The SSF process of ethanol production from pulp from w heat straw - - PowerPoint PPT Presentation

The SSF process of ethanol production from pulp from w heat straw

“Development

• f

a process for the utilization both the carbohydrate and the lignin content from lignocellulosic materials of annual plants for the production of valuable products”

10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 1

ERA-IB-project EIB.10.013:

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Overall process for the utilization of wheat straw

10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014

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SSF-process of ethanol production from pulp

10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014

SSF-process from the technical point of view

Advantage of SSF-process Requirem ents

Reduction and simplification of the process steps

• Lower investment costs
• Stability of the cellulase

complex in the SSF process during the entire fermentation period

• Overall simplified process

execution when the cellulase complex is produced on the basis of the lignocellulosic substrates in the ethanol plant

• Less inhibition of the cellulase

complex by ethanol and by- products of the lignocellulose- pre-treatment such as lignin

• Optimal supply of the required

amount of pulp in the fermentation process taking into account the high intrinsic viscosity of pulp suspension

• Yeast strains, stable to by-

products of the pre-treatment process

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The following main tasks are studied:

Pre-treatm ent of w heat straw

• Investigation on different properties of the pulp depending on

the method for pre-treatment

• Investigation on different properties of the lignin depending on

the method for pre-treatment Penicillium verruculosum cellulase-com plex

• Production of the P

. v.-cellulase using substrates of the pre-treatment process

• Saccharification of pulp
• Inhibition by lignin and ethanol

SSF-process

• Influence of pre-treatment on yield of ethanol
• Stability of the cellulase in the SSF-process
• Supply of the required amount of pulp by pre-hydrolysis, fed-

batch feeding and SSF-process in solid-state-fermentation

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Pre-treatm ent of lignocellulose

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• Alkaline pre-treatment with NaOH
• Natural Pulping pre-treatment with formic acid / H2O2
• Autohydrolysis

• 1. Charging the digester
• 2. Pulping procedure

3 . Separation of pulp

• 4. Lignin precipitation
• 5. Lignin separation

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Scale-up of alkaline pre-treatm ent at Fraunhofer Center for Chemical- Biotechnological Processes CBP, Leuna

wheat straw pulping with formic acid and H2O2 separation, washing black liquor distillation of formic acid precipitation with water pH=1,5 „black acid“

(water, formic acid, XOS)

lignin

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Natural pulping of wheat straw

residue formic acid pulp

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Scale-up of Natural Pulping pre-treatment

• 600-L Reactor (enameled)
• Agitation: 1 impeller
• Including distillation-unit

Lab-scale Pilot-scale

(SIAB) (Lanxess Deutschland GmbH, Group Function Innovation and Technology)

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Hydrothermal pre-treatment (autohydrolysis)

20 40 60 80 100 180 190 200 210 220

g/100 g hydrolysate Temperature (ºC) Furans Acetic acid Monosaccharides GlcOS AcO XOS

20 40 60 80 100 180 190 200 210 220

g/100g processed solids Temperature (ºC) Acetyl groups Xylan Glucan Acid insoluble lignin

Liquid phase Solid phase

Optimization of xylo-oligosaccharides production

• Autohydrolysis is highly selective towards hemicellulose enabling a high

recovery of xylo-oligosaccharides (XOS)

• An important glucan and lignin enrichment of the solid phase was possible

making the solids very attractive for further processing (i.e. enzymatic saccharification)

from EIB.10.013: Florbela Carvalheiro, LNEG, Lisbon

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I nfluence of pre-treatm ent on properties

• f pulp and lignin

Analytic of pulp:  I ntrinsic viscosity

• Determination of crystallinity by X-ray

diffractometry

• Scanning electron microscopy
• Com position of pulp; lignin, cellulose

and holocellulose content

• Xylo-oligosaccharides at autohydrolysis

Analytic of lignin:

• Influence of pulping duration / liquid ratio
• Influence of formic acid concentration (NP)
• Influence of NaOH-concentr. (alkaline p.)
• Functional groups
• IR-spectroscopy
• Molecular weight
• Klason lignin

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Comparison of all pre-treatments

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Pre-treatm ent Advantage Disadvantage

Natural Pulping Liquor ratio 1: 14

• Recovery of formic acid
• High purity of lignin
• Non-pressurised process
• Pulp has a lower intrinsic

viscosity

• High content of lignin in

pulp

• Low solid content (1: 14!)
• Corrosion protection

(e.g. enameled steel)

Alkaline Pulping Liquor ratio 1: 6

• Low lignin content in

pulp

• High technological

readiness

• Recovery of sodium

hydroxide

• Pressure of 6 bar

Auto-hydrolysis Liquor ratio 1: 8

• No chemicals needed
• Recovery of

hemicellulose

• High energy consumption
• Pressure of 20-25 bar

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Penicillium verruculosum enzym e com plex

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Production of the P. verruculosum cellulase

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0,1 1,3 1,1 0,5 0,6 1,4

0,4 0,8 1,2 1,6

• P. v.
• T. r.

0,9 6,4 7,4 1,7 1,3 1,1

2 4 6 8 10

• P. v.
• T. r.

15 g/L WB 20 g/L WB 30 g/L WB 5 g/L MCC 5 g/L MCC 5 g/L MCC 5 g/L G 5 g/L G 5 g/L G 15 g/L WB 20 g/L WB 30 g/L WB 5 g/L MCC 5 g/L MCC 5 g/L MCC 5 g/L G 5 g/L G 5 g/L G

from the Bachelor-Thesis Denise Lachmann, 2013

Cellulase (FPU/ml) ß-Glucosidase (IU/ml)

Production of cellulase based on wheat bran, glucose and MCC as substrate

(lab-scale in shake flask)

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Production of cellulase from P. verruculosum M28-10

• 600L Bioreactor
• VF = 400L
• Agitation: Rushton (x3)
• Aeration 0,2-0,8 vvm (air)
• Medium composition:

glucose, wheat bran, MCC

• fed-batch technique:

feeding of MCC

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Fermentation of P. verruculosum - course of enzyme activity

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Saccharification of pulp by

• P. verruculosum cellulase

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25 50 75 100 125 150 175 200 25 50 75 100 125

Red.

• ed. suga

ugar [g [g/l /l] Ti Time e [h] h]

Enzymatic saccharification of NP- and alkaline pulp: Feeding of pulp in fed-batch technique

125 FPU/ g pulp

(dry weight)

• -- alkaline pulp
• -- NP-pulp

4 1 2 3 1 feeding of pulp (in both experiments) 2 feeding of pulp (in both experiments) 3 feeding of NP pulp 4 feeding of alkaline P.

from the Bachelor-Thesis Robert Koksch, 2012

Comparison between T. reesei- and P. verruculosum-cellulase in hydrolysis of untreated and pre-treated wheat bran

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Inhibition of P. verruculosum cellulase by lignin and ethanol

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5 10 15 20 25 30 12 24 36 48

reducing sugar [g/L] time [h] saccharification of microcrystalline cellulose (MCC) in presence of lignin from natural pulping

MCC control MCC+25 g/L NPL MCC+50 g/L NPL MCC+75 g/L NPL

Study on inhibition of P. verruculosum cellulase by NP-lignin

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Study on inhibition of P. verruculosum cellulase by lignin from alkaline and hydrothermal treatments

5 10 15 20 25 30 35 12 24 36 48

reducing sugar [g/L] time [h]

saccharification of microcrystalline cellulose (MCC) in presence

• f lignin from alkaline and hydrothermal pulping

MCC control MCC+25 g/L alkaline lignin MCC+25 g/L hydrotherm. lignin

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5 10 15 20 25 30 35 40 6 12 18 24 30 36 42 48

Reducing sugar [g/l] Time [h]

Saccharification of MCC by P. verruculosum cellulase in presence of lignin from wheat bran by Natural Pulping (FPU= 50 U/gDM; substrate 50 g/LDM MCC)

MCC control MCC+25 g/L wheat bran lignin

I nvestigations on the SSF-process

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Investigations in 3-L-bioreactor

Experimental setup - process parameters

Working volume 1,700 ml FPU/ g DM 50 Yeast inoculum

(dry yeast, ZFT)

5 g/ L pulp content (g/ L)

• natural pulping
• alkaline pulping

50 - 75 75 - 100 NH4 Cl 2 g KH2PO4 1 g pH 5 temperature 35°C trial time ~ 72 h pre-hydrolysis (45°C) 6 h / 8 h

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pulp with 75 gDM/L

5 10 15 20 25 30 10 20 30 40 50 60 70 80 time [h] ethanol concentration [g/L] natural pulping alkaline pulping

Comparison of SSF between pulp from natural and alkaline pulping

pulp with 50 gDM/L

5 10 15 20 10 20 30 40 50 60 70 80 time [h] ethanol concentration [g/L] natural pulping alkaline pulping

without pre-hydrolyses with pre-hydrolyses (6h, 45°C)

10 20 30 40 50 60 70 80 90 100 natural pulp. 50g/L natural pulp. 75 g/l (prehydr.) alkaline pulp. 50 g/L alkaline pulp. 75 g/L (prehydr.)

ethanol yield/gDM [%] ethanol yield in ssf process with pulp from natural and alkaline pulping

yield ethanol per cellulose content

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SSF process with feeding of yeast in fed batch

15 30 45 60 75 90 20 40 60 80 100 timet [h]

• red. saccharides [g/]

0,0 0,8 1,6 2,4 3,2 4,0 4,8 EtOH [%] w/v

• red. saccharides

EtOH

1 2 Inoc. 10g/L DY +5g/L DY +5g/L DY

1, 2 = Dosage pulp + 2x50g/L (7h hydrolysis, 45°C)

• 5L stirred bioreactor, VF = 2L, 200g/L pulp (natural pulping), 30FPU/gDM
• EtOH-conc. = 4% (w/v) after 72h
• EtOH-yield = 35% (Pulp), 44% (Cellulose)
• Pulp-reduction = 57% (pulp-residue = 43%)

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SSF-Process in technical scale at CBP Leuna

• 220 L Reactor
• Agitation: anchor stirrer
• Medium composition:
• 100 g/ L alkaline pulp,
• 50 FPU/ g Cellulose (DM),
• 7,5 g/ L yeast, + inorganic

compounds

• Pre-hydrolysis 45°C
• Fermentation 35°C; pH = 4,5 – 5,5

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Results:

• EtOH-max: 3,84 Vol.%
• EtOH-yield: 73,7%

(related to cellulose-content in pulp)

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Course of the anaerobic fermentation at CBP

• final EtOH-conc. ~ 3.8% (w/v) after 15h
• EtOH-yield = 55% (based on Pulp), =73% (based on Cellulose)
• successful fermentation on technical scale (further optimization necessary)

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12 24 36 48 60 72 84 5 10 15 20 25 30 timt [h]

• red. Saccharides [g/L],

Glucose [g/L] 12 24 36 48 60 72 84 Yield (EtOH) on Cellulose [%]

• red. Saccharides

Glucose EtOH 2 1

before 1 = Hydrolysis of pulp (2,5h at 45°C) 1= Inoculation 7,5g/L yeast + 1/3 residual pulp 2= 2nd dosage of Cellulase (30 --> FPU/gDM)

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10 20 30 40 50 60 6 12 18 24 30 36 42 48 c(Glukose) in [g/L] Time [h]

Influence of NP-lignin on glucose degradation by yeast

Kontrolle ohne Lignin 5 g/L 10 g/L 25 g/L 10 20 30 40 50 60 6 12 18 24 30 36 42 48

c(Glukose) in [g/L] Time [h]

Influence of alkaline lignin on glucose degradation by yeast

Kontrolle Ohne Lignin 5 g/L 10 g/L 25 g/L

Influence of lignin on glucose utilization by yeast

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31 5 10 15 20 25 30 35 40 45 50 55 60 6 12 18 24 30 36 42 48

Glucose [g/l] Time [h]

3,33 g/L 1 g/L 0,75 g/L 0,5 g/L 0 g/L

Inhibition of aerobe glucose utilization by formic acid

Concentration: 0.5 g/ L - 3.33 g/ L formic acid

Influence of formic acid on yeast growth

Stability of P. verruculosum cellulase in the SSF-process

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42-L-reactor; P. verruculosum-cellulase, 50 FPU/ g DM Cellulase activity in the course of the SSF-process with a-cellulose as substrate

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Cellulase activity in the course of the SSF-process with NP pulp as substrate 3-L-reactor; P. verruculosum-cellulase, 50 FPU/ g DM

10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014

Cellulase activity in the course of the SSF-process with wheat bran as substrate

from Master thesis Manuel Meißner, 2013

42-L-reactor; P. verruculosum-cellulase, 15 FPU/ g DM

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2 %(w/v) 5 %(w/v) 10 %(w/v)

Problems to supply the required amount of pulp for > 10% ethanol in the SSF process

In stirred bioreactor max. 7.5 % (w/ v) solid concentration

SSF-process at different solid content

from EIB.10.013: Doreen Steffien, FUMT, Freiberg

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• economical ethanol production:

ethanol conc. > 4 % ( v/ v) required  > 1 0 % ( w / v) solid concentration necessary

• High viscosity  “free-fall-mixing”

required  15 L solid state bioreactor

Solid state reactor for high pulp concentration

10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014

High solid content

SSF-process in solid state reactor

from EIB.10.013: Doreen Steffien, FUMT, Freiberg

Comparison of different yeast strains

Shake flask culture: 50 g/ L MCC, 30 FPU/ g DM initial OD600= 12, Temp.= 35°C

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0,0 0,3 0,6 0,9 1,2 1,5 10 20 30 40 50 60 70 80 EtOH % (w/v) time [h]

• S. cerevisae - Fresh Yeast, Germany
• S. cerevisae - Dry Yeast, Austria

t [ h] FPA [ I U/ m L] aerob Yeast + enzyme + yeast extract / peptone 144 1 ,0 6 yeast + enzyme 144 0 ,0 0 anaerob yeast + enzyme + yeast extract/ peptone 144 0 ,9 5 yeast + enzyme 144 0 ,0 0 yeast + enzym e + stillage 9 3 0 ,8 5

Interaction between yeast and enzyme

Kluyveromyces marxianus; 37 °C; 90 rpm, pH= 5.0, control: 1.0 FPU/ mL

from the Diploma thesis: Anne-Catrin Letzel, 2010, FUMT, Freiberg

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Cellulase com plex

• The P. verruculosum-cellulase complex leads to higher hydrolysis

rates in comparison with T. reesei

• No or only low inhibition of P. verruculosum cellulase by lignin

SSF-Process

• Pulp from NP process generate more ethanol in SSF process than

pulp from alkaline pulping

• Stirred bioreactors (CSTR) allow a maximum of about 7.5 % (w/ v)

pulp, therefore a partial pre-hydrolyses of pulp or feeding in fed- batch-technique must be performed to realize economic concentrations of ethanol

• Yield ethanol / g NP-cellulose > 75% (laboratory conditions)
• Pre-hydrolyses with P. verruculosum cellulase improves particularly

strong the SSF-process with pulp from Natural Pulping pre-treatment

• Solid state fermenter: 50 % (w/ v) pulp  max. 12.8 % (w/ v) ethanol
• The yeast needs to be optimized for the SSF process

Results to the SSF-process

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Pre-treatm ent of lignocellulose:

The different pre-treatment processes are economical to compare with respect to the particular application, e.g. pulp for ethanol and lignin for basic chemicals or for composites.

SSF-process:

The provision of the necessary amount of pulp for > 10% ethanol in the SSF-process must be optimized. The possibilities for this are partial pre- hydrolysis

• f

the pulp, feeding

• f

pulp in fed-batch-technique

• r

fermentation of high pulp concentration in a solid-state-fermenter.

• P. verruculosum production strain:

The P. verruculosum-enzyme complex is favoured for the SSF-process in “second generation”. For industrial scale, the used P. verruculosum production strain must be improved, in particular to eliminate the carbon catabolite repression by classical genetic methods. This has the advantage that the strain can be produced in the ethanol plant without the requirements for GMOs.

Open questions still to be worked:

10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014

Partner of the EIB.10.013-Consortium:

(www.era-ib-lignocellulose.eu) SI AB, UL

SIAB and Leipzig University, Leipzig

I PW C

Technical University of Dresden

I W PT FUMT

Freiberg University of Mining and Technology

VTT

VTT, Technical research centre of Finland

LNEG

Laboratório Nacional de Energia e Geologia, Lisbon

W UR-FBR

WUR-FBR, Wageningen, Netherlands

Biotehnol

Biotehnol, Bucharest, Romania

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Consortium at project meeting in Bucharest, 2013

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Thank you for your attention!

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Notes to the presentation

pre-treated wheat straw pulping using NaOH (aq.) separation, washing pulp black liquor precipitation at pH= 1 “black acid“ lignin

Fakultätsname XYZ Fachrichtung XYZ Institutsname XYZ, Professur XYZ

alkaline pulping of wheat straw

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10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014

Scale-up of alkaline pre-treatment in CBP Leuna

Scale

Sample

Cellulose [ % ] Hemi- cellulose [ % ] KLASON- Lignin [ % ] In/ Output [ kg atro]

Yield [ % ] 350-L Wheat straw 41,9 31,7 21,7 25 Pulp 75,2 18,0 6,5 11,5 46,1 Lignin

• 61,7

10,0 25,0 2-L Pulp 76,5 12,3 4,3 0,07 55,2 Lignin

• 66,0

0,03 19,3

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Alkaline pulping Natural Pulping

• ptimal pulping

conditions c(NaOH)= 3 wt-% T= 160 °C t= 30 min c(HCOOH)= 60 % T= 103-105°C t= 40 min after H2O2 addition (30 % ) yield of pulp*

• ca. 55 %

Contains: 2 % Klason-lignin 79 % cellulose 19 % hemicellulose

• ca. 45-50 %

Contains: 11 % Klason-lignin 83 % cellulose 6 % hemicellulose yield of lignin precipitation product*

• ca. 20 %
• contains

70 % Klason-lignin

• ca. 60 % of original

lignin is obtained

• ca. 10 %
• contains

80 % Klason-lignin

• ca. 40 % of original

lignin is obtained * in relation to wheat straw

Comparison of the pre-treatment:

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To the SSF-process

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Ferm entation

• 37 °C
• pH 7.0, tap water
• 200 mg nitrogen

  1: 1 (NH4) 2SO4 : thin stillage

• 3 % (w/ v) yeast

(S. cerevisiae)

Preliminary tests for SSF-process

Conclusion:

• Yeast grows on hydrolysate,
• no sterilization necessary

10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014

Enzymatic hydrolysis with P. verruculosum

• Influence of solid concentration

21.66 g/L 26.59 g/L 10.98 g/L 53

SSF-process

• 5 % (w/ v) solid concentration

 max. 1.85 %(v/v) ethanol  Hydrolysis: yield = 86 %  Fermentation: yield = 71 %

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TMP– w heat straw

from TU Dresden Com ponent s percent Dry m atter 97.8 Ash« 2.7 Extract 2.5 Cellulose 49.0 Polyosen 76.4 Hemicellulose 27.4 Lignin 22.8