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The use of microalgae as method for phosphorus removal from a human derived wastestream. From a lab scale to a household scale cultivation systemVeele, Willemien

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CIO, Center for Isotope Research

IVEM, Center for Energy and Environmental Studies

Master Programme Energy and Environmental Sciences

The use of microalgae as method for phosphorus

removal from a human derived waste stream From a lab scale to a household scale cultivation system

Willemien Veele

EES 2012-148 T

University of Groningen

Training report of Willemien Veele

Supervised by: Dr. H.J.van der Strate (Ocean Ecosystems)

Dr. P. Boelen (Ocean Ecosystems)

Dr.ir. S. Nonhebel (IVEM)

University of Groningen

Ocean Ecosystems

Nijenborgh 7

9747AG Groningen

The Netherlands

http://www.rug.nl/fmns-research/oe

University of Groningen

CIO, Center for Isotope Research

IVEM, Center for Energy and Environmental Studies

Nijenborgh 4

9747 AG Groningen

The Netherlands

http://www.rug.nl/fmns-research/cio

http://www.rug.nl/fmns-research/ivem

ACKNOWLEDGEMENT

I would like to thank Peter Boelen and Han van der Strate for their supervision throughout the experimental part of this research. Especially their enthusiasm for my project, the trips to DeSaH and their confidence in me during the entire research. Furthermore, I would like to thank Nico Elzinga and Brendo Meulman at DeSaH for providing precious information and data to me and for giving a nice tour throughout the city of Sneek. Finally, I would like to thank Sanderine Nonhebel for her supervision during the entire project and her interesting comments and ideas for this research and report.

TABLE OF CONTENTS

Summary ................................................................................................................................................. 7

Samenvatting........................................................................................................................................... 9

1. Readers guide & introduction ......................................................................................................... 11

2. Phosphorus .................................................................................................................................... 13

2.1 Disturbances of the natural phosphorus cycle ............................................................................... 13

2.1.1 The introduction of water based sanitation ............................................................................. 13

2.1.2 The introduction of artificial fertilizers .................................................................................. 13

3. Wastewater treatment .................................................................................................................... 15

3.1 Chemical precipitation ................................................................................................................. 15

3.2 Biological removal ....................................................................................................................... 15

3.3 New sanitation concept ................................................................................................................ 15

3.3.1 The DESAR concept ............................................................................................................. 15

4. Algae and phosphorus .................................................................................................................... 17

4.1 Algae ........................................................................................................................................... 17

4.2 Microalgae growth and cultivation ............................................................................................... 17

4.3 Phosphorus and algae ................................................................................................................... 17

4.4 Applications of algal biomass ...................................................................................................... 18

4.5 Phosphorus removal from anaerobic digested waste streams......................................................... 18

4.5.1. Cyanobacteria ...................................................................................................................... 18

4.5.2 Freshwater algal species ........................................................................................................ 18

4.5.3 Microalgae as biofertilizers ................................................................................................... 19

5. Research questions and methods .................................................................................................... 21

5.1 Aim and main research question................................................................................................... 21

5.2 Research methodology ................................................................................................................. 21

5.2.1 Experimental research ........................................................................................................... 21

5.2.2 Household scale cultivation system ....................................................................................... 21

6. Experimental part – Methods & materials ..................................................................................... 23

6.1 Experimental design .................................................................................................................... 23

6.2 Microalgae selection .................................................................................................................... 23

6.3 Cultivation media ........................................................................................................................ 23

6.4 Cultivation conditions .................................................................................................................. 24

6.5 Daily measurements ..................................................................................................................... 24

6.6 Other measurements .................................................................................................................... 24

6.7 Data analysis ............................................................................................................................... 25

7. Experimental part – Results .......................................................................................................... 27

7.1 Results D. tertiolecta ................................................................................................................... 27

7.1.1 Relative growth and nutrient concentrations control medium ................................................. 27

7.1.2. Relative growth and nutrient concentrations 10% effluent medium ....................................... 28

7.1.3. Relative growth and nutrient concentrations 20% effluent medium ....................................... 29

7.1.4. Relative growth and nutrient concentrations 50% effluent medium ....................................... 29

7.2 Results P. tricornutum ................................................................................................................. 30

7.2.1 Relative growth and nutrient concentrations control medium ................................................. 30

7.2.2. Relative growth and nutrient concentrations 10% effluent medium ....................................... 31

7.2.3. Growth and nutrient removal 20% effluent medium .............................................................. 32

7.2.4. Growth and nutrient removal 50% effluent medium .............................................................. 33

7.3 Light transmission ....................................................................................................................... 34

7.3.1 Percentage of light transmission through different media ....................................................... 35

7.3.2. Effect of filtering effluent on light transmission .................................................................... 35

7.4. Average growth rates and phosphorus removal ............................................................................ 36

8. Experimental part – conclusions & discussion ............................................................................... 37

8.1 Phosphorus removal ..................................................................................................................... 37

8.2 Ammonium toxicity ..................................................................................................................... 37

8.3 Effluent turbidity ......................................................................................................................... 38

9. Scaling up ..................................................................................................................................... 39

9.1 Present and past sanitary systems ................................................................................................. 39

9.2 Phosphorus quantity of the Dutch households .............................................................................. 39

9.3 Technical design .......................................................................................................................... 40

9.3.1. System design ...................................................................................................................... 40

9.3.2 Production of 10% effluent .................................................................................................... 41

9.3.3 Cultivation system ................................................................................................................. 41

9.3.4. Algae stock culture ............................................................................................................... 41

9.3.5 Cultivation conditions ........................................................................................................... 41

9.3.6 Area needed .......................................................................................................................... 41

10. System analysis and optimalization ............................................................................................ 43

10.1 Pathogens and odour nuisance .................................................................................................... 43

10.1.1 Anaerobic digestion............................................................................................................. 43

10.1.2 Effluent filtration ................................................................................................................. 43

10.1.3 Salt water ............................................................................................................................ 43

10.2 Water usage and residue ............................................................................................................. 43

10.3 Societal acceptance .................................................................................................................... 44

11. Discussion, conclusions and future challenges ............................................................................ 45

11.1 The Dutch possibility ................................................................................................................. 45

11.2 The World possibility................................................................................................................. 45

11.3 Algae as biofertilizers ................................................................................................................ 46

11.4 The value of human excreta ....................................................................................................... 46

11.5 Research at different levels of scale ............................................................................................ 46

References ............................................................................................................................................. 47

Appendix A – Microalgae and media ..................................................................................................... 53

A.1 Microalgae species ...................................................................................................................... 53

A.2 Cultivation conditions ................................................................................................................. 53

A.3 Culture media ............................................................................................................................. 53

Appendix B – Measurements and protocols ........................................................................................... 55

B.1 Cell density ................................................................................................................................. 55

B.2 Average Growth rate ................................................................................................................... 55

B.3 Relative growth ........................................................................................................................... 55

B.4 Efficiency of photosystem II........................................................................................................ 55

B.5 Nutrient concentrations ............................................................................................................... 56

B.6 Phosphorus measurements ........................................................................................................... 56

B.7 Ammonium measurements .......................................................................................................... 57

B.8 Silicate measurements ................................................................................................................. 58

B.9 Salinity and pH measurements..................................................................................................... 59

B.10 Light transmission measurements .............................................................................................. 59

Appendix C – Effluent and media composition ...................................................................................... 61

Appendix D – Calculations upscaled system .......................................................................................... 63

D.1 Calculations P-removal using D. tertiolecta on 10% effluent medium .......................................... 63

D.2 Calculations area needed for cultivation system ........................................................................... 63

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SUMMARY In this research the possibility of two different microalgae species to remove phosphorus (P) from anaerobically digested human excreta has been investigated. This research was performed on two

different levels of scale. First research was performed within a lab scale setting, and then the data derived from these experiments was used to design a household scale cultivation system. The significance of this research is that the phosphate rock reserves, which are used for the production of artificial fertilizers, are going to be depleted within the next 50-100 years. Artificial fertilizers are needed for the production of high yielding crop varieties to feed the ever growing human population. By human

activity the natural phosphorus cycle has been disturbed leading to large losses of phosphorus into waterways. This has resulted in eutrophication of waterways, which is a worldwide problem. Since we depend strongly on phosphorus for our food supply and to prevent the eutrophication of waterways, there is a necessity to recover this essential element from sinks and to close the phosphorus cycle again. Especially urban areas are point sources of phosphorus losses caused by the high phosphorus content of humane excreta. With the use of a new sanitation concept, developed by the company of DeSaH, this phosphorus rich stream is being concentrated and used for biogas production through anaerobic digestion. The residue that remains after digestion, called effluent, still contains a significant amount of phosphorus. The cultivation of algae on anaerobically digested animal waste streams to remove phosphorus has proven successful. The produced algal biomass can be used as a biofertilizer to partially close the phosphorus cycle again. Less research has been done on the ability of algae to remove phosphorus from human derived waste streams. Therefore the following main research question has been formulated: What is the possibility of microalgae, cultivated on anaerobically digested human excreta, as method for phosphorus removal? At the experimental level, P. tricornutum and D. tertiolecta were cultured at 10%, 20% and 50% effluent medium. The growth and phosphorus removal of these algae on these three media was compared to the growth and phosphorus removal on a standard growth medium. Both P. tricornutum and D. tertiolecta were able to grow and remove phosphorus from the 10% effluent medium. P. tricornutum was also able to grow and remove phosphorus from the 20% effluent medium. Both species were not able to grow on the 50% effluent medium. The highest phosphorus removal was achieved by D. tertiolecta within 4 days on the 10% effluent medium. When theoretically enlarging this cultivation system for all the households of the Netherlands using outdoor raceway ponds, around 4 million kg op P could be removed from anaerobically digested human excreta. The area needed for such a system would comprise of 2.6 m2 per household. On an experimental scale algae are able to remove phosphorus from anaerobically digested human excreta. However, this concept is technically not feasible within the Netherlands for the next 20-50 years. This is due to the already existing improved sanitation infrastructure and the expected societal resistance

to such an outdoor system, because of health and odor concerns. Within developing countries this concept could help improve accessibility to improved sanitations. However, the water demand and area needed by this cultivation system is also seen as a bottleneck within densely populated areas. In order to overcome technical and societal bottlenecks further research is needed on 1) the implementation of large scale algae cultivation using different cultivation technologies and algae, and 2)

the ability of algal biomass to be used as biofertilizers in order to close the phosphorus cycle, especially concerning the health aspects and the transferability of pathogens.

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SAMENVATTING In dit onderzoek is de mogelijkheid van twee microalgen om fosfor (P) te verwijderen uit een reststroom van vergiste menselijke uitwerpselen onderzocht. Hiervoor is onderzoek gedaan op twee verschillende

schaalniveaus. Eerst op experimenteel schaalniveau en vervolgens is met behulp van de gegevens uit dit experimentele onderzoek een kweeksysteem op huishoudelijke schaal berekend. Dit onderzoek is van belang aangezien de wereldwijde reserves van fosfaatgesteenten binnen 50 tot 100 jaar uitgeput zullen raken. Deze fosfaatgesteenten worden gebruikt voor de productie van kunstmest. Kunstmest is nodig voor de productie van hoogwaardige voedselgewassen om de groeiende menselijke

populatie te blijven voorzien van voedsel. Door menselijk toedoen is de fosfaatcyclus verstoord en treden er grote verliezen van fosfaten op. Hierdoor treedt eutrofiëring van waterwegen op, wat een wereldwijd probleem is. Aangezien we sterk afhankelijk zijn van fosfor voor onze voedselvoorziening en om de eutrofiëring van wateren te voorkomen is het een noodzaak om dit essentiële element terug te winnen uit afvalstromen. Vooral stedelijke gebieden zijn puntbronnen van fosfaatverliezen. Dit komt door de hoge fosfaat concentraties in menselijke uitwerpselen. Met behulp van een nieuw sanitair concept (DESAR concept), ontworpen door het bedrijf DeSaH, wordt deze fosfaat rijke afvalstroom nu geconcentreerd verzameld en vergist voor de productie van biogas. Na de vergisting blijft er een reststroom over, genaamd effluent, die aanzienlijk veel fosfor bevat. Het kweken van algen op vergiste dierlijke uitwerpselen voor het verwijderen van fosfaat is succesvol gebleken. De geproduceerde biomassa uit algen kan als biokunstmest gebruikt worden om zo de fosfaat kringloop weer deels te sluiten. Aangezien er nog weinig bekend is over de mogelijkheid van algen om fosfaten te verwijderen uit menselijke reststromen, is de volgende hoofdvraag geformuleerd: Wat zijn de mogelijkheden van microalgen, gekweekt op vergiste menselijke uitwerpselen, als methode om fosfor terug te winnen? Op experimenteel niveau zijn P. tricornutum en D. tertiolecta gekweekt op 10%, 20% en 50% effluent medium. De groei en fosfaat opname van de algen op deze drie media is vergeleken met groei en fosfaat opname in een controle medium. De resultaten van het experimentele werk tonen aan dat zowel P.

tricornutum als D. tertiolecta kunnen groeien en fosfaten verwijderen uit het 10% effluent medium. P.

tricornutum vertoonde ook groei en fosfaat opname uit 20% effluent medium. Beide soorten konden niet groeien op 50% effluent medium. De hoogste fosfor verwijdering werd bereikt door D. tertiolecta, in 4 dagen uit het 10% effluent medium. Als dit kweeksysteem zou worden opgeschaald voor alle huishoudens in Nederland door gebruik te maken van outdoor raceway ponds, dan zou er theoretisch ongeveer 4 miljoen kg P te verwijderen zijn uit vergiste menselijk uitwerpselen. Dit vereist een oppervlak van 2,6 m2 per huishouden. Op een experimentele schaal kunnen algen gebruikt worden om fosfaten terug te winnen uit vergiste

menselijke uitwerpselen. In Nederland is dit concept technisch niet haalbaar binnen 20 tot 50 jaar mede door de al aanwezige sanitaire infrastructuur en de verwachte maatschappelijk weerstand tegen een open kweeksysteem. Deze weerstand heeft vooral betrekking op de gezondheidsaspecten en eventuele stankoverlast. In ontwikkelingslanden zou het DESAR concept kunnen leiden tot de toegang tot verbeterde sanitaire voorzieningen. Echter, het waterverbruik en de oppervlakte die nodig zijn voor een

opschaal kweeksysteem worden hier ook als problemen gezien, zeker binnen dichtbevolkte gebieden. Om technische en maatschappelijke knelpunten te overwinnen is meer onderzoek nodig naar 1) de grootschalige uitvoering van dergelijke kweeksystemen waarbij gebruikt gemaakt wordt van verschillende kweekmethoden en algen, en 2) de mogelijkheid om de geproduceerde algen biomassa in te zetten als biokunstmest om te fosfaatcyclus te sluiten, vooral met betrekking tot gezondheidsaspecten en

de overdraagbaarheid van ziektes.

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1. READERS GUIDE & INTRODUCTION

Within this research the removal of the essential element of phosphorus from a human derived waste stream using microalgae has been investigated. Since this research has a broad scope, from a global scale to a cellular level, research has been performed on two different levels of scale and at two different

research departments. First research was performed on an experimental scale within in a lab setting at the department of Ocean Ecosystems. Then a household scale cultivation system was designed at the department of IVEM. Furthermore, within this research different subjects are combined. In specific, the element of phosphorus, wastewater treatment & sanitation and algae cultivation. Therefore the structure of this report is rather unconventional.

In order to give a clear overview of the performed research the following structure has been applied to this report. Chapters 2 until 4 explain the environmental context and significance of this research. Starting

with the importance of the element of phosphorus, followed by the current and new method for wastewater treatment and completed with the use of microalgae as method for phosphorus removal from waste streams. Chapter 5 contains the research questions answered within this research. Chapters 6 until 8 contain the experimental part of this research. In chapters 9 until 11 the experimental research is combined with the environmental research.

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2. PHOSPHORUS Phosphorus (P) is an element that is essential for all life forms which cannot be substituted by any other element (Cordell et al., 2009; EFMA, 2000; Smil, 2000; Steen, 1998). It is a key element in many physiological and biochemical processes, like the formation of DNA and within photosynthesis. The element of phosphorus does not occur in nature as a free element due to its high reactivity (Ashley et al.,

2011). Phosphorus is always combined with other elements, like oxygen, to form phosphates which occur in many different complex forms (EFMA, 2000). It is not a rare element, being the eleventh most abundant element in the lithosphere (EFMA, 2000; Smil, 2000). However, the form in which it is present in the biosphere is often a form that is unavailable for plants (Ashley et al., 2011; Smil, 2000). Plants can only absorb the soluble inorganic form of phosphorus, orthophosphate (PO4), dissolved in soil solution

(Ashley et al., 2011). This form of phosphorus is an essential nutrient for plant growth, hence it is a vital element for our food production and thus for life.

2.1 Disturbances of the natural phosphorus cycle The flows of phosphorus within the natural phosphorus cycle have been disturbed due to human interferences. The global environmental problem of this is the eutrophication of waterways caused by the leakage of excess phosphorus from saturated soils (Ashley et al., 2011; EFMA, 2000; Smil, 2000). There are two main causes of this disturbance of the natural phosphorus cycle. The first is the introduction of water based sanitation concepts and the second is the introduction of artificial fertilizers.

2.1.1 The introduction of water based sanitation

Historically, humans relied on natural levels of soil phosphorus for crop and food production, with the addition of organic matter like animal or human manure and crop residues (Ashley et al., 2011). With the local production and consumption of food, phosphorus was mainly recycled, using the locally produced manure as fertilizer. This way the nutrient cycle was closed.

Within the first cities, human excreta or ‘night-soil’ was transported to the agricultural lands using a tonnage system to retain soil fertility (van Zon, 1986). The Industrial Revolution (~1760) triggered the mass movement of humans into cities, which as a result started to expand in size (Ashley et al., 2011). The ‘night-soil’ was no longer transported back to agricultural lands, because of the disease-carrying properties of mainly faeces, the foul odours, the increased volumes, transportation costs and the increased distance to agricultural lands (Ashley et al., 2011; van Zon, 1986). With the ‘Sanitation Revolution’ there was a transition from a land based to a water based disposal of human wastes, using flush toilets and

extended sewage systems (Ashley et al., 2011). Phosphorus rich human excreta was and is no longer recycled and returned to agricultural lands, but transported into waterways. This is one of the causes of the currently open phosphorus cycle.

Urban sewages currently represent the largest point source of phosphorus losses (EFMA, 2000; Smil, 2000). This is caused by the high phosphorus content within human excreta (urine and faeces) (Kirchmann & Pettersson, 1995). Total phosphorus from human excreta is expected to increase within

urban areas, due to the estimated increase in urbanization. By the year 2050 it is estimated that 70% of the world population will reside in cities (UNFPA, 2007), making urban areas phosphorus ‘hotspots’ (Cordell et al., 2009). Thus, within this human phosphorus stream lie options for reducing phosphorus losses.

2.1.2 The introduction of artificial fertilizers

It was Justus von Liebig (1840) who confirmed that the fertilizing effect of humus on plant growth was due to inorganic salts of phosphorus and nitrogen and not organic matter (Liebig, 1840). This discovery, together with increasing soil degradation and famines in Europe in the 17th and 18th century, started the search for external sources of phosphorus (Ashley et al., 2011). Sources of phosphorus were discovered in crushed bones, guano and eventually also in phosphate rock deposits. Phosphate rock was seen as a cheap

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and abundant source of phosphorus and it became widely used in favour of organic sources of phosphorus (Ashley et al., 2011; Smil, 2000).

In order to keep up with the ever growing human population, increasing food shortages and urbanization, high-yielding crop varieties were developed. In combination with the usage of artificial fertilizers, pesticides, extended irrigation infrastructures and mechanised labour, this was also known as ‘The Green Revolution’. Through the Green Revolution agriculture was reformed, largely abandoning organic fertilizers (Ashley et al., 2011).

With the discovery of artificial fertilizers in the mid-nineteenth century, we have become more dependent on phosphorus for our food supply. The world fertilizer consumption has increased tenfold since 1930 and almost six fold from 1950 until 1995, from 5 Mt of phosphates until 30 Mt of phosphates (Steen, 1998). It is estimated that the global phosphate consumption will rise until approximately 44 Mt in 2014 (Heffer & Prud'Homme, 2010). Not only will the expected population growth increase the demand for

agricultural phosphate. There is also an expected increase in wealth and therefore an increase in the demand for higher dietary standards, like eating meat. Increasing meat consumption will increase the need for cereals to feed live-stock, consequently increasing the demand for phosphate fertilizers (Steen, 1998).

The largest problem concerning our food supply is that the phosphate rock reserves are estimated to be depleted within the next 50-100 years (Cordell et al., 2009; Smit et al., 2009). Therefore there is a necessity to recover phosphorus and in addition, reduce the losses of phosphorus into waterways. The focus of this research is on the human derived waste streams, since this is the largest point source of phosphorus losses (EFMA, 2000; Smil, 2000). Within the next chapter the current methods of phosphorus removal from human waste streams within waste water treatment plants will be discussed. Furthermore, a new sanitation concept will be introduced.

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3. WASTEWATER TREATMENT Wastewaters are a combination of streams which are transported via an extended sewage system, often together with rain water, to be treated in centralised waste water treatment plants (Kujawa-Roeleveld & Zeeman, 2006b). Water is the main component of wastewaters. Even the most highly polluted

wastewaters consist for 99.9% out of water (McKinney et al., 2007). Before being discharged into other water bodies, wastewaters need to be treated due to their high pollutant content and their hazard for the human population and the environment (de-Bashan & Bashan, 2010; McKinney et al., 2007). Policies have been implemented to reduce the levels of phosphorus entering surface water, because of their contribution to worldwide eutrophication (de-Bashan & Bashan, 2004). To remove phosphorus from domestic and industrial wastewaters, different technologies have been implemented. There are two main methods to remove phosphorus from wastewaters, which are chemical precipitation and biological removal (Stratful et al., 1999).

3.1 Chemical precipitation The most common chemical method for phosphorus removal is through metal precipitation using calcium, aluminium or iron (Donnert & Salecker, 1999). However, the resulting precipitate is unrecoverable for possible industrial processing and reuses in for example fertilizers (Donnert &

Salecker, 1999). The most promising chemical method for phosphorus removal is the use of struvite, or magnesium ammonium phosphate hexahydrate (MgNH4PO4.6H2O). Struvite forms spontaneously in environments where high concentrations of soluble phosphorus, magnesium and ammonium are present (de-Bashan & Bashan, 2004). Additional conditions for struvite precipitation are a low concentration of suspended solids and a pH above 7.5. Furthermore, the components of struvite need to be available in a

molecular ratio of 1 (Mg2+):1(NH4+):1 (PO4

3-) (de-Bashan & Bashan, 2004). A downside to this method of phosphorus removal is the spontaneous formation of struvite which contaminates pipes and other inner surfaces of the water treatment plants, therefore being the major cause of operational breakdown (Stowa, 2005a). For removal of this precipitate sulphuric acid and/or physical removal is needed (Williams, 1999). The clogging of pipes reduces the efficiency of the plant and makes phosphorus removal a costly process (de-Bashan & Bashan, 2004; Jaffer et al., 2002).

3.2 Biological removal Biological methods for phosphorus removal from urban wastewaters are constructed wetland systems or absorption by various microorganisms (bacteria and microalgae). Microorganisms also remove various toxic elements present in the highly polluted wastewater. This however makes the end-product unusable for direct reuse (de-Bashan & Bashan, 2010; Stratful et al., 1999). Furthermore, the harvesting and disposal of the microorganisms is very difficult (de-Bashan & Bashan, 2010).

3.3 New sanitation concept Instead of combining different wastewater streams and diluting the content, the company of DeSaH (Sneek, The Netherlands) has developed a new concept for domestic wastewater treatment by separating and concentrating different domestic wastewater streams. Domestic wastewaters contain significant amounts of phosphorus, pathogens and micro pollutants, due to the high content of human excreta (Kirchmann & Pettersson, 1995; Kujawa-Roeleveld & Zeeman, 2006b). The intention of the new concept is that concentrating the risks of high nutrients and pathogens in small volumes enables better control and limits the negative environmental effects (Kujawa-Roeleveld & Zeeman, 2006b).

3.3.1 The DESAR concept

The concept that is developed by DeSaH is called the DESAR concept, which stands for Decentralised Sanitation and Reuse. The goal is to separate waste streams according to their type of pollution and re-use potential of their resources (Kujawa-Roeleveld et al., 2006a). Within this concept three main resources are considered: 1) Bio-energy from the transformation of organic material, 2) nutrients (phosphorus,

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nitrogen, potassium and sulphur) and 3) water from advanced treatment of cleaner wastewater streams. Treatment of wastewater streams is selected in such a way that the reuse potential is preserved and the quality of the end product meets the local requirements (Kujawa-Roeleveld et al., 2006a).

In the city of Sneek, DeSaH has started a pilot project with 32 households containing the DESAR concept, separately collecting domestic wastewaters and dividing it into two streams (see figure 3.1). The first stream is concentrated black water, consisting of urine and faeces and optionally kitchen waste. The

human excreta are collected with the use of vacuum toilets, using only 1 litre of water per flush. The second stream is low concentrated grey water, composed of shower-, bath-, kitchen-, and laundry-water. By diverting black water from grey water, 80-95% of the nutrients from households can be recovered, because these mostly reside in black water (Kujawa-Roeleveld & Zeeman, 2006b).

The organic black water of the 32 households is transported to an anaerobic mesophilic digestion tank of 35°C, in which biogas (CH4) is produced (see figure 3.2). The residue from the digestion process, called effluent, still contains significant amounts of phosphorus and nitrogen which can be recovered. The exact composition of the effluent is unknown, but it still contains a certain amount of solids, pathogens, micro-pollutants, heavy metals, pharmaceuticals and hormones (Kujawa-Roeleveld et al., 2006a; Kujawa-Roeleveld & Zeeman, 2006b; Stowa, 2011b). Nevertheless, through the anaerobic digestion process the number of pathogens is reduced (Stowa, 2011b). Therefore, the anaerobic digestion process could be seen as a purification step of the human excreta.

Currently, DeSaH uses struvite for phosphorus removal. Nitrogen is removed using anammox bacteria (Anaerobic Ammonium Oxidation) (Zeeman et al., 2008). Another option for the removal of phosphorus (and nitrogen) from the anaerobically digested human excreta could be the use of microalgae. Microalgae have been cultivated successfully on different anaerobically digested waste streams. The use of microalgae as method for phosphorus removal will be further introduced within the next chapter.

Figure 3.2: Anaerobic digester producing biogas and

effluent. Figure 3.1: Separated collection of domestic waste

streams, black- and grey water.

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4. ALGAE AND PHOSPHORUS Algae are known for their strong growth potential in phosphorus rich waters, producing large quantities of algal biomass. The cultivation of algae on concentrated waste streams and effluent from anaerobic digestion of different wastes is not an unfamiliar concept. Before the cultivation of algae on anaerobically

digested waste streams is discussed, first a small introduction of algae, algae cultivation and the importance of phosphorus for algae is given.

4.1 Algae Algae are ancient eukaryotic organisms that occur in many different forms, unicellular and multicellular, and in almost every ecosystem of the biosphere. There are nine phyla of algae based on molecular sequence information, (Graham & Wilcox, 2000). These phyla are the Cyanobacteria (prokaryote), Glaucophyta, Euglenophyta, Cryptophyta, Haptophyta, Dinophyta, Ocrophyta, Rhodophyta (red algae)

and Chlorophyta (green algae). Algae can be divided into macroalgae (e.g. seaweeds) and microalgae (e.g. phytoplankton). Microalgae are the focus of this report. The biodiversity of microalgae is immense and represents an almost untapped resource (Pulz & Gross, 2004). It has been estimated that between 200,000 and several million species exist (Norton et al., 1996), compared to around 250,000 species of higher plants (Pulz & Gross, 2004).

4.2 Microalgae growth and cultivation The main ingredients for microalgae growth are light energy and CO2. Other important factors for successful algae cultivation are pH, alkalinity, cultural cell density, temperature, contamination and competition by other microorganisms, which are all genera, cultivation type and strain dependent (Markou & Georgakakis, 2011). Furthermore, adequate amounts of macronutrients (carbon, phosphorus and nitrogen), major ions (sodium, potassium, magnesium, calcium, sulphur and chloride) and micronutrient metals (iron, zinc, copper and manganese) need to be supplied to algal cultures (Andersen, 2005; Graham & Wilcox, 2000; Markou & Georgakakis, 2011). The major nutrients need to be available in the correct nutrient ratio’s to obtain maximum growth rate, also known as the Redfield ratio of 106 C: 16 N: 1 P (Redfield, 1958).

4.3 Phosphorus and algae Especially phosphorus is essential for the growth of algae, limiting growth when not available in sufficient quantities (Graham & Wilcox, 2000; Jansson, 1988). Low phosphorus concentrations are related to low cell densities. Phosphorus deficient algae take up phosphate at higher rates than phosphorus sufficient algae (Jansson, 1988; S. K. Singh et al., 2007). The form of phosphorus which is utilized by microalgae is orthophosphate (PO4) (Correll, 1998). The available organic phosphorus in a solution is hydrolysed to PO4 by the extracellular enzyme alkaline phosphatase, of which the production is stimulated by phosphorus deficiency (Correll, 1998; Graham & Wilcox, 2000). Algae commonly possess an internal inorganic phosphorus pool, in specific a polyphosphate (PolyP) pool, which is filled when the phosphorus is present in excess (Cembella et al., 1984; Jansson, 1988). These internal storage pools can sustain growth by storing enough phosphate to provide for as many as 20 cell divisions in the complete absence of an external phosphorus sources (Graham & Wilcox, 2000; Jansson, 1988). The uptake of phosphate is energy dependent and the uptake rate is slower in dark than in light environments (Jansson, 1988; S. K. Singh et al., 2007). Furthermore, the uptake of phosphate is influenced by pH. Uptake rates decrease in acidic and relatively alkaline environments (Rigby et al., 1980). Lack of ions such as K+, Na+ and Mg+ also decrease the phosphate uptake rate (Correll, 1998;

Markou & Georgakakis, 2011; Rigby et al., 1980). Both algae and bacteria use PO4 for their nutrition, therefore they compete for the available PO4 (Jansson, 1988). The presence of other microorganisms within a culture influences the available amount of phosphorus for algae.

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4.4 Applications of algal biomass Algae have been used as food by humans for thousands of years (Milledge, 2011). Over the last decades the commercial application of microalgae has grown strongly. Microalgae have been used in the production of fish- and live-stock feed, human nutrition and food, pharmaceutical production

(antioxidants, enzymes, vitamins), lipids and poly unsaturated fatty acids (PUFA’s), pigments and colours (carotenoids), polysaccharides, biodiesel production and as biofertilizers (Adamsson, 2000; Banerjee et al., 1997; Milledge, 2011; Mulbry et al., 2005; Perez-Garcia et al., 2011; Pulz & Gross, 2004). Especially the use of algae as biofertilizer is interesting when thinking of the depletion of the phosphate rock reserves for the production of artificial fertilizers and closing the phosphorus cycle.

4.5 Phosphorus removal from anaerobic digested waste streams Microalgae have been used in wastewater treatment systems to recover nutrients like phosphorus and nitrogen (Ashley et al., 2011; Aslan & Kapdan, 2006; de-Bashan & Bashan, 2004). They have also been successfully cultivated on anaerobically digested animal waste streams. The following studies support the option of cultivating algae on effluent from anaerobically digested wastes. 4.5.1. Cyanobacteria

Wu & Pond (1981) cultivated cyanobacteria (Spirulina maxima) on the effluent from anaerobically digested swine-, cattle- and poultry faeces, sewage sludge and swine blood slurry. The aim of the study of Wu & Ponds was to grow algae on effluent streams to reclaim the nutrients within these streams and use the algae biomass as animal feed or human food. Wu & Pond (1981) also examined the high microorganism content of the effluent, stating that exotoxins or metabolites from some microorganisms

might inhibit algal growth. However, they also suggested that ammonia could favour growth of microorganisms in the algae culture tank. Wu & Pond (1981) conclude that “the use of fermented waste to grow algae on for animal feed and human food is of considerable potential importance, but that additional studies need to be done to determine the public health hazard in using waste-grown algae as a nutrient source for animals and humans”. The review of Markou & Georgakakis (2011) discusses several articles on the use of cyanobacteria to remove organic and inorganic pollutants from agro-industrial wastes and waste streams. The most common method to treat agro-industrial wastes and waste streams are by aerobic or anaerobic digestion, removing mainly the organic pollutants (Markou & Georgakakis, 2011). The removal of the inorganic pollutants requires expensive chemical methods (Benemann, 1979). Phosphorus is the most difficult element to remove (de-Bashan & Bashan, 2010). Cyanobacteria and other microalgae can be used as a cheaper and an alternative secondary treatment of waste waters to remove organic and inorganic pollutants. For example, Spirulina platensis can remove up to 99,4% of phosphorus content of digested sago starch effluent (Phang et al., 2000) and 87% of phosphorus from anaerobic effluents from pig waste (Olguin et al., 2003). From the review it can be derived that the dilution of the effluent on which the algae are cultivated is of importance for the nutrient removal by the algae from the effluent. A serious disadvantage of the effluents from wastes and waste waters as cultivation medium is their strong (seasonal) variation in composition and volume (Markou & Georgakakis, 2011). Finally, the reviewers conclude that the cyanobacteria can make a significant contribution to the treatment of agro-industrial wastes and waste streams, reducing their inorganic and organic pollutant significantly. 4.5.2 Freshwater algal species

Wilkie & Mulbry (2002) cultivated benthic freshwater algae on digested and undigested dairy manure. The objective of their research was to assess the ability of benthic freshwater algae to recover nutrients from digested and undigested dairy manure and to evaluate the nutrient uptake rate, dry matter and protein yields in comparison to conventional cropping systems. Increasing the concentration of the nutrients supplied to the algae cultures increased the nutrient content and protein content of the algae. From their research it was concluded that the growth of benthic freshwater algae on digested and

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undigested dairy manure has a potential for nutrient recovery and high protein yields from dairy manures. Wilkie and Mulbry (2002) suggest the use of these algae as a replacement for high-protein feed supplements, due to the high protein yields of the algae.

Kebede-Westhead et al. (2003 & 2004) also cultivated freshwater algae on different concentrations of dairy manure effluent. Mainly filamentous green algae were used, but also some cyanobacteria and diatoms arose during cultivation (2003). The algae were cultivated on laboratory scale algal turf scrubbers (ATS). The authors were not only interested in the nutrient uptake by the algae, but also in the elemental composition of the algal biomass with regard to the uptake of heavy metals by the algae. From the

research performed in 2003 it was concluded that higher irradiance levels and higher concentrations enhanced algal growth. Around 30-59% of phosphorus was recovered from the digested dairy manure (2003). From the research performed in 2004 it was concluded that around 35% of phosphorus was recovered in algal biomass at the highest loading rate. Furthermore, it was concluded that the absolute amount of different elements and heavy metals recovered in the algal biomass generally increased with a higher loading rate, because of higher productivity. The amount of elements within the algae exceeded the maximum tolerable dietary levels in dairy cow feed. However, when using the algal biomass as a potential feed component, it would only constitute as a small portion of the total feed. When using the algae as fertilizers, the loadings of the heavy metals from the algal biomass would be below regulatory limits, when applied in correct volumes. The authors recommend the use of the algal biomass within animal feed, aquaculture and as fertilizer (Kebede-Westhead et al., 2003; Kebede-Westhead et al., 2004). Poultry litter is also a rich source of phosphorus. Singh et al. (2011) cultivated three freshwater microalgae species on effluent from anaerobically digested poultry litter. The algae removed up to 80% of phosphorus from the effluent. Important factors for successful algae cultivation and phosphorus removal were effluent concentration and medium depth. The produced biomass could be used as supplement within animal feed or within bio-energy applications (M. Singh et al., 2011). 4.5.3 Microalgae as biofertilizers

Microalgae can also be used as biofertilizers. This was researched by Mulbry et al. (2005), who produced algal biomass on the anaerobically digested dairy manure with the use of laboratory ATS. The dried algal biomass was applied to soils as slow release fertilizers, resulting in plant growth by the uptake of the algal nitrogen and phosphor. Algal biomass contained approximately 50-70% of phosphor from the original manure. Corn and cucumber seedlings, grown on a potting mixture that was amended with algal biomass, contained 38-60% of the algal phosphorus. This study suggests that the dried algal biomass produced on anaerobically digested dairy manure can substitute commercial fertilizers used for potting systems (Mulbry et al., 2005).

Algae could have a potential as biofertilizers or as soil-conditioning product. An advantage of algae as biofertilizers is that they concentrate the nutrients in a smaller volume than in the original (manure) wastewaters (Wilkie & Mulbry, 2002). Blue-green algae have been evaluated for improving the cultivation of rice (Banerjee et al., 1997). Furthermore, algae application to soils improve soil qualities by

improving soil aggregate stability, minimizing erosion and optimizing aeration, water movement, root development, fertilizer use and water holding capacity (Mackenzie & Pearson, 1979; Metting, 1987; Wilkie & Mulbry, 2002). Using algae as fertilizers, could hence reduce irrigation demand, improve nutrient retention and reduce the potential for groundwater contamination (Wilkie & Mulbry, 2002). Furthermore, using algae as biofertilizers would be an option to close the phosphorus cycle again.

The previously discussed research shows the possibility to cultivate algae on the effluent of anaerobically digested animal waste streams in order to remove phosphorus. The option of cultivating microalgae on the effluent of anaerobically digested human excreta to remove phosphorus has not fully been investigated yet. Within the next chapter the main research questions and methods will be presented.

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5. RESEARCH QUESTIONS AND METHODS In this research the possibility of microalgae to remove the phosphorus from anaerobically digested human excreta has been researched. The reasons for this are 1) phosphorus removal by microalgae from anaerobically digested animal waste streams has been proven successful 2) that phosphorus removal from

anaerobically digested human waste streams with the use of microalgae has not been researched fully yet on and 3) microalgae have broad applicability’s, using the algal biomass as biofertilizers could serve as an option on closing the phosphorus cycle.

5.1 Aim and main research question The aim of this research is to give more insight into the possibility of microalgae as method to remove phosphorus from anaerobically digested human excreta. From this aim the following main research question can be derived:

What is the possibility of microalgae, cultivated on anaerobically digested human excreta, as method for

phosphorus removal?

5.2 Research methodology To answer the main research question research has been performed at two different levels of scale and at two different research departments. The first level was at a small laboratory scale. The data derived from this experimental part of the research was used as input to design a household scale cultivation system. 5.2.1 Experimental research

The experimental part of this research has been executed at the department of Ocean Ecosystems under supervision of Dr. P. Boelen and Dr. H.J. van der Strate. In total 10 weeks of research have been executed at this department. Experimental research was needed, since there has not been much research done on the cultivation of microalgae on anaerobically digested human excreta. Therefore this research can be seen as a pilot study. The department of Ocean Ecosystems has much experience and knowledge on the cultivation and experimentation of different algae species. In order to answer the main research question the following sub-question were defined, which could be answered using experimental methods. a) Can microalgae grow on and remove phosphorus from the effluent?

b) What is the best effluent concentration to grow these microalgae on in terms of phosphorus removal

and growth rate?

5.2.2 Household scale cultivation system

For the second part of this research a household scale cultivation system was designed. Herein the data obtained from the experiments was used as input. This household scale cultivation system was based on the pilot project of the company of DeSaH concerning 32 households. The modelling part of this research has been performed at the department of IVEM (Institute for Energy and Environment) under supervision of Dr. Ir. S. Nonhebel. At the department of IVEM there is much experience on the analysis of natural resource systems and models. The boundary of this part of the research concerned all the households within the Netherlands. The household scale system was used in order to answer the following sub-questions: a) How much phosphorus can theoretically be recovered from the effluent of all the Dutch households

with the use of microalgae?

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b) What would be the technical design of such a household scale system?

c) How does this system compare to the current and past sanitary systems?

Within the following chapters first the experimental part (chapter 6-8) and then the household scale

cultivation system (chapter 9-10) will be described.

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6. EXPERIMENTAL PART – METHODS & MATERIALS 6.1 Experimental design With the use of literature study and advice of experts at the department of Ocean Ecosystems an experimental design was constructed, see figure 6.1. An elucidation of this design is described below.

Figure 6.1: Experimental design

6.2 Microalgae selection The selection of microalgae was based on 1) previously obtained results at the department of Ocean Ecosystems and DeSaH and 2) from literature. Two marine algae species were selected. In specific, the

diatom Phaeodactylum tricornutum (P. tricornutum) and 2) the green algae Dunaliella tertiolecta (D.

tertiolecta). These marine species were selected because fresh water is becoming scarce in the next few decades (UNDP, 2006) and salt water is a more stable medium to cultivate algae on (Provasoli et al., 1957; Provosoli, 1959). The department of Ocean Ecosystems has a lot of experience with the cultivation of these two species. Both species are relatively easy to cultivate and have high growth rates. Phosphorus removal from standard growth media has been achieved with these two species (van den Heuvel, 2011; Wijers, 2011). Additionally, P. tricornutum is an interesting species, since its entire genome has been sequenced (Bowler et al., 2008). Finally, it is interesting to observe the possible differences between the different species and compare these with each other.

6.3 Cultivation media For this research the following media were composed. The first medium is a standard growth medium used at the department of Ocean Ecosystems, which is the f/2-medium (Guillard R.R.L., 1975; Veldhuis

& Admiraal, 1987). This medium was used to cultivate the microalgae stocks and it was used as a control medium. The growth and phosphorus uptake of the microalgae on this control medium were compared to the growth and phosphorus uptake from the other media.

The other media were dilutions of the human derived effluent in natural or artificial sea water. The human derived effluent was provided by the company of DeSaH. This effluent originated from the anaerobic mesophilic digester of the DeSaH pilot project of 32 households. According to measurements of DeSaH

the phosphorus content of the effluent used within the experiments was 89 mg P/L (see appendix C) (Nico Elzinga, 2011-2012). This is 80 times more phosphorus than there is within the control medium. The effluent was diluted using natural or artificial seawater, to create three different media. In specific, 10%, 20% and 50% effluent medium. The 20% and 50% effluent media were constructed using artificial seawater, to keep the salinity around 35‰. These dilutions were chosen, because the company of DeSaH

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had obtained successful results using a 10% effluent medium. In order to see what the optimal concentration of effluent is to cultivate algae on in terms of phosphorus removal, the other concentrations were chosen. For the exact construction and composition of the different media and the effluent see appendix A.2 and appendix C.

6.4 Cultivation conditions Important factors for microalgae growth are light energy and temperature. Therefore, the algae were cultivated under constant light and temperature conditions within a culture chamber. Also a dark:light cycle of 8 hours of darkness and 16 hours of light was implemented, modelling natural conditions. The temperature and light intensity were kept constant to assure that these parameters would not affect the growth of the species. For the specific cultivation conditions see appendix A.3.

The microalgae were cultivated in small 250 ml Erlenmeyer flasks. For every medium type each microalgae was cultivated in triplicate. Medium was added to each Erlenmeyer flask and then the

microalgae from a stock culture were added. These algae from the stock culture were at the end of their growth period, and depleted of phosphorus. This was done, because P-depleted algae have the tendency to take up phosphorus from a medium at a higher rate than P-sufficient algae (Jansson, 1988; S. K. Singh et

al., 2007). The duration of the cultivation of the algae on the different media was based on the growth progression of the different species. The maximum cultivation period was set to 10 days due to the time

frame of 10 weeks in which all the experiments had to be performed.

6.5 Daily measurements Daily samples were taken from each culture to measure for cell density, efficiency of photosystem II, medium concentrations of orthophosphate (PO4), silicic acid (Si(OH)4) and ammonium (NH4). Cell density was measured to calculate the average growth rate and relative growth rate of the species. For the exact methods and calculations used see appendix B.1 until B.3. The efficiency of photosystem II is an indicator of the ability of the algae to perform photosynthesis. This results in a value between 0 (no ability to perform photosynthesis) and 1 (maximum photosynthesis). Yet, the value of 1 is hardly ever reached. For D. tertiolecta and P tricornutum the highest value reached will be around 0.6-0.7. Furthermore, this measurement is a standard procedure used at the department of Ocean Ecosystems. For the exact methods used see appendix B.4.

Daily samples of the medium were taken to assess the concentration of orthophosphate (PO4), silicic acid (Si(OH)4) and ammonium (NH4) within the medium. These concentrations were measured before the algae were added to the medium and directly after the algae were added to the medium. This was done to assess the removal of the nutrients from the medium by the algae. Orthophosphate was measured, since

this is the form of phosphorus readily taken up by algae (Jansson, 1988). Unfortunately, it was technically not possible to measure the phosphorus content within the algae. Silicic acid was measured, because this is a nutrient which is essential for P.tricornutum and to make sure this was not affecting the growth of the algae. Ammonium was measured, because this is also an important nutrient for growth (Guillard R.R.L., 1975; Levasseur et al., 1993) and very high levels of ammonium are present within the effluent. For the

exact methods used to measure these nutrients see appendix B.5 until B.8.

6.6 Other measurements Other factors that could affect growth are pH, salinity and light availability (Graham & Wilcox, 2000). Therefore these factors were also measured. During the cultivation of the algae the salinity and pH of the culture was checked once or twice to see if changes occurred. For the exact methods used, see appendix B.9. Furthermore, the light transmission through all the different media was measured. This was done, because the effluent used within the medium has a brownish colour and contains solids of unknown materials that are making the effluent turbid. The colour and these solids could have light blocking

25

properties. Furthermore, there could be an effect of the storage of the effluent over a longer time period, for example changes in the colour of the medium and precipitation of the materials. Therefore the light transmission through the medium was measured over a light spectrum of 300 until 800 nm. This is the light wavelength range at which algae can absorb light and use this for photosynthesis (Markou &

Georgakakis, 2011). For the salinity and pH of the different media see appendix C. For the protocol used to measure the light transmission, see appendix B.10.

6.7 Data analysis The data obtained from al the measurements were implemented in SigmaPlot. This is a graphical design program in which the graphs presented within the result section were constructed. No statistical analyses were performed, due to time limitations.

In total, 8 successful cultivation experiments were executed. Within the following chapter the results of all these experiments will be presented.

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7. EXPERIMENTAL PART – RESULTS Within this chapter the results from the experiments are presented. First, the growth and nutrient concentration within the different media of D. tertiolecta are revealed, followed by the results of P.

tricornutum. Then the results from the light transmission through the different media will be displayed. Finally, the growth rates and the phosphorus removed by the different species will be presented.

7.1 Results D. tertiolecta Within the following graphs the relative growth and the efficiency of photosystem II of D. tertiolecta are presented in graph A. The concentration of phosphorus (PO4) and ammonium (NH4) are presented in graph B. Note that the NH4 concentration was not measured for the control medium, because this medium contains a different form of nitrogen (NO3). Furthermore, silicic acid (Si(OH)4) was not measured, since this is only an essential nutrient for P. tricornutum. In appendix C the salinity, pH and the nutrient concentrations compared to the control medium within the different media are presented.

7.1.1 Relative growth and nutrient concentrations control medium

In the figure 7.1A the relative growth and the efficiency of photosystem II of D. tertiolecta are presented. In figure 7.1B the PO4 concentration within the control medium is shown.

Figure 7.1: A) Relative growth and efficiency of photosystem II and B) Nutrient concentrations within control medium of D. tertiolecta.

Time in days

0 2 4 6 8

[Pho

sph

ate

] (µ

Μ P

O4)

0

5

10

15

20

25

30

Concentration of phosphate

in medium (µΜ PO4

)

Time in days0 2 4 6 8

Re

lative g

row

th

0

10

20

30

40

50

Eff

icie

ncy p

ho

tosyste

m I

I

0,0

0,2

0,4

0,6

0,8Relative growth

Efficiency of photosystem II

A

B

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What is visible from figure 7.1B is that PO4 is completely removed from the medium after day 1 and then the algae start to grow, as visible from graph 7.1A. The efficiency of photosystem II decreases over time.

7.1.2. Relative growth and nutrient concentrations 10% effluent medium

In figure 7.2A the relative growth and the efficiency of photosystem II of D. tertiolecta is presented. In figure 7.2.B the PO4 and NH4 concentration within the 10% effluent medium are visualised.

Figure 7.2A demonstrates that D. tertiolecta is able to grow in the 10% effluent medium. This was expected, since the company of DeSaH also obtained successful results with the cultivation of algae on 10% effluent medium. The efficiency of photosystem II remains relatively stable around 0.6, indicating that the culture has a good efficiency of photosystem II and is able to perform photosynthesis. In figure 7.2B it is visible that PO4 is removed completely from the medium within 4 days. Furthermore, the NH4 concentration also declines over time.

Figure 7.2: A) Relative growth and efficiency of photosytem II. B) Nutrient concentrations

within 10% effluent medium with D. tertiolecta.

Time in days

0 2 4 6 8

[Ph

osph

ate

] (µ

Μ P

O4)

0

50

100

150

200

250

[Am

mon

ium

] (µ

Μ N

H4)

0

1000

2000

3000

4000

5000

Concentration of phosphate

in medium (µΜ PO4)

Concentration of ammonium in

medium (µΜ NH4)

Time in days0 2 4 6 8

Rela

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gro

wth

0

10

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40

50

Eff

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Relative growth

Efficiency of photosystem II

A

B

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Time in days

0 2 4 6 8

[Pho

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te]

(µΜ

PO

4)

0

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200

300

400

[Am

mo

niu

m]

(µΜ

NH

4)

0

1000

2000

3000

4000

5000

6000

Concentration of phosphate

in medium (µΜ PO4)

Concentration of ammonium in

medium (µΜ NH4)

Time in days0 2 4 6 8

Re

lative

gro

wth

0

10

20

30

40

50

Eff

icie

ncy p

hoto

syste

m I

I

0,0

0,1

0,2

0,3

0,4

0,5

0,6

Relative growth

Efficiency of photosystem II

A

B

7.1.3. Relative growth and nutrient concentrations 20% effluent medium

In figure 7.3A the relative growth and the efficiency of photosystem II of D. tertiolecta are presented. In figure 7.3B the PO4 and NH4 concentrations within the 20% effluent medium are revealed.

As visible from figure 7.3A D. tertiolecta is not able to grow on 20% effluent medium. This corresponds to the results from the efficiency of photosystem II. However, PO4 decreases in the medium as shown in figure 7.3B. Also a decline of NH4 in the medium is visible. Nevertheless, there is no growth of the algae.

7.1.4. Relative growth and nutrient concentrations 50% effluent medium

In figure 7.4A the relative growth and the efficiency of photosystem II of D. tertiolecta are presented. In

figure 7.3B the PO4 and NH4 concentrations within the 50% effluent medium are visualized.

Figure 7.3: A) Relative growth and efficiency of photosystem II. B) Nutrient concentrations

within 20% effluent medium with D. tertiolecta.

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Time in days

0 1 2 3 4 5 6 7

[Ph

osph

ate

] (µ

Μ P

O4)

0

200

400

600

800

1000

1200

1400

1600

1800

[Am

mon

ium

] (µ

Μ N

H4)

0

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10000

15000

20000

25000

30000

35000

Concentration of phosphate

in medium (µΜ PO4

)

Concentration of ammonium in

medium (µΜ NH4)

Time in days0 1 2 3 4 5 6 7

Rela

tive

gro

wth

0

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tosyste

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0,00

0,05

0,10

0,15

0,20

0,25

Relative growth

Efficiency of photosystem II

A

B

Demonstrated in figure 7.4A, is the inability of D. tertiolecta to grow on 50% effluent medium. This

consists with the results of the efficiency of photosystem II. The concentration of PO4 declines rapidly before the algae are added to the medium, from 1.65 mmol P/L until 0.31 mmol P/L. This sharp decline before the algae are added also applies to the NH4 concentration. After the algae are added to the medium, there is a less steep drop in the concentration. The NH4 concentration remains stable after adding the algae.

7.2 Results P. tricornutum In the following graphs the growth and the efficiency of photosystem II of P. tricornutum are presented in graph A. The concentration of PO4, NH4 and Si(OH)4 are presented in graph B. Since the control medium does not contain NH4 this was not measured within this media. Furthermore, NH4 was not measured correctly in the 20% medium, therefore this data is absent. In appendix C the salinity, pH and the nutrient concentrations compared to the control medium within the different media are presented.

7.2.1 Relative growth and nutrient concentrations control medium

In figure 7.5A the relative growth and the efficiency of photosystem II of P. tricornutum are presented. In figure 7.5B the PO4 concentrations within the control medium are visualised.

Figure 7.4: A) Relative growth and efficiency of photosystem II. B) Nutrient concentrations within 50% effluent medium with D. tertiolecta.

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A Time in days

0 2 4 6 8

Re

lative

gro

wth

0

10

20

30

40

50

Eff

icie

ncy p

ho

tosyste

m I

I

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

Relative growth

Efficiency of photosystem II

B Time in days0 2 4 6 8

[Ph

osp

hate

] (µ

Μ P

O4

)

0

5

10

15

20

25

Concentration of phosphate in

medium (µΜ PO4)

From figure 7.5A it can be derived that the algae grow on the control medium. Over time the efficiency of photosystem II decreases slowly. Graph 7.5B shows that PO4 is removed quickly from the medium within 2 days. After two days the algae also start to grow.

7.2.2. Relative growth and nutrient concentrations 10% effluent medium

In figure 7.6A the relative growth and the efficiency of photosystem II of P. tricornutum are presented. In figure 7.6B the nutrient concentrations within the 10% effluent medium are shown.

Figure 7.5: A) Relative growth and efficiency of photosystem II. B) Nutrient

concentrations within control medium with P. tricornutum.

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P. tricornutum is able to grow on 10% effluent medium, as presented in graph 7.6A. Furthermore, a sudden drop in the efficiency of photosystem II is visible from graph 7.6A. The results from graph 7.6B illustrate that PO4 is removed from the medium completely within 7 days. NH4 also decreases slowly over time.

7.2.3. Growth and nutrient removal 20% effluent medium

In figure 7.7A the relative growth and the efficiency of photosystem II of P. tricornutum are presented. In figure 7.7B the nutrient concentrations within the 20% effluent medium are shown.

A Time in days

0 2 4 6 8

Re

lative

gro

wth

0

10

20

30

40

50

Eff

icie

ncy p

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tosyste

m I

I

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

Relative growth

Efficiency of photosystem II

B Time in days0 2 4 6 8

[Pho

sp

ha

te]

(µΜ

PO

4)

0

50

100

150

200

250

[Am

mo

niu

m]

(µΜ

NH

4 )

0

1000

2000

3000

4000

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[Sili

ca

te]

(µΜ

Si(O

H) 4

)

0

20

40

60

80

100

120

140

160

180

Concentration of phosphate in medium (µΜ PO4)

Concentration of ammonium in medium (µΜ NH4)

Concentration of silicate in medium (µΜ Si(OH)4)

Figure 7.6: A) Relative growth and efficiency of photosystem II. B) Nutrient concentrations within 10% effluent medium with P. tricornutum.

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A Time in days

0 2 4 6 8

Re

lative g

row

th

0

10

20

30

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50

Eff

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ncy p

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tosyste

m I

I

0,0

0,1

0,2

0,3

0,4

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0,6

Relative growth

Efficiency of photosystem II

B Time in days0 2 4 6 8

[Ph

osp

hate

] ( µ

Μ P

O4

)

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[Sili

ca

te]

(µΜ

Si(

OH

) 4)

0

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Concentration of phosphate in medium (µΜ PO4)

Concentration of silicate in medium (µΜ Si(OH)4)

From figure 7.7a it is visible that P. tricornutum is able to grow on 20% effluent medium. Again there is a sharp drop in the efficiency of photosystem II. However, it increases again after day 5. Figure 7.7b shows a decrease in the concentration of PO4 within the medium over time. Also a slow decrease in the Si(OH)4 concentration is visible, which rises again after day 3.

7.2.4. Growth and nutrient removal 50% effluent medium

In figure 7.8A the relative growth and the efficiency of photosystem II of P. tricornutum are presented. In

figure 7.8B the nutrient concentrations within the 50% effluent medium are shown.

Figure 7.7: A) Relative growth and efficiency of photosystem II. B) Nutrient concentrations within 20% effluent medium with P. tricornutum.

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Figure 7.8: A) Relative growth and efficiency of photosystem II. B) Nutrient concentrations in 50% effluent

medium with P. tricornutum.

The results in graph 7.8A indicate that P. tricornutum was not able to grow on 50% effluent medium.

This also coincides with the results of the efficiency of photosystem II. The PO4 concentration decreases strongly before the algae are added, from 1.32 mmol P/L until 0.39 mmol P/L. After the algae are added to the medium the PO4 concentration decreases more slowly. The NH4 and Si(OH)4 concentrations stay relatively constant.

7.3 Light transmission As mentioned in the previous chapter, algae need light to grow. The effluent used within the medium has a brownish colour and contains organic materials that are floating around within the effluent. The colour and these organic materials could have light blocking properties. Furthermore, there could be an effect of the storage of the effluent over a longer time period, for example changes in the colour of the medium and

precipitation of the materials. Therefore the light transmission through the medium was measured over a light spectrum of 300 until 800 nm. This was done for every medium type. These results are shown in figure 7.9. In order to see the effect of filtering the effluent, the light transmission through 100% effluent versus 100% filtered effluent was measured. These results are presented in figure 7.10.

A Time in days

0 1 2 3 4 5 6 7

Re

lative

gro

wth

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Eff

icie

ncy p

ho

tosyste

m I

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Relative growth

Efficiency of photosystem II

B Time in days0 1 2 3 4 5 6 7

[Pho

sp

ha

te]

(µΜ

PO

4)

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600

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[Am

mo

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m]

(µΜ

NH

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[Sili

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te]

(µΜ

Si(O

H) 4

)

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Concentration of phosphate in medium (µΜ PO4)

Concentration of ammonium in (µΜ NH4)

Concentration of silicate in medium (µΜ Si(OH)4)

35

7.3.1 Percentage of light transmission through different media

In figure 7.9 the light transmission trough the different media is presented. What is obvious from this graph is that effluent medium has a lower percentage of light transmission than control medium. Secondly, when the dilution of the effluent is higher, a larger percentage of light is transmitted through

the medium. Furthermore, at a lower wavelength, the percentage light transmission decreases.

Figure 7.9: Light transmission through the different media over 1 cm.

7.3.2. Effect of filtering effluent on light transmission

From figure 7.10 the effect of filtering the effluent is presented. This figure shows that filtrating, and thus removing solids from the effluent, strongly increases the percentage of light transmission over 1 cm through the effluent. At 800 nm the light transmission trough 1 cm of unfiltered 100% effluent is 65% compared to 93% of filtered 100% effluent.

Wavelength (nm)

300 400 500 600 700 800

% lig

ht tr

an

sm

issio

n o

ve

r 1

cm

0

20

40

60

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100

Water

Control medium

10% effluent medium D. tertiolecta

10% effluent medium P. tricornutum

20% eflfuent medium D. tertiolecta

20% effluent medium P. tricornutum

50% effluent medium D. tertiolecta

50% effluent medium P. tricornutum

100% effluent

36

Figure 7.10: Effect of filtering effluent on percentage of light transmission.

7.4. Average growth rates and phosphorus removal In table 7.1 the average growth rates and in table 7.2 the phosphorus removal over 4 days are presented.

The average growth rates were calculated over the exponential part of the growth curves of the species. This was mainly between day 1 until day 4. For the exact calculations see appendix B.1 until B.3. The phosphorus removal is calculated from before the algae are added to the medium until day 4, because within this time period the largest share of phosphorus is removed from the medium. This is visible in the previous presented graphs in paragraph 7.1 and 7.2.

Table 7.1: Average growth rate in µ of D. tertiolecta and P. tricornutum on different media.

Medium Type

Species Control (f2) 10% effluent 20% effluent 50% effluent

D. tertiolecta 0.53 0.43 0 0

P. tricornutum 0.84 0.64 0.39 0

Table 7.2: Phosphorus removal from different media over 4 days in mmol P/L and in percentage of total.

Medium Type

Species Control (f2) 10% effluent 20% effluent 50% effluent

D. tertiolecta 0.03 (100%) 0.20 (97.8%) 0.21 (59.4%) (No growth)

1.63 (98.8%) (No growth)

P. tricornutum 0.02 (100%) 0.16% (82.6%) 0.25 (69.9%) (No growth)

1.28 (97.5%) (No growth)

Within the 10% effluent medium D. tertiolecta has the largest phosphorus removal. However, P.

tricornutum was also able to grow on the 20% effluent medium. From this medium even more phosphorus was removed than within the 10% medium by D. tertiolecta. P. tricornutum also has the highest average growth rates on the different media. Even though D. tertiolecta did not grow on the 20% effluent media and both the algae were not able to grow on the 50% effluent medium, phosphorus was removed from the media within these experiments.

Wavelenght (nm)

300 400 500 600 700 800

% lig

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100% filtered effluent

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8. EXPERIMENTAL PART – CONCLUSIONS & DISCUSSION From the results it can be concluded that both D. tertiolecta and P. tricornutum are able to grow and remove phosphorus from anaerobically digested human excreta within a lab scale setting.

The 10% effluent medium is the best concentration to grow these algae on. Within this medium the algae show the highest growth rates and high phosphorus removal. What is especially clear from the results is that the algae start to grow after they have taken up the phosphorus from the medium. This corresponds to the growth results of the algae on the control medium. Furthermore, within the 10% effluent medium the largest share of phosphorus was removed from the medium by D. tertiolecta. Even though P. tricornutum has a higher amount of phosphorus removal from the 20% effluent medium within 4 days, the remaining phosphorus within the 10% effluent medium is lowest with D. tertiolecta. This is an advantage for waste water treatment companies, since they are obliged to remove as much phosphorus as possible from effluent in order to prevent eutrophication. Therefore, it is concluded that D. tertiolecta on 10% effluent medium is the best method to remove as much phosphorus as possible from this human derived waste stream.

When cultivating the algae on higher concentrations of effluent other results are obtained. Within the 20% effluent experiments not all the phosphorus was removed from the medium. Furthermore, P. tricornutum was able to grow on this medium, but D. tertiolecta was not. This indicates that there are other factors within the effluent influencing the phosphorus removal and the growth of the algae. Similar results were obtained within the 50% effluent medium. Within this medium both species were unable to grow.

However, before the algae were added to the 50% effluent medium a sharp drop in phosphorus concentration was observed. In order to explain what factors could have caused this phosphorus removal and inability to grow a literature study was performed.

8.1 Phosphorus removal Factors influencing phosphorus removal are three-fold. Firstly, the PO4 within the medium could be bound and/or converted into an unavailable form of phosphorus by solids, pathogens or other unknown materials present within the effluent (Jansson, 1988). This makes the phosphate unavailable for the algae. Secondly, when the pH increases, PO4 can precipitate, making it unavailable for the algae to take up (Adamsson, 2000; Olsen et al., 2006). Thirdly, when soluble ammonium and phosphorus are available in high concentrations, together with magnesium and a pH above 7.5, struvite forms spontaneously and precipitates (de-Bashan & Bashan, 2004). Within the 20% and 50% effluent experiments artificial seawater was used in order to maintain salinity, containing high amounts of magnesium (see Appendix A.3). Within these experiments the pH during cultivation was 7.7 at its lowest (see appendix C). The combination of the magnesium, high pH and high nutrient concentrations could have resulted in the precipitation of phosphorus in the form of stuvite. Since only the PO4 concentration within the medium was measured it cannot be determined which of these factors could have caused the phosphorus removal. Therefore it is recommended that in future research other forms of phosphate within the medium are measured, together with the concentration of the PO4 within the algae. This needs to be measured to assess if the PO4 is converted into other forms of phosphate and to assess which part of the PO4 has been removed by the algae and which part has possibly precipitated.

8.2 Ammonium toxicity The inability to grow could be caused by ammonium toxicity. The effluent contains very high amounts of NH4 compared to a standard growth media with NH4 (or h/2- medium) (Guillard R.R.L., 1975). The 50% effluent contains 75 times as much NH4 when compared to the h/2-medium. In such amounts, the ammonium concentration could be toxic for the algae (Källqvist & Svenson, 2003; Thomas et al., 1980).

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According to Olguìn and Vigueras (1981) there was a need to dilute the anaerobic effluent of animal waste in order to avoid toxicity caused by NH4 and to avoid excess of turbidity (Olguin & Vingueras, 1981). The amount in which ammonium is toxic for an algae is specie dependent (Guillard R.R.L., 1975; Källqvist & Svenson, 2003). This could explain the inability of the algae to grow in higher concentrations

of effluent. Therefore this NH4 toxicity needs more attention in future research.

8.3 Effluent turbidity As mentioned by Olguìn and Vigueras (1981), it was needed to dilute the effluent to decrease the turbidity of the effluent. Although the light transmission through the medium decreases strongly when the concentration of the effluent medium increases, this is not seen as indicator for growth failure. Even when a 100% unfiltered effluent would be used, the light transmission over 1 cm is still above 50%. The decreased light transmission could however influence the growth rate (Martinez et al., 2012). Therefore it is recommended that in future research the effluent is filtered prior to use as medium. Filtering the effluent decreases the turbidity of the effluent and possibly also the number of micro-organisms within the medium. This could reduce the conversion and/or binding of PO4 by these microorganisms. Furthermore, filtering the effluent increases the light transmission through the effluent perhaps causing an increase the growth rate of the algae. The effect of filtrated effluent medium on algae growth and

phosphorus removal needs to be further investigated. P. tricornutum and D. tertiolecta show promising results as method for phosphorus removal from anaerobically digested human. The ability of other microalgae species to grow and remove phosphorus from human derived effluent also needs more attention. Within this research two marine microalgae were

used, but with an estimated species variety of 200,000 until several million species (Norton et al., 1996) other algae species should also be investigated. Especially cyanobacteria could be interesting to research, since these are known for their high phosphorus uptake. Different cyanobacteria have already been successfully cultivated on digested animal derived waste streams (Markou & Georgakakis, 2011; Olguin

et al., 2003; Wu & Pond, 1981). Additionally, more research is needed to define the optimal effluent concentration at which a certain algae species has an optimal growth rate and the highest phosphorus removal.

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9. SCALING UP The results obtained from the experimental part of this research indicate that microalgae are able to remove phosphorus from a human derived waste stream within a small lab setting. What are the effects when this system would be implemented within all the households of the Netherlands? In this chapter the

possibility of enlarging such a cultivation system within the Netherlands is presented.

9.1 Present and past sanitary systems Within the current sanitary system in the Netherlands human excreta is flushed away with the use large amounts of water. It is collected within a sewage system and transported over large distances to a centralised waste water treatment plant. Here the wastewater is treated to remove phosphorus, using chemical and biological methods. However, through the combination with other streams, this stream is highly polluted and diluted. Therefore the removal of phosphorus of this stream mainly results in

unusable end-products. The current water based sanitation concept was introduced within the 19th century in order to reduce the transmission of diseases through the use of human excreta on agricultural lands, the stench and the unacceptable working conditions when using a bucket system (van Zon, 1986). However, due to the water

based sanitation system the phosphorus cycle has opened up, resulting in eutrophication of waterways and losses of valuable phosphorus. Especially, urbanised areas are becoming phosphorus hotspots (Cordell et

al., 2009). The amount of phosphorus excreted per person per year is 0.3-0.5 kg P through excreta and 0.7-1.0 kg P

through urine (Kirchmann & Pettersson, 1995). The amount of nutrients within human excreta varies per region, diet and culture (Kirchmann & Pettersson, 1995; Mihelcic et al., 2011). Close to 100% of the phosphorus eaten in food is excreted (Jönsson et al., 2004). Globally this leads to around 3 to 3.3 Mt of phosphorus excreted in urine and faeces by the global population (Cordell et al., 2009) With the use of the DESAR concept human excreta, or black water, from a household is concentrated and anaerobically digested for the production of biogas. The theoretical daily black water production consists of the daily number of defecation and urination, which is respectively 1 and 5 times, with an average volume of urine (1.25 L) and faeces (0.25 L) per person per day (Zeeman et al., 2008). With the use of vacuum toilets, using approximately 1 litre water per flush, this results in around 6 litres of flush water per person per day and a total amount of 7.5 litres of black water per person per day (Zeeman et al., 2008). This is a significant reduction in water use compared to the traditional water based sanitation concept, which uses 33.7 litres of water per person per day (Foekema & van Thiel, 2011). This is around 5.5 litres of water per flush. The intention of this new sanitation concept is that concentrating the risks of high nutrients and pathogens in small volumes enables better control and limits the negative environmental effects (Kujawa-Roeleveld & Zeeman, 2006b).

9.2 Phosphorus quantity of the Dutch households In the city of Sneek, DeSaH has started a pilot project concerning 32 households with the DESAR concept. The daily effluent production of these 32 households in Sneek is around 500 L (Nico Elzinga, 2011-2012). The effluent contains nitrogen and phosphorus which are mainly available in the soluble forms of ammonium (NH4) (1100 mg N/L) and orthophosphate (PO4) (90 mg P/L) (Kujawa-Roeleveld et

al., 2006a; Nico Elzinga, 2011-2012; Zeeman et al., 2008). With a daily effluent production of 500 L the total amount of phosphorus present in the effluent is 45 g of P/day. When using D. tertiolecta with a

removal rate of 98%, 44 g P/day can be removed from the effluent. For all the 7.5 million households in the Netherlands (CBS, 2011a), this would result in a phosphorus removal of around 4.0 million kg of P per year. This is equal to the annual artificial P-based fertilizer usage within the Netherlands (CBS, 2011b). For the exact calculation see appendix D.1.

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From this calculation it is derived that the use of the DESAR concept, in combination with microalgae as method for phosphorus removal, could have a significant contribution to the Dutch fertilizer demand. Therefore it is interesting to see what is technically needed when this system is enlarged. Furthermore, it is interesting to analyse if this household scale cultivation system is feasible within the Netherlands.

9.3 Technical design To give an idea of the feasibility of the use of microalgae as method for phosphorus removal, a household scale cultivation system has been designed with the use of a literature study. This system is designed for the 32 households of DeSaH. The data derived from this system will be extrapolated to all the households in the Netherlands. This will give an idea of the size and feasibility of enlarging a small scale lab system into a household based system.

9.3.1. System design

The design of the household scale cultivation system is mainly derived from research performed by Stowa (2011) on effluent polishing with the use of algae (Stowa, 2011a; Stowa, 2011b) and other literature sources. It is based on the best results from the experimental research, which is D. tertiolecta on 10% effluent for 4 days. The following system was designed, see figure 9.1, which will be explained within the next paragraphs.

Figure 9.1: Graphical overview of upscale system design.

Raceway ponds

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9.3.2 Production of 10% effluent

In this system the produced effluent by the anaerobic digester of 32 households is diluted to 10% effluent medium using seawater. This results in 5000 L of medium. To this medium 250 L of D. tertiolecta stock-culture is added, resulting in a total culture volume of 5.25 m3. The amount of stock-culture is added in the same proportion as used within the lab setting (see appendix A.2).

9.3.3 Cultivation system

As a cultivation system the open raceway pond is chosen, since it is the most widely used algal production system and due to the low construction and maintenance costs (Norsker et al., 2011; Stowa, 2011b). An open raceway pond is a shallow outdoor pond system for algal biomass production, in which water is propelled using a paddlewheel (see figure 9.1) (Stowa, 2011b). Within this model a depth of 0.3 meters is selected, because from calculations with a model of Stowa (2011b) the highest algal production and phosphorus removal was achieved with this depth.

9.3.4. Algae stock culture

An onsite algae stock culture is needed in this system. Therefore every raceway pond has its own algae stock, derived from one back-up algae stock culture (see figure 9.1). These small algae stocks are created adding 250 L of algae stock culture to 250 L of medium, resulting in a total volume of 0.5 m3 per algae stock. In total this results in 1000 L of algae stock culture divided over 4 algae stocks for every raceway pond. Therefore a volume of 1.5 m3 is chosen for the large algae stock culture. This algae stock culture is maintained and used as a back-up system in case of algae stock contamination or failure. In order to give a rough impression of the area needed for these algae stocks, it is assumed that all the algae stocks are kept in square containers.

Within this cultivation system also phosphorus deplete algae are used to obtain a high phosphorus removal rate (Jansson, 1988; S. K. Singh et al., 2007). Every time a raceway pond is filled, 250 L of algae stock culture is added. Then the algae stock will receive 250 L of new medium and the algae within the stock culture will grow for 4 days. Adding 250 L of fresh medium, thus a 1:1 ratio of algae stock and fresh medium, will result in a fast phosphorus uptake, since less phosphorus is available in relation to the algae. It can be assumed that after 4 days the algae will be phosphorus depleted and are ready to be used within the raceway ponds (see figure 7.1A&B).

9.3.5 Cultivation conditions

Even though the open raceway pond is an open outdoor system, the same cultivation conditions (light and temperature) as within the closed lab setting are assumed. This is done, because changing factors as light and temperature would change the outcome of the entire experiment, since these are important factors for algal growth (see chapter 4.2 & 4.3). Most important, there is no data available on the effect of these parameters on the growth and phosphorus uptake by the algae from the effluent. The algae culture of D. tertiolecta is kept within the pond for 4 days to achieve the highest phosphorus removal. At the end of day 4 the produced algal biomass is harvested from the pond using a drum filter containing a filter with a mesh size of 10-20 µM (Stowa, 2011b). After the algal biomass is harvested, the pond can be reused for a new batch of medium and algae. The remaining medium can partly be reused, saving water use. In total 4 raceway ponds are needed within this system, since every day 500 L of effluent is produced.

9.3.6 Area needed

With a depth of 0.3 m and a volume of 5.25 m3, the area needed for one raceway pond is 17.5 m2. In the system designed four raceway ponds are used, resulting in a total area needed of 70 m2 for 32 households.

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The total area needed for all the algae stocks with a total volume of 3.5 m3 is 12.25 m2. The total area needed for the 32 households is 82.25 m2. Thus per households an area of 2.6 m2 is needed. When applying all the 7.5 million households in the Netherlands with this system, a total land area of 19 km2 is needed. This is 0.06% of the total land area of the Netherlands (Index Mundi, 2011), around 1/4th of the

city of Groningen (CBS, 2012). For the exact calculations see appendix D.2.

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10. SYSTEM ANALYSIS AND OPTIMALIZATION From the previously presented results, the household scale cultivation system seems to be technically feasible. However, when analysing and comparing the DESAR system to the past and present sanitary systems, the following remarks and recommendations for optimization can be made. This comparison was

performed with the use of literature study.

10.1 Pathogens and odour nuisance One of the largest problems with the 19th century bucket system was the transmission of diseases by human excreta and the odour nuisance (van Zon, 1986). Within the DESAR concept, the anaerobic digester, effluent filtration and salt water usage can have an effect on these aspects.

10.1.1 Anaerobic digestion

Anaerobic digestion reduces the presence of pathogens available within digested source-separated black water (Stowa, 2005b). This compares to results found from anaerobically digesting animal manure

(Bonetta et al., 2011; Wagner et al., 2008; Wilkie, 2000). The retention time, digester type and temperature within the digester have an influence on the pathogen availability and survival rate (Bonetta

et al., 2011; Stowa, 2005b; Wagner et al., 2008). The survival rate of bacteria is also negatively affected by the sudden change from anaerobic conditions to aerobic conditions (Allison, 2011). Furthermore, odour nuisance is also reduced through the anaerobic digestion process (Wilkie, 2000). Therefore, the

digestion step within this system could be seen as a “purification step” of the human excreta. Further research could be performed using a different anaerobic digester with higher temperatures, for example an anaerobic thermophilic digester. This type of digester operates at temperatures higher than 50°C, making it even harder for pathogens to survive (Wagner et al., 2008).

10.1.2 Effluent filtration

Even though the anaerobic digestion process reduces the presence of pathogens, the effluent still contains micro-organisms, pharmaceutical residues, hormones and heavy metals (Kujawa-Roeleveld et al., 2006a;

Kujawa-Roeleveld & Zeeman, 2006b; Stowa, 2005b). Since algae are able to absorb heavy metals (Kebede-Westhead et al., 2003; Kebede-Westhead et al., 2004) this could serve as a problem when the algal biomass is reused for secondary purposes, for example as biofertilizer. The number of pathogens could be reduced by filtering the effluent prior to use as medium (Stowa, 2005b). Filtration is also of importance for the light transmission trough the medium, as presented within the experimental part of this

research (see chapter 7.3.2). Furthermore, storage time is again of importance for the reduction of pathogens (Karak & Bhattacharyya, 2011; Stowa, 2005b). Therefore, options for further research are filtering and storing the effluent prior to use as medium. This would require additional storage facilities, enlarging the area needed for this system. Another option to reduce the available pathogens within the effluent could be treating the effluent with UV-lighting (Chang et al., 1985). The effect of the

pharmaceutical residues and hormones on the algae, their growth, possible uptake and transmission of these elements also needs further attention and research.

10.1.3 Salt water

From literature study it is derived that salt water has a negative effect on the viability and colony formation ability of enteric bacteria, like Escherichia coli (E.coli) (Rozen & Belkin, 2001). The retention time within the salt water is of importance for the survival rate of bacteria (Jamieson et al., 1976). However, according to Rozen (2001) the pathogenicity of enteric bacteria in salt water is still disputed and inconclusive. Therefore, caution is needed before discharging the salt water residue from cultivation into surface waters.

10.2 Water usage and residue One of the problems with the current sanitary systems is the large fresh water usage. With the use of the vacuum toilet within the DESAR concept, water is saved. However, for the cultivation of the algae on the

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effluent large amounts of water are needed. The advantage of using salt water is that it is not as scarce as fresh water. Nevertheless, salt water needs to be transported to the cultivation location, since this is not always locally available. This will require a logistic infrastructure. Otherwise, salt water can be produced with the use of fresh water and aquarium salts (see appendix A.2). Then the question rises on how water

saving this concept really is. In order to reduce water usage within this system other algae species need to be researched which can grow on a higher effluent concentration. This will require a lower dilution of the effluent, thus less water usage.

Another problem that arises when enlarging the system is the salt water residue that remains after cultivation. This residue cannot be discharged onto surface water due to its salinity. It could be reused again to dilute a new batch of effluent. Therefore the pH of this residue needs to be assessed, because this can become high (pH 10- 11) (Stowa, 2011b). This is also a reason for not discharging this water into surface waters (Stowa, 2011b). Therefore, the treatment of this residue needs further attention.

10.3 Societal acceptance It can be expected that the outdoor cultivation of algae on digested human excreta in (densely) urbanised areas will not be easily accepted. Causes for this are the expected stench derived from the ponds, the area needed for the system and mainly health concerns. We are a highly hygienically society, and the chances that people will accept living next to an ‘open sewer’ are very unlikely. The NIMBY-effect (Not In My

back Yard) will occur easily with such an open outdoor system. Therefore the cultivation plants would have to be located outside populated areas, resulting in the transportation of effluent and other resources. This will require the implementation of an infrastructure and further technical planning.

In order to address this problem of societal acceptance, odour nuisance, water usage and space reduction, other cultivation system could be used, like a tubular photobioreactor. This is a closed system in which the cultivation conditions are controllable, less space and water is needed and the biomass productivity is higher (Norsker et al., 2011). Additionally, it is also the cheapest system when looking at biomass productions costs (Norsker et al., 2011). Furthermore, a closed system is also less susceptible for contamination by other algal species and climatic factors (light and temperature) affecting growth and possibly phosphorus uptake (Stowa, 2011b). In regard to odour nuisance a closed system would also be more preferable. Therefore this needs further research. The societal aspect of enlarging such a system will need further attention, as well as the technical consequences caused by this.

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11. DISCUSSION, CONCLUSIONS AND FUTURE CHALLENGES

This study has researched the possibility of microalgae, cultivated on a human derived waste stream, as method for phosphorus removal. This has been researched on two different levels of scale, from a lab scale to a household scale cultivation system.

11.1 The Dutch possibility The results from both scale levels show it is possible to use microalgae as method for phosphorus removal. At the experimental scale, a phosphorus removal rate of 98% was reached by D. tertiolecta within 4 days on a 10% effluent medium. Thus it is concluded that microalgae are a possible method for phosphorus removal. When transferring this data to a household scale cultivation system, this results in a theoretical possibility to remove 4 million kg of P from all the households in the Netherlands by the use of microalgae. This is equal to the amount of artificial P-based fertilizers applied to the Dutch agricultural

lands. Theoretically it would therefore be possible to replace almost the entire artificial P-based fertilizer usage within the Netherlands with microalgae cultivated on the anaerobically digested excreta produced from all the households within the Netherlands.

A small remark needs to be placed by this statement. In this research the data from 32 households was used to calculate the phosphorus removal from all the households within the Netherlands. Even though some large steps in scale were taken, this research does give an indication of the possibility of microalgae

as method for phosphorus removal. It also indicates the phosphorus potential of the excreta of the Dutch. Certainly when mentioning that the amount of excreta produced within large companies or institutions were not taken into account within this research. This indicates that the possible amount of phosphorus removable from digested Dutch excreta is even larger.

The Netherlands is a country which has an improved sanitary system with a large and complex infrastructure. The introduction of the DESAR concept will therefore only be implementable within newly build residential areas. The largest bottlenecks of the outdoor raceway pond system used in this

research are the societal acceptance and the technical challenges. It is expected that the acceptance of such an outdoor system will be low, especially in densely urbanised areas. This is mainly caused by the expected odour nuisance and health hazards of this system, possibly resulting in the NIMBY effect. Therefore it is suggested that if such a system is introduced, it should be placed outside of residential areas. Another option is to introduce this concept only in non-urban areas, since enough space is

available. Also here the societal acceptance remains a hurdle that must be overcome. This requires some technical improvements of the cultivation system, like the usage of photobioreactors instead of outdoor raceway ponds and the implementation of an infrastructure for the salt water supply.

Due to these technical and societal challenges of an enlarged cultivation system, it is concluded that the household scale cultivation of microalgae on anaerobically digested human excreta, as method for phosphorus removal, is technically not feasible within the Netherlands within the next 20-50 years.

11.2 The World possibility Even though the large scale implementation of the source-separated sanitation concept is not feasible

within the Netherlands, it could be implemented within developing countries that are not equipped with such an improved sanitary system. Worldwide 2.6 billion people have no access to improve sanitation facilities, of which 72% lives in Asia (World Health Organisation and UNICEF, 2010). In countries like Sub Saharan Africa, open defection is still widely practiced by 27% of the population (World Health Organisation and UNICEF, 2010). The sanitation concept by DeSaH could serve more than just one

purpose. When installing this sanitation concept in a cheaper version societies would improve their access to improved sanitation. This would also contribute to achieving the millennium goals on improved sanitation (World Health Organisation and UNICEF, 2010), reduce the spreading of disease through human excreta and would also generate energy by the production of biogas. Furthermore, by preventing

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the implementation of water-based sanitation concepts such as we have in the Netherlands, the loss of phosphorus rich human excreta to waterways is reduced. This will contribute to decreasing the eutrophication of waterways. Finally, the algal biomass generated could be applied to agricultural lands as biofertilizers, thus creating a self-sustainable process.

However, the water demand of the household scale system used in this research is rather high. Such amounts of water may not be available for these countries. Even though salt water supply is readily available at sea-side areas, this supply becomes a problem when moving more inland. This also requires the implementation of a technical infrastructure. Perhaps the outdoor raceway ponds are more suitable for

non-urban areas when thinking of the cultivation system needed and the area this requires. Large cities within developing countries like India are even more densely populated than Dutch cities. Therefore it is concluded that this system will not be technical feasible within densely populated cities, due to the area needed for this system and the water demand of the system. Also here the conclusion is drawn that other cultivation technologies need to be investigated which strongly reduce the area and the water demand of this system. This also requires research for other algae species which are able to grow on higher concentrations of the effluent, hereby reducing the water demand of the system.

11.3 Algae as biofertilizers The usage of microalgae cultivated on a human derived waste stream as biofertilizers needs to be further investigated. The ability of the algae to release the recovered phosphorus to surrounding soils and plants needs to be researched. Additionally, the complete composition of the effluent needs to be determined prior to using it as a cultivation medium. This is necessary to assess the effect of possible pathogens,

heavy metals, hormones and pharmaceutical residues on the algae, as well as the ability of the algae to take up these elements and transfer them to other locations when the algal biomass is used for secondary purposes. This form of biofertilizers is preferably used for non-food crops. Finally, the societal acceptance of using biofertilizers produced on digested human excreta also needs further attention.

11.4 The value of human excreta Another conclusion that is derived from this research is that human excreta as source of phosphorus needs serious attention. Especially when thinking of the upcoming phosphorus shortages, the growing human population and its demand for food. The element of phosphorus is of worldwide importance and this research supports the use of algae as method the recover this vital element from the largest point source of phosphorus losses. The phosphorus cycle is disturbed, partly due to water based sanitation concepts causing eutrophication. The new sanitation concept by DeSaH in combination with the use of microalgae is a potential method to reduce the phosphorus losses from human derived waste streams into waterways and therefore needs further research.

11.5 Research at different levels of scale The final conclusion is that research on different levels of scale, as performed in this research, is of great importance. Experimental research at a small scale is fundamental as input for an upscale system. The results derived from a small scale experiment can result in completely different outcomes when implemented at a larger scale, as shown by this research. This gives more insight into the practical

feasibility of implementing an enlarged system. Hence, new research questions are formulated which can be investigated at both levels of scale. The most important future challenges that need to be faced on both scales are 1) putting the large scale cultivation of microalgae on human derived effluent into practice using different cultivation technologies and algae and 2) research on the ability of algal biomass to be used as biofertilizers in order to close the vital phosphorus cycle.

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APPENDIX A – MICROALGAE AND MEDIA A.1 Microalgae species For these experiments two microalgae were used. The salt water diatom Phaeodactylum tricornutum,

(Bacillariophyceae) (strain CCMP Bigalow 632) and the salt water green algae Dunaliella tertiolecta

(Chlorophyceae) (strain CCAP Bigalow 19/27).

A.2 Cultivation conditions The microalgae were cultured in sterile 250 ml Erlenmeyer flask under fluorescent lighting with a light intensity of 100 µmol fotons m-2 sec-1. Using a Quantum/Radiometer/Photometer by Li-Cor (model Li-250 light meter) the light intensity was assessed. This was the maximum capacity of the light installation used. A light-dark cycle of 16:8 hours was implemented using an electric time clock (Ragni & D'Alcala, 2007). The temperature in the cultivation room was kept constant at a temperature of 21°C (Guillard R.R.L., 1975). To each sterile 250 ml Erlenmeyer, 150 ml of cultivation medium was added and 7.5 ml of microalgae stock-culture (ratio 20:1). The cultures were aerated, stirring them daily by hand before taking samples.

A.3 Culture media Different culture media were prepared throughout the experiments. As basis for the media filtered natural seawater (North Sea, Marsdiep) was used. When the concentration of effluent within a medium increased, artificial seawater was used instead of natural seawater. This was due to the fact that the effluent had a salinity of 5.5‰, decreasing the salinity of the natural seawater when added in a higher volume. To ensure that the salinity was not affecting growth, artificial seawater was used instead of which the salinity could be determined on beforehand. Another adjustment was added to the media. After the 10% effluent experiments, the concentration of Na2SiO3.9H2O (53.5 mM) in the media for P. tricornutum was increased. This was adjusted to ensure that this nutrient was available in excess, not becoming a limiting factor for growth. - Natural seawater: Natural sea water from the North Sea (Marsdiep) was filtered using a 0.22 µM paper Whatman filter. The filtered natural seawater was transferred into 1 litre glass bottles which were sterilized by autoclaving.

- Artificial seawater: Aquarium salt (Ao Aqua Medic- No Reef Salt) was used. In order to achieve the required salinity, the amount of aquarium salt that needed to be dissolved in a certain volume of MiliQ was calculated. This differed per medium type. When dissolved in desalted water at 35‰ the artificial seawater contained the following major elements (mg/L): Na (11.000), Mg (1200), Ca (420), K (350), Cl (19700), SO4 (2200), HCO3 (180) and Sr (16).

- f/2 medium: adapted from Veldhuis & Admiraal (1987) and Guillard (1975). This a standard medium used to cultivate microalgae on (Guillard R.R.L., 1975; Veldhuis & Admiraal, 1987). To a sterilized 1 liter glass bottle of natural seawater with a salinity of 35‰, 0.5 ml minor salts, 0.5 ml trace elements and 1.0 ml vitamins were added. The following nutrients were added: 1.0 ml of KNO3 (880 mM) and 1.0 ml of NaH2OPO4 (36 mM). For the medium of P. tricornutum 2 ml of Na2SiO3.9H2O (53.5 mM) was added, since this is a vital nutrient for diatoms. The medium was stirred well and put in the climate chamber to adjust to temperature before it was used.

- Minor salt: 92.4 µM KBr, 11.98 µM SrCl.6H2O, 0.105 µM AlCl3, 0.252 µM RbCl, 0.070 µM LiCl, 0.060 µM KI, 8.09 µM H3BO3

- Trace elements: 5.86 µM Na2EDTA.2H2O, 5.83 µM FeCl3.6H2O, 0.02 µM CuSO4, 0.038 µM ZnSO4.6H2O, 0.021 µM CoCl2.6H2O, 0.455 MnCl2.4H2O, 0.012 Na2MoO4.2H2O

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- 10% effluent medium: 100 ml of effluent was added to 900 ml of sterilized natural seawater (salinity of 35‰) in a 1 liter glass bottle. Minor salts (0.5 ml) and trace elements (0.5 ml) were added as well as vitamins (1.0 ml). No additional nutrients were added, except for the medium of P. tricornutum to which 2 ml of Na2SiO3.9H2O (53.5 mM) was added. The medium was stirred well and put in the climate

chamber to adjust to temperature before it was used.

- 20% effluent medium: 200 ml of effluent was added to 800 ml of artificial seawater (35 g aquarium salt dissolved in 800 ml MiliQ; salinity of 32.3 ± 0.3‰). Vitamins (1.0 ml) were added. No additional nutrients were added, except for the medium of P. tricornutum to which 10 ml of Na2SiO3.5H2O (53.5 mM) was added. The medium was stirred well and put in the climate chamber to adjust to temperature before it was used.

- 50% effluent medium: 500 ml of effluent was added to 500 ml of artificial seawater (32.25 g aquarium salt dissolved in 500 ml MiliQ; salinity of 30.4 ± 0.1 ‰) in a 1 liter glass bottle. Vitamins (1.0 ml) were added. No additional nutrients were added, except for the medium of P. tricornutum to which 10 ml of Na2SiO3.5H2O (53 mM) was added. The medium was stirred well and put in the climate chamber to adjust to temperature before it was used.

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APPENDIX B – MEASUREMENTS AND PROTOCOLS Daily samples were taken from each culture to measure for cell density, efficiency of photosystem II, medium concentrations of orthophosphate (PO4), silicate (Si(OH)4) and ammonium (NH4).

B.1 Cell density Using a flow cytometer (Coulter Epics XL-MCL, Beckman Coulter) the relative cell density (cells ml-1) within each culture was measured. For each microalgae species a separate flow cytometer protocol was constructed. The following formula was used to calculate the number of cells ml-1:

Cell density = (1 / (∆W) – V) * N * D) (1) In which: ∆W = Difference in tube weight before and after flowcytometry. V = Dead volume of flow cytometer. N = Number of cells counted during flowcytometry. D = Dilution of culture for use in flow cytometer.

B.2 Average Growth rate The average growth rate (a), expressed in µ, of the algae was calculated by taking the natural logarithm

(Ln) from the cell densities and fitting a linear trend line (� � �� � �� through the exponential growth phase of the algae. The exponential growth phase lay almost always between day 1 and 4.

B.3 Relative growth The relative growth of the algae was calculated by the following formula:

Relative growth = Ct/C0 (2)

In which:

Ct= Cell density at time t. C0= Cell density at time 0.

B.4 Efficiency of photosystem II The maximum quantum yield of photosystem II (Fv/Fm) was measured daily using the Pulse Amplitude Modulated (PAM) (Walz Mess und Regel technik: PAM-control S/N: UKEA0231 & Water-ED/B S/N: EDEJ0116). For the experiments with f/2-medium, 10% effluent medium and 50% effluent medium the PAM-control was used. Then this was replaced by the Water-ED/B PAM. This water PAM was much more sensitive than the control PAM. Furthermore, the protocol was adjusted when during the 50% effluent medium experiment it was discovered that the medium itself exhibited fluorescence lighting. See the following protocols.

PAM-control protocol: This protocol was used within the experiments with the f/2-medium, 10% effluent medium and 50% effluent medium.

1) Take a 5 ml sample of the culture. 2) Keep this in darkness for 15 minutes. 3) Take some natural seawater and use this to auto-zero the PAM. Notate the F offs value. 4) Put the sample into a clean PAM-cuvette.

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5) Notate the Ft value. This is the background fluorescence or steady-state yield of fluorescence (Maxwell & Johnson, 2000). The Ft value has to be between 200 and 500. The optimal Ft value is 300. Therefore, adjust the Pm-gain and Out-gain in the right order (see manual PAM).

6) If the Ft is too high dilute the sample using the natural seawater and in that way that the Ft values

lies between the 200 and 500. 7) When the Pm-gain and/or Out-gain is changed, auto-zero the PAM again using the natural

seawater. 8) Give the sample the light shock. 9) Notate the Pm-gain, Out-gain, F, Fm and Yield (Fv/Fm) values.

10) Clean the used cuvettes by washing them three times with MiliQ and if they contained effluent also once with alcohol.

Water PAM protocol: This protocol was used within the 20% effluent experiments.

1) Take a 7 ml sample of the culture. 2) Keep this in darkness for 15 minutes. 3) Take 3.5 ml of the sample and filtrate this using a 0.45 µM Whatman filter. This filtrated sample

is used to auto-zero the PAM. Notate the Ft value of the filtrate and notate the F offs value. 4) Put the left over 3.5 ml sample into a clean PAM-cuvette. 5) Notate the Ft value. This is the background fluorescence or steady-state yield of fluorescence

(Maxwell & Johnson, 2000). The Ft value has to be between 200 and 500. The optimal Ft value is 300. Therefore, adjust the Pm-gain and Out-gain in the right order (see manual PAM).

6) If the Ft is too high dilute the sample using the filtrate. In that way you take into account the fact that the medium itself has a Ft value and you don’t need to correct for this again. If you would dilute the sample using natural seawater, then you would also have to dilute the filtrate with natural seawater and measure the Ft again of this medium and auto-zero the PAM again.

7) When the Pm-gain and/or Out-gain is changed, auto-zero the PAM again using the filtrate. 8) Give the sample the light shock. 9) Notate the Pm-gain, Out-gain, F, Fm and Yield (Fv/Fm) values. 10) Clean the used cuvettes by washing them three times with MiliQ and if they contained effluent

also once with alcohol.

B.5 Nutrient concentrations To assess the phosphorus, ammonium and silicate concentration within the different media, every day a 5 ml sample was taken from the medium and filtered using a 0.2 µM or 0.45 µM Whatman filter and a

plastic syringe. The sample was collected in a 5 ml plastic vial, labelled and stored at 4°C before further analyses.

B.6 Phosphorus measurements To determine the orthophosphate concentration within the medium, the molybdenum blue method was

used by Murphy and Riley (1962) (Murphy & Riley, 1962). For these measurements the Lambda 25 UV/VIS spectrometer from Perkin Elmer Instruments was used. When this one was defect the Cary 3E UV-Visible spectrophotometer by Varian was used. Both instruments have a double beam setting which was used. As a blanco MiliQ was used. For each spectrophotometer a new calibration series was made according the following protocol.

Phosphate protocol: Preparation of reagents:

A) Ammonium molybdate ((NH4)6Mo7O24.4H2O): 8 g in 100 ml MilliQ (dissolves slowly)

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B) Sulfuric acid 10N: 140 ml concentrated sulfuric acid in 500 ml MilliQ (Recommended: place the

beaker in ice bath, the reaction is exothermix) C) Ascorbic acid 0.2 M: 2.64 g in 75 ml MilliQ. Store in fridge. D) Potassium timonyltartrate (tartar emetic): (KSbO.C4H4O6) 0.5486 g in 100 ml MilliQ. Store in

fridge. To complete the reagent, mix the liquids in the following ratio prior to use: A:B:C:D - 3:10:6:1. The reagent mixture has a light-yellow colour.

Measurements: 1. Ad 0.1 ml reagent mix to 1 ml sample, shake well on a vortex 2. Leave for 30 minutes 3. Measure the absorption with the spectrophotometer at 882 nm (auto-zero with MiliQ) 4. Calculate the total amount of phosphate by creating a calibration curve Calibration curve: - Dry around 1 g of KH2PO4 on a petri dish in a stove at 60°C for 30 min - Weigh 0.0340 g of this dried KH2PO4 and dissolve it in 50 ml MiliQ (end concentration of 50

mM) - This 50 mM stock is diluted 100x in MiliQ (1.0 ml Stock in 100 ml MiliQ) to obtain a 50 µM

PO4 stock-solution. - The following calibration series is made in 15 ml plastic tubes adding the following amounts of

MiliQ and 50 µM PO4-stock.

Table B.1: Calibration series phosphorus measurements

ml 50 µM PO4-stock

0 0.5 1.0 2.0 2.5 3.0 5.0 7.5 10.0

ml MiliQ 10 9.5 9.0 8.0 7.5 7.0 5.0 2.5 0

µM PO4 0 2.5 5.0 10.0 12.5 15.0 25.0 37.5 50.0

- Apply all the steps which are mentioned within the measurement section on the samples from the

calibration series to obtain which absorption corresponds to a certain concentration of PO4.

B.7 Ammonium measurements To measure the ammonium concentration a NH4 kit was used by JBL (GmbH & Co. KG). The kit has a measurement range of 0.25 mg N/L – 6.0 mg N/L, contains three solutions and a colour scale with 8 colours, ranging from light green to dark olive. For this NH4 measurement, only the samples of one culture series per experiment were used instead of all the three culture series of a species. This was done due to the limited quantity of the kit. The samples needed to be diluted on beforehand, since most of the samples exceeded the measurement range of the kit.

Protocol ammonium kit 1) Dilute the sample until it is within the range of the kit. 2) Add to 5 ml sample, 4 drops of solution 1 and shake.

3) Add 4 drops of solution 2 and shake. 4) Add 5 drops of solution 3, shake and leave for 15 min. 5) Compare the colour of the sample with the provided colour scale and determine the concentration

of sample.

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B.8 Silicate measurements In order to assess the silicate concentration within the medium of the cultures with P. tricornutum the following protocol was used. This protocol is also based on an ammonium molybdate reaction.

Silicate protocol Use only plastic bottles and vials for this measurement and its preparations.

Preparation of reagents: Molybdate Reagent

Fill a plastic container with 300 ml MilliQ and ad 4.0 g ammonium paramolybdate. Ad 12 ml 37% HCl and fill up to 500 ml with MilliQ. Protect bottle against light (Note: if white precipitation forms on the

sides of the container the reagent should be discarded) Reducing Reagent A) Sulfuric acid - Ad 250 ml H2SO4 to 250 ml MillQ in a beaker. When cooled make sure the total is 500 ml, if not add extra MilliQ to fill up. (Recommended: place the beaker in ice bath, the reaction is

exothermix) B) Oxalic acid - Dissolve 50 g oxalic acid in 500 ml MilliQ in a plastic container. Shake for 10 minutes. C) Metol - Dissolve 0.6 g Na2SO3 in 50 ml MilliQ in a plastic container. Once dissolved ad 1.0 g 4-methylaminophenol Sulfate. Mix well on vortex, dissolves slowly. D) MilliQ To complete the reagent mix the liquids in the following ratio: A:B:C:D - 10:10:17:13. Measurements:

1. Ad 1.2 ml MilliQ, 0.6 ml Molybdate Reagent and finally 0.3 ml sample to a 5 ml vial 2. Leave for 10 minutes 3. Ad 0.9 ml Reducing Reagent, mix well 4. Incubate for 2 hours 5. Measure the absorption with the spectrophotometer at 810 nm (auto-zero with MilliQ) 6. Calculate the total amount of silicate by creating a calibration curve

Calibration curve:

- Dry ±0.3 g of Na2SiF6 on a petri dish in a stove at 85°C for 1.5 hours. ATTENTION: Toxic! - Stock I: Weigh 0.2351 g of dried Na2SiF6 and put it in a 1 litre plastic bottle. Add 300 ml of

MiliQ and shake well to dissolve. Add 2 NAOH tablets and add MiliQ until 500 ml is reached (end concentration of 1.25 mM)

- Stock II: Add 25 ml of stock I to 225 ml of MiliQ in a plastic bottle (end concentration 125µM) - The following calibration series was made in 15 ml plastic tubes adding the following amounts of

MiliQ and stock II.

Table B.2: Calibration series of silicate measurements

ml stock II 0 0.5 1.0 2.0 2.5 3.0 5.0 7.5 10.0

ml MiliQ 10.0 9.5 9.0 8.0 7.5 7.0 5.0 2.5 0

µM Si(OH)4 0 6.25 12.5 25 31.25 37.5 62.5 93.75 125

- Apply all the steps which are mentioned within the measurement section on the samples from the

calibration series to obtain which absorption corresponds to a certain concentration of Si(OH)4.

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B.9 Salinity and pH measurements The salinity and the pH of the effluent and cultures were measured once or twice during the experiments.

The salinity was measured using the TetraCon 325 by WTW (order number: 2C20-0011). The pH was measured using the 780 pH meter by Metrohm. The pH meter was calibrated, before each measurement of the pH.

B.10 Light transmission measurements To determine the light transmission through the effluent and the different media, an undiluted sample of the effluent and the media was taken and put into a 1 cm3 plastic cuvette. The light transmission through the sample was measured using the Lambda 25 UV/VIS spectrometer from Perkin Elmer Instruments. When this one was defect the Cary 3E UV-Visible spectrophotometer by Varian was used. Both instruments have a double beam setting which was used. As a blanco MiliQ was used. The light transmission over 1 cm was measured from a wavelength range of 800 until 300 nm. The data was collected using a floppy disk and transferred into an excel file.

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APPENDIX C – EFFLUENT AND MEDIA COMPOSITION Table C.1: Effluent values from the effluent obtained bu DeSaH. The effluent has a salinity of 5.5‰ and pH of 7.5.

Component mg/L g/mol mM 100% µM 10% µM 20% µM 50%

COD t* 1030 -

COD f** 874 -

P total 96.5 31 3.11 311.29 622.58 1556.85

PO4-P 89.75 31 2.90 289.52 579.04 1447.60

N total 1245 14 88.93 8892.86 17785.71 44464.3

NH4-N 1040 14 74.29 7428.57 14857.14 37142.85

NO3-N 3.88 14 0.28 27.50 55.00 137.5

NO2-N 0.32 14 0.02 2.29 4.57 11.45

VFA*** 173 -

* COD t stands for Chemical Oxygen demand of total ** COD f stands for Chemical Oxygen demand of filtered fraction *** VFA stands for Volatile Fatty Acids Table C.2: Salinity, pH and nutrient concentrations within the different media for D. tertiolecta.

Medium Type

Control (f2) 10% effluent 20% effluent 50% effluent

Salinity (‰) 35 33 30.5 30.3

pH - 8.7 8.5 7.7

P-PO4 (µM) 25 204 355 1646

N-NH4 (µM) - 4285 5357 30357

Table C.3: Salinity, pH and nutrient concentrations within different media for P. tricornutum.

Medium Type

Control (f2) 10% medium 20% medium 50% medium

Salinity (‰) 35 33 30.7 30.5

pH - 9.1 8.5 7.7

P-PO4 (µM) 21 197 356 1316

N-NH4 (µM) - 3214 - 21428

Si-Si(OH)4 (µM) - 155 328 386

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APPENDIX D – CALCULATIONS UPSCALED SYSTEM

D.1 Calculations P-removal using D. tertiolecta on 10% effluent medium The parameters used to calculate the amount of phosphorus removed by D. tertiolecta from the digested human excreta per household and for all the households in the Netherlands are presented in table D.1.

Table D.1: Parameters used within calculations for P-removal from Dutch households.

Parameters

Number of households DeSaH (Nico Elzinga, 2011-2012)

32

Effluent production (Nico Elzinga, 2011-2012) 500 L/day

P-content effluent (Nico Elzinga, 2011-2012) 89.75 mg P/L

P-removal D. tertiolecta 97.6%

Number of households Netherlands 2010 (CBS, 2011a)

7,473,438

Amount of artificial P-based fertilizers applied to agricultural lands of the Netherlands in 2010 (CBS, 2011b)

4.0 *106 kg P

D.1.1 Calculations P-removal Dutch households

(1) The 32 households of DeSaH produce 500 litres of effluent per day with a phosphorus

concentration of 89.75 mg P/L. These 500 litres of effluent contain: 500* 0.08975 = 44.88 g P/day.

(2) Within the lab setting a removal of 97.8% of the phosphorus within 4 days by D. tertiolecta. This results in: (97.8* 44.88)/100= 44.3 g P removal per day.

(3) Per household per day this results in: 44.3/32= 1.38 g of P.

(4) Per household per year this is 1.38 g * 365 = 0.5 kg P. (5) For all the households in the Netherlands in 2010 this would result in 7,473,438* 0.5 kg =

3.74*106 kg P removal per year. (6) The amount of artificial P-based fertilizers applied to agricultural lands in 2010 is 4 million kg P.

Thus the percentage of phosphorus removed from digested human excreta using D. tertiolecta, compares to (3.74*106/ 4.0 *106)*100 = 93.5% of the artificial P-based fertilizers applied to agricultural lands within the Netherlands.

D.2 Calculations area needed for cultivation system To calculate the area needed for the upscale cultivation system the following parameters were used. See table D.2.

Table D.2: Parameters used for calculations.

Parameters

Height raceway pond 0.3 m

Volume raceway pond 5.25m3

Number of households DeSaH (Nico Elzinga, 2011-2012)

32

Number of households Netherlands 2010 (CBS, 2011a)

7,473,438

Land area of the city of Groningen (CBS, 2012) 78.05 km2

Land area the Netherlands (Index Mundi, 2011) 33,893 km2

Volume all algae stocks 3.5 m3

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D.2.1 Calculations

(1) The area needed for 1 pond is 5.25 m3/ 0.3 m = 17.5 m2 (2) For 4 ponds: 4* 17.5 m2 = 70 m2 (3) The area needed for all the algae stocks with a volume of 3.5 m3: 3.5 m * 3.5 m= 12.25 m2

(4) The area needed per household is (70 + 12.25)/ 32= 2.57 m2 (5) The area needed for all the households in the Netherlands = 7,473,438 * 2.57 m2 = 19,209,052 m2

= 19.21 km2. This is around 1/4th of the area of Groningen (78.05/19.21= 4.06) (6) This compares to (19.21 / 33,893)* 100= 0.06% of the total land area of the Netherlands.