ORIGINAL ARTICLE Biotechnological production of poly(3-hydroxybutyrate) with Wautersia eutropha by application of green grass juice and silage juice as additional complex substrates M. KOLLER, R. BONA, C. HERMANN, P. HORVAT, J. MARTINZ, J. NETO, L. PEREIRA, P. VARILA, & G. BRAUNEGG Institute of Biotechnology & Bioprocess Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria (Received 3 December 2004; accepted 30 March 2005) Abstract Alternative inexpensive complex nitrogen- and phosphate sources from agriculture, green grass juice (GGJ) and silage juice (SJ), were added to cultivation medium in order to investigate their impact on growth of the well-known polyhydroxyalk- anoate (PHA) accumulating strain Wautersia eutropha . The influence of these additives was directly compared with cultivations on defined minimal mineral medium (M) as well as on the same medium supplemented with more expensive complex additives: corn steep liquor (CSL) and casamino acids (CA). It turned out that the supplementation with most complex additives results in shortening of lag-phases of bacterial growth and in higher end-concentrations of residual biomass compared with M-medium. Finally, higher volumetric productivities for poly(3-hydroxybutyrate) (3-PHB) were achieved. The effect of the inexpensive additive SJ on volumetric productivity was similar to the result for the expensive CA (0.653 vs. 0.619 g L 1 h 1 ). The same was found for the biomass concentration (7.00 vs. 7.44 g L 1 respectively). Together with an economic appraisal presented in this study, the results suggest it is possible to make the sustainable process of microbial PHA-production more economically feasible. A survey of the thermal characteristics and molecular mass properties of the isolated polymers completes this work. Keywords: Complex additives, green grass juice, polyhydroxyalkanoates, Wautersia eutropha, silage juice Introduction Polyhydroxyalkanoates (PHAs) are biodegradable polyesters with physical properties of thermoplastics and/or elastomers. Therefore, they can be applied as alternatives to common plastic materials produced from mineral oils (Holmes 1988; Brandl et al. 1990; Steinbu ¨chel & Valentin 1995; Lee et al. 1996). PHAs are synthesized in the cytoplasm of various prokaryotic strains usually from carbohydrates, but also from other renewable resources (Hocking & Marchessault 1994; Bourque et al. 1995; Ramsay 1995). Under unfavourable growth conditions (sur- plus of carbon source plus limitation of an essential substrate, e.g. nitrogen, phosphate or oxygen), some strains are able to divert the usual carbon flux (Acetyl-CoA synthesized in the central metabolic pathways of the microorganism) from biosynthesis of protein (biomass) constituents to the formation of compounds acting as precursors for the production of PHA, mainly poly-3-hydroxybutyric acid (3- PHB). Due to a high degree of polymerization, their molecular weights can reach several million (Wang & Lee 1997; Steinbu ¨chel & Lu ¨ tke-Eversloh 2003). These polymers serve as an intracellular reserve carbon- and energy source which normally will be degraded if the external carbon source is depleted. Because of their outstanding property of complete degradation to water and CO 2 , they are embedded into natures carbon cycle. If applied instead of fossil oil derived polymers, the mass balance of carbon from biomass will be closed, but its durability will be prolonged compared with the usual biological life cycle of PHA-carbon. The fossil fuel energy demand during the life span of PHAs will not exceed the amount necessary for the industrial production and Correspondence: M. Koller, Institute of Biotechnology & Bioprocess Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria. Tel:  /43-316-8738411. Fax:  /43-316-8738434. E-mail: martin.koller@tugraz.at Biocatalysis and Biotransformation, September  /October 2005; 23(5): 329  /337 ISSN 1024-2422 print/ISSN 1029-2446 online # 2005 Taylor & Francis DOI: 10.1080/10242420500292252