MAB2.0 project: Integrating algae production into wastewater treatment

Wastewater and treatment related streams can have fluctuating and sub-optimal composition, as well as include toxic/inhibitory components. One-time sampling and measurement may lead to biased results. At the case study location, sampling of the effluent of different dewatering devices happens on a daily basis (except weekends) and 6 parameters are measured: pH, chemical oxygen demand (COD), total nitrogen and ammonium (NH4+),$\left( \text{N}{{\text{H}}_{4}}^{+} \right),$total phosphorus and phosphates (PO43−),$\left( \text{P}{{\text{O}}_{4}}^{3-} \right),$as reported in Table 1.

A protocol implying a statistical analysis was developed that can be used as a basis for assessing the historical parameters. The protocol highlights the effect of seasonality on concentration profiles and the levels of concentration and their variability in time, as well as confirms the suitability of these effluents for microalgae growth. A set of analytical tools are included in the protocol to analyse the database of historically measured data. As microalgae growth and productivity are highly dependent on a satisfactory (C:)N:P ratio, the set of data is plotted versus the general formula for microalgae composition (23) as a function of time. The data are compared to thresholds of micronutrients to highlight any imbalance in the samples. Nevertheless, considering its origin imbalances are expected, but the reason for those should be addressed for example by influencing pH to prevent phosphate precipitation (24).

Different WWTPs use different units and parameters, and statistical analysis can reveal some inconsistencies in the datasets, hence verification of the data and crosschecking the analytical methods are also advised. However, the composition of AD effluent is not measured and registered at every WWTP, as it is directed to the start of the water line as return flow, and not a natural discharge with legal requirements. Thus, lack of historical data on the AD effluent composition can become an obstacle in the design process.

The stream intended as substrate for microalgae must be characterized not only by COD (and N and P) contents but also by its biological oxygen demand (BOD) and inorganic carbon (IC) content to assess its biodegradability under aerobic conditions. As AD effluent comes from the biogas plant, COD was revealed as non- or low-biodegradable. Thus, meeting the legal threshold by application of microalgae to discharge in the environment (<150 mg/L) is not a realistic expectation. Presence of biodegradable compounds can also imply possibilities for mixotrophic conditions.

Tests with samples can also indicate the need for additional treatment. For example, in the case study an extra step of settling was needed as after centrifugation, the effluent still contained suspended solid. Settling can result in up to 95% removal of total suspended solids which is most likely due to the flocculation agent added during dewatering. A filtration step could be also required to remove further particles and coarse colloids (Fig. 2), as they can impede light penetration and provide shelters for bacteria. The need for treatment has implications on the actual design of integration, introduced in the next chapter.

Figure 2

In order to select the most appropriate strains for a given stream, a robust, high-throughput, low cost, low labour and easy to apply strain selection protocol is needed to find the most suitable algae species. For these reasons, a microplate based protocol was developed for short-listing of strains, while further investigations for temperature optimum determination are advised in flask scale (around 0.1 L).

Literature, culture collections and own isolations are a good basis to inquire a starting set of up to 50 strains to investigate and to prepare a short list. In the case study, the initially tested strains were provided by the University of West Hungary, Mosonmagyaróvár Algal Culture Collection (MACC) and complemented with strains collected from different wastewater streams. Interestingly, those isolated strains were outperformed by the ones from the culture collection.

The strain selection protocol has two important practical aspects; to screen strains against the temperature optimum and medium strength. Considering the features of the wastewater streams, it is possible that without adaptation the strains could not survive in undiluted feedstock, thus different dilutions were tested and also the adaptive capacity of strains investigated. In the case study, the results of strain selection highlighted the importance of an adaptation period to the AD effluent.

Outdoor production of microalgae is subject to seasonal temperature fluctuations which can affect the growth of algae. Because the incident sunlight is also heating the reactors, temperatures over 40°C can be reached in photobioreactors. In open ponds, since evaporation contributes to cooling, the temperature is lower than 40˚C in most geographic locations. This temperature is too high for the optimal range described for most commercial algae species and at temperatures exceeding the optimum, microalgae growth rate sharply declines (25).

Cooling is often applied to keep the culture medium at a lower temperature, to optimize the efficiency and prevent crashes of algae cultures (26). However, cooling represents a major energy and cost factor. The use of a higher cultivation temperature, will not only save energy for cooling, but is a selective factor that can reduce the probability of local algae contamination making possible the maintenance of a monoculture.

The developed protocol in the case study showed a good reproducibility. Complementing the AD effluent by micronutrients resulted in a balanced growth in semi-continuous cultivation mode. In order to provide more accurate information on the conditions for optimal biomass cultivation, a lab scale photobioreactor can be used to simulate an average daily light condition at the location of the wastewater treatment unit (Budapest for the case study), operating under continuous mode. This experiment provides information needed to setup the operation protocol for algae growth at the location.

Though the European policy points towards circular economy and nutrient recycling, the current legal framework on wastewaters is outside the scope of the waste legislation. The Waste Framework Directive defines ’waste’ and clearly excludes sewage and wastewater from this definition (27). As a result, usually national legislation regulates wastewater and its treatment separately from waste regulations. Thus, circular economy aspects are usually not considered and implemented.

From a legal point of view, there is only wastewater and clean water that meets the threshold, so from this aspect AD effluent is considered as wastewater. As being defined as wastewater, stricter regulations apply with regards to transportation and transfer of ownership compared to regulations on waste. Moreover, there is no legal way to ’redefine’ wastewater or some of its separated substances as by-product similar to what is available in case of waste with all its further possibilities of evolving into a product and commercialization.

This regulatory framework considerably narrows the commercial viability of an otherwise microbiologically sound and widely researched method of producing microalgae and cleaning the AD effluent. According to current laws wastewater flows in a one-way direction. Wastewater (including AD effluent by legal means) can only leave the point of origin by direct transport (pipeline or collecting truck) to the treatment plant. Also, whenever it has reached the treatment plant there is no legal way to transfer any part of that to any third party (to set up algae production outside the WWTP). This way the only two places where algae producing investments can take place are the point of origin or the wastewater treatment plant which is the same for AD effluent. This considerably limits the feasibility of any similar investment given certain limitations including available area at the WWTP for own microalgae production. This has to be considered in the planning process.

The proposed integration design has three tailorable technology modules (Fig. 3).

Figure 3

The upstream part, referred as interface, aims to integrate and condition incoming streams of AD effluent and flue gas to make them appropriate for microalgae growth. The interface is responding the need for treatment as concluded by the preliminary evaluation. It is designed to reduce exposure to parameter fluctuation, decrease suspended solid content and toxicity, as well as mix flue gas with air. Prerequisite for the interface is that the WWTP has sludge dewatering and flue gas (as CO₂ source and heating medium) available. Technology elements that may be part of the interface include settling tanks, sand filters, storage tanks and flue gas heat exchanger and blower. The parameters and features of the interface depend significantly on the composition of the AD effluent, the hydraulic endowments of the hosting plant, the existing infrastructure (i.e. pipelines and wells), as well as the composition and temperature of the flue gas. Hence, some of these elements are optional in the interface design, the decision on exact design needs to be taken based on the preliminary evaluations. In the case study location, the AD effluent comes from dewatering centrifuges (Table 3) and still contains large amount of suspended solids removable by settling and filtering as indicated during the preliminary evaluation.

The next step is the reactor that secures the appropriate conditions and habitat for microalgae, thus a set of technical criteria was defined. The microalgal culture needs to be in optimum environment to successfully outcompete the bacteria present in the feedstock (whether it is AD effluent, raw wastewater or any related stream). The presence of bacteria, and organic matter can promote the formation of biofilm on surfaces, thus leading to biofouling and wall growth that can block light penetration. In order to minimize it, the selected reactor needs to have an optimal mixing and an easy cleaning procedure. As the AD effluent has a higher nutrient content than raw wastewater or other usual algae media, in order to significantly reduce nitrogen and phosphorus concentration, a reactor with high volumetric productivity is required. From a biological point of view, this means that the light intensity per unit of volume needs to be maximized. Nevertheless, other factors may compromise the biological performance such as limited area available, economic factors, tailorable design, as well as easy maintenance, cleaning and accessibility of internal surfaces.

The downstream step aims to concentrate the microalgae, for which technology options and their relation to the final products need to be accessed. The selection of the microalgal harvesting method is crucial (28) since harvesting costs can reach up to 20-30% of the total production costs (29). This selection is dependent on many factors including cell type, density and size, downstream processing requirements and the value of the end product (30). Therefore, developing an efficient harvesting strategy is a major challenge in the commercialization of products from microalgae. Davis et al. point at the strong economy of scale sensitivities for the downstream processing operations, which should be taken into account when extrapolating results (31).

In view of the algae end product criteria and targeted market, a protocol to access harvesting alternatives and process parameters was developed. The application fields of the produced algae determine the wanted final concentration in the algal paste, and also the state of the microalgae (if destructive methods can be applied or cells need to stay intact). The value of the end product is crucial in the final economic analysis, allowing or banning more expensive harvesting and drying methods.

This protocol also relies on findings of the preliminary evaluation phase. The selection of a strain best adapted to the given wastewater stream limits the harvesting options; certain species of algae are much easier to harvest than others. For example, Spirulina, which is a long microalga (20-100 μm) can be harvested by microscreening. This technique cannot be applied to smaller cells like Chlorella sp. which performed best in the case study strain selection process.

The protocol also investigates membrane unit inclusion scenarios. According to Bilad et al initial dewatering via membrane filtration followed by a further dewatering step via centrifuges could most probably be optimal (32) since membranes offer a cost-effective strategy for a dewatering step before centrifugation. Application of membrane unit decreases the flow that needs to be set into rotation, thus reducing energy costs and increasing the solids content in the input and output of the centrifuge. An advantage of membrane dewatering is that its efficacy has been proven at large scale over sustained operations, although fouling – and, in general, system performance – will depend on the strain, the state of the culture and the water matrix. The protocol contains specific experiments to assess membrane filtration performance and determine the optimal working conditions. The case study includes a membrane filtration unit as a harvesting or pre-harvesting step, without any recirculation of the biomass into the reactor.

After harvesting, with further drying processes it is possible to obtain a dry biomass with water content as low as 3%. How ever, these are expensive processes, which require costly equipment and have a high energy demand. The removal of 1 kg of water during drying requires more than 0.9 kWh of energy, and thus any reduction of water content by previous dewatering techniques is beneficial from energetic and cost standpoints (33). It is estimated that the drying techniques can only be applied to obtain products with a high market price (above 1 $/kg) (31). Since some of these techniques make use of steam, hot water or hot gases, the presence or absence of such utilities at WWTPs are taken into account for the economic balance of the processes, together with the heat tolerance or volatility of compounds of interest.

In order to assess the techno-economic features of algae integration scale-up options for WWTPs, as well as to learn about the environmental impact, standard protocols were developed. For assessing the technical performance, process models were developed in Aspen Plus v8.4 (Aspen Technology, Inc., USA), and later exported into Microsoft Excel spreadsheets. Several elements of a biorefinery system are not available in the databases of Aspen Properties, therefore an in-house property database of the National Renewable Energy Laboratory was used based on the work of Wooley and Putsche (34). The model can be easily tailored to different reactor types by changing the relevant parameters; in the case study the model was prepared to compare 3 different reactor types (raceway pond, tubular reactor and flat panel) on 2 scales (100 and 1 000 m³ input a day).

Independently of the type of algae cultivation units, the configurations are modelled following the same processing steps as described before: an interface, a growth and pre-harvesting step, and a harvesting step where biomass is dewatered. Fig. 4 shows a diagram of the flowsheet used to calculate the mass and energy balances.

The protocol offers the possibility for WWTPs to tailor the model to their location and study how the climate conditions affect the algae production. To this end, a simple microalgal growth model by Blackman (35) in combination with a simple light model by Lambert-Beer (36) was included into the modelling. Calculations are based on the monthly averaged irradiance values (Fig. 5 for the case study location).

Figure 4

Figure 5

The biological characteristics of the selected microalga strains are considered too, and based on the main aspect of the strain selection protocol the optimal temperature is assumed to be 35 °C. The model is tailorable to the specific characteristics of the selected photobioreactor such as volume to area ratio and different concentrations. Productivity can vary depending on the specific parameters of each reactor by modifying the hydraulic retention time (HRT) and thus specific microalgae concentration, and/or by modifying the number of months of operation (37). Additionally, a membrane unit is also included in the modelling attached to the reactor as pre-harvesting step.

As output, the model delivers mass and energy balances, and costs (CAPEX, OPEX) for the selected scale. The economic advantage for the WWTP operator embodies in reducing the nutrient content of the return flow which is difficult to quantify due to combined treatment of return flow with the incoming raw wastewater. Further research is needed to calculate the direct saving considering the ratio of return flow. Optimization of the design can happen through the available sensitivity analyses. In the case study, the sensitivity analyses suggest to look for ways in reducing the energy costs as being the main contributor to OPEX. Investigating the impact of different energy costs can encourage WWTP operators to understand and exploit energy integration options with biogas and other on-site sources, as well as to research options when algae production can deliver more revenues per energy unit as electricity wholesale market (which is lower than the feed-in-tariff schemes for biogas based electricity). Due to lack of actual large-scale algae integration into WWTPs there is large uncertainty in capital costs.

The model is also flexible because it is possible to switch on/off operating months and process sections (e.g. to change where to remove the microalgae from the system: as algae suspension or dried biomass).

The outputs of the technoeconomic analysis are linked to the LCA model. The LCA aims to assess the potential environmental impacts of the production of microalgae biomass from wastewater treatment effluents. The protocol is a prospective Cradle-to-Gate Analysis following ISO standard (38) and using ReCiPe impact characterization method (39). Assuming that the microalgae system will always be part of WWTP, inputs such as AD effluent and flue gas are burden free, as upstream environmental impacts are assumed to be allocated to treated water for consumers.

Case study analysis compared different functional units (e.g. unit bioplastics, unit fertilizer and unit wastewater treated). Similar to the techno-economic analysis, results indicate that integration within the wastewater treatment, including the use of on-site produced utilities, enhances the environmental performance of microalgae production. Case study results indicate that environmental impact depends on the selection of the functional unit, thus as hint for WWTP operators, interpretation needs to be carefully revised, as the functional unit might provide a misleading message.

As indicated during the preliminary evaluation, the significant amount of TSS content (Table 1) represented a challenge in pilot scale too, but removing it was necessary due to its black colour absorbing light. The interface module of the case study contains a 11 m³ settling tank with conical bottom operated in batch mode. The AD effluent is sent through a Honeywell type flow-through water fine filter to separate the particles still present after settling.

The storage tanks for the settled effluent allows the balancing of the feedstock supply for algae growth in case of fluctuations, toxic content or other errors in the operation of the plant, and additionally helps polyelectrolyte decomposition which is added to dewatering to assist flocculation (40). Polyelectrolyte can be present also in the AD effluent and certain types can, if above a concentration (which depends on dosing and dewatering), aggregate also algae cells. Nevertheless, after 72 hours (or less) these compounds decompose.

In line with the caution to assess the statistical parameters of datasets, the averaged composition of AD effluent (Table 4) shows large difference between each batch indicating the changing quality and different settling properties. This fluctuation may be caused by the integrated rain water sewage system in Budapest influencing sludge quality and external organic material added to the biogas plant (Table 2). As expected during the preliminary evaluation phase, imbalances in the medium conditions exist leading to suboptimal composition for algae growth. Precipitation of phosphate salts due to the high pH also contributes to the unavailability of micronutrients.

The fluctuations in the AD effluent volume and quality made the settling process unpredictable, thus the retention time in the settler was varied for each batch (Table 5). Additionally, during settling the AD effluent separates to three phases indicating that the clean AD effluent must be taken from the middle phase. In order to improve the treatment of the AD effluent, further investigations are needed on the large-scale design of the interface module. The optimisation needs to reduce the long residence time in the settling tank that may influence the composition of the AD effluent too. These experiences highlight the importance of proper integration with the process of wastewater treatment for scale-up and continuous operation.

An open cultivation algal system, such as the used raceway pond, is highly exposed to predation and contamination by undesired microorganisms due to unsterilized wastewater media (41). In order to reach a sufficient background concentration of

microalgae able to compete and grow in this medium, inoculation of the system was performed with a batch of selected microalgal culture in high cell density. This resulted in a starting TSS concentration of 400 mg L^-1. The inoculum was prepared under sterile heterotrophic conditions to have the inoculum ready in a short time, as Chlorella is able to consume organic carbon too (42). The two-stage inoculation strategy was an appropriate method to inoculate large-scale algal ponds (43).

The pond had operated in semi-continuous way for 32 weeks and treated AD effluent was fed manually. Usually, 1-2 m³ effluent was added into the pond each week, but on occasions, no AD effluent was added for two-three weeks. Operation of the pond was tracked by daily sampling for determination of TSS, nitrogen and phosphorus forms, and microscopic analysis. The aims of the experiments were to investigate the impact of varying AD effluent qualities, validate findings in protocol developments and conclude operational strategy for scale-up due to WWTP specific factors. For these reasons, the long run operation allowed to obtain findings that a WWTP operator should be aware of when scaling-up algae cultivation.

Main findings include that i) maximal dry matter concentration was 800 mg L^-1, ii) predation and competition influenced algae production largely, iii) thick foam layer formed in months with lower temperature and illumination, iv) the creation and implementation of a pH control strategy is necessary, v) phosphorus precipitation caused challenges in harvesting, vi) most of the ammonium was stripped out, vii) presence of nitrifying bacteria can lead to nitrate and nitrite accumulation. Impact of those findings are detailed herein.

Based on agar plate culture assays, microscopic and Denaturing Gradient Gel Electrophoresis (DGGE) examinations, the main microorganism was the selected Chlorella vulgaris, but cyanobacteria, fungi, ammonium oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB) and other eukaryotic algae were detected too. The presence of AOB was also confirmed from the conversion of ammonium in the pond. Due to the high pH and presence of AOB, ammonium in the added AD effluent got almost completely stripped out and turned into nitrite and nitrate within a few days after feeding. The pH profile also indicates those processes, the feeding of AD effluent results in peaks of around pH 8 which sharply falls to around 6.5 where it stabilizes (Fig. 6). This acidification is a result of consumption of ammonium by microalgae and the nitrification process by nitrifying bacteria. A lower pH helped to reduce the phosphorous precipitation (44) and ammonia stripping (45), but it also harmed the culture. Main experience puts an emphasis on the pH control, as it is needed to find the balance between the pH level and the solubility of phosphorus and ammonium in the presence of competitive microorganisms. The initial pH control through flue gas that was set to a threshold pH value of 7.5 was never activated due to the quick drop to 6-6.5 by ammonium conversion.

Figure 6

During the months of operation, a quickly settling and coarse sand like solid matter content had appeared in the pond. Analysis confirmed (Table 6), that this solid matter is made of calcium, magnesium and phosphorus indicating precipitation of salts either formed in the pond or previously. Presence of carbon indicates organic matter, probably cell debris and external biomass. As consequence, determination of the amount of algae biomass by dry matter content may lead to biased results, and those particles can cause problems in harvesting technologies. Regular shut down and cleaning of the open pond, as well as adding extra filtration both in sampling and harvesting process can help to reduce errors.

The above experiences are also reflected in the mass balances for nitrogen and phosphorus (Table 7) at the end of the run. Based on the input as form of AD effluent feeding (aggregated), into the pond, current nutrient concentrations in the pond and

output as microalgae (harvests and in the pond at the end of the experiments) the balances were defined. The loss in case of both elements is a significant portion of the balance which may be due to ammonium stripping and phosphate precipitation.