RFDMW, SFDMW, and SM
FDMW was collected from three manure storage lagoons located in three different dairy farms in Merced, Glenn, and Tulare Counties of California, USA. These dairy farms house ≈3000–5000 dairy cows including both milking and non-milking cows. In these dairy farms, the FDMW passes through a solid separator before entering into the lagoon. Once collected from the lagoon, the FDMW was stored at 4 °C prior to starting the experiment. The FDMW average total nitrogen (TN), total solid, carbon, and pH were 2950 (± 429) mg/L, 1.27 (± 0.74) %, 0.36 (± 0.20) %, and 7.7 (± 0.05), respectively. Total phosphorous (TP) of FDMW in wastewater of similar lagoons in the same regions are reported to vary from 141 to 3263 mg/L (with median of 972 mg/L) (Pettygrove 2010). The FDMW was centrifuged (ThermoFisher Sci.: Sorvall Legend X1R) at 10,000 rpm for 15 min. Subsequently, the supernatant was used as RFDMW feedstock for growing C. vulgaris. The TN and TP of initial RFDMW were 156.4 and 12.7 mg/L, respectively. RFDMW was sterilized at 121 °C for 15 min to inactivate manure-borne microbial population, and this sterilized manure was used as SFDMW feedstock for growing C. vulgaris. The TN and TP of initial SFDMW were 56.6 and 12.7 mg/L, respectively. Established procedures (APHA 1999) were used for observing TN and TP.
To test the effect of supplementation with SM on C. vulgaris, we used a blue-green medium (BG-11), a recipe commonly used for growing freshwater algae including C. vulgaris (FACC 2014; UTEX 2014). The BG-11 (i.e., SM) was prepared by mixing 958 mL of distilled water, NaNO3 (0.25 g), K2HPO4·3H2O (0.075 g), MgSO4·7H2O (0.075 g), CaCl2·2H2O (0.025 g), KH2PO4 (0.175 g), NaCl (0.025 g), 40 mL of soil extract solution, FeCl3·6H2O (0.005 g), 1.0 mL of Fe-EDTA solution, and 1.0 mL of A5 solution. The SM was autoclaved and stored in 4 °C before using it for growing C. vulgaris. To prepare soil extract solution for mixing into SM, we used 200 g unfertilized garden soil and 1000 mL distilled water, heating in a water bath (at 100 °C) for 3 h, and then cooling for 24 h. Then the solution was filtered (0.45 µm) and supernatant was used as a soil extract solution. The Fe-EDTA solution was prepared by mixing 50 mL distilled water, Na2EDTA (1.0 g), FeCl3·6H2O (81 mg) and 0.1 N HCl (50 mL). The composition of the A5 solution was H3BO3 (2.86 g/L), MnCl2·4H2O (1.86 g/L), ZnSO4·7H2O (0.22 g/L), Na2MoO4·2H2O (0.39 g/L), CuSO4·5H2O (0.08 g/L) and Co(NO3)2·6H2O (0.05 g/L).
Experiment design
The growth of C. vulgaris was assessed in RFDMW, SFDMW, and SM using 500 mL conical flasks under controlled temperature conditions (25 ± 1 °C). The strain of C. vulgaris (UTEX-2714) was obtained from the culture collection of algae, University of Texas, Austin, USA. The pre-cultured C. vulgaris (OD 680 ≈ 0.355) was inoculated into a 300 mL volume of medium (in 500 mL conical flasks) with a proportion of 20 % (v/v) under sterile conditions. In order to avoid the potential ambient contamination, the experiments were conducted in a biological controlled environment (i.e., inside a bio-safety cabinet level II (SterilGARD Hood, Baker Company)). The bio-safety cabinet was converted into a photo-bioreactor by equipping it with controlled light (two 4 ft. T12 40-w Cool White Supreme (4100 K) Alto Linear Fluorescent Light Bulb with brightness of 2600 lumens) and a temperature control facility. The temperature of bio-reactor was controlled using a heating/cooling tower (Dyson-AM09 Fan, Model: 302198-01) equipped with a sensor for controlling heating and cooling precisely. The growth of C. vulgaris in RFDMW, SFDMW, and SM was monitored over 10 days at 25 ± 1 °C. The growth experiment was conducted in dark (12 h) and light (12 h) cycle conditions using 300 mL of growth media in 500 mL conical flasks. The experiment was continued to 30 days, but no increase in cell density was observed beyond 10 days. Previous studies have used a similar growth period of 10 days for assessing the growth of algal biomass in various wastewater sources (Hena et al. 2015; Kothari et al. 2013; Passero et al. 2015). The light and dark conditions were controlled using an electric timer (CUTNSTK624, Prime). To mix the growth environment, intermittent shaking was performed twice a day (by hand) for the first 6 days of cultivation.
The growth of C. vulgaris in RFDMW and SM was assessed for 10 days. During the 10 day cultivation period, samples of C. vulgaris were collected daily for biomass analysis. Biomass analysis was used to compare the growth of C. vulgaris in RFDMW and SM. Subsequently, a series of experiments was conducted to determine the effect of supplementing RFDMW with SM. Three mixtures with RFDMW and SM ratio (volumetric basis) of 20:80, 40:60 and 70:30 were used to evaluate the effect of C. vulgaris biomass production. To assess the impacts of animal waste-borne microbial population on C. vulgaris growth, we compare the growth of C. vulgaris in RFDMW and SFDMW in identical growth conditions. Further, a series of experiments (as described previously for RFDMW) was conducted to identify the optimal growth environment for SFDMW feedstock.
Algal growth and biomass
The growth of C. vulgaris was monitored by measuring the OD at a wavelength of 680 nm using previously published approaches (Mulbry and Wilkie 2001; Mulbry et al. 2008; Wang et al. 2010). Colored dissolved organic matter (CDOM) occurs naturally in wastewater because of tannins released from decaying matter. Both CDOM and chlorophyll a absorb in the same spectral range, which poses challenges in differentiating absorbance caused by chlorophyll a and wastewater. In order to resolve this issue, we have used controls of each level of RFDMW, SFDMW, and SM prior to measurement. First, the OD of these controls were measured and zeroed, and then the OD of actual sample was measured. This process resolved the differentiation issue of CDOM and C. vulgaris optical density.
For algal biomass analysis, a 10 mL sample volume was centrifuged at 8000 rpm for 10 min, and the centrifuged pellets were washed twice with distilled water to remove the salts and solids. Subsequently, each pellet was resuspended in distilled water and filtered through a 47 mm membrane filter (HAWG047S6, Millipore). The C. vulgaris biomass retained in the filter was dried overnight at 60 °C and the final biomass weight was measured. An empirical equation (Eq. 1) was developed (R2 = 0.98) for calculating the biomass using OD 680 readings where BM
d
is biomass dry weight (g/L).
$$ BM_{d} = 0.3386 \cdot OD680.$$
(1)
In addition to BM
d
, volumetric biomass productivity (P
b
) (g/L/d) and specific growth rate (µ) (1/d) were estimated using the reported methods (Blair et al. 2014).
$$P_{b} = \frac{{X_{2} - X_{1} }}{{t_{2} - t_{1} }}$$
(2)
$$\mu = \frac{{\ln \left[ {X_{2} /X_{1} } \right]}}{{t_{2} - t_{1} }}.$$
(3)
where, X
1
and X
2
are the biomass concentration (g/L) on days t
1
and t
2
, respectively.
Ultrastructure analysis of C. vulgaris using TEM
To understand the impacts of RFDMW, SFDMW, and SM on the ultrastructure of C. vulgaris, we used TEM analysis. The pellets of C. vulgaris were fixed in 2 % paraformaldehyde + 2.5 % glutaraldehyde in 0.1 M sodium phosphate buffer. Subsequently, the pellets were rinsed in buffer and then fixed in 2 % OsO4 in the same buffer for 1.5 h. The samples were then dehydrated in a graded series of acetone in PBS (10, 30, 50, 70, and 90 %) for 10 min at each level of acetone. Subsequently, at 100 % acetone, samples were dehydrated for 30 min. A mixture of acetone and resin (1:1) was used for 1 h resin infiltration, which was followed by overnight infiltration with 100 % resin. The next day, fresh resin (100 %) was used for a 2 h infiltration before final embedding and polymerization. Ultrathin sections (50 nm thick) were cut using a Diatome diamond knife and picked up onto copper (carbon coated) grids (200 mesh) then stained with 0.5 % uranyl acetate for 2 h and 3 % lead citrate for 5 min before viewing in a Philips CM120 electron microscope. An accelerating voltage of 80 kV and magnification of 13.90 kx were used for examining the specimen.
