Spirulina - Science topic

Dear 王嘉麟 Wangjialin

I hope this message finds you 王嘉麟 Wangjialin well! I wanted to dive into the topic of Spirulina culture in open ponds, particularly concerning the potential for contamination. Given the use of Zarrouk medium, it seems like a prime candidate for various algae species to invade, especially since outdoor settings can introduce airborne or waterborne spores.

From my observations, the most common contaminants we might encounter include Chlorella sp., which is often green and round-shaped, making it a frequent visitor in outdoor cultures. Then there’s Scenedesmus sp., which tends to form small colonies with distinctive spines, and Ankistrodesmus sp., known for its elongated or crescent shapes. Lastly, we should keep an eye out for Anabaena or other filamentous cyanobacteria, as they can be particularly harmful due to their toxin production.

Identifying these species can be tricky without a closer look at their morphology, especially under magnification. If you notice any distinctive shapes—like round cells for Chlorella or spined clusters for Scenedesmus—those could help us narrow it down. For more precise identification, consulting an algal identification key or considering molecular marker analysis might be worthwhile if we have access to those resources.

Looking forward to hearing your thoughts on this!

Continuous aeration, especially if it's too vigorous, can cause mechanical damage to the algae, leading to fiber breakage and clumping.

While Spirulina benefits from continuous light, excessive intensity or improper light distribution can stress the cells, causing them to clump and break.

A sudden change or imbalance in nutrients could weaken the algae, making them more susceptible to breaking.

Although the pH range you maintain is generally suitable for Spirulina, slight fluctuations or sudden changes might stress the algae, leading to clumping and broken fibers.

The presence of contaminants (like other microorganisms or debris) could cause physical or chemical stress, leading to clumping and fiber breakage.

Although Spirulina can tolerate temperatures up to 36°C, sustained high temperatures close to this upper limit might cause stress and damage to the cells.

You might want to check the aeration intensity, light intensity, and nutrient levels, as well as ensure the culture is free from contamination.

Hello, although I do not work with Spirulina, I have studies based on the production of C-phycocyanin with other isolates. If this information will help you, I can help you.

This answers helped me a lot, thanks! Can you recommend any articles on this subject?

How many algal species are recorded globally

i saw the article, but spirulina surely does not produce mitotic spindles that are the target of colchicine. That means that the effect cannot be linked to the normal effect of colchicine. In the article i saw also strange variance values in the diameter measurement....

Dear Megan,

It is usual for an air pump to produce bubbles when it is turned on, as this is the function of the pump. However, the bubbles should not be so strong as to create a disturbance in the water that could damage your spirulina culture. Here are some suggestions to help you address this issue:

Spirulina requires aeration to grow, so running the air pump is essential. However, ensuring that the bubbles do not damage your culture is equally important. With some experimentation, you should be able to find a solution that works for you.

Yours sincerely,

Edgar M Cambaza

In my opinion it is the long pale filaments that are the anomaly : unusually long filaments (>10 spires) are usually caused by a lack of iron, pale color comes with a lack of chlorophyll (an indication of stress), and healthy spirulina usually tend to float whereas stressed spirulina tends to sink.

You may also have 2 different strains in your medium. Try to spike the medium with iron and apply a very very gentle agitation, to see if you go back to a single phenotype (otherwise you have 2 strains)

It is not yet clear why some spirulina cultures are more "linear" than others. Growth conditions are certainly the main cause of the morphological change of spirulina, but sometimes it is also due to a specific growth phase of the biomass.

I would check if there are any problem with culture media, or if is present a contamination by other cyanobacteria.

8/20/22

Dear Hooi,

Do you see this in every smear that you examine w/ the microscope? Or is this the only one you've seen? At first, I thought it was a small length of hyphal (fungal) filament, but it looks more like a tiny stand of cotton fiber than a microorganism.

Bill Colonna Iowa State University, Ames, Iowa, USA wcolonna@iastate.edu

ribulose bisphosphate carboxylase oxygenase

I expect yes

Use 0,4g/l Na2CO3

Hi Tomas, OK thanks. All the best, Jules

5/26/22

Dear Justus,

See the reference below. This may answer your questions.

I hope this information helps you.

Bill Colonna

Iowa State University, Ames, Iowa, USA wcolonna@iastate.edu

J Anim Physiol Anim Nutr (Berl). 2020 Jan; 104(1): 310–321.

Published online 2019 Nov 3. doi: 10.1111/jpn.13239

PMCID: PMC7004008

PMID: 31680348

A two‐enzyme constituted mixture to improve the degradation of Arthrospira platensis microalga cell wall for monogastric diets

Diogo Coelho, 1 Paula A. Lopes, 1 Vânia Cardoso, 2 Patrícia Ponte, 2 Joana Brás, 2 Marta S. Madeira, 1 Cristina M. Alfaia, 1 Narcisa M. Bandarra, 3 Carlos M. G. A. Fontes, 1 , 2 and José A. M. Prates📷 1 , 2

Author information Article notes Copyright and License information Disclaimer

Cycloheximide in concentration of 100 µg/ml is toxic for eukaryotic algae including diatoms and not toxic for cyanobacteria. Several passages on plates with cycloheximide should help to eliminate diatoms.

Check https://www.fao.org/3/y3781e/y3781e.pdf

It is not Arthrospira. It is a green alga (one could see chloroplasts), so you have a contamination problem. We could discuss further details (reasons, solutions) in private chat if you are interested.

Thank you for all of yours suggestions, Schott proveide really high tech products but it is really expensive. BBI is no longer produce reactors in pilot or industrial scale. I didnt know algoliner, I will contact them. Thnaks again.

Dear Lukas Behr,

Firstly, Spirulina (Arthrospira platensis) is a filamentous and multicellular blue-green alga. Eventually, it doesn't require too much effort for harvesting.

Secondly, using chemical flocculants for harvesting microalgae is not advisable for food/pharmaceutical applications because the chemicals might have toxic effects on the final products. On the other hand, physical harvesting, for instance, moving net, could be promising.

Please check the video link below:

Thank you

Best of luck

Chandan

Thanks for all the suggestions Virendra Kumar Saxena Kiprotich Kiptum Grigorios Kyriakopoulos

Is the bottle kept in agitation? You should mix the culture, but avoiding using magnets within the broth, otherwise it causes destruction of filaments. Try using a tilting plate instead, if you are not already doing it. In addition, try to pump air into the liquid by using a "straw": this would improve CO2 solubilization.

Of course you should first check medium, temperature, light, state of the inoculum etc...

It is an interesting question. We are not sure to get full yield with organic inputs. But along with biostimulants, organic inputs could help in realising good yield potential.

I need to know too

Anh Nguyen: although the NaHCO3 is a good buffer it can not raise the pH. In fact, when algae use It as a carbon source It can turn into NaHCO3 and raise the pH but in low density, there isn't enough biomass. to increase the start pH the best option is NaCO3. If you don't have access to NACO3 don't be worry! you can heat NaHCO3 in the oven at 200 C for 2h or simply put the NaHCO3 in a pan and heat it until the color turns whiter and the particles pop up.

please see the following link

Hamid Reza Roozban

where

Abstract

A convenient small-scale extraction method for lyophilized micro-algae is described that dispenses with labor-intensive homogenization and is widely applicable to algae from different phyla. The procedure employs an optimized sequential extraction in trichloroacetic acid (TCA) and NaOH to achieve chemical lysis. Conditions were tested using several micro-algal strains to develop a method that was generally applicable. Incubation of lyophilized material in 24% (w/v) TCA at 95 °C followed by a hot alkaline treatment was found to be effective for strains that are resistant to conventional extraction approaches, such as the Chlorella and the Eustigmatophycean species. The single-tube extraction procedure can be complete in 4 h and is conveniently followed by the Lowry assay, requiring a further 30 min. Overall, this method proved to be generally applicable and ideal either for single samples or for high-throughput screening of multiple algal strains for protein content.

Just replace Lowry with Bradford- make sure solution is roughly neutral before assay. Caveat: Bradford assay will be false positive if you have detergent in your preparation.

Check https://www.sciencedirect.com/science/article/pii/S0963996921002131

Dear Menuja Wijewickrama

It's maybe due to oxidation of pigment.

Thank you

It Is smaller than standard salinity for spirulina. The standard amount is 30 g/L which about 5 grams of this come from NaCl and the other part is from Soda, Soda ash, and other nutrients. from of my view, you didn't use enough Soda (16 g/L) and Soda ash (8g/L) which are very important for preventing predators and competitors. When evaporation occurs the salinity could rise higher and higher, in this situation you have to keep watching the salinity doesn't go more than 60 g/L and by adding freshwater keep it at 30 g/L points.

Hi Andri. It is normal for spirulina culture. After stopping agitation they will form big colonies and of course, this colony regarding spirulina composition and water salinity can be lighter or heavier than water and they will float or sink in the bottom.

According to international standards in OECD-GLP Guideline better to feed Yeast to Daphnia at early stages it will grow fast.

The world of micronutrients still has many gaps, and the specific case of vitamin b12 is particularly complex given the clinical importance of its levels, which are low, but also high. In this sense, in complement to the debate question, I want to share with you the following manuscript detailing the aspects associated with high levels of vitamin b12.

first spirulina unlike other cyanobacteria doesn't have heterocyst so it doesn't have capability of using atmospheric N and its completely depended on water oriented N. the available nitrogen sources for spirulina consist of NH3, urea and Nitrate. excess NH3 is harmfull for spirulina and easilly can kill it. also urea after dissolving in water degrade to NH3 and CO2 and again harms spirulina in excess amount. but Nitrate is completely safe. You can add nitrate to your culture in the first day and add cheep urea daily. At first, spirulina use urea as the N source and after depletion of urea if spirulina need more N it will use more expensive nitrate.

Well done. As I explained before the total area is very important. the average production is 10 g/m2 per day. So, if you have 1 m3 system with 1 m depth so its need 100 days to reach the 1g/l! and if you have 1 m3 system with 1 cm depth its just need 10 days.

Please follow this link:

1.

2.

Dear Chamari Coswatte , in the attachment you can find your answer. Good luck!

Hi Ramón Kenayfati

I believe that this phenomenon is related to its inoculum, but we can consider two phases:

i) first exponential growth phase: fragmented cells of A. platensis are reproducing rapidly.

ii) second exponencial growth phase: the fragmented cells of A. platensis now grow in size rather than reproducing, due to some environmental variation (probably lack of nutrient or change in the pH).

Filamentous algae commonly exhibit this behavior. You must know these phases well in order to justify choosing a kinetic growth model.

I have gathered a few recipes from artisanal spirulina producers, and it seems hard to go below 8 g/L of sodium bicarbonate. However, it is possible to run in fed-batch or continuous mode with diluted media : we once ran a 30-L batch inoculated at 1 g/L spirulina, in a Zarrouk medium diluted 5 times, and had quite good growth curves during 3 days without adding any nutrients.

You may replace sodium bicarbonate by some other source of alkalinity (sodium carbonate for example) if you have the possibility to inject CO2 in the reactor. But in my opinion, the best solution to reduce nutrient costs is to recycle the medium after harvesting.

Dear Behnam. If protozoa cant infect Spirulina directly It means there is another micro algae present in your culture! so the best way is to cut other micro algae! good news is the protozoa presentation will help you to cut other unwanted algae so I suggest don't try to kill them but if still you persist you can use some small dose of ammonia to kill them all too.

Hi Nasrin. you need to use the sodium bicarbonate just once and after harvest just add another nutrient except sodium bicarbonate. Also if you use 8 g/l of sodium bicarbonate and 8 g/l of sodium carbonate you will achieve better results.

In addition to the factors that Mr. Matthew has already reported, I suggest some punctual and more accurate information:

- ideal pH for most cyanobacteria is slightly alkaline (between 8.5-9.0)

- nitrogen level offered should prevent the lush phases

- if you have CO2 available, use a 0.3-0.5% enrichment

- Homogenization should be moderate for filamentous species.

I hope I was helpful.

In this assumption it is essential to take into consideration the quality of the final product. For it is well known that harvesting (and storage) influences metabolite recovery.

Thank you all for your contributions. As I'm running batch cultures, It was not possible for me to regarly add new culture medium; What i used to do is adding each day, the equivalent of distilled water assuming that the evaporated water is free of any nutrient.

Please take a look at this useful RG link.

Are you sure that you observe contamination or it could be just morphological changes?

The answer to your question is "Yes"! Please see the following RG link.

I think you can increase the power and breakdown time of sonication, Or break it with glass beads of smaller diameter. And you can replace HCl and NaOH solution with a phosphate buffer. Wish you success！

Thank you very much dear Dr. Mishra

The blue green micro-algae Spirulina platensis is an important source of nutrients. As important obstacle for the production of Spirulina is Rotifer. A method is presented for automated identification and recognition of rotifer contamination in Spirulina using image processing techniques. Some tools are employed effectively for online monitoring density of microorganism in water. This research has shown that automatic identification of contamination in algae through the processing of mobile phone images is the easiest way for improving and evaluating the growth of Spirulina for effective cultivation of algae. For details consult Indian Jounrnal of Science and Technology. Vol 8 ( 8 ), 702 -706. 2015

Dear,

we used native microalgae. Now I am using a native mcroalgal-bacteria consortium in a pilot raceway pond.

Have a nice day,

gae

Thank you Norhidayah Mohd Taufek for your excellent article attachment. Its help me a lot. I am really grateful to you.

usually 14% dry weight which is 140 mg....

Henrikson, R. (2009). Earth food Spirulina: How this remarkable blue-green algae can transform your health and our planet. Hana, Maui County, HI: Ronore Enterprises, Inc.

mine i got 13.59% dry weight.

Nima Hajinajaf thank you so much for the answer. It s a clear explanation

spirulina has more medicinal value ,i don't recommend for gasification

Dear Reham,

Are you talking about genetic transformation? If you mean it, I would recommend you to read very detailed studies on this subject. You also need to have a very good genetic laboratory to do this. In recents, electroporation is the most common method in literature I think. May be you can focus on electroporation. Good luck.

Dear Sir. Concerning your issue about the procedure for extracting tryptophan amino acid from spirulina. Tryptophan is one of 8 essential amino acids that our bodies need. The body uses tryptophan to produce serotonin, which can then used to make melatonin, our “sleep hormone.” Individual free amino acids were extracted and quantified according to the method of Park et al. (2014) with modifications. Briefly, a 100 mg portion of a fine powdered sample was mixed with 1.2 mL of 5% trichloroacetic acid (TCA) in a 2 mL Eppendorf tube and vigorously shaken for 5 min. The slurry sample was incubated at room temperature for 60 min, and the upper layer was then separated by centrifugation. The collected samples were diluted with 0.1 M HCl and then filtered through a 0.45 μm PTFE syringe filter. The filtrate was then analysed by HPLC (Agilent Technologies, Palo Alto, CA). The HPLC analyses of free amino acids were conducted according to the “rapid, accurate, sensitive, and reproducible HPLC analysis of amino acids analysis” method with Zorbax Eclipse-AAA columns using an Agilent 1100 HPLC system. Briefly, the separation of the free amino acids was performed on a Zorbax Eclipse AAA analytical column. The oven temperature of the column was set at 40°C, and the detection wavelength was set a 338 nm. The injection volume was 10 μL. The mobile phase consisted of a mixture of 40 mM NaH2PO4 (pH 7.8, solvent A), and solvent B (ACN, MeOH, and water at a 45 : 45 : 10 v/v/v ratio) was passed at a rate of 2.0 mL/min. The HPLC separation parameters were as follows: 0 min, 0% B; 0–1.9 min, 0% B; 1.9–21.1 min, 57% B; 21.1–21.6 min, 100% B; 21.6–25 min, 100% B; 25–25.1 min, 0% B; and 25.1–30 min, 0% B. A sample with an amino acid content of 50 pmoL/μL was used as the standard. The quantifications of the different amino acids were based on the peak areas and were calculated as equivalents of the standard compounds. All contents are expressed as milligrams ∗ gram/fresh weight (FW). I think the following below links may help you in your analysis:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4737012/

Thanks

In case of microalgae growth in raceway ponds Co2 is absorbed by the microalge and pH increases in this case we sparge CO2/air in the reactor to bring down the pH and control it by continuous sparging of Co2. In general pH is controlled by the flow of CO2 in raceway ponds because using buffers will be uneconomical also instead of pure CO2 air is sparged.

Maria, Did you measure medium pH before the inoculation and after its crush? What was the ratio between "old culture" used for the inoculation and new medium?

It is likely that 100 uE were too strong for the newly prepared culture. I would recommend you to use deem light (< 30 uE) for young cultures of cyanobacteria with low density. I will continue when I got your information about pH dynamics. IB

A simpler method that worked well for us was the freeze thawing method. 

Dear Diego, 

The kind of water depends on the type of study you are doing. Is it necessary to follow the trace elements concentration? Or you just want a good microalgal production? 

Spirulina medium calls for alkalinization (usually with sodium carbonate/bicarbonate), and addition of nitrate, phosphate, magnesium and micronutrients. Because the commercial-grade salts *already carry* contaminants, you will have metals such as Ca Fe, K and Mg carried to the solution by the simple addition of "premium" grade NaHCO3. So, using very pure water is uncommon. We use deionized water (for lab cultures), and tap water for larger cultures.

Spirulina is best food for boosting energy and immunity.

Hi Ariyan,

You may want to try to grow them immobilized. Using e.g. the Twin-Layer system has advantages especially for pigment extraction. 

In this system the algae are immobilized on a membrane and the bulk medium flows on the other side of it. The algae are supplied with water and nutrients, but are not suspended. Harvesting can be done by simply scraping off the biomass. It contains only a fraction of the water supplied. You can then directly go on with pigment extraction without any centrifugation or filtration. 

Another advantage is the direct exposure to light. Because the algae are not covered by a layer of water all radiation reaches the biomass (the upper cell layers). Pigment composition will differ within the biofilm. 

Have a look at the attached publication and let me know if you need additional information.

Spirulina is an aquatic micro-organism often referred to as an algae, though it more closely resembles bacteria. It is used as a food supplement to combat malnutrition. 1 gram of spirulina is said to be as nutritious as 100g of spinach or carrots, and is cheaper. It has an extremely high protein content, with 60-70% of its dry weight consisting of a balanced mix of various essential amino acids. Further it is very rich in beta carotene (to produce vitamin A), iron, vitamin B12, ganna-linolenic acid and other micronutrients. It has no cell wall and is therefore very easy to digest.

Spirulina has very high micronutrient content, is easy and cheap to produce locally. It is therefore a very realistic and also sustainable solution to the problem of malnutrition as opposed to food fortification or distribution programs. Fortification programs try to improve the quality of food by for example adding vitamin A and D were to milk and margarine. These are not always effective since this food is usually not available to the people in rural areas who suffer most.

Spirulina grows in solutions of specific minerals with the correct chemical balance and a pH of 8-11. There are various different recipes for this, depending on the budget available and the conditions. It needs a minimum of 20˚C to grow substantially, though a temperature of 35-37˚C is most effective. 

Spirulina is most nutritious in its wet form. However this lasts at most for a few days if refrigerated, and only a few hours at room temperature. Hence if it needs to be transported or stored it must be dried. If dried and packaged well it can be stored for at least a year without losing nutritional value. However if dried it acquires an unpleasant smell and taste, and is inconvenient to use. It can then also be combined with various other food products or simply packaged on its own.The production of Spirulina requires manufacturing of a tank. The size of this depends on the scale of production, and the number of tanks. 1 tank of 18m2 produces approximately 150g of Spirulina per day.

This study aimed to provide a cost effective medium to large scale production of Spirulina platensis. This intention was implemented by substituting all the nutrients present in Zarrouk’s medium (SM) with cheaper and locally available commercial fertilizers and chemicals. The Reduced Cost medium contained single super phosphate (SSP), commercial sodium bicarbonate, Muriate of potash (MOP) and crude sea-salt, (Syahat salt). Four grades of nitrogen concentrations representing 10%, 20%, 30% and 40% of SM nitrogen concentration (29.42 mM-N) were taken from ammonium nitrate (Treatments 1–4) or urea (Treatments 5–8) respectively, for testing. The alga was grown for 33 days at 30 ± 2 °C, pH 9, 30 μEm2 s−1 irradiance. The growth characteristics (maximum biomass Xm, cell productivity Px, specific growth rate μm and chlorophyll concentration), and biochemical composition (proteins, carbohydrates and lipids) of the alga grown in these media were compared with that cultivated in SM. Significant differences in the growth parameters and biochemical composition were observed for the different nitrogen sources and concentrations. The results revealed that S. platensis could utilize ammonium nitrate most efficiently and that growth was enhanced with increasing the concentrations of ammonium nitrate giving maximum biomass at 0.353 g/L (Treatment 3). Further increasing the concentration limited growth. The growth parameters in urea showed a significant decrease associated with increasing urea concentrations. The maximum biomass, chlorophyll and protein yield (0.813 ± 0.018 mg/L, 0.0685 ± 0.0024 μg/L and 52.62%, respectively) were recorded using Treatment 3 which was comparable with that of SM (0.840 ± 0.008 mg/L, 0.0701 ± 0.0089 μg/L and 52.95%, respectively). The results indicated that the newly prepared medium can be used profitably for large-scale mass production of protein-rich Spirulina and yields similar performance with cost effective to Zarrouk’s medium. Source ; The Egyptian Journal of Aquatic Research, Volume 38, Issue 1, 2012, Pages 51-57

Kindly see the following link:

Review this information;

ALGAL CELLS DISRUPTION

I have analyzed the purple solution and this is the UV spectrum chart.

thank you all, very much.

Initially you have to fix the sample using any biological fixatives like formaldehyde, alcohol, gluteraldehyde etc. you can go for gluteraldehyde because it preserves the fine structure of the sample down to macromolecular resolution. if you want to cross link the lipids also use osmium tetroxide but its costly. 

wash the algal sample and fix in a solution of gluteraldehyde in phosphate buffer for 24 hrs. again wash in phosphate buffer and rinse with distilled water. dehydrate in 100 % ethanol. critical dry the sample and mount on the stub covered with carbon stub with a neat coating of aluminium foil. the sample is ready for SEM observation.

Spirulina is a blue green alga which contains the pigments C phycocyanin (blue in colour) and C pycoerythrin (red in colour). Since the C phycocyanin dominate over C phycoerythrin in blue green algae hence they appear blue green in colour. However, under certain circumstances like change in wave length of light there is increased synthesis of C pycoerythrin imparting red colour to blue green algae. 

Please check the following link.

Dear, friend

can follow this method for  fatty acid (One-step method for quantitative and qualitative analysis of fatty acids in marine animal samples by Sakdullah Abdulkadira, , , , Makoto Tsuchiyaa.  Journal of Experimental Marine Biology and Ecology, Volume 354, Issue 1, 4 January 2008, Pages 1–8.

I think you need to consider the pH of the media...Spirulina likes to grow at a culture medium with a basic pH (pH 10 or 11). 

Regular swirling or continuous mixing at slow speed  of culture medium in Spirulina culture tanks will help in reducing the bubble formation.

Hi Anna,

I found this in a pdf called GROW YOUR OWN SPIRULINA by Jean-Paul JOURDAN

"The amount of gas, sugar or bicarbonate to be fed is adjusted so as to control the pH at around 10.4. A pH lower than 10.2 may cause an overproduction of undesirable, but not dangerous, exopolysacharide (EPS). A good practical dose of carbon feed is the equivalent of 40 % of the spirulina produced (i.e. about 0.8 kg of CO2 or 0.4 kg of sugar per kg of dry spirulina harvested). Sugar may induce some degradation of the culture medium, so actually it is recommended to feed less than 0.3 kg/kg, and as regularly as possible."

Are there separate batches of media prepared for the culture?  Are the downstream unit operations done continuously as well?

A good guidance to follow is OECD Test No 201 (http://www.oecd-ilibrary.org/environment/test-no-201-alga-growth-inhibition-test_9789264069923-en)