WO2019171316A1

WO2019171316A1 - Method for the production of polyhydroxyalkanoates (phas) from high solid content organic waste

Info

Publication number: WO2019171316A1
Application number: PCT/IB2019/051844
Authority: WO
Inventors: Mauro Majone; Francesco Valentino; Paolo Pavan; David BOLZONELLA; Federico MICOLUCCI; Marco GOTTARDO
Original assignee: Universita' Degli Studi Di Roma "La Sapienza"; Universita' Ca' Foscari; Universita' Degli Studi Di Verona
Priority date: 2018-03-07
Filing date: 2019-03-07
Publication date: 2019-09-12
Also published as: EP3762502A1; IT201800003322A1

Abstract

A process for the biosynthesis of PHAs from the organic fraction of metropolitan solid waste. The process uses a mixed microbial consortium (MMC) and combines three stages in sequence (Stage I: anaerobic fermentation, Stage II: sequential aerobic fermentation, Stage III: batch aerobic fermentation). The material leaving the fermentation Stage I reactor is initially conveyed through a filter which reduces its content of suspended solids without significantly interfering with the concentration of VFAs and nutrients, such as phosphorus (P) and nitrogen (N), elements that in Stage II, favour the production of the MMC biomass specialized in the synthesis of PHAs, used in the subsequent Stage III. The flow is then divided between the Stage II reactor and a second filter with membrane that eliminates its suspended solid particulate matter and reduces the concentration of P and N, thus favouring the synthesis of PHAs by the microorganisms during Phase III.

Description

METHOD FOR THE PRODUCTION OF POLYHYDROXYALKANOATES (PHAS) FROM HIGH SOLID CONTENT ORGANIC WASTE

FIELD OF THE INVENTION

The present invention relates to the field of biopolyester production via a microbial synthesis biological process.

STATE OF THE ART

The use of renewable resources for the production of chemicals, fuels, and innovative materials has had a growing interest in recent years, in view of the increasingly growing need to limit the use of fossil resources. The production of a specific class of biopolyesters, named polyhydroxyalkanoates (PHAs), naturally produced by aerobic microorganisms and accumulated within the cell wall, fits perfectly in the scope of this approach. PHAs are fully biodegradable thermoplastic polymers, ideal candidates to fill a part of the market currently occupied by traditional synthetic thermoplastic materials. PHAs represent a family of copolymers having widely variable properties, obtainable from numerous feedstock that can be exploited and microbiologically synthesized in relatively mild and easy to manage process conditions. The most common and relatively easy to produce copolymer is Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)).

Based on these characteristics, PHAs have a high potential to be competitive on the market; in fact, market analyses forecast a quadrupled production capacity in the 201 1 -2020 decade. This assumption, however, is valid as long as the following three objectives are at least partially achieved: a) the current market price decreases; b) their properties have to be modulated according to the specific application and/or use; c) the environmental impact associated with the production process is reduced (energy consumption, CO2 emissions, etc..).

The current market price for PHAs is of between 5-8€/kg (Valentino et al. , Carbon recovery from effluent through bioconversion into biodegradable polymers; New Biotechnology 37:9-23, 2017); the main reason for these high costs is given by the intrinsic characteristics of the current industrial production processes, based on the cultivation of single strains of microorganisms (the most used being Cupravidus Necator, originally known as Ralstonia eutropha or Alcaligenes eutrophus). These operating conditions require ad-hoc formulated substrates ( e.g . glucose from fermentation of starch from agricultural crops) and rather high energy consumption (e.g. sterilization of substrate and equipment, high oxygen transfer rates due to the typical high cell density in bioreactors). WO2012/166822 describes a PHA production process based on the culture of single strains of microorganisms (specifically, Delftia acidovorans), wherein the initial organic feedstock is first subjected to acidogenic anaerobic fermentation, and subsequently subjected to removal of at least a part of the solid residue before being subjected to fermentation with a single strain of Delftia acidovorans for the production of PHAs.

However, there are widely studied and applied strategies on a laboratory scale, and more recently on a pilot scale, aimed at improving the economy of the whole process; these strategies involve the use of mixed microbial consortium (MMC), rather than pure cultures, fed with zero-cost (or low-cost) feedstock that do not compete with the food chain.

CN104651420 describes a method for the production of PHAs by using MMC, said method, wherein the initial organic feedstock is first subjected to acidogenic anaerobic fermentation and subsequently subjected to centrifugation in order to remove the solid parts and obtain a fermentation solution to be fermented using MMC to obtain PHAs.

In general, any easily fermentable organic substrate may be converted into PHAs through a multi-stage biological process. Specifically, the most widely studied and applied scheme is the three-stage process illustrated in Figure 1 (EP1400569). This process includes three different units: the acidogenic fermentation reactor (Anaerobic Section, AnS), wherein the organic carbon or COD (Chemical Oxygen Demand) is converted into volatile fatty acids (VFAs), thus producing effluent with concentrated VFAs (preferably with a high VFAs/COD ratio), to be fed in the two subsequent aerobic stages (Aerobic Section 1 and 2, AS1 and AS2). Section AS1 consists of a sequential batch reactor (SBR), inoculated with activated sludge, used for the production of a PHA-producing mixed microbial consortium (MMC) through a dynamic feeding regimen of the fermented organic effluent. The VFA-rich mixture produced in AnS and the MMC mixed consortium produced in AS1 are then conveyed to the third stage (AS2), wherein the intracellular content of PHAs is maximized and, therefore, the actual production takes place. In particular, the MMC culture selection stage plays a crucial role on the overall process performance, and it is typically performed in SBR reactors, particularly suitable for operating under conditions of feast-famine regimen, also known as aerobic dynamic feeding (ADF). To date, this technology has not been tested yet in full-scale plants; the most recent examples of started prototypes are on a pilot scale: within the 7th Framework Program (FP7), several projects included (and in some cases were entirely dedicated to) the implementation of the process described above; more recently, as part of the ResUrbis FI2020 project (2017-2019), a pilot platform has been implemented, and it is currently operating at the Treviso purification plant (TV), designed to maintain a productivity of between 0.5-1 .0 kgPFIA/d using metropolitan organic waste (in particular, the Organic Fraction of Metropolitan Solid Waste, OFMSW). Within the scope of this platform, it is clearly necessary to consider the characteristics of the effluent to be used, in particular the high solids content (Total Suspended Solid, TSS > 100 g/L) typical of OFMSW. A non-negligible fraction of these solids, often inert, do not undergo degradation after acidogenic fermentation (AnS), the first step of the process. This feature does not allow the direct use of this matrix in the PFIA line of the platform since, if the high solids content is not drastically reduced, the operation of the subsequent steps in the process would be compromised, particularly in the downstream section. The presence of solids and/or inerts could, in fact, make the extraction more difficult, and compromise the final purity of the extracted PHAs.

As described above, the high solids content (TSS or TVS) present in OFMSW, or other organic waste matrices, makes their direct use for PHA production practically difficult. In fact, the process based on the MMC production and selection, and the subsequent PHA production is usually carried out with feedstock characterized by a TSS content that is never higher than 5.0-6.0 g/L (e.g. whey, molasses, or other food processing waste, wherein most of the COD is present in a soluble form). Therefore, although PHA production rate and efficiency depend on the soluble COD (and particularly on the VFA fraction), any non-fermentable solid residue migrates throughout the process and then remains concentrated together with the PHA-rich biomass at the end of the stage AS2; this phenomenon prevents the obtaining of high purity PHAs in the final downstream stage.

The object of the present invention is to provide a process, and the related plant, for the production of PHAs using OFMSW as the starting organic matrix.

DEFINITIONS AND ABBREVIATIONS

Aerobic Dynamic Feeding (ADF)

Chemical Oxygen Demand (COD)

Organic Fraction of Metropolitan Solid Waste (OFMSW)

Mixed Microbial Consortium (MMC)

Polyhydroxyalkanoates (PHA)

Poly-3-hydroxybutyrate-co-3-hydroxyvalerate [P(3HB)-co-(3HV)j

Total Solids (TS)

Total Volatile Solids (TVS)

Total Suspended Solids (TSS)

Volatile Suspended Solids (VSS)

Volatile Fatty Acids (VFAs)

Total Kjeldahl Nitrogen (TKN)

Hydraulic Retention Time (HRT)

Sludge Retention Time (SRT)

Organic Load Rate (OLR)

SUMMARY OF THE INVENTION

The present invention solves the aforementioned problems by means of a process for the production of one or more polyhydroxyalkanoates (PHAs), said process, with reference to Figure 2, comprising:

a first stage of anaerobic fermentation (AnS), wherein a fermentable organic waste having a TSS greater than 10.0 g/L is subjected to acidogenic anaerobic fermentation, wherein the organic carbon or COD (Chemical Oxygen Demand) is converted into volatile fatty acids (VFAs), thus producing a fermented organic effluent (HS) concentrated in VFAs; subjecting HS to a solid/liquid separation by filtration (C1 ) with a filter having a porosity of 5-10 pm to obtain a filtered fermented organic effluent (LS1 ) having a suspended solids content lower than 10 g/L and higher than 1 g/L;

subjecting at least a portion of LS1 to a solid/liquid separation by filtration (M1 ) with a membrane module having a porosity of 0.2-1 .0 pm, at an operating pressure of 4-5 bar, to obtain a doubly-filtered fermented organic effluent (LS2) having a suspended solids content lower than 0.1 g/L;

a first stage of aerobic fermentation (AS1 ), wherein the remaining portion of LS1 , or, when the whole LS1 is subjected to solid/liquid separation by filtration (M1 ), a portion of LS2 is subjected, in a sequential batch reactor (SBR), inoculated with activated sludge, to an aerobic dynamic feeding (ADF) regimen to obtain the production of a PHA-producing mixed microbial consortium (MMC);

a second stage of aerobic fermentation (AS2) wherein LS2 or, when the whole LS1 is subjected to solid/liquid separation by filtration (M1 ), a portion of LS2 and at least a portion of MMC are brought into contact to obtain the production of one or more PHAs by the MMC.

According to the present invention, the scheme known in the state of the art, and illustrated in Figure 1, has been modified by dividing the flow from the anaerobic fermentation into two distinct sub-flows, characterized by a different solids content obtainable by means of two different and sequential solid/liquid separations. This strategy allows to use a first sub-flow, with a solids content at least below 10.0 g/L, exclusively for AS1 , and a second sub-flow having a solids concentration lower than 0.1 g/L, and preferably equal to zero, for exclusive use in AS2.

Furthermore, a good filtering system M1 , ensuring total solids removal for use of the matrix in AS2, may also partially reduce the nutrient content of the matrix itself. This is particularly indicated for limiting cell growth in AS2, thus allowing a higher intracellular PHA accumulation. Given the partial limitation of nutrients, in fact, soluble carbon is mostly directed towards the synthesis of PHAs rather than the formation of new cells; this allows to obtain a relatively high PHA content inside the cells, corresponding to 50-60% based on cellular dry weight, with consequent simplification of the downstream section, and obtaining a product of higher purity. Although it is known that the solids content must be necessarily reduced for a better use of the COD contained in the fermented matter, in order to maximize the conversion to PHAs and obtain a high PHA content in the biomass after the accumulation stage in AS2, this is not equally true to maximize the AS1 operation because:

- together with the solids, a large COD content would also be removed, that could be used to support the aerobic growth of biomass (or MMC) in AS1 ;

- together with the solids, a substantial nutrient content (nitrogen and phosphorus, N and P) would be removed, that could be used to support the aerobic growth of biomass (MMC) in AS1

On the other hand, AS2 does not require nutrients, and the VFA/COD ratio should be kept as high as possible.

Therefore, since the operating conditions of AS1 and AS2 are different, the presence of two different solids removal steps (i.e. C1 and M1 ) on the fermented effluent HS, which can be set on two different removal levels and/or degrees, is distinctive and advantageous to the present invention. Depending on the inevitable variability of the starting feedstock (since it is waste), the presence of the double separation system, C1 and M1 , in series allows in each case to direct the supply towards the stages AS1 and AS2, so as to optimize the process performance. Object of the present invention is also an integrated system for the production of one or more polyhydroxyalkanoates (PHAs) by the process as described above, said system comprising:

a section for the anaerobic fermentation (AnS), said section AnS comprising an inlet to receive a fermentable organic waste and an outlet to discharge a fermented organic effluent (HS);

a first filtering unit (C1 ) comprising a filter having a porosity of 5-10 pm, said unit C1 comprising an inlet in fluid connection with the outlet of the zone AnS and an outlet to discharge the filtered fermented organic effluent (LS1 );

a sequential batch reactor (SBR), inoculated with activated sludge, for the first stage of aerobic fermentation (AS1 ), said SBR comprising an inlet in fluid connection with the outlet of C1 and an outlet to discharge a PHA-producing mixed microbial consortium (MMC);

a second filtering unit with membrane (M1 ) comprising a membrane having a porosity of 0.2-1 .0 pm, said unit M1 comprising an inlet in fluid connection with the outlet of C1 and an outlet to discharge a doubly-filtered fermented organic effluent (LS2);

an aerobic fermentation stage section (AS2), said section AS2 comprising a first inlet in fluid connection with the outlet of M1 , a second inlet in fluid connection with the outlet of SBR, and an outlet to discharge a biomass containing one or more PHAs.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Three-stage process known in the state of the art and mostly applied for the production of PHAs from MMC and organic effluents. AnS = anaerobic fermenter; AS1 = 1 ^st aerobic step for production/selection of the mixed consortium (MMC); AS2 = 2^nd aerobic step for intracellular accumulation of PHAs.

Figure 2. Multi-stage process according to the present invention for the production of PHAs from MMC and organic effluent.

AnS = anaerobic fermenter; AS1 = 1 ^st aerobic step per la production/selection of the mixed consortium (MMC); AS2 = 2^nd aerobic step for intracellular accumulation of PHAs, C1 and C2 = filter bag centrifuge for solid/liquid separation; M1 and M2 = membrane unit for solid/liquid separation, F = coagulation/flocculation unit,

LS = effluent with low solids content (low solids); HS = effluent with high solids content (high solids)

Figure 3. illustrates the trend of all the different VFAs produced after starting the AnS fermentation process according to an embodiment of the process of the invention.

Figure 4. shows the trends of PHAs, VFA and dissolved O2 following detailed analysis performed on a typical operation cycle, in optimal operating conditions, of the process according to an embodiment of the process of the invention. Figure 5 illustrates the trends of suspended solids (TSS and VSS), at cycle end and at feast phase end (minimum and maximum concentration, respectively) according to an embodiment of the process of the invention.

Figure 6 illustrates the trends of PHAs at cycle end and at feast phase end (minimum and maximum concentration, respectively) according to an embodiment of the process of the invention.

Figure 7 shows the storage potential of the selected biomass, that was tested during batch accumulation tests, carried out with LS2 permeate (Batch 1 P and 2P) according to an embodiment of the process of the invention.

DETAILED DESCRIPTION OF THE INVENTION

To date, the scheme of the process according to the present invention has been tested starting from OFMSW, however its use could be extended to any type of fermentable organic waste whose content of solid residues in the fermented matrix exceeds 10.0 g/L. This strategy includes another intrinsic operational advantage. The main feature of the first filtering unit, C1 , is to significantly reduce the solids content (and the slowly biodegradable COD) without interfering with the initial VFAs and nutrients (N, P) concentration. In the specific case of OFMSW, the LS1 efflux is characterized by a suspended solids content of less than 10 g/L, which has been shown to be sufficiently low to generally allow a suitable operation of AS1 . On the other hand, the VFA content remains unchanged, thus increasing the VFAs/COD ratio, therefore allowing to obtain a shorter“feast” phase, and consequently a more efficient selection of PHA-producing microorganisms. Finally, the N and P content is not significantly altered, and this involves the presence of nutrients in a sufficient amount to allow for the growth of the PHA-producing biomass during the entire “famine” phase, at the end of which the same biomass (or part thereof) is conveyed to AS2 for the accumulation stage. Beyond this specific example, the unit C1 must allow for an effective separation of the solids from the fermented liquid, by removing all solids having a particle size bigger than the mesh size of the filtering basket (5- 10 pm).

Preferably the first solid/liquid separation unit“C1” is a vertical axis centrifuge equipped with a filter bag having a porosity in the range of 5-10 pm. Preferably the LS1 portion sent to the stage AS1 is equal to 35-45% by volume with respect to the whole LS1 .

The second separation unit“M1” is a membrane module characterized by a porosity in the range of 0.2-1 .0 pm and an operating pressure equal to 4-5 bar. It should be noted that the level of solids in LS1 would still be too high to ensure the best performance in AS2. In particular, by feeding HS1 or LS1 in the accumulation step AS2, the final percentage of PHAs compared to TSS may be too low to obtain a polymer with a high degree of purity in the downstream stages. Therefore, LS1 is sent to the second solid/liquid separation unit,“M1”, in order to further reduce TSS and VSS. In the best case, the effluent LS2 does not contain any suspended solids while the VFA content is still kept constant, thus increasing the VFAs/COD ratio to around 0.90. Furthermore, given the low porosity, a not-negligible reduction of N and P may be detected, to the advantage of the storage response over the cell growth in AS2. In this way, the level of nutrients in the fermented product would be partially limiting growth (COD:N:P = 100:3.5:0.7, approximately), which makes this particular, doubly filtered, fermented matter a renewable feedstock perfectly suited for maximizing PFIA in AS2.

If a better control of the COD:N:P ratio is required in LS2, a coagulation/flocculation unit“F” may preferably be installed between“C1” and“M1”; such unit provides for a coagulation/flocculation treatment, wherein LS1 is added with calcium hydroxide up to a pH within the range of 10.0-1 1 .0. This treatment involves a further removal of P from LS2 through the precipitation of calcium phosphate, thus further limiting the growth response and maximizing the storage (accumulation) in AS2.

Furthermore, depending on the feedstock composition variation and/or the AnS stage effluent and/or the C1 and M1 stages effluents, the process scheme provides for the possibility of operating the AS1 stage feed with the effluent from stage M1 rather than the previous stage C1 . In an embodiment of the process according to the invention, 100% of LS1 is subjected to the second separation unit, M1 , and the resulting liquid LS2 is used for both the aerobic stages, AS1 and AS2, preferably according to the volumetric percentages provided for by the process (35-45% in AS1 and 55-65% in AS2). In accordance with this embodiment of the process, the system according to the invention provides that SBR has a second inlet in fluid communication with the M1 outlet.

Since the secondary flows (S1 and S2) generated in both units, “C1” and“M1”, contain biodegradable COD, an anaerobic digestion unit“AD” may preferably be installed for recovering energy from the anaerobic digestion, particularly to support the plant energy needs. Since these secondary flows contain a high solids content, their hydrolysis is encouraged thanks to the thermophilic operating conditions adopted. In this case, the integrated system object of the present invention comprises a first filtering unit (C1 ) further comprising a second outlet to discharge a solid effluent (S1 ); a second filtering unit with membrane (M1 ) further comprising a second outlet to discharge a concentrated effluent (S2); and a section (AD) for the anaerobic digestion comprising at least one inlet in fluid connection with the second outlet of C1 and/or M1.

Preferably, the process and system according to the present invention further comprise a third separation unit, which may be a filter bag centrifuge (C2) or a membrane module (M2), installed to quickly separate the PHA high-content biomass (exiting from AS2) from the liquid medium. The liquid medium thus recovered may be used as a means of diluting the slurries in stages AnS, AS1 , and AD. Through water recirculation, the slurries composition in the stages AnS, AS1 , and AD may be handled independently, without generating untreated flows exiting the entire platform. Furthermore, the downstream processing section is carried out with a rather concentrated biomass flow having a high PHA content.

The present invention provides an integrated closed-cycle process, wherein each flow is recovered and exploited without any generation of untreated secondary waste streams.

The present invention may be better understood in the light of the following embodiment examples.

EXPERIMENTAL PART

1a. Calculation of Characteristic Parameters in AnS The solids (TS and TVS) reduction was calculated as the percentage ratio between the solids measured in the AnS output, and the input solids typical of the feedstock (TSo and TVSo).

The organic matter solubilization was calculated as the ratio between the CODSOL produced in AnS (net of those already present in the unfermented mixture), and the total COD (CODTOT(O)), or the TVS(O) of the unfermented feedstock.

The fermentation products yield (YVFA) was calculated as the ratio between the VFAs produced only in AnS, and CODSOL/O), CODTOT/O), and TVS(O) of the unfermented feedstock.

1b. Calculation of Characteristic Parameters in AS1 and AS2

To express the observed growth rate, the storage yield, and the specific substrate consumption and polymer formation rates, it was preferred to convert the experimental data in terms of COD, by unifying all the concentrations in a single unit of measurement. That is, the active biomass (XA) or non-polymer biomass expressed as:

XA = (VSS-PHA)*1 .42.

The specific soluble COD (CODSOL) removal rate was calculated as the ratio between the amount of removed CODSOL (AS) and the length of the feast phase (t), per unit of active biomass XA:

(_q_S ^feast) = AS/(t-X_A).

The polymer stored (DRHA) during the feast phase was calculated as the difference between the PHA concentration at the end of the feast phase and at the end of the cycle. The specific production rate was calculated from the ratio between the stored PHAs and the length of the feast phase (h) per unit of XA:

qpfeast ₌ DR HA/(Ϊ·CA).

The storage yield during the feast phase was determined by the ratio between stored PHAs and the removed COD: Yp/s^feast = APHA/AS.

The observed yield (YOBS^sbr) was determined from the ratio between the VSS concentration representative of the medium and the applied OLR load:

YOBS^SBR = VSS/OLR. The polymer content in the biomass was calculated as the ratio between the concentration of PHAs and VSS (the latter as a sum of XA and stored PHAs): %PHAs = PHAsA/SS = PHAS/(XA+PHAS). 2. Feedstock Characterization

The mixture used to feed the acidogenic fermenter (AnS) was sampled weekly in the Treviso water purification plant. The withdrawal of OFMSW and secondary sludge was carried out separately; the mixing took place in a 1.0 m³ container and the total amount withdrawn was of between 0.6-0.7 m³, consisting of a thickened secondary sludge volumetric percentage of between 55-60% v/v, and pressed OFMSW in a percentage equal to 40-45% v/v.

In the following Table 1 , the main chemical parameters characterizing the aforesaid mixture are reported.

TABLE 1. Mean Values and standard deviations of the main parameters characterizing the feedstock used for the AnS acidogenic fermentation stage.

As it can be seen from the data in the table, the solids content in the mixture was just under 100 gTS/Kg, of which more than 80% consisted of volatile solids of biodegradable organic matrix. The mixture was also subject to a partial and rapid fermentation, before the AnS fermentation process itself, as shown by the VFA content, equal to about 15% with respect to the soluble COD.

3. Acidogenic Fermentation (AnS)

The fermentation of the organic mixture was carried out in an anaerobic reactor with an operating volume of 380 L, a CSTR (Continuous Stirred Tank Reactor) configuration, and thermostated in thermophilic conditions (55°C). The operating conditions provided for an HRT (equal to SRT) of 6 d, with an applied organic load (OLR) equal, in average, to 9.2 ± 0.3 KgVS/m³ d; this value varied based on the solids content in the feedstock that, as mentioned above, was sampled weekly and was, therefore, subject to a variation (albeit limited) in the characteristics thereof. Moreover, the fermentation stage was not equipped with a pH control system, being the alkalinity of the feedstock higher than 2000 mgCaCCb/L and, therefore, sufficient to buffer the system during the acid fermentation stage.

Figure 3 illustrates the trend of all the different VFAs produced after starting the fermentation process; after a start-up phase of around 14 days (i.e. 2.3 HRT), the acid effluent was characterized by an approximately constant VFA concentration, and above 20 gCOD/L, with a significant predominance of butyric acid, equal to about 50% of CODVFA (10.2 ± 0.6 gCOD/L). In decreasing order, the spectrum of the other VFA products was the following: acetic acid (4.8 ± 0.3), propionic acid (2.4 ± 0.3), caproic acid (1 .7 ± 0.1 ), isocaproic acid (1 .1 ± 0.3), pentanoic acid (0.89 ± 0.08), isopentanoic acid (0.7 ± 0.2), heptanoic acid (0.7 ± 0.1 ), isobutyric acid (0.4 ± 0.1 ).

In terms of degree of solids reduction and solubilization of the COD in the feedstock performances, the thermophilic operating conditions ensured a solids reduction close to 50%, 45% and 47% for TS and TVS, respectively. The degree of solubilization of the organic matrix was calculated based on CODTOT and TVSs entering the fermenter and expressed as a function of the quantified CODSOL: 0.1 7 ± 0.01 CODSOL/CODTOT and 0.16 ± 0.01 CODSOL/CODTVS. AS regards to VFA yield (YVFA), calculated based on CODTOT and TVS in the feedstock entering AnS, was equal to 0.27 ± 0.01 CODVFA/CODTOT and 0.24 ± 0.01 CODVFA/CODTVS. The following Table 2 shows the the fermentation stage performances and the chemical characteristics of the fermented matter used after solid/liquid separation through coaxial centrifuge and membrane, in the subsequent aerobic stages of the platform according to the process of the present invention.

TABLE 2. Mean Values and standard deviations of the fermented acid exiting AnS; fermentative performance in terms of solubilization, solids reduction and VFA yield

The fermentation stage also led to about 50% increase in the concentration of ammoniacal nitrogen (625 mgN-NH₄ ⁺/L), therefore in a soluble form, with respect to the initial amount in the mixture; unlike soluble phosphorus (orthophosphate), whose concentration remained constant between entering and exiting the fermentation stage (about 450 mgP-P04³ /L). Therefore, based on the concentrations of nitrogen and phosphorus in a soluble or readily bioavailable form, it can be concluded that the acid fermentation was characterized by a [CODVFA: N: P] ratio equal to [100:2.7: 1 .9], therefore partially limiting in ammoniacal nitrogen, based on the reference [100:5: 1 ] characteristic of balanced aerobic growth media. 4. Mixed microbial consortium (MMC) selection and enrichment - Stage AS1

To inoculate the selection reactor, about 30 L of thickened aerobic sludge, from the Treviso wastewater treatment plant, was used. The SBR reactor, with a working volume of 120 L, was fed under ADF regimen, using an operation cycle lasting 6 hrs (4 cycles per day). The feeding took place at the beginning of each cycle, for a relatively short period of 2 min. No sedimentation phase was provided for, the Hydraulic Retention Time (HRT) was equal to the Sludge Retention Time (SRT), i.e. 1 .0 d. Therefore, since the SBR reactor is continuously aerated, all the excess biomass was purged for 1 min, immediately before the end of each cycle. The OLR load, subject to fluctuations based on the AnS acidogenic fermenter performance, averaged 5.2 ± 0.2 gCODsoiVL d, and 2.8 ± 0.4 gCODvFA/L d. The SBR reactor was not equipped neither with a pH control nor with a temperature control; it was aerated by means of special blowers, whose flow rate guaranteed dissolved O2 concentrations that were never limiting and of between 2.0 - 8.5 mg/L.

The feeding solution exiting from AnS was conveyed into the first solid/liquid separation unit, consisting of a coaxial centrifuge (C1 ), equipped with a filtering bag having a porosity of between 5 - 10 pm. Section C1 allowed to obtain an LS1 filtrate with a solids content reduced by about 83% and 80% based on TS and TVS, respectively, with respect to the fermented HS. The solids concentration (measured as a suspended form) in LS1 was 1 1 ± 1 gTSS/L and 10 ± 1 gVSS/L. In terms of recovered volumetric load, LS1 had a volume of between 80-85% with respect to HS entering section C1 . Furthermore, the centrifugation step did not alter the chemical characteristics of the filtrate, i.e. CODSOL, CODVFA and nutrients (N-NH⁴⁺ and P-PO4³ ) concentrations.

Figure 4 allows to view the trends of PHAs, VFAs and dissolved O2 following a detailed analysis carried out on a typical operation cycle, in optimal operating conditions of the process. In fact, in the specific case shown, the feast phase had a duration of 35 minutes, equal to 9.7% of the length of the cycle. Therefore, this short duration ensured a selective pressure adequate for selection of a specific PHA- producing microbial consortium. Furthermore, since the early AS1 starting phases, the system has shown a rapid adaptability to the enforced ADF process conditions, then maintaining a constant selective pressure throughout the entire operation, shown as an illustrative example. The fluctuations observed in terms of storage response, quantified as rate and efficiency, were minimal, thus demonstrating that the system was characterized by a certain reliability and continuity in the enforced selective pressure, over the entire operation duration (about 80 days).

Figures 5 and 6 illustrate the trends of suspended solids (TSS and VSS), and PFIAs at the end of the cycle and at the end of the feast phase (respectively minimum and maximum concentration).

With the exception of the first week, corresponding to start-up of AS1 , and the short period at day 65, the solids concentration in AS1 may be defined with a good constant approximation. TSS and VSS mean value was 2561 ± 156 mgTSS/L and 2036 ± 121 mgVSS/L, respectively, with a VSS/TSS ratio of 0.80 ± 0.01 . This ratio indicates the presence of a solids amount equal to 20%, in AS1 , and different from the organic and/or cellular matrix. Part of this amount was due to the inert fraction present in the fermented feedstock LS1 , which was not completely removed following the centrifuge C1 step.

The PHA concentration in AS1 , on the other hand, is subject to greater fluctuations; however, since the start of the aerobic process, the difference between the PHA concentration at the feast end and at cycle end was clear (with the exception of a short period close to day 30). This difference was the clear evidence of the stimulated storage response in the selected microbial consortium, and therefore of the correct operation of the selection reactor.

The following Table 3 shows the main parameters and performance monitored and quantified during the whole operation of the AS1 aerobic reactor.

TABLE 3. Mean Values and standard deviations of the main parameters and process performance monitored in the SBR selection reactor (AS1 )

As it can be seen from the reduced standard deviation, the length of the feast phase did not undergo significant fluctuations and the mean value thereof was equal to 50 minutes, or 14% with respect to the total length of the cycle; a value low enough to guarantee adequate selective pressure on the mixed microbial consortium.

The produced PHA is the P(3-HB)-co-(3HV) copolymer, consisting of two constituent monomers, as expected on the basis of the VFA spectrum of the fermented acid: 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV); the latter, produced starting from VFAs having an odd number of carbon atoms (essentially C3-C5), was present in a percentage, by weight, of 12.4% gFIV/gPFIA.

The MMC consortium selection/enrichment step further contributed to a substantial reduction in CODSOL, reduced by about 83 ± 1 %; the high percentages of reduction of ammoniacal nitrogen and orthophosphate ion, equal to 71 ± 5% e 94.9 ± 0.7%, respectively, are also to be reported.

5. PHAs Accumulation (AS2) The fermented acid, subjected to centrifugation (C1 ), and having therefore significantly reduced solids content (LS1 ), was sent to the membrane ultrafiltration section (M1 ) to obtain a permeate with zero or negligible solids content (LS2). The ultrafiltration treatment also had a second substantial advantage: that is, the reduction of CODSOL content by an amount equal to 10 ± 2%, this figure, compared to a negligible decline in the VFA content (less than 1 % decrease), led to a 13.0 ± 0.9%, increase in the CODVFA/CODSOL ratio, thus making LS2 particularly suitable for conducting the accumulation stage.

The storage potential of the selected biomass was tested during accumulation batch tests, carried out with LS2 permeate (Batch 1 P and 2P, Figure 7), by applying an OLR 4-5 times higher than that applied in AS1. From the PFIA concentration trend in the figure, it can be seen that the selected biomass had not reached the polymer saturation over the 6 hours of testing, especially in the test 1 P, wherein the increase was roughly linear. This observation may be justified by the presence of a growth response, albeit lower than the storage one, in the PHA-producing microorganisms selected in the previous aerobic step.

At the end of the two tests, the PHA concentration in the aerobic reactor was of about 1500 mg/L (test 1 P) and 1950 mg/L (test 2P), corresponding to a PHA weight percentage in the biomass of 38% gPHA/gTSS in both tests, and 44% (1 P) and 49% (1 P) gPHA/gVSS.

Claims

1 . A process for the production of one or more polyhydroxyalkanoates (PHAs), said process comprising:

a first stage of anaerobic fermentation (AnS), wherein a fermentable organic waste having a Total Suspended Solids (TSS) greater than 10.0 g/L is subjected to acidogenic anaerobic fermentation, wherein the organic carbon or COD (Chemical Oxygen Demand) is converted into volatile fatty acids (VFAs), thus producing a fermented organic effluent (HS) concentrated in VFAs;

subjecting HS to a solid/liquid separation by filtration (C1 ) with a filter having a porosity of 5-10 pm to obtain a filtered fermented organic effluent (LS1 ) having a suspended solids content lower than 10 g/L and higher than 1 g/L;

2. The process according to claim 1 , wherein the organic fermentable waste is OFMSW (Organic Fraction of Metropolitan Solid Waste).

3. The process according to any one of the preceding claims, wherein the portion of LS1 sent to stage AS1 is equal to 35-45% by volume with respect to the whole LS1 and the remaining portion of LS1 is subjected to solid/liquid separation by filtration (M1 ).

4. The process according to any one of claims 1 -2, wherein the whole LS1 is subjected to solid/liquid separation by filtration (M1 ) and the fermentation step AS1 is fed with a portion of LS2 equal to 35-45% by volume with respect to the total of LS2.

5. The process according to any one of the preceding claims further comprising, before the solid/liquid separation by filtration (M1 ), subjecting the remaining portion of LS1 to a coagulation/flocculation treatment.

6. The process according to any one of the preceding claims, wherein the solid effluents (S1 and S2) resulting from C1 and M1 separations, respectively, are subjected to anaerobic digestion (AD) for the recovery of energy from anaerobic digestion.

7. The process according to any one of the preceding claims, wherein the mixture exiting from AS2 is subjected to separation (C2 or M2) to separate the PHA-rich biomass from the liquid medium.

8. An integrated system for the production of one or more polyhydroxyalkanoates (PHAs) by a process according to the preceding claims, said system comprising: a section for the anaerobic fermentation (AnS), said section AnS comprising an inlet to receive a fermentable organic waste and an outlet to discharge a fermented organic effluent (HS);

a second filtering unit with membrane (M1 ) comprising a membrane having a porosity of 0.2-1.0 pm, said unit M1 comprising an inlet in fluid connection with the outlet of C1 and an outlet to discharge a doubly-filtered fermented organic effluent (LS2);

9. The system according to claim 8, wherein the first filtering unit (C1 ) is a vertical axis centrifuge equipped with a filter bag having a porosity in the range of 5-10 pm.

10. The system according to any one of the claims 8-9 further comprising a coagulation/flocculation unit (F) comprising an inlet in fluid connection with the outlet of C1 and an outlet in fluid connection with the inlet of M1.

11. The system according to any one of the claims 8-10 further comprising an anaerobic digestion unit (AD) comprising a first inlet in fluid connection with a second outlet of C1 and a second inlet in fluid connection with a second outlet of M1.

12. The system according to any one of the claims 8-11 , wherein SBR comprises a second inlet in fluid connection with the outlet of M1.

13. The system according to any one of the claims 8-12 further comprising a third separation unit (C2 or M2) comprising an inlet in fluid connection with the outlet of AS2 and an outlet to discharge the PHA-rich biomass and a second outlet to discharge the liquid medium.