PHA Polymers Research Articles

Production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Bacillus megaterium LVN01 Using Biogas Digestate

The Bacillus megaterium LVN01 species native to Colombia has demonstrated the ability to metabolize different coproducts or industrial waste (such as fique juice, cane molasses, and residual glycerol) and accumulate polyhydroxybutyrate (PHB), giving it potential in the bioplastics industry. In this research, the potential of liquid digestate as a carbon source for the production of PHA polymers in fermentation processes with this bacterial strain was evaluated. Favorably, it was found that B. megaterium utilizes the nutrients from this residual substrate to multiply appropriately and efficiently synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Bench-scale aerobic batch fermentation, under the operational conditions of this research [volume: 3 L; temperature: 30.8 °C; agitation: 400 rpm; pH: 7.0 ± 0.2; dissolved oxygen: 100% saturation; antifoam: 10% (v/v)], generated maximum values of dry cell weight (DCW) (0.56 g cell L−1) at 60 h, while the maximum PHBV yield (360 mg PHBV L−1) occurred at 16 h, which is very favorable for sustainable degradable bioplastics production. Additionally, GC–MS and NMR analyses confirmed that the PHBV copolymer synthesized by B. megaterium is made up of the monomers 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). Furthermore, the thermal properties determined by TGA (Tonset = 283.1 °C; Tendset = 296.98 °C; Td = 290.114 °C) and DSC (Tm = °C 155.7 °C; ΔHf = 19.80 J g−1; Xcr = 18.17%) indicate that it is a thermally stable biopolymer with low percentages of crystallinity, providing flexibility that facilitates molding, adaptation, and application in various industrial sectors.

Biosynthesis and properties of polyhydroxyalkanoates synthesized from mixed C5 and C6 sugars obtained from hardwood hydrolysis

Glucose and xylose are fermentable sugars readily available from lignocellulosic biomass, and are a sustainable carbon substrate supporting industrial biotechnology. Three strains were assessed in this work – Paraburkholderia sacchari, Hydrogenophaga pseudoflava, and Bacillus megaterium – for their ability to uptake both C5 and C6 sugars contained in a hardwood hydrolysate produced via a thermomechanical pulping-based process with concomitant production of poly(3-hydroxyalkanoate) (PHA) biopolymers. In batch conditions, B. megaterium showed poor growth after 12 h, minimal uptake of xylose throughout the cultivation, and accumulated a maximum of only 25 % of the dry biomass as PHA. The other strains simultaneously utilized both sugars, although glucose uptake was faster than xylose. From hardwood hydrolysate, P. sacchari accumulated 57 % of its biomass as PHA within 24 h, whereas H. pseudoflava achieved an intracellular PHA content of 84 % by 72 h. The molecular weight of the PHA synthesized by H. pseudoflava (520.2 kDa) was higher than that of P. sacchari (265.5 kDa). When the medium was supplemented with propionic acid, the latter was rapidly consumed by both strains and incorporated as 3-hydroxyvalerate subunits into the polymer, demonstrating the potential for production of polymers with improved properties and value. H. pseudoflava incorporated 3-hydroxyvalerate subunits with at least a 3-fold higher yield, and produced polymers with higher 3-hydroxyvalerate content than P. sacchari. Overall, this work has shown that H. pseudoflava can be an excellent candidate for bioconversion of lignocellulosic sugars to PHA polymers or copolymers as part of an integrated biorefinery.

A Review on Biological Synthesis of the Biodegradable Polymers Polyhydroxyalkanoates and the Development of Multiple Applications

Polyhydroxyalkanoates, or PHAs, belong to a class of biopolyesters where the biodegradable PHA polymer is accumulated by microorganisms as intracellular granules known as carbonosomes. Microorganisms can accumulate PHA using a wide variety of substrates under specific inorganic nutrient limiting conditions, with many of the carbon-containing substrates coming from waste or low-value sources. PHAs are universally thermoplastic, with PHB and PHB copolymers having similar characteristics to conventional fossil-based polymers such as polypropylene. PHA properties are dependent on the composition of its monomers, meaning PHAs can have a diverse range of properties and, thus, functionalities within this biopolyester family. This diversity in functionality results in a wide array of applications in sectors such as food-packaging and biomedical industries. In order for PHAs to compete with the conventional plastic industry in terms of applications and economics, the scale of PHA production needs to grow from its current low base. Similar to all new polymers, PHAs need continuous technological developments in their production and material science developments to grow their market opportunities. The setup of end-of-life management (biodegradability, recyclability) system infrastructure is also critical to ensure that PHA and other biobased biodegradable polymers can be marketed with maximum benefits to society. The biobased nature and the biodegradability of PHAs mean they can be a key polymer in the materials sector of the future. The worldwide scale of plastic waste pollution demands a reformation of the current polymer industry, or humankind will face the consequences of having plastic in every step of the food chain and beyond. This review will discuss the aforementioned points in more detail, hoping to provide information that sheds light on how PHAs can be polymers of the future.

Optimizing a Fed-Batch High-Density Fermentation Process for Medium Chain-Length Poly(3-Hydroxyalkanoates) in Escherichia coli.

Production of medium chain-length poly(3-hydroxyalkanoates) [PHA] polymers with tightly defined compositions is an important area of research to expand the application and improve the properties of these promising biobased and biodegradable materials. PHA polymers with homopolymeric or defined compositions exhibit attractive material properties such as increased flexibility and elasticity relative to poly(3-hydroxybutyrate) [PHB]; however, these polymers are difficult to biosynthesize in native PHA-producing organisms, and there is a paucity of research toward developing high-density cultivation methods while retaining compositional control. In this study, we developed and optimized a fed-batch fermentation process in a stirred tank reactor, beginning with the biosynthesis of poly(3-hydroxydecanoate) [PHD] from decanoic acid by β-oxidation deficient recombinant Escherichia coli LSBJ using glucose as a co-substrate solely for growth. Bacteria were cultured in two stages, a biomass accumulation stage (37°C, pH 7.0) with glucose as the primary carbon source and a PHA biosynthesis stage (30°C, pH 8.0) with co-feeding of glucose and a fatty acid. Through iterative optimizations of semi-defined media composition and glucose feed rate, 6.0 g of decanoic acid was converted to PHD with an 87.5% molar yield (4.54 g L–1). Stepwise increases in the amount of decanoic acid fed during the fermentation correlated with an increase in PHD, resulting in a final decanoic acid feed of 25 g converted to PHD at a yield of 89.4% (20.1 g L–1, 0.42 g L–1 h–1), at which point foaming became uncontrollable. Hexanoic acid, octanoic acid, 10-undecenoic acid, and 10-bromodecanoic acid were all individually supplemented at 20 g each and successfully polymerized with yields ranging from 66.8 to 99.0% (9.24 to 18.2 g L–1). Using this bioreactor strategy, co-fatty acid feeds of octanoic acid/decanoic acid and octanoic acid/10-azidodecanoic acid (8:2 mol ratio each) resulted in the production of their respective copolymers at nearly the same ratio and at high yield, demonstrating that these methods can be used to control PHA copolymer composition.

Impact of Agricultural Weathering on Physicochemical Properties of Biodegradable Plastic Mulch Films: Comparison of Two Diverse Climates Over Four Successive Years

Biodegradable plastic mulch films (BDMs) are essential in the production of vegetable and specialty crops due to their promotion of increased crop yield and quality. Unlike conventional polyethylene (PE) mulches, BDMs can be tilled into the soil after crop harvest to undergo biodegradation, thereby leading to minimal environmental impact. Agricultural weathering impacts both the performance of BDMs during crop production as a barrier to weeds and biodegradability of BDMs in the soil. To better understand the relative importance of climatic factors, the change of physicochemical properties of BDMs during single-season, 3–4 month, field trials for vegetable production at two diverse climates (Knoxville, TN and Mount Vernon, WA) across four successive years (2015–2018) was evaluated. Mulch treatments consisted of four commercially available BDMs composed primarily of polybutylene co-adipate- co-terephthalate (PBAT) that differed in color and polymeric feedstock, a black experimental BDM prepared from polylactic acid/polyhydroxybutyrate (PLA/PHA) blend, and conventional PE mulch. Solar radiation, an important factor to degradation of mulches, was higher in WA than TN in most sampling years. Yet, degradation occurred more greatly for BDMs in TN, which is attributable to higher temperatures in TN. Mulch deterioration did not very extensively between years. Loss of mechanical properties and color was greater than chemical property changes. Differences in the extent of molecular weight decrease between years correlated significantly with solar radiation exposure at the two locations. A black-colored PBAT-based BDM was less susceptible to degradation than equivalent clear and white-on-black films, due to carbon black acting as a photostabilizer. The impact of weathering also differed between three commercially available PBAT-based films. The PLA/PHA mulch was more susceptible to degradation than PBAT-based BDMs, particularly in the warmer location, TN, partially due to a leaching out of PHA and lower-molecular weight polymer molecules. The extent of change for physicochemical properties of BDMs due to agricultural weathering is greatly affected by polymeric composition, and is greater in warmer climates.

Machine-Learning-Based Predictive Modeling of Glass Transition Temperatures: A Case of Polyhydroxyalkanoate Homopolymers and Copolymers.

Polyhydroxyalkanoate-based polymers-being ecofriendly, biosynthesizable, and economically viable and possessing a broad range of tunable properties-are currently being actively pursued as promising alternatives for petroleum-based plastics. The vast chemical complexity accessible within this class of polymers gives rise to challenges in the rational discovery of novel polymer chemistries for specific applications. The burgeoning field of polymer informatics addresses this challenge via providing tools and strategies for accelerated property prediction and materials design via surrogate machine-learning models built on reliable past data. In this contribution, we use glass transition temperature Tg as an example target property to demonstrate promise of the data-enabled route to accelerated learning of accurate structure-property mappings in PHA-based polymers. Our analysis uses a data set of experimentally measured Tg values, polymer molecular weights, and a polydispersity index for PHA-based homo- and copolymers that was carefully assembled from the literature. A fingerprinting scheme that captures key properties based on topology, shape, and charge/polarity of specific chemical units or motifs forming the polymer backbone was devised to numerically represent the polymers. A validated statistical learning model is then developed to allow for a mapping of the polymer fingerprints onto the property space in a physically meaningful and reliable manner. Once developed, the model can not only rapidly predict the property of new PHA polymers but also provide uncertainties underlying the predictions. The model is further combined with an evolutionary-algorithm-based search strategy to efficiently identify multicomponent polymer compositions with a prespecified Tg. While the present contribution is focused specifically on Tg, the surrogate model development approach put forward here is general and can, in principle, be extended to a range of other properties.

Colonization and degradation of polyhydroxyalkanoates by lipase-producing bacteria.

Biodegradation of short-chain-length polyhydroxyalkanoates (scl-PHAs) and medium-chain-length polyhydroxyalkanoates (mcl-PHAs) was studied using 2 bacteria, Pseudomonas chlororaphis and Acinetobacter lwoffii, which secrete an enzyme, or enzymes, with lipase activity. These bacteria produced clear zones of depolymerization on Petri plates containing colloidal solutions of PHA polymers with different monomer compositions. Lipase activity in these bacteria was measured using p-nitrophenyl octanoate as a substrate. In liquid medium, scl-PHA (e.g., PHBV) and mcl-PHA (e.g., PHO) films were used as the sole carbon source for growth, and after 7 days, 5%-18% loss in mass of PHA films was observed. Scanning electron microscopy of these films revealed bacterial colonization of the polymers, with cracks and pitting in the film surfaces. Degradation of polymers released 3-hydroxyhexanoate, 3-hydroxyoctanoate, and 3-hydroxydecanoate monomers into the liquid medium, depending on the starting polymer. Genes encoding secretory lipases, with amino acid consensus sequences for lipase boxes and oxyanion holes, were identified in the genomes of P. chlororaphis and A. lwoffii. Although amino acid consensus sequences for lipase boxes and oxyanion holes are also present in PHA depolymerases identified in the genomes of other PHA-degrading bacteria, the P. chlororaphis and A. lwoffii lipases had low homology with these depolymerases.

Role of PhaC Type I and Type II Enzymes during PHA Biosynthesis.

PHA synthases (PhaC) are grouped into four classes based on the kinetics and mechanisms of reaction. The grouping of PhaC enzymes into four classes is dependent on substrate specificity, according to the preference in forming short-chain-length (scl) or medium-chain-length (mcl) polymers: Class I, Class III and Class IV produce scl-PHAs depending on propionate, butyrate, valerate and hexanoate precursors, while Class II PhaC synthesize mcl-PHAs based on the alkane (C6 to C14) precursors. PHA synthases of Class I, in particular PhaCCs from Chromobacterium USM2 and PhaCCn/RePhaC1 from Cupriavidus necator/Ralstonia eutropha, have been analysed and the crystal structures of the C-domains have been determined. PhaCCn/RePhaC1 was also studied by X-ray absorption fine-structure (XAFS) analysis. Models have been proposed for dimerization, catalysis mechanism, substrate recognition and affinity, product formation, and product egress route. The assays based on amino acid substitution by mutagenesis have been useful to validate the hypothesis on the role of amino acids in catalysis and in accommodation of bulky substrates, and for the synthesis of PHB copolymers and medium-chain-length PHA polymers with optimized chemical properties.

In Vivo and In Vitro Degradation Studies for Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Biopolymer

Studies have shown that the copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] possesses favorable mechanical properties for use in medical supplies and products (e.g., sutures, scaffolds, bone plates). One of the major under-addressed issues associated with the use of biodegradable, bio-based PHA polymers in resorbable medical products is the correlation between the mechanical properties and the in vivo material degradation over time. In this study, P(HB-co-17 mol% HHx) matrices were mechanically tested after either incubation in cultures of human embryonic kidney cells (HEK) for in vitro degradation studies for up to 4 weeks, or inserted into Danio rerio (zebrafish) tissues for in vivo degradation studies for up to 7 weeks. The mechanical properties and scanning electron microscopy (SEM) images of the degraded materials were examined and later correlated to understand the degradation phenomenon. Our results show that Young’s modulus of P(HB-co-17 mol%HHx) during in vitro studies decreased from 3.26 to 2.42 GPa within 4 weeks, and in vivo breakdown resulted in a significant decrease in Young’s modulus with a decrease from 3.26 to 0.51 GPa and a mass loss of 59 % within 7 weeks. SEM images showed the development of pores and cracks on the surface of the material over time. Plasticization and recrystallization were observed and likely play a role in the alteration of mechanical properties.

Chemically Intractable No More: In Vivo Incorporation of "Click"-Ready Fatty Acids into Poly-[(R)-3-hydroxyalkanoates] in Escherichia coli.

Poly-[(R)-3-hydroxyalkanoate] biopolymers, or PHAs, are biocompatible and biodegradable polyesters that can be produced by diverse microbial strains. PHA polymers have found widespread uses in applications ranging from sustainable replacements of nonbiodegradable bulk-commodity plastics to biomaterials. However, further expansion into other markets and industries has generally been limited by the inability to chemically modify these polymers. Recently, our lab engineered E. coli LSBJ, a microbial strain able to produce PHA copolymers with controlled unit compositions from simple and accessible fatty acid feedstocks. We envisioned meaningfully broadening the application spectrum of these materials via production of chemically tractable PHA biopolymers containing "click"-ready chemical functionalities. With a myriad of applications in mind, in this study we demonstrate the synthesis and biopolymerization of a panel of ω-azido fatty acids and take the first exploratory steps toward demonstrating their conjugation via a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. The convenience of accessing these materials will open the door to new applications for functionalized PHA polymers.

Bioplastics from waste glycerol derived from biodiesel industry

AbstractPolyhydroxyalkanoates (PHAs) are polyesters that can be biologically synthesized by many microorganisms and engineered plants that have been investigated by microbiologists, biochemists, polymer scientists, material engineers, and medical researchers for several decades. Research on microbial production of PHAs has been extensively focused on using pure carbon sources, such as sugars and fatty acids. Practical considerations of production costs of PHAs have resulted in research efforts to use alternative renewable and inexpensive feedstocks. One potential feedstock for the production of PHA polymers is the glycerol waste byproduct of biodiesel production. The major focus of this review is the production of PHA polymers from glycerol. A review of biosynthetic pathways for PHAs production from glycerol, current production of waste glycerol in biodiesel industry, physical and mechanical properties of PHAs, and applications of PHAs in the areas of packaging industry, implant materials, drug carrier, biofuels, are covered. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013

Enzymatic synthesis of 6-O-glucosyl-poly(3-hydroxyalkanoate) in organic solvents and their binary mixture

The effects of organic solvents and their binary mixture in the glucose functionalization of bacterial poly-3-hydroxyalkanoates catalyzed by Lecitase™ Ultra were studied. Equal volume binary mixture of DMSO and chloroform with moderate polarity was more effective for the enzyme catalyzed synthesis of the carbohydrate polymer at ≈38.2 (±0.8)% reactant conversion as compared to the mono-phasic and other binary solvents studied. The apparent reaction rate constant as a function of medium water activity (aw) was observed to increase with increasing solvent polarity, with optimum aw of 0.2, 0.4 and 0.7 (±0.1) observed in hydrophilic DMSO, binary mixture DMSO:isooctane and hydrophobic isooctane, respectively. Molecular sieve loading between 13 to 15gL−1 (±0.2) and reaction temperature between 40 to 50°C were found optimal. Functionalized PHA polymer showed potential characteristics and biodegradability.

Reward for Bdellovibrio bacteriovorus for preying on a polyhydroxyalkanoate producer

Bdellovibrio bacteriovorus HD100 is an obligate predator that invades and grows within the periplasm of Gram-negative bacteria, including mcl-polyhydroxyalkanoate (PHA) producers such as Pseudomonas putida. We investigated the impact of prey PHA content on the predator fitness and the potential advantages for preying on a PHA producer. Using a new procedure to control P. putida KT2442 cell size we demonstrated that the number of Bdellovibrio progeny depends on the prey biomass and not on the viable prey cell number or PHA content. The presence of mcl-PHA hydrolysed products in the culture supernatant after predation on P. putida KT42Z, a PHA producing strain lacking PhaZ depolymerase, confirmed the ability of Bdellovibrio to degrade the prey's PHA. Predator motility was higher when growing on PHA accumulating prey. External addition of PHA polymer (latex suspension) to Bdellovibrio preying on the PHA minus mutant P. putida KT42C1 restored predator movement, suggesting that PHA is a key prey component to sustain predator swimming speed. High velocities observed in Bdellovibrio preying on the PHA producing strain were correlated to high intracellular ATP levels of the predator. These effects brought Bdellovibrio fitness benefits as predation on PHA producers was more efficient than predation on non-producing bacteria.

Biosynthesis of Poly[(R)-3-hydroxyalkanoate] Copolymers with Controlled Repeating Unit Compositions and Physical Properties

As applications for biodegradable and biologically produced poly[(R)-3-hydroxyalkanoates] (PHAs) grow into more specialized areas, the need to precisely control the repeating unit composition and consequently the physical properties of these polymers has become essential. A previous study reported our development of Escherichia coli LSBJ in order to produce PHA polymers composed of single repeating units ranging from 4 to 12 carbon atoms. This investigation expands the scope of our effort toward controlling the repeating unit composition of a variety of PHA copolymers. The sizes for the repeating units within the copolymers were modulated by feeding specific ratios of fatty acids with defined carbon lengths to E. coli LSBJ, which resulted in defined mole ratios for the repeating units. Various physical properties of the copolymers (including the Young's modulus, elongation to break, and glass-transition temperature) were shown to be strongly dependent upon the mole ratios of repeating units. This work demonstrates that copolymers of PHAs with repeating units from 4 to 12 carbons can be incorporated accurately to obtain any desired mole ratio within the PHA copolymers. Our methodology may thus be extended to generate tailor-made PHA copolymers with prescribed values for key sets of physical properties.

Water soluble poly(histamine acrylamide) with superior buffer capacity mediates efficient and nontoxic in vitro gene transfection

AbstractWater‐soluble cationic polymers, poly(histamine acrylamide)s (PHAs), with superior buffer capacity at the endosomal pH range were designed, prepared, and investigated for non‐viral gene transfection. PHAs were obtained with molecular weights ranging from 9.2 to 28.7 kDa through controlled radical polymerization of histamine acrylamide (HA). Acid–base titration results displayed that all PHA polymers had a remarkably high buffer capacity of about 70% at pH 5.1–7.2. 12.7–28.7 kDa PHAs were able to effectively condense DNA into nano‐sized (<220 nm) polyplexes with moderate positive surface charges (+13–+19 mV) at N/P ratios ≥10/1. CCK assays indicated that polyplexes of 12.7 and 17.5 kDa PHAs were non‐toxic to COS‐7 cells up to a tested N/P ratio of 20/1. Interestingly, the in vitro transfection using pCMV‐Luc and pEGFP‐C1 plasmid DNA as reporter genes showed that polyplexes of 12.7 kDa PHA formed at an N/P ratio of 20/1 mediated efficient transfection in COS‐7 cells under 10% serum conditions, with transfection efficiencies comparable to that of 25 kDa polyethylenimine control. Their versatile design of structures, controlled synthesis, low cytotoxicity, and high transfection activity render PHA‐based cationic polymers particularly interesting for the development of safe and efficient non‐viral gene delivery systems. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011.

Process analysis of the conversion of styrene to biomass and medium chain length polyhydroxyalkanoate in a two‐phase bioreactor

The improvement and modeling of a process for the supply of the volatile aromatic hydrocarbon, styrene, to a fermentor for increased biomass production of the medium chain length polyhydroxyalkanoate (mcl-PHA) accumulating bacterium Pseudomonas putida CA-3 was investigated. Fed-batch experiments were undertaken using different methods to provide the styrene. Initial experiments where styrene was supplied as a liquid to the bioreactor had detrimental effects on cell growth and inhibited PHA polymer accumulation. By changing the feed of gaseous styrene to liquid styrene through the air sparger a 5.4-fold increase in cell dry-weight was achieved (total of 10.56 g L(-1)) which corresponds to a fourfold improvement in PHA production (3.36 g L(-1)) compared to previous studies performed in our laboratory (0.82 g L(-1)). In addition this final improved feeding strategy reduced the release of styrene from the fermentor 50-fold compared to initial experiments (0.12 mL total styrene released per 48 h run). An unstructured kinetic model was developed to describe cell growth along with substrate and oxygen utilization. The formation of dispersed gas (air) and liquid (styrene) phases in the medium and the transfer of styrene between the aqueous and dispersed liquid droplet phases was also modeled. The model provided a detailed description of these phase transitions and helped explain how the feeding strategy led to improved process performance in terms of final biomass levels. It also highlighted the key factors to be considered during further process improvement.

Poly(hydroxyalkanoate) Biosynthesis from Crude Alaskan Pollock (Theragra chalcogramma) Oil

Six strains of Pseudomonas were tested for their abilities to synthesize poly(hydroxyalkanoate) (PHA) polymers from crude Pollock oil, a large volume byproduct of the Alaskan fishing industry. All six strains were found to produce PHA polymers from hydrolyzed Pollock oil with productivities (P; the percent of the cell mass that is polymer) ranging from 6 to 53% of the cell dry weight (CDW). Two strains, P. oleovorans NRRL B-778 (P = 27%) and P. oleovorans NRRL B-14682 (P = 6%), synthesized poly(3-hydroxybutyrate) (PHB) with number average molecular weights (Mn) of 206,000 g/mol and 195,000 g/mol, respectively. Four strains, P. oleovorans NRRL B-14683 (P = 52%), P. resinovorans NRRL B-2649 (P = 53%), P. corrugata 388 (P = 43%), and P. putida KT2442 (P = 39%), synthesized medium-chain-length PHA (mcl-PHA) polymers with Mn values ranging from 84,000 g/mol to 153,000 g/mol. All mcl-PHA polymers were primarily composed of 3-hydroxyoctanoic acid (C8:0) and 3-hydroxydecanoic acid (C10:0) amounting to at least 75% of the total monomers present. Unsaturated monomers were also present in the mcl-PHA polymers at concentrations between 13% and 16%, providing loci for polymer derivatization and/or crosslinking.

Enzymatic processes for biodegradation of poly(hydroxyalkanoate)s crystals

Poly(hydroxyalkanoate)s (PHAs) have attracted much attention as environmentally compatible polymeric materials that can be produced from renewable carbon resources. Biodegradation of PHA materials occurs by the function of extracellular PHA depolymerase secreted from microorganisms. Thus, elucidation of the enzymatic degradation mechanism for PHA materials is important to design PHA materials with desirable properties and controlled biodegradability. The solid PHA polymer is a water-insoluble substrate but PHA depolymerases are soluble in water. Therefore, the enzymatic degradation of PHA materials is a heterogeneous reaction on the material’s surface. Two distinct processes are involved during the degradation, namely, adsorption of the enzyme on the surface of PHA material and the subsequent hydrolysis of polymer chains. Atomic force microscopy (AFM) is a powerful tool that has been used for the quantitative analysis of PHA crystal degradation. AFM enables the characterization of the crystal surface nanostructure in a buffer solution. By using in-situ (real-time) AFM observations, we recently succeeded in observing the degradation processes of PHA crystals. Subsequently, we were also able to investigate the degradation rates of PHA crystals using the same technique. In this review, we have attempted to give an overview concerning the direct visualization of the adsorption, as well as the hydrolysis reactions of PHA depolymerases at the nanometer scale. In addition, we present other analytical techniques besides AFM as a complimentary approach to analyze the effect of enzyme adsorption on PHA crystals.Key words: poly(hydroxyalkanoate) (PHA), enzymatic degradation, lamellar crystal, PHA depolymerase.

Topographical imaging as a means of monitoring biodegradation of poly(hydroxyalkanoate) films

Poly(hydroxyalkanoates) (PHAs) are a class of bacterially-derived polymers that are naturally biodegradable through the action of extracellular depolymerase enzymes secreted by a number of different bacteria and fungi. In this paper we describe the development of topographical imaging protocols (by both scanning electron microscopy; SEM, and confocal microscopy; CM) as a means of monitoring the biodegradation of solution cast films of poly(3-hydroxybutanoate-co-3-hydroxyhexanoate) (P3HB/3HHx) and medium-chain-length (mcl-) PHA. Pseudomonas lemoignei and Comamonas P37C were used as sources for PHA depolymerase enzymes as these bacteria are known to degrade at least one of the polymers in question. SEM revealed the bacterial colonization of the film surfaces while CM permitted the comparative assessment of the roughness of the film surfaces upon exposure to the two bacterial strains. By dividing the total surface area of the film (A′) by the total area of the scan (A) it was possible to monitor biodegradation by observing differences in the topography of the film surface. Prior to inoculation, P3HB/3HHx films had an A′/A ratio of 1.06. A 24-h incubation with P. lemoignei increased the A′/A ratio to 1.47 while a 48- and 120-h incubation with Comamonas resulted in A′/A ratios of 1.16 and 1.33, respectively. These increases in the A′/A ratios over time demonstrated an increase in the irregularity of the film surface, indicative of PHA polymer breakdown.

Conversion of agricultural feedstock and coproducts into poly(hydroxyalkanoates)

Aside from their importance to the survival and general welfare of mankind, agriculture and its related industries produce large quantities of feedstocks and coproducts that can be used as inexpensive substrates for fermentative processes. Successful adoption of these materials into commercial processes could further the realization of a biorefinery industry based on agriculturally derived feedstocks. One potential concept is the production of poly(hydroxyalkanoate) (PHA) polymers, a family of microbial biopolyesters with a myriad of possible monomeric compositions and performance properties. The economics for the fermentative production of PHA could benefit from the use of low-cost agricultural feedstocks and coproducts. This mini-review provides a brief survey of research performed in this area, with specific emphasis on studies describing the utilization of intact triacylglycerols (vegetable oils and animal fats), dairy whey, molasses, and meat-and-bone meal as substrates in the microbial synthesis of PHA polymers.