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Karin Larsdotter (2006): Microalgae for phosphorus removal from wastewater in a Nordic climate. A doctoral thesis from the School of Biotechnology, Royal Institute of Technology, Stockholm, Sweden. ISBN: 91-7178-288-5
Abstract As part of a research project aiming to develop and evaluate a hydroponic system for wastewater treatment in Sweden, extended nutrient removal by microalgae was tested. The hydroponic/microalgal wastewater treatment system was built in a greenhouse in order to improve growth conditions for plants and algae. Studies on the treatment step with microalgae showed that phosphorus removal could be successfully accomplished owing to the combined effect of phosphorus assimilation and biologically mediated chemical precipitation of calcium phosphates. This precipitation was mainly induced by the increased pH in the algal cultures, and the pH increase was in turn a result of the inorganic carbon assimilation by the algae. The results showed that the algal growth was mainly light limited which resulted in higher algal biomass density and also lower residual nutrients in the water at longer hydraulic retention times (HRT). In contrast the phosphorus removal rate was load limited, i.e. shorter HRT gave higher removal rates. This load dependency was due to the chemical precipitation, whereas the phosphorus assimilation was dependent on algal growth. Furthermore, results from an intensive study during summer showed that culture depths of 17 cm gave higher removal efficiencies (78% ‐ 92%) than cultures of 33 cm (66% ‐ 88%). On the other hand, the removal rate per area was higher in the deeper cultures, which implies that these may be preferred if area is of concern.
Nitrogen removal was achieved mainly by the assimilation of nitrate to algal biomass, and removal efficiencies of around 40% (nitrate) could be reached for most parts of the year although the nitrogen removal performance was quite uneven. Up to 60% ‐ 80% could however be reached during summer in the shallow cultures. A net removal in total nitrogen of up to 40% was observed in the shallow cultures during summer, which was most probably a consequence of grazing zooplankton and subsequent urea excretion and ammonia volatilisation as a result of the high pH values.
Over the year, there were large fluctuations in algal growth and removal efficiency as a result of the seasonal variations in light and temperature. During winter, phosphorus removal efficiencies lower than 25% were observed in the shallow tanks and lower than 10% in the deep tanks. Additional illumination during winter improved the phosphorus removal in the shallow cultures but did not have a significant effect on the deep cultures. Such additional illumination increases the total energy demand of the system, and hence alternative methods for phosphorus removal during winter would probably be more economical unless the algal biomass produced had great commercial value.
Key words: assimilation, hydroponics, light, microalgae, nitrogen, phosphorus, plants, precipitation, wastewater treatment
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Sammanfattning Som del i en studie där hydroponisk avloppsrening undersökts testades även mikroalgers förmåga att rena avloppsvatten från fosfor och kväve. Avlopps‐reningssystemet byggdes i ett växthus för att under en större del av året kunna tillhandahålla en god miljö för växterna i den hydroponiska odlingen och även för algerna. Studier av reningssteget med mikroalger visade att algerna kunde användas framförallt för fosforrening, eftersom algernas fosforassimilation kombinerades med utfällning av kalciumfosfater. Denna utfällning var en direkt följd av det höga pH‐värdet i algkulturerna som orsakats av algernas upptag av oorganiskt kol. Resultaten visade vidare att algtillväxten var ljusbegränsad vilket gav högre algtäthet samt lägre kväve‐ och fosforkoncentrationer vid längre hydraulisk uppehållstid (HRT). Fosfor‐avskiljningen å andra sidan var flödesbegränsad, d.v.s. kortare HRT gav högre reningshastighet. Detta berodde på att den kemiska utfällningen var flödesberoende, medan fosforassimileringen var begränsad av algtillväxt. Grunda kulturer (17cm) visade sig under en intensivstudie på sommaren rena mer fosfor (78 % ‐ 92 %), än lite djupare kulturer (33cm, 66 % ‐ 88 %). Å andra sidan hade de djupare kulturerna en högre reningshastighet per ytenhet, vilket medför att dessa kan vara att föredra om man har begränsade yttillgångar.
Kvävereningen var främst en effekt av algernas nitratupptag, och en reningseffektivitet på cirka 40 % (av nitrat) visade sig vara möjlig under större delar av året även om kvävereningen var ganska ojämn. Reningseffekter på upp till 60 % ‐ 80 % erfors även i de grunda algkulturerna på sommaren. Sommartid kunde också en reduktion i totalkväve med upp till 40 % ses i de grunda tankarna. Detta var förmodligen en följd av att zooplankton betat alger, och att deras ureautsöndring följts av ammoniakavgång till luften som effekt av de höga pH‐värdena. Över året sågs stora fluktuationer i algtillväxt och fosforreningseffektivitet, vilket var en följd av de årstidsberoende variationerna i värme och ljus. Under vintern avskiljdes mindre än 25 % av fosforn i de grunda kulturerna och under 10 % i de djupare kulturerna. Tilläggsbelysning visade sig förbättra reningseffekten något i de grunda tankarna men inte i de djupa. Elektriskt ljus på vintern ökar förstås den totala energiförbrukningen, och alternativa metoder för fosforrening bör därför övervägas under vintern om inte algerna som produceras kompenserar energiförbrukningen genom ett högt marknadsvärde.
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List of papers This thesis is based on the following papers, which in the text are referred to by their Roman numerals. Papers I‐III are reproduced with the kind permission of the publishers.
I. Norström, A., Larsdotter, K., Gumaelius, L., la Cour Jansen, J. and Dalhammar, G. (2003). A small scale hydroponics wastewater treatment system under Swedish conditions. Wat. Sci. Technol. 48(11‐12): 161‐167.
II. Larsdotter, K., Norström, A. and Dalhammar, G. (2004) Theoretical energy requirements for hydroponic wastewater treatment. Vatten 60: 187‐191. III. Larsdotter, K., Söderbäck, E. and Dalhammar, G. (2004). Phosphorus removal from wastewater by microalgae in a greenhouse in Sweden. Water Environ. Manag. Ser. 3: 183‐188. IWA Publishing.
IV. Larsdotter, K., la Cour Jansen, J. and Dalhammar, G. Microalgae as a phosphorus trap after hydroponic wastewater treatment. Submitted manuscript.
V. Larsdotter, K., la Cour Jansen, J. and Dalhammar, G. Biologically mediated phosphorus precipitation in wastewater treatment with microalgae. Submitted manuscript.
VI. Larsdotter, K., la Cour Jansen, J. and Dalhammar, G. Phosphorus removal from wastewater by microalgae in a Nordic climate – a year‐round perspective. Submitted manuscript.
Related paper Larsdotter, K. (2006) Wastewater treatment with microalgae – a literature review. Vatten 62
Related conference contribution Larsdotter, K., Oliviusson, B., Söderbäck, E. and Dalhammar, G. (2002) Phosphorus removal from wastewater by microalgae in a greenhouse in Sweden during winter. Poster presentation at the 1st Congress of the International Society for applied Phycology, 9th International Conference on Applied Algology, Almeria, Spain.
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Abbreviations
BOD Biochemical oxygen demand Ca2+ Calcium CO32‐ Carbonate COD Chemical oxygen demand EBPR Enhanced Biological Phosphorus Removal HCO3‐ Bicarbonate HRAP High rate algal pond HRT Hydraulic retention time Mg2+ Magnesium N Nitrogen P Phosphorus
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Contents 1. Introduction.................................................................................................................................. 1 2. Aim and scope ............................................................................................................................. 3 3. Background .................................................................................................................................. 4
Wastewater and wastewater treatment .............................................................................. 4 Hydroponics and other green treatment processes .......................................................... 6 Wastewater treatment with microalgae .............................................................................. 7
Growth in light limited cultures .................................................................................... 8 Phosphorus removal in algal cultures......................................................................... 11 Nitrogen removal in algal cultures .............................................................................. 12 Harvesting ....................................................................................................................... 13 Algal wastewater treatment at higher latitudes ........................................................ 14
4. Results and discussion.............................................................................................................. 16 The hydroponic wastewater treatment plant at Överjärva, a description (Paper I)... 16 Energy requirements of hydroponic wastewater treatment (Paper II) ........................ 18 The pilot study on microalgae in the greenhouse (Paper III) ........................................ 20 The microalgal treatment step (Paper IV)......................................................................... 23 The phosphorus removal mechanisms, a lab‐scale study (Paper V) ............................ 27 The whole‐year study – performance variations over the year (Paper VI) ................. 30 Discussion concerning the applicability of the technique .............................................. 33
5. Conclusions ................................................................................................................................ 35 6. Acknowledgements / Tack....................................................................................................... 37 7. References ................................................................................................................................... 39
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1. Introduction Swedish water and sanitation systems are functioning well from a technical point of view; most people have access to clean water, and the wastewater in urban areas is taken care of and treated centrally in modern wastewater treatment plants. From an international viewpoint, Sweden is very well developed regarding drinking water safety and wastewater treatment. However, in order to secure a sustainable development of the Swedish water and sanitation systems and to meet future demands regarding function, health and environmental aspects it may be necessary to reassess the solutions.
The Swedish research programme Sustainable Urban Water Management, or Urban Water in short, has aimed to develop support for strategic decisions on future water and wastewater systems (Urban Water, 2002). The main questions in the programme have been: How should the urban water and wastewater systems be designed and operated in a future sustainable Sweden? Will the sustainable water and wastewater systems of the future be improved versions of those that exist today, or will there be some radical changes? A conceptual framework was developed that describes the interplay between users, technology and organisation, and five groups of criteria for sustainability were also defined: health, environment, economy, socio‐culture and technical function (Figure 1). Each group of criteria has a number of indicators which are quantifiable (Hellström et al., 2000). The project described in this thesis fits in mainly in the technology sub‐system, and deals primarily with the environmental criteria concerning eutrophication and use of natural resources.
Figure 1. The Urban Water conceptual framework (Urban Water, 2002).
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Although the Swedish urban wastewater treatment systems of today are efficient, some drawbacks are sludge production and the need for chemicals. In one of the projects within the Urban Water programme, an alternative system was therefore suggested which combines hydroponics with the biological steps used in conventional wastewater treatment. The main advantages were expected to be that no chemicals were used in the process, that green biomass was produced instead of sludge and that valuable products could be recovered from the nutrient rich wastewater. The cultivation of valuable plants as an integrated part of wastewater treatment sounded attractive, and the question to answer was rather whether this kind of ecotechnological approach is suitable in the Swedish climate, and if it could contribute to a more sustainable development of the water and wastewater sector. In the project, which was called “Integrated product recovery and wastewater treatment”, a hydroponic wastewater treatment pilot plant was built in a greenhouse at Överjärva gård, a farm close to Stockholm. Several species of green plants were successfully cultivated and harvested, and it was obvious from the start that hydroponic cultivation of plants in wastewater is relatively easy. However, since the effluent standards imposed on the treatment plant, for phosphorus in particular, were hard to achieve completely with the current design of the treatment plant, an additional green treatment was also proposed – microalgae. The microalgae functioned mainly as a phosphorus trap after the hydroponics, but algal biomass could potentially also be used for other purposes, e.g. production of pharmaceutical substances (Borowitzka, 1988; Glombitza and Koch, 1989), food (Grobbelaar, 1982), animal feed (Becker, 1988), fertilizers (Benemann, 1979), fuel (Calvin and Taylor, 1989) and hydrogen gas production (Benemann, 2000) (a review covering most applications is also given by Richmond (1990)).
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2. Aim and scope
Besides the construction and evaluation of the hydroponic wastewater treatment system, the aim of the work presented in this thesis has mainly been to construct a microalgal treatment step for the polishing of residual nutrients in the effluent from the hydroponics, and to evaluate the suitability of the process in Sweden. Some questions have been:
• Can microalgae be used for extended nutrient removal from wastewater in
hydroponic treatment systems? • What sorts of microalgae work well in the treatment process? • What phosphorus and nitrogen removal rates and efficiencies can be
expected? • What are the mechanisms for phosphorus removal?
• How does the performance vary over the year? • How can the nutrient removal be optimised?
• What is the area demand?
The results are naturally dependent on the wastewater composition and climate at the specific site, and also on the greenhouse and bioreactor design. Nevertheless, the results presented give useful guidance for the suitability of similar light dependent wastewater treatment techniques in a Nordic climate.
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3. Background
Wastewater and wastewater treatment Domestic wastewater contains high concentrations of organic and inorganic nutrients. If released untreated, receiving waters such as lakes and streams may suffer from oxygen depletion as a direct effect of the microbial oxidation of organic pollutants and ammonium, and indirectly as an effect of eutrophication. Such eutrophication is caused mainly by the plant nutrients nitrogen and phosphorus that promote algal blooms in the receiving water, which in turn consumes oxygen when the algae are degraded. If severe oxygen depletion occurs, the benthic fauna and fish may die. Nevertheless, all degrees of eutrophication have an impact on the ecological communities and in many cases cause decreased biodiversity.
To cope with the negative effects of wastewater on the recipient, different steps of wastewater treatment have been developed, and these are described by e.g. Bitton (1990), Henze et al. (2002) and Ødegaard (1992). Primary treatment aims at removing large particles in the sewage by means of grids or sedimentation. Secondary treatment reduces the biochemical oxygen demand (BOD) in the wastewater by oxidizing organic compounds and ammonium. This process, which is often carried out in aerated tanks with so called activated sludge, involves both heterotrophic bacteria and protozoa. The bacteria degrade the organic material and the protozoa graze the bacteria, and in both cases organic material is converted to carbon dioxide and water. In addition to activated sludge, secondary treatment may also be performed with e.g. trickling filters or oxidation ponds.
Tertiary wastewater treatment mainly aims at removing the plant nutrients nitrogen and phosphorus. Nitrogen may be removed by nitrification‐denitrification processes, where the ammonium is first oxidized to nitrate by nitrifying bacteria in aerobic reactors, and thereafter recycled to an anoxic (oxygen free) reactor where it is converted to nitrogen gas (N2) by denitrifying bacteria. The first step in the nitrification is oxidation of ammonium to nitrite (1) and the second step is the oxidation of nitrite to nitrate (2):
1. NH4+ + 3⁄2 O2 → NO2‐ + 2H+ + H2O
2. NO2‐ + 1⁄2 O2 → NO3‐
Nitrification is performed by autotrophic bacteria belonging to the family Nitrobacteriaceae. Denitrification, on the other hand, is carried out by a large number of
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heterotrophic bacteria, which all need organic carbon for growth. The denitrification reaction also has nitrite as an intermediary step, and is performed according to the following reaction:
NO3‐ → NO2‐ → NO → N2O → N2
Phosphorus in wastewater is most often removed by chemical precipitation with aluminium or iron salts to form aluminium or ferric phosphate:
Me3+ + PO43‐ → MePO4
The metal (Me) salts are generally added in excess to compete with natural alkalinity, mainly according to the following reaction (Henze et al., 2002):
Me3+ + 3 HCO3
- → Me(OH)3 + 3 CO2
However, the precipitation product may be a relatively complicated combination of the metal added, calcium, carbonate and phosphate (Henze et al., 2002). In addition to chemical precipitation with metal salts, chemical phosphorus removal can also be achieved by adsorption to filter materials containing positively charged surfaces, e.g. limestone, zeolites, blast furnace slag or LECA (Westholm, 2006).
Besides chemical means for phosphorus removal, biological methods also exist. One way is the Enhanced Biological Phosphorus Removal (EBPR) process. In this process, bacteria in the activated sludge over‐accumulate phosphorus as a response to shifts between aerobic and anaerobic conditions. When the environment is anaerobic, the bacteria use their phosphorus storage as energy source to pick up substrate, and the phosphorus is thereby released (Christensson, 1997; Henze et al., 2002). When the conditions are shifted to aerobic, however, more phosphorus than was released is taken up by the cells and a net removal is achieved. Magnesium and potassium are co‐transported with phosphate, and the excess phosphorus is stored in the cells as polyphosphate granules (Bitton, 1990). Moreover, chemical precipitation of phosphorus may also occur in the EBPR process as a result of the increased phosphorus concentration in the water during the anaerobic phase (Arvin, 1983). Such biologically mediated phosphorus precipitation can also be promoted by denitrification, which is an alkalinity producing process that increases the pH, which in turn induces precipitation of phosphates (Arvin, 1983).
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Hydroponics and other green treatment processes Apart from the tertiary treatment processes described above, other biological nutrient removal processes also exist, e.g. wetlands, hydroponics and algal cultivation. These treatment systems primarily concern nutrient assimilation into green biomass, but other nutrient stripping processes are also involved. In wetlands, nitrification and denitrification may polish the wastewater from nitrogen, particularly if the water level is varied which promotes alternately aerobic and anoxic conditions (McBride and Tanner, 1999). Furthermore, phosphorus removal may in addition to biological assimilation be achieved by sorption to soil particles or precipitation with cations in the water (White et al., 1999). Sedimentation is often the most important removal mechanism for phosphorus in wetlands.
In hydroponic wastewater treatment, which means cultivation of green plants in wastewater without any solid support material, the nutrients are assimilated directly from the water by the plant roots (Jewell, 1994; Ayaza and Saygin, 1996; Ghaly et al., 2005). However, since most plant roots need aerobic conditions to thrive, adequate aeration of the hydroponic tanks is crucial. In addition to the direct nutrient uptake by vascular plants, microbial biofilms on the roots may also contribute to the wastewater treatment by e.g. BOD degradation and nitrification (Todd and Josephson, 1996; Polprasert and Khatiwada, 1998; Todd et al., 2003; Vaillant et al., 2003; Norström, 2005b). In order to optimize the wastewater treatment performance in hydroponic systems, fast‐growing plant species which assimilate large amounts of nutrients, as well as species with large root systems which maximize the surface area for biofilms should be selected. However, if the system is integrated with product recovery, the choice of species may also be based upon the type of products that are of interest.
Hydroponic wastewater treatment has been tested in some different locations at higher latitudes, e.g. in Denmark (Hinge and Stewart, 1997; Jungersen, 1997), South Burlington, USA (Todd et al., 2003), at Stensund, Sweden (Guterstam, 1996; Guterstam et al., 1998) and also at Överjärva, Sweden, which is where the project described in this thesis is located (Figure 2) (Norström et al., 2003). A comparative study of six hydroponic wastewater treatment plants located in greenhouses at higher latitudes, however, have concluded that the main mechanisms for nutrient removal in such systems are due to microbial activity rather than assimilation by plants (Norström, 2005a). BOD and nitrogen removal is often satisfactorily achieved by microbial processes, but the phosphorus removal is not very efficient.
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Figure 2. Pictures of the hydroponic cultivation of green plants at the Överjärva wastewater treatment plant. (Photo: Kaj Kauko)
Wastewater treatment with microalgae Microalgae are microscopic photosynthesising organisms which are found primarily in aquatic habitats. The idea of using microalgae for wastewater treatment originally developed in the 1950s in California by William Oswald (Oswald and Gotaas, 1957; Oswald, 1963). The role of the algae was both to assimilate plant nutrients and to support bacteria with oxygen. The bacteria, in turn, were involved in the degradation of organic material in the wastewater, the same process which is utilised in activated sludge (Figure 3).
Organicwaste
Excessbacteria
Excessalgae
Solarenergy
CO2 + H2O + NH4+
Chlorophyll
Dissolved oxygen
Bacterialoxidation
Algalphotosynthesis
Organicwaste
Excessbacteria
Excessalgae
Solarenergy
CO2 + H2O + NH4+
Chlorophyll
Dissolved oxygen
Bacterialoxidation
Algalphotosynthesis
Figure 3. The main processes involved in a high rate algal pond, extracted from Oswald and Gotaas (1957).
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The process was developed in shallow ponds, less than a meter deep and continuously stirred with paddle wheels. These, so‐called high rate algal ponds (HRAP), were aerobic throughout their volumes in contrast to facultative ponds which are anoxic near the bottom (Oswald, 1988a). The HRAPs are often built as so‐called raceways, with a meandering configuration (Figure 4). Properly designed and operated HRAPs are capable of removing more than 90% of the BOD and up to 80% of the nitrogen and phosphorus (Oswald, 1988b).
Water in
Water out Figure 4. Schematic picture of a raceway pond. Apart from open ponds, microalgae can also be cultivated in closed photobioreactors such as tubes or flat panels (Richmond, 1990; Borowitzka, 1996; Pushparaj, 1997; Grima et al., 1999; Mirón, 1999). These often have better light penetrating characteristics which makes it possible to sustain higher biomass and productivity with less hydraulic retention time (HRT) than is possible in open ponds (Borowitzka, 1998). However, closed bioreactors are often more sophisticated and operation demanding than ponds, and are mostly used for commercial production of microalgae rather than for wastewater treatment.
Growth in light limited cultures Since wastewater is a very nutrient rich medium, most algal cultures grown in wastewater are growth limited by light or carbon rather than by plant nutrients (Oswald, 1988b; de la Noüe et al., 1992). Light limited cultures behave in a manner quite different from nutrient limited cultures, which is described by Pipes and Koutsoyannis (1962). The growth of an organism cultured in a nutrient limited culture is a function of the concentration of the limiting nutrient in the culture. The higher concentration in the medium, the higher cell density can be achieved at steady state. The cell density is thus constant and not a function of the medium flow rate or the average HRT in the culture. Cultures limited by light or inorganic carbon supply, however, show a somewhat different growth pattern both in batch and continuous cultures. In a batch culture with
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algae in a nutrient rich medium, e.g. wastewater, the growth will be exponential in the beginning when the cell density is low, and this is similar in nutrient limited cultures (Figure 5). As the cell density increases, however, mutual shading of the cells causes the light intensity seen by each cell to decrease (Raven, 1988; Borowitzka, 1998; Wood et al., 1999). At a certain cell density the average light intensity seen by each cell is less than saturation light intensity, and light then becomes the factor limiting growth. The growth rate of every single cell thereafter decreases as the cell density increases, and the cell density increases linearly; the linear growth phase of the culture has started. The algal biomass production hereafter is proportional to the light intensity provided, and thus constant if the light intensity is constant. In a nutrient limited culture, on the other hand, the growth rate decreases and there will be no net growth of the population as the limiting nutrient is depleted. Figure 5. Batch culture of the cyanobacterium Anabaena PCC 7120 in synthetic wastewater. a) Cell density versus time and b) the same but with logarithmic scale. During the first week, the growth was exponential, which can be seen as a linear increase in b). Thereafter, the culture is becoming light limited. This can be seen in b) where the linear increase levels off. After this, the growth is linear with a constant biomass production (g dry weight per litre and day) which can be seen in a). (Unpublished results from studies within the doctoral project) In light‐limited continuous cultures, the cell density at steady state will be proportional to the amount of photons captured during the average HRT. The cell density per area hence mainly depends on two factors:
1. HRT. The longer the HRT, the higher the cell density (Figure 6).
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2. Light intensity. The higher the intensity, the higher the cell density (up to a certain limit, after which light starts inhibiting growth, so called photoinhibition (Krüger and Elnoff, 1981; Borowitzka, 1998; Oliver and Ganf, 2000)).
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Figure 6. a) cell density and b) algal biomass production as a function of HRT. As can be seen, the cell density increases with increased HRT, whereas the production remains constant. This indicates that the culture was limited by light (or by external inorganic carbon supply). (Extracted from Paper V) However, since the light intensity decreases exponentially with depth in water, the cell density per volume will also be lower the deeper the culture is. Consequently, in order to achieve dense algal cultures the depth is an important parameter to work with. As the residual nutrient concentration in the medium decreases with increased cell density, high‐density cultures are then favourable from a wastewater treatment perspective since effluent limits of nitrogen and phosphorus must usually be met. However, light is not always completely necessary for algal growth, and some microalgae have the ability to grow without light on organic carbon sources instead of carbon dioxide, such as organic acids, sugars, acetate or glycerol (Fogg, 1975; Kawaguchi, 1980; Borowitzka, 1998; Wood et al., 1999; Ogbonna et al., 2000). Some studies have indicated that about 25 ‐ 50% of the algal carbon in high rate algal ponds is derived from heterotrophic utilisation of organic carbon (Borowitzka, 1998). Nevertheless, in order to have dense cultures of microalgae at higher latitudes, the light conditions should be the most important operational parameter to optimize.
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Phosphorus removal in algal cultures Assimilation Microalgae, like all organisms, need phosphorus to grow. The phosphorus is used in the cells mainly for production of phospholipids, ATP and nucleic acids. Algae assimilate phosphorus as inorganic orthophosphate, preferably as H2PO4‐ or HPO4
2-, and the uptake process is active, i.e. it requires energy (Becker, 1994). Organic phosphates can be converted to orthophosphates by phosphatases at the cell surface, and this occurs especially when orthophosphates are in short supply. Moreover, microalgae are able to assimilate phosphorus in excess, which is stored within the cells in the form of polyphosphate (volutin) granules. These reserves can be sufficient for prolonged growth in the absence of available phosphorus (Fogg, 1975; Oliver and Ganf, 2000). The growth rate of an alga may therefore not respond at once to changes in the external concentration of phosphorus, as opposed to the immediate responses to temperature and light. Mostert and Grobbelaar (1987) found that the phosphorus concentration in cells varied with supply concentration, from approximately 1 mg P per g dry mass at a supply concentration of 0.1 mg P l‐1 to 100 mg P per g dry mass at supplies of 5 mg P l‐1 and greater. On average, an algal cell contains 13 mg P per g dry weight (Oswald, 1988a). Algae cultivated in wastewater, which normally has a concentration of between 10 and 20 mg P L‐1, may hence contain much higher amounts of phosphorus than is needed for growth.
In order to maximize the phosphorus assimilation, it is important to optimize the growth conditions for the algae since the assimilation of phosphorus generally depends on the algal carbon assimilation, and hence on the algal growth. Since algae starved of nutrients take up phosphorus much faster than non‐starved algae, there is also the possibility of using an operational strategy where starved algae are added to the reactor. Precipitation When microalgae photosynthesise, they take up inorganic carbon from the water. The inorganic carbon species normally used by microalgae are carbon dioxide (CO2) and bicarbonate (HCO3‐) (Oswald, 1988a; Borowitzka, 1998), the latter requiring the enzyme carbonic anhydrase to convert it to CO2. When bicarbonate is used as carbon source, the pH in the medium increases due to the reaction:
HCO3‐ → CO2 + OH-
This pH increase, which can elevate the pH in algal cultures to values above 11, strongly affects the water chemistry. Phosphorus may as a result precipitate with available cations to form metal phosphates, where calcium phosphates are the most common. Besides being promoted by high pH values, precipitation of calcium phosphates is also
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favoured by increased temperature as well as high concentrations of calcium and phosphorus (Song et al., 2002). Precipitation from wastewater is also possible at neutral pH values, but then the phosphate concentration must be at least 50 mg P L‐1 and the calcium concentration 100 mg Ca L‐1 (Carlsson et al., 1997). In soft water, i.e. with a calcium concentration of less than 50 mg L‐1, the phosphate concentration must be even higher in order to induce precipitation. At low aqueous calcium and phosphate concentrations, precipitated calcium phosphates may also be redissolved.
There are several different calcium phosphates that may form from wastewater, which all have Ca/P molar ratios between 1 and 1.67. Some salts, like amorphous calcium phosphate (ACP), dicalcium phosphate and octacalcium phosphate may be precursors to hydroxyapatite (HAp), which in turn is the most thermodynamically stable (Arvin 1983). Such HAp formation is, however, inhibited by high concentrations of magnesium, carbonate and pyrophosphates (P2O74‐) (Fergusson et al., 1973; Arvin, 1983). The effect of magnesium is pronounced when the Mg/Ca ratio exceeds 0.45. At pH levels above 10.5, however, magnesium precipitates with hydroxide ions and consequently loses its effect on the phosphorus solubility (Jenkins et al., 1971). Carbonate both decreases the crystallinity of calcium phosphates and promotes the formation of ACP, but also competes with phosphates in precipitating with calcium to form calcite (CaCO3) at pH values above 8 (Fergusson and McCarty, 1971; Arvin, 1983). As a result, the phosphorus precipitation rate is inversely correlated to the carbonate concentration (Fergusson et al., 1973).
Chemical precipitation of phosphorus may comprise a significant part of the total phosphorus removal in algal wastewater treatment (Doran and Boyle, 1979; Moutin et al., 1992; Proulx et al., 1994; Mesple et al., 1996; Tam and Wong, 2000). Particularly in areas with hard water, i.e. water with high concentrations of calcium and magnesium, this effect may be pronounced. The chemical stripping of phosphorus may be regarded as an advantageous by‐effect of the algal growth, with enhanced phosphorus removal as a result. Another advantage is that the chemical sludge which is produced is easier to harvest than free‐floating cells due to the tendency of the precipitate to sink.
Nitrogen removal in algal cultures Nitrogen removal in microalgal wastewater treatment processes is mainly achieved by assimilation to algal cells. Nitrogen is the second most important nutrient after carbon, and may comprise more than 10% of the biomass (Becker, 1994). Nitrogen exists in many forms, and the most common nitrogen compounds assimilated by microalgae are ammonium (NH4+) and nitrate (NO3‐) (Oliver and Ganf, 2000). The preferred compound is ammonium, and when this is available, no alternative nitrogen sources will be assimilated (Bhaya et al., 2000). However, high ammonium concentrations (>20 mg NH4‐N L‐1) are not recommended since in combination with high pH values, the cells may
12
suffer from ammonia toxicity (Borowitzka, 1998). In addition to ammonium and nitrate, urea ((NH2)2CO) and nitrite (NO2‐) can also be used as nitrogen sources. However, the toxicity of nitrite at higher concentrations makes it less convenient (Becker, 1994). Cyanobacteria are also able to assimilate the amino acids arginine, glutamine and asparagine, and some species of cyanobacteria can fix nitrogen gas (N2) (Bhaya et al., 2000). Of all nitrogen sources, this nitrogen fixation is the most energy demanding and only occurs in some cyanobacteria when no other nitrogen compounds are available in sufficient amounts (Benemann, 1979). Several microalgae can take up nitrogen in excess of the immediate metabolic needs, so called luxury consumption. This can be used later in the case of nitrogen starvation. For wastewater treatment purposes, the nitrogen assimilation may be optimized by maximizing algal growth. However, in addition to assimilation of nitrogen, ammonia may also be stripped to the air as a result of the increased pH values often found in algal cultures (Tam and Wong, 2000; Nunez et al., 2001). Denitrification, which is the major microbial process utilised in conventional wastewater treatment, is not often considered as a significant process in algal cultures though, since the high oxygen levels mostly found do not promote that reaction.
Harvesting Since a large part of the nutrients in algal wastewater treatment systems will end up as biomass, harvesting is crucial in order to separate both nutrients and BOD from the water. However, because microalgae are very small and usually have slow settling rates, this is not easily achieved and is consequently a cost expensive part of the cultivating process (Benemann, 1989; de la Noüe et al., 1992). Even though harvesting effectively can be accomplished by e.g. filtration or centrifugation, such methods may be too difficult or costly to implement (Mohn, 1988; de la Noüe et al., 1992; Tang et al., 1997). Sedimentation without the addition of chemicals is the most common method in full‐scale facilities (García et al., 2000), but previous flocculation is mostly desirable. Flotation of unicellular algae without flocculation may also be very difficult due to the hydrophilic cell surface on which air bubbles will not attach (personal unpublished experiences). Examples of chemical flocculants to use with flotation or sedimentation are alum, lime, FeCl3, cationic polyelectrolytes and Ca(OH)2, and some more toxically safe flocculating agents are e.g. potato starch derivatives and chitosans (Moraine et al., 1980; Mohn, 1988; de la Noüe et al., 1992). Some microalgae may flocculate naturally, so‐called bioflocculation. The process is often induced by turbulence stress and results in formation of biopolymers by extracellular enzymes. The polymers can also be produced by bacteria associated with the algal cells, but in both cases this leads to changes in the surface charge that in turn causes the cells to aggregate (Lincoln and Koopman, 1986; Mohn, 1988). In particular some species of cyanobacteria form flocs spontaneously and
13
some cyanobacteria are also able to form gas vacuoles that makes them accumulate at the water surface, and both these features make them easy to harvest (Benemann, 1979). For wastewater treatment purposes, fast mixing of the algal culture and increased HRT may be recommended since this increases the harvestability of the algae due to bioflocculation (Benemann et al., 1980). However, longer HRT also makes the culture more susceptible to grazing zooplankton; with an HRT of four days or longer, regular crashes of the algal culture have been experienced (Benemann et al., 1980). This may, however, not be very problematic since the algae rapidly recover after such crashes. On the other hand, grazing zooplankton may also be utilized for the harvest of algae, which is known as biological filtration. With this method, easily harvested filter feeders are fed with algae and it is consequently a form of aquaculture. Well known filter feeders are mussels and cladocerans like Daphnia spp. (de la Noüe et al., 1992). Complete food chains starting with wastewater or urine have been studied in order to develop integrated systems able to generate useful biomass simultaneously with effluent purification (Etnier and Guterstam, 1991; Adamsson, 2000).
A solution in order to avoid the need for harvest is to immobilize the algal cells in gelatinous media such as alginates or synthetic polymers (Brouders et al., 1989; Kaya et al., 1995; Robinson, 1997). The algae‐medium mixture is often shaped like beads, but can even cover screens or surfaces (Borowitzka, 1998). The medium allows the nutrients in the wastewater to diffuse to the cells, and the nutrient uptake rates have been shown to be similar for free and immobilized cells (de la Noüe et al., 1992).
Algal wastewater treatment at higher latitudes Since microalgal cultures are often light limited, cultivation at higher latitudes may be problematic during the darker period of the year. Even at latitudes lower than Sweden, e.g. Spain 37°N, variations over the year are observed with several times higher biomass productivity during summer than during winter (Mirón, 1999). At higher latitudes, this effect is naturally even more pronounced. Not many studies on microalgae for wastewater treatment have been conducted in colder climates; however, some results regarding the performance during summer have been reported. For example, in an HRAP at 63°N latitude, a removal of total phosphorus of up to 49% was reported (Grönlund, 2004). Algal cultivation has also been tested in greenhouses e.g. on diluted human urine at 57°N where the algae removed between 37% and 97% of the phosphorus during summer and autumn (Adamsson, 2000). Some researchers have suggested that algal and cyanobacterial species isolated from high‐latitude ecosystems may perform well in wastewater treatment since they are adapted to growth in colder climates; however, even if the strains tolerate low temperatures, they always grow faster at higher temperatures, i.e. they are cold tolerant (psychrotrophic) rather than cold loving (psychrophilic) (Talbot et al., 1991; Tang et al., 1997; Chevalier et al., 2000). Generally,
14
temperatures between 15°C and 25°C seem to suit most algal species, even those which are adapted to growth at lower temperatures. Nevertheless, cold tolerant species may be important in wastewater treatment processes in a Nordic climate, even if the growth conditions are not optimal.
15
4. Results and discussion
The hydroponic wastewater treatment plant at Överjärva, a description (Paper I) During the years 2000‐2001, a hydroponic wastewater treatment plant was constructed at Överjärva farm in Solna, close to Stockholm. The treatment plant treats municipal wastewater from the farm which includes two resident families, a cafeteria, biology education for children, different shops and other activities. A thorough description of the evolution of the wastewater treatment plant is given in the doctoral thesis by Anna Norström (2005b), and results from the first study on the treatment plant are presented in Paper I. The plant basically involves a series of microbiological and hydroponic steps, and the purpose has been to treat the wastewater without using chemicals and to integrate the treatment with recovery of valuable products. The plants cultivated in the hydroponics can be used for many purposes; as ornamental plants, food, feed or production of substances just to give some examples. However, this aspect of the project is not covered within the scope of this thesis.
The pilot plant is composed of several steps which are illustrated in Figure 7. After a collection tank underground outside the greenhouse, the water is pumped to a first anoxic tank (AN) (Figure 8). This is filled with Kaldnes carriers (AnoxKaldnes, Norway), on which microbial biofilms are attached. In addition to the raw wastewater, recycle flow from the hydroponics also loads the reactor, and the nitrate rich recycle flow in combination with the carbon rich raw wastewater promotes denitrification.
Figure 7. Overview of the tanks and water flows in the hydroponic wastewater treatment process, including sample points 1‐12 as described in Paper I. The design of the process train was later somewhat modified.
16
The effluent from the anoxic tank flows by gravity to the next tank, an aerated reactor (CA) in which the nitrification of ammonium starts and the organic compounds are further mineralised. The tank is covered by an earth filter (Figure 8), which functions as an air cleaner that purifies the air from bad smelling gases. Since the air‐filled space in the anoxic reactor is also connected to this tank, gases produced in the anoxic tank are also purified. Moreover, the earth layer is planted with good‐smelling plants, and as a result, the environment in the greenhouse is pleasant and the occurrence of sewage smell is uncommon.
Figure 8. The two first tanks in the treatment process, the anoxic tank (behind to the left) and the covered aerobic tank (in front), which has a planted soil layer on top. (Photo: Kaj Kauko) From the first two “closed” tanks, the water enters the three consecutive aerated hydroponic tanks (HP1 ‐ HP3) (See also Figure 2). These are wider than the closed tanks, and constructed from transparent plastic. At the water surface, racks with plastic nets support vascular plants. These have large root‐systems which hang down in the water for nutrient uptake. The roots also support biofilms, which treat the water further by BOD removal and nitrification.
After the last hydroponic tank, there is a clarifier (CL) for the recycling of easily settled material to the anoxic tank. However, no significant amount of sludge has been
17
produced, and the recycle flow is for that reason not in operation. From the clarifier, the water enters a sump (P1), from where water is pumped to the algal step. This consisted of two tanks (A1 and A2) connected in parallel at the time for the study described in Paper I, but was later increased by two more tanks (See Paper IV). From the algal tanks, the water flows by gravity to an algae clarifier (ACL) and then two final sand filters (S1 and S2).
The study described in Paper I mainly aimed to describe the design and to evaluate the performance of the whole treatment plant. The paper presents results from a sample period of 22 consecutive days during July 2002.
The major findings in the study were that:
• 90% COD removal was achieved. • the ammonium was completely nitrified in the hydroponics. Therefore the plant
roots were good growth substrate for nitrifying biofilms. • denitrification through recirculation of nitrate rich water from the hydroponics
to the anoxic tank was successful, and was responsible for the majority of the 72% nitrogen removal in the system.
• 47% of the phosphorus was removed, mainly in the algal tanks and the sand
filters. • further optimisation of the treatment process was still necessary in order to
reach the effluent standards at the site: 10 mg N L‐1, 0.5 mg P L‐1 and 15 mg BOD L‐1.
Energy requirements of hydroponic wastewater treatment (Paper II) As part of the overall evaluation of the suitability of hydroponic wastewater treatment in Sweden, a theoretical study of the energy requirements was conducted. Since the greenhouse at Överjärva was built according to the original construction drawing from 1860, the empirical data from the operation of the greenhouse were not relevant for future implementation of similar systems. Instead, data from modern greenhouses and greenhouse operation were used in order to calculate the need for heating and illumination during different parts of the year, which is presented in Paper II. Two operational strategies were compared, one giving light and heat sufficient to maintain
18
winter resting plants without growth (minimum 10°C, 400 lux 16 h day ), the other sufficient to maintain good growth conditions for plants with moderate light requirements (minimum 20°C, 2000 lux 16 h day ) (Christensson, 1988). Data regarding incident light intensities during the year and outdoor temperatures (SMHI) were used to calculate the need for additional illumination and heat in order to fulfil the two requireme
‐1
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nts for five locations at different latitudes in Sweden, from Lund 55°72’ N to Kiruna 67°83’N. The energy requirements for aeration of the wastewater were not included in the model, but were assumed to be the same as in conventional wastewater treatment with activated sludge. The energy requirements calculated were rather the additional energy that is needed in order to treat the wastewater with hydroponics instead of with conventional activated sludge systems. The results, which are presented in Figure 9 showed that:
• the heat demand was generally larger than the light demand independent of growth condition and latitude.
• on a yearly basis, 30% more light energy is needed in Kiruna than in Lund for
both growth conditions. • the heat demand showed much more pronounced latitude dependence than
light. In order to reach the 10°C growth condition, the heat demand is 700% higher in Kiruna than in Lund.
B: 2000 lux, 20°C A: 400 lux, 10°C
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Another conclusion from the study was that large energy savings could be achieved by letting the system go down to the low light and heat conditions during winter but still have the higher temperature and light conditions during the rest of the year. Such a resting period between October and March in Kiruna would result in a reduction of the energy demand of 37% compared to the high light and temperature conditions. In Lund, an even shorter period of low temperature and light intensity, between November and February, would save 54% of the annual energy requirements. Moreover, the contribution to the temperature inside the greenhouse by the heat in the wastewater was not included in the calculations, which could save even more energy.
The pilot study on microalgae in the greenhouse (Paper III) Since the effluent from the hydroponics still contained some residual nutrients, particularly phosphorus, microalgae were suggested as a green method to complement the phosphorus removal in the system. The first study on microalgae was conducted during summer 2001 in order to see if the method was appropriate, and another aim was to see which algal species would dominate the treatment step. In order to investigate whether different types of algae appeared as a result of different nutrient concentrations and N/P ratios, a train of four tanks was constructed. The first tank in the train received the effluent from the hydroponics, and the overflow from that tank flowed into the next tank etc (Figure 10). Between each tank, the algal biomass was harvested with filters. The first hypothesis was that the high nutrient concentration in the first tank would favour fast growing green algae, whereas later in the train, when nitrogen was depleted, there would be a selection for nitrogen fixing cyanobacteria. In other words, the suggestion in the beginning of the experiment was that nitrogen would be the limiting factor for the algal growth as a result of the low N/P ratio in the effluent from the hydroponics, which was sometimes as low as 1. This ratio is usually around 7 on a mass basis in algae, the so called Redfield ratio (Redfield, 1958) (not 10 as is incorrectly typed in Paper III).
20
Figure 10. Photo of the pilot study on microalgae. The effluent from the hydroponics was poured into the first tank, A, from where it flowed by gravity to next tank B etc. Between each algal tank, the algal biomass was collected in filters. (Photo: Kaj Kauko)
Interestingly, the results showed something totally different; instead of nitrogen depletion, a rapid phosphorus removal was seen in the first tank of the train (Figure 11). Moreover, there was no difference in algal selection between the different tanks, and nitrogen fixing cyanobacteria were not observed at all. The high phosphorus removal rate in the first tank was not seen in the following tanks of the train, although the algal production was the same in all tanks. The conclusion was that as a result of the increased pH in the algal tanks which was mediated by the photosynthetic carbon assimilation, phosphorus in the effluent from the hydroponics was rapidly precipitated in the first tank with available cations, probably calcium. As a result, there was less calcium and phosphorus left to precipitate in the water in the following tanks, and slower removal rates were therefore experienced.
21
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Figure 11. The dissolved phosphorus concentration in incoming water and in the four algal tanks A‐D. The first pilot study was extended to winter‐spring 2001‐2002, and was then composed of two similar trains of tanks where one of them achieved extra light from fluorescent lamps. This part is presented in Paper III. The results showed that the additional illumination increased the algal production and phosphorus removal rate during winter, but that in real terms this difference was small compared to the impact of natural light during spring. Even though the average phosphorus removal rate was 100% higher in the tanks with additional illumination than in the tanks with natural light during November‐December, the rate was almost three times higher during February‐April than during winter in the tanks with additional illumination. The light intensity by the extra illumination was 55‐150 μmol quanta m‐2 s‐1, and was achieved with plant lighting fluorescent lamps. These light intensities are reported to be sufficient to support moderate to good growth by green and flowering plants (Christensson, 1988), but obviously were too low to fully support an algal culture. Higher incident light intensities are usually needed for algal cultivation to compensate for the light absorption by the water and the algal cells themselves (Ogbonna and Tanaka, 2000). Naturally, a higher algal growth rate would have been expected if a stronger lamp had been chosen, but this would have demanded even more energy.
The major conclusions from the pilot study on microalgae were that:
• a large proportion of the removed phosphorus was due to chemical
precipitation rather than assimilation into cells. This conclusion was based upon two observations;
22
a) the low ratio between removed nitrogen and phosphorus, lower than 0.2
compared to the theoretical ratio of 7 indicates that a lot more phosphorus than was needed for algal growth was removed.
b) the phosphorus removal rate was strikingly higher in the first tank of each
train than in the following tanks, whereas the algal biomass production did not vary between the tanks. This suggests that there was a rapid precipitation in the first tank as a result of the increased pH and the high calcium and phosphorus concentrations. In the following tanks, the ion concentrations were lower and hence there was less precipitation.
• although extra illumination by fluorescent lamps increased the phosphorus removal capacity of the algae, there was still a large difference in performance between winter and summer.
The microalgal treatment step (Paper IV) Since the pilot studies had shown that microalgae had a great potential to remove residual phosphorus from the hydroponics effluent, a microalgal treatment step was constructed as an extended part of the treatment process during summer 2002. At first, this step consisted of two glass tanks connected in parallel, since the results from the pilot study had indicated that the highest phosphorus removal rate was achieved in the first tank in a train of tanks, where chemical precipitation was promoted. These tanks were denoted tank 1a and tank 1b, they had a culture depth of 33 cm and were continuously aerated for agitation with the same system as that which aerated the covered aerobic tank and the hydroponics. A clarifier was connected to the effluent from the algal tanks in order to allow algae to sediment. However, since the unicellular green algae that were dominating the treatment step were not easily settled, the clarifier did not remove all biomass produced in the algal tanks. For that reason, a dairy centrifuge was connected to the effluent from the clarifier which was able to remove more than 90% of the cells.
As mentioned, the algae that evolved in the treatment step were mainly unicellular green algae (Chlorophyceae), in particular of the genus Monoraphidium, but also other genera were occasionally present in relatively high amounts, e.g. Scenedesmus. In addition to the algae in the free water, biofilms also evolved on the inside of the tank surfaces. These consisted of many forms of microalgae, e.g. diatoms and cyanobacteria.
23
These biofilms were, however, removed regularly in order to allow light to penetrate the reactors from the sides.
After start‐up, an HRT of 1.9 days was used, which was too short to retain enough algal biomass in the tanks. This was the main reason for the relatively weak performance of the treatment step which is presented in Paper I. At the end of July 2002, the flow to the algal tanks was decreased to give an HRT of 4 days. This gave immediate results with a clear increase in pH and a decrease in both total and dissolved phosphorus in the algal cultures compared to the effluent from the hydroponics (Figure 12). In the influent to the algal cultures (=effluent from the hydroponics), the total and dissolved phosphorus concentrations were equally high, which indicates that the phosphorus was completely mineralised in the previous treatment steps. The dissolved phosphorus is the filtered orthophosphate fraction, whereas total phosphorus is analysed on unfiltered samples where cells and other organic material are included. In contrast to the mineralised influent, a small difference between total and dissolved phosphorus could be seen in the algal cultures, which represents the phosphorus assimilated into algal biomass. What is more interesting, though, is the much larger difference between total phosphorus in the influent and in the algal culture, which indicates that phosphorus has disappeared in some way. The explanation is that since the samples from the algal step were taken at the water surface (grab samples), the precipitated phosphorus, which was found at the bottom of the tank, was not included in the samples. The difference between total phosphorus in the incoming water and in the algal culture may therefore be interpreted as the chemical precipitation of phosphorus.
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Figure 12. a) pH and b) total (tot‐P) and dissolved phosphorus (PO43‐) in the effluent from the hydroponics (HP) and in one of the algal cultures (culture 1a) during August 2002.
24
The three fractions of phosphorus that were found in the algal step could hence be described as: • Paq = the dissolved orthophosphate fraction • Pprec = precipitated phosphorus = Ptot in – Ptot out • Pcell = phosphorus assimilated into algal cells = Ptot out – Paq out
During summer 2003, an intensive study on the algal step was performed, which is presented in Paper IV. In addition to tanks 1a and 1b, two other tanks which had previously served as filter tanks were converted to algal tanks (Figure 13). These two were denoted 2a and 2b and were shallower than the original algal tanks, with a culture depth of 17 cm.
Figure 13. The four tanks used for algal cultivation in the effluent from the hydroponics. (Photo: Kaj Kauko) In the experiment, the four algal tanks were connected in parallel to the effluent from the hydroponics, and the removal rates of nitrogen and phosphorus in the deep and the shallow tanks were compared. In addition to the two culture depths, four different HRTs (3, 4, 5 and 6 days HRT) were tested in order to see how the performance varied
25
with load. For practical reasons, each HRT was tested for one week and the cultures could adapt to the new flows adjusted during Friday to Monday, which is too short a time for a new hydraulic steady state to develop. Despite this, the results were quite clear and new knowledge on the potential of the treatment step was in fact gained.
The main findings from the summer study were that:
• longer HRT gave higher algal biomass concentration and higher phosphorus
removal efficiency. Hence, the residual phosphorus concentrations were lower with longer HRT.
• the shallower cultures had higher phosphorus removal efficiency (78‐92%) than
the deeper cultures (66‐88%).
• the algal growth was mainly light limited whereas the phosphorus removal was load limited (i.e. shorter HRT gave higher removal rates). The removal rates were between 1.2 and 2.2 mg P L day .‐1 ‐1
• the deeper cultures had a higher phosphorus removal rate per area (0.4 ‐ 0.6 g P
m‐2 day‐1) than the shallower cultures (0.2 ‐ 0.4 g P m‐2 day‐1). • between 29% and 77% of the phosphorus removal was due to chemical
precipitation rather than algal assimilation. The shallower cultures had a somewhat higher proportion of precipitated phosphorus than the deeper cultures.
• nitrogen removal efficiencies between 8% and 63% were experienced, where the
lowest nitrate levels were found in the shallow culture at the longest HRT. In the shallow tanks with 5 and 6 days HRT, a reduction in total nitrogen of between 26.6% and 40.3% was seen, which can probably be ascribed to the grazing of algae by zooplankton and subsequent ammonia stripping.
A suggestion was also that the phosphorus assimilation was dependent on growth, which implies that it was mainly light limited, whereas the phosphorus precipitation was limited by phosphorus load. However, these assumptions as well as the conclusion that there was a significant precipitation of phosphorus had to be tested in a more controlled experiment. For that reason the next experiment was designed.
26
The phosphorus removal mechanisms, a lab‐scale study (Paper V)
In order to investigate the chemical precipitation of phosphorus in the microalgal treatment step, a lab‐scale experiment was performed which is presented in Paper V. Several microalgal strains were isolated from the treatment step, and one strain was selected for the experiment on the assumption that it was one of the dominant species found in the treatment step. This strain was a unicellular green alga (Chlorophyceae), preliminarily determined to the Monoraphidium genus (Figure 14a). Since the aim was to study the precipitation conditions in the treatment step, sterile filtered wastewater from the inlet point to the algal tanks was used as growth medium.
A small culture vessel consisting of a separation funnel was set up in a culture chamber (Figure 14b). In the culture chamber, the algae were provided with artificial light from fluorescent plant lighting lamps. In contrast to the algal tanks in the greenhouse, the precipitate was not allowed to sediment due to the aeration from the bottom of the funnel, and samples taken from the bottom hence consisted of a representative amount of both cells and precipitate. During the experiment, a continuous in and outflow was provided by peristaltic pumps.
Effluent AerationInfluent
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Figure 14: a) Strain Y6, a unicellular green alga isolated from the microalgal treatment step and b) the experimental setup. The algal culture was grown in a separation funnel with continuous in‐ and outflow. Aeration from the bottom of the funnel prevented cells and precipitate from settling. Samples were taken from the bottom of the funnel. (Photo: Kaj Kauko)
a) b)Effluent Aeration
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27
The samples were then treated to separate the three different phosphorus fractions:
• Pcell, which was the phosphorus assimilated by the algae, • Pprec, which was the phosphorus fraction that was chemically precipitated, and • Paq, which was the soluble orthophosphate fraction, i.e. the residual phosphorus
in the culture.
Before the experiment began, batch cultures were grown in order to evaluate the fractionation method and to see how fast the precipitate was formed. One batch culture of 100 ml was grown for three weeks to give a dense algal culture. After sampling of the culture, 200 ml new sterile‐filtered wastewater was added to the culture. Immediately after addition (or at least within two minutes), new samples were taken. Additional samples were then taken after 15, 45 and 90 minutes. The samples were treated for the fractions described above, which gave the results presented in Figure 15.
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Figure 15. Phosphorus fractions during batch experiment. 200 ml wastewater (= P influent) was added to a culture of 100 ml. On the Y‐axis, the total amount of phosphorus in the culture is given, not the concentration.
However, since some phosphorus may have precipitated during the centrifugation of the samples (see Methods section Paper V), the samples taken immediately after addition of new wastewater may have been able to allow precipitation during maximum ten minutes.
28
Hence, the conclusions from the batch experiment were that:
• within ten minutes after the addition of new wastewater, a significant part of the added phosphorus precipitated. The precipitation should thus be recognised as a fast reaction in relation to the time scales used in the continuous experiment described below.
• the amount of cellular and precipitated phosphorus increased during 90
minutes after the wastewater addition, whereas the aqueous phosphorus fraction decreased.
In the experiment with a continuous culture, which is presented in Paper V, the HRT was varied in order to clarify the assimilation and precipitation dependency on load. In this experiment, the HRTs were 2, 3, 4 and 5 days. From the phosphorus fractions, the average phosphorus assimilation and precipitation rates respectively could be calculated. In addition to phosphorus, magnesium and calcium were also measured in the precipitate in order to roughly estimate the precipitate composition.
The main conclusions from the fractionation study were that:
• the average precipitation rate was load dependent whereas the assimilation rate
was growth dependent (Figure 16).
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Figure 16. Phosphorus removal rate as the sum of assimilation and precipitation during the four different HRTs tested. The average precipitation rate increased with decreased HRT and was hence load dependent. The phosphorus assimilation rate, on the other hand, did not vary as a result of HRT.
29
• the algal biomass production was light limited or limited by the carbon supply
from the aeration. • the precipitate was probably composed of an amorphous calcium phosphate
with magnesium included, and also by calcite and magnesium hydroxide. • the pH dependency of the chemical precipitation was clear, when a significantly
higher pH was experienced during 3 days HRT, a larger proportion of the phosphorus was also precipitated.
• a significant nitrogen removal was experienced.
The whole‐year study – performance variations over the year (Paper VI)
So, bearing in mind the results from peak performance during summer and the mechanisms for phosphorus removal, how suitable is the treatment process in a year‐round perspective? Since algae are phototrophs and hence depend on light intensity to thrive, it is obvious that such a process would be largely affected by season at higher latitudes. The question is rather for how long a period of the year we can expect a significant contribution by the algae to the overall wastewater treatment performance. To answer that question, the summer study was extended to a one‐year study, which is presented in Paper VI. From November to March, the effect of extra illumination during winter was also tested. The flows were adjusted to be as high as possible without washout‐effects of the treatment step; i.e. the flows were lower during winter than during summer to compensate for the lower algal growth rate. Generally the HRT was between 3 and 5 days during summer and between 5 and 7 days during winter.
The results from the whole‐year study showed that:
• there were large fluctuations in algal biomass production and phosphorus
removal as a result of season (Figure 17). The phosphorus removal efficiency was between 60% and 100% during summer but lower than 25% during winter except in the shallow cultures with extra illumination where efficiencies of up to 60% ‐ 80% were experienced even during winter.
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Shallow tank 2a
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a) b) Figure 17. a) Algal biomass production as milligram dry weight per litre and day, and b) phosphorus removal efficiency over an annual cycle in one of the four cultures in the treatment step (shallow culture 2a, with no additional illumination during winter).
• there was a clear correlation between phosphorus removal efficiency and pH (Figure 18). This correlation is due to two inter‐connected phenomena:
1. Higher phosphorus assimilation to algal cells is coupled to higher algal
biomass production, which in turn requires more inorganic carbon. This results in elevated pH, and phosphorus assimilation therefore indirectly gives a higher pH.
2. The increased pH induces chemical precipitation of phosphorus. The higher
the pH, the more precipitation.
• the shallower cultures generally had both higher algal biomass production and
phosphorus removal efficiency than the deeper cultures. This can be ascribed to different factors:
1. The higher light conditions allowed a higher growth rate of the algae and
hence a higher nutrient assimilation rate. 2. The higher light conditions resulted in 1‐2°C higher temperature in the
shallow cultures than in the deeper cultures. Higher temperature is well known to favour algal growth (Soeder, 1981; Grobbelaar, 1982; Borowitzka, 1998).
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3. The higher algal production resulted in higher pH and as a consequence
more chemical precipitation of phosphorus.
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Figure 18. Phosphorus removal efficiency against pH during the whole‐year study of the treatment step. The removal efficiency shows a correlation to pH up to pH 10 approximately.
• between 50% and 90% of the phosphorus removal in the shallow cultures was due to chemical precipitation, and the proportion in the deeper cultures was between 40% and 80%. There was, however, no clear correlation between the size of the precipitated fraction and season.
• a pronounced effect by the extra illumination on phosphorus removal during
winter was observed in the shallow cultures but not in the deeper. The illumination hence was too low to increase the average light intensity in the deeper algal cultures sufficiently to have a pronounced effect on algal productivity and phosphorus removal.
• a nitrogen (nitrate) removal efficiency of around 40% could be reached for most
parts of the year, and efficiencies of up to 60% ‐ 80% were achieved during summer in the shallow cultures. A removal of total nitrogen of up to 40% was observed in the shallow tanks during summer, and this was probably a result of grazing zooplankton and subsequent ammonia volatilisation as a result of the high pH in the algal cultures.
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Discussion concerning the applicability of the technique To evaluate the suitability of using microalgae for phosphorus removal in Sweden, we have to consider the treatment performance as well as area and energy demand, but also the fact that precipitation chemicals can be saved and that there is a possibility of using the algal biomass for different purposes. The results have clearly shown that microalgae are interesting for phosphorus removal due to the combination of assimilation and biologically induced chemical precipitation. However, since wastewater from only one location has been used in the studies, other results would probably be achieved for other wastewater compositions. The water at the inlet point to the algal step at Överjärva gård is hard, around 200 mg CaCO L , and chemical precipitation of phosphates is therefore favoured. This chemical stripping effect by the microalgae can be utilised particularly in areas with hard water, but in places with soft water, the removal efficiency would be more dependent on the algal phosphorus assimilation. Compared to conventional phosphorus removal with ferric or aluminium salts, the removal rates are relatively low since the HRT needed for algal cultivation is a minimum of approximately three days, whereas the HRT needed for chemical precipitation and subsequent sedimentation is rather measured in hours. However, compared to the hydroponics, which were found to remove very little phosphorus, microalgae are efficient. Hence, if the purpose is to cultivate green biomass in the wastewater, then microalgae would be a good alternative to chemical precipitation.
3 ‐1
The dependency on light limits the performance over the year at higher latitudes and also results in a large area demand for two reasons; firstly, the reactors must be shallow to allow good algal growth. Culture depths larger than 50 cm are not recommended for algal cultures, and at places with lower incident light intensities or during winter, depths smaller than 20 cm are recommended (Benemann, 1979; Fontes et al., 1987). Secondly, the light dependency by the algal biomass production requires that the HRT is long enough to keep enough biomass in the reactors, and this further increases the area demand. When it comes to the experimental setup in this project, an HRT of 3 days and a culture depth of 17 cm would require an area of 18 m2 per m3 water treated per day, and for 33 cm deep cultures the required area would be 9 m2 .
During summer when the incident light intensities were high, a phosphorus removal efficiency of up to 100% could be reached with 17 cm deep cultures. During winter, however, the performance with the bioreactor design used in the studies was much weaker and more uneven. Properly working phosphorus removal during winter would therefore require either i) more additional illumination than was used or ii) shallower cultures than those used plus the illumination used in the studies. As mentioned above however, shallower cultures demand more area, and illumination requires energy. If the algal biomass could render a product of very high economic
33
value, to use artificial light is a good idea, but otherwise, such energy demand may be too expensive (Borowitzka, 1996). Commercial cultivation of microalgae for the production of high‐value products is most often performed using closed systems with strict monocultures, and such systems may be difficult to maintain with wastewater as the growth medium. Consequently, it is worth considering whether another technique for phosphorus removal would save energy and land during the darkest period of the year. However, even if the algal method is restricted to operation during spring to autumn, an appreciable amount of precipitation chemicals could still be saved on a yearly basis.
With these facts in mind, microalgae can be used for phosphorus removal from wastewater in Sweden particularly in one or several of the following circumstances:
• during summer when high incident light intensities are experienced, since the
performance is directly correlated to light availability. • in areas with hard water, since the biologically mediated phosphorus
precipitation can be utilised which gives higher removal efficiency. • if greenhouses can be provided, since this has a positive effect of the algal
growth due to higher temperatures. Outdoor cultivation may give a shorter growth season.
• if land is not expensive, since the method demands larger area than
conventional wastewater treatment. This also makes the method more promising for small scale treatment rather than in large centralised urban treatment plants.
• if the biomass can be used for any purpose, then the economy in the system
would be improved. Finally, does this green technique contribute to a more sustainable development of the Swedish water and sanitation systems? Well, it certainly contributes to the range of solutions for wastewater treatment, especially for small scale solutions. It is also appealing since it reduces the need for chemicals, even though the main contribution to the performance is in fact due to chemistry!
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5. Conclusions The results from the studies performed in the doctoral project described in this thesis can be summarised as follows:
• Microalgal cultivation could efficiently provide extended phosphorus and
nitrogen removal in a hydroponic wastewater treatment system. The algal growth was mainly light limited, which resulted in higher cell densities and lower residual nutrients at longer HRT. The effluent limit for phosphorus of 0.5 mg P L‐1 was reached frequently during summer with shallow algal cultures (17 cm) but more occasionally with deeper cultures (33 cm).
• The phosphorus removal was a combination of assimilation to algal cells and
precipitation of calcium phosphates as a result of the increased pH in the algal cultures. This, in turn, was a result of the assimilation of inorganic carbon by the algae. The phosphorus assimilation rate was dependent on algal growth and was consequently independent on HRT. The average rate for precipitation, on the other hand, was load dependent, i.e. the removal rate increased with decreased HRT. Between 50% and 90% of the phosphorus removal in the shallow cultures was due to chemical precipitation, and the proportions in the deeper cultures were between 40% and 80%. There was, however, no clear correlation between the size of the precipitated fraction and season.
• In an intensive study during summer, phosphorus removal efficiencies between
78% and 92% were achieved with the shallow algal cultures, which correspond to phosphorus removal rates of up to 2.2 mg P L‐1 day-1 (0.4 g P m day‐2 ‐1). The removal efficiencies were lower in the deeper cultures, between 66% and 88%; however, higher removal rates per area than in the shallow cultures were observed, up to 0.6 g P m day (1.9 ‐2 ‐1 mg P L‐1 day-1)
• During winter, the algal growth and phosphorus removal efficiency was
considerably lower than during summer. Without extra illumination, phosphorus removal efficiencies lower than 25% were observed in the shallow cultures and even lower efficiencies were experienced in the deeper cultures. Additional illumination, however, increased the efficiency in the shallow cultures but not in the deeper.
• The nitrogen removal was mainly a result of nitrate assimilation by the algae. A
nitrogen (nitrate) removal of 40% was possible during most parts of the year,
35
but the removal performance was more uneven than the phosphorus removal. Efficiencies of up to 60% ‐ 80% were however observed during summer in the shallow tanks. A reduction in total nitrogen of up to 40% in the shallow tanks during summer was also experienced, which suggests ammonia volatilisation as a subsequent phenomenon after zooplankton grazing and urea excretion.
• Microalgae are appropriate for nutrient removal from wastewater in Sweden
particularly during summer, in areas with hard water, if greenhouses can be provided, if land is not expensive or if the biomass can be used for any purpose.
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6. Acknowledgements / Tack Efter åren som doktorand ser jag nu tillbaka på en mycket intressant och utvecklande tid, och tänker även på alla er som betytt mycket för mig och för forskningsprojektet. Den jag först och främst vill tacka är min handledare Gunnel Dalhammar. Tack för att du anställde mig, en grön biolog, till KTH där jag aldrig trott att jag skulle hamna. Tack även för din stora optimism och entusiasm och för att du gett mig så gott som helt fria händer att undersöka det jag tyckte var mest intressant. Sedan vill jag tacka min biträdande handledare Jes la Cour Jansen, Lunds Tekniska Högskola, för din otroliga energi och snabba feedback samt för dina ovärderliga kunskaper om avloppsrening. En av Jes doktorander som jag också vill passa på att tacka är Michael Ljunggren som ställde upp och gjorde flotationsförsök på algerna (resultaten blev kanske inte så bra, men vi blev mycket klokare på kuppen!). Tack Erik Söderbäck, Stockholms Universitet, som var biträdande handledare under en tid, för att jag fick komma till botaniska institutionen och vara på ert lab samt för den biologiska vinkeln på algers och cyanobakteriers tillväxt. I samma veva vill jag även tacka Sara Jonasson och alla de andra doktoranderna på SU växtfysiologi. Det var en jättetrevlig tid när jag fick vara hos er och leka lite biolog igen. Tack även Marianne Pedersén som var min första kontakt på botan samt Maria Greger och Åsa Fritioff för ert intresse för reningsverket. Ett jättestort tack vill jag rikta till hela Urban Water‐programmet, framför allt Per‐Arne Malmqvist och Henriette Söderberg. Det har varit jätteintressant att få vara med i ett så stort forskningsprogram med så många olika kompetenser. Ett superstort tack till Jan‐Olof Drangert också som höll i forskarskolan som jag tycker var toppen! Men vad vore den utan alla Urban‐doktorander: Anna, Kristina, Helena K, Helena A(P), Mattias, Therese, Jakob, Annika, Jonas, Pernilla, Frank, Gerald, Mats, Edgar och Stefan. Alla forskarskolekurser och konferenser har varit en fröjd tack vare er! Ett stort tack till Anna Norström för alla åren i växthuset och på KTH med gemensamma arbetsbekymmer och trevliga fikaraster, samt inte minst för dina petiga genomläsningar av mina artiklar. Tack Björn Oliviusson för att du tog hand om växthuset och gjorde det till en grön oas, samt för hjälp med vattenanalyser och annat. Tack Helen Vallhov för din hjälp under intensivstudien. Tack Kaj för alla mikroskopbilder och andra foton. Thank you all the other members of the microbiology group: Sofia, Guna, Lydia, Pelle, Karl‐Johan, Karin E., Erik, Fredrik, Guro, Joseph, Seyoum, Lena, Jacky, Peter, Helena and all the other former members, PhD students, ex‐
37
jobbare mm. It has been a great time working with all of you! Thank you all the nice people at floor 2 also, especially Lotta and Alphonsa. Tack alla på Överjärva gård, främst Ninni och Elisabet för ert engagemang i växthuset och reningsverket och för den mycket trevliga arbetsmiljön. Ett specialtack till min alg‐kollega Erik Grönlund i Östersund som vågade sig på att odla alger i norrland! Supertack till mamma, pappa och Eva för er totala support och lojalitet, och brorsan också, tackelitacktack! Tack alla kära vänner för avkopplande stunder under dessa långa år som jag traskat i avloppsträsket. Oändligt mycket tack till Andreas för kärleksfullt stöd, omtanke, otippat engagemang i fosforrening och mikroalger, ett litet dataprogram som kunde räkna ut reningshastigheter från den mest komplexa mätserie samt att stort antal middagar. Tack också kära lilla Rita som kom så lämpligt, jag fick nästan ett helt år utan forskning precis innan avhandlingsskrivandet, en välbehövlig paus med stora tankeställare om vad som är viktigast här i livet!
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