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.
In contrast, PHA separates from lysed cell matter in an aqueous sodium hypochlorite solution through sedimentation because it is insoluble in water. Furthermore, sodium hypochlorite is not volatile or combustible and requires therefore less safety measures than most solvents. As studies on PHA recovery using sodium hypochlorite suggested a separation of the polymer by centrifugation (Berger et al.
1989, Hahn et al.
1994, Ribera et al.
2001) one has to consider the total volume of a large-scale extraction process. Since the maximum concentration of cell matter that can be digested in a sodium hypochlorite solution is much lower than in solvents, separation through centrifugation would exceed available capacities. Therefore, we show that digestion of cell matter using a relatively high concentrated sodium hypochlorite solution with the subsequent addition of water mediates efficient digestion of non-PHA biomass and clear separation, which is due to the insolubility of PHAs in water. Thereby, the volume to be centrifuged is reduced to a minimum. A sodium hypochlorite concentration of 13% is well suited for the process, as it is commercially available and efficiently digests non-PHA biomass without generating excessive, non-manageable heat, the use of a higher concentrated solution would lead to. Additional steps such as solving the polymer in chloroform with a subsequently required precipitation (Hahn et al.
1994, Sayyed et al.
2009) are not necessary in order to achieve a reasonable purity and product recovery. Alternatively, the polymer can be simply washed with e. g. isopropanol to remove remaining lipids. In addition, we observed that instead of an increase of the temperature to 30°C or 37°C (Hahn et al.
1994, Ribera et al.
2001), in- or external cooling is required since the digestion of biomass using sodium hypochlorite is strongly exothermic.
In contrast to previous studies, our protocol applies a biomass to hypochlorite ratio ideal for a large scale extraction of poly(3HB). The concentration of dried R. eutropha H16 cells, which are digested in a sodium hypochlorite solution is three-fold higher than the concentration of 1% (w/v) used by Berger et al. (
1989), thereby reducing the total volume as well as the amount of chemicals and energy required for the extraction process considerably. Whereas Hahn et al. (
1994) suggested a biomass concentration of 4% (w/v) to be digested in a 30% (v/v) sodium hypochlorite solution, we showed that a lower sodium hypochlorite concentration is sufficient to achieve a complete digestion of an only slightly lower concentration of non-poly(3HB) biomass. A lower hypochlorite concentration is advantageous in order to reduce the temperature of the process and also to limit the degradation of the polymer (Berger et al.
In addition, our study also showed that it is inevitable for the extraction process to grind lyophilized cell matter thoroughly in order to present a maximum surface area and thereby provide the basis for the complete digestion of the non-PHA biomass. Of the seven large scale batches, only the last batch, where pulverized dried cell matter from the bottom of the container was extracted, resulted in a reasonably high recovery rate. In contrast, batches with chunks of cell matter displayed less surface area to the hypochlorite solution, so that much of the cell matter remained undigested. Therefore, only the recovery rate, but not the purity of the isolated polymer varied in the seven different batches. In order to confirm this, further extraction experiments with pulverized cell matter could be carried out.
Assuming chloroform extraction does not lead to degradation of poly(3HB), GPC analyses revealed a decrease of the original molecular weight of poly(3HB) isolated through treatment with sodium hypochlorite by 50 – 70%. Additionally, the isolation with sodium hypochlorite led to a polymer of higher dispersity. As most applications demand polymers of high molecular weights and low dispersities, the polymer degradation through treatment with hypochlorite is a drawback of the process. However, our protocol does not lead to a more severe degradation than in previous studies, where poly(3HB) caused a decrease in molecular weight of up to 50% (Hahn et al.
1995) and even 80% (Berger et al.
1989) when extracted with sodium hypochlorite at comparable parameters.
In summary, we developed an ideal method for large scale isolation of poly(3HB) from dried cell mass, which (i) recovers a product of high purity with little turnover, (ii) proves to be simple and cost-saving by mediating efficient separation of the polymer from the lysed cells without the use of additional chemicals or a large scale centrifuge, (iii) limits the total volume of the extraction process with an optimized ratio of biomass to hypochlorite creating a manageable amount of heat and foam and (iv) presents technical solutions to overcome challenges that emerge with the upscale of an extraction process with sodium hypochlorite.