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. 2023 Nov 23:11:1325019.
doi: 10.3389/fbioe.2023.1325019. eCollection 2023.

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

Tobias Karmainski  1 , Marie R E Dielentheis-Frenken  1 , Marie K Lipa  1 , An N T Phan  1 , Lars M Blank  1 , Till Tiso  1
Affiliations

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

Tobias Karmainski et al. Front Bioeng Biotechnol. .

Abstract

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.

Keywords: acetate; alkanes; bioremediation; biosurfactant; glycolipid; hydrocarbonoclastic bacteria; hydrocarbons; membrane aeration.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

FIGURE 1
FIGURE 1
Basic structure of glycine-glucolipid of A. borkumensis SK2. The glycolipid consists of four 3-hydroxy fatty acids linked by ester bonds, glycosidically linked to the C1 atom of glucose, and linked to glycine via an amide linkage at the terminal fatty acid.
FIGURE 2
FIGURE 2
Growth Profiler cultivation of A. borkumensis SK2 on pyruvate in ONR7a and modified ONR7a media. (A) A lin-lin plot of the growth curves. (B) A log-lin plot of the growth curves between 4 and 20 h (same data). Error bands indicate deviation from the mean (n = 4). Cultivation conditions: ONR7a or modified ONR7a medium, 24-well white plate, N = 225 rpm, T = 30°C, ODstart = 0.1, VL = 1.5 mL, 10 g L-1 pyruvate.
FIGURE 3
FIGURE 3
Growth behavior and glycolipid production of A. borkumensis SK2 at various temperatures with pyruvate. (A) BioLector backscatter growth curves at different temperatures, (B) growth rates (µmax), and normalized glycolipid production of A. borkumensis SK2 at temperatures between 27°C–35°C. Error bands/bars indicate deviation from the mean (n = 4 for 31°C–35°C and n = 7 for 27°C–30°C). Normalized glycolipids: the peak area of glycolipids produced per biomass at 30°C was set to 100%. All other amounts were set in relation to this value. Cultivation conditions: modified ONR7a medium, BioLector 48-well FlowerPlate (MTP-48-B), T = 27°C–35°C, N = 1,000 rpm, ODstart = 0.1, VL = 1.0 mL, 10 g L-1 pyruvate.
FIGURE 4
FIGURE 4
Physiology of A. borkumensis SK2 with pyruvate as the sole carbon source in shake flask. Pyruvate concentration in an abiotic control shake flask is also shown (Pyruvate (control) without inoculum) (n = 3). Cultivation conditions: modified ONR7a medium, 500 mL shake flask, T = 30°C, N = 300 rpm, ODstart = 0.1, VL = 50 mL, 10 g L-1 pyruvate.
FIGURE 5
FIGURE 5
Growth of A. borkumensis SK2 on hydrophilic carbon sources. (A) Test for growth on 0.17 Cmol L-1 pyruvate, acetate, propionate, citrate, succinate, lactate, formate, methanol, ethanol, glycerol, ethylene glycol, and terephthalate. (B) Test for substrate inhibition at different pyruvate (Pyr), acetate (Ac), and propionate (Prop) concentrations (5–20 g L-1). Error bands indicate deviation from the mean (n = 4) (A) or range (n = 2) (B). Cultivation conditions: modified ONR7a medium, Growth Profiler, white 24-well plate, N = 225 rpm, T = 30°C, ODstart = 0.1, VL = 1.0 mL.
FIGURE 6
FIGURE 6
Comparison of growth and glycolipid production of A. borkumensis SK2 on five different carbon sources in shake flask cultivations. (A) Time course of oxygen transfer rate (OTR), and (B) cell dry weight (CDW), glycolipid concentration, and product-to-biomass yield (YP/X) at the end of the cultivation. Error bands/bars indicate deviation from the mean (n = 2). Cultivation conditions: modified ONR7a medium, 500 mL TOM shake flask, T = 30°C, N = 300 rpm, ODstart = 0.2, VL = 25–50 mL, 0.34 Cmol L-1 substrate.
FIGURE 7
FIGURE 7
Batch fermentation of A. borkumensis SK2 with n-tetradecane as carbon source. (A) Time course of oxygen transfer rate (OTR), carbon dioxide transfer rate (CTR), and respiratory quotient (RQ); (B) glycolipid concentration course. Error bands/bars indicate deviation from the mean (n = 2). Cultivation conditions: modified ONR7a medium, 3 L stirred-tank bioreactor, T = 30°C, pH = 7.3, N = 300–1,200 min-1 (cascaded), DO = 30%, FAir = 24 L h-1, ODstart = 0.2, VL = 1.2 L, 4.83 g L-1 n-tetradecane.
FIGURE 8
FIGURE 8
Βatch fermentation of A. borkumensis SK2 with acetate as carbon source. (A) Time course of oxygen transfer rate (OTR), carbon dioxide transfer rate (CTR), and respiratory quotient (RQ); (B) cell dry weight (CDW), glycolipids, acetate, and ammonium concentration course. Error bands/bars indicate deviation from the mean (n = 2). Cultivation conditions: modified ONR7a medium, 3 L stirred-tank bioreactor, T = 30°C, pH = 7.3, N = 300–1,200 min-1 (cascaded), DO = 30%, FAir = 32.4 L h-1, ODstart = 0.2, VL = 1.2 L, 10 g L-1 acetate.
FIGURE 9
FIGURE 9
Membrane-aerated batch fermentation of A. borkumensis SK2 with acetate as carbon source. (A) Time course of O2 and CO2 volume concentration; (B) cell dry weight (CDW), glycolipid, acetate, and ammonium concentration course. Error bands/bars indicate deviation from the mean (n = 2). Cultivation conditions: modified ONR7a medium, 3 L stirred-tank bioreactor, BioThrust static membrane module 2 L, T = 30°C, pH = 7.3, N = 300 min-1, DO = 30%, FGas = 60 L h-1, TMP = 0.3 bar, XO2 = 21–100% (cascaded), ODstart = 0.2, VL = 2.0 L, 10 g L-1 acetate.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. LMB and TT thank the German Federal Ministry of Education and Research (BMBF) for the project GlycoX (grant no. 161B0866B). The laboratory of LMB has been partially funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy—Exzellenzcluster 2186 The Fuel Science Center” ID: 390919832.

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