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.