Functionalization of Polyhydroxyalkanoates (PHA)-Based Bioplastic with Phloretin for Active Food Packaging: Characterization of Its Mechanical, Antioxidant, and Antimicrobial Activities.

The formulation of eco-friendly biodegradable packaging has received great attention during the last decades as an alternative to traditional widespread petroleum-based food packaging. With this aim, we designed and tested the properties of polyhydroxyalkanoates (PHA)-based bioplastics functionalized with phloretin as far as antioxidant, antimicrobial, and morpho-mechanic features are concerned. Mechanical and hydrophilicity features investigations revealed a mild influence of phloretin on the novel materials as a function of the concentration utilized (5, 7.5, 10, and 20 mg) with variation in FTIR e RAMAN spectra as well as in mechanical properties. Functionalization of PHA-based polymers resulted in the acquisition of the antioxidant activity (in a dose-dependent manner) tested by DPPH, TEAC, FRAR, and chelating assays, and in a decrease in the growth of food-borne pathogens (Listeria monocytogenes ATCC 13932). Finally, apple samples were packed in the functionalized PHA films for 24, 48, and 72 h, observing remarkable effects on the stabilization of apple samples. The results open the possibility to utilize phloretin as a functionalizing agent for bioplastic formulation, especially in relation to food packaging.

An Alternative Exploitation of Synechocystis sp. PCC6803: A Cascade Approach for the Recovery of High Added-Value Products.

Microalgal biomass represents a very interesting biological feedstock to be converted into several high-value products in a biorefinery approach. In this study, the cyanobacterium Synechocystis sp. PCC6803 was used to obtain different classes of molecules: proteins, carotenoids and lipids by using a cascade approach. In particular, the protein extract showed a selective cytotoxicity towards cancer cells, whereas carotenoids were found to be active as antioxidants both in vitro and on a cell-based model. Finally, for the first time, lipids were recovered from Synechocystis biomass as the last class of molecules and were successfully used as an alternative substrate for the production of polyhydroxyalkanoate (PHA) by the native PHA producer Pseudomonas resinovorans. Taken together, our results lead to a significant increase in the valorization of Synechocystis sp. PCC6803 biomass, thus allowing a possible offsetting of the process costs.

Upgrading cardoon biomass into Polyhydroxybutyrate based blends: A holistic approach for the synthesis of biopolymers and additives.

Cardoon, Cynara cardunculus L. represents a biorefinery crop with a great potential in the bioplastic field. This work investigates the valorization of different cardoon components into high added value products, finally recombined into novel upgraded bioplastics. Bioprocesses for Polyhydroxybutyrate (PHB) and medium-chain-length Polyhydroxyalkanoates (mcl-PHA) production were set up starting from root inulin and seed oil respectively, highlighting the effect of process conditions on polymer properties. The ternary blend, in which the PHB polymer matrix was added with mcl-PHA and epoxidized cardoon oil, evidenced a synergic effect of both additives in modulating PHB structural and thermal properties, promoted by the physical interaction occurring among the components. This proof-of concept frames the paper in the holistic approach of circular economy applied to bioplastic production.

Polyhydroxyalkanoates-Based Nanoparticles as Essential Oil Carriers.

Plant-derived essential oils (EOs) represent a green alternative to conventional antimicrobial agents in food preservation. Due to their volatility and instability, their application is dependent on the development of efficient encapsulation strategies allowing their protection and release control. Encapsulation in Polyhydroxyalkanoate (PHA)-based nanoparticles (NPs) addresses this challenge, providing a biodegradable and biobased material whose delivery properties can be tuned by varying polymer composition. In this work, EO from Mexican oregano was efficiently encapsulated in Polyhydroxybutyrate (PHB) and Poly-3-hydroxybutyrate-co-hydroxyhexanoate (PHB-HHx)-based NPs by solvent evaporation technique achieving high encapsulation efficiency, (>60%) and loading capacity, (about 50%). The obtained NPs displayed a regular distribution with a size range of 150-210 nm. In vitro release studies in food simulant media were fitted with the Korsmeyer-Peppas model, indicating diffusion as the main factor controlling the release. The cumulative release was affected by the polymer composition, possibly related to the more amorphous nature of the copolymer, as confirmed by WAXS and DSC analyses. Both the EO-loaded nanosystems displayed antimicrobial activity against Micrococcus luteus, with PHB-HHx-based NPs being even more effective than the pure EO. The results open the way to the effective exploitation of the developed nanosystems in active packaging.

The power of two: An artificial microbial consortium for the conversion of inulin into Polyhydroxyalkanoates.

One of the major issues for the microbial production of polyhydroxyalkanoates (PHA) is to secure renewable, non-food biomass feedstocks to feed the fermentation process. Inulin, a polydisperse fructan that accumulates as reserve polysaccharide in the roots of several low-requirement crops, has the potential to face this challenge. In this work, a "substrate facilitator" microbial consortium was designed to address PHA production using inulin as feedstock. A microbial collection of Bacillus species was screened for efficient inulinase producer and the genome of the selected strain, RHF15, identified as Bacillus gibsonii, was analysed unravelling its wide catabolic potential. RHF15 was co-cultured with Cupriavidus necator, an established PHA producer, lacking the ability to metabolize inulin. A Central Composite Rotary Design (CCRD) was applied to optimise PHA synthesis from inulin by the designed artificial microbial consortium, assessing the impact of species inoculum ratio and inulin and N-source concentrations. In the optimized conditions, a maximum of 1.9 g L-1 of Polyhydroxybutyrate (PHB), corresponding to ~80% (gpolymer/gCDW) polymer content was achieved. The investigated approach represents an effective process optimization method, potentially applicable to the production of PHA from other complex C- sources.

Optimization of Inulin Hydrolysis by Penicillium lanosocoeruleum Inulinases and Efficient Conversion Into Polyhydroxyalkanoates.

Inulin, a polydisperse fructan found as a common storage polysaccharide in the roots of several plants, represents a renewable non-food biomass resource for the synthesis of bio-based products. Exploitation of inulin-containing feedstocks requires the integration of different processes, including inulinase production, saccharification of inulin, and microbial fermentation for the conversion of released sugars into added-value products. In this work paper, a new microbial source of inulinase, Penicillium lanosocoeruleum, was identified through the screening of a fungal library. Inulinase production using inulin as C-source was optimized, reaching up to 28 U mL-1 at the 4th day of growth. The fungal inulinase mixture (PlaI) was characterized for pH and temperature stability and activity profile, and its isoenzymes composition was investigated by proteomic strategies. Statistical optimization of inulin hydrolysis was performed using a central composite rotatable design (CCRD), by analyzing the effect of four factors. In the optimized conditions (T, 45.5°C; pH, 5.1; substrate concentration, 60 g L-1; enzyme loading, 50 U gsubstrate -1), up to 96% inulin is converted in fructose within 20 h. The integration of PlaI in a process for polyhydroxyalkanoate (PHA) production by Cupriavidus necator from inulin was tested in both separated hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). A maximum of 3.2 g L-1 of PHB accumulation, corresponding to 82% polymer content, was achieved in the SSF. The proved efficiency in inulin hydrolysis and its effective integration into a SSF process pave the way to a profitable exploitation of the PlaI enzymatic mixture in inulin-based biorefineries.

In vivo and Post-synthesis Strategies to Enhance the Properties of PHB-Based Materials: A Review.

The transition toward "green" alternatives to petroleum-based plastics is driven by the need for "drop-in" replacement materials able to combine characteristics of existing plastics with biodegradability and renewability features. Promising alternatives are the polyhydroxyalkanoates (PHAs), microbial biodegradable polyesters produced by a wide range of microorganisms as carbon, energy, and redox storage material, displaying properties very close to fossil-fuel-derived polyolefins. Among PHAs, polyhydroxybutyrate (PHB) is by far the most well-studied polymer. PHB is a thermoplastic polyester, with very narrow processability window, due to very low resistance to thermal degradation. Since the melting temperature of PHB is around 170-180°C, the processing temperature should be at least 180-190°C. The thermal degradation of PHB at these temperatures proceeds very quickly, causing a rapid decrease in its molecular weight. Moreover, due to its high crystallinity, PHB is stiff and brittle resulting in very poor mechanical properties with low extension at break, which limits its range of application. A further limit to the effective exploitation of these polymers is related to their production costs, which is mostly affected by the costs of the starting feedstocks. Since the first identification of PHB, researchers have faced these issues, and several strategies to improve the processability and reduce brittleness of this polymer have been developed. These approaches range from the in vivo synthesis of PHA copolymers, to the enhancement of post-synthesis PHB-based material performances, thus the addition of additives and plasticizers, acting on the crystallization process as well as on polymer glass transition temperature. In addition, reactive polymer blending with other bio-based polymers represents a versatile approach to modulate polymer properties while preserving its biodegradability. This review examines the state of the art of PHA processing, shedding light on the green and cost-effective tailored strategies aimed at modulating and optimizing polymer performances. Pioneering examples in this field will be examined, and prospects and challenges for their exploitation will be presented. Furthermore, since the establishment of a PHA-based industry passes through the designing of cost-competitive production processes, this review will inspect reported examples assessing this economic aspect, examining the most recent progresses toward process sustainability.

Conversion of no/low value waste frying oils into biodiesel and polyhydroxyalkanoates.

A sustainable bioprocess was developed for the valorization of a no/low value substrate, i.e. waste frying oils (WFOs) with high content of free fatty acids (FFAs), otherwise unsuitable for biodiesel production. The bioprocess was verified using both recombinant (Escherichia coli) and native (Pseudomonas resinovorans) polyhydroxyalkanoates (PHAs) producing cell factories. Microbial fermentation of WFOs provided a 2-fold advantage: i) the reduction of FFAs content resulting into an upgrading of the "exhausted waste oils" and ii) the production of a bio-based microbial polymer. Proper strain designing and process optimization allowed to achieve up to 1.5 g L-1 of medium chain length, mcl-PHAs, together with an efficient conversion (80% yield) of the treated WFO into biodiesel.