Elsevier

Bioresource Technology

Volume 123, November 2012, Pages 272-278
Bioresource Technology

Electrofiltration as a purification strategy for microbial poly-(3-hydroxybutyrate)

https://doi.org/10.1016/j.biortech.2012.07.039Get rights and content

Abstract

The biodegradable polyester poly-(3-hydroxybutyrate) (PHB), produced by Ralstonia eutropha in batch and fed-batch processes, was purified by electrofiltration. The protein film on PHB granules determines their high negative zeta potential, enabling the application of electrofiltration as an integrated technology in the downstream processing of PHB. In order to determine the optimal purification parameters, various pressure and electric field strength conditions were tested. Electrofiltration of PHB at 4 bars and 4 V/mm provided an up to four times higher concentration factor than conventional filtration. FT-Raman spectroscopy demonstrated that electrofiltration did not result in structural changes to the products. The study demonstrates the efficiency and practical advantages of electrofiltration as a promising downstream step in the PHB production technology.

Highlights

► Due to high phb surface charge, electrofiltration can advantageously be applied. ► Short filtration times were achieved by application of electric field and pressure. ► Application of different cultivation strategies affected the filtration behavior. ► No unwanted modifications of PHB molecules were observed. ► Electrofiltration opens new possibilities for PHB production.

Introduction

The disposal of plastics is a worldwide environmental problem, requiring an effective and urgent industry response. A permanent and global solution emerges from the development and implementation of biodegradable materials as alternatives to petroleum-based plastics such as polypropylene. High biodegradability, advantageous physical and mechanical properties (Lenz and Marchessault, 2005) and a variety of special applications define poly-(3-hydroxybutyrate) (PHB) as an attractive product with commercial significance (Orts et al., 2008). Many different bacteria species synthesize the polyester intracellularly under limited nutrient conditions as a carbon and energy storage material (Hazer and Steinbüchel, 2007). Among PHB-producing bacteria, Ralstonia eutropha has high accumulation capacity resulting in PHB formation of up to 80% of the cell dry weight (Tavares et al., 2004).

The downstream processing of PHB, involves enzymatic, chemical or mechanical cell disruption (Jacquel et al., 2008). Generally, after cell disruption, PHB is precipitated by adding a suitable solvent. For further purification, PHB can be dissolved in chloroform and precipitated with hexane or diethyl ether (Hocking and Marchessault, 1998); however, addition of large amounts of organic solvents is not environmentally friendly and increases the total cost (Khosravi-Darani et al., 2004). Although possessing no electrostatic charge, PHB granules demonstrate a high negative zeta potential which is caused by their protein surface layer (Asran et al., 2010). Electrofiltration, a hybrid process combining dead-end filtration and electrophoresis, enables purification of PHB due to this protein layer. The innovative strategy in the downstream processing of PHB leads to a reduction in processing time, energy and respectively purification costs.

The aim of the present work was to investigate the application of electrofiltration and establishment of an alternative downstream processing step, overcoming previous limitations and drawbacks in PHB production. In order to determine the efficiency of the purification strategy, process dynamics and material characteristics were determined.

Section snippets

Batch process

Batch cultivation of R. eutropha ATCC 17699 (DSM 428) was carried out in a 15-L bioreactor (B. Braun Biotech, Melsungen, Germany) with a working volume of 6 L by using a mineral medium which consisted of 20 g/L sodium gluconate as carbon source, 9.0 g/L Na2HPO4·10H2O, 1.5 g/L KH2PO4, 1.0 g/L NH4Cl, 0.2 g/L MgSO4·7H2O, 17.6 mg/L CaCl2·2H2O, 1.2 mg/L ammonium ferric citrate (C6H11FeNO7), and 0.1 mL/L of 100X SL6 trace element solution (Pfennig, 1974). Sodium gluconate, MgSO4·7H2O, CaCl2·2H2O and ammonium

Microbial production of PHB in batch and fed-batch processes

During the batch process, samples were periodically withdrawn after the lag phase for ammonia assays and optical density (OD) measurements. Fig. 1 presents cell growth, measured as OD at 550 nm. Furthermore, the maximum reduction of pO2 and the maximum increase of pCO2 demonstrated the maximal growth rate. The cell dry weight at the end of fermentation was 2.2 g/L and nitrogen limitation was reached after 20 h.

By using a fed batch process with crude glycerine as C-source, a by-product of biodiesel

Conclusions

This research presents an innovative approach for the purification of PHB and introduces electrofiltration as a perspective method in the production chain of biopolymers. The application of different cultivation strategies including different media, strains and conditions affected the structural and dynamical properties of PHB dispersions causing changes in filtration behavior. The lack of unwanted modifications of the final product facilitates the practical applications of PHB, opening new

Acknowledgements

The authors express their gratitude to BMBF (Federal Ministry of Education and Research) for financial support.

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