Fig. 3. Limonene production enables cell aggregation in UTEX 2973.

Cell aggregation is observed in L524 (b) but not in wild-type (WT, a). Quantification analysis suggests 91% of L524 cells are found in aggregates (c). Such aggregation could result from limonene production in L524. Indeed, putative limonene droplets are found on L524 cells (e) but not in wild-type (d) as shown by TEM images, and limonene production is only detected in L524 at ~1.4 mg/L/day/OD₇₃₀ in normalized productivity by GC-MS (f). SRS chemical imaging was used to identify chemical compositions in the droplets. A significantly higher limonene signal is found in L524 (h) compared to wild-type (g). This observation is more evident at the L524 cell surface, where limonene droplets appear to attach to the outer cell surface. In L524 cell aggregation, the surface-attaching limonene appears to form inter-cell junctions bridging cells (i). Moreover, cell surface hydrophobicity was measured by BATH assay. The significantly reduced fluorescence signal in L524 (j) suggests over 40% of L524 cells bind to hydrophobic hydrocarbon, confirming their increased cell surface hydrophobicity. Scale bar, 2 µm. N.D. not detected. **p = 8.4 × 10⁻¹⁰ (two-tailed Student’s t test, n = 3 independent samples with three technical replicates). Original data points of bar figures (c, f, and j) are shown on the plot with blue dots. Data are presented as mean values ±standard deviations (n = 3 independent samples with three technical replicates). Source data are provided as a Source Data file.