Microalgal Bioprocessing: Process Technologies, Modelling and Optimization

MBT – Fall 2014

October 28th, 2014 Hector De la Hoz Siegler

Department of Chemical and Petroleum Engineering University of Calgary

h.siegler@ucalgary.ca

Hector De la Hoz Siegler. PhD.

Outline of Today’s Lecture

I. Introduction to microalgae – What and why

– Applications: • Biofuels

• Nutraceuticals

II. Culturing techniques – Medium requirements

– Open ponds and photobioreactors

– Phototrophic and heterotrophic

III. Optimization of heterotrophic cultures IV. Summary

INTRODUCTION TO MICROALGAL BIOTECHNOLOGY

Part I

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Microalgae: what are they?

• Microalgae are plant-like unicellular organisms capable of producing several end-products that can be used as, or converted into, fuels: hydrogen, ethanol, oil, starch, lignocellulose.

• The term microalgae comprises a polyphyletic group of photosynthetic eukaryotes. Microalgae have a great capacity for adapting to changing environmental conditions as well as using different substrates.

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Microalgae as efficient organisms

• Benefits: • Highly efficient microorganisms

• Nutrient flexibility

• Stress adaptability

• Produce and store high amounts of oil

• Other valuable byproducts

• Challenges • Low culture density

• Slow growth: low productivity

• High production cost

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Applications

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Microalgae

Biofuels

Fine chemicals Waste-water treatment / Remediation

Pharma- and nutraceuticals

Human and animal food

CO2 Capture

Some Commercial Applications

Species/group Product Application areas Production facilities

References

Haematococcus pluvialis / Chlorophyta

Carotenoids, astaxanthin

Health food, feed additives and pharmaceuticals

Open ponds, PBR

Del Campo et al. (2007)

Odontella aurita / Bacillariophyta

Fatty acids Pharmaceuticals, 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

Yongmanitchai and 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)

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Currently, applications of microalgal biotechnology are limited to niche (small) markets. Though high value! We expect to move into large scale markets.

Biofuels: a renewable energy source

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Energy reserves / Energy consumption

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Biofuels

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• 1st Generation: derived from food-crops, i.e. ethanol from sugar cane or corn, biodiesel from canola or soybeans.

• 2nd Generation: produced from lignocellulosic materials, i.e. ethanol from wood chips, switch grass.

• 3rd Generation: fuels from microalgae

• 4th Generation: from crops designed for fuels in combination with highly efficient microbes.

Tim

e to

real

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Land

requ

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for s

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The microalgal bio-fuels portfolio

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 Photosynthesis Intermediates Fuels

Pyrolysis

Hydrolysis

Hydrodeoxygenation

Hydrotreating

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Biodiesel from Micro-algae

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

• Biodiesel derived from oil crops is a potential renewable and carbon neutral alternative to petroleum fuels.

• Biodiesel from oil crops, waste cooking oil and animal fat cannot realistically satisfy the demand for transport fuels.

Crop land requirement by different oil crops to replace 50% of all transport fuel needs of the US. Chisti (2007). Too optimistic to be true!

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Algae as a source of oil 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) 13

Oil and Biodiesel

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Triglycerides:

Biodiesel Production:

Gly

cero

l Fatty Acids

Polyunsaturated Fatty Acids (PUFA)

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• Fatty acids with multiple double bonds

• C18:3 and longer are essential: mammals cannot synthesize C18:3. Need to take them from their diet

• Multiple biological functions as signalling molecules or building blocks

DHA: C22:6

EPA: C20:5

Microalgae as a Source of ω-3 PUFA

• Fish oil has been used for the commercial production of EPA and DHA.

• Factors that limit fish oil as a source of ω-3 fatty acids include: taste, odour and stability problems. High purification cost.

• Fish obtain ω-3 fatty acids from their diet.

• Several species of microalgae are primary producers of long chain PUFA.

• US$ 1.5 billion/year generated from the production of DHA (Pulz and Gross, 2004).

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PUFA proportions in Microalgae (% TFA)

Organisms 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 — 17

General Process Diagram

Harvesting

Dryer

Culture Extraction

Crude Product

debris

S/L Separator

Solvent recovery

Cell disrupter

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MICROALGAL CULTURING TECHNIQUES

Part II

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Nutritional requirements

• Depends on application – Food or health oils: food grade chemicals – Otherwise industrial chemicals or seawater / wastewater

• Carbon source: CO2, sugars, acetate, ethanol • Macronutrients: Nitrogen and phosphorus • 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, Ni

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Solar radiation in Canada

Solar radiation in Alberta Fort McMurray: 4181 MJ/m2∙y Edmonton: 4510 MJ/m2∙y Medicine Hat: 5221 MJ/m2∙y Munich (GE): 4044 MJ/m2∙y Naples (IT): 5293 MJ/m2∙y Kuala Lumpur: 5622 MJ/m2∙y Orlando (FL): 5922 MJ/m2∙y Acapulco (MX): 7261 MJ/m2∙y Phoenix (AZ): 7621 MJ/m2∙y

Solar radiation data taken from: U.S. Department of Energy - EnergyPlus Weather Data. http://apps1.eere.energy.gov/buildings/energyplus/cfm/weather_data.cfm

Culturing techniques: Open Ponds

• By far, the most common production system.

• Low installation cost • Lagoons or artificial ponds • High risk of contamination • Application limited to few

species (extremophiles). • Unmixed ponds: area range from

1 - 200 Ha, depth 20-30 cm • Raceway ponds are up to 1 Ha.

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Culturing: Close Ponds and Tanks

• Simpler designs similar to open ponds, with a cover (greenhouses).

• Aim to reduce contamination risks.

• Control CO2 looses. • Tanks are usually mixed by

aeration. • Deep tanks are inefficient. Bad

light transmission. • Easy to operate, low cost.

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Culturing: Photobioreactors

Tubular Photobioreactor - Algae and Biofuels Facility, South Australian Research and Development Institute

Flat Panel photobioreactor

Arizona Center for Algal Technology and Innovation

Flexible plastic film Photobioreactor - Algenol, Florida 25

Culturing: Photobioreactors

• Better culture control • Higher productivity, and culture

density • Minimal contamination risk • Well mixed • Excellent temperature control • Oxygen control is an issue • High capital investment • Frequent cleaning required • Cooling required

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Heterotrophic Production of Algae

• Some algae species can grow using an organic carbon source.

• Conventional bioreactors can be used.

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Phototrophic vs. Heterotrophic?

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Specie Oil 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)

Phot

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MODEL-BASED OPTIMIZATION OF HETEROTROPHIC ALGAL CULTURES

Part III

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Bioprocess Optimization

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Strain selection

Media formulation

Process conditions

Continuous / Real-time

Genetic modification

The Objective for Optimization

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Stress Oil storing is a metabolic response to stress, particularly nitrogen deficiency. At nitrogen deficient conditions, algal cells over-accumulate lipids.

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 cell functioning. Therefore, conditions that favored oil accumulation constraint productivity.

Understanding algal growth

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Nitrogen uptake

Lipid production

Cellular growth

An algal growth model

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Cellular growth

Nitrogen uptake

Oil production

Macroscopic balances

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Optimization: Problem formulation

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Subject to:

Simulation results

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Biomass productivity in continuous cultures

Lipid productivity in continuous cultures

Experimental results

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Biomass productivity and growth rate

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Lipid productivity and production rate

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Comparative study: growth on glucose

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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)

Bact

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Optimization: closing remarks

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Model-based 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 (40–60% 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

• Algae are promising organisms: highly efficient

• Good source of oil: PUFA, biodiesel precursor

• Algae can growth on simple inexpensive media

• Several reactor types and geometry. Application will limit

reactor choice

• Several successful commercial applications currently working.

• A lot of research is still needed! 42