Académique Documents
Professionnel Documents
Culture Documents
Universidad de Valladolid
INFORME DE PRCTICAS
TRABAJO BIBLIOGRFICO
GRUPO: B
PAREJA: 7
TIEMPO EMPLEADO
ALUMNO
EN INFORME
10 h
10 h
FECHA: 24/10/2011
INDICE
4. Bibliografa ........................................................................................................... 12
5. Anexo .................................................................................................................. 15
1. DEFINICIN
Son aquellos reactores que combinan la reaccin y la separacin en una sola unidad;
la membrana selectivamente remueve una (o ms) de las especies reactantes o
productos.
Estos reactores han sido comnmente usados para incrementar el rendimiento y la
selectividad de reacciones enzimticas y catalticas influyendo a travs de la
membrana sobre la concentracin de una o ms especies intermedias, removindolas
selectivamente o ayudando a mantenerlas en una concentracin baja mediante el
filtrado continuo de productos, evitando la posibilidad de que dichos compuestos
envenenen o desactiven el catalizador y para proveer una interfase controlada entre
dos o ms reactantes.
2. TIPOS DE REACTORES DE MEMBRANA
Reactor de membrana densa Pd-Ag
Es un tipo destacado de membrana inorgnica. El paladio y sus aleaciones son
permeables al hidrgeno, en su forma de biocombustible para producir energa.
Existen numerosos estudios y casos documentados del uso de este tipo de
membranas.[11-13]
stos se emplean en diversas reacciones para producir, y a la vez separar los
productos generados en esa reaccin. Especialmente tiles son las membranas Pd-Ag
densas, ya que permiten conseguir directamente un producto ultrapuro sin ser
necesario el uso de algn otro mtodo de purificacin [14-18]
Estos reactores tambin se pueden emplear en el reformado de metanol y etanol de
forma simultnea
Reactor de membrana orgnica
La membrana de stos es de carbono normalmente polimrico, tienen gran inters ya
que son estables a temperaturas elevadas y resisten bastante bien los ataques
qumicos, sobre todo medios muy cidos y medios bsicos [21-23]
Reactor de membrana inorgnica
Este tipo de reactores se utiliza mayoritariamente en reacciones en fase lquida con
disolvente inorgnico. Debido a la excelente estabilidad trmica de la membrana, estos
reactores pueden ser empleados en reacciones a altas temperaturas. Estas
membranas son muy resistentes al ataque qumico y existen una gran cantidad de
materiales que pueden ser utilizados en la fabricacin de estas membranas. Para
entornos muy hostiles son el nico tipo de membrana que se puede utilizar [21-23]
(1)
(2)
(3)
Sealar por otra parte, que los resultados que se mostrarn a continuacin provienen
del estudio del proceso de reformado basado en un modelo terico, hecho que no
haba sido desarrollado anteriormente.
Resultados y discusin
En primer lugar se procede a validar el modelo terico utilizado para comparar ambos
reactores y se emplea la grfica 1. Usando los datos experimentales proporcionados
[2]. para reactores tradicionales se pudo validar dicho modelo para el rango de
temperaturas desde 673 K a 873 K y una relacin molar entre agua y etanol de 0 a 17
kg catalizador/(mol/s). Una vez realizada la experimentacin se demuestra que el
modelo terico y los resultados experimentales muestran una gran concordancia para
todo el rango de parmetros investigados.
Sin embargo la comparacin entre reactores de membrana y tradicionales no es
posible por carecer de datos de el reformado de etanol en reactores de membrana.
Atendiendo a la temperatura
Atendiendo a la presin
Claves en la operacin
Una solucin podra ser usar reactores de membrana que mediante el uso de enzimas
no inmovilizadas en la membrana del reactor y retirando continuamente los productos
de reaccin se consigue evitar en gran medida los problemas de inhibicin.
La grfica nos [29] permite observar que para tiempos de hidrlisis elevados la
inhibicin es baja, ya que el rendimiento de la operacin (ordenada) aumenta con el
tiempo segn la relacin molar de la concentracin de inhibidor y la concentracin de
glucosa.
No obstante, a tiempos de reaccin bajos el rendimiento es menor por lo cual los
mtodos para operar en continuo con elevado rendimiento, como aqu se habla, son
muy apreciados.
El empleo de un reactor de membrana para este proceso, permite una alimentacin
continua de substrato y una retirada continua de producto sin prdida de enzimas[3335] E incluye[30]:
a) El uso de enzimas celulticas por largos periodos de tiempo al ser retenidas en
el sistema.
b) La obtencin de una mayor conversin de los productos al reducir su tasa de
inhibicin.
c) La obtencin de productos de hidrlisis puros, libres de contaminantes como
enzimas, sustrato sin convertir y otros residuos e impurezas del proceso que de
otra manera disminuiran el rendimiento de la hidrlisis.
d) La posibilidad de mantener una corriente de concentracin constante de
productos sin aadir nuevas enzimas.
Por otra parte, el usar reactores de membrana nos es muy adecuado en cuanto al
proceso de retirada de glucosa ya que a concentraciones muy bajas revierte en una
concentracin final de etanol baja en la mezcla de fermentacin y provoca unos costes
elevados de destilacin para la recuperacin de etanol.
Para que la operacin se mantenga con unas concentraciones de etanol finales
aceptables el volumen de reaccin debe ser constante; lo cual, obliga a mantener una
operacin continua y simultnea de alimentacin y filtrado en el reactor. Tambin
obliga a una carga de alimentacin no demasiado elevada ya que puede haber
problemas con la resistencia de la membrana y con la polarizacin de las enzimas, lo
cual revierte en una reduccin del flujo a travs de la membrana.
La alta viscosidad de la alimentacin de lignocelulosa entorpece la mezcla perfecta y
la transferencia de materia y es un problema grave a altas concentraciones de
alimentacin.
10
Por estas razones dadas, no se puede hidrolizar de forma continua y a gran escala la
lignocelulosa.
El empleo de reactores de membrana produce un beneficio en cuanto a conversin ya
que comparndolo con procesos batch, la diferencia de conversiones es superior a un
40%. En la mayora de los casos, el grado final de conversin ronda el 70-90%[35-36]c.
11
4. BIBLIOGRAFA
[1] K Liguras D, Kondarides DI, Verykios XE. Production of hydrogen for fuel cells by
steam reforming of ethanol over supported noble metal catalysts. Appl Catal 2003;43:
34554.
[2] Sahoo DR, Shilpi Vajpai, Sanjay Patel, Pant KK. Kinetic modelling of steam
reforming of ethanol for the production of hydrogen over Co/Al2O3 catalyst. Chem Eng
J 2007;125(3):13947.
[3] Freni S, Mondello N, Cavallaro S, Cacciola G, Pardon VN, Sobyanin VA. Hydrogen
production by steam reforming of ethanol: a two process. React Kinet Catal Lett
2000;71:14352.
[4] Srinivas D, Satyanarayana CVV, Potdar HS, Ratnasamy P. Structural studies on
NiOCeO2ZrO2 catalysts for steam reforming of ethanol. Appl Catal 2003;246:323
34.
[5] Llorca J, Homs N, Sales J, de la Piscina PR. Efficient production of hydrogen over
supported cobalt catalysts from ethanol steam reforming. J Catal 2002;209:30617.
[6] Haga F, Nakajima T, Yamashita K, Mishima S. Effect of crystallite size on the
catalysis of alumina-supported cobalt catalyst for steam reforming of ethanol. React
Kinet Catal Lett 1998;63:2539.
[7] Kaddouri A, Mazzocchia C. A study of the influence of the synthesis conditions upon
the catalytic properties of Co/SiO2 or Co/Al2O3 catalysts used for ethanol steam
reforming. Catal Commun 2004;5:33945.
12
13
[24]Liu, J., 2004. Biodiesel Production from Canola Oil Using a Membrane Reactor.
M.A.Sc. Thesis, Department of Chemical Engineering, University of Ottawa, Canada0
[25]Liu, K., 1994. Preparation of fatty acid methyl esters for gas-chromatographic
analysis of lipids in biological materials. J. Am. Oil Chem. Soc. 71, 11791187.
[26]Freedman, B., Pryde, E.H., Mounts, T.L., 1984. Variables affecting the yields of
fatty esters from transesterified vegetable oils. J. Am. Oil Chem. Soc. 61, 16381643
[27]Gan et al., 2003 Q. Gan, S.J. Allen and G. Taylor, Kinetic dynamics in
heterogeneous enzymatic hydrolysis of cellulose: an overview, an experimental study
and mathematical modeling. Process Biochem, 38 (2003), pp. 10031018.
[28]Gusakov et al., 1987 A.V. Gusakov, A.P. Sinitsyn and A.A. Klyosov, Factors
affecting the enzymatic hydrolysis of cellulose in batch and continuous reactors :
computer simulation and experiment. Biotechnol Bioeng, 40 (1987), pp. 663671.
[29]Andri et al., 2010b P. Andri, P.A. Jensen, A.S. Meyer and K. Dam-Johansen
Reactor design for minimizing product inhibition during enzymatic lignocellulose
hydrolysis: I. Significance and mechanism of glucose inhibition on cellulolytic enzymes
(2010).
[30]Cantarel et al., 2009 B.L. Cantarel, P.M. Coutinho, C. Rancurel, T. Bernard, V.
Lombard and B. Henrissat, The carbohydrate-active enzymes database (CAZy): an
expert resource for glycogenomics. Nucleic Acids Res, 37 (2009), pp. D233D238.
[31]Hahn-Hgerdal et al., 1981 B. Hahn-Hgerdal, E. Andersson, M. Lpez-Leiva and
B. Mattiasson, Membrane biotechnology, co-immobilization, and aqueous two-phase
systems: alternatives in bioconversion of cellulose. Biotechnol Bioeng Symp, 11
(1981), pp. 651661.
[32]Blafi-Bak et al., 2006 K. Blafi-Bak, A. Koutinas, N. Nemestthy, L. Gubicza
and C. Webb, Continuous enzymatic cellulose hydrolysis in a tubular membrane
bioreactor. Enzyme Microb Technol, 38 (2006), pp. 155161.
[33]Hong et al., 1981 J. Hong, G.T. Tsao and P.C. Wankat, Membrane reactor for
enzymatic hydrolysis of cellobiose. Biotechnol Bioeng, 23 (1981), pp. 15011506.
[34]Yang et al., 2006 S. Yang, W. Ding and H. Chen, Enzymatic hydrolysis of rice straw
in a tubular reactor coupled with UF membrane. Process Biochem, 41 (2006), pp.
721
[35]Alfani et al., 1983 F. Alfani, M. Cantarella and V. Scardi, Use of a
membrane reactor for studying enzymatic hydrolysis of cellulose. J Membr Sci, 16
(1983), pp. 407416.
[36]Henley et al., 1980 R.G. Henley, R.Y.K. Yang and P.F. Greenfield, Enzymatic
saccharification of cellulose in membrane reactors . Enzyme Microb Technol, 2
(1980), pp. 206208.
[37]Knutsen & Davis, 2004 J.S. Knutsen and R.H. Davis, Cellulase retention and sugar
removal by membrane ultrafiltration during lignocellulosic biomass hydrolysis. Appl
Biochem Biotechnol, 113116 (2004), pp. 585599.
14
5.- ANEXO
15
Biotechnology Advances
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b i o t e c h a d v
a r t i c l e
i n f o
Article history:
Received 14 October 2009
Received in revised form 26 January 2010
Accepted 13 February 2010
Available online 19 February 2010
Keywords:
Membrane reactors
Glucose removal
Enzyme inhibition
Hydrolysis kinetics
a b s t r a c t
Product inhibition of cellulolytic enzymes affects the efciency of the biocatalytic conversion of
lignocellulosic biomass to ethanol and other valuable products. New strategies that focus on reactor designs
encompassing product removal, notably glucose removal, during enzymatic cellulose conversion are
required for alleviation of glucose product inhibition. Supported by numerous calculations this review
assesses the quantitative aspects of glucose product inhibition on enzyme-catalyzed cellulose degradation
rates. The signicance of glucose product inhibition on dimensioning of different ideal reactor types, i.e.
batch, continuous stirred, and plug-ow, is illustrated quantitatively by modeling different extents of
cellulose conversion at different reaction conditions. The main operational challenges of membrane reactors
for lignocellulose conversion are highlighted. Key membrane reactor features, including system set-up,
dilution rate, glucose output prole, and the problem of cellobiose are examined to illustrate the quantitative
signicance of the glucose product inhibition and the total glucose concentration on the cellulolytic
conversion rate. Comprehensive overviews of the available literature data for glucose removal by
membranes and for cellulose enzyme stability in membrane reactors are given. The treatise clearly shows
that membrane reactors allowing continuous, complete, glucose removal during enzymatic cellulose
hydrolysis, can provide for both higher cellulose hydrolysis rates and higher enzyme usage efciency
(kgproduct/kgenzyme). Current membrane reactor designs are however not feasible for large scale operations.
The report emphasizes that the industrial realization of cellulosic ethanol requires more focus on the
operational feasibility within the different hydrolysis reactor designs, notably for membrane reactors, to
achieve efcient enzyme-catalyzed cellulose degradation.
2010 Elsevier Inc. All rights reserved.
Contents
1.
2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.
Inuence of product inhibition on enzyme-catalyzed rates . . . . . . . . . . . . . . . . . . .
1.2.
Dimensioning of ideal continuous reactors for enzymatic degradation of (ligno)cellulose . . . .
1.3.
Glucose formation rates at different lignocellulose dry matter contents (DM%) in a batch reactor
Design of membrane reactors for hydrolysis products removal . . . . . . . . . . . . . . . . . . . .
2.1.
Membrane bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Membrane bioreactors for lignocellulose hydrolysis: key issues . . . . . . . . . . . . . . . .
2.3.
Quantitative effects of product removal on the cellulolytic hydrolysis rates and extent of cellulose
Membrane reactors operation strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Product removal strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
System set-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
Enzyme retention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.
Specic design features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
conversion
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
408
408
410
411
412
412
412
413
414
414
414
415
416
408
4.
Key factors inuencing membrane reactor performance for enzymatic cellulose hydrolysis . . . .
4.1.
Glucose output prole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
The effect of dilution rate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
The problem of cellobiose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.
Factors affecting the membrane ux . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
Fouling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
Reaction slurry properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.
Molecular cut-off . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.
Concentration polarization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.
Membrane reactors for glucose removal during (ligno)cellulose hydrolysis: operational challenges
6.1.
The glucose concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.
Fed-batch operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3.
Continuous operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4.
Enzyme activity retainment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.
Membrane reactors and hydrolysis kinetic studies . . . . . . . . . . . . . . . . . . . . . . .
7.1.
General kinetic studies using membrane reactors . . . . . . . . . . . . . . . . . . . .
7.2.
Product inhibition studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.
Other techniques for glucose removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.1.
Two-phase systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.2.
Dialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.3.
Simultaneous hydrolysis and fermentation (SSF) and removal of ethanol . . . . . . . . .
9.
Membrane bioreactor design for glucose removal: conclusions and recommendations . . . . . .
9.1.
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.2.
Advantages and challenges of membrane reactors . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Product inhibition of cellulases by cellobiose and glucose has long
been known to signicantly retard the rates of enzyme-catalyzed
cellulose hydrolysis (Gan et al., 2003; Gusakov et al., 1987). This
inhibition constitutes a main obstacle for achieving efcient enzymatic degradation of cellulose and high glucose yields in current
lignocellulose-to-ethanol processing schemes (Andri et al., 2010a;
Bla-Bak et al., 2006; Xiao et al., 2004). The product inhibition of
cellulolytic enzymes also affects the efciency of other processes
involving conversion of lignocellulosic biomass to valuable products.
Alleviation of this product inhibition, notably the inhibition by the
hydrolysis end-product glucose, is therefore a key prerequisite for
achieving cost-efcient conversion of lignocellulosic biomass to
biofuels notably bioethanol and biobutanol and other valuable
products such as platform biochemicals. A number of glucose tolerant
fungal -glucosidases, produced by various Aspergillus spp. and e.g.
Humicola insolens, have been identied relatively recently (Decker
et al., 2001; Sonia et al., 2008), but the prospects of developing and
using glucose tolerant enzymes seem to receive surprisingly limited
attention in the commercial enzyme development for biomass
utilization. Rather, the industrial focus has mainly been on reducing
the enzyme costs by improving the efciency of known enzymes,
identifying new, more active enzymes, creating optimal enzyme
mixtures for selected pre-treated substrates, and on minimizing the
enzyme production costs (Merino and Cherry, 2007; Rosgaard et al.,
2007b). A careful analysis of the mechanisms and kinetics of the
product inhibition induced by glucose and cellobiose on microbial
cellulases and -glucosidase has substantiated that reactor designs
which involve continuous or semi-continuous product removal
notably glucose removal must be at the core of future-directed
design strategies for lignocellulose-to-ethanol processes (Andri et al.,
2010b).
Simultaneous saccharifaction and fermentation (SSF), with or
without separate fermentation of pentose monosaccharides, is
considered a main technology scenario in current biomass-to-ethanol
processes (Hahn-Hgerdal et al., 2006; Lynd et al., 2008). Although
alleviation of product inhibition is a rationale for SSF, it seems to have
been overlooked that the efciency of this technology is restricted by
the inhibition that the ethanol exerts on the cellulolytic enzymes
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
416
417
417
417
418
418
418
418
418
419
419
419
419
420
421
421
421
421
421
422
422
423
423
423
424
cl
= 1
Ecl
1
= 1
I
E0
1+
KI
f pr = 1
Epr
1
= 1
P
E0
1+
KIP
dG
dt
dG
dt
nkcat
E0 S
KM + S 1 +
nkcat
E0 S
KM + S 1 +
P
KIP
I
KI
and
409
pr
E
E
0
=
=
S
P
P
1+
1+
1+
KM
KIP
KIP
These two types of inhibitors will exhibit the same effect on the
catalysis rate at the point where P = I (in Fig. 1 this point is 0.17 g/g or
0.01 M). Since the product concentration increases during the
enzyme-catalyzed reaction starting from virtually 0 and then
gradually approaching a maximal concentration at the maximum
extent of conversion the enzyme will experience a range of product
concentrations in succession that are (usually) higher than the
concentration of I. The efciencies of enzyme-catalyzed reactions at
high extents of substrate conversion are thus signicantly affected in
both batch and continuous processes when sensitive to product inhibition. Since high product concentrations are required in the
prospected large scale lignocellulosic conversion processes it must
be anticipated that the product inhibition will signicantly retard the
hydrolysis reaction rates.
It is important to note that the progressive feature of product
inhibition is the reason why it is often neglected in initial rate enzyme
kinetics inhibition studies. Nevertheless, the signicant inuence of
the product concentration increment in product inhibition is exactly
the reason why design of reactors that involve continuous or semicontinuous removal of the products from the enzyme-catalyzed
reaction during the reaction must be considered in industrial-scale
biomass processing demanding high conversion degrees.
Cellobiose exerts the strongest inhibition effect on cellulase
activity with typical KI ranges between 0.01 and 6 g/L (Andri et al.,
2010b). However, this inhibition is usually alleviated by adjusting the
dosing of -glucosidase (EC 3.2.1.21) so that the cellobiose is rapidly
hydrolysed to glucose. Unfortunately, glucose also inhibits cellulase
activities, with reported overall KI ranges varying widely from 0.1 to
70 g/L the variation depending mainly on the experimental
conditions. Glucose also exerts a strong inhibition on the activity of
-glucosidases with reported KI values typically ranging from 0.1 to
0.8 g/L (Andri et al., 2010b).
The effect that glucose exerts as a product of cellulose degradation
on the lignocellulose enzymatic conversion may formally be classied
as medium when the molar ratio KM/KIP = 0.14, or very strong, with
mass ratio KM/KIP = 58 where IP indicates glucose as product
inhibitor. These effect estimates are based on parameters published
previously (Andri et al., 2010a) and a classication given by Riebel
and Bommarius (2004). Even for the medium effect (molar KM/KIP =
0.14) the effect of the inhibition on the hydrolysis rate and the glucose
yield is considerable (Fig. 2). When KM/KIP = 10, the reaction is almost
halted, requiring a massively extended reaction time to increase the
yields (Fig. 2).
The physical meaning of the inhibition constant KI or KIP may be
interpreted in a similar fashion as the Michaelis constant KM.
The KM designates the initial substrate concentration (S0) at which
the initial reaction rate (v0) is exactly equal to of the maximal rate
Vmax:
on the enzymatic
reaction model simulation of a batch reaction. The product formation rates are given
as the percentage of the initial uninhibited rate (t = 0; I, P (yield) = 0). Model
parameters and constants: k c at = 12 h 1 , K M = 0.9 mM, K IP = K I = 6.4 mM;
S0 = 0.14 mM, E0 = 0.01 mM, MS = 73566 g/mol, MP = MI = 180 g/mol; I = 0.01 M.
Figure legend:
I classical inhibitor
P product inhibitor
No
inhibition.
v0 =
kcat E0 S0
k E S
V
= cat 0 0 = max
KM + S0
S0 + S0
2
410
Fig. 2. Effect of supposed glucose inhibition power on glucose yield. Model simulation.
Model parameters and constants are given in Fig. 1, except KI which is varied according
to a desired molar KM/KI ratio. The real molar KM/KI ratio is equal to 0.14 (Andri et al.,
2010a). Figure legend:
KM/KI = 10
KM/KI = 1
KM/KI = 0.14
KM/
KM/KI = 0.01
KM/KI = 0.
KI = 0.1
dP
kcat E0 S0
=
dt 0
KM + S0 1 +
P0
KIP
=
KM
kcat E0 S0
+ S0 1 +
P0
P0
=
vP;0
2
kcat E0 S0
KM + S0
411
Table 1
Comparison of ideal reactor sizes for conversion of lignocelluloses (hydrothermally pre-treated wheat straw) based on experimental results in batch reactor: 30% cellulose
conversion, 3.6 g/L glucose outlet/nal concentration, r = 1.1v = 0.23 g/(L h) (Andri et al., 2010a). The CSTR and PFR were calculated from the design equations based on the
experimentally determined r from the batch reactor (at t = 6 h). The predetermined ratio of the reactors' height to diameter (H/D) for BR/CSTR and reactor's length to diameter (L/D)
for PFR is equal to 1. Reaction conditions: pH 5, 50 C, enzyme dosage 8 FPU/gDM and 13 CBU/gDM; substrate 2% DM content (48% cellulose), S0 = 10 g/L.
Ideal reactor type
Batch
(BR)
Design equation and reaction/residence time [h]
Continuous stirred
(CSTR)
S
tBR = dS = 6 h
S0
CSTR =
S0 S
v
Plug-ow
(PFR)
S
= 15:6 h
PFR = dS = 6 hr
S0
1 10 2
1
1000
1 10 2
1
1000
Volumeb [L]
0.06 c
6
6000
0.16
15.6
15 600
0.06
6
6000
0.6 d
0.23
0.6
0.043
0.2
2
0.058
0.27
2.7
0.043
0.2
2
1.2 10 6
9.1 10 6
9.1 10 5
a
b
c
d
Fixed.
From reactors design equation.
Approximate size of BR used in inhibition study (Andri et al., 2010a).
Productivity in batch mode.
Table 2
Comparison of ideal continuous reactor sizes for conversion of lignocelluloses at
different conversion degrees and inuence of glucose inhibition on reactor dimension,
for the given ow rate of 1000 L/h. Model simulation. Model parameters and constants
are given in Fig. 1. For a special case where inhibition is excluded from simulation, KIP
(for glucose) = 0. The reaction conditions and other data are given in Table 1. rcalc
glucose formation rate obtained from non-competitive kinetic model (Andri et al.,
2010a).
Conversion
[%]
Glucose
[g/L]
rcalc
[g/(L h)]
Plug-ow (PFR)
0.51
0.27
0.03
2.9
11.5
293
1.9
6.5
75
1.3
3.2
31
1.2
2.7
12.8
412
rough rule of thumb is, therefore, that the glucose should be removed to
at least below 10 g/L in order to drastically regain a heavily inhibited
glucose production rate. It is important to note that this extent of
removal will be independent of the employed substrate concentration. If
the remaining glucose concentration is much above a glucose level of
10 g/L even with glucose removal, the relative ratio of glucose:enzyme
will usually be so high that the positive effect of the glucose removal will
be insignicant (Fig. 3). These relations may explain why many product
removal studies have failed to obtain prominent effects on the
cellulolytic hydrolysis rates (see discussion below).
2. Design of membrane reactors for hydrolysis products removal
In-situ product removal by integration of the biocatalysis reactor
with a separation unit (reactionseparation hybrids) has shown
promising results with product inhibited or equilibrium limited
enzyme-catalyzed conversions (Ahmed et al., 2001; Gan et al., 2002).
On this background, the introduction of membrane (bio)reactors seems
to be one of the obvious approaches to accomplish simultaneous in-situ
removal of glucose during enzymatic hydrolysis of lignocellulose.
2.1. Membrane bioreactors
A membrane (bio-)reactor is a multifunction reactor that combines the reaction with a separation, namely in this case product
removal by membrane separation, in one integrated unit, i.e. in-situ
removal, or alternatively in two or more separate units. In practice,
the hitherto used membrane bioreactors in enzyme technology have
mainly employed ultra- and nanoltration for the separation (Drioli,
2004; Pinelo et al., 2009).
Ultraltration membrane reactors were rst used in conjunction
with development of novel enzyme immobilization techniques.
However, immobilized enzymes are not suitable for insoluble,
polymeric substrates, and this will include lignocellulose, due to the
necessary enzyme adsorption on the macromoleculer substrate
particles that becomes severely mass transfer limited with immobilized enzymes (Alfani et al., 1983). The use of free, un-immobilized,
enzymes conned in membrane reactors avoids some of these
problems, and still allows continuous product removal (HahnHgerdal et al., 1981). Membrane reactors have been investigated for
use in inorganic catalytic reactions for a very long time (Sun and Khang,
1988), but have also been employed for a range of very different
biocatalysis based reactions. These applications include e.g. classical
production of citric acid by fermentation in which the product has to be
removed to maintain high production rates (Chekhova et al., 2000);
selective production of physiologically active chitosan oligosaccharides
by continuous hydrolysis of chitosan (Kuroiwa et al., 2009); production
of whey hydrolysates with low contents of phenylalanine (CabreraPadilla et al., 2009), and continuous production of pure and sterile
glucose solutions from tapioca starch powder (Sarbatly et al., 2007).
Membrane reactors also nd use in the pharmaceutical industry, e.g.
for production of S-ibuprofen (Cauwenberg et al., 1999) as well as in
waste water treatment (Meng et al., 2009). However, apart from a few
seminal studies discussed below, there is a surprising scarcity of data
on membrane reactor performance for enzymatic conversion of
lignocellulose in potential lignocellulose-to-ethanol processes.
2.2. Membrane bioreactors for lignocellulose hydrolysis: key issues
The molecular weight of glucose is 180 g/mol while the molecular
weight of most of the currently used fungal cellulases used for lignocellulose hydrolysis range from 35,000 to 65,000 g/mol (Cantarel
et al., 2009). Several studies using various fungal cellulase systems
and different cellulose substrates have conrmed that it is possible,
via membrane technology, to retain the enzymes present in the
system while allowing the transfer of low-molecular weight reaction
413
Table 3
Overview of the studies of glucose removal by UF membranes.
Enzyme feeding
Substrate feeding
MF unit/buffer
replacement
Removal method
Dilution rate
[h 1]
Cut-off
[kDa]
Vreactor
[L]
Gmax
[g/L]
Source
Start
Fed-batch
Start
Continuous1
Fed-batch2
Continuous
No/yes
In-situ, continuous
In-situ, intermittent
Separate, continuous
0.38
N/A
N/A9
30
7531
6332
N/A
0.61.9
0.310
0.20.5
0.61.3
0.060.6
N/A11
0.060.312
0213
0.120.1614
10
30
10
10
20
4/5
10
0.2
5.518
0.22 + 0.119
0.22 + 0.0320
0.260.3321,24
7
0.065
0.250.325
0.05
1026
0.1
10
N/A
0.060.25
0.4415
T
[C]
4.25.260
4.85
4.8
4.8
4.8
50
Avicel
No/yes
Start
Start
Start
Start
Start
Start
Start
Continuous
Start/continuous
Start
Start/fed-batch
Fed-batch
Fed-batch
Start/continuous
No/yes
No/yes
No/yes
No/yes
No/yes
Yes/yes6
No/yes
In situ, continuous
Separate/continuous
In situ, continuous
In situ, continuous
In-situ/continuous
Separate, intermittent
In situ, continuous
Start
Start
No/yes
Start
Start
Start
Start/fed-batch
Start4
Continuous5
Yes/yes
No/yes
No/yes7
In situ, intermittent
and continuous
Separate, intermittent
Separate, continuous
In situ, continuous
Enzyme source
Substrate type
pH
T. viride37
Solca Floc50
Solca Floc51
Solca Floc52
Cellobiose
Solca Floc53
T. viride38
T. viride39
T. reesei38,40l
A. phoenicis41
T. viride42
Sweet almonds43
T. reesei38
A. niger44
Sporotrichum
cellulophilum45
T. viride46
T. viride
T. reesei47
N/A48
T. reesei45
T. reesei49
50
33
127
1.9
3.5
0.08
9.534
18
N/A35
1112,34
2513
9.5 34
5016
10
N/A17
0.0522,28
0.0629
0.2730
2736 60
3534
N/A
DMmax
[% w/v]
E0
[% w/v]
treaction
[h]
Xmax
[%]
90
50
50
50
10
1063
0.04564
0.265
1
0.07773
0.000274
0.02275,76
81
243
0.20.584
2033
200
77
71
92
9133
80
4.8
45
0.1166
0.003377
25
5.2
Sallow54
4.8
40
1067
178
20
9485
KC Floc
55.561
37
1067
279
120
N 90
4.7
45
0.5
5062
40
45
50
50
568
2.569
1570
18.571
2.5
0.180
0.0181
3.282
3.483
588
49
82
7286
53
N 6070
44
50
207059
4.862
4.7
4.7
4.8
4.8
192
240
48
48
16836408
25
25
55
Steamed hardwood
Hardwood kraft pulp
-cellulose
-cellulose56
Corn stover57
Rice straw58
Solca Floc53
Mavicell59
23
45
67
24
46
68
250300 rpm.
810 rpm, magnetic bar.
25
500 rpm, 4-blade propeller.
26
Flat-blade impeller.
27
5090 rpm.
28
Shaking.
29
No stirring.
30
Recirculation.
31
7.5 w/v%.
32
6.3 w/v%.
33
3-stage reactor.
34
Reducing sugar.
35
3.5%, 19 h.
36
No fed-batch.
37
QM 9123.
38
SP122.
39
Isolated cellobiase, Miles labs.
40
Powder.
41
QM329 alumina immobilized.
42
B.D.H. Italia.
43
Cellobiase, BBR.
44
Novozym 188.
414
The operation of integrated membrane reactors for the simultaneous cellulose hydrolysis and removal of inhibitory product can be
performed using several different congurations (Figs. 4 and 5). The
Fig. 4. Schematic representation of the process ow sheet for simultaneous lignocellulose hydrolysis and removal of produced glucose. STR (stirred reactor) is needed for achieving
additional conversion due to available volume and/or for unconvertible fraction discharge.
415
Fig. 5. Strategies for operation of membrane reactors: 1. Integrated reaction vessel (stirred reactor) with UF membrane (Alfani et al., 1982; Alfani et al., 1983; Gan et al., 2002; Ghose
and Kostick, 1970; Hong et al., 1981; Kinoshita et al., 1986; Lee and Kim, 1993; Ohlson et al., 1984); 2. Separate reaction vessel with: (2a) UF membrane (Henley et al., 1980; model
system 2 from Ghose and Kostick (1970); combined with PBR in Yang et al. (2006)) , (2b) ordinary (model system 1 from Ghose and Kostick (1970) and special membrane reactor
(with adsorption of substrate and enzymes from Bla-Bak et al., 2006), (2c) MF and UF for recovery of bounded and soluble enzymes, respectively (Knutsen and Davis, 2004),
(2d) UF and MF membrane for recovery of enzymes and removal of lignin reach fraction, respectively (Ishihara et al., 1991); 3. Separate reaction vessel with UF membrane with shell
immobilized -glucosidases (BG), in (3a) 1-stage, and (3b) 2-stages (Klei et al., 1981). PBR packed bed reactor.
stirred cell (UF) or hollow-ber cartridge (HFC) into the CSTR-UF and
CSTR-HFC system, respectively (Henley et al., 1980) (Fig. 5, 2a).
In some of the few reports describing lignocellulose hydrolysis in
membrane reactors, an additional microltration unit has exceptionally been used to retain the unconverted lignin-rich solid fraction due
to the present tightly bound enzymes (Knutsen and Davis, 2004) or
has been employed to remove the unconverted substrate from the
reactor. These set-ups result in slightly complex process layouts for
the hydrolysis (Fig. 5, 2c and 2d).
Ishihara et al. (1991) accomplished a semi-continuous hydrolysis
reaction by using a continuously stirred reservoir tank, connected to a
suction lter unit for the removal of the lignin-rich residue and an
ultraltration membrane unit (tubular module), through which the
ltrate was pumped in order to separate the hydrolysis products from
the ltrate containing cellulases (Figs. 5, 2d). The concentration of the
lignocellullose substrate in the reactor was maintained almost
constant by addition of fresh substrate at appropriate intervals; the
lter and ultraltration units were operated intermittently, while the
enzymes were added at the start, recovered in the UF module and
recycled back into the reactor (Ishihara et al., 1991).
3.3. Enzyme retention
The retention of enzymes in the reaction system, either in situ or in
separate units, is as a rule accomplished by the membrane which is at
the same time permeable for the products. In order to retain the
enzymes in the reacting system, the combination of MF and UF units
may be used to recover the enzymes that are not rmly bound to the
substrate. Knutsen and Davis (2004) did a batch saccharication using
416
dt
dt produced in reaction 1
dt consumed in reaction 2
dG
dG
= DGout + 1:05
dt
dt produced in reaction 2
dC
dt
=
produced in reaction 1
k1 Ec SS et
1K1L L
1 + C = K1C + G = K1G KE + EC
k2 EG Eg* C
dG
1K2L L
=
dt produced in reaction 2
KM 1 + G = K2G + C
FS designates the substrate feeding rate (kg s 1), VR is the reaction volume (m3), D is the dilution rate (h 1), Ss is the concentration
of the available cellulose surface (m2 m 3) which is related to the
cellulose concentration S (kg m 3) by the specic area (m2 kg 1), C
is the cellobiose concentration (kg m 3), G is the glucose concentration (kg m 3), EC and EG are the concentration of the cellulases
and -glucosidases in the solution, respectively (kg m 3). The k1
(kg m 2 h 1) and k2 (kg CBU 1 h 1) are specic rates of reactions
1 and 2, respectively, KE is the equilibrium constant for cellulose
adsorption to cellulose (kg m 3), K1L and K2L are constants for the
cellulase and -glucosidase adsorption to lignin (m3 kg 1), respectively, L is the concentration of lignin (kg m 3), K1C, K1G and K2G are
cellobiose (C) and glucose (1G and 2G) inhibition constants for
cellulases and -glucosidases (kg m 3), E*g is -glucosidase specic
activity (CBU kg 1), KM is the MichaelisMenten constant for
cellobiose (kg m 3), and is the specic rate of decrease of effective
cellulose specic surface area (h 1) which denes the quality of the
cellulosic substrate, but can as well in general designate the
exponential decrease of the enzymatic activity with time. The above
models highlight the signicance of the product inhibition constants
for the rate of the cellobiose and glucose formation, respectively, and
are some of the few models, that include the unproductive
adsorption of the enzymes to the lignin.
417
Fig. 6. Typical glucose concentration prole in the permeate from membrane bioreactor in a: (1a) batch (Lee and Kim, 1993; Ohlson et al, 1984, Yang et al., 2006); (1b) fed-batch
(Knutsen and Davis, 2004; Kinoshita et al., 1986); (1c) batch with optimal dilution rate prole (Lee and Kim, 1993); (2a), (2b) continuous operation mode (Alfani et al., 1982; Ghose
and Kostick, 1970; Hong et al., 1981; Klei et al., 1981); and (3) the typical change of glucose permeate prole with increase in dilution rate in the batch mode (Kinoshita et al., 1986;
Lee and Kim, 1993; Ohlson et al., 1984; Yang et al., 2006). The relative position of the curves is due only to the overview and it is not related to the absolute value of the glucose
concentration.
418
5.1. Fouling
With dead-end ltration and crossow microltration, Mores et al.
(2001) found that the water ux ( 14 000 L/(m2 h)) through a
polysulfone 0.2 m (P = 0.7 bar) membrane was appreciably reduced by pre-treated yellow poplar (5% (w/v), 1500 L/(m2 h); 0.2%
(w/v), 1300 L/(m2 h), respectively), cellulases (0.3% (w/v), 300 and
400 L/(m2 h) , respectively) and their mixture (100 and 360 L/(m2 h) ,
respectively). The authors speculated that the cellulaselignocellulose
mixture showed increased fouling due to the stickiness of enzymebounded particles, especially since the ux was dramatically recovered by backushing of the membrane (11 20011 300 L/(m2 h)).
c
2 c
c
= DS 2 Jv
t
x
x
in which Jv is the solvent ux (m3 m 2 h 1) and DS is the solute
diffusion coefcient (m2 h 1).
If the solute molecules are completely retained by the membrane,
the convective ow of the solute molecules towards the membrane
surface will be equal to the diffusive transport back to the bulk phase,
at steady-state conditions. The concentration polarization will be
more pronounced at higher dilution rates, i.e. at higher product
removal rates or at higher permeate ux. Unfortunately, the negative
effect of increased dilution rate is not counterbalanced by the benet
of the corresponding reduction in the concentration of the reaction
product in the reactor (Alfani et al., 1982). An illustration of this was
problem was reported in a modeling study with a xed boundary
polarization layer thickness of 25 m: In this case a doubling of the
dilution rate from 0.8 to 1.6 h 1 decreased the fractional bulk
concentration of enzyme from 0.97 to 0.12 (Hong et al., 1981). Since
the cellulose is insoluble and requires the enzymes to be adsorbed in
order for the reaction to take place, concentration polarization will
decrease the effective concentration of enzyme and correspondingly
the conversion rate.
The enzyme concentration polarization level mainly depends on
solute accumulation due to membrane rejection, which is very strong
for enzymes, and back diffusion, which is slow for enzymes, into the
bulk solution. It can therefore be reduced and partially avoided by
vigorous stirring in the region next to the membrane surface to
decrease the polarization layer or by increasing the trans-membrane
pressure for a higher permeate ux. However, the high local shear rate
can deactivate the enzymes that are rejected and accumulated in a
polarization lm region immediately next to the membrane surface.
This deactivation is accelerated by the exchange of the deactivated
enzyme in the polarization layer where shear from stirring is high
with the enzyme in the bulk solution through the diffusion and
convection (Alfani et al., 1982; Hong et al., 1981). Thus, the conversion
rate is affected both by the decrease of bulk concentration of enzyme
and at the same time by the shear deactivation of the accumulated
enzyme. Due to simultaneous effect of 2 distinct phenomena (product
inhibition and concentration polarization), Alfani et al. (1982) found
that the cellulose conversion rate was increased for small and medium
values of the dilution rate (0.170.33 h 1) and reached a maximum
(0.38 h 1). This increase in the conversion rate for the relatively small
dilution rates was mainly due to the lower product accumulation in
the reactor, at which there is a negligible enzyme inactivation by
accumulation in the laminar sub-layer on the membrane surface that
can be avoided by agitation of the reactor contents. Hong et al. (1981)
determined that, when operating CSTR membrane cell for cellobiose
419
420
Table 4
Overview of enzyme deactivation studies in membrane reactors. CBH is cellobiohydrolase; EG is endoglucanase; BG is -glucosidase.
Source
CBH1
EG4
BG
5667
4450
0.2
No
None of the enzyme found in efuent; maybe inadequacies in enzyme assay procedure; some
other inactivation
CBH
EG
BG
035
1060
No
CBH
EG
BG
Yes10
29
Deactivation due to passage through hollow bers or adsorption onto substrate; soluble BG easily
Klei et al. (1981)
shear-deactivated; 40% of BG activity lost on immobilization, but stability better than in the soluble form
CBH
EG
BG
10
10
No
CBH
EG
BG
906,7
606,7
Yes10
CBH3
EG2
BG
20508
5158
45808
Yes
CBH1
EG2
BG
85
75
65
15
No
Signicant loss after 67 days; proteolytic modication T, pH; in reactor irreversible adsorption on
insoluble residue and UF tubular unit physical deactivation
CBH1
EG2
BG5
55
10
30
No
Thermal deactivation
CBH
EG
BG
Yes10
Small9
CBH1
EG
BG
50
30
Yes10
CBH
EG
BG
0.9
Yes10
N/A
Bla-Bak et al.
(2006)
421
Fig. 7. Inuence of added glucose on glucose formation rate in a study where glucose is
externally added (040 g/L) prior to reaction (2%DM); the formation rates are
calculated from the non-competitive inhibition model (Andri et al., 2010a). Figure
legend:
0 g/L
10 g/L
20 g/L
40 g/L.
422
Fig. 8. Summary of inhibition studies from Andri et al., 2010a. The dashed lines designate the beginning of transglycosylsation effect observed in Andri et al., 2010a. The marked
studies have employed lignocellulosic substrates the dashed-dotted arrows point at real cellulosic substrate-to-added glucose ratio. Frenneson et al., 1985 have used a membrane
reactor. Figure legend:
Cellulases
Cellulases + -glucosidases
-glucosidases.
Fig. 10. Dialysis of model solutions. The performance of membrane with 10 g/L glucose
solution. The glucose is collected in a buffer reservoir surrounding the membrane; the
concentration is measured and used to calculate the glucose concentration in a dialysis
membrane, which was furthermore measured at the start and end of dialysis. Starting
volume was 25 mL. The green and dark blue solid lines are the ts of the calculated
glucose concentration in dialysis membrane and in the reservoir, respectively. Figure
legend:
glucose in dialysis membrane_exp
glucose in dialysis membrane_calc
glucose in reservoir_exp.
Control hydrolysis
In-situ glucose removal
24 h
48 h
72 h
96 h
0.54
0.54
0.65
0.71
0.70
0.93
0.76
0.94
ethanol, all of which are inhibitory for the enzymes. Therefore, this
concept from the theoretical point of view represents the most
desired processing option, as minimizes all inhibitory effects on the
rate of the enzyme- and yeast-cell-catalyzed reactions. The concept is
further based on producing fermentations broths with concentrated
ethanol that is less energy demanding to further purify by distillation
to the product grade than the ordinary distillation, which is in turn
typically done after the fermentation of the hydrolyzate (Cardona and
Sanchez, 2007). Furthermore, the ethanol may increase volatility
when found in the fermentation broth containing also the enzymes
(Roychoudhury et al., 1986). One attractive option within this concept
is to evaporate the waterethanol mixture from the SSF vessel by
rapid vacuum shortly applied in cycles while the substrate can be
semi-continuously fed (Roychoudhury et al., 1992). Another interesting solution is to remove the ethanol by the membranes; in a SSF
process coupled with the pervaporation membranes or the membrane
distillation modules (Cardona and Sanchez, 2007). It is our impression
that this potentially attractive subject has not received a major
attention in the literature. In general, the search for the feasible
integrated reactionseparation technology for the lignocellulose
degradation is currently ongoing and the summary of these concepts
e.g. vacuum fermentation, fermentation with gas stripping, fermentation coupled with pervaporation, extractive fermentation, etc, is
presented in Cardona and Sanchez (2007).
The SSF coupled with ethanol removal is conceptually advantageous conguration to SHF due to the reduced cellobiose, glucose and
ethanol inhibition, but on the other hand, represents a heavy
compromise of two optimal reaction conditions (pH and temperature) and includes another (vulnerable) biocatalyst i.e. fermenting
microorganism like yeast cells, in the reaction vessel containing highly
heterogeneous and viscous mixture (lignocellulose, cellulases, yeasts,
nutrients, buffers, sugars, etc.). Furthermore, if it is desired to
423
Fig. 11. Dialysis of model solutions, Buffer back-ux into the membrane with 2% DM
real lignocellulosic substrate (pre-treated wheat straw). Starting volume was 25 mL.
The solid line is the t of the measured amount of buffer which penetrated into the
dialysis membrane. Figure legend: weight of buffer penetrated [g] buffer back-ux
[g/(min cm2)].
424
425
Tjerneld F, Persson I, Albertsson P-. Enzymatic hydrolysis of cellulose in aqueous twophase systems. II. Semicontinous conversion of a model substrate, Solca Floc BW
200. Biotechnol Bioeng 1985;27:104450.
Wyman C. Cellulose bioconversion technology: the simultaneous saccharication and
fermentation process. Model formulation. Handbook on bioethanol: production
and utilization. Taylor & Francis; 1996. p. 2656.
Xiao Z, Zhang X, Gregg DJ, Saddler JN. Effects of sugar inhibition on cellulases and glucosidase during enzymatic hydrolysis of softwood substrates. Appl Biochem
Biotechol 2004;113116:111526.
Yang S, Ding W, Chen H. Enzymatic hydrolysis of rice straw in a tubular reactor coupled
with UF membrane. Process Biochem 2006;41:7215.
ARTICLE IN PRESS
I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y
Available at www.sciencedirect.com
CNR-ITM, c/o University of Calabria, Via Pietro Bucci, Cubo 17C, 87030 Rende (CS), Italy
Universita` di Roma La Sapienza, Dip. Ing. Chimica e Materiali, Via Eudossiana 18, 00184 Roma, Italy
c
ENEA, Dipartimento Fusione, Tecnologie e Presidio Nucleare, C.R. ENEA Frascati, Via E. Fermi 45, 00044 Frascati (RM), Italy
b
ar t ic l e i n f o
abs tra ct
Article history:
The ethanol steam-reforming reaction for the production of synthesis gas has been studied
theoretically. A mathematical model has been formulated for a traditional reactor packed
with a Co-based catalyst and then applied to a membrane reactor (MR) in which the
27 September 2007
hydrogen production is increased by removing the hydrogen produced from the reaction
results show that with MR it is possible to obtain both higher conversions of ethanol and
Keywords:
Ethanol steam reforming
Membrane reactor
Pd membranes
Hydrogen production
Modelling
1.
Introduction
producing hydrogen. However, the use of fossil fuels increases the global warming as a consequence of the large
releases of carbon dioxide. Therefore, the hydrogen production starting from biofuels, such as the ethanol obtained from
biomasses, could contribute to stabilize the carbon dioxide
concentration and to reduce the greenhouse effect.
Many authors [914] studied the ethanol steam reforming
using traditional reactors. It is an endothermic catalysed
reaction whose conversion increases with the temperature:
C2 H5 OH H2 O 3 2CO 4H2 ,
DH298 K 239:47 kJ=mol.
If the water gas shift reaction is used for converting the CO,
the complete reaction is
C2 H5 OH 3H2 O 3 2CO2 6H2 ,
DH298 K 157:09 kJ=mol.
ARTICLE IN PRESS
I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y
Nomenclature
A
CTs1
D
Ea
JH2
Kj
kr , kw ,
MR
N0i
membrane area, m2
total surface concentration of site 1, mol=m2 ,
membrane diameter, m
apparent activation energy, kJ/mol
hydrogen flux through the membrane,
mol=m2 s
adsorption constant for the species j, dimensionless
kd rate constants for the reactions 1, 2 and 3,
depending on the equation
membrane reactor, dimensionless
flow rate of the component i, mol/s
Usually, catalysts based on Rh, Ru, Pd, Pt, Ni, Co and Cu are
used on supports of Al2O3, SiO2, MgO and La2O3. The reaction
conversion and selectivity of the products (mainly H2, CO, CO2
and in minor part, CH4, CH3CHO and C2H4) depend on the
catalyst used as well as on the operating temperature. A large
excess of water in the feed stream increases the hydrogen
selectivity and reduces the coke formation that may poison
the catalyst particularly when the reaction is operated at low
temperature.
Using a traditional reactor, for example Liguras et al. [9]
obtained the complete ethanol conversion at 800 C with a
5%-Ru on alumina catalyst. In order to increase the ethanol
conversion at lower temperature, the use of membrane
reactors for carrying out the steam reforming is proposed in
this work. In fact, a membrane reactor is a device in which a
reaction and a selective separation take place simultaneously:
in this way, the continuous removal of one of the product (i.e.
hydrogen) gives reaction conversion beyond the thermodynamic equilibrium which is an upper limit to be considered in
a traditional reactor (shift effect) [1517]. In practice, when a
dehydrogenation reaction is performed, the use of a membrane selectively permeable to the hydrogen permits to build
a reactor producing and separating pure hydrogen with high
reaction conversions. The palladium and its alloys are
permeable to the hydrogen and are used for manufacturing
permeators and membrane reactors aimed at separating and
producing hydrogen: several studies report the use of these
membranes [1820]. Especially, the use of dense PdAg
membranes permits to maximize the shift effect and produce
directly ultra pure hydrogen without using any other purification unit.
The application of dense PdAg membrane reactors was
also suggested for carrying out methanol and ethanol steam
reforming reactions [2125]. The kinetic expressions for a Cubased catalyst is available in literature for the methanol
steam reforming. The use of PdAg membrane reactor was
also considered from a simulation viewpoint giving the
optimal set of operating parameters for maximising the COfree hydrogen recovery [26,27]. Such an optimisation was not
possible for the ethanol steam reforming since no reasonable
kinetic expression were available in the specialised literature.
In a recent paper, Sahoo et al. [28] presented, for the first
time, useful kinetic expressions for the ethanol steam
645
P0
pj
plumen
H2
pshell
H2
R
ri
T
TR
V
W=F
W
ni;j
2.
Theoretical model of ethanol steam
reforming
The model has been developed using all the thermodynamic
values for the species involved in the reaction and the kinetic
expressions; hydrogen removal through a self supported
PdAg membrane having a 23% Ag content was calculated
using experimental permeabilities [26].
The equation considered in the ethanol reaction system
carried out on a Co-based catalyst are as follows:
C2 H5 OH 3H2 O 3 2CO2 6H2 ,
(2)
CO H2 O 3 CO2 H2 ,
(3)
C2 H5 OH 3 CO CH4 H2 ,
DH298 K 33:18 kJ=mol.
ARTICLE IN PRESS
646
I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y
The rate equations are taken from Sahoo et al. [28], as well
as kinetic and adsorption parameters:
r1
0
1 "
pCH3 CH2 OH
A 1
kr KCH3 CH2 O1 @
1=2
pH2
!#
p4H2 p2CO2
Kr p2H2 O pCH3 CH2 OH
CTS1 2 ,
DEN
(5)
"
kw KHCOO1 pCO2 1
r2
!#
pH2 O pCO
Kw pH2 pCO2
CTS1 2 ,
DEN
p3H2 p2CO2
kd KCH3 CHO1
! "
1
p2H2 O
r3
(6)
!#
p3H2 p2CO2
CTS1 2 ,
DEN
(7)
v r Ji
V j1 ij i
A
dz
Kr
KHO1 pH2 O
p3H2 O
1=2
pH2
1=2
pH2
(9)
kw
KHCHO1
kw
,
Kw
KOH1 KCO1
KCH3 CHO1
z 0.
Reaction zone (lumen side):
kd
kd
(10)
N01 ;
N02
N03
N04
N05
N06
KCH4 1 KCO1
(11)
H2 O
107 N01 ;
H2
107 N01 ;
CO2
107 N01 ;
CO
107 N01 ;
CH4
N03 0 ) ri ! 1,
where m is the H2 O=CH3 CH2 OH molar feed ratio and the sum
of N0i is the feed molar flow rate F.
Permeation zone (shell side):
N07 N0sweep
gas ;
N08 0;
Products
Stainless
Steel Tube
(13)
Boundary conditions:
kr
kr
,
KCH3 CHO1 KH2 1
Kd
dNi
pD
.
Ji
A
dz
KCH3 CH2 O1
(12)
Permeation zone:
1=2
Glass Spheres
z
0:5
0:5 pshell
JH2 P0 e RT plumen
H2
H2 ,
(14)
Permeate Outlet
Catalyst Pellets
(lumen side)
Retentate Stream
Feed
Thermocouple
z
Stainless
Steel Tube
Graphite
Gasket
ARTICLE IN PRESS
I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y
where the values of the apparent activation energy and preexponential factor are 29.16 kJ/mol and 1:12 105 mol m=
s m2 Pa0:5 , respectively.
A non reactive sweep gas (nitrogen or steam) has been
considered in the simulations. The sweep gas ratio, defined as
the sweep gas molar rate/ethanol feed molar rate, has been
set at 0.1 except for the last simulations in which the effect of
the sweep ratio was studied.
The set of differential equations has been solved using a IV
order RungeKutta method with variable step in order to
overcome the instabilities in the first part of the reactor. The
code was written by using the Salford Plato3 FORTRAN95
platform.
In order to compare the results for the two reactors, the
ethanol conversion, the hydrogen production and the hydrogen recovery are defined as follows:
CH3 CH2 OH conversion; %
CH3 CH2 OHIN CH3 CH2 OHOUT
100,
CH3 CH2 OHIN
3.1.
3.2.
3.2.1.
(16)
H2;outshell
100,
H2;outlumen H2;outshell
3.
whole range of parameters investigated. In fact, the maximum deviation between the model and the experimental
results in terms of ethanol conversion is 5.8% at 773 K and
W=F 15 kgcat =mol s.
Unfortunately, to the best of our knowledge, there are no
data in literature regarding the use of membrane reactors for
the ethanol steam reforming on Co-based catalyst. For this
reason, a direct comparison of the modelling results and
membrane reactor experimental results is not possible.
15
H
H2;outshell
H2 produced; % 2;outlumen
100,
6 CH3 CH2 OHIN
H2 recovery; %
(17)
100
MR , 873K
Ethanol conversion, %
Ethanol Conversion, %
100
80
60
40
673 K
773 K
873 K
Model
20
MR , 773K
80
MR , 673K
60
TR , 873K
TR , 773K
40
TR , 673K
20
TR
MR
0
0
0
0
6
8
10
12
W/F, kg cat/(mol/s)
647
14
16
18
6
8
10
12
W/F, kg cat/(mol/s)
14
16
18
ARTICLE IN PRESS
648
I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y
3.2.2.
100
8 bar
Ethanol conversion, %
4 bar
2 bar
1 bar
80
1 bar
60
2 bar
4 bar
8 bar
40
20
TR
MR
0
0
6
8
10
12
W/F, kg cat/(mol/s)
14
16
18
1 bar
1 bar
2 bar
Ethanol conversion, %
80
4 bar
60
8 bar
40
20
TR
MR
0
0
4 5
9 10 11 12 13 14 15 16 17 18
W/F, kg cat/(mol/s)
ARTICLE IN PRESS
I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y
100
Hydrogen recovery, %
4 bar
80
2 bar
60
40
1 bar
20
MR
90
90
TR
80
70
60
60
50
50
40
40
30
30
6
8
10
12
W/F, kg cat/(mol/s)
14
16
18
Hydrogen produced, %
100
8 bar
4 bar
2 bar
80
1bar
60
40
20
0
0
10
W/F, kg cat/(mol/s)
15
20
3.2.3.
20
0
80
70
20
0
100
Hydrogen recovery, %
Ethanol conversion, %
8 bar
100
649
3 4 5 6 7 8 9 10 11 12
Steam/ethanol molar ratio, -
3.2.4.
ARTICLE IN PRESS
650
I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y
100
Ethanol Conversion, %
Sweep ratio
20
80
1
60
40
20
0
0
6
8
10
12
W/F, kg cat/(mol/s)
14
16
18
4.
Conclusions
ARTICLE IN PRESS
I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y
651
Abstract
The immiscibility of canola oil in methanol provides a mass-transfer challenge in the early stages of the transesterication of canola oil
in the production of fatty acid methyl esters (FAME or biodiesel). To overcome or rather, exploit this situation, a two-phase membrane
reactor was developed to produce FAME from canola oil and methanol. The transesterication of canola oil was performed via both
acid- or base-catalysis. Runs were performed in the membrane reactor in semi-batch mode at 60, 65 and 70 C and at dierent catalyst
concentrations and feed ow rates. Increases in temperature, catalyst concentration and feedstock (methanol/oil) ow rate signicantly
increased the conversion of oil to biodiesel. The novel reactor enabled the separation of reaction products (FAME/glycerol in methanol)
from the original canola oil feed. The two-phase membrane reactor was particularly useful in removing unreacted canola oil from the
FAME product yielding high purity biodiesel and shifting the reaction equilibrium to the product side.
2006 Elsevier Ltd. All rights reserved.
Keywords: Biodiesel; Methanol; Acid-catalyzed transesterication; Base-catalyzed transesterication; Two-phase membrane reactor
1. Introduction
Biodiesel is a clean-burning diesel fuel produced from
vegetable oils, animal fats, or grease. Its chemical structure
is that of fatty acid alkyl esters (FAAE). Commercially,
biodiesel is produced by transesterication. This reaction
consists of transforming triglyceride (TG) into FAAE, in
the presence of an alcohol (e.g. methanol, ethanol) and a
catalyst (e.g. alkali, acid, enzyme) with glycerol as a major
by-product. The reaction scheme is shown in Fig. 1.
In Fig. 1, X represents the alkyl group of the alcohol
(e.g. CH3 for methanol) while R is a carbon chain typically
on the order of 1120 carbon atoms long. The conversion
of TG to FAAE comprises three consecutive reversible
reactions with diglyceride (DG) and monoglyceride (MG)
as intermediate products. Biodiesel is being used increasingly in public transportation in Europe, Japan and North
America.
*
Due to diminishing petroleum reserves and the environmental consequences of exhaust gases from petroleumderived fuels, such as gasoline and diesel, biodiesel has
attracted attention during the past decade as a renewable
and environmentally friendly fuel. Because biodiesel is
made entirely from vegetable oil or animal fats, it is renewable, environmentally benign (biodegradable), and does
not contain any sulphur, aromatic hydrocarbons, metals
or crude oil residues.
Like petroleum diesel, biodiesel operates in compressionignition engines such as those used in farm equipment, and private and commercial vehicles. Essentially no
engine modications are required, and biodiesel maintains
the payload capacity and range of diesel. Because biodiesel
is oxygenated, it is a better lubricant than diesel fuel,
increasing the life of engines, and is combusted more completely. Indeed, many countries are introducing biodiesel
blends to replace the lubricating eect of sulfur compounds
in low-sulfur diesel fuels (Anastopoulos et al., 2001; Dmytryshyn et al., 2004). The higher ash point of biodiesel
makes it a safer fuel to use, handle and store. With its
relatively low emission prole, it is an ideal fuel for use
640
H2C-O-H
H 2C-O-R
Catalyst
HC-O-R
H 2C-O-R
3X-OH
HC-O-H
3RCOOX
H 2C-O-H
641
3. Experimental
3.1. Materials
Methanol (99.85% Reag. Grade containing <0.1%
water) was supplied by (Commercial Alcohols Inc., Brampton, ON, Canada) and the canola oil by (No Name, Toronto, ON, Canada, purchased at a local foodstore). Fatty
acid methyl esters (FAME or biodiesel) used for miscibility
testing was produced from the acid-catalyzed transesterication of waste oils from a previous study (Zheng, 2003).
Sulfuric acid (9598%, Reagent Grade) and tetrahydrofuran (99.95%, Chromatography Grade) were supplied by
(EMD Chemicals Inc., Gibbstown, NJ, USA).
3.2. Membrane reactor design and experimental design
A 300 mL membrane reactor system was constructed and
is shown schematically in Fig. 2. A carbon membrane (Koch
Membrane Systems, Inc., Wilmington, DE, USA) was used
in the reactor. The pore size of the membrane was 0.05 lm.
The inside and outside diameters of the membrane were 6
and 8 mm, respectively. The length of carbon membrane
tube was 1200 mm giving a surface area of 0.022 m2 for
the entire membrane. A controlled volume pump (Milton
Roy Company, Ivyland, PA, USA) was used to feed the
oil and methanol/catalyst mixtures to the system while a
seal-less centrifugal canned motor pump (Labcor Inc. Concord, ON) was used to circulate the mixture at a speed of
15.2 mL/min. A heat exchanger (Neslab Instruments, Inc.,
Portsmouth, NH, USA) coupled with LabViewTM software
was used to control the reaction temperature.
Experiments were carried out at 60, 65 and 70 C in a
300 mL membrane reactor for 6 h, 0.5, 2, 4 and 6 wt.% concentrations of sulfuric acid catalyst were investigated (see
Table 1). Canola oil (100 g) was used in each run. Pressure
was controlled at 138 kPa between the permeation side and
reaction side of the membrane. All experiments and sample
analyses were carried out in random order to minimize any
potential experimental errors. Several replicate runs also
were performed (see Table 1). Additional experiments were
conducted to verify the eect of methanol feed ow rate
and the use of a base catalyst.
3.3. Membrane reactor experiments procedure
The methanol and sulfuric acid were pre-mixed and
charged into the membrane reaction system prior to each
reaction. Canola oil (100 g) was charged into the membrane reactor, the membrane reactor was sealed and the
642
Table 1
Experimental conditions
Temperature (C)
Catalyst concentration
(wt.%)
# of replicates
60
65
70
60
65
70
60
65
70
60
65
70
0.5
0.5
0.5
2
2
2
4
4
4
6
6
6
2
2
2
3
3
2
2
2
2
4
4
4
Retention time
(min)
Relative retention
time
Triolein (TG)
Diolein (DG)
Monoolein (MG)
Methyl oleate (FAME)
Glycerol
24.57
25.45
27.12
28.68
30.95
1
1.04
1.10
1.17
1.26
M oilt0 M oiltt
M oilt0
Table 2
Retention time of standards
Standard
643
80
Diolein
Monoolein
70
Triolein
60
Methyl oleate
Glycerol
mV
50
40
30
20
10
0
22
23
24
25
26
27
28
29
30
31
32
33
644
mV
1500
1400
1300
1200
1100
1000
900
800
700
600
500
400
300
200
100
0
22
23
24
25
26
27
28
29
Elution time (min)
30
31
32
33
Fig. 5. HPLC chromatogram of the phase-separated oil phase of the emulsion in the reactor after 6 h of operation (reaction at 65 C, 2 wt.% acid catalyst).
mV
1500
1400
1300
1200
1100
1000
900
800
700
600
500
400
300
200
100
0
22
23
24
25
26
27
28
29
Elution time (min)
30
645
31
32
33
Fig. 6. HPLC chromatogram of permeate after washing (reaction at 65 C, 2 wt.% acid catalyst).
Table 3
Eect of ow rate on conversion
1
0.9
0.8
Conversion at 6 h
0.7
Expt.
Flow rate
(mL/min)
Temperature
(C)
Conversion via
acid-catalyst (%)
Conversion via
base-catalyst (%)
1
2
3
2.5
3.2
6.1
65
65
65
35
48
64
95
96
96
0.6
0.5
0.4
0.3
0.50%
2%
0.2
4%
0.1
6%
0
58
60
62
64
66
Temperature (C)
68
70
72
the acid-catalyzed case shows that the base catalyst provided a much higher conversion, than that of acid catalyst.
Freedman et al. (1984) studied the eect of the type of catalyst on the reaction. It was found that 98% conversion was
observed at 1 wt.% sodium hydroxide. They also found
that greater than 90% of the oil was converted to methyl
esters at 1 wt.% sulphuric acid. In our base-catalyzed
experiments, small amounts of soap were detected in the
wash waters. These were not found in the acid-catalyzed
runs. One possible reason was that the canola oil may have
contained signicant amounts of FFA that were converted
to soaps rather than FAME by the base catalyst. This
would have implications for the use of an acid catalyst
which, despite the slower reaction rate, may provide both
a technological and economic advantage for the use of
lower cost waste feedstock, which contain higher levels of
FFA (Zhang et al., 2003a,b).
646
5. Conclusions
The purication of FAME from a canola oil/methanol/
catalyst reaction mixture poses some important challenges
in the production of biodiesel. This work has demonstrated
that a membrane reactor can be used to alleviate many of
these diculties and successfully carry out the transesterication of triglycerides to FAME.
In the present semi-batch process, a large methanol:oil
ratio was employed. From visual observations, the concentration of FAME in the permeate was not constant as the
reaction progressed. Initially, the permeate was quite
concentrated but as the reaction proceeded, the permeate
concentration decreased. In a continuous process, oil and
methanol would be fed to the reactor at a xed ratio resulting in the continuous production of a concentrated permeate. The present work illustrates that oil and methanol can
readily co-exist in the reactor at a volume ratio of 1:2 without plugging the membrane pores. This allows for the reaction to be carried out in an emulsion where oil and reacted
products can be continuously separated in order to produce a TG-free FAME.
In most commercial processes, as the reaction progresses, the formed FAME will eventually behave as a
mutual solvent for the TG and alcohol phases. Noureddini
and Zhu (1997) have discussed the benets of the formation of a homogeneous alcohol/TG/FAME phase as
FAME is formed in the reaction. As discussed in Section
4.1, maintaining a two-phase system in the membrane reactor inhibits the transfer of TG and non-reacting lipids to
the product stream. One of the benets of producing a
TG-free FAME is a simplication of the often onerous
downstream purication of FAME. This, of course, leads
to the production of high quality FAME. The membrane
reactor allows a phase barrier which limits the presence
of TG and non-reacting lipids in the product. This is highly
desirable in maintaining quality assurance in the production of biodiesel. Maintaining a phase barrier prohibits
the transfer of highly hydrophobic molecules to the product. This provides a limiting barrier in the production of
biodiesel. This parallels the advantages of using distillation
in maintaining product quality in the petroleum processing
industries.
Acknowledgements
The authors thank the support from the Premiers
Research Excellence Award of Ontario and the University
of Ottawa.
References
Anastopoulos, G., Lois, E., Karonis, D., Zanikos, F., Kalligeros, S., 2001.
A preliminary evaluation of esters of monocarboxylic fatty acid on the
lubrication properties of diesel fuel. Ind. Eng. Chem. Res. 40, 452456.
Armor, J.N., 1989. Catalysis with permselective inorganic membranes.
Appl. Cat. 49, 125.
647
Zhang, Y., Dube, M.A., McLean, D.D., Kates, M., 2003a. Biodiesel
production from waste cooking oil. 1: Process design and technological
assessment. Biores. Tech. 89, 116.
Zhang, Y., Dube, M.A., McLean, D.D., Kates, M., 2003b. Biodiesel
production from waste cooking oil. 2: Economic assessment and
sensitivity analysis. Biores. Tech. 90, 229240.
Zheng, S., 2003. Biodiesel Production from Waste Frying Oil: Conversion
Monitoring and Modeling. M.A.Sc. Thesis, Department of Chemical
Engineering, University of Ottawa, Canada.
2.6.134
Screening of yeast producer of fructosyltransferase
E. Setsuko Kamimura 1, , G.S. Silva 1 , L.M. Covre 1 , S. Campos
Hossri 1 , A.M. Pinheiro Santos 2
1
ABSTRACTS
2.6.135
Membrane reactors for bioethanol production of enzymatically pretreated wheat straw
M.C. Sgua , S.M. Paixo, L. Baeta-Hall, B. Ribeiro, J. Pereira, A.M.
Anselmo, J.C. Duarte
INETI, Lisboa, Portugal
www.elsevier.com/locate/nbt S243
ABSTRACTS
2.6.137
Improving yield and quality of recombinant proteins in
Escherichia coli by stress minimisation and mutant selection
Y. Sevastsyanovich , S. Alfasi, L. Grifths, T. Overton, J. Cole
University of Birmingham, Birmingham, United Kingdom
2.6.136
Bioprocess development to produce rabies virus glycoprotein in bioreactor using insect S2 cells
A. Tonso , D.C. Ventini, P.B. Vieira, R.M. Astray, K.N. Greco, S.A.C.
Jorge, C.A. Pereira
Escola Politcnica da Universidade de So Paulo, So Paulo, Brazil
Drosophila melanogaster Schneider 2 (S2) cells have become increasingly utilized over the past few years for the expression of
heterologous proteins. High levels of protein expression with pharmacological and biotechnological interest can be achieved using
Drosophila Expression System procedure. Insect cell cultures are
easier to handle than mammalian cells, being capable to multiply in monolayers or in suspension at temperatures ranging from
22 to 30 C. The recombinant rabies virus glycoprotein (RVGP) is
an interesting biotechnological product as it is responsible for the
induction of protective immune response against rabies infection.
The aim of this work is the development of a bioprocess using a
bioreactor to produce RVGP. Two cell constructions were tested,
where the gene vectors were constructed in order to be under the
control of metalotionein or actin promoter. The rst is inducible
by CuSO4 and the second is constitutive. The cells were grown
in different conditions in two bioreactors using SF-900II medium
and varying temperature, agitation and aeration. Kinetic studies
of S2 cells were made by analyzing cell growth, RVGP expression,
consumption of glucose and glutamine and production of lactate. Storage tests were performed using different cryoprotectors
(threalose, sucrose and glycerol) in a buffered solution containing
protease inhibitor (PMSF). Cell lyses and RVGP extraction were
made either by mechanic stress (homogenization) followed by
detergent treatment, or just by detergent treatment. Accordingly
to runs with different cell constructions and conditions, the best
results of cell growth and RVGP expression were obtained with
inducible cells in the following culture conditions: at 28 C, 90 rpm
agitation, pitched blade impellers, dissolved oxygen of 10% of air
saturation and 4 gas sparging aeration. The solution with 20% of
glycerol led to the best storage performances and the homogenization associated with detergent was the best lysis-extraction
protocol. The bioreactor cultivation of S2 cells expressing RVGP
is a very promising way to obtain great quantities of the protein,
showing the possibility of performing a well controlled and reproducible process. The storage, lyses and extraction studies are the
preliminary steps to the development of an efcient preservation
and purication procedures.
doi:10.1016/j.nbt.2009.06.239
S244
www.elsevier.com/locate/nbt
The general stress response is strongly induced during recombinant protein production in bacterial hosts. This problem is
particularly severe when T7 promoter-based expression systems
are used, such as Novagens pET vectors that require E. coli BL21 or
its derivatives as production hosts. In a typical production protocol, high concentrations of IPTG are used for induction, resulting
in rapid but transient burst of recombinant gene expression. Due
to the limited folding capacity of a cell, misfolded protein species
begin to accumulate and are deposited into inclusion bodies.
This together with depletion of cellular recourses due to excessive foreign protein synthesis results in growth arrest, induction
of RpoH-dependent stress response, ppGpp-dependent stringent
response, RpoS-dependent stationary phase response, and nally
overgrowth of the culture by plasmid-free, non-productive bacteria. We showed using recombinant E. coli BL21*(DE3) producing
CheYGFP fusion protein that growth arrest is accompanied
by >95% loss of cultivability of the productive population, so
plasmid-free bacteria are able to grow without competition and
by 24 h post-induction comprise up to 8090% of the culture. We
then demonstrated that even challenging recombinant proteins,
such as gonococcal cytochromes could be accumulated successfully by redesigning fermentation conditions that minimised stress
response. These include decreasing the inducer concentration and
keeping low constant temperature throughout the fermentation
in order to avoid both drastic changes in growth rate and stress
from temperature shift upon induction. This method has been
successfully reproduced in shake asks, batch and fed-batch fermentations, in which cell densities up to 30 g dry mass per litre of
culture have been achieved. Using this approach, culture growth
and productivity were sustained for at least 70 h and CheYGFP
yields were typically 2530% of total cellular protein. Unlike
the original production protocol, the majority of this protein (up
to 90%) was found in the soluble cell fraction and showed signicantly higher uorescence indicating correct protein folding.
Flow cytometric analysis also showed that virtually all bacteria in
the nal population accumulated recombinant protein of good
conformational quality and in high yield. Western blotting analysis suggested that the major reason for success was decreased
accumulation of the T7 RNA polymerase. We have used different
techniques, including uorescence-activated cell sorting, for isolation of mutants that are resistant to IPTG-induced stress, but also
accumulate high levels of good quality recombinant protein when
the normal induction protocol is followed. The molecular basis of
this improvement is currently being investigated.
doi:10.1016/j.nbt.2009.06.240