Autoclaving, Cleaning / glassware preparation, Stock solutions and salts, Seawater source and treatment, Notes for Aquaculturists Media Comparison (constituents in µmoles)
Major nutrients, Carbon, Nitrogen, Phosphorus, Silica
Other nutrients, Boron, Buffers, Chelators,
Potassium, Soil Extract, Vitamins
The media used in CMARC are predominantly
from published recipes, some are well known and widely used in the phycological
and aquaculture communities such as f/2.
All the media have several components in common : sources of nitrogen,
phosphorus, vitamins and trace metals.
However the specific types of these nutrients, their concentrations and
ratios vary between the media. Some
media used in CMARC have been developed by us over many years, for instance our
medium of choice for freshwater cyanobacteria is MLA (Bolch and Blackburn,
1996) which is a modification of the ASM media developed by Gorham et al (1964). Some media have relatively unusual trace
metals such as selenium in GSe (A modification of GPM, Loeblich, 1975 and vanadium in MJ medium. Because of their unusual nature it may be
tempting to leave these out if stocks are difficult to come by but this would
defeat the very purpose of the media. A
detailed description of the common nutrients, buffers and chelators can be
found by following this link
Proper cleaning of cultureware is as critical to successful maintenance of microalgal cultures as it is for accurate results in analytical chemistry. Some species, such as certain benthic diatoms, adhere to the glass and autoclaving cultures before disposal can also lead to a crust on the glassware at the meniscus which is not removed simply by a detergent bath but requires scrubbing with a bristled brush. Our glassware preparation involves a preparatory rinse in hot tap water, soaking overnight in a detergent bath (e.g. Pyroneg), scrubbing, a wash/rinse cycle in a Laboratory dishwasher (wash cycle at 85 0C, 2 tapwater rinses, 2 Reverse Osmosis water rinses and a drying cycle), followed by 3 hand rinses in Milli Q water and drying in a laboratory oven.
Non-disposable plasticware such as ploycarbonate carboys and polypropylene
fittings are soaked in a compatible detergent such as Nalgene L-900.
We use autoclaving as the prime method
for sterilizing media and particularly for making media for axenic
cultures. Many of our media recipes
refer to fully autoclaved media, where the last stage in media preparation is
for individual culture flasks to be autoclaved so that the flasks remain
totally unopened until culture transfer.
However where maintaining axenic cultures is not as critical then filter
sterilizing particular components of the media and adding them aseptically to
presterilized seawater (or freshwater) can be used. Concentrated nutrient solutions can be
prepared in this way so that phosphate stocks do not precipitate and vitamin activity is not impaired.
While
autoclaving at 121 0C for 15 minutes appears to be a common
mantra, the required duration of autoclaving can cause some confusion. It should be noted that the autoclave time
refers to the duration the goods or total volume of liquid is maintained at the
set temperature, and not simply the time in the autoclave or the time after 121 0C is reached.
Autoclaves come with different chamber sizes and with different
capacities for pressurization, air removal, vacuum generation, steam generation
and air exhaust, and all these factors influence the rate of temperature
increase, holding capacity and decrease.
Therefore it is difficult to give predefined autoclave times for
anything other than hard goods such as pipettes and empty glassware or containers
with up to 100mL of media where the typical 121 0C
for 15 minutes is probably appropriate.
Note, however that a carboy with 10 L of media may require in excess of 60 minutes
once the autoclave reaches the set temperature of 121 0C. The only reliable means of monitoring
temperature in bulk media is to have a thermistor within a reference container
(e.g. PT100) and this would only have to be done periodically to “calibrate”
the system. Relatively small benchtop
pressure cookers may be the sole autoclave in many aquaculture facilities or
used as backup autoclaves in laboratories.
In this case, where for example the largest container that will fit may
be a 1 L culture bottle, it is rare for more than 800mL to be autoclaved and in
this instance a time of 25-30 minutes may be appropriate. Some confusion also exists around pressure
and steam, with a perception that these factors are important in
lethality. This is not true, temperature
above 100 0C is the lethal factor and pressure and
steam are simply the means of delivering that temperature to the entire
contents of the autoclavable goods.
Without steam, air pockets and voids can exist in both glassware and
within autoclave bags and these can persist at lower temperatures than the
chamber temperature but steam penetration into these voids ensures the the
required temperature is met.
Autoclaving drives off carbon dioxide, needed for algal photosynthesis, and raises the pH to undesirable levels. Leaving the media for at least 1-2 days before use will allow sufficient time for CO2 equilibration.
Autoclaving is also a
mandatory method for disposing of our microalgal stock cultures under our
Approved Premise for Quarantine Permit (provided by the Australian Quarantine
and Inspection Service, AQIS). Minimum
draft regulations set by AQIS June 2004 are 121 0C
at 15psi for 30mins. Note these
guidelines are more strict than those for preparing media. Other countries may
have other regulations. The following
two links are on autoclaving culture media and on the myths of autoclaving – commercial source note
http://www.lab-m.com/TAutoclavingMedia.htm
http://www.800ezmicro.com/microBiology.asp?mb=88
In our recipes we refer to “working stocks” and “primary stocks”. Working stocks are those whose aliquots contribute directly to making the final media. Primary stocks are normally made where several single substance solutions are then combined to form the working stock, eg. CuSO4.5H2O and ZnSO4.7H2O are two of the primary stocks used to make up the Trace Metal working stock in F/2 medium.
Culture Collections and scientists undertaking experimental research will use chemicals that are at least Analytical Reagent Grade, both to reduce trace contaminants that may be harmful to some microalgae and also to ensure experimental rigour. While we suggest all stock or starter cultures be grown with AR grade chemicals it is understandable that in mass culture applications (> 20 - 50 L) , particularly for aquaculture, these chemicals may be too expensive when bought in bulk quantities.
Stock solutions are made up by
accurately weighing the prescribed amount of nutrient and dissolving in a
specified volume of distilled water, if possible in a volumetric flask. Some nutrients will readily dissolve, others
need heat and stirring to fully dissolve.
In contrast vitamin stocks are heat sensitive and should not be
subjected to heat treatment and should also be kept in the dark. Failure to fully dissolve the primary stocks
of some nutrients such as EDTA can lead to gross precipitation when these
stocks are combined to make the media.
Nutrients
come with different salts and hydration. For example, while copper and zinc may
be two desired active constituents they are readily obtained from suppliers
with either SO4 or Cl2 salts (ie CuSO4 or CuCl2 and
ZnSO4
or ZnCl2). Some nutrients
also come with different hydrations, ie the .nH2O
suffix. Substituting one form for another may have no effect on the growth of
some microalgae species, but it can lead to poor growth in others and also lead
to unwanted and time consuming precipitation problems as the overall ratio of
salts in the medium has changed.
Therefore deviating from the prescribed recipes is to be avoided and
ordering the correct form is recommended.
The marine microalgae species maintained in CMAR are grown using unpolluted oceanic seawater therefore, strictly speaking , we are using an enrichment medium. Artificial seawater media in contrast is composed of marine salts and nutrients added to pure freshwater. Artificial seawater is only necessary where a clean natural seawater source is unavailable or in particular research studies (e.g. trace metal studies) where the exact composition needs to be controlled. Our collection source takes advantage of CSIRO Hydrochemistry group's monitoring station on the seaward side of Maria Island off the continental shelf along the east coast of Tasmania. The salinity at this site is ~33-36 Practical Salinity Units. This off-shore site has very low concentrations of metal and organic pollutants therefore making it suitable as the base medium for a wide range of marine microalgae species.
The seawater is collected in 20L black polyethylene carboys acid washed prior to collection and then stored at 4 0C until needed. Then it is treated using a filtration series incorporating three 12” Millipore™ cartridge filters (5 µm prefilter, activated charcoal filter for organics removal and a Durapore 0.45µm filter) and finally a Millipak 40TM 0.22 µm disc filter.
The
recipes and preparation protocols used in CMARC are predominantly designed for
laboratory scale culturing. Even the concentrated nutrient solutions may not be
of sufficient volume for regular mass algal cultivation. It is up to the user to modify the quantities
of material needed and determine the cost-effectiveness of using our proposed media
versus commercial alternatives. As an
example in the procedure to make concentrated f2 media 5mL
of the 1L working stocks are used to make 100mL of concentrated media. As the usage rate is 1mL per 100mL of
seawater, the 100mL stock is sufficient for a 10L culture. As there are 200 aliquots of working stock
available (1L stock/5mL), the total culture volume that can be supported is
2000L. The simplest modification to
obtain useful volumes for aquaculture is to increase the volumes of the
concentrated stock solution and its constituent working stocks e.g. for f2
Lab scale concentrated stock : 6 stocks x
5 ml each = 30 mL, made up to 100 ml with distilled H2O (d.H2O) This provides enough concentrated stock for
10 L f2 medium.
Largescale : 6 stocks x 500 ml each = 3000 mL, made up to
10,000 ml (10 L) with (d.H2O)
. This provides enough
concentrated stock for 1000 L f2 medium.
Note
in our recipe only 100 ml of vitamin stock is made at any one time. For large scale purposes that volume would
need to be increased to 1000 ml in accordance with the other stocks.
Microalgae do not require equal amounts of all nutrients,
therefore they are added in different quantities. Natural seawater will supply enough nutrients
for limited growth of some marine species.
Most media recipes supply nutrients vastly in excess of the
concentrations normally found in order to support high biomass cultures. Different species require different amounts
of some nutients but the requirement for the major nutrients is relatively
uniform according to the Redfield Ratio of 106:16:1 of inorganic
carbon:nitrogen:phosphate (by moles).
According to this ratio microalgae will
need roughly 6 times more carbon than nitrogen.
For marine species the carbon requirement in small batch cultures is met
by the 2 mM contained within seawater, and by allowing atmospheric exchange then
the carbon requirement can be supplied over time. Carbon may be lost directly after
autoclaving, therefore it is sensible to allow freshly autoclaved media to
stand for at least 1-2 days before innoculating with microalgae. Large batch cultures, generally greater than
500 mL to 1 L may need to be aerated with either air or an air/CO2
mix to prevent carbon limitation. Typical CO2 concentrations are in
the range 0.5 - 5.0 % v/v
Nitrogen is most commonly added as
nitrate. However some algal species grow
better on ammonium (Thompson et al, 1989). Unfortunately ammonium is frequently
toxic at >100 mM so that much lower concentrations of
ammonium must be used. Guillard
specifies 500 mM (Guillard, 1983) but this concentration
should be tested prior to widespread use.
In some situations it is desirable to have ammonium available. This can be relatively easily accomplished by
adding it to the nitrate stock solution to provide both nitrogen sources, for
example 50 mM NH4 and standard NO3. Because algae preferentially remove NH4
they are likely to deplete all NH4 prior to using nitrate for
growth.
Silica
is only really required by diatoms (excepting the rarely cultured
silicoflagellates) and is therefore often left out of media which are selective
for other organisms. Diatoms only need
trace quantities of silica because they are extremely efficient at scouring
silica from the environment. Some
diatoms may grow for many generations in seawater media which has no added
silica and some may continue to divide for several weeks, albeit with poor
frustule development. Media such as the G
variety (GP, GSe) where the only added silica, apart from the
seawater, comes from soil extract may readily support the growth of
some diatom species. Enrichment cultures
for isolating microalgae devoid of added silicate may still become rich in
diatoms therefore if selection is for non-diatom species a diatom inhibitor is
advantageous. Germanium dioxide (GeO2)
is toxic to diatoms because it disrupts silica deposition and the addition of
low concentrations (5 mg / L) of GeO2 to a culture medium can inhibit diatom growth.
The
silica stock may precipitate if made up in glass bottles so teflon bottles can
be used instead. Precipitation is a
greater problem when the stock is first added to seawater especially if
autoclaved and in glass containers.
However it will slowly disassociate and be available for uptake. Polycarbonate carboys for larger volumes or
teflon bottles (1 L) may be used to limit the precipitation if it is problematic,
noting that teflon is especially expensive.
Other
Nutrient components
Carrano etal, (2009) present a review on boron and marine life. Many studies have shown boron is an essential trace element for both terrestrial higher plants and freshwater and marine algae. Lewin (1965, 1966) showed that Boron is essential for marine diatom growth based on results with 16 centric and pennate species and essential for some but not all marine flagellate species. Diatom cell division was much reduced at boron concentrations less than 0.5 mgL−1 or ~0.05mM (~ 10% of the typical 4.5 mg/L natural seawater concentration). While boron is a component of many culture media it is not present in some common media such as Guillards F or F/2 which have been used widely to support marine microalgal growth including diatoms. The presence of boron leaching into media from borosilicate glassware may account for some of this positive diatom growth but scale-up of diatom growth in plastic bags (e.g. 100 - 500 L) in mariculture operations argues that boron may not be essential for all species. Boron is required by heterocystous cyanobacteria through its role in nitrogenase activity in dinitrogen fixation and heterocyst morphology may alter without boron. Non-heterocystous cyanobacteria display no alteration in growth or nitrogen fixation. (Bonilla et al, 1990).
Bonilla, I., M. Garcia-González, et al. (1990). Boron requirement in cyanobacteria. Its possible role in the early evolution of photosynthetic organisms. Plant Physiol. 94: 1554-1560.
Carrano, C. J., S. Schellenberg, et al. (2009). Boron and Marine Life: A New Look at an Enigmatic Bioelement. Marine Biotechnology 11(4): 431-440.
Lewin, J. C. (1965). Boron Requirement of a Marine Diatom. Naturwissenschaften 52(3): 70
Lewin, J. (1966). Boron as a Growth Requirement for Diatoms. Journal of Phycology 2(4): 160-163
Microalgal consumption of CO2
in batch culture systems (without added CO2) will increase the pH,
and levels above pH 9 are toxic to many microalgal species (cyanobacteria
generally have higher tolerances for elevated pH which may give them a
competitive advantage under such conditions).
Buffers may give some control over the rise in pH and are suitable to low
volume stock cultures, however in large scale dense cultures pH control is
probably more readily met by the addition of CO2 back into the
culture by aeration (air only or air/CO2 mix ..see Carbon).
TRIS buffer (tris (hydroxymethyl) aminomethane): Generally TRIS is used at concentrations up to 5 mM but some microalage find it toxic. An example of varying susceptibility is shown by a comparison between two frewshwater cyanobacteria, Microcystis and Anabaena. TRIS at 10 mM provides good buffering between pH 7.4-7.7 in Microcystis; with increased tolerance towards supraoptimal concentrations of monovalent but not divalent cations (McLachlan and Gorham, 1961). However TRIS is toxic in Anabaena flos-aquae at concentrations too low to provide suitable buffering but filament coiling, indicative of good growth, is promoted by addition of 1 mM TRIS (Gorham et al, 1964).
Glycylglycine: is used at concentrations up to 3.8 mM, however it is both expensive and rapidly metabolised by bacteria and should only be used in axenic cultures
Sodium bicarbonate (NaHCO3): buffers well in the pH range 7.5-8.5 and is used in the freshwater medium MLA for cynaobacteria cultures.
Metals are often insoluble in seawater,
and if added may rapidly form insoluble hydroxides and not be available for
algal uptake. Various chelators (organic
chemicals) are available which bind with the free metal ions forming a metal
complex which may be utilized by microalgae.
Ethylenediaminetetraacetic acid (EDTA) is commonly used because it is
not readily metabolized by bacteria. The
di-sodium salt, Na2EDTA.2H20 has improved solubility
characteristics and is the most widely used.
However when making trace metal solutions where EDTA is required it is
paramount to completely dissolve the EDTA before adding further trace metals as
precipitation can still be a problem.
Citrate (or citric acid) has been recommended because of its lower
affinities for metals commonly found in seawater (Ca, Mg). Thus citrate should produce a more constant
degree of chelation in situations where salinity fluctuates. A limitation of citrate is its high
biodegradability which also provides organic material for bacterial growth. The
theoretical chelator:metal ratio should be 1:1 but as some chelator will be
bound to Ca and Mg in the seawater, more chelator should be added to ensure all
metals stay in solution (ratios ranging up to 3:1; McLaughlin 1973).
Soil extract is an adaptation of E.G. Pringsheim's biphasic
soil-water medium and is a component of some of our media. As the chemical
composition is not well-defined and may vary from batch to batch its use in
experimental situations is not recommended, however in our experience certain
species will not grow well without it and soil extract is added for both
culture maintenance and experimental studies.
Soil must be collected from a natural uncultivated environment or a rich
garden loam may be suitable. No fungicides, insecticides, garden fertilizers or
fresh manure should be present. At
CSIRO, topsoil from a local sandy bushland environment has proved to have
particularly beneficial growth promoting properties. Soil from clay or other soil types are less
suitable in our experience. Soil should not be stored or processed in the algal
culture laboratory, since it is a potent source of unwanted microorganisms.
Soil should be aged under moist conditions (preferably for 6 months or more)
and then kept dry and away from light.An extensive list of modified Soil Water
media developed for a range of microalgae from specific environments can be
found on the UTEX website (The Culture Collection of Algae at the University of
Texas) http://www.utex.org/.
The three most widely used vitamins in order of significance to algae are vitamin B12 (cobalamin or cyanocobalamin), thiamine and biotin. They are most often prepared as a single stock solution.
Vitamin
While vitamin
Croft, M.T., Lawrence, A.D., Raux-Deery, E., Warren, M.J. and Alison G. Smith, A.G. (2005) Algae acquire vitamin B12 through a symbiotic relationship with bacteria. Nature 438 (3)90-93.
Droop, M. R. (2007). Vitamins, phytoplankton and bacteria: symbiosis or scavenging? Journal of Plankton Research 29:107-13.
Thiamine........
Biotin..........
Vitamins are known to be heat and light sensitive, primary stocks of Vitamin B12 and biotin are dispensed in 10-20 mL aliquots in polycarbonate tubes and frozen, where they may keep for long periods and may be refrozen. The working stock solution, including the added thiamine, is normally only kept for 3 months in alfoil wrapped refrigerated bottles before new solutions are prepared. Despite the known degradation of vitamins when heat sterilized, many media types call for total autoclaving. Autoclaving is regularly employed for making media for axenic cultures and rarely have we encountered growth impedance that can be traced to vitamin deficiency. Some algae may be able to use the decomposition products incurred after autoclaving (Provasoli and Carlucci, 1974), however we recommend that for important new isolates or poorly growing species 0.2 um filter-sterilized vitamins should be added aseptically to presterilized media.
Provasoli, L. and Carlucci, A. F. (1974). Vitamins and growth regulators. In : W. D. P. Stewart (ed) Algal Physiology and Biochemistry. Blackwell Scientific, UK, 741-87
Effect of nutrients on species
Filament coiling in Anabaena
flos-aquae : promoted by 4x > [Fe] or deficiency
in Manganese; filament straightening promoted by 4x > [Mn] or iron deficiency. Too high
a concentration of either causes filament fragmentation into short pieces. Best
growth occurs with slight coiling (Gorham et al, 1964).