Spirulina growing: inoculum preparation and maintenance.

Inoculum characteristics

• Cell density at inoculation: Spirulina grows best when inoculated at OD680 ≥0.6 (optical density at 680 nm, ~0.3–0.4 g/L dry weight). This density ensures rapid cell division and fast colonisation of fresh medium within 3–5 days. Lower-density inocula (OD680 <0.3) take longer to establish (~7–10 days) and are more vulnerable to contamination during the lag phase. For a 100 L production batch, inoculate at ~10–15 L of high-density culture (OD680 0.8–1.0), yielding final starting OD680 ~0.08–0.15 (diluted in fresh medium).
• Purity assessment: Examine inoculum under microscope at 40× or 100× (oil immersion). Spirulina cells are helical filaments (coils, 5–8 µm diameter). “Pure” culture is >95% spirulina; contaminating bacteria (rod-shaped or coccoid, <1 µm) may be visible but are slow-growing at pH 9.5–10.5 (alkaline suppresses most bacteria). Algal contaminants (chlorophyll-containing, green-coloured, may be unicellular or filamentous) must be <1% (excluded). If >5% of the field contains green algal cells, the strain is contaminated; discard and restart from archive stock or purchase new strain.
• Motility: Fresh, healthy spirulina cells are motile (actively swimming via flagella in liquid culture, visible as slow movement under microscope). Non-motile cells indicate senescence (age-related decline) or cell death. >90% motile cells indicates healthy inoculum ready for scaling; <50% motile suggests inoculum fatigue (subculture or replace).

Inoculum subculturing protocol

• Subculture interval: Continuously maintained liquid cultures (without cryopreservation) require subculturing every 4–6 weeks. At week 4–5, take ~10–15% of the culture (transfer to fresh medium at 1:10 dilution ratio), place in clean container under 12–16 hour light, 35–37°C. The remaining culture (~85–90%) can remain in the original container and be used for inoculation or harvest. Subculture every 4–6 weeks prevents senescence (loss of motility, colour fading) and reduces contamination accumulation (slow-growing contaminants multiply over months).
• Growth medium for inoculum: Use standard Zarrouk or modified Zarrouk medium (see Nitrogen management post). Volume: start small (250 mL to 1 L, in clean glass bottle or sealed plastic container). Aerate gently (air pump through aquarium stone, ~0.5–1 bubble/sec; not aggressive bubbling, which causes shearing stress). Light: 12–16 hours/day from fluorescent lamp or cool white LED at ~100 µmol/m²/s (minimum to sustain growth without photoinhibition). Temperature: 35–37°C (use aquarium heater with thermostat for precision).
• Sterility during subculturing: Use sterile technique: work near a clean area (Bunsen burner optional but helpful). Sterilise transfer pipette or small cup with 70% ethanol or brief flame. Allow to cool. Aseptically transfer inoculum from old culture to fresh medium. Close containers immediately after transfer. Avoid prolonged exposure to air. This minimises bacterial ingress during the transfer.

Viability testing

• Optical density (OD680) measurement: Use a simple spectrophotometer or colorimeter set to 680 nm (chlorophyll absorption peak). Take a small sample (3–5 mL) from the inoculum, place in a clean cuvette, and measure absorbance. OD680 <0.4 = inoculum is dilute, subculture or supplement with concentrated spirulina to reach OD680 ≥0.6. OD680 0.6–1.2 = healthy, ready for inoculation. OD680 >1.2 = very dense, dilute with fresh medium before inoculation to avoid osmotic stress from overconcentrated cells.
• Motility assessment: Observe under 40× or 100× microscope objective. Healthy spirulina filaments exhibit slow but visible movement. Count 10 fields of view; estimate percentage of cells with detectable motion. >80% motile = healthy. 50–80% = acceptable but aging. <50% = poor condition, subculture or replace immediately.
• Colour assessment: Fresh spirulina is vivid blue-green (phycocyanin + chlorophyll). Fading to dull green-brown indicates phycocyanin loss (age, pH drift, excessive light stress). If colour is substantially faded despite high OD680, the strain may be stressed; subculture and monitor for recovery.

Cryopreservation (long-term storage)

• DMSO protocol: Prepare healthy inoculum at OD680 0.8–1.0. Add sterile DMSO (dimethyl sulfoxide, molecular biology grade, not pure solvent) to 10% final concentration. Mix gently. Aliquot into sterile cryovials (1–2 mL per vial). Freeze immediately at −20°C (laboratory freezer) or −80°C (ultra-low freezer for longer stability). At −20°C, viability is typically >80% after 1–2 years. At −80°C, viability remains >90% for 5+ years.
• Thawing and revival: Remove cryovial from freezer; thaw at 37°C in water bath or incubator for 2–3 minutes (rapid thaw minimises ice crystal damage). Immediately transfer to 50 mL of fresh Zarrouk medium at room temperature. Leave at room temperature for 1 hour (osmotic equilibration). Then place at 35–37°C under light. Within 24–48 hours, motility should increase and OD680 should begin rising. If no recovery by 72 hours, the vial is non-viable (discard).
• Why cryopreservation is superior to continuous subculturing: Continuous subculturing over years accumulates genetic drift (selection for faster-growing clones, loss of minor traits) and slow-growing contaminant multiplication. Cryopreserved stock at your starting point (year 1) remains genetically identical and contaminant-free indefinitely. You can subculture for 2–4 years from the same cryovial, then restart from a fresh vial. This ensures consistent strain performance across production seasons.

Contaminant screening before scaling

• Bacterial agar plate assay: Before using inoculum to scale to your 100+ L production raceway, screen for heterotrophic bacteria that grow at neutral pH. Plate 100 µL of inoculum (undiluted or diluted 1:10 in sterile water) onto standard nutrient agar (peptone 5 g/L, beef extract 3 g/L, agar 15 g/L, pH 7.0). Incubate plates at 37°C for 48–72 hours. <5 colonies per plate (100 µL) indicates low bacterial load, acceptable for production. >20 colonies indicates significant bacterial contamination; do not scale from this inoculum. >50 colonies: culture is heavily contaminated; discard and start fresh.
• Algal contaminant screening (microscopy): View fresh inoculum under 100× microscope with proper illumination. Spirulina cells are blue-green (phycocyanin) helical filaments. Any cells that are pure green (chlorophyll only, no phycocyanin) or round/ovoid (non-helical) are contaminant algae (likely chlorella or scenedesmus). <1% algal contaminants = acceptable. 1–5% = monitor closely (may increase with time in production). >5% = exclude this inoculum.

Inoculation rates for production

• Static culture (100–500 L tanks): Inoculate at 10–15% by volume. E.g., 100 L tank requires 10–15 L of high-density inoculum (OD680 0.8–1.0). This yields starting OD680 ~0.08–0.15, which doubles every 2–3 days to 0.8–1.0 within 7–10 days (harvest window).
• Paddlewheel raceway (continuous harvest): Maintain 5–10% inoculum in the raceway at all times via regular subculturing. Each 3–4 days, remove ~20–30% of the culture, harvest for product, and replace the volume with fresh medium. This keeps OD680 in the 0.8–1.2 range continuously.

Strain preservation best practices

• Archive strategy: Maintain three levels of inoculum: (1) production culture (current batch, ~50–100 L), (2) backup liquid subculture (250–500 mL, refreshed every 4–6 weeks), (3) cryopreserved master stock (multiple vials at −20°C or −80°C). If production crashes from contamination, you can restart from backup liquid or cryopreserved vial within 1–2 weeks.
• Seasonal restart: If outdoor spirulina operations shut down in winter, cryopreserve a vial of your active strain in autumn. Spring: thaw and restart production from the cryopreserved vial, ensuring you have the exact same strain (not a drifted clone from continuous subculturing over winter).