The Handbook of Polyhydroxyalkanoates

Microbial Biosynthesis and Feedstocks

Martin Koller

• Publisher: CRC Press
• Year: 2020
• eBook ISBN: 9781000173659
• Edition: 1
• Topic: Medicine
• Subtopic: Biotechnology in Medicine

The first volume of the "Handbook of Polyhydroxyalkanoates (PHA): Microbial Biosynthesis and Feedstocks" focusses on feedstock aspects, enzymology, metabolism and genetic engineering of PHA biosynthesis. It addresses better understanding the mechanisms of PHA biosynthesis in scientific terms and profiting from this understanding in order to enhance PHA biosynthesis in bio-technological terms and in terms of PHA microstructure. It further discusses making PHA competitive for outperforming established petrol-based plastics on industrial scale and obstacles for market penetration of PHA. Aimed at professionals and graduate students in Polymer (plastic) industry, wastewater treatment plants, food industry, biodiesel industry, this book

Covers the intracellular on-goings in PHA-accumulating bacteria

Assesses diverse feedstocks to be used as carbon source for PHA production including current knowledge on PHA biosynthesis starting from inexpensive waste feedstocks

Summarizes recent relevant results dealing with PHA production from various organic by-products

Presents the key elements to understand and fine-tune the microstructure and sequence-controlled molecular architecture of PHA co-polyesters

Discusses the use of CO-rich syngas, sourced from various organic waste materials, for PHA biosynthesis

The Handbook of Polyhydroxyalkanoates, Volume 1: Introduction by the Editor

Nowadays, it is generally undisputed that we need alternatives for various fossil-resource based products such as plastics, which make our daily life indeed comfortable. Plastics, per definitionem a group of synthetic polymeric materials typically not produced by Mother Nature, are currently produced at increasing quantities, now in a magnitude of about 400 Mt per year. Such plastics, which are manufactured by well-established technologies, are used in innumerable fields of application, such as packaging materials, parts in the automotive industry, sports articles, biomedical devices, electronic parts, and many more. Despite their undoubted contribution to facilitating our all-day life, current plastic production is associated with essential shortcomings, such as the ongoing depletion of fossil resources, growing piles of waste consisting of non-degradable full-carbon-backbone plastics, microplastics accumulating in marine and other aquatic environments and also in food, and elevated CO₂ and toxin levels in the atmosphere generated by plastic incineration.

The last few decades have been dedicated to finding a way out of the fatal “Plastic Age” we live in today and to overcoming the above-mentioned evils. This goes in parallel with current political regulations in diverse global regions, such as the European Strategy for Plastics in a Circular Economy by the European Commission, or the forthcoming ban on disposable plastic items in megacities of PR China, which was announced just the other day (January 2020).

Switching from petrol-based plastics to bio-alternatives with plastic-like properties, which are based on renewable resources, and which can be subjected to biodegradation and composting, is regarded as one of these exit strategies. In this context, polyhydroxyalkanoates (PHA), microbial storage materials produced by numerous eubacterial and archaeal prokaryotes, are, to an increasing extent, considered auspicious candidates to replace traditional plastics in several market sectors, such as the packaging field, or even in sophisticated biomedical applications.

However, to make PHA competitive, they must cope with petrol-based plastics both in terms of quality and in economic aspects. Quality improvement of PHA-based materials is currently achieved by advanced microbial feeding strategies during the biosynthesis, by the generation of (nano)composites with diverse compatible and often cost-efficient (nano)filler materials, by blending with other suitable polymers, or development of novel (nano)composite materials. Importantly, the entire PHA production chain, encompassing the isolation of new robust production strains, improvement of the strains by means of genetic engineering, understanding the enzymatic machinery of PHA anabolism and catabolism, feedstock selection, fermentation technology, process engineering, and bioreactor design, and, last but not least, the downstream processing, needs to meet the criteria of sustainability. Hence, despite the myriad of premature praise articles found in the current literature, PHA and other “plastic-like” biopolymers cannot be regarded as the one and only panacea to solve the global plastic problem! The previously often-cited myth of biopolymers being intrinsically more sustainable than established petrochemical plastics nowadays has finally been abandoned by most serious scientists. This means that without considering and conceiving the entire life cycle of biopolymers like PHA and the products produced thereof, it is impossible to conclude a priori if they inherently outperform their petrochemical counterparts in terms of environmental benefit. This is only possible by using modern tools of cradle-to-grave life cycle assessment and holistic cleaner production studies.

Such economic, sustainability, and quality aspects are dealt with in the 42 chapters of this Handbook of Polyhydroxyalkanoates, which consists of carefully selected contributions by differently focused research groups, all of them belonging to the top global cohort regarding their individual PHA-related expertise. In general, the book consists of chapters each dedicated to one of the subsequently listed three major objectives:

1. a) How to better understand the mechanisms of PHA biosynthesis in scientific terms (genetics background, enzymology, metabolomics, “synthetic biology” approaches for engineering PHA production strains in a more effective way, etc.), and profiting from this understanding in order to enhance PHA biosynthesis in biotechnological terms and terms of PHA microstructure?
2. b) How to make smart materials based on PHA to be used for defined applications, both in the bulk and niche sector?
3. c) How to make PHA competitive for outperforming established petrol-based plastics on the industrial scale? What are the obstacles to market penetration of PHA?

In principle, these three major questions, especially (a) and (c), are treated by 15 contributions in the present volume 1 of The Handbook of Polyhydroxyalkanoates, which are dedicated to the subsequent central thrusts of PHA research.

Enzymology/Metabolism/Genome Aspects for Microbial PHA Biosynthesis

This broad topic covers the intracellular processes in PHA-accumulating microorganisms. As a core part of the entire book, it is covered by a total of seven chapters, which, of course, are also somehow related to substrate aspects described in the subsequent feedstock-focused chapters.

The first chapter in this section, provided by Maierwufu Mierzati and Takeharu Tsuge, describes the action of the enzymatic machinery involved in PHA biosynthesis by different microbial production strains. This encompasses a range of different biocatalysts, which finally provide the activated building blocks (monomers) that undergo polymerization by different PHA synthase enzymes; here, the focus is dedicated to hydroxyacyl-coenzyme A (HA-CoA) generation and subsequent polymerization of the hydroxyacyl moiety in HA-CoA as the two most essential elements in PHA biosynthesis. This chapter, for sure, is pivotal to understand both the interrelation between the synthetic mechanism of PHA formation and PHA’s material properties.

A comprehensive chapter by Mariela P. Mezzina, Daniela S. Alvarez, and M. Julia Pettinari deals with physiological and metabolic aspects of PHA granules (“carbonosomes”), which constitute de facto organelles in prokaryotic microbes; these granules possess fascinating, complex attitudes regarding their composition and formation. The readers will learn that these “carbonosomes” are by far more than just simple “bioplastic inclusions in bacteria,” and will obtain a deep insight into the broad range of PHA granule-associated proteins essential for in vivo PHA formation, such as synthases, polymerases, or phasins.

A genetically focused chapter by Parveen K. Sharma et al. from David Levin’s team reviews the genomics and genetics of short-chain-length (scl-) PHA synthesis by Cupriavidus necator H16 (formerly Ralstonia eutropha H16) with the completely deciphered genome, PHA synthesis by recombinant C. necator strains, the genomics and genetics of medium-chain-length (mcl-) PHA synthesis by Pseudomonas putida and other Pseudomonas species, PHA synthesis by recombinant Pseudomonas species, and the genomics and genetics of PHA synthesis by Halomonas sp. and recombinant Escherichia coli strains. Special emphasis is dedicated to the different groups of PHA synthases found in individual PHA production strains. It is shown that genome analysis of PHA producers steadily identifies new genes; in the future, this knowledge should definitely be tapped for manipulating and advancing PHA production!

In the case of mcl-PHA, the enzymatic machinery and the metabolic pathways toward mcl-PHA differ considerably if compared to the events observed during scl-PHA biosynthesis; therefore, a specialized chapter by Maria Tsampika Manoli and other researchers associated with Auxiliadora Prieto is dedicated to the molecular basis of the PHA machinery in the best-known mcl-PHA producer, Pseudomonas putida, focusing on the involved genes, and diverse factors involved in the expression of the genes relevant for mcl-PHA biosynthesis.

Beyond that, PHA production by Paraburkholderia and Burkholderia species is reviewed in a separate chapter written by Natalia Alvarez-Santullano and colleagues from Michael Seeger’s team. Here, the genes encoding enzymes and proteins involved in PHA metabolism by these powerful strains are presented, and the metabolic routes of PHA homo- and heteropolyester synthesis and the metabolism of substrates that are used by Paraburkholderia and Burkholderia to produce PHA are presented. Biotechnological applications, including biomedical uses of bacterial PHA produced by exactly these microbial species, are discussed.

The next highly specialized chapter by Lorenzo Favaro and colleagues from the team of Marina Basaglia and Sergio Casella summarizes recent relevant results dealing with PHA production from various organic byproducts by means of genetically engineered microbial strains. The most relevant and recent genomic tools for the genetic modification are initially described, with emphasis on hosts, genes, plasmids, promoters, and gene copy numbers. This chapter deals with two principal approaches in this direction, namely the engineering of highly efficient PHA producing microorganisms for their use of waste streams (“make the PHA producer convert an inexpensive substrate”), and the engineering of bacteria naturally able to use complex and inexpensive carbon sources, but unable to produce PHA (“make a converter of an inexpensive substrate accumulate PHA”).

Because the composition of both scl-PHA and mcl-PHA on the molecular level and the exact microstructure of PHA (blocky structured PHA vs. random distribution of the monomers in heteropolyesters) are of major significance for the material properties and workability of PHA, an individual chapter by Camila Utsunomia, Nils Hanik, and Manfred Zinn covers the biosynthesis and sequence control of scl-PHA and mcl-PHA. This chapter presents the key elements to be considered in order to understand and fine-tune the microstructure and sequence-controlled molecular architecture of PHA copolyesters, including feeding regimes, genetic engineering of production strains, and artificial genetic networks.

Feedstocks

Eight chapters deal with the assessment of diverse feedstocks to be used as a carbon source for PHA production. Importantly, these feedstocks constitute carbonaceous (agro)industrial waste streams (lignocelluloses, waste glycerol, starchy waste, surplus whey, molasses, CO₂, CH₄, etc.) or their volatile follow-up products like syngas or biogas.

In this context, a comprehensive overview chapter by Sebastian Riedel and Christopher Brigham summarizes the current knowledge on PHA biosynthesis, starting from inexpensive waste feedstocks. Focus is dedicated to available industrial waste from agriculture and food processing as inexpensive feedstocks for PHA production, the types of polymers that are made of them, and the possibility of upscaling these processes to enable large-scale, low-cost PHA production.

The second chapter in this section, provided by Neha Rani Bhagat et al. from the team of Geeta Gahlawat addresses the fact that crude glycerol is a byproduct of many industrial processes, such as biodiesel production, and huge surplus amounts of it are released into the environment as waste, thereby necessitating the search for new methods of its utilization. These authors present the challenges, benefits, and drawbacks of PHA biosynthesis based on crude glycerol stemming from diverse, inexpensive resources by application of different microbial production strains and focus on the different types and properties of the generated PHA biopolyesters.

Another chapter by Manoj Lakshmanan and colleagues from the group of Kumar Sudesh discusses the use of vegetable oils and its byproducts, including oils without nutritional value, by various bacterial strains for PHA biosynthesis. This includes the production of PHA by both wild-type and genetically engineered bacterial strains. The potential application of these strain-substrate combinations for large-scale PHA production at low cost is also discussed in this chapter.

One specialized chapter in this feedstock section, provided by Chris Dartiailh and other associates of David Levin, reviews the synthesis of mcl-PHA using long-chain fatty acids (LCFAs) from different waste oils, highlighting the influence of selected substrates, carbon loading, and bioreactor systems on the yields of mcl-PHA in bacteria, the monomer composition, and the properties of synthesized biopolyesters. Emphasis is dedicated to functionalization and cross-linking of vinyl moieties present in obtained mcl-PHA to generate new biomaterials, and optimization of mcl-PHA production using LCFAs as feedstocks.

As a rather exotic, but currently emerging topic, even follow-up products of spent petrochemical plastics treated by chemo-biotechnological processes can be used as raw materials for microbial “bioplastic” production. Such “upcycling” of plastic waste to biodegradable polymers could indeed be part of the new circular economy paradigm. This chapter by Tanja Narancic and colleagues from Kevin E. O’Connor’s team also provides a detailed, unprecedented analysis of the metabolic background of microbes being able to convert follow-up products of traditional spent plastics to PHA biopolyesters.

Among gaseous C1-substrates used for PHA biosynthesis, CO₂ from industrial effluent gases is of increasing interest and therefore is handled in a chapter comparing chemoheterotrophic with solar-based photoautotrophic PHA production. This chapter by Ines Fritz, Katharina Meixner, Markus Neureiter, and Bernhard Drosg provides an intriguing insight into the current state of autotrophic PHA biosynthesis by cyanobacteria, with a strong focus dedicated to the comparison of photoautotrophic and chemoheterotrophic PHA biosynthesis. Most of all, a frank discussion on the potential of CO₂-based PHA production for industrial implementation is provided.

In the context of gaseous substrates, the next chapter focuses on the use of the C1-compound methane for PHA production by type II methane-oxidizing α-proteobacteria. Here, it is shown by Yadira Rodríguez and colleagues from the research group of Raúl Muñoz how biopolyester production can be coupled to biogas generation based on anaerobic digestion of inexpensive organic waste materials. Importantly, it is shown in this chapter by techno-economic an...

Table of contents

• Cover
• Half-Title
• Title
• Copyright
• Dedication
• Contents
• Foreword I
• Foreword II
• About the Editor
• Contributors
• The Handbook of Polyhydroxyalkanoates, Volume 1: Introduction by the Editor
• PART I Enzymology/Metabolism/Genome Aspects for Microbial PHA Biosynthesis
• PART II Feedstocks
• Index