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POLYHYDROXYALKANOATE (PHA) PRODUCTION FROM METHANE & NATURAL GAS - LITERATURE REVIEW PART 4

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POLYHYDROXYALKANOATE (PHA) PRODUCTION FROM METHANE & NATURAL GAS - LITERATURE REVIEW PART 4

Wed, 04/03/2019 - 17:33
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PHA Polyester
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Section 4: Polyhydroxyalkanoates (PHAs)

4.1 Introduction

Polyhydroxyalkanoates (PHAs) are a family of natural-occurring polyesters synthesized by a wide variety of bacteria through the fermentation of sugars, alkanes, alkanoic acids and lipids. These polymers appear as discrete cytoplasmic insoluble granules inside the bacterial cell. The number and the size of the granules per cell depends on the PHA producers and the stage of the growth cycle [81]. PHAs are biodegradable, biocompatible and recyclable, and when extracted from the cell, exhibit excellent mechanical properties making them great replacements for petroleum-based polymers. PHA can be used for several applications such as packaging material, cosmetic products, medical implants and in drug delivery. Since its discovery, PHAs have attracted much research and commercial interest due to its ability to address the rising concern about synthetic plastic pollution in our environment.

 

4.2 Brief History of PHAs

In 1923, French Scientist Maurice Lemoigne observed an aerobe, Bacillus megaterium, accumulating 3-hydroxybutyric acid under anaerobic conditions. In a later investigation after successfully quantifying the amount of 3-hydroxybutyric acid formed, he performed a chloroform extraction of the accumulating substance and proved that the material was a polymer of 3-hydroxybutyric acid. After several decades, poly(3-hydroxbutyrate) or P(3HB) became the first PHA to be properly synthesized, isolated and produced on a commercial scale, and also used to fabricate materials such as sutures and prosthetic devices. This pioneer work by Baptist and Werber at W.R Grace and Co. (USA) earned them several patents. Unfortunately, due to low fermentation yields, significant impurities in the polymer product and the high cost of polymer extraction, its production was terminated. When the oil crisis hit in the 1970s, a need for alternative plastics arose. ICI (UK) found new processing conditions to produce P(3HB) at a yield of about 70% of its dry weight, but P(3HB) was very brittle and had poor mechanical properties with very high production costs. ICI later discovered a novel polymer they called BIOPOL, which is a co-polymer of 3-hydroxybutryate and 3-hydroxyvalerate (or PHBV). This polymer exhibits better properties, such as low crystallinity and more flexibility, than P(3HB). After a series of breaks and readjustments to the company structure, Metabolix Inc. finally acquired the product license and jointly formed a new company called Tepha Inc. with the Children’s Hospital in Boston. Tepha now leads the technology in developing medical devices from biodegradable polymers. To date, several research institutions and industries are intensively carrying out research to produce more cost-effective and environmentally friendly PHAs [8,82]. 

 

4.3 The Nature of the PHA Polymer

PHAs are polymers of carbon, hydrogen and oxygen, which have a general structure as seen in Figure (6). These molecules are typically made up of thousands of (R)-hydroxy fatty acid monomer units where the carboxyl group of one monomer forms an ester bond with the hydroxyl group of the neighboring monomer. The R group side chain on each monomer unit usually consists of a hydrogen atom or a saturated alkyl group, but in other less common instances this side chain could carry an unsaturated alkyl group, a branched alkyl group or a substituted alkyl group. The ‘x’ symbol in the repeating monomer unit refers to the size of the alkyl group. Together the R group and ‘x’ symbol determine the type of hydroxyalkanoate present in the polymer. The total number of carbon units within the monomer differs across PHA polymers and the different chain lengths can be used to classify them, as seen in Table (5) below [81,8,83]. When these monomer units are incorporated into the polymer, they can either form homopolymers like P(3HB) or copolymers like PHBV with various physical properties.

 

Approximately, 150 different PHA polymers have been identified so far and this has been made possible due to the broad substrate specificity of PHA synthase (the key enzyme in PHA biosynthesis) alongside the diverse metabolic pathways active within the cell [81]. This number continues to grow as new PHA discoveries occur across the globe.

 

Figure 6: General PHA Structure

PHA Fig 6

Table 5: Polyhydroxyalkanoate (PHA) Classification

Types of Chain Length

Number of Carbon Atoms

Examples

Short Chain-Length

3-5

Poly(3-hydroxybutrate) or P(3HB)

Poly(4-hydroxybutrate) or P(4HB)

Poly(3-hydroxyvalerate) or PHV

Medium Chain-Length

6-14

Poly(3-hydroxyhexanoate) or PHHx

Poly(3-hydroxyheptanoate) or P(3HHP)

Poly(3-hydroxyoctanoate) or P(3HO)

Poly(3-hydroxydodecanote) or P(3HDD)

Long Chain-Length

15 or more

Poly(3-hydroxypentadecanoate) or P(3HPD)

Poly(3-hydroxyhexadecanote) or P(3HHD)

 

 

Table 6: Examples of PHA Copolymers

PHA Fig 7

4.4 The Biology, Occurrence & Biosynthesis of PHAs

A wide variety of gram-negative, gram-positive and certain archaea are capable of synthesizing PHAs. These microorganisms often respond to unexpected changes in the quantities of their essential nutrients by storing up relevant nutrients for survival while subjected to long periods of starvation [81]. PHAs are one of such storage compounds, which in most cases function as intracellular carbon and energy storage compounds. These PHAs are usually accumulated when the bacterial culture is subjected to unbalanced growth conditions with excess carbon and limited nutrient availability such as nitrogen, phosphorous, etc. [8]. Under these conditions, the carbon is assimilated and converted into hydroxyalkanoates, which is further polymerized to form the desirable high molecular weight PHA compound. PHAs are water insoluble and appear as discrete, almost spherical granules in the cytoplasm of the cell. This excellent storage property permits the granules to be non-disruptive to the osmotic pressure of the cell, regardless of the quantity present. The number and size of the granules per cell differs from one PHA-producing microorganism to another and is dependent on the growth stage. The average size of the PHA granule ranges from 0.2-0.6 μm & an average number of 8-13 granules of PHA are often present per cell. The PHA granule is often covered with a phospholipid monolayer containing certain granule-associated proteins such as PHA synthase, depolymerase, structural proteins, regulatory proteins and cytosolic proteins that may be attached to granule through hydrophobic interactions [81]. 

 

The PHA biosynthesis process consists of two major steps: (1) The carbon source is assimilated and incorporated into a hydroxyacyl-CoA & (2) The hydroxyacyl-CoA intermediate is polymerized into a PHA by the PHA synthase enzyme encoded by the phaC gene [8]. To date, several PHA biosynthesis pathways have been discovered and studied extensively over the years. These pathways are basically linked up with several other central bacterial metabolic pathways such as glycolysis, the TCA cycle (or Krebs cycle), β-oxidation, the serine cycle, the Calvin cycle, de novo fatty acid synthesis and amino-acid metabolism. Many intermediates are shared between these pathways with acetyl-CoA being one of the key intermediates. As previously mentioned, PHA accumulation is stimulated during unbalanced growth conditions with excess carbon and nutrient limitation. During these unbalanced growth conditions when a nutrient such as nitrogen or phosphorus is limiting, the Krebs cycle produces lower levels of coenzyme A due to the inhibition of citrate synthase. This allows acetyl-CoA to be directed towards the PHA synthetic pathway for PHA production. Meanwhile, during balanced growth and nutrient-rich conditions, the Krebs cycle produces higher amounts of coenzyme A, hence blocking PHA synthesis by inhibiting 3-ketothiolase (phaA) such that acetyl-CoA is channeled back to the Krebs cycle for energy production and cell growth [81] [83]. This mechanism enables the microorganisms to maximize their resources and adapt to stress conditions while imposed to fluctuating environmental conditions.

 

Detailed reviews on the different PHA biosynthesis pathways and enzymes involved have been outlined in several articles [81,82,83,8,84]. Figure (7) shows a few of these pathways, most of which are relevant to this report. Nonetheless, there is still much about PHA biosynthesis that is yet to be uncovered. Upcoming research will clarify some doubts about current pathways and also bring to light new pathways that could possibly optimize the production of PHAs for tailor made applications.

 

Figure 7: Metabolic Routes for PHA Biosynthesis

PHA Fig 8

Note 2: Adapted from Djonlagic et al. (2011)

 

4.5 PHA Biosynthesis in Methanotrophic Bacteria

Methanotrophic bacteria are also very capable of producing PHAs. A recent study showed that the production of PHB is linked to the serine cycle through which acetyl-CoA is produced [48]. The aim of this study was to test for PHA production in methanotrophic bacteria. Twelve strains from different genera, which represented both Type I & II methanotrophs were screened for the PHA synthase enzyme, which encodes the phaC gene. It was observed that Type I methanotrophs, which assimilate carbon via the RuMP pathway did not posses the phaC gene and hence were incapable of producing PHB. Meanwhile, Type IIs on the other hand were capable of producing PHB from methane gas via the serine pathway and hence possessed the phaC gene [48]. Downstream of the serine pathway, three main enzymes (β-ketothiolase, Acetoacetyl-CoA reductase & PHA synthase) are crucial to the synthesis to PHA. β-ketothiolase encoded by phaA, condenses two acetyl-CoA molecules to from acetoacetyl-CoA. Acetoacetyl-CoA reductase encoded by phaB, is then reduced to (R)-3-hydroxybutryl-CoA which finally gets polymerized by an esterification process. With the aid of the PHA synthase enzyme (phaC), poly(3-hydroxybutrate), PHB is produced as a result [48] [8]. A schematic of this biological pathway can be seen in Figure (8). 

 

In a mixed culture containing both Type I & Type II methanotrophs, understanding the best selection strategies needed to maintain a stable culture of Type II methanotrophs for PHB production is crucial. As explained in section 3 of this report, low copper concentration favors the growth of Type II methanotrophs given that they all possess the sMMO enzyme, which is activated in the presence of low copper in the media. Nitrogen fixation might be a useful selector as Type I methanotrophs have been reported to grow much slower on nitrogen gas than their Type II counterparts. Other factors such as pH and varying nutrient concentration in the media have also been reported to play a major role in selecting for the best methane-utilizers capable of producing PHB. These selection pressures were tested and verified by a few recent studies with a conclusion that one of the best strategies is through nitrogen starvation alongside alternating the nitrogen source between ammonia and nitrate during the growth phase [48,49]. 

 

Figure 8: PHB Production from Methane via the Serine Cycle

PHA Fig 8

Note 3: [1]-Methane monooxygenase (mmo); [2]-Methanol dehydrogenase; [3]-Formaldehyde dehydrogenase; [4]-Formate dehydrogenase; [5]-3-ketothiolase; [6]-Acetoacetyl-CoA reductase; [7]-PHA Synthase

 

4.6    Properties of PHAs

The physical, mechanical and material properties of PHAs are highly dependent on their monomer composition and chemical structure. This could cause the polymers to either be hard and crystalline in nature or elastic and rubbery. P(3HB) for example has a melting temperature (Tm) of 180 oC and glass transition temperature (Tg) of 4 oC. This P(3HB) homopolymer is a typical thermoplastic which means the polymer resin becomes highly viscous and moldable at temperatures above its Tm [82,8]. P(3HB) has mechanical properties similar to those of polypropylene (PP) with a Young’s modulus of 3.5 GPa & tensile strength of 40 MPa. However, PP has a higher elongation to break (400%) compared to P(3HB) (5%) [81]. This is because P(3HB) is very brittle and stiff, and highly crystalline in nature. This brittleness in P(3HB) is caused by the presence of large crystalline spherical-like bodies that form during processing when the material is being molded into product [85]. 

 

Incorporating a co-monomer unit into the polymer backbone can greatly affect and improve the polymer properties by increasing flexibility, and decreasing toughness and crystallinity of the material. For example, when 3-hydroxyvalerate (3HV) is introduced into a P(3HB) polymer, its properties are greatly impacted. A steady decrease in the Young’s Modulus & tensile strength are observed as the 3HV concentration increases from 0-25 mol%, indicating that the polymer’s flexibility and ductility increase respectively. At the same time, the impact strength increases with increasing 3HV concentration implying that the copolymer gets tougher. Meanwhile, the decrease in Tm widens the processing gap within which the polymer can be melted without degrading. Consequently, the decrease in Tg allows the co-polymer material to be used at lower temperatures without experiencing any brittleness [81] [85]. 

 

Poly(4-hydroxybutyrate) is a very flexible homopolymer with a high elongation to break of 1000%. This polymer is very strong and malleable with a Young’s modulus of 149 GPA & tensile strength of 104 MPa. P(4HB) has a glass transition (Tg) and melting temperature (Tm) relatively lower (-48 oC and 53 oC respectively) than several other PHAs which permits the material to be very ductile. Incorporating the 4HB monomer unit results in copolymers with desirable properties such as reduced crystallinity, low tensile strength and great elastomeric properties. Varying the concentration of 4HB in the copolymer modifies the polymer’s material properties permitting for the synthesis of tailor-made polymers. P(4HB) and its copolymers are therefore very promising for future applications, especially in the medical field. Many PHAs are also known to possess piezoelectric properties useful in bone growth and wound healing through the electrical stimulation that arises from the diversity of the polymer’s chemical structure [82,86,8,81,87]. Table (7) below summarizes all the physical and mechanical properties discussed in this section.

Table 7: Polyhydroxyalkanoate (PHA) Properties

PHA table 7

Note 4: Polypropylene (PP); Polyethylene (PE); Polystyrene (PS)

 

4.7 Detection & Quantification of PHAs

Several methods have been developed and are available for the detection and quantification of PHA in the microbial cell. Depending on the detection focus, these methods can reveal the presence or absence of PHAs in the cell, identify the microorganism’s PHA producing capacity, visualize the PHA granule in the cell, distinguish between different PHAs present and provide quantitative values of how much PHA the cell is producing. To obtain high-throughput screening and identification of microbes with a PHA production capacity, polymerase chain reaction (PCR) gene detection and cell-staining methods could be employed. PCR gene detection uses primers to amplify the phaC gene, which encodes for PHA synthase. This gene is expected to be present only in PHA-producers even though it is possible for non-specific PCR amplification or other possible errors to occur [83]. For cell-staining methods, dyes like Nile red or Nile blue A are directly added to growth media, liquid cell culture or onto fixed cells, and under UV limitations, these dyes fluoresce allowing for detection of PHA in the cells. A drawback to this method is the possibility of the dyes staining other non-PHA lipid storage compounds. The PCR gene detection and cell-staining methods are both easy ways to detect the presence of PHA in the cells effectively and efficiently. However, other methods need to be employed in order to obtain a qualitative and quantitative analysis of PHA composition, and also to differentiate between the types of PHAs being accumulated. Visualization of the polymer directly in the cell is also possible by the use of Transmission Electron Microscopy (TEM), which provides imaging proof of the accumulation of PHA. The main down side with TEM is the tedious sample preparation process, which uses toxic chemicals that are lethal to the cell [83].

 

A more quantitative and qualitative analysis of the intracellular PHA can be achieved through methods such as Fourier transform infrared spectroscopy (FTIR), liquid chromatography (LC) and gas chromatography (GC). The GC technique however continues to be the most preferred method due to its ability to provide accurate quantification information and its high detection sensitivity [83]. Braunegg et al developed one of the first GC methods for accurate and reproducible determination of the PHA content in the microbial biomass [88]. This method which does not require an extraction step, consists of a methanolysis process to convert P(3HB) into its methyl ester derivatives for further quantification using a GC with flamed ionization detector (GC-FID). The method enables determination of PHB in the cell to levels as low as 10-5 g/L. The GC-FID method has now been expanded for the determination of copolymers and other mcl-PHA. However, exact analytical standards are needed to obtain precise measurements while utilizing the GC-FID method. Others have coupled GC to mass spectroscopy detectors (GC-MS) specifically to identify new PHA monomers in the absence of analytical standards [83].

 

4.8 Separation & Purification of the PHA Polymer

The most important step in the PHA production process is the extraction of the intracellular PHA granule from the cell. However, this recovery process is very expensive, laborious and energy intensive. Therefore, a cost effective recovery process is imperative to keep the PHA polymer competitive in the market compared to the more standard synthetic polymers. Several methods have been developed for the extractions of PHAs from the cell. These methods include solvent extraction methods, digestion methods, mechanical disruption, supercritical (SC) fluid techniques, floatation techniques, gamma irradiation methods, spontaneous liberation methods and cell secretion methods. Table (8) shows an outline of most of the methods that have been developed so far while Table (9) details some advantages and disadvantages of these methods.

 

4.8.1 Solvent Extraction

The use of solvents for the extraction of PHA is one of the most conventional methods studied to date. This method can be broken down into two stages: (1) first the cell membrane is modified to encourage permeability, which facilitates the release of the PHA polymer, and (2) a non-solvent precipitation process follows. The PHA extraction solvents commonly used are chlorinated hydrocarbons such as chloroform, chloroethane, chloropropanes; cyclic carbonates such as ethylene carbonate, 1,2-propylene carbonate; and ketones such acetone. Tetrahydrofuran methyl cyanide, tetrahydrofuran ethyl cyanide and acetic anhydride are also other solvents that have been tested. The non-solvent precipitation compounds most frequently used are methanol and ethanol [89,90]. 

 

Solvent extraction methods are very efficient and most of them result in very high levels of purity (>95%) of the PHA polymer. However, the major drawback is their high cost of operation. P(3HB) in particular has been reported to be highly viscous especially when its concentration exceeds 5% (w/v). Therefore removal of the cell debris is very difficult as it requires larger quantities of solvent and takes longer for the separation process to be completed. Potentially, this process poses a great environmental concern as highly toxic and volatile chemicals are being released as waste. That is why solvent extraction has had limited success in large-scale processes and the method is widely use only in laboratories [89,90]. 

 

Contrary to many extraction methods, solvent extraction does not degrade the polymer and it eliminates endotoxins commonly found in most gram-negative bacteria, hence rendering the polymer even more attractive for medical applications. However, the use of solvents destroys the natural morphology of the PHA granule, impacting its use for certain applications such as the production of strong fibers [90]. 

 

In order to address some of the issues of solvent extraction highlighted above, the use of waste solvents or recyclable solvents could minimize the cost of production. Furthermore, using non-halogenated or organic solvents could also address some of the hazardous waste and environmental concerns associated with most solvent extraction methods [89,90].

 

4.8.2 Digestion Methods

Digestion methods have been widely used as an alternative method to solvent extraction and unlike solvent extraction, this method focuses on dissolving the non-PHA cell material surrounding the polymer granule. These digestion methods could be carried out either enzymatically or chemically, with chemical digestion being the most studied due to the well-known properties of most chemicals [89].

 

The chemical digestion method makes use of sodium hypochlorite, which is non-selective and has strong oxidizing properties, or surfactants such as sodium dodecyl sulfate (SDS) are also normally used. These chemicals are used to solubilize the non-PHA biomass in-order to achieve PHA separation and the polymer is recovered by centrifugation. However, the PHA obtained through the extraction of either of these compounds is not of good quality. Sodium hypochlorite specifically degrades the cell resulting in lower molecular weights while SDS produces lower purity polymers. A number of studies investigated combination methods comprising of surfactant-hypochlorite & chloroform-hypochlorite. Of these combinations studied, the surfactant-hypochlorite method demonstrated higher PHA recovery and the cost of extraction was 50% less than that from the chloroform-hypochlorite method. However, the high cost of the chemical agents required still remains a problem [89,90,83]. Surfactant-chelate digestion methods have been tested resulting in high quality product and low environmental pollution. However, large volumes of wastewater are produced during the recovery process [90]. 

 

PHA recovery by enzymatic digestion requires a variety of suitable enzymes such as proteases to digest and lyse the cell, usually through a number of processes such as heat treatment, enzymatic hydrolysis & surfactant washing. This method is very attractive due to its environmental impact advantages, simplicity in process operation and high level of enzyme specificity for the target reaction. However, the high cost of enzymes makes the method infeasible for large-scale processes [89]. 

 

4.8.3 Mechanical Disruption

Mechanical cell disruption is commonly used for releasing intracellular proteins. This process could either undergo solid shearing (bead milling) or liquid shearing (high pressure homogenization) [90]. 

 

Bead mill disruption is based on the use of beads to shear cells and transferring the energy from the beads to the cells in the contact zone. The method is independent of the biomass concentration, it requires less power supply, and is not susceptible to blockages. However, the disruption process is affected by the bead loading, as it requires several passes to obtain complete disruption [90,89]. 

 

Alternatively, high-pressure homogenization is one of the widely used disruption techniques for large-scale production. It is carried out by disrupting a cell suspension under high pressure through an adjusted, restricted orifice discharge valve. However, reports have stated that the PHA recovery efficiency with homogenization is much less compared to bead milling, mostly because microbial physiological parameters together with cell concentrations could affect the disruption efficiency. Furthermore, high-pressure homogenization could potentially thermally degrade the polymer and produce cell debris in the product that could impact downstream processing.

 

In general, mechanical disruption methods do not require chemicals and therefore do no pollute the environment. They do not significantly contaminate the products and are only reported to cause very mild damage to the products being tested. However, the upfront cost for the process is very high due to the high capital investment, processing times are very long and scale-up is quite difficult [89].

 

4.8.4 Cell Secretion

Research has recently been published on the secretion of PHB from E. coli using synthetic biology techniques [91]. In this study, phasins – which are low molecular weight proteins that bind to PHA granule surface and play a role in the formation of granule – are targeted for the secretion process since PHB is a non-proteinaceous polymer and cannot be targeted for genetic secretions. The phasin is overexpressed and channeled through a type 1 secretion pathway through genetic fusion with signal peptide targeting sequences. The type 1 secretion system then translocates the protein from the cytoplasm of the cell to the external media without any interaction with periplasm. This study demonstrated that after a 48-hour cell incubation and PHB induction period, 28.3% of PHB in dry mass was secreted, accounting for 36% of the total PHB produced [91]. This process is quite promising for downstream PHA processing as (1) it eliminates the need to disrupt the cell by not affecting the cell vitality, (2) it has a potential to improve upon current PHA recovery and purification techniques, and (3) if properly understood, it could be implemented for scaled up systems. However, extensive knowledge of synthetic biology is required to better understand the process operation in order to accomplish these goals. 

 

Table 8: Summary of PHA Extraction Methods. Adapted from G.A Tan et al. (2014); N. Jacquel et al. (2008) & Kunasundari and Sudesh (2011)

Method

Chemicals Used

Bacterial Strain(s)

Result Ranges

Solvent Extraction

Chloroform; chlorinated hydrocarbon; cyclic carbonates; acetone; tetrahydrofuran methyl cyanide, tetrahydrofuran ethyl cyanide and acetic anhydride

Bacillus megaterium; Ralstonia eutropha; Cupriavidus necator 545

Purity: 93%-100%

Recovery: 65%-95%

Digestion by surfactants

SDS; Palmitoyl carnitine

A. lactus; A. eutrophus; Ralstonia eutropha; Recombinant E. coli

Release rate: 70%-85%

Purity: 95%

Recovery: 90%

Digestion by sodium hypochlorite (NaOCl)

sodium hypochlorite (NaOCl)

Cupriavidus necator 545; Ralstonia eutropha; Recombinant E. coli; Pseudomonas putidia KT2442

Purity: 86%-99%

Recovery: 78-94%

Digestion by sodium hypochlorite (NaOCl) & surfactant

 

Azotobacter chroococcum G-3; Cupriavidus necator 545 

Purity: 98%

Recovery: 86%

Enzyme Digestion

Bromelain; Pancreatin; Papain; Trysin

Ralstonia eutropha; Cupriavidus necator 545; Burkholderia sp. PTU9

Purity: 87%-90%

Recovery: 78%

Mechanical Disruption

Bead milling & High pressure homogenization

A. latus

 

Supercritical fluid

Superficial CO2

Cupriavidus necator 545

Recovery: 89%

Dissolved air flotation

Enzymatic hydrolysis; Sonication; Floatation

Pseudomonas putidia KT2442

Purity: 86%

Gamma irradation

Radiation-chloroform

Bacillus flexus

Recovery: 45%-54%

 

Table 9: Advantages and Disadvantages of PHA Extraction Methods. Adapted from N. Jacquel et al. (2008) & Kunasundari and Sudesh (2011)

Methods

Advantages

Disadvantages

Solvent Extraction

Endotoxin removal; High purity; Limited polymer degradation; High molecular weight

Requires large amount of toxic & volatile solvents; Possible disruption to the native order of polymer chains in the PHA granule; Low recovery rates; Laborious process. 

Digestion by surfactants

High molecular weight; No disruption to the native order of polymer chains

Low purity; Large amounts of surfactants required; Wastewater treatment required.

Digestion by sodium hypochlorite (NaOCl)

High purity of PHA

Reduction to molecular weight; Degradation of polymer.

Digestion by sodium hypochlorite (NaOCl) & surfactant

Limited degradation to polymer; High purity; Low operating cost compared to solvent extraction; Simple and rapid process

High cost chemicals; Wastewater treatment required

Enzyme Digestion 

Excellent recovery; Good quality product

High cost of enzymes; Complex process

Mechanical disruption methods

No chemical required; Less contamination

Bead milling requires several passes; Homogenization results in poor disruption rates for low biomass levels

Supercritical fluid (e.g. CO2)

Inexpensive; Low toxicity; Simple process

Difficulties in dealing with natural samples and extracting polar analytes; Dependent on process parameters

Dissolved air floatation

No chemical required; Less contamination

Several sequential floatation steps required

Gamma irradiation

No chemical required; Less contamination; Low degree of cross-linking leads to retention of solvent solubility

High initial investment cost; Extended length of irradiation time

 

4.9 Characterization of PHAs

Due to the diversity of the PHA’s chemical composition caused by their numerous and extremely different monomer unit incorporations, it is important to characterize the polymer to pinpoint suitable downstream applications. 

 

The composition of the monomer unit can be determined using GC, LC and nuclear magnetic resonance spectroscopy (NMR). The chromatographic methods are similar to those used for detecting the polymer, as mentioned previously. NMR looks at the whole PHA polymer, differentiating between PHA blends and co-polymers, and providing details of the arrangements and functional groups in the molecule [83]. The 1H-NMR & 1C-NMR are the two techniques used for this analysis. Nonetheless, 1H-NMR is most preferred due to the high proton abundance in the PHA molecule making the technique very sensitive, and the short analysis time (~1hr) makes the technique even more desirable. However, the two techniques could be applied in parallel to obtain a broader analysis of the PHA polymer. The intensity of the signals obtained from the NMR spectra could be used for a more quantitative estimation of PHA monomers present [83]. A positive aspect with the NMR analysis is the non-destructive manner of the analytical tool and there is no need for analytical standards, which allows for new and unknown PHA polymers to be discovered.

 

Determining the PHA polymer’s average molecular weight (Mw), molecular weight distribution (Mn) and polydispersity Index (PDI) are all important to understand the polymer’s physical properties such as strength, toughness and transition temperatures. When the Mw is too low, then the polymer’s strength is too low for it to be useful. Polymers usually have uneven chain lengths and therefore will have their molecular mass distributed over a certain range. The PDI then measures the broadness of the molecular mass distribution (Mn) where larger PDIs imply that the molecular weight is broader. However, a PDI=1 means the polymer is monodisperse - having equal chain lengths [92]. These characteristics can be measured using a Gel Permeation Chromatography (GPC), which uses polystyrene standards and separates the polymer sample in different columns on a size basis. Typical PHA molecular weights usually range from 2x105 to 3x106 Da [93].

 

In order to understand the processing window for a better determination of the appropriate application of the PHAs, it is important to obtain the glass transition (Tg) and melting (Tm) temperatures. Differential Scanning Calorimetry (DSC) and Differential Thermal Analysis (DTA) are the two methods commonly used for this characterization. However, the DSC method is preferable because it provides both qualitative and quantitative thermal information while DTA provides only qualitative thermal information [83].

 

Other mechanical properties usually evaluated for PHAs are Young’s modulus (sometimes called the flexural modulus), elongation to break, and the tensile strength. These metrics provide measurements on stiffness, the ability of the polymer to stretch and the force required to pull the material, respectively. A tensile teaser instrument using standardized tests is used to perform such analyses [83]. An overview of these techniques can be seen in Table (10) below.

 

Table 10: Characterization Techniques for PHA polymers. Adapted from G.A. Tan et al. (2014)

Technique

Property

Indicator

Sample Requirement

Gas Chromatography (GC) & Liquid Chromatography (LC)

PHA monomeric units

Chemical composition of PHA monomers

5-15 mg of biomass

Nuclear Magnetic Resonance (NMR)

PHA polymeric units

Topology and functional groups of PHA molecules

5-50 mg of PHA 

Gel Permeation Chromatograph (GPC)

Molecular distribution

Polydispersity index (PDI), Molecular mass and Molecular weight

0.1-1 mg of PHA 

Differential Scanning Calorimetry (DSC) & Differential Thermal Analysis (DTA)

Thermal properties

Glass transition and melting temperatures

5-10 mg of PHA

Thermogravimetric Analysis (TGA)

Thermal properties

Thermodegradation temperatures

10mg of PHA

Fourier Transform Infrared Spectroscopy (FTIR) & X-ray diffraction

Crystallinity

Infrared absorption bands and diffraction intensity correlated to crystallinity 

5-10 mg of PHA

Mechanical Testing Instruments like a tensile teaser

Mechanical properties

Tensile strength, tensile stress, elongation to break and Young’s modulus

Polymer thickness: 1-14mm, width: 19-29 mm, length: 165-246 mm

 

4.10 Biodegradation of PHAs

PHAs have the ability to degrade biologically under both aerobic and anaerobic conditions, making them an attractive alternative for synthetic polymers. Other than biological degradation, PHAs can also be degraded hydrolytically or thermally. However, degradation by hydrolysis is very slow due to the high crystallinity of the polymer and thermal degradation is reported to be an issue with respect to the ability to process the material [86].

 

Most PHA-producing organisms are capable of the intracellular biodegradation of the PHA polymer by using the PHA depolymerase enzyme, which most of them possess. For example, when P(3HB) undergoes intracellular biodegradation, PHB depolymerase breaks PHB into 3-hydroxybutric acid, which is further converted by a series of enzymes including β-kethiolase (phaA) to form acetyl-CoA. Under aerobic conditions, acetyl-CoA is fed back into the Krebs cycle and oxidized to produce CO2 [82]. Extracellular biodegradation is also possible by microorganisms (such as bacteria, fungi, algae) present in several natural environments, which possess extracellular depolymerases that are secreted upon attacking the polymer. These depolymerases solubilize the polymer and these soluble products become available for them to absorb as a growth substrate [82,86]. The microbial population in the environment contributes enormously to the biodegradation activity of the polymer.

 

PHA biodegradation in the environment is controlled by a number of key factors such as its physicochemical properties (pH, moisture content, temperature), microbiological parameters (population microbial diversity, microbial adaptability), the material properties of the PHA material itself (molecular weight, polymer composition, crystallinity) and the material processing (surface area, additives, material thickness) [82,86,93]. Reports have shown that PHAs with lower molecular weight are more liable to biodegradation and in cases where the melting temperatures are significantly high, the enzymatic activity responsible for biodegradation is very slow [82]. For effective biodegradation, it is important that the environment contains the suitable microbial population, its conditions are favorable for the appropriate microbial activity to take place, and the polymers present are susceptible to microbial attack [93].

 

PHA biodegradation in living systems is important especially when designing materials for medial applications. Studies have showed that PHA degradation occurs in many stages. First the amorphous regions break apart randomly, then the polymer chains are disrupted and its crystallinity increases forming smaller molecules with lower molecular weights. Finally the polymer erodes leading to loss in the polymer mass. This degradation process period varies depending upon the environmental conditions and the material properties of the polymer [82].

 

Very recently, it has been discovered that wax worms are capable of degrading polymers in their gut [94]. This fairly new finding is very intriguing and has been examined with food packaging made from both polyethylene (PE) and polystyrene (PS). The study showed that (1) the insect larva can grow on both PS and PE, (2) a short retention time of only 12-24 hrs was needed for the polymers to be degraded in the larva’s gut, and (3) PS degradation stopped after subjected to antibiotic treatment, demonstrating that microbes in the gut played an integral role in the polymer degradation. This discovery proposes a new route for polymer degradation and therefore suggests another approach to improve upon polymer biodegradation [94]. Current research has started to look at the ability of bacterial community in the wax worm’s gut to degrade PHA polymers and conditions favorable for this transformation to take place.

 

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