Development of microbial and/or enzymatic systems for the valorization of lignocellulosic wastes

The future trend of white biotechnology is the seek of renewable resources for the production of goods, traditionally obtained from petrol. Shifting society’s dependence away from petroleum to renewable biomass resources is generally viewed as an important contribute to the development of a sustainable industrial society and the reduction of greenhouse gas emissions. Among the available resources that are exploitable for biofuel production, there are the residues coming from different human activities such as agriculture (i.e.: cereals straws), food processing-industry, forestry, green and organic fractions of urban wastes. Their exploitation can contribute to the reduction of the price of the bioethanol production process, raw materials being responsible of almost half of the total production cost. A further element contributing to soaring biofuels costs, is constituted by enzymes. Their use roughly doubles the cost of cellulosic ethanol production and lessens the economic advantages of using waste materials. A valide alternative to purified/commercial enzymes may be the direct production of the enzymes of interest on the lignocellulosic material to be converted through its microbial fermentation. At this purpose, a powerful technique is solid state fermentation (SSF). This fermentative technique reproduces conditions really close to the natural environment in which many higher filamentous fungi have evolved. These microorganisms can be exploited both for wastes pretreatment, for the removal of lignin and hemicellulose hydrolysis, and for the production of industrially relevant enzymes (oxidative as well as hydolytic activities) for further applications. As a matter of fact, lignocellulosic wastes may contain significant concentrations of soluble carbohydrates and inducers of enzyme synthesis ensuring efficient production of lignino-cellulolytic enzymes. In the present study the residues from tomato and apple processing were chosen as raw materials. On the other side, as conversion microorganisms, the two white rot fungi Pleurotus ostreatus and Trametes versicolor, were selected. Culture conditions were set up, allowing waste colonization and transformation. This study showed the good potential of tomato pomace as substrate for laccases production by P. ostreatus and T. versicolor SSF, considering that significant enzyme activity levels were achieved without any optimization of culture conditions, neither by nutrient addition nor by O2 enrichment. Furthermore, SSFs on tomato pomace hold enormous potential for protease production, giving activity levels higher than those reported for fungi typically considered as the best protease producers. A process of fungal SSF was developed on apple waste, identifying the parameters allowing fast substrate colonization by both fungi. It was shown that apple pomace induced high levels of xylanases, with P. ostreatus secreting higher levels than T. versicolor. Both P. ostreatus and T. versicolor secreted levels of laccase activities that are lower or comparable with those obtained on tomato pomace. T. versicolor was shown to produce Manganese peroxidase, even if at low levels. On this substrate, low levels of protease activity were obtained, for both microorganisms. Furthermore, both tomato and apple pomace SSFs were shown to be better systems than liquid culture for the production of high laccases levels by P. ostreatus. Moreover, as one of the most significant results of this study, the developed P. ostreatus SSF processes provide the production of two laccase isoforms not detected in any other liquid culture conditions analysed so far. Finally, a strategy for P. ostreatus xylanase enrichment was investigated. The first trials for the identification of xylanolytic enzymes allowed the identification of an α-galactosidase. This enzyme even though not involved in xylan main chain break-down, plays its role in the removal of galactose units from both galactomannans and arabinoxylans, acting as ancillar xylanolytic enzyme

Electrical stimulation of adipose-derived stem cells in 3D nanofibrillar cellulose increases their osteogenic potential

Due to the ageing population, there is a steadily increasing incidence of osteoporosis and osteoporotic fractures. As conventional pharmacological therapy options for osteoporosis are often associated with severe side effects, bone grafts are still considered the clinical gold standard. However, the availability of viable, autologous bone grafts is limited making alternative cell-based strategies a promising therapeutic alternative. Adipose-derived stem cells (ASCs) are a readily available population of mesenchymal stem/stromal cells (MSCs) that can be isolated within minimally invasive surgery. This ease of availability and their ability to undergo osteogenic differentiation makes ASCs promising candidates for cell-based therapies. Recent studies have suggested that both exposure to electrical fields and cultivation in 3D can positively affect osteogenic potential of MSCs. To elucidate the osteoinductive potential of a combination of these biophysical cues on ASCs, cells were embedded within anionic nanofibrillar cellulose (aNFC) hydrogels and exposed to electrical stimulation (ES) for up to 21 days. ES was applied to ASCs in 2D and 3D at a voltage of 0.1 V/cm with a duration of 0.04 ms, and a frequency of 10 Hz for 30 min per day. Exposure of ACSs to ES in 3D resulted in high alkaline phosphatase (ALP) activity and in an increased mineralisation evidenced by Alizarin Red S staining. Moreover, ES in 3D aNFC led to an increased expression of the osteogenic markers osteopontin and osteocalcin and a rearrangement and alignment of the actin cytoskeleton. Taken together, our data suggest that a combination of ES with 3D cell culture can increase the osteogenic potential of ASC. Thus, exposure of ASCs to these biophysical cues might improve the clinical outcomes of regenerative therapies in treatment of osteoporotic fractures

Conducting Polymer Scaffolds Based on Poly(3,4-ethylenedioxythiophene) and Xanthan Gum for Live-Cell Monitoring.

Conducting polymer scaffolds can promote cell growth by electrical stimulation, which is advantageous for some specific type of cells such as neurons, muscle, or cardiac cells. As an additional feature, the measure of their impedance has been demonstrated as a tool to monitor cell growth within the scaffold. In this work, we present innovative conducting polymer porous scaffolds based on poly(3,4-ethylenedioxythiophene) (PEDOT):xanthan gum instead of the well-known PEDOT:polystyrene sulfonate scaffolds. These novel scaffolds combine the conductivity of PEDOT and the mechanical support and biocompatibility provided by a polysaccharide, xanthan gum. For this purpose, first, the oxidative chemical polymerization of 3,4-ethylenedioxythiophene was carried out in the presence of polysaccharides leading to stable PEDOT:xanthan gum aqueous dispersions. Then, by a simple freeze-drying process, porous scaffolds were prepared from these dispersions. Our results indicated that the porosity of the scaffolds and mechanical properties are tuned by the solid content and formulation of the initial PEDOT:polysaccharide dispersion. Scaffolds showed interconnected pore structure with tunable sizes ranging between 10 and 150 μm and Young's moduli between 10 and 45 kPa. These scaffolds successfully support three-dimensional cell cultures of MDCK II eGFP and MDCK II LifeAct epithelial cells, achieving good cell attachment with very high degree of pore coverage. Interestingly, by measuring the impedance of the synthesized PEDOT scaffolds, the growth of the cells could be monitored

Optically Transparent Anionic Nanofibrillar Cellulose Is Cytocompatible with Human Adipose Tissue-Derived Stem Cells and Allows Simple Imaging in 3D.

The anti-inflammatory and immunomodulatory properties of human mesenchymal stromal cells (MSCs) are a focus within regenerative medicine. However, 2D cultivation of MSCs for extended periods results in abnormal cell polarity, chromosomal changes, reduction in viability, and altered differentiation potential. As an alternative, various 3D hydrogels have been developed which mimic the endogenous niche of MSCs. Nevertheless, imaging cells embedded within 3D hydrogels often suffers from low signal-to-noise ratios which can be at least partly attributed to the high light absorbance and light scattering of the hydrogels in the visible light spectrum. In this study, human adipose tissue-derived MSCs (ADSCs) are cultivated within an anionic nanofibrillar cellulose (aNFC) hydrogel. It is demonstrated that aNFC forms nanofibres arranged as a porous network with low light absorbance in the visible spectrum. Moreover, it is shown that aNFC is cytocompatible, allowing for MSC proliferation, maintaining cell viability and multilineage differentiation potential. Finally, aNFC is compatible with scanning electron microscopy (SEM) and light microscopy including the application of conventional dyes, fluorescent probes, indirect immunocytochemistry, and calcium imaging. Overall, the results indicate that aNFC represents a promising 3D material for the expansion of MSCs whilst allowing detailed examination of cell morphology and cellular behaviour.Peer Reviewe

Biomimetic and electroactive 3D scaffolds for human neural crest-derived stem cell expansion and osteogenic differentiation

Abstract: Osteoporosis is a skeletal disease characterized by bone loss and bone microarchitectural deterioration. The combination of smart materials and stem cells represents a new therapeutic approach. In the present study, highly porous scaffolds are prepared by combining the conducting polymer PEDOT:PSS with collagen type I, the most abundant protein in bone. The inclusion of collagen proves to be an effective way to modulate their mechanical properties and it induces an increase in scaffolds’ electrochemical impedance. The biomimetic scaffolds support neural crest-derived stem cell osteogenic differentiation, with no need for scaffold pre-conditioning contrarily to other reports

Organic nanofibers embedding stimuli-responsive threaded molecular components

While most of the studies on molecular machines have been performed in solution, interfacing these supramolecular systems with solid-state nanostructures and materials is very important in view of their utilization in sensing components working by chemical and photonic actuation. Host polymeric materials, and particularly polymer nanofibers, enable the manipulation of the functional molecules constituting molecular machines, and provide a way to induce and control the supramolecular organization. Here, we present electrospun nanocomposites embedding a self-assembling rotaxane-type system that is responsive to both optical (UV-visible light) and chemical (acid/base) stimuli. The system includes a molecular axle comprised of a dibenzylammonium recognition site and two azobenzene end groups, and a dibenzo[24]crown-8 molecular ring. The dethreading and rethreading of the molecular components in nanofibers induced by exposure to base and acid vapors, as well as the photoisomerization of the azobenzene end groups, occur in a similar manner to what observed in solution. Importantly, however, the nanoscale mechanical function following external chemical stimuli induces a measurable variation of the macroscopic mechanical properties of nanofibers aligned in arrays, whose Young's modulus is significantly enhanced upon dethreading of the axles from the rings. These composite nanosystems show therefore great potential for application in chemical sensors, photonic actuators and environmentally responsive materials.Comment: 39 pages, 16 figure

Controlling the electrochromic properties of conductive polymers using UV-light

The phenomenon of electrochromism in conductive polymers is well known and has been exploited in many scientific reports. Using a newly developed patterning technique for conductive polymers, we manufactured high-resolution electrochromic devices from the complementary polymers PEDOT and polypyrrole. The technique, which combines UV-light exposure with vapor phase polymerization, has previously only been demonstrated with the conductive polymer PEDOT. We further demonstrated how the same technique can be used to control the optical properties and the electrochromic contrast in these polymers. Oxidant exposure to UV-light prior to vapor phase polymerization showed a reduction in polymer electrochromic contrast allowing high-resolution (100 mu m) patterns to completely disappear while applying a voltage bias due to their optical similarity in one redox state and dissimilarity in the other. This unique electrochromic property enabled us to construct devices displaying images that appear and disappear with the change in applied voltage. Finally, a modification of the electrochromic device architecture permitted a dual image electrochromic device incorporating patterned PEDOT and patterned polypyrrole on the same electrode, allowing the switching between two different images.Funding Agencies|European Research Council [307596]; Advanced Functional Materials Center at Linkoping University</p

Conducting Polymer Scaffolds Based on Poly(3,4-ethylenedioxythiophene) and Xanthan Gum for Live-Cell Monitoring.

Conducting polymer scaffolds can promote cell growth by electrical stimulation, which is advantageous for some specific type of cells such as neurons, muscle, or cardiac cells. As an additional feature, the measure of their impedance has been demonstrated as a tool to monitor cell growth within the scaffold. In this work, we present innovative conducting polymer porous scaffolds based on poly(3,4-ethylenedioxythiophene) (PEDOT):xanthan gum instead of the well-known PEDOT:polystyrene sulfonate scaffolds. These novel scaffolds combine the conductivity of PEDOT and the mechanical support and biocompatibility provided by a polysaccharide, xanthan gum. For this purpose, first, the oxidative chemical polymerization of 3,4-ethylenedioxythiophene was carried out in the presence of polysaccharides leading to stable PEDOT:xanthan gum aqueous dispersions. Then, by a simple freeze-drying process, porous scaffolds were prepared from these dispersions. Our results indicated that the porosity of the scaffolds and mechanical properties are tuned by the solid content and formulation of the initial PEDOT:polysaccharide dispersion. Scaffolds showed interconnected pore structure with tunable sizes ranging between 10 and 150 μm and Young's moduli between 10 and 45 kPa. These scaffolds successfully support three-dimensional cell cultures of MDCK II eGFP and MDCK II LifeAct epithelial cells, achieving good cell attachment with very high degree of pore coverage. Interestingly, by measuring the impedance of the synthesized PEDOT scaffolds, the growth of the cells could be monitored

Organic Matrix and Secondary Metabolites in Nacre

International audienceNacre, also called mother-of-pearl, is a naturally occurring biomineral, largely studied by chemists, structural biologists, and physicists to understand its outstanding and diverse properties. Nacre is constituted of aragonite nanograins surrounded by organic matrix, and it has been established that the organic matrix is responsible for initiating and guiding the biomineralization process. The first challenge to study the organic matrix of nacre lays in its separation from the biomineral. Several extraction methods have been developed so far. They are categorized as either strong (e.g., decalcification) or soft (e.g., water, ethanol) and they allow specific extractions of targeted compounds. The structure of the nacreous organic matrix is complex, and it provides interesting clues to describe the mineralization process. Proteins, sugars, lipids, peptides, and other molecules have been identified and their role in mineralization investigated. Moreover, the organic matrix of nacre has shown interesting properties for human health. Several studies are investigating its activity on bone mineralization and its properties for skin care. In this review, we focus on the organic constituents, as lipids, sugars, and small metabolites which are less studied since present in small quantities