CHAPTER 1
Bio-based Polymers and Materials
NATHALIE BEREZINA AND SILVIA MARIA MARTELLI
1.1 Introduction
Biomaterials have gained attractiveness in the last decades due to both ecological and economic concerns. Increased pollution, and especially visible pollution, has first driven the scientific and industrial communities to look at biofragmentable and biodegradable substitutes for traditional petroleum-based non-biodegradable materials. Then the dramatic increase of oil prices before the economic crisis of 2007 influenced the move from the biodegradable to the bio-based. Finally, the compliance of the obtained materials with thermo-mechanical constraints has turned interest to the partially bio-based materials.
Bio-based materials can be obtained mainly by two different ways: the direct production of polymers or the production of bio-based monomers and their further (bio)chemical polymerization. The direct production of biopolymers can be achieved by microorganisms (polyhydroxyalkanoates, PHA), by algae (alginate etc.), by superior plants (pectin etc.) or by several types of producers, e.g. cellulose is produced by superior plants but also by bacteria, chitosan is produced by crustacean but also by fungi.
Whatever the producer of biopolymers, the main difficulty is to trigger its composition. Indeed, the obtained material has to comply with the thermomechanical constraints of its anticipated usages, and these characteristics are strongly related to the monomeric composition of the polymer and its size. This common problem of biopolymers does not have a unique solution. In the case of PHA, several microorganisms can produce the same polymer and the modification of the feeding substrates influences the monomeric composition of the polymers produced by the same microorganism, whereas important differences are found in the monomeric composition of the poly/ oligo-saccharides produced by different (micro)organisms. The regulation of the size of the biopolymers appears even more complicated; although PHA can be obtained with important masses, polysaccharides are actually mainly oligosaccharides. In any case, the main difficulty consists in making the (micro)organism perform the biosynthesis we want consistently and repeatedly.
To circumvent this problem, one can be tempted to make more controlled chemical polymerization with the bio-based monomers. Thus the production of bio-based monomers was also developed. However, the polymerization of bio-based monomers often asks for more development, as in the case of polylactic acid (PLA) and of polybutylene succinate (PBS); moreover, the thermo-mechanical needs for the expected applications are hardly reached with these polymers. Therefore, two more options can be foreseen: the production of partially bio-based materials (Sorona®) or the production of bio-monomers identical to the already existing and improved petroleum-based (ethylene, isobutylene, caprolactam etc.).
In this chapter we discuss the main biomaterials produced by these different methods as well as the achieved improvements and remaining bottlenecks in their production, modifications and applications.
1.2 Direct Production of Biopolymers
1.2.1 PHA
Polyhydroxyalkanoates (PHA) were discovered in 1926 by Maurice Lemoigne as energy storage materials in Bacillus megatherium and Bacillus mesentericus vulgatis. Still, they had to wait until the 1960s and for Cupriavidus genera (previously referred as Hydrogenomonas, Alcaligenes, Ralstonia and Wautersia) to be extensively studied. Indeed, the accumulation of the PHA by this genera has appeared to be more effective. Moreover, the first petrol crisis and further ecological issues increased the awareness and the interest in the bio-based materials.
Several PHA producing microorganisms as well as several types of PHA were discovered. The whole PHA family can be sub-divided into three main categories: the short-chain length PHA (PHASCL), the medium-chain length PHA (PHAMCL) and rarer PHA (Figure 1.1). The structural differences inside the PHA family imply deep differences in their thermo-mechanical properties. Thus, PHASCL mainly composed by polyhydroxybutanoates (PHB) and poly(hydroxybutanoate-co-valerate) (PHBV), are crystalline polymers, which are rather brittle and stiff, with high melting points (near 160–180 °C) and low glass transition temperature (between -5 and 0 °C), whereas the PHAMCL are thermoplastic elastomers with low crystallinity and tensile strength with high elongation to break (400–700%).
1.2.1.1 PHASCL
PHASCL are the most studied biopolymers among the PHA family. Numerous improvements of their production have been achieved during these last decades. These improvements mainly concerned the selection of wild-type strains (121 g L-1 of PHB was thus achieved using Cupriavidus genera), the engineering of strains (161 g L-1 of PHB was reported with E. coli (XL1-Blue) strain), the feeding strategy 'nutrient limited' versus 'nutrient sufficient' conditions, with the latter having been recently proved to be most efficient for the main producing strains (33 times productivity enhancement).
Also, the growth and the PHA accumulation on wastes and by-products have been paid important attention in recent years, in order to enhance the economic and sustainable efficiencies. Thus, different alternative substrates were tested – such as vinasse, oil palm frond juice, soybean oil, waste glycerol and other by-products from the biodiesel industry. Unfortunately up to now these strategies have not shown comparable productivities as an artificial carbon source (only 67.2 g L-1 of PHB were produced when soybean oil was used as a substrate).
Even if the Cupriavidus genera remains predominant in the production of PHASCL, other genera were also discovered and studied in recent years: Bacillus cereus, Brevundimonas vesicularis, Sphingopyxis macrogoltabia, Nostoc muscorum, Synechocystis sp., Herbaspirillum seropedicae, Haloferax mediterranei etc.
The important issues of the control of the biopolymer composition, the relative abundance of the 3-hydroxybutanoate (3-HB) and 3-hydroxyvalerate (3-HV), was also addressed by different feeding strategies, namely the choice of the 3-HV inducing substrates (up to 80% of 3-HV content was obtained with 1 g L-1 mixture of levulinic acid and sodium propionate) as well as the choice of one-time initial versus sequential addition of those substrates, the latter been found more efficient.
Finally, bioprocess improvements such as solid-state fermentation (SSF), continuous and two-stage culture systems, down-stream processing (DSP) and purification were studied.
1.2.1.2 PHAMCL
PHAMCL are mainly produced by the Pseudomonads. They are usually synthesized as copolymers of two or three or even more monomers, obtained by β-oxidation of fatty acids used as feeding substrates, the monomeric parts usually bear n [+ or -] 2 carbons. One noticeable exception to this general rule is the recently reported Pseudomonas mendocina strain able to produce pure homopolymers of poly-3-hydroxyoctanoates (PHO).
Although several strategies, such as multiple nutrient limitation, batch and chemostat strategies or strain engineering, were attempted for improving the PHAMCL productivity, it remains rather low compared to the results of PHASCL. Thus PHA production of 0.2 g L-1 h-1 was observed for Pseudomonas oleovorans grown on octanoic acid, or 47% of PHA inside the cells of Pseudomonas putida grown on 11-phenoxydecanoic acid, or 53–58% of the conversion of raw materials by Comomonas testosterone grown on vegetable oil.
1.2.1.3 Rarer PHA
With PHAMCL presenting more interesting thermo-mechanical properties and PHASCL being more easily produced it was tempting to try to combine the advantages by synthesizing the PHASCL-co-PHAMCL. The most studied among these copolymers is poly(3-hydroxybutanoate-co-3-hydroxyhexanoate) (P(HB-co-HHx)). Aeromonas caviae seems to be one of the rare bacteria to naturally produce such copolymers, the main results being obtained with engineered strains. The best results so far (up to 70% of 3-HHx content) was obtained with the Cupriviadus necator engineered with the Rhodococcus aetherivorans PHA synthase, grown on crude kernel oil.
Other rare PHA are mainly composed of P3HA-co-P4HA and the thiopolyesters polyhydroxybutanoate-co-polymercaptoprionate (PHB-co-PMP), however, the whole PHA family contains more than hundred different polymers, and is still growing.
1.2.1.4 Applications and Industrial Production of PHA
Applications of PHA have evolved. Initially foreseen applications in packaging have been recently replaced by more promising and cost-compatible medical applications. Numerous devices (patches, scaffolds etc.), wound management tools (suture, dressings), drugs delivery systems and pro-drugs were based on these biopolymers.
The observed shift in the applications of PHA has significant importance on the production of those polymers. More particularly the high purity required for the final products designed for medical application may not be compatible with the use of wastes and by-products as raw materials. Thus, in the near future we will observe a shift from the study of cheap raw materials (in order to lower the overall cost of PHA) to purification processes in order to separate the PHA from the enzymes and proteins linked to the PHA granules inside the cells.
Although present worldwide, the total industrial production of PHA remains tiny (Table 1.1). However, very recently one of the main historical producers of PHA, Metabolix, has achieved for the first time a $3.6 million profit in 2011. It now becomes reasonable to foresee more success stories in the future for this family of biopolymers.
1.2.2 Polysaccharides and Oligosaccharides
Polysaccharides and oligosaccharides are widely produced in nature. Animals and plants are the most important producers in terms of volume, whereas microorganisms produce much wider diversity. Also, the small-scale structure, the proportion of different sugars and the type of linkage play an important role in the final properties of these biopolymers. Thus, applications of these poly- and oligo-saccharides are also very different, from low- to high-added value products.
1.2.2.1 Polysaccharides Produced by Plants
Plants are the most important producers of polysaccharides. Their main products are cellulose, hemicellulose, starch, inulin and pectin. Cellulose is by far the most abundant renewable polymer available worldwide, its occurrence was estimated at some 10-10 tons per annum.
Cellulose and starch are both homopolymers, only composed by D-glucose units (Figure 1.2). The only difference between them is the type of linkage between the sugar units. Cellulose is composed by β-D-glucose units, whereas starch has α-D-glucose units. This tiny structural difference makes a huge difference in the properties of these two polysaccharides. Starch is digestible product used by many organisms on earth, whereas the digestion of cellulose is very difficult as it is often requiring both physical and chemical steps. Starch, used by humans for centuries for food and feed (as well for its nutritional value as thickener and emulsifier), has found application also in textile and paper industries and as a biodegradable packaging material. More recently it has been investigated as a first-generation bio-fuel, but raised ethical issues due to the famine problem in different parts of the world. On the other hand, the traditional usage of cellulose was in the paper and textile industries, but more technical applications have been recently explored using nanocrystals or grafted cellulosic materials. Nowadays it is also abundantly studied as a second-generation bio-fuel.
Inulin is another homopolymer produced by plants. It is composed mainly of fructose units, even if a starting glucose moiety can be present (Figure 1.3). The main producers of inulin are either chicory and artichokes or biocatalytically synthesized fructo-oligosaccharides. Inulin is mainly used in the food industry for both its nutritional and technological advantages. Indeed, being composed of fructose units, inulin is hardly hydrolysed during the digestion process. The non-food applications of inulin have also been recently investigated; until now they concern merely modified inulin, thus carboxymethylinulin (CMI) was successfully used as dispersing agent and dicarboxyinulin (DCI) as a builder or co-builder in detergent formulation to replace polyacrylates.
Pectins are polysaccharides bearing methyl-esterified galacturonic acid and rhamnose units. The proportions of these units as well as the presence of other constitutive units depend on the plant from which the studied pectin was isolated. Pectins are biosynthetically produced in the Golgi apparatus of plants. Although many plants are able to produce pectins, industrial pectin is mainly extracted from citrus peel and apple pomace under mildly acidic conditions. The main applications of pectins remain in the food industry as gelling agents, while more technical applications could arise from more homogeneous polysaccharides obtained by chemical modification or gene technology.
1.2.2.2 Polysaccharides Produced by Animals
Chitin and chitosan are the main polysaccharides produced by animals. Those molecules are part of insects' and crustaceans' exoskeletons, even if some mushrooms and fungi are also able to produce them. Chitin and chitosan are aminoglucopyranans composed of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) units (Figure 1.4). Currently the most important source of chitin and chitosan remains chemical processing of the waste fraction of the shellfish industry, even if some biotechnological and entomological studies are in progress. The main applications of these polysaccharides are, until now, based on their antimicrobial properties as applied to either the food or the cosmetic industries.
1.2.2.3 Polysaccharides Produced by Microorganisms
Compared to higher plants and animals, microorganisms are characterized by a wide diversity of the poly- and oligo-saccharides they produce (Table 1.2). Among the microorganisms, bacteria have the widest spread of possibilities. Polysaccharides produced by bacteria are mainly extracellular polysaccharides (EPS), also called exopolysaccharides, whereas those produced by algae are mainly cell wall and structural constituents.
Intracellular bacterial polysaccharides have not yet found proper applications; they are, however, extensively studied as storage materials similar to those in humans (glycogen) and as specific targets for the drug attacks of pathogens (murein, teichoic and teichonic acids).
The main applications of EPS are in the food and cosmetic industries as thickeners, gelling agents and emulsifiers or in pharmacy and medicine, with some of them being used as the active principles (schizophyllan).
Some of the microbial polysaccharides are also produced by higher organisms, such as cellulose in plants or chitin and chitosan in animals; however, microbial production is often better controlled and offers the possibility of higher-added value applications.
Among other applications we can underline are the use as oil-drilling agents (dextran and derivatives, xanthan, sphingan, scleroglucan), immobilization supports (curdlan, alginate), cement-based materials (sphingan99), adhesives (pullulan) and can sealing (alginate).
1.2.3 Others
Several other biopolymers are directly synthesized in nature, such as proteins, poly(amino acid)s, lignin, humic substances or sporopollenin. Until now, they are under-used and under-studied compared to the previously detailed two main families of directly produced biopolymers. Thus in this paragraph we will only mention some recent developments in the field of proteins and poly(amino acid)s, and lignin.
1.2.3.1 Proteins and Poly(amino acid)s
Proteins are composed of amino acids linked by peptide bonds. Two main biosynthetic pathways for protein production have been identified so far: the ribosomal and the non-ribosomal-multi-enzyme paths. The proteins are mainly heteropolymers composed by a variety of amino acids; however, three poly(amino acid)s can be obtained through the multi-enzyme pathway: cyanophycin (aspartic acid–arginine dipeptide), ε-poly-L-lysine and poly-α,β-aspartic acid (Figure 1.5).
The main applications of proteins are in the nutraceutical and pharmaceutical industries. The specific antimicrobial properties of ε-poly-L-lysine promoted its utilization in the food industry in Japan, whereas poly-α,β-aspartic acid is mainly used as a polydispersant in detergents.
Some proteins were studied for a long time for their materials applications: soy protein, wheat gluten and collagen (the denatured form being called gelatine). In all the cases, the stability of the proteins and their sensitivity to moisture require strengthening by plasticization, compatibilization, cross-linkage or production of protein–nanoclay composites.
