(231u) From Cellular Storage Compounds to Value-Added Products: Model-Based Design of PHB Production

Energy storage compounds, common amongst all living organisms, give the host cell that contains them a biological advantage over other cells not so well endowed to survive in starving conditions. Because of the chemistry of these compounds, polysaccharides and lipids mainly, they can also be materials of interest to replace oil derived macromolecules such as plastics and fuels. Cells synthetize energy storage molecules when carbon is present in excess and, simultaneously, they lack another essential nutrient for growth (N, P, Mg, etc.). Thus, the selection of the operational conditions that yield high productivities is not trivial. A predictive model is used throughout this work to guide the process development and optimization of poly-3-hydroxybutyrate (PHB) production in bacteria.

PHB is a biodegradable bioplastic, which on its own or as part of a heteropolymer, finds application in everyday products, competing directly with oil-derived resins in terms of physical and mechanical properties. PHB has the potential to replace conventional plastics like polyester, hence, roosting out the oil dependency as well as eliminating the problematic disposal of the traditional counterparts. The carbon dioxide released when PHB naturally degrades is balanced with the CO₂up-taken by biomass, fermentation feedstock for PHB synthesis, during growth. This makes the process carbon footprint very close to zero.

The scarce implementation of PHB production at large scale is attributed to expensive raw materials, insufficient fermentation optimization and poor product recovery. Its high selling price, combined with the current low oil prices, can only justify application in limited fields, basically within the medical and pharmaceutical sectors. However, providing its producing costs are minimized, its use could be extended to areas as varied as food and beverage, packaging, textiles, agricultural products or electronic components to name a few.

The industries that are already manufacturing PHB around the world rely mainly on sucrose-based derivatives as fermentation feedstock. For tackling the production cost reduction, this project has chosen glycerol as substrate for the fermentation process. Glycerol is a by-product of biodiesel industry. Considering the increase of biodiesel production around the world and that every ton of biodiesel yields 100 kg of glycerol, it is not surprising that there is a surplus of glycerol in the market. Thus, the price is of crude glycerol has dramatically dropped. By employing glycerol as a feedstock not only will the upstream cost of PHB be reduced, but the biodiesel manufacturers will benefit from this major subproduct, which was starting to be seen as a problematic waste to treat before disposal.

We have conducted experimental studies to confirm that glycerol is a suitable feedstock and can act as a single carbon source for PHB production by Cupriavidus necator DSM 545.PHB accumulation has been triggered by applying nitrogen limiting conditions and its content inside the cells was as high as 80 % (on cell mass basis), being the main fermentation product. Besides, concentration above 50 g/l glycerol can be applied without affecting growth, which encourages its scalability potential. A deep study in cell adaptation to glycerol has demonstrated that undesirable lag phases can be greatly reduced by serial sub-cultivation of cells in glycerol rich media. Also the glycerol uptake is improved, probably due to a natural mutation which causes an overexpression of the genes that regulates the mechanisms of glycerol entry inside the cytosol. The cell preference of glucose over glycerol can be reverted after the right bio-training period.

In order to achieve an effective fermentation technology, a macroscopic model has been built based on fundamental knowledge of the main phenomena governing PHB production. This predictive model combines microbial kinetics and mass balances to describe the dynamic behaviour of the system. The field of intracellular products requires separate and special attention and kinetics models for them have not received sufficient attention in the past. Newly formulated equations have been applied here and considerable amount of time and resources are saved as simulation can replace empirical tests to investigate the best operational conditions.

In this way, the model has been used to select the initial concentrations that support maximum growth rate and highest productivity in batch operation. Due to the different condition required for cell growth and product formation, two-stage fermentation is the preferred configuration. Again, the model can be used here in the analysis of different feeding strategies. Oxygen has proved a growing limiting role in bench top bioreactor studies and thereby, aeration flow rate can become a variable to control and trigger PHB production at the right time.

The importance of the approach adopted is that, the model has first served for assessing our understanding of the bio-process until it has become a tool for design. It is believed that this case of study can shed light on similar systems and ease their study. Lastly, simulations of the biodiesel plant coupled with the PHB production process provide useful information about the economic feasibility of such integrated biorefinery.