May 23, 2025
Version 2
Auxenochlorella ApM1/ApRM1 media recipes and stocks V.2
- 1University of California, Berkeley
- Merchant Lab UC Berkeley
Protocol Citation: Jeffrey Moseley 2025. Auxenochlorella ApM1/ApRM1 media recipes and stocks. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g71pq8gwz/v2Version created by Jeffrey L Moseley
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 22, 2025
Last Modified: May 23, 2025
Protocol Integer ID: 218816
Keywords: algae, homologous recombination, triacylglyceride, synthetic biology, auxenochlorella apm1, auxenochlorella protothecoide, heterotrophic growth of auxenochlorella protothecoide, aprm1 media recipe, apm1, aprm1 medium, heterotrophic growth, excessive acidification, high phosphate, recipe
Funders Acknowledgements:
US Department of Energy Biological and Environmental Research
Grant ID: DE-SC0023027
Abstract
This protocol will make 1 L of ApM1 or ApRM1 medium, optimized for heterotrophic growth of Auxenochlorella protothecoides. This is high phosphate buffered medium that prevents excessive acidification. Use the attached spreadsheet to adjust masses and volumes as required 
Auxenochlorella protothecoides media.xlsx114.6KB . The recipe is adapted from US Patent US20140178950A1 (pg. 28), US Patent US8927522B2 (pg. 37) and US Patent US12037630B2 (pg. 18 & 20).
Auxenochlorella protothecoides media.xlsx114.6KB . The recipe is adapted from US Patent US20140178950A1 (pg. 28), US Patent US8927522B2 (pg. 37) and US Patent US12037630B2 (pg. 18 & 20).Guidelines
Add phosphate salts to water; do not add water to phosphate salts or they may form an insoluble slag
Ammonium sulfate concentration is 7.5 mM, ammonium ion concentration is 15 mM
Ammonium sulfate is added post-sterilization to avoid precipitation
Sugar is added post-sterilization to avoid caramelization
Vitamins and antibiotics are added post-sterilization to avoid degradation
Materials
1 L beaker
1 L autoclavable media storage bottle
2 L Erlenmeyer flask
Milli-Q water
potassium phosphate dibasic anhydrous (Fisher P288-500)
sodium phosphate monobasic monohydrate (Fisher S369-1)
magnesium sulfate heptahydrate (Fisher M63-500)
citric acid (Fisher BP339-500)
calcium chloride dihydrate (Fisher C79-500)
agar (Fisher (BP1423-500)
agarose (Fisher BP1356-500)
yeast extract (Fisher BP9727-500)
ammonium sulfate (Fisher A702-500)
glucose (Fisher D19-212)
thiamine-HCl (Sigma T1270-25G)
sucrose (Fisher S2-500)
melibiose (CHEM-IMPEX 00808)
G418-sulfate (VWR E859-5G)
hygromycin B (VWR K547-20ML)
copper sulfate pentahydrate (Fisher BP346-500)
boric acid (Sigma B9645-500G)
zinc sulfate heptahydrate (Sigma Z0251-500G)
manganese chloride tetrahydrate (Sigma M3634-100G)
sodium molybdate dihydrate (Sigma M1651-100G)
iron sulfate heptahydrate (MP Biochemicals 194663)
Fill beaker to about 700 mL with MilliQ water
Add magnetic stir bar and spin to mix
Add ingredients
4.2 g potassium phosphate dibasic anhydrous (Fisher P288-500) 24.1 millimolar (mM)
3.57 g sodium phosphate monobasic monohydrate (Fisher S369-1) 25.9 millimolar (mM)
240 mg magnesium sulfate heptahydrate (Fisher M63-500) 974 micromolar (µM)
250 mg citric acid (Fisher BP339-500) 1.3 millimolar (mM)
1.7 mL 100 mM calcium chloride dihydrate (Fisher C79-500) 170 micromolar (µM)
10 mL 100X Ap trace element solution
Optional for solid media
15 g agar (Fisher (BP1423-500) 1.5 Mass / % volume
OR
8 g agarose (Fisher BP1356-500) 0.8 Mass / % volume (use agarose for minus thiamine selection)
Optional for rich media (ApRM1)
4 g yeast extract (Fisher BP9727-500) 0.4 Mass / % volume
Adjust volume to 909 mL with MilliQ water and autoclave
55m
Autoclave 121 °C 00:55:00 1.24 Bar
55m
Post-sterilization additions
Cool to 50 °C in water bath
50 mL 150 mM ammonium sulfate (Fisher A702-500) 7.5 millimolar (mM)
40 mL 50% glucose (Fisher D19-212) 2 Mass / % volume
1 mL 2 mM thiamine-HCl (Sigma T1270-25G) 2 micromolar (µM)
Optional for minus-thiamine media - don't add thiamine-HCl
Optional for sucrose media
40 mL 50% sucrose (Fisher S2-500) 2 Mass / % volume
Optional for melibiose media
40 mL 50% melibiose (CHEM-IMPEX 00808) 2 Mass / % volume
Optional for G418 media
1 mL 100 mg/mL G418-sulfate (VWR E859-5G) 100 Mass Percent
Optional for hygromycin B media
6 mL 50 mg/ mL hygromycin B (VWR K547-20ML) 300 Mass Percent
Storage
Store liquid medium without sugar, thiamine or antibiotics at Room temperature or 4 °C
Store liquid medium with sugar, thiamine or antibiotics at 4 °C
Store solid medium with sugar, thiamine or antibiotics at 4 °C
Stock solutions
100X Ap trace element solution (1 L)
2.743 g citric acid (Fisher BP339-500) 14.3 millimolar (mM)
11 mg copper sulfate pentahydrate (Fisher BP346-500) 44.1 micromolar (µM)
330 mg boric acid (Sigma B9645-500G) 340.5 micromolar (µM)
1.4 g zinc sulfate heptahydrate (Sigma Z0251-500G) 4.89 millimolar (mM)
948.5 mg manganese chloride tetrahydrate (Sigma M3634-100G) 4.79 millimolar (mM)
3.9 mg sodium molybdate dihydrate (Sigma M1651-100G) 161 micromolar (µM)
110 mg iron sulfate heptahydrate (MP Biochemicals 194663) 396 micromolar (µM)
Add citric acid first, then salts to 700 mL of MilliQ water with a magnetic stir bar and mix to dissolve
Adjust volume to 1 L
Store at 4 °C
100 mM calcium chloride (1 L)
14.701 g calcium chloride dihydrate (Fisher C79-500)
Dissolve in 700 mL MilliQ water with a magnetic stir bar
Adjust volume to 1 L
Store at 4 °C
150 mM ammonium sulfate (1 L)
19.82 g ammonium sulfate (Fisher A702-500) 150 millimolar (mM)
Dissolve in 700 mL MilliQ water with a magnetic stir bar
Adjust volume to 1 L
Filter sterilize into a sterile bottle with a bottle top filter (Fisherbrand FB12566510)
Store at Room temperature
2 mM thiamine hydrochloride (1L)
675 mg Sigma (T1270-25G) 2 millimolar (mM)
Dissolve in 700 mL MilliQ water with a magnetic stir bar
Adjust volume to 1 L
Filter sterilize into a sterile bottle with a bottle top filter (Fisherbrand FB12566510)
Store at 4 °C
50% glucose (1L)
500 g (Fisher D19-212) 50 Mass / % volume
Slowly add to 400 mL MilliQ water with a magnetic stir bar, mix with heat until fully dissolved
Adjust volume to 1 L
Filter sterilize into a sterile bottle with a bottle top filter (Fisherbrand FB12566510)
Store at Room temperature
50% sucrose (1L)
500 g (Fisher S2-500) 50 Mass / % volume
Slowly add to 400 mL MilliQ water with a magnetic stir bar, mix with heat until fully dissolved
Adjust volume to 1 L
Filter sterilize into a sterile bottle with a bottle top filter (Fisherbrand FB12566510)
Store at Room temperature
50% melibiose (500 mL)
250 g melibiose monohydrate (CHEM-IMPEX 00808) 50 Mass / % volume
Slowly add to 200 mL MilliQ water with a magnetic stir bar, mix with heat until fully dissolved
Adjust volume to 500 mL
Filter sterilize into a sterile bottle with a bottle top filter (Fisherbrand FB12566510)
Store at Room temperature
100 mg/mL G418 sulfate (10 mL)
5 g G418 Sulfate (VWR E859-5G)
Dissolve in 40 mL MilliQ water
Adjust volume to 50 mL
Filter sterilize with a 0.22 μm syringe filter (MlliporeSigma SLGVR33RB)
Aliquot 1 mL into sterile eppendorf tubes
Store aliquots at -20 °C
Protocol references
Coragliotti, A. et al. MICROALGAL POLYSACCHARIDE COMPOSITIONS. (2015). US Patent US20140178950, filed March 29, 2010 and granted January 6, 2015.
Franklin, S. et al. GENETICALLY ENGINEERED MICROBIAL STRAINS INCLUDING CHLORELLA PROTOTHECOIDES LIPID PATHWAY GENES. US Patent US20140178950A1, filed December 6, 2013 and granted December 13, 2016.
Moseley, J. et al. PRODUCTION OF LIPIDS AND TERPENOIDS IN AUXENOCHLORELLA
PROTOTHECOIDES. US Patent US12037630B2, filed November 5, 2021 and granted July 16 2024.
Dueñas, M.A. et al. (2025) ‘Leaky ribosomal scanning enables tunable translation of bicistronic ORFs in green algae’, Proceedings of the National Academy of Sciences, 122(9), p. e2417695122.