11
Saccharification of Concentrated Brewing Bagasse Slurries with Dilute Sulfuric Acid for Producing Acetone- Butano1 by Clostridium acetobutylicum Josep Juanbaro and Lluis Puigjaner" Biotechnolog y Group, Departament dlEnginyeria Quimica, ETSEIB, Universitat Politecnica de Catalun ya, 647 Diagonal, 08028, Barcelona Accepted for publication November 25, 1985 A comprehensive kinetic study of the acid hydrolysis of concentrated brewing bagasse slurries was performed. The use of the simple series reaction model was found to be suitable when a "heterogeneous correction" (pseudo- substrate-inhibition) is taken into account in slurries with low liquid-to-biomass ratios. Rate constants are shown to be dependent not only on temperature and acid con- centration but essentially also on the initial biomass con- centration. Actual rate constants, activation energies, and acid and substrate reaction orders are reported for xylan, arabinan, and a-glucan acid saccharification. There is a threshold acid loading necessary to overcome the 80% conversion, but no threshold has been found to over- come the "neutralizing" property of cellulosic materials. Reversible acid capture from brewing bagasse has been postulated. The highest monosaccharide concentration into hydrolyzates has been found (65 g/L)after 10 h treat- ment, but economic considerations led us to treat a mean- concentrated slurry (156 g/L) with 0.3M H2S0, at 96"C, thus obtaining 45.5 glL monosaccharides in 5 h with 50% less furfural content. After pH regulation only, growth of Clostridiurn aceto butylicurn has been o bta i ned, a It h ou g h complete sugar consumption has not been achieved. Ex- periments are now underway to reach complete diges- tion and to investigate the increase of enzymic accessi- bility into residual substrate rich in cellulose. INTRODUCTION Agricultural by-products containing cellulose, hemi- celluloses, and lignin, if available in large amounts at a low price, appear to be some of the most relevant raw materials for producing renewable fuel, food, and chemical feedstocks.'-' Enzymatic hydrolysis of the cellulose is one of the most promising processes for converting these by-product^,^,^ but its performance is restricted by the chemical structure itself and cellulose association in nature with hemicelluloses and lignin,6 and therefore sugar yields thus obtained are generally * To whom all correspondence should be addressed. low unless pretreatments are Hemicelluloses have less crystalline and more branched structures than cel- lulose, and the glycosidic linkages are less stable and more readily hydrolyzed by acid than the linkages in cellulose.s A pretreatment with diluted acids extracts hemicelluloses a s p e n t ~ s e s , ~ - ' ~ thus improving the cel- lulase accessibility and glucose yield in the subsequent enzymatic h y d r o l y s i ~ . ~ ~ ~ ~ ' ~ ~ ' ~ Hemicellulosic sugars comprise about one-half the total carbohydrate that can be obtained from Iignocellulosic raw materials.2.16 While hexoses and glucose in particular serve as the most suitable substrates for microbial activity, pen- toses are somewhat less favored by microorganisms.I6 Unlike a great number of yeasts such as Saccharo- myces cerevisiae strains, many bacteria are capable of using pentoses. Specifically, Clostridium acetobutyli- cum strains are capable of utilizing pentose sugars to produce n-butanol with acetone and, to a lesser extent, ethanol." Considering that the cost of the conversion of bio- mass to sugars and solvents is highly dependent on the cost of b i o m a ~ s ~ , ' ~ we have selected a lignocellulosic agroindustrial by-product that has an insignificant cost in most countries: residues of beer manufacturing, oth- erwise named brewing bagasse. Brewing bagasse rep- resents, on a dry basis, about 30% of the solid raw materials used in brewing, and therefore it is free from expenses derived from collection. Additionally, this by-product presents serious problems of storage in the factory itself since it consists of a medium that is very suitable for the development of lactic acid bacteria, whose activity may easily contaminate the brewing process. Therefore, brewing bagasse has been chosen as an appropriate biomass source for producing n-bu- tanol, a cosolvent used in the blending of gasohols. l9 The first step considered in the conversion process has been dilute acid hydrolysis of brewing bagasse as Biotechnology and Bioengineering, Vol. XXVIII, Pp. 154-1554 (1986) 0 1986 John Wiley & Sons, Inc. CCC 0006-3592/86/10154411$04.00

Saccharification of concentrated brewing bagasse slurries with dilute sulfuric acid for producing acetone–butanol by Clostridium acetobutylicum

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Saccharification of Concentrated Brewing Bagasse Slurries with Dilute Sulfuric Acid for Producing Acetone- Buta no1 by Clostridium acetobutylicum

Josep Juanbaro and Lluis Puigjaner" Biotechnolog y Group, Departament dlEngin yeria Quimica, ETSEIB, Universitat Politecnica de Catalun ya, 647 Diagonal, 08028, Barcelona

Accepted for publication November 25, 1985

A comprehensive kinetic study of the acid hydrolysis of concentrated brewing bagasse slurries was performed. The use of the simple series reaction model was found to be suitable when a "heterogeneous correction" (pseudo- substrate-inhibition) is taken into account in slurries with low liquid-to-biomass ratios. Rate constants are shown to be dependent not only on temperature and acid con- centration but essentially also on the initial biomass con- centration. Actual rate constants, activation energies, and acid and substrate reaction orders are reported for xylan, arabinan, and a-glucan acid saccharification. There is a threshold acid loading necessary to overcome the 80% conversion, but no threshold has been found to over- come the "neutralizing" property of cellulosic materials. Reversible acid capture from brewing bagasse has been postulated. The highest monosaccharide concentration into hydrolyzates has been found (65 g/L) after 10 h treat- ment, but economic considerations led u s to treat a mean- concentrated slurry (156 g/L) with 0.3M H2S0, at 96"C, t h u s obtaining 45.5 glL monosaccharides in 5 h with 50% less furfural content. After pH regulation only, growth of Clostridiurn aceto butylicurn has been o bta i ned, a It h ou g h complete sugar consumption has not been achieved. Ex- periments are now underway to reach complete diges- tion and to investigate the increase of enzymic accessi- bility into residual substrate rich in cellulose.

INTRODUCTION

Agricultural by-products containing cellulose, hemi- celluloses, and lignin, if available in large amounts at a low price, appear to be some of the most relevant raw materials for producing renewable fuel, food, and chemical feedstocks.'-' Enzymatic hydrolysis of the cellulose is one of the most promising processes for converting these by-product^,^,^ but its performance is restricted by the chemical structure itself and cellulose association in nature with hemicelluloses and lignin,6 and therefore sugar yields thus obtained are generally

* To whom all correspondence should be addressed.

low unless pretreatments are Hemicelluloses have less crystalline and more branched structures than cel- lulose, and the glycosidic linkages are less stable and more readily hydrolyzed by acid than the linkages in cellulose.s A pretreatment with diluted acids extracts hemicelluloses as p e n t ~ s e s , ~ - ' ~ thus improving the cel- lulase accessibility and glucose yield in the subsequent enzymatic h y d r o l y s i ~ . ~ ~ ~ ~ ' ~ ~ ' ~ Hemicellulosic sugars comprise about one-half the total carbohydrate that can be obtained from Iignocellulosic raw materials.2.16 While hexoses and glucose in particular serve as the most suitable substrates for microbial activity, pen- toses are somewhat less favored by microorganisms.I6 Unlike a great number of yeasts such as Saccharo- myces cerevisiae strains, many bacteria are capable of using pentoses. Specifically, Clostridium acetobutyli- cum strains are capable of utilizing pentose sugars to produce n-butanol with acetone and, to a lesser extent, ethanol."

Considering that the cost of the conversion of bio- mass to sugars and solvents is highly dependent on the cost of b i o m a ~ s ~ , ' ~ we have selected a lignocellulosic agroindustrial by-product that has an insignificant cost in most countries: residues of beer manufacturing, oth- erwise named brewing bagasse. Brewing bagasse rep- resents, on a dry basis, about 30% of the solid raw materials used in brewing, and therefore it is free from expenses derived from collection. Additionally, this by-product presents serious problems of storage in the factory itself since it consists of a medium that is very suitable for the development of lactic acid bacteria, whose activity may easily contaminate the brewing process. Therefore, brewing bagasse has been chosen as an appropriate biomass source for producing n-bu- tanol, a cosolvent used in the blending of gasohols. l9

The first step considered in the conversion process has been dilute acid hydrolysis of brewing bagasse as

Biotechnology and Bioengineering, Vol. XXVIII, Pp. 154-1554 (1986) 0 1986 John Wiley & Sons, Inc. CCC 0006-3592/86/10154411$04.00

a pretreatment of the cellulosic fraction to be later enzymatically hydrolyzed and subsequently as a means of extracting the hemicellulosic fraction. Very often, proposed flowsheets of plants to convert biomass to solvents either include costly evaporation units for concentrating diluted hydrolyzates7 or simply do not consider the economic aspects of this dilution in sol- vent r e ~ o v e r y . ’ ~ . ’ ~ It can be inferred that an increase in sugar concentration in the hydrolyzates will reduce the fermentor size and energy requirements for sub- sequent recovery of solvents. One approach for in- creasing the sugar concentration of the hydrolyzate is to carry out the hydrolysis in a concentrated slurry.I0 On the other hand, it is necessary to achieve the highest conversion of the pentosans to pentoses, which means complete conversion of the pentosans with the least decomposition to furfural, thus reducing at one and the same time the volume-of-reactor-to-biomass-treated ratio. Finally, it is essential to determine the optimal hydrolysis conditions for the specific vegetable mate- rial used, since major variations occur in the hydrolysis rate of the specific glycosidic bonds present as well as in the stability of the actual monosaccharides released.l2

The studies reported here were conducted to deter- mine, for the brewing bagasse, the best conditions to attain both purposes: the highest sugar concentration in hydrolyzates and the most satisfactory conversion of brewing bagasse, which may be in turn fermented by C . acetobutylicum to obtain n-butanol. Hemicel- lulose acid hydrolysis was carried out considering (1) different liquid-to-biomass ratios, (2) various dilute sul- furic acid concentrations, and (3) an appropriate range of temperatures at atmospheric pressure.

MATERIALS AND METHODS

Substrate

Fresh brewing bagasse containing lignocellulosic residues from brewery was supplied by DAMM, S.A. (Barcelona) in a wet, hot form immediately after fil- tering a malt slurry heated to 74°C for extracting and hydrolyzing starches of germinated barley grains. The wet brewing bagasse was packed into plastic bags and then frozen and stored at -20°C. Dry matter content was determined by drying samples at 60°C (71% mois- ture) in an oven provided with a blow-through system.

Brewing bagasse is basically composed of ground husks of malted barley grains and other fractions re- tained during the brewing process, and contains, in a dry basis, about 5 1% carbohydrates (18% cellulose, 25% hemicellulose, 7% starch, 1% soluble sugars), 15% lignin, 23% protein, 7% fat matter and 4% mineral matter.20

The dry brewing bagasse was screen analyzed in a Retsch Testing Sieve Shaker using a series standard screens of 0.090, 0.120, 0.177,0.250, 0.400,0.500,0.750,

r s

10 2.0 3.0 Particle Diameter / m m

Figure 1. bagasse used in the experiments.

Accumulative diagram for the screen-analyzed brewing

0.999, 1.200, 2.000, and 2.800 mm. An accumulative diagram was obtained from screen analysis (Fig. l), and the mean diameter was determined to be 0.980 mm (+ 16 mesh). This relatively small size of particle in the raw material avoided a pretreatment by milling that would have weighed heavily on the process costs with- out adding substantial improvement to the overail conversion.

Experiments were carried out with the dried brewing bagasse. The moisture content was determined, and appropriate corrections were made to all concentra- tions used.

Dilute Sulfuric Acid Hydrolysis

Hydrolysis experiments were carried out in a I-L refluxed stirred tank reactor with 300 mL dilute sulfuric acid (0.1, 0.2, 0.3, and 0.4M concentrations) at differ- ent temperatures under atmospheric pressure (80, 90, 96, and lOOOC) using samples with 15.2,29.5,47.4,58.1, and 67.1 g (dry basis) of brewing bagasse, i.e., working at low liquid-to-biomass ratios (19.7, 10.2,6.4,5.2, and 4.6 mL/g, respectively). Once measured out, the brew- ing bagasse samples to be treated were added quickly to the 300-mL preheated H2S04 at zero time of the hydrolysis.

Hydrolysis kinetics were monitored by withdrawing

JUANBARO AND PUIGJANER: ACETONE BUTANOL BY C. ACETOBUTYLICUM 1545

5-cm3 aliquots at different time intervals and immedi- ately cooling them on ice. Hydrolyzates were centri- fuged at 7500 rpm for 30 min to remove the residual substrate. Sugar analysis was conducted on the trans- parent supernatant fluid to determine the rate of release of individual sugars.

Bioconversion of Hydrolysis Sugars by C. acetobutylicum

Clostridiurn acetobutylicurn ATCC 824 was main- tained on 38 g/L Oxoid RCM (Reinforced Clostridial Medium) solutions. Cultures were grown for a week at 37°C and, once sporulated, stored at 5°C.

From a test tube containing C. acetobutylicurn spores, a 10-mL sterile RCM aliquot was inoculated and grown for 32 h. Thereafter 5 mL of the latter was inoculated into 50 mL sterile brewing bagasse hydrolyzates. Fi- nally, 500 mL bagasse hydrolyzate in a 1-L New Bruns- wick fermentor was inoculated with the 50-mL culture after growing for 24 h. Anaerobic conditions were ob- tained by degassing the contents of the inoculum tubes by immersion in boiling water for 15 min and by flush- ing the fermentor contents with sterile N2 (laboratory grade).

Acid hydrolyzates were prepared for fermentation as follows. Two 79-g (dry basis) samples of brewing bagasse were hydrolyzed with 500 mL 0.3M H2S04 at 96°C for 5 h in a 1-L refluxed stirred tank reactor. Hydrolyzates were centrifuged at 7500 rpm for 30 min to remove the residual substrate and then combined to obtain 800 mL transparent fluid. The initial pH of the sugar solution was 0.9. The solution was adjusted to pH 7.0 with solid calcium hydroxide, centrifuged again to remove precipitated calcium sulfate, and adjusted to pH 6.0 with acetic acid. Sterile conditions were obtained by heating sugar solutions at 75°C for 45 min in a thermostatic bath.

End Product Analysis

Sugar analysis was done by HPLC (Waters, with Refractive Index detector) without neutralizing or re- moving acid.]' Clear samples were injected into a Waters Sugarpack column at 90°C using a 20-pL loop and Milli- Q water as the mobile phase at a flow rate of 0.5 mL/min. To calibrate the HPLC unit, standard solutions were injected and amount-to-area ratios were found using a C-R2AX Shimadzu integrator. Furfural could also be determined by this procedure.

Solvents (butanol, acetone, ethanol) and acetic and butyric acids were determined by gas chromatography (Dani, provided with flame detector) following the ad- dition of a 0.1-mL 50% HCl aliquot to 2 mL clarified samples. Acidified samples of 1 p L from anaerobic fermentation were injected into a 80/100 mesh Pora- pack column using N2 at a flow rate of 30 mL/min and

column temperature programming (160°C for 340 s to 190°C for 700 s using a gradient of 30°C min-I). As above, amount-to-area ratios were found by using stan- dard solutions and an automatic integrator of the same kind.

RESULTS AND DISCUSSION

HPLC sugar analysis of samples withdrawn from a refluxed stirred tank reactor (RSTR) showed three main monosaccharides (xylose, arabinose, and glucose) be- sides different amounts of sugars with DP 2 2 and furfural in varying concentrations. The xylose and arabinose appearance was attributed to hemicellulose decomposition, the glucose to starch saccharification, and the furfural as an oxidation product from pentoses, primarily xylose.

Since all polysaccharide decomposition into their constituent monosaccharides may be represented by

(Ct,,), + (n - 1)H2O nCm [rn = 5 (pentosans), 6 (hexosans)] and since n + m,

(1)

the substrate composition in hexosans and in pento- sans will be, respectively, 0.90- and 0.88-fold the po- tential hexose and pentose contents. Experimental data obtained from acid hydrolysis allowed us to fix the dry basis composition of brewing bagasse at 6.8% starch, 7.7% arabinan, 16.9% xylan, and 0.2% free glucose. Therefore, potential glucose, arabinose, and xylose yields used in all calculations were taken to be 7.6, 8.8, and 19.2 g/100 g brewing bagasse, respectively.

then n 2- n - 1

Extent of Acid Hydrolysis

Figure 2 shows the releasable sugar yield as a func- tion of time for different temperatures at a liquid-to- biomass ratio of 6.4 mL/g (or 156 g/L solids concen- tration) and a sulfuric acid concentration of 0.1 M. At this low ratio the results given in Figure 2 show a strong effect of temperature.

Monosaccharide yields are presented in Figure 3 as a function of time for different acid concentrations at a liquid-to-solid ratio of 6.4 mL/g and at 96°C. For these conditions results given in Figure 3 show two important effects of acid concentration, i.e., when the acid con- centration is increased: (1) the rate of hydrolysis in- creases significantly and (2) the hydrolysis yield in- creases to a certain extent. When acid concentration goes beyond 0.1 to 0.2M, there is a substantial increase in the monosaccharide yield, attaining a value that re- mains practically constant even at higher acid concen- tration (from 0.2 to 0.3M) or even falling slightly at 0.4M.

Figure 4 shows the effect of liquid-to-biomass ratio on monosaccharide yields at 96°C and 0.1M sulfuric acid concentration. Experimental data indicate a sharp

1546 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 28, OCTOBER 1986

loot

o t I I I I I

80!

Ybc

I I I I I

0 20 40 60 80 t / h

Figure 2. temperatures (liquid-to-biomass ratio of 6.4 mL/g)

Releasable sugar yield as a function of time at different

lO0l------

80

60

LO

040 M 20 0 030M

A 0.20 M A O.1OM

I i I 1

0 10 20 30 40 t / h

Figure 3. Monosaccharide yield as a function of time for different acid concentrations at a liquid-to-biomass ratio of 6.4 mL/g and 96°C.

100, 1 1

Figure 4. at 96°C and 0.1M H,SO, concentration.

Effect of liquid-to-biomass ratio on monosaccharide yield

decrease in hydrolysis yield when the slurry goes to higher concentrations.

At present, the results shown in Figures 3 and 4 seem to be dependent on the same parameter. In the first case we have kept constant the substrate concentration varying the acid concentration and in the second, keep- ing constant the acid concentration, we have varied the substrate concentration, to obtain qualitatively analogous results in both cases. It appears that the hypothetical common parameter must be related to the acid loading lo or the acid-to-biomass ratio.20 In Figure 5 releasable sugar yield is presented as a function of acid-to-biomass ratio and, at the same time, as a func- tion of acid concentration for different hydrolysis times at a liquid-to-biomass ratio of 6.4 mL/g and at 96°C. Figure 6 also presents the releasable sugar yield as a function of acid-to-biomass ratio and liquid-to-biomass ratio for different hydrolysis times at a sulfuric acid concentration of 0.1M and at 96°C. From the analysis of both figures we can state that the effects of acid loading (r) are only quantitatively comparable at low r values. When working at 0.1M H,SO, concentration, an increment in acid loading results in an increasing yield, and then time lines do not cross; that is, an increase of acid loading and an increase in hydrolysis time will lead to a 100% releasable sugar yield. On the contrary, an increase in acid loading, when keeping

JUANBARO AND PUIGJANER: ACETONE BUTANOL BY C. ACETOBUTYflCUM 1547

Acid Concentration / rnol.L-' 0.30 0.40

1:s /ml;g-'

0 0.10 0.20 030 0 0.10 0-20 0-30 Acid Loading, r / gH so . g-l Acid Loading, r / g 9 -l 2 4 brewing bagasse H2% brewing bagasse

Figure 5. Releasable sugar yield as a function of acid-to-biomass ratio and acid concentration for different hydrolysis times (liquid-

Figure 6 . Releasable sugar yield as a function of acid-to-biomass ratio and liquid-to-biomass ratio for different hydrolysis times (0. IM

to-biomass ratio of 6.4 mL/g, 96°C). HZSO,, 96°C).

the liquid-to-biomass ratio at 6.4 m Wg, eventually leads to a maximum yield well under 100%. Then there is a slight loss in yield, which increases at prolonged hy- drolysis times. Our initial assumptionz0 that at higher biomass concentration higher acid concentration is needed to reach the same yield, i.e., thus maintaining the threshold value on acid loading, seems to be in- correct. This would be valid if monosaccharides did not experience decomposition reactions to furfural or hydroxymethylfurfural, both with rate constants di- rectly dependent on bulk acid concentration.2-2' This acid concentration effect can be observed in the falling sugar yield between two sets of experimental data with practically the same acid loading ( r ) : 19.6 mL/g ( r =

0.192) and 6.4 mL/g ( r = 0.185) but at 0.1 and 0.3M H2S04 concentration, respectively.

Therefore, we can assume as a first approximation that a minimum acid loading value exists over which we can reach a maximum releasable sugar yield higher than 80% and at a rate increasing with increasing acid concentration as long as the sulfuric acid concentration is less than 0.4 molar. This minimum value, indepen-

dent of substrate concentration, can be taken at 0.10 g H2S04/g brewing bagasse.

Although acid loading seems to play an important role in acid hydrolysis,i0 from present results we can state that at low liquid-to-biomass ratios initial sub- strate concentration and H2S04 molarity individually influence the hydrolysis kinetics of brewing bagasse polysaccharides.

Mild Acid Hydrolysis Kinetics

Kinetic models suggested for polysaccharide hy- d r ~ l y s i s ~ ~ , * ~ and for h e m i c e l l ~ l o s e ~ ~ ~ ~ ~ may be sum- marized as follows:

h r ( C A or C , - C,,,

(2) (polysaccharide) (monosaccharide)

D (degradation products)

!.1 +

where D represents furfural for C5 or hydroxymethyl- furfural for C,. Although the hemicellulose acid hy-

7 548 BIOTECHNOLOGY A N D BIOENGINEERING, VOL. 28, OCTOBER 1986

drolysis reaction model suggested by Mehlberg and Tsao” includes three steps between hemicellulose and xylose branches (oligomers formation), Chambers’s ~implification’~ reflected in equation (2) has been con- sidered sufficient in the present work. All researchers depict the acid hydrolysis process as a homogeneous pseudo-first-order sequential process2:

d d t - (D) = YzkzC,

(4)

The Arrhenius form of rate constants in these equa-

(6) where preexponential factors are usually reported as a function of acid concentration C raised to some power m,:

and where activation energies are independent of acid concentration and temperature.26

If the hydrolysis analysis is limited to short reaction times, i.e., neglecting monosaccharide degradation, the above set of equations can be reduced to equation ( 3 ) . This is a good approximation when the hydrolysis con- version is limited to 10-20%. Equation ( 3 ) can be in- tegrated and rearranged to

tions can be expressed as

k, = Ai exp( - Ei/RT)

A, = AFC”! (7)

ln(1 - X ) = - k , t (8)

10

F

I \ 1 x- U

t

v) t 0 0

CI 0 0.1

2! 0.01 z 0.001

I I I o ARABINOSE 0 XYLOSE A GLUCOSE

369 363 353 Temperature, T/K

Figure 7. Plot of the kinetic constant ( k , ) vs. the inverse of tem- perature ( l / T ) to obtain the preexponential factors (0.1M H2S04, 156 g/L slurry concentration): 0, arabinose; 0, xylose; A , glucose.

Table 1. H2S04 molarity and 156 g/L brewing bagasse concentration.

Polysaccharide Preexponential factor Activation energy

Kinetic parameters for polysaccharide hydrolysis at 0.1

j A , 0 - l ) E l , (10’ calimol)

Xylan 1.49 x 1023 41.1 Arabinan 4.29 x 11.3 a-Glucan 3.04 x 10” 22.0

where X is the polysaccharide conversion. By plotting ln(1 - X ) vs. t , a straight line will result and its slope will be the kinetic “constant” kI.’O Note that this con- stant is actually a function of temperature and acid concentration.

From HPLC data and using equation (8), we have obtained all kl for glucose, arabinose, and xylose re- lease for all hydrolyses carried out at specific condi- tions of temperature and acid and substrate concen- trations. Figure 7 represents the plot of In k , vs. 1/T [eq. (6)] for obtaining preexponential factors at O.1M sulfuric acid concentration and 156 g/L slurry as well as activation energies for xylan, arabinan, and a-glucan decomposition into their corresponding monosaccha- rides. Table I summarizes the values obtained. Com- parable activation energy data with that found in the literaturez6 for the case of xylan as well as new infor- mation about activation energies for arabinose and glu- cose release from hemicellulose and a-glucan can be noted. Moreover, the values obtained for the reaction activation energy E (in all cases greater than 10 kcalhole) indicate that a chemical reaction is the con-

100

7

10 \

x-

u 1 b U c v) c 0 0

2 0-1 8

0.01

1 I 1 1 1 1 o ARABINOSE 0 XYLOSE A GLUCOSE - -

I 1 I r l I 0.1 05 1.0

Acid Concentration, c/rnd-l! Figure 8. Logarithmic plot of kinetic constant k , vs. acid concen- tration C for obtaining the acid reaction order. (Legend for symbols is as in Fig. 7.)

JUANBARO AND PUIGJANER: ACETONE BUTANOL BY C. ACETOBUTYLlCUM 1549

Table 11. and 156 g/L brewing bagasse concentration.

Kinetic parameters for polysaccharide hydrolysis at 96°C

I I

P I o ARABINOSE LZ 10 - 0 XYLOSE -

5 1 E

\

x- -

- I k 0 0

b w a, 0-1 U

0-01

Table 111. and 0.1 H,S04 molarity.

Kinetic parameters for polysaccharide hydrolysis at 96°C

rate constants found from equation ( 3 ) and those pre- dicted by using equation (10).

Therefore, the heterogeneous correction postulated in equation (10) can be assumed to be a pseudo-sub- strate-inhibition, and it can be asserted that acid load- ing is not a characteristic parameter as suggested by Horwath et al.,’Oexcept when mlJ + nlJ = 0. Although kinetic parameters k, have been found as a classical function of acid concentration and temperature, their strong dependence on initial substrate concentration has also been demonstrated. In fact, K, is presented as an actual kinetic constant only dependent on the nature and structure of substrate.

The “neutralizing” property of biomass has also been found, as described by Horwath et al.,” to be due to surface hydroxyl group, but no threshold acid loading,

Polysaccharide Preexponential acid Acid reaction j factor el (h-l) order rn,

Xylan 8.36 2.06 Arabinan 169.7 2.29 a-Glucan 2.89 2.00

Preexponential Polysaccharide polysaccharide Polysaccharide

factor KiI (h-I) reaction order nI, j

Xylan 31.34 - 1.93 Arabinan 19.21 - 1.86 a-Glucan 2.43 - 1.93

trolling mechanism. Figure 8 shows the plot of In k l vs. In C, as equations (6) and (7) suggest, for obtaining the reaction order of acid concentration, mi. Table I1 summarizes values obtained from regression between both variables.

The preceding work represents the results obtained from a typical treatment of polysaccharide acid hy- drolysis by studying the kinetics at only one substrate concentration. From the analysis of Figure 4 we can state that the initial rate of monosaccharide release decreases when the initial substrate concentration is increased. Figure 9 demonstrates this effect by plotting In kl vs. In CQ, where CQ is the initial individual poly- saccharide concentration in the acidic brewing bagasse slurry under mild conditions of acid concentration and temperature (0.1M and 96°C). The value of C:, can be obtained as

(9)

where CO,, is the initial brewing bagasse concentration

c;, = co,,-p, 0

and p,” is the initial polysaccharide percentage. Param- eters of resulting straight lines have been compiled in Table 111.

Therefore, it can be inferred that acid hydrolysis kinetic constants of monosaccharide release are ex- ponentially dependent on temperature and also on acid concentration as well as on initial polysaccharide con- centration. This triple dependency has been summa- rized in

k IJ = K IJ .C(mIWo(niJ)*eXp( XJ - EIJ/RT) (10)

or

k ~ j = KIJ-ZIJ (1 1)

Typical regressions obtained between k, and 2, for xylan, arabinan, and a-glucan saccharification have demonstrated an exact proportionality by plotting kIJ vs. Z,, where the slopes are K,, with the independent terms of straight lines being practically zero. Table IV

1550 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 28, OCTOBER 1986

Table IV. Kinetic parameters for xylan, arabinan, and a-glucan in sulfuric acid hydrolysis.

Polysaccharide Actual constant Acid reaction Polysaccharide reaction Activation energy j K , W') order nil, order n, , El, (lo3 ca lho l )

Xylan 0.875 x loz8 2.03 Arabinan 0.828 x lo1' 2.29 a-Glucan 0.300 x loB6 2.00

- 1.93 - 1.86 - 1.93

41.1 11.3 22.0

capture takes place but in no case as in irreversible adsorption.

Monosaccharide Concentration in Hydrolyzates

Experimental data showed the feasibility of achiev- ing a practically 100% conversion when less concen- trated slurries are hydrolyzed at O.1M sulfuric acid concentration, where the presence of decomposition products was found to be practically zero for a long period of time (Fig. 10). When slurry concentration was increased, a maximum was maintained at over 80% conversion by increasing acid concentration at the same time. The highest total monosaccharide concentration (65 g/L) was achieved by using 191 g/L slurry (Fig. 1 I ) , where losses were attributed to monosaccharide degradation. When a similar acid-to-biomass ratio was maintained but slurry concentrations increased to 220 g/L (Fig. 12) or when slurry and acid concentrations (191-220 g/L and 0.3-0.4M) were maintained but tem- peratures increased to 100°C (data not shown), a dra- matic loss in sugar yield due to formation of degra- dation products was observed.

All observations are well explained from the se- quential process represented by equations (3), (4), and (5) when equation (10) is taken into account. Decom- position reactions of xylose and glucose have been

investigated and kinetic constants (m2, E2) are reported elsewhere.2'.26 Thus, from equation (4), when acid con- centration is relatively low, monosaccharide concen- tration increases sharply for a short period of time remains constant thereafter (Fig. 10). An increase in temperature leads to an increase in monosaccharide concentration (Fig. 2) as well as an increase in furfural concentration (data not shown) because of the similar activation energies for monosaccharide release and by- product formation. At higher substrate concentrations the rate of monosaccharide release decreases (Fig. 4) and furfural concentration also decreases (data not shown). However, an increase in acid concentration increases both the monosaccharide release rate and monosaccharide degradation. Thus, when bulk mono- saccharide concentration is increased (Fig. I I ) , the in- crement in the degradation kinetic constant due to acid concentration leads to a rapid monosaccharide dis- appearance.

It is shown in Table VI how shorter hydrolysis times will lead to both lower monosaccharide and furfural yield. The latter should be considered in the necessary trade-off between such critical parameters as temper- ature, initial substrate, and acid concentration and the residual hemicellulose associated to cellulose to be treated thereafter by enzymatic hydrolysis. Therefore, by selecting slurry and acid concentrations as well as

Table V. Comparison between experimentally determined and predicted polysaccharide kinetic constants from brewing bagasse sulfuric acid hydrolysis.

Xylan kinetic Arabinan kinetic Glucan kinetic constants (h-l) constants (h-') constants (h-I)

Brewing bagasse Sulfuric acid Hydrolysis concentration concentration temperature Observed Predicted Observed Predicted Observed Predicted CO,, (dL) C (mol/L) t ("C) k IJ k*, k l J kX, k I, k*,

51 98

156 156 156 156 156 156 191" 220 220"

0.10 0.10 0.10 0.10 0.10 0.20 0.30 0.40 0.30" 0.10 0.40"

96 96 80 w 96 96 96 96 96" 96 96"

0.445 0.147 0.00605 0.0237 0.0804 0.299 0.764 1.299 0.334 0.0219 0.464

0.626 0.176 0.00564 0.0283 0.0713 0.291 0.661 1.119 0.449 0.0370 0.615

6.701 1.518 0.42 1 0.644 0.851 4.546

10.347 20.826 6.488 0.747 9.450

6.557 1.926 0.401 0.625 0.807 3.945 9.881

19.285 6.876 0.424

10.240

0.226 0.0573 0.00687 0.0167 0.0267 0.141 0.246 0.441 0.206 0.0128 0.224

0.244 0.0685 0.00710 0.0169 0.0278 0.111 0.250 0.444 0.170 0.0144 0.230

a Hydrolysis whose measured kinetic constants are left out of regression analysis.

JUANBAR0 AND PUIGJANER: ACETONE BUTANOL BY C. ACETOBUTYLICUM 1551

20

too

s

I! c 5= 'L 10 2 L 2 50 $ a, r/, EI a, z

U TOTAL MONOSAC. 0 ARABINOSE

XYLOSE D GLUCOSE

15

\ '-I

\ a,

0

- CT X FURFURAL

c 0

0 V

5 3 d

0 0 0 20 40 60

t / h

Figure 10. Releasable sugar conversion vs. time at 0.1M sulfuric acid concentration. Symbols: 0, total monosaccharides; 0, arabi- nose; 0, xylose; A, glucose; x , furfural.

80

60 - 2 \ c 0

0

c a, u c 0 V

._ CI

40

20

0

r I 1 1

0 TOTAL MONOSAC.

100 o ARABINOSE XYLOSE

A GLUCOSE x FURFURAL

s

9 jr:

E 50 5

\

al

b D

al 3

- H

0 0 20 40 60

t /h

Figure 11. Releasable sugar conversion vs. time using a 191 glL slurry. (Symbols are as in Fig. 10.)

loo '

0 200 400

8 rewi ng 8agass e Concentration/ g - C' Figure 12. Total monosaccharide concentration as a function of brewing bagasse concentration.

appropriate temperatures, an optimum can be reached with minimum hydrolysis times and minimum concen- trations of degradation products. This optimum should benefit the overall yield at the fermentation stage.

Growth of C. acetobutylicum on Neutralized Acid Hydrolyzates

Fermentations have been carried out on neutralized sugar syrups as described above. Hydrolyzates from acid hydrolysis at 0.3M sulfuric acid concentration and using 156 g/L brewing bagasse at 96°C after 5 h have been selected because of the short hydrolysis times and small amount of furfural present (0.5 mg/mL after neutralization). Furfural has been described as possi- bly toxic for growing C. acetobutylicurn. 17,*' The me- dium contains 8.8 g/L glucose, 24.7 g/L xyiose, and 12.0 g/L arabinose.

Results are shown in Table VII and compare with those obtained by Volesky and Szczesny17 using a me- dium of monosaccharides (50 g/L), ammonium sulfate (9.6 g/L), ferrous sulfate (0.25 g/L), Difco yeast extract (7.5 g/L), calcium phosphate (0.22 g/L), and magne- sium sulfate (1 .O g/L).

From these results we can affirm that no toxic sub- stances are present in high enough concentration in the neutralized sugar syrup to completely arrest the fer- mentation. These substances did inhibit complete glu- cose utilization. Conversely, by comparison with Vo- lesky and Szczesny's results,I7 problems may be due to relatively low initial glucose concentration com- pared to high initial pentose concentration.

7 552 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 28, OCTOBER 1986

Table VI. polysaccharide conversion and furfural concentration at 96°C for r = 0.10 g H2S04/g brewing bagasse.

Highest monosaccharide concentrations obtained from different hydrolysis cases with regard to minimum time for reaching the

Initial Highest Minimum substrate Acid Acid monosaccharide hydrolysis Polysaccharide Furfural

concentration concentration loading concentration time conversion concentration ~ B B (dL) C (mol/L) r (gAigBB) c!L (g/L) ti: (h) (%I c, (dL)

51 0.10 0.192 17.8 20 98 98 0.10 0.100 28.8 20 83

156 0.20 0.123 47.0 7.5 85 I56 0.30 0.185 45.5 5 82 156 0.40 0.247 45.6 10 82 191 0.30 0.151 65.0 10 96 220 0.40 0. I74 57.5 7.5 73

0.25 0.65 0.50 0.50 2.05 0.90 1.20

Table VII. Results from C. acetobutylicum growth on neutralized hydrolyzates compared with data taken from literature.

Acids Initial sugars mix Sugars utilized Solvents produced produced

( d L ) ( m d U Total Total Glu Xyl Ara Total But. Ace. Eth. ~

Glu Xyl Ara (g/L) (%) (%) (%) (%) (dL) (%) (%) (%) Acet. Buty. Reference

8.8 24.7 12.0 11.6 25.5 60 16 19 1.3 72.1 19.5 8.4 2095 2565 This work 5.0 45.0 - 11.6 23.2 100 14.6 - 1.6 65.1 30.0 4.9 2937 2710 12

CONCLUSIONS

The use of the simple series reaction model was found to be suitable for describing the acid hydrolysis kinetics of brewing bagasse. Thus, the Arrhenius form of the rate constants in correlating the effect of tem- perature where preexponential factors have also been demonstrated to be a function of acid concentration raised to a power can be taken into account. Never- theless, the acid hydrolysis process can only be de- picted as a homogeneous pseudo-first-order sequential process when heterogeneous correction (pseudo-sub- strate-inhibition) is taken into account in slurries with low liquid-to-biomass ratios. Therefore, a new de- pendency has been found for rate constants besides temperature and acid concentration: initial biomass concentration. Actual rate constants are reported for xylan, arabinan, and a-glucan acid saccharification as well as their activation energies and acid and substrate reaction orders.

There is a threshold acid loading necessary to over- come the 80% conversion (0.10 g H2S04/g brewing ba- gasse), but no threshold has been found to overcome the “neutralizing” property of cellulosic materials. Re- versible acid capture from brewing bagasse has been postulated.

The highest monosaccharide concentration in the hydrolyzates has been found (65 g/L) from 191 g/L slurry after 10 h treatment with H2S04 0.3M at 96”C, keeping a high conversion and a low furfural content. Economic considerations of treatment time have led us to propose a concentrated slurry (156 g/L), treated

as before, for obtaining in 5 h a hydrolyzate containing 45.5 g/L monosaccharides and 50% less furfural con- centration than the case cited above so that the hy- drolyzate can be neutralized and inoculated with C . acetobutylicum.

Without adding anything else, other than pH regu- lators, C . acetobutylicum growth has been obtained, although complete sugar consumption has not been achieved. Interactions will have to be determined be- tween the relatively low glucose contents and the pres- ence of some inhibitor of the solvent production phase. Experiments are now underway to reach complete digestion and to investigate the increase in enzymic accessibility of the residual substrate, which is rich in cellulose.

We are grateful to DAMM S.A. for providing brewing bagasse free of charge.

NOMENCLATURE

preexponential factors (h- ’) sulfuric acid concentration (mol/L) initial brewing bagasse concentration (g/L) furfural concentration (g/L) monosaccharide concentration or the selfsame mono- saccharide (g/L) highest monosaccharide concentration reached at time

pol ysaccharide polysaccharide concentration or the selfsame polysac- charide (g/L) initial polysaccharide j concentration (g/L)

ti: ( d L )

JUANBARO AND PUIGJANER: ACETONE BUTANOL BY C. ACETOBUTYLlCUM 1553

degradation product concentration or the selfsame prod- ucts (mol/L) activation energies (cal/mol) experimentally determined rate constants (h- I )

predicted rate constants (h-]) actual rate constant for polysaccharide j in reaction 1 (h-I) preexponential acid factor for pol ysaccharide j in reac- tion 1 (h-’) preexponential substrate factor for polysaccharide j in reaction 1 (h-’) acid reaction orders substrate reaction orders for polysaccharide j in reac- tion 1 initial percentage of polysaccharide j in brewing bagasse acid loading or acid-to-biomass ratio (g/g) universal gas constant (calimol K) time (hPC) minimum hydrolysis time for reaching concentration C2 (h) absolute temperature (K) polysaccharide conversion

Subscripts 1 reaction i, i = 1 (monosaccharide formation) and i = 2

(monosaccharide degradation) polysaccharide j , j : xylan, arabinan, a-glucan j

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1554 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 28, OCTOBER 1986