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Potential use of sludge from El Ferrol Bay (Chimbote, Peru) for the production of lipids in the culture of Scenedesmus acutus (Meyen, 1829) – Scientific Reports

Medium chemical profile

Chemical characterization of the AES medium revealed that it incorporates all the nutrients needed for microalgae cultivation, as well as other highly toxic elements. At least 20 elements were quantified with levels (ge) 0.05 ppm. It is crucial to highlight that some of the nutrients with high values occur naturally in seawater, such as sodium, potassium, magnesium, and calcium. Here, we did not consider analyzing aluminum as it is the container material used in the thermal process and there could be detachment during the preparation of the medium. Besides, we identified Mg, Si, Ca, Fe, P, N, B, Sr, As, Li, Ba, and, Zn as the elements with the highest concentration. Other elements with concentrations between 0.09 and 0.05 ppm were Mn, Pb, Cu, V, and Mo; some of which are toxic, like Pb and Cu. Interestingly, the medium showed a balanced ratio of nitrogen and phosphorus, whose values are 9.06 and 10.94 ppm, respectively (Fig. 2).

Figure 2
figure 2

Chemical profile of the AES culture medium. The main elements quantified in the AES medium and their respective individual contributions are presented in ppm. The values are plotted using a logarithmic scale. *Nitrogen was quantified as ammonia nitrogen (NH(_3)).

This study has used a heat treatment approach for the preparation of the culture medium. Thermal treatments are widely used in different technologies, as high temperatures increase the mobility and facilitate the extraction of organic contaminants52. In addition, it induces the disruption of cellular components and other complex molecules, such as polymers, proteins, and carbohydrates53. The dissociation of polymers to simpler chemical structures is the result of the disruption of chemical bonds, triggered by the high kinetic energy of heating. These low molecular weight compounds, such as sugars, amino acids, or short lipid chains, are more easily incorporated by microalgae and therefore allow for enhanced growth54,55. An additional advantage of this methodology is the elimination of microorganisms that may infect microalgae or simply compete for nutrients. Other research has pointed out the possibility of using the waste effluent directly since it contains a greater organic load, as well as microorganisms that produce chemical species that favor the growth of microalgae56. According to our criteria, this would produce contamination in the cultures. However, it is possible to use the sludge directly in cases where the objective is only the removal of nutrients and not the production of algal biomass. Therefore, a temperature-based separation methodology to obtain an aqueous medium is the most appropriate.

The culture medium described in this research has been shown to provide elements such as N, P, K, Mg, and Fe, essential for microalgae reproduction57. In addition, trace metals, such as Mg, Mn, B, Mo, K, Co, and Zn, are also required as co-factors for cellular functions58. All these nutrients, excluding Co, have been quantified in the AES medium with levels > 0.01, whose concentrations are not balanced as in traditional mediums. It is important to note that the nutrient balance could not be controlled since the medium was elaborated from a unique organic component collected from the natural environment. However, having a balanced culture medium is necessary to support a continuous increase in biomass; furthermore, microalgae cultures can be improved only if nutrient deprivation can be controlled throughout cell growth<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 59" title="Mandalam, R. K. & Palsson, B. Elemental balancing of biomass and medium composition enhances growth capacity in high-density chlorella vulgaris cultures. Biotechnol. Bioeng. 59, 605–611.
https://doi.org/10.1002/(SICI)1097-0290(19980905)59:53.0.CO;2-8

(1998).” href=”#ref-CR59″ id=”ref-link-section-d147993116e1737″>59. Therefore, not having a balanced culture medium is a limitation for our procedure, as well as for other studies involving residual material since it is not possible to balance the medium in such cases. Besides, the incorporation of complementary nutrients to balance the medium could be a cost-increasing option. Despite this, the AES medium allowed us to obtain important growth and, in the case of the lowest concentration, high levels of lipids. Further studies could also perform calculations from the unbalanced media to predict biomass yields by theoretical calculations based on the composition of microalgae cells.

It is worth noting that, we identify some elements in the medium that may be limiting the growth due to its toxicity. For instance, As, Cd, Pb, Ba, and Ag. Previous reports indicate that high concentrations of these metals induce oxidative stress, changes in metabolism and structure, and degradation of vesicle cells60,61. Although, microalgae possess adaptive mechanisms to bioaccumulate these compounds; among which vacuolization, accumulation in the cell wall, and other processes of resistance induced by metallochaperones62,63. Under these circumstances, it is possible to sustain high growth even in the presence of high concentrations of toxic elements.

Scenedesmus spp. are well known for their adaptability to different conditions and culture mediums. Toxicity studies suggest that they are able to tolerate a wide range of heavy metals; such as up to 200 ppm Pb64, 10 ppm Cr65, 100 ppm As66, 20 ppm Cd67, 5 ppm Ba68, and 0.0385 ppm Ag69. Growth under these conditions is limited, but possible. S. acutus is, therefore, one of the most promising microalgae to cultivate with AES medium.

Table 1 Chemical analysis of the inorganic nutrients, COD, and TOC of the sludge, AES medium, and water at the end of the experiment.

On the other hand, the analysis of inorganic nutrients demonstrated the reduction of nutrients by the microalga S. acutus. In relation to COD, the highest level was observed in the EAS medium (1766.67 ± 82 mg/L), while, after 7 days of culture a maximum reduction of 30.37% was achieved in the treatment with 60% EAS medium (1230 ± 20 mg/L). Interestingly, ammonium levels were significantly reduced (p < 0.05), on the last day, with the treatment with 20% AES medium, showing the highest reduction (98.27%). Meanwhile, nitrites remained relatively low (<0.20 (mu)/L), and nitrates in higher concentration, as normally occurs due to the nitrifying activity of the bacteria70. In contrast, phosphates were reduced only in the 20% AES medium treatment by 30.28%, while in the 40 and 60% AES medium treatment, they were reduced by less than 5.1%. Surprisingly, phosphates in the 80% AES medium treatment increased by 82.14%, which we attribute to slow growth and subsequent culture death (Table 1). Likewise, TOC was slightly reduced only in the 20% medium treatment; however, it increased in the other treatments, probably attributed to cell death and the presence of bacteria in the water.

The high concentration of these elements in the AES medium demonstrates dramatic levels of pollution in the sampling area. Additionally, it is essential to note that the concentration of chemical elements present in the sludge may fluctuate; therefore, the final chemical composition of the medium will depend on the characteristics of the sample in the collecting area71. Nevertheless, it is expected that the use of our methodology with other samples from nearby places to the collecting area will provide similar results to the obtained data. In this sense, future studies will be required to expand the sampling area and determine the variations in the characteristics of the medium, in function of the geographic location and depth.

Population growth and biomass production

Our experiments demonstrate that the AES medium allows microalgae growth in all treatments. However, none of them exceeded the population growth obtained with HM medium (Fig. 3A). Maximum growth was obtained with 60% of AES medium (833.61 ± 24.54 x 104 cells/mL), which represents almost half of that obtained with HM medium (1515.67 ± 371.98 x 104 cells/mL). Interestingly, treatments with 20 and 40% of AES showed similar growth trends (629.00 ± 59.81 and 638.33 ± 28.22 x 104 cells/mL), while the lowest growth was achieved with 80% AES (244.00 ± 52.20 x 104 cells/mL). It is important to note that treatments showed different tonalities attributed to the synthesis of lipids (yellowish) and pigments (greenish), both of which are associated with nutrient availability, mainly nitrogen (Fig. 3B).

Figure 3
figure 3

Population growth of the microalgal cultures. (A) Growth curves of the average population density. (B) Different tonalities of the experimental treatments during the 7 days of cultivation are related to lipids and pigment synthesis depending on nutrient concentration (control treatment, 20, 40, 60, and 80% of AES medium, from left to right, each with 3 replicates).

Besides, the statistical analyses of the growth variables were found to correlate with the doses of nutrients supplied (Table 2). We highlight that the specific growth rates ((mu)) increase with the amount of medium up to 60%. Beyond this concentration, the cultures exhibit a significant decrease in all growth parameters ((mu), Div/day, DT, and biomass). Moreover, it is noteworthy that the treatments of 20, 40, and 60% did not present significant statistical differences among them (p < 0.05); however, in all cases, the HM medium provides better performance.

Both population density and biomass have been influenced by the AES medium and the conditions that it generates. It has been established that the availability of nitrogen and phosphorus are the main growth-limiting factors72; therefore, the increase observed at higher concentrations was expected. Although we have evaluated wide ranges of medium concentrations, we have noted that no experimental concentrations were able to outperform the HM medium.

The cultures supplemented with HM medium presented the best performance, both in population growth and biomass. This medium incorporates 4 nutrients: urea, potassium chloride, phosphoric acid, and iron. The better yields of the control cultures can be attributed to the better balance of this medium, in addition to the fact urea incorporates carbon as a nutrient73. In other experimental studies, the biomass of Scenedesmus ranges from 0.5674 to 1.76 g/L75. Those fluctuations depend mainly on physical-chemical factors, as well as on whether the work was performed, indoors or outdoors. Therefore, our results suggest that HM medium leads to 1.69 ± 0.14 g/L, while 60% AES leads to a maximum of 0.75 ± 0.07 g/L, which is about half. Nevertheless, the biomass obtained is considerable enough to justify the use of the AES medium. Likewise, better growth could be obtained by modifying the cultivation parameters or the cultivation strategy.

In regards to growth phases, the lag phase of the cultures was similar in all treatments up to day 2. On the other hand, the 60 and 80% treatments maintained a lag phase up to day 4. This difference between the lag phases could be associated with the toxic elements found, since the 20 and 40% treatments, which contained fewer contaminant concentrations, presented a lower lag phase. This reinforced the excellent adaptability of S. acutus. In both cases, the period may seem significantly long for production purposes; thus, initial cultures with higher cell density could overcome this disadvantage. Several studies have established that inoculum size influences microalgal cultures76,77. In addition, the prolonged lag phase could also be explained by the use of a quiescent inoculum. Considering this, we project that the use of our medium in real scenarios of scaling could lead to higher population growth in less time, and therefore better yields.

On the other hand, the growth variables (mu), Div./day, and DT, are in agreement with the nutrient supply. The 80% treatment presented the lowest (mu), Div./day, and the highest DT, as a result of the higher amount of contaminants. However, we can also mention that these variables together with the growth could be affected by the turbidity that generates a lower light transmission in the photobioreactor. On the other hand, the 20, 40, and 60% treatments did not show significant differences between these variables. Despite this, we noted other aspects of cellular composition that denoted a change in the metabolism of the microalgae in those.

Population structure

The population structure of S. acutus was also strongly influenced by nutrient availability, which can be differentiated on the last day of cultivation. A greater presence of 8-cell cenobium (48.24%) was observed in the control treatment (Fig. 4A and F). Whereas, cells of 1-cenobium are found in a major percentage (84.95%) in the cultures with 20% of AES (Fig. 4B and G). Cultures with 40% medium similarly presented a high percentage (72.85%) of 1-cell cenobium (Fig. 4C). Meanwhile, cultures with 60% AES (Fig. 4D) also presented a higher percentage of 1-cell cenobium; although, 8-cell cenobium was in a high percentage (28.15%) as well. In cultures with 80% of the medium, 4-cell cenobium dominated, despite the low population growth (Fig. 4E).

Figure 4
figure 4

Population structure of the microalgal cultures. The population structure of S. acutus expressed in percentage, where (AE) represent the control, 20, 40, 60, and 80% treatments, respectively. Clear differences between 8-cell and 1-cell cenobium populations for the control and 20% AES treatment can be discerned in figures (F) and (G), respectively.

An interesting event observed was the different population structures of S. acutus in the experimental and control treatments. S. acutus is a microalga that grows forming cenobium of 1, 2, 4, and 8 cells. It has been suggested that the formation of cenobium is a defense mechanism against grazers78. Moreover, it has been reported that the formation of larger cenobium is an indicator of optimal growth79. This leads to better biomass production. The number of cenobium can also be related to the number of divisions since it is plausible to hypothesize that cells with “n” cenobium will produce “n” generations; therefore, the higher the number of cenobium, the higher the number of daughter cells.

Although the molecular mechanisms of cenobium formation have not been fully understood, we infer that they are mainly influenced by the adaptability of the microalgae to the environment. We observed that 8-cell cenobium was more related to biomass and pigment production than to lipid production. Therefore, we propose that the metabolic pathways that produce the structural machinery that maintains the formation of cenobium (cell linked to cell) are affected by the lack of nitrogen or phosphorus. This requires further study. In addition, quantifying population structure may have relevance to the direction of microalgae production. In this regard, cells with larger cenobium present larger areas and therefore can be more easily centrifuged and/or sedimented, reducing the cost of harvesting.

Lipid content

We found that the highest lipid percentage (59.42 ± 6.16) was achieved with 20% AES medium, being in agreement with the lowest level of nutrients supplied. The treatments with 40, 60, and 80% of AES produced lower lipid percentages (33.01 ± 1.37, 29.25 ± 1.83, and 25.66 ± 3.42%, respectively), with no significant statistical difference ((p <) 0.05). On the other hand, the control treatment presented a total lipid content of 17.19 ± 3.97%, the lowest of all the experimental treatments (Fig. 5).

Figure 5
figure 5

Total lipid content of the microalgal cultures. Total lipid content is shown as a percentage for each of the experiments. The letters above the bars represent the statistical differences between treatments (p < 0.05). Cultures with 20 % AES provide high amounts of lipids.

Lipid production in microalgae can be induced by physiological stress. Reports of S. obliquus cultures under low levels of nitrogen indicate that it can reach a total of 30.77%80. Under this condition, microalgae tend to exhibit structural changes such as increased volume for better nitrogen absorption81. We did not observe changes in the morphology of individual cells, but we did observe changes in the population structure and pigmentation of the cultures. Therefore, we can propose that the formation of cenobium and lipid synthesis are inversely related events. Moreover, our findings suggest that the studied strain of S. acutus posses a higher lipid synthesis capacity, up to 59.42 ± 6.16% of total lipids with 20% AES medium, compared to other Scenedesmus species. However, this fact could also be influenced by the characteristics of the experimental medium.

Even though lipid production is mainly affected by N and P, it has been claimed that it is possible to force lipid synthesis by altering other parameters such as light intensity and salinity82. Other research indicates that phosphorus deprivation in the medium (less than 2 ppm) causes a decrease in the formation of pyrenoids, the CO(_2)-fixing machinery83. This drives us to infer that the characteristics of low pigmentation and increased lipid content are gaited in part by this event, strongly demonstrated in the treatment with 20% of AES medium ((approx)2.18 ppm of P). Further ultrastructural examinations may corroborate this.

Furthermore, microalgae are a promising source of lipids, such as EPA and DHA, which are currently obtained mainly from fish oil84. The use of residual material however limits the use of these microalgae lipids for human consumption. Nevertheless, such lipids can be used in the production of biofuels. The quality of the lipids form with AES medium needs to be determined since higher amounts of oleic acid are preferable for biofuel production purposes85.

Parameters and correlations

The average initial pH in the control cultures was 7.20 units, while in the experimental cultures, the initial pH was > 8.08 units. In all cultures, the pH tended to increase, except in the 20% AES treatment, where the pH decreased moderately after day 4 (Fig. 6A). Meanwhile, temperature maintained a slight fluctuation within the appropriate range, between 21.57 and 23.07 (^circ)C (Fig. 6B).

Figure 6
figure 6

pH and temperature of the microalgal cultures. (A) There can be observed marked differences in the initial pH between the control and experimental treatments. (B) Temperature fluctuations are slight and within the appropriate range for optimal growth.

Figure 7
figure 7

Correlation analysis. The correlations between the main parameters collected indicate that the initial pH of the cultures correlates inversely with growth (cells/mL and biomass). A slight correlation between biomass and lipid content is also observed.

Table 2 Statistical analysis of the main growth variables in day 7.

In the correlation analysis, we found strong and medium correlations (Fig. 7). The highest correlation found was between biomass (cells/mL) and pH-initial of the culture (-0.89, C.I. = [-0.35;-0.96]). Similarly, cell density (cells/mL) showed a high inverse correlation with initial pH (-0.83, C.I. = [-0.41;-0.95]). Both correlations were significant at a level of p < 0.05. An important correlation to note is that given between biomass and lipids (-0.46, C.I. = [0.09;-0.75]), since it denotes a certain level of inverse correlation that agrees with the theory, although not statistically significant was found (Table 3).

Table 3 Correlation coefficients and confidence intervals of the main parameters.

Determining correlations between any cultivation parameter and yields is of vital importance to optimize cultures. From our analyses, the highest correlation was found with initial pH and growth (inverse correlation), expressed in cell density and biomass. Scenedesmus spp. has an optimal pH range between 7.0 and 8.086; although it tolerates pH ranges up to 9.087. Therefore, the experimental cultures that showed pH values higher than 8.0 affect growth. Furthermore, the bootstrapping analysis indicates that the correlation coefficients can be between -0.35 and -0.96. Based on these results, we can infer that in some cases a not-so-strong correlation could be observed. Thus, we assume that other undetermined factors, such as the effect of toxic elements and/or the absence of some micronutrients (not determined in this study) may be affecting growth.

On the other hand, we have not identified a significant correlation between biomass and lipid quantity, unlike other studies that suggest that biomass is limited by lipid synthesis as a consequence of nitrogen limitation88. Hence, the factor inducing lipid synthesis must be more related to the chemical composition of the medium.