Sustainable fish feeds: optimization of levels of inorganic fertilizers for mass production of Oocystis sp. for climate smart aquaculture

Use of microalgae as a source of food in aquaculture production is gaining recognition due to their rapid growth rate that promises high biomass generation within a short time. The challenge faced is getting good and inexpensive nutrients source to be used in mass production of the required microalgae. This study investigated the effect of different nutrient combination in influencing the growth rate of the of the green algae Oocystis sp. which has been identified as a possible protein source for the raising of Orechromis niloticus fingerlings for fish farming. Modified Bolds 3 N Medium and commercial agricultural fertilizers (urea, NPK and DAP) media were compared to establish the appropriate combinations that would result into high biomass generation but at the lowest cost possible. The Modified Bold 3 N Medium acted as the control, at a cost of 11.28 KSh per litre; the other media were derived from urea, NPK, and DAP (varying the ratio of each) at a cost of treatment 1 (0.14 KSh per litre), treatment 2 (0.18 KSh per litre), and treatment 3 (0.22 KSh per litre). The algae was cultured for 5 weeks with samples taken daily for biomass analyses using chloropyhll-a concentration as the surrogate for Oocystis sp. biomass for 30 days, from each treatment was determined. The growth rate, doubling time, and divisions per day were then estimated based on this chlorophyll-a concentration. The results showed that the mean concentrations of chlorophyll-a in treatment 1 was highest (7.715 ± 0.667 µg/ml), while treatment 3 (6.441 ± 0.555 µg/ml) had the least. There were no significant differences in the mean concentrations of chlorophyll-a in the four treatments (Kruskal–Wallis H test: P > 0.05). The chlorophyll-a concentration varied significantly in each treatment with time (Kruskal–Wallis H test: P < 0.001). There was no significant difference in the growth rate (Kruskal–Wallis H test: P > 0.05), divisions per day (Kruskal–Wallis H test: P > 0.05), and doubling time (Kruskal–Wallis H test: P > 0.05) from the different treatments. The results of this study showed that inorganic fertilizers can be used as cost-effective media in the mass scale culture of Oocystis sp.


Introduction
Microalgae are associated with diverse applications such as biodiesel production and animal feed sources due to their faster growth rates that yield high biomass within a short time (Mata et al. 2010;Khan et al. 2018). Some species of microalgae have high lipid accumulation in their dry cell weight ranging between 50 and 60% that makes them good candidates for use as energy sources (Hu et al. 2008;Mata et al. 2010). Saadaoui et al. (2021) observed that some microalgae have been used as food in livestock, poultry, and aquaculture production due to their diverse nutritional properties. For example, Chlorella vulgaris was used as a substitute for fish meal in feed for Clarias gariepinus (Enyidi 2017). In another study, El-Sheekh et al. (2014) used Arthrospira platensis as feed for hybrid red tilapia (Oreochromis niloticus × Oreochromis mossambicus). These studies revealed that the inclusion of algae in the feeds was beneficial to the cultured fish through an increase in the feed conversion ratio.
Some other species of such as Oocystis sp. has been reported to have varying amounts of proteins in the cells, ranging between 15 and 40% ( Lee and Picard 1982). The differences in the protein contents have been attributed to the culture medium. However, Oocystis sp., incorporation in fish feeds has not yet been done investigated comprehensively.
The expansion of microalgae production is critical in any perceived applications, hence the need for development of cost-effective media for microalgae cultivation. The factors that affect microalgae growth are namely light, temperature, and nutrients (da Silva Ferreira and Sant'Anna 2017; Gani et al. 2019;Kazbar et al. 2019;Chowdury et al. 2020). For their nutrient requirements, this varies with each algal species having a specific requirement to optimize growth (Ghafari et al. 2018;Khan et al. 2018). Nitrogen and phosphorus are essential as they are limiting factors to microalgae growth (Yaakob et al. 2021). Consequently, nitrogen and phosphorus sources are usually emphasized while upscaling the culture of microalgae.
Raising interest in the use of inorganic fertilizers in microalgae culture, as a source of nutrients, which stands out as a major limitation in large-scale production of algae (Hu et al. 2008;Mata et al. 2010;Ravindran et al. 2016), is critical. Various studies have been done assessing the use of inorganic fertilizers (either singly or combined) in microalgae culture (Ashraf et al. 2011;Nayak et al. 2016;Michael et al. 2019).
The inorganic fertilizers commonly used in mass-scale production are urea, diammonium phosphate (DAP), and NPK (Ansari et al. 2017;Arenas et al. 2017;Win et al. 2018;Renuka et al. 2018). Urea is a nitrogenous fertilizer that consists of 46% of nitrogen (having the highest nitrogen content). DAP is important as it provides both nitrogen and phosphorus for growth, found having different ratios depending on the fertilizer's end-use. NPK is also an inorganic fertilizer that is wholesome as it provides nitrogen, phosphorus, and potassium nutrients for the growth of organisms, with individual concentrations differing based on the end-use. Different concentrations of the inorganic fertilizers affect the growth of microalgae; further, a combination of different fertilizers is used to provide microalgae with all the nutrients required for growth.
The aim of this study was to establish the optimum level of inorganic fertilizers combination that could be used in the mass production of Oocystis sp. to give maximum yield, by focusing on urea level inclusion, with the ultimate goal of upscaling microalgae culture using cost-effective media.

Study area
The study was conducted at Egerton University in the Biological Sciences Department laboratories. The university is located in Nakuru County, Njoro Sub-County, and is approximately 25 km south-west of Nakuru city, at an altitude of 1890-2190 m above sea level. The area's temperature ranges between 17 and 27 °C (Waithaka et al. 2017).

Culturing of Oocystis sp.
The algae was cultured using the Modified Bolds 3 N Medium and commercial agricultural fertilizers (urea, NPK and DAP) media for a period of 5 weeks. The inoculum for culturing Oocystis sp. was obtained from the University of Texas Culture Collection of Algae (UTEX). The Modified Bolds 3 N Medium (UTEX, n.d.) was used as a control. The other treatments were as shown in Table 1. Pure cultures were raised through isolation and growing the culture in the incubators in the laboratory to raise suitable biomass before transferring them into 3-L plastic bottles as growth chambers or bioreactors containing the culture media.

Sampling and biomass estimation
Samples were taken daily after 24 h for 30 days, where 20 ml was filtered through glass fibre carbon filters from each treatment bottle. The Oocystis sp. biomass was analysed through determination of chlorophyll-a concentration which was done according to the standard method as given in the American Public Health Association (APHA) (2005). Extraction of the chlorophyll-a was done using acetone, where the filters and seston were folded and covered by aluminium foil and stored in a freezer overnight to aid in the bursting of the cells. The seston and the filters were then homogenized in a tissue grinder (Heidolph, 637 69, Germany) at around 5000-rpm where 5 ml of 90% aqueous acetone was added. The samples were then transferred into a centrifuge tube, the grinder rinsed with 90% acetone (volume used was noted), and the rinsed slurry was added to the extraction slurry. The volume was then adjusted to 10 ml with 90% acetone and left for at least 8 h in the dark at 4 °C for full chlorophyll-a extraction. After incubation, the samples were centrifuged for 10 min at 3000 rpm. The clarified extract was decanted into a clean test tube. The light absorbance of the chlorophyll-a extract was measured with a spectrophotometer (Pharmacia Biotech Novaspec II, Sweden) at 750 nm and 663 nm.
Chlorophyll-a concentration was calculated using the following equation according to Talling and Driver (1963): After which, the chlorophyll-a concentration was used to derive the growth rate, divisions per day, and the generation time as critical parameters of importance. These were all derived from the equations developed by Levasseur et al. (1993): where N1 and N2 is the biomass at time 1 (t1) and time 2 (t2) respectively.
Divisions per day and the doubling time were calculated based on the specific growth rate.
where Ln2 is the natural logarithm to the base 2

Statistical analyses
The biomass, growth rate, divisions per day, and doubling time of Oocystis sp. cultured in the different treatments were compared using the Sigma Plot software (version 14) through the Kruskal-Wallis H test, as the data failed normality test (Shapiro-Wilk: P < 0.050).

Water quality
Mean temperature during the study ranged from 24.23 to 27.33 °C. Temperature variations during the study period were not significant among the treatments (Kruskal-Wallis H test: P > 0.05). The pH for treatment 1 ranged from 6.8 to 7.7, where there was a steady increase from the beginning of the study. On the other hand, pH for treatment 2 ranged from 6.6 to 7.4, and treatment 3 ranged from 6.4 to 7.2, all following a similar trend as that of treatment 1 characterized by an increase in the pH. Finally, the control treatment had a pH ranging from 6.1 to 7.1, also having a similar trend in time with the other three treatments. (1)

Comparison of chlorophyll-a concentration at different fertilizer treatments
The concentrations o of chlorophyll-a in treatment 1 was highest (7.715 ± 0.667 µg/ml) followed by the control (6.963 ± 0.788 µg/ml) and treatment 2 (6.862 ± 0.617 µg/ml), with the least in treatment 3 (6.441 ± 0.555 µg/ml). There were no statistically significant differences in mean concentrations of chlorophyll-a among different treatments (Kruskal-Wallis H test: P > 0.05).

Temporal variations in chlorophyll-a concentration
Generally, the concentration increased gradually in all treatments until it reached its peak on different days for all the treatments. The control treatment (Modified Bolds 3 N Medium) achieved the highest chlorophyll-a concentration on the 25th day (12.977 ± 0.788 µg/ml). Treatment 1 followed, reaching its highest chlorophyll-a concentration on the 17th day (11.4437 ± 0.667 µg/ml). Treatment 2 achieved its highest concentration on the 21st day (11.3696 ± 0.617 µg/ml), and finally, treatment 3 had the least concentration, which was achieved on the 16th day (10.2714 ± 0.555 µg/ ml). Overall, the chlorophyll-a concentration in all the treatments fluctuated once the optimum concentration was achieved. At the end of the experiment, the concentration of chlorophyll-a in the treatments followed a similar pattern, with the control having the highest (11.2442 ± 0.788 µg/ ml), while treatment 3 had the least (5.8197 ± 0.555 µg/ml) (see Figs. 1, 2, 3 and 4).
The chlorophyll-a concentration varied significantly in each treatment with time (Kruskal-Wallis H test: P < 0.001) for all the treatments. At the beginning of the study, all the treatments had equal chlorophyll-a concentration (where 100 ml of stock Oocystis sp. cultured having a concentration of 10.0719 µg/ml was introduced into 2.49 L of respective treatment media).

Growth rate, divisions per day, and doubling time
The maximum, mean, and least values of the growth rate, divisions per day, and doubling time in different treatments are shown in Table 2. There was no significant difference in the mean growth rate (Kruskal-Wallis H test: P > 0.05), divisions per day (Kruskal-Wallis H test: P > 0.05), and doubling time (Kruskal-Wallis H test: P > 0.05) for the treatments.

Media cost and biomass
The control (Modified Bold 3 N Medium) was produced at a cost of 11.28 KSh per litre; the other media were derived from urea, NPK, and DAP at a cost of 0.14 KSh per litre for treatment 1, 0.18 KSh per litre for treatment 2, and 0.22 KSh per litre for treatment 3. Distilled water was generated by a distiller in the laboratory for use in generating the control media (Modified Bold 3 N Medium), while harvested rain water was used for the experiment for treatments 1, 2, and 3; hence, their costs were not captured in the above calculation.

Discussion
The nature of the culture media is the main determining factor in the growth of microalgae, their productivity, and ultimately their biomass as long as the pH, light intensity, and temperature needs have been fulfilled (da Silva Ferreira and Sant'Anna, 2017; Gani et al.   Latala et al. (1991) found that the growth of Oocystis sp. was completely inhibited at high light intensities ranging between 270 and 380 μE/m 2 s. pH influences the quantity of free carbon in the culture media and the balance between carbonate and bicarbonate. In this study, the pH was adjusted to range between 6.0 and 8.0, as reported by RAo (1963), to suit the growth of Oocystis sp. optimally. Mayo (1997) found similar results while culturing Chlorella sp. with the maximum growth rate at pH ranging from 6.31 to 6.84. Changes in the media pH were attributed to the photosynthetic and decomposition process of the cultured Oocystis sp. ( Tucker and D'Abramo 2008;Rahardini et al. 2018).
During the study, the water temperature range was in the optimal range as reported by Nalley et al. (2018), where they found the best temperature for optimum growth of Oocystis sp. ranged between 26.60 and 27.78 °C. Chowdury et al. (2020) state that temperature is critical in microalgae culture as it affects the algal growth rate, cell size, biochemical composition, and nutrient requirements.
The temporal chlorophyll-a concentration varied amongst the treatments, mainly due to the different constituents in the culture treatment media, with the differences being significant with time in all the treatments. Sutkowy et al. (2019) state that the composition of culture media influences the growth of micro-algae in vitro. Overall, all treatments followed a similar trend where there was a decrease in the chlorophyll-a concentration once it was at its highest biomass. The pattern achieved is similar to that obtained by Rahardini et al. (2018) while assessing the growth of Chlorella sp., which is also found in the Chlorophyceae class of green algae.
At the beginning of the experiment, there was a steady increase (exponential phase) in the chlorophyll-a concentration due to sufficient nutrient concentration in all the treatments. There was no lag phase observed during the study, which can be attributed to the conditions of the inoculum (Spencer 1954). Talling (1966) notes that an inoculum obtained from a healthy exponentially growing population is unlikely to have a lag phase when transferred to a freshly prepared media under similar growth conditions (light, temperature, and pH); consequently, there is no need for physiological adaptations for growth. The lack of a lag phase is crucial as it reduces the time required for upscaling the culture, allowing the harvesting of cultured cells sooner. The exponential phase is characterized by the production of materials that are also capable of growth (Fogg and Thake 1987), with its length depending on the size of the inoculum, the growth rate, and the capacity of the medium and culturing conditions to support algal growth. The exponential phase was followed by the stationary phase, characterized by a decline in population growth, resulting from reduced nutrient concentration in the culture media, causing the number of cells to remain constant. The stationary phase is characterized by zero net growth, as the rate of the growth of the cells is equal to the rate of cell death (Nyström 2004;Maier and Pepper 2015). Finally, the death phase follows, as nutrients in the culture media run out, resulting in the death of cultured algal cells; therefore, a decline in the chlorophyll-a concentration was witnessed. The death rate of cells in the media became higher than the rate of cell growth (Maier and Pepper 2015).
The study demonstrated that the inorganic fertilizers' specific compositions and comparison between the culture media did not have any significant impact on the growth rate, divisions per day, and the doubling time of cultured Oocystis sp. Similar results were also obtained by Rahardini et al. (2018) who also found no significant differences in the growth rate and doubling time of Chlorella sp. cultured in media with different compositions. The divisions per day obtained was higher than those from Csavina et al. (2011), which can be attributed to the continuous agitation of the algal cells during culture, consequently not allowing the clamping of cells together, resulting in deaths and ultimately decline in the growth rate and divisions per day. Research carried out by Sobczuk et al. (2006) showed an increase in the growth rate of microalgae with an increase with agitation; it should also be noted that excessive agitation can result in cell death.

Conclusion
This study found that inorganic fertilizers can be used as an option of cost-effective media in the culture of Oocystis sp., with the ratio of 3:1:1 while using NPK, urea, and DAP fertilizers respectively to achieve the highest biomass within the shortest period. The finding that there were no significant differences in the Oocystis sp. biomass using either medium is an encouraging result as it offers opportunities to explore the use of low-cost media which is equally competitive with the standard media in mass production of Oocystis sp. for potential use in fish feeds in Aquaculture.