WO2011070544A2 - Procédé pour accumuler des polyhydroxyalcanoates dans une biomasse avec contrôle en ligne pour la commande de la vitesse d'alimentation et l'arrêt du processus - Google Patents

Procédé pour accumuler des polyhydroxyalcanoates dans une biomasse avec contrôle en ligne pour la commande de la vitesse d'alimentation et l'arrêt du processus Download PDF

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WO2011070544A2
WO2011070544A2 PCT/IB2010/055745 IB2010055745W WO2011070544A2 WO 2011070544 A2 WO2011070544 A2 WO 2011070544A2 IB 2010055745 W IB2010055745 W IB 2010055745W WO 2011070544 A2 WO2011070544 A2 WO 2011070544A2
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Prior art keywords
biomass
substrate
feeding
pha
concentration
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PCT/IB2010/055745
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English (en)
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WO2011070544A3 (fr
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Alan Gideon Werker
Simon Olof Harald Bengtsson
Carl Anton Börje KARLSSON
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Veolia Water Solutions & Technologies Support
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Priority to CN201080063546.1A priority Critical patent/CN102770549B/zh
Priority to CA2783591A priority patent/CA2783591C/fr
Priority to RU2012128820/10A priority patent/RU2535341C2/ru
Priority to BR112012013882A priority patent/BR112012013882A2/pt
Priority to IN5193DEN2012 priority patent/IN2012DN05193A/en
Priority to US13/514,660 priority patent/US8748138B2/en
Priority to EP10809241.2A priority patent/EP2510103B1/fr
Priority to NZ600542A priority patent/NZ600542A/xx
Priority to AU2010329473A priority patent/AU2010329473B2/en
Publication of WO2011070544A2 publication Critical patent/WO2011070544A2/fr
Publication of WO2011070544A3 publication Critical patent/WO2011070544A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/30—Aerobic and anaerobic processes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids

Definitions

  • This invention relates to the accumulation of polyhydroxyalkanoates (PHAs) in biomass in conjunction with biological organic waste treatment.
  • PHAs polyhydroxyalkanoates
  • the invention concerns the art of combining wastewater rich in readily biodegradable chemical oxygen demand (RBCOD) with biomass enriched in PHA-accumulating bacteria (PAB), and monitoring the process.
  • RBCOD readily biodegradable chemical oxygen demand
  • PAB PHA-accumulating bacteria
  • PHAs Polyhydroxyalkanoates
  • VFAs volatile fatty acids
  • the present invention may be useful for solving a number of problems associated with process quality objectives of PHA accumulation in PAB-rich biomass and wastewater treatment as explained further below.
  • the present invention provides methods of producing and accumulating PHAs in a PAB-rich biomass from RCBOD.
  • the invention provides a method for producing polyhydroyxalkanoates (PHAs) in biomass, comprising feeding an organic carbon-containing substrate to the biomass by intermittently supplying the substrate to the biomass over a period of time and controlling the frequency of the intermittent supply and amount of the intermittent supply of the substrate to the biomass such that the average molecular weight of the PHAs produced is at least 400,000 g mole.
  • PHAs polyhydroyxalkanoates
  • the invention provides a method of promoting the accumulation of PHAs in biomass, comprising feeding an organic carbon-containing substrate to the biomass by mixing substrate containing readily biodegradable chemical oxygen demand (RBCOD) with the biomass to form a biomass-substrate mixture, controlling the concentration of the RBCOD in the biomass-substrate mixture such that during PHA accumulation the concentration of RBCOD in the biomass-substrate mixture is generally maintained between 1,000 mg-COD/L and 10 mg-COD/L, and wherein the method of feeding RBCOD to the biomass and controlling the concentration of RBCOD in the biomass-substrate mixture produces PHAs having an average molecular weight above 400,000 g/mole.
  • RBCOD readily biodegradable chemical oxygen demand
  • the invention provides a method of producing high molecular weight PHAs in biomass including feeding an organic carbon-containing substrate to a PHA-accumulating biomass by intermittently supplying the substrate to the biomass over a period of time, and controlling the frequency and amount of the supply of substrate such that the respiration rate of the biomass does not drop more than 70% relative to the maximum respiration rate achieved by the biomass in response to the most recent supply of substrate.
  • Figure 1 Normalized experimental data from 6 studies in relation to an empirical model (Equation 1) for the biomass response to a VFA-rich feed stimulus.
  • Figure 2 Representative dissolved oxygen (DO) concentration signal used to control automatic substrate addition (Equation 3) in experiments for PHA accumulation in PAB-rich biomass with pulses to achieve a constant stimulus of 100 mg-COD/L of VFA.
  • DOE dissolved oxygen
  • Figure 4 Results from replicate accumulation experiments using a concentrated fermented dairy wastewater as substrate for fed-batch accumulation of PHA.
  • Figure 7 Summary of accumulation results of numerous experiments considering the resultant estimated average pulse minimum respiration rate reported with respect to the extant maximum pulse respiration rate, and weight average molecular weight of the PHA accumulated.
  • Figure 8 Representative results of pilot scale (100 L) accumulation of PHA in PAB-rich biomass using a fermented dairy industry wastewater as substrate.
  • Figure 9 Representative example of a variable volume aerobic PHA accumulation process.
  • Figure 10. Representative example of a variable volume aerobic PHA accumulation process.
  • Figure 11. Representative example of a constant volume aerobic PHA accumulation process.
  • the present invention includes novel but practical engineering solutions for the process of accumulation of PHAs in biomass. Objectives that can be satisfied by employing the present invention include:
  • Wastewaters are often characterized by organic content in terms of chemical oxygen demand (COD).
  • COD chemical oxygen demand
  • the total chemical oxygen demand (TCOD) of a wastewater can be further categorized by standard methods in terms of soluble (SCOD) and biodegradable (BCOD) fractions.
  • SOD forms part of the soluble COD in a wastewater and can generally be thought as those organic compounds comprising the BCOD that can be assimilated for biomass growth without requiring intervening steps of hydrolysis.
  • the respective concentrations of TCOD, SCOD, BCOD, RBCOD and so forth, in the wastewater may be reported as mg-COD per liter or mg-COD/L where the mass of COD being reported is with direct reference to the component of organic content being considered.
  • Microbial activity and other forms of physical-chemical processes can be used to increase the soluble, biodegradable, and even the RBCOD fraction of the wastewater TCOD.
  • the wastewater is suitably characterized and the chemical identity of the RBCOD is known, then the RBCOD content of the wastewater can be explicitly expressed, for example, as total VFAs measured as mg-COD/L.
  • RBCOD can also be operationally defined by standardized methods of respirometry that consider the fraction of the wastewater COD that is rapidly utilized by a biomass when an aliquot of wastewater is pulse fed to the biomass under controlled conditions (Henze et al., 2000).
  • the results of such an operational measurement of RBCOD based on methods of respirometry may vary depending on how well the biomass is acclimatized to the organic compounds that otherwise may be generally understood to be readily assimilated into metabolism of biomass growth, for example RBCOD.
  • the RBCOD of interest for the present invention is the RBCOD which when combined with a suitably acclimated biomass can be assimilated by that biomass and stored intracellularly as PHA.
  • VFAs are well established substrates for producing PHAs but other forms of RBCOD are also known to be substrates that mixed cultures can convert into PHAs.
  • the present invention includes PHA production from VFAs and RBCOD in general.
  • the present invention further provides a fed-batch wastewater treatment process for the biological removal of RBCOD using a biomass that is enriched with PHA accumulating bacteria and the controlled conversion of RBCOD into PHAs.
  • the addition of wastewater to the biomass is controlled so as to achieve a negligible buildup of RBCOD in the mixed liquor by the end of the accumulation process.
  • Mixed liquor is generally understood to those familiar with the practice of biological wastewater treatment as the mixture of raw or settled wastewater and activated sludge contained in an aeration basin in the activated sludge process.
  • Mixed liquor suspended solids (MLSS) is the concentration of total suspended solids (TSS) in mixed liquor as measured by standard methods, usually expressed in milligrams per litre (mg L).
  • MVSS Mixed liquor volatile suspended solids
  • SLS volatile suspended solids
  • mixed liquor is used to express the liquid contents of the accumulation process comprising but not limited to suspended solids of active biomass and dissolved solids of RBCOD. Since the biomass in the practice of this invention may be waste activated sludge from a biological wastewater treatment process, the accumulation process mixed liquor is considered and expressed as mixed liquor even before any RBCOD is added for purposes of PHA accumulation.
  • Process monitoring can include strategies of direct measurement of water quality, biomass activity, or biomass characteristics. Process monitoring can also include so- called soft-sensor signals that in combination with specific process knowledge can be used for indirect interpretation of water quality, biomass activity, biomass growth, PHA accumulation, and biomass characteristics. Examples of process monitoring parameters for the process control are as follows:
  • UVYVis Ultraviolet/visible spectroscopy for indirect indication for substrate concentration (COD), and/or biomass concentration (TSS).
  • Respirometry based on, for example, dissolved oxygen, carbon dioxide and/or redox potential measurements for the control of the process aeration and to monitor and respond to changes in the extant biomass respiration rate during sequential fed-batch wastewater inputs.
  • Hydrogen ion concentration (pH) as a soft-sensor of biomass response and metabolic activity to fed-batch RBCOD inputs.
  • One embodiment of the invention is a method to produce PHAs with an average molecular weight (M w ) of at least 400,000 g mol, preferably greater than 600,000 g mol and more preferably greater than 1,000,000 g/mol.
  • M w average molecular weight
  • PHA accumulation in pure culture fermentation as well as in open mixed-culture processes with VFAs is the result of a complex chain of metabolic processes. Not being bound by any particular theory, it is believed that by controlling the rate of one or more of these metabolic processes in the biomass, high average molecular weight PHAs can be produced.
  • the kinetics of PHA accumulation may be considered to be controlled by:
  • VFAs entering into the cell cytoplasm can be utilized for three possible metabolic functions. The following rates of such functions may be affected by the intracellular VFA concentration:
  • Non- PHA biomass can be in the form of active microorganisms as well as other storage products such as extra-cellular polysaccharides.
  • R e the rate of catabolic conversion of VFAs into 3 ⁇ 40, and C0 2 in order to drive metabolic activity for ongoing maintenance, growth and PHA accumulation respiration.
  • R e requirements can be satisfied by this non-PHA precursor source up to 100% of the biomass energy requirements.
  • R r for the case of a RBCOD- only feed, R r , or the removal rate of VFAs from the wastewater, can be assumed to be dependent on R gJ R p and R e when R t and R c are sufficiently rapid:
  • VFA catabolic rate may be considered to be limited to a function of growth and polymerization rates:
  • R r depends on R g and R p or
  • R t and R c are relatively rapid, the rate limiting steps for VFA removal are R p and/or R g .
  • R g is negligible due to, for example, a period of famine, and/or a limitation of an essential growth element like nitrogen or even oxygen, then the rate limiting step or "bottle-neck" for VFA removal is the rate of PHA polymerization or R p .
  • R p limiting conditions are created when conditions are such that R t and are, relatively speaking, significantly more rapid than R p such that R p is independent of R t and/or ⁇ .
  • R p limiting conditions or "PHA polymerization limiting kinetics" as used herein are created when the biomass is able to maintain an intracellular pool of PHA- monomers (substrate) that supply the polymerization process for making PHAs (product), such that the polymerization rate (R p ) is unaffected by PHA-monomer concentration. Further, when R p is the rate limiting step, the kinetics of VFA removal from the mixed liquor is zero order, namely independent of the concentration ofVFAs in the mixed liquor.
  • the molecular weights of the PHAs produced in the biomass are predicted to be greater as the probability of chain termination reactions during the accumulation process decreases.
  • chain termination probability is minimized by maintaining the level of PHA-monomer precursors during the accumulation process so as to not limit the rate of PHA polymerization.
  • level of PHA-monomer precursors may be indicated by zero order VFA removal kinetics, or similarly by the maximum respirometric rate achieved in the biomass and maintained in the biomass during the accumulation process.
  • the kinetics of VFA removal, oxygen consumption, carbon dioxide production, and pH change are examples of parameters that can be monitored in the accumulation process as a means to determine the maximum respirometric rate that has been achieved in the biomass and by what percentage it attenuates subsequent to each supply of RBCOD. Notwithstanding these theoretical interpretations and the potential for other such interpretations, the present invention provides the timing of and supply of sufficient RBCOD to the mixed liquor in order to stimulate a maximum biomass response for PHA accumulation to achieve high molecular weight PHA polymers in parallel with wastewater treatment.
  • the present invention comprises PHA production with open mixed cultures of biomass for treating waste organic matter. Notwithstanding, the principles and techniques employed by the invention can be used in processes involving pure or constrained mixed cultures of bacteria and/or more refined RBCOD or other feedstocks for biomass and/or PHA production.
  • Biomass harvested from wastewater treatment facilities can be made to accumulate PHA when fed with RBCOD-containing wastewater.
  • Biomass that is enriched in PHA- accumulating bacteria can accumulate typically in excess of 50 % PHA of its total dry weight (active biomass plus PHA).
  • the RBCOD feed should be supplied in order to achieve an initial concentration high enough to stimulate a PHA storage response in the biomass but not too high in concentration so as to cause any form of metabolic inhibition that would be detrimental to the process with respect to polymer yield and productivity.
  • the present invention provides the controlled addition of RBCOD to the biomass to optimize for higher polymer molecular weight distribution.
  • One aspect of the present invention is a method of producing high molecular weight PHAs in a biomass comprising:
  • the working range will be mass inputs of RBCOD that periodically expose the biomass to peak stimulating concentrations preferably between 20 and 500 mg-COD/L, and more preferably within the range between 40 and 200 mg-COD/L.
  • RBCOD input amounts can be constant but preferable will be higher initially and attenuated over time as the polymerization kinetics decrease during the PHA accumulation process.
  • RBCOD inputs to the biomass are provided with sufficient frequency such that the extant biomass respiration rate does not drop more than 70 percent and preferably not more than 30 percent relative to the respiration rate achieved in response to the most recent wastewater RBCOD input.
  • the acceptable respiration rate decrease for the biomass between RBCOD inputs depends on the number of input events. The greater the number of input events or interruptions the less the extant respiration can decrease between events if molecular weight is to be maximized.
  • Active biomass concentration in the accumulation process can be defined as the total biomass concentration (measured as volatile suspended solids) minus the PHA concentration.
  • PHA average molecular weight may be less affected by the frequency of stimulus events when extant respiration does not decrease more than 30 percent of its extant maximum potential.
  • the RBCOD may be consumed by the biomass only for cellular maintenance and endogenous respiration.
  • the PHA productivity diminishes and the RBCOD removal rate becomes significantly reduced.
  • mixed culture accumulation processes are terminated.
  • the suspended biomass and water phases are separated and the product is a biomass with high levels of accumulated PHA.
  • the effluent from such an accumulation process may contain high levels of residual RBCOD necessitating further treatment before final discharge of the effluent is allowable.
  • the RBCOD may be consumed by the biomass to support processes of non-PHA related growth and maintenance, in addition to concurrent PHA storage. At this stage it is possible to maintain the accumulation process until such point when the PHA mass in the reactor has reached an optimal level and/or buildup of residual soluble COD in the mixed liquor reaches a selected maximum level.
  • One embodiment of the invention is to drive a PHA-accumulation process in open mixed cultures where the aqueous discharge from the accumulation process is biologically treated at least with respect to its RBCOD content.
  • Another embodiment of the invention is to drive PHA-accumulation processes with real wastewaters where levels of nutrients other than RBCOD used for PHA production promotes a process of combined PHA storage and biomass non-PHA growth metabolic activities.
  • Feed-stocks that are good for PHA accumulation may not always be significantly concentrated and therefore, the problem may be to attain sufficient concentrations of RBCOD in order to reach optimum PHA accumulation rates and PHA quality.
  • Lower feed input amounts are in any event more desirable because the higher the feed frequency is, the more opportunities exist for tighter process control, manipulative shifts in feed composition, as well as the avoidance of excess RBCOD in solution at the end of the PHA accumulation batch run.
  • Fed-batch operation with a higher frequency of fed-batch inputs begins to approach conditions of a continuous feed strategy. As the frequency of feed increases the distinction between a fed-batch and a continuous feed strategy become blurred. Thus the volumetric feed rate rather than the dose input frequency becomes an equivalent parameter in the control and process operation.
  • Fed-batch operation can be accomplished by distinct dose inputs of selected volume applied to all the biomass in a completely mixed reactor.
  • Fed-batch operation can also be accomplished by bringing the biomass in a side stream or within distinct reactor zones in contact with RBCOD input such that organisms in the biomass experience distinct stimuli of substrate supply and distinct periods of substrate interruption.
  • the feed may be supplied in pulses or continuously.
  • a feed interruption may be defined by conditions where the microorganisms in the biomass experience feed supply reduction in time or space due to being exposed to an environment where a negative gradient in substrate concentration exists.
  • the concept embodied in the present method for accumulating PHAs in biomass entails pulse feeding as well as feed interruptions.
  • Certain terms are used herein to describe both pulse feeding and feed interruptions.
  • “intermittently supplying the substrate to the biomass” or “interrupting the feed” includes pulse feeding as well as providing for feed interruptions and specifically includes pulse feeding the substrate to the biomass or circulating a portion of the biomass from a zone having a relatively low concentration of substrate to a zone having a relatively high concentration of substrate while the substrate is fed continuously or non-continuously.
  • the method or process is intermittently supplying substrate to the biomass.
  • the PHA accumulation process is continued until practical limitations are reached, feeding targets have been achieved, signs of saturation for PHA-accumulation are apparent, and/or the total mass of PHA in the reactor has reached a targeted level. Indications of these events include:
  • Feeding targets including the addition of wastewater RBCOD based on established or case-specific observed norms of the biomass conversion yields which may vary from wastewater to wastewater.
  • a typically observed conversion yield for a fermented dairy wastewater is 0.4 kg-PHA produced per kg-VFA-COD consumed. Therefore, if the biomass is with known capacity for PHA accumulation to 100% of the initial biomass dry weight, then the targeted wastewater addition would be 2.5 kg-COD per kilogram of initial biomass.
  • the time required for biomass to accumulate its maximum potential in PHA may vary from batch to batch. Fluctuations may occur with respect to kinetics and other characteristics of the mixed culture process due to, for instance, microbial population dynamics, shifts in metabolic condition (physiological state) and variations in influent feed characteristics.
  • Suitable PHA accumulation feed-stocks can include feed-stocks selected from RBCOD sources other than those resident in the wastewater used to produce the PHA-accumulating biomass. Such disparate feed-stocks may enhance the overall process economics or may tailor the RBCOD composition for producing different kinds of PHAs.
  • the 1 day HRT was based on 2 x 12 hours cycles per day where a cycle was defined with a starting point of 2 L reactor mixed liquor volume.
  • a cycle was defined with a starting point of 2 L reactor mixed liquor volume.
  • 2 L of wastewater were rapidly fed under aerobic conditions.
  • the wastewater and mixed liquor were reacted aerobically with dissolved oxygen levels in excess of 1 mg-0 2 /L for approximately 11 hours.
  • aeration and mixing were stopped and the activated sludge in the mixed liquor was allowed to settle for 30 minutes under quiescent conditions. After settling 2 L of supernatant were decanted.
  • 500 mL of mixed liquor were pumped out (wasted) for the SRT control just before aeration and mixing were stopped for sedimentation.
  • the wasted biomass was subjected to pulse inputs of either concentrated acetic acid or the fermented dairy wastewater.
  • the objective was to measure biomass response to a "feast" stimulus under conditions where extant PHA content in the biomass was negligible.
  • the bioniass response to these substrate inputs was monitored based on dissolved oxygen trends and also based on more detailed water quality analyses over the course of the biomass response to respective pulse inputs of organic substrate.
  • the biomass response could be modeled by a function of form (Figure 1):
  • s the substrate initial concentration providing the stimulus (mg-COD/L)
  • Deviations from the model at higher stimulus concentrations are believed to be due to PHA accumulation in the biomass after a series of stimuli.
  • the VFA removal rate (R r ) was observed to follow zero order kinetics to well below the estimated Sf concentration, and thus q s was approximately constant after each of the respective stimulated response level (s). This outcome indicated that sufficient substrate (s f ) was required to stimulate a measureable feast response, and the metabolic response was sensitive to the level of stimulus (s) for stimuli that were below the
  • the concentration s m ranged from 40 to 115 mg-COD/L. In direct proportion, Sf ranged from 3 to 9 mg-COD/L and q m ranged from 8 to 21 mg-COD/g-biomass/minute. If these experimental results are to be considered typical, then fed-batch VFA additions achieving in excess of approximately 150 mg-COD/L should be sufficient to drive the process of PHA accumulation such that the accumulation process kinetics are limited only by the intracellular rate of PHA polymerization.
  • VFA used to stimulate theoretical PHA polymerization limiting kinetics can vary. Based on our current body of experimental results, a conservative RBCOD-pulse input would be one that achieves an initial stimulus level of 200 mg-COD/L for biomass with negligible stored PHA.
  • a subsequent VFA-pulse input was triggered by a relative increase in DO representing a decrease in biomass respiration rate due to substrate depletion.
  • DO t a relative increase in DO in time
  • ⁇ * a pre-determined critical threshold
  • the average decrease in biomass respiration rate between fed-batch input events is a substantial controlling parameter.
  • the biomass respiration rate is maintained at more than 30 percent of the extant maximum.
  • the biomass respiration rate is maintained in excess of 40 percent of the extant maximum.
  • the average biomass respiration rate is maintained in excess of 70 % of the extant maximum.
  • Up to a 30 percent decrease in relative respiration rates between fed-batch input events is preferred so as to reduce the negative impact of increased theoretical chain termination probability during the PHA accumulation process.
  • a high probability of chain termination during PHA accumulation in biomass is generally understood to result in lower average molecular weight.
  • fed-batch input control is based on a respiration trigger point rather than solely on dissolved oxygen.
  • Figure 6 illustrates how selection of a fed-batch input trigger based on equation 3 translated to a corresponding relative decrease in relative respiration between RBCOD stimulus events.
  • the ⁇ * set point under estimated the actual ⁇ * upon feeding due to delay in the feed-back and control ( ⁇ ). Differences between set and actual ⁇ * values were highest in the beginning of an accumulation due to the fact that the biomass was more active at the start of an accumulation.
  • the resultant relative decrease in extant respiration rates before the next feed-input was estimated
  • the fed-batch input control is quantitatively calibrated to actual respirometry rate shifts for the biomass.
  • Average molecular weight reflects the average size of the polymer chain lengths. In most cases PHA is a polymer with a relatively broad molecular weight distribution. M n is the number average molar mass and it is defined as:
  • Ni is the number of molecules with molar mass Mj.
  • M w The weight average molar mass, M w , is defined as:
  • PDI polydispersity index
  • M w is always larger than M n so the PDI will always be greater than 1.
  • PDI for PHA-resin is typically around 2 and M w has been observed to generally range from 10,000 to 3,000,000 Da.
  • Molecular weight distribution can be influenced by the method of accumulating PHA in the biomass, the method for recovering the PHA resin and the method of further processing the resin into end-user products.
  • PHA was extracted from distilled water-rinsed and dried biomass with acetone (20 mg- biomass containing nominally 10 mg-PHA per mL acetone) at 125°C for 2 hours.
  • the extracted polymer in acetone was decanted from the residual biomass and the solvent was evaporated.
  • SEC size exclusion chromatography
  • the SEC was performed with a pump (Viscotek VE 1122), a dual refractometer/viscometer-detector (Viscotek Model 250) and three linear columns coupled in a series (Shodex KF-805, Shodex KF-804 and Shodex KF802.5).
  • the detector temperature was 37°C, while the studies were carried out at room temperature.
  • the solvent used was chloroform (Merck pro analysis >99%) having a flow rate of 1 mL/min.
  • the injection volume was 100 ⁇
  • the sample examined by SEC was dissolved in chlorofonn to a concentration of 5 mg/mL at 100°C for 10 minutes. Before injecting the sample into the column the polymer solution was filtered (PALL Life Sciences Acrodisc ® CR 25 mm Syringe Filter with 0.45 ⁇ in pore size). From the resolved distribution of molecular weight for the PHA from each sample, the characteristic quantities of M w , M n and PDI were calculated.
  • the weight average molar mass (weight) or M has been the adopted metric for PHA molecular weight for the present invention. Determination of maximum molecular weight and accumulation rate
  • a challenge for fed-batch PHA accumulation is to achieve the maximum molecular weight by sustaining a high rate of polymerization with low chain termination probability.
  • the following methodology can be used to determine the expected maximum molecular weight and accumulation rate that is obtainable for a particular biomass under a particular set of environmental conditions. This maximum can serve as a point of reference for fed-batch accumulations that are conducted with the same biomass and RBCOD combination under more readily achievable fed-batch conditions in a larger scale system.
  • Biomass with substantial potential for PHA-accumulation but with a low initial PHA content (below 5 % of the total suspended solids) is used.
  • the biomass is adequately stirred and aeration is provided such that the concentration of dissolved oxygen is always above 2 mg/L.
  • Carbon substrate in the form of RBCOD is added to the biomass such that a substantial amount of PHA is produced without the need of more than three fed-batch inputs of substrate.
  • concentration of RBCOD exposed to the biomass will not go below 100 mg L more than a maximum of three times during the course of the accumulation experiment.
  • These feeding interruptions are systematically kept to a minimum using, for example, the control strategy of equation 3.
  • Adequate fed-batch input concentrations of RBCOD are in the range 0.5 to 2 g/L.
  • the molecular weight of the PHA extracted from the biomass at the end of such an experiment can reference the practically achievable maximum obtainable with the given biomass and substrate combination.
  • the highest specific rate of PHA accumulation observed over the course of such an experiment (expressed as g-PHA per g-'active biomass' per hour) can be used to indicate the maximum rate of PHA accumulation of the biomass under representative environmental conditions. (See, for example, Lemos et al., 2006, Serafim et al., 2004, Serafim et al., 2008).
  • Higher PHA molecular weight appears to be associated with maintaining, on average, higher respiration rates in between events of fed-pulse stimuli as shown in Figure 7.
  • a PHA accumulation process was performed with PAB-rich biomass and fermented dairy wastewater that is restricted in nitrogen content but still contains sufficient nitrogen, phosphorus and other trace nutrients to support a non-PHA growth response in the biomass during the PHA accumulation process.
  • the biomass achieved a nominal PHA content of 50% of the total suspended solids, but after that point combined growth and PHA storage lead to an overall increase of both active biomass and PHA content.
  • the relative PHA content of the biomass remained constant or increased slightly. In such cases a high respirometry in the biomass continues long after a maximum PHA content has been achieved since the total biomass in the process continues to increase.
  • the method of the present invention includes the monitoring of the mixed liquor suspended solids concentration as well as the relative change in suspended solids reflectivity (color) to follow the PHA accumulation process.
  • biomass reflectivity or color change is monitored with the technique of light back-scatter at near infrared wavelengths.
  • Figure 8 An example of such monitoring data is provided in Figure 8.
  • the initial biomass concentration was 1.5 g-VSS/L and feed pulse inputs resulted in a theoretical nominal maximum RBCOD concentration of 55 mg-COD/L for each feeding event.
  • the process can be terminated upon one or more thresholds being achieved.
  • the termination criteria can be established related to practical process capacity, wastewater discharge limits, PHA-volumetric productivity, PHA yields on substrate and the like.
  • One skilled in the art can recognize the practical, technical, economic and/or environmental performance constraints for determining when to terminate a PHA accumulation process.
  • the PHA accumulation process described in these examples and throughout the specification can be carried out as a part of a biological wastewater treatment process, or as an adjunct to a wastewater treatment process, or entirely separate from a wastewater treatment process.
  • wastewater influent or a wastewater stream is directed into a wastewater treatment system that typically comprises one or more reactors, a solids separator, and other complimentary components.
  • Activated sludge is used to biologically treat the wastewater influent.
  • the activated sludge is mixed with the wastewater influent to form mixed liquor and the mixed liquor is biologically treated.
  • the mixed liquor is subjected to aerobic, anoxic, and/or anaerobic conditions to carry out various biological treatment processes.
  • a solids separator such as a clarifier is used to separate the activated sludge from the wastewater and the separated activated sludge is recycled and mixed with incoming wastewater influent while a portion of the activated sludge is wasted.
  • the biomass that forms a part of the activated sludge or waste activated sludge can then be utilized in the PHA accumulation processes described herein.
  • the biomass is separated from the activated sludge or the waste activated sludge and directed to one or more reactors where the PHA accumulation process is employed.
  • the feed for the biomass can be taken from the wastewater influent which typically includes RBCOD. There are cases where the RBCOD concentration or the type of RBCOD in the wastewater influent is not appropriate for the PHA accumulation process. Therefore, in certain cases the biomass is fed with an alternative or augmented wastewater stream or another feed stream having an appropriate concentration and type of RBCOD.
  • the first example is a fed-batch PHA accumulation process wherein the active liquid (mixed liquor) volume increases over the course of the cycle ( Figure 9 and 10).
  • the second example is analogous except that a reactor configuration is shown where the active liquid volume is constant over the accumulation cycle ( Figure 11).
  • the accumulation cycle is briefly described below.
  • Both examples include monitoring strategies for the assessment of the specific substrate consumption rate (q s , Eqn. 2).
  • q s can be assessed by measurement of:
  • the mass or RBCOD fed to the process with each input of wastewater can be quantified. If only the RBCOD concentration is known, the mass fed into the process is equal to the volume fed times the RBCOD concentration.
  • the stimulating RBCOD concentration, s, for each fed-batch input must be greater than or equal to s m .
  • Methods for employing this concept can include the following steps: 1. Using a constant conservative fed-batch input to achieve theoretical PHA polymerization limiting kinetics. For example, an initial stimulation, s, of 200mg- RBCOD/L is conservative based on currently available experimental data.
  • s m Given the estimated q s to a fed-batch input stimulus of s, s m can be estimated due to an observed relationship between s m and k s (Eqn. 1). The relation can be calibrated specifically for different wastewater feeds as was performed for the results shown in Figure 1.
  • the system is shown at the start of the accumulation process before the first wastewater feed from P2.
  • Just tank T2 is shown in Figure 10.
  • the system is shown at the end of the accumulation process (A) where the DAF feed is supplied (B) in order to separate the effluent from the concentrated biomass (C).
  • Tl - an aerated constant volume tank used for rapidly combining wastewater influent (P2) with the recirculated mixed liquor (P3).
  • This tank is a completely mixed stirred tank reactor that may be aerated.
  • T2 - an aerated tank that can accommodate an increase of volume due to the wastewater influent added in Tl .
  • mixed liquor is recirculated back into reactor T2 from Tl.
  • This tank is an aerated completely mixed stirred tank reactor.
  • DAF - a reservoir for supplying dissolved air in order to achieve dissolved air flotation (DAF) in T2.
  • DAF dissolved air flotation
  • P5 - a pump and valve assembly for the discharge of effluent and or thickened biomass after biomass separation by DAF.
  • Chemicals may be used for conditioning the biomass for reasons of improved DAF separation and/or for reasons of conditioning the biomass for improved PHA recovery after accumulation.
  • the accumulation process proceeds as follows:
  • Waste activated sludge (WAS) of PAB-rich biomass from a wastewater treatment plant (WWTP) is fed into T2 with P4.
  • T2 and Tl aeration are started as is the recirculation pump P3.
  • the initial conditions for the biomass concentration, dissolved organic matter concentration, and respiration rates are assessed based on monitoring from Ml and M2.
  • a targeted peak concentration is achieved in Tl in order to provide a sufficient accumulation stimulus for the biomass.
  • the stimulus is sustained such that the biomass respiration rate is maintained at its extant maximum due to the selected pumping rates for P2 and P3 with on-line monitoring Ml and M2.
  • Wastewater influent input and recirculation flow rates may be based on measured depletion of dissolved organic matter concentration, estimated depletion rates of dissolved organic matter concentration, and/or a shift down in respiration rates based on Ml and M2.
  • Wastewater input is continued until the available reactor volume of T2 has been used up, the target BCOD mass has been fed, and/or the biomass exhibit signs of saturation in PHA due to, for example, a plateau or target in the trends of the biomass reflectivity.
  • Biomass is concentrated in reactor T2 by DAF. Generally, after the PHA accumulation process has begun and reached a steady state condition, the volume of mixed liquor in reactor Tl is maintained at a generally constant level while the volume of mixed liquor in reactor T2 varies. Note also that reactor Tl in this embodiment is smaller than reactor T2 and consequently the volume of mixed liquor in reactor Tl is less than the volume of mixed liquor in reactor T2.
  • the reactor is ready for the next PHA-accumulation cycle.
  • a - an internal airlift providing rapid mixing of wastewater influent and the reactor mixed liquor in a localized zone of reduced volume.
  • the airlift provides for aeration and mixing in the reactor.
  • the airlift is an internal open cylinder.
  • B - a downcomer which is in this illustration a concentric cylinder where the reactor contents are recirculated down back to the entrance of the airlift.
  • P1-P4 Pumps for control of WAS influent, wastewater influent, chemical addition, final effluent discharge (effluent-2), biomass harvesting.
  • the initially empty reactor is pumped full with WAS (P2).
  • Aeration is started.
  • the initial conditions for the biomass concentration, dissolved organic matter concentration, and respiration rates are assessed based on monitoring from Ml and M2.
  • a targeted peak concentration is achieved in A in order to provide a sufficient accumulation stimulus for the biomass.
  • the stimulus is sustained such that all the biomass has been exposed based on online monitoring Ml and M2, and/or the assessed internal recirculation rate.
  • Subsequent fed-batch wastewater influent inputs can be based on measured depletion of dissolved organic matter concentration, estimated depletion rates of dissolved organic matter concentration, and/or a shift down in respiration activity based on Ml and M2.
  • Wastewater input is continued until the target RBCOD mass has been fed, and/or the biomass exhibit signs of saturation in PHA due to, for example, a plateau in the trends of the biomass reflectivity.
  • aeration may briefly continue to maintain mixing while chemicals are added (PI). Chemicals may be added in order to inhibit degradation of the stored PHA and to improve biomass separation by gravity settling.
  • Biomass is concentrated in the reactor by gravity settling.
  • the reactor is ready for the next PHA-accumulation cycle.

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Abstract

L'invention concerne un procédé ou un processus de production de polyhydroxyalcanoates (PHA) dans une biomasse. Le procédé implique l'alimentation par un substrat contenant du carbone organique d'une biomasse enrichie en bactéries accumulant les PHA. En particulier, le procédé implique l'apport intermittent du substrat à la biomasse au moins trois fois séparément sur une période déterminée. L'objectif du procédé est de produire des PHA de poids moléculaire relativement élevé, au moins 400 000 g/mole. En commandant la fréquence à laquelle le substrat est apporté à la biomasse et en alimentant la biomasse par une quantité suffisante de substrat, le procédé produit des PHA de poids moléculaire relativement élevé.
PCT/IB2010/055745 2009-12-10 2010-12-10 Procédé pour accumuler des polyhydroxyalcanoates dans une biomasse avec contrôle en ligne pour la commande de la vitesse d'alimentation et l'arrêt du processus WO2011070544A2 (fr)

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CN201080063546.1A CN102770549B (zh) 2009-12-10 2010-12-10 使用在线监测进料速率控制和过程终止在生物质中积累聚羟基烷酸酯的方法
CA2783591A CA2783591C (fr) 2009-12-10 2010-12-10 Procede pour accumuler des polyhydroxyalcanoates dans une biomasse avec controle en ligne pour la commande de la vitesse d'alimentation et l'arret du processus
RU2012128820/10A RU2535341C2 (ru) 2009-12-10 2010-12-10 Способы аккумуляции полигидроксиалканоатов в биомассе со слежением в масштабе времени (варианты)
BR112012013882A BR112012013882A2 (pt) 2009-12-10 2010-12-10 métodos para produzir poli-hidroxialcanoatos (phas), e, para promover o acúmulo de poli-hidroxialcanoatos (phas) em biomassa
IN5193DEN2012 IN2012DN05193A (fr) 2009-12-10 2010-12-10
US13/514,660 US8748138B2 (en) 2009-12-10 2010-12-10 Method for accumulation of polyhydroxyalkanoates in biomass with on-line monitoring for feed rate control and process termination
EP10809241.2A EP2510103B1 (fr) 2009-12-10 2010-12-10 Procédé pour accumuler des polyhydroxyalcanoates dans une biomasse avec contrôle en ligne pour la commande de la vitesse d'alimentation et l'arrêt du processus
NZ600542A NZ600542A (en) 2009-12-10 2010-12-10 Method for accumulation of polyhydroxyalkanoates in biomass with on-line monitoring for feed rate control and process termination
AU2010329473A AU2010329473B2 (en) 2009-12-10 2010-12-10 Method for accumulation of polyhydroxyalkanoates in biomass with on-line monitoring for feed rate control and process termination

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WO2013022874A1 (fr) * 2011-08-09 2013-02-14 Hsin-Ying Liu Production de polyhydroxyalcanoate pendant le traitement des eaux usées
WO2013140013A1 (fr) * 2012-03-23 2013-09-26 Universidade Da Coruña Procédé d'obtention de polyhydroxyalcanoates à partir d'eaux résiduaires provenant de l'industrie de la fabrication de la bière
WO2014108878A1 (fr) 2013-01-11 2014-07-17 Veolia Water Solutions & Technologies Support Procédé permettant d'obtenir une productivité accrue de polyhydroxyalcanoates (pha) dans des procédés d'alimentation programmée pour biomasse dérivée du traitement des eaux usées
WO2014108864A1 (fr) 2013-01-11 2014-07-17 Veolia Water Solutions & Technologies Support Procédés de traitement biologique des eaux usées qui améliorent la capacité d'accumulation des polyhydroxyalcanoates (pha) dans une biomasse en culture mixte
WO2014125422A1 (fr) 2013-02-14 2014-08-21 Veolia Water Solutions & Technologies Support Procédé d'extraction de polyhydroxyalcanoates (pha) à partir d'une biomasse
WO2016020884A1 (fr) 2014-08-07 2016-02-11 Veolia Water Solutions & Technologies Support Procédé pour améliorer l'accumulation de polyhydroxyalcanoates d'une biomasse de boues activées
WO2016020816A2 (fr) 2014-08-04 2016-02-11 Veolia Water Solutions & Technologies Support Procédé utilisant un biofilm pour le traitement de l'eau et caractérisé par une production continue ou semi-continue de biomasse à teneur renforcée en polyhydroxyalcanoates
WO2016081902A1 (fr) * 2014-11-20 2016-05-26 Full Cycle Bioplastics Inc. Production de copolymères de polyhydroxyalcanoates à partir de déchets organiques
CN109312372A (zh) * 2016-06-23 2019-02-05 株式会社钟化 聚羟基链烷酸的制造方法
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EP3176132A1 (fr) * 2015-12-03 2017-06-07 Paques I.P. B.V. Procédé de production d'un composé de stockage microbien
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US6737263B2 (en) * 2000-09-08 2004-05-18 E. I. Du Pont De Nemours And Company Polyhydroxyalkanoate levels as an indicator of bioreactor health
RU2201453C1 (ru) * 2001-10-18 2003-03-27 Бонарцева Гарина Александровна СПОСОБ ПОЛУЧЕНИЯ ПОЛИ-β-ОКСИБУТИРАТА ЗАДАННОЙ МОЛЕКУЛЯРНОЙ МАССЫ
ITRM20020444A1 (it) * 2002-09-06 2004-03-07 Univ Roma Processo per la sintesi di polimeri biodegradabili a partire da rifiuti e fanghi attivati arricchiti in condizioni non stazionarie.
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WO2013022874A1 (fr) * 2011-08-09 2013-02-14 Hsin-Ying Liu Production de polyhydroxyalcanoate pendant le traitement des eaux usées
US9150445B2 (en) 2011-08-09 2015-10-06 Hsin-Ying Liu Polyhydroxyalkanoate production during wastewater treatment
US10807893B2 (en) 2011-08-09 2020-10-20 Hsinying Liu Polyhydroxyalkanoate production during wastewater treatment
WO2013140013A1 (fr) * 2012-03-23 2013-09-26 Universidade Da Coruña Procédé d'obtention de polyhydroxyalcanoates à partir d'eaux résiduaires provenant de l'industrie de la fabrication de la bière
JP2016509532A (ja) * 2013-01-11 2016-03-31 ヴェオリア・ウォーター・ソリューションズ・アンド・テクノロジーズ・サポート 混合培養バイオマスにおいてポリヒドロキシアルカノエート蓄積容量を向上する、生物的廃水処理プロセス
WO2014108878A1 (fr) 2013-01-11 2014-07-17 Veolia Water Solutions & Technologies Support Procédé permettant d'obtenir une productivité accrue de polyhydroxyalcanoates (pha) dans des procédés d'alimentation programmée pour biomasse dérivée du traitement des eaux usées
WO2014108864A1 (fr) 2013-01-11 2014-07-17 Veolia Water Solutions & Technologies Support Procédés de traitement biologique des eaux usées qui améliorent la capacité d'accumulation des polyhydroxyalcanoates (pha) dans une biomasse en culture mixte
JP2016510214A (ja) * 2013-01-11 2016-04-07 ヴェオリア・ウォーター・ソリューションズ・アンド・テクノロジーズ・サポート 廃水の処理から得られる、バイオマスのためのフェドバッチプロセスにおけるポリヒドロキシアルカノエート（ｐｈａ）の生産性向上のための方法
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WO2016020884A1 (fr) 2014-08-07 2016-02-11 Veolia Water Solutions & Technologies Support Procédé pour améliorer l'accumulation de polyhydroxyalcanoates d'une biomasse de boues activées
WO2016081902A1 (fr) * 2014-11-20 2016-05-26 Full Cycle Bioplastics Inc. Production de copolymères de polyhydroxyalcanoates à partir de déchets organiques
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EP3476943A4 (fr) * 2016-06-23 2020-03-04 Kaneka Corporation Procédé de fabrication d'acide polyhydroxyalcanoïque
CN109312372B (zh) * 2016-06-23 2022-06-28 株式会社钟化 聚羟基链烷酸的制造方法
US11440823B2 (en) 2016-06-23 2022-09-13 Kaneka Corporation Method for producing polyhydroxyalkanoic acid

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EP2510103A2 (fr) 2012-10-17
BR112012013882A2 (pt) 2019-09-24
NZ600542A (en) 2013-10-25
RU2012128820A (ru) 2014-01-20
RU2535341C2 (ru) 2014-12-10
WO2011070544A3 (fr) 2011-08-11
US20130029388A1 (en) 2013-01-31
CA2783591C (fr) 2016-02-02
AU2010329473B2 (en) 2013-07-04
CN102770549A (zh) 2012-11-07
US8748138B2 (en) 2014-06-10
AU2010329473A1 (en) 2012-07-05

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