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Recovering PHA from mixed microbial biomass: Using non-ionic surfactants as a pretreatment step

Bianca Colombo, Joana Pereira, Margarida Martins, Mario . Torres-Acosta, Ana. Dias, Paulo C. Lemose, Sónia P.M. Ventura, Giorgio Eisele, Anna Alekseeva, Fabrizio Adani, Luísa S. Serafim. Separation and Purification Technology 253 DOI: 10.1016/j.seppur.2020.117521

Polyhydroxyalkanoates (PHA) are biodegradable plastics of microbial origin, whose biodegradability and thermochemical properties make them greener alternatives to conventional plastics. Despite their high industrial potential, the PHA’ high production costs still hinder their application. Mixed microbial biomass combined with agro-industrial wastes are being used to strategically reduce these costs. However, it is still necessary to optimize the downstream processing, where the extraction process amounts to 30–50% of the total costs. Conventional processes apply chlorinated solvents to recover PHA from microbial biomass but cannot be implemented industrially due to environmental regulations. Alternative solvents, with good results of purity and recovery yields, usually have a negative impact on the molecular weight of the final polymer. In this work, the addition of a pretreatment based on non-ionic surfactants (Tween® 20, Brij® L4, and Triton™ X-114) to extract PHA from mixed microbial biomass selected on fermented agro-industrial wastes was investigated. The best results were obtained with Tween® 20 allowing for an increase in 50% compared with the use of dimethylcarbonate without any pretreatment (from 38.4 ± 0.8% to 53 ± 2%) and very close to those obtained with chloroform (63%). The extracted polymer was analysed and characterized, revealing a PHA of high purity (> 90%) and low molecular weight loss (under 24%). Additionally, a material-focused economic and a carbon footprint analysis were performed and supported the selection of the method as one of the cheapest options and with the lowest carbon footprint.

In-depth structural characterization of pentosan polysulfate sodium complex drug using orthogonal analytical tools

Anna Alekseeva, Rahul Raman, Giorgio Eisele, Thomas Clark, Adam Fisher, Sau (Larry) Lee, Xiaohui Jiang, Giangiacomo Torri, Ram Sasisekharan, Sabrina Bertini. Carbohydrate Polymers 234:115913 DOI: 10.1016/j.carbpol.2020.115913

Rapid advances have been made in developing analytical technologies for characterization of highly heterogeneous active ingredients of complex drugs, such as pentosan polysulfate (PPS), active ingredient of the drug Elmiron®, approved by the Food and Drug Administration and marketed in the United States to treat interstitial cystitis. PPS sulfated polysaccharides comprise of a repeat unit of β(1–4)‐D‐xylopyranoses randomly substituted by 4‐O-methyl-glucopyranosyluronic acid. To define the critical quality attributes (CQAs) of such a complex drug, it is critical to develop an approach that integrates data from orthogonal analytical methodologies. Here, we developed an approach integrating diverse analytical tools including gel permeation chromatography, LC/ ESI-MS and NMR to measure CQAs of PPS. The proposed mathematical framework integrates the data from these diverse analytical methods as function of PPS chain length and building blocks. Our approach would facilitate in establishing a scientific foundation for comparative characterization of drug products with complex active ingredients.

Heparanase as an Additional Tool for Detecting Structural Peculiarities of Heparin Oligosaccharides

Anna Alekseeva ,Elena Urso ,Giulia Mazzini and Annamaria Naggi. Molecules 2019, 24(23), 4403;; doi:10.3390/molecules22071116.

Due to the biological properties of heparin and low-molecular-weight heparin (LMWH), continuous advances in elucidation of their microheterogeneous structure and discovery of novel structural peculiarities are crucial. Effective strategies for monitoring manufacturing processes and assessment of more restrictive specifications, as imposed by the current regulatory agencies, need to be developed. Hereby, we apply an efficient heparanase-based strategy to assert the structure of two major isomeric octasaccharides of dalteparin and investigate the tetrasaccharides arising from antithrombin binding region (ATBR) of bovine mucosal heparin. Heparanase, especially when combined with other sample preparation methods (e.g., size exclusion, affinity chromatography, heparinase depolymerization), was shown to be a powerful tool providing relevant information about heparin structural peculiarities. The applied approach provided direct evidence that oligomers bearing glucuronic acid–glucosamine-3-O-sulfate at their nonreducing end represent an important structural signature of dalteparin. When extended to ATBR-related tetramers of bovine heparin, the heparanase-based approach allowed for elucidation of the structure of minor sequences that have not been reported yet. The obtained results are of high importance in the view of the growing interest of regulatory agencies and manufacturers in the development of low-molecular-weight heparin generics as well as bovine heparin as alternative source.

Characterization of Danaparoid Complex Extractive Drug by an Orthogonal Analytical Approach

Cristina Gardini , Elena Urso , Marco Guerrini , René van Herpen , Pauline de Wit and Annamaria Naggi. Molecules 2017, 22, 1116; doi:10.3390/molecules22071116.

Danaparoid sodium salt, is the active component of ORGARAN, an anticoagulant and antithrombotic drug constituted of three glycosaminoglycans (GAGs) obtained from porcine intestinal mucosa extracts. Heparan sulfate is the major component, dermatan sulfate and chondroitin sulfate being the minor ones. Currently dermatan sulfate and chondroitin sulfate are quantified by UV detection of their unsaturated disaccharides obtained by enzymatic depolymerization. Due to the complexity of danaparoid biopolymers and the presence of shared components, an orthogonal approach has been applied using more advanced tools and methods. To integrate the analytical profile, 2D heteronuclear single quantum coherence (HSQC) NMR spectroscopy was applied and found effective to identify and quantify GAG component signals as well as those of some process signatures of danaparoid active pharmaceutical ingredient (API) batches. Analyses of components of both API samples and size separated fractions proceeded through the determination and distribution of the molecular weight (Mw) by high performance size exclusion chromatographic triple detector array (HP-SEC-TDA), chain mapping by LC/MS, and mono- (1H and 13C) and bi-dimensional (HSQC) NMR spectroscopy. Finally, large scale chromatographic isolation and depolymerization of each GAG followed by LC/MS and 2D-NMR analysis, allowed the sequences to be defined and components to be evaluated of each GAG including oxidized residues of hexosamines and uronic acids at the reducing ends.

Enhanced polyhydroxyalkanoate (PHA) production from the organic fraction of municipal solid waste by using mixed microbial culture.

Colombo B., Favini F., Scaglia B., Sciarria TP., D'Imporzano G., Pognani M., Alekseeva A., Eisele G., Cosentino C., Adani F. Biotechnol Biofuels. 2017 Aug 22;10:201. doi: 10.1186/s13068-017-0888-8. eCollection 2017.

BACKGROUND: In Europe, almost 87.6 million tonnes of food waste are produced. Despite the high biological value of food waste, traditional management solutions do not consider it as a precious resource. Many studies have reported the use of food waste for the production of high added value molecules. Polyhydroxyalkanoates (PHAs) represent a class of interesting bio-polyesters accumulated by different bacterial cells, and has been proposed for production from the organic fraction of municipal solid waste (OFMSW). Nevertheless, until now, no attention has been paid to the entire biological process leading to the transformation of food waste to organic acids (OA) and then to PHA, getting high PHA yield per food waste unit. In particular, the acid-generating process needs to be optimized, maximizing OA production from OFMSW. To do so, a pilot-scale Anaerobic Percolation Biocell Reactor (100 L in volume) was used to produce an OA-rich percolate from OFMSW which was used subsequently to produce PHA.
RESULTS: The optimized acidogenic process resulted in an OA production of 151 g kg-1 from fresh OFMSW. The subsequent optimization of PHA production from OA gave a PHA production, on average, of 223 ± 28 g kg-1 total OA fed. Total mass balance indicated, for the best case studied, a PHA production per OFMSW weight unit of 33.22 ± 4.2 g kg-1 from fresh OFMSW, corresponding to 114.4 ± 14.5 g kg-1 of total solids from OFMSW. PHA composition revealed a hydroxybutyrate/hydroxyvalerate (%) ratio of 53/47 and Mw of 8∙105 kDa with a low polydispersity index, i.e. 1.4.
CONCLUSIONS: This work showed how by optimizing acidic fermentation it could be possible to get a large amount of OA from OFMSW to be then transformed into PHA. This step is important as it greatly affects the total final PHA yield. Data obtained in this work can be useful as the starting point for considering the economic feasibility of PHA production from OFMSW by using mixed culture.


Risonanza Magnetica Nucleare

  • High-resolution 600 MHz Bruker Avance III 600 Quadruple resonance (H/C/N/2H) High-sensitivity CryoProbe TCI 5mm
  • High-resolution 500 MHz Bruker Avance NEO 500 Quadruple resonance (H/C/N/2H) High-sensitivity CryoProbe TCI 5mm
  • High-resolution 500 MHz Bruker Avance III Inverse detection multinuclear BB probe 10mm TXI 5mm probe
  • Solid-state High-resolution Bruker Avance300 WB High-resolution magic angle spinning probe heads up to 18KHz multinuclear CP and DD X/1H probes, 4 and 7mm rotors.


Spettrometria di massa

  • UHR-QqTOF (Ultra-High Resolution Qq-Time-Of-Flight), Impact II Bruker Daltonics, (UHPLC Platin Blue Knauer)
  • ESI-Q-TOF MS, micrOTOFQ Bruker Daltonics (HPLC Dionex Ultimate 3000)
  • ESI-FT MS, Solarix high mass accuracy and resolution, Bruker Daltonics (HPLC Agilent 1100)


Determinazione pesi molecolari

  • TDA 305 Viscotek equipped with autosampler HTA 110
  • TDA 302 Viscotek
  • Omnisec System (Omnisec Resolve/Reveal) Malvern Panalytical


  • Viscotek TDA 305
  • Viscotek TDA 302
  • OMNISEC Malvern