Culturing Chlamydomonas reinhardtii - ALG App008

  • By algenuity
  • 17 Aug, 2016


Chlamydomonas reinhardtii is a motile, unicellular green microalga typically measuring around 10 µm in diameter. It is widely distributed, and is often isolated from soil and freshwater samples. C. reinhardtii has been used as a model organism for over 70 years for both basic and applied research, largely due to its ease of cultivation, rapid doubling time of 6-8 h, and established molecular toolbox (Harris, 2009). Noted areas of study include photosynthesis, phototaxis, cell wall biogenesis, cell cycle events, flagella assembly, mating processes, and nuclear/chloroplast interactions (Rochaix, 1995; Shimogawara et al., 1998; Merchant et al., 2007). Annotated sequences are available for the nuclear, chloroplast and mitochondrial genomes (Merchant et al., 2007, Scaife et al., 2015), and several extensive libraries of mutants have been generated. Transformation of all three genomes have been demonstrated, with nuclear and chloroplast manipulation becoming routine (Boynton et al. 1988; Kindle et al. 1989; Sodeinde and Kindle. 1993).

Recently C. reinhardtii has gained attention as a platform for commercial applications; these include recombinant protein expression (Mayfield et al., 2007; Rosales-Mendoza et al., 2012), biohydrogen production (Torzillo et al., 2015), and as a model testbed for biofuel technologies prior to shuttling into more industrially relevant, but less easy to manipulate, biofuel production strains.

Cultivation of C. reinhardtii is typically conducted mixotrophically on TAP (tris acetate phosphate) medium. Although suitable for lab-scale work, TAP medium is not appropriate for scale up due to its relatively high cost and susceptibility to contamination.


To compare the growth of C. reinhardtii CC1690 under mixotrophic (TAP medium) and phototrophic (HSM medium) conditions in the Algem photobioreactor.

Experimental Design

Two starter cultures of C. reinhardtii CC1690 were inoculated from plates in either TAP or HSM. Cultures were incubated at 20°C, with 40 µE/m²/s continuous light, and 120 rpm agitation (Innova 44, New Brunswick Scientific, New Jersey) for four days, then sub-cultured. Starter cultures in mid-logarithmic phase were used to inoculate duplicate 400 ml cultures of HSM and TAP medium which were then cultured in Algems at 25°C, with 200 µE/m²/s continuous light (85: 15 white: red mix), at 100 rpm agitation and 10cc/minute aeration with 5 % CO for a period of five days.


Growth curves of algae cultures growing on two different  media
Figure 1 - C. reinhardtii CC 1690 cultured in HSM and TAP medium at 25°C, 200 µE/m²/s continuous light (85: 15 white; red mix), 100 rpm, with 10 cm³/min aeration with 5 % CO, (n=2)
Graphical display of limited temperature variation during algae growth

Figure 2 - Temperature variation over the course of the above experiment (n=4)

Graphical display of pH of algae cultures with two different media
Figure 3 - pH variation over the course of the above experiment (n=2)


It was observed that C. reinhardtii grew more productively in TAP than HSM; however, it is unclear from these data whether this is due to the available carbon source (acetate and 5% CO₂ vs. 5% CO₂ only), or the buffering effects of acetate consumption. When algal cells are grown on NH₄+ a drop in pH is generally seen, as the ammonium symporter pumps out a proton into the medium for each ammonium ion transported into the cell. Conversely, when acetate is consumed, the pH increases as the tris: acetate buffer equilibrium is shifted towards the basic. In TAP these two processes occur simultaneously resulting in a relatively stable pH, whereas in HSM only the acidifying uptake of NH₄ + occurs, resulting in a significant drop in the pH. In both cases, OD, pH, and measured temperature were shown to vary very little between replicates as shown in the figures 1, 2, and 3 respectively: error bars in each figure represent standard deviation.

C. reinhardtii CC1609 was observed to reach stationary phase after around 3 days independent of growth medium; however, a higher final OD 740nm was achieved in TAP. Due to the economic benefits associated with HSM over TAP, the optimisation of C. reinhardtii growth in HSM would be worth pursuing, with investigations into NO₃- as a nitrogen source as oppose to NH₄+ presenting the next logical step in this process.


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