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 ERA-IB-project EIB.10.013: “Development of 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

Overall process for the utilization of wheat straw 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 2

SSF-process of ethanol production from pulp 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 3

SSF-process from the technical point of view Advantage of SSF-process Requirem ents Reduction and simplification of • Stability of the cellulase the process steps complex in the SSF process during the entire fermentation • Lower investment costs period • Overall simplified process • Less inhibition of the cellulase execution when the cellulase complex by ethanol and by- complex is produced on the products of the lignocellulose- basis of the lignocellulosic pre-treatment such as lignin substrates in the ethanol plant • 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 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 4

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 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 5

Pre-treatm ent of lignocellulose • Alkaline pre-treatment with NaOH • Natural Pulping pre-treatment with formic acid / H 2 O 2 • Autohydrolysis 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 6

Scale-up of alkaline pre-treatm ent at Fraunhofer Center for Chemical- Biotechnological Processes CBP, Leuna 1. Charging the digester 2. Pulping procedure 3 . Separation of pulp 4. Lignin precipitation 5. Lignin separation 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 7

Natural pulping of wheat straw wheat straw pulping with formic acid and H 2 O 2 separation, washing black liquor pulp distillation of formic acid formic acid residue precipitation with water pH=1,5 „black acid“ lignin (water, formic acid, XOS) 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 8

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) 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 9

Hydrothermal pre-treatment (autohydrolysis) Optimization of xylo-oligosaccharides production Solid phase Liquid phase 100 100 g/100g processed solids 80 80 g/100 g hydrolysate 60 60 40 40 20 20 0 0 180 190 200 210 220 180 190 200 210 220 Temperature (ºC) Temperature (ºC) Acetyl groups Xylan Glucan Acid insoluble lignin Furans Acetic acid Monosaccharides GlcOS AcO XOS  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 10

I nfluence of pre-treatm ent on properties of 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  Influence of pulping duration / liquid ratio Analytic of lignin:  Influence of formic acid concentration (NP)  Influence of NaOH-concentr. (alkaline p.)  Functional groups  IR-spectroscopy  Molecular weight  Klason lignin 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 11

Comparison of all pre-treatments Pre-treatm ent Advantage Disadvantage   Recovery of formic acid High content of lignin in Natural Pulping  High purity of lignin pulp Liquor ratio 1: 14   Non-pressurised process Low solid content (1: 14!)   Pulp has a lower intrinsic Corrosion protection viscosity (e.g. enameled steel)   Alkaline Pulping Low lignin content in Recovery of sodium pulp hydroxide Liquor ratio 1: 6   High technological Pressure of 6 bar readiness   Auto-hydrolysis No chemicals needed High energy consumption   Recovery of Pressure of 20-25 bar Liquor ratio 1: 8 hemicellulose 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 12

Penicillium verruculosum enzym e com plex Production of the P. verruculosum cellulase 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 13

Cellulase (FPU/ml) ß-Glucosidase (IU/ml) P. v. T. r. P. v. T. r. 1,6 10 1,4 1,3 6,4 7,4 1,1 8 1,2 6 0,8 0,6 0,5 4 1,7 0,1 1,3 1,1 0,4 2 0,9 0 0 15 g/L WB 20 g/L WB 30 g/L WB 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 MCC 5 g/L MCC 5 g/L MCC 5 g/L G 5 g/L G 5 g/L G 5 g/L G 5 g/L G 5 g/L G Production of cellulase based on wheat bran, glucose and MCC as substrate (lab-scale in shake flask) from the Bachelor-Thesis Denise Lachmann, 2013 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 14

Production of cellulase from P. verruculosum M28-10 - 600L Bioreactor - V F = 400L - Agitation: Rushton (x3) - Aeration 0,2-0,8 vvm (air) - Medium composition: glucose, wheat bran, MCC - fed-batch technique: feeding of MCC 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 15

Fermentation of P. verruculosum - course of enzyme activity 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 16

Saccharification of pulp by P. verruculosum cellulase 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 17

Enzymatic saccharification of NP- and alkaline pulp: Feeding of pulp in fed-batch technique 200 125 FPU/ g pulp (dry weight) 175 4 3 150 2 --- alkaline pulp 1 /l] [g/l 125 ugar [g ed. suga --- NP-pulp 100 Red. 75 1 feeding of pulp (in both experiments) 50 2 feeding of pulp (in both experiments) 25 3 feeding of NP pulp 4 feeding of alkaline P. 0 0 25 50 75 100 125 Time Ti e [h] h] 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 18

Comparison between T. reesei- and P. verruculosum -cellulase in hydrolysis of untreated and pre-treated wheat bran from the Bachelor-Thesis Robert Koksch, 2012 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 19

Inhibition of P. verruculosum cellulase by lignin and ethanol 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 20

Study on inhibition of P. verruculosum cellulase by NP-lignin saccharification of microcrystalline cellulose (MCC) in presence of lignin from natural pulping 30 reducing sugar [g/L] MCC control 25 MCC+25 g/L NPL 20 15 MCC+50 g/L NPL 10 MCC+75 g/L NPL 5 0 0 12 24 36 48 time [h] 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 21

Study on inhibition of P. verruculosum cellulase by lignin from alkaline and hydrothermal treatments saccharification of microcrystalline cellulose (MCC) in presence of lignin from alkaline and hydrothermal pulping MCC control 35 reducing sugar [g/L] 30 25 MCC+25 g/L alkaline lignin 20 15 MCC+25 g/L hydrotherm. 10 lignin 5 0 0 12 24 36 48 time [h] 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 22

Saccharification of MCC by P. verruculosum cellulase in presence of lignin from wheat bran by Natural Pulping (FPU= 50 U/g DM ; substrate 50 g/L DM MCC) 40 35 Reducing sugar [g/l] 30 25 20 15 10 5 0 0 6 12 18 24 30 36 42 48 Time [h] MCC control MCC+25 g/L wheat bran lignin 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 23

I nvestigations on the SSF-process 10th Bioethanol and Bioconversion Technology Meeting, Detmold 2014 24

Investigations in 3-L-bioreactor Experimental setup - process parameters Working volume 1,700 ml FPU/ g DM 50 Yeast inoculum 5 g/ L (dry yeast, ZFT) pulp content (g/ L) -natural pulping 50 - 75 -alkaline pulping 75 - 100 NH 4 Cl 2 g KH 2 PO 4 1 g pH 5 temperature 35°C trial time ~ 72 h pre-hydrolysis (45°C) 6 h / 8 h 25