Livestock wastewater bioremediation through indigenous microalgae culturing as a circular bioeconomy approach as cattle feed

Algal Research 78 (2024) 103424 Available online 8 February 2024 2211-9264/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/bync/4.0/). Livestock wastewater bioremediation through indigenous microalgae culturing as a circular bioeconomy approach as cattle feed M. Blanco-Vieites a , M. ´ Alvarez-Gil b , c , F. Delgado b , c , L. García-Ruesgas d , E. Rodríguez c , * a GEMAP (GI1243), CRETUS, Departamento de Edafoloxía e Química Agrícola, Facultade de Farmacia, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain b Neoalgae Micro Seaweed Products, Calle Carmen Leal Mata, 191, 33211 Gijon, Spain c Department of Construction and Manufacturing Engineering, University of Oviedo, Pedro Puig Adam, s/n, 33203 Gijon, Spain d Department of Engineering Graphics, University of Seville, 41092 Seville, Spain ARTICLE INFO Keywords: Microalgae Bioremediation Wastewater Livestock Circular economy ABSTRACT Growing human population demands the enhancement intensive livestock activities, which lead to severe environmental issues like water pollution. Cattle wastewater is rich in some inorganic pollutants, mainly nitrogen and phosphorous, which might constitute a risk to water bodies. Considering this scenario, microalgae’s capacity to develop within manure wastewater is attracting interest, mainly for simultaneous wastewater bioremediation and biomass production. The aim of this work was evaluating the response of several microalgae strains to increasing percentages of unsterilized cattle farm wastewater. Moreover, this study also analyses the possibility of enhancing the development of indigenous native microalgal strains, to create a bioremediation system based on previously adapted species, which specific diversity was determined through taxonomic techniques. Two monospecific Chlorophyceae strains were used: Chlorella sp. and Coelastrella cogersae; and one Cyanobacteria strain: Arthrospira platensis. Experimental assays were conducted in 6 groups and triplicates with increasing percentages of Livestock wastewater (LW): control, 15, 25, 35, 50 and 70 %. All the strains tested showed that cattle wastewater represented a source of metabolic stress for microalgae development. Nevertheless, the autochthonous microalgal bloom showed better results in terms of tolerance to wastewater percentage and development under same conditions Laboratory scale results showed that microalgae could develop with optimum results in presence of 15 % of wastewater, reaching absorbance values close to 90 % of the achieved by control cultures. Moreover, indigenous microalgae cultures were scaled up to open type industrial systems (raceways) and influence of their growth on wastewater composition was evaluated. Results showed a severe reduction of the concentration of nitrates to 43.4 (mg⋅L −1 ) (20 % of initial measurements). Moreover, the obtained biomass showed a biochemical profile similar to traditional crops, in terms of proteins and carbohydrates (48 and 41 % respectively), suggesting that this biomass could be further valuated into cattle feed as a circular economy approach. 1. Introduction Nowadays, it is estimated that 56 billion cattle animals are raised, annually, for human consumption, which might be doubled by year 2050 to fulfill the needs of the growing global population [1]. On this basis, the water demand of cattle industry is also expected to increase. Previous studies estimate that 76.7 L of water is required to produce 1 kg of beef, which is mainly discharged into the environment as livestock wastewater that represent a risk for eutrophication of water bodies [2,3]. This residue mainly includes the effluents derived from a mixture of excrements and maintenance duties, as the water used for cattle sheds cleaning, that has elevated concentrations of nitrogen and phosphorus. Moreover, it also has high levels of organic matter, which is usually expressed as biochemical oxygen demand (BOD) and chemical oxygen demand (COD) [4]. According to previous studies, these pollutants have a wide range based on species, age and feed composition, among others. Nevertheless, usual concentrations range as follows: 2000–30,000 mg⋅L −1 BOD, 200–2055 mg⋅L −1 nitrogen (N) and 100–620 mg⋅L −1 total phosphorus (TP). These contaminants might become an issue of public concern if untreated, especially due to their passive influence on * Corresponding author. E-mail address: [email protected] (E. Rodríguez). Contents lists available at ScienceDirect Algal Research journal homepage: www.elsevier.com/locate/algal https://doi.org/10.1016/j.algal.2024.103424 Received 28 December 2023; Received in revised form 31 January 2024; Accepted 5 February 2024 Algal Research 78 (2024) 103424 2 groundwater and air, that even threat public health [5]. Thus, proper environmental disposal and adequate treatment of these residue is crucial for a sustainable development. Several chemical, physical and biological methods have been developed to treat these types of wastewaters. According to previous studies, the main chemical treatments include the use of toxic chemicals such as chlorine, hydrogen peroxide and ozone to sterilize wastewater or even control pH [6]. On the same basis, some limes are used to coagulate, and precipitate determined pollutants. Hence, several physical treatments were developed to face the growing issue of wastewater generation in farming industry. These methods refer to the removal of suspended solids without influencing the chemical or biological composition of the targeted wastewater. Among the developed methods, aeration, adsorption, membrane separation and sedimentation are considered the most popular physical methods for livestock wastewater remediation. These techniques are commonly used as pre-treatments for subsequent systems, due to the fact that they do not eliminate compounds of emerging concern such as biological or chemical pollutants [7,8]. Furthermore, several authors proposed the use of biological systems with good acceptance by farming industry, specially based on the fact that biological methods are considered as more cost-effective than chemical or physical methods [9]. Biological wastewater treatments are classified in three broad categories: aerobic, anaerobic and mixed systems. Overall, the functionality of these systems is based on cellular processes and depend on organisms such as nematodes and bacteria, among others, that uses their metabolism to breakdown organic wastes [10]. Their performance involves different components such as bioaccumulation and biodegradation of pollutants, among others, that are involved in the wholescale of bioremediation. According to previous studies, bioremediation has shown to be very effective in term of wastewater treatment due to its adsorption potential and low time needed [11,12]. Until now, livestock farming industry has used a wide variety of these approaches, however, there is still scope and need to develop new sustainable procedures. Novel systems should consider these residues as an origin of resources that could be further utilized, like water and nutrients, making and approach to circular bioeconomy. In view of the above situation, photosynthetic organisms such as microalgae can offer a novel tool for wastewater recycling, specially due to their large requirements of water and nutrients [13]. In this regard, livestock wastewater is highly concentrated in nutrients, mainly nitrogen and phosphorous, which are considered as main compounds needed for microalgae cultivation [14]. Previous studies have demonstrated the efficiency of these microorganisms to recover heavy metals, sulphates and other complex substances, such as phenolic compounds [15,16]. This technology not only reduces the pollutants concentration of the targeted wastewater but also converts this residue into reusable water and recovers highly valuable nutrients. Thus, microalgal bioremediation could represent a feasible substitute for expensive and less sustainable traditional chemical treatments. Moreover, consortia based on bacteria and microalgal microorganisms have been proved to enhance nitrogen assimilation into microalgal biomass from nutrient-rich wastewater medium [17]. Furthermore, microalgal bioremediation of wastewaters has also been proved to be a valuable tool for circular economy development, specially due to the biomass synthesis and further processing into novel biofuels ([18,19]). Nevertheless, tolerance to wastewater presence is a bottleneck in the industrial application of this technology. Based on previous works, dilution of livestock wastewater is necessary to evade inhibition of microalgae development. Thus, this step requires the addition of additional nutrients and great amounts of fresh water, that reduce efficiency and increases the final cost [13]. This drawback can be partially avoided by selecting the most adequate microalgal strain through tolerance assays. Nevertheless, based on the available bibliography, all previous works study monospecific cultures and seem to discard the idea of enhancing the growth of indigenous strains, that might be better adapted to the reign conditions in wastewater [20–22]. In this context, the goal of this work is to analyze the viability of livestock bioremediation through microalgal cultivation. For this purpose, the main objectives of this research were: i) study tolerance of 2 eukaryotic microalgae (Chlorella sp. and Coelastrella cogersae) and 1 cyanobacteria (Arthrospira platensis) to livestock wastewater, in order to evaluate which species are the most tolerant to this residue; ii) evaluate the possible presence of indigenous microalgal species and their specific variety through taxonomic techniques, iii) escalate the best adapted strains to industrial scale open-type culture systems (raceways), in order to study the feasibility of the developed technology at industrial scale. Finally, cultures were harvested and the influence of microalgae presence on wastewater was studied through the quantification of determined targeted pollutants. Moreover, in order to stablish an approach to circular bioeconomy, biomass biochemical profile was analyzed to evaluate the ratio of proteins, carbohydrates and lipids and compared to feedstock crops. 2. Materials and methods This study was carried out in the facilities of Neoalgae Micro Seaweed Products (Gij´ on, Spain). The investigation was developed in two phases: tolerance to increasing percentages of wastewater and scaling up of the developed technology. 2.1. Microalgal strains The selection of strains was done according to the outcomes of previous studies focused on wastewater bioremediation [15,23]. All of the selected strains were maintained in the repository of Neoalgae Micro Seaweed Products catalogue (Gij´ on, Spain) along the duration of the experimentation. Two eukaryotic green microalgae were chosen, Chlorella sp., which was previously isolated by Su´ arez-Montes et al. [24] and Coelastrella cogersae, which was also isolated by [24]. Moreover, a cyanobacteria was also selected for the study: Arthrospira platensis (obtained from the Spanish Bank of Algae, BEA). Moreover, an indigenous microalgal bloom was obtained from wastewater. This bloom was generated by diluting samples of livestock wastewater to 20 % (with addition of 80 % of sterile fresh water) and exposing these samples to the same conditions as the selected eukaryotic strains, as suggested by [25]. Once this bloom was obtained, initial cultures were maintained in exponential growth by periodic renewals, in order to keep healthy inoculum for the rest of the experimentation. 2.2. Culture media The selection of the culture media was done according to the results observed by previous studies, specially the outcomes recorded by [24,26]. On this basis, BG-11 medium was selected for eukaryotic microalgae growth, which composition and dosage was taken as described by [23]. Moreover, as suggested by [27], cyanobacteria of Arthrospira genre grow under optimum conditions with addition of Zarrouk medium. On this basis, this culture media was used as described in their work and dosage was done by adding 1 mL of stock solution × L −1 . Chemical compounds were purchased to Labbox Labware (Barcelona, Spain), being all of them considered as analytical grade. 2.3. Morphological analysis of the microalgal bloom Samples of the selected wastewater were taken by using 2 L inert plastic bottles. In order to discover the microalgal variability in such an extreme environment, classical morphological studies through microscopy examination were done. Accordingly, aliquots of the sampled wastewater were characterized by specific morphological features. Thus, optical observation of the obtained cultures was done by using an inverted phase-contrast optical microscope (BioBlue, S/N-EU-1612189, Euromex, Holland). Taxonomic identification of the present phototrophs was carried out by means of morphological characters, such as shape, M. Blanco-Vieites et al. Algal Research 78 (2024) 103424 3 presence of organelles and flagella, among others. Observed features were then compared to previous published works with taxonomic descriptions [28,29]. 2.4. Livestock wastewater composition The selected wastewater (WW) was not sterilized by any physical or chemical methods, expecting to preserve original characteristics. WW was provided by a piggery livestock farm located in the northwest of Spain, whose information will be settled as confidential, and samples were kept in 20 L inert plastic bottles under dark and cool conditions. According to the aim of this study, it was decided to analyze main parameters of concern: BOD, COD, nitrates, total aerobes and total enterobacteria. Due to obtained outcomes, it was decided to analyze LW at a dilution of 15 % in culture media, based on the fact that this was the maximum tolerated percentage during tolerance assays. BOD was analyzed by following the OXITOP method, according to the standards indicated by the UNI EN ISO 1899-1:2001, showing a mean value of 669 (O 2 ⋅mL −1 ). Moreover, COD was measured following the APAT/CNR method, according to the IRSA Manuals 117/2014 - Method 513 [30], with a mean value of 962 (O 2 ⋅mL −1 ). Regarding nitrates, mean concentration was 350 (mg⋅L −1 ). This value was measured following the protocol described by [31], based on ultraviolet spectrophotometric screening methodology. Bacterial presence evaluation was done by colony-count techniques, as described in ISO 21528-2:2017 (Corrected version 2018-06-01) [32]. Mean value of this parameter was 1⋅10 3 (ufc⋅mL −1 ). 2.5. Laboratory conditions Laboratory scale cultures were maintained in a chamber with controlled conditions of irradiance (70–100 μ Em −2 s −1 ), temperature (25 ±1 ◦C) and photoperiod (16 h light:8 h dark), as suggested by [33]. Microalgae monospecific cultures were submitted to a preliminary acclimatation phase by growing the selected strains in borosilicate flasks for 10 days, in order to obtain a homogeneous initial inoculum. For all developed assays, experimental cultures were performed in triplicate, including a control group. In addition, the cultures were subjected to continuous agitation by aeration using air pumps with a flow rate of 100 L⋅h −1 . 2.6. Pilot scale conditions After obtaining results from laboratory scale assays, it was decided to scale up to pre-industrial systems the best adapted strain (to presence of wastewater). Accordingly, 2 L were used as initial inoculum for 10 L plastic bottles (Nalgene, USA), which were maintained inside a greenhouse with uncontrolled conditions (Gij´ on, Spain). Temperature was an uncontrolled parameter along the experimental assay, anyway, it was periodically measured, showing an average of 27.6 ◦C, which was considered in the optimum range. Based on the study of [34], meteorological variations in environmental conditions could negatively affect growth rates. Thus, the step from controlled conditions chamber to uncontrolled conditions greenhouse was considered as critical phase due to the possible sources of stress. Based on this, it was decided to periodically renew the cultures 5 times every 10 days to acclimate the cultures to the controlled conditions. Afterwards, 10 L culture bottles were used as inoculum for closed type photobioreactors (PBRs) (Aqualgae, Spain), which consisted of vertical closed methacrylate columns with 3 m high and 30 cm wide. In these systems, agitation of the cultures was provided through the addition of aeration at a flow rate of 100 L⋅h −1 . Once PBR cultures reached the exponential phase, they were used as inoculum for opentype culturing ponds (known as raceways, RW) (Fibrastur, Spain). RW consisted of oval open ponds that include a wheeled paddle which continuously moves and agitates the entire working volume of this system (600 L), so homogenizes cultures and facilitates gas exchange. RW dimensions were: 3 m long, 2 m wide and 0.3 m deep. Pilot-scale set up is shown in Fig. 1. 2.7. Microalgal growth measurement It was decided to measure the development of the microalgal populations using spectrophotometric methods by measuring samples of experimental cultures at 680 nm [35]. On this basis, it was decided to sample 1 mL of each replicate along the experimental duration and measure it using a spectrophotometer Libra S11 (BioChrom, United Kingdom). Samples were taken under sterile conditions and were homogenized through agitation with vortex for 5 s before measurements. Background fluorescence was determined by measuring un-cultured wastewater with culture medium and using this as a blank. Along PBR assays, absorbance measurements showed irregular results, so growth measurement was also studied through the extraction of the chlorophyll present in the cultures. Thus, samples were taken and exposed to an extraction protocol based on sequential centrifugation and washing steps for further extraction with organic solvents, as described by [36]. Extracted solvents were then measured through spectrophotometry at 663, 645 and 630 nm. Values were then exposed to Eq. (1) to evaluate the concentration of chlorophyll in the cultures, as a measurement of viable cells: μ g Chlorophyll mL culture = (11.64A663 −2.16A645 −0.1A630)v/(lV)(1) where A663, A645 and A630 are the absorbance measurements at 663, 645 and 630 nm, respectively. Moreover, v relates to the solvent volume (mL), V is the sample volume and l is the length of the spectrophotometer cell. 2.8. Pollutant removal quantification After finalizing the tolerance study at pilot-scale, cultures were harvested through centrifugation at 10,000 rpm with an industrial centrifuge STC 3–06-107 (Gea Westfalia Separators, Germany) [15]. This process separated the microalgal biomass from the culturing liquid, which was considered as “centrifugation effluent”. On this basis, 100 mL samples of this effluent were analyzed as described in Section 2.4 for the quantification of the influence of microalgal development on the LW biochemical profile. Thus, initial and final measurements were exposed to Eqs. (2)–(5) as described by [37]. Total BOD =(TBOD0 −TBOD1) TBOD0 ×100 (2) Total COD =(TCOD0 −TCOD1) TCOD0 ×100 (3) Total nitrates removal =(TN0 −TN1) TN0 ×100 (4) Total aerobes removal =(TAe0 −TAe1) TAe0 ×100 (5) where BOD0 equals initial values of BOD and BOD1 equals the values measured in the centrifugation effluent. On the same basis, COD0 refers to initial values of COD and COD1, to final values after harvesting. Also, TN0 equals initial nitrates values and TN1, final values; and TAe0, initial total aerobes measurements, and TAe1, final values. 2.9. Nutritional profile of the obtained microalgal biomass In order to evaluate the potential of the obtained microalgal biomass, it was decided to develop a nutritional analysis of it. Thus, samples of biomass were exposed to Gas chromatography/Mass spectrometry (GC/ M. Blanco-Vieites et al. Algal Research 78 (2024) 103424 4 MS), due to its high selectivity and accuracy [38]. The cited measurements were taken by an external laboratory (Anaqua, Spain) under the standards of UNE-EN ISO 9001:2015. Moreover, it was decided to develop a nutritional analysis according to the regulations of the European Union as stated on [39], so findings could be further compared to another feedstock biomass. 2.10. Statistical analysis Experimental assays were conducted in triplicates and shown results are presented as averages, with exception of the industrial-scale raceway ponds, due to the limited facilities. PAST Statistical Analysis Software was used for study the significance of the obtained data through oneway analysis of variance (ANOVA) and Tukey’s test. Significance of the given results was considered at p <0.05. Fig. 1. Pilot-scale set up for culture assays, being a) the PBR set up and b) the raceway technology. Fig. 2. OD 680 measurements of tolerance assay of: a) Chlorella sp., b) Coelastrella cogersae and c) Arthrospira platensis to increasing percentages of LW. M. Blanco-Vieites et al. Algal Research 78 (2024) 103424 5 3. Results and discussion 3.1. Tolerance of the selected strains to livestock wastewater in laboratory scale For this assay, experimental and control cultures were inoculated with 25 % of inoculum with an average OD 680 of 1.858, which turned into an initial absorbance of 0.465 for control group, which was considered as optimum based on the study of [35]. Subsequent to this inoculation, experimental assay consisted of 5 groups of experimental cultures with increasing percentages of Livestock wastewater (LW): 15, 25, 35, 50 and 70 % of LW in culture media, as is shown in Fig. 2. Despite the fact that inoculum absorbance was pretended to be similar, we observed initial variations in terms of OD 680 measurements at day 0 on groups with presence of LW superior to 15 %. These variations were attributed to the elevated density of the LW, whose opacity difficulted the accuracy of initial measurements of chlorophyll through spectrophotometry. Observing control group, we determined that culture conditions remained within the optimum range for this specie, even doubling initial measurements by day 10. In spite of this good results, absorbance measurements were low if compared to the outcomes obtained by [40], whose study aimed to optimize the culturing techniques for this specie. Nevertheless, culture optimization was considered as outside of our research scope, so control measurements were assumed as optimum within the presented conditions. Based on the experimental outcomes, it was observed that presence of LW induced a slight exponential increase during first 48 h of experimentation. This increasement was generalized among the experimental samples but was not observed in control cultures. This outcome may be explained by the presence of elevated concentration of critical nutrients, such as nitrogen, for microalgae culturing, as observed by [41]. Nevertheless, perhaps we saw an initial increasement of growth, even superior to what control group showed along initial 48 h (Fig. 2, 15 % group, orange line), next measurements clearly demonstrated a negative influence of LW. This tendency was sightlier but also present in the assays with C. cogersae culturing. Thus, OD 680 measurements descended for the next measurement (day 4) for both sampled eukaryotic strains. This tendency was considered as consolidated by day 6, were 35, 50 and 70 % of LW cultured showed absorbance close to 0, suggesting cellular death, in the assay with Chlorella sp. Contrary to what was observed, C. cogersae cultures with elevated concentrations of LW (25, 35, 50, 70 %) seemed to enter a stationary growth phase, suggesting that this strain could get acclimated to this residue with better results than Chlorella. This outcome supports what was observed by [42], who suggested Coelastrella sp. as the optimum microalgae for raw piggery wastewater bioremediation. Nevertheless, the observed results support the outcomes of [43], who demonstrated the inhibitory effect of excessive concentration of determined nitrogen-based compounds. On the same basis, due to the elevated opacity of the selected residue, light deprivation could be the reason behind the inhibitory effect of LW presence. On this basis, [44] recently studied the effects of light intensity on nitrogen-based compounds removal, stating that low light intensities inhibited the metabolic pathways for proper nitrogen assimilation and, as a consequence, tolerance. Regarding the prokaryotic selected strain, A. platensis, was selected based on its popularity in the microalgae culturing sector, being the most popular in terms of annual volume [45]. As well as in previous trials, we saw an initial variation of OD 680 , perhaps all of the experimental groups started with same inoculum of 25 % from an initial culture with absorbance values of 2.9 approximately. According to the results obtained on control group, it was observed that initial values equaled a maximum measurement of 0.765, which was doubled by the end of experimentation at day 10. Despite the fact that this growth is not considered as optimum for biomass production, as shown in [46], we do consider these values as reference since LW was not included in this culture medium. Moreover, as well as happened with eukaryotic strains, A. platensis presented a brief exponential growth phase that lasted for the first 48 h of experimentation. Nevertheless, contrary to what was observed on previous assays, percentages of LW superior to 15 % did not induce this exponential growth phase, as can be seen on Fig. 2c). Based on the obtained results, it was observed that none of the experimental groups presented a growth rate significantly similar to what was observed on control samples. Accordingly, all of the experimental cultures presented low growth rates. Contrary to what we observed, [46] demonstrated the elevated tolerance of this cyanobacterium to elevated concentrations of nitrates. Thus, we could not assess that this was the reason behind the observed development inhibition. Moreover, the effect of opacity and, hence, light limitation on Arthrospira development, as well as for eukaryotic strains, is a well-known phenomenon [47]. On this basis, we suggest that the negative influence of LW on the growth of determined phototrophs could be produced by the opacity of the selected residue. Also, light deprivation could act in synergy with presence of nitrogen-based compounds, as suggested by [48]. These statements support our findings along the tolerance assays. Nevertheless, as suggested by [49], these limitations could be avoided by including light sources to enable photosynthesis, such as LED systems. 3.2. Tolerance of indigenous microalgae to LW Characterization of the generated microalgal bloom was carried out by morphological characterization. On this basis, it was decided to use light microscopy for observation of diagnostic features that allowed an identification of the present strains. Based on the observed characteristics, 3 genres of eukaryotic microalgae: Chlamydomonas, Scenedesmus and Coelastrella (see Fig. 3). Moreover, based on the morphological plasticity of a severe range of the known microalgal strains, it is interesting to supplement morphological data with DNA sequencing techniques, as suggested by [50]. Nevertheless, the aim of this study was to compare the use of autochthonous microalgae blooms to the results yielded by monospecific strains, so molecular identification of the found strains is out of the scope of this work. However, this information was found to be highly valuable, so will be deepened in further works. Based on the identification results obtained, Scenedesmus and Coelastrella strains are usually found in contaminated sites such as wastewater and waste landfills [24]. This outcome supports the findings of [51,52], who suggested the suitability of this genera of microalgae to tolerate and bioremediate wastewater. Moreover, the presence of Chlamydomonas sp. in the sampled LW was also expected, specially due to the high tolerance of this genera to organic wastewater [53]. Based on the obtained results, it was decided to culture the obtained microalgae bloom, without presence of wastewater, through periodic renewals in 200 mL flasks (5 renewals periodically each 10 days). Afterwards, cultures were exposed to presence of 15 % of LW presence in ½ L systems, based on the fact that this was the maximum percentage tolerated along the single-strain assays and results are shown in Fig. 4. In spite of the natural existence of the cited strains in the LW, results indicate this residue possess inhibitory capacity on the optimum development of these microalgae, which might be explained by the difference between tolerance and optimum development. As well as seen on Fig. 1, the microalgal bloom experienced an initial exponential phase that lasted the first 48 h of experimentation. Nevertheless, from day 2 to day 4 it was observed the opposite tendency, which could be derived from culture opacity and lack of acclimatation to the reign conditions. Based on previous studies, it was found that microalgal acclimatation to wastewater presence is a fast process that could be lost along the several renewals that were made before the tolerance assay [54]. On this basis, future steps of the experimental assay will be done taking these findings into consideration. Moreover, after day 4, experimental cultures showed a slight growth tendency that could be related to the cited acclimatation process. Subsequently, experimental cultures entered an exponential growth phase. M. Blanco-Vieites et al. Algal Research 78 (2024) 103424 6 This exponential growth led experimental cultures to show absorbance measurements that could not be demonstrated as significantly different from the results yielded by control group (p <0.05). These results might be explained by the synergy effect among indigenous bacteria and microalga. Interaction between these 2 taxa has shown to comprise compounds stimulation such as oxidation of organic substances into inorganic forms, by bacteria, that can be further metabolized by microalgae [55]. On this basis, further steps of the experimental assay were focused on the influence of these indigenous microalgal bloom on LW chemical profile. Fig. 3. Images of the identified strains under optical microscopy: a) Chlamydomonas, b) Scenedesmus and c) Coelastrella. Pictures were taken after 14 days of culturing 20 % of diluted LW into BG-11 culture media and 80 % sterilized fresh water. Fig. 4. Response of indigenous microalgal bloom to presence of 15 % of LW in culture media. M. Blanco-Vieites et al. Algal Research 78 (2024) 103424 7 3.3. Scaling-up process to pilot plant conditions Based on the above results, it was decided to develop the obtained outcomes to pilot-scale, to verify the functionality in in-situ conditions. Thus, based on the findings from Sections 3.1 and 3.2, it was decided to culture the indigenous microalgal bloom to industrial conditions. Accordingly, cultures from laboratory conditions were used as inoculum for 10 L bottle-like systems in 2 groups: control group (absence of LW) and experimental group (cultured in presence of 15 % of LW). These cultures were grown under uncontrolled conditions of temperature, light intensity and photoperiod, inside a greenhouse located inside the Neoalgae facilities (Gij´ on, Spain). Growth development of the cited assay is shown in Fig. 5. Based on the obtained results, development rate of control groups was considered as optimum, since growth curve presented a clear standard trend [56]. Both control and experimental cultures initial absorbance measurements presented similar values, without significant differences (p <0.05). On this basis, it was considered that initial variables for both assays were equal, with exception of the presence of LW. From day 0 to day 2, both groups showed similar OD 680 values. Nevertheless, at day 4, lower absorbance values if compared to control group, which entered an exponential growth phase. Experimental cultures also entered a slighter exponential phase that lasted up to day 6 of experimentation, while control group reached day 8. By the end of the experiment, control group reached OD 680 values 35 % higher than what was observed for experimental group. These results contrast to the ones obtained during laboratory scale trials. [57] studied the influence of temperature control over microalgae cultures, finding it to be a critical parameter to optimize optimum growth. Also, [48] evaluated the influence of light availability on microalgae growth, which appeared to be other critical parameter in microalgae culturing. Based on the above, our results could be derived from the absence of controlled conditions like in laboratory scale experiments. Despite the fact that experimental cultures did not develop with expected results, as laboratory trials, final absorbance measurements reached up to 275 % higher OD 680 values. On this basis, we considered the obtained results as satisfactory. Accordingly, 10 L cultures were used as inoculum for vertical PBR systems with a working volume of 100 L, which were exposed to the same conditions as cited previously. Nevertheless, based on the high opacity of the LW, absorbance measurements showed irregular results. Accordingly, it was decided to elaborate an extraction of viable chlorophylls as a measurement of survival of microalgal populations, as suggested by [58]. Results are shown in Fig. 6. Perhaps previous results, it was observed that experimental cultures presented an initial slight decrease in terms of chlorophyll content, which indicates a phase of cytotoxicity. This was found to be opposite to what was observed for control group cultures. On the same basis, from day 4 to day 8, experimental cultures showed a dramatic descent in Fig. 5. Absorbance measurements of the initial trials without controlled conditions (10 L assay). Blue line (“C”) relates to control group and orange line (“15 %”) relates to experimental groups cultured with 15 % of LW. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 6. Chlorophyll concentration ( μ g⋅mg −1 ) in PBR cultures. The blue line (“C”) refers to the control group and the orange line (“E”) refers to the experimental cultures, which were supplemented with 15 % of LW. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) M. Blanco-Vieites et al. Algal Research 78 (2024) 103424 8 terms of chlorophyll concentration. On the other hand, control cultures showed a clear exponential growth phase that lasted from day 4 to day 8. From day 8 and ahead, experimental cultures presented chlorophyll content levels reached levels below the initial measurements, reaching down to 19 % of initial values. Based on the study of [59], cattle, swine and poultry wastewater represent a toxic environment for several microalgae, whose tolerance vary among strains. Moreover, previous works have analyzed the potential of microalgae bioremediation in outdoor cultivation conditions, which have shown to be much more challenging than laboratory-controlled conditions. As [60] observed, day-night variations on environmental temperature can reach situations that become incompatible with presence of other stressors such as LW. On this basis, our study was carried out along winter season, which is recognized as the most unsuitable season for industrial scale cultivation, so presence of LW might be synergic this stressor. Moreover, [54] demonstrated that elevated concentrations of organic nutrients can have an inhibitory effect on microalgal growth. Also, [61] proved that Chlamydomonas sp. can grow in presence of 50 % of nutrient-rich wastewater, suggesting that our tolerance results could be further improved. Also, as suggested by [62] microalgae survival in wastewater might be further improved through genetic engineering techniques, which is an interesting option that deserves to be deeply studied. Furthermore, their work also analyzed the low resistance of Scenedesmus sp. towards presence of industrial wastewater, which was identified among the strains belonging to the found microalgae bloom. Also, their conclusions suggest that microalgae cultures need long periods of time in order to adapt to the culture medium (and wastewater presence), as well to low light conditions, before becoming a viable tool for bioremediation of aqueous residues. On this basis, we recommend that future works focus on optimizing the minimum time needed for developing the maximum performance in terms of microalgae tolerance to determined wastewater. Based on the above outcomes, it was decided to scale control group cultures (absence of LW) to pilot-scale open systems, raceways (RW), in order to obtain a healthy culture in terms of cellular density. Once reached the end of the exponential phase of growth, 15 % of culture was harvested and replaced with 15 % of LW. This decision was taken under the assumption that highly concentrated cultures resist with better results presence of wastewater. OD 680 measurements are shown in Fig. 7. PBR with control cultures were used as inoculum for RW systems, accordingly, average initial OD 680 was 0.127. Contrary to what was observed in previous assays, cultures showed an initial lag phase that lasted up to day 4 of culturing. Perhaps the unexpected result, this outcome was also observed by [63], which also measured an extended lag phase after scaling up cultures to industrial scale systems. Based on the above, winter conditions of temperature, light intensity and photoperiod represent a source of stress for microalgae culturing. Thus, we strongly believe that the observed lag phase was due to these uncontrolled conditions. Moreover, RW cultures entered an exponential phase that reached a maximum OD 680 at day 12 (0.782), almost 7 times superior to initial measurements at day 0. At this point of experimentation, renewal with 15 % of LW was done. Despite the presence of this residue, cultures continued an exponential growth phase that continued until day 14 of experimentation. Accordingly, cultures entered a semilag phase with discrete growth, which could not be found as statistically significant (p <0.05). Moreover, cultures entered a second exponential growth phase by the 18th day of culturing, that lasted until the end of the experimentation. As an overall analysis of the results obtained, the addition of LW as a renewal of 15 % total volume did not represent a negative stress on microalgae development. Despite the good results obtained, the overall performance could be further improved as suggested by [63], who probed that Chlorella sp. cultures could be used as bioremediatory organisms in, semi-continuous systems, by periodic renewals of 80 % of the total volume. On this basis, our renewal percentage could be further improved, which will be taken into account for future works. Moreover, the obtained results agree with [64], who already observed the advantages of semi-continuous culturing, instead of batch mode, for microalgal bioremediation systems. Despite previous studies, [65], evaluated the bioremediatory potential of batch and continuous microalgae cultures, finding out greater biomass synthesis in batch systems. On the same basis, [66] obtained great results in terms of microalgal growth with presence of LW, specially during assays with supplementation of CO 2 . Nevertheless, mentioned previous works used pretreated wastewater (by chemical or physical means), which involves a lower source of stress if compared to untreated LW, like the one used in our study. Based on the available bibliography, this study is the first attempt to develop a microalgae bioremediation system at pilot scale that involves raw LW and an indigenous microalgal bloom. 3.4. Pollutants removal efficiency of microalgae By the end of experimentation, cultures were harvested by centrifugation and samples of the effluent were analyzed to evaluate the influence of microalgal growth over LW composition. Thus, it was decided to take an initial sample at renewal day in RW system (day 12 in Fig. 7) and compare it to the observed values by the end of the assay (day 20). Results are shown as averages in Table 1. After the addition of 15 % LW to the microalgal culture, it was observed that it did not induce any negative effect on the development. Nevertheless, presence and growth of these photosynthetic organisms indeed had an effect on LW biochemical profile: all of the targeted parameters were reduced significantly (p <0.05). LW is rich in organic carbon, that can be mineralized by microalgal metabolism. This source of carbon is usually measured in terms of BOD and COD, which are related to exploitation of the dissolved oxygen and eutrophication of water bodies. Hence, these two parameters are critical to evaluate in bioremediation systems of wastewaters. On this basis, BOD was measured, showing a reduction of almost 30 % in 8 days, which was considered as a good result. Nevertheless, according to the findings of [67], Desmodesmus sp. can reduce this parameter up to 76 % within 20 days. On the same basis, [68] achieved better remotion yield of this parameter when culturing different microalgal strains (Nostoc commune, Oscillatoria limosa and Chlorella vulgaris) in industrial wastewater. Their study reached up to 80 % of maximum BOD removal, which is also clearly superior to the outcomes we observed during our study. Nevertheless, their works proved that culture time is a critical parameter in terms of BOD remediation and their experimental assay indeed took 2 Fig. 7. Response of indigenous microalgal bloom to renewal with 15 % of LW. Table 1 Overall influence of indigenous microalgal growth on LW composition. Parameters Initial values Final values Total removal (%) BOD 669 (O 2 ⋅mL −1 ) 477 (O 2 ⋅mL −1 ) 29.3 COD 962 (O 2 ⋅mL −1 ) 690 (O 2 ⋅mL −1 ) 29.2 Nitrates 350 (mg⋅L −1 ) 43.4 (mg⋅L −1 ) 83.6 Total aerobes 1⋅10 3 (ufc⋅mL −1 ) 6.2⋅10 2 (ufc⋅mL −1 ) 38 M. Blanco-Vieites et al. Algal Research 78 (2024) 103424 9 times more than ours. On this basis, taking into account the 8 days of culture time of our study, our results are superior to the ones yielded by these authors in such time. However, their culture time approach is highly valuable to optimize this technique and will be used on further studies. Regarding COD, our analysis after 8 days of culture with LW showed a maximum reduction of 29.2 %. Despite the fact that this was considered as a good result, previous works like [69] achieved higher removal rates, but with longer culture times and controlled conditions. COD decrease is usually difficult to evaluate with exactitude based on the fact that some microalgal strains release metabolic and polymeric compounds to the culture media. Nevertheless, our outcomes are still superior to the ones obtained by [70], who analyzed the influence of microalgae on synthetic wastewater. Their outcomes showed that 7-day cultures could reduce COD from initial concentrations of 125 mg⋅L −1 , 250 mg⋅L −1 , and 500 mg⋅L −1 to 27.4, 19.3, and 48.0 %, respectively. On the same basis, [71] also studied the influence of phototrophic cultures on COD values, achieving a maximum decrease of 65.85 %, which is clearly superior to what was obtained by our study. Their work analyzed the enhancement of bioremediation of COD by Chlorella cultures through their culturing in membrane photobioreactors through continuous cultivation. Accordingly, we consider that this technique could be applied to optimize our results. Thus, further works should deepen this topic to approach the results obtained to what was observed by previous works that achieved COD removal rates close to 80 % [72]. Other parameter of interest was nitrates concentration. Previous studies have demonstrated that microalgae cultures have preference for nitrates when assimilating nitrogen-based compounds [17]. Thus, after evaluating the obtained data, our experimental design showed a reduction of almost 84 % in 8 days of culturing, showing a maximum daily rate of 38.3 mg⋅L −1 [73] already examined the ability of a certain strain of Scenedesmus sp. for nitrates removal, showing a maximum removal rate of 22.4 and 15.5 mg⋅L −1 in autotrophic and mixotrophic conditions, respectively. Nevertheless, as shown by [74], nitrate uptake yield and ratio are highly dependent on the initial concentration so systems with fewer quantity of nitrates will suffer slighter decreases if compared with saturated systems. On this basis, we consider our outcomes as good but not optimum. Furthermore, the presented results of nitrate uptake could be further optimized as done by [16], who achieved a maximum yield of nitrate uptake of 100 %. On the same basis, as stated by [17], microalgal nitrates consumption could be enhanced by using activated sludge as a pre-treatment of the selected wastewater. This technology facilitates the conversion of different nitrogen-based compounds into nitrates, which are easier to assimilate. Regarding the total aerobes recount, the results shown in Table 1 clearly shown that microalgae culturing produces a significant descent (p <0.05) on the total aerobes count. On this basis, our pilot-scale assay showed that microalgae culturing reduced total aerobes by 40 % under the mentioned circumstances. This result supports the findings of [75], who deepened the bacteria-microalgae dynamics when cultured in high-rate algal ponds (similar to RW). Also, [76] also evaluated raw piggery wastewater as a novel residue for microalgal bioremediation, including pathogen bacteria as targeted pollutant, and their findings showed that microalgae could decrease down to 60 % of the initial bacterial concentration. Despite the fact that most of the current published works deepen the synergic effect of microalgae and bacteria for pollutants removal, few papers do investigate the antibiotic effect of microalgae [77]. Based on the previous statements, we strongly believe that microalgae represent a novel tool for LW remediation, especially for simultaneous removal of several pollutants of interest. Nevertheless, based on the high dependance on biochemical profile of the residue of interest, specific works must be carried for optimization of each. 3.5. Nutritional profile of the obtained microalgal biomass Once tolerance and scaling-up assays were finished, RW were harvested to separate liquid phase (effluent) from solid phase (microalgal biomass). Effluent was exposed to pollutants analysis, as described in Section 2.9, and microalgal biomass used for nutritional analysis, which results are shown as averages in Table 2. Based on the fresh biomass composition analysis, it was observed that protein and fat content was equilibrated, being fat content slightly superior (average 7 %). Based on previous studies that estimate soybean protein content, the algal biomass obtained along the bioremediation assay presented almost 15 % more protein if compared to average standard soybean [78]. Moreover, the obtained protein percentage on biomass was significantly close (p <0.05) to the results observed by [79] for some insect’s protein content, such as Protaetia brevitarsis. Nevertheless, it is considered that insect-based protein is the main novel protein source for a sustainable development, nevertheless, microalgae have the advantage that can be included into the circular economy paradigm as bioremediation organism and protein synthesizers. Furthermore, according to the USDA database (2015), the protein content of the indigenous microalgal bloom presented higher protein percentage than what was observed for average beef (40.5 %) and pork (27.7 %) meat. Accordingly, we assume that the obtained biomass fulfills the protein percentage required to be used in animal dietary products, since protein content is superior to the observed on standard meat sources. On the same basis, [80] analyzed fungal-based protein products as an innovative source of protein. Thus, they studied the “Mycoprotein”, which a novel product made of Fusarium veneatum biomass and stated it as the future of nutritious non-meat protein. Despite their promising results, we found that the obtained algal biomass showed significantly similar protein content (p <0.05), less fat content and higher percentage of carbohydrates. Nevertheless, according to the findings of [81] chlorophytes fat concentration can be enhanced through the optimization of culturing parameters. As an example, this mentioned work achieved an impressive 50.4 % of maximum lipids in Chlorella sp. biomass. On the same basis, [82] also investigated the possibilities of lipid accumulation of Chlorella strains, finding that mixotrophic culturing techniques would be the most appropriate for this purpose. In addition, the bioremediation approach done in this study develops this biomass as an interesting novel source of protein with a sustainable added value as wastewater living filter. Based on the nutritional profile shown in Table 2, the obtained biomass could be used as ingredient in standard livestock diets, recovering lost nutrients in wastewater and including them again into farming production chain. Moreover, previous works highlighted the effects of including algal biomass into cattle feed, such as a severe methane reduction during enteric digestion [83]. Nevertheless, due to the possible presence of contaminants of concern, such as heavy metals and pathogenic microbes, specific analysis and legislation review will be needed before considering the obtained biomass as livestock feeding. Based on the above, we consider that microalgal-based wastewater treatment systems could represent a novel tool for water depuration simultaneous to cattle feed synthesis, but further works are needed to support this hypothesis. 4. Conclusions This investigation reports, for the first time, the high efficiency of native microalgal populations in livestock wastewater as bioremediation organisms and biomass producers at pilot-scale. Based on the tolerance assays, we concluded that indigenous strains possessed better Table 2 Nutritional profile of the harvested biomass of the indigenous microalgal bloom. Parameters Wet biomass Percentage on dry weight (%) Total carbohydrates 9.07 (g⋅100 g −1 ) 41 Total fat 2.21 (g⋅100 g −1 ) 10 Protein 10.6 (g⋅100 g −1 ) 48 Total energy 98.6 (Kcal⋅100 g −1 ) – M. Blanco-Vieites et al.