Karin Larsdotter (2006): Microalgae for phosphorus removal from wastewater in a

Nordic climate. A doctoral thesis from the School of Biotechnology, Royal Institute of
Technology,Stockholm,Sweden.
ISBN: 91-7178-288-5

Abstract
As part of a research project aiming to develop and evaluate a hydroponic system for
wastewater treatment in Sweden, extended nutrient removal by microalgae was tested. The
hydroponic/microalgal wastewater treatment system was built in a greenhouse in order to
improvegrowthconditionsforplantsandalgae.Studiesonthetreatmentstepwithmicroalgae
showed that phosphorus removal could be successfully accomplished owing to the combined
effect of phosphorus assimilation and biologically mediated chemical precipitation of calcium
phosphates. This precipitation was mainly induced by the increased pH in the algal cultures,
andthepHincreasewasinturnaresultoftheinorganiccarbonassimilationbythealgae.The
results showed that the algal growth was mainly light limited which resulted in higher algal
biomass density and also lower residual nutrients in the water at longer hydraulic retention
times (HRT). In contrast the phosphorus removal rate was load limited, i.e. shorter HRT gave
higherremovalrates.Thisloaddependencywasduetothechemicalprecipitation,whereasthe
phosphorus assimilation was dependent on algal growth. Furthermore, results from an
intensive study during summer showed that culture depths of 17 cm gave higher removal
efficiencies(78%92%)thanculturesof33cm(66%88%).Ontheotherhand,theremovalrate
perareawashigherinthedeepercultures,whichimpliesthatthesemaybepreferredifareais
ofconcern.
Nitrogen removal was achieved mainly by the assimilation of nitrate to algal biomass,
and removal efficiencies of around 40% (nitrate) could be reached for most parts of the year
althoughthenitrogenremovalperformancewasquiteuneven.Upto60%80%couldhowever
bereachedduringsummerintheshallowcultures.Anetremovalintotalnitrogenofupto40%
wasobservedintheshallowculturesduringsummer,whichwasmostprobablyaconsequence
ofgrazingzooplanktonandsubsequentureaexcretionandammoniavolatilisationasaresultof
thehighpHvalues.
Overtheyear,therewerelargefluctuationsinalgalgrowthandremovalefficiencyasa
resultoftheseasonalvariationsinlightandtemperature.Duringwinter,phosphorusremoval
efficiencieslowerthan25%wereobservedintheshallowtanksandlowerthan10%inthedeep
tanks.Additionalilluminationduringwinterimprovedthephosphorusremovalintheshallow
culturesbutdidnothaveasignificanteffectonthedeepcultures.Suchadditionalillumination
increasesthetotalenergydemandofthesystem,andhencealternativemethodsforphosphorus
removalduringwinterwouldprobablybemoreeconomicalunlessthealgalbiomassproduced
hadgreatcommercialvalue.
Key words: assimilation, hydroponics, light, microalgae, nitrogen, phosphorus, plants,
precipitation,wastewatertreatment

Sammanfattning
Som del i en studie dr hydroponisk avloppsrening underskts testades ven
mikroalgers frmga att rena avloppsvatten frn fosfor och kvve. Avlopps
reningssystemet byggdes i ett vxthus fr att under en strre del av ret kunna
tillhandahlla en god milj fr vxterna i den hydroponiska odlingen och ven fr
algerna. Studier av reningssteget med mikroalger visade att algerna kunde anvndas
framfrallt fr fosforrening, eftersom algernas fosforassimilation kombinerades med
utfllning av kalciumfosfater. Denna utfllning var en direkt fljd av det hga pH
vrdet i algkulturerna som orsakats av algernas upptag av oorganiskt kol. Resultaten
visade vidare att algtillvxten var ljusbegrnsad vilket gav hgre algtthet samt lgre
kvve och fosforkoncentrationer vid lngre hydraulisk uppehllstid (HRT). Fosfor
avskiljningen andra sidan var fldesbegrnsad, d.v.s. kortare HRT gav hgre
reningshastighet. Detta berodde p att den kemiska utfllningen var fldesberoende,
medanfosforassimileringenvarbegrnsadavalgtillvxt.Grundakulturer(17cm)visade
sigunderenintensivstudiepsommarenrenamerfosfor(78%92%),nlitedjupare
kulturer (33cm, 66 % 88 %). andra sidan hade de djupare kulturerna en hgre
reningshastighetper ytenhet,vilketmedfr attdessakanvaraattfredraom man har
begrnsadeyttillgngar.
Kvvereningen var frmst en effekt av algernas nitratupptag, och en
reningseffektivitetpcirka40%(avnitrat)visadesigvaramjligunderstrredelarav
retvenomkvvereningenvarganskaojmn.Reningseffekterpupptill60%80%
erfors ven i de grunda algkulturerna p sommaren. Sommartid kunde ocks en
reduktion i totalkvve med upp till 40 % ses i de grunda tankarna. Detta var
frmodligenenfljdavattzooplanktonbetatalger,ochattderasureautsndringfljts
avammoniakavgngtillluftensomeffektavdehgapHvrdena.

verretsgsstorafluktuationerialgtillvxtochfosforreningseffektivitet,vilket
var en fljd av de rstidsberoende variationerna i vrme och ljus. Under vintern
avskiljdes mindre n 25 % av fosforn i de grunda kulturerna och under 10 % i de
djupare kulturerna. Tillggsbelysning visade sig frbttra reningseffekten ngot i de
grunda tankarna men inte i de djupa. Elektriskt ljus p vintern kar frstsden totala
energifrbrukningen, och alternativa metoder fr fosforrening br drfr vervgas
under vintern om inte algerna som produceras kompenserar energifrbrukningen
genometthgtmarknadsvrde.

ii

Listofpapers
This thesis is based on the following papers, whichin the textare referred to by their
Roman numerals. Papers IIII are reproduced with the kind permissionof the
publishers.

I.
Norstrm, A., Larsdotter, K., Gumaelius, L., la Cour Jansen, J. and Dalhammar,
G. (2003). A small scale hydroponics wastewater treatment system under Swedish
conditions.Wat.Sci.Technol.48(1112):161167.

II.
Larsdotter, K., Norstrm, A. and Dalhammar, G. (2004) Theoretical energy
requirementsforhydroponicwastewatertreatment.Vatten60:187191.

III.
Larsdotter, K., Sderbck, E. and Dalhammar, G. (2004). Phosphorus removal
fromwastewaterbymicroalgaeinagreenhouseinSweden.WaterEnviron.Manag.Ser.
3:183188.IWAPublishing.

IV.
Larsdotter,K.,laCourJansen,J.andDalhammar,G.Microalgaeasaphosphorus
trapafterhydroponicwastewatertreatment.Submittedmanuscript.

V.
Larsdotter, K., la Cour Jansen, J. and Dalhammar, G. Biologically mediated
phosphorus precipitation in wastewater treatment with microalgae. Submitted
manuscript.

VI.
Larsdotter, K., laCourJansen,J. andDalhammar, G.Phosphorusremovalfrom
wastewater by microalgae in a Nordic climate a yearround perspective. Submitted
manuscript.

Relatedpaper
Larsdotter,K.(2006)Wastewatertreatmentwithmicroalgaealiteraturereview.
Vatten62

Relatedconferencecontribution
Larsdotter,K.,Oliviusson,B.,Sderbck,E.andDalhammar,G.(2002)Phosphorus
removalfromwastewaterbymicroalgaeinagreenhouseinSwedenduringwinter.
Posterpresentationatthe1stCongressoftheInternationalSocietyforapplied
Phycology,9thInternationalConferenceonAppliedAlgology,Almeria,Spain.
iii

Abbreviations
BOD
Ca2+
CO32
COD
EBPR
HCO3
HRAP
HRT
Mg2+
N

Biochemicaloxygendemand
Calcium
Carbonate
Chemicaloxygendemand
EnhancedBiologicalPhosphorusRemoval
Bicarbonate
Highratealgalpond
Hydraulicretentiontime
Magnesium
Nitrogen

Phosphorus

iv

Contents
1.Introduction.................................................................................................................................. 1
2.Aimandscope ............................................................................................................................. 3
3.Background .................................................................................................................................. 4
Wastewaterandwastewatertreatment .............................................................................. 4
Hydroponicsandothergreentreatmentprocesses .......................................................... 6
Wastewatertreatmentwithmicroalgae .............................................................................. 7
Growthinlightlimitedcultures .................................................................................... 8
Phosphorusremovalinalgalcultures......................................................................... 11
Nitrogenremovalinalgalcultures .............................................................................. 12
Harvesting ....................................................................................................................... 13
Algalwastewatertreatmentathigherlatitudes ........................................................ 14
4.Resultsanddiscussion.............................................................................................................. 16
Thehydroponicwastewatertreatmentplantatverjrva,adescription(PaperI)... 16
Energyrequirementsofhydroponicwastewatertreatment(PaperII) ........................ 18
Thepilotstudyonmicroalgaeinthegreenhouse(PaperIII) ........................................ 20
Themicroalgaltreatmentstep(PaperIV)......................................................................... 23
Thephosphorusremovalmechanisms,alabscalestudy(PaperV) ............................ 27
Thewholeyearstudyperformancevariationsovertheyear(PaperVI) ................. 30
Discussionconcerningtheapplicabilityofthetechnique .............................................. 33
5.Conclusions ................................................................................................................................ 35
6.Acknowledgements/Tack....................................................................................................... 37
7.References................................................................................................................................... 39

vi

1.Introduction
Swedish water and sanitation systems are functioning well from a technical point of
view; most people have access to clean water, and the wastewater in urban areas is
taken care of and treated centrally in modern wastewater treatment plants. From an
internationalviewpoint,Swedenisverywelldevelopedregardingdrinkingwatersafety
and wastewater treatment. However, in order to secure a sustainable development of
the Swedish water and sanitation systems and to meet future demands regarding
function,healthandenvironmentalaspectsitmaybenecessarytoreassessthesolutions.
The Swedish research programme Sustainable Urban Water Management, or
Urban Water in short, has aimed to develop support for strategic decisions on future
water and wastewater systems (Urban Water, 2002). The main questions in the
programme have been: How should the urban water and wastewater systems be
designedandoperatedinafuturesustainableSweden?Willthesustainablewaterand
wastewatersystemsofthefuturebeimprovedversionsofthosethatexisttoday,orwill
therebesomeradicalchanges?Aconceptualframeworkwasdevelopedthatdescribes
theinterplaybetweenusers,technologyandorganisation,andfivegroupsofcriteriafor
sustainability were also defined: health, environment, economy, socioculture and
technicalfunction(Figure1).Eachgroupofcriteriahasanumberofindicatorswhichare
quantifiable(Hellstrmetal.,2000).Theprojectdescribedinthisthesisfitsinmainlyin
the technology subsystem, and deals primarily with the environmental criteria
concerningeutrophicationanduseofnaturalresources.

Figure1.TheUrbanWaterconceptualframework(UrbanWater,2002).

AlthoughtheSwedishurbanwastewatertreatmentsystemsoftodayareefficient,some
drawbacks are sludge production and the need for chemicals. In one of the projects
within the Urban Water programme, an alternative system was therefore suggested
whichcombineshydroponicswiththebiologicalstepsusedinconventionalwastewater
treatment.Themainadvantageswereexpectedtobethatnochemicalswereusedinthe
process,thatgreenbiomasswasproducedinsteadofsludgeandthatvaluableproducts
couldberecoveredfromthenutrientrichwastewater.Thecultivationofvaluableplants
as an integrated part of wastewater treatment sounded attractive, and the question to
answer was rather whether this kind of ecotechnological approach is suitable in the
Swedish climate, and if it could contribute to a more sustainable development of the
water and wastewater sector. In the project, which was called Integrated product
recovery and wastewater treatment, a hydroponic wastewater treatment pilot plant
wasbuiltinagreenhouseatverjrvagrd,afarmclosetoStockholm.Severalspecies
ofgreenplantsweresuccessfullycultivatedandharvested,anditwasobviousfromthe
start that hydroponic cultivation of plants in wastewater is relatively easy. However,
since the effluent standards imposed on the treatment plant, for phosphorus in
particular, were hard to achieve completely with the current design of the treatment
plant, an additional green treatment was also proposed microalgae. The microalgae
functionedmainlyasaphosphorustrapafterthehydroponics,butalgalbiomasscould
potentially also be used for other purposes, e.g. production of pharmaceutical
substances (Borowitzka, 1988; Glombitza and Koch, 1989), food (Grobbelaar, 1982),
animal feed (Becker,1988), fertilizers (Benemann, 1979), fuel (Calvin and Taylor, 1989)
andhydrogengasproduction(Benemann,2000)(areviewcoveringmostapplicationsis
alsogivenbyRichmond(1990)).

2.Aimandscope

Besides the construction and evaluation of the hydroponic wastewater treatment

system, the aim of the work presented in this thesis has mainly been to construct a
microalgaltreatmentstepforthepolishingofresidualnutrientsintheeffluentfromthe
hydroponics, and to evaluate the suitability of the process in Sweden. Some questions
havebeen:

Canmicroalgaebeusedforextendednutrientremovalfromwastewaterin
hydroponictreatmentsystems?

Whatsortsofmicroalgaeworkwellinthetreatmentprocess?

What phosphorus and nitrogen removal rates and efficiencies can be

expected?

Whatarethemechanismsforphosphorusremoval?

Howdoestheperformancevaryovertheyear?

Howcanthenutrientremovalbeoptimised?

Whatistheareademand?

Theresultsarenaturally dependent onthe wastewater compositionand climateat the

specificsite,andalsoonthegreenhouseandbioreactordesign.Nevertheless,theresults
presentedgiveusefulguidanceforthesuitabilityofsimilarlightdependentwastewater
treatmenttechniquesinaNordicclimate.

3.Background
Wastewaterandwastewatertreatment
Domesticwastewatercontainshighconcentrationsoforganicandinorganicnutrients.If
releaseduntreated,receivingwaterssuchaslakesandstreamsmaysufferfromoxygen
depletion as a direct effect of the microbial oxidation of organic pollutants and
ammonium,andindirectlyasaneffectofeutrophication.Sucheutrophicationiscaused
mainlybytheplantnutrientsnitrogenandphosphorusthatpromotealgalbloomsinthe
receivingwater,whichinturnconsumesoxygenwhenthealgaearedegraded.Ifsevere
oxygendepletionoccurs,thebenthicfaunaandfishmaydie.Nevertheless,alldegrees
of eutrophication have an impact on the ecological communities and in many cases
causedecreasedbiodiversity.
Tocopewiththenegativeeffectsofwastewaterontherecipient,differentstepsof
wastewater treatment have been developed, and these are described by e.g. Bitton
(1990), Henze et al. (2002) and degaard (1992). Primary treatment aims at removing
largeparticlesinthesewagebymeansofgridsorsedimentation.Secondarytreatment
reducesthebiochemicaloxygendemand(BOD)inthewastewaterbyoxidizingorganic
compounds and ammonium. This process, which is often carried out in aerated tanks
withsocalledactivatedsludge,involvesbothheterotrophicbacteriaandprotozoa.The
bacteria degradethe organicmaterialand the protozoa graze the bacteria, andinboth
casesorganicmaterialisconvertedtocarbondioxideandwater.Inadditiontoactivated
sludge, secondary treatment may also be performed with e.g. trickling filters or
oxidationponds.
Tertiary wastewater treatment mainly aims at removing the plant nutrients
nitrogen and phosphorus. Nitrogen may be removed by nitrificationdenitrification
processes, where the ammonium is first oxidized to nitrate by nitrifying bacteria in
aerobic reactors, and thereafter recycled to an anoxic (oxygen free) reactor where it is
convertedtonitrogengas(N2)bydenitrifyingbacteria.Thefirststepinthenitrification
isoxidationofammoniumtonitrite(1)andthesecondstepistheoxidationofnitriteto
nitrate(2):

NH4++32O2NO2+2H++H2O

1.

2.
NO2+12O2NO3

Nitrification is performed by autotrophic bacteria belonging to the family

Nitrobacteriaceae.Denitrification,ontheotherhand,iscarriedoutbyalargenumberof
4

heterotrophic bacteria, which all need organic carbon for growth. The denitrification
reaction also has nitrite as an intermediary step, and is performed according to the
followingreaction:

NO3NO2NON2ON2

Phosphorus in wastewater is most often removed by chemical precipitation with

aluminiumorironsaltstoformaluminiumorferricphosphate:

Me3++PO43MePO4

The metal (Me) salts are generally added in excess to compete with natural alkalinity,
mainlyaccordingtothefollowingreaction(Henzeetal.,2002):

Me3+ + 3 HCO3- Me(OH)3 + 3 CO2

However,theprecipitationproductmaybearelativelycomplicatedcombinationofthe
metal added, calcium, carbonate and phosphate (Henze et al., 2002). In addition to
chemical precipitation with metal salts, chemical phosphorus removal can also be
achieved by adsorption to filter materials containing positively charged surfaces, e.g.
limestone,zeolites,blastfurnaceslagorLECA(Westholm,2006).
Besides chemical means for phosphorus removal, biological methods also exist.
One way is the Enhanced Biological Phosphorus Removal (EBPR) process. In this
process, bacteria in the activated sludge overaccumulate phosphorus as a response to
shifts between aerobic and anaerobic conditions. When the environment is anaerobic,
thebacteriausetheirphosphorusstorageasenergysourcetopickupsubstrate,andthe
phosphorus is thereby released (Christensson, 1997; Henze et al., 2002). When the
conditionsareshiftedtoaerobic,however,morephosphorusthanwasreleasedistaken
up by the cells and a net removal is achieved. Magnesium and potassium are co
transported with phosphate, and the excess phosphorus is stored in the cells as
polyphosphategranules(Bitton,1990).Moreover,chemicalprecipitationofphosphorus
may also occur in the EBPR process as a result of the increased phosphorus
concentration in the water during the anaerobic phase (Arvin, 1983). Such biologically
mediatedphosphorusprecipitationcanalsobepromotedbydenitrification,whichisan
alkalinityproducingprocessthatincreasesthepH,whichinturninducesprecipitation
ofphosphates(Arvin,1983).

Hydroponicsandothergreentreatmentprocesses
Apart from the tertiary treatment processes described above, other biological nutrient
removal processes also exist, e.g. wetlands, hydroponics and algal cultivation. These
treatmentsystemsprimarilyconcernnutrientassimilationintogreenbiomass,butother
nutrient stripping processes are also involved. In wetlands, nitrification and
denitrificationmaypolishthewastewaterfromnitrogen,particularlyifthewaterlevelis
variedwhichpromotesalternatelyaerobicandanoxicconditions(McBrideandTanner,
1999). Furthermore, phosphorus removal may in addition to biological assimilation be
achievedbysorptiontosoilparticlesorprecipitationwithcationsinthewater(Whiteet
al., 1999). Sedimentation is often the most important removal mechanism for
phosphorusinwetlands.
Inhydroponicwastewatertreatment,whichmeanscultivationofgreenplantsin
wastewater without any solid support material, the nutrients are assimilated directly
from the water by the plant roots (Jewell, 1994; Ayaza and Saygin, 1996; Ghaly et al.,
2005). However, since most plant roots need aerobic conditions to thrive, adequate
aerationofthehydroponictanksiscrucial.Inadditiontothedirectnutrientuptakeby
vascular plants, microbial biofilms on the roots may also contribute to the wastewater
treatment by e.g. BOD degradation and nitrification (Todd and Josephson, 1996;
PolprasertandKhatiwada,1998;Toddetal.,2003;Vaillantetal.,2003;Norstrm,2005b).
Inordertooptimizethewastewatertreatmentperformanceinhydroponicsystems,fast
growing plant species which assimilate large amounts of nutrients, as well as species
withlargerootsystemswhichmaximizethesurfaceareaforbiofilmsshouldbeselected.
However, if the system is integrated with product recovery, the choice of species may
alsobebaseduponthetypeofproductsthatareofinterest.
Hydroponicwastewatertreatmenthasbeentestedinsomedifferentlocationsat
higher latitudes, e.g. in Denmark (Hinge and Stewart, 1997; Jungersen, 1997), South
Burlington,USA(Toddetal.,2003),atStensund,Sweden(Guterstam,1996;Guterstam
etal.,1998)andalsoatverjrva,Sweden,whichiswheretheprojectdescribedinthis
thesis is located (Figure 2) (Norstrm et al., 2003). A comparative study of six
hydroponic wastewater treatment plants located in greenhouses at higher latitudes,
however, have concluded that the main mechanisms for nutrient removal in such
systems are due to microbial activity rather than assimilation by plants (Norstrm,
2005a). BOD and nitrogen removal is often satisfactorily achieved by microbial
processes,butthephosphorusremovalisnotveryefficient.

Figure 2. Pictures of the hydroponic cultivation of green plants at the verjrva wastewater treatment
plant.(Photo:KajKauko)

Wastewatertreatmentwithmicroalgae
Microalgaearemicroscopicphotosynthesisingorganismswhicharefoundprimarilyin
aquatic habitats. The idea of using microalgae for wastewater treatment originally
developed in the 1950s in California by William Oswald (Oswald and Gotaas, 1957;
Oswald, 1963). The role of the algae was both to assimilate plant nutrients and to
supportbacteriawithoxygen.Thebacteria,inturn,wereinvolvedinthedegradationof
organic material in the wastewater, the same process which is utilised in activated
sludge(Figure3).

Organic
Dissolved
Excess
waste
oxygen
algae

Algal
Bacterial

photosynthesis
oxidation

Excess
CO2 + H2O + NH4+
Solar

bacteria
energy
Chlorophyll

Figure 3. The main processes involved in a high rate algal pond, extracted from Oswald and Gotaas
(1957).

Theprocesswasdevelopedinshallowponds,lessthanameterdeepandcontinuously
stirredwithpaddlewheels.These,socalledhighratealgalponds(HRAP),wereaerobic
throughout their volumes in contrast to facultative ponds which are anoxic near the
bottom (Oswald, 1988a). The HRAPs are often built as socalled raceways, with a
meandering configuration (Figure 4). Properly designed and operated HRAPs are
capable of removing more than 90% of the BOD and up to 80% of the nitrogen and
phosphorus(Oswald,1988b).

Water out

Water in
Figure 4. Schematic picture of a raceway pond.

Apart from open ponds, microalgae can also be cultivated in closed photobioreactors
suchastubesorflatpanels(Richmond,1990;Borowitzka,1996;Pushparaj,1997;Grima
etal.,1999;Mirn,1999).Theseoftenhavebetterlightpenetratingcharacteristicswhich
makes it possible to sustain higher biomass and productivity with less hydraulic
retention time (HRT) than is possible in open ponds (Borowitzka, 1998). However,
closed bioreactors are often more sophisticated and operation demanding than ponds,
and are mostly used for commercial production of microalgae rather than for
wastewatertreatment.

Growthinlightlimitedcultures
Since wastewater is a very nutrient rich medium, most algal cultures grown in
wastewater are growth limited by light or carbon rather than by plant nutrients
(Oswald,1988b;delaNoeetal.,1992).Lightlimitedculturesbehaveinamannerquite
differentfromnutrientlimitedcultures,whichisdescribedbyPipesandKoutsoyannis
(1962).Thegrowthofanorganismculturedinanutrientlimitedcultureisafunctionof
theconcentrationofthelimitingnutrientintheculture.Thehigherconcentrationinthe
medium,thehighercelldensitycanbeachievedatsteadystate.Thecelldensityisthus
constantandnotafunctionofthemediumflowrateortheaverageHRTintheculture.

Cultureslimitedbylightorinorganiccarbonsupply,however,showasomewhat
differentgrowthpatternbothinbatchandcontinuouscultures.Inabatchculturewith
8

LN cell density (g/L)

cell density (g/L)

algaeinanutrientrichmedium,e.g.wastewater,thegrowthwillbeexponentialinthe
beginning when the cell density is low, and this is similar in nutrient limited cultures
(Figure5).Asthecelldensityincreases,however,mutualshadingofthecellscausesthe
lightintensityseenbyeachcelltodecrease(Raven,1988;Borowitzka,1998;Woodetal.,
1999).Atacertaincelldensitytheaveragelightintensityseenbyeachcellislessthan
saturationlightintensity,andlightthenbecomesthefactorlimitinggrowth.Thegrowth
rate of every single cell thereafter decreases as the cell density increases, and the cell
densityincreaseslinearly;thelineargrowthphaseoftheculturehasstarted.Thealgal
biomass production hereafter is proportional to the light intensity provided, and thus
constantifthelightintensityisconstant.Inanutrientlimitedculture,ontheotherhand,
the growth rate decreases and there will be no net growth of the population as the
limitingnutrientisdepleted.

b)
a)
0,5
1,4

0,0
1,2

-0,5
1,0
-1,0

0,8
-1,5
0,6
-2,0
-2,5
0,4
-3,0
0,2
-3,5
0,0
-4,0
10
20
30
40
0
10
20
30
40
0
days
days

Figure5.BatchcultureofthecyanobacteriumAnabaenaPCC7120insyntheticwastewater.a)Celldensity
versus time and b) the same but with logarithmic scale. During the first week, the growth was
exponential,whichcanbeseenasalinearincreaseinb).Thereafter,thecultureisbecominglightlimited.
Thiscanbeseeninb)wherethelinearincreaselevelsoff.Afterthis,thegrowthislinearwithaconstant
biomassproduction(gdryweightperlitreandday)whichcanbeseenina).(Unpublishedresultsfrom
studieswithinthedoctoralproject)

Inlightlimitedcontinuouscultures,thecelldensityatsteadystatewillbeproportional
to the amount of photons captured during the average HRT. The cell density per area
hencemainlydependsontwofactors:

1. HRT.ThelongertheHRT,thehigherthecelldensity(Figure6).

2. Light intensity. The higher the intensity, the higher the cell density (up to a
certainlimit,afterwhichlightstartsinhibitinggrowth,socalledphotoinhibition
(KrgerandElnoff,1981;Borowitzka,1998;OliverandGanf,2000)).

a)

b)

1400

300
-1

Production (mg DW L d )

1200

250

-1

-1

Cell density (mg DW L )

1000
800
600
400
200
0

200
150
100
50
0

HRT (days)

HRT (days)

Figure 6. a) cell density and b) algal biomass production as a function of HRT. As can be seen, the cell
densityincreases with increased HRT,whereas theproduction remains constant. This indicates that the
culturewaslimitedbylight(orbyexternalinorganiccarbonsupply).(ExtractedfromPaperV)

However,sincethelightintensitydecreasesexponentiallywithdepthinwater,thecell
densitypervolumewillalsobelowerthedeeperthecultureis.Consequently,inorder
toachievedensealgalculturesthedepthisanimportantparametertoworkwith.Asthe
residual nutrient concentration in the medium decreases with increased cell density,
highdensityculturesarethenfavourablefromawastewatertreatmentperspectivesince
effluentlimitsofnitrogenandphosphorusmustusuallybemet.
However, light is not always completely necessary for algal growth, and some
microalgaehavetheabilitytogrowwithoutlightonorganiccarbonsourcesinsteadof
carbon dioxide, such as organic acids, sugars, acetate or glycerol (Fogg, 1975;
Kawaguchi, 1980; Borowitzka, 1998; Wood et al., 1999; Ogbonna et al., 2000). Some
studieshaveindicatedthatabout2550%ofthealgalcarboninhighratealgalpondsis
derived from heterotrophic utilisation of organic carbon (Borowitzka, 1998).
Nevertheless,inordertohavedenseculturesofmicroalgaeathigherlatitudes,thelight
conditionsshouldbethemostimportantoperationalparametertooptimize.

10

Phosphorusremovalinalgalcultures
Assimilation
Microalgae,likeallorganisms,needphosphorustogrow.Thephosphorusisusedinthe
cells mainly for production of phospholipids, ATP and nucleic acids. Algae assimilate
phosphorusasinorganicorthophosphate,preferablyasH2PO4or HPO42-,andtheuptake
process is active, i.e. it requires energy (Becker, 1994). Organic phosphates can be
converted to orthophosphates by phosphatases at the cell surface, and this occurs
especiallywhenorthophosphatesareinshortsupply.Moreover,microalgaeareableto
assimilate phosphorus in excess, which is stored within the cells in the form of
polyphosphate(volutin)granules.Thesereservescanbesufficientforprolongedgrowth
intheabsenceofavailablephosphorus(Fogg,1975;OliverandGanf,2000).Thegrowth
rate of an alga may therefore not respond at once to changes in the external
concentration of phosphorus, as opposed to the immediate responses to temperature
and light. Mostert and Grobbelaar (1987) found that the phosphorus concentration in
cellsvariedwithsupplyconcentration,fromapproximately1mgPpergdrymassata
supplyconcentrationof0.1mgPl1to100mgPpergdrymassatsuppliesof5mgPl1
and greater. On average, an algal cell contains 13 mg P per g dry weight (Oswald,
1988a).Algaecultivatedinwastewater,whichnormallyhasaconcentrationofbetween
10 and 20 mg P L1, may hence contain much higher amounts of phosphorus than is
neededforgrowth.
Inordertomaximizethephosphorusassimilation,itisimportanttooptimizethe
growthconditionsforthealgaesincetheassimilationofphosphorusgenerallydepends
onthealgalcarbonassimilation,andhenceonthealgalgrowth.Sincealgaestarvedof
nutrients take up phosphorus much faster than nonstarved algae, there is also the
possibilityofusinganoperationalstrategywherestarvedalgaeareaddedtothereactor.

Precipitation
When microalgae photosynthesise, they take up inorganic carbon from the water. The
inorganic carbon species normally used by microalgae are carbon dioxide (CO2) and
bicarbonate(HCO3) (Oswald,1988a;Borowitzka,1998),thelatterrequiringtheenzyme
carbonicanhydrasetoconvertittoCO2.Whenbicarbonateisusedascarbonsource,the
pHinthemediumincreasesduetothereaction:

HCO3 CO2+ OH-

ThispHincrease,whichcanelevatethepHinalgalculturestovaluesabove11,strongly
affects the water chemistry. Phosphorus may as a result precipitate with available
cations to form metal phosphates, where calcium phosphates are the most common.
BesidesbeingpromotedbyhighpHvalues,precipitationofcalciumphosphatesisalso

11

favoured by increased temperature as well as high concentrations of calcium and

phosphorus(Songetal.,2002).Precipitationfromwastewaterisalsopossibleatneutral
pH values, but then the phosphate concentration must be at least 50 mg P L1 and the
calcium concentration 100 mg Ca L1 (Carlsson et al., 1997). In soft water, i.e. with a
calciumconcentrationoflessthan50mgL1,thephosphateconcentrationmustbeeven
higher in order to induce precipitation. At low aqueous calcium and phosphate
concentrations,precipitatedcalciumphosphatesmayalsoberedissolved.
Thereareseveraldifferentcalciumphosphatesthatmayformfromwastewater,
which all have Ca/P molar ratios between 1 and 1.67. Some salts, like amorphous
calcium phosphate (ACP), dicalcium phosphate and octacalcium phosphate may be
precursors to hydroxyapatite (HAp), which in turn is the most thermodynamically
stable(Arvin1983).SuchHApformationis,however,inhibitedbyhighconcentrations
of magnesium, carbonate and pyrophosphates (P2O74) (Fergusson et al., 1973; Arvin,
1983). The effect of magnesium is pronounced when the Mg/Ca ratio exceeds 0.45. At
pH levels above 10.5, however, magnesium precipitates with hydroxide ions and
consequently loses its effect on the phosphorus solubility (Jenkins et al., 1971).
Carbonate both decreases the crystallinity of calcium phosphates and promotes the
formationofACP,butalsocompeteswithphosphatesinprecipitatingwithcalciumto
formcalcite(CaCO3)atpHvaluesabove8(FergussonandMcCarty,1971;Arvin,1983).
As a result, the phosphorus precipitation rate is inversely correlated to the carbonate
concentration(Fergussonetal.,1973).
Chemicalprecipitationofphosphorusmaycompriseasignificantpartofthetotal
phosphorus removal in algal wastewater treatment (Doran and Boyle, 1979; Moutin et
al., 1992; Proulx et al., 1994; Mesple et al., 1996; Tam and Wong, 2000). Particularly in
areas with hardwater, i.e.waterwithhighconcentrationsof calcium andmagnesium,
thiseffectmaybepronounced.Thechemicalstrippingofphosphorusmayberegarded
asanadvantageousbyeffectofthealgalgrowth,withenhancedphosphorusremovalas
a result. Another advantage is that the chemical sludge which is produced is easier to
harvestthanfreefloatingcellsduetothetendencyoftheprecipitatetosink.

Nitrogenremovalinalgalcultures
Nitrogenremovalinmicroalgalwastewatertreatmentprocessesismainlyachievedby
assimilationtoalgalcells.Nitrogenisthesecondmostimportantnutrientaftercarbon,
and may comprise more than 10% of the biomass (Becker, 1994). Nitrogen exists in
manyforms,andthemostcommonnitrogencompoundsassimilatedbymicroalgaeare
ammonium(NH4+)andnitrate(NO3)(OliverandGanf,2000).Thepreferredcompound
is ammonium, and when this is available, no alternative nitrogen sources will be
assimilated(Bhayaetal.,2000).However,highammoniumconcentrations(>20mgNH4
N L1) are not recommended since in combination with high pH values, the cells may
12

sufferfromammoniatoxicity(Borowitzka,1998).Inadditiontoammoniumandnitrate,
urea((NH2)2CO)and nitrite(NO2)canalsobeusedasnitrogen sources. However, the
toxicity of nitrite at higher concentrations makes it less convenient (Becker, 1994).
Cyanobacteria are also able to assimilate the amino acids arginine, glutamine and
asparagine, and some species of cyanobacteria can fix nitrogen gas (N2) (Bhaya et al.,
2000).Ofallnitrogensources,thisnitrogenfixationisthemostenergydemandingand
onlyoccursinsomecyanobacteriawhennoothernitrogencompoundsareavailablein
sufficientamounts(Benemann,1979).Severalmicroalgaecantakeupnitrogeninexcess
oftheimmediatemetabolicneeds,socalledluxuryconsumption.Thiscanbeusedlater
inthecaseofnitrogenstarvation.

Forwastewatertreatmentpurposes,thenitrogenassimilationmaybeoptimized
bymaximizingalgalgrowth.However,inadditiontoassimilationofnitrogen,ammonia
mayalsobestrippedtotheairasaresultoftheincreasedpHvaluesoftenfoundinalgal
cultures (Tam and Wong, 2000; Nunez et al., 2001). Denitrification, which is the major
microbialprocessutilisedinconventionalwastewatertreatment,isnotoftenconsidered
as a significant process in algal cultures though, since the high oxygen levels mostly
founddonotpromotethatreaction.

Harvesting
Sincealargepartofthenutrientsinalgalwastewatertreatmentsystemswillendupas
biomass, harvesting is crucial in order to separate both nutrients and BOD from the
water.However,becausemicroalgaeareverysmallandusuallyhaveslowsettlingrates,
this is not easily achieved and is consequently a cost expensive part of the cultivating
process (Benemann, 1989; de la Noe et al., 1992). Even though harvesting effectively
can be accomplished by e.g. filtration or centrifugation, such methods may be too
difficultorcostlytoimplement(Mohn,1988;delaNoeetal.,1992;Tangetal.,1997).
Sedimentation without the addition of chemicals is the most common method in full
scale facilities (Garca et al., 2000), but previous flocculation is mostly desirable.
Flotationofunicellularalgaewithoutflocculationmayalsobeverydifficultduetothe
hydrophilic cell surface on which air bubbles will not attach (personal unpublished
experiences).Examplesofchemicalflocculantstousewithflotationorsedimentationare
alum, lime, FeCl3, cationic polyelectrolytes and Ca(OH)2, and somemore toxically safe
flocculatingagentsaree.g.potatostarchderivativesandchitosans(Moraineetal.,1980;
Mohn,1988;delaNoeetal.,1992).Somemicroalgaemayflocculatenaturally,socalled
bioflocculation. The process is often induced by turbulence stress and results in
formationofbiopolymersbyextracellularenzymes.Thepolymerscanalsobeproduced
bybacteriaassociatedwiththealgalcells,butinbothcasesthisleadstochangesinthe
surface charge that in turn causes the cells to aggregate (Lincoln and Koopman, 1986;
Mohn,1988).Inparticularsomespeciesofcyanobacteriaformflocsspontaneouslyand
13

some cyanobacteria are also able to form gas vacuoles that makes them accumulate at
thewatersurface,andboththesefeaturesmakethemeasytoharvest(Benemann,1979).

Forwastewatertreatmentpurposes,fastmixingofthealgalcultureandincreased
HRT may be recommended since this increases the harvestability of the algae due to
bioflocculation (Benemann et al., 1980). However, longer HRT also makes the culture
more susceptibleto grazingzooplankton; withan HRTof fourdaysorlonger, regular
crashes of the algal culture have been experienced (Benemann et al., 1980). This may,
however,notbeveryproblematicsincethealgaerapidlyrecoveraftersuchcrashes.On
theotherhand,grazingzooplanktonmayalsobeutilizedfortheharvestofalgae,which
isknownasbiologicalfiltration.Withthismethod,easilyharvestedfilterfeedersarefed
with algae and it is consequently a form of aquaculture. Well known filter feeders are
mussels and cladocerans like Daphnia spp. (de la Noe et al., 1992). Complete food
chains starting with wastewater or urine have been studied in order to develop
integrated systems able to generate useful biomass simultaneously with effluent
purification(EtnierandGuterstam,1991;Adamsson,2000).
Asolutioninordertoavoidtheneedforharvestistoimmobilizethealgalcellsin
gelatinousmediasuchasalginatesorsyntheticpolymers(Broudersetal.,1989;Kayaet
al.,1995;Robinson,1997).Thealgaemediummixtureisoftenshapedlikebeads,butcan
evencoverscreensorsurfaces(Borowitzka,1998).Themediumallowsthenutrientsin
thewastewatertodiffusetothecells,andthenutrientuptakerateshavebeenshownto
besimilarforfreeandimmobilizedcells(delaNoeetal.,1992).

Algalwastewatertreatmentathigherlatitudes
Since microalgalcultures areoftenlight limited,cultivation at higher latitudesmaybe
problematicduringthedarkerperiodoftheyear.EvenatlatitudeslowerthanSweden,
e.g.Spain37N,variationsovertheyearareobservedwithseveraltimeshigherbiomass
productivityduringsummerthanduringwinter(Mirn,1999).Athigherlatitudes,this
effect is naturally even more pronounced. Not many studies on microalgae for
wastewater treatment have been conducted in colder climates; however, some results
regarding the performance during summer have been reported. For example, in an
HRAP at 63N latitude, a removal of total phosphorus of up to 49% was reported
(Grnlund,2004).Algalcultivationhasalsobeentestedin greenhousese.g.ondiluted
humanurineat57Nwherethealgaeremovedbetween37%and97%ofthephosphorus
during summer and autumn (Adamsson, 2000). Some researchers have suggested that
algal and cyanobacterial species isolated from highlatitude ecosystems may perform
well in wastewater treatment since they are adapted to growth in colder climates;
however,evenifthestrainstoleratelowtemperatures,theyalwaysgrowfasterathigher
temperatures, i.e. they are cold tolerant (psychrotrophic) rather than cold loving
(psychrophilic) (Talbot et al., 1991; Tang et al., 1997; Chevalier et al., 2000). Generally,
14

temperaturesbetween15Cand25Cseemtosuitmostalgalspecies,eventhosewhich
areadaptedtogrowthatlowertemperatures.Nevertheless,coldtolerantspeciesmaybe
important in wastewater treatment processes in a Nordic climate, even if the growth
conditionsarenotoptimal.

15

4.Resultsanddiscussion
The hydroponic wastewater treatment plant at verjrva, a description
(PaperI)

Duringtheyears20002001,ahydroponicwastewatertreatmentplantwasconstructed
at verjrva farm in Solna, close to Stockholm. The treatment plant treats municipal
wastewater from the farm which includes two resident families, a cafeteria, biology
education for children, different shops and other activities. A thorough description of
theevolutionofthewastewatertreatmentplantisgiveninthedoctoralthesisbyAnna
Norstrm(2005b),andresultsfromthefirststudyonthetreatmentplantarepresented
inPaperI.Theplantbasicallyinvolvesaseriesofmicrobiologicalandhydroponicsteps,
and the purpose has been to treat the wastewater without using chemicals and to
integratethetreatmentwithrecoveryofvaluableproducts.Theplantscultivatedinthe
hydroponics can be used for many purposes; as ornamental plants, food, feed or
productionofsubstancesjusttogivesomeexamples.However,thisaspectoftheproject
isnotcoveredwithinthescopeofthisthesis.
The pilot plant is composed of several steps which are illustrated in Figure 7.
Afteracollectiontankundergroundoutsidethegreenhouse,thewaterispumpedtoa
first anoxic tank (AN) (Figure 8). This is filled with Kaldnes carriers (AnoxKaldnes,
Norway),onwhichmicrobialbiofilmsareattached.Inadditiontotherawwastewater,
recycle flow from the hydroponics also loads the reactor, and the nitrate rich recycle
flowincombinationwiththecarbonrichrawwastewaterpromotesdenitrification.

Figure 7. Overview of the tanks and water flows in the hydroponic wastewater treatment process,
includingsamplepoints112asdescribedinPaperI.Thedesignoftheprocesstrainwaslatersomewhat
modified.

16

Theeffluentfromtheanoxictankflowsbygravitytothenexttank,anaeratedreactor
(CA) in which the nitrification of ammonium starts and the organic compounds are
furthermineralised.Thetankiscoveredbyanearthfilter(Figure8),whichfunctionsas
anaircleanerthatpurifiestheairfrombadsmellinggases.Sincetheairfilledspacein
theanoxicreactorisalsoconnectedtothistank,gasesproducedintheanoxictankare
alsopurified.Moreover,theearthlayerisplantedwithgoodsmellingplants,andasa
result, the environment in the greenhouse is pleasant and the occurrence of sewage
smellisuncommon.

Figure8.Thetwofirsttanksinthetreatmentprocess,theanoxictank(behindtotheleft)andthecovered
aerobictank(infront),whichhasaplantedsoillayerontop.(Photo:KajKauko)

From the first two closed tanks, the water enters the three consecutive aerated
hydroponictanks(HP1HP3)(SeealsoFigure2).Thesearewiderthantheclosedtanks,
and constructed from transparent plastic. At the water surface, racks with plastic nets
supportvascularplants.Thesehavelargerootsystemswhichhangdowninthewater
for nutrient uptake. The roots also support biofilms, which treat the water further by
BODremovalandnitrification.
Afterthelasthydroponictank,thereisaclarifier(CL)fortherecyclingofeasily
settledmaterialtotheanoxictank.However,nosignificantamountofsludgehasbeen

17

produced,andtherecycleflowisforthatreasonnotinoperation.Fromtheclarifier,the
waterentersasump(P1),fromwherewaterispumpedtothealgalstep.Thisconsisted
of two tanks (A1 and A2) connected in parallel at the time for the study described in
PaperI,butwaslaterincreasedbytwomoretanks(SeePaperIV).Fromthealgaltanks,
thewaterflowsbygravitytoanalgaeclarifier(ACL)andthentwofinalsandfilters(S1
andS2).
The study described in Paper I mainly aimed to describe the design and to
evaluatetheperformanceofthewholetreatmentplant.Thepaperpresentsresultsfrom
asampleperiodof22consecutivedaysduringJuly2002.

Themajorfindingsinthestudywerethat:

90%CODremovalwasachieved.

theammoniumwascompletelynitrifiedinthehydroponics.Thereforetheplant
rootsweregoodgrowthsubstratefornitrifyingbiofilms.

denitrificationthroughrecirculationofnitraterichwaterfromthehydroponics
to the anoxic tank was successful, and was responsible for the majority of the
72%nitrogenremovalinthesystem.

47% of the phosphorus was removed, mainly in the algal tanks and the sand
filters.

further optimisation of the treatment process was still necessary in order to

reach the effluent standards at the site: 10 mg N L1, 0.5 mg P L1 and 15 mg
BODL1.

Energyrequirementsofhydroponicwastewatertreatment(PaperII)

Aspartoftheoverallevaluationofthesuitabilityofhydroponicwastewatertreatment
in Sweden, a theoretical study of the energy requirements was conducted. Since the
greenhouseatverjrvawasbuiltaccordingtotheoriginalconstructiondrawingfrom
1860, the empirical data from the operation of the greenhouse were not relevant for
futureimplementationofsimilarsystems.Instead,datafrommoderngreenhousesand
greenhouse operation were used in order to calculate the need for heating and
illumination during different parts of the year, which is presented in Paper II. Two
operational strategies were compared, one giving light and heat sufficient to maintain
18

winter resting plants without growth (minimum 10C, 400 lux 16 h day1), the other
sufficient to maintain good growth conditions for plants with moderate light
requirements(minimum20C,2000lux16hday1)(Christensson,1988).Dataregarding
incidentlightintensitiesduringtheyearandoutdoortemperatures(SMHI)wereusedto
calculate the need for additional illumination and heat in order to fulfil the two
requirementsforfivelocationsatdifferentlatitudesinSweden,fromLund5572Nto
Kiruna 6783N. The energy requirements for aeration of the wastewater were not
includedinthemodel,butwereassumedtobethesameasinconventionalwastewater
treatment with activated sludge. The energy requirements calculated were rather the
additional energy that is needed in order to treat the wastewater with hydroponics
insteadofwithconventionalactivatedsludgesystems.

Theresults,whicharepresentedinFigure9showedthat:

the heat demand was generally larger than the light demand independent of
growthconditionandlatitude.

onayearlybasis,30%morelightenergyisneededinKirunathaninLundfor
bothgrowthconditions.

the heat demand showed much more pronounced latitude dependence than
light. In order to reach the 10C growth condition, the heat demand is 700%
higherinKirunathaninLund.
450
400

kWh m -2

350
300
250

heat

200

light

150
100
50

A: 400 lux, 10C

Kiruna

stersund

Stockholm

Visby

Lund

Kiruna

stersund

Stockholm

Visby

Lund

B: 2000 lux, 20C

Figure 9. Theoretical total energy requirements for heat and light in one year for five Swedish cities at
differentlatitudes.

19

Anotherconclusionfromthestudywasthatlargeenergysavingscouldbeachievedby
lettingthesystemgodowntothelowlightandheatconditionsduringwinterbutstill
have the higher temperature and light conditions during the rest of the year. Such a
restingperiodbetweenOctoberandMarchinKirunawouldresultinareductionofthe
energydemandof37%comparedtothehighlightandtemperatureconditions.InLund,
anevenshorterperiodoflowtemperatureandlightintensity,betweenNovemberand
February, would save 54% of the annual energy requirements. Moreover, the
contributiontothetemperatureinsidethegreenhousebytheheatinthewastewaterwas
notincludedinthecalculations,whichcouldsaveevenmoreenergy.

Thepilotstudyonmicroalgaeinthegreenhouse(PaperIII)

Since the effluent from the hydroponics still contained some residual nutrients,
particularlyphosphorus,microalgaeweresuggestedasagreenmethodtocomplement
the phosphorus removal in the system. The first study on microalgae was conducted
during summer 2001 in order to see if the method was appropriate, and another aim
was to see which algal species would dominate the treatment step. In order to
investigate whether different types of algae appeared as a result of different nutrient
concentrationsandN/Pratios,atrainoffourtankswasconstructed.Thefirsttankinthe
train received the effluent from the hydroponics, and the overflow from that tank
flowed into the next tank etc (Figure 10). Between each tank, the algal biomass was
harvested with filters. The first hypothesis was that the high nutrient concentration in
the first tank would favour fast growing green algae, whereas later in the train, when
nitrogenwasdepleted,therewouldbeaselectionfornitrogenfixingcyanobacteria.In
otherwords,thesuggestioninthebeginningoftheexperimentwasthatnitrogenwould
bethelimitingfactorforthealgalgrowthasaresultofthelowN/Pratiointheeffluent
fromthehydroponics,whichwassometimesaslowas1.Thisratioisusuallyaround7
on a mass basis in algae, the so called Redfield ratio (Redfield, 1958) (not 10 as is
incorrectlytypedinPaperIII).

20

Figure10.Photoofthepilotstudyonmicroalgae.Theeffluentfromthehydroponicswaspouredintothe
first tank, A, from where it flowed by gravity to next tank B etc. Between each algal tank, the algal
biomasswascollectedinfilters.(Photo:KajKauko)

Interestingly, the results showed something totally different; instead of nitrogen

depletion,arapidphosphorusremovalwasseeninthefirsttankofthetrain(Figure11).
Moreover, there was no difference in algal selection between the different tanks, and
nitrogen fixing cyanobacteria were not observed at all. The high phosphorus removal
rateinthefirsttankwasnotseeninthefollowingtanksofthetrain,althoughthealgal
production was the same in all tanks. The conclusion was that as a result of the
increased pH in the algal tanks which was mediated by the photosynthetic carbon
assimilation,phosphorusintheeffluentfromthehydroponicswasrapidlyprecipitated
in the first tank with available cations, probably calcium. As a result, there was less
calcium and phosphorus left to precipitate in the water in the following tanks, and
slowerremovalrateswerethereforeexperienced.

21

12
influent
A
B
C
D

mg P L-1

10
8
6
4
2
0
15-Mar 25-Mar

4-Apr

14-Apr 24-Apr

4-May 14-May

Figure11.ThedissolvedphosphorusconcentrationinincomingwaterandinthefouralgaltanksAD.

Thefirstpilotstudywasextendedtowinterspring20012002,andwasthencomposed
of two similar trains of tanks where one of them achieved extra light from fluorescent
lamps. This part is presented in Paper III. The results showed that the additional
illumination increased the algal production and phosphorus removal rate during
winter, but that in real terms this difference was small compared to the impact of
natural light during spring. Even though the average phosphorus removal rate was
100% higher in the tanks with additional illumination than in the tanks with natural
light during NovemberDecember, the rate was almost three times higher during
FebruaryAprilthan duringwinterin the tankswith additional illumination. Thelight
intensity by the extra illumination was 55150 mol quanta m2 s1, and was achieved
withplantlightingfluorescentlamps.Theselightintensitiesarereportedtobesufficient
to support moderate to good growth by green and flowering plants (Christensson,
1988),butobviouslyweretoolowtofullysupportanalgalculture. Higherincidentlight
intensitiesareusuallyneededforalgalcultivationtocompensateforthelightabsorption
by the water and the algal cells themselves (Ogbonna and Tanaka, 2000). Naturally, a
higheralgalgrowthratewouldhavebeenexpectedifastrongerlamphadbeenchosen,
butthiswouldhavedemandedevenmoreenergy.

Themajorconclusionsfromthepilotstudyonmicroalgaewerethat:

a large proportion of the removed phosphorus was due to chemical

precipitationratherthanassimilationintocells.Thisconclusionwasbasedupon
twoobservations;

22

a) the low ratio between removed nitrogen and phosphorus, lower than 0.2
comparedtothetheoreticalratioof7indicatesthatalotmorephosphorus
thanwasneededforalgalgrowthwasremoved.

b) thephosphorusremovalratewasstrikinglyhigherinthefirsttankofeach
trainthaninthefollowingtanks,whereasthealgalbiomassproductiondid
not vary between the tanks. This suggests that there was a rapid
precipitation in the first tank as a result of the increased pH and the high
calcium and phosphorus concentrations. In the following tanks, the ion
concentrationswerelowerandhencetherewaslessprecipitation.

although extra illumination by fluorescent lamps increased the phosphorus

removalcapacityofthealgae,therewasstillalargedifferenceinperformance
betweenwinterandsummer.

Themicroalgaltreatmentstep(PaperIV)

Since the pilot studies had shown that microalgae had a great potential to remove
residual phosphorus from the hydroponics effluent, a microalgal treatment step was
constructedasanextendedpartofthetreatmentprocessduringsummer2002.Atfirst,
this step consisted of two glass tanks connected in parallel, since the results from the
pilotstudyhadindicatedthatthehighestphosphorusremovalratewasachievedinthe
first tank in a train of tanks, where chemical precipitation was promoted. These tanks
were denoted tank 1a and tank 1b, they had a culture depth of 33 cm and were
continuously aerated for agitation with the same system as that which aerated the
coveredaerobictankandthehydroponics.Aclarifierwasconnectedtotheeffluentfrom
thealgaltanksinordertoallowalgaetosediment.However,sincetheunicellulargreen
algae that were dominatingthe treatment step were not easilysettled,theclarifier did
notremoveallbiomassproducedinthealgaltanks.Forthatreason,adairycentrifuge
was connected to the effluent from the clarifier which was able to remove more than
90%ofthecells.
As mentioned, the algae that evolved in the treatment step were mainly
unicellular green algae (Chlorophyceae), in particular of the genus Monoraphidium, but
alsoothergenerawereoccasionallypresentinrelativelyhighamounts,e.g.Scenedesmus.
Inadditiontothealgaeinthefreewater,biofilmsalsoevolvedontheinsideofthetank
surfaces.Theseconsistedofmanyformsofmicroalgae,e.g.diatomsandcyanobacteria.

23

algal tank
8,0

7,5

7,0
1

ug
-A

g
Au
6-

ug
-A
11

ug
ug
ug
ug
-A
-A
-A
-A
31
26
21
16

S
5-

ep

tot-P algal tank

PO4 algal tank

4
2
0
1Au
g
6Au
g
11
-A
ug
16
-A
ug
21
-A
ug
26
-A
ug
31
-A
ug
5Se
p

pH

-1

Total and dissolved phosphorus (mg P L )

Thesebiofilmswere,however,removedregularlyinordertoallowlighttopenetratethe
reactorsfromthesides.
After startup, an HRT of 1.9 days was used, which was too short to retain
enough algal biomass in the tanks. This was the main reason for the relatively weak
performanceofthetreatmentstepwhichispresentedinPaperI.AttheendofJuly2002,
theflowtothealgaltankswasdecreasedtogiveanHRTof4days.Thisgaveimmediate
results with a clear increase in pH and a decrease in both total and dissolved
phosphorusinthealgalculturescomparedtotheeffluentfromthehydroponics(Figure
12).Intheinfluenttothealgalcultures(=effluentfromthehydroponics),thetotaland
dissolved phosphorus concentrations were equally high, which indicates that the
phosphoruswascompletelymineralisedintheprevioustreatmentsteps.Thedissolved
phosphorus is the filtered orthophosphate fraction, whereas total phosphorus is
analysedonunfilteredsampleswherecellsandotherorganicmaterialareincluded.In
contrast to the mineralised influent, a small difference between total and dissolved
phosphorus could be seen in the algal cultures, which represents the phosphorus
assimilated into algal biomass. What is more interesting, though, is the much larger
difference between total phosphorus in the influent and in the algal culture, which
indicates that phosphorus has disappeared in some way. The explanation is that since
the samples from the algal step were taken at the water surface (grab samples), the
precipitatedphosphorus,whichwasfoundatthebottomofthetank,wasnotincluded
inthesamples.Thedifferencebetweentotalphosphorusintheincomingwaterandin
the algal culture may therefore be interpreted as the chemical precipitation of
phosphorus.

a)
b)

14
9,5

12

9,0
10

tot-P influent water

8,5
8

PO4 influent water

influent water

Figure12.a)pHandb)total(totP)anddissolvedphosphorus(PO43)intheeffluentfromthehydroponics
(HP)andinoneofthealgalcultures(culture1a)duringAugust2002.

24

Thethreefractionsofphosphorusthatwerefoundinthealgalstepcouldhencebe
describedas:

Paq=thedissolvedorthophosphatefraction

Pprec=precipitatedphosphorus=PtotinPtotout

Pcell=phosphorusassimilatedintoalgalcells=PtotoutPaqout

During summer 2003, an intensive study on the algal step was performed, which is
presented in Paper IV. In addition to tanks 1a and 1b, two other tanks which had
previously served as filter tanks were converted to algal tanks (Figure 13). These two
weredenoted2aand2bandwereshallowerthantheoriginalalgaltanks,withaculture
depthof17cm.

Figure 13. The four tanks used for algal cultivation in the effluent from the hydroponics. (Photo: Kaj
Kauko)

In the experiment, the four algal tanks were connected in parallel to the effluent from
thehydroponics,andtheremovalratesofnitrogenandphosphorusinthedeepandthe
shallow tanks were compared. In addition to the two culture depths, four different
HRTs(3,4,5and6daysHRT)weretestedinordertoseehowtheperformancevaried

25

with load. For practical reasons, each HRT was tested for one week and the cultures
could adapt to the new flows adjusted during Friday to Monday, which is too short a
time for a new hydraulic steady state to develop. Despite this, the results were quite
clearandnewknowledgeonthepotentialofthetreatmentstepwasinfactgained.

Themainfindingsfromthesummerstudywerethat:

longer HRT gave higher algal biomass concentration and higher phosphorus
removalefficiency.Hence,theresidualphosphorusconcentrationswerelower
withlongerHRT.

theshallowercultureshadhigherphosphorusremovalefficiency(7892%)than
thedeepercultures(6688%).

thealgalgrowthwasmainlylightlimitedwhereasthephosphorusremovalwas
load limited (i.e. shorter HRT gave higher removal rates). The removal rates
werebetween1.2and2.2mgPL1day1.

thedeepercultureshadahigherphosphorusremovalrateperarea(0.40.6gP
m2day1)thantheshallowercultures(0.20.4gPm2day1).

between 29% and 77% of the phosphorus removal was due to chemical
precipitation rather than algal assimilation. The shallower cultures had a
somewhat higher proportion of precipitated phosphorus than the deeper
cultures.

nitrogenremovalefficienciesbetween8%and63%wereexperienced,wherethe
lowest nitrate levels were found in the shallow culture at the longest HRT. In
the shallow tanks with 5 and 6 days HRT, a reduction in total nitrogen of
between 26.6% and 40.3% was seen, which can probably be ascribed to the
grazingofalgaebyzooplanktonandsubsequentammoniastripping.

A suggestion was also that the phosphorus assimilation was dependent on growth,
which implies that it was mainly light limited, whereas the phosphorus precipitation
waslimitedbyphosphorusload.However,theseassumptionsaswellastheconclusion
that there was a significant precipitation of phosphorus had to be tested in a more
controlledexperiment.Forthatreasonthenextexperimentwasdesigned.

26

Thephosphorusremovalmechanisms,alabscalestudy(PaperV)

In order to investigate the chemical precipitation of phosphorus in the microalgal

treatment step, a labscale experiment was performed which is presented in Paper V.
Several microalgal strains were isolated from the treatment step, and one strain was
selectedfortheexperimentontheassumptionthatitwasoneofthedominantspecies
found in the treatment step. This strain was a unicellular green alga (Chlorophyceae),
preliminarilydeterminedtotheMonoraphidiumgenus(Figure14a).Sincetheaimwasto
studytheprecipitationconditionsinthetreatmentstep,sterilefilteredwastewaterfrom
theinletpointtothealgaltankswasusedasgrowthmedium.
A small culture vessel consisting of a separation funnel was set up in a culture
chamber (Figure 14b). In the culture chamber, the algae were provided with artificial
light from fluorescent plant lighting lamps. In contrast to the algal tanks in the
greenhouse, the precipitate was not allowed to sediment due to the aeration from the
bottom of the funnel, and samples taken from the bottom hence consisted of a
representative amount of both cells and precipitate. During the experiment, a
continuousinandoutflowwasprovidedbyperistalticpumps.

Effluent
Aeration
a)
b)

Influent

Samples

Figure 14: a) Strain Y6, a unicellular green alga isolated from the microalgal treatment step and b) the
experimentalsetup.Thealgalculturewasgrowninaseparationfunnelwithcontinuousinandoutflow.
Aerationfromthebottomofthefunnelpreventedcellsandprecipitatefromsettling.Samplesweretaken
fromthebottomofthefunnel.(Photo:KajKauko)

27

Thesampleswerethentreatedtoseparatethethreedifferentphosphorusfractions:

Pcell,whichwasthephosphorusassimilatedbythealgae,
Pprec,whichwasthephosphorusfractionthatwaschemicallyprecipitated,and
Paq,whichwasthesolubleorthophosphatefraction,i.e.theresidualphosphorus
intheculture.

Before the experiment began, batch cultures were grown in order to evaluate the
fractionationmethodandtoseehowfasttheprecipitatewasformed.Onebatchculture
of100mlwasgrownforthreeweekstogiveadensealgalculture.Aftersamplingofthe
culture, 200 ml new sterilefiltered wastewater was added to the culture. Immediately
after addition (or at least within two minutes), new samples were taken. Additional
sampleswerethentakenafter15,45and90minutes.Thesamplesweretreatedforthe
fractionsdescribedabove,whichgavetheresultspresentedinFigure15.
2,5

2,0

Pcell
Pprec
Paq
P influent

mg P

1,5

1,0

0,5

0,0
before

15

45

90

time (min)

Figure15.Phosphorusfractionsduringbatchexperiment.200mlwastewater(=Pinfluent)wasaddedto
a culture of 100 ml. On the Yaxis, the total amount of phosphorus in the culture is given, not the
concentration.

However, since some phosphorus may have precipitated during the centrifugation of
the samples (see Methods section Paper V), the samples taken immediately after
addition of new wastewater may have been able to allow precipitation during
maximumtenminutes.
28

3,5

-1

-1

Phosphorus removal rate (mg P L d )

Hence,theconclusionsfromthebatchexperimentwerethat:

within ten minutes after the addition of new wastewater, a significant part of
the added phosphorus precipitated. The precipitation should thus be
recognisedasafastreactioninrelationtothetimescalesusedinthecontinuous
experimentdescribedbelow.

the amount of cellular and precipitated phosphorus increased during 90

minutes after the wastewater addition, whereas the aqueous phosphorus
fractiondecreased.

In the experiment with a continuous culture, which is presented in Paper V, the HRT
wasvariedinordertoclarifytheassimilationandprecipitationdependencyonload.In
thisexperiment,theHRTswere2,3,4and5days.Fromthephosphorusfractions,the
average phosphorus assimilation and precipitation rates respectively could be
calculated.Inadditiontophosphorus,magnesiumandcalciumwerealsomeasuredin
theprecipitateinordertoroughlyestimatetheprecipitatecomposition.

Themainconclusionsfromthefractionationstudywerethat:

theaverageprecipitationratewasloaddependentwhereastheassimilationrate
wasgrowthdependent(Figure16).

3,0
2,5
P precipitation
P assimilation

2,0
1,5
1,0
0,5
0,0
2

HRT (days)

Figure16.Phosphorusremovalrateasthesumofassimilationandprecipitationduringthefourdifferent
HRTs tested. The average precipitation rate increased with decreased HRT and was hence load
dependent.Thephosphorusassimilationrate,ontheotherhand,didnotvaryasaresultofHRT.

29

thealgalbiomassproductionwaslightlimitedorlimitedbythecarbonsupply
fromtheaeration.

the precipitate was probably composed of an amorphous calcium phosphate

withmagnesiumincluded,andalsobycalciteandmagnesiumhydroxide.

thepHdependencyofthechemicalprecipitationwasclear,whenasignificantly
higher pH was experienced during 3 days HRT, a larger proportion of the
phosphoruswasalsoprecipitated.

asignificantnitrogenremovalwasexperienced.

Thewholeyearstudyperformancevariationsovertheyear(PaperVI)

So, bearing in mind the results from peak performance during summer and the
mechanisms for phosphorus removal, how suitable is the treatment process in a year
roundperspective?Sincealgaearephototrophsandhencedependonlightintensityto
thrive, it is obvious that such a process would be largely affected by season at higher
latitudes. The question is rather for how long a period of the year we can expect a
significant contribution by the algae to the overall wastewater treatment performance.
Toanswerthatquestion,thesummerstudywasextendedtoaoneyearstudy,whichis
presentedinPaperVI.FromNovembertoMarch,theeffectofextrailluminationduring
winter was also tested. The flows were adjusted to be as high as possible without
washouteffects of the treatment step; i.e. the flows were lower during winter than
duringsummertocompensatefortheloweralgalgrowthrate.GenerallytheHRTwas
between3and5daysduringsummerandbetween5and7daysduringwinter.

Theresultsfromthewholeyearstudyshowedthat:

there were large fluctuations in algal biomass production and phosphorus

removal as a result of season (Figure 17). The phosphorus removal efficiency
wasbetween60%and100%duringsummerbutlowerthan25%duringwinter
except intheshallowcultures withextra illumination where efficiencies ofup
to60%80%wereexperiencedevenduringwinter.

30

P-removal efficiency

-1

-1

Algal production (mg DW l d )

Shallow tank 2a
Shallow tank 2a
a)
b)

35
100%

30
80%

25

60%

20

40%
15

20%

10

0%
5

-20%
0
jun
sep
dec
mar
jun
sep

jun
sep
dec
mar
jun
sep

Figure 17. a) Algal biomass production as milligram dry weight per litre and day, and b) phosphorus
removalefficiencyoveranannualcycleinoneofthefourculturesinthetreatmentstep(shallowculture
2a,withnoadditionalilluminationduringwinter).

there was a clear correlation between phosphorus removal efficiency and pH

(Figure18).Thiscorrelationisduetotwointerconnectedphenomena:

1. Higher phosphorus assimilation to algal cells is coupled to higher algal

biomass production, which in turn requires more inorganic carbon. This
results in elevated pH, and phosphorus assimilation therefore indirectly
givesahigherpH.

2. TheincreasedpHinduceschemicalprecipitationofphosphorus.Thehigher
thepH,themoreprecipitation.

theshallowerculturesgenerallyhadbothhigheralgalbiomassproductionand
phosphorusremovalefficiencythanthedeepercultures.Thiscanbeascribedto
differentfactors:

1. The higher light conditions allowed a higher growth rate of the algae and
henceahighernutrientassimilationrate.

2. The higher light conditions resulted in 12C higher temperature in the

shallow cultures than in the deeper cultures. Higher temperature is well
knowntofavouralgalgrowth(Soeder,1981;Grobbelaar,1982;Borowitzka,
1998).
31


3. The higher algal production resulted in higher pH and as a consequence
morechemicalprecipitationofphosphorus.

100%

P-removal efficiency

80%
60%

1a
1b
2a
2b

40%
20%
0%
-20%
7

10

11

pH

Figure18.PhosphorusremovalefficiencyagainstpHduringthewholeyearstudyofthetreatmentstep.
TheremovalefficiencyshowsacorrelationtopHuptopH10approximately.

between50%and90%ofthephosphorusremovalintheshallowcultureswas
due to chemical precipitation, and the proportion in the deeper cultures was
between 40% and 80%. There was, however, no clear correlation between the
sizeoftheprecipitatedfractionandseason.
a pronounced effect by the extra illumination on phosphorus removal during
winter was observed in the shallow cultures but not in the deeper. The
illumination hence was too low to increase the average light intensity in the
deeper algal cultures sufficiently to have a pronounced effect on algal
productivityandphosphorusremoval.
anitrogen(nitrate)removalefficiencyofaround40%couldbereachedformost
parts of the year, and efficiencies of up to 60% 80% were achieved during
summerintheshallowcultures.Aremovaloftotalnitrogenofupto40%was
observedintheshallowtanksduringsummer,andthiswasprobablyaresultof
grazing zooplankton and subsequent ammonia volatilisation as a result of the
highpHinthealgalcultures.

32

Discussionconcerningtheapplicabilityofthetechnique
ToevaluatethesuitabilityofusingmicroalgaeforphosphorusremovalinSweden,we
havetoconsiderthetreatmentperformanceaswellasareaandenergydemand,butalso
thefactthatprecipitationchemicalscanbesavedandthatthereisapossibilityofusing
thealgalbiomassfordifferentpurposes.Theresultshaveclearlyshownthatmicroalgae
are interesting for phosphorus removal due to the combination of assimilation and
biologicallyinducedchemicalprecipitation. However, since wastewater from onlyone
locationhasbeenusedinthestudies,otherresultswouldprobablybeachievedforother
wastewatercompositions.Thewaterattheinletpointtothealgalstepatverjrvagrd
ishard,around200mgCaCO3L1,andchemicalprecipitationofphosphatesistherefore
favoured.Thischemicalstrippingeffectbythemicroalgaecanbeutilisedparticularlyin
areas with hard water, but in places with soft water, the removal efficiency would be
more dependent on the algal phosphorus assimilation. Compared to conventional
phosphorusremovalwithferricoraluminiumsalts,theremovalratesarerelativelylow
sincetheHRTneededforalgalcultivationisaminimumofapproximatelythreedays,
whereas the HRT needed for chemical precipitation and subsequent sedimentation is
rathermeasuredinhours.However,comparedtothehydroponics,whichwerefoundto
remove very little phosphorus, microalgae are efficient. Hence, if the purpose is to
cultivategreenbiomassinthewastewater,thenmicroalgaewouldbeagoodalternative
tochemicalprecipitation.

Thedependencyonlightlimitstheperformanceovertheyearathigherlatitudes
and also results in a large area demand for two reasons; firstly, the reactors must be
shallow to allow good algal growth. Culture depths larger than 50 cm are not
recommended for algal cultures, and at places with lower incident light intensities or
duringwinter,depthssmallerthan20cmarerecommended(Benemann,1979;Fonteset
al.,1987).Secondly,thelightdependencybythealgalbiomassproductionrequiresthat
the HRT is long enough to keep enough biomass in the reactors, and this further
increasestheareademand.Whenitcomestotheexperimentalsetupinthisproject,an
HRTof3daysandaculturedepthof17cmwouldrequireanareaof18m2perm3water
treatedperday,andfor33cmdeepculturestherequiredareawouldbe9m2.
During summer when the incident light intensities were high, a phosphorus
removal efficiency of up to 100% could be reached with 17 cm deep cultures. During
winter, however, the performance with the bioreactor design used in the studies was
muchweakerandmoreuneven.Properlyworkingphosphorusremovalduringwinter
would therefore require either i) more additional illumination than was used or ii)
shallower cultures than those used plus the illumination used in the studies. As
mentioned above however, shallower cultures demand more area, and illumination
requires energy. If the algal biomass could render a product of very high economic

33

value,touseartificiallightisagoodidea,butotherwise,suchenergydemandmaybe
too expensive (Borowitzka, 1996). Commercial cultivation of microalgae for the
production of highvalue products is most often performed using closed systems with
strictmonocultures,andsuchsystemsmaybedifficulttomaintainwithwastewateras
the growthmedium.Consequently,itis worth considering whether anothertechnique
forphosphorusremovalwouldsaveenergyandlandduringthedarkestperiodofthe
year. However, even if the algal method is restricted to operation during spring to
autumn, an appreciable amount of precipitation chemicals could still be saved on a
yearlybasis.
Withthesefacts inmind,microalgaecanbe used for phosphorusremoval from
wastewaterinSwedenparticularlyinoneorseveralofthefollowingcircumstances:

duringsummerwhenhighincidentlight intensities areexperienced, since the

performanceisdirectlycorrelatedtolightavailability.

in areas with hard water, since the biologically mediated phosphorus

precipitationcanbeutilisedwhichgiveshigherremovalefficiency.

if greenhouses can be provided, since this has a positive effect of the algal
growth due to higher temperatures. Outdoor cultivation may give a shorter
growthseason.

if land is not expensive, since the method demands larger area than
conventional wastewater treatment. This also makes the method more
promising for small scale treatment rather than in large centralised urban
treatmentplants.

if the biomass can be used for any purpose, then the economy in the system
wouldbeimproved.

Finally,doesthisgreentechniquecontributetoamoresustainabledevelopmentofthe
Swedish water and sanitation systems? Well, it certainly contributes to the range of
solutions for wastewater treatment, especially for small scale solutions. It is also
appealingsinceitreducestheneedforchemicals,eventhoughthemaincontributionto
theperformanceisinfactduetochemistry!

34

5.Conclusions
The results from the studies performed in the doctoral project described in this thesis
canbesummarisedasfollows:

Microalgal cultivation could efficiently provide extended phosphorus and

nitrogen removal in a hydroponic wastewater treatment system. The algal
growth was mainly light limited, which resulted in higher cell densities and
lowerresidualnutrientsatlongerHRT.Theeffluentlimitforphosphorusof0.5
mgPL1wasreachedfrequentlyduringsummerwithshallowalgalcultures(17
cm)butmoreoccasionallywithdeepercultures(33cm).

The phosphorus removal was a combination of assimilation to algal cells and

precipitationofcalciumphosphatesasaresultoftheincreasedpHinthealgal
cultures. This, in turn, was a result of the assimilation of inorganic carbon by
the algae. The phosphorus assimilation rate was dependent on algal growth
andwasconsequentlyindependentonHRT.Theaveragerateforprecipitation,
on the other hand, was load dependent, i.e. the removal rate increased with
decreased HRT. Between 50% and 90% of the phosphorus removal in the
shallowcultureswasduetochemicalprecipitation,andtheproportionsinthe
deeper cultures were between 40% and 80%. There was, however, no clear
correlationbetweenthesizeoftheprecipitatedfractionandseason.

Inanintensivestudyduringsummer,phosphorusremovalefficienciesbetween
78%and92%wereachievedwiththeshallowalgalcultures,whichcorrespond
tophosphorusremovalratesofupto2.2mgPL1 day-1 (0.4gPm2day1).The
removalefficiencieswerelowerinthedeepercultures,between66%and88%;
however, higher removal rates per area than in the shallow cultures were
observed,upto0.6gPm2day1(1.9mgPL1day-1)

During winter, the algal growth and phosphorus removal efficiency was
considerably lower than during summer. Without extra illumination,
phosphorusremovalefficiencieslowerthan25%wereobservedintheshallow
cultures and even lower efficiencies were experienced in the deeper cultures.
Additional illumination, however, increased the efficiency in the shallow
culturesbutnotinthedeeper.

Thenitrogenremovalwasmainlyaresultofnitrateassimilationbythealgae.A
nitrogen (nitrate) removal of 40% was possible during most parts of the year,
35

buttheremovalperformancewasmoreuneventhanthephosphorusremoval.
Efficienciesofupto60%80%werehoweverobservedduringsummerinthe
shallowtanks.Areductionintotalnitrogenofupto40%intheshallowtanks
during summer was also experienced, which suggests ammonia volatilisation
asasubsequentphenomenonafterzooplanktongrazingandureaexcretion.

Microalgae are appropriate for nutrient removal from wastewater in Sweden

particularly during summer, in areas with hard water, if greenhouses can be
provided,iflandisnotexpensiveorifthebiomasscanbeusedforanypurpose.

36

6.Acknowledgements/Tack
Efter ren som doktorand ser jag nu tillbaka p en mycket intressant och utvecklande
tid, och tnker ven p alla er som betytt mycket fr mig och fr forskningsprojektet.
DenjagfrstochfrmstvilltackarminhandledareGunnelDalhammar.Tackfratt
duanstlldemig,engrnbiolog,tillKTHdrjagaldrigtrottattjagskullehamna.Tack
venfrdinstoraoptimismochentusiasmochfrattdugettmigsgottsomheltfria
hnderattunderskadetjagtycktevarmestintressant.

Sedan vill jag tacka min bitrdande handledare Jes la Cour Jansen, Lunds Tekniska
Hgskola, fr din otroliga energi och snabba feedback samt fr dina ovrderliga
kunskaper om avloppsrening. En av Jes doktorander som jag ocks vill passa p att
tacka r Michael Ljunggren som stllde upp och gjorde flotationsfrsk p algerna
(resultatenblevkanskeintesbra,menviblevmycketklokarepkuppen!).

TackErikSderbck,StockholmsUniversitet,somvarbitrdandehandledareunderen
tid,fratt jag fickkomma till botaniska institutionenoch vara pertlab samt fr den
biologiska vinkeln p algers och cyanobakteriers tillvxt. I samma veva vill jag ven
tacka Sara Jonasson och alla de andra doktoranderna p SU vxtfysiologi. Det var en
jttetrevlig tid nr jag fick vara hos er och leka lite biolog igen. Tack ven Marianne
PedersnsomvarminfrstakontaktpbotansamtMariaGregerochsaFritiofffrert
intressefrreningsverket.

EttjttestorttackvilljagriktatillhelaUrbanWaterprogrammet,framfralltPerArne
MalmqvistochHenrietteSderberg.Detharvaritjtteintressantattfvaramedietts
stort forskningsprogram med s mnga olika kompetenser. Ett superstort tack till Jan
OlofDrangertockssomhlliforskarskolansomjagtyckervartoppen!Menvadvore
den utan alla Urbandoktorander: Anna, Kristina, Helena K, Helena A(P), Mattias,
Therese, Jakob, Annika, Jonas, Pernilla, Frank, Gerald, Mats, Edgar och Stefan. Alla
forskarskolekurserochkonferenserharvaritenfrjdtackvareer!

Ett stort tack till Anna Norstrm fr alla ren i vxthuset och p KTH med
gemensamma arbetsbekymmer och trevliga fikaraster, samt inte minst fr dina petiga
genomlsningar av mina artiklar. Tack Bjrn Oliviusson fr att du tog hand om
vxthusetochgjordedettillengrnoas,samtfrhjlpmedvattenanalyserochannat.
Tack Helen Vallhov fr din hjlp under intensivstudien. Tack Kaj fr alla
mikroskopbilderochandrafoton.Thankyoualltheothermembersofthemicrobiology
group: Sofia, Guna, Lydia, Pelle, KarlJohan, Karin E., Erik, Fredrik, Guro, Joseph,
Seyoum,Lena,Jacky,Peter,Helenaandalltheotherformermembers,PhDstudents,ex
37

jobbare mm. It has been a great time working with all of you! Thank you all the nice
peopleatfloor2also,especiallyLottaandAlphonsa.

Tackallapverjrvagrd,frmstNinniochElisabetfrertengagemangivxthuset
ochreningsverketochfrdenmyckettrevligaarbetsmiljn.

EttspecialtacktillminalgkollegaErikGrnlundistersundsomvgadesigpattodla
algerinorrland!

Supertack till mamma, pappa och Eva fr er totala support och lojalitet, och brorsan
ocks,tackelitacktack!Tackallakravnnerfravkopplandestunderunderdessalnga
r som jag traskat i avloppstrsket. Ondligt mycket tack till Andreas fr krleksfullt
std,omtanke,otippatengagemangifosforreningochmikroalger,ettlitetdataprogram
somkunderknautreningshastigheterfrndenmestkomplexamtseriesamtattstort
antalmiddagar.TackockskralillaRitasomkomslmpligt,jagficknstanettheltr
utan forskning precis innan avhandlingsskrivandet, en vlbehvlig paus med stora
tankestllareomvadsomrviktigasthrilivet!

38

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