non-food biotechnology in department of bioprocess

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Non-food Biotechnology in Department of Bioprocess Engineering at TU Lodz

Poland

Non-food Biotechnology in Department of Bioprocess Engineering at TU Lodz

Poland

Stanisław Ledakowicz

Technical University of Lodz,

Department of Bioprocess Engineering

e-mail: [email protected]

Industrial biotechnology: novel high added-value bio -products and bio-processes, BXL, 26.10.2010

2

1. Staff of the Department

2. Research Equipment

3. Fields of interest

4. Biomercury

5. Integration of nanofiltration with biological

treatment of textile wastewater

6. Biogas from energetic plants

7. Lovastatin

8. Laccase

9. Proposal of new EU project Helati

Contents of presentation

DEPARTMENT OF BIOPROCESS ENGINEERING

Faculty of Process and Environmental Engineering, Technical University of Lodz, http://wipos.p.lodz.pl

Industrial biotechnology: novel high added-valuebio-products and bio-processes, BXL, 26.10.2010

3Staff of the Department

Head:Professor Stanisław Ledakowicz, PhD,

D.Sc,Representative of Poland in the Working

Party of Bioreactor Performance, EFB

Associate professor:

Liliana Krzystek, PhD, D.Sc

Assistant Professors:

Anna Antecka, PhD

Marcin Bizukojć, PhD, D.Sc.

Paweł Głuszcz, PhD

Teresa Jamroz, PhD

Jacek Miller, PhD

Beata Pawłowska, PhD

Paweł Stolarek, PhD

Katarzyna Paździor, PhD

Barbara Sencio, MSc

Anna Klepacz-Smółka, MSc

Technical staff 3

PhD students 12




4List of research equipment

Bioreactors with impellers 2, 10, 42 & 100 dm3 & air

lift bioreactor 15, 32 & 200dm3, with computer-aided

process control (B. Braun/Sartorius)

Photobioreactor (Sartorius)

Laminar chamber HBB 2448 (Holten)

Cascade incubation shaker Certomat BS1 (B. Braun)

(from -10 to +70°C)

System of Simulated Moving Bed Chromatograph

(6 IE columns Spectra/Chrom)

Biomass filter FUNDABAC TSD & Ultra-filtration

modulus (Milipore)

Microscope BX 40 (Olympus) with a fluorescence

unit & image analysing




5List of research equipment

GC (Varian), HPLC (Waters), UHPLC/MS/MS –

purchase in progress

Ionic chromatography system ( Dionex)

Solid Phase Extractor ( Baker)

Two elementary analysers (CHNS+O) NA 2500, (CE Instruments)

Gas analysers (Servomex-1400 (CO2, O2) & Gas-Data (CH4, CO2, O2, H2S)

IR & UV-VIS Spectrophotometer UV 300 (Unicam) with a flow cell

TOC - TN analyser (Hach Lange)

AAS Atomic absorption spectrometer (Perkin Elmer)

QMS 200 Quadrupole mass spectrometer (Balzers)




6Projects

International projects

3-STEPS (Starter To Environmental Problem Solution), - Tempus II within the V Frame

Programme of the European Union (FP EU).

BIOMERCURY – “Worldwide remediation of mercury hazards through

biotechnology”, No. NMP2-CT-2004-505561 within the VI FP EU

REMOVALS – Reduction, modification & valorisation of sludge, FP6-2004-Global

Change & Ecosystem No.18525 505561 within VI FP EU

Enzymes for industrial application. Scientific & technological cooperation joint

project for year 2007–2008 with CSIR Biosciences, Pretoria, South Africa

IGRE - Development of species index and optimisation of energetic plants

production technology POIG.01.03.01-00-132/08-00 financed by the Ministry

of Regional Development and co-financed by the European Union. 2007-2013

35 National research projects (Ministry of Science & Higher Education)




7Environmental Biotechnology

Biological methods of degradation, decolorisation and detoxication of

industrial wastewater and landfill leachates

Biodegradation of organic fraction of solid wastes and

reclamation of old landfills

Integration of biological and chemical (AOP) methods

for decomposition of xenobiotics

Biological removal of mercury from contaminated

soil and wastewater




8Worldwide remediation of mercury hazards through biotechnology Acronym: Biomercury

Sixth Framework Programme

Priority 3 NMP

Nanotechnology and Nanosciences,

knowledge based multifunctional materials, new

production processes and devices

Project NMP2-CT-2004-505561

Specific Support Action

Duration 01.03.2004 - 28.02.2007

www.biomercury.de

Metallic mercury




W.D. Deckwer et al. ( 2004): Microbial Removal of Ionic Mercury in a Three-Phase Fluidized Bed Reactor, Environ. Sci. Technol., 38, 858-865.P. Gluszcz, et al..(2007): Thermodynamic and kinetic aspects of mercury sorption on activated carbon in the process of mercury bioreduction. Chem. Biochem. Eng. Quat., 21 (4), 307-314.P.Gluszcz, et al. (2007): Integrated process of ionic mercury bioreduction and adsorption from wastewater, Chem. Process Eng., 28, 909-920.P. Gluszcz, et al..( 2007): Engineering aspects of microbial remedaiation of mercury from wastewater.J. Biotechnol. 2007, 131, 118.P. Gluszcz, et al.. (2008): Bioreduction of Ionic Mercury from Wastewater in a Fixed-bed Bioreactor with Activated Carbon. Chem. Papers 62, 232-238.

9Idea of the bioremediation with bacteria possessin gmer operon encoded mercury resistance

outer membraneouter membraneperiplasmperiplasm

inner membraneinner membrane

cytoplasmcytoplasm

Hg0

Hg0

Hg2+

merA mercuric reductase

merP

merT

merA

NADP + 2H +

NADPH2

2e-




10New Technology for Bioremediation of Industrial Wast ewater Containing Mercury Nitrogen Works Tarn ów S.A ., Poland

Hg < 50 µg/l

Hg2+<50 mg/l

Hg< 0.1-0.5 mg/l

In operation since 2008In operation since 2008In operation since 2008In operation since 2008




Gluszcz P. et al. (2010): The integrated process of mercury bioremediation in the industrial bioreactor, Bioprocess Biosystem Eng.33,

11Integration of nanofiltration and biological proces ses for textile wastewater treatment




Dye house

Wastewater

Nanofiltration

Reverse osmosis

Bioreactor SBR

Bioreactors An/Ae BrineProcess

water

Pazdzior et. al. (2009) Integration of nanofiltration and biological degradation of textile wastewater containing azo dye Chemsphere, 76, 250-255.

Sojka-Ledakowicz et.al. (2010), Application of membrane processes in closing of water cycle in a textile dye-house, Desalination, 250, 634-638.

12Biogas production from energetic plants and wastes

Substrates for biogas

Glycerol from

vegetable oil

cooking oil

vegetable oil treated with 80% acetic acid

rapeseed oil after repeated transestrification

Cheese whey

Corn silage of energetic plants




13Lovastatin to decrease endogenous cholesterol level

Aspergillus terreus ATCC20542

CH3

HO

CH3

CH3

CH3

H

H

H3C

H

H

CH3

H

H3C

O

O

H3C

OH

HO

OH

O

CH3

Statins were discoveredby Akira Endo in 70’s of 20 th

lovastatincholesterol

CoA

C

HOOH

O

CH3

O

S CoAreductase 3-HMG-CoA(EC1.1.1.34)

2NADPH

mevalonate(S)-3-hydroxy-3-methylglutaryl-CoA

com

petit

ive

inhi

bitio

n

AcCoA




14Evolution of Aspergillus terreus ATCC20542 in a submerged culture

from a spore to a pellet. Visualization of the infrastructure of the pellets




preculture

production culture

Bizukojć M., Ledakowicz S., (2010):The morphological & physiological evolution of A. terreus mycelium in the submerged culture and its relation to formation of secondary metabolitesWorld J.Microbiol. Biotechnol. 26:41-54

15Lovastatin production in batch culture




0 25 50 75 100 125 150 175 200

0

5

10

15

20

0

10

20

30

40

50

60

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0

2

4

6

8

10

12

14 LAC

Lact

ose

(g l

-1)

Time (h)Lo

vast

atin

(m

g l

-1)

LOV

Org

anic

nitr

ogen

(g

l-1)

N

Bio

mas

s (g

l-1)

X

0 20 40 60 80 100 120 140 160 1800

2

4

6

8

10

12

14

16

18

0

10

20

30

40

50

60

70

0.0

0.2

0.4

0.6

0.8

1.0

0

2

4

6

8

10

12

Lact

ose

(g l-1

)

Time (h)

LAC LOV

Lova

stat

in (

mg

l-1)

N

Org

anic

nitr

ogen

(m

g l-1

)

X

Bio

mas

s (g

l-1)

Publications:

1. Bizukojć M., Ledakowicz S. (2009) Physiological, morphological and kinetic aspects of lovastatin biosynthesis by Aspergillus terreus. Biotechnol. J. 4: 647-664.

2. Bizukojć M., Ledakowicz S. (2008) Biosynthesis of lovastatin and (+)-geodin by Aspergillus terreus in batch and fed-batch culture in the stirred tank bioreactor. Biochem. Eng. J. 42: 198-207.

3. Bizukojć M., Ledakowicz S. (2007) Simultaneous biosynthesis of (+)-geodin by a lovastatin-producing fungus Aspergillus terreus. J.Biotechnol. 132: 453-460.

4. Bizukojć M., Ledakowicz S. (2007) A macrokinetic modelling of the biosynthesis of lovastatin by Aspergillus terreus. J. Biotechnol. 130: 422-43.

5. Bizukojć M., Pawłowska B. Ledakowicz S. (2007) Supplementation of the cultivation media with B-group vitamins enhances lovastatin biosynthesis by Aspergillus terreus. J. Biotechnol. 127: 258-268.

Shake flask

Bioreactor

16Laccase - extra-cellular, a very robust enzyme (EC 1.10.3.2)




First laccase from lacquer tree was described by Yo shida (1883)

Abundant in white rot fungi but also in plants and bacteria

Involved in the biodegradation of lignin, humic acid s and

biosynthesis of new compounds (e.g. dyes); don’t re quire NADH

Used in diverse biotechnological

applications, like bioremediation,

biotransformation of many persistent

environmental pollutants, like azo dyes,

PAHs, endocrine disruptors or

polycyclic musk fragrances

Thermotolerant & thermostable

A. Michniewicz et al. (2006): The white-rot fungus Cerrena unicilor produces two laccase isoenzymes with different physico-chemical and catalytical properties, Applied Microbiology & Biotechnology, 69, 682A. Antecka et al. (2009): Kinetic model of laccase biosynthesis by C. unicolor, Chem. Proc. Eng., 30, 403

Laccase

Fig.4 Kinetic curves of H 2O sorption on linen woven fabric after treatment with laccase enzyme

0

10

20

30

40

50

60

70

0 5 10 15 20 25 30 35 40time (s)

sorp

tion

valu

e µl

/cm

2after alkali treatment2,4 U/g fabric(30min)2,4 U/g fabric(60min)5,0 U/g fabric(30min)5,0 U/g fabric(60min)7,5 U/g fabric(30min)7,5 U/g fabric(60min)

4,13

3,63

3,823,71

3,2

3,4

3,6

3,8

4

4,2

ligni

n co

nten

ts

[%dr

y m

atte

r]

Fig.6 The influence of enzymatic treatment of linen woven fabric onto the contents of lignin d ry matter

raw woven fabric after alkali treatment2,4 U/g fabric;30min 2,4 U/g fabric;60min

4,72

4,00

4,524,29

3

4

5

hem

icel

lulo

se

cont

ents

[%

dry

mat

ter]

Fig.5 The influence of enzymatic treatment of linen woven fabric onto the contents of hemicellulose dry matter

raw woven fabric after alkali treatment2,4 U/g fabric;30min 2,4 U/g fabric;60min;

Sójka-Ledakowicz J. et al. (2007)Bioscouring of linen by laccase fromCerrena unicolor. Fibres & Textiles in Eastern Europe, 15 (4), 86-89.




1717

Before enzyme treatment

After

Bio-scouring of linen fabrics co-operation with Textile Research Institute




1818Biotreatment of linen fabrics with multi-enzyme complex

Kinetic curves of H2O sorption on linen woven fabrics after inegrated

enzymatic treatment with laccase enzyme and multi-enzyme complex

Microscopic image (SEM) of linen woven fabric (600x):

a) raw woven fabric,

b) fabric after alkali scouring,

c) fabric after I stage enzymatic treatment with laccase

enzyme

d) fabric after II stage enzymatic treatment with laccase

enzyme and multi-enzymatic complexes

Bio-treatment of textile fabrics enhances economic effectivennes (decreased water and energy consumption) and at the same time reduces harmful impacts on the environment (wastewater discharge) hence it is the pro-ecological alternative to conventional technologies.

19




HIGHEST PRODUCERS

BATCH FERMENTATION

DOWNSTREAM PROCESSING

LOW COPY

� MANNANASE ACTIVITY 1.

� DCW� GLUCOSE� OD� pH

MULTI-COPY

HIGHEST PRODUCER

SCREENING

Gene transfer

Optimising biosynthesis

Industrial

ENZYME

Overexpression of laccase

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Thanks foryour kind attention

Stanislaw Ledakowicz TU Lodz, Depart. Bioproc. Eng., [email protected]

non-food biotechnology in department of bioprocess

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