Enzyme and Microbial Technology 46 (2010) 177–184

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Enzyme and Microbial Technology

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Enzymatic hydrolysis and fermentation of palm kernel press cake for production
of bioethanol
José María Cerveró a , Pernille Anastasia Skovgaard b , Claus Felby b ,
Hanne Risbjerg Sørensen c,1 , Henning Jørgensen b,∗
a
Department of Chemical Engineering and Environmental Technology, University of Oviedo, C/Julián Clavería, 8, 33071, Oviedo, Spain
b
Danish Centre for Forest, Landscape and Planning, University of Copenhagen, Rolighedsvej 23, DK-1958 Frederiksberg C, Denmark
c
Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsværd, Denmark

a r t i c l e i n f o a b s t r a c t

Article history: Palm kernel press cake (PKC) is a residue of palm oil extraction, which was found to contain 48.5% of
Received 29 June 2009 total carbohydrates of which 35.2% was mannan. The present study examines enzymatic hydrolysis of
Received in revised form polysaccharides from the cell-wall material present in PKC to obtain monosaccharides that can be sub-
25 September 2009
strate in various fermentation processes such as ethanol production. The requirements for pretreatment
Accepted 27 October 2009
were investigated and it was found that mannan in PKC was readily hydrolysed without any pretreat-
ment. Several enzyme preparations were tested and Mannaway 25L was found as the best for releasing
Keywords:
mannose, and Gammanase 1.0L worked well in degrading cellulose and mannose. Binary mixtures of
Bioethanol
Palm kernel press cake
enzymes were tested to increase the conversion, and 1:1 mixture of Mannaway 25L and Gammanase
Mannan 1.0L showed good synergistic effect releasing 30% more mannose than the sum obtained using these
Saccharomyces cerevisiae enzymes individually. Using an enzyme loading of 2.3 mg protein/g PKC resulted in 63% of mannan in
PKC being hydrolysed to mannose in 24 h, and in 96 h a total of 365 g mannose and glucose could be
produced per kg PKC. Finally, PKC was hydrolysed and fermented using Saccharomyces cerevisiae with an
ethanol yield of 125 g/kg PKC.
© 2009 Elsevier Inc. All rights reserved.

1. Introduction yeast Saccharomyces cerevisiae normally applied in ethanol fermen-

Bioethanol produced by fermentation of carbohydrates is
increasingly used as fuel in the transportation sector. It is a promis-
ing substitute to fossil fuels due to the carbon dioxide neutrality
and the fact that it can be a domestic renewable energy source. The
raw materials used for industrial bioethanol production are cur-
rently sugar cane or starch containing materials such as corn or
grain. However, as a result of the growing demand for bioethanol
world wide, there is an interest to look for alternative sources of
fermentable carbohydrates.
The most abundant source of carbohydrates can be found in the
structural parts of plants, i.e. stems and leaves, commonly known
as lignocellulose, which consists mainly of cellulose, hemicellu-
loses and lignin. Non-starch polysaccharides such as cellulose are
often difficult to process into fermentable sugars. Usually, the pro-
cess involves an extensive thermal/chemical pretreatment prior
to enzymatic hydrolysis and fermentation [1]. Furthermore, the
∗ Corresponding author. Tel.: +45 3533 1704; fax: +45 3533 1508.
E-mail address: hnj@life.ku.dk (H. Jørgensen).
1
Present address: BioGasol ApS, Lautrupvang 2A, DK-2750 Ballerup, Denmark.
tations is not capable of naturally fermenting pentose (C5) sugars
such as xylose and arabinose originating from the hemicelluloses.
A significant part of the sugar in many lignocellulosic materials can
therefore not be efficiently utilised [2].
Mannose is the principal carbohydrate present in palm oil ker-
nels with a content in the range of 30–35%, but also 7–9% glucose
is present [3,4]. Palm oil kernels are available as palm kernel press
cake (PKC), which is a residue from extraction of oil from palm
kernels. The extraction can be either mechanical or solvent extrac-
tion resulting in a residue containing around 50% carbohydrate and
15–20% protein [4]. The high content of polysaccharides in PKC and
the favourable composition of the sugars with a high percentage
of fermentable hexose (C6) sugars make it a potential raw material
for bioethanol production.
Malaysia and Indonesia are the two largest producers of palm
oil with a world marked share of 42–44% each in 2005 [5]. Due to
increased demand for plant oils globally there has been a tremen-
dous increase in palm oil production in recent years. From 2000 to
2005 the palm oil production was doubled in Indonesia [5], and is
expected to increase also in following years. Due to the increase in
palm oil production, the amount of PKC available has also increased.
The production of PKC in Malaysia was according to the Malaysian

0141-0229/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2009.10.012
178 J.M. Cerveró et al. / Enzyme and Microbial Technology 46 (2010) 177–184
Palm Oil Board 2.2 million tons in 2007, and the amount available
world wide can be estimated to 5 million tons. At present PKC is
only used for feeding purposes, but low nutritive value, moderate
protein content and poor amino acid profile (deficiency in lysine,
methionine and tryptophan) makes PKC only a medium quality feed
[6,7]. Fast deterioration combined with the increased production
results in large amounts of PKC being discarded, which could be
environmental problematic in future in countries like Indonesia [8].
The use of PKC for ethanol production could, besides production
of renewable energy, likely increase the nutritional value, as the
by-product after the fermentation would have even higher protein
content due to removal of complex polysaccharides and enrich-
ment with yeast cell protein. Furthermore, excess PKC could be
more efficiently utilised.
Most mannan in PKC consists of a (1 → 4)-linked ␤-d-
mannopyranose backbone, but small amounts of mannan with
(1 → 6)-linked ␣-d-galactopyranose (galactomannan) are also
present. However, the galactose substitution is low (12–20%)
[4,9]. Due to the low substitution, mannan in PKC resembles
very much cellulose by being crystalline, hard and water insol-
uble [8–10]. In addition to mannans, PKC also contains some
cellulose and low amounts of 4-O-methyl-glucoronoxylan and ara-
binoxylan [10]. Enzymatic hydrolysis of mannan/galactomannan
requires the action of endo-1,4-␤-mannosidases (E.C. 3.2.1.78), ␤-
mannosidases (E.C. 3.2.1.25) and ␣-galactosidases (E.C. 3.2.1.22)
for removal of the galactose side groups. Cellulose can be
hydrolysed by exo-1,4-␤-d-glucanases or cellobiohydrolases (EC
3.2.1.91), endo-1,4-␤-d-glucanases (EC 3.2.1.4) and 1,4-␤-d-
glucosidases (EC 3.2.1.21). The xylan backbone is hydrolysed by
endo-1,4-␤-d-xylanases (EC 3.2.1.8) and 1,4-␤-d-xylosidases (EC
3.2.1.37) and removal of side groups requires the action of ␣-
l-arabinofuranosidases (EC 3.2.1.55) and ␣-glucuronidases (EC
3.2.1.139). Efficiently releasing all fermentable sugars in PKC
therefore requires the combined action of a large number of dif-
ferent enzymes. Furthermore, some of these enzymes could work
synergistically and increase overall amount of monosaccharides
released.
The aim of the present work was to determine the composi-
tion of PKC, and identify the most suitable enzyme preparations
among the ones tested for hydrolysis of PKC into monosaccharides.
This also involved testing binary mixtures to find possible syner-
gies between enzyme preparations. The necessity for pretreatment
of PKC prior to enzymatic hydrolysis was also tested. The hydrol-
ysed material was used for fermentation with S. cerevisiae to verify
the possibility of using PKC for ethanol production.
2. Materials and methods
2.1. Substrate
Palm kernel (Elaeis guineenis) press cake was from PT. Musin Mas (Indonesia).
The PKC was grinded in a mortar to ensure homogeneity. Dry matter content of PKC
was 95.7%.
2.2. Enzymes
Novozym NS 51054 batch SE-2005-00217 (NZ 51054), Gammanase 1.0L batch
SE-2002-00049 (Gammanase), Mannaway 25L batch KHP30007 (Mannaway), Ultra-
flo XL SE-2005-00219 (Ultraflo), Pectinex Ultra SP-L batch KRN05501 (Pectinex),
Celluclast 1.5 FG L batch CCN03110 (Celluclast) and Novozym 188 batch DCN00211
(NZ 188) were a gift from Novozymes (Bagsværd, Denmark). ␤-mannanosidase
(Mannosidase) from C. fimi was purchased from Megazyme International Ire-
land Ltd. (Bray, Ireland), diluted 10-fold in 50 mM sodium acetate buffer pH
6.5 and stored frozen. Enzyme names in bracket are short form used in the
text.
Filter paper activity was determined at pH 4.8 according to [11]. Man-
nanase activity was measured at pH 5.4 according to [12] using locust bean
gum (Sigma G-0753) as substrate. The activity of ␤-glucosidase, ␤-mannosidase
and ␣-galactosidase was measured at pH 5.4 using 5 mM p-nitrophenyl-
␤-d-glucopyranoside, p-nitrophenyl-␤-d-mannopyranoside, p-nitrophenyl-␣-d-
galactopyranoside, respectively, in 50 mM sodium citrate buffer as substrate. The
assay was performed in microtiter plates by incubating 20 ␮l of sample with 100 ␮l
of substrate solution at 50 ◦ C for 15 min in a Grant Scientific QBD2 heating block.
The reaction was terminated by addition of 120 ␮l stop solution (0.5 M glycin/NaOH
buffer pH 10.0, 2 mM EDTA) and absorbance read at 405 nm. Table 1 summarizes
the enzyme preparations used and their characteristics.
Protein content in the enzyme preparations was quantified using the Bio-Rad
Protein Assay. The assay was carried out according to the manufacture’s instructions
and Bovine Serum Albumin was used as standard.
2.3. Pretreatment of PKC
Pretreatment tests were performed in which PKC either was used without any
pretreatment, pretreated using an autoclave at 126 ◦ C for 11 min or pretreated at
180 ◦ C for 10 min. Pretreatment in autoclave was performed in 100 ml blue cap bot-
tles closed with caps with PTFE lining. PKC was mixed with sodium acetate buffer
pH 5.4 in order to give a final solids concentration of 5% (w/w) after addition of
enzyme solution. The closed bottles were autoclaved at 126 ◦ C for 11 min in a Tut-
tnauer 2540EL autoclave. After cooling, enzymes were added thereby giving a total
filling of 40 g. This procedure was adapted as the general pretreatment mainly to
ensure sterility in experiments.
The unpretreated samples were prepared similarly to the autoclaved but with-
out autoclavation.
Pretreatment at 180 ◦ C was performed by heating a 5% (w/w) solution of PKC
in sodium acetate buffer pH 5.4 in a stainless steal tube with temperature probe to
180 ◦ C in an oil bath, keeping it at 180 ◦ C for 10 min and then cooling in at water
bath. The total slurry was then transferred to 100 ml blue cap bottles.
Evaluation of the pretreatment effect on hydrolysis efficiency was done using a
1:1 mixture of Gammanase/Mannaway at a loading of 10 ml enzyme/100 g PKC dry
weight.
2.4. Enzymatic hydrolysis
The enzymatic hydrolysis was performed in 100 ml blue cap bottles with 5%
dry matter (w/w) of PKC in 50 mM sodium acetate buffer pH 5.4. After pretreat-
ment/autoclavation as given above, enzyme was added to give a 10% (v/w) enzyme
loading (10 ml enzyme preparation per 100 g of PKC dry weight). For testing the
effect of mixtures, enzyme preparations were tested alone using a 5% (v/w) enzyme
loading and in mixtures using 5% of each enzyme preparation. The bottles were
placed in a water bath at 50 ◦ C and stirring by a magnetic stirrer at 200 rpm. Samples
were withdrawn and the reaction was terminated by boiling the samples for 10 min.
Samples were stored at −20 ◦ C until analysis. All experiments were performed in
duplicate.
2.5. Combined enzymatic hydrolysis and fermentation
Baker’s yeast from the supermarket (S. cerevisiae, De Danske Spritfabrikker,
Grenaa, Denmark) was used for the fermentation. The yeast was maintained on
YPD-agar plates to ensure a pure culture. A preculture was inoculated with one
loopfull of yeast into a 500-ml shake-flask with 150 ml of YPD-medium and placed
at 32 ◦ C on an Infors HT Ecotron rotary shaker for 24 h. The YPD-medium consisted
of per l: 10 g yeast extract, 20 g peptone and 20 g glucose.
Hydrolysis and fermentation studies were performed in 100-ml blue cap bot-
tles with a total filling of 100 g using 10% dry matter (w/w) of PKC in 50 mM sodium
acetate buffer pH 5.4. After pretreatment, enzymes (1:1 mixture of Mannaway and
Gammanase) were added to give a total enzyme loading of 10% (10 ml enzyme prepa-
ration per 100 g of PCK dry weight). The bottles were placed in a water bath at 50 ◦ C
and stirred by a magnetic stirrer at 200 rpm. After 24 h, the temperature was low-
ered to 30 ◦ C and 2 ml of either sterile water or a 10% solution of yeast extract was
added. The fermentation was started by addition of 1 ml of the preculture (resulting
in a final total filling of 100 g and a initial yeast concentration of 0.1 g/l). The bottles
were closed with a rubber stopper equipped with two needles—one for sampling
and one for venting of carbon dioxide. The saccharification and fermentation was
continued for a total of 165 h. Samples were withdrawn at regular intervals and
boiled for 10 min to end the enzyme activity in sealed tubes to avoid evaporation.
Samples were stored at −20 ◦ C until analysis.
2.6. Analysis of PKC
Dry matter was determined using a Sartorius MA 30 moisture analyzer at 105 ◦ C.
The content of lignin, sugars (arabinose, galactose, glucose, mannose and xylose)
was analysed using two-step acid hydrolysis according to the procedure pub-
lished by NREL [13] and sugars were determined by HPLC (see below). Before
hydrolysis the PKC was dried at 45 ◦ C for one day and grinded to homogeny in a
mortar.
Total ash was determined by incineration at 550 ◦ C for 3 h.
Protein was estimated as total nitrogen determined by Kjeldahl and multiplied
by 6.25.
All analyses of composition were performed in triplicate.
 J.M. Cerveró et al. / Enzyme and Microbial Technology 46 (2010) 177–184 179

Table 2

Filter paper activity

Composition of PKC (on dry basis).

FPU/ml (FPU/mg)
Component Composition (%)

Glucose 7.7

75 (1.3)

12 (1.7)
Xylose 2.6

3 (0.2)
Arabinose 1.1

nd

nd
<2
0
0
Galactose 1.9
Mannose 35.2
Protein 15.0
Lignin 15.1
Ash 5.0
nkat/ml(nkat/mg)

Sugars reported as anhydrous form.

6040 ± 550 (151)

␤-Glucosidase

450 ± 50 (7.8)

580 ± 16 (29)
530 ± 7 (88)

37 ± 1 (5.3)
2.7. HPLC Analysis

Sugars (arabinose, galactose, glucose, mannose and xylose) were separated on

a Dionex BioLC system fitted with a CarboPac PA1 column (4 mm × 250 mm) and a
0

CarboPac PA1 precolumn (4 mm × 50 mm). The separation was performed at 25 ◦ C,

a flow of 1 ml/min and an eluent of 2 mM KOH for 35 min, 60 mM for 5 min and
2 mM for 10 min. Quantification was done using a pulsed electrochemical detector
in pulsed amperiometric detection mode. Prior to analysis samples were filtered
through a 0.2 ␮m filter and diluted by MilliQ-water.
nkat/ml(nkat/mg)

Ethanol, acetate, glycerol were separated on a Dionex Summit system fitted

␣-Galactosidase

550 ± 40 (13.8)

740 ± 40 (106)

with a Phenomenex Rezex RHM column (7.8 mm × 300 mm) and quantified on a
240 ± 60 (40)

Shimadzu RID-6A refractive index detector. Separation was performed at 80 ◦ C with

8 ± 1 (0.1)

an eluent of 5 mM H2 SO4 and a flow of 0.6 ml/min. Prior to analysis samples were
filtered through a 0.2 ␮m filter and diluted by eluent.
0

2.8. Statistical analysis

Statistical analyses were performed in SAS JMP 7, SAS Institute.

3. Results and discussion

nkat/ml(nkat/mg)
␤-Mannosidase

3.1. Palm kernel cake composition

146 ± 5 (24)

85 ± 5 (2.1)

10 ± 1 (1.4)
20 ± 0 (2.5)

The main component of the PKC used was mannan, constituting

2±0

35.2% on dry weight basis (Table 2). The glucan content was 8% and
0

the remaining sugars accounted for less than 6%. In total, carbo-
hydrates accounted for almost 50% of PKC. In most other studies,
the content of carbohydrates has also been determined to approx-
imately 50% [3,4]. The majority of glucose in PKC has previously
51,200 ± 1900 (8500)
81,800 ± 6600 (2050)

92,100 ± 5900 (8400)

36,800 ± 2100 (5300)

been verified not to be starch but rather cellulose [3,4]. This was
nkat/ml (nkat/mg)

not analysed in the present study. The lignin content in PKC was
1100 ± 90 (55)
380 ± 30 (9.5)

15%, which is lower than some other lignocellulosic materials such

2240 ± 0 (39)
Mannanase

as wood or straw [2]. Low lignin content is generally favourable

for enzymatic hydrolysis as lignin can adsorb enzymes and result
in irreversible loss of enzyme activity [14]. It is generally believed
0

that the lignin is from shell fragments present in the PKC [6]. The
protein content was 15%, which is the reason why PKC is at present
used as animal feed, although this is still a rather low-protein feed
compared to Distillers Dry Grain with Solubles (DDGS) or soy pro-
Specific activities are given in brackets. Nd—not determined.
Total protein

tein feed [15]. The oil/fat content was not determined, but it was
reported by the supplier to be up to 9%. This is accordance with
List of enzyme preparations and main enzyme activities.

10
3
1

1
2
0

Name given in brackets is short form used in text.

what has been published, but this value is much depending on the
±
±
±
±
±
±
±
±
g/l

production method [6,16].

8
58
6
40
40
11
7
20

3.2. Effect of pretreatment conditions

Lignocellulosic materials such as straw and wood require in gen-

Gammanase 1.0L (Gammanase)

eral a rather severe pretreatment in order to open up the material

␤-Mannosidase (mannosidase)
Celluclast 1.5 FG L (Celluclast)

Pectinex Ultra SP-L (Pectinex)

Mannaway 25L (Mannaway)

Novozym 51054 (NZ51054)

and make the carbohydrates readily accessible for enzymes [17].

It was not known how PKC would respond to a thermal pretreat-
Novozym 188 (NZ188)

ment. Three different conditions were applied in order to clarify the

Ultraflo XL (Ultraflo)
Commercial namea

extent of pretreatment needed to make PKC (mainly mannan and

01 cellulose) ready for enzymatic hydrolysis. Condition A was the con-
trol employing the raw material directly without any pretreatment.
d agung isnaini
Condition B was autoclaving at 126 ◦ C for 11 min and C was heating
Table 1

to 180 ◦ C and holding it for 10 min. The results show that the high-
a

est amount of all sugars after enzymatic hydrolysis was obtained

180 J.M. Cerveró et al. / Enzyme and Microbial Technology 46 (2010) 177–184

Table 3
Effect of pretreatment conditions on enzymatic hydrolysis of PKC.

Pretreatment temperature Pretreatment time Mannose Glucose Total

(◦ C) (min) (%)a (%)a (g/l)b

Condition A – – 66.0 ± 0.4 13.8 ± 0.0 12.9 ± 0.1

Condition B 126 11 71.7 ± 0.7 15.0 ± 0.1 13.9 ± 0.1
Condition C 180 10 58.4 ± 6.4 13.6 ± 1.4 11.4 ± 1.2

All reactions carried out at 5% (w/w) of PKC. Enzymatic hydrolysis was for 24 h with a 1:1 mixture of Mannaway and Gammanase with total 10% (v/w) enzyme loading.
a
Relative to maximum theoretical based on composition as given in Table 2.
b
Sum of released sugars (mannose and glucose).

Table 4
Sugars released in 24 h hydrolysis of 5% PKC using individual enzymes with 10% (v/w) enzyme loading.

Arabinose Galactose Glucose Xylose Mannose Total

(%)§ (%)§ (%)§ (%)§ (%)§ (g/l)#

NZ51054 0e 0d 2d 8cd 4d 0.96cd

Ultraflo 12c 0d 10bc 12a 1d 0.91cd
Gammanase 36a 21b 11b 0f 24b 5.65a
Mannaway 0e 0d 2d 7d 30a 6.30a
Celluclast 7d 0d 17a 11ab 1d 1.26c
Pectinex 35a 31a 10c 2e 11c 3.14b
NZ188 24b 14c 11bc 2e 8c 2.48b
Mannosidase* 0e 0d 2d 10bc 0d 0.24d

Mean values marked by different letters in same column are significantly different at ˛ = 0.05 level.
#
Sum of released sugars.
§
Relative to maximum theoretical based on composition as given in Table 2.
*
Enzyme loading was 5% (v/w).

employing just a simple autoclavation (Condition B) (Table 3). The
amount of mannose and glucose released with conditions B corre-
sponded to 72% and 15%, respectively, of the maximum theoretical.
Even without any pretreatment it was possible to reach high degree
of hydrolysis of mannan (Table 3). It is important to realise that the
processing method applied during the production of palm kernel
oil might act as a pretreatment of the PKC. Usually, the process-
ing involves many steps with heating of the oil palm fruit/kernel,
such as steam sterilization, digestion, pressing and kernel separa-
tion [18]. Most of these unit operations can be regarded as sort of
pretreatment and are likely to alter the structure and convertibility
of the material.
The lowest sugar yield was obtained using the highest severity
(180 ◦ C). It is well known from pretreatment of lignocellulosics that
sugar degradation takes place at these elevated temperatures [19].
In addition, the high protein content in combination with sugars
could potentially result in Maillard reaction. Already at 126 ◦ C these
reactions might take place, but at a lower rate.
Although only three temperatures were tested and not fur-
ther optimisation undertaken, the results seem to indicate that
for hydrolysis of mannan no extensive pretreatment is required.
Using the autoclavation method appeared to be a good trade off
between enhancing the accessibility of the PKC (mainly mannan)
for enzymes and to avoid too high sugar loss. Furthermore, the auto-
clavation functioned as a sterilisation thereby minimised the risk of
microbial contamination. This pretreatment was therefore selected
for subsequent experiments.
3.3. Enzymatic hydrolysis of PKC with individual enzyme
preparations
In a first approach, screening of several enzyme preparations
was carried out in order to determine the amount of sugars that
could be released from PKC by these enzymes individually. The
main activities and protein content of these enzymes are listed in
Table 1. The preliminary test was carried out at 10% enzyme loading
(v/w of substrate). It was chosen to load enzymes based on volume
since the enzyme preparations were very different with respect
to purity and presence of activities, e.g. Mannaway was a mono-
component endo-mannanase whereas others such as Pectinex and
Novozym 188 had activity against many substrates and there-
fore presence of multiple enzymes. Addition based on a common
activity, e.g. mannanases activity, was not found feasible as some
enzymes were mainly tested for their auxiliary enzyme activities
that could improve overall hydrolysis yields.
It was chosen to evaluate the enzyme preparations based on
their production of monosaccharides, as main focus of the work
was to produce fermentable sugars. Moreover, S. cerevisiae can only
utilise C6-monosaccharides. The sugar profiles produced by the
enzyme preparations are shown in Table 4.
The highest release of mannose was obtained using Mannaway,
which yielded 30% of theoretical mannose within 24 h (Table 4).
However, Mannaway produced only very low amounts of other
sugars. Mannaway is from the producer given as being a mono-
component enzyme preparation and this is also consistent with the
enzyme activities measured in the enzyme preparation (Table 1).
Although being an endo-acting enzyme, and no activity towards
p-nitrophenyl-␤-d-mannopyranoside was detected, it efficiently
produced free mannose. The Mannaway mannanase must there-
fore be able to work on even small oligasaccharides or close to
chain ends thereby liberating mannose. Opposite to Mannaway, the
other monocomponent endo-mannanase NZ 51054 produced only
modest amounts of mannose (Table 4) despite almost similar man-
nanase activity when measured as reducing sugars produced from
locust bean gum (Table 1). The two mannanases were also very dif-
ferent with respect to specific activity as NZ 51054 had a 4-fold high
specific activity (Table 1). The Mannaway endo-mannanase is given
to be cloned from a Bacillus strain, and previously it has been shown
that some Bacillus endo-mannanases are able to release mannose
[20]. However, many other endo-mannanases are mainly releasing
mannobiose, mannotriose or higher and are not able to hydrolyse
short chain manno-oligosaccharides [21–23].
Gammanase was slightly less efficient compared to Mannaway
for mannose release (24% of theoretical), but was capable of also
releasing glucose (11% of theoretical) as well as arabinose and
galactose (Table 4). The lower mannose release by Gammanase is
consistent with the lower mannanase loading compared to Mann-
away (5120 and 8180 nkat/g PKC, respectively). The enzyme protein
 J.M. Cerveró et al. / Enzyme and Microbial Technology 46 (2010) 177–184
loading was with Mannaway 4 mg protein/g PKC and with Gam-
manase 0.6 mg protein/g PKC. Based on the actual enzyme protein
loading Gammanase was more effective than Mannaway. This is
most likely due to that Gammanase contains ␤-mannosidase and
␣-galactosidase activity, which assist in hydrolysis of galactoman-
nan. The ␤-mannosidase preparation released no mannose from
PKC, which was likely due the absence of suitable substrates for
the enzyme, i.e. di- tri-, or oligosaccharides of mannose.
Celluclast, being mainly a cellulase preparation, gave the high-
est level of glucose (17% of theoretical) among all tested enzymes
(Table 4). However, considering that the cellulase loading corre-
sponded to 97 FPU/g cellulose, which is a very high enzyme loading
[2,24], this was a low conversion. The mannose activity present
in Celluclast was among the lowest (Table 1), and consequently
limited amounts of mannose were released (1% of theoretical).
The pectinase preparation Pectinex contained relatively high
mannanases activity and the highest ␣-galactosidase activity of all
tested enzymes (Table 1). Consistent with the high ␣-galactosidase
activity, Pectinex gave the highest galactose release (31%). Pectinex
was also able to release significant amounts of mannose, arabinose
and glucose and gave the third best overall sugar release (Table 4).
NZ 188 showed similar broad specificity compared to Pectinex,
although at a lower level.
Ultraflo, a glucanase and xylanase preparation [25], was the best
for releasing xylose (12%) and among the best for releasing glu-
cose (10%). Despite the 25-fold lower cellulase activity compared
to Celluclast, the two enzyme preparations released almost similar
amounts of glucose. Either this glucose originates from other glu-
cans than cellulose or the remaining cellulose is not accessible to
the enzymes. The majority of the glucose in PKC should, however,
be in the form of cellulose [3].
Overall, Mannaway releases most total sugar, 23% of theoretical,
corresponding to 122 g/kg PKC. Although Gammanase was less effi-
cient at releasing mannose, the total sugar release was only slightly
less (21%) due to the more efficient release of other sugars, espe-
cially glucose. In addition, the enzyme protein loading was almost
7-fold lower using Gammanase compared to Mannaway.
3.4. Hydrolysis with enzymes mixtures
Mannaway and Gamannase were both efficient at releasing
mannose, but Mannaway very poorly released other sugars. Based
on these results, mixtures of most enzyme preparations were tested
with Mannaway or Gammanase in a 1:1 ratio to see the possi-
ble synergistic effects between these enzymes and Mannaway or
Gammanase. The test was performed at 50 ◦ C, pH of 5.4 and 24 h of
reaction, with a 10% of total enzyme loading based on dry matter.
If a synergistic effect between two enzymes preparations is seen,
the sum of the mixture should be higher than the theoretical sum
based on the individual enzymes when acting alone.
From Table 5 it can be seen that the mixture of Mannaway and
Gammanase resulted in 30% more mannose liberated than expected
from the sum of the two when used separately. Actually, more man-
nose was released than expected from the sum of the two when
added individually at a 10% enzyme loading (Table 4). The mixture
hydrolysed 63% of mannan into mannose in 24 h. Mixing the two
enzymes therefore clearly shows a synergistic action on mannan.
Based on the measured enzyme activities (Table 1), the resulting
mannanase activity should be 6655 nkat/g PKC (or 18,900 nkat/g
mannan) in the mixture compared to 8180 and 5120 nkat/g PKC
when using Mannaway and Gammanase alone, respectively. Since
Mannaway did not contain any ␤-mannosidase or ␣-galactosidase
activity, the improved hydrolysis by mixing Mannaway and Gam-
manase is likely the effect of combining the Mannaway mannanases
with the ␤-mannosidase and ␣-galactosidase activity from the
Gammanase preparation.
181
The only other combination that clearly showed synergistic
effect on mannan hydrolysis was Celluclast and Gammanase, which
released 45% more mannose than expected from the sum of the
two alone (Table 5). According to the measured enzyme activities
(Table 1), both contain all the enzyme activities needed for hydro-
lysis of galactomannan, although Celluclast in general had lower
activities. It is thus puzzling how mannan hydrolysis is improved
by mixing the two enzyme preparations.
Mixing either Mannaway or Gammanase with the ␤-
mannosidase from Megazyme did not improve mannose pro-
duction (Table 5). Addition of extra ␤-mannosidase activity was
expected to improve the mannose yield especially in combination
with Mannaway as it does not contain ␤-mannosidase activity.
However, this was not observed. The reason likely being that the
␤-mannosidase activity under the given conditions was rather low
(Table 1). According to the product information from the supplier,
the mannosidase enzyme has pH optimum around 6.5 and temper-
ature optimum around 35 ◦ C. In the current experiment, at 50 ◦ C,
the activity could therefore be lost completely already after few
hours. More suitable ␤-mannosidases thus have to be used in future
experiments.
Part of the mannan in PKC is believed to be galactomannan, but
other parts are likely crystalline in structure [8]. As only a fraction
of the mannan is substituted, the actual degree of substitution on
this fraction cannot be estimated. Based on the measured compo-
sition (Table 2), the overall galactose substitution was 0.05. Mixing
of different enzyme preparations did not reveal that release of
galactose was coupled with higher conversion of mannan into man-
nose (Table 5). The mixture of Pectinex and Gammanase had the
same mannanase activity as Celluclast and Mannaway (4400 and
4202 nkat/g PKC, respectively) but very different ␣-galactosidase
activity (49 and 0.4 nkat/g PKC, respectively). Despite the Cellu-
clast and Mannaway mixture was not able to remove galactose
side groups, these two mixtures resulted in the same mannan
conversion (21 and 24%, respectively). At the conversion levels
obtained after 24 h of hydrolysis, the galactose substitution might
not yet be hindering hydrolysis, which is why no clear effect of
␣-galactosidase activity was observed.
Celluclast was the best enzyme preparation to hydrolyse cel-
lulose into glucose but only limited amounts of mannose was
produced. However, mixing Celluclast with either Mannaway or
Gammanase significantly improved cellulose hydrolysis (Table 5).
The yield of glucose was 1.8–2.5 times higher than expected from
the individual enzyme preparations thereby showing synergistic
action in the binary mixtures. In the best case (Celluclast and
Gammanase), the cellulose conversion into glucose was 53% of
theoretical. This yield was obtained with a cellulase loading of
50 FPU/g cellulose, which is still high compared to what is com-
monly used for other lignocellulosic materials [2,24]. The other
combinations did not show significant synergistic effect on release
of the major sugars mannose and glucose and only the combination
Celluclast–Gammanase and Pectinex–Mannaway showed syner-
gistic effect on release of arabinose and galactose, respectively.
3.5. Hydrolysis reaction profile
In order to further study the synergistic effect between Man-
naway, Gammanase and Celluclast a more thorough experiment
with these three enzyme preparations was set up. Again the three
enzyme preparations were tested alone at 5% loading and together
also with 5% loading of each enzyme, but the hydrolysis was then
followed for 96 h to see the effect at more extensive levels of hydro-
lysis.
After 96 h, Mannaway, Gammanase and the mixture of the
two enzymes yielded 39, 44 and 87%, respectively, conversion
of mannan into mannose (Fig. 1A). The sum of the two individ-
182 J.M. Cerveró et al. / Enzyme and Microbial Technology 46 (2010) 177–184

Table 5
Release of sugars after hydrolysis of 5% PKC for 24 h using mixtures of enzymes in 1:1 ration and a total enzyme loading of 10% (v/w).

Enzymes Arabinose Galactose Glucose Xylose Mannose Totalb

(%)a (%)a (%)a (%)a (%)a (g/l)

Ultraflo + Gammanase Measured 39 19 24 5 21 5.81

Sumc 47 21 21 13 23 6.16
Celluclast + Gammanase Measured 59 31 53 7 33 9.61
Sum 37 20 29 14 23 6.43
Pectinex + Gammanase Measured 40 34 19 0 21 5.64
Sum 71 49 23 2 30 7.92
Mannosidase + Gammanase Measured 31 13 11 0 18 4.31
Sum 37 20 15 10 22 5.59
Ultraflo + Mannaway Measured 5 0 22 9 30 7.00
Sum 9 1 10 20 27 6.13
Celluclast + Mannaway Measured 0 0 46 11 24 6.77
Sum 0 0 18 21 27 6.40
Pectinex + Mannaway Measured 40 62 11 0 33 7.91
Sum 33 28 12 9 34 7.89
Mannosidase + Mannaway Measured 0 0 2 6 25 5.03
Sum 0 0 4 17 26 5.56
Mannaway + Gammanase Measured 45 42 14 0 63 13.7
Sum 37 20 15 7 48 10.7
a
Relative to maximum theoretical based on composition as given in Table 2.
b
Sum of released sugars.
c
Theoretical sum is based on sum of sugar released by individual enzyme preparations using 5% enzyme loading.

ually was therefore almost equal to the result obtained in the
mixture. Celluclast and Gammanase showed synergistic effect on
mannan hydrolysis also at 96 h (Fig. 1A), but here the final con-
version level was only 52%. The apparent lack of synergistic effect
with Mannaway and Gammanase could be due to that almost all
Fig. 1. Conversion of mannan and glucan into mannose (A) and glucose (B) dur-
ing hydrolysis of PKC with individual enzymes (closed symbols) or mixtures
(open symbol, solid line). Individual enzymes were Gammanase (䊉), Mannaway
() and Celluclast (). The mixtures were Gammanase + Mannaway (), Gam-
manase + Celluclast () and Mannaway + Celluclast (). Dashed line with open
symbol is theoretical conversion with mixtures calculated from the conversion
obtained with the individual enzymes. Enzyme loading with individual enzymes
was 5% (v/w) and in mixtures 5% (v/w) of each enzyme. Substrate loading was 5%
(w/w) PKC.
substrate was hydrolysed at this point. During the first 24 h, the
Mannaway–Gammanase mixture released mannose at a higher
rate than the sum of the individual enzymes. In the last part of
the hydrolysis the difference between the measured and theoreti-
cal sum narrowed down as the substrate started to get depleted.
It is interesting to note that although Mannaway alone is very
effective initially, the hydrolysis seems to almost stop after 48 h
(Fig. 1A). Gammanase on the other hand releases mannose with
an almost constant rate. The lack of auxiliary enzymes such as ␣-
galactosidase, ␤-mannosidase and cellulases might explain such a
difference. Mannaway lack these enzymes, and as hydrolysis pro-
ceeds, substituted manno-oligosaccharides that are not substrate
for the endo-mannanase starts to accumulate.
Mixing of either Gammanase or Mannaway with Celluclast
significantly enhanced the hydrolysis of cellulose into glucose
(Fig. 1B). The highest conversion (72%) was obtained using the
mixture of Mannaway and Celluclast. Mannaway had no cellulase
activity (filter paper activity, Table 1), but Mannaway in combina-
tion with Celluclast show strong synergistic effect, almost doubling
the glucose release (Fig. 1B). This synergistic effect upon combina-
tion with Celluclast indicates that removal of mannan increases
accessibility of cellulases to cellulose. Similar effects have been
observed with addition of hemicellulases during hydrolysis of
cellulose with other lignocellulosic materials [24,26]. The same
rationale could possibly explain the synergistic effect between Cel-
luclast and Mannaway/Gammanase in hydrolysing more mannan
as the mannanase activity in Celluclast was very minor.
Employing a 1:1 mixture of Gammanase and Mannaway was
effective for hydrolysis of mannan and 74% of potential man-
nose was produced already after 48 h. Increasing the hydrolysis
time to 96 h increased the mannose yield to 87%, which cor-
responds to production of 340 g mannose/kg of PKC. In total,
the Gammanase–Mannaway mixture produced around 365 g fer-
mentable C6 sugar (galactose, glucose, mannose) per kg of PKC
in 96 h, which is 75% of theoretical. This was obtained using a
total enzyme protein loading of 2.3 mg/g PKC, which is rather low
compared to what is commonly used for hydrolysis of other ligno-
cellulosic materials [24].
The binary mixtures were only tested in a 1:1 ratio. But since
part of the effect is likely due supplementing the Mannaway man-
nanases with ␤-mannosidase and ␣-galactosidase activity from
Gammanase another ratio could be more effective. Mannaway and
Gammanase contained only limited cellulase activity. Cellulose
 J.M. Cerveró et al. / Enzyme and Microbial Technology 46 (2010) 177–184
Fig. 2. Concentration of mannose (), glucose () and ethanol (䊉) during the
fermentation of 10% (w/w) PKC using Saccharomyces cerevisiae after 24 h. of pre-
hydrolysis using a 1:1 mixture Mannaway and Gammanase (10% (v/w) enzyme
loading).
hydrolysis could thus be improved by including also Celluclast in
the mixture. Balancing the ternary mixture for optimum hydrol-
ysis of both mannan and cellulose would require more extensive
studies, but could result in even higher conversion yields than
reported here.
3.6. Hydrolysis and fermentation of PKC
S. cerevisiae is capable of fermenting glucose, mannose and
galactose into ethanol and was therefore used to assess the fer-
mentability of the PKC hydrolysate. PKC can contain compounds
inhibitory to the yeast which would reduce the fermentation yield.
The fermentation was therefore tested with 10% PKC to verify if
higher concentrations of PKC would negatively affect the fermen-
tation performance. The experiment was performed using a 1:1
mixture of Mannaway and Gammanase, pre-hydrolysis for 24 h at
50 ◦ C and subsequent fermentation at 32 ◦ C. The need for extra
nutrients for the yeast was tested by inclusion of yeast extract in
the medium.
Using 10% PKC without addition of yeast extract resulted in
the production of 16.2 g/l mannose and 3.0 g/l of glucose during
the pre-hydrolysis (Fig. 2). Within the first 19 h of fermentation,
free mannose and glucose was rapidly metabolised into ethanol.
Throughout the rest of the fermentation, the concentration of
glucose and mannose remained low. The ethanol concentration
continued to increase indicating that more glucose and/or man-
nose were indeed liberated (Fig. 2). After 113 h of fermentation, the
ethanol concentration reached 12.5 g/l. This corresponds to around
52% of the maximum theoretical ethanol that can be achieved based
on the total amount of mannose and glucose present in PKC. No
differences in the course of the fermentations were seen between
fermentation with and without addition of yeast extract (data not
shown). With addition of yeast extract as a nitrogen source 12.4 g/l
of ethanol was obtained after 113 h.
The ethanol yield (ethanol produced per g consumed sugar)
during the first 19 h was on average (with/without yeast extract)
0.48 g/g or 95% of theoretical, based on the initial sugar and sugar
after 19 h. However, during this period monosaccharides were
also produced by the action of the enzymes and the true ethanol
yield is therefore somewhat lower. Extrapolating from the ethanol
production in the period 19–48 h, a more correct yield could be
estimated to around 0.4 g/g or 80% of theoretical. The low yield
could indicate that some inhibitors are present in the PKC. How-
183
ever, the initial sugars were rapidly fermented within the first
19 h.
In the hydrolysis experiments with 5% substrate consistency,
almost 90% conversion of mannan was obtained in 96 h. Assum-
ing an ethanol yield of 0.4 g/g throughout the entire fermentation
gives that around 65–70% of the mannan and cellulose in PKC was
hydrolysed in 137 h. The lower conversion could be due to the lower
temperature, inhibitory effect of ethanol on the enzyme perfor-
mance or an effect of the higher substrate concentration. Similar
effects have been observed for conversion of wheat straw into
ethanol [17]. It should therefore be tested if the process is best oper-
ated as separate hydrolysis and fermentation or as simultaneous
saccharification and fermentation. It should also be investigated
if mannases are affected by product inhibition by mannose and
mannobiose as is well known from cellulases [1].
Another important observation was that yeast extract did not
improve the fermentation yield, neither in the amount of ethanol
produced nor in the reaction speed. This implies that PKC con-
tains sufficient nutrients to make the yeast grow and ferment. PKC
contains 15% proteins or amino acids which might sustain good
fermentation performance of the yeast. This is quite important
because this adds an important economical saving to the process
in terms of fewer chemicals to purchase
Based on 65–70% of the carbohydrates in PKC being converted
into ethanol implies that the residue after fermentation has protein
content around 22%, as the original protein in PKC gets concen-
trated. The yeast could further contribute with protein making the
actual protein content in the solids after fermentation even higher.
Ethanol production from carbohydrates in PKC therefore has the
potential to make a more concentrated protein product, which
could be beneficial when used as a protein feed.
4. Conclusions
PKC was demonstrated to be a potentially good raw material for
bioethanol production. The hydrolysis tests showed that the yield
of monosaccharides obtained represented nearly 75% of the total
polysaccharides content in PKC. Although autoclavation was used
to pretreat the material, results showed that even without pretreat-
ment it was possible to obtain high mannan conversion. Mannaway
was found to be the single best enzyme preparation for hydrolysis
of mannan, whereas Celluclast was the best performing enzyme
for cellulose hydrolysis. Preliminary testing of various blends of
enzyme preparations revealed that positive synergistic effects were
observed among some of the tested enzyme preparations. A 1:1
mixture of Gammanase and Mannaway resulted in 2.8-fold more
mannose being released compared to the individual enzyme prepa-
rations in 24 h. Using this mixture, 365 g of fermentable hexose
sugar could be produced in 96 h applying a modest enzyme protein
loading of 2.3 mg/g PKC.
Successful fermentation of the mannose and glucose from PKC
was obtained with traditional yeast S. cerevisiae without any use
of additional nutrients. This shows that the carbohydrates in PKC
could efficiently be utilised for ethanol production. From the palm
oil fruit it is therefore possible to produce liquid fuel in the form
of biodiesel (from the oil), bioethanol and a protein rich animal
feed from the fermented PKC residue. The residue after extracting
sugars for fermentation has a higher protein content, which makes
this residue a more valuable feed product than PKC.
Acknowledgments
Authors want to acknowledge Novozymes A/S for their support
of the research. José María Cerveró wants to acknowledge Spanish
Government for funding his research and Faculty of Life Sciences,
Copenhagen, Denmark, for the research resources provided.
184 J.M. Cerveró et al. / Enzyme and Microbial Technology 46 (2010) 177–184
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Annotations

Enzymatic hydrolysis and fermentation of palm kernel press

cake for production of bioethanol
Cerveró, José María; Skovgaard, Pernille Anastasia; Felby, Claus; Sørensen,
Hanne Risbjerg; Jørgensen, Henning

01 muhamad agung isnaini Page 3

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