Microalgal biomass production

The microalgal consortium-obtained from the Department of Agroindustrial Engineering of the University of Guanajuato, Mexico-was subjected to an adaptation process under controlled conditions (pH 7.0, room temperature 25 ºC, constant aeration and photoperiod 16/8 h) for 30 days, placing the microalgal inoculum in flat-plate reactors (5 L), with 80:20 v/v Bold’s Basal Medium (BBM) and inoculum (^{Barsanti and Gualtieri 2014}). After the adaptation period, microalgal biomass production was performed, using 60% v/v BBM culture medium (^{Barsanti and Gualtieri 2014}), 40% v/v DWW, in incubation periods of 10 days under mixotrophic conditions, with the availability of organic and inorganic C during the process. The 21 physical and chemical indicators evaluated for DWW characterization are listed in table I.

Microalgal growth

Growth and nutrient consumption kinetics were conducted during the production of microalgal biomass. The microalgal biomass concentration was established by spectrophotometry, following the methodology of ^{García-Gozalbes et al. (2015)}), reported in g/L. The indicators pH, chemical oxygen demand (COD), soluble organic C (SOC), N-NO₃ ⁻, and phosphates (P-PO₄ ⁻) were determined as reported in table II. All indicators were evaluated in triplicate.

Characterization of the microalgal biomass

The microalgal biomass was collected by flocculation with 1.0 g of chitosan (β-(1-4)-2-amino-2-desoxy-D-glucopyranose), 20% glacial acetic acid (CH₃COOH) and 80% drinking water (H₂O) v/v (^{Terkula Iber et al. 2022}). Subsequently, 40 mL of flocculating agent per liter of microalgal culture was subjected to agitation, first at 200 rpm for 30 s, then at 100 rpm for 10 min, and finally it was left to stand for 10 min. Afterward, it was centrifuged in 50 mL Falcon tubes at 6000 rpm for 10 min, using a Thermo Scientific® Myspin 12 centrifuge. The precipitate was oven-dried at 80 ºC for 72 h, and then pulverized using an agate mortar. The biomass obtained was characterized using the 17 physical, chemical and nutritional indicators described in table III.

Sampling and physical chemical characterization of the soil

The samples of soil were obtained in Guanajuato, Mexico, at coordinates 20º60’22.22’’ N, 101º02’30.56’’ W, in compliance with the standards described in ISO 18400-104:2018 (^{ISO 2018}). The sampled soil was divided into three subplots of 600 m². In each area, a systematic zig-zag sampling pattern was followed starting from one corner of each subplot and performing excavations with an auger drill every 18 m, with 30 cm depth and 40 cm diameter. At each sampling point, 2 kg of soil was taken. In total, 30 kg of composite samples were taken per subplot. The samples were transported in plastic bags to the laboratory and stored at 4 ºC until analysis. The soil characterization was carried out with the determination of 34 physical and chemical indicators, as shown in table IV.

Short-term C and N mineralization dynamics of soil-microalgae systems

Statistical analysis

Statistical analyses were performed using R statistical software version 3.6.2 (^{RCT 2019}). It began with a Shapiro normality analysis, followed by a Kruskall-Wallis nonparametric analysis of variance, and subsequent Dunnett’s mean contrast test with the Bonferroni adjustment method at a significance level p ≤ 0.05. Correlations between the indicators analyzed were established using a Spearman correlation matrix (r), with those indicators with a Spearman correlation (r ≥± 0.6) being considered significant correlations (^{Zhang et al. 2016}). Finally, a Friedman analysis was performed with a significant difference of p ≤ 0.05 (^{Zimmerman and Zumbo 1993}).

Characterization of dairy wastewater

The results of DWW characterization can be found in table V, where the indicators pH, COD, and biological oxygen demand at 5 days (BOD₅) presented values considered normal for DWW: pH of 4.7-11, COD of 80-95 000 mg/L, and BOD₅ of 40-48 000 mg/L (^{Daneshvar et al. 2019}).

Microalgal growth and microalgal biomass

The values for the indicators that were monitored during the microalgal growth kinetics are shown in table VI. The pH ranged from 8.6 to 9.2, being 9.02 the initial value and 9.10 the final value. An inverse trend was observed between the amount of biomass (which increased from 0.43 to 0.80 g/L) and the values of N-NO₃ ^-, P-PO₄ ^-, SOC, and COD, all of which decreased during the 10 days of growth as a consequence of the metabolism of the microalgal consortium.

Characterization of the microalgal biomass

The microalgal biomass presented TN values of 8.3%, TOC of 50.6%, a C/N ratio of 6.1, and 2.03% P (Table VII). Regarding the macronutrient content, the microalgal biomass presented 3000 mg/kg of dry microalgae for K, 23 900 mg/kg of dry microalgae for Ca, and 4600 mg/kg of dry microalgae for Mg. The presence of these elements is of great importance because, in their cationic form, they replace the Na⁺ ions present in the soil aggregates, improving their structure (^{Leogrande and Vitti, 2019}).

Soil characterization

According to the physical and chemical characterization of the soil (Table VIII), it presented alkaline pH conditions (9.4), low EC (0.37 < 2.0 dS/m), and high ESP (41 > 15%). The OM present was considered within the normal range for agricultural soils (4.95%) but with a high C/N ratio (47.9), which indicated an N deficit. Macro- and micronutrients and their various ratios were found in amounts considered low.

C and N mineralization dynamics

Figure 1 shows the results for C and N mineralization dynamics. The evolution of pH (Fig. 1A) ranged from 8.09 to 9.16, remaining in an alkaline range in all treatments. There were significant differences between T2, T3, and T4, with respect to T0 during all the C and N mineralization dynamics (p ≤ 0.05), but no differences between T1 and T0 at 168 h (p > 0.05).

Fig. 1 Response of the physical and chemical indicators (A-F) evaluated during the 360 h mineralization dynamics for the different treatments tested. Red dotted line for T0 (50 g soil + 0 mg microalgae [control]), yellow dotted line for T1 (50 g soil + 500 mg microalgae [25 mg N-NH₄ ⁺/kg dry soil]), blue dotted line for T2 (50 g soil + 1000 mg microalgae [50 mg N-NH₄ ⁺/kg dry soil]), green dotted line for T3 (50 g soil + 2000 mg microalgae [100 mg N-NH₄ ⁺/kg dry soil]), orange dotted line for T4 (50 g soil + 3000 mg microalgae [150 mg N-NH₄ ⁺/kg dry soil]). Gray area refers to standard deviation with 95% confidence interval. A: pH; B: EC (electrical conductivity); C: N-NO₃ ^-; D: N-NH₄ ⁺; E: N-NH₃ ⁺; F: C-CO₂. 

The EC indicator (Fig. 1B) presented values in the range of 0.37-1.04 dS/m, being T3 the one with the highest EC. Despite the increase in EC observed in T3, in general, the addition of microalgal biomass did not significantly (p > 0.05) increase the EC.

The dynamics of C-CO₂ emission (Fig. 1F) showed values in the range of 0-240 mg C-CO₂/kg dry soil, with an increase in C-CO₂ observed in all treatments as a function of the application rate and over time. T0 (control) showed lower C-CO₂ emissions than the rest of the treatments.

The N-NH₄ ⁺ indicator (Fig. 1D), showed values in the range of 35.18-1071.92 mg N-NH₄ ⁺/kg dry soil, with an increase in N-NH₄ ⁺ over time for all treatments, except for T0 (1.96 mg N-NH₄ ⁺/kg dry soil). In the same context, T4 and T3 presented higher N-NH₄ ⁺ values at most experimental time points (1071 mg N-NH₄ ⁺/kg dry soil for both). No loss of N-NH₃ ⁺ by volatilization was observed with p > 0.05 (3.46-5.46 mg N-NH₃ ⁺/kg dry soil), in figure 1E.

Regarding N-NO₃ ^- dynamics (Fig. 1C), values in the range of 33.8-1.45 mg N-NO₃ ^-/kg dry soil were observed, showing a decrease in N-NO₃ ^- in all treatments over time. There were no significant differences (p > 0.05) between T0 and the other treatments at any of the treatment time points, and the concentration decreased or remained equal in the following order: T1 > T0 > T2 = T3 = T4.

The values of TOC, TN, and C/N are shown in figure 2. The TOC results (Fig. 2A) were in the range of 0.41-2.95%, and its increase was directly proportional to the amount of microalgal biomass that had been added: T4 > T3 > T2 > T1 > T0. The TN results (Fig. 2B) presented values in the range of 508.41-2375.65 mg/kg dry soil, which implied an increase in TN at the beginning of the experiment, which was also directly proportional to the amount of microalgae added: T4 > T3 > T2 > T1 > T0. The variation of C/N (Fig. 2C) was in the range of 8.17- -20.44, and the concentration decreased in the following order: T2 > T3 > T1 > T4 > T0, highlighting the absence of significant differences (p > 0.05). In all treatments, the C/N ratios were below or close to 24, which is considered optimum for applying organic amendments (^{Ammar et al. 2022}).

Fig. 2 Kruskall Wallis analysis of variance with subsequent Dunnett’s mean contrast test with Bonferroni adjustment for the treatments used. Dotted black line denotes the mean of the indicator. A: TOC (Total organic C); B: TN (Total N); C: C/N ratio. 

Microalgal biomass production

The production of microalgae in wastewater is of great relevance worldwide due to the high content of nutrients present in wastewater, which facilitates the growth of microalgal consortia. During their growth, these consortia undergo bioremediation processes, metabolizing large amounts of N and P (^{Brar et al. 2019}, ^{Talapatra et al. 2021}). However, the physical and chemical characteristics of DWW may vary, and this will influence the production of microalgal biomass. For example, Chlorella vulgaris grown in DWW reached 1.2 g/L of biomass in 18 days (^{Handayani et al. 2020}), whereas Scenedesmus quadricauda and Tetraselmis suecica in DWW reached 0.39 g/L and 0.51 g/L in 12 days, respectively (^{Daneshvar et al. 2019}), and Chlorella pyrenoidosa and Scenedesmus abundans in DWW reached 0.043 g/L in 312 h and 0.019 g/L in 6 days (^{Brar et al. 2019}). Therefore, the biomass obtained in the present work of 0.8 g/L in 10 days (Table VI) is within the range of values reported in previous studies.

Furthermore, during their growth, microalgae consume N-NO₃ ^- and P-PO₄ ^-, which are important compounds to eliminate from wastewater before its final disposal. Regarding N-NO₃ ^- removal, values of 89.52% have been reported for Anabaena ambigua, 88.91% for C. pyrenoidosa, and 84.72% for S. abundans after 25 days in DWW (^{Brar et al. 2019}). Concerning P-PO₄ ^- removal, values of 87.83% have been reported for A. ambigua, 79.02% for C. pyrenoidosa, and 86.51% for S. abundans after 25 days in DWW (^{Brar et al. 2019}). In the case of S. quadricauda and T. suecica, values of 71.20% and 41.63%-respectively-have been reported for the removal of P-PO₄ ^- in a period of 12 days (^{Daneshvar et al. 2019}). In the present study, in 10 days the percentage of N-NO₃ ^- removal by the microalgal consortium was 79.88% (going from 62.63 to 12.60 mg/L) and the P-PO₄ ^- removal was 30.17% (going from 89 to 62.15 mg/L) (Table VI). N and P removal has been related to microalgal biomass production because these elements are vital for protein and nucleic acid biosynthesis (^{Patel et al. 2020}). Specifically, microalgae perform the assimilation process of N-NO₃ ^-, reducing it to N-NH₄ ⁺, which is finally incorporated into amino acids (Daneshvar et al. 2019, ^{Handayani et al. 2020}). In addition, P is used to generate compounds such as astaxanthin and polyunsaturated fatty acids through orthophosphate assimilation (^{Brar et al. 2019}).

Another important indicator is the chemical oxygen demand (COD). COD removal is an indicator directly related to the mixotrophic cultivation of microalgae, where, apart from utilizing C-CO₂, they can metabolize more organic compounds (^{Brar et al. 2019}). In other studies, Tetraselmis indica showed 77.03% COD removal in 10 days (^{Talapatra et al. 2021}) and C. vulgaris 92% removal in 10 days (^{Handayani et al. 2020}). The microalgal consortium analyzed in this study presented a percentage of COD removal between the values previously mentioned, with 85.8% (going from 674 to 95.5 mg/L) in 10 days as well (Table VI).

P and N contents in microalgal biomass have been reported to be 1.3% and 6.6%, respectively (^{Lage et al. 2018}). In the case of the microalgal biomass obtained in this study, the values of the indicators N and P exceeded what had been reported by 0.73% and 1.70%, respectively (Table VII). Likewise, there are reports of organic fertilizers with 0.7% P and 1.09% N (^{Mahapatra et al. 2018}). Therefore, the use of microalgal biomass as an organic amendment would be a good alternative, since it supplies the soil with a higher amount of P and N than an organic fertilizer.

Finally, when applying microalgal biomass to soils, high contents of C and N and a low C/N ratio of < 25 (Table VII), can favor microbial activity (^{Liyanage et al. 2022}), which is essential for the improvement of soil quality, increasing the C of microbial biomass (CMB) and improving the nutrient cycling and enzyme production (^{Alvarez et al. 2021}). Moreover, the contents of macro- and micronutrients found in this study (Table VII) suggests that microalgal biomass could be a complementary amendment to traditional chemical fertilizers, since the latter do not include this type of micronutrients in their formulation (^{Yilmaz and Sönmez 2017}).

Sampled soil and dynamics of C and N mineralization

The sampled soil in this study could be classified as sodic, with values corresponding to: EC < 4 dS/m, ESP > 15%, SAR > 13 (^{Osman 2018}, ^{Brusseau et al. 2019}) (Table VIII). Such values of EC, ESP, and SAR compromise soil structure, reducing its WHC, as well as its ability to supply nutrients to microorganisms and crops. Besides, the growth and development of microorganisms and plants can be affected by the high C/N ratio (^{Brusseau et al. 2019}, ^{Medina-Herrera et al. 2020}). In sodic soils, a high Na⁺ concentration increases osmotic stress in plants and microorganisms, affecting both systems’ correct development and activities (^{Srivastava 2020}, ^{Noori et al. 2021}). In addition, a deficit of nutrients, such as Mg, K, P, Fe, and B (Table VIII), can affect the growth of the plant cover, preventing it from carrying out essential functions such as photosynthesis (^{Villegas Hurtado et al. 2016}). In this regard, organic amendments can solve this deficit due to their macro- and micronutrient contents, which are available to improve soil fertility (^{Sulok et al. 2021}). Concerning the results of the dynamics of C and N mineralization, a lower pH (Fig. 1A) in the treatments with respect to T0 can be explained by their high microbial activity, since microbes produce organic acids during the mineralization of additional sources of C and N due to their metabolism (^{Leogrande and Vitti 2019}). The increase in pH at 168 h in treatments T2 and T4 was related to the mineralization processes of nitrogenous compounds, carbonates, and silicates (^{Castro-Alonso et al. 2019}, ^{Oviedo 2020}). When the initial pH of the soil is alkaline, it is beneficial if the applied amendment does not promote an increase in soil pH, an effect reported in the application of compost, Gliricidia, charged biochar, tea waste, and raw biochar (^{Liyanage et al. 2022}), as well as organic amendments made from residues of Phaseolus vulgaris L. and Cajanus cajan (^{Abera et al. 2012}), all of which increased the soil pH. Likewise, the importance of avoiding an increase in soil pH lies in the fact that alkaline values (pH > 8) promote N-NH₃ ⁺ release and inactivation of enzymes, disfavoring the activity of actinomycetes and bacteria (whose ideal pH is 6.5-8), which is important in nutrient cycling (^{Zhao et al. 2018}, ^{Neina, 2019}). In contrast, not increasing the pH favors the mineralization of the added OM, as well as that of the soil, thanks to the breaking of its bonds with clays and its high solubility, thus increasing the mineral N and the release of C-CO₂ (^{Neina 2019}).

The values of the EC indicator increased, possibly due to the rapid decomposition of OM and the release of salts contained in the added microalgal biomass. The increase in EC has been considered a disadvantage in organic amendments (^{Noori et al. 2021}); therefore, monitoring this indicator is suggested for applying microalgal biomass as an amendment to not considerably affect the soil with the effects of salinity. In this regard, the increase in EC concentration in T3 (Fig. 1B) could be an atypical form of accumulation or release of salts in the soil sample. However, it remained at an EC value < 4 dS/m, which is expected not to affect the mineralization of added OM (^{Osman 2018}). With respect to C-CO₂ release (Fig. 1F), the addition of microalgal biomass biostimulated microbial development and metabolism, which could be observed in the decrease in N-NO₃ ^- and the increase in C-CO₂ production. This could probably lead to an increase in soil quality indicators of the microbial biomass, such as C and N, microbial diversity, and enzymatic production (^{Alvarez et al. 2021}).

Regarding N-NH₄ ⁺, the results shown in figure 1D suggest that the addition of microalgal biomass promoted ammonification processes. The added N could be exploited by microorganisms present in the soil for the development of cellular structures and production of intra- and extracellular enzymes (^{Liyanage et al. 2022}). As for N-NH₃ ⁺ volatilization (Fig. 1E), it involves chemical reactions where N-NH₄ ⁺-in the presence of high concentrations of OH^- ions-releases protons, resulting in the production of N-NH₃ ⁺ and water molecules. Such reactions are maintained in equilibrium when the pH is neutral, however, at an alkaline pH and above, the release of N-NH₃ ⁺ is favored, with maximum volatilization peaks at pH 8.6; in contrast, when the pH decreases, the opposite reaction is favored (^{Neina 2019}). On the other hand, the organic N applied and mineralized (via production of N-NH₄ ⁺ and N-NO₂ ^-) into N-NO₃ ^- (Fig. 1C) can be quickly taken advantage of by the microorganisms through either biotic immobilization or abiotic immobilization, thus avoiding its loss via leaching processes, which explains the decrease in N-NO₃ ^- in the period evaluated in this study. Similarly, according to the pH values obtained (9.16-8.09), N-NO₃ ^- could also be used by denitrifying bacteria, which grow at pH values of 6.5 to 8.3, and from this compound, they can release N₂ or accumulating NO₂ ^- and producing N₂O (^{Neina 2019}). In the same context, an alkaline pH in the soil, if there is no increase in N-NO₃ ^- concentration over time during mineralization dynamics, could result in low activity by nitrifying microorganisms (^{Zhao et al. 2018}).

For basal respiration (Fig. 2), as well as for C and mineralizable N concentrations, it was confirmed that the values of these indicators increase in soils treated with microalgal biomass, which corresponds to what had been reported by ^{Aytenew and Bore (2020)}. The advantage of applying microalgal biomass to the soil was thus highlighted because C and N appear in bioavailable forms to be used immediately by microorganisms, a situation sometimes not observed with other amendments such as composts, in which C can be found in the form of lignocellulose or molecules that are difficult to degrade and be used by microorganisms (^{Sulok et al. 2021}). Moreover, the utilization of the N applied to the soil by the microbial biomass is evidenced by the decrease of N-NO₃ ^-, the increase of N-NH₄ ⁺ and the release of C-CO₂ (Fig. 1).

Since the indicators analyzed during the short-term C and N mineralization dynamics did not present a normal distribution, non-parametric analyses (Spearman correlation and Friedman analyses) were performed. Figure 3 shows the Spearman correlation analysis, with the highest positive association between N-NH₄ ⁺ and C-CO₂ (0.85), and the lowest positive association for N-NH₃ ⁺ and N-NH₄ ⁺ (0.26), the latter confirming the reversible chemical reaction between both compounds. The existence of a positive correlation of 0.43 between pH and N-NH₃ ⁺-indicators which are noteworthy-would indicate that, at a higher pH, soils tend to lose N by volatilization, a characteristic of sodic soils. The fact that the addition of microalgal biomass did not increase the pH reduced the N loss from the soil by volatilization. In addition, there are positive correlations between the C-CO₂ indicator with the N-NH₄ ⁺ and N-NH₃ ⁺ indicators, and a negative one with the N-NO₃ ⁻ indicator, which would support what was mentioned in previous paragraphs about possible ammonification and denitrification processes carried out by the microorganisms present in the soil (^{Liyanage et al. 2022}), since both microbiological processes are confirmed by the negative relationship (0.56) between N-NH₄ ⁺ and N-NO₃ ^-. This negative relationship also confirms the absence of nitrifying bacteria mentioned in previous paragraphs and explains why N-NH₄ ⁺ is not being transformed into N-NO₃ ⁻, and thus the transformation rates between the two indicators differ.

Fig. 3 Spearman correlation matrix for analyzed indicators (+1: Perfect positive association, -1: Perfect negative association, 0: No association). EC: electrical conductivity. 

A Friedman analysis was performed for the various treatments with respect to the indicators analyzed against time and the results of this analysis are shown in figure 4. An increase in the concentration of the TOC, TN, and C/N indicators can be observed as a function of the treatment. It is worth noting that T0 presented a very marked area in reference to the pH and N-NH₃ ⁺ indicators; as the dose of microalgal biomass added increases, the area expands towards the other indicators. This would indicate an adjustment in relation to the nutritional conditions of the soil, allowing for the development of microorganisms and nutrient cycling processes (^{Alvarez et al. 2021}).

Fig. 4 Friedman analysis for the treatments used in the dynamics of C and N mineralization. A: T0, B: T1, C: T2, D: T3, E: T4. Different letters between the same vertices refer to significant differences (p ≤ 0.05). N-NO₃ ^-, Nitrate N-NH₄ ⁺, Ammonium N-NH₃ ⁺, Ammonia EC: Electrical conductivity, TN: Total N, TOC: Total organic C, C/N ratio. 

Likewise, as can be seen in figure 4, all treatments presented significant differences in pH; where the highest alkalinity was observed in T0 and the pH decreased as a function of the dose of microalgal biomass applied. The above result could be related to the production of organic acids by microbial activity, which was favored by the increase in the application dose of the organic amendment (^{Leogrande and Vitti 2019}). Also, the initial concentration of Ca²⁺ present in the microalgal biomass could help to lower the pH by replacing part of the H⁺ present in the soil solution, as well as reacting with bicarbonates (Ca(HCO₃)₄), precipitating calcium carbonates (CaCO₃), and releasing H⁺ (^{Noori et al. 2021}).

There were differences between T1 and T2 when compared to the other treatments in terms of N-NO₃ ^-, showing a tendency to decrease with increasing doses of organic amendment application (Fig. 4). This suggests that, due to the low concentration of available N in the soil, microorganisms could be using the available N-NO₃ ^- and maintaining the N immobilization processes by the microbial biomass (^{Leogrande and Vitti 2019}, ^{Liyanage et al. 2022}). The above is supported by the Spearman correlation matrix shown in figure 3, specifically by the negative association between N-NO₃ ^- and the N-NH₄ ^- and C-CO₂ indicators.

In the case of N-NH₄ ^- (Fig. 4), it was observed that the greater the addition of microalgal biomass, the greater the production of this compound. Its release through ammonification processes allows it to be absorbed by plants via a simple route with low energy consumption (^{Liyanage et al. 2022}). It should be noted that microalgal biomass has been reported to be a quick-use amendment for crops. Furthermore, there was no significant difference in N volatilization via N-NH₃ ⁺ production, which prevented the release of greenhouse gasses (^{Chávez-García and Siebe 2019}).

Finally, in figure 4, the increase in C-CO₂ concentration as a function of the increase in the application rate of microalgal biomass could be due to the biostimulation of microbial activity in soils when organic amendments are applied as sources of rich and more available C and N (^{Medina-Herrera et al. 2020}). This biostimulation is the basis for improving soil conditions because it contributes to nutrient cycling, since the microbial biomass generated will become part of the available OM and the C from the microbial biomass will be available for use (^{Alvarez et al. 2021}).