Nitrogen removal from wastewater by an immobilized consortium of microalgae–bacteria in hybrid hydrogels

Inorganic ions such as ⁠, ⁠, ⁠, ⁠, and are considered relevant pollutants in water once their concentration in water exceeds the permissible established limits (Singh et al. 2020). These ions are present in many water bodies as a result of discharges of municipal and industrial wastewater (treated and untreated), such as those from tanneries, mining, refining, and manufacture of fertilizer and paper (Akizuki et al. 2020; Kwon et al. 2020; Singh et al. 2020). High loads of these ions, especially those based on phosphorus and nitrogen, cause serious eutrophication and a decrease in dissolved oxygen concentration of water bodies, threatening aquatic organisms, balance of ecosystems, and human health (Kwon et al. 2020; Nguyen et al. 2020).

Treatment of wastewater with high content of nitrogen, mainly ammonium, brings some operational and economical issues to conventional wastewater treatment plants (WWTPs), such as that based on activated sludge. These issues include high energy consumption due to aeration required for nitrification, adaptation of facilities with additional basins and recirculation flows, management of the excess biomass, and the low nitrification rates for un-controlled and un-monitored treatment systems (Kwon et al. 2020; Nguyen et al. 2020). Among cost-effective alternatives for water treatment with high ammonium loads is the shortcut nitrification process. This technology consists of two steps: partial nitrification and anammox process. First, ammonium is partially oxidized to nitrite under aerobic conditions by the ammonium oxidizing bacteria (AOB). Then, the anammox bacteria (AMB) oxidize the remaining ammonium to gaseous nitrogen involving nitrite as an electron acceptor. Although AMB requires lower energy levels in terms of successful maintenance of the two-stage systems (conventional WWTP), a vital point is the achievement of an effective nitrite oxidizing bacteria (NOB) suppression. This step is necessary because of the competition between NOB and AMB for available nitrite, as well as the limitation of nitrate production, which increases the operating costs for nitrogen elimination (Shourjeh et al. 2020). Little research has been done so far regarding the suppression of NOB in the main nitrification/denitrification systems (Zajac et al. 2022), and optimizing the nitrification process of other microorganisms (Otondo et al. 2018). Therefore, research addressed to develop efficient technologies that contribute to remove nitrogen from wastewater is needed.

Immobilization of microorganisms has gained importance in wastewater treatment, especially to upgrade conventional WWTPs because this enables to overcome technical and economical limitations, e.g. cost of aeration, management of biosolids, and besides it is considered a biotechnological strategy to optimize nitrification and denitrification in wastewater treatment (Bouabidi et al. 2019; Nguyen et al. 2020). Among several approaches to immobilize microorganisms, hydrogels of organic and inorganic material are promising because they can embed microorganisms, are permeable, and have affinity to microorganisms (Subhan et al. 2021), while other carriers, e.g. those for biofilm growth, can take a long time to develop the biofilm and are sensitive to changes in the organic loading. Hydrogels have been used to immobilize microorganisms such as microalgae (Cao et al. 2020; Kube et al. 2021), bacteria (Dolejš et al. 2019), and even activated sludge (Cruz et al. 2020), which maintained their metabolic functions under controlled environment and model solutions; thus evaluations of these hydrogels in real environments are needed. Hydrogels containing selected microorganisms and exposed to real conditions of bioprocess could improve the removal of conventional contaminants in wastewater such as carbon, nitrogen, and phosphorus (Kumar et al. 2022). This is because microorganisms immobilized in hydrogels can present higher tolerance for external stressors, higher residence times, as well as an effective spatial–temporal control of biomass (Mehrotra et al. 2021), compared to the suspended microorganisms.

However, a limitation in the use of hydrogels for wastewater treatment is the short duration before disintegration, which is a result of the interaction between the components used for their synthesis and complex water media (Tsai et al. 2019). Therefore, hydrogels may present drawbacks such as poor durability alongside low mechanical strength (Pham & Tho Bach 2014). For hydrogels to acquire long operating time and high effectiveness, the polymer matrix must be strong enough to immobilize biomass, powders, and others within the structure (Tuyen et al. 2018), inducing the synthesis of multicomponent hydrogels, namely hybrid hydrogels. Hence, it is important to combine polymeric materials, e.g. synthetic and natural, to develop the best matrix corresponding to its purpose (Kumar et al. 2022).

Few experiments have evaluated the performance of hydrogels with microorganisms that perform a specific process, e.g. nitrification or nitrogen removal, in complex matrices as wastewater. Majority of experiments have been addressed only embedding microalgae, and hydrogel provides a controlled environment to promote microalgae growth, and protection from undesirable biological species, as well as from a series of pollutants that come from different harmful effluents (Cao et al. 2020; Kube et al. 2021). Kube et al. (2021) immobilized Chlorella vulgaris, Scenedesmus abundans, Selenastrum capricornutum, and Coelastrum microporum in hydrogel beads based on sodium alginate (SA) for N and P uptake and removal. Results indicated that the increased concentration of microorganisms in reactors and the hydraulic retention time (HRT) lower than 12 h, will allow the design of compact systems that simultaneously remove N and P (Kube et al. 2021). In addition to pollutants removal, when hydrogels with microalgae are in membrane bioreactors, there is a delay in membrane fouling due to limited organic matter in the mixed liquor (Cao et al. 2020). However, nitrogen removal via microalgae can be enhanced up to two times when bacteria interact with microalgae in photo-bioreactors (Rada-Ariza et al. 2017).

Microalgae and bacteria consortium is a very attractive alternative for nitrogen removal from wastewater in photo-reactors (González-Camejo et al. 2021). During photosynthesis, microalgae produce oxygen by using light as a source of energy, CO₂ as a source of carbon, and a partial consumption of nitrogen and phosphorous for their cell functions. Oxygen generated by the microalgae is used by the nitrifying bacteria, which provide the CO₂ in return to microalgae (Akao et al. 2021). This results in the oxidation of ammonium, an increase of nitrate content in wastewater (Otondo et al. 2018; González-Camejo et al. 2021), and in a higher survival rate than individual cultures (Phong Vo et al. 2020). Hence, the implementation of microalgae–bacteria systems for wastewater treatment results in lower cost, better effluent quality, and higher nitrogen and phosphorus removal, when compared to activated sludge (Morillas-España et al. 2021). However, microalgae and bacteria systems have operating conditions that must be properly controlled for efficient application in real wastewater treatment, such as biomass/nutrient ratio (Kwon et al. 2020), HRT (Jiang et al. 2018), and sludge retention time (SRT) (Fallahi et al. 2021). Hydrogels containing microalgae–bacteria can contribute to control these operating conditions and enhance the nitrogen removal.

Immobilized co-cultivation of microalgae with bacteria has mainly been investigated as biofilm in conventional carriers for degradation of pollutants in wastewater (Akao et al. 2021,) and not in a hydrogel. Although synergy between microalgae and nitrifying bacteria has been successfully applied for the nitrification process, nitrification rates when co-culture is in biofilm have been low compared to suspended consortium (Mehrotra et al. 2021). This has been attributed to the inhibitory effect of heavy metals and recalcitrant organic compounds over bacteria (Yu et al. 2020), and due to direct exposure of biofilm (Grandclément et al. 2017). Enhancement of the nitrification process can occur in presence of activated carbon (AC), which improves nitrifying activity (Ng & Stenstrom 1987). This is because AC can adsorb organic compounds responsible for inhibiting nitrification, which are characterized by their resistance to biological degradation as well as being adsorbable by AC (Ng & Stenstrom 1987). To decrease the toxic effects of the inhibitors over nitrifying microorganisms and increase the nitrification rates, addition of AC to bioreactors can be considered. The aim of this study was to prepare stable hydrogels made of PVA and SA, and to evaluate for the first time the performance of hydrogels embedding a consortium of microalgae–bacteria at different biomass concentrations, and AC as the adsorbent of inhibitory substances, in the nitrification process during treatment of real wastewater.

Hydrogels containing biomass (microalgae and nitrifying bacteria) were produced from a solution of 10 wt% polyvinyl alcohol (PVA), 2 wt% SA, and a fraction (5 or 16 wt%) of biomass in deionized water (DI). SA was selected as it provides an ideal environment, it is permeable, non-toxic, and can protect organisms from extreme physical and chemical conditions (Xue et al. 2020). On the other hand, PVA has been widely used for the immobilization of microorganisms and its implementation in wastewater treatment for its accessibility, low toxicity, porosity, and ability to diffuse oxygen and substrate (Bouabidi et al. 2019).

For hydrogel preparation, PVA and SA were first dissolved in 50 mL of distilled water on a hot plate magnetic stirrer at 40 °C. Resulting solution was autoclaved for 15 min at 121 °C. Subsequently, the solution was poured into a 100-mL beaker, and temperature was maintained at 40 °C. Water was added to reach 10 wt% of PVA and 2 wt% of SA. Biomass was collected from the adapted culture (see details in Section 2.2), and centrifuged at F = 1,792 g, and 28 °C for 20 min to obtain 5 or 16 g of wet biomass, equivalent to 1.35 and 4.35 g/L of TSS and to 5 and 16 wt%, which have been plausible concentrations of biomass embedded in hydrogels to treat wastewater (Wang et al. 2019). Then, biomass was incorporated to the hydrogel solution. 0.44 g of AC (<45 μm, HYCEL) was also added to hydrogel solution to minimize the effect of inhibitory substances to the nitrification process, obtaining hydrogels with AC. The hydrogel solution containing biomass (with or without AC) was dripped into a crosslinking solution, containing 5.6 wt% of boric acid and 2 wt% of calcium chloride at room temperature. The hydrogel beads with 5 and 16 wt% of biomass and with or without AC were kept in the crosslinking solution for 24 h under constant stirring at 150 rpm. After, the hydrogel beads were transferred to a 7.1 wt% of sodium sulfate buffer solution where they were stirred again (150 rpm) for 48 h. Then, hydrogels were transferred to DI water until experimentation.

The stability of the synthetized hydrogels without biomass was tested before the nitrification experiment according to methodologies using both tap water and DI water (Tuyen et al. 2018; Wang et al. 2019). Stability of the hydrogels was judged based on the two parameters of mechanical strength (durability exposed to mechanical stirring) and agglomeration of the gel beads. The test consisted of 150 hydrogels beads that were placed in a 250-mL beaker with 100 mL of DI water and 150 beads in 100 mL of tap water. Each beaker was put on a stirring plate where the hydrogels were exposed to 200, 400, 600 and 800 rpm for 15 min. At the end, the hydrogels were counted and determined how many beads remained undamaged and if they were agglomerated.

The enriched microalgae–nitrifying bacteria consortium was obtained from microalgae–bacteria cultures that treat wastewater in the laboratory (Akizuki et al. 2020), and it was adapted to treat raw wastewater. Microalgae consisted C. vulgaris while nitrifying bacteria were collected from the full-scale oxidation ditch WWTP in Guanajuato, Mexico. For adaptation, 250 mL of seed culture was added to four Erlenmeyer flasks of 1 L. The consortium was adapted to treat wastewater (350 mL) from the WWTP of the Tecnológico de Monterrey Campus Puebla, whose characteristics are shown in Table 1. Flasks operated as sequential batch reactors (SBRs), settling biomass of flasks, and replacing 350 mL of supernatant by wastewater every third day. SRT was complete during adaptation period (14 days). The flasks containing the consortium were placed in an incubator (incubator AlgaeTron AG130) at an average temperature of 27 °C with a constant stirring at 150 rpm, White LED light was irradiated at an intensity of 160 μmol/m²/s, with a cycle of 12 h of light and 12 h of darkness. Flasks were characterized every 2 or 3 days as the function of pH, electrical conductivity (EC), ammonium (NH₄), and nitrate (NO₃).

The parameters analyzed in the experiment were pH, electrical EC, temperature, ammonium, nitrate, chemical oxygen demand (COD), total suspended solids (TSS), volatile suspended solids (VSS), total solids (TS), and total volatile solids (TVS). The pH, temperature, and EC values of all flasks were measured with a portable meter using the corresponding probes (HACH HQd). COD, ammonium, and nitrate content in the samples were analyzed according to the commercial methods (HACH). TSS, VSS, TS, TVS concentrations in the consortium were determined according to Standard Methods (APHA 2005), respectively. Light intensity was directly measured using a solar radiation meter (PCE-SPM 1).

A three-way ANOVA analysis was carried out with Minitab using a p-value of 0.05, through a factorial design (2 × 2 × 3), where the response variables were ammonium and nitrate. Results of response variables are shown as the average value ± standard deviation. Parameters were measured every 24 h in a period of 72 h to quantify ammonium removal and nitrate production. Biomass (5 and 16 wt%), AC (without and with), and time (24, 48 and 72 h) were the factors analyzed with their respective levels and the effect on each of the response variables through principal effect plots.

For the establishment of a suitable immobilization method, stability of hydrogels was tested in DI and tap water under a constant agitation with different stirring speeds (200–800 rpm) (VELP, AREC Heating Magnetic Stirrer), as function of persistence and agglomeration (Table 2).

In tap water at 200 and 400 rpm, all the hydrogel beads maintained their original shape without agglomeration. At 600 rpm, the damage of the beads started to appear, and approximately 12% of beads were disintegrated and dissolved, while at 800 rpm, hydrogels were completely dissolved. Whereas, in DI water, approximately 17% of the beads were already disintegrated at 400 rpm and even mild agglomeration was also observed, which was worse at higher speed, reaching complete dissolution at 800 rpm.

The results showed that hydrogels in tap water had higher mechanical durability and resistance for agglomeration than those in DI water. This could be a result of the chemical nature of ions present in tap water, which can stabilize the structure of the hydrogels, while DI water, due to lack of these ions, only caused rapid disintegration and provoke agglomeration (Alcântara et al. 2009). Although complete dissolution was observed for both cases at 800 rpm, this speed is an extremely severe condition to test mechanical durability within a short period of time. The best performance of hydrogels was observed at 200 rpm, hence the nitrification experiment was carried out below this speed, at 150 rpm. It can be concluded that the prepared hydrogels had the mechanical durability and resistance for agglomeration suitable for nitrification experiments.

Treatments with AC had a similar downward trend for ammonium concentration, reaching around 20 mg/L, regardless content. At 48 h, ammonium concentration did not change significantly, while at 72 h, the final concentration of ammonium in assays with AC and both content of biomass reached a value of around 14 mg/L. According to the statistical results (Supplementary material, Figure S2), with a p-value greater than 0.05 in ammonium removal, all factors alone, as well as their respective interactions, showed not to have an effect on the response variable. Therefore, it can be concluded with statistical evidence that H₀ is not rejected. This means that biomass, content, presence of AC, and time had no influence on the ammonium removal.

In the presence of AC, the hydrogel with 5% of biomass at 24 h had greater production of nitrates (10 mg/L), which was maintained up to 48 h. However, at 72 h concentration had a decrease of 78% resulting in 2.17 mg/L. Hydrogel with AC and 16% of biomass presented 4.3 mg/L of nitrate at 24 h, then increased to 9.6 mg/L at 48 h the concentration. However, similar to the hydrogel with 5% of biomass, the final concentration of nitrate had a decrease of 86%, ending with 1.37 mg/L. This suggests that the decrease in nitrate concentration at the end of the experiment may have resulted from the presence of AC in treatments (Najmi et al. 2020). Unlike treatments without AC, nitrate accumulation was evident. AC has presented high adsorption capacity of nitrate, more than 65% when 100 mg/L was fed, in short periods (300 min), which was attributed to positive charge and micro-pores on its surface (Azhdarpoor et al. 2019).

According to the statistical results in nitrate production (Supplementary material, Figure S3), the AC factor was shown to have an effect in the response variable with a p-value of 0. Therefore, it can be concluded with statistical evidence that H₀ is rejected. The rest of the factors alone, as well as their respective interactions, resulted in p-values greater than 0.05, therefore it can be concluded that biomass and time had no effect in the nitrate production.

Nitrate concentration at the end of the experiments in all treatments confirm that the nitrification process was carried out, but at different levels for treatments with and without AC. The hydrogels with 16% biomass without AC induced the highest production with 39.77 mg/L. Conversely, the results of treatments with AC, showed lower nitrate concentration. This is in line with the low ARE, 80.7 and 81.7% for 5 and 16% of biomass content, respectively (Figure 4(a)). This demonstrates an adverse effect of AC during the nitrification process by hydrogels containing microalgae–bacteria, regardless biomass content. The main potential reason is because the available space within the hydrogel matrix may have been saturated with the presence of AC, resulting in the inability of microorganisms to develop the nitrification process (Wu et al. 2014).

Considering the nitrification rate per mass of microorganisms values in experiments without AC, hydrogels with 5% of biomass showed better performance than those with 16%. Figure 4(b) shows the highest nitrification rate of 0.43 mg N/g TSS·h for hydrogels containing 5% of biomass without AC addition, while for hydrogels with 16%, it was 0.20 mg N/g TSS·h at the experimental endpoint. It can be concluded that hydrogels without AC and with 5% biomass were more efficient among all treatments. This means that embedded consortium in hydrogel was able to produce oxygen using light as a source of energy and CO₂ as a source of carbon, while oxygen generated by the microalgae was used by the nitrifying bacteria, which provide the CO₂ in return to microalgae, and oxidize the ammonium to nitrate. Besides, high content of biomass did not result in higher efficiency. This can be attributed to a self-shading of microorganisms and, therefore, reduction of the penetration of light necessary for photosynthetic and microbial activity (Foladori et al. 2020). Therefore, it could be considered that the microorganisms of the upper layers of the hydrogel were able to perform metabolic activities, limiting the performance of the microorganisms in the core of the hydrogels.

A clear effect of AC over ammonium removal and nitrate production was observed, which can be attributed to the adsorption of these compounds rather than the shortcut nitrification process. Considering that the nitrification process was performed during first 48 h, the low nitrate concentration in AC treatments may have resulted from their adsorption in AC hydrogel (Mehrotra et al. 2021). However, change in the microbial community responsible for nitrogen removal caused by the presence of AC has been already reported (Yu et al. 2020), and it could be explored as a potential explanation. Hence, more research involving the reusability of hydrogels, together with microbial diversity analysis can be performed.

In this study, microalgae and nitrifying bacteria were immobilized together in hydrogels in different biomass concentrations, with or without AC. The effectiveness of the treatments was analyzed throughout the 72-h experiment, monitoring ammonium removal as well as nitrate production. With the results obtained it can be concluded that the most effective hydrogel was that with 5% of biomass concentration and without AC, which presented a nitrification rate of 0.43 mg N/g TSS·h, and up to 95% of ammonium removal. Presence of AC in hydrogels containing microalgae–bacteria may have interfered the nitrification process of microalgae-nitrifying bacteria, which can be attributed to the sorption of some compounds in the surface of AC. The persistence of hybrid hydrogels can be extended as a result of the components used for synthesis, as well as the type of water media to which it is exposed. However, during application there is the possibility of slow release of some compounds, e.g. PVA or SA. This leachate may affect the quality of the treated effluent in terms of COD concentration. Nonetheless, hybrid hydrogels with immobilized consortium are a feasible option to perform the nitrification process in wastewater.

P.C.-A. acknowledges CONACYT (330129). Authors acknowledge the Grant in Aid for Scientific Research (C), JSPS KAKENHI (award number JP18K11716).

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.