Fodder Yeast and Biogas Production

54 Pages·2016·5.02 MB·English

Faculty of Natural Resources and
Agricultural Sciences
Fodder Yeast and Biogas Production:
A Fruitful Symbiosis?
– Optimization of a Single-Cell Protein Process and
Examination of its Impact on Biogas Output
Jonas Ohlsson
Department of Microbiology
Master's Thesis • 30 hec • Second cycle, A2E
Agriculture Program – Food Science • Examensarbete/Sveriges lantbruksuniversitet,
Institutionen för mikrobiologi, 2016:5 • ISSN 1101-8151
Uppsala 2016

Fodder Yeast and Biogas Production: A Fruitful
Symbiosis? - Optimization of a Single-Cell Protein Process
and Examination of its Impact on Biogas Output
Jonas Ohlsson
Supervisor: Su-lin Hedén, Swedish University of Agricultural Sciences,
Department of Microbiology
Assistant Supervisor: Matilda Olstorpe, Swedish University of Agricultural Sciences,
Department of Microbiology
Examiner: Volkmar Passoth, Swedish University of Agricultural Sciences,
Department of Microbiology
Credits: 30 hec
Level: Second cycle, A2E
Course title: Independent project/degree project in Food Science – Master’s thesis
Course code: EX0425
Programme/education: Agriculture Programme – Food Science
Place of publication: Uppsala
Year of publication: 2016
Cover picture: Jonas Ohlsson
Title of series: Examensarbete/Sveriges lantbruksuniversitet, Institutionen för
mikrobiologi No: 2016:5
ISSN: 1101-8151
Online publication: http://stud.epsilon.slu.se
Keywords: fodder yeast, biogas, nutrient recycling, Wickerhamomyces anomalus, Pichia
kudriavzevii, Blastobotrys adeninivorans
Sveriges lantbruksuniversitet
Swedish University of Agricultural Sciences
Faculty of Natural Resources and Agricultural Sciences
Uppsala BioCenter
Department of Microbiology

Abstract
Duetoagrowingworldpopulationandstrainedwildfishstocks,aquaculture
isexpectedtoprovidealargepartoftheincreasedglobaldemandforanimal
protein,asthemajorityofwildfishstocksarealreadybeingfishedatorabove
their sustainable capacity. However, fish feed often contains a significant
amount of fish meal, produced from wild-caught fish. Thus, increasing
farmedfishproductionmaynotbesustainableaslongasfishmealremainsa
majoringredient.
Microbialbiomass,commonlyknownassingle-cellprotein(SCP),can
replacefishmealinfeedformulations. ThefinancialviabilityofSCPpro-
duction is dependent on the availability of cheap growth media. Biogas
substrates,oftenmixturesofwastederivedfromfoodindustry,agriculture,
andhouseholds,couldbeidealmediaduetotheirlowcostandubiquitous
availability.
Theaimsofthisthesiswere: first,toinvestigatewhetheryeastSCPcan
beproducedonabiogassubstrateconsistingofhouseholdandagricultural
waste; second, to screen several yeast strains and growth conditions for
optimal biomass production; and third, to evaluate the effects on biogas
productionifpartofinputsubstratestreamisdivertedintoSCPproduction
priortofurtherdigestioninthebiogasreactor.
SeveralyeaststrainswerescreenedforbiomassyieldatseveralpHlev-
els and temperatures. After screening, best-performing strains were culti-
vatedonbiogassubstrateincontinuously-stirredbioreactors,analyzingthe
resultant biomass. Strains were Wickerhamomyces anomalus J121, Pichia
kudriavzeviiJ550,andBlastobotrysadeninivoransJ564. Harvestedbiomass
rangedfrom7.0–14.8gdrymatterperlsubstrate,andproteincontentswere
22.6–32.7%. Levelsoftheaminoacidlysinewerehighinallbiomasssamples,
whichisimportantforreplacingfishmealinfeedformulations.
Supernatants from the bioreactor fermentations were combined with
wholebiogassubstrate,andtheeffectsonbiogasproductionwereevaluated,
asmethaneproducedpergofvolatilesolids,usingabatchmethanepotential
assay. At the highest inclusion levels (1:1 control/supernatant (w/w)) P.
kudriavzeviiprovidedanincreasedoutputcomparedtoW.anomalus(25%,
p = 0.04). P. kudriavzevii provided a 24% increase compared to control,
butresultswerenotsignificant(p =0.06). Filtered,untreatedsupernatant
providedthelargestincreaseinbiogasproductioncomparedbothtocontrol
substrate(30%, p =0.02)andtoW.anomalus(31%, p =0.01).
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Sammanfattning
I takt med en ständigt växande världsbefolkning förväntas efterfrågan på
animalisktproteinöka.Akvakultur,ellerfiskodling,kommersannoliktatt
bidratillenstordelavdenförväntatökandeproduktionen.Produktionen
från majoritetenavvilda fiskbestånd beräknasintekunnaöka ytterligare,
ochenstorandelklassasredansomöverfiskade.Dåfiskmjöl,somframställs
frånvildfångadfisk,utgörenviktigingrediensifiskfoderärdetintesäkertatt
enutökadproduktionavodladfiskärhållbar,ochalternativaproteinkällor
ärdärförefterfrågade.
En sådan proteinkälla utgörs av mikrobiell biomassa, eller singelcell-
protein (SCP). För storskalig produktion av SCP krävs tillgång till billigt
tillväxtmedium.Biogassubstrat,oftaenblandningavavfallfrånlivsmedels-
och jordbrukssektorn samt hushåll, skulle kunna utgöra ett idealt sådant
mediump.g.a.desslågakostnadsamthögatillgänglighet.
Syftet med detta arbete var tredelat: att undersöka om SCP från jäst
kanproduceraspåbiogassubstratbeståendeavhushålls-ochjordbruksavfall;
vidareattutvärderaflerajäststammarsamtodlingsbetingelserföroptimal
tillväxt; och slutligen att utvärdera effekten på biogasproduktion om jäst
odlaspåendelavbiogassubstratetföredessintroduktionibiogasreaktorn.
Ett antal jäststammar utvärderades för biomasseproduktion vid flera
temperaturersamtpH-nivåer.Demesthögproducerandestammarnaodlades
sedanpåbiogassubstratifermentorer,ochdenproduceradebiomassananaly-
serades.DessavarWickerhamomycesanomalusJ121,PichiakudriavzeviiJ550
ochBlastobotrysadeninivoransJ564.Fermentationernaresulteradeislutlig
biomassapå7.0–14.8gperlsubstrat,ochinnehöll22.6–32.7%protein.
Supernatanternafrånodlingarnablandadesmedorörtbiogassubstratoch
utvärderadesförmetanproduktion,sommängdmetanpergglödförlust,iett
batchförsök.Viddenhögstagradeninblandning(obehandlatsubstratoch
supernatantiett1:1-förhållandebaseratpåvikt)produceradeproverbase-
radepåP.kudriavzevii25%merbiogasjämförtmedmotsvarandefrånW.
anomalus(p =0.04).En24%-igökningkundesesdåsupernatantfrånP.kud-
riavzeviiJ550jämfördesmedkontrollsubstrat,mendettaresultatuppnådde
inte statistisk signifikans (p = 0.06). Den största ökningen av biogaspro-
duktion sågs vid tillsats av obehandlat, filtrerat substrat, som presterade
bättreänbådeobehandladkontroll(30%), p =0.02)ochW.anomalus(31%,
p =0.01).
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Contents
1 Introduction 5
1.1 Aquacultureandfishfarming 6
1.1.1 FishfarminginEuropeandtheNordiccountries 7
1.1.2 Feedcompositionandingredients 7
1.2 Single-cellprotein 8
1.2.1 Productionofsingle-cellprotein 8
1.2.2 Commercialexamples 9
1.2.3 Microbialfishfeed 10
1.3 Theanaerobicdigestionprocess 11
1.3.1 Overview 11
1.3.2 Evaluatingmethanepotential 12
2 Overviewandcommonmethods 13
2.1 Overviewofexperiments 13
2.2 Commonmaterialsandmethods 13
2.2.1 Biogassubstrateandinoculum 13
2.2.2 Culturemediaandchemicals 14
2.2.3 Yeaststrains 14
2.2.4 Yeastinoculumpreparation 15
2.2.5 Viablecellcountsandbiomassmeasurements 16
2.2.6 Bioreactoroperation 17
2.2.7 Chemicalanalyses 17
2.2.8 Statisticalanalyses 17
3 Yeastscreening 18
3.1 Materialsandmethods 18
3.2 Results 19
4 Effectonmethanepotentialbyyeasttreatmentofsubstrate 19
4.1 Materialsandmethods 20
4.2 Results 22
5 Scale-upexperiments 24
5.1 Materialsandmethods 24
5.2 Results 24
6 Discussion 27
7 Conclusions 31
3
Appendices 36
List of Tables
1 Chemicalcharacterizationofthebiogassubstrate 14
2 Culturemediaandsolutions 16
3 Yeaststrainsusedinthisthesis 16
4 Overviewoftreatmentsincludedinthemethanepotential
assay 21
5 Nutritionalcharacteristicsoftheyeastbiomass 25
6 Biomassconcentration,proteincontent,andproductivity 29
D Aminoacidcompositionofthefinalwashedpellets 46
E Chemical characterization of the biogas substrate during
differentstagesofprocessing 47
List of Figures
1 Worldfisheriesandaquacultureproductionbyyear,1950–2013 7
2 World fisheries, aquaculture, and livestock production by
year,1961–2012 8
3 Overviewoftheanaerobicdigestionprocess 12
4 Biogassubstratebeforeandafterfiltration 15
5 Example96-wellplatelayoutusedinscreeningexperiment 18
6 LightmicrographsofW.anomalusJ121fermentersamples 22
7 Specific methane potential for untreated whole substrate
withaddedsupernatant 23
8 Relativeessentialaminoacidcompositionofyeastpellets 26
A1 Screeningresultsfor30°C 36
A2 Screeningresultsfor25°C 36
A3 Screeningresultsfor37°C 37
A4 Screeningresultsfor30°C 37
A5 Screeningresultsfor30°C 38
A6 ScreeningresultsfordeterminingoptimumpH 38
C1 CFUcountsforcultivationofyeaststrainsonfilteredbiogas
substrate,BMPassay 43
C2 CFUcountsfromthescale-upfermentations 44
C3 Drymatterestimatesfromthescale-upfermentations 45
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Acronyms and abbreviations
AA Aminoacid
AD Anaerobicdigestion
BMP Biomethanepotential
CFU Colonyformingunit
CL Crudelipid
CP Crudeprotein
CSTR Continuouslystirredtankreactor
EAA Essentialaminoacid
FM Fishmeal
FO Fishoil
GHG Greenhousegas
IHT Inter-specieshydrogentransfer
OD Opticaldensityat600nm
600
RNA Ribonucleicacid
QPS Qualifiedpresumptionofsafety
SCP Single-cellprotein
TS Totalsolids
VS Volatilesolids
1 Introduction
Withaneverincreasingworldpopulation,resourcescarcityisexpectedto
become a global problem. In 2050, the world population is expected to
reach9.6billion(FAO,2014),whichwilllikelyputastrainontheworld’s
foodandenergysupplies. Tomeettheheighteneddemand,effortstowards
increasingfoodandenergyoutputsarerequired. Increasingtheseoutputs
does come with its own cost however, as the world’s supply of natural
resources (including, but not limited to, fossil fuels and phosphorus) are
furtherdiminishedwhilepromotinghigherratesofgreenhousegas(GHG)
emissionsbytheburningoffossilfuels.
Aquaculture,andespeciallythepracticeoffishandshellfishfarming,has
suppliedasubstantialportionoftheincreaseddemandforanimalprotein
during the last few decades, and is expected to continue doing so as the
world’s population grows larger. It is the fastest growing primary animal
food producing sector, exhibiting an average annual rate of expansion of
6.2%intheyears2000–2012(FAO,2014). Althoughfarmedfishandshellfish
exhibitgoodfeedconversionratioscomparedtomanyotherfarmedanimals,
alargepartofaquacultureisstilldependentondecreasingmarinefishstocks
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forfeed. Dwindlingsuppliesandincreasingpricesmeanthatalternativesto
fishmeal(FM),suchassoyameal,arebeingusedasreplacements.
AnotherFMalternativeismicrobiallyproducedbiomass,anovelsource
ofcaloriescommonlyknownassingle-cellprotein(SCP;MatelbsandTan-
nenbaum(1968)). Althoughnotentirelycomprisedofprotein,thismacronu-
trienthasusuallybeenthefocusofsuchproducts. Theseproductsmaybe
marketedeitherforhumanconsumption,orasingredientsofanimalfeed.
SCP has several benefits compared to other feedstocks: large amounts of
biomass can be manufactured in short time; organisms used for SCP pro-
ductionnormallyhavelownutrientrequirements;oftenthesubstratesused
arelow-valuecommodities,orsidestreamsandwasteproductsfromother
manufacturingprocesses.
ToreduceGHGemissionsandpromoteenergyindependenceandenergy
securityasfossilfuelsbecomescarcer,methodsforbioenergyproduction
are the focus of much research. Biogas production is one such method,
wherebywasteproductsareconvertedintoenergy-richmethane(CH )and
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nutrient-richfertilizer(digestate)inananaerobicdigestion(AD)process.
Nutrient recycling and efficient use and generation of energy are im-
portant factors for sustaining the world in light of what’s been discussed
above,andtheirimportancewillincreasewithresourcescarcity. Feedinga
growingworldpopulationusingfewerresourceswilllikelybecomeoneof
themostpressingissuesofthiscentury. Theaimofthisthesisistoinvesti-
gateacombinedSCPandbiogasproductionusingthesamesubstrate,and
assesswhetheritrepresentsanovelwayofincreasingresourceutilization
andnutrientrecycling.
1.1 Aquaculture and fish farming
Aquaculturereferstothecultivationofwater-livingcreaturessuchasfishes
andmollusks,butalsoaquaticplants. However,thefocusonaquaculture
in this thesis refers exclusively to finfish, as this is the group of aquacul-
ture organisms that are the intended recipients of the SCP product being
developed.
Sincethebeginningofthe1980s,theincreaseinworldfishproductionis
entirelyduetofarmedfish(FAO,2014;seeFig.1). Ifcurrenttrendscontinue,
itwillbecomethelargestanimalfoodproducingsectorinafewyears. In
Fig.2,bothfishandlivestock(includingchicken)productionaredisplayed
forcomparison(FAO(2014,2016).
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