Introduction 2 2007 2005a b 2006 l cphA Now that cyanophycin can be produced in sufficient amounts by pilot scale fermentations for studying its material properties, it appears of biotechnological interest because purified cyanophycin can be chemically converted into a polymer with a reduced arginine content, which might be used like poly-aspartic acid as a biodegradable substitute for synthetic polyacrylate in various technical processes. In addition, cyanophycin might also be of interest for other applications when the hitherto unknown physical and material properties of this polymer will be revealed. On the other hand, cyanophycin is a convenient source of the constituent amino acids that may be regarded as nitrogen-functionalized precursor chemicals. In the current paper, conditions will be discussed for the technological and economic feasibility of cyanophycin production by microbial fermentation and by cyanophycin production directly in plants. The conditions for fermentative cyanophycin production will be based upon the use of cheap substrates derived from agricultural waste streams and the possible cyanophycin production simultaneously with other fermentation products like ethanol. This aspect is denoted process integration. Biorefinery and its place in the production of chemicals 2 2 2004 1 2005 2007 Table 1 Different applications and contributions of biomass Contribution Integral cost prices (€/GJ end product) Raw material cost fossil (€/GJ) Percentage of total energy in the Netherlands (3.000 PJ) consumed per application (%) Heat 4 3 (Coal) ±20 Electricity 22 6 (Coal) ±20 Transport fuel 10 8 (Oil) ±20 Average bulk chemicals 75 30 (Oil) ±20 Rest of industry   ±20 To obtain a good net income for biomass, an effective biorefinery system is required for the separation of the harvested crop into fractions for use in (several of) these applications. These may be used directly as the desired product or undergo conversion by chemical, enzymatic, and/or microbial means to obtain other products. Biorefinery systems are well established for a number of crops. For example, soybeans are the raw materials for large biorefineries to produce oil (for biofuels), proteins and valuable nutraceuticals. 2007 2 2007 General introduction on NRPs and especially cyanophycin 1887 2002 l l l 1976 2004 1 1990 1982 Fig. 1 Chemical structure of the cyanophycin monomer  Cyanobacteria 1971 1976 1973 1980 1990 1996 1990 2002 2002 Acinetobacter Desulfitobacterium hafniense 1998 1998 1998 cphA Anabaena variabilis Anabaena Synechocystis Synechocystis Synechococcus elongatus Synechococcus Escherichia coli 2000 2000 1999 1999 1998 cphA Ralstonia eutropha Corynebacterium glutamicum Pseudomonas putida 2001 1976 cphA 2001 1998 in silico 2007 E. coli E. coli 1996 E. coli trp lac L 1998 E. coli cphA Synechocystis 2002 1976 1971 1976 1976 cphA 1998 2000 2000 2002 cphB cph cph 2002 2004 2004 2005a b Acinetobacter 1976 cphA 2001 2001 2002 2006 R. eutropha C. glutamicum P. putida 2001 2006 In conclusion, large-scale fermentation processes for cyanophycin production and downstream processing are available for a number of different microorganisms able to grow in different substrates, including the potato waste stream Protamylasse™, and also, low cyanophycin yields were reported in plants. Production and economic aspects of fermentative cyanophycin production An important contribution to sustainability can be made by the use of a considerable plant waste stream for the production of renewable, biodegradable, and biocompatible polymers and/or valuable chemicals that are now produced on large scale from petroleum. Some of the polymer classes to be developed may be expected to replace some existing mineral petroleum-based polymers as soon as competitive production prices can be obtained and/or supporting measures will be taken to promote the use of renewable resources. On the other hand, completely novel types of biopolymers may be developed for completely novel applications. 3 v v v v E. coli 3 2005a b v v E. coli cphA w w E. coli 3 3 3 3 3 E. coli w w v v E. coli S. cerevisiae w w 3 v v 3 S. cerevisiae http://sec.edgar-online.com/ 2004/06/14/0001104659-04-016859/Section7.asp 2 Table 2 Economical and technological bottlenecks and proposed measures Bottleneck Proposed measure(s) Investments, including costs for fermentation and downstream processing equipment The calculation provided here suggests that these may be acceptable Costs for the production of cyanophycin, cyanophycin-derived products and for downstream processing of biomass Construction of a sufficiently productive microbial strain to convert or simply utilize constituents of plant waste streams like Protamylasse™ and to incorporate these compounds, presumably amino acids, into the cyanophycin polymer chain during cyanophycin biosynthesis E. coli cphA 6803 Construction of stable strains with integrated copies of the cyanophycinsynthesis genes E. coli Since not all components present in the current source of Protamylasse™ may have the proper concentration for current laboratory strain(s), an optimization may require the addition of substrates other than Protamylasse™, for example other plant waste streams. Sufficient provision of amino acids like arginine should be ensured during the production phase Optimization of microbial biomass formation S. cerevisiae Pichia pastoris Sub-optimal fermentation processes Fermentation technology and feeding regimes have to be developed for optimum amino acid utilization or biosynthesis from Protamylasse™ or other plant waste streams Generation of valuable side stream particle fraction of Protamylasse™ Alternative use of the side stream particle fraction of Protamylasse™, e.g. by using cyanophycin producing filamentous fungi Co-production with, e.g., ethanol S. cerevisiae Costs for cyanophycin extraction Development of alternative cheap cyanophycin extraction methods using, e.g., hydro-cyclone equipment for the non-soluble fraction Cost-efficient production of cyanophycin in plants cphA Efficacy of downstream processing Downstream processing has to be adapted and optimized for cyanophycin or cyanophycin derivatives containing biomass, which will be either bacterial cells or eukaryotic (mostly plant) cells or tissues Lack of insight in possible modifications of cyanophycin, their impact on cyanophycin properties and market potential The diverse possibilities to modify the cyanophycin molecule chemically or enzymatically has to be exhaustingly explored to identify all potential key applications for cyanophycin-derived products and to find the most suitable products with regard to market potential and the possibility of their commercialization Lack of knowledge concerning properties of known cyanophycin synthetases and their genetic engineering The possibility to modify the active sites of the cyanophycin synthetases in order to change its substrate specificity and to allow the production of cyanophycin derivatives has to be determined Insufficient insight in all possible applications for cyanophycin as a polymer or as a starting material for chemical syntheses The exploitation of cyanophycins and cyanophycin-derived molecules as substitutes for well established industrial products or as renewable raw materials has to be determined precisely Cyanophycin production in plants 2 1992 2002 2003 2005 2005 Thermosynechococcus elongatus E. coli Production of the cyanophycin biopolymer in potato is of high interest to the potato starch industry. Production in this plant does not require any additional infrastructure. After processing of the potatoes, cyanophycin can be isolated from the Protamylasse™. However, for commercial application, the efficiency of cyanophycin accumulation in potato has to be significantly improved. 2005 2005 2005 2005 Additional strategies Priming cyanophycin elongation in vitro 3 In planta chpB chpE cphI cphI It might be possible to use a poly-Asp backbone as primer for cyanophycin biosynthesis. This peptide can be produced by ribosomal protein biosynthesis. The gene should be under the control of a low-level promoter to prevent the production of many peptides, and thus, the production of many low molecular weight polymers. Optimization of amino acid biosynthesis 2000 It is possible that the availability of substrates (Asp and Arg) in plants is limiting or off-balance. Therefore, it is important to identify the organs that have the highest concentrations of available substrates and to investigate whether the substrate supply can be enhanced by introduction of genes involved in substrate production. Comparison of economics of cyanophycin production by fermentation or in plants 2 Fig. 2 in planta Gray square filled square open square  Assuming that a typical fermentative production of a bulk product, such as lysine, citric acid or glutamic acid costs about € 1,500 per ton and that these costs consist of: € 500 for the raw materials, € 500 for the fermentation process, and € 500 for recovery and purification. The advantage of producing in plants is that both the raw material costs and the fermentation costs can almost be neglected. On the other hand, recovery costs could be much higher. For the sake of the reasoning, it is assumed that recovery costs for cyanophycin production in plants will be the same as in the case of fermented production, i.e., € 500 per ton. In case of a fermentation process, a typical production volume for a company would be in the order of magnitude of 100,000 tons/year. The turnover would then be 150 million euros per year (i.e., € 1,500 per ton × 100,000 tons/year). In the case of production in plants, as raw material and fermentation costs are negligible, cost savings would be 100 million euros per year. This would be the maximum advantage, as this calculation does not include any costs for the production of the crop nor any additional costs for biorefining of the crop and treatment of side products. A similar reasoning would bring a maximum advantage for a product with a typical company production volume of 20,000 tons per year, such as the case with a medium-sized monomer. In this case, cost savings per ton would be higher, but as the volume is much smaller, the maximum advantage could be around 70 million euros. For specialty products with a volume of 300 tons per year (enzymes), a maximum advantage is estimated to be in the order of 5–10 million euros. For pharmaceutical production with a volume of 10 tons per year, the advantage would be around 0.5–1.5 million euros, as the volume would be small and production costs would mainly be ascribed to recovery. Other plant side streams 2 2006 Cyanophycin-derived bulk chemicals Arginine may be converted to 1,4-butanediamine. 1,4-diaminobutane, derived from petrochemistry, is currently used as a co-monomer in the production of nylon-4,6. The volume of production is not known, but is estimated to be in the range of 10,000s tons per year with a value of >€ 1,600 per ton. 6 Other chemicals which could be obtained from cyanophycin but are currently prepared from fossil resources include, e.g., 1,4-butanediol and urea. The production of cyanophycin by plants will drastically reduce the cost price, potentially below € 1,000 per ton. This will enable the production of functionalized bulk chemicals such as 1,4-diaminobutane, and possibly, also acrylonitrile. The transition from mineral oil to plant-based precursor production has a considerable impact. The use of ammonia for the incorporation of nitrogen into chemicals is very important, but also very energy-intensive. Therefore, if the incorporation of nitrogen can be realized in systems based on plant (rest) streams in the form of protein or amino acid precursors, then this will yield considerable energy savings. Cyanophycin-based biopolymers Poly-aspartic acid is derived from cyanophycin after the hydrolytic removal of arginine. This polymer has properties that are very similar to poly-acrylic acid. The cost price of this polymer can be set on 1,000€/ton in the cost calculations for arginine here above. As the volumes of these products will be similar, only 1,000 tons/year will be manufactured. Higher market prices might be obtained for special applications in food and/or pharmaceutical applications. This might change the cost structure of arginine in a way that market volumes might double or triple. Cyanophycin as such might have applications as a polymer. Furthermore, derivatives obtained by enzymatic/genetic and/or chemical modifications might give valuable properties. Without thorough investigations, we cannot anticipate on the value of these polymers. Utilization in this area can be expected after 10 to 12 years after the beginning of the proposed research approaches for research and development. Possible cyanophycin modifications, applications for bulk chemicals and for polymers E. coli E. coli Saccharomyces cerevisiae Outlook Given the anticipated cost development for fossil energy carriers and environmental regulations, the chemical industry is facing increasing financial pressure and is thus looking for possibilities to broach new resources as a basis for polymer production. Important considerations in this search are to lower energy costs and prices of raw material and to develop cheaper and more sustainable production processes. Unlike poly-γ-glutamic acid and poly-ɛ-lysine, cyanophycin has not been commercialized yet. Cyanophycin can be broken down into the individual amino acids that can be used as building blocks in various industrial processes. Because of its homogeneous structure and composition, the cyanophycin polymer and its derivatives also appear to be good candidates as starting materials for the production of nitrogen-rich commodity products, which are based on nitrogen-rich chemicals, like, for example, nylons. For example, for poly(aspartic acid) which is the polymer backbone of cyanophycin, various applications have been developed ranging from water-softening or detergent applications to applications in the paper, building material, petroleum or leather industry, in cosmetics, as well as many dispersant applications. 1998 It can be expected that economical activities can be developed within the following areas, such as: fermentation industry, biopolymer production, processing, modification and product development (also for medical technology), packaging industry, food and feed supplementation industries and, last but not least, state-of-the-art technology (which, in turn, will attract additional financial sources and economical activities). It should be emphasized, however, that the mentioned applications are still uncertain and that these are so far only potential applications. On the one hand, this development will lead to the substitution of chemicals that are now produced at the cost of fossil raw materials, such as oil. As oil may be depleted in about 50 years, and as there seems to be a correlation of the use of fossil raw materials with climate changes, it is essential to develop alternatives. The anticipated alternatives can be produced by fermentation and, in principle, by plant production systems, and so, giving a new economic and knowledge intensive value to the fermentation industry and/or to agriculture. On the other hand, novel types of polymers will be developed that do not simply replace existing applications but that will enter novel product markets. 2006