Factors influencing the growth of Spirulina platensis in closed photobioreactors under CO 2

Abstract

Since there is growing interest throughout the world in photosynthetic microbes as a potential source of food or food supplements, an assessment of factors which influence the biomass obtained in bioreactors, protein contents and constituents is important. This work reviews the autotrophic cultivation conditions of Spirulina platensis especially the dependency on the strain, the composition of the nutrient solution, pH, temperature of the medium, light intensity and color as well as exposure rhythm, the flow rate and composition of the aerating gas mixture and the bubble size, the content of oxygen, CO₂ and HCO₃ in the medium and last but not least from the optical density of the spirulina suspension during growth.

Keywords

Spirulina platensis influencing factors temperature light intensity wave length

1 Introduction

Algae such as Spirulina platensis have been part of human food for centuries. Because spirulina algae thrive undemandingly in salt and brackish water, they are also considered an opportunity in the fight against hunger. On 1 January 2019, the world population comprised 7.67 billion people [1]. For the period 2015 to 2020 a population growth of about 80 million people per year is expected, so that the United Nations expects about 9.7 billion people in 2050 [1]. One of the greatest global challenges associated with this is to sustainably supply a growing world population with food in times of climate change. This situation is aggravated by the overfishing of the oceans. The drastic collapse of cod stocks of Newfoundland since 1992 has not been able to recover, despite an absolute ban on fishing and rearing measures. The ecological balance has obviously already shifted permanently [2]. The situation is similar with cod stocks in the North Sea and the Baltic.

A previously largely neglected option for the production of proteins as a renewable resource is the photoautotrophic cultivation of microalgae, which can take place in water bodies or bioreactors without competing with agricultural land. Microalgae such as Spirulina platensis enable products with unique properties, which have been on the market for a long time in the form of high-quality products for food and feed supplementation, but which are also of great interest for pharmacy and agriculture [3].

Spirulina is a multicellular, filamentous cyanobacterium. On microscopic observation it appears as blue-green filaments composed of cylindrical cells arranged in unbranched trichomes characterized by helical shape (Fig. 1).

Fig.1

Typical form of spirulina platensis.

Spirulina is considered the “food of the future” because it contains over fifty healthy nutrients, including vitamins, minerals and amino acids. Compared to other foods or by weight, spirulina is considered one of the most nutritious foods in the world: rich in proteins containing all essential amino acids, including B vitamins, iron, magnesium, potassium and many other vitamins and minerals, as well as antioxidants. Spirulina has a high protein content (55–70 percent of dry weight), which is more than other common plant sources such as dried soybeans (35 percent), peanuts (25 percent) or cereals (8–10 percent) [4]. Spirulina was therefore declared the best food of the future by the United Nations World Food Conference already in 1974. The World Health Organization described spirulina as one of the best super foods in the world and NASA considers it an excellent compact food for space travel, as small amounts can provide a wide range of nutrients [5]. Spirulina has the advantage over other microalgae that, as a bacterium, it does not have a cell wall of cellulose, so no chemical or physical processing is required to be digested [6].

Algae formed on Earth over three billion years ago and produced the first oxygen on our planet. The photosynthesis potential is so great that the Japan Aerospace Exploration Agency (JAXA) uses them in an air purification system in the space station. Spirulina platensis is capable of reducing greenhouse gases such as CO₂ and thereby releasing O₂, which, however, requires specific process control.

Despite many publications on spirulina (a literature search under the search word “Spirulina” resulted in 1,820 publications in the database “Pubmed"), many questions remain unanswered. However, various factors are discussed which influence the biomass obtained, the constituents, the protein content, etc. The results of the study are not yet conclusive:

the strain of Spirulina platensis

composition of the nutrient solution

pH value of the medium

temperature of the medium

light intensity and light color

exposure rhythm

flow rate and composition of the aerating gas mixture (CO₂-air mixture)

bubble size of the aerating gas mixture

O₂, CO₂ and ${HCO}_{3}^{-}$ content of the medium

optical density of spirulina suspension during growth

1) Selection of the strain Spirulina platensis

In the literature there is no comprehensive comparative assessment of the biomass productivity of the different known Spirulina platensis strains. The Göttingen algae collection offers Spirulina platensis cultures from 3 different regions (Namibia, Peru and Lake Chad), which are all listed under the taxonomical name “Nordstaedt Gomont”. Also, the “American Type Culture Collection” (ATCC) offers the Spirulina platensis type “Nordstaedt”. A study with Spirulina platensis strains collected in different regions in China showed that they differed in comparative genome analyses [7]. Another study investigated biomass productivity for three Spirulina platensis strains from different regions of India under identical conditions. Significant differences in yield were found, the lowest concentration after 30 days was 0.217 g/l, the highest concentration 0.362 g/l [8]. Since no genetic data are available for many of the published Spirulina platensis studies, it is unclear whether the different yields, protein and ingredient contents were caused by different strains.

2) Composition of the nutrient solution

The cultivation medium has a great impact on the productivity of biomass and other compounds of interest. Zarrouk’s medium, Spirul, Conway-, BG11-, Hiri’s-, Jourdan’s - as well as the F/2 medium or sea water with different additives are reported as nutrient solution for Spirulina platensis [9 –12]. Mostly, Zarrouk’s medium or slightly modified or diluted Zarrouk’s medium is used [9]. It was noticeable that a dilution of the Zarrouk’s medium by a factor of 5 did not result in a significant decrease of the biomass yield (at least as long as the nutrients were available in sufficient quantities) [9]. Later (after day 15), however, there was less growth compared to the full medium. The composition of the different media is quite comparable, which results from the requirements of the Spirulina bacteria. Media with reduced cost can be as effective as Zarrouk’s medium in terms of final biomass concentration, chlorophyll and protein content [13].

Spirulina require a pH value of approx. 9–9.5 for optimal growth, which can be adjusted by adding carbonate-based buffer substances. In addition, nitrates and phosphates as well as sulphates are required. For example, nitrogen concentration in the medium [14] (optimum at 2.5 g/L) and also nitrogen source (urea better than ammonium or nitrate) [15] has a great effect on Spirulina productivity. Additionally, a phosphate concentration of 250 mg/L in the form of K₂HPO₄ was found to optimize biomass production [16]. These can be found in various concentrations in all nutrient solutions.

3) pH-value of the medium

There is broad agreement in the literature regarding the pH value of the medium in the photobioreactor. The desired pH range is between 9 and 10 [17, 18]. Belkin et al. indicate a larger range. They described a good growth up to a pH value of 11.5, while the Spirulina bacteria stopped the growth at a pH value of 7. The adjustment of the pH is very different [19]. Some working groups adjust the pH value via added acids and bases [17, 20], while others control this via the supply of the CO₂-air mixture, since the added CO₂ in aqueous solutions leads to a reduction of the pH value due to the carbon dioxide dissociation equilibrium [17, 21]. Since spirulina consumes CO₂ from the medium when exposed to light (see Equation 1 [22]), $12 H_{2} O + 6 {CO}_{2} \to C_{6} H_{12} O_{6} + 6 O_{2} ↑ + 6 H_{2} O$ (1) the pH value increases with consumption. This allows the pH value to be controlled by the targeted addition of CO₂ (see Fig. 2).

Fig.2

pH value control by adding CO₂ to the photobioreactor (taken from [17]).

Troschl et al. operated a photobioreactor with a volume of 200 l for the production of bioplastics from power plant exhaust gases. A total of 118 g or 59 l pure CO₂ was used for the regulation of the pH value shown in Fig. 2 [21]. A pH value of at least 9.5 should also be maintained to prevent contamination with other - possibly toxic - cultures.

4) Temperature of the medium

The data in the literature differ with regard to the operating temperature (see Fig. 3). Here one can find data for the optimum process temperature between 25 and 38°C [18 , 24]. At higher temperatures the biomass yield seems to decrease, as described for lower temperatures [25]. However, Spirulina platensis and Spirulina maxima can tolerate temperatures as high as 40°C for a few hours without appreciable adverse effects [26]. Spirulina cultures survived temperatures of up to 60°C in total.

Fig.3

Growth dependence (chlorophyll content was measured) of Spirulina platensis on temperature [20].

Vonshak showed that the maximum growth rate for three different spirulina strains differed significantly depending on temperature [24]. While the maximum growth rate for DA was 30–32 degrees C, it was 40–42 degrees C for strain EY-5. He considers temperature to be the most important factor influencing spirulina growth and therefore prefers a strain that - like SPL-2 - has a wide maximum between 25 and 40 degrees C (which makes it easier to control the reactor temperature, especially in outdoor applications).

5) Light intensity and light frequency

In the literature there are some studies on the light intensity at which relative maximum yields of spirulina were found. However, the data differ considerably in some cases. These differences may occur due to different light sources with different emission spectra, different photobioreactor designs and different concentrations of the spirulina cultures and the spirulina strains themselves. In addition, light transmission and reflection behavior of the photobioreactors can have an influence. The literature contains information on optimal light intensity between 1200 lux and 162000 lux (conversion according to http://www.egc.com/useful_info_lighting.php, [27 –31]).

Kumar et al. demonstrated with Spirulina platensis that the biomass productivity at 2000 lux was at a higher level than at 3500 lux, but that at 3500 lux the carotenoid content was increased [25].

However, Pandey et al. showed that for Spirulina platensis a light density of 5000 lux or more showed an optimum in biomass productivity [20].

These different results of the listed investigations show that in order to ensure an optimal yield, the light intensity should be continuously adjusted to the respective optical density and the bioreactor. It must be ensured that excessive illumination is excluded or at least minimized in order to minimize photolysis/photoinhibition [27].

The rate of photosynthesis depends on the rate of absorbed light energy. For very low light intensities, spirulina has a net consumption of O₂ or a production of CO₂ according to Equation 1 corresponding to respiration. With increasing light intensity, photosynthetic production increases so that at a certain compensation light intensity, Ic, production compensates for respiration and net positive photosynthesis is achieved. As a rule, production (P) increases linearly with light intensity (I) up to a certain value. Typically, the curve gradually flattens out and finally levels off at the maximum photosynthetic rate Pmax. Extrapolating the linear part of the curve gives the start of saturation of the irradiance Is. With increasing irradiance, the photoinhibition starts and the production (P) starts to decrease. The light intensity at which this decrease occurs is called inhibition threshold Ib. As the light intensity continues to increase, photoinhibition begins and the biomass yield decreases.

In addition to the light intensity, the emission spectrum of the light source used also plays an important role both for the ingredients and for the yield. Hua-Bing Chen et al. found the highest content of chlorophyll in lighting with yellow light, and the highest content of phycocyanin in lighting with blue light [29].

Chih-Yu Wang achieved the highest biomass productivity when illuminated with red light, while a significantly lower yield was achieved when illuminated with blue light [30].

Wen-qing Shi et al. investigated the biomass productivity of spirulina under different spectra and found that a mixture of red and blue has a higher efficiency than red alone, which in turn has a higher efficiency than blue alone [23].

6) Exposure rhythm

Exposure times differ considerably in the literature. Usually cycles of 12 to 12 hours, but also 18 to 6, 20 to 4 and up to 24 to 0 hours of illumination per day are given [10 , 33]. Comparative studies on productivity and energy balance are not yet available.

7) Flow rate and composition of aerating gas mixture

A gas flow through the photobioreactor is used for two reasons:

i) to achieve a gentle circulation of the liquid in the bioreactor and thus to avoid photoinhibition of the side of the photobioreactor close to the edge and facing the light sources, and

(ii) remove the oxygen formed during photosynthesis from the algae solution and thereby prevent oxidative damage to the culture.

When CO₂ consumption is targeted, the gas stream is also used to supply the nutrient solution with fresh CO₂ again. In terms of design, the residence time of the bubbles in the medium can be considerably extended by e.g. countercurrent flow and thus bring more CO₂ into solution. Often a volume flow between 500 and 50,000 ml/min is provided [34]. In addition to the volume flow itself, the CO₂ content of the gas flowing through also determines the pCO₂ in the culture medium. Kim et al. showed that a CO₂ content of the gas flow of 3% led to a higher biomass yield than a content of 6% [34].

8) Bubble size of the aerating gas mixture

In addition to the size of the volume flow and the gas composition, the bubble size for the pCO₂ in the culture medium also plays a role [35]. The smaller the bubble size, the larger the exchange area for the transition of CO₂ molecules into the solution. This means that more CO₂ molecules are available for the spirulina bacteria. The gas flow with the smaller bubbles (100 μm diameter) led already on the third day to a growth rate twice as high as with the larger bubble diameters (5000 μm) [35].

9) O₂, CO₂ and ${HCO}_{3}^{-}$ content of the medium

In order to avoid the release of CO₂ into the atmosphere, a closed system must be used. The gas stream that has flowed through the culture medium is returned to the culture medium through a hose. The CO₂ absorbed by the spirulina bacteria from the gas stream must be replaced regularly to ensure maximum biomass yield. The CO₂ content of the gas volume flow is continuously measured in the air volume of the bioreactor and kept constant at a specified nominal value. With the consumption of CO₂, the oxygen content in the culture medium increases accordingly through photosynthesis. Without appropriate process control (control of exposure intensity and gas volume flow), the oxygen content in the culture medium can rise to values that lead to oxidative damage to the bacteria and thus to a decrease in biomass. To avoid this, exposure intensity and gas volume flow must be adjusted to the respective optical density and thus to the concentration of spirulina bacteria during the entire growth phase. A reduction of the oxygen partial pressure in the air volume of the bioreactor can be controlled e.g. by using oxygen-permeable membranes in combination with the addition of CO₂.

In the aqueous culture medium, ${HCO}_{3}^{-}$ is formed from CO₂ and H₂O passed through, depending on the dissociation equilibrium, which in turn depends on the pH value. The process of conversion to ${HCO}_{3}^{-}$ is very slow. However, with the pH value and the CO₂ concentration in the culture medium kept constant, a constant HCO₃^- concentration should gradually develop. ${HCO}_{3}^{-}$ is only absorbed by spirulina bacteria when CO₂ is depleted [36].

10) Optical density of the spirulina suspension during growth

The optical density of the culture medium correlates linearly with the concentration (including the dry matter harvested) of the spirulina bacteria. As the number of spirulina bacteria per unit volume increases, the optical density increases (see Fig. 4).

Spirulina bacteria stop photosynthesis in the dark. Therefore, the growth curve (Fig. 5) becomes saturated at a certain concentration or optical density.

Fig.4

Typical association between biomass production and light intensity (P-I curve) (taken from [32]).

Fig.5

Relationship between the dry matter harvested per liter of spirulina culture and the optical density (taken from [37]).

It is therefore common to stop the culture at the latest when the saturation phase of the growth curve is reached and to harvest spirulina. Two strategies are pursued here: firstly, harvesting the entire contents of the photobioreactor (batch harvest (Fig. 6)) and secondly, semi-continuous harvesting with replacement of the removed volume by nutrient medium at the time of saturation (as can be seen in Fig. 7).

Fig.6

Optical density in relation to the biomass concentration (taken from [9]).

Fig.7

Biomass yield during semi-continuous harvesting (taken from [38]).

2 Conclusion

A closed system is mostly used to obtain spirulina as a source of protein or as a food supplement for humans in order to avoid contamination. The key factors for the growth of Spirulina platensis are i) sufficient light intensity, ii) a pH of the culture medium around 9.5, iii) a temperature of 30°C to 35°C and iv) sufficient availability of all required nutrients.

A constant temperature should be ensured by a control system, either from the outside or by a heat exchanger from the inside. The amount of irradiated light should be dynamically adjusted to the optical density of the increasing concentration of spirulina bacteria in the cultivation medium, respectively, to ensure optimum growth while avoiding photoinhibition. This should also be controlled by measuring the optical density during the cultivation period.

The regulation of the pH value is closely linked to the CO₂ concentration in the medium (and in the gas volume flow as well as through the bubble size), via feedback of the pH value itself with the production of ${HCO}_{3}^{-}$ , the oxygen concentration in the medium and the gas volume and the light intensity. These values have to be measured continuously and compared in the process computer in such a way that the pH value fluctuates around the nominal value.

Conflict of interest

The authors have no conflict of interest to report.

Footnotes

Acknowledgments

The authors have no acknowledgments.

References

https://www.br.de/themen/wissen/weltbevoelkerung-bevoelkerungswachstum-menschen-erde-welt-100.html.

Boudreau

, Shackell

, Carson

, den Heyer

. Connectivity, persistence, and loss of high abundance areas of a recovering marine fish population in the Northwest Atlantic Ocean. Ecol Evol. 2017;7(22):9739–49.

Jung

, Krüger-Genge

, Waldeck

, Küpper

J-H

. Spirulina platensis, a super food? J Cell Biotechnol. 2019;5:43–54.

Habib

MAB

, Parvin

, Huntington

TCH

, Hasan

. A review on culture, production and use of spirulina as food for humans and feeds for domestic animals and fish. Food and Agriculture Organization of The United Nations. Retrieved November 20, 2008, Circular 1034.

Khan

, Bhadouria

, Bisen

. Nutritional and therapeutic potential of Spirulina. Curr Pharm Biotechnol. 2005;6:373–9.

Dillon

, Phuc

, Dubacq

. Nutritional value of the alga Spirulina. World Rev Nutr Diet. 1995;77:32–46.

, Qin

, Hu

, Song

, Yin

, Lee

, Dong

, Zhao

, Yang

, Bao

. Hole genomic DNA sequencing and comparative genomic analysis of Arthrospira platensis: High genome plasticity and genetic diversity. DNA Research. 2016;23:325–38.

Devanathan

, Ramanathan

. Utilization of seawater as a medium for mass production of spirulina platensis – a novel approach. International Journal of Recent Scientific Research. 2013;4:597–602.

Delrue

, Alaux

, Moudjaoui

, Gaignard

, Fleury

, Perilhou

, Richaud

, Petitjean

, Sassi

J-F

. Optimization of Arthrospira platensis (Spirulina) Growth: From Laboratory Scale to Pilot Scale. Fermentation. 2017;3:59.

10.

Dineshkumar

, Narendran

, Sampathkumar

. Cultivation of Spirulina platensis in different selective media. Indian Journal of Geo Marine Sciences. 2016;45:1749–54.

11.

Rajasekaran

, Ajeesh

CPM

, Balaji

, Shalini

, Siva

, Das

, Fulzele

, Kalaivani

. Effect of modified zarrouk’s medium on growth of different spirulina strains. Agriculture Technology and Biological Sciences. 2015;13(1):67–75.

12.

Gami

, Naik

, Patel

. Cultivation of spirulina species in different liquid media. J Algal Biomass Utln. 2011;2:15–26.

13.

Madkour

, El-Wahab Kamil

, Nasr

. Production and nutritive value of Spirulina platensis in reduced cost media. Egypt J Aquat Res. 2012;38:51–7.

14.

Çelekli

, Yavuzatmaca

. Predictive modeling of biomass production by Spirulina platensis as function of nitrate and NaCl concentrations. Bioresour Technol. 2009;100:1857–1.

15.

Soletto

, Binaghi

, Lodi

, Carvalho

JCM

, Converti

. Batch and fed-batch cultivations of Spirulina platensis using ammonium sulphate and urea as nitrogen sources. Aquaculture. 2005;243:217–24.

16.

Markou

, Chatzipavlidis

, Georgakakis

. Effects of phosphorus concentration and light intensity on the biomass composition of Arthrospira (Spirulina) platensis. World J Microbiol Biotechnol. 2012;28:2661–70.

17.

Chen

C-Y

, Kao

P-C

, Tan

, Show

, Cheah

, Lee

W-L

, Ling

, Chang

J-S

. Using an innovative pH-stat CO₂ feeding strategy to enhance cell growth and C-phycocyanin production from Spirulina platensis. Biochemical Engineering Journal. 2016;112:78–85.

18.

Ogbonda

, Aminigo

, Abu

. Influence of temperature and pH on biomass production and protein biosynthesis in a putative Spirulina sp. Bioresource Technology. 2007;98:2207–11.

19.

Belkin

, Boussiba

. Resistance of Spirulina platensis to Ammonia at High pH Values. Plant and Cell Physiology. 1991;32:953–8.

20.

Pandey

, Pathak

, Tiwari

. Standardization of pH and light intensity for the biomass production of spirulina platensis. Journal of Algal Biomass Utilisation. 2010;1(2):93–102.

21.

Troschl

, Meixner

, Drosg

. Cyanobacterial PHA production-review of recent advances and a summary of three years’ working experience running a pilot plant. Bioengineering. 2017;4:26.

22.

Wilhelm

, Jakob

. Balancing the conversion efficiency from photon to biomass. In: Microalgal Biotechnology: Potential and production. De Gruyter Berlin/Boston. 2012;39–53.

23.

Shi

, Li

, Wang

, Chen

, Li

, Ling

X-W

. Investigation of main factors affecting the growth rate of Spirulina. Optik. 2016;127:6688–94.

24.

Vonshak

. Spirulina: Growth, Physiology and Biochemistry. In: Spirulina platensis (Arthrospira): Physiology, Cell-Biology and Biotechnology (Ed. Vonshak

). Taylor & Francis, London, 1997, pp. 43–65.

25.

Kumar

, Kulshreshtha

, Singh

. Growth and biopigment accumulation of cyanobacterium Spirulina platensis at different light intensities and temperature. Brazilian Journal of Microbiology. 2011;42:1128–35.

26.

Torzillo

, Pushparaj

, Bocci

, Balloni

, Materassi

, Florenzano

. Production of spirulina biomass in closed photobioreactors. Biomass. 1986;11:61–74.

27.

Carvalho

, Silva

, Baptista

, Malcata

. Light requirements in microalgal photobioreactors: An overview of biophotonic aspects. Applied Microbiology and Biotechnology. 2011;89:1275–88.

28.

Ravelonandro

, Ratianarivo

, Joannis-Cassan

, Isambert

, Raherimandimby

. Influence of light quality and intensity in the cultivation of Spirulina platensis from Toliara (Madagascar) in a closed system. Journal of Chemical Technology and Biotechnology. 2008;83:842–8.

29.

Chen

H-B

, Wu

J-Y

, Wang

C-F

, Fu

C-C

, Shieh

C-J

, Wang

C-C

, Liu

Y-C

. Modeling on chlorophyll a and phycocyanin production by Spirulina platensis under various light-emitting diodes. Biochemical Engineering Journal. 2010;53:152–56.

30.

Wang

C-Y

, Fu

C-C

, Liu

Y-C

. Effects of using light-emitting diodes on the cultivation of Spirulina platensis. Biochemical Engineering Journal. 2007;37:21–5.

31.

Subramaniyan

, Bai

. Effect of different nitrogen levels and light quality on growth, protein and synthesis in Spirulina fusiformis. In. Proc. Spirulina ETTA National Symposium, MCRC, Madras. 1992, pp. 97–9.

32.

Ross

. Algal Motility in Variable Turbulence. PhD thesis, University of Southampton, 2004.

33.

Jetley

, Choudhary

, Fatma

. The impact of physical stresses on the growth of cyanobacterium Spirulina platensis-S5. Journal of Environmental Science & Engineering. 2004;46(4):303–11.

34.

Kim

, Lee

S-H

. Quantitative analysis of Spirulina platensis growth with CO2 mixed aeration. Environmental Engineering Research. 2018;23(2):216–22.

35.

Kim

, Choi

, Ji

, Park

, Do

, Hwang

, Lee

, Holzapfel

. Impact of bubble size on growth and CO2 uptake of Arthrospira (Spirulina) platensis KMMCC CY-007. Bioresource Technology. 2014;170:310–5.

36.

Kaplan

. Photoinhibition in Spirulina platensis: Response of photosynthesis and HCO₃^- uptake capability to CO2 depleted conditions. Journal of Experimental Botany. 1981;32:669–77.

37.

Tadros

. NASA Contractor NCC 2-501. Characterization of Spirulina Biomass for CELSS Diet Potential, CELSS Program, Alabama University, 1988.

38.

Moreira

, Terra

ALM

, Costa

JAV

, Morais

. Utilization of CO2 in semi-continous cultivation of Spirulina sand Chlorella fusca and evaluation of biomass composition. Brazilian Journal of Chemical Engineering. 2016;33:691–8.

Factors influencing the growth of Spirulina platensis in closed photobioreactors under CO 2 – O 2 conversion

Abstract

Keywords

1 Introduction

Conflict of interest

Footnotes

Acknowledgments

References