Metabolic engineering of Pseudomonas putida and enhancement of a two-phase partitioning bioreactor for degradation of phenol

Two-phase partitioning bioreactors (TPPBs) present an effective method for the biological treatment of highly concentrated pollutants without the need for prior dilution through incorporation of an immiscible and biocompatible solvent phase to partition the xenobiotic to the cell containing aqueous phase. A key component of system design involves identification of an appropriate delivery solvent, and this has been one of the main challenges in designing and operating TPPB systems. Desired characteristics of the solvent phase include a requirement that the solvent itself not be bioavailable to the microbial catalyst. In this work transposon mutagenesis was used to address this issue through elimination of microbial bioavailability for medium chain length alcohols. The growth capabilities of the resulting modified strain, AVP2, were assessed, resulting in identification of six previously exempted solvents that could now be applied as the delivery phase within the TPPB. The growth capability (on phenol) and kinetic analysis verified that this genetic alteration was made at no cost to degradative efficiency or other microbial features, such as solvent tolerance or auxotrophy, that would negatively influence xenobiotic degradation or increase medium formulation and operating costs.; Fermentations employed a 4 l total volume with either 1:1 (2 l of each phase) or 3:1 aqueous:solvent ratios. Decanol and Adol 85 NF were selected as model solvents, with phenol as the xenobiotic substrate to specifically demonstrate the efficiency of AVP2 and the newly utilizable solvents within the TPPB. In both systems, 36 g of phenol were degraded within approximately 37 h. A positive effect on degradation rates was observed for TPPB operation with higher solvent ratios. Stability analysis of the mutant's alcohol non-utilizing phenotype under operating conditions was confirmed through stability testing under selection pressure and assessment of growth throughout fermentations. Phenol degradation within the reactor was shown to be equivalent to that of the wild type, but operation with AVP2 presented operating advantages associated with decreased solvent losses.; The influence of operating with the two phases dispersed was evaluated and revealed improvement in volumetric consumption rates relative to previous systems operated as two distinct phases. Extended fed-batch operation of the TPPB demonstrated the potential for long-term application of TPPB. Another potential application of this bioremediation process was illustrated by the introduction of a highly concentrated phenol solution (3000 mg/l), into the reactor and use of the solvent to recover the majority of the xenobiotic. This concentration was selected to simulate a phenol-contaminated industrial effluent. Microbial addition and controlled feeding through xenobiotic equilibrium partitioning enabled biological treatment with the entire mass of phenol being degraded within 15 h. The process was shown to be repeatable with 6 l of the contaminated solution being treated with only 1 l of solvent.; Bioprocesses, such as TPPB, present powerful solutions to various pollution issues, but efforts to enhance operation have often relied solely on engineering principles acting on a microbial black-box. This work demonstrated the potential for the catalyst itself to become a modifiable parameter in overcoming process limitations and expanding potential application. Furthermore, the versatile possibilities for application of TPPB technology and the potential for enhancing operation through alterations in both physical and microbial parameters were illustrated.