**5. Nutrients depletion and biomass yield in the pilot test**

This case study showed that the N and P residues (mg L<sup>1</sup> range) in the tertiary treated wastewater could be effectively removed by the inoculated co-culture (**Figure 3**). In the 1st and 2nd batches, most of NH4 <sup>+</sup> and P were depleted within 1 day. However, the depletion rate became slower in the 3rd batch. Moreover, the nutrient depletion followed a similar pattern. The depletion of NH4 <sup>+</sup> preceded the other inorganic N nutrients. Then, nitrate and nitrite started to deplete once NH4 <sup>+</sup> was close to 0. This is consistent with the previous reports that algae have a preference on inorganic N: NH4 <sup>+</sup> over other sources of NO3 [17, 18]. Regardless of the difference in 3rd batch, it seems that the total N could be reduced by 1.5 mg L<sup>1</sup> in 3 days. As this rate was achieved by the experimental amount of inoculated co-culture, perhaps the nutrient depletion could be accelerated with more co-culture inoculum.

Albeit of a similar amount of algae inoculum used in each batch (**Table 2**), their production was different. As there was a low level of nutrients (especially P) in the

**Figure 3.** *Macronutrients during WWTP effluent treatment with algal co-culture.*


#### **Table 2.**

*The summary of algae production and nutrient consumed in the pilot tests.*

treated wastewater, the exposed biomass was likely to confront prolonged P starvation with each subsequent batch. This is contradictive to findings that initial P-starvation can be implemented on microalgae to maximize the P uptake in wastewater [19]. This could happen in the 2nd pilot test but did not occurr in the 3rd batch. Although it was attempted to recycle the produced algal biomass for continuous effluent purification, the results support that the efficiency turned unsustainable after 14 days (duration of first two batches) even with CO2 supplement. It is thereby deduced that the fresh algae inoculum is imperative to the rapid nutrient recovery in municipal tertiary wastewater.

Overall, the biomass of co-culture increased by 330, 250, and 200%, respectively, in three consecutive pilot tests (**Table 2**). The obtained algae productivity was consistent with the reported range of 0.8– 50.0 g m2 d<sup>1</sup> on filamentous algae [4]. The results showed that the algae grew slower after day 3 in the first two pilot tests, when the N and P became deficient afterward (**Figure 3**). It is indicated that the real productivity of co-culture would be above the average level over 7 days. The effective *A New Insight of Phycoremdiation Study: Using Filamentous Algae for the Treatment… DOI: http://dx.doi.org/10.5772/intechopen.104253*

#### **Figure 4.**

*Microscopy picture of co-culture: A taken from the 1st batch pilot test on day 6. B taken from the 3rd batch pilot test on day 7. (*Spirogyra *sp.—Wider, spiral chlorophyll;* Klebsormidium *sp.—Thinner filaments; and air bubble—Empty round cycle).*

retention time for the wastewater reclamation could be less than 3 days as well. Obviously, the biomass yield can be elevated purposely by using an optimal amount of algae inoculum. Given the more biomass yield (vs both N and P consumed) in the first two batches (especially in the 1st cycle), this also underpins the above suggestion on fresh inoculum preparation for tertiary wastewater reclamation. Therefore, how to optimize the algae inoculum for the pilot treatment will be vital for further (semi-) continuous treatment process.

During the pilot test, both algae species grew well in the municipal treated wastewater. There was no sign of growth inhibition (dead cells or faded color) identified in the microscopy examinations (**Figure 4**). Interestingly, these two filamentous species clumped together. The co-culture formed numerous small algae colonies (about 1 cm) in the raceway, making biomass harvested much easier and quicker via a simple filtration on a 35 mm plankton net. Apparently, this can benefit to the practicability and scalability of selected co-culture cultivation for wastewater treatment. Only a few ciliates *Vorticella* were visualized to attach to the filamentous algae colonies in the 3rd batch, and they were possibly the "carryover" with the consecutive cultivation. However, they were not a predator to the co-culture as filamentous algae were too big to be the prey.

In this case study, big variations were obtained for algae growth measurements between sampling days (data not shown) by filtering 2 L of water samples. So, the biomass quantification was only based on the total algae production at the end of each pilot batch. The cell density of *Klebsormidium sp*. appeared gradually increase in the colonies along with the tests according to the microscope observation. In order to identify the change of combination ratio in the co-culture, the sensitive gene sequencing techniques (e.g. QPCR) or laser scanning confocal microscope (LSCM) with appropriate probes could be considered for future study.
