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Bioresource Technology60 (1997) 143-151

© 1997 Elsevier Science Limited
All rights reserved. Printed in Great Britain
0960-8524/97 $17.00


Mats Larsson, a Mats G a l b e a & G u i d o Z a c c h i a*
aDepartment of Chemical Engineering 1, Lund University, Lund, Sweden

(Received 4 June 1996; revised version received 13 December 1996; accepted 20 December 1996)

Abstract using ethanol instead of petrol is the removal of
In the production of ethanol from lignoceUulosics, aromatic hydrocarbons from the emissions. Ethanol
large quantities of water are needed in various process from renewable sources is usually produced from
steps. In industrial processes, recycling of process starch, and the production cost is very dependent on
streams is necessary to minimise fresh water require- the market for cattle feed, since the main byproduct
ments and decrease the amount of wastewater is a protein-rich residue. It is expected that in the
produced. This leads to increases in various sub- future, cheaper raw materials, such as lignocellu-
stances, both volatile and non-volatile, which inhibit losics, will be used.
fermentation. In the present study ethanol was pro- Lignocellulose as a raw material for ethanol pro-
duced from softwood in a bench-scale process duction costs less than starch and larger quantities
development unit. The stillage stream from the distilla- are available, allowing for large-scale production
tion step was fractionated by evaporation and various (Lou, 1994). The dependence on the market for
evaporation fractions characterised and their inhibitory byproducts is less than for starch-based production,
effect on fermentation investigated. It was found that since the main byproduct, lignin, is a valuable solid
the volatile fractions have no negative effect on the fuel with very low ash content. Lignocellulosic
fermentation, while the non-volatile fraction inhibited materials are consequently preferable to starch as a
fermentation and resulted in a decreased yield. Simula- raw material for the production of fuel ethanol (The
tion of a six-effect evaporation unit based on the Swedish National Board for Industrial and Technical
experimental data shows that the intermediate evapora- Development, 1994).
tion condensate fractions are most suited for disposal Several processes based on enzymatic hydrolysis
The chemical and biological oxygen demands are only on lignocellulosic materials have been suggested in
about 10% of the value in the stiUage stream. Evap- the literature (Jurasek, 1979; Saddler et al., 1982;
oration has been shown to be an effective way to Grethlein et al., 1984; Mes-Hartree et al., 1988; Lynd
drastically diminish the build-up of inhibitory com- et al., 1991). Most of these have a similar process
pounds when process streams are recirculated, but the configuration consisting of prehydrolysis of hemicel-
energy demand is high. Various process configurations lulose, enzymatic hydrolysis of cellulose and
to reduce the energy demand are suggested. © 1997 fermentation of the liberated sugars to ethanol.
Elsevier Science Ltd, Techniques involved in the various production steps,
such as prehydrolysis by steam pretreatment (Clark
Key words: Ethanol, recirculation, inhibitory, bench-
and Mackie, 1987), enzymatic hydrolysis (Esterbauer
scale process development unit, evaporation.
et al., 1983) and simultaneous saccharification and
fermentation (SFF) (Wright et al., 1988), have been
INTRODUCTION improved over the years. For ethanol to be competi-
tive with petrol, the utilisation of the raw material
Ethanol from renewable sources has gained in must be high, since the raw material contributes
interest during the last 20 years. One reason for this more than 30% of the production cost (von Sivers
is that provided no fossil fuel is used in the produc- and Zacchi, 1996; Busche et al., 1992; US Depart-
tion, ethanol produced from biomass does not ment of Energy, 1993; Hinman et al., 1992). This
contribute to the greenhouse effect since the carbon requires high yields in the saccharification steps.
dioxide formed in the production and the combus- Furthermore, micro-organisms which ferment
tion of ethanol is taken up by photosynthesis during glucose to ethanol with high yields and which can
the growth of new biomass. Another benefit with withstand potential inhibitors present in the raw
material and formed in the preceding steps are
*Author to whom correspondence should be addressed. required. The yeast Saccharomyces cerevisiae has

6 w t % and the ash content around In the present study. ethanol in a bench-scale process development unit (Palmqvist et al. the material Environmental impact was estimated by measuring was transferred to a plastic bag and impregnated the chemical oxygen demands (COD) and the bio.ati. using steam of volatile compounds assayed by fermentation.Yeast Evaporation [ J . with gaseous sulphur dioxide (SO2). tion of the mixture is shown in Table 1 (STORA culation in terms of fermentability and Teknik AB.. (1996a). Galbe.. The amount of logical oxygen demands (BODT) of various fractions. Immediately after presteaming. both volatile and non-volatile. 1997). By using the occur at different locations in the process. In industrial processes. atrnent Steam 20-28 bar Cydone ~_1 3 hydrolysis E.. waste stream for softwood as raw material are com- ments hexoses (Palmqvist et al.. Water Ethanol Condensate~ ~ Distillation [. The composi- determine process streams that are suited for recir. the minimum pretreatment. The different units are not con- Zacchi. !983). SO2 added was approximately 5 wt% of the DM. 1991) has shown that more than 10-fold nected physically so the material is transferred higher concentrations of non-volatile substances can manually from one step to the next. 30 min in a small pressure vessel. Larsson. The non-volatile fraction has been found to Substrate inhibit fermentation greatly and hydrolysis modera. the range 2 . 2 bar. a real inhibitory effects on enzymatic hydrolysis and fer.r_~ Steam L~nln Residue _ i L~ ~-. Zacchi proved to be very resistant to the inhibitors present The inhibitory effects and the characteristics of the in lignocellulosic hydrolysates and it efficiently fer. process para- meters can be carefully controlled and usually pure METHODS water or buffer solutions are employed to optimise the conditions. The material was chopped and process must be taken care of in the best possible screened and fractions from 2 to 5 mm selected for way.nzym. the amount of extractives to that present in the raw material has to be dis. In the latter. and an Zacchi. M. 1. Industrial-scale processes differ from small-scale laboratory investigations. requirement is that an amount of water equivalent For similar substrates.c Washing . It is thus of considerable importance to and paper mill was used as substrate. This recycling leads to increases in ethanol. A previous investigation (Galbe and evaporation unit. Schematic flowsheet for the bench-scale unit. The stillage stream from Pretreatment the distillation was fractionated by evaporation and Here 2454 g of softwood (DM) was presteamed for the inhibitory effects of the volatile and the non. a fermentor. The dry matter (DM) content environmental impact. a distillation column. Wood chips Steam1bar '~b'e PS:~u~~~.144 M. The equip- mentation of the volatile and non-volatile ment is described in more detail elsewhere compounds released from willow during steam pre. The bench-scale unit consists of a steam various substances. For a totally closed process. using a bench-scale process requirements and decrease the amount of waste. The same material throughout the entire process. A mixture of pine and spruce from a Swedish pulp tely. (Palmqvist et al. development unit for converting softwood to water produced. Sweden). 1996). softwood was converted to 0. in pretreatment unit. 1993). recycling of Figure 1 shows a schematic flowsheet of the process process streams is necessary to minimise fresh water configuration studied. G.5 wt% (Ladisch. pared with those of willow. The waste streams from the was 48wt%. 1996b). process can be experimentally simulated. ~ Fermentation Stiflage Fig._~ SO2 . (not measured) is reported in the literature to be in posed of. a reactor for enzymatic hydro- the hydrolysis and fermentation steps (Galbe and lysis. . treatment have been studied by Palmqvist et al.

The boiling point for fermentability of these fractions. material of 5 wt%. fermentation broth was stripped in the evaporation unit prior to distillation. Fermentation was per- Enzymatic hydrolysis formed in a shake-incubator for 5 h at 30°C. Sweden). 6. was filtered in a filter press and the solid residue Japan) equipped with a refractive index detector removed.5 with Ca(OH)2 and 0. Glucose night at 4°C. 24. USA) at 80°C Fermentation with water. A Hydrolysis was performed on the fibrous material reference fermentation containing only 50 g/l and the filtrate at a dry-matter content of fibrous glucose and nutrients was run for comparison.5 condensates Mannan 11.5 before inoculation. Denmark) per gram substrate (DM).8. in order to evaporation residue. 301 tank containing 25 1 medium. these conditions have been found fractions and the evaporation residue. 0. Recirculating process water in ethanol production 145 Table 1. the non-volatile specific heat and the boiling point elevation were fraction from the stripping stage was mixed with the determined for various dry-matter contents in the stillage stream from the distillation stage. were assessed by fer- Xylan 5. 48. 1989). The ethanol. Denmark) and 0. The SO2 impregnated material was at a concentration of 50 g/l was added to samples of then subjected to steam pretreatment at 210°C for the original stillage.2mm filters (MFS-13. hexosan and pentosan contents of the raw material and the ethanol yield is based on the theoretically Distillation and evaporation available amounts of fermentable sugars. The nutrients used in the fermentation of the hydrolysate were also added.2 be recycled in the process. The atmospheric pressure was determined at the Depart- mixture was evaporated and the condensate col. glycerol. 16. Composition of the raw material the stillage stream and evaporation residue. to Arabinan 1. the hydrolysate USA) and analysed on an HPLC unit (Shimadzu. 1974).8 The inhibitory effects of evaporation condensates. 1. laboratory Cenox AB (Lund.5 ml/min as eluent.6 mentation using S.5 g/l. supplemented with 0. 0. After distillation. 188 (Novo AS.5 ml/min as eluent. at a flow rate of 0. The softwood hydrolysate was fermented in non. The hydrolysis Analysis was performed for 96 h at 40 + 1°C. Rotebro. 2. Prior to fermenta. at a flow rate of tion. which were to be optimal in terms of the sugar yield in then inoculated to a final cell concentration (DM) of enzymatic hydrolysis (Saddler et al. 72. wood Galactan 1.25 g/1 (NH4)EPO4. USA) at 65°C using 5 mM H2SO4. 1. 5 and finally 8 h.. Standard 02 81 43 (SIS.0g/l yeast COD and BOD7 were determined at the certified extract.05 g Novozym 30 min for 5 h. 40. corresponding to 12 FPU/g substrate. were wt% of dry determined. Saccharomyces cere. as con. visiae (J/istbolaget. Sugars were separated using an Aminex HPX-87P column (Biorad. Lund University lected in five fractions. 0. Samples were taken Yields at 0. Samples were withdrawn at the start and then every clast 2L (Novo AS. The medium was inoculated methods of Dr Lange. All samples from the hydrolysis. 1. fermentation and trolled by titration with 10wt% NaOH solution. 10 g/1. Japan).5. cerevisiae. BOD7 and (Suurkuusk and Wads6. The specific heat at 25°C and maintain the process configuration in Fig. The volume of the fermentation broth was 75 ml and the The plastic bag with its contents was stored over- pH was adjusted to 5. After hydrolysis. to avoid problems associ. Sweden) using the and 12 g/l NaHzPO4. pH 4. and acetic acid were sepa- sterile conditions at 30°C in an intermittently stirred rated on an Aminex HPX-87H column (Biorad. The COD. glucose Since the distillation was operated continuously the and mannose. Specific heat and boiling point elevation ated with suspended particles in the packed The physical data necessary for computer simulation distillation column.025 gfl MgSO4-7H20. and 96 h. at which time all All yields are based on the theoretically available glucose had been consumed.9 Assessment of the inhibitory effects of evaporation Glucan 40. the five evaporation condensate 5 min. Micro Filtration Systems.1 Lignin 27. LCK Cuvettes and Swedish with compressed baker's yeast. 3. Fermentation of both total and fractionated stillage was carried out in 100 ml glass flasks sealed with cotton stoppers. ment of Thermochemistry. 4. For spruce. (Shimadzu. as well as those of the evaporation residue at atmospheric pressure was . the hydrolysate supplemented with 1. the pH was adjusted to 5. to a final cell concentration (DM) of 7. 3. 1990). distillation/evaporation steps were filtered through Samples were withdrawn after 0. the then distilled. The evaporated fraction was of the evaporation of the distillation stillage.2 g Cellu.

.5 wt% of the ethanol present in the fermentation broth was Yields obtained in the distillate. COD and BOD7 for the volatile fraction from the After enzymatic hydrolysis. cerevisiae used could not ferment these sugars. glycerol. Pentoses and galactose do not contribute to the yield since the strain of S. 2. con. In this study the loss 3°t 25 ~ Glucose . wood.41 g/g.1 g/1 mannose prior to fermentation. 31.146 M. The loss of ethanol in the distilla- tile compounds which. Time [h] Fig. the evaporation. oration. approximately 98. Sugar concentrations versus time during enzymatic hydrolysis.. based on the impact of discharging these streams... 20% of the stillage has been evaporated. Data for willow obtained by achieved. consumed.. In the latter of 15. Sixty-four wt% of the material ended up as fibrous material Characterisation of recycling and waste streams (cellulose and lignin) and the rest. to some extent. . 7. which drops to below 3000 mg/l when hydrolysis as a function of time are shown in Fig. evaporation step as a function of the relative retical glucose yield based on raw material was amount evaporated. the five condensate fractions from the evap- hemicellulose and degraded lignin. 65 wt% of the theo. . The BOD7 The hydrolysate contained 26. in the condensate.. RESULTS After distillation. The maximum productivity in fermentation was amounts of liquid are to be concentrated.6 g ethanol/100 g of original dry matter. This was partially due to the formation of vola. .2g/1 glucose and for softwood and willow is 65-75% of the COD.. the first condensate has a COD around centrations of glucose and other sugars in the 9000 mg/1. corresponding to 80% of explained by entrainment of non-volatile com- the theoretical value and resulting in an overall yield pounds.0 g ethanol/(l h) with a final productivity of 3. . Based on The slight increases in COD and BOD7 for willow at total fermentable sugars in the hydrolysate.8 g loss will be relatively greater. respectively. determined in order to estimate the environmental ment were 21 and 71 wt%. leave the tion step can be lowered with improved control. G. The yields of oration and the evaporation residue were glucose and mannose in the liquid after pretreat..4wt%.6 wt% of the material was present in the two first condensate fractions from lost. since the 7. The missing ethanol was During pretreatment 4. enzymatic hydrolysis since most of the hemicellulose Both COD and BOD7 decrease rapidly. The con. For soft- is hydrolysed during the pretreatment step.. 2. • / o ° ... M. The concentrations of sugars and the separator. e. . Figure 4 shows theoretical amount available in the raw material. Larsson.. the high relative amounts of evaporated liquid are ethanol yield was 0. The amounts of COD and the BOD7 in the stillage sisted of dissolved solids. This stage of the concentration of the liquid in the evap- corresponds to 53% of the theoretical yield based on orator. Galbe.. The problem is more noticeable when small 3... thereby increasing the loss by evap- ethanol produced in fermentation are shown in Fig. Zacchi determined relative to that of pure water using a ethanol/(l h) when all the fermentable sugars were reflux boiler. the viscosity of the liquid increased. The mannose yield is not affected by Palmqvist et al.. which the amount of fermentable sugars available in the resulted in deposition of the solutes on the walls of raw material.g. mainly hydrolysed stream.. cyclone together with the flash vapour. (1996) are included for comparison.

The COD and BOD7 for the stillage stream and the evaporation residue for sol~ood and willow Stream COD BOD7 Percentage of (mg/I) (mg/l) stillage Stillage. softwood 26500 12800 -- Residue... and willow are shown in Table 2. . Recirculating process water in ethanol production 147 25. .o.7 . ~ .O°. I ~6000 .00 -~ .. 4.00 & ~ | o Arabhlos.-X 1000 . Amounts of COD and BOD7 in the evaporation condensates for softwood (this study) and willow (Palmqvist et al. -_.o.. e. . . . a fall. # CO0-Softwood I .g.. -x.3 wt%. .. . willow 33 300 19 800 -- Residue. I 20.+. 2~able 2.. . 3. Concentration of sugars and ethanol as functions of time during fermentation. Bhsnol II l 1 Time [hi Fig..o. BOD-Soflw ood I . stream and the evaporation residue for softwood ing-film evaporator. softwood 182000 86900 17.. . BOD-WIIow l 5000 4000 '\. 1996). was 6. ~3000 0 2000 . willow 208 000 95 400 10. The problem would be avoided in a The values of COD and BOD7 for the stillage continuous commercial evaporation unit.. . -& l. 0 I l l I I I I I 0 10 20 30 40 50 60 70 80 90 Rnlatlve m o u n t of evlporlted liquid [%] Fig. coo. A • ..5 Stillage...

39 Fraction 4 10. xylan is hydrolysed into xylose.41 have the same area. Ethanol productivity and yield in the fermenta. evaporation residue. corresponding to a wood intake Reference -. Zacchi Due to the problem with the evaporation The specific heat at various dry-matter contents in described earlier.0 g ethanol/l h.2 0.40 the lowest for effect 6. cerevisiae used in of the boiling point (BPE) of two evaporation resi- the fermentation. Specific heat for the evaporation residue as function of dry-matter content. Data were interpolated using the expression tion condensate fractions and the evaporation residue were measured by fermentation. Ethanol BPE = 3. From 3. The temperature of the cooling water is assumed to be 55°C which. Galbe.5 4 3.5 I ~ I I I 0.2 0. G. unfortuna. Percentage of Productivity Yield Saturated live steam of 3 bar (133. with the highest value for effect 1 and Fraction 5 47. Fig.35 of 100 metric ton/h. resulting in a non-volatile concentration only the unit.148 M.5 0.4 0. with a non-diluted residue. Larsson.5 1 0.10x 2 (2) yields and final productivities are listed in Table 3. The higher values of lowing expression: COD and BOD7 for willow compared with softwood can be accounted for by a higher content of xylan in Cp = 4.3 0. 5. M. dues with different dry-matter contents are shown in The inhibitory effects of stillage. yielding the fol- of evaporation condensate. 6.2 0. effect 6 the liquid is taken to effect 3 and then counter-currently to effect 1.0 kW/m2°C. During pretreatment.7 0.0 5. a pulp and paper mill in the south of which had been stored at 4°C and resulted in a yield Sweden. 4.419x 4 (1) the raw material.5 6. The feed enters effect number 4 and the of 0.9 5. Cp: kJ/kg°C). Data were fitted to a between the amount of stillage and the total amount polynomial using a least-squares fit. This was actually used for the evaporation of black liquor by repeated 3 months later.5 times that of the stillage stream.40 cients were assumed to vary between 3. .5 3 ~2. The calcu- glucose) lations are based on the assumption that all effects Fraction 1 5.2 0. which cannot be converted (x: kg DM/kg solution. 7.5 5. Simulation The fermentability of the residue was.3798x+ 13. is based on the unit 1. Stillage -.675x+5. with a Table 3.6 Dry m alter content [kg/Izg] Fig. the five evapora. The configuration of times. Nym611a AB.20 bar in effect number 6.1 5. Due to difficulties in the glucose analysis of the (x: kg DM/kg solution. 6. tion of fractions from the evaporation stage results in a pressure of 0.5°C) has been stillage (g EtOH/I h) (g EtOH/g chosen as the heating medium in effect 1. temperature difference of 5°C in the condenser.2 and Fraction 3 11. 5.37 (DM) of 100000 metric ton/year and an ethanol pro- 4.38 2.5 12 ~ V) 1.6 0. The elevations into ethanol with the strain of S. BPE: °C). shown in Fig. The experimental data were used in the simulation tely.41 g ethanol/g glucose and a productivity of liquid passes co-currently to effect number 6. performed with a residue which was diluted five of a six-effect evaporation unit. the percentage of stillage for the the evaporation residue at 25°C and atmospheric evaporation residue is defined as the difference pressure are shown in Fig.1 0. the ethanol yield for the resi- due is based on a calculated initial glucose concentration.180-4.1 0.7 0.5 0.722x3-3.0 4. The overall heat transfer coeffi- Fraction 2 8.34 The feed to the evaporation unit has a flow rate Residue 17.

Recirculating process water in ethanol production 149 6 os == 3 2 1 m 0 0. The and COD for the various condensate streams leaving second test with evaporation residue that has been each effect are shown in Table 4. however.3 0. but to investigate the inhibitory effect on fer- specific heat at 25°(2 and assuming that the variation mentation when process streams are recycled. resulting in a productivity but an increase in ethanol yield.2 0. present optimal conditions to obtain a high ethanol The enthalpy for the feed is calculated using the yield. The live steam stored at 4°C for 3 months showed a decrease in consumption is 23. not to metric tons/h with a dry-matter content of 65 wt%. The The first fermentation test with diluted evapora- boiling point elevation is assumed to be dependent tion residue showed that the ethanol yields for the only on the dry-matter content in the liquid. with its negative impact on fermentation.6 Dry matter content [kg/kg] Fig. 4wt%. The product flow rate is 6 The main object with this study was. temperatures obtained by Palmqvist et al.1 0.5 metric ton/h. This shows that something had happened areas were found to be 380 m 2 for each effect. The results were similar to those The calculated values of flow rates. Cooling Live steam water Fig. so fresh residue. The overall ethanol yield obtained in this study. in specific heat with temperature is negligible. stillage energy calculations the condensate streams are and residue was lower than for the volatile fractions treated as pure water. Enhanced ethanol yield is expected lage contains compounds inhibitory to fermentation. In an industrial process.5 0. A higher pounds that inhibit fermentation.4 0. and the reference. The temperature when the hydrolysis will be performed in a fed-batch and dry-matter content of the feed are 40°C and mode and the fermentation in a continuous mode. respectively. Schematic flowsheet for the evaporation unit. duction of 18400 metric tons/year. We have found yield is required in order to produce ethanol to a that the non-volatile fraction of the distillation stil- competitive cost. Boiling point elevations of evaporation residues at atmospheric pressure. must be excluded from the process. For these operating conditions. 53% Further research is necessary to identify the com- of theoretical. . (1996) for willow. 7. must be considered as low. The steam economy of 4 kg evaporated vapour/kg live yield was of the same magnitude as for the volatile steam. 3 months of storage is not an economical DISCUSSION alternative. with the residue during storage and that the inhibi- tors might have been degraded. In the fractions containing non-volatile compounds. 6. the heating fractions.

R. & Zacchi. The integration of a stripper in the evaporation G. M. & Schurz. Biotechnol. centration of the distillation stillage with reverse 19. Jurasek. P. Zacchi Table 4. (1983). L. M. (1984). R. 639-649.0 to 5.8 metric ton/metric ton of A comparative study of the enzymatic hydrolysis of the ethanol. W. M. & Zacchi. utilise the raw material efficiently and the reduction Clark. . E. . H. E. Jungschaffer. Ind. O.. technol. B. and Zacchi. 261. E. J.. however. TechnoL. B. Bio- used as fuel for the generation of process steam. Results from simulation of the evaporation unit osmosis. Further reduction in steam consumption can Lou. (1991). ethanol fermentation. Bergeron. from the simulation of a six-effect evaporator unit Esterbauer. Enzymatic hydrolysis of pretreated aspen wood. The effect of water soluble inhibitors from unit is an alternative which will further reduce total steam-pretreated willow on enzymatic hydrolysis and steam consumption. Palmqvist. Depending on 251. ysing at higher dry-matter content.. If the evaporated Development (NUTEK) for their financial support. Steam explosion of the softwood Pinus radiata with sulphur dioxide addi- of COD and BOD7 in the disposal stream decreases tion. (1988). (1983). J. Enzymatic hydrolysis of biomass. Fuel ethanol from cellulosic biomass. 373-403. R. C. The engineering and economic evaluation be achieved by increasing the dry-matter content in for chemical utilisation of biomass.4 11000 6 11304 70. 10. these might also be an alternative. Hahn-H~gerdal. D. T. evaporation unit is. G.. R. 1498. M. chem. 3. Galbe. Biochem. Total optimisation of the Jernqvist. Biochem. A. (1994).1 3000 5 10260 77. as well as alternative configurations of the evapora. (1993). H. A. Microbiol. The values of COD and BOD7 for these 3(3). Larsson. Results 7(3).7 3000 balance in the process. K. Various energy. ethanol production for SSF technology. 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