Pavlo Bohutskyi, Ph.D. CandidateDepartment of Geography & Environmental Engineering,

Johns Hopkins University, Baltimore, MD

Wednesday, October 1, 20148th Annual Algae Biomass Summit

San Diego, CA, USA

Enhancing Performance and Assaying Nutrient Recycling Potential for a Sequential Photoautotrophic-Heterotrophic Algal Biofuel Production System

Presented at 8th Annual Algae Biomass Summit September 29 – October 2, 2014 | San Diego, CA, USA

Lipid Content and Biomass Conversion to Energy

High lipid content is essential

Conversion of Lipid Extracted Algae(LEA) to energy is important

biodiesel vs. anaerobic digestion (methane) vs. combined

Fertilizer Demand for Algal Fuel Production

A – Pate et al. (2011) Resource demand implications for US algae biofuels production scale-up. Applied Energy 88/10:3377–3388

Nutrient Recycling is Possible and Required

• Food production and prices

• Only C and H are part of fuel

Fertilizer demand for 20 BGY ( ~6% of US liquid fuel consumption)A

Algal biomass 70-170 M mt/yr

Nitrogen 6.1-15 M mt/yr

Phosphorus 0.8-2.1 M mt/yr

Fertilizer demand for 20 BGY ( ~6% of US liquid fuel consumption)A

Algal biomass 70-170 M mt/yr

Nitrogen 6.1-15 M mt/yr 44-107% of US use in 2006

Phosphorus 0.8-2.1 M mt/yr 20-50% of US use in 2006

Presentation Outline

Part 1. Description of the mixed trophic system

Part 2. Fate of nutrients and their availability for recycling with spent medium

Part 3. Anaerobic digestion (AD) of LEA for methane production and nutrient recovery from algal

air/CO2

air/CO2

glucose

CO2

algal

culture

conce

ntr

ated

alg

ae

heterotrophic

spen

t g

row

th

med

ium

photoautotrophic

1 2 3 4

pH dO2

A750 Temp.

NaOH

air

centrifugeaseptic conditions

lipid-rich

algae

0.025L

800L15cm(depth)

0.25L 2L 40L 5L

nutrients

nu

trie

nts

nu

trie

nts

nu

trie

nts

Mixed Trophic Algal Cultivation Process

• Microalga: Auxenochlorella (formerly Chlorella) protothecoides UTEX 25

• Phototrophic Medium: minerals, ammonium chloride (0.5 g/L) as nitrogen source

• Heterotrophic Medium: minerals, glucose maintained at 20 g/L, no nitrogen

• Centrifugation: stacked-disc centrifuge

*Growth medium: 1.44 g K2HPO4, 0.72 g KH2PO4, 50 mg Tetrasodium EDTA, 20 mg MgSO47H2O, 10 mg CaCl22H2O, 10 mg FeCl36H2O, 5 mg H3BO3, 1.25 mg ZnSO47H2O, 0.38 MnSO4H2O, 0.25 mg CoCl26H2O, 0.25 mg Na2MoO42H2O, 0.08 mg CuSO45H2O. NH4Cl (0.5 g/L).

air/CO2

air/CO2

glucose

CO2

algal

culture

conce

ntr

ated

alg

ae

heterotrophic

spen

t g

row

th

med

ium

photoautotrophic

1 2 3 4

pH dO2

A750 Temp.

NaOH

air

centrifugeaseptic conditions

lipid-rich

algae

0.025L

800L15cm(depth)

0.25L 2L 40L 5L

nutrients

nu

trie

nts

nu

trie

nts

nu

trie

nts

55

Photoautotrophic Step Heterotrophic StepAseptic Conditions

Results: Photoautotrophic Cultivation

Condition Unit Step 1 Step 2 Step 3Step 4 (bag)

Step 5 (pond)

Starting density

g/L <0.03 0.06 0.05 0.07 0.07

Final density

g/L 0.69 0.37 1.08 1.49 0.28

Duration days 1.78 0.96 6.1 6.26 5.01

Total

lipids% dw 15

Results: Heterotrophic Cultivation

Phototrophically grown non-stressed A. protothecoides has unacceptably low culture density and lipid content (15 %dw)

A second heterotrophic stage boosts the biomass concentration over 115 g/L and lipid content over 55%

Aseptic conditions are not essential for last steps of cultivation (including heterotrophic stage)

Part I: Conclusions

Nutrient Consumption in the Photoautotrophic Growth Medium (Pond)

Initial medium concentration

Final medium concentration

Nutrient Consumption in the Heterotrophic Growth Medium (Fermenter)

growth medium – red circles; µg/L or mg/L; algal biomass – blue crosses; µg/g or mg/g.

algal biomass – blue crosses

growth medium – red circles

Overall Process Nutrient Distribution -Potential for Reuse with Spent Growth Medium

air/CO2

air/CO2

glucose

alga

lcu

ltu

re

con

cen

trat

ed

alga

e

heterotrophicstage

spen

tg

row

th

med

ium

photoautotrophicstage

NaOH

air

centrifuge

asepticconditions

CO2 800L,15cm(depth)0.25L 2L 40L

racewaypond

bag

5L

lip

id-r

ich

al

gae sp

ent

gro

wth

m

ediu

m

centrifuge

1 2 3 4 5 6

air/CO2

air/CO2

glucose

alga

lcu

ltu

re

con

cen

trat

ed

alga

e

heterotrophicstage

spen

tg

row

th

med

ium

photoautotrophicstage

NaOH

air

centrifuge

asepticconditions

CO2 800L,15cm(depth)

potentialnutrientrecycling

0.25L 2L 40L

racewaypond

bag

5L

nu

trie

nts

lip

id-r

ich

al

gae sp

ent

gro

wth

m

ediu

m

centrifuge

potentialnutrientrecycling

1 2 3 4 5 6

Composition of A. protothecoides biomass (% dw)

ElementPhotoautotrophic

Stage

Heterotrophic

Stage (120 h)

Carbon 49.12 59.2

Oxygen 32.26 26.1

Hydrogen 7.026 9.57

Nitrogen 8.068 1.54

Phosphorus 1.176 0.31

Magnesium 0.19 0.025

Sulfur 0.11 0.012

Iron 0.12 0.018

Calcium 0.045 0.0057

Zinc 5.1x10-3 0.64x10-3

Copper 2.7x10-3 0.2 x10-3

Manganese 4.1x10-3 0.58x10-3

Cobalt 0.31x10-3 0.045x10-3

Molybdenum 0.093x10-3 0.054x10-3

Boron 0.04x10-3 0.003x10-3

ElementPhotoautotrophic

Stage

Heterotrophic

Stage (120 h)

% Difference

Heterotrophic/

Photoautotrophic

Required for 1 L

of produced TAGs

Carbon 49.12 59.2 20.5 1.32

Oxygen 32.26 26.1 (-19.1) 0.58

Hydrogen 7.026 9.57 36.2 0.21

Nitrogen 8.068 1.54 (-80.0) 0.034

Phosphorus 1.176 0.31 (-73.6)0.0069

Magnesium 0.19 0.025 (-86.8)0.56x10-3

Sulfur 0.11 0.012 (-89.1) 0. 27x10-3

Iron 0.12 0.018 (-85.0) 0. 40x10-3

Calcium 0.045 0.0057 (-87.3) 0. 13x10-3

Zinc 5.1x10-3 0.64x10-3 (-87.5) 0.014x10-3

Copper 2.7x10-3 0.2 x10-3 (-92.6) 0. 0044x10-3

Manganese 4.1x10-3 0.58x10-3 (-85.9)0. 013x10-3

Cobalt 0.31x10-3 0.045x10-3 (-85.5) 0. 0010x10-3

Molybdenum 0.093x10-3 0.054x10-3 (-41.1)0. 0012x10-3

Boron 0.04x10-3 0.003x10-3 (-92.5) 0. 067x10-6

Part II: Conclusions

>99% overall nutrients supplied to photoautotrophic stage - significantly larger volume

Photoautotrophic stage nutrient consumption –

• 10-35% Mn, S, Fe, N, Mg, Cu

• < 5% of the P, Mo, Co, B, Zn, and Ca

Heterotrophic stage - Zn, Mo, Mn, Mg, Ca and N, were exhausted (90-99% removal) during the first 25 h.

Total for both stages:

• 10-20% of S, Mn, Fe, N, Cu and less than 5% of Ca, Zn, Mo, Co, P, B were assimilated into algal biomass

Fertilizer demand for algal biofuel maybe overestimated if composition of low-lipid algae is used for estimation

Anaerobic Digestion (AD) of Lipid Extracted Algae

Semi-continuous AD system:

Bioreactor - 3 L spinner flasks at 35C;

Biogas flow - wet-tip gas meter;

Methane - Shimadzu GC-TCD, Hayes Q80/100 column.

Effluent nutrients - filtered (0.45 µm),

N & P Hach TNTplus kits, other by ICP-MS

Run HRT, days Feed, gVS/L OLR, gVS/L-day

1 20 40.91.5 0.970.06

2 20 38.02.0 2.050.08

Operational parameters

Biogas and Methane Production

Energy: Biodiesel vs. Methane vs. Combined

Nutrient Recovery from LEA with the AD Effluent

Recovered Fraction of Nutrient from LEA content

Part III: Conclusions

Lipid extracted algal biomass is a suitable substrate for AD but the

conversion has been limited to ~50% possibly due to recalcitrance;

Inhibition effects from the solvent residues;

Integrated biodiesel-biogas process increased energy yield from

biomass by 30%;

Up to 40-60% of N, P, Mg, Ca, S, and 15-25% of Mn and Fe

contained in the LEA are soluble in AD effluent and potentially

available for recycling;

Soluble Zn, Mo, Co did not exceed 5% in general.

Phycal Inc.:

Ben A. Kessler

Thomas Kula

Dr. F. C. Thomas Allnutt

Funding was provided by: U.S. NSF CBET Program. Grant #1236691 to JHU and by U.S. DOE CCS Program Grant No.DE-FE0001888 to Phycal Inc.

JHU:

Kexin Liu, DOGEE

Dr. Edward J. Bouwer, DOGEE

Dr. Michael J. Betenbaugh, ChemBE

Dr. Yongseok Hong, DOGEE

Our Team

JOHNS HOPKINS

UNIVERSITY