U.S. patent application number 14/420765 was filed with the patent office on 2015-07-23 for process for enzymatic hydrolysis of lignocellulosic material and fermentation of sugars.
The applicant listed for this patent is DSM IP ASSETS B.V.. Invention is credited to Bertus Noordam.
Application Number | 20150203885 14/420765 |
Document ID | / |
Family ID | 49622788 |
Filed Date | 2015-07-23 |
United States Patent
Application |
20150203885 |
Kind Code |
A1 |
Noordam; Bertus |
July 23, 2015 |
PROCESS FOR ENZYMATIC HYDROLYSIS OF LIGNOCELLULOSIC MATERIAL AND
FERMENTATION OF SUGARS
Abstract
The invention relates to a process for the preparation of a
fermentation product from ligno-cellulosic material, comprising the
following steps: a) optionally pre-treatment of the
ligno-cellulosic material; b) optionally washing of the optionally
pre-treated ligno-cellulosic material; c) enzymatic hydrolysis of
the optionally washed and/or optionally pre-treated
ligno-cellulosic material using an enzyme composition comprising at
least two cellulase and whereby the enzyme composition at least
comprises GH61; d) fermentation of the hydrolysed ligno-cellulosic
material to produce a fermentation product; and e) optionally
recovery of a fermentation product; wherein before and/or during
the enzymatic hydrolysis oxygen is added to the ligno-cellulosic
material.
Inventors: |
Noordam; Bertus; (Echt,
NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DSM IP ASSETS B.V. |
Heerlen |
|
NL |
|
|
Family ID: |
49622788 |
Appl. No.: |
14/420765 |
Filed: |
November 7, 2013 |
PCT Filed: |
November 7, 2013 |
PCT NO: |
PCT/EP2013/073250 |
371 Date: |
February 10, 2015 |
Current U.S.
Class: |
435/43 ; 435/108;
435/109; 435/113; 435/115; 435/136; 435/139; 435/140; 435/144;
435/145; 435/146; 435/158; 435/159; 435/160; 435/162; 435/167;
435/189; 435/193; 435/198; 435/200; 435/202; 435/209; 435/219;
435/232; 435/47; 435/99 |
Current CPC
Class: |
C12P 19/14 20130101;
Y02E 50/16 20130101; Y02E 50/10 20130101; C12P 19/02 20130101 |
International
Class: |
C12P 19/14 20060101
C12P019/14; C12P 19/02 20060101 C12P019/02 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 9, 2012 |
EP |
12191957.5 |
Jul 2, 2013 |
EP |
13174656.2 |
Jul 15, 2013 |
EP |
13176500.0 |
Sep 17, 2013 |
EP |
13184702.2 |
Claims
1. Process for preparing a sugar product from ligno-cellulosic
material, comprising: a) optionally pre-treatment of the
ligno-cellulosic material; b) optionally washing of the optionally
pre-treated ligno-cellulosic material; c) enzymatic hydrolysis of
the optionally washed and/or optionally pre-treated
ligno-cellulosic material using an enzyme composition comprising at
least two cellulase and whereby the enzyme composition at least
comprises GH61; and d) optionally recovery of a sugar product;
wherein after the pre-treatment and before and/or during the
enzymatic hydrolysis oxygen is added to the ligno-cellulosic
material.
2. Process for preparing a fermentation product from
ligno-cellulosic material, comprising: a) optionally pre-treatment
of the ligno-cellulosic material; b) optionally washing of the
optionally pre-treated ligno-cellulosic material; c) enzymatic
hydrolysis of the optionally washed and/or optionally pre-treated
ligno-cellulosic material using an enzyme composition comprising at
least two cellulase and whereby the enzyme composition at least
comprises GH61; d) fermentation of the hydrolysed ligno-cellulosic
material to produce a fermentation product; and e) optionally
recovery of a fermentation product; wherein after the pre-treatment
and before and/or during the enzymatic hydrolysis oxygen is added
to the ligno-cellulosic material.
3. Process according to claim 1, wherein during the enzymatic
hydrolysis c) oxygen is added to the ligno-cellulosic material.
4. Process according to claim 1, wherein the oxygen is added in the
form of bubbles.
5. Process according to claim 1, wherein the reactor for the
enzymatic hydrolysis has a volume of 1 m.sup.3 or more.
6. Process according to claim 2, wherein the enzymatic hydrolysis
time is 5 to 150 hours.
7. Process according to claim 1, wherein the enzyme composition
used retains activity for 30 hours or more.
8. Process according to claim 1, wherein the hydrolysis is
conducted at a temperature of 45.degree. C. or more, optionally
50.degree. C. or more and optionally at a temperature of 55.degree.
C. or more.
9. Process according to claim 1, wherein the enzyme composition is
derived from a fungus, optionally microorganism of the genus
Rasamsonia or the enzyme composition comprises a fungal enzyme, a
Rasamsonia enzyme.
10. Process according to claim 1, wherein the dry matter content in
the hydrolysis c) is 10 wt % or more, optionally is 14 wt % or more
and optionally is 14 to 33 wt %.
11. Process according to claim 9, wherein the dry matter content in
the hydrolysis c) is 14 to 33% wt %.
12. A process according to claim 1 in which the enzymatic
hydrolysis takes place in a batch, fed batch and/or continuous
culture reactor.
13. A process according to claim 1 in which oxygen is introduced as
an oxygen-containing gas optionally air.
Description
FIELD OF THE INVENTION
[0001] The invention relates to a process for the enzymatic
hydrolysis of lignocellulosic material and fermentation of
sugars.
BACKGROUND OF THE INVENTION
[0002] Ligno-cellulosic plant material, herein also called
feedstock, is a renewable source of energy in the form of sugars
that can be converted into valuable products e.g. sugars or
bio-fuel, such as bio-ethanol. During this process, (ligno or
hemi)-cellulose present in the feedstock, such as wheat straw, corn
stover, rice hulls, etc., is converted into reducing sugars by
(hemi)-cellulolytic enzymes, which then are optionally converted
into valuable products such as ethanol by microorganisms like
yeast, bacteria and fungi.
[0003] Since the (hemi)-cellulose is crystalline and entrapped in a
network of lignin the conversion into reducing sugars is in general
slow and incomplete. Typically, enzymatic hydrolysis of untreated
feedstock yields sugars <20% of theoretical quantity. By
applying a chemical and thermo-physical pre-treatment, the
(hemi)-cellulose is more accessible for the (hemi)-cellulolytic
enzymes, and thus conversions go faster and at higher yields.
[0004] A typical ethanol yield from glucose, derived from
pre-treated corn stover, is 40 gallons of ethanol per 1000 kg of
dry corn stover (Badger, P, Ethanol from cellulose: a general
review, Trends in new crops and new uses, 2002, J. Janick and A.
Whipkey (eds.) ASHS Press, Alexandria, Va.) or 0.3 g ethanol per g
feedstock. The maximum yield of ethanol on cellulose base is
approximately 90%.
[0005] Cellulolytic enzymes--most of them are produced by species
like Trichoderma, Humicola and Aspergillus--are commercially used
to convert pre-treated feedstock into a mash containing insoluble
(hemi)cellulose, reducing sugars made thereof, and lignin.
Thermostable cellulolytic enzymes derived from Rasamsonia, have
been used for degrading ligno-cellulosic feedstock and these
enzymes are known for their thermostability, see WO2007091231. The
produced mash is used in a fermentation during which the reducing
sugars are converted into yeast biomass (cells), carbon dioxide and
ethanol. The ethanol produced in this way is called
bio-ethanol.
[0006] The common production of sugars from pre-treated
ligno-celullosic feedstock, the hydrolysis also called
liquefaction, pre-saccharification or saccharification, typically
takes place during a process lasting 6 to 168 hours (Kumar, S. ,
Chem. Eng. Technol. 32 (2009) 517-526) under elevated temperatures
of 45 to 50.degree. C. and non-sterile conditions. During this
hydrolysis, the cellulose present is partly (typically 30 to 95%,
dependable on enzyme activity and hydrolysis conditions) converted
into reducing sugars. In case of inhibition of enzymes by compounds
present in the pre-treated feedstock and by released sugars; and to
minimize thermal inactivation, this period of elevated temperature
is minimized as much as possible.
[0007] The fermentation following the hydrolysis takes place in a
separate preferably anaerobic process step, either in the same or
in a different vessel, in which temperature is adjusted to 30 to
33.degree. C. (mesophilic process) to accommodate growth and
ethanol production by microbial biomass, commonly yeasts. During
this fermentation process, the remaining (hemi) cellulosic material
is converted into reducing sugars by the enzymes already present
from the hydrolysis step, while microbial biomass and ethanol are
produced. The fermentation is finished once (hemi) cellulosic
material is converted into fermentable sugars and all fermentable
sugars are converted into ethanol, carbon dioxide and microbial
cells. This may take up to 6 days. In general the overall process
time of hydrolysis and fermentation may amount up to 13 days.
[0008] The so obtained fermented mash consists of non-fermentable
sugars, non-hydrolysable (hemi) cellulosic material, lignin,
microbial cells (most common yeast cells), water, ethanol,
dissolved carbon dioxide. During the successive steps, ethanol is
distilled from the mash and further purified. The remaining solid
suspension is dried and used as, for instance, burning fuel,
fertilizer or cattle feed.
[0009] WO2010080407 suggests treating cellulosic material with a
cellulase composition under anaerobic conditions. Removal or
exclusion of reactive oxygen species may improve the performance of
cellulose-hydrolyzing enzyme systems. Hydrolysis of cellulosic
material, e.g., lignocellulose, by an enzyme composition can be
reduced by oxidative damage to components of the enzyme composition
and/or oxidation of the cellulosic material by, for example,
molecular oxygen.
[0010] WO2009046538 discloses a method for treating lignocellulosic
feedstock plant materials to release fermentable sugars using an
enzymatic hydrolysis process for treating the materials performed
under vacuum and producing a sugar rich process stream comprising
reduced amounts of volatile sugar/fermentation inhibiting compounds
such as furfural and acetic acid. Apart from removing volatile
inhibitory compounds, other compounds and/or molecules that are
also removed include nitrogen, oxygen, argon and carbon
dioxide.
[0011] With each batch of feedstock, enzymes are added to maximize
the yield and rate of fermentable sugars released from the
pre-treated ligno-cellulosic feedstock during the given process
time. In general, costs for enzymes production, feedstock to
ethanol yields and investments are major cost factors in the
overall production costs (Kumar, S. Chem. Eng. Technol. 32 (2009)
517-526). Thus far, cost of enzyme usage reduction is achieved by
applying enzyme products from a single or from multiple microbial
sources (WO 2008/008793) with broader and/or higher (specific)
hydrolytic activity which use aims at a lower enzyme need, faster
conversion rates and/or a higher conversion yields, and thus at
overall lower bio-ethanol production costs. This requires large
investments in research and development of these enzyme products.
In case of an enzyme product composed of enzymes from multiple
microbial sources, large capital investments are needed for
production of each single enzyme compound.
[0012] It is therefore desirable to improve the above process
involving hydrolysis and fermentation.
SUMMARY OF THE INVENTION
[0013] An object of the invention is therefore to provide a process
in which the hydrolysis step is conducted at improved conditions.
Another object of the invention is to provide a process involving
hydrolysis having a reduced process time. Further object of the
invention is to provide a process, wherein the dosage of enzyme may
be reduced and at the same time output of useful hydrolysis product
is maintained at the same level or even increased. Another object
is to provide a process involving hydrolysis, wherein the process
conditions of the hydrolysis are optimized. A still further object
of the invention is to provide a process involving hydrolysis,
wherein the output of useful hydrolysis product is increased using
the same enzyme dosage. One or more of these objects are attained
according to the invention.
[0014] The present invention provides a process for the preparation
of a sugar product from ligno-cellulosic material, comprising the
following steps: [0015] a) optionally pre-treatment of the
ligno-cellulosic material; [0016] b) optionally washing of the
optionally pre-treated ligno-cellulosic material; [0017] c)
enzymatic hydrolysis of the optionally washed and/or optionally
pre-treated ligno-cellulosic material using an enzyme composition
comprising at least two cellulase and whereby the enzyme
composition at least comprises GH61; and [0018] d) optionally
recovery of a sugar product; wherein after the pre-treatment and
before and/or during the enzymatic hydrolysis oxygen is added to
the ligno-cellulosic material.
[0019] Furthermore the present invention provides a process for the
preparation of a fermentation product from ligno-cellulosic
material, comprising the following steps: [0020] a) optionally
pre-treatment of the ligno-cellulosic material; [0021] b)
optionally washing of the optionally pre-treated ligno-cellulosic
material; [0022] c) enzymatic hydrolysis of the optionally washed
and/or optionally pre-treated ligno-cellulosic material using an
enzyme composition comprising at least two cellulase and whereby
the enzyme composition at least comprises GH61; [0023] d)
fermentation of the hydrolysed ligno-cellulosic material to produce
a fermentation product; and [0024] e) optionally recovery of a
fermentation product; wherein after the pre-treatment and before
and/or during the enzymatic hydrolysis oxygen is added to the
ligno-cellulosic material.
[0025] Preferably the oxygen is added during the enzymatic
hydrolysis step c).
[0026] In a preferred embodiment the oxygen is added in the form of
(gaseous) bubbles.
[0027] Surprisingly, according to the invention, by the addition of
oxygen it is possible to attain many process advantages, including
optimal temperature conditions, reduced process time, reduced
dosage of enzyme, re-use of enzymes, higher yields and other
process optimizations, resulting in reduced costs.
[0028] In one embodiment of this process, the fermentation time is
5 to 120 hours. In an embodiment the stable enzyme composition used
retains activity for 30 hours or more. According to a further
embodiment the hydrolysis is preferably conducted at a temperature
of 45.degree. C. or more, more preferably at a temperature of
50.degree. C. or more and still more preferably at a temperature of
55.degree. C. or more. In a preferred embodiment, the enzyme
composition is derived from a fungus, preferably a microorganism of
the genus Rasamsonia or the enzyme composition comprises a fungal
enzyme, preferably a Rasamsonia enzyme. The process of the
invention will be illustrated in more detail below.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIG. 1: The effect of sparging nitrogen or air through a 10%
aCS feedstock before hydrolysis, on the total amount of glucose
(g/l) released by the TEC-210 mix (1), 4E-GH61 mix (2) and 4E-EG
mix (3).
[0030] FIG. 2: The enzymatic hydrolysis in a 270 liter reactor
(pilot plant scale) whereby the glucan conversion (%) is shown in
20% aCS as function of the process time (hours) for 3.75 mg
TEC210/g feedstock DM for low DO (--.quadrature.--) and high DO
(--.box-solid.--).
[0031] FIG. 3: The effect of the dissolved oxygen concentration
(DO) on glcan hydrolysis in pretreated lignocellulosic feedstock as
function of hydrolysis time for 2.5mg/g of enzyme and DO=0.030
mol/m.sup.3 (--.diamond-solid.--) and 3.5mg/g of enzyme and
DO=0.005 mol/m.sup.3 (--.box-solid.--).
DETAILED DESCRIPTION OF THE INVENTION
[0032] Throughout the present specification and the accompanying
claims, the words "comprise" and "include" and variations such as
"comprises", "comprising", "includes" and "including" are to be
interpreted inclusively. That is, these words are intended to
convey the possible inclusion of other elements or integers not
specifically recited, where the context allows. The articles "a"
and "an" are used herein to refer to one or to more than one (i.e.
to one or at least one) of the grammatical object of the article.
By way of example, "an element" may mean one element or more than
one element.
[0033] In the context of the present invention "improved",
"increased", "reduced" is used to indicate that the present
invention shows an advantage compared to the same situation,
process or process conditions except that no extra oxygen is added.
Within the context of the present invention "measured under the
same conditions" or "analysed under the same conditions" etc. means
that the process of the invention and the same process without
addition of oxygen are performed under the same conditions (except
the oxygen addition) and that the results of the present process,
if compared to the process without oxygen addition, are measured
using the same conditions, preferably by using the same assay
and/or methodology, more preferably within the same or parallel
experiment. Conditions of the hydrolysis are an example of such
conditions.
[0034] In prior art it is suggested to improve the hydrolysis of
cellulolytic material by using anaerobic (WO2010/080407) or vacuum
(WO2009/046538) conditions during the enzymatic hydrolysis. In the
processes of both documents the oxygen level was decreased. It has
now surprisingly been found that the hydrolysis of the present
invention shows results in an improved reaction product that gives
higher amounts of (reduced) sugar products and/or desired
fermentation products in the fermentation following the hydrolysis
as compared to a process wherein no oxygen is added. In general an
increase of the glucose conversion is observed of 5 to 15 w/w
%.
[0035] Oxygen can be added in several ways. For example oxygen can
be added as oxygen gas, oxygen enriched gas such as oxygen enriched
air or air. Oxygen can be added continuously or dis-continuously.
By oxygen "is added" is meant that oxygen is added to the liquid
phase (comprising the lingo-cellulosic material) in the hydrolysis
reactor and not that oxygen is present in the headspace in the
reactor above the liquid phase whereby the oxygen has to diffuse
from the headspace to the liquid phase. So preferably the oxygen is
added as bubbles, most preferably as small bubbles.
[0036] In case the enzyme may be damaged by the presence or
addition of oxygen, milder oxygen supply may be used. In that case
a balance can be found between the improved glucose production and
the enzyme performance. The addition of the oxygen to the
cellulolytic material can be done before and/or during the
enzymatic hydrolysis. In case oxygen is added in gaseous form,
oxygen-containing gas can be introduced, for example blown, into
the liquid hydrolysis reactor contents of cellulolytic material. In
another embodiment of invention the oxygen-containing gas is
introduced into the liquid cellulolytic material stream that will
enter the hydrolysis reactor. In still another embodiment of the
invention the oxygen containing gas is introduced together with the
cellulolytic material that enters the hydrolysis reactor or with
part of the liquid reactor contents that passes an external loop of
the reactor. In most cases the addition of oxygen before entering
the hydrolysis reactor is not sufficient enough and oxygen addition
may be done during the hydrolysis as well. In another embodiment of
the invention the gaseous phase present in the upper part of the
reactor (head space) is continuously or dis-continuously refreshed
with the oxygen-containing gas. In the latter case (vigorous)
mixing or stirring is needed to get the oxygen as bubbles and/or by
diffusion into the liquid reactor contents preferably in
combination with over-pressure in the reactor. In general flushing
the headspace with air in combination with (vigorous) mixing or
stirring may introduce sufficient oxygen into the cellulosic
material in the hydrolysis reactor for reactors up to a size of 100
liter to 1 m.sup.3. At larger scale, for example in a reactor of 50
m.sup.3 or more, for example 100 m.sup.3, so much energy is needed
for vigorous stirring that from economic point of view this will
not be applied in a commercially operating process.
[0037] According to the present invention the oxygen may be added
before the hydrolysis step, during part of the hydrolysis step,
during the whole hydrolysis step or a combination of before or
during the hydrolysis step. Advantageously the oxygen is added
during the first half in time of the hydrolysis step. The addition
of oxygen during only part of the hydrolysis may be done for
example in case of oxidation damage of the enzyme(s) occurs. In
case the oxygen present in the hydrolysis reactor contents or the
sugar product or the hydrolysate formed in the hydrolysis step
might influence or disturb in the subsequent fermentation step,
oxygen addition may be done except for the last part of the
hydrolysis and thus (most of) the oxygen is consumed before the
hydrolysed biomass enters the fermentation reactor.
[0038] Several examples of aeration during the enzymatic hydrolysis
process are given in the Examples to show the beneficial effect of
the present invention. This beneficial effect is found for several
substrates or feedstocks and therefore believed to be present for
the hydrolysis of all kind of substrates or feedstocks.
[0039] Several examples of enzyme compositions for the enzymatic
hydrolysis process are given in the Examples to show the beneficial
effect of the present invention. This beneficial effect is found
for several enzyme compositions and therefore believed to be
present for all kind of hydrolysing enzyme compositions.
[0040] To a further preferred embodiment of the invention the
oxygen concentration in the liquid phase (DO), wherein the
ligno-cellulosic material is present during the enzymatic
hydrolysis, is at least 0.001 mol/m.sup.3, preferably at least
0.002 mol/m.sup.3, more preferably at least 0.003 mol/m.sup.3 and
even more preferably more than 0.01 mol/m.sup.3,for example more
than 0.02 mol/m.sup.3 or 0.03 mol/m.sup.3. In reactors of less than
1 m.sup.3 DO values of below 0.01 mol/m.sup.3 or 0.02 mol/m.sup.3
will be obtained by slow stirring. Vigorous mixing or stirring at
such scale introduces part of the gas phase of the headspace into
the reaction liquid. For example the mixing or stirring may create
a whirlpool that draws oxygen into the liquid. In general flushing
the headspace with air in combination with (vigorous) mixing or
stirring will introduce sufficient oxygen into the cellulosic
material in the hydrolysis reactor for reactors up to a size of 100
liter to 1 m.sup.3. At larger scale, for example in a reactor of 50
m.sup.3 or more, for example 100 m.sup.3, so much energy is needed
for vigorous stirring that from economic point of view this will
not be applied in a commercially operating process. In general in
large reactors, stirring or mixing without introducing air or
oxygen will result in DO values of less than 0.01 mol/m.sup.3.
[0041] To still another preferred embodiment of the invention
during the oxygen generation or production the oxygen concentration
in the liquid phase (aeration or addition of oxygen), the oxygen
concentration in the liquid phase wherein the ligno-cellulosic
material is present during the enzymatic hydrolysis, is preferably
at most 80% of the saturation concentration of oxygen under the
hydrolysis reaction conditions, more preferably at most 0.12
mol/m.sup.3, still more preferably at most 0.09 mol/m.sup.3, even
more preferably at most 0.06 mol/m.sup.3, even still more
preferably at most 0.045 mol/m.sup.3 and most preferably at most
0.03 mol/m.sup.3. Temperature and pressure will influence the DO.
The preferred and exemplary mol/m.sup.3 values given above relate
to normal atmospheric pressure and a temperature of about
62.degree. C. The skilled person in the art will appreciate
favourable DO values on basis of the present teachings.
[0042] The oxygen addition in the form of air or other
oxygen-containing gas according to the invention may also be used
to at least partially stir or mix the hydrolysis reactor
contents.The present process of the invention shows especially on
pilot plant and industrial scale advantages. Preferably the
hydrolysis reactor has a volume of 1 m.sup.3 or more, preferably of
more than 10 m.sup.3 and most preferably of 50 m.sup.3 or more. In
general the hydrolysis reactor will be smaller than 3000 m.sup.3 or
5000 m.sup.3. The inventor poses the theory that especially at
large scale insufficient oxygen is available for the hydrolysis
which might be due to oxygen transfer limitations in the reactor
for example in the cellulolytic biomass. On lab-scale experiments
this oxygen insufficiency may play a less important role. The
surface area (or oxygen contact area of the reactor content) to
reactor volume ratio is more favourable for small scale experiments
than in large scale experiments. Moreover mixing in small scale
experiments is relatively easier than at large scale. During those
small scale experiments also the transport of oxygen from the
headspace of the hydrolysis reactor is faster than compared to the
situation in large scale experiments. This theory is only given as
possible explanation of the effect noticed by the inventors, and
the present invention does not fall or stands with the correctness
of this theory. According to a further embodiment of the invention
the addition of oxygen may be used to control at least partially
the hydrolysis process.
[0043] The process of the invention is advantageously applied in
combination with the use of thermostable enzymes.
[0044] A "thermostable" enzyme means that the enzyme has a
temperature optimum 60.degree. C. or higher, for example 70.degree.
C. or higher, such as 75.degree. C. or higher, for example
80.degree. C. or higher such as 85.degree. C. or higher. They may
for example be isolated from thermophilic microorganisms, or may be
designed by the skilled person and artificially synthesized. In one
embodiment the polynucleotides may be isolated or obtained from
thermophilic or thermotolerant filamentous fungi or isolated from
non-thermophilic or non-thermotolerant fungi but are found to be
thermostable.
[0045] By "thermophilic fungus" is meant a fungus that grows at a
temperature of 50.degree. C. or above. By "themotolerant" fungus is
meant a fungus that grows at a temperature of 45.degree. C. or
above, having a maximum near 50.degree. C.
[0046] Examples of thermophilic fungal strains are Rasamsonia
emersonii (formerly known as Talaromyces emersoni; Talaromyces
emersonii, Penicillium geosmithia emersonii and Rasamsonia
emersonii are used interchangeably herein).
[0047] Suitable thermophilic or thermotolerant fungal cells may be
a Humicola, Rhizomucor, Myceliophthora, Rasamsonia, Talaromyces,
Thermomyces, Thermoascus or Thielavia cell, preferably a Rasamsonia
emersonii cell. Preferred thermophilic or thermotolerant fungi are
Humicola grisea var. thermoidea, Humicola lanuginosa,
Myceliophthora thermophila, Papulaspora thermophilia, Rasamsonia
byssochlamydoides, Rasamsonia emersonii, Rasamsonia argillacea,
Rasamsonia eburnean, Rasamsonia brevistipitata, Rasamsonia
cylindrospora, Rhizomucor pusillus, Rhizomucor miehei, Talaromyces
bacillisporus, Talaromyces leycettanus, Talaromyces thermophilus,
Thermomyces lenuginosus, Thermoascus crustaceus, Thermoascus
thermophilus Thermoascus aurantiacus and Thielavia terrestris.
[0048] Thermophilic fungi are not restricted to a specific
taxonomic order and occur all over the fungal tree of life.
Examples are Rhizomucor in the Mucorales, Myceliophthora in
Sordariales and Talaromyces, Thermomyces and Thermoascus in the
Eurotiales (Mouchacca 1997). The majority of Talaromyces species
are rnesophiles but exceptions are species within sections
Emersorii and Thermophila. Section Emersonii includes Talaromyces
emersonii, Talaromyces byssochlamydoides, Talaromyces bacillisporus
and Talaromyces leycettanus, all of which grow well at 40.degree.
C. Talaromyces bacillisporus is thermotolerant, T. leycettanus is
thermotolerant to thermophilic, and T. emersonii and T.
byssochlamydoides are truly thermophilic (Stolk and Samson 1972).
The sole member of Talaromyces section Thermophila, T.
thermophilus, grows rapidly at 50.degree. C. (Evans and Stolk 1971;
Evans 1971; Stolk and Samson 1972). The current classification of
these thermophilic Talaromyces species is mainly based on
phenotypic and physiological characters, such as their ability to
grow above 40.degree. C., ascospore color, the structure of
ascornatal covering and the formation of a certain type of
anamorph. Stolk and Samson (1972) stated that the members of the
section Emersonii have anamorphs of either Paecilomyces (T.
byssochlamydoides and T. leycettanus) or Penicillium cylindrosporum
series (T. emersonii and T. bacillisporus). Later, Pitt (1979)
transferred the species belonging to the Penicillium cylindrosporum
series to the genus Geosmithia, based on various characters such as
the formation of conidia from terminal pores instead of on collula
(necks), a character of Penicillium and Paecilomyces. Within the
genus Geosmithia, only G. argillacea is thermotolerant, and Stolk
et al. (1969) and Evans (1971) proposed a connection with members
of Talaromyces sect. Emersonii. The phylogenetic relationship of
the themophilic Talaromyces species within Talaromyces and the
Trichocomaceae is unknown. See J. Houbraken, Antonie van
Leeuwenhoek 2012 February; 101(2): 403-21.
[0049] Rasamsonia is a new genus comprising thermotolerant and
thermophilic Talaromyces and Geosmithia species (J. Houbraken et al
vida supra). Based on phenotypic, physiological and molecular data,
Houbraken et al proposed to transfer the species T. emersonii, T.
byssochlamydoides, T. eburneus, G. argillacea and G. cylindrospora
to Rasamsonia gen. nov. Talaromyces emersonii, Penicillium
geosmithia emersonii and Rasamsonia emersonii are used
interchangeably herein.
[0050] Preferred thermophilic fungi are Rasamsonia
byssochlamydoides, Rasamsonia emersonii, Thermomyces lenuginosus,
Talaromyces thermophilus, Thermoascus crustaceus, Thermoascus
thermophilus and Thermoascus aurantiacus.
[0051] "Filamentous fungi" include all filamentous forms of the
subdivision Eumycota and Oomycota (as defined by Hawksworth et al.,
In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,
1995, CAB International, University Press, Cambridge, UK). The
filamentous fungi are characterized by a mycelial wall composed of
chitin, cellulose, glucan, chitosan, mannan, and other complex
polysaccharides. Vegetative growth is by hyphal elongation and
carbon catabolism is obligately aerobic. Filamentous fungal strains
include, but are not limited to, strains of Acremonium, Agaricus,
Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus,
Filibasidium, Fusarium, Geosmithia, Humicola, Magnaporthe, Mucor,
Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,
Penicillium, Piromyces, Panerochaete, Pleurotus, Rasamsonia,
Schizophyllum, Talaromyces, Thermoascus, Thermomyces, Thielavia,
Tolypocladium, and Trichoderma.
[0052] Several strains of filamentous fungi are readily accessible
to the public in a number of culture collections, such as the
American Type Culture Collection (ATCC), Deutsche Sammlung von
Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor
Schimmelcultures (CBS), and Agricultural Research Service Patent
Culture Collection, Northern Regional Research Center (NRRL).
Examples of such strains include Aspergillus niger CBS 513.88,
Aspergillus oryzae ATCC 20423, IFO 4177, ATCC 1011, ATCC 9576,
ATCC14488-14491, ATCC 11601, ATCC12892, P. chrysogenum CBS 455.95,
Penicillium citrinum ATCC 38065, Penicillium chrysogenum P2,
Talaromyces emersonii CBS 393.64, Acremonium chrysogenum ATCC 36225
or ATCC 48272, Trichoderma reesei ATCC 26921 or ATCC 56765 or ATCC
26921, Aspergillus sojae ATCC11906, Chrysosporium lucknowense C1,
Garg 27K, VKM F-3500-D, ATCC44006 and derivatives thereof.
[0053] An advantage of expression and production of the enzymes
(for example at least two, three or four different cellulases) in a
suitable microorganism may be a high enzyme composition yield which
can be used in the process of the present invention.
[0054] According to the invention, by the addition of oxygen it is
possible to attain many process advantages, including optimal
temperature conditions, reduced process time, reduced dosage of
enzyme, re-use of enzymes and other process optimizations,
resulting in reduced costs. Advantageously the invention provides a
process in which the hydrolysis step is conducted at improved
conditions. The invention also provides a process involving
hydrolysis having a reduced process time. Furthermore the invention
provides a process, wherein the dosage of enzyme may be reduced and
at the same time output of useful hydrolysis product is maintained
at the same level. Another advantage of the invention is that the
present process involving hydrolysis may result in process
conditions which are optimized. A still further advantage of the
invention is that the output of useful hydrolysis product of the
process involving hydrolysis is increased using the same enzyme
dosage.
Stable Enzyme Composition
[0055] Stable enzyme composition herein means that the enzyme
composition retains activity after 30 hours of hydrolysis reaction
time, preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%,
75%, 80%, 90%, 95%, 96% 97%, 98%, 99% or 100% of its initial
activity after 30 hours of hydrolysis reaction time. Preferably the
enzyme composition retains activity after 40, 50, 60, 70, 80, 90
100, 150, 200, 250, 300, 350, 400, 450, 500 hours of hydrolysis
reaction time.
[0056] The enzyme composition may be prepared by fermentation of a
suitable substrate with a suitable microorganism, e.g. Rasamsonia
emersonii or Aspergillus niger wherein the enzyme composition is
produced by the microorganism. The microorganism may be altered to
improve or to make the cellulase composition. For example the
microorganism may be mutated by classical strain improvement
procedures or by recombinant DNA techniques. Therefore the
microorganisms mentioned herein can be used as such to produce the
cellulase composition or may be altered to increase the production
or to produce an altered cellulase composition which might include
heterologous cellulases, thus enzymes that are not originally
produced by that microorganism. Preferably a fungus, more
preferably a filamentous fungus is used to produce the cellulase
composition. Advantageously a thermophilic or thermotolerant
microorganism is used. Optionally a substrate is used that induces
the expression of the enzymes in the enzyme composition during the
production of the enzyme composition.
[0057] The enzyme composition is used to release sugars from
lignocellulose, that comprises polysaccharides. The major
polysaccharides are cellulose (glucans), hem icelluloses (xylans,
heteroxylans and xyloglucans). In addition, some hemicellulose may
be present as glucomannans, for example in wood-derived feedstocks.
The enzymatic hydrolysis of these polysaccharides to soluble
sugars, including both monomers and multimers, for example glucose,
cellobiose, xylose, arabinose, galactose, fructose, mannose,
rhamnose, ribose, galacturonic acid, glucoronic acid and other
hexoses and pentoses occurs under the action of different enzymes
acting in concert. By sugar product is meant the enzymatic
hydrolysis product of the feedstock or ligno-cellulosic material.
The sugar product will comprise soluble sugars, including both
monomers and multimers, preferably will comprise glucose. Examples
of other sugars are cellobiose, xylose, arabinose, galactose,
fructose, mannose, rhamnose, ribose, galacturonic acid, glucoronic
acid and other hexoses and pentoses. The sugar product may be used
as such or may be further processed for example purified.
[0058] In addition, pectins and other pectic substances such as
arabinans may make up considerably proportion of the dry mass of
typically cell walls from non-woody plant tissues (about a quarter
to half of dry mass may be pectins).
[0059] Cellulose is a linear polysaccharide composed of glucose
residues linked by .beta.-1,4 bonds. The linear nature of the
cellulose fibers, as well as the stoichiometry of the .beta.-linked
glucose (relative to a) generates structures more prone to
interstrand hydrogen bonding than the highly branched
.alpha.-linked structures of starch. Thus, cellulose polymers are
generally less soluble, and form more tightly bound fibers than the
fibers found in starch.
[0060] Enzymes that may be included in the stable enzyme
composition used in the invention are now described in more
detail:
[0061] GH61, Endoglucanases (EG) and exo-cellobiohydrolases (CBH)
catalyze the hydrolysis of insoluble cellulose to products such as
cellooligosaccharides (cellobiose as a main product), while
.beta.-glucosidases (BG) convert the oligosaccharides, mainly
cellobiose and cellotriose to glucose.
[0062] Hemicellulose is a complex polymer, and its composition
often varies widely from organism to organism and from one tissue
type to another. In general, a main component of hemicellulose is
.beta.-1,4-linked xylose, a five carbon sugar. However, this xylose
is often branched at 0 to 3 and/or 0 to 2 atom of xylose, and can
be substituted with linkages to arabinose, galactose, mannose,
glucuronic acid, galacturonic acid or by esterification to acetic
acid (and esterification of ferulic acid to arabinose).
Hemicellulose can also contain glucan, which is a general term for
.beta.-linked six carbon sugars (such as the .beta.-(1,3)(1,4)
glucans and heteroglucans mentioned previously) and additionally
glucomannans (in which both glucose and mannose are present in the
linear backbone, linked to each other by .beta.-linkages).
[0063] Xylanases together with other accessory enzymes, for example
.alpha.-L-arabinofuranosidases, feruloyl and acetylxylan esterases,
glucuronidases, and .beta.-xylosidases) catalyze the hydrolysis of
hemicelluloses.
[0064] Pectic substances include pectins, arabinans, galactans and
arabinogalactans. Pectins are the most complex polysaccharides in
the plant cell wall. They are built up around a core chain of
.beta.(1,4)-linked D-galacturonic acid units interspersed to some
degree with L-rhamnose. In any one cell wall there are a number of
structural units that fit this description and it has generally
been considered that in a single pectic molecule, the core chains
of different structural units are continuous with one another.
[0065] The principal types of structural unit are: galacturonan
(homogalacturonan), which may be substituted with methanol on the
carboxyl group and acetate on O-2 and O-3; rhamnogalacturonan I
(RGI), in which galacturonic acid units alternate with rhamnose
units carrying (1,4)-linked galactan and (1,5)-linked arabinan
side-chains. The arabinan side-chains may be attached directly to
rhamnose or indirectly through the galactan chains;
xylogalacturonan, with single xylosyl units on O-3 of galacturonic
acid (closely associated with RGI); and rhamnogalacturonan II
(RGII), a particularly complex minor unit containing unusual
sugars, for example apiose. An RGII unit may contain two apiosyl
residues which, under suitable ionic conditions, can reversibly
form esters with borate.
[0066] A composition for use in a method of the invention comprises
preferably at least two activities, although typically a
composition will comprise more than two activities, for example,
three, four, five, six, seven, eight, nine or more. Typically, a
composition of the invention may comprise at least two different
celulases or one cellulase and at least one hemicellulase. A
composition of the invention may comprise cellulases, but no
xylanases. In addition, a composition of the invention may comprise
auxiliary enzyme activity, i.e. additional activity which, either
directly or indirectly leads to lignocellulose degradation.
Examples of such auxiliary activities are mentioned herein.
[0067] Thus, a composition for use in the invention may comprise
GH61, endoglucanase activity and/or cellobiohydrolase activity
and/or .beta.-glucosidase activity. A composition for use in the
invention may comprise more than one enzyme activity in one or more
of those classes. For example, a composition for use in the
invention may comprise two endoglucanase activities, for example,
endo-1,3(1,4)-.beta. glucanase activity and
endo-.beta.-1,4-glucanase activity. Such a composition may also
comprise one or more xylanase activities. Such a composition may
comprise an auxiliary enzyme activity.
[0068] A composition for use in the invention may be derived from
Rasamsonia emersonii. In the invention, it is anticipated that a
core set of (lignocellulose degrading) enzyme activities may be
derived from Rasamsonia emersonii. Rasamsonia emersonii can provide
a highly effective set of activities as demonstrated herein for the
hydrolysis of lignocellulosic biomass. That activity can then be
supplemented with additional enzyme activities from other sources.
Such additional activities may be derived from classical sources
and/or produced by a genetically modified organism.
[0069] The activities in a composition for use in the invention may
be thermostable. Herein, this means that the activity has a
temperature optimum of about 60.degree. C. or higher, for example
about 70.degree. C. or higher, such as about 75.degree. C. or
higher, for example about 80.degree. C. or higher such as
85.degree. C. or higher. Activities in a composition for use in the
invention will typically not have the same temperature optima, but
preferably will, nevertheless, be thermostable.
[0070] In addition, enzyme activities in a composition for use in
the invention may be able to work at low pH. For the purposes of
this invention, low pH indicates a pH of about 5.5 or lower, about
5 or lower, about 4.9 or lower, about 4.8 or lower, about 4.7 or
lower, about 4,6 or lower, about 4.5 or lower, about 4.4 or lower,
about 4.3 or lower, about 4.2 or lower, about 4,1 or lower, about
4.0 or lower about 3.9 or lower, or about 3.8 or lower, about 3.7
or lower, about 3.6 or lower, or about 3.5 or lower.
[0071] Activities in a composition for use in the invention may be
defined by a combination of any of the above temperature optima and
pH values.
[0072] The composition used in a method of the invention may
comprise, in addition to the activities derived from Rasamsonia, a
cellulase (for example one derived from a source other than
Rasamsonia) and/or a hemicellulase (for example one derived from a
source other than Rasamsonia) and/or a pectinase.
[0073] A composition for use in the invention may comprise one,
two, three, four classes or more of cellulase, for example one, two
three or four or all of a GH61, an endoglucanase (EG), one or two
exo-cellobiohydrolase (CBH) and a .beta.-glucosidase (BG). A
composition for use in the invention may comprise two or more of
any of these classes of cellulase.
[0074] A composition of the invention may comprise an activity
which has a different type of cellulase activity and/or
hemicellulase activity and/or pectinase activity than that provided
by the composition for use in a method of the invention. For
example, a composition of the invention may comprise one type of
cellulase and/or hemicellulase activity and/or pectinase activity
provided by a composition as described herein and a second type of
cellulase and/or hemicellulase activity and/or pectinase activity
provided by an additional cellulose/hemicellulase/pectinase.
[0075] Herein, a cellulase is any polypeptide which is capable of
degrading or modifying cellulose. A polypeptide which is capable of
degrading cellulose is one which is capable of catalysing the
process of breaking down cellulose into smaller units, either
partially, for example into cellodextrins, or completely into
glucose monomers. A cellulase according to the invention may give
rise to a mixed population of cellodextrins and glucose monomers
when contacted with the cellulase. Such degradation will typically
take place by way of a hydrolysis reaction.
[0076] GH61 (glycoside hydrolase family 61 or sometimes referred to
EGIV) proteins are oxygen-dependent polysaccharide monooxygenases
(PMO's) according to the latest literature. Often in literature
these proteins are mentioned to enhance the action of cellulases on
lignocellulose substrates. GH61 was originally classified as
endogluconase based on measurement of very weak
endo-1,4.beta.-d-glucanase activity in one family member. The term
"GH61" as used herein, is to be understood as a family of enzymes,
which share common conserved sequence portions and foldings to be
classified in family of the well-established CAZY GH classification
system (http://www.cazy.org/GH61.html). The glycoside hydrolase
family 61 is a member of the family of glycoside hydrolases EC
3.2.1. GH61 is used herein as being part of the cellulases.
[0077] Herein, a hemicellulase is any polypeptide which is capable
of degrading or modifying hemicellulose. That is to say, a
hemicellulase may be capable of degrading or modifying one or more
of xylan, glucuronoxylan, arabinoxylan, glucomannan and xyloglucan.
A polypeptide which is capable of degrading a hemicellulose is one
which is capable of catalysing the process of breaking down the
hemicellulose into smaller polysaccharides, either partially, for
example into oligosaccharides, or completely into sugar monomers,
for example hexose or pentose sugar monomers. A hemicellulase
according to the invention may give rise to a mixed population of
oligosaccharides and sugar monomers when contacted with the
hemicellulase. Such degradation will typically take place by way of
a hydrolysis reaction.
[0078] Herein, a pectinase is any polypeptide which is capable of
degrading or modifying pectin. A polypeptide which is capable of
degrading pectin is one which is capable of catalysing the process
of breaking down pectin into smaller units, either partially, for
example into oligosaccharides, or completely into sugar monomers. A
pectinase according to the invention may give rise to a mixed
population of oligosacchardies and sugar monomers when contacted
with the pectinase. Such degradation will typically take place by
way of a hydrolysis reaction.
[0079] Accordingly, a composition of the invention may comprise any
cellulase, for example, a GH61, a cellobiohydrolase, an
endo-.beta.1,4-glucanase, a .beta.-glucosidase or a
.beta.-(1,3)(1,4)-glucanase.
[0080] Herein, a cellobiohydrolase (EC 3.2.1.91) is any polypeptide
which is capable of catalysing the hydrolysis of
1,4-.beta.-D-glucosidic linkages in cellulose or cellotetraose,
releasing cellobiose from the ends of the chains. This enzyme may
also be referred to as cellulase 1,4-.beta.-cellobiosidase,
1,4-.beta.-cellobiohydrolase, 1,4-.beta.-D-glucan
cellobiohydrolase, avicelase, exo-1,4-.beta.-D-glucanase,
exocellobiohydrolase or exoglucanase.
[0081] Herein, an endo-.beta.-1,4-glucanase (EC 3.2.1.4) is any
polypeptide which is capable of catalysing the endohydrolysis of
1,4-.beta.-D-glucosidic linkages in cellulose, lichenin or cereal
.beta.-D-glucans. Such a polypeptide may also be capable of
hydrolyzing 1,4-linkages in .beta.-D-glucans also containing
1,3-linkages. This enzyme may also be referred to as cellulase,
avicelase, .beta.1,4-endoglucan hydrolase, .beta.-1,4-glucanase,
carboxymethyl cellulase, celludextrinase,
endo-1,4.beta.-D-glucanase, endo-1,4-.beta.-D-glucanohydrolase,
endo-1,4-.beta.-glucanase or endoglucanase.
[0082] Herein, a .beta.-glucosidase (EC 3.2.1.21) is any
polypeptide which is capable of catalysing the hydrolysis of
terminal, non-reducing .beta.-D-glucose residues with release of
.beta.-D-glucose. Such a polypeptide may have a wide specificity
for .beta.-D-glucosides and may also hydrolyze one or more of the
following: a .beta.-D-galactoside, an .alpha.-L-arabinoside, a
.beta.-D-xyloside or a .beta.-D-fucoside. This enzyme may also be
referred to as amygdalase, .beta.-D-glucoside glucohydrolase,
cellobiase or gentobiase.
[0083] Herein a .beta.-(1,3)(1,4)-glucanase (EC 3.2.1.73) is any
polypeptide which is capable of catalyzing the hydrolysis of
1,4-.beta.-D-glucosidic linkages in .beta.-D-glucans containing
1,3- and 1,4-bonds. Such a polypeptide may act on lichenin and
cereal .beta.-D-glucans glucans, but not on .beta.-D-glucans
containing only 1,3- or 1,4-bonds. This enzyme may also be referred
to as licheninase, 1,3-1,4-.beta.-D-glucan 4-glucanohydrolase,
.beta.-glucanase, endo-.beta.-1,3-1,4 glucanase, lichenase or mixed
linkage .beta.-glucanase. An alternative for this type of enzyme is
EC 3.2.1.6, which is described as endo-1,3(4)-beta-glucanase. This
type of enzyme hydrolyses 1,3- or 1,4-linkages in beta-D-glucanse
when the glucose residue whose reducing group is involved in the
linkage to be hydrolysed is itself substituted at C-3. Alternative
names include endo-1,3-beta-glucanase, laminarinase,
1,3-(1,3;1,4)-beta-D-glucan 3 (4) glucanohydrolase; substrates
include laminarin, lichenin and cereal beta-D-glucans.
[0084] A composition of the invention may comprise any
hemicellulase, for example, an endoxylanase, a .beta.-xylosidase, a
.alpha.-L-arabionofuranosidase, an .alpha.-D-glucuronidase, an
acetyl xylan esterase, a feruloyl esterase, a coumaroyl esterase,
an .alpha.-galactosidase, a .beta.-galactosidase, a
.beta.-mannanase or a .beta.-mannosidase.
[0085] Herein, an endoxylanase (EC 3.2.1.8) is any polypeptide
which is capable of catalyzing the endohydrolysis of
1,4-.beta.-D-xylosidic linkages in xylans. This enzyme may also be
referred to as endo-1,4-.beta.-xylanase or 1,4-.beta.-D-xylan
xylanohydrolase. An alternative is EC 3.2.1.136, a
glucuronoarabinoxylan endoxylanase, an enzyme that is able to
hydrolyse 1,4 xylosidic linkages in glucuronoarabinoxylans.
[0086] Herein, a .beta.-xylosidase (EC 3.2.1.37) is any polypeptide
which is capable of catalyzing the hydrolysis of
1,4-.beta.-D-xylans, to remove successive D-xylose residues from
the non-reducing termini. Such enzymes may also hydrolyze
xylobiose. This enzyme may also be referred to as xylan
1,4-.beta.-xylosidase, 1,4-.beta.-D-xylan xylohydrolase,
exo-1,4-.beta.-xylosidase or xylobiase.
[0087] Herein, an .beta.-L-arabinofuranosidase (EC 3.2.1.55) is any
polypeptide which is capable of acting on
.alpha.-L-arabinofuranosides, .alpha.-L-arabinans containing (1,2)
and/or (1,3)- and/or (1,5)-linkages, arabinoxylans and
arabinogalactans. This enzyme may also be referred to as
.alpha.-N-arabinofuranosidase, arabinofuranosidase or
arabinosidase.
[0088] Herein, an .beta.-D-glucuronidase (EC 3.2.1.139) is any
polypeptide which is capable of catalyzing a reaction of the
following form: alpha-D-glucuronoside+H(2)O=an
alcohol+D-glucuronate. This enzyme may also be referred to as
alpha-glucuronidase or alpha-glucosiduronase. These enzymes may
also hydrolyse 4-O-methylated glucoronic acid, which can also be
present as a substituent in xylans. Alternative is EC 3.2.1.131:
xylan alpha-1,2-glucuronosidase, which catalyses the hydrolysis of
alpha-1,2-(4-O-methyl)glucuronosyl links.
[0089] Herein, an acetyl xylan esterase (EC 3.1.1.72) is any
polypeptide which is capable of catalyzing the deacetylation of
xylans and xylo-oligosaccharides. Such a polypeptide may catalyze
the hydrolysis of acetyl groups from polymeric xylan, acetylated
xylose, acetylated glucose, alpha-napthyl acetate or p-nitrophenyl
acetate but, typically, not from triacetylglycerol. Such a
polypeptide typically does not act on acetylated mannan or
pectin.
[0090] Herein, a feruloyl esterase (EC 3.1.1.73) is any polypeptide
which is capable of catalyzing a reaction of the form:
feruloyl-saccharide+H(2)O=ferulate+saccharide. The saccharide may
be, for example, an oligosaccharide or a polysaccharide. It may
typically catalyze the hydrolysis of the
4-hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified
sugar, which is usually arabinose in `natural` substrates.
p-nitrophenol acetate and methyl ferulate are typically poorer
substrates. This enzyme may also be referred to as cinnamoyl ester
hydrolase, ferulic acid esterase or hydroxycinnamoyl esterase. It
may also be referred to as a hemicellulase accessory enzyme, since
it may help xylanases and pectinases to break down plant cell wall
hemicellulose and pectin.
[0091] Herein, a coumaroyl esterase (EC 3.1.1.73) is any
polypeptide which is capable of catalyzing a reaction of the form:
coumaroyl-saccharide +H(2)O=coumarate+saccharide. The saccharide
may be, for example, an oligosaccharide or a polysaccharide. This
enzyme may also be referred to as trans-4-coumaroyl esterase,
trans-p-coumaroyl esterase, p-coumaroyl esterase or p-coumaric acid
esterase. This enzyme also falls within EC 3.1.1.73 so may also be
referred to as a feruloyl esterase.
[0092] Herein, an .alpha.-galactosidase (EC 3.2.1.22) is any
polypeptide which is capable of catalyzing the hydrolysis of of
terminal, non-reducing .alpha.-D-galactose residues in
.alpha.-D-galactosides, including galactose oligosaccharides,
galactomannans, galactans and arabinogalactans. Such a polypeptide
may also be capable of hydrolyzing .alpha.-D-fucosides. This enzyme
may also be referred to as melibiase.
[0093] Herein, a .beta.-galactosidase (EC 3.2.1.23) is any
polypeptide which is capable of catalyzing the hydrolysis of
terminal non-reducing .beta.-D-galactose residues in
.beta.-D-galactosides. Such a polypeptide may also be capable of
hydrolyzing .alpha.-L-arabinosides. This enzyme may also be
referred to as exo-(1.fwdarw.4)-.beta.-D-galactanase or
lactase.
[0094] Herein, a .beta.-mannanase (EC 3.2.1.78) is any polypeptide
which is capable of catalyzing the random hydrolysis of
1,4.beta.-D-mannosidic linkages in mannans, galactomannans and
glucomannans. This enzyme may also be referred to as mannan
endo-1,4-.beta.-mannosidase or endo-1,4-mannanase.
[0095] Herein, a .beta.-mannosidase (EC 3.2.1.25) is any
polypeptide which is capable of catalyzing the hydrolysis of
terminal, non-reducing .beta.-D-mannose residues in
.beta.-D-mannosides. This enzyme may also be referred to as
mannanase or mannase.
[0096] A composition of the invention may comprise any pectinase,
for example an endo polygalacturonase, a pectin methyl esterase, an
endo-galactanase, a beta galactosidase, a pectin acetyl esterase,
an endo-pectin lyase, pectate lyase, alpha rhamnosidase, an
exo-galacturonase, an expolygalacturonate lyase, a
rhamnogalacturonan hydrolase, a rhamnogalacturonan lyase, a
rhamnogalacturonan acetyl esterase, a rhamnogalacturonan
galacturonohydrolase, a xylogalacturonase.
[0097] Herein, an endo-polygalacturonase (EC 3.2.1.15) is any
polypeptide which is capable of catalyzing the random hydrolysis of
1,4-.alpha.-D-galactosiduronic linkages in pectate and other
galacturonans. This enzyme may also be referred to as
polygalacturonase pectin depolym erase, pectinase,
endopolygalacturonase, pectolase, pectin hydrolase, pectin
polygalacturonase, poly-.alpha.-1,4-galacturonide glycanohydrolase,
endogalacturonase; endo-D-galacturonase or
poly(1,4-.alpha.-D-galacturonide) glycanohydrolase.
[0098] Herein, a pectin methyl esterase (EC 3.1.1.11) is any enzyme
which is capable of catalyzing the reaction: pectin+n H.sub.2O=n
methanol+pectate. The enzyme may also been known as pectinesterase,
pectin demethoxylase, pectin methoxylase, pectin methylesterase,
pectase, pectinoesterase or pectin pectylhydrolase.
[0099] Herein, an endo-galactanase (EC 3.2.1.89) is any enzyme
capable of catalyzing the endohydrolysis of
1,4-.beta.-D-galactosidic linkages in arabinogalactans. The enzyme
may also be known as arabinogalactan endo-1,4-.beta.-galactosidase,
endo-1,4-.beta.-galactanase, galactanase, arabinogalactanase or
arabinogalactan 4-.beta.-D-galactanohydrolase.
[0100] Herein, a pectin acetyl esterase is defined herein as any
enzyme which has an acetyl esterase activity which catalyzes the
deacetylation of the acetyl groups at the hydroxyl groups of GaIUA
residues of pectin
[0101] Herein, an endo-pectin lyase (EC 4.2.2.10) is any enzyme
capable of catalyzing the eliminative cleavage of
(1.fwdarw.4)-.alpha.-D-galacturonan methyl ester to give
oligosaccharides with
4-deoxy-6-O-methyl-.alpha.-D-galact-4-enuronosyl groups at their
non-reducing ends. The enzyme may also be known as pectin lyase,
pectin trans-eliminase; endo-pectin lyase, polymethylgalacturonic
transeliminase, pectin methyltranseliminase, pectolyase, PL, PNL or
PMGL or (1.fwdarw.4)-6-O-methyl-.alpha.-D-galacturonan lyase.
[0102] Herein, a pectate lyase (EC 4.2.2.2) is any enzyme capable
of catalyzing the eliminative cleavage of
(1.fwdarw.4)-.alpha.-D-galacturonan to give oligosaccharides with
4-deoxy-.alpha.-D-galact-4-enuronosyl groups at their non-reducing
ends. The enzyme may also be known polygalacturonic transeliminase,
pectic acid transeliminase, polygalacturonate lyase, endopectin
methyltranseliminase, pectate transeliminase, endogalacturonate
transeliminase, pectic acid lyase, pectic lyase,
.alpha.-1,4-D-endopolygalacturonic acid lyase, PGA lyase, PPase-N,
endo-.alpha.-1,4-polygalacturonic acid lyase, polygalacturonic acid
lyase, pectin trans-eliminase, polygalacturonic acid
trans-eliminase or (1.fwdarw.4)-.alpha.-D-galacturonan lyase.
[0103] Herein, an alpha rhamnosidase (EC 3.2.1.40) is any
polypeptide which is capable of catalyzing the hydrolysis of
terminal non-reducing .alpha.-L-rhamnose residues in
.alpha.-L-rhamnosides or alternatively in rhamnogalacturonan. This
enzyme may also be known as .alpha.-L-rhamnosidase T,
.alpha.-L-rhamnosidase N or .alpha.-L-rhamnoside
rhamnohydrolase.
[0104] Herein, exo-galacturonase (EC 3.2.1.82) is any polypeptide
capable of hydrolysis of pectic acid from the non-reducing end,
releasing digalacturonate. The enzyme may also be known as
exo-poly-.alpha.-galacturonosidase, exopolygalacturonosidase or
exopolygalacturanosidase.
[0105] Herein, exo-galacturonase (EC 3.2.1.67) is any polypeptide
capable of catalyzing:
(1,4-.alpha.-D-galacturonide).sub.n+H.sub.2O=(1,4-.alpha.-D-galacturonide-
).sub.n-1+D-galacturonate. The enzyme may also be known as
galacturan 1,4-.alpha.-galacturonidase, exopolygalacturonase,
poly(galacturonate) hydrolase, exo-D-galacturonase,
exo-D-galacturonanase, exopoly-D-galacturonase or
poly(1,4-.alpha.-D-galacturonide) galacturonohydrolase.
[0106] Herein, exopolygalacturonate lyase (EC 4.2.2.9) is any
polypeptide capable of catalyzing eliminative cleavage of
4-(4-deoxy-.alpha.-D-galact-4-enuronosyl)-D-galacturonate from the
reducing end of pectate, i.e. de-esterified pectin. This enzyme may
be known as pectate disaccharide-lyase, pectate exo-lyase,
exopectic acid transeliminase, exopectate lyase,
exopolygalacturonic acid-trans-eliminase, PATE, exo-PATE, exo-PGL
or (1.fwdarw.4)-.alpha.-D-galacturonan
reducing-end-disaccharide-lyase.
[0107] Herein, rhamnogalacturonan hydrolase is any polypeptide
which is capable of hydrolyzing the linkage between
galactosyluronic acid acid and rhamnopyranosyl in an endo-fashion
in strictly alternating rhamnogalacturonan structures, consisting
of the disaccharide
[(1,2-alpha-L-rhamnoyl-(1,4)-alpha-galactosyluronic acid].
[0108] Herein, rhamnogalacturonan lyase is any polypeptide which is
any polypeptide which is capable of cleaving
.alpha.-L-Rhap-(1.fwdarw.4)-.alpha.-D-GalpA linkages in an
endo-fashion in rhamnogalacturonan by beta-elimination.
[0109] Herein, rhamnogalacturonan acetyl esterase is any
polypeptide which catalyzes the deacetylation of the backbone of
alternating rhamnose and galacturonic acid residues in
rhamnogalacturonan.
[0110] Herein, rhamnogalacturonan galacturonohydrolase is any
polypeptide which is capable of hydrolyzing galacturonic acid from
the non-reducing end of strictly alternating rhamnogalacturonan
structures in an exo-fashion.
[0111] Herein, xylogalacturonase is any polypeptide which acts on
xylogalacturonan by cleaving the .beta.-xylose substituted
galacturonic acid backbone in an endo-manner. This enzyme may also
be known as xylogalacturonan hydrolase.
[0112] Herein, an .alpha.-L-arabinofuranosidase (EC 3.2.1.55) is
any polypeptide which is capable of acting on
.alpha.-L-arabinofuranosides, .alpha.-L-arabinans containing (1,2)
and/or (1,3)- and/or (1,5)-linkages, arabinoxylans and
arabinogalactans. This enzyme may also be referred to as
.alpha.-N-arabinofuranosidase, arabinofuranosidase or
arabinosidase.
[0113] Herein, endo-arabinanase (EC 3.2.1.99) is any polypeptide
which is capable of catalyzing endohydrolysis of
1,5-.alpha.-arabinofuranosidic linkages in 1,5-arabinans. The
enzyme may also be know as endo-arabinase, arabinan
endo-1,5-.alpha.-L-arabinosidase, endo-1,5-.alpha.-L-arabinanase,
endo-.alpha.-1,5-arabanase; endo-arabanase or
1,5-.alpha.-L-arabinan 1,5-.alpha.-L-arabinanohydrolase.
[0114] A composition of the invention will typically comprise at
least one cellulase and/or at least one hemicellulase and/or at
least one pectinase (one of which is a polypeptide according to the
invention). A composition of the invention may comprise a GH61, a
cellobiohydrolase, an endoglucanase and/or a .beta.-glucosidase.
Such a composition may also comprise one or more hemicellulases
and/or one or more pectinases.
[0115] In addition, one or more (for example two, three, four or
all) of an amylase, a protease, a lipase, a ligninase, a
hexosyltransferase, a glucuronidase or an expansin or a cellulose
induced protein or a cellulose integrating protein or like protein
may be present in a composition of the invention (these are
referred to as auxiliary activities above).
[0116] "Protease" includes enzymes that hydrolyze peptide bonds
(peptidases), as well as enzymes that hydrolyze bonds between
peptides and other moieties, such as sugars (glycopeptidases). Many
proteases are characterized under EC 3.4, and are suitable for use
in the invention incorporated herein by reference. Some specific
types of proteases include, cysteine proteases including pepsin,
papain and serine proteases including chymotrypsins,
carboxypeptidases and metalloendopeptidases. "Lipase" includes
enzymes that hydrolyze lipids, fatty acids, and acylglycerides,
including phospoglycerides, lipoproteins, diacylglycerols, and the
like. In plants, lipids are used as structural components to limit
water loss and pathogen infection. These lipids include waxes
derived from fatty acids, as well as cutin and suberin.
[0117] "Ligninase" includes enzymes that can hydrolyze or break
down the structure of lignin polymers. Enzymes that can break down
lignin include lignin peroxidases, manganese peroxidases, laccases
and feruloyl esterases, and other enzymes described in the art
known to depolymerize or otherwise break lignin polymers. Also
included are enzymes capable of hydrolyzing bonds formed between
hemicellulosic sugars (notably arabinose) and lignin. Ligninases
include but are not limited to the following group of enzymes:
lignin peroxidases (EC 1.11.1.14), manganese peroxidases (EC
1.11.1.13), laccases (EC 1.10.3.2) and feruloyl esterases (EC
3.1.1.73).
[0118] "Hexosyltransferase" (2.4.1-) includes enzymes which are
capable of catalyzing a transferase reaction, but which can also
catalyze a hydrolysis reaction, for example of cellulose and/or
cellulose degradation products. An example of a hexosyltransferase
which may be used in the invention is a
.beta.-glucanosyltransferase. Such an enzyme may be able to
catalyze degradation of (1,3)(1,4)glucan and/or cellulose and/or a
cellulose degradation product.
[0119] "Glucuronidase" includes enzymes that catalyze the
hydrolysis of a glucoronoside, for example .beta.-glucuronoside to
yield an alcohol. Many glucuronidases have been characterized and
may be suitable for use in the invention, for example
.beta.-glucuronidase (EC 3.2.1.31), hyalurono-glucuronidase (EC
3.2.1.36), glucuronosyl-disulfoglucosamine glucuronidase
(3.2.1.56), glycyrrhizinate .beta.-glucuronidase (3.2.1.128) or
.alpha.-D-glucuronidase (EC 3.2.1.139).
[0120] A composition for use in the invention may comprise an
expansin or expansin-like protein, such as a swollenin (see
Salheimo et al., Eur. J. Biohem. 269, 4202-4211, 2002) or a
swollenin-like protein.
[0121] Expansins are implicated in loosening of the cell wall
structure during plant cell growth. Expansins have been proposed to
disrupt hydrogen bonding between cellulose and other cell wall
polysaccharides without having hydrolytic activity. In this way,
they are thought to allow the sliding of cellulose fibers and
enlargement of the cell wall. Swollenin, an expansin-like protein
contains an N-terminal Carbohydrate Binding Module Family 1 domain
(CBD) and a C-terminal expansin-like domain. For the purposes of
this invention, an expansin-like protein or swollenin-like protein
may comprise one or both of such domains and/or may disrupt the
structure of cell walls (such as disrupting cellulose structure),
optionally without producing detectable amounts of reducing
sugars.
[0122] A composition for use in the invention may be a cellulose
induced protein, for example the polypeptide product of the cip1 or
cip2 gene or similar genes (see Foreman et al., J. Biol. Chem.
278(34), 31988-31997, 2003), a cellulose/cellulosome integrating
protein, for example the polypeptide product of the cipA or cipC
gene, or a scaffoldin or a scaffoldin-like protein. Scaffoldins and
cellulose integrating proteins are multi-functional integrating
subunits which may organize cellulolytic subunits into a
multi-enzyme complex. This is accomplished by the interaction of
two complementary classes of domain, i.e. a cohesion domain on
scaffoldin and a dockerin domain on each enzymatic unit. The
scaffoldin subunit also bears a cellulose-binding module (CBM) that
mediates attachment of the cellulosome to its substrate. A
scaffoldin or cellulose integrating protein for the purposes of
this invention may comprise one or both of such domains.
[0123] A composition for use in a method of the invention may be
composed of a member of each of the classes of enzymes mentioned
above, several members of one enzyme class, or any combination of
these enzymes classes or helper proteins (i.e. those proteins
mentioned herein which do not have enzymatic activity per se, but
do nevertheless assist in lignocellulosic degradation).
[0124] A composition for use in a method of the invention may be
composed of enzymes from (1) commercial suppliers; (2) cloned genes
expressing enzymes; (3) complex broth (such as that resulting from
growth of a microbial strain in media, wherein the strains secrete
proteins and enzymes into the media; (4) cell lysates of strains
grown as in (3); and/or (5) plant material expressing enzymes.
Different enzymes in a composition of the invention may be obtained
from different sources.
[0125] The enzymes can be produced either exogenously in
microorganisms, yeasts, fungi, bacteria or plants, then isolated
and added, for example, to lignocellulosic feedstock.
Alternatively, the enzymes are produced, but not isolated, and
crude cell mass fermentation broth, or plant material (such as corn
stover or wheat straw), and the like may be added to, for example,
the feedstock. Alternatively, the crude cell mass or enzyme
production medium or plant material may be treated to prevent
further microbial growth (for example, by heating or addition of
antimicrobial agents), then added to, for example, a feedstock.
These crude enzyme mixtures may include the organism producing the
enzyme. Alternatively, the enzyme may be produced in a fermentation
that uses (pre-treated) feedstock (such as corn stover or wheat
straw) to provide nutrition to an organism that produces an
enzyme(s). In this manner, plants that produce the enzymes may
themselves serve as a lignocellulosic feedstock and be added into
lignocellulosic feedstock.
[0126] In the uses and methods described herein, the components of
the compositions described above may be provided concomitantly
(i.e. as a single composition per se) or separately or
sequentially.
[0127] The invention thus relates to methods in which the
composition described above are used and to uses of the composition
in industrial processes.
Ligno-Cellulosic Material
[0128] Lignocellulosic material herein includes any lignocellulosic
and/or hemicellulosic material. Lignocellulosic material suitable
for use as feedstock in the invention includes biomass, e.g. virgin
biomass and/or non-virgin biomass such as agricultural biomass,
commercial organics, construction and demolition debris, municipal
solid waste, waste paper and yard waste. Common forms of biomass
include trees, shrubs and grasses, wheat, wheat straw, sugar cane
bagasse, switch grass, miscanthus, corn, corn stover, corn husks,
corn cobs, canola stems, soybean stems, sweet sorghum, corn kernel
including fiber from kernels, products and by-products from milling
of grains such as corn, wheat and barley (including wet milling and
dry milling) often called "bran or fibre" as well as municipal
solid waste, waste paper and yard waste. The biomass can also be,
but is not limited to, herbaceous material, agricultural residues,
forestry residues, municipal solid wastes, waste paper, and pulp
and paper mill residues. "Agricultural biomass" includes branches,
bushes, canes, corn and corn husks, energy crops, forests, fruits,
flowers, grains, grasses, herbaceous crops, leaves, bark, needles,
logs, roots, saplings, short rotation woody crops, shrubs, switch
grasses, trees, vegetables, fruit peels, vines, sugar beet pulp,
wheat midlings, oat hulls, and hard and soft woods (not including
woods with deleterious materials). In addition, agricultural
biomass includes organic waste materials generated from
agricultural processes including farming and forestry activities,
specifically including forestry wood waste. Agricultural biomass
may be any of the aforementioned singularly or in any combination
or mixture thereof.
Pre-Treatment
[0129] The feedstock may optionally be pre-treated with heat,
mechanical and/or chemical modification or any combination of such
methods in order to to enhance the accessibility of the substrate
to enzymatic hydrolysis and/or hydrolyse the hemicellulose and/or
solubilize the hemicellulose and/or cellulose and/or lignin, in any
way known in the art. In one embodiment, the pre-treatment is
conducted treating the lignocellulose with steam explosion, hot
water treatment or treatment with dilute acid or dilute base.
Washing Step
[0130] Optionally, the process according to the invention comprises
a washing step. The optional washing step may be used to remove
water soluble compounds that may act as inhibitors for the
fermentation step. The washing step may be conducted in known
manner.
Enzymatic Hydrolysis
[0131] The enzyme composition used in the process of the invention
can extremely effectively hydrolyze lignocellulolytic material, for
example corn stover or wheat straw, which can then be further
converted into a useful product, such as ethanol, biogas, butanol,
lactic acid, a plastic, an organic acid, a solvent, an animal feed
supplement, a pharmaceutical, a vitamin, an amino acid, an enzyme
or a chemical feedstock. Additionally, intermediate products from a
process following the hydrolysis, for example lactic acid as
intermediate in biogas production, can be used as building block
for other materials. The present invention is exemplified with the
production of ethanol but this is done as exemplification only
rather than as limitation, the other mentioned useful products can
be produced equally well.
[0132] The process according to the invention comprises an
enzymatic hydrolysis step. The enzymatic hydrolysis includes, but
is not limited to, hydrolysis for the purpose of liquification of
the feedstock and hydrolysis for the purpose of releasing sugar
from the feedstock or both. In this step optionally pre-treated and
optionally washed ligno-cellulosic material is brought into contact
with the enzyme composition according to the invention. Depending
on the lignocellulosic material and the pre-treatment, the
different reaction conditions, e.g. temperature, enzyme dosage,
hydrolysis reaction time and dry matter concentration, may be
adapted by the skilled person in order to achieve a desired
conversion of lignocellulose to sugar. Some indications are given
hereafter.
[0133] In one aspect of the invention the hydrolysis is conducted
at a temperature of 45.degree. C. or more, 50.degree. C. or more,
55.degree. C. or more, 60.degree. C. or more, 65.degree. C. or
more, or 70.degree. C. or more. The high temperature during
hydrolysis has many advantages, which include working at the
optimum temperature of the enzyme composition, the reduction of
risk of (bacterial) contamination, reduced viscosity, smaller
amount of cooling water required, use of cooling water with a
higher temperature, re-use of the enzymes and more.
[0134] In a further aspect of the invention, the amount of enzyme
composition added (herein also called enzyme dosage or enzyme load)
is low. In an embodiment the amount of enzyme is 6 mg protein/g dry
matter weight or lower, 5 mg protein/g dry matter or lower, 4 mg
protein/g dry matter or lower, 3 mg protein/g dry matter or lower,
2 mg protein/g dry matter or lower, or 1 mg protein/g dry matter or
lower (expressed as protein in mg protein/g dry matter). In an
embodiment, the amount of enzyme is 0.5 mg enzyme/g dry matter
weight or lower, 0.4 mg enzyme composition/g dry matter weight or
lower, 0.3 mg enzyme/g dry matter weight or lower, 0.25 mg enzyme/g
dry matter weight or lower, 0.20 mg enzyme/g dry matter weight or
lower, 0.18 mg enzyme /g dry matter weight or lower, 0.15 mg
enzyme/g dry matter weight or lower or 0.10 mg enzyme/g dry matter
weight or lower (expressed as total of cellulase enzymes in mg
enzyme/g dry matter). Low enzyme dosage is possible, since because
of the activity and stability of the enzymes, it is possible to
increase the hydrolysis reaction time.
[0135] In a further aspect of the invention, the hydrolysis
reaction time is 5 hours or more, 10 hours or more, 20 hours or
more, 40 hours or more, 50 hours or more, 60 hours or more, 70
hours or more, 80 hours or more, 90 hours or more, 100 hours or
more, 120 hours or more, 130 h or more. In another aspect, the
hydrolysis reaction time is 5 to 150 hours, 40 to 130 hours, 50 to
120 hours, 60 to 120 hours, 60 to 110 hours, 60 to 100 hours, 70 to
100 hours, 70 to 90 hours or 70 to 80 hours. Due to the stability
of the enzyme composition longer hydrolysis reaction times are
possible with corresponding higher sugar yields.
[0136] The pH during hydrolysis may be chosen by the skilled
person. In a further aspect of the invention, the pH during the
hydrolysis may be 3.0 to 6.4. The stable enzymes of the invention
may have a broad pH range of up to 2 pH units, up to 3 pH units, up
to 5 pH units. The optimum pH may lie within the limits of pH 2.0
to 8.0, 3.0 to 8.0, 3.5 to 7.0, 3.5 to 6.0, 3.5 to 5.0, 3.5 to 4.5,
4.0 to 4.5 or is about 4.2.
[0137] In a further aspect of the invention the hydrolysis step is
conducted until 70% or more, 80% or more, 85% or more, 90% or more,
92% or more, 95% or more of available sugar in lignocellulosic
material is released.
[0138] Significantly, a process of the invention may be carried out
using high levels of dry matter (of the lignocellulosic material)
in the hydrolysis reaction. Thus, the invention may be carried out
with a dry matter content of about 5 wt % or higher, about 8 wt %
or higher, about 10 wt % or higher, about 11 wt % or higher, about
12 wt % or higher, about 13 wt % or higher, about 14 wt % or
higher, about 15 wt % or higher, about 20 wt % or higher, about 25
wt % or higher, about 30 wt % or higher, about 35 wt % or higher or
about 40 wt % or higher. In a further embodiment, the dry matter
content in the hydrolysis step is 14 wt %, 15 wt %, 16 wt %, 17 wt
%, 18 wt %, 19 wt %, 20 wt %, 21 wt %, 22 wt %, 23 wt %, 24 wt %,
25 wt %, 26 wt %, 27 wt %, 28 wt %, 29 wt %, 30 wt %, 31 wt %, 32
wt %, 33 wt % or more or 14 to 33 wt %.
Fermentation
[0139] The process according to the invention comprises a
fermentation step. In a further aspect, the invention thus includes
in step fermentation processes in which a microorganism is used for
the fermentation of a carbon source comprising sugar(s), e.g.
glucose, L-arabinose and/or xylose. The carbon source may include
any carbohydrate oligo- or polymer comprising L-arabinose, xylose
or glucose units, such as e.g. lignocellulose, xylans, cellulose,
starch, arabinan and the like. For release of xylose or glucose
units from such carbohydrates, appropriate carbohydrases (such as
xylanases, glucanases, amylases and the like) may be added to the
fermentation medium or may be produced by the modified host cell.
In the latter case the modified host cell may be genetically
engineered to produce and excrete such carbohydrases. An additional
advantage of using oligo- or polymeric sources of glucose is that
it enables to maintain a low(er) concentration of free glucose
during the fermentation, e.g. by using rate-limiting amounts of the
carbohydrases. This, in turn, will prevent repression of systems
required for metabolism and transport of non-glucose sugars such as
xylose. In a preferred process the modified host cell ferments both
the L-arabinose (optionally xylose) and glucose, preferably
simultaneously in which case preferably a modified host cell is
used which is insensitive to glucose repression to prevent diauxic
growth. In addition to a source of L-arabinose, optionally xylose
(and glucose) as carbon source, the fermentation medium will
further comprise the appropriate ingredient required for growth of
the modified host cell. Compositions of fermentation media for
growth of microorganisms such as yeasts or filamentous fungi are
well known in the art.
[0140] The fermentation time may be shorter than in conventional
fermentation at the same conditions, wherein part of the enzymatic
hydrolysis still has to take part during fermentation. In one
embodiment, the fermentation time is 100 hours or less, 90 hours or
less, 80 hours or less, 70 hours or less, or 60 hours or less, for
a sugar composition of 50g/l glucose and corresponding other sugars
from the lignocellulosic feedstock (e.g. 50 g/l xylose, 35 g/l
L-arabinose and 10 g/l galactose. For more dilute sugar
compositions the fermentation time may correspondingly be
reduced.
[0141] The fermentation process may be an aerobic or an anaerobic
fermentation process. An anaerobic fermentation process is herein
defined as a fermentation process run in the absence of oxygen or
in which substantially no oxygen is consumed, preferably less than
5, 2.5 or 1 mmol/L/h, more preferably 0 mmol/L/h is consumed (i.e.
oxygen consumption is not detectable), and wherein organic
molecules serve as both electron donor and electron acceptors. In
the absence of oxygen, NADH produced in glycolysis and biomass
formation, cannot be oxidised by oxidative phosphorylation. To
solve this problem many microorganisms use pyruvate or one of its
derivatives as an electron and hydrogen acceptor thereby
regenerating NAD.sup.+. Thus, in a preferred anaerobic fermentation
process pyruvate is used as an electron (and hydrogen acceptor) and
is reduced to fermentation products such as ethanol, lactic acid,
3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,
citric acid, malic acid, fumaric acid, an amino acid,
1,3-propane-diol, ethylene, glycerol, butanol, .beta.-lactam
antibiotics and a cephalosporin. In a preferred embodiment, the
fermentation process is anaerobic. An anaerobic process is
advantageous since it is cheaper than aerobic processes: less
special equipment is needed. Furthermore, anaerobic processes are
expected to give a higher product yield than aerobic processes.
Under aerobic conditions, usually the biomass yield is higher than
under anaerobic conditions. As a consequence, usually under aerobic
conditions, the expected product yield is lower than under
anaerobic conditions.
[0142] In another embodiment, the fermentation process is under
oxygen-limited conditions. More preferably, the fermentation
process is aerobic and under oxygen-limited conditions. An
oxygen-limited fermentation process is a process in which the
oxygen consumption is limited by the oxygen transfer from the gas
to the liquid. The degree of oxygen limitation is determined by the
amount and composition of the ingoing gas flow as well as the
actual mixing/mass transfer properties of the fermentation
equipment used. Preferably, in a process under oxygen-limited
conditions, the rate of oxygen consumption is at least 5.5, more
preferably at least 6 and even more preferably at least 7
mmol/L/h.
[0143] The fermentation process is preferably run at a temperature
that is optimal for the modified cell. Thus, for most yeasts or
fungal cells, the fermentation process is performed at a
temperature which is less than 42.degree. C., preferably less than
38.degree. C. For yeast or filamentous fungal host cells, the
fermentation process is preferably performed at a temperature which
is lower than 35, 33, 30 or 28.degree. C. and at a temperature
which is higher than 20, 22, or 25.degree. C.
[0144] In an embodiment of the invention, in step the fermentation
is conducted with a microorganism that is able to ferment at least
one C5 sugar. In an embodiment the process is a process for the
production of ethanol whereby the process comprises the step
comprises fermenting a medium containing sugar(s) with a
microorganism that is able to ferment at least one C5 sugar,
whereby the host cell is able to ferment glucose, L-arabinose and
xylose to ethanol. In an embodiment thereof the microorganism that
is able to ferment at least one C5 sugar is a yeast. In an
embodiment, the yeast is belongs to the genus Saccharomyces,
preferably of the species Saccharomyces cerevisiae, in which
genetic modifications have been made. An example of such a
microorganism and its preparation is described in more detail in WO
2008/041840 and in European Patent Application EP10160622.6, filed
21 Apr. 2010. In an embodiment, the fermentation process for the
production of ethanol is anaerobic. Anaerobic has already been
defined earlier herein. In another preferred embodiment, the
fermentation process for the production of ethanol is aerobic. In
another preferred embodiment, the fermentation process for the
production of ethanol is under oxygen-limited conditions, more
preferably aerobic and under oxygen-limited conditions.
Oxygen-limited conditions have already been defined earlier
herein.
[0145] In such process, the volumetric ethanol productivity is
preferably at least 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0 or 10.0 g
ethanol per litre per hour. The ethanol yield on L-arabinose and
optionally xylose and/or glucose in the process preferably is at
least 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 95 or 98%. The
ethanol yield is herein defined as a percentage of the theoretical
maximum yield, which, for glucose and L-arabinose and optionally
xylose is 0.51 g. ethanol per g. glucose or xylose.
[0146] In one aspect, the fermentation process leading to the
production of ethanol, has several advantages by comparison to
known ethanol fermentations processes:
[0147] anaerobic processes are possible;
[0148] oxygen limited conditions are also possible;
[0149] higher ethanol yields and ethanol production rates can be
obtained;
[0150] the strain used may be able to use L-arabinose and
optionally xylose.
[0151] Alternatively to the fermentation processes described above,
at least two distinct cells may be used, this means this process is
a co-fermentation process. All preferred embodiments of the
fermentation processes as described above are also preferred
embodiments of this co-fermentation process: identity of the
fermentation product, identity of source of L-arabinose and source
of xylose, conditions of fermentation (aerobical or anaerobical
conditions, oxygen-limited conditions, temperature at which the
process is being carried out, productivity of ethanol, yield of
ethanol).
[0152] The fermentation process may be carried out without any
requirement to adjust the pH during the process. That is to say,
the process is one which may be carried out without the addition of
any acid(s) or base(s). However, this excludes a pretreatment step,
where acid may be added. The point is that the composition of the
invention is capable of acting at low pH and, therefore, there is
no need to adjust the pH of acid of an acid pretreated feedstock in
order that saccharification or hydrolysis may take place.
Accordingly, a method of the invention may be a zero waste method
using only organic products with no requirement for inorganic
chemical input.
Overall Reaction Time
[0153] According to the invention, the overall reaction time (or
the reaction time of hydrolysis step and fermentation step
together) may be reduced. In one embodiment, the overall reaction
time is 300 hours or less, 200 hours or less, 150 hours or less,
140 hours or less, 130 or less, 120 hours or less, 110 hours or
less, 100 hours of less, 90 hours or less, 80 hours or less, 75
hours or less, or about 72 hours at 90% glucose yield.
Correspondingly lower overall times may be reached at lower glucose
yield.
Fermentation Products
[0154] Fermentation products which may be produced according to the
invention include amino acids, vitamins, pharmaceuticals, animal
feed supplements, specialty chemicals, chemical feedstocks,
plastics, solvents, fuels, or other organic polymers, lactic acid,
and ethanol, including fuel ethanol (the term "ethanol" being
understood to include ethyl alcohol or mixtures of ethyl alcohol
and water).
[0155] Specific value-added products that may be produced by the
methods of the invention include, but not limited to, biofuels
(including biogas, ethanol and butanol); lactic acid;
3-hydroxy-propionic acid; acrylic acid; acetic acid;
1,3-propane-diol; ethylene; glycerol; a plastic; a specialty
chemical; an organic acid, including citric acid, succinic acid and
maleic acid; a solvent; an animal feed supplement; a pharmaceutical
such as a .beta.-lactam antibiotic or a cephalosporin; a vitamin;
an amino acid, such as lysine, methionine, tryptophan, threonine,
and aspartic acid; an enzyme, such as a protease, a cellulase, an
amylase, a glucanase, a lactase, a lipase, a lyase, an
oxidoreductase, a transferase or a xylanase; a chemical feedstock;
or an animal feed supplement.
Separation of Fermentation Product
[0156] The process according to the invention optionally comprises
recovery of fermentation product. A fermentation product may be
separated from the fermentation broth in any known manner. For each
fermentation product the skilled person will thus be able to select
a proper separation technique. For instance ethanol may be
separated from a yeast fermentation broth by distillation, for
instance steam distillation/vacuum distillation in conventional
way.
[0157] Certain embodiments of the invention will below be described
in more detail, but are in no way limiting the scope of the present
invention.
Use of Thermostable Enzymes under Optimal Temperature
Conditions
[0158] In one embodiment, the invention relates to the use of
thermostable enzymes such as cellulolytic enzymes of Rasamsonia for
the production of reducing sugars from pre-treated ligno-cellulosic
feedstock in, but not limiting to, ethanol production. Cellulolytic
enzymes of Rasamsonia applied on pre-treated ligno-cellulosic
feedstock showed maximal conversion rates at temperature within the
range of 50 to 70.degree. C. The enzyme remains active under these
circumstances for 14 days and more without complete cessation of
activity.
[0159] By using optimal temperature conditions, maximal amount of
reducing sugars can be released from feedstock (total hydrolysis)
within the shortest possible hydrolysis time. In this way, 100%
conversion of cellulose in glucose is achieved in less than 5
days.
[0160] The theoretical maximum yield (Yps max in g product per gram
glucose) of a fermentation product can be derived from textbook
biochemistry. For ethanol, 1 mole of glucose (180 g) yields
according to normal glycolysis fermentation pathway in yeast 2
moles of ethanol (=2.times.46=92 g ethanol. The theoretical maximum
yield of ethanol on glucose is therefore 92/180=0.511 g ethanol/g
glucose.
[0161] For butanol (MW 74 g/mole) or iso butanol, the theoretical
maximum yield is 1 mole of butanol per mole of glucose. So Yps max
for (iso-)butanol=74/180=0.411 g (iso-)butanol/g glucose.
[0162] For lactic acid the fermentation yield for homolactic
fermentation is 2 moles of lactic acid (MW=90 g/mole) per mole of
glucose. According to this stoichiometry, the Yps max=1 g lactic
acid/g glucose.
[0163] For other fermentation products a similar calculation may be
made.
[0164] The cost reduction achieved with applying cellulolytic
enzymes of Rasamsonia will be the result of an overall process time
reduction.
Compensation of Lower Enzyme Dosage with Extended Hydrolysis Time
Using Rasamsonia Enzymes
[0165] Due to the high stability of the stable enzymes, the
activities do not cease in time, although less reducing sugars are
liberated in the course of the hydrolysis. It is possible to lower
the enzyme dosage and extend the use of the enzyme by prolonging
the hydrolysis times to obtain similar levels of released reducing
sugars. For example, 0.175 mL enzyme/g feedstock dry-matter
resulted in release of approximately 90% of the theoretical maximum
of reducing sugars from pre-treated feedstock within 72 h. When
using 0.075 mL enzyme/g feedstock dry-matter, approximately 90%
conversion of the theoretical maximum is achieved within 120 h. The
results show that, because of the stability of the enzyme activity,
lowering the enzyme dosage can be compensated by extending the
hydrolysis time to obtain the same amount of reducing sugars. The
same holds for hydrolysis of pre-treated feedstock at dry-matter
contents higher than 10% shows that compensating effect of extended
hydrolysis time at 15% dry matter feedstock.
[0166] The cost reduction achieved by using stable cellulolytic
enzymes, such as of Rasamsonia, results from requiring less enzyme
dosage, resulting in similar hydrolysis conversion yields.
Lowering the Risk on Contamination with Stable Enzymes
[0167] In a common process for converting ligno-cellulosic material
into ethanol, process steps are preferably done under septic
conditions to lower the operational costs. Contamination and growth
of contaminating microorganisms can therefore occur and result in
undesirable side effects, such lactic acid, formic acid and acetic
acid production, yield losses of ethanol on substrate, production
of toxins and extracellular polysaccharides, which may affect
production costs significantly. A high process temperature and/or a
short process time will limit the risk on contamination during
hydrolysis and fermentation. Thermostable enzymes, like those of
Rasamsonia, are capable of hydrolysing ligno-cellulosic feedstock
at temperatures of higher than 60.degree. C. At these temperatures,
the risk that a contaminating microorganism will cause undesired
side effects will be little to almost zero.
[0168] During the fermentation step, in which ethanol is produced,
temperatures are typically between 30 to 37.degree. C. and will
preferably not be raised because of production losses. By applying
fermentation process times as short as possible the risks and
effects of contamination and/or growth of contaminants will be
reduced as much as possible. With stable enzymes, like those of
Rasamsonia a short as possible fermentation times can be applied
(see description above), and thus risks on contamination and/or
growth of contaminants will be reduced as much as possible. The
cost reduction achieved with applying thermostable cellulolytic
enzymes of Rasamsonia in this way will result from lower risk of
process failures due to contamination.
Stable Enzymes Reduce Cooling Costs and Increase Productivity of
Ethanol Plants
[0169] The first step after thermal pretreatment will be to cool
the pretreated feedstock to temperatures where the enzymes are
optimal active. On large scale, this is typically done by adding
(cooled) water, which will, besides decreasing the temperature,
reduce the dry-matter content. By using thermos stable enzymes,
like those of Rasamsonia, cost reduction can be achieved by the
fact that (i) less cooling of the pretreated feedstock is required
since higher temperatures are allowed during hydrolysis, and (ii)
less water will be added, which will increase the dry-matter
content during hydrolysis and fermentation and thus increase the
ethanol production capacity (amount produced per time unit per
volume) of an ethanol plant. Also, by using thermostable enzymes
according to the invention, like those of Rasamsonia, cost
reduction may also be achieved by using cooling water having higher
temperature that the water that is used in a process with
non-thermostable enzyme.
Enzyme Recycling after Hydrolysis with Stable Enzymes
[0170] At the end of the hydrolysis, enzyme activities appear to be
low since little reducing sugars are released once almost all
cellulose is converted. The amount of enzymatic activity present,
however, has decreased only a little, assumingly mainly due to
absorption of the enzymes to the substrate. By applying
solid-liquid separation after hydrolysis, such as centrifugation,
filtration, sedicantation, etcetera, 60% or more e.g. 70% of the
enzyme activity in solution can be recovered and re-used for
hydrolysis of a new pre-treated ligno-cellulosic feedstock during
the next hydrolysis.
[0171] Moreover, after solid-liquid separation the enzyme in
solution can be separated from the solution containing reducing
sugars and other hydrolysis products from the enzymatic actions.
This separation can be done by, but not limiting to, (ultra and
micro)filtration, centrifugation, sedicantation, sedimentation,
with or without first adsorption of the enzyme to a carrier of any
kind.
[0172] For example, after hydrolysis of pre-treated feedstock with
0.175 mL/g feedstock dry matter enzyme load for 20 h, 50% of the
theoretical maximum amount of reducing sugars is liberated and
after the same hydrolysis for 72 h, 90% of the theoretical maximum
amount of reducing sugars is liberated. By centrifugation and
ultrafiltration, 60-70% of the enzyme activity was recovered in the
retentate, while the filtrate contained more than 80% of the
liberated reducing sugars. By re-using the retentate, either as it
is or after further purification and/or concentration, enzyme
dosage during the next hydrolysis step can be reduced with 60 to
70%. The cost reduction achieved by using stable cellulolytic
enzymes, such as of Rasamsonia, in this way results from requiring
less enzyme dosage.
Enzyme Recyclina after Hydrolysis in Combination with Enzyme
Production and Yeast-Cell Recyclina with Stable Enzymes
[0173] The process including enzyme recycling after hydrolysis, as
described above, can be combined with recycling of the ethanol
producing microorganism after fermentation and with the use of the
reducing sugars containing filtrate as a substrate (purified and/or
concentrated or diluted) in enzyme-production fermentation and as
substrate for the cultivation of the ethanol-producing
microorganism.
Enzyme Recyclina after Vacuum Distillation with Stable Enzymes
[0174] The thermo stability of enzymes, like those from Rasamsonia,
causes remaining cellulolytic activity after hydrolysis,
fermentation and vacuum distillation in the thin stillage. The
total activity of the enzyme is reduced during the three successive
process steps. The thin stillage obtained after vacuum distillation
can thus be re-used as a source of enzyme for a newly started
hydrolysis-fermentation-distillation process cycle of pre-treated
wheat straw conversion into ethanol. The thin stillage can be used
either in concentrated or (un)diluted form and/or purified and with
or without additional enzyme supplementation.
Enzyme Recycling in Combination with Enzyme Supplementation after
Vacuum Distillation with Thermostable Enzymes
[0175] In an optimal process, an amount of enzyme is supplemented
into the thin stillage, before its re-use in a new process cycle,
equal to the amount of activity lost during the three successive
process steps of the previous process cycle. In this way
over-dosage of enzyme is avoided and thus most efficient use of
enzyme is obtained.
[0176] Moreover, by providing high enzyme dosage in the first
process cycle, and supplementing enzyme equal to the amount of
activity lost during the three successive process steps in the
following process cycles, highest possible hydrolysis rates can be
obtained in each process cycle resulting in short hydrolysis times
of less than 48 h in combination with most efficient use of
enzymes.
Use of Stable Enzymes in Mixed Systems
[0177] By applying mixing during hydrolysis, enzymes come more
often in contact with substrates, which results in a more efficient
use of the catalytic activity. This will result in a lower enzyme
dosages and thus in lower costs, unless the mixing has a negative
effect on the enzymes. Stable enzymes, like the thermostable
enzymes from Rasamsonia, are robust and can resist circumstances of
(locally) high shear and temperatures, which is the case during
intensive mixing of slurries. The use of it in mixed systems is
therefore beneficial and will lead to dosage and thus costs
reduction.
[0178] The invention is further described by the following
examples, which should not be construed as limiting the scope of
the invention.
EXAMPLES
Experimental Information
Strains
[0179] Rasamsonia (Talaromyces) emersonii strain was deposited at
CENTRAAL BUREAU VOOR SCHIMMELCULTURES, Uppsalalaan 8, P.O. Box
85167, NL-3508 AD Utrecht, The Netherlands in December 1964 having
the Accession Number CBS 393.64. Other suitable strains can be
equally used in the present examples to show the effect and
advantages of the invention. For example TEC-101, TEC-147, TEC-192,
TEC-201 or TEC-210 are suitable Rasamsonia strains which are
described in WO2011/000949.
Preparation of Acid Pre-Treated Corn Stover Substrate.
[0180] Dilute-acid pre-treated corn stover (aCS) was obtained as
described in Schell, D. J., Applied Biochemistry and Biotechnology
(2003), vol. 105-108, pp 69-85. A pilot scale pretreatment reactor
was used operating at steady state conditions of 190.degree. C., 1
min residence time and an effective H2SO4 acid concentration of
1.45% (w/w) in the liquid phase.
Protein Measurement Assays
1.Total Protein
TCA Biuret
[0181] The method was a combination of precipitation of protein
using trichloro acetic acid (TCA) to remove disturbing substances
and allow determination of the protein concentration with the
colorimetric Biuret reaction. In the Biuret reaction, a copper (II)
ion is reduced to copper (I), which forms a complex with the
nitrogens and carbons of the peptide bonds in an alkaline solution.
A violet color indicates the presence of proteins. The intensity of
the color, and hence the absorption at 546 nm, is directly
proportional to the protein concentration, according to the
Beer-Lambert law. The standardisation was performed using BSA
(Bovine Serum Albumine) and the protein content was expressed in g
protein as BSA equivalent/L or mg protein as BSA equivalent /ml.
The protein content was calculated using standard calculation
protocols known in the art, by plotting the OD.sub.546 versus the
concentration of samples with known concentration, followed by the
calculation of the concentration of the unknown samples using the
equation generated from the calibration line.
2. Individual Proteins Using PAGE
Sample Pre-Treatment SDS-PAGE
[0182] Based on the estimated protein concentration of the samples
the following samples preparation was performed. To 10 .mu.l sample
40 .mu.l MilliQ water and 50 .mu.l TCA (20%) was added to dilute
the sample five times (.about.1 mg/ml) and precipitate the
proteins. After 1 hour on ice the sample was centrifuged (10
minutes, 14000 rpm). The pellet was washed with 500 .mu.l Aceton
and centrifuged (10 minutes, 14000 rpm). The pellet was treated as
described below.
SDS-PAGE
[0183] The pellet was dissolved in 65 .mu.l of the MilliQ water, 25
.mu.l NuPAGE.TM. LDS sample buffer (4.times.) Invitrogen and 10
.mu.l NuPAGE.TM. Sample Reducing agent (10.times.) Invitrogen.
Prior to the the deanuarion step the sample was diluted 5 timnes
using a mix of MilliQ; NuPAGE.TM. LDS sample buffer and 10 .mu.l
NuPAGE.TM. Sample Reducing in the ratio of 65:25:10. After mixing,
the samples were incubated in a thermo mixer for 10 minutes at
70.degree. C. The sample solutions were applied on a 4-12% Bis-Tris
gel (NuPAGE.TM. BisTris, Invitrogen). A sample (10.mu.l ) of marker
M12 (Invitrogen) was also applied on the gel. The gel was run at
200 V for 50 minutes, using the XCELL Surelock, with 600 ml 20
.times.diluted SDS buffer in the outer buffer chamber and 200 ml
20.times. diluted SDS buffer, containing 0.5 ml of antioxidant
(NuPAGE.TM. Invitrogen) in the inner buffer chamber. After running,
the gel was rinsed twice with demineralised water the gels were
fixed with 50% methanol/7% acetic acid solution for one hour and
stained with Sypro Ruby (50 ml per gel) overnight. An image was
made using the Typhoon 9200 (610 BP 30, Green (532 nm), PMT 600V,
100 micron) after washing the gel with MilliQ water.
Quantitative Analysis of the Protein
[0184] Using the Typhoon scanner the ratio between protein bands
within a lane was determined using standard methods known in the
art. The sample was applied in triplicate and the gray values were
determined using the program Image quant. Values are expressed as
relative % protein to the total protein, calculated using the gray
value of the selected protein band relative to the total gray value
all the protein bands.
[0185] Glucan Conversion Calculation:
glucan conversion(%)=(glucose(g/l).times.100%)/(glucan(fraction on
DM).times.dm(g/kg).times.1.1) [0186] Wherein: [0187] glucose
(g/l)=glucose concentration in supernatant after hydrolysis. [0188]
glucan (fraction on dm)=glucan content of the substrate before
pretreatment. [0189] dm (g/kg)=dry matter of hydrolysis (f.i. 20%
dm=200 g/kg). [0190] 1.1=weight increase due to water incorporation
during hydrolysis.
[0191] Example Calculation: [0192] glucose=60 g/l [0193] glucan
fraction=0.40 (is 40% on dry matter) [0194] dm=200 g/kg
[0194] glucan conversion
example=(60*100)/(0.4.times.200.times.1.1)=68% conversion
Example 1
Evaluation of the Effect of the Absence of Oxygen During Hydrolysis
on the Cellulolytic Activity of Cellulase Enzyme Cocktails
[0195] The effect of oxygen absence during hydrolysis on the
cellulolytic activity of three different enzyme cocktails was
evaluated according to the procedures described below. The
hydrolysis reactions were performed with acid pretreated cornstover
(aCS) feedstock at a final concentration of 10 w/w % DM. This
feedstock solution was prepared via the dilution of a concentrated
feedstock solution with water. Subsequently the pH was adjusted to
pH 4.5 with a 4M NaOH solution. The elimination of oxygen from the
feedstock was accomplished in two steps. First, the feedstock
solution was degassed via sonication under vacuum in a sonication
bath (Bransonic 5510E-DTH, setting; Degas) for 15 minutes. In the
second step, the oxygen was further removed by continuous sparging
of a nitrogen flow through a 500 ml solution of the 10% DM
feedstock for a period of 3 hours. Prior to being sparged through
the feedstock solution, the nitrogen flow was sparged through water
in order to saturate it with water vapour and prevent evaporation
of the water from the feedstock solution. In parallel, 500 ml of
the same batch 10 w/w % DM aCS was sparged with air as an
oxygen-containing control sample in a similar set-up and according
to the same protocol.
[0196] The hydrolysis of the oxygen-depleted (nitrogen sparged) and
the oxygen-saturated (air-sparged) 10 w/w % aCS feedstock solutions
were conducted in air-tight, 30-ml centrifuge bottles (Nalgene
Oakridge) in a total reaction volume of 10 ml. The bottles, already
containing the cellulase solution, used for the oxygen-depleted
experiment were sparged with nitrogen prior to- and during filling
them with feedstock. Each hydrolysis was performed in duplicate
with 7.5 mg/g DM cellulase enzyme cocktail added in a total volume
not larger than 375 .mu.l. The three cellulase enzyme cocktails
tested included: a TEC-210 mix (mixture of cellulases), a 4E-GH61
mix (consisting of 9 w/w % of total protein BG, 30 w/w % of total
protein CBHI, 25 w/w % of total protein GBHII and 36 w/w % of total
protein GH61) and a 4E-EG mix (consisting of 9 w/w % of total
protein BG, 30 w/w % of total protein CBHI, 25 w/w % of total
protein CBHII and 36 w/w % of total protein EG). TEC-210 was
fermented according to the inoculation and fermentation procedures
described in WO2011/000949. The 4E mix (as described in
WO2011/098577) was used.
[0197] The centrifuge bottles containing the feedstock and enzyme
solution were placed in an oven incubator (Techne HB-1 D
hybridization oven) and incubated for 72 hours at 65.degree. C.
while rotating at set-point 3 (12 rpm per minute). Following
hydrolysis, the samples were cooled on ice and immediately 50 .mu.l
of each supernatant was diluted in 1450 .mu.l grade I water. The
diluted supernatant was subsequently filtered (0.45 .mu.m filter,
Pall PN 454) and the filtrates were analysed for sugar content as
described below.
[0198] The sugar concentrations of the diluted samples were
measured using an HPLC equipped with an Aminex HPX-87P column
(Biorad #1250098) by elution with water at 85.degree. C. at a flow
rate of 0.6 ml per minute and quantified by integration of the
glucose signals from refractive index detection (R.I.) calibrated
with glucose standard solutions.
[0199] The data presented in Table 1/FIG. 1 show that the glucose
released from the nitrogen-sparged feedstocks is lower than the
glucose released from the feedstocks sparged with air for both the
TEC-210 mix and the 4E-GH61 mix incubations. There is no difference
in glucose release detectable between the nitrogen and air sparged
feedstocks for samples hydrolyzed by the 4E-EG mix.
[0200] Based on these results we conclude that the presence of
oxygen improves the cellulolytic performance of cellulase mixtures
that contain GH61 enzymes.
TABLE-US-00001 TABLE 1 The effect of sparging nitrogen or air
through a 10% aCS feedstock before hydrolysis, on the total amount
of glucose released by three different cellulase mixes. Sparged
with air Average glucose Sparged with N.sub.2 Cellulase (g/l) stdev
Average glucose (g/l) stdev TEC-210 34.5 0.8 31.9 1.1 4E-GH61 mix
31.7 1.4 27.4 0.1 4E-EG mix 22.7 0.1 23.3 1.7
Example 2
The Effect of the Dissolved Oxygen Concentration on the
Cellulolytic Activity of Cellulase Enzyme Compositions During
Hydrolysis of Lignocellulosic Feedstock on Pilot Scale (270
liter)
[0201] The effect of the dissolved oxygen concentration on the
cellulolytic activity of the enzyme composition or enzyme cocktail
during the hydrolysis of lignocellulosic feedstock on pilot scale
(270 liter) is shown in this example. The hydrolysis reactions were
performed with acid pretreated cornstover (aCS) feedstock at a
(final) concentration of 20 w/w % DM. The feedstock solution was
prepared by the dilution of concentrated feedstock slurry with
water. The pH was adjusted to pH 4.5 with a 25% (w/w) NH.sub.4OH
solution.
[0202] The enzymatic hydrolysis was done in a 270 liter pilot
reactor which was pH and temperature controlled with a working
volume of 150 liter. The dissolved oxygen during the process was
controlled by adjusting impeller speed at a given airflow and
overpressure. The enzymatic hydrolysis was performed with a dosage
of 3.75 mg TCA protein/g DM of TEC-210 cellulase enzyme
composition. TEC-210 was produced according to the inoculation and
fermentation procedures described in WO2011/000949.
[0203] The following experiments were done: [0204] 1. 150 l of 20%
aCS, pH 4.5, temperature 62.degree. C., no overpressure, no
airflow, 3.75 mg TCA/g dm of TEC-210 cellulase composition,
incubation time 120 hours in a 270 liter pilot reactor. The
dissolved oxygen concentration (DO) of the reaction mixture was
measured constantly using a DO electrode. The DO was controlled at
a level of 0.01 mol/m3 by adjusting the impeller speed. [0205] 2.
150 l of 20% aCS, pH 4.5, temperature 62.degree. C., 1 bar
overpressure, 10 kg/h airflow in the headspace of the reactor, 3.75
mg TCA/g dm of TEC-210 cellulase composition, incubation time 120
hours in a 270 liter pilot reactor The dissolved oxygen
concentration (DO) of the reaction mixture was measured constantly
using a DO electrode. The DO was controlled at a level of 0.2
mol/m3 by adjusting the impeller speed.
[0206] During the enzymatic hydrolysis, samples were taken daily
for carbohydrate analysis (glucose, cellobiose) by NMR and
viscosity and pH measurement.
[0207] Composition analysis of the acid pretreated Corn Stover
(aCS) was done by chemical hydrolysis of the sample and
determination of the mono saccharides by NMR.
[0208] Samples taken during enzymatic hydrolysis were analysed for
(oligo)sugars, organic acids and inhibitors by flow NMR.
[0209] Conversion was calculated based on the measured glucan
concentration (g/kg) at the start of the enzymatic hydrolysis and
glucose concentration (g/l) which was measured during the enzymatic
hydrolysis.
[0210] The results are presented in FIG. 2.
[0211] This example shows that at larger reactor volumes, for
example 100 liter or more the addition of oxygen (in the form of
air) strongly improves the hydrolysis of pretreated lignocellulosic
material. On scales of 1 m.sup.3 or more, or 10 m.sup.3 or more or
50 m.sup.3 or more reactor content similar improvement in
conversion during hydrolysis will be found.
Example 3
The Effect of Oxygen on the Cellulolytic Activity of Cellulase
Enzyme Compositions During Hydrolysis of Lignocellulosic Feedstock
using a Low Enzyme Dosage
[0212] The effect of oxygen on the cellulolytic activity of the
enzyme composition using a low enzyme dosage during the hydrolysis
of lignocellulosic feedstock is shown in this example. The
hydrolysis reactions are performed with acid pretreated cornstover
(aCS) feedstock at a final concentration of 20 w/w % DM. This
feedstock solution is prepared via the dilution of a concentrated
feedstock solution with water. Subsequently the pH is adjusted to
pH 4.5 with a 10% (w/w) NH.sub.4OH solution. The glucan content of
the applied corn stover was 37% on dry matter.
[0213] The hydrolysis is done in a stirred, pH controlled and
temperature controlled reactor with a working volume of 1 l. Each
hydrolysis is performed in duplicate with 2.5 mg/g DM of TEC-210
cellulase enzyme composition (or cocktail). TEC-210 was produced
according to the inoculation and fermentation procedures described
in WO2011/000949. [0214] The following experiments are done: [0215]
1. 1 l of 20 % aCS, pH 4.5, temperature 62.degree. C., stirrer
speed 60 rpm, 3.5 mg TEC-210 cellulase composition per gram
feedstock (on dry matter), incubation time 120 hours (reference
experiment) in a closed reactor. The dissolved oxygen level of the
reaction mixture was measured constantly using a DO electrode. This
slow stirring resulted in a dissolved oxygen level 0.005
mol/m.sup.3. [0216] 2. As experiment 1 but using and enzyme dosage
of 2.5 mg TEC-210 per gram feedstock (on dry matter) and a stirrer
speed of 250 rpm a head space over the reaction mixture which is
constantly refreshed with fresh air. The higher stirring speed in
combination with refreshment of the head space with fresh air
resulted in a dissolved oxygen level of. 0.030 mol/m.sup.3 in the
reaction mixture.
[0217] During hydrolysis samples were taken for analysis. The
samples were cooled on ice and immediately 50 .mu.l of each
supernatant is diluted in 1450 .mu.l grade l water. The diluted
supernatant is subsequently filtered (0.45 .mu.m filter, Pall PN
454) and the filtrates are analysed for sugar content as described
below.
[0218] The sugar concentrations of the diluted samples are measured
using an HPLC equipped with an Aminex HPX-87P column (Biorad
#1250098) by elution with water at 85.degree. C. at a flow rate of
0.6 ml per minute and quantified by integration of the glucose
signals from refractive index detection (R.I.) calibrated with
glucose standard solutions.
[0219] The results are presented in FIG. 3.
[0220] The glucan conversions are listed in Table 2.
TABLE-US-00002 TABLE 2 glucan conversion levels. Enzyme dosage DO
level Glucan conversion Experiment (mg/g dm) mol/m.sup.3 (%) 1 2.5
0.030 75 2 3.5 0.005 70
* * * * *
References