Coupling an Electroactive Pseudomonas putida KT2440 with Bioelectrochemical Rhamnolipid Production

Abstract

Sufficient supply of oxygen is a major bottleneck in industrial biotechnological synthesis. One example is the heterologous production of rhamnolipids using Pseudomonas putida KT2440. Typically, the synthesis is accompanied by strong foam formation in the reactor vessel hampering the process. It is caused by the extensive bubbling needed to sustain the high respirative oxygen demand in the presence of the produced surfactants. One way to reduce the oxygen requirement is to enable the cells to use the anode of a bioelectrochemical system (BES) as an alternative sink for their metabolically derived electrons. We here used a P. putida KT2440 strain that interacts with the anode using mediated extracellular electron transfer via intrinsically produced phenazines, to perform heterologous rhamnolipid production under oxygen limitation. The strain P. putida RL-PCA successfully produced 30.4 ± 4.7 mg/L mono-rhamnolipids together with 11.2 ± 0.8 mg/L of phenazine-1-carboxylic acid (PCA) in 500-mL benchtop BES reactors and 30.5 ± 0.5 mg/L rhamnolipids accompanied by 25.7 ± 8.0 mg/L PCA in electrode containing standard 1-L bioreactors. Hence, this study marks a first proof of concept to produce glycolipid surfactants in oxygen-limited BES with an industrially relevant strain.

Keywords: Pseudomonas putida; bioelectrochemical system; electrobiotechnology; metabolic engineering; microbial electrosynthesis; phenazines; redox mediator; rhamnolipid.

Figure 1

Schematic representation of the (a) rhamnolipid production and (b) phenazine biosynthesis pathways in P. putida KT 2440. The proteins needed for the heterologous production are highlighted in orange and were expressed via two plasmids.

Figure 2

Growth behavior (a–d) and mono-rhamnolipid production (e–h) as well as phenazine production (g–h) of eight clones from each strain used in this study (P. putida RL, P. putida RL-MS, P. putida RL-PCA, P. putida RL-PYO; for detailed information about the strains see text). Cultures where grown in a micro-cultivation platform (n = 3 for each clone) in Delft media with 10 g/L glucose. For the growth curves (a–d) the means of the triplicates without the standard deviation are shown to ensure readability of the data. “Green value” corresponds to the biomass density. The stars indicate the clone selected to be further characterized in shake flasks and bioelectrochemical system (BES) experiments (where applicable).

Figure 3

Fully aerobic flasks cultivation of different P. putida strains for mono-rhamnolipid and phenazine production (PCA and PYO). Non-phenazine producing control strains shown in black traces (circles). Phenazine production strains are shown in green (triangles). *: doubled amount of salicylate inducer (2 mM vs. normally 1 mM; orange squares). Growth measurement via optical density at 600 nm for (a) P. putida RL-PCA strain and P. putida RL control and (b) P. putida RL-PYO and P. putida RL control in Delft minimum media containing 10 g/L glucose. (c,d) show the respective rhamnolipid production (solid traces), PCA production (dashed traces) and PYO production (dotted traces) of these cultivations. (n = 3; error bars represent standard deviations).

Figure 4

Passively aerated BES reactors of P. putida RL-PCA at an applied potential of 0.2 V. (a–c) Benchtop BES reactors (n = 3), showing data for cell density (OD₆₀₀) and current production, glucose consumption and 2-ketogluconate production, formation of PCA and rhamnolipids (RL). (d–f) Electrobioreactors (n = 2), with the equivalent data shown as for the benchtop reactors. The dotted lines indicate the length of the benchtop BES reactor experiment. Gluconate and acetate were not detected in these experiments and are therefore not shown.