Visualization of Polyhydroxyalkanoate Accumulated in Waste Activated Sludge.

Polyhydroxyalkanoates (PHAs) can be produced with municipal waste activated sludge from biological wastewater treatment processes. Methods of selective fluorescent staining with confocal laser scanning microscopy (CLSM) were developed and optimized to evaluate the distribution of PHA storage activity in this mixed culture activated sludge microbial communities. Selective staining methods were applied to a municipal activated sludge during pilot scale PHA accumulation in replicate experiments. Visualization of stained flocs revealed that a significant but limited fraction of the biomass was engaged with PHA accumulation. Accumulated PHA granules were furthermore heterogeneously distributed within and between flocs. These observations suggested that the PHA content for the bacteria storing PHAs was significantly higher than the average PHA content measured for the biomass as a whole. Optimized staining methods provided high acuity for imaging of PHA distribution when compared to other methods reported in the literature. Selective staining methods were sufficient to resolve and distinguish between distinctly different morphotypes in the biomass, and these observations of distinctions have interpreted implications for PHA recovery methods. Visualization tools facilitate meaningful insights for advancements of activated sludge processes where systematic observations, as applied in the present work, can reveal underlying details of structure-function relationships.

Calcium enhances polyhydroxyalkanoate production and promotes selective growth of the polyhydroxyalkanoate-storing biomass in municipal activated sludge.

Activated sludge from municipal wastewater treatment processes can be used directly for the production of biodegradable polyesters from the family of polyhydroxyalkanoates (PHAs). However, municipal activated sludge typically cannot accumulate PHAs to very high levels and often low yields of polymer produced on substrate are observed. In the present work, it was found that the presence of calcium promotes selective growth and enrichment of the PHA-storing biomass fraction and significantly improved both PHA contents and yields. Calcium addition resulted in PHA contents of 0.60 ± 0.03 gPHA/gVSS and average PHA yields on substrate of 0.49 ± 0.03 gCODPHA/gCODHAc compared to 0.35 ± 0.01 gPHA/gVSS and 0.19 ± 0.01 gCODPHA/gCODHAc without calcium addition. After 48 h, three times more PHA was produced compared to control experiments without calcium addition. Higher PHA content and selective biomass production is proposed to be a consequence of calcium dependent increased levels of passive acetate uptake. Such more efficient substrate uptake could be related to a formation of calcium acetate complexes. Findings lead to bioprocess methods to stimulate a short-term selective growth of PHA-storing microorganisms and this enables improvements to the techno-economic feasibility for municipal waste activated sludge to become a generic resource for industrial scale PHA production.

Process conditions affect properties and outcomes of polyhydroxyalkanoate accumulation in municipal activated sludge.

The developments of mixed culture polyhydroxyalkanoate production has been directed to maximize the biomass PHA content with limited attention to polymer quality. Direct comparison of PHA accumulation literature is challenging, and even regularly contradicting in reported results, due to underlying differences that are not well expressed. A study was undertaken to systematically compare the commonly reported process conditions for PHA accumulation by full-scale municipal activated sludge. A biomass acclimation step combined with a pulse-wise feeding strategy resulted in maximum average PHA contents and product yields. pH control and active nitrification did not result in observable effects on the PHA productivity. Under these conditions a high molecular weight polymer (1536 ± 221 kDa) can be produced. Polymer extraction recoveries were influenced by the PHA molecular weight. A standard protocol for an activated sludge PHA accumulation test including downstream processing and standardized extraction has been developed and is available as supplementary material.

Exploring the Limits of Polyhydroxyalkanoate Production by Municipal Activated Sludge.

Municipal activated sludge can be used for polyhydroxyalkanoate (PHA) production, when supplied with volatile fatty acids. In this work, standardized PHA accumulation assays were performed with different activated sludge to determine (1) the maximum biomass PHA content, (2) the degree of enrichment (or volume-to-volume ratio of PHA-accumulating bacteria with respect to the total biomass), and (3) the average PHA content in the PHA-storing biomass fraction. The maximum attained biomass PHA content with different activated sludge ranged from 0.18 to 0.42 gPHA/gVSS, and the degree of enrichment ranged from 0.16 to 0.51 volume/volume. The average PHA content within the PHA-accumulating biomass fraction was relatively constant and independent of activated sludge source, with an average value of 0.58 ± 0.07 gPHA/gVSS. The degree of enrichment for PHA-accumulating bacteria was identified as the key factor to maximize PHA content when municipal activated sludge is directly used for PHA accumulation. Future optimization should focus on obtaining a higher degree of enrichment of PHA-accumulating biomass, either through selection during wastewater treatment or by selective growth during PHA accumulation. A PHA content in the order of 0.6 g PHA/g VSS is a realistic target to be achieved when using municipal activated sludge for PHA production.

Quantification of polyhydroxyalkanoate accumulated in waste activated sludge.

Polyhydroxyalkanoate accumulation experiments at pilot scale were performed with fullscale municipal waste activated sludge. Development of biomass PHA content was quantified by thermogravimetric analysis. Over 48 h the biomass reached up to 0.49 ± 0.03 gPHA/gVSS (n=4). Samples were processed in parallel to characterise the distribution of PHA in the biomass. Selective staining methods and image analysis were performed by Confocal Laser Scanning Microscopy. The image analysis indicated that nominally 55% of this waste activated sludge was engaged in PHA storage activity. Thus even if the biomass PHA content reached 0.49gPHA/gVSS, the accumulating fraction of the biomass was estimated to have attained about 0.64gPHA/gVSS. The combination of quantitative microscopy and polymer mass assessment enabled to distinguish the effect of level of enrichment in PHA storing bacteria and the average PHA storage capacity of the accumulating bacteria. The distribution of microbial 16S rRNA levels did not follow a measurable trend during PHA accumulation.

Modelling Mixed Microbial Culture Polyhydroxyalkanoate Accumulation Bioprocess towards Novel Methods for Polymer Production Using Dilute Volatile Fatty Acid Rich Feedstocks.

Volatile fatty acid (VFA) rich streams from fermentation of organic residuals and wastewater are suitable feedstocks for mixed microbial culture (MMC) Polyhydroxyalkanoate (PHA) production. However, many such streams have low total VFA concentration (1-10 gCOD/L). PHA accumulation requires a flow-through bioprocess if the VFAs are not concentrated. A flow through bioprocess must balance goals of productivity (highest possible influent flow rates) with goals of substrate utilization efficiency (lowest possible effluent VFA concentration). Towards these goals, dynamics of upshift and downshift respiration kinetics for laboratory and pilot scale MMCs were evaluated. Monod kinetics described a hysteresis between the upshift and downshift responses. Substrate concentrations necessary to stimulate a given substrate uptake rate were significantly higher than the concentrations necessary to sustain the attained substrate uptake rate. A benefit of this hysteresis was explored in Monte Carlo based PHA accumulation bioprocess numerical simulations. Simulations illustrated for a potential to establish continuous flow-through PHA production bioprocesses even at a low (1 gCOD/L) influent total VFA concentration. Process biomass recirculation into an engineered higher substrate concentration mixing zone, due to the constant influent substrate flow, enabled to drive the process to maximal possible PHA production rates without sacrificing substrate utilization efficiency.

Simultaneous nitrification and denitrification in microbial community-based polyhydroxyalkanoate production.

Microbial community-based polyhydroxyalkanoate (PHA) production has been demonstrated repeatedly at pilot scale. Ammonium, normally present in waste streams, might be oxidized by nitrifying bacteria resulting in additional aeration energy demand. The use of low dissolved oxygen (DO) concentrations allowed to reduce nitrifying rates by up to 70% compared to non-oxygen limiting conditions. At lower DO concentrations nitrate was used as alternative electron acceptor for PHA production and therefore, a constant PHA production rate could only be maintained if nitrate was sufficiently available. An optimum DO concentration (0.9 mgO2/L) was found for which nitrification was mitigated but also exploited to supply requisite heterotrophic nitrate requirements that maintained maximum PHA production rates. PHA accumulations with such DO control was estimated to reduce oxygen demand by about 18%. This work contributes to establish fundamental insight towards viable industrial practice with the control and exploitation of nitrifying bacteria in microbial community-based PHA production.

Scaling-up microbial community-based polyhydroxyalkanoate production: status and challenges.

Conversion of organic waste and wastewater to polyhydroxyalkanoates (PHAs) offers a potential to recover valuable resources from organic waste. Microbial community-based PHA production systems have been successfully applied in the last decade at lab- and pilot-scales, with a total of 19 pilot installations reported in the scientific literature. In this review, research at pilot-scale on microbial community-based PHA production is categorized and subsequently analyzed with focus on feedstocks, enrichment strategies, yields of PHA on substrate, biomass PHA content and polymer characterization. From this assessment, the challenges for further scaling-up of microbial community-based PHA production are identified.

Experimental data for extrusion processing and tensile properties of poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) polymer and wood fibre reinforced PHBV biocomposites.

This article features a large database on different extrusion processing conditions and the resulting tensile properties of poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and wood fibre reinforced biocomposites. The data presented here corresponds to a comprehensive design of experiments conducted separately for both neat PHBV polymer and wood-PHBV composites, in which the effects of temperature profile, screw speed, feeding rate, feeding method, screw configuration, and wood contents (wood-PHBV composites only) of 10, 20, 30, and 40 wt% wood content were examined. For each processing condition, 5 specimens were tested under uniaxial tensile loading. Here we provide the complete set of extrusion parameters, including the observed screw torque, residence time and material output. Individual stress-strain curves for each specimens are provided, along with their calculated elastic modulus, strength, and strain at maximum load. The data is also provided as support material for the research article: "Extrusion of wood fibre reinforced Poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) biocomposites: statistical analysis of the effect of processing conditions on mechanical performance" (Vandi et al., 2018).

Application of dissolved oxygen (DO) level control for polyhydroxyalkanoate (PHA) accumulation with concurrent nitrification in surplus municipal activated sludge.

Mixed microbial cultures are a viable means for polyhydroxyalkanoate (PHA) production, which can produce polymers of commercial quality with high yields. Various PHA co-polymer blends can be produced by surplus full-scale municipal activated sludge fed with fermented waste feedstocks. In biological nutrient removal, ammonia is converted to nitrate by ammonia and nitrite oxidizing bacteria (AOBs and NOBs) through nitrification and removed as nitrogen gas through denitrification. Activated sludge can be enriched with significant PHA storage potential alongside nitrogen removal by denitrifying heterotrophic and nitrifying autotrophic bacteria. The latter adds complexity and aeration demand during the aerobic side-stream PHA accumulation stage since fermented organic residuals often contain significant amounts of ammonia. In the present work, the influence of dissolved oxygen (DO) levels on both PHA accumulation and nitrification rates for a municipal activated sludge were evaluated. The objective was to identify potential for a DO control strategy for PHA accumulation, which would mitigate the unnecessary nitrification activity during PHA production. A much higher apparent Michaelis-Menten DO affinity for volatile fatty acid (VFA) consumption (KDO _VFA 0.1 ± 0.06 mg/L) was found as compared to nitrification (KDO _NH4 2.87 ± 1.31 mg/L). Consequently, with lower DO levels, PHA production was not limited by oxygen supply, while nitrogen was removed by simultaneous nitrification and denitrification processes. This study suggests a method for PHA accumulation using nitrifying activated sludge, while feeding ammonia-containing organic feedstocks by means of DO level control where: (1) NOB activity and growth are both mitigated, (2) nitrogen removal is facilitated, (3) alkalinity is controlled through simultaneous denitrification, and (4) energy demand for aeration is reduced.

Consistent production of high quality PHA using activated sludge harvested from full scale municipal wastewater treatment - PHARIO.

Production of polyhydroxyalkanoate (PHA) biopolymers by mixed microbial cultures concurrent to wastewater treatment is a valorization route for residual organic material. This development has been at pilot scale since 2011 using industrial and municipal organic residuals. Previous experience was the basis for a PHA production demonstration project: PHARIO. PHARIO was centred on processing surplus activated sludge biomass from the Bath full-scale municipal wastewater treatment plant in the Netherlands to produce PHA. Full-scale surplus activated sludge was fed to a pilot facility to produce PHA rich biomass using fermented volatile fatty acid (VFA) rich liquors from industry or primary sludge sources. A PHA rich biomass with on average 0.41 gPHA/gVSS was obtained with reproducible thermal properties and high thermal stability. A routine kilogram scale production was established over 10 months and the polymer material properties and market potential were evaluated. Surplus full-scale activated sludge, over four seasons of operations, was a reliable raw material to consistently and predictably produce commercial quality grades of PHA. Polymer type and properties were systematic functions of the mean co-polymer content. The mean co-polymer content was predictably determined by the fermented feedstock composition. PHARIO polymers were estimated to have a significantly lower environmental impact compared to currently available (bio)plastics.

Wood-PHA Composites: Mapping Opportunities.

Polyhydroxyalkanoate (PHA) biopolymers are emerging as attractive new sustainable polymers due to their true biodegradability and highly tuneable mechanical properties. However, despite significant investments, commercialisation barriers are hindering the capacity growth of PHA. In this work, we investigated the market potential for wood plastic composites (WPCs) based on PHAs. We considered the latest global production capacity of PHAs, estimated at 66,000 tonnes/year, and examined the implications of using PHAs for WPC production on the WPC market. Results indicate that a hypothetical usage of the current global PHA production for WPC manufacture would only represent the equivalent of 4.4% of the global WPC market, which is currently experiencing a 10.5% compounded annual growth rate. An economic assessment revealed that a wood-PHA composite as a drop-in alternative WPC product could cost as little as 37% of the cost of its neat PHA counterpart. Thus, WPCs with PHA offer a means to access benefits of PHA in engineering applications at reduced costs; however, further developments are required to improve strain at failure. The successful adoption of wood-PHA composites into the market is furthermore reliant on support from public sector to encourage biodegradable products where recycling is not a ready solution.

The Evolution of Polymer Composition during PHA Accumulation: The Significance of Reducing Equivalents.

This paper presents a systematic investigation into monomer development during mixed culture Polyhydroxyalkanoates (PHA) accumulation involving concurrent active biomass growth and polymer storage. A series of mixed culture PHA accumulation experiments, using several different substrate-feeding strategies, was carried out. The feedstock comprised volatile fatty acids, which were applied as single carbon sources, as mixtures, or in series, using a fed-batch feed-on-demand controlled bioprocess. A dynamic trend in active biomass growth as well as polymer composition was observed. The observations were consistent over replicate accumulations. Metabolic flux analysis (MFA) was used to investigate metabolic activity through time. It was concluded that carbon flux, and consequently copolymer composition, could be linked with how reducing equivalents are generated.

A process for polyhydroxyalkanoate (PHA) production from municipal wastewater treatment with biological carbon and nitrogen removal demonstrated at pilot-scale.

A process was developed for biological treatment of municipal wastewater for carbon and nitrogen removal while producing added-value polyhydroxyalkanoates (PHAs). The process comprised steps for pre-denitrification, nitrification and post-denitrification and included integrated fixed-film activated sludge (IFAS) with biofilm carrier media to support nitrification. In a pilot-scale demonstration (500-800L), wastewater treatment performance, in line with European standards, were achieved for total chemical oxygen demand (83% removal) and total nitrogen (80% removal) while producing a biomass that was able to accumulate up to 49% PHA of volatile suspended solids with acetic acid or fermented organic residues as substrates. Robust performance in wastewater treatment and enrichment of PHA-producing biomass was demonstrated under realistic conditions including influent variability during 225days of operation. The IFAS system was found to be advantageous since maintaining nitrification on the biofilm allowed for a relatively low (2days) solids retention time (SRT) for the suspended biomass in the bulk phase. Lower SRT has advantages in higher biomass yield and higher active fraction in the biomass which leads to higher PHA productivity and content. The outcomes show that production of added-value biopolymers may be readily integrated with carbon and nitrogen removal from municipal wastewater.

Carbon recovery from wastewater through bioconversion into biodegradable polymers.

Polyhydroxyalkanoates (PHA) are biodegradable polyesters that can be produced in bioprocesses from renewable resources in contrast to fossil-based bio-recalcitrant polymers. Research efforts have been directed towards establishing technical feasibility in the use of mixed microbial cultures (MMC) for PHA production using residuals as feedstock, mainly consisting of industrial process effluent waters and wastewaters. In this context, PHA production can be integrated with waste and wastewater biological treatment, with concurrent benefits of resource recovery and sludge minimization. Over the past 15 years, much of the research on MMC PHA production has been performed at laboratory scale in three process elements as follows: (1) acidogenic fermentation to obtain a volatile fatty acid (VFA)-rich stream, (2) a dedicated biomass production yielding MMCs enriched with PHA-storing potential, and (3) a PHA accumulation step where (1) and (2) outputs are combined in a final biopolymer production bioprocess. This paper reviews the recent developments on MMC PHA production from synthetic and real wastewaters. The goals of the critical review are: a) to highlight the progress of the three-steps in MMC PHA production, and as well to recommend room for improvements, and b) to explore the ideas and developments of integration of PHA production within existing infrastructure of municipal and industrial wastewaters treatment. There has been much technical advancement of ideas and results in the MMC PHA rich biomass production. However, clear demonstration of production and recovery of the polymers within a context of product quality over an extended period of time, within an up-scalable commercially viable context of regional material supply, and with well-defined quality demands for specific intent of material use, is a hill that still needs to be climbed in order to truly spur on innovations for this field of research and development.

Fluxes in PHA-storing microbial communities during enrichment and biopolymer accumulation processes.

The use of mixed microbial cultures for the production of polyhydroxyalkanoates (PHAs) is emerging as a viable technology. In this study, 16S rRNA gene amplicon pyrosequencing was used to analyse fluctuations in populations over a 63-day period within a PHA-storing mixed microbial community enriched on fermented whey permeate. This community was dominated by the genera Flavisolibacter and Zoogloea as well as an unidentified organism belonging to the phylum Bacteroidetes. The population was observed to cycle through an increase in Zoogloea followed by a return to a community composition similar to the initial one (highly enriched in Flavisolibacter). It was found that the PHA accumulation capacity of the community was robust to population flux during enrichment and even PHA accumulation, with final polymer composition dependent on the overall proportion of acetic to propionic acids in the feed. This community adaptation suggests that mixed culture PHA production is a robust process.

Polyhydroxyalkanoate (PHA) storage within a mixed-culture biomass with simultaneous growth as a function of accumulation substrate nitrogen and phosphorus levels.

The response of a mixed-microbial-culture (MMC) biomass for PHA accumulation was evaluated over a range of relative nitrogen (N) and phosphorus (P) availabilities with respect to the supply of either complex (fermented whey permeate - FWP) or simpler (acetic acid) organic feedstocks. Fed-batch feed-on-demand PHA accumulation experiments were conducted where the feed N/COD and P/COD ratios were varied ranging from conditions of nutrient starvation to excess. A feast-famine enrichment (activated sludge) biomass, produced in a pilot-scale aerobic sequencing batch reactor on FWP and with a long history of stable PHA accumulation performance, was used for all the experiments as reference material. FWP with N/COD ratios of (2, 5, 15, 70 mg/g all with P/COD = 8 mg/g) as well as simulated FWP with nutrient starvation (N/COD = P/COD = 0) conditions were applied. For the acetic acid accumulations, nutrient starvation as well as N/COD variations (2.5, 5, 50 mg/g all with P/COD = 9 mg/g) and P/COD variations (0.5, 2, 9, 15 mg/g all with N/COD = 10 mg/g) were evaluated. An optimal range of combined N and P limitation with N/COD from 2 to 15 mg/g and P/COD from 0.5 to 3 mg/g was considered to offer consistent improvement of productivity over the case of nutrient starvation. Productivity increased due to active biomass growth of the PHA storing biomass without observed risk for a growth response overtaking PHA storage activity. PHA production with respect to the initial active biomass was significantly higher even in cases of excess nutrient additions when compared to the cases of nutrient starvation. The 24-h PHA productivities were enhanced as much as 4-fold from a base value of 1.35 g-PHA per gram initial active biomass with respect nutrient starvation feedstock. With or without nutrient loading the biomass consistently accumulated similar and significant PHA (nominally 60% g-PHA/g-VSS). Based on results from replicate experiments some variability in the extant biomass maximum PHA content was attributed to interpreted differences in the biomass initial physiological state and not due to changes in feedstock nutrient loading. We found that the accumulation process production rates for mixed cultures can be sustained long after the maximum PHA content of the biomass was reached. Within the specific context of the applied fed-batch feed-on-demand methods, active biomass growth was interpreted to have been largely restricted to the PHA-storing phenotypic fraction of the biomass. This study suggests practical prospects for mixed culture PHA production using a wide range of volatile fatty acid (VFA) rich feedstocks. Such VFA sources derived from residual industrial or municipal organic wastes often naturally contain associated nutrients ranging in levels from limitation to excess.

Sludge minimization in municipal wastewater treatment by polyhydroxyalkanoate (PHA) production.

An innovative approach has been recently proposed in order to link polyhydroxyalkanoates (PHA) production with sludge minimization in municipal wastewater treatment, where (1) a sequencing batch reactor (SBR) is used for the simultaneous municipal wastewater treatment and the selection/enrichment of biomass with storage ability and (2) the acidogenic fermentation of the primary sludge is used to produce a stream rich in volatile fatty acids (VFAs) as the carbon source for the following PHA accumulation stage. The reliability of the proposed process has been evaluated at lab scale by using substrate synthetic mixtures for both stages, simulating a low-strength municipal wastewater and the effluent from primary sludge fermentation, respectively. Six SBR runs were performed under the same operating conditions, each time starting from a new activated sludge inoculum. In every SBR run, despite the low VFA content (10% chemical oxygen demand, COD basis) of the substrate synthetic mixture, a stable feast-famine regime was established, ensuring the necessary selection/enrichment of the sludge and soluble COD removal to 89%. A good process reproducibility was observed, as also confirmed by denaturing gradient gel electrophoresis (DGGE) analysis of the microbial community, which showed that a high similarity after SBR steady-state had been reached. The main variation factors of the storage properties among different runs were uncontrolled changes of settling properties which in turn caused variations of both sludge retention time and specific organic loading rate. In the following accumulation batch tests, the selected/enriched consortium was able to accumulate PHA with good rate (63 mg CODPHA g CODXa(-1) h(-1)) and yield (0.23 CODPHA CODΔS(-1)) in spite that the feeding solution was different from the acclimation one. Even though the PHA production performance still requires optimization, the proposed process has a good potential especially if coupled to minimization of both primary sludge (by its use as the VFA source for the PHA accumulation, via previous fermentation) and excess secondary sludge (by its use as the biomass source for the PHA accumulation).

Fractionation of microbial populations in a PHA accumulating mixed culture and associated PHA content and composition.

The uniformity of PHA composition and content across groups of organisms in mixed cultures was considered. An activated sludge microbial community, with an average PHA content of 20wt%, was fractioned by Percoll assisted buoyant density separation. The microbial community in the two principal fractions was characterised using amplicon pyrosequencing. While organisms were common to both fractions, the relative abundances of species were found to be different between the two fractions. The average PHA content in one of the fractions was found to be higher (24wt%) than the other (16wt%); separation was considered to be in part driven by the density difference associated with PHA content, but also by other factors such as cell dimension and cellular morphology. But while differences in PHA content were observed, the PHA composition in both fractions was found to be approximately the same (43-44mol% HV), which shows that distinct groups of microbial populations within mixed cultures may generate PHA with similar average copolymer composition.

Crystallisation and fractionation of selected polyhydroxyalkanoates produced from mixed cultures.

Poly[R-3-hydroxybutyrate-co-(R-3-hydroxyvalerate)] (PHBV) copolymers were produced from mixed cultures of biomass (activated sludge) fed with acetic acid (HAc) and propionic acid (HPr). Feeding was performed in such a way as to produce materials with a wide range of monomer compositions and microstructures. Solvent-cast thin films of these materials have recently been shown to exhibit a narrow range of mechanical properties similar to those of the homopolymer poly(R-3-hydroxybutyrate) (PHB) [1]. In this work, more detailed analyses of the thermal and crystallisation properties of these mixed-culture polyesters have revealed that they like comprise complex blends with broad compositional distribution of random and/or blocky copolymers of very different 3-hydroxyvalerate (3HV) contents and melting temperatures and thus have very different respective crystallisation kinetics. This blend complexity was confirmed by solvent fractionation of selected samples. The findings support the hypothesis that overall mechanical properties of these complex copolymer blend materials will be strongly influenced by the more rapidly crystallising components that form the matrix within which the slower crystallising components exist as microdomains. New opportunities in the material development of PHAs are likely to be found in establishing and exploiting such structure-function relationships.