Biomass conversion to butanediol by simultaneous saccharification and fermentation

Trends in Biotechnology, VoL 3, No. 4, 1985

100

Butanediol is also an appealing product because of its diverse potential uses: its heating value of 27.2 kJ/g compares favorably with other liquid fuels (methanol, 22.1; ethanol, 29.1)1; and it has a higher octane number and can therefore be used as octane booster for Ernest K. C. Yu and John N. Saddler gasoline or as high-grade aviation fueP. We are, however, more interested i n The efficient biological conversion o f all the available sugars in b i o m a s s the potential of butanediol as a residues to fuels and c h e m i c a l s is crucial to the efficiency o f any process chemical feedstock, since its projected intended to c o m p e t e e c o n o m i c a l l y with p e t r o c h e m i c a l products. Both value as a polymeric substrate is h e m i c e i l u l o s e - and cellulose-derived carbohydrates f r o m w o o d and generally about two to three times its agricultural wastes can be converted to 2,3-butanediol by simultaneous value as a fuel9'~°. Butanediol can be saccharification and fermentation. This approach results in i m p r o v e d readily dehydrated to methyl ethyl butanediol yields and process productivities, and also enables b i o m a s s ketone, an industrial solvent, and to substrates, after a s i m p l e p r e t r e a t m e n t (steam-explosion), to be directly butadiene (and styrene) for manufacused for efficient butanediol production. turing synthetic rubber s. More recent studies suggest that fermentationThe recent global energy crisis and the sidered (Table 1). The ability of the derived 2,3-butanediol can also replace projected future energy shortage have organism to use hexoses (glucose, galac- 1,4-butanediol in the production of led to renewed interest in the use of tose, mannose), pentoses (xylose and Table I. Cellulose and hemicellulose in aspensuch biomass as forest and agricultural arabinose), certain disaccharides (such wood and wheat straw residues as alternative energy sources 1. as cellobiose and xylobiose), as well as % Total dry weight HemiceUulose can account for 20-35% uronic acids and acetate 3-5 can maxiAspenwood Wheat of the total dry weight ofbiomass2 and mize the conversion efficiency and im- Components $tTGW constitutes an inexpensive and prove the economic outlook of the proGlucan 48.0 35.0 abundant source of fermentable sugars cess. 17.0 19.0 There are several other advantages to Xylan for conversion to fuels and chemicals. Arabinan 0.5 4.4 butanediol fermentation by K. pneuTo date, energy from biomass proGalactan 2.0 0.7 cesses where cellulose alone is con- moniae which render the process attrac- Mannan 2.1 0.4 verted cannot compete economically tive. The organism is a fast-growing Uronicanhydride 4.3 2.7 with the energy from petrochemicals. facultative anaerobe, and is con- Acetyl 3.7 2.9 The efficient use of hemicellulose as sequently easy to work with. It can prowell as cellulose components could duce butanediol at near theoretical both speciality and commodity grades significantly improve the competitive yields, i.e. 0.5 g ofbutanediol per g of of polyesters and polyurethanes11. To edge of any potential commercial bio- sugar used 6'7. Moreover, because diols ensure that biomass conversion proconversion process. Research in our are less toxic than alcohols, final pro- cesses can be cost-effective when comlaboratory has therefore placed con- duct concentrations in excess of pared with conventional petrochemical siderable emphasis on using both hemi- the 80-100 g/1 considered desirable processes, we have initiated studies to cellulose- and cellulose-derived carbo- for economical product recovery develop a simple and effective process hydrates for producing a variety of can be achieved through acclimati- for the conversion of both cellulose and fuels and chemicals, including 2,3- zation (adaptation) and fed-batch hemicellulose sugars from biomass to approaches 6,7. butanediol. butanediol.

Biomass conversion to butanediol by simultaneous saccharification and fermentation

Reasons for interest in K.

Endocellulase .

pneumoniae butanediol

INSOLUBLE CELLULOSE

fermentation

(DP>6)

Klebsiella pneumoniae can use all the major substrates known to be present in the cellulose and hemicellulose components of wood and agricultural 34 residues'. This is a significant asset for any potential biomass conversion process when the heterogeneous nature of lignocellulosic substrates, such as aspenwood and wheat straw, is con-

F

~

•

. . .

Exocellulase

Exocellulase

SOLUBLE CELLULOSE (OP-
Endproduct Inhibition

E n d o c e l l u ~ C~ellobiase Cellobiase

MOSTLY CELLOBIOSE (DP=2)

1

E. K. C. Yu and J. N. Saddler are at the Bio- GLUCOSE (DP=I) technology and Che~nistry Department, Forintek Canada Corporation, Ottawa, Fig. I. The enzymic hydrolysisof cellulose. Ontario, Canada. © 1985,Elsevier Science Fublishers B.V.j Amsterdam 0166 9430/85/$02.00

I--~.on,.,ooi.g °.d

101

Trends in Biotechnology, VoL 3, No. 4, 1985

hydrolysis proceeds, i.e. glucose on cellobiase and cellulases, and xylose I PENTOSES xylobiase and xylanases ~s'~9. D-Xy~ose 1 on L-Arabinose Disaccharides also accumulate and inhibit the cellulase and xylanase Isomerases systems. Kinases The end-product inhibition has been Epi m erases successfully relieved using a process CELLOBIOSE MONOPHOSPHATE known as simultaneous saccharificaD-XYLULOSE-5-(~ tion and fermentation (SSF)2° 23. In this Phosphoapproach, the hydrolysis of cellulose to glucose is carried out in the presence of glucosidase (EMBDEN'MEYERHOF1 a microorganism, such as a yeast or GLUCOSE~ GLUCOSE-6-(~ ~- t GlycolyticPathway,/J " bacterium which can simultaneously % ferment the sugars produced during hydrolysis to products, in the same f reactor. The continual fermentation of ORGANICACIDS~ ...... PYRUVATE~ ACETYL the released sugars to ethanol effeck. J tively prevents them from accumulating in inhibitory amounts and thereby ] Acetolactatesynthase relieves the end-product inhibition of OL-ACETOLACTATE the hydrolytic enzymes. A significant improvement in the overall efficiency ~Acetolactatedecarboxylase of the bioconversion process results. Similar approaches can be readily I ACETOIN 1 adapted for the conversion of hemicellulose to fuels and chemicals. The SSF approach can also be I lAcet°in reductase economic, as only one reactor vessel is required for both the hydrolysis and fermentation steps. These potential I BUTANEDIOL1 benefits clearly justify further exploration of SSF, particularly ira single Fig. 2, Simplified metabolic pathways in K. pneumoniae of the major sugars in biomass process can be used for hemicellulose hydrolysates. The pathways involved in using o-xylobiose have not been established. and cellulose together.

I

,,, CELLOBIOSE

1

I

HEXOSES 1 O-GNcose D-Mannose

O-a,acoe

1

/

Rationale for SSF As a first step before fermentation, it is necessary to break down biomass polysaccharides to respective monomers. This can be accomplished by chemical or enzymic means. Recently, we demonstrated efficient production ofbutanediol from various aspenwood fractions which had been hydrolysed by dilute or concentrated sulphuric acid 7'12-14 or anhydrous hydrogen fluoride 15. However, acid hydrolysis has been shown to inactivate the lignin and degrade the hemicellulose and, to a lesser extent, cellulose components, resulting in the loss of fermentable sugars and the formation of fermentation inhibitors. Consequently we have concentrated our efforts on the enzymic saccharification of various lignocellulosic substrates. Earlier, it had been shown that the cellulose and hemicellulose components of wood and agricultural residues could be broken down by the extracellular enzymes of various wooddecaying fung£ 3,a6. However, the

1

hydrolytic efficiency of the enzymes decreases with increasing substrate concentration 16, a phenomenom well documented for hydrolysis of cellulose by fungal cellulases (Fig. 1), and generally believed to apply to the enzymic hydrolysis of hemicellulose. Most of the fungal cultures studied have low cellobiase and xylobiase activities, relative to their cellulase and xylanase activities 5,17. Consequently, conversion of disaccharides to monosaccharides is usually the rate-limiting step in the hydrolysis of both cellulose and xylan. Enzymic hydrolysis is further complicated by end-product inhibition of hydrolytic enzymes as

Prerequisites of SSF Several criteria would have to be met before an efficient SSF process for both cellulose and hemicellulose carbohydrates could be used industrially: the hydrolytic enzymes should be produced by the fungus when it is grown on inexpensive substrates, such as wood or agricultural wastes; the complete enzyme complex should be excreted by the fungus into the culture medium to facilitate harvesting, handling, concentrating and sterilizing (if necessary) of the enzyme preparations for scaled-up commercial operations; the enzyme preparations should contain a full spectrum of active

Table 2. Comparison of butanediol production using simultaneous saccharification and fermentation (SSF)or sequential hydrolysis and fermentation (SHF) Substrate

Xylan Solkafloc

Substrate concentration (g/l)

50 100 50 100

Butanediol produced (g/l) SHE SSF

5.7 9.9 6.4 10.4

8.2 16.6 9.1 15.0

102

Trends in Biotechnology, VoL 3, No. 4, 1985 Uninoculated (control) • Inoculation

Inoculated (SSF) • 1Or

-,° r "~ tr

0

-~"

15

8~ ><

10

0"

'

6

i

5

°

4

m

2

i I

1

2

3

4

0

1

2

3

4

Incubation time (days)

Fig. 3. Simultaneous saccharification and fermentation (SSF) of xylan (5%, w/v), using crude fungal culture filtrates as the xylanase enzyme preparation. cellulolytic and xylanolytic enzymes to ensure efficient substrate hydrolysis. We have shown that most of these prerequisites can be met by using culture filtrates of Trichoderma harzianum (strain E58) 5,24. Once the lignocellulosic substrates have been hydrolysed to their component sugars, the fermentative member of the SSF system should be able to use both the hemicellulose- and cellulosederived sugars. This requirement is fully met by 1£. pneumoniae which can directly use glucose and xylose, as well as cellobiose and xylobiose3'5, thereby ensuring that all the end-products of hydrolysis are effectively removed (Fig. 1). The ability of the organism to use the disaccharides directly circumvents the requirement for active cellobiase and xylobiase enzymes, which are absent in most cellulase and xylanase systems. K. pneumoniae also has the advantage of being able to use all the other major components of the cellulose and hemicellulose fractions for additional butanediol production (Fig. 2). The hydrolytic enzyme preparations and the fermentative organisms used in the process must also be compatible. Compatibility has been established by showing that the activities of the fungal cellulase and xylanase enzymes were not adversely affected by the presence ofK. pneumoniae throughout the SSF process 24, and that the material associated with the fungal enzyme prepara-

tions which had previously inhibited butanediol fermentations 24-26, was removed when ultrafiltration was used instead of rotary evaporation to concentrate the enzymes 5,24,26.

typical SSF profile for butanediol production from xylan is shown in Fig. 3. In the absence of the fermentative organism, the xylanase enzymes in the original culture filtrates of T. harzianum E58 could readily hydrolyse xylan to reducing sugars consisting of mainly xylose and xylobiose25. The low concentrations of enzymes required for this process (equivalent to one fifth of the original enzyme concentration in the crude fungal culture filtrates, or 4 mg of crude protein per g of substrate used) demonstrated the potential of this approach. Upon inoculation of the organism, the released sugars were readily removed and converted to butanediol. The final solvent yields (g/l) and the process productivities (g 1-1 h-l) could be significantly improved by using enzymes concentrated by ultrafiltration. SSF, using the concentrated enzyme preparations, resulted in final solvent yields which were 40-68% higher than the yields obtained with conventional sequential hydrolysis and fermentation (Table 2), while the duration of incubation was significantly reduced 5'24'25. Under optimum conditions, solvent yields of 15.4 and 23.4 g/1 (or 65 and 86% of the theoretical conversion efficiencies) could be obtained in 3-5 days from 50 g/1 of xylan and solka floc, respectively5.

Technical a s s e s s m e n t of S S F The technical feasibility&using SSF to convert lignocellulosic residues to butanediol was first assessed using aspenwood xylan and solka floc as rep- Practical applications of S S F in resentatives of the hemicellulose and b i o m a s s conversion cellulose fractions respectively. A The feasibility of using SSF for Table 3. Solvent production from steam-exploded substratesby SSF Substrate Yields (g/lO0g of original substrate)

Theoretical conversion (%)

Aspenwood WS WI SE

1.9 8.8 23.0

34.1 48.9 65.4

Wheat straw WS WI SE

3.2 12.9 19.5

107.8 49.9 50.0

Barleystraw WS WI SE

0.4 9.4 13.8

8.2 38.2 38.9

Corn stover WS WI SE

1.1 9.5 11.7

36.4 54.3 42.9

-

64.8

Xylan

Solkafloc 85.9 WS, steam-explodedsubstrate,hemicellulose-richwater-solublefraction;WI, steam-explodedsubstrate, cellulose-richwater-insolublefraction;SE, steam-explodedsubstrate,unfractionated.

103

Trends in Biotechnology, VoL 3, No. 4, 1985 i

converting natural biomass substrates to butanediol was then examined. Earlier, k had been shown that steamexplosion was an effective pretreatment for facilitating enzymic hydrolysis of various lignocellulosic substrates2~t Steam-explosion, when combined with simple water-and-alkali extraction, resulted in the breakdown of wood and agricultural residues into a watersoluble hemicellulose-rich fraction and an alkali-soluble lignin-rich fraction leaving an insoluble cellulose-rich residue. Hemicellulose can be hydrolysed by dilute acid or by fungal xylanases, and cellulose by a more drastic acid treatment or by fungal cellulases. Each hydrolysate can in turn be separately fermented to butane-

WOOD or AGRICULTURAL RESIDUES

El STEAM-EXPLOSION 10% of steamed residues 90% of steamed residues

Enzyme Production

dio17,12,13,14,16

Alternatively, the two streams of carbohydrates can be separately converted to butanediol using SSF ~'24'25. We have subsequently shown that steam-exploded biomass containing both cellulose and hemicellulose carbohydrates, which has not been subjected to water-arid-alkali extraction, could be used directly for producing butanediol using the SSF process (Table 3). The final solvent yields exceeded the combined yields obtained when the separate cellulose and hemicellulose components were used 5 (Table 3). This work has resuked in a reassessment of the overall process. It is now possible to eliminate the fractionation of substrates, the concentration of the dilute hemicellulose sugars in the water-soluble extracts, and the separate hydrolysis and fermentation steps involved in cellulose and hemicellulose utilization. A simplified process scheme is therefore proposed, as illustrated in Fig. 4. Further simplification of the current SSF scheme has also been investigated. Since production of hydrolytic enzymes is a major expenditure 3° it is desirable to extend SSF to a co-culture system where a single reactor vessel can be used for enzyme production, enzymatic hydrolysis and subsequent fermentation of the substrates to useful products. This would then eliminate the steps necessary for separate production, harvesting, handling, sterilization and storage of the enzyme preparations. The technical feasibility of such an improved process was recently demonstrated using a sequential culture approach where K. pneumoniae was cultured with a cellulolytic fungus, T.

ENZYMES

[I Simultaneous Saccharification

BUTANEDIOL + ETHANOL

Fig. 4. A simplified process for converting biomass celluloseand hemicellulose to butanediol using SSF.

harzianum E58, or with a cellulolytic reeseO. To minimize the problem of bacterium, Clostridium thermocellum end-product inhibition, the cellulase (unpublished results). Further work is enzyme preparations used are usually required, however, before this supplemented by cellobiase enzymes approach can be better exploited for from other sources such as Aspergillus species. Although this procedure can commercial conversion ofbiomass. As a result of this work we can now provide some useful information, the use SSF for assessing the effectiveness ultimate goal of the pretreatment which of various pretreatment methods used is to use biomass substrates to form ferin processes involving enzymic hydro- mentation products, is not assessed. lysis and subsequent fermentation of Moreover, the use of hemicellulose lignocellulosic substrates. At present, sugars, another important aspect in biothe effectiveness ofa pretreatment pro- mass conversion, is not considered. cess is usually assessed by quantitating Using SSF, the direct conversion of the ease of hydrolysis of the cellulose pretreated biomass to fermentation component of the substrates by certain products can be assessed, including the 'standard' enzyme preparations (e.g. improvement in the hydrolysis of cellucellulase enzymes of Trichoderma lose and hemicellulose and the fermen-

104

Trends in Biotechnology, Vol. 3, No. 4, 1985

tation of the released hexose and p e n tose sugars to products. Together with the results on the recovery ofsubstrates following pretreatment, SSF can provide an assay for overall conversion yields, i.e. g of product per g of original untreated biomass substrates, which is the single most important criterion for determining the feasibility ofa biomass conversion processk Outlook T h e current global oil glut casts doubt on the economic viability of obtaining fermentation products from biomass substrates in the near future. However, the long-term outlook for producing butanediol from residues is more promising. The major hurdle appears to be the cost-effectiveness of production on an industrial scale. Recent economic studies 9 have suggested that biomass-derived butadiene from butanediol can completely displace butadiene derived from nbutane and n-butylene by conventional routes if the cost of producing butanediol can be reduced to 60% of the projected price of the petroleum-derived chemical. Other studies H, based on the production of polyurethane resins from butanediol derived from corn starch, have indicated that the biological route can compete with the conventional 1,4-butanediol process if the plants produce more than 18 million kg of product per annum. The economics could be drastically improved if cellulosederived glucose is used, since substrate costs could be reduced by two-thirds ~. Ifhemicellulose-derived sugars are also

used and a commercial use is developed for the lignin component, biomass conversion processes will begin to look even more attractive. Thus, converting lignocellulosic residues to butanediol is technically feasible. The range of substrates that can be used for making butanediol and the diversity of products that can be derived from it should make it a leading candidate as the first bulk chemical derived from lignocellulosic residues. References

1 Flickinger, M. C. (1980) Biotechnol. Bioeng. 22S, 27-48 2 Timell, T. E. (1967) Wood Sci. Technol. 1, 45-70 3 Yu, E. K. C. and Saddler, J. N. (1982) BiotechnoL Lett. 4, 121-126 4 Yu, E. K. C. and Saddler, J. N. (1982) AppL Environ. Mierobiol. 44, 777-784 sion of Cellulosic Substances into Energy, 5 Yu, E. K. C., Deschatelets, L. and Chemicals and Microbial Protein (Ghose, Saddler, J. N. Biotechnol. Bioeng. Symp. T. K., ed.), pp. 551-572, ITT, New 14, in press Delhi 6 Yu, E. K. C. and Saddler, J. N. (1983) 24 Yu, E. K. C., Deschatelets, L. and Appl. Environ. Microbiol. 46, 630-635 Saddler, J. N. (1984) Appl. Microb. Bio7 Yu, E. K. C. and Saddler,J. N. Proc. Biotechnol. 19, 365-372 mass Conversion TechnoL, Waterloo, 25 Yu, E, K. C., Deschatelets, L. and Ontario, Canada, in press Saddler, J. N. (1984) Proc. 5th Bioenergy 8 Long, S. K. and Patrick, R. (1963) in R & D Seminar (Ottawa, Ontario, Advances in Applied Microbiology Canada), Vol. 5, pp. 267-271 (Umbreit, W. W., ed.), vol. 5, pp. 26 Yu, E. K. C., Deschatelets, L., Tan, 135-155, Academic Press L. U. L. and Saddler, J. N. Biotechnol. Lett., in press 9 Palsson, B. O., Fathi-Afshar, S., Rudd, D. F. and Lightfoot, E. N. (1981)Science 27 Saddler, J. N., Brownell, H. H., 213, 513-517 Clermont, L. P. and Levitin, N. (1982) Biotechnol. Bioeng. 24, 1389-1402 10 Jansen, N. B. (1982) Ph.D. Thesis, Purdue University, USA 28 Saddler, J. N., Mes-Hartree, M., Yu, 11 Eur. Chem. News (1984) June 4/11, 19 E. K. C. and Brownell, H. H. (1983) Bio12 Yu, E. K. C., Levitin, N. and Saddler, technol. Bioeng. Symp. 13, 225-238 J. N. (1982) Biotechnol. Lett. 4, 741-746 29 Yu, E. K. C. and Saddler, J. N. (1984) 13 Yu, E. K. C., Levitin, N. and Saddler, Proc. Bioenergy Specialists' Meeting J. N. (1984) Dev. Ind. Microbiol. 25, (Waterloo, Ontario, Canada), in press 613-620 30 Wilke, C. R., Yang, R. D. and Von 14 Yu, E. K. C., Deschatelets, L. and Stockar, U. (1976) Biotechnol. Bioeng. Saddler, J. N. (1984) Bioteehnol. Lett. 6, Symp. 6, 155-175

Conserving the genetic heritage CROP GENETIC RESOURCES: CONSERVATION AND EVALUATION edited by J. H. W. Holden and J. T. Williams, George Allen & Unwin, 1984. £9.95 (pbk)/£20.O0 (hbk). (xvi + 296 pages) I S B N 0 04 581018 4 (pbk), 0 04 581017 6 (hbk)

It has been argued that the single most crucial stage in the evolution of human civilization was acquisition of the

327-332 15 Yu, E. K. C., Deschatelets, L., Levitin, N. and Saddler, J. N. (1984) Biotechnol. Lett. 6, 611 614 16 Saddler, J. N., Yu, E. K. C., MesHartree, M., Levitin, N. and Brownell, H. H. (1983)Appl. Environ. Microbiol. 45, 153-160 17 Saddler, J. N. (1982) Enzyme Microb. Technol. 4, 414-418 18 Bisaria, J. S. and Ghose, T. K. (1981) Enzyme Microb. Technol. 3, 90-104 19 Dekker, R. F. H. (1983) Biotechnol. Bioeng. 25, 1127-1146 20 Emert, G. H. and Katzen, R. (1980) Chem. Tech. Oct., 610-614 21 Saddler, J. N., Hogan, C., Chan, M. K.-H. and Louise-Seize, G. (1982) Can. J. Microbiol. 28, 1311-1319 22 Ghose, P., Pamment, N. B. and Martin, W. R. B. (1982)EnzymeMicrob. Teehnol. 4, 425-430 23 Takagi, M., Abe, S., Suzuki, S., Emert, G. H. and Yata, N. (1977) in Bioconver-

ability to breed plants. This allowed nomadic, hunter-gatherers to progress to a culture on fLxed agriculture and stable settlements. With fixed geographical location and more secure food supply, there were opportunities for development of architecture, education and scientific investigation. Since these early beginnings, mankind has exploited the genetic resources of crops and related wild species on an ever-increasing scale. The planet now

supports a population of the dominant mammalian species which is orders of magnitude above that possible for hunter-gatherers, and ever increasing demands are placed on agricultural productivity to keep up with the population growth rate which it has largely created. Two trends have been apparent in the history of plant breeding. There has been an increase in diversity, by harnessing in crop species genes for characters such as disease resistance from related wild species. In contrast, largely as a result of modern high-technology farming, there has been a trend towards narrowing of the genetic diversity of crops. International markets and the placing of emphasis on high yield