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Biomediated production of structurally diverse poly(hydroxyalkanoates) from surplus streams of the animal processing industry

Koller M., Braunegg G.

Polimery

2015

T. 60, nr 5

298--308

For commercial success, enhanced poly(hydroxyalkanoate) (PHA) production must address both material performance and economic aspects. Conventional PHA production consumes expensive feedstocks dedicated to nutrition. Switching to carbon-rich (agro)industrial side-streams alleviates industrial disposal problems, preserves food resources, and can be economically superior. Processes developed in the recently performed EU-FP7 project ANIMPOL resort to lipid-rich surplus streams from slaughterhouses and the rendering industry; these materials undergo chemical transformation to crude glycerol phase (CGP) and biodiesel. The saturated biodiesel share (SFAE) counteracts its applicability as abiofuel but, in addition to CGP, can be converted biotechnologically to PHAs. Depending on the applied microbial production strain and the selected carbon source (SFAE or CGP), thermoplastic short chain length PHA (scl-PHA), as well as elastomeric to latex-like medium chain length PHA (mcl-PHA), can be produced from these inexpensive feed stocks. The article illustrates the biotechnological conversion of animal-based CGP and SFAE towards poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), respectively, by Cupriavidus necator strain DSM 545. SFAE conversion towards mcl-PHAs consisting of various saturated and unsaturated building blocks by two pseudomonades, Ps. citronellolis DSM 50332 and Ps. chlororaphis DSM 50083, are also shown. Together with the kinetics of the bioprocesses, the results from the characterization of isolated samples of these structurally diverse biopolyesters are compared; data demonstrate the high versatility of biopolymer properties making them applicable in various fields of the plastic market. In addition to the need for inexpensive carbon feed stocks, the article points to further hot spots of the PHA-production chain that must be considered in order to lower the overall PHA production costs, and to enhance product quality. The benefits arising from multistage continuous cultivation production set-ups, namely high-throughput production of PHA of predefined composition and constant quality, are especially discussed. Finally, contemporary approaches towards environmentally and ecologically sustainable PHA recovery from biomass are summarized.

Artykuł stanowi przegląd literatury dotyczącej biosyntezy poli(hydroksyalkanianów) (PHA) z wykorzystaniem jako surowców odpadów z przemysłu rolno-spożywczego. Omówiono wyniki uzyskane podczas realizacji projektu ANIMPOL (7. PR UE), w którym jako surowiec do syntezy PHA stosowano bogate w tłuszcze produkty uboczne z rzeźni i zakładów utylizacji odpadów zwierzęcych, przekształcone chemicznie w surową fazę glicerynową (CGP) i biodiesel (estry nasyconych kwasów tłuszczowych, SFAE). Zależnie od użytego szczepu bakterii oraz źródła węgla (SFAE lub CGP) otrzymano termoplastyczne krótkołańcuchowe PHA (scl-PHA) lub średniołańcuchowe PHA (mcl-PHA). Zaprezentowano biotechnologiczną konwersję CGP iSFAE pochodzenia zwierzęcego do poli(3-hydroksymaślanu) (PHB) i poli(3-hydroksymaślanu-co-3-hydroksywalerianu) (PHBV) za pomocą szczepu bakteryjnego Cupriavidus necator strain DSM 545, syntezę mcl-PHA zawierających nasycone i nienasycone elementy strukturalne za pomocą bakterii z rodzaju Pseudomonas (Ps. citronellolis DSM 50332 i Ps. chlororaphis DSM 50083). Omówiono kinetykę bioprocesów oraz charakterystykę otrzymanych biopoliestrów, przedyskutowano elementy cyklu produkcyjnego PHA kluczowe z punktu widzenia zmniejszenia kosztów i poprawy jakości produktów oraz korzyści wynikające z zastosowania układów ciągłej wielostopniowej hodowli w wysokowydajnej produkcji PHA o założonym składzie i stabilnej jakości. Omówiono też nowe metody odzyskiwania PHA z biomasy, zgodne z wymaganiami ochrony środowiska.

On the nature of silver ions in complex media

Loza K., Diendorf J., Sengstock C., Koller M., Epple M.

Engineering of Biomaterials

2014

Vol. 17, no. 128-129

Antimicrobial biocides are commonly used to present the growth of bacteria on surfaces and within materials. They are typically added in small quantities to many applications to prevent bacterial growth on the treated object. Silver is increasingly used in many applications due to the aim to replace organic chemical agents by inorganic additives. Examples of applications are bacteriostatic water filters for household use or swimming pool algaecides and numerous devices, ranging from consumer commodities like mobile phones, refrigerators, and clothes to medical devices like catheters, implant surfaces, and plasters. To meet the diversity of application types, many different forms of silver compounds have been developed to serve this market. In particular, there is little information on the types of transformations that silver nanoparticles will undoubtedly undergo in real, complex environments during long-term aging, and the impact of these transformations on their distribution in the environment, bioavailability, and toxicity potential. The biocidal action results from the interaction of silver ions with bacteria. The most potent compounds for a high silver release are soluble silver salts like silver nitrate or silver acetate. These are fully water soluble with a high silver ion release rate. Therefore they are often used as control in cell experiments to elucidate the biological effect of silver nanoparticles. However, in the case of free silver nanoparticles the interactions can be more complex and catalytic reactions on the particle surface which depend on the size and shape of the nanoparticles can render the system very complex. If AgNO3 is used as control, it is tacitly assumed, that the free silver ion concentration is the same as that in the added AgNO3. This obviously cannot be true because of the presence of a whole set of proteins, biomolecules and inorganic ions like Cl- and H2PO4- in the biological medium. These will react with the silver ions in one or the other way. We report on experiments on the behaviour of silver ions in biologically relevant concentrations in different media, from physiological salt solution over phosphate-buffered saline solution to cell culture media. For dissolution and immersion experiments PVP-coated silver nanoparticles were synthesized by reduction with glucose in the presence of PVP. The final silver concentration in all dispersions was determined by atomic absorption spectroscopy. The dissolution of silver nanoparticles was followed in long-term experiments out of a dialysis tube which was permeable only for silver ions. In case of immersion experiments, the nanoparticles and all precipitates were isolated by ultracentrifugation, redispersed in pure water and again subjected to ultracentrifugation. The particles were analyzed by scanning electron microscopy, energy-dispersive X-ray spectroscopy and X-ray powder diffraction. The dissolution requires the presence of dissolved oxygen. If no oxygen is present, only a very small fraction of silver is dissolved, possibly by traces of oxygen in the experimental setup. An oxidizing agent like H2O2 clearly enhances the dissolution. The presence of NaCl, either in pure form or as PBS, strongly slows down the dissolution, probably due to silver chloride formation. Cysteine has a clearly inhibiting effect with almost no dissolution of the silver nanoparticles whereas glucose has a decelerating effect but leads to a similar final dissolved fraction. This suggests that cysteine adsorbs onto the silver nanoparticle surface with its thiol group and prevents the oxidation. In contrast, glucose slows down the dissolution, but clearly did not prevent the oxidation on a longer time scale. We have extended the studies by mixing silver nanoparticle dispersions with different media of increasingly biological nature. The solutions/dispersions were stirred for equilibration and then subjected to ultracentrifugation. All precipitates and nanoparticles were isolated by this way and then analyzed. The results show that both initially present silver ions and released silver ions are mainly precipitated as AgCl if chloride is present. Only in the absence of chloride, glucose is able to reduce Ag+ to Ag0. The initially present silver nanoparticles were recovered in all cases. Silver phosphate was not observed in any case, probably due to the moderate pH (around 7) at which phosphate is mostly protonated to hydrogen phosphate and dihydrogen phosphate. We can conclude that released silver ions precipitate mostly as AgCl in biological media, and that most cell culture studies where silver ions are used as control are in fact studying the effect of colloidal silver chloride on the cells. To prove this assumption, human mesenchymal stem cells (hMSC) were cultured in the presence of silver chloride nanoparticles (diameter 120 nm), and the viability of the cells was analyzed by fluorescence microscopy. In general, we clearly observed that pure silver nanoparticles have lower toxicity to hMSC compared to silver chloride nanoparticles with a comparable total silver dose. Silver acetate in the biological medium had a comparable toxicity to hMSC compared to silver chloride nanoparticles with the same total silver dose.