Alan Werker

Production of polyhydroxyalkanoates in open, mixed cultures from a waste sludge stream containing high levels of soluble organics, nitrogen and phosphorus

In this study, the production of polyhydroxyalkanoates (PHAs) from waste activated sludge (WAS) was evaluated. PHAs were produced from fermented WAS pretreated via high-pressure thermal hydrolysis, a stream characterised by high levels of nutrients (approximately 3.5 g N L(-1) and 0.5 g P L(-1)) and soluble organics. PHA-storing organisms were successfully enriched at high organic loading rates (6 g COD(sol) L(-1) d(-1)) under aerobic dynamic feeding in sequencing batch reactors at a sludge retention time of 6 d with a short feast length less than 20% of the cycle, and a maximum substrate concentration during feast of 1 g COD(VFA) L(-1). The biomass enrichment, characterised by a decrease in species evenness based on Lorenz curves, provided a biomass that accumulated 25% PHA on a dry-biomass basis with yields on VFA of 0.4 Cmol Cmol(-1) in batch tests. The PHA consisted of ∼70 mol% 3-hydroxybutyrate and ∼30 mol% 3-hydroxyvalerate, and presented high thermal stability (T(d) = 283-287 °C) and a molecular mass ranging from 0.7 to 1.0 × 10(6) g mol(-1). Overall PHA storage was comparable to that achieved with other complex substrates; however, lower PHA storage rates (0.04-0.05 Cmol PHA(-1) Cmol X(-1) h(-1)) and productivities (3-4 Cmol PHA L(-1) h(-1)) were probably associated with a biomass-growth and high-respiration response induced by high levels of non-VFA organics (40-50% of COD(sol) in feed) and nutrients. PHA production is feasible from pretreated WAS, but the enrichment and accumulation process require further optimisation. A milder WAS pretreatment yielding lower levels of non-VFA organics and readily available nutrients may be more amenable for improved performance.

Production of volatile fatty acids by fermentation of waste activated sludge pre-treated in full-scale thermal hydrolysis plants

This work focuses on fermentation of pre-treated waste activated sludge (WAS) to generate volatile fatty acids (VFAs). Pre-treatment by high-pressure thermal hydrolysis (HPTH) was shown to aid WAS fermentation. Compared to fermentation of raw WAS, pre-treatment enabled a 2-5x increase in VFA yield (gVFA(COD)gTCOD(-1)) and 4-6x increase in VFA production rate (gVFA(COD) L(-1) d(-1)). Three sludges, pre-treated in full-scale HPTH plants, were fermented. One was from a plant processing a mix of primary sludge and WAS and the other two from plants processing solely WAS. The HPTH plants solubilised suspended matter, evidenced by a 20-30% decrease in suspended solids and an increase of soluble COD : total COD from 0.04 to 0.4. Fermentation of the three sludges yielded similar VFA concentrations (15-20gVFA(COD) L(-1)). The yields were largely independent of retention time (1 d-6 d) and temperature (42°C, 55°C). Also, the product spectrum depended mostly on the composition of the sludge rather than on operating conditions.

Production of polyhydroxyalkanoates by glycogen accumulating organisms treating a paper mill wastewater

A process for production of polyhydroxyalkanoates (PHA) by activated sludge treating a paper mill wastewater was investigated. The applied strategy was to select for glycogen accumulating organisms (GAOs) by alternating anaerobic/aerobic conditions. Acidogenic fermentation was used as pretreatment to convert various organic compounds to volatile fatty acids which are preferable substrates for PHA production. Enrichment resulted in a culture dominated by GAOs related to Defluviicoccus vanus (56%) and Candidatus Competibacter phosphatis (22%). Optimization of PHA accumulation by the enriched GAO culture was performed through batch experiments. Accumulation of PHA under anaerobic conditions was limited by the intracellular glycogen stored. Under aerobic conditions significant glycogen production (to 25% of sludge dry weight) was observed alongside PHA accumulation (to 22% of sludge dry weight). By applying a subsequent anaerobic period after an initial aerobic, the produced glycogen could be utilized for further PHA accumulation and by this strategy PHA content was increased to 42% of sludge dry weight. The PHA yield over the entire process was 0.10 kg per kg of influent COD treated which is similar to what has been achieved with a process applying feast/famine enrichment strategy with the same wastewater.

Polyhydroxyalkanoate (PHA) production from sludge and municipal wastewater treatment.

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters with comparable properties to some petroleum-based polyolefins. PHA production can be achieved in open, mixed microbial cultures and thereby coupled to wastewater and solid residual treatment. In this context, waste organic matter is utilised as a carbon source in activated sludge biological treatment for biopolymer synthesis. Within the EU project Routes, the feasibility of PHA production has been evaluated in processes for sludge treatment and volatile fatty acid (VFA) production and municipal wastewater treatment. This PHA production process is being investigated in four units: (i) wastewater treatment with enrichment and production of a functional biomass sustaining PHA storage capacity, (ii) acidogenic fermentation of sludge for VFA production, (iii) PHA accumulation from VFA-rich streams, and (iv) PHA recovery and characterisation. Laboratory- and pilot-scale studies demonstrated the feasibility of municipal wastewater and solid waste treatment alongside production of PHA-rich biomass. The PHA storage capacity of biomass selected under feast-famine with municipal wastewater has been increased up to 34% (g PHA g VSS(-1)) in batch accumulations with acetate during 20 h. VFAs obtained from waste activated sludge fermentation were found to be a suitable feedstock for PHA production.

Integrated production of polyhydroxyalkanoates (PHAs) with municipal wastewater and sludge treatment at pilot scale

A pilot-scale process was operated over 22 months at the Brussels North Wastewater Treatment Plant (WWTP) in order to evaluate polyhydroxyalkanoate (PHA) production integration with services of municipal wastewater and sludge management. Activated sludge was produced with PHA accumulation potential (PAP) by applying feast-famine selection while treating the readily biodegradable COD from influent wastewater (average removals of 70% COD, 60% CODsol, 24% nitrogen, and 46% phosphorus). The biomass PAP was evaluated to be in excess of 0.4gPHA/gVSS. Batch fermentation of full-scale WWTP sludge at selected temperatures (35, 42 and 55 °C) produced centrate (6-9.4 gCODVFA/L) of consistent VFA composition, with optimal fermentation performance at 42 °C. Centrate was used to accumulate PHA up to 0.39 gPHA/gVSS. The centrate nutrients are a challenge to the accumulation process but producing a biomass with 0.5 gPHA/gVSS is considered to be realistically achievable within the typically available carbon flows at municipal waste management facilities.

Rapid quantification of intracellular PHA using infrared spectroscopy: An application in mixed cultures

Fourier transform infrared (FT-IR) spectroscopy is proposed for a method for rapid quantification of polyhydroxyalkanoates (PHA) in mixed culture bacterial systems. Spectra from 122 samples from a wide range of PHA production systems were studied. The spectra were collected in a library that was used to calibrate a partial least squares (PLS) model linking FT-IR spectra with PHA content in the biomass. The library of spectra contained samples with a range of total PHA content (0.03-0.58 w/w) as well as varying compositions (poly-(3-hydroxyvalerate) (3HV) content of 0-63% in Cmol basis). A robust PLS model was developed using calibration data from a diverse range of systems and PHA content. Coupling this model with FT-IR spectra has been shown to be applicable for predicting PHA content in mixed culture production systems. The method was used to reliably determine PHA content in biomass from a new, independent PHA production system with a standard error of prediction (RMSEP) value of 0.023 w/w, despite the complexity of the matrices. This method reduces the analytical time for PHA quantification down to under 30 min (5 min handling time was achieved when FT-IR equipment was immediately available), and eliminates hazardous waste by-products. The work has demonstrated a level of accuracy and reproducibility in quantifying PHA in mixed culture systems similar to that obtained from the GC analytical technique. Further work is required to enable the use of the method to analyze crystallinity related factors that may be useful towards quantifying poly-(3-hydroxybutyrate) and poly-(3-hydroxyvalerate) (3HB/3HV) composition. The method has been shown to be suitable for rapid quantification in large scale applications and in its present form is reliable for routine process monitoring.

Community Structure Evolution and Enrichment of Glycogen-Accumulating Organisms Producing Polyhydroxyalkanoates from Fermented Molasses

ABSTRACT An open mixed culture was enriched with glycogen-accumulating organisms (GAOs) by using a sequencing batch reactor and treating an agroindustrial waste (sugar cane molasses) under cyclic anaerobic-aerobic conditions. Over a 1-year operating period, the culture exhibited a very stable GAO phenotype with an average polyhydroxyalkanoate (PHA) content of 17% total suspended solids. However, the GAO microbial community evolved over the course of operation to a culture exhibiting unusual characteristics in producing PHAs comprised of short-chain-length monomers, namely, 3-hydroxybutyrate, 3-hydroxy-2-methylbutyrate, 3-hydroxyvalerate, and 3-hydroxy-2-methylvalerate, and also, up to 31 mol% of the medium-chain-length (MCL) monomer 3-hydroxyhexanoate (3HHx). Microbial community analysis by fluorescence in situ hybridization revealed a concurrent long-term drift in the GAO community balance, from mainly “Candidatus Competibacter phosphatis” to mainly Defluviicoccus vanus-related organisms. The production of 3HHx was confirmed by 13C nuclear magnetic resonance (NMR) and appeared to be related to the increased presence of D. vanus-related GAOs. These results suggest a broadened spectrum of material, chemical, and mechanical properties that can be achieved for biopolymers produced by open mixed cultures from fermented waste. The increased spectrum of polymer properties brings a wider scope of potential applications.

Inhibition by fatty acids during fermentation of pre-treated waste activated sludge

Fermentation of waste activated sludge produces volatile fatty acids (VFAs), which can be used as the carbon sources for numerous biological processes. However, product inhibition can limit extent of fermentation to VFAs. In this study, product inhibition during fermentation of waste activated sludge pre-treated by a thermal hydrolysis process (THP-WAS) was investigated. Product inhibition was confirmed as spiking reactors with high levels of a mix of VFAs prevented fermentation taking place. Various inhibition models were trialled and it was found that a threshold model (based on thermodynamics) provided the best fit between model and data. This is the first time that threshold type inhibition has been shown for a mixed substrate, mixed population system. Batch fermentations carried out with THP-WAS of different dilutions were used to evaluate the impact of different organic loadings. The threshold VFA concentration for the systems studied was determined to be 17±1gCOD(VFA)L(-1). Inhibition was shown to be due to the presence of a combination of VFAs containing 2-6 carbon atoms each. When evaluated individually, by spiking individual VFAs, all VFAs except for acetate had the same impact at this threshold; acetate being approximately 50% as inhibitory as the other organic acids (COD basis). Based on this, a weighted model could be proposed to better represent the data. Strategies to improve overall yield could be increased production of acetate, or dilution to below the inhibitory level.

Chlorinated-solvent-free gas chromatographic analysis of biomass containing polyhydroxyalkanoates.

An extraction and derivatization method was developed for more environmentally friendly routine quantification of polyhydroxyalkanoates (PHAs) in activated sludge biomass by gas chromatography (GC). This method can be further applied to assess relative changes in biomass carbohydrate levels relating to, for example, glycogen or extracellular polysaccharides. Further, co-extracted principal membrane fatty acids are indicative of relative changes in active biomass. The protocol is attractive for routine assessment because it does not require any chlorinated solvents, which are still almost exclusively used today for PHA analysis by GC. Acidic alcoholysis of dried microbial biomass using 3:1 butanol to concentrated (37%) hydrochloric acid at 100 degrees C for 8 h will hydrolyse and derivatize microbial storage products and membrane lipids. Esters of the hydroxyalkanoates, carbohydrates converted to levulinic acid, and long chain microbial fatty acids are reliably extracted into hexane for gas chromatographic analysis and quantification. Calibration can be achieved with benzoic, 2-hydroxyvaleric, or 2-hydroxycaproic acids as the method reference standards.

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.