Characterization of Dicarboxylic Acids for Cellulose Hydrolysis

Nathan S. Mosier,
,
Ayda Sarikaya,
,
Christine M. Ladisch,

and
Michael R. Ladisch*
,,,
Department of Agri cul tural and Bi ol ogi cal Engi neeri ng, Laboratory of Renewabl e Resources Engi neeri ng,
Department of Bi omedi cal Engi neeri ng, and Texti l e Sci ence, Department of Consumer Sci ences and Retai l i ng,
Purdue Uni versi ty, West Lafayette, I ndi ana 47907
I n thi s paper, we show that di l ute mal ei c aci d, a di carboxyl i c aci d, hydrol yzes cel l obi ose,
the repeat uni t of cel l ul ose, and the mi crocrystal l i ne cel l ul ose Avi cel as effecti vel y as
di l ute sul furi c aci d but wi th mi ni mal gl ucose degradati on. Mal ei c aci d, superi or to
other carboxyl i c aci ds reported i n thi s paper, gi ves hi gher yi el ds of gl ucose that i s
more easi l y fermented as a resul t of l ower concentrati ons of degradati on products.
These resul ts are especi al l y si gni fi cant because mal ei c aci d, i n the form of mal ei c
anhydri de, i s wi del y avai l abl e and produced i n l arge quanti ti es annual l y.
Introduction
Li mi ted fossi l fuel suppl i es and ri si ng oi l pri ces al ong
wi th i ncreasi ng concern about the envi ronmental i mpact
of thei r use has prompted emphasi s on research and
devel opment for the use of renewabl e resources for the
generati on of fuel s and other chemi cal s now produced
from petrol eum. Bi omass materi al s, consi sti ng l argel y of
cel l ul ose, are a promi si ng renewabl e resource for the
producti on of fuel s and i ndustri al chemi cal s.
A number of processes for hydrol yzi ng cel l ul ose i nto
gl ucose have been devel oped over the years. The two most
common processes uti l i ze ei ther cel l ul ol yti c enzymes
harvested from fi l amentous fungi such as Trichoderma
sp. or sul furi c aci d of varyi ng strengths from di l ute to
concentrated. Hi stori cal l y, enzymes have been too ex-
pensi ve for economi cal producti on of fuel ethanol from
bi omass (1). Sul furi c aci d i tsel f i s l ess expensi ve than
cel l ul ol yti c enzymes, al though di sposal costs associ ated
wi th the use of sul furi c aci d si gni fi cantl y i ncrease i ts cost.
However, the si ngl e l argest drawback to usi ng sul furi c
aci d i s that i t al so readi l y degrades gl ucose at the hi gh
temperatures requi red for cel l ul ose hydrol ysi s (2-4).
Gl ucose degradati on not onl y l owers the yi el d of ferment-
abl e sugars from bi omass but forms the degradati on
products hydroxymethyl furfural , l evul i ni c aci d, and
formi c aci d, whi ch themsel ves are i nhi bi tory to yeast
fermentati on (5-8). Al though concentrated mi neral aci ds
at l ower temperatures have been used wi th some success
(9, 10), the cost of aci d recovery has i mpeded thei r
wi despread use.
Thi s paper addresses the use of carboxyl i c aci ds as
cel l ul ose-hydrol yzi ng catal ysts as part of a l arger re-
search effort to devel op organi c mol ecul es that mi mi c the
speci fi ci ty of enzymes (11). I t i s known that strong
mi neral aci ds hydrol yze cel l ul ose more effecti vel y than
weak aci ds (12) and that carboxyl i c aci d al one i s a weak
aci d (hi gh pK
a
). However, compounds wi th mul ti pl e
carboxyl i c aci d moi eti es are stronger aci ds than mono-
carboxyl i c aci ds, both i n number of protons avai l abl e for
donati on to a base and l owered pK
a
s for the i ndi vi dual
carboxyl i c aci d moi eti es (compare the pK
a
of aceti c aci d
to succi ni c and mal ei c aci ds, Tabl e 1). The di carboxyl i c
aci ds, mal ei c and succi ni c aci ds, were eval uated by
hydrol ysi s of the cel l ul ose di sacchari de repeat uni t cel -
l obi ose. Resul ts were compared agai nst aceti c aci d, a
monocarboxyl i c aci d, and sul furi c aci d, a mi neral aci d,
as wel l as water al one. Mal ei c and sul furi c aci ds gave
the l argest extents of hydrol ysi s. These were then eval u-
ated for the hydrol ysi s of the mi crocrystal l i ne cel l ul ose
Avi cel .
Materials and Methods
Al l chemi cal s used i n these experi ments were pur-
chased from Si gma-Al dri ch, St. Loui s, MO. General l ab
and HPLC suppl i es were obtai ned from Fi sher Sci enti fi c,
Pi ttsburgh, PA. Stai nl ess steel tubi ng and Swagel ok
fi tti ngs were purchased from I ndi anapol i s Val ve and
Fi tti ng Co., I ndi anapol i s, I N.
Carbohydrate HPLC Analysis. Sampl e anal ysi s
uti l i zed a Bi o-Rad HPX-87H organi c aci d col umn (Bi o-
Rad Laboratori es I nc., Hercul es, CA) i n a HPLC system
consi sti ng of a Mi l ton Roy mi ni pump (Mi l ton Roy Co.,
I vyl and, PA), Waters 717 pl us autosampl er, Waters R401
di fferenti al refractometer (Waters Corp., Mi l ford, MA),
Hewl ett-Packard 3396 seri es I I i ntegrator (Hewl ett-
Packard, Pal o Al to, CA), and a personal computer for
data storage. The mobi l e phase was 5 mM sul furi c aci d
i n di sti l l ed, dei oni zed water fi l tered to 0.2 m. The
operati ng condi ti ons for the HPLC col umn were 60 C
wi th a fl ow rate of 0.6 mL/mi nute. Compl ete sampl e
el uti on coul d be accompl i shed wi thi n 35 mi n per i njec-
ti on.
* E-mai l : l adi sch@ecn.purdue.edu.

Department of Agri cul tural and Bi ol ogi cal Engi neeri ng.

Laboratory of Renewabl e Resources Engi neeri ng.

Department of Bi omedi cal Engi neeri ng.

Texti l e Sci ence, Department of Consumer Sci ences and Retai l -

i ng.
Table 1. pKa for Acid Catalysts at 20C
pKa
aci d catal yst mol ecul ar wt 1 2
sul furi c aci d 98 -3 2.0
mal ei c aci d 116 1.9 4.4
succi ni c aci d 118 4.2 5.6
aceti c aci d 60 4.7
water 18 7.0
474 Biotechnol. Prog. 2001, 17, 474480
10.1021/bp010028u CCC: $20.00 2001 American Chemical Society and American Institute of Chemical Engineers
Published on Web 05/01/2001
Standard curves were generated for gl ucose and cel -
l obi ose wi th pure sampl es di ssol ved i n mobi l e phase.
Chemi cal sampl es were dri ed at 70 C for 24 h before
wei ghi ng to el i mi nate moi sture error. Fracti onal di l uti ons
of the standard sol uti on, 5.00 mg/mL, were made to gi ve
a standard curve range of 0.25-5.00 mg/mL.
Glucose Analyzer. Gl ucose concentrati ons for al l
sampl es were confi rmed wi th an enzymati c anal ysi s
usi ng a Beckman gl ucose anal yzer 2 (Beckman Coul ter,
I nc., Ful l erton, CA). Al l sampl es from a gi ven experi ment
were equal l y di l uted when necessary to gi ve maxi mum
concentrati ons bel ow 2.5 mg/mL to keep the measure-
ments wi thi n the l i near range of the i nstrument. I nstru-
ment cal i brati on was carri ed out usi ng a 150 mg/dL
gl ucose standard purchased from Beckman.
pH Measurement. The pH of each carbohydrate and
catal yst sol uti on was measured usi ng a Cal omel pH
el ectrode 5990-95 from the Col e-Parmer I nstrument
Company (Vernon Hi l l s, I L) attached to a Corni ng pH/
i on meter 150 (Corni ng Medi cal and Sci enti fi c I nstru-
ments, Hal stead, Essex, Engl and). Before each use, the
pH meter was cal i brated at two poi nts usi ng pH 7.00 and
4.00 buffer sol uti ons obtai ned from the Fi sher Chemi cal
Company (Pi ttsburgh, PA).
Each carbohydrate and catal yst sol uti on was equi l i -
brated to room temperature (23 C) before the pH probe
was i nserted i nto the sol uti on. The sol uti on was gentl y
sti rred unti l the pH readi ng stabi l i zed, and the resul t
was recorded to the nearest pH (0.01.
Initial Screening of Varying Acid Catalyst Con-
centrations. I ni ti al screeni ng of di fferent catal ysts used
di fferent concentrati ons i n order to determi ne reasonabl e
concentrati ons for more detai l ed ki neti c anal ysi s. Aque-
ous sol uti ons of cel l obi ose at 10 g L
-1
were hydrol yzed
wi th varyi ng concentrati ons of aci d. A concentrati on of
10 g L
-1
represents the maxi mum of the l i near range for
detecti on by HPLC wi thout sampl e di l uti on for both
cel l obi ose and gl ucose. Experi ments uti l i zed sampl es of
100 mL i n a 200-mL conti nuous-sti r autocl ave reactor
(Autocl ave Engi neers I nc., Eri e, PA). Al l experi ments
fol l owed the same temperature profi l e (Fi gure 1) i n whi ch
the target temperature of 160 C was reached i n 60 mi n
and then hel d constant for an addi ti onal 30 mi n. Cool
down at the end of each experi ment was achi eved by
runni ng tap water through cool i ng coi l s i n the reactor.
Reactor temperature fel l bel ow 100 C i n l ess than 60 s
for each experi ment.
I n addi ti on to the carboxyl i c aci d catal yst candi dates,
a control wi th no aci d (water wi th 10 g L
-1
cel l obi ose)
and varyi ng concentrati ons of sul furi c aci d, the standard
aci d for cel l ul ose hydrol ysi s, were used. Each experi ment
was dupl i cated to gi ve two data poi nts per concentrati on.
Reactant and product concentrati ons before and after
hydrol ysi s were determi ned usi ng HPLC and Beckman
gl ucose anal yzer 2. Gl ucose concentrati ons i n sol uti ons
contai ni ng mal ei c aci d were determi ned sol el y by Beck-
man gl ucose anal yzer 2 because mal ei c aci d and gl ucose
coel ute (Fi gure 2).
Cellulose Hydrolysis. Avi cel (FMC Corporati on,
Phi l adel phi a, PA), si eve cut 200-270 (53-75 m), was
used as the mi crocrystal l i ne cel l ul ose for hydrol ysi s. To
200 mL of 50 mM mal ei c aci d and sul furi c aci d aqueous
sol uti ons was added 8.0 g of the si eved Avi cel , gi vi ng an
approxi mated cel l ul ose concentrati on of 35 g L
-1
. Thi s
concentrati on of Avi cel was sel ected to gi ve measurabl e
gl ucose wi thi n 30 mi n of hydrol ysi s at 175 C. The
sol uti on pH was measured before and after the addi ti on
of the cel l ul ose. The Avi cel and aci d sol uti on mi xture was
vi gorousl y shaken i mmedi atel y before 4.0 mL of l i qui d
was wi thdrawn by pi pet to fi l l the reactor tubes.
Reactor tubes were constructed from 0.375 i n. 0.065
i n. 316 stai nl ess steel tubi ng and 0.375 i n. Swagel ok caps
(Swagel ok Cos., Sol on, OH). Each reactor tube was 6 i n.
i n l ength gi vi ng a total vol ume of 5.5 mL. However, a
sampl e vol ume of onl y 4.0 mL (at room temperature) was
used i n each tube to al l ow for l i qui d expansi on from
heati ng.
Temperature control was achi eved uti l i zi ng a Tecam
SBL-1 fl ui di zed sand bath. As a resul t of si ze constrai nts
and temperature vari ati on on the verti cal axi s of the sand
bath, onl y two reactor tubes coul d be i mmersed at one
ti me. I nternal reactor tube temperatures duri ng heat-
up for varyi ng bath temperatures were determi ned usi ng
an Omega thermowel l wi th thermocoupl e (Omega Engi -
neeri ng, I nc., Stamford, CT). The thermowel l and ther-
mocoupl e were i nserted i nto the reactor tube to the
verti cal and axi al center and hel d i n pl ace wi th a
Swagel ok fi tti ng and ferul e to mai ntai n pressure. Heat-
up ti mes were determi ned usi ng two reactor tubes each
fi l l ed wi th 4 mL of water, one bl ank and one wi th
thermocoupl e i nserted. A repl i cate of each temperature
experi ment was performed (data shown i n Fi gure 3).
Heat-up ti mes of 2 mi n were requi red to achi eve 175 C
at the correspondi ng saturati on water vapor pressure
Figure 1. Heat-up profi l e for 200-mL autocl ave reactor to 160 C i n 60 mi n wi th 30 mi n hol d ti me.
Biotechnol. Prog., 2001, Vol. 17, No. 3 475
(930 kPa), so that the contents of the tube were mai n-
tai ned i n a l i qui d state. The reactor tubes were cool ed
by i mmersi on i n i ce water. I nternal tube temperature
dropped bel ow 100 C i n l ess than 10 s for al l tested sand
bath temperatures (data not shown).
Four ti me poi nts were tested, i ncl udi ng a zero ti me,
whi ch represented heat-up of the reactor tube to the
desi red temperature and i mmedi ate cool -down i n i ce
water. The order of reacti on ti mes was randomi zed for
each experi ment. I n addi ti on, al l ti me poi nt experi ments
were repeated. The two repl i cates gave four data poi nts
per reacti on ti me (two sets of two tubes).
After cool -down, the contents of the reactor tubes were
fi l tered through Whatman PVDF centri fuge fi l ters, 0.2
m pore si ze, 6.5 mm di ameter (Fi sher Sci enti fi c, Pi tts-
burgh, PA) that were dri ed at 60 C for 24 h and
i ndi vi dual l y wei ghed pri or to fi l trati on. The supernatant
was retai ned for anal ysi s. The sampl e bottl es were ri nsed
to recover resi dual Avi cel that was then fi l tered through
the same fi l ters. The recovered Avi cel i n the centri fuge
fi l ters was then dri ed at 60 C for 72 h and wei ghed. The
i ni ti al mass of Avi cel was determi ned by averagi ng the
recovered Avi cel masses from the four zero-ti me sampl es.
Concentrati ons of di ssol ved sacchari des, ol i gosacchari des,
and degradati on products were determi ned by HPLC and
Beckman gl ucose anal yzer 2 as descri bed above.
Theory
Modeling Cellulose Saccharification by Dilute
Sulfuric Acid. Cel l ul ose i s a heterogeneous substrate
that makes model i ng cel l ul ose hydrol ysi s di ffi cul t. Cel -
l ul ose i s composed of chai ns of gl ucose connected by -
(1-4) gl ycosi di c bonds. One chai n end i s termed the
reduci ng end because the hemi acetal i s abl e to open to
expose the reduci ng al dehyde. The other chai n end i s
cal l ed the non-reduci ng end because the 1-carbon i n the
hemi acetal i s i nvol ved i n the (1-4) bond, preventi ng
ri ng openi ng. Li ke starch chai ns, thi s gi ves cel l ul ose
Figure2. Chromatograms of sol uti ons contai ni ng cel l obi ose and gl ucose, cel l obi ose and 0.05 M mal ei c aci d, and al l three compounds.
Mal ei c aci d and gl ucose coel ute, and thus al l gl ucose concentrati ons i n mal ei c aci d sol uti ons determi ned sol el y by Beckman gl ucose
anal yzer 2.
Figure 3. Heat-up profi l es for reactor tubes fi l l ed wi th water.
476 Biotechnol. Prog., 2001, Vol. 17, No. 3
chai ns di recti onal i ty. Unl i ke starch, havi ng gl ucose as
i ts repeat uni t, the repeat uni t for cel l ul ose i s cel l obi ose,
a gl ucose di mer. Thi s i s because the (1-4) bond causes
the gl ucose mol ecul es to al i gn wi th al ternati ng di recti on-
al i ty, unl i ke the R(1-4) bound gl ucose mol ecul es i n starch
(13).
Begi nni ng wi th Saeman i n 1945 (2) and confi rmed by
many others (12, 14-18) cel l ul ose hydrol ysi s by aci d
catal ysts has been model ed as a pseudo-fi rst-order ho-
mogeneous sequenti al reacti on of cel l ul ose hydrol ysi s
fol l owed by gl ucose degradati on:
where C )cel l ul ose, G )gl ucose, HMF )hydroxymethyl
fufural , LA ) l evul i ni c aci d, FA ) formi c aci d, k
hyd
)
ki neti c constant of cel l ul ose hydrol ysi s, and k
deg
) ki neti c
constant of gl ucose degradati on. However, cel l ul osi c
materi al s are not homogeneous sol i ds. Thei r physi cal and
chemi cal properti es change over the course of hydrol ysi s,
whi ch makes effecti ve general i zed model i ng of cel l ul ose
hydrol ysi s di ffi cul t. The cel l ul ose hydrol ysi s ki neti c
constants must be determi ned empi ri cal l y for each bi o-
mass materi al , aci d catal yst, and set of reacti on condi -
ti ons.
CellobioseHydrolysisandGlucoseDegradation.
Cel l obi ose, the repeat uni t of cel l ul ose, was chosen for
the screeni ng and i ni ti al ki neti c experi ments of the aci d
catal ysts. The use of cel l obi ose served to si mpl i fy the
experi ments si nce there was onl y one hydrol yzabl e bond
for each mol ecul e of reactant. Furthermore, cel l obi ose i s
sol ubl e and can be obtai ned i n greater puri ty (>99.9%)
than pl ant cel l ul ose (97-99%), si mpl i fyi ng i nterpretati on
of the data. There i s al so l ess homogenei ty for reacti on
sl urri es contai ni ng cel l ul ose than cel l obi ose because the
crystal l i ni ty and accessi bi l i ty of the cel l ul ose to the
catal yst can vary substanti al l y between sol i d parti cl es
of cel l ul ose, as wel l as wi thi n the i ndi vi dual parti cl es
themsel ves. I t must al so be noted that di rect uti l i zati on
of cel l obi ose hydrol ysi s ki neti cs for predi cti ng cel l ul ose
hydrol ysi s i s l i mi ted by the varyi ng accessi bi l i ty and
crystal l i ni ty of the cel l ul ose, whi ch changes the reacti vi ty
of the i ndi vi dual (1-4) gl ycosi di c bonds. However,
cel l obi ose hydrol ysi s data show i mportant trends that
hel p focus further study wi th puri fi ed cel l ul ose and
bi omass materi al s of possi bl e i ndustri al i mportance.
Hydrol ysi s of cel l obi ose occurs by the fol l owi ng formul a
where G
2
) cel l obi ose and G ) gl ucose:
Gl ucose degradati on has been shown to generate
products by
where G ) gl ucose, HMF ) hydroxymethyl furfural , LA
) l evul i ni c aci d, FA ) formi c aci d, and k
deg
) ki neti c
constant of gl ucose degradati on.
Di sappearance of cel l obi ose from the reacti on l i qui d i s
reported i n thi s paper as a percent of the measured
ori gi nal concentrati on. The measured gl ucose concentra-
ti ons can be i nterpreted i n two di fferent ways. The fi rst
expressi on of the gl ucose concentrati on data i s gl ucose
yi el d. Gl ucose yi el d i s the observed fi nal gl ucose concen-
trati on over the stoi chi ometri c maxi mum gl ucose from
the measured di sappearance of cel l obi ose:
where Y
G
) percent expected gl ucose yi el d, [G] ) mol ar
gl ucose concentrati on, [G
2,0
] ) i ni ti al mol ar cel l obi ose
concentrati on, and [G
2,f
] ) fi nal mol ar cel l obi ose concen-
trati on
The second expressi on, theoreti cal gl ucose yi el d, i s
cal cul ated as percent fi nal gl ucose concentrati on over
total theoreti cal gl ucose from i ni ti al cel l obi ose:
where, Y
G/G2
) percent gl ucose yi el d, [G] ) mol ar con-
centrati on of gl ucose, and [G
2
] ) mol ar concentrati on of
cel l obi ose.
Results and Discussion
Cellobiose Hydrolysis. Mal ei c aci d hydrol yzed 95-
99%of the cel l obi ose (Fi gure 4a) wi th a maxi mum gl ucose
yi el d of 90% achi eved at 50 mM aci d concentrati on
(Fi gure 4b). The gl ucose yi el d gi ves a measure of the
quanti ty of hydrol yzed cel l obi ose that i s found as gl ucose
at the end of the reacti on. Succi ni c aci d gave conversi ons
of 60-80% wi th gl ucose yi el ds of 85-90%. Sul furi c aci d
gave cl ose to 100%hydrol ysi s but onl y 80%gl ucose yi el d
i n the best case (compare Fi gure 4a and b). The mono-
carboxyl i c aci d, aceti c aci d, resul ted i n the l owest conver-
si on for al l exami ned aci ds, wi th gl ucose yi el ds fal l i ng
between sul furi c aci d and the di carboxyl i c aci ds. When
cel l obi ose i s cooked i n water al one, 15% i s hydrol yzed
C 98
k
hyd
G 98
k
deg
HMF + H
2
O f LA + FA (1)
G
2
+ H
2
O 98
k
2G (2)
G 98
k
deg
HMF + H
2
O f LA + FA (3)
Figure4. Cel l obi ose (10 g L
-1
) hydrol ysi s (a) and gl ucose yi el d
(b) for varyi ng aci d concentrati ons at 160 C, 30 mi n hol d ti me.
Y
G
)
[G]
2([G
2,0
] - [G
2,f
])
100% (4)
Y
G/ G
2
)
[G]
2[G
2
]
100% (5)
Biotechnol. Prog., 2001, Vol. 17, No. 3 477
wi th a gl ucose yi el d of 55%. Water al one i s capabl e of
both hydrol yzi ng cel l obi ose and degradi ng gl ucose, con-
si stent wi th the l i terature resul ts (19, 20). Chromato-
graphi c anal ysi s of al l control s and aci ds tested showed
the presence of si gni fi cant l evel s of gl ucose degradati on
products such as l evul i nc aci d, formi c aci d, and hy-
droxymethyl furfural i n the sampl es wi th l ow gl ucose
yi el d.
The resul ts show stri ki ng di fferences among the cata-
l yti c abi l i ty of the four aci ds tested. The percent hydrol y-
si s of cel l obi ose (Fi gure 4a) shows some i mportant trends.
Al though the aceti c aci d and succi ni c aci d sol uti ons buffer
water to approxi matel y the same pH, 2.5-2.9, the
percent hydrol ysi s i s substanti al l y di fferent. For the other
group, both mal ei c aci d and sul furi c aci d achi eve near
compl ete hydrol ysi s for the reacti on temperature and
ti me. The basel i ne hydrol ysi s of cel l obi ose i n water al one,
shown for compari son, i s by far the l owest. Both succi ni c
aci d and mal ei c aci d show very hi gh (82-95%) gl ucose
yi el d i n the fi nal sol uti ons (Fi gure 4b). Aceti c aci d had
moderate l evel s of expected gl uocose as di d l ower con-
centrati ons of sul furi c aci d. However, water al one and
hi gher concentrati ons of sul furi c aci d showed si gni fi cant
l oss of the desi red product.
For both mal ei c and sul furi c aci d, the theoreti cal
gl ucose yi el ds (Fi gure 5) have a l ocal maxi mum at
approxi matel y 50 mM concentrati ons. Theoreti cal gl ucose
yi el d (eq 5) descri bes percent total conversi on to gl ucose
of the i ni ti al cel l obi ose i n sol uti on. Thi s shows that, for
the reacti on durati on and temperature profi l e, 50 mM
aci d concentrati on i s near opti mal si nce near 100%
cel l obi ose hydrol ysi s was achi eved. These resul ts l ed to
the sel ecti on of 50 mM mal ei c and sul furi c aci d as
concentrati ons for i ni ti al ki neti c studi es of cel l obi ose
hydrol ysi s and gl ucose degradati on. For succi ni c and
aceti c aci ds, the upward trend i n yi el d cl osel y mi rrors
the upward trend i n percent hydrol ysi s for i ncreasi ng
concentrati ons (Fi gure 4a). Thi s can be i nterpreted that
the reacti on condi ti ons, temperature and ti me, as wel l
as aci d concentrati ons, are far from opti mi zed.
I n summary, mal ei c aci d produced the hi ghest gl ucose
yi el ds of al l of the tested aci ds. Al though sul furi c aci d
produced sl i ghtl y hi gher cel l obi ose hydrol ysi s than mal ei c
aci d, l ess subsequent gl ucose degradati on by the mal ei c
aci d produced hi gher gl ucose yi el ds than sul furi c aci d.
Succi ni c aci d gave superi or gl ucose yi el ds compared wi th
sul furi c aci d when both aci ds were at the hi ghest tested
concentrati ons.
Al though succi ni c and mal ei c aci d had hi gh gl ucose
yi el ds from hydrol yzed cel l obi ose (Fi gure 4b), HPLC
resul ts al so show smal l l evel s of gl ucose degradati on
products that account for the l ost gl ucose. These resul ts
suggest that the reacti on rates for cel l obi ose hydrol ysi s
are greater than the rates of gl ucose degradati on for
succi ni c aci d and mal ei c aci d when compared to aceti c
aci d and sul furi c aci d.
The resul ts for mal ei c aci d are especi al l y si gni fi cant
si nce i t i s a chemi cal commodi ty wi del y used i n the form
of mal ei c anhydri de. Mal ei c anhydri de wi l l hydrol yze i nto
mal ei c aci d at room temperature i n aqueous sol uti on (21).
Al though mal ei c anhydri de i tsel f has no consumer uses,
i ts deri vati ves are uni versal l y known and used. Mal ei c
anhydri de i s a requi red reactant for the producti on of
unsaturated pol yester resi ns used to produce fi bergl ass,
casti ng resi ns, and auto repai r putty. The US. producti on
of mal ei c anhydri de was i n excess of 189,000 tons i n 1992
wi th the major producers bei ng Amoco and Huntsman
Speci al ty Chemi cal s, formerl y of Monsanto (21). I t i s
currentl y sol d as a bul k commodi ty for $0.44-0.53 per
pound (22).
Hydrolysisof Cellulose(Avicel). Al though hydrol y-
si s of cel l obi ose i s useful for screeni ng catal ysts for
cel l ul ose hydrol ysi s, further testi ng of the promi si ng
candi dates wi th cel l ul ose i s requi red to confi rm the
cel l obi ose hydrol ysi s resul ts. Avi cel was chosen as the
cel l ul ose substrate. Avi cel i s a hi ghl y crystal l i ne form of
cel l ul ose produced by aci d refl ux hydrol ysi s of wood. The
hi gh crystal l i ni ty of Avi cel makes i t resi stant to hydrol y-
si s (23). Cel l ul ol yti c enzymes are abl e to achi eve a
maxi mum conversi on of 90%(10%not degradabl e by the
enzymes) after more than 100 hours at 50 C (24). Of
the aci ds screened, mal ei c aci d was chosen as the most
promi si ng carboxyl i c aci d because of the hi gh theoreti cal
gl ucose yi el ds obtai ned from cel l obi ose hydrol ysi s. Mal ei c
aci d at 50 mM concentrati on was chosen as the test case
si nce i t gave the hi ghest gl ucose yi el d at 160 C (Fi gure
4b). As a control , Avi cel was al so hydrol yzed by sul furi c
aci d at 50 mM, the sul furi c aci d concentrati on that gave
the hi ghest gl ucose yi el d at 160 C. However, the hy-
drol ysi s was carri ed out at 175 C because mi crocrystal -
l i ne cel l ul ose requi res pretreatment at temperatures
above 160 C to open the crystal l i ne structure to hydrol y-
si s (19, 25). Hydrol ysi s was carri ed out for three hol d
ti mes, 30, 60, and 180 mi n, at 175 C. To better control
the temperature, these reacti ons were carri ed out i n 5.5-
mL, 316 stai nl ess steel reactor tubes pl aced i n a fl ui di zed
sand bath that al l owed the reacti on mi xture to reach 175
C i n 2 mi n, as descri bed i n Materi al s and Methods. After
the hol d ti me, the reacti on tubes were quenched i n i ce
water to reach ambi ent temperature i n l ess than 10 s.
Tabl e 2 shows the sol i d cel l ul ose and di ssol ved gl ucose
resul ts from four repl i cates. The fi nal percent hydrol ysi s
i s nearl y i denti cal for both aci ds. However, there i s
sl i ghtl y more cel l ul ose hydrol yzed at 60 mi n i n mal ei c
aci d (13.8%) than i n sul furi c aci d (8.23%). The resul ts of
greatest i mportance are % gl ucan as gl ucose (i .e., %
yi el d col umn). Thi s number represents the mol ar mass
of measured gl ucose di vi ded by the change i n mol ar mass
of cel l ul ose. The mol ar mass i s cal cul ated by descri bi ng
cel l ul ose as gl ucan, or anhydrogl ucose, to si mpl i fy the
varyi ng degrees of pol ymeri zati on of the i ndi vi dual
cel l ul ose chai ns wi thi n each parti cl e:
The remai ni ng percent change i n cel l ul ose mol ar mass
i s l ost as gl ucose degradati on products. Al though the
percent hydrol ysi s i s nearl y i denti cal for both aci ds, the
Figure5. Measured gl ucose yi el d from decomposed cel l obi ose
vs aci d concentrati on at 160 C, 30 mi n hol d ti me.
MW
gl ucan
) MW
gl ucose
- MW
water
;
162.2 ) 180.2 - 18.0 (6)
478 Biotechnol. Prog., 2001, Vol. 17, No. 3
percent gl ucose yi el ded from hydrol ysi s i s much hi gher
for mal ei c aci d. The data suggest that the rate of Avi cel
hydrol ysi s i s nearl y i denti cal for both 50 mM mal ei c and
sul furi c aci ds, whereas the rate of gl ucose degradati on
i s much l ower for mal ei c aci d than sul furi c aci d. Thi s i s
cl ear evi dence of the superi ori ty of mal ei c aci d for the
hydrol ysi s of cel l ul ose compared wi th sul furi c aci d. Fi gure
6 shows thi s l arge di fference i n gl ucose from the nearl y
equi val ent hydrol ysi s of Avi cel between the two aci ds.
At 30 mi n, 65.6%of the decrease i n cel l ul ose mol ar mass
i s recovered as gl ucose through mal ei c aci d hydrol ysi s
compared wi th 25.7% for sul furi c aci d hydrol ysi s. At the
60 and 180 mi n i nterval s the percent recovered as gl ucose
decreases for both aci ds. Thi s i s presumabl y because
hydrol ysi s rate sl ows as the more easi l y hydrol yzed
cel l ul ose i s removed l eavi ng i ncreasi ngl y recal ci trant
materi al . Al though the rate of hydrol ysi s that generates
gl ucose i s reduced, the rate of gl ucose degradati on
remai ns unchanged. At the poi nt the rate of gl ucose
generati on i s l ess than the rate of gl ucose degradati on,
the gl ucose concentrati on decreases (note the drop i n
gl ucose concentrati on after reachi ng a peak i n Fi gure 6).
However, the l arge vari abi l i ty i n gl ucose concentrati ons
at 60 and 180 mi n wi thi n the four repl i cates prompted
further i nvesti gati on. To determi ne i f sul furi c aci d de-
graded gl ucose more si gni fi cantl y than mal ei c aci d, the
same experi mental apparatus was used to test four
repl i cates of each aci d on sol uti ons of pure gl ucose. After
30 mi n at 175 C, between 84% and 93% of the gl ucose
(5 g L
-1
i ni ti al concentrati on) was degraded i n the
presence of 50 mM sul furi c aci d, whereas onl y between
13%and 17%of the gl ucose was degraded i n the presence
of 50 mM mal ei c aci d.
Conclusion
Di l ute mal ei c aci d, a di carboxyl i c aci d, has been shown
to hydrol yze cel l obi ose and cel l ul ose as effecti vel y as
di l ute sul furi c aci d. Hi gher gl ucose yi el ds from cel l ul ose
hydrol ysi s al so suggest that mal ei c aci d does not degrade
gl ucose as easi l y as sul furi c aci d. Thi s i s si gni fi cant
because l ess gl ucose degradati on by mal ei c aci d woul d
resul t i n hi gher gl ucose yi el ds from a cel l ul osi c bi omass,
whi ch woul d be more easi l y fermented as a resul t of l ower
concentrati ons of fermentati on-i nhi bi ti ng degradati on
products. Further work i nto the ki neti cs of cel l obi ose and
cel l ul ose hydrol ysi s and the ki neti cs of gl ucose degrada-
ti on for these aci ds i s needed to confi rm these hypotheses
and to devel op a model for predi cti ng gl ucose yi el ds from
bi omass hydrol ysi s. Such work i s requi red for opti mi zi ng
the cost of bi omass conversi on usi ng mal ei c aci d.
Acknowledgment
We woul d l i ke to thank Crai g Kei m and Kyl e Beery
for thei r hel pful comments on thi s manuscri pt. Thi s
materi al i s based upon work supported by the Nati onal
Sci ence Foundati on under grant BES-9727096.
References and Notes
(1) Lynd, L. R.; El ander, R. T.; Wyman, C. E. Li kel y Features
and Costs of Mature Bi omass Ethanol Technol ogy. Appl.
Biochem. Biotechnol. 1996, 57-8, 741-761.
(2) Saeman, J. F. Ki neti cs of Wood Sacchari fi cati on: Hydrol ysi s
of Cel l ul ose and Decomposi ti on of Sugars i n Di l ute Aci d at
Hi gh Temperatures. I nd. Eng. Chem. 1945, 37(1), 43-52.
Figure 6. Compari son of measured Avi cel di sappearance and gl ucose appearance from hydrol ysi s at 175 C.
Table 2. Results of Avicel Hydrolysis at 175C
ti me
(mi n) compound
concn
(g/L)
-gl ucan
(g/L) std dev
%
hydrol ysi s
%
yi el d
50 mM Mal ei c Aci d
0 cel l ul ose 35.10 0.00 1.7977 0.0
30 cel l ul ose 32.30 2.80 0.8203 8.0
60 cel l ul ose 30.26 4.84 0.4656 14.0
180 cel l ul ose 28.21 6.89 0.6385 20.0
0 gl ucose 0.00 0.00 0.0000 n/a
30 gl ucose 2.27 2.04 0.2256 73.0
60 gl ucose 2.55 2.30 0.4538 48.0
180 gl ucose 2.35 2.12 0.9712 31.0
50 mM Sul furi c Aci d
0 cel l ul ose 32.74 0.00 0.0129 0.0
30 cel l ul ose 30.54 2.20 0.0096 7.0
60 cel l ul ose 30.04 2.69 0.0040 8.0
180 cel l ul ose 26.76 5.98 0.0112 18.0
0 gl ucose 0.19 0.18 0.0559 n/a
30 gl ucose 0.82 0.57 0.4715 26.0
60 gl ucose 0.45 0.23 0.1299 9.0
180 gl ucose 0.37 0.16 0.0245 3.0
Biotechnol. Prog., 2001, Vol. 17, No. 3 479
(3) McKi bbi ns, S. W.; Harri s, J. F.; Saeman, J. F.; Nei l l , W. K.
Ki neti cs of the Aci d-Catal yzed Conversi on of Gl ucose to
5-Hydroxymethyl -2Fural dehyde and Levul i ni c Aci d. Forest
Products J . 1962, 12, 17.
(4) Bi enkowski , P. R.; Ladi sch, M. R.; Narayan, R.; Tsao, G.
T.; Eckert, R. Correl ati on of Gl ucose (Dextrose) Degradati on
at 90 to 190 C i n 0.4 to 20% Aci d. Chem. Eng. Commun.
1987, 51, 179-192.
(5) Del genes, J. P.; Mol etta, R.; Navarro, J. M. Effects of
Li gnocel l ul ose Degradati on Products on Ethanol Fermenta-
ti ons of Gl ucose and Xyl ose by Saccharomyces cerevisiae,
Zymomonas mobilis, Pichia stipitis, and Candida shehatae.
EnzymeMicrob. Technol. 1996, 19, 220-225.
(6) Taherzadeh, M. J.; Ni kl asson, C.; Li nden, G. Conversi on of
Di l ute Aci d Hydrol yzate of Spruce and Bi rch to Ethanol by
Fed-Batch Fermentati on. Bioresour. Technol. 1999, 69, 59-
66.
(7) Larsson, S.; Pal mqvi st, E.; Hahn-Hagerdal , B.; Tengborg,
C.; Stenberg, K.; Zacchi , G.; Ni l vebrant, N.-O. The Generati on
of Fermentati on I nhi bi tors Duri ng Di l ute Aci d Hydrol ysi s of
Softwood. EnzymeMicrob. Technol. 1999, 24, 151-159.
(8) Jeffri es, T. W.; Sreenath, H. K. Producti on of Ethanol from
Wood Hydrol yzate by Yeasts. Bioresour. Technol. 2000, 72,
253-260.
(9) Gol dstei n, I . S.; Easter, J. M. An I mproved Process for
Converti ng Cel l ul ose to Ethanol . TAPPI J . 1992, 75(8), 135-
140.
(10) Gol dstei n, I . S.; Perei ra, H.; Pi ttman, J. L.; Strouse, B. A.;
Scari ngel l i , F. P. The Hydrol ysi s of Cel l ul ose wi th Supercon-
centrated Hydrochl ori c Aci d. Biotechnol. Bioeng. 1983, 13,
17-25.
(11) Mosi er, N. S.; Hal l , P.; Ladi sch, C. M.; Ladi sch, M. R.
Reacti on Ki neti cs, Mol ecul ar Acti on, and Mechani sms of
Cel l ul ol yti c Protei ns. Adv. Biochem. Eng./ Biotechnol. 1999,
65, 23-40.
(12) Mal ester, I . A.; Green, M.; Shel ef, G. Ki neti cs of Di l ute Aci d
Hydrol ysi s of Cel l ul ose Ori gi nati ng from Muni ci pal Sol i d
Wastes. I nd. Eng. Chem. 1992, 31, 1998-2003.
(13) Ott, E.; Spurl i n, H.; Graffl i n M.; Mark, H. Structures and
Properti es of Cel l ul ose Fi bers. I n Cellulose and Cellulose
Derivatives, 2nd ed.; I ntersci ence Publ i shers: New York,
1954; Vol . 1, Chapter 4, pp 217-300.
(14) Fagan, R. D.; Converse, O.; Grethl ei n, H. E.; Porteous, A.
Ki neti cs of the Aci d Hydrol ysi s of Cel l ul ose Found i n Paper
Refuse. Environ. Sci. Technol. 1971, 5(6), 545-547.
(15) Church, J. A.; Wool dri dge, D. Conti nuous Hi gh-Sol i ds Aci d
Hydrol ysi s of Bi omass i n a 1.5 i n. Pl ug Fl ow Reactor. I nd.
Eng. Chem. Prod. Res. Dev. 1981, 20(2), 371-378.
(16) McParl and, J. J.; Grethl ei n, H. E.; Converse, A. O. Ki neti cs
of Aci d Hydrol ysi s of Corn Stover. Solar Energy 1982, 28(1),
55-63.
(17) Bhandari , N.; MacDonal d, D. G.; Bakhshi , N. N. Ki neti c
Studi es of Corn Stover Sacchari fi cati on Usi ng Sul phuri c Aci d.
Biotechnol. Bioeng. 1984, 26, 320-327.
(18) Si di ras, D. K.; Kouki os, E. G. Aci d Sacchari fi cati on of Bal l -
Mi l l ed Straw. Biomass 1989, 19(4), 289-306.
(19) Baugh, K. D.; Levy, J. A.; McCarty, P. L. Thermochemi cal
pretreatment of l i gnocel l ul ose to enhance methane fermenta-
ti on: I I . Eval uati on and appl i cati on of pretreatment model .
Biotechnol. Bioeng. 1988, 31, 62-70.
(20) Bobl eter, O.; Bonn, G. The Hydrothermol ysi s of Cel l obi ose
and i ts Reacti on Product D-Gl ucose. Carbohydr. Res. 1983,
124, 185-193.
(21) Fel thouse, T. R.; Burnett, J. C.; Mi tchel l , S. F.; Mummey,
M. J. Mal ei c Anhydri de, Mal ei c and Fumari c Aci d. I n Kirk-
Othmer Encyclopedia of Chemical Technology, 4th ed.;
Kroschwi tz, J. I ., Howe-Grant, M., Eds.; John Wi l ey & Sons:
New York, 1995; Vol . 15, pp 893-928.
(22) Chemical Market Reporter; Feb 14, 2000 through Jun 5,
2000.
(23) Lee, Y.-H.; Fan, L. T. Ki neti c Studi es of Enzymati c
Hydrol ysi s of I nsol ubl e Cel l ul ose I I . Biotechnol. Bioeng. 1983,
25, 939-966.
(24) Ni detzky, B.; Stei ner, W. A New Approach for Model i ng
Cel l ul ase-Cel l ul ose Adsorpti on and the Ki neti cs of the En-
zymati c Hydrol ysi s of Mi crocrystal l i ne Cel l ul ose. Biotechnol.
Bioeng. 1993, 42, 469-479.
(25) Kohl mann, K. L.; Sari kaya, A.; Westgate, P. J.; Wei l , J.;
Vel ayudhan, A.; Hendri ckson, R.; Ladi sch, M. R. Enhanced
enzyme acti vi ti es on hydrated l i gnocel l ul osi c substrates. I n
EnzymaticDegradation of I nsolubleCarbohydrates; Saddl er,
J. N., Penner, M. H. Eds.; ACS Symposi um Seri es 618;
Ameri can Chemi cal Soci ety: Washi nton DC, 1995; pp 237-
255.
Accepted for publ i cati on March 27, 2001.
BP010028U
480 Biotechnol. Prog., 2001, Vol. 17, No. 3