Protocols for isolation and analysis of polyhydroxyalkanoates

Some storage compounds such as polyhydroxyalkanates have been drawing considerable attention due to their similar properties to those of conventional plastics. Therefore many efforts have been done to develop and improve analytical methods to isolate and characterize these biopolymers. This chapter pretends to give easy guidelines in all the steps for recovering and characterizing these compounds

Natural polymers ::a source of inspiration

An understanding of how natural materials perform and how their assembly processes work is being applied to guide the design and performance of medical materials at the macro-, micro-, and nanoscale levels. This improved knowledge of natural and hierarchical structures along with advances in polymer science and nanotechnology have become the drivers for the synthesis of biomimetic materials. Highly sophisticated material performances can be achieved, eg, material self-assembly and self-healing, specific adhesion or repelling properties, biocompatibility in combination with biodegradability that broaden the specific uses in medicine. In this chapter, we describe key materials and assembly processes in nature and also cover their applications in three fields of bioengineering, namely, scaffolds for tissue engineering, biomedical adhesives, and smart drug delivery systems

Robust at-line quantification of poly(3-hydroxyalkanoate) biosynthesis by flow cytometry using a BODIPY 493/503-SYTO 62 double-staining

Poly(3-hydroxyalkanoates) (PHAs) are bio-based and biodegradable polyesters which have been considered as a promising alternative to petrol-based plastics. Their bacterial production is a dynamic process in which intracellular polymerization and depolymerization are closely linked and depend on the availability of carbon substrates and other nutrients. These dynamics require a fast and quantitative method to determine the optimal harvest-time of PHA containing cells or to adjust carbon supply. In principle, flow cytometry (FCM) is an ideal tool that suits these requirements and, in addition, provides data on the PHA content of different cell populations. However, FCM-based PHA quantification methods have often relied on laborious sample preparation including washing steps and long incubation times. Here, we introduce a fast method based on double-staining using BODIPY 493/503 for PHA staining and SYTO 62 for DNA that allows acquiring reliable fluorescence and cell count data in < 10 min. Finally, fed-batch experiments with Pseudomonas putida KT2440 and Rhodospirillum rubrum S1 revealed that the method was robust and independent of the strain and type of PHA (medium-chain-length [mcl-] and short-chain-length [scl-] PHA, respectively). Interestingly, the specific PHA fluorescence was in case of mcl-PHA larger than for scl-PHA, probably reflecting the different material properties (e.g., specific density, hydrophilicity and crystallinity)

Tight coupling of polymerization and depolymerization of polyhydroxyalkanoates ensures efficient management of carbon resources in Pseudomonas putida

Environmental microbes oscillate between feast and famine and need to carefully manage utilization, storage and conversion of reserve products to exploitable sources of carbon and energy. Polyhydroxyalkanoates (PHAs) are storage polymers that serve bacteria as sources of food materials under physiological conditions of carbon demand. In order to obtain insights into the role of PHA depolymerase (PhaZ) and its relationship to a PHA polymerase (PhaC2) in the carbon management activity of Pseudomonas putida strain U, we created a polymerase hyperexpression strain and a depolymerase knockout mutant of this strain, and examined their synthesis of PHA and expression of their PHA genes. This study revealed that hyperexpression of PhaC2 led to the accumulation of higher amounts of PHA (44%wt) than in the wild-type strain (24%wt) after 24 h of cultivation, which then returned to wild-type levels by 48 h, as a result of elevated depolymerization. The phaZ mutant, however, accumulated higher levels of PHA than the parental strain (62%wt), which were maintained for at least 96 h. Transcriptional analysis of the pha cluster by RT-PCR revealed that PHA operon proteins, including depolymerase, are expressed from the beginning of the growth phase. Hyperexpression of the PhaC2 polymerase was accompanied by an increase in the expression of the PhaZ depolymerase and a decrease in expression of another PHA polymerase, PhaC1. This suggests tight regulatory coupling of PHA polymerase and depolymerase activities that act in synergy, and in concert with other PHA proteins, to provide dynamic PHA granule synthesis and remodelling that rapidly and sensitively respond to changes in availability of carbon and the physiological-metabolic needs of the cell, to ensure optimal carbon resource management

Identification of oxylipins with antifungal activity by LC-MS/MS from the supernatant of Pseudomonas 42A2.

In microorganisms hydroxy fatty acids are produced from the biotransformation of unsaturated fatty acids. Such compounds belong to a class of oxylipins which are reported to perform a variety of biological functions such as anti-inflammatory or cytotoxic activity. These compounds have been found in rice and timothy plants after being infected by specific fungus. When grown in submerged culture with linoleic acid, Pseudomonas 42A2 accumulated in the supernatant several hydroxy fatty acids. In this work LC-MS/MS has been used to elucidate the structure of the components form the organic extract: 9-hydroxy-10,12-octadecadienoic acid; 13-hydroxy-9,11-octadecadienoic acid; 7,10-dihydroxy-8E-octadecenoic acid; 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid. Antimicrobial activity against several pathogenic fungal strains is presented: MIC (microg/mL) Verticillium dhaliae, 32; Macrophonia phaesolina, 32; Arthroderma uncinatum, 32; Trycophyton mentagrophytes, 64

MIM of nickel-free nitrogen strengthened austenitic stainless steel from bipolymer based feedstock

Nickel-free stainless steels are of high interest for medical and dental applications, as well as consumer goods where contact with the skin is a consideration. In this paper Prof Efraín Carreño-Morelli and colleagues from the University of Applied Sciences and Arts Western Switzerland evaluate the MIM processing of the high-nitrogen content austenitic stainless steel P558. This is processed using a novel environmentally friendly binder that incorporates a polymer produced by bacterial fermentation

Chemical modification of polyhydroxyalkanoates (PHAs) for the preparation of hybrid biomaterials

Polyhydroxyalkanoates (PHAs) are biopolyesters produced by bacteria as intracellular granules under metabolic stress conditions. Many carbon sources such as alkanes, alkenes, alcohols, sugars, fatty acids can be used as feedstock and thus a wide variety of polyesters and monomer units can be potentially synthetized. The work presented here describes the process to chemically modify such biopolymers in order to render them readily available for the preparation of bio-molecular conjugates as promising new classes of biocompatible biomaterials. Such hybrid biomaterials belong to the rapidly growing class of biocompatible polymers, which are of great interest for medical and therapeutic applications. In this work, the biosynthesis of a new PHA homopolymer and the chemical modification, an epoxidation reaction, are described

Design and characterization of conductive biopolymer nanocomposite electrodes for medical applications

Metal-based electrodes, despite being the most widely used for biomedical applications, are limited by a poor reliable skin-surface interface and patients suffer from comfort issues. The most common problems/inconveniences are caused by stiff electrodes, skin irritation, allergic reaction or corrosion. In order to overcome these problems, we produced and tested flexible electrodes involving biopolymer nanocomposite materials. Conductive polymers have been intensively studied and applied in the field of organic photovoltaics and flexible organic electronics. Recently, the use of conductive biopolymer nanocomposite has also emerged as an interesting and promising material for biomedical applications. In this study, we have designed and characterized electrodes made of a flexible and conductive nanocomposite material using a biocompatible and biodegradable polymeric matrix of poly (3- hydroxyalkanoate) (PHA, in particular poly (3-hydroxybutyrate), PHB) containing conductive nanowires. The biopolymer nanocomposites and their electrical conductivities were investigated by optical microscopy, scanning electron microscopy (SEM) and electrical four-point probing. The electrical conductivities obtained in the different PHA-polymer nanocomposites containing different concentrations of conductive additives is discussed in relation to the nanocomposite structure at the microscopic level. Finally, our developed biopolymer nanocomposite prototype electrodes have successfully been tested for transcutaneous electrical nerve stimulation (TENS) and electrocardiography ECG applications in comparison to conventional electrodes

A systematic experimental and computational analysis of commercially available aliphatic polyesters

Aliphatic polyesters, such as polylactic acid (PLA), polyglycolic acid (PGA), and their copolymer polylactic-co-glycolic acid (PLGA) have become an established choice in the biomedical field in a wide range of applications, from nanoparticles for local drug delivery to bone fixation screws, and, hence, in a huge spectrum of uses in different medical devices currently available on the market worldwide. The reason for their popularity lies in their combination of interesting peculiarities: in situ degradation, intrinsic biocompatibility (degradation products are recognized and metabolized), processability with standard industrial technologies, and tailorable properties. The knowledge of the degradation rate is an essential requirement for optimal device design when, e.g., fast adsorption time is required, or mechanical properties must be assured over a given time span. In this regard, experimental studies can be time- and money-consuming, due to the time scales (weeks–months) involved in the hydrolysis process. This work aims at providing to both industry and academia robust guidelines for optimal material choice through a systematic experimental and computational analysis of most commonly used PLGA formulations (selected from commercially available products), evaluating the degradation kinetics and its impact on polymer properties

Novel RP-HPLC based assay for selective and sensitive endotoxin quantification

The paper presents a novel instrumental analytical endotoxin quantification assay. It uses common analytical laboratory equipment (HPLC-FLD) and allows quantifying endotoxins (ETs) in different matrices from about 109 EU / mL down to about 40 EU / mL (RSE based). Test results are obtained in concentration units (e.g. ng ET / mL), which can then be converted to commonly used endotoxin units (EU / mL) in case of known pyrogenic activity. During endotoxin hydrolysis, the endotoxin specific rare sugar acid KDO is obtained quantitatively. After that, KDO is stoichiometrically reacted with DMB, which results in a highly fluorescent derivative. The mixture is separated using RP-HPLC followed by KDO-DMB quantification with a fluorescence detector. Based on the KDO content, the endotoxin content in the sample is calculated. The developed assay is economic and has a small error. Its applicability was demonstrated in applied research. ETs were quantified in purified bacterial biopolymers, which were produced by Gram-negative bacteria. Results were compared to LAL results obtained for the same samples. A high correlation was found between the results of both methods. Further, the new assay was utilized with high success during the development of novel endotoxin specific depth filters, which allow efficient, economic and sustainable ET removal during DSP. Those examples demonstrate that the new assay has the potential to complement the animal-based biological LAL pyrogenic quantification tests, which are accepted today by the major health authorities worldwide for the release of commercial pharmaceutical products