Microalgal Bioprocessing - Introduction

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MicroalgalBiotechnology:

CultivationandProcessing

MBT Fall2015

October29th,2015 HectorDelaHozSiegler

DepartmentofChemicalandPetroleumEngineering

UniversityofCalgary

[email protected]

HectorDelaHozSiegler,Ph.D.,P.Eng.

Outlineoftodaystalk

I. Introduc tion to microalga e

What and why

Applications

II. Renewable energy from microalga e

Motivation

Biofuel portfolio

Biodiesel

III. Culturing techniques

Medium requirements

Open ponds and photobioreactors

Phototrophic and heterotrophic

IV. Optimization of heterotrophic culturesV. Summary

INTRODUCTIONTOMICROALGAL

BIOTECHNOLOGY

PartI

3


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Microalgae:whatarethey?

Microalgaeareplantlikeunicellularorganisms:apolyphyletic group

ofphotosyntheticeukaryotes.

Sunlight-driven cell factories able to convert carbon dioxide to

potential biofuels, food, and high-value products

4

ProductsfromMicroalgae

Image source: Rosenberg, J.N., Oyler, G.A., Wilkinson, L., Bet enbaugh, M.J.Agreen light for engineeredalgae: redirecting metabolismto fuel abiotechnology

revolution ,Current Opinion in Biotechnology, 19 (5), pp. 430-436 (2008)

Applications

6

Microalgae

Biofuels

Finechemicals:

e.g.antioxidants

Wastewater

treatment/

Remediation

Pharmaand

nutraceuticals

Humanand

animalfood

CO2Capture


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Microalgaeascellfactories

Benefits:

Highlyefficient

Simplenutritional

requirements

Easilyadaptabletoenvironmentalstresses

Produceandstorehighamountsofoil

Othervaluablebyproducts

Eukaryotic!

Challenges

Lowculturedensity

Slowgrowth:lowproductivity(comparedtobacteriaandyeast)

Highproductioncosts

7

SomeCommercialApplications

S pe ci es/ gr ou p Pro du ct A pp li ca ti on a re as Pr od uc ti on

facilities

References

Haematococcus

pluvialis /

Chlorophyta

Carotenoids,

astaxanthin

Health food, feed additives

and pharmaceuticals

Open ponds,

PBR

Del Campo et al. (2007)

Odontella aurita

/ Bacillariophyta

Fat ty acids Pha rm aceu ti ca ls ,

cosmetics, baby food

Open ponds Pulz and Gross (2004)

Isochrysis

galbana /

Chlorophyta

Fatty acids Animal nutrition Open ponds,

PBR

Molina Grima et al.

(1994); Pulz and Gross(2004)

Phaedactylum

tricornutum /

Bacillariophyta

Lipids, fatty

acids

Nutrition, fuel production Open ponds,

basins, PBR

Yongmanitchaiand Ward(1991); Acien-

Fernandez et al. (2003)

Muriellopsis sp.

/ Chlorophyta

Carotenoids,

Lutein

Health food, food

supplement, feed

Open ponds,

PBR

Blanco et al. (2007); Del

Campo et al. (2007)

Crypthecodinium

cohnii

DHA Food additive Fermenters

(heterotrophic)

Carvalho et al.

(2006)

8

Currently,applications

of

microalgal

biotechnology

are

limited

to

niche

(small)

markets.

Though

highvalue!Weexpecttomoveintolargescalemarkets.

ALGAEASASOURCEOF

RENEWABLEENERGY

PartII

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Energy,economy,andGlobalwarming

Poland

Uzbekistan

Chad

Ghana

Vanuatu

India

China

Russia

Brazil

UK

France

US

Norway

Japan

South

Korea

Canada

China (2002)

India(2002)

US(2002)

0.01

0.1

1

10

100

100 1,000 10,000 100,000

PrimaryEnergyConsumption(kWyear/p

erson)

GDP($/person/year)

Energyreserves/Energyconsumption

11

Climatechangedebate


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Thenaturalcarboncycle

G. A. Olahand colleagues, J.Am.Chem.Soc, 133, 12881-12898, 2011

Carbon CaptureandUtilisation(CCU)

T. Shirvani, X. Yan, O. R. Inderwildi, P. P. Edwards and D. A. King, Energy Environ. Sci., 2011, 4, 37733778

Biofuels

15

1stGeneration: derived from food-crops, i.e.ethanol from sugar cane or corn, biodieselfrom canola or soybeans.

2ndGeneration

: produced from lignocellulosicmaterials, i.e. ethanol from wood chips,switch grass.

3rdGeneration: fuels from microalgae

4thGeneration: from crops designed for fuelsin combination with highly efficient microbes.

Timetorealworldapplication

Landrequiredforsatisfydemand


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Themicroalgalbiofuelsportfolio

Algal Biomass:

- Oil/Lipids

- Sugars/Starch

- Lignocellulose

Excreted products:

- Hydrogen

- Alcohols

Sugars

Bio-oil

SynGas

Biodiesel

Green Diesel

Gasoline

Hydrogen

Alcohols

CO2

Water

Sunlight

Trace elements

Feedstocks Photosynthe sis Inte rme diates Fu els

Pyrolysis

Hydrolysis

Hydrodeoxygenation

Hydrotreating

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BiodieselfromMicroalgae

Crop Oil yield

(L/Ha)

Land area needed

(M Ha)

% of existing US

cropping area

Corn 172 1540 846

Soybean 446 594 326

Canola 1190 223 122

Oil Palm 5950 45 24

Microalgae(70% oil w/w)

136900 2 1.1

Microalgae(30% oil w/w)

58700 4.5 2.5

Biodieselderivedfromoilcropsisapotentialrenewableand

carbonneutralalternativetopetroleumfuels.

Biodieselfromoilcrops,wastecookingoilandanimalfatcannot

realisticallysatisfythedemandfortransportfuels.

Croplandrequirement

bydifferentoilcropsto

replace50%ofall

transportfuelneedsof

theUS.Chisti(2007).

Toooptimistic

to

be

true!

17

Algaeasasourceofoil

Species Oil content

(% dw)

Reference

Ankistrodesmus TR-87 28 40 Ben-Amotz and Tornabene (1985)

Botryococcus braunii 25 75 Sheehan et al. (1998); Banerjee et al. (2002); Metzger and Largeau (2005)

Chlorella sp. 28 32 Sheehan et al. (1998), Chisti (2007)

Chlorella protothecoides 15 55 Xu et al. (2006)

Cyclotella DI-35 42 Sheehan et al. (1998)

Dunaliella tertiolecta 36 42 Kishimoto et al. (1994); Tsukahara and Sawayama (2005)

Hantzschia DI-160 66 Sheehan et al. (1998)

Isochrysis sp. 7 33 Sheehan et al. (1998); Valenzuela-Espinoza et al. (2002)

Nannochloris 20 -35 (6 -63) Ben-Amotz and Tornabene (1985); Negoro et al. (1991); Sheehan et al. (1998)

Nannochloropsis 46 (31 -68) Sheehan et al. (1998); Hu et al. (2006)

Nitzschia TR-114 28 50 Kyle DJ, Gladue RM. WO 91/14427 (Patent)

Phaeodactylum tricornutum 20 31 Sheehan et al. (1998), Chisti (2007)

Scenedesmus TR-84 45 Sheehan et al. (1998)

Stichococcus 33 (9 -59) Sheehan et al. (1998)

Tetraselmis suecica 15 32 Sheehan et al. (1998); Zittelli et al. (2006); Chisti (2007)

Thalassiosira pseudonana (21 -31) Brown et al. (1996)18


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OilandBiodiesel

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Triglycerides:

BiodieselProduction:

Glycerol

FattyAcids

PolyunsaturatedFattyAcids(PUFA)

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Fattyacidswithmultipledoublebonds

C18:3andlongerareessential:mammalscannotsynthesize

C18:3.Needtotakethemfromtheirdiet

Multiplebiologicalfunctionsassignallingmoleculesorbuilding

blocks

DHA: C22:6

EPA: C20:5

MicroalgaeasaSourceof3PUFA

FishoilhasbeenusedforthecommercialproductionofEPAandDHA.

Factorsthatlimitfishoilasasourceof3fattyacidsinclude:taste,odourandstabilityproblems.Highpurificationcost.

Fishobtain3fattyacidsfromtheirdiet.

SeveralspeciesofmicroalgaeareprimaryproducersoflongchainPUFA.

US$1.5billion/yeargeneratedfromtheproductionofDHA(Pulz andGross,2004).

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PUFAproportionsinMicroalgae(%TFA)

Organ is ms ARA ( 20 :4 ) EPA ( 20 :5 ) DHA ( 22 :6 )

Gymnodinium splendens 8 30

Cricosphaera elongata 2 28

Isochrysis galbana 15 7.5

Monodus subterraneus 4.7 33

Nannochloropsis sp. 35

Schizochytrium sp. 1.0 2.3 40.9

Chlorella minutissima 5.7 45

Hetermastrix rotundra 1 28 7

Chromonas sp. 12.0 6.6

Cryptomonas sp. 16 10

Rhodomonas sp. 8.7 4.6

Asterionella japonica 11 20

Biddulphia sinensis 24 1

Crypthecodinium cohnii 30

Nitzschia laevis 6.2 19.1

Phaeodactylum Tricornutum 34.5

Skeletonema costatum 29.2 22

GeneralProcessDiagram

Harvesting

Dryer

CultureExtraction

Crude

Product

debris

S/L Separator

Solvent

recovery

Cell disrupter

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MICROALGALCULTURING

TECHNIQUES

PartIII

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Nutritionalrequirements

Dependsonapplication

Foodorhealthoils:foodgradechemicals

Otherwiseindustrial

chemicals

or

seawater

/wastewater

Carbonsource:CO2,sugars,acetate,ethanol

Macronutrients:Nitrogenandphosphorus

Micronutrients:Fe,Mg,Si,S,K

Traces:Ca,Mn,Zn,Co,Se,Cu,Mo

Vitamins:B1,B12,B6,B2

Seawater:Na,K,Mg,Ca,Cl,SO4,HCO3,BO3

Br,F,IO3,Li,Rb,Sr,Ba,Mo,V,Cr,As,Se

NO3,PO4,Fe,Zn,Mn,Cu,Co,Si,Ni25

Multiplewaysofgrowing

26

Mixotrophic

FixCO2

Fast growth

Productivealldaylong

Complexand

unknowncellregulation

CO2 capture

Waste

water

treatment Highvalueproducts

Proteins

EnergySource

Applications

Phototrophic

FixCO2

Slowgrowth

Noactivityduringnight

CO2capture

Heterotrophic

Fast growth

Productivealldaylong

Dontfixgreenhousegases

ProduceCO2

Wastewatertreatment

Highvalue

products

Proteins

SolarradiationinCanada


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SolarradiationinAlberta

FortMcMurray: 4 181MJ/m2y

Edmonton: 4510MJ/m2y

MedicineHat: 5221MJ/m2y

Munich(GE): 4044MJ/m2y

Naples(IT): 5293MJ/m2y

KualaLumpur: 5622MJ/m2y

Orlando(FL): 5922MJ/m2y

Acapulco(MX): 7261MJ/m2y

Phoenix(AZ): 7621MJ/m2y

Solarradiation datatakenfrom:U.S.Department ofEnergyEnergyPlusWeather Data.

http://apps1.eere.energy.gov/buildings/energyplus/cfm/weather_data.cfm

Culturingtechniques:OpenPonds

Byfar,themostcommonproductionsystem.

Lowinstallationcost

Lagoonsorartificialponds

Highriskofcontamination: Unwantedalgae

Grazers

Applicationlimitedtofewspecies(extremophiles).

Unmixedponds:arearangefrom1200Ha,depth2030cm

Racewaypondsareupto1Ha.

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Culturing:ClosePondsandTanks

Simplerdesignssimilartoopen

ponds,withacover

(greenhouses).

Aimtoreducecontamination

risks. ControlCO2looses.

Tanksareusuallymixedby

aeration.

Deeptanksareinefficient.Bad

lighttransmission.

Easytooperate,lowcost.

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Culturing:Photobioreactors

TubularPhotobioreactor AlgaeandBiofuelsFacility, SouthAustralianResearchandDevelopmentInstitute

FlatPanelphotobioreactor

ArizonaCenterforAlgalTechnology andInnovation

FlexibleplasticfilmPhotobioreactorAlgenol,Florida 31

Culturing:Photobioreactors

Betterculturecontrol

Higherproductivity,andculture

density

Minimalcontaminationrisk

Wellmixed

Excellenttemperaturecontrol

Oxygencontrolisanissue

Highcapitalinvestment

Frequentcleaningrequired

Coolingrequired

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HeterotrophicProductionofAlgae

Somealgaespeciescangrow

usinganorganiccarbonsource.

Conventionalbioreactorscanbe

used.

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Phototrophicvs.Heterotrophic?

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SpecieOil content

(%)

Cell conc.

(g/L)

Oil Prod.

(mg/L d)References

Ettlia oleoabundans 36 42 2.9 164 Griffiths et al (2009);

Li et al. (2008)

Nannochloropsis sp. 31 68 2.1 204 Rodolfi et al. (2009)

Amphora 40 51 - 593 Sheehan et al. (1998)

Chlorella sp. 28 32 1.1 139 Hsieh and Wu (2009)

Chlorella vulgaris 25 42 1.7 54 Liang et al. (2009)

Chlorella zofingiensis 25.8 1.9 35 Liu et al. (2010)

Chlorella zofingiensis 51.1 9.6 354 Liu et al. (2010)

Nitzschia laevis 16.5 22.1 914 Wen and Chen (2003)

S. Limacinum (DHA) 17.3 37.9 656 Chi et al. (2009)

A. protothecoides 38.3 53.0 8.4 820 Cheng et al. (2009)

A. protothecoides 50.3 57.8 51.1 3320 Xiong et al. (2008)

Phototrophic

Heterotrophic

MODELBASEDOPTIMIZATIONOF

HETEROTROPHICALGALCULTURES

PartIV

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BioprocessOptimization

36

Strainselection

Mediaformulation

Processconditions

Continuous/

Realtime

Geneticmodification


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TheObjectiveforOptimization

37

StressOil sto ring is a metabol ic response to stress,

particularly nitrogen def iciency. At nitrogen

deficient conditions, algal cells over-accumulatelipids.

The challenge is to maximize biomass production

while keeping a high oil content. It is necessary to

determine the nitrogen supplementation strategy

to achieve this.

Nitrogen

As nitrogen is required for protein synthesis, its

deficiency negatively affects growth and cel l

functioning. Therefore, conditions that favored oil

accumulation constraint productivity.

Understandingalgalgrowth

38

Nitrogen uptake

Lipid production

Cellular growth

Analgalgrowthmodel

39

Cellular growth

Nitrogen uptake

Oil production


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Macroscopicbalances

40

Optimization:Problemformulation

41

Subject to:

Simulationresults

42

Biomassproductivityin

continuouscultures

Lipidproductivityin

continuouscultures


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Experimentalresults

43

Biomassproductivityandgrowthrate

44

Lipidproductivityandproductionrate

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Comparativestudy:growthonglucose

46

Specie Lipid content

(%, w/w)

Oil Productivity

(g/L h)

References

E. coli (gen. modified) 25.4 0.246 Elbahoul et al (2010)

R. opacus PD630 38.4 0.171 Kurosawa et al (2010)

M. ramanniana 67.7 0.17 Hiruta et al (1997)

C. echinulata 26.9 0.07 Kosa et al (2011)

R. toruloides 67.5 0.54 Li et al. (2007)

L. starkeyi 56.0 0.04 Kosa et al. (2011)

C. curvatus 82.7 0.47 Zhang et al. (2011)

Schizochytrium sp. 30 0.096 Ganuza et al (2007)

C. vulgaris 9.7 0.12 Doucha et al. (2011)

A. protothecoides 50.3 0.14 Xiong et al. (2008)

A. protothecoides 49.4 0.43 0.84 De la Hoz et al (2012)

Bacteria

Molds

Yeasts

Microalgae

Optimization:closingremarks

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Modelbased optimization of heterotrophic microalgal cultures

allowed to reach very high densities, with biomass productivity

greater than 30 g/L d, and as high as 70 g/L d.

High oil content (4060% w/w) can be sustained with a lipid

productivity around 20 g/L d.

High quality monitoring and control is essential to achieve high

productivities.

Better control / sensors = higher productivity.

Summary

Algaearepromisingorganisms:highlyefficient

Goodsourceofoil:PUFA,biodieselprecursor

Algaecan

growth

on

simple

inexpensive

media

Severalreactortypesandgeometry.Applicationwilllimit

reactorchoice

Severalsuccessfulcommercialapplicationscurrentlyworking.

Alotofresearchisstillneeded!

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Microalgal Bioprocessing - Introduction