A biotechnological process for producing pha

Abstract

A biotechnological process for producing PHA is described, comprising the steps of: subjecting an initial mixture containing a mixed bacterial mass to a biological selection process, so as to obtain a first intermediate mixture containing mainly selected bacteria able to store a carbonaceous source (for example VFA) in the form of PHA; introducing said first intermediate mixture in a tank reactor (120, 215) operating under anoxic con-ditions; introducing in said tank reactor (120, 215) also an aqueous nutrient solution, containing both the carbonaceous source (for example VFA) and nutrients, so as to ob-tain within said tank reactor (120, 215) a second intermediate mixture comprising PHA-containing bacteria; treating said second intermediate mixture with a solid-liquid separa-tion apparatus (125, 220), for example a vertical axis centrifuge, a decanter centrifuge or any other device adapted to execute a centrifugal separation of the second interme-diate mixture, so as to obtain a final mixture having a higher solids concentration with respect to that of the second intermediate mixture; subjecting the final mixture to a PHA extraction process.

Description

[0001] A BIOTECHNOLOGICAL PROCESS FOR PRODUCING PHA

[0002] Technical Field

[0003] The present invention relates to a biotechnological process for producing poly hydroxyalkanoates, commonly referred to with the acronym PHA.

[0004] Prior art

[0005] Polyhydroxyalkanoates (PHA) are biodegradable, biocompatible polyesters with physi- cal / chemical properties of interest for a significant number of industrial applications.

[0006] PHAs are stored as granules in the cell cytoplasm of certain types of microorganisms when the latter are subjected to stress conditions caused, for example, by the limitation of a nutrient, dissolved oxygen or electron donor / acceptor (Anderson and Dawes 1990; Majone et al. 1996; Satoh et al. 1998; Gujeret al. 1999).

[0007] All the industrial processes implemented to date for producing PHA use pure microbial cultures (single or genetically modified strains), which can accumulate PHA up to 90% of cell dry weight (Lee and Choi 1998; Madison and Huisman 1999; Grothe et al. 1999).

[0008] However, these processes generally have a rather high cost, typically four to nine times greater than that of synthetic plastics, which is mainly attributable to the fermentation (energy consumed for sterilisation and cost of the substrate) and aeration required for the biological process.

[0009] For this reason, although PHAs are relatively well characterised in terms of properties and market applications, the dissemination and industrial production of these polymers is still low with respect to other biodegradable polymers and synthetic plastics.

[0010] Therefore, the need is felt to develop more efficient and cost-effective technologies to make PHA production costs competitive with respect to products with similar characteristics and fields of application.

[0011] In order to meet this need, the possibility of producing PHA from mixed microbial cultures (MMC) was evaluated, especially in the context of studying the functioning mechanism of bacterial consortia involved in wastewater purification (activated sludge plants and / or secondary treatments).

[0012] Several research groups have shown that under certain conditions, viz., where there are very wide fluctuations in substrate availability, activated sludge bacterial communities in civil and industrial sewage treatment plants tend to utilise (and accumulate) large amounts of PHA as an energy reserve to survive in feast and famine (F&F) regimes. In order to induce this (ecological) adaptation in a controlled manner, it is necessary to set up within an aerated system, for example within a sequencing batch reactor (SBR), an inoculum of a mixed bacterial culture, which can consist of activated sludge from wastewater purification processes.

[0013] This aerated system is then fed with nutrients, for example nitrogen and phosphorous, and an easily degradable substrate containing a carbonaceous source, for example volatile fatty acids, commonly referred to with the acronym VFA, which can be obtained by fermenting waste substrates or effluents.

[0014] In particular, the feeding of VFAs and nutrients into the sequencing batch reactor can occur intermittently, in a manner commonly referred to as dynamic aerobic feeding.

[0015] Thereby, following nutrient feeding, a feast condition occurs, during which the PHA-accu- mulating organisms in the mixed bacterial culture quickly consume the carbonaceous source (for example the VFAs) contained in the substrate and accumulate it in the form of PHA within their own cells.

[0016] When the supply of VFAs and nutrients is suspended, a famine condition ensues, during which only organisms that have been able to store PHA in the feast step are in a position to grow, consuming precisely the accumulated PHA, while the other organisms tend to succumb.

[0017] Repeatedly this feast&famine cycle several times therefore results in the selection of microbial communities, including for example prokaryotes of the genera Azoarcus sp., Am- aricoccus sp., Tahuera sp., Rhodobacter sp., Alcaligenes sp., Pseudomonas sp., which are indeed capable of accumulating PHA effectively and in large amounts.

[0018] The microbial communities thus selected can then be transferred into a second aerated sequencing batch reactor, with the introduction of the substrate containing the carbonaceous source (for example VFA), in order to cause the growth and multiplication of the selected organisms and the accumulation of PHA under aerobic conditions.

[0019] Subsequently, the biomass obtained in the second sequencing batch reactor is withdrawn and first subjected to a concentration process, so as to increase the solids concentration thereof, and then to a PHA extraction process, which can finally be used for various applications, not exclusively for producing biodegradable plastics and / or plasticisers.

[0020] A drawback of this solution lies in the fact that the PHA accumulation step, which is performed within the second sequencing batch reactor operating under aerobic conditions, must occur without nutrient supply and only with the supply of the carbonaceous source (for example VFA), which implies the need to have two separate streams available: a first stream dedicated to the supply of the carbonaceous source only (for example VFA), which can be fed to both the first and the second reactor, and a second stream dedicated to the supply of nutrients, which can only be fed to the first reactor.

[0021] However, if the carbonaceous source (for example VFA) originates from fermentation processes of waste substrates or effluents, or regardless from similar processes, it is generally difficult to separate the carbonaceous source from the nutrients, if not by means of the use of additives, for example MgO, to cause the nutrients to precipitate (for example phosphorous) and allow the removal thereof by means of filtration.

[0022] Therefore, the need to create these two separate streams represents a very significant cost factor which, added to the costs required for the continuous supply of oxygen within both sequencing batch reactors operating under aerobic conditions, contributes to making the entire PHA production process economically uncompetitive.

[0023] The same need to use two separate streams (carbonaceous source and nutrient source) is also found in other solutions, including the one described in Italian patent No. 102014902318854, although in the latter case, the process in the first sequencing batch reactor occurs by alternating anoxic and aerobic steps which allow to remove ammoniacal nitrogen with the aim of purifying the wastewater of such a pollutant.

[0024] In addition to this, it should be noted that the approaches outlined above, which utilise sequencing batch reactors (SBR), apply well to industrial processes which produce the fermented effluent (viz., the carbonaceous source) discontinuously or, at least, in amounts which can be stored during the famine steps of the PHA production process.

[0025] However, when the fermented effluent comes from wastewater treatment plants or industrial plants operating continuously, it would be preferable to implement a system for producing PHA which is also capable of operating continuously, so that it can be more easily integrated with existing treatment plants.

[0026] However, the selection of PHA-accumulating microorganisms in a continuous system, viz., within continuous stirred-tank reactors (CSTR) has only been tested on a laboratory scale (Brison et al.2023), showing that, by using effluent from municipal sewage treatment plants within CSTRs, an average of 30% PHA accumulation can be achieved (0.3 grams of PHA for every gram of biomass), but has not yet provided solutions applicable on an industrial scale.

[0027] Disclosure of the invention

[0028] In light of the above, an aim of the present invention is to provide a biotechnological process for producing PHA which can be more economical overall than those currently available.

[0029] Another aim is to provide a process which can possibly, although not necessarily, be performed continuously, in this case in continuous stirred tank reactors (CSTR).

[0030] These and other objects are achieved thanks to the characteristics of the invention which are reported in the independent claim. The dependent claims outline preferred and / or particularly advantageous aspects of the invention which however are not strictly required for the implementation thereof.

[0031] In particular, an embodiment of the present invention provides a biotechnological process for producing PHA, which comprises firstly the step of subjecting an initial mixture containing a mixed bacterial mass to a biological selection process, so as to obtain a first intermediate mixture containing mainly selected bacteria capable of storing a carbonaceous source (for example, VFA) in the form of PHA.

[0032] According to an aspect of the invention, the initial mixture can comprise activated sludge, for example activated sludge comprising sewage sludge obtained from civil and / or industrial and / or livestock effluents.

[0033] It should be specified herein that activated sludge is intended as a suspension of a biomass (for example bacteria, protozoa, amoebae, rotifers and other microorganisms) in water, typically but not necessarily in flakes, of the type used in wastewater treatment.

[0034] This biomass can contain both autotrophic organisms, viz., those capable of synthesising their own organic molecules from inorganic substances and using energy not derived from assimilated organic substances, and heterotrophic organisms, viz., those which consume organic compounds previously synthesised by other organisms.

[0035] Sewage sludge is defined as a material separated, for example by sedimentation, from sewage from a sewage treatment plant, for example sewage deriving from wastewater, municipal and / or industrial effluents, livestock effluents or leachate from a MSW (municipal solid waste) landfill.

[0036] According to another aspect of the invention, the first intermediate mixture obtained from the biological selection process can have a concentration of bacteria capable of storing PHA comprised between 1 and 15 g / l (inclusive), viz., an amount of bacteria comprised between 1 and 15 grams of bacteria (inclusive) per litre of the first intermediate mixture obtained from the selection process, for example comprised between 1 and 10 g / l (inclusive).

[0037] Subsequently, the biotechnological process of the present invention comprises the steps of:

[0038] - introducing the aforesaid first intermediate mixture into a tank reactor operating under anoxic conditions, and

[0039] - introducing in said tank reactor also an aqueous nutrient solution containing both the carbonaceous source (for example VFA) and nutrients, so as to obtain within said tank reactor a second intermediate mixture comprising bacteria containing PHA.

[0040] According to an aspect of the invention, the aqueous nutrient solution can be obtained by acidogenic or acetogenic fermentation of wet biomass.

[0041] The term 'wet biomass' generally means fermentable organic matrices.

[0042] In the specific case, the wet biomass can comprise one or more materials selected from the group consisting of:

[0043] - sewage sludge obtained from civil and / or industrial and / or livestock effluents,

[0044] - waste and / or organic waste from the agri-food industry,

[0045] - organic fraction of municipal solid waste.

[0046] Thanks to this solution, the PHA production process is effectively capable of exploiting waste products or regardless by-products generated by other existing biotechnological processes in a cost-effective manner.

[0047] For example, the aforesaid aqueous nutrient solution can be obtained through the anaerobic digestion phase of wet biomasses which already occurs in the anaerobic digesters found in common wastewater and / or agri-food waste and / or organic waste treatment plants.

[0048] However, it is not ruled out that in other embodiments, the aqueous nutrient solution can be obtained by other technological processes, including, for example, fermentation processes for producing hydrogen (H2), chemical processes for producing VFA, and other equivalent processes.

[0049] To provide an indication of the composition of the aqueous nutrient solution, the parameter called COD can be used.

[0050] COD (acronym for Chemical Oxygen Demand) represents the amount of oxygen required to convert all the organic substances present in a sample of an aqueous solution into carbon dioxide and water, and is therefore one of the parameters commonly used to indirectly measure the content of organic substances in a solution.

[0051] The COD value can be expressed in grams per litre (g / l), viz., grams of oxygen (O2) consumed to convert all the organic substances present in one litre of the solution to be measured.

[0052] The measurement is generally performed using a strong oxidising agent (dichromate or permanganate) and determining the amount of residual oxidiser at the end of all the oxidation reactions.

[0053] If the test is executed on a sample of solution which has been previously filtered, typically through a 0.45 mm filter, mention is more appropriately made of 'soluble COD'.

[0054] In the context of the present discussion, 'soluble COD', viz., the amount of oxygen required to convert all the organic substances present in a sample of a previously filtered aqueous solution into carbon dioxide and water, will be referred to as 'total soluble COD' in order to distinguish it from 'specific soluble COD', which will instead represent the amount of oxygen required to convert only one (specific) of the organic substances present in a sample of a previously filtered aqueous solution into carbon dioxide and water. 'Specific soluble COD' is thus an indirect measurement of the content of that specific organic substance within the solution.

[0055] Having made this premise, a preferred aspect of the invention is that the aqueous nutrient solution can have a total soluble COD value greater than or equal to 5 g / l, for example comprised between 5 and 50 g / l (inclusive), more preferably greater than or equal to 50 g / i-

[0056] In other words, for each litre of the aqueous nutrient solution, 5 or more grams of oxygen (O2) is required to convert all the organic substances in the aforesaid litre of the aqueous nutrient solution into carbon dioxide and water, for example between 5 and 50 grams of oxygen, more preferably 50 or more grams of oxygen.

[0057] According to another aspect of the invention, the aqueous nutrient solution can contain a concentration of the carbonaceous source (for example, of VFA) corresponding to a specific soluble COD value, viz., attributable to the conversion of the carbonaceous source alone (for example, of VFA alone), greater than or equal to 2 g / l, for example comprised between 2 and 25 g / l (inclusive), more preferably greater than or equal to 25 g / l.

[0058] In other words, for each litre of the aqueous nutrient solution, 2 or more grams of oxygen (O2) are required to convert all and only the molecules of the carbonaceous source (for example VFA) present in said litre of the aqueous nutrient solution into carbon dioxide and water, for example between 2 and 25 grams of oxygen, more preferably 25 or more grams of oxygen.

[0059] From a different point of view, the aqueous nutrient solution can contain a concentration of the carbonaceous source (for example VFAs) corresponding to a value greater than or equal to 20% of the total soluble COD value of the aqueous nutrient solution itself, for example comprised between 20% and 50% (inclusive) of the total soluble COD value of the aqueous nutrient solution, more preferably greater than or equal to 50% of the total soluble COD value of the aqueous nutrient solution.

[0060] In other words, given the amount of oxygen (O2) globally required to convert all the organic substances present in the aqueous nutrient solution into carbon dioxide and water, a portion equal to or greater than 20% of said amount is used to convert only the molecules of the carbonaceous source (for example of VFA) into carbon dioxide and water, for example a portion comprised between 20% and 50% (inclusive) of said amount, more preferably a portion greater than or equal to 50% of said amount.

[0061] Another parameter which can be used to provide an indication of the composition of the aqueous nutrient solution can be TSS.

[0062] TSS (acronym for Total Suspended Solids) represents the amount of solids, measured for example in grams per litre (g / l), present in suspension and which can be separated by mechanical means such as filtration or centrifugation of a sample of the suspension.

[0063] An aspect of the invention therefore envisages that the aqueous nutrient solution can have an amount of suspended solids corresponding to a TSS value less than or equal to 2 g / l.

[0064] Another aspect of the invention is that the aqueous nutrient solution can have a pH value greater than or equal to 6, preferably comprised between 6.5 and 8.8 (inclusive).

[0065] Turning to more specific aspects, as anticipated, the carbonaceous source present in the aqueous nutrient solution can comprise (or consist of) volatile fatty acids, viz., VFA, preferably short-chain volatile fatty acids, for example selected from the group consisting of acetic acid, propionic acid and butyric acid.

[0066] The nutrients in the aqueous nutrient solution can instead comprise substances belonging to (or selected from) the group consisting of:

[0067] - nitrogen and compounds thereof, for example nitrates and ammoniacal nitrogen,

[0068] - phosphorus and compounds thereof, for example phosphates,

[0069] - sulphur and compounds thereof, for example sulphates,

[0070] - potassium and compounds thereof,

[0071] - magnesium and compounds thereof,

[0072] - calcium and compounds thereof.

[0073] Specifically, the aqueous nutrient solution can contain a nitrogen concentration greater than or equal to 1.4% of the total soluble COD value of the aqueous nutrient solution itself, for example comprised between 1.4% and 20% of the total soluble COD value of the aqueous nutrient solution (inclusive).

[0074] In other words, the ratio of the total soluble COD in the aqueous nutrient solution to the concentration (expressed in grams per litre) of nitrogen in the same aqueous nutrient solution is less than or equal to about 70, for example comprised between 5 and about 70 (inclusive).

[0075] The aqueous nutrient solution can also contain a phosphorous concentration greater than or equal to 0.1% of the total soluble COD value of the solution, for example comprised between 0.1% and 2.7% of the total soluble COD value of the aqueous nutrient solution (inclusive).

[0076] In other words, the ratio of the total soluble COD of the aqueous nutrient solution to the concentration (expressed in grams per litre) of phosphorus in the same aqueous nutrient solution is less than or equal to 1000, for example comprised between approximately 38 and 1000 (inclusive).

[0077] According to a different aspect of the invention, the second intermediate mixture obtained in the tank reactor can have a concentration of PHA-containing bacteria comprised between 1 and 15 g / l (inclusive).

[0078] The biotechnological process of the present invention further comprises the step of treating the second intermediate mixture, obtained in the tank reactor, with a solid-liquid separation apparatus, so as to obtain a final mixture having a higher solids concentration with respect to that of the second intermediate mixture. In particular, the solid-liquid separation apparatus can obtain the final mixture by performing a solid / liquid separation, for example by centrifugation of the second intermediate mixture.

[0079] For example, the solid-liquid separation apparatus can comprise a device selected from:

[0080] - a vertical axis centrifuge,

[0081] - a decanter centrifuge.

[0082] According to an aspect of the invention, the final mixture obtained with the solid-liquid separation apparatus can have a concentration of PHA-containing bacteria comprised between 50 and 250 g / l (inclusive), or a mass concentration of PHA-containing bacteria comprised between 5% and 25% of the total mass of the mixture (inclusive).

[0083] Another aspect of the invention is that the final mixture can have a mass concentration of PHA comprised between 10% and 70% of the total mass of bacteria present in the final mixture (inclusive), for example comprised between 10% and 60% (inclusive).

[0084] The biotechnological process according to the present invention lastly comprises the step of subjecting the final mixture to a PHA extraction process.

[0085] The PHA extraction process can be achieved by various methods individually known in the art, through the integration of various technologies, for example by extraction with organic solvents, followed by separation with physical and mechanical methods, for example but not necessarily through the use of ultrasound.

[0086] One of the most advantageous features of the biotechnological process according to the invention is that, especially due to the anoxic conditions under which the PHA accumulation step occurs, it is advantageously possible to feed the tank reactor not only with the carbonaceous source (for example with VFA), but also with nutrients.

[0087] That is, the carbonaceous source (for example VFA) and the nutrients are administered in the form of a single stream of a mixture containing both, without the need to arrange any nutrient removal system or, at most, using much simpler and consequently significantly cheaper removal systems than those currently envisaged.

[0088] The fact that the PHA accumulation step occurs under anoxic conditions also means that less air can be supplied to the tank reactor, thus also reducing the costs associated with aerating the biological process.

[0089] Ultimately, therefore, the biotechnological process for producing PHA which is the subject-matter of the present invention is advantageously cheaper with respect to those proposed so far.

[0090] It should be clarified herein that, in the context of the present discussion, the term 'operating under anoxic conditions' is intended to indicate a reactor in which the amount of possibly insufflated air (aeration) and / or other process parameters are controlled so that the redox potential of the solution / mixture contained therein has a value less than or equal to +50 mV (millivolts), and / or so that the amount of oxygen (O2) dissolved in said solution / mixture is less than or equal to 1 mg / l, viz., less than or equal to one milligram of dissolved oxygen per litre of the solution / mixture.

[0091] On the other hand, the term 'operating under aerobic conditions' refers to a reactor in which the amount of any air insufflated (aeration) and / or other process parameters are controlled so that the redox potential of the solution / mixture contained therein has a value greater than +50 mV (millivolts), and / or so that the amount of oxygen (O2) dissolved in said solution / mixture is greater than 1 mg / l.

[0092] With specific reference to the tank reactor operating under anoxic conditions, it is preferable that the redox potential of the second intermediate mixture contained therein is maintained at a value comprised between +50 mV and -200 mV (inclusive), for example, comprised between +50 mV and -150 mV (inclusive), preferably approximately equal to -80 mV, and / or that the amount of oxygen (O2) dissolved in said second intermediate mixture contained within said tank reactor is maintained at or below 1 mg / l.

[0093] From an implementation point of view, the biotechnological process outlined above can be carried out in accordance with at least two alternative embodiments (or configurations). Another aspect of the present invention envisages that the tank reactor can be an aerated reactor, viz., a reactor provided with an air flow insufflation system therein.

[0094] Thereby, it is advantageously possible to properly regulate the oxygen supply within the tank reactor to maintain a controlled anoxic condition.

[0095] The tank reactor can also be a stirred-tank reactor, viz., the contents of which are subjected to continuous stirring.

[0096] Thereby, it is advantageously possible to achieve greater uniformity of the second intermediate mixture contained therein.

[0097] To achieve this effect, the tank reactor can be supplied with airflows and / or other streams in input with a fairly high velocity, or it can be provided with suitable stirring means therein, such as an impeller. According to a first embodiment, the biological selection process can envisage:

[0098] - arranging an inoculum of the initial mixture within a first continuous flow selection reactor, preferably operating under anoxic conditions,

[0099] - feeding said first selection reactor with a continuous stream of said aqueous nutrient solution, so as to obtain a first suspension,

[0100] - conveying a continuous stream of said first suspension into at least a second continuous flow selection reactor, preferably operating under aerobic conditions and / or preferably operating without the further supply of any carbonaceous source (for example VFA) and / or any nutrient, so as to obtain a second suspension,

[0101] - subjecting a continuous stream of said second suspension to a clarification step so as to obtain a continuous stream of a third suspension having a higher solids concentration with respect to that of the second suspension,

[0102] - conveying the continuous stream of the third suspension within the first selection reactor so as to obtain the first intermediate mixture.

[0103] This embodiment configures a continuous-type selection process, in which the alternation of feast and famine steps, responsible for biological selection, is achieved 'spatially' in the transition between the various continuous-flow reactors.

[0104] Specifically, within the first selection reactor, thanks to the feeding of the carbonaceous source (for example VFA) and nutrients, the bacterial mass will be subjected to a feast step, in which bacteria capable of storing PHA will degrade the carbonaceous source and accumulate it in the form of PHA therein; whereas within the second selection reactor, in which neither nutrients nor the carbonaceous source (for example VFA) are fed, the bacterial mass will be subjected to a famine step, in which only those organisms which were capable of storing PHA in the feast step will be in conditions to grow, consuming precisely the previously accumulated PHA, while the other organisms tend to succumb.

[0105] By returning the organisms obtained at the outlet of the second selection reactor back into the first selection reactor, the latter will then be subjected to a new feast step and so on, until an initial intermediate mixture of selected bacteria capable of storing PHA effectively and in large amounts is obtained within the first selection reactor.

[0106] Thanks to this continuous selection process, the process of the invention lends itself to being effectively integrated with any process capable of producing the carbonaceous source (for example VFA) in a continuous manner, for example with the aforementioned fermentation processes used in wastewater treatment plants or industrial plants.

[0107] Furthermore, since in the first selection reactor the carbonaceous source (for example VFA) is administered in a mixture with the nutrients, the selection process also does not require the use of nutrient removal systems capable of providing two separate streams (carbonaceous source and nutrients).

[0108] Lastly, the possibility of operating the first selection reactor under anoxic conditions has the advantage of reducing the oxygen supply required to perform the selection step, which reduces costs.

[0109] In this regard, a preferred aspect of this first configuration envisages that the redox potential of the first solution contained in the first selection reactor is maintained at a value comprised between -450 mV and -120 mV (inclusive), for example equal to approximately -350 mV, and / or that the amount of oxygen (O2) dissolved in said first solution contained in the first selection reactor is maintained at a value less than or equal to 0.2 mg / l (milli- grams / litre).

[0110] On the other hand, the possibility of operating the second selection reactor under aerobic conditions has the effect of enhancing, during the famine step, the growth of bacteria which, in the previous feast step, were able to store PHA in their cells.

[0111] In this regard, a preferred aspect of this first configuration envisages that the amount of oxygen (O2) dissolved in the second solution contained in the second selection reactor is maintained at a value greater than or equal to 3 mg / l.

[0112] According to another aspect of this configuration, the first selection reactor can be sized to have an overall hydraulic retention time comprised between 1 hour and 12 hours (inclusive), generally varying depending on the characteristics of the aqueous nutrient solution in input.

[0113] Hydraulic retention time is intended as the average time a solution spends inside a flow through reactor.

[0114] This retention time allows the reactions involved in the biological process to occur more efficiently.

[0115] A further aspect of this configuration envisages that the first and / or second selection reactor can be a stirred tank reactor, viz., the contents of which are subjected to continuous stirring.

[0116] It is thereby advantageously possible to achieve greater uniformity of the solutions which are contained within the first and / or second selection reactor.

[0117] To achieve this effect, the first and / or second selection reactor can be fed with a stream in input having a fairly high velocity, or it can be provided with suitable stirring means therein, such as an impeller.

[0118] The first and / or the second selection reactor can also be aerated reactors, viz., reactors which are provided with a system for the insufflation of air therein.

[0119] Thereby, it is advantageously possible to suitably regulate the oxygen supply inside the reactors.

[0120] According to a different aspect of this configuration, the continuous flow of the first suspension exiting the first selection reactor can be conveyed, instead of into a single second selection reactor, into a system comprising a plurality of said second continuous flow selection reactors connected in series with each other, preferably each operating under aerobic conditions and / or preferably each operating without further supply of any carbonaceous source (for example VFA) and / or any nutrient.

[0121] Thereby, when passing through two or more second selection reactors in series, the bacterial mass is subjected to a longer and more intense famine step, as the amount of carbonaceous source (for example VFA) and nutrients which can possibly come from the first selection reactor is gradually reduced.

[0122] Regardless of the number of second selection reactors, an aspect of the invention envisages that said second selection reactors are sized to have a total hydraulic retention time comprised between 1 hour and 12 hours (inclusive), generally varying depending on the characteristics of the first suspension exiting the first selection reactor.

[0123] 'Total hydraulic retention time' is intended as the average time which elapses from when the first suspension from the first selection reactor is fed into the first of the 'second selection reactors' to when the same exits (obviously with different characteristics and following treatment) the last of the 'second selection reactors'.

[0124] Also in this case, this retention time allows the reactions involved in the biological process to occur more efficiently.

[0125] According to another aspect of this configuration, the clarification step of the second suspension can occur by sedimentation, for example with a sedimentation speed comprised between 0.05 m3 / hour and 1 m3 / hour (inclusive).

[0126] In any case, the clarification step of the second suspension can be achieved by means of a device selected from:

[0127] - a natural sedimentation tank, with or without the use of polyelectrolytes for flocculation, having for example a truncated cone-shaped tank, preferably not aerated and with a wall slope greater than or equal to 45°,

[0128] - a scraper bridge clarifier, using polyelectrolytes for flocculation,

[0129] - a sedimentation tank, with or without the use of polyelectrolytes for flocculation, connected to a centrifuge.

[0130] The clarification step of the second suspension naturally produces not only the more concentrated third suspension, which is returned to the first selection reactor, but also a continuous stream of a fourth aqueous suspension with a (much) lower solids concentration with respect to that of the second suspension.

[0131] In particular, this fourth suspension can have an amount of volatile suspended solids, also commonly referred to with the acronym VSS, less than or equal to 1 g / l (grams / litre), viz., it can comprise an amount of volatile suspended solids less than or equal to 1 gram per litre of the fourth suspension.

[0132] The fourth suspension can also have a total COD value less than or equal to 1.2 g / l, preferably equal to 1 g / l (grams / litre).

[0133] Finally, the fourth suspension can have an ammoniacal nitrogen concentration comprised between 200 mg / l and 1000 mg / l (milligrams / litre), including the extreme values.

[0134] According to an aspect of this embodiment, the stream of the fourth suspension obtained from the clarification step can be delivered to a water treatment plant.

[0135] Returning to the tank reactor used downstream of the selection process, a preferred aspect of this first configuration envisages that it too can be a flow through reactor, which receives a continuous stream of the first intermediate mixture (for example from the first selection reactor) and a second continuous stream of said aqueous nutrient solution to provide a continuous stream of the second intermediate mixture.

[0136] Thanks to this solution, the PHA accumulation step in the previously selected bacterial mass also occurs continuously, perfectly integrating with the selection process.

[0137] This flow through tank reactor can be sized to have a hydraulic retention time comprised between 3 hours and 24 hours (inclusive), for example comprised between 5 hours and 15 hours (inclusive), preferably between 8 hours and 12 hours (inclusive).

[0138] This retention time allows the reactions involved in the biological process to occur more efficiently.

[0139] Since the effectiveness of the reactions involved in the biological process can also depend on the temperature, another aspect of this first embodiment of the invention envisages that, within the flow through tank reactor, the second intermediate mixture is maintained at a temperature of less than or equal to 42°C, preferably comprised between 20°C and 35°C, for example approximately equal to 25°C.

[0140] Turning finally also to the solid-liquid separation apparatus placed downstream of the tank reactor, this first embodiment of the invention envisages that it too can be of a continuous type, for example a vertical axis centrifuge or a decanter centrifuge, so as to receive the continuous stream of the second intermediate mixture from the tank reactor and provide a continuous stream of the final mixture in output.

[0141] In doing so, essentially the entire PHA production process becomes continuous.

[0142] Using the solid-liquid separation apparatus, not only is the final mixture with a high PHA concentration naturally obtained, but also a liquid or supernatant fraction having a (much) lower solids concentration with respect to that of the final mixture.

[0143] According to an aspect of this first embodiment, the liquid fraction obtained in output from the solid-liquid separation apparatus can be fed within the first selection reactor.

[0144] In this way, it is advantageously possible to recover the residual amount of carbonaceous source (for example VFA) that can possibly be present in the aforesaid liquid fraction.

[0145] In accordance with a second embodiment of the present invention (or configuration), the biological selection process can envisage:

[0146] - arranging an inoculation of the first mixture within a sequencing (or batch) selection reactor, preferably operating under aerobic conditions, and

[0147] - pulsed (or intermittent) feeding into said sequencing batch selection reactor an amount of said aqueous nutrient solution.

[0148] In practice, feeding the aqueous nutrient solution into the sequencing batch selection reactor occurs over preset periods of time, interspersed with periods of time in which the nutrient solution is not fed.

[0149] This embodiment therefore configures a batch type of selection process, in which the alternation of feast and famine steps, responsible for biological selection, is achieved in a temporal sequence within the sequencing batch selection reactor.

[0150] Specifically, when the aqueous nutrient solution is administered, the bacterial mass inside the sequencing batch selection reactor is subjected to a feast condition, in which bacteria capable of accumulating PHA degrade the carbonaceous source and accumulate it in the form of PHA therein.

[0151] When, on the other hand, the aqueous nutrient solution is not administered in the sequencing batch selection reactor, the bacteria rapidly consume the remaining carbonaceous source until a famine condition is reached, in which only those organisms which were able to store PHA in the feast step are in a position to grow, consuming the previously accumulated PHA, while the other organisms tend to succumb.

[0152] By performing this cycle of feeding and suspension one or more times, it is advantageously possible to obtain, within the sequencing batch selection reactor, a mixture of selected bacteria capable of accumulating PHA effectively and in large amounts.

[0153] An advantage of this batch selection process is that, as in the previous case, no filtration systems are required, as the carbonaceous source (for example VFA) is fed into the sequencing batch selection reactor in a mixture with the nutrients.

[0154] With respect to the continuous case, this batch selection process can however be carried out with plants having a lower structural and construction complexity, as they consist of a smaller number of tanks and fewer elements (for example pumps) of a large size, making it more adaptable also to smaller scale plants (for example less than 5000 tonnes / year of aqueous input).

[0155] Furthermore, the possibility of operating the sequencing batch selection reactor under aerobic conditions has the effect of enhancing, during the famine steps, the growth of bacteria which, during the previous feast step, were able to store PHA in their cells.

[0156] In this respect, a preferred aspect of this embodiment is that the amount of oxygen (O2) dissolved in the first intermediate mixture contained in the sequencing batch selection reactor is maintained at a value greater than or equal to 1 mg / l, for example greater than or equal to 2 mg / l, and / or that the redox potential of said first intermediate mixture contained in the sequencing batch selection reactor is greater than or equal to 0 mV.

[0157] According to an aspect of this second embodiment, the sequencing batch selection reactor can be an aerated reactor, viz., a reactor provided with an air flow insufflation system therein.

[0158] Thereby, it is advantageously possible to suitably regulate the oxygen supply within the sequencing batch selection reactor. Another aspect of this embodiment is that the sequencing batch selection reactor can be a stirred tank reactor, viz., the contents of which can be subjected to continuous stirring. Thereby, it is advantageously possible to achieve greater uniformity of the solution contained therein.

[0159] To achieve this effect, the sequencing batch selection reactor can, for example, be fed with airflows having a fairly high velocity, or it can be provided with suitable stirring means therein, such as an impeller.

[0160] According to a different aspect of this embodiment, the biological selection process can also envisage feeding an amount of supplementary nutrients into the sequencing batch selection reactor at the same time as the introduction of the aqueous nutrient solution.

[0161] Thereby, it can be more reliably ensured that the nutrient supply is sufficiently high to support the biological reactions within the sequencing batch selection reactor.

[0162] In the context of this second embodiment, it is preferable that the tank reactor also be a sequencing batch reactor, which can receive the first intermediate mixture coming from the sequencing batch selection reactor, for example by overflow from a weir opening of the sequencing batch selection reactor, at the same time as the nutrient aqueous solution is fed within the latter.

[0163] According to another aspect of this embodiment, after a preset time from the introduction of the nutrient solution, the contents of the sequencing tank reactor can be allowed to settle, for example by suspending the air supply, so as to achieve a separation between the second intermediate mixture, which concentrates at the bottom, and a supernatant.

[0164] A final preferred aspect of this embodiment is that at least part of the supernatant can be withdrawn and fed into the sequencing batch selection reactor.

[0165] Thereby, it is possible to recover at least part of the carbonaceous source (for example VFA) and / or nutrients which were not consumed in the sequencing tank reactor.

[0166] Brief description of the drawings

[0167] Further features and advantages of the invention will be more apparent after reading the following description provided byway of a non-limiting example, with the aid of the figures illustrated in the accompanying tables.

[0168] Figure 1 is a block diagram illustrating a 'continuous1PHA production process according to a first embodiment of the present invention.

[0169] Figure 2 is a block diagram illustrating a 'batch' PHA production process according to a second embodiment of the present invention.

[0170] Figure 3 illustrates the sequence of steps occurring within the selection reactor of the diagram of figure 2.

[0171] Figure 4 illustrates the sequence of steps occurring within the tank reactor of the diagram of figure 2.

[0172] Detailed description

[0173] With the help of the above-mentioned figures, the two embodiments of implementation of the invention which were outlined above are now described in more detail.

[0174] A common characteristic of both embodiments is that they use, as 'raw material', an initial mixture containing a mixed bacterial mass (generally comprising both heterotrophic and autotrophic bacteria) and an aqueous nutrient solution containing both a carbonaceous source (for example VFA) and nutrients.

[0175] As explained at length above, the initial mixture can comprise activated sludge, for example activated sludge comprising sewage sludge from civil and / or industrial and / or livestock effluents.

[0176] In particular, the initial mixture can have a bacteria concentration comprised between 1 and 15 g / l (inclusive).

[0177] On the other hand, the aqueous nutrient solution can be obtained by acidogenic or aceto- genic fermentation of wet biomasses, such as sewage sludge from civil and / or industrial and / or livestock effluents, organic scrap and / or waste from the agri-food industry, as well as organic fraction of municipal solid waste.

[0178] For example, the aqueous nutrient solution can be obtained through the anaerobic digestion phase of wet biomasses which already occurs in the anaerobic digesters found in common wastewater and / or agri-food waste and / or organic waste treatment plants.

[0179] However, it is not ruled out that in other embodiments, the aqueous nutrient solution can be obtained by other technological processes, including, for example, fermentation processes for producing hydrogen (H2), chemical processes for producing VFA, and other equivalent processes.

[0180] The aqueous nutrient solution can have a total soluble COD value greater than or equal to 5 g / l, for example comprised between 5 and 50 g / l (inclusive), more preferably greater than or equal to 50 g / l.

[0181] The concentration of the carbonaceous source (for example, of VFA) in the aqueous nutrient solution can be such that, of this total soluble COD, a portion equal to or greater than 20% is attributable to the conversion of only the molecules of said carbonaceous source (for example, of VFA), for example, a portion comprised between 20% and 50% (inclusive) of the total soluble COD, more preferably a portion greater than or equal to 50% of the total soluble COD.

[0182] Alternatively, it is possible to state that, preferably, the aqueous nutrient solution can contain a concentration of the carbonaceous source (for example of VFA) corresponding to a specific soluble COD value, viz., attributable to the conversion of the carbonaceous source alone (for example of VFA alone), greater than or equal to 2 g / l, for example comprised between 2 and 25 g / l (inclusive), more preferably greater than or equal to 25 g / l.

[0183] The aqueous nutrient solution can also have an amount of suspended solids corresponding to a TSS value of 2 g / l or less.

[0184] The pH of the aqueous nutrient solution can be greater than or equal to 6, preferably comprised between 6.5 and 8.8 (inclusive).

[0185] As mentioned above, the carbonaceous source in the aqueous nutrient solution can comprise (or consist of) volatile fatty acids, or VFAs, preferably short-chain volatile fatty acids, for example selected from the group consisting of acetic acid, propionic acid and butyric acid.

[0186] The nutrients present in the aqueous nutrient solution can comprise substances belonging to (or selected from) the group consisting of: nitrogen and compounds thereof, for example nitrate and ammoniacal nitrogen, phosphorus and compounds thereof, for example phosphates, sulphur and compounds thereof, for example sulphates, potassium and compounds thereof, magnesium and compounds thereof, calcium and compounds thereof, sodium and compounds thereof.

[0187] In particular, the aqueous nutrient solution can contain a nitrogen concentration greater than or equal to 1.4% of the total soluble COD value of the aqueous nutrient solution itself, for example comprised between 1.4% and 20% of the total soluble COD value of the aqueous nutrient solution (inclusive).

[0188] The aqueous nutrient solution can also contain a phosphorous concentration greater than or equal to 0.1% of the total soluble COD value of the solution, for example comprised between 0.1% and 2.7% of the total soluble COD value of the solution (inclusive).

[0189] Starting with these 'raw materials', a first embodiment (or configuration) of the biotechnological process for producing PHA is illustrated in figure 1.

[0190] This first embodiment envisages subjecting the initial mixture, containing the mixed bacterial mass, to a biological selection process by means of at least a first continuous flow selection reactor 100 and at least a second continuous flow selection reactor 105, more preferably a plurality of continuous flow selection reactors 105 connected in series with each other and with the first continuous flow selection reactor 100.

[0191] Both the first selection reactor 100 and each of the second selection reactors 105 can be an aerated and / or stirred tank reactor.

[0192] The first selection reactor 100 can be controlled so as to operate in anoxic conditions.

[0193] For example, the first selection reactor 100 can be controlled so that the redox potential of the solution contained therein is maintained at a value comprised between -450 mV and -120 mV (inclusive), for example equal to approximately -350 mV, and / or so that the amount of oxygen (O2) dissolved in the solution contained therein is maintained at a value less than or equal to 0.2 mg / l (milligrams / litre).

[0194] In the first selection reactor 100, an inoculum of the initial mixture is first arranged, after which the first selection reactor 100 is fed with a continuous stream (viz., uninterrupted as long as the process is running) of the aqueous nutrient solution.

[0195] This continuous stream of the aqueous nutrient solution can come from one of the processes mentioned above or from any other source, as schematically indicated with reference number 110.

[0196] At steady state, a first bacterial suspension will thus be obtained within the first selection reactor 100, a continuous stream of which flows out of the first selection reactor 100 and is conveyed within the second selection reactor 105, viz., within the first of the 'second selection reactors' 105 which are connected in series.

[0197] It should be noted that the first selection reactor 100 can be sized to have a total hydraulic retention time comprised between 1 hour and 12 hours (inclusive), which generally varies depending on the characteristics of the aqueous nutrient solution in input.

[0198] Each of the second selection reactors 105 can be controlled to operate under aerobic conditions.

[0199] For example, each of these second selection reactors 105 can be controlled so that the amount of oxygen (O2) dissolved in the solution contained therein is maintained at a value greater than or equal to 3 mg / l. Furthermore, irrespective of the carbonaceous source (for example, VFA) and any nutrients transported by the stream of the first bacterial suspension from the first selection reactor 100, each of the second selection reactors 105 is configured so that it cannot receive any further carbonaceous source and / or any further nutrients.

[0200] For example, each of the second selection reactors 105 can be configured to receive, as an affluent, only the first bacterial suspension coming, either directly or through one or more of the other second selection reactors 105, from the first selection reactor 100.

[0201] Thereby, at steady state, at the outlet of the second selection reactor 105, viz., at the outlet of the last of the 'second selection reactors' 105 which are connected in series, a continuous stream of a second bacterial suspension having different characteristics from the first will be obtained.

[0202] It should be noted that the second selection reactors 105 can be sized to have a total hydraulic retention time comprised between 1 hour and 12 hours (inclusive), which generally varies depending on the characteristics of the first suspension in output from the first selection reactor 100.

[0203] The continuous stream of the second bacterial suspension is then subjected to a clarification step, for example within a device 115, preferably also operating at continuous stream, which can be selected from: a natural sedimentation reactor, for example with a truncated cone-shaped section tank, preferably not aerated and with a wall slope greater than or equal to 45°, a scraper bridge clarifier, and a reactor connected to a centrifuge.

[0204] If the clarification occurs by sedimentation, the sedimentation rate can be comprised between 0.05 m3 / hour and 1 m3 / hour (inclusive).

[0205] By means of the clarification step, the continuous stream of the second bacterial suspension from the second selection reactor(s) 105 is separated into two continuous streams, one continuous stream of a third bacterial suspension which is more concentrated with respect to the second, and one continuous stream of a fourth bacterial suspension which is less concentrated with respect to the second.

[0206] In particular, the fourth bacterial suspension can have an amount of volatile suspended solids (VSS) less than or equal to 1 g / l (grams / litre).

[0207] The fourth bacterial suspension can also have a total COD value of less than or equal to 1.2 g / l, preferably equal to 1 g / l (grams / litre), and / or an ammoniacal nitrogen concentration comprised between 200 mg / l and 1000 mg / l (inclusive). The continuous stream of this fourth bacterial suspension, after exiting from the clarifier 115, for example by overflow, can be delivered to a water treatment plant.

[0208] Conversely, the continuous stream of the third bacterial suspension can be conveyed within the first selection reactor 100.

[0209] Thereby, at steady state, a first intermediate mixture will be obtained in the first selection reactor 100, which will in fact also substantially coincide with the first bacterial suspension mentioned above, which will mainly contain selected bacteria capable of storing a carbonaceous source (for example VFA) in the form of PHA.

[0210] Specifically, within the first selection reactor 100, thanks to the feeding of the carbonaceous source (for example VFA) and nutrients, the bacterial mass will be subjected to a feast step, in which bacteria capable of storing PHA will degrade the carbonaceous source and accumulate it in the form of PHA therein; whereas within each of the second selection reactors 105, in which neither nutrients nor the carbonaceous source (for example VFA) are fed, the bacterial mass will be subjected to a famine step, in which only those organisms which were capable of storing PHA in the feast step will be in conditions to grow, consuming precisely the previously accumulated PHA, while the other organisms tend to succumb.

[0211] In particular, this famine step will be longer and more intense (selective) as the bacterial mass passes through the various second selection reactors 105 in the series, as the residual amount of carbonaceous source (for example VFA) and nutrients gradually becomes smaller.

[0212] By then returning the organisms obtained in output from the second selection reactor 105, viz., from the last of the second selection reactors of the series 105, back inside the first selection reactor 100 (after clarification), these organisms will be subjected to a new feast step and so on, until a first intermediate mixture of selected bacteria capable of accumulating PHA effectively and in large amounts is obtained inside the first selection reactor 100.

[0213] Specifically, the first intermediate mixture obtained at steady state in the first selection reactor 100 can have a bacteria concentration comprised between 1 and 15 g / l (inclusive). In particular, it can have a concentration of bacteria capable of storing PHA comprised between 1 and 10 g / l (inclusive).

[0214] The biotechnological process according to this embodiment therefore envisages conveying a continuous stream of the first intermediate mixture, which was obtained in the first selection reactor 100, into a tank reactor 120, also preferably of the continuous stream type.

[0215] The tank reactor 120, which can be of the aerated and / or stirred tank type, operates under anoxic conditions.

[0216] In particular, the tank reactor 120 can be controlled such that the redox potential of the solution contained therein is maintained at a value comprised between +50 mV and -200 mV (inclusive), for example comprised between +50 mV and -150 mV (inclusive), preferably approximately equal to -80 mV, and / or that the amount of oxygen (O2) dissolved in said solution contained in the tank reactor 120 is maintained at less than or equal to 1 mg / l.

[0217] In addition to the continuous stream of the first intermediate mixture, this tank reactor 120 is also fed with a continuous stream of the aqueous nutrient solution, for example the same aqueous nutrient solution which is also fed into the first selection reactor 100.

[0218] Thereby, the previously selected bacteria grow within the tank reactor 120, transforming the carbonaceous source (for example VFA) into PHA and accumulating them within their own cells.

[0219] When fully operational, the tank reactor 120 will therefore contain a second intermediate mixture comprising bacteria containing PHA, in a concentration for example comprised between 1 and 15 g / l (inclusive), a continuous stream of which will flow in output.

[0220] It should be noted that the tank reactor 120 can be sized to have a hydraulic retention time comprised between 3 hours and 24 hours (inclusive), for example comprised between 5 hours and 15 hours (inclusive), preferably between 8 hours and 12 hours (inclusive).

[0221] Since the choice of hydraulic retention time can be temperature-dependent, it is also preferable that the mixture within the tank reactor 120 be kept at a temperature of less than or equal to 42°C, preferably comprised between 20°C and 35°C, for example approximately equal to 25°C.

[0222] Specifically, the tank reactor 120 can be sized to ensure a hydraulic retention time of approximately 5 to 15 hours at a temperature of 25°C (better if between 8 and 12 hours, preferably around 10 hours).

[0223] The continuous stream of the second intermediate mixture, exiting from the tank reactor 120, is then conveyed to a solid-liquid separation apparatus 125, also preferably operating continuously, so as to obtain a continuous stream of a final mixture having a higher solids concentration with respect to that of the second intermediate mixture.

[0224] This solid-liquid separation apparatus 125 can obtain the final mixture by performing a centrifugal separation of the second intermediate mixture and, for this purpose, can comprise a vertical axis centrifuge, a decanter centrifuge or other similar devices.

[0225] The final mixture obtained with the solid-liquid separation apparatus 125 can have a concentration of PHA-containing bacteria comprised between 50 and 250 g / l (inclusive), or a mass concentration of PHA-containing bacteria comprised between 5% and 25% of the total mass of the mixture (inclusive).

[0226] The final mixture can also have a PHA mass concentration comprised between 10% and 70% of the total mass of bacteria present in the final mixture (inclusive).

[0227] By means of the solid-liquid separation apparatus 125, not only is the final mixture with a high PHA concentration naturally obtained, but also a continuous stream of a liquid (or supernatant) fraction with a (much) lower solids concentration with respect to that of the final mixture.

[0228] This continuous flow of the liquid fraction in output from the solid-liquid separation apparatus 125 can preferably be conveyed within the first selection reactor 100, so as to recover the residual amount of carbonaceous source (for example VFA) which can still be present in the aforesaid liquid fraction.

[0229] The continuous stream of the final mixture exiting the solid-liquid separation apparatus 125, after being possibly collected, is instead subjected to a PHA extraction process (not illustrated).

[0230] This PHA extraction process can be carried out by methods known in the art, for example by means of extraction with organic solvents (for example chloroform), or preferably with physical methods, for example but not necessarily through the use of ultrasound.

[0231] The biotechnological process subject-matter of the present invention can also be implemented in a second embodiment (or configuration), which is illustrated in figure 2.

[0232] This second embodiment also envisages first subjecting the initial mixture containing the mixed bacterial mass to a biological selection process.

[0233] In this case, however, the selection process is carried out within a sequencing batch selection reactor 200, for example an aerated and / or stirred tank sequencing batch reactor. The selection reactor 200 can be controlled so as to operate under aerobic conditions.

[0234] For example, the selection reactor 200 can be controlled so that the amount of oxygen (O2) dissolved in the solution contained therein is maintained at a value greater than or equal to 1 mg / l, for example greater than or equal to 2 mg / l, and / or that the redox potential of said solution in the sequencing batch reactor 200 is greater than or equal to 0 mV.

[0235] In the selection reactor 200, an inoculum of the initial mixture is first arranged, after which the first selection reactor 200 is pulsed (or intermittently) fed with a predetermined amount of the aqueous nutrient solution from any generic source 205, including those mentioned above.

[0236] In practice, the introduction of the aqueous nutrient solution into the sequencing batch selection reactor 200 occurs for preset periods of time, so as to reach the desired amount, interspersed with periods of time when the nutrient solution is not introduced.

[0237] For example, a certain amount of the nutrient solution can be fed / introduced into the selection reactor 200 once every 24 hours, then with increased frequency to once every 12 hours, and thereafter once every period of time comprised between 4 hours and 10 hours. If necessary, an (additional) amount of supplementary nutrients (for example water-soluble nitrogen and phosphorous salts) can also be fed into the sequencing batch selection reactor 200 from any general source 210, for example nitrogen- and phosphorous-rich industrial wastewater, at the same time as the introduction of the aqueous nutrient solution.

[0238] As will be explained more clearly below, the sequencing selection batch reactor 200 can possibly also be fed with an alkaline solution recirculated from a tank reactor 215 placed downstream.

[0239] Thereby, the selection process is configured as a batch process, in which the alternation of feast and famine steps, responsible for biological selection, is achieved in a time sequence within the sequencing selection batch reactor 200.

[0240] Specifically, when the aqueous nutrient solution is administered, the bacterial mass inside the selection reactor 200 is subjected to a feast condition, indicated with SA in figure 3, in which the PHA-accumulating bacteria degrade the carbonaceous source and accumulate it in the form of PHA therein.

[0241] When the aqueous nutrient solution is instead not administered in the selection reactor 200, the bacteria rapidly consume the residual carbonaceous source until they reach a famine condition, indicated with SB in figure 3, in which only those organisms which were able to store PHA in the feast step are in a position to grow, consuming the previously accumulated PHA, while the other organisms tend to succumb.

[0242] Thereby, it is advantageously possible to obtain an initial intermediate mixture of selected bacteria within the selection reactor 200 which can accumulate PHA effectively and in large amounts.

[0243] For example, this first intermediate mixture can have a concentration of bacteria comprised between 1 and 15 g / l (inclusive), of which a concentration of bacteria capable of storing PHA comprised between 1 and 10 g / l (inclusive).

[0244] The first intermediate mixture obtained in the selection reactor 200 is then fed into the aforementioned tank reactor 215, which is also preferably a sequencing batch reactor and can be an aerated and / or stirred tank type reactor.

[0245] In particular, the transfer of the first intermediate mixture from the selection reactor 200 to the tank reactor 215 can occur by overflow from a weir opening of the selection reactor 200, concurrently with the introduction of the aqueous nutrient solution within the latter.

[0246] The tank reactor 215 is preferably operated under anoxic conditions, for example controlled so that the redox potential of the solution contained therein is maintained at a value comprised between +50 mV and -200 mV (inclusive), for example, comprised between +50 mV and -150 mV (inclusive), preferably approximately equal to -80 mV, and / or so that the amount of oxygen (O2) dissolved in said solution contained in the tank reactor 200 is kept less than or equal to 1 mg / l.

[0247] The tank reactor 215 is also fed, for example in pulsed (intermittent) mode, with an amount of the aqueous nutrient solution, for example the same aqueous nutrient solution which is also fed into the selection reactor 200.

[0248] In particular, the aqueous nutrient solution can be fed during the step indicated with SC in figure 4.

[0249] Thereby, for the duration of a subsequent step SD, within the tank reactor 215, the previously selected bacteria transform the carbonaceous source (for example VFA) into PHA and accumulate this PHA within their own cells.

[0250] After a preset time from the introduction of the aqueous nutrient solution, the tank reactor 215 will therefore contain a suspension comprising bacteria containing PHA (step SE).

[0251] In particular, within this suspension, the total weight of PHA can be comprised between 15% and 50% of the total weight of the entire bacterial mass present in the same suspension.

[0252] After the aforesaid preset time from the introduction of the aqueous nutrient solution, the suspension contained in the sequencing batch tank reactor 215 can be allowed to settle (step SF), for example by interrupting the administration of air, so as to obtain, on the bottom of the reactor, the accumulation of a second, more concentrated, intermediate mixture substantially separated from a supernatant which remains on the surface.

[0253] The second intermediate mixture obtained at the bottom of the tank reactor 215 can have a concentration of PHA-containing bacteria comprised between 1 and 15 g / l (inclusive). At the end of the sedimentation, at least part (or all) of the supernatant can be withdrawn from the tank reactor 215 and, as mentioned above, returned to the selection reactor 200. The settled suspension, viz., the second intermediate mixture, is instead withdrawn from the tank reactor 215 and sent to a solid-liquid separation apparatus 220, so as to obtain a final mixture which is even more concentrated with respect to the second intermediate mixture.

[0254] This solid-liquid separation apparatus 220 can obtain the final mixture by performing centrifugation separation of the second intermediate mixture.

[0255] For example, the solid-liquid separation apparatus can comprise a device selected from a vertical axis centrifuge, a decanter centrifuge or other equivalent devices.

[0256] The final mixture obtained from the solid-liquid separation apparatus 220 can have a suspended solids content comprised between 50 g / l and 250 g / l (inclusive), for example comprised between 50 g / l and 200 g / l (inclusive), or a bacteria mass concentration comprised between 5% and 25% of the total mass of the final mixture (inclusive).

[0257] The final mixture can also have a PHA mass concentration comprised between 10% and 70% of the total mass of bacteria present in the final mixture (inclusive), for example comprised between 10% and 60% (inclusive).

[0258] In addition to the final (concentrated) mixture, the solid-liquid separation apparatus 220 will also produce a liquid fraction, viz., a suspension with a (much) lower solids content than the final mixture, which can be disposed of as waste water, possibly together with that part of the supernatant obtained in the tank reactor 215 which was not returned and introduced into the tank reactor 200.

[0259] Instead, the final (concentrated) mixture is subjected to a PHA extraction process (not shown), which can be carried out by methods known in the art, for example by means of extraction with organic solvents, or preferably with physical methods, for example but not necessarily through the use of ultrasound.

[0260] The PHA obtained with the two embodiments (configurations) outlined above has entirely comparable characteristics.

[0261] In particular, it can consist mainly of the monomers HB-hydroxybutyrate, for example in percentages varying between 80% and 95%, more usually between 85 and 90%, and HV-hydroxyvalerate, for example in percentages less than 50%, more usually between 10% and 15%. The following table shows the more in-depth characterisation of the PHA extracted at the end of the process according to the invention in comparison with that of a commercialgrade polyhydroxybutyrate (PHB) where Mw denotes the molecular weight expressed in Mega-Dalton (MDa), PDI represents the polydispersion index and Tm denotes the melting temperature expressed in degrees centigrade.

[0262] Obviously, an expert in the art can make several technical-applicative modifications to all that above, without departing from the scope of the invention as hereinbelow claimed.

Claims

CLAIMS1. A biotechnological process for producing PHA, comprising the steps of:- subjecting an initial mixture containing a mixed bacterial mass to a biological selection process, so as to obtain a first intermediate mixture containing mainly selected bacteria capable of storing a carbonaceous source (for example VFA) in the form of PHA, said first intermediate mixture having, for example, a concentration of bacteria capable of storing PHA comprised between 1 and 15 g / l,- introducing said first intermediate mixture into a tank reactor (120, 215) operating under anoxic conditions, for example an aerated and / or stirred tank reactor (120, 215),- also introducing into said tank reactor (120, 215) an aqueous nutrient solution, containing both the carbonaceous source (for example, VFA) and nutrients, so as to obtain within said tank reactor (120, 215) a second intermediate mixture comprising PHA-containing bacteria, said second intermediate mixture having, for example, a concentration of PHA-containing bacteria comprised between 1 and 15 g / i,- treating said second intermediate mixture with a solid-liquid separation apparatus (125, 220), for example a vertical axis centrifuge, a decanter centrifuge or any other device capable of executing a solid / liquid separation of the second intermediate mixture, so as to obtain a final mixture having a higher solids concentration with respect to that of the second intermediate mixture, for example a concentration of PHA-containing bacteria comprised between 50 and 250 g / l,- subjecting the final mixture to a PHA extraction process, for example a PHA extraction process carried out by means of extraction with organic solvents in addition to physical and mechanical methods, for example through the use of ultrasound.

2. A process according to claim 1 , wherein the initial mixture comprises activated sludge, for example activated sludge comprising sewage sludge obtained from civil and / or industrial and / or livestock effluents.

3. A process according to any one of the preceding claims, wherein the aqueous nutrient solution is obtained by acidogenic or acetogenic fermentation of wet biomasses, for example wet biomasses comprising one or more materials selected from the groupconsisting of: sewage sludge from civil and / or industrial and / or livestock effluents, organic scrap and / or waste from the agri-food industry, organic fraction of municipal solid waste.

4. A process according to any one of the preceding claims, wherein the carbonaceous source present in the aqueous nutrient solution comprises volatile fatty acids, preferably short chain volatile fatty acids, for example selected from the group consisting of acetic acid, propionic acid and butyric acid, and wherein the nutrients present in the aqueous nutrient solution comprise substances selected from the group consisting of: nitrogen and compounds thereof, for example nitrate and ammoniacal nitrogen, phosphorus and compounds thereof, for example phosphates, sulphur and compounds thereof, for example sulphates, potassium and compounds thereof, magnesium and compounds thereof, calcium and compounds thereof, sodium and compounds thereof.

5. A process according to any one of the preceding claims, comprising at least one of the following characteristics:- the redox potential of the second intermediate mixture contained in the tank reactor (120, 215) is maintained at a value comprised between +50 mV and -200 mV (inclusive), for example comprised between +50 mV and -150 mV (inclusive), preferably approximately equal to -80 mV,- the amount of oxygen (O2) dissolved in said second intermediate mixture in the tank reactor (120, 215) is kept less than or equal to 1 mg / L6. A process according to any one of the preceding claims, wherein the biological selection process envisages:- arranging an inoculum of the initial mixture within a first continuous flow selection reactor (100), for example stirred and / or aerated tank, and preferably operating under anoxic conditions,- feeding said first selection reactor (100) with a continuous stream of said aqueous nutrient solution, so as to obtain a first suspension,- conveying a continuous stream of said first suspension into at least a second continuous flow selection reactor (105), for example with a stirred and / or aerated tank, and preferably operating under aerobic conditions and / or preferably operating without further supply of any carbonaceous source (for example VFA) and / or any nutrient, so as to obtain a second suspension,- subjecting a continuous stream of said second suspension to a clarification step,for example by means of a natural sedimentation reactor, a scraper bridge clarifier or a reactor connected to a centrifuge, so as to obtain a continuous stream of a third suspension having a higher solids concentration with respect to that of the second suspension,- conveying the continuous stream of the third suspension within the first selection reactor (100) so as to obtain the first intermediate mixture, the tank reactor (120) also being a flow through reactor, which receives a continuous stream of the first intermediate mixture and a second continuous stream of said aqueous nutrient solution to provide a continuous stream of the second intermediate mixture.

7. A process according to claim 6, comprising at least one of the following characteristics:- the redox potential of the first solution in the first selection reactor (100) is maintained at a value comprised between -450 mV and -120 mV, for example equal to approximately -350 mV,- the amount of dissolved oxygen in said first solution contained in the first selection reactor is maintained at a value less than or equal to 0.2 mg / l,- the amount of dissolved oxygen in the second solution contained in the second selection reactor (105) is maintained at a value greater than or equal to 3 mg / l.

8. A process according to claim 6 or 7, wherein said continuous stream of the first suspension is conveyed into a system comprising a plurality of said second continuous flow selection reactors (105) connected in series with each other, preferably each operating under aerobic conditions and / or preferably each operating without further supply of any carbonaceous source (for example, VFA) and / or any nutrient.

9. A process according to any one of claims 6 to 8, wherein the first selection reactor (100) is sized to have a total hydraulic retention time comprised between 1 hour and 12 hours, wherein said second selection reactor is sized to have a total hydraulic retention time comprised between 1 hour and 12 hours, and wherein the tank reactor (120) is sized to have a hydraulic retention time comprised between 3 hours and 24 hours, for example, comprised between 5 hours and 15 hours, preferably between 8 hours and 12 hours.

10. A process according to any one of claims 6 to 8, wherein the second intermediate mixture within the tank reactor (120) is maintained at a temperature less than or equal to42°C, preferably comprised between 20°C and 35°C, for example approximately equal to 25°C.

11. A process according to any one of claims 6 to 10, wherein a liquid fraction obtained in output from the solid-liquid separation apparatus (125) is conveyed within the first selection reactor (100).

12. A process according to any one of claims 1 to 5, wherein the biological selection process envisages:- arranging an inoculum of the first mixture within a sequencing batch selection reactor (200), for example of the aerated and / or stirred tank type, preferably operating under aerobic conditions, and- pulsed feeding into said sequencing batch selection reactor (200) an amount of said aqueous nutrient solution, the tank reactor (215) being a sequencing batch reactor, which receives the first intermediate mixture from the sequencing batch selection reactor (200), for example by overflow from a weir opening of the sequencing batch selection reactor (200), for example at the same time as the nutrient aqueous solution is introduced within the latter.

13. A process according to claim 12, comprising at least one of the following characteristics:- the amount of oxygen (O2) dissolved in the first intermediate mixture in the sequencing batch selection reactor (200) is maintained at a value greater than or equal to 1 mg / l, for example greater than or equal to 2 mg / l,- the redox potential of said first intermediate mixture in the sequencing batch selection reactor (200) is greater than or equal to 0 mV.

14. A process according to claim 12 or 13, wherein the biological selection process comprises also introducing an amount of supplementary nutrients within said sequencing batch selection reactor (200) at the same time as introducing the aqueous nutrient solution.

15. A process according to any one of claims 12 to 14, wherein, after a preset time from the introduction of the nutrient solution, the contents of the sequencing batch tank reactor (215) are allowed to settle, for example by suspending the administration of air, so as to achieve a separation between the second intermediate mixture, which concentrates at the bottom, and a supernatant.

16. A process according to claim 15, wherein at least part of said supernatant is withdrawn taken and introduced into the sequencing batch selection reactor (200).

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