PHARIO - stepping stone to a sustainable value chain for PHA bioplastic using municipal activated sludge

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Report number: STOWA 2017-15, ISBN 978.90.5773.752.7, Affiliation: Aiforo
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Abstract
Production of biopolymers of the polyhydroxyalkanoate (PHA) family by mixed microbial cultures (MMC) in association with wastewater treatment (WWT) has been identified as a valorization route of residual organic material. Since 2011, AnoxKaldnes has been benchmarking technologies for MMC PHA production from WWT at pilot scale at three different sites and with both industrial as well as municipal organic residuals as feedstock. These previous experiences served as a basis to a PHA production and biobased value chain demonstration project, PHARIO, together with the Dutch water authorities under STOWA and lead by water authorities Brabantse Delta, de Dommel, Fryslân and Scheldestromen. Other project partners were AnoxKaldnes (Veolia Water Technologies AB), sludge incinerator SNB and KNN. PHARIO was centred on processing surplus biomass from the Bath full-scale municipal wastewater treatment plant (WWTP) in the Netherlands to produce PHA polymers. For the PHARIO project, the full-scale surplus activated sludge was fed into a pilot facility at Bath to consistently produce PHA rich biomass with on average 0.41 gPHA/gVSS. To produce PHA, the biomass was fed with VFA rich liquors derived from a local candy industry or primary sludge. As a benchmark mixtures of pure acetic and propionic acids were also fed. The PHA in the biomass was recovered in a pilot extraction process located at AnoxKaldnes in Lund Sweden. A routine of a weekly kilogram scale batch wise production was established over a 10-month period and the recovered polymers were evaluated for their material properties and market potential. The results of the project show that the harvested activated sludge can consistently produce a high quality PHA polymer that has interesting and meaningful application potentials. Additionally, the polymer has a significantly lower environmental impact compared to currently available (bio)plastics.
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POSTBUS 2180 3800 CD AMERSFOORT
PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
2017 15
PHARIO
STEPPING STONE TO A SUSTAINABLE
VALUE CHAIN FOR PHA BIOPLASTIC
USING MUNICIPAL ACTIVATED SLUDGE
RAPPORT
2017
15
STOWA 2017 15 omslag.indd 1 05-04-17 13:23
stowa@stowa.nl
www
.stowa.nl
TEL 033 460 32 00
Stationsplein 89 3818 LE Amersfoort
POSTBUS 2180 3800 CD AMERSFOORT
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PHARIO:
STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR
PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
2017
15
RAPPORT
ISBN 978.90.5773.752.7
STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
UITGAVE Stichting Toegepast Onderzoek Waterbeheer
Postbus 2180
3800 CD Amersfoort
AUTHORS
Simon Bengtsson, Veolia Water Technologies / Promiko
Alan Werker, Veolia Water Technologies / Promiko
Cindy Visser, KNN Advies
Leon Korving, Aiforo
PROJECT MANAGEMENT GROUP
Yede van der Kooij, Wetterskip Fryslan
Etteke Wypkema, Waterschap Brabantse Delta
Leon Korving, Waterschap Brabantse Delta, Aiforo
Aad Oomens, Waterschap De Dommel
Jarno de Jonge, Waterschap De Dommel
Luc Sijstermans, N.V. Slibverwerking Noord-Brabant
Martin Tietema, KNN Bioplastics
Alan Werker, AnoxKaldnes AB/Veolia Water Technologies AB, Promiko
Cora Uijterlinde, STOWA
DRUK Kruyt Grafisch Adviesbureau
STOWA STOWA 2017-15
ISBN 978.90.5773.752.7
COLOFON
COPYRIGHT Teksten en figuren uit dit rapport mogen alleen worden overgenomen met bronvermelding.
DISCLAIMER Deze uitgave is met de grootst mogelijke zorg samengesteld. Niettemin aanvaarden de auteurs en
de uitgever geen enkele aansprakelijkheid voor mogelijke onjuistheden of eventuele gevolgen door
toepassing van de inhoud van dit rapport.
STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
TEN GELEIDE
De Nederlandse waterschappen werken hard aan het terugwinnen van grondstoffen uit
rioolwaterzuiveringen. Dat gebeurt in het kader van een Green Deal Grondstoffen die de
waterschappen en het Rijk in november 2014 sloten. De ambities op dit gebied zijn in januari
2017 nog eens vastgelegd in het nationale Grondstoffenakkoord dat mede werd ondertekend
door de Unie van Waterschappen.
Waar dat harde werken toe kan leiden, laat het PHARIO-project (PHA uit RIOolwater) goed
zien. In dit project is in de praktijk aangetoond dat actief slib uit rioolwaterzuiveringen kan
worden ingezet als grondstof voor de productie van PHA, een hoogwaardige kwaliteit biolo-
gisch afbreekbaar én biobased plastic.
PHA is een bijzonder bioplastic, omdat het relatief snel afbreekt in een waterig milieu. Deze
eigenschap maakt het zeer interessant voor het bestrijden van microplastics in oppervlakte-
water, de zogenoemde plastic soup. Daarmee dragen de waterschappen zelf ook bij aan een
goede oppervlaktewaterkwaliteit, waar zij medeverantwoordelijk voor zijn.
De milieu-impact van PHA-plastic uit actief slib is fors lager dan plastics van petrochemische
oorsprong en zelfs lager dan vergelijkbare bioplastics die nu op de markt aanwezig zijn.
PHA bioplastic gemaakt uit actief slib concurreert bovendien niet met de voedselketen. Het
huidige PHA bioplastic, afkomstig van landbouwproducten, doet dat wel.
De resultaten van dit project geven meer dan voldoende reden om te onderzoeken hoe we
dit concept verder kunnen opschalen. Die opschaling moet vermoedelijk in stappen plaats-
vinden, waarbij de productie eerst op demonstratie schaal wordt beproefd, waarna de stap
naar een commerciële schaalgrootte gezet kan worden.
De benodigde investeringen voor deze opschaling zijn fors. Het is daarom belangrijk in
overleg met waterschapsbestuurders de rol van het waterschap bij het terugwinnen en
verwaarden van grondstoffen uit afvalwater verder uit te werken. Door slimme samenwer-
kingen en gebruik van bestaande faciliteiten kunnen de kosten van deze stappen hopelijk
verlaagd worden, zodat we met elkaar deze veelbelovende ontwikkeling met beheersbare
risico’s verder kunnen verkennen.
Joost Buntsma,
Directeur STOWA
STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
SUMMARY
Production of biopolymers of the polyhydroxyalkanoate (PHA) family by mixed microbial
cultures (MMC) in association with wastewater treatment (WWT) has been identified as a valo-
rization route of residual organic material. Since 2011, AnoxKaldnes has been benchmarking
technologies for MMC PHA production from WWT at pilot scale at three different sites and
with both industrial as well as municipal organic residuals as feedstock Eslöv (Sweden),
Brussels (Belgium) and Leeuwarden (the Netherlands). This experience has lead to methods to
up-scale unit processes for MMC PHA production with various strategies of technology inte-
gration to existing infrastructures for WWT.
These milestones of technical success have lead in 2015 and 2016 to a PHA production and
biobased value chain demonstration project, PHARIO, together with the Dutch water authori-
ties under STOWA and lead by water authorities Brabantse Delta, de Dommel, Fryslan and
Scheldestromen. Other project partners were AnoxKaldnes (Veolia Water Technologies AB),
sludge incinerator SNB and KNN. PHARIO was centred on processing surplus biomass from
the Bath full-scale municipal wastewater treatment plant (WWTP) in the Netherlands to
produce PHA polymers.
The biological municipal WWT at the Bath site is a biological nitrogen removal process with
anoxic pre-denitrification and chemical phosphorus removal. The bioprocess facilitates feast
and famine conditions, which have been shown to favor the selection of a biomass with PHA
accumulating potential. A PHARIO pre-investigation using the pilot scale facility in Brussels
and using the full-scale secondary activated sludge from Bath WWTP have similarly been
shown to produce a biomass with a PHA content of up to 0.47 gPHA/gVSS. Generally, a PHA
accumulation potential above 0.40 gPHA/gVSS has been identified as a threshold for achieving
a promising business case of integrating PHA production in the material flows of municipal
and/or industrial wastewater treatment plants.
FIGURE 1 BUSINESS CARD HOLDER MADE WITHIN THE PHARIO PROJECT USING A BIOPLASTIC FORMULATION CONTAINING 74% PHARIO PHA
(ACKNOWLEDGEMENT: MADE POSSIBLE BY COURTESY OF PEZY)
STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
For the PHARIO project, the full-scale surplus activated sludge was fed into a pilot facility at
Bath to consistently produce PHA rich biomass with greater than 0.40 gPHA/gVSS. In order to
produce PHA, the biomass was fed with VFA rich liquors derived from a local candy industry
or primary sludge. As a benchmark mixtures of pure acetic and propionic acids were also fed.
The PHA in the biomass was recovered in a pilot refinery process located at AnoxKaldnes in
Lund Sweden. A routine of a weekly kilogram scale batch wise production was established
over a 10-month period and the recovered polymers were evaluated for their material proper-
ties and market potential.
The results of the project show that the harvested activated sludge can consistently produce
a high quality PHA polymer that has interesting and meaningful application potentials.
Within the PHARIO project a large set of material property data (thermal, mechanical) were
generated, thus making it possible for PHA end-users to evaluate the potential of the material.
The project showed that PHA can be produced at a competitive cost prize, compared to current
market prices. Further cost reductions are possible. Additionally, the produced polymer has a
70% lower environmental impact compared to currently available PHA bioplastic.
STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
DE STOWA IN BRIEF
The Foundation for Applied Water Research (in short, STOWA) is a research platform for
Dutch water controllers. STOWA participants are all ground and surface water managers in
rural and urban areas, managers of domestic wastewater treatment installations and dam
inspectors.
The water controllers avail themselves of STOWA’s facilities for the realisation of all kinds
of applied technological, scientific, administrative legal and social scientific research activi-
ties that may be of communal importance. Research programmes are developed based on
requirement reports generated by the institute’s participants. Research suggestions proposed
by third parties such as knowledge institutes and consultants, are more than welcome. After
having received such suggestions STOWA then consults its participants in order to verify the
need for such proposed research.
STOWA does not conduct any research itself, instead it commissions specialised bodies to do
the required research. All the studies are supervised by supervisory boards composed of staff
from the various participating organisations and, where necessary, experts are brought in.
The money required for research, development, information and other services is raised by
the various participating parties. At the moment, this amounts to an annual budget of some
6,5 million euro.
For telephone contact number is: +31 (0)33 - 460 32 00.
The postal address is: STOWA, P.O. Box 2180, 3800 CD Amersfoort.
E-mail: stowa@stowa.nl.
Website: www.stowa.nl.
PHARIO:
STEPPING STONE TO A SUSTAINABLE
VALUE CHAIN FOR PHA BIOPLASTIC
USING MUNICIPAL ACTIVATED SLUDGE
STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
INHOUD
TEN GELEIDE
SUMMARY
DE STOWA IN BRIEF
1 INTRODUCTION 1
1.1 Guide to the reader 1
1.2 Acknowledgements 2
2 THE PHARIO CONCEPT 4
3 PHA ACCUMULATION POTENTIAL OF WWTPS 7
3.1 Introduction 7
3.2 Selection of plants 7
3.3 PHA accumulation potential tests 9
3.3.1 Method 9
3.3.2 Calculations 9
3.4 Results 10
3.4.1 General behaviour of the accumulation experiments 10
3.4.2 Accumulation results 10
3.4.3 Factors influencing PHA accumulation potential 12
3.4.4 Separate COD-rich streams 15
3.5 Approaches to improve PHA accumulation potential 16
3.5.1 Establishing distinct feast conditions for the biomass 16
3.5.2 Integration of EBPR and PHA production 18
3.6 Potential for PHA production 19
3.7 Summary and conclusion 20
4 TESTING THE PHARIO PROCESS 21
4.1 Pilot plant operation 21
4.1.1 VFA Feedstocks 21
4.1.2 PHA accumulation 22
4.1.3 PHA extraction 23
4.1.4 Production batches and coding 24
4.2 PHA chemistry and characterization 24
4.2.1 Introduction to PHA polymer chemistry 24
4.2.2 Thermal characterization of PHA 26
4.3 PHA yields and substrate consumption 29
4.4 Stability of the produced PHA 32
STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
4.4.1 Stability during storage 32
4.4.2 Thermal stability 34
4.5 Steering the PHA quality 37
4.5.1 Effect of the feedstock composition 37
4.5.2 VFA feed composition determines co-polymer composition 39
4.5.3 Consistent polydispersity 41
4.5.4 Consistent thermal stability 41
4.5.5 Varying molecular weight but consistent mechanical properties 42
4.5.6 Homogeneous blends of copolymers: one glass transition temperature 43
4.5.7 Different PHA batches can be blended 44
4.5.8 Consistent melt and crystallization behaviour 45
4.6 Conclusion 49
5 VALORIZATION OF THE PRODUCED PHA 51
5.1 Introduction 51
5.2 Pilot scale PHA extraction 51
5.2.1 Procedure 51
5.2.2 Tuning PHA extraction to 3HV content 52
5.2.3 Influence of pilot scale extraction on molecular weight 53
5.3 Mechanical properties of the extracted PHA 55
5.4 Testing applications of PHARIO PHA 57
5.4.1 Biomer-like thermoplastics from PHARIO PHA 58
5.4.2 PHARIO PHA to improve impact resistance of PLA 61
5.4.3 Injection moulding of PHARIO PHA 64
5.4.4 PHARIO PHA in film applications 65
5.4.5 Other commercial contacts for downstream processing 67
6 SUSTAINABILITY OF THE PHARIO PROCESS 70
6.1 Introduction 70
6.2 Environmental impact 70
6.2.1 Introduction LCA study 70
6.2.2 Method and LCA framework 70
6.2.3 Substituted product: pure culture PHA 71
6.2.4 Scenario’s 73
6.2.5 Results 75
6.2.6 Sensitivity analysis 76
6.3 Economics of a future value chain 77
6.3.1 Market size and scale of commercial production 77
6.3.2 Availability of VFA feedstock 78
6.3.3 PHA production cost for a first commercial reference 79
6.3.4 PHARIO business case 80
7 A DEMONSTRATION FACILITY FOR PHARIO 82
7.1 Introduction 82
7.2 Design approach 82
7.3 Cost estimate 83
7.4 Conclusion 83
8 SUMMARIZING CONCLUSION 84
9 ABBREVIATIONS 86
10 REFERENCES 87
1
STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
1
INTRODUCTION
Production of biopolymers of the polyhydroxyalkanoate (PHA) family by mixed microbial
cultures (MMC) in association with wastewater treatment (WWT) has since long been identi-
fied as a valorisation route of residual organic material. Since 2011, AnoxKaldnes has been
benchmarking technologies for MMC PHA production from WWT at pilot scale at three
different sites and with both industrial as well as municipal organic residuals as feedstock
– Eslöv (Sweden), Brussels (Belgium) and Leeuwarden (the Netherlands). This experience has
lead to methods to up-scale unit processes for MMC PHA production with various strategies of
technology integration to existing infrastructures for WWT.
All these previous projects showed the potential for production of PHA using activated sludge
harvested from municipal wastewater treatment plants. However more information was
needed about the quality of the PHA that can be produced in this way. This has led to a PHA
production and biobased value chain demonstration project, PHARIO. Project partners in the
project were water authorities Brabantse Delta, de Dommel, Fryslan and Scheldestromen and
the other Dutch water authorities under STOWA. Other project partners were AnoxKaldnes
(Veolia Water Technologies AB), sewage sludge incinerator SNB and KNN Advies. This project
was made possible through a financial contribution from the Dutch government, repre-
sented by RVO under the subsidy program “Subsidieregeling energie en innovatie, Biobased
Economy: Innovatieprojecten”.
PHARIO was centred on processing surplus biomass from the Bath full-scale municipal waste-
water treatment plant (WWTP) in the Netherlands to produce PHA polymers.
Main objectives of the PHARIO project were to:
Prove that a high quality PHA product can consistently be produced using biomass harve-
sted from a full-scale municipal wastewater treatment plant.
To provide enough PHA material to be able to evaluate product quality and identify pos-
sible applications.
In this way the PHARIO project is a stepping-stone towards further up scaling of the tech-
nology to a demonstration phase where the technology can be demonstrated on a continuous
basis producing enough material to test market application.
1.1 GUIDE TO THE READER
Within the scope of the PHARIO project several work packages were defined covering different
aspects of the scope of the PHARIO project.
Work package 1 (WP1) was the core of the project and involved a 10 month pilot operation
at the waste water treatment plant of Bath. During this period activated sludge was continu-
2
STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
ously harvested from the full scale waste water treatment plant and then fed with different
sources of volatile fatty acids (VFA). As a benchmark synthetic VFAs were fed to evaluate the
performance of the biomass without other influences. Furthermore waste water was obtained
from a local candy factory and this waste water was fermented on site at Bath and the fed to
the biomass. Also primary sludge was obtained from the sewage treatment plant of Tilburg of
water authority De Dommel. The primary sludge was fermented at Bath and then fed to the
secondary sludge of Bath.
In this way typically two batches of PHA rich biomass were produced per week. These batches
were dried and then transported to Anoxkaldnes in Lund, Sweden. Here part of the batches
were extracted in a pilot extraction unit to produce pure PHA.
The quality of the extracted PHA was characterized and part of the extracted PHA was used
to test potential applications of the PHA and to assess mechanical properties of the PHA. The
results of the work in workpackage 1 are discussed in chapters 4 and 5.
In work package 2 (WP2) the scalability of the process to other wastewater treatment
plants was tested. Based on previous knowledge the wastewater treatment plant of Bath was
selected because it had shown high PHA accumulation potential in lab scale tests. Within
WP2 a large number of different wastewater treatment plants were sampled and tested at lab
scale for their accumulation potential to understand how often high accumulation potentials
are encountered at waste water treatment plants and to learn more about the factors that
favour high accumulation potentials. The results of this work are reported in chapter 3.
In work package 3 (WP3) a basic engineering design and cost estimate was made for a first
full scale reference plant (FFSR) with a capacity of 30-100 ton PHA/year. This plant would be
a next step in scale after the PHARIO project and has as objective to demonstrate the value
chain for the produced PHA and test all unit operations at such a scale that the testing would
allow further up scaling to a commercial plant. The FFSR-facility would not yet be a commer-
cial operation but demonstrates the potential of the technology. The results of this work
package are discussed in chapter 7.
Work package 4 (WP4) studied the business case for a first commercial reference (FCR) plant
with an anticipated capacity of 5000 ton PHA/year. A basic engineering design of all process
elements was made within this framework and based on that design operational and capital
costs were estimated. Furthermore a Life Cycle Analysis study was performed to evaluate the
environmental benefit of the PHARIO concept. The results of this work package are discussed
in chapter 6.
1.2 ACKNOWLEDGEMENTS
This report summarizes the results of the PHARIO project and is based on a set of deliverables
with further and more detailed information on the different results of the PHARIO project.
PHARIO was a large team and a multifaceted coordinated effort from the AnoxKaldnes Cella™
team (Veolia Water Technologies AB) including Simon Bengtsson, Markus Hjort, Simon
Anterrieu, Lamija Karabegovic, Peter Johansson, Per Magnusson, Tomas Alexandersson, Anton
Karlsson, and Fernando Morgan-Sagastume. There were many who contributed and made a
difference. The project was managed by a project management group with participation of
3
STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
Leon Korving (project manager, Aiforo), Alan Werker (AnoxKaldnes), Etteke Wypkema (water-
schap Brabantse Delta), Yede van der Kooij (wetterskip Fryslan), Cora Uijterlinde (STOWA),
Martin Tietema (KNN Advies and Bioplastics), Jarno de Jonge (waterschap De Dommel) and
Luc Sijstermans (SNB).
Many more contributed to the succes. We also thank: Waterschap Brabantse Delta: Lennert De
Graaf, Martijn Gebraad, Gijs Doornbusch, Louise Johann-Deusing, Sean van der Meulen, Kees
van Hoof, Ruben de Wild, Danny Tak, Leon Maas, Jan van Eekelen, Tomas van Eekeren, Eric
Groenewald, Jack Eversdijk, Levien van Dixhoom. Waterschap De Dommel: Doy Schellekens,
Peter van Horne, Alexandra Deeke, Victor Claessen, Aad Oomens. KNN Advies and KNN
Bioplastics: Yme Flapper, Peter Dijkstra. Waterschap Scheldestromen: Jo Nieuwlands, Avans
Hogeshool: Koen van Beurden, Jack van Schijndel. Kruger AS: Hans Erik Madsen. Pezy Group:
Jan Hoekstra, Joop Onnekink, Abel Hartlief, Thijs Feenstra. Biomer: Urs Hänggi. Bioplastech:
Kevin O’Connor, Ramesh Padama. Wageningen University and Research Centre: Gerald
Schennink, Richard op den Kamp, Hans Mooibroek, Frans Kappen. Veolia: Eric Train, Corrine
Jamot, Carina Roselius, Gitte Andersen, Stig Stork. The list of names, in spite of best efforts
and intentions, may not be all-inclusive. So, it is with much thanks to all who have provided
wind in the sails of the PHARIO project along its way.
PHARIO is financially supported by a subsidy from the Topsector Energy program of the
Dutch ministry of Economic Affairs (TKI Biobased Economy) and contributions by the
PHARIO project partners: Veolia Water Technologies, the Dutch water authorities Brabantse
Delta, De Dommel, Fryslan and Scheldestromen, STOWA, KNN and Slibverwerking Noord-
Brabant.
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STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
2
THE PHARIO CONCEPT
During the past decade, the technical feasibility of the production of biodegradable thermo-
plastic polyesters, poly-hydroxyalkanoates (PHAs), by open mixed microbial cultures (MMCs)
has been repeatedly demonstrated using waste and residual carbon sources as substrates.
These MMC systems generally include three biological process elements (PEs): PE1 - acido-
genic fermentation, PE2 - enrichment and production of biomass with PHA-storing capacity,
and PE3 - PHA accumulation using PE2 surplus biomass and feedstocks with easily degrad-
able organics. Polymers may be recovered from a PHA-rich mixed culture biomass in a fourth
process element (PE4) by means of solvent extraction.
FIGURE 2 MUNICIPAL WASTEWATER TREATMENT PLANT BATH. THE PLANT TREATS WASTEWATER FROM 500.000 PEOPLE EQUIVALENTS AND HAS THE
POTENTIAL TO SUPPLY BIOMASS TO PRODUCE 2000-2500 TON PHA/YEAR.
The PHARIO project is based on the understanding that full-scale municipal wastewater treat-
ment plants can serve as process units that produce functional biomass (activated sludge)
with PHA storing capacities without modifications to the sewage treatment plant (they
already are PE2s). In this way the need of a separate process element is avoided, as well as the
need to dedicate raw material to the production of the functional biomass (Figure 3).
5
STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
FIGURE 3 SIMPLIFIED DIAGRAM COMPARING THE PHARIO APPROACH TO THE PRODUCTION OF PHA RICH BIOMASS USING PURE CULTURES.
PHARIO
PHARIO final reportpublic version 28/3/2017 Page 8
2 THE PHARIO CONCEPT
During the past decade, the technical feasibility of the production of biodegradable thermoplastic polyesters,
poly-hydroxyalkanoates (PHAs), by open mixed microbial cultures (MMCs) has been repeatedly demonstrated
using waste and residual carbon sources as substrates. These MMC systems generally include three
biological process elements (PEs): PE1 - acidogenic fermentation, PE2 - enrichment and production of
biomass with PHA-storing capacity, and PE3 - PHA accumulation using PE2 surplus biomass and feedstocks
with easily degradable organics. Polymers may be recovered from a PHA-rich mixed culture biomass in a
fourth process element (PE4) by means of solvent extraction.
Figure 2: Municipal wastewater treatment plant Bath. The plant treats wastewater from 500.000 people equivalents and has the
potential to supply biomass to produce 2000-2500 ton PHA/year.
The PHARIO project is based on the understanding that full-scale municipal wastewater treatment plants can
serve as process units that produce functional biomass (activated sludge) with PHA storing capacities without
modifications to the sewage treatment plant (they already are PE2’s). In this way the need of a separate
process element is avoided, as well as the need to dedicate raw material to the production of the functional
biomass (Figure 3).
Figure 3: simplified diagram comparing the PHARIO approach to the production of PHA rich biomass using pure cultrues
A value chain based on this concept is shown in Figure 4. Secondary sludge is harvested from a municipal
sewage treatment plant and then used as functional biomass to produce PHA. Organic residuals from the
region around the sewage treatment plant can be collected, fermented (PE1) and fed to the sludge (PE3) to
produce a PHA rich biomass, with a typical PHA content of 40-50% of the total volatile solids. The water
authorities themselves can supply part of the required organic waste in the form of the primary sludge they
A value chain based on this concept is shown in Figure 4. Secondary sludge is harvested
from a municipal sewage treatment plant and then used as functional biomass to produce
PHA. Organic residuals from the region around the sewage treatment plant can be collected,
fermented (PE1) and fed to the sludge (PE3) to produce a PHA rich biomass, with a typical PHA
content of 40-50% of the total volatile solids. The water authorities themselves can supply part
of the required organic waste in the form of the primary sludge they produce. Normally the
accumulation potential of the secondary sludge is much larger than the amount of fatty acids
that can be produced from the primary sludge. Therefore it is advantageous to also resource
regional organic waste streams as feed to make optimal use of the PHA accumulation poten-
tial of the secondary sludge.
FIGURE 4 PROCESS STEPS TO PRODUCE PHA USING BIOMASS HARVESTED FROM A MUNICIPAL WASTEWATER TREATMENT PLANT.
PHARIO final reportpublic version 28/3/2017 Page 9
The PHA rich biomass is acidified to conserve the PHA and then dewatered in centrifuges or
belt filter presses. The dewatered PHA rich biomass is then dried in a thermal dryer to a dry
matter content of at least 90%. Following the drying the PHA is then extracted in an extrac-
tion facility where solvents like butanol are used to extract the PHA from the biomass. The
extraction takes place at elevated temperature to dissolve the PHA in the solvent. Through
cooling of the solvent the PHA can be recovered from the solvent and the solvent reused (see
Werker, 2015). An important advantage of this type of extraction is that it safeguards the
6
STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
quality of the PHA and provides opportunities to control the quality and blend different PHA
batches to a compound.
The residual matter after extraction is then incinerated in a similar way as the original sludge
would have been. There is future potential for the extraction of lipids from this residue, but
this was not investigated within the framework of the PHARIO project.
7
STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
3
PHA ACCUMULATION POTENTIAL OF
WWTPS
3.1 INTRODUCTION
Production of polyhydroxyalkanoate (PHA) biopolymers integrated with municipal waste-
water treatment may be accomplished with four process elements (PE1-4). In PE2, a surplus
biomass with the potential to accumulate PHA is produced while wastewater is concurrently
treated. The possibility to produce such a biomass with synthetic streams and process waters
having relatively high concentrations of readily biodegradable chemical oxygen demand
(RBCOD) has been already studied for a long time. More recently, was found that the rela-
tively low concentrations of RBCOD generally found in municipal wastewater are sufficient
to produce biomass with a high PHA accumulation potential (PAP) given that a selective pres-
sure for storage of RBCOD is established in the biological treatment process. It has been esti-
mated that the PAP should be at least 40 % g-PHA/g-VSS for economical down-stream recovery
of the polymer.
Enrichment of a biomass with high PAP in municipal wastewater treatment has been observed
at laboratory, pilot and, in one case, at full-scale treatment, namely at the wastewater treat-
ment plant (WWTP) Bath. However, until now it has been unclear how common enrichment
of PAP is at existing wastewater treatment plants and what factors with respect to process
configuration and operation could influence the PAP level. Therefore, this study aimed to
clarify the wider scope for sourcing municipal activated sludge for PHA production by inves-
tigating fifteen wastewater treatment plants across the Netherlands with respect to PAP
and the related process operating conditions. Activated sludge biomass grab samples were
obtained from all the plants and assessed for PAP under standardized conditions in Sweden.
Information about the biological treatment processes was gathered from the ten associated
water boards and site visits were made to most of the treatment plants as part of the survey.
3.2 SELECTION OF PLANTS
Fifteen wastewater treatment plants were selected with preference for large plants treating
over 100 000 PE with variety of process configurations spread over a range of geographic
locations within the Netherlands. Efforts were made to both include plants with factors that
are known to be favorable for enrichment of PAP and some without. Consideration was given
to factors such as instances with and without primary treatment, high and low fractions of
industrial discharge, and high and low influent organic concentrations.
Information was gathered from the ten water authorities responsible for each of the respective
treatment plants. The type of information that was requested included process configurations,
process flow diagrams, water quality data (influent and effluent concentrations), operating
8
STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
conditions and additional information such as separate streams discharged to the plant.
Concentrations of total suspended solids (TSS) and solids retention times (SRTs) were
obtained and nominal values for volumetric and specific organic loading rates (OLR and
SOLR, respectively) were estimated. The sludge management procedures were determined to
identify for any potential influence of return dewatering streams or similar. Special attention
was devoted to identifying zones in the existing bioprocess that would tend to stimulate a
feast-response in the biomass.
In addition to the desktop evaluations based on the information received from the water
boards, dedicated site-visits were made to thirteen of the fifteen treatment plants. These visits
gave the benefit of direct and visual impressions of the process operations, a chance for identi-
fication of undocumented quirks of operations, and more direct interaction with the respon-
sible engineers and operators.
Most of the WWTPs included in the survey were of large size of 100 000 – 1 000 000 P.E.
(TOD-150) although a few smaller plants were also included such as WWTPs Kootstertille and
Workum. The WWTPs were operated at between 65 and 115 % of their respective design load.
The influent total chemical oxygen demand concentrations were in the range 288 to 977 mg/L
and the biochemical oxygen demand (BOD) concentrations were in the range 113 to 324 mg/L.
The specific organic loading rates were between 0.08 and 1.7 g- COD/g-TSS/d and the SRTs
between 0.3 and 29 days. The specific consumption of precipitation chemicals for P removal
with respect to incoming P (mol-Me/mol-Pin) was from zero to 1.05 mol-Me/mol-Pin. The types
of process configurations at the respective WWTPs are summarized in Table 1 as well as some
key performance data. In all the selection embraced a wide range of process types, influent
water qualities, and operating conditions. Detailed process information and descriptions are
available in the full WP2 report.
TABLE 1 MAIN CHARACTERISTICS OF THE SAMPLED WASTEWATER TREATMENT PLANTS.
WWTP Water authority Actual capacity
(P.E.-150 g TOD)
Process Primary
settling
SRT
(days)
SOLR
(g-COD/g-TSS/d)
Almere Zuiderzeeland 200 000 Selector-Carrousel No 16 0.18
Amsterdam W Waternet 999 371 Modified UCT Yes 12 0.15
Bath Brabantse Delta 415 346 Predenitrification-nitrification Yes 20 0.15
Beverwijk HH Hollands
Noorderkwartier
207 651 Predenitrification-nitrification Yes 15 0.10
Dokhaven Hollandse Delta 442 806 AB-system with predenitrification No 0.3 * 6.2
Dordrecht Hollandse Delta 203 264 A2O with carrousel for polishing No 18 0.13
Ede Vallei en Veluwe 311 694 Modified Biodenipho™ Yes 21 0.11
Eindhoven Dommel 610 286 UCT Yes 18 0.13
Heerenveen Fryslan 94 310 Selector-Carrousel No 26 0.08
Kootstertille Fryslan 42 857 A2ONo 29 0.09
Land van Cuijk Aa en Maas 158 957 A2O Yes 17 0.18
Nijmegen Rivierenland 333 078 A2O/Predenitrification-nitrification Yes 16 n.a.
Sint Oedenrode Dommel 91 224 Selector-Carrousel No 15 0.13
Workum Fryslan 14 663 Carrousel No 25 0.08
Zaandam O HH Hollands
Noorderkwartier
109 059 UCT with carrousel Yes n.a. n.a.
* A-stage
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3.3 PHA ACCUMULATION POTENTIAL TESTS
3.3.1 METHOD
Activated sludge grab samples were collected from the aeration tanks at the 15 different
WWTPs. The samples were transported to Lund and stored refrigerated (4°C) pending the PAP
evaluation. Assessments for PHA accumulation potential were started within three days after
the sampling.
The activated sludge samples (1.6-1.7 L) were incubated in a PLC-controlled fed-batch feed-
on-demand reactor system. The temperature of the mantled 2-L reactors was controlled at
22±1°C. Mixing was provided with magnetic stirrer and the reactors were aerated through a
glass membrane diffuser. DO as well as pH were monitored continuously. A substrate of acetic
acid (100 g/L) supplemented with NH4Cl and KH2PO4 was dosed in pulses based on a well-
established respiration feed-on-demand control (Valentino et al., 2015a; Werker et al., 2011a)
during the incubation of the biomass. The nutrient composition COD:N:P was 100:1:0.05
(mass basis) except with the biomass from WWTP Kootstertille that was without N (COD:N:P
= 100:0:0.05).
Repeated feed pulses generated 90 - 165 mg-COD/L peak concentrations to maintain the
biomass under feast conditions. The trends in DO signal were used to indicate depletion of
substrate which triggered the feed pump to add the next pulse of substrate. The pH of the
substrate was regulated to 5.0±0.5 by addition of NaOH. Allylthiourea (10 mg/L) was added to
the activated sludge samples (except the biomass samples from WWTPs Workum, Kootstertille
and Heerenveen) in order to inhibit nitrification for the laboratory based evaluation (note
that allylthiourea is not added in any pilot scale work).
Acclimation of the biomass to the substrate was conducted according to a patent pending
method (Werker et al., 2016) before each PAP assessment test to establish the same biomass
history prior to the same applied accumulation conditions. The substrate for this acclimation
was the same as for the accumulation. The acclimation consisted of a feast period with an
initial reactor substrate concentration of 50 mg COD/L followed by a period of famine that
was 3 times the duration of the feast. This acclimation procedure was repeated three times
and lasted, in total, 2 to 4 h. The DO signal was used to monitor and control these feast and
the famine acclimation periods. The PHA accumulation assessments were started immedi-
ately after the acclimation. Acclimation was not conducted before the accumulations with
the biomass samples from WWTPs Workum, Kootstertille and Heerenveen.
The PHA accumulation assessments were conducted over at least 46 hours and grab samples
were taken at selected times to monitor trends in biomass PHA content and water quality
(COD, VFA and ammonium).
3.3.2 CALCULATIONS
Trends in biomass PHA content over time (PAPt in g-PHA/g-VSS) were fitted by least-squares
regression analysis to the empirical function:
PAPt = A0+A(1-e-kt)
with A0, A and k as constants to facilitate quantitative comparison of the progress in biomass
PHA accumulation as a function of time. A time constant was defined as τ = 1/k [h].
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STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
Trends in VSS and substrate consumption (St in g-COD) over time were fitted to linear and
quadratic empirical functions by least squares regression, respectively. Yields and specific
production rates of PHA were calculated as initial values as well as average values. Initial
values were calculated over 0.2τ (typically around 2 h of accumulation) and average values
were calculated over 3τ. The period 3τ represents a time at which the biomass was nearly satu-
rated and had nominally reached 95 % of its maximum PHA content. The initial (i) and average
(a) yields of PHA on substrate (YiP/S and YaP/S in g-COD/g-COD) were calculated according to:
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PHARIO final reportpublic version 28/3/2017 Page 12
The PHA accumulation assessments were conducted over at least 46 hours and grab samples were taken
taken at selected times to monitor trends in biomass PHA content and water quality (COD, VFA and
ammonium).
3.3.2 Calculations
Trends in biomass PHA content over time (PAPt in g-PHA/g-VSS) were fitted to the empirical function:
PAPt = A0+A(1-e-kt)
with A0, A and k as constants to facilitate quantitative comparison of the progress in biomass PHA
accumulation as a function of time. A time constant was defined as τ = 1/k [h].
Trends in VSS and substrate consumption (St in g-COD) over time were fitted to linear and quadratic
empirical functions by least squares regression, respectively. Yields and specific production rates of PHA
were calculated as initial values as well as average values. Initial values were calculated over 0.2τ (typically
around 2 h of accumulation) and average values were calculated over 3τ. The period 3τ represents a time at
which the biomass was nearly saturated and had typically reached 95 % of its maximum PHA content. The
initial and average yields of PHA on substrate (YiP/S and YaP/S in g-COD/g-COD) were calculated according to:
!" =
(
!
!)
!
with Pt and P0 being the mass of PHA produced at time t and time 0, respectively, and t being 0.2τ and 3τ for
YiP/S and YaP/S, respectively. The conversion factor for polyhydroxybutyrate mass to COD, kp, is equal to 1.67
g-COD/g-PHB.
The active biomass (X in g/L) was calculated by subtracting the concentration of PHA from the measured
VSS. The initial (maximum) specific PHA production rate (qiPHA in mg-PHA/g- X/h) and the average specific
PHA production rate (qaPHA) were calculated by estimating increase in the PHA concentration over time and
dividing by the active biomass concentration for time periods of 0.2τ and 3τ for qiPHA and qaPHA, respectively.
3.4 Results
3.4.1 General behaviour of the accumulation experiments
The PHA content in the biomass samples gradually increased over the course of the accumulation tests until
saturation levels of PHA content were reached. The majority of the PHA storage was achieved in the first 24
h. The 46 h accumulation time was applied to be conservatively long enough to reach PHA saturation for all
biomass samples. With two biomass samples, from RWZIs Nijmegen and Eindhoven the PHA content
reached a saturation level, that then became consumed by the biomass. The reason for this consumption was
most likely related to a shift in physiological state in the biomass from PHA storage to active biomass growth.
The external substrate was consumed alongside the consumption of the internally stored PHA and thus, this
observed onset of PHA consumption was not related to an interruption in supplied external substrate
consumption in these cases.
The PAP observed for RWZI Bath was 39 % g-PHA/g-VSS. Testing of the RWZI Bath biomass was an
experimental control given the wealth of experience for the same biomass during pilot testing. This particular
PAP result confirmed that the standardized laboratory-scale PAP assessments were representative of
outcomes at pilot scale under realistic field conditions. The 39 % PAP level observed with the RWZI Bath
biomass matched very closely the 39-42 % g-PHA/g-VSS (Hjort et al., 2016) PHA content obtained with the
Cella™ pilot on site and during the same month of production.
3.4.2 Accumulation results
Table 2 summarizes the results from the PAP assessments for the different WWTP’s. Figure 5 shows the full
curves for the seven best performing WWTP’s.
with Pt and P0 being the mass of PHA produced at time t and time 0, respectively, and t being
0.2τ and 3τ for YiP/S and YaP/S, respectively. The conversion factor for polyhydroxybutyrate mass
to COD, kp, is equal to 1.67 g-COD/g-PHB.
The active biomass (X in g/L) was calculated by subtracting the concentration of PHA from the
measured VSS. The initial (maximum) specific PHA production rate (qiPHA in mg-PHA/g- X/h)
and the average specific PHA production rate (qaPHA) were calculated by estimating increase
in the PHA concentration over time and dividing by the active biomass concentration for time
periods of 0.2τ and 3τ for qiPHA and qaPHA, respectively.
3.4 RESULTS
3.4.1 GENERAL BEHAVIOUR OF THE ACCUMULATION EXPERIMENTS
The PHA content in the biomass samples gradually increased over the course of the accumula-
tion tests until saturation levels of PHA content were reached. The majority of the PHA storage
was achieved in the first 24 h. The 46 h accumulation time was applied to be conservatively
long enough to reach PHA saturation for all biomass samples. With two biomass samples,
from WWTPs Nijmegen and Eindhoven the PHA content reached a saturation level, that then
became consumed by the biomass. The reason for this consumption was most likely related
to a shift in physiological state in the biomass from PHA storage to active biomass growth.
The external substrate was consumed alongside the consumption of the internally stored
PHA and thus, this observed onset of PHA consumption was not related to an interruption in
supplied external substrate consumption in these cases.
The PAP observed for WWTP Bath was 39% g-PHA/g-VSS. Testing of the WWTP Bath biomass
was an experimental control given the wealth of experience for the same biomass during
pilot testing. This particular PAP result confirmed that the standardized laboratory-scale PAP
assessments were representative of outcomes at pilot scale made under realistic field condi-
tions. The 39 % PAP level observed with the WWTP Bath biomass matched very closely the
39-42 % g-PHA/g-VSS (Hjort et al., 2016) PHA content obtained with the Cella™ pilot on site and
during the same month of production.
3.4.2 ACCUMULATION RESULTS
Table 2 summarizes the results from the PAP-assessments for the different WWTPs. Figure 5
shows the full curves for the seven best performing WWTPs.
The PAP-assessments in the present study give a “snapshot” of the surplus activated sludge
from the different locations and at the time of sampling. The PAP of a biomass may vary over
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STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
time as a function of variations in environmental and operational conditions at the WWTP. It
may also be influenced by conditions of accumulation as well as the feedstock VFA composi-
tion. For example, the experience with biomass from WWTP Bath is that it exhibits a consis-
tently high PAP although the PAP has varied from between 40 to as high as 55% g-PHA/g-VSS
over the course of the pilot operation.
It is estimated that for an economical viable PHA recovery the biomass should have a PAP of
at least 40 % g-PHA/g-VSS. The outcomes show clearly that high PAP in biomass at existing
WWTPs is not a feature that is unique for one specific plant but is relatively wide spread.
Among the fifteen plants tested in this study, four plants (27 %) had a PAP level of at least
38 % g-PHA/g-VSS. The PAP level for the biomass from WWTP Beverwijk was well above the
threshold criterion and is therefore already today a very interesting resource for PHA produc-
tion. It is noteworthy that these positive outcomes from the survey exist without any specific
attention being applied at the respective WWTPs for maximizing PAP. A well-tuned municipal
WWTP could possibly achieve up to 60 % PAP given that specific attention is placed on the
existing bioprocess to ensure an optimal selection pressure for PHA accumulation.
Besides the biomass from WWTPs Beverwijk and Bath, the biomasses from WWTPs Dordrecht,
Workum, Dokhaven and Heerenveen should also be considered as already potential sources of
biomasses for PHA production since they exhibited PAP levels that are approximately at the
40 % threshold for an economic polymer recovery. The other biomasses exhibited PAP levels
that are lower, and these facilities would therefore require the inclusion of potentially rather
simple adjuncts for bioprocess optimization in order to stimulate for PAP as will be discussed
later.
FIGURE 5 PAP DURING THE ACCUMULATION ASSESMENTS FOR THE SEVEN BEST PERFORMING WWTPS. CURVES FOR THE OTHER WWTPS ARE AVAILABLE IN THE
DETAILED WP2 REPORT.
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PHARIO final reportpublic version 28/3/2017 Page 13
The PAP assessments in the present study give a “snapshot” of the surplus activated sludge from the different
locations and at the time of sampling. The PAP of a biomass may vary over time as a function of variations in
environmental and operational conditions at the WWTP. It may also be influenced by conditions of
accumulation as well as the feedstock VFA composition. For example, the experience with biomass from
RWZI Bath is that it exhibits a consistently high PAP although the PAP has varied from between 40 to as high
as 55 % g-PHA/g-VSS over the course of the pilot operation.
It is estimated that for an economical viable PHA recovery the biomass should have a PAP of at least 40 % g-
PHA/g-VSS. The outcomes show clearly that high PAP in biomass at existing WWTPs is not a feature that is
unique for one specific plant but is relatively wide spread. Among the fifteen plants tested in this study, four
plants (27 %) had a PAP level of at least 38 % g-PHA/g-VSS. The PAP level for the biomass from RWZI
Beverwijk was well above the threshold criterion and is therefore already today a very interesting resource for
PHA production. It is noteworthy that these positive outcomes from the survey exist without any specific
attention being applied at the respective WWTPs for maximizing PAP. A well-tuned municipal WWTP could
possibly achieve up to 60 % PAP given that specific attention is placed on the existing bioprocess to ensure
an optimal selection pressure for PHA accumulation.
Besides the biomass from RWZIs Beverwijk and Bath, the biomasses from RWZIs Dordrecht, Workum,
Dokhaven and Heerenveen should also be considered as already potential sources of biomasses for PHA
production since they exhibited PAP levels that are approximately at the 40 % threshold for an economic
polymer recovery. The other biomasses exhibited PAP levels that are lower, and these facilities would
therefore require the inclusion of potentially rather simple adjuncts for bioprocess optimization in order to
stimulate for PAP as will be discussed later.
Figure 5: PAP during the accumulation assesments for the seven best performing WWTP’s. Curves for the other WWTP’s are
available in the detailed WP2 report.
The initial (maximum) specific PHA storage rate was between 12 and 47 mg-PHA/g-X/h and the average
specific storage rate was between 4.6 and 21 mg-PHA/g-X/h for all the assessments. Overall, there was no
correlation between PAP and storage rates. Therefore, the rate of PHA accumulation by a biomass is not
necessarily tied to the amount of polymer the biomass can accumulate.
The yields of PHA on substrate in the PAP assessment were generally initially high (0.37-0.70 g-COD/g-COD)
to then decrease when the biomass approached saturation of PHA. The theoretical maximum yield is about
0.7 g-COD/g-COD in complete absence of growth (Beun et al., 2000). Since the PHA storage rate is generally
lower when the biomass approaches PHA saturation levels, the PHA yields also tend to decrease over time.
Average PHA yields, measured until near saturation, were in the range 0.19 to 0.39 g-COD/g-COD.
It should be noted that PHA yields and production rates may vary with the type of substrate. A biomass can
have a higher yield and production rate when using a real fermented substrate than with a synthetic and
single model substrate such as applied in these assessments (Bengtsson et al., 2017; Morgan-Sagastume et
al., 2015). Therefore the values of yields and rates with the WP2 bioassays are reflective of differences
between locations but not necessarily absolute values for the respective biomass sources.
010 20 30 40 50
0.0
0.1
0.2
0.3
0.4
0.5
Dordrecht
PAP (g-PHA/g-VSS)
Time (h)
Beverwijk
Bath Workum
010 20 30 40 50
0.0
0.1
0.2
0.3
0.4
0.5
Eindhoven
PAP (g-PHA/g-VSS)
Time (h)
Nijmegen
Heerenveen
Dokhaven
The initial (maximum) specific PHA storage rate was between 12 and 47 mg-PHA/g-X/h and
the average specific storage rate was between 4.6 and 21 mg-PHA/g-X/h for all the assessments.
Overall, there was no correlation between PAP and storage rates. Therefore, the rate of PHA
accumulation by a biomass is not necessarily tied to the amount of polymer the biomass can
accumulate.
The yields of PHA on substrate in the PAP assessment were generally initially high (0.37-0.70
g-COD/g-COD) to then decrease when the biomass approached saturation of PHA. The theo-
retical maximum yield is about 0.7 g-COD/g-COD in complete absence of growth (Beun et al.,
2002). Since the PHA storage rate is generally lower when the biomass approaches PHA satura-
tion levels, the PHA yields also tend to decrease over time. Average PHA yields, measured until
near saturation, were in the range 0.19 to 0.39 g-COD/g-COD.
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STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
It should be noted that PHA yields and production rates may vary with the type of substrate.
A biomass can have a higher yield and production rate when using a real fermented substrate
than with a synthetic and single model substrate such as applied in these assessments
(Bengtsson et al., 2017; Morgan-Sagastume et al., 2015). Therefore the values of yields and rates
with the WP2 bioassays are reflective of differences between locations but not necessarily
absolute values for the respective biomass sources.
TABLE 2 RESULTS FROM THE PAP ASSESSMENTS REGARDING THE MAXIMUM PHA CONTENT (PAP), INITIAL PHA YIELD (YIP/S), AVERAGE PHA YIELD (YAP/S),
INITIAL SPECIFIC PHA PRODUCTION RATE (QIPHA) AND AVERAGE SPECIFIC PHA PRODUCTION RATE (QAPHA).
Biomass PAP YiP/S YaP/S qiPHA qaPHA
g-PHA/g-VSS g-COD/g-COD g-COD/g-COD mg-PHA/g-X/h mg-PHA/g-X/h
Beverwijk 0.52 0.70 0.32 35 21
Dordrecht 0.42 0.46 0.23 17 12
Bath 0.39 0.52 0.30 41 21
Workum 0.38 0.57 0.29 25 15
Heerenveen 0.36 0.53 0.22 26 8.8
Dokhaven 0.36 0.41 0.30 42 19
Nijmegen 0.30 0.61 0.27 47 18
Eindhoven 0.27 0.37 0.30 36 10
Ede 0.26 0.47 0.23 20 7.5
Kootstertille 0.24 0.56 0.39 27 10
Land van Cuijk 0.24 0.49 0.22 17 6.3
Amsterdam West 0.24 0.59 0.27 23 9.2
Sint-Oedenrode 0.21 0.46 0.23 16 5.6
Almere 0.17 0.40 0.19 12 4.6
Zaandam Oost 0.15 0.47 0.34 33 11
3.4.3 FACTORS INFLUENCING PHA ACCUMULATION POTENTIAL
3.4.3.1 PROCESS CONFIGURATIONS
The WWTPs were categorized with respect to process configuration according to the following:
Predenitrification-nitrification (WWTPs Bath and Beverwijk)
A2O (Anaerobic-anoxic-aerobic) process (WWTPs Dordrecht, Kootstertille, Land van Cuijk
and Nijmegen)
UCT (University of Cape Town) or Modified UCT process (WWTPs Amsterdam-West,
Eindhoven and Zaandam-Oost)
Carrousel with or without selector (WWTPs Almere, Heerenveen, Sint-Oedenrode and
Workum)
Other (AB-system, WWTP Dokhaven and Modified Biodenipho™, WWTP Ede)
When comparing process configurations with PAP outcomes (Figure 6), it was found that PAP
levels were generally higher for biomasses from processes with only predenitrification and
nitrification than for biomasses from processes with anaerobic and anoxic tanks in series.
The latter category includes A2O and UCT/MUCT processes.
The principal difference is that in the predenitrification process, nitrate becomes available
to the biomass together with the influent RBCOD and thus, an anoxic feast response may
be more readily established. In the A2O and UCT/MUCT processes, RBCOD from the waste-
water is first exposed to the biomass under anaerobic conditions. Although storage of RBCOD
occurs also under anaerobic conditions, anaerobic storage metabolism is distinctly different
compared to those under aerobic and anoxic conditions.
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STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
RBCOD supplied to a biomass under anaerobic conditions promotes enrichment for phos-
phate-accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) in the
biomass. These organisms also store PHA but with a metabolism that also includes storage
of glycogen and, in case of PAOs, polyphosphate. PAOs and GAOs are generally considered to
utilize specific low-molecular-mass organic compounds as substrates such as VFAs (Oehmen et
al., 2007) and glucose (Kristiansen et al., 2013)how similar their ecophysiology is to ‘Candidatus
Accumulibacter phosphatis’ is unclear, although they may occupy different ecological niches
in EBPR communities. The genomes of four Tetrasphaera isolates (T. australiensis, T. japonica,
T. elongata and T. jenkinsii. In contrast, anoxic storage may occur with a broader range of
organic compounds that contribute to the RBCOD fraction of municipal wastewater influent.
Therefore, it can be hypothesized that predenitrification on the incoming wastewater under
anoxic conditions can be made to promote for a stronger feast stimulation when compared
to anaerobic conditions. Notwithstanding, enrichment for high PAP with A2O and UCT/MUCT
processes may still be possible but with some further optimization or minor process modifi-
cations (see also paragraph 3.5.2).
FIGURE 6 PERFORMANCE IN PAP ASSESSMENTS FOR BIOMASS FROM DIFFERENT TYPES OF PROCESS CONFIGURATIONS.
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Figure 6: Performance in PAP assessments for biomass from different types of process configurations.
3.4.3.2 Influent wastewater concentrations
Since it is known that exposure of relatively high concentrations of RBCOD to the biomass stimulates storage,
it could be expected that higher influent COD and BOD concentrations generally lead to higher PAP.
However, and surprisingly, there was no such correlation found in the present investigation (Figure 7).
Actually, the highest PAP levels that were observed (RWZIs Beverwijk and Dordrecht) were from treatment
plants with influent organic concentrations in the lower range (around 425 mg-COD/L and 180 mg-BOD/L).
And the high influent COD concentration at RWZI Almere (977 mg/L) due to the almost storm-water-free
effluent, did not at least on its own, promote a high PAP level.
Figure 7: Performance in PAP assessments for biomass from different types of process configurations.
These observations strengthen the impression that rather than the concentration of organic matter in the
wastewater itself, it is the RBCOD level that the biomass is periodically exposed to that is crucial for a feast
stimulation and, feast stimulation is a critical factor controlling the level of enrichment with respect to PAP.
Predenitr/nitr A2O UCT/MUCT Carrousel Others
0.0
0.1
0.2
0.3
0.4
0.5
0.6
PAP
(g-PHA/g-VSS),
YP/S
(g-COD/g-COD)
qPHA
*10 (g-PHA/g-X/h)
PAP Y
P/S
q
PHA
0200 400 600 800 1000
0.0
0.1
0.2
0.3
0.4
0.5
0.6
COD
BOD
PAP (g-PHA/g-VSS)
COD and BOD (mg/L)
3.4.3.2 INFLUENT WASTEWATER CONCENTRATIONS
Since it is known that exposure of relatively high concentrations of RBCOD to the biomass
stimulates storage, it could be expected that higher influent COD and BOD concentrations
generally lead to higher PAP. However, and surprisingly, there was no such correlation found
in the present investigation (Figure 7). Actually, the highest PAP levels that were observed
(WWTPs Beverwijk and Dordrecht) were from treatment plants with influent organic concen-
trations in the lower range (around 425 mg-COD/L and 180 mg-BOD/L). And the high influent
COD concentration at WWTP Almere (977 mg/L) due to the almost storm-water-free effluent,
did not at least on its own, promote a high PAP level.
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STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
FIGURE 7 PERFORMANCE IN PAP ASSESSMENTS FOR BIOMASS FROM DIFFERENT TYPES OF PROCESS CONFIGURATIONS.
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PHARIO final reportpublic version 28/3/2017 Page 15
Figure 6: Performance in PAP assessments for biomass from different types of process configurations.
3.4.3.2 Influent wastewater concentrations
Since it is known that exposure of relatively high concentrations of RBCOD to the biomass stimulates storage,
it could be expected that higher influent COD and BOD concentrations generally lead to higher PAP.
However, and surprisingly, there was no such correlation found in the present investigation (Figure 7).
Actually, the highest PAP levels that were observed (RWZIs Beverwijk and Dordrecht) were from treatment
plants with influent organic concentrations in the lower range (around 425 mg-COD/L and 180 mg-BOD/L).
And the high influent COD concentration at RWZI Almere (977 mg/L) due to the almost storm-water-free
effluent, did not at least on its own, promote a high PAP level.
Figure 7: Performance in PAP assessments for biomass from different types of process configurations.
These observations strengthen the impression that rather than the concentration of organic matter in the
wastewater itself, it is the RBCOD level that the biomass is periodically exposed to that is crucial for a feast
stimulation and, feast stimulation is a critical factor controlling the level of enrichment with respect to PAP.
Predenitr/nitr A2O UCT/MUCT Carrousel Others
0.0
0.1
0.2
0.3
0.4
0.5
0.6
PAP
(g-PHA/g-VSS),
YP/S
(g-COD/g-COD)
qPHA
*10 (g-PHA/g-X/h)
PAP Y
P/S
q
PHA
0200 400 600 800 1000
0.0
0.1
0.2
0.3
0.4
0.5
0.6
COD
BOD
PAP (g-PHA/g-VSS)
COD and BOD (mg/L)
These observations strengthen the impression that rather than the concentration of organic
matter in the wastewater itself, it is the RBCOD level that the biomass is periodically exposed
to that is crucial for a feast stimulation and, feast stimulation is a critical factor controlling
the level of enrichment with respect to PAP.
3.4.3.3 OPERATING AND ENVIRONMENTAL CONDITIONS
Operating conditions such as SRT (solids retention time or sludge age) and organic loading
rates were evaluated for a potential correlation with PAP. Here it as also found that no such
correlations existed within the present study, neither for SRT nor for volumetric or specific
organic loading rates (Figure 8). Biomass from treatment plants operated under interme-
diate loading rates (0.3-0.5 g-COD/L/day) exhibited PAP levels varying over a wide range from
15 to 52 % g-PHA/g-VSS. The biomass from the A-stage of WWTP Dokhaven, that is operated
under extremely low SRT (0.3 days) and high organic loading rates (9.3 g-COD/L/day and 6.2
g-COD/g-TSS/day) compared to the other plants, exhibited a much less extreme PAP level of 36
% g-PHA/g-VSS. Again, while low SRT and high organic loading rates can be understood to be
helpful towards PAP and PHA accumulation kinetics, these factors are not essential criteria
towards producing a surplus activated sludge from municipal wastewater treatment with
high PAP.
FIGURE 8 LEFT: PAP LEVELS VERSUS SOLIDS RETENTION TIME (SLUDGE AGE) IN THE TREATMENT PLANTS. RIGHT: PAP LEVELS VERSUS VOLUMETRIC AND
SPECIFIC ORGANIC LOADING RATES IN THE TREATMENT PLANTS.
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3.4.3.3 Operating and environmental conditions
Operating conditions such as SRT (solids retention time or sludge age) and organic loading rates were
evaluated for a potential correlation with PAP. Here it as also found that no such correlations existed within
the present study, neither for SRT nor for volumetric or specific organic loading rates (Figure 8). Biomass from
treatment plants operated under intermediate loading rates (0.3-0.5 g-COD/L/day) exhibited PAP levels
varying over a wide range from 15 to 52 % g-PHA/g-VSS. The biomass from the A-stage of RWZI Dokhaven,
that is operated under extremely low SRT (0.3 days) and high organic loading rates (9.3 g-COD/L/day and 6.2
g-COD/g-TSS/day) compared to the other plants, exhibited a much less extreme PAP level of 36 % g-PHA/g-
VSS. Again, while low SRT and high organic loading rates can be understood to be helpful towards PAP and
PHA accumulation kinetics, these factors are not essential criteria towards producing a surplus activated
sludge from municipal wastewater treatment with high PAP.
Figure 8. Left: PAP levels versus solids retention time (sludge age) in the treatment plants. Right: PAP levels versus volumetric
and specific organic loading rates in the treatment plants.
Thus, based on these observations, and somewhat unexpectedly as an exciting outcome from PHARIO, we
find that enrichment for PAP should be feasible within a wide range of loading rates and process operating
conditions. The broad range of loading rates and sludge age that are typically applied for municipal
wastewater treatment are generally sufficient to drive selection pressures for a biomass with significant PAP.
Nevertheless, a low SRT for the biomass may have advantages in the context of PHA production due to a
higher fraction of active cells in the biomass which ultimately may lead to a higher PAP and/or more rapid
accumulation kinetics. Short SRTs also mean greater biomass yield and lower oxygen demands. Thus, a
shorter SRT can lead to a higher PAP and increase the amount of surplus biomass that is available as a
resource for PHA production. With the pace of development for mainstream Anammox nitrogen treatment, a
high rate heterotrophic biomass can be produced for carbon removal (Morgan-Sagastume et al., 2015), while
the Anammox processes manages the effluent nitrogen water quality (Laureni et al., 2016).
Five of the plants were operated under temperatures that were considered to have been elevated compared
to the norm. In some of these cases, treated effluent is used as cooling water by nearby facilities and returned
to the biological process (RWZIs Beverwijk, Dordrecht and Nijmegen). In case of RWZI Almere, the
temperature is elevated due to the high extent of sewer separation between wastewater and storm water
flows. The relatively high temperature at RWZI Workum is due to an influence of the relatively warm dairy
effluent discharged directly to the plant. From the survey, there was nothing to suggest a difference for
treatment plants operated at relatively higher temperatures (36±13 % g-PHA/g-VSS) and the other plants
(27±8 % g-PHA/g-VSS). Thus, high temperature is not a prerequisite for enrichment of PAP.
3.4.3.4 Primary treatment
It is generally considered that primary treatment of influent wastewater is a benefit to the goals of producing a
biomass for PHA production value chains. In the first instance, primary treatment creates for an opportunity to
recover suspended solids for bioenergy or VFA production and cellulose as a renewable resource. For PAP
enrichment, primary treatment is advantageous since otherwise, influent solids that are not degraded in the
process (fibers, inert organic solids, etc.), and that end up associated with the biomass solids, detract from the
0 5 10 15 20 25 30 35
0.0
0.1
0.2
0.3
0.4
0.5
0.6
PAP (g-PHA/g-VSS)
SRT (days)
0.0 0.2 0.4 0.6 0.8 1.0 8 9 10
0.0
0.1
0.2
0.3
0.4
0.5
0.6
PAP (g-PHA/g-VSS)
Volumetric organic loading rate, OLR ( g-COD/L/d)
0.0 0.1 0.2 6 7
Specific organic loading rate, SOLR (g- COD/g-TSS/d)
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STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
Thus, based on these observations, and somewhat unexpectedly as an exciting outcome from
PHARIO, we find that enrichment for PAP should be feasible within a wide range of loading
rates and process operating conditions. The broad range of loading rates and sludge age that
are typically applied for municipal wastewater treatment are generally sufficient to drive
selection pressures for a biomass with significant PAP.
Nevertheless, a low SRT for the biomass may have advantages in the context of PHA production
due to a higher fraction of active cells in the biomass which ultimately may lead to a higher
PAP and/or more rapid accumulation kinetics. Short SRTs also mean greater biomass yield and
lower oxygen demands. Thus, a shorter SRT can lead to a higher PAP and increase the amount
of surplus biomass that is available as a resource for PHA production. With the pace of develop-
ment for mainstream Anammox nitrogen treatment, a high rate heterotrophic biomass can be
produced for carbon removal (Morgan-Sagastume et al., 2015), while the Anammox processes
manages the effluent nitrogen water quality (Laureni et al., 2016)including over 5 months at 15
degrees C. The two systems consisted of a moving bed biofilm reactor (MBBR).
Five of the plants were operated under temperatures that were considered to have been
elevated compared to the norm. In some of these cases, treated ef fluent is used as cooling
water by nearby facilities and returned to the biological process (WWTPs Beverwijk, Dordrecht
and Nijmegen). In case of WWTP Almere, the temperature is elevated due to the high extent of
sewer separation between wastewater and storm water flows. The relatively high temperature
at WWTP Workum is due to an influence of the relatively warm dairy effluent discharged
directly to the plant. From the survey, there was nothing to suggest a difference for treatment
plants operated at relatively higher temperatures (36±13 % g-PHA/g-VSS) and the other plants
(27±8 % g-PHA/g-VSS). Thus, high temperature is not a prerequisite for enrichment of PAP.
3.4.3.4 PRIMARY TREATMENT
It is generally considered that primary treatment of influent wastewater is a benefit to the
goals of producing a biomass for PHA production value chains. In the first instance, primary
treatment creates for an opportunity to recover suspended solids for bioenergy or VFA
production and cellulose as a renewable resource. For PAP enrichment, primary treatment
is advantageous since otherwise, influent solids that are not degraded in the process (fibers,
inert organic solids, etc.), and that end up associated with the biomass solids, detract from
the harvested “functional” biomass quality. Quality is reduced since the active fraction of
the biomass may become “diluted out” by associated inert suspended solids. Furthermore,
without primary treatment, more colloidal COD that is degraded more slowly can reduce the
extent to which the biomass will be disposed to sufficiently stringent famine conditions. A
stringent famine condition is the compliment to feast that creates for a selective advantage
for PHA-storing microorganisms in the biomass.
Eight of the plants in the present survey were with primary clarifiers. These plants had average
PAP levels (31±11 % g-PHA/g-VSS) and this average was similar to those without primary clari-
fiers (29±9 % g-PHA/g-VSS). Thus, even though primary settling is generally considered advan-
tageous for high PAP, it was not a determining factor for the surveyed facilities and a signifi-
cant PAP may still be possible for an activated sludge without the benefit of a primary clarifier.
3.4.4 SEPARATE COD-RICH STREAMS
The eight treatment plants that receive contributions in form of separate streams with relatively
high concentrations of COD are listed below (Table 3). Although it is not possible to determine
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STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
the influence of these separate streams on the enrichment for PAP, it is clear from this and
previous work that significant contributions from such streams are not necessary for high PAP as
evidenced by the relatively high PAP levels observed for WWTPs Bath, Beverwijk and Dordrecht.
This principle is perhaps most convincingly demonstrated by observations from the pilot
plant project carried out at WWTP Leeuwarden before the PHARIO project. Treatment of
the Leeuwarden wastewater, which is dominated by domestic household wastewater, in the
Cella™ pilot plant resulted in a PAP level of 49 % g-PHA/g-VSS. This high PAP level may be
compared to 15 % for the surplus activated sludge produced from treating the same waste-
water in the full-scale wastewater treatment plant (Bengtsson et al., 2017).
TABLE 3 SUMMARY OF CONTRIBUTIONS FROM SEPARATE COD-RICH STREAMS TO THE TREATMENT PLANTS.
WWTP Type of contribution Fraction of TCOD loading (%) PAP (g-PHA/g-VSS)
Bath Condensate from SNB 3 0.39
Beverwijk 1-6 Condensate/SHARON 13 0.52
Dokhaven Centrate/Anammox n.a. 0.36
Dordrecht Condensate from HVC 6 0.42
Eindhoven Centrate from Mierlo n.a. 0.27
Heerenveen Filtrate from SOI 22 0.36
Nijmegen Dewatering supernatant n.a. 0.30
Workum Dairy industry 50 0.38
Others PHARIO None 0 0.22 ± 0.04
Leeuwarden Full-scale None 0 0.151
Leeuwarden Cella™ pilot None 0 0.491
1 (Bengtsson et al., 2017)
Although such separate RBCOD rich streams may not be necessary, they can still serve to
benefit the PAP enrichment in the biomass. It appears likely that separate streams facilitated
the enrichment in two cases. For WWTP Heerenveen and Workum, the process configurations
alone were not expected to provide for a strong selective pressure but PAP levels were still rela-
tively high. These plants both receive contributions from the separate streams that contribute
to relatively high fractions of the COD load to the plants, namely 22 and 50 %. If the process
configuration does not by design provide for PAP enrichment, a separate COD-rich stream
may facilitate an improvement in the selective pressure by generating a feast zone. Hence,
although a separate COD-stream is not necessary for enrichment of PAP, if such a stream is
used purposefully and strategically to stimulate a periodic feast response for at least some of
the biomass for some of the time, then the process will enrich a biomass with significant PAP.
3.5 APPROACHES TO IMPROVE PHA ACCUMULATION POTENTIAL
3.5.1 ESTABLISHING DISTINCT FEAST CONDITIONS FOR THE BIOMASS
From the comparison of PAP outcomes and pilot plant operating conditions, it became clear
that in order to achieve enrichment of high PAP, there is no need for very unusual or special
conditions with respect to SRT, loading rates or temperature. Furthermore, enrichment is
feasible with different types and concentrations of wastewater. Separate COD-rich streams
may improve the enrichment for PAP but this is by no means a necessary factor. Wastewater
dominated by domestic household contributions have sufficient levels of readily biodegrad-
able COD to promote PAP enrichment given that the process configuration is somehow made
to be favorable for enrichment.
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STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
Based on the observations made from all the surveyed treatment plants, it furthermore was
considered that the most crucial factor for PAP enrichment regardless of the process configu-
ration is how the influent RBCOD in the wastewater is brought in contact with the biomass. It
seems as if the contact zone for RBCOD and biomass is such that at least some of the biomass
and some of the time periodically experiences a relatively high concentration of RBCOD, then
storage is induced which ultimately leads to a selection pressure for a higher PAP in the
surplus biomass.
Process configurations that lead to feast-stimulating conditions can be exemplified by WWTP
Beverwijk, the plant that exhibited the highest PAP levels from in this survey. At this plant,
the return activated sludge (RAS) is directed to the initial part of the biological treatment
(Figure 9). The main part of the RAS is not brought in contact with the influent wastewater
but directed to a large tank for predenitrification (and intermittently nitrification for treat-
ment lines 1-2). A minor part of the RAS is directed to a small contact zone in which the
influent wastewater also enters. By only bringing a minor part of the RAS in contact with
the influent RBCOD rather than all the R AS, a higher RBCOD concentration can be periodi-
cally established for at least some of the biomass than otherwise would be the case. In this
way, fractions of the biomass will repeatedly be exposed to relatively high concentrations of
RBCOD. Over time, all the biomass contained in the process will eventually experience this
relatively high RBCOD concentration, which will lead to a general benefit from a storage
response and ultimately enrichment for PAP.
FIGURE 9 SCHEMATIC DRAWING AS TOP VIEW OF TREATMENT LINES 1-2 AT WWTP BEVERWIJK.
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3.5 Approaches to improve PHA accumulation potential
3.5.1 Establishing distinct feast conditions for the biomass
From the comparison of PAP outcomes and pilot plant operating conditions, it became clear that in order to
achieve enrichment of high PAP, there is no need for very unusual or special conditions with respect to SRT,
loading rates or temperature. Furthermore, enrichment is feasible with different types and concentrations of
wastewater. Separate COD-rich streams may improve the enrichment for PAP but this is by no means a
necessary factor. Wastewater dominated by domestic household contributions have sufficient levels of readily
biodegradable COD to promote PAP enrichment given that the process configuration is somehow made to be
favorable for enrichment.
Based on the observations made from all the surveyed treatment plants, it furthermore was considered that
the most crucial factor for PAP enrichment regardless of the process configuration is how the influent RBCOD
in the wastewater is brought in contact with the biomass. It seems as if the contact zone for RBCOD and
biomass is such that at least some of the biomass and some of the time periodically experiences a relatively
high concentration of RBCOD, then storage is induced which ultimately leads to a selection pressure for a
higher PAP in the surplus biomass.
Process configurations that lead to feast-stimulating conditions can be exemplified by RWZI Beverwijk, the
plant that exhibited the highest PAP levels from in this survey. At this plant, the return activated sludge (RAS)
is directed to the initial part of the biological treatment (Figure 9). The main part of the RAS is not brought in
contact with the influent wastewater but directed to a large tank for predenitrification (and intermittently
nitrification for treatment lines 1-2). A minor part of the RAS is directed to a small contact zone in which the
influent wastewater also enters. By only bringing a minor part of the RAS in contact with the influent RBCOD
rather than all the RAS, a higher RBCOD concentration can be periodically established for at least some of
the biomass than otherwise would be the case. In this way, fractions of the biomass will repeatedly be
exposed to relatively high concentrations of RBCOD. Over time, all the biomass contained in the process will
eventually experience this relatively high RBCOD concentration, which will lead to a general benefit from a
storage response and ultimately enrichment for PAP.
Figure 9: Schematic drawing as top view of treatment lines 1-2 at RWZI Beverwijk.
Although all treatment plants may not have configurations that are purposeful in order to create feast-
stimulating zones, process modifications that are relatively minor and simple can achieve the same effect and
thereby provide significant improvements in enrichment performance without changing configuration or water
quality management strategy. Examples of how a feast-stimulating zone can be established are:
A small tank up-front of the main treatment volume where influent wastewater and RAS, or a fraction
of the RAS, are brought in contact.
Modification of the confluence point between return biomass and influent such that the contact zone
is well defined and small enough to provide an elevated RBCOD concentration exposed to the
biomass.
A side-stream sequencing feast reactor (SFR) in which return biomass is mixed with influent
wastewater batch-wise at given intervals.
The principle behind the side-stream SFR relies on intermittently feeding return biomass to the reactor,
followed by feeding wastewater to the reactor and thereafter allowing the biomass to take up the RBCOD for
Although all treatment plants may not have configurations that are purposeful in order to
create feast-stimulating zones, process modifications that are relatively minor and simple can
achieve the same effect and thereby provide significant improvements in enrichment perfor-
mance without changing configuration or water quality management strategy. Examples of
how a feast-stimulating zone can be established are:
A small tank up-front of the main treatment volume where influent wastewater and RAS,
or a fraction of the RAS, are brought in contact.
Modification of the confluence point between return biomass and influent such that the
contact zone is well defined and small enough to provide an elevated RBCOD concentra-
tion exposed to the biomass.
A side-stream sequencing feast reactor (SFR) in which return biomass is mixed with influ-
ent wastewater batch-wise at given intervals.
The principle behind the side-stream SFR relies on intermittently feeding return biomass
to the reactor, followed by feeding wastewater to the reactor and thereafter allowing the
18
STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
biomass to take up the RBCOD for storage (Figure 10). The relatively high concentration of
RBCOD will stimulate the biomass to feast conditions. Each sequence can be terminated after
depletion of RBCOD. At this point, the reactor contents can be pumped back to the main
treatment volume, in which famine conditions are inherently maintained and thereafter, a
new cycle can be initiated. The influent to the SFR may be the main wastewater or a separate
stream rich in RBCOD. The latter alternative may help to further improve PAP enrichment if
such a stream is available.
The side-stream SFR approach may in many cases represent a flexible and cost effective means
of upgrade for improved PAP. The advantages include relatively small modifications and large
operational flexibility. Since the reactor is controlled by an operational sequence, the feeding
of biomass and wastewater can be individually controlled in order to establish an optimized
feast response in the biomass. A cost effective and tunable adjunct to the process permits to
optimize the selection pressure without influencing the main hydraulic flows in treatment.
FIGURE 10 A SIDE-STREAM SEQUENCING FEAST REACTOR AS AN EXAMPLE OF A MINOR PROCESS MODIFICATION TO IMPROVE SELECTIVE PRESSURE FOR PAP
ENRICHMENT.
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PHARIO final reportpublic version 28/3/2017 Page 19
storage (Figure 10). The relatively high concentration of RBCOD will stimulate the biomass to feast conditions.
Each sequence can be terminated after depletion of RBCOD. At this point, the reactor contents can be
pumped back to the main treatment volume, in which famine conditions are inherently maintained and
thereafter, a new cycle can be initiated. The influent to the SFR may be the main wastewater or a separate
stream rich in RBCOD. The latter alternative may help to further improve PAP enrichment if such a stream is
available.
The side-stream SFR approach may in many cases represent a flexible and cost effective means of upgrade
for improved PAP. The advantages include relatively small modifications and large operational flexibility.
Since the reactor is controlled by an operational sequence, the feeding of biomass and wastewater can be
individually controlled in order to establish an optimized feast response in the biomass. A cost effective and
tunable adjunct to the process permits to optimize the selection pressure without influencing the main
hydraulic treatment.
Figure 10: A side-stream sequencing feast reactor as an example of a minor process modification to improve selective pressure
for PAP enrichment.
3.5.2 Integration of EBPR and PHA production
The treatment plants in this survey rely to a varying degree on enhanced biological phosphorus removal to
meet their effluent P demands. Four of the plants have no design elements that favor EBPR such as a
selector that could be expected to become anaerobic, namely RWZIs Bath, Beverwijk, Dokhaven and
Workum. As described above, the plants with A2O or UCT/MUCT process configurations were generally with
lower observed PAP levels.
Feast stimulation for PAP enrichment with a municipal wastewater appears to be most favorable under
aerobic or anoxic conditions. Anaerobic feast conditions will favor polyphosphate-accumulating organisms
(PAOs) and PAOs are a subpopulation of the PHA-storing organisms that are clearly not required for
achieving high PAP. The anaerobic uptake of RBCOD that occurs by PAOs in the EBPR process have been
observed to rely mainly on some specific organic compounds such as VFAs (Oehmen et al., 2007) and
glucose (Kristiansen et al., 2013). Anoxic storage, on the other hand, can occur with a broader range of
organic compounds that make up the RBCOD. It may be suggested that within the UCT/MUCT process,
stronger selective pressure for PHA storage could be established if it would be possible to utilize the “break
through” residual RBCOD after the initial RBCOD uptake by PAOs for a second, anoxic, feast stimulation.
Such feast stimulation would require a well-defined contact zone in the initial part of the anoxic volume of the
UCT/MUCT process. Improvement of the PAP in surplus biomass from EBPR processes require further
optimization. However, given that storage is already an integral component of the EBPR mechanism,
necessary process modifications are most likely to be minor.
Integration of PHA production with EBPR require specific consideration and strategies for P management.
When subjected to aerobic PHA accumulation (in PE3), PAOs with stored poly-P will release ortho-P back into
the water phase (Anterrieu et al., 2014; Guisasola et al., 2004; Pijuan et al., 2005; Rodgers and Wu, 2010). If
this P release is strategically placed then, P can be harvested for recovery by precipitation as an integral part
of the PHA accumulation process.
3.5.2 INTEGRATION OF EBPR AND PHA PRODUCTION
The treatment plants in this survey rely to a varying degree on enhanced biological phos-
phorus removal to meet their effluent P demands. Four of the plants have no design elements
that favor EBPR such as a selector that could be expected to become anaerobic, namely
WWTPs Bath, Beverwijk, Dokhaven and Workum. As described above, the plants with A2O or
UCT/MUCT process configurations were generally with lower observed PAP levels.
Feast stimulation for PAP enrichment with a municipal wastewater appears to be most favorable
under aerobic or anoxic conditions. Anaerobic feast conditions will favor polyphosphate-
accumulating organisms (PAOs) and PAOs are a subpopulation of the PHA-storing organisms
that are clearly not required for achieving high PAP. The anaerobic uptake of RBCOD that
occurs by PAOs in the EBPR process have been observed to rely mainly on some specific organic
compounds such as VFAs (Oehmen et al., 2007) and glucose (Kristiansen et al., 2013). Anoxic
storage, on the other hand, can occur with a broader range of organic compounds that make
up the RBCOD. It may be suggested that within the UCT/MUCT process, stronger selective
pressure for PHA storage could be established if it would be possible to utilize the “break
through” residual RBCOD after the initial RBCOD uptake by PAOs for a second, anoxic, feast
stimulation. Such feast stimulation would require a well-defined contact zone in the initial
part of the anoxic volume of the UCT/MUCT process. Improvement of the PAP in surplus
biomass from EBPR processes require further optimization. However, given that storage is
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STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
already an integral component of the EBPR mechanism, necessary process modifications are
most likely to be minor.
Integration of PHA production with EBPR require specific consideration and strategies for P
management. When subjected to aerobic PHA accumulation (in PE3), PAOs with stored poly-P
will release ortho-P back into the water phase (Anterrieu et al., 2014; Guisasola et al., 2004;
Pijuan et al., 2005; Rodgers and Wu, 2010). If this P release is strategically placed then, P can be
harvested for recovery by precipitation as an integral part of the PHA accumulation process.
3.6 POTENTIAL FOR PHA PRODUCTION
Estimations were made to assess the quantity of PHA that may be produced from the WWTPs
included in the survey given regionally available VFA feedstocks. The calculations were made
based on the assumption that the enrichment for PAP could be conservatively improved such
that the harvested excess biomass from the treatment plants was able to accumulate at least
40% g-PHA/g-VSS. Conservative assumptions of yields for biomass and PHA as well as COD
removal were made based on previous experience (Table 4).
It was estimated that most of the plants could potentially supply biomass for the production
of between 1 000 and 6 000 ton PHA per year (Table 5), provided enough VFA feedstocks can
be sourced from the region. The total potential considering all the 15 plants is in the order
of 25 000 ton-PHA/y. Thus, each of the large plants (around 100 000 P.E. and above) could be
providing raw material in form of surplus biomass for production of commercially signifi-
cant quantities of PHA.
TABLE 4 ASSUMPTIONS MADE BASED ON EXPERIENCE FOR CALCULATING POTENTIAL PHA PRODUCTION.
Parameter Unit Value
Sludge yield. Biomass harvested per COD removed g-VSS/g-COD 0.25
PHA content in the biomass after PE3 g-PHA/g-VSS 0.40
COD removal efficiency in PE2 g-COD/g-COD 0.93
PHA yield on VFA in PE3 g-PHA/g-COD 0.187
TABLE 5 ESTIMATED POTENTIAL PHA PRODUCTION FROM THE WWTP INCLUDED IN THE STUDY BASED ON THE ASSUMPTIONS IN TABLE 4.
PE ton-PHA/y
Almere 200 000 1 700
Amsterdam-West 999 371 5 800
Bath 415 346 2 700
Beverwijk 207 651 1 200
Dokhaven 442 806 2 500
Dordrecht 203 264 1 100
Ede 311 694 1 600
Eindhoven 610 286 3 600
Heerenveen 94 310 450
Kootstertille 42 857 250
Land van Cuijk 158 957 1 000
Nijmegen 333 078 2 300
Sint-Oedenrode 91 224 600
Workum 14 663 75
Zaandam-Oost 109 059 700
Total 4 234 566 25 575
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3.7 SUMMARY & CONCLUSION
It was found that four of the investigated plants (27 %) already exhibited PAP levels close to
the threshold level of 40%. The high PAP level in biomass from WWTP Bath (39% g-PHA/g- VSS)
was confirmed which proved the relevance of the standardized test procedures that were
applied for larger scale PHA accumulations. Two other WWTPs, namely WWTPs Dordrecht
and Workum, exhibited similar PAP levels and the biomass from WWTP Beverwijk exhibited
the most significant PAP of 52%. Therefore, even without specific optimization of the biopro-
cess, a relevant high PAP for PHA production may be possible at several locations.
Plant configurations based on pure predenitrification and nitrification were generally
associated with higher PAP than plants with anaerobic and anoxic tanks in series. This
confirmed, as expected, that feast stimulation of the biomass with wastewater RBCOD under
anoxic (or aerobic) conditions more naturally leads to enrichment of high PAP whereas other
configurations may need some optimization for periodic feast to at least some of the biomass
some of the time.
No apparent correlations were found between operating conditions such as sludge age and
loading rates and PAP or between influent concentrations and PAP. Thus, enrichment for high
PAP was found, to our surprise, to be generally feasible and for a broad range of operating
conditions, bioprocess configurations, and with different types of wastewaters. Based on the
outcomes and the observations made on the sites, we consider that the initial contact zone
for biomass and wastewater RBCOD is a critical factor for driving PAP enrichment. Several
alternative and relatively simple strategies are available for process modifications to improve
feast stimulation of the biomass. Optimization for PAP is likely to be readily possible with case
to case attention to detail, and only minor process modification.
With 4 of the 15 plants investigated having PAP levels near or above 40%, the potential for
water board biomass for PHA value chain development is already significant. Already these
four plants show a production potential of 7.500 ton PHA/year which is more than suffi-
cient for a commercial production scale for which a minimum scale of 5.000 ton PHA/year is
suggested to be required.
One can estimate that PHA production from just the treatment plants surveyed for the present
investigation could be brought to at least conservative improvements in PAP levels to reach
40% g-PHA/g-VSS. In this way, a potential annual PHA production capacity of 1 000 – 6 000 ton/
year for most of the surveyed plants would be possible and this translates to a production of
25 000 ton PHA per year for a total treatment capacity of 4,2 million people equivalents. Thus,
each of the water boards involved in this survey could be stakeholders within a biobased
biopolymer supply hub providing surplus biomass as a raw material to produce commercially
relevant amounts of PHA. Given that methods for improved PAP are expected to be possible
with only minor process modifications, this potential could be readily expanded and spread
nationally.
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4
TESTING THE PHARIO PROCESS
4.1 PILOT PLANT OPERATION
Production of PHA in PHARIO involved 4 process elements, or PE1 to PE4 (Figure 3 to
Figure 5). PE1 was a pilot scale (1200 L) well-stirred and thermostated batch anaerobic
fermentation vessel called BioVAP that was operated on site at WWTP Bath. BioVAP had
been previously employed in a collaborative project with AnoxKaldnes, KNN and Wassenaar
(https://www.nom.nl/wassenaar-maakt-bioplastic-van-plant-enresten-2/, Karlsson et al. 2014).
PE2 was a full-scale WWTP process for active biomass production (WWTP Bath). PE3 was a
pilot-scale (500 L) fed-batch process for PHA-rich biomass production using surplus PE2
activated sludge, and fermentation filtrate from PE1 as feedstocks (Morgan-Sagastume et al.
2015, Bengtsson et al. 2017). PE3 was also operated on site at WWTP Bath. PE4 was a pilot scale
(10-L) batch green-solvent PHA recovery process located in Sweden (Magnusson et al. 2016).
In the normal routine of production operations, two accumulation batches were performed
each week from June 2015 to March 2016. For each batch, a grab sample of freshly thickened
(50 to 70 gTS/ kg) surplus WWTP Bath activated sludge was obtained for the biomass supply.
The grab sample was assessed for solids content and about 1 kilogram of thickened activated
sludge (as dry volatile solids) was delivered to the accumulation process along with dilution
water. The accumulation pilot process comprised a working aeration volume of about 400 L
coupled to a clarifier volume of 120 L (Morgan-Sagastume et al. 2015). The aerated volume was
mechanically stirred and air was supplied via a blower with coarse bubble aeration. Active
pumping to, and return from, the clarifier maintained a short solids residence time in the
clarifier. Depletion of dissolved oxygen for the biomass while in the clarifier maintained
oxygen limiting kinetics which are known to prolong the metabolic activity associated with
accumulation processes (Pratt et al. 2012). Therefore, time for the fractions of biomass in the
clarifier at any one time was simply an accumulation dead time.
There is no specific requirement that the activated sludge biomass source should be thick-
ened. Thickened biomass was used as the source for PHARIO to simplify the material flow
control and to decouple the pilot operations from the full-scale wastewater treatment plant
operations. Furthermore, in the process scale up, an accumulation process may not be directly
adjacent to the wastewater treatment plant.
4.1.1 VFA FEEDSTOCKS
PHA was accumulated in the biomass by semi-continuous supply of a VFA rich feedstock.
Organic sources for VFA feedstocks used in the project were either a fermented carbohydrate
rich process effluent delivered from a local candy factory, fermented primary sludge centrate
from primary municipal sludge delivered from Waterschap De Dommel (Tilburg facility), and
defined mixtures of acetic and propionic acids.
The process water from the candy factory was delivered batch wise once per week to WWTP
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STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
Bath in IBC containers. The BioVAP pilot fermenter was used to maximize the fermentation
product content in a well stirred and temperature controlled volume of 1200 L. The process
water was trimmed with dilution water to about 18 gCOD/L and was incubated at 37°C for
up to 7 days with a pH controlled nominally between 5.5 and 6.0. A 7-day incubation was
not required for the fermentation, but this was time given conservatively and for simplicity
with respect to all other aspects of the hands-on piloting production activities. The pH was
maintained with a PID controller and pulse wise additions of concentrated NaOH (45 % w/w).
The fermentation process biomass (nominally about 1.2 gTSS/L) was maintained within the
process at an SRT of between 7 and 10 days. Suspended solids from the candy factory process
water fermentation effluent were removed by means of pre-settling with added flocculent
polymer (Flopam FO 4800 SH, SNF Floerger), followed by a Hydrotech drum filter model 801
(HDF 801, with 10 µm screen). Suspended solids levels in and out of the HDF were on average
0.6 and 0.2 g/L, respectively. The fermentation product water for the accumulation process
contained on average 16 gCOD/L as soluble RBCOD (readily biodegradable COD). Decrease
in COD across the fermentation process was primarily due to water entrainment from the
pilot HDF backwash operations. The feedstock nutrient balance of COD:N:P was 100:0.5:0.1
(by weight).
Primary sludge was also delivered fresh to WWTP Bath from the De Dommel Tilburg facility
in IBC containers. Solids content was variable, but on average it was 45 g/L. The batches of
delivered primary sludge were fermented with continuous mechanical stirring for 6 days at
37°C, with pH monitoring but with no pH control. The sludge matrix was self-buffering and
generally the pH was inherently maintained between 4.8 and 5.5. No solids were retained in
the BioVAP process between batches because unlike the process water from the candy factory,
such sludge is already richly inoculated for anaerobic fermentation microbial activity. The
fermenter batch was first discharged to a holding tank and then under automation control
pumped to a centrifuge decanter (Morgan-Sagastume et al. 2015) with rotating bowl (3000
rpm) and a relative velocity between bowl and screw of 10 rpm. Cationic polymer (Flopam
FO4800SH, SNF/ FLOERGER) was added in-line for coagulation/flocculation to improve the
solids/liquid separation. The polymer addition was set and controlled by PLC to 50 g-polymer/
kg-TSS. The solids reject cake was collected and disposed to the full-scale anaerobic digestion
facility on site. Centrate was collected in a holding tank and iron chloride was added from
concentrated solution (44% w/w) to precipitate excess soluble phosphorus based on a molar
dosing ratio of 1.42 mol Fe:mol PO4. The centrate was further processed through the pilot
Hydrotech drum filter (HDF 801, with 18 µm screen) to produce feedstock batches for the
pilot-scale accumulation. This fermented primary sludge feedstock was about 7 gCOD/L with
a COD:N:P balance of 100:5:0.1 (by weight).
So-called “synthetic” feedstocks were generated by diluting and blending proportions of
concentrated acetic and propionic acids in IBC containers to about 10 gCOD/L. A targeted
COD:N:P (by weight) of 100:1:0.05 nutrient balance was obtained by chemical additions of
NH4Cl and KH2PO4. pH was adjusted to 5 by addition of NaOH (33 or 45% w/w).
4.1.2 PHA ACCUMULATION
Prior to each accumulation the process biomass was subject to an acclimation phase. This
acclimation comprised a sequence of three feast and famine cycles. The feast in each of the
cycles was generated by stimulating the biomass to near maximal respiration with a pulse
input of substrate with sufficient volume to a targeted maximum COD concentration of
200 mgCOD/L. The feast respiration response duration was measured based on changes in
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STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
dissolved oxygen concentration. A period of famine was provided to last 4 times as long as the
time of feast. The acclimation phase was designed as a controlled assessment of the biomass
respiration response from batch to batch over the 10 months of operations, and as a means
to provide all the biomass batches with the same perturbation history before the onset of
the accumulation response. Previous work suggests that feast-famine acclimation before an
accumulation response generally results in an improved PHA accumulation potential (Werker
et al. 2016).
At the end of the last acclimation famine period, the accumulation process was started auto-
matically. Accumulations were at 25°C and were sustained using semi-continuous feedstock
supply based on feed-on-demand biomass respiration control methods (Werker et al. 2013a).
The control method objectives were designed to sustain a prolonged period of near maximal
and continuous feast respiration with pulse wise inputs of substrate. There is no requirement
that the temperature should be 25°C and equally successful results have been demonstrated
for mixed culture accumulations over a wide temperature range from 15 to 30°C (De Grazia
et al. in progress). The accumulation or “PHA production process” was typically maintained
from 16 to 20 hours in duration.
The accumulations were performed with constant process volume, meaning the influent
volume inputs displaced an equal volume that was produced as effluent from the process
clarifier. Therefore, some loss of added substrate was to be expected due to hydraulic short-
circuiting. Accumulation process operating strategies exist to minimize “substrate leakage”
but these were not implemented as part of the present investigation. In fact, this and several
other bioprocess optimisation and performance efficiency improvements were not included
nor undertaken during the present PHARIO investigation because the main goal was to
produce polymer as routinely, and as often, as possible. All efforts were focused on producing
materials and evaluating the material quality with respect to the routine of production that
was applied.
With exceptions of a few abnormal events that were detected by the process automatic
control, the accumulation process was terminated based on a set time of accumulation.
Frequently the automated termination occurred in the middle of the night. After accumula-
tion termination, the aeration was automatically turned off and the biomass was collected
in the main process volume and allowed to settle by gravity. Due to the scheduling and logis-
tics of production, as well as the permitted timing for access to the site, process termination
usually occurred in the middle of the night. With return of operating personnel to the site in
the morning, and now under manual control, about 100 to 150 L of mixed liquor containing
gravity thickened solids of PHA-rich biomass were pumped over to a 200 L holding tank and
the mixed liquor pH was adjusted to 2 by titration with concentrated H2SO4 (Werker et al.
2013b). Following acidification, the solids were further thickened by floatation within the
200 L volume and these thickened solids were then dewatered by means of a filter bag centri-
fuge after adding sludge dewatering chemicals. The mixed liquor with added dewatering
chemicals (Flopam EM 840 TBD) with a bag centrifuge (at 980×g with filter bags defined with
7 L/dm2/min at 200 Pa) were dewatered on average to about 19% dry solids. Under optimized
dewatering conditions it is anticipated to reach approximately 25% dry solids content. The
sludge cake was transferred manually to drying trays and then set to dry at 70°C.
4.1.3 PHA EXTRACTION
The dried materials were packaged and sent by courier to AnoxKaldnes in Sweden where
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the biomass, PHA-in biomass and recovered and purified PHA qualities were assessed quan-
titatively (Werker et al. 2013b, Werker et al. 2015). Over the course of the process operations
including fermentation, accumulation and downstream processing, selected samples were
obtained for basic water quality and solids analyses. From these analyses the process mass
balances were evaluated and potential influences for the process operations on the recovered
polymer product quality were examined.
4.1.4 PRODUCTION BATCHES AND CODING
The WP1 efforts for PHA production thereby comprised 59 activated sludge batches Axx (A01
to A59), accumulated with 3 different types of feedstocks Sxx, Cxx and Pxx over ten months
of pilot operations. For Sxx, the xx denotes the propionic weight fraction (as COD) in the
mixture of acetic and propionic acids. Thus, S05 denotes 5 percent propionic acid and 95
percent acetic acid as COD in the feedstock. Cxx refers to the fermentation batches with the
process water from the candy factory (C01 to C26) where C01 to C06 were made during the
initial commissioning and benchmarking period (Hjort et al. 2015). Pxx refers similarly to
the primary sludge fermentation centrate batches P01 to P12 that were produced in the final
and third phase of operations (Hjort et al. 2015). Exception events happened for the following
Axx batches:
A01 was a first “dry run”.
A07 was terminated due to loss of alkalinity causing undue pH decrease
A08 was terminated due to pumping errors
A09 was abnormally terminated early
A10 required corrections to the PLC program on the that were made on the fly
A25 was stopped early due to feed running out (higher biomass loading)
A53 was terminated due to feed running out during accumulation
These batches were not included in the evaluation of product quality and variability. Fifty-two
batches of PHA-rich biomass were successfully completed out of the 59 accumulation runs.
The quality of the polymer in the biomass (PHA-in biomass quality) and the recovered polymer
quality in relation to the feedstock and the operations form the basis of the assessment were
performed and are reported.
Out of the 52 successful accumulation trials:
20 were with defined mixtures of acetic and propionic acids, or so-called synthetic (“S”)
feedstock.
27 were with the supply of 26 batches of fermented candy factory process water filtrate
feedstock, or “C” feedstock.
5 were with the supply of 12 batches of fermented primary sludge centrate, or “P”
feedstock.
4.2 PHA CHEMISTRY AND CHARACTERIZATION
4.2.1 INTRODUCTION TO PHA POLYMER CHEMISTRY
The purpose of this section is to introduce to non-polymer science engineering professionals
the kinds of PHA polymers produced in PHARIO and the parameters evaluated for quality.
Polyhydroxyalkanoates or PHAs are a family of linear polyesters produced by many species
of bacteria found in nature (Sudesh et al. 2000, Janarthanan et al. 2016). For the bacteria,
they represent an efficient means to store energy and carbon intracellularly. More than
150 different monomers can be combined within the PHA family, wherein the respectively
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STOWA 2017-15 PHARIO: STEPPING STONE TO A SUSTAINABLE VALUE CHAIN FOR PHA BIOPLASTIC USING MUNICIPAL ACTIVATED SLUDGE
different combinations result in extremely different materials with properties as thermo-
plastic or elastomeric materials and with melting points ranging from 40 to 180°C (Laycock
et al. 2012). The polymers are biodegradable (Arcos-Hernandez et al. 2012) and they can
be used in formulations to make bioplastics for which such things as processing and
mechanical properties, as well as features of biodegradability, can be significantly modulated.
The mechanical properties and compatibility of PHA can be influenced through surface
modification and/or blending and combining PHA with other polymers, enzymes, and/or
inorganic materials. Thus, from this interesting class of biopolymer, comes a very a wide and
largely unexplored range of possible applications. Most people refer to these biopolymers
simply as PHAs, but PHA is a polymer family, wherein the kinds of PHAs produced in PHARIO
are specific and well defined, and these specific polymers will be described in this chapter.
In PHARIO polymers accumulated by the biomass comprised two building blocks (Figure 11)
poly-(3 hydroxybutyrate) (or PHB ) and poly-(3 hydroxyvalerate) or (or PHV). The mixture of
these monomer building blocks (3 hydroxybutyrate and 3 hydroxyvalerate, or 3HB and 3HV)
on a polymer chain is the co-polymer poly-(3 hydroxybutyrate-co-3 hydroxyvalerate) or PHBV.
The intermediary pool of metabolites that provide the precursors to 3HB and 3HV and the
microbial storage or “accumulation” of PHB, PHV, and/or PHBV, are related to the type of
substrates that are fed to the biomass.
FIGURE 11 THE POLYMERS ACCUMULATED BY THE BIOMASS COMPRISED TWO BUILDING BLOCKS POLY-(3 HYDROXYBUTYRATE) OR PHB AND POLY-(3
HYDROXYVALERATE) OR PHV. THE MIX OF THESE BUILDING BLOCKS ON A POLYMER CHAIN IS POLY-(3 HYDROXYBUTYRATE-CO-3 HYDROXYVALERATE) OR
PHBV. N AND M REPRESENT THE NUMBER OF RESPECTIVE REPEATING UNITS.
PHARIO
PHARIO final reportpublic version 28/3/2017 Page 25
4.2 PHA chemistry and characterization
4.2.1 Introduction to PHA polymer chemistry
The purpose of this section is to introduce to non-polymer science engineering professionals the kinds of PHA
polymers produced in PHARIO and the parameters evaluated for quality. Polyhydroxyalkanoates or PHAs are
a family of linear polyesters produced by many species of bacteria found in nature (Sudesh et al. 2000,
Janarthanan et al. 2016). For the bacteria, they represent an efficient means to store energy and carbon
intracellularly. More than 150 different monomers can be combined within the PHA family, wherein the
respectively different combinations result in extremely different materials with properties as thermoplastic or
elastomeric materials and with melting points ranging from 40 to 180°C (Laycock et al. 2012). The polymers
are biodegradable (Arcos-Hernandez et al. 2012) and they can be used in formulations to make bioplastics for
which such things as processing and mechanical properties, as well as features of biodegradability, can be
significantly modulated. The mechanical properties and compatibility of PHA can be influenced through
surface modification and/or blending and combining PHA with other polymers, enzymes, and/or inorganic
materials. Thus, from this interesting class of biopolymer, comes a very a wide and largely unexplored range
of possible applications. Most people refer to these biopolymers simply as PHAs, but PHA is a polymer family,
wherein the kinds of PHAs produced in PHARIO are specific and well defined, and these specific polymers
will be described in this chapter.
In PHARIO polymers accumulated by the biomass comprised two building blocks (Figure 11) poly-(3
hydroxybutyrate) (or PHB ) and poly-(3 hydroxyvalerate) or (or PHV). The mixture of these monomer building