Pilot and prototyping scale investigations were undertaken in order to evaluate the technical feasibility of producing value-added biopolymers (polyhydroxyalkanoates (PHAs)) as a by-product to essential services of wastewater treatment and environmental protection. A commonly asked question concerns PHA quality that may be expected from surplus biomass produced during biological treatment for water quality improvement. This paper summarizes the findings from a collection of investigations. Alongside the summarized technical efforts, attention has been paid to the social and economic networks. Such networks are needed in order to nurture circular economies that would drive value chains in renewable resource processing from contaminated water amelioration into renewable value-added bioplastic products and services. We find commercial promise in the polymer quality and in the process technical feasibility. The next challenge ahead does not reside so much any more in fundamental research and development of the technology but, rather, in social-economic steps that will be necessary to realize first demonstration scale polymer production activities. It is a material supply that will stimulate niche business opportunities that can grow and stimulate technology pull with benefit of real life material product market combinations.
Plastics have come to play an essential role in the fabric of our lives because of the extraordinary range of properties available in polymeric materials. Owing to a low density, resistance to natural breakdown and the vast scale of plastic production, plastic waste pollutes the environment and has become a threat to the health of aquatic ecosystems. Efforts to alleviate this pollution have brought to the market developments for bio-based biodegradable plastics derived from renewable resources at competitive prices (bioplastics). Polyhydroxyalkanoates (PHAs) are bio-based biodegradable polymers, produced by many species of naturally occurring bacteria, which can be compounded into such bioplastics.
PHAs have properties comparable with polymers used in conventional plastics (Laycock et al. 2013a). To date, pure culture microbial PHA production methods (the use of axenic cultures) have had limited market penetration because these sources for PHA are still considered to be relatively expensive. The price of PHA can be reduced if the production costs are reduced. Production costs can be decreased through the integration of the polymer production into a synergistic network of existing essential services of environmental protection. This investigation has been concerned with the production of PHA as a natural outcome of municipal and industrial wastewater treatment and sludge management services. This coupling of renewable resource generation in parallel to classical ‘waste management services’ is increasingly being recognized as an attractive opportunity.
A growing body of scientific literature finds that both pure and mixed culture PHA production strategies can become more economical through the use of low-value carbon-rich raw material feedstocks such as effluents from industrial and municipal activities (Nikodinovic-Runic et al. 2013). Mixed culture PHA production has a further benefit, which is that sterilization, of equipment and feedstocks, is not required. In particular it has been found that mixed culture PHA production can be seamlessly integrated into the essential services of water pollution control with biological wastewater or process water treatment (Anterrieu et al. 2013). However, the anticipated challenge of mixed culture PHA is the quality control of the polymer production. In spite of repeated examples advocating technical feasibility of mixed culture PHA production as a by-product of wastewater treatment, it has not been possible until recently to generate sufficient quantities of PHA from mixed culture systems in order to adequately evaluate the quality and commercial potential for the PHA that can be expected.
As part of the Australian Research Council (ARC) Linkage Program (ARC LP0990917), AnoxKaldnes and the University of Queensland (Chemical Engineering and Centre for High Performance Polymers) established a 5-year trans-disciplinary research and development project (2009–2014) that was focused on bridging fundamental knowledge of mixed culture PHA production to the development of products and services of the produced polymers. The motivation of this project stemmed from a similar collaboration wherein laboratory scale studies were used to consider technical feasibility for production of PHA using food industry effluents as well as fermented sludge liquors (Gurieff 2007). Hydrolysis and fermentation of sludge as well as production of PHA with the fermented streams in the frame of a European Union sponsored project, NEPTUNE, were also part of this effort to consider practical real life case studies with bioprocesses for PHA production (Morgan-Sagastume et al. 2010, 2011).
In parallel, business development activities have been ongoing especially in the Netherlands toward the technology implementation and commercialization goals (de Vegt et al. 2012). Dutch Water Boards are advancing in leading edge developments that consider wastewater treatment plants as production sites of renewable raw materials as well as, more traditionally, green energy sources. It is within this forward-thinking strategy that ideas of a bio-based society are being considered in practical case studies within the water industry services and technology framework. Such activities have been put forward through the development of a number of exciting stakeholder networks. The purpose of this article is to summarize cumulative findings of technical feasibility across different collaborative efforts and in so doing bring forward collated perspectives on the future of the integration of PHA production strategies into mainstream wastewater treatment services. The positive achievements of the different collaborative technical efforts highlight the fact that the remaining challenge is chiefly a social-economical one.
PROCESS DESCRIPTION AND RESEARCH ACTIVITIES
The cumulative findings reported herein are a product of a suite of collaborative technical investigative efforts; however they naturally build together a larger picture of integrating water quality management with renewable resource value chains. The general manner of such value chain integration for the case of PHA production is described below in terms of four process elements (PE1–PE4) and the related research activities. The research activities entailed both fundamental investigations with pilot-scale facilities located in Sweden and the Netherlands. The work in the Netherlands further involved addressing social and economic considerations for the value chain stakeholders.
Integration of PHA production is to be within a context of a bio-based value chain whereby value-added renewable resources are generated out of industrial and municipal environmental engineering services. The technology integration involves four principal process elements, or PEs (Figure 1). PE1 avails sources of short chain volatile fatty acids (VFAs) from management of industrial, agricultural, and/or municipal organic carbon-rich residues. VFAs are platform chemicals for PHA production by mixed microbial cultures. PE2 provides a source of biomass with PHA accumulation potential (PAP) as a by-product of treating process waters and/or wastewaters. PE3 exploits the PAP of the harvested surplus biomass from PE2 to produce a PHA-rich-biomass and PE3 is a surrogate to today's waste activated sludge management practices. PE4 is a refinery that enables the recovery of PHA as well as other value-added resources such as nutrients (nitrogen and phosphorus), lipids, and platform chemicals or energy from the non-PHA biomass fraction. PE4 requires scales of economy and to this end it is to be a strategically located facility to serve a network of PE3 s producing PHA-rich-biomass. For the purposes of the ARC linkage project, we have been mainly focused on the factors that may influence polymer quality as part of the process elements (PE3 and PE4).
PE1 – sources of VFA for PHA accumulation
Different VFA feedstocks were applied for a series of campaigns of PHA production. The substrates employed for the enrichment (PE2) stage were typically sourced from the acidogenic fermentation of dairy and food industry effluents. The VFA feedstocks for the accumulation (PE3) stage comprised fermented dairy and food industry process effluents as well as synthetic substrates. ARC activities included fundamental studies with objectives to manipulate the copolymer composition and morphology, whereby mixtures of acetic and propionic acids in different proportions were applied. A wide range of copolymers in the group of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with varying hydroxyvalerate (HV) content were produced. Our objectives were to better understand the potential for manipulation of polymer production in order to asses for scope of influence with respect to material properties that mixed cultures could be expected to achieve in commercial practice. Developments of the bio-based society and residual material value chains include a diversity of sources of VFAs and here it is anticipated that a suite of PHBVs will be produced in first commercial implementations. Since VFAs are a common platform chemical for methods of mixed culture PHA production, acidogenic fermentation of waste streams, waste activated sludge (Gurieff 2007; Morgan-Sagastume et al. 2010, 2011) and food industry waste streams (Gurieff 2007) were also investigated in detail.
PE2 – sources of process waters and wastewaters for producing a functional biomass
PHAs were produced in surplus activated sludge biomass (mixed cultures) that was harvested from a number of laboratory and pilot-scale wastewater treatment processes treating a variety of industry or municipal process or wastewater effluents.
In all cases, a two-step process approach was employed whereby activated sludge was firstly submitted to a dynamic feeding regime to select for biomass with high PAP. Generally, we have used either an aerobic or anoxic-aerobic dynamic feeding strategy (ADF, or AnADF). The dynamic feeding consisted of submitting biomass to alternating periods of substrate abundance (feast) and scarcity (famine). By ADF both feast and famine are aerobic environments. For AnADF, the feast environment was anoxic. When bacteria are submitted to such an environment of an intermittent feeding, those bacteria that store substrate quickly during feast as intracellular PHA tend to become enriched and dominant within the biomass ecology. Surplus activated sludge biomass that is enriched in PAP from ADF or AnADF was harvested and exploited in a second step of aerobic fed-batch accumulation (PE3).
PE3 – production of PHA from harvested activate sludge biomass
VFA feedstocks have been applied in laboratory scale academic studies to stimulate maximal accumulation of PHA in activated sludge biomass (Serafim et al. 2004; Lemos et al. 2006; Johnson et al. 2009; Albuquerque et al. 2011). Our challenge was to establish up-scalable industrial process methods. We have applied such methods with feed-on-demand control at laboratory and pilot scales (Werker et al. 2013). The basic challenge with accumulation strategies has been to establish viable industrial process methods of accumulation of a PHA with high molecular weight using low strength VFA containing feedstocks which are found widely in effluent streams from industries and within municipal waste management activities. After accumulation, the PHA-rich-biomass was dewatered and dried. The polymer was recovered from the biomass by methods of solvent extraction as presented further below.
In particular, the main source of biomass with high PAP for the ARC collaboration was from a prototype pilot plant (Lund, Sweden) treating a dairy wastewater. The ADF wastewater treatment pilot plant was in continuous service of biomass production from 2008 to 2013. In addition, the other practical trials with other sources of ADF biomass were performed in different facilities of a technology prototype pilot plant treating food industry effluent (Eslöv, Sweden) by ADF that was in operation from 2010 to 2013. An ADF pilot prototype treating a municipal influent (Brussels, Belgium) that has been in operation since 2010 was also part of the source of biomass for these studies. In the Netherlands, the project BioTrip (2011–2013) made use of biomass produced from process water management by AnADF methods for a sugar factory (de Vegt et al. 2012). More recently prototype pilot-scale demonstration activities of AnADF methods were situated in Leeuwarden (Netherlands) (2013–2014). Biomass with significant PAP from municipal wastewater treatment for carbon and nitrogen removal verified BioTRIP laboratory trial findings as published in Anterrieu et al. (2014).
In the ARC linkage project a suite of fundamental investigations into the PHA accumulation process were conducted with systematic adjustments to the feeding strategy. These manipulations were made in order to challenge the scope to which the nature of the polymer properties could be influenced. Dynamic feeding strategies were aimed to vary the copolymer sequence distribution toward producing random, nominally diblock (A–B) blended with random copolymer, and nominally repeating multiblock (A–B)n copolymers. Here, ‘A–B’ represents segments of homopolymer blocks (hydroxybutyrate (HB) and HV). Further details on the feeding strategies and resultant PHBVs can be found in Arcos-Hernández et al. (2012, 2013a) and Laycock et al. (2013b, 2014). In selected accumulation experiments, the microbial community ecology was assessed by means of the biomass DNA extraction and amplicon pyrosequencing methods (Janarthanan 2014).
PE4 – PHA recovery from PHA-rich-biomass
The biomass after accumulation was processed according to the methods described in WO 2012/022998(A1) in order to generate a PHA-rich-biomass with a high ‘in-biomass’ PHA thermal stability. The thermal stability of the PHA in the biomass was improved by reducing the cationic content of the biomass before final dewatering and drying as part of the downstream processing of the PHA-rich-biomass. The polymer was extracted from the biomass using non-chlorinated solvents at bench and pilot scales (Werker et al. 2012; Laycock et al. 2013b).
The polymer chemical and mechanical properties were systematically evaluated (Arcos-Hernández et al. 2013a; Laycock et al. 2013b). The properties of the PHAs were compared to analogous commercially available material that had been produced by pure culture methods. Criteria for comparison included molecular weight, thermal properties and thermal degradation during melt processing as previously described (Arcos-Hernández et al. 2013b; Montaño-Herrera et al. 2014).
The glass transition temperature (Tg), melting temperature (Tm), enthalpy of fusion (ΔHm) and crystallization temperature (Tc) were determined using differential scanning calorimetry (DSC). Thermogravimetric analysis was used for determination of decomposition temperature (Td). Weight average molecular weight (MW), number average molecular weight (Mn) and polydispersity index (PDI= MW/Mn) were quantified using gel permeation chromatography.
Chemical compositional distribution and overall co-polymer distribution were determined through analysis of quantitative 1H and 13C high resolution one-dimensional nuclear magnetic resonance (NMR) spectra that were acquired from selected samples. The parameters D (Kamiya et al. 1989) and R (Žagar et al. 2006) were used to estimate monomer sequence distribution of PHBV produced. Average monomer unit compositions were also determined by gas chromatography/mass spectrometry by methods as described in Werker et al. (2008) and Arcos-Hernández et al. (2013a).
Extracted materials were solvent cast and tested according to ASTM-D882-02 standard methods for tensile property measurement. Atomic force microscopy images were obtained in pulsed force mode in order to study the surface distribution of the polymer films. Biodegradability and toxicity of degradation compounds were also measured in controlled soil environments using ASTM standard method D 5988-03 and standard test method OECD 208, respectively. Infrared spectroscopy (IR) was used as a complementary technique to assess material purity and PHA content in biomass, and as an aid to evaluate the material crystallinity. Thermal degradation during melt processing was followed using near-IR. Further details have been published (Arcos-Hernández et al. 2010, 2012, 2013a, 2013b; Laycock et al. 2013b; Montaño-Herrera 2014).
The mixed culture PHAs were found to be co-polymer blends and the blend nature could be analysed by DSC but also by fractionating the blend components with a chloroform/n-hexane mixed solvent at ambient temperature. Additionally, isothermal spherulite growth behavior was investigated in selected solvent cast films of fractionated and non-fractionated samples (Laycock et al. 2013b; Laycock et al. 2014).
RESEARCH OUTCOMES AND TECHNOLOGY OUTLOOK
The specific research outcomes described herein are themselves covered in greater technical detail from cited peer-reviewed disseminations. Hence, the reader is referred to related publications for further reading. The goal of this presentation has been to link these technical outcomes into a perspective of anticipation for the future technology outlook.
To date, mixed culture PHA research and development has been heavily focused on enrichment of the PHA producing phenotype in an activated sludge (PE2) and achieving high biomass PHA content in an accumulation process (PE3). Systematic investigations toward fundamental insight of mixed culture polymer quality and its control have been otherwise lacking (PE3 and PE4).
Analyses of bacterial PHA properties have mostly relied on the assumption that bacterial polyesters would be produced as random co-polymers with narrow polydispersity. Thus, one has typically assumed that well-defined and reproducible properties would be achieved in commercial practice (Laycock et al. 2013a). Hence, the introduction of other monomeric units has generally been the principal objective of recent studies focused on yielding a mixed culture PHA with novel or diversified chemo-mechanical properties (Bengtsson et al. 2010; Albuquerque et al. 2011). However, the efforts of the ARC linkage have shown, in contrast, that the assumption of a random co-polymer is not necessarily valid for mixed cultures, and even for pure culture PHA production methods. It is important to know the compositional distribution of the PHA in order to appropriately understand challenges and also as yet unexplored opportunities for novel material product market combinations (Laycock et al. 2013a).
The compositional distribution can be manipulated in a mixed culture PHA accumulation process and the use of dynamic feedstock feeding strategies have been shown to successfully manipulate copolymer sequence distributions. Consequently it was found that production of novel co-polymer blends that avail microstructures yielding desirable material mechanical properties is possible. The identification of controlling factors influencing microstructure morphologies resulting in target mechanical properties is of relevance in order to guarantee a product with reproducible quality and high value (Laycock et al. 2013a). Physico-chemical properties of mixed culture PHBVs were analyzed with a goal to discover structure-function relationships by studying the effect of manipulation of accumulation strategies on copolymer morphologies, which in turn govern microstructure and resulting mechanical properties (Laycock et al. 2014).
Influence of monomer proportions
Campaigns of production of copolymers comprising 3-HB and 3-HV monomer units of varying composition (PHBV) and copolymer blends of these copolymers were made. A range of overall 3-hydroxyvalerate (3-HV) content was achieved from 12 to 91 mol %. This range of monomer composition was possible by changing proportions of odd and even chain length VFAs in the feedstock used for the accumulation process. Many sources of VFA rich feedstocks from industry have been found to be rich in precursors for HB and HV monomers before and/or after acidogenic fermentation. Most of the prototype and pilot activities previously described confirmed this outcome in practice by yielding PHBV copolymers with different HB and HV fractions governed chiefly by the composition of the VFA rich feedstocks utilized.
The manipulation of sequences of monomeric units within the polymer chain can result in modification of mechanical properties (Arcos-Hernández et al. 2013a and Laycock et al. 2013b). The modulation of feed composition during an accumulation process has also been shown to promote production of non-random distributions of monomer units in pure culture PHA copolymers (McChalicher & Srienc 2007; Li et al. 2011). In our work, we found that PHA copolymers with higher and lower degree of randomness and blockiness could be produced by manipulating the feeding strategies during accumulation (Arcos-Hernández et al. 2013a; Laycock et al. 2013b). Thus, HV content alone is insufficient in itself to infer the material mechanical properties.
The analysis of thermal properties of the recovered PHBV indicated the presence of at least two different crystalline phases. Multiple glass transition temperatures were also observed and this indicated phase segregation, probably due to the presence of blend systems where some of the component copolymers were not miscible in the melt and/or had different rates of crystallization. Fractionation and thermal characterization confirmed that mixed culture PHBV are in fact co-polymer blends. Some immiscibility given a sufficient difference in HV content could be purposefully produced (Arcos-Hernández et al. 2013a; Laycock et al. 2013b; Laycock et al. 2014). More detailed analyses of the thermal and crystallization properties of these polyesters have revealed the possibility to establish complex blends with broad compositional distribution of random and/or blocky copolymers of very different 3-HV contents and melting temperatures and these blend elements have very different respective crystallization kinetics (Laycock et al. 2013b).
Opportunities for novel structure-function relationships were to be found in tailored blends with sufficiently different respective crystallization rates. Multiphasic microstructures can be formed due to the presence of blends of different PHBV copolymers in the same matrix thus in the morphological level many distinct microstructures may be created (Figure 2) so as to influence the material mechanical properties (Laycock et al. 2013b).
The effect of such complex multiphasic systems on mechanical properties was systematically explored. It was shown that the tensile properties of lower HV content PHBV materials (less than 20 mol %) yielded anticipated material properties (i.e. low elongation to break and high Young's modulus). However, higher HV content materials (greater than 40 mol %) could possess unique and steerable material properties. However, higher HV content PHBVs come with a challenge for processing due to slower crystallization kinetics (Arcos-Hernández et al. 2013a and Laycock et al. 2013b). In particular, two of the materials we produced demonstrated unique crystallization and thermal properties in their fractions, which were considered to have contributed to superior elasticity (Laycock et al. 2013b).
The evidence in discovered scope to be able to establish complex multiphase polymer domains from mixed culture PHA processes suggests a richness of potential that we could not have anticipated a priori from the research literature. Recent work has revealed that the overall co-monomer composition is robust regardless of microbial community changes (Janarthanan 2014). In on going research and development, we explore further the opportunity to manipulate and tailor material composition and microstructures in order to design for engineered polymer mechanical properties such as elongation to break. Studies on the biodegradation in soil of PHBV films of varying compositions and microstructure found that the mechanisms of biodegradation are also greatly influenced by the complex multiphasic systems of mixed culture PHA, nevertheless the rates of biodegradation of native uncompounded materials were proved to be within time frames adequate for applications in agriculture. The biodegradation products were also found to be non-toxic (Arcos-Hernández et al. 2012). These fundamental studies on polymer microstructure and biodegradation indicate, by extension and in anticipation, the nature and the opportunities for PHA to be recovered from the scaling up of such mixed culture pilot and prototype polymer production activities.
Comparisons to pure culture PHA
It is a natural assumption to consider that mixed culture PHA is of a lower quality when compared to pure culture PHA. Notwithstanding, we find from the range of materials that we have produced, chemical-mechanical properties of mixed culture PHAs that are comparable to those produced using pure cultures based on commercially available materials (Arcos-Hernández et al. 2012; Laycock et al. 2013b; Montaño-Herrera et al. 2014). For example Montaño-Herrera et al. (2014) found that mixed culture PHAs could be more thermally stable when compared with the commercial pure culture PHAs. The thermal stability of these mixed culture PHA was improved by reducing the cationic content of the biomass before final dewatering and drying as described in WO 2012 022998 A1 (Werker et al. 2012). These outcomes indicated that the quality of the polymers on the market today might often be more related to the nature of the impurities associated with the recovered product. The methods of recovery may have more influence on the recovered polymer quality rather than the distinction of if the polymer was recovered from a pure or mixed culture.
Through these research and development efforts, fundamental insight was gained especially with regard to methods to tailor an accumulation process to influence polymer production towards bioplastics with advanced chemical/mechanical characteristics. The piloting experience suggested that in practice consistency in product quality is technically feasible for mixed culture methods. In a production situation, conditions of accumulation would be reproduced through selection of optimal process operating parameters and these selected conditions will be important towards ensuring a consistent product quality.
Although the results from the ARC linkage investigations suggested challenges due to a complexity arising from the formation of copolymer blends, we have found that this complexity is also an exciting opportunity towards establishing unique materials by manipulation of the microstructures in melt processing. At the same time we found, for example, reproducible practical outcomes from pilot prototype PHA production campaigns with biomass produced from municipal wastewater treatment and VFAs sourced from municipal sludge fermentation. So in spite of the ill-defined nature, for example, of a sludge sourced for VFAs, and the anticipated diversity of the ecology from a mixed culture, consistent polymer monomeric compositions, thermal behavior, and an average molecular weight in excess of 400 kDa were replicated. Next to the importance of providing for a consistent raw material we have found that an improved demonstrated thermal stability (WO 2012/022998A1) widens the potential in opportunities for the methods of polymer recovery and in producing high value low volume material-product-market combinations.
Therefore, in principle the key challenge for integrating PHA production from mixed culture processes and methods is not a fundamental research question any more, it is a social-economic challenge. The economic drivers to invest in moving up the value-added pyramid compete with the status quo of wasting biomass and producing biogas in order to reduce sludge disposal costs. Biogas production enjoys subsidies today that weigh against perceived risks for investments towards channeling organic carbon residuals toward less established value-added renewable resources. The markets for PHA from mixed cultures can only be reliably established from first full-scale reference projects from which sufficient quantities of PHA can be produced in order to generate real commercial value chains with real world material-product-market combinations. Subsidies to represent a ‘biogas equivalent’ also can serve to bring all renewable resources to a level playing field. Currently, there is also no burden in price for conventional plastics that are associated with a now well-documented risk for environmental impact as plastic trash in oceans and other ecosystems. PHAs, which cannot be made as cheaply but which do not share the same risk for environmental impact as conventional plastics, have difficulty competing only on the basis of purchase price versus fossil fuel-based plastics as substitutive raw materials today. Therefore, early success in mixed culture PHA production would be best served by entrepreneurial niche applications where the unique properties of these polymers in processing and in application are exploited in ways that sets them apart in their own category of useful products and services. It is perhaps a less strategic approach to place PHAs as directly substitutive materials for plastics as we know and use them today.
Pilot-scale production campaigns of PHA from activated sludge treatment can yield PHBV with comparable chemical and mechanical properties to similar commercially available pure culture PHAs. In fundamental studies we identified scope in untapped opportunity to influence PHBV microstructures with resulting desirable mechanical properties. Meaningful fundamental, practical and market advancements of the technology requires that commercial relevant quantities of the mixed culture PHA be produced and this step necessitates investment in first reference demonstration scale value chains. Such value chains can open as yet unexpected opportunities for renewable resource management activities that further drive a game change in environmental engineering community waste management practices within circular economies of a bio-based society.
The authors would like to acknowledge the Australian Research Council for funding this work through project LP0990917. We also thank Veolia Water Technologies Sweden and Veolia Water Corporate in co-funding through the grant ARC LP09900917, and in vision and commitment to the wider scope of initiatives represented by the research and development efforts presented in this article. We are especially grateful to the Dutch Water Boards of Fryslân, De Dommel and Brabantse Delta, as well as to Suiker Unie (Groningen), Kenniscentrum Papier & Karton (KCPK) and the Provinces of Frysân and Groningen for support, engagement and encouragement to tackle the real life challenges and see practical feasibility as well as economic potential for the technology integration within concrete contexts of a local social fabrics.