Microalgae biomass from swine wastewater and its conversion to bioenergy.

Ever-increasing swine wastewater (SW) has become a serious environmental concern. High levels of nutrients and toxic contaminants in SW significantly impact on the ecosystem and public health. On the other hand, swine wastewater is considered as valuable water and nutrient source for microalgae cultivation. The potential for converting the nutrients from SW into valuable biomass and then generating bioenergy from it has drawn increasing attention. For this reason, this review comprehensively discussed the biomass production, SW treatment efficiencies, and bioenergy generation potentials through cultivating microalgae in SW. Microalgae species grow well in SW with large amounts of biomass being produced, despite the impact of various parameters (e.g., nutrients and toxicants levels, cultivation conditions, and bacteria in SW). Pollutants in SW can effectively be removed by harvesting microalgae from SW, and the harvested microalgae biomass elicits high potential for conversion to valuable bioenergy.


Introduction
Pork is the most widely consumed meat in the world according to the report named "Global Pork Meat Market 2017-2021" (Reportlinker, 2017). The rapid increase in pork demand has prompted the development of concentrated swine feeding operations, resulting in large quantities of SW production. SW contains not only high levels of suspended solids, organic matters, nutrients, but also toxicants, for example heavy metals, antibiotics and hormones (Cheng et al., 2018b). Concentrations of the above mentioned pollutants in SW are shown in Fig. 1  Anaerobic digestion is the most widely used technology for treating SW because this helps convert organic matter from SW into biogas, yet the removal of nutrients is curtailed For this reason, the development of renewable and sustainable alternatives is urgent, so that further aggravation of the energy crisis and global climate change can be avoided (Zhu et al., 2013b). Biofuels derived from biomass have been identified as alternatives to fossil fuels for alleviating world energy demands and reducing greenhouse emissions (Amin, 2009).
However, the large-scale generation of biomass is to some extent restricted, because their continuous growth process requires large quantities of water and nutrients like nitrogen and phosphorus (Poorter & Nagel, 2000). The addition of fresh water and commercial fertilizers to meet economic growth requirements can significantly increase the production costs, which is not cost-effective for biofuel production and energy recovery (Mata et al., 2010). Hence, cultivation, high concentrations of pollutants in SW (see Fig. 1) might adversely affect the microalgae growth rate and biomass production. Generally, microalgae growth could be affected by initial nutrition levels, cultivation conditions (e.g., light intensity, temperature, pH and cultivation modes), heavy metals, antibiotics, hormones and bacteria in SW (Li et

Microalgae growth and biomass production
The selection of robust strains that can consistently grow in SW is important for better production of bioenergy and SW purification (Ji et al., 2013a). The chosen microalgae should have high tolerance to the pollutants in SW, high growth rate, high recovery rate of nutrients, and robust growth properties with improved tolerance for varied environmental conditions (Salama et al., 2017). Several microalgae species (for instance, Chlorella sp., Scenedesmus sp., Neochloris sp., Chlorellaceae sp. and Coelastrella sp.) have been studied for SW treatment coupled with biomass production under various cultivation conditions (see Table   1).
Species in the genera Chlorella and Scenedesmus are the most employed microalgae cultivated in SW due to their high environmental tolerance, high biomass and lipid accumulation potential, Chlorella vulgaris and Scenedesmus obliquus in particular (Wang et al., 2016b). The maximum biomass concentration of Chlorella vulgaris (4.25 g/L) is higher than Scenedesmus obliquus (2.3 g/L) when they are cultivated in SW. As stated in Table 1, the microalgae biomass production and growth rate highly depend on microalgae species, cultivation conditions and SW compositions. The biomass concentrations of Chlorella vulgaris and Scenedesmus obliquus have a wide range in response to different culture conditions, with 0.2-4.25 g/L and 0.24-2.3 g/L, respectively. Biomass productivity and specific growth rate are important parameters for assessing the microalgae growth scenario for all these studies microalgae species, which ranged from 29 to 890 mg/L/d and 0.074-1.73 d -1 , respectively. SW has been regarded as suitable media for the growth of microalgae species for biomass production, despite the fact that comparing cultivation in different types of wastewater is not straight forward. This is due to the wide range of cultivation conditions applied (like cultivation mode, working volume, light intensity and temperature) that can affect biomass production (Kuo et al., 2015). Nevertheless, Ferreira et al. (2018) observed the maximum biomass production in SW by comparing the microalgae growth in different sources of wastewater (swine, poultry, cattle breeding, brewery, dairy, industrial and municipal wastewater) under the same operating conditions.

Impact factors for microalgae growth and biomass production
To enhance the economic feasibility of microalgae biofuel production, efficient growth and high biomass production of microalgae are required (Mata et al., 2010). The efficient growth of microalgae in SW depends significantly on cultivation conditions, nutrient and toxicant concentrations in SW (Amini et al., 2016). It can be seen from Table 1 that there is a wide range of growth and biomass production under different conditions, even the same species, which indicated that the culture medium and conditions are significant impact factors.

Impact of nutrients in swine wastewater
From Table 1, it should be noted that most microalgae species used in previous studies are cultivated in pretreated and/or diluted SW with either synthetic media or distilled water (Marjakangas et al., 2015). This is mainly attributed to: firstly, the high salinity as measured by ion conductivity in undiluted SW may induce osmotic stress and oxidative stress in the algal cells to inhibit their growth (Deng et al., 2018); secondly, suspended matter or impurities in the SW could compromise the water's clarity and interfere with light penetration (Wang et al., 2012); thirdly, the concentrated organic/inorganic compositions might inhibit the growth of microalgae (Park et al., 2010). Thus, the original SW wielded an adverse effect on the growth of microalgae species resulting in a very low biomass production (Franchino et al., 2016).
The microalgae biomass production and growth rate are affected by the SW dilution rate, because they strongly depend on the nutrients concentration of growth media. As summarized in Table 1, based on the initial nutrients concentration in SW, having a suitable dilution rate is important for biomass production and growth rate of microalgae. To be specific, microalgae growth could be inhibited if extremely high levels of nutrient are present in SW, so that more dilution is required. Conversely, greater dilution of SW can result in restricting the microalgae growth due to nutrient deficiencies. For example, Chlorella vulgaris grew better in the cultivation media with 5% of wastewater (initial nutrient concentrations: TN=3355, NH4-N=2050 and TP=318.5 mg/L), and had higher biomass concentration (1.47 g/L) and productivity (229 mg/L) when compared to cultivation in media with 10% of SW; no growth was observed in the media with 20% and  Wang et al. (2017) all concluded that the highest values of biomass production and microalgae growth were obtained in undiluted SW with the initial concentrations of TN<300 mg/L and TP<30 mg/L, respectively. Low phosphorus concentrations in the SW may be a limiting factor for microalgae biomass production (Ji et al., 2013a). Thus, increasing phosphate concentration could improve the biomass production by avoiding phosphate limitation . This outcome has been confirmed by Kuo et al. (2015), who observed that the maximum microalgae biomass production and specific growth rate were achieved in undiluted SW (550 mg/L TN, 490 mg/L NH4-N and 20 mg/L P) due to the deficiency of phosphorus. Hence, the appropriate dilution ratio of SW is essential to achieve the best cultivating conditions for microalgae biomass production.

Impact of cultivation conditions
In addition to nutrient levels in SW, the microalgae growth and biomass production depend significantly on cultivation conditions. Impact factors like pH, temperature, growth  Table 1), the applied temperature and light intensity are in the 20 -27 °C and 45 -300 μmol/m 2 /s ranges, respectively. This is in consistent with Singh and Singh's (2015) findings, who concluded that the optimum temperature and light irradiance for the growth of different microalgae species ranged from 20 -30 °C and 33 -400 μmol/m 2 /s. In another study, Wang et al. (2017) analysed the growth of Neochloris aquatic Cl-M1 in undiluted SW in various temperatures (15 °C, 25 °C, 37 °C and 40 °C). The maximum biomass concentration (3.7 g/L) and maximum carbohydrate content (33.2%) were obtained at 25 °C. Therefore, relatively lower and/or higher temperature both demonstrate negative effects on biomass production. One possible explanation is that low temperature could reduce the metabolic rates of microalgae, while high temperatures may trigger oxidative stress (Ali et al., 2005). A positive correlation between biomass production/carbon dioxide fixation and duration hours of the light has been

Impact of toxic contaminants in swine wastewater
Heavy metals, antibiotics and hormones are usually used in concentrated swine farms worldwide to treat/prevent pig diseases and promote growth (Li et

Impact of bacteria in swine wastewater
As summarized in Table 1

SW purification by harvesting microalgae
High concentrations of organic matter, nutrients and toxic contaminants in SW have raised many concerns over their adverse effects on the environment and people's health

Removal of nutrients
Carbon, nitrogen and phosphate are important sources of nutrients required for the growth of microalgae. Inorganic nitrogen, including ammonium (NH4 + ), nitrate (NO3 − ) and nitrite (NO2 − ), and organic nitrogen (urea and amino acids) in SW are required by microalgae for synthesis of proteins, nucleic acid, enzymes, chlorophylls, and genetic material (Cao et al., 2018). Phosphorus is also a key factor in the energy metabolism of algae and is found in nucleic acids, lipids, proteins, and the intermediates of carbohydrate metabolism (Cai et al., 2013). Hence, TN and TP in SW can be reduced by microalgae via uptake (Xu et al., 2015).
It is also evident that the removal of TN and TP is partly due to abiotic processes, such as chemical precipitation and ammonia stripping at high pH (Cai et (Norvill et al., 2017). However, the leaching of these toxicants from algae ponds may lead to contamination of ground water, which become a high risk to human health.

Bioenergy production potential by harvesting microalgae from swine wastewater
A number of studies have confirmed that microalgae can be used as feedstock for biofuel production, including biodiesel, biomethane and biohydrogen (Kadir et al., 2018; Wieczorek et al., 2014). The creation of biofuel from microalgae has garnered much attention due to the rising energy demand worldwide, and the need to greatly reduce greenhouse gas emissions into the atmosphere (Udaiyappan et al., 2017). Microalgae harvested from SW exhibit favorable properties for biofuel production due to its tolerance and high growth rate in SW, as well as high lipid and carbohydrate content.

Biodiesel production potential
Biodiesel is a mixture of long chain fatty acid methyl esters (FAME) and is obtained by  Fig. 3 (a, b). The values

Biomethane and biohydrogen production potential
The harvested microalgae biomass from SW can also be used as a substrate to generate biomethane and biohydrogen through anaerobic digestion and dark fermentation (Wieczorek et al., 2014). Carbohydrates contained in microalgae (mainly in the form of glucose and some polysaccharides like starch, agar and carrageenan), are ideal feedstock for green gaseous biofuel production (Ho et al., 2012). Compared to the general carbohydrate contents in microalgae (10-20%), large quantities of carbohydrates in the microalgae from SW confirmed their potential for gaseous biofuel production (from 27.6% to 58.3%, shown in Fig. 3 (d)). Microalgae Chlorella and Scenedesmus may be the favored species for SW treatment and biofuel production due to their fast growth in SW culture and high

Future perspectives
Though SW is considered to be a valuable source for microalgae-based biomass production, high concentrations of nutrients and toxic pollutants could inhibit the microalgae growth. Based on the above discussion, it is obvious that the dilution of SW with water or synthetic media is usually necessary for efficient microalgae biomass production, although an increase in costs is currently unavoidable. Therefore, developing an efficient process allowing algae to grow well in undiluted SW is necessary in the future, considering its feasible and economical application to full-scale wastewater treatment and bioenergy production. To avoid the toxic effects of bacteria on microalgae growth, SW is usually sterilized through autoclaving, which also increases the operational costs and limits the largescale application of microalgae-based systems. As discussed earlier in section 2.2.4, the impact of bacteria on microalgae growth still requires further and long-term investigation.
Research is required to establish the minimal inhibitory concentration of toxic contaminants in SW on microalgae growth and nutrient removal, as well as what their combined effects are. Microalgae are effective in removing toxic contaminants from wastewater, but little research has been done on their removal from SW. Moreover, the dominant and preferred mechanism for removing contaminants by microalgae must be further clarified so that the efficiency in removing contaminants is enhanced.
Meanwhile, problems from the cultivation of microalgae in swine wastewater also require further assessment. For example, the volatile organic carbon (VOC) emissions from algae ponds lead to the production of tropospheric ozone (O3) and thus they have adverse effects for humans by affecting the respiratory problems. Overall, there are still some obstacles limiting the wide application of SW purification and recovering bioenergy via microalgae cultivation in SW, so further research is still required in this field.

Conclusions
The key conclusions from this review are: (1) cultivating microalgae in SW is an alternative method for SW treatment and bioenergy production; (2) microalgae can alive and grow well in swine wastewater for biomass production; (3) nutrients and toxic contaminants in SW can be removed effectively from wastewater through harvesting microalgae; and (4) the lipid and carbohydrate contents in microalgae harvested from SW are comparable with the common values, indicating their potential for bioenergy production. Technol. 129, 7-11.