Cultivation of Brackish Water Microalgae for Pig Manure Liquid Digestate Recycling

Abstract

Sustainability and recycling of agricultural and animal husbandry waste are important. Pig manure contains relatively high concentrations of organic matter, such as nitrate-nitrogen, ammonia nitrogen, and phosphate, and a direct discharge can cause environmental pollution. This study successfully culturing four brackish water microalgae, including Tetraselmis chuii, Isochrysis galbana, Chlorella vulgaris, and Proteomonas sulcata, by using a diluted digestate solution of pig manure (NH₃ concentration is lower than 10 mg/L). These microalgae can reach their highest cell concentration within 3–7 days of cultivation. The small microalgae, C. vulgaris and I. galbana, reached a cell density of 2.5 × 10⁷ and 1.5 × 10⁷, respectively, whereas lower cell densities were documented for large microalgae T. chuii (1.4 × 10⁶) and P. sulcata (1.6 × 10⁶). Our findings highlight the feasibility of sustainable treatment of animal manure using brackish water microalgae. These results provide opportunities to reduce freshwater usage and environmental pollutions, and support microalgae production for further aquaculture application.

1. Introduction

The rapid expansion of the livestock industry, notably pig farming, poses significant environmental challenges due to the substantial generation of waste [1,2,3]. In Taiwan, pig farming is a major contributor to the issue of waste generation; approximately 5 million metric tons of waste are produced annually, predominantly from pig manure [4,5]. This waste, rich in organics, nitrogen, and phosphates, poses serious environmental risks if not properly managed, affecting water, soil, and air quality [1,2,3].

Pig farm wastewater is especially problematic, characterized by high concentrations of organic matter, suspended solids, and potentially harmful substances, like antibiotics and hormones [6,7,8,9]. Traditional biotechnologies for treating this wastewater, such as anaerobic digestion and various physical or chemical methods, are effective, but they often involve high costs and resource wastage [6,7,8,9]. Smaller-scale farms in Taiwan, which typically follow a three-stage wastewater treatment process, face issues like high electricity costs and residual nitrogen and phosphorus in effluents. These factors underscore the importance of exploring alternative solutions for manure management [4,5].

In response to these challenges, the concept of circular agriculture, which emphasizes recycling agricultural resources, has emerged as a crucial strategy for sustainability [10,11,12,13]. This approach includes transforming waste into valuable resources through innovative treatment methods. For instance, countries like Denmark and Italy have successfully utilized animal waste for agricultural fertilization [10,11,12,13].

A promising aspect of circular agriculture is the cultivation of microalgae using nutrient-rich effluents from pig farming [14,15,16,17,18]. This not only treats the effluent effectively but also produces valuable biomass that can be economically leveraged. Despite its potential, there are significant challenges, such as the variability of digestate composition, the cost of cultivation, and safety concerns, which hinder the large-scale application of this technology [17,18,19,20,21]. Microalgae, recognized for their high lipid content and efficiency in treating various pollutants, present a viable solution for wastewater treatment [22,23,24]. However, challenges like efficient harvesting and the high water content in suspension cultures necessitate innovative approaches, such as direct use in aquaculture, to circumvent the costs of harvesting [25,26,27].

Aquaculture often employs fish meal, agricultural fertilizers, organic fertilizers, or fish slurry from lesser-value fish for anaerobic fermentation in fertilization ponds [28]. If pig manure digestate could be utilized to cultivate aquaculture-specific algae, it would save on input costs for raw materials and fertilizers and offer a novel utilization model for the digestate. In Taiwan, where brackish aquaculture predominates, and many pig farms are located in coastal areas, there is an opportunity to integrate pig manure digestate with saltwater microalgae cultivation. This study explores the potential of using pig manure and urine digestion liquid to cultivate four saltwater microalgae species, aiming to produce feed organisms for shellfish and aquaculture.

2. Materials and Methods

2.1. Microalage Culture

Four pure-strain algae, Tetraselmis chuii, Isochrysis galbana, Chlorella vulgaris, and Proteomonas sulcata, were sourced from the Taiwan International Algae Research Fund, National Taiwan Ocean University. These microalgae were cultured in 250 mL conical flasks in static, each containing 100 mL of filtered and autoclaved natural seawater (salinity 30–33) enriched with F/2 medium (https://utex.org/, accessed on 1 October 2023) and maintained in a climate-controlled room at 26 ± 1 °C with a 12-h light-dark cycle at an illumination of 4000 lux. The cultures, utilized during their exponential growth phase, were inoculated every 10–14 days, ensuring a consistent supply of active biomass for the experiments.

2.2. Collection and Preparation of Pig Manure Liquid Digestate

From March to August 2022, pig manure liquid digestate was sourced from pig farms in Chang-hua’s coastal region, Taiwan, each housing 500–1000 pigs. This digestate was processed through solid–liquid separation and anaerobic digestion. The liquid was filtered using a 100-mesh filter net to remove suspended solids, stored in chemical barrels, and then transported to the laboratory. A comprehensive analysis of the liquid digestate was completed within three days of collection to maintain data accuracy and relevance (Supplementary SA1).

2.3. Water Quality Parameters

When using pig manure liquid digestate as a nutrient source for cultivating microalgae, the deep brown color of the digestate can interfere with the determination of algal color and the outcomes of colorimetric water quality assessments, necessitating calibration of colorimetric measurements. Tests were conducted using a Lovibond XD7500 spectrophotometer (The Tintometer Limited, Amesbury, England) in conjunction with proprietary reagents, then diluting samples to concentrations within the ranges recommended in the manual. In practice, once the digestate was diluted 50–100-fold, its original color was no longer pronounced, and the test results were minimally impacted. Additionally, the digestate, when collected, often contained high levels of suspended solids and continued to undergo digestion and nitrification reactions before algae cultivation. Therefore, water quality tests were conducted after each preparation to ensure the reliability of our experimental results. In field applications, where using the Lovibond XD7500 spectrophotometer is impractical, the WaterLink Spin Touch FX photometer was employed for rapid testing. Despite variations between the two testing methods, data obtained from the WaterLink Spin Touch FX photometer (LaMotte Company, Chestertown, MD, USA) was adjusted based on the results from the Lovibond XD7500 spectrophotometer for calibration.

2.4. Digestate Application in Microalgae Cultivation

Initial tests on the four algae strains established the optimal digestate dilution ratio for cultivation, finding a 50 to 100-fold dilution with 2–10 mg/L ammonia nitrogen most effective. Post-preparation, the culture medium was sterilized, and following algae inoculation, water quality parameters (NH₃, PO₄³⁻, NO₂⁻, NO₃⁻) were assessed using an Lovibond XD7500 spectrophotometer (The Tintometer Limited, Amesbury, England) and standard test kits. Algae concentration was determined using hemocytometer-based cell counts conducted in triplicate.

2.4.1. Four Algae Strains in the Same Digestate Medium

Over 14 days, the four algae strains were cultured under identical conditions, with water quality and cell counts assessed on days 0, 1, 2, 3, 4, 5, 7, and 14. An additional 7-day period followed to observe potential aging effects on the algae. The experiment was conducted in a climate-controlled room at 26 ± 1 °C with a 12 h light–dark cycle at an illumination of 4000 lux.

2.4.2. Four Algae Strains in Different Digestate Mediums

In parallel, C. vulgaris and T. chuii were grown across eight different digestate mediums, and I. galbana and P. sulcata were grown in 10 other mediums. All were grown over 14 days and were evaluated at similar intervals. Separately, T. chuii was cultivated in 40 distinct digestate mediums over 7 days, with assessments conducted on days 0 and 7. All these study subsets maintained the consistent cultivation conditions outlined in Section 2.4.1.

2.5. Field Application of Digestate-Cultivated Algae

Field tests (outdoor) ranged from 200 and 500 L (microalgae and zooplankton) to 10 tons (bivalves) and involved species like rotifers, copepods, Japanese clam (Corbicula japonica), and oyster seedlings (Magallana angulata). The digestate and seawater were not disinfected or sterilized during cultivation but were filtered (300-mesh). Cultivation commenced with a mixture of T. chuii and C. vulgaris, anticipating some contamination over time. Initial concentrations were 10 rotifer and 0.1 copepods per mL, and bivalve seedlings were fed the cultivated algae daily. Water quality parameters, including NH₃ (0–4.0 mg/L), NO₃⁻ (0–60 mg/L), NO₂⁻ (0–2.0 mg/L), and PO₄³⁻ (0–2.0 mg/L), were regularly measured using a WaterLink Spin Touch FX photometer (LaMotte Company, Chestertown, MD, USA).

2.6. Statistical Analysis

The average values of three independent replicates of all trials are presented. One-way analysis of variance (ANOVA) was used to analyze differences among growth based on cell numbers in all trials. The SPSS software (SPSS Statistics 29) was used for all the statistical analyses.

3. Results

3.1. Four Algae Strains in the Same Digestate Medium

The microalgae T. chuii, I. galbana, C. vulgaris, and P. sulcata were cultivated using pig manure liquid digestate in the experiments. The digestate was optimally diluted 50–100 times (keeping NH₃ concentrations 2–3 mg/L), with an initial algae inoculation ratio of 8 × 10⁴ to 2.5 × 10⁶. In Experiment 2.4.1, the NH₃ concentration was measured at 2.7 mg/L, and the NO₃⁻ concentration (186 mg/L) was relatively high. After 1–3 days of cultivation, the NH₃ was almost entirely depleted, with NO₃⁻ predominating after that point. By the 7th day of cultivation, I. galbana and P. sulcata reached their peak concentrations and then declined (1.5 × 10⁷ and 1.6 × 10⁶, respectively; Figure 1). In contrast, T. chuii and C. vulgaris did not reach their highest concentrations until day 14 of cultivation (1.4 × 10⁶ and 2.5 × 10⁷, respectively; Figure 1). Notably, NO₂⁻ levels increased over time, while PO₄³⁻ was scarcely consumed.

3.2. Chlorophyta Algae Cultivation in Eight Different Digestate Mediums

In Experiment 2.4.2, the cultivation concentrations of C. vulgaris ranged between 2.86 × 10⁶ and 1.65 × 10⁷ (ANOVA, p < 0.001, Supplementary SA2). In contrast, those of T. chuii ranged from 7.15 × 10⁵ to 1.09 × 10⁶ (ANOVA, p < 0.001, Supplementary SA2), exhibiting a wide adaptation range to nutrient salts (NH₃ from 6.8 to 10.8, NO₃⁻ from 0 to 210; Figure 2 and Figure 3). Similar to Experiment 2.4.1, there were no significant effects on C. vulgaris and T. chuii when NH₃ was below 10 mg/L and NO₃⁻ was under 200. However, when NH₃ was higher (exceeding 10 mg/L), even after 14 days of cultivation, NH₃ was not wholly utilized, leaving higher residues, which might be the reason for the lower final concentrations of C. vulgaris (Figure 2, trials 7–8) The excess of NO₃⁻ and PO₄³⁻ had no significant effects on algal growth. Still, the accumulation of unused nutrients could potentially affect the subsequent use of algae in feeding zooplankton and bivalves.

3.3. Chromista Algae Cultivation in 10 Different Digestate Mediums

In Experiment 2.4.2, the cultivation concentrations of I. galbana ranged from 3.8 × 10⁶ to 1.5 × 10⁷ (ANOVA, p < 0.001, Supplementary SA2), while those of P. sulcata were between 1.3 × 10⁶ and 8.8 × 10⁶ (ANOVA, p < 0.001, Supplementary SA2), demonstrating a broad adaptability to nutrient salts (NH₃ from 5.4 to 12.6, NO₃⁻ from 3 to 95; Figure 4 and Figure 5). Similar to Experiment 2.4.1, there were no notable impacts on I. galbana and P. sulcata when NH₃ was less than 10 mg/L and NO₃⁻ was below 200. However, when NH₃ levels were higher (more than 10 mg/L), even after 14 days of cultivation, not all NH₃ could be consumed entirely, leaving a higher residue, which may explain the lower final concentrations of I. galbana (Figure 4, trials 8). The effects of excessive NO₃⁻ and PO₄³⁻ on algal growth were less clear, but the accumulation of nutrients that are not fully utilized might affect the subsequent application of these algae in feeding zooplankton and bivalves.

3.4. Tetraselmis Algae Cultivation in 40 Different Digestate Mediums

A total of 40 trials were further conducted on T. chuii. The cultivation concentrations of T. chuii ranged from 3.75 × 10⁵ to 1.5 × 10⁶ (ANOVA, p < 0.001, Supplementary SA2), showcasing a wide adaptability to nutrient salts (NH₃ from 4.1 to 47.2, NO₃⁻ from 0 to 4.2; Figure 6), with the highest concentrations similar to those in other group experiments. Consistent with other tests, when NH₃ was above 10 mg/L, NH₃ could not be fully utilized even after 7 days of cultivation, leaving higher residues. However, T. chuii exhibited a higher tolerance to elevated NH₃ levels, resulting in a lesser impact on cell concentrations after 7 days (Figure 6, trials 10, 20, 30, and 40). Additionally, when NH₃ was below 7 mg/L and PO₄³⁻ was under 3 mg/L, T. chuii could absorb most of the nutrients within 7 days (Figure 6, trials 10, 20, 30 and 40).

3.5. Field Application of Digestate-Cultivated Algae

Outdoor cultivation of a mixture of microalgae (T. chuii and C. vulgaris) was conducted in 200–500 L volumes, achieving concentrations between 1.3 and 2.5 × 10⁵ (Figure 7). Following cultivation to higher densities, protozoans, diatoms, and dinoflagellates began to appear. When using 50–100-fold diluted digestate (with NH₃ concentration at 5.9 mg/L, NO₃⁻ at 3 mg/L, and PO₄³⁻ at 1.5 mg/L), NH₃ was wholly consumed after 7 days, while varying residues of NO₃⁻ and PO₄³⁻ remained (Figure 7). The mixed algae culture could sustain itself for 3–7 days without additional digestate, depending on the outdoor weather conditions. Prolonged maintenance was possible with multiple additions of diluted digestate (50 to 100 times), but this led to increased algal residue on the walls and bottom of the containers.

In outdoor cultures, both adult and juvenile, zooplankton (rotifers and copepods) and bivalves (clams and oysters) showed average growth, with no significant difference compared to chemical fertilizers. When applying digestate as fertilizer in the field, keeping NH₃ concentrations below 10 mg/L did not result in toxicity or excessive accumulation of nutrients for the microalgae. A clear impact was observed for copepods when NH₃ concentrations exceeded 5 mg/L. Subsequently, the cultivated low-density mixed algae culture could be transferred to FRP barrels for further microalgae expansion or feeding bait animals. Ammonia nitrogen levels were monitored to stay below 0.1 mg/L in bivalve tanks, below one mg/L in zooplankton tanks, and under ten mg/L in algae tanks (Figure 8). Post-feeding, algae concentrations in bivalve tanks were kept below 1.0 × 10⁴, in zooplankton tanks below 1.0 × 10⁵, and in algae tanks typically between 1.0 × 10⁴ and 5.0 × 10⁵, varying with algal strains and climate conditions. After a week of cultivating the mixed algae, rotifers introduced into the culture reached their peak density (32–54 ind/mL) approximately one week later. In comparison, copepods took two weeks to get their highest density (0.52–0.7 ind/mL).

4. Discussion

The rapid growth of the livestock industry, especially in developing countries, has led to a significant increase in animal waste, posing environmental challenges [28,29]. In 2019 in Taiwan, there were a substantial number of pig farms (5991) with about 5.5 million pigs, resulting in a large volume of pollutant wastewater [4,5]. Anaerobic digestion (AD) is widely used for its environmental benefits, notably in reducing methane emissions and producing biogas energy. In Taiwan, AD is primarily adopted by large-scale pig farms, as setting up biogas plants requires significant investment [4,5,30]. Smaller-scale farms in Taiwan, which typically have from 500 to 1000 pigs, often use a standard three-stage wastewater treatment that includes solid–liquid separation, anaerobic digestion, and aerobic aeration. While this method is effective in some aspects, it incurs high electricity and maintenance costs. It does not fully address the issue of nutrient-rich effluents, which can contribute to eutrophication. Thus, while AD technology helps reduce greenhouse gas emissions, its effectiveness in mitigating the environmental impact on water resources remains limited, underscoring the need for alternative manure management strategies [1,2,3].

The use of microalgae for treating wastewater from various sources is being increasingly researched. Wastewater treatment systems based on algae primarily employ eukaryotic green algae and prokaryotic cyanobacteria [31], with several studies highlighting the efficiency of Chlorella [18,19,20,21,22,32]. For instance, Safi et al. [22] compiled the effects of Chlorella vulgaris on different types of wastewater (agricultural, municipal), reporting nitrogen removal of 45–97% and phosphorus removal of 28–96%. Silva et al. [31] found that Chlorella sorokiniana and Chlorocaccum sp. could grow in tubular photobioreactors (PBRs) and remove nitrogen and phosphorus from anaerobically digested blackwater, with removal rates of 28–62 mg.L⁻¹.d⁻¹ for nitrogen and 2.3–5.4 mg.L⁻¹.d⁻¹ for phosphorus. Other green algae, such as Scenedesmus, have also shown promising results [16,33,34,35]. Figler et al. [33] used Coelastrum morus to remove nitrogen and phosphorus from high-saline wastewater. The four brackish microalgae used in this study have not been applied in wastewater treatment, partly due to the fact that most wastewater treatments focus on fresh water, with limited demand for saline wastewater treatment [35]. The green algae Chlorella vulgaris is a marine type with a lower tolerance and treatment capacity for ammonium nitrogen than other studies. Moreover, Tetraselmis chuii performed better with higher levels of ammonium nitrogen (Figure 1 and Figure 6).

Although the brackish water microalgae used in our study showed a lower tolerance to ammonium nitrogen than freshwater microalgae, there is a practical need for treating these types of livestock wastewater in Taiwan, an island nation with many livestock farms near the coast. In Chang-hua, the location of digestate collection, coastal areas are often a mix of livestock farms and brackish aquaculture. Wastewater discharged from livestock farms frequently causes eutrophication in drainage channels, serving as water inlets and outlets for brackish ponds. In these areas, bivalve farming (hard clam; Meretrix taiwanica) is a major product. High ammonium nitrogen and phosphorus levels in the inlet make water quality control difficult. Therefore, treating this livestock wastewater with brackish water microalgae meets a real need.

Beyond nitrogen, phosphorus is a crucial factor limiting algal growth in water. A study of 227 lakes in northwestern Ontario, Canada, found that bodies of water remained eutrophic as long as they contained sufficient phosphorus [36]. When phosphorus input is limited, microorganisms cannot acquire phosphorus through alternative pathways, and the amount of algae in the water rapidly decreases. When nitrogen input is limited, the water stimulates the growth of nitrogen-fixing bacteria, with little effect on the amount of algae [36,37,38]. Studies have shown that, even under aerobic conditions in the ocean and coastal seas, certain non-heterocystous filamentous cyanobacteria and unicellular cyanobacteria can contribute significantly to nitrogen fixation [37,38]. Thus, phosphorus is the main factor regulating algal numbers in the water [37,38]. In our study, ammonia was the first to be utilized, and excess phosphorus was rarely wholly consumed, so over long-term serial cultivation, the accumulation of too much phosphorus could affect subsequent applications (Figure 1, Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6).

Nutrient concentrations in wastewater vary greatly. However, nutrient concentrations and imbalanced N:P ratios can result in insufficient growth or nutrient removal [39]. Numerous studies have reported the significance of the N:P ratio from ecological and biotechnological approaches. However, significant variations exist among different phytoplankton species [37]. It is generally acknowledged that this ratio could represent the ideal nutrient proportion essential for growth. Still, algae-specific differences exist: eukaryotic algae’s average optimal N:P ratio ranges from 16–23 N to 1 P [37,39]. In contrast, the discovered optimal ratio for cyanobacteria is 10–16 N to 1 P [38]. The interaction between single or multiple algal species and bacteria can influence the optimal nitrogen-to-phosphorus ratio [37,38,39].

In the study, significant production, accumulation, or disappearance of NO₂⁻ was observed (Figure 1, Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6). Generally, algae do not participate in nitrification processes. This is because ammonia-oxidizing bacteria, primarily belonging to the β- and γ-proteobacteria, perform the oxidation of NH₄⁺ to NO₂⁻. Subsequently, nitrite-oxidizing bacteria carry out the oxidation of NO₂⁻ to NO₃⁻ [40]. In the non-sterile algae cultivation process, bacterial growth is rapid, leading to the swift production, accumulation, or disappearance of NO₂⁻. Due to the limited total amount of ammonia throughout the cultivation process, it is challenging for ammonia-oxidizing bacteria to dominate and persist [41]. This could be one of the reasons for the slow initial growth of microalgae.

Four microalgae strains could inoculate smoothly in our year-long microalgae cultivation when utilizing digestate as a nutrient source. Compared to the F/2 medium, it had a shorter preservation period under conditions containing more organic matter and ammonia. However, digestate could replace complex vitamins and soil extracts with only a minimal addition. This could be because the digestate originates from a difficult nutrient source and organic matter composition [17,31]. While the digestate was sterilized in the small-scale cultivation experiments, it can be used directly in outdoor cultivation. In addition to removing nitrogen and phosphorus from wastewater, the two types of green algae cultivated are the most suitable for outdoor cultivation. They are widely used in bivalve cultivation and for producing zooplankton for fish larvae [42]. The other two types of Chromista algae are primarily cultured indoors and supplied for aquaculture mollusk larvae [43]. Therefore, despite the less efficient wastewater treatment effect, the application of using digestate for cultivating microalgae for aquaculture purposes has been demonstrated (Figure 7 and Figure 8).

Further application can be divided into three stages. First, the microalgae are cultured in small-scale tanks with digestate. The second stage involves large-scale outdoor bivalve farming (Figure 7 and Figure 8). Finally, the large-scale outdoor cultivation system is connected with livestock farms, and wastewater treatment can be conducted on-site. This approach helps circumvent the challenge of transporting large volumes of digestate over long distances and eliminates the need to harvest microalgae. Furthermore, it is anticipated to decrease transport costs and greenhouse gas emissions. Enhancing the system’s sustainability, this method holds the potential to achieve a more significant energy surplus [44]. Digestate was utilized instead of the chemical media we initially employed in practical aquaculture applications, achieving favorable results. Digestate proves to be a convenient and straightforward fertilization source, providing effective control of ammonia nitrogen content. Traditionally, bivalves and planktonic fauna and flora in Taiwan were cultivated using the direct application of animal manure for fertilization and fermentation. However, general consumers no longer widely accept the current method of applying raw manure [45]. The early practice of bivalve–livestock–tilapia co-production in Taiwan has disappeared due to the rejection of natural manure. Utilizing digestate as a replacement for non-anaerobically digested manure, especially in the initial cultivation of algae, offers the advantage of reducing harmful bacteria and antibiotics, thereby enhancing the safety of aquatic products [46]. Our study shows the potential of digestate for on-site fertilization in the livestock–saltwater bivalves–fish circular agriculture production system.

As global awareness of sustainable agriculture and environmental protection grows, the technology of treating livestock wastewater with microalgae is gaining broader attention. Cultivating microalgae in brackish/seawater combinations with livestock wastewater might supply some of the missing nutrients and reduce freshwater use, offering a unique and potentially sustainable approach. Our current methods require digestate dilution for these algae to grow normally. However, it is possible that, through gradual adaptation methods or the selection of specific algal strains, the dilution rate can be reduced or even without dilution [47,48]. Future research should optimize microalgae growth conditions, improve nutrient removal efficiency, and develop economic systems to treat diverse wastewater types concurrently. This integrated approach will contribute to the sustainable development of coastal areas where livestock and aquaculture coexist.

5. Conclusions

Microalgae have tremendous potential in the treatment of livestock wastewater. Although there are still some challenges, such as cost, system optimization, and scaled-up production, with the advancement of technology and increased environmental awareness, microalgae technology will become an integral part of sustainable agriculture in the future. The potential of saltwater microalgae for use in livestock wastewater treatment and saline aquaculture has been demonstrated. By integrating innovative technologies and optimized management strategies, saltwater microalgae can address the wastewater treatment issue in the livestock industry, reduce freshwater use, and contribute to the new concept of circular agriculture. This holds significant importance for promoting environmental sustainability and ecological stability.