Effects of residues on the degradation of PHA produced from mixed microbial cultures and processed in extrusion

Thermal degradation upon melting is one of the major drawbacks reported for polyhydroxyalkanoates (PHA). However, the role of residues originating from the fermentation and the extraction steps on the thermal stability of this class of biopolymers still needs to be clarified. In the particular case of PHA produced from mixed microbial cultures (MMC), this topic is even less documented in the literature. Here, two polyhydroxy(butyrate-co-valerate) (PHBV) produced from MMC enriched in PHA accumulating organisms and fed with cheese whey were studied. A micro extrusion line is used to produce filaments and assess the processability and the degradation of processed PHBV. The prototype micro extrusion line allows for studying grams of materials. The two PHBV contain 18 mol% HV. PHBV was recovered with 11 wt% residues, and further submitted to a purification procedure resulting in a second biopolyester containing less than 2 wt% impurities. The thermorheological characterization of the two PHBV is first presented, together with their semicrystalline properties. Then the processing windows of the two biopolyesters are presented. Finally, the properties of extruded filaments are reported and the thermomechanical degradation of PHBV is extensively studied. The structure was assessed by wide angle X-ray diffraction, mechanical and rheological properties are reported, thermal properties are studied with differential scanning calorimetry and thermogravimetric analysis, whereas Fourier Transform Infrared spectroscopy was used to assess the impact of the extrusion on PHBV chemical structure. All results obtained with the two PHBV are compared to assess the effects of residues on both PHBV processability and degradation.

Effects of fermentation residues on the melt processability and thermomechanical degradation of PHBV produced from cheese whey using mixed microbial cultures

"Available online 28 March 2016"

Effect of sulfate and iron (III) on LCFA degradation by a methanogenic community

[Excerpt] Under anaerobic conditions long chain fatty acids (LCFA) can be converted to methane by syntrophic bacteria and methanogenic archaea. LCFA degradation was also reported in the presence of alternative hydrogenotrophic partners, such as sulfate-reducing bacteria (SRB) and iron-reducing bacteria (IRB), which generally show higher affinity for H2 than methanogens and are more resistant to LCFA [1,2,3]. Their presence in a microbial culture degrading LCFA can be advantageous to reduce LCFA toxicity towards methanogens, although high concentrations of external electron acceptor (EEA) can lead to outcompetition of methanogens and cease methane production. In this work, we tested the effect of adding sub-stoichiometric concentrations of sulfate and iron(III) to methanogenic communities degrading LCFA. (...)

Public service culture collections of microorganisms: are they important for biotechnology?

Microbiology as a scientific discipline recognised the need to preserve microorganisms for scientific studies establishing from its very beginning research culture collections (CC). Later on, to better serve different scientific fields and bioindustries with the increasing number of strains of scientific, medical, ecological and biotechnological importance public service CC were established with the specific aims to support their user communities. Currently, the more developed public service CC are recognised as microBiological Resources Centres (mBRC). mBRC are considered to be one of the key elements for sustainable international scientific infrastructure, which is necessary to underpin successful delivery of the benefits of biotechnology, whether within the health sector, the industrial sector or other sectors, and in turn ensure that these advances help drive economic growth. In more detail, mBRCs are defined by Organisation for Economic Co-operation and Development (OECD) as service providers and repositories of the living cells, genomes of organisms, and information relating to heredity and functions of biological systems. mBRCs contain collections of culturable organisms (e.g., microorganisms, plant, animal cells), replicable parts of these (e.g. genomes, plasmids, virus, cDNAs), viable but not yet culturable organisms, cells and tissues, as well as database containing molecular, physiological and structural information relevant to these collections and related bioinformatics. Thus mBRCs are fundamental to harnessing and preserving the world’s microbial biodiversity and genetic resources and serve as an essential element of the infrastructure for research and development. mBRCs serve a multitude of functions and assume a range of shapes and forms. Some are large national centres performing a comprehensive role providing access to diverse organisms. Other centres play much narrower, yet important, roles supplying limited but crucial specialised resources. In the era of the knowledge-based bio-economy mBRCs are recognised as vital element to underpinning the biotechnology.

Fueling the bio-economy: european culture collections and microbiology education and training

A survey of European Microbial Biological Resource Centers and their users provided an overview on Microbiology education and training. The results identified future increases in demand despite several shortcomings and gaps in the current offer. Urgent adjustments are needed to match users' needs, integrate innovative programs, and adopt new technologies.

Development of Janus hydrogel microcapsules as novel biomaterials-stem cells construct for 3D co-culture applications

Co-cultures of two or more cell types and biodegradable biomaterials of natural origin have been successfully combined to recreate tissue microenvironments. Segregated co-cultures are preferred over conventional mixed ones in order to better control the degree of homotypic and heterotypic interactions. Hydrogel-based systems in particular, have gained much attention to mimic tissue-specific microenvironments and they can be microengineered by innovative bottom-up approaches such as microfluidics. In this study, we developed bi-compartmentalized (Janus) hydrogel microcapsules of methacrylated hyaluronic acid (MeHA)/methacrylated-chitosan (MeCht) blended with marine-origin collagen by droplet-based microfluidics co-flow. Human adipose stem cells (hASCs) and microvascular endothelial cells (hMVECs) were co-encapsulated to create platforms of study relevant for vascularized bone tissue engineering. A specially designed Janus-droplet generator chip was used to fabricate the microcapsules (<250â μm units) and Janus-gradient co-cultures of hASCs: hMVECs were generated in various ratios (90:10; 75:25; 50:50; 25:75; 10:90), through an automated microfluidic flow controller (Elveflow microfluidics system). Such monodisperse 3D co-culture systems were optimized regarding cell number and culture media specific for concomitant maintenance of both phenotypes to establish effective cell-cell (homotypic and heterotypic) and cell-materials interactions. Cellular parameters such as viability, matrix deposition, mineralization and hMVECs re-organization in tube-like structures, were enhanced by blending MeHA/MeCht with marine-origin collagen and increasing hASCs: hMVECs co-culture gradient had significant impact on it. Such Janus hybrid hydrogel microcapsules can be used as a platform to investigate biomaterials interactions with distinct combined cell populations.

Microbial respiration with chlorine oxyanions: diversity and physiological and biochemical properties of chlorate- and perchlorate-reducing microorganisms

Chlorine oxyanions are valuable electron acceptors for microorganisms. Recent findings have shed light on the natural formation of chlorine oxyanions in the environment. These suggest a permanent introduction of respective compounds on Earth, long before their anthropogenic manufacture. Microorganisms that are able to grow by the reduction of chlorate and perchlorate are affiliated with phylogenetically diverse lineages, spanning from the Proteobacteria to the Firmicutes and archaeal microorganisms. Microbial reduction of chlorine oxyanions can be found in diverse environments and different environmental conditions (temperature, salinities, pH). It commonly involves the enzymes perchlorate reductase (Pcr) or chlorate reductase (Clr) and chlorite dismutase (Cld). Horizontal gene transfer seems to play an important role for the acquisition of functional genes. Novel and efficient Clds were isolated from microorganisms incapable of growing on chlorine oxyanions. Archaea seem to use a periplasmic Nar-type reductase (pNar) for perchlorate reduction and lack a functional Cld. Chlorite is possibly eliminated by alternative (abiotic) reactions. This was already demonstrated for Archaeoglobus fulgidus, which uses reduced sulfur compounds to detoxify chlorite. A broad biochemical diversity of the trait, its environmental dispersal, and the occurrence of relevant enzymes in diverse lineages may indicate early adaptations of life toward chlorine oxyanions on Earth.

Tapping the wealth of microbial data in high-throughput metabolic model reconstruction

Genome-scale metabolic models are valuable tools in the metabolic engineering process, based on the ability of these models to integrate diverse sources of data to produce global predictions of organism behavior. At the most basic level, these models require only a genome sequence to construct, and once built, they may be used to predict essential genes, culture conditions, pathway utilization, and the modifications required to enhance a desired organism behavior. In this chapter, we address two key challenges associated with the reconstruction of metabolic models: (a) leveraging existing knowledge of microbiology, biochemistry, and available omics data to produce the best possible model; and (b) applying available tools and data to automate the reconstruction process. We consider these challenges as we progress through the model reconstruction process, beginning with genome assembly, and culminating in the integration of constraints to capture the impact of transcriptional regulation. We divide the reconstruction process into ten distinct steps: (1) genome assembly from sequenced reads; (2) automated structural and functional annotation; (3) phylogenetic tree-based curation of genome annotations; (4) assembly and standardization of biochemistry database; (5) genome-scale metabolic reconstruction; (6) generation of core metabolic model; (7) generation of biomass composition reaction; (8) completion of draft metabolic model; (9) curation of metabolic model; and (10) integration of regulatory constraints. Each of these ten steps is documented in detail.

Influence of oxygen conditions on bacterial interactions within biofilms related with cystic fibrosis

Dissertação de mestrado em Bioengineering

Osteogenic differentiation of adipose stem cells by endothelial cells co-culture within liquified capsules

Inspired by the native co-existence of multiple cell types and from the concept of deconstructing the stem cell niche, we propose a co-encapsulation strategy within liquified capsules. The present team has already proven the application of liquified capsules as bioencapsulation systems1. Here, we intend to use the optimized system towards osteogenic differentiation. Capsules encapsulating adipose stem cells alone (MONO-capsules) or in co-culture with endothelial cells (CO-capsules) were maintained in endothelial medium with or without osteogenic differentiation factors. The suitability of the capsules for living stem and endothelial cells encapsulation was demonstrated by MTS and DNA assays. The osteogenic differentiation was assessed by quantifying the deposition of calcium and the activity of ALP up to 21 days. CO capsules had an enhanced osteogenic differentiation, even when cultured in the absence of osteogenic factors. Furthermore, osteopontin and CD31 could be detected, which respectively indicate that osteogenic differentiation had occurred and endothelial cells maintained their phenotype. An enhanced osteogenic differentiation by co-encapsulation was also confirmed by the upregulation of osteogenic markers (BMP-2, RUNX2, BSP) while the expression of angiogenic markers (VEGF, vWF, CD31) revealed the presence of endothelial cells. The proposed capsules can also act as a growth factor release system upon implantation, as showed by VEGF and BMP-2 quantification. These findings demonstrate that the co-encapsulation of stem and endothelial cells within liquified injectable capsules provides a promising strategy for bone tissue engineering.  

In vitro chondrogenic commitment of human Wharton's jelly stem cells by co-culture with human articular chondrocytes.

Wharton's jelly stem cells (WJSCs) are a potential source of transplantable stem cells in cartilage-regenerative strategies, due to their highly proliferative and multilineage differentiation capacity. We hypothesized that a non-direct co-culture system with human articular chondrocytes (hACs) could enhance the potential chondrogenic phenotype of hWJSCs during the expansion phase compared to those expanded in monoculture conditions. Primary hWJSCs were cultured in the bottom of a multiwell plate separated by a porous transwell membrane insert seeded with hACs. No statistically significant differences in hWJSCs duplication number were observed under either of the culture conditions during the expansion phase. hWJSCs under co-culture conditions show upregulations of collagen type I and II, COMP, TGFβ1 and aggrecan, as well as of the main cartilage transcription factor, SOX9, when compared to those cultured in the absence of chondrocytes. Chondrogenic differentiation of hWJSCs, previously expanded in co-culture and monoculture conditions, was evaluated for each cellular passage using the micromass culture model. Cells expanded in co-culture showed higher accumulation of glycosaminoglycans (GAGs) compared to cells in monoculture, and immunohistochemistry for localization of collagen type I revealed a strong detection signal when hWJSCs were expanded under monoculture conditions. In contrast, type II collagen was detected when cells were expanded under co-culture conditions, where numerous round-shaped cell clusters were observed. Using a micromass differentiation model, hWJSCs, previously exposed to soluble factors secreted by hACs, were able to express higher levels of chondrogenic genes with deposition of cartilage extracellular matrix components, suggesting their use as an alternative cell source for treating degenerated cartilage.

Non emergency patients transport - a mixed integer linear programming

This work presents a model and a heuristic to solve the non-emergency patients transport (NEPT) service issues given the new rules recently established in Portugal. The model follows the same principle of the Team Orienteering Problem by selecting the patients to be included in the routes attending the maximum reduction in costs when compared with individual transportation. This model establishes the best sets of patients to be transported together. The model was implemented in AMPL and a compact formulation was solved using NEOS Server. A heuristic procedure based on iteratively solving problems with one vehicle was presented, and this heuristic provides good results in terms of accuracy and computation time.

Biofunctionalization of titanium surfaces for dental implants: osteogenic, anti-microbial and tribocorrosion resistant surfaces

PhD thesis in Biomedical Engineering

Seismic-resistant building practices resulting from local seismic culture

Considering that vernacular architecture may bear important lessons on hazard mitigation, this chapter focuses on the European Mediterranean countries and studies traditional seismic-resistant architectural elements and techniques that local populations developed to prevent or repair earthquake damage. This area was selected as a case study because, as a highly seismic region, it has suffered the effect of many earthquakes along the history and, thus, regions within this area are prone to have developed a Local Seismic Culture. After reviewing seismic resistant construction concepts, a wide range of traditional construction solutions that, in many cases, have shown to improve the seismic performance of vernacular constructions of these regions is presented, as a contribution to the general overview of retrofitting building systems provided in this book. The main motivation is that most of these techniques can be successfully applied to preserve and to retrofit surviving examples without prejudice for their identity.

Quantitative image analysis as a tool for Yarrowia lipolytica dimorphic growth evaluation in different culture media

Yarrowia lipolytica, a yeast strain with a huge biotechnological potential, capable to produce metabolites such as γ-decalactone, citric acid, intracellular lipids and enzymes, possesses the ability to change its morphology in response to environmental conditions. In the present study, a quantitative image analysis (QIA) procedure was developed for the identification and quantification of Y. lipolytica W29 and MTLY40-2P strains dimorphic growth, cultivated in batch cultures on hydrophilic (glucose and N-acetylglucosamine (GlcNAc) and hydrophobic (olive oil and castor oil) media. The morphological characterization of yeast cells by QIA techniques revealed that hydrophobic carbon sources, namely castor oil, should be preferred for both strains growth in the yeast single cell morphotype. On the other hand, hydrophilic sugars, namely glucose and GlcNAc caused a dimorphic transition growth towards the hyphae morphotype. Experiments for γ-decalactone production with MTLY40-2P strain in two distinct morphotypes (yeast single cells and hyphae cells) were also performed. The obtained results showed the adequacy of the proposed morphology monitoring tool in relation to each morphotype on the aroma production ability. The present work allowed establishing that QIA techniques can be a valuable tool for the identification of the best culture conditions for industrial processes implementation.