(8d) Biogas Production and Nutrient Recovery from Batch Anaerobic Digestion of Lipid Extracted Freshwater and Marine Algal Biomass: Chlorella vulgaris and Cyclotella Sp

Biogas production and nutrient recovery from batch anaerobic digestion of lipid extracted freshwater and marine algal biomass: Chlorella vulgaris and Cyclotella sp.

1 Introduction
Anaerobic digestion (AD) is a biological process that uses anaerobic bacteria to break the complex organic components into biogas or other nutrients (Mattocks, 1984; Zamalloa et al., 2011). It has been widely applied for the effective treatment of municipal wastewater, industrial wastewater, and biomass. Various kinds of biomass have been used as feedstocks for bioenergy, including crops, forest, and marine biomass (Chynoweth, 1987). Algal biomass is promising feedstock for biofuel production due to it fast growth rate and high lipid content. However, to realize these advantages anaerobic digestion is required to recycle nutrients and recover energy from excess (non-lipid) biomass. The biogas from digestion is composed of mostly methane and carbon dioxide, as well as a small amount of nitrogen, sulfur and other trace components (Mattocks, 1984). Depending on the algae feedstock and the conditions of the digestion, the methane concentration in the biogas ranges from 45% to 70% (Ishida, 1982). Most of methane in the process is evolved and recovered since methane is a sparingly soluble gas (Grady et al., 1999).
For large scale algae cultivation, the management of huge quantities of residual biomass must be considered after the lipid extraction process. Anaerobic digestion is a key process to convert large quantities of residual biomass into biogas, which is similar to natural gas (Davis et al., 2012). In addition, algae contain large quantities of nitrogen and phosphate that are valuable nutrients and should be recovered and recycle to the algal cultivation process. Anaerobic digestion can mineralize and recycle nitrogen and phosphorus as fertilizer or other valuable products (Sialve et al., 2009). This research aim is to identify scalable anaerobic digestion strategies that enable biogas production and nutrient recovery from spent algae. Two types of algal substrate for anaerobic digestions were investigated: a fresh water species commonly proposed for algal biofuels (Chlorella) and a marine species (Cyclotella) that could be used in approaches to reduce water demands or produced unique coproducts.

2 Methods
2.1 Collection and characterization of substrate and inoculum
The seed Chlorella vulgaris strains (UTEA 2714) in this study are obtained from University of Texas Culture Collection of Algae. BG-11 medium was inoculated with cells in 100 ml flasks with orbital shaking. The cells were then grow in 1 L polycarbonate tubular photobioreator (PBR) (3.8 cm of diameter) for 11-14 days. The columns are illuminated with indoor 16/8 hours period light at 50 μmol m-2s-1. Chlorella culture were grown at 21 °C and aerated with an air flow rate of 0.2 L min-1. Due to pH increase during algae cultivation, pure CO2 was provided to the PBR to lower the pH to 8. Algae productivity was 0.13 ± 0.07 g/l. During the first 1 to 2 days, the cells adapt to growth conditions and grew slowly. Algae reached concentrations of 1.1 ± 0.2 g/l at 9 to 10 days. At stationary phase, algae concentrations remained approximately constant. The initial cultivation contained 17.6 mM of nitrate that was depleted in about 12-13 days. At Chlorella harvesting, all of the nitrogen and phosphorus nutrients were consumed by algae.
The seed Cyclotella sp. are marine diatom that were also obtained from the University of Texas Culture Collection of Algae. Cyclotella was cultivated with Harrison’s and Guillard’s f/2 enrichment Artificial Seawater Medium and grow at the same environmental conditions as Chlorella vulgaris.
The anaerobic inoculum was obtained from the anaerobic digester at the Corvallis wastewater treatment plant in Oregon (in US). The mean concentration for total solid (TS) and volatile solid (VS) content in TS are 2.1 g/L and 60% in the inoculum.

2.2 Analytical methods
Algae substrate was mixed with specific amounts of anaerobic inoculum to obtain a different loading rates of algae. The reactors were placed in a constant temperature at 30 °C in incubator and constantly agitated by a shaker. The experiment involved two series of samples. One set contains different loading rates of lipid-extracted Chlorella vulgaris from 0.21-0.62 mg-VS/reactor. Another set involved compare the biogas generation and nutrient transformation between lipid-extracted Chlorella vulgaris and lipid-extracted Cyclotella. Each digester case include triplicate of samples for biogas and nutrients test.
Gas chromatography equipped with thermal conductivity detector was used to determine the content of methane. Biogas volume was measured with dry 20 ml Micro-Mate glass syringe. Total solids and volatile solids were determined according to standard methods. Total nitrogen and carbon was measure by elementary analyzer. Total phosphorus was assayed using phosphor-vanadomolybdate method. Soluble nitrate, nitrite and phosphate are measured by ion chromatography. Sample from the digestion were also collected for the measurement of COD, pH, alkalinity and ammonium.

3 Results and Discussion
In the first set of experiments, lipid extracted Chlorella had a loading rate range from 0.21 to 0.62 mg-VS/reactor with same amount of inoculum in the reactor. The ratio for inoculum/substrate ranged from 1.9 to 5.8. For 15 days of retention time, reactors has the same methane yield of 0.16-0.21 L/g-VS and methane content of 60-64%. Results show that inoculum/substrate ratio is not low enough to cause any digestion inhibition. For 30 days of digestion, methane yield is 0.20-0.23 L/g-VS with more algae digested. Methane content in biogas range from 61 to 65% which is same as 15 days retention time. For 30 days of anaerobic digestion, 67-74% of biogas and methane are generated from first 10 days of digestion. However, for competitive digestion process, high loading rate and short retention time will reduce the economic cost.
Based on mass balance of carbon, 80-100% of total carbon is converted to biogas and 46-56% of VS in algae is digested to biogas in 15 days. During anaerobic digestion, the solid from both inoculum and algae release nitrogen as ammonium which was recoverable in solution. Liquid phase nitrate and nitrite concentration did not change in reactor. For 30 days of digestion, about 12-15% of nitrogen in solid was transformed to ammonium. During digestion, more phosphate was released into the liquid phase with higher algae loading rate and more biogas formed. However, there is no specific events to show whether inoculum or algae is the source for phosphate.
In the second set of experiment, with same amount of VS loading rate, Chlorella and Cyclotella biomass have the similar methane yield for 15 days of digestion. Methane yield is 0.15-0.21 L/g-VS for Chlorella sp. and 0.16-0.17 L/g-VS for Cyclotella, respectively. However, Cyclotella has more biogas generation than Chlorella during the first week of digestion. One reason is due to the processes of harvesting and drying of Cyclotella may lyse and disintegrate the cell resulting in efficient digestion. After about one week, Cyclotella generates less biogas than Chlorella. This is expected as Cyclotella contains less carbon than Chlorella. Studies shows that Cyclotella contains 34% of carbon in VS, while Chlorella has 51% of carbon in VS. With same loading rate in anaerobic digestion, ammonium transformation rate are same for both algae.

3 Conclusion
This project is to investigate nutrient (nitrogen and phosphorus) transformation and recovery, carbon transformation and biogas production on batch anaerobic digestion of lipid extracted Chlorella vulgaris and Cyclotella sp. biomass. Methane yield for lipid extracted Chlorella algae is about 0.17 L/g-VS for 15 days retention time and 0.22 L/g-VS for 30 days retention time. Although with different carbon content, Chlorella vulgaris and Cyclotella sp. generated similar amount of biogas and methane volume. Based on mass balance of carbon, results show that 80-100% of total carbon is converted to biogas and 51% of carbon in algae is digested to biogas in 15 days. With higher algae loading rate, more nitrogen and phosphorus are transformed to ammonium and phosphate respectively in solution. The results from this study will allow the incorporation of anaerobic digestion into algal biorefinery techno-economic models for nutrient recovery and excess non-lipid biomass utilization.