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Materials and Methods
Experiments were carried out using wastewater generated in Indian Institute of Science (IISc) Campus, Bangalore, India. The characteristics and features of the raw wastewater are summarized in Table 1.
Table 1: Physico-chemical Characteristics of domestic wastewaters used for experiments
| Physico-chemical Parameters |
Mean ± standard deviation (SD) |
| Total Nitrogen (TN) mg L-1 |
32±1.72 |
| Ammonium-Nitrogen (NH4-N) mg L-1 |
25±1.44 |
| Nitrate-Nitrogen (NO3-N) mg L-1 |
0.1±0.021 |
| Nitrite-Nitrogen (NO2-N) mg L-1 |
0.8±0.066 |
| Total Phosphorus (TP) mg L-1 |
18±1.22 |
| Ortho-phosphates (OP) mg L-1 |
16±1.45 |
| Total Organic Carbon (TOC) mg L-1 |
250±21 |
| Chemical Oxygen Demand (COD) mg L-1 |
660±64 |
| Total Solids (TS) mg L-1 |
1400±130 |
| Total Suspended solids(TSS) mg L-1 |
540±84 |
| Total Volatile Solids (TVS) mg L-1 |
344±66 |
| pH |
6.8±0.1 |
| Redox Potential (ORP) mV |
-120±34 |
Municipal wastewater used for Euglena sp. (cultivation): The values of Ammonium Nitrogen (NH4-N), Total Nitrogen (TN), Nitrate Nitrogen (NO3-N), Nitrite Nitrogen (NO2-N), Ortho-Phosphates (OP), Total Phosphorus (TP), Total Organic Carbon (TOC) in domestic wastewater culture broth were determined following the HACH protocols. Physico-chemical parameters were analysed as per standard protocol (APHA, 2005). C and N analysis were performed by CHN elemental analyser (LECO, True Spec CHNS). Domestic wastewater (sewage) used as the medium for culturing microalgae was first allowed to settle for several hours. Then the supernatant was sterilised, filtered and then used as the culture media (as per Zhou et al. 2012). All experiments were carried out in triplicate and mean with standard deviation were computed to understand variability.
Algal species culturing and growth conditions: Euglena sp. was isolated from the sewage collected from treatment firms (between 12.273681°-12.270031° N and 76.650737°-76.655947° E), at Mysore Karnataka and was maintained in the Bolds Basal (BB) media (Richmond. 2004). Inocula of the algae were prepared and then added to the algal reactors (10 L) (~106 cells mL-1; inoculum volume 20 mL). Reactors were kept on the rooftop under sunlight (12:12 h light/dark period) and cultures were mixed periodically. Algal cultures were maintained for 9 days at ambient temperatures and the growth was monitored. Everyday 100 mL of broth was collected and centrifuged, which was replaced with deionised water. The supernatant was used for analysis of nutrients and the algal pellet was washed repeatedly and was then used for dry weight estimation, total lipid content, spectroscopic and elemental analysis. Algal broth of 100 mL was centrifuged and pellets formed were then dried and weighed to assess the algal biomass (cell dry weight in the culturing medium, g L-1). The specific growth rate (µ) was calculated by equation 1, considering dry weight of cells during 9 days of culture:
µ (d-1) = (ln N/N0)/T2 –T1 …………………………..1
Where T2–T1 (day): Time between the two measurements,
N and N0 (g L-1): Concentrations of biomass at day T2 (next day for 8 days) and T1 (1st day),
The biomass productivity at the time of culturing is given by equation 2:
Bio. Prod. (g L-1 d-1) = (N-N0)/T …………………………2
Where N (g L-1): concentration of biomass at the end of the cultivation,
N0 (g L-1): concentration of biomass at the beginning, and
T: the duration of cultivation (9 days).
Biochemical Transitions through FTIR Analysis: Macromolecular biochemical compositions of algal cells were monitored through FTIR spectroscope (Alpha Bruker). Algal dry biomass after freeze drying were analysed with attenuated total reflectance through ATR-FTIR Spectroscope in the absorbance mode (range 1800-800 cm-1 wave numbers) with 128 scans at a spatial resolution of 2 cm-1. IR absorption spectra were collected for the daily monitoring. The data analysis was carried out with Origin Pro software with an initial base line correction, and scaled upto Amide I max (Stehfast et al. 2005). L/P and C/P required for biochemical composition assessment were derived from the area under the curve (Giordano et al. 2001; Murdock et al. 2009).
Lipid Measurements: The total lipids were determined by following Bligh and Dyer method (1959). Cell suspensions from cultures were centrifuged at 1162 g for 10 min. Then the algal pellet was stored at -20 ºC until use. The calculation of the Total lipid content is given by equation 3.
Lipid content Algae (g g-1) = Wt. (Lipids)/ Wt. (Dry biomass) ………………….3
The lipid productivity was given by the equation 4
Lipid Prod. (g L-1 d-1) = (LfN – L0N0)/T ..…………………4
Where L0 (g g-1): Lipid content in the beginning
Lf (g g-1): Lipid content at the end of the batch culture
N0 (g L-1): Biomass concentration at the beginning
N (g L-1): Biomass concentration at the end of the batch culture (8 days)
Fatty acid composition analysis (FAME): Disruption of cells was done by sonication using ultrasonic bath (frequency 35 kHz) for 30 minutes. Lipids were extracted with chloroform-methanol (2:1, v/v), and separated into chloroform and aqueous methanol layers by the addition of methanol and water to give a final solvent ratio (chloroform: methanol: water as 2:1.1:0.9). 20 mL of 5% NaCl solution was used for washing off the chloroform layer, and was then evaporated using a rotary vacuum evaporator with a water bath temperature of 60ºC. Methylation of lipids was performed using Boron-trifluoride-Methanol (BF3-MeOH) that converts all fatty acids to their corresponding Methyl esters. The methylated sample was loaded onto silica column with helium gas as carrier in split-less mode. The total run time was calculated to be 47.667 min. Fatty acids were identified by comparing the retention time obtained to that of known standards. The composition of the FAME was assessed through gas chromatograph mass spectrometry (Agilent Technologies 7890C, GC System; Agilent Technologies 5975C insert MSD with Triple-Axis Detector). Both the initial column temperature and the injection port temperature were maintained at 250ºC. Detector temperature was 280ºC, and this was increased to 300ºC at a temperature gradient of 10ºC min-1. The oven temperature was then raised to 230ºC at a ramp rate of 3ºC min-1 and finally it was raised to 300ºC at a ramp rate of 10ºC min-1 and this temperature was maintained for 2 minutes. The components were identified based on their retention time, abundance and fragmentation patterns by comparing with a known standard. These experiments were carried out in triplicate and average values are reported. Results were analysed using PAST 1.86b. Correlation tests, ANOVA analysis and Tukey’s post hoc analysis were used to determine the significance of difference wherever applicable.
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