1. Development of integrated bioprocess for ethanol production from sugar beet Dr. sc. Božidar Šantek, Full Professor Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierotijeva 6/IV, HR-10000, Zagreb, Croatia bsantek@pbf.hr

2. Introduction - biofuels are viable and realistic alternative in the energy market due to rising environmental concerns and oil prices - biodiesel from plant oils and from animal waste fats - bioethanol from grains, sugar cane and sugar beet - bioethanol production from lignocellulose raw materials at the beginning of the large- scale commercial production (e.g. new plants in the USA) - the production costs of biofuels are higher than production costs of gasoline from fossil oil ( e.g. gasoline is 2 times cheaper than bioethanol )

3. Figure 1. General unit operations in bioethanol production from sugar beet. LCA - bioethanol prodution from sugar beet

4. Table 1. Unit operations incorporated in different ethanol production plants used for determination of total and specific area for embedding processes. Unit operationsProcess assignation 11a2345678 Washing /slicing+++++++++ Diffusion+++++--AA Evaporation+++------ Sterilization---++---- Juice fermentation+++++---- Beet pulp fermentation-----++AA Centrifugal separation+++++---- Distillation+++++++AA Ethanol dehydration+++++++++ Stillage evaporation++++-+--- Pulp pressing-----+-+- Pellets forming/drying++++---+- Biogas production----+-+-+ Heat recuperation-+*+----+- (+) incorporated in the process; (-) not incorporated in the process; (*) the calculation with the 50 % of heat recuperation; (A) simultaneous sugar extraction, fermentation and product recovery.

5. Table 2. Total energy consumption for different processes of bioethanol production from sugar beet Process No. Specific energy consumption (kWh/kg) 114.875 1a7.538 23.148 37.121 44.467 ( 1 ) 53.281 60.619 ( 1 ) 73.068 80.411 ( 1 ) ( 1 ) Energy obtained from biogas counted in as goodness Figure 2. The ecological footprint (specific area; a tot ) of examined processes for ethanol production.

6. Experimental set-up of stirred tank bioreactor Sugar beet juice - addition of NH 4 H 2 PO 4 as N and P suorce (1 g/L) - medium sterilization at 121 o C for 20 min prior to inoculation with yeast Saccharomyces cerevisiae - ethanol production in STB (5 L) at 28 o C with 10 % v/v yeast suspension - batch and fed batch cultivation techniques were used - pH value was maintained in the range of 4.5 - 5.0 by the addition of 0.1 M NaOH and 0.1 M H 2 SO 4 - feeding in the fed batch process started when carbon source was almost completely depleted by the addition of a few portions of concentrated sugar beet juice (200 mL; approx. 800 g/L of sugar)

7. Figure 3. Alteration of substrate (S, ● ), ethanol (P1, ▲ ) and glycerol (P2,∆) concentration during batch fermentation of raw sugar beet juice in stirred tank bioreactor

8. Figure 4. Alteration of substrate (S, ■ ), ethanol (P1, ▲ ), glycerol (P2, □ ) and biomass (X, ● ) concentration, pH ( ○ ) and broth absorbance (A600nm,∆) during fed batch fermentation of raw sugar beet juice in stirred tank bioreactor. Arrows represent the addition of concentrated fresh medium during the fed batch process.

9. Experimental set-up of HRTB Sugar beet cossettes - ethanol production at room temperature after bioreactor sterilization at 121 o C for 30 min. - 5 kg of non-sterile raw sugar beet cassettes (23 % dry matter) - addition of NH 4 H 2 PO 4 (1 g/kg raw sugar beet cossettes) - inoculum Saccharomyces cerevisiae - different quantities of inoculum (9.1-23.7 % V/m of raw sugar beet cossettes) in order to define the broth minimal liquid content - different operational conditions of HRTB [rotation, the working volume of bioreactor (range 20 – 70 %)]

10. Figure 6. Alteration of substrate (S, ● ), ethanol (P 1, ▲ ), glycerol (P 2,∆), acetate (P 3, □ ) concentration and dry mass of sugar beet cossettes (DM, ○ ) inside raw sugar beet cossettes during fermentation in HRTB with the inoculum of 16.7 % V/m by periodical rotation

11. Figure 7. Alteration of substrate (S, ● ), ethanol (P 1, ▲ ), glycerol (P 2,∆), acetate (P 3, □ ) concentration in the liquid part of the fermentation broth in the HRTB with inoculum of 16.7 % V/m by periodical rotation

12. Table 3. Comparison among different systems for ethanol production from intermediates of sugar beet processing Production system t / hY P/S / g/gE /% Pr / g/(L·h) RSBJ+STB+BP98.50.42478.80.608 RSBJ+STB+FBP2340.50293.40.503 RSBC+HRTB (9.1 % V/m of INM) 930.38671.70.497 RSBC+HRTB (13 % V/m of INM) 990.36968.60.464 RSBC+HRTB (16.7 % V/m of INM) 1020.42879.50.476 RSBC+HRTB (20 % v/m of INM) 900.28152.30.357 RSBC+HRTB (23.1 % V/m of INM) 680.30857.30.467 RSBJ - raw sugar beet juice, RSBC - raw sugar beet cossettes, STB - stirred tank bioreactor, BP - batch process, FBP - fed batch process, INM - inoculum

13. Rotational set-up of HRTB during ethanol production from raw sugar beet cossettes Table 4. Investigation of the manner of HRTB rotation Table 5. Investigation of optimal rotation interval and speed of HRTB Interval of HRTB rotation (min/h) 3691215 HRTB rotation speed (min -1 ) 5+++++ 10+++++ 15+++++ Manner of HRTB rotationPeriodical (3-4 rotation/ day) Constant rotation HRTB rotation speed (min -1 ) 5++ 10++ 15++

14. Figure 8. Alteration of substrate (S, ● ), ethanol (P 1, ▲ ), glycerol (P 2,∆), acetate (P 3, □ ) concentration and dry mass of sugar beet cossettes (DM, ○ ) inside raw sugar beet cossettes the fermentation broth in HRTB with the inoculum of 16.7 % V/m by 3 min/h rotation and the rotation speed of 15 min -1

15. Figure 9. Alteration of substrate (S, ● ), ethanol (P 1, ▲ ), glycerol (P 2,∆), acetate (P 3, □ ) concentration in the liquid part of the fermentation broth in HRTB with the inoculum of 16.7 % V/m by 3 min/h rotation and the rotation speed of 15 min -1

16. Conclusions - sugar beet juice and cossettes can be successfully used for ethanol production. - the use of raw sugar beet cossettes in ethanol production eliminates extraction process of sugar beet cossettes by hot water, which considerably reduces energy demand for bioethanol production and final ethanol price. - further research of ethanol production from the raw sugar beet cossettes is required combined with improvement of sampling techniques due to the system heterogeneity

17. Thank you for your attention !!!