Iron availability for microalgal uptake seems to be largely dependent on levels of chelation. It is highly recommended that iron be added as the chemically prepared chelated iron salt of EDTA rather than as iron chloride or other iron salts; the formation of iron chelates is relatively slow, and iron hydoxides will form first in seawater, leading to precipitation of much of the iron in the culture medium.
Apparently as a result of the extreme scarcity of copper in anaerobic waters, copper did not begin to be utilised by organisms until the earth became aerobic and copper increased in abundance. Consequently copper does not seem to be an obligate requirement, algae either not needing it, or needing so little that free ionic copper concentrations in natural seawater are sufficient to maintain maximum growth rates Brand, Copper may indeed be toxic, particularly to more primitive algae, and hence copper, if added to culture media at all, should be kept at low concentrations.
The provision of manganese, zinc and cobalt in culture medium should not be problematical since even fairly high concentrations are not thought to be toxic to algae. It is recommended that these vitamins are routinely added to seawater media. No other vitamins have ever been demonstrated to be required by any photosynthetic microalgae. Soil extract has historically been an important component of culture media. The solution provides macronutrients, micronutrients, vitamins, and trace metal chelators in undefined quantities, each batch being different, and hence having unpredictable effects on microalgae.
With increasing understanding of the importance of various constituents of culture media, soil extract is less frequently used. Soil extract should only be used on a non-experimental basis. The control of pH in culture media is important since certain algae grow only within narrowly defined pH ranges, and in order to prevent the formation of precipitates.
Except under unusual conditions, the pH of natural seawater is around 8. Because of the large buffering capacity of natural seawater due to a bicarbonate buffering system, HCO 3 being present at c. The buffer system is overwhelmed only during autoclaving, when high temperatures drive CO 2 out of solution and hence cause a shift in the bicarbonate buffer system and an increase in pH, or in very dense cultures of microalgae, when enough CO 2 is taken up to produce a similar effect. As culture medium cools after autoclaving, CO 2 reenters solution from the atmosphere, but certain measures must be taken if normal pH is not fully restored:.
The pH of seawater may be lowered prior to autoclaving adjustment to pH Certain media recipes include additions of extra buffer, either as bicarbonate, Tris Tris-hydroxymethyl-aminomethane , or glycylglycine to supplement the natural buffering system. Tris may also act as a Cu buffer, but has occasionally been cited for its toxic properties to microalgae.
Glycylglycine is rapidly metabolized by bacteria and hence can only be used with axenic cultures. These additions are generally not necessary if media are filter sterilized, unless very high cell densities are expected. The problem of CO 2 depletion in dense cultures may be reduced by having a large surface area of media exposed to the atmosphere relative to the volume of the culture, or by bubbling with either air CO 2 concentration c.
Unless there is a large amount of biomass taking up the CO 2 , the higher concentrations could actually cause a significant decline in pH. When bubbling is employed, the gas must first pass through an in line 0. Millipore Millex GS to maintain sterile conditions. For many microalgal species, aeration is not an option since the physical disturbance may inhibit growth or kill cells. Always use reagant grade chemicals and bidistilled or purer water to make stock solutions of enrichments. When preparing a stock solution containing a mixture of compounds, dissolve each individually in a minimal volume of water before mixing, then combine and dilute to volume.
Seawater, stock solutions of enrichments and the final media must be sterile in order to prevent or more realistically minimize biological contamination of unialgal cultures. Several methods are available for sterilization:. A commercial autoclave is best, but pressure cookers of various sizes are also suitable.
Ensure the heating elements are covered with distilled water, and the escape valve should not be closed until a steady stream of steam is observed. Autoclaving is a process which has many effects on seawater and its constituents, potentially altering or destroying inhibitory organic compounds, as well as beneficial organic molecules. Because of the steam atmosphere in an autoclave, CO 2 is driven out of the seawater and the pH is raised to about 10, a level which can cause precipitation of the iron and phosphate added in the medium.
Some of this precipitate may disappear upon re-equilibriation of CO 2 on cooling, but both the reduced iron and phosphate levels, and the direct physical effect of the precipitate may limit algal growth. The presence of EDTA and the use of organic phosphate may reduce precipitation effects.
Algal Culturing Techniques
The best solution, however, if media are autoclaved, is to sterilize iron and phosphate or even all media additions seperately and add them aseptically afterwards. Autoclave steam may contaminate the media i. Autoclaving also produces leaching of chemicals from the medium receptacle into the medium silica from glass bottles, toxic chemicals from plastics. Autoclaving in well-used Teflon or polycarbonate vessels reduces leaching of trace contaminants. Autoclaving will cause evaporation of water, and hence an increase in salinity usually of c.
Distilled water can be added prior to autoclaving to compensate for this increase. Pasteurization does not, however, completely sterilize the seawater; it kills all eukaryotes and most bacteria, but some bacterial spores probably survive. Ultraviolet radiation can be used to sterilize seawater, but very high intensities are needed to kill everything in the seawater W lamp, h for culture media in quartz tubes. Such intense UV light necessarily alters and destroys the organic molecules in seawater and generates many long lived free radicals and other toxic reactive chemical species Brand, Seawater exposed to intense UV light must, therefore, be stored for several days prior to use to allow the level of these highly reactive chemical species to decline.
Sterilization by commercial microwave apparatus is another option. Microwave sterilization has not, as yet, been widely employed in culture media preparation due to uncertainties about sterilization efficiency. Trials should be conducted before use of this method to ensure sterility of seawater.
Basic Methods for Isolating and Culturing Microalgae | Springer Nature Experiments
Sterile filtration is probably the best method of sterilizing seawater without altering the chemistry of the seawater, as long as care is taken not to contaminate the seawater with dirty filter apparatus. Sterilization efficiency is, however, to some extent reduced compared with heat sterilization methods. Membrane filters of 0. Millipore Millex GS can be used; for volumes up to 1 litre reusable autoclavable self-assembly filter units glass or polycarbonate with 47mm cellulose ester membrane filters eg.
Millipore HA can be used with suction provided by a vacuum pump; for larger volumes an in-line system with peristaltic pump and cartridge filters may be the best option.
Filter units particularly disposable plastic systems , and the membrane filters themselves can also leak toxic compounds into the filtrate. The first portion of filtrate eg. Most stock solutions of culture medium additions can be sterilized separately by autoclaving, although vitamin stock solutions are routinely filtered through 0.
Millipore Millex GS , since heat sterilization will denature these organic compounds. Filter sterilization of all additions may reduce uncertainties about stability of the chemical compounds and contamination from autoclave steam, but absolute sterilization is not guaranteed. The recipes of 3 media which have proved successful for the culture of coccolithophores are given. A variety of alternative marine culture media recipes are given by Stein , and on the web pages of the major culture collections eg.
CCMP, Utex. Quantity Compound Stock solution sterile Final conc. Quantity Compound Stock solution Final conc. B 12 cyanocobalamin 0. Make up to 1 litre with distilled H 2 O, sterilize autoclave or filter and store in fridge. B 12 cyanocobalamin The agar and culture medium should not be autoclaved together, because toxic breakdown products can be generated.
Research and environmental applications, provision of educational materials and algal depository. CCBA at Institute of Oceanography UG maintains the strains of Baltic and a more limited number of freshwater microalgae and small multicellular seaweeds collected from a wide range of habitats. The culture collection specializes in the Polish region and conducts continuous enlargement of the resources by addition of new strains.
The cultures are unialgal, mostly non-axenic. Strains are available for research and education purposes without any fee. They are available for purchase by commercial enterprises. ABSTRACT: Global increases in atmospheric CO2 and climate change are drawing considerable attention to identify sources of energy with lower environmental impact than those currently in use. Biodiesel production from microalgae lipids can, in the future, occupy a prominent place in energy generation because it represents a sustainable alternative to petroleum-based fuels.
Several species of microalgae produce large amounts of lipids per biomass unit. Triacylglycerol is the fatty acid used for biodiesel production and the main source of energy reserves in microalgae. The current literature indicates that nutrient limitations can lead to triacylglycerol accumulation in different species of microalgae. Further efforts in microalgae screening for biodiesel production are needed to discover a native microalgae that will be feasible for biodiesel production in terms of biomass productivity and oil.
This revision focuses in the biotechnological potential and viability of biodiesel production from microalgae.