Nitrogen and Phosphorous fertilizers on Nannochloropsis occulata Growth

Justin Chan, Year 2 Research

Abstract

Algae have numerous pharmaceutical, dietary, agricultural, and environmental uses. Microalgae Nannochloropsis occulata has shown this potential for use in agricultural feed, a dietary supplement for omega-3 fatty acids and the synthesis of pharmaceutical compounds used to combat cancer. Because of these important properties, it is increasingly important that we understand the cultivation requirements of these photosynthetic organisms. In this experiment, different amounts of nitrogen (Ca(NO₃)₂) and phosphorous (P₂O₅) were used to cultivate Nannochloropsis occulata. Solutions were derived from nutrient levels during algal blooms and followed a 7:1 nitrogen to phosphorous ratio deemed suitable for algal growth. Concentrations of samples were obtained by manual cell count using a hemocytometer. Results of the experiment tentatively suggest that nitrogen has a greater impact on algal growth than phosphorous. The results of this experiment contribute to existing knowledge and pave many more directions of study for growing algae in closed system.

Introduction

Algae are a diverse group of autotrophic organisms that can inhabit “inhospitable” environments anywhere from parched deserts to the brittle cold of polar regions (Cannell, 1993). However, these photosynthetic organisms are most commonly found in aquatic environments (Tan et al, 2020). Despite dismissive names like “seaweed”, “rockweed”, and “pond scum” (Chapman, 2010) used to describe some of the known 30 000 species (Cannell, 1993), algae —more specifically the unicellular microalgae— have numerous useful properties. These properties include: the extraction of heavy metals (El-Baroty et al. 2007; Abdel-Raouf et al. 2012), secondary and tertiary wastewater filtration (Rani et al. 2019; L. Wang et al. 2009; S. Wang et al. 2018), production of over half of the world’s oxygen (Chapman 5-12), CO₂ capture to limit the effects of pollution (Alami et al, 2021; Moreira and Pires 2016; Chung et al. 2010), use in livestock diets as a nutritional supplement or as an economical meal substitution (Sarker et al. 2018; Kholif et al. 2020), cytotoxic and antimicrobial abilities (El-Baroty et al. 2007; Gnanakani et al. 2019), and have potential to produce useful biochemicals and biomolecules like omega 3 fatty-acids (Lane et al. 2014; König and Wright 1993; Cardozo et al. 2007; Metting and Pyne 1986; Cannell, 1993). Furthermore, algae has seized the attention environmental scientists due to the global uptick in harmful algal blooms (Hallegraeff, 1993; Peperzak, 2005; Hallegraeff et al. 2004), which has the potential to negatively affect the economy (Larkin and Adams; 2005) and health (Heisler et al. 2008) of surrounding communities. Currently, large-scale cultivation of these photosynthetic organisms commonly relies on outdoor, open systems like ponds. These open systems do not protect the culture from contamination from chemicals or other microorganisms, making them unsuitable for the subsequent production of temperamental biochemicals (Johnson et al. 2018; Rocha et al. 2003). While the closed system of photobioreactors (PBRs) (systems that grow algae in closed glass tubes or panels) provides solution for the issue of contamination, the specifics of nutrients, hydrodynamics, and light requirements are not fully understood (Rocha et al. 2003; Fang et al. 1993). Information of the nutritional needs for algae growth is also needed as both the effects of nitrogen and phosphorus on harmful algal blooms is not clearly understood for specific microalgal species (Conley et al. 2009; Fang et al. 1993). This study aims to gather data on the effect of macronutrients nitrogen and phosphorous in the forms of calcium nitrate (Ca(NO₃)₂) and phosphorous pentoxide (P₂O₅) respectively, on microalgal species Nannochloropsis occulata that share many of the pharmaceutical, agricultural, and ecological potentialities of other microalgae species (Gomaa et al. 2018; Wild et al. 2018; Kholif et al. 2020; Sarker et al. 2018; Gnanakani et al. 2019). The results of this study will be especially pertinent towards those looking to design a nutritional plan to maximize algal growth within a closed culturing system.

Materials and Methods

Three experimental groups and one control group medium were formulated in this experiment. Common to all three solutions was 1.5 L of sea water collected at a shoreline park located in Port Moody, British Columbia and a modified f/2 growth media formula (“f/2 Nutrient Solution”) (Algae Research Supply). The 1.5 L of water was boiled for five minutes to ensure sterilization and cooled to room temperature prior to the addition of nutrient solution to prevent the degradation of heat sensitive vitamins.

The ratio of nitrogen (N) to phosphorous (P), 7:1, was derived from the “Klamath River Modelling Project” report that also suggests that 0.01 mg/L of phosphorous concentration will support algal species (Deas and Orlob 56-61). Another study by Nuzzi and Waters was consulted in which the researchers studied Aureococcus anophagefferens biomass and nitrogen concentrations during algal blooms. In their data, there was a relationship between a nitrogen level of 0.4 mg/L and the largest biomass of Aureococcus anophagefferens during the year (Nuzzi and Waters, 2004). 0.4 mg/L would be the highest tested nitrogen amount for the samples. Using the 7:1 ratio, the highest phosphorous amount would be 0.06 mg/L.

Two fertilizers were used. Calcium Nitrate with a 15-0-0 macro-nutrient ratio (Jon’s Plant Factory) and Phosphorous Pentoxide with a ratio of 0-9-0 (Gaia Green). To measure the correct amount of nutrients for each sample, the fertilizer ratios —that indicated the percentage of those nutrients in the dry weight of fertilizers— were calculated and weighed using a mechanical balance (Ohaus). Fertilizers were then boiled in the collected sea water to allow the nutrients to properly dissolve. This concentrate fertilizer solution was then added to the f/2 and “Algae growth medium” solution to achieve dilution for the appropriate amount of nutrients. 100 mL was poured into clear, 295.7 mL cups (Hefty) that were covered tightly with food film (Resinite) to prevent evaporative loss and simulate a closed growing chamber. Three samples per group were made.

Cups were placed in four separate rows and a 24W Led grow light (Moniois) was placed on top of the cups such that at least one cup from each group sat directly under the light. The light was said to be full spectrum by the company however the photosynthetic active radiation (PAR) was not measured. The colonies only received light from this source aside from ambient room lighting. Room temperature would ranged from 14ᵒC-22ᵒC. Cells were lightly swirled twice: the first time was three days after cultures were placed into the cups and the second occurred on day 18 after noticing the clumping algae sediment.A manual cell count was performed 23 days later through a hemocytometer and microscope. To prepare cultures for placement on the hemocytometer, cells were suspended into the water column by pipetting and stirring the water; this was done for 30 seconds for each sample.  12 squares were counted and chosen from each corner of the hemocytometer. Clumps of cells were counted by counting the visible number cells in the clump. Data was recorded using a digital table (Table 1).

Results

As can be seen in Fig. 1, the group with 0.40 N/0.01 P or an elevated amount of nitrogen and the standard level of phosphorous, experienced the most algal growth as it had an average density of 613 889 cells/mL. The group with 0.07N/0.06P that had an elevated amount of phosphorous and an ordinary amount of nitrogen, did not experience the same increase in growth as seen with the higher nitrogen groups, having the second lowest cell densities. The control group (no fertilizers added) had the least growth, averaging 316 667 cells/mL almost have that of 0.07N/0.06P. Between each of the groups, the group with 0.40N/0.06 P (elevated amounts of both nutrients) had the most variance in samples in which both the lowest (sample 1) and highest (sample 2) concentrations of all samples tested were present in the same group; this discrepancy between samples can be visualized by Table 1 and Figure 2.

Table 1. Number of squares used for algal counts.

Figure 1. Average number of cells per mL in the groups.

Figure. 2 Concentration of Nannochloropsis occulata in each sample

Discussion

The results showed that group 2 with increased nitrogen, had the most algal growth while group 1 with increased phosphorous, had the least algal growth compared to the control group. These results suggest that nitrogen may spur algal growth more than phosphorous. However, this conclusion is made tentatively, as there have been other studies have shown that phosphorous plays an important role in algal growth, and that the absence of it can prevent algal blooms from happening (Schindler 1974; Edmondson 1970). Conversely, there is also literature that supports the importance of nitrogen in algal growth (Soto 2002; Ahlgren 1978; Schindler 2006). The studies mentioned above, were conducted on open systems, such as lakes, ponds, or other bodies of water. Research on nutrients in closed systems like photobioreactors is scarce, so information on algal nutrient requirements must be extrapolated from these studies that show both nutrients —nitrogen and phosphorous— contribute to the growth of algae. Interestingly, this experiment did not reflect the positive effect of phosphorus on N. occulata growth.

Upon further analysis of the results in this study, there appears to be inconsistencies between the samples in the group with 0.40N/0.06P (elevated amounts of both nutrients. This may have been due to errors during the mixing of media nutrients or improper suspension of cells before placement on the hemocytometer. This outlier may have skewed the results of group 3 as only the average was calculated from each group. More samples are needed to decide if phosphorous pentoxide has a negative or no effect on algal growth. If anything conclusive can be drawn from the results of this experiment, it is that more research is needed to explore the relationship of nitrogen and phosphorous on algal growth. To approach this question, future experiments would test the optimal ratios of nitrogen to phosphorous. This would be done, by changing the increasing or decreasing the amount of nitrogen added to a fixed amount of phosphorous. Data would then be collected to over three-day intervals to calculate the rate of growth of the algae and determine when algal growth plateaus. It may also be beneficial to study different sources of nutrients such as whether an ammonia supply of nitrogen would be more beneficial than a nitrate-based fertilizer. Further, the method of data collection would also need to be improved in order to find a more reliable way to measure growth beyond mechanical cell counting. In this experiment, there is a degree of human error which may skew results such as the over or undercounting of cells, changes in the location in the water column in which cells were pipetted for viewing, and the perceivers ability to distinguish cells from other contaminant particles. During the experiment, concerns of the subjectivity of cell counting came into fruition when the unicellular Nannocropolis occulata clumped into indistinguishable masses. While counting, only the cells visible, usually the cells around the perimeter, were counted. In addition, during the actual mixing process of the growing medium and the handling and sourcing of the salt water was not done in a sterile environment, therefore there may have been contaminants unknowingly introduced into the samples. The limitations of this study are the long-term growth behaviour, of these photosynthetic organisms, that would provide valuable data on the longevity of these nutrients in closed systems like photobioreactors and how often they would need to be replenished. Further, the relationships of the nutrients in the growing media. While this study has information on the growing media and the N and P levels of each sample, the initial composition of the collected sea water remains unknown. The inability to measure the nutrients in the water, presence severe limitations in addressing whether it is N, P, or another nutrient that is limiting the growth of these algal cultures.

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