Characterizing the Anoxic Phenotype of Pseudomonas putida Using a Bioelectrochemical System.

Bin Lai,Jens Krömer,Anh Nguyen

doi:10.3390/mps2020026

Abstract

Industrial fermentation in aerobic processes is plagued by high costs due to gas transfer limitations and substrate oxidation to CO2. It has been a longstanding challenge to engineer an obligate aerobe organism, such as Pseudomonas putida, into an anaerobe to facilitate its industrial application. However, the progress in this field is limited, due to the poor understanding of the constraints restricting its anoxic phenotype. In this paper, we provide a methodological description of a novel cultivation technology for P. putida under anaerobic conditions, using the so-called microbial electrochemical technology within a bioelectrochemical system. By using an electrode as the terminal electron acceptor (mediated via redox chemicals), glucose catabolism could be activated without oxygen present. This (i) provides an anoxic-producing platform for sugar acid production at high yield and (ii) more importantly, enables systematic and quantitative characterizations of the phenotype of P. putida in the absence of molecular oxygen. This unique electrode-based cultivation approach offers a tool to understand and in turn engineer the anoxic phenotype of P. putida and possibly also other obligate aerobes.

Highlights

Pseudomonas putida (P. putida) is a promising industrial host for the production of harsh chemicals due to its resilience against environmental stresses that will quickly diminish other industrial microorganisms [1,2,3,4]
Nikel et al [9] engineered a fermentative pathway towards acetate and ethanol into P. putida KT2440 and another group reported an approach of introducing a nitrate/nitrite-based respiration pathway in the same strain [10]. Both systems were active in the recombinant strains; the mutants only showed limited metabolic activity and were not able to grow without oxygen
We present a detailed protocol for the cultivation of P. putida in a bioelectrochemical system (BES) reactor without oxygen as well as the general results to be expected

Summary

Introduction

Pseudomonas putida (P. putida) is a promising industrial host for the production of harsh chemicals due to its resilience against environmental stresses (e.g., solvents) that will quickly diminish other industrial microorganisms [1,2,3,4]. Oxygen-dependent metabolism can obtain higher energy yields, a high metabolic turnover and product purity [5,6], but it suffers significantly from the comparatively low product yield and high operating cost for heat removal and oxygen supply [7,8] These limitations pose a strong economic barrier for the industrial application of P. putida, as well as other obligate aerobic strains and limits feasible reactor sizes. The redox power from an anode and a redox mediator can successfully drive the anoxic sugar metabolism of P. putida which allows further quantitative physiology characterizations This electrode-based cultivation approach provides a unique platform to quantitatively assess the anaerobic phenotype of P. putida cells, which cannot be achieved by any other technique so far. We believe that this lays the basis for rationally developing an anaerobically growing P. putida, and similar approaches may be transferred to cultivate other obligate aerobic microbes without oxygen present

Experimental Design

Methods