pulsed electric field processing of microalgae benefits and ......2016/12/04 · pulsed electric...

Institute for Pulsed Power and Microwave Technology (IHM)

www.kit.edu KIT – University of the State of Baden-Wuerttemberg and

National Research Center of the Helmholtz Association

Pulsed Electric Field Processing of Microalgae

Benefits and Limitations W. Frey, C. Gusbeth, A. Silve, R. Straessner, G. Mueller - IHM

C. Posten, M. Schirmer - BVT

P. Nick - Botanics I

Auxenochlorella

protothecoides

20 kW PEF facility Flat Panel Photobioreactor

Lipid bodies in A.p.

1 December 2016 Institute for Pulsed Power and

Microwave Technology

W. Frey - AlgaEurope 2016

Challenges of Microalgae Processing for Economic Component Recovery

Energy-efficient and, more important, fractionating techniques are needed for microalgae processing

Energy [MJ/kgdw]

% of energy stored in microalgae

Energy required for cultivation (closed PBR systems) 10 - 15 ~ 50 %

Harvesting/separation/concentration 2 - 4 ~ 10 %

Conventional cell disruption

3 – 5 ~ 15 %

Bead milling:

~ 3 MJ/kgdw (Postma, 2015, Biores. Technol. )

High pressure homogenization:

~ 4 MJ/kgdw (Günerken, 2015, Biotechnol. Adv.)

Cultivation Concentration Cell disruption Extraction Separation

Refining

lowest reported values:

p GEA-Westfalia

dw: dry weight PBR: photobioreactor

Biorefinery approaches must strive for complete biomass valorization:

…disintegrates the biomass, thus impeding separation




untreated

Pulsed Electric Field (PEF)Treatment Permeabilizes Phosopholipid Bilayers

5 µm

Example: Red Grape Skin :

Red pigment is enclosed by the vacuole's membrane

10 min after PEF treatment

Pulsed Electric Field Treatment

(Electroporation)

Primary function of membranes in cell biology is to encase and protect inner substances

PEF treatment enables transport of molecules and ions across phospholipid bilayers (membranes)

a




Pore Formation Immediately Follows Upon Plasmamembrane Charging


(Electroporation)

a

0 90 180 270 360

Me

mb

rane

Volta

ge

VM

Eext

t

M extV 1.5aE sin [1 e ]

Onset of strong Permeabilization

Frey, Biophys.J. 90 2006

Flickinger, Protoplasma 247 2010

A wide angular range of the cell's surface has to be permeabilized

for efficient component flow across the cell boundary




Pore Formation Immediately Follows Upon Plasmamembrane Charging


(Electroporation)

a

0 90 180 270 360

Me

mb

rane

Volta

ge

VM

Eext

t

M extV 1.5aE sin [1 e ]

cell type 2a [µm] Eext [kV/cm] application

bacteria 2-3 45...200 bacterial decontamination

microalgae 5-10 17...80 component extraction

plant cells in tissue 100 1.5...6.5 sugar extraction

Required electric field strength Eext for large-cell-surface permeabilization (µs-pulses):

: time constant of membrane charging, << pulse duration for microalgae in culture medium

Onset of strong Permeabilization

Frey, Biophys.J. 90 2006

Flickinger, Protoplasma 247 2010




Basic Effects of Plasmamembrane Permeabilization: Pore Formation is a short-term effect and predominantly happens during the electric field pulse

Pore formation:

Water fingers enter into the

fatty acid chain space

of the phospholipid (PL) bilayer

PL-headgroups turn towards

water finger boundary

Pore resealing time is

in the sub-microsecond range

Long-lasting and persisting

permeabilization cannot be

explained by pore formation

Molecular Dynamics Simulation, Courtesy of Tom Vernier, Old Dominion University, Norfolk, VA

Red - O White - H Blue - N Gold - P

duration: 5 ns

E




Basic Effects of Plasmamembrane Permeabilization: Cytoskeleton Dissolution A mechanism that induces long-term membrane permeabilization

Actin cytoskeleton consists of a meshwork underneath the plasmamembrane

It mechanically stabilizes the cell and the plasmamembrane in particular

Upon PEF exposure, the cortical actin meshwork dissolves (4 min ff)

which results in membrane permeabilization evident long after pulse termination

Other long-term effects under discussion: edocytosis, PL-peroxidation, membrane disordering

0 min (time after pulse) 4 min 8 min 16 min

co

rtex

10 µm

Th. Berghöfer et al.: BBRC 387 (2009) 590-595

BY-2, actin is tagged with green fluorescent protein (GFP)




Typical Set-up for Continuous-Flow PEF-Treatment

0 1000 2000

0

5

10

15

[ns]

[kV

]

treatment

chamber

cross-linear treatment chamber

lab-scale continuous flow setup

Cell suspension to be processed is pumped through the treatment chamber along two electrodes

Voltage across the electrodes induces an electric field in the cell suspension




Spontaneous Release of Intracellular Components after PEF Treatment water-soluble fraction

Auxenochlorella protothecoides (100 gdw/l)

0 1000 2000

0

5

10

15

[ns]

[kV

]

treatment

chamber

1 µs, 34 kV/cm, square pulses

Instantaneous release of intracellular ions increases suspension conductivity after PEF treatment

Goettel, M., Frey, W. et.al., Algal Research, vol. 2, 2013, pp. 401–408.

0 30 60 90 120

0,4

0,6

0,8

1,0

1,2

1,4

1,6

Time [s]

Ce

ll su

sp

en

sio

n c

on

du

ctivity [

mS

/cm

]

suspension enters treatment chamber

dw: dry weight

Conductivity of microalgae suspension ~ 1 mS/cm

before PEF treatment

permeabilization affects conductivity



Proteins[mg/l]

Carbohydrates[g/l]

TOC[g/l]

externalTotal Solids

[g/l]

0

2

4

6

8

10

12

14

16

Co

nce

ntr

atio

n

Untreated

PEF Treated


Spontaneous Release of Intracellular Components after PEF Treatment water-soluble fraction


0 1000 2000

0

5

10

15

[ns]

[kV

]

treatment

chamber


Goettel, M., Frey, W. et.al., Algal Research, vol. 2, 2013, pp. 401–408. TOC: total organic carbon

14 % of the total biomass is released into extracellular medium within minutes

40 x increase of extracellular carbohydrates




Spontaneous Release of Intracellular Components after PEF Treatment water-soluble fraction, energy requirements

Control


Goettel, M., Frey, W. et.al., Algal Research, vol. 2, 2013, pp. 401–408.

0 1000 2000

0

5

10

15

[ns]

[kV

]

treatment

chamber

A. protothecoides: Extraction yield starts to saturate at 150 kJ/lsus ►

Energy demand related to dry biomass is 1.5 MJ/kgdw


TOC: total organic carbon dw: dry weight




Influence of Biomass Concentration on Component Release Efficiency

50 100 150 200

1

2

3

4

5

6

100(cTOC-PEF

- cTOC-0

)/cx

cultivation #1 cultivation #2

Biomass concentration cx [g

DCW/l]

TO

C R

ele

ase

fa

cto

r

TOC Release factor:

50 100 150 200

2

4

6

8

10

cultivation #1 cultivation #2

Biomass concentration cx [g

DCW/l]

TO

C C

on

ce

ntr

atio

n c

TO

C [

g/l]

Specific component yield does not decrease at high biomass concentrations

This is an important condition for further reducing required PEF treatment energy

Control

PEF-treated:

155 kJ/lsus

32 kV/cm

cTOC-PEF - cTOC-0

A.protothecoides.: 1µs, 32kV/cm, 155 kJ/lsus

PEF treatment of suspensions of different biomass densites, @ constant treatment energy

No

rma

lize

d T

OC

Re

lea

se

DCW: dry weight




PEF-Processing Energy and Biomass Density in Suspension

1.5

0.75

𝑃𝐸𝐹 𝑝𝑟𝑜𝑐𝑒𝑠𝑠𝑖𝑛𝑔 𝑒𝑛𝑒𝑟𝑔𝑦 𝑀𝐽

𝑘𝑔𝑑𝑤=

𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 𝑒𝑛𝑒𝑟𝑔𝑦 𝑘𝐽

𝑙𝑠𝑢𝑠

𝑏𝑖𝑜𝑚𝑎𝑠𝑠 𝑑𝑒𝑛𝑠𝑖𝑡𝑦 𝑐𝑥𝑔𝑑𝑤𝑙𝑠𝑢𝑠

For energy-efficient PEF processing, high microalgae biomass density in suspension is required

Example: specific treatment energy delivered to the suspension is 150 kJ/lsus

Treatment energy minimum

for mechanical cell disruption




PEF Treatment Facilitates Excellent Biomass Separability

Separability of biomass

by centrifugation

High

Pressure

Homogenization

Pulsed

Electric

Field treatment

HPH PEF

p

GEA-Westfalia

Cell shape is not affected by PEF-treatment, no debris

biomass 100g/l

PEF

aqueous comp. separation

residual 1




Protein Recovery by PEF Treatment

biomass 100g/l

PEF


residual 1

alkal. extraction

proteins separation

residual 2

membrane-associated

proteins

trans-

membrane

proteins

cytosolic

proteins

cell wall proteins

PEF-induced component flow across the cell boundary is based on diffusion-driven processes

The missing diagram

shows, that approx. 50%

of the water-soluble protein

fraction could be recovered

by application of a protein extraction

buffer containing DTT. Positive control

was HPH, 5 passes, 2kbar.

Recovery without alkal. extraction buffer

was about 5 times less.

(publication is pending!)




Major components released extracellularly by water-based extraction methods

- Low-molecular-weight organic acids

- Mono- and di-sacccharides

- Amino acids and proteins

No spontaneous release of lipids could be detected after PEF-treatment

Conditions for PEF-assisted Lipid Recovery

SEM-image of the cell wall of microalgae, C. r.

The Journal of Cell Biology, vol.101, 1985, pp.1550-1568.

1

Lipid bodies

5µ A. protothecoides

Fluorescence image of lipid bodies in A.p. after Nile Red staining




PEF-Assisted Recovery of Lipids

95 % of recovery of total lipids from Auxenochlorella protothecoides

previous protein extraction does not affect the lipid yield of subsequent solvent extraction

Chlorella vulgaris biomass 100g/l

PEF


residual 1

alkal. extraction

proteins separation

residual 2

wet EtOH/Hex

residual 3

lipids

1

2

The missing diagram

shows lipid yields in %dw

control w/o pretreatment

was about 7%,

yield route 1: 18%,

route 2: about 19%

(publication is pending!)




Ethanolic Extraction of Lipids from Wet Biomass

Extraction characteristics depends on algae strain

Wet-route processing is more efficient

After drying, the permeabilizing effect is still evident

C.v. requires less treatment energy for high lipid yield (50 kJ/lsus► 0.5 MJ/kgdw)

treatment conditions:

100 gdw/l, 1µs, 34kV/cm, 150 kJ/lsus square pulses

wet processing of

EtOH/hexane Extraction



Desirable pulse parameters for large-scale processing facilities:

low pulse voltage amplitudes (means: low field values)

and long pulses (means: moderate repetition rates)

(lower costs for HV-insulation, switching, power supply)


Continuous-Flow PEF-Processing of Microalgae Towards Large-Scale

cross-linear treatment chamber

lab-scale continuous flow setup

20 kW PEF facility




Biomass Deposition at the Anode of the Treatment Chamber is caused by electrophoresis and can be avoided by short pulse application (duration: 1 µs)

R. Straessner, et.al., Innovative Food Science and Emerging Technologies (2016), DOI: 10.1016/j.ifset.2016.07.008

Cleaned anode

before PEF experiment Precipitated wet biomass

A. protothecoides

Treatment parameters:

200 µs, 5 kV/cm, 50 kJ/lsus,

Biomass density: 97.5 gdw/lsus

Dried biomass cake

for weight determination

►Please see Poster of Ralf Straessner




Summary

Current understanding of the

fractionating properties of PEF-processing

PEF-Processing of Microalgae

enables externalization of water-soluble products

improves intracellular access of extraction buffers

and solvents

can provide fractionating component recovery

maintains cell shape and mechanical separability

energy-efficient: ~ 1 MJ/kgdw

"cold" processing technology

cannot detach molecules from intracellular structures

will be applied in SABANA Project

cell wall

plasma

membrane

water-soluble

components

solvents

(EtOH)

aqueous buffers

pulsed electric field processing of microalgae benefits and ......2016/12/04 · pulsed electric...

Documents