Three different scenarios were defined in which microalgae are integrated in a municipal wastewater treatment plant (WWTP), as shown in Figure 1.

In Scenario 1 the microalgal biofilm system is used as a post-treatment system for effluent from an activated sludge process. In Scenario 2 the first stage of wastewater treatment removes the bulk of the COD. Nitrification is prevented in this stage by operating at a short sludge retention time (SRT; 2.5 days). The second stage consists of a microalgal biofilm system removing N and P. In contrast to Scenario 1, N is mainly present as NH₄⁺ rather than NO₃⁻. In Scenario 3 the microalgae are used in a symbiotic process of algae and bacteria. N is removed by combined nitrification and denitrification and via assimilation by microalgae, COD is removed by heterotrophs, and P is mainly assimilated by microalgae.

In all scenarios the target effluent values were 2.2 mg/L total N and 0.15 mg/L total P. These values are the maximum tolerable risk (MTR) guidelines which are used by the Dutch water boards, as the classification of the good chemical and ecological status of surface water of the Water Framework Directive is not yet known.

The results of this scenario analysis showed that it was not possible to simultaneously remove the N and P in the wastewater to the target values of 2.2 mg N/L and 0.15 mg P/L. In the post-treatment scenario P, while in Scenarios 2 and 3 N was limiting the microalgae growth. Indeed, given the ratio of N:P in wastewater, N will always be the limiting nutrient, if the molar ratio of C:N:P of 100:12:1 represents the real average elemental composition of microalgae grown in such systems. However, elemental composition in microalgae is known to be highly variable. Compositions with C:P ratios between 34:1 and 418:1, and N:P ratios between 3.5:1 and 38:1 are reported in literature for different species of microalgae [20,22]. Also the growth conditions, with respect to nutrient and/or light limitation, influence the elemental composition. Molar N:P ratios as low as 3:1 have been reported under N limiting conditions, while under conditions of P limitation a N:P ratio of 100:1 is possible [23,24]. At low growth rates, the N:P ratio of the biomass composition can sometimes match the supply ratio. Especially luxury uptake of P with storage as polyphosphate, is known to take place in microalgae [25,26]. This luxury uptake might make it possible to not only achieve MTR quality with respect to N, but also with respect to P.

In the scenario analysis only N and P removal by microalgal assimilation was taken into account. Additional removal of P by precipitation with cations like Ca²⁺ and Mg²⁺ is also possible. This precipitation occurs at higher pH levels caused by microalgae changing the CO₂/HCO₃⁻/CO₃²⁻equilibrium when more CO₂ is taken up than can be supplied via absorption from the atmosphere [9,26]. With such a pH rise expected within the biofilm, it is likely that in practice a larger P removal occurs than was calculated.

This study found area requirements for the three scenarios between 0.32 and 2.1 m²/PE. A conventional WWTP is estimated to have an area requirement around 0.2–0.4 m²/PE. Hence, with 0.32 m²/PE, the microalgal post-treatment requires a similar area to that of the activated sludge plant. The two-stage system of Scenario 2 requires the largest area of 2.1 m²/PE. However, the first activated sludge stage will be considerably smaller than in a conventional WWTP, because nitrification is absent. In addition, more sludge is produced, and this sludge will have a higher energy value. This implies that more methane can be produced when digesting the sludge anaerobically.

The symbiotic system of Scenario 3 has an area requirement of 0.76 m²/PE, which is smaller than the area of the system of Scenario 2, and similar to the conventional WWTP combined with microalgal post-treatment in Scenario 1. Moreover, this system has the advantage that the O₂ production by microalgae and O₂ consumption by heterotrophs can be balanced. This balance implies no need for an external oxygen supply, giving energy and cost savings. Although very attractive, the technology required to support a symbiotic biofilm system still needs to be developed.

The area requirements are based on the calculated uptake capacities of 1.85 g N/m²/d and 0.34 g P/m²/d in the post-treatment scenario and 2.2 g N/m²/d and 0.41 g P/m²/d in Scenarios 2 and 3. These calculated uptake capacities are higher than the 0.1–0.6 g N/m²/d and 0.006–0.09 g P/m²/d measured in lab-scale biofilm systems [13,14], but lower than the 0.7–2.1 g P/m²/d measured in other pilot scale microalgal biofilms systems [11,27]. This indicates that the calculated uptake capacities in this study can be considered a good average estimation.

The area requirement calculated for the different microalgal systems depends on the elemental composition of microalgal biomass, as well as on the irradiance and the photosynthetic efficiency. As mentioned above, the elemental composition can vary in microalgae. Clearly a higher N and P content in the microalgae will result in a lower area requirement, while a lower content will increase the required area. This again illustrates the importance of knowing the real elemental composition of the microalgae when growing on wastewater.

The effect of irradiance on the area requirement can only be changed by either moving the system to another location, or by applying artificial illumination. However, the addition of artificial light yields no substantial area reduction. If all produced biomass, both activated sludge as well as microalgal biomass, would be converted into biogas to produce electricity for artificial light, the area reduction for the three scenarios is at most 0.8% (see Appendix B). This extremely low reduction is related to the energy losses in the process of methanogenesis, in converting biogas into electricity, electricity into light and light into new biomass via photosynthesis. Clearly these large scale microalgae based processes can only be fueled by sunlight.

In this analysis the oxygen quantum efficiency was assumed to be 0.05 mol O₂/mol photons. This value is not reached in horizontal microalgal systems, but has been reached in vertical panel photobioreactors [17]. This efficiency has been determined with light as the limiting substrate. However, it is likely that CO₂ limitation will occur when the biofilm is only exposed to ambient air. Modeling of microalgal biofilms has shown that CO₂ limitation can easily occur, the level of limitation depending on the bulk pH and alkalinity [28]. Therefore, the assumed efficiency can only be reached in a vertical biofilm system, if CO₂ limitation can be prevented. Using heterotrophic microorganisms to directly supply the CO₂ to the microalgae as in Scenario 3, may therefore be a very attractive way to accomplish this.

Both microalgal growth and uptake of N and P decrease at lower temperatures and light intensity [29]. Therefore, the capacity of the microalgal system will change throughout the day and the seasons. Low uptake of nutrients by microalgae during winter may be one of the main limitations of the application of microalgal wastewater treatment systems in a country like the Netherlands. In this scenario analysis, the system was assumed to be running only in the tourist season, from May until October. The application of the microalgal system in places where a much higher capacity is needed during summer is the most interesting application of this technology in The Netherlands. The microalgal system will provide additional capacity during summer, whereas during winter the existing WWTP capacity will be sufficient. Such a microalgal system may be applied on the islands in the Wadden Sea of the Netherlands, being a tourist location during summer. On the island Ameland for example, wastewater production during summer can be more than three times the amount during the rest of the year. With these conditions a microalgal biofilm system is an interesting option to treat the additional wastewater in summer, instead of increasing the size of the wastewater treatment plant to be able to treat the summer wastewater load.

The microalgae production was based on the total irradiation received in five months, and therefore the diurnal light cycle was not taken into account. In general, uptake of N and P by microalgae changes throughout the day and is faster during daytime than in the dark [24,25]. N-limited microalgae, on the other hand, are known to take up either NO₃⁻ or NH₄⁺ in the dark. Uptake of NH₄⁺ can be more than 50% of the daylight value in N-limited microalgae [30]. Consequently, when N is the limiting nutrient for microalgae in the treatment of municipal wastewater, considerable uptake of N during darkness might be expected. In addition, the wastewater loading rate of N and P is expected to be lower during the night, possibly compensating for the reduced nutrient uptake.

When using microalgal biofilm systems in wastewater treatment, substantial amounts of microalgal biomass are produced. In Scenario 1 the microalgal biomass production was 24 g/m²/d based on the consumption of NO₃⁻ and in Scenarios 2 and 3 the production was 28 g/m²/d based on the consumption of NH₄⁺. These biomass productions are slightly higher than biomass production of 11–18 g/m²/d measured in pilot photobioreactors [31,32] and in the range of biomass production of 24–31 g/m²/d measured in other biofilm pilot systems [11,27]. With this biomass production, a WWTP for 100,000 inhabitants with the microalgal post-treatment of Scenario 1 produces 200 ton (dry weight) microalgal biomass during the five summer months of operation. In Scenarios 2 and 3 this amount of biomass is even larger. With such a substantial amount of biomass, it is important to find an efficient way to harvest the biomass, and a proper destination.

To ensure an actively growing biofilm, and to prevent washout of valuable biomass with the effluent, the biofilm will need to be harvested regularly. This regular harvesting will reduce respiratory biomass losses or even cell death, which otherwise would lead to release of N and P from the biofilm. Two ways of harvesting can be distinguished, passive and active. Passive harvesting entails collecting the microalgal biomass that naturally detaches from the top of the biofilm when it ages. This might involve the addition of a settler tank, resulting in extra area requirement. Active harvesting techniques currently applied to remove biofilms in other systems include pH shock [33], backwashing and scraping. Active harvesting appears more attractive as it gives the possibility to harvest very regularly, hereby reducing the respiratory losses as much as possible.

Nitrogen was shown to be the limiting nutrient when integrating microalgal biofilms in the wastewater treatment (Scenarios 2 and 3). In this case, it might be possible to accumulate lipids in the microalgal biomass, as microalgae start to accumulate these under conditions of N-limitation [34,35]. To achieve this N-starvation and induce lipid accumulation the C:N ratio should be twice as high as was assumed in the scenario analysis. Although this increased ratio would result in an area requirement twice as large, the amount of produced biomass will also be doubled and thus larger amounts of lipids may be produced. In Scenario 1 these lipids would approximately amount to 79 ton during the five summer months of operation (see Appendix C). However, further research is still needed to induce the accumulation of specific desired lipids, to obtain a stable (mixed) culture of the desired species in the system, and to set up the biorefinery needed to extracts these lipids as well as valorize the remaining biomass constituent [30,31].

Depending on the microalgal biomass composition other products might also be possible. Using the biomass as fertilizer is very attractive but is only possible when no heavy metals or other recalcitrant compounds are present in the wastewater and accumulated by microalgae. Anaerobic digestion for biogas production is another possibility, although afterwards still autotrophic N and P removal or recovery will be necessary. The CO₂ that is produced during digestion might be recycled to the microalgal biofilm system as an additional CO₂ supply [36,37].