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The ocean is an excellent source for new biocatalysts due to the tremendous genetic diversity of marine microorganisms, and it may contribute to the development of sustainable industrial processes. In a recent paper published in Microorganisms, Carlos J.C. Rodrigues and Carla C.C.R. de Carvalho (iBB-BERG) used a marine bacterium for the conversion of benzaldehyde to benzyl alcohol, which is an important chemical employed as a precursor for producing esters for cosmetics and other industries. A stirred reactor, using a fed-batch approach, enabled a 1.5-fold increase in benzyl alcohol productivity when compared with batch mode. However, product accumulation in the reactor hindered the conversion. The use of a continuous flow reactor packed with immobilized cells enabled a 9.5-fold increase in productivity when compared with the fed-batch stirred reactor system.

Greener, cost-effective methodologies for food processing and production have been actively looked after because of increased demand due to growing population. Enzymes, as green and highly efficient catalysts, have been a mainstay of food technology. However, the advantageous features of enzymes are negatively impacted by their long-term instability, harsh industrial operational conditions and challenges for enzyme recovery and reuse. Immobilized enzyme formulations have been developed to address these limitations. In a recently published book chapter, Filipe Carvalho and Pedro Fernandes (iBB-BERG) present the latest finding and trends on the design and application of immobilized enzyme for food production and processing. The chapter was published in the book Value Addition in Food Products and Processing using Enzyme Technology, published by Academic Press.

Pathogenic Vibrio species are responsible for human and animal illness and one of the main causes of human infection is related to the ingestion of undercooked seafood. Due to their filter-feeding habit, marine invertebrates, such as clams, are known to be a natural reservoir of specific microbial communities. In a recently published study carried out by researchers of ESTM, Polytechnic of Leiria, and Carla C.C.R. de Carvalho (BERG-iBB), the profile of antimicrobial resistance and the presence of virulence genes in the bacterial isolates from clams were evaluated. The presence of the mobilisable colistin resistance gene mcr-1 in three Vibrio spp. isolates highlights a potential threat to public health.

A new strategy to modify cellulose with the short antimicrobial hexapeptide MP196 (RWRWRW-NH2) is proposed by BERG and BSIRG researchers that uses fusions of Cys-terminated derivatives of MP196 and a carbohydrate binding module (CBM). CBM3-MP196-modified cellulose hydrogels displayed antibacterial activity that was significantly higher when compared with controls. This versatile concept offers a toolbox for the functionalization of different cellulose materials with a broad choice in peptides. the paper was published in Acta Biomaterialia. The work was funded by project CBM-X.

Lactococcus lactis is a food-grade, and generally recognized as safe, bacterium, which making it ideal for producing plasmid DNA (pDNA) or recombinant proteins for industrial or pharmaceutical applications. A paper published in Microorganisms by Sofia Duarte and Gabriel Monteiro from BERG-iBB reviews the major findings from L. lactis transcriptome and proteome studies, with an overexpression of native or recombinant proteins. These studies provide important insights on how to engineer the plasmid vectors and/or the strains in order to achieve high pDNA or recombinant proteins yields, with high quality standards. 

In recent years, there has been an increased interest in exploring the potential of micro-and mesoscale milling technologies for developing cost-effective microfluidic systems with high design flexibility and a rapid microfabrication process that does not require a cleanroom. Nevertheless, the number of current studies aiming to fully understand and establish the benefits of this technique in developing high-quality microsystems with simple integrability is still limited. In a recent paper published in Micromachines, BERG-iBB researches, Ana Azevedo and Amin Javidanbardan, in collaboration, with João Pedro Conde and Virginia Chu from INESC-MN, describe a systematic methodology for developing 3D high-quality thermoplastic-based molds for microfluidic structures based on micromilling technology. The work was performed under the Marie Skłodowska-Curie grant agreement No. 812909 CODOBIO, within the Marie Skłodowska-Curie European Training Networks framework.

Developing viable biorefineries is urgent to support the transition to a sustainable society. Lignocellulosic biomass is a crucial renewable feedstock, but usually a combination of pretreatments is necessary to extract both sugars and lignin. In a recent paper published in the journal Sustainability, the M.Sc. student Manuel Dias and Carla C.C.R. de Carvalho (BERG-iBB) collaborated with researchers from Technische Universität Wien (Vienna, Austria) under supervision of Prof Anton Friedl, to study liquid-hot-water (LHW) as pretreatment for hemicellulose hydrolysis. Assessment of different LHW conditions allowed the determination of those leading to high sugar concentration and low concentration of degradation products. The lignin remaining in the solid phase could be extracted and hydrolysed. By including the analysis of lignin hydrolysis into the standard sugar and degradation products in LHW, it was possible to make an integrated decision to valorize the different lignocellulosic components of wheat straw.

Carboxylic acids (CAs) are considered key players in the implementation of more sustainable industrial processes due to their potential to replace a set of oil-derived commodity chemicals and there is growing interest in producing them through microbial processes. While many CAs are intermediates of microbial central carbon metabolism, and therefore envisaging their production in a host of choice is relatively straightforward; for other CAs this approach is difficult. This could be because they do not occur naturally (as is the case for levulinic acid) or because the described production pathway cannot be easily ported (as it is the case for adipic, muconic or glucaric acids). In a review published in Journal of Fungi, Nuno Mira’s team (iBB) reviewed the synthetic biology approaches that have been made towards enabling the production of non-natural CAs in microbes, with a large emphasis on metabolic retrobiosynthesis methodologies. Additionally, the authors also reviewed the pathway prospecting studies towards microbial levulinic, poly-lactic and methacrylic acid production, as two case-studies where there is a need to bridge the gap between natural CAs and their non-natural industrial derivatives.

The nutritional composition and bioactive properties of roots and rhizomes of Asparagus stipularis were evaluated to demonstrate its potential in the food and pharmaceutical industries. HPLC-DAD-ESI/MS characterization of infusions allowed the identification and quantitation of 7 hydroxycinnamoyl derivatives, with caffeic acid as the most abundant. Roots infusion contained the highest amounts of these compounds. It also exhibited the highest antioxidant activity in all assays, with EC50 values of 0.44 ± 0.01, 0.98 ± 0.03 and 0.64 ± 0.01 mg/mL for DPPH, ABTS and FRAP assays, respectively, with no toxicity towards PLP2 primary cell cultures (GI50 > 400 μg/mL). The extract was encapsulated into PLGA nanoparticles obtaining a size of 260 nm and a polydispersity index around 0.1, with a zeta potential of -36 mV, as well as a good encapsulation efficiency of approximately 83%. The particles had a spherical morphology and smooth surface. FTIR and DSC assays confirmed the efficacy of the encapsulation methodology. This paper was published in Food Bioscience by Pedro Fonte from BERG-iBB in a transnational collaboration with researchers from Portugal, Spain, Italy and Tunisia. The developed systems will be used as delivery systems for bioactive compounds of A. stipularis and as an innovative dietary supplement.

Cassava (Manihot esculenta) cultivation is of great importance in many economies, particularly in Colombia. About 630,000 tons of C-rich cassava waste is produced annually and applications to high value products, applying the circular economy concept, must be developed. A recent publication in Journal of Polymers and the Environment assesses the potential use of cassava peel for the production of polyhydroxyalkanoates (PHAs) by Cupriavidus necator. A copolymer of P3HB-3HV was produced and processed into electrospun meshes of random and aligned microfibers, allowing the development of structures that can be applied in the context of tissue engineering. This work involved Manuela Fonseca, Frederico Ferreita and Teresa Cesário form BERG-IBB and has been done in collaboration with researchers from the University of Antioquia, Medellin-Colombia.

Lupanine is used as a building block in the synthesis of sparteine, a chiral selector in drug synthesis. This alkaloid is found in wastewaters derived from the debittering process that makes lupin beans edible. In a recently published work, carried out by researchers of the Faculty of Sciences and Faculty of Pharmacy from the University of Lisbon, and Teresa Esteves, Frederico Ferreira, Flávio Ferreira and Ana Mota from BERG-iBB, a computational chemistry approach was taken to design molecularly imprinted polymers (MIPs) selecting itaconic acid, a biobased building block, as a functional monomer that can provide higher affinities for lupanine. In this work, lupanine was concentrated from lupin bean wastewater by nanofiltration, extracted with ethyl acetate, and purified using the synthesized MIP, which was able to selectively recognize lupanine and improve its purity to 88%, with 82% recovery of the alkaloid, showing the potential application of this strategy to render the industrial process more sustainable.

When an idea in a coffee break in an international meeting turns into a paper, Pedro Fonte from BERG-iBB in collaboration with researchers from UFVJM, Brazil just published a work in JSFA Reports performing an optimization of the brewing parameters on coffee extraction using a central composite rotatable design. The effects of extraction time, particle size of ground coffee, extraction temperature, coffee-to-water ratio, stirring on caffeine yield, and soluble solids on caffeine concentrations were studied. Optimized parameters showed 45 min sufficed to perform a cold extraction at 4°C and 24°C. The parameters selected for validation were 24°C, 30% coffee-to-water ratio, a stirring of 400 rpm resulting in 3.98 mg/ml of extracted caffeine, 11.20 °Brix, and 93.9% of caffeine yield. The smaller particle size (595 μm) displayed the higher caffeine extraction of about 4 mg/ml. This study reveals the high efficiency of cold brew extraction and its potential at the industrial scale, decreasing costs with energy and extraction time, and producing a coffee rich in caffeine and soluble solids.

The development of new scale-down models for accelerated and cost-effective biopharmaceutical process development has been receiving considerable attention in the last decade. Microfluidic systems have emerged as a particularly attractive candidate by offering miniaturization, parallelization, and allowing continuous and precise pumping and online monitoring possible. In a collaborative work, published in Journal of Chromatography A, BERG-iBB researchers Ana Azevedo and Amin Javidanbardan, together with INESC-MN researchers João Pedro Conde and Virginia Chu, propose a simple and cost-effective method for developing microfluidic chromatographic systems with integrated sensors. Fabrication and integration of microsensors, in this case, an impedance sensor, in each microcolumn provided the possibility of parallelizing experiments with separate online monitoring. The work was performed under the Marie Skłodowska-Curie grant agreement No. 812909 CODOBIO, within the Marie Skłodowska-Curie European Training Networks framework.

Consumer acceptance of microalgae products is often hampered by their colour, “fishy” smell and flavor. An international project with research teams from ISA (PI Prof. Anabela Raymundo), IST (Carla de Carvalho, iBB-BERG), Norce (Norway) and the company Pagarete Microalgae solutions (Portugal) is aimed at the use of enzymes to improve the overall sensory quality of microalgal ingredients. The project, entitled “YUM ALGAE - enzYmes for improved sensory qUality of MicroALGAE ingredients in foods”, is funded in ca. 1 million euros by the Iceland, Liechtenstein, Norway grants (EEA grants, Blue Growth programme).

The mild operational conditions typically required by biobased production processes, together with the selective nature of biocatalysts, microbial diversity and tunability, and the century-long record of fermentation, clearly place bioprocesses as the primary contenders, by far, in achieving the generalized implementation of efficient, green manufacturing strategies. Moreover, by overlapping with the biorefinery approach, the foundations needed for bioprocesses to embrace the circular economy concept are set. In this Special Issue on “Bioprocess Engineering: Sustainable Manufacturing for a Green Society", guest edited by Pedro Fernandes and Carla de Carvalho (iBB-BERG) in Processes, the papers will discuss new bioproducts and concomitant bioengineering approaches to achieve a sustainable and environmentally friendly economy.

Many studies that apply PnD in pre-clinical cutaneous wound healing models show large variations in the choice of the animal species (e.g., large animals, rodents), the choice of diabetic or non-diabetic animals, the type of injury (full-thickness wounds, burns, radiation-induced wounds, skin flaps), the source and type of PnD (placenta, umbilical cord, fetal membranes, cells, secretomes, tissue extracts), the method of administration (topical application, intradermal/subcutaneous injection, intravenous or intraperitoneal injection, subcutaneous implantation), and the type of delivery systems (e.g., hydrogels, synthetic or natural biomaterials as carriers for transplanted cells, extracts or secretomes). In a collaborative work coordinated by Prof. Pedro Fonte under the COST Action SPRINT (CA17116), the Postdoc researcher Ana Macedo and Master student Francisca Mendes from BERG-iBB, provided a comprehensive and integrative overview of the application of PnD in wound healing to assess its efficacy in preclinical animal models. The review was published in Frontiers in Bioengineering and Biotechnology.

The cyclic anionic lipopeptide daptomycin (DAP) is used in the treatment of severe infections caused by Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). DAP resistance, though rare, often results in treatment failure. Paradoxically, in MRSA, DAP resistance is usually accompanied by a concomitant decrease in b-lactam resistance in what is known as the ‘see-saw effect’. In a recent publication in the Journal of Antimicrobial Chemotherapy, Carla CCR de Carvalho (iBB-BERG), Agustina Taglialegna and Adriana E. Rosato (currently at Riverside University Health System, USA), showed that the post-translocational protein PrsA has a major role in orchestrating the cell membrane lipid adaptation associated with the see-saw effect, in addition to its function required for the posttranscriptional maturation of PBP 2a. This study contributes greatly to the understanding of MRSA biology and of the impact of DAP/b-lactam on the cell membrane/cell wall machinery.

Wound care is clinically demanding due to treatment inefficiency and represents an economic burden for healthcare systems. A promising therapeutic strategy is the use of exogenous growth factors that are decreased at the wound site and hence limit recovery of the skin. Insulin is one of the cheapest growth factors in the market able to accelerate the re-epithelialization and stimulate angiogenesis and cell migration. However, the effectiveness of topical insulin in wound healing is hampered by the proteases in the wound bed. The encapsulation into nanoparticles improves its stability in the wound, providing adhesion to the mucosal surface and allowing its sustained release. In a paper published in Materials, Pedro Fonte from BERG-iBB in a collaboration with researchers from CCMAR, University of Algarve and LAQV, University of Porto performed a standing point about a promising strategy to treat different types of wounds by the topical delivery of insulin-loaded nanocarriers.