DoE-based medium optimization for improved biosurfactant production with Aureobasidium pullulans.

Polyol lipids (a.k.a. liamocins) produced by the polyextremotolerant, yeast-like fungus Aureobasidium pullulans are amphiphilic molecules with high potential to serve as biosurfactants. So far, cultivations of A. pullulans have been performed in media with complex components, which complicates further process optimization due to their undefined composition. In this study, we developed and optimized a minimal medium, focusing on biosurfactant production. Firstly, we replaced yeast extract and peptone in the best-performing polyol lipid production medium to date with a vitamin solution, a trace-element solution, and a nitrogen source. We employed a design of experiments approach with a factor screening using a two-level-factorial design, followed by a central composite design. The polyol lipid titer was increased by 56% to 48 g L-1, and the space-time yield from 0.13 to 0.20 g L-1 h-1 in microtiter plate cultivations. This was followed by a successful transfer to a 1 L bioreactor, reaching a polyol lipid concentration of 41 g L-1. The final minimal medium allows the investigation of alternative carbon sources and the metabolic pathways involved, to pinpoint targets for genetic modifications. The results are discussed in the context of the industrial applicability of this robust and versatile fungus.

High-quality physiology of Alcanivorax borkumensis SK2 producing glycolipids enables efficient stirred-tank bioreactor cultivation.

Glycine-glucolipid, a glycolipid, is natively synthesized by the marine bacterium Alcanivorax borkumensis SK2. A. borkumensis is a Gram-negative, non-motile, aerobic, halophilic, rod-shaped γ-proteobacterium, classified as an obligate hydrocarbonoclastic bacterium. Naturally, this bacterium exists in low cell numbers in unpolluted marine environments, but during oil spills, the cell number significantly increases and can account for up to 90% of the microbial community responsible for oil degradation. This growth surge is attributed to two remarkable abilities: hydrocarbon degradation and membrane-associated biosurfactant production. This study aimed to characterize and enhance the growth and biosurfactant production of A. borkumensis, which initially exhibited poor growth in the previously published ONR7a, a defined salt medium. Various online analytic tools for monitoring growth were employed to optimize the published medium, leading to improved growth rates and elongated growth on pyruvate as a carbon source. The modified medium was supplemented with different carbon sources to stimulate glycine-glucolipid production. Pyruvate, acetate, and various hydrophobic carbon sources were utilized for glycolipid production. Growth was monitored via online determined oxygen transfer rate in shake flasks, while a recently published hyphenated HPLC-MS method was used for glycine-glucolipid analytics. To transfer into 3 L stirred-tank bioreactor, aerated batch fermentations were conducted using n-tetradecane and acetate as carbon sources. The challenge of foam formation was overcome using bubble-free membrane aeration with acetate as the carbon source. In conclusion, the growth kinetics of A. borkumensis and glycine-glucolipid production were significantly improved, while reaching product titers relevant for applications remains a challenge.

Identification and quantification of biosurfactants produced by the marine bacterium Alcanivorax borkumensis by hyphenated techniques.

A novel biosurfactant was discovered to be synthesized by the marine bacterium Alcanivorax borkumensis in 1992. This bacterium is abundant in marine environments affected by oil spills, where it helps to degrade alkanes and, under such conditions, produces a glycine-glucolipid biosurfactant. The biosurfactant enhances the bacterium's attachment to oil droplets and facilitates the uptake of hydrocarbons. Due to its useful properties expected, there is interest in the biotechnological production of this biosurfactant. To support this effort analytically, a method combining reversed-phase high-performance liquid chromatography (HPLC) with high-resolution mass spectrometry (HRMS) was developed, allowing the separation and identification of glycine-glucolipid congeners. Accurate mass, retention time, and characteristic fragmentation pattern were utilized for species assignment. In addition, charged-aerosol detection (CAD) was employed to enable absolute quantification without authentic standards. The methodology was used to investigate the glycine-glucolipid production by A. borkumensis SK2 using different carbon sources. Mass spectrometry allowed us to identify congeners with varying chain lengths (C6-C12) and degrees of unsaturation (0-1 double bonds) in the incorporated 3-hydroxy-alkanoic acids, some previously unknown. Quantification using CAD revealed that the titer was approximately twice as high when grown with hexadecane as with pyruvate (49 mg/L versus 22 mg/L). The main congener for both carbon sources was glc-40:0-gly, accounting for 64% with pyruvate and 85% with hexadecane as sole carbon source. With the here presented analytical suit, complex and varying glycolipids can be identified, characterized, and quantified, as here exemplarily shown for the interesting glycine-glucolipid of A. borkumensis.

A novel membrane stirrer system enables foam-free biosurfactant production.

Bioreactors are the operative backbone, for example, for the production of biopharmaceuticals, biomaterials in tissue engineering, and sustainable substitutes for chemicals. Still, the Achilles' heel of bioreactors nowadays is the aeration which is based on intense stirring and gas sparging, yielding inherent drawbacks such as shear stress, foaming, and sterility concerns. We present the synergistic combination of simulations and experiments toward a membrane stirrer for the efficient bubble-free aeration of bioreactors. A digital twin of the bioreactor with an integrated membrane-module stirrer (MemStir) was developed with computational fluid dynamics (CFD) studies addressing the determination of fluid mixing, shear rates, and local oxygen concentration. Usability of the MemStir is shown in a foam-free recombinant production process of biosurfactants (rhamnolipids) from glucose with different strains of Pseudomonas putida KT2440 in a 3-L vessel and benchmarked against a regular aerated process. The MemStir delivered a maximal oxygen transfer rate (OTRmax ) of 175 mmol L-1 h-1 in completely foam-free cultivations. With a high space-time yield (STY) of 118 mgRL L-1 h-1 during a fed-batch fermentation, the effectiveness of the novel MemStir is demonstrated. Simulations show the generic value of the MemStir beyond biosurfactant production, for example, for animal cell cultivation.

The Glycine-Glucolipid of Alcanivorax borkumensis Is Resident to the Bacterial Cell Wall.

The marine bacterium Alcanivorax borkumensis produces a surface-active glycine-glucolipid during growth with long-chain alkanes. A high-performance liquid chromatography (HPLC) method was developed for absolute quantification. This method is based on the conversion of the glycine-glucolipid to phenacyl esters with subsequent measurement by HPLC with diode array detection (HPLC-DAD). Different molecular species were separated by HPLC and identified as glucosyl-tetra(3-hydroxy-acyl)-glycine with varying numbers of 3-hydroxy-decanoic acid or 3-hydroxy-octanoic acid groups via mass spectrometry. The growth rate of A. borkumensis cells with pyruvate as the sole carbon source was elevated compared to hexadecane as recorded by the increase in cell density as well as oxygen/carbon dioxide transfer rates. The amount of the glycine-glucolipid produced per cell during growth on hexadecane was higher compared with growth on pyruvate. The glycine-glucolipid from pyruvate-grown cells contained considerable amounts of 3-hydroxy-octanoic acid, in contrast to hexadecane-grown cells, which almost exclusively incorporated 3-hydroxy-decanoic acid into the glycine-glucolipid. The predominant proportion of the glycine-glucolipid was found in the cell pellet, while only minute amounts were present in the cell-free supernatant. The glycine-glucolipid isolated from the bacterial cell broth, cell pellet, or cell-free supernatant showed the same structure containing a glycine residue, in contrast to previous reports, which suggested that a glycine-free form of the glucolipid exists which is secreted into the supernatant. In conclusion, the glycine-glucolipid of A. borkumensis is resident to the cell wall and enables the bacterium to bind and solubilize alkanes at the lipid-water interface. IMPORTANCE Alcanivorax borkumensis is one of the most abundant marine bacteria found in areas of oil spills, where it degrades alkanes. The production of a glycine-glucolipid is considered an essential element for alkane degradation. We developed a quantitative method and determined the structure of the A. borkumensis glycine-glucolipid in different fractions of the cultures after growth in various media. Our results show that the amount of the glycine-glucolipid in the cells by far exceeds the amount measured in the supernatant, confirming the proposed cell wall localization. These results support the scenario that the surface hydrophobicity of A. borkumensis cells increases by producing the glycine-glucolipid, allowing the cells to attach to the alkane-water interface and form a biofilm. We found no evidence for a glycine-free form of the glucolipid.

Corrigendum: Killing Two Birds With One Stone - Strain Engineering Facilitates the Development of a Unique Rhamnolipid Production Process.

[This corrects the article DOI: 10.3389/fbioe.2020.00899.].

Integration of Genetic and Process Engineering for Optimized Rhamnolipid Production Using Pseudomonas putida.

Rhamnolipids are biosurfactants produced by microorganisms with the potential to replace synthetic compounds with petrochemical origin. To promote industrial use of rhamnolipids, recombinant rhamnolipid production from sugars needs to be intensified. Since this remains challenging, the aim of the presented research is to utilize a multidisciplinary approach to take a step toward developing a sustainable rhamnolipid production process. Here, we developed expression cassettes for stable integration of the rhamnolipid biosynthesis genes into the genome outperformed plasmid-based expression systems. Furthermore, the genetic stability of the production strain was improved by using an inducible promoter. To enhance rhamnolipid synthesis, energy- and/or carbon-consuming traits were removed: mutants negative for the synthesis of the flagellar machinery or the storage polymer PHA showed increased production by 50%. Variation of time of induction resulted in an 18% increase in titers. A scale-up from shake flasks was carried out using a 1-L bioreactor. By recycling of the foam, biomass loss could be minimized and a rhamnolipid titer of up to 1.5 g/L was achieved without using mechanical foam destroyers or antifoaming agents. Subsequent liquid-liquid extraction was optimized by using a suitable minimal medium during fermentation to reduce undesired interphase formation. A technical-scale production process was designed and evaluated by a life-cycle assessment (LCA). Different process chains and their specific environmental impact were examined. It was found that next to biomass supply, the fermentation had the biggest environmental impact. The present work underlines the need for multidisciplinary approaches to address the challenges associated with achieving sustainable production of microbial secondary metabolites. The results are discussed in the context of the challenges of microbial biosurfactant production using hydrophilic substrates on an industrial scale.

Killing Two Birds With One Stone - Strain Engineering Facilitates the Development of a Unique Rhamnolipid Production Process.

High-titer biosurfactant production in aerated fermenters using hydrophilic substrates is often hampered by excessive foaming. Ethanol has been shown to efficiently destabilize foam of rhamnolipids, a popular group of biosurfactants. To exploit this feature, we used ethanol as carbon source and defoamer, without introducing novel challenges for rhamnolipid purification. In detail, we engineered the non-pathogenic Pseudomonas putida KT2440 for heterologous rhamnolipid production from ethanol. To obtain a strain with high growth rate on ethanol as sole carbon source at elevated ethanol concentrations, adaptive laboratory evolution (ALE) was performed. Genome re-sequencing allowed to allocate the phenotypic changes to emerged mutations. Several genes were affected and differentially expressed including alcohol and aldehyde dehydrogenases, potentially contributing to the increased growth rate on ethanol of 0.51 h-1 after ALE. Further, mutations in genes were found, which possibly led to increased ethanol tolerance. The engineered rhamnolipid producer was used in a fed-batch fermentation with automated ethanol addition over 23 h, which resulted in a 3-(3-hydroxyalkanoyloxy)alkanoates and mono-rhamnolipids concentration of about 5 g L-1. The ethanol concomitantly served as carbon source and defoamer with the advantage of increased rhamnolipid and biomass production. In summary, we present a unique combination of strain and process engineering that facilitated the development of a stable fed-batch fermentation for rhamnolipid production, circumventing mechanical or chemical foam disruption.