Basics Figure 4:
Basics Figure 4: Electron microscopic image of microorganisms containing PHA (© Metabolix) A lack of carbon or energy will cause the degradation of the PHA storage polymers. The choice of microorganisms for industrial applications depends on the microorganism’s stability and biological safety, its PHA production rates, PHA extractability, the molecular weight of the agglomerated PHA, as well as the spectrum of useable carbon sources. The maximum known production rate lies in the range of 5 g per liter fermenter volume and hour. In general, two different types of microorganism can be used to generate PHB. One type produces PHB continuously, the other type only when basic growth supporting substances are depleted while there is still an oversupply from a carbon source available, i.e., discontinuously. The following process steps can be distinguished in bacteria fermentation: a) Continuous synthesis (e.g., alcaligenes latus): 1) Inoculation, i.e., multiplication and growth of the production organism and parallel PHA synthesis by continuously synthesizing microorganisms 2) Isolation/production of the biopolymer, i.e., separation from biomass and purification 3) Compounding and granulation b) Discontinuous synthesis (e.g., alcaligenes eutrophus): 1) Inoculation, i.e., multiplication and growth of the production organism 2) PHA synthesis under altered fermentation conditions 3) Isolation/production of the biopolymer, i.e., separation from biomass and purification References [1] Endres, H.-J., Siebert-Raths, A., Engineering Biopolymers, MArkets, Manufacturing and Applications, Carl Hanser Verlag, München, 2011 [2] Hocking, P., et al. Enzymatic Degradability of Poly(beta-Hydroxybutyrate) as a Function of Tacticity. Macromolecular Raprid Communication. Jg. 15 , 2003, H. 6, S. 447 – 452. [3] Wolf, O. (Ed.), et al. Techno-economic Feasibility of Largescale Production of Biobased Polymers in Europe. Brüssel : s.n., 2005. Technical Report EUR 22103 EN. [4]76. Kaplan, D. L. Biopolymers from renewable resources. Berlin : Springer-Verlag, 1998. [5] Doi, Y. et.al.:. Environmental life cycle comparison of polyhydroxyalkanoates produced from renewable carbon resources by bacterial fermentation. 11/2002. [6] Stevens, E. S. Green plastics. An introduction to the new science of biodegradable plastics. Princeton : s.n., 2002. [7] Smith, R. Biodegradable polymers for industrial applications. Boca Raton Fl : CRC Press LLC, 2005. [8] Fakirov, S. und Bhattacharyya, D. Handbook of engineering biopolymers. Homopolymers, blends, and composites. Cincinnati : s.n., 2007. 4) Compounding and granulation For PHAs, much as with PLA, inoculation is the first step of the bacterial fermentation process. Here, the bacteria required for the subsequent metabolization process multiply and grow in an aqueous medium enriched with a balanced nutrition supply (C, N, P, S, O, Mg, Fe) and air under optimum physical conditions. In the next step, the actual PHA synthesis begins under conditions not conducive to growth and multiplication (e.g., phosphate limitation) and a relative oversupply of C. The PHAs are usually stored in intracellular inclusion bodies and can account for up to 90% of dry cellular weight. Their molecular weight generally ranges from 100,000−500,000 g/mol. However, molecular weights of considerably more than 1,000,000 g/mol are obtained under special conditions (ultra-high molecular weight PHAs). The complete fermentation process typically takes approx. two days [7, 8]. Glucose and sugar-containing substrates, e.g., molasses, lactose, cellulose, starch, and whey hydrolysates, serve as nutrient sources for intracellular PHA generation. Other sources such as alcohols (e.g., methanol or glycerol), alkanes (hexane or dodecane), vegetable oils, or organic acids are also suitable nutrient sources. The enzymes involved in the fermentation process are quite unspecific. Thus, a tailored substrate supply allows for the production of a wide variety of short (4−5 × C) or medium chain-length monomers (6−16 × C); PHA copolymers or, in the future, PHA terpolymers, can also be 44 bioplastics MAGAZINE [03/11] Vol. 6
Basics generated. For example, hydroxyvaleric acid can be incorporated by breeding the cells on glycose with additions of, e.g., propionic, methylpropionic, or valeric acid. A variety of copolymers can be generated by varying the fermentation conditions and the substrate supply. Other than with chemical (or man-made) synthesis, biosynthesis does not require catalysts or other auxiliary substances for polymerization. Thus, the microbial polyesters present in the cells are characterized by extremely high purity. Often there is no spatial separation between the two processing steps of bacterial growth/ multiplication and actual PHA generation. Different fermenters are not required because the transition from bacterial growth to PHA generation is initiated by a change in nutrient supply and fermentation conditions in a single fermenter. PHAs are usually manufactured in batch or fed-batch processes because optimum conditions for the individual process steps in the growth and production phases can be achieved most easily in batch processes. They provide higher intracellular PHA contents than continuous processes. On the other hand, the potential variation in product quality is a disadvantage of batch-wise manufacture. In the next step, the polymer-containing microorganisms are isolated from the fermentation broth and the intracellular agglomerated PHAs are purified. Classical mechanical separation techniques, such as centrifugation and filtration, are used in a first sub-step to separate the cells from the culture medium. In the second sub-step, the cells are destroyed and the raw polymer is isolated. PHA extraction can be carried out by various solvent extraction methods, but also by solvent-free, so called LF-methods. The solvents used are returned to the process in a closed circuit. Separation and lysis of bacterial cells and the subsequent separation of raw PHA essentially determine cost and quality of the final product and the ecology of the production method. (…) Although solvent-free methods are fundamentally more ecological than methods using solvents, they do not achieve similarly high product purity. Here, a new development using genetically modified bacteria represents progress: after fermentation has taken place at 28°C, the cell membranes are lysed by a virus incorporated into the bacteria genome and activated only above 42°C. Subsequent to isolation, the PHAs are usually further purified and dried in vacuum processes. Further research is required to determine beneficial uses for the cell residue and/or biomass accruing during PHA production. Some potential options include conversion to biogas, production of animal feed, using it as substrate for further PHA production, or catalytical enzyme production from the biomass protein content. In a final step, PHA powder is extrusion-granulated for further processing to plastics on injection molding machines. Simultaneously, additives such plasticizers and nucleation agents can be incorporated for targeted improvement of processing properties. Compared to other biopolymers, the price of PHAs (…) is relatively high. (…). Initial manufacturers of various PHAs on a small scale include Biomer, Mitsubishi Gas Chemical Company, PHB Industrial Brazil S.A., Tianan Biologic Material Co., Ltd., and Kaneka Corporation. Meredian Inc. is also working on the development of a PHA material (Nodax). The US biotech company Metabolix bought all the rights to ICI materials patents from Monsanto. Metabolix (or Telles, a joint venture formed by Metabolix and ADM) (… started a production plant of 50,000 tonnes/ year in Clinton, Iowa, USA in 2010 - MT). Another approach of Metabolix Inc. is the utilization of genetically engineered tobacco to produce polyhydroxyalkanoates. A number of companies in the Brazilian bioethanol industry (besides PHB Industrial SA) are interested in expanding their product range. Fermentative, sugarcane-based PHA generation offers a product with higher added value and synergy effects. Not only is sugar obtained as substrate, but incidental manufacturing byproduct, bagasse or cane-trash, can be used to provide processing energy for PHA production. bioplastics MAGAZINE [03/11] Vol. 6 45
