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.

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.

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.

Integration of biopolymer production with process water treatment at a sugar factory.

The present investigation has focused on generating a surplus denitrifying biomass with high polyhydroxyalkanoate (PHA) producing potential while maintaining water treatment performance in biological nitrogen removal. The motivation for the study was to examine integration of PHA production into the water treatment and residuals management needs at the Suiker Unie sugar beet factory in Groningen, the Netherlands. At the factory, process waters are treated in nitrifying-denitrifying sequencing batch reactors (SBRs) to remove nitrogen found in condensate. Organic slippage (COD) in waters coming from beet washing is the substrate used for denitrification. The full-scale SBR was mimicked at laboratory scale. In two parallel laboratory scale SBRs, a mixed-culture biomass selection strategy of anoxic-feast and aerobic-famine was investigated using the condensate and wash water from Suiker Unie. One laboratory SBR was operated as conventional activated sludge with long solids retention time similar to the full-scale (SRT >16 days) while the other SBR was a hybrid biofilm-activated sludge (IFAS) process with short SRT (4-6 days) for the suspended solids. Both SBRs were found to produce biomass with augmented PHA production potential while sustaining process water treatment for carbon, nitrogen and phosphorus for the factory process waters. PHA producing potential in excess of 60 percent g-PHA/g-VSS was achieved with the lab scale surplus biomass. Surplus biomass of low (4-6 days) and high (>16 days) solids retention time yielded similar results in PHA accumulation potential. However, nitrification performance was found to be more robust for the IFAS SBR. Assessment of the SBR microbial ecology based on 16sDNA and selected PHA synthase genes at full-scale in comparison to biomass from the laboratory scale SBRs suggested that the full-scale process was enriched with a PHA storing microbial community. However, structure-function relationships based on RNA levels for the selected PHA synthases could not be established and, towards this ambition, it is speculated that a wider representation of PHA synthesases would need to be monitored. Additionally at the factory, beet tail press waters coming from the factory beet residuals management activities are available as a carbon source for PHA accumulation. At pilot scale, beet tail press waters were shown to provide a suitable carbon source for mixed culture PHA production in spite of otherwise being of relatively low organic strength (≤ 10 g-COD/L). A copolymer of 3-hydroxybutyrate with 3-hydroxyvalerate (PHBV with 15% HV on a molar basis) of high thermal stability and high weight average molecular mass (980 kDa) was produced from the beet tail press water. The mixed culture accumulation process sustained PHA storage with parallel biomass growth of PHA storing bacteria suggesting a strategy to further leverage the utilization of surplus functional biomass from biological treatment systems. Integration of PHA production into the existing factory water management by using surplus biomass from condensate water treatment and press waters from beet residuals processing was found to be a feasible strategy for biopolymer production.