The role of biotechnology in the transition from plastics to bioplastics: an opportunity to reconnect global growth with sustainability.

Building new value chains, through the valorization of biomass components for the development of innovative bio-based products (BBPs) aimed at specific market sectors, will accelerate the transition from traditional production technologies to the concept of biorefineries. Recent studies aimed at mapping the most relevant innovations undergoing in the field of BBPs (Fabbri et al. 2019, Final Report of the Task 3 BIOSPRI Tender Study on Support to R&I Policy in the Area of Bio-based Products and Services, delivered to the European Commission (DG RTD)), clearly showed the dominant position played by the plastics sector, in which new materials and innovative technical solutions based on renewable resources, concretely contribute to the achievement of relevant global sustainability goals. New sustainable solutions for the plastic sector, either bio-based or bio-based and biodegradable, have been intensely investigated in recent years. The global bioplastics and biopolymers market size is expected to grow from USD 10.5 billion in 2020 to USD 27.9 billion by 2025 (Markets and Markets, 2020, Bioplastics & Biopolymers Market by Type (Non-Biodegradable/Bio-Based, Biodegradable), End-Use Industry (Packaging, Consumer Goods, Automotive & Transportation, Textiles, Agriculture & Horticulture), Region - Global Forecast to 2025), and this high growth is driven primarily by the growth of the global packaging end-use industry. Such relevant opportunities are the outcomes of intensive scientific and technological research devoted to the development of new materials with selected technical features, which can represent feasible substitutes for the fossil-based plastic materials currently used in the packaging sectors and other main fields. This article offers a map of the latest developments connected to the plastic sector, achieved through the application of biotechnological routes for the preparation of completely new polymeric structures, or drop-in substitutes derived from renewable resources, and it describes the specific role played by biotechnology in promoting and making this transition faster.

Conversion of waste cooking oil into biogas: perspectives and limits.

Each year, large quantities of waste cooking oils are produced worldwide which are currently reused mainly for biodiesel production. Since lipids have a very high potential for biomethanation, the production of biogas is a possible alternative for the recycling of edible used oils. The digestion of fats is hindered mainly by their hydrophobicity, which implies a biphasic system with problems of floating and foaming of the oily materials, and by the accumulation of long-chain fatty acids, which are toxic to microbial consortia. The objectives of this review were to highlight the recycling potential of waste cooking oil to biogas production and to facilitate the application of the technology by identifying solutions to overcome biological and engineering limits to its diffusion. Particular attention was paid to the microbial populations involved, to the process factors whose control is important to improve the digestion of fats such as lipid concentration, pH, temperature, and agitation, and to technological solutions whose application also aims to improve digestion, such as pretreatment of raw materials and co-digestion of fats with other feedstocks. The state of the art in reactor designs suitable for lipid digestion was also examined.

Containment of a genetically modified microorganism by an activated sludge system.

The effectiveness of physical, chemical and biological barriers to the diffusion of genetically modified microorganisms (GMMs) to prevent their release into the environment is currently under scrutiny worldwide because of the associated potential ecological impacts. An industrial discharge of a non-sterilized fermentation broth containing GMM biomass into a conventional municipal wastewater treatment plant would deliver the GMMs into the activated sludge system process (ASSP). The present work aimed to model and evaluate the containment capability of a small ASSP (part of a 20,000 people equivalent municipal plant) in the event of receiving GMM biomass from a medium-small biotechnological plant dedicated to the production of polyhydroxyalkanoates (3000 t/year of biopolymer). An actual GMM (Pseudomonas putida KTOY06) was injected into a bench-scale ASSP (ASSPLab) in a quantity proportional to the relative dimensions of the plants mentioned. The experimental and model results indicated that the ASSP of the target municipal treatment plant would not be capable of holding back such a sudden input of GMM; 6 h after the discharge, 11-15 % of injected GMM cells were released through the clarified stream of the ASSPLab, with the rest being gradually released over time. Since the GMM employed did not exhibit any growth in the ASSPLab, its concentration in the clarified water stream would not represent a substantial risk of release into the environment if appropriate tertiary treatments were integrated. This study confirmed the necessity of a thorough risk assessment of biotechnological processes prior to their implementation.

Hydraulic retention time effects on wastewater nutrient removal and bioproduct production via rotating algal biofilm reactor.

Rotating algal biofilm reactor (RABR) technology was successfully employed in an effective strategy to couple the removal of wastewater nutrients with accumulation of valuable bioproducts by grown algae. A secondary stage municipal wastewater was fed to the developed system and the effects of the hydraulic retention time (HRT) parameter on both nutrient removal and bioproduct production were evaluated under fed-batch operation mode. Two sets of bench scale RABRs were designed and operated with HRTs of 2 and 6days in order to provide competitive environment for algal growth. The HRT significantly affected nitrogen and phosphorus uptakes along with lipid and starch accumulations by microalgae in harvested biofilms. Domination of nitrogen removal in 2-day HRT with higher lipid accumulation (20% on dried weight basis) and phosphorus removal in 6-day HRT with higher starch production (27% on dried weight basis) was observed by comparing the performances of the RABRs in duplicate runs.

High impact biowastes from South European agro-industries as feedstock for second-generation biorefineries.

Availability of bio-based chemicals, materials and energy at reasonable cost will be one of the forthcoming issues for the EU economy. In particular, the development of technologies making use of alternative resources to fossil fuels is encouraged by the current European research and innovation strategy to face the societal challenge of natural resource scarcity, fossil resource dependence and sustainable economic growth. In this respect, second- generation biorefineries, i.e. biorefineries fed with biowastes, appear to be good candidates to substitute and replace the present downstream processing scheme. Contrary to first-generation biorefineries, which make use of dedicated crops or primary cultivations to achieve such a goal, the former employ agricultural, industrial, zootechnical, fishery and forestry biowastes as the main feedstock. This leaves aside any ethical and social issue generated by first-generation approaches, and concomitantly prevents environmental and economical issues associated with the disposal of the aforementioned leftovers. Unfortunately, to date, a comprehensive and updated mapping of the availability and potential use of bioresources for second-generation biorefineries in Europe is missing. This is a lack that severely limits R&D and industrial applications in the sector. On the other hand, attempts at valorizing the most diverse biowastes dates back to the nineteenth century and plenty of information in the literature on their sustainable exploitation is available. However, the large majority of these investigations have been focused on single fractions of biowastes or single steps of biowaste processing, preventing considerations on an integrated and modular (cascade) approach for the whole valorization of organic leftovers. This review aims at addressing these issues by gathering recent data on (a) some of the main high-impact biowastes located in Europe and in particular in its Southern part, and (b) the bio-based chemicals, materials and fuels that can be produced from such residues. In particular, we focused on those key compounds referred to as "chemical platforms", which have been indicated as fundamental to generate the large majority of the industrially relevant goods to date.

Uncoupled hydrogen and volatile fatty acids generation in a two-step biotechnological anaerobic process fed with actual site wastewater.

Among agro-wastes, olive mill wastewater (OMW) truly qualifies as a high impact organic residue due to its biochemical-rich composition and high annual production. In the present investigation, dephenolized OMW (OMWdeph) was employed as the feedstock for a biotechnological two-stage anaerobic process dedicated to the production of biohydrogen and volatile fatty acids (VFAs), respectively. To this end, two identically configured packed-bed biofilm reactors were operated sequentially. In the first, the hydraulic retention time was set to 1 day, whereas in the second it was equal to 5 days. The rationale was to decouple the hydrolysis of the organic macronutrients held by the OMWdeph, so as to quantitatively generate a biogas enriched in H2 (first stage aim), for the acidogenesis of the residual components left after hydrolysis, to then produce a highly concentrated mixture of VFAs (second stage aim). Results showed that the generation of H2 and VFAs was effectively split, with carbohydrates and lipids, respectively, being the main substrates of the two processes. About 250 ml H2 L(-1) day(-1) was produced, corresponding to a yield of 0.36 mol mol(-1) of consumed carbohydrates (expressed as glucose equivalents). The overall concentration of VFAs in the acidogenic process was 13.80 g COD L(-1), so that 2.76 g COD L(-1) day(-1) was obtained. Second generation biorefineries use a selected fraction of an organic waste to conduct a microbiologically-driven pathway towards the generation of one target molecule. With the proposed approach, a greater value of the waste was attained, since the multi-purpose two-stage process did not entail competition for substrates between the first and the second steps.

Acclimation to hypoxia in Chlamydomonas reinhardtii: can biophotolysis be the major trigger for long-term H2 production?

In anaerobiosis, the microalga Chlamydomonas reinhardtii is able to produce H2 gas. Electrons mainly derive from mobilization of internal reserves or from water through biophotolysis. However, the exact mechanisms triggering this process are still unclear. Our hypothesis was that, once a proper redox state has been achieved, H2 production is eventually observed. To avoid nutrient depletion, which would result in enhanced fermentative pathways, we aimed to induce long-lasting H2 production solely through a photosynthesis : respiration equilibrium. Thus, growing cells were incubated in Tris Acetate Phosphate (TAP) medium under low light and high chlorophyll content. After a 250-h acclimation phase, a 350-h H2 production phase was observed. The light-to-H2 conversion efficiency was comparable to that given in some reports operating under sulphur starvation. Electron sources were found to be water, through biophotolysis, and proteins, particularly through photofermentation. Nonetheless, a substantial contribution from acetate could not be ruled out. In addition, photosystem II (PSII) inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) showed that it actively contributed to maintaining a redox balance during cell acclimation. In appropriate conditions, PSII may represent the major source of reducing power to feed the H2 evolution process, by inducing and maintaining an ideal excess of reducing power.

Effect of the organic loading rate on the production of polyhydroxyalkanoates in a multi-stage process aimed at the valorization of olive oil mill wastewater.

Mixed microbial culture polyhydroxyalkanoates (PHA) production has been investigated by using olive oil mill wastewater (OMW) as no-cost feedstock in a multi-stage process, also involving phenols removal and recovery. The selection of PHA-storing microorganisms occurred in a sequencing batch reactor (SBR), fed with dephenolized and fermented OMW and operated at different organic loading rates (OLR), ranging from 2.40 to 8.40gCOD/Ld. The optimal operating condition was observed at an OLR of 4.70gCOD/Ld, which showed the highest values of storage rate and yield (339±48mgCOD/gCODh and 0.56±0.05 COD/COD, respectively). The OLR applied to the SBR largely affected the performance of the PHA-accumulating reactor, which was fed through multiple pulsed additions of pretreated OMW. From an overall mass balance, involving all the stages of the process, an abatement of about 85% of the OMW initial COD (chemical oxygen demand) was estimated whereas the conversion of the influent COD into PHA was about 10% (or 22% by taking into account only the COD contained in the pretreated OMW, which is directly fed to the PHA production stages). Overall, polymer volumetric productivity (calculated from the combination of both the SBR and the accumulation reactor) accounted for 1.50gPHA/Ld.

Recovery of amorphous polyhydroxybutyrate granules from Cupriavidus necator cells grown on used cooking oil.

Used cooking oil (UCO) was employed as the sole carbon source for the production of polyhydroxybutyrate (PHB) by cultivation in batch mode of Cupriavidus necator DSM 428. The produced biomass was used for extraction of the PHB granules with a solvent-free approach using sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), and the enzyme Alcalase in an aqueous medium. The recovered PHB granules showed a degree of purity higher than 90% and no crystallization (i.e., granules were recovered in their 'native' amorphous state) as demonstrated by wide angle X-ray diffraction (WAXS). Granules were characterized according to their thermal properties and stability by differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Results show that UCO can be used as a renewable resource to produce amorphous PHB granules with excellent properties in a biocompatible manner.

Biodegradation of low-ethoxylated nonylphenols in a bioreactor packed with a new ceramic support (Vukopor ® S10).

This work was aimed at studying the possibility of biodegrading 4-nonylphenol and low ethoxylated nonylphenol mixtures, which are particularly recalcitrant to microbial degradation, by employing a biofilm reactor packed with a ceramic support (Vukopor® S10). A selected microbial consortium (Consortium A) was used to colonize the support. 4-Nonylphenol and ethoxylated nonylphenol degradation and mineralization capabilities were studied both in batch and continuous mode. The results showed that Vukopor® S10 was able to be colonized by an active biofilm for the degradation of the target pollutants with the reactor operating both in batch and continuous mode. On the other hand, pollutant adsorption on the support was negligible. FISH showed equal proportion of Alphaproteobacteria and Gammaproteobacteria in the Igepal CO-520 degrading reactor. A shift towards high proportion of Gammaproteobacteria was observed by supplying Igepal CO-210. PCR-density gradient gel electrophoresis (DGGE) analyses also evidenced that the biofilm evolved with time by changing the mixture applied and that Proteobacteria were the most represented phylum in the biofilm. Taken together, the data obtained provide a strong indication that the biofilm reactor packed with Vukopor® S10 and inoculated with Consortium A could potentially be used to develop a technology for the decontamination of 4-nonylphenol and low ethoxylated nonylphenol polluted effluents.

Innovative two-stage anaerobic process for effective codigestion of cheese whey and cattle manure.

The valorisation of agroindustrial waste through anaerobic digestion represents a significant opportunity for refuse treatment and renewable energy production. This study aimed to improve the codigestion of cheese whey (CW) and cattle manure (CM) by an innovative two-stage process, based on concentric acidogenic and methanogenic phases, designed for enhancing performance and reducing footprint. The optimum CW to CM ratio was evaluated under batch conditions. Thereafter, codigestion was implemented under continuous-flow conditions comparing one- and two-stage processes. The results demonstrated that the addition of CM in codigestion with CW greatly improved the anaerobic process. The highest methane yield was obtained co-treating the two substrates at equal ratio by using the innovative two-stage process. The proposed system reached the maximum value of 258 mL(CH4) g(gv(-1), which was more than twice the value obtained by the one-stage process and 10% higher than the value obtained by the two-stage one.

A continuous-flow approach for the development of an anaerobic consortium capable of an effective biomethanization of a mechanically sorted organic fraction of municipal solid waste as the sole substrate.

An effective mesophilic continuous anaerobic digestion process fed only with a mechanically sorted organic fraction of municipal solid waste (MS-OFMSW) was developed. During a preliminary 3-month experimental phase, the microbial consortium was acclimated toward MS-OFMSW by initially filling the reactor with cattle manure and then continuously feeding it with MS-OFMSW. The Hydraulic Retention Time (HRT) and Organic Loading Rate (OLR) were 23 days and 2.5 g/L/day, respectively. After 4 weeks, the reactor reached stationary performances (84% COD removal yield, 0.15 L(CH4)/g(COD removed) methane production yield). The acclimated consortium was then employed in a second run in which the reactor was operated under steady state conditions at the previous HRT and OLR for 73 days. The COD removal and the methane production yield increased up to 87% and 0.25 L(CH4) /g(CODremoved), respectively. The capability of the acclimated consortium to biomethanize MS-OFMSW was further studied via batch digestion experiments, carried out by inoculating the target waste with reactor effluents collected at the beginning of first run and at the end of the first and second run. The best normalized methane production (0.39 L(CH4) /g(initial COD)) was obtained with the inoculum collected at the end of the second run. Molecular analysis of the microbial community occurring in the reactor during the two sequential runs indicated that the progressive improvement of the process performances was closely related to the selection and enrichment of specific hydrolytic and acidogenic bacteria in the reactor.

A physicochemical-biotechnological approach for an integrated valorization of olive mill wastewater.

An integrated physicochemical-biotechnological approach for a multipurpose valorization of olive mill wastewaters was studied. More than 60% of the wastewater natural polyphenols were recovered through a solid phase extraction procedure, by employing Amberlite XAD16 resin as the adsorbent and ethanol as the biocompatible desorbing phase. Thereafter, the dephenolized effluent was fed to a mesophilic anaerobic acidogenic packed-bed biofilm reactor for the bioconversion of the organic leftover into volatile fatty acids (VFAs). A VFAs concentration of 19 gCODL(-1) was obtained, representing more than 70% of the COD occurring in the anaerobic effluent. The biotechnological process was assessed by means of bio-molecular analyses, which showed that the reactor packed bed was mostly colonized by bacteria of the Firmicutes phylogenetic group. The biorefinery scheme developed in this study allowed the obtainment of 1.59 g of polyphenols per liter of wastewater treated and 2.72 gCODL(-1) day(-1) of VFAs.

Biotransformation of a highly chlorinated PCB mixture in an activated sludge collected from a Membrane Biological Reactor (MBR) subjected to anaerobic digestion.

The role of anaerobic digestion (AD) on the decontamination and biomethanization of a PCB-spiked sludge obtained from a Membrane Biological Reactor (MBR) pilot plant was investigated throughout a 10-month batch experiment. The study was carried out under mesophilic (35°C) and thermophilic (55°C) conditions and was monitored by means of an integrated chemical, microbiological and molecular biology strategy. Remarkable PCB depletions (higher than 50% of the overall spiked PCBs) and dechlorinations were achieved under methanogenic conditions. The process was not affected by yeast extract addition. Both acetoclastic and hydrogenotrophic methanogens, together with some fermentative eubacteria, were found to persist in all PCB biodegrading microcosms. This finding, together with those obtained from parallel microcosms where specific populations were selectively inhibited, suggested that native methanogens played a key role in the biodegradation and dechlorination of the spiked PCBs. Taken together, the results of this study indicate that AD is a feasible option for the decontamination and the efficient disposal (with the production of a CH(4)-rich biogas) of contaminated MBR sludge, which can be then employed as a fertilizer for agricultural purposes.

Anaerobic acidogenic digestion of olive mill wastewaters in biofilm reactors packed with ceramic filters or granular activated carbon.

Four identically configured anaerobic packed bed biofilm reactors were developed and employed in the continuous acidogenic digestion of olive mill wastewaters to produce volatile fatty acids (VFAs), which can be exploited in the biotechnological production of polyhydroxyalkanoates. Ceramic porous cubes or granular activated carbon were used as biofilm supports. Aside packing material, the role of temperature and organic loading rate (OLR) on VFA production yield and mixture composition were also studied. The process was monitored through a chemical, microbiological and molecular biology integrated procedure. The highest wastewater acidification yield was achieved with the ceramic-based technology at 25 degrees C, with an inlet COD and an OLR of about 17 g/L and 13 g/L/day, respectively. Under these conditions, about the 66% of the influent COD (not including its VFA content) was converted into VFAs, whose final amount represented more than 82% of the influent COD. In particular, acetic, propionic and butyric acids were the main VFAs by composing the 55.7, 21.5 and 14.4%, respectively, of the whole VFA mixture. Importantly, the relative concentrations of acetate and propionate were affected by the OLR parameter. The nature of the packing material remarkable influenced the process performances, by greatly affecting the biofilm bacterial community structure. In particular, ceramic cubes favoured the immobilization of Firmicutes of the genera Bacillus, Paenibacillus and Clostridium, which were probably involved in the VFA producing process.

Nonylphenol polyethoxylate degradation in aqueous waste by the use of batch and continuous biofilm bioreactors.

An aerobic bacterial consortium (Consortium A) was recently obtained from textile wastewater and was capable of degrading 4-nonylphenol and nonylphenol polyethoxylates (NPnEOs). In the perspective of developing a biotechnological process for the treatment of effluents from activated sludge plants fed with NPnEO contaminated wastewater, the capability of Consortium A of biodegrading an industrial mixture of NPnEOs in the physiological condition of immobilized cells was investigated. Two identically configured packed bed reactors were developed by immobilizing the consortium on silica beads or granular activated carbon. Both reactors were tested in batch and continuous mode by feeding them with water supplemented with NPnEOs. The two reactors were monitored through chemical, microbiological and molecular integrated methodology. Active biofilms were generated on both immobilization supports. Both reactors displayed comparable NPnEO mineralization under batch and continuous conditions. FISH analyses evidenced that the biofilms evolved with time by changing the reactor operation mode and the organic load. Taken together, the data collected in this study provide a preliminary strong indication on the feasibility of Consortium A-based biofilm technology for the decontamination of NPnEO containing effluents.

Microbial processes associated to the decontamination and detoxification of a polluted activated sludge during its anaerobic stabilization.

Xenobiotic compounds accumulate in activated sludge resulting from wastewater treatment plants serving both civil and industrial areas. The opportunity to use anaerobic digestion for the decontamination and beneficial disposal of a contaminated activated sludge was investigated in mesophilic and thermophilic microcosms monitored through an integrated chemical, microbiological and ecotoxicological procedure. The 10 months anaerobic sludge incubation at 35 degrees C resulted in an extensive production of a methane-rich biogas, a marked reduction of pathogenic cultivable bacteria and, importantly, a marked biodegradation of the sludge-carried organic pollutants, including some polychlorinated biphenyls and polycyclic aromatic hydrocarbons, along with a relevant sludge detoxification. The sludge decontamination seemed to occur mostly under methanogenic conditions and was not significantly affected by the addition of yeast extract or molasses. Lower bioremediation and biomethanization yields were observed under thermophilic conditions.

Performances and microbial features of an aerobic packed-bed biofilm reactor developed to post-treat an olive mill effluent from an anaerobic GAC reactor.

BACKGROUND: Olive mill wastewater (OMW) is the aqueous effluent of olive oil producing processes. Given its high COD and content of phenols, it has to be decontaminated before being discharged. Anaerobic digestion is one of the most promising treatment process for such an effluent, as it combines high decontamination efficiency with methane production. The large scale anaerobic digestion of OMWs is normally conducted in dispersed-growth reactors, where however are generally achieved unsatisfactory COD removal and methane production yields. The possibility of intensifying the performance of the process using a packed bed biofilm reactor, as anaerobic treatment alternative, was demonstrated. Even in this case, however, a post-treatment step is required to further reduce the COD. In this work, a biological post-treatment, consisting of an aerobic biological "Manville" silica bead-packed bed aerobic reactor, was developed, tested for its ability to complete COD removal from the anaerobic digestion effluents, and characterized biologically through molecular tools. RESULTS: The aerobic post-treatment was assessed through a 2 month-continuous feeding with the digested effluent at 50.42 and 2.04 gl(-1)day(-1) of COD and phenol loading rates, respectively. It was found to be a stable process, able to remove 24 and 39% of such organic loads, respectively, and to account for 1/4 of the overall decontamination efficiency displayed by the anaerobic-aerobic integrated system when fed with an amended OMW at 31.74 and 1.70 gl(-1)day(-1) of COD and phenol loading rates, respectively. Analysis of 16S rRNA gene sequences of biomass samples from the aerobic reactor biofilm revealed that it was colonized by Rhodobacterales, Bacteroidales, Pseudomonadales, Enterobacteriales, Rhodocyclales and genera incertae sedis TM7. Some taxons occurring in the influent were not detected in the biofilm, whereas others, such as Paracoccus, Pseudomonas, Acinetobacter and Enterobacter, enriched significantly in the biofilter throughout the treatment. CONCLUSION: The silica-bead packed bed biofilm reactor developed and characterized in this study was able to significantly decontaminate anaerobically digested OMWs. Therefore, the application of an integrated anaerobic-aerobic process resulted in an improved system for valorization and decontamination of OMWs.

Anaerobic digestion of olive mill wastewaters in biofilm reactors packed with granular activated carbon and "Manville" silica beads.

Anaerobic digestion is one of the most promising technologies for disposing olive mill wastewaters (OMWs). The process is generally carried out in the conventional contact bioreactors, which however are often unable to efficiently remove OMW phenolic compounds, that therefore occur in the effluents. The possibility of mitigating this problem by employing an anaerobic OMW-digesting microbial consortium passively immobilized in column reactors packed with granular activated carbon (GAC) or "Manville" silica beads (SB) was here investigated. Under batch conditions, both GAC- and SB-packed-bed biofilm reactors exhibited OMW COD and phenolic compound removal efficiencies markedly higher (from 60% to 250%) than those attained in a parallel anaerobic dispersed growth reactor developed with the same inoculum; GAC-reactor exhibited COD and phenolic compound depletion yields higher by 62% and 78%, respectively, than those achieved with the identically configured SB-biofilm reactor. Both biofilm reactors also mediated an extensive OMW remediation under continuous conditions, where GAC-reactor was much more effective than the corresponding SB-one, and showed a tolerance to high and variable organic loads along with a volumetric productivity in terms of COD and phenolic compound removal significantly higher than those averagely displayed by most of the conventional and packed-bed laboratory-scale reactors previously proposed for the OMW digestion.