Development of Low-Cost Growing Media with Mungbean as A Source of Carbon for Spirulina

Spirulina is a multicellular, photosynthesis, filamentous blue-green algae which is found naturally in a wide range of fresh, marine, and brackish waters. It is an excellent source of protein, vitamins, minerals, lipids, carbohydrates, and pigments thus considered as the “superfood” of the century. The commercial production of Spirulina depends on many factors such as nutrient availability, temperature, and light. Zarrouk’s medium is known to be the standard media (SM) which contains all the macro and micro-nutrients required for the growth of Spirulina . The cost of nutrients is however found to be the second limiting factor next to labor cost affecting the commercial-scale production of Spirulina . The cost of carbon source is higher than that of N and P sources. Therefore, this study aimed at developing a low-cost medium for large-scale production of Spirulina . This intention was implemented by substituting carbon sources present in SM with cheaper and locally available carbon sources. Two separate experiments were conducted using a complete randomized design (CRD) with 3 replicates. The conventional carbon source in Zarrouk’s medium (NaHCO 3 ) was substituted with low-cost carbon sources i.e., Mungbean flour (before and after germination separately). The carbon content in SM was replaced by 100 %, 75 %, 50 % and 25 % of Mungbean flour. Zarrouk’s medium was used as the control. The culture was maintained at 30 0 C ± 2 0 C under 4000 Lux, continuous illumination using a white, fluorescent tube for 16 days. Growth was measured using a spectrometer and optical density (OD) values were recorded at 560 nm with two days interval. Data were analyzed using SAS version 9.4. The best growth of Spirulina was recorded at the 50 % replacement of carbon in SM by Mungbean flour (before germination). Mungbean flour (after germination) could also replace 25 % of carbon in Zarrouk’s media. Taking the cost factor into consideration, 50 % replacement of carbon in Zarrouk’s medium by Mungbean flour (before germination) can be recommended for commercial scale cultivation. The comparative cost reduction of this replacement is estimated to be 50 %.


Introduction
Spirulina (Arthrospira sp.) spiral-shaped, edible filamentous cyanobacteria is commonly known as blue-green microalgae.(Jiang et al., 2021).Spirulina, a microalga that naturally grows in lakes, is known for its nutritional benefits and is widely recognized as a valuable source of nutrients.It is a type of unicellular blue-green microalgae that can be grown in large quantities with a high level of carbonate and bicarbonate in tropical and subtropical water bodies (Shen et al., 2021).The filaments are up to 500 mm long and have a diameter of 3-5 mm (Nosratimovafagh et al., 2023).In commercial cultivations, Spirulina can successfully be grown on freshwater, saltwater, and brackish water.It thrives in a pH range of 10-12, which is highly alkaline (Wijayanti et al., 2020).

Although
this microorganism grows photosynthetically, an organic substrate can encourage its growth (Jung et al., 2022).Spirulina has a photosynthetic conversion rate of 8 % to 10 % (García-Lópeza & Olguína, 2020).The composition of microalgal biomass is highly determined by environmental conditions (Mulokozi et al., 2019).Mexicans have been using Spirulina for over 1000 years, dating back to the Aztec civilization.Dangeard, a French phycologist, documented the utilization of Spirulina as a food source by the indigenous people in the Lake Chad region, as reported by Ibrahim et al. (2023).The rediscovery of Spirulina by J. Leonard and Compere in the 1960s paved the way for commercial production in the late 1970s (Ibrahim et al., 2023).Spirulina is now commercially cultivated in several countries, with an annual production of a few hundred tons (Manogar et al., 2020).Because of its potential applications in human food additives, animal feed, and pharmaceuticals, Spirulina's commercial production has gained a considerable attention (Torky et al., 2023).Spirulina has a huge potential not only for making conventional food but also for obtaining useful chemicals like β-carotene and phycocyanin.The use of Spirulina pigments as feed for tropical fish is popular in aquaculture.It's also used in agriculture and wastewater treatments (Matufi & Choopani, 2020).Vegetable production in most developed countries is insufficient to fulfill the population's dietary needs.Spirulina with its high content of β-carotene could be considered as a healthy substitute source of vitamin A (Fernandes et al., 2023).
Spirulina was considered as "the best food for tomorrow" by the United Nations World Food Conference, and it has gained attention as a food supplement in recent years.The WHO has called Spirulina "the greatest superfood on earth" (Ramírez- Rodrigues et al., 2021).Spirulina has a unique combination of nutrients that no other food can supply.Vitamin B-complex, nutrients, high-quality proteins, gamma-linolenic acid, and the super antioxidants, beta-carotene, vitamin E, and trace elements are all present in Spirulina (Suyoso et al., 2022).Every day, 40 000 children succumb to death due to malnutrition and its associated diseases, underscoring the urgent need for global efforts to address and alleviate this devastating public health issue (World Health Organization [WHO], 2013).One of the aims for growing Spirulina was to produce a new source of complete food supplement for a starving planet.One tablespoon per day has major health benefits (Al Hinai et al., 2019).The growth in the world's population and forecasts of a protein crisis in the early 1950s sparked a search for new complementary and unconventional protein sources (de Morais et al., 2019).Spirulina spp. is well recognized for its richness in proteins due to its exceptional value when compared with other microorganisms.Spirulina's protein level is high and accounts for between 60 % and 70 % of the dry matter, more than fish (25 %) or soybean (35 %).The amino acids composition of the protein is balanced in Spirulina and more than 40 % of amino acids are found such as methionine, threonine, isoleucine, tryptophan, leucine, lysine, and valine.The composition of Spirulina is too low in sulfur-containing amino acids (Cysteine & methionine) which are essential for people who are malnourished, particularly children.Spirulina must be substituted with other amino acid-rich protein sources, it contains a significant amount of essential amino acids (Shao et al., 2019).Microalgae contain carbohydrates in the form of starch, glucose, sugar, and other polysaccharides (Álvarez & Otero, 2020).
Carbohydrates represent approximately 15 % to 25 % of the dry weight of Spirulina.Both in S. platensis and S. maxima, the overall total lipid content is 5.6 % -7 % of the dry weight.By using a better extraction method can obtain more than 11 % of lipids.
For several years, Zarrouk's medium has successfully utilized as the conventional and standard media for Spirulina culture (Fanka et al., 2022).It contains all the nutrients required for the growth of Spirulina including C, N, and P. In the standard media, NaHCO 3 is used as the source of carbon.The cost of carbon source is higher than phosphorous and nitrogen source.Therefore, high purchasing cost of carbon source is a limiting factor in large scale cultivation.Cost-effective Spirulina cultivation is necessary when large-scale cultivation is done for industrial purposes.Therefore, to reduce the cost of growing medium, (NaHCO 3 ) should be replaced with a cheaper and locally available carbon source.
The high cost of the growing medium limits large-scale production around the world, which is highly dependent on Zarrouk's medium.Due to the analytical grade materials used to produce the medium, the medium is expensive (Michael et al., 2019).When considering large-scale production for industrial purposes, it is important to grow Spirulina at a lower cost.After the labor costs, the cost of nutrients is the second most significant factor affecting the cost of Spirulina biomass production (Rodrigues et al., 2020).The major objective of this research is to identify a possible low-cost alternative source of carbon that could substitute NaHCO 3 in Zarrouk's medium.In this study carbon source was substituted with Mungbean flour (before and after germination).

Experiment location
The research was conducted at the Research laboratory, Department of Crop Science, Faculty of Agriculture, University of Ruhuna, Mapalana, Sri Lanka.The experiment was conducted for a five-month period starting from November 2022 to May 2023.The average annual temperature of the area is 30 o C, with an average humidity of 79 % and 1813 mm annual precipitation.

Mother stock
To prepare the mother stock the microalgae Spirulina was stored in the faculty's culture collection center.The mother stock was prepared in a 5-liter conical flask which was initially sterilized using the autoclave.Distilled water added to the conical flask was boiled and kept at room temperature for 24 hrs.until cooling.All constitutes of Zarrouk's medium were added to the conical flask (according to the calculated weight required for the 5-liter medium) and dissolved using a magnetic stir.After 24 hrs., the media was inoculated with 250 mL of Spirulina inoculum which was taken from the culture collection.Mother stock was maintained at 32 o C ± 2 o C, pH 8.5, with continuous illumination using 35 W fluorescent tubes which is equivalent to 300 to 500 lux.

Experiment design, experimental layout, data recording and sampling
Experiment 1: Preparation of low-cost media using Mungbean flour (before germination) as a carbon source.Experiment 2: Preparation of low-cost media using Mungbean flour (after germination) as a carbon source and Experiment 3: Preparation of growth enhancement media using coconut water.Fifteen conical flasks with the four modified concentrations and controls, which containing the Zarrouk's medium were placed according to Completely Randomized Design (CRD) with three replicates.Growth of Spirulina was measured using a spectrophotometer at 560 nm wavelength within two days intervals and optical density (OD) values were recorded.
Samples were withdrawn from the culture flask with a pipette for spectrophotometer readings.

Amount of Mungbean calculation steps
The amount of Mungbean flour (before/after germination) needed to replace the NaHCO 3 in Zarrouk's medium was calculated as follows.

Preparation of low-cost media
For the experiment, two liters of Zarrouk's medium (control solution) and Mungbean flour solution (before germination), after germination and coconut water were separately prepared.Distilled water was used for the preparation of all the medium.Experiment 1 -Sundried Mungbean was ground using a grinder and sieved using a 1mm sieve to get a fine powder  1).

Culture condition
Culture medium (200 mL) was transferred into 250 mL of conical flasks and 50 mL of algal culture (5×10 6 cells in the 50 mL culture) was inoculated separately for each conical flask.
Physical conditions such as temperature, light, mixing rate were maintained at standard levels.
The experiment was carried out with three replicates for 16 days at room temperature, under 4000 Lux white, fluorescent lamps, with daily handshaking.

Calculation of the Dry Weight (g/L)
The mean values from the statistical analysis were converted into dry weights using the equation, DW = Dry Weight (g/L), OD = Optical Density and (r=0.9927)(Arunakumara et al., 2007).

Pigment analysis
Chlorophyll a and β-carotene content was determined using 90 % acetone method.A sample of 10 ml was kept in a tube with 10 mL of acetone and the tube was immersed in an ice -bath.An Ultrasonic -vibrator was used to break the cells.After the completion of the extraction process, visible two layers can be observed.5 mL of acetone extract (upper layer) was taken and evaluated.

Chlorophyll a
The optical density was measured using a spectrophotometer at 663 nm and 645 nm wavelength to calculate the chlorophyll content.Optical density values were converted into the chlorophyll a (mg/L) content using the following equation,

β-carotene
The optical density was measured using a spectrophotometer at 450 nm wavelength to calculate the β-carotene content.Optical density values were converted into the β-carotene (mg/l) content using the following equation,

Statistical analysis
Data were analyzed using one-way analysis of variance (ANOVA) with the help of statistical analysis system (SAS) version 9.4 computer package, Mean separation was done with Dunnett's Multiple Range Test (DMRT).

Results
Spirulina spp. was cultured at 33 0 C, under 400 Lux illumination for 16 days.On the 1 st day of inoculation, no significant growth variations were found among different treatments (Pr = 0.2075).At the end of the 7 th day of incubation, a significant reduction in the growth of the algae treated with T4 and T5 was evident.Spirulina that was in T4 and T5 died 13 th days after incubation.However, as incubation progressed significant differences among other treatments could be observed (T1, T2, and T3).On the 16 th day, T2 and T3 recorded the highest growth compared to the control (Table 2).Significantly higher growth was recorded in T2 and T3 compared to the control indicating that 25 %, 50 % of the carbon in Zarrouk's media could be replaced by the Mungbean flour (before germination) as a source of carbon (Table 3).
On the 1 st day of incubation, the growth of Spirulina was not found to be varied among the different treatments (Pr = 0.1094).However, as incubation progressed significant differences among treatments were observed, implying that supplementation of Mungbean flour (after germination) as a source of carbon could be done.As incubation progressed, optical density value was gradually increased in T1 and T2 due to growth increment.On the 16 th day of incubation, significant reductions in the growth of algae were observed in T4 and T5.On the 16 th day, T2 recorded significantly higher growth compared to the control (Table 2) Significantly higher growth was recorded in T2 compared to the control, indicating that 25 % of the carbon in Zarrouk's media could be replaced by the Mungbean flour (after germination) as a source of carbon (Table 3).
As incubation progressed, optical density values gradually increased due to growth increment.T4 and T5 treatment showed the lowest optical density values compared to the control.T2 and T3 showed significantly higher optical density values compared to the control.According to the results, incorporating 5 to 10 percent replacement level of coconut water could be deemed a viable option for enhancing the growth of Spirulina.
After the 16 th day of incubation significantly higher chlorophyll a content was observed in T2 and T3 compared to the control.The lowest chlorophyll a content was recorded at control (T1).According to the results 25 % and 50 % of carbon in Zarrouk's media could be replaced by the Mungbean flour (before germination).After the 16 th day of incubation significantly higher β-carotene content was shown in T2 compared to the control.T3 showed the second highest β-carotene content which was however not significantly different from the control.The lowest β-carotene content was shown in T1.According to the results, 25 % of carbon in Zarrouk's media could be replaced by the Mungbean flour (before germination) (Table 4).
After 16 th day of incubation, significantly higher chlorophyll a content was recorded in T2 compared to the control.T3 showed the second highest chlorophyll a content.The lowest chlorophyll a content was observed in control (T1).According to the results, 25 % of carbon in Zarrouk's media could be replaced by Mungbean flour (after germination).After the 16th day of incubation, significantly higher β-carotene content was shown in T2 compared to the control.T3 showed the second higher β-carotene content.
The lowest β-carotene content was shown in T1.
According to the results, 25 % carbon could be replaced by Mungbean flour (after germination) (Table 4).It clearly shows the difference growth of Spirulina on different media is a significantly differ (Figure 1).

Discussion
One of the most important factors attributed to the growth and development of microalgae is the nutrition condition of the growing media.The present investigation was carried out with the objective of developing a low-cost media for culturing Spirulina.low-cost medium for largescale production of Spirulina was implemented by substituting Zarrouk's medium with cheaper and locally available organic sources.Ragaza et al. (2020) assessed the ability to utilize glycerol as a source of carbon for Spirulina platensis   Chlorophyll a content was measured as OD value at 663 nm and 645 nm (Pr ˂ 0.05) (N= 3) β-carotene content was measured as OD value at 450 nm (Pr ˂ 0.05) (N= 3).**Means are significantly higher than the control (Ragaza et al., 2020).Glycerol was added as an alternative carbon source (2.5 mM -l level) in the first experiment and sodium bicarbonate was reduced from 16.8 g.L -1 to 2 g.L -1 .In the second trial instead of bicarbonate, glycerol was used as the sole carbon source with the concentrations ranging from 1.25 mM -1 to 12.
5 mM -1 .Results showed that Spirulina grown on glycerol medium decreased in pigment content of phycocyanin and chlorophyll-a compared to the control.The lipid content of the glycerolgrown groups was like that of the control, though there were differences in the fatty acid profile.The amount of g-linolenic acid (GLA) in the Neutral lipid (NL) fraction was also increased.Sukanya et al., (2020) conducted a study with five cultures of Spirulina platensis using Na 2 CO 3 , D-glucose, Mannitol, (NH4) 2 CO 3 , CaCO 3 , urea, and sucrose as a sole carbon source, and sodium bicarbonate containing growth medium served as the control (Sukanya et al., 2020).When NaHCO 3 in the standard Zarrouk's medium was substituted with Na 2 CO 3 optimum biomass production was observed.Among these various carbon sources, Na 2 CO 3 greatly increased biomass production, while CaCO 3 produced the least biomass production.The death of culture was detected when NaHCO 3 was substituted with some organic carbon source within 10 days of inoculation.According to them, insufficient quantity of carbon for the growth of Spirulina provided with an inorganic form of carbon explained the results.Also, they observed the maximum growth when Zarrouk's medium was replaced with glucose (2 g.L -1 ) confirming that glucose can be used as a supplement to enhance growth, but not as a sole carbon source.
Many studies have shown that limitation in nutrients and/or organic carbon supplementation decreases the production of single high-value products from microalgal cultivation.Cultures grown mixotrophically with organic carbon sources typically found in agricultural and industrial waste streams (acetate, formate, glycerol, and oxalate) and under nutrient restriction did not show a clear pattern of growth.Supplementing glycerol and acetate to Arthrospira platensis cultured in media devoid of both N and P resulted in a substantial increase in biomass productivity relative to a photoautotrophic reference culture but had a negative impact on both phycocyanin and polyhydroxy butyrate production.According to the findings, a minimum amount of N is needed to maintain phycocyanin level and a higher concentration of the organic C supply is required to achieve a redox balance within the cells that increase PHB production (Moradi et al., 2021).Almomani & Bhosale (2021) conducted research to see whether the production of Chlorella vulgaris microalgal biomass and biochemical components including photosynthetic pigments, lipids, soluble carbohydrates, and proteins using glycerol and glucose as a complex carbohydrate substrate (Almomani & Bhosale, 2021).The findings suggested that C. vulgaris can use glycerol as a single carbon substrate though a lower effect than a combination of glycerol and glucose was reported.The effect of glycerol and glucose on the algal cell growth rate, biomass content, and volumetric productivity could compensate for the lower biomass production when glycerol is used as the sole organic carbon source in a mixotrophic culture medium.Bandara & Arunakumara (2020) substituted the carbon content in Zarrouk's medium with different concentrations of (25 %, 50 %, 75 %, and 100 %) of alternate carbon sources.They observed the maximum dry weight (0.747 g/ L) from 25 % replacement level at the end of the 16-day incubation, while the lowest (0.419 g/L) dry weight at 100 % replacement level of table sugar.In this study, cassava flour was also used as a source alternative carbon source.The 50 % replacement level has produced the maximum dry weight (1.174 g/L) at the end of the 16day incubation, followed by 25 % replacement level.Cassava flour which replaced the carbon source in Zarrouk's medium at 75 % and 100 % replacement level significantly decreased in dry weights implying that the carbon source in Zarrouk's medium could not be replaced by cassava flour at high replacement levels (Bandara & Arunakumara, 2020).
In the mixotrophic production of Arthrospira platensis, the effects of various organic carbon sources glucose, ethanol, and acetic acid at different concentrations (0.1 g/L, 0.5 g/L, 1.0 g/L for batch and 1.0 g/L, 2.0 g/L, and 3.0 g/L for fed-batch) were investigated by Mohammad et al., (2022).Increasing the concentrations of glucose, ethanol, and acetic acid in the batch media resulted in an improvement in biomass, lipid, and linolenic acid content.Carbon sources in fed-batch media at concentrations greater than 1.0 g/L reported no significant impact on the above parameters.It was also shown that biomass, lipid, and linolenic acid production using ethanol and acetic acid were like those found with conventional glucose-based culture media (Pérez-Juárez et al., 2022).
Al Mahrouqi et al., (2022) investigated the possibility of cultivating microalgae Spirulina platensis using molasses as organic substrate under mixotrophic cultivation (Al Mahrouqi et al., 2022).The effects of molasses concentration and light levels on mixotrophic biomass production by Spirulina platensis were investigated in this study.Zarrouk's medium supplemented with 0.25 g/L, 0.5 g/L, or 0.75 g/L of molasses.According to the results, increased molasses concentration favored biomass production, though it was unfavorable for the specific growth rate.After 11 days, the light intensity started to influence growth parameters but showed smaller effects than molasses concentration.These results highlighted the potential of molasses as an organic substrate for the mixotrophic cultivation of Spirulina platensis and the possibility of using low-cost agricultural by-products for the growth of this microorganism (AlFadhly, et al., 2022).Johny (2022) stated that cassava starch hydrolysate is a good source of carbon to replace carbon source in growing media (Jhony, 2022).
In the present study, higher replacement level of Mungbean flour (before and after germination) showed a significantly negative impact on the growth of Spirulina which might be due to the nature of carbon source and level of absorption.Higher replacement levels of Mungbean flour cause to change the pH of the growth media and create an acidic condition.Spirulina needs an alkaline condition for their growth (pH 8-9).Coconut water contains many micro and macro nutrients and hormones.Minerals such as Calcium, Copper, Magnesium, Manganese, Potassium, Sodium, Zinc, and hormones such as auxin, cytokinin's, gibberellins are among them.These elements are a good source for the growth of Spirulina.But higher replacement level of coconut water showed a negative impact on the growth of Spirulina possibly due to the acidic conditions created in the media.

Cost Analysis
NaHCO 3 is the inorganic carbon source present in Zarrouk's medium.The cost of production is higher due to the high purchasing cost of the inorganic carbon source (NaHCO 3 ).Therefore, replacing NaHCO 3 in standard medium with Mungbean is suggested.The production cost of 1L Zarrouk's medium with NaHCO 3 could be calculated as follows,

Recommendation
Growth of Spirulina under alternative carbon sources should be further evaluated with their different concentrations.

Figure 1 .
Figure 1.Photographic presentation of the effect of Mungbean (Before germination), Mungbean (After germination), and Coconut water on the growth of Spirulina as a low-cost source of growth enhancement

Table 1 .
The composition of the Zarrouk's media with Mungbean flour (before germination), Mungbean flour (after germination) and coconut water

Table 2 .
Effect of Mungbean flour (before germination), Mungbean (after germination) and coconut as a low-cost alternative carbon source on growth of Spirulina

Table 3 .
DryGrowth was measured as OD value at 560 nm (Pr ˂ 0.05) (N= 3) Depicted values of relevant dry weight for the OD values in each treatment.**Means are significantly higher than the control *Means are significantly lower than the control weight (g/l) of Spirulina when Mungbean flour (before germination), Mungbean (after germination) and coconut as a low-cost alternative carbon source

Table 4 .
Chlorophyll a content (mg/L) and β-carotene content (mg/L) of Spirulina treated with different levels of Mungbean.
Price of 1Kg of NaHCO 3 = Rs.2057.31(1g=Rs 2.05) Price of 1Kg of Mungbean including processing cost = Rs.300.00 (1 g= Rs 0.3)ConclusionAlternative carbon sources prepared with Mungbean seeds (before germination) can be used in replacing 50 % carbon in Zarrouk's medium.Alternative carbon sources prepared with Mungbean seeds (after germination) can be used in replacing 25 % carbon in Zarrouk's medium.Comparatively high chlorophyll-a and β-carotene contents can be obtained from 25 % replacement level of carbon with Mungbean flour (before and after germination).Taking the economic benefits into account, 50 % replacement of carbon in Zarrouk's medium by Mungbean flour (before germination) can be recommended for the commercial cultivation of Spirulina.Spirulina.