Light management by algal aggregates in living photosynthetic hydrogels

Significance Light distribution within algal cultures is one of the primary limitations to scalable and efficient biomass growth, a pertinent issue given the increasing interest in nonplanktonic growth methods, such as biofilms. Within these, cells experience uneven illumination via either overexposure on the outer surface or underexposure inside the film. We show how light distribution is altered upon cell aggregation, which naturally occurs under confinement, and enhanced through the incorporation of scatterers. Our work provides insights into how future photobioreactors could be engineered to optimize light delivery, allowing efficient cultivation of microalgae at scale. Last, our work also provides a better understanding of light propagation through gel-encapsulated biomass, a key area given the rise of research interest in engineered living materials.


Extraction of optical parameters using optical coherence tomography (OCT)
Inherent optical properties for the gel immobilised algal aggregates, i.e., the scattering coefficient µs [cm -1 ] and the anisotropy of scattering g, were obtained using theoretical models of light propagation based on the inverse Monte Carlo method (1).A more detailed description of extraction of optical properties from OCT scans can be found elsewhere (2,3).
Briefly, OCT B-scans were acquired with a resolution of 581×1024 pixels, over a fixed depth of 2.8 mm, and variable distance in the X-plane.The setup was optimised to yield the highest signal at a fixed distance of 0.4 mm from the top of the scan.Before measurements, the OCT reflectivity (R) was calibrated (SI Figure 2A) using homemade reflectance standards with an immersion oil-glass, a water-glass, and an air-glass interface.R values from the standards were determined using Fresnel's equation: using the refractive index (n) for air (1), water (1.33), immersion oil (1.46), and quartz glass (1.52).The OCT signal (in decibel, dB), from the samples, was then converted to the depth-dependent R via a linear fit of log10(R) versus OCT intensity values (see reference (3) for details).
The focus function of the objective lens was calibrated by measuring the OCT signal fall off, in steps of 0.1 mm, from either side of the focal plane (z = 0.4 mm) to z = 0 mm and 0.8 mm, respectively.The signal loss from the focal plane follows an exponential decay function.The determined R values from the sample scans were corrected by dividing with the exponential fit.The corrected R values were then plotted against sample depth (z, distance from focal volume) and fitted to the exponential decay function (SI Figure 2B): where ρ (dimensionless) is the light intensity and µ is the signal attenuation (cm -1 ) from the focal volume.The fit was considered satisfactory if R 2 > 0.5.
Using the grid method (4), values of ρ and µ were mapped to g and µs based on the theory described in previous studies (3,5).It was assumed that the sample absorption at 930 nm was negligible and that the absorption was dominated by water (µa ~ 0.43 cm -1 ).S1. Cell density of Chlamydomonas reinhardtii in Tris-minimal and in TAP media with and without cellulose microparticles (CMP), before and after 4 days of incubation in the dark.The values correspond to the mean of nine replicates, in the unit of 10 6 cells per mL, with their respective standard error.

Figure S1 .
Figure S1.Simulation of the attenuation of scalar irradiance with depth in a hydrogel with microalgal aggregates of different oblateness (O = − ), where a and c denote the major and minor axis, respectively.

Figure
Figure S2.(A) Calibrated reflectivity versus OCT signal intensity in dB; (B) Reflectivity profile from an OCT scan of an algal aggregate illustrating the exponential decay of backscattered light with depth into the aggregate, which could be fitted with an empirical model (5).

Figure S3 .
Figure S3.Confocal imaging (maximum intensity projection from a 100 µm deep Z stack) of Chlorella vulgaris aggregates after 7 days of growth within an agarose hydrogel.

Figure S4 .Figure S5 .
Figure S4.Light response curve obtained experimentally using microsensor measurements of photon scalar irradiance (400-700 nm) and oxygen concentration across an isolated Chlamydomonas reinhardtii aggregate within Tris-minimal medium, fitted to the empirical model of Platt et al.(6).

Figure S8 .
Figure S8.The effect of cellulose microparticles (CMP) doping on the transmission and scattering within 1 mm (A) and 2 mm (B) thick agarose hydrogels, across different levels of embedded CMP.Transmission spectra are shown with solid lines, haze spectra of scattered light are shown with dashed lines.