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6 p. ; Global warming, market and production capacity are being the key drivers for selecting the main players for the next decades in the market of bio-based plastics. The drop-in bio-based polymers such as the bio-based polyethylene terephtalate (PET) or polyethylene (PE), chemically identical to their petrochemical counterparts but having a component of biological origin, are in the top of the list. They are followed by new polymers such as PHA and PLA with a significant market growth rate since 2014 with projections to 2020. Research will provide improved strains designed through synthetic and systems biology approaches; furthermore, the use of low-cost substrates will contribute to the widespread application of these bio- based polymers. The durability of plastics is not considered anymore as a virtue, and interesting bioprospecting strategies to isolate microorganisms for assimilating the recalcitrant plastics will pave the way for in vivo strategies for plastic mineralization. In this context, waste management of bio-based plastic will be one of the most important issues in the near future in terms of the circular economy. There is a clear need for standardized labelling and sorting instructions, which should be regulated in a coordinated way by policymakers and material producers. ; Research on polymer biotechnology at the lab of Auxi Prieto is supported by grants from the European Union's Seventh Framework Programme and for research, technological development and demonstration under grant agreement nº. 311815, and the Horizon 2020 research and innovation Programme under grant agreement nº 633962 and nº. 679050. We also acknowledge the Community of Madrid (P2013/MIT2807) and the Spanish Ministry of Economy (BIO201344878R, BIO2014-61515-EXP). ; Peer reviewed

1 p. ; Biopolymers such as polyhydroxyalkanoates (PHAs) are considered as an alternative to conventional plastics, but their production can only be economically feasible by waste valorization approaches. The project Afterlife1, funded by the Bio-Based Industries Joint Undertaking under the European Union's Horizon 2020 research and innovation program, aims a flexible, cost- and resource-efficient process for recovering and valorizing the relevant fractions from wastewater (WW). The AFTERLIFE process will isolate the different components of value using a cascade of membrane filtration units that will separate all the solids in the WW. These will then be treated to obtain highly-pure extracts and metabolites or, alternatively, to be converted into PHA. Volatile fatty acid (VFA) fractions with different compositions were obtained by the acidogenic fermentation of the WW from three food industries. In this study, we present a strategy to revalorize those VFAs for the production of PHA using pure cultures. The model strain Cupriavidus necator H16 is a non-pathogenic bacterium that has been the subject of intensive research due to its metabolical versatility and its ability to store up to 90 % of its cell dry weight as polyhydroxybutyrate (PHB). The tolerance studies of H16 strain to VFAs and the determination of key physiological parameters have been applied to optimize the fermentation conditions using three fermented industrial WW on a lab-scale up to 5 liters. The highest PHA concentration (5 g L-1 ), PHA productivity (0.1 g L-1 h-1), and PHA content (80 %) have been achieved by modulating the carbon pulses in a fed-batch system, based on the specific consumption and biomass/product produced. 1https://www.bbi.europa.eu/projects/afterlife. ; Peer reviewed

26 p.-3 fig.-2 tab. ; Medium-chain-length polyhydroxyalkanoates (mcl-PHA) are biotechnologically useful natural products found in many bacteria. This biopolymer functions as a carbon and energy storage reservoir in cells but has physical and mechanical properties that make it a promising bioplastic with applications ranging from adhesives to medical implants. Therefore, there is much interest in understanding the biology of mcl-PHA synthesis and metabolism. Increased knowledge of PHA biology serves as a foundation for the bioengineering of PHA and its eventual use as a biologically derived product. This chapter covers the state of knowledge on mcl-PHA, including its synthesis and its central role in cellular metabolism. Moreover, this chapter discusses methods for bioengineering mcl-PHA production in bacteria as well as synthetic biology methods for its study and production in the natural mcl-PHA producer, Pseudomonas putida. ; Research on polymer biotechnology in the laboratory of M. Auxiliadora Prieto is supported by funding from the European Union's Horizon 2020 research and innovation program under grant agreements number 633962 and 679050. We also acknowledge support from the Community of Madrid (P2013/MIT2807) and the Spanish Ministry of Economy (BIO201344878R, BIO2014-61515-EXP). ; Peer reviewed ; Postprint

19 p.-9 fig.-1 tab. ; Polyhydroxyalkanoates (PHAs) are polyesters produced by numerous microorganisms for energy and carbon storage. Simultaneous synthesis and degradation of PHA drives a dynamic cycle linked to the central carbon metabolism, which modulates numerous and diverse bacterial processes, such as stress endurance, pathogenesis, and persistence. Here, we analyze the role of the PHA cycle in conferring robustness to the model bacterium P. putida KT2440. To assess the effect of this cycle in the cell, we began by constructing a PHA depolymerase (PhaZ) mutant strain that had its PHA cycle blocked. We then restored the flux through the cycle in the context of an engineered library of P. putida strains harboring differential levels of PhaZ. High-throughput phenotyping analyses of this collection of strains revealed significant changes in response to PHA cycle performance impacting cell number and size, PHA accumulation, and production of extracellular (R)-hydroxyalkanoic acids. To understand the metabolic changes at the system level due to PHA turnover, we contextualized these physiological data using the genome-scale metabolic model iJN1411. Model-based predictions suggest successive metabolic steady states during the growth curve and an important carbon flux rerouting driven by the activity of the PHA cycle. Overall, we demonstrate that modulating the activity of the PHA cycle gives us control over the carbon metabolism of P. putida, which in turn will give us the ability to tailor cellular mechanisms driving stress tolerance, e.g., defenses against oxidative stress, and any potential biotechnological applications. ; This research was funded by the European Union's Horizon 2020 research and innovation program under grant agreement number 633962 (P4SB) and 870294 (MIX-up). We also acknowledge financial support from the Spanish Ministry of Science, Innovation, and Universities through projects BIO2017-83448-R_TECMABIO and RobExplode PID2019‐108458RB‐I00 (AEI/10.13039/501100011033). ; Peer reviewed

10 p.-2 fig. ; Human activity has been altering many ecological cycles for decades, disturbing the natural mechanisms which are responsible for re-establishing the normal environmental balances. Probably, the most disrupted of these cycles is the cycle of carbon. In this context, many technologies have been developed for an efficient CO2 removal from the atmosphere. Once captured, it could be stored in large geological formations and other reservoirs like oceans. This strategy could present some environmental and economic problems. Alternately, CO2 can be transformed into carbonates or different added-value products, such as biofuels and bioplastics, recycling CO2 from fossil fuel. Currently different methods are being studied in this field. We classified them into biological, inorganic and hybrid systems for CO2 transformation. To be environmentally compatible, they should be powered by renewable energy sources. Although hybrid systems are still incipient technologies, they have made great advances in the recent years. In this scenario, biotechnology is the spearhead of ambitious strategies to capture CO2 and reduce global warming. ; Research on polymer biotechnology at the laboratory of Auxiliadora Prieto is supported by grants from the European Union's Horizon 2020 Research and Innovation Programme under grant agreements, no 633962, no. 679050, no. 745791 and no. 745737. We also acknowledge the Community of Madrid (P2013/MIT2807) and the Spanish Ministry of Economy (BIO2013-44878R, BIO2014- 61515-EXP). ; Peer reviewed

The gasification of organic waste materials to synthesis gas (syngas), followed by microbial fermentation provides a significant resource for generating bioproducts such as polyhydroxyalkanoates (PHA). The anaerobic photosynthetic bacterium, Rhodospirillum rubrum, is an organism particularly attractive for the bioconversion of syngas into PHAs. In this study, a quantitative physiological analysis of R. rubrum was carried out by implementing GC-MS and HPLC techniques to unravel the metabolic pathway operating during syngas fermentation that leads to PHA production. Further, detailed investigations of the central carbon metabolites using 13C-labeled substrate showed significant CO2 assimilation (of 40 %) into cell material and PHA from syngas carbon fraction. By a combination of quantitative gene expression and enzyme activity analyses, the main role of carboxylases from the central carbon metabolism in CO2 assimilation was shown, where the Calvin Benson-Bassham Cycle (CBB) played a minor role. This knowledge sheds light about the biochemical pathways that contribute to synthesis of PHA during syngas fermentation being valuable information to further optimize the fermentation process. ; This work has been funded by the EU project SYNPOL (grant agreement n° 311815) under the European Union's Seventh Framework Programme. ; Peer reviewed

16 p.-5 fig.-6 tab. ; Background: Rhodospirillum rubrum is a purple non-sulphur bacterium that produces H2 by photofermentation of several organic compounds or by water gas-shift reaction during CO fermentation. Successful strategies for both processes have been developed in light-dependent systems. This work explores a dark fermentation bioprocess for H2 production from water using CO as the electron donor. ; Results: The study of the influence of the stirring and the initial CO partial pressure (pCO) demonstrated that the process was inhibited at pCO of 1.00 atm. Optimal pCO value was established in 0.60 atm. CO dose adaptation to bacterial growth in fed-batch fermentations increased the global rate of H2 production, yielding 27.2 mmol H2 l−1 h−1 and reduced by 50% the operation time. A kinetic model was proposed to describe the evolution of the molecular species involved in gas and liquid phases in a wide range of pCO conditions from 0.10 to 1.00 atm. ; Conclusions: Dark fermentation in R. rubrum expands the ways to produce biohydrogen from CO. This work optimizes this bioprocess at lab-bioreactor scale studying the influence of the stirring speed, the initial CO partial pressure and the operation in batch and fed-batch regimes. Dynamic CO supply adapted to the biomass growth enhances the productivity reached in darkness by other strategies described in the literature, being similar to that obtained under light continuous syngas fermentations. The kinetic model proposed describes all the conditions tested. ; The European Union's Horizon 2020 research and innovation program under grant agreement no. 679050 (CELBICON), the Community of Madrid (P2018/NMT4389) and the Spanish Ministry of Science, Innovation and Universities (BIO2017-83448-R) supported this work. ; Peer reviewed

17 p.-7 fig. ; Polyhydroxyalkanoates (PHA) are polyesters produced intracellularly by many bacterial species as energy storage materials, which are used in biomedical applications, including drug delivery systems, due to their biocompatibility and biodegradability. In this study, we evaluated the potential application of this nanomaterial as a basis of inhaled drug delivery systems. To that end, we assessed the possible interaction between PHA nanoparticles (NPs) and pulmonary surfactant using dynamic light scattering, Langmuir balances, and epifluorescence microscopy. Our results demonstrate that NPs deposited onto preformed monolayers of DPPC or DPPC/POPG bind these surfactant lipids. This interaction facilitated the translocation of the nanomaterial towards the aqueous subphase, with the subsequent loss of lipid from the interface. NPs that remained at the interface associated with liquid expanded (LE)/tilted condensed (TC) phase boundaries, decreasing the size of condensed domains and promoting the intermixing of TC and LE phases at submicroscopic scale. This provided the stability necessary for attaining high surface pressures upon compression, countering the destabilization induced by lipid loss. These effects were observed only for high NP loads, suggesting a limit for the use of these NPs in pulmonary drug delivery. ; Research in the laboratory of the authors is currently funded by grants from the Spanish Ministry of Science, Universities and Innovation (RTI2018-094564-B-I00, BIO2017-83448-R) and the Regional Government of Madrid (P2018/NMT-4389). ; Peer reviewed

14 pag.- 8 fig.- 5 tab. ; Pseudomonas putida KT2440 has evolved a tightly regulated system for metabolizing glycerol implying a prolonged growth lag-phase. We have learnt that this fact can be avoided by the addition of small amounts of some growth precursors. The addition of 1mM octanoic acid as co-feeder completely eliminated the lag-phase, resulting in an improvement, in terms of invested time, of both growth and polyhydroxyalkanoates (PHA) accumulation. To investigate this phenomenon, we have followed co-metabolic approaches combined with mutations of the specific and global regulatory networks that connect glycerol catabolism and PHA synthesis. By using mutant strains in metabolic genes from the PHA and tricarboxylic acid (TCA) cycles, we have demonstrated that the co-feeding effect is independent of PHA accumulation, but driven on active glyoxylate shunt and Entner-Doudoroff (ED) routes. These findings suggested that the effect of octanoate on glycerol metabolism could rely, either on a global activation of the cell energy state, or on the generation of specific metabolites or cofactors needed for the activation of glycerol metabolism. Our results addressed GlpR as the key factor controlling the efficient utilization of glycerol as growth precursor in P.putida KT2440. Accordingly, a glpR knockout mutant of P.putida KT2440 showed an elimination of the lag-phase when cultured on glycerol in the absence of co-feeder. Besides, the production of PHA in this strain was increased near twofold, resulting in a higher final yield in terms of PHA accumulation. The repressor activity of the GlpR protein over the glp genes in the absence of glycerol was finally demonstrated by qRT-PCR. This work contributed to unravel the physiological causes of the long lag-phase produced by glycerol in the model strain P.putida KT2440 that hinders its use as carbon source in biotechnological applications for generating valuable products. ; This work was supported by 597 grants from the Comisión Interministerial de Ciencia y Tecnología and CSIC598 (BIO2010-21049 and 201120E092), and by the European Union Grant (NMP2-CT-599 2007-026515). Isabel F. Escapa is a recipient of CSIC-I3P predoctoral fellowship. ; Peer reviewed

Phasins are intracellular polyhydroxyalkanoat4e (PHA)-associated proteins involved in the stabilization of these bacterial carbon storage granules. Despite its importance in PHA metabolism and regulation, only few reports have focused so far on the structure of these proteins. In this work we have investigated the structure and stability of the PhaF phasin from Pseudomonas putida KT2440, a protein that is involved in PHA granule stabilization and distribution to daughter cells upon cell division. A structural, three-dimensional model of the protein was built from homology modeling procedures and consensus secondary structure predictions. The model predicts that PhaF is an elongated protein, with a long, amphipathic N-terminal helix with PHA binding capacity, followed by a short leucine zipper involved in protein oligomerization and a superhelical C-terminal domain wrapped around the chromosomal DNA. Hydrodynamic, spectroscopical and thermodynamic experiments validated the model and confirmed both that free PhaF is a tetramer in solution and that most part of the protein is intrinsically disordered in the absence of its ligands. The results lay a molecular basis for the explanation of the biological role of PhaF and, along with an exhaustive analysis of phasin sequence databases, suggest that intrinsic disorder and oligomerization through coiled-coils may be a widespread mechanism among these proteins. © 2013 Maestro et al. ; Spanish Ministry of Economy and CompetitivenessBFU2010-17824, BIO2010-21049, BIO2007-67304-C02 (, http:// www.mineco.gob.es/portal/site/mineco?lang_choosen = en) Valencian Government, Spain, http://www.gva.es/en/inicio/presentacion FPA/2010/055 ; Peer Reviewed

31 p.-9 fig.-3 tab. ; This study deals with the optimization and scaling up of the production of poly(3-hydroxybutyrate), PHB, nanocomposites containing biosynthesized silver nanoparticles (AgNPs) to generate materials with antimicrobial performance. First, a comparative study of the chemical and biological synthesis of AgNPs during the fermentation process of Cupriavidus necator at shake flask-scale was carried out. These experiments demonstrated the inherent capacity of C. necator to reduce the silver salt and produce AgNPs without the need for adding a reducing agent and, that the method of synthesis (with or without reducing agent) affects the dispersion of the AgNPs and their antimicrobial performance. Finally, the process was scaled-up to a 10Liters bioreactor and the relevant physical properties of the PHB-AgNPs nanocomposites pressed into films were determined. From the characterization work, the AgNPs were found to be well dispersed and distributed into the polymer matrix, having a maximum frequency of particles with average diameter of 76-95nm. Moreover, the presence of AgNPs did not cause any effect on the thermal properties of the biopolymer, although a slight reduction in crystallinity was seen. The developed materials presented a strong antimicrobial activity against the food-borne pathogens Salmonella enterica and Listeria monocytogenes, which makes them potentially suitable for active coatings and packaging applications. Complete biodisintegration of the samples occurred during composting conditions within the first 40days. Interestingly, the presence of the AgNPs did not impair the profile of biodegradation of the microbial polymer. ; The authors would like to thank to the Active and intelligent fibre-based packaging - innovation and market introduction (ActInPak) COST Action FP1405 for the funding of the Short Therm Scientific Mission, to Octavio Cedenilla from the Instituto de Ciencias Agrarias-CSIC for the ICP-OES analysis and to Dr. Luis Cabedo, from Universitat Jaume I for his support with mechanical testing, WAXD analyses and biodisintegration tests.Research leading to these results has received funding from the Comunidad de Madrid (P2013/MIT2807) and the Spanish Ministerio de Economía y Competitividad, (BIO2013-44878-R and AGL2015-63855-C2-1-R). Jinneth Lorena Castro-Mayorga is supported by the Administrative Department of Science, Technology and Innovation (Colciencias) of the Colombian Government. ; Peer reviewed

4 p. ; The authors have received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement no. 633962 for the project P4SB. ; Peer reviewed

13 p.-5 fig. ; Komagataeibacter medellinensis ID13488 (formerly Gluconacetobacter medellinensis ID13488) is able to produce crystalline bacterial cellulose (BC) under high acidic growth conditions. These abilities make this strain desirable for industrial BC production from acidic residues (e.g. wastes generated from cider production). To explore the molecular bases of the BC biosynthesis in this bacterium, the genome has been sequenced revealing a sequence of 3.4 Mb containing three putative plasmids of 38.1 kb (pKM01), 4.3 kb (pKM02) and 3.3 Kb (pKM03). Genome comparison analyses of K. medellinensis ID13488 with other cellulose-producing related strains resulted in the identification of the bcs genes involved in the cellulose biosynthesis. Genes arrangement and composition of four bcs clusters (bcs1, bcs2, bcs3 and bcs4) was studied by RT-PCR, and their organization in four operons transcribed as four independent polycistronic mRNAs was determined. qRT-PCR experiments demonstrated that mostly bcs1 and bcs4 are expressed under BC production conditions, suggesting that these operons direct the synthesis of BC. Genomic differences with the close related strain K. medellinensis NBRC 3288 unable to produce BC were also described and discussed. ; The authors thank for the financial support from the Foundation Domingo Martınez (2015-Area Materiales 2), the Spanish Ministry of Science, Innovation and Universities (MAT2016-76294-R, BIO2017-83448-R), the Community of Madrid (P2013/MIT2807), the Basque Government in the frame of Grupos Consolidados (IT-776-13). L.U. PIF PRE_2014_1_371. ; Peer reviewed

The massive production of urban and agricultural wastes has promoted a clear need for alternative processes of disposal and waste management. The potential use of municipal solid wastes (MSW) as feedstock for the production of polyhydroxyalkanoates (PHA) by a process known as syngas fermentation is considered herein as an attractive bio-economic strategy to reduce these wastes. In this work, we have evaluated the potential of Rhodospirillum rubrum as microbial cell factory for the synthesis of PHA from syngas produced by microwave pyrolysis of the MSW organic fraction from a European city (Seville). Growth rate, uptake rate, biomass yield and PHA production from syngas in R. rubrum have been analysed. The results revealed the strong robustness of this syngas fermentation where the purity of the syngas is not a critical constraint for PHA production. Microwave-induced pyrolysis is a tangible alternative to standard pyrolysis, because it can reduce cost in terms of energy and time as well as increase syngas production, providing a satisfactory PHA yield. ; Research leading to these results has received funding from the European Union's Seventh Framework Programme for Research, Technological Development and Demonstration under grant agreement no. 311815 (SYNPOL), and from the Comunidad de Madrid (P2013/MIT2807). D. B. also acknowledges the financial support received from PCTI and FICYT of the Government of the Principado de Asturias. ; Peer reviewed

The obligate predator Bdellovibrio bacteriovorus HD100 shows a large set of proteases and other hydrolases as part of its hydrolytic arsenal needed for its predatory life cycle. We present genetic and biochemical evidence that open reading frame (ORF) Bd3709 of B. bacteriovorus HD100 encodes a novel medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase (PhaZ Bd). The primary structure of PhaZ Bd suggests that this enzyme belongs to the α/β-hydrolase fold family and has a typical serine hydrolase catalytic triad (serine-histidine-aspartic acid) in agreement with other PHA depolymerases and lipases. PhaZ Bd has been extracellularly produced using different hypersecretor Tol-pal mutants of Escherichia coli and Pseudomonas putida as recombinant hosts. The recombinant PhaZ Bd has been characterized, and its biochemical properties have been compared to those of other PHA depolymerases. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. It is also affected by the reducing agent dithiothreitol and nonionic detergents like Tween 80. PhaZ Bd is an endoexohydrolase that cleaves both large and small PHA molecules, producing mainly dimers but also monomers and trimers. The enzyme specifically degrades mcl-PHA and is inactive toward short-chain-length polyhydroxyalkanoates (scl-PHA) like polyhydroxybutyrate (PHB). These studies shed light on the potentiality of these predators as sources of new biocatalysts, such as an mcl-PHA depolymerase, for the production of enantiopure hydroxyalkanoic acids and oligomers as building blocks for the synthesis of biobased polymers. ; This work was supported by the Ministry of Economía y Competitividad (BIO2007-67304, BIO2010-21049, CSD2007-00005; CISC 201120E050) and by European Union Grants (NMP2-CT-2007-026515). ; Peer Reviewed