In the Laboratory
Bacterial Production of Poly(3-hydroxybutyrate) An Undergraduate Student Laboratory Experiment Kristi L. Burns, Charlie D. Oldham, and Sheldon W. May* School of Chemistry and Biochemistry and the Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA 30332-0400; *
[email protected] Polyhydroxyalkanoates are a relatively large class of thermoplastic biopolyesters of which poly(3-hydroxybutyrate) (PHB) is the simplest: O
∙
O
CH3 H (R) O
O
CH3 (R) H O
O
n
CH3 H (R) OH
PHB is an intracellular polymer that accumulates in the cytoplasm of bacterial cells when a carbon source is provided and at least one nutrient, such as nitrogen, is limited (1–6). Because it is a biodegradable polyester, there is much interest in its production. In addition to being biodegradable, this polyester has characteristics similar to polypropylene (crystallinity, tensile strength, and mechanical properties), which makes it suitable for a wide range of applications (7). Recently, biocompatible PHB copolymers have been investigated for use in tissue engineering (8) and in sustained-release drug delivery systems (9), and there has been progress in optimization of PHB production (10, 11). Consequently, biodegradable biopolymers produced from renewable resources may represent a viable and environmentally friendly alternative to traditional plastics. As part of an undergraduate multidisciplinary course that is cross-listed among five departments,1 we have developed a hands-on student laboratory experiment for culturing, isolating, and purifying PHB. This experiment is a valuable addition to the conventional polymer science curriculum, which generally focuses on traditional petrochemically derived polymers. Given the current commercial interest in biopolymers and the future promise of “bio-refineries”, students in a polymer chemistry laboratory are well served by being exposed to techniques such as cell culture and biological polymer extraction, which this laboratory incorporates. The traditional methodology for PHB extraction is so time consuming that students could not complete the experiment within a typical undergraduate laboratory time block. We therefore developed a technique that allows students to complete the PHB production and extraction in several short sessions. An important aspect of this laboratory experiment is that it introduces students to biological methods such as sterile technique, bacterial cell culture in shake flasks, centrifugation, and autoclaving, in addition to employing more familiar chemical methodologies such as refluxing, precipitations, and vacuum filtration. In addition to chemistry and polymer curricula, this laboratory experiment would be suitable for a variety of biology, biochemistry, or biotechnology curricula. Overview This laboratory experiment is designed in such a way that the students can perform the PHB cultivation and extrac-
tion processes in three stages. In the first 1.5 hour session, the students pellet, wash, and transfer a supplied cultured mass of Cupriavidus necator cells to the nitrogen-limited medium for PHB production. For convenience, the resulting PHB-laden cell mixture can then be stored in a refrigerator for up to one week before PHB extraction and purification. Finally, the students perform the PHB extraction and purification in two 3 hour sessions. Experimental Procedure Initial Growth of C. necator Many microorganisms (e.g., alcaligenes, azotobacter, bacillus, pseudomonads, and cupriavidus) are known to accumulate PHB. For the purposes of this laboratory, we chose to utilize Cupriavidus necator, which is commercially available.2 Prior to the first laboratory, the cells are initially grown according to product instructions to amplify the number of cells available. The cell suspension is then divided into aliquots and stored frozen at –75 °C. A few days prior to the first laboratory session, a frozen aliquot of the C. necator cell suspension is rapidly thawed in a water bath at 30 °C and 250 μL is used to inoculate a 125 mL flask containing 35 mL of growth medium that is cultivated aerobically at 30 °C and 325 rpm. (The composition of the media is available in the online material.) One flask of cultivated cells is prepared for each student by the instructor or assistant. When the culture reaches an A600 of 15–16 the cells are harvested by centrifugation. Production of PHB In the first laboratory session, PHB production is induced by resuspending the C. necator cell paste in 35 mL of nitrogenlimited medium and cultivated as previously described until the A600 reaches 30–40. A 10 mL aliquot of the cellular mixture is removed and dried in a 60 °C oven for determination of the dry cellular mass and the cells in the remaining 25 mL are harvested by centrifugation. Extraction of PHB from C. necator Cells In the second laboratory session, the PHB-laden cell pellet is resuspended in 25 mL of deionized water and 0.2 g of SDS and 0.18 g of EDTA is added, mixed thoroughly, and autoclaved for 30 min. After autoclaving, the cell mixture is centrifuged at 13,800g for 10 min, and the crude PHB mixture is washed by resuspending the pellet in 25 mL of deionized water and centrifuging again. The crude PHB is then resuspended in a minimal quantity of water (
