Results and discussion
The phototrophic microalgal chemostat enrichment cultures reached a
steady-state for each nitrogen loading rate formerly detailed analysis
was performed. The nitrogen concentration in the effluent dropped below
the method detection limit for the NLRs of 10, 20, 32.5 and 42
mgN.l-1.d-1 which indicates that
nitrogen supply limited growth. Therefore, these NLRs are considered
nitrogen-limited conditions. On the other hand, the increase of the NLR
to 63
mgN.l-1.d-1 caused an incomplete
nitrogen uptake. At the NLR of 63
mgN.l-1.d-1 and in the presence of
other nutrients (the phosphate concentration was measured every day,
data are not shown), mixed microalgae could not consume all nitrogen
supplied to the reactor, which indicates a different growth limitation
in the system rather than nutrient deficiency. This was likely a light
limitation which is attributed to the high biomass concentration that
limits the light penetration into the microalgal culture. Therefore, NLR
of 63 mgN.l-1.d-1 is considered as
light-limited condition (for more explanation, see supplementary data
1). Similarly, Klok et al. (2013) reported that when residual nitrogen
was observed in the effluent of Neochloris oleoabundanscontinuous culture system (0.066±0.002 g.l-1 at the
dilution rate of 1.15 ± 0.10 d-1), the growth was
limited by the light supplied to the system . At lower nitrogen supply
rates, residual nitrate was non-detectable, indicating nitrogen-limited
growth conditions.
Effects of Nitrogen Loading Rates on Present
Species
Microscopic observations of species grown under the five nitrogen
regimes are represented in Figure 2. According to the observed
microscopic morphology, a coculture of species including Chlorella
sp. , Arthrospira platensis, and Scendesmus was identified
at both NLR of 10 and 20 mgN.l-1.d-1(Figure 2. A & B). Chlorella sorokiniana and Chlorella
vulgaris were identified as dominant species at
NLR of 32.5 and 42
mgN.l-1.d-1, respectively (Figure 2.
C & D). Different species ofArthrospira platensis , Chlorella sp. , Rhopalodiacoexisted under the light-limited condition at
NLR of 63
mgN.l-1.d-1 (Figure 2. E). Genomic
DNA was extracted to confirm the identity of microscopically observed
species. The amplicons of extracted DNA were amplified by 18S rRNA and
16S rRNA. According to the 16S
rRNA and 18S rRNA sequences, the morphologically recognized microalgal
species were closely related to PCR-DGGE identified species (Table 2).
Besides some minor species were identified by the amplicon sequencing.
The coexistence of different species at NLRs of 10, 20, and 63
mg.l-1.d-1 can be illustrated by
biodiversity neutral theory which contends that diversity is due to the
equivalent competitive ability of all species within the same functional
group.
Table 2.
Regan et al. (1984) investigated the dominant microalgal species under
different nitrogen and light limitations in continuous culture and
reported dominance and coexistence of various species including diatoms,
blue-green algae, and green flagellates. For dilution rates of 0.1-0.4
d-1, at low inlet nitrate concentrations (7.5
mgN.l-1), pennate diatoms (Nitzschia) were dominated
(Regan and Ivancic, 1984). However, at higher inlet nitrate
concentrations (37 mgN.l-1), pennate diatoms
(Nitzschia) and green flagellates (Tetraselmis) coexisted (Regan and
Ivancic, 1984). Similarly, an N-limited chemostat culture was dominated
by picocyanobacteria and a diverse group of diatoms using multispecies
inoculum. In our study, cyanobacteria existed in F5culture in which their presence may be attributed to their capacity to
produce accessory pigments to harvest light more efficiently under light
limiting conditions . This result also supports the suggestion that
cyanobacteria can be introduced as shade organisms. This characteristic
of cyanobacteria helps them compete with other species present in
culture under light limitation. Aligned with this, coculture of
different species of cyanobacteria, green algae and diatoms were
enriched at the nitrogen loading rates of 20 and 10
mgN.l-1.d-1 (Table 2), which may
conclude that the decrease of nitrogen loading rate and higher light
availability also leads to the growth of nitrogen-fixing algae such as
cyanobacteria in the algal community. In fact, the specific nature of
cyanobacteria’s water-soluble light-harvesting antenna complexes,
phycobilisomes (PBs), dictates their photoprotection mechanisms. PBs
gather light in a wide spectral range and transfer excitation energy to
the photosystems. Cyanobacteria use the photoactive orange carotenoid
protein (OCP) to control this energy flow . Diatoms species ofNavicula sp ., Nitzschia thermalis, and Rhopalodia
gibba could stay in the competition of F1,
F2, and F5 cultures, respectively. It
can be pointed that diatoms contain the accessory pigments Chl
c1 + c2, and the xanthophyll,
fucoxanthin. Light energy absorbed by fucoxanthin utilized in
photosynthesis with the same quantum efficiency as Chl a which makes
diatoms be adaptable to low light growth conditions (NLR of 63
mg.l-1.d-1) (Tanada, 1951; Friedman
and Alberte, 1984). Another study explained in detailed the
photoprotection general features of diatoms exposed by harmless
dissipation of excess energy which can be the purpose of diatoms
presence at NLRs of 10 and 20
mg.l-1.d-1 .
Based on the microscopic images (Figure 2, right pictures which are
under fluorescence light), the accumulation of lipids is obvious at NLRs
of 32.5 and 42 mgN.l-1.d-1, which
are related to Chlorella sorokiniana and Chlorella
vulgaris , the dominant species of F3 and
F4 culture, respectively.
Figure 2
Effects of Nitrogen Loading Rates on Cell Growth, Lipids and
Starch
Accumulation
Figure 3 illustrates the consumed nitrogen to dry weight and active
biomass ratios for the NLR of F1 to F5cultures. Active biomass is calculated by subtracting the values of ash,
lipid, and starch from dry weight. The ratios of consumed nitrogen and
biomass dry weight are approximately identical throughout the tested
experimental conditions (F1 to F4). This
may be attributed to the fact that the amount of nitrogen that is
assimilated per produced biomass is approximately constant for these
experiments. However, the consumed nitrogen to dry weight ratio for the
nitrogen loading rate of 10
mgN.l-1.d-1 dropped considerably
which indicates that the diatoms and/or cyanobacteria fix nitrogen (See
Figure 3). Moreover, the approximately identical pattern was noticed for
the consumed nitrogen to active biomass ratio (Figure 3).
Figure 3.
The dry weight concentration was measured to be 342 ± 11
mg.l-1 at a NLR of 10
mgN.l-1.d-1 and increased
approximately linearly to 868 ± 12 mg.l-1 at NLR of 42
mgN.l-1.d-1 (Table 3). However, the
microalgae growth was limited by light supply for the nitrogen loading
rate of 63 mgN.l-1.d-1 in which dry
weight slightly increased to 895 ± 14 mg.l-1.
Table 3.
According to Table 3, the content of lipids increased from 5.5 ± 0.3%
to 14.0 ± 0.8% on the basis of dry weight, simultaneous with increasing
NLR from 10 to 42 mgN.l-1.d-1 up to
where light became the growth limiting factor. The highest amount of
lipids were measured at NLR of 42
mgN.l-1.d-1, which is confirming the
observation of lipid bodies by fluorescence microscopy (Figure 2).
Contrary to our findings, Klok et al. reported the increase of lipids
bodies inside of N. oleoabundans with a decrease in the nitrogen
supply rate. Apparently, the lipid enhancement can be attributed to the
decrease of growth rate which was implemented by dilution rate reduction
in the experiments of Klok et al. (2013). Another pure culture study
indicated a higher accumulation of lipids in C. subellipsoidea at
lower nitrate addition rates, which was similarly induced by a decreased
dilution rate . There was no significant difference in the starch,
lipids and biomass concentration of F4 and
F5 at 5% significant level (P-value>0.05).
The highest lipids and starch concentration 121.2 ± 1.7
mg.l-1 (14.0 ± 0.8 % on the basis of dry weight), 42
± 4.5 mg.l-1 (4.8 ± 0.5 % of dry weight),
respectively were achieved under nitrogen limiting conditions of
experiment F4 (Table 3). The results confirm the
hypothesis that chemostat cultures under nitrogen limiting conditions
can lead to the accumulation of lipids and therefore it can be a good
strategy to enrich the lipids accumulating algal communities.
Interestingly we obtained significant lipids accumulation in our
enrichment culture. This was not observed for algal enrichments under
dynamic cultivation in a day-night cycle . Xiao et al. (2013) culturedNannochloropsis oceanica using a medium with different nitrogen
concentrations at constant dilution rates fed to a photobioreactor. They
revealed that nitrogen limitation induced by a continuous medium with a
nitrogen concentration of 0.23
mgN.l-1.d-1 at a dilution rate of
0.27 d-1 showed the highest lipids accumulation inNannochloropsis oceanica cells up to 56.17 ± 0.21 % of dry
weight with a value of 179.26 mg.l-1.
Other studies were based on decreasing the nitrogen feeding rate by
reduction of dilution rate of pure culture studies such asNeochloris oleoabundans , Chlorella pyrenoidosa , andCoccomyxa subellipsoidea . Klok et al. (2013) reported the
accumulation of 14.8% total fatty acid on the basis of dry weight inNeochloris oleoabundans at a dilution rate of 0.33
d-1. Another report revealed that Coccomyxa
subellipsoidea under the lowest nitrate loading rate of 64.7
µg.l-1.h-1 with a dilution rate of
0.06 d-1 resulted in the highest lipids accumulation
up to 30.7±1.3 mg.l-1 . Moreover, Wen et al. (2014)
observed that the lipids content of Chlorella pyrenoidosa under
low specific nitrogen loading rate of 7.9
mg.l-1.d-1 at a dilution rate of
0.48 d-1 was 34.7 % of DW. The main and notable
advantage of this study is that the lipids and starch are measured in
entire experiments which provide the chance to conclude that continuous
mode cultivation resulted in higher lipid accumulation than the starch
synthesis for all NLRs. Therefore, lipids are considered as dominant
storage compounds in our chemostat enrichment system. The highest ratio
of lipids to starch concentration was calculated for experiment
F3 with a value of 4.2. It corroborates the results of
the continuous growth of Nannochloropsis sp. in which the content
of lipids was doubled under nitrogen limitation . However, the greater
increase of carbohydrates than lipids occurred for Scenedesmus sp.
AMDD under nitrogen-limited continuous culture .
For Chlorellasorokiniana andChlorella vulgaris , the dominant species of experiment
F3 and F4, the trade-off between starch
and lipids accumulation was reported . The lipids and starch contents ofChlorella sorokiniana were reported 7.2% and 16.2% on a dry
weight basis in a complete medium batch mode cultivation. The latter
species could increase simultaneously starch and lipids content to 40%
and 30% of DW, respectively under batch sulfur-deficient condition .
Another study revealed that under nonexistence of nitrogen and moderate
aeration and light intensity in batch culture, the lipid content
increased in Chlorella vulgaris cells up to 43% of dry cell
weight which equates to lipids productivity of 77.8
mg.l-1.d-1.
Table 4.
The starch content of experiment F4 in whichChlorella vulgaris was dominant decreased to 4.8±0.5% of DW and
the lipids content increased up to 14.0±0.8% based on DW.Chlorella sorokiniana which was enriched at a nitrogen loading
rate of 32.5 mgN.l-1.d-1 had a
starch and lipids content of 3.2±0.2% and 13.5±0.9% on a dry weight
basis, respectively. Therefore, additionally, it can be concluded that
continuous mode can promote the enrichment of species which tend to
enhance the lipids content under unfavorable culture conditions . As
illustrated in Table 4, there is a significant positive relationship
between the NLRs, biomass, and lipids productivity
(mg.l-1d-1) of F1 to
F4 culture. Furthermore, a comparison between the
biomass and lipids productivity of continuous pure microalgal production
and that of our study revealed that mixed microalgae could provide
admissible biomass and lipids productivity without the high expense of
sterilization (Table 4). Mazzuca Sobczuk and Chisti (2010) reported the
biomass and lipids productivity of 351 and 83
mg.l-1.d-1 for Choricystis
minor at a dilution rate of 0.336 d-1. Another study
showed the maximum biomass productivity of 242.2
mg.l-1.d-1 and 82.5
mg.l-1.d-1 lipids productivity for
the continuous production ofNannochloropsis oceanica .
In the latter mentioned study, the nitrogen supply rate was 8.3
mgN.l-1.d-1 with a dilution rate of
0.27 d-1. The maximum biomass and lipids productivity
obtained in this study were 433.9 and 60.6
mg.l-1.d-1, respectively which are
quite high. However, the process conditions optimizing are required
which may result in higher lipid content, comparable to those reported
for pure culture.