various fermentation parameters, such as pH, DO, organic acids, carbon content, FFA content, glycerol content, PHA concentration, biomass growth, cell mass, and the like.

[0109] In some implementations, the real-time monitoring can further include determination of the carbon content and / or the nutrient content based on the probe measurement. Such determination is performed by correlating carbon consumption and / or nutrient consumption of the PHA-producing microorganism to monitored PHA production.EXPERIMENTAL RESULTS

[0110] Experimental bacterial growth and PHA production was performed and realtime monitoring of the culture medium was performed using a wavelength having an excitation range from 360 nm to 460 nm using an in situ bioptic probe.General method

[0111] Different FFA-containing and micronutrient-containing substrates were used as a source of carbon and micronutrients for PHA production respectively. Characterization was performed for each source to analyze the carbon, micronutrients, macronutrients, and other compounds. After characterization, shake flask studies were conducted to optimize the carbon content of the FFA-containing substrates with micronutrients containing substrates, and achieve a predetermined optimized total carbon content.

[0112] Then, PHA production was tested in the shake flasks by using pure culture (one type of bacteria) under optimum conditions of temperature 30 °C (range 25-35 degrees), agitation 200 rpm (range 150-700) and pH 6.8 (range 6.4-7.2). The samples were collected and analyzed for colony forming unit (CFU), biomass concentration g / L, biomass PHA content % (w / w), carbon concentration and macronutrients concentration.

[0113] The shake flask studies were further validated using 5 L or 7 L and later 50L and 150 L size fermenters.Preparation of the micronutrient-containing substrate, e.g., when using activated sludge such as PPM AS

[0114] The activated sludge (AS) was concentrated by gravity settling for 8h and supernatant was separated. Before further processing, the sludge was filtered through screens (500 mm mesh size) in order to remove the large particles. The sludge was washed by centrifugation at 8000 rpm for 10 min. The supernatant was discarded, and the sludge solids were re-suspended using tap water and centrifuged again. The supernatant was discarded, and SS concentration was measured only after washing. The obtained suspended solids (washed) were re-suspended in tap water to obtain an optimized 15 g / L of concentration of sludge solids. The suspended solids (SS) concentration was measured after washing only and not before washing.Strain maintenance and Inoculum development

[0115] Cupriavidus necator 11599 was used for the testing. It was sub-cultured and streaked on mineral media agar (Marope et al., 2015) plates. Mineral media (Modified palleroni and doudoroff mineral base medium) consists of (g / L): 6.00 Na2HPO4.12H2O, 2.4 KH2PO4, 1.00 NH4CI, 0.50 MgSO4'7H2O, 0.01 FeCI3.6H2O, 0.01 CaCI2.6H2O, 20 Agar. Glucose (20 g / L) was supplemented to the mineral media. The agar was sterilized with glucose. The solution of FeCh.QFW and CaCh.eFLO was filter sterilized through 0.2 pm Polyethersulfone filter (Abidin et al., 2013). The FeCh.QFLO and CaCh.eFLO were filter sterilized for pre-culture1 as these two trace elements are unstable in nature and get denatured when subjected to high temperature; therefore, steam sterilization was not done for FeCh.QFLO and CaCl2.6H2O. These two components were used while preparation of plates and pre-culture 1 (synthetic media). Otherwise, for pre-culture 2 and production media (with sludge) the FeCh.QFLO and CaCh.eFLO were not added. Na2HPO4.12H2O, KH2PO4, NH4CI were autoclaved together and MgSO4.7H2O was autoclaved separately at 121°C for 15 min and these solutions were mixed aseptically after cooling. Medium pH was maintained at 6.8. The culture was revived in every 15 days and glycerol stocks were prepared in every 6 months after careful analysis of characteristics (cell growth pattern, physical parameters such as shape, size on agar plate, microscopic analysis) of the culture. The culture purity was examined carefully under microscope and morphology of the bacterial cells when plated on agar plates.Culture conditions or Inoculum developmentPre-culture 1

[0116] The dormant pure culture of C. necator stored in the refrigerator (4 °C) was revived by cultivating in a 100 mL pre-culture 1 (or PC-1) synthetic media. A loopful of Cupriavidus necator from plates (MMA) was used to inoculate in Erlenmeyer flasks containing sterilized medium. The medium for Pre-culture-1 was: (g / L):6.00 Na2HPO4.12H2O, 2.4 KH2PO4, 1.00 NH4CI, 0.50 MgSO4'7H2O, 0.01 FeCI3.6H2O, 0.01 CaCl2.6H2O. Glucose of 20 g / L was supplemented to the mineral media. The solution of FeCI3.6H2O and CaCl2.6H2O was filter sterilized through 0.2 pm Polyethersulfone (PES) filter. Glucose, NH4CI and MgSO4'7H2O were autoclaved separately at 121 °C for 15min. and these solutions were mixed aseptically after cooling. The flasks were incubated in a rotary shaker at 200 revolutions per min (rpm) at 30 °C for 24 h.Pre-culture 2

[0117] The treated micronutrient containing substrate, FFA-containing substrate and macronutrients are sterilized 121 °C for 30 min.

[0118] After cooling, these solutions were mixed aseptically. More particularly, the macronutrient-containing substrate and the FFA-containing substrate are added in the micronutrient-containing substrate flask under aseptic conditions.

[0119] Actively growing cells from pre-culture-1 were used as inoculum for the preculture-2. The medium pH was adjusted (6.8) and pre-culture 1 is used to inoculate the pre-culture 2. After inoculation, the flasks were incubated in a rotary shaker at 200 revolutions per min (rpm) at 30 °C for 24 h.Production media preparation (in-case of PPM AS)

[0120] Sludge suspended solids (SS) (15 g / L) was added as the micronutrientcontaining substrate to the fermenter followed by addition of calcium hydroxide (0.11 g / g of solids) to form a sludge medium which was then autoclaved for 30 min at 121 °C for 15 psi. After the sludge medium was autoclaved, the macronutrient-containing substrate having an optimum concentration of macronutrients (6.00 g / L Na2HPC>4.12H2O, 2.4 g / L KH2PO4, 0.5 g / L Urea and 0.50 MgSO4'7H2O, and the FFA-containing substrate were added as a carbon source (sterilized in the same way as discussed for pre-culture 1 and 2) to the sludge medium (autoclaved 15g / L sludge with 0.11 g / g calcium hydroxide) to form the production media. The pH was adjusted at 6.80 using 4N H2SO4 or 4N NaOH at 30 °C.Operational conditions

[0121] The inoculum was added to the production media after calibration to start PHA production, and PHA production and biomass growth were supported by batch, fed- batch or continuous fermentation. During fermentation, the FFA-containing substrate was intermittently added depending upon the monitored carbon consumption, or was continuously added to maintain the carbon content at an optimized carbon content orwithin an optimized carbon content range. In the same way, the macronutrient-containing substrate was added with the optimized concentration of macronutrients at TO (for a batch fermentation) and from T24 (for continuous addition in continuous fermentation).

[0122] The PHA was produced using fed-batch fermentation mode from TO to T12 with continuous feeding strategies from T12 to T84. For the continuous feeding, the FFA- containing substrate was fed at the rate of 0.06 * Vf and nutrimix feed fed at 0.20 Vf over 72h, where Vf is a final working volume used for production of PHA.

[0123] The continuous monitoring and real-time data are collected using sensors, probes, bio-optic probes, and sample analysis for any troubleshooting required.

[0124] For example, the fermenter was equipped with accessories and programmable logic control (PLC) system for dissolved oxygen (DO), pH, anti-foam, impeller speed, aeration rate and temperature. The software allowed automatic set-point control and integration of all parameters via PLC. Before each sterilization cycle, the polarographic pH-electrode (Mettler Toledo, USA) was calibrated using buffers of pH 4 and 7 (VWR, Canada). The oxygen probe was calibrated to zero with 5 % Na2SOs solution and 100 % with air-saturated water. After sterilization, DO probe was recalibrated to zero by sparging N2 gas and 100% saturation by sparging air at an agitation rate of 500 rpm. During fermentation, DO was maintained between 10% and 40 % saturation by adjusting agitation rate (300-700 rpm) and air flow rate (1.3-2.5 L / min). The temperature was maintained at 30 °C by circulating water through the jacket. Real-time monitoring of the carbon content and the macronutrient content was performed using a wavelength having an excitation range from 360 nm to 460 nm via an in situ bioptic probe.Results 1

[0125] The culture medium was prepared and fermented as per the above method and process steps, and real-time monitoring included using a wavelength having an excitation range from 360 nm to 460 nm using an in situ bioptic probe. It was shown that a particularly good correlation can be found between signals measured by the probe and PHA production. This seems especially true for excitation at 420 nm and 450 nm (detection ~ 510nm). Referring to Figures 1 and 2 providing fluorometric data of the culture medium during fermentation using a mixture of free fatty acids (FFA-containing substrate) as an additional carbon source, one can see that the produced PHA is wellcorrelated to fluorescence emissions at a wavelength of 510 nm when the culture medium is excited at an excitation wavelength between 420 nm and 450 nm.

[0126] Referring to Figures 3 and 4, it was shown that excitation of the culture medium at an excitation wavelength of 365 nm can also be relevant to the monitoring because it is modulated differently in two different fermentations (BOSK-01 and BOSK-02) using different types of additional carbon sources. BOSK-01 culture medium sample results from fermentation using waste cooking oil as the FFA-containing substrate and BOSK-02 culture medium sample resulted from fermentation using a waste substrate obtained from a biodiesel refinery as the FFA-containing substrate. For BOSK-01 fermentation, response to excitation at 365 nm is high, lower and rise again, while in BOSK-02, response to excitation at 365 nm is low at the beginning and increase after. Knowing the culture media was not the same in those two batches, and detection trend shows the concentration of PHA inside production media. The response to the excitation at 365 nm trends also provides real-time consumption profile of the carbon source by the bacteria which means if PHA producing bacteria is unable to metabolize an additional carbon source or there is substrate inhibition inside the system then it starts consuming its own produced PHA. Hence, the Figures 3 and 4 signifies the drop in PHA concentration at certain time points which are also co-related with the concentration of available carbon source in the production media.Resu / ts 2Based on below Table 5, the main difference between the FFA-containing and non FFA- containing substrates (non-fat substrate) is the process efficiency and feasibility to produce high microbial biomass, PHA content and PHA yield. The free fatty acids present in the FFA-containing substrate, such as waste cooking oil or waste stream from biodiesel refinery, lead to the accumulation of more PHA inside cells and almost negligible quantity of non-PHA cellular mass. The enhanced PHA yield while using FFA- containing substrates is attributed to high organic carbon present in FFA-containing substrates and the micronutrients present in nutrient rich substrates obtained from different industries such as wastewater of pulp and paper mill, cheese industries, sugar and molasses industries, starch, lignocellulose, other food industries, agro-industries and activated sludge of industries such as pulp and dapper mill activated sludge.Table 5

[0127] It is worth mentioning that throughout the following description when the article “a” is used to introduce an element it does not have the meaning of “only one” it rather means of “one or more”. It is to be understood that where the specification states that a component, feature, or characteristic “may”, “might”, “can” or “could” be included, that particular component, feature, or characteristic is not required to be included.

[0128] In the following description, the term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. It is commonly accepted that a 10% precision measure is acceptable and encompasses the term “about”.

[0129] In the above description, an embodiment is an example or implementation of the inventions. The various appearances of “one embodiment,” “an embodiment” or “some embodiments” do not necessarily all refer to the same embodiments. Although various features of the invention may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the invention may be described herein in the contextof separate embodiments for clarity, the invention may also be implemented in a single embodiment.

Claims

CLAIMS1 . A process for producing PHA comprising: providing a culture medium comprising a substrate mixture and PHA- producing microorganisms; subjecting the culture medium to fermentation conditions to sustain biomass growth and to trigger production of PHA by the PHA-producing microorganisms; and recovering PHA from the culture medium; wherein the substrate mixture comprises an FFA-containing substrate and a nutrient-containing substrate; and wherein the FFA-containing substrate has a carbon content accounting for between about 50 wt% to 100 wt% of a total carbon content of the culture medium.

2. The process of claim 1, wherein the FFA-containing substrate has a weight ratio of unsaturated fat to saturated fat between 3 and 10.

3. The process of claim 1 or 2, wherein the FFA-containing substrate has a weight ratio of unsaturated FFA to saturated FFA between 20 and 200.

4. The process of any one of claims 1 to 3, wherein the FFA-containing substrate has a saturated fat content between 10 wt% and 20 wt% with respect to a total weight of the FFA-containing substrate.

5. The process of any one of claims 1 to 4, wherein the FFA-containing substrate has a trans fat content of at most 0.05 wt% with respect to the total weight of the FFA-containing substrate.

6. The process of any one of claims 1 to 5, wherein the FFA-containing substrate has at least one of: a C16:0 saturated FFA content of at most 50 g / kg of FFA-containing substrate;a C16:1 unsaturated FFA content between 5 g / kg and 200 g / kg of FFA-containing substrate; a C18:3 unsaturated FFA content between 150 g / kg and 400 g / kg of FFA- containing substrate; and a C18:0 saturated FFA content of at most 20 g / kg of FFA-containing substrate.

7. The process of any one of claims 1 to 6, wherein the FFA-containing substrate has a viscosity of between 40 and 60 CP as measured with ASTM D445 standard at 40°C.

8. The process of any one of claims 1 to 7, wherein the nutrient-containing substrate comprises at least one of a micronutrient-containing substrate and a macronutrient-containing substrate.

9. The process of claim 8, wherein the micronutrient-containing substrate provides between 30 wt% and 100 wt% of a total micronutrient content of the culture medium.

10. The process of any one of claims 1 to 9, wherein providing the culture medium comprises: providing the substrate mixture as at least part of a production media; and inoculating the production media with an inoculum comprising the PHA- producing microorganisms to the produce the culture medium.

11. The process of any one of claims 1 to 10, further comprising pre-treating the substrate mixture prior to being provided as part of the culture medium to prevent contamination of the microorganisms.

12. The process of any one of claims 1 to 11 , comprising measuring and adjusting a pH of the culture medium prior to or during fermentation to maintain the pH between 6 and 8.

13. The process of any one of claims 1 to 12, further comprising:monitoring at least one of a carbon content, a nutrient content, a biomass growth, and a cell mass of the culture medium during fermentation; and adding at least one of the FFA-containing substrate and the nutrientcontaining substrate in accordance with monitored information.

14. The process of claim 13, wherein the at least one of the FFA-containing substrate and the nutrient-containing substrate is continuously added in an adjusted amount in accordance with the monitored information to maintain PHA concentration at at least about 60 wt% of the total weight of suspended solids in the culture medium.

15. The process of claim 13 or 14, wherein the monitoring is performed in real time in the culture medium during fermentation.

16. The process of any one of claims 13 to 15, wherein the monitoring comprises: measuring at least one fermentation parameter of the culture medium during fermentation, the at least one fermentation parameter comprising an optical density, total suspended solids, PHA concentration or any combinations thereof; and based on the at least one fermentation parameter, determining at least one of the carbon content, the nutrient content, the biomass growth, and the cell mass of the culture medium.

17. The process of claim 16, wherein measuring the at least one fermentation parameter comprises probing the culture medium for in situ spectroscopic measurement to collect a fluorescent response of the culture medium that is correlated to the at least one fermentation parameter.

18. The process of any one of claims 1 to 17, comprising adding the FFA-containing substrate to the culture medium during fermentation to maintain a total carbon content in the culture medium at the optimal total carbon content of the culture medium.

19. The process of claim 18, wherein the optimal total carbon content of the culture medium is between 3 g / L and 12 g / L of the culture medium.

20. Use of an FFA-containing substrate for the production of PHA as part of a culture medium, the FFA-containing substrate having at least one feature being defined in any one of claims 1 to 7.

21. The use of claim 20, wherein the FFA-containing substrate is used as a production media prior to fermentation or as a continuous feed during fermentation.

22. The use of claim 20 or 21 , wherein the substrate mixture is used to provide between 50 wt% and 100 wt% of the total carbon content consumed by the PHA- producing microorganisms to yield at least 60 wt% of PHA with respect to a total weight of the suspended solids.

23. A method for optical monitoring and analysis of a PHA production process in a culture medium comprising microorganisms, the method comprising: exciting the culture medium with excitation light; detecting fluorescent light emitted by the culture medium in response to the excitation light; and analyzing the detected fluorescent light using a response model relating fluorescent spectrum data with fermentation parameter data to derive at least one fermentation parameter of the culture medium; wherein the at least one fermentation parameter comprises at least one of an optical density parameter, a suspended solids parameter, or a PHA concentration parameter.

24. The method of claim 23, further comprising controlling the PHA production process based on the analysis to adjust or maintain the at least one fermentation parameter.

25. The method of claim 24, wherein controlling the PHA production process comprises adding at least one of a nutrient source or a carbon source to the culture medium based on the at least one fermentation parameter.

26. The method of any one of claims 23 to 25, wherein the excitation light lies within a wavelength range of 280 nm to 490 nm.

27. The method of any one of claims 23 to 26, wherein the fluorescent light is detected within a wavelength range of 250 nm to 595 nm.

28. The method of any one of claims 23 to 27, wherein the at least one fermentation parameter comprises the optical density parameter, and the method further comprises determining cell growth information from the optical density parameter.

29. The method of claim 28, wherein the optical density parameter is determined from fluorescent light detected within a wavelength range of 360 nm to 595 nm in response to excitation light within a wavelength range of 340 nm to 490 nm.

30. The method of any one of claims 23 to 29, wherein the at least one fermentation parameter comprises the suspended solids parameter, and the method further comprises determining cell mass information from the suspended solids parameter.

31. The method of claim 30, wherein the suspended solids parameter is determined from fluorescent light detected within a wavelength range of 305 nm to 595 nm in response to excitation light within a wavelength range of 280 nm to 490 nm.

32. The method of any one of claims 23 to 31 , wherein the at least one fermentation parameter comprises the PHA concentration parameter, the method further comprising determining nutrient and / or carbon content information about the culture medium from the PHA concentration parameter.

33. The method of claim 32, wherein the PHA concentration parameter is determined from fluorescent light detected within a wavelength range of 480 nm to 595 nm in response to excitation light within a wavelength range of 450 nm to 490 nm.

34. The method of any one of claims 23 to 33, wherein the response model is a partial least squares model.

35. The method of any one of claims 23 to 34, wherein excitation and detection are performed using a light source and a spectral detector contained within a single probe.

36. A PHA production process in a culture medium comprising PHA-producing microorganisms comprising monitoring in real time at least one fermentation parameter comprising at least one of an optical density parameter, a suspended solids parameter or a PHA concentration parameter, wherein the monitoring comprises using the method as defined in any one of claims 23 to 35.

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