Comparative Analysis of Livestock Wastewater Reuse Under Summer and Winter Conditions at a Scale-Down Microalgae Culture

Abstract

Microalgae-based wastewater treatment systems are an environmentally friendly technology for reuse of polluted water produced in livestock farming. Since pollution removal depends on light availability, the performance should be evaluated under different seasonal conditions, even in reduced lab scale systems. This study evaluates the treatment of livestock digestate in an experimental High-Rate Algae Pond (HRAP) that recreates outdoor conditions. Chemical and biological pollution removal were analyzed, as well as the response of photosynthetic activity of the culture. Pollutant removal varied between seasons, while summer was characterized by higher nitrogen and phosphorus removal (81 and 69%, respectively), on the other hand, winter presented higher elimination of organic matter (91%) and pathogens. In this sense, P. aeruginosa removal was notably higher in winter (100%) than in summer (50%). Higher light penetration and increased photosynthetic efficiency in winter, along with greater fluctuations in pH and dissolved oxygen concentrations, contributed to higher levels of pathogen decay. Photosynthetic response tests indicated higher oxygen production per unit biomass in winter, suggesting physiological adaptations to lesser light conditions. This adaptation was correlated with the relative high pH and dissolved oxygen values registered. The findings highlight the adaptation and robustness of algae cultures as a solution for wastewater treatment and reuse in the primary sector.

1. Introduction

There is an increasing deficit of pure water suitable for consumption as consequence of climate change, which is affecting temperature and precipitation regime [1]. In recent years, less rainfall occurs, or it occurs in the form of storms, affecting the availability of drinking water. Besides this, the increasing global population, along with the intensive farming and industrial activity reduces water quality [2]. Alterations in chemical composition of water supply, such as presence of contaminants, appear with increasing frequency. Livestock wastewaters usually presents elevated concentrations of organic matter, nitrogen and phosphorus. Consequently, untreated discharge of these effluents results in decreased water quality and eutrophication events that finally involved risks for human health and ecosystems [3]. In addition, biological pollution linked to sewage and manure discharge, limits the water utilization in the primary sector. Enterobacteria and coliform bacteria present in the aquifers or water bodies have been related to public health problems when used for crop irrigation. According to the WHO [4], it is estimated that one in ten people contract diseases from consuming spoiled food which has been watered or has encountered contaminated water. Every year, 420,000 people die from consuming food contaminated by microbial agents [5]. By 2050, water demand is expected to increase by 40% and energy demand by 50%. Besides this, food production should increase by 60% to cover global demand [6]. In this contest, it is necessary to apply economical and environmentally friendly solutions to wastewater purification and reuse [7]. The culture of microalgae in wastewater can be an alternative solution to conventional purification technologies since it can eliminate inorganic nutrients, such as nitrogen and phosphorus, together with removal of pathogens [8,9,10]. Opposite to conventional aerobic treatments based on aeration (activated sludge), that consume important amounts of energy and do not allow nitrogen recovery, microalgae assimilate ammonia and nitrate as proteins, in a biological process driven by solar energy. In addition, this process fixes carbon dioxide (CO₂), the main compound that causes the greenhouse effect. [11]. Microalgae culture broth is characterized by high pH values and elevated dissolved oxygen concentrations, both conditions that reduce pathogen survival. Besides this, microalgae secrete inhibitory substances that reduce bacteria growth, and the high light exposition of cultures creates a hostile environment for many microorganisms. Then, microalgae culture provides disinfection of high loaded wastewaters, such as digestates from anaerobic digestion of manure. This process is especially adequate for livestock farming wastewater that can be reused for irrigation after adequate treatment [12]. In this sense, water demand could be reduced when organic effluents are disinfected and used for crop irrigation [7,10,13].

Microalgae systems for wastewater treatment have been tested at different scales [14,15,16]. However, most of the documented experiences have been carried out at lab or bench scale, that fail in reproducing the outdoor conditions [17]. In this manner, the light sources applied at reduced scale and the mass transfer characteristics differ from algae ponds applied in real scale [17]. In must be considered that high-rate algae ponds (HRAPs), shallow channels in meandering configuration mixed by paddles or pumps, are the only sustainable microalgae culture technique appropriate for wastewater applications [15]. On the other hand, lab scale experiments are normally conducted in glass flasks or tanks (named photobioreactors) that exhibit different light availability for the algae cells and dissimilar mass transfer conditions [14,17,18]. Besides this, artificial illumination systems used in lab experiments are conventionally based on constant irradiation that are not equivalent to day cycles of outdoor systems, characterized by a parabolic profile in intensity. In this work, the potential of microalgae culture for pollutant removal (pathogens, organic matter and nutrients) was studied in a scale-down bioreactor simulating outdoor conditions. The lab experimental system allowed for reproducing daily light profiles, temperature and mass transfer conditions of HRAPs. Seasonal variations were studied and reuse of the effluent after algae culture was evaluated according to the limits established by the European regulations. Additionally, photosynthesis activity of the culture was measured to assess the impact of seasonality over the biological process.

2. Materials and Methods

2.1. Cultivation Conditions

Microalgae cultivation was carried out in a lab scale microalgae culture reactor designed with the aim of reproducing the conditions of outdoor HRAP treating digested pig manure. The substrate was withdrawn from an experimental digester working at 15 days of hydraulic retention time in mesophilic conditions. Digestate was diluted with tap water resulting in dilution factors between 1:20 and 1:40, then organic matter concentration was kept at relative stable concentrations, with a final COD concentration between 0.5 and 1.0 g L⁻¹. The experimental system was a stainless-steel tank reproducing a small-scale raceway high-rate algae pond (HRAP) system. Water depth was maintained at 25 cm, simulating standard working conditions in microalgae lagoons (Figure 1). The total volume was 32 L. A peristaltic pump with adjustable flow rate was used to feed the reactor and a propeller agitator for mixing. Raw wastewater was kept in a refrigerator at 4 °C. Mixing of the reactor was applied by means of 2 centrifugal recirculation pumps placed at the bottom, EHEIM CompactON 15 W, working at a flow rate between 400–1000 L h⁻¹ and maximum head of 1.4 m. As a light source, 2 Philips LED light plates with a power of 478.6 W were used to simulate sunlight cycles.

The LED lamp was programmed to reach the light intensity variations of the daylight cycle corresponding to summer and winter conditions in latitude 40°. Two power drivers and an Arduino microcontroller were used for regulation of light intensity along the day cycle (Figure 2a). Similar light source was described by Gonzalo-Ibrahim et al. (2023) and Ruiz et al. (2024) [17,19]. A thermoregulator bath (Selecta Frigiterm, Barcelona, Spain) was used to adjust temperature. The experimental HRAP was semi-submerged in the bath to reproduce temperature variation of outdoor conditions. A conical decantation system of 8 L was placed to separate the algae biomass from the water treated by sedimentation. The scale-down HRAP was operated at 8 days of hydraulic retention time during a total time of 100 days. Mass transfer coefficient was determined using the dynamic method for oxygen exchange. In this sense, dissolved oxygen was removed with the addition of NaSO₃, and the re-oxygenation was determined using a dissolved oxygen probe (Vernier, Beaverton, OR, USA).

2.2. Analytics and Measurement

Throughout the trial, the following parameters were continuously monitored by (Vernier sensors and data logger LabQuest Mini, Beaverton, OR, USA): O₂, photosynthetically active radiation (PAR), temperature and pH. Vernier Graphical Analysis^® software was used for data acquisition, and Microsoft Excel was used for data processing and the elaboration of the figures. Total and volatile suspended solids (TSS) were determined according to APHA 2540 B and D methodology, and volatile suspended solids (VSS), according to APHA 2540 E. Chemical Oxygen Demand (COD) was determined according to APHA 5220 D, ammonia/ammonium (NH₃/NH₄⁺) according to APHA 4500-NH₃ C, and phosphate (PO₄³⁻) according to APHA 4500-P C. Samples were analyzed twice a week in the inlet and outlet effluent (downstream of the settling tank) [20]. Biomass concentration in the reactor, microalgae and bacteria, was measured as total and volatile suspended solids [20]. The sensor that measures the PAR was placed on the surface of the culture water to monitor the amount of light received by the algae under normal working conditions. Average PAR value was calculated measuring 10 different points of the surface tank. Vernier sensors and data loggers were used for data collection.

2.3. Disinfection Analysis

The removal of pathogens was evaluated under the different seasonal conditions. Samples were taken from the inlet and outlet water of the experimental HRAP, when the process reached steady state conditions (after 3 hydraulic retention times). Data collected during each trial were compared with the limits established for the colony-forming units (CFU) of the E. coli species, according to the EU regulation [21]. In addition, other coliform species, such as Citrobacter freundii, Enterobacter aerogenes and Klebsiella pneumoniae, as well as the non-coliform species Pseudomonas aeruginosa. For the disinfection analysis, the water was filtered by means of a kitasato and vacuum pump, using grid filters. Filters were placed on Petri dishes with Chromocult coliform agar for bacterial determination. After 18 to 24 h of incubation, the number of CFU were counted [22].

2.4. Light Response Tests

Light response tests were carried out with a Vernier dissolved oxygen probe and a Vernier light intensity probe (PAR) in microalgae culture samples taken under both experimental conditions. Oxygen production slopes were obtained at different distances to the visible light source, corresponding to different light intensities. Then a transparent test tube containing 10 mL of microalgae culture broth was irradiated obtaining different dissolved oxygen production rates. Microalgae concentration of the samples was determined by means of total suspended solid (TSS) concentration and the photosynthetic activity was expressed in terms of oxygen produced per mass of microalgae and time (mg O₂ g TSS⁻¹ s⁻¹). This method is based on the previously reported experiences by Costache et al. (2012), Barreiro et al. (2021) and Ruiz et al. (2024) [19,23,24]. As a result, a point spread with the values of slope and light intensity was obtained

3. Results and Discussion

Two trials were conducted simulating two conditions: summer and winter considering the light cycle and temperatures corresponding to temperate climate at latitude 40°. Systems were operated during a total of 100 days with evaluation of pollution removal (chemical and biological) and determination of light response tests in microalgae biomass.

3.1. Microalgae Culture Monitorization

The microlgae culture was monitored through the parameters pH, dissolved oxygen (D.O.), temperature and incident photosynthetic active radiation (PAR) reaching the surface. Microalgae photosynthesis increases the concentration of dissolved oxygen and rises the levels of pH due to the up-take of dissolved carbon dioxide and bicarbonate. In this sense, nictimeral variations detected along the day serve as indicator of the microlgae biological activity that results in the pollution removal. Beside this, the daily variations of temperature and incident radiation allowed for a fair comparision of the experimental set up with the outdoor systems [25].

Maximum values of PAR of 1800 μmol m⁻² s⁻¹ and 750 μmol m⁻² s⁻¹ at midday were detected in summer and winter conditions, respectively (Figure 2a,b). The temperature presented a variable pattern along the day. During summer conditions ranged from 25 °C to 33 °C and in winter between 7 °C and 14 °C. Both conditions of irradiance and temperature are assimilable to outdoor HRAP placed in temperate climate. These values correspond to the average temperatures of an inner Mediterranean climate. Regarding the mass transfer coefficient, that determines the exchange of oxygen with the atmosphere, a value of the gas-liquid volumetric oxygen mass transfer coefficient of 0.41 ± 0.02 h⁻¹ were found. This value is in the same range that those reported for outdoor HRAPs or raceway systems used for microalgae culture and/or wastewater treatment.

The concentration of dissolved oxygen, which depends on the photosynthetic activity of the microalgae, bacterial consumption and mass transfer conditions, presented significant differences in both conditions. Values above the oxygen saturation limit, higher than 8 mg L⁻¹, were reached under both conditions. However, during summer dissolved oxygen decreased to anoxic conditions during more than 9 h at night (Figure 2b). The higher solubility of oxygen in cold water maintained aerobic conditions in winter test even during night cycle, with values above 4 mg O₂ L⁻¹. In the same manner, greater variations in pH values were detected during winter experiments. While the variation between day and night was of 1.5 pH units in summer, winter was characterized by a sharper variation, reaching 2.5 units of difference and values close to 9.5 at midday. These findings are not in agreement with some of the previouly repoted experiences that measured higher pH values during summer conditions [25]. pH raise in a direct consequence of biocarbonate depletion due to microlgae assimilation as carbon source [26]. Although summer conditions are characterized by a considerible higher light availability, this fact may not neccesarally result in higher photosynthetic activity. According to light response test (see Section 3.2) photosaturation is reached at moderate light irridiances (from 500 μmol m⁻² s⁻¹), therefore a considerable high proportion of the light period in the summer experiment could be characterized by low light utilization by microalgae cells. Notice that, the decreased photosynthesis under summer conditions is also evidenced by the sharp drop in dissolved oxygen that is detected after the maximum (Figure 2b). Altough HRAP are the most economic way to cultivate microalgae, these systems do not achieve a high light utilization by cells since the bulk liquid present a relative low disperssion and reduced mass transfer coefficients [25,27]. In addition, the differences in the photosynthetic activity detected between both seasons could explain the lower pH values reached at the central hours of the day in the summer, since winter samples presented a considerable higher light utilization by microalgae biomass (see Figure 6 and Section 3.2). Moreover, it is important to notice that other authors have reported elevated pH values along to the year in outdoor systems [28].

3.2. Chemical Pollution Removal

The performance of the experimental system in chemical pollution removal is shown in Figure 3 and Figure 4. Concentration of microalgal biomass was notably higher in the photobioreactor during the summer period with average value of 1.61 g TSS L⁻¹, while the winter period presented a value of 0.60 g TSS L⁻¹. These values are in the range of the previously reported for open-air HRAP operated under temperate or Mediterranean climate, highlighting the robutness of the experimental set up reproducing outdoor systems [28,29,30]. Figure 4a shows the concentration of total suspended solids contained in the water entering the photobioreactor and the water leaving the settler during both seasons. The concentration of total solids in the intake water in the summer period presented a higher average value than winter samples, 0.8 vs. 0.3 g TSS L⁻¹, respectively. During both periods there was a notable reduction in the concentration of solids of 96% and 48%, in winter and summer, respectively. The higher removal of the winter experimentation is directly related with the higher rates of settling of particles at lower temperature [31]. At this point, it must highlight that a wide rage of efficiencies have been reported in case of gravidity settlers used in microalgae separation [32]. These fluctuations have been atributed to different factors, like changes in microbial communities, bulking and biomass flotation [26]. However, the results herein presented and the visual observation of the settler performance under both conditions, evidenced the strong impact of water temperature in the biomass separation by gravidity. The ammonia concentration in the summer inflow water was 4 times higher than in the winter. These fluctuations in the chemical composition are due to the different farming practices and the amount of water used for cleaning the pits. Higher removal of ammoniacal nitrogen was observed under summer conditions, (81% vs. 31%) (Figure 4b), which is in agreement with the results reported by other authors [12,33,34] and directly related to the higher biomass assimilation promoted by elevated temperatures and biomass formation [28]. Similarly to nitrogen, the phosphate concentration was twice in summer conditions compared to the winter and also the removal efficiency was noticeable higher (69% vs. 34%) [34,35,36]. In case of organic matter removal (determined as chemical oxygen demand), the winter essay was characterized by higher performance (91% vs. 65%). (See Figure 4d). This fact is directly related with the increased settling rates as consequence of low temperatures [31]. Very low nitrifying activity was detected under both conditions, with stable concentrations of nitrite and nitrate in the final effluent in both seasons (less than 5 mg L⁻¹). Substrate limiting conditions found in microalgae cultures prevent from high levels of ammonia conversion to nitrite and nitrate In this sense, the active uptake of inorganic carbon and ammonia by algae cells prevent the growth of nitrifying bacteria that also use this substrate for their primary metabolism [37].

3.3. Bacterial Pathogen Removal

Pathogen removal was divided into microbial types, creating 3 blocks. First, E. coli removal was evaluated and then compared to the limits established for water reuse according to the European regulations. Secondly, the rest of the coliforms detected by the biological analysis were grouped (C. freu, E. aero and K. pneu) and P. aeruginosa species were evaluated in the last block. Figure 5 shows the removal of bacteria in colony forming units CFU mL⁻¹ in the summer and winter periods.

The percentage of removal of pathogen has been calculated as an average for both periods. The main parameters are presented in

Table 1. Percentage of elimination of the main parameters measured in the water, in summer and winter conditions.

Parameter	Initial Concentration in Summer	Summer Removal	Initial Concentration in Winter	Winter Removal
TSS	0.71 (g L−1)	48.94 ± 78.20	0.32 (g L−1)	96.20 ± 2.01
NH3	110.18 (mg L−1)	78.26 ± 20.77	22.81 (mg L−1)	16.82 ± 54.11
PO43−	21.72 (mg L−1)	69.83 ± 48.28	12.29 (mg L−1)	34.83 ± 42.50
DQO	1.38 (g L−1)	65.34 ± 25.04	0.54 (g L−1)	91.08 ± 4.57
E. coli	6000 (UFC mL−1)	99.6	210 (UFC mL−1)	100.0
C. freu., E. aero., K. pneu.	1700 (UFC mL−1)	98.5	55 (UFC mL−1)	98.2
P. aeruginosa	20,000 (UFC mL−1)	50.0	35 (UFC mL−1)	100.0

. The concentration of E. coli in the water during the summer decreases from 600,000 CFU 100 mL⁻¹ to 2500 CFU 100 mL⁻¹, complying with the regulation for type “D” waters (≤10,000 CFU 100 mL⁻¹). Winter conditions were characterized by a lower biological load of pathogen, then the concentration passed from 2100 CFU 100 mL⁻¹ in the inlet wastewater to 1 CFU 100 mL⁻¹. Therefore, effluents generated during winter conditions comply with the regulations for type “A” waters (≤10 CFU/100 mL). Type D water can be used for irrigation of industrial crops or trees without direct contact between water and fruits. Other uses include industrial applications out of food production, energy generation and seed production. Type A water is suitable for irrigation of food crops that are consumed raw, especially those where the edible part is in direct contact with the reclaimed water, as well as for irrigation of private gardens or specific industrial applications that require absence of pathogens, such as cooling towers [21].

While the percentage of elimination of E. coli and the other coliform bacteria achieved by the microalgae culture was similar in winter and in summer (higher than 98%), in the case of the non-coliform bacteria species P. aeruginosa the elimination presented notably differences between both periods, with very low removal detected 50% in summer and total removal in winter. Although water reuse regulation is exclusively determined by the levels of E. coli, the Pseudomonas genus is a pathogen that must be considered since it is related to multiple infectious diseases. The eliminations recorded under the summer period for this pathogen were considerably lower than values detected in outdoor experience. For instance, Fallowfield et al. (2018) [38] reported very high removals (>99%) in a 200 m² under average irradiance of 810 µmol m⁻² s⁻¹ and temperatures between 7.6 and 23.1 °C. At this point, it must be noticed that the disinfection capacity of the microalgae culture may be due to several factors, abiotic and biotic. In this sense, the segregation of bactericidal substances by the microalgae, together with the high pH and oxygen concentration creates a hostile environment for bacteria. The pH in both periods presented alkaline values, being slightly higher in winter (8–11) than in summer (7.5–9.5). This fact could be related to the considerable higher photosynthetic efficiency of algae detected during winter, according to the light response tests and photosaturation effect that takes place during a long part of the light period (see Section 3.3). Regarding oxygen concentrations, it is widely accepted that very high concentrations prevent the survival of this pathogen. Therefore, the anoxic conditions found during dark periods in summer experiments could contribute to the pathogen survival of pathogens, resulting in higher levels of P. aeruginosa in the effluent (Figure 2). The removal of coliforms and E. coli can be attributed to variations in the pH of the culture broth, driven by the photosynthetic activity of the microalgae during periods of higher and lower irradiance, as well as by nocturnal respiration. This pH imbalance between day and night leads to inactivation of coliforms and other pathogens [39]. In this case, fluctuations in pH are more pronounced in winter than in summer, which may hint at the greater elimination of pathogens under cold conditions. Finally, light mediated mechanisms have been described as one of the factors contributing to pathogen decay. In this sense, elevated concentrations of microalgae biomass found during summer reduce light penetration in the culture broth and then decreasing the overall elimination [40]. This indirect effect could explain the considerable reduced elimination of P. aeruginosa during summer. At this point it must be stressed that the Pseudomonas genus is sensitive to light and the decay of this organism could be related to the amount of photons received. Although the disinfection levels achieved in both conditions are compatible with water reuse (of different qualities), the differences found between both seasons and the high survival of P. aeruginosa in summer suggest the high impact of light mediated mechanisms in the disinfection process.

3.4. Light Response Test

Samples of the culture broth were withdrawn from both tests (summer and winter) and the oxygen production rate were evaluated at different light intensity conditions. Figure 6 shows the oxygen production at different levels of light intensity. Photolimitation conditions were detected between 0 and 500 μmol m⁻² s⁻¹, characterized by a direct correlation between irradiance levels and oxygen produced per biomass, in both seasonal conditions (positive slope). Photosaturation conditions were found from 500 to 2500 μmol m⁻² s⁻¹ with relative constant values of oxygenation. Suprisingly, biomass withdrawn at wither presented a considerable higher photosynthetic activity: 0.025 mg O₂ g TSS⁻¹ s⁻¹ vs. 0.014 mg O₂ g TSS⁻¹ s^−1, measured in summer samples. This fact could be related with the physiological addaptations of microalgae cells to light limiting conditions [41]. These results are directly related with the high levels of dissolved oxygen and pH found during the winter conditions, evidencing the active photosynthetic activity in all the conditions. The possible adaptation of the microalgae culture to harst environmental conditions during winter could overcome the light limitiation, and then resulting in a efficient treatment in terms of pollution removal. At this point, it must be notice that elevated photosynthetic activity also conduct to pathogen decay and can explain the higher performance detected during winter conditions, as discussed in the previous section. The oxygenation capacity and the light utilization determined in this essay was in the range of previously reported experiences of microalgae cultured in outdoor conditions: between 50 and 90 mg O₂ g TSS⁻¹ s⁻¹, in winter and summer conditions vs. 88 mg O₂ g TSS⁻¹ s⁻¹ measured by [24]. The considerable elevated light utilization by microalgae cells detected during the winter conditions, driven by cell adaptation, is regarded as an evidence of the robutness of the algae based systems. In this sense, even in the light limiting conditions of winter, the high ligth utilization provided sufficient chemical and biological pollution removal.

4. Conclusions

The treatment of manure digestate in an indoor high-rate algae pond recreating outdoor system was tested under summer and winter conditions. While summer experiment presented a better performance in terms of nutrient removal, the elimination of organic matter and suspended solids was higher in case of winter conditions, as consequence of the higher settling rates of microalgae biomass at low temperatures. In terms of disinfection, effluents with low levels of contamination and consistent with the water reuse normative were achieved, with a considerable higher quality in winter essays, reaching the total absence of the microbial indicator E. coli. Winter conditions also resulted in total removal of the pathogen P. aeruginosa, while summer effluents presented a considerable concentration of these organisms. The reduced light penetration due to the elevated biomass concentrations of summer could explain the lower elimination of E. coli. The determination of the photosynthetic response under both conditions revealed a higher microalgae activity per unit of biomass in winter samples, revealing the existence of biological adaptations to the light limiting conditions. This study demonstrates the possible livestock wastewater reuse for irrigation of crops using a simple microalgae culture under the different seasonal conditions found at intermediate latitudes.