Biodegradability and toxicity of monorhamnolipid biosurfactant diastereomers

Abstract

Synthetic monorhamnolipids differ from biologically produced material because they are produced as single congeners, depending on the β-hydroxyalkanoic acid used during synthesis. Each congener is produced as one of four possible diastereomers resulting from two chiral centers at the carbinols of the lipid tails [(R,R), (R,S), (S,R) and (S,S)]. We compare the biodegradability (CO₂ respirometry), acute toxicity (Microtox assay), embryo toxicity (Zebrafish assay), and cytotoxicity (xCELLigence and MTS assays) of synthetic rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (Rha-C10-C10) monorhamnolipids against biosynthesized monorhamnolipid mixtures (bio-mRL). All Rha-C10-C10 diastereomers and bio-mRL were inherently biodegradable ranging from 34 to 92% mineralized. The Microtox assay showed all Rha-C10-C10 diastereomers and bio-mRL are slightly toxic according to the US EPA ecotoxicity categories with 5 min EC₅₀ values ranging from 39.6 to 87.5 μM. The zebrafish assay showed that of 22 developmental endpoints tested, only mortality was observed at 120 h post fertilization; all Rha-C10-C10 diastereomers and bio-mRL caused significant mortality at 640 μM, except the Rha-C10-C10 (R,R) which showed no developmental effects. xCELLigence and MTS showed IC₅₀ values ranging from 103.4 to 191.1 μM for human lung cell line H1299 after 72 h exposure. These data provide key information regarding Rha-C10-C10 diastereomers that is pertinent when considering potential applications.

Keywords: Biodegradation; Biosurfactant; Rhamnolipid; Stereochemistry; Toxicity.

Figure 1

Structures of bio-mRL (1) and synthetic (2–5) monorhamnolipids utilized in this study. The varying chain lengths of bio-mRL are represented by ‘m’ and ‘n’ values which vary from 4 to 12.

Figure 2

CO₂ production due to mineralization of (A) Rha-C10-C10 (R,R), (R,S), (S,S), and (S,R) and (B) Rha-C10-C10 (R,R), Rha-C10-C10 mixture, and bio-mRL. C_mRL represents the moles of carbon added as monorhamnolipid. The filled symbols indicate the estimated transition from exponential to stationary phase at which point mineralization was calculated. Increases in CO₂ after this time are attributed to endogenous decay. Error bars represent the standard deviation.

Figure 3

Zebrafish mortality (n = 32) at 120 hpf for monorhamnolipid treatments from 0 to 640 μM. Stars indicate a significant difference in mortality compared to the monorhamnolipid-free control.

Figure 4

Growth inhibition curves for monorhamnolipids as measured by the xCELLigence RTCA (A) or MTS assay (B). Error bars indicate the standard error.