The authors gratefully acknowledge the partial funding from the TecNM under the Call for Scientific Research, Technological Development, and Innovation (16898.23-P) for the Institutos Tecnologicos Federales. They also appreciate the support from the Instituto de Ciencia, Tecnolog&#237;a e Innovaci&#243;n del Estado de Michoac&#225;n de Ocampo (FCCHTI23_ME-4.1.-0001).

1. Culture media preparation and inoculum preparation
<ol>
	<li>Prepare 1 L of 3N-BBM+ V(CCAP) growth medium (Macronutrients: NaNO3 [0.75 g L-1], CaCl2 [0.019 g L-1], MgSO4 [0.019 g L-1], K2HPO4 [0.057 g L-1], NaCl [0.025 g L-1], KH2PO4 [0.175 g L-1]; micronutrients: Na2 EDTA [0.0186 g L-1], FeCl3 [0.0024 g L-1], MnCl<...

Enhancing Chlorophyll Production in Microalgae via Growth Factor Optimization

<ol>
	<li>Khan, M. I., Shin, J. H., Kim, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promising+future+of+microalgae:+Current+status,+challenges,+and+optimization+of+a+sustainable+and+renewable+industry+for+biofuels,+feed,+and+other+products.">The promising future of microalgae: Current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products.</a> Microb Cell Fact. 17 (1), 36 (2018).</li><li>Otero-Paternina, A. S. M., Cruz-Casallas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+hydrocarbon+phenanthrene+on+Chlorella+vulgaris+(Chlorellaceae)+growth.">Effect of the hydrocarbon phenanthrene on Chlorella vulgaris (Chlorellaceae) growth.</a> Acta Biol Col. 18 (1), 87-98 (2013).</li><li>Pancha, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salinity-induced+oxidative+stress+enhanced+the+biofuel+production+potential+of+microalgae+Scenedesmus+sp.+CCNM+1077.">Salinity-induced oxidative stress enhanced the biofuel production potential of microalgae Scenedesmus sp. CCNM 1077.</a> Bioresour Techno. 189, 341-348 (2015).</li><li>Ortiz, M. L., Cort&#233;s, C. E., S&#225;nchez, J., Padilla, J., Otero, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+microalgae+Chlorella+sorokiniana+growth+in+different+culture+mediums+in+autotrophic+and+mixotrophic+conditions.">Evaluating microalgae Chlorella sorokiniana growth in different culture mediums in autotrophic and mixotrophic conditions.</a> Orinoquia. 16 (1), 11-20 (2012).</li><li>Camacho Kurmen, J. E., Gonz&#225;lez, G., Klotz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astaxanthin+production+in+Haematococcus+pluvialis+under+different+stress+conditions.">Astaxanthin production in Haematococcus pluvialis under different stress conditions.</a> Nova. 11 (19), 94-104 (2013).</li><li>. Production of chlorophyll and astaxanthin from Haematococcus pluvialis under nitrogen deficiency stress in a 5-liter Biostat Aplus bioreactor Available from: <a target="_blank" href="https://repositorio.unicolmayor.edu.co/handle/unicolmayor/2735">https://repositorio.unicolmayor.edu.co/handle/unicolmayor/2735</a> (2019)</li><li>Bischoff, H. W., Bold, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phycological+studies+IV.+Some+soil+algae+from+Enchanted+Rock+and+related+algal+species.">Phycological studies IV. Some soil algae from Enchanted Rock and related algal species.</a> University of Texas. 6318, 95 (1963).</li><li>Cerezo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LED+characterization+through+the+use+and+programming+of+a+spectrometer.">LED characterization through the use and programming of a spectrometer.</a> Universidad Polit&#233;cnica de. 22323, (2013).</li><li>Couso, I., Vila, M., Vigara, J., Cordero, B. F., Vargas, M. &. #. 1. 9. 3. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+carotenoids+and+regulation+of+the+carotenoid+biosynthesis+pathway+in+response+to+high+light+stress+in+the+unicellular+microalga+Chlamydomonas+reinhardtii.">Synthesis of carotenoids and regulation of the carotenoid biosynthesis pathway in response to high light stress in the unicellular microalga Chlamydomonas reinhardtii.</a> Eur J Phycol. 47 (3), 223-232 (2012).</li><li>Vera-L&#243;pez, F., Mart&#237;nez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pigments+in+microalgae:+Functions,+applications,+and+overproduction+techniques.">Pigments in microalgae: Functions, applications, and overproduction techniques.</a> BioTechnology. 25 (5), 35-51 (2021).</li></ol>

The authors have nothing to disclose.

The comparative study between H. pluvialis and C. sorokiniana revealed significant differences in chlorophyll production dynamics. While H. pluvialis exhibited a decrease in chlorophyll concentration throughout the experiment, C. sorokiniana showed a steady increase. Additionally, there was initially a lower proportion of chlorophyll a in both species, but this ratio reversed in particular growth conditions, which may give indications of an induction of the production of said ...

In recent years, the growing environmental problems caused by anthropogenic activities and their adverse effects on the health and balance of ecosystems have driven the search for more efficient and environmentally friendly production systems. This has accelerated processes in industries and fostered the implementation of bioremediation treatments and the development of bio compounds to mitigate these harmful effects1.
This context has led to a significant growth in the study of microalgae, driven by the need to find innovative solutions to current environmental and economic challenges. Microalgae thrive in aquatic environments, using sunlight and carbon dioxide as sources of energy and carbon, respectively. This characteristic makes them a sustainable and promising alternative for producing various valuable products. Research in this field has focused on understanding the physiology and metabolism of these cells, as well as developing efficient technologies for their cultivation and processing2.
There is a clear need to develop accessible and reliable tools for studying microalgae to accelerate research processes and deepen the understanding of their physiology, metabolism, and potential applications. These tools should enable rapid analysis of the effect of environmental and production factors on key parameters, such as chlorophyll concentration, which is a fundamental indicator of the health and development of these organisms. A decrease in chlorophyll content may indicate environmental stress, nutritional deficiencies, or diseases.
These green pigments play a crucial role in photosynthesis, capturing sunlight energy to convert carbon dioxide and water into glucose and oxygen and constitute between 0.5% and 5% of the biomass of microalgae3. Beyond their essential role in sustaining life processes, chlorophylls have found applications in various industries. Chlorophyll extracts are utilized in food and beverage processing as natural colorants, imparting vibrant green hues to products while also providing antioxidant properties. Moreover, chlorophyll-based supplements are gaining popularity in the health and wellness sector due to their purported detoxifying and anti-inflammatory effects. By harnessing the multifaceted properties of chlorophyll, industries can develop innovative products that contribute to both visual appeal and consumer well-being1.
In this regard, one microalgae of great biotechnological relevance is C. sorokiniana. This microorganism stands out for its rapid growth rate, making it highly efficient in biomass production. Additionally, C. sorokiniana exhibits a diverse content of highly nutritional compounds, including proteins, lipids, and vitamins, which renders it valuable for various applications in food, feed, and biofuel production2. Moreover, this microalgae species has been found to produce extracellular enzymes with diverse functions, opening up possibilities for biotechnological applications such as wastewater treatment, bioremediation, and pharmaceuticals4. In addition to its rapid growth and versatile applications, C. sorokiniana also demonstrates significant potential for chlorophyll production. As a photosynthetic microorganism, C. sorokiniana possesses the machinery necessary for the synthesis of chlorophyll, which constitutes between 0.5% and 5% of the biomass of microalgae3. This capability makes C. sorokiniana an attractive candidate for commercial-scale chlorophyll production and promises to be a sustainable solution to urgent environmental and nutritional challenges5.
On the other hand, another microalgae species of significant interest is H. pluvialis. This microalga is renowned for its production of astaxanthin, a potent antioxidant pigment with numerous industrial applications. Astaxanthin serves as a protective mechanism for H. pluvialis photosystem, shielding it from oxidative stress induced by environmental factors. This pigment is highly sought after in cosmetics, nutraceuticals, and aquaculture industries, owing to its antioxidant properties and potential health benefits. With its abundant ability to produce astaxanthin, H. pluvialis presents a promising avenue for developing innovative products catering to various industrial and consumer needs6.
Recent studies have also highlighted H. pluvialis1 potential for chlorophyll production, further enhancing its industrial significance. For instance, a study investigated the chlorophyll content of H. pluvialis under varying growth conditions and found that under optimal cultivation parameters, H. pluvialis exhibited remarkable chlorophyll production rates, surpassing those of other microalgae species7. This finding underscores the potential of H. pluvialis as a sustainable source of chlorophyll, offering novel opportunities for its utilization in various industrial sectors.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>C3H6O</td>
			<td>Meyer</td>
			<td>67-64-1</td>
			<td>Acetone 90%</td>
		</tr>
		<tr>
			<td>15 mL tube</td>
			<td>Biologix</td>
			<td>10-9502</td>
			<td>Test tube</td>
		</tr>
		<tr>
			<td>2510-DTH</td>
			<td>Branson</td>
			<td>D-73595</td>
			<td>Sonicator</td>
		</tr>
		<tr>
			<td>5 mL screw cap test tube</td>
			<td>Kimax</td>
			<td>45066-13100</td>
			<td>Test tube</td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>Biologix</td>
			<td>10-9151</td>
			<td>Test tube</td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Reynolds</td>
			<td>611 standard, 12&#34; x 1000 feet</td>
			<td>Test tube cover&#160;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Meyer</td>
			<td>0925-250</td>
			<td>Calcium Chloride</td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Dynamica</td>
			<td>14 R</td>
			<td>Centrifuge Refrigerated</td>
		</tr>
		<tr>
			<td>CoCl2</td>
			<td>Merck</td>
			<td>1057-100</td>
			<td>Cobalt dichloride</td>
		</tr>
		<tr>
			<td>FeCl3</td>
			<td>Merck</td>
			<td>157740</td>
			<td>Iron(III) Chloride</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Meyer</td>
			<td>2051-250</td>
			<td>Dipotassium Phosphate</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Meyer</td>
			<td>2055-250</td>
			<td>Monopotassium Phosphate</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Meyer</td>
			<td>1605-250</td>
			<td>Magnesium Sulphate</td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>LabNet</td>
			<td>Model Beta-Pette</td>
			<td>Micropipette</td>
		</tr>
		<tr>
			<td>MnCl2</td>
			<td>Merck</td>
			<td>429449</td>
			<td>Manganese(II) Chloride&#160;</td>
		</tr>
		<tr>
			<td>Na2 EDTA&#160;</td>
			<td>Merck</td>
			<td>200-449-4</td>
			<td>Edatamil, Edetato Disodium Salt Dihydrate</td>
		</tr>
		<tr>
			<td>Na2MoO4</td>
			<td>Merck</td>
			<td>243655</td>
			<td>Sodium Molybdate</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Meyer</td>
			<td>2365-500</td>
			<td>Sodium Chloride</td>
		</tr>
		<tr>
			<td>NaNO3</td>
			<td>Meyer</td>
			<td>2465-250</td>
			<td>Sodium Nitrate</td>
		</tr>
		<tr>
			<td>RGB LED stripe</td>
			<td>Steren</td>
			<td>GAD-LED2</td>
			<td>Light source</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>PerkinElmer</td>
			<td>Model Lambda35</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr>
			<td>spectroradiometer</td>
			<td>Gigahertz-Optik</td>
			<td>model BTS256</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Scientific Industries</td>
			<td>Vortex-Genie&#174; 2</td>
			<td>Vortex</td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>Merck</td>
			<td>208086</td>
			<td>Zinc Chloride</td>
		</tr>
	</tbody>
</table>

evaluation of the effect of growth factors on chlorophylls a and b production from microalgae

Microalgae contain two main groups of pigments: chlorophylls and carotenoids. Chlorophyll is a green pigment that absorbs light energy and transforms it into chemical energy to facilitate the synthesis of organic compounds. This pigment serves as a valuable primary source for biotechnological input products in the food, pharmaceutical, and cosmetic industries due to its high antioxidant properties and coloring capabilities. The objective of this research was to evaluate the effect of growth factors (CO2 concentration, light color, and light intensity) through a Taguchi L4 experimental design on cell growth and the cellular content of chlorophyll a and b in Chlorella sorokiniana, followed by validation of the method using Haematococcus pluvialis microalgae as an additional study model. Cell growth was quantified using the optical density spectrophotometric technique at a wavelength of 550 nm. For the quantification of chlorophylls, a cell extract was obtained using a 90% pure acetone solution, and subsequently, the concentrations of chlorophylls a and b were quantified using spectrophotometric techniques at wavelengths of 647 nm and 664 nm, according to the method described by Jeffrey and Humphrey. The experimental results indicated that controlling conditions of low CO2 addition, purple light, and low light intensity increases both cell growth and the concentration of chlorophylls a and b within the cells. The implementation of this chlorophyll quantification method allows for quick, simple, and precise determination of chlorophyll content, as the wavelengths used are at the absorbance peaks of both types of chlorophylls, making this technique easily reproducible for any microalgae under study.

Author Spotlight: Optimizing Growth Factors for Production of Biotechnologically Relevant Secondary Metabolites

Evaluation of the Effect of Growth Factors on Chlorophylls a and b Production from Microalgae

Our project evaluates how growth factor control affects the production of biotechnologically-relevant secondary metabolites such as chlorophyll from microalgae such as Chlorella sorokiniana and Haematococcus pluvialis at different scales. We have focused on establishing an analytical method to quantify chlorophylls in microalgae. Some emerging technologies for advancement in this are the modeling and optimization of processes using computational technique.However, in order to carry them out, it is necessary to obtain experimental information on the processes. The growth factors evaluated in Chlorella sorokiniana that have the greatest effect on chlorophyll production are the light color and the carbon dioxide supply flow. The changing light intensity does not significantly influence the production of this metabolite.The findings in this field of study can be advanced by implementing growth factors that induce microalgae to increase the production of secondary metabolites in conjunction with rapid, simple, and reliable analysis techniques. Our work provide those with guidelines to investigate and understand the biochemical and the energetic processes carried out by microalgae with different substrates, evaluating the effect of environmental variables to optimize the production of your mass and cellular compounds for various applications.

To observe the efficiency of the technique detecting variations in chlorophyll cellular concentration and evaluate the effect of growth factors in C. sorokiniana, a Taguchi L4 experimental design was established, evaluating CO2 volume addition, light color, and light intensity. Each factor was assessed at low and high levels, as shown in Table 1, under the conditions defined by the experimental design in Table 2.
Once the experim...

The present study highlights the advantages of employing the method developed by Jeffrey and Humphrey for extracting and quantifying fat-soluble pigments from microalgae. This method serves as a valuable tool for assessing the influence of growth factors on chlorophyll production and cellular content in these organisms.

Microalgae contain two main groups of pigments: chlorophylls and carotenoids. Chlorophyll is a green pigment that absorbs light energy and transforms it ...

evaluation-effect-growth-factors-on-chlorophylls-b-production-from

Research

JoVE Journal

Biochemistry

554 Views.  National Institute of Technology of Mexico. Our project evaluates how growth factor control affects the production of biotechnologically-relevant secondary metabolites such as chlorophyll from microalgae such as Chlorella sorokiniana and Haematococcus pluvialis at different scales. We have focused on establishing an analytical method to quantify chlorophylls in microalgae. Some emerging technologies for advancement in this are the modeling and optimization of processes using computational technique.However, in order to carry the...