*A New Insight of Phycoremdiation Study: Using Filamentous Algae for the Treatment… DOI: http://dx.doi.org/10.5772/intechopen.104253*

the algae screening and tests. Based on the previous knowledge on local rivers ecosystem [e.g., 14, 15], inclusion of filamentous algae selection was to avoid the potential risk of introducing invasive species to the local environment. The VEAS tertiary wastewater was freshly sampled and filtered (0.2 μm) on the day for the laboratory test below. All the selection process was conducted following the NORCCA's standard protocols. There were two selection criteria, (1) being filamentous and (2) at least 100% of increase in chlorophyll fluorescence. The filamentous strains with the highest growth rates were selected for co-culture combination studies.

Among these candidates, only K-1454 (*Spirogyra* sp.) and NIVA-CHL142 (*Klebsormidium* sp.) passed the criteria, and their growth rates (109–137%) were comparable to the level of unicellular strains (129–195%) (**Figure 1**). Although *Trichormus* sp. was selected as the third candidate for the subsequent co-culture tests, the growth of monoculture was only detected on *Spirogyra sp*. and *Klebsormidium sp*. when these three strains were exposed directly to the VEAS effluent, rather than on *Trichormus sp.* (**Figure 2**). In the following combination model that simulating the coculture conditions, the results showed that the co-culture of *Spirogyra sp*. and *Klebsormidium sp*. could grow in the effluent, based on the detection of fluorescence increment in the end. Actually, the different sized algae cells have different uptake and scaled uptake affinities for the nutrient utilization [16], so the algae mixture in different cell sizes will be better for the purpose of nutrient recovery from wastewater reclamation. As the cell of *Spirogyra sp*. was much bigger than that of *Klebsormidium sp*. (approx. 10 time of difference), their co-culture was supposed a matched combination. Moreover, mixed culture would have a better resilience to the variable conditions and complex microbial community in the wastewater. Therefore, the co-culture of *Klebsormidium sp*. and *Spirogyra sp.* was selected as a model filamentous combination to treat the tertiary VEAS effluent.

At NIVA's Solbergstrand Algae R&D Facility, these two filamentous algae of *Klebsormidium* sp. and *Spirogyra* sp. were scaled up to 100 liters separately. All the algae cells were harvested by filtration (35 mm plankton net) when the inoculum biomass reached to the level of 2 g L<sup>1</sup> , and then washed by clean water, and weighed (after 10 min of air-drying) for the pilot test. A few grams of biomass were sampled from each species and lyophilised for a benchmark study, and the rest was used for

#### **Figure 1.**

*The comparison of fluorescence increments among different selected algae in the screening test (mean SD). (note: The first 3 days were omitted to allow inoculated cells for acclimation, and thereby the results were obtained between days 3 and 10 during their exponential growth phase. The chlorophyll fluorescent was measured at a wavelength of 685 nm with excited emission at 450-550 nm.)*

*A New Insight of Phycoremdiation Study: Using Filamentous Algae for the Treatment… DOI: http://dx.doi.org/10.5772/intechopen.104253*

#### **Figure 2.**

*Monoculture and co-culture flask experiments (mean SD;* Kleb: *Klebsormidium sp.* Tric: *Trichormus sp.* Spir: *Spirogyra sp.)*

pilot tests. The pilot test was performed in three consecutive cycles with about 1500 L of municipal tertiary effluent (non-filtered, provided by VEAS) on each, which was conducted on 1:15 (v/v) with each indoor inoculum in a raceway. The 1st batch was inoculated with about 242 g wet *Klebsormidium* (equal to 22.3 g of dry weight, DW) and 547 g wet *Spirogyra* (about 39.9 g DW). With an attempt to continue algae cultivation, a similar amount (wet weight, WW) of co-culture was taken from the final produced biomass and subsequently used as the new inoculum for second batch, and so did on third cycle. The outdoor pilot test was carried out at 10–15°C (similar to the conditions at VEAS), with supplemental 24 hr. of LED light radiation (30–95 μE m<sup>2</sup> s 1 ). The three batches were all monitored for 7 days, with 2 L of water sampling on each day. As the pH value in the raceway increased from 7 to 9 within 24 hr in the first 2 cycles (personal communication), the pH in the third batch was controlled at 7.5 with automatic CO2 addition after day 2. In the end, the total co-culture biomass in the raceway was filtrated via a 80-mm plankton net, rinsed with tap water, and quantified for the yield measurement. The biomass was freeze-dried for various analytical analyses, including protein, starch, and lipids.
