## 6 Specific Applications of Wastewater Bioprocessing with Simultaneous Biofuel Production
### Livestock Waste and Bioethanol Production  
In addition to microalgae that accumulate lipids in their biomass when grown in livestock waste, there are also species that accumulate increased amounts of carbohydrates and may be utilized as a fermentation feedstock for bioethanol production. Pig farm waste (manure) is one of the most heavily burdened wastes worldwide due to its high concentration in organic load and greenhouse gas emissions. In Mexico, about 50% of methane and nitrogen oxide emissions come from the decomposition processes of this waste. Due to the very high concentrations of total nitrogen (9000 to 18,000 mg/L) and total phosphorus (6200 to 39,000 mg/L), anaerobic digestion is the best available technology for the management of wastewater from pig waste. From this process, biogas and digestion residue is produced, which is a liquid rich in nutrients (nitrogen and phosphorus) and organic acids, and can therefore be used for microalgae cultures [47]. The green microalgae _Chlorococc_ sp. was isolated from waste treatment plants in Mexico and cultivated in anaerobic digestion residue of pig farm waste via two approaches. Firstly, growing conditions were controlled for lighting and temperature, while secondly, there was no intervention to control conditions. In the 27-day controlled culture, lighting was maintained consistently at 46 \(\pm\) 3 \(\upmu\)mol photons/m\({}^{2}\)/s and temperature at 30 \({}^{\circ}\)C. In the uncontrolled culture, lighting ranged from 94 \(\upmu\)mol photons/m\({}^{2}\)/s to 357 \(\upmu\)mol photons/m\({}^{2}\)/s with an average of 24 days of cultivation, 98 \(\upmu\)mol photons/m\({}^{2}\)/s. In addition, the temperature inside the greenhouse ranged from 25 \({}^{\circ}\)C to 39 \({}^{\circ}\)C, with an average of 30 \({}^{\circ}\)C. The results showed that under controlled conditions, the highest biomass production was found in the 8% digestate sample and amounted to 0.85 g/L, while in the uncontrolled conditions it reached 0.64 g/L in the 5.6% digestate sample, which is very close to the 0.64 g/L reached in the reference sample (modified Bold's Basal Medium). In relation to lipid formation, controlled cultures ranged from 5.4 to 9.8% on a dry basis, while non-controlled cultures ranged from 9.6 to 12.9% with a content of C18:3n6 (gamma-linolenic acid), which exhibited very high levels (26-35%). At the same time, while the carbohydrate content from the inoculum stage was 198 mg/g, it reached 454.1 \(\pm\) 2 mg/g on a dry basis, a value higher than the reference sample (425.6 \(\pm\) 10 mg/g), with the increase occurring when ammoniacal nitrogen declined. The FAME profile in the two cases was different and did not meet the requirements of EN 14214 for biodiesel production. On the contrary, it was found that the strain _Chlorococcum_ sp., under conditions of nitrogen deficiency from the 12 days of cultivation, accumulated carbohydrates and not lipids. The same phenomenon has been observed in cultures of _Chlorella vulgaris_. According to the above, the authors consider that the cultivation of _Chlorococcum_ sp. under uncontrolled conditions, in a properly diluted anaerobic digestion residue of pig farm waste, is a promising system for bioethanol production within a microalgae biorefinery. The lipids produced can potentially be high-added-value products, since gamma-linolenic, palmitic, and linoleic acids, accounting for about 40-60% of the total lipid content of the biomass of Chlorococcum sp., are of great value in aquaculture [47].