Hydroponic Fertilizers as a Growth Media for Chlorella vulgaris for Producing Affordable Protein in Developing Countries

doi:10.21203/rs.3.rs-4198065/v1

Chlorella vulgaris stands out as a promising microalga for single-cell protein production, particularly due to its potential to address the need for affordable nutrition in less developed regions. Given this context, the quest for more economical fertilization methods is essential. Among the viable alternatives, hydroponic fertilizer (HF) emerges as a widely available option, particularly in regions such as Indonesia. Typically, HF is supplied in two distinct forms: powdered Mix-A and Mix-B to prepare the stock solutions. This study investigates the impact of varying concentrations and stock solution ratios on both the growth rate and biomass yield of Chlorella vulgaris. Additionally, an economic analysis of cultivation activities is conducted, with a specific focus on comparing the feasibility of utilizing hydroponic fertilizer against conventional methods. The findings reveal that specific ratios and concentrations of the tested growth media outperform standard mediums in promoting the growth rate and biomass yield of cultivated Chlorella vulgaris. Furthermore, the economic analysis assesses two primary cost components: fertilizer and energy. Remarkably, the study demonstrates that all tested hydroponic mediums offer a more cost-effective cultivation solution compared to the conventional methods, primarily attributed to the lower cost of the hydroponic fertilizers.

Protein, being one of the essential macronutrients, plays an essential role in maintaining the overall health and well-being of individuals (Loveday 2019; Zohoori 2020). However, the accessibility and affordability of protein sources remain a challenge for many, especially in less developed regions (Wu et al. 2014; Cheng et al. 2019). As the global population continues to increase, there is an urgent need for alternative and affordable protein sources to meet the nutritional demands of people in these areas.

Single-cell protein (SCP) presents a promising solution to the shortage of essential protein (Abdel-Azeem and Sheir 2020; Sharif et al. 2021). SCP refers to protein derived from microbial organisms such as microalgae, yeast, fungi, and bacteria. Chlorella vulgaris, in particular, has gained significant attention as a potential source of SCP (Caporgno and Mathys 2018; Grossmann et al. 2020). Their ability to produce proteins makes them an excellent candidate for SCP.

The cultivation of Chlorella vulgaris for human consumption, however, requires a suitable growth medium that provides the necessary nutrients for their growth. Currently, the cost of growth media represents a significant portion of the overall production cost (Zabochnicka-Świątek et al. 2019; Ibanez et al. 2020). Finding an affordable and effective growth medium is important to make microalgae-based SCP more economically viable to those in need.

Hydroponic fertilizer (HF) is a simple-to-prepare fertilizer and is a widely affordable nutrient, particularly in Indonesia. It includes essential macronutrients (e.g. nitrogen, phosphorus, and potassium) and micronutrients (e.g. iron, magnesium, and zinc) for plant growth (Phibunwatthanawong and Riddech 2019; Kano et al. 2021). The nutritional requirements of microalgae cultivation closely resemble those of terrestrial plants. Despite not being initially designated as the primary crop, microalgae have proven to thrive in hydroponic systems (Barone et al. 2019; Supraja et al. 2020; Ajeng et al. 2022). It is essential to investigate the application of HF for microalgae culture and determine the optimal ratio and concentration of macro- and micronutrients. The successful application of the HF for microalgae will open numerous options to deliver more affordable cost.

This study aims to explore an alternative media for cultivating microalgae for human consumption with HF as the examined media. An economic evaluation comparing the standard media and the alternative ones was compared as well.

2.1. Strain and cultivation medium

The Chlorella vulgaris was collected from the Government Research Center for Brackish Water Aquaculture (BBAP), Situbondo, Indonesia. Originally, the microalgae were cultivated in Bold Bassal Medium (BBM) with 30 ppt salinity. BBM was the controlled media in this study and was prepared following the standards established by CCAP, UK with a minor adjustment (CCAP UK).

The HF powder was manufactured in Indonesia, and consists two distinct components: Mix-A and Mix-B. Each component contains a combination of macronutrients and micronutrients (Table 1). To ensure optimal growth, microalgae underwent acclimatization in HF culture, gradually decreasing salinity from 30 ppt to approximately 20 ppt by systematically reducing seawater addition.

Table 1. Content of the concentrated solutions of BBM and hydroponic fertilizer (HF)

Bold Bassal Medium (BBM		Hydroponic Fertilizer (HF)
Content	Concentration (g/L)	Content	Concentration (g/L)
Stock-A		Mix-A
NaNO₃	25.0	KNO₃	42
MgSO₄.7H₂O	7.5	Ca(NO₃)₂	27
NaCl	2.5	Fe-chelate	3
K₂HPO₄	7.5
KH₂PO₄	17.5
CaCl₂.H₂O	2.5
Stock-B		Mix-B
ZnSO₄	8.82	KH₂PO₄	20
MnCl₂	1.44	MgSO₄	20
MoO₃	1.4	K₂SO₄	15.5
CuSO₄	1.57	KNO₃	14.5
Co(NO₃)₂	1.57	MnSO₄	0.15
H₃BO₃	11.42	ZnSO₄	0.23
EDTA	50	B	0.23
KOH	31	CuSO₄	0.03
FeSO₄	5.0	MoO₄	0.01
H₂SO₄	1

2.2. Experiment Apparatus

In this experimental laboratory-scale study, Chlorella vulgaris was cultivated in a 1000 ml glass photobioreactor with a working volume of 700 ml. A ceramic diffuser was placed at the base of each reactor served to induce and disperse a CO₂ fine bubble from the ambient air, which also served the mixing purposes. Airflow was regulated at a rate of 1.5+0.2 liters per minute via an air pump. The schematic representation of each bioreactor is presented in Figure 1. Alongside natural daylight, microalgae received continuous illumination from a 24-hour LED light strip, emitting approximately 4000 lux at the photobioreactor surface. Including indirect sunlight, daylight luminance could reach up to 5040 lux. While no strict temperature control was implemented, periodic measurements of the liquid temperature revealed that it was approximately + 2-3^oC (higher or lower) than the room temperature, which was about 22-25^oC since it was air conditioned.

2.3. Experimental set-up

Variables examined were the ratio and concentration of the Mix-A to Mix-B stock solution of the HF. The experimental design matrix is presented in Table 2. The experiment was conducted in three replications with additional control.

Table 2. Experimental design matrix: the volume of concentrated solution of the hydroponic fertilizer added into the medium

Concentration	Ratio
Concentration	Ratio-A (1:1)	Ratio-B (1:2)	Ratio-C (2:1)
Conc-1	3.5ml Mix-A; 3.5ml Mix-B	3.5ml Mix-A; 7ml Mix-B	7ml s Mix-A; 3.5ml Mix-B
Conc-2	4.9ml Mix-A; 4.9ml Mix-B	4.9ml Mix-A; 9.8ml s Mix-B	9.8ml Mix-A; 4.9ml s Mix-B
Conc-3	7ml Mix-A; 7ml Mix-B	7ml Mix-A; 14ml s Mix-B	14ml Mix-A; 7ml Mix-B

The cultivation was initiated by adding a certain volume of inoculum in 700 ml of liquid media to reach an initial optical density (OD) of 0.2+0.05, equivalent to a biomass dry weight (DW) of 2 g/L.

2.4. Growth rate and biomass yield

Biomass was sampled daily and its OD was measured at 680 nm using a UV-VIS spectrophotometer. The cultivation was conducted until the microalgal showed a decrease in three to four consecutive days. Due to the inability to measure the biomass DW in real-time at their highest density, estimation was conducted using a calibration curve correlating OD with biomass DW. The calibration and associated equations are detailed in Supplement 1.

The average growth rate was estimated by fitting the data using the Gompertz equation as presented by Equation 1 in Origin 2019b software using SGompertz function with Levenberg Marquardt iteration algorithm. Y is the calculated OD, A is the asymptote, k is the growth rate, X is the time (day) and Xc is the time when it reaches the point of inflection in which microalgae reaches its highest growth rate.

To ensure the fitting accuracy, data points corresponding to the decreasing growth phase were excluded. In their place, three data points were introduced, each having the same value as the peak point. This adjustment was made to prevent any potential underestimation or overestimation of the growth rate (k) and the point of inflection (Xc) parameters, thereby enhancing the reliability of the analysis.

2.5. Proximate analysis

The composition of carbohydrates, lipids, proteins, and ash was analyzed concurrently using the method outlined by Chen and Vaidyanathan (2013) with some necessary modifications. Ash content analysis involved placing the dried biomass in a furnace at 560°C for a duration of 6 hours, followed by the weighing of the residual ash.

2.6. Statistical analysis

A statistical analysis was performed only on the growth rate and density of the microalgae. This analytical investigation was carried out in Origin 2019b software, employing a Two-Way Analysis of Variance (ANOVA) followed by Tukey's post hoc test, with a significance level set at 0.05.

3.1. General cultivation condition

The temperature range (22°C to 28°C) is conducive to the growth of Chlorella vulgaris (Dahmani et al. 2016), as indicated in Table 3, although an optimum temperature was suggested in the range 15°C to 20°C (Zhang et al. 2020). Exceeding a temperature of 30°C might render the conditions less favorable for microalgae growth (González-Camejo et al. 2019). Media temperature has an impact the on solubility of CO₂, which serves as the primary carbon source (Al-Anezi et al. 2008).

Table 3. Environmental parameters measured periodically during the cultivation

Treatment	Measured parameters
Treatment	Temperature (°C)	Salinity (ppt)	pH
Control (BBM)	22-28	20-25	7.3 - 8.1
A1	22-28	20-23	5.5 - 5.8
A2	22-28	20-24	5.5 - 6.0
A3	22-28	20-26	5.6 - 6.2
B1	22-28	20-23	5.5 - 5.8
B2	22-28	20-24	5.5 - 6.4
B3	22-28	20-26	5.5 - 6.5
C1	22-28	20-23	5.2 - 6.0
C2	22-28	20-24	5.6 - 6.3
C3	22-28	20-26	5.5 - 6.2

The pH levels of the growth media ranged from 5.2 to 6.3 for HF and from 7.3 to 8.1 for the control. All tested media were in acidic pH, with higher HF concentrations tended to give higher pH value. No pH buffer was applied to the media with an intention of observing the authentic response of microalgae, thereby reducing the additional cultivation expenses, particularly in economically disadvantaged regions. Numerous studies have indicated a preference for Chlorella vulgaris for alkaline pH levels (Choi and Lee 2011; Yu et al. 2018), with few reports of acidic pH (Sarker and Salam 2020). The pH value observed in BBM demonstrated a more ideal for promoting the growth of Chlorella vulgaris.

The cultivation commenced at 20 ppt and gradually rose to 26 ppt. Chlorella vulgaris is known to thrive in saline environments, with the optimal salinity range from 25 to 30 ppt (Adenan et al. 2013; Pandit et al. 2017). Evaporation due to constant aeration during the cultivation process might be the reason for this salinity increase.

3.2. Growth of the microalgae

Figure 2 illustrates the appearance of the microalgae during different time intervals for treatment in A3, B3, and C3. On day 30, microalgae exhibited a notably high cell density. Among treatments in Concentration-3, A3 entered the death phase earlier than the other two.

Figure 3 illustrates the growth of Chlorella vulgaris under varying HF ratios and concentrations with their non-linear fitting included. Microalgae cultivation in BBM lasted for 19 days, with the highest density observed on day 16, which was slightly different from those in treatments A2, B2, and C2. The shortest time required to reach the highest density was observed in treatments A1, B1 and C1 (12 days), while longer ones were observed in all treatments of Concentration-3.

The non-linear fitting using the Gompertz model conducted on the data shows the correlation coefficient (R²) ranged from 0.927 to 0.991, with an average R² value of each treatment was 0.961. All parameter values in Equation 1 were obtained by the software.

In general, the duration of microbial growth is notably impacted by the concentration of the growth media, regardless of the specific HF ratio employed. Higher concentrations of the added HF extended the cultivation period prior to entering the death phase, as evidenced by the extended duration required to reach peak density in each HF ratio. Cultivation in BBM media continued for 19 days, with the peak biomass concentration observed on day 16. This pattern only slightly differed from the results in treatments A2, B2, and C2, where the average time ranged from 15 to 17 days to reach the peak biomass concentration. Conversely, the shortest time required to reach peak cell density was observed in treatments A1 and C1, occurring on day 12, with a one-day delay for treatment B1.

Comparing the growth rate value resulting from Equation 1, it shows that the growth rate of microalgae cultivated in control media (0.166.day^-1) exceeded that in most of the tested media, except for treatments B1, B2, and C1 (Figure 4). While examining individual treatments, it was noted that the highest growth rate was observed in one of the B1 samples (0.313 day^-1). However, when having a look at the average growth rate in each treatment, B2 shows slightly higher than that of B1, with both showing no significant difference. Specifically, the growth rate for B1 was measured at 0.2625, while B2 had a growth rate of 0.2644. A long treatment duration tends to have a negative impact on the overall growth rate, as evidenced by much lower growth rates in all treatments at Concentration-3.

By applying the ANOVA test (Table 4), ratio of the HF significantly influences the growth rate of the microalgae, which is shown by P Value of 2.09E^-04. Although the overall ANOVA test results shows that the concentration and the interaction between the two tested parameters did not significantly influence the growth rate, the Tukey’s post-hoc test comparing a more detail comparison within the same factor shows that the growth rate of all Concentration-3 treatment shows significantly lower, as can be conclude visually by the data. The detail of this post-hot test can be found in the Supplement 2.

Table 4. Two-way ANOVA test for the growth rate

	DF	Sum of Squares	Mean Square	F Value	P Value
Ratio	3	0.0534	0.0178	11.3318	2.09E-04
Concentration	2	0.0067	0.0033	2.1309	0.1477
Interaction	6	0.0179	0.003	1.8928	0.1374
Model	11	0.0994	0.009	5.7511	5.77E-04
Error	18	0.0283	0.0016	--	--
Corrected Total	29	0.1277	--	--	--

Figure 5 illustrates that the earliest inflection point was observed in treatment B2, occurring at 3.4 days, followed closely by treatment B1 at 3.6 days. Conversely, all Concentration-3 treatments exhibited the latest times to reach the inflection point in each HF ratio, with values slightly exceeding 10 days. Given that the inflection point signifies the phase of fastest microalgal growth, making it a crucial indicator in microalgal culture for determining the optimal time to harvest cell biomass, particularly when biomass is the primary product (Richmond 2003).

3.3. Biomass yield

The calibration graph presenting DBW shows the linear regression equation y = 3.1388x + 1.9755 with an R² value of 0.974 (Supplement 1). Figure 6 illustrates the biomass yield at its highest density. Treatment B3 exhibits the highest biomass yield, approximately 9.75g.l^-1. Unsurprisingly, all treatments in Concentration-3 (i.e. A3, B3, and C3) demonstrate distinctively higher biomass yield, which can be attributed to their extended cultivation period as a result of more available nutrients. In a comparable time to reach the peak density of the BBM (15-16 days), only treatment A2 slightly surpassed the biomass yield of the control. The two-way ANOVA followed by Tukey's post hoc test concluded that while the ratio did not exert a significant impact, the concentration significantly affected the biomass yield (Supplement 3).

3.4. Proximate value (protein, lipid, carbohydrate, and ash)

The proximate analysis of microalgal cells identifies major components in dried biomass, essential for assessing nutritional value and suitability for consumption. This analysis typically determines key nutrients: carbohydrate, protein, and lipid. In this study, the proximate analysis was conducted only for BBM and Concentration-3 of each HF ratio.

Chlorella vulgaris cultivated in Ratio-B of HF demonstrates the highest protein content, accounting for approximately 33.93% of the dry weight of biomass (Figure 7). This figure does not significantly differ from those of other treatments, which ranged between 30% and 34%. Microalgae cultivated in BBM exhibit the highest lipid (35.30%), slightly surpassing that of microalgae cultivated in HF. As for carbohydrates, microalgae cultivated in BBM contained the lowest percentage (28%), contrasting with other treatments that ranged from 35% to 42%. Similarly, the ash content in the various treatments exhibits relatively comparable percentages.

3.5. Cultivation cost

The cultivation cost discussed in this study refers to the expenses involved in producing one kilogram of dried microalgae biomass. This cost estimation is tailored for SCP production within households, small and medium-scale enterprises. This estimation was originally conducted in the local currency of Indonesia (IDR), considering the specific cultivation location, with a specific emphasis on the East Java Province. To enhance the clarity, the cost figures presented in this paper have been converted into Euro currency (EUR). The 'dried biomass' in this context refers to biomass with no moisture content, unlike dried microalgae powder available in the market that has moisture content ranges from 2% to 9% (Amin et al. 2021; Zhang et al. 2022). In other words, the production cost per unit mass could potentially be lower if the moisture content of market-available microalgae powder were applied as the benchmark.

The cultivation cost estimation primarily focuses on two pivotal components: fertilizer and energy cost. A 10-liter effective working volume of a vertical tubular photobioreactor serves as the basis for the estimation. The energy cost is associated with 24-hour daily aeration and LED lighting. Notably, expenditure categories such as investment, labor, and downstream processing cost have been intentionally excluded from this analysis, considering for less developed regions, where home production or micro- or middle enterprises may handle the production process, or if it is intended for direct consumption.

Figure 8 shows the cultivation cost estimation of all treatments. More detail of each cost component, number of photobioreactors and energy required for the cultivation can be seen in Supplement 4. In comparison to the control media cost (EUR 8.24), all tested media exhibited lower cultivation costs, primarily attributed to the considerably lower price of fertilizer. While there was no significant difference in fertilizer costs among the tested samples, the lowest fertilizer cost was presented by all treatments in Ratio-A, with the standard HF ratio (A1) presented the lowest one (EUR 1.42).

The cost for energy of all treatments was significantly higher than that of the fertilizer cost, except for cultivation in BBM that was only slightly lower, which was EUR 4.39 and EUR 3.86 for fertilizer and energy cost, respectively. Energy cost was influenced by the number of photobioreactors utilized for the cultivation, as each requires its own aerator and a set of LED light strips. Another influencing factor is the duration of treatment, which was proportional to the cost incurred for lighting and aeration. While a higher biomass yield may reduce the number of bioreactors needed to produce a kilogram of biomass, an extended cultivation duration negatively impacted the total energy cost (Table 5). Comparing the energy costs of all treatments, the control media shows a moderate cost. For a more detail estimation of cultivation cost, please refer to Supplement 4. The energy cost of the control was comparable to that of most of the tested media, except for the concentration-3 of HF that shows relatively higher (> EUR 4.5).

Table 5. Factors influence the cultivation cost

Parameters	BBM	Ratio-A			Ratio-B			Ratio-C
Parameters	BBM	A1	A2	A3	B1	B2	B3	C1	C2	C3
Biomass productivity (g DW.L-1.day-1)	0.42	0.47	0.42	0.31	0.44	0.49	0.36	0.48	0.42	0.33
Biomass density at peak (g DW. L-1)	6.71	5.65	7.07	9.00	5.78	6.83	9.75	5.77	6.34	8.96
Mass of biomass in 10l PBR	67.11	56.54	70.68	89.95	57.85	68.33	97.45	57.73	63.36	89.65
Number of PBR required to produce 1 kg DW of Biomass	14.90	17.69	14.15	11.12	17.29	14.63	10.26	17.32	15.78	11.16
Time to reach the peak density (day)	16	11	17	29	13	14	27	11	15	27

Regarding microalgae growth rates, the HF ratio shows a significant influence. Notably, the B-ratio treatment demonstrates the highest growth rate, except for the Concentration-3. Table 1 reveals that Ratio-B contains a higher proportion of trace elements compared to other ratios and even BBM. A greater availability of trace metals in the media might have positively correlated to the growth rate as they are essential for microalgal growth (Liu et al. 2017; Silva et al. 2021), although there is no solid opinion regarding the certain amount of trace metals that should be provided. Unexpectedly, higher concentrations of hydroponic fertilizer exhibit a negative impact on growth rates, possibly due to extended cultivation durations.

Despite focusing solely on the ratio and concentration of HF, this study acknowledges the likelihood of several environmental factors impacting growth rates. Firstly, the acidity of the HF was contrast with the alkaline pH of BBM, in which the microalgae are optimally cultivated. Given that Chlorella vulgaris thrives optimally in alkaline pH conditions (Choi and Lee 2011; Yu et al. 2018), the acidic nature of the HF medium during the cultivation study might have contributed to the relatively low growth rates. Secondly, a higher medium temperature during this study gave another drawback for Chlorella vulgaris, which ideally grows within a temperature range of 15-20°C (Zhang et al. 2020).

Addressing the acidic environment, one potential strategy to enhance growth rates in HF cultivation is by adjusting the pH. Rather than introducing alkaline buffers, incorporating an ammonium-based nitrogen source could achieve dual objectives of pH adjustment and growth rate enhancement. Ammonium-based nitrogen sources are known to facilitate faster nutrient uptake and significantly boost growth rates while also increasing microalgae protein content (Li et al. 2019; Lachmann et al. 2019; Salbitani and Carfagna 2021; Villaró-Cos et al. 2024). As for temperature concerns, it is noteworthy that this study was conducted and addressed for cultivation in a tropical region with daily temperatures ranging from 20°C to slightly above 30°C. While lowering temperatures could indeed enhance growth rates, the increase in cultivation costs poses a significant trade-off. Given the study's objective of optimizing production costs for lower-income regions, prioritizing cost-effectiveness outweighs the potential benefits of heightened growth rates.

Regarding the economic analysis of the cultivation, it is obvious that the fertilizer price has made the overall cost of using HF considerably more economically feasible compared to the standard medium. EC is primarily influenced by cultivation duration, with lower initial fertilizer concentrations resulting in shorter cultivation periods and thus reduced energy costs. While the number of PBR required to produce every kilogram of microalgal biomass correlated to the energy expense, the impact of cultivation duration appears to be more substantial.

Surprisingly, growth rate appears to have less influence on this cost analysis. However, it is important to specify that this analysis was conducted exclusively on costs that were directly associated with the cultivation activities and accounted for a single instance of cultivation. Conducting a comprehensive study on an annual basis covering all expenditures including investment costs, production costs, and fixed costs may give different results, and potentially highlight the greater influence of growth rates on the overall SCP production over extended periods.

In comparison to BBM, the utilization of HF as a growing medium for Chlorella vulgaris has demonstrated promising outcomes, provided that the fertilizer's ratio and concentration are appropriate. While the growth rate was significantly influenced solely by the fertilizer ratio, the biomass yields unsurprisingly influenced by the medium concentration, that additionally extend the cultivation period. The cost analysis results suggested that the predominant factor influencing the economic viability of cultivating Chlorella vulgaris in HF is the cost of the fertilizer itself.

Several key points warrant attention for future research endeavors. Firstly, since applying an original formula of HF has given an acidic environment, it is advisable to explore methods for pH adjustment using ammonia-based nitrogen sources. Secondly, while considering the prospective implementation of HF in scenarios involving large-scale production, it is essential to perform a comprehensive economic study that encompasses all conceivable expenses on an annual basis. Such an analysis may clarify the significance of optimizing growth rates within the broader context of economic considerations.

The authors declare no conflict of interest.

Ethical Approval

Not applicable

This research did not involve human and animal.

Funding

This research was supported by funding from the Faculty of Agricultural Technology, Brawijaya University, Indonesia.

Availability of data and materials

Not Applicable.

Main data can be found in the main article. Some additional data related to this research have been submitted as a “Supplement Materials”.

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No competing interests reported.

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Hydroponic Fertilizers as a Growth Media for Chlorella vulgaris for Producing Affordable Protein in Developing Countries

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Abstract

Figures

1. Introduction

2. Materials And Methods

3. Results

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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