Date Published: November 19, 2012
Author(s): Daniel Heinrich, Mohamed H Madkour, Mansour A Al-Ghamdi, Ibraheem I Shabbaj, Alexander Steinbüchel.
Isolation of polyhydroxyalkanoates (PHAs) from bacterial cell matter is a critical step in order to achieve a profitable production of the polymer. Therefore, an extraction method must lead to a high recovery of a pure product at low costs. This study presents a simplified method for large scale poly(3-hydroxybutyrate), poly(3HB), extraction using sodium hypochlorite. Poly(3HB) was extracted from cells of Ralstonia eutropha H16 at almost 96% purity. At different extraction volumes, a maximum recovery rate of 91.32% was obtained. At the largest extraction volume of 50 L, poly(3HB) with an average purity of 93.32% ± 4.62% was extracted with a maximum recovery of 87.03% of the initial poly(3HB) content. This process is easy to handle and requires less efforts than previously described processes.
Today, plastic materials are part of humanities everyday life and are indispensible for numerous consumer goods and applications. Currently, they are mostly produced from fossil resources (Chanprateep 2010). In addition to persistent, non-biodegradable plastics, an increasing demand for biodegradable plastics exists (Poirier et al. 1995). As a potential substitute for conventional petrochemically produced plastics, biodegradable polymers (biopolymers) synthesized from renewable resources have attracted considerable interest in the past decades (Steinbüchel 2001). Among the different types of biopolymers, polyhydroxyalkanoates (PHAs) present a promising group of polymers, as they are biocompatible, thermoplastic and nontoxic, which makes them suitable for various applications in industry, medicine or agriculture (Anderson and Dawes 1990, Steinbüchel 2001, Van der Walle et al. 2001). Although PHAs with equivalent functionality to conventional plastics can be produced by bacterial fermentation at a large scale, their production accounts so far for only a small fraction of the global plastic production, which is mostly due to the comparably high production costs (Keshavarz and Roy 2010).
Fed-batch cultivation of R. eutropha H16 (Figure
1) was carried out in mineral salt medium with an initial content of 30 g L-1of sodium gluconate and 2.0 g L-1 of (NH4)2HPO4 . A pH of 7 was automatically maintained throughout the process by adding 4 M HCl or NaOH. A total yield of 10.8 kg of dried cell mass was obtained through a single fermentation of R. eutropha H16 at the 400 L scale with subsequent harvesting of the cells with a continuous centrifuge. The poly(3HB) content of the cells was 65.2% ± 1.6% (w/w), as measured by GC analysis. The cell density at the point of harvest was 24.2 g L-1 and the productivity regarding accumulated poly(3HB) for the period of the main fermentation was 0.23 g L-1 h-1. At various time points during fermentation, samples of cells were stained with the hydrophobic dye nile red. The stained poly(3HB) granules were examined with a fluorescent microscope. Upon nitrogen limitation of the culture after 40 h of cultivation cells increasingly displayed large granules of poly(3HB).
Through fermentation of R. eutropha H16, a sufficient amount of biomass for a large scale extraction experiment was produced. However, the productivity regarding accumulated poly(3HB) was below average, which was due to the long lag phase of the culture. The aim of this study, was to develop a simple and efficient downstream process for the production of poly(3HB). The most common methods for PHA recovery from bacterial cells involve the use of (halogenated) solvents (Ramsay et al.
1994, Elbahloul and Steinbüchel
2009). After the solvent modifies the cell membrane and dissolves PHA, separation of the polymer from the solvent is necessary. This can either be mediated by evaporation of the solvent, or precipitation of PHA by a non-solvent, such as ethanol, methanol or even water (Zinn et al.
1990). Although extractions involving the use of solvents have accomplished purities of higher than 98% and recoveries of more than 95% (Zinn et al.
2003, Lafferty and Heinzle
1978), a complex setup for the execution of successive steps is required.
The authors declare that they have no competing interests.