# Effects of different nitrogen/phosphorus ratios on the growth and metabolism of the microalgae Scenedesmus obliquus cultivated in the wastewater mixture from primary clarifiers and sludge thickeners (2023)

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Banda 170,

February 2023

, pages 824-833

## Resume

Microalgae have great potential in wastewater treatment and resource use, but few studies have examined the mechanisms of action of pollutants in their biochemical compositions. In this study, the effects of different ratios of nitrogen to phosphorus (N/P) on microalgae were investigated.let's go to the sideSubstances that grew in the mixed primary clarifier (PSW) and sludge thickener (STW) wastewater are examined. The optimal N/P ratio for the growth ofS. obliquawas determined by nutrient utilization rates and the growth curve. Based on this, the effects of different N/P ratios on their biochemical compositions were studied and the ratios were further analyzed using proteomics. The results showed that at N/P=18, the growth ofS. obliquaoptimum of war (OD680=2.34). And the dry weight, lipid production and accumulation of chlorophyll-a were the highest (1.70 g L-1, 0,49gL-1, 7,43 mg·l-1), while protein production was the lowest (0.10 g L-1). The proteomic results showed that the metabolic activities ofS. obliquaCells were more active at N/P=18. Productivity processes were involved in up-regulating proteins, causing more energy to be produced. In addition, lipid metabolism and fatty acid synthesis pathways were strengthened. The ribosome synthesis activity increased significantly, but the protein content was low, which may be due to the energy generated by the production activity for other activities.

## Introduction

With population growth and the rapid development of the global economy, energy shortages and environmental pollution have become the main challenges facing the world (Ding et al., 2019, Jacob et al., 2021). Clean and green energy research has become an important research focus that urgently needs to be addressed (Duarah et al., 2022). Biodiesel as a non-toxic, renewable and degradable fuel has attracted much attention (Ong et al., 2021). However, the application of biodiesel is limited due to the low lipid content of traditional raw materials and the high demands for water, land and fertilizers (Khan et al., 2018).

As a third generation biomass energy feedstock, microalgae have the advantages of high lipid content, fast growth rate and high tolerance to contaminants (Ananthi et al., 2021, Li et al., 2022). Furthermore, microalgae can use carbon, nitrogen and phosphorus in wastewater as primary nutrients to support their own growth and achieve pollutant removal in wastewater, helping to offset environmental pressures and reduce costs (Clarens et al., 2010, Nie et al., 2020, Su, 2021, Zhou et al., 2023). It is a viable way of using resources to combine microalgae cultivation with wastewater treatment to produce biodiesel, which has great research potential and application perspectives (Bohutskyi et al., 2015, Gao et al., 2021).

Most of the waste water is collected through the network of pipes to the waste water treatment plants (ARA). The focus of wastewater treatment is naturally on wastewater treatment plants. The pursuit of efficient, low-carbon and energy-saving resource recovery has become a new direction (Jin et al., 2014, Liu et al., 2021). Studies have reported that wastewater from various treatment units can be used for microalgae growth (Abdelfattah et al., 2023, Kehrein et al., 2020, Mahapatra and Murthy, 2021). Thickened sludge effluents (STW) are rich in nitrogen and phosphorus (Liu et al., 2022, Pittman et al., 2011, Zhang et al., 2021), making them difficult to manage and their equipment costs are high (Wild et al., 2021). al., al., 1997; Liu et al., 2022). The studies by Min et al. (2011) showed that under a 25% harvest rateChlorella pirenoidosachemical oxygen demand (COD), total Kjeldahl nitrogen (TKN) and total dissolved phosphorus (TDP) in STW were reduced by 70%, 61% and 61%, respectively. In contrast, wastewater from the primary tailings pond (PSW) exhibits the characteristics of a large water volume, high organic carbon content, and a relatively high nitrogen to phosphorus atomic mass ratio (N/P ratio) (Bohutskyi et al. al., 2015). Our previous study measured the utilization rates of total nitrogen (TN), total phosphorus (TP) and total lipid production of microalgae grown in PSW at 99.8%, 83.1% and 0.38 g·L, respectively.-1(Han et al., 2021). Bohutskyi et al. (2015) we will cultivatescenedesmsp. in individual PSW and PSW supplemented with 5% pool concentrate concentrate and then found biomass concentrations at 0.25 gdw L-1y 0,35 gpd L-1, separately. Meanwhile, the TN utilization rate by microalgae grown in a single PSW was higher than in mixed culture conditions, while the phosphate utilization rate (PO43-) was opposite. Therefore, STW and PSW have the potential to cultivate microalgae. Nitrogen and phosphorus in wastewater are essential elements for microalgae growth, as they are involved in the anabolism of many functional cell components (Arora et al., 2016, Yu et al., 2017). Microalgae showed different nutrient removal efficiencies and growth stages at different N/P ratios (Rasdi and Qin, 2015, Liu et al., 2011). Hosai et al. (2022) showed that the maximum nitrogen and phosphorus utilization rate ofChlorella kesslerican be obtained at N/P=6. Liu et al. (2022) studied that under the optimal N/P ratio of 20, the light use efficiency ofChlorellaThe density of microalgae was the highest (2.96 × 107ml cell-1) was the highest and clearance rates for TN and TP were 57.0% and 75.9%, respectively. When these two types of sewage PSW and STW are mixed in a certain ratio, the N/P ratio suitable for microalgae growth can be obtained, which promotes microalgae growth and saves the cost of microalgae cultivation. At the same time, it can also reduce nitrogen and phosphorus removal pressure in sewage treatment plants.

Proteomic approaches reveal the protein responses of organisms to different environmental conditions, studying and analyzing their metabolic pathway (Tu et al., 2020). Isobar tags for relative and absolute quantification (iTRAQ) are the newest and most accurate analysis tool in proteomics (Nagarajan et al., 2022). read on (2015) demonstrated that high concentrations of nitrogen and phosphorus stimulateEichornia crassipesto elicit a range of stress responses: to promote intracellular pathways involved in metabolism, nitrogen and phosphorus synthesis and secretion, photosynthetic biosynthesis and energy metabolism, and to enhance other functions of proteins to support excessive nutritional environments, eliminate nitrogen and phosphorus, and inhibit the growth of seaweed. Che et al. (2018) who studied protein changes inThalassiosira pseudonanaCells in the absence of macronutrients such as nitrogen, phosphorus and silicon revealed specific protein responses to various macronutrient deficiencies. Zhang et al. (2015) reported the glutamine synthase (GS) activity ofProro Zentrum DonghaienseCells were more active under nitrogen deprivation. However, few studies have examined the impact of N/P ratios on microalgae culture from a proteomics perspective.

In this studio,S. obliquawas grown in various mixed wastewater with different N/P ratios by mixing PSW and STW. Changes in nitrogen and phosphorus content in the culture medium and microalgae growth were analyzed. iTRAQ was used for the first time to analyze the effect of N/P ratios on microalgae metabolism and demonstrated that the N/P ratio plays an important role in regulating the biochemical composition, influencing the process of protein synthesis in microalgae cells. It can be used to control the efficient harvesting of microalgae resources.

## Microalgae and culture conditions.

microalgaS. obliqua276 was purchased from the freshwater algae culture collection of the Institute of Hydrobiology (FACHB, Wuhan, China). The strain was preserved in a 250 ml Erlenmeyer flask with BG11 medium at 25 ± 2 ℃.

STW was taken from the supernatant of the sludge concentration tank of a sewage treatment plant (Shenzhen, China) and used for the test after 2 hours of rest. The PSW used in the experiment was obtained from domestic sewage (Shenzhen, China) in the underground pipeline.

## Effects of different N/P ratios on nutrient utilization ofS. obliqua

Changes in nutrient concentrations in the culture medium withS. obliquaCulture times are shown in Figure 1 and final nutrient utilization rates are shown in Table 2. Nutrient concentrations in the culture medium gradually decreased as the culture time increased. Nutrient utilization rates in STW and N/P=11 were significantly lower than in other growing conditions (P value < 0.01).

S. obliquahad the lowest nutrient utilization rate in STW. That

## Conclusion

In this study, PSW and STW were mixed in a culture medium with different N/P for culture.S. obliqua; measuring the tendency of nitrogen and phosphorus change in the culture medium and the growth state ofoblique pointthe best N/P was selected. The mechanism of the effect of different N/P on the growth ofS. obliquawas examined by proteomic analysis. The main conclusions are as follows:

The optimal N/P ratio for the cropS. obliquawas 18 years old and TN, NH usage fees4+

## Declaration of conflict of interest

The authors declare that they are not aware of any competing financial interest or personal relationship that may have influenced the work described in this article.

## Gracias

This work was supported byGuangdong Foundation for Basic Research and Applied Basic Research(grant no.2019A1515110969);Innovation projects characteristic of universities in Guangdong province(2022KTSCX213);Shenzhen Science Foundation(Not.GXWD20201230155427003-20200821174135002).

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