Polyhydroxyalkanoates (PHA): Study Notes

This article provides a study-note on polyhydroxyalkanoates (PHA)

Polyhydroxyalkanoates (PHA) are intracellular carbon and energy storage compounds, produced by many microorganisms. They are biodegradable polymers, and are elastic in nature depending on the polymer composition. PHA are well suited for the synthesis of plastics, the biodegradable packing materials.

The conventional plastics, made from coal or oil, are not biodegradable. They survive hundreds of years and are a major source of environmental pollution, often resulting in ecological imbalance. A heavy demand for biodegradable plastic materials exists. There are some attempts to chemically synthesize biodegradable polyesters e.g. polylactic acid and poly-glycolic acid. The production of polyhydroxyalkanoates by fermentation is preferred for use as biodegradable plastics.

PHA-Chemistry and Properties:

PHA serve as lipid reserve materials in bacteria. Their function in bacteria is comparable to that of fats and oils in yeasts and fungi. The granules of PHA, stored within the cells are clearly visible under electron microscope. Some bacteria may accumulate huge quantities (up to 80% of dry weight) of PHA. Polyhydroxyalkanoates are linear polyester polymers composed of hydroxy-acid monomers (Fig. 30.1 A). The most commonly found monomers are 3 hydroxy acids with a carbon length ranging from C₃ to C₁₄.

Homo-Polymer PHA-Polyhydroxybutyrate:

The most common PHA is polyhydroxybutyrate which is frequently referred to as PHB. It is a polyester with 3-hydroxybutyrate as the repeating unit (Fig. 30.1 B). PHB is the homo-polymer PHA. PHB, as such, is rather hard and inflexible.

Being a high molecular weight compound, the accumulation of PHB in huge quantities also does not significantly effect the osmotic pressure within the cell. The reserve carbon compound PHB can be oxidized to carbon dioxide and water, releasing large amount of energy. Bacteria require energy, although they are not growing, to maintain pH gradient and concentration gradient of several compounds. This energy, referred to as maintenance energy essential for the survival of the cells, is met by the reserve material PHB.

Heteropolymers of PHA:

Majority of polyhydroxyalkanoates, with the exception of PHB, contain two or more different monomers and are referred to as heteropolymers. These heteropolymers are usually composed of a random sequence of monomers, and not different monomers, in different chains.

Besides 3-hydroxy acids, several other hydroxy acids are found in the structure of PHA. e.g. 4-hydroxybutyrate (4HB). In fact, it has been found that the bacteria are capable of incorporating more than 100 different hydroxylated monomers into PHA. This depends on the organism and the nature of the carbon source supplied during the accumulation of the polymer. The properties of PHA mostly depend on the nature of the monomers it contains. In general, PHA with longer side chains (e.g. 3-hydroxy- octanoate containing PHA) and heteropolymeric PHA are more flexible and soft.

3-Hydroxybutyrate-co- 3-Hydroxy Valerate:

As already stated, by selecting a specific organism and by manipulating the composition of the medium, the chemical construction of the PHA can be altered. Thus, when the medium contains glucose and propionic acid, the organism Ralstonia eutrophia produces a copolymer of 3-hydroxy­butyrate and 3-hydroxyvalerate (abbreviated PHB/ V).

PHB as such is hard and brittle but the presence 3-hydroxy valerate monomers makes PHB/V flexible and stronger. The properties of PHB/V are similar to those of polypropylene, and therefore it is commercially more useful.

Polyhydroxybutyrates (PHB):

Biosynthesis of PHB:

Among the several PHA, the pathway for the biosynthesis of polyhydroxybutyrate (PHB) has been thoroughly investigated. Starting with acetyl CoA, PHB is synthesized in three reaction steps (Fig. 30.2). Acetyl CoA is converted acetoacetyl CoA by the enzyme 3-ketothiolase which is then reduced to 3-hydroxybutyryl CoA by acetoacetyl CoA reductase. The reducing equivalents are supplied by NADPH. The enzyme PHA synthase is responsible for the addition of 3-hydroxybutyrate residues to the growing PHB chain.

Regulation of PHB Biosynthesis:

The outline pathways concerned with the biosynthesis and degradation of PHB in Ralstonia eutrophia are depicted in Fig. 30.3. The enzymes of PHB biosynthesis are constitutive in nature. Thus the enzyme machinery for PHB synthesis is present all the time in the cell. When the growth of the microorganism is restricted by limiting an essential nutrient, PHB synthesis rapidly occurs.

During the active growth phase, acetyl CoA gets oxidized through the Krebs cycle. Further, free coenzyme A inhibits 3-ketothiolase and therefore very little PHB is synthesized. When the growth ceases or is restricted by limiting a nutrient, the operation of Krebs cycle (i.e. oxidation of acetyl CoA) decreases. This is mainly due to the inhibition of citrate synthase by NADH.

A decrease in the concentration of coenzyme A (as citrate is not formed from acetyl CoA, CoA is low) relieves the inhibition of 3-ketothiolase. This enables the diversion of acetyl CoA for the production of PHB. The supply of reducing equivalents (NADPH) required by the enzyme acetoacetyl CoA reductase also regulates the synthesis of PHB.

PHB, the storage energy reserve compound, can be degraded to acetyl CoA and metabolised via Krebs cycle. This occurs when the organism is deprived of energy supplying carbon sources. PHB is degraded by PHB de-polymerase to form 3-hydroxybutyrate. NADH is the inhibitor of the subsequent reaction, catalysed by the enzyme 3-hydroxybutyrate dehydrogenase i.e. formation of acetoacetate from 3-hydroxybutyrate. The biosynthesis and breakdown of PHB form a cyclic process, as depicted in Fig. 30.3.

Production of PHB:

Polyhydroxybutyrate is mostly manufactured by batch culture. PHB production occurs when there is an excess supply of carbon source, and limitation of some other essential nutrient such as nitrogen, phosphorus or sulfur source. The production/ accumulation of PHB is depicted in Fig. 30.4.

There are two distinct phases—a growth phase and a polymer accumulation phase. As the growth phase ceases, due to nutrient exhaustion, synthesis of polymer (PHB) commences. It is also possible to produce PHB by restricting the oxygen supply to aerobic bacteria.

Applications of PHB:

PHB can be implanted in the human body without rejection. This is because PHB does not produce any immune response and thus it is biocompatible. PHB has several medical applications e.g. as durable bone implants, for wound dressings. Attempts to use PHB as degradable sutures and other implants has not met with success due to very slow degradation of PHB.

Biosynthesis of Other PHA:

The biosynthesis of other PHA is quite comparable to that described for PHB. Some important features with regard to the synthesis of two important PHA are described.

Biosynthesis of PHB/V:

Some strains of Ralstonia eutropha (formerly known as Alcaligenes eutrophus) are capable of synthesizing poly (hydroxybutyrate-co-hydroxy- valerate) (PHB/V). For the formation of PHB/V, glucose and propionic acid are required as substrates. Propionic acid as propionyl CoA is responsible for the synthesis of 3-hydroxy valerate.

The three enzymes involved in the synthesis of 3- hydroxybutyrate (3-HB) also participate in the formation of 3-hydroxy valerate (3-HV). The polymer PHB/V contains 3-HB and 3-HV monomers in a random sequence (no individual polymers of 3-HB or 3-HV exist). The relative concentrations of glucose (precursor for 3-HB) and propionic acid (precursor for 3-HV) in the culture medium determine the chemical composition of PHB/V.

The biosynthetic pathway for the formation of PHB/V is depicted in Fig. 30.5.

Biosynthesis of PHA with long side chains:

It is generally observed that when a PHA producing organism is grown on a carbon source containing n carbons, 3-hydroxy monomers with n- 2 or n + 2 carbons are produced. This suggests the involvement of P-oxidation of fatty acids (which sequentially removes 2-carbon fragment) in the biosynthesis of PHA.

The organism Pseudomonas oleovorans can synthesize PHA when organic acids and alkanes are supplied in the medium as carbon sources. For instance, PHA with high content of 3-hydroxyoctanoate can be produced from n-octane or n-octanoic acid.

Biopol-A Biodegradable Plastic:

Biopol was the trade name used by ICI pic in UK for the biodegradable plastics composed of PHB and PHB/V. The Gram-negative soil bacterium, Ralstonia eutropha was employed for the manufacture of biopol.

Production of Biopol:

The fermentation process for the production of biopol involves two distinct phases-biomass production phase or growth phase, and polymer accumulation phase. This is comparable to the production of PHB (Refer Fig. 30.4).

When the bacterium Ralstonia eutropha is cultured on a simple medium containing glucose and salts, growth phase is observed. By restricting the essential nutrient phosphate (preferred since it is expensive compared to other nutrients), growth phase can be stopped. Now, glucose and propionic acid are continuously fed for large scale production of PHA i.e. PHB/V. The relative concentration of glucose and propionic acid in the culture medium determines the proportion of 3-HB and 3-HV monomers in biopol. Biopol containing around 10% 3-HV is found to possess the desired characteristics.

Recovery of biopol:

The cells can be disrupted and subjected to solubilisation of components other than biopol. The polymer can be washed and recovered by centrifugation.

Limitations in the production of biopol:

The manufacture of biopol by fermentation is very expensive when compared to the production of conventional non-biodegradable plastics. For this reason, many companies have stopped the commercial production of biopol by fermentation.

Applications of Biopol:

Biopol is approved for use as a food contact material. It is being used in the manufacture of paper cups and food trays. It is also used as a thin water proof layer in food packaging. Biopol, can be moulded to produce bottles and several other items.

Genetic Engineering for PHA Production:

It is possible to commercially produce PHB, PHB/V and other polyhydroxyalkanoates by fermentation of R. eutropha. This organism, however, grows very slowly and utilizes only a limited number of carbon sources for growth. For these reasons, the production of PHA by R. eutropha is expensive.

Fortunately, the genes responsible for the synthesis of important types of PHA by R. eutropha have been characterized and cloned. These genes have been transferred to E. coli (a bacterium that does not normally synthesize PHA). The resultant trans-formants, E. coli cells, grow rapidly and produced large quantities of PHB.

But the major limitation is that about half of the E. coli cells lose the plasmid constructs in about 50 generation’s. This poses a big problem, particularly for large scale continuous cultures. In recent years, some workers have tried to genetically manipulate and produce stable plasmids with some success.

Recombinant strains of E. coli have been used for the production of PHB, PHB/V and other types of PHA. There are some other advantages of producing PHA by E. coli instead of R. eutropha. The polymers are produced in a crystalline state by E. coli (they are in amorphous state in R. eutropha), and their extraction and recovery are much easier.

PHA Production by Plants:

The major limitation of producing PHA by fermentation is the cost factor. Approximately, it is ten times more expensive to produce PHA by bacteria compared to the manufacture of petrochemical plastics. Plants are attractive sources for a less expensive production of PHA. A transgenic plant of Arabidopsis thaliana, containing the bacterial genes for PHB genes, was developed in 1992. However, the yield of PHB was rather low.

Oil seed plants which have plenty of acetyl CoA (for oil biosynthesis) are attractive sources for PHA production. This is because the acetyl CoA is the key substrate for PHA synthesis. No significant success has been achieved so far for PHA production by oil seed plants.