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Polyhydroxyalkanoates (PHA) are a sustainable alternative to conventional plastics that can be obtained from industrial wastes/by-products using mixed microbial cultures (MMC). MMC PHA production is commonly carried out in a 3-stage process of acidogenesis, PHA culture selection and accumulation. This research focused on the possibility of tailoring PHA by controlling the acidogenic reactor operating conditions, namely pH, using cheese whey as model feedstock. The objective was to investigate the impact that dynamically varying the acidogenic pH, when targeting different PHA monomer profiles, had on the performance and microbial community profile of the anaerobic reactor. To accomplish this, an anaerobic reactor was continuously operated under dynamic pH changes, ranging from pH 4 to 7, turning to pH 6 after each change of pH. At pH 6, lactate and acetate were the dominant products (41-48% gCOD basis and 22-44% gCOD basis, respectively). At low pH, lactate production was higher while at high pH acetate production was favoured. Despite the dynamic change of pH, the fermentation product composition at pH 6 was always similar, showing the resilience of the process, i.e. when the same pH value was imposed, the culture produced the same metabolic products independently of the history of changes occurring in the system. The different fermentation product fractions led to PHAs of different compositions. The microbial community, analysed by high throughput sequencing of bacterial 16S rRNA gene fragments, was dominated by Lactobacillus, but varied markedly when subjected to the highest and lowest pH values of the tested range (4 and 7), with increase in the abundance of Lactococcus and a member of the Candidate Division TM7. Different bacterial profiles obtained at pH 6 during this dynamic operation were able to produce a consistent profile of fermentation products (and consequently a constant PHA composition), demonstrating the community's functional redundancy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The yeast Komagataella pastoris was cultivated under different fed-batch strategies for the production of chitin-glucan complex (CGC), a co-polymer of chitin and β-glucan. The tested fed-batch strategies included DO-stat mode, predefined feeding profile and repeated fed-batch operation. Although high cell dry mass and high CGC production were obtained under the tested DO-stat strategy in a 94h cultivation (159 and 29g/L, respectively), the overall biomass and CGC productivities were low (41 and 7.4g/Lday, respectively). Cultivation with a predefined profile significantly improved both biomass and CGC volumetric productivity (87 and 10.8g/Lday, respectively). Hence, this strategy was used to implement a repeated fed-batch process comprising 7 consecutive cycles. A daily production of 119-126g/L of biomass with a CGC content of 11-16wt% was obtained, thus proving this cultivation strategy is adequate to reach a high CGC productivity that ranged between 11 and 18g/Lday. The process was stable and reproducible in terms of CGC productivity and polymer composition, making it a promising strategy for further process development., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Production of polyhydroxyalkanoates (PHAs) by open mixed microbial cultures (MMCs) has been attracting increasing interest as an alternative technology to PHA production by pure cultures, due to the potential for lower costs associated with the use of open systems (eliminating the requirement for sterile conditions) and the utilisation of cheap feedstock (industrial and agricultural wastes). Such technology relies on the efficient selection of an MMC enriched in PHA-accumulating organisms. Fermented cheese whey, a protein-rich complex feedstock, has been used previously to produce PHA using the feast and famine regime for selection of PHA accumulating cultures. While this selection strategy was found efficient when operated at relatively low organic loading rate (OLR, 2g-CODL -1 d -1 ), great instability and low selection efficiency of PHA accumulating organisms were observed when higher OLR (ca. 6g-CODL -1 d -1 ) was applied. High organic loading is desirable as a means to enhance PHA productivity. In the present study, a new selection strategy was tested with the aim of improving selection for high OLR. It was based on uncoupling carbon and nitrogen supply and was implemented and compared with the conventional feast and famine strategy. For this, two selection reactors were fed with fermented cheese whey applying an OLR of ca. 8.5g-CODL -1 (with 3.8g-CODL -1 resulting from organic acids and ethanol), and operated in parallel under similar conditions, except for the timing of nitrogen supplementation. Whereas in the conventional strategy nitrogen and carbon substrates were added simultaneously at the beginning of the cycle, in the uncoupled substrates strategy, nitrogen addition was delayed to the end of the feast phase (i.e. after exogenous carbon was exhausted). The two different strategies selected different PHA-storing microbial communities, dominated by Corynebacterium and a Xantomonadaceae, respectively with the conventional and the new approaches. The new strategy originated a more efficient PHA-production process than the conventional one (global PHA productivity of 6.09g-PHAL -1 d -1 and storage yield of 0.96 versus 2.55g-PHAL -1 d -1 and 0.86, respectively). Dissociation between the feast to famine length ratio (F/F) and storage efficiency was shown to be possible with the new strategy, allowing selection of an efficient PHA-storing culture with complex feedstock under high organic loading rates., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Exopolysaccharides (EPS) are polymers excreted by some microorganisms with interesting properties and used in many industrial applications. A new Pseudoalteromonas sp. strain, MD12-642, was isolated from marine sediments and cultivated in bioreactor in saline culture medium containing glucose as carbon source. Its ability to produce EPS under saline conditions was demonstrated reaching an EPS production of 4.4g/L within 17hours of cultivation, corresponding to a volumetric productivity of 0.25g/Lh, the highest value so far obtained for Pseudoalteromonas sp. strains. The compositional analysis of the EPS revealed the presence of galacturonic acid (41-42mol%), glucuronic acid (25-26mol%), rhamnose (16-22mol%) and glucosamine (12-16mol%) sugar residues. The polymer presents a high molecular weight (above 1000kDa). These results encourage the biotechnological exploitation of strain MD12-642 for the production of valuable EPS with unique composition, using saline by-products/wastes as feedstocks., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Olive oil distillate (OOD), biodiesel fatty acids-byproduct (FAB) and used cooking oil (UCO) were tested as inexpensive carbon sources for the production of polyhydroxyalkanoates (PHA) with different composition using twelve bacterial strains. OOD and FAB were exploited for the first time as alternative substrates for PHA production. UCO, OOD and FAB were used by Cupriavidus necator and Pseudomonas oleovorans to synthesize the homopolymer poly-3-hydroxybutyrate, while Pseudomonas resinovorans and Pseudomonas citronellolis produced mcl-PHA polymers mainly composed of hydroxyoctanoate and hydroxydecanoate monomers. The highest polymer content in the biomass was obtained for C. necator (62 wt.%) cultivated on OOD. Relatively high mcl-PHA content (28-31 wt.%) was reached by P. resinovorans cultivated in OOD. This study shows, for the first time, that OOD is a promising substrate for PHA production since it gives high polymer yields and allows for the synthesis of different polymers (scl- or mcl-PHA) by selection of the adequate strains., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Haloferax mediterranei was cultivated in highly saline medium using cheese whey as the substrate for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV). Acid hydrolysis provided a simple inexpensive method to obtain a cheese whey hydrolysate that was used for cultivation of H. mediterranei. Batch bioreactor cultivation of H. mediterranei resulted in the production of an active biomass concentration of 7.54 g L(-1) with a polymer content of 53%, and a volumetric productivity of 4.04 g L(-1) day(-1). Supplementation of the cultivation medium with micronutrients favored galactose consumption that was used for polymer synthesis after exhaustion of the available glucose. P(3HB-co-3HV) with a 3-hydroxyvalerate content of 1.5 mol% was extracted from the biomass by hypo-osmotic shock. The polymer presented a molecular mass of 4.4×10(5), with a polydispersity index of 1.5. This work demonstrated the feasibility of using cheese whey for the production of a value-added biopolymer with high volumetric productivity, by using a glucose- and galactose-rich substrate obtained by acid hydrolysis of cheese whey. The use of H. mediterranei as the producing strain avoids the need for strict sterility due to the culture's high salinity requirements and, also, allows for polymer extraction by simply contacting the biomass with water., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Chitin-glucan complex (CGC) is a valuable biomaterial that can be extracted from the cell wall of several yeast and fungi. In this work, the yeast Komagataella (Pichia) pastoris was grown on glycerol as the sole carbon source in batch cultivation experiments to evaluate the effect of pH (3.5-6.5) and temperature (20-40°C) on CGC production and polymer composition. The CGC content in the biomass and the volumetric productivity (rp) were not significantly affected within the tested pH and temperature ranges. Nevertheless, both parameters could be maximized (CGC ≥14wt% and rp ≥ 3.0 gCGC L(-1)day(-1)) for temperatures within 27-34°C and pH above 6.0 or below 4.0. In contrast, the effect of pH and temperature on the polymer's chitin:β-glucan molar ratio was more pronounced. The highest chitin:β-glucan molar ratio (>14:86) was obtained for the mid-range pH (4.5-5.8) and temperatures (26-33°C), while a drastic reduction of chitin to ≤ 6%mol was observed outside those ranges. Therefore, a compromise between maximal CGC production and the synthesis of polymers enriched in chitin must be achieved, depending on the final application of this product., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Polyhydroxyalkanoates (PHAs) are polyesters that can be produced from industrial wastewater or surplus products by mixed microbial cultures (MMC). To optimise PHA production by MMCs, the link between the microbial structure and function of these enrichments must be better established. This study investigates, for the first time, the impact of operational changes on the microbial community and the associated process performance of PHA producing MMCs. It was found that a PHA producing community fed with fermented molasses was dominated by a combination of Azoarcus, Thauera and Paracoccus, where the former two groups were present in highest abundance. Dominance of either Thauera or Azoarcus seemed to be determined by the organic loading rate imposed in the selection reactor. While higher Azoarcus enrichments led to higher PHA production yields and lower biomass growth yields as compared to Thauera, the Thauera abundance was strongly linked to higher hydroxyvalerate (HV) fractions. Paracoccus abundance was correlated with a lower PHA production capacity as compared to Azoarcus, and produced lower HV fractions than Thauera and Azoarcus. The findings of this study suggest that MMCs targeting the enrichment of Azoarcus as the primary biomass fraction with Thauera as a minor fraction lead to optimal specific PHA production and polymers with high HV content, which is likely to improve their mechanical properties., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In this work, we present a dynamic metabolic model that describes the uptake of complex mixtures of volatile fatty acids (VFA) and respective conversion into PHA by mixed microbial cultures (MMC). This model builds upon a previously published flux balance analysis model [1] that identified the minimization of TCA cycle activity as the key metabolic objective to predict PHA storage fluxes and respective composition. The model was calibrated either with experimental data of PHA production from fermented sugar cane molasses or from synthetic mixtures of VFA. All PHA production experiments were performed using a MMC selected with fermented sugar cane molasses under feast and famine regimen. The model was able to capture the process dynamics denoted by an excellent fit between experimental and computed time profiles of concentrations with the regression coefficients always above 0.92. The introduced VFA uptake regulatory factor reflects the decrease of acetyl-CoA and propionyl-CoA available to TCA cycle in conformity with the hypothesis that the minimization of TCA cycle is a key metabolic objective for MMC subjected to feast and famine regimen for the maximization of PHA production., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Polyhydroxyalkanoates (PHA) can be produced by mixed microbial cultures (MMC) using a three-stage process. An attractive feature of MMC for PHA production is the ability to use waste/surplus feedstocks. In this study, the effect of a feedstock shift, mimicking a seasonal feedstock scenario and/or as a strategy for controlling polymer composition, on a MMC PHA production process was assessed using cheese whey (CW) and sugar cane molasses (SCM) as model feedstocks. The acidogenic stage responded immediately to the feedstock shift by changing the fermented products profile, with acetate and butyrate being the main acids produced from CW, while for SCM propionate and valerate were the dominant products. The fermentation process was then quite stable during long term operation. The PHA culture selection stage also responded quickly to the fermented feestocks shift, generating a polymer whose composition was linearly dependent on the concentration of HV and HB precursors produced in the acidogenic stage. The selected culture reached a maximum PHA content of 56% and 65% with fermented SCM and CW, respectively. Mixing fermented CW and SCM, in equal volume proportions, demonstrated the possibility of using different fermented feedstocks for tailoring polymer composition., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Glycerol, a highly abundant byproduct of the biodiesel industry, constitutes today a cheap feedstock for biobased succinic acid (SA) production. Actinobacillus succinogenes is one of the best SA producers. However, glycerol consumption by this biocatalyst is limited because of a redox imbalance during cell growth. The use of an external electron acceptor may improve the metabolism of SA synthesis by A. succinogenes in glycerol. In this study, the effect of dimethylsulfoxide (DMSO), an electron acceptor, on glycerol consumption and SA production by A. succinogenes under controlled fermentation conditions was investigated. Concentrations of DMSO between 1 and 4% (v/v) greatly promoted glycerol consumption and SA production by A. succinogenes. During fed-batch cultivation, SA concentration reached 49.62 g/L, with a product yield of 0.87 gSA/gGLR and a maximum production rate of 2.31 gSA/Lh, the highest values so far reported in the literature for A. succinogenes using glycerol as carbon source. These results show that using DMSO as external electron acceptor significantly promotes glycerol consumption and succinic acid production by A. succinogenes and may be used as a co-substrate, opening new perspectives for the use of glycerol by this biocatalyst., (Copyright © 2013 Elsevier B.V. All rights reserved.)