Materials and methods
Feed composition
Different ecological water-bodies of Netherlands ponds were inoculated
in chemostat feeding medium with composition as follows (concentration
in mg.l-1): MgSO4.7H2O (394.4); KCl (18.2);
K2HPO4 (156.8);
CaCl2.2H2O (22.0);
FeCl3.6H2O (38.0);
NaEDTA.2H2O (100.5);
H3BO3 (46.4);
NaSiO3.9H2O (85.3) and 1 ml/L trace
element solution. Except for the nitrogen source, all other nutrients
concentrations were adjusted in a way to ensure the nitrogen limitation
was the only possible nutrient limitation occurred in all experiments.
For different NLRs, the concentration of NaNO3 is shown
in table 1.
The final trace elements composition in medium for all different
nitrogen feeding regimes was as follows (concentration on µmol/L):
ZnSO4.7H2 O (1.7); MnCl2.4H2O (19.3); CuSO4.5H2O (0.1); CoCl2.6H2 O
(1.1); NaMoO4.2H2O (2.0);
Na2SeO4 (0.3);
Na3VO4 (0.02).
Table 1
A non-sterile 2 L bioreactor (Applikon, Schiedam, the Netherlands) was
used in a continuous mode for three months with average light intensity
at photobioreactor’s inner surface 375
µmol.m-2.s-1 provided by an LED
lamp. The working volume of 1.5 L was used for all nitrogen loading
rates. The pH of the culture was maintained at 7.5 using 1 M HCl and 1 M
NaOH. The medium was aerated with enriched CO2 (5%, v/v
in N2) at a flow rate of 46 ml.min-1controlled by a mass flow controller (Brooks Instruments, Ede, the
Netherlands). The outflow gas stream was cooled to 4 °C and sent to the
Rosemount NGA off-gas analyzer (Emerson, USA). The speed of stirrer and
temperature were maintained at 350 rpm and 26 °C, respectively.
Masterflex pump (Cole-Parmer, Vernon Hills, IL, USA) with two head was
used at inflow and outflow rate of 0.52 ml.min-1 to
provide the constant dilution rate of 0.5 d-1 and HRT
of 2 d. A Biocontroller ADI 1030 (Applikon, Schiedam, The Netherlands)
continuously measured pH and DOT (Dissolved Oxygen Tension). All data
were stored by PC with MFCS_win software (Sartorius Stedim Systems,
Goettingen, Germany). A schematic overview of the experimental setup is
shown in Figure 1.
Figure 1
Photobioreactor operation
First, the fresh medium of F1 was inoculated as
described in the Materials and methods section. The initial OD was 0.05
and the photobioreactor was run in batch mode for two days to reach the
OD of 1.8. Then the fresh medium with the composition of
F1 was supplied to the reactor continuously to reach the
steady-state point. Off-gas compositions and the biomass concentration
were used for monitoring the steady-state condition, in which the
variation of these parameters should be less than 10% for three
hydraulic retention times. The different nitrogen loading rates were
applied by supplying different nitrate concentrations in the fresh
medium, F1, F2, F3,
F4, and F5.
Analytical methods
Samples were collected from the photobioreactor every day. All
experiments were conducted in triplicate. Data were analyzed by an
analysis of variance (P<0.05) and results were processed by a
computer program: Excel software.
Nitrate measurement
The content of nitrate in the reactor was measured
spectrophotometrically using Dr. Lange LCK 339 Nitrate cuvette tests
(Hach Lange, Dusseldorf, Germany) .
Biomass measurements
The effluent from the reactor was collected in an ice bath for measuring
biomass dry weight (DW). 300 ml of microalgae suspension was
centrifuged, the supernatant was removed and the pellet was dried
overnight in pre-weighed aluminum foil container at 104 °C. The optical
density was measured at the wavelength of 680 nm every day. Biomass
productivity was calculated by the following equation:
Biomass Productivity (mg.l-1.d-1) =
DW (mg.l-1) ×D (d-1) (1)
where DW and D are dry weight and dilution rate, respectively.
Lipids and starch
analysis
The content of Lipids was measured following the PHB extraction protocol
described by Johnson et al. with minor modification. Formaldehyde was
not added to the samples and Myristic, palmitic, palmitoleic, stearic,
oleic, linoleic and linolenic acid were applied as standards. Into
digestion tubes, 50 µl of internal standard benzoic acid in 1-propanol
(1 g in 50 mL), 1.5mL of 1-propanol: hydrochloric acid (4:1 v/v) and 1.5
ml of 1,2-dichloroethane were added to weighed freeze-dried biomass. The
tubes were placed in a digester block at 100 °C for 2 h. Gas
chromatography (model 6890N, Agilent, USA) equipped with a flame
ionization detector on an HP Innowax column was used for extracted
lipids analysis. For starch measurement, approximately 4-mg freeze-dried
biomass heated with 0.6 M HCl for 3 h at 100 °C. After centrifugation
and filtration with a 0.45-μm pore size filter (PVDF Membrane,
Millipore, Ireland), the poly glucose concentration was measured by
high-performance liquid chromatography with the specification of an
Aminex HPX-87H column from Bio-Rad (USA) (t=60 °C) coupled to an
ultraviolet and a refractive index detector . The productivity of starch
and lipids are calculated according to the following equations:
Starch Productivity (mg.l-1.d-1) =
Starch Content(mg.l-1)×D(d-1) (2)
Lipid Productivity (mg.l-1.d-1) =
Lipid Content (mg.l-1)×D(d-1) (3)
Staining of microalgal
cells
4 μl BODIPY 505/515 (4,4-difluoro-1,3,5,7-tetramethyl-4-
bora-3a,4a-diazasindacene) in anhydrous dimethyl sulfoxide (DMSO; 1
mg.ml-1) was added to 0.2 mL of algal culture for
visualizing the lipids droplets in the cells by Leica DM500B light
microscope (Leica Microsystems, Wetzlar, Germany) equipped with
fluorescence filtercube A. The excitation and emission wavelengths for
monitoring BODIPY 505/515 fluorescence were 488 and 515 nm, respectively
as described by Klock et al. (2016). Starch staining was conducted by
adding 4µl of Lugol’s solution to 0.2 ml of microalgal suspension .
Different species present in the five NLR cultures were categorized
based on the morphological microscopic cellular appearance by taking
pictures with an above-mentioned microscope using 100 times
magnification. Identification was based on the morphology of the
individual cells following microscopic examination. The objects which
were not morphologically consistent with algae, such as debris,
bacteria, and particles were discarded.
PCR-DGGE Analysis
The microbial composition of chemostat enriched culture at different
nitrogen loading rates was analyzed using the PCR-DGGE technique as
described by Danesh et al. .