Anna Salerno

Mathematical modeling of poly((R)-3-hydroxyalkanoate) synthesis by Cupriavidus necator DSM 545 on substrates stemming from biodiesel production

Two low structured mathematical models for fed-batch production of polyhydroxybutyrate and poly[hydroxybutyrate-co-hydroxyvalerate] by Cupriavidus necator DSM 545 on renewable substrates (glycerol and fatty acid methyl esters-FAME) combined with glucose and valeric acid, were established. The models were used for development/optimization of feeding strategies of carbon and nitrogen sources concerning PHA content and polymer/copolymer composition. Glycerol/glucose fermentation featured a max. specific growth rate of 0.171 h(-1), a max. specific production rate of 0.038 h(-1) and a PHB content of 64.5%, whereas the FAME/valeric acid fermentation resulted in a max. specific growth rate of 0.046 h(-1), a max. specific production rate of 0.07 h(-1) and 63.6% PHBV content with 4.3% of 3-hydroxyvalerate (3HV) in PHBV. A strong inhibition of glycerol consumption by glucose was confirmed (inhibition constant ki,G=4.28×10(-4) g L(-1)). Applied concentration of FAME (10-12 g L(-1)) positively influenced on PHBV synthesis. HV/PHBV ratio depends on applied VA concentration.

Novel Description of mcl-PHA Biosynthesis by Pseudomonas chlororaphis from Animal-Derived Waste

A novel description of mcl-PHA biosynthesis by Ps. chlororaphis from tallow-based biodiesel as an inexpensive carbon feed stock is presented. Fermentation protocols, kinetic analysis, an efficient product recovery strategy, and product characterization are included. Maximum specific growth rates (μmax.) of 0.08 h(-1), 0.10 h(-1) and 0.13 h(-1), respectively, were achieved in three different fermentation set-ups. Volumetric productivity for mcl-PHA amounted to 0.071 g/L h, 0.094 g/L h and 0.138 g/L h, final intracellular PHA contents calculated from the sum of active biomass and PHA from 22.1 to 29.4 wt.-%, respectively. GC-FID analysis showed that the obtained biopolyester predominantly consists of 3-hydroxyoctanoate and 3-hydroxydecanoate, and, to a minor extent, 3-hydroxydodecanoate, 3-hydroxynonanoate, 3-hydroxyhexanoate, and 3-hydroxyheptanoate monomers. The overall distribution of the monomers remained similar, regardless to working volumes, biodiesel concentrations and pre-treatment of the inoculum.

Novel precursors for production of 3-hydroxyvalerate-containing poly[(R)-hydroxyalkanoate]s

Abstract Polyhydroxyalkanoates (PHAs) with tailored properties are needed to meet consumer demands regarding the use of eco-compatible biobased polymeric materials and relevant plastic items. Inserting 3-hydroxvalerate (3HV) monomeric units in PHA biopolyesters results in poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate] (PHBHV) copolyesters aimed at their conversion into production of biodegradable eco-friendly plastic items. As inexpensive novel precursor substrate structurally related to 3HV, a mixture of odd-numbered carboxylic acids with 9–17 carbon atoms was produced by oxidative ozonolysis of alkenes. This mixture was successfully applied for biomediated PHBHV production by Cupriavidus necator. Applying this mixture as carbon substrate, a molar 3HV fraction exceeding 0.12 was obtained. The isolated copolyesters featured a low degree of crystallinity, narrow molar mass distribution, and low melting temperatures. These properties should make application of the novel 3HV-precursors interesting for large-scale production of easily processable copolyesters.

Whey Lactose as a Raw Material for Microbial Production of Biodegradable Polyesters

Whey, a by-product of diary and cheese industry, constitutes the watery portion after the separation of fat and caseins from whole milk. Cheese whey is a surplus material produced in volumes almost equal to the milk processed in cheese manufactories, therefore its disposal as a waste causes serious pollution problems in the surrounding environment where it s discarded. This is due to its enormous biochemical oxygen demand that is mainly caused by its high lactose content; as a consequence a large amount of industrial capital is requested for whey disposal. During the last years, the amounts of whey increased to such an extent that they cannot be simply used as animal feed as the most common application. To overcome these problems a sustainable alternative is to upgrade whey and its derivates to a resource for many value added industrial products, making whey not only a waste but also a valuable resource.

In silico optimization and low structured kinetic model of poly[(R)-3-hydroxybutyrate] synthesis by Cupriavidus necator DSM 545 by fed-batch cultivation on glycerol.

Glycerol was utilized by Cupriavidus necator DSM 545 for production of poly-3-hydroxybutyrate (PHB) in fed-batch fermentation. Maximal specific growth rates (0.12 and 0.3h(-1)) and maximal specific non-growth PHB production rate (0.16 g g(-1)h(-1)) were determined from two experiments (inocula from exponential and stationary phase). Saturation constants for nitrogen (0.107 and 0.016 g L(-1)), glycerol (0.05 g L(-1)), non-growth related PHB synthesis (0.011 g L(-1)) and nitrogen/PHB related inhibition constant (0.405 g L(-1)), were estimated. Five relations for specific growth rate were tested using mathematical models. In silico performed optimization procedures (varied glycerol/nitrogen ratio and feeding) has resulted in a PHB content of 70.9%, shorter cultivation time (23 h) and better PHB yield (0.347 g g(-1)). Initial concentration of biomass 16.8 g L(-1) and glycerol concentration in broth between 3 and 5 g L(-1) were decisive factors for increasing of productivity.

The Wnt5a Receptor, Receptor Tyrosine Kinase-Like Orphan Receptor 2, Is a Predictive Cell Surface Marker of Human Mesenchymal Stem Cells with an Enhanced Capacity for Chondrogenic Differentiation

Multipotent mesenchymal stem cells (MSCs) have enormous potential in tissue engineering and regenerative medicine. However, until now, their development for clinical use has been severely limited as they are a mixed population of cells with varying capacities for lineage differentiation and tissue formation. Here, we identify receptor tyrosine kinase‐like orphan receptor 2 (ROR2) as a cell surface marker expressed by those MSCs with an enhanced capacity for cartilage formation. We generated clonal human MSC populations with varying capacities for chondrogenesis. ROR2 was identified through screening for upregulated genes in the most chondrogenic clones. When isolated from uncloned populations, ROR2+ve MSCs were significantly more chondrogenic than either ROR2–ve or unfractionated MSCs. In a sheep cartilage‐repair model, they produced significantly more defect filling with no loss of cartilage quality compared with controls. ROR2+ve MSCs/perivascular cells were present in developing human cartilage, adult bone marrow, and adipose tissue. Their frequency in bone marrow was significantly lower in patients with osteoarthritis (OA) than in controls. However, after isolation of these cells and their initial expansion in vitro, there was greater ROR2 expression in the population derived from OA patients compared with controls. Furthermore, osteoarthritis‐derived MSCs were better able to form cartilage than MSCs from control patients in a tissue engineering assay. We conclude that MSCs expressing high levels of ROR2 provide a defined population capable of predictably enhanced cartilage production. Stem Cells 2017;35:2280–2291

The Wnt5a receptor ROR2 is a predictive cell surface marker of human mesenchymal stem cells with an enhanced capacity for chondrogenic differentiation

Multipotent mesenchymal stem cells (MSCs) have enormous potential in tissue engineering and regenerative medicine. However, until now, their development for clinical use has been severely limited as they are a mixed population of cells with varying capacities for lineage differentiation and tissue formation. Here, we identify receptor tyrosine kinase‐like orphan receptor 2 (ROR2) as a cell surface marker expressed by those MSCs with an enhanced capacity for cartilage formation. We generated clonal human MSC populations with varying capacities for chondrogenesis. ROR2 was identified through screening for upregulated genes in the most chondrogenic clones. When isolated from uncloned populations, ROR2+ve MSCs were significantly more chondrogenic than either ROR2–ve or unfractionated MSCs. In a sheep cartilage‐repair model, they produced significantly more defect filling with no loss of cartilage quality compared with controls. ROR2+ve MSCs/perivascular cells were present in developing human cartilage, adult bone marrow, and adipose tissue. Their frequency in bone marrow was significantly lower in patients with osteoarthritis (OA) than in controls. However, after isolation of these cells and their initial expansion in vitro, there was greater ROR2 expression in the population derived from OA patients compared with controls. Furthermore, osteoarthritis‐derived MSCs were better able to form cartilage than MSCs from control patients in a tissue engineering assay. We conclude that MSCs expressing high levels of ROR2 provide a defined population capable of predictably enhanced cartilage production. Stem Cells 2017;35:2280–2291

Microalgae for Sustainable Energy Production

Carbon-neutral liquid fuels are doubtlessly needed for future sustainable transport. Biodiesel produced from oil crops, animal waste, and used cooking oil potentially is an alternative for mineral oils used preferably today, but the quantities available are by far too small to satisfy the needs of a future market. Microalgae constitute powerful unicellular factories with enormous potential for mitigation of miscellaneous pollutants from effluent gases and waste waters. Most of all their outstanding capacity for photosynthetic CO2 fixation underlines their high potential for diminishing current ecological problems. Together with these contributions to beneficial environmental development, various microalgae accumulate high concentrations of oils and even hydrocarbons (30–80 % of cell dry mass formed) beside other high-value marketable products (e.g., polyunsaturated fatty acids or pigments like astaxanthin) as cell constituents. Low cell densities and moderate growth rates typical for algal cultivation are known as the major obstacles toward a broad market penetration of microalgal products: Here, high cell densities are required to obtain reasonable volumetric productivities. For some microalgal strains, mixotrophic cultivation by providing organic carbon substrates together with CO2 results in increased biomass concentration in a first cultivation step. For this purpose, numerous organic waste streams can be applied as substrate. In a second step, the fresh catalytically active algal biomass accumulates desired products via CO2 fixation, e.g., from industrial effluent gases, as the sole carbon source. This can be realized by two-stage, continuously operated closed photo-bioreactor systems. After cell harvest and optimized product recovery, the value-added conversion of residual algal biomass for generation of sustainable energy sources, e.g., in biogas plants, constitutes another challenge.