2.1 Microalgae cultivation
A microalgae consortium was bioprospected from a water body (pH 2.8) on an active mine site and identified to be predominantly (91%) a single acid-tolerant Coccomyxa sp. taxon while grown at pH 2.5 based on high throughput SSU rRNA gene amplicon sequencing on the Illumina MiSeq platform using the 18S primer set and methods described in Bradley et al. . The remaining 9% was made up of other green microalgae, protozoa and fungi. The microalgae were inoculated in 2 L flasks (triplicated) containing 1.2 L of acidified (pH 2.5) Bold’s basal media (BBM; ) with a starting density of approximately 100 mg L-1 dry weight biomass. The pH of the flasks was lowered from neutral pH using 1 M sulfuric acid and measured daily using a calibrated Oakton™ pHTestr™ 30 (Fisher Scientific; CAT#13200263). The flasks were placed on a gyratory shaker (Model G2, New Brunswick Scientific Co.) at 125 rpm and maintained under red and blue LEDs (approximately 22 mmol s-1 m-2) with a 12-hour light/dark cycle. A simulated flue gas mixture of 6% CO2 and 94% N2 was continuously bubbled through the flasks for eight hours, starting at the beginning of the 12-hour light photoperiod. This mixture, along with the pH modification was used to simulate acidic flue gas produced by a nickel smelter.
Daily biomass productivity was measured based on the daily increase of dry weight biomass throughout the growth period and expressed as mgdw L-1 day-1. Average biomass productivity (mgdw L-1 day-1) was measured based on the increase of dry weight biomass from the beginning of the growth period to the end of exponential phase, and specific growth rate (day-1) calculated using Eq. (1):
where X1 and X2 are the biomass densities (mgdw L-1) at the beginning and end of the exponential phase, respectively, t1 and t2 are the time in days at which exponential phase begins and ends, respectively.
Individual samples for lipid analysis (duplicated) and elemental analysis (duplicated) were removed from the flasks once the highest density was achieved (end of exponential phase), to be used as controls. The CO2 biofixation rate was calculated for the control sample using Eq. (2):
where P is the average biomass productivity, Ccarbon the carbon content (fractional), which was determined by elemental analysis (described in section 2.4), MCO2 the molecular weight of CO2, and MC the molecular weight of carbon.
2.2 Microalgae under light-dark and dark conditions
After exponential phase had been reached under the same growing conditions as previously mentioned (1.2 L of BBM, 12-hour light/dark cycle, pH 2.5, 6% CO2), the biomass experienced either light-dark or dark treatments. During light-dark treatment, the flasks remained on the gyratory shaker and the biomass after the exponential phase was kept under 12-hour light/dark cycle with 6% CO2 for 7 days. Individual samples for lipid analysis (duplicated) and elemental analysis (duplicated) were removed on days 1, 2, 3, 4, and 7. These samples represent ordinary conditions for cells during stationary and/or decline phase, and were used as comparison against the dark-treated cells. All samples were stored at -80°C until the biochemical and elemental analyses were conducted.
During dark treatment, biomass obtained after the exponential phase was placed on a gyratory shaker at 125 rpm under constant darkness for 7 days. Unlike the flasks exposed to the normal 12-hour light/dark cycle, these flasks did not receive any CO2 since photosynthesis does not occur in the dark for photosynthetic green microalgae. After the flasks were placed in the dark, individual samples for lipid analysis (duplicated) and elemental analysis (duplicated) were removed from the flasks on days 1-4, and 7 and stored as noted above.
2.3 Lipid and elemental analysis
For lipid analysis, frozen, wet samples were sent to Lipid Analytical Laboratories Inc. at the University of Guelph, Ontario, Canada. Lipids were extracted based on the Bligh and Dyer method  using tridecanoin and methyl tricosanoate (NuChek Prep, Elysian, MN, USA). The total lipid extract was quantified via transmethylation  to produce fatty acid methyl esters (FAMEs). Analysis of FAMEs was completed using an Agilent 7890B gas-liquid chromatograph (GC-FID) with a 60 m DB-23 capillary column (internal diameter of 0.32mm). Determination of the fatty acids was carried out by a 90-110% match of the peak area of the known internal standards from Sigma-Aldrich (Oakville, ON, Canada) and Nu Chek Prep.
For elemental analysis, samples were lyophilized and ground down using a mortar and pestle before being sent to the Perdue Central Analytical Facility at Laurentian University, Ontario, Canada, for analysis. Vanadium pentoxide was added to samples weighing 3 – 3.5 mg to help facilitate sulfur combustion, and a Thermo ScientificTM FlashSmartTM elemental analyzer with a 2m PTFE column was used to determine the relative abundance of C, H, N, and S in the biomass. Calibration curves were created using BBOT (2,5-Bis(5-tert-butyl-benzoxazol-2yl)thiophene), and cysteine was used as a certified reference material to evaluate recovery.
Data presented are the averages ± standard errors within their respective technical replicates. Statistical analysis of the data was conducted in R , and all graphical representations of the data were created using the R package ggplot2 . The statistical significance threshold was placed at 0.05.