Process Development for Ethanol Production based on ...Process Development for Ethanol Production based on Enzymatic Hydrolysis of Cellulosic Biomass S. K. Tangnu" c The utilization

Process Development for Ethanol Production based on Enzymatic Hydrolysis of Cellulosic Biomass

S. K. Tangnu" c

The utilization of biomass to provide a source of liquid fuels, such as ethanol, requires the conversion of al l available sugars to ethanol.

This paper deals with the various physical and chemical pretreatments employed for the treatment of cellulosic materials, and then describes the production of cellulase in batch and continuous systems. This enzyme (cellulase), when contacted with pretreated material yields glucose, which is a starting material for the production of ethyl alcohol.

The performance of various processes used for alcohol production, including the immobilized'systems is compared. Economic analysis and suggestions for the future are detailed.

The cellulosic materials which can be util- ised in biomass production include grains, agricultural crop residues, processing wastes and energy crops. In general, biomass con- sists of cellulose, hemicellulose and lignin in the ratio of approximately 4:3:2. The net world production of biomass has been estimated a t 1841 X109 tonslyear, out of which cellulose production is 100 billion tonslyear. If biomass i s chosen to convert this annually replenishable resource to energy-rich products (Figure 1) that can be stored, shipped and used anywhere, then it is highly desirable to hydrolyse cellulose to primary glucose molecules.

The annual amount of solar radiation used by the earth's plant l i fe varies between 0.1-03% (3.2 X102'J energy i r l 2 X10" tons of carbon fixed per 3 X J of total radiation on the earth's surface per year). This production of fixed carbon i s ten times greater than the present consumption of energy.

Energy consumption in the United States has been estimated at 7 to 8 X lo6 Btulyear. This energy is obtained from oil (43%). gas (35%) and coal (19%). The United States, with 6% of the world's population, consumes one-third of the world's energy. To achieve the ultimate goal of energy independence, we will have to harness effectively and economically the inexhaustible energy of the sun.

Ethanol has received the most attention recently as a fuel source because: It can be produced from several surplus agricultural residues; alcohol production for fuel pur- poses is in itself not new, and conversion systems of biomass-toethanol are available; the fuel properties of gasoline-ethanol blends are often considered to be better

The author is with the Dept. of Chemical Engineering, University of California. Berkeley. CA 94720, USA.

Table 1. Effect of compression milling pretreatment on the enzymatic hydrolysis. of various cellulosic substrates.

Substrate Milling time (min.) 4h 24h

Total reducing sugar (mg mi-'

Pure celluloses Solka Floc (SW40)

Wastes Urban waste

Hardwoods Maple

Softwoods Eastern Spruce

Agricultural residues Corn stover

Sugar cane' Baggase

0 3

0 1.5

0 5

0 4

0 4

0 4

10.5 28.5

10.7 20.0

1.2 21.8

2.0 14.8

4.6 20.0

1.6 13.2

16.8 48.1

18.5 30.0

1.6 28.6

3.8 22.4

8.0 26.5

2.5 19.6

' T . reesei cellulase (QM 9414). 19 IU g-' substrate, pH 4.8,50°C, 5% substrate slurries.

than those of gasolinemethanol blends. merisation of the cellulose. A good pre- The purpose of this paper is to report, treatment must make material more acces-

step by step, the various processes for the sible to by the enzyme and must also be production of ethanol. economicai.

Physical Pretreatment: Large increases in enzymatic hydrolysis'-3 rates and yields

Pretreatment (Table 1) were obtained when newspaper Pretreatment of cellulosic materials is neces- and other substrates were subjected to the sary to enhance the overall enzyme kinetics compressive and shearing forces of steel of the cellulose degradation process, by rollers. Likewise, ball milled newsprint reducing the crystallinityanddegree of poly- (-200 mesh) resulted in a t least 84% carbo-

Process Biochemistry, May/June 1982 36

hydrate conversion. Delignified wheat straw, bell milled to < 105 mesh increased the conversion to 67%.

CHmical Pretreatment: Figure 2 compares enzymatic hydrolysis' of corn stover with acid treatment, acid treatment followed by enzymatic hydrolysis, and acid-base treat- ments followed by enzymatic hydrolysis. For acid treatment the milled agricultural residue was boiled in 5 wt96 suspension in 0.WM sulphuric acid for 5'Ah. For base treatment, the acid treated material was boiled for 3h at 100°C with 1 wt% ( 0 . 2 5 ~ ) .

HUMAN FOOD -

Tablo 2. Alkali treatment on previously acid oxtrwtd matorials

I CHFNICAL Material Ib NaOH used Alkali

Ib-' glucose used produced (%)

Barley straw 0.341 55.2 Corn stover 0.388 63.4 Cotton gin trash 0.696 50.2 Rice hulls 0.484 43.9 Rice straw 0.253 40.0 Sorghum straw 0.347 59.9 Wheat strew 0.320 57.6

It can be seen when comparing the glu- eosa yields (Figure 2) from enzymatic hydrolysis of acid-base treated materials with the acid pretreated alone, that there is an increase from 46.2 g glucose to 66.4 g glucose. The alkali treatment performed on the acid pretreated materials appears to remove lignin. Since the sodium hydroxide used per pound of glucose (Table 2) produced is excessive, the economics of this treatment are not favorable. For example, it is possible to produce (Table 3) 1 kg of glucose from 2.66 kg of corn s tow a t 100% efficiency. whrn enzymatically hydrolysing the acid h a treated material it is possible to pro- dum 1 kg of glucose, from 4.6 kg of sub- strata at an additional cost of 0.388 kg of $odium hydroxide.

Other procerses for the pretreatment of cellulosic materials are briefly described in Table 4.

cdlulpra production" The overall conversion of biomass to ethanol through !he enzymatic conversion of cellu- loso to gluco~e'~-*~ and the subsequent fermentation of the glucose syrups to ethanol hac been hampered by two economic 'bottlenecks' - the high cost associated with dolignification and enzyme Troduc- tionl"' .

It is possible to double the cellulase productivity by increasing the cellulose concentration1"" and controlling pH during growth''. Bacause of the resulting increase in enzyme activity, culture filtrates from 2.5% cellulose cultures can reduce the hydrolysis time for practical saccharification to one half that required by culture filtrates from 1.0% cellulose cultures.

Until recently. most of the process dwebpment work in this area utilized strain Tr&hodrmr. viride QM 9414 as a source of crNulaae for enzymatic hydrolysis. The dmlopment of hyper producing and cat* Wit# rsprrrrion mistant strain T. reesei

I ENZYME HYDROLYSIS

1 I-

RAW MATERIALS

s. c . P. FUEL

SOLVENTS

CHEMICALS

ANTIBIOTICS

ENZYMES

Figure 1. Conversion of cellulose to energy rich products.

H= 2.9 (7.2%) , P= 14.6 (81%)

Hexoses d0.1 Original Acid

Pentoses = Solid , 1 Treatment I Liquid

Lignin = 15.1 L= 3 .3

H = Hexoses

Sol id H= 11.1 (33.0%) I (16.13) *p= 1.8 (64%)

H= 33.0 P= 2.8 L= 1.7

L= 1.4 P= Pentoses' L= Lignin

Figure 2. Distributions of compounds as a result of the various pretreatments.

Rut C-3022, has led to a reevaluation of these processes.

The opportunity of improving enzyme productivities from the cellulose fermentation process has been examined". The maximum productivity obtained in batch and continuous (cell recycle) systems was 20.6 and 30.00 U I-' h-' respectively. The objec- tive was to demonstrate that the fed-batch process is more desirable than a normal batch process for production of cellulases. Slow addition of cellulose feed medium in feed-batch culture improved enzyme productivity by as much as 33%. In continuous culture it was possible to increase the productivity of the system by 15-20% through

recycling operations, and not by higher inlet feed cellulose concentrations.

Cultivation of Trichoderma reesei QM 9414 has been attemptedm on 3% (WlV) cellulose medium (C/N= 8.51, which produced 4.5 IU ml-' cellulase in 180 h a t a cell growth rate of 8.0 g I-' (0.266 g cell g-' cellulose). This corresponded to an average cellvlase productivity of 25.00 IU

(3.5 IU g cell-hl. In the same medium 9.5g I-' cell mass (0.316 g cell g-' cellulose), 6.2 IU ml-I cellulase, and 38.75 IU I-' h-I (4.0 IU g-' cell-h) cellulase productivity could be obtained using a pH cycling condition during cultivation. Cell mass. cellulase yield and productivity were

1-1 h-'

~ ~~

INSOLUBLE E N W G L U C W A S E S INSOLUBLE AND

C E L L W S E i SOLUBLE ( W 6 )

CELLOOLIGO-

SACCHARIDES

( D P p 6 ) 4

EXOGLUCANASES ( I N H I B I T I O N BY t - - - - - PRODUCTS OF

HYDROLYSIS ) - - - - CELLOBIOSE (Dp.2)

! I I 1 I I - - - - - * I B -GLUCOSIDASE!

I+ - - - - - G L U C O S E (DP=l)

ENDOGLUCANASESi G-G-G-0-G-G-0-G t CX

RANWM ACTION

EXOGLUCANASESi G-G-G-G-G-G F c 1

ENDWISE ACTION

-GLUCOSIDASES8 0-0 .F

0 4 HycxoLrsis OF C ~ L O B I O S E

TO GLUCOSE

Figure 3. Mode of action of cellulase.

further increased to 10.009 I-', 7.2 IU mi-' and 44.00 IU I-' h-' respectively, by simultaneous pH recycling and temperature pro- filing.

The biosyntheses of cellulase in a medium containing cellulose as the carbon source is greately influenced by the overall profile of the batch fermentation. A complex interaction exists between the medium composition, initial pH, inoculum size and condition, and aeration capacity. The formation of foam is one of the recurring problems when carrying out fermentations. A considerable loss of volume can also occur with changes in the agitation pattern of the fermentation. The modified inoculum build- up scheme" resulted in a faster rate of enzyme production and a decrease of foam during the fermentation. The various disadvantages of uncontrolled foam formation, which is a main consideration in the scale-up of fermentation, were resolved.

The initial lag i s common to all fermentations and can be controlled by the inoculum age and size. The initial rapid growth rate is due to the proteins in the peptone which are more available than cellulose. The ratio of carbon to nitrogen addition, and the

TIME

1 - XYLOSE

0

0 CELLOBIOSE I

8 16 24 TIME

Figure 4. Sugar concentration versus time.

form in which nitrogen is supplied, is of special significance, Ammonium sulphate and proteose peptone are the best nitrogen sources, as they increase the enzyme activity and cell mass and reduce the generation time, Addition of urea to the media resulted in an increase of C, and CX activities from the start of the fermentation, while filter paper activity was found to be low. The cellulase production of T. viride QM-9414 or T. reesei Rut C-30 is reduced unless Tween80 is added. For Rut C-30, 0.02% Tween80 concentration was optimum: higher concentration (0.1%) resulted in a decrease in the filter paper activities. By use of environmental control it is possible to pioduce an enzyme mixture with different ratios of FPA, C,, CX, p-glucosidase and xylanase.

Since T. reesai can provide excellent yields of p-glucosidase, production of p-glu- codiase by Awergillus niger, for example, as a supplement to the cellulase mixture or immobolizing 0-glucosidase on a carrier and conducting enzymatic hydrolysis using a recycle system, may not be necessary.

Table 3. Solids treated per unit wt. of glucosa produced

Original Acid Enz. hyd. of Enz. hyd. of Enz. hyd. of material extract original acid treated acid base (A) material material treated material

Barley 2.44 31 12.8 7.4 5.4 straw

Corn 2.56 34 8.5 5.9 4.6 stover

Cotton 5.00 143 17.2 15.4 11.5 gin trash

Rice hulls 2.77 20 17.5 20.8 11.5

Rice straw 2.44 26 5.7 4.7 4.2

Sorghum 2.77 38 9.1 6.7 4.2 straw

Wheat straw 2.74 27 11.2 7.3 5.5

(A) 100% Conversion Limit

38 . .

Agricultural residues such as corn stover, rice straw, and so on can be hydrolysed to sugars by a single enzyme preparation. Glucose can be fermented to alcohol by yeast, while xylose can be converted to alcohol by a variety of organisms, Fusarium oxysporum; 1.4 butandiol or acetic acidfdr example. Thus, the possibility of using mixed culture system for conversion of all sugars to ethanol may be an attractive option.

Table 5 provides a batch comparison of Rut 12-30 with T. viride OM-9414. The higher activities of the cellular enzymes and soluble protein in run 3 as compared to run 2 were primarily as a result of pH being allowed to remain 5.0 and not controlled (pH=5.0) a t 5.0. The release of ammonium ions when pH was allowed to freely rise above 5.0 may have affected the release of enzyme into the broth during the fermentation. With the increase in substrate concentration from 25 to 50 SI-' (runs 4 & 5). there was no substantial increase in cellulase activity, although soluble protein was twice higher. Thus, in the same fermentation time, using Rut C-30, as opposed to T. viride QM-9414 it is possible to produce cellulase enzyme of about three times the strength, which indicates the cost advantages.

When solka floc was used as the substrate for different strains, as shown in Table 6, only Rut C-30 was capable of growing and producing xylanase, while others needed xylan as the carbon source. These results suggest that xylanase and cellulase in the Rut C-30 are constitutive and adaptive enzymes.

Multi stage single stream production of cellulasez1 - l7 The typical batch fermentation process can be divided into two phases. In phase 1, most of the cellulose is consumed, with a corres- ponding increase in cell dry weight. The cellulase activity was half that of the total. During a rapid fall in pH or the consumption of (NH, Iz SO,, saccharifying and endo-

Process Biochemistry, May/June 1982

portion utilized most slowly. The dilution rate is thus considerably reduced thereby diminishing the productivity of t h e system.

Tabk 4. Various procacg for the pretreatment of cellulosic materials

Pretreatment Conditions Remarks

Nitric Oxide'

Explosive Steam Decompression6-'

Wet Oxidation Procers'"

Ozone'O

High temperature alcohol'"1z

5 parts nitric oxide per 100 parts of solid (20% moisture). Reaction time, 24 h a t 20°C.

Sequential mode of gas addition gave Mode ,,f action of cellulase better results. Overall enzymatic conversion of treated material was 60%. Native cellulose undergoing an attack by Extracted liauor was rich in xvlose. cellulase . . . exhibits extensive changes in

, . pnysicai properties prior to producing a measurable quantity of reducing sugar.

with sizable amount of lignin.

Woods chips heated with Urban waste lost 40% of i ts reactivity 550 Ibf/inz steam for 5-10 min. (poplar) and bagwe was improved by Then pressure was released by a factor of 11 and 6 respectively. dumping the whole load into Temperature and cook time are a large bin. responsible for conversion of pen-

toses to furfural. Yield of about 60% can be achieved by enzymatic hydrolysis.

a) Batch (Two-Stage Process) Liquid phase

Solid 4

Glucose yields of 45%. with a sugar concentration of about 4.6 wt% of treated material can be achieved on enzymatic hydrolysis.

Hydrolysis a t pH 2, 160" C, 225 Ibf/in" 60 min,

Further oxidation a t 180°C (2nd Stage)

b) Continuous

50% (VI alcohol on wheat straw at pH6 and 1850 C and 23OoC (for two samples).

Long chain structures of lignin and hem ice1 I u lose are attacked.

Enzymatic hydrolysis of treated wood at 185" and 23OoC yielded glucose and cellobiose of the order of 39.6 and 37.1% respectively. Final lignin content (original 14.5) was 12.8 and 3.7% at 185" and 230°C.

cellulase are induced. The rate of acid production was directly related to the rate of carbohydrate consumption. In phase 2 there was an increase in filter paper activity a t the expeme of the decrease in cell weight and substrate exhaustion. Autolysis and sporu- lation mre the main features of this phase.

Since both the phases cannot be carried out in a single fermentor, the importance of two stage single stream production of cellulase ww not ruled out. In first stage (or single stafp) "wash wt" can occur i f dilution rate (0) approeches the maximum. The inherent instability of feed devices and the sensitivity of the call maul X and the substrate S near "wash wt" to'slight changes in dilution rate, D, should be taken into accoua. I f the first stage i s operated below maximum then the problem of "wash out" does not arise. In that case it is possible to operate second s t q p at dilution rata above maximum, an inherent property of second stage and hence its importance in the continuous culture.

The two phase phenomenon and the nature of substrate, indicate that it is better to run a continuous fermentation in two sagsr for the following reasons: Cellu- lore is e multiple substrate with a metabo- l@n which is rapid only in the amorphous fmction. The crystalline fraction is meta- bdizdd very s(owly and only after consider- &@ Won to convert it to a more reactive fonn. I t ww assumed that moqt of the

amorphous and part of the crystalline cellulose will be consumed in the first stage, while the rest of the crystalline portion will be utilized in the second stage, provided cellulose concentration is not above 25 g I-' . If the same goal is to be achieved in a single stage continuous fermentation, the dilution rate should be adapted to the crystalline

- - These changes include fragmentation, swelling, considerable loss in tensile strength, transverse cracking and lowering of the degree of polymerisation.

Reesez* and his co-workers have proposed the so-called (C, -CX) hypothesis to explain the mechanism of cellulase action. The first component, C,, activates or deaggregrates the cellulase chains in preparation for attack by the next hydrolytic component of the cellulase complex. In the next step, enzyme CX hydrolyses soluble derivatives of cellulose. Later w ~ r k ' ~ ' ~ ~ has shown, that C, is a p-1;Qglucan cellobio- hydrolase, and thus acts on the chains formed by CX action (Figure 4). contrary to previous reports.

It is now well established that cellulase is a multicomponent enzyme complex, and that crystalline cellulase is hydrolysed by synergism3' of these cellulase components (Table 7).

Enzymatic hydrolysis Enzymatic hydrolysis of cellulose is attractive because of its specificity and absence of the competitive degradation which normally accompanies acid hydrolysis. Hydrolysis yields and rates are affected by substrate concentration and composition, pH, temperature, reaction time, extent and type of pretreatment, degree of agitation during the hydrolysis, enzyme source and i t s strength.

Effect of solid concentrationJ2. Figure 4a shows a reducing sugar concentration against time profile for hydrolysis, using cellulase of 5.2 IU ml-' activity and pretreated substrate (Corn Stover) concentrations ranging from 5-25% by weight. A total sugar concentration of up to 9% i s possible under these conditions. Figure 4b presents a break-

Table 5. Comparison of Rut C302 '~as and QM 941426~2'~46

Run So Strain FPA p-Glucosidase Soluble Remarks No. (g I-') (U I-' h-' ) (U I-'h-' ) protein

(mg mi-' )

1 50 Rut C-30 75.00 135.41 20 pH25.0, Tween-80

2 25 Rut C-30 27.00 52.08 8.2 pH=5.0, Tween -80

3 25 Rut C-30 38.00 65.10 10.3 pH25.0, Tween-80

=0.02%, 25OC

=0.02%, 25'C

=0.02%, 25OC (0-1 day) pH-allowed to fall to 4.0

4 50 QM9414 26.56 5.21 12.7 (1-2 day) pH-allowed to fall to 2.9 (2 day) pH raised to 3.3 and controlled not to go below pH 3.3

5 25 QM9414 22.4 6.25 5.9 Same as above

down of the sugar components correspond- ing to 25% solid loading. Approximately 80% of the sugar produced is glucose. Xylose concentration levels off to a value of 8 g I-' after approximately 8 h. Cellobiose under- goes maximum change early in the reaction and decreases to a low value after approximately 16 h.

Figure 5 shows the glucose yield for the hydrolysis process described above. Yields range from 60 to 43%. decreasing with increasing solid concentration. Cellulase with an activity of 7 IU ml-' gave total sugar concentration rates from 12-1 5%. and conversion rates from 7046%. Substrate nature and type of pretreatment: Steam pretreatment was found to be very effective with agricultural residues and hardwoods (Table 8 ) but not with softwoods or wastes derived from softwoods. Urban waste lost 40% of i t s reactivity as a result of steam pretreatment while poplar was improved by a factor of 11 and sugar cane baggase by a 6 fold factor.

If we compare steam pretreatment (Table 8) with compression (Table 1) milling pretreatment for corn stover and baggase it can be seen that milling gives better results. Table 9 lists the fermentation types available.

Enzyme source: Figure 6 shows the effect of different mutant strains on erlzymatic hydrolysis. For ball milled newspaper, it is apparent that the Rut C-30 enzyme is the most effective followed by MCG 77 and T. viride QM 9414 enzymes. However, other hydrolysis data using a high xylan containing chemical pulp and compression milled cotton indicate that the MCG 77 enzyme is the best followed by OM 9414 and Rut C-3033. Hence the cellulase enzyme com- plexes synthesized by these strains are different and this is reflected during hydrolysis on different substrates.

Cellulase of Trichodenne viride was concentrated in various molecular cut-off membranes by Ghose and Kostick". Flux rates and retention of activity were studied under ultrafiltration conditions. Little or no cellulase was discharged through the membranes wed. Theconcentrated (5 -8 fold) enzymes

C-30 A C T I V I T Y , 5.2 IU/ml

SUBSTRATEi ACID TREATED

CORN STOVER

10 20 30

SUBSTRATE CONC. (*.%I

Table 6. Comparison of xylan- producing strains

Run Substrate Strain Xylanase Remarks No. activity

(I U I-' h-' )

1. Solka Floc Rut C30 593.75 pH>5.0, temp.= 260C, proteose (10 g I-,) peptone=l .Og I- ' . Produced 90 IU ml-I

(469 I U I-' h-' ) xylanase activity when glucose was used as a substrate

2. Larchwood Streptomyces 538.9 pH 7.4, temp. = 30°C. xylan xylophegusa3 bactopeptone = 3g I-' (10 g I-' )

3. Larchwood Chaetomium 137.3 pH 7.0. temp = 28°C. xylan trilaterale24 y e w extract = 1 g I-' (10 g 1-1

were used to saccharify finely ground sub. strate (Solka Floc) in stirred tank and membrane reactors. Nearly 14% glucose concentration was achieved in less than 50 h in STR by digestinga 30% cellulose suspension. Based on experimental data a model system was proposed for the continuous steady state saccharification of ground substrate in which there was a continuous removal of concentrated glucose syrup, and feedback of enzyme.

In addition to the use of a conventional stirred tank reactor for hydrolysis, applica- tion of other processes like simultaneous saccharification and fermentation (SSF) (separate enzyme prod~ct ion)~~" ' mixed culture ~ y s t e m . 3 ~ - ~ ~ fixed packed bed reactor, 40-4a fluidized be&' and fluidized tampered bed has been attemp- ted by various workers.

Enzyme recovery4' : During the hydrolysis process, enzyme can be lost in two ways. Part of the enzyme is strongly adsorbed on unhydrolyzed cellulose and some of it remains in the solution containing glucose.

Methods of enzyme desorption" such as those using phosphate ion gradient on cellulose and/or using urea may permit greater enzyme recovery. Fresh solids can be brought into contact with the hydrolysate to adsorb some of the enzyme remaining

S U P P L E a W T p -GLUCOSIDASE ( I U h l L )

Figure 5. Glucose yield at 24 h versus substrate concentration.

Figure 6. Effect of different mutants on hydrolysis.

the solution. Workers a t Natick Laboratories have found that enzyme recovery is not economical.

Ethanol production Under anerobic conditions yeast liberates two moles of ethanol and carbon dioxide from every gramme of glucose consumed.-

C,H,,O, + 2C,H,OH+ (1 9) (0.460 g) 2C0, + Energy (stored as ATP) (0.460g COJ + 0.1 g new cells

However observed values are less than those shown in the equation above. Under aerobic conditions, glucose is converted to carbon dioxide and cell mass, with no ethanol being formed. Even though ethanol fermentation i s an anaerobic process, trace amounts of oxygen are required for biosynthesis. Recent

using Saccharomyces cerevisiae (ATCC 4126) has shown that an oxygen tension of 0.07 mm Hg i s optimal for ethanol production.

Fermentor ethanol productivities in both batch and continuous culture are limited by two factors4* : Ethanol inhibition and a low cell mass concentration. This is illustrated in Figure 7 for continuous culture. As the sugar feed concentration was increased, the specific ethanol productivity (g EtoH produced/g cells-h) decreased because more ethanol was produced a t high46 sugar concentrations and ethanol inhibition increased. At low sugar concentration, ethanol inhibition was less, but the cellmass concentration decreased. These two counterbalancing effects' produced an optimum fermentor productivity a t 10% sugar feed.

Table 7. Effect of cross-synargi~m~'

Component Relative cellulase activity (%I

Original solution 100 C, 1 CX 5 C' + cx 102 CMC-ase 4 Cellobiase 1 CMC-ase + cellobiase 2 C, +CMC-ase 35 C, + cellobiase 20 C, + CMC-ase + cellobiase 104

Tablo 8. E- of steam pretreatment on the enzymatic hydrolysis' of various cellulosic substrates

Substrate Pretreatment Total reducing sugar (mg ml-I 1 4h 24h

Waste Urban Waste None

Steam

Softwoods Eastern Spruce None

Steam

Hardwoods Poplar None

Steam

Agricultural residues Corn stover None

0.5 18.0 6.2 10.8

2.0 3.8 3.5 6.4

1.4 2.4 5.3 25.8

4.9 7.8 Steam 15.7 22.5

Baggase None 1.5 2.5 Steam 9.5 16.1

"T. reesei cellulase (QM 9414). 19 IU/gm substrate, 5% substrate slurries, pH 4.8, m0C, steamed substrates washed prior to enzymatic hydrolysis.

Ethanol fermentations have been operated batchwise, even though continuous fermentation would produce substantial savings by eliminating fermentor down- time between the batch fermentations. The two main reasons for continuous fermentations not having been used more extensively are firstly, the risk of possible mutations and, secondly, the problem of maintaining a high fermentation rate during continuous fermentation. Continuous ethanol fermentations have been maintained in the laboratory in excess of 60 days without any signs of deleterious mutations. During these extended experiments ethanol yield of over 90% of the theoretical yield was obtained, although the product did have a noxious taste.

Various process for alcohol production are listed in Table 10.

1979, in Japan, alcohol produ~tion'~ using S. Carlsbergensis cells, immobilised by entrapment into kcarrageenan gel (a poly- saccharide from seaweeds used as a non- toxic food additive) was studied. Gel beads containing a small number of cells were incubated in a complete medium. The number of living cells per ml of gel increased to ten times that of free cells per ml of culture medium. The generation time of cells in gel was estimated to be 3.0 h in the expo- nential growth phase, and this is similar to that of free cells in medium. Steady state operation of immobilised growing cells could be maintained a t a retention time of 15 min to 1 h (twice the washout of free cells), without a decrease of cell number in the gel. The amount of cells in the solution was lo6 to lo7 cells ml-' , while in the gel the level was maintained a t lo9 cells ml-' .

Trends in alcohol technology Processes based on immobilized living microbial cells have several advantages. Due to increased cell density (- loLa cells ml-' ) it is possible to: Achieve higher specific productivity; conduct operation on a continuous basis a t higher dilution rates without washout; and eliminate costly fermentor design and provide better and easier control of process.

When cofactors are required the use of cells is preferred to enzymes. The methods of immobilisation66 of yeast cells can be divided into three groups: lmmobilisation by adsorption, entrapment, covalent cross- linking.

Production of ethanol by yeast immobilised within a solid matrix has, primarily, three requirements that need to be considered: The immobilised conditions must be mild to ensure that the required activity is retained; if growth is allowed to continue there will be an undesirable concentration of free cells; and the matrix can be disrup- ted by CO, gas bubbles i f their diffusion is hindered.

Several research group^^*-^^ in different parts of the world are working on alcohol production using immobilised yeast cells. In

42

The volumetric productivity was ten timer that of the conventional ethanol fermenta-. tion. However, when a medium containing 25% glucose was fed, the growth of yeast cells in gel was inhibited. The new concentration of 114 gm I-' was achieved at e retention time of 2.6 h.

For large scale production, Chotani designed a fermentation process based on biologically active fluidised bed6'. In a fluidised bed reactor, gel immobilised yeast cells as films or colonies on inert particles are kept in relative motion. The accumula- tion of cells leads to particle distribution, wlth the largest, overgrown particles a t the top and the smallest a t the bottom of the column. In this process, an aerobic atmospheric (I) pressure fermentation stage pre- cedeLthe vacuum fermentor. The yeast are able to actively ferment during anaerobic conditions (11) in the vacuum fermentor after being aerobically grown in the pre- sence of essential nutrients.

Ghose and Bandyopadhyay" carried out continuous ethanol fermentation in an immobjlised yeast cell packed reactor using mollases syrup as the carbon source. With 30% reducing sugar, fermentation could be carried out within 12 h. However, the maximum productivity of 24.9 g l-'h-' WBS attained within a period of 2.86 h using mollases containing 19.7% reducing sugar. Considering the productivity data, this process appears to be superior to those of Divies" and Ghose and Tyag?' and com- parable with the process of Wick and Popper-. In this type of reactor CO, hold- up has been a problem and has resulted in a dead space in the reactor. Owing to gas hold-up, the substrate mean residence time is also less than the space time and this results in a decrease in the conversion rate.

Discussion If the sugar syrup produced from enzymatic hydrolysis of cellulose is to be used for

18

14

s : 4

v; 10

v 6

2

FERMENTOR

I I 1 1 1 I 1 I

4 8 12 16

PER CMT SUGAR IN FEED I Figure 7. Effect of glucose concentration on continuous fermentation.

Process Biochemistry, May/June 1982

athandl manufacture, then it is imperative that rasearch efforts be directed towards intagrating and optimiiing ethanol production with the other parts of the process.

Fedstuck: There is s t i l l a considerable dsbate on a number of issues relating to the production and usage of gasohol. One of the mOIt important issues is the availability and cost of feedstocks. While practically any biomass material can be readily converted to alcohol, the overall economics of each raw material will be governed by the extent of i ts availability and easa of conversion.

Energy production in terms of biomass production, harvest and transport and conversion to energy should be examined simultaneously with continual exchange of information. It is totally inadequate to simply state that there is a lot of biomass and that it has e high annual production. It is of vital importance to know exactly where tho biomass is, how much is in a particular area, if it is available and how much it will cost to deliver the biomass to tb energy conversion plant. It is worth noting that the socslled agricultural waste and residue am at pesent used as an organic material in the soil and if these materials are

diverted to alcohol production, the long term agricultural implications of this approach should be considered.

The development of plants that have high dry matter yields with a low lignin content to make them more biodegradable could significantly improve the energy production. Intensive cultivation of coni- ferous and broadleaved forest ecosystems could more than quadruple biomass production when compared to natural strands. The development of alternative substrates that are not part of the present food or feed supply chain would help the gasohol industry evolve successfully.

are 55% to 65% starch. A bushel (56 Ib) that is 60% starch will yield about 2.6 gal of alcohol weighing 6.61 Ib gal-' . Grain with a higher starch percentage will yield more alcohol and visaversa.The starch in potatoes has equal value to that in corn for making alcohol. A hundredweight of potatoes will yield about 1.4 gal of alcohol. Sugar beets can be processed to yield about 27 gal ton-' of alcohol.

The net energy gain is dependent on the energy required to both grow and process

Cereal grains such as corn and

the crop. One of the primary reasons why cereal grains do not provide the net energy returns of sweet sorghum and sugar cane is because of the lower production levels of fermentable sugar.

Although the studies of manioc, sorghum and babassu utilisation as raw materiaP present interesting long term possibilities sugar cane is the raw material that will allow the attaintment in short and medium term, of the goals set up by the Brazilian govern- ment. Being an old and traditional crop, sugar cane is linked with Brazil's economic history and is widely cultivated throughout the country on a large scale. The average national sugar cane production i s about 50 tons ha-', with three crops over a four year period. The cost breakdown of a 60000 I day-' ethanol plant from sugar cane shows that 65.2d constitutes the cost of raw material alone, when the total cost of alcohol is 1 1 5.3 USd gal-'. In the agricultural area work currently being carried out aims to select more productive plant varieties and to study the behaviour of known varieties, mainly in "cerrado" zones, with savanna type vegetation. With respect to manioc the first experimental plantations

Tabla 9. Alcohol fermentation process types

P- Remerks

C l d system (Batch)49

Overallso*'' productivity: I .8-2.5g ethanol/l-' h-' . High labour costs because of the continual start up/ shut down. Wil l need about eighteen 100,000 gal farmentors" for 25 million gal of alcohol per year. Capital cost will be approximately 518 million.

Overall productivity: -6 g ethanol I-'' h-' . Specific ethanol productivitys3 i s limited by ethanol inhibition. Economic analysis'O has shown a 50% reduction in capital costs and a 53% reduction in operating costs as compared to batch fermentation. Fermentor volumes required are 1/3

those required for batch process.

It has been shown that the productivity of a two- stage CSTR systemU is 2.3 times that of a single CSTR. Productivity of the first fermentor is high, because ethanol concentration is low. The second one is a lower productivity fermentor because it converts less sugar than if it was operated alone.

P r o d u c t i ~ i t y " ~ ~ ' ~ ' ~ ~ ~ and cell density are of the order of 30-40 g ethanol I-' h-' and 83 g I-' respectively. It is similar to CSTR fermentation system. except a centrifuge or cell settler is used to separate the yeast from product overflow and the return of yeast tg the fermentor.

Fermentor was continually sparged with air to maintain the oxygen tension a t a very low level, while in the reactorsWl1 no air was sparged and the oxygen tension was essentially zero. The fermentor would act as a continuous source of cells, wherws reactors (1) would beheve like fluidized beds with hetrogenous character. With this arrangement the residual glucose level was reduced to less than 0.3 g I-' at an inlet dilution rate of 0.3 h-' . The product concentration increased from 30 to 44 g I-' (when inlet sugar concentration was 100 g I-' as compared to the conventional single stage CSTR.

P r o d ~ c t i v i t y ~ ~ ' ~ ~ and cell concentration are 80 g "ation'"'%thanoI I-' h-' and 120 g I-' respectively. Vacuum

operation direct into the fermentor can lead to contamination end oxygen limitation. Pure oxygen

Open system (CSTR) continuous stirred tank fermentor

Multi-rtage CSTR5sn."

CSTR (RecycId4~" men-61

CSTR-Plug Flow (Racycle)6a

.

Vmum

(0.12 vlvlm) is sparged through the fermentor. Carbon dioxide produced during fermentation must be compressed up to atmospheric pressure. To keep the concentration of non-volatiles a t a level which did not inhibit yeast growth or ethanol production, a bleed of fermented broth was con- tinuallv withdrawn from the fermentor.

Flash Fermentation is carried out in an atmosphere Fermentation6' pressure and needs sparged air. To remove ethanol, (Figure 8) beer is rapidly cycled through a small auxillary

flash vessel where it boils and is collected in a separate vessel with the aid of vacuum. Contami- nation chances are reduced because the fermentation is no longer directly under vacuum, instead a small flask vessel serves the purpose. Like vacuum fermentation bleed of fermented broth is continually withdrawn. To remove ethanol, 3.5% ethanol beer is rapidly cycled between the fermenter and a small vacuum flask vessel where ethanol is boiled away and 2.5% (wt) ethanol beer is returned to the fermenter. Only the small amount of CO, dissolved in the cycling beer is carried into the flash vessel, which must be processed through the compressors. Because the ethanol concentration in the flash vessel must be maintained a t less than 3.5% wt% of that which is desired in the fermenter, the equilibrium amount of water carried overhead with the ethanol product is increased relative to that in the vaco-ferm process. Very l i t t le ethanol (less than 1 % of the total product) is carried away with the vented CO,, and this can be largely recovered by sparging the CO, back through the dilute beer solution in the fermenter. The costly absorber is thus eliminated. Ethanol manufacturing (exclusive of feed) is reduced to 6.97d gal-' as compared to 7.63d I-' for the vacu-ferm and 13.6d I-' for the batch process. The overall energy requirement (including feed sterilization and yeast product drying) i s 8.36 X IO6 J/L, which is reduced from the vacuum energy requirement of 1.09 X IO' J I-' but is s t i l l higher than the energy requirement for a batch process which requires no vacuum compressors. The super high productivities (BO g ethanol I-' h-') of the vacu-ferm process are maintained while many disadvantages are eliminated.

FEED

A I R * CARBON D I O X I D Z VACUUM

COMPRESSOR

I *DYpHANOL/

WAT EH

VAPOR

PRODUCT

FLASH

V E S S E L

%BEER RECYCLE 0.24

I+I H I G H COMPRESSION

S T E R I L E PUMP

A I R

Figure 8. Flash-Ferm process.

covering large areas have been started, using several soil types and varieties. With respect to banassu and sorghum studies, surveys and investigations are now going on for the purpose of gaining more reliable knowledge as to the farming productivites of known species.

Pretreatment: A good level of enzymatic hydrolysis could be obtained with ball milled materials. However, this type of milling does not appear to be practical for large scale processing due to prohibitive equipment and energy costs. Chemical pretreatment by 0.09M sulphuric acid of milled (2" Wiley) material, steam decompression and compression (two roll) milling are promising methods.

Cellulase Production: During the past three years promising new mutant strains have been developed at Natick laboratory and Rutgers Univelsity by exposing the wild strain (OM 6a) and the mutant OM 9414 to mutagenic agents such as nitrosoguanidine and ultraviolet light. These new mutants have been responsible for significantly higher enzyme activity and productivity. The highest filter paper activity obtained to date by Tangnu e t al" was from a fermentation on Solka floc BM-200 (50g I - ' ) with the Rut C-30 (Rutgers) mutant. However, a thermophillic and a constitutive mutant would be the ideal strain.

Most cellulase synthesis studies have been conducted with relatively expensive "pure" cellulose. From a practical stand- point a cheap cellulosic substrate must be sought for cellulase production. The refer- ence carbon source, solka floc, Avicel or lactose, are in fact too expensive for industrial production of cellulase.

Employing a single stream two-stage continuous system with cell recycle can cut the down-time and reduce the production costs of glucose. Tangnu e t a1 were able to operate such a system continuously for about 3'/* months at an overall dilution rate of 0.017 (dilution rate in the first fermentor was 0.02 while in the second it was 0.06h-'). If instead of T. viride QM 9414, Rut (2-30 mutant is used, then urea can be easily removed from the medium. The cost of the peptone (used commonly for cellulase production) is $1.40 Ib-' while that of CSL i s 1 . Id Ib-' . If the latter is used the cost or organic nitrogen source will be

44

ture system of yeastClostridium Thenno- cellum could be used.

Enzymatic hydrolysis is governed by the inhibition of an intermediate product (cellobiose) or the final product (glucose) formed during the process. A ratio of C,, Cx and pglucosidese in the mixture can make the reaction move forward a t a faster rate. However, glucose inhibition can be reduced either by simultaneous saccharification and alcohol fermentation or removing glucose from the system by a physical process such as ultrafiltration, reverse osmosis or molecular sieve membrane. Physical processes are governed by their own limitation espe- cially when dealing with bigger volumes. Another approach is to use a cellqase producing strain capable of producing concen- tratebugar solutions during hydrolysis.

reduced by It can be seen from this that type of strain and the nature of the organic nitrogen source can reduce the cost of enzyme production.

Enzymatic hydrolysis The results obtained suggest that cellulase in the Rut C-30 is an inducible enzyme, and that i t s formation is not controlled by so- called catabolite repression. This organism produced, together with higher cellulase activity, considerable amounts of xylanases. Thus, an enzyme mixture of Rut C-30 can conduct a simultaneous enzymatic hydrolysis of the cellulose and hemicellulose of an agricultural residue.

I f yeast is taken and fermentation carried out on such a mixture of su'gar solution then obviously xylose will be left in the aqueous phase after alcohol i s removed by distillation. In order that xylose and glucose can be fermented simultaneously with alcohol, Clostridium Thermosaccharolyticum should be used. Alternatively, a mixed cul-

Alcohol production Using qll recycle in conjunction with the vacuum system, ethanol productivities of almost 12 times have been achieved. Also, the production of a concentrated product (16 to 20% ethanol) could reduce distillation costs for the final recovery of ethanol.

The most energy intensive operation in the overall alcohol production process is the separation of alcohol and concentrating it to a purity level of 99.5%. By prudent recycling and proper design and operation of distillation columns, the energy requirement has been reduced to as low as 14 to 18 US gal-' of alcohol.

In conventional ethanol production from biomass feed two distillation steps are normally used. Vacuum distillation and vapour compression could replace the convectional thermal distillation step. The eight processes examined by Batelle could replace one or both distillation steps. It has been suggested that they could use as little as 13% of the energy that a convectional distillation uses.

An efficient immobilized growing yeast cell system with provision for removing alcohol inhibition could result in better economical ethanol production in the near future.

Table 10. Suaar production cost

cost Milling Acid Hydrolysis Enzyme Enzyme Total pretreatment recovery make-up

Fixed capital 3.375 5,150 8,684 1,937 10,261 29.407 X l O o o 8

Annual capital X l O O O 8

Annual labour 96 191 191 96 191 768 X l O O O 8

Annual utilities 109 451 657 62 x 1000 8

Annual material 1,238 51 X l O O O 8

Annual manufacture 1,015 3,116 2.697 623 6,623 14.100 X l O O O 8

Glucose, d lb-' 0.72 2.21 1.91 0.44 4.69 10.00

810 1,236 1,798 465 2,463 6.771

809 2,088

3,106 4,452

Process Biochemistry, MaylJune 1982

. , T.M. 11. Ethanol production cost Table 12. Cost of sugar produeion cellulase QM 9414

against Rut C-30 at various com stover cost

Processing cost distribution d gal-' % Total

Corn stover cost: $0 Ton-' 825 Ton-' 350 Ton-' 95% EtoH

Sugar concentration 5.2 2.9 Fermentation 7.6 4.2 Distillation 3 .O 1.7 Sugarcost QM9414 14.9 22.9 30.9

3 - - s B s :: E 2 0 :

16

12

10

Medium chemicals Glucose

of Californie/Lawrence Berkeley Laboratory Report 7879.. Sep. 1978.

6. Personel communication, J. Noble, lotech Corporation, 220 Laurier Avenue West, Ottawa, Ont; Canada.

7. Galloway, D., Pulp b Paper. 1975, 104. 8. Schaleger. L. L., and Brink. D. L.. TAPPI, 1978, 65, 61

9. Bicho, J. G., end Brink, D. L.: Sanitary Eng. Report,

10. Pelloni, L.. Brown-Boveri Co. Ltd., Zurich, CH 8060,

11. Kleinert. T. N., TAPPI, 197437, 8, 99. 12. Wilke, C. R., Univ. of California, Rerkeley/Lawrence

Berkeley Report 6860. June, 1977. 13. Wilke. C. R.. and Mitra, G.. "Cellulose as e chemical

and energy resource.: 1975, 5, C. R. Wilke, Ed. p. 253. (New York: Interscience).

14. Wilke. C. R., and Yang. A. D., Proceedings of the

0 25DON (1 978).

1971, 71-1, ppl.

Switzerland.

J/ -

-

- -6 I I I I 1

21.4 135.9

0 10 20 30

SUBSTRATE CONCMTRATION (hT.$)

12.0 75.7

symposium on enzymatic hydrolysis of cellulose, M. Bailey, T. M. Enari,andM. Linko. Eds. 1975.(Aulanko, Finland: SITRA). p.485).

15. Wilke, C. R., and Yang, R. D.. J. Appl. Polym. Sci.

(d 1b-l) C30 10.5 14.6 18.7

Methane generation 6.3 3.5

179.4 100.0 % Difference 30 36 40

shorter period of time; reducing the cost by having a simultaneous cellulase and saccharification step in one reactor; using less energy during the distillation step by processes like

Economic assessment The cost analysis is based on a plant with a manufacturing capacity of 10 x106 gat year-' of 95%fuel grade ethanol. A material balance of the plant shows that each day 1376 tons of cellulose waste containing 58% glucose equivalent is supplied. Using 7 IU ml-' of cellulase (from batch), 40% of the substrate is hydrolysed to fermentable sugars (1 2-1 5%) which in turn are converted by yeast to ethanol in 46% yields. Contin- uws countercurrent recovery of the remaining enzyme is accomplished by adsorption on fresh pretreated corn stover. Following filtration, spent solids from the hydrolyser are fed to furnace and steam power plant to provide steam and electricity for the process.

The economic analysis based on this, as rummarised in Table 10, suggests a manu- ficturingcostof sugar of 10.0d Ib-' .

Modification of the enzyme production operation by using a single stream two stage system and involving use of cell recycle system (cell recycle fraction=0.70) can l o w the fixedcapital cost to approximately 24 million dollars and the sugar cost to 7.314 Ib-' .

Using the glucose cost in Table 10, the processing cost and fixed capital distribution for ethanol production are given in Table 11. The predominant portion (76%) of the final ethanol coot is due to glucose cort Every 1.06 Ib-' of glucose cost contri- butes 13.6% gal-' to ethanol cost. When using a continuous two stage system with call recycle system for cellulase production the glucose cost decreases from lO.0d Ib-' to 7.314 Ib-'. In other words the alcohol cost could be expected to be 3% gal-' less. Y e w cake (from alcohol production) and mycellium (from cellulase unit) assumed to have a market value of 13.3d and 24.7d Ib-' respectively may together reduce the ethanol cost by a further 26d gal-' .

Figure 9 is a sensitivity analysis+ the cost of producing sugar at various corn stover colts. The minimum production costs shift to lower substrate concentration, as the prim of corn stover is increased. This i s due to higher yields under these conditions.

Table 12 gives a comparison between the lowest manufacturing cost obtained using celhrlllre from Rut C-30 and that obtained from QM 9414. Processing with Rut C-30 n#tltt in a cost w i n g of 3040% over that M n e d with QM 9414.

If the corn stover is free then the cost of alcohol per gallon is equal to 51.80. I f a 8in0I0 stream two stage continuous system With cell recycle system is used, the cost is tWuCd by 3U; and if credit for mycellium

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Conclusion Industrial ethanol production in the United States is presently kept low by the petro- chemical industry. To produce one gallon of ethanol by fermentation, 12.88 Ib of sugar are required. To synthesize one pallon of ethanol from ethylene, four pounds of ethylene are needed. Based on materials costs along, the price of fermentable sugars must be reduced to approximately one-third the price of ethylene which is 10 to 20 d Ib-' .

Technoloaical asDects which must be

Proceedings of the Bioconversion Symposium.T. K: Ghose, Ed. 1977 (Delhi. India: In). p.249.

19. Sternberg. 0.. Riotechnol. Rioeng., 1975, 18, 1751. 20. Wilke. C. R.. and Blanch, H. W., Lawrence Berkeley

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Riotechnol. Rioeng., 1981, 23, 1837. 22. Montencourt. 8. S.. and Eveleigh. 0. E., "Advencesin

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23. Tangnu, S. K., Blench, H. W., and Wilke C. R.. Acta Riotechnologia, 1980. 1. 31.

24. Tangnu, S. K., Blanch. H. W., and Wilke C. R., "Retch Multistage Production of Xylanases by Cheetomium trilaterale", to be published.

26. Tangu S. K.. Blanch. H. W.. Wi1ke.C. R.,"Kineticsand Dvnamics of Cellulase Production bv Rut C-30. -. to be - published.

and Muhistage single stream Production of cellulase by T. viride QM 9414': to be published in Riotechnol.

taken into account to achieve a viable

of an effective and economical pretreatment economical process include: ~~~~l~~~~~~ 26. Tangnu. s. K.. Blanch H. W.. and Wilke. C. R., "Ratch

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hydrolysis; development of a constitutive and thermophilic mutant for cellulase pro-

27. Wilke. C. R.. and Blanch, H. W., Lewrence Berkeley Laborerory, California, Report 9989. 1980.

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30. Li, L. H.. et. SI.. Arch. Riochem. Riophys., 1965, 11 1, 439.

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33. Allen. A. L., and Blodgett. C. R.. Proceedings of 87th

continued on page 4.9

. . . - extraction fermentor in which the ethanol is removed from the medium by a specific extraction ~ubstance.3~ Recently, the appli- cation of immobilized yeast cells in continuous fermentation procedures was investigated. The immobilization of cells may be obtained for instance by their embedding in a gel matrix or by covalent binding at specific

All procedures for biotechnological ethanol production have one disadvantage: the concentration of the end product in a solution i s only between 6 and 20 ~01%. Albeit the energy quotient of the procedure is positive: it is however decreased by the distillation required to obtain the end product. For instance: from 100 kg pure starch one may only obtain 54 kg of 95.6 ~01% ethanol with an energy value of 1400 MJ. For the distillation 1040 MJ are needed: this leaves a gain of energy of only 360 MJ."

The end concentration of the ethanol in the fermentor is of essential importance with regard to the energy needed for distillation. In order to concentrate a 25 vol% ethanol solution one needs only half of the energy which is required for the concentration of 10 vol% solution.?

Furthermore, ethanol may be concentrated by distillation up to a maximum of 95.6 ~01%. In this range of concentration ethanol forms a binary azeotropic mixture with water, the boiling point (78.15"C) of which is lower than that of absolute ethanol (78.3"):' The production of alcoholus absolufus is only possible by the exploita- tion of a ternary azeotrope which one may obtain, for instance, by addition of benzol to the ethanol-water mixture. Following distillation of this azeotrope the ethanol-water ratio in the gas phase is modified (7.5 % H,O, 18.5 % ethanol, 74 %

benzol) so that pure alcohol remain^.^" In conclusion, Figure 3 is a flow diagram

which summarizes the single steps leading from the substrate to the product.

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