Francesco Valentino

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.

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.

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.

Polyhydroxyalkanoates production with mixed microbial cultures: from culture selection to polymer recovery in a high-rate continuous process.

Polyhydroxyalkanoates (PHA) production with mixed microbial cultures (MMC) has been investigated by means of a sequential process involving three different stages, consisting of a lab-scale sequencing batch reactor for MMC selection, a PHA accumulation reactor and a polymer extraction reactor. All stages were performed under continuous operation for at least 4 months to check the overall process robustness as well as the related variability of polymer composition and properties. By operating both biological stages at high organic loads (8.5 and 29.1 gCOD/Ld, respectively) with a synthetic mixture of acetic and propionic acid, it was possible to continuously produce PHA at 1.43 g/Ld with stable performance (overall, the storage yield was 0.18 COD/COD). To identify the optimal operating conditions of the extraction reactor, two digestion solutions have been tested, NaOH (1m) and NaClO (5% active Cl2). The latter resulted in the best performance both in terms of yield of polymer recovery (around 100%, w/w) and purity (more than 90% of PHA content in the residual solids, on a weight basis). In spite of the stable operating conditions and performance, a large variation was observed for the HV content, ranging between 4 and 20 (%, w/w) for daily samples after accumulation and between 9 and 13 (%, w/w) for weekly average samples after extraction and lyophilization. The molecular weight of the produced polymer ranged between 3.4 × 10(5) and 5.4 × 10(5)g/mol with a large polydispersity index. By contrast, TGA and DSC analysis showed that the thermal polymer behavior did not substantially change over time, although it was strongly affected by the extraction agent used (NaClO or NaOH).

Feed frequency in a Sequencing Batch Reactor strongly affects the production of polyhydroxyalkanoates (PHAs) from volatile fatty acids

The production of polyhydroxyalkanoates (PHAs) by activated sludge selected in a sequencing batch reactor (SBR) has been investigated. Several SBR runs were performed at the same applied organic load rate (OLR), hydraulic retention time (HRT) and feed concentration (8.5 g COD L(-1) of volatile fatty acids, VFAs) under aerobic conditions. The effect of the feeding time was only evaluated with a cycle length of 8h; for this particular cycle length, an increase in the storage response was observed by increasing the rate at which the substrate was fed into the reactor (at a fixed feeding frequency). Furthermore, a significantly stronger effect was observed by decreasing the cycle length from 8h to 6h and then to 2h, changing the feed frequency or changing the organic load given per cycle (all of the other conditions remained the same): the length of the feast phase decreased from 26 to 20.0 and then to 19.7% of the overall cycle length, respectively, due to an increase in the substrate removal rate. This removal rate was high and similar for the runs with cycle lengths of 2h and 6h in the SBR. This result was due to an increase in the selective pressure and the specific storage properties of the selected biomass. The highest polymer productivity after long-term accumulation batch tests was 1.7 g PHA L(-1)d(-1), with PHA content in the biomass of approximately 50% on a COD basis under nitrogen limitation. The DGGE profiles showed that the good storage performance correlated to the development of Lampropedia hyalina, which was only observed in the SBR runs characterized by a shorter cycle length.

Effect of hydraulic and organic loads in sequencing batch reactor on microbial ecology of activated sludge and storage of polyhydroxyalkanoates

Reactor on microbial ecology of activated sludge and storage of polyhydroxyalkanoates Marianna Villano, Silvia Lampis, Francesco Valentino, Giovanni Vallini, Mauro Majone, Mario Beccari Dipartimento di Chimica, Sapienza Universita di Roma, P.le Aldo Moro 5, 00185 Roma, Italia Dipartimento di Biotecnologie, Universita di Verona, Strada Le Grazie 15, 37134 Verona, Italia corresponding author: mario.beccari@uniroma1.it

Fate of β-hexachlorocyclohexane in the mixed microbial cultures (MMCs) three-stage polyhydroxyalkanoates (PHA) production process from cheese whey

This work aimed to study the fate and effect of β-hexachlorocyclohexane (β-HCH) during several steps of PHA production and purification, by using an artificially contaminated cheese whey (CW) as the feedstock. Most of β-HCH (around 90%) was adsorbed on CW solids and it was removed after the acidogenic fermentation step, when residual CW solids are separated along with anaerobic biomass from the liquid-phase. Purification steps also contributed strongly to the removal of residual β-HCH; overall, the PHA production process removed about 99.9% of initial β-HCH content. Moreover, it has been shown that β-HCH has neither detrimental effect on acidogenic fermentation nor on PHA accumulation, that were performed by using unacclimated mixed microbial cultures.

Impact of nitrogen feeding regulation on polyhydroxyalkanoates production by mixed microbial cultures.

A sequencing batch reactor (SBR) is typically used for selecting mixed microbial cultures (MMC) for polyhydroxyalkanoate (PHA) production. Since many waste streams suitable as process feedstock for PHA production are nitrogen-deficient, a nutrient supply in the SBR is typically required to allow for efficient microbial growth. The scope of this study was to devise a nitrogen feeding strategy which allows controlling the nitrogen levels during the feast and famine regime of a lab-scale SBR, thereby selecting for PHA-storing microorganisms. At the beginning of the cycle the reactor was fed with a synthetic mixture of acetic and propionic acids at an overall organic load rate of 8.5gCODL-1d-1 (i.e. 260CmmolL-1d-1), whereas nitrogen (in the form of ammonium sulphate) was added either simultaneously to the carbon feed (coupled feeding strategy) or after the end of the feast phase (uncoupled feeding strategy). As a main result, PHA production was more than doubled (up to about 1300±64mgCODL-1) when carbon and nitrogen were separately fed and the higher PHA production also corresponded to an 82% increase in the polymer HV content (up to 20±1%, wtwt-1). Three SBR runs were performed with the uncoupled carbon and nitrogen feeding at different carbon to nitrogen (C/N) ratios (of 14.3, 17.9, and 22.3CmolNmol-1, respectively) which were varied by progressively reducing the concentration of the nitrogen feeding. In spite of a comparable PHA storage yield at 14.3 and 17.9CmolNmol-1 (0.41±0.05 gCODPHA gCODVFA-1 and 0.38±0.05 gCODPHA gCODVFA-1, respectively), the storage response of the selected MMC significantly decreased when the C/N ratio was set at the highest investigated value. Notably, an increase in this parameter also resulted in a change in the HV content in the polymer regardless the composition of the organic acids solution.

Extraction of polycyclic aromatic hydrocarbons from polyhydroxyalkanoates before gas chromatography/mass spectrometry analysis

Among the organic contaminants that could pass from waste to polyhydroxyalkanoates (PHAs), there are the polycyclic aromatic hydrocarbons (PAHs). For this reason, we have developed a rapid analytical method for the determination of sixteen PAHs in PHAs. PAHs were extracted by n-hexane, after matrix dispersion and crumbling into sand; the extract was purified by solid phase extraction using florisil as adsorbent. Recoveries in the range of 89-101% were obtained for the deuterated analytes, except for the two with the lowest molecular weight. Trueness between 92% and 108% and within-laboratory precision (expressed as relative standard deviation) ≤ 18% were estimated for all the analytes. Gas chromatography/mass spectrometry was used for analyte determination. Method limits of quantification were suitable to assure that PAH presence in PHA biolpolymers is much below the limits set by European law for plastic materials. Indeed, analysis of two different PHA samples showed that contamination is limited to few compounds at non-concerning levels.

Effect of culture residence time on substrate uptake and storage by a pure culture of Thiothrix (CT3 strain) under continuous or batch feeding

A pure culture of the filamentous bacterium Thiothrix, strain CT3, was aerobically cultured in a chemostat under continuous acetate feeding at three different culture residence times (RT 6, 12 or 22 d) and the same volumetric organic load rate (OLR 0.12gCOD/L/d). Cells cultured at decreasing RT in the chemostat had an increasing transient response to acetate spikes in batch tests. The maximum specific acetate removal rate increased from 25 to 185mgCOD/gCOD/h, corresponding to a 1.8 to 8.1 fold higher respective steady-state rate in the chemostat. The transient response was mainly due to acetate storage in the form of poly(3-hydroxybutyrate) (PHB), whereas no growth response was observed at any RT. Interestingly, even though the storage rate also decreased as the RT increased, the storage yield increased from 0.41 to 0.50 COD/COD. This finding does not support the traditional view that storage plays a more important role as the transient response increases. The transient response of the steady-state cells was much lower than in cells cultured under periodic feeding (at 6 d RT, from 82 to 247mgCOD/gCOD/h), with the latter cells showing both storage and growth responses. On the other hand, even though steady-state cells had no growth response and their storage rate was also less, steady-state cells showed a higher storage yield than cells cultured under dynamic feeding. This suggests that in Thiothrix strain CT3, the growth response is triggered by periodic feeding, whereas the storage response is a constitutive mechanism, independent from previous acclimation to transient conditions.