Processes for producing polyhydroxyalkanoates (PHAs) and triacylglycerides (TAGs)

Abstract

A single-unit biotechnological method for the simultaneous recovery of polyhydroxyalkanoates (PHAs) and triacylglycerides (TAGs) is disclosed. The present invention relates to a method for producing biomass in the form of a mixed microbial culture containing PHAs and TAGs. More specifically, the method comprises certain steps that take place in a single discontinuous sequential reactor.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a biotechnological process for producing biomass containing intracellular PHAs and TAGs. More specifically, the process comprises specific steps carried out in a single sequencing batch reactor. [Background technology]

[0002] Only certain microorganisms can synthesize and intracellularly accumulate polyhydroxyalkanoates (PHAs) and triacylglycerides (TAGs) under stress conditions (Carsanba et al., Crit. Rev. Biotechnol. 2018, 38, 1230-1243). Therefore, a mixed microbial culture (MMC) incorporating microorganisms capable of intracellularly accumulating PHA and microorganisms capable of intracellularly accumulating TAG has been developed. When working with microbial cultures, an enrichment step must be carried out to select for the populations with the greatest ability to accumulate these compounds.

[0003] For this purpose, sequencing batch reactors (SBRs) are usually used. There are various strategies for implementing the selection process, but the most common is the so-called aerobic dynamic feeding (ADF) strategy. This is based on applying feast / starvation cycles under aerobic conditions, in which the culture is exposed to a short-term excess (feast) and a long-term shortage (starvation) of a carbon source (Kourmentza et al., Bioengineering, 2017, 4, 55). In this regard, microorganisms capable of accumulating substrate during the feast phase have a competitive advantage over other microorganisms, since they can use intracellular carbon as a growth and energy source during the starvation phase. On the other hand, non-accumulating microorganisms spend long periods without substrate and, after applying successive feast / starvation cycles, are unable to self-replicate and drop out of the system (Valentino et al., N. Biotechnol. 2017, 37, 9-23).

[0004] Over the past decade, various variations on the ADF strategy have been proposed to optimize culture enrichment. Among these, uncoupling feeding from carbon and nitrogen sources stands out, known as the double growth limitation (DGL) strategy. Adding excess carbon during the nitrogen-limited feast phase prevents the growth of non-accumulating populations. Only microorganisms capable of metabolizing the substrate and accumulating it intracellularly can consume the carbon source. Therefore, adding nitrogen at the end of the feast phase (in the absence of carbon, since carbon was consumed during the feast) allows populations that accumulated PHA and / or TAG during the feast phase to utilize the nitrogen for growth, resulting in faster and more efficient MMC selection (Kourmentza et al., Bioengineering, 2017, 4, 55).

[0005] The biomass enriched in the SBR is then used as inoculum in the second unit, a fed-batch reactor (FBR)-type accumulation reactor in which the substrate is available in excess to maximize the amount of intracellular compounds accumulated before extraction and purification. This involves exposing the biomass to a prolonged feeding period under aerobic conditions and in the absence of nitrogen, as long as the substrate allows, to prevent the growth of non-accumulating populations and force the conversion of the substrate to PHAs and / or TAGs. The preferred feeding regime in this type of system is a pulse-feeding strategy, as it avoids the effects of substrate inhibition and allows for high accumulation efficiency. This consists of adding successive pulses of carbon after the previous carbon is consumed until maximum accumulation capacity is reached (Kourmentza, 2014). et al.,,Bioengineering, 2017, 4, 55;Mannina et al. al.,,Bioresour.Technol.2020, 297, 122478). The most common way to know when to add substrate is to monitor the dissolved oxygen (DO) concentration in the system. After the addition of a carbon pulse, DO decreases, indicating that the substrate is being consumed, and returns to saturation values ​​when it is depleted. At this point, a new pulse is added until a decrease in DO concentration is observed after the addition of the carbon source, indicating that the maximum accumulation capacity of MMC has been reached.

[0006] In addition to these two reactors, if a stream containing organic compounds that are difficult to metabolize, such as lipids, is used, a third reactor is used to pretreat the substrate to obtain easily metabolized soluble organic acids (usually volatile fatty acids (VFAs), the most commonly used precursors for the synthesis of PHAs). This is an acid fermentation process carried out in a Continuous Stirred Tank Reactor (Mannina et al. , Bioresour. Technol. 2020, 297, 122478).

[0007] To date, obtaining high-value intracellular compounds from lipids has relied mainly on the use of pure microbial cultures and vegetable oils as substrates (Basnett et al. al.Sci. Mater.Med.2018, 29.; Donot et al., Biomass and Bioenergy 2014, 68, 135-150; Perez-Arauz et al., Food Packag. Shelf Life 2019, 20, 100297). However, these processes are not competitive on a large scale due to their high costs. Therefore, in recent years, research has been carried out aiming to develop processes based on the use of MMC, which does not require aseptic conditions and can use waste streams as substrates (De Donno Novelli et al., Bioresources. Technol. 2021, 331, 124985).

[0008] So far, existing research on the production of PHAs and / or TAGs using mixed cultures and from waste streams is very scarce.

[0009] On the other hand, Campanari et al. (Int. J. Biol. Macromol. (2014, 71, 34-41) demonstrated the feasibility of producing PHAs from wastewater generated in olive oil production using a three-step system consisting of pretreatment, concentration, and accumulation in multiple reactors in series. The pretreatment step consisted of two steps: 1) solid-phase extraction for the removal and recovery of polyphenols, and 2) anaerobic acidification of the dephenolated wastewater to convert the remaining organic matter into volatile fatty acids (VFAs), which are the only precursors for PHA synthesis. MMCs were concentrated in an SBR-type reactor fed with pretreated wastewater rich in VFAs. The concentrated biomass in the SBR was then used as inoculum for a FBR-type accumulation reactor, which was also fed with the pretreated waste stream.

[0010] Furthermore, Tamis et al. (Biotechnol. Biofuels 2015, 8, 1-11) demonstrated the feasibility of recovering TAGs from wastewater using MMCs fed with vegetable oil-water emulsions as a model substrate. To do this, they investigated a system consisting of two consecutive steps using two reactors: concentration and accumulation were carried out in an SBR and an FBR-type reactor, respectively. In the first step, MMCs with the ability to accumulate TAGs were selected. Subsequently, the FBR reactor was inoculated with the biomass concentrated in this first step to maximize the amount of TAG accumulated.

[0011] Finally, the present inventors have been able to produce primarily PHAs, or primarily TAGs, from fatty wastewater generated in the canning industry using a process comprising two reactors: an SBR-type concentration unit and another FBR-type accumulation unit (Argiz et al., Sci. Total Environ. 2021, 763, 142944).

[0012] Therefore, there remains a need to achieve a simpler and cheaper method that allows obtaining TAGs, PHAs, or both simultaneously, avoiding the pretreatment of readily available streams, preferably waste lipid streams. Summary of the Invention

[0013] Brief description of the invention The inventors of the present invention have developed a single-unit biotechnological process for the recovery of polyhydroxyalkanoates (PHAs) and triacylglycerides (TAGs) from lipid streams using biomass in the form of mixed microbial cultures (MCCs). In particular, the inventors have designed a method advantageous for the development of MMCs that can simultaneously accumulate PHAs and TAGs intracellularly in a simple process that is easy to implement in industry.

[0014] A further advantage is that the method is carried out in a single reactor, and the process installation for its operation requires a smaller footprint than that required for currently used processes requiring three reactors in series.

[0015] Another advantage of the present invention is that it avoids the need for prior pre-treatment of the biomass source (substrate), thereby simplifying the process and reducing start-up and run time.

[0016] Another advantage is that when implemented in a single unit, even low intracellular accumulation rates can result in higher daily productivity (expressed as g PHA and TAG produced per day and per liter of reactor). This is because, in the present invention, by performing a volume exchange within the system at the end of the feasting period, the resulting stream already contains biomass containing accumulated biopolymers, which can be extracted and purified. However, conventional processes require the operation of a second reactor, which increases processing time and reduces productivity.

[0017] Another advantage is that reducing the number of reaction units reduces investment costs. Operating costs are also expected to be lower, for example, because costs associated with energy consumption in pretreatment and accumulation reactors (agitation, aeration, pumping, etc.) are avoided.

[0018] From a conceptual point of view, it is a simpler process than traditional processes, which minimizes errors and makes it easier to train non-specialist staff to operate it.

[0019] Compared to existing mixed culture systems, the present invention is unique in that it allows: 1) using lipid streams as substrates without the need for pretreatment to obtain VFAs in separate units; 2) performing the traditional pretreatment steps (in this case only hydrolysis), concentration, and accumulation in the same unit; and 3) obtaining two high-value intracellular compounds, PHA and / or TAG, from the same carbon source.

[0020] Thus, one aspect of the present invention relates to a process for the intracellular production of polyhydroxyalkanoates (PHAs) and triacylglycerides (TAGs) in a mixed microbial culture, characterized in that all steps are carried out in a single sequencing batch reactor, and further comprising the steps of: a) inoculating a reactor with activated sludge; b) Steps of the reactor operation cycle including: i. feeding the microorganisms present in the reactor with a lipid carbon source, the feeding being pulsed under nitrogen-limited conditions; ii. removing 50% of the mixture from the reactor, wherein the mixture comprises biomass having intracellular PHAs and TAGs; iii. Adding a stream containing nitrogen at 0.9 to 2 times the stoichiometric amount of nitrogen required for microbial growth; c) Repeating the process from step b) in successive cycles. [Brief explanation of the drawings]

[0021] [Figure 1] Figure 1 shows a simplified diagram of a single-unit process operated at laboratory scale to recover PHAs and TAGs from a lipid stream. It includes: (1) a sequencing batch reactor (SBR), (2) a lipid stream (carbon source) feed pump, (3) a discharge pump, (4) a water stream (nitrogen source) feed pump, (5) a blower, (6) a thermostatic bath, (7) oxygen and temperature probes, (8) a programmable logic controller (PLC), (9) a reservoir for the lipid substrate used as a carbon source (see Table 1 for composition), (10) a reservoir for the aqueous mixture containing the nitrogen source (see Table 2 for composition), and (11) a reservoir for the reactor effluent containing the biomass with accumulated intracellular compounds (PHAs and TAGs). [Figure 2(a)] FIG. 2(a) compares the operating cycle configuration of the SBR of the present invention (a) with the concentration and subsequent accumulation steps of a conventional SBR (b). [Figure 2(b)] FIG. 2(b) compares the operating cycle configuration of the SBR of the present invention (a) with the concentration and subsequent accumulation step of a conventional SBR (b). [Figure 3] Figure 3 shows a simplified diagram of the various configurations of the operating cycle evaluated in the SBR reactor. C indicates the time point at which a pulse of lipid substrate used as a carbon source is added, and N indicates the time point at which a water stream containing a nitrogen source is added. [Figure 4]Figure 4 shows the complete characteristics of three operating cycles of the SBR reactor: (a) a 12-hour cycle corresponding to day 43 of operation, (b) an 18-hour cycle corresponding to day 72 of operation, and (c) a 24-hour cycle corresponding to day 133 of operation. The plots show pH (filled circles), X concentration (line only), TN concentration (filled diamonds), % accumulated biopolymer (Biop) (open circles), and C concentration (open squares). Here, Biop refers to the sum of PHA and TAG accumulated within the cells, expressed as a percentage of the dry cell weight (% dw). C refers to the cumulative amount of substrate added in the pulse, expressed in grams of chemical oxygen demand (COD). TN refers to the total nitrogen concentration in g / L. X refers to the concentration of active biomass present in the reaction medium, in C mmol / L. DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description of the Invention In the present invention, "activated sludge" is understood to mean a flocculent mixture of microorganisms grown in an aeration tank under controlled conditions, which is used in biological wastewater treatment processes for the degradation of organic compounds.

[0023] In the context of the present invention, a "mixed microbial culture (MMC)" is understood to be one that is found in non-sterile conditions and consists of a mixture of species belonging to different microbial groups (bacteria, fungi, yeasts).

[0024] In the present invention, the term "biomass" refers to the collection of microorganisms (bacteria, fungi, and yeasts) present in the MMC that were initially present in the activated sludge used as inoculum and subsequently enriched into a population capable of hydrolyzing the lipidic carbon source used as a substrate and accumulating PHAs and TAGs.

[0025] In the present invention, the term "oleaginous microorganisms or oleaginous populations" refers to microorganisms or populations of microorganisms that can accumulate 20% or more of their dry weight in triacylglycerides. Among the various classes of oleaginous microorganisms (microalgae, bacteria, yeast, and fungi), only certain species, particularly yeast, can survive in hydrophobic environments. This means that they have the ability to hydrolyze and metabolize lipidic carbon sources for intracellular accumulation. Some examples include the oleaginous yeasts Yarrowia, Cryptococcus, Rhodosporidium, Geotrichum, and Trichosporon (Patel and Matsakas, Ultrason. Sonochem. 2019, 52, 364-374).

[0026] In the present invention, "microbial activity" refers to the ability of heterotrophic microorganisms to decompose dissolved organic matter and simultaneously form microbial biomass.

[0027] In the present invention, "substrate" is understood as the waste lipid stream containing the carbon source that MMC uses to grow and accumulate PHA and TAG.

[0028] In the present invention, "lipidic carbon source" is understood as a stream that is used by microorganisms as a carbon source for growth and has a lipid content of 85% or more, in particular 85-90%, of COD.

[0029] In the present invention, "long chain fatty acids" are understood to be those made up of a chain of 14 or more carbon atoms, such as oleic acid, linoleic acid, palmitic acid, stearic acid, etc. Preferably, they have 14 to 32 carbon atoms, more preferably 14 to 26 carbon atoms.

[0030] In the present invention, "hydrolysis" is understood as the process by which the lipid components of the substrate are solubilized in the aqueous phase and become available for transformation by the microorganism.

[0031] In the present invention, "enrichment" or MMC selection is understood as a process in which specific operating conditions (called selection pressure) are imposed to promote the growth of specific microorganisms initially present in the inoculum (activated sludge) in order to maximize the presence of microorganisms in the enriched MMC. The intention is to promote the survival of populations capable of performing specific functions in place of specific microorganisms. The present invention promotes the growth of microorganisms capable of: 1) hydrolyzing lipid streams used as a carbon source; and 2) metabolizing the compounds resulting from the hydrolysis (long-chain fatty acids and glycerol) and accumulating them intracellularly as PHAs and / or TAGs.

[0032] In the present invention, "accumulation" is understood as a process in which the synthesis of PHA and TAG is promoted within the cells of microorganisms present in concentrated MMC, and the amounts thereof increase.

[0033] "Limiting nitrogen conditions" is understood to mean conditions in which nitrogen is present in the reaction broth in a minimal amount, in other words in an amount less than 20 mg total nitrogen (TN) / L, so that microbial growth is limited.

[0034] The "stoichiometric amount of nitrogen necessary for microbial growth" is determined by the elemental composition (CH 1.8 O 0.5 N 0.2 ) is understood as the number of moles of nitrogen required to produce 1 mole of biomass (Popovic, Heliyon, 2019, 5, 6). Therefore, stoichiometrically, 0.2 moles of N are required per mole of biomass present in the reaction medium.

[0035] In the present invention, the term "feast" refers to the period of reactor operation time during which MMC has excess extracellular carbon available in the reaction medium and under nitrogen-limited conditions.

[0036] In the present invention, "famine" is understood as a period during which the MMC does not have excess extracellular carbon available in the reaction medium, but does have nitrogen.

[0037] In the process of the present invention, the hydrolysis, concentration, and accumulation steps are carried out in one unit, in other words, one reactor, whereas conventional processes use three reactors in series (CSTR+SBR+FBR).

[0038] In a preferred embodiment, the process is carried out in a single sequential reactor of the sequencing batch reactor (SBR) type, operated in batch mode.

[0039] The process of the present invention comprises a first step a) characterized by inoculating activated sludge in a sequencing batch reactor.

[0040] In a preferred embodiment, the sludge of the present invention comprises a microbial population capable of performing the following functions:

[0041] 1) The fats present in the lipid stream used as a carbon source are hydrolyzed to long chain fatty acids and glycerol.

[0042] 2) Accumulating compounds resulting from the hydrolysis process, such as PHA and TAG, intracellularly.

[0043] In a more preferred embodiment, the activated sludge of the present invention comprises accumulating microorganisms, such as Acidovorax, Acmetobacter, Pandoraea, Azospirillum, and Pseudomonas, and such TAG accumulating microorganisms as Candida, Yarrowia, Geotrichum, Mortierella, and Rhodoccocus.

[0044] In another preferred embodiment, the activated sludge of the present invention comprises microorganisms capable of hydrolyzing lipids. Generally, these microorganisms are oleaginous populations of fungi and yeasts, although some bacterial populations with this function can also be found. These microorganisms with the ability to hydrolyze lipids can be, for example, Candida, Yarrowia, Fonsecaea, Capronia, Acinetobacter, Cryptococcus, Rhodosporidium, Geotrichum, and Trichosporon.

[0045] In a further preferred embodiment, the activated sludge of the present invention comprises PHA and TAG accumulating microorganisms and microorganisms capable of hydrolyzing lipids.

[0046] The activated sludge used in the present invention can have different origins: in a preferred embodiment, the sludge used in the present invention is sludge from a municipal wastewater treatment plant (WWTP), and in a more preferred embodiment, sludge from the aeration tank of the secondary treatment of a WWTP.

[0047] Another advantage of the present invention is that the culture is enriched in accumulating microorganisms and also enriched in microorganisms capable of hydrolyzing the lipid stream used as a carbon source. Thus, after adding a carbon source to the reaction medium, microorganisms capable of secreting extracellular lipases catalyze the hydrolysis, releasing long-chain fatty acids and glycerol (precursors in the synthesis of PHA and TAG).

[0048] In a preferred embodiment, the process of the present invention does not include a pretreatment or hydrolysis step for the lipidic carbon source. In this way, the lipid stream can be used directly without the need for an additional reactor for pretreatment. A prior hydrolysis step to obtain long-chain fatty acids and glycerol is not required. Furthermore, it is not necessary to convert this stream into a more easily metabolized carbon source, such as volatile fatty acids (obtained by acidifying the lipidic carbon source). This is possible through the enrichment performed in the step of selecting accumulating microorganisms and lipid-hydrolyzing microorganisms. Another advantage of the process of the present invention over conventional techniques is that the reactor cycle configuration is designed to enable the following in a single reactor: i) selecting a culture capable of hydrolyzing the substrate and accumulating PHA and TAG from activated sludge used as inoculum; ii) regenerating the culture present in the reaction liquid during each operating cycle; and iii) maximizing the amount of accumulated PHA and TAG. i) and ii) are conventionally performed in an SBR-type thickening reactor, and iii) in an FBR-type thickening reactor. FIG. 2 shows a comparison of the operating cycle configuration of the SBR designed in this invention (FIG. 2(a)) with the configuration of the thickening SBR and subsequent accumulation step of the conventional process (FIG. 2(b)).

[0049] The process of the present invention further comprises step b) characterized by an operating cycle of the reactor.

[0050] This step b) comprises (i) feeding a lipidic carbon source to the microorganisms present in the reactor, the feeding being pulsed and under nitrogen-limited conditions, i.e., the nitrogen content is less than 20 mg total nitrogen (TN) / L. Preferably, the nitrogen content is between 5 and 20 mg TN / L. More preferably, the nitrogen content is 5 mg TN / L or less.

[0051] Step (i) is a prolonged feast phase, in which a carbon source is added in pulses over an extended period under nitrogen-limited conditions. The intent of this feeding strategy is to maximize the amount of intracellular compounds accumulated at the end of the feast phase. This feast phase is designed to maximize the amount of intracellular compounds accumulated at the end of the feast phase, and the biomass removed includes the PHAs and TAGs produced in the system.

[0052] The feast stage design of the present invention avoids the need to operate a second FBR-type storage reactor.

[0053] In the present invention, the feast phase is designed to accumulate the maximum possible amount of biopolymer within the cells (accumulation), and the biomass obtained during the feast phase can be processed to obtain the biopolymer. In contrast, in conventional processes, the feast phase is designed simply to supply MMC and concentrate it in the microorganism of interest, and accumulation to obtain the maximum possible amount of biopolymer must be carried out in a separate reactor, usually of the FBR type, requiring more steps and a much longer process than the present invention.

[0054] Therefore, to obtain biomass containing intracellular biopolymers suitable for extraction and purification (30-40% or more intracellular dry weight (dw) of PHA and TAG), the present invention avoids the need to inoculate an FBR-type reactor with a culture pre-enriched in an SBR and subject the microorganisms to a prolonged starvation phase to maximize the amount of accumulated biopolymers. Unlike the starvation phase of a conventional SBR-type enrichment reactor, which requires only a certain amount of carbon to be accumulated after extracellular carbon is depleted (starvation phase), the present invention further maximizes the amount accumulated in the SBR, thereby avoiding the need to operate a second accumulation unit.

[0055] Thus, the first step (i) of this cycle simultaneously performs the pretreatment and accumulation steps, which are conventionally performed in two reactors, and this step also contributes to the selection of MMCs, since only certain microorganisms are able to accumulate PHA and TAG and thus grow and remain in the system during starvation periods using these intracellular compounds as a carbon source.

[0056] The reactor operating cycle of step b) of the process of the present invention further comprises step ii) in which 50% of the mixture is removed from the reactor, said mixture comprising biomass with intracellular PHA and TAG.

[0057] A further advantage of the present invention is that, as shown in Examples 1, 2 and 3, biomass can be obtained that accumulates PHAs and TAGs in amounts greater than 30% by intracellular dry weight (dw) of PHAs and TAGs, and can therefore be directly carried forward to the subsequent PHA and TAG extraction and purification steps.

[0058] In a preferred embodiment, this step ii) is carried out immediately after step i). This is a relevant difference with respect to conventional systems, where the biomass discharge occurs at the end of the SBR enrichment reactor's operating cycle, in other words, after the starvation period. Thus, after the starvation period, the microorganisms have no accumulated intracellular compounds, since they used the intracellular compounds as a carbon source for growth. The biomass removed from the reactor after the starvation period is brought into the FBR-type reactor and used as inoculum for the accumulation process, which aims to maximize the amount of accumulated intracellular compounds. This is one of the reasons why conventional processes require the use of multiple reactors.

[0059] The reactor operating cycle of step b) of the process of the present invention further comprises step iii) of adding a stream containing nitrogen in an amount from 0.9 to 2 times the stoichiometric amount required for microbial growth.

[0060] Therefore, in this step iii), the starvation phase begins, and the microorganisms are left without extracellular carbon in the liquid medium for a long period of time. Due to the lack of external carbon, only the microorganisms that accumulated PHA and TAG during the feast phase can grow using these compounds as a carbon source. Conversely, non-accumulating populations cannot replicate and are dropped out of the system. In this regard, by repeating several operating cycles, the system gradually and naturally enriches for accumulating populations, and MMC is enriched in this step.

[0061] The amount of nitrogen added at the beginning of the starvation phase should be at most the stoichiometric amount required for microbial growth. In a particular embodiment, the stream used in this step is an aqueous solution containing a nitrogen compound. In another particular embodiment, the nitrogen compound is selected from ammonium sulfate, ammonium nitrate, and ammonium chloride. In a more particular embodiment, the aqueous solution contains ammonium chloride at a concentration of 5 mg / L to 280 mg / L.

[0062] In a preferred embodiment, at the end of step (iii), the total nitrogen content in the reaction medium is less than 20 mg total nitrogen per liter (<20 mg TN / L), preferably less than 5 mg total nitrogen per liter (<5 mg TN / L). At this point, the next cycle, in which the feast phase begins by supplying a carbon source, begins when nitrogen levels are below 20 mg total nitrogen per liter (<20 mg total nitrogen per liter). The growth of the lipid stream is preferably carried out in the presence of less than 5 mg total nitrogen per liter (<5 mg TN / L), to favor accumulation and prevent the lipid stream from being used as a carbon source for growth. Best results were obtained when step (i) was carried out at a nitrogen concentration close to 5 mg TN / L, as shown in Example 1. Thus, preferably, the nitrogen concentration at the end of step (iii) is 5 mg TN / L or less, and step (i) is carried out at a nitrogen concentration close to 5 mg TN / L. This is done at a nitrogen concentration of TN / L or less.

[0063] The process of the present invention further comprises step c), which is the repetition of the process from step b) in successive cycles.

[0064] In certain embodiments, the process of the present invention comprises a variable length operating cycle with different operating periods of 12 hours, 18 hours, and 24 hours in length. In another particular embodiment, in step (i), the feast phase lasts from 6 hours to 12 hours. In another particular embodiment, in step (iii), the starvation phase lasts from 6 hours to 12 hours.

[0065] Depending on the operating conditions, the method of the present invention may yield biomass with different ratios of PHA and TAG, thus allowing enrichment of the culture of one microorganism or another.

[0066] In a specific embodiment, the process of the present invention enables intracellular production of PHA and TAG at a PHA:TAG ratio of 20:80 to 80:20. In this regard, the method of the present invention comprises enriching the culture with microorganisms capable of accumulating PHA, as well as microorganisms capable of hydrolyzing the substrate and accumulating TAG. Specifically, the method comprises feeding a lipid substrate in pulses (the feast phase) separated from the nitrogen source (added at the end of the feast and consumed during starvation). Excess carbon must be limited to ensure that the culture is partially enriched with PHA. To this end, in a preferred embodiment, during the feast phase, each feeding pulse contains a carbon content of 1,200 mg COD or less. In another more preferred embodiment, a pulse is added when 85% of the carbon from the previous pulse has been consumed or depleted, leaving 15% of the carbon remaining unconsumed, corresponding to the non-biodegradable fraction of the carbon source. Carbon consumption or depletion can be monitored by the amount of dissolved oxygen (DO). In another preferred embodiment, the carbon content at the start of the starvation phase is 15% or less. Furthermore, the consumption of added nitrogen for microbial growth during the starvation phase must be such that the next operating cycle begins under nitrogen-limited conditions, i.e., below 20 mg TN / L.

[0067] Specific embodiments illustrating the present invention are described below.

[0068] Methods and experimental design The process was carried out in an SBR-type reactor operated at laboratory scale. The reaction vessel (1, see Figure 1) was made of methacrylate, equipped with an external coil for temperature control, and had a working volume of 10 L (total volume 15 L). In addition, the system included: one fixed-flow peristaltic pump (2) to supply the lipid stream used as a carbon source, two variable-flow peristaltic pumps with corresponding controllers for volume exchange within the system (a pump (3) discharging 50% of the reaction volume and a feed pump (4) for the water stream filling the reactor, respectively), one blower (5) to supply air to the reaction medium, a thermostatic bath (6) for temperature control, and an oxygen probe (HQ40d HACH) for continuous monitoring of dissolved oxygen (DO) and temperature. Lange, USA) (7), a programmable logic controller (PLC) Siemens S7-1200 (8) for programming the operating cycle (activating the pump at the desired time), a 1 L container for the lipid source (9), a 20 L bag for the water flow (10), and a 30 L container for waste collection (11).

[0069] For its implementation, the reactor was inoculated at the beginning of the process with 10 L of activated sludge from the Calo-Milladoiro wastewater treatment plant (A Coruña). The initial concentration of these sludge was 3.63 ± 0.16 g of total suspended solids (TSS). The total sugar content (TSS) / L, volatile suspended solids (VSS) were 3.01±0.17 g TSS / L, and the pH was 7.5.

[0070] The reactor was continuously aerated and thoroughly mixed by introducing air with an air pump through a pair of diffusers located at the bottom of the reactor. No mechanical stirring system was used. Temperature was controlled at 30 ± 2 °C with an external coil and a thermostatic bath. The pH of the medium was not controlled by an automatic control system but was maintained near neutral (7.0 ± 0.3) throughout the entire operating cycle by adding bicarbonate buffer to the water stream.

[0071] The activated sludge used as inoculum contained a variety of microbial populations, and it was necessary to select cultures that exhibited the following functions.

[0072] 1) The fats present in the lipid stream used as a carbon source are hydrolyzed into long-chain fatty acids and glycerol. 2) Compounds resulting from the hydrolysis process, such as PHA and / or TAG, accumulate intracellularly.

[0073] To this end, the system was operated under a feast / starvation regime, a modification of the conventional strategy (ADF) that involves supplying carbon and nitrogen sources in a decoupled manner, i.e., the DGL strategy (Oliveira, et al., N. Biotechnol. 2017, 37, 69-79). In this regard, lipid flow (carbon source) was added in the absence of nitrogen at the beginning of the cycle, creating a feast phase, and once complete, nitrogen source was added in the absence of carbon, creating a starvation phase. Carbon source addition was performed in pulses throughout the feast phase.

[0074] As shown in Figure 3, three different process configurations consisting of 12-, 18-, and 24-h operation cycles were tested and distributed as follows: (I) feast phase (360–720 min): carbon source feeding interval (2 min) + aerobic reaction (58–118 min), (II) 50% exchange of reaction volume (emptying + filling), (III) starvation phase (348–708 min): aerobic reaction after nitrogen source addition (348–708 min).

[0075] Feast phase: The carbon source consisted of the fatty fraction of wastewater from the canning industry for tuna production, which has a high oleic acid content (Table 1).

[0076] Table 1. Characteristics of the lipid stream used as substrate: C (carbon), COD (chemical oxygen demand), H (hydrogen), N (nitrogen), O (oxygen), S (sulfur), TS (total solids), and VS (volatile solids).

[0077] [Table 1]

[0078] This lipid fraction, obtained from canning industry wastewater, was added in pulses (every 1 to 2 hours) from the start of the cycle until the end of the feast phase. After each substrate pulse, a new substrate pulse was added. The periodicity between pulses was set as a function of the DO concentration, which was continuously measured in the reactor using an oxygen probe.

[0079] - Upon addition of carbon source, DO concentration decreased as a result of substrate metabolism and then increased again until it reached saturation value, which suggested carbon source depletion. The time elapsed between the addition of the carbon pulse and the return to saturation defined the time required for substrate consumption, in other words the periodicity between pulses. - Once this time is defined, the moment at which each pulse is added is programmed into a programmable logic controller (PLC). - The evolution of DO concentrations was recorded during continuous monitoring and the profiles were reviewed daily in case the substrate consumption time changed and required readjustment of the PLC programming.

[0080] Replacement: At the end of the feast phase, half of the reactor volume (5 L containing the biomass where PHA and TAG had accumulated) was removed for biopolymer extraction and purification. Then, the same volume (5 L) of water containing nutrient solution (nitrogen source) was added, and the starvation phase began.

[0081] Starvation phase: The nitrogen source used consisted of a synthetic solution containing the minimum amount of nitrogen in the form of NH4Cl required for the replication (growth) of the microorganisms present in the reaction medium, a NaHCO3 buffer to maintain the pH of the system near neutral, allylthiourea to inhibit nitrification activity, and other macro- and micronutrients (aqueous stream, Table 2).

[0082] [Table 2]

[0083] The minimum amount of nitrogen required for microbial growth, in other words the stoichiometric amount of nitrogen for microbial growth, is determined as follows.

[0084] - The empirical formula for biomass is given in the literature (Popovic, Heliyon, 2019, 5, 6) (C X H Y N W O Z) and taking into account the type of microbial population present in the system (PHA and TAG accumulators). Once the biomass equation is defined, the number of moles of nitrogen required to form one mole of biomass is known. Therefore, if the number of moles of biomass present in the reactor is known, the number of moles of nitrogen required to double the biomass can be calculated by stoichiometry. That is, - Biomass C a H b N c O d According to the formula, c moles of nitrogen are required to form 1 mole of biomass. - If M is the number of moles of active biomass in the reactor, then 0.2 × M × c moles of nitrogen are required to double the biomass. In the example shown, the following elemental formula for biomass was considered: CH 1.8 O 0.5 N 0.2 Therefore, 0.2 moles of nitrogen were required to form 1 mole of biomass.

[0085] - The actual amount of nitrogen required by the microorganisms for growth in each cycle is then readjusted based on the nitrogen concentration remaining at the end of the starvation phase (which should be zero). This value is obtained by analyzing the total nitrogen concentration present in the liquid phase of a sample taken of the reaction medium at the end of the starvation phase (see the Sampling and Analysis section).

[0086] Sampling and analysis During the characterization of the SBR operating cycle, periodic samples of the reaction volume were taken. During the feast phase, samples were taken immediately before each pulse of carbon source addition.

[0087] pH was measured using a pH & ion meter (GLP 22 Crison, Spain). Total suspended solids (TSS), volatile suspended solids (VSS), and total chemical oxygen demand (COD) were measured. t ), and soluble chemical oxygen demand (COD s) concentrations were measured using the Standard Method for the Examination of Water and Wastewater (Baird, R., & Bridgewater, L. 2017.Standard methods for the examination of water and wastewater.23 rd edition.Washington, DC: American Public Health Association; Methods 2540 E pages 4-6 (TSS,VSS), and 5220 B pages 2-3 (COD). Ion concentrations were measured by ion chromatography (861 Advanced Compact IC Metrohm, Switzerland), and total organic carbon, inorganic carbon, and total nitrogen (TOC, IC, and TN, respectively) by catalytic combustion (TOL-L analyzer with TNM module, TOC-5000 Shimadzu, Japan). s The concentrations of ions, TOC, IC, and TN were measured in the soluble fraction of the samples after centrifugation (5430 Eppendorf centrifuge, USA) and filtration (0.45 μm pore size filter with cellulose ester membrane, Advantech, Japan) of the raw samples. To characterize the carbon source, conductivity was measured with a portable conductivity meter (Probe Sension + EC5 HACH-Lange, USA), and the concentrations of total solids and volatile solids (TS and VS, respectively) were measured according to the Standard Methods for the Examination of Water and Wastewater (Baird, R., & Bridgewater, L. 2017). methods for the examination of water and wastewater,23rd edition. Washington, The elemental composition was measured as described in the American Public Health Association's Methods 2540 B p. 2 (TS) and 2540 D p. 6 (VS). The elemental composition was measured using an elemental analyzer (FlashEA 1112 Thermo Scientific, USA). The fatty acid profile was measured by gas chromatography according to ISO standard 12966-2:2011(4.2) - rapid method and ISO standard 12966-4:2015.

[0088] The PHA and TAG accumulated intracellularly in the biomass were analyzed by Smolders et al., Biotechnol. The concentrations were determined by gas chromatography (Innovax HP column equipped with a flame ionization detector (FID) (Agilent, USA)) according to the method described by [Bioeng. 1994, 44, 837-848]. To do so, a sample of the reaction volume was taken, centrifuged (5420 Eppendorf Centrifuge, USA), and lyophilized to obtain a solid phase. The accumulated compounds were quantified using commercially available calibration standards (Sigma-Aldrich, USA) of PHA (a copolymer containing 88% hydroxybutyrate (HB) and 12% hydroxyvalerate (HV)) and TAG (palmitic acid, stearic acid, oleic acid, and linoleic acid).

[0089] calculation The content of intracellular compounds (PHAs and TAGs) in the biomass was expressed as a percentage of the measured VSS and as a percentage of the dry weight of the cells (dw%). The active biomass concentration (X) was calculated by the CH 1.8 O 0.5 N 0.2 Considering the following empirical formula for biomass, it was calculated as the difference between the mass of VSS and the sum of accumulated intracellular compounds (Biop = PHA + TAG).

[0090] The maximum carbon and nitrogen consumption (respectively, -q S , -q N), and the maximum production of PHA, TAG, and active biomass (q PHA , q TAG , q X ) were determined based on the maximum slope of the curves representing the time course of each parameter concentration. These parameters were referenced to the substrate (S) and expressed as Cmmol / (Cmmol x q defined as x Except for Cmmol / (Cmmol S The production yield (Y, Cmmol / Cmmol S ) was calculated by dividing the maximum production rate by the maximum consumption rate of S. The maximum daily productivity refers to the mass (g) of intracellular compounds (PHA and TAG) accumulated per day per L of substrate volume supplied to the system. This value was calculated by dividing the yield (previously converted from C mmol to g) by the lipid flow (L) supplied to the system per day.

[0091] Three specific examples of the system's behavior in the various cycle configurations that were tested are given below.

[0092] Example 1 The reactor was operated in a 12-hour cycle for 57 days, with the cycle illustrated here corresponding to day 43 of operation.

[0093] The carbon source (lipid stream) was added in pulses during the feast phase (first 6 h): a total of six pulses of 0.6 ml (1.20 g COD / pulse, considering that the lipid stream contained 2,000 g COD / L) with a frequency of 1 h between pulses (2 min carbon source feeding + 58 min aerobic reaction), which meant a flow rate of 3.42 Cmmol / (L·h) during this first phase.

[0094] After that, 50% (5 L) of the reaction volume was removed, and the nitrogen concentration was about 16 mg 5 L of NTZL water stream was added.

[0095] The starvation period took place during the remaining 6 hours.

[0096] Figure 4(a) shows the evolution of the main operating parameters monitored in the reactor. Table 3 shows the intracellular accumulation values, the composition of accumulated compounds, the main kinetic parameters, and the achieved yield. After 6 h (end of the feast phase), the intracellular accumulation amount reached 50.45 ± 0.22 dw. % (ratio PHA:TAG 50.5:49.5). At this point, the maximum accumulation rates of PHA and TAG were 0.088 cm mmol, respectively. PHA / (Cmmol S ·h) and 0.073 Cmmol TAG / (Cmmol S ·h), and the consumption rate of lipid substrates is −0.458 Cmmol S / Cmmol X The production yield of PHA and TAG was 0.231 cmmol PHA / Cmmol S and 0.192 Cmmol TAG / Cmmol S The total yield was 0.423 Cmmol Biop / Cmmol S It was.

[0097] [Table 3]

[0098] Biop (intracellular accumulated PHA + TAG); C (lipid substrate); LIN (linoleic acid); OL (oleic acid); PAL (palmitic acid); PHA (polyhydroxyalkanoic acid); PHB (polyhydroxybutyrate); PHV (polyhydroxyvalerate); q (specific maximum production); -q (specific maximum consumption); STE (stearic acid); TAG (triacylglyceride); X (active biomass); Y (performance)

[0099] Example 2 The reactor was operated on an 18-hour cycle starting on day 58 of operation. At this point, the feast phase was extended from 6 hours to 12 hours, while the starvation phase remained at 6 hours. The example discussed here corresponds to day 72 of operation.

[0100] The carbon source (lipid flow) was added in pulses during the feast phase (first 12 h). A total of six pulses of 0.6 ml (1.20 g COD / pulse, considering that the lipid flow was 2,000 g COD / L) were added, with a 1-h pulse interval (2 min for carbon source supply + 118 min for aerobic reaction). This means that a flow rate of 1.71 Cmmol / (L·h) was supplied during the first phase.

[0101] After that, 50% (5 L) of the reaction volume was removed, and the nitrogen concentration was about 18 mg 5 L of TN / L water flow was added.

[0102] The starvation period took place during the remaining 6 hours.

[0103] Figure 4(b) shows the evolution of the main operating parameters monitored in the reactor. Table 4 shows the intracellular accumulation values, the composition of accumulated compounds, the main kinetic parameters, and the achieved yield. The carbon and nitrogen consumption rates during the feast and starvation phases were lower than those in the 12-h cycle (Example 1), resulting in lower carbon and nitrogen consumption during each period. Therefore, the proportion of nitrogen in the reaction solution was higher (6 vs. 13 mg / L at the end of the feast phase in the 12-h and 18-h cycles, respectively), and the amount of extracellular carbon was also higher during the starvation phase (80 vs. 400 mg COD / L). Therefore, the feast phase was less nitrogen-limiting, while the amount of carbon was higher during the starvation phase (more than the 10-15% of COD added to the reaction solution during the feast phase, which corresponds to the non-biodegradable COD of the substrate), allowing the growth of non-accumulating populations. As a result, a decrease in the accumulation capacity of the system was observed. Between day 43 (Example 1) and day 72 (Example 3), the maximum intracellular accumulation level increased from 50.24±0.44 (TAG:PHA = 51:49) to 30.75±1.67 (TAG:PHA = 65:35) and the maximum production of intracellular compounds was 0.429 Cmmol Biop / Cmmol S to 0.299 Cmmol Biop / Cmmol S It decreased to.

[0104] [Table 4]

[0105] Biop (intracellular accumulated PHA + TAG); C (lipid substrate); LIN (linoleic acid); OL (oleic acid); PAL (palmitic acid); PHA (polyhydroxyalkanoic acid); PHB (polyhydroxybutyric acid); PHV (polyhydroxyvalerate); q (specific maximum production); -q (specific maximum consumption); STE (stearic acid); TAG (triacylglyceride); X (active biomass); Y (performance)

[0106] Example 3 The reactor described above was operated in a 24-hour cycle starting on day 96 of operation. In this example, the cycle corresponding to day 133 of operation is analyzed.

[0107] To increase the carbon and nitrogen consumption during the feast and starvation phases, respectively, the operating cycle configuration was modified compared to that described in Example 2. To achieve this, the feast phase was maintained at 12 h, but the amount of substrate added during this phase was reduced to avoid the presence of extracellular carbon during the starvation phase. Instead of six 0.6 ml pulses, five pulses of 0.60 ml each (1.20 g COD / pulse, considering the lipid stream contained 2,000 g COD / L) were added, resulting in a flow rate of 1.45 Cmmol / (L·h). The MMC was supplied with carbon source as follows: four intervals of 2 min carbon source supply + 118 min aerobic reaction; and a final interval of 2 min carbon source supply + 236 min aerobic reaction.

[0108] Afterwards, 50% (5 L) of the reaction volume was removed and 5 L of a water stream with a nitrogen concentration of approximately 10 mg / L was added.

[0109] A starvation period was carried out during the remaining 12 hours. The duration of the starvation period was extended from 6 hours to 12 hours compared to Example 2, and the nitrogen addition rate was readjusted based on the total nitrogen (TN) concentration measured at the end of the run cycle to ensure more limiting nitrogen conditions than in Example 2.

[0110] In this way, it was possible to reduce the amount of extracellular carbon available in the reaction medium at the end of the feast phase (50 vs. 400 mg COD / L) and to further limit the concentration of nitrogen available in the reaction medium at the end of the starvation phase (5 vs. 13 mg TN / L), which had a positive impact on the accumulation capacity of the culture compared to the 18-h cycle.

[0111] Figure 4(c) shows the evolution of the main operational parameters monitored in the reactor. Table 5 shows the intracellular accumulation values, the composition of accumulated compounds, the main kinetic parameters, and the achieved yields. From day 72 (Example 2) to day 133 (Example 3) of operation, the intracellular accumulation at the end of the feast phase (12 h) increased from 30.75 ± 1.67 (TAG:PHA = 65:35) to 45.31 ± 2.18 (TAG:PHA = 25:75) dw% despite the addition of a low amount of carbon source. As a result, the production yield of intracellular compounds increased by 96% (from 0.299 to 0.586 cm mmol Biop / Cmmol S ).

[0112] [Table 5]

[0113] Biop (intracellular accumulated PHA + TAG); C (lipid substrate); LIN (linoleic acid); OL (oleic acid); PAL (palmitic acid); PHA (polyhydroxyalkanoic acid); PHB (polyhydroxybutyric acid); PHV (polyhydroxyvalerate); q (specific maximum production); -q (specific maximum consumption); STE (stearic acid); TAG (triacylglyceride); X (active biomass); Y (performance)

[0114] Comparison with literature Table 6 shows a comparison of the results obtained in the present invention (Example 1) with three representative publications from the literature that used mixed cultures: (a) accumulation of PHA from lipid streams, (b) accumulation of TAG from lipid streams, and (c) preferential accumulation of PHA or TAG from lipid streams.

[0115] [Table 6]

[0116] Biop (total of PHA and TAG), FBR (Fed-batch reactor), PHA (Polyhydroxyalkanoic acid), SBR (Sequencing batch reactor)

[0117] In a previous run using the same carbon source in a two-unit process (SBR enrichment reactor + FBR accumulation reactor) (Argiz et al., Sci. Total Environ. 2021, 763, 142944), when PHAs were the primary target, 12 hours of operation in the SBR and 30 hours of operation in the FBR were required to achieve an accumulation of 82.30% dry weight PHA and 3.33% dry weight TAG (1.8 g VSS / L, 0.45 g X / L). When TAG accumulation was prioritized, 12 hours of operation in the SBR and an additional 27 hours of operation in the FBR were required to achieve an accumulation of 39.55% dry weight TAG and 5.80% dry weight PHA (2.1 g VSS / L, 1.1 g X / L). Thus, maximum production rates of 0.88 and 0.67 g Biop / (L d), respectively, were reached, whereas in the present invention, a maximum production rate of 1.21 g Biop / (L d) was reached in the single-unit process, despite lower biomass concentrations (0.48 g VSS / L, 0.22 g X / L).

Claims

1. A process for the intracellular production of polyhydroxyalkanoates (PHAs) and triacylglycerides (TAGs) in a mixed microbial culture, characterized in that all steps are carried out in a single sequencing batch reactor, and further characterized in that: a) initially inoculating the reactor with activated sludge, the activated sludge comprising PHA and TAG accumulating microorganisms and microorganisms capable of hydrolyzing lipids; b) Next, a step in the operation cycle of the reactor, i. initially feeding a lipidic carbon source to the microorganisms present in the reactor, said feeding being pulsed and under nitrogen-limited conditions; ii. then removing 50% of the mixture from the reactor, the mixture comprising biomass with intracellular PHAs and TAGs; and iii. thereafter adding a stream containing nitrogen in an amount of 0.9 to 2 times the stoichiometric amount of nitrogen required for microbial growth; wherein step (ii) occurs immediately after step (i); c) thereafter repeating the process of step b) in successive cycles; A process with.

2. the activated sludge is enriched with a PHA-accumulating microorganism selected from Acidovorax, Acinetobacter, Pandoraea, Azospirillum, and Pseudomonas, and a PHA-accumulating microorganism selected from Candida, Yarrowia, Geotrichum, Mortierella, and Rhodoccocus; 2. The process of claim 1, comprising a TAG-accumulating microorganism to be treated and a microorganism capable of hydrolyzing lipids selected from among Candida, Yarrowia, Fonsecaea, Capronia, Acinetobacter, Cryptococcus, Rhodosporidium, Geotrichum, and Trichosporon.

3. 3. The process of claim 1 or 2, wherein the activated sludge is obtained from a municipal wastewater treatment plant (WWTP).

4. 3. The process of claim 1 or 2, wherein the lipidic carbon source has a lipid content of greater than 85% of the chemical oxygen demand (COD).

5. 3. The process of claim 1 or 2, wherein each feed pulse contains a carbon content of 1,200 mg COD or less.

6. 3. The process of claim 1 or 2, wherein the amount of nitrogen in step (i) is less than 20 mg total nitrogen (TN) / L.

7. 3. The process of claim 1 or 2, wherein the amount of nitrogen in step (i) is 5 to 20 mg TN / L.

8. 3. The process of claim 1 or 2, wherein step (i) lasts from 6 to 12 hours.

9. 3. The process of claim 1 or 2, wherein step (iii) lasts from 6 to 12 hours.

10. 3. The process of claim 1 or 2, wherein the stream of step (iii) is an aqueous solution containing a nitrogen compound.

11. 11. The process of claim 10, wherein the nitrogen compound is selected from ammonium sulfate, ammonium nitrate, and ammonium chloride.

12. 11. The process of claim 10, wherein the aqueous solution comprises ammonium chloride at a concentration of 5 mg / L to 280 mg / L.

13. 3. The process of claim 1 or 2, wherein at the end of step (iii), the total amount of nitrogen in the reaction medium is less than 20 mg per liter.

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