Enzymatic hydrolysis and fermentation of palm kernel press cake for production of bioethanol

(1)

Contents lists available atScienceDirect

Enzyme and Microbial Technology

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / e m t

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^,∗

aDepartment of Chemical Engineering and Environmental Technology, University of Oviedo, C/Julián Clavería, 8, 33071, Oviedo, Spain

bDanish Centre for Forest, Landscape and Planning, University of Copenhagen, Rolighedsvej 23, DK-1958 Frederiksberg C, Denmark

cNovozymes A/S, Krogshøjvej 36, DK-2880 Bagsværd, Denmark

a r t i c l e i n f o

Article history:

Received 29 June 2009 Received in revised form 25 September 2009 Accepted 27 October 2009

Keywords:

Bioethanol

Palm kernel press cake Mannan

Saccharomyces cerevisiae

a b s t r a c t

Palm kernel press cake (PKC) is a residue of palm oil extraction, which was found to contain 48.5% of total carbohydrates of which 35.2% was mannan. The present study examines enzymatic hydrolysis of polysaccharides from the cell-wall material present in PKC to obtain monosaccharides that can be substrate in various fermentation processes such as ethanol production. The requirements for pretreatment were investigated and it was found that mannan in PKC was readily hydrolysed without any pretreatment. Several enzyme preparations were tested and Mannaway 25L was found as the best for releasing mannose, and Gammanase 1.0L worked well in degrading cellulose and mannose. Binary mixtures of enzymes were tested to increase the conversion, and 1:1 mixture of Mannaway 25L and Gammanase 1.0L showed good synergistic effect releasing 30% more mannose than the sum obtained using these 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 usingSaccharomyces cerevisiaewith an ethanol yield of 125 g/kg PKC.

1. Introduction

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, hemicelluloses and lignin. Non-starch polysaccharides such as cellulose are often difﬁcult to process into fermentable sugars. Usually, the process 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:[email protected](H. Jørgensen).

1Present address: BioGasol ApS, Lautrupvang 2A, DK-2750 Ballerup, Denmark.

yeastSaccharomyces cerevisiaenormally applied in ethanol fermentations 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 kernels 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 extraction 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.

doi:10.1016/j.enzmictec.2009.10.012

(2)

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 different 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 composition 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 hydrolysed material was used for fermentation withS. cerevisiaeto 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) fromC. 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 1summarizes 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 bottles 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 without 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 pretreatment/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 bottles 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 preparation 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 published 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.

(3)

Table1 Listofenzymepreparationsandmainenzymeactivities. CommercialnameaTotalproteinMannanase␤-Mannosidase␣-Galactosidase␤-GlucosidaseFilterpaperactivity g/lnkat/ml(nkat/mg)nkat/ml(nkat/mg)nkat/ml(nkat/mg)nkat/ml(nkat/mg)FPU/ml(FPU/mg) ␤-Mannosidase(mannosidase)8±0020±0(2.5)00nd Celluclast1.5FGL(Celluclast)58±32240±0(39)2±08±1(0.1)450±50(7.8)75(1.3) Gammanase1.0L(Gammanase)6±151,200±1900(8500)146±5(24)240±60(40)530±7(88)<2 Mannaway25L(Mannaway)40±1081,800±6600(2050)0000 Novozym188(NZ188)40±3380±30(9.5)85±5(2.1)550±40(13.8)6040±550(151)0 Novozym51054(NZ51054)11±092,100±5900(8400)000nd PectinexUltraSP-L(Pectinex)7±136,800±2100(5300)10±1(1.4)740±40(106)37±1(5.3)12(1.7) UltrafloXL(Ultraflo)20±21100±90(55)00580±16(29)3(0.2) Specificactivitiesaregiveninbrackets.Nd—notdetermined. aNamegiveninbracketsisshortformusedintext.

Table 2

Composition of PKC (on dry basis).

Component Composition (%)

Glucose 7.7

Xylose 2.6

Arabinose 1.1

Galactose 1.9

Mannose 35.2

Protein 15.0

Lignin 15.1

Ash 5.0

Sugars reported as anhydrous form.

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 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.

Ethanol, acetate, glycerol were separated on a Dionex Summit system fitted with a Phenomenex Rezex RHM column (7.8 mm×300 mm) and quantified on a Shimadzu RID-6A refractive index detector. Separation was performed at 80^◦C with an eluent of 5 mM H2SO4and a flow of 0.6 ml/min. Prior to analysis samples were filtered through a 0.2␮m filter and diluted by eluent.

2.8. Statistical analysis

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

3. Results and discussion 3.1. Palm kernel cake composition

The main component of the PKC used was mannan, constituting 35.2% on dry weight basis (Table 2). The glucan content was 8% and the remaining sugars accounted for less than 6%. In total, carbohydrates 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 been veriﬁed not to be starch but rather cellulose[3,4]. This was not analysed in the present study. The lignin content in PKC was 15%, which is lower than some other lignocellulosic materials such 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 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 protein 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 what has been published, but this value is much depending on the production method[6,16].

3.2. Effect of pretreatment conditions

Lignocellulosic materials such as straw and wood require in general a rather severe pretreatment in order to open up the material and make the carbohydrates readily accessible for enzymes[17].

It was not known how PKC would respond to a thermal pretreatment. Three different conditions were applied in order to clarify the extent of pretreatment needed to make PKC (mainly mannan and cellulose) ready for enzymatic hydrolysis. Condition A was the con- trol employing the raw material directly without any pretreatment.

Condition B was autoclaving at 126^◦C for 11 min and C was heating to 180^◦C and holding it for 10 min. The results show that the highest amount of all sugars after enzymatic hydrolysis was obtained 01

muhamad agung isnaini

(4)

Table 3

Effect of pretreatment conditions on enzymatic hydrolysis of PKC.

Pretreatment temperature (^◦C)

Pretreatment time (min)

Mannose (%)^a

Glucose (%)^a

Total (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.

aRelative to maximum theoretical based on composition as given inTable 2.

bSum 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 0^e 0^d 2^d 8^cd 4^d 0.96^cd

Ultraﬂo 12^c 0^d 10^bc 12^a 1^d 0.91^cd

Gammanase 36^a 21^b 11^b 0^f 24^b 5.65^a

Mannaway 0ê 0^d 2^d 7^d 30â 6.30â

Celluclast 7^d 0^d 17^a 11^ab 1^d 1.26^c

Pectinex 35â 31â 10^c 2ê 11^c 3.14^b

NZ188 24^b 14^c 11^bc 2^e 8^c 2.48^b

Mannosidase^* 0^e 0^d 2^d 10^bc 0^d 0.24^d

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 inTable 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 processing involves many steps with heating of the oil palm fruit/kernel, such as steam sterilization, digestion, pressing and kernel separation[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 further 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 autoclavation 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 therefore 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. cerevisiaecan only utilise C6-monosaccharides. The sugar proﬁles produced by the enzyme preparations are shown inTable 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 monocomponent 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 therefore 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 mannanase activity when measured as reducing sugars produced from locust bean gum (Table 1). The two mannanases were also very different 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 aBacillusstrain, and previously it has been shown that someBacillusendo-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

(5)

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 galactomannan. 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 highest 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 glucose (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, especially 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 possible 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.

FromTable 5it 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 mannose 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.

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 hydrolysis 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 production (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 temperature 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 composition (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 mannose (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 cellulose 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 commonly 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 synergistic 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 hydrolysis.

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-

(6)

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

(%)^a

Galactose (%)^a

Glucose (%)^a

Xylose (%)^a

Mannose (%)^a

Total^b (g/l)

Ultraﬂo + Gammanase Measured 39 19 24 5 21 5.81

Sum^c 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

aRelative to maximum theoretical based on composition as given inTable 2.

bSum 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 conversion 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) during 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 theoretical 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 combination with Celluclast show strong synergistic effect, almost doubling the glucose release (Fig. 1B). This synergistic effect upon combination 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 mannose was produced already after 48 h. Increasing the hydrolysis time to 96 h increased the mannose yield to 87%, which corresponds to production of 340 g mannose/kg of PKC. In total, the Gammanase–Mannaway mixture produced around 365 g fermentable 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 lignocellulosic 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 mannanases with ␤-mannosidase and ␣-galactosidase activity from Gammanase another ratio could be more effective. Mannaway and Gammanase contained only limited cellulase activity. Cellulose

(7)

Fig. 2.Concentration of mannose (), glucose () and ethanol (䊉) during the fermentation of 10% (w/w) PKC usingSaccharomyces cerevisiaeafter 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 hydrolysis 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 fermentation 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 mannose 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-

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 performance 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 contains 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 concentrated. 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 pretreatment 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 preparations 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 yeastS. cerevisiaewithout 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.

(8)

References

[1] Jørgensen H, Kristensen JB, Felby C. Enzymatic conversion of lignocellulose into fermentable sugars: challenges and opportunities. Biofuels, Bioprod Bioref 2007;1:119–34.

[2] Olsson L, Jørgensen H, Krogh KBR, Roca C. Bioethanol production from lignocellulosic material. In: Dumitriu S, editor. Polysaccharides: structural diversity and functional versatility, 2. New York: Marcel Dekker; 2005. p. 957–93.

[3] Düsterhöft EM, Voragen AGJ, Engels FM. Nonstarch polysaccharides from sunﬂower (Helianthus annuus) meal and palm kernel (Elaeis guineenis) meal preparation of cell-wall material and extraction of polysaccharide fractions. J Sci Food Agric 1991;55:411–22.

[4] Knudsen KEB. Carbohydrate and lignin contents of plant materials used in animal feeding. Anim Feed Sci Technol 1997;67:319–38.

[5] Basiron Y. Palm oil production through sustainable plantations. Eur J Lipid Sci Technol 2007;109:289–95.

[6] O’Mara FP, Mulligan FJ, Cronin EJ, Rath M, Caffrey PJ. The nutritive value of palm kernel meal measured in vivo and using rumen ﬂuid and enzymatic techniques.

Livest Prod Sci 1999;60:305–16.

[7] Ramachandran S, Singh SK, Larroche C, Soccol CR, Pandey A. Oil cakes and their biotechnological applications—a review. Bioresour Technol 2007;98:2000–9.

[8] Sundu B, Kumar A, Dingle J. Palm kernel meal in broiler diets: effect on chicken performance and health. Worlds Poult Sci J 2006;62:316–25.

[9] Düsterhöft EM, Posthumus MA, Voragen AGJ. Nonstarch polysaccharides from sunﬂower (Helianthus-Annuus) meal and palm-kernel (Elaeis-Guineensis) meal investigation of the structure of major polysaccharides. J Sci Food Agric 1992;59:151–60.

[10] Aspinall GO. Mannans, Galactomannans and Glucomannans. In: Aspinall GO, editor. Polyscaaharides, 1. Oxford: Pergamon Press Ltd; 1970. p. 85–94.

[11] Wood TM, Bhat KM. Methods for measuring cellulase activities. In: Wood WA, Kellogg ST, editors. Biomass—part A: cellulose and hemicellulose. San Diego:

Academic Press; 1988. p. 87–112.

[12] Stålbrand H, Siika-Aho M, Tenkanen M, Viikari L. Puriﬁcation and characterization of two ␤-mannanases from Trichoderma reesei. J Biotechnol 1993;29:229–42.

[13] Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D. Deter- mination of structural carbohydrates and lignin in biomass.http://devafdc.

nrel.gov/pdfs/9572.pdf; 2006.

[14] Eriksson T, Börjesson J, Tjerneld F. Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose. Enzyme Microb Technol 2002;31:353–

64.

[15] Balan V, Rogers CA, Chundawat SPS, Sousa LD, Slininger PJ, Gupta R, et al.

Conversion of extracted oil cake ﬁbers into bioethanol including ddgs, canola, sunﬂower, sesame, soy, and peanut for integrated biodiesel processing. J Am Oil Chem Soc 2009;86:157–65.

[16] Iluyemi FB, Hanaﬁ MM, Radziah O, Kamarudin MS. Fungal solid state culture of palm kernel cake. Bioresour Technol 2006;97:477–82.

[17] Jørgensen H, Vibe-Pedersen J, Larsen J, Felby C. Liquefaction of lignocellulose at high-solids concentrations. Biotechnol Bioeng 2007;96:862–70.

[18] Jacquemard JC. Oil extraction. In: Tindall HD, editor. Oil Palm. London: MacMil- lan; 1998. p. 102–16.

[19] Larsson S, Palmqvist E, Hahn-Hagerdal B, Tengborg C, Stenberg K, Zacchi G, et al. The generation of fermentation inhibitors during dilute acid hydrolysis of softwood. Enzyme Microb Technol 1999;24:151–9.

[20] Ooi T, Kikuchi D. Puriﬁcation and some properties of␤-mannanase fromBacillus sp. World J Microbiol Biotechnol 1995;11:310–4.

[21] Jiang Z, Wei Y, Li D, Li L, Chai P, Kusakabe I. High-level production, puriﬁcation and characterization of a thermostable␤-mannanase from the newly isolated Bacillus subtilisWY34. Carbohydr Polym 2006;66:88–96.

[22] Gübitz GM, Hayn M, Sommerauer M, Steiner W. Mannan-degrading enzymes from Sclerotium rolfsii: characterisation and synergism of two endo-␤-mannanases and a␤-mannosidase. Bioresour Technol 1996;58:127–

35.

[23] Kurakake M, Komaki T. Production of ␤-mannanase and ␤-mannosidase fromAspergillus awamoriK4 and their properties. Curr Microbiol 2001;42:

377–80.

[24] Kumar R, Wyman CE. Effect of enzyme supplementation at moderate cellulase loadings on initial glucose and xylose release from corn stover solids pretreated by leading technologies. Biotechnol Bioeng 2009;102:457–67.

[25] Sørensen HR, Pedersen S, Viksø-Nielsen A, Meyer AS. Efﬁciencies of designed enzyme combinations in releasing arabinose and xylose from wheat arabi- noxylan in an industrial ethanol fermentation residue. Enzyme Microb Technol 2005;36:773–84.

[26] Berlin A, Maximenko V, Gilkes N, Saddler J. Optimization of enzyme complexes for lignocellulose hydrolysis. Biotechnol Bioeng 2007;97:287–96.

(9)

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

Enzymatic hydrolysis and fermentation of palm kernel press cake for production of bioethanol

Enzyme and Microbial Technology

Enzymatic hydrolysis and fermentation of palm kernel press cake for production of bioethanol

José María Cerveró

, Pernille Anastasia Skovgaard

, Claus Felby

, Hanne Risbjerg Sørensen

, Henning Jørgensen

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

muhamad agung isnaini Page 3

17/3/2024 15:49