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Article

Co-Cultivation between the Microalga Tetradesmus obliquus and Filamentous Fungus Cunninghamella echinulata Improves Tertiary Treatment of Cheese Whey Effluent in Semicontinuous Mode

by
Leandro Monteiro dos Santos
1,
Joyce Camila Barbosa da Silva
1,
Carlos Eduardo de Farias Silva
1,*,
Brígida Maria Villar da Gama
1,
Josimayra Almeida Medeiros
1,
Giorgos Markou
2,
Renata Maria Rosas Garcia Almeida
1 and
Ana Karla de Souza Abud
3
1
Technology Center, Federal University of Alagoas, Maceió 57072-900, Brazil
2
Institute of Technology of Agricultural Products, Hellenic Agricultural Organization—DIMITRA, 14123 Athens, Greece
3
Food Technology Department, Federal University of Sergipe, São Cristóvão 49100-000, Brazil
*
Author to whom correspondence should be addressed.
Processes 2024, 12(8), 1573; https://doi.org/10.3390/pr12081573 (registering DOI)
Submission received: 29 June 2024 / Revised: 23 July 2024 / Accepted: 24 July 2024 / Published: 27 July 2024
(This article belongs to the Special Issue Advances in Bioremediation and Biosorption Processes)
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Abstract

:
Cheese whey (CW), a byproduct resulting from dairy processing, requires proper treatment and disposal. The use of microalgae during tertiary treatment emerges as a promising option due to its efficiency to remove chemical oxygen demand (COD), total nitrogen (TN), and total phosphorus (TP) from effluents obtained after secondary treatment processes. The present study was focused on treating CW effluent at two organic loads (C1 and C2—with different concentrations of COD, TN, and TP) (550–2200 mg·L−1 of COD, 14–56 mg·L−1 of TN, and 4.5–18 mg·L−1 of TP) using the microalga Tetradesmus obliquus alone (CM1 and CM2) and in a consortium with the fungus Cuninghamella echinulata (CMF1 and CMF2), evaluating the residual values of COD, TN, and TP and removal efficiency. The experiments were carried out in an open system with a volumetric replacement ratio (VRR) of 40 and 60%. The CM treatment showed residual values of COD in the range of 190–410 mg·L−1 (removal efficiency: 57–68%), TN in the range of 6–24 mg·L−1 (removal efficiency: 29–35%), and TP in the range of 0.90–3.0 mg·L−1 (removal efficiency: 65–68%), after 7 days of volumetric replacement time (VRT) in a semicontinuous mode. In contrast, the consortium (CMF) showed greater stability and efficiency in contaminant removal compared to the treatment system containing only the microalga, showing residual values of COD in the range of 61–226 mg·L−1 (removal efficiency: 75–77%), TN in the range of 1.8–9.5 mg·L−1 (removal efficiency: 70–74%), and TP in the range of 0.6–3.5 mg·L−1 (removal efficiency: 66–70%), applying a lower VRT of 3 days and reaching the legislation standard for discharge to CMF1 (VRR: 40 and 60%) and CMF2 (VRR: 40%). The cell dry weight of 290–850 mg·L−1 was obtained (microalga and microalga–fungus cultivation), which can be a valuable biomass for biotechnological applications. Finally, during microalga–fungus co-cultivation, there was greater system buffering (with less pH variation), ensuring a better system stability.

1. Introduction

Among the main sectors that generate wastewater with high organic load, and consequently, pollutant power, the dairy industry stands out. Most processes to treat dairy wastewater require one or more stages during secondary treatment (stage to remove substantial contaminant concentration) and a sequential polishing step (tertiary treatment) to meet legislation. The process carried out in batch mode was previously studied applying a consortium of Tetradesmus obliquus and Cunninghamella echinulata [1]. This manuscript presents a semicontinuous process that applies this co-cultivation to the tertiary treatment of cheese whey effluent. It is important to highlight that this operation mode can present improvements in the process such as no need for inoculation, higher contaminant removal efficiency, and lower treatment time.
The dairy industry is one of the main sectors in the food and beverages industries, generating 21.6 billion tons of whey annually as a byproduct of cheese production [2,3]. Although whey can be used in the production of ricotta, dairy beverages, as a mineral source in strengthening processed meats, permeate concentrates, and proteins, or even animal feed, a substantial portion is often discarded into water bodies without any treatment, representing about 50% of whey produced worldwide (especially acid whey) [4,5].
Whey can cause a significant environmental problem if it is improperly disposed, due to its high COD (40,000–100,000 mg·L−1), considerable pH variations, TN (800–2500 mg·L−1), and TP (300–600 mg·L−1) [6,7,8], presenting a pollutant potential 100 times higher than that caused by domestic wastewater [9].
As a result of their high pollutant load, effluents containing whey require more treatment stages to comply with the limits established by the European Union (EU) for discharging these effluents into receiving bodies. These limits include chemical oxygen demand (COD) (less than 125 mg·L−1), total nitrogen (TN) (10–15 mg·L−1), and total phosphorus (TP) (1–2 mg·L−1) [10]. Thus, primary and secondary treatments are used for a more substantial removal of initial pollutants, while tertiary or advanced treatment (polishing step) is responsible for removing residual contaminants to meet the discharge standards required by national and international regulations, with emphasis on the European Union (EU) standards, which are among the most stringent [11,12].
Given this scenario, bioremediation (biological treatment) using microorganisms emerges as a sustainable, valid, and effective alternative compared to physical and chemical treatment processes, which depend on the costly and intensive use of chemicals or materials (such as membranes) [13,14]. Additionally, biological methods enhance the efficiency in effluent remediation, help the production of high-value biomass, and promote the development of a circular economy [15,16].
Regarding biological treatment, despite the existence of traditionally applied methods, especially during secondary treatment in which aerobic and anaerobic processes are applied such as activated sludge and anaerobic digestion, respectively; significant amounts of COD, TN, and TP still remain after the secondary treatment, requiring a sequential stage (or even more than one) of polishing (also called tertiary or advanced treatment) to meet the required discharge standards into water bodies. Residual values of COD in the range of 1200–46,000 mg·L−1, TN in the range of 50–2500 mg·L−1, and TP in the range of 5–80 mg·L−1 have been observed after secondary treatment of dairy wastewater and other effluents in several studies [17,18,19]. In addition, after anaerobic processes, nitrogen and phosphorus are mostly mineralized, requiring disposal after the process [20].
Among the classes of microorganisms used in tertiary treatment, microalgae are highly considered due to their high capacity for nitrogen and phosphorus remediation from effluents and are applied either alone or in co-cultivation with other microbial groups [21]. Several studies have reported the use of microalgae in the treatment of domestic, industrial, and agro-industrial effluents [22,23].
Microalgae cultivation can be carried out through open systems (tanks or ponds) or closed systems, such as photobioreactors [24,25]. Open systems have advantages such as being more economical and easier to operate [26]. Additionally, the energy demand for microalgae cultivation in a photobioreactor is 3.7 times higher than in a pond [27,28]. Open raceway ponds (ORPs) and closed tubular photobioreactors (PBRs) are currently widely recognized as the most viable configurations for large-scale microalgae cultivation [29].
The treatment of effluents by microalgae can be enhanced by symbiotic associations with other microorganisms, such as bacteria and filamentous fungi. There are many studies on the application of consortia between microalgae and bacteria, whereas information on microalgae and fungi combination (molds and yeasts) is scarce, requiring further studies to understand the nutritional, environmental, and operational aspects for this symbiosis when different types of effluents are applied [21,30].
Considering the co-cultivation of microalgae and filamentous fungi, a mutually beneficial relationship between both microbial groups is observed, such as the gas exchange between the fungal respiration and the microalgae’s photosynthetic process. Additionally, filamentous fungi can mineralize organic matter more efficiently in the effluent, making the absorption of nutrients/contaminants easier for both microbial groups. Microalgae are excellent nitrogen and phosphorus remediators. Also, this consortium leads better with pH fluctuations in the treatment process, because fungi tend to make the environment acidic during their growth, while microalgae make it alkaline because of the photosynthesis. Lastly, filamentous fungi, through the formation of pellets, provide greater protection to microalgae, facilitating their autoflocculation (harvesting stage) and recovery after the process [31,32]. The semicontinuous process has some advantages in comparison to a batch process: lower cultivation time related to the volumetric replacement time (VRT), higher biomass concentration between the cycles which can improve contaminant removal efficiency, control of the process with a single variable (replacement volume) related to the volumetric replacement ratio (VRR), and no need for inoculum replacement between the cycles because the microbial community remaining in the system is adapted and carries out the new cycle efficiently. It is worth noting that the volumetric replacement time (VRT) is directly proportional to the hydraulic retention time (RT); therefore, as the VRT increases, the RT increases [33,34,35].
The microalga Tetradesmus obliquus was already used to treat dairy wastewater [31,32,33,34,35,36], but studies applying a semicontinuous mode in a consortium with a filamentous fungus were not found in the literature. In addition, the fungus Cunninghamella echinulata presented greater efficiency in the treatment of effluents when compared to other fungi traditionally used in the literature, such as the genera Aspergillus and Penicillium. Also, a few studies using this species of fungus in a consortium with microalgae are described in the literature [1,37,38,39].
In this sense, the work aimed to study the tertiary treatment of CW wastewater in an open reactor using the microalga Tetradesmus obliquus alone and in co-cultivation with the fungus Cunninghamella echinulata, carried out in a semicontinuous mode, evaluating the volumetric replacement ratio (VRR) and the volumetric replacement time (VRR and VRT, respectively) for the removal of COD, TN, and TP, and consequently, producing microbial sludge.

2. Materials and Methods

2.1. Cheese Whey and Physicochemical Characterization

The cheese whey (CW) was collected at the Federal Institute of Alagoas—Campus Satuba—Brazil. The effluent was filtered using qualitative filter paper and was subsequently centrifuged at 3500 rpm for 15 min (Centrifuge ACB Labor, São Paulo, Brazil, Dry-Bloch) to remove solid particles, and finally, the effluent was stored in plastic containers and frozen (−18 to −12 °C).
The wastewater characterization was carried out before and after the bioremediation process (tertiary treatment) through analyses of COD, TN, TP, pH, and dry cell weight (after treatment). In order to remove any solids present in the wastewater, the samples underwent filtration using hydrophilic PES (polyethersulfone) filters with porosities of 0.22 and 0.45 µm.
The COD was determined using the dichromate digestion method and using a UV–vis spectrophotometer (Shimadzu, Kyoto, Japan, UVmini 1240) at 600 nm [40]. Total Kjeldahl nitrogen (TN or TKN) (organic nitrogen and ammonium) was determined based on the Kjeldahl method adapted for agro-industrial effluents [41], carried out in three steps: (1) sample digestion (TECNAL, Piracicaba, Brazil, digestion block, model TE007 MP); (2) distillation (TECNAL nitrogen distiller, model TE-0363); and (3) titration with standardized HCI. Total phosphorus (TP) was determined using the Murphy and Riley colorimetric method (ascorbic acid method) [42], using a UV–vis spectrophotometer (Shimadzu, UVmini 1240) at 706 nm. The pH was controlled during the entire experiment using a pH meter (TECNAL) previously calibrated with pH 4.0 and 7.0 standards. Cell dry weight (microbial sludge) was determined from the solids separated during centrifugation at 3200 rpm for 20 min (ACB Labor Dry-Bloch Centrifuge), and after supernatant removal, the solid part was transferred to pre-weighed capsules, and dried in an oven at 105 °C until a constant weight was reached. The sample preparation and treatment analyses are described in Figure 1.

2.2. Microorganisms

The microalga used was Tetradesmus obliquus LCE-01 (indigenous species from the Northeast of Brazil), isolated and characterized by Andrade and collaborators [37]. The microorganism was maintained on a solidified nutrient agar medium (Kasvi®, Pinhais, Brazil) and in a liquid medium for use as an inoculum in BG-11 (blue–green medium) [43]. The microalga was inoculated at an approximate concentration of 50 mg·L−1 of microalga.
The filamentous fungus used in the co-cultivation was obtained from the URM Mycoteque at the Federal University of Pernambuco (UFPE). The species Cunninghamella echinulata (Thaxt) URM 7150 was selected and maintained on PDA medium (Potato Dextrose Agar). The culture medium consisted of an infusion of 140 g·L−1 of potato water, 20 g·L−1 of glucose, 10 g·L−1 of yeast extract, 20 g·L−1 of peptone, and 20 g·L−1 of agar, with a pH adjusted to 5.6 and sterilized at 121 °C and 1 atm for 15 min in an autoclave. After an average growth period of 10 days, the spores were collected using a sterile solution of 0.1% (v/v) Triton and an Ni-Cr alloy to obtain a spore suspension. In each reactor, the fungus was inoculated with a concentration of 2 × 103 spores·mL−1.

2.3. Experimental Setup

Acrylic transparent vessels (PoliControl®, Diadema, Brazil) with dimensions of 18.7 cm × 12 cm × 12 cm were used as bioreactors. Artificial illumination was provided throughout the experiment (Plafon LED, TASCHIBRA, Indaial, Brazil, 25 W 6500 K), measured using a radiometer (HD 2302.0—Delta OHM), applying 100 µmol photons.m−2·s−1 in one side of the reactor, as shown in Figure 2. The effective volume in each trial was 1.7 L. The bioreactor was magnetically stirred (SP LABOR®, Prudente, Brazil) and operated at a room temperature of approximately 30–35 °C, with a pH of 8.0–8.5 when the microalga was used alone and 7.0–7.5 when co-cultivated with the fungus.
The initial organic load of cheese whey had the following characteristics: COD—67,014.48 mg·L−1; TN—1377.05 mg·L−1; and TP—625.60 mg·L−1). Simulated residual concentrations after the secondary treatment presented concentrations of COD in the range of 550–2200 mg·L−1; TN in the range of 14–56 mg·L−1; and TP in the range of 4.5–18 mg·L−1 which were applied in tertiary treatment to meet legislation requirements for discharge in water bodies. The effluent was sterilized at 121 °C for 15 min in an autoclave to start the process without microorganisms other than the inocula.
The experiments consisted of monitoring the growth of the microalga and fungus, as well as contaminant removal, in batch mode (1st–7th day), necessary for the conditioning of the microbial cultures, followed by three cycles of the process in semicontinuous mode, the main objective of this work. In the first stage, only the microalgae were used under surface aeration, adopting volumetric replacement ratios (VRR) of 40% and 60% (v/v) and a volumetric replacement time (VRT) of 7 days. The VRRs of 20% and 80% had been tested in preliminary studies and did not show technical advantages for the system (20%—small volume to be treated and 80%—stability problems due to the imbalance of remaining microbial mass in the reactor to carry out the next semicontinuous cycle).
In the second stage, during microalga–fungus co-cultivation, VRRs of 40% and 60% were also applied, but with a shorter VRT, equivalent to 3 days (it had been previously verified that after this period, contaminant removal was not significant). Aeration was required because filamentous fungi are aerobic, and an effort was made to verify if there was an improvement in the treatment process efficiency. Aeration was provided by a compressor (JEBO, Zhongshan, China, 2 L·min−1), applying a rate of approximately 1.5 vvm. Dissolved oxygen was measured during the experiment (Mod Mo-920 Instrutherm) and maintained in the range of 6–9 mg·L−1. A summary of the experimental stages and their purposes is presented in Table 1.
Analysis of variance (ANOVA), Bonferroni multiple comparison, and Tukey test were carried out using the ASTATSA Calculator (https://astatsa.com/OneWay_Anova_with_TukeyHSD/ (accessed on 8 July 20244) considering significant difference between the treatments if p-value < 0.05.

3. Results and Discussion

3.1. Physical–Chemical Characterization of Cheese Whey Wastewater

The characterization of the CW wastewater used with different organic loads aimed at tertiary treatment is presented in Table 2. Two organic loads, C1 and C2, were used and named CM1 and CM2 when applied for treatment solely with the microalga, and CMF1 and CMF2 when applied for treatment with the microalga–fungus consortium.
After the tertiary treatment, the levels of COD, TN, and TP still exceed the legally established limits for discharge into water bodies [10,44,45], and if only the microalga were applied, an additional polishing treatment (bioremediation) would be necessary. Control experiments were not performed since previous studies were carried out and published by the research group showing that in an open system, the microalga, fungus, or microalga–fungus consortium presented higher efficiency than the control [1,31].
It is worth mentioning that microalgae prefer pH values of 7.5–10 during the dairy wastewater treatment process, with the optimum pH for microalgae of the species T. obliquus being between 7.0 and 8.0. On the other hand, Filamentous fungi prefer acidic pHs, so a range suitable for both microbial groups (pH 7.5–8.0) was considered with the aim of maintaining both species growth, and, consequently, an efficient treatment process [46,47,48].

3.2. Optimizing VRR Applying Microalgae in VRT for 7 Days Using Surface Aeration

The residual values of the evaluated contaminants, the cell dry weight, and pH after the tertiary treatment process of cheese whey wastewater with different organic loads exclusively using the microalga Tetradesmus obliquus under surface aeration and in semicontinuous mode (values are the average of the three cycles applied) are shown in Table 3.
The wastewater discharge standard by the European Union (European Commission 91/271/EEC 2014) advocates for final concentrations and minimum percentage removal in the treatment for COD of 125 mg·L−1 or a 75% reduction, for TN in the range of 10–15 mg·L−1 or 70–80% removal; and for TP in the range of 1–2 mg·L−1 or a minimum of 80% removal [10]. Requirements for COD are more focused on secondary treatment but can be extended to tertiary treatment, while the TN and TP residuals are directly linked to tertiary treatment.
It can be observed that increasing the volumetric replacement rate (VRR) in the analysis of COD and TN caused a decrease in the contaminant removal efficiency for CM1 and CM2. For COD, residual concentrations of 200–400 mg·L−1 were obtained (except for CM1 with a VRR of 60%), still exceeding the European standard. Regarding TN, the VRRs obtained for CM1 or CM2 ranged between 6–7 and 19–24 mg·L−1, respectively. CM1 achieved the discharge parameter, but the minimum treatment percentage could not be reached. Finally, for TP, only the process carried out with CM1 reached the European limit. It is important to highlight that the semicontinuous process was conducted with 7 days of VRT (cycle), which is still high for a polishing step.
After dairy wastewater and sewage secondary treatment processes, several studies showed values of COD in the range of 246–931 mg·L−1, BOD (biochemical oxygen demand) in the range of 80–180 mg·L−1, TN in the range of 10–34 mg·L−1, and TP in the range of 0.5–18 mg·L−1 from initial concentrations of COD, BOD, TN, and TP of 1800–3700 mg·L−1, 1100–2900 mg·L−1, 68–109 mg·L−1, and 14–27 mg·L−1, respectively, with Chlorella sorokiniana, Scenedesmus obliquus, Chlorella vulgaris, Chlorella pyrenoidosa, and Scenedesmus abundans being used [19,49,50].
Regarding the algae concentration (cell dry weight), it was observed that the microbial biomass concentration reached values of 200–440 mg·L−1, with higher concentrations obtained for CM2 due to the higher availability of organic load. According to Choi et al. [51], the microbial growth of microalgae is proportional to the removal of contaminants, especially organic carbon, nitrogen, and phosphorus; therefore, it is necessary to compare the results with the amount of contaminant removal. Considering pH behaviour, there was a wide variation in the range of 4–10, requiring daily adjustment throughout the process. It is important to highlight that the initial phase of decomposition of organic matter is generally acidogenic, leading to a drop in pH that can compromise cell viability, especially for microalgae. After that, when photosynthetic activity is higher, an alkaline pH can be developed [52,53,54].
The adequacy of pH levels obtained are within the range required by CONAMA standards in Resolution No. 430 of 2011 (pH 5–9) [44]. In addition, Leng et al. [55] suggest that there is a flocculation process of the microorganisms at alkaline or slightly alkaline pH levels.
Additionally, forced aeration benefits the removal of COD, TN, and TP, favouring the supply of oxygen or carbon dioxide, increasing biomass performance. The use or non-use of forced aeration depends on effluent and system characteristics, symbiosis with other microorganisms, and operational parameters, as the literature shows studies using microalgae in open systems that are cultured or used to treat effluents by introducing forced aeration [56,57] or surface aeration [58,59]. The main disadvantage of implementing aeration is the energetic cost of the process, but, if necessary, it needs to be considered.
Other studies have emphasized the use of microalgae in COD removal from wastewater, especially when combined with aeration. For example, Afifah, Suryawan, and Sarwono [60] applied the microalga Nannochloropsis sp. for wastewater treatment and observed an improvement with the implementation of aeration in COD removal, achieving removal efficiencies of 73.88% ± 2.05 (aeration of 12 h/d; COD: from approximately 175 mg·L−1 to 50 mg·L−1) and 75.2% ± 3.97 (aeration of 24 h/d; COD: from approximately 175 mg·L−1 to 45 mg·L−1) compared to a removal efficiency of 69.86% ± 5.69 (no aeration; COD: from approximately 75 mg·L−1 to 23 mg·L−1). Salgueiro et al. [61] implemented aeration (14/10 h) in the treatment of effluents in three different types of wastewaters (COD: 720–2240 mg·L−1; PO4−3: 1.5–4.53 mg·L−1) using Chlorella vulgaris, in a semicontinuous process for 15 days with a VRR of 25, 50, 75, and 90% applied on days 4, 7, 9, 10, and 13, respectively. The study showed a removal efficiency of 69% of organic matter, 88% of COD, and nearly complete elimination of phosphates.
Similar results were obtained by Medeiros et al. [1], Choi et al. [51], Mendonça et al. [62], Muthuraman, Murugappan, and Soundharajan [63], Kiran and Mohan [64], and Wang et al. [65]. Medeiros et al. [1] evaluated the removal capacity of COD, TN, and TP from dairy wastewater (COD: 223–1663 mg·L−1; TN: 13–61 mg·L−1; TP: 3–26 mg·L−1) through bioremediation with the fungus Cunninghamella echinulata, the microalga Tetradesmus obliquus, and their consortium. The experiments were carried out using aerated bioreactors (1.5 vvm) for 7 days in batch mode, with a light intensity of 100 µmol photon·m−2·s−1. The results demonstrated the superiority of the consortium, obtaining removal percentages (and rates) of up to 93.7% (429 mg·L−1·d−1), 78.8% (16 mg·L−1·d−1), and 93.4% (11 mg·L−1·d−1) for COD, TN, and TP, respectively.
In Choi et al. [51], the treatment of dairy wastewater (COD: 356 ± 124.7 mg·L−1; TN: 28.9 ± 9.4 mg·L−1; TP: 9.4 ± 4.6 mg·L−1) was studied using the microalga Chlorella vulgaris. The authors reported that after 10 days of treatment with aeration, 80.62% of COD (69.02 mg·L−1), 85.47% of TN (4.20 mg·L−1), and 65.96% of TP (6.2 mg·L−1) was removed.
Also, Mendonça et al. [62] conducted a batch process for treating urban wastewater (COD: 2913 mg·L−1; NH4+: 498 mg·L−1; PO4−3: 23.5 mg·L−1) using the microalga Scenedesmus obliquus. The study achieved removal efficiencies ranging from 65 to 70% (1019–874 mg·L−1) for COD, in the range of 98–99% (9.96–4.98 mg·L−1) for NH4+, and in the range of 69–77.5% (7.30–5.30 mg·L−1) for PO4−3 after 12 days. On the other hand, in a continuous process, the values were 57–61% (1252–1136 mg·L−1) for COD, 94–96% (29.88–19.92 mg·L−1) for NH4+, and 65–70% (8.22–7.05 mg·L−1) for PO4−3 with a hydraulic retention time of 12 days.
In the work of Muthuraman, Murugappan, and Soundharajan [63], the microalga Coelastrella sp. was used to treat pig wastewater with and without aeration. Aeration favoured the removal of TN and TP, achieving removal percentages of 80% (from 25 to 5 mg·L−1) for TP, and 64% (from 110 to 40 mg·L−1) for TN. On the other hand, the non-aeration process resulted in a removal efficiency of 31% (from 65 to 45 mg·L−1) for TN, and 58% (from 12 to 5 mg·L−1) for TP. The average biomass productivity obtained was 81.5 mg·L−1·d−1. In addition, the work developed by Kiran and Mohan [64] using Tetradesmus sp. SVMIICT4 in the treatment of dairy effluent achieved the removal efficiencies of 95% for COD (3600 to 160 mg·L−1), 65.2% for TN (145.93 to 50.69 mg·L−1), and 57.35% for TP (175.97 to 75.04 mg·L−1) with aeration of 35 L·min−1 for 12 days.
Finally, Wang et al. [65] used the microalga Oedogonium sp. for treating livestock wastewater (COD: 90.7 mg·L−1; TN: 59.4 mg·L−1; TP: 6.2 mg·L−1) for 7 days with aeration at 0.5 L·min−1, achieving removal efficiencies for COD, TN, and TP of 62.5% (34 mg·L−1), 95.9% (2.43 mg·L−1), and 92.9% (0.44 mg·L−1), respectively. According to Magro et al. [66], COD removal is a mixotrophic process specific to each species of microalga. The autotrophic part is generally more efficient than the heterotrophic part when CO2 and light are available, revealing a limitation to apply the treatment in very high organic loads when microalgae are applied alone.
Based on this, an attempt was made to optimize the preceding step by applying a microbial consortium consisting of the microalga (Tetradesmus obliquus) and a filamentous fungus (Cunninghamella echinulata) to observe if greater system stability, increased efficiency in contaminant removal, and a reduction in treatment time under semicontinuous conditions could be achieved (this outcome can be a result of the synergistic contribution of the filamentous fungus to the microalga, combined with forced aeration applied to the system).

3.3. Evaluation of Microalga–Fungus Co-Cultivation Using VRT = 3 Days with VRRs of 40 and 60% under Forced Aeration

It is noted that in both organic loads applied, the residual values exhibited high percentages of removal for COD. These data were more evident at the 40% VRR (both CMF1 and CMF2), achieving 77.0% and 74.1%, respectively (Table 4), which were higher and with a shorter VRT (3 days) than in the previous stage when only the microalga was used with a VRT of 7 days.
The residual concentration of COD in the CMF1 effluent showed a variation in the range from 61.86 to 88.17 mg·L−1 (removal efficiency of 70 and 74%, respectively), remaining within the limits established by the European standard (European Commission 91/271/EEC 2014), which stipulates a maximum concentration of 125 mg·L−1 [10], considering both VRRs of 40% and 60%. In CMF2, the system managed to stay within the established limits for residual COD by the European standard for 40% of VRR and by the percentage reduction for CMF2 in 60% of VRR. It is noticeable that a significant amount of COD was successfully reduced in CMF2, and it came very close to the established limit for the 60% VRR, which could be easily achieved with a sequential stage.
Nitrogen and phosphorus are crucial for biomass formation in microbial communities, and their efficient removal is essential to meet legal requirements due to eutrophication problems in water bodies [67]. Considering TN and the parameters established for discharge into water bodies, compliance with European standards is observed in both treatments, with residual values from 1.82 to 2.71 mg·L−1 (removal efficiency of 74 and 72%, respectively) for CMF1 treatment and from 6.50 1 to 9.49 mg·L−1 (removal efficiency of 72 and 70%, respectively) for CMF2 treatment, results much lower than those obtained for CM1 and CM2. Additionally, the data are in line with the minimum percentage and residual concentration stipulated by European legislation.
Analyzing the results for TP, lower residual concentrations of total phosphorus were achieved compared to the previous stage, ranging from 0.65 to 0.88 mg·L−1 for CMF1 and between 1.98 and 3.38 mg·L−1 for CMF2 (Table 4). A maximum removal efficiency of 65–71% was observed in both effluents (CMF1 and CMF2). It is important to highlight that both processes carried out with CMF1 effluent in both VRRs and CMF2 with a 40% VRR met European regulatory standards (residual concentration). In this context, alternatives such as introducing aeration to reduce the volumetric replacement time (VRT) and/or microalga–fungus co-cultivation proved to be promising strategies to enhance process efficiency.
Since TN was the only parameter (both VRRs of 40 and 60% and organic loads CMF1 and CMF2) to achieve residual values compatible with discharge standards under all tested conditions, it may be present in limiting concentrations, hindering the removal efficiency of COD and total phosphorus from the system. The COD:N:P balance (DQO:N and also N:P ratios) is necessary for effective effluent treatment and depends on the microorganism used as well as the natural characteristics of the effluent, especially in agro-industrial effluents. In this study, a DQO:N ratio of around 50 was applied, but an evaluation between 10–50 is recommended during co-cultivation [31], and it can be explored in future studies.
The microalga–fungus consortium is carried out by aggregating microalgae on the meshes of fungal hyphae through absorption and encapsulation, leading to increased biomass production and causing a synergistic effect between these two microorganisms as explained by Wang et al. [32] and Chu et al. [68].
Additionally, the bioflocculation occurs in the consortium between microalgae and filamentous fungi and it can be explained by three mechanisms: (1) charge neutralization (neutral pH) on the surface of negatively charged microalgae cell wall functional compounds interacting with the positively charged surface of fungi, behaving as cationic flocculants that tend to neutralize the negative charges of microalgae, intertwining with these microorganisms; (2) interaction between surface proteins; and (3) adhesion of microalgae to the exopolysaccharides excreted by fungi [55,69].
Other studies also demonstrate that the consortium between microalgae and/or other microbial classes enhances wastewater treatment. For example, Hu et al. [70] acclimatized a consortium with microalgae, including C. vulgaris, to efficiently remove organic pollutants and nutrients from meat processing wastewater. Mujtaba, Rizwan, and Lee [71] demonstrated that the co-cultivation of C. vulgaris and Pseudomonas putida enhanced the removal of nutrients and COD from wastewater. Similarly, Guo et al. [72] verified a higher efficiency of a co-culture of Ganoderma lucidum and C. vulgaris in reducing COD, total nitrogen, and total phosphorus from swine wastewater.
In the present work, the presence of exogenous microorganisms did not inhibit the growth of the microalga–fungus consortium. Also, the semicontinuous process reached lower contaminant concentrations in lower cultivation time and without inoculum replacement with stability during three cycles.

3.3.1. Microalga–Fungus Cell Dry Weight

The biomass concentration at the end of semicontinuous cycles reached values of 350.66 ± 13.61 and 319.33 ± 7.02 mg·L−1 for CMF1 and 778.00 ± 15.02 and 690.66 ± 28.59 mg·L−1 for CMF2 to VVRs of 40 and 60%, respectively. A lower biomass concentration for a VRR of 60% compared to 40% was obtained (p < 0.05). The synergy with fungi ensures greater stability for the microalgae and consequently higher production of microbial biomass (cell dry weight) with pellet formation (Figure 3). The growth of microalgae is indeed proportional to the removal of contaminants, particularly organic carbon, nitrogen, and phosphorus [73,74]. Similarly, other microbial groups during microalgae cultivation in wastewater can significantly impact biomass production and nutrient removal [75].
The use of microalgae and/or fungi in dairy wastewater treatment have been shown in the literature. For example, the study by Kiran and Mohan [64] using Tetradesmus sp. SVIICT4 in the treatment of dairy wastewater (COD: 3600 mg·L−1; TN: 158.69 mg·L−1; TP: 175.97 mg·L−1) in a flat-screen photobioreactor (light intensity of 200 µmol photon m−2·s−1) and applying a daily photoperiod of 12/12 h (light/dark) for 12 days, with aeration of 35 L·min−1 (air), resulted in a biomass concentration of 2.38 g·L−1. Djelal and Amrane [76] used a consortium of fungi Aspergillus niger, Mucor hiemalis, and Galactomyces geotrichum in the treatment of dairy wastewater (COD: 2000 mg·L−1) in an aerated bioreactor carried out in batch operation mode, achieving final biomass concentrations in the range of 1.65–1.9 g·L−1 after 160 h of cultivation time. In addition, Daneshvar et al. [36] verified that two cultivation cycles of the microalgae Scenedesmus quadricauda and Tetraselmis suecica in dairy industry wastewater resulted in cell dry weights of 360 and 650 mg·L−1, respectively. Finally, Ding et al. [77] demonstrated the feasibility of cultivating the microalga Chlorella sp. in dairy wastewater, producing a cell dry weight of 860 mg·L−1. All authors mentioned that microalgal and/or fungal growth is proportional to the availability of organic carbon, nitrogen, and phosphorus in the effluents [73,74].
Furthermore, in some cases, an increase in biomass production was found when the reactors were supplemented with an additional nitrogen concentration such as (NH4)2SO4 and can be justified by a good correlation between the ratio of available organic carbon, nitrogen, and phosphorus [78]. A consortium between microalgae and fungi for the tertiary treatment of dairy wastewater in batch mode was proposed by Medeiros et al. [1] with the filamentous fungus Cunninghamella echinulata, the microalga Tetradesmus obliquus, and their co-cultivation (COD: 223–1663 mg·L−1; TN: 13–61 mg·L−1; TP: 3–26 mg·L−1). The study was carried out using aerated bioreactors for 7 days and applying light intensity (100 µmol·m−2·s−1). The cell dry weight obtained was 65–267 mg·L−1 using the microalga, 66–307 mg·L−1 with the filamentous fungus, and 124–533 mg·L−1 with the microalga–fungus consortium, proving that the consortium can produce a higher biomass concentration, as was also verified in this work in semicontinuous mode.

3.3.2. pH Behaviour during Microalga–Fungus Co-Cultivation

Observing the pH, it was possible to highlight greater pH stability when there is co-cultivation between microalgae and fungi compared to microalgae alone, with lower pH variation, in the range of 6–8.5 but without significantly affecting the microbial classes involved, mostly remaining in the range of 7.5–8.5 (Figure 4). pH regulation during the treatment stage proved to be fundamental due to its influence on microorganism activity and the overall process performance.
Additionally, microalgae prefer pH values between 7.5–10 during wastewater treatment [46]. On the other hand, filamentous fungi prefer acidic pH levels, so it is important to consider a range that is suitable for these microbial groups. It is noticeable that the range used, 7.5–8.0, ensured a good treatment process.
According to Leng et al. [55] and Zhang and Zhang [79], the influences of pH on microalga–fungus co-cultivation systems have been observed through their interactions, highlighting the difference in zeta potentials (negative charge for microalgae and positive charge for fungi), increasing buffering capacity, and facilitating the aggregation and pellet formation. Additionally, it is mentioned that microorganism flocculation processes are favoured at alkaline pH levels.
It is also important to note that the initial phase of organic matter decomposition, driven by fungi, mainly, is acidogenic, leading to a pH drop that can compromise cell viability, especially for microalgae [52,53]. However, it was observed in this work that during co-cultivation, pH drops were less pronounced compared to microalgae applied alone in the first stage of the study (from 4–10 to a 5–8 pH range), indicating increased buffering capacity when these microorganisms are cultivated together. Depending on effluent characteristics, the buffering capacity in the fungi–microalga co-culture may exist without requiring pH adjustment [80], which was not the case in this study.

4. Conclusions

The symbiosis between the fungus Cunninghamella echinulata and the microalga Tetradesmus obliquus proved to be a promising strategy for the efficient removal of contaminants from CW wastewater during tertiary treatment, notably COD, total nitrogen, and total phosphorus. The study addressed different operational conditions of the bioreactors in semicontinuous mode, such as volumetric replacement time and ratio, in addition to co-cultivation between the species. With co-cultivation, volumetric replacement rates (VRRs) of 40–60% over a replacement time (VRT) of 3 days were sufficient to achieve discharge standards required by legislation for CW effluents with initial concentrations of 550–2200 mg·L−1 for COD, 14–56 mg·L−1 for TN, and 4.5–18 mg·L−1 for TP. However, it is important to highlight that a larger scale validation of the process is necessary, seeking a reduction in treatment time and higher contaminant removal efficiency.

Author Contributions

Conceptualization, L.M.d.S., C.E.d.F.S. and J.A.M.; methodology, L.M.d.S., C.E.d.F.S. and J.A.M.; formal analysis, L.M.d.S., C.E.d.F.S. and B.M.V.d.G.; investigation, L.M.d.S., J.C.B.d.S. and C.E.d.F.S.; writing—original draft preparation, L.M.d.S., C.E.d.F.S. and B.M.V.d.G.; resources, J.C.B.d.S. and C.E.d.F.S.; visualization, J.C.B.d.S., C.E.d.F.S., A.K.d.S.A., R.M.R.G.A. and G.M.; software, C.E.d.F.S. and B.M.V.d.G.; validation, C.E.d.F.S.; data curation, C.E.d.F.S., B.M.V.d.G., A.K.d.S.A., R.M.R.G.A. and G.M.; writing—review and editing, C.E.d.F.S., B.M.V.d.G., A.K.d.S.A., R.M.R.G.A. and G.M.; supervision, C.E.d.F.S.; project administration, C.E.d.F.S.; funding acquisition, C.E.d.F.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research work was funded by CNPq—Brazil (National Council for Scientific and Technological Development) (project numbers: 313195/2019-6, 440070/2019-8, 407274/2018-9 and 312996/2022-5) and FAPEAL (Foundation for Research Support of Alagoas—Brazil) (project numbers: E:60030.0000002360/2022 and E:60030.0000000318/2023).

Data Availability Statement

Data available on request.

Acknowledgments

The authors would like to thank CNPq—Brazil (National Council for Scientific and Technological Development) and FAPEAL (Foundation for Research Support of Alagoas—Brazil).

Conflicts of Interest

The authors declare no conflicts of interest.

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Processes 12 01573 g001
Figure 1. Flowchart of effluent conditioning and analyses performed in the experiments.
Figure 1. Flowchart of effluent conditioning and analyses performed in the experiments.
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Figure 2. Schematic representation of the bioreactor.
Figure 2. Schematic representation of the bioreactor.
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Figure 3. Macroscopic aspect and optical microscopy of the microalga–fungus consortium (40×).
Figure 3. Macroscopic aspect and optical microscopy of the microalga–fungus consortium (40×).
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Figure 4. pH during the treatment of CMF1 (A,B) and CMF2 (C,D) as a function of VRRs of 40% and 60% (microalga–fungus co-cultivation).
Figure 4. pH during the treatment of CMF1 (A,B) and CMF2 (C,D) as a function of VRRs of 40% and 60% (microalga–fungus co-cultivation).
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Table 1. Detailing the experimental stages.
Table 1. Detailing the experimental stages.
StageOperation Mode (VRT *)VRR *Microorganism/
Representation		Forced AerationFunction
1stBatch +
Semicontinuous ** (7 d)		40 and 60%Microalga/CMNoEvaluate VRR * during microalga cultivation
2ndBatch +
Semicontinuous ** (3 d)		40 and 60%Microalga +
fungus/CMF		YesImprove the treatment efficiency applying co-cultivation and decrease VRT *
* VRR—volumetric replacement ratio and VRT—volumetric replacement time. ** In the semicontinuous mode, 3 cycles were carried out to evaluate the system’s stability. The batch phase lasted for a period of 7 days to allow the necessary microbial growth to apply the semicontinuous mode.
Table 2. Characterization of CW wastewater.
Table 2. Characterization of CW wastewater.
ParameterMicroalgaMicroalga–Fungus Co-Cultivation
CM1CM2CMF1CMF2
COD (mg·L−1)558.41 ± 7.472224.41 ± 3.73546.46 ± 3.082288.87 ± 2.56
TN (mg·L−1)14.38 ± 1.0755.76 ± 3.2114.03 ± 1.1456.10 ± 2.37
TP (mg·L−1)4.75 ± 0.0317.84 ± 0.064.65 ± 0.0117.92 ± 0.01
pH3.29 ± 0.022.97 ± 0.023.25 ± 0.032.93 ± 0.02
Table 3. Residual values and removal efficiency of COD, TN, and TP during semicontinuous treatment for CM1 and CM2 (microalga only).
Table 3. Residual values and removal efficiency of COD, TN, and TP during semicontinuous treatment for CM1 and CM2 (microalga only).
VRR *COD TNTPCell Dry WeightpHadjusted
(mg·L−1)(%) **(mg·L−1)(%) **(mg·L−1)(%) **(mg·L−1) ***
CM1
40%190.1 ± 24.00 a56.9 ± 5.26.92 ± 0.35 a29.5 ± 8.90.98 ± 0.19 a65.2 ± 8.30348.7 ± 38.2 a8.38
60%107.37 ± 32.27 b68.2 ± 3.46.17 ± 0.18 b46.5 ± 3.81.64 ± 0.24 b54.5 ± 7.50298.7 ± 18.2 b8.40
CM2
40%409.24 ± 47.52 a68.2 ± 4.823.52 ± 3.05 a34.6 ± 8.32.86 ± 0.76 a68.5 ± 10.40862.0 ± 8.0 a8.38
60%198.02 ± 59.85 b83.6 ± 5.919.20 ± 2.95 b37.2 ± 3.78.00 ± 1.55 b40.60 ± 9.80539.3 ± 10.2 b8.32
* VRR—volumetric replacement ratio. ** (%) Process efficiency considering initial and final contaminant concentrations after volume replacement during 3 cycles. Residual contaminant values are more representative to analyze process efficiency because they are directly related to the legislation standards. *** Microalga-based sludge. Different letters above the residual values of the contaminants (and final dry cell weight) indicate a significant statistical difference between the experiments according to Bonferroni multiple comparison and Tukey test (p < 0.05). CM1 with VRR: 40% was compared to VRR: 60%, as well as CM2 with VRR: 40% was compared to VRR: 60%.
Table 4. Residual values and removal efficiency of COD, TN, and TP in batch and in semicontinuous mode for CMF1 and CMF2 (microalga–fungus co-cultivation).
Table 4. Residual values and removal efficiency of COD, TN, and TP in batch and in semicontinuous mode for CMF1 and CMF2 (microalga–fungus co-cultivation).
VRR *Operation ModeCODTNTP
(mg·L−1)(%) **(mg·L−1)(%) **(mg·L−1)(%) **
CMF1
40%Batch187.62 ± 5.0866.43.09 ± 1.0378.00.62 ± 0.0688.7
Semicontinuous1st cycle49.45 ± 3.0874.01.96 ± 0.5475.40.63 ± 0.0171.8
2nd cycle33.83 ± 1.0386.71.40 ± 0.3680.00.60 ± 0.0172.9
3rd cycle 52.29 ± 1.0372.21.50 ± 0.4467.40.70 ± 0.0166.9
Average45.19 ± 9.94 b77.0 ± 6.51.61 ± 0.28 b74.3 ± 4.60.65 ± 0.04 b70.5 ± 2.4
60%Batch175.32 ± 7.1867.92.52 ± 0.3882.00.54 ± 0.0886.7
Semicontinuous1st cycle92.98 ± 2.0568.13.09 ± 0.5569.90.92 ± 0.0170.6
2nd cycle64.59 ± 1.0380.62.24 ± 0.2275.80.83 ± 0.0171.7
3rd cycle78.94 ± 1.0373.62.81 ± 0.7668.80.90 ± 0.0067.3
Average78.51 ± 13.71 a74.1 ± 4.32.71 ± 0.43 a71.5 ± 2.90.88 ± 0.04 a69.9 ± 1.7
CMF2
40%Batch320.39 ± 2.5686.18.84 ± 1.3684.32.76 ± 0.0584.6
Semicontinuous1st cycle215.3 ± 0.00 ***76.57.29 ± 1.0470.52.23 ± 0.0171.3
2nd cycle116.61 ± 1.0379.65.75 ± 1.4775.61.68 ± 0.0174.5
3rd cycle113.28 ± 5.1377.36.45 ± 0.8569.72.02 ± 0.0167.1
Average115.07 ± 3.49 b77.8 ± 1.26.50 ± 0.53 b71.9 ± 2.41.98 ± 0.20 b71.0 ± 2.6
60%Batch335.77 ± 12.5685.210.1 ± 0.5882.02.80 ± 0.0184.3
Semicontinuous1st cycle299.89 ± 2.26 ***75.69.82 ± 0.9471.13.58 ± 0.0167.2
2nd cycle238.37 ± 3.0677.79.54 ± 1.2770.63.01 ± 0.0169.9
3rd cycle212.74 ± 5.1673.69.12 ± 0.3368.03.55 ± 0.0361.2
Average225.33 ± 13.04 a75.6 ± 1.49.49 ± 0.25 a69.9 ± 1.33.38 ± 0.25 a66.1 ± 3.3
* VRR—volumetric replacement ratio. ** (%) Process efficiency considering initial and final contaminant concentrations in the operation mode. Residual contaminant values are more representative to analyze process efficiency because they are directly related to the legislation standards. *** Not considered in the average calculation because the system was not stable at that moment. Different letters above the residual values of the contaminants (and final dry cell weight) indicate a significant statistical difference between the experiments according to Bonferroni multiple comparison and Tukey test (p < 0.05). CMF1 with VRR: 40% was compared to VRR: 60%, as well as CMF2 with VRR: 40% was compared to VRR: 60%. Also, batch experiments were compared between them to emphasize the reproducibility of the experiments before the semicontinuous mode application and did not show a significant statistical difference (p < 0.05).
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MDPI and ACS Style

Monteiro dos Santos, L.; Barbosa da Silva, J.C.; de Farias Silva, C.E.; Villar da Gama, B.M.; Almeida Medeiros, J.; Markou, G.; Rosas Garcia Almeida, R.M.; Abud, A.K.d.S. Co-Cultivation between the Microalga Tetradesmus obliquus and Filamentous Fungus Cunninghamella echinulata Improves Tertiary Treatment of Cheese Whey Effluent in Semicontinuous Mode. Processes 2024, 12, 1573. https://doi.org/10.3390/pr12081573

AMA Style

Monteiro dos Santos L, Barbosa da Silva JC, de Farias Silva CE, Villar da Gama BM, Almeida Medeiros J, Markou G, Rosas Garcia Almeida RM, Abud AKdS. Co-Cultivation between the Microalga Tetradesmus obliquus and Filamentous Fungus Cunninghamella echinulata Improves Tertiary Treatment of Cheese Whey Effluent in Semicontinuous Mode. Processes. 2024; 12(8):1573. https://doi.org/10.3390/pr12081573

Chicago/Turabian Style

Monteiro dos Santos, Leandro, Joyce Camila Barbosa da Silva, Carlos Eduardo de Farias Silva, Brígida Maria Villar da Gama, Josimayra Almeida Medeiros, Giorgos Markou, Renata Maria Rosas Garcia Almeida, and Ana Karla de Souza Abud. 2024. "Co-Cultivation between the Microalga Tetradesmus obliquus and Filamentous Fungus Cunninghamella echinulata Improves Tertiary Treatment of Cheese Whey Effluent in Semicontinuous Mode" Processes 12, no. 8: 1573. https://doi.org/10.3390/pr12081573

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