The potential of modified karst water as a substitute for microalgae culture media

Karst water concocted culture medium which contains high concentration of complex minerals was presumed to support production of microalgae, such as Spirulina sp. (Arthrospira sp.), Chlorella sp., and Nannochloropsis sp. This study aims to evaluate the potential of enriched karst water medium to grow Spirulina sp., Chlorella sp., and Nannochloropsis sp. There were two batches of culture with different treatments of nutrient supplementary; the first was for Spirulina sp. and the second was for Chlorella sp. and Nannochloropsis sp. The results showed that all treatments were suitable for Spirulina sp. with different growth rates. The medium with 20:1 N/P ratio showed the best productivity for Chlorella sp., and 10:1 N/P ratio for Nannochloropsis sp. The pure karst water and all level as the nutrient modified karst water media showed a relatively similar growth rate of Spirulina sp. Furthermore, in the best medium for Chlorella sp. and Nannochloropsis sp. gave increasing density until the end of observation, instead of a death phase of commercial addition batch culture. Generally, modified karst water has the potential to be used as the microalgae growing medium.


Introduction
Spirulina sp., Chlorella sp., and Nannochloropsis sp.grow quickly and are adapted to a hostile environment, hence the cultivation or production is relatively simple [1].However, on the contrary, the production of microalgae needs costly process [2].The highest cost is obtained from the culture media as nutrient source [3].Generally, to support the productivity, most of the culture media are developed as manufactured fertilizer.Many efforts are developed and many studies were carried out about comparison and evaluation of microalgal growth and its biochemical composition in various media [4,5].
In mass culturing, there are commercial media substitution, such as Chu-12, Guillard F/2, and Walne as nutrient source [6][7][8][9].Culture is also carried out by modifying the conditions of raw water for culture media.For example, light conditioning [10], recycling the culture system [11], or a new media preparation [12].Moreover, the low nitrogen and phosphorus in seawater could be substituted by various waste water or agricultural technical fertilizers [13].Another potential nutrient source is karst water that can be used as growth media of fungus species, Ganoderma iucidum [14].
Karst rocks have a high content of various minerals, such as Ca and Mg [15], that function in the process of forming cell walls and plant chlorophyll [16].Meanwhile, lime is used as neutralizer for the pH value of soil and water [17].Karst water is sourced from ground-water with dilution of calcareous rocks [18,19].It contains high concentration and complex nutrients, with the most of calcium, magnesium, carbon, and oxygen [20]; anions as CO3 2-, SO4 2-, OH, F -, and Cl -; cations, as Fe, Mn, and Zn; and trace elements as B, Ba, P, Mg, Ni, Cu, Fe, Zn, Mn, V, Na, U, Sr, Pb, and K [21,22].It shows a high potential to be utilized as an algae culture media.Although, in high concentration, those minerals inhibit the light absorption of microalgae [23].
Karst area is also widespread in West Java, such as Ciampea and Ciseeng region, with uniqueness of some karst water that the salinity could reach about 30-35 ppt, such as Ciseeng karst water [24].Salinity is a limiting element in the culture of marine microalgae.With the high and complex content of nutrients and the high salinity, Ciseeng karst water provide high potential of developing inland culture of commercial marine microalgae.However, there is no research about utilization of saline karst water as culture media of any marine microalgae.
Therefore, this research was conducted to get an idea of the possible use of karst water, as modifying water conditions and nutrients source of marine microalgae culture medium.The suitable media for the growth of the microalgae is indicated by the productivity or growth rate.To determine the potential of karst water as a medium for supporting microalgal growth, this study was designed to analyze the potential of enriched karst water medium to grow.Spirulina sp., Chlorella sp., and Nannochloropsis sp.

Materials and methods
Preliminary tests were conducted to study the capacity of marine microalgae to live in two karst water sources with different characteristics, the karst water of Ciampea (normal condition with about 26 °C of temperature and 0.0 ppt of salinity) and Ciseeng (high temperature, up to 40 °C and salinity, 30-35 ppt).For the preliminary experiment, modifications were made without significant changes in the physical and chemical properties because karst water has minerals (Table 1) to grow Spirulina sp., which was stocked in saline Spirulina Media (SM) [36,37].Spirulina sp. can survive and grow in the karst waters of Ciseeng.Moreover, to comprehend the productivity of Spirulina sp., Chlorella sp. and Nannochloropsis sp., the Ciseeng karst water was utilized as media in the primary experiment and a simple random in times design [38] was implemented.Two separated batches [39] of experiment based on different initial stocked media (SM for Spirulina sp. and Walne for two others), were set.For Spirulina sp., the treatments were: pure karst water (K100), 75% karst water and 25% SM (KSM1), 50% karst water and 50% SM (KSM2), 25% karst water and 75% SM (KSM3), and pure SM (SM100); with three replicates in 18 days of observation (Day 1 to Day 18).For Chlorella sp. and Nannochloropsis sp., the nutrient treatment given in Walne media were several different compositions of N and P ratios through the addition of technical fertilizers urea and TSP [40,41].The ratio was set with different N concentrations and constant P concentration, at 0.5 mg L -1 .The fertilizer treatment dosages in karst media consist of karst water with N:P ratio of 4:1 (C-K1/N-K1), 10:1 (C-K2/N-K2), 20:1 (C-K3/N-K3), and karst water with Walne culture media (C-KW/N-KW); C code for Chlorella sp. and N code for Nannochloropsis sp. with three replications and 12 days of observation (Day1 to Day12).
The density of microalgae was obtained from spectrophotometric optical density (OD) approach [42].According to preliminary research findings, the optimal wavelengths for Spirulina sp., Chlorella sp. and Nannochloropsis sp. are 680 nm, 684 nm, and 686 nm, respectively.Since Spirulina sp. is a filamentousmicroalgae, no cell count was performed, but rather indirect biomass determination based on OD values [43].Spirulina sp.biomass weighing is not carried out directly because the OD approach is more equated with dry biomass.Meanwhile, the determination of dry biomass directly needs to be combined with wet biomass first, which creates a greater bias.While the density of the two singled cell microalgae were also counted directly using haemocytometer [35].Inoculum of Spirulina sp., Chlorella sp., and Nannochloropsis sp. which were stocked one day prior to the start of observation or day 0 (Day 0) were 38.93 mg dry biomass, 5 × 10 5 cells mL -1 , and 6 × 10 5 cells mL -1 , respectively.In addition, specific growth rate and doubling time [44] of the three cultivated microalgae were calculated, as follows.

μ = (
( ln X ̅ expi -ln X ̅ exp0 ) t ) with: μ = growth rate (day -1 ) X ̅ expi = biomass or density of final day in the exponential phase (g L -1 ) X ̅ exp0 = biomass or density of first day in the exponential phase (g L -1 ) t = time intervals from X ̅ exp-0 to X ̅ exp-i (days) and T 2 = 0.6931 μ with: T2 = coefficient of doubling time (day) μ = growth rate (day -1 ) Water quality measured during the experiment included temperature, pH, salinity, light intensity, and nutrients (ammonia, nitrite, nitrate, and orthophosphate).The nutrient analysis method was carried out based on the standard methods [45].
ANOVA was used to assess the difference in response to each treatment and to ascertain the most efficient treatment, followed by Duncan's multiple-distance test analysis [46].In addition, a matrix approach was utilized to find the best modified karst water to use as an alternative medium for microalgae.A weight and score matrix was also utilized to determine the optimum modified karst water.
The overall weighted value is 20, which is a combination of the last biomass yield value (2), specific growth (2), harvesting time (4), harvested quantity or biomass (4), cost (4), and price (4).A score between 1 and 5 is assigned to each parameter based on its condition.Therefore, the highest value of the multiplication between score and weight is 100.

Results
Increased biomass of Spirulina sp.occurred in all treatments and did not show a downward trend in the last day, with the SM100 treatment had the highest average value (315.449mg L -1 ) and the lowest in KSM1 (218.765mg L -1 ) (Figure 1a).Besides, the density of single cell microalgae increased sharply the day after stocking (Figure 2), with the highest density was reached in Walne media.However, at the end of the observation, there was a sharp mortality phase in the culture with the media.On the other hand, in culture with karst water and technical fertilizers, a stationary phase appeared with a certain pattern; the higher the N:P ratio, the higher the density achieved.In addition, the density of Nannochloropsis sp.relatively higher than Chlorella sp.The changes in biomass and density (Figure 1b, and 3) show the ability of cells to utilize nutrients and the environment to maintain and increase their population, or their inability to maintain their existence, resulting in mortality; represent the growth pattern of cultured microalgae [47].Spirulina sp. has a relatively high growth rate at the beginning of maintenance (Day1 to Day6), but then decreases until the end of the observation (Figure 4a).Spirulina sp. had the highest peak biomass in KSM2 treatment, showed the lowest value in KSM1, and showed significantly different growth rates between treatments (P<0.05).Furthermore, all treatments were statistically significantly different from the control treatment (SM100).While, there were no significant difference for all karst treatment, except KSM2.
Chlorella sp.shows differences in density, both based on treatment and observation time (P<0.05).Whereas, in Nannochloropsis sp. the difference was shown in observation time (p <0.05).A significant difference in density (p<0.05) was found between Chlorella sp. in C-KW with all karst water treatment (C-K1-3).Furthermore, the highest density was achieved on the 5 th day for Chlorella sp. and on the 7 th day for Nannochloropsis sp.(Figure 4b).
The presence of nutrients appears to be suitable for the growth of all algae, which is indicated by the strong relationship between biomass or density and available nutrients (Figure 5-7).This shows that all microalgae are able to utilize the available nutritional sources in each treatment, as needed.Meanwhile, the environment, which was an important factor in culture media [48] of all treatments, was controlled according to culture activity (Table 2).

Discussion
The microalgae community is relatively responsive to water quality changing [49,50] that were controllable to support microalgae growth at all treatments.This lead high photosynthetic activation, support the optimum growth [51], and avoid the lysis of microalgae cells, although there was an increase as a result of the addition of SM to the Spirulina sp.culture medium.
The salinity in karst water were set at 20 and 28 ppt to support the optimum growth of marine microalgae [52].Salinity conditions must be regulated to avoid hyperosmotic which results in lysis of microalgae cells [53].Furthermore, the measured pH value in the range of indicates the optimum range [54].However, there was an increasing of the pH that related to enhancement of SM in the Spirulina sp.culture.
Microalgae growth must be supported by the availability of dissolved inorganic nutrients in karst water media [55].Spirulina Media (SM) and technical fertilizers, such as urea and TSP are added to ensure the inorganic nutrients requirements of microalgae.SM is a commercial fertilizer specially formulated for Spirulina with a complex nutrient composition.Urea is a nitrogen substance, while TSP (Triple Super Phosphate) is a phosphorus source.The change of nitrogen or phosphorous composition will lead the variation of the ratio, that will influence the growth of microalgae [56].
In all treatments, Spirulina sp.demonstrated the ability to utilize nutrients.Microalgae require ammonium, nitrate, nitrite, and orthophosphate as primary nutrients.The growth of Spirulina sp.indicated to be effective in nutrients utilization, and there was not any declining phase for all treatments.Limnetic (lakes or ponds) of tropical area [57], estuary, and saltwater environments are places which Spirulina sp. is usually found.Spirulina sp. is one of dominant species in bloom occurrence [58].In this experiment, the Spirulina sp.inoculum was classified as brackish to marine organisms able to survive in media with the salinity of 15-20‰.The Spirulina Media (SM) is a manufactured culture medium that provides salinity of 20‰ and pH of 10 [36].
Differences in media conditions in each treatment affect differences in the growing rate of cultivated Spirulina sp.The pH of the medium decreases as the SM concentration in the modified karst water decreases.Therefore, the treatment P1 (media containing pure karst water) had the lowest pH.However, the pH condition was not a key factor for Spirulina sp.since the setting of culture media with pH range of 6-10 is suitable for the algae growth [48].
The population of Spirulina sp. which tends to increase in all treatment media appears to continue until the end of the observation, hence no specific best time to harvest [47].The growth of a type of algae is strongly influenced by conditions of the environment or the conditions of the culture media [59,60].The exponential phases of Spirulina sp. were started on day one of the culture periods and ended at different times (Figure 4a).The difference in biomass and growth rate between SM culture and nutrientmodified karst water media was not statistically significant (P>0.05).This is thought to be related to nutritional adequacy in all treatments [61,62].This suggests that Spirulina sp. is able to utilize the nutrients contained in pure karst medium without requiring additional nutrients, and have relatively the same productivity as those grown in nutrient-modified karst water media.The relationship between dry biomass and nutrients is shown in Figure 5, 6, and 7.
The abundance of Chlorella sp. and Nannochloropsis sp. on karst media using Urea and TSP was still lower than the use of Walne media.Minerals Ca(NO3)2, MgSO4, KH4PO4, KNO3, and FeCl3 are available in Walne media [63], so they are beneficial for growing Chlorella sp. and Nannochloropsis sp.However, in this batch culture, the availability of nutrients from Walne's media was not sufficient by day 12 (Figure 4b).Both types of microalgae show a mortality phase, while the population grown on technical fertilizer media tends to increase.
The highest density of Chlorella sp. was indicated by medium with an N:P of 20:1, which differed significantly from other treatments (P<0.05); as well as Nannochloropsis sp., but did not show a significant difference from other treatments (P>0.05).Thus, the karst water media with the addition of urea and TSP fertilizers with an N: P ratio of 20:1 became a proper culture media for both types of single cell microalgae.Furthermore, days 5 and 7 indicate the best time to harvest.
The relationship between cell density in culture and the availability of nitrogen and phosphorus is close, as shown in Figure 5-7.It can describe the relationship between these two components well and show the close relationship.However, there was an inconsistent relationship in the treatment of N:P 20:1 for Chlorella sp. and the treatment N:P 10:1 for Nannochloropsis sp.
Nitrate and orthophosphate are limiting factors in microalgae culture.The nitrate and orthophosphate content in this treatment was at the optimum level for Chlorella sp. and Nannochloropsis sp.growth [64].The optimal growth of microalgae requires nitrate concentration of 0.9-3.5 mg L -1 and orthophosphate of 0.09-1.80mg L -1 [65].If less than 0.02 mg L -1 , orthophosphate will be a limiting factor [66,67].The decrease in nitrate and orthophosphate concentrations can be caused by the utilization of microalgae as essential nutrients for growth [64].
Chlorella sp. was able to adapt faster than Nannochloropsis sp.The culture of Chlorella sp. in this study had a stationary initial time of less than one day, as the results of other researches [68,69].The doubling time value of this study that ranged from 1.33 to 2.11 days were close to the value of other research result [68].
Ciseeng karst water has a significant mineral content, making it a unique freshwater.The minerals come from dissolving eroded rocks [15,20], and the seeped mineral could rise the salinity to 30 until 35‰.Furthermore, Ciseeng karst water temperature is relatively warm at approximately 40°C [70].Ciseeng karst water with these characteristics, which are similar to sea water, has been shown to support the growth of marine microalgae.However, the temperature should be adjusted to suit the requirements of microalgae life.
The highest biomass was reached by Spirulina in treatment SM100 with less growth rate than in KSM2 and KSM3.The biomass performance of Spirulina was not significantly different in those three treatments, with a lower value than SM100.It showed that karst water without enrichment was suitable for growing algae.
A similar pattern is also found in single-cell microalgae cultures.Although the highest density was reached in karst water and Walne media, the Chlorella sp. and Nannochloropsis sp.sharply dropped at the 12 th day.Space-dependent was not shown in the cultivation without the addition of commercial fertilizers.The microalgae density showed an increase on the 12 th day with a slight difference in the pattern between the two.Best performance for Chlorella sp. is shown in N:P 20:1 (C-K3), while Nannochloropsis sp. with an N:P of 10:1 (N-K1).
There is an assumption that the culture media sourced from karst water give rise to the phenomenon of slow release of nutrients needed by microalgae to grow.The lifetime of microalgae becomes longer than those cultured with commercial fertilizers.This is suggested that pure karst water can be used as a culture media for filamentous-microalgae, while karst water with the addition of urea and TSP can be used as a media for single-cell microalgae culture.The application of potential natural resources, such as karst water, can be an alternative to reduce the commercial fertilizer.
The most effective culture media were chosen based on the ranks of final values according to matrix.The value was calculated by the multiplication of the weight and score (Table 3).The highest final matrix value was determined from the pure karst media for Spirulina sp., the karst water with N:P of 20:1 for Chlorella sp., and the karst water with N:P of 10:1 for Nannochloropsis sp.It is concluded that karst water could function as an alternative medium for growing marine microalgae as a substitute of manufactured commercial media.

Figure 1 .
Figure 1.Dry biomass and biomass difference of Spirulina sp. in all modified karst water cultures.

Figure 6 .
Figure 6.Relationship between density of Chlorella sp. and nutrients (N and P) in all N:P ratios.

Figure 7 .
Figure 7. Relationship between density of Nannochloropsis sp. and nutrients (N and P) in all N:P ratios.

Table 1 .
Comparation between seawater characteristics karst water of Ciseeng and Ciampea.

Table 2 .
Water quality of culture media.