Introduction

At present, there is an exponential increase in swine farms around the world to meet the increasing demand for proteins, resulting in a significant amount of swine/piggery wastewater (Nagarajan et al., 2019), which has become a serious environmental concern, due to the high levels of nutrients and toxic contaminants that significantly impact on the ecosystem and public health (Cheng et al., 2019).

Swine wastewater is categorized as one of the agricultural wastewaters with high contents of organics and nutrients including nitrogen and phosphorus, which may lead to environmental eutrophication. So too in slaughterhouse process, and wastewaters are considered as a hotspot for antibiotic-resistant bacteria and antimicrobial residues and may thus play an important role for their dissemination into the environment (Banach et al., 2018; Li et al., 2018; Savin et al., 2020). In this sense, insufficient technologies to remove those nutrients could lead to environmental problems after discharge. Several physical and chemical methods have been applied to treat the swine wastewater, the degradation capacity of these conventional treatment technologies is limited, especially regarding heavy metals, nutrients, and xenobiotics, steering the researchers to bioremediation using microalgae (Phycoremediation). In this sense, bioremediation can be defined as the use of microalgae for removal or biotransformation of pollutants and CO₂ from wastewater with concomitant biomass production. Bioremediation with microalgae is particularly effective because of their capabilities of converting solar energy into useful biomasses and assimilate nutrients such as phosphorus and nitrogen which cause eutrophication in the photosynthesis process. In tertiary wastewater treatment, microalgae offer an effective low-cost approach to remove contaminants and excess nutrients, and produce potentially valuable biomass, because of its high ability for inorganic nutrient uptake, showing high ability to remove inorganic nutrients from artificial domestic secondary effluents (Wang et al., 2016; Feng et al., 2020; Lu et al., 2020). Thus, biological treatments are considered as the promising methods due to the cost effectiveness and performance efficiency, along with the production of valuable products and bioenergies (Cheng et al., 2020; Chen et al., 2020 a,b).

The use of a microalgal biomass is a powerful tool to adequately operate wastewater treatment processes, providing valuable information to model wastewater treatment systems (Sánchez-Zurano et al., 2020). So too, several types of microalgae such as Chlorella and Dunaliella have proved their applicability in wastewaters treatment, reporting average removals of NH₄-N (40 - 51 %), PO₄-P (33 - 43 %) and Chemical Oxygen Demand (38 - 63 %) (Wang et al., 2014; Cai et al., 2017; Li et al., 2018; Chen et al., 2020). However, it is important to identify other microalgae with potential for bioremediation, which could achieve higher removal values, since each microalgal morphotype responds differently under specific growth conditions, so also with respect to its tolerance and resistance to the growth medium composition (Pei et al., 2018).

Due to the foregoing, the present research work aimed to evaluate the microalgal biomass of Verrucodesmus verrucosus in bioremediation of wastewater from pig farms, as well as the removal of Escherichia coli as a pathogenic microorganism.

Material and methods

Sampling

The samples were taken from the pig farm called “El Chano” in the municipality of Suchiapa, Chiapas, located at 16°37′05″N 93°05′39″W. One liter of residual water was collected, from the piglet and fattening growth stages, accumulated during the day, considering that cleaning is carried out daily. The residual water was stored in previously sterilized plastic bottles, at 4 °C (NMX-AA-034-SCFI-2015).

Treatment establishment

Nine wastewater treatments were established from the collection of two growth stages: piglet (P) and fattening (F), including a combination of these (50/50); the wastewater was evaluated as it was collected (crude), so too, wastewater was filtered (f) using a 15 µm diameter sieve and sterilized (s) in autoclave at 121°C for 15 min, as established in Table 1.

After having identified the treatments, they were characterized by the quantification of ammonium, nitrate, phosphorus, phosphate and nitrogen, to subsequently inoculate Verrucodesmus verrucosus and evaluate for 40 days its behavior, through growth kinetics and its subsequent removal capacity as well as its antagonistic capacity against Escherichia coli, as described below and shown in Figure 1.

Physical and chemical analyses of water samples

For the characterization of residual water, the total nitrogen (N) was analyzed by Kjeldahl method (Stafilov et al., 2020), potential of hydrogen (pH) with the use of a potentiometer (HANNA HI 2211-01) (NMX-AA-008-SCFI-2011), according to Jin et al. (2019). Also, ammonium, nitrate, total phosphorous and phosphate were analyzed by quantitative colorimetric analysis according to Pei et al. (2018), Zhu et al. (2008)), Feng et al. (2020) and Wilfert et al. (2018), respectively. Finally, the determinations of Chemical Oxygen Demand (COD) and Biochemical Oxygen Demand (BOD) were performed according to NMX-AA-030/1-SCFI-2012 and NMX-AA-028-SCFI-2000, respectively.

Microorganism and culture conditions

Verrucodesmus verrucosus was provided by the UAMI Applied Fiction Laboratory (Universidad Autónoma Metropolitana Iztapalapa). The cells are elliptical, without appendages and curved edges. V. verrucosus can form cenobium obes containing 4, 8, 16, or more cells between 4 and 6 µm in size. The specie was recently described by Hegewald et al. (2013) and López-Mendoza et al. (2015), with GenBank accession number JQ240289. Verrucodesmus verrucosus was evaluated in the nine wastewater treatments in a period of 40 d with an initial inoculation of 25 CFU mL^-1 at a temperature of 25 °C + 2.

Escherichia coli quantification

The quantity of the coliform group microorganisms was determined according to the Mexican Standard NMX-AA-42-1987, and according to the total coliform quantification protocols established by Banach et al. (2018).

Productivity and growth kinetics

Growth kinetics were performed by cell count in a Neubauer chamber, and plate count by colony forming units (CFU). The cultures were shaken in each of the flasks to allow a uniform distribution of cells, and 1 mL samples were taken and placed in previously washed tubes. The camera was observed in the microscope with 100X objective. The cell concentration (CFU mL^-1) was calculated using the following formula, Eq. (1):

Where C is the cell concentration (CFU mL^-1), N is the average of cells present in 1mm² (0.1μL), dil the dilution factor and 10⁴ the conversion factor from 0.1μL to 1mL. From these data the specific growth rate was determined, which was calculated with the following exponential equation (Wang et al., 2014), Eq. (2):

Where, μ is the specific growth rate in days, ln is the natural logarithm, N₀ corresponds to the initial population concentration (CFU mL^-1), N_t concentration after the final growth time (CFU mL^-1), t₁ initial time for the growth interval of interest in days, and t₂ final time of growth of the interval in days.

Statistical analysis

The parameters of the evaluations were estimated using a sigmoidal model, and Gompertz (Sigma plot ® v.11.0) was used for the correlation evaluation between points. An analysis of variance (ANOVA) was performed in order to evaluate the differences between groups. The means of each treatment were compared using the Tukey method (p≤0.05). The analyzes were carried out using the statistical software Minitab18®.

Results

Wastewater characterization

The characterization of the residual water from a pig farm was carried out at two stages of growth (piglet and fattening) and the mixture between these (50/50), subjected to two treatments (sterilization and filtering), which is why it is possible to identify the higher concentrations in the fattening crude stage for ammonium, nitrate, phosphorous, phosphate, nitrogen, chemical oxygen demand and biochemical oxygen demand in the values of 1992.38 mg mL^-1, 9546.76 mg mL^-1, 1410.14 mg mL^-1, 2999.58 mg mL^-1, 49%, 1629.56 mg mL^-1 and 896.25 mg mL^-1, respectively (Table 2).

Mineral kinetics

In the kinetics of minerals, the progressive removal of the evaluated minerals is observed (ammonium, nitrate, phosphorous, phosphate, nitrogen), however it is important to mention that Verrucodesmus verrucosus demonstrated to start the decrease of minerals in the medium at day eight, reaching levels of highest removal at day 32 of evaluation, so it was determined in this research that the average removal time for these microalgae is 32 to 40 days (Figure 2 a to e).

On the other hand, the growth stage with the highest removal was the piglet stage, for the minerals phosphorus and phosphate with 97.98 %, 82.18 % respectively, in the filtered and sterilized treatments. However, the fattening stage has the highest removals in nitrate and nitrogen with 85.65 % and 85.71% respectively, in the sterilized treatments for nitrate. So, it is also important to mention that the decrease in nitrogen is significantly higher compared to the other treatments in its crude form (Table 3 and Figure 2).

At the conclusion of the mineral kinetics, it was determined that V. verrucosus has, on average, the ability to remove ammonia, nitrate, phosphorus, phosphates and nitrogen at percentages of 54.13 %, 77.51 %, 57.73 %, 54.22 % and 53.06 % respectively, so that this microalgae has an average mineral removal capacity in pork wastewater of 66.24 %, benefiting the removal processes by applying sterilization and filtration treatments (Table 3).

COD and BOD removal kinetics

During the kinetics evaluation of the chemical and biochemical oxygen demand, a significant removal is observed at 32 days after V. verrucosus inoculation, thus also identifying that residual water from the lechon growth stage reaches higher removal rates of 92 and 96 % for BOD and COD, respectively (Figure 3 and Table 3).

Escherichia coli removal

During the growth kinetics of V. verrucosus the start of the exponential stage is observed at 8 days, demonstrating the maximum growth rate of 0.14 CFU / Day in the sterile fattening treatment and the shortest doubling time of 4.31 d, therefore the removal values begin to be important, however the exponential stage ends between 24 and 32 d, data that correlates the higher removal values for the minerals as well as the chemical and biochemical oxygen demands. On the other hand, it is observed how kinetics of Escherichia coli decreases significantly after 8 days of inoculating V. verrucosus, decreasing the bacterial population in its entirety at 32 d. Therefore, V. verrucosus demonstrated 100 % removal of the E.coli bacterial population within 24 d (Figure 4 and Table 4).

Discussion

Wastewater characterization

Swine wastewater is rich in nitrogen and organic carbon, however, in the present research it was shown that the wastewater from fattening pigs, corresponding to an adult age, showed the highest concentrations in the different nitrogen and phosphorus forms evaluated (Table 2), data similar to that reported byWen et al. (2017), they evaluated the resistance condition of Chlorella vulgaris in undiluted swine slurry and artificial wastewater, identifying concentrations higher than 2000 mg / L of the different forms of TN and TP.

This is because fattening pigs consume a more complete diet accompanied by chemical compounds that modulate feed conversion, associated with digestibility coefficient problems, such as bad absorption or bacterial infections that force the use of antibiotics, generating feces loaded with organic and inorganic matter that convert wastewater into a complex mixture. It is also important to mention that the nitrogen and phosphorous retention capacity decreases with the age of the animal (Wang et al., 2017; Likiliki et al., 2020).

Mineral kinetics

During the mineral removal kinetics, it was determined that V. verrucosus has the ability to decrease the concentration of phosphorus and phosphate present in wastewater of piglets, from 82 to 97.98 % of removal respectively. Thus, removal concentrations of 85.71% were demonstrated for nitrogen evaluated in this research work, it is also important to mention that the highest total nitrogen removals are found in crude treatments (Table 3). Similar data has been reported by Garcia et al. (2018), who mentioned that results revealed a high diversity and rapid variations in the structure of microalgae populations, Chlorella sp., were recorded in the removal efficiencies (REs) of total organic carbon (86 - 87 %), inorganic carbon (62 - 71 %), total nitrogen (82 - 85 %) and total phosphorous (90 - 92 %).

According to the aforementioned, Sudiarto et al. (2019) studied the removal of nitrogen and phosphorus from the effluent of treated swine wastewater by Eichhornia crassipes, Pistia stratiotes, Limnobium laevigatum, and Lemna sp. Pistia stratiotes showed the highest total nitrogen removal (63.15 %) from the treated effluent, Lemna sp. showed the highest phosphorus removal of 36.15 % from the treated effluent. Caí et al. (2019) also showed that Spirulina platensis removal efficiency of ammonium was 99 %, which provides an alternative method for the utilization of Digested Piggery Wastewater (DPW).

However, Chen et al. (2020) evaluated Desmodesmus sp., a microalgae belonging to the same family of V. verrucosus, showing that Desmodesmus sp. PW1grew well in diluted and undiluted piggery wastewater, and could effectively remove nitrogen and phosphorus with removal rates up to 90 % and 70 %, respectively. At laboratory scale by 30L photobioreactor, microalgae also performed well in TN (65.3 %) and TP (83.5 %) removal.

It is important to mention that the treatment of fattening without sterilization showed the highest removal values (Table 3), so it is not necessary to implement a treatment to improve the removal capacity of V. verrucosos, data that agrees with the work carried out by Wang et al. (2016) who evaluated an UV irradiation-based method to treat the piggery wastewater before inoculating the microalgae biomass, in order to reduce microbial competition and benefit the degradation of compounds. However, the microalgae grew well in slurry without treating and achieved outstanding removal efficiencies in total nitrogen (TN) and total phosphorus (TP), with 89.5 % and 85.3 %, respectively (Rasoul-Amini et al., 2014).

The assimilation of nutrients is variable and depends on the level of daily consumption or intake, which are affected by factors such as genetics and mainly age or growth phase.

It has been verified that in piglets from fetal life, the gastrointestinal tract is in charge of supplying in its entirety of metabolites and protective substances through nutrients absorption, the endocytosis of immunoglobulins from colostrum and milk, an activity that guarantees the high absorption and retention of nutritional substances. Another reason is associated with the lumen surface of the small intestine, which is made up of numerous villi at the base where are tubular glands (Lieberkühn crypts) that descend to the muscularis mucosa benefiting their high absorption, unlike fattening pigs that with advancing age, a certain percentage of intestinal mucosa velocities atrophy, reducing the efficiency of nutrient absorption, associated with competition for the substrate and problems caused by the presence of Enterobacteriaceae (Bibbal et al., 2018).

With all of the above, it was possible to demonstrate the potential of V. verrucosus to remove minerals in mixotrophic conditions, since the swine wastewater is rich in nitrogen and organic carbon which are essential macronutrients for microalgal growth (Chen et al., 2020; Sánchez et al., 2020).

The ability of microalgae to feed on minerals present in pigs’ wastewater has benefited the accumulation of compounds of biotechnological interest, as mentioned by Li et al. (2018), who demonstrated that Coelastrella sp. could remove nutrients from anaerobically digested swine wastewater (ADSW) effectively, if its responses to the stress of Cu (II) were less. However, they showed that finding high copper concentrations in the medium increases the concentration of the superoxide dismutase enzyme responsible for pigment synthesis and oxidative stability of lipids synthesized by the microalgae.

COD and BOD removal kinetics

In this research an 83 % average removal of chemical and biochemical oxygen demand by V. verrucosus was demonstrated (Table 3). These data agree with those obtained by Chen et al. (2020), who evaluated the remove capacity from Chlorella sorokiniana in swine wastewater, and demonstrated the COD, TN and TP removal efficiency for the swine wastewater was 90.1, 97.0 and 92.8 %, respectively (Zhou et al., 2018).

These results tend to be promising when considering the V. verrucosus microalgal biomass, as a potential alternative to remove, since removal efficiencies of 50 % have been demonstrated by facultative aerobic bacteria, which are not very effective with respect to microalgae. As demonstrated by Chen et al. (2020), who evaluated the COD removal efficiency (55.46 %) in anaerobic digestion treatment of real piggery wastewater: Treatment efficiency and bacterial diversity.

Escherichia coli removal

Through the evaluation of the microalgal biomass growth kinetics, it was observed that the exponential stage of V. verrocosus is coupled to the stationary and death stage of the growth kinetics of Escherichia coli (Figure 4). Said aforementioned behavior was observed in other investigations, as mentioned by Wen et al. (2017), they identified the stress tolerance of Chlorella vulgaris in wastewater, which was verified in artificial wastewater containing different levels of chemical oxygen demand (COD), corresponding to a high bacterial load.

The microalgae V. verrucosus demonstrated antagonistic capacity by removing 100 % of E. coli (Figure 4 and Table 4), an enterobacterium present at a high concentration in feces deposited in wastewater, as shown in the present work, since slaughterhouse process and wastewater are considered as a hotspot for antibiotic resistant bacteria and antimicrobial residues, which may thus play an important role for their dissemination into the environment (Savin et al., 2020).

This phenomenon has been observed previously, for instance, Li et al. (2020) identified the effect of antibiotic residue pollution from swine feedlots to nearby groundwater environment, analyzing the presence of residues from most commonly used antibiotics, including tetracyclines (TCs), fluoroquinolones (FQNs), sulfonamides (SAs), macrolides, and fenicols, therefore, antibiotics discharged from swine feedlots through wastewater could disseminate into surrounding groundwater environments, generating antibiotic resistance genes (AGRs) in different microorganisms.

Also, Yang et al. (2020) demonstrated that in the wastewater of pig farms was a higher abundance of Proteobacteria, including the potential human pathogenic bacteria (HPB) (Escherichia, Shigella, Bordetella and Morganella), crop pathogen (Pectobacterium) and denitrifying bacteria (Zobellella); the results showed that the bacteria played an important role in the denitrification, which can provide a new point of penetration for improving the bioremove at pig farms, because eradicating the nitrogen present in the wastewater would eliminate a large population of pathogenic bacteria. For this reason, since V. verrucosus is efficient in removing nitrogen forms and is also metabolically more active, it is a disadvantage for pathogenic bacteria that try to compete for the substrate (Wu et al., 2020).

Thus, it is also important to mention that the highest biomass production was reached in wastewater from the fattening stage, identifying a direct relationship between biomass and the concentration of pollutants (Table 4, Figure 4). This effect was identified in an evaluation on C. pyrenoidosa, where, the results showed the maximum nutrient removal efficiencies at 50 % in the fattening pig wastewater, since the fattening pig wastewater is rich in nutrients, which can be utilized for algal biomass production (Azam et al., 2020).

Finally, it is important to mention that according to the aforementioned results, the residual water from pig farms previously treated with V. verrucosus can be used for the irrigation of fruit and vegetable crops.

Conclusions

The use of V. verrucosus is a powerful tool to adequately manage and operate wastewater treatment processes, providing valuable information to model wastewater treatment systems with these microalgae, on the other hand, it was shown that this microalgae species grows well in swine wastewater with large amounts of biomass being produced, despite the impact of various parameters (e.g., nutrients and toxicants levels, cultivation conditions, and bacteria in swine wastewater).

It is important to mention that the harvested microalgae biomass elicits high potential for conversion to bioenergy, because the lipids can be used for biodiesel production, and according to this, the development of renewable and clean energy as well as bio-based fine chemicals technologies are the keys to overcome the problems such as fossil depletion, global warming, and environment pollution.

The strategy is therefore a promising method for microalgae to purify piggery slurry containing high nutrient contents, reducing the environmental impact of contaminated waters that damage soils and consumers of food irrigated with these waters.

Acknowledgments

This study was supported by the Universidad Politécnica de Chiapas.

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Author notes

^*Author for correspondence: Yolanda del Carmen Pérez Luna, Yazmin Sánchez Roque. e-mail: yperez@upchiapas.edu.mx; ysanchez@ia.upchiapas.edu.mx

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