Exploiting RNA thermometer-driven molecular bioprocess control as a concept for heterologous rhamnolipid production

A key challenge to advance the efficiency of bioprocesses is the uncoupling of biomass from product formation, as biomass represents a by-product that is in most cases difficult to recycle efficiently. Using the example of rhamnolipid biosurfactants, a temperature-sensitive heterologous production system under translation control of a fourU RNA thermometer from Salmonella was established to allow separating phases of preferred growth from product formation. Rhamnolipids as bulk chemicals represent a model system for future processes of industrial biotechnology and are therefore tied to the efficiency requirements in competition with the chemical industry. Experimental data confirms function of the RNA thermometer and suggests a major effect of temperature on specific rhamnolipid production rates with an increase of the average production rate by a factor of 11 between 25 and 38 °C, while the major part of this increase is attributable to the regulatory effect of the RNA thermometer rather than an unspecific overall increase in bacterial metabolism. The production capacity of the developed temperature sensitive-system was evaluated in a simple batch process driven by a temperature switch. Product formation was evaluated by efficiency parameters and yields, confirming increased product formation rates and product-per-biomass yields compared to a high titer heterologous rhamnolipid production process from literature.

Burkholderia thailandensis E264 as a promising safe rhamnolipids’ producer towards a sustainable valorization of grape marcs and olive mill pomace

Within the circular economy framework, our study aims to assess the rhamnolipid production from winery and olive oil residues as low-cost carbon sources by nonpathogenic strains. After evaluating various agricultural residues from those two sectors, Burkholderia thailandensis E264 was found to use the raw soluble fraction of nonfermented (white) grape marcs (NF), as the sole carbon and energy source, and simultaneously, reducing the surface tension to around 35 mN/m. Interestingly, this strain showed a rhamnolipid production up to 1070 mg/L (13.37 mg/g of NF), with a higher purity, on those grape marcs, predominately Rha-Rha C14-C14, in MSM medium. On olive oil residues, the rhamnolipid yield of using olive mill pomace (OMP) at 2% (w/v) was around 300 mg/L (15 mg/g of OMP) with a similar CMC of 500 mg/L. To the best of our knowledge, our study indicated for the first time that a nonpathogenic bacterium is able to produce long-chain rhamnolipids in MSM medium supplemented with winery residues, as sole carbon and energy source. Key points • Winery and olive oil residues are used for producing long-chain rhamnolipids (RLs). • Both higher RL yields and purity were obtained on nonfermented grape marcs as substrates. • Long-chain RLs revealed stabilities over a wide range of pH, temperatures, and salinities

Process Simulation and Economic Analysis of Rhamnolipid Production by Pseudomonas aeruginosa Using Oil Palm Empty Fruit Bunch as Substrate

Surfactants are amphiphilic molecules that have been used in widely used as emulsifier and cleaning agent in various industries. These broad industrial applications made the global surfactant production increased annually. In 2020, the amount of global surfactant production estimated would reach more than 24 million per year. However, the currently used surfactants are synthetically produced from chemical or petroleum-derived products, so it is often toxic and non-biodegradable. Rhamnolipids are “surfactant like” molecules produced by Pseudomonas aeruginosa, which could be alternatives for more environmentally friendly surfactants. The use of rhamnolipids is quite limited due to its expensive production cost. The production cost of rhamnolipid could be reduced using by agro-industrial by-product as a substrate. One of the abundant agro-industrial by-products in Indonesia that can be used as a substrate for rhamnolipid production is Oil Palm Empty Fruit Bunch (OPEFB). This study employed SuperPro Designer v9.5 to performed process simulation and economic assessment of rhamnolipid production using OPEFB as a substrate through two different purification methods, which are solvent extraction and chromatography adsorption. Based on the process simulation that has been done, the process that used adsorption chromatography purification methods more efficient in terms of the usage of the feedstock and energy. The economic assessment also showed that these methods were more profitable and economically attractive, with the value of NPV 11.400.000 USD, IRR 11,64%, and Payback Period 6,21 years.

Coupling an Electroactive Pseudomonas putida KT2440 with Bioelectrochemical Rhamnolipid Production

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.