Recent Advances in Biotechnology

The present book chapter is aimed at providing an outlook about the synthesis, properties and potential applications of bacterial Poly[(R)-3-hydroxybutyrate)] (PHB) and its copolymer with (R)-3-hydroxyvalerate (PHBHV). Selected reports were analyzed in order to provide strategies aimed at producing standard formulations based on PHB and PHBHV by given emphasis on packaging applications. Modification of PHB and PHBHV properties by using reinforcing agents and blends with biodegradable and non-biodegradable polymers has been reviewed. The strategies for modulation and/or standardization of PHB and PHBHV, in composite formulations and blends, were done by taking into account thermo-mechanical properties, dimensional stability and melt flow index data. Odor control in the end-products by using additives and potential methodologies to mitigate it as well as guidelines for the production of odorless, standard and biodegradable PHB and PHBHV based products for packaging were also provided.

To fulfill the many expectations placed upon polymers and plastics to remain competitive and acceptable compared to other materials, they must constantly improve in their functional properties and their cost/benefit ratio. More recently it has also become imperative that the environmental burdens caused by polymers and plastics be reduced and that overall sustainability of these materials be raised. Today the environmental aspect of a material are one of the key factors for assessing its acceptability and for making decisions about its use.

Thus there is now a greater emphasis on evaluating the environmental impacts of polymers and plastics. Using the life cycle assessment method polymers and plastics are being compared to each other and to other materials. The environmental emphasis has also prompted active development of new, or in some cases reengineered, biobased and biodegradable polymers and plastics. During the last decade these materials have moved from research laboratories into commercial production and represents one of the fastest growing niche segments in plastics, although the overall quantity still remains relatively low.

Now that bioplastics have been successfully launched and real-life experiences from early applications have been obtained, their sustainability is undergoing a thorough examination. The results show that these new biomaterials cause environmental burdens similar to those caused by conventional plastics. In general, bioplastics offer reductions in emissions of greenhouse gases and the use of fossil resources, whereas the production of bioresources from farming contributes to higher acidification and eutrophication burdens. End-of-life waste management can also strongly influence the overall results. It is expected that future developments in technology and organization and the use of second generation bioresources will improve the environmental profile of bioplastics. Bioplastics can contribute to reaching policy goals e.g., regarding the reduction of greenhouse gases but they must be used properly to achieve the desired sustainability benefits.

Process/strain optimization procedures are unavoidable in the improvement of large-scale bioproduction processes. These procedures are necessary on macroscopic (bioreactor) level and on biochemical/genetic level of producing strains. Inter alia, mathematical modeling is one of the applicable and useful methods in such procedures. Mathematical models applied for PHA biosynthesis (classified as both structured and unstructured) are denominated as formal kinetic, low-structured, dynamic, metabolicor high-structured, cybernetic, and hybrid type models, or neural networks. In the chapter at hand, each specific group of models is discussed in the light of its applicability and benefit for increased productivity, enhancing of the specific PHA biosynthesis rate, and better understanding of the intracellular metabolic regulation systems. Characteristics of production strains, particularities of mixed microbial cultures and features of industrial-scale plants cannot be described by a single type of mathematical model since it is not possible to address all the different requirements by a single model type. Therefore, it is more than necessary to fine-tune the modeling approach to each actual case in a sophisticated way. Formal-kinetic and “lowstructured” models that are relatively simple and display low computational demand, are applicable for simple cases and are beneficial for mathematical embodiment of “standard microbial cultivations and practices”. Hybrid models are used by some authors to address certain deficiencies of diverse types of models. In this context, satisfying, compromised, perhaps most promising solutions can be reached if mechanistic, cybernetic, computational fluid dynamics (CFD) and neural models were combined. A hybrid modeling strategy like this generates a holistic representation of solutions for the total PHA biosynthesis process, including all advantages of the different modeling schemes. For example, application of growth media of complex composition usually entails a higher degree of model organization. For the future, really existent biotechnological systems are expected to be expressed by hybrid models of high organization.

Major attention was dedicated to the use of elementary flux modes (EFMs) and yield space analysis (YSA) to develop metabolic models of PHA biosynthesis. The implementation of these methods is reported for numerous case studies which involve modeling of metabolic networks. The chapter concludes with some case studies, where the implementation of EFMs and YSA performed as a powerful modeling tool. It includes the description of intracellular PHA generation and mobilization in the organism Cupriavidus necator, the limitation-based picture of the steady-state flux cone of the organism´s metabolic network, the detailed analysis of a multi-stage bioreactor cascade dedicated to continuous PHA production, and metabolic flux investigation in the metabolism of C. necator cultivated using glycerol.

Considerable interests and widespread studies in the development of biodegradation of plastic materials have been carried out in order to overcome the environmental problems associated with petrochemical plastics waste. Among the various biodegradable polymers, poly(3-hydroxybutyrate) (PHB) is an attractive substitute for conventional petrochemical plastics due to similar properties to thermoplastics and elastomers, and complete biodegradability upon disposal under various environments. PHB is the most famous member of polyhydroxyalkanoates (PHAs) and can be accumulated as an intracellular carbon/energy source for various microorganisms. Synthesis of these distinct granules occurs when there is a growth limiting component in the presence of excess carbon source. The use of PHB in a wide range of applications has been hampered mainly by their high production cost compared with petrochemical based polymers. The fermentation performance, carbon substrate, the isolation of new microorganisms with high growth rate and potential of production as well as yield and recovery method affect the production cost of PHB. To overcome mentioned problems, knowing the specific strategies such as controlled systems is necessary. In controlled cultivation- and production systems like bioreactors, high cell density occurred. Hydrodynamic and mass transfer behaviors are important in gas liquid bioreactors. Oxygen transfer and residence time distribution must be monitored on-line. The mixing characteristics of gas liquid bioreactors are often intermediates between the characteristics of plug-flow and well mixed flow. This phenomenon is modeled using two methods: the axial dispersion model and tanks-in series model. In this chapter after a glance introducing of PHB and producing bacteria which grow on different carbon sources in batch, fed-batch, and continuous systems have been reviewed and compared to each other from the productivity point of view. Also, a special interest has been done on inhibition kinetics as well as modelling of gas and mass transfer in different bioreactors.

Polyhydroxyalkanoate (PHA) production cost is dependent on several factors like substrate, chosen production strain, cultivation strategy and, to a still underestimated extent, on the downstream processing needed to recover PHA from microbial biomass. The availability of green and cheap technologies for PHA recovery is crucial for the development of a reliable and sustainable PHA production chain. Inexpensive and scalable recovery schemes need to be devised to achieve low-cost production that is competitive with traditional thermoplastics. Hence, in order to maximize both biomass growth and PHA productivity, one has to carefully optimize the operation conditions; this aspect of bioprocesses is of major economic importance in a bioprocess. PHA, a group of biobased microbial biopolyesters subjectable towards biodegradation constitute promising candidates to potentially substitute diverse conventional petrol-based plastics. As a drawback, PHA´s too high production cost still hampers their success on the on the market. The most prominent cost generating factors are the upstream processing, the bioprocess (fermentation), and, last, but not least, thus downstream processing dedicated to PHA recovery from microbial biomass. Downstream processing severely accounts to the economics of the overall process. A broad range of diverse recovery techniques can be found in the literature, most of them studied exclusively on small bench scale, with some exceptions which performed well also during industrial operation. Recovery by solvent extraction, chemical or enzymatic digestion of non-PHA cellular material, mechanical disintegration of cells, supercritical fluid extraction, flotation techniques, gamma irradiation and, more recently, the use of aqueous two-phase systems are reported. The present chapter summarizes investigated recovery methods and compares them in terms of efficacy and resulting product quality (e.g., purity and impact on molar mass).

Microbial polyester polyhydroxyalkanoate (PHA) is a carbon-neutral and environmental-friendly material with high commercial value due to its biodegradability and biocompatibility properties. PHA was originally thought to comprise of a single monomeric repeat unit of 3-hydroxybutyrate, but this notion has been since overthrown with the discovery of other chemically-distinct PHA monomers. To date, more than 150 PHA monomers have been documented. A PHA molecule may consist of two or more PHA monomers, endowing PHA polymer with high chemical diversity. This enables PHA materials of varied properties to be produced and tailored for applicationspecific purposes. However, the sheer number of chemically-diverse PHA monomers has also made the task of PHA analysis an extremely challenging one. Numerous techniques have been exploited for the detection, quantification, and characterization of microbial intracellular PHA and PHA polymers. New techniques are also continuously being developed with advancing instrumentation capabilities. This book chapter introduces the basic working principles underlying current and emerging PHA analytical techniques, and summarizes key protocols and information related to these techniques. The potential applications of emerging techniques are also highlighted and discussed.

Polyhydroxyalkanoates (PHAs) are bio-based polyesters synthesized by bacteria as an intracellular storage material. PHAs can be produced from renewable biomass without using fossil resources, and thus are environmentally friendly plastics. The most common PHA, poly[(R)-3-hydroxybutyrate] [PHB or P(3HB)], can be synthesized in large quantities by bacterial fermentation, but has rigid and brittle properties. Therefore, attempts have been made to improve its material properties. This chapter focuses on the recent progress in improving two types of PHA, 3HB-based copolymers and unusual PHA homopolymers, which show improved material properties and/or cannot be synthesized in nature. 3HB-based copolymers, that not only includes 3-hydroxyvalerate, 3-hydroxyhexanoate, and long chain 3-hydroxyalkanoatecontaining copolymers, but also 3-hydroxy-4-methylvalerate, 3-hydroxy- 3-phenylpropionate, 3-hydroxy-2-methylbutyrate, and lactate-containing copolymers have been reviewed. Additionally, ultrahigh-molecular-weight PHB and mediumchain- length PHA homopolymers are highlighted as unusual homopolymers. These polymers have notable characteristics and are expected to expand the range of PHA applications.

Crystalline structure and morphology of the solid state of thermoplastic semi-crystalline polymers strongly affect their properties and performances. This is particularly evident on mechanical properties of poly[(R)-3-hydroxybutyrate] (PHB), and its copolymers, blends, composites, and nanocomposites. The organization and the morphology of the crystals and the interconnection between the crystalline and the amorphous regions can influence and govern the mechanical properties of PHB based materials.

The presence of a rigid amorphous fraction, which produces a stiffening of the amorphous segments at the crystal/amorphous interface, can contribute to the progressive change of the mechanical properties in PHB based materials. Properties of PHB based materials may be tuned and controlled by blending with other polymers, by addition of properly selected plasticizers, nucleating agents, as well as by addition of fillers and nanofillers.

In the present Chapter the influence of the morphology and the crystalline content on the mechanical properties of PHB based materials is analyzed and discussed.