List of limiting components leading to PHA formation

• Ammonia - Alcaligenes eutrophus, also others
• Carbon - Spirillum spp., Hypomicrobium spp.
• Iron, Mg - Pseudomonas spp.
• Mn, O₂ - Azospirillum, Rhodobacter spp.
• PO₄ - Rhodospirillum, Rhodobacter spp.
• Potassium sulfate Bacillus, Rhodospirillum, Rhodobacter etc.

Biopol of ICI is produced by using Alcaligenes eutrophus. Polymer separation and purification is accomplished by using a proprietary aqueous wash process followed by drying. PHB is too stiff and brittle for most applications, so the ICI add s a small amount of simple organic acid to the sugar feed stock to make the plastic stronger and more flexible. In technical terms, bacteria then produce co-polymers composed of PHB with varying amounts of hydroxyvalerate (HV). In this way ICI can produce a range of thermoplastic polymers which can be processed with conventional techniques to make bottles, mouldings, fibres and films. High grade Biopol is being made for medical applications, including woven patches for use inside the body to protect tissue s from scarring after surgery. After the wound is healed, enzymes in the blood dissolve away the patch. ICI's biodegradable plastic (thermoplastic Biopol) has found its North American applications as a blow-molded bottle and injection-molded caps for hair c are products. The Berlin Packaging corporation, Chicago will produce bottles and caps. Biopol currently sells for $ 8-10 per lb but is expected to sell for $ 4 per lb. The ICI company has currently a capacity of 600,000 lb per year in Billingham, UK. Annual production of 11 billion lbs is predicted by late 1990s. Biotehnologische Forschungsgesellschaft mbH in Austria produces PHB on a technical scale from cells of Alcaligenes latus with sucrose as a carbon source. 

Biopol has been used in Germany since 1990 to make the bottles of Wella's Sanara Shampoo. Biopol's US launch came in 1995, in the form of bottles for Brocanto International's Evanesce shampoo and material is being tested in the UK for cosmetic containers.
Japan also has shown interest in Biopol. Biopol has been introduced there in 1991 as a container for Ishizawa Kenkyujo's Earthic Alga shampoos and conditioners. Now three more hair care companies of Japan have started using Biopol containers and shortly Kai will use it for disposable razor with B iopol handle. Rubbish bags, disposable nappies, paper plates, cups coated with thin plastic film can be made with Biopol and will get degraded when thrown in landfill. Biopol shampoo bottle disappears in two years in typical dung.
With development in yields, productivity, use of newer strains PHB concentration and productivity of 100 g.l^-1 and 2.5 g.l^-1 h^-1 can be reached.
Chemi Linz AG began to develop a process for PHB production by fermentation in 1980s. Their polymer group Petrochemia Danubia (PCD) carries out the fermentation process through btF, a biotechnological research unit. The PCD process is different in that it uses Alcaligenes lactus as the producing organism and sucrose as the substrate. PHB formation is growth associated and nutrient limitation is not used to induce polymer accumulation. Solvent extraction is used to extract the product. It is a PHB homopolymer which is made. PCD-Polymere, an Austrian polyolefin producer developed a production process for PHB and processing technology for injection moulding and blow moulding. An Austrian biotechnology research company has developed a process for PCD-Polymere.
The fermentation process is based on a unique bacterial strain Alkaligenes latus. The strain is isolated from soil in California and Au stralia. It produces PHB in large amounts (80% of cell dry weight) during unlimited growth. Proprietary fermentation process developed is scaled upto 10 m3 of fermentation volume. In a fed batch mode more than 1 ton of PHB can be produced in less than a week. These fermentations are carried out in common stirred tank reactors, but other reactors like air lift or bubble column may also be used to get similar good results. The fermentation is carried out in mineral salts medium, sucrose or glucose as source of carbon. If precursors like propionic acid are added then co-polymers of PHB/HV can be produced as well as co-polymers of 3HB/4HB (3-hydroxybutyrate-co-4hydroxybutyrate) if 1,4 butanediol is added as precursor.
After fermentation cells are harvested, was hed with tap water and a concentrated suspension of 200 g/l is prepared. This suspension is directly used for the extraction process. The cell suspension is treated with solvent (methylene chloride). After this extraction step solvent is separated by cent r ifugation. The dissolved PHB is precipitated in water, recovered as white powder and dried. After one extraction step and one precipitation step PHB of 99% purity is obtained. This powder is directly used for compounding and further processing. Biomass can be recovered after the extraction process. It was tested as soil enhancer.
PCD-Polymere films and fibres are under development. Good results were obtained for injection moulding and blow moulding parts. PCD also helps others to develop further the technology.
The cost of producing PHB can be substantially reduced if methanol is used as substrate. Methanol is one of the cheapest noble substrates available, and has several advantages as fermentation substrate (purity, solubility, availability etc.) and is non-food substrate. Methanol can be easily obtained from natural gas and may also be obtained from biomass if necessary.
Methylobacterium extorquens is the isolate (gram negative, motile, pink pigmented bacterium can be used. Average PHB contents are found to be 25-30% of dry weight. It was also capable of producing PHV with the ratio of PHV to PHB of 0.2. Good process control was essential in the development of high cell-density fed-batch fermentation process for PHB production from methanol. Biomass leve l of 120 g/l and PHB levels of 60 g/l could be reached.