Enzymic saccharification of alfalfa fibre after liquid hot water pretreatment

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Process Biochemistry 35 (1999) 33-41
Enzymic saccharification of alfalfa fibre after liquid hot water
Hassan K. Sreenath a,*, Richard G. Koegelb, Ana B. Moldes c,d, Thomas W. Jeffriesc,
Richard J. Strauba
a Department of Biological Systems Engineering, University of Wisconsin, Madison, WI 53706, USA
b Dairy Forage Research Center, US Department of Agriculture, Madison, WI 53706, USA
c Forest Products Laboratory, Forest Service, US Department of Agriculture, Madison, WI 53705, USA
d Chemical Engineering Department, Universidade de Vigo, Campus de Ourense, As Lugaos 32004, Ourense, Spain
Received 25 November 1998; received in revised form 8 February 1999; accepted 15 February 1999
Liquid hot water (LHW) at a high temperature has been advocated as a pretreatment for herbaceous and lignocellulosic
materials prior to enzymic saccharification. The focus of our research was the suitability of LHW pretreatment of alfalfa
(Medicago saliva) fibre in the presence and absence of mild acid for optimum saccharification using enzymes. Enzymic
saccharification was optimised in terms of substrate and enzyme concentrations. The main components of the enzymic
hydrolyxates were sucrose, glucose, xylose, arabinose, and low amounts of uronic acid. After LHW pretreatment, both the soluble
hemicellulose-rich ‘extract’ fraction and the insoluble residue ‘raffinate’ fraction (which contains predominantly cellulose) of
alfalfa were recovered; the yield was 48 and 41%, respectively. Enzymic saccharification released more reducing sugars from
pretreated fibre than from untreated fibre. For untreated alfalfa, a commercial pectinase and cellulase mixture caused maximum
release of reducing sugars; using 2 and 4% (w/v) enzyme, a maximum of 51 g/l reducing sugars was released from 100 g/l untreated
alfalfa. The soluble extract, LHW-pretreated alfalfa, was clarified and saccharified with the same cellulase and pectinase mixture
releasing 8.4 g/l reducing sugars from 15.5 g/l solids. Release of reducing sugars from the insoluble raffinate ranged from 59 to
65 g/l from 100 g/l substrate, using 2 and 4% (w/v) cellulase. Addition of 0.07% sulphuric acid to the LHW pretreatment
facilitated hemicellulose solubilisation; 9.9 g/l reducing sugars were released from the alfalfa extract by enzymic saccharification.
However, pretreatment with sulphuric acid reduced sugar release and decreased fibre degradation of the raffinate. © 1999 Elsevier
Science Ltd. All rights reserved.
Keywords: Untreated alfalfa; Liquid hot water; Raffinate; Extract; Cellulases; Pectinases; Saccharification
1. Introduction
Alfalfa is a vigorous and productive farm crop that
makes excellent ruminant feed [1]. However, its use has
¶The Forest Products Laboratory is maintained in cooperation
been limited because ruminants are able only partially
with the University of Wisconsin. This article was written and
to digest the fibre component of the herbage. These
prepared by US Government employees on official time, and it is
fibres mainly consist of celluloses, hemicelluloses,
therefore in the public domain and not subject to copyright. The use
lignin, and small amounts of pectin and proteins [2].
of trade or firm names in this publication is for reader information
The potential biotechnological importance of alfalfa
and does not imply endorsement by the US Department of Agricul-
biomass has been increased by the production of low
ture of any product or service.
fibre juice-derived co-products such as particulate
* Corresponding author. Present address: USDA Forest Service,
Forest Products Laboratory, One Gifford Pinchot Drive, Madison,
(chloroplastic) protein concentrates, soluble protein
WI 53705-2398, USA Tel.: + 1-608-231-9409; fax: + 1-608-231-
concentrates, carotenoids, vitamins, minerals, plant and
animal growth factors, pharmaceutical agents, cosmetic
E-mail address: [email protected] (H.K. Sreenath)
products, and transgenic enzymes [3-5].
0032-9592/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0032-9592(99)00029-1

H.K. Sreenath et al. /Process Biochemistry 35 (1999) 33-41
The major lignocellulosic or fibrous fraction of al-
2.2. Enzymes
falfa can be used directly as fuel either by direct com-
bustion or by first gasifying and then burning the gas.
Commercial enzyme preparations of pectinase (SP-
However, there is a great deal of interest in utilising
249) and xylanase (Pulpzyme HC) were procured from
and converting the lignocellulosic fraction as feedstock
Novo (Franklinton, NC). Cellulase (Multifect B) was
material for ethanol and other chemicals [6]. In all of
procured from Genencore (Rochester, NY). Enzyme
these applications, component fractionation is playing
activity on pectin, xylan, filter paper, carboxymethylcel-
an increasingly important role in the complete and
lulose, and microcrystalline cellulose was estimated [17].
efficient utilisation of biomass. The fractionation of
herbage biomass by various pretreatments involving
2.3. Enzymic saccharification
acids or bases coupled with elevated temperatures or
softening the fibre with steam or liquid ammonia for
One gram of untreated alfalfa residue (5%) in 0.01 M
efficient hydrolysis of fibre have been extensively dis-
potassium phosphate buffer or distilled water, pH 5.4,
cussed [7-9].
was treated with 0.1-0.8 ml of cellulase, pectinase,
In this study, liquid hot water (LHW; 220°C for 2
xylanase, or an enzyme mixture (1:1 pectinase, cellulase,
min) without chemicals was chosen as the pretreatment
or xylanase). Twenty millilitres of this reaction mixture
for total solubilisation of hemicelluloses and partial
was incubated at 50°C for 75-96 h with occasional
hydrolysis of lignin [10, 11]. Enzymic saccharification
stirring. The raffinate fibre was incubated with various
was necessary for reducing high molecular weight
enzymes under identical conditions. Suitable substrate
oligosaccharides to low molecular weight monomers
and enzyme controls were determined. Samples (1.5 ml)
consisting of a mixture of pentoses and hexoses, and
were removed periodically and centrifuged at 16000 × g
finally fermenting to ethanol [12-16].
for 3 min; clear samples were saved for reducing sugar,
The objective of our work is to optimise enzymic
uronic acid, and high performance liquid chromatogra-
saccharification of alfalfa fractions obtained from
phy (HPLC) analysis. Unsaccharified residue was
LHW pretreatment and ultimately to ferment the sug-
washed and dried, and dry weight was determined
ars to ethanol and/or other chemicals. In this paper, we
report on the optimisation of enzymic saccharification
2.4. Screening of enzyme and substrate concentration
of alfalfa fractions with commercial cellulases and
2.4.1. Saccharification of untreated alfalfa and raffinate
Cellulase was mixed with pectinase (1:1) and xylanase
(1:1) separately. Similarly, xylanase was mixed with
pectinase (1:1). A mixture of cellulase, pectinase, and
2. Materials and methods
xylanase (1: 1: 1) was also made. Various concentrations
of these single enzymes and enzyme mixtures, 0.25-4%
(w/v), were used to saccharify 2.5- 15% (w/v) untreated
2.1. Alfalfa fibre
alfalfa (enzyme:substrate 1:1.25-1:40). Cellulase was
used only to saccharify raffinate. Concentration of raffi-
Alfalfa (Medicago sativa) fibre was obtained from
nate used in saccharification ranged from 2.5 to 15%.
wet fractionation of freshly cut alfalfa herbage. The
Cellulase concentrations ranged from 1 to 4% (w/v) for
herbage was macerated using a rotary impact device
enzyme:substrate ratio of 1:2.5-1:10. Other reaction
and immediately dejuiced by a screw press. This mate-
conditions were similar to those described in the previ-
rial was air dried and stored prior to LHW pretreat-
ous subsection on enzymic saccharification.
ment. Pretreatment consisted of flowing preheated
water at 220°C through 30 g of untreated alfalfa for 2
2.4.2. Saccharification of alfalfa extract
min [6]. The water was held at sufficient pressure to
Single enzymes (pectinase, cellulase, and xylanase)
ensure that it remained in the liquid phase. After treat-
and 1:1 enzyme combinations (cellulase + pectinase,
ment, the fibrous insoluble residue (‘raffinate’) was
cellulase + xylanase, pectinase + xylanase, and pecti-
removed, oven-dried, and stored for further treatment.
nase + cellulase + xylanase) were used to saccharify the
The liquid ‘extract’ was weighed and sampled to deter-
alfalfa extract. The LHW-pretreated alfalfa extract (20
mine dry matter content before storage at 4°C. The
ml), pH 5, was incubated with 0.5-2% (w/v) enzyme at
raffinate and extract fractions were saccharified further
50°C with occasional stirring. Samples (1.5 ml) were
using enzymes. In some experiments, 0.07% sulphuric
removed periodically and centrifuged at 16000 × g for
acid was added to untreated alfalfa fibre during LHW
3-4 min; clear samples were saved for reducing sugar,
pretreatment at 175-225°C to study the nature of the
uronic acid, and HPLC analysis. Dry weight of the
soluble extract and insoluble raffinate obtained for
alfalfa extract was measured before and after enzymic
subsequent enzymic saccharification.

H.K. Sreenath et al. /Process Biochemistry 35 (1999) 33-41
2.5. Analysis
3.2. Enzymic saccharification
After complete hydrolysis with sulphuric acid, the
3.2.1. Effect on reducing sugar release
carbohydrate content of alfalfa fractions was deter-
The selection of commercial enzyme preparations for
mined by HPLC using pulsed amperometric detection
saccharification of untreated and LHW-pretreated al-
[18]. The organic acid content of the alfalfa extract was
falfa was carefully based on their activity on pectin,
also determined by HPLC using a Biorad Aminex ion
xylan, carboxymethylcellulose, filter paper, and Avicel
exclusion column HPX-87H (300 × 7.8 mm; Biorad,
microcrystalline celluloses [17]. The commercial prepa-
Hercules, CA) [19]. Reducing sugars were estimated by
ration of pectinase contained both carboxymethyl cellu-
dinitrosalicylic acid (DNS) reagent [20]. The uronic acid
lase and xylanase activities as impurities (data not
content was measured using the 3-phenylphenol
shown). Similarly, the commercial cellulase preparation
method with galacturonic and glucuronic acids as stan-
contained pectinase and xylanase as impurities, whereas
dards [21]. Sugars released during saccharification were
the commercial xylanase preparation was fairly pure.
identified by HPLC using an Aminex Carbohydrate
Addition of pectinase or cellulase to untreated alfalfa
HPX-87C (300 × 7.8 mm) column (Biorad) [22].
and extract released more reducing sugars than did the
addition of xylanase. Addition of a pectinase and cellu-
lase mixture further enhanced sugar release (Table 2).
3. Results and discussion
During raffinate saccharification, cellulase released
more sugars than did pectinase or xylanase.
3.1. Chemical composition of alfalfa fibre fractions
The primary sugars released during alfalfa saccharifi-
cation were glucose, xylose, and arabinose. Sucrose and
Dejuiced alfalfa herbage fibre consisted of 70-75%
cellobiose were also found in all saccharified alfalfa
dry matter of the original herbage. This air-dried un-
fractions. Low amounts of galactose, mannose, and
treated alfalfa material contained 33% cellulose, 18%
rhamnose were found as well, and were not routinely
hemicellulose, 8% lignin, 11% protein, 9% ash, and 22%
separated. When used alone, 0.5% (w/v) pectinase re-
solubles [6].
leased 2-3% galacturonic acid from untreated alfalfa
After 44 LHW-pretreatment runs, average yields of
on a dry weight basis. However, when 0.25% (w/v)
48% liquid extract and 41% insoluble raffinate were
pectinase was mixed with 0.25% (w/v) cellulase, the
obtained. On average, 11%. material was lost as
mixture released less galacturonic acid from untreated
volatiles in the LHW pretreatment process. Table 1
alfalfa (1.8-2.l%). After enzymic saccharification, the
shows the amount of monomers (hexoses, pentoses, and
insoluble raffinate fraction did not contain uronic acid
uronic acids) derived on complete hydrolysis of alfalfa
residues. However, 1.0 and 0.8% galacturonic acid was
herbage fractions with H
released from the saccharified extract by pectinase
2SO4 [18]. The alfalfa fibre
fraction contained pectin residues as well as various
alone and in combination with cellulase, respectively.
hexose and pentose polymers. The insoluble raffinate
fraction contained predominantly glucose and small
3.2.2. Optimisation of saccharification of untreated
amounts of xylose, mannose, and galactose. Uronic
alfalfa fibre
acids were absent in the raffinate. The extract fraction
Pectinase and cellulase when used individually re-
contained predominantly xylose, glucose,. arabinose,
leased greater amounts of reducing sugar from un-
and galactose as well as uronic acids. The extract
treated alfalfa than did xylanase (Fig. l(a)). The
fraction had a pH of 4.4-4.6 and contained acetic acid
pectinase and cellulase mixture was more advantageous
(0.85-1 g/l) and formic acid (0.29-0.32 g/l). The latter
than other enzyme combinations in enhancing sacchar-
is reported to hinder yeast growth and fermentation
ification of untreated alfalfa; with the 1% (w/v) pecti-
nase and cellulase mixture, approximately 37-40 g
Table 1
Carbohydrate composition of untreated and LHW-pretreated alfalfa fractions.

Carbohydrate (g%)
Alfalfa fibre
Arabinose Xylose Glucose Galactose
Mannose Rhamnose Galacturonic acid
Glucuronic acid
a Analysed after complete hydrolysis with H2SO4 [18]; expressed in percentage of dry weight of sample.
b From trial batch of LHW-treated samples.

Table 2
Analysis of sugars obtained during enzymic hydrolysisa of various alfalfa residues at 75 h
Enzyme treatment
Sugar (g/100 g substrate)b
Untreated alfalfa
Reducing sugarc
Reducing sugarc
Reducing sugarc
Enzyme mixtured
a One gram of untreated alfalfa (before LHW) and raffinate (after LHW) from a trial batch in 20 ml 0.01-M potassium phosphate buffer, pH 5.5, was incubated for 75 h with 1.0% v/v enzyme
at 50°C. Alfalfa extract, 20 ml (15.5 g/l solids) was incubated with 1.0% enzyme under identical conditions. All values were subtracted from respective enzyme and substrate controls.
b Analysed by HPLC.
c Reducing sugar estimated by DNS reagent.
d Xylanase+pectinase+cellulase (1:1:1).

H.K. Sreenath et al./Process Biochemistry 35 (1999) 33-41
was enhanced by an increase in substrate concentration
(Fig. 3(a)). However, above 10% (w/v) concentration,
mixing the substrate and liquid became difficult. The
amount of glucose released during saccharification of
untreated alfalfa residue was two to three times that of
xylose (Fig. 3(b)). Small fractions of unhydrolyzed
oligosaccharides were present in the alfalfa hydrolyzate
sample, even at 96 h of saccharification. This suggests
that enzymic saccharification was not complete; it was
probably inhibited by the reaction product or slow
enzyme inactivation or both.
3.2.3. Clarification and saccharification of alfalfa

Alfalfa extract obtained from various LHW pretreat-
ment trials contained 15-17.0 g solids per litre on a dry
weight basis. The use of a pectinase and cellulase
mixture accelerated clarification of the extract, and a
sediment was produced in 6 h. After 24 h, incubation
with the enzyme mixture produced a 4.2-g sediment per
litre extract on a dry weight basis. This also suggests
that 75% of extract carbohydrates were hydrolyzed in
the first 24 h of treatment with the 1:1 pectinase and
Fig. 1. Effect of various enzymes on saccharification of untreated
cellulase mixture.
alfalfa: (a) single enzymes, (b) enzyme mixtures at 1:1 ratio. Alfalfa
was incubated with 1% (w/v) enzyme, pH 5.4, at 50°C. Abbreviations:
C, cellulase; P, pectinase; X, xylanase.

reducing sugars was released from 100 g untreated
alfalfa at 96 h (Fig. 1(b)). A greater degree of synergism
of pectinase and cellulase was found during saccharifi-
cation of untreated alfalfa fibre (Table 2, Fig. 2). In-
creasing the concentration of the 1:1 pectinase and
cellulase mixture from 0.5 to 2% (w/v) with 10% (w/v)
untreated alfalfa resulted in an increase in reducing
sugar release (Fig. 2). This enhanced saccharification
from substrate depended on the amount of pectinase
and cellulase present in the mixture. In the initial 30 h,
reducing sugar release was directly proportional to all
concentrations of pectinaSe and cellulase mixture (Fig.
2(a)). However, on prolonged incubation, the increase
in reducing sugar release remained unchanged, particu-
larly at higher concentrations of pectinase and cellulase
mixture (Fig. 2(b)). The 2% (w/v) pectinase and cellu-
lase mixture released a maximum of 51 g/l reducing
sugars from 100 g/l untreated alfalfa at 96 h (Fig. 2(b)).
No further increase in release of reducing sugars or
fibre degradation was observed with higher enzyme
(4%, w/v) concentration. The degradation of untreated
alfalfa was about 40% during this saccharification.
Various concentrations of untreated alfalfa (2.5, 5.0,
10.0 and 15.0% (w/v); E:S ratio of 1:1.25, 1:2.5, 1:5 and
1:7.5, respectively) were treated with the 2% (w/v) pecti-
nase and cellulase mixture for a period of 70 h and the
Fig. 2. Effect of various concentrations of pectinase, cellulase, and
their mixture on saccharification of untreated alfalfa: (a) release of

amount of reducing sugar release and sugar analysis
reducing sugars at 30 h; and (b) release of reducing sugars at 96 h.
determined by HPLC. The release of reducing sugars
Untreated alfalfa (10% w/v) was incubated with 0.25-2.0% (w/v)
from untreated alfalfa during enzymic saccharification
enzyme, pH 5.4, at 50°C.

H.K. Sreenath et al. /Process Biochemistry 35 (1999) 33-41
gradually increased and at 75 h, the level of glucose was
almost the same as that of xylose in the extract (Table
2). Low levels of galacturonic acid were also released in
the first 24 h. The pectinase activity immediately en-
hanced the release of reducing sugars from extract
compared to cellulase in the first 6 h of incubation (Fig.
5(a)). However, on prolonged incubation, a lower in-
crease in sugar release was noticed due to the combined
action of cellulase and pectinase (Fig. 5(b)).
3.2.4. Saccharification of raffinate residue
The LHW pretreatment yielded 40% insoluble raffi-
nate residue (dry weight). Because of its predominantly
cellulosic nature, the raffinate was liquefied and saccha-
rified using only cellulase. Glucose was the primary
sugar released by cellulolytic saccharification (Table 2).
Galacturonic acid was absent in the enzyme hy-
drolyzate. Saccharification was poor when pectinase or
xylanase was used alone (Table 2). The effect of sub-
strate concentration on release of reducing sugars dur-
ing cellulolytic saccharification of raffinate is shown in
Fig. 6. A 10% substrate was adequate to release opti-
mum reducing sugars. Substrate concentrations higher
than 10% was not advantageous because the mixing of
liquid and substrate became difficult. On a dry weight
basis, the reducing sugar release was 21-22 g/100 g
substrate using 1% (w/v) cellulase at 96 h (Table 2). The
amount of raffinate degraded by cellulase was 24-25%.
The 2 and 4% (w/v) concentrations of cellulase in-
Fig. 3. Effect of substrate concentration on cellulolytic and pecti-
creased release of reducing sugars to 59 and 65 g/100 g
nolytic saccharification of untreated alfalfa: (a) release of reducing
raffiate at 96 h, respectively (Fig. 6(c)).
sugars (S, substrate; E, enzyme mixture); (b) HPLC analysis of sugars
at 70 h. Untreated alfalfa (2.5-15% w/v) was incubated with 2%
(w/v) cnxyme, pH 5.4, at 50°C.

The extract was only partially clarified when pecti-
nase was used alone. In the first 6 h of incubation, the
use of cellulase or xylanase alone failed to clarify the
extract; the extract was cloudy like the control. Pro-
longed incubation (48 h) with pectinase clarified the
extract almost completely. Partial clarification was
recorded at 72 h with cellulase. When used alone,
xylanase did not clarify the extract, even after 96 h of
The organic acid components of the extract did not
affect enzymic saccharification. When used alone, pecti-
nase released more reducing sugars from the extract
than did cellulase or xylanase (Fig. 4(a)). On extended
incubation, the 1% (w/v) pectinase and cellulase mix-
ture released greater amounts of reducing sugars than
did single enzymes or other enzyme combinations (Fig.
4(b)). A 1-2% (w/v) pectinase and cellulase mixture
released 7-8.6 g/l reducing sugars (dry weight basis)
from alfalfa extract (15.5 g/l solids). The ratio of xy-
Fig. 4. Effect of various enzymes on saccharification of alfalfa
1ose:glucose:arabinose released from the extract by the
extract: (a) single enzymes; (b) enzyme mixtures at 1:1 ratio. A total
pectinase and cellulase mixture was 2:1:0.5 in the first
of 20 ml alfalfa extract (15.5 g/l solids) was incubated with 1%
24 h of incubation (data not shown); glucose content
enzyme, pH 5.0, at 50°C.

H.K. Sreenath et al./Process Biochemistry 35 (1999) 33-41
cellulolytic attack. Cellulolytic saccharification of raffi-
nate was the same at various LHW pretreatment tem-
peratures, releasing 63 g reducing sugars per 100 g
substrate (Fig. 7(a)).
The addition of 0.07% sulphuric acid resulted in the
production of more extract and raffinate during LHW
pretreatment and had a profound effect on the release
of reducing sugars. The acid changed the colour of the
raffinate fibre to dull black compared to the dark
brown colour of fibre pretreated without acid. Enzymic
saccharification released 10% more reducing sugars (9.9
g/l) from extract pretreated with sulphuric acid com-
pared to extract pretreated without the acid (Table 3).
However, the addition of sulphuric acid to the pretreat-
ment lowered the release of reducing sugars from the
raffinate fraction by 60% (Fig. 7(b)). In addition, the
raffinate was degraded 10-12%. This effect was proba-
bly due to the severity of the LHW pretreatment at
high temperature.
The enzymic hydrolysis yield undergoes a remarkable
change when substrates are pretreated by different
steam-aqueous conditions [7]. Acid impregnation of
Fig. 5. Effect of various concentrations of pectinase, cellulase, and
their mixture on saccharification of extract: (a) release of reducing
sugars at 6 h; (b) release of reducing sugars at 96 h. Twenty millititres
of extract (15.5 g/l solids) was incubated with 1% (w/v) enzyme, pH
5.0, at 50°C.

Technology for using cellulases in conjunction with
pectic enzymes for partial or complete plant tissue
liquefaction has been described [24-26]. Similar to flax
and kenaf, the major fibres in soft parenchymatous
tissue of alfalfa are protected by pectin [27]. Hence,
addition of pectinase was’ crucial to the degradation of
pectin and softening of fibre tissue. After pectinase was
added, cellulase synergistically liquefied the substrate
and enhanced sugar release.
3.3. Effect of pretreatment on addition of 0.07%
sulphuric acid
Data on release of reducing sugars after various
treatments as a function of time are shown in Table 3.
Warm water extraction did not facilitate hemicellulose
solubilisation. In contrast, LHW pretreatment facili-
tated both hemicellulose solubilisation and cellulose
fractionation. After enzymic saccharification, the raffi-
nate fraction released more reducing sugars than did
Fig. 6. Effect of various substrate and cellulase concentrations on
saccharification of raffinate obtained from LHW pretreatment: (a)

untreated alfalfa (Table 3, Fig. 6(c)). Increasing LHW
2.5% (w/v) raffinate, (b) 5% (w/v) raffinate, and (c) 10% (w/v)
pretreatment temperature from 175 to 225°C did not
raffinate. Raffinate was incubated with 1, 2 and 4% (w/v) cellulolytic
alter the production of raffinate and its susceptibility to
enzyme (E), pH 5.4, at 50°C.

H.K. Sreenath et al. /Process Biochemistry 35 (1999) 33-41
Table 3
Release of reducing sugars from alfalfa fibre fractions during saccharificationa

Time (h)
Reducing sugar (g/100 g substrate)
Warm water
LHW + 0.07% acid
l Reaction mixture (20 ml) consisting of 2 g untreated alfalfa fibre or raffinate (10%) was incubated with 2% enzyme, pH 5.4, at 50°C. Untreated
and LHW-treated fibre were incubated with mixture of pectinase and cellulase. Raffinate was treated with only cellulase. Extract 20 ml (15.5 g/l
solids) was incubated with a 1% pectinase and cellulase mixture, pH 5, at 50°C.

Fig. 7. Effect of LHW pretreatment at various temperatures, with or
without 0.07% sulphuric acid, on cellulolytic saccharification of raffi-
nate: (a) LHW pretreatment without sulfuric acid; (b) LHW pretreat-
ment with sulfuric acid. 10% (w/v) raffinate was incubated with 4%
(w/v) cellulase, pH 5.4, at 50°C.

lignocellulosic biomass before steam treatment was re-
ported to produce a negative effect on hydrolysis yield
[28]. Other causes may also underlie the inhibitory effect
of pretreatment on enzyme penetration. Other fa-
vourable conditions such as product recovery and re-
moval during saccharification of alfalfa fractions would
be helpful in such applications. Therefore, future work
will address sequential and simultaneous saccharification
and fermentation of alfalfa fractions with and without
pretreatment for producing ethanol and other chemicals.
This work was supported by NRIC Grant 94-34142-
0426 and by the USDA Agricultural Research Service.

H.K. Sreenath et al. / Process Biochemistry 35 (1999) 33-41