Galdieria · Aim 1 · Heterotrophic screen · results
Heterotrophic screen — live results
Seven isolates, now growing in the dark on a kiwifruit-sugar blend (glucose / fructose / sucrose, ~10 g/L). Day 0 (T0) is logged below; growth curves and phycocyanin re-accumulation populate as timepoints come in. Beneath that sits the carbon-starvation prep that set the starting line.
Screen
T0 logged
7 isolates × 4 flasks inoculated on kiwifruit sugar.
Densest start
CM1.2Y
OD₇₅₀ 0.053; KP1.2Y lowest at 0.008.
Baseline phycocyanin
RTK37.1
Highest per-biomass PC — the one to watch on regrowth.
Heterotrophic screen — Day 0 (T0)
The screen's starting line: seven starved isolates inoculated into ~10 g/L kiwifruit sugar (42.9% glucose / 47.2% fructose / 9.9% sucrose). Each isolate runs as 3 sugar-fed (+C) replicates plus a no-carbon (−C) control — the −C is what will prove any growth is sugar-driven, not residual reserves. Values are blank-subtracted neat OD₇₅₀; total biomass = +C OD × flask volume (50 mL V4 + 2 mL sugar + inoculum; no evaporation at t=0). Replicate precision is excellent where signal allows — CM1.2Y's +C flasks agree to ~1%, while the dilute isolates carry ~10–12% CV purely because they sit just above the reader's floor. (1:3 / 1:9 wells are at noise — neat only until cultures climb past ~OD 0.4.)
| Isolate | +C OD₇₅₀ (n=3) | CV % | +C total biomass | −C ctrl OD₇₅₀ |
|---|---|---|---|---|
| CM1.2Y | 0.054 | 1 | 3.29 | 0.053 |
| RK1.2 | 0.037 | 11 | 2.18 | 0.040 |
| CM1.1G | 0.035 | 12 | 2.03 | 0.041 |
| CM1.3Y | 0.026 | 11 | 1.43 | 0.032 |
| RTK37.1 | 0.023 | 9 | 1.25 | 0.020 |
| RK1.3 | 0.016 | 10 | 0.89 | 0.018 |
| KP1.2Y | 0.008 | 6 | 0.41 | 0.010 |
These t=0 values are the denominators for every later metric — specific growth rate (µ) and per-biomass phycocyanin re-accumulation. Flask weights are banked as the t=0 reference; their change over coming days gives per-flask evaporation for the volume correction. Watch the −C controls stay flat (true heterotrophy) and for two-phase growth as the cells work glucose → fructose → (acid-inverted) sucrose. Growth curves render here as Day 1+ arrives.
Heterotrophic growth — Day 1 (~24 h)
The first growth point. Every sugar-fed (+C) culture has grown 1.1–2.0× in 24 h while the no-carbon (−C) controls held flat — genuine, sugar-driven heterotrophy across all seven isolates. Phycocyanin here is the direct fluorescence read (ex 590 / em 670), not the spectral index used for the starvation phase. Absolute phycocyanin is roughly constant while biomass climbs, so specific PC dips on Day 1 — the cells are dividing faster than they re-make pigment; the question is whether it rebounds toward stationary.
| Isolate | D0 +C | D1 +C | +C fold | −C ctrl D1 | PC (RFU) | specific PC |
|---|---|---|---|---|---|---|
| RK1.3 | 0.016 | 0.032 | 2.00× | 0.010 | 42 | 1302 |
| RTK37.1 | 0.023 | 0.041 | 1.78× | 0.027 | 157 | 3821 |
| RK1.2 | 0.037 | 0.062 | 1.68× | 0.037 | 143 | 2312 |
| CM1.3Y | 0.026 | 0.042 | 1.62× | 0.033 | 11 | 262 |
| KP1.2Y | 0.008 | 0.013 | 1.62× | 0.009 | 0 | 0 |
| CM1.2Y | 0.054 | 0.070 | 1.30× | 0.050 | 168 | 2395 |
| CM1.1G | 0.035 | 0.038 | 1.09× | 0.035 | 128 | 3377 |
- Heterotrophy is real and general — all seven +C cultures grew, the −C controls did not. Leaders: RK1.3 (2.0×), RTK37.1 (1.8×), RK1.2 (1.7×); CM1.1G slowest (1.1×).
- RTK37.1 is the early front-runner — top per-biomass phycocyanin and 2nd-fastest grower.
- KP1.2Y makes zero phycocyanin (0 RFU both days) yet grows on sugar — likely a pigmentless heterotroph, not a producer. CM1.3Y is a genuine low producer (~10× below the main group).
Phycocyanin is now read directly by fluorescence (corrects an earlier report that said pigment data was missing — it was present, just stacked under each Biomass row). Total-biomass / total-PC volume corrections follow once Day-1 flask weights are logged.
Biomass — the starve worked
Volume-corrected total biomass. Flat-to-declining for every isolate is the starvation signature: internal reserves are spent and nothing is growing, so the no-carbon control will read as a true negative. The Day-1 dip is an evaporated-state sampling artifact (raw OD over-reads when a flask has dried down); dividing by volume removes it.
| Isolate | Day 0 | Day 1 | Day 2 | Day 3 | Verdict |
|---|---|---|---|---|---|
| CM1.3Y | 8.5 | 7.3 | 8.8 | 7.3 | plateaued |
| CM1.1G | 9.0 | 8.1 | 9.3 | 7.7 | plateaued |
| CM1.2Y | 14.6 | 13.6 | 13.2 | 11.3 | declining |
| RK1.2 | 10.4 | 8.0 | 8.0 | 7.7 | plateaued |
| RK1.3 | 3.0 | 3.5 | 3.5 | 3.2 | plateaued |
| RTK37.1 | 6.0 | 4.6 | 4.8 | 4.8 | plateaued |
| KP1.2Y | 2.9 | 3.1 | 2.9 | 2.6 | declining |
Phycocyanin — who's carrying the product
Phycocyanin is the target product. Because the isolates started at different biomass, the metric that matters now is specific PC — pigment per unit biomass — not the total. On that measure RTK37.1 leads through Days 0–1, so if it grows well it could out-produce a bigger but pigment-poorer isolate. Every isolate bleaches to near-zero by Day 3 as phycobilisomes are degraded for reserves, so the per-biomass ranking is only meaningful Days 0–2 (Day 3 is dividing noise by noise).
Reading the field so far
What the starting line says — and what it does not.
- Specific PC is the metric that matters now. The isolates started at different biomass, so per-biomass pigment — not the total — is the fair comparison at this stage. RTK37.1 carries the highest specific PC (Days 0–1), so the working hypothesis is that once it gets growing it out-produces the others on pigment.
- CM1.2Y still carries the most total product today (biomass × content, ~2× the next) and sits second on specific PC — a strong all-rounder and the one to beat on sheer output.
- KP1.2Y carries no phycocyanin. The direct fluorescence read (Day 0–1 screen) shows a flat 0 RFU — it makes none, even while growing on sugar. This corrects an earlier note here: the spectral A₆₂₀ index wrongly placed it mid-pack (scatter, not signal). It is best treated as a pigmentless heterotroph, not a producer.
- This is a baseline, not a verdict. Phycocyanin is degraded during the dark starve (everything bleaches to near-zero by Day 3), so the real ranking comes from the heterotrophic growth phase, just inoculated — who regrows fastest and re-accumulates the most pigment on sugar. That is the test of the RTK37.1 hypothesis.
Methods: total biomass = least-suppressed OD₇₅₀ (neat or dilution back-calc) × flask volume at sampling. Phycocyanin index = blank- and scatter-corrected A₆₂₀ − A₇₅₀ from the plate-reader wavelength scan; total PC = index × volume. Day 3 decoded from a 180°-rotated plate. Reads captured OD₇₅₀ + spectra only — pigment values are spectral indices, not calibrated concentrations.